sbornik_s_2011.pdf

XI. Woorkshop 

of Physsical 

Chemists and a Electrochemmists´11 

XXI. 

PPraco 

ovní setk kání fyzik f kální ích 

chhemi 

iků a elek ktroc chemmiků 

11th WWorkshop 

p of Physsical 

Che emists an nd Electrrochemi 

ists 

Sborník 

př říspěv vků 

1. – 2. 6. 2011 2 

Přírodo ovědecká ffakulta 

Ma asarykovy y univerzityy 

a 

Agronommická 

faku ulta MEND DELU 

Brno o 

- 1 - 

Brnoo

XI. Workshop oof 

Physical Cheemists 

and Electrochemists´11 

Přírodovvědecká 

ffakulta 

MU M V Brněě 

Ústav chemmie 

Kotlářská 2 

611 37 Brnno 

http://wwww.sci.munii.cz/ 

Agronommická 

fakkulta 

MEN NDELU v BBrně 

Ústav chemmie 

a biochhemie 

Zemědělskká 

1 

613 00 Brnno 

http://ucb. .af.mendeluu.cz 

Libuše Trnnková 

libuse@chhemi.muni.ccz 

(Ústav ch hemie, RECCETOX, 

Př řF MU) 

René Kizek 

kizek@sci.muni.cz 

(ÚÚstav 

chem mie a biocheemie 

MEND DELU, REC CETOX, PřFF 

MU) 

Vojtěch AAdam, 

Sylvie 

Holubová, 

Kristina Nádeníčko ová, Olga Kr ryštofová, JJiří 

Sochor, Petr 

Koudelka a Ondřej Zíítka 

Publikace neprošla jaazykovou 

kontrolou. k Jednotlivé příspěvky jsou publiikovány 

tak k, jak 

byly dodáány 

autoryy. 

Za věcn nou a odbbornou 

spr rávnost jsou 

plně oddpovědni 

autoři a 

příspěvků. . 

Podrobné informacee 

včetně sb borníku přííspěvků 

jso ou k dispoz zici na inteernetové 

adrese 

a 

http://labiffel.byethosst24.com/ 

ISBN 9788‐80‐73755‐514‐0 

ORRGANIZA 

ACE POŘŘÁDAJÍC 

CÍ KONFE ERENCI 

ORGGANIZAČ 

ČNÍ ZABEEZPEČEN 

NÍ KONFERENCEE 

- 2 - 

Brno

XI. Woorkshop 

of Physsical 


PPracovvní 

setk kání byylo 

podpořeno 

výzzkumn 

nými 

projekty 

Spo onzoři pracov vního setkán s ní 

- 3 - 

Brnoo




Úvodemm 

Milí přátellé, 

pro konánní 

letošníhoo 

XI. Pracov vního setkáání 

fyzikáln ních chemi iků a elektrrochemiků 

Workshopp 

of Physiccal 

Chemist ts and Elect ctrochemist ts) bylo zvo oleno datumm 

1. a 2. če ervna 

roku 2011. 

Místem kkonání 

je op pět jihomorravská 

met tropole, Brn no. Na účasstníky 

z Če eské a 

Slovenské republiky čekají dva konferenční 

dny nap plněné blok kem plenárn rních předn nášek, 

přednášekk 

nadaných studentů (Sekce ( mladdých) 

a pře ednášek, které 

shrnujíí 

nové pozn natky 

v oblasti ffyzikální 

a biofyzikál lní chemie, , elektroch hemie a bio oelektrocheemie, 

analy ytické 

chemie a chemie noových 

či po okročilých materiálů. . Již druhý ým rokem jje 

do prog gramu 

kromě přííspěvků 

zaaměřených 

na výukuu 

zařazen také t blok přednášek k věnovaný ých v 

současnostti 

hojně diskutova aným témmatům, 

jako 

je nanověda, 

nanomate eriály, 

nanotechnnologie 

a naanobiotech 

hnologie. Vššichni 

se mohou m těšit na vědeckké 

diskuse, které 

budou iniccializoványy 

nejen těmito 

přednášškami, 

ale i prezentace emi velkéhoo 

počtu pos sterů. 

Záštitu nnad 

konferencí 

převzali 

jak rektoři obou univ verzit (Prrof. 

PhDr. 

Petr 

Fiala, Ph.DD., 

LL.M. a Prof. Ing g. Jaroslav Hlušek, CS Sc., Dr.h.c. .), tak děkkani 

přísluš šných 

fakult: za Přírodověědeckou 

fakultu 

MU doc. RND Dr. Jaromír r Leichmannn, 

Ph.D. a za 

Agronomiickou 

fakulttu 

MENDE ELU Prof. Inng. 

Ladislav v Zeman, CSc. 

Počeet 

konferennčních 

příspěvků 

s poostupujícím 

mi ročníky neustále rooste. 

V leto ošním 

roce je jicch 

více nežž 

osmdesát. . Je nejen vvíce 

poster rových sděl lení, ale taaké 

i plenárních 

přednášekk. 

Pozvání nna 

letošní ročník PS FCH a EL LCH přijalo celkem šeest 

význam mných 

českých věědců 

z obooru 

fyzikáln ní chemie a z oborů, kde fyzikál lní chemie hraje důle ežitou 

roli – bioffyzikální 

chhemie, 

biof fyzika, bioeelektrochem 

mie, biome edicína (Inng. 

Tomáš Fessl, 

Dr. Michaael 

Heyrovsský, 

Prof. Emil E Palečeek, 

Prof. Iv van Švancar ra, Prof. Moojmír 

Šob, Prof. 

Jiří Zíma). . Postupnýý 

nárůst př říspěvků zaaznamenává 

á i Sekce mladých, m vee 

které stu udenti 

prezentujíí 

a obhajují 

výsledky y své prácee 

v anglické ém jazyce. Jako každdý 

rok, vyb braná 

komise buude 

hodnotiit 

jejich výkon 

a tři nnejlepší 

bud dou oceněni 

věcnými dary a dipl lomy. 

Hodnocenní 

nebude vynechán no ani v ppřípadě 

po osterů a z důvodů objektivně ějšího 

hodnoceníí 

bude poster 

doplněn n krátkou prrezentací 

je ejího autora a, popř. autoorů. 

Jak ssi 

jistě všimmnete, 

vše echna abstrrakta 

jsou v jazyce an nglickém, mmají 

rozšíř řenou 

podobu a obsahují bbarevné 

ob brázky či ggrafy. 

Abstr rakta jsou součástí sbborníku 

s ISBN 

(sborník ppříspěvků 

odpovídá kritériím k ppro 

hodnoc cení VaV v kategoriii 

„Příspěve ek do 

sborníku“) ). Mohou být 

základem m připravovvané 

publik kace pro ča asopis Internnational 

Journal 

of Electrocchemical 

Science 

– IJES 

(http://wwww.electr 

rochemsci. org/) s IF 22.175 

(z 2009). 

2 

Podrobnossti 

(odevzdáání 

rukopis su a výše pooplatku) 

bu udou sdělen ny během ko konference. 

- 4 - 

Brno 

ů (11 th

XI. Workshop of Physical Chemists and Electrochemists´11 Brno 

Vítáme všechny účastníky XI. Pracovního setkání fyzikálních chemiků a 

elektrochemiků a přejeme všem úspěšnou prezentaci, která může být spolu s diskusními 

příspěvky velmi užitečným pomocníkem v jejich bádání. 

Organizační a vědecký výbor: 

doc. RNDr. Libuše Trnková, CSc. 

doc. Ing. René Kizek, Ph.D. 

doc. Ing. Jaromír Hubálek, Ph.D. 

RNDr. Vojtěch Adam, Ph.D. 

Technické zabezpečení Pracovního setkání: 

Mgr. Sylvie Holubová, 

Mgr. Olga Kryštofová, 

Kristina Nádeníčková 

Ing. Jiří Sochor 

- 5 - 

Libuše Trnková




Orgaanizátoři 

děěkují 

všem letošním ssponzorům 

za podpor ru, která ummožnila 

po ořádat 

tuto, již trradiční, 

akcci: 

ANTON PAAR spool. 

s r.o., EN NVINET sp pol. s r.o., CCHROMSERVIS 

spol. s r. o., MANEKKO 

spol. s r. o., METTROHM 

sp pol. s r. o. , PRAGOLLAB 

spol. s r.o., 

RADANAL 

spol. s r. . o., 2-THE ETA spol. s r. o., TRIG GON PLUS S spol. s r. o., též ČE ESKÁ 

SPOLEČNNOST 

CHEMMICKÁ 

(po obočka Brnoo). 

Věda V máá 

svůj sm mysl, 

pokkud 

je chápána c a jako cesta c k pravdě p 

- 6 - 

Brno


ELECTROCHEMISTRY OF 

METALLOTHIONEINS 

Vojtěch ADAM 1 , Ondřej BLASTIK 1 , Ivo FABRIK 1 , Pavlína ŠOBROVÁ 1 , Jitka 

PETRLOVÁ 1 , Petr MAJZLÍK 1 , Libuše TRNKOVÁ 1 , René KIZEK 1 

1 Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, CZ 

Abstract 

Metallothioneins (MT) are a family of ubiquitous, biologically interesting proteins which 

have been isolated and studied in a wide variety of organisms, including prokaryotes, 

plants, invertebrates and vertebrates. Due to the property of MT being metal-inducible 

and, also, due to their high affinity to metal ions, homeostasis of heavy metal levels is 

probably their most important biological function. In addition, MT are involved in other 

important biochemical pathways including scavenging of reactive oxygen species, 

activation of transcription factors or participation in carcinogenesis. Detection and 

quantification of MT is not simple due to the high content of cysteine and relatively low 

molecular mass. These proteins can be detected very sensitively by electrochemical 

methods. Moreover, MT can be used as a part of biosensors. 

1. INTRODUCTION 

Metallothioneins (MT) belonging to the group of intracellular and low molecular 

mass proteins (from 2 to 16 kDa) were discovered in 1957, when Margoshes and Valee 

isolated them from a horse renal cortex tissue [1]. These proteins have been isolated and 

studied in a wide variety of organisms, including prokaryotes, plants, invertebrates and 

vertebrates [2]. Concerning their primary structure, they are rich in cysteine and have no 

aromatic amino acids. The metal binding domain of MT consists of 20 cysteine residues 

juxtaposed with basic amino acids (lysine and arginine) arranged in two thiol-rich sites 

called α and β. The cysteine sulfhydryl groups can bind 7 moles of divalent metal ions per 

mol of MT, while the molar ratio for monovalent metal ions (Cu and Ag) is twelve. 

Although the naturally occurring protein has Zn2+ in both binding sites, this ion may be 

substituted for another metal ion that has a higher affinity for thiolate such as Pb, Cu, Cd, 

Hg, Ag, Fe, Pt and/or Pd [3,4]. 

Besides the metalthiolate clusters and the absence of aromatic amino acids, MT do 

not have other characteristic structural features. The primary structure is extremely 

variable, whereas it is only conserved within closely related species, which makes the 

classification of MT problematic [5]. Based on their metal-inducible properties and their 

high affinity for metal ions, homeostasis of heavy metal levels is probably MT's most 

important biological function. MT can also serve as “maintainers” of the redox pool of a 

cell [6]. In mammals, these proteins may serve as a reservoir of metals (mainly zinc and 

copper) for synthesis of apoenzymes and zinc-finger transcription regulators. Moreover, 

new roles of these proteins have been discovered including those needed in the 

carcinogenic process [7]. 

- 7 -


It is not surprising that techniques using for detection and determination of MT 

have been reviewed several times [8-14] . Isolation, separation, detection and/or 

quantification of MT are not easy tasks for modern bioanalytical chemistry. Thanks to MT 

low molecular mass and unique primary structure, commonly used methods for detection 

of proteins suffer from many deficiencies including insufficient specificity and sensitivity. 

The most frequent methods used for detection of these proteins are indirect and based on 

quantification of heavy metal ions occurring in their structure or on high content of 

sulfhydryl groups. 

2. ISOLATION PROCEDURES 

2.1 Blood, blood serum and cells 

Isolation and consequent detection of MT in blood and/or blood serum samples is 

not so frequently carried out in comparison with tissues analysis. Heat treatment of a 

sample (app. 100 °C for more than five minutes) to denature and remove high molecular 

mass proteins from samples proposed by Erk et al. [15] is nowadays successfully applied to 

blood and blood serum samples [16,17]. Moreover, Petrlova et al. showed that using of tris 

(2-carboxyethyl)phosphine as a reducing agent could be beneficial for quantification of 

MT. The modified method was utilized for preparation of blood and blood serum samples 

of patients with various tumour diseases [18,19]or preparation fish sperm [20], in which 

these proteins have not been quantified before. Caulfield et al. used heat treatment for 

preparation of human red blood cells [21]. Cells were disrupted by repeated freezethawing 

cycles. The lysates obtained were heat treated and analysed. The authors had 

drawn blood from patients by venipuncture into tubes containing heparin. The presence 

of heparin or others compounds such as ethylenediaminetetraacetic acid (EDTA) can 

seriously influence quantification of MT in blood, blood serum or blood fractions, when 

electrochemical methods are used. Adam et al. showed that the presence of EDTA 

influenced voltammetric signals markedly [22]. 

2.2 Animal tissues 

To isolate MT from animal tissues, a preparation of crude extract from a tissue and 

purification of such extract by using gel filtration is one of the most commonly used 

protocols [23]. Tissue extract is prepared in the presence of Tris HCl with added sucrose 

[24], glucose and antioxidant specie (mercaptoethanol, dithiothreitol and/or TCEP) [25]. 

This extract is centrifuged or heat treated with subsequent centrifugation. Erk et al. 

reported on comparison of different procedures to purify MT from the digestive glands of 

mussels (Mytilus galloprovincialis) exposed to cadmium: heat treatment (at 70 and 85 °C), 

solvent precipitation, and gel-filtration [15]. They found that the most convenient was 

using heat treatment for the preparation of both heavy metal stressed and non-stressed 

tissues with consequent voltammetric detection. Moreover, Beattie et al. successfully 

utilized solid phase extractors for MT isolation [26]. 

2.3 Plant tissues 

Preparation of plant tissues, cells and parts to isolate phytochelatins (included into 

MT Class III) have been shown in many papers and reviewed several times [27]. MT Class 

I and II cannot be found in plant tissues without genetic modification of a plant genome. 

- 8 -


Macek et al. inserted MT genes from yeast and human into tobacco to enhance their 

ability to accumulate metal ions [28]. To detect MT, Diopan et al. prepared crude extracts 

from these plants and heat treated the extracts. Results on content of MT were similar to 

those detecting expression of mRNA [29]. 

3. ELECTROCHEMICAL METHODS 

Determination of MT by electrochemical methods is based on electroactivity of –SH 

moieties, which tend to be oxidized or catalyze evolution of hydrogen from a supporting 

electrolyte. To prevent interferences and lower detection limits, an adsorptive transfer 

stripping technique (AdTS) is often coupled with electrochemical methods. The main 

improvement of AdTS is based on removing the electrode from a solution after 

accumulation of a target molecule on its surface, rinsing of the electrode and transferring 

it to a pure supporting electrolyte, where no interferences are present [22]. To detect MT, 

linear sweep, cyclic, differential pulse and square wave voltammetry have been used. 

Usage of these techniques was reviewed by Sestakova and Navratil [30]. Besides the 

previously mentioned voltammetric methods, differential pulse voltammetry with a 

modification called after its founder “Brdicka reaction” is the most commonly used 

electrochemical method for detection of MT in various types of samples since Olafson 

optimized it on fish tissues [31]. Over several decades, the method has been optimized 

with detection limit under fM [16]. Temperature of the supporting electrolyte (app. 5 °C) 

and concentration of cobalt(III) ions (app. 1 mM) play the key role in the reaching the 

lowest detection limit. Raspor attempted to elucidate the exact mechanisms of this 

reaction [32]. Based on these results, Raspor and her colleagues have done a lot of work to 

propose physical and chemical conditions to achieve comparable results in various 

laboratories [33]. Moreover, sample-preparation-steps including heat treatment 

(mentioned in chapter 2) must precede a measurement. Measurements can be also 

automated and thus used for larger set of samples, as was shown by Fabrik et al. [34]. In 

spite of the fact that Brdicka reaction is commonly used for detection of MT, Pedersen et 

al. showed that differential pulse polarography was found to be unsuitable for crustacean 

tissues due to unidentified interfering compounds which led to 5- to 20-fold 

overestimation of metallothionein levels [35]. The interfering compounds such as other 

low molecular mass thiols, ionic strength or surfactants contained in a sample can be 

considered [36]. 

Besides voltammetric methods, chronopotentiometric stripping analysis (CPSA) can 

be also utilized for detection of MT. This method is the most sensitive analytical tool for 

detection and determination of MT with detection limits estimated as units of aM [17]. 

Reaction and therefore sensitivity of determination depends on many parameters such as 

pH and ionic strength of a supporting electrolyte, and isoelectric point of measured 

protein. Temperature is not a concern compared to Brdicka reaction. Another study 

discovered that addition of [Co(NH3)6]Cl3 to a supporting electrolyte can increase 

sensitivity up to 30 % [37]. The signal amplification is probably caused by complex 

inorganic salt-protein formation. AdTS coupled with the CPSA method was used for 

detection of MT expressed in yeast Yarrowia lipolytica exposed to Zn, Ni, Co and Cd [38]. 

However, Petrlova et al. found that the CPSA signal of MT is dependent on content of 

- 9 -


metals in the sample, because MT-metal complex gives lower CPSA signal compared to 

metal free MT [39]. However, the results can be re-calculated on the content of metals. 

Detection limits of electrochemical methods are summarized in Table 1. It is obvious that 

electrochemical methods are the most sensitive, however, they can suffer from 

misinterpreting of the measured data or inadequate preparation of a sample. 

4. BIOSENSORS 

The wide spectrum of metal-binding proteins from naturally occurred to artificial 

one, prepared using protein engineering, which is mostly specific for one metal ion, is 

used for non-enzymatic or affinity based biosensors employed for heavy metal ions 

determination. Heavy metal binding proteins including metallothioneins are mostly used 

for biosensors construction for different heavy metal ions e.g. mercury(II), copper(II), 

cadmium(II), zinc(II) and lead(II) in wide concentration range from fM to mM. These 

biosensors have good sensitivity and selectivity, and also acceptable stability time 

(approximately 2 weeks) besides wide concentration intervals (Castillo et al. 2004). The 

other approach used in biosensing of heavy metal ions is fusion of SmtA metallothionen 

from nostoc (families of cyanobacteria) with glutathione-S-transferase. Such modified 

metallothionein demonstrated wide selectivity to heavy metals (Zn(II), Cd(II), Cu(II) and 

Hg(II)) with high sensitivity up to fM. Glutathione-S-transferase-SmtA electrode was 

based on electric capacity determination and it was regenerated with EDTA and stored for 

16 days (Corbisier et al. 1999). Rabbit metallothionein was successfully employed as 

biological agent of biosensor for cadmium(II) and zinc(II) ions (Adam et al. 2007b), 

palladium(II) ions (Adam et al. 2007a), silver(I) ions (Krizkova et al. 2010), and cisplatin 

(Huska et al. 2009) determination. 

5. ACKNOWLEDGEMENT 

The work was by GA AV IAA401990701. 

6. REFERENCES 

[1] M. Margoshes, B.L. Vallee, J. Am. Chem. Soc. 79 (1957) 4813. 

[2] P. Coyle, J.C. Philcox, L.C. Carey, A.M. Rofe, Cell. Mol. Life Sci. 59 (2002) 627. 

[3] T.T. Ngu, M.J. Stillman, IUBMB Life 61 (2009) 438. 

[4] M. Nordberg, G.F. Nordberg, Cell. Mol. Biol. 46 (2000) 451. 

[5] J.H.R. Kagi, Method Enzymol. 205 (1991) 613. 

[6] A. Viarengo, B. Burlando, N. Ceratto, I. Panfoli, Cell. Mol. Biol. 46 (2000) 407. 

[7] T. Eckschlager, V. Adam, J. Hrabeta, K. Figova, R. Kizek, Curr. Protein Pept. Sci. 10 (2009) 360. 

[8] J.C. Amiard, C. Amiard-Triquet, S. Barka, J. Pellerin, P.S. Rainbow, Aquat. Toxicol. 76 (2006) 160. 

[9] A.T. Miles, G.M. Hawksworth, J.H. Beattie, V. Rodilla, Crit. Rev. Biochem. Mol. Biol. 35 (2000) 35. 

[10] K. Das, V. Debacker, J.M. Bouquegneau, Cell. Mol. Biol. 46 (2000) 283. 

[11] T. Minami, S. Ichida, K. Kubo, J. Chromatogr. B 781 (2002) 303. 

[12] A. Prange, D. Schaumloffel, Anal. Bioanal. Chem. 373 (2002) 441. 

[13] J. Szpunar, Analyst 125 (2000) 963. 

[14] J. Szpunar, Analyst 130 (2005) 442. 

[15] M. Erk, D. Ivanković, B. Raspor, J. Pavicić, Talanta 57 (2002) 1211. 

[16] J. Petrlova, D. Potesil, R. Mikelova, O. Blastik, V. Adam, L. Trnkova, F. Jelen, R. Prusa, J. Kukacka, R. 

Kizek, Electrochim. Acta 51 (2006) 5112. 

[17] R. Kizek, L. Trnkova, E. Palecek, Anal. Chem. 73 (2001) 4801. 

- 10 -


[18] I. Fabrik, S. Krizkova, D. Huska, V. Adam, J. Hubalek, L. Trnkova, T. Eckschlager, J. Kukacka, R. 

Prusa, R. Kizek, Electroanalysis 20 (2008) 1521. 

[19] S. Krizkova, I. Fabrik, V. Adam, J. Kukacka, R. Prusa, G.J. Chavis, L. Trnkova, J. Strnadel, V. Horak, 

R. Kizek, Sensors 8 (2008) 3106. 

[20] I. Fabrik, Z. Svobodova, V. Adam, S. Krizkova, L. Trnkova, M. Beklova, M. Rodina, R. Kizek, J. Appl. 

Ichthyol. 24 (2008) 522. 

[21] L.E. Caulfield, C.M. Donangelo, P. Chen, J. Junco, M. Merialdi, N. Zavaleta, Nutrition 24 (2008) 1081. 

[22] V. Adam, S. Krizkova, O. Zitka, L. Trnkova, J. Petrlova, M. Beklova, R. Kizek, Electroanalysis 19 

(2007) 339. 

[23] Y. Li, W. Maret, J. Anal. At. Spectrom. 23 (2007) 1055. 

[24] J.H. Beattie, M.P. Richards, R. Self, J. Chrom. 632 (1993) 127. 

[25] H. Vodickova, V. Pacakova, I. Sestakova, P. Mader, Chem. Listy 95 (2001) 477. 

[26] J.H. Beattie, R. Self, M.P. Richards, Electrophoresis 16 (1994) 322. 

[27] C. Cobbett, P. Goldsbrough, Annu. Rev. Plant Biol. 53 (2002) 159. 

[28] T. Macek, M. Mackova, D. Pavlikova, J. Szakova, M. Truksa, S. Cundy, P. Kotrba, N. Yancey, W.H. 

Scouten, Acta Biotech. 22 (2001) 101. 

[29] V. Diopan, V. Shestivska, V. Adam, T. Macek, M. Mackova, L. Havel, R. Kizek, Plant Cell Tissue 

Organ Cult. 94 (2007) 291. 

[30] I. Sestakova, T. Navratil, Bioinorg. Chem. Appl. 3 (2003) 43. 

[31] R.W. Olafson, P.E. Olsson, Method Enzymol. 205 (1991) 205. 

[32] B. Raspor, J. Electroanal. Chem. 503 (2001) 159. 

[33] B. Raspor, M. Paic, M. Erk, Talanta 55 (2001) 109. 

[34] I. Fabrik, Z. Ruferova, K. Hilscherova, V. Adam, L. Trnkova, R. Kizek, Sensors 8 (2008) 4081. 

[35] K.L. Pedersen, S.N. Pedersen, J. Knudsen, P. Jerregaard, Environ. Sci. Technol. 42 (2008) 8426. 

[36] S. Krizkova, I. Fabrik, V. Adam, J. Kukacka, R. Prusa, L. Trnkova, J. Strnadel, V. Horak, R. Kizek, 

Electroanalysis 21 (2008) 640. 

[37] M. Tomschik, L. Havran, E. Palecek, M. Heyrovsky, Electroanalysis 12 (2000) 274. 

[38] M. Strouhal, R. Kizek, J. Vecek, L. Trnkova, M. Nemec, Bioelectrochemistry 60 (2003) 29. 

[39] J. Petrlova, S. Krizkova, O. Zitka, J. Hubalek, R. Prusa, V. Adam, J. Wang, M. Beklova, B. Sures, R. 

Kizek, Sens. Actuator B-Chem. 127 (2007) 112. 

- 11 -


ELECTROCHEMISTRY IN SPACE 

René KIZEK 1 , Vojtěch ADAM 1 , Jaromír HUBÁLEK 2 


2 Brno University of Technology, Technicka 10, CZ-616 00 Brno, CZ 

Abstract 

The exploration of Mars has been an important part of the space exploration programs of 

many countries in the world. Dozens of robotic spacecraft, including orbiters, landers, and 

rovers, have been launched toward Mars since the 1960s. These missions were aimed at 

gathering data about current conditions and answering questions about the history of 

Mars as well as a preparation for a possible human mission to Mars. The questions raised 

by the scientific community are expected to not only give a better appreciation of the red 

planet but also yield further insight into the past, and possible future, of Earth. It seems 

that electrochemistry could play a key role in the development of well portable and 

sensitive analyzers. 


In 2008, researchers from Brno University of Technology, Mendel University in 

Brno and Masaryk Universtiy succeeded in the program Nanotechnology for Society 

supported by the government of the Czech Republic and the ongoing project is 

successfully solving the priority tasks. This project is focused on development of new 

nanosystems applicable in medicine as biosensors for online monitoring particular 

physiological characteristics and treatment progression in general. To further reinforce 

the collaboration of Brno Universities (Masaryk University, Brno University of 

Technology, and Mendel University in Brno) the centre of excellence called CEITEC 

(Central European Institute of Technology) and Regional research centre SIX: Sensors, 

information and communication systems have been established to create an effective 

platform for research in nanotechnnology and nanoscience comprising materials and 

functional structures suitable for nanoelectronics and nanophotonics in general, 

addressing both the preparation and the characterization of nanostructures applicable in 

bio-medical areas, energetic and information and communication technologies. This 

platform based on specific experiences of members will enable the participation on 

important projects founded by European Union, which have begun by fruitful 

participation in ongoing project MAS (Nanoelectronics for Mobile AAL-Systems). During 

the last five years, the results of Laboratory of Metallomics and Nanotechnologies have 

been published as 128 publications in ISI-indexed journals with a total impact factor 

241,664. The most important results can be found in highly impacted journals as TRAC- 

Trends in Analytical Chemistry, Analytical Chemistry, Current Medical Chemistry, PLoS 

ONE, Biochemical Pharmacology and the others 

- 12 -


2. ADVANCED ROBOTIC NANOBIOTECHNOLOGIES FOR EXPLORATION 

OF MARS’S SURFACE 

The ARNEMS project is focused on the development of a unique multi-purpose 

detector called Eurydica for analysis of surface and environment at Mars. The 

multipurpose detector Eurydica uses the latest lab-on-chip nanobiotechnologies. The 

developed detector will be controlled by our developed software, to which neural 

networks and artificial intelligence will be implemented with regard to do the most 

sensitive evaluation and processing of signals. Eurydica detector will be also implemented 

in the reconnaissance robot Orpheus. Moreover, the robot will carry additional 

equipments for sampling of Mars environment and for cultivating and handling with 

bacteria able to live on the Mars and create oxygen atmosphere. This feature will serve as 

so called incubator for the microorganisms. Using sensing devices, we will be able to 

analyse simultaneously outer environment and inner conditions in bacteria colony. 

ARNEMS will be equipped with: 

• fully automatic r reconnaissance robotic systems for exploring of a plant surface and 

carrying additional equipments, 

• the robotic system enables wireless data transfer and telepresentation based user 

interface, 

• chemical and biochemical analyser based on electrochemical lab-on-chip techniques, 

• dock for bacteria and their cultivation medium. 

ARNEMS will enable to us! 

• ability to detect living species, 

• ability to analyze water, 

• ability to detect desired gases, 

• to maintain and monitor conditions for cultivation of bacteria. 

3. NOWADAYS TECHNOLOGIES - PHOENIX 

Phoenix was a robotic spacecraft on a space exploration mission on Mars under the 

Mars Scout Program. The Phoenix lander descended on Mars on May 25, 2008. Mission 

scientists used instruments aboard the lander to search for environments suitable for 

microbial life on Mars, and to research the history of water there. The multi-agency 

program was headed by the Lunar and Planetary Laboratory at the University of Arizona, 

under the direction of NASA's Jet Propulsion Laboratory. The program was a partnership 

of universities in the United States, Canada, Switzerland, Denmark, Germany, the United 

Kingdom, NASA, the Canadian Space Agency, the Finnish Meteorological Institute, 

Lockheed Martin Space Systems, MacDonald Dettwiler & Associates (MDA) and other 

aerospace companies. It was the first mission to Mars led by a public university in NASA 

history. The mission underscored the value of university-led management. It was led 

directly from the University of Arizona's campus in Tucson, with project management at 

the Jet Propulsion Laboratory in Pasadena, Calif., and project development at Lockheed 

- 13 -


Martin in Denver, Colorado. The operational funding for the mission extended through 

November 10, 2008 [1-6]. 

Phoenix is NASA's sixth successful landing out of seven attempts and is the most 

recent spacecraft to land successfully on Mars as well as the first successful landing in a 

Martian polar region. The lander completed its mission in August 2008, and made a last 

brief communication with Earth on November 2 as available solar power dropped with 

the Martian winter. The mission was declared concluded on November 10, 2008, after 

engineers were unable to re-contact the craft. After unsuccessful attempts to contact the 

lander by the Mars Odyssey orbiter up to and past the Martian summer solstice on May 

12, 2010, JPL declared the lander to be dead. Like the two Mars Exploration Rovers, the 

program was considered a success because it exceeded its planned mission length by 

several months [7,8]. 

On June 24, 2008, NASA's scientists launched a major series of tests. The robotic arm 

scooped up more soil and delivered it to 3 different on-board analyzers: an oven that 

baked it and tested the emitted gases, a microscopic imager, and a wet chemistry lab. The 

lander's Robotic Arm scoop was positioned over the Wet Chemistry Lab delivery funnel 

on Sol 29 (the 29th Martian day after landing, i.e. June 24, 2008). The soil was transferred 

to the instrument on Sol 30 (June 25, 2008), and Phoenix performed the first wet 

chemistry tests. On Sol 31 (June 26, 2008) Phoenix returned the wet chemistry test results 

with information on the salts in the soil, and its acidity. The wet chemistry lab was part of 

the suite of tools called the Microscopy, Electrochemistry and Conductivity Analyzer 

(MECA) [9]. 

Preliminary wet chemistry lab results showed the surface soil is moderately alkaline, 

between pH 8 and 9. Magnesium, sodium, potassium and chloride ions were found; the 

overall level of salinity is modest. Chloride levels were low, and thus the bulk of the 

anions present were not initially identified. The pH and salinity level were viewed as 

benign from the standpoint of biology. TEGA analysis of its first soil sample indicated the 

presence of bound water and CO2 that were released during the final (highesttemperature, 

1,000°C) heating cycle. Results published in the journal Science after the 

mission ended reported that chloride, bicarbonate, magnesium, sodium potassium, 

calcium, and possibly sulfate were detected in the samples. The pH was narrowed down to 

7.7±0.5. Perchlorate (ClO4), a strong oxidizer at elevated temperatures, was detected. This 

was a significant discovery. The chemical has the potential of being used for rocket fuel 

and as a source of oxygen for future colonists. Under certain conditions perchlorate can 

inhibit life; however some microorganisms obtain energy from the substance (by 

anaerobic reduction). The chemical when mixed with water can greatly lower freezing 

points, in a manner similar to how salt is applied to roads to melt ice. So, perchlorate may 

be allowing small amounts of liquid water to form on Mars today. Gullies, which are 

common in certain areas of Mars, may have formed from perchlorate melting ice and 

causing water to erode soil on steep slopes [10]. 

- 14 -


4. WET CHEMISTRY LAB 

The wet chemistry lab (WCL) sensor assembly and leaching solution were designed 

and built by Thermo Fisher Scientific. The WCL actuator assembly was designed and built 

by Starsys Research in Boulder, Colorado. Tufts University developed the reagent pellets, 

barium ISE, ASV electrodes, and performed the preflight characterization of the sensor 

array [9-13]. 

The robotic arm scooped up some soil, put it in one of four wet chemistry lab cells, 

where water was added, and while stirring, an array of electrochemical sensors measured 

a dozen dissolved ions such as sodium, magnesium, calcium, and sulfate that have leached 

out from the soil into the water. This provided information on the biological compatibility 

of the soil, both for possible indigenous microbes and for possible future Earth visitors 

[14]. Every wet chemistry cell has 26 chemical sensors and a temperature sensor. The 

polymer Ion Selective Electrodes were able to determine the concentration of ions by 

measuring the change of electric potential within the sensor, which is separated from the 

wet chemistry cell by an ion selective membrane. The two gas sensing electrodes for 

oxygen and carbon dioxide work on the same principle and are separated from the wet 

chemistry cell by a gas permeable membrane. A gold micro-electrode array is used for the 

Cyclic voltammetry and Anodic Stripping Voltammetry. Cyclic voltammetry is a method 

to study ions by applying a waveform of varying potential and measuring the currentvoltage 

curve. Anodic Stripping Voltammetry first deposits the metals onto the gold 

electrode with an applied potential. After the potential is reversed, the current is 

measured while the metals are stripped off the electrode. The first measurement indicated 

that the surface layer contained water soluble salts and had a pH between 8 and 9. 

Additional tests on soil composition revealed the presence of perchlorate [14]. 

Later publication of results in the journals Science and JGR reported that chloride, bicarbonate, 

magnesium, sodium potassium, calcium, and possibly sulfate were detected in the samples. The pH 

was narrowed down to 7.7 + or – 0.5 [9-11]. Further data analysis has indicated that the soil contains 

soluble sulfate at a minimum of 1.1% wt % SO3 and provided a refined formulation of the soil [9]. 


The work was by NanoBioTECell GA ČR P102/11/1068. 

6. REFERENCES 

[1] R.A. Kerr, Science 329 (2010) 1267. 

[2] R.G. Bonitz, L. Shiraishi, M. Robinson, R.E. Arvidson, P.C. Chu, J.J. Wilson, K.R. Davis, G. Paulsen, 

A.G. Kusack, D. Archer, P. Smith, Journal of Geophysical Research-Planets 113 (2008) 10. 

[3] J.R. Guinn, M.D. Garcia, K. Talley, Journal of Geophysical Research-Planets 113 (2008) 16. 

[4] J.H. Hoffman, R.C. Chaney, H. Hammack, Journal of the American Society for Mass Spectrometry 19 

(2008) 1377. 

[5] H.U. Keller, W. Goetz, H. Hartwig, S.F. Hviid, R. Kramm, W.J. Markiewicz, R. Reynolds, C. 

Shinohara, P. Smith, R. Tanner, P. Woida, R. Woida, B.J. Bos, M.T. Lemmon, Journal of Geophysical 

Research-Planets 113 (2008) 15. 

[6] P.A. Taylor, D.C. Catling, M. Daly, C.S. Dickinson, H.P. Gunnlaugsson, A.M. Harri, C.F. Lange, 

Journal of Geophysical Research-Planets 113 (2008) 8. 

[7] R.E. Arvidson, R.G. Bonitz, M.L. Robinson, J.L. Carsten, R.A. Volpe, A. Trebi-Ollennu, M.T. Mellon, 

P.C. Chu, K.R. Davis, J.J. Wilson, A.S. Shaw, R.N. Greenberger, K.L. Siebach, T.C. Stein, S.C. Cull, W. 

- 15 -


Goetz, R.V. Morris, D.W. Ming, H.U. Keller, M.T. Lemmon, H.G. Sizemore, M. Mehta, Journal of 

Geophysical Research-Planets 114 (2009) 21. 

[8] R. Bonitz, L. Shiraishi, M. Robinson, J. Carsten, R. Volpe, A. Trebi-Ollennu, R.E. Arvidson, P.C. Chu, 

J.J. Wilson, K.R. Davis, Ieee, in 2009 Ieee Aerospace Conference, Vols 1-7, Ieee, New York, 2009, p. 

42. 

[9] S.P. Kounaves, M.H. Hecht, S.J. West, J.M. Morookian, S.M.M. Young, R. Quinn, P. Grunthaner, 

X.W. Wen, M. Weilert, C.A. Cable, A. Fisher, K. Gospodinova, J. Kapit, S. Stroble, P.C. Hsu, B.C. 

Clark, D.W. Ming, P.H. Smith, Journal of Geophysical Research-Planets 114 (2009) 20. 

[10] W.V. Boynton, D.W. Ming, S.P. Kounaves, S.M.M. Young, R.E. Arvidson, M.H. Hecht, J. Hoffman, 

P.B. Niles, D.K. Hamara, R.C. Quinn, P.H. Smith, B. Sutter, D.C. Catling, R.V. Morris, Science 325 

(2009) 61. 

[11] M.H. Hecht, S.P. Kounaves, R.C. Quinn, S.J. West, S.M.M. Young, D.W. Ming, D.C. Catling, B.C. 

Clark, W.V. Boynton, J. Hoffman, L.P. DeFlores, K. Gospodinova, J. Kapit, P.H. Smith, Science 325 

(2009) 64. 

[12] S.P. Kounaves, Chemical & Engineering News 86 (2008) 8. 

[13] S.R. Lukow, S.R. Kounaves, Electroanalysis 17 (2005) 1441. 

[14] S.P. Kounaves, S.R. Lukow, B.P. Comeau, M.H. Hecht, S.M. Grannan-Feldman, K. 

Manatt, S.J. West, X.W. Wen, M. Frant, T. Gillette, Journal of Geophysical 


- 16 -


ANATOMICAL STUDY OF VEGETATIVE 

ORGANS OF RHUS HIRTA (L.) SUDW. 

(ANACARDIACEAE) 

Petr BABULA 1 , Anna KORVASOVÁ 1 , Vojtěch ADAM 2 , René KIZEK 2 

1 Department of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences 

Brno, Palackeho 1/3, CZ-61242 Brno, Czech Republic. 

2 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University, Zemedelska 1, CZ- 

61300 Brno, Czech Republic 

Abstract 

Family Anacardiaceae (cashew family, sumac family) consists of about 700 species of 

shrubs and trees classified in 82 genera. Some species are used for production of fruit 

(mango, pistachio, cashew nuts), some species are important toxicologically – for example 

poison ivy (Toxicodendron radicans). Rhus hirta (L.) Sudw. (staghorn sumac), small 

deciduous tree native to Southeastern Canada, is widely cultivated in gardens as 

ornamental plant. Despite this fact, anatomical study of individual vegetative organs of R. 

hirta is still missing. This study is focused on the anatomy of vegetative organs – roots, 

stems and leaves – of Rhus hirta (L.) Sudw. using methods of light and fluorescence 

microscopy. 


Family Anacardiaceae Lindl. comprises evergreen or deciduous shrubs and trees 

usually with latex of tropical and subtropical regions (Tingshuang, et al.). Some plants are 

widely cultivated due to edible fruits or seeds – mango (Mangifera indica), pistachio 

(Pistacia vera), cashew nuts (Anacardium occidentale) or pink pepper (Schinus 

terebinthifolius). Wood of species of South American genus Schinopsis - “quebracho 

colorado” - is used for its hardness. Some plants of this family have toxicological 

importance due to ability to irritate skin and induce dermatitis after contact. Especially 

species of genus Toxicodendron, which are placed also in the genus Rhus, are responsible 

for cases of poisoning – poison ivy (T. radicans, T. rydbergii), Eastern poison oak (T. 

toxicarium), Western poison oak (T. versilobum) and poison sumac (T. vernix). Chinese 

and Japanese species Toxicodendron vermicifluum - “Japanese lac tree” and Metopium 

toxiferum - “poison wood” - a shrub native to South Florida and Northern West Indies are 

also responsible for contact dermatitis (Lampe; Beaman, Hurtado). Urushiol, a mixture of 

3-alkylcatechols and 3-alkencatechols, is the main component of above-mentioned 

species, however, next chemically relative compounds have been found in different 

species of Anacardiaceae family (Moyo et al., . Chemical formulas of these compounds are 

summarized in Tab. 1. 

- 17 -




(1) 

COOH H 

( (2) 

Fig. 1: Cheemical 

formmulas 

of ph henolic lipiddes 

in Anac cardiaceae. 1 – anacarrdic 

acids, 2 – 3- 

alkyylphenols 

(cardanol, anacardol) ), 3 – 5-al lkylresorcin nols (cardools) 

and 4 – 3- 

alkyylcatecholss 

(urushiols s). R = C13, C15 or C17 7, usually alkan a or alkken. 

Somee 

memberrs 

of fam mily Anacaardiaceae 

are a used in n folk meedicines 

du ue to 

antimicrobbial, 

antivirral 

(anti-ret troviral), annti-parasiti 

ical and ant ti-inflammaatory 

prope erties 

(Amphipte terygium ad adstringens – (Mexicoo), 

Astroni ium urund deuvea – BBolivia, 

Co otinus 

coggygria – Bulgariaa, 

Harpephy hyllum caffr frum – Africa, 

Heeria insignis - Africa, La annea 

corromanddelica 

– Inddia, 

Ozoroa a insignis (AAfrica), 

Rhu hus spp. (Afr rica), Schinnus 

molle (S South 

America), Spondyis sspp. 

(South America, AAfrica), 

and d others). 

Rhus us hirta (L.) Sudw. (syn. 

Rhus typ yphina L.) is i a small tr ree native to Southea astern 

Canada. Itt 

grows to 3 – 10 m tall, 

pinnatelly 

compoun nds leaves are a 25 – 55 cm long. Plants P 

are dioecioous, 

flowerrs 

form characteristic 

clusters of male/female 

flowers ( (see Fig. 2) . The 

fruits of RR. 

hirta are in some co ountries coollected 

and d used for making of pink lemonade. 

Native Ammerican 

tribbes 

mix lea aves of this species with 

leaves of o tobacco ffor 

smoking g. All 

parts of plants 

exceptt 

of roots ar re used as a natural dy ye and as a mordant du due to conte ent of 

tannins (ggallotanninns). 

Other secondary metabolit tes (except of gallotaannins) 

include 

flavonoidss, 

and terpeenes 

- espec cially monooterpenes, 

sesquiterpen 

nes and tritterpenes. 

Fig. 2. Femmale 

(A) andd 

male (B) plant of Rhhus 

hirta Rhus Rh hirta (L L.) Sudw. 

- 18 - 

(3) 

(4) 

Brno


2. EXPERIMENT 

Plant material (roots, stems, leaves) was collected in the botanical garden of 

University of Veterinary and Pharmaceutical University Brno. For the anatomical study, 

transversal, radial and tangential hand-made sections were used. For the visualization of 

lignified plant tissues, ethanolic (50 %, v/v) mixture of acid fuchsine and malachite green 

(both 1%, w/w; Sigma-Aldrich, USA) was used. Sections were stained for four minutes 

and after it washed with acidic ethanol (60 %, v/v, with 37% hydrochloric acid 0.1 %, v/v, 

Sigma-Aldrich, USA). At once, sections, which were not stained, were used for 

microscopic analysis using fluorescence microscope (Carl Zeiss Axioscop 40, Zeiss, 

Germany). In addition, sections were stained by aqueous solution of acridine orange (1 %, 

w/w, Sigma-Aldrich, USA) for detection of non-lignified and lignified structures. 

3. RESULTS AND DISCUSSION 

The object of this study was focused on vegetative organs of Rhus hirta (L.) Sudw. 

Roots were observed only in the secondary state because of very early formation of 

vascular cambium, which produces elements of secondary xylem and secondary phloem. 

Secondary xylem of roots is typical due to presence of high number of vessels of large 

diameter. Amount of libriform is low; in addition, secondary cell walls if libriform are 

very thin and almost without lignification. Secondary phloem consists of sieve tubemembers, 

which are arranged in tangential groups, radial parenchyma forms well visible 

rays. Axial parenchyma of secondary phloem are associated with sieve tube-members. In 

the older parts of secondary phloem, there are large schizogenous intercellular cavities. 

Secondary dermal tissue periderm consists of layers of phellem cells with suberinised cell 

walls and 1-2-layered cork cambium (phellogene). Phelloderm is reduced to only several 

cells, which undergo sclerification under formation of sclereids (see Fig. 3). 

Stems as well as roots very early undergo process of secondary thickening due to 

activity of vascular cambium and cork cambium. However, epidermis persists after 

formation of vascular cambium. Some epidermal cells are modified into trichomes. 

Glandular trichomes, which are associated with production of some secondary 

metabolites, are typically multicellular. Cortex consists of collenchymatous hypodermis, 

parenchymatous mesodermis and indistinct endodermis- starch sheath. Vascular bundles 

are arranged in one ring (eustele) and are typically collateral. Elements of protophloem 

undergo sclerification under formation of sclerenchyma on the periphery of vascular 

bundles. Metaphloem is connected with formation of large schizogenous secretory 

cavities, each vascular bundle contains one cavity. In comparison with roots, secondary 

xylem of stem contains less vessels and higher amount of libriform with distinct secondary 

cell walls. Axial parenchyma is associated with vessels, ray parenchyma forms 

heterocellular rays. Pith is parenchymatous, some cells undergo sclerification and form 

individual sclereids. In addition, pith contains schizogenous intercellular cavities with 

distinct epithelial cells (see Fig. 4). 

- 19 -




Anattomy 

of peetiole 

is sim milar to struucture 

of stem s includ ding format ation of vas scular 

cambium. Leaves aree 

bifacial wi ith leaf meesophyll 

dif fferentiated d into palisaade 

parench hyma 

and sponggy 

parenchhyma. 

Vasc cular bunddles 

(collat teral) contain 

distincct 

schizom matous 

secretory ccavities 

in mmetaphloem 

m (Fig. 5). 

Fig. 3: Traansversal 

aand 

tangent tial sectionns 

of roots: general an natomy (A) ), magnific cation 

x40, and ddetailed 

strructure 

of secondary s pphloem 

(B) ), magnifica ation x200, , and secon ndary 

xylem, mmagnificationn 

x200 (C C) and x4000 

(D). Un nstained se ection (A) ), acid fuc chsin- 

malachite green stainning 

(B, D) and acridinne 

orange under u DAP PI filter (C) . Descriptio on A: 

1 – periderrm, 

2 – corrk 

cambium m, 3 – seconndary 

phloe em, 4 – schi izogenous ssecretory 

ca avity, 

5 – vascullar 

cambiuum, 

6 – sec condary xyylem, 

7 – ray parenc chyma; B: 1 – sieve tube- 

members, 2 – axial parenchym ma, 3 – raddial 

parench hyma - ray y; C: 1 – vvessel, 

2 – axial 

parenchymma, 

3 – libriiform; 

D: detail 

of pittted 

and scal lariform ve essels of seccondary 

xyl lem. 

- 20 - 

Brno

XI. Woorkshop 

of Physsical 


Fig. 4: Transveersal 

sectio on of stem in primar ry state (A A), magnificcation 

x100, 

detailedd 

struccture 

of trichomes 

(B), 

magnificcation 

x400 0, structure e of the steem 

in secon ndary statee 

(C), magnificattion 

x40, an nd radial section 

of stem s in sec condary staate 

of thick kening (D), , 

magnnification 

xx40. 

Acridi ine orange staining using u FITC (A) and TTexas 

Red (D) filters, , 

unstaained 

section, 

autoflu uorescence – DAPI (B B) and FITC 

(C) filterr. 

Descript tion A: 1 – 

epideermis 

withh 

cuticle, 2 – colllenchymat 

tous hypodermis, 

3 – paren nchymatouss 

mesoodermis, 

4 – collatera al vascular bbundle 

- metaphloem 

m m, 5 – schizzogenous 

in ntercellularr 

cavitty 

with disstinct 

epith helial cells, 6 – vessel ls of metax xylem; B: 1 – base of f glandularr 

trichhome, 

2 – mmulticellula 

ar head; C: 1 – phellem m, 2 – cork k cambium with phell loderm, 3 – 

corteex, 

4 – secoondary 

phlo oem, 5 – scchizogenou 

us intercellu ular space, 6 – sclerifie ed primaryy 

phloem, 

7 – seccondary 

xyl lem, 8 – vaascular 

cam mbium; D. 1 – secondarry 

phloem, 2 – vessel, , 

3 – rray 

parenchhyma 

– hete erocellular ray, 4 – libriform, 

5 –p primary xyylem, 

6 - pit th. 

- 21 - 

Brnoo




Fig. 5: Trransversal 

sections of f petiole (AA, 

B, C) and a leaf (D D): generaal 

anatomy y (A), 

magnificattion 

x40, detailed structure of vascula ar tissue with largee 

schizoge enous 

intercellullar 

cavity ( B), magnifi ication x2000, 

detail of f schizogen nous interceellular 

cavi ity in 

pith (C), magnificattion 

x400 and a transvversal 

section 

of leaf (D), magnnification 

x200. 

Autofluoreescence 

unnder 

DAPI I filter (A) ), unstaine ed section (B) and aacridine 

or range 

staining uunder 

FITCC 

filter (C C, D). Desccription 

A: A 1 – epid dermis witth 

cuticle, 2 – 

hypodermmis, 

3 – messodermis, 

4 – sclerifiedd 

primary phloem p wit th sclerifiedd 

interfasci icular 

parenchymma, 

5 – mettaphloem 

with w schizoogenous 

int tercellular cavity, c 6 – metaxylem m, 7 – 

pith, 8 – sschizogenoous 

intercel llular cavityy; 

B: 1 – schizogenou 

us intercelllular 

cavity y, 2 – 

epithelial cells, 3 – metaphloem, 

4 – rayy 

parenchy yma, 5 – sc clerified paarenchyma 

a, 6 – 

secondary phloem, 7 – vascula ar cambiumm, 

8 – sec condary xy ylem; C: 1 - schizoge enous 


cavity, 2 – epithel lial cells; DD: 

1 – epid dermis, 2 – palisade pparenchyma 

a, 3 – 

spongy paarenchymaa, 

4 – epi idermis, 5 – primar ry xylem of vasculaar 

bundle, 6 - 

schizogenoous 

intercellular 

cavity, 

7 – primmary 

phloem m. 

4. CONNCLUSIONN 

Worrk 

demonstrated 

anato omy roots, stems and leaves of widely w cultiivated 

tree Rhus 

hirta (L.) Sudw. (synn. 

Rhus typ phina L.) byy 

the use of o methods s of light an and fluoresc cence 

microscoppy. 

Work shhows 

not on nly to the aanatomical 

structures of individuual 

plant or rgans, 

but also too 

the possibbility 

of usag ge of fluoreescence 

mic croscopy in n these typees 

of studies s. 

- 22 - 

Brno



The work has been supported by REMEDTECH GA ČR 522/10/P618 a FRVŠ 

2506/F4/2011/VFU. 

6. REFERENCES 

[1] Tingshuang, Y., et al., (2004): Phylogenetic and biogeographic diversification of Rhus 

(Anacardiaceae) in the Northern Hemisphere, Molecular Phylogenetics and Evolution, 33: 861-879 

[2] Lampe, K. F. (1986): Dermatitis-producing Anacardiaceae,of the Caribbean area, Clinics in 

Dermatology, 4: 171-182 

[3] Beaman, J. H. (1986): Allergenic Asian Anacardiaceae, Clinics in Dermatology, 4: 191-203 

[4] Hurtado, I. (1986): Poisonous Anacardiaceae,of South America, Clinics in Dermatology, 4: 183-190 

[5] Moyo, M., et al: Phenolic composition, antioxidant and acetylcholinesterase inhibitory activities of 

Sclerocarya birrea and Harpephyllum cafrum (Anacardiaceae), Food Chemistry, 123: 69-76 

- 23 -


ANATOMICAL STUDY OF VEGETATIVE 

ORGANS OF PHARMACEUTICALLY AND 

TOXICOLOGICALLY IMPORTANT PLANT 

ACONITUM NAPELLUS L. EM. SKALICKY 

(RANUNCULACEAE) 

Petr BABULA 1 , Simona KVAŠŇÁKOVÁ 1 , Vojtěch ADAM 2 , René KIZEK 2 





Abstract 

Aconitum napellus L. em. Skalicky (Ranunculaceae) is one of the most poisonous plant in 

Europe. In the Aconitum genus there are toxicologically important diterpene (C20) and 

nor-diterpene (C19) alkaloids represented mainly by aconitine presented in primarily in 

roots. However, species contains other chemical constituents, such as flavonol glycosides, 

which are in the focus of interest. Despite the well known toxicological aspects of this 

plant, anatomical study of individual vegetative organs is still missing. This study is 

focused on the demonstration of anatomy of vegetative organs – roots, stems and leaves – 

of Aconitum napellus L. em. Skalický using both methods of light and fluorescence 

microscopy. 


Aconitum napellus L. em. Skalický, “Monk´s hood” is a perennial herb 0.5 – 1.5 m in 

height with tuberous fleshy roots and erect stout stems with leaves dissected into 5-7 

segments, violet-blue flowers with helmet-shaped hood arranged in dense racemes and 

multiple fruit of follicles containing black triangular seeds. Plants are distributed mainly 

in Alps and other mountainous regions in Europe. Monk’s hood is considered to be one of 

the most poisonous plants in Europe, which toxicity is exceeded only by Indian Aconitum 

ferox Wall. used by as an arrow poison [1-3]. However, classification of members of 

Aconitum genus is very difficult due to genetic instability [4, 5]. Numerous studies have 

dealt with the diterpene (C20) and nor-diterpene (C19) alkaloids [6-8]. The main alkaloids 

of Aconitum napellus aggregate are aconitine, picroaconitine, isoaconitine, benzaconitine, 

aconine, neopelline, eoline, napelline, mesaconitine, and hypaconitinine (see Fig. 1). 

Symptoms of poisoning include burning and tingling in the mouth and also in the fingers 

and toes. After it, paraesthesia extends over the whole body, accompanied by bouts of 

sweating and shivering; this gradually changes to feelings of roughness, insensibility and 

ice coldness. This stage is followed by the vomiting, colicky diarrhea, paralysis of the 

- 24 -

XI. Woorkshop 

of Physsical 


skeleetal 

muscullature 

and intense paiin. 

Death occurs o after r one hourrs 

through respiratoryy 

parallysis 

or heaart 

failure [3 3, 9-11]. 

However, , some studies 

have demonstra ated also phenolic 

coonstituents, 

especiallyy 

flavoonol 

and hyydroxyphen 

netyl glycoosides 

with antioxidan nt propertiees 

[12-14]. Flavonoidss 

havee 

been fouund 

in some 

subspeecies 

of Aconitum A napellus n – A. napel llus subsp. . 

neommontanum 

(Wulfen) ) Bayer (flowers - querceti in 7-O-(66-trans-caff 

feoyl)-beta- 

glucoopyranosyll-(1,3)-alpha-rhamnoppyranoside-3-O-beta-g 

glucopyrannoside, 

kaem mpferol7- O-(66-trans-cafffeoyl)-beta-glucopyrannosyl-(1,3)- 

-alpha-rham mnopyranooside-3-O-b 

beta 

glucoopyranosidde 

and kae empferol 77-O-(6-tran 

ns-p-coumaroyl)-beta-glucopyran 

nosyl-(1,3)- 

alphaa-rhamnoppyranoside 

3-O-beta-gglucopyrano 

oside together t 

with beta-3,4- 

dihyydroxyphennethyl 

beta a-glucopyraanoside) 

an nd A. nape ellus subspp. 

tauricum m (Wulfen) ) 

Bayeer, 

species distributed 

in Easteern 

Alps and a southe ern Carpatthians 

(3-O O-(6-trans- 

caffeeoyl)-beta-gglucopyranosyl-(1,2)-bbeta-glucop 

pyranoside-7-O-alphaa-rhamnopy 

yranoside, 

kaemmpferol 

3-O-(6-trans 

s-caffeoyl)-bbeta-glucop 

pyranosyl-( (1,2)-beta-gglucopyran 

noside-7-O- 

alphaa 

-rhamnoopyranosid 

de, quercettin 

3-O-(6 6-trans-p-co oumaroyl)-beta-gluco 

opyranosyl- 

(1,2) -beta-glucoopyranoside 

e-7-O-alphha-rhamnop 

pyranoside aand 

beta-3,4- 

dihyydroxyphennethyl 

beta-glucopyrannoside) 

[15 5, 16]. Flav vonoids havve 

been fou und also inn 

other 

members of Aconitu um genus [117]. 

Fig. 

1: The most 

import tant diterppene 

alkalo oids of Aco onitum nap apellus aggr regate. 1 - 

napellinne, 

Fig. 2: aconitinne 

- (R 

hypacon 

1 = - 

nitine (R1 -CH2CH3, R 

= CH3, R2 R 

= H 

2 = -OH), mesakonit tine (R 

H) 

1 = --CH3, 

R2 = -OH) andd 

Plants of Aconitum genus are uused 

in tra aditional Ch hinese meddicine 

after processingg 

to ddecompose 

toxic alka aloids to the less toxic t deriv vatives andd 

for prep paration off 

hommeopathic 

prreparations 

s [18-20]. 

2. EXPERIMMENT 

Plant maaterial 

(roo ots, stems, leaves) was w collecte ed in the botanical garden off 

Univversity 

of VVeterinary 

and a Pharmmaceutical 

University U Brno. For tthe 

anatom mical study, , 

transsversal, 

raddial 

and tan ngential hannd-made 

se ections wer re used. Foor 

the visua alization off 

ligniified 

plant ttissues, 

ethanolic 

(50 % %, v/v) mix xture of acid 

fuchsinee 

and malac chite greenn 

(bothh 

1%, w/ww; 

Sigma-Al ldrich, USAA) 

was use ed. Sections s were stainned 

for fou ur minutess 

and aafter 

it wasshed 

with acidic a ethannol 

(60 %, v/v, v with 37 7% hydrochhloric 

acid 0.1 %, v/v, , 

- 25 - 

Brnoo




Sigma-Alddrich, 

USAA). 

At once e, sections, , which were w not st tained, werre 

prepared 

for 

fluorescennce 

microsccopic 

analysis 

(Carl Zeeiss 

Axiosc cop 40, Zeiss, 

Germanny). 

In addition, 

sections wwere 

stainedd 

by aqueo ous solutionn 

of acridin ne orange (1 ( %, w/w, Sigma-Ald drich, 

USA). 

3. RESSULTS 

ANND 

DISCU USSION 

Usinng 

acid fuchsine 

– malachite m grreen 

stainin ng, individ dual anatommical 

struc ctures 

were welll 

distinguisshable. 

Wh hereas stemms 

were ob bserved onl ly in the pprimary 

sta ate of 

growth duue 

to absennce 

of vascu ular cambiuum, 

roots were w observ ved only inn 

the secon ndary 

state. Basicc 

stem struucture 

is ba ased on the eustele. In n compariso on with thee 

youngest stem 

parts anatoomy, 

anatoomy 

of olde er stem partts 

is based on o the scler rification oof 

interfasci icular 

parenchymma 

and pericycle 

under 

formattion 

of ver ry mechan nically hardd 

structure es. In 

addition, aanatomy 

of 

tuber wa as describedd. 

Its anato omical stru ucture is abbnormal 

du ue to 

absence off 

formationn 

of cork cam mbium, whhich 

produc ces seconda ary dermal ttissue 

perid derm. 

Rhizodermmis 

is replaaced 

by the e outer parrt 

of cortex x. Secondar ry thickeniing 

of roots s and 

tubers is bbased 

on thhe 

activity of o vascular cambium, which produces 

especcially 

secon ndary 

phloem wwith 

predoominant 

ph hloem pareenchyma 

with w stora age functioon. 

Sieve tube- 

members ooccur 

in smmall 

groups interdisperrsed 

in phlo oem parenc chyma. Leavves 

are typi ically 

bifacial wwith 

leaf mmesophyll 

differentiaated 

into palisade and a sponggy 

parench hyma. 

Anatomicaal 

structurre 

of petio ole is simiilar 

to ana atomy of stem; s howwever, 

extensive 


space att 

the cente er is well oobservable. 

Individual l details arre 

introduced 

in 

Fig. 2 (anaatomy 

of root), 

Fig. 3 (anatomyy 

of tuber), Fig. 4 (anatomy 

of sstem) 

and Fig. F 5 

(anatomy of leaf). 

A 

C 

- 26 - 

B 

D 

Brno

XI. Woorkshop 

of Physsical 



sections 

of rootss: 

general anatomy ( A, B), maggnification 

x100, andd 

detaiiled 

structuure 

of end dodermis aand 

vascul lar cylinde er (C, D), magnifica ation x200. . 

Unsttained 

sectiion 

(A), acr ridine orannge 

staining g using FIT TC filter (B) ) and Texas s Red filterr 

(D) aand 

autofluuorescence 

of unstainned 

section under FIT TC filter (CC). 

Descript tion A: a – 

rhizoodermis, 

b – exoderm mis, c – meesodermis, 

d – endode ermis with Casparian strips, e – 

periccycle, 

f – vvascular 

ca ambium, g – secondary 

phloem, 

h – seconndary 

xyle em, i – rayy 

parennchyma; 

j – primary xylem; x B: a – mesoder rmis, b – en ndodermis wwith 

Caspa arian strips, , 

c – ppericycle, 

d – secondar ry phloem - groups of f sieve-tube e-members in axial pa arenchyma, , 

e – pprimary 

xyllem, 

f – secondary 

xyllem, 

g – ray y parenchym ma containning 

starch grains; g C: a 

– enddodermis 

wwith 

Caspar rian strips, b – pericyc cle, c – pass sage cell, d – secondary 

phloem, , 

e – vvascular 

cambium, 

f – secondaryy 

xylem, g – primary xylem; D. . a – mesod dermis, b – 

endoodermis, 

c – pericycle e, d – seconndary 

phlo oem, e – va ascular cammbium, 

f – secondaryy 

xylemm 

(vessel), g – primary y xylem, h – ray paren nchyma. 

A 

C 


sectio ons of tubeers: 

genera al anatomy y (A), maggnification 

x100, andd 

detaiiled 

structuure 

of corte ex and outter 

part of vascular cy ylinder (B) , magnifica ation x200, , 

sievee-tube-memmbers 

(C), magnificaation 

x400 0, and secondary 

vvascular 

ti issues (D), , 


xx400. 

Acrid dine orangee 

staining using u FITC (A, B) andd 

DAPI filte ers (D) andd 

acid fuchsin – mmalachite 

green g stainiing. 

Descrip ption A: a – groups off 

sieve tube e-members, , 

b – aaxiall 

parennchyma 

con ntaining staarch 

grains, c – primar ry xylem, d – secondar ry xylem, e 

– vasscular 

cambbium, 

f – pith p containning 

starch grains; B: a – mesodeermis, 

b – endodermis e s 

withh 

Caspariann 

strips, c – pericycle, , d – secon ndary phloe em; C: a – groups of sieve s tube- 

- 27 - 

B 

D 

Brnoo




members, b – axiall 

parenchy yma; D. a – groups of sieve tube-membbers, 

b – axial 

parenchymma, 

c – initiials 

of vascu ular cambiuum, 

d – ray y parenchym ma, e –seconndary 

xylem. 

A 


sections 

of st tem: generaal 

anatomy (A), magni ification x100, 

and det tailed 

structure oof 

epidermmis 

and out ter part of cortex (B) , magnifica ation x200. . In compa arison 

with abovve-mentioned 

staining g, combinedd 

staining (safranine, gentian vi violet, orang ge G, 

brilliant green 

and cchrysoidine) 

(A) and cchrysoidine 

e staining (B) ( were ussed. 

Description 

A: a – epiidermis 

witth 

cuticle, b – hypodermis, 

c – mesodermis, 

d – enddodermis 

(s starch 

sheath), e – pericyccle, 

f – vascular 

bunndle 

– collateral, 

g – pith, h – interfasci icular 

parenchymma; 

B: a – cuuticle, 

b – epidermis, e c – hypode ermis - colle enchyma, d –sclerench hyma 

cells with plasmodessmata, 

e – mesodermiis 

– parenc chyma, f – pericycle, g – endode ermis 

(starch sheeath); 

- 28 - 

B 

Brno

XI. Woorkshop 

of Physsical 


A 

C 


sectio ons of petiole 

(A) an nd lamina (B – D): ggeneral 

ana atomy (A), , 


xx100 

and x2 200 (B, C), aand 

detailed 

structure lamina (D) ), magnification 

x200. . 

Acriddine 

orangge 

staining using FITCC 

(A, C) an nd DAPI fi ilters (B, DD). 

Descript tion A: a – 

epideermis, 

b – cuticle, c – hypoderrmis, 

d – ground g tissu ue - parencchyma, 

e – sclerifiedd 

interrfascicular 

pparenchym 

ma, f – scleri rified protop phloem, g – metaphlooem, 

h – me etaxylem, i 

– prootoxylem, 

j – central cavity – iintercellula 

ar space; B: a – cuticlle, 

b – epid dermis, c – 

palissade 

parencchyma, 

d – epidermiss, 

e – inter rcellular spa aces of spoongy 

parenchyma, 

f – 

sponngy 

parenchhyma 

cells; C: a – guarrd 

cells, b – spongy pa arenchyma cells, c – ep pidermis, d 

– coollateral 

vaascular 

bun ndle, e – pparenchym 

ma cells sur rrounding vascular bundle, b f – 

epideermis 

withh 

cuticle, g – palisadde 

parench hyma; D. a – palisadde 

parench hyma, b – 

epideermis, 

c – collateral vascular bbundle, 

d – parenchy yma cells surroundin ng vascularr 

bunddle, 

e – sppongy 

pare enchyma, f – epider rmis, g – intercellullar 

spaces of spongyy 

parennchyma, 

h – mechanical 

tissue – collenchym ma, I - epid dermis. 

4. CONCLUUSION 

Work deemonstrated 

d anatomyy 

roots, ste ems and le eaves of ppharmaceut 

tically andd 

toxiccologically 

important plant Aconnitum 

nape ellus L. em. Skalický (RRanunculac 

aceae) usingg 

methhods 

of lighht 

and fluo orescence mmicroscopy 

y. Work sho ows not onnly 

to the anatomicall 

strucctures 

of inndividual 

plant 

organss, 

but also to the possibility 

of uusage 

of flu uorescencee 

micrroscopy 

in tthese 

types of studies. 

5. ACKNOWWLEDGEM 

MENT 

The workk 

has been n supporteed 

by REM MEDTECH GA ČR 5522/10/P61 

18 a FRVŠŠ 

25066/F4/2011/VVFU 

- 29 - 

B 

D 

Brnoo


6. REFERENCES 

1. Feldkamp, A.; Koster, B.; Weber, H. P., Fatal Poisoning by Monks-Hood (Aconitum-Napellus). 

Mon.schr. Kinderheilkd. 1991, 139, (6), 366-367. 

2. Haas, C., Monk's hood, Aconitum napellus, poison and medecine. Ann. Med. Interne 1999, 150, (5), 

446-447. 

3. Pullela, R.; Young, L.; Gallagher, B.; Avis, S. P.; Randell, E. W., A case of fatal aconitine poisoning by 

monkshood ingestion. J. Forensic Sci. 2008, 53, (2), 491-494. 

4. Mitka, J., Phenetic and geographic pattern of Aconitum sect. napellus (Ranunculaceae) in the Eastern 

Carpathians - A numerical approach. Acta Soc. Bot. Pol. 2002, 71, (1), 35-48. 

5. Le Cadre, S.; Boisselier-Dubayle, M. C.; Lambourdiere, J.; Machon, N.; Moret, J.; Samadi, S., 

Polymorphic microsatellites for the study of Aconitum napellus L. (Ranunculaceae), a rare species in 

France. Mol. Ecol. Notes 2005, 5, (2), 358-360. 

6. Chen, Y.; Koelliker, S.; Oehme, M.; Katz, A., Isolation of diterpenoid alkaloids from herb and flowers 

of Aconitum napellus ssp vulgare and electrospray ion trap multiple MS study of these alkaloids. J. 

Nat. Prod. 1999, 62, (5), 701-704. 

7. Csupor, D.; Forgo, P.; Csedo, K.; Hohmann, J., C-19 and C-20 diterpene alkaloids from Aconitum 

toxicum RCHB. Helv. Chim. Acta 2006, 89, (12), 2981-2986. 

8. Forgo, P.; Borcsa, B.; Csupor, D.; Fodor, L.; Robert, B.; Molnar, A. V.; Hohmann, J., Diterpene 

Alkaloids from Aconitum anthora and Assessment of the hERG-Inhibiting Ability of Aconitum 

Alkaloids. Planta Med. 2011, 77, (4), 368-373. 

9. Weijters, B. J.; Verbunt, R.; Hoogsteen, J.; Visser, R. F., Salade malade: malignant ventricular 

arrhythmias due to an accidental intoxication with Aconitum napellus. Neth. Heart J. 2008, 16, (3), 

96-+. 

10. Chan, T. Y. K., Aconite poisoning. Clin. Toxicol. 2009, 47, (4), 279-285. 

11. Strzelecki, A.; Pichon, N.; Gaulier, J. M.; Amiel, J. B.; Champy, P.; Clavel, M., Acute Toxic Herbal 

Intake in a Suicide Attempt and Fatal Refractory Ventricular Arrhythmia. Basic Clin. Pharmacol. 

Toxicol. 2010, 107, (2), 698-699. 

12. Braca, A.; Fico, G.; Morelli, I.; De Simone, F.; Tome, F.; De Tommasi, N., Antioxidant and free radical 

scavenging activity of flavonol glycosides from different Aconitum species. J. Ethnopharmacol. 2003, 

86, (1), 63-67. 

13. Diaz, J. G.; Ruiz, J. G.; Dias, B. R.; Sazatornil, J. A. G.; Herz, W., Flavonol 3,7-glycosides and 

dihydroxyphenethyl glycosides from Aconitum napellus subsp lusitanicum. Biochem. Syst. Ecol. 

2005, 33, (2), 201-205. 

14. Luis, J. C.; Valdes, F.; Martin, R.; Carmona, A. J.; Diaz, J. G., DPPH radical scavenging activity of two 

flavonol glycosides from Aconitum napellus sp lusitanicum. Fitoterapia 2006, 77, (6), 469-471. 

15. Fico, G.; Braca, A.; De Tommasi, N.; Tome, F.; Morelli, I., Flavonoids from Aconitum napellus subsp 

neomontanum. Phytochemistry 2001, 57, (4), 543-546. 

16. Fico, G.; Braca, A.; Bilia, A. R.; Tome, F.; Morelli, I., New flavonol glycosides from the flowers of 

Aconitum napellus ssp tauricum. Planta Med. 2001, 67, (3), 287-290. 

17. Vitalini, S.; Braca, A.; Passarella, D.; Fico, G., New flavonol glycosides from Aconitum burnatii Gayer 

and Aconitum variegatum L. Fitoterapia 2010, 81, (7), 940-947. 

18. Singhuber, J.; Zhu, M.; Prinz, S.; Kopp, B., Aconitum in Traditional Chinese Medicine-A valuable 

drug or an unpredictable risk? J. Ethnopharmacol. 2009, 126, (1), 18-30. 

19. Piltan, D.; Rist, L.; Simoes-Wust, A. P.; Saller, R., Test of a Homeopathic Dilution of Aconitum 

napellus A Clinical, Randomized, Double-Blind, Controlled Crossover Study in Healthy Volunteers. 

Forsch. Komplement.med. 2009, 16, (3), 168-173. 

20. Thurneysen, A., On: Piltan D, Rist L, Simoes-Wust A, Saller R: Test of a homeopathic dilution of 

Aconitum napellus. A clinical, randomized, double-blind, controlled crossover study in healthy 

volunteers. Forsch Komplementmed 2009;16:168-173. Forsch. Komplement.med. 2009, 16, (5), 349- 

349. 

- 30 -


ANATOMICAL STUDY OF 

PHARMACEUTICALLY IMPORTANT PLANT 

MACLURA POMIFERA (RAF.) C.K. SCHNEID. 

(MORACEAE) 

Petr BABULA 1 , Jana TKADLEČKOVÁ 1 , Vojtěch ADAM 2 , René KIZEK 2 





Abstract 

Maclura pomifera (Raf.) C.K.Schneid. (Moraceae), “Osage orange”, is the tree native to the 

mid-western and south-eastern United States. Plant contains many different secondary 

metabolites with interesting pharmaceutical properties, such as flavonoids, isoflavonoids, 

triterpenes, xanthones and stilbenes. Despite the fact that plant is in the focus of interest 

due to above-mentioned compounds, anatomical study of this plant is still missing. This 

study is focused on the demonstration of anatomy of vegetative organs – roots, stems and 

leaves – of Maclura pomifera. 


Maclura pomifera (Raf.) C.K.Schneid. (Moraceae), “Osage orange”, is a very common 

tree native to the mid-western and south-eastern United States, however, species has been 

introduced into many countries including Czech Republic. The types of secondary 

metabolites isolated from different parts of Osage orange belong to the different classes, 

such as flavonoids and isoflavonoids (especially prenylated), xanthones, stilbenes and 

triterpenes. Many phytochemical studies are focused on the multiple fruits due to 

presence of pharmacologically interesting isoflavonoids osajin and pomiferin [1]. 

Pomiferin is studied as a potent histone deacetylase inhibitor [2]. However, 

pharmacologically active secondary metabolites have been detected also in other plant 

parts. Xanthones (osajaxanthone, alvaxanthone, macluraxanthone, 8prenyltoxyloxanthone) 

and flavanones (euchrestaflavanone B and euchrestaflavanone C) 

have been found in bark of Osage orange [3]. Stilbenes (such as oxyresveratrol) have been 

found in heartwood [4]. 

Type of compound Plant part Pharmacological properties Citation 

Prenylated isoflavones – 

pomiferin, osajin 

Multiple 

fruits 

Histone deacetylase inhibitor 

(pomiferin) 

Antioxidant properties 

Cytotoxic properties 

Prenylated isoflavones – Multiple Anti-inflammatory and [8] 

- 31 - 

[2] 

[1] 

[5] 

[6] 

[7]


scandenone, auriculasin fruits antinociceptive activity 

Prenylated flavones Leaves, 

stems 

Antioxidant properties [9] 

Xanthones - Root bark Antimicrobial, anti- [3] 

osajaxanthone, 

alvaxant-hone, 

inflammatory, antidepressive [10] 

macluraxanthone, 8prenyltoxyloxanthone 

Flavanones - Root bark Antioxidant properties [3] 

euchrestaflavanone B 

and euchrestaflavanone 

C 

Stilbenes 

oxyresveratrol 

- Heartwood Antioxidant properties [4] 

Aliphatic compouds, Multiple - [11] 

especially alcohols fruits 

Phenylpropane Multiple - [11] 

derivates 

fruits 

Monoterpenes and Multiple - [11] 

sesquiterpenes 

different types 

– fruits 

Triterpenes/Phytosterols seeds Antioxidant together with [12] 

– beta-sitosterol, 

tocopherols detected in seeds [13] 

campesterol, 

stigmasterol, lupeol, 

Proteins – 

seeds Protease inhibitor [14] 

MPI 

Serine proteinase 

[15] 

Tab. 1: The most important chemical constituents isolated from different parts of Maclura 

pomifera 

Both flavonoids and xanthones demonstrate interesting properties – antioxidant, 

antimicrobial, antifungal, antitumoral and antidepressive properties have been 

determined. The most important compounds isolated from Maclura pomifera are 

summarized in Tab. 1 chemical formulas are introduced in Fig. 1. 

- 32 -


A B C 

O O 

O O 

OH 

O 

OH 

D E F 

O 

OH 

O 

O 

OH 

OH 

O 

OH 

OH 

Fig. 1: The most important chemical constituents of Maclura pomifera. Osajin (A), 

pomiferin (B), scandenone (C), auriculasin (D), alvaxanthone (E) and 8prenyltoxiloxanthon 

(F) 

Family Moraceae consists of shrubs, trees and woody wines with lactifers containing 

milky latex. Some species are important for “fruits”, respectively multiple fruits – figs 

(Ficus carica), mulberry (Morus spp.), and bread-fruit (Artocarpus faltitis). Members of 

genus Ficus are studied because potential pharmacological usage [16, 17]. Some plants are 

important as ornamental, widely cultivated plants (Ficus benjamina). Some plants are 

poisonous. Antiaris toxicaria, plant native to Australia, Asian countries and some African 

countries, such as Uganda or Sudan, is used for hunting. The latex contains cardiac 

glycosides, such as antiarin, antiarosides and antiarotoxin [18, 19]. Latex is used as an 

arrow poison called upas. Despite above-mentioned facts, knowledge about anatomy of 

these plants is still missing. Submitted work is focused on the anatomical study of 

vegetative organs Maclura pomifera (Raf.) C.K.Schneid. 

2. EXPERIMENT 

Plant material (roots, stems, leaves) was collected in the botanical garden of 

University of Veterinary and Pharmaceutical University Brno. For the anatomical study, 

transversal, radial and tangential hand-made sections were used. For the visualization of 

lignified plant tissues, ethanolic (50 %, v/v) mixture of acid fuchsine and malachite green 

(both 1%, w/w; Sigma-Aldrich, USA) was used. Sections were stained for four minutes 

and after it washed with acidic ethanol (60 %, v/v, with 37% hydrochloric acid 0.1 %, v/v, 

Sigma-Aldrich, USA). At once, sections, which were not stained, were prepared for 

fluorescence microscopic analysis (Carl Zeiss Axioscop 40, Zeiss, Germany). In addition, 

sections were stained by aqueous solution of acridine orange (1 %, w/w, Sigma-Aldrich, 

USA). 

O 

O 

O 

- 33 - 

OH 

OH 

OH 

OH 

O 

O 

OH 

O 

O 

O 

O 

OH 

OH 

OH




3. RESSULTS 

ANND 

DISCU USSION 

Usinng 

acid fuchsine 

– malachite m grreen 

stainin ng, individ dual anatommical 

struc ctures 

were welll 

distinguisshable. 

Stem ms and rooots 

were observed 

on nly in the secondary state 

because off 

the very rapid form mation of vvascular 

cam mbium and d productioon 

of secon ndary 

vascular tissues – secondary y phloem and sec condary xy ylem. Diffferentiation 

n of 

sclerenchyyma 

fibers in seconda ary phloemm 

was obser rved in bot th stems annd 

roots. In n the 

comparisoon 

of the sttructure 

of secondary y xylem of roots and stems, s secoondary 

xyle em of 

stems conntains 

highher 

rate of o libriformm, 

which is respons sible for tthe 

mecha anical 

properties. 

Lactifers wwere 

obser rved especiaally 

in the cortex of the t stem. GGeneral 

ana atomy 

is describeed 

in under-mentione 

ed figures ( (Fig. 2 – an natomy of root, Fig. 3 – anatom my of 

stem, Fig. 4 – anatommy 

of petiole e and laminna). 

A 

C 

Fig. 2: Trransversal 

ssections 

of f roots: genneral 

anato omy (A, B), 

magnificcation 

x40, 

and 

detailed sttructure 

of secondary phloem (CC) 

and secon ndary xylem m (D), maggnification 

x200. 

Acid fuchssine 

– malaachite 

gree en staining (A, C, D), acridine orange o stainning 

using FITC 

filter (B). DDescriptionn 

A, B: 1 – secondary s ddermal 

tissu ue periderm m, 2 – seconndary 

phloe em, 3 

– secondarry 

xylem, 4 – cork cam mbium, 5 – vascular cambium, c 6 – ray pareenchyma 

(ra ay), 7 

– vessel, 8 – sclerencchyma 

fiber rs of seconddary 

phloem m, 9 – grou ups of sievee-tube 

mem mbers; 

C: 1 – rayy 

parenchymma, 

2 – sie eve-tube mmembers, 

3 – groups of o sclerenchhyma 

fibers s, 4 – 

axial pareenchyma; 

DD: 

1 – ra ay parenchhyma 

(hete erocellular) ), 2 – axiial 

parench hyma 

associated with vesseel, 

3 – vessel, 

4 – groupps 

of librifo orm fibers. 

- 34 - 

B 

D 

Brno

XI. Woorkshop 

of Physsical 


A 

C 

Fig. 3: Transverrsal 

(A, B) and radial (C, D) sect tions of stem ms. Magniffication 

x40 0 (A), x1000 

(B, DD) 

and x2000 

(C). Aci id fuchsinee 

– malachite 

green staining s (AA, 

C), acridine 

orangee 

stainning 

using FFITC 

filter (B, ( D). Desccription 

A: 1 – epiderm mis with cuuticle, 

2 – periderm, p 3 

– corrtex, 

4 – coortex 

with lactifers inncluding 

pr rimary phlo oem, 5 – seecondary 

phloem, 

6 – 

vascuular 

cambiium, 

7 – secondary xylem, 8 – primar ry xylem, 9 – pith, 10 – rayy 

parennchyma, 

111 

– vessel, 12 1 – sclerifi fied primary y phloem; B: B 1 - epideermis 

with cuticle, 2 – 

phelllem, 

3 – cork camb bium, 4 – hypoderm mis, 5 – mesodermis m s with lactifers, 

6 – 

protoophloem, 

7 – sclerif fied metapphloem/the 

e oldest pa art of secoondary 

phloem, 

8 – 

seconndary 

phlooem, 

9 – va ascular cammbium, 

10 – secondar ry xylem; C: detail of o cortex, 1 

indiccates 

drusees 

of calciu um oxalate in hypode ermis; D: 1 – epidermmis 

with cuticle, c 2 – 

phelllem, 

3 – ccork 

cambiu um, 4 – phhelloderm, 

5 – cortex x, 6 –innerr 

part of cortex 

withh 

lactiffers, 

7 – scllerified 

prim mary phloeem, 

8 – seco ondary phlo oem, 9 – vaascular 

cam mbium, 10 – 

seconndary 

xylemm, 

11 – ray y parenchymma, 

12 – scl lerenchyma a fibers of pprotophloem 

m. 

- 35 - 

B 

D 

Brnoo




A 

C 


seections 

of petiole (A, , B) and la amina (C, D). D Magnifi fication x40 0 (A), 

x100 (B, CC) 

and x2000 

(D). Acrid dine orangee 

staining using u FITC filter (B), TTexas 

Red filter 

(A, D) andd 

DAPI filtter 

(C). Des scription AA: 

1 – epide ermis with cuticle, 2 – hypoderm mis as 

mechanicaal 

tissue, 3 – ground tissue/paren t nchyma, 4 – protophloem, 

5 – mmetaphloem 

m, 6 – 

metaxylemm, 

7 – prottoxylem; 

B: B 1 – detaail 

of tricho ome; C: 1 – epidermmis, 

2 – pal lisade 

parenchymma, 

3 – spoongy 

parenc chyma withh 

intercellu ular spaces, , 4 –epidermmis; 

D: det tail of 

stomata – 1 – guard ccells. 


Worrk 

demonstrated 

anato omy roots, stems and leaves of pharmaceutiically 

impo ortant 

plant Macclura 

pomi mifera (Raf.) ) C.K.Schnneid. 

(Mor raceae) usin ng methodds 

of light t and 

fluorescennce 

microsccopy. 

Work k shows nott 

only to th he anatomic cal structurres 

of indiv vidual 

plant orgaans, 

but alsoo 

to the pos ssibility of usage of flu uorescence microscoppy 

in these types 

of studies. 

5. ACKKNOWLEDDGEMENT 

T 

The work has been sup pported by REMEDT TECH GA ČR 522/100/P618 

a FRVŠ F 

2506/F4/20011/VFU 

6. REFFERENCESS 

1. Diopaan, 

V.; Babulaa, 

P.; Shestivs ska, V.; Adamm, 

V.; Zemlick ka, M.; Dvorska, 

M.; Hubaalek, 

J.; Trnko ova, L.; 

Havell, 

L.; Kizek, R., Electroch hemical and sspectrometric 

c study of an ntioxidant acttivity 

of pom miferin, 

isopommiferin, 

osajiin 

and catalpo oside. J. Pharmm. 

Biomed. Anal. A 2008, 48 8, (1), 127-1333. 

2. Son, II. 

H.; Chung, I. M.; Lee, S. 

I.; Yang, H. D.; Moon, H. H I., Pomiferi in, histone deeacetylase 

inh hibitor 

isolateed 

from the ffruits 

of Maclu ura pomifera. . Bioorg. Med d. Chem. Lett t. 2007, 17, (177), 

4753-4755 5. 

- 36 - 

B 

D 

Brno


3. Teixeira, D. M.; da Costa, C. T., Novel methods to extract flavanones and xanthones from the root 

bark of Maclura pomifera. J. Chromatogr. A 2005, 1062, (2), 175-181. 

4. Djapic, N.; Djarmati, Z.; Filip, S.; Jankov, R. M., A stilbene from the heartwood of Maclura pomifera. 

J. Serb. Chem. Soc. 2003, 68, (3), 235-237. 

5. Hamed, S. F.; Hussein, A. A., Effect of Maclura pomifera total acetonic extract, pomiferin and osajin 

on the autooxidation of purified sunflower triacylglycerols. Grasas Aceites 2005, 56, (1), 21-24. 

6. Tsao, R.; Yang, R.; Young, J. C., Antioxidant isoflavones in Osage orange, Maclura pomifera (Raf.) 

Schneid. J. Agric. Food Chem. 2003, 51, (22), 6445-6451. 

7. Huang, T. T.; Liu, F. G.; Wei, C. F.; Lu, C. C.; Chen, C. C.; Lin, H. C.; Ojcius, D. M.; Lai, H. C., 

Activation of Multiple Apoptotic Pathways in Human Nasopharyngeal Carcinoma Cells by the 

Prenylated Isoflavone, Osajin. PLoS One 2011, 6, (4). 

8. Kupeli, E.; Orhan, I.; Toker, G.; Yesilada, E., Anti-inflammatory and antinociceptive potential of 

Maclura pomifera (Rafin.) Schneider fruit extracts and its major isoflavonoids, scandenone and 

auriculasin. J. Ethnopharmacol. 2006, 107, (2), 169-174. 

9. Lee, S. J.; Wood, A. R.; Maier, C. G. A.; Dixon, R. A.; Mabry, T. J., Prenylated flavonoids from 

Maclura pomifera. Phytochemistry 1998, 49, (8), 2573-2577. 

10. da Costa, C. T.; Dalluge, J. J.; Margolis, S. A.; Horton, D., Liquid chromatography atmosphericpressure 

chemical-ionization mass spectrometry (LC-APCI-MS) of C-glycosylxanthones from the root 

bark of the Osage orange (Maclura pomifera) tree. Abstr. Pap. Am. Chem. Soc. 1999, 217, 015-CARB. 

11. Jerkovic, I.; Mastelic, J.; Marijanovic, Z., Bound volatile compounds and essential oil from the fruit of 

Maclura pomifera (Raf.) Schneid. (osage orange). Flavour Frag. J. 2007, 22, (1), 84-88. 

12. Lee, S. J.; Ahmed, A. A.; Wood, A.; Mabry, T. J., New lupane triterpene fatty acid ester from leaves of 

Maclura pomifera. Nat. Prod. Lett. 1997, 10, (4), 313-317. 

13. Saloua, F.; Eddine, N. I.; Hedi, Z., Chemical composition and profile characteristics of Osage orange 

Maclura pomifera (Rafin.) Schneider seed and seed oil. Ind. Crop. Prod. 2009, 29, (1), 1-8. 

14. Lazza, C. M.; Trejo, S. A.; Obregon, W. D.; Pistaccio, L. G.; Caffini, N. O.; Lopez, L. M. I., A Novel 

Trypsin and alpha-Chymotrypsin Inhibitor from Maclura pomifera Seeds. Lett. Drug Des. Discov. 

2010, 7, (4), 244-249. 

15. Rudenskaya, G. N.; Bogdanova, E. A.; Revina, L. P.; Golovkin, B. N.; Stepanov, V. M., Macluralisin - a 

Serine Proteinase from Fruits of Maclura-Pomifera (Raf) Schneid. Planta 1995, 196, (1), 174-179. 

16. Aghel, N.; Kalantari, H.; Rezazadeh, S., Hepatoprotective Effect of Ficus carica Leaf Extract on Mice 

Intoxicated with Carbon Tetrachloride. Iran. J. Pharm. Res. 2011, 10, (1), 63-67. 

17. Mostafaie, A.; Mansouri, K.; Norooznezhad, A. H.; Mohammadi-Motlagh, H. R., Anti-Angiogenic 

Activity of Ficus carica Latex Extract on Human Umbilical Vein Endothelial Cells. Yakhteh 2011, 12, 

(4), 525-528. 

18. Dai, H. F.; Gan, Y. J.; Que, D. M.; Wu, J.; Wen, Z. C.; Mei, W. L., A New Cytotoxic 19-Norcardenolide 

from the Latex of Antiaris toxicaria. Molecules 2009, 14, (9), 3694-3699. 

19. Shi, L. S.; Liao, Y. R.; Su, M. J.; Lee, A. S.; Kuo, P. C.; Damu, A. G.; Kuo, S. C.; Sun, H. D.; Lee, K. H.; 

Wu, T. S., Cardiac Glycosides from Antiaris toxicaria with Potent Cardiotonic Activity. J. Nat. Prod. 

2010, 73, (7), 1214-1222. 

- 37 -




VOLTTAMMMETRIC 

C MOONITO 

ORING OF 

RIBOOFLAVVIN 

RE EDUCTTION 

ON MERCU M URY 

MENISCUSS 

MOD DIFIED 

SILV VER SOLID S D 

AMAALGAMM 

ELECTROODE 

Lenka BANNDŽUCHOOVÁ1 

, Rená áta ŠELEŠO 

OVSKÁ 1 

1Institute off 

Environmenntal 

and Chem mical Engineeering, 

Univer rsity of Pardub ubice, Students tská 573, Pard dubice, 

532210, 

Czech Re epublic 

Abstractt 

Voltammeetric 

reducttion 

of rib boflavin (RRF) 

on mer rcury meni iscus modif 

amalgam eelectrode 

( m-AgSAE) has been studied in this paper r. It was ob 

provides 1 reduction peak (Ep = -150 mV) suitable for r its determ mination. D 

voltammettry 

(DPV) proved to o be a suittable 

volta ammetric method m for 

riboflavin reduction. The limit of detection 

was foun nd as 4.89×1 10 

method inn 

conjuncttion 

with m-AgSAEE 

was also o successfu 

determinaation 

in twwo 

types of pharmaceuuticals. 

All l results we 

obtained oon 

hanging mercury drop 

electroode 

(HMDE E). 

-9 fied silver solid 

bserved tha at RF 

Differential pulse 

r monitorin ng of 

M (tacc = 5 s). The DPV 

lly aplliedd 

for ribof flavin 

ere comparred 

with re esults 

7. INTRRODUCTIION 

Ribooflavin 

(6,77-dimethyl-9-(1-D-ribbityl)isoallo 

oxazin, Fig. . 1) is an essential water w 

soluble vittamin, 

whoose 

aqueou us solutionss 

are very sensitive of 

light. RFF 

has two active a 

forms: flavvin 

mononnukleotide 

(FMN) andd 

flavin ade enine mononukleotidde 

(FAD). These T 

two coenzzymes 

playy 

importan nt role in the organ nism. They y participat ate many redox r 

processes llike 

oxidative 

phosph horylation oor 

synthesi is and degradation 

of fatty acids. 

The 

recommennded 

daily iintake 

of RF R is for addults 

1.3 – 1.7 1 mg. The e food richh 

in RF are eggs, 

livers, meeet, 

milk andd 

milk prod ducts 0,0. 

Fig g. 1 The struucture 

of ri iboflavin 

Due to its bioloogical 

impo ortance andd 

light insta 

of the lighht 

of RF annd 

other fl lavins has bbeen 

invest 

Birss et al. . studied thhe 

photolysis 

of adsorbbed 

flavins 

studies, whhich 

describe 

the electrochemicaal 

behavior 

electrodes, 

have beenn 

provided 0-7]. Mechhanism 

of el 

described by Linquisst 

and Farro oha 0 as a 2e- /2H + ability the photolysis p aand 

the stability 

tigated for example inn 

literature e 0,0. 

on mercury 

electrodees 

0. Some other 

and determ mination off 

RF on mer rcury 

lectrochem mical behaviior 

on DME E was 

rev versible red dox processs. 

RF provi ides 1 

- 38 - 

Brno


reduction and 1 oxidation peak on mercury electrodes 0. Nowadays, due to the toxicity of 

liquid mercury, new non toxic electrode materials are still looked for. RF has been 

successfully determined using glassy carbon electrode 0, modified gold electrode 0 or 

diamond electrode 0. 

The m-AgSAE is one of the modifications of silver solid amalgam electrodes, which 

were introduced by Novotný and Yosypchuk in 2000 0. These electrodes are made from 

non toxic silver amalgam and have been successfully used for voltammetric determination 

of various compounds. This paper has been focused on application of mercury meniscus 

modified silver solid amalgam electrode for voltammetric monitoring of riboflavin 

reduction and its determination in pharmaceuticals. 

8. EXPERIMENT 

The stock solution of RF had to be stored in the dark and all analysis had to be 

provided in a covered polarographic cell due to the light instability of RF. All 

measurements were provided by computer controlled Eco-Tribo Polarograph PC-ETP in 

3-electrodes set up (m-AgSAE or HMDE as a working electrode, saturated argentchloride 

as a reference and platinum wire as an auxiliary electrode). DPV with optimized 

parameters (Ein = 100 mV, Efin = -800mV, v = 20 mV.s -1 Eacc= 100 mV, tacc depends on 

concentration of RF, height of pulse -50 mV, width of pulse 80ms) was found as a suitable 

method for RF determination. The optimal conditions for the m-AgSAE`s surface 

regeneration was found as 30 potential jumps between 0 and -1500 mV. 


It was found that RF provides 1 reduction and 1 oxidation peak on m-AgSAE as well 

as on HMDE in wide range of pH (3-12), which consists with literature 0,0. The highest 

reduction response on both used working electrodes was observed in medium of pH 5 that 

is why the 0.05M acetate buffer of pH 5 was used for all subsequent analysis. It was also 

found, using direct current voltammetry, that heigh of the reduction peak increased 

linearly with the scan rate, which shows that the reduction is realized in adsorbed state. 

Some statistical parameters were determined for the reduction of RF. The relative 

standard deviation of 11 repeated measurements was calculated as RSDM(11) = 2.73 % (m- 

AgSAE) resp. 1.36 %(HMDE). The relative standard deviations of 5 repeated 

determinations were calculated for various concentrations levels using ADSTAT 0 (Tab. 

1). The limit of detection is equal to 4.89×10 -9 (tacc = 5 s) for m-AgSAE and 5.16×10 -10 

(tacc = 120 s) for HMDE. The linear dynamic range was found as 1×10 -8 to 1×10 -6 for m- 

AgSAE and 2×10 -9 to 1×10 -6 for HMDE. The example of concentration dependence 

obtained on m-AgSAE is shown in Fig. 2. 

Tab. 1: Comparison of relative deviations and relative standard deviations of repeated 

determinations obtained on m-AgSAE and HMDE 

Added 

[M] 

5×10 -8 

m-AgSAE HMDE 

Found RD RSDD(5) Added Found RD RSDD(5) 

[M] [%] [%] [M] [M] [%] [%] 

4.95×10 -8 -1 0.65 1×10 -8 1.04×10 -8 +4 3.65 

- 39 -


2×10 -8 2.06×10 -8 +2.9 0.84 5×10 -9 5.01×10 -9 +0.2 0.78 

1×10 -8 1.00×10 -8 0 1.11 2×10 -9 2.05×10 -9 +2.4 1.17 

I [nA] 

-8,5 

-8 

-7,5 

-7 

-6,5 

-6 

-5,5 

-5 

-4,5 

-4 

0 

-50 

-100 

-150 

Fig. 2 The concentration dependence of RBF on m-AgSAE, sup. electrolyte: acetate buffer 

(pH 5), method: DPV with optimized parameters, c(RF) = 2×10 -8 - 16×10 -8 M 

The content of RF was also determined in two pharmaceuticals using standard 

addition method. Both pharmacetuticals content 10 mg RF in 1 tablet. The determined 

average content (9,8 resp. 9,6 mg/tbl.) is consistent with content from the producer and 

differs of 2 resp. 4 %. Almost the same results were obtained using HMDE. 

10. CONCLUSION 

It can be concluded, that the m-AgSAE can replace mercury electrodes in 

voltammetric determination of RF due to its sufficient sensitivity and good reproducibility 

of measurement. DPV in combination with m-AgSAE was successfully applied for 

determination RF in two types of pharmaceuticals. 


The work has been supported by Ministry of Education, Youth and Sports of the 

Czech Republic by the Research Centre LC06035 and by the project MSM 0021627502. 

12. REFERENCES 

[1] Vávrová, J.: Vitaminy a stopové prvky 2007,1 st edition, Racek, J., Dastych, M., 2007, Pardubice, 16, 

27. 

[2] Ahmad, I., Fasihullah, Q., Noor, A., Ansari, I.A., Manzar Ali, Q.N.: Int. J. Pharm., 280 (2004), 1-2, 

199. 

[3] Sikorska, E., Khmelinskii, I., Komas, A., Koput, J., Ferreira, L.F.V., Herance, J.R., Bourdelance, J.L., 

Williams, S.L., Worrall, D.R., Insinska-Rak, M., Sikorski, M.: Chem. Phys., 314 (2005), 1-3, 239. 

[4] Birss, V.I., Guha-Thakurta, S., McGarvey, C.E., Quach, S., Vanysek, P.: J. Electroanal. Chem., 423 

(1997), 1-2, 13. 

[5] Villamil, M.J.F, Ordieres, A.J.M, Garcia, A.C., Blanco, P.T.: Anal. Chim. Acta, 273 (1993), 1-2, 377. 

[6] Lindquist, J., Farroha, S.M.: Analyst, 100 (1975), 1191, 377. 

[7] Wang, J., Luo, D.-B., Farias, P.A.M., Mahmoud J.S.: Anal. Chem, 57 (1985), 1, 158. 

- 40 - 

I p [nA] 

-3 

-2 

-1 

0 

I p [nA] = -0,018 c [nM] + 0,021 

R = 0,997 

0 100 200 

c [nM] 

-200 -250 

E [mV]


[8] Marian, I.O., Bonciocat, N., Sandulescu, R., Filip, C.: J. Pharmaceut. Biomed., 24 (2001), 5-6, 1175. 

[9] Qijin, W., Nanjung, Y., Haili, Z., Xinpin, Z., Bin, X.: Talanta, 55 (2001), 3, 459. 

[10] Chatterjee, A., Foord, J.S.: Diam. Relat. Mater., 18 (2009), 5-8, 899. 

[11] Yosypchuk, B., Barek, J.: Crit. Rev. Anal. Chem., 39 (2009), 3, 189. 

[12] Trilobyte statistical software, TriloByte s.r.o., Pardubice (1995). 

- 41 -


PHOTOINDUCED OXYGEN ACTIVATION IN 

THE PRESENCE OF 4- 

ANILINOQUINAZOLINES 

Zuzana BARBIERIKOVÁ 1 , Jana BALÁŽIKOVÁ 2 , Soňa JANTOVÁ 2 , Vlasta BREZOVÁ 1 

1 Institute of Physical Chemistry and Chemical Physics, 

2 Institute of Biochemistry, Nutrition and Health Protection, Faculty of Chemical and Food Technology 

Slovak University of Technology in Bratislava, Radlinského 9, SK-812 37 Bratislava, Slovak Republic 

Abstract 

To evidence photoinduced molecular oxygen activation in dimethylsulfoxide or 

acetonitrile solutions of 4-anilinoquinazoline derivatives, in situ photochemical EPR 

experiments were performed. The ability of investigated compounds to generate 

paramagnetic intermediates and superoxide radical anion upon photoexcitation was 

studied applying the EPR spin trapping technique. The generation of singlet oxygen was 

evidenced via the oxidation of 4-hydroxy-2,2,6,6-piperidine to paramagnetic 4-hydroxy- 

2,2,6,6-tetramethylpiperidine N-oxyl (TEMPOL). Photobiological studies evaluated 

antiproliferative/ cytotoxic effect in the dark and in the presence of UVA light using 

cancer cell lines Hela, HT-29 and L1210. 


4-Anilinoquinazolines represent a class of epidermal growth factor receptor tyrosine 

kinase inhibitors widely used to treat cell lung cancer and other tumours [1]. Majority of 

recent studies related to anilinoquinazolines deals with the development of novel 

synthesized derivatives and further investigations of their anticancer activity. However 

possible phototoxic response of these compounds is not sufficiently examined. 

Consequently also their potential photosensitizer properties remain open. Hence this 

study focuses on the photochemical investigations of three 4-anilinoquinazoline 

derivatives (Tab. 1), in order to find out whether these compounds may activate molecular 

oxygen upon specific, mostly UVA, irradiation. In biological specimens, dissolved oxygen 

represents a very effective quenching agent for a molecule in the excited triplet state. The 

ground state oxygen molecule ( 3 O2) can be excited to the reactive singlet states, leading to 

reactions that exhibit a phototoxic effect on living cells. We recognise two mechanisms of 

the photosensitizer triplet excited state quenching by molecular oxygen, either by energy 

transfer from the excited sensitizer molecule to 3 O2, resulting in the formation of singlet 

oxygen ( 1 O2), or by electron transfer, forming superoxide radical anion (O2 − ) and other 

radical intermediates. 

- 42 -


Tab. 1: Overview of investigated 4-anilinoquinazolines. 

A B C D 

-Br 

-Br 

-Cl 

2. EXPERIMENT 

EPR in situ photochemical experiments were performed upon UVA irradiation of 4anilinoquinazolines 

in dimethylsulfoxide (DMSO) applying an EPR spin trapping 

technique to observe short living radical intermediates. 5,5-Dimethyl-1-pyrroline N-oxide 

(DMPO) and 5-ethoxy carbonyl-5-methyl-1-pyrroline N-oxide (EMPO) were used as 

spin trapping agents. The photoinduced production of singlet oxygen during the 

excitation of 4-anilinoquinazolines was followed in acetonitrile (ACN) solutions in the 

presence of 4-hydroxy-2,2,6,6-piperidine (TMP). The solutions of 4-anilinoquinazolines 

containing spin traps or TMP were mixed directly before the EPR measurements, then 

carefully saturated with air and immediately transferred to a small quartz flat cell 

optimized for the TE102 cavity of the EPR spectrometer EMX (Bruker, Germany). The 

samples were irradiated at 295 K directly in the EPR resonator, and the EPR spectra 

recorded in situ during continuous photoexcitation. 


The generation of paramagnetic species upon photoexcitation of studied 

anilinoquinazoline derivatives (Tab. 1) was investigated in DMSO solutions by in situ EPR 

spin trapping technique. Applying DMPO as a spin trapping agent dominating 

photoinduced twelve line EPR signal was observed. This signal was attributed to the 

superoxide radical anion spin adduct ( DMPO-O2 – ), considering good agreement of spin 

Hamiltonian parameters, obtained from simulation analysis, with the hyperfine coupling 

constants of DMPO-O2 – in DMSO. In addition to the major spin adduct, DMPO-OCH3 

- 43 - 

-H -H 

- 

NO 

2 

-H 

-H 

- 

CH 

3 

N-phenyl-6bromo-2morpholinoquinazoline-4amine 

(QA1) 

N-(2nitrophenyl)-6bromo-2morpholinoquinazoline-4amine 

(QA2) 

N-(4methylphenyl)-6chloro-2-phenylquinazoline-4amine 

(QA3)




was also ppresent. 

In 

DMPO, EMMPO 

spin t 

spectra obbtained 

in D 

DMPO annd 

EMPO. T 

correspondding 

to ind 

attributed to 

simulation 

 

EMPO-CR 

order to v 

trap was us 

DMSO solu 

The simula 

dividual spin 

EMPOO-O2 

n analysis, 

R’’. 

– and erify the aassignment 

of the spin n adducts mmonitored 

using 

sed. Fig. 1 suummarizes 

s the experi imental andd 

simulated d EPR 

ution of QAA2 

upon continuous 

ex xposure in the presen nce of 

ated spectraa 

represent t a linear co ombinationn 

of EPR si ignals 

n adducts. TThe 

major spin adducts 

found wwith 

EMPO were 

EMPO-OC E CH3 [2]. The e other spin n adducts, assigned du uring 

 

present in relatively low concen ntrations, account a to EMPO-O OR’ or 

(a) 

Fig. 1: Expperimental 

(black) an nd simulated 

(red) electron 

param magnetic sp spectra obse erved 

upoon 

continuuous 

photo oexcitation of aerate ed 1.3 mM M QA2 dimmethylsulfo 

foxide 

soluutions 

in thhe 

presence e of two diiferent 

spin n trapping agents (a) 55,5-dimeth 

hyl-1- 

pyrrroline 

N-ooxide 

(DM MPO) and ( (b) 5-ethox xycarbonyl l-5-methyl-1-pyrrolin 

neN- oxide 

(EMPOO). 

Simulati ions repressent 

linear combinati ions of thee 

correspon nding 

spinn 

adducts. 

Phottoinduced 

generation n of singleet 

oxygen upon continuous 

irr rradiation of o 4- 

anilinoquiinazolines 

in ACN so olutions wwas 

monitor red via the 

oxidationn 

of TMP to a 

semistablee 

nitroxide radical TE EMPOL whhich 

is char racterized by b a three-line 

EPR signal s 

[3]. Continnuous 

photoexcitation 

n of the derrivatives 

in the presenc ce of TMP under the given g 

experimenntal 

conditiions 

caused d an increease 

in the relative in ntensity off 

EPR sign nal of 

TEMPOL. 

The photobiollogical 

inv vestigationss 

were oriented 

mainly m on the stud dy of 

biological/ /photobioloogical 

effect ts of the deerivatives 

on o cancer cell 

lines. Thhe 

sensitivity 

of 

Hela, HT-229 

and L12210 

cells to UVA irradiiation 

was evaluated. 

4. CONNCLUSION 

The EPR exper 

anilinoquiinazolines 

paramagneetic 

specie 

generatingg 

O2 

the format 

N 

riments con nfirmed thhat 

UVA (λ λmax = 365 nm) n photooexcitation 

of 4- 

in the pre esence of mmolecular 

oxygen results 

in thhe 

formatio on of 

s coupled with electtron 

or en nergy trans sfer to moolecular 

ox xygen 

– and 1O2. The ap pplication oof 

different t spin trapp ping agentss 

also evide enced 

tion of otheer 

oxygen- and carbonn-centered 

radical spin n adducts. RResults 

obta ained 

- 44 - 

(b) 

Brno


in photobiological experiments evidenced the increase in the sensitivity of all tested cell 

models on investigated derivatives. Leukemia L1210 cells were the most sensitive to the 

effect of non-photoactivated and photoactivated 4-anilinoquinazolines. In conclusion our 

investigations show that 4-anilinoquinazolines behave as photosensitive compounds, with 

potential application as photosensitizers. 


This study was financially supported by Scientific Grant Agency (VEGA Project 

1/0018/09) and Research and Development Agency (contract No. APVV-0339-10). 

6. REFERENCES 

[1] Sun H.-Y., Guan S., Bi H.-C., Su Q.-B., Huang W.-L., Chowbay B., Huang M., Chen X., Li C.-G., Zhou S.- 

F.: Journal of Chromatography B, 854 (2007), 320-327 

[2] Stolze K., Udilova N., Nohl H.: Biological Chemistry, 383 (2002), 813-820 

[3] Zang L., van Kuijk F., Misra B., Misra H.: Biochemistry and Molecular Biology International, 37 (1995), 

283-293 

- 45 -


ELECTROCHEMICAL DETECTION OF RNA 

USING A COMPLEX OF SIX-VALENT 

OSMIUM 

Martin BARTOŠÍK 1 , Mojmír TREFULKA 1 , Emil PALEČEK 1 

1 Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno, 

Czech Republic 

Abstract 

Currently, nucleic acid electrochemistry is mainly focused on development of DNA 

hybridization biosensors. After a discovery that short, gene-regulating RNA molecules - 

microRNAs (miRNAs) - might play a role in biomedicine, particularly in cancer 

development, new assays for the miRNA detection have been sought. We have previously 

demonstrated that electroactive complexes of six-valent osmium with nitrogenous ligands 

(Os(VI)L) preferentially bind to polysaccharides. Recently, Os(VI)L was shown to bind 

also to ribose-containing oligonucleotides (ONs), if the ribose was located at the 3’-end, 

but not to deoxyribose. Such Os(VI)L-ON adducts were analyzed at carbon and mercury 

electrodes (HgE), including solid amalgam electrodes, and were easily discriminated from 

oligodeoxynucleotides. Moreover, application of HgE enabled picomolar determination of 

ribose-containing ONs due to an electrocatalytic nature of the signal provided by the 

adduct. This finding might lead to a new, sensitive method for miRNA detection. 


Nucleic acids (NAs) are electroactive species undergoing oxidation and reduction 

processes at carbon and mercury electrodes, producing analytically useful electrochemical 

(EC) signals [1]. Moreover, NAs can be labeled to render them electroactive at electrodes, 

if (a) they do not produce any oxidation or reduction signals or (b) to discriminate 

between different NAs, e.g. unlabeled probe and target DNA. The fifty years history of 

NA electrochemistry was recently reviewed [2]. In the first three decades, only small 

number of laboratories studied NAs from EC point of view. However, fast progress in 

genomics strongly influenced the NA electrochemistry and particularly the research and 

development of the EC DNA sensors, making it a booming field with > 700 papers in 2010. 

Although EC determination of nucleotide sequences or DNA point mutations (single-base 

mismatches) after PCR amplification is well-established, detection of a specific (nonrepetitive), 

amplification-free sequence in eukaryotic genomes, as well as EC sensing of 

DNA-protein interactions still represent important challenges. Recently it was shown that 

short microRNAs (miRNA) play significant roles in molecular biology processes. For 

instance, miRNA was shown to be potential cancer biomarker, but its role is being 

investigated also in other diseases. Literature dealing with an EC analysis of RNA, as 

compared to DNA hybridization sensors, is still rather scarce [3, 4]. 

- 46 -

XI. Woorkshop 

of Physsical 



All voltammmetric 

me easurementts 

were per rformed wi ith an AUTTOLAB 

(Ec co Chemie, , 

Nethherlands) 

inn 

connectio on with VAA-Stand 

663 

(Metrohm m, Switzerlland), 

using g standard, , 

threee-electrodee 

system co omprising wworking 

el lectrode (h hanging meercury 

drop p electrodee 

(HMMDE), 

solid amalgam electrode e (SSAE) 

or pyr rolytic grap phite electroode 

(PGE)) ), referencee 

electtrode 

(Ag/AAgCl/3 

M KCl) K and aauxiliary 

el lectrode (pl latinum wiire). 

Oligon nucleotidess 

(ONss) 

having eeither 

all deoxyribose 

d e residues (ODN), or deoxyriboose 

residues s with onee 

ribosse 

residue at the 3’-e end of the ON (ODR RN), were used. ONs were mod dified withh 

Os(VVI)L 

compllexes, 

whe ere L was nitrogenou us ligand, usually biipyridine 

(bipy). ( Forr 

modification 

deetails, 

see [5 5]. 

3. RESULTS 

AND DISCUSSIO 

D ON 

In differeence 

to Os s(VIII)L coomplexes, 

modifying m DNA and RNA base es, Os(VI)LL 

compplexes 

speccifically 

modify m sugaar 

residues s in ribosi ides [6]. WWe 

used Os(VI)bipy O y 

compplexes 

for mmodificatio 

on of ONs, aas 

shown in n Fig. 1. Os(VI)bipy 

mmodified 

on nly ODRN, , 

containing 

riboose 

at the 3‘-end, andd 

not ODN Ns without any ribosee, 

in agreement 

withh 

propposed 

specifficity 

of Os(VI)L O to rribose 

resid dues. At HMDE, H welll-develope 

ed catalyticc 

peakk 

(peak Cat at) was pro oduced by Os(VI)bipy y-ODRN ( Fig. 2A,B), , allowing picomolarr 

deterrmination 

oof 

the ODR RN [7]. Simmilar 

results were obtai ined also wwith 

SAE (Fi ig 2C). Thee 

meassurement 

ccan 

be don ne not onlyy 

in situ, but b also ex x situ, afterr 

transfer of ODRN- 

modified 

electtrode 

to the t blank backgroun nd electrolyte. 

In eex 

situ ex xperiments, , 

Os(VVI)bipy-ODDRN 

was adsorbed a frrom 

L vo olumes of solution, s mmaking 

it possible p too 

deterrmine 

femmto- 

and su ubfemtomoole 

amount ts of ODR RNs. At PGGE 

(not sh hown), noo 

catallytic 

peak wwas 

produce ed. Instead, , well-deve eloped anod dic peaks and were e observed, , 

againn 

highly specific 

to rib bose-contaiining 

ONs. Due to a no on-catalytiic 

nature of f the signal, , 

highher 

concenttrations 

we ere necessarry 

to deter rmine ODR RN (~ 250 nM). End-labeling 

off 

ODRRN 

is not sequence 

sp pecific - almmost 

identi ical responses 

were oobtained 

wi ith ODRNss 

havinng 

different 

nucleot tide sequeences 

(not t shown). Moreoverr, 

ODRNs could bee 

sensiitively 

deteected 

in an excess of OODNs. 

Fig. 

1. Modificcation 

of a ribose at the 3’-end d of an olig gonucleotidde 

with electroactivee 

Os(VI)L. 

Oligodeox xynucleotidde 

without ribose resid due is not mmodified. 

- 47 - 

Brnoo




Fig. 2. Diifferential 

pulse volt tammogramms 

of Os(V VI)bipy-modified 

oliggonucleotid 

des at 

HMMDE 

and SAAE. 

(A) Pea ak Cat of 220 

nM ODR RN (green) and ODN ( (without ri ibose, 

redd) 

at HMDDE. 

(B) OD DRN peak CCat 

(green) 

at the co oncentratioon 

of 50 pM p at 

HMMDE. 

(C) 4400 

nM ODRN O (greeen) 

measur red at SAE E. Backgroound 

electr rolyte 

(blaack 

dashed) ). 


It wwas 

shown that ribose e residue aat 

the 3’-en nd of the ON O can bee 

modified with 

electroactiive 

Os(VI) )L comple exes, produucing 

sign nals at both 

mercurry 

and ca arbon 

electrodes. 

Advantagge 

of mercury 

electroodes 

lies in n a catalyti ic nature oof 

the resp ponse, 

allowing hhighly 

sensitive 

determ mination oof 

ONs at picomolar 

le evel. It is encouraging 

g that 

the catalyytic 

peak wwas 

obtain ned also att 

SAE, wh hich is mo ore convennient 

for se ensor 

applicationns. 

We beliieve 

that ou ur new appproach 

could 

be utiliz zed in EC aanalysis 

of RNA, R 

especially miRNA. 


T 

This work wass 

supported d by MEYSS 

CR ME09 9038 and GACR G P3001/11/2055 

(EP), 

GACR 3001/10/P5488 

(MT), Research R 

AV0Z500440507 

and AAV0Z50040 

0702. 

Centre LC C06036 an nd IBP RResearch 

Plans 


[1] Paleček, 

E., Jelen, F.: Electrochemistry 

of nuucleic 

acids an nd proteins. Towards T electtrochemical 

sensors s 

for geenomics 

and pproteomics, 

Paleček, P E., Sccheller, 

F. W., W Wang, J. (e eds), 2005, Ammsterdam: 

Elsevier, 

73. 

[2] Paleček, 

E.: Electrooanalysis, 

21 (2009), 239. 

[3] Lusi, E. A., Passammano, 

M., Gua arascio, P., Scaarpa, 

A., Schi iavo, L.: Anal. 

Chem., 81 (22009), 

2819. 

[4] Poehllmann, 

C., Spprinzl, 

M.: An nal. Chem., 822 

(2010), 4434 4. 

[5] Trefuulka, 

M., Palečček, 

E.: Electr roanalysis, 211 

(2009), 1763 3. 


M., Ostatná, 

V., Havran, 

L., Fojta MM., 

Paleček, E.: E Electroana alysis, 19 (20007), 

1281. 


M., Bartoošík, 

M., Pale eček, E.: Electtrochem. 

Com mmun., 12 (20 010), 1760. 

- 48 - 

Brno


THE SPECTROSCOPIC STUDY OF 

PHOTOINDUCED REACTIONS OF N- 

HETEROCYCLIC COMPOUNDS 

Miroslava BOBENIČOVÁ 1 , Jana TABAČIAROVÁ 1 , Kristína PLEVOVÁ 2 , Dana 

DVORANOVÁ 1 

1 Institute of Physical Chemistry and Chemical Physics, Faculty of Chemical and Food Technology, Slovak 

University of Technology in Bratislava, Radlinského 9, SK-812 37 Bratislava, Slovak Republic 

2 Institute of Organic Chemistry, Catalysis and Petrochemistry, Faculty of Chemical and Food Technology, 

Slovak University of Technology in Bratislava, Radlinského 9, SK-812 37 Bratislava, Slovak Republic 

Abstract 

The work is focused on the photochemical transformation of novel fluoroquinolones in 

aprotic media by means of UV-Vis spectroscopy and EPR spin trapping technique. 

Investigated molecules behave as photosensitizers and UVA irradiation in the presence of 

air led to the formation of reactive oxygen species. 


N-Heterocyclic compounds, mainly quinoline derivatives, are known for their 

importance in therapeutical treatment. Although these substances contain basic quinoline 

skeleton, their biological activity depends on their substitution character and position. 

The 4-oxoquinoline derivatives represent one of the largest classes of antimicrobial agents 

and they are known for their inhibition of Topoisomerase II. From them, the 4oxoquinoline 

derivatives possessing a fluorine atom at position 6 as a substituent have 

been classified as most efficient in biological activity [1,2]. The molecules contain 

extended π-electron system which could be activated by UVA irradiation and molecules 

could behave as photosensitizers. The photoactivation in the presence of oxygen leads to 

the formation of reactive oxygen species (ROS) as superoxide radical anion, singlet oxygen 

or hydroxyl radical [3,4], which could activated the undesirable side reactions, frequently 

coupled with drop of biological activity of drug. Our study was focused on novel 

fluoroquinolnoes (Table 1) with main attention on their photochemical transformation 

using EPR and UV-Vis spectroscopy. 

Tab. 1: Structures of investigated fluoroquinolones 

F 

O 

N 

R2 

R1 

- 49 - 

R1 R2 

F7Q2 COOC2H5 H 

F7Q3 COOCH3 H 

F7Q4 COOH H 

F7EQ2 COOC2H5 C2H5


2. EXPERIMENT 

Four fluoroquinolones (Table 1) were synthesized in our laboratory and applied in 

photochemical experiments. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was used as spin 

trapping agent and 2,2,6,6-tetramethyl-4-piperidin-N-oxyl (TEMPOL) was applied as spin 

label. The oxidation of 4-hydroxy-2,2,6,6-tetramethylpiperidine (TMP) via singlet oxygen 

to the paramagnetic nitroxyl radical (TEMPOL) was utilized for 1 O2 detection by EPR 

spectroscopy. The stock solutions of fluoroquinolones were prepared in dimethylsulfoxide 

(DMSO) and acetonitrile (ACN) and directly mixed with solution of spin trap/spin label 

prior to irradiation. The prepared solutions were saturated by argon or air, filled in the 

quartz flat cell and the X-band EPR spectra were recorded at EPR Bruker EMX 

spectrometer. Samples were irradiated at 295 K in situ using HPA 400/30S lamp (400 W, 

max = 365 nm, Philips, UVA irradiance 5 mW cm –2 ). A Pyrex glass filter was applied to 

eliminate the radiation wavelengths below 300 nm. The EPR spectra obtained were 

processed, analyzed and simulated using the Bruker software WinEPR and SimFonia and 

the Winsim2002 software. The UV-Vis spectra of the investigated compounds were 

recorded using a UV-3600 UV-Vis spectrometer (Shimadzu, Japan) with a 1 cm square 

quartz cell. 


The UV-Vis spectra of F7Q2, F7Q3, F7Q4 and F7EQ2 in DMSO confirmed their 

absorption of the UVA region. The flouroquinolones have two absorption maxima in the 

region 310-330 nm. Therefore, by the photoactivation with UVA irradiation the 

molecules may behave as photosenzitizer producing reactive oxygen species. The EPR 

spin trapping technique is suitable tool for the evidence the reactive short-lived free 

radicals adding them to spin trapping agent under the formation of more stable 

paramagnetic products (spin adducts). The EPR spectrum of adducts brings information on 

type of reactive radical trapped [3-5]. Figure 1 represents the experimental and simulated 

EPR spectra of irradiated aerated DMSO solution of F7Q2 in the presence of DMPO spin 

trap. 

- 50 -


Fig.1 Experimental (solid line) and simulated (dotted line) EPR spectra (magnetic field 

sweep 8 mT) obtained after 25 minutes of continuous UVA irradiation of system 

F7Q2/DMPO/DMSO/air. Initial concentration: c0(F7Q2) = 0.80 mmol dm –3 and 

c0(DMPO) = 42 mmol dm –3 . 

The continuous UVA irradiation led to the formation of reactive radical species 

DMPO-O2 – (rel. conc. 60 %); DMPO-OCH3 (23 %); DMPO-OR (12 %) and DMPO-CR 

(5 %). The formation of superoxide radical anion upon photoexcitation of F7Q2 in DMSO 

solvent was confirmed by the addition of superoxide dismutase (SOD) into the solution, 

which caused a significant decrease of DMPO-O2 – EPR intensity by the competitive 

reaction of SOD with O2 – . The generation of DMPO-OCH3 has been observed in systems 

containing DMSO and DMPO as product of the reaction of ROS with solvent producing 

CH3 radicals, trapped under air as methoxy radical to DMPO spin trap. The UVA 

irradiation of other fluoroquinolones (F7Q3, F7Q4 and F7EQ2) in the DMPO/DMSO/air 

reaction system confirmed formation of identical type of spin trap adducts with different 

concentration. Replacing air by inert atmosphere led to the generation mainly carboncentered 

radicals originated probably from the molecule decomposition. 

Alternative technique to detect the reactive free radical formation is based on 

monitoring the decrease EPR intensity of nitroxide radical, e. g. TEMPOL, resulting from 

the interaction of its >N-O group with the generated reactive radical species, as well as 

singlet oxygen. The application TEMPOL in inert atmosphere and under air led to the 

changes of three-line EPR signal intensity upon irradiation reaction systems of 

fluoroquinolones, which sensitively reflects the influence of experimental conditions 

(solvent, atmosphere). The photoinduced generation of singlet oxygen upon continuous 

irradiation of quinolones in aerated ACN solutions was monitored via the oxidation of 

diamagnetic TMP. 

4. CONCLUSION 

The results of our study confirmed evidence that investigated molecules behave as 

photosensitizers possessing the ability to photoactivate molecular oxygen via an 

electron/energy transfer mechanism generating reactive oxygen species. 

- 51 -



This study was financially supported by Scientific Grant Agency of the Ministry of 

Education of the Slovak Republic (Projects VEGA 1/0018/09) and Research and 

Development Agency of the Slovak Republic (contracts No. APVV 0339-10 and SK-AT- 

0016-08). 

6. REFERENCES 

Drlica, K., Hiasa, H., Kerns R., Malik, M., Mustaev, A., Zhao X.: Curr. Top. Med. Chem. 9 (2009) 981. 

Boteva, A., Krasnykh, O.: Chem. Heterocycl. Compd. 45 (2009) 757. 

Brezová, V., Valko, M., Breza, M., Morris, H., Telser, J., Dvoranová, D., Kaiserová, K., Varečka, Ľ., Mazúr, 

M., Leibfritz, D.: J. Phys. Chem. B 107 (2003) 2415. 

Brezová, V., Gabčová, S., Dvoranová, D., Staško A.: Photochem. Photobiol. B Biol. 79 (2005) 121. 

Rimarčík, J., Lukeš, V., Klein, E., Kelterer, A.M., Milata, V., Vrecková, Z., Brezová, V.: J. Photochem. 

Photobiol. A Chem. 211 (2010) 47. 

- 52 -


TECHNICAL SOLUTION OF PLANET 

EXPLORATION 

Jaromír HUBÁLEK 1 

1 Brno University of Technology, Technická 10, CZ-616 00 Brno, CZ 

Abstract 

The exploration of Mars has been an important part of the space exploration programs of 

many countries in the world. Dozens of robotic spacecraft, including orbiters, landers, and 

rovers, have been launched toward Mars since the 1960s. These missions were aimed at 

gathering data about current conditions and answering questions about the history of 

Mars as well as a preparation for a possible human mission to Mars. These missions 

performed huge technology level and human craft in area of space research. Environment 

condition of Mars are extremely harder than on the Earth which front the scientist to 

many technical challenges to be solved in order to enable work of explorers in extreme 

conditions. 


In 2008, researchers from Brno University of Technology, Mendel University in 

Brno and Masaryk Universtiy succeeded in the program Nanotechnology for Society 

supported by the government of the Czech Republic and the ongoing project is 

successfully solving the priority tasks. This project is focused on development of new 

nanosystems applicable in medicine as biosensors for online monitoring particular 

physiological characteristics and treatment progression in general. To further reinforce 

the collaboration of Brno Universities (Masaryk University, Brno University of 

Technology, and Mendel University in Brno) the centre of excellence called CEITEC 

(Central European Institute of Technology) and Regional research centre SIX: Sensors, 

information and communication systems have been established to create an effective 

platform for research in nanotechnnology and nanoscience comprising materials and 

functional structures suitable for nanoelectronics and nanophotonics in general, 

addressing both the preparation and the characterization of nanostructures applicable in 

bio-medical areas, energetic and information and communication technologies. 

2. TODAYS KNOWLEDGE ABOUT RED PLANET 

The Mars is fourth planet of solar system, second the smallest planet after the 

Mercury. The Mars has ten times lighter then Earth. One day is 24 our, 39 minutes which 

is very similar to Earth but one year is 1,88 of Earth year. The surface is covered by iron 

oxides creating red colour of the planet. Gravitation is 0,376 G, it means aprox. 3 times 

less. The temperature on the surface can be extremely different from -143°C to 27°C with 

average value below 0°C. Athosphere is containing mainly CO2 with low pressure 600 Pa. 

The water has been found on the Mars. In these conditions the water is still frozen. Also 

CO2 is frozen on the poles of the planet together with water. 

- 53 -




The bigggest 

crater HHellas 

Planiti ia is 7 km deeep. 

On the bottom the atmosphericc 

pressure can c be 

1155 Pa and 

scientist eexpect 

that te emperature ccan 

rise abov ve 0°C and liquid l water ccan 

be found d. It is 

very importaant 

for humaan 

mission. [1 1] 

Fig. 1 Frozen 

poles of f the Mars 

3. NOWWADAYS 

TECHNO OLOGIES 

Phoeenix 

was a robotic spa acecraft on a space ex xploration mission m on Mars unde er the 

Mars Scouut 

Program. 

The Phoe enix landerr 

descended d on Mars on May 255, 

2008. Mi ission 

scientists used instruuments 

abo oard the laander 

to search 

for environme e ents suitabl le for 

microbial life on MMars, 

and to o research the histor ry of wate er there. TThe 

robotic c arm 

scooped uup 

more soil 

and deli ivered it too 

3 differen nt on-boar rd analyzerrs: 

an oven n that 

baked it annd 

tested thhe 

emitted gases, a miicroscopic 

imager, i and d a wet cheemistry 

lab b. The 

lander's RRobotic 

Armm 

scoop wa as positioneed 

over the e Wet Chem mistry Lab delivery fu unnel 

on Sol 29 (the 29th MMartian 

day y after landding, 

i.e. Jun ne 24, 2008 8). The soil was transf ferred 

to the insstrument 

oon 

Sol 30 (June 25, 2008), and 

Phoenix performedd 

the first t wet 

chemistry tests. On SSol 

31 (June e 26, 2008) Phoenix re eturned the e wet chemmistry 

test re esults 

with inforrmation 

on the salts in n the soil, annd 

its acidi ity. The wet 

chemistryy 

lab was part 

of 

the suite of tools caalled 

the Microscopy M , Electroch hemistry an nd Conducctivity 

Ana alyzer 

(MECA) [22]. 

Marss 

Science Laboratory 

as a Mars Expploration 

Rover R (MER R)is intendeed 

to be the e first 

planetary mission (pllaned 

arriva al 2012) to use precisi ion landing g techniquees, 

steering itself 

toward thee 

Martian ssurface 

similar 

to the wway 

the sp pace shuttle controls itts 

entry thr rough 

the Earth's 

upper atmmosphere. 

In this waay, 

the spacecraft 

wil ll fly to a ddesired 

loc cation 

above the surface of f Mars befo ore deployinng 

its para achute for the t final laanding. 

Lik ke the 

twin roverrs 

now on tthe 

surface of Mars, MMars 

Science e Laborator ry will havee 

six wheel ls and 

cameras mmounted 

onn 

a mast. Un nlike the twwin 

rovers, 

it will car rry a laser ffor 

vaporiz zing a 

thin layerr 

from the surface of f a rock annd 

analyzin ng the elem mental commposition 

of o the 

underlyingg 

materials. 

It will be able to colllect 

rock an nd soil samp ples and disstribute 

the em to 

on-board test chambbers 

for ch hemical annalysis. 

Its design includes 

a suuite 

of scientific 

- 54 - 

Fig. F 2 Crate er Hellas Pllanitia 

Brno


instruments for identifying organic compounds such as proteins, amino acids, and other 

acids and bases that attach themselves to carbon backbones and are essential to life as we 

know it. It can also identify features such as atmospheric gases that may be associated with 

biological activity. Using these tools, Mars Science Laboratory will examine Martian rocks 

and soils in greater detail than ever before to determine the geologic processes that formed 

them; study the martian atmosphere; and determine the distribution and circulation of 

water and carbon dioxide, whether frozen, liquid, or gaseous. 

NASA plans to select a landing site on the basis of highly detailed images sent to 

Earth by the Mars Reconnaissance Orbiter, in addition to data from earlier missions. The 

rover will carry a radioisotope power system that generates electricity from the heat of 

plutonium's radioactive decay. This power source gives the mission an operating lifespan 

on Mars' surface of a full martian year (687 Earth days) or more while also providing 

significantly greater mobility and operational flexibility, enhanced science payload 

capability, and exploration of a much larger range of latitudes and altitudes than was 

possible on previous missions to Mars. [3] 



5. REFERENCES 

[1] CARR, Michael H. The surface of Mars. New York : Cambridge University Press, 2006 

[2] H.U. Keller, W. Goetz, H. Hartwig, S.F. Hviid, R. Kramm, W.J. Markiewicz, R. Reynolds, C. 

Shinohara, P. Smith, R. Tanner, P. Woida, R. Woida, B.J. Bos, M.T. Lemmon, Journal of Geophysical 


[3] Mars Science Laboratory: Program and Missions source [http://mars.jpl.nasa.gov/programmissions/ 

missions/present/msl/] 

- 55 -


ELECTROCHEMICAL DATA FROM SPACE 

René KIZEK 1 , Vojtěch ADAM 1 , Jaromír HUBÁLEK 2 



Abstract 

There have been developing numerous analytical methods for the most sensitive detection 

of target molecules under the lowest demands on dimension, consumables and operation 

The one analytical technique that has many of the requisite characteristics to meet such a 

challenge is electroanalysis. Several electrochemical instruments have been constructed, 

tested and successfully used in space research. This short contribution will give you short 

overview of the most important ones. 

1. THE MARS ENVIRONMENTAL COMPATIBILITY ASSESSMENT (MECA) 

The MECA instrument was originally designed, built, and flight qualified for the 

2001 Mars Lander mission. The mission was subsequently cancelled due to the loss of the 

Mars Polar Lander in 1999. The MECA, and a newer version, the Robotic Chemical 

Analysis Laboratory (RCAL), have been proposed for the 2007 launch opportunity. MECA 

was designed to evaluate potential geochemical and environmental hazards to which 

future Mars explorers might be exposed and to return data that would help in 

understanding the geology, geochemistry, paleoclimate, and exobiology of Mars. The 

MECA instrument package contained wet chemistry laboratory, an optical and atomic 

force microscope, an electrometer to characterize the electrostatics of the soil and its 

environment, and an array of material patches to study the abrasive and adhesive 

properties of soil grains. Because of payload limitations, the entire MECA package was 

limited to amass of 10 kg, a peak power of 15 W, and a volume of 35 × 25 × 15 cm 3 . The 

development of MECA for analyzing the surface material in a remote hostile environment 

posed a unique set of challenges, especially for remote chemical analysis and more 

specifically for electrochemical analysis [1,2]. 

2. THE CRYOSCOUT MARS INORGANIC CHEMICAL ANALYZER (MICA) 

CryoScout has been defined as a deep subsurface mission to the north polar cap of 

Mars, which will explore the stratigraphic record of recent climate change in the 

underlying layered terrain. A “cryobot” thermally driven probe will penetrate the ice to 

examine the borehole wall's stratigraphy and the chemistry of the melt water and 

entrained dust. Together with surface-station observations, these data will provide for a 

new understanding of the Martian polar meteorology; present climate and polar water 

exchange; recent polar volatile deposition and erosion; the scale, texture, structure, dust, 

and volatile content of subsurface layers; their accumulation rates; the origin of the 

entrained dust; the evolution of the polar cap; and the role of orbital variations in this 

evolution. These high scientific priority goals, and this type of probe and landing site, 

represent important contribution to the Mars and planetary exploration program [2]. 

- 56 -


The “bubble“ of melt water that surrounds the cryobot while it descends will 

contain dissolved gases, dust, and soluble species extracted from the dust. The dust is 

derived from many sources, including ancient hydrothermal mineralization, chemical 

precipitation in lake beds, floodwaters episodically disgorged from the upper crust, or 

from moisture-driven mineral differentiation in the pedogenic surface. The MICA system 

is an electrochemically based flow-through variant of the MECA WCL. Similar to the 

MECA, the MICA will contain a similar array of sensors that will analyze the soluble 

components of the Martian dust by measuring a variety of ionic species and properties, 

including conductivity, pH, cations (sodium, potassium, magnesium, calcium, and 

ammonium using ISEs), anions (chloride, nitric and hydrochloric), metals (copper, 

cadmium, mercury and lead), reversible and irreversible oxidants in the melt water (using 

cyclic voltammetry), dissolved CO2, dissolved O2, oxidation reduction potential, and 

temperature [2]. 

3. AN ELECTROCHEMICAL MICROBIAL GROWTH DETECTION SYSTEM 

(LIDA) 

Bacterial growth in culture media has typically been monitored optically, by 

measuring turbidity, or electrochemically, by conductivity, pH, or capacitance [3,4]. 

Although never flown, several of these methods have been proposed for detection of 

extraterrestrial microbial life [5,6]. Optical turbidity does not appear to be a viable 

technique because of the problems associated with analyzing a particulate soil sample. 

Modifications have been proposed to resolve this dilemma [5], but very little is gained and 

the final results may still allow ambiguous interpretation. Even though more reliable, each 

of the electrochemical techniques by themselves may also be prone to interferences or 

ambiguous interpretation. However, we propose that integrating the conductivity, pH, 

and several ion selective electrodes as a sensor array and incorporating them into a 

multisample micro-laboratory will provide a reliable, robust, low-mass, and low-power 

device for monitoring microbial growth – the Life Detection Array (LIDA). LIDA is based 

on several crucial components, which ensure a definitive conclusion that changes in the 

growth chambers were biologically induced. These components include two chambers 

containing a differentially monitored pair of sensor arrays with one chamber for control 

and the other for the inoculation, a special sterilization and inoculation procedure, use of 

the local soil as a growth medium, and multiple replications. The chambers are identical 

and each contains sensors for conductivity, oxidation ± reduction potential (ORP), pH, 

sodium cations, potassium cations, calcium cations, magnesium cations, chloride anions 

and nitric anions. More sensors can be added but, as will be seen from our preliminary 

experiments, they may not be needed. Any global changes due to temperature, pressure, 

or soil chemistry should affect both chambers identically and thus the differential 

measurements will remain “zeroed” [1,2]. 



- 57 -


5. REFERENCES 

[1] S.R. Lukow, S.R. Kounaves, Electroanalysis 17 (2005) 1441. 

[2] S.P. Kounaves, Chemphyschem 4 (2003) 162. 

[3] M. Lanzanova, G. Mucchetti, E. Neviani, Journal of Dairy Science 76 (1993) 20. 

[4] R.E. Madrid, C.J. Felice, M.E. Valentinuzzi, Medical & Biological Engineering & Computing 37 (1999) 

789. 

[5] E.L. Merek, V.I. Oyama, Applied Microbiology 16 (1968) 724. 

[6] M.P. Silverman, E.F. Munoz, Applied Microbiology 28 (1974) 960. 

- 58 -


SPECTROPHOTOMETRIC ACID-BASE 

CHARACTERIZATION OF ADENINE 

ANALOGUES 

Pavel BUČEK 1 , Přemysl LUBAL 1 , Petra NETROUFALOVÁ 1 , Libuše TRNKOVÁ 1 

1 Department of Chemistry, Faculty of Science, Masaryk University, Brno. 

Abstract 

Purine based DNA/RNA bases adenine (6-aminopurine) and guanine (2-aminopurin-6one), 

serve in live bodies as building blocks of high physiological importance. For in vitro 

research applications they are replaced by 2-aminopurine and 2,6-diaminopurine. These 

aminopurines are used either “free” as replacements for adenine/guanine in nucleic acid 

strands or are subject to further derivatization and use (e.g. enzyme function research [1]). 

Despite their frequent use, very few is known about the substances’ physico-chemical 

properties. We studied acid-base properties of 2-aminopurine and 2,6-diaminopurine with 

use of advanced chemometric tools applied to UV/Vis spectroscopy data and since the 2aminopurine 

is highly fluorescent, fluorescence spectroscopy data, too. 


Beside the use as (fluorescent) replacements of original bases in DNA strands. 2aminopurine 

(2-AP) and 2,6-diaminopurine (2,6-DAP) are used for research purposes 

such as enzyme blockersChyba! Záložka není definována., enhanced bioavailability drugs 

[2] after further derivatization or agents against both severe and common viral diseases 

[3]. Despite various fields and quite numerous application of 2-AP and 2,6-DAP, data 

describing their acid-base properties are not almost available (Adenine about 40, 2,6-DAP 

about 4, 2-AP no papers – NIST database v. 7). Continuously increasing computing 

capacity of computers allows us to use advanced chemometric process high amounts of 

data (in this case spectra) almost real-time. We used the programs Equispec [4], a hardmodel-based 

routine for Matlab, and Opium. 

2. EXPERIMENT 

For experiment, acidic and basic solutions of aminopurines were used for acidobasic 

titration where concentration of both aminopurines was 0.1 mM. The measurements were 

carried out at temperature 25°C and ionic strength adjusted to 0,1M. The UV/Vis 

absorption and luminescence spectra were recorded after pH solution equilibration (Orion 

SA 720 pH meter with combined glass electrode) in 10mm path-length quartz cuvette on 

Agilent HP8452 A and Aminco Bowman series2 (excitation wavelength 295nm) 

spectrophotometers. 

Spectra were processed using standard MatLab routines and analyzed with Equispec 

program. Spectral data in form 3D matrix D (1D=wavelength,2D=absorbance,3D=pH) 

were decomposed according to equation (1) [5]: D=CS T +E (1) where C and S T are the data 

- 59 -


matrices of the distribution diagram and the pure spectra for each of the spectroscopic 

active conformations (species) present in the experiment. E represents the data matrix of 

residuals not associated with the proposed conformations in C and S T . Target of the 

analysis is to describe the system by the best possible way with C and S T while keeping the 

value of residuals‘ matrix E as low as possible. The hard-model approach to such data 

analysis is by defined chemical model of reaction which is kept by the program during 

calculation. The program then iteratively calculates the best results possible under given 

conditions, e.g. for acidobasic properties of 2-AP and 2,6-DAP we used only two types of 

constraints: estimates of the equilibrium constants and number of present species. 


For comparison of results, the UV/Vis and luminiscence spectral data were analyzed 

separately and also simultaneously. The values of protonation constants obtained by both 

techniques are almost the same. It means that ligand protonation does not have influence 

on excite state of ligand and it is taking place in ground state. The first pKa of 2-AP is 

lower while this value is higher for 2,6-DAP than parent Adenine. This fact is probably 

consequence of different polarity as was shown by HPLC of studied ligands on reverse 

stationary phase [6]. 

Absorbance (A.U.) 

Absorbance (A.U.) 

0.8 

0.6 

0.4 

0.2 

2-AP UV/Vis spectra 

0 

250 300 350 400 

Wavelength (nm) 

1.5 

1 

0.5 

2,6-DAP UV/Vis spectra 

0 

250 300 350 400 


Fig. 1: The experimental data for 2-AP (upper) and 2,6-DAP (lower) obtained 

by molecular absorption (left) and emission (right) spectroscopy. 

- 60 - 

Intensity (A.U.) 

Intensity (A.U.) 

3 

2 

1 

0 

2-AP emission spectra 

-1 

300 350 400 


450 500 

0.2 

0.15 

0.1 

0.05 

0 

2,6-DAP emission spectra 

-0.05 

300 350 400 450 500 

Wavelength (nm)


4. CONCLUSION 

Acidobasic properties of widely used adenine analogues/derivatives were studied by 

means of UV/VIS and fluorescence spectroscopy combined with advanced chemometric 

data analysis. Both methods are useful for determination of protonation constants. 


The work has been supported by Ministry of Education of the Czech Republic 

(projects LC06035, ME09065, MUNI/A/0992/2009). 

6. REFERENCES 

[1] Sasaki, M., Ikeda, H., Sato, Y., Nakanuma, Y.: Free Radical Research, 42 (2008), 625 

[2] Arimili et al. - US patent no. 5922695 

[3] Tracerco Instruments - European Patent EP0343133 

[4] Dyson, R., Kaderli, S., Lawrence, G.A., Maeder, M., Zuberbühler, A.D.: Anal. Chim. Acta, 353 

(1997), 381 

[5] del Toro, M., Bucek, P., Avino, A., Jaumot, J., Gonzalez, C., Eritja, R., Gargallo, R.: Biochemie, 91 

(2009), 894 

[6] Kristova, P., Diploma Thesis, Masaryk University, Brno 2010. 

- 61 -


ROBOTIC MODULE EURYDICE 

Vojtěch ADAM 1 , Jaromír HUBÁLEK 2 , René KIZEK 1 



Abstract 

The development of robotic module eurydice is focused on the development of a unique 

multi-purpose detector for detection and identification of dangerous pathogens. The 

multipurpose detector Eurydica uses the latest lab-on-a-chip nanobiotechnologies mainly 

based on the nanoparticles. The proposed detector will be controlled by our developed 

software, to which neural networks and artificial intelligence will be implemented with 

regard to do the most sensitive evaluation and processing of signals. Eurydica detector will 

be also implemented in the rescue robot Orpheus for use in situations threatening national 

security. 

1. NANOPARTICLES 

Generally, 200 nm is considered as an upper limit for the size of nanoparticles, while 

the minimal diameter should be about 10 nm. Certainly, nanoparticle properties 

requirements also depend on tumour characteristics including cancer type, stage of the 

disease, location in the body, tumour vascularisation and properties of the interstitial 

matrix or host species. These requirements are summarized in a review of Adiseshaiah et 

al. [1]. Nanoparticles designed for tumour targeted therapies consist of various 

components, in most cases from nanocarrier (in some literature called nanovector) and an 

active agent (drug) [2]. Drug-carrier nanoparticles are considered as submicroscopic 

colloidal systems that may act as a drug vehicles, either as nanospheres (matrix system in 

which the drug is dispersed) or nanocapsules (reservoirs in which the drug is confined in a 

hydrophobic or hydrophilic core surrounded by a single polymeric membrane) [3]. 

Nanoparticle carriers are mostly composed of iron oxides, gold, biodegradable polymers, 

dendrimers, lipid based carriers such as liposomes and micelles, viruses (viral 

nanoparticles) and even organometallic compounds [4-6]. A detailed review about 

nanocarriers was recently published by Peer et al [7]. Several such engineered drugs are 

already in clinical practice, over 20 nanoparticle therapeutics have been approved by the 

FDA for clinical use including liposomal doxorubicin and albumin conjugate paclitaxel 

[8,9]. There was described that doxorubicin or paclitaxel nanoparticules drug delivery 

systems overcome chemoresistance with decreased side effects not only in experiment but 

also in clinical studies[10,11]. Micelles composed of pluronic polymers has been studied 

for its effect on multidrug reversion[12] and lipid-based nanoparticles offer a promising 

approach to the delivery of cytostatics that pass though blood brain barrier for brain 

tumours and brain metastases therapy [13]. However, the potential success of these 

particles in the clinical application relies on the consideration of above mentioned 

important parameters, most importantly, minimum toxicity of the carrier itself [14]. 

Concerning the nanoparticle shape, following nanostructures are frequently cited in 

- 62 -


literature: nanoshells, nanorods, nanocages, nanocubes or nanotubes. Nanoparticles have 

the ability to treat as well as to diagnose the disease therefore a new term “theranostic 

nanoparticles” (therapeutic and diagnostic) is currently commonly used in the 

literature.[15-18] Magnetic nanoparticles are well-established elements that offer 

controlled size, ability to be manipulated externally, and enhancement of contrast in 

magnetic resonance imaging (MRI). Therefore, these nanoparticles could have many 

applications in biology and medicine, including drug delivery [16,19-22], and medical 

imaging [23-29]. Particularly iron-based nanoparticles have been used as therapeutic 

agents with specific application as contrasting agents for MRI and magnetically targeted 

drug delivery to the tumour cell. Imunomagnetic nanoparticules can be used for positive 

or negative selection of cells used for cell therapy and their presence on transplanted cells 

can be used as a magnetic cell label for in vivo cell visualization by MRI[30]. Iron oxides 

have several crystalline polymorphs known as hematite, maghemite and magnetite 

(Fe3O4) and some others. However, only maghemite and magnetite found the interest in 

bioapplications. Numerous procedures of the MNPs synthesis have widely been studied 

and different synthetic mechanisms have been established over past decades. The most 

popular methods, such as co-precipitation, microemulsion, flame-assisted thermal 

decomposition, etc. are based on chemical principles. Co-precipitation of Fe 3+ and Fe 2+ 

salts is a classical method used to prepare iron oxide NPs. Reaction temperature, pH and 

precursor type; have to be precisely optimized to control NP morphology, size, and 

quantity. Other methods based on physical principles including mechanical grinding, 

biopolymerization processes, gel templating and solvent-free methods such as chemical 

vapour deposition (CVD), electrical explosion and mechanical milling have been reported. 

CVD, mechanical milling and other solvent-free methods are especially useful for the 

synthesis of other interesting materials such as NdFeB and carbon nanotubes for which 

there are no established “wet” synthesis routes. To date, NPs prepared by CVD have been 

used almost exclusively for heterogeneous catalysis, magnetic data storage and 

nanoelectronic devices rather than biomedicine.[31] In comparison with physical 

methods and biopolymerization processes, the chemical methods especially the solution 

based synthetic routes are generally more suitable for superparamagnetic MNPs for MRI 

due to the controlled parameters such as particle size, size distribution and purity. To 

meet the requirements of biocompatibility and desired functionality the surface 

modification is a key step for MNP applications. It is provided by coating of MNP surface 

by biocompatible layer enabling further interaction with functional coating. 

Functionalization of the nanoparticle surface with DNA fragments is used for gene 

therapy as more safe alternative to commonly used viral vectors.[32,33] Genetic material 

such as DNA plasmids, RNA and/or siRNA can be encapsulated inside or conjugated to the 

surface of the nanoparticle. In bionanotechnology, specific functions of proteins such as 

antibody-antigen detection and receptor-substrate recognition such as the biotin-avidin 

interaction are very valuable. Proteins can be incorporated into NPs during the 

coprecipitation step. More sophisticated bioconjugation techniques are desirable to assure 

that the biomolecule is bound to the NP surface in correct orientation and biological 

activity is preserved [34-43]. Both natural as well as synthetic peptide ligands have 

- 63 -


potential for the NP stabilization and biofunctionalisation. Besides targeted drug delivery, 

imaging of all kinds is an extremely important field of nanoparticle application. 

2. BIOSENSORS BASED ON THE PARTICLES 

Detection. Human population is globally threatened by expansive pandemics caused 

by viruses particularly HIV, HBA, HBV, HBC and influenza viruses. All these viral 

diseases are distinguished by the rapid spreading in population, considerable morbidity 

and also mortality. Approximately 5% of the world population is infected by the hepatitis 

B virus (HBV) and more than 10% by the hepatitis A virus that cause a 

necroinflammatory liver disease of variable duration and severity. Chronically infected 

patients with active liver disease carry a high risk of developing cirrhosis and 

hepatocellular carcinoma. Diagnostic possibilities are relatively limited till now and they 

are focused in the direct cultural proof of identity or they result from the detection of 

viral antigen by the use of antibodies or of antiviral antibodies. All above mentioned 

methods are expensive and time consuming - cultivation, detection of viral antigens or are 

not so specific - antiviral antibodies. Detection of viral nucleic acid presence in biological 

sample represents another approach in the therapeutic strategy. Isolation of nucleic acid 

by magnetic beads is widely used method of extraction for complex biological sample. 

Magnetic particles responding to an external magnet field are providing an elegant 

method of separation of targeted molecule from the solution. Taking advantage of their 

magnetic characteristic and low cost of synthesis, magnetic particles (MPs) have been 

widely used as a universal separation tool for biologically active compounds such as 

nucleic acids (i.e., DNA and RNA), proteins and peptides, as well as intact cells [44]. 

Magnetic particles have been explored in many other biomedical and bioengineering 

applications including magnetic resonance imaging (MRI), tissue repairing, detoxification 

of biological fluids, drug delivery, magnetic hyperthermia and chemotherapeutic agents’ 

delivery. Miniaturization has become an important factor in all areas of modern society, 

reflected strongly in scientific research. Particularly in chemistry, new direction was 

given a in the 1990s when lab-on-a-chip and micro-total analysis approach was 

introduced. Nowadays, microfluidics is a thriving multidisciplinary area. Integration of 

the sample preparation procedure straight to the analytical process is increasing the 

analytical throughput and therefore decreasing the analysis time as well as its costs. In line 

with the general trend in miniaturization utilizing microfluidic chips has been gaining 

popularity especially due to their advantages including extremely low consumption of 

samples as well as other chemical compounds, ultrafast separations and/or in many cases 

cost effective mass fabrication of disposable chips. 



4. REFERENCES 

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[2] M. Ferrari, Nat. Rev. Cancer 5 (2005) 161. 

[3] L. Juillerat-Jeanneret, Drug Discov. Today 13 (2008) 1099. 

- 64 -


[4] F.X. Gu, R. Karnik, A.Z. Wang, F. Alexis, E. Levy-Nissenbaum, S. Hong, R.S. Langer, O.C. Farokhzad, 

Nano Today 2 (2007) 14. 

[5] K.J. Cho, X. Wang, S.M. Nie, Z. Chen, D.M. Shin, Clin. Cancer Res. 14 (2008) 1310. 

[6] B. Mishra, B.B. Patel, S. Tiwari, Nanomed.-Nanotechnol. Biol. Med. 6 (2010) 9. 

[7] D. Peer, J.M. Karp, S. Hong, O.C. FaroKhzad, R. Margalit, R. Langer, Nat. Nanotechnol. 2 (2007) 751. 

[8] B. Haley, E. Frenkel, Urol. Oncol.-Semin. Orig. Investig. 26 (2008) 57. 

[9] R.K. Jain, T. Stylianopoulos, Nature Reviews Clinical Oncology 7 (2010) 653. 

[10] Y.Z. Wang, L. Yu, L.M. Han, X.Y. Sha, X.L. Fang, International Journal of Pharmaceutics 337 (2007) 

63. 

[11] M.E.R. O'Brien, N. Wigler, M. Inbar, R. Rosso, E. Grischke, A. Santoro, R. Catane, D.G. Kieback, P. 

Tomczak, S.P. Ackland, F. Orlandi, L. Mellars, L. Alland, C. Tendler, C.B.C.S. Grp, Annals of 

Oncology 15 (2004) 440. 

[12] T. Minko, E.V. Batrakova, S. Li, Y.L. Li, R.I. Pakunlu, V.Y. Alakhov, A.V. Kabanov, Journal of 

Controlled Release 105 (2005) 269. 

[13] J.L. Arias, B. Clares, M.E. Morales, V. Gallardo, M.A. Ruiz, Curr. Drug Targets in press (2011). 

[14] A. Puri, K. Loomis, B. Smith, J.H. Lee, A. Yavlovich, E. Heldman, R. Blumenthal, Crit. Rev. Ther. 

Drug Carr. Syst. 26 (2009) 523. 

[15] M.Z. Ahmad, S. Akhter, G.K. Jain, M. Rahman, S.A. Pathan, F.J. Ahmad, R.K. Khar, Expert Opinion 

on Drug Delivery 7 (2010) 927. 

[16] M.S. Bhojani, M. Van Dort, A. Rehemtulla, B.D. Ross, Molecular Pharmaceutics 7 (2010) 1921. 

[17] S.M. Janib, A.S. Moses, J.A. MacKay, Advanced Drug Delivery Reviews 62 (2010) 1052. 

[18] J. Xie, S. Lee, X.Y. Chen, Advanced Drug Delivery Reviews 62 (2010) 1064. 

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62 (2010) 144. 

[20] D.K. Kim, J. Dobson, Journal of Materials Chemistry 19 (2009) 6294. 

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B.S. Tang, K. Xia, J.H. Xia, Chinese Science Bulletin 50 (2005) 2323. 

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- 66 -


CHEMORESITANCE OF CANCER CELLS- 

MODELS FOR STUDY IN VITRO AND IN VIVO 

Tomáš ECKSCHLAGER 1 , Jan HRABĚTA 1 , M.A.RAHMAN 1 , Jitka POLJAKOVÁ 2 , Marie 

STIBOROVÁ 2 

1 Department of Pediatric Hematology and Oncology, 2 nd Medical School, Charles University and University 

Hospital Motol, V Úvalu 84, 150 06 Prague 5, Czech Republic 

2 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech 

Republic 

Abstract 

Cancer is one of the main causes of death in developed countries. Resistance to cytostatics 

is a major cancer therapy problem. The behaviour may be explained by a selection of 

subclones within tumor that have the ability to survive the cytotoxic effects of cytostatics. 

At the cellular level, a number of resistance mechanisms operate. They include drug efflux 

via membrane pumps, failure to activate a prodrug, alteration in the abundance of the 

target protein, mutation of the target protein and/or inactivation of apoptotic pathways. 

Other causes of chemoresitance are hypoxia or increased number of cancer stem cells 

(CSC). Hypoxia enhances resistance to chemotherapy. Some studies demonstrated that 

hypoxia-induced chemoresistance is through the HIF pathway. CD133+ cells (CSC) are 

more resistant to some chemotherapeutics compared to CD133- cells. We discuss our 

experience with resistant neuroblastoma cell lines, with chemoresistance induced by 

hypoxia and with CSC. 

Introduction 

The development of resistance to cytostatic agents is a major cancer therapy problem 

and has been the focus of many research efforts [1]. Tolerance to one agent is often 

accompanied by cross-resistance to a variety of others, often unrelated compounds. The 

behaviour may be explained by a selection of subclones of cells within the original tumor 

that have the ability to survive the cytotoxic effects of anticancer drugs [1]. The 

description of chromosomal aberrations in resistant tumors is an clue in the identification 

of genes participating in chemoresistance. 

At the cellular level, a number of resistance mechanisms can operate. These 

mechanisms include drug efflux via membrane pumps (ABC transportes such as Pglycoprotein), 

drug metabolism, including inactivation or failure to activate a prodrug, 

alteration in the abundance of the target protein, for example the topoisomerase II 

enzyme, mutation of the target protein and/or inactivation of pathways leading to cell 

death, such as apoptotic signaling [1, 2]. 

Neuroblastoma /NBL/ is the third most common pediatric cancer and is responsible 

for approximately 15% of all childhood cancer deaths [1]. It is a malignant tumor 

consisting of neuronal crest derived undifferentiated neuroectodermal cells. The clinical 

hallmark of NBL is heterogeneity. Some of the tumors undergo spontaneous regression or 

- 67 -


differentiate into benign ganglioneuromas, some are curable with surgery and little or no 

adjuvant therapy and some progress despite intensive multimodal therapy. This clinical 

diversity is closely correlated with the molecular biological features of the tumor. Among 

the most prominent are MYCN amplification, gain of 17q, loss of 1p36.2-36.3, loss of 

11q23 and less frequently, gains at 2p, 3q 4q and 6p and losses at 14q, 3p, 4p, 5q, 9p and 

18q. Tumors with 1p loss often have MYCN amplification and highly malignant clinical 

behavior 

5. METHODS 

The NBL cell lines UKF-NB-2, UKF-NB-3 and UKF-NB-4 were established from 

bone marrow metastases of high risk neuroblastoma harvested at relapse in three patients. 

The Vincristin, Doxorubicin, Cisplatin and Ellipticine resistant cell sublines designated 

UKF-NB-2 VCR , UKF-NB-2 DOX , UKF-NB-2 CDDP , UKF-NB-3 VCR , UKF-NB-3 DOX , UKF-NB-3 CDDP , 

UKF-NB-4 VCR , UKF-NB-4 DOX and UKF-NB-4 CDDP were established by incubation of 

parental cells with increasing concentrations of the respective drug. Examination by 

fluorescent in situ hybridization (FISH), comparative genomic hybridization (CGH), MTT 

tests in normoxic and hypoxic (1% O2) conditions 


Analysis of our results suggests that chemoresistance is a very complex phenomenon 

and those multiple gains and losses indicate that drug resistance is thought to be mediated 

through multiple complementary pathways. Drug resistance to Doxorubicin and 

Vincristin was mediated not only by over expression of the MDR1 gene, but also by 

amplification of the gene [3]. In the cell line UKF-NB-4 where amplification on 

chromosome 7 occurred in the parental cell line the drug resistant daughter cell line had 

even greater amplification in the region where the MDR1 gene is located. Cell lines 

resistant to CDDP did not show any amplification of the MDR1 gene, which corresponds 

with YASUNO et al findings [4]. We found in neuroblastoma cells resistant to CDDP but 

not in sensitive ones metallothionein overexpression which was induced by cisplatin or 

carboplatin. We described that UKF-NB-4 CDDP cells lost MYCN gene copies in comparison 

to UKF-NB-4 [4]. 

MTT tests that compared effect of cytostatics (CDDP, Ellipticine) in normoxia and 

hypoxia resulted in a decrease in toxicity to NBL cells both sensitive and resistant to 

respective drug. It correlated with lower levels of DNA adducts in experiments with 

ellipticine [5]. 

It has been demonstrated that CD133+ cells (cancer stem cells) are more resistant to 

hypoxia, irradiations and some chemotherapeutics compared to CD133- cells [6]. 

7. CONCLUSIONS 

Taken together, we showed that the treatment of NBL cells with cytostatics resulted 

in development of drug resistance. We have seen that in addition to epigenetic 

mechanisms, the acquired karyotypic changes in NBL may lead to up and down 

modulation of genes related to drug resistance. The established cell sublines provide a 

- 68 -


useful model that can be used to examine and characterize signaling mechanisms that 

account for induced resistance to cytostatics. Moreover, new chemotherapeutic strategies 

for overcoming drug resistance in malignant cells may be tested using chemoresistant 

sublines. 

8. REFERENCES 

Supported by GACR P301/10/0356. 


1. GOLDIE JH, COLDMAN AJ. Drug resistance in cancer. Mechanisms and models. Cambridge: Cambridge 

University Press, 1998 

2. POLAND J, SCHADENDORF D, LAGE H et al. Study of therapy resistance in cancer cells with functional 

proteome analysis. Clin Chem Lab Med 2002; 40: 221–234. 

3. BEDRNICEK J, VICHA A, JAROSOVA M et al. Characterization of drug-resistant neuroblastoma cell 

lines by comparative genomic hybridization. Neoplasma. 2005;52:415-9 

4.YASUNO T, MATSUMURAT, SHIKATAT et al. Establishment and characterization of a cisplatin-resistant 

human neuroblastoma cell line. Anticancer Res 1999;19: 4049–4057 

4. PROCHAZKA P, HRABETA J, VÍCHA A, ECKSCHLAGER T. Expulsion of amplified MYCN from 

homogenously staining chromosomal regions in neuroblastoma cell lines after cultivation with 

cisplatin, doxorubicin, hydroxyurea, and vincristine. Cancer Genet Cytogenet. 2010;196:96-104 

5. POLJAKOVÁ J, ECKSCHLAGER T, HRABETA J et al.The mechanism of cytotoxicity and DNA adduct 

formation by the anticancer drug ellipticine in human neuroblastoma cells. Biochem Pharmacol. 

2009;77:1466-79 

6. VANGIPURAM SD, WANG ZJ, LYMAN WD. Resistance of stem-like cells from neuroblastoma cell lines 

to commonly used chemotherapeutic agents. Pediatr Blood Cancer. 2010;54:361-8 

- 69 -


PROCESSING OF ELECTROCHEMICAL 

SIGNALS FOR DETERMINATION OF 

METALLOTHIONEIN CONCENTRATION 

Vladimíra KUBICOVÁ 1 , Petr MAJZLÍK 2 , Ivo PROVAZNÍK 1 , Martin VALLA 1 , René 

KIZEK 2 

1 Department of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno 

University of Technology, Kolejni 4,CZ 612 00 Brno, Czech Republic 

2 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, 

Zemedelska 1, CZ 613 00 Brno, Czech Republic 

Abstract 

Metallothioneins are the low molecular weight proteins localized to the membrane of the 

Golgi apparatus. The function of metallothioneins is not clear, but experimental data 

suggests the connection of metallothioneins with many diseases. Automated 

electrochemical detection allows analyzing of metallothioneins in the very small volume 

with the excellent sensitivity, reliability and reproducibility. The main aim of this work is 

to process electrochemical catalytic signals and to calculate the height of a peak in the 

signal, which corresponds with the concentration of metallothioneins in the substance. 


Metallothioneins (MT) belong to the group of the intracellular proteins and they 

were discovered in 1957, when Margoshes and Valee isolated them from the horse kidney 

[1]. The main role of MTs is probably the homeostatic control and the detoxification of 

metal ions in an organism. MTs are divided into classes (isoforms) depending on their 

primary structure and organism, in which they were isolated [2]. The MT-I isoform 

includes the mammalian metallothioneins built from 61 aminoacids. The synthesis of 

human MT may be inducted by the increasing metals concentration. Relation between 

the concentration of MT and carcinogenesis, spontaneous mutagenesis and the 

participation in the mechanics of the action of anti-tumour drugs and pharmaceutical 

products was proved. Overexpression of MTs is under investigation as a new diagnostic 

and prognostic marker in many types of malignant tumors [3]. 

There are several ways how to determinate the amount of MTs in a specimen 

described in literature: Cd-hem method, enzyme-linked immunosorbent assay, method 

based on the detection of mRNA, capillary zone electrophoresis, electrochemical 

determination and others [4]. The electrochemical determination is based on the catalytic 

processes which proceed at the negative potentials on mercury electrodes. These processes 

are accompanied by evolution of hydrogen from the supporting electrolyte components. It 

was found that –SH groups present in MTs are responsible for catalytic processes [5]. For 

the determination of the concentration of the substances which involve –SH groups is 

used Brdicka procedure. This method is very sensitive because of catalytic process and 

thus it allows determination of nanomolar concentration. The Brdicka solution contains 

- 70 -


Co(NH3)6Cl3 complex and ammonia buffer (NH4Cl + NH4OH) [6]. The determination of 

MT realizes at potentials from -1.9 V to 0 V. The proteins are represented polarographic 

peaks in the resulting voltammogram. Height of the first peak represents concentration of 

MT. The concentration of Co(NH3)6 has to be sufficient towards the concentration of MTs. 

If this condition is fulfilled, then the height of the peak will be linearly dependent on the 

concentration of MTs. The shape of the catalytic signal curve depends on the number of 

cysteines in the MT molecule, the size of this molecule and its concentration [5]. 

2. EXPERIMENT 

The signals for the analysis were obtained by Brdicka procedure using the 

differential pulse voltammetry (DPV) and adsorptive transfer stripping technique (AdTS). 

The measurement of each specimen was making four or five times and the results of each 

analysis were exported to a pure text file (extension *.txt). The algorithm described in this 

paper was created and verified in the Matlab computing environment (MathWorks, Inc., 

USA). The aim of the algorithm is to calculate the height of the peak which represents 

concentration of MT. 


Measured signals represent the dependence of the current on the voltage in the form 

of voltammograms. To calculate the height of the peak, positions of voltammogram 

minima must be identified. Height of the peak is defined as maximum vertical distance 

between the signal curve and the line which is created by connection of two detected 

surrounding minima (see Figure 1). 

Measured signals were interpolated using cubic splines for improved detection of the 

minima. It is based on the observation that if each pair of knots is connected by a cubic 

curve, the second derivative within each interval is a straight line. Samples of the signal in 

equal distances were obtained by interpolation and it allowed further processing. The 

interpolated signals were not smooth enough. It caused a number of false detections 

resulted in errors of peak height determination. To improve quality of detection, the 

interpolated signals were filtered by a digital FIR filter (low-pass filter). The output of this 

filter is a weighted sum of the current and a finite number of previous values of the input. 

The filter was experimentally designed with the most suitable filter order of 500. Cutoff 

frequency of the filter was 25 V -1 (the sampling frequency was calculated by interpolation 

step and it was equal to 1000 V -1 ). 

The minima were detected from the interpolated and filtered signal close to the 

voltage -1.65 V and -1.4 V. Every sample was measured five or more times. The height of 

the peak for every sample was calculated by two methods: 

1. An average signal was calculated and for this signal the height of the peak was 

found out, 

2. The height of the peak was found out for every measured signal and then the 

average height was calculated. 

For described algorithm, the user interface was created and it allowed changing the 

computation parameters e.g. order filter and cutoff frequency (see Figure 2). The results of 

- 71 -




the analyssis 

– calculaated 

heights s (voltage) and their locations 

(cu urrent) – wwere 

export ted to 

an Excel ffile. 

The pprogram 

wa as tested oon 

94 signa als and the e detectionn 

efficiency y was 

98.9 % in the case of the me ethod basedd 

on evalu uation of averaged a siignal. 

Dete ection 

efficiency was slightlly 

lower for r the secondd 

method ( less than 1 %). 

Fig. 1: 

The expla anation of tthe 

calculat tion of the peak p heightt 

Fig. 2: Thhe 

graphicaal 

user inte erface, the example of o the win ndow for ssetting 

filtr ration 

parameterrs 

- 72 - 

Brno


4. CONCLUSION 

The designed and realized program can detect and calculate the height of the peak in 

voltammogram. Detection efficiency was 98.9 %. The height was calculated by two 

methods and these results were similar (difference around 1 %). The graphical user 

interface allowed changing the calculation parameters for optimization of the method 

efficiency. Results of analysis can be exported into MS Excel file (extension *.xls) for 

further processing. The automated detection of the height of the peak and the calculation 

of the concentration of MT can be applied in diagnostics of diseases, in which MT may 

play role. 


This work was supported by GAČR 102/09/H083, GAČR P102/11/1068. 

6. REFERENCES 

[1] Margoshes M., Vallee B.: Chem. Soc. (1957), 4813, 79 

[2] Kojima Y.: Methods Enyzmol (1991), 205, 8-10 

[3] Krizkova S., Blahova P.: Comparison of metallothioneins detection by using Brdicka reaction and 

enzyme-linked immunosorbent assay employing chicken yolk antibodies, Electroanalysis, 21 (2009), 

2575-2583 

[4] Petrlová J., Svoboda M.: Přehled Analytických metod pro stanovení metalothioneinu v tkáních. XXX. 

Brněnské onkologické dny a XX. Konference pro sestry a laboranty, 2006, 187. 

[5] Vacek, J., Trnkova, L., Jelen, F. and Kizek, R.: Electrochemical determination of cysteine-containing 

biomolecules by means of catalytic reactions on a mercury electrode. In Kokkinidis, G. (ed.), ISE 

Annual meeting. International Society of Electrochemistry (2004), Vol. 55 

[6] Brdicka, R.: Coll.Czech. Chem. Commun., 5 (1933), 148-164 

- 73 -


STRUCTURE AND MAGNETISM OF CLEAN 

AND IMPURITY-DECORATED GRAIN 

BOUNDARIES IN NICKEL FROM FIRST 

PRINCIPLES 

Monika VŠIANSKÁ 1,2,3 , Mojmír ŠOB 1,2,3 

1 Department of Chemistry, Faculty of Science, Brno, Czech Republic 

2 Central European Institute of Technology, CEITEC MU, Masaryk University, Brno, Czech Republic 

3 Institute of Physics of Materials, Academy of Sciences of the Czech Republic, Brno, Czech Republic 

Abstract 

We present a detailed theoretical study of segregation and strengthening/embrittling 

energy of sp-elements from the 3rd, 4th and 5th period (Al, Si, P, S, Ga, Ge, As, Se, In, Sn, 

Sb and Te) at the Σ5(210) grain boundary (GB) in fcc ferromagnetic nickel. Whereas there 

is a slight enhancement of magnetization at the clean GB and FS with respect to bulk 

nickel (3–7% and 24%, respectively), the studied impurities entirely kill or strongly 

reduce ferromagnetism at the GB and in its immediate neighbourhood so that 

magnetically dead layers are formed. We determine the preferred segregation sites at the 

Σ5(210) GB for the sp-impurities studied, their segregation enthalpies and 

strengthening/embrittling energies with their decomposition into the chemical and 

mechanical components. We find interstitially segregated Si as a GB cohesion enhancer, 

substitutionally segregated Al and interstitially segregated P with none or minimum 

strengthening effect and interstitially segregated S, Ge, As, Se and substitutionally 

segregated Ga, In, Sn, Sb and Te as GB embrittlers in nickel. As there is very little 

experimental information on GB segregation in nickel most of the present results are 

theoretical predictions which may motivate future experimental work. 


Grain boundaries (GB) represent an important class of two-dimensional extended defects 

and macroscopic strength of polycrystalline materials depends strongly on GB cohesion. It 

was found that the impurities in ppm concentration can drastically change material 

properties. Intergranular embrittlement which is usually associated with segregation of 

impurities on the GB can result in a dramatic reduction of the ductility and strength. The 

purpose of the present research is to advance our fundamental understanding of structure, 

magnetism and embrittlement of impurity-segregated grain boundaries (GB) in 

ferromagnetic nickel. 

2. METHODS 

Spin-polarized electronic structure calculations were performed within the density 

functional theory using the Vienna ab-initio Simulation Package (VASP). The studied 

Σ5(210) GB was modelled by rotating two fcc grains around the [001] axis by 36.9°. It was 

- 74 -


represented by a supercell containing 60 nonequivalent atoms with two equivalent, 

reversely oriented GBs. Substitutional impurity atoms were considered to occupy the 

whole GB plane. Interstitial impurity atoms were placed in larger spaces at the GBs 

capable to accommodate them {1}. 


The effect of segregated impurities on GB magnetism in nickel is illustrated in Fig. 1 

[1]. It may be seen that except of case of sulphur, the magnetic moments of nickel atoms 

adjacent to the substitutionally segregated impurities are reduced to about 1/3–2/3 of the 

bulk value. In case of most substitutionally segregated impurities from the 3rd and 4th 

period, the nickel atoms in the 3rd layer have even a lower magnetic moment than those 

in the 2nd layer (in the immediate neighbourhood of the impurity layer). In more distant 

layers, the magnetic moments mostly increase and reach the bulk value from the 6th layer 

on. Much stronger reduction of nickel magnetic moments may be observed for 

interstitially segregated impurities (Fig. 1). Except of interstitially segregated Al, Ga and 

In, the magnetic moments of the nickel atoms lying in the GB layer are reduced nearly to 

zero (




Fig. 1. Local maagnetic 

moments 

for GGB 

and FS with segregated 

impuurities 

as well w as 

for isolatedd 

impuritiees 

(the righ ht column) in nickel. For comparison, 

the rresults 

for clean 

GB, FS and 

unperturrbed 

bulk are a also inccluded. 

Mag gnetic mom ments inducced 

on imp purity 

atoms are denoted byy 

the full symbols, s emmpty 

symbo ols correspo ond to nickkel 

atoms. For a 

better clarrity, 

the maagnetic 

mo oments of immpurities 

in i case of interstitial 

ssegregation 

n (the 

second collumn 

from the left) are 

put aside 

(as a laye er denoted by I), althoough 

they lie in 

the GB layyer. 

For FSS 

segregatio on (the seccond 

colum mn from th he right), wwe 

interpre et the 

results as iif 

we addedd 

an impuri ity layer onnto 

the FS layer; l this added a layerr 

is also den noted 

as I and thhe 

magneticc 

moments of atoms frrom 

the first 

nickel layer 

are commpared 

wit th the 

magnetic moments ffor 

clean FS F accordinngly. 

The straight 

line es betweenn 

the point ts are 

drawn as a guide forr 

eyes. In case of thee 

GB segregation, 

the ey also indiicate 

wher re the 

impurity pprefers 

to seegregate 

su ubstitutionaally 

or inter rstitially. In n the first ccolumn 

from m the 

left, whichh 

displays tthe 

results for substituutional 

seg gregation, st traight linees 

for impu urities 

which turrned 

out tto 

prefer substitution s nal positio ons are ful ll and for the impu urities 

preferring interstitiaal 

positions the conneecting 

lines s are dashe ed. In the second column 

from the lleft 

showing 

the result ts for intersstitial 

segre egation, the e lines are ddrawn 

the other 

way arounnd. 

In this way, dashe ed lines maark 

energet tically unfa avourable ccases 

which h will 

not take pllace 

in reallity. 


As iimpurity 

ssegregation 

in nickell 

was not studied so s much aas 

in iron, 

the 

experimenntal 

data arre 

scarce an nd most off 

our findin ngs have a character of a theoretical 

predictionn. 

Segregatiion 

phenom mena are iimportant 

for f technol logical appplications 

of o the 

- 76 - 

Brno


nickel-based materials. We hope that our results may motivate experimentalists to 

perform more measurements on impurity segregation in nickel. 


This research was supported by the Grant Agency of the Czech Republic (Projects 

No. 202/09/1786 and 106/09/H035), the Grant Agency of the Academy of Sciences of the 

Czech Republic (Project No. IAA100100920), by the Research Projects AV0Z20410507 

and MSM0021622410 and by the Project CEITEC–Central European Institute of 

Technology (CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund. 

The access to the MetaCentrum computing facilities provided under the Research Project 

MSM6383917201 is greatly appreciated. We thank Prof. P. Lejček, Dr. V. Paidar, Prof. I. 

Turek and Prof. V. Vitek for fruitful discussions. 

6. REFERENCES 

[1] Všianská M, Šob M.: Prog Mater Sci, 56 (2011), doi:10.1016/j.pmatsci.2011.01.008. 

- 77 -


USE OF LIGAND STEP GRADIENT 

FOCUSING IN COMBINATION WITH 

ISOTACHOPHORESIS (LSGF-ITP) FOR THE 

EFFECTIVE PRE-CONCENTRATION AND 

ANALYSIS OF HEAVY METALS 

Eliska GLOVINOVA 1 , Jan POSPICHAL 1 , Eliska SISPEROVA 1 


Abstract 

The new method was developed for the concentration and separation of metal ions called 

ligand step gradient focusing- LSGF. The principle of the method is a stationary pH 

discontinuity –neutralization reaction boundary, inside an ITP capillary which creates 

distinctly different complexing regions when a ligand is placed throughout the capillary. 

By selecting conditions such that the metal is uncomplexed at low pH and complexed at 

high pH in such a way that it forms a negative complex, the metals can be focused around 

the pH change. 

1. ANALYTICAL PART 

The analytical procedure consisted of pre-concentration, mobilization and detection 

of analytes. The low-concentrated metal ions in the cationic form were electrokinetically 

continuously dosed into the column where they were selectively trapped on the 

stationary ligand step gradient in the form of unmoving zones of chelate complexes with 

effectively zero charge. After a detectable amount of analyte was accumulated, the 

accumulated zones were mobilized to the analytical column, where they were analyzed 

by the conventional ITP method using conductivity detection. 

- 78 -

XI. Woorkshop 

of Physsical 


Fig. I. Graf deppicting 

frac ction of diff fferent ionic c species in n dependennce 

on elect trolyte pH. . 

Note, thhat 

around d pH 4,2 cadmium is present ted in thee 

form of unchargedd 

complexxes. 

Fig.II. 

Scheme of the flow ws on neuttralization 

boundary b with w ligandd 

step gradi ient duringg 

the focuusing. 

Note e, that metaal 

is presen nted in alka aline electroolyte 

/left side/ s in thee 

anionic form and in the aciddic 

electrol lyte /right side/ in thhe 

metal or r cathionicc 

complexx, 

both form ms are migrrating 

to the e centre of column. 

2. 

MATERIAL 

Usedd 

electrolyyte 

system m 

PE: 0,01M NHH4Ac, 

0,01M M NH4OH, 2.10-3M (N NH4)2C6H H6O7, pH=99,24 

/alcalin ne focusingg 

electtrolyte/ 

LE: 0,02M NH4Ac, 

0,0 01CH3COOOH, 

2.10-3 3M (NH4) 2C6H6O7, 

/anallytical 

leasiing 

el./ 

TE: 00,01M 

CH33COOH, 

pH H=3,44 /termminating 

el lectrolyte/ 

- 79 - 

Brnoo 

1 PEG, 

pH=4,955




DE: 0,0011M 

CH3COOOH, 

0,00 001M NH44Ac, 

pH=5, ,05+ 2.10-5 5M metalss 

/acidic dosing d 

electrolytee/ 

3. RESSULTS 

Deteermination 

of MT by electrochem 

e mical methods 

is based d on electrooactivity 

of f –SH 

moieties, wwhich 

tendd 

to be The trapping seelectivity 

was w set-up by b proper chhoice 

of pH H and 

complexinng 

agents. A mixture of o heavy mmetals 

- Pb and Cd we ere used as model analytes, 

citrate weere 

used ass 

complexing 

agents. Using a 2000 2 sec. dosing d timee, 

the prop posed 

method immproved 

thhe 

detection n limit by 1100-150 

tim mes in com mparison to analysis by y ITP 

with classiical 

injectioon. 

Fig. 

IV. Depenndence 

of zone z lengthh 

of the focused 

metal on the dossing 

time. 

Fig. V. AAnalysis 

of tthe 

model mixture m of Cdd, 

Pb (2x10-5 5 Mol/l) by regular 

ITP aanalysis-C, 

by b ITP 

with 500secc 

of focusingg-B 

and with h 1200sec oof 

focusing-A A. Note, tha at with 1200ssec 

of focus sing is 

analyte 50times 

pre-conncentrated. 


T 

The work was bby 

GA ČR 206/10/1219 

2 9. 

- 80 - 

Brno


LIGNITE – A LOW QUALITY SOLID FUEL 

WITH ATTRACTIVE SORPTION ABILITIES 

Petra BUŠINOVÁ 1 , Miloslav PEKAŘ 2 , Jaromír HUBÁLEK 1 

1 Brno University of Technology, Faculty of Electrical Engineering and Communication, Department of 

Microelectronics, Technická 3058/10, 616 00 Brno, Czech Republic 

2 Brno University of Technology, Faculty of Chemistry, Institute of Physical and Applied Chemistry, 

Purkyňova 118, 612 00 Brno, Czech Republic 

Abstract 

Lignite was considered just as a low quality solid fuel for a long time. More recently, 

various non-fuel applications, especially based on sorption processes, have been 

investigated. This contribution reports on the ability of the South Moravian lignite to 

adsorb basic textile dyes from aqueous solutions. 


Lignite as the youngest brown coal with a low degree of coalification has unique 

chemical composition and specific properties. Therefore, it can be used as a versatile 

material in several non-energy applications especially in sorption technologies. Lignite, 

even in its natural state, is reported to have significant sorption affinity for metal ions [1] 

as well as for some organic molecules, e.g. dyes [2], phenol [3] or non-ionic surface active 

agent [4]. Dyes, especially the synthetic ones, are extensively used in various fields of 

everyday life including e.g. textile, paper or printing industries. Most of the solutions used 

in dyeing processes are discharged as effluents and may cause certain hazards and 

environmental problems. The removal of dyes from wastewaters is extremely important 

because most of these dyes can be toxic, mutagenic and carcinogenic [5].Our research is 

focused on the sorption abilities of lignite mined in the region of South Moravia (Czech 

Republic). The affinity of this material for heavy metal ions (e.g. Pb 2+ , Zn 2+ , Cu 2+ , Cd 2+ ) and 

fluoride ion has previously been published [6,7]. This paper introduces results on the 

sorption of seven textile dyes on the powdered South Moravian lignite. 

2. EXPERIMENT 

Sorption studies were performed using lignite mined in the South Moravia, 

Mikulčice locality. Its detailed characterization is published elsewhere [6]. Fraction of 

lignite particles smaller than 0.2 mm, dried at 105 °C for 24 hours with final moisture 

content about 6 % was used. Adsorption of seven basic dyes (see Tab. 1) from aqueous 

solutions was studied. UV-VIS spectroscopy was used to determine residual dye 

concentration in solutions. The wavelengths of the absorption maxima of all seven dyes 

are given in Tab. 1. A change in the intensity of these maxima was used in order to 

characterize the removal of dyes from solutions. In each experiment 0.1 g of powdered 

lignite was mixed with 25 mL of aqueous dye solution with initial dye concentration of 

1000 mg·L –1 in 50 mL screw capped plastic centrifuge tubes with the conical bottom. 

Mixtures were agitated at constant agitation speed (27 rpm) on rotary shaker for given 

- 81 -


time period. After the required time period, samples were centrifuged at 4000 rpm for 15 

min and residual dye concentration in supernatant was determined. Experiments were 

conducted at laboratory temperature (25±2 °C). 


In this study, sorption of seven basic textile dyes (see Tab. 1) from aqueous solutions 

using natural powdered lignite as a sorbent was investigated. The residual dye 

concentration (cR in mg.L –1 ), the percentage removal of dye (R in percent) and the amount 

of dye adsorbed per gram of lignite (qt in mg.g –1 ) after 24 hours were determined and 

results are given in Tab. 1. It can be seen that all studied dyes can be removed from 

aqueous solution by adsorption on the South Moravian lignite, although with different 

efficiency. 

Tab. 1: The wavelengths of the absorption maxima of used dyes and residual dye 

concentration (cR), percentage removal of dye (R) and amount of dye adsorbed per gram of 

lignite (qt) after 24 hours 

Dye max (nm) cR (mg.L – 

1 

) 

- 82 - 

gt (mg.g – 

1 ) 

Methylene blue (MB) 664 511 122 49 

Astrazon blue 3GL (AB) 593 624 94 38 

Bezacryl blue FBS (BB) 598 511 122 49 

Maxilon red M-4GL (MR) 505 10 247 99 

Bezacryl red GRL 180 % (BR) 529 356 161 64 

Maxilon yellow M-3RL (MY) 422 10 247 99 

Bezacryl golden yellow GL 200 % 

199 80 

437 

(BY) 

204 

In addition, the kinetics of removal of MR and MY from solutions was studied for 

144 hours. Several kinetic models were investigated in order to choose the best model for 

the dye/lignite adsorption system. Variation of dye concentration in solutions with time 

during the 144 hours is shown in Fig. 1. This figure indicates that in the case of both dyes 

the removal of dyes is rapid in the initial stages of contact time and gradually decreases 

with time. The pseudo-second-order equation kinetic model was successfully used in 

determining the sorption rate and describing the reaction mechanism of MR and MY onto 

powdered lignite. Linear plots t/qt against time t for tested dyes can be seen in Fig. 2 and 3. 

Linear regressions of these functions provide the rate constant of the pseudo-second-order 

sorption (k2 in g.mg –1 .hour –1 ), the amount of dye adsorbed at equilibrium (qe in mg.g –1 ) and 

the initial sorption rate (h in mg.g –1 .hour0.5 ) for adsorption of MR and MY on powdered 

lignite. Results together with correlation coefficient R2 can be seen in Tab. 2. Good 

agreement between experimental data and pseudo-second-order model was observed as 

illustrated by values of R2 close to unity. 

R 

(%)


Fig.1 Variation of dye concentration in solutions MR and MY with time during adsorption 

on powdered lignite 

t/q t (h.g.mg -1 ) 

c R (mg.dm -3 ) 

0,6 

0,5 

0,4 

0,3 

0,2 

0,1 

0 

100 

80 

60 

40 

20 

0 

0 12 24 36 48 60 72 84 96 108 120 132 144 

Fig.2 Pseudo-second-order kinetics for 

sorption of MR on lignite 

- 83 - 

Fig.3 Pseudo-second-order kinetics for 

sorption of MY on lignite 

Tab. 2: Comparison of the pseudo-second-order sorption rate constants (k2), amounts of 

dye sorbed at equilibrium (qe), initial sorption rates (h) and correlation coefficients 

(R 2 ) of studied dyes MR and MY 

Dye R 2 k2 (g.mg -1 .hour -1 ) qe (mg.g -1 ) h (mg.g -1 .hour 0.5 ) 

MR 1 

6.83×10 -2 248 4.21×10 3 

MY 1 

t (h) 

0 24 48 72 96 120 144 

t (h) 

5.99×10 -2 248 3.69×10 3 

4. CONCLUSION 

Sorption abilities of the South Moravian lignite for seven basic dyes were explored. 

Furthermore, the adsorption kinetics of two selected dyes was investigated. It was 

t/q t (h.g.mg -1 ) 

0,6 

0,5 

0,4 

0,3 

0,2 

0,1 

0 

MR 

MY 

0 24 48 72 96 120 144 

t (h)


observed, that lignite is able to adsorb various basic dyes and the adsorption process 

followed a pseudo-second-order kinetic model. 


The financial support from the grant KAN 208130801 and the frame of Research 

Plan MSM 0021630503 is highly acknowledged. 

6. REFERENCES 

[1] Mizera, J.,et al.: Water Res., 41 (2007), 620. 

[2] Allen, S. J., Mckay, G., Khader, K.Y.H.: J. Chem. Tech. Biotechnol., 45 (1998), 291. 

[3] Polat, H., Molva, M., Polat, M.: Int. J. Miner. Process., 79 (2006), 236. 

[4] Aktaş, Z.: Turk J Chem., 25 (2001) 

[5] Pavan, F. A., et al.: Bioresource Technol., 99 (2008), 3162. 

[6] Havelcová, M., et al.: J. Hazard. Mater., 161 (2009), 559. 

[7] Pekař, M.: Petroleum and Coal, 48 (2007), 3, 1. 

- 84 -


APPLICATIONS OF IRON OXIDE 

NANOPARTICLES 

Petra BUŠINOVÁ 1 , Jana CHOMOUCKÁ 1 , Jaromír HUBÁLEK 1 


Microelectronics, Technicka 3058/10, 616 00 Brno, Czech Republic 

Abstract 

In this work, a short summary of various applications of magnetic iron oxide nanoparticles 

is presented. Today, magnetic nanoparticles can be used in important biomedical 

applications as well as as magnetic recording devices, magnetic data storage devices, gas 

sensors, catalysts or wastewater treatment adsorbents. 


In recent years, magnetic nanoparticles are of great interest for researchers from a 

broad range of disciplines. There are unique magnetic properties such as 

superparamagnetic, high coercivity, low Curie temperature, high magnetic susceptibility, 

etc., which allows magnetic nanoparticles for various nanotechnology applications. 

Magnetic ferrofluids and data storage led to the integration of magnetic nanoparticles in a 

numerous of commercial applications. Today, magnetic nanoparticles are also used in 

important biomedical applications. Therefore, it is crucial to choose the suitable materials 

for the preparation of nanoparticles with adjustable physical and chemical properties. For 

this purpose, magnetic iron oxide nanoparticles became the strong candidates due to its 

biocompatibility [1, 2]. 

Magnetite (Fe3O4) and its oxidised form maghemite (-Fe2O3) are the most common 

forms of iron oxide, which can be used in various fields of nanotechnology, especially in 

biomedical applications. Both of them are ferrimagnetic and in addition Fe3O4 show 

superparamagnetic properties when particle size is less than 15 nm [1, 3]. Furthermore, 

their surface can be easily modified through the creation of few atomic layers of organic 

polymer or inorganic metallic (e.g. gold) or oxide surfaces (e.g. silica or alumina), suitable 

for further functionalization by the attachment of various bioactive molecules [4]. 

This contribution briefly reports on possibilities of utilization of iron oxide magnetic 

nanoparticles in wide range of applications in many different fields. 

2. BIOMEDICAL APPLICATIONS 

Biomedical applications of iron-based magnetic nanoparticles are classified into two 

categories according to their application inside (in vivo) or outside (in vitro) the body. In 

vivo applications could be further divided in therapeutic (hyperthermia and drug 

targeting) and diagnostic applications (nuclear magnetic resonance (NMR) imaging). For 

in vitro applications, the main use is in diagnostic and separation/labeling of biomolecules, 

such as protein, cell, DNA/RNA and microorganism. [5, 6] 

- 85 -


Magnetic separation. The basic principle of magnetic separation is very simple. 

Magnetic particles with an immobilized affinity or hydrophobic ligand or ion-exchange 

groups are mixed with a sample containing the target compound. Following an incubation 

period, when the target compound binds to the magnetic particles, the magnetic complex 

is easily and rapidly separated from the sample using an appropriate magnetic separator. 

After washing out the contaminants, the target can be eluted and used for further work. 

This method has wide application in biotechnology and biomedicine, such as 

immunomagnetic cell separation and purification, immunoassays, isolation, purification 

and recognition of proteins. In molecular biology, it can be used for DNA/RNA 

purification. In addition, the isolation and detection of microorganisms is easily possible 

using magnetic separation [6]. 

Magnetic target drug delivery. This system of drug delivery is considered the most 

popular and efficient. In this technique, the drug carrying magnetic materials like Fe3O4 

will be led to the cancer areas by outside magnetic field after taken orally or injected 

through vein. After absorption by human body, the remanent magnetic particles can be 

safely excreted through skin, bile, kidney, etc. [7, 8] 

Hyperthermia. It is one of the promising approaches in cancer therapy. This idea is 

based on the principle that a magnetic particle can generate heat by hysteresis loss under 

an alternating magnetic field (AMF). Magnetic particles embedded around a tumor site 

and placed within an oscillating magnetic field will heat up to a temperature dependent 

on the magnetic properties of the material, the strength of the magnetic field, the 

frequency of oscillation and the cooling capacity of the blood flow in the tumor site. 

Cancer cells are destroyed at temperatures higher than 43 °C, whereas the normal cells 

can survive at higher temperatures [4, 6]. 

Magnetofection. The fundamental principle of magnetofection is simple and 

comprises the steps of formulating a magnetic vector composed of a therapeutic gene and 

surface modified magnetic nanoparticles, adding it to the medium covering cultured cells 

or injecting it systemically via the blood stream or applying it locally to a target tissue, and 

in addition applying a magnetic field in order to direct the vector towards the target cells 

or retain it in the target tissue, respectively [6]. 

Magnetic resonance imaging (MRI). Clinical diagnostics with MRI has become a 

popular noninvasive method for diagnosing mainly soft tissue or recent cartilage 

pathologies, because of the different relaxation times of hydrogen atoms. 

Superparamagnetic nanoparticles were developed as contrast agents for MRI and 

increased the diagnostic sensitivity and specificity due to modifications of the relaxation 

time of the protons [6]. 

3. NON-BIOMEDICAL APPLICATIONS 

There are also other branches where iron oxide nanoparticles are or could be used. 

Some of them are mentioned below. Catalyst. Materials based on iron oxides have been 

found to be good, efficient and cheap catalysts, especially in environmental catalysis (e.g. 

for catalytic oxidation removal phenolic and aniline compounds from wastewater [9]). 

- 86 -


Iron oxide nanoparticle-based catalysts can be more effective than those conventional 

from micron-sized particles. These effects could be derived from the high activity of 

nanoparticles that have high BET surface areas and more coordination of unsaturated sites 

on the surfaces. Chemical and electronic properties, such as phase changes, OH content, 

band gap changes etc., could also have contributed to their high reactivity. Iron oxide 

(usually mixed with other metal oxides) in particular, has been shown to be a very active 

(although unstable) catalyst for the oxygen evolution process as well as other related 

processes, such as water splitting, chlorine evolution, the oxidation of organic molecules, 

the oxygen reduction process and for the hydrogen peroxide decomposition. Even more 

important are iron oxide-based catalysts in non-electrochemical processes [5]. 

Gas sensor. A number of studies have focused on maghemite (-Fe2O3), which 

exhibits good sensing characteristics towards hydrocarbon gases, carbon monoxide and 

alcohol. The advantages of -Fe2O3 gas sensors are its high response and low cost. 

Furthermore, the sensitivity of these sensors can be enhanced by various doping schemes 

and a number of different dopants such as Ni, Pd, Sn, Ti, Zn etc.. While doping is an 

important factor for controlling the sensing characteristics, the sensor structure, and 

especially the thickness of its active layer, also has a great influence on the sensitivity. In 

fact, bulk and thick-film type sensors exhibit a relatively low sensitivity, which 

substantially improves when the same sensing material is used in a thin-film type sensor. 

The improvement is even greater when a nanosized material is used [10]. 

Impurity control agent. Iron oxides have relatively high surface area and surface 

charge, and they often regulate free metal and organic matter concentrations in soil or 

water through adsorption reactions. Many toxic cations (e.g. Co, Zn, Pb, Cd, Cs, U, Sr) and 

anions (e.g. AsO4 3− , CrO4 2− , PO4 3− , CO3 2− ) are removed using various phases of iron oxide. 

At the nanoscale these materials are potentially highly efficient for binding metal ions. 

Selective adsorption of different metal ions can be achieved by tailoring the composition 

of the iron oxides. Use of iron oxide nanoparticles is thus becoming very attractive in the 

area of adsorption or recovery of metal ions from industrial wastes or natural water 

streams [5]. 

Electro magnetic material. Magnetic nanoparticles, including ferrites, have been 

studied for many years for their application as magnetic storage media and ferro-fluids. 

Over the recent years, maghemite (-Fe2O3), a ferromagnetic material, is widely used as 

magnetic storage media in audio and video recording, magneto-optical devices and 

magnetic refrigeration. Furthermore, iron-based nano compounds as positive cathode 

materials for Li-ion secondary batteries are of interest due to the low-cost and nontoxicity 

[5]. 

Colouring and coating material. Finally, iron oxide nanoparticles can be found in 

transparent iron oxide pigments, which can be used in automotive paints, wood finishes, 

construction paints, industrial coatings, plastic, nylon, rubber and print ink. The excellent 

weather fastness, UV absorption properties, high transparency and color strength makes 

trans-oxide to enrich the colors, increase color shades when combined with organic 

pigments and dyes [5]. 

- 87 -


4. CONCLUSION 

In this paper we have briefly reviewed the possibilities of iron oxide-based magnetic 

nanoparticles utilization. From the information given above, it can be concluded that iron 

oxide nanoparticles can be used for numerous in vivo or in vitro biomedical applications, 

such as contrast agents for magnetic resonance imaging, magnetofection reagent, 

hyperthermia media for tumors treatment, etc.. Besides, iron oxides have been explored as 

low-cost and non-toxic magnetic materials for e.g. wastewater treatment, magnetic 

storage devises, catalysis or gas sensing. 




6. REFERENCES 

[1] Wu, W., He, Q., Jiang, Ch.: Nanoscale Res. Lett., 3 (2008), 397 

[2] Drbohlavova, J., et al.: Sensors, 9 (2009) , 4, 2352 

[3] Qiang, Y., et al.: J Nanopart. Res., 8 (2006), 489 

[4] Gupta, A. J., Gupta, M.: Biomaterials, 26 (2005), 3995 

[5] Mohapatra, M., Anand, S.: Int. J. Eng. Sci. Tech., 2 (2010), 8, 127 

[6] Ma, Z., Liu, H.: China Particuology, 5 (2007), 1 

[7] Zhao, Y., Qiu, Z., Huang, J.: Chin. J. Chem. Eng., 16 (2008), 3, 451 

[8] Chomoucka, J., et al.: Pharmacol. Res., 62 (2010), 2, 144 

[9] Zhang, S., et al.: J. Hazard. Mater., 167 (2009), 560 

[10] Jasinski, J., et al.: Sensor. Actuat. B-Chem., 109 (2005),1, 19 

- 88 -

XI. Woorkshop 

of Physsical 


EXXPRESSSING 

G OF BBACTE 

ERIAL L 

DIHYDRRODIP 

PICOLLINAT 

TE SYN NTHAASE 

IN 

GRRAIN 

OOF 

TR RANSGGENIC 

C BAR RLEY 

Nataalia 

CERNE 

GALLUSZKA 

BAB 

4 , Ja 

BULA6 EI 

, Ren 

1 , Ondřej Z 

ana VASKO 

né KIZEK1 ZÍTKA1 , Lu 

OVA2 udmila OHN NOUTKOV 

, Markk 

A. SMED DLEY4 VÁ 

, Wen 

2,3 , Katarrina 

MRIZO 

ndy A. HARRWOOD5 

OVÁ 

, 

4 , Petr 

Petr 

1Depaartment of Chhemistry 

and 

1, 1, CZ-613 00 BBrno, 

Czech R 

Czech Reppublic; 

Oloomouc, 

Slecht 

of MMolecular 

Bio 

Crop p Genetics, Joh 

Faculty of Ph 

3 d Biochemistry 

Republic; 

Depar 

titelu 11, 7837 

iology, Faculty 

hn Innes Cen 

harmacy, Uni 

2 ry, Faculty of Agronomy, A M 

Insstitute 

of Expe perimental Bot 

rtment of Celll 

Biology and d Genetics, Fa 

371 Olomouc-H -Holice, Czech h Republic; 

y of Science, PPalacky 

Univ 

ntre, Norwich h Research Par 

iversity of Veeterinary 

and 

4 Mendel Unive 

tany, v.v.i., A 

aculty of Scien 

Department D o 

versity in Olo omouc, Czech 

ark, United Ki ingdom, 

d Pharmaceuti 

6 ersity in Brno 

Academy of Sc 

ence, Palacky 

of Biochemist 

h Republic; 

Dep 

ical Sciences, 

5 o, Zemedelskaa 

Sciences of thee 

University inn 

try – Divisionn 

Department D off 

partment of Natural N Drugs, s, 

Palackeho 1- -3, CZ-612 422 

Brno, Cz zech Republicc 

Absstract 

Nutritionnal 

quality of human aand 

animal l foodstuffs is determinned 

by the content off 

essenntial 

aminoo 

acids. Bar rley is the fourth mo ost importa ant cereal oof 

the wor rld and thee 

seconnd 

most immportant 

ce ereal grownn 

in the Cz zech Repub blic. Cereal grains such h as barleyy 

contain 

insufficcient 

levels of some essential 

ami ino acid, esp pecially lyssine. 

1. INTRODDUCTION 

Provision of sufficie ent quality food and fodders f is still s presennt 

appeal. World W foodd 

crisiss 

brings to border of famine f milllions 

of peo ople. Food supplement s tation for biologically b y 

impoortant 

commpounds, 

su uch as vittamins, 

am mino-acids and essenntial 

fatty acids mayy 

contribute 

-to solve thi is problemm. 

Modern n methods of moleccular 

biolo ogy enablee 

introoducing 

of new prope erties into plants. Lys sine is biol logically veery 

importa ant amino-- 

acid, 

which iis 

essential for human n. Adults nneed 

about 1-1.5 g off 

lysine 

per dayy, 

children about 44 mg m of lysinne 

per day. Generally, , 

L-l lysine is ann 

essential structural s amino-acid a d, which is crucial forr 

all proteins. Lysine pla ays an imp portant rolle 

in calciu um uptakee 

abs sorption, paarticipates 

in biosynth hesis of horrmones, 

en nzymes andd 

ant tibodies annd 

its impo ortance is also a in mettabolism 

of 

muscularr 

tissue. 

Positive effect of lysine l is deemonstrated 

in processes 

of heaaling 

of wo ounds afterr 

surgical interventio ons or inju uries. Due to these properties, , 

supplemeentation 

of f fodders for 

livestockk 

by lysine e is a veryy 

importannt 

factor for r improving g of daily inncrease 

in weight. w 

2. EXXPERIMEN 

NT 

Bioological 

exp periment: Transgenic T bbarley 

plan nts of the T11 

generatioon 

were evaluated e by b PCR, RReal-Time 

PCR andd 

Westernn 

blot. In 2010, grad dually matturing 

and d graduallyy 

- 89 - 

Brnoo


regenerate transformed plants, the grain was harvested in July 2010. Plants were divided 

based on the RT-PCR analysis in four groups: high expression of the desired gene dapA 

and selective gene hpt (hygromycin phophotransferase), intermediate expression, low 

expression and different expression. The activity of DHPDS protein and lysine content. 

Fig1.MW 3000 Anton Paar 

The selected plants with high protein expression were determined. Grain was 

harvested from 336 plants. In winter there was a significant slowdown in growth of plants 

and extending the growing season. The plants differed in height- even the number of 

offsprings and grain and number of grain per spike . Transgenic plants were observed in 

relatively large numbers of sterile flower, which has not in corn. An average of all plants 

was 20 grains per spike, plants usually had five offsprings. 

Analysis: Amino acids content was analyzed by Aminoacid Analyzer AAA400 with 

post column derivatization by ninhydrin. Leave samples were prepared by HCl hydrolysis: 

0.25 g of homogenized solid sample was mineraralized with 0.5 ml 6M HCl in MW Aton 

Paar 3000, power-80,ramp-15,hold-90 (Fig.2). 


Two constructs pBract214::sTPdapA and pBract214::mdapA containing the dapA 

gene from Escherichia coli coding bacterial DHPS were used for transformation of barley. 

The vector pBract214::sTPdapA in addition includes the transit peptide Rubisco Hordeum 

vulgare ribulose-1,5-bisphosphate carboxylase small subunit, Genbank U43493. An 

Agrobacterium-mediated technique was used for transformation of immature embryos of 

barley cv. Golden Promise. Plants were analysed with respect to expression of cloned 

genes by qRT-PCR technique. On a basis of this analysis, four groups of plants were 

established (low, medium, high and unequal gene expression). Due to this fact, we decided 

to carry out an analysis of lysine as a product of gene transcription. Ionex chromatography 

was used for analysis. Signal of lysine was well-separated with limits of detection of 500 

nM and limit of quantification about 5 μM with deviation of 5%.(Fig.2) Real samples were 

hydrolyzed by HCl at elevated temperature; obtained extracts were subsequently injected 

into chromatographic system in triplicates (R.S.D. was 6.5%). Concentration of lysine in 

control group of plants was about 303.5 mM, the concentration of total content of 2 

amino-acids. From obtained results follows very good correlation between the rate of 

cloned gene expression and lysine concentration (R 2 =0.99). 

- 90 -

XI. Woorkshop 

of Physsical 


Fig.22: 

Standard of Lysine after a hydrollysis 

in MW W Anton Pa aar 

Fig. 33: 

Calibrattion 

curve of o lysine 

Pictures 44-7 

show th hat the aveerage 

value of the stud died amino o acids did not n changee 

signiificantly 

deepending 

on o the leveel 

of gene expression n. Howeverr 

in negati ive controll 

grouup 

(no vectoor 

transform med) the ammino 

acid content 

was s lower. 

- 91 - 

Brnoo




Fig.4: Thee 

amount 

exppression. 

concentration [mM] 


8000 

7000 

6000 

5000 

4000 

3000 

2000 

1000 

0 

600 

500 

400 

300 

200 

100 

0 

of studied d amino aacids 

deter rmined in 

mediuum 

expressio on 

PCR 3 PC CR 28 PCR 311PCR 

52 PCR 

70 PCR134 4 

Fig.5: Thee 

amount oof 

studied amino aciids 

determi ined in sam mples withh 

medium gene 

exppression. 

low expresssion 

PCR 7 PCR R 16 PCR 477 

PCR 55 PCR R 88 PCR 96 

- 92 - 

samples wwith 

high 

Lysin 

Threoninn 

Methionnin 

Lysin 

Threoninn 

Methionnin 

Brno 

gene


Fig.6: The amount of studied amino acids determined in samples with low gene 

expression. 


800 

700 

600 

500 

400 

300 

200 

100 

0 

Fig.7: The amount of studied amino acids determined in samples with unequal gene 

expression 


400 

350 

300 

250 

200 

150 

100 

50 

0 

unequal expression 

PCR 6 PCR 39 PCR 73 PCR 84 PCR 95 PCR 

123 

negative control 

1 

Golden promise 

Fig.8: The amount of studied amino acids determined in samples without gene expression. 

4. CONCLUSION 

Amino acid content was analyzed by Aminoacid analyzer AAA400 with post 

column derivatization by ninhydrin. 26 samples were measured with the gene expression 

of lysine. The result was converted to mM concentration of lysine, methionine and 

therionine. Lysine content had no effect on other amino-acids. The selected T1 progene 

was sown in October in a greenhouse and characterized by PCR at the level of expression 

and gain detection dapA. 


This work was supported by project 1M06030 of the Ministry of Education, Youth 

and Sports of the Czech Republic. 

- 93 - 

Lysin 

Threonin 

Methionin 

Lysin 

Threonin 

Methionin


6. REFERENCES 

[1] Krishnakumar V, Manohar S, Nagalakshmi R.: Spectrochimica Acta Part A-Molecular And 

Biomolecular Spectroscopy, 75 (2010), 5, 1394-1397 

[2] Veriter S, Mergen J, Goebbels RM.: Tissue Engineering Part A, 16 (2010), 5, 1503-1513 

[3] Vyroubalova S, Ohnoutkova L, Galuszka P, In Vitro Cellular Developmental Biology-Animal, 44 

(2008), S68-S68 

[4] Serhantova V, Ehrenbergerova J, Ohnoutkova L, Plant Soil and Environment, 50 (2004), 456-462 

[5] Halamkova E, Vagera J, Ohnoutkova L Biologia Plantarum, 48 (2004), 313-316 

[6] Ohnoutkova L,Vaškova J,Mrizova K,Smedley M, Harwood W, XXIVth Genetic Day, Book of 

Abstracts 55,1-3 September, 2010 

- 94 -

XI. Woorkshop 

of Physsical 


DEETERMMINAT 

TION PROS STATE E SPECCIFIC 

ANNTIGEEN 

AN ND TESSTOSTERO 

ONE INN 

TUUMOUUR 

CELL 

LINNES 

WITH W ENCO E ORE ZINC 

ANND 

SAARCOS 

SINE AAS 

WE ELL AS A IN PPROST 

TATE 

CAANCERR 

PAT TIENTSS 

Nataalia 

CERNE 

SOCHOR1 

EI 

, Petr 

1 , Michal M 

r BABULA3 MASAŘÍK 2 , Jaromír G 

3 , René KIZZEK 

1 

2 Dep 

3 Dep 

11 

Department t of Chemistry y and Biochemmistry 

Mende 

partment of PPathological 

Physiology, P Fa Faculty of Med 

CZ-662 2 43 Brno, Czeech 

Republic 

partment of NNatural 

Drugs, s, Faculty of P 

3 

el University, Zemedelska a 1, CZ-613 00 0 Brno, Czechh 

Republicc 

dicine, Masary ryk University ty, Komenskeh eho namesti 2, 2, 

3Department t of Natural Dr Drugs, Faculty y of Pharmacyy 

Pharmacy, Un niversity of Veterinary Ve andd 

Pharmaceut tical Sciences, , 

Palackeho ho 1-3, CZ-6122 

42 Brno, Cz zech Republicc 

Absstract 

Tummour 

markerrs 

are bioch hemical inddicators 

of malignant m tumour t prooliferation. 

They servee 

not oonly 

for diaagnostics 

of f malignantt 

tumour diseases, d but t also for mmonitoring 

of effect off 

theraapy 

and reecurrence 

of o disease. One of th he most important 

inddicators 

of f quality off 

oncoological 

maarkers 

is the eir sensitivvity; 

it mea ans the possibility 

to detect dise eases at thee 

earlyy 

stages, annd 

specificity 

to givenn 

type of tu umour. In the case oof 

prostate carcinoma, , 

prosttatic 

acid pphosphatase 

e (PAP) waas 

usually used u in the e past. PAPP 

was repla aced at thee 

end of eighties of last cent tury by serrum 

prostat te specific antigen a (PSSA), 

which belongs too 

a grooup 

of the bbest 

tumour r markers, ccurrently 

available. a 


Seruum 

prostate e specific antigen a (PSSA) 

was us sed for thee 

first timee 

at 1969 by Haura et al. PSSA 

is a gl lycoproteinn 

composedd 

of 237 amino-acids 

s, which ooriginates 

in i prostatee 

gland andd 

is necess sary to normal 

physsiological 

function fu off 

sperm. Att 

the physio ological sta ate, most off 

PSA is sec creted intoo 

sperm. Onnly 

small amount a of PSA is traansported 

into i blood, , 

due to thiss 

fact, PSA is detectab ble in bloodd 

serum. In the case off 

disorganizzation 

of inner 

archite ecture of prrostate 

gland, 

majorityy 

of PSA iss 

transporte ed into blo ood, whichh 

results in n increasedd 

PSA level in bllood 

serum m. It was deetermined 

that t increas sed PSA levvel 

is assoc ciated withh 

tumoour 

diseases 

of prostate 

gland. DDeterminati 

ion of PSA A level is att 

the presen nt the bestt 

prosttate 

tumouur 

marker, , which iss 

presently y known and used.TTestosteron 

ne is malee 

hormmone 

- haas 

no direc ct relationsship 

to tu umour form mation, maay 

contribu ute to thee 

deveelopment 

off 

clinical manifestatio 

m ons. It was found f that the incidennce 

of prostate 

cancerr 

is muuch 

lower iin 

eunuchs - they lackk 

testosterone. 

- 95 - 

GUMULEC 2 

2 , Ondřej ZZÍTKA1 

, Jiří í 

Brnoo


2. EXPERIMENT 

Samples were analysed by the use of apparatus Immune Enzymatic Automated 

Analyzer AIA 600 ΙΙ, which serves for measurement of immunochemical parameters in 

biological liquids; it uses set of reagents AIA–PACK PSA and fPSAThe AIA -600II 

Automated Enzyme immunoassay. The AIA –PACK reagent series test cups are disposable 

plastic cups containing the necessary reagents and analytes for each test, one specimen 

immunoreaction.The contents of the test cups are freeze-dried and sealed for long term 

storage. All reactions and fluorescent measurements are performed in the test cups. An 

antigen-antibody reaction begins by combining a patient sample, control, or calibrator 

with solvent in an immunoreactions test cup from the AIA-PACK reagent series. In the 

EIMA assay, during the incubation period, the antibodies are attached to two distinct 

epitopes on the antigen forming a sandwich. Samples are incubated at 37°C with antibody 

bound to the surface of magnetic particles. Separation of the bound antibody is achieved 

by washingthe beads with a washsolution that removes any conjugates. 

After washing, a substrate, 4-methyumbelliferyl phosphate, is added to the test cup. 

The remaining enzyme activity on the solid phase (magnetic beads) is then measured 

using fluorescent rate method. A set of reagents AIA-PACK TES was used 

formeasurement of testosterone. Cell lines were derived from normal prostate tissue 

(PNT1A) and tumour tissue - tumour cell lines RVL1. 


In our experiment, we used fully automated immunochemical detection of serum 

prostate specific antigen. Reaction itself is initiated by pipetting of sample (10 μl) 

consisting of blood serum or cell lysate with solvents in testing pot AIA-PACK. Samples 

were incubated at 37°C, antibodies were bonded on surface of paramagnetic particles. 

Separation of bonded and unbounded antibodies is obtained by washing by rinsing 

solution - unbounded antibodies were washed out. After washing step, substrate - 4methylumbelliferyl 

phosphate was added into testing pot and enzymatic activity on 

paramagnetic particles was measured. Calibration curve for PSA was designed (R 2 = 

0.9993, standard deviation 0.2 %) and for fPSA (R 2 = 0.9990, standard deviation 0.5 %) 

(Fig. 1). 

- 96 -

XI. Woorkshop 

of Physsical 


Fig.11: 

Calibratioon 

curves for f determiination 

of total t serum m prostate sspecific 

ant tigen (PSA) ) 

and free PSA (fPSA A). 

We analyysed 

18 sa amples of patients ag 

adennocarcinomma 

and 3 controls ( (healthy y 

deterrmined 

usiing 

two procedures. p Correlatio 

methhod 

fPSA) wwas 

very close c with R 

valuee 

of determmined 

PSA (method P 

PSA values deetermined 

by b PA me 

compparison 

witth 

PSA2 method. 

At l 

by PPSA2 

methood 

was obse erved (enha 

PA mmethod). 

Leevels 

of PSA A of patien 

was 1.50-3.19 nng/ml. 

Testo osteron leve 

2 ged 62-80 years, sufffering 

from m prostatee 

young men n). Levels of total PSA weree 

on of obta ained data (method PSA2 andd 

=0.990. Newly N intro oduced meethod 

PSA2 2 decreasedd 

PA) by 1.5 ng/ml at average. a Addditionally, 

in higherr 

ethod, PSA A level was 

for abouut 

25-30 % lower inn 

low PSA le evels, highe er sensitivitty 

of PSA determined d d 

ancement of o PSA for about a 5-7 % in compa arison withh 

nts were in the range 4.78-12.50 ng/ml, levels 

of fPSAA 

el of patien nts was 278-666 

ng/dl. 

- 97 - 

Brnoo




Fig.2: Conncentration 

PSA2 and fPSA f of pattients 


testosteron ne of patiennts 

Propposed 

methodology 

for 

determinnation 

of PS SA and fPSA 

was subssequently 

tested t 

on cell lyssates. 

In noon-tumour 

cell lines, PPSA 

levels were abou ut 0.02 ng/mml 

and for fPSA 

0.02 ng/mll. 

In the casse 

of tumou ur cell liness 

- for tumo our cell line e PC-3, PSAA 

level was s 0.05 

ng/ml andd 

level of fPPSA 

was 0.0 02 ng/ml; tuumour 

cell line RVL 22 2 with enccore 

sarcosi ine 0- 

500μM deemonstratedd 

significan nt enhanceement 

of PSA P levels to 9.58 nng/ml 

and fPSA 

to11.40 ngg/ml 

(Fig.44).Testoster 

rone level in cell lines 

RVL 1 22 was 36617- 

4205 ng/dl 

(Fig.5). 


PSA and fP PSA of cell lines 22 RV VL1 and PN NT 1A 

- 98 - 

Brno

XI. Woorkshop 

of Physsical 


Fig.55: 

Concentrration 

testosterone 

of ccell 

lines 22 2 RVL1 and d PNT 1A 

Along witth 

testoster rone levels in the pros state cancer r cells the iinfluence 

of o sarcosinee 

on ccells 

lines RVL1 wa as detectedd. 

To cell ls lines RVL1, 

10.500 

μM and d 500 μMM 

conccentrations 

of sarcosin ne were addded. 

Figure es 6 shows that encorre 

of sarco osine didn’tt 

havee 

any effectt 

on levels of PSA andd 

fPSA. Ho owever Figu ure 7 showws 

significan nt effect off 

sarcoosine 

on tesstosterone 

levels. l 

Fig.66: 

Concentrration 

PSA and fPSA oof 

cell lines 22 RVL1 with w encoree 

sarcosine 0-500μM 

- 99 - 

Brnoo





testosteron ne of cell linnes 

22 RVL L 1with enc core sarcosiine 

0-500 μM M 


Deteermination 

of PSA in blood b serumm 

of patien nts is comm mon diagnosstic 

method d. We 

demonstraated 

in ourr 

work tha at fully auttomated 

tec chnique of f PSA immmunodetecti 

ion is 

possible allso 

in cell lyysates.Determination 

oof 

testoster rone in cell lines RVL11 

and PNT 1A is 

possible allso 

in cell llysates, 

con ntent testossterone 

is very v high.T The level off 

testosterone 

in 

men rangees 

from 3500 

to 1000 ng/dl n after tthe 

fortieth h year of lif fe is this noormal 

level 

will 

decrease bby 

about 1% 

per year. 

A historry 

of prosta ate cancer has been a long stan nding 

contraindiication 

to the use of testosterrone 

thera apy due to o the beliief 

that higher h 

serum testtosterone 

caauses 

more rapid prosttate 

cancer growth. 


T 

The work has bbeen 

suppo orted by graants: 

GACR R 301/09/P4 436, IGA MMZ 

10200-3 3 and 

NANOSEMMED 

GA AAV 

KAN208 8130801, IGGA 

MZ 10200-3. 


[1] Caire AA, Sun L, RRobertson 

CN N.:, J.: UROLOOGY, 

75(2010 0), 5,1122-112 27 

[2] Williaams 

SA, Xu YY, 

De Marzo AM.:, A J.: PROOSTATE, 

70(2010 

), 7, 788-796 

[3] Naritaa 

D, Anghel AA, 

Cimpean AM.:,J.: A NEOPPLASMA, 

57 7(2010 ), 3, 19 98-206 

[4] Lee TTK, 

Miller JS, Epstein JI.:,J. : PATHOLOGGY, 

42(2010) , 4, 319-324 

[5] Morggentaler 

A, LippshultzLI,Ben 

nnett R, et al. .JOURNAL OF O UROLOGY Y,185(2011),44,1256-1260 

- 100 - 

Brno


FABRICATION AND CHARACTERIZATION 

OF TIO2 QUANTUM DOTS ARRAY 

Jana DRBOHLAVOVÁ 1 , Dmitry SOLOVEI 1 , Jaromír HUBÁLEK 1 


Microelectronics, Údolní 53, 602 00 Brno, Czech Republic 

Abstract 

In this work, we report on the electrochemical preparation of TiO2 quantum dots (QDs) 

on Ti layer deposited on Si wafer with the aim to investigate the different physicochemical 

properties. This QDs array may be widely used for biosensing purposes in 

medicine, allowing the detection of DNA and proteins in vitro. The prepared TiO2 QDs 

were less than 10 nm in size (characterized with SEM), predominantly composed of 

anatase phase (observed with Raman spectroscopy), and exhibited broad emission band in 

VIS range (spectra taken by fluorescence spectroscopy). 


Nonlitographic nanopatterning through thin nanoporous anodic alumina 

membranes used as template enables the fabrication of large-scale ordered arrays of 

nanostructures on various surfaces. Compared to traditionally employed techniques for 

nanostructures synthesis, such as photolithography or e-beam lithography, which are 

time-consuming and expensive processes, this technique allows the above mentioned aims 

in cheap, fast and well reproducible way. Nowadays, there are lots of papers dealing with 

ordered nanotubes array fabricated using template methods, but only very few works 

concerning the application of anodization technique for nanodots preparation. Sometimes, 

the scientists combine the usage of nanoporous template and other ways of nanodots 

deposition, like ion beam evaporation, electron gun evaporation, nanoscale selective 

epitaxial growth, selective anodization using AFM tip, electrodeposition etc. 

In order to achieve nanocrystals directly grown by anodization with sizes in the 

range of 1 to 10 nm which is essential for their quantum effect, the diameter of pores in 

the template must also be in this range. Therefore the experimental conditions must be 

tuned by choice of electrolyte, temperature, anodization voltage and time, which strictly 

depend on aluminium layer thickness. Except quantum effect requirements, the other one 

concerns the biocompatibility of QDs. Most of traditionally prepared QDs are toxic, hence 

there is a demand for non-toxic material such titanium dioxide [1]. The accomplishment 

of both requirements is not easy feasible. For example, Chen et al. prepared TiO2 nanodots 

using anodization technique, however they did not reach the size needed for quantum 

effect which is strictly below 15 nm (for anatase phase) [2]. Here, new approach of 

anodization process application deals with synthesis of QDs array with high 

photoluminescence on silicon substrate. Such prepared QDs array is highly promising for 

biological imaging and detection of various biomolecules [3]. 

- 101 -


2. EXPERIMENT 

The sample preparation involves the deposition of 50 nm Ti layer (using magnetron 

sputtering) on Si wafer with SiO2 thermal oxide and subsequent evaporation of Al layer 

(700 nm). The anodization procedure takes place in the utility model equipment for 

electrochemical post-processing deposition fabricated in our laboratory. Thanks to 

different anodizing behavior of Al and Ti layers, the same electrolyte during the whole 

process can be applied. Sulphuric acid was chosen as electrolyte since it is known to 

provide smaller pore diameter in template compared to other commonly used electrolytes. 

The anodization process ran in constant potential mode under various conditions 

(electrolyte concentration, temperature, anodization voltage) to obtain as smallest QDs as 

possible. After QDs fabrication, the alumina template was removed by etching in a 

mixture of H3PO4 (50 ml L –1 ) and CrO3 (30 g L –1 ) for 5 min at 60 °C. 

Phase composition, surface topology and photoluminescence studies were performed 

through Raman spectroscopy (T64000, Jobin-Yvon), SEM (Mira, Tescan) and fluorescence 

spectroscopy (Horiba, Jobin-Yvon), resp. Before Raman and fluorescence characterization, 

the samples were annealed at 390 °C for 1h in vacuum furnace. 


The smallest size of TiO2 QDs was reached under anodic oxidation of Ti in 3 M 

sulphuric acid under 4 V at 9 °C. Fig. 1 left shows SEM image of TiO2 QDs array with 

approx. diameter and height less than 10 nm created on Ti layer. The nanodots densely 

covered titanium surface with interdot distance from 5 to 10 nm. These TiO2 QDs were 

found in anatase crystallographic form, as can be seen in Raman spectrum (Fig. 2) with 

two characteristic anatase peaks at 537 cm -1 (corresponding to A1g and B1g vibrational 

mode) and at 640 cm -1 (corresponding to Eg vibrational mode). Fig. 3 represents the 

emission spectra of annealed and non-annealed TiO2 QDs taken with filter cut-off 

(400 nm). No photoluminescence was observed for as-prepared QDs (see inset image), 

while a broad emission peak appeared in visible range in the case of heat-treated QDs. 

XRD analysis was also performed to characterize the sample composition but it brought 

no results probably due to texture. Hence electron backscatter diffraction measurement is 

now under process. 

- 102 -

XI. Woorkshop 

of Physsical 


Fiig.1 

SEM im mage of TiOO2 

QDs after 

alumina template t diissolving 

Fig. .2 Raman sp pectrum off 

TiO2 QDs with dominating 

anattase 

phase 

Fig.33 

Emission spectrum of annealeed 

TiO2 QD Ds with the 

inset ima mage corresp ponding too 

emissionn 

of as-prep pared TiO2 QQDs 

without 

thermal treatment 


Ordered arrays of titania QDDs 

with siz ze below 10 nm achhieved 

by successivee 

anoddization 

off 

aluminiu um and tiitanium 

la ayers in su ulphuric aacid 

revealed 

strongg 

- 103 - 

Brnoo


luminescence. After further surface modification with suitable biomolecules such as DNA, 

these arrays may be used for biosensing purposes and in vitro imaging. Thanks to this 

sensors arrangement, where each sensor can be created from QDs emitting the light at the 

different wavelength, it could be possible to easily detect many different biomolecules at 

the same time. 


The financial support from the grants GACR P102/10/P618 and KAN 208130801 is 

highly acknowledged. 

6. REFERENCES 

[1] Bao, S.J., Li, C.M.; Zang, J.F., Cui, X.Q., Qiao, Y.: Adv. Func. Mater., 18 (2008) 591 

[2] Chen, P.L., Kuo, C.T., Pan, F.M., Tsai, T.G.: Appl. Phys. Lett. 84 (2004) 3888 

[3] Drbohlavova, J., Adam, V., Kizek, R., Hubalek, J.: Int. J. Mol. Sci., 10 (2009) 656 

- 104 -

XI. Woorkshop 

of Physsical 


DEETERMMINAT 

TION OF SA ARCO OSINE AS 

POOSSIBBLE 

TU UMOUUR 

MA ARKER R OF PPROS 

STATE 

TUUMOUURS 

AND A ROUTINE 

BIOCHEEMICA 

AL 

TEESTS 

IN 

URINE 

SAAMPL 

LES 

Nataalia 

CERNE 

SOCHOR1 

EI 

, Petr 

1 , Michal M 

r BABULA3 MASAŘÍK 2 , Jaromír G 

3 , René KIZZEK 

1 

2 Dep 

Absstract 

Aminno 

acid sarrcosine, 

kn nown also aas 

N-methy ylglycine, may m be esta tablished as s new veryy 

impoortant 

markker 

in prostate 

malignnant 

tumou urs and may y be determmined 

by very v simplee 

test. Cancer of prostate is s one of thee 

most incident 

types s of malignnant 

tumou urs in men. . 

Moree 

than one thousand men m in Czeech 

Republ lic die due to this diseease. 

As we ell as in thee 

case of other malignant 

tu umours, for initiation of o treatmen nt well timeed 

diagnosis 

of diseasee 

is neecessary. 

1. 

11 

Department t of Chemistry y and Biochemmistry 

Mende 

partment of PPathological 

Physiology, P Fa Faculty of Med 

CZ-662 2 43 Brno, Czeech 

Republic 3 

el University, Zemedelska a 1, CZ-613 00 0 Brno, Czechh 

Republicc 

dicine, Masary ryk University ty, Komenskeh eho namesti 2, 2, 

3Department t of Natural Dr Drugs, Faculty y of Pharmacyy 

INTRODDUCTION 

Sarccosine 

is naturally n occurring 

nnon-toxic 

amino a acidd 

soluble inn 

water. Sarcosine S may be pplaced 

into o group off 

biogenic aamines, 

wh hich occur in differennt 

types of foods f (fish, , 

meat, beer, 

wine, cabbage, olives). Inn 

foods, sa arcosine iss 

origiinated 

durinng 

process of proteins fermentati ion by actio on of variouus 

kinds of organisms. . 

At iits 

consummmation, 

sa arcosine paasses 

to urine u in un nchanged form. Sarc cosine alsoo 

origiinates 

in livver 

and kidneys 

as inteermediate 

of o choline metabolism m m. At the be eginning off 

20099, 

study demmonstrating 

g diagnostiic 

potential l of this am mino acid wwas 

publish hed in veryy 

presttigious 

jourrnal 

Natur re. Sarcosinne 

is very reputable marker als lso in early y stages off 

tumoours. 

Very important is also facct 

that sarcosine 

was not presennt 

in urine of healthyy 

peopple. 

Due to this fact, probabilityy 

of false positivity p of f investigattion 

is min nimized. Inn 

accorrdance 

witth 

published d results, saarcosine 

is significant tly preferabble 

marker of prostatee 

canccer 

to prosstate-specif 

fic antigenee, 

whose presence p in 

blood iss 

routinely y analysed. . 

Preseence 

of ammino 

acid sarcosine s inndicates 

rig ghtly aggre essive, highh-malignan 

nt tumours. . 

Aim of our woork 

consiste ed in desiggn 

of chrom matographi ic method for determ mination off 

sarcoosine 

in uriine 

sample. 

Identificaation 

and determinati d on was donne 

by spiki ing and byy 

methhod 

of stanndard 

addi ition of saarcosine 

to the samp ple of urinne. 

We we ere able too 

deterrmine 

ultrra 

low sarcosine 

cconcentrati 

ions as well w becauuse 

we used u ionexx 

chroomatographhy 

on apparatus 

Aminoo 

Acid Ana alyzer AAA 400. 

- 105 - 

GUMULEC 2 

2 , Ondřej ZZÍTKA1 

, Jiři i 

Brnoo




2. EXPPERIMENTT 

The sarcosine oof 

99% puri ity was obttained 

from m Fluka Bio oChemika ( (Switzerlan nd). A 

solution off 

the sarcossine 

for pre eparation oof 

the calibr ration curv ve was preppared 

in a buffer b 

Na:TDG ( (N3Na-0.10 g,NaCl-11 1.5 g, C6H88O7-14g 

in 1 l of H2O O). The sepparation 

of 

the 

sarcosine wwas 

carriedd 

out with utilizationn 

of the ion nex chroma atography oon 

an appa aratus 

Amino Accid 

Analyzeer 

AAA 40 00 (Ingos, CCzech 

Rep public). The e AAA anaalyser 

work ks on 

basis of medium ppressure 

li iquid chroomatograph 

hy with ionex i coluumn 

ninhy ydrin 

derivatizattion 

and phhotometric 

detection aat 

520 nm. 

Fig. 1 Caliibration 

cuurve 

of sarco osine 

Absorbance 

75 

50 

25 

0 

0 

Cys 

Sar 

H-Cys 

Linear 

Linear 

Linear 

200 400 

CCalibration 

curve 

y = 0.0658x 

R2 x + 0.0154 

= 0.99986 

CConcentration 

(mmM) 

Fig. 2 Calibbration 

currve 

sarcosin ne in presennce 

of cyste eine and ho omocysteinee. 

- 106 - 

600 

y = 0.0566x - 0 

R2 .3913 

= 0.99244 

y = 00.0266x 

- 0.279 

R2 94 

= 0.9984 

800 1000 

1200 

Brno

XI. Woorkshop 

of Physsical 


3. RESULTS 

AND DISCUSSIO 

D ON 

In the exxperimental 

l part, we ffirstly 

focu used on an analytical determinat tion of thee 

sarcoosine. 

Ionex 

chromato ography wwas 

chosen as the mos st suitable analytical technique. . 

The sarcosine wwas 

analyse ed in a testted 

concen ntration sca ale from 5 tto 

1000 μM M. Error off 

deterrmination 

wwas 

about 8%. 8 

Well resoolved 

signal l of sarcosi 

The calibrationn 

curve was s linear in 

and correlationn 

coefficien nt R 

500 nnM 

(3S/N). The limit o 

2 ine with migration m tim me of 32 mminutes 

was 

obtained. . 

entire test ted range with w equatioon: 

y=0.0266x-0.27944 

=0.99884. 

Limit of f detection of sarcosinne 

was dete ermined ass 

of quantificcation 

of sa arcosine wa as determinned 

as 5 μM (10 S/N). 

Analysis oof 

sarcosine e in presennce 

of two hardly dete erminable aamino 

acid ds (cysteinee 

and homocysteeine) 

was ca arried out. Presence of o these tw wo amino aacids 

had no 

effect onn 

chroomatographhic 

signals of o sarcosinee. 

All chro omatograph hic signals wwere 

resolv ved (Fig. 1 

and Fig.2). Freee 

amino acids a are prresent 

in biological b samples s – due to thi is fact it iss 

absollutely 

neceessary 

to propose p suuitable 

tech hnique for determinaation 

of sa arcosine inn 

preseence 

of free 

amino ac cids. In ourr 

next expe eriment, sa arcosine at concentrat tion 10 μMM 

was added intoo 

a mixture of 17 aminno 

acids of f 20 μM con ncentrationn. 

Fig. 3 demonstratess 

typiccal 

chromaatographic 

record. TThe 

individ dual amino o acids aree 

well sep parated; noo 

interrferences 

wwith 

determ mined sarcossine 

were observed. o 

Fig. 3 Typical chromatogra 

aphic recorrd 

of comm mon amino acids a with aaddition 

of f sarcosine 

Humman 

urine samples collection 

c n and prep paration 

Patients: UUrine 

samp ples were oobtained 

fro om patients s hospitalizzed 

at the Department 

D t 

of UUrology 

oof 

St. An nne´s Uniiversity 

Hospital H Br rno diagnnosed 

with h prostatee 

adennocarcinomma 

(n=11). Average A agge 

was 62-76 

years. The sarcossine 

of con ncentrationn 

15 μMM 

-1480 μMM 

was dete ected in urrine 

sample es from pat tients (Fig.33). 

The sam mples fromm 

curedd 

patients ddiagnosed 

with w prostaate 

adenoca arcinoma (n n=11) (Depaartment 

of Urology off 

St. AAnne´s 

Univversity 

Hos spital Brno) ) were anal lysed. 

- 107 - 

Brnoo


Samples from healthy volunteers (n=30, average age 17-31 years) were obtained 

from students Faculty of Agronomy, Mendel University in Brno. In the samples from 

healthy volunteers sarcosine wasn’t detected. The urine samples were primarily intended 

for routine biochemical tests at the Department of Chemistry Biochemistry, Faculty of 

Agronomy, Mendel University in Brno. Urine samples were diluted 10 times (100μl 

sample urine+900μl buffer) in buffer Na:TDG (N3Na-0.10 g,NaCl-11.5 g, C6H8O7-14g in 1 l 

of H2O). 

Fig.4.: Sarcosine contents in patients and control group 

Fig.5.: Uric acid contents in patiens, cured and control group 

- 108 -

XI. Woorkshop 

of Physsical 


Fig.6.: Ur rea contentts 

in patiens s, cured and d control grroup 

Fig.7.: Glucose 

contennts 

in patiens, 

cured and 

control group 


Chromatoographic 

method m enaabling 

sim multaneous determinat ation of sa arcosine inn 

preseence 

of 17 free amino o acids was designed in n our exper rimental wwork. 

In pat tients urinee 

sampple 

sarcosinne 

was det tected and in urine samples 

from 

healthy y volunteers 

sarcosinee 

wasnn´t 

detecteed. 

In cure ed patientss 

samples sarcosine wasn’t dettected, 

bec cause afterr 

hemiioterapy 

deetection 

of f sarcosine is not possible. 

Amo ong others biochemic cal markerss 

sarcoosine 

is thee 

only one e exhibitingg 

statistical lly significa ant differennce 

betwee en patientss 

and control grroup. 

Increasing 

levell 

of glucose 

is also observed, o hhowever 

th his trend iss 

statisstically 

incconclusive 

due d to the small num mber of peo ople in eacch 

group. In 

contrast, , 

urea and uric accid 

are inde ependent prrostate 

can ncer. 

It shhould 

be nooted 

that elevated e leevels 

of cer rtain bioma arkers mayy 

be assoc ciated withh 

age of patientss 

and the possible ppresence 

of o other diseases. 

Inn 

contrast, sarcosinee 

conttent 

is age independe ent and itss 

associatio on with oth her diseasees 

will be the 

subjectt 

- 109 - 

Brnoo


of further research. Compared to other biomarkers, sarcosine adds even more 

diagnostic potential for prediction of CaP. Further validation experiments and 

optimization for the strategy of constructing this model are warranted. 


The work has been supported by grants: GACR 301/09/P436, IGA MZ 10200-3 and 

NANOSEMED GA AV KAN208130801, IGA MZ 10200-3 

6. REFERENCES 

[1] Jamaspishvili T, Kral M, Khomeriki I:, J.: PROSTATE CANCER AND PROSTATIC DISEASES , 13 

(2010),, 12-19,1. 

[2] Gonzalez DE, Covitz KMY, Sadee W.:, J.:CANCER RESEARCH, 58 

[3] 

(1998), 519-525,3. 

WoganGN, Paglialunga S, Archer MC.J: CANCER RESEARCH,35 (1975), 1981-1984 , 8. 

[4] Sreekumar A, Poisson LM, Rajendiran TM, et al.: Nature (2009), 457, 910-914 

[5] Lucarelli G, Larocca A, Fanelli M, et al. EUROPEAN UROLOGY SUPPLEMENTS(MAR 2011) 10 ,2 

,205-205 

[6] Cao DL, Ye DW, Zhang HL, et al. PROSTATE (MAY 15 2011) 71 , 7 , 700-710 

[7] Issaq HJ, Waybright TJ, Veenstra ELECTROPHORESIS(APR) 32 Issue: 9 , SI, 967-975 

[8] Jentzmik F, Stephan C, Lein M, et al. JOURNAL OF UROLOGY(FEB 2011 ),185,2,706-711 

- 110 -


PHOTOCURRENT GENERATION IN INKJET 

PRINTED TIO2 LAYERS 

Petr DZIK 1 , Magdalena MOROZOVÁ 2 , Michal VESELÝ 1 

1 Brno University of Technology, Faculty of Chemistry, Purkyňova 118, 612 00 Brno, Czech Republic 

2 Institute of Chemical Process Fundamentals of the ASCR, v.v.i., Department of Catalysis and Reaction 

Engineering, Rozvojová 135, 165 02 Prague 6, Czech Republic 

Abstract 

TiO2 layers were prepared by inkjet printing of reversal micelles sol onto ITO substrates. 

Printed films of various thickness were thermally gelled and calcined. Layer homogeneity, 

thickness and morfology was studied by optical microscopy, SEM and AFM. The 

generation of charge carriers upon UV irradiation was studied by electrochemical 

methods. 


TiO2 photocatalysis has received much attention during the last two decades. When 

TiO2 is irradiated by photons of sufficient energy ( < 380 nm) [1], an electron excitation 

takes place followed by the creation of an electron-hole pair [2]. The electrochemical 

potentials of resulting charge carriers are sufficient to reduce molecular O2 and oxidize 

water to hydroxyl radicals. Subsequent reactions in aerated aqueous environment are 

somewhat complex producing the so called reactive oxygen species (ROS) as the main 

product. Despite their obscure origin, nature and short lifetime, ROS are very powerful 

oxidizing agents. They would quickly attack any organic matter in their proximity until it 

is totally cleaved to CO2 and water 0. Such process can be exploited for photocatalytic 

purification of water and air. ROS also attack the organic matter, including living 

organisms (viruses, bacteria, yeast and fungi), thus exhibiting deactivating and disinfecting 

effect. Unlike other disinfection processes, photocatalysis is capable of both inactivating 

bacteria and removing its toxins [4]. Moreover, a photoinduced reversible superhydrophilic 

conversion takes place on irradiated surface of TiO2, yielding a very low water 

contact angle. The combined effects of photocatalysis, photodisinfection and 

photoinduced superhydrophilicity form the platform for the design of smart self-cleaning 

surfaces. 

Sol-gel technique is one of the popular synthetic routes to thin coatings of TiO2. In 

sol-gel processes, TiO2 is usually prepared by hydrolysis and polycondensation of titanium 

alkoxides, which are then transformed into inorganic oxide by thermal calcination. The 

traditional methods of sol application include dip-coating, spin-coating and spray coating. 

These methods are widely used, yet they are burdened by several significant disadvantages 

(limited coated area, sensitivity to surface deffects etc). The authors have shown earlier 0 

that inkjet printing is an elegant method for sol delivery to substrate. It provides a 

complete control over the deposition process parameters together with an excellent 

efficiency of precursor use. Moreover, the possibility of precise patterning and the ease of 

- 111 -


upscaling make this deposition method very appealing for the production of sensors, solar 

cells etc. 

The photoexcitation properties of the prepared TiO2 layers and the ability to 

photocurrent generation were investigated by electrochemical methods – linear 

voltammetry and amperometry. The values of generated photocurrent were recalculated 

to Incident Photon to Current conversion Efficiency (IPCE) using the relation IPCE = i 

/(F×P), where i denotes for the photocurrent density [A cm -2 ], F for the Faraday constant 

(96.485 C mol -1 ) and P is the incident photon flux intensity [Einstein cm -2 s -1 ]. 

2. EXPERIMENT 

Reverse-micelle sol was prepared according to 0. However, this time Triton X-102 

and xylen were used as surfactant and solvent. Sol deposition and patterning onto 

commercial ITO-coated glass was performed by a dedicated experimental inkjet printer 

Fujifilm Dimatix 2831. Prepared sol was sonicated for 5 minutes and then loaded into a 

syringe. A 0.2 m membrane filter and a blunt needle were attached to the syringe luer 

port and the sol was filtered and filled into the Dimatix ink tank in once. A Dimatix 10 pL 

printing head was attached to the the tank and these were mounted into the Dimatix 

printer. Since the sol is a true analytical solution and does not contain any solid 

components, the jetting performace was excellent and all 16 nozzlez were used for 

printing. 

Printed layers were dryed and gelled at 110 °C for 30 min and calcined in an 

furnance by heating at 3 °C min –1 to 450 °C and keeping at this temperature for 4 hours. 

However, two distinctive processing ways were adopted for the multilayered samples: 

Single calcinated (SC-n) series of samples was prepared by printing 1-4 layers of 

sol. After printing, the sample was gelled and calcined. 

Repeatedly calcined (RC-n) samples were prepared by repeated printing, gelling 

and calcination after printing each layer. In both cases, n indicates the number of 

layers. 

Microphotograps of printed layers were recorded using Nikon Eclipse E200 optical 

microscope equipped with a Nikon D5000 digital camera and Nikon Camera Control Pro 2 

software. Captured RAW images were processed by Adobe Lightroom. The surface 

morphology and layers thickness was also investigated by the SEM and AFM. 

Photoelectrochemical measurements were performed in a three-electrodes cell 

(working el. TiO2/ITO, reference el. Ag/AgCl/KCl(sat), Pt counter el.). As a supporting 

electrolyte the water solution of Na2SO4 (0.1M) was used and the wavelength of the 

incident light was focused on 365 nm by an interference filter. 


SEM imaging was employed to determine the layer thickness. Fig. 1 clearly depicts 

that the thickness of 3 layers is approx. 340 nm, giving cca 110 nm per single layer. AFM 

scan shows the globular structure of the layer originating from the micellar nature of the 

sol. The RMS roughness was 5.7 nm. 

- 112 -

XI. Woorkshop 

of Physsical 


Fig.11 

AFM scann 

of sample SC-1 and SSEM 

cross-s 

The electtrochemical 

l response of prepared 

layers to irradiationn 

was imm mediate andd 

compparable 

to ddip-coated 

layers of simmilar 

thick kness. Howe ever, the reeached 

valu ues of IPCEE 

tendd 

to be sommewhat 

low wer than inn 

the case of o dip-coat ting (IPCE was 0,035 for 3 dip- 

coateed 

layers hhaving 

thic ckness apprr. 

330 nm) . Worth noting 

is thhe 

growing differencee 

betwween 

RC annd 

SC laye ers of the same thick kness. Resu ults from oother 

analy ysis (XRD) ) 

indiccate 

that thhe 

RC laye ers are madde 

of large er crystallit tes due to repeated calcination, c , 

durinng 

which the re-hea ated materrial 

forming 

lower la ayer rearraanges 

to fo orm biggerr 

crysttallites. 

IPCE 

0.022 

0.01 

0.000 

-0.01 

-400 -200 

0 200 400 600 

Potential (mV) 

SC, 1layeer 

RC, 1 layyer 

SC, 2 layers 

RC, 2 layyers 

SC, 3 layers 

RC, 3 layyers 

ITO 

800 1000 

Fig.22-left: 

Polarization 

cu urves of linnear 

voltam 

light/darrk 

period. Right: R The photocurre 

0.6 V (AAg/AgCl) 

at 10 mW/cmm 

2 mmetry, irr radiation att 

5 s interv vals of thee 

ent-time be ehaviour wwith 

constan nt potentiall 

irradiatio on. 


Thin oxidde 

semicond ductor layeers 

have be een prepare ed by inkjeet 

printing and sol-gell 

process. 

Not onnly 

the laye er thicknesss 

but also the therma al history hhas 

signific cant impactt 

on chharge 

generration, 

as th hicker and repeatedly y calcined la ayers give hhigher 

values 

of IPCE. . 

section of R 

IPCE 

- 113 - 

0.02 

0.01 

0.00 

0 20 40 

RC-3 samplles 

60 80 100 

120 140 160 1880 

200 220 240 

Time 

(s) 

Brnoo 

SC, 1 layer 

RC, 1 layer 

SC, 2 layers 

RC, 2 layers 

SC, 3 layers 

RC, 3 layers 

ITO


5. 4.ACKNOWLEDGEMENT 

This work has been supported by project 104/09/P165 of the Czech Science 

Foundation. 

6. REFERENCES 

[1] Blount, M. C., Kim, D. H., Falconer, J. L., Environmental Science & Technology 35 (2001), 14, 2988- 

2994. 

[2] Han, F., et al., Applied Catalysis A: General, 359 (2009), 1-2, 25-40. 

[3] Mills, A., Le Hunte, S., Journal of Photochemistry and Photobiology A: Chemistry, 108 (1997), 1, 1- 

35. 

[4] G. S. Shephard, et al., Water Research, 36 (2002), 1, 140-146 

[5] Dzik P., Veselý M., Chomoucká J., Journal of Advanced Oxidation Technologies, 13 (2010), 2, 172- 

183 

[6] Klusoň, P., Lusková, H., Cajthaml, T., Šolcová, O. Thin Solid Films, 495 (2006), 1-2, 18-23 

- 114 -


OPTICAL SPECTROSCOPY IN MODERN 

BIOPHYSICAL RESEARCH 

Tomáš FESSL 1 

1 Tomáš Fessl, Institute of Physical Biology, University of South Bohemia, Zamek 136, Nove Hrady 

Abstract 

UV/VIS optical spectroscopy evolved in last few decades into a powerful and sensitive tool 

widely used in biophysical, chemical and biological sciences. Rapid development of new 

technologies implemented into experimental instruments led to distinct improvement of 

spatial and temporal sensitivity, hence improved applicability to solve complex problems. 

Here, we show the advantages of Single-Molecule Fluorescence Spectroscopy (SMFS) and 

Fluorescence Correlation Spectroscopy (FCS) employed in detection of CHD4 protein 

translocating along DNA as ATP-driven molecular motor and in detection of MS2 

bacteriophage capsid assembly, respectively. On the example of fast charge transfer 

between DNA and fluorescent dye QSY 21, we demonstrate the ultrasensitive temporal 

resolution of femtosecond transient absorption spectroscopy. 

1. CONTENT 

Huge development in electronics, computer science and the consequent 

implementation of this technology into scientific instrumentation facilitated birth or 

distinct improvement of many novel concepts in UV/VIS spectroscopy. Here we will 

show the main advantages and applications of two advanced fluorescence and one 

absorption technique. 

Fluorescence techniques take advantage of great spatial and decent temporal 

resolution. Here we report FCS and Förster Resonance Energy Transfer (FRET) on 

individual molecules. FCS is one of the fluctuation techniques, detecting via 

autocorrelation or crosscorrelation changes in fluorescence intensity in approximately 

femtolitre volume. Via these fluctuations, FCS provides information about diffusion 

coefficients, triplet state lifetime and dye photophysics, such as blinking, fluorescence 

quenching and FRET kinetics. Here we utilize FCS to create simple tool for detection of 

virus capsid assembly on the model virus. During long assembly time, we measure FCS 

record on labelled MS2 bacteriophage's simplified assembly components and calculate the 

probability distribution of diffusion coefficients as a function of assembly time via inverse 

Laplace transform (Fig. 1). 

- 115 -




Fig. 1 Capsid 

formatiion 

during 400 minutees 

of assem mbly time. The T curves were calcu ulated 

fromm 

inverse Laplace tr ransforms oof 

individu ual FCS re ecords. Twoo 

datasets with 

diffferent 

initial 

assembly y conditionns 

are show wn, their as ssembly traajectories 

visibly 

difffer, 

hence wwe 

claim, the t methodd 

is capable to detect complex c sysstem 

behav viour, 

succh 

as fallingg 

into kinetic 

traps andd 

subsequen nt escape. 

To inntroduce 

siingle-molec 

cule FRET, we presen nt the study y of humann 

CHD4 pr rotein 

translocatiion 

activityy. 

Due to changes oof 

the FRE ET efficienc cy, we cann 

track lab belled 

protein sttepping 

aloong 

DNA molecule, , reveal it ts directionality 

andd 

elucidate e the 

dependencce 

of translocation 

mo ovement onn 

ATP meta abolism (Fig g. 2). 

Fig 2. Trannslocation 

activity of f human CHHD4 

protei in in the presence 

of ATP. From m the 

FREET 

(or prooximity 

ra atio) time trajectories s, direction nality, dweell 

times (Δt1), ( 

trannslocation 

ttimes 

(Δt2) and step siize 

(Δp1 an nd Δp2) can be revealed. 

Ultraafast 

transient 

absorpt tion spectrroscopy 

as an a example e of absorpption 

techn niques 

offers the possibility to study kinetics 

of eexcited 

stat te absorptio on, ground state bleac ching, 

stimulatedd 

emission, charge tra ansfer and other phot tophysical processes oon 

femtose econd 

- 116 - 

Brno

XI. Woorkshop 

of Physsical 


time scale. Heree 

we emplo oyed this teechnique 

to o explore th he interactioon 

between n DNA andd 

termminally 

attaached 

non-fluorescentt 

quencher r QSY 21 (Fig. 3) annd 

the pho otophysicall 

propperties 

of thhe 

quencher r itself. 

Fig. 

3. 

[1] 

3 Two maain 

binding g motifs obbtained 

fro om molecular 

dynamiics 

simulat tion of thee 

DNA-QS QSY 21 com mplex [1]. TThe 

dye is covalently c attached too 

DNA via six carbonn 

flexible llinker. 

Dur ring the timme, 

QSY 21 spend in binding b mottif 

captured d at the topp 

photo-innduced 

elec ctron transffer 

is highly y probable. 


MENT 

The workk 

has been n supported 

by “Bioa active Bioc compatible Surfaces and Novell 

Nanoostructuredd 

Composites 

for AApplication 

ns to Me edicine annd 

Drug Delivery”, , 

KANN2001008011. 

REFERENNCES 

Kabelac, M. .: Phys. Chem m. Chem. Phyys., 

12 (2010), 9677-9684 

- 117 - 

Brnoo


AN ELECTROCHEMICAL SPM UNDER FULL 

ENVIRONMENTAL CONTROL 

Fiona FREHILL 1 

1 Fiona Frehill, Agilent Technologies, 610 Wharfedale Road, IQ Winnersh, Wokingham, Berkshire, RG41 

5TP, UK, fiona_frehill@agilent.com 

For over two decades, scanning probe microscopy (SPM) has provided scientists a 

unique tool to study in situ electrochemical (EC) processes with atomic/molecular 

resolution. New discoveries continue to be reported as the research boundary expands 

wider and deeper. While the field steadily advances, the requirements for electrochemical 

scanning probe microscopes also become greater. A substantial effort has been made by 

Agilent over the past years to provide an EC SPM device which - in addition to clean 

liquid imaging and atomic and molecular resolution – provides an oxygen-free 

environment. This has been considered critical for many EC SPM studies, highlighting the 

oxygen-elimination capability of our standard environmental chamber, plus the optional 

use of a fully integrated glove box.Examples presented range from studies of metallic and 

molecular layers under electrochemical and environmental control (temperature, solvent, 

atmosphere), studies of electron-transfer, electro-polymerization of poly-electrolytes 

using cyclic voltammetry, up to studies of “real-world” samples like corrosion studies on 

steel alloys or applications in non-aqueous battery technology (Lithium-and Zinc-based 

substrates). 

In summary several techniques and examples will be shown that demonstrate the 

capabilities of electrochemical SPM for topographical imaging on the nanometer scale and 

simultaneously modifying a surface under potentiostatic and environmental control. 

- 118 -


HEAVY METALS IN PROSTATE CANCER 

CELL LINES 

Jaromír GUMULEC 1 , Marian HLAVNA 1 , Markéta SZTALMACHOVÁ 1 , Šárka 

KUCHTICKOVÁ 1 , Roman HRABEC 2 , Arne ROVNÝ 2 , Soňa KŘÍŽKOVÁ 4 , Petr BABULA 3 , 

Vojtěch ADAM 4 , René KIZEK 4 , Michal MASAŘÍK 1 

1 Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Kamenice 5, CZ-625 00 

Brno, Czech Republic 

2 Department of Urology, St. Anne´s University Hospital, Pekarska 53, CZ-65691, Brno 


Brno, Palackeho 1/3, CZ-612 42 Brno, Czech Republic 

4 Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, 


Abstract 

Prostate cancer is second leading cause of death among men. In the present time, prostate 

specific antigen (PSA) is the only tumor marker used for carcinoma detection. This tumor 

marker has high level of sensitivity, but lower level of specificity, so it can be increased 

either in inflammation or in prostate hyperplasia. It is desirable to find a new markers or 

genes with higher level of specificity. The aim of this study is to describe the level of 

metallothionein using electrochemical methods and real time PCR in prostatic cell lines 

after stimulation by various heavy metals. 


Prostate tissue is specific in zinc metabolism – it accumulates high level of zinc(II) – 

up to ten times higher compared to other tissues. In such concentration, zinc(II) affects 

energetic metabolism, antioxidative processes and apoptosis. High zinc(II) content affect 

apoptosis through the formation of BAX pores on the outer mitochondrial membrane. 

Cytochrome-C can further be released from mitochondria and can trigger cascade of 

caspases resulting in increased apoptosis.[1] 

Metallothioneins play a key role in metabolism, transport and storage of heavy 

metals. Due to zinc(II) high affinity to proteins following the Irving-Williams series, 

metallothioneins bind mostly zinc(II).[2, 3] When zinc(II) ions get into a cytosol through 

zinc transporters, it is immediately buffered by metallothionein, thus the free zinc(II) ions 

level is maintained on very low level. Due to signalling roles of free zinc(II) ions, 

metallothionein plays an important role in this process because of its level regulation. 

Metallothioneins also protect cells against oxidative stress,[2] because they cooperate with 

reduced glutathione (GSH).[4] Due to these features it is not surprising that MTs are 

overexpressed under conditions with increased risk of reactive oxygen species formation 

such as cell proliferation or embryonic development.[4] A lot of recent studies indicate 

elevated serum levels of metallothioneins in number of malignancies such as breast, 

bronchial, urogenital, colorectal, prostate carcinomas, melanoma and several 

lymphoma.[2] 

- 119 -


However, it was found that the level of intracellular zinc(II) of lateral lobes of 

prostate is significantly lower in patients suffering from prostate cancer.[5]This is due to 

the loss of ability to accumulate zinc(II) of prostate cancer cells.[6] Together with the 

finding that lateral lobes are also the major site of initiation of malignancy and zinc(II) has 

effects on apoptosis and proliferation, it is expectable that zinc(II) may play an important 

role in prostate cancer pathogenesis. According to results from recent studies, in zincimport 

transporter family, reduced expression of ZIP1 protein was identified.[7]The 

importance of ZIP1 transporter in prostate (cancer) is emphasized by finding that this 

transporter does uptake the majority of zinc(II) from the circulation. Furthermore, 

another phenomenon of ZIP1 in prostate cancer was described: the increased expression 

of this transporter reduces the ability of prostate cancer to metastasize.[8] ZIP1 may 

therefore be regarded as a tumor suppressing gene. ZIP1 is down-regulated by RREB-1 

due to up-regulated RAS-RAF-MEK-ERK cascade.[9] However, this prostate cancerspecific 

zinc(II) dyregulation is still not fully clarified and needs further research that will 

lead to a more complex view on zinc(II) fluxes. As a consequence of zinc(II) 

downregulation, metallothionein level is significantly decreased. 

As a result of zinc(II)/metallohionein down-regulation, apoptic/antiapoptic balance, 

energetic metabolism and antioxidative capacity is changed. Cancer cells can become 

more energy-efficient compared to healthy "energy unefficient" prostate cells.[10]This 

phenomenon can also be used in prostate cancer to support tumor cell growth. In terms of 

apoptosis and proliferation of prostate cancer cells, overexpression of Bcl-2 was observed. 

Antiapoptic gene Bcl-2 is overexpressed in 30–60% of prostate cancer within the luminal 

epithelium in high-grade prostatic intraepithelial neoplasia lesions.[11, 12]. Bcl-2 inhibits 

caspase activity either by preventing the release of Cytochrome C from the mitochondria 

and/or by binding to the apoptosis-activating factor (APAF-1). Moreover, due to 

decreased zinc(II) level, BAX-Cytochrome C apoptic cascade is significantly silenced.[13] 

The aim of this study is to describe the level of metallothionein using 

electrochemical methods in prostatic cell lines after stimulation by various heavy 

metals. 

5. EXPERIMENT 

In this study, two cell lines were used: (1) 22Rv1, derived from a xenograft from 

androgen-dependent primary tumor of Gleason sum 9), (2) PNT1A, established by 

immortalisation of normal adult prostatic epithelial cells by transfection with SV40). Both 

cell lines used in this study were purchased from HPA Culture Collections (Salisbury, 

UK). Once the cells grew up to 50-60% confluence of the culture, the grow mediums were 

replaced by fresh medium for 24h to synchronize cell growth. The cells were then treated 

with or without zinc(II) (0–800μM), Copper (0–800 μM) and cadmium (0–150 μM) in 

fresh medium for 48h. Subsequently, thermal-mechanical lysates were prepared at 99 °C 

in a thermomixer (Eppendorf 5430, Germany) for 15 min with shaking. This is possible 

due to thermostable characteristics of metallotihioneins. For RNA isolation was used High 

pure total RNA isolation kit from Roche (Basel, CH), RNA lysates were prepared 

according to instruction manual. 

- 120 -


RT-PCR was performed in triplicate using the TaqMan gene expression assay system 

with the 7500 real-time PCR system (Applied Biosystems), and the amplified DNA was 

analyzed by the comparative Ct method using β-actin as an endogenous control. The 

primer and probe sets for β-actin (Assay ID: Hs00185826_m1), MT1 Assay ID: 

Hs00185826_m1) and MT2 (Hs00794796_m1) were selected from TaqMan gene. 

Metallothionein content was analysed electrochemically using differential pulse 

voltammetry. Measurements were performed with 747 VA Stand instrument connected to 

746 VA Trace Analyzer and 695 Autosampler (Metrohm, Switzerland), using a standard 

cell with three electrodes and cooled sample holder (4 °C). A hanging mercury drop 

electrode (HMDE) with a drop area of 0.4 mm2 was the working electrode. An 

Ag/AgCl/3M KCl electrode was the reference and glassy carbon electrode was auxiliary. 

For data processing GPES 4.9 supplied by EcoChemie was employed. The analyzed 

samples were deoxygenated prior to measurements by purging with argon (99.999 %), 

saturated with water for 120 s. Brdicka supporting electrolyte containing 1 mM 

Co(NH3)6Cl3 and 1 M ammonia buffer (NH3(aq) + NH4Cl, pH = 9.6) was used. The 

supporting electrolyte was exchanged after each analysis. The parameters of the 

measurement were as follows: initial potential of –0.7 V, end potential of –1.75 V, 

modulation time 0.057 s, time interval 0.2 s, step potential 2 mV, modulation amplitude - 

250 mV, Eads = 0 V, volume of injected sample: 20 μl (100 × diluted sample with 0.1 M 

phosphate buffer pH 7.0). All experiments were carried out at temperature 4°C employing 

thermostat Julabo F25 (Labortechnik GmbH, Germany). 

Software Statistica 9 was used to perform statistical analysis and visualization of 

results. Correlations were performed to evaluate dependency between variables, 

visualized results are those with p < 0.05 and those where strong dependence is present, 

i.e. with |r| > 0.85 and p is barely significant, i.e. close to 0.05. 


We have observed statistically significant strong correlation between zinc in 

medium and in cells (p = 0,001, r = 0,99) in 22Rv1 cancer cell line and, interestingly, we 

found statistically significant negative correlation, in PNT1A cell line suggesting 

significant differences in zinc transport between healthy and cancer cells. 

In terms of metallothionein, we found significantly elevated metallothionein protein 

level in healthy cell line, which is in contrary to previous studies (fig. 2 right). Moreover, 

we’ve observed significant correlations in PNT1A cell line between metallothionein level 

and zinc(II) (positive correlation r = 0,90 at p = 0,04) and copper(II) (negative correlation r 

= -0,98 at p = 0,002). We found no correlation between zinc(II) and metallothionein in 

cancer cell line 22Rv1. We found negative correlation between intracellular free zinc and 

metallothionein in PNT1A cell line (-0,93 at p = 0,02), not in 22Rv1 cell line (fig.1). 

In mRNA analysis, we found elevation of MT2A class of metallothionein as 

compared to MT1A class. Moreover, we found elevation of booth MT classes in cancer 

cell line (fig.2). This finding is not consistent with protein level of metallohionein. This is 

evidenced by finding, that MT protein level does not correlate with booth mRNA classes 

- 121 -


of metallothioneins except zinc-treated PNT1A cell line and MT2A gene (r = 0,94 at p = 

0,01). No similar evidence was observed in 22Rv1 cell line (data not shown). 

Intracellular free metal level 

Metallothionein/total protein (µg/mg) 

Metal: Cd 

Metal: Cu 

Metal: Zn 

Metal: Cd 

Metal: Cu 

Metal: Zn 

30 

20 

10 

0 

0,0 

0 40 80 120 0 20 40 60 

28 

24 

20 

16 

12 

8 

4 

0 

2,0 

1,6 

1,2 

0,8 

0,4 

60 

40 

20 

0 200 400 600 800 

0 

0 200 400 600 800 

Cell line: 22Rv1 

Metal in medium (µM) 

0,0 

0 40 80 120 160 

1,6 

1,2 

0,8 

0,4 

0,0 

0 200 400 600 800 

1,6 

1,2 

0,8 

0,4 

0,0 

0 200 400 600 800 

Cell line: 22Rv1 

Fig.1 Correlations of metal in medium vs. intracellular metal level (left), between metal in 

medium vs. MT (right) 

- 122 - 

2,4 

2,0 

1,6 

1,2 

0,8 

0,4 

60 

40 

20 

Metal in medium (µM) 

0 

0 100 200 300 400 

6 

4 

2 

0 

0 40 80 120 160 

40 

30 

20 

10 

Cell line: PNT1A 

0 

0 20 40 60 

40 

30 

20 

10 

0 

0 100 200 300 400 

30 

20 

10 

0 

0 40 80 120 160 

Cell line: PNT1A


relative transcript level x PNT1A 

rel. transcript level 

200% 

190% 

180% 

170% 

160% 

150% 

140% 

130% 

120% 

110% 

100% 

90% 

16000% 

14000% 

12000% 

10000% 

8000% 

6000% 

4000% 

2000% 

0% 

MT1A expression 

22Rv1 PNT1A 

Cell line 

22Rv1 Metallothionein 

MT1A MT2A 

mRNA 

Metallothionein/total protein (µg/mg) 

35 

30 

25 

20 

15 

10 

5 

0 

relative transcript level x PNT1A 

rel. transcript level 

260% 

250% 

240% 

230% 

220% 

210% 

200% 

190% 

180% 

170% 

160% 

150% 

140% 

130% 

120% 

110% 

100% 

90% 

7500% 

5000% 

2500% 

750% 

500% 

250% 

22Rv1 PNT1A 

Cell line 

MT2A expression 

22Rv1 PNT1A 

Cell line 

PNT1A Metallothionein 

MT1A MT2A 

mRNA 

Fig.2 Metallothionein mRNA level: Difference in classes (first row left), difference in cell 

lines (second row left), metallothionein protein level (below) 

7. CONCLUSION 

In this study we have shown different behavior of cancer and healthy prostate 

tissue, as demonstrated on the cell lines. We have shown, that zinc buffering significantly 

differs between cell lines, that metallothionein level is dependent on the zinc(II) level 

more significantly in healthy tissue. Furthermore, we have demonstrated a discrepancy 

between metallothionein RNA and protein level and, at last, we identified MT2A as a 

major metallothionein class in prostate tissue. 


The work has been supported by grants GACR 301/09/P436 and NSI0200-3 

- 123 -


9. REFERENCES 

1. Feng, P., et al., The involvement of bax in zinc-induced mitochondrial apoptogenesis in malignant 

prostate cells. Molecular Cancer, 2008. 7. 

2. Eckschlager, T., et al., Metallothioneins and Cancer. Current Protein & Peptide Science, 2009. 10(4): 

p. 360-375. 

3. Colvin, R., et al., Cytosolic zinc buffering and muffling: Their role in intracellular zinc homeostasis. 

Metallomics, 2010. 2(5): p. 306-317. 

4. Krizkova, S., et al., Metallothionein - a promising tool for cancer diagnostics. Bratislavske Lekarske 

Listy, 2009. 110(2): p. 93-97. 

5. Kambe, T., et al., Overview of mammalian zinc transporters. Cellular and Molecular Life Sciences, 

2004. 61(1): p. 49-68. 

6. Costello, L. and R. Franklin, The intermediary metabolism of the prostate: a key to understanding the 

pathogenesis and progression of prostate malignancy. Oncology, 2000. 59(4): p. 269-282. 

7. Hogstrand, C., et al., Zinc transporters and cancer: a potential role for ZIP7 as a hub for tyrosine 

kinase activation. Trends in Molecular Medicine, 2009. 15(3): p. 101-111. 

8. Golovine, K., et al., Overexpression of the Zinc Uptake Transporter hZIP1 Inhibits Nuclear Factor- B 

and Reduces the Malignant Potential of Prostate Cancer Cells In vitro and In vivo. Clinical Cancer 

Research, 2008. 14(17): p. 5376. 

9. Milon, B.C., et al., Ras Responsive Element Binding Protein-1 (RREB-1) Down-Regulates hZIP1 

Expression in Prostate Cancer Cells. Prostate, 2010. 70(3): p. 288-296. 

10. Costello, L. and R. Franklin, The clinical relevance of the metabolism of prostate cancer; zinc and 

tumor suppression: connecting the dots. Molecular Cancer, 2006. 5(1): p. 17. 

11. Humbert, L. and M. Chevrette, Somatic Molecular Genetics of Prostate Cancer, in Male Reproductive 

Cancers Epidemiology, Pathology and Genetics, W.D. Foulkes and K.A. Cooney, Editors. 2009, 

Springer Verlag: New York, Dordrecht, Heidelberg, London. 

12. DiPaola, R.S., J. Patel, and M.M. Rafi, Targeting apoptosis in prostate cancer. Hematology-Oncology 

Clinics of North America, 2001. 15(3): p. 509-+. 

13. Franklin, R. and L. Costello, The important role of the apoptotic effects of zinc in the development of 

cancers. Journal of cellular biochemistry, 2009. 106(5): p. 750-757. 

- 124 -


NEW POSSIBILITIES IN 

FUNCTIONALIZATION AND LABELING OF 

DNA FOR ELECTROCHEMICAL ANALYSIS 

Luděk HAVRAN 1 , Petra HORÁKOVÁ 1 , Hana PIVOŇKOVÁ 1 , Milan VRÁBEL 2 , Hana 

MACÍČKOVÁ-CAHOVÁ 2 , Michal HOCEK 2 , Miroslav FOJTA 1 

1 Institute of Biophysics ASCR v.v.i., Královopolská 135, 612 65 Brno, Czech Republic 

2 Institute of Organic Chemistry and Biochemistry ASCR v.v.i., Flemingovo nám. 2, 16610 Prague 6, Czech 

Republic 

Abstract 

A chemically modified DNA probes labelled by different electroactive tags were prepared 

by incorporation of modified deoxynucleoside triphosphates (dNTP) into the 

oligonucleotide chain by using enzymes. Modified dNTPs were synthesized by simple 

cross-coupling reaction in aqueous media. Electroactive modified DNA probes were used 

in development of electrochemical sensors for the analysis of nucleotide sequences and for 

DNA-protein interaction assays. 


Electrochemical analysis of DNA has been one of the most dynamically developing 

fields in bioanalytical electrochemistry in last two decades [1]. This progress starts mainly 

due to try to develop electrochemical sensors for detection of DNA hybridization, 

interactions, and damage. Application of signaling DNA probes (short oligonucleotide 

molecules) modified by some electroactive tags belong to techniques frequently used in 

this area. Beside “classical” methods – using modified dNTPs in solid-phase 

oligonucleotide synthesis or post-synthetic modification of synthetic oligonucleotide, a 

new approach was introduced recently [2] based on enzymatic incorporation of modified 

dNTPs in to oligonucleotide chain. Modified dNTPs can be synthesized by simple crosscoupling 

reaction in aqueous media [2]. In this contribution we demonstrate using of the 

above mentioned method for the preparation of a set of signaling DNA probes labeled 

with different electroactive tags (ferrocene [3], amino or nitro groups [4,5], complexes of 

Ru 2+ or Os 2+ [6] and alkylsulfanylphenyl group [7]), their incorporation into DNA and 

application in the development of electrochemical sensors for the analysis of nucleotide 

sequences and DNA protein interactions. 

2. EXPERIMENT 

Modified dNTPs were synthesized by the direct single-step aqueous-phase 

crosscoupling reactions of halogenated dNTPs. Based on our previous experience that 8substituted 

purine dNTPs are not efficiently incorporated we have chosen 7-substituted 7deaza-adenine 

and 7-deaza-guanine as substitute for the adenine and guanine bases. 

Labelled DNA signalling probes was prepared by means of primer extension (PEX) 

catalysed by different DNA polymerases (Klenow (exo-) DNA polymerase fragment, 

- 125 -




DyNAzymme, 

Vent(exxo-), 

Pwo - New Enggland 

Biolab bs (UK), PE EQLAB (SRRN), 

Finnz zymes 

(Finland)) . Results off 

PEX reacti ion were mmonitored 

by b polyacry ylamide gel electropho oresis. 

PEX prodducts 

were separated by Qiagenn 

Nucleotid de Remova al kit or bby 

streptav vidine 

coated maagnetic 

beads 

(Dynabeads® 

MM-270 

Strep ptavidin, Dynal D A.S. Norway). The 

building bblocks 

andd 

PEX pro oducts weree 

analysed d by mean ns of in siitu 

and ex x situ 

(adsorptivee 

transfer stripping) cyclic andd/or 

square e-wave vol ltammetry (CV, SWV V) at 

hanging mmercury 

drrop 

electro ode (HMDEE) 

or pyro olytic graphite 

electrrode 

(PGE) ). All 

voltammettric 

measurrements 

were w performmed 

at am mbient temp perature ussing 

an Au utolab 

analyzer (EEcoChemiee, 

The Neth herlands) inn 

a three-ele ectrode set-up. 

3. RESSULTS 

ANND 

DISCU USSION 

By crosscouplinng 

reactions 

were synnthetized 

dN 

or alkylsullfanylphenyyl 

groups, and a compleexes 

of Ru2+ NTPs bearing 

ferrocenne, 

amino, nitro 

+ or Os2+ (Fig g.1) 

Fig.1. Struuctures 

of ddNTPs 

mod 

(B) , [Ru(bipy) 3] 2+ dified by: fe ferrocenylet thynyl group 

(A), nittrophenyl 

group g 

(C), and d benzylsullfanyl 

group p (D). 

Electtrochemicaal 

behaviou ur of thesee 

dNTPs was w studied d at mercuury 

and ca arbon 

electrodes by differeent 

voltam mmetric meethods. 

Con nstant curr rent chronoopotentiom 

metric 

stripping was used in case of f dNTPs annd 

PEX products 

be earing alkyylsulfanylph 

henyl 

groups. Laabelled 

dNTTPs 

were in ncorporatedd 

in to DN NA by PEX reaction. PProducts 

of f PEX 

reaction wwas 

electrocchemically 

analysed ssame 

way as a dNTPs. Some S modiified 

dNTPs s was 

used for ellectrochemmical 

analysis 

of nucleootide 

seque ence or for study of DNNA 

interac ctions 

with proteeins 

(Fig.2). . 

- 126 - 

Brno

XI. Woorkshop 

of Physsical 


Fig. 22. 

Electrochhemical 

“m multicolour” ” labelling of o DNA usi ing dNTPs modified by b differentt 

electroacctive 

tags. 


DNA proobes 

labele ed by elecctroactive 

tags t were prepared by incorp poration off 

diffeerent 

chemiically 

modi ified dNTP. 

These pro obes were used u for eleectrochemic 

cal analysiss 

of DDNA 

sequeences 

(inclu uding anallysis 

of sin ngle nucle eotide polyymorphisms) 

and forr 

monnitoring 

of DDNA 

– prot tein interacctions. 


MENT 

The workk 

was suppo orted by Grrant 

Agency 

of the Ac cademy of SSciences 

of f the Czechh 

Repuublic 

(IAA 400040903 3), MEYS CR (LC060 035) and th he Academmy 

of Scien nces of thee 

Czecch 

Republicc 

(AV0Z500 040507, AVV0Z5004070 

02). 

6. 

[1] 

[2] 

[3] 

[4] 

[5] 

[6] 

[7] 

REFERENNCES 

Paleček, E.: Electroanaly ysis, 21 (20099), 

239 

Hocek, M., Fojta, M.: Or rg. Biomol. CChem., 

6 (2008 8), 2233 

Brázdilová, P., Vrábel, M., M Pohl, R., PPivoňková, 

H., 

Havran, L., Hocek, M., FFojta, 

M.: Che em. Eur. J. 133 

(2007), 95277 

Cahová, H., , Havran, L., Brázdilová, PP., 

Pivoňková á, H., Pohl, R., 

Fojta, M., HHocek, 

M.: An ngew. Chem. . 

Int. Ed. 47 ( (2008), 2059 

Horáková, PP., 

Macíčkov vá-Cahová, HH., 

Pivoňková á, H., Špaček, , J., Havran, L., Hocek, M., M Fojta, M.: : 

Org. Biomool. 

Chem. 9 (2011), 

1366 

Vrábel, M., Horáková, P., P Pivoňkováá, 

H., Kalacho ová, L., Černo ocká, H., Cahhová, 

H., Poh hl, R., Šebest, , 

P., Havran, L., Hocek, M., M Fojta, M.: CChem. 

Eur. J. 15 (2009), 11 144 

Macíčková--Cahová, 

H., Pohl, R., Hooráková, 

P., Havran, H L., Šp paček, J., Fojtta, 

M., Hocek k, M.: Chem. . 

Eur. J. 2011 in press 

- 127 - 

Brnoo


REVIEW OF ELECTROREDUCTION OF 

HYDROGEN IONS ON MERCURY 

Michael HEYROVSKÝ 1 

1 J.Heyrovský Institute of Physical Chemistry, Academy of Sciences of Czech Rep.,v.v.i., Dolejškova 3, 182 23 

Prague 8 

Electroreduction of hydrogen ions occurs on mercury with highest overpotential of 

all metals. This has the advantage in electroanalysis that mercury electrodes provide the 

widest potential range for reduction processes. The high hydrogen overpotential gives 

also the possibility to catalyze hydrogen evolution by a variety of chemical entities - they 

can be ions of rare metals, cobalt complexes containing sulphur, some nanoparticles, 

peptides, proteins, nucleic acids, polysaccharides. The hydrogen evolution catalysis has 

been utilized in polarography and in voltammetry, however, the most sensitive reactions 

of hydrogen catalysis can be achieved in chronopotentiometry, where during fast change 

of electrode potential can become catalytically active even short interactions between the 

catalyst and the electrode. 

- 128 -


QUANTUM DOTS WITH FUNCTIONALIZED 

SHELL LAYERS FOR ENHANCED 

BIOCONJUGATION AND 

FLUOROIMMUNOASSAYS 

Antonín HLAVÁČEK 1 , Petr SKLÁDAL 1 

1 Masaryk University, Faculty of Science, Department of Biochemistry, Kotlářská 2, 611 37 Brno 

Abstract 

Quantum dots (QD) are widely proposed as useful fluorescent labels with potential to rival 

classic organic fluorophores. Their optical properties ensure low background what is 

desired in variety of immunochemical assays. Here, the application of highly stable 

biotinylated QD conjugates prepared at our laboratory is presented. The preparation of 

QDs was optimized with aim to reach storage stability (two months in buffer without any 

decay of signal) and high signal/noise ratio when used in fluorescent bioaffinity assays. 

Such optimized QDs were successfully applied in the competitive flouroimmunoassay of 

human serum albumin in urine samples. Characteristics of this assay (LOD 1.9 μg/ml, 

working range from 2.8 to 10.4 μg/ml) are relevant to clinically important limits for 

microalbuminuria. 


Fluorescent semiconductor nanocrystals known as quantum dots (QD) used as labels 

in various bioconjugates belong to the most highlighted applications of nanotechnology in 

bioanalytical chemistry [1,2]. QDs exhibit size-tuneable photoluminescence properties, 

broader excitation spectra and narrower emission bandwidth in comparison with the 

classic organic fluorophores [3]. Such properties provide significantly higher signal/noise 

ratio in the fluorescent techniques. In bioanalytical chemistry, QDs were used for 

multiplexed fluoroimmunoassays (FIA) [4,5] enabling sensitive detection of specific 

oligonucleotides and proteins using fluorescently encoded microbeads [6]. The 

dependence of QDs fluorescence intensity in aqueous solutions on metal ion 

concentration was used for their determination [7]. Electrochemiluminescent properties 

of QDs were used for construction of novel sensors for determination of proteins [8], DNA 

[9], cells [10], ascorbic acid and others analytes [11]. Here, biotinylated QDs were used for 

competitive fluoroimmunoassays of human serum albumin in urine samples. 

2. EXPERIMENT 

Competitive FIA utilizing fluorescent biotin-QD conjugate was developed. The 384 

well microplates were coated with HSA (80 μl per well, 0.3 mg/ml HSA in Pi, incubated 

overnight at refrigerator). The sodium phosphate buffer with 0.05 % Tween-20 (Pi, 50 

mM phosphate, pH 7.4) was used for washing of the plates which were stored in a 

desiccator at cold. The mixtures of biotinylated antibody and either HSA standard or 

- 129 -


diluted urine samples in PBS were prepared and incubated for 15 min at laboratory 

temperature. Next, 20 μl of the resulting solution were added to the microplate wells, 

incubated for 40 min at laboratory temperature and the plates were washed with 

Pi/Tween. The microplate wells were filled with 20 μl of avidin solution (40 μg/ml in Pi), 

incubated for 25 min and washed. Biotinylated QDs were added (20 μl, 5 nM in Pi) and 

incubated for 25 min. Finally, fluorescence of the washed and dried wells was measured 

by Synergy 2 (excitation / emission at 360 / 620 nm, respectively). 


The calibration curve (Fig. 1) was generated using repeated assays of five 

concentrations of the HSA standard (1.6, 4, 8, 20 and 100 μg/ml in Pi) which are around 

the threshold value of 20 μg/ml HSA in urine indicating the clinical complications. The 

concentrations of HSA in the analyzed urine samples were determined using this 

calibration curve, significant matrix effects were not observed. However, the background 

level (fluorescence around 970) was significantly higher than zero; it was due to the nonspecific 

binding of the reagents during the assay. 

The concentrations of HSA in samples were compared with the values estimated by 

the common clinical procedure – the automatic turbidimetric analyser COBAS Integra 

800 (Fig. 2). Parameters of the developed method were: LOD 1.9 μg/ml (corresponding to 

90% fluorescence), IC50 5.5 μg/ml (corresponding to 50% fluorescence) and working range 

from 2.8 to 10.4 μg/ml (corresponding to 80% - 20% fluorescence). The total time of 

analysis was 120 min, which is not that much critical due to the parallel processing of 

samples in the microplates. 

The proposed assay of HSA using QD as labels seems useful for clinical application 

although further assay optimisation is required mainly to lower background fluorescence 

and to increase precision. Nephropathy becomes developed in 45% of the patients with 

insulin dependent diabetes mellitus and the onset of this disease is accompanied with 

slightly increased level of albumin in urine known as microalbuminuria. The 

concentration range typical for microalbuminuria is from 20 to 200 μg/ml in the 24-h 

specimens. In this respect, the reported analytical parameters of the presented FIA are 

sufficient to discriminate positive microalbuminuria samples. 

- 130 -

XI. Woorkshop 

of Physsical 


Fig.11 

Calibratioon 

curve of f the develooped 

fluore escence imm munoassay utilizing biotinylated 

b d 

QD. Parrameters 

of f the approoximating 

si igmoidal eq quation aree 

indicated d inside thee 

graph. 

Fig.22 

Correlatioon 

of the lev vels of HSAA 

in urine samples s det termined ussing 

the pro oposed FIAA 

method and the standard iimmunotur 

rbidimetric c assay (CCOBAS 

Int tegra 800). . 

Parametters 

of the linear 

regreession 

fit are e given insi ide the grapph. 


Biotinylatted 

QDs were w successsfully 

used d for fluore escence immmunoassay 

of humann 

serumm 

albumin in urine sa amples. Parrameters 

of f the fluore escence immmunoassay 

(LOD 1.966 

μg/mml 

and workking 

range 2.86-10.4 μμg/ml) 

are sufficient s fo or detectionn 

of slightly y increasedd 

levells 

of HSA inn 

urine (mi icroalbumiinuria). 

The e biotinylat ted quantumm 

dots are consideredd 

to bee 

generally applicable for fluoresccence 

affinity 

assays. 

- 131 - 

Brnoo



The work has been supported by the ESF OPVK grant CZ.1.07/2.3.00/09.0167 

"Nanobiotechnologies and biosensors for biointeraction studies" and by the Ministry of 

Education project no. LC06030 "Biomolecular centre". 

6. REFERENCES 

[1] Katz, E., Willner, I.: Angew. Chem. Int. Ed., 43 (2004), 45, 6042 

[2] Michalet, X., Pinaud, F.F., Bentolila, L.A., Tsay, J.M., Doose, S., Li, J.J., Sundaresan, G., Wu, A.M., 

Gambhir, S.S., Weiss, S.: Science, 307 (2005), 5709, 538 

[3] Resch-Genger, U., Grabolle, M., Cavaliere-Jaricot, S. Nitschke, R., Nann, T.: Nat. Methods, 5 (2008), 

9, 763 

[4] Goldman, E.R., Clapp, A.R., Anderson, G.P., Uyeda, H.T., Mauro, J.M., Medintz, I.L., Mattoussi, H.: 

Anal. Chem., 76 (2004), 3, 684 

[5] Peng, C., Li, Z., Zhu, Y., Chen, W., Yuan, Y., Liu, L., Li, Q., Xu, D., Qiao, R., Wang, L., Zhu, S., Jin, 

Z., Xu, C.: Biosens. Bioelectron., 24 (2009), 12, 3657 

[6] Ma, Q., Wang, X., Li, Y., Shi, Y., Su, X.: Talanta, 72 (2007), 4, 1446 

[7] Xia, Y.S., Zhu, C.Q.: Talanta, 75 (2008), 1, 215 

[8] Wang, G.L., Xu, J.J., Chen, H.Y., Fu, S.Z.: Biosens. Bioelectron., 25 (2009), 4, 791 

[9] Willner, I., Patolsky, F., Wasserman, J.: Angew. Chem. Int. Ed., 40 (2001), 10, 1861 

[10] Qian, Z., Bai, H.J., Wang, G.L., Xu, J.J., Chen, H.Y.: Biosens. Bioelectron., 25 (2010), 9, 2045 

[11] Chen, H., Li, R., Lin, L., Guo, G., Lin, J.M.: Talanta, 81 (2010), 4, 1688 

- 132 -


USING OF TEMPLATES BASE METHOD FOR 

FARICATION OF NANORODS STRUCTURES 

FOR ELECTROCHEMICALSENZING 

ELEMENTS 

Radim HRDÝ 1 , Marina VOROZHTSOVÁ 1 , Jana DRBOHLAVOVÁ 1 , Jana CHOMOUCKÁ 1 , 

Jan PRÁŠEK 1 , Petra BUSINOVÁ 1 , Jaromír HUBÁLEK 1 


Microelectronics, Technicka 3058/10, 616 00 Brno, Czech Republic 

Abstract 

The porous alumina attracts attention because of its self-ordered hexagonal structure. It 

can be used as a template nanosize structure, for many devices such as magnetic, 

electronic and optoelectronic. The aim of this method is preparation of the mask for 

electrodepositing nanowires directly on Si substrate. The presented technique without 

utilization of high-resolution electron-beam lithographs belongs to low-cost technology 

in the microelectronic industry. Anodic alumina has been prepared in several electrolytes 

by the anodization process and the characteristics of pore structures have been studied in 

different anodizing conditions. The thickness of aluminum film for anodization was 1-2 

um. The two methods for deposition of aluminum thin film on Si substrate are thermal 

evaporating and sputtering. The prepared alumina structures have 15-30 nm pore 

diameters and 30-110 nm interpore distances. 


Many techniques have existed for preparing nanostructures. Lithographic methods 

have the best chance under the control of formation and sizes of single samples of 

nanostructures, but they are not low cost technologies. A cheaper technique exists for the 

deposition of ordered hexagonal nanostructures; aluminum can be transformed to selfordered 

alumina pore structure by special conditions [1-3]. These qualities of aluminum 

are of interest to many scientists. The purpose of this method is creating a template for 

electro deposition nanowires directly on n-type Si substrate. Semiconductor, 

optoelectronic and sensor devices use this template without using thick aluminum sheet. 

We used 1-2 μm thin films of aluminum for the fabrication of the template. The purpose 

of this method is anodic oxidation of aluminum film which is anode [4]. Cathode is 

graphite electrode. Diffusion of ions of oxygen and aluminum through the alumina layer 

is controlled by an electric field. The speed of growth of alumina film is exponential to the 

relationship of the electric field intensity. Al3+ ions have moved through the oxide barrier 

and they have increased the thickness of the barrier. The array of hexagonal ordered pores 

is the result of this method. This technique is not a simple process. The first problem is 

deposition of homogenous aluminum film on Si substrate. We used two methods: 

sputtering and evaporating. Both methods have advantages and disadvantages [5]. 

- 133 -




Evaporatedd 

aluminuum 

film is homogenoous 

but it has bad adhesion. In compar rison, 

sputtered film has saatisfactory 

adhesion a buut 

sputterin ng has crea ated nanocrrystal 

struc ctures 

during depposition. 

Thhey 

avoid fabricating f a self-order red pore str ructure. A ffilm 

prepar red in 

this way should bee 

annealed in vacuuum. 

Theref fore, evapo orating hass 

been use ed in 

preferencee 

to sputtering 

and 100 

nm thiin 

Au film is used as s an intermmetalic 

laye er for 

improvingg 

adhesion. 

The seco ond probleem 

was fin nding cond ditions for r fabricatin ng an 

ordered poore 

templatte. 

Next op peration consists 

of fil lling the po ores with mmetal 

by el lectro 

depositionn 

method, in order to fabricatted 

nanow wires or na anotubes. TThin 

film gold 

electrodes with alummina 

templa ate are appplied 

like base b for dep posited nannoparticles. 

The 

fabricationn 

of nanommachining 

surface s deppends 

on va arious param meters likee 

temperatu ure of 

solution, ppH 

and currrent 

densit ty. Last opeeration 

is dissolving d the 

aluminaa 

template. As a 

result we obtained mmetal 

nano owires on ccomb 

electr rodes whic ch are objeect 

of addit tional 

research. 

Fig.1 Model 

of preparring 

ordere ed alumina template. 

2. EXPPERIMENTT 

To bbegin 

withh, 

high pur rity (99,99 

substrate iin 

vacuum. 

The alum minum film 

was degreeased 

in aceetone 

and cleaned in 

10 wt % ssulfuric 

aciid 

at 10 °C C temperat 

potenciosttatic 

mode. . The volta age was 22 

current vaalue 

decreaased 

by les ss than 1 m 

sample weere 

signalized 

by the depletion d of 

prepared ppores 

were opened an nd increased 

was 10 - 115 

nm andd 

the interpore 

dista 

applicationn 

of the oppened 

alum mina, the t 

nanowiress. 

The mettal 

used fo or experim 

(componennt 

of electrrolyte: 

6 g.L 

density off 

depositionn 

over the 

nanostructtures 

and tthe 

depositi 

was approox. 

50 °C. Inn 

the next 

Results it iis 

seen on FFig.2. 

. 

-1 99%) alumi inum was evaporated 

m was 2 μm m. Subseque ently, the a 

n deionized d-water. Th he sample w 

ture. Porou us alumina a film was 

2 V and cu urrent 5 mA. m After 3 

mA. This decline d and d change o 

f all alumin num for for rmation por 

d in 5% H3 3PO4 solutio on. The res 

ance was 30 – 50 nm. 

Concer 

template was w used fo or the elect 

ments on the t nanostr ructure gro 

of K[AAu(CN)2] 

an nd 2.32 g.L L 

total area of nanopores 

was usu 

ion time wwas 

10 secon nds. The te 

phase, thee 

filled tem mplate was 

-1 d on n-typ 

aluminum 

was placed 

s conducte 

35 minutes 

of colour o 

re alumina 

sulting diam 

rning, the 

trodepositio 

rowth was 

of H3BOO3). 

The cu 

ually 0.25 mA.cm 

emperature 

dissolved in 

2 pe Si 

layer 

d into 

ed in 

s, the 

of the 

a. The 

meter 

next 

on of 

gold 

urrent 

fo or Au 

e of plating bath 

n 5% H3PO O4 [6] 

- 134 - 

Brno

XI. Woorkshop 

of Physsical 


Fig.22 

SEM imagges 

of fabri icated gold nanowires s, on the left/ 

nanowirres 

which are a hold inn 

the alummina 

porous s templates, 

right / cro oss section of o nanowire res structure e. 

3. RESULTS 

AND DISCUSSIO 

D ON 

Several saamples 

of thin t aluminnum 

were prepared in i differentt 

condition ns but onlyy 

somee 

of them aare 

suitable e for fabricaation 

of na ano templat tes. It is neecessary 

to use a highh 

hommogenous 

suubstrate. 

Th he sputterinng 

method ds are not acceptable a for the deposition 

off 

thin aluminum films beca ause they crreate 

nanoc crystals in the aluminnum 

layer. The size off 

the nnanocrystalls 

grows wi ith time. Thhe 

layer thickness 

1-2 2 μm is mouulded 

by much m biggerr 

crysttals 

than inn 

thin layer r 100 nm. MMany 

defec cts can be found f betwween 

nanocrystals. 

Onn 

the ggrain 

bounndary, 

the current deensity 

prev vails to cur rrent densiity 

in nano ocrystal byy 

anoddization 

proocess 

and it i avoids crreating 

ord dered pore structures. . The solut tion of thee 

issuee 

is in usinng 

of CVD. . The alumminum 

laye er which was w depositeed 

by sput ttering wass 

turbiid. 

On the other hand, 

the aluuminum 

films 

which h have been 

prepare ed by thee 

evapporating 

tecchnique 

are a brilliant polish. 2 μm 

film is pr repared by overlaying g of a singlee 

layerr. 

Thereforre 

the nan nocrystals ddo 

not gro ow continu ually as theey 

do by sputtering. . 

Bordders 

betweeen 

nanocry ystals are evvident 

but they t do no ot affect thee 

anodizatio on process. . 

The pore structture 

is regu ular withouut 

defects. This T anodized 

aluminaa 

is useful for f using ass 

a maask 

for depoosition 

orde ered nanowwires 

or for etching tec chniques. TThe 

lack of adhesion iss 

onlyy 

one problem 

caused d by evapoorating. 

Th he layers have h usuallly 

been cr racked andd 

severred 

by anoodization. 

The T solutioon 

of the is ssue is usin ng an interrmetalic 

Au u layer andd 

prehheating 

of thhe 

substrate e. The widtth 

and the density of created c nannostructure 

es are givenn 

by thhe 

templatee. 

The length 

of nanosstructures 

depends d on n the amounnt 

of depos sited metal, , 

thus the requirred 

length of o nanostruuctures 

can be achieve ed if the cuurrent 

dens sity (with a 

certaain 

limitattion 

of the 

diffusionn, 

electron n transfer, electrical potential, , chemicall 

potenntial, 

the pprocessing 

temperaturre 

which can c influen nce the moobility 

of io ons, crystall 

growwth, 

etc.) aand 

the tim me of electrrodepositio 

on are adeq quate. Metaal 

nanostru uctures aree 

fabriicated 

by ellectrodepos 

sition of thhe 

required metal into o the nanoppores 

of the e template. . 

Metaal 

ions are attracted to o the cathoode 

(conduc ctive substr rate of the template) leaving l thee 

insullant 

aluminna 

template e. 

- 135 - 

Brnoo


4. CONCLUSION 

The aim of this work is to find the conditions of the anodization process. The 

optimal conditions for anodization processes have been determined. The self-organized 

process of thin films is dependent on high purity and the homogenity of aluminum film. 


This research has been supported by Grant Agency of the Czech Republic under the 

contract GA 102/08/1546 and Grant Agency of the Czech Academy of Science, Czech 

Republic under the contract KAN 208130801. 

6. REFERENCES 

[1] Li X, Chin E, Sun H, Kurup P, Gu Z:. Sens Actuators, B 2010, 148:404-412. 

[2] Klosova K, Hubalek J: Phys Status Solidi A-Appl Mat 2008, 205:1435-1438 

[1] Mozalev A, Smith AJ, Borodin S, Plihauka A, Hassel AW, Sakairi M, Takahashi H: Electrochim Acta 

2009, 54:935-945. 

[4] Rabin O, Herz PR, Lin YM, Akinwande AI, Cronin SB, Dresselhaus MS: Adv Funct Mater 2003, 

13:631-638. 

[5] Hubalek J, Hrdy R, Vorozhtsova M: In Proceedings of the Eurosensors XXIII Conference. Volume 1. 

Edited by Brugger J, Briand D. Amsterdam: Elsevier Science Bv; 2009: 36-39: Procedia Chemistry 

[6] Hrdy R, Vorozhtsova M, Drbohlavova J, Prasek J, Hubalek J: Electrochemical transducer utilizing 

nanowires surface. In.; 2010: 510-514 

- 136 -


TRENDS IN CHEMICAL SENSORS 

Jaromír HUBÁLEK 1 

1 Laboratory of Microsensors and Nanotechnologies, Brno University of Technology, Technická 10, 616 00 

Brno 

Abstract 

Principals of chemical sensors have been widely developed during last two decades. 

Technology advancing brought many materials and structures involved into transducer 

which is the most important part of chemical sensors. The chemical sensing based on 

these materials where chemical reaction is transformed to physical quantity is most 

widespread and it can be called the first generation of chemical tranducers. As the second 

generation the optical principles can be called. In this case chemical properties are 

observed using light and the transducer contain light generator and detector as physical 

part of optical transducers. 


Chemical sensors are becoming more and more important in any area where the 

measurement of concentrations of volatile compounds is relevant for both control and 

analytical purposes. They have also found many applications in sensor systems called 

electronic noses and tongues. This chapter will first consider fundamentals of sensor 

science including a brief discussion on the main terms encountered in practical 

applications, such as: sensor, transducer, response curve, differential sensitivity, noise, 

resolution and drift. Basic electronic circuits employed in the sensor area will be discussed 

with a particular emphasis on the noise aspects, which are important for achieving high 

resolution values in those contexts where measurement of the lowest concentration values 

of chemicals is the main objective. All the most relevant transducers such as: MOSFET, 

CMOS, Surface Plasmon Resonance device, Optical Fibre, ISFET, will be covered in some 

detail including their intrinsic operating mechanisms and showing their limitation and 

performance. Shrinking effects of these transducers will also be commented on. The 

electronic nose and electronic tongue will be described as systems able to give olfactory 

and chemical images, respectively, in a variety of applications fields, including medicine, 

environment, food and agriculture. [1] 

Electronic nose’ systems involve various types of electronic chemical gas sensors 

with partial specificity, as well as suitable statistical methods enabling the recognition of 

complex odours. As commercial instruments have become available, a substantial increase 

in research into the application of electronic noses in the evaluation of volatile 

compounds in food, cosmetic and other items of everyday life is observed. At present, the 

commercial gas sensor technologies comprise metal oxide semiconductors, metal oxide 

semiconductor field effect transistors, organic conducting polymers, and piezoelectric 

crystal sensors. Further sensors based on fibreoptic, electrochemical and bi-metal 

principles are still in the developmental stage. Statistical analysis techniques range from 

simple graphical evaluation to multivariate analysis such as artificial neural network and 

- 137 -




radial bassis 

functionn. 

The int troduction of electronic 

noses into the aarea 

of food 

is 

envisaged for qualiity 

contro ol, process monitorin ng, freshn ness evaluaation, 

shel lf-life 

investigatiion 

and autthenticity 

assessment. a Considerab ble work ha as already bbeen 

carrie ed out 

on meat, ggrains, 

coffe fee, mushro ooms, cheesse, 

sugar, fi ish, beer an nd other beeverages, 

as s well 

as on the oodour 

qualiity 

evaluation 

of food packaging material. [2 2] 

The most relevvant 

contri ibutions inn 

the field of fiber-op ptic chemiical 

sensors s and 

biosensorss 

in the lastt 

five years s are reviewwed. 

Gas optodes o (inc cluding oxyygen, 

hydrogen, 

carbon diooxide 

and aammonia), 

humidity h seensors, 

mon nitors for pH, p cations and anions s, and 

sensors forr 

organic ccompounds 

constitutee 

the differe ent section ns. Optical fiber biose ensors 

based on eenzymes, 

anntibodies, 

nucleic n acidds 

and who ole microor rganisms seerve 

to illus strate 

the state-of-the-art 

in this active areea. 

Selecte ed examples 

of abbsorbance-b 

based, 

luminescent, 

evanesscent 

wave e, Fabry-Perot, 

chem miluminesce ent and suurface 

plasmon 

resonance-based 

senssors 

and biosensors, 

aamong 

othe er techniques 

used forr 

interrogat te the 

sensitive ppart 

of the ddevices 

are methods using 

in analysis. 

[3] 

The main typess 

of modern n multisenssor 

systems s of the elec ctronic tonggue 

type as s well 

as the sennsitive 

materials 

and sensors (trransducers) 

) are also commonly c used analy ytical 

applicationns 

as recognnition 

and classificatioon 

of variou us liquid media, m quanttitative 

ana alysis, 

process moonitoring 

and 

taste ass sessment off 

foodstuffs. 

[4] 

2. PRINNCIPAL 

MMETHODS 

S 

Princciples 

utilizzed 

for che emical sensoors 

are resi istive transd ducers - MOOS 

(metal oxide 

semiconduuctor), 

and CP (Condu ucting Polyymers), 

grav vimetric transducers 

– SAW (Su urface 

Acoustic WWave) 

and BAW (Bulk 

Acoustic Wave), it is the same e as QCM ( Quartz Cou upled 

Microbalances), 

semiiconductor 

transducerr 

– MOSFE ET (Metal Oxide O Semiiconductor 

Field 

Effect Traansistor) 

seee 

Fig.1. Ele ectrochemiical 

transdu ucer – usin ng electrodees 

with sol lid or 

liquid eleectrolytes 

and stand dards metthods 

as potentiom metric, volttammetric 

and 

amperomeetric. 

Last pprincipals 

use u optical transducers 

(Fig.2) - hollow h wavveguides 

HWGs H 

(hollow wwaveguidess), 

SPR (Surface ( PPlasmon 

Resonance) R see Fig.33, 

fluoresc cence 

transducerrs 

– optical transducer r with chemmiluminisce 

ent layer, quantum q doots, 

fluoresc ceins, 

etc. 

Resitivve 

QCM ( (BAW) 

Fig.1 Princcipal 

transdducers 

using g sensing mmaterials 

- 138 - 

SAW 

Brno 

FET 

F

XI. Woorkshop 

of Physsical 


Fig.33 

Sensing paart 

of optical 

transduccer 

using SP PR method 


The trendds 

are going g to advancce 

today kn nown princ ciples usingg 

new mate erials to bee 

miniiaturized 

innto 

very small 

chemicaal 

analyzers s of very wide 

spectruum 

of specie es. 

5. 

[1] 

[2] 

[3] 

[4] 

REFERENNCES 

Figg. 

2 Optical 

transduce er 


MENT 

The workk 

has been supported by project t GACR 10 02/08/1546 (NANIME EL) and thee 

framme 

of researcch 

plan MS SM 00216300503. 

Orsini A., DD´Amico 

A., Conference AAdvances 

in sensing s with security Appplications, 

July y 2005, Italy, , 

1-26 

Schaller E., et al.: Lebens sm.-Wiss. u.- Technol., 31 (1998), 305–3 316 

Current Annalytical 

Chem mistry, Vol. 4 (2008), 273-2 295 

Yuri G Vlassov 

et al 2006 Russ. Chem. . Rev. 75 (200 06), 1-125 

- 139 - 

Brnoo


NANOTECHNOLOGY FOR SENSORS AND 

DIAGNOSIS 

Jaromír HUBÁLEK 1 , René KIZEK 2 

1 Laboratory of Microsensors and Nanotechnologies, Brno University of Technology, Technická 10, 616 00 

Brno 

2 Laboratory of Metalomics and Nanotechnoogies, Mendel University in Brno, Zemědělská 1, 613 00 Brno 

Abstract 

Nanotechnologies are going to spread in many disciplines including sensors and devices 

for diagnosis in medicine because of promising properties advancing of small integrated 

devices. Usually increasing sensitivity and specificity is expected. Also level of integration 

can be increased, it means the smaller and smaller devices can be prepared for common 

use not only in specialized laboratories. Different nanomaterials are nowadays used as 

nanopillars, nanowires and nanotubes mostly prepared template based method on 

substrates, and colloidal nanoparticles for in vitro or in-vivo applications in analysis. 

Nanopillars or nanotubes are usually used in nanostructuring of sensing part of chemical 

sensors. Quantum dots or magnetic nanoparticles are used for bio-probe employing as 

separation tool or imaging tool in DNA, RNA or another desired biological material 

analysis. 


Large scale of nanoparticles can be used for detection of important molecules 

including quantum dots, magnetic beads, carbon nanotubes or metal nanostructures such 

as nanorods, nanowires and nanotubes formed on various solid supports (electrodes). 

These nanostructures are particularly convenient for bio/chemosensing purposes because 

the sensors show significantly higher sensitivity due to increasing of active sensing surface 

and in addition often modified/functionalized for desired purpose. The most important 

advantage of this techniques of nanostructures is its low cost [1]. Carbon nanotubes 

(CNTs) belong to the most promising nano-materials, because they show unique 

electronic, mechanical and chemical properties [2] that lead to many applications. 

The magnetic nanoparticles can be conjugated further with biologically active 

compounds in order to use them in controlled drug delivery, as agents in magnetic 

resonance imaging as well as for magnetic-induced tumour treatment via hyperthermia. 

Namely, streptavidin protein is one of the functionalization agent which can be attached 

to magnetic nanoparticle surface for its special affinity to vitamin biotin and hence for 

detection of diverse biomolecules in immunoassays and DNA hybridization procedures 

[3]. Viral infections pose a threat for mankind [4]. Certain viruses such as Human 

Immunodeficiency Virus (HIV) or influenzas viruses (mainly influenza A virus subtype 

H5N1) can evoke pandemics with high mortality. The time is very important in these 

cases of pandemics because the rapid detection can safe many lives. This magnetic bioprobe 

can be smart tool obtain this property of rapid technique. 

- 140 -

XI. Woorkshop 

of Physsical 


Quantum dots, which 

also fouund 

usage for biomed dical intenttions, 

can be utilizedd 

eitheer 

in colloiidal 

phase mainly as labels in fl luorescence e imaging oor 

in epitaxial 

grownn 

formm 

as biosennsors 

for in n situ detecction 

of bio omolecules s [5]. Geneerally, 

they y consist off 

semiiconductorss 

like CdSe e, CdTe, CCdHgTe, 

bu ut most fre equently thhey 

are fab bricated ass 

core/ /shell mateerial, 

e.g. CdSe/ZnS. 

Since 

they are a intende ed to be useed 

in biosy ystems, twoo 

key pparameters 

must be sa atisfied: no toxicity and 

solubility y in aqueouus 

medium. 

2. NANOSTTRUCTUR 

RES AND TTHEIR 

AP PPLICATIO ONS 

Results annd 

experien nces obtainned 

in nano otechnology y using in ssensing 

are e nowadayss 

very y wide. Thhe 

non-lit thography template- -based tech hniques of electrod de surfacess 

nanoostructuringg 

were dev veloped [6] . Nickel na anopillars as a example are shown n in Fig.1a, , 

gold nanotubes in Fig.1b, CNTs C in Figg.1c. 

aa) 

Fig. 1 Nickel naanopillars 

(a a), gold nannotubes 

(b) , carbon na anotubes (CCNTs) 

(c). 

The nanoostructures 

on electroode 

can be 

used for r increasingg 

specific affinity orr 

selecctivity 

moleecules 

in diagnosis d inn-vitro 

from m human fl luids e.g. uurease 

[7] or o it can bee 

b) b 

- 141 - 

c) c 

Brnoo




covered bby 

oxide naanoparticle 

es for gas ddetection 

[8], [ where significanttly 

increase e the 

sensing arrea 

resultinng 

to highe er signal ouutput 

and lower l detec ction limitts 

regarding g S/N 

ration risinng. 

The carbon eleectrode 

wa as substitutted 

by car rbon nanot tubes (CNTTs) 

[9]. Sui itable 

using andd 

modificattion 

of CN NTs deposiited 

on electrode 

ca an enhancee 

the dete ection 

properties. 

Patterninng, 

sprayin ng with oorganic 

vehicle, 

dire ect grow iin 

PECVD D are 

nowadays used for deeposition. 

CNTs C are ussed 

in sensi ing pure or r functionallized 

to inc crease 

specific seensitivity 

ffor 

exampl le in case of heavy metals an nd other ttoxic 

substa ances 

detection [10]. 

Fig. 2 Quaantum 

dotss 

functiona alized by biotin-glutat 

thione and d streptaviddin 

as bio-p probe 

for labelling oof 

DNA. 

Nowwadays 

in medical diagnosis d nnew 

approaches 

emp ploying naanoparticles 

s are 

utilized. QQuantum 

doots 

are obje ective of thhe 

research h as bio-pro obe with seeveral 

func ctions 

[11]. Currrent 

way is focuse ed on labbeling 

for r in-vivo imaging. Technique es of 

functionallization 

of quantum dots by biiotinated 

glutathione g were alsoo 

developed 

for 

sensing appplications 

[12]. Their r principal l function is i presented 

in Fig. 22. 

Another very 

interestingg 

applicatioon 

of nan noparticles is using modified m paramagnet p tic particle es for 

separationn 

of desiredd 

molecules 

[13] fromm 

body flu uids brings perspectivee 

tool for rapid 

techniquess 

of virusees, 

bacteria 

or interresting 

biomolecules 

detection [14]. Also o the 

technique can be used 

for heavy y metal deteection 

[15] and drug delivery d [166]. 


The paper deaals 

about techniquess 

and usin ng of new nanomateerials 

in se ensor 

constructioon 

and in medicine applicationns. 

Metal nanostruct tures verticcally 

aligne ed to 

surfaces oof 

electrodes 

have been 

foundd 

to be su uitable in chemical c ssensing. 

Se everal 

techniquess 

can be ussed 

for incr reasing of ssensitivity 

as a modifica ation and fuunctionaliz 

zation 

of nanostrructures 

to iincrease 

th he affinity too 

detected ions or molecules. 

Quuantum 

dot ts and 

magnetic nanoparticcles 

were shown as very prom mising tool ls in mediccine 

as in n-vivo 

diagnosis aand 

drug deelivery. 


T 

The work has been supported 

by pproject 

GAA AV KAN20 08130801 ( (NANOSEM MED) 

and the fraame 

of reseearch 

plan MSM M 00216630503. 

- 142 - 

Brno


5. REFERENCES 

[1] Shingubaras S., Journal of Nanoparticle Research, ISSN: 1388-0764, Vol. 5, No. 1-2 

[2] Ajayan P.M.; Chem. Rev., 99 (1999), pp 1787-1799. 

[3] Liu H.L., Sonn C.H., et al., Biomaterials. 29 (2008) 4003-4011. 

[4] Gessain A. and Calattini S., Future Virol., 2008, pp. 71-81. 

[5] Drbohlavova J., Adam V., Kizek R., Hubalek J., Int. J. Mol. Sci. 10 (2009) 656-673 

[6] Klosova, K., Hubalek, J., physica status solidi Vol. 205, No. 6, 2008, pp. 1435 – 1438 

[7] Hubalek J., et al., Sensors Vol. 7, No. 7, 2007, pp. 1238-1255 

[8] Drbohlavova, J.,et al., Chemické listy Vol. 102, No. 15, 2008, pp. S1043-2 

[9] Prasek J., et al., IEEE Sensors Vol. 1-3, 2006, pp. 1257-1260 

[10] Majzlik P., et al., Listy cukrovarnicke a reparske Vol. 126, No. 11, 2010, pp. 413-414 

[11] Drbohlavova, J., et al., International Journal of Molecular Sciences Vol. 10, No. 2, 2009, pp. 656 – 673 

[12] Chomoucka J., 32nd International Spring Seminar on Electronics Technology, 2009 pp. 653-657 

[13] Drbohlavova J., et al., Sensors Vol. 9, No. 4, 2009, pp. 2352-2362 

[14] Kukacka, J., et al., Clinical Chemistry Vol. 55, No. 6, 2009, pp. A42 

[15] Huska D., et al., FEBS Journal Vol. 276, 2009, pp. 281-281 

[16] Chomoucka, J.; Drbohlavova, et. al., Pharmacological Research Vol. 62, No. 2, 2010, pp. 144 – 149 

- 143 -


ELECTROCHEMICAL ANALYSIS OF DNA 

AND CADMIUM IONS INTERACTION 

Dalibor HŮSKA 1 , Jan SLAVÍK 2 , Vojtěch ADAM 1 , Libuše TRNKOVÁ 3 , René KIZEK 1 

1 Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, 



University of Technology, Kolejni 4, CZ-612 00 Brno, Czech Republic 

3 Department of Chemistry Faculty of Science, Masaryk University, Kotlarska 2, CZ-611 37 Brno, Czech 

Republic 

Abstract 

Cadmium (Cd) is a highly toxic environmental pollutant dangerous especially due to the 

ability of accumulation. Cd is emitted to the environment mostly by metallurgy, mining 

industry and production of nickel-cadmium batteries. The intoxication is caused mostly 

by cigarette smoke, foodstuffs and air pollution. The connection between cadmium and 

prostate, lung and testicles cancer as well as kidney and liver failure, lung oedema and 

spondylomalacia was proven. (Tokumoto 2011). 

Cd also generates reactive oxygen forms, damage DNA influence the function of proteins 

connected with an antioxidant defence of the organism. This work is focused on 

monitoring of the interactions between Cd ions and chicken genome DNA by square wave 

voltammetry on hanging mercury drop electrode. Significant signal decrease of 25% and 

80% was observed in DNA exposed to Cd ions for 1 hour and 3 days, respectively. 


Cd toxicity was observed already in 1955 in Japan (Itai-itai dinase) and negative 

impacts on human health were proven since then. The most serious effects of Cd on 

human health include immune failure, osteoporosis, prostate and lung cancer and/or 

kidney and liver failure. Also DNA damage and gene expression disruption as well as 

influence of antioxidant proteins are other impacts of Cd exposure. At the cell level Cd 

influence the progresses in cell cycle and methylation inhibition. The effects on DNA 

synthesis and cell proliferation are dependent on Cd concentration. It was found that 

already at 1 μM concentrations these processes are inhibited. However at lower 

concentrations the DNA synthesis and cell proliferation is stimulated. The main effect of 

Cd on DNA is in creation of single strand breaks (SSB) and chromosomal aberrations, 

sister chromatid exchanges and DNA–protein binding failures in several types of 

mammalian cells (Bertin a Averbeck, 2006, Huska, a kol., 2009, Palecek a Jelen, 2002, 

Tokumoto, a kol., 2011). The aim of this work is to monitor the interaction between DNA 

and Cd ions by electrochemical methods. 

- 144 -

XI. Woorkshop 

of Physsical 




me easurementts 

were performed 

w 

connnected 

to 7446 

VA Trac ce Analyzerr 

and 695 Autosample A 

a standard 

cell with three electrodes s and cooled d sample ho 

dropp 

electrode (HMDE) with w a droop 

area of 0.4 mm 

Ag/AAgCl/3M 

KKCl 

electrod de was the reference 

electtrode. 

GPPES 

4.9 suppliedd 

by 

EcoCChemie 

waas 

employe ed. The annalysed 

sampples 

were deoxygen nated prioor 

to 

softwware 

measuurements 

by b purgingg 

with 

argonn 

(99.999% %) saturated d with watter 

for 

120 s. Electrocchemical 

measuremen 

m nt An 

acetaate 

buffer ( (0.2 M CH H3COOH + 0.2 M 

CH3CCOONa; 

pH5) was w used for 

electtrochemical 

measure ement. Chhicken 

genoome 

DNA aand 

Cd(NO3 3)2 were ussed 

for 

expeeriments. 

2 with 747 VVA 

Stand instrument i t 

r (Metrohmm, 

Switzerland), 

usingg 

older (4 °C) ). A hangin ng mercuryy 

was w the wo working elec ctrode. Ann 

and glassy carbon eleectrode 

wa as auxiliaryy 

3. RESULTS 

AND DISCUSSIO 

D 

First, the dependenc cy of the si 

was iinvestigated. 

Signal in ncreased int 

range 

from 0,2 to 6,25 μg/ ml was obe 

3.48224 

with ccoefficient 

(Fig. 1). 

R2 ON 

ignal response 

on the concentraation 

of chi icken DNAA 

the concen ntration range 

0.2 – 50 0 μg/ml and d the linearr 

erved. The calibration n curve equaation 

is: y = 193.64x + 

of 0.9975 

Based onn 

these ex xperimentss 

DNA 

conccentration 

of 3 μg/ml l was chossen 

for 

furthher 

expeeriments. 

Cd(NO3) )2 at 

conccentratios 

oof 

1 μM, 5 μM and 10 μM 

was used. All of these Cd C concenttrations 

decreeased 

the signal 

of DN NA for abouut 

35% 

in 600 

minutes oof 

interactio on (Fig. 2). 

Fig. 22. 

Interacti ion of chick ken DNA wwith 

Cd2+ af fter 60 min. . 

The signiificantly 

hi igher decreease 

in the e DNA sign nal was obbserved 

afte er 72 hourr 

expoosure 

to thee 

Cd ions. One O μM Cd ions caused 

80% decr rease in thee 

signal in comparison c n 

- 145 - 

Brnoo 

Fig.1. Calibration 

cuurve 

of chic cken DNA. .




to pure DNNA. 

The deecrease 

was 60% and 990% 

in case e of 5 μM Cd C and 10 μμM, 

respect tively 

(Fig. 3). 

Subssequently 

tiime-depend 

dency of DDNA 

signal in presence 

of Cd ionns 

was expl lored. 

Twelve meeasurementts 

of the DN NS signal innteracting 

with 5 M Cd ions wwas 

perform med in 

6-minute intervals. The decrea ase of 10-115% 

was observed o be etween 5 aand 

30 mi inute. 

Between 330 

and 70 mminute 

the e signal deccreased 

to the 50% of 

the originnal 

value. In I 72 

hours the signal of onnly 

20% of the originaal 

value was s observed (= 80% deccrease) 

The impact of tthe 

Cd ions 

on a shorrt 

nucleotid de (GAAGG GAACAGGGACAAGCT 

TGC) 

was also thhe 

interest. 

Therefore e the same experimen nt as describ bed previouusly 

was ca arried 

out. Besidees, 

no decreease 

in the DNA signaal, 

but even n about 10% % increase wwas 

observe ed. 

Fig. . 3. Interact tion of chiccken 

DNA with w Cd2+ after a 72 h. 


Fromm 

the obtainned 

results s follow thaat 

electroch hemical methods 

suchh 

as square wave 

voltammettry 

using hhanging 

me ercury dropp 

electrode are able to o monitor cchanges 

in DNA 

signal caussed 

by the toxic eleme ents such aas 

cadmium m It can be concluded c that Cd res strain 

the adeninne 

and cytoosine 

reduct tion on thee 

electrode and therefo ore lower ccurrent 

resp ponse 

is detectedd. 

On the other hand d it is inteeresting 

tha at such a decrease d inn 

the signal l was 

observed iin 

the anaalysis 

of th he chicken genome DNA D but not n in the analysis of o the 

synthetic oligonucleootide, 

wher re the signnal 

even inc creased. Th his phenommenon 

is no ot yet 

clear and rrequires 

furrther 

invest tigation. 


T 

The work has bbeen 

suppor rted by NAANIMEL 

GA A ČR 102/08/1546. 

- 146 - 

Brno


6. REFERENCES 

[1] BERTIN, G.; AVERBECK, D. Cadmium: cellular effects, modifications of biomolecules, modulation of 

DNA repair and genotoxic consequences (a review). Biochimie, 2006, roč. 88. č. 11, s. 1549-1559. ISS 

0300-9084. 

[2] HUSKA, D.; ADAM, V.; BABULA, P.; HRABETA, J.; STIBOROVA, M.; ECKSCHLAGER, T.; 

TRNKOVA, L.; KIZEK, R. Electroanalysis, 2009, roč. 21. č. 3-5, s. 487-494. ISS 1040-0397. clanek IF 

2.630 

[3] PALECEK, E.; JELEN, F.. Crit. Rev. Anal. Chem., 2002, roč. 32. č. 3, s. 261-270. ISS 1040-8347. 

[4] TOKUMOTO, M.; OHTSU, T.; HONDA, A.; FUJIWARA, Y.; NAGASE, H.; SATOH, M. Journal of 

Toxicological Sciences, 2011, roč. 36. č. 1, s. 127-129. ISS 0388-1350. 

- 147 -


FABRICATION OF COPPER 

MICROPARTICLES BASED WORKING 

ELECTRODES FOR ELECTROCHEMICAL 

DETECTION OF ADENINE 

Jana CHOMOUCKÁ 1 , Jan PRÁŠEK 1 , Petra BUSINOVÁ 1 , Libuše TRNKOVÁ 2 , Jaromír 

HUBÁLEK 1 

1 LabSensNano, Department of Microelectronics, Faculty of Electrical Engineering and Communication, 

Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno, Czech Republic 

2 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, CZ-625 00 Brno, Czech 

Republic 

Abstract 

This paper is focused on the preparation and characterization of Cu2O nanoparticles via 

simple wet chemical route. Samples were characterized by SEM and XRD. These 

nanoparticles were used for fabrication of spraying working electrodes for electrochemical 

detection of purine bases in DNA. 


Cuprous oxide (Cu2O) is a p–type metal oxide semiconductor with a direct band gap 

of 2.0–2.2 eV. It has attracted increasing interest due to its promising application in 

magnetic devices, solar energy conversion and catalysts [1]. 

New types of solid electrodes are necessary for small device technologies contrary to 

standard electrochemical analysis, where mercury drop electrodes are commonly used. 

The performance of solid electrode is determined by its surface modifications to make it 

sensitive and selective towards a certain analyte, to obtain either chemically or 

biochemically modified electrodes [2]. Solid electrodes can be fabricated by thick–film 

technology (TFT) process. The advantage of TFT is its flexibility, low production costs, 

good reproducibility and good electrical and mechanical properties of electrodes. 

2. EXPERIMENT 

Cu2O microparticles preparation 

The preparation method of Cu2O nanoparticles is based on the procedure reported in 

[3]. Nanoparticles were prepared by two-step synthesis. At first, 0.0035 mol 

Cu(CH3COO)2·H2O was dissolved in 100 mL absolute ethanol under ultrasonic to form the 

deep green solution. The solution was heated to 60 °C. Than 50 mL glucose aqueous 

solution (0.1 mol/L) was slowly added into the solution under vigorous stirring. After 

that, 50 mL NaOH aqueous solution (0.5 mol/L) was also added to the solution at the same 

speed. Then some light yellow precipitates was occurred. The particles in the suspension 

were then separated by centrifugation at 4000 rpm for 8 min. The particles were 

- 148 -


resuspended in absolute ethanol followed by distilled water. The centrifugation was 

repeated thrice to remove CH3COO − , glucose and NaOH. The light yellow precipitate was 

then dried under nitrogene atmosphere overnight. 

Electrodes fabrication 

Working mikroelectrodes were fabricated using standard thick-film technology 

process on the alumina substrate. Thick-film pastes used for contact and covering layers 

were ESL 9562-G and ESL 4917 both from ESL Electroscience, UK. Carbon paste BQ 221 

(Dupont) was screen-printed over Ag/Pd/Pt based contact layer to avoid the contact 

layer to be present in electrochemical reaction. 

The working electrode was fabricated by spray coating deposition using Cu2O 

microparticles powder as the filling material N-Methyl-Pyrrolidone (NMP) as a carrier 

and binding material. For spray coating deposition 0.4 g of Cu2O microparticles with 10 

ml of NMP and the mixture was dispersed in an ultrasonic bath for 30 minutes. 

The spray-coating process was done using standard airbrush gun Fengda BD-208. To 

ensure good shape of the electrode a precise template was used for the deposition and the 

substrate was heated to 250 °C. The spray-coating process was repeated until the carbon 

layer was totally covered with the nanotubes to prevent later impact to electrochemical 

analysis. 

Electrochemical measurement 

Electrochemical measurements were performed with PalmSens handheld 

potentiostat/galvanostat (Palm Instruments BV, Netherlands). The device was connected 

to a personal computer for measurement setup and response evaluation. A three–electrode 

system was used, Cu2O electrode was employed as the working electrode, an Ag/AgCl/3M 

KCl electrode served as the reference electrode and Pt electrode was used as the auxiliary 

electrode. Cyclic voltammetry (CV) were carried out in the presence of 20 ml 0,2 M 

acetate buffer pH 5.0 and in the presence of various concentration of adenine. CV 

parameters: scan rate 100 mV/s, potential range -0.5 to 0.5 V. 


In our experiment, glucose was used as reductant. Reduction of Cu(II) tartrate 

complex by glucose can yield stable sols of cuprous oxide, whose particle size and 

morphology are strongly dependent on the concentration of reactants As shown in Fig. 1, 

sample has a spherical aggregation with a diameter of 1000 nm. But they are 

conglomerated by smaller particles with the mean size of 150 nm. According to the XRD 

measurement, we prepared Cu2O/CuO nanoparticles consisted of 70 % Cu2O and 30 % 

CuO. 

- 149 -




The electrocheemical 

oxid dation of aadenine, 

gu uanine or other puriine 

derivat tes at 

carbon eleectrode 

is wwell 

known n. Formationn 

of complexes 

of these 

compouunds 

with metals m 

including copper has 

been studied. 

Speciies 

Cu(II) can be red duced to CCu(I) 

and in n the 

presence of adeninee, 

Cu(I) re eacts with adenine to form in nsoluble ccompounds 

that 

accumulatte 

on the electrode 

sur rface [4] and 

cause dec creasing of current ressponse 

(Fig.2). 

Current [uA] 

200 

150 

100 

50 

0 

‐50 

‐100 

‐150 

‐0,66 

‐0,4 

Fig.1 SEM S image of Cu2O na anoparticle es 

1.2 2.10‐6 M Adde 

2.4 4.10‐6 M Adde 

4.8 8.10‐6 M Adde 

0 M Ade 

‐0,2 0 

PPotential 

[V] 

Fig.1 Cycliic 

voltammmograms 

rep presenting the electro ochemical behaviour b oof 

Cu2O working 

elecctrode. 

CVV 

conditions; 

100 mV/ /s scan rate e in potent tial range bbetween 

+0 0.5 V 

andd 

−0.5 V. 


Cu2OO 

microparrticles 

for preparatioon 

of thic ck film pa astes weree 

prepared and 

characterizzed. 

Prepaared 

microp paricles weere 

sprayed 

on prev viously preppared 

electrode 

substrate. These eleectrodes 

were w succeessfully 

used 

as the working electrodes s for 

electrocheemical 

detecction 

of ade enine. 

- 150 - 

0,2 

0,4 

0,6 

Brno



The financial support from the grant GAČR 102/08/1546 and the frame of Research 


6. REFERENCES 

[1] Zahmakiran, M., Ozkar, S., et al.: Mater. Lett., 2009, 63, 400-402. 

[2] Pravda, M., O'Meara, C., et al.: Talanta, 2001, 54, 887-892. 

[3] Huang, L., Peng, F., et al.: Solid State Sciences, 2009, 11, 129-138. 

[4] Trnkova, L., Zerzankova, L., et al.: Sensors, 2008, 8, 429-444. 

- 151 -


PREPARATION OF WATER SOLUBLE 

GLUTATHIONE-COATED CDTE QUANTUM 

DOTS 

Jana CHOMOUCKÁ 1 , Markéta RYVOLOVÁ 2 , Libor JANU 2 , Jana DRBOHLAVOVÁ 1 , 

Vojtěch ADAM 2 , René KIZEK 2 , Jaromír HUBÁLEK 1 

1 Department of Microelectronics, Faculty of Electrical Engineering and Communication, Brno University of 

Technology, Technicka 3058/10, CZ-616 00 Brno, Czech Republic 


613 00 Brno, Czech Republic 

Abstract 

In this paper, water soluble glutathione-coated CdTe quantum dots were prepared by a 

simple one step method using Na2TeO3 and CdCl2. Obtained QDs were separated from the 

excess of the glutathione by capillary electrophoresis employing 300 mM borate buffer 

with pH 7.8 as a background electrolyte. 


Quantum dots (QDs) are nanometer-sized crystals made of metallic or mostly of 

semiconductor materials with dimensions in the range of 2–10 nm. QDs are characterised 

by a number of unique physical and optical properties like strong light absorbance, sizetuneble 

emission, bright fluorescence, high quantum yield, narrow symmetric emission 

bands, high photostability and low photobleaching rates [1]. Their broad absorption 

spectrum allows the simultaneous excitation of QDs of all sizes by a single excitation light 

source in the UV to violet part of spectrum. QDs play an important role mainly in the 

imaging and as fluorescent probes for biological sensing [2]. The most popular types of 

QDs include CdTe, CdSe, ZnSe, ZnS, however also other semiconductor metals such as In, 

Ga, and many others can be used. Majority of sensing techniques employing QDs in 

biological systems are applied in solution (colloidal form) [3]. The organometallic way 

produces QDs, which are generally capped with hydrophobic ligands (e.g. 

trioctylphosphine oxide - TOPO or trioctylphosphine - TOP) and hence cannot be 

directly employed in bioapplications. Such hydrophobic QDs have to be transferred from 

the organic phase to aqueous solution, which is quite complicated procedure and often 

associated with the significant loss of quantum yield and stability. The second way is the 

aqueous synthesis route, producing QDs with excellent water solubility, biological 

compatibility, and stability. Thiol-capped QDs could be prepared directly in aqueous 

solution with thiols as efficient stabilizers. Mercaptopropionic acid and reduced 

glutathione (GSH) are the most popular coatings among thiols. GSH is not only an 

important water-phase antioxidant and essential cofactor for antioxidant enzymes, but 

also plays roles in catalysis, metabolism, signal transduction and gene expression [4]. GSH 

due to its key function in detoxification of heavy metals in organisms provides an 

additional functionality to the QDs. In addition, GSH-QDs exhibit high sensitivity to H2O2 

- 152 -


produced from the glucose oxidase catalyzing oxidation of glucose and therefore glucose 

can be sensitively detected by the quenching of the GSH-QDs florescence. Beside the 

application as simple sensors, QDs have much higher impact as unique fluorescent labels. 

Various specific labeling strategies are known and most of these approaches are based on 

bioconjugation with other biomolecule exhibiting some specific affinity to the target 

compound [5]. 

QDs can be applied in molecule tracking in immunochemistry, where they replace 

the fluorescent beads used for study of dynamics of neurotransmitter receptors. Due to 

their much smaller size compared to latex beads (approx. 500 nm), the lateral movement 

of individual receptor can be studied in great detail. Another example of QDs application 

is in genetic disease screening and diagnostics, where, in combination with stage-scanning 

confocal microscopy, they provide the imaging of QDs chromatic free aberrations, with 

resolution better than 10 vnm. Infrared QDs were also found to be useful probes for noninvasive 

detection in vivo, mainly inside small animals, where they can substitute 

conventional organic fluorophores emitting in the IR, which suffer from poor stability 

and quantum yield. 

2. EXPERIMENT 

Synthesis of glutathione coated CdTe QDs 

The procedure for synthesis of glutathione coated CdTe QDs was adapted from the 

work of Duan et al. [6]. Sodium telluride was used as the Te source. Due to the fact that 

sodium telluride is air stable, all of the operations were performed in the air avoiding the 

need for inert atmosphere. The synthesis of CdTe QDs and their subsequent coating were 

as follows: 2 mL of the CdCl2 solution (c = 0.04 mol/L) was diluted with 21 mL of water. 

During constant stirring, 50 mg of sodium citrate, 2 mL of Na2TeO3 solution (c = 0.01 

mol/L), 150 mg of GSH and 20 mg of NaBH4 were added into water-cadmium(II) solution. 

The mixture was kept at 95°C under the reflux cooling for 2.5 hours. As a result, yellow 

solution of the GSH-QDs was obtained. 

Capillary electrophoresis 

Synthesized GSH-QDs were analyzed by capillary electrophoresis (Beckman 

Coulter, PACE 5500) with absorbance detection at 214 nm and with the laser-induced 

fluorescence detection (Ar + , λex - 488 nm/ λem - 530 nm). Separation of the excess of B-GSH 

and GSH was carried out using uncoated fused silica capillary with 50 μm internal 

diameter and 375 μm b outer diameter. Total length was 47 cm and the effective length 

was 40 cm. Borate buffer (300 mmol/L, pH 7.8) was used as a background electrolyte. 


We prepared water soluble glutathione-coated CdTe QDs by a simple one step 

method using Na2TeO3 and CdCl2. The prepared GSH-CdTe QDs at 520 nm (Fig.1) and 

their emission spectrum showed quite symmetric and narrow shape. An inset in Fig. 1 

shows the solution of the GSH-QDs under the ambient light (left) and the fluorescence 

under the illumination by the UV lamp is shown on the right. 

- 153 -




Fig.1 Fluoorescence 

sspectrum 

of o GSH-CdTTe 

QDs. In nset: GHS-QDs 

underr 

ambient light 

(lefft), 

GSH-QD QDs under UV U light illuumination 

(right) ( 

The typical elecctropherog 

gram of the GSH CdTe e QDs solut tion is showwn 

in the Fig F 2. 

The identtification 

GSH signal 

was doone 

by th he standar rd additionn 

method and 

identificattion 

of the GGSH-QDs 

signal s was ddone 

by CE E-LIF. 

GSH 

Fig.2 Electtropherograam 

of GSH-QDs, 

UV detection at a 214 nm, LIF L detectiion 

(488 nm m/530 

nmm) 


It folllows 

fromm 

the results 

obtained that GSH 

synthesis of thiol sttabilized 

Cd dTe QDs. Obtained 

fluorimetrric 

detection 

with the e excitationn 

by Ar 

emission oof 

530 nm. 

+ is suitable coating forr 

the single e step 

QDs are of o good prooperties 

for 

the 

las ser at the wavelength w h of 488 nm m and 

- 154 - 

GSH‐QDs 

Brno





6. REFERENCES 

[1] Drummen, G.: Int. J. Mol. Sci., 11 (2010), 154-163. 

[2] Chomoucka, J., Drbohlavova, J. et al.: Procedia Engineering, 5 (2010), 922 

[3] Drbohlavova, J., Adam, V. et al.: Int. J. Mol. Sci., 10 (2009), 656 

[4] Liu, Y.F., Yu, J.S.: J. Colloid Interface Sci., 333 (2009), 690 

[5] Ryvolova, M., Chomoucka, J. et al.: Electrophoresis, 32 (2011), 1 

[6] Duan, J. L., Song, L. X. et al.: Nano Res., 2 (2009), 61 

- 155 -


APPLICATION OF CITP FOR BIOMINERAL 

ANALYSIS 

Zdeňka JAROLÍMOVÁ 1 , Přemysl LUBAL 1 

1 Department of Chemistry, Faculty of Science, Masaryk University, Brno 

Abstract 

This contribution concerns the possibility of application of capillary isotachophoresis 

(CITP) for analysis of biominerals concerning of less soluble compounds (calcium oxalate, 

hydroxyapatite). 


CITP is an analytical electromigration technique, which enables ion’s separation 

based on their different mobilities [1]. The goal of this work was to develop analytical 

method for simultaneous determination of anions such as oxalates and phosphates and 

their species which can be found in biominerals (e.g. renal stones). The developed method 

was utilized for the study of composition and solubility of those compounds in order to 

follow the experimental conditions driving their solubility. 

2. EXPERIMENTAL 

Method optimization for separation and determination of analytes was carried out 

on electrophoretic equipment EA 102 (Villa Labeco, Spišská Nová Ves, Slovakia) on PTFE 

capillary (diameter 0.3 mm, length 200 mm) joined with conductivity detector under 

laboratory temperature. The samples were analyzed under following conditions: 10 mM 

HCl + histidine (leading electrolyte, pH = 5.50), 10 mM hexanoic acid + imidazole 

(terminating electrolyte, pH = 7.00). 0.1% solution of hydroxyethylcellulose was added to 

eliminate electro-osmotic flow. 


The analytical procedure was optimized for simultaneous determination of mixture 

of anionic oxalate and phosphate species. Detection limits for anion determination were 

calculated from their calibration lines: phosphate 0.9 M, diphosphate 5.0 μM, 

triphosphate 1.9 μM, oxalate 0.7 μM. Then this procedure was applied for analysis of 

samples of renal stones (see Fig. 1) and it was shown that developed approach can 

distinguish the kind of stone consisting of calcium phosphate and calcium oxalate[2]. This 

approach was also employed for the study of solubility of these compounds (calcium 

oxalate, hydroxyapatite) which are present in biominerals [2]. 

- 156 -

XI. WWorkshop 

of Phyysical 

Chemists and Electrocheemists´11 

Fig. . 1: The exxample 

of ITP I recordd 

of renal stone s of ox xalate class. . The first zone is 

oxalate, 

the second d zone is phhosphate. 

4. CONCLLUSION 

This conntribution 

is i related tto 

biomine eral analysi is by meanns 

of CITP P which 

seemms 

to be mmore 

suitabl le alternativve 

to other r analytical l methods eemployed 

in i anion 

analysis 

(CZE, 

ionic chr romatograpphy). 

Also it can be used for study of physico- p 

chemical 

proceesses 

leadin ng to formaation 

of these 

biominerals. 

5. ACKNOOWLEDGE 

EMENT 

The worrk 

was sup pported byy 

Ministry of Educat tion of thee 

Czech Republic R 

(proojects 

ME009065 

and d LC060355) 

and Gr rant agenc cy of the Czech Republic R 

(2033/09/1394). 

. 

6. 

REFEREENCES 

[1] BBoček 

P., Demml 

M., Gebaue er P., Dolník V., Analytick ká kapilární iz zotachoforézaa 

(Analytical Capillary 

Isotachophhoresis), 

Acad demia, Praha 1987. 

[2] KKönigsberger 

E., Königsbe erger L.C., Biiomineralizati 

ion: Medical Aspects of SSolubility, 

Wi iley, New 

York 20066. 

- 157 - 

Brno


UTILIZATION OF FRACTIONAL 

EXTRACTION FOR CHARACTERIZATION 

OF THE INTERACTIONS BETWEEN HUMIC 

ACIDS AND METALS 

Michal KALINA 1 , Martina KLUČÁKOVÁ 1 , Petr SEDLÁČEK 1 

1 Brno University of Technology, Faculty of Chemistry, Centre for Materials Research 

CZ.1.05/2.1.00/01.0012, Purkyňova 464/118, 612 00 Brno, e-mail: xckalina@fch.vutbr.cz 

Abstract 

The aim of this work is the study of interactions between the humic acids and 

copper(II) in diffusion experiments. The diffusion experiments were combined with 

selective extractions of the diffused copper(II) ions. The results showed that the 

diffused ions are in humic gel presented in more forms according to the bond strength 

towards humic acids. Their distributions into the individual fractions developed in 

time and were stabilized. 


Humic acids (HA) are natural organic compounds, which can be found in soils, 

waters, sediments and coal. In previous works 0, 0, 0 easy diffusion experiments in 

humic hydrogels were described as a suitable approach for the study of reactivity of 

humic acids. Metals in nature may exist in different chemical forms and can be 

bonded on various matrices with different bond strength. For the determination of 

the individual ion fractions the application of selective extractions of the metals seems 

to be suitable. The aim of this work is the deeper exploration of the interactions 

between the HA and metals. 

2. EXPERIMENT 

The isolation of humic acids from South-Moravian lignite and preparation of 

humic hydrogel is listed elsewhere 0, 00. For the diffusion experiments prepared 

humic hydrogel was packed into the cylindrical glass tubes (length 3 cm, diameter 

1 cm). These tubes were sunk into the diffusion solution of 0.05M CuCl2. The chosen 

method for the diffusion experiments was the non-stationary diffusion from the 

solution with time variable concentration 0, 0. After passing the defined time of the 

diffusion experiments (1, 3, 5, 7, 9, 11, 14 and 20 days) humic gels were sliced and 

each slice was separately extracted with the leaching solutions. All slices originating 

from the gel of the same glass tube were leached with the same extraction solution. 

The used leaching solutions were water, 1M MgCl2 and 1M HCl 0. All prepared 

extracts were after diluting measured by the means of electrochemical analyzer 

EcaFlow 150 GLP. From obtained data the concentration profiles of copper(II) ions 

in the tubes and diffusion fluxes were calculated. These diffusion fluxes were 

- 158 -


compared to the diffusion fluxes calculated from decrease of the concentration of 

cupric ions in diffusion solution. 


Fig. 1 a) presents the concentration profiles of copper(II) ions in humic hydrogel 

for three fractions of diffused ions, which corresponds to three used leaching 

solutions. The concentration profiles have the same symmetrical shape with the 

minimum in the middle. The highest concentrations of cupric ions were obtained by 

extraction with 1M HCl, on the other side the lowest with water. The concentration 

profile for 1M MgCl2 lies between these two extremes. 

a) b) 

concentration of copper(II) ions 

(mol dm -3 ) 

0,06 

0,05 

0,04 

0,03 

0,02 

0,01 

0,00 

0 5 10 15 20 25 30 

distance from interface gel/solution (mm) 

- 159 - 

diffusion flux (mol.m –2 ) 

1,00 

0,80 

0,60 

0,40 

0,20 

0,00 

. 

water 1 M MgCl2 1 M HCl 

Fig.1 a) Concentration profiles of diffused ions extracted by water (black cirles), 1M 

MgCl2 (grey circles), 1M HCl (white circles), b) calculated diffusion fluxes of 

used fractions of diffused ions (colour identification of fractions is same as 

previous) 

It is assumed that water can leach out only free mobile fraction of diffused ions, 

1M MgCl2 can extract previous plus ion-exchangeable fraction and 1M HCl extracts 

all diffused ions 0. Fig. 1b) represents the comparison of diffusion 

fluxes of three above mentioned fractions of diffused ions. The fluxes are 

increasing in order water, 1M MgCl2, 1M HCl. The both data in Fig.1 a) and b) are for 

the duration of the diffusion 1 day. The concentration profiles and the diffusion fluxes 

for other duration of diffusion experiments show similar results. The differences can 

be found only in the shape of the profiles. With increasing duration of diffusions the 

profiles become more constant, from the duration 9 day the profiles were straight.


diffusion flux (mol.m –2 ) 

1,4 

1,2 

1,0 

0,8 

0,6 

0,4 

0,2 

0,0 

0 5 10 15 20 25 

square root of time (hour 0,5 ) 

Fig.2 Diffusion fluxes in dependence on square root of duration of the diffusions for 

ions extracted by water (black cirles), 1M MgCl2 (grey circles), 1M HCl (white 

circles) and calculated from decrease of concentrations in diffusion solutions 

(black triangles) 

According to the mathematical apparatus 0, 0 6the dependence of diffusion flux 

on the square root of time was constructed (Fig. 2). These results confirm that water 

extracts the least amounts of diffused ions – only the mobile fractions. 1M MgCl2 has 

higher affinity to cupric ions. It extracts higher amounts of diffused ions, which 

corresponds to mobile plus ion-exchangeable fractions. The highest affinity has 1M 

HCl, which leach out previous plus strongly bonded and residual fractions of diffused 

ions. The comparison of diffusion flux of fraction extracted with 1M HCl and the 

diffusion flux calculated from decrease of the concentration of cupric ions in diffusion 

solution shows that 1M HCl extracts at used concentrations all diffused ions. 

Simultaneously, the results of Fig. 2 show, that the dependence of diffusion fluxes on 

square root of time for extraction with water corresponds to the changes of diffusion 

flux for mobile fractions of ions during the experiments. The difference between the 

dependences for extraction with 1M MgCl2 and water corresponds to changes of 

diffusion flux of ion-exchangeable fraction of cupric ions and the difference between 

the dependences for 1M HCl and 1M MgCl2 corresponds to the changes 

of the diffusion flux of the strongly bonded and residual fraction of diffused cupric 

ions. The results also show the constant distribution of cupric ions between the three 

fractions, after passing a certain time. It indicates equilibrium between the fractions, 

which is created and then maintained during the experiments. 

4. CONCLUSION 

The combination of the diffusion experiments with the selective extraction of 

diffused ions showed to be suitable for the study of interactions, which take place 

between humic matrices and metals in diffusion experiments. The diffused ions were 

divided according to the bond strength towards humic acids. The results indicate that 

after passing a certain time the distribution between the fractions becomes constant. 

- 160 -


5. ACKNOWLEDGMENT 

This work was supported by the project “Centre for Materials Research at FCH 

BUT” No. CZ.1.05/2.1.00/01.0012 from ERDF. 

6. REFERENCES 

[1] Sedláček, P., Klučákova. M.: Geoderma, 153 (2009), 1–2, 286 – 292 

[2] Sedláček, P., Klučáková, M.: Collect. Czech. Chem. C., 74 (2009), 9, 1323 – 1340 

[3] Klučáková, M. Pekař M. Colloid Surface A, 349 (2009), 1-3, 96-101 

[4] Klučáková, M.: CHEMagazín, 3 (2004), 14, 9 

[5] Cranck, J. The Mathematics od Diffusion, 2 nd ed., Oxford: Claredon Press 1975 

[6] Tessier, A. et al.: Analytical Chemistry 51 (1979) 844 

- 161 -


MONITORING OF STRESS MARKERS IN 

MAIZE (ZEA MAYS L.) EXPOSED TO 

CADMIUM AND ZINC IONS 

Andrea KLECKEROVÁ 1 , Olga KRYŠTOFOVÁ 1 , Pavlína ŠOBROVÁ 1 , Natalia 

CERNEI 1 , David HYNEK 1 , Vojtěch ADAM 1 , René KIZEK 1 , 


Zemědělská 1, Brno, Czech Republic 

Abstract 

Heavy metals are classified as priority pollutants monitored in environments. Heavy 

metals are not biodegradable, having ability to accumulate in organisms. Cadmium is 

one of the most toxic heavy metal ions in the environment due to its high mobility 

and severe toxicity to the organisms. Zinc is an essential micronutrient for plants but 

can be highly toxic when present at excessive concentration. We aimed at 

investigation of detoxification mechanisms of maize plants (Zea mays L.) treated with 

the following combinations of zinc(II) and cadmium(II) ions 0 M Zn 2+ + 0 M Cd 2+ ; 

100 M Zn 2+ + 0 M Cd 2+ ; 0 M Zn 2+ + 100 M Cd 2+ ; 10 M Zn 2+ + 100 M Cd 2+ ; 50 M 

Zn 2+ + 100 M Cd 2+ ; 75 M Zn 2+ + 100 M Cd 2+ and 100 M Zn 2+ + 100 M Cd 2+ for 10 

days. Based on fresh weight of plants we observed the all treated experimental groups 

had decrease production of both aboveground biomass and root parts compared with 

control. In addition to growth parameters, we electrochemically determined the metal 

content in plants. 


Cadmium is one of the most toxic heavy metal in the environment due to its 

high mobility and severe toxicity to the organisms [1, 2]. Zinc is an essential 

micronutrient for plants but can be highly toxic when present at excessive 

concentration. Anthropogenic inputs of Cd and Zn to soils occur via short or longrange 

of mining, atmospheric depositions, and use of fertilizers/manures, municipal 

sewage-wastes, compost and industrial sludge. Cadmium and zinc are elements having 

similar geochemical and environmental properties. Zinc ores normally contain 0.1– 

5% of Cd, and the processing and subsequent release of Zn to the environment is 

normally accompanied by Cd [3]. 

Symptoms of phytotoxicity were reflected in concentrations much lower than 

that of other toxic metals. The most frequently reported symptoms of Cd toxicity 

include browning of root hairs and root tips of plants, reddish-brown tint, reddishbrown 

necrosis on young leaves and especially reduction of growth [4, 5]. Zinc plays 

an important role in the metabolism of plants, because they contribute to the 

activation of many enzymes and provides a link between enzyme and substrate. 

- 162 -


However, the surplus will limit plant growth, especially roots, turgor decrease and 

toxicity are similar chlorosis [6]. 

The association of cadmium and zinc in the environment and their chemical 

similarity can lead to interactions between these two ions, resulting in the lowering of 

cadmium toxicity. Cadmium is a non-essential ion and it is toxic at a lower 

concentration than zinc. Consequently, the uptake and translocation of zinc by plants 

is higher than that of cadmium. This antagonistic effect is due to the smaller ionic 

radius of Zn 2+ than the Cd 2+ [3, 4, 7]. 

Phytoremediation, i.e. the use of green plants to remove pollutants from the 

environment or to render them harmless has been proposed as an environment 

friendly and cost-effective technique for soil and water remediation. In case of heavy 

metal contaminated soil, the biological process of phytoextraction includes metal 

mobilization as well as acquisition and transport. Root system is the main interface of 

ion exchange between plants and their environment, thus in each process roots play a 

central role [8]. Excessive metal has marked effects on root growth of plants; however, 

there is less knowledge about the root morphological changes in hyperaccumulators 

after exposure to heavy metal stress. Various previous studies were limited to toxic 

effects of metal on the shoot with less attention to root system, and the data about 

toxic effects of heavy metal on hyperaccumulator roots were mostly limited to root 

biomass. Studies on the interactions between heavy metal conducted have been 

focused so far on the conventional plant species, little information is available about 

the interactions in the hyperaccumulator, especially on root morphological changes. 

In hyperaccumulators, root length determines the capacity to acquire water and 

nutrients, and therefore metal uptake capacity is more strongly related to root length 

than root weight. When looking at phytoextraction, not only quantitative root 

parameters (biomass) should be considered but qualitative root characteristics may 

also be looked into. Root length, surface area, length–diameter distribution and 

volume could serve as valuable parameters when describing and comparing root 

systems [9]. 

2. EXPERIMENT 

PLANT CULTIVATION 

Maize (Zea mays L.) CE 220 (hybrid) was used in our experiment. Five-day 

seedlings were placed in hydroponic culture vessels, which were available for seven 

days filled with Richter’s nutrient solution. After seven days, the contents of the 

containers were exchanged for a solution of zinc and cadmium with these 

concentrations: 0 μM Zn 2+ + 0 μM Cd 2+ ; 100 μM Zn 2+ + 0 μM Cd 2+ ; 0 μM Zn 2+ + 100 μM 

Cd 2+ ; 10 μM Zn 2+ + 100 μM Cd 2+ ; 50 μM Zn 2+ + 100 μM Cd 2+ ; 75 μM Zn 2+ + 100 μM Cd 2+ 

and 100 μM Zn 2+ + 100 μM Cd 2+ . For the preparation of chemical solutions of the 

metal ions, Zn(NO3)2 and Cd(NO3)2 were used (Sigma-Aldrich, USA). The experiment 

was conducted for 10 days under constant conditions (temperature 20°C, humidity 

65%, 12h light). Five plants were harvested from each experimental variant. 

Harvested plants were rinsed in distilled water, and then were divided into above- 

- 163 -

XI. Workshhop 

of Physical l Chemists and Electrochemists 

E 

s´11 

groundd 

part and rroots. 

The fresh f weighht 

of the sa amples was s measuredd 

on a Sarto orius 

scale immmediatelyy 

after rinsin ng. 

ELECTR 

E 

(EcoCh 

three e 

with w 

1 KCl) a 

(EcoCh 

mol/l-1 ROCHEMICCAL 

DETERMINATIONN 

OF CADM 

lectrochemmical 

meas surements were per 

hemie, Nethherland) 

with w VA-Staand 

663 (M 

lectrode syystem, 

whic ch consisteed 

of hangin 

working elecctrode 

surfa ace 0.4 mmm 

as referencee 

electrode e and platin 

hemie, Nethherland) 

wa as used for 

CH3COOHH 

+ CH3CO OONa) was 

corn keernels 

weree 

deoxygen nated by arg 

cadmiuum 

was mmeasured 

using u diffe 

(DPASVV). 

Anodicc 

scan was started at 

accumuulated 

on tthe 

HMDE E potential 

temperrature. 

The solution was w mixed 

importaant 

methodd 

parameters 

were: m 

modulaation 

amplittude 

49.5 mV. m 

2 MIUM 

rformed on o the deevice 

Aut 

Metrohm, Switzerland S d). It was u 

ng mercury y drop elecctrode 

(HM 

, silver-ch hloride elect trode (Ag / AgCl / 3 m 

num wire as 

auxiliary electrode. GPES softw 

processing raw data. Acetate A bufffer 

pH 3.6 

used as the 

supportin ng electrolyyte. 

Sample 

gon (99.999 9%) for 120 

s. The cooncentratio 

erential pu ulse anodic c strippingg 

voltamm 

-0.7 V and 

stopped at -0.4 V. Cadmium 

at 0.7 V with w a 120 s accumul ulation at r 

during exc cretion (145 50 rev minn 

odulations time 0.02 s, step pote 

-1 tolab 

used 

MDE) 

mol.l 

). Other u 

ential 1.05 

- 

ware 

(0.2 

es of 

on of 

metry 

was 

oom 

used 

mV, 

3. RRESULTS 

AND DIS SCUSSIONN 

In our studdy, 

we stud died the inffluence 

of different d co oncentratioons 

of cadm mium 

and zinnc 

ions on thhe 

growth of maize. 

m ( (g FW) 

days 

0 ZZn 

0 

Cd 

0 ZZn 

1000 

Cd 

Fig. 1a. 

Fi 

part an 

concen 

Cd2+ Growth cu 

irstly, we s 

nd roots of 

ntrations 0 μ 

; 100 

μM Zn 

and 10 

observe 

abovegr 

signific 

2+ + 

0 μM Zn2+ urve – above 

studied the 

maize. Ma 

μM Zn 

ed the all 

round biom 

cant growth 

2+ eground pa 

e influence 

aize (Zea m 

+ 0 μM Cd 

+ 100 μM C 

+ + 100 μM 

treated e 

mass and ro 

h inhibitio 

2+ ; 

Cd2+ ; 50 μM 

M Cd2+ arts Fig. 

e of metal i 

mays L.) we 

100 μM Zn 

Zn 

for 

experimenta 

oot parts c 

on was obs 

2+ 1b.Growth 

ions on the 

re exposed 

n 

+ 100 

10 days. B 

al groups 

compared w 

served in g 

2+ + 0 μM 

0 μM Cd2+ h curve – ro 

e fresh wei 

d to Cd 

; 7 

Based on fr 

had decre 

with contro 

groups in w 

2+ an 

Cd2+ ; 0 μM 

75 μM Zn2+ oots 

ight of gro 

nd Zn 

resh weigh 

ease produ 

ol (Fig. 1 a 

which we 

2+ ion 

Zn2+ ound 

ns in 

+ 100 0 μM 

+ + 100 μM Cd 

ht of plants 

uction of b 

a, b). The m 

combined 

2+ 

s we 

both 

most 

the 

- 164 - 

Brno


highest concentrations of Zn 2+ and Cd 2+ ions. At these plants were also observed 

procumbention of plants and chlorosis. 

In addition to growth parameters, we electrochemically determined the metal 

content in plants. It was found that content of both metal ions enhanced in 

aboveground parts and in roots during the experiment (Fig. 2a - d). 

ng/g DW 

ng/g DW 

2a 

350 

300 

250 

200 

150 

100 

50 

140 

120 

100 

80 

60 

40 

20 

0 

0 

0Zn 

0Cd 

0Zn 

0Cd 

0Zn 

100Cd 

0Zn 

100Cd 

2. day 10. day 

100Zn 

0Cd 

10Zn 

100Cd 

50Zn 

100Cd 

Applied concentration (M) 

100Zn 

0Cd 


10Zn 

100Cd 

50Zn 

100Cd 


75Zn 

100Cd 

75Zn 

100Cd 

100Zn 

100Cd 

Fig 2a, b. Zinc content in the aboveground parts (a) and roots (b) of maize plants in 

the 2 nd and 10 th day of cultivation 

100Zn 

100Cd 

- 165 - 

ng/g DW 

700 

600 

500 

400 

300 

200 

100 

0 

0Zn 

0Cd 

0Zn 

100Cd 

100Zn 

0Cd 


10Zn 

100Cd 

50Zn 

100Cd 


75Zn 

100Cd 

Fig 2c, d. Cadmium content in the aboveground parts (c) and roots (d) of maize plants 

in the 2 nd and 10 th day of cultivation 

ng/g DW 

1400 

1200 

1000 

2b 

800 

600 

400 

200 

0 

0Zn 

0Cd 

0Zn 

100Cd 

100Zn 

0Cd 


10Zn 

100Cd 

50Zn 

100Cd 


75Zn 

100Cd 

100Zn 

100Cd 

100Zn 

100Cd


As we can see in Fig. 2 a - d, plants accumulated more Zn 2+ than Cd 2+ , which as 

the essential element it isacceptable for them. We also determined a higher 

concentration of metal ions in root sections which shows on the fact that roots are the 

first plant tissue, which is in close contact with heavy metal ions. 

It was found that the group treated with 100 M Zn 2+ + 0 M Cd 2+ took less Zn in 

the second collection day on average 0.8 - 6 times and in the tenth day of collection 

0.1 - 1.1 times in aboveground parts and in the second day of sampling averaged 0.3 - 

4.8 times and in the tenth day of collection 0.7 - 2.2 times the root sections (Fig. 2a, 

b). In the case of Cd it was found that the groups treated with a combination of Zn 

and Cd had enhancing concentration of Cd with increasing concentrations of Zn in 

aboveground parts (Fig. 2c) in the second and tenth day of collection and root parts of 

the tenth day of sampling (Fig. 2d). An interesting finding in the experiment was that 

the presence of Zn in the culture solution increases the intake of Cd maize plants. 

Based on the comparison of results obtained from the second and tenth day of 

the experiment it can be concluded that despite the growth inhibition caused by 

metals, accumulation of Zn and Cd occurs in the aboveground parts and in roots. 

4. CONCLUSION 

The increasing amount of pollutants of various kinds and origin, 

environmental organisms leads to the activation of detoxification mechanisms. 

Learning how to adapt to different types of pollution is a very important task of 

modern analytical chemistry, biochemistry and molecular biology. 

Total inhibition of growth of maize plants was probably due to the activation of 

defensive reactions, preferably plant protection compounds synthesized, instead of the 

biosynthesis of substances necessary for growth. Furthermore, we can conclude that 

the inhibition of root part is probably associated with the intake of cadmium root 

system. In the plant, the content of metal ions was increasing with increasing time of 

cultivation. An interesting finding in the experiment was that the presence of Zn in 

the culture solution increases the intake of Cd by maize plants. 


This work was supported by grant IGA AF MENDELU TP 7/2011. 

6. REFERENCES 

[1] Das, P., S. Samantaray, and G.R. Rout, Studies on cadmium toxicity in plants: A review. 

Environmental Pollution, 1997. 98(1): p. 29-36. 

[2] Matusik, J., T. Bajda, and M. Manecki, Immobilization of aqueous cadmium by addition of 

phosphates. Journal of Hazardous Materials, 2008. 152(3): p. 1332-1339. 

[3] Chakravarty, B. and S. Srivastava, Effect of cadmium and zinc interaction on metal uptake and 

regeneration of tolerant plants in linseed. Agriculture Ecosystems & Environment, 1997. 61(1): 

p. 45-50. 

[4] Gabbrielli, R. and L.S. di Toppi, Response to cadmium in higher plants. Environmental and 

Experimental Botany, 1999. 41(2): p. 105-130. 

[5] Adriano, D.C., Trace elements in terrestrial environments: Biogeochemistry, bioavailability, and 

risks of metals. 2001, New York: Springer-Verlag. 

- 166 -


[6] Pandey, N., et al., Enzymic changes in response to zinc nutrition. Journal of Plant Physiology, 

2002. 159(10): p. 1151-1153. 

[7] Shrivastava, G.K. and V.P. Singh, Uptake accumulation and translocation of cadmium and zinc 

in Abelmoschus esculentus L. Moench. Plant Physiol. Biochem, 1989. 16: p. 17-22. 

[8] Marschner, H., Mineral Nutrition of Higher Plants. second ed. 1995, London: Academic Press. 

[9] Li, T.Q., et al., Effects of zinc and cadmium interactions on root morphology and metal 

translocation in a hyperaccumulating species under hydroponic conditions. Journal of Hazardous 

Materials, 2009. 169(1-3): p. 734-741. 

- 167 -


SYNDROM OF NEWLY FILLED RESERVOIR 

FROM THE MERCURY POINT OF VIEW 

Kamila KRUŽÍKOVÁ 1 , Renata KENSOVÁ 1 , Lenka GAJDOVÁ 1 , Zdeňka 

SVOBODOVÁ 1 . 

1 University of Veterinary and Pharmaceutical Sciences, Faculty of Veterinary Hygiene and Ecology, 

Department of Veterinary Public Health and Toxicology 

Abstract 

Želivka is a typical canyon-shaped reservoir. After imundation, unexpectedly high 

level of mercury was found in fish from Želivka. That is why the long-term 

monitoring of mercury contamination by some of most frequent kind of fish started. 

Monitoring is in progress from 1974. After flooding, there are a good and available 

conditions for microbial convertion (because of anoxic condition and decay of organic 

matter) of inorganic Hg to organic mercury which is accumulate into food chain. In 

the first years, the mercury level is high and then is gradually decline. It pointed to 

that mercury level in fish tissues is stabilized and do not exceed hygienic limit set by 

European Union. 


The accumulation of mercury in the fish tissues found in recently impounded 

reservoirs has been known for forty years (Brinkmann and Rasmussen, 2010). Because 

of the risks to human consumers of these fish, it is important to determine how long 

after impoundment this will continue to occur. In the manmade reservoir for drinking 

water, Želivka (Czech Republic), built from 1970 to 1974, the systematic investigation 

of bioaccumulation of mercury in fish from 1974 to 1997 and in 2009 was performed. 

2. EXPERIMENT 

In the Želivka reservoir, the main fish predator species were sampled and 

mercury concentration in the fish muscle and liver were analyzed using method of 

cold vapor atomic absorption spectrometry (AMA 254 analyzer). The data for Esox 

lucius, Perca fluviatilis, Sander lucioperca and Aspius aspius were evaluated. Muscle 

and liver of fish were analyzed. 


In the period 1974–1988 have been founded a quite high value (about 1 mg/kg) 

of mercury content in fish tissues. Although there is not any source of mercury 

contamination in the Želivka reservoir, about 55% of analyzed sampled (predatory 

species) exceeded 0.6 mg/kg. An expressive reduction have been found after twenty 

years and in 2009 following mercury content in fish tissues was determined most for 

Aspius aspius 0,197 mg/kg in the muscle. The hypothesis has been pronounced that in 

the newly filled reservoir are suitable physico-chemical and biological conditions for 

- 168 -

XI. WWorkshop 

of Phyysical 


change 

of anoorganic 

me ercury to oorganic 

mer rcury and their ingreession 

to the 

food 

chain. 

Mercuryy 

levels in fish tissues are establish 

in time. 

The resuults 

of merc cury contennt 

in the predator p fish 

(perch, aasp, 

pikeperch 

and 

pikee) 

muscle and liver r are shoow 

in Gra aph 1. Af fter floodiing, 

the mercury m 

conntaminationn 

was for up p to 5timess 

higher tha an hygienic c limit is (00.5 

mg/kg). As time 

go oon 

(11 yearrs 

after), mercury m conntamination 

n was decli ine to levell 

about 0.2 mg/kg) 

andd 

stood dowwn. 

It pointe ed to that mmercury 

lev vel in fish tissues 

is staabilized 

and d do not 

exceeed 

hygiennic 

limit set by European 

Union. 

Fig. . 1 Mercuryy 

and methy ylmercury content in perch and asp. 

Fig. . 2 Mercuryy 

and methy ylmercury content in pike perch h and pike. 

- 169 - 

Brno


4. CONCLUSION 

Very high level of mercury contamination was found in the muscle and liver of 

the sampled fish in spite of any mercury source (like chemic factory). After flooding, 

there are a good and available conditions for microbial convertion (because of anoxic 

condition and decay of organic matter) of inorganic Hg to organic mercury which is 

accumulate into food chain. In the first years, the mercury level is high and then is 

gradually decline. Our results are agreeable with data from literature. 

5. AKNOWLEDGEMENT 

The work has been supported by project MSM 6215712402 

6. REFERENCES 

[1] Brinkmann, L., Rasmussen, J.B.: High levels of mercury in biota of a new Prairie irrigation 

reservoir with a simplified food web in Southen Alberta, Canada. Hydrobiologia, 64 (2010), 11- 

22. 

- 170 -


UTILIZATION OF TOTAL ANTIOXIDANT 

CAPACITY TO EVALUATE THE EFFECT OF 

MULTIWALL CARBON NANOTUBES AND 

MAGNETIC NANOPARTICLES ON 

SUSPENSION TOBACCO CULTURE 

Sona KRÍŽKOVA 1 , Olga KRYŠTOFOVÁ 1 , Zuzana HRDINOVÁ 1 , Jiří SOCHOR 1 , Libor 

VYSLOUŽIL 2 , Ondřej JASEK 2 , Vít KUDRLE 2 , Vojtěch ADAM 1 , René KIZEK 1 

1 Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-613 00 


2 Department of Physical Electronics, Faculty of Science, Masaryk University, Kotlarska 2, CZ-611 37 


Abstract 

Nanotechnology and nanoscience are new areas of research and human cognition. 

The effects of nanomaterials on humans and the environment are still poorly 

understood. Because of their much larger surface there is a possibility to occur toxic 

effects unknown in “normal” materials. 

The aim of this work was to use the set of the methods for antioxidants capacity 

determination to compare the effect of multiwall carbon nanotubes (MWCNTs) and 

magnetic microparticles (MNPs) in concentrations 0, 1 and 10 μg/ml on suspension 

tobacco culture. After 7-day cultivation, compared to controls, the weight of the cells 

exposed to 10μg/ml MWCNTs was 3× higher. MNPs did not influence the cells 

growth, but they induced free radicals and synthesis of antioxidative compounds, 

especially thiols. In cells exposed to MNPs the total antioxidant capacity was 30 % 

increased compared to controls, in cells exposed to MWCNTs a slight decrease was 

observed. This indicates possible changes in whole plants metabolism. 


Due to the increasing utilization of nanoparticles and nanomaterials it is 

necessary to obtain data about their influence of living organisms. According to actual 

knowledge, commonly used tests of toxicity provide misleading information. 

Therefore new methods to evaluate the toxicity of nanomaterials and nanoparticles 

are necessary. 

Nanoparticles can be released into soil, air or ground waters. Then they can be 

either degraded, biotransformed or accumulated in the organisms and consequently 

along the food chain [1]. Plants are involved in all abovementioned events and 

moreover they are potential producers of nanoparticles [2]. Nanotechnology can be 

applied also in production of fertilizers. 

- 171 -


It is known, that nanoparticles have an effect on plants. Their action is 

dependent on the material, shape and size of the nanoparticles. The same type of the 

nanoparticles can have an adverse effect of different plant species. 

Oxidation stress is one of the possible effects of the nanoparticles both in 

animals and plants. Determination of total antioxidant capacity is a relatively cheap 

and non time-consuming method of toxicity evaluation. Using of suspension plant 

cultures is an alternative to the long-term whole-plant experiments. 

The aim of this work was to use the set of the methods for antioxidants capacity 

determination to compare the effect of multiwall carbon nanotubes (MWCNTs) and 

magnetic microparticles (MNPs) on suspension tobacco culture. 

2. EXPERIMENT 

Tobacco cells in suspension culture were cultivated in Murashige-Skoog 

medium with concentration of 0, 1 and 10 μg/ml MWCNTS (diameter 50 nm) or 

MNPs (40% / 60% (magnetid Fe3O4/magnetid gama Fe2O3) for 7 days in 25°C with 

shaking. After the cultivation the cells were peleted and stored in -80°C. For analysis 

0.1 g of the pellet was used. The sample was disintegrated in liquid nitrogen using the 

semiautomatic homogenizer Schuett homgenplus (Schuett-biotec, Germany) and 

resuspended in 0.1 M phosphate buffer pH = 7.5. After the disintegration the sample 

was vortexed for 30 min and centrifuged (16000 RPM, 30 min). The supernatant was 

then used for all following analyses: total protein determination by pyrrogal red, 

activity of glutathione-S transferase, total thiol compounds determination by Ellman’s 

method, DPPH, ABTS, DPMD and Blue CrO5 test. 


After the 7-day cultivation the cells were peleted and weighed. The growth 

curve was plotted from weight of the pellet and NPs concentrations. The growth of 

the cells exposed to MWCNTS was stimulated markedly. Compared to controls the 

weight of the cells exposed to 1 μg/ml MWCNTS the weight of the cells was 2 × 

higher and the weight of the cells exposed to 10μg/ml MWCNTS was 3× higher. At 

MNPs no influence on the cells growth was observed. 

In the cells the activity of the enzyme glutathione-S transferase was determined. 

This enzyme is involved in conjugation reaction in pollutants and ROS detoxication. 

This enzyme was markedly inhibited in cells exposed to MWCNTs, while in cells 

exposed to MNPs the activity of this enzyme was stimulated. Total thiol content, 

which is connected to the activity of GST was consistent with this observation, e. g. at 

carbon nanoparticels the content of total thiols was decreased and in ferric 

nanoparticles the total thiols content was increased. 

The total antioxidant capacity in cells exposed to ferric MNPs was 30 % 

increased and in cells exposed to carbon nanoparticles a slight decrease was observed. 

- 172 -


Total antioxinant activity [% of 

control] 

160 

140 

120 

100 

80 

60 

40 

20 

0 

MWCNTs MNPs 

0 1 10 

Concentration of nanoparticles [μg/ml] 

Fig.1 Comparison of total antioxidant activity in tobacco cells exposed to MWCNTs 

and MNPs 

Those data are consistent with previous results [3]. It is known, that both 

MWCNTs [4] and MNPs [5] induce free radicals. In cells exposed to MNPs the total 

antioxidant capacity was 30 % increased compared to controls, in cells exposed to 

MWCNTs a gradual decrease in total antioxidant capacity was observed. 

4. CONCLUSION 

From the obtained results flows, that the both MWCNTs and MNPs have a toxic 

effect on tobacco, but the mechanisms of the toxicity are different. After the 

exposition to MWCNTs the growth and metabolism of the cells was stimulated 

markedly, but detoxication pathways were inhibited. MNPs did not influence the cells 

growth, but they induced free radicals and synthesis of antioxidative compounds, 

especially thiols. The activity of glutathione-S transferase was also increased. This 

indicates possible changes in whole plants metabolism. 


The work has been supported by grant NanoBioTECell GA ČR P102/11/1068, 

MSM0021622411. 

6. REFERENCES 

[1] Navarro, E., Baun, A., Behra, R., et al.: Ecotoxicology 17 (2008), 5, 372-386 

[2] Thakkar, K.N., Mhatre, S.S., Parikh, R.Y.: Nanomedicine-nanotechnology biology and medicine, 

6 (2010), 2. 257-262 

[3] Xingmao, M.G-L., Yang Deng, C. Kolmakov, A.: Science of the total environment 408 (2010), 16, 

3053-3061 

[4] Tan, X., Lin, C., Fugetsu, B.: Carbon, 47 (2009), 15, 3479-3487 

[5] Wang, H.H.: Nanotoxicology 5 (2010), 1, 30-42 

- 173 -


EPR AND UV-VIS 

SPECTROELECTROCHEMICAL STUDIES 

OF NOVEL SYNTHESIZED COPPER, 

NICKEL AND COBALT-BASED COMPLEX 

DERIVATIVES 

Karol LUŠPAI 1 , Peter RAPTA 1 , Peter MACHATA 1 , Peter HERICH 1 

1 Institute of Physical Chemistry and Chemical Physics, Faculty of Chemical and Food Technology, 

Slovak University of Technology in Bratislava, Radlinského 9, SK-81237 Bratislava 

Abstract 

Using different spectroelectrochemical techniques the redox properties of the new 

dithiolate-based complex derivatives with Cu(III), Ni(III) and Co(III) as a central atom 

were determined. Changes in EPR and UV-VIS-NIR spectra during electrochemical 

reduction have been analysed. Electrochemical reversibility of studied derivatives was 

dependent on solvent used. 


In recent years dithiolate-based complex compounds have become interesting 

for research and development, because of their potential application in the role of new 

superconductors, pesticides, compounds with unusual magnetic properties, and biocatalysts 

in biochemistry [1]. 

Some of these compounds were studied by computational methods. For example 

benzene-1,2-dithiolate complexes with Cu, Ni, Co were calculated by M. Breza et al.. 

and stability of various charge and spin states was determined [2]. 

2. EXPERIMENT 

Cyclovoltammetric measurements were performed using HEKA PG 284 

(Lambrecht, Germany) potentiostat/galvanostat using PotPulse 8.53 software package. 

A standard three electrode system was used. Platinum wire as working and counter 

electrode, and silver oxidized wire as pseudo-reference electrode was chosen. 

Measured potentials were recorded at 100 mV.s -1 potential rate. Concentration of 

sample was appx. 0.5 mM in solution of 0.2 M TBAPF6 supporting electrolyte. All 

samples were purged by argon, and measured under inert argon atmosphere. Because 

of using pseudo-reference electrode, internal standard (Fc + /Fc) was used. 

Spectroelectrochemical measurements were performed in spectroelectrochemical flat 

cell using Pt mesh as a working electrode. Pt wire served as a counter electrode and 

silver wire as a pseudo-reference electrode. This cell was inserted in optical EPR 

resonator and EPR spectra were recorded simultaneously during voltammetric cycle. 

Bruker EMX X-band EPR spectrometer (Bruker, Germany) and WinEPR 4.40 

- 174 -


software package was used. In-situ recorded UV-VIS spectra were recorded using 

diode-array PC2000 spectrometer (Ocean Optics, Inc.) equipped with deuteriumhalogen 

light source. For UV-VIS-NIR spectroelectrochemical measurements 

Shimadzu UV 3600 spectrophotometer was used. In this case the electrode system 

was immersed in 1 mm quartz cell. 


Electrochemical studies were performed using different solvents. Cyclic 

voltammetry shows one reversible or quasireversible cathodic peak associated 

probably with one-electron transfer for all investigated complexes. Half-wave 

potentials E1/2 vs. Fc + /Fc were -1.02 V for copper-, -0.95 V for nickel-, and -1.27 V for 

cobalt-complex. This peak is associated with electron transfer to M III central atom, and 

coupled with reduction to M II form. 

Current response (norm.) 

-1.4 -1.2 -1.0 -0.8 -0.6 

Potential / V vs. Fc + / Fc 

Fig. 1 Cyclic voltammograms of (MePh3P)[M(bdt)2] complexes ( M = Cu solid line; M 

= Ni dotted line; M = Co dashed line ) in dichloromethane 

Upon this reduction process UV-VIS-NIR spectra of (MePh3P)[Ni(bdt)2] 

complex in dimethylformamide shows a new band at 310 nm and absorbance decrease 

in bands at 361 and 876 nm. The existence of isosbestic point serves as an evidence, 

that the compound is reduced directly from an oxidized form to reduced without 

follow-up reactions, and it indicates also a good stability of its reduced form in 

dimethylformamide. However, a decrease of intensity of new bands 

is evident after longer time, as is shown in Fig. 2 (dark grey-solid and blacksolid). 

That means, the reduced form is stable, but slowly undergo the follow-up 

reactions also in dimethylformamide. 

The EPR spectra of (MePh3P)[Ni(bdt)2] complex show a broad singlet with 

isotropic g = 2.0089. During electrochemical reduction decrease of intensity was 

observed, that means, a reduced form of this complex is diamagnetic. 

- 175 -


0.30 

A 

0.25 

0.20 

0.15 

0.10 

0.05 

I EPR 

0.00 

300 400 500 600 700 800 900 

/ nm 

Fig. 2 UV-VIS-NIR spectroelectrochemistry of complex derivative (MePh3P)[Ni(bdt)2] 

in dimethylformamide (evolution of spectra during reduction corresponds the 

colour from gray-dashed to black-solid). The inset shows EPR spectrum of the 

sample before reduction in dimethylformamide 

Spectroelectrochemistry of (MePh3P)[Ni(bdt)2] complex in dichloromethane 

shows decrease of intensity of all of bands in electronic spectra, and no new bands are 

formed in observed spectral region. This indicates the low stability of its reduced form 

in dichloromethane. 

4. CONCLUSION 

Spectroelectrochemical investigation of dithiolate metalo-complex derivatives is 

presented in this work. Samples were studied both by cyclic voltammetry and EPR- 

UV-VIS-NIR spectroelectrochemistry. Results show that reduction of all investigated 

complexes is associated with the change of oxidation state of the central metal atom 

and stability of their reduced forms strongly dependents on solvent used. 


The financial support of the Slovak Grant Agency VEGA (contracts No. 

1/0679/11 and 1/0018/09) is gratefully acknowledged. 

6. REFERENCES 

[1] Mrkvová, K., Kameníček, J., Šindelář, Z., Kvítek L., Mrozinski J., Nahorska M., Žák Z.: 

Trans. Met. Chem., 29 (2003), 238 

[2] Šoralová, S., Breza, M., Gróf, M.: Polyhedron, 30 (2011), 307 

- 176 - 

100 G


VARIOUS MICROWAVE DIGESTION 

PROCEDURE OF SAMPLES FOR HEAVY 

METALS ELECTROCHEMICAL 

DETERMINATION 

Petr MAJZLÍK 1 , David HYNEK 1 , René KIZEK 1* 

1 Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, CZ (e-mail: kizek@sci.muni.cz); 

Abstract 

Electrochemical determination of heavy metals is very useful and important 

analytical method. Determination of these polutants is very important in environment 

too. Electrochemical determination of samples (dry matter) is primarily affected by 

sample praparation. Two ways of mineralisation (microwave digestion) first with 

HNO3 and second with mixture HNO3 + H2O2 are presented. Use of microwave system 

Multiwave 3000 for samples mineralisation is shown too. 


Electrochemical determination of heavy metals is very useful and important 

analytical method. Determination of these polutants is very important in environment 

too. Electrochemical determination of dry matter samples is primarily affected by 

sample praparation. Various ways of sample praparation can affect results from 

electrochemical determination of heavy metals. Preparation of samples by microwave 

digestion is very often use too. Very often there are used two basic mineralisation 

mixtures - HNO3 and HNO3 + H2O2. 

2. EXPERIMENT 

Mineralisation 

Microwave system Multiwave3000 (Anton-Paar GmbH) was used for 

mineralisation of samples. Two various mineralisation mixtures were used. Each of 

mixtures had unique composition and microwave program, see table 1 and 2. 

Table 1: Composition of mineralisation mixture 

Composition of 

mixture 

HNO3 

69% 

H202 

30% 

1 HNO3 1000µl 10mg 

2 HNO3 + H2O2 700µl 300µl 10mg 

- 177 - 

Ammount of sample (dry 

matter)

XI. Workshhop 


E 

s´11 

Table 22: 

Microwavve 

program m 

CCompositioon 

of 

mmixture 

1 HHNO3 

2 HHNO3 

+ H2O 

O2 

Microwavve 

program m 

power 1000W, 

ramp 10 min, hold 

99 minn, 

cooling 10 

min. 

power 125W, 

ramp p 8 min. – power 2200W, 

ram mp 

5min. – poower 

225W W, ramp 8 min. m – coolling 

10min. 

Fig. 

1: Multiwwave3000 

(A Anton-Paar r GmbH) 

10mg off 

sample were w put into 

bottle MMG5 

and filled f 

wit th 900μl cooncentrated 

d nitric acid 

(mixturee 

1) or mix xture 

HN NO3 + H2O22 

(mixture 2). The samples s weere 

placed into 

roto or 64MG5 and than the microw wave decoomposition 

was 

carried 

out. MMicrowave 

programs p ar re presenteed 

in Table 2. 

Electroochemical 

determin nation 

MMeasuring 

ssystem 

Met trohm was used for th he determin nation of hheavy 

meta als – 

Autosammpler 

813 Compact and a measuuring 

unit VA V Compu utrace 797. Samples were w 

prepareed 

in this mmanner: 

15 l of samplees 

were mix xed with 98 85μl of acettic 

buffer (p pH = 

5). Difeerential 

pullse 

voltamet try was useed 

for the measuremen 

m nt with theese 

paramet ters: 

pootencial 

of deposition -1,3V, timme 

of depos sition 240s, range of p 

1,3V too 

0,15V, pulse 

amplitu ude 0,025V, , pulse time e 0,04s, uni it step 0,00 

unit steep 

0,3s. A sstandard 

ce ell with thrree 

electrod des was use ed for the m 

hangingg 

mercury drop electr rode (HMDDE) 

with a drop d area of f 0.4 mm2 potencial fro om - 

05035V, tim me of 

measurmen nt. A 

wwas 

employ yed 

ass 

the workiing 

electrod de. An Ag/AAgCl/3M 

KCl K electrod de served aas 

the refere ence 

electrodde. 

Pt electtrode 

was used u as the aauxiliary 

el lectrode. 

3. RRESULTS 


Comparison 

of two mineralisati m ion mixture es were exa amined at corn samples 

- 

roots aand 

stipes. These part ts of plantts 

were dri ied and dis ssolved in mineralisa ation 

mixturee 

1 or 2 (TTable 

1). Th he sampless 

were plac ced into rotor 

64MG55 

and than n the 

microwwave 

decommposition 

was w carried out. Comp parison of two t voltammograms 

sh hows 

Fig. 2. Diference between mineralisati m ion 1 and 2 is obvio ously. Proccess 

2 (HNO O3 + 

H2O2) hhas 

better resolution 

of o peaks andd 

no cumul lative behav viour at pottencial 

-1,4 4 to - 

0,8V. IIn 

general, behaviour r of curve 1 (HNO3) is so major, 

that finee 

resolution 

of 

voltamoogram 

2 (HHNO3 

+ H2 2O2) is hiddden 

and de eterminatio on of indivvidual 

met tal is 

forbiddden 

too. 

- 178 - 

Brno


I [nA] 

300 

250 

200 

150 

100 

50 

0 

-1.4 -1.2 -1 -0.8 -0.6 

Potencial [V] 

-0.4 -0.2 0 0.2 

Fig. 2: Voltamograms of two various ways of minearlisation. 

Electrochemical determination of heavy metals in standard (IAEA 413) was 

made too. Comparison of amount of metals in standard with electrochemical 

determination and reference sheet represent Fig. 3. As it seems, correlation between 

electrochemical determination connecting with mineralisation 2 and data in reference 

sheet of standard is very good (standard deviation is 0,0494). 100% repersent amount 

of metals introduced in data sheet. 

100% 

90% 

80% 

70% 

60% 

50% 

40% 

30% 

20% 

10% 

0% 

Zn Cd Pb Cu 

Fig. 3: Relative amount of heavy metals in standard IAEA 413; mineralisation 2 

- 179 - 

HNO3 + H2O2 

HNO3

XI. Workshhop 


E 

s´11 

CCalibration 

curves, see e Fig. 4, reppresent 

dep pendence of 

concentraation 

of me etals 

on amoount 

of IAEEA 

413. Al ll four curvves 

have go ood depend dence of linnearity 

(hig gher 

than 999%). 

Concentration of metals [uM] 

455 

400 

355 

300 

255 

200 

155 

100 

5 

Zn 

Cd 

Pb 

Cu 

0 

0 5 10 

Fig. 4: CCalibrationn 

curves of heavy h metaals 

in standa ard IAEA 413. 4 

15 

20 225 

30 

Amount t of IAEA 413 sttandard 

[mg] 

Fig 5: MMicrowave 

program – temperaturre 

depende ence – mine eralisation HHNO3. 

- 180 - 

35 

40 

Brno 

45


Fig 5 and Fig 6 show temperature dependence by microwave program for every 

mineralisation. Mineralisation 1 has constant linear behaviour from 1200 to 6700s by 

60°C. Temperature by mineralistaion 2 growing to 90°C at 1250s and than cooling is 

started. Mineralisation 2 is shorter three times by higher temperature, which has good 

influence for lifetime of bottles MG5. 

temperature (°C) 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

1 251 501 751 1001 

time (sec) 

1251 1501 1751 

Fig 6: Microwave program – temperature dependence – mineralisation HNO3 + H2O2. 

4. CONCLUSION 

Two various ways of mineralisation samples (dry matter) were published in this 

paper. First, mineralisation with conc. HNO3, second mineralisation with mix – HNO3 

+ H2O2. Both types of operations were effected by Microwave system Multiwave3000 

(Anton-Paar GmbH). Better from two ways of mineralisation is variation 2 (HNO3 + 

H2O2), because this variation has better peak resolution, time of procedur is shorter 

but samples is exposed higher temperature. Next improvement of mineralisation 

procedure is recommendated. 


The work has been supported by NANIMEL GA ČR 102/08/1546. 

6. REFERENCES 

Guerin T, Chekri R, Vastel C, Sirot V, Volatier JL, Leblanc JC, Noel L, Food Chemistry, 127, 3, 2011, 

932-934 

Joksimovic D, Tomic Ilija, Stankovic A, Jovic M, Stankovic S, Food Chemistry, 127, 2, 2011, 632-637 

Recknagel S, Richter A, Richter S, Waste management, 29, 3, 2009, 1213-1217 

- 181 -


ELECTROCHEMICAL DETERMINATION OF 

HEAVY METALS IN RAINWATER 

Petr MAJZLÍK 1 , David HYNEK 1 , René KIZEK 1* 


Abstract 

Electrochemical determination of heavy metals in rain water is the aim of this 

paper. Determination of heavy metals in rainwater from weather-station Bořitov in 

course of one year was carried out. For electrochemical determination measuring 

system Metrohm (Autosampler 813 Compact and measuring unit VA Computrace 

797) was employed. 


Electrochemical determination of heavy metals in rain water is simple, practic 

and useful way how to check the amount of heavy metals in environment. Zinc, 

cadmium, lead and copper belong to the group of heavy metals. There are two insights 

into pollution of living environment by heavy metals. First represents occurrence of 

these metals in rocks, from which can be gradually released and entry the plants, 

respectively food chain. Second possibility of occurrence of heavy metals in living 

environment represents human itself due to anthropogenic activity (mining and 

processing of ore, chemical industry). Heavy metals represent significant problem, 

especially because of their toxicity, which may cause acute as well as chronic 

intoxication and lead to origination of malignant tumour disease. Due to these facts, 

improving of methods of their qualification and quantification using various 

measuring procedures is very advisable. 

2. EXPERIMENT 

Electrochemical determination 

Measuring system Metrohm was employed for the determination of heavy 

metals – Autosampler 813 Compact and measuring unit VA Computrace 797. Samples 

were prepared in this manner: 15 l of sample were mixed with 985 l of acetic buffer 

(pH = 5). Diferential pulse voltametry was used for the measurement with these 

parameters: potencial of deposition -1,3V, time of deposition 240s, range of potencial 

from -1,3V to 0,15V, pulse amplitude 0,025V, pulse time 0,04s, unit step 0,005035V, 

time of unit step 0,3s. A standard cell with three electrodes was used for the 

measurment. A hanging mercury drop electrode (HMDE) with a drop area of 0.4 mm 2 

was employed as the working electrode. An Ag/AgCl/3M KCl electrode served as the 

reference electrode. Pt electrode was used as the auxiliary electrode. 

- 182 -

XI. WWorkshop 

of Phyysical 


3. RESULTTS 

AND DISCUSSI 

D ION 

Measurrement 

of heavy h mettals 

by DPV V techniqu ue has beenn 

described 

many 

times 

over in literature; therefore, it is not necessary n to o representt 

accomplis shments 

andd 

negatives of this tec chnique. MMain 

aim of 

our work k consistedd 

in propos sing and 

reallization 

of aautomated 

measuring g procedure e for analysis 

of defineed 

small vo olume of 

sammple 

for dettermination 

n of above mmentioned 

heavy met tals in rainwwater. 

Prep paration 

of ssample 

prooceeded 

in accordancce 

with following 

pr rocedure: vvolume 

of 15 l of 

sammple 

for measurement 

of heavy mmetals 

(Zn, Cd, and Cu u) was placced 

into Eppendorf 

miccro 

test tube 

togethe er with 9885 

l of ace etate buffe er (pH 5.000). 

Sample es were 

subssequently 

placed into o Autosammpler 

813; in enclosed d software, 

list of measured m 

sammples 

was crreated. 

Fig. 1 demonstrate 

es typical vvoltammog 

gram of sim multaneous s determina ation of 

zincc, 

cadmiumm, 

lead and copper ionss. 

Dependeence 

of buff fer pH and d concentrat tion of hea avy metals iillustrates 

Fig. F 2. It 

is obbvious 

thatt 

the best detection d off 

heavy met tals is by pH H 5.00. Thaat 

is the rea ason for 

use of acetic buuffer 

pH 5. 00 for otheer 

measurem ments. 

Fig. . 1: Typicall 

voltammo ogram of siimultaneou 

us determin nation of zinnc, 

cadmiu um, lead 

and coppper 

ions. 

I (nA) 

33,5 

3 

22,5 

2 

1,5 

1 

00,5 

0 

4 

Cu Zn 

4,2 

4,4 4,6 

Acetic c buffer (pH) 

Fig. . 2: Dependdence 

of buf ffer pH andd 

system response 

Cd 

- 183 - 

Pb 

4,8 

5 

Brno


Rainwater samples were measured for present of heavy metals. Furthermore 

conductivity and pH were measured. Average characteristic values for each season in 

one year are presented in Table 1. Concentration of pollutants which are presented in 

table is increasing in summer months. Opposite in winter the concentration of 

pollutants is higher. Probably it is due to human activity, especially heating and 

pollutants emitted into air). Dependence of pH in one year is increasing from spring 

to winter as well as conductivity. 

Table 1: Comparison of various characterictic values in course of one year. 

1. spring 

(6,53) 

2. summer 

(6,37) 

3. autumn 

(6,90) 

4. winter 

(7,15) 

pH conductivity 

(µS/cm) 

spring 

(56,12) 

summer 

(63,38) 

autumn 

(71,74) 

winter 

(88,47) 

conc. Zn 

(µM) 

spring 

(28,05) 

summer 

(29,32) 

autumn 

(90,41) 

winter 

(98,80) 

- 184 - 

conc. Cd 

(µM) 

spring 

(0,54) 

summer 

(0,12) 

autumn 

(5,66) 

winter 

(6.00) 

conc. Pb 

(µM) 

spring 

(1,29) 

summer 

(1,44) 

autumn 

(4,55) 

winter 

(7,06) 

conc. Cu 

(µM) 

spring 

(21,58) 

summer 

(19,91) 

autumn 

(37,27) 

winter 

(51,06) 

4. CONCLUSION 

Monitoring of amount of heavy metals in rainwater is presented in this paper. 

Electrochemical detection of heavy metals represents analytical method with good 

reproducibility, effortless service and relatively rapid determination. It seems to be 

good analytical tool for periodic determination of heavy metals in rainwater. 

Monitoring of pollutants in rainwater is very useful tool for environment control. 



6. REFERENCES 

[1] Vacek J, Petrek J, Kizek R, et all.: Bioelectrochemistry, 63 (2004), 1-2, 347-351 

[2] Strouhal M, Kizek R, Vacek J, et all.: Bioelectrochemistry, 60 (2003), 1-2, 29-36 

[3] Adam V, Petrlova J, Potesil D, et all.: Electroanalysis, 17 (2005), 18, 1649-1657 

[4] Kovarova J, Kizek R, Adam V, et all.: Sensors, 9 (2009), 6, 4789-4803 

[5] Adam V, Sileny J, Hubalek J, et all.: Toxicology Letters, 180 (2008), Suppl.1, S227-S228 

[6] Adam V, Fabrik I, Kohoutkova V, et all.: International Journal of Electrochemical Science, 5 

(2010), 429-447 

[7] Krystofova O, Trnkova L, Adam V, et all.: Sensors, 10, 6, 5308-5328



MT1 AND MT2 ISOFORMS 

Petr MAJZLÍK 1 , David HYNEK 1 , Tereza REICHLOVÁ 2 , René KIZEK 1* 


2 Brno University of Technology, Faculty of electrical engineering and communication, Department of 

biomedical engineering 

Abstract 

Metallothioneins (MTs) are low molecular, cysteine-rich proteins that have naturallyoccurring 

Zn 2+ in both clusters. They may serve as a reservoir of metals for synthesis 

of apoenzymes and zinc-finger transcription regulators. New biological roles for these 

proteins have been identified including those needed in the carcinogenic process. In 

this study, electrochemical determination of metallothionein isoforms MT1 and MT2 

was carried out. Metallothionein analysis was carried out by adsorptive transfer 

stripping differential pulse voltammetry Brdicka reaction. Determination and 

resolution of isomers MT1 and MT2 with electrochemical determination is aim of this 

work. 


Metallothioneins (MTs) are metal-binding proteins 6–7 kDa in size. They are 

induced by heavy metals, and they actively regulate and detoxify metals in numerous 

cells and organs [1-4]. MTs are composed of four isoform classes: MT-I, -II, -III, and - 

IV. MT-I and MT-II are the main MT isoform classes expressed in many organs. MT- 

III is localized in the brain and MT-IV in the squamous epithelium. In addition, it is 

becoming clear that MTs are not only involved in metal regulation but also have 

radicalscavenging and reactive oxygen species (ROS)-quenching effects. MTs are 

reported to be induced by a wide spektrum of stressors such as steroids, carcinogens, 

chemical substances, ionizing radiation and UV radiation [5–9]. In this study, 

electrochemical determination of metallothionein isoforms MT1 and MT2 [10-13] was 

carried out. Metallothionein analysis was carried out by adsorptive transfer stripping 

differential pulse voltammetry Brdicka reaction [14-16]. 

2. EXPERIMENT 

Electrochemical determination of Metallothionein 

Using our modified MT determination with Brdicka reaction four signals were 

measured (Fig. 1). It can be concluded that catalytic signals of hydrogen ions 

reduction are measured at potential about -1.5 V. Next signal which is appearing at 

potential about -1.3 V is result of reduction of RS2Co complex. There are shown 

catalytic signal at -1.5 V and less developed signal of RS2Co complex in 

voltammogram. Another signals are probable due to reduction of [Co(H2O)6] 2+ at -1.2 

- 185 -

XI. Workshhop 


E 

s´11 

V (marrked 

as Co1) 

and -0.8 V (Co2). CCo2 

signal with w decrea asing concenntration 

of f MT 

decreasses 

and mmoves 

to 

potentiial. 

Fig. 1: TTypical 

volltammogram 

ms of MT saample 

Electrochemmical 

measu urements wwere 

perfor rmed using g an AUTOOLAB 

anal lyser 

(EcoChhemie, 

The Netherlands) 

conneccted 

to VA-Stand 

663 (Metrohmm, 

Switzerla and), 

using a standard ccell 

with three 

electrrodes. 

The three-elect trode systeem 

consiste ed of 

hangingg 

mercury drop electr rode as worrking 

electr rode, an Ag g/AgCl/3 M KCl refere ence 

electrodde 

and a glassy car rbon auxilliary 

electr rode. For smoothingg 

and base eline 

correction 

the sofftware 

GPE ES 4.4 suppllied 

by Eco oChemie wa as employeed. 

The Brd dicka 

supportting 

electrrolyte 

cont taining 1 mmM 

Co(NH3)6Cl3 

and 

1 M ammmonia 

bu uffer 

(NH3(aqq) 

+ NH4Cll, 

pH = 9.6) was used; surface-act tive agent was w not addded. 

AdTS DPV D 

Brdickaa 

reaction parameters s were as ffollows: 

ini itial potent tial -0,7 V, , purgetime e 5s, 

duratioon 

240s, stteppotentia 

al 1,05mV, 

scan rat te 4,2mV/s s, modulattion 

amplit tude 

25,05mmV. 

Temperrature 

of the 

supportinng 

electroly yte was 4 °C C. 

MMethod 

for polarograp phic determmination 

of f proteins containing c –SH group p on 

mercurry 

electrodde 

was pub blished by Brdicka and a this method m wass 

improved d by 

Palečekk 

and otheers. 

Method d is based on catalytic 

reaction n of proteins 

in Brd dicka 

solutionn 

which 

[Co(NHH3)6]Cl3. 

is prepar red from ammonia buffer (N NH4OH + NH4Cl) and 

1 

negative 

2 

MT 

Adsorpttion 

of proteein 

Fig. 3: Scheme off 

adsorptive e stripping technique e (AdST). Working W ellectrode 

is first 

inserted innto 

small volume v of ssample. 

Me etallothione ein adsorbss 

on surfac ce of 

mercury ellectrode 

and 

it is separrated 

from other therm mostabel thhiols 

in sam mple. 

- 186 - 

3 

Washing W 

of 

4 

Electrochemmic 

al detectioon 

Brno


Then is electrode washed and finally is inserted into measuring vessel with 

supporting electrolyte. 

Materials 

MT-1 M5267-5MG, 014K7053 Sigma - Aldrich 

MT-2 M5392-5MG, 052K7002 Sigma - Aldrich 

ACS water Lot: S43109-287 Sigma - Aldrich 

miliQ water Millipore 

EDTA Lot: 042K0087 Sigma - Aldrich 


I [nA] 

300 

250 

200 

150 

100 

50 

0 

Fig. 4: Calibration curve of MT 1 

- 187 - 

y = 0.2677x - 10.204 

R 2 = 0.9909 

0 100 200 300 400 500 600 700 800 900 1000 

Concentration of MT 1 [nM]

XI. Workshhop 


E 

s´11 

I [nA] 

3300 

2250 

2200 

150 

100 

50 

0 

0 1000 

200 

Fig. 5: CCalibrationn 

curve of MT M 2 

300 4000 

500 

Concenntration 

of MT 2 [nM] 

Fig. 4 and 5 show ca alibration ccurves 

of both b isoform ms MT1 aand 

MT2. Both 

B 

calibrattion 

curves have linea ar behaviouur 

with depe endability higher h thann 

99%. 

Fig. 6: PPosition 

of Cat2 C peak ffor 

MT1 an nd MT2 

- 188 - 

600 700 

y = 0.2414x - 11.08 

R 2 86 

= 0.9931 

800 9900 

1000 

Brno


Differences in position of peaks Cat2 by MT1 and MT2 were discovered. Five 

series (each of ten steps) of measurement for MT1 and MT2 were determined. 

Average values of position peak Cat2 represent Fig. 6. Average value of peak position 

Cat2 for MT1 is -1,479V and for MT2 -1,472V. Peak position values for determination 

average values had standard deviation 0.005. The difference between both isoforms 

is 0.005V and this difference is apparent by larger number of measurement. 

4. CONCLUSION 

Electrochemical determination of MT1 and MT2 isoforms is aim of this work. 

Metallothionein analysis was carried out by adsorptive transfer stripping differential 

pulse voltammetry with Brdicka reaction. Comparison of peak position Cat2 for MT1 

and MT2 was made. Average value of peak position Cat2 for MT1 is -1,479V and for 

MT2 -1,472V. The difference between both isoforms is 0.005V. 



6. REFERENCES 

[1] J.H.R. Kagi, A. Schaffer, Biochemistry 27 (1988) 8509. 

[2] M. Margoshes, B.L.A. Vallee, J. Am. Chem. Soc. 79 (1957) 4813. 

[3] M. Studnickova, J. Turanek, H. Zabrsova, M. Krejci, M. Kysel, J. Electroanal.Chem. 421 (1997) 

25. 

[4] R.D. Palmiter, Proc. Natl. Acad. Sci. USA 91 (1994) 1219. 

[5] Angel P, Poting A, Mallick U, Rahmsdorf HJ, Schorpp M, Herrlich P. Mol Cell Biol 1986;6:1760– 

1766. 

[6] Bauman JW, Liu J, Liu YP, Klaassen CD. Toxicko Appl Pharmacol 1991;110:347–354. 

[7] Fornace AJ Jr, Schalch H, Alamo I Jr. Mol Cell Biol 1988;8:4716–4720. 

[8] R. Kizek, L. Trnkova, E. Palecek, Anal. Chem. 73 (2001) 4801. 

[9] R. Prusa, R. Kizek, J. Vacek, L. Trnkova, J. Zehnalek, Clin. Chem. 50 (2004) A28. 

[10] M. Strouhal, R. Kizek, J. Vacek, L. Trnkova, M. Nemec, Bioelectrochemistry 60 (2003) 29. 

[11] L. Trnkova, R. Kizek, J. Vacek, Bioelectrochemistry 56 (2002) 57. 

[12] R. Brdicka, Coll. Czech. Chem. Commun. 5 (1933) 148. 

[13] R. Brdicka, Coll. Czech. Chem. Commun. 5 (1933) 112. 

[14] R. Bdrdicka, Coll. Czech. Chem. Commun. 8 (1936) 366. 

- 189 -


INFLUENCE OF PtCl4 ON MAIZE AND PEA 

Hana MIKULÁŠKOVÁ 1 , Olga KRYŠTOFOVÁ 2 , David HYNEK 2 , Natalia CERNEI 2 , 

Pavlína ŠOBROVÁ 2 , Miroslava BEKLOVÁ 1 , Vojtěch ADAM 2 , René KIZEK 2 

1 Department of Veterinary Ecology and Environmental Protection, University of Veterinary and 

Pharmaceutical Sciences, Palackeho 1-3, CZ-612 42 Brno, Czech Republic 



Abstract 

The aim of this work was to study the influence of platinum ions on the germination 

of selected plant species, which are an important part of the food chain of livestock 

and humans. Peas (Pisum sativum L.) and maize (Zea mays L.) were chosen for the 

experiment. Seeds of plants were exposed to different concentrations of platinum ions 

0, 5, 10, 25, 50, 100 μM for 8 and 12 days. In the end of the experiment, the changes in 

plant growth, content of zinc and enzyme activity of GST (glutathione S-transferase) 

were monitored. Measuring system consisting of potentiostat Autolab TypeIII 

(EcoChemie, Netherlands) connected with the measuring unit VA Stand 663 

(Metrohm, Switzerland) was used for determination of zinc, and spectrofotometric 

analyzer BS-200 (Mindray, China) for determination of GST activity. 


Environmental pollution by heavy metals is an increasing problem worldwide. 

Because of the accumulation effect of some heavy metals, especially through the food 

chain, their bioavailability needs to be monitored (Kouba et al., 2010). The present 

literature survey shows that the concentration of these metals has increased 

significantly in the last decades in diverse environmental matrices; like airborne 

particulate matter, soil, roadside dust and vegetation, river, coastal and oceanic 

environment (Appenroth, 2010; Nagajyoti et al., 2010). However, the increasing uses 

of platinum in vehicle exhaust catalysts, in addition to some other applications (e.g. 

industry, jewellery, anticancer drugs) cause their anthropogenic emission and spread 

in the environment and thus represent the risk in human health. The diseases 

described in human are respiratory and skin diseases, in some cases may support the 

possibility of cancer. Some Pt salts, like hexachloro platinate and tetrachloro platinite, 

are among the most potent allergens and sensitizers (Ravindra et al., 2004). 

2. EXPERIMENT 

Seeds of pea (Pisum sativum L.) and maize (Zea mays L.) were used to determine 

the effects of platinum (Pt). The seeds were exposed to PtCl4 at concentrations of 0, 5, 

10, 25, 50, and 100 μM. For each variant were selected 100 germinated seeds, which 

were placed on plates and covered with cellulose. The ends of cellulose were dipped 

in vessels with solution with different metal concentrations in volume 300 ml. 

Subsequently, the seeds sprouted in the culture box for 8 and 12 days in the dark at 25 

° C and humidity of 60%. In the 8 and 12 day of the experiment the harvested germs 

- 190 -

XI. WWorkshop 

of Phyysical 


werre 

carried oout 

and met trics parammeters 

(lengt th, weight, dry weightt) 

and biochemical 

marrkers 

(zinc, activity of f GST) of abboveground 

d and root parts p were oobserved. 

3. RESULTTS 

AND DISCUSSI 

D ION 

Influencee 

of platinu um in formm 

Pt 

seleected 

plantts 

species was obser 

incrreasing 

conncentration 

of PtCl4 in 

andd 

roots of ppeas 

and corn. 

The gro 

andd 

12 day 775% 

in the 

group w 

commparison 

wiith 

the con ntrol group 

deteermined 

zinnc 

content. 

In pea pla 

incrreasing 


of applied 

(Figg. 

1, b). A ssimilar 

tren nd is shown 

the experimennt 

observed increase in 

IV+ on growth 

of ro oot and abooveground 

parts of 

rved. It wa as found by b measurrement, 

that 

with 

ncrease the growth inh hibition of abovegroun nd parts 

owth inhib bition in 8 day of expperiment 

was 

60 % 

with highest 

concent tration (1000 

μM) of Pt 

(0 μM). In addition to o growth chharacteristi 

ants, we ob bserved an increase i of f zinc conte 

metal PtCl l4 and durat tion of expoosure 

to th 

n on maize (Fig. 1 c, d), d where wwe 

in the 12 

n zinc conte ent in comp parison witth 

control. 

V+ in 

ics were 

ent with 

his metal 

2 day of 

Moreoveer, 

enzyme marker GSST 

(glutath hione-S-transferase) 

wwas 

measur red. The 

obtaained 

resultts 

showed that t the GSST 

activity increase, i mainly m at thee 

second sa ampling, 

withh 

increasinng 

concentr ration of appplied 

meta al PtCl4. It was observved 

the inc crease of 

GSTT 

activity uup 

to five times 

of abooveground 

parts p and fo our at the rroot 

of the plant in 

commparison 

wwith 

the con ntrol groupp. 

The high hest GST activity 

was recorded in n plants 

exposed 

to conncentration 

ns of metalss 

100 μM. The T GST activity 

noticceable 

incre eased in 

maiize, 

mainlyy 

at the fir rst samplinng 

in comp parison with 

the secoond 

samplin ng. The 

incrrease 

in GSST 

activity y in the firrst 

sampling 

was thre ee times hiigher 

comp pared to 

conntrol. 

In the 

case of the t second sampling, the activit ty of GST was at all applied 

conncentrationss 

of metal equivalent. e 

Figuure 

1: influuence 

of Pt tCl4 on zinnc 

content in pea in abovegroun a nd (a) and root (b) 

parts off 

plants and d maize in aabovegroun 

nd (c) and root 

(d) partt 

- 191 - 

Brno


4. CONCLUSION 

Heavy metals belong in present to the major environmental pollutants. The 

increasing uses of platinum in vehicle exhaust catalysts, in addition to some other 

applications cause their anthropogenic emission and spread in the environment and 

thus represent the risk in human health. The bioavailability of platinum PtCl4, which 

was used in our experiment, was demonstrated. Our results show, that the influence 

of platinum on the growth of pea and maize are comparable 


The work has been supported by IGA 83/2011/FVHE, IGA MENDELU 2/2011 

and MSM 6215712402 

6. REFERENCES 

[1] Appenroth, K. J.: Acta Physiologiae Plantarum 32 (2010) 615-619. 

[2] Kouba, A., M. Buric, and P. Kozak.: Water Air and Soil Pollution 211 (2010) 5-16. 

[3] Nagajyoti, P. C., K. D. Lee, and T. V. M.: Environmental Chemistry Letters 8 (2010) 199-216. 

[4] Ravindra, K., L. Bencs, and R. Van Grieken.: Science of the Total Environment 318 (2004) 1-43. 

- 192 -


PLATINUM GROUPS ELEMENTS AND 

THEIR INFLUENCE ON PEA SEEDLINGS 

Hana MIKULÁŠKOVÁ 1 , Olga KRYŠTOFOVÁ 2 , David HYNEK 2 , Natalia CERNEI 2 , 

Pavlína ŠOBROVÁ 2 , Miroslava BEKLOVÁ 1 , Vojtěch ADAM 2 , René KIZEK 2 

1 Department of Veterinary Ecology and Environmental Protection, University of Veterinary and 

Pharmaceutical Sciences, Palackeho 1-3, CZ-612 42 Brno, Czech Republic 



Abstract 

In the last ten years there has been noted the significant incensement in 

concentrations of platinum group elements in the environment. The main emission 

sources of platinum group elements to the environment are automotive catalysts, 

industrial ones and hospital wastes. Platinum content of road dusts can be soluble; 

consequently, it enters the waters, sediments, soil and finally, the food chain. Our 

work was aimed at investigation of influence of platinum and palladium on the seeds 

of pea (Pisum sativum L.) that were exposed to PtCl4 PdCl2 in concentrations 0, 5, 10, 

25, 50, 100 μM. In the 12th day-long experiment, we evaluated the inhibition effects 

of platinum (Pt and Pd) on the growth of plants in their early stages of growth. 

Moreover, we monitored the influence of platinum on chosen biochemical markers 

(content of protein and zinc). 


Platinum group metals (PGE), (Pt, Pd, Rh and more rarely Ir, Os and Ru) can be 

naturally found only at very low concentration in the earth crust. The best-known 

representative of this group, platinum, was used in ancient Egypt, for the manufacture 

of jewelry by the natives. Another platinum metals Rh, Pd, Ir, Os, Ru were discovered 

at 18th century. Worldwide production of PGEs has been steadily increasing since 

1970 (WHO, 1991). Nowadays, these metals found their application in the large field 

of industry. In chemical industry are PGE used as catalysts for chemical synthesis or 

for production of chemical-resistant glass. In medicine Pt complexes has been 

observed as highly effective anti-tumour or ‘anti-neoplastic’ drugs for treating 

testicular tumours, ovarian carcinomas, bladder tumours and tumours of the head and 

neck [1]. PGE also are abundantly used in jewellery. PGE contamination initially 

occurs in airborne particulate matter (PM), roadside dust, soil, sludge and water, etc.; 

which finally results in bioaccumulation of these elements in the living organisms 

through diverse pathways [2]. Generally, PGEs are referred to behave in an inert 

manner and to be immobile. However, there is an evidence of spread and 

bioaccumulation of these elements in the environment. Platinum content of road 

dusts can be soluble; consequently, it enters the waters, sediments, soil and finally, the 

food chain [3]. The effect of chronic occupational exposure to Pt compounds is welldocumented, 

and certain Pt species are known to exhibit allergenic potential. 

- 193 -


However, the toxicity of biologically available anthropogenic Pt is not clear. Hence, 

there is a need to study the effect on human health of long-term chronic exposure to 

low levels of Pt compounds [4]. 

2. EXPERIMENT 

In our experiment we determined the effects of platinum metals on pea seeds 

(Pisum sativum L.). Our work was aimed at investigation of influence of platinum and 

palladium on the seeds of pea (Pisum sativum L.) that were exposed to PtCl4 PdCl2 in 

concentrations 0, 5, 10, 25, 50, 100 μM. For each variant were selected 100 germinated 

seeds, which were placed on plates and covered with cellulose. The ends of cellulose 

were dipped in vessels with solution with different metal concentrations in volume 

300 ml. Subsequently, the seeds sprouted in the culture box for 8 and 12 days in the 

dark at 25 ° C and humidity of 60%. In the 8 and 12 day of the experiment the 

harvested germs were carried out and metrics parameters (length and biomass 

weight), total content of protein and electrochemical determination of zinc content 

were determined. 


The literature shows that the amount of platinum metals in the bodies of plants 

and animals in is in the order in ng/g [4-7]. Therefore, we firstly studied the influence 

of PtCl4 and PdCl2 on growth of pea seeds. The obtained results show that the 

application of metals caused significant growth inhibition in root and aboveground 

parts of pea in 8th and 12th day of experiment with increasing concentrations of 

applied metal. The highest growth inhibition compared to control was observed at 

concentrations 50 and 100 μM. The growth inhibition was in the second sampling 

triple in aboveground and four in the root part at platinum and to five times the 

aboveground part and the root of seven times at the Palladium. These trends were 

similar in the weight of biomass determination. Based on these initial results we can 

conclude that the PdCl2 has stronger inhibitive effect than PtCl4. 

In addition, selected interested stress markers were studied in our experiment. 

One of them is the protein content in plants. Proteins are generally involved in the 

regulation of stress factors caused by foreign substances. In our experiment, Pt 4+ and 

Pd 2+ ion as foreign substances were used. We found that PtCl4 and PdCl2 reduced 

protein synthesis at all concentrations applied in aboveground and root parts of pea 

seeds in 8th and 12th day of experiment. The highest inhibition of protein synthesis 

was observed at concentrations 100 μM. (Fig.1) 

Moreover, we evaluated total zinc content, which is an important chemical 

compound in enzyme activation and protein synthesis. We found that with the 

increasing concentration of applied metal occurred to increasing content of zinc in 

comparison with the control group in the case of PtCl4. Conversely, the pea seeds 

exposed to PdCl2, demonstrated reduction of zinc after exposition of increasing metal 

concentration in 12 day in comparison with the control group. 

- 194 -

XI. WWorkshop 

of Phyysical 


Figuure 

1: influuence 

of PtC Cl4 on proteein 

content 

in pea in abovegrounnd 

(a) and root (b) 

parts off 

plants and d PdCl2 on pprotein 

con ntent in pea a in abovegground 

(a) and a root 

(b) partts 

of plants 


Due to ssignificant 

environmeental 

degrad dation associated 

withh 

high traf ffic, it is 

necessary 

havve 

a deeper r interest iin 

the fate of polluta ants producced 

by traf ffic and 

poteential 

healtth 

and envi ironmental risks associated 

with them. 

In the caase 

of plati inum (PtCll4) 

and palla adium (PdC Cl2), whichh 

were used d in the 

experiment, 

thhe 

influenc ce on seleccted 

bioche emical mark kers (proteein 

and zin nc) were 

demmonstrated. 

5. 

6. 

[1] 

[2] 

[3] 

[4] 

[5] 

[6] 

[7] 

[8] 

ACKNOOWLEDGE 

EMENT 

The workk 

has been supported by IGA 83/ /2011/FVHE 

and MSMM 

62157124 402 

REFEREENCES 

WHO.Envvir 

onmental Health Critteria 

125–Pla atinum. Geneva: 

World Health Orga anization, 

Internationnal 

Programm me on Chemiccal 

Safety, 19 991. 

K. Kummeerer, 

E. Helme ers, Science oof 

the Total Environment 

193 1 (1997) 1779. 

S. Rauch, GG.M. 

Morriso on, Elements 4 (2008) 259. 

M. Moldovvan, 

Anal. Bio oanal. Chem. 388 (2007) 537. 

K. Ravindrra, 

L. Bencs, R. R Van Griekeen, 

Sci. Total Environ. 318 8 (2004) 1. 

R. Djingovva, 

P. Kovacheva, 

G. Wagnner, 

B. Marke ert, Science of f the Total Ennvironment 

308 3 (2003) 

235. 

K.H. Ek, SS. 

Rauch, G.M M. Morrison, P. Lindberg, 

Science of the t Total Env nvironment 334 

(2004) 

149. 

S.H. Pan, G. Zhang, Y.L. Y Sun, P. CChakraborty, 

Science of the t Total Envvironment 

40 07 (2009) 

4248. 

- 195 - 

Brno


UTILIZATION OF ELECTROCHEMICAL 

METHODS IN STUDIES OF 

TRANSPORTING PROCESSES ACROSS 

THE PHOSPHOLIPID BILAYERS 

Tomáš NAVRÁTIL 1 , Ivana ŠESTÁKOVÁ 1 , Vladimír MAREČEK 1 

1 J. Heyrovský Institute of Physical Chemistry of AS CR, v.v.i., Dolejškova 3, 182 23 Prague, Czech 

Republic, E-mail: navratil@jh-inst.cas.cz 

Abstract 

This contribution deals with studying and characterization of transporting processes 

of heavy metals (lead and cadmium) in the form of their free ions and of their 

complexes with small organic ligands across biological membranes. These membranes 

are represented by model phospholipid bilayers, formed on the surface a suitable flat 

support material or in its pores. Phosphatidyl choline and phosphatidyl ethanolamine 

were used as the membrane building elements. Voltammetry and electrochemical 

impedance spectrometry (EIS) have been used for characterization of the transporting 

processes. 


To secure normal functioning of living (plant or animal) cells, it is necessary to 

realize transport of various inorganic and organic compounds (nutrients, etc.), across 

the cell membrane (composed from phospholipids and other parts) into or out of the 

cells or various sub-cellular structures. Not only the useful and usual metabolic 

compounds are transported into the cells across the membranes; unfortunately, the 

above mentioned undesired ions, compounds and particles, which are connected with 

the pollution of the environment, also participate in the transporting processes [1,2]. 

The principles, on which the transporting processes are based, have been studied for 

many years in many laboratories all over the world [3-5]. 

Lipid bilayers (LBs) (or phospholipid bilayers (PLBs)) are thin, flat membranes 

consisting of two layers of lipid molecules, with their hydrophobic parts, usually fatty 

acid tails, directed toward the centre of the membrane, and with hydrophilic parts 

located at the inner and outer borders [6-9]. There are many different principles, on 

which the molecules, ions, or the particles are transported across them. Gases like 

oxygen, CO2 and nitrogen – small molecules hardly interacting with solvents – diffuse 

easily across the hydrophobic part of the membrane [5]. Lipid molecules, e.g., 

steroidal hormones, permeate the bilayer easily [5]. Compounds insoluble in fats are 

transported across amphipathic proteins and can be dipped into equally oriented lipid 

bilayer. The proteins form channels for ions and small molecules and serve for 

transport of bigger molecules, which would not be otherwise able to pass across the 

- 196 -


bilayer [1]. Transport can be passive or supplied by some external energy. There are 

some other utilized principles of transport; we can mention endocytosis and 

exocytosis (e.g., in cases of larger objects and particles, such as bacteria, viruses), 

electroporation etc. [1,5]. 

On the other hand, the (mostly negative) role of heavy metals in living cells has 

been studied very intensively. The principles of their fate and transport inside such 

cells are very well known too (e.g., under participation of phytochelatins and 

metallothioneins [10-14]). Nevertheless, the principles of their transport across the 

cell membranes have remained unelucidated and this contribution should contribute 

to their explaination. 

2. EXPERIMENT 

The experiments described in this contribution were realized using porous 

membranes constructed of two types of phospholipids: 1,2-dipalmitoyl-sn-glycero-3phosphocholine 

(lecithin, DPPC, GPCho (16:0/16:0), CAS No. 63-89-8) (Avanti Polar 

Lipids, Alabaster, USA), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine 

(DPPE, GPEtn(16:0/16:0), CAS No. 923-61-5) (Sigma-Aldrich, Prague, Czech 

Republic). The PLBs were formed by self-assembling in the holes of the Isopore 

Membrane Filters (Millipore, USA) polycarbonate, hydrophilic 8.0 μm, and the 

supporting membrane thickness amounted to 7-22 m. The area of one pore 

amounted to 50 m 2 , the experimentally found porosity of the membranes was about 

25-45 %. 

The other type of supported membranes was prepared on the surface of agar 

electrode, which was prepared from a Teflon tube (diameter 1 mm) filled with agar. 

Contact was realized by a silver wire covered by silver chloride. 

The way used to preparation of supported PLBs (SPBLs) on porous membranes 

was described in detail, e.g., in [1,7]. We mention the basic points here: A 

phospholipid solution (20 mg.mL -l in n-heptane) was applied to both sides of the 

porous polycarbonate membrane and the solvent was evaporated on the air. The 

ionophores were added to the phospholipid solutions before their application on the 

membrane surfaces. To prevent contamination in the experiments, all the parts 

(polycarbonate membrane, cup, upper and lower part) were exchanged for new ones 

prior to each experiment [15]. 

Two types of electrical equivalent circuits were utilized to characterize the 

formed SPLBs bilayers and the corresponding transport processes. The simpler one 

(composed of one resistor in serial combination with parallel combination of a resistor 

and a capacitor) was applicable for characterization of the free polycarbonate 

membranes. The other one was more suitable for characterization of SPLBs formed on 

the polycarbonate membrane pores. This circuit was similar to the simpler one, but 

additionally, a parallel combination of one capacitor and one resistor was added to the 

first capacitor (series-connected) [15]. 

- 197 -



The transporting processes of heavy metals – lead and cadmium - across the 

model SPLB will be characterized in this contribution. The described experiments 

were realized using model membranes, however, the conditions of the experiments 

were very similar to those in real nature. The investigated cations were transported 

across the membrane using ion channels, which were formed by addition of 

ionophores calcimycin and valinomycin to the phospholipid solution. We investigated 

the roles and effects of presence of some other cations (K + , Ca 2+ , etc.) and of some 

small organic molecules on the transport. 

4. CONCLUSION 

In correspondence with the earlier published results [1,3,4,6,7] and with the 

results published in this contribution, it can be concluded that the model membranes 

in the form of SPLBs can be used for simulation of real cell membranes. The SPLB can 

be considered as completely formed in about 30-60 minutes after application of 

phospholipids on the support holder. In case of agar support are the times a bit 

shorter. The values of its capacitances increase after the SPLB exposure to the aqueous 

phase till steady state is reached. Application of voltage equal or higher than ±0.5 V 

can destroy the consistency of the SPBL irreversibly. The results achieved using DPPC 

are almost equivalent to those achieved using DPPE. The results from both tested cell 

types (“U-cell” and “Insert”) differ only negligibly and from the statistical point of 

view they are equivalent. 

It was proved that the transporting processes are relatively complicated, because 

they can be affected by many parameters (e.g., pH, applied voltage, composition of the 

intracellular and extracellular solutions, and presence of other cations). The presence 

Ca2+ cations plays very important role in case of the incorporation calcimycin 

ionophore channels. 


The work has been supported by the GA AV CR by the project No. 

IAA400400806 and by the GA ČR by the project No. P206/11/1638. 

6. REFERENCES 

[1] Navratil, T.; Sestakova, I.; Jaklova Dytrtova, J.; Jakl, M.; Marecek, V.: WSEAS Transactions on 

Environment and Development, 6 (2010), 3, pp. 208-19. 

[2] Navratil, T.; Sestakova, I.; Jaklova Dytrtova, J.; Jakl, M.; Marecek, V. In 7th WSEAS International 

Conference on Environment, Ecosystems and Development; Otesteanu, M.; Celikyay, S.; 

Mastorakis, N.; Lache, S.; Benra, F. K., Eds.; World Scientific and Engineering Acad. and Soc.: 

Puerto de la Cruz, SPAIN, 2009, pp. 212-7. 

[3] Navratil, T.; Sestakova, I.; Stulik, K.; Marecek, V.: Electroanalysis, 22 (2010), 17-18, pp. 2043-50. 

[4] Sestakova, I.; Jaklova Dytrtova, J.; Jakl, M.; Navratil, T. In Development, Energy, Environment, 

Economics (DEEE '10); Mladenov, V.; Psarris, K.; Mastorakis, N.; Caballero, A.; Vachtsevanos, 

G., Eds.: Puerto de la Cruz, 2010, pp. 186-91. 

[5] Murray, R. K.; Granner, K. D.; Mayes, P. A.; Rodwell, V. W. Harper's Biochemistry; Appleton 

and Lange: Stamford, 1996, 873. 

- 198 -


[6] Navratil, T.; Sestakova, I.; Marecek, V. In Modern Electrochemical Methods XXIX; Barek, J.; 

Navratil, T., Eds.; BEST Servis: Jetrichovice, 2009, pp. 74-6. 

[7] Navratil, T.; Sestakova, I.; Marecek, V.; Stulik, K. In Modern Electrochemical Methods XXX; 

Barek, J.; Navratil, T., Eds.; BEST Servis: Jetrichovice, 2010, pp. 119-23. 

[8] Tien, H. T. Bilayer Lipid Membranes; Marcel Dekker, Inc.: New York, 1974. 

[9] Tien, H. T.; Salamon, Z.; Guo, D. L.; Ottovaleitmannova, A. In Active Materials and Adaptive 

Structures; Knowles, G. J., Ed.; Iop Publishing Ltd: Bristol, 1992, pp. 27-32. 

[10] Serrano, N.; Sestakova, I.; Diaz-Cruz, J. M.; Arino, C.: Journal of Electroanalytical Chemistry, 

591 (2006), 1, pp. 105-17. 

[11] Dorcak, V.; Sestakova, I.: Bioelectrochemistry, 68 (2006), 1, pp. 14-21. 

[12] Fojta, M.; Fojtova, M.; Havran, L.; Pivonkova, H.; Dorcak, V.; Sestakova, I.: Analytica Chimica 

Acta, 558 (2006), 1-2, pp. 171-8. 

[13] Yosypchuk, B.; Sestakova, I.; Novotny, L.: Talanta, 59 (2003), 6, pp. 1253-8. 

[14] Sestakova, I.; Navratil, T.: Bioinorganic Chemistry and Applications, 3 (2005), 1-2, pp. 43-53. 

[15] Navratil, T.; Sestakova, I.; Marecek, V. In Development, Energy, Environment, Economics 

(DEEE '10); Mladenov, V.; Psarris, K.; Mastorakis, N.; Caballero, A.; Vachtsevanos, G., Eds.: 

Puerto de la Cruz, 2010, pp. 192-7. 

- 199 -


OPTICAL CHARACTERIZATION OF 

ORGANIC SEMI-CONDUCTORS 

Imad OUZZANE 1 , Martin ŠEDINA 1 , Patricie HEINRICHOVÁ 1 , Martin VALA 1 , 

Martin WEITER 1 . 

1 Brno University of Technology, Faculty of Chemistry, Purkyňova 118, Brno, Czech Republic, 

ouzzane@fch.vutbr.cz 

Abstract 

Several derivatives of diphenyl-diketo-pyrrolopyrrole and metal complex 

phtalocyanines where newly synthesized and proposed to be used in optical and 

optoelectronic devices. Our task was to afford valuable measuring methods for their 

intrinsic optical investigation and to compare the compounds properties according to 

their inner structure. For this optical characterization purposes, optical process were 

investigated like: Time resolved fluorescence, amplified spontaneous emission, one 

and two photon absorption and fluorescence quenching. 


Organic semiconductors are nowadays very attractive molecules to work with in 

industry as well as in research as their chemical advantages (flexibility, solubility, 

recyclability, and affordable materials etc.) are to be combined with their 

advantageous physical characteristics. Diphenyl-diketo-pyrrolopyrrole or DPP 

derivatives [1-3], phtalocyanines [4,5] like other newly synthesized possible 

chromophores are of a great interest for scientific community. Their synthetically 

changeable pendant substituents play a role on their optical properties and 

characteristics and this is the main task of our work as we draw out therefore and 

according to the results new model molecules to be synthesized towards optical or 

electrical needs. Indeed, as modern measuring and analyzing apparatus are getting 

more performing, we have the possibility to study and know better, optical as well as 

electronic characteristics of the relevant organic compounds. Here we will present 

some specific optical characteristics like two photon absorption, amplified 

spontaneous emission and fluorescence quenching of the proposed newly synthesized 

organic compounds. 

2. EXPERIMENT 

For the one and two photon emission spectra of DPP derivatives experiment, we 

prepared different concentrated solutions of DPP solubilized in different organic 

solvents. The exciting wavelength used for two photon excitation was the 

fundamental wavelength from a picosecond laser at 1064nm. The beam was then 

concentrated trough a focalizing lens to the very middle of the sample and the 

emission signal was then collected via hyperbolic mirrors that focused emitted 

photons to the spectrograph and ICCD camera input slit. 

- 200 -


For the amplified spontaneous emission [6,7] experiment we had to prepare thin 

layers of common transparent polymer doped with DPP derivatives. As for and to do 

so, we diluted a maximum amount of DPP with the housing polymer in a minimum 

amount of solubilizing solvent. The thin layer were then prepared by spin coating and 

then measured with an exciting angle of 90 degrees formed between the incident 

beam (3rd harmonics at 355nm) and the planar thin film serving as a waveguide. 

For the dynamic and static quenching [8] of phtalocynines, compounds were 

prepared in solution with a growing concentration of quencher C60. 


The emission spectra after one and two photon excitation and amplified 

spontaneous emission of DPP derivatives are shown in Figure 1. The results obtained 

clearly show that the quadrupolar donor-acceptor-donor character of the substituted 

DPP led to the observable fluorescence signal after two photon absorption. The nonlinear 

optical behavior was therefore achieved by this modification. 

The ASE signal (Figure 1 right) is obtained by illuminating a narrow stripe of the 

thin film (serving as waveguide) with laser pulse. In the longer direction of the 

waveguide, the light emission is synchronized, causing lasing action. With the 

substituted donating groups R1R2 derivative (see Figure 1 left) we could obtain 

significantly reasonable spectral narrowing (the FWHM was found to be around 12 

nm). 

The fluorescence quenching experiments were used to evaluate the extent of 

charge transfer from the electron donor (pthalocyanine in this case) and electron 

acceptor (C60 in this study). The outcomes can serve as a guide in order to predict the 

ability of the system to convert light into electricity in bulk heterojunction organic 

solar cells. The experiments are demonstrated in Figure 2. We achieved strong 

fluorescence quenching comparable with the conventional P3HT:PCBM organic solar 

cells. 

R 1 

O 

R3 N N 

O 

R 4 

A, Pl EMM (-) 

1 

0.9 

0.8 

0.7 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

2PE 

1PE 

Abs 

0 

400 450 500 550 600 650 700 750 

wavelength (nm) 

R 2 

Fig.1 (Left) General formula of the basic diketo-pyrrolo-pyrrole used as parent 

molecule in this study. (Middle) Absorption, one photon and two photon 

fluorescence of substituted DPP with donating groups R1R2. (Right) One 

photon fluorescence and amplified spontaneous emission signal of the 

substituted DPP. 

- 201 - 

Normalized Intensity 

1,0 

0,8 

0,6 

0,4 

0,2 

0,0 

-0,2 

300 400 500 600 700 800 900 1000 

Wavelength 

DPP Input 4 

DPP Input 40


Fluorescence (arb.u.) 

4 

2 

650 700 750 800 


- 202 - 

F 0 /F 

3 

2 

1 

experimental data 

after calibration 

0 1 2 3 

[C ]x10 60 5 (mol/l) 

Fig.2 (left) Fluorescence quenching of phtalocynines compounds (Pc): The 

fluorescence decrease with increasing concentration of quencher (C60) and the 

Stern-Volmer plot of mixture of Pc and water-soluble derivative of C60 

obtained from the fluorescence emission (right) before (circles) and after 

correction on the re-absorption effect (squares). 

4. CONCLUSION 

We have modified several organic molecules in order to obtain their non-linear 

optical behavior, amplified spontaneous emission and to use them in organic solar 

cells. The NLO properties were demonstrated for donating groups substituted in R1R2 

position of DPP. The lasing action was also achieved for this particular substituted 

derivative. Furthermore, the utilization of fluorescence quenching method to predict 

solar conversion efficiency was demonstrated. 


The work has been supported by MŠMT (OP VaVpI) via project Centres for 

materials research at FCH BUT No. CZ.1.05/2.1.00/01.0012. 

6. REFERENCES 

[1] M. Vala, M. Weiter, J. Vynuchal, P. Toman, S. Lunak Jr., J. Fluoresc (2008) 18:1181-1186. 

[2] M. Vala, J. Vynuchal, P. Toman, M. Weiter, S. Lunak Jr., Dyes and Pig. 84 (2010) 176-182. 

[3] S. Lunak Jr., J. Vynuchal, M. Vala, L. Havel, R. Hrdina. 

[4] Nalwa, H. S.; Shirk, J. S. In Phtalocyanines, Properties and Applications, Leznoff, C. C., Lever, A. 

B. P., Eds.; New York, 1996; Vol. 4, pp 83-181 and references therein. 

[5] K. Rieko, K. Masahiro, Mol. Cryst., 1998, Vol. 315, pp. 169-174. 

[6] G. H. Gelinck, J. M. Warman, M. Remmers, D. Neher, Chemical Physics Letters 265 (1997) 320- 

326. 

[7] M. McGehee, R. Gupta, S. Veenstra, E. K. Miller, M. A. Diaz-Garcia, and A. J. Heeger, Physical 

Review B, Volume58, Number 11. 

[8] Principles of Fluorescence Spectroscopy, Third Edition, Joseph R. Lakowicz


ELECTROCHEMISTRY OF 

BIOMACROMOLECULES. TRENDS IN 

PROTEIN ANALYSIS 

Emil PALEČEK 1 , Veronika OSTATNÁ 1 , Hana ČERNOCKÁ 1 , Mojmír TREFULKA 1 , 

Martin BARTOŠÍK 1 

1 Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65 


Abstract 

In recent decades electrochemistry of proteins has dealt mainly with conjugated 

proteins containing non-protein redox centers, being thus limited to a small fraction 

of proteins investigated by proteomics. Recently we have shown that using the 

constant current chronopotentiometric stripping (CPS) in combination with bare 

mercury electrodes practically all proteins produce the so-called peak H, which is due 

to the catalytic hydrogen evolution reaction. Peak H differs from the previously 

studied electrochemical responses of proteins (i) by its ability to detect proteins down 

to nanomolar and subnanomolar concentrations (ii) by high sensitivity to changes in 

protein structures; (iii) by small requirement for the sample volumes - in ex situ 

experiments, few l drops of protein solution are sufficient for the analysis, allowing 

protein determination at femtomole or attomole levels. DTT-modified electrodes were 

recently applied for studies of wt and mutant p53 proteins. 

1. ELECTROCHEMICAL ANALYSIS IN GENOMICS AND PROTEOMICS 

Recent advances in genomics, proteomics and glycomics require large amounts 

of data regarding human genomes, protein expression, posttranslational modification 

of proteins, DNA and RNA, etc. Among the current techniques used in genomics, 

electrochemical (EC) sensing of DNA is gaining ground while EC analyses of proteins 

and carbohydrates for proteomics or glycomics, respectively have developed rather 

slowly. 

2. ELECTROCHEMISTRY OF PROTEINS 

In recent decades electrochemistry of proteins focused on conjugated proteins 

containing non-protein redox centers [1,2]. This interesting part of 

bioelectrochemistry has been limited to a small fraction of thousands proteins studied 

by proteomics. 

2.1 Constant current chronopotentiometric stripping with mercury 

electrodes 

The first paper on electrochemistry of proteins was published more than 80 

years ago (3). It was based on the ability of proteins to catalyze hydrogen evolution at 

the dropping mercury electrode (DME) yielding the d.c. polarographic “presodium 

- 203 -


wave”. This wave appeared too close to the background discharge and was thus poorly 

developed and considered of little use in protein analysis. The history of this discovery 

was already reviewed (e.g. 4 and references therein). 

Recently we have shown that using the constant current chronopotentiometric 

stripping (CPS) in combination with bare mercury electrodes practically all proteins 

produce the so-called peak H, which is due to the catalytic hydrogen evolution 

reaction (CHER) [5, 6]. Peak H differs from the previously studied electrochemical 

responses of proteins (i) by its ability to detect proteins down to nanomolar and 

subnanomolar concentrations; (ii) by high sensitivity to changes in protein structures; 

(iii) by small requirement for the sample volumes - in ex situ experiments, few l 

drops of protein solution are sufficient for the analysis, allowing protein 

determination at femtomole or lower levels. 

2.1.1 Denaturation of proteins at mercury surfaces can be avoided 

Until recently an opinion prevailed in the literature that proteins are denatured 

when adsorbed at bare mercury and other metal surfaces [7]. We have shown that 

proteins are not denatured when adsorbed at bare HMDE at open current potential 

but that their denaturation may occur when the electrode is charged to potentials 

negative to the potential of zero charge (p.z.c.) [6, 8, 9]. Such surface denaturation can 

be avoided if high speed of the electrode charging to negative potentials and proper 

ionic conditions are used [8]. For example, we have found that bovine serum albumin 

(BSA) and other proteins behaved in CPS experiments as native in 50 mM but not in 

200 mM sodium phosphate, pH 7 (Fig. 1A,B). We used CPS to study aggregation of - 

synuclein (the protein important in Parkinson’s disease), and detected changes in the 

interfacial behavior of this protein preceding the fibril formation [10]. 

CPS analysis of proteins at DTT-modified Hg electrodes 

Dithiothreitol (DTT)-mercury and DTT-solid amalgam electrodes were proposed 

for protein microanalysis by means of CPS (11). At the DTT-modified hanging 

mercury drop electrode (DTT-HMDE) proteins produced CPS peak H, due to the 

CHER. Self-assembled monolayers (SAMs) of DTT at the electrode protected surfaceattached 

proteins from the electric field-driven denaturation, but did not interfere 

with the CHER. Using CPS peak H denatured (using urea or guanidium chloride) and 

native forms of BSA and of other proteins were easily distinguished. In contrast, in 

usual voltammetric measurements (scan rates between 50 mV and 1 V/s) the adsorbed 

BSA behaved as fully or partially denatured. In CPS, which uses highly negative 

stripping currents causing high current densities (~-7.5 mA/cm 2 at Istr -30 mA), 

changes in potential are very fast (e.g. ~250 V/s) and the protein denaturation at 

negatively charged surfaces (occurring in the voltammetric experiments) is prevented. 

BSA-modified DTT-HMDE was exposed to different potentials, EB, for 60 s, followed 

by CPS measurement. Three EB regions were found, in which either BSA remained 

native (A, -0.1 to -0.3 V), was denatured (B, -0.35 V to -1.4 V) or underwent 

desorption (C, at potentials more negative than -1.4 V). At potentials more positive 

than the reduction potential of the DTT Hg-S bond, the densely packed DTT SAM 

- 204 -


was impermeable to [Ru(NH3)6] 3+ . At more negative potentials the DTT SAM was 

disturbed but under conditions of CPS this SAM still protected the protein from the 

denaturation. Thiol-modified Hg electrodes in combination with CPS appear 

attractive as a new tool for protein analysis in biomedicine and proteomics. 

2.1.3 Peak H responds to changes in p53 resulting from structural changes 

in mutants 

CPS peak H obtained with DTT-HMDE has been applied in studies of tumor 

suppressor wild type (wt) and mutant p53 proteins [12]. CPS responses of wt and 

mutant p53 showed correlation with structural and stability data and provided 

additional insights into the differential dynamic behavior of the proteins immobilized 

at electrically charged surfaces. The loss of zinc ion either due to mutation in R175H 

or to treatment of wt p53 with a chelating agent were monitored. It is expected that 

this CPS method can be useful in screening of p53-specific drugs for future cancer 

treatment. 

2.1.4 Solid amalgam electrodes for protein analysis 

Sensing of proteins is based on their ability to catalyze hydrogen evolution at Hg 

electrodes. Liquid mercury electrodes are excellent tools for basic electrochemical 

research but their application in sensors and arrays for highly parallel analysis is 

rather inconvenient. We showed that some solid mercury amalgam electrodes (SAE) 

can be used in the analysis of proteins and oligo- and polysaccharides. Recently 

developed silver SAE arrays [13] in diameter of 400 mm and thickness of < 1 mm are 

as little toxic as dental amalgams but compared to the tooth filling, the amount of Hg 

in the whole array is negligible. Our preliminary results suggest that these arrays can 

be modified with DTT and other thiols representing an alternative to the DTT- 

HMDE. Thus SAEs, with the unique potential window of Hg electrodes allowing 

studies at highly negative potentials necessary for CHER, open the door for protein 

analysis in biomedicine and proteomics using biosensors and arrays. 

3. CONCLUSION 

Electroactivity of proteins and nucleic acids has been known for more than 80 

(3,4) and 50 years, respectively, while until recently poly- and oligosaccharides were 

considered as non-electroactive and attracted little attention of electrochemists. 

Recently we have demonstrated electroactivity in some polysaccharides (14). We have 

also shown that using Os(VI) complexes electroactive labels can be easily introduced 

in various poly- and oligosaccharides (15-17). CPS analysis of proteins with liquid and 

solid Hg electrodes opens the door for application of electrochemical methods in 

biomedicine and proteomics. Chemical modification of these electrodes makes them 

particularly attractive for protein structure analysis. 

In this lecture I wish to present our recent results dealing with CPS analysis of 

proteins obtained with bare and DTT-modified Hg electrodes and briefly summarize 

our studies based on chemical modification and EC analysis of poly- and 

oligosaccharides and ribose residues in RNA shown in companion posters and papers 

(15-18). 

- 205 -



Support from the GACR P301/11/2055, Res. Centre LC06035 and the IBP 

Research Plans Nos. AV0Z50040507 and AV0Z50040702 is acknowledged. 

5. REFERENCES 

[1] O. Hammerich and J. Ulstrup, Bioinorganic Electrochemistry, Dordrecht: Springer, 2008. 

[2] E. Palecek, F. Scheller and J. Wang, Electrochemistry of nucleic acids and proteins. Towards 

electrochemical sensors for genomics and proteomics, 1 ed., Vol. 1. Amsterdam: Elsevier, 2005. 

[3] J. Heyrovsky and J. Babicka, "Polarographic studies with the dropping mercury cathode. Part XIII. 

Effect of albumins", Coll. Czech. Chem. Commun., Vol. 2, p. 370, 1930. 

[4] E. Palecek, M. Heyrovsky, B. Janik, D. Kalab, Z. Pechan, "From dc polarographic presodium wave of 

proteins to electrochemistry of biomacromolecules," Coll. Czech. Chem. Commun., Vol. 74, pp. 

1739-1755, 2009. 

[5] E. Palecek, "Electroactivity of proteins. Possibilities in biomedicine and proteomics," in 

Electrochemistry of Nucleic Acids and Proteins. Towards Electrochemical Sensors for Genomics 

and Proteomics, E. Palecek, F. W. Scheller and J. Wang, Eds. Amsterdam: Elsevier, 2005, pp. 

690-750. 

[6] E. Palecek and V. Ostatna, "Electroactivity of nonconjugated proteins and peptides. Towards 

electroanalysis of all proteins," Electroanalysis, Vol. 19, pp. 2383-2403, 2007. 

[7] F. A. Armstrong, "Voltammetry of proteins," in Bioelectrochemistry, Vol. 9, G. S. Wilson, Ed. 

Weinheim: Wiley-VCH, 2002, pp. 11-29. 

[8] E. Palecek and V. Ostatna, "Ionic strength-dependent structural transition of proteins at electrode 

surfaces," Chem. Commun., pp. 1685-1687, 2009. 

[9] E. Palecek and V. Ostatna, "Potential-dependent surface denaturation of BSA in acid media," 

Analyst, Vol. 134, pp. 2076-2080, 2009. 

[10] E. Palecek, V. Ostatna, M. Masarik, C. W. Bertoncini and T. M. Jovin, "Changes in interfacial 

properties of -synuclein preceding its aggregation," Analyst, Vol. 133, pp. 76-84, 2008. 

[11] V. Ostatna, H. Cernocka and E. Palecek, "Protein Structure-Sensitive Electrocatalysis at 

Dithiothreitol-Modified Electrodes," J. Am. Chem. Soc., Vol. 132, pp. 9408-9413, 2010. 

[12] E Paleček, V Ostatná, H Černocká, A C. Joerger, A R. Fersht, Electrocatalytic monitoring of 

metal binding and mutation-induced conformational changes in p53 at picomole level. J. 

Am. Chem Soc. 2011, in press. 

[13] P. Juskova, V. Ostatna, E. Palecek and F. Foret, "Fabrication and Characterization of Solid 

Mercury Amalgam Electrodes for Protein Analysis," Anal. Chem., Vol. 82, pp. 2690-2695, 

2010. 

[14] S. Strmečki, M. Plavšić, B. Ćosović, V. Ostatná and E. Palecek, "Constant current 

chronopotentiometric stripping of sulphated polysaccharides," Electrochem. Commun., Vol. 

11, pp. 2032-2035. 2009. 

[15] M. Trefulka and E. Palecek, "Voltammetry of Os(VI)-modified polysaccharides at carbon 

electrodes," Electroanalysis, Vol. 21, pp. 1763 – 1766, 2009. 

[16] M. Trefulka and E. Paleček, "Voltammetry of Os(VI)-Modified Polysaccharides," 

Electroanalysis, Vol. 22, pp. 1837-1845, 2010. 

[17] E. Paleček and M. Trefulka, "Electrocatalytic detection of polysaccharides at picomolar 

concentrations," Analyst, Vol. 136, pp. 321-326, 2011. 

[18] M. Trefulka, M. Bartošík, E. Paleček, "Facile end-labeling of RNA with electroactive Os(VI) 

complexes," Electrochem. Commun., Vol. 12, pp. 1760-1763, 2010. 

- 206 -


THE STUDY OF 6 – 

BENZYLAMINOPURINE AND ITS 

DERIVATIVES BY ELECTROCHEMICAL 

AND SPECTRAL METHODS 

Iveta PILAŘOVÁ 1 , Sylvie HOLUBOVÁ 1 , Zdeňka BALCAROVÁ 1 , Núria SERRANO 2 , 

Libuše TRNKOVÁ 1 

1 Department of Chemistry, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech 

Republic 

2 Departament de Química Analítica, Facultat de Química, Universitat de Barcelona, Martí i Franquès 1- 

11, E – 08028 – Barcelona, Spain 

Abstract 

Electrochemical and spectral behaviors of 6 – benzylaminopurine (6 – BAP) and its 

chlorine and methoxy derivatives have been studied by voltammetry, potentiometry 

and spectrophotometry. Using linear sweep voltammetry (LSV) in connection with a 

mercury electrode the reduction peaks of BAPs were observed. On the other hand, 

using elimination voltammetry with linear scan (EVLS) the differences in the 

reduction processes of derivatives studied were found. The EVLS confirmed the 

electron transfer in an adsorbed state. The pH value of water-methanol solutions was 

chosen according to the protonation constants of BAPs determined by both 

potentiometric and UV-Vis spectrophotometric titrations. The effect of methanol 

content and temperature on pKa values of 6-BAP was monitored. 


Cytokinins (CKs) are phytohormones which play very important role in the 

development and growth processes of plants. Together with auxins are involved in the 

regulation of cell division 1 . One of the most important aromatic cytokinins, 6 – 

benzylaminopurine (6 – BAP), has been recently investigated electrochemically on a 

mercury electrode 2 . Nowadays, the attention is focused on its methoxy and chlorine 

derivatives due to their antitumor activity. These derivatives can be considered as 

potential cytostatics 3-6 . 

The aim of our research was the studying of electrochemical and spectral 

behavior of 6 – BAP and its methoxy and chlorine derivatives. We used voltammetric 

methods (linear sweep and elimination voltammetry on a mercury electrode) allowing 

determining the reduction potentials of benzylaminopurines and predicting their 

electrode process mechanisms. To investigate of the electrode process, elimination 

voltammetry with linear scan (EVLS) is suitable. It is able to eliminate some selected 

current components (diffusion, charging or charging) and conserve the other ones 

from the total voltammetric current. The procedure is represented by EVLS current 

function as a linear combination of total currents measured at different scan rates. The 

- 207 -


best EVLS sensitivity is achieved by the function eliminating kinetic and charging 

current components {f (I) = 17.485 I - 11.657 I1/2 - 5.8584 I2 } for the reduction or 

oxidation process proceeding in an adsorbed state 7-10 . For the study of redox behavior 

of 6-BAP and its derivatives on mercury electrode it is important to know their 

protonation equilibria and due to this fact we determined the protonation constants 

(pKa) using potentiometric and spectral methods (UV-Vis). 

2. EXPERIMENT 

Using linear sweep voltammetry (LSV) the reduction behavior of 6 - 

benzylaminopurine and its chlorine and methoxy derivatives has been investigated. 

The measurements were performed using the analyzer Autolab 20 EcoChemie 

connected with the VA Stand 663 and controlled by GPES manager software. The 

main part of this apparatus was electrochemical vessel equipped with three electrodes, 

in which all voltammetric measurements were carried out. The mercury drop 

electrode with effective area 0.4 mm 2 performed the function of working electrode, an 

argent chloride electrode (Ag/AgCl/3MKCl) and a platinum wire were the reference 

and counter electrode, respectively. The samples of analyzed derivatives (most often 

1·10 -5 M) were prepared in the phosphate – acetate buffer with pH ranging from 4.53 

to 6.69 and with addition of 10 (v/v) percentages of CH3OH. The ionic strength was 

set to 0.1 M by NaCl. For the study of concentration dependences of 6-BAP, the 

concentration range from 1·10 -5 to 0.5· 10 -4 M of 6-BAP in phosphate - acetate buffer 

(pH 2.8) with 10(v/v) percentages of CH3OH and with ionic strength 0.1 M (NaCl) was 

used. Experimental conditions of LSV were following: the scan potential was from - 

1.1 V to -1.7 V, the time of conditioning of electrode was 2 seconds, the potential step 

was 2 mV. The LSV curves were measured at different scan rates from 100 to 800 

mV/s. All voltammetric measurements were performed in inert atmosphere (argon) 

and at room temperature. Three of these curves with integer 2 (1/2, 1, and 2 multiple 

of reference scan rate) were taken for EVLS procedure. 

The potentiometric titrations were carried out using an automatic titration 

apparatus Titrando 835 controlled with Tiamo 1.2 software. The potential change 

during the titration was measured with the combined ISE electrode LL Ecotrode plus. 

All measurements were carried out in a tempered 50 mL tube in inert argon 

atmosphere. The potentiometric titrations were proceeded using the standard titration 

solution of 0.1 M NaOH, prepared in CH3OH (from 5 to 20 v/v percentages of 

CH3OH), in the temperature range from 15 to 37 °C. The ionic strength was set to I = 

0.1 M with NaCl. The concentration of analyzed samples was 1·10 -4 M. 

The spectrophotometric titration was acidimetric titration of alkaline sample 

(pH 11, I=0.1 M) by acidic sample (pH 2, I=0.1M) in 1 cm quartz cuvette. The starting 

volume was 1.5 mL and after each addition of the acidic sample the absorption 

spectrum (UV-Vis) using spectrophotometer UNICAM UV 4, connected with Vision 

32 software, was measured. After measuring of each spectrum, the actual pH value 

using LL Biotrode and pH meter CyberScan PCD 6500 was determined. The 

spectrophotometric titrations were performed in 10 (v/v) percentages of CH3OH. The 

- 208 -

XI. WWorkshop 

of Phyysical 


sammples 

of 6-BBAP 

and its derivativess 

were prep pared with concentrattion 

of 3.66 6·10 

Spectroscopic 

data were e exportedd 

to EQUI ISPEC (MA ATLAB) an and the va 

prottonation 

coonstants 

(pK Ka) were caalculated. 

-5 M. 

alues of 

3. RESULTTS 

AND DISCUSSI 

D ION 

6-BAP annd 

its deriv vatives (Cl-and 

-OCH3 3) were stud died by botth 

equilibri ium and 

dynnamic 

electtrochemica 

al methodss 

(potentiometry 

and d voltamme metry) and by UV 

absoorption 

speectra. 

While 

potentiommetric 

and spectral ex xperiments provided the t data 

for the deterrmination 

of pKa, vvoltammetri 

ic experim ment (LSV and EVLS) 

gave 

eviddence 

abouut 

the electr ron transferr 

in the pro ocess of redu uction. 

LSVV 

AND EVLLS 

VOLTAM MMETRY 

The LSVV 

experiment 

was perrformed 

in phosphoric 

– acetic buffer (pH H 4.1) in 

10% % (v/v) CH33OH. 

The pH 

value waas 

chosen according 

to o pKa valuees 

ensuring the fact 

thatt 

derivativees 

are in protonated 

foorms 

requir red for thei ir reductionn. 

Reductio on BAPs 

signnals 

were oobserved 

at t the potenntial 

app. -1300 

mV. Figs. 1 andd 

2 show LSV L and 

EVLLS 

curves oof 

2-, 3-, an nd 4- Cl BAAPs, 

Figs.3 and a 4 show w LSV and EEVLS 

curves 

of 2-, 

3-, aand 

4- OCHH3 

BAPs. Both 

types oof 

derivativ ves yield EV VLS peak–coounterpeak 

k signals 

indiicating 

the electrode process p in aan 

adsorbed 

state. Mo oreover, in the case of f 2- and 

4-mmethoxy 

deerivatives 

we w can obbserve 

the second sm mall peak in more negative n 

poteentials. 

Thiis 

new proc cess will be studied in our further r research. 

Fig. .1: LSV of cchlorine 

der rivatives off 

6 – BAP 

Fig. .2: EVLS oof 

chlorine e derivativves 

of 6 – 

in 10% (v/v) CH3O OH (pH 4.1) 

- 209 - 

BAP in 10% 1 (v/v) 

Brno 

CH3OH (p pH 4.1)

XI. Workshhop 


E 

s´11 

Fig.3: LLSV 

of methhoxy 

deriva atives of 6 – BAP 

Fig.4: EEVLS 

of mmethoxy 

de erivatives of 6 – BA AP in 10% % (v/v) CHH3OH 

(pH 4.1) 

in 10% (v/vv) 

CH3OH (pH ( 4.1) 

POTENNTIOMETRRIC 

TITRAT TION 

The 

protonaation 

consta ants pKa1 annd 

pKa2 of 6-BAP 6 were 

influenceed 

by methanol 

contentt 

and tempperature 

(T Tab.1) and with incre easing of th hese parammeters 

both pKa 

values decrease. TThe 

tempe erature deppendence 

of o pKa allow wed the thhermodyna 

amic 

evaluattion 

of prottonation-de 

eprotonatioon 

equilibri ium. Therm modynamicc 

functions ∆H, 

∆S and ∆G in the dependenc ce on the mmethanol 

co ontent are presented p inn 

Tab.2. While W 

the vallues 

∆G aree 

practicall ly same witth 

v/v perc centages of f methanoll, 

enthalpy and 

entropyy 

of the deeprotonatio 

on process oof 

BAP, in ndicating an n exothermmic 

process, , are 

changed 

with diffferent 

cont tent of metthanol. 

Acc cording calc culated valuues 

of enth halpy 

and enttropy 

is alsoo 

evident th hat increasiing 

content t of methan nol causes aan 

increasin ng of 

disordeered 

protonnation 

state and the neegative 

valu ues of entropy 

are obtaained 

(Table 

2). 

Tab. 1: : The influuence 

of pK Ka values oon 

the tem mperature for f 6 – BAAP 

in diffe erent 

content of CH3OH at I = 0.1 M 

t 

[°C] 

15 

20 

25 

30 

37 

pKa1 

4.16 ± 

0.03 

4.10 ± 

0.03 

4.08 ± 

0.03 

4.02 ± 

0.02 

3.98 ± 

0.03 

5% CH3OH 

pKa2 2 pKKa1 

10.96 ± 4.002 

± 

0.06 0. .03 

10.80 ± 3.996 

± 

0.06 0. .02 

10.75 ± 3.888 

± 

0.08 0. .04 

10.70 ± 3.880 

± 

0.07 0. .01 

10.57 ± 3.771 

± 

0.07 0. .02 

10% CH3O OH 

- 210 - 

pKa2 

10 0.76±0.0 

8 

10 0.71±0.0 

8 

10 0.63±0.0 

7 

10 0.59±0.0 

6 

10 0.49±0.0 

8 

pKa1 

3.98 ± 

0.01 

3.93 ± 

0.02 

3.83 ± 

0.02 

3.77 ± 

0.03 

3.67 ± 

0.02 

20% % CH3OH 

pKa2 2 

10.55 ± 

0.07 7 

10.44 ± 

0.06 6 

10.36 ± 

0.08 8 

10.33 ± 

0.07 7 

10.22 ± 

0.07 7 

Brno


Tab.2: Thermodynamic parameters of 6 – BAP at I = 0.1 M 

∆Hexp 

[kJ.mol - 

1 ] 

∆Sexp 

[J.K -1 .mol - 

1 ] 

∆Gexp 

[kJ.mol - 

1 ] 

SPECTROPHOTOMETRIC TITRATION 

The protonation constants pKa1 and pKa2 of 6-BAP and its derivatives 

determined by means of spectrophotometric titration and presented in Table 3 are in 

good accordance with pKa1 and pKa2 determined by means of potentiometric 

titrations. The table illustrates not only the effect of Cl or OCH3 substituent but also 

the effect of its position. 

Tab.3: pKa values calculated from measured UV – Vis spectra 

compound pKa1 pKa2 

BAP 4.22 ± 0.07 9.84 ± 0.05 

2´- Cl BAP 4.13 ± 0.05 9.84 ± 0.04 

3´- Cl BAP 4.06 ± 0.06 9.80 ± 0.04 

4´- Cl BAP 4.14 ± 0.05 9.83 ± 0.04 

2´- OCH3 BAP 4.56 ± 0.06 9.98 ± 0.04 

3´- OCH3 BAP 4.33 ± 0.05 9.83 ± 0.04 

4´- OCH3 BAP 4.51 ± 0.05 9.87 ± 0.03 

The spectral data processing is illustrated by the experiment of 2‘- Cl-BAP 

(Figs.5 and 6). Fig.5 shows the spectra recorded during the titration of sample and 

Fig.6 shows its concentration profile calculated by EQUISPEC in MATLAB 

environment. 

Absorbance [a.u.] 

1 

0.9 

0.8 

0.7 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

4. CONCLUSION 

For the study of electrochemical and spectral behavior of 6 – BAP and its 

methoxy and chlorine derivatives voltammetry, potentiometry and 

spectrophotometry have been used. In linear sweep voltammetry (LSV) in connection 

- 211 - 

v/v 

percentages 

of CH3OH 

-13.89 31.33 -23.23 5 

-24.67 -8.53 -22.13 10 

-24.69 -9.33 -21.91 20 

Spectrum 

0 

220 230 240 250 260 270 280 290 300 310 320 

Wavelength [nm] 

Relative concentration 

3.5 

3 

2.5 

2 

1.5 

1 

0.5 

x 10-5 

4 

Concentration profile 

0 

3 4 5 6 7 8 9 10 

pH


with a mercury electrode the reduction peaks of BAPs were observed. The pH value 

of water-methanol solutions was chosen according to the protonation constants of 

BAPs determined by both potentiometric and UV-Vis spectrophotometric titrations. 

Using elimination voltammetry with linear scan (EVLS) the differences in the 

reduction processes of derivatives in adsorbed state were found. In the case of the 

methoxy derivatives of 6 – BAP LSV curves (measured at pH 4.1) indicate a new 

process which is visible only at 2‘-OCH3 BAP and at 4‘ – OCH3 BAP. The EVLS 

confirmed the electron transfer in an adsorbed state (peak – counter peak form). The 

effect of methanol content and temperature on pKa values of 6-BAP was monitored. 

According to the dependence of the content of methanol on pKa is evident that with 

increasing of the molar fraction of methanol in solution leads to decreasing of pKa 

values. Based on the dependence of pKa on the temperature it was possible to 

determine the thermodynamic parameters (enthalpy and entropy). It was found that 

ΔS becomes more negative values with increasing content of methanol. Protonation 

constants determined by potentiometric titrations were compared with pKa values 

calculated from UV – Vis spectra. 


This work has been supported by projects INCHEMBIOL MSM0021622412, 

BIO-ANAL-MED LC06035 and MUNI/A/0992/2009 by the Ministry of Education, 

Youth and Sports of the Czech Republic. 

6. REFERENCES 

[1] Tarkowski, P.; Dolezal, K.; Strnad, M. Chem. Listy (2004), 98, 834. 

[2] Tarkowska, D.; Kotoucek, M.; Dolezal, K. Collect. Czech. Chem. Commun. (2003), 68, 1076. 

[3] Klanicova, A.; Travnicek, Z.; Vanco, J.; Popa, I.; Sindelar, Z. Polyhedron, 29, 2582. 

[4] Malon, M.; Travnicek, Z.; Marek, R.; Strnad, M. J. Inorg. Biochem. (2005), 99, 2127. 

[5] Malon, M.; Travnicek, Z.; Marysko, M.; Marek, J.; Dolezal, K.; Rolcik, J.; Strnad, M. Transit. Met. 

Chem. (2002), 27, 580. 

[6] Malon, M.; Travnicek, Z.; Marysko, M.; Zboril, R.; Maslan, M.; Marek, J.; Dolezal, K.; Rolcik, J.; 

Krystof, V.; Strnad, M. Inorg. Chim. Acta (2001), 323, 119. 

[7] Trnkova, L. Chem. Listy (2001), 95, 518. 

[8] Trnkova, L.; Jelen, F.; Petrlova, J.; Adam, V.; Potesil, D.; Kizek, R. Sensors (2005), 5, 448. 

[9] Trnkova, L.; Jelen, F.; Postbieglova, I. Electroanalysis (2003), 15, 1529. 

[10] Trnkova, L.; Kizek, R.; Dracka, O. Electroanalysis (2000), 12, 905. 

- 212 -


SPRAY-COATED WORKING ELECTRODES 

FOR ELECTROCHEMICAL SENSORS 

Jan PRÁŠEK 1 , Petra BUSINOVÁ 1 , Jana CHOMOUCKÁ 1 

1 Dept. of Microelectronics, Brno University of Technology, Technicka 10, 616 00 Brno, Czech Republic 

Abstract 

This paper is related to an area of electrochemical analytical systems. The problematic 

of three electrode planar systems miniaturization is discussed. Standard working 

electrode of three electrode thick film sensor have been modified using carbon 

nanotubes and deposited on the alumina substrate using spray-coating technique. 

Fabricated electrode was compared with commercial planar electrode and standard 

glassy carbon electrode resulting in better detection limit and sensitivity. 


Recently, the small analytical systems for electrochemical analyses are in the 

point of investigation [1]. Small, usually planar three-electrode systems are used 

instead of standard electrodes. The size of electrode area varies from units of square 

millimetres in case of thin film electrodes systems to tens of square millimetres in case 

of other electrodes systems (e.g. thick film electrodes on alumina substrate). In 

electrochemical analysis, the electrode systems miniaturization brings many problems 

as it was studied in [2]. The main problem is reduction of output signal which is equal 

to active size of working electrode due to active electrode area reduction. One 

possibility, how to solve this problem is to increase the active size of the electrode 

preserving the geometrical size of the working electrode (WE). It could be achieved 

by creation of nanostructures on electrode surface [3-5] which could increase the 

active electrode size several times. Electrochemical sensors with such nanostructures 

could be also used as a base for intelligent sensors [6]. 

In this work we fabricated carbon nanotubes (CNTs) based spray-coated 

working microelectrodes for electrochemical sensors. Fabricated electrodes were 

examined optically using scanning electron microscopy and electrochemically on the 

detection of heavy metal ions in acetate buffer solution using differential pulse 

voltammetry (DPV). Obtained results were compared with standard glassy carbon 

electrode and commercial CNTs based electrochemical sensor. 

2. EXPERIMENT 

Working microelectrodes were fabricated using standard thick-film technology 

process on the alumina substrate. Thick-film pastes used for contact and covering 

layers were ESL 9562-G and ESL 4917 both from ESL Electroscience, UK. The 

working electrode was fabricated by spray-coating deposition using multiwalled 

carbon nanotubes (MWCNTs) powder as the filling material (parameters: OD=40-70 

nm, ID=5-40 nm, length=0.5 2 μm) and N-Methyl-Pyrrolidone (NMP) as a carrier and 

- 213 -

XI. Workshhop 


E 

s´11 

bindingg 

material. Sample of o fabricateed 

electrod de is show wn in the 

microgrraph 

of fabricated 

elec ctrode is shhown 

in the e figure 2. 

AAll 

of used chemicals were purcchased 

from 

Sigma Aldrich A (Stt. 

Louis, USA), U 

unless noted othherwise. 

Differential 

pulse volt tammetry (DPV) in range of the 

potentiial 

from -1 to 0 V wit th scan ratee 

of 50 mV V/sec was performed 

uusing 

PalmS Sens 

handheeld 

potenntiostat/galvanostat 

(Palm Instrument ts BV, Netherlan nds). 


eexperiments 

were carr rried out in n a 5 mL voltammetriic 

cell at room 

temperrature 

(25°CC), 

using a three-elecctrode 

conf figuration system s withh 

the stand dard 

Ag/AgCCl 

referencce 

electrode e type 6.07726.100 

and d platinum m auxiliary electrode type t 

6.0343. 000 (both ffrom 

Metro ohm, CH). 

Fig.1 FFabricated 

sample of the t MWNTTs 

Fig g.2 SEM mic crograph off 

fabricated d 

based 

sppray-coated 

d working electrode el 

electrode e 

3. RRESULTS 


MMWNTs 

baased 

solutio on was useed 

for wor rking elect trodes of eelectrochem 

mical 

sensorss 

fabricationn. 

SEM ima age of the ffabricated 

electrode are a shown iin 

the figur re 2. 

From thhe 

SEM figgures 

of spr ray-coated eelectrode 

is s clear that the homoggenous 

laye er of 

openedd 

MWCNTss 

was achieved. 

It parttially 

satisf fied our pre esumption oof 

suitabilit ty of 

NMP uutilization 

aas 

the bindin ng materiall. 

The 

electroddes 

were ele ectrochemiically 

meas sured in ace etate bufferr 

solution using u 

differenntial 

pulse vvoltammetry. 

Lead ions 

were used 

as the detected 

maatter. 

Sampl le of 

DPV reesponse 

of fabricated electrode tto 

lead ions s is shown in the figuure 

3. From m the 

figure 3 is clear that the current c ressponse 

to lead l ions addition a is good and the 

electrodde 

could bee 

used for lead l ions deetection 

wi ith good lin nearity as iis 

shown in n the 

inset inn 

the figure 3. 

Foor 

better eevaluation, 

the fabriccated 

MW WNTs based d working electrode was 

comparred 

with staandard 

glas ssy carbon ( (GC) electr rode type 6.1204.300 

( (Metrohm, CH) 

and woorking 

elecctrode 

from m commerrcial 

sensor r DS 110CNT 

(DropSSens, 

ES). The 

calibrattion 

curvess 

compariso on for Pb ioons 

of all measured m electrodes 

iss 

shown in n the 

figure 44. 

- 214 - 

Brno 

figure 1. SEM S

XI. WWorkshop 

of Phyysical 


From thhe 

figure 4 is clear thhat 

with considering c of the ressults 

recou unted to 

currrent 

densitty 

for bette er evaluatioon 

of differ rently sized d electrodees, 

the resp ponse of 

fabrricated 

elecctrode 

to le ead ions is almost two o times hig gher than thhe 

response 

of GC 

elecctrode 

and little bit higher 

than the respon nse of comm mercial DSS 

110CNT working w 

elecctrode. 

Thee 

linearity of o our electtrode 

is als so better th han in case of commer rcial DS 

1100CNT 

working 

electrod de. 

Another experimen nt confirmeed 

that our r electrode without anny 

modific cation is 

ablee 

to detect tthe 

concentrations 

froom 

1 mol/ /L of lead io ons. 

Fi Fig.1 DPV respponse 

of prepa pared electrodde 

to Pb Fig.1 Fi Calibratio on curves commparison 

of fa abricated 

ions ( (inset: calibrat ation curve) 

MWNTs M based d electrode, sttandard 

GC electrode e 

an nd MWNTs based b WE froom 

DS 110CN NT sensor 

on the lead ionss 

detection 


In this work, the ere was faabricated 

spray-coate s ed MWNTTs 

based working w 

elecctrode 

for tthick-film 

electrochem 

e mical senso ors as a mix xture of CNNTs 

with NMP 

and 

commpared 

withh 

standard GC electroode 

and WE W from com mmercial ssensor 

DS 210CNT 2 

on eelectrochemmical 

detection 

of leadd 

ions in a three t electr rode systemm. 

Obtainedd 

results sh hown in chhapter 

3 con nfirmed th he suitabilitty 

of our el lectrode 

for electrochemmical 

analy ysis. Considdering 

the results r reco ounted to cuurrent 

dens sity, the 

respponse 

of faabricated 

electrode 

too 

lead ions s is almost two timess 

higher th han the 

respponse 

of GGC 

electrode 

and littlee 

bit highe er than the e response of commer rcial DS 

1100CNT 

working 

electrode. 

The linnearity 

of ou ur electrode e is also bettter 

than in n case of 

commmercial 

DDS 

110CN NT workinng 

electro ode. We were ablee 

to dete ect the 


from 1 m mol/L of leaad 

ions with hout any electrode 

moodification. 

Finally ccould 

be con ncluded thaat 

the fabri icated electrodes 

couldd 

be used as 

a good 

basee 

for other experimen nts with eleectrodes 

modification 

to be suitaable 

for bio osensing 

appplications. 


EMENT 

Funding for this wo ork was proovided 

by the t Grant agency a of thhe 


undder 

the conntracts 

GA ACR 102/088/1546, 

and d Czech Ministry M of Education n in the 

framme 

of Reseaarch 

Plan MSM M 00216630503 

MIK KROSYN. 

- 215 - 

Brno


6. 6.REFERENCES 

[1] Prasek, J., et al.: Sensors, Vol. 6, No. 11, 2006, pp. 1498-1512 

[2] Krejci, J., et al.: Microelectronics International, Vol. 21, No. 3, 2004, pp. 20-24 

[3] Prasek J., et al.: 5th IEEE Sensors Conference, 2006, pp. 1257-1260 

[4] Klosova, K, et al.: Phys. stat. sol. A, Vol. 205, No. 6, 2007, pp. 1435-1438 

[5] Prasek, J., et al.: 33rd International Spring Seminar on Electronics Technology, 2010, pp. 1-4 

[6] Fujcik, L., et al.: Microelectronics International, Vol. 27, No. 1, 2010, pp. 3-10 

- 216 -


IMPROVEMENT OF SENSING PROPERIES 

OF THE THICK-FILM ELECTROCHEMICAL 

SENSORS BY SPECIFICATION 

MEASUREMENT WITH THE ROTATING 

VESSEL CELL 

Zdenek PYTLÍČEK 1 , Jan PRÁŠEK 1 


Abstract 

This paper solves sensing properties optimization of thick-film electrochemical 

sensors for detection of substances in aqueous solutions using new electrochemical 

analytical device. The device uses rotating vessel cell for defined and reproducible 

measurement. The sensing properties have been examined using cyclic voltammetry 

method in a standard electrochemical solution of potassium ferro-ferricyanide. 

Distribution of response and noise in dependence on sensor position in vessel cell 

have been obtained and studied. Finally a possible sensor’s shapes influence to sensor 

response improvement is mentioned and discussed. 


Electrochemical analysis of species that are dissolved in water solutions is one of 

the cheapest analyses in this field. Generally the electrochemical analysis is provided 

by laboratory potentiostats with use of various electrochemical arrangements. 

Commonly used arrangements are unstirred cells, rotating disk electrodes, channel 

cells or wall-jet arrangements. All of mentioned electrochemical arrangements are 

designed to be used with solid electrodes, which is in contradiction with standard 

electrochemical analysis that commonly uses mercury drop electrode due to its very 

good sensing properties. The commercial standard solid electrodes are usually of high 

dimensions and cannot be used in small systems. The miniaturized solid electrodes in 

a form of more electrodes sensor system can be fabricated using thick-film technology 

(TFT). Such thick-film electrochemical systems (sensors) could be used as a base for 

another electrodes enhancement using nanotechnologies [1-3] or in complex sensors 

[4]. This work solves the optimization of new electrochemical analytical arrangement 

with rotating vessel [5] that was designed especially for use with thick-film 

electrochemical sensors. The device was designed to ensure reproducible mass 

transport of the solution to the electrodes. The optimization of the sensor and its 

position in the rotating vessel with respect to signal noise ratio was studied in this 

work. 

- 217 -

XI. Workshhop 


E 

s´11 

2. EXPERIMEENT 

Inn 

our previous 

work [5] was shhown 

that the new electrochem 

e mical analy ytical 

arrangeement 

is working 

wel ll and can bbe 

used for r electrochemical 

anaalysis. 

Princ ciple 

and firsst 

steps of ssensing 

pro operties of the sensor evaluation n using thiss 

device and 

its 

comparrison 

with other syste ems have bbeen 

alread dy described 

[5]. It wwas 

shown, that 

there iss 

a possibility 

to vary output currrent 

respo onse of the sensor in ddependence 

e on 

sensor position inn 

the vessel l that can bbe 

changed d in three axes 

accordiing 

to Figu ure 1 

(left). TThis 

new electrochemi 

ical analytiical 

devices is designed d especiallyy 

for work with w 

thick-fiilm 

sensorss 

fabricated on aluminna 

substrate shown in the t Figure 1 (right). 

Fig. 1 RRotating 

veessel 

cell with w possiblle 

changes (left) and real TFT ssensor 

used d for 

measuremeents 

(right) 

AAll 

measureements 

have 

been carrried 

out us sing cyclic voltammettry 

in rang ge of 

the pottential 

fromm 

-100 to 350 3 mV. MMeasuremen 

nt with sca an rate of 225 

mV/sec was 

performmed 

using tthe 

Voltala ab PST050 (Radiomete er analytica al, Denmarrk). 

The de evice 

was connnected 

to a personal l computerr 

for measu urement me ethod setupp 

and respo onse 

evaluattion. 

As aan 

electro ochemical standard solution a 5 mmool/L 

potass sium 

ferrocyyanide 

K4Fee(CN6) 

and 5 mmol/L ppotassium 

ferricyanide 

f e K3Fe(CN66) 

was prepared 

using 18 

M deioonized 

and redistilled water take en from Dir rect-Q Watter 

Purifica ation 

System (Milliporee). 

All used d chemicalls 

were fro om Sigma Aldrich A (Stt. 

Louis, USA). U 


eexperiment 

ts were caarried 

out in rotatin ng vessel ccell 

(18 ml m of 

solutionn) 

at room temperatur re (25 °C), uusing 

a thre ee-electrode 

system coonfiguration 

n. 

3. 3.RESULTSS 

AND DISCUSSIO 

D ON 

Fiirst 

measurrements 

we ere done too 

know the level of th he noise duuring 

chang ge of 

rotationn 

speed. Thhe 

rotation speed was cchanged 

fro om 0 to 450 0 rpm. The dependenc ce of 

output current reesponse 

an nd its corrresponding 

noise leve el measureed 

at poten ntial 

aroundd 

300 mV onn 

the chang ge of rotatioon 

speed is shown in the t Figure 2 a). 

Seecond 

measurements 

were donee 

with two different position p of f the sensor r. In 

the firsst 

one the ssensor 

elect trode systeem 

was orie ented outside 

and thee 

second on ne to 

the cenntre 

of the vvessel. 

All of measureements 

wer re done at the t tree levvels 

of z axi is (0, 

1, 2 mmm). 

En exammple 

of best 

results obbtained 

for first positio on of the seensor 

electr rode 

system is shown inn 

the Figur re 2 b) and FFigure 

2 c). . 

- 218 - 

referennce 

electrode (A Ag) 

workinng 

electrode (Au u) 

Brno

XI. WWorkshop 

of Phyysical 


c) 

Fig. . 2 Dependeence 

of out tput currennt 

response and its corr respondingg 

noise leve el on the 

change of rotation 

speed (aa), 

Sensor oriented o ou utside of tthe 

rotating g vessel 

currentt 

response for the z = 0 an nd rotation n speed 1125 

rpm (b) ( and 

correspponding 

noi ise level (c) ) 

The last experimen nt was donne 

with mo odified sensor 

edges ( (see Fig. 3 left) to 

ensuure 

better mass flow w along thhe 

electrod des with minimum m tturbulences 

s. From 

commparison 

off 

unchanged d and modiified 

sensor r response (see Fig. 3 right) is cl lear that 

withh 

use of mmodified 

sen nsor was acchieved 

hig gher curren nt response but the no oise was 

alsoo 

increased. 

It conclud des that thhe 

presump ption of lov ver noise leevel 

with modified m 

senssor 

was nott 

confirmed d. 

Fig. . 3 Modifiication 

of edges of ssensor 

(left ft) and com mparison oof 

unchang ged and 

modifieed 

sensor re esponse (rigght) 


There wwas 

made new n electroochemical 

analytical device opptimization 

in this 

worrk. 

For the experimen nts the rotattion 

speed of o the vesse el 125 rpm was chosen n due to 

- 219 - 

a) 

b) 

Brno


high output current response with low level of noise at this value. During the 

investigation of the best sensor positions in the vessel were studied two different 

orientations of the sensor. In both cases was found that the best results were achieved 

when the sensor was put in its deepest position (z axis = 0). From the comparison of 

both sensors orientation is clear that better results in all xy range can be achieved 

with sensor oriented outside of the vessel, although the position with best ration 

signal/noise was found in sensor electrodes to the centre orientation. The last 

experiment was done with modified sensor edges to ensure better mass flow along the 

electrodes with minimum turbulences. From comparison of unchanged and modified 

sensor is clear that with use of modified sensor was achieved higher current response 

but the noise was also increased. It concludes that the presumption of lover noise level 

with modified sensor was not confirmed. 


Funding for this work was provided by the Czech grant agency under the 

contract GACR 102/08/1546 and Czech Ministry of Education in the frame of 

Research Plan MSM 0021630503 MIKROSYN. 

6. REFERENCES 

[1] Prasek J., et al.: IEEE Sensors, Vol. 1-3, 2006, pp. 1257-1260 

[2] Prasek J., et al.:, 33rd International Spring Seminar on Electronics Technology, 2010, pp. 1-4 

[3] Majzlik P., et al.: Listy cukrovarnicke a reparske, Vol. 126, No. 11, 2010, pp. 413-414 

[4] Fujcik L., et al.: Microelectronics International, Vol. 27, No. 1, 2010, pp. 3-10 

[5] Prasek J, et al. Proceedings of the IEEE Sensors, Vol. 1-3, 2004, pp. 749-752 

- 220 -


FLUORO/NITRO DERIVATIVES OF 

QUINOLONES: THEORETICAL AND 

SPECTROSCOPIC STUDY 

Ján RIMARČÍK 1 , Kraiwan PUNYAIN 2 , Erik KLEIN 1 , Vladimír LUKEŠ 1 , Dana 

DVORANOVÁ 1 , Anne-Marie KELTERER 2 , Vlasta BREZOVÁ 1 

1 Slovak University of Technology, Radlinského 9, SK-812 37 Bratislava, Slovak Republic 

2 Graz University of Technology, Stremayrgasse 9/I, A-8010 Graz, Austria 

Abstract 

In this work, we have studied fluoro and/or nitro substituted oxoquinolines 

theoretically as well as spectroscopically. Characterization of these novel potential 

drugs was oriented on oxo/hydroxy conformation stabilities, UV/vis and IR spectra, 

and EPR study of the radical species obtained upon photoinduced electron transfer 

from photoexcited TiO2 particles. 


1,4-Dihydro-4-oxoquinoline derivatives (4-quinolones) substituted at 4pyridone 

or benzene rings represent molecules possessing a variety of biological 

activities including antimicrobial, antiviral, antimycotic, antiprotozoal and 

antimalarial effects. Nowadays, the fluoro-substituted 1,4-dihydro-4-oxoquinoline-3carboxylic 

acids (fluoroquinolones) play a specific role in medicine. 

This study is oriented on the theoretical and spectroscopic characterization of 

conformation stabilities of new potential quinolone drugs [1]. The optimized 

geometrical parameters of ethyl 1,4-dihydro-4-oxoquinoline-3-carboxylate (Q) and its 

6-fluoro and 8-nitro derivatives (FQ, QN, FQN, see Fig. 1) in the gas phase, polar 

(acetonitrile and dimethylsulfoxide) and non-polar (toluene) organic aprotic solvents 

have been studied [2]. 

The presence of nitro-substitution has an influence on the redox properties of 

quinolones, and allows generation and observation of corresponding radical anions 

(QN – and FQN –) using EPR. Theoretical study of the radical species obtained upon 

photoinduced electron transfer from photoexcited TiO2 particles to nitroquinolones 

represents another aim of this work. 

Quinolone R R' 

Q H H 

FQ F H 

QN H NO2 

FQN F NO2 

- 221 -


Fig.1 Structure, atom numbering and denotation of studied quinolones 

2. COMPUTATIONAL DETAILS 

DFT/B3LYP/6-31+G(d) calculations were performed using Gaussian 03 program 

package [3]. Solvent contribution was calculated employing the polarizable 

continuum model within IEF-PCM method [4]. Theoretical spectra of studied 

quinolones were constructed from the first 25 vertical excitations (TD-DFT) and their 

transition dipole moments using the Orca_Asa program [5]. Experimental details are 

summarized in ref 2 and references therein. 


The free rotation of COOEt group at C3 atom exhibits various structures 

determined by the value of torsion angle . This dihedral angle defined between the 

carbonyl group of COOEt and the C3–C4 bond within the pyridone moiety (see Fig. 1), 

provides two planar or roughly planar stable conformers with = 0 or 180 degrees, 

respectively. In the case of nitro-substituted hydroxy-tautomers (QN, FQN), NO2 

group is twisted out of the plane of quinolone moieties, and the existence of two 

stable conformers may be considered. However, both conformers exhibit almost the 

same properties. The gas-phase B3LYP/6-31+G(d) calculations with the inclusion of 

the Zero Point Energy (ZPE) corrections indicate that the hydroxy-form is clearly 

preferred in the case of Q and FQ quinolones. The presence of hydrogen 

intramolecular interaction between the OH group and oxygen atom of the 

neighboring COOEt substituent stabilizes this structure. In the case of the QN and 

FQN quinolones, the presence of electron withdrawing NO2 group is responsible for 

the stabilization of the oxo-tautomeric form via the intramolecular interaction 

between N1–H…O2N groups. However, the potential population of both tautomers in 

the gas phase should be assumed, due to the low values of relative B3LYP+ZPE 

energies of 7 and 4 kJ mol –1 for QN and FQN, considering most stable hydroxy-form 

and the most stable oxo-form. 

The geometrical changes induced by the fluorine and nitro groups are also 

reflected in the vertically excited singlet electronic states of studied quinolones. In the 

case of oxo-forms of the studied quinolones, in all environments, the first selected 

excitation energies lie in the range from 2.76 eV to 4.21 eV. In the hydroxy-forms, the 

first selected excitation energies appear in narrower interval from 3.22 eV to 3.99 eV. 

The mutual comparison of excitation energies obtained using the IEF-PCM 

model shows that the obtained energies are shifted up to 0.57 eV (QN in DMSO, 

second significant transition) with respect to the gas phase. The presence of the 

solvent causes small changes in oscillator strengths, too. In general, hydroxy forms of 

nitro-substituted quinolones, QN and FQN, show the highest sensitivity of excitation 

energies with respect to the solvent used. Despite the certain shifts of the calculated 

TD-DFT B3LYP(IEF-PCM=ACN)/6-31+G(d) convoluted absorption bands, the 

- 222 -

XI. WWorkshop 

of Phyysical 


simuulated 

UV/ /vis spectra a obtained iin 

ACN are e in good ac ccordance wwith 

the do ominant 

pressence 

of oxxo-tautomer 

rs of all studdied 

quinol lones. 

In orderr 

to under rstand thee 

effect of f the centr ral 4-pyriddone 

unit on the 

absoorption 

speectra, 

it is useful to eexamine 

th he relevant t frontier mmolecular 

orbitals, o 

whiich 

play a ddominant 

role r in electtronic 

transitions 

of the 

quinoloones 

Q and FQN in 

oxoo-forms. 

Thhe 

calculate ed first elecctron 

transi itions at 4. 21 eV for Q and 2.81 1 eV for 

FQNN 

are basedd 

on the ele ectron excittation 

from m the highest 

occupiedd 

molecular r orbital 

(HOOMO) 

to thhe 

lowest un noccupied mmolecular 

orbital o (LUMO). 

The UVVA 

photoexc citation of f nitroquin nolones QN 

titannia 

suspennsion 

resul lted in thee 

formatio on of EPR 

maxximal 

spin density at 

nitro subbstituent 

on o benzene 

proppose 

that ssuitable 

red dox potentiial 

values of o 8-nitroqu 

trannsfer 

of electrons 

(pho otogenerateed 

upon Ti iO2 irradiat 

prodducing 

thee 

correspon nding radiccal 

anions QN 

deteected 

radiccal 

anion st tructures oobtained 

fro 

andd 

FQN quinnolones 

were 

also studdied 

at the 

calcculated 

B33LYP(IEF-P 

PCM=DMSOO)/6-31+G( 

seleected 

atomss 

were calculated 

and d visualizati 

2. Itt 

can be coonsidered 

that 

spin deensities 

for 

are spread maiinly 

over NO2 N group aand 

benzene 

N and FQN 

R signals c 

e moiety o 

uinolone de 

tion) to qui 

– and FQN 

om the ph 

B3LYP/6- 

(d) hyperf 

ion of spin 

r both radic 

e ring. 

N in a de-aerated 

characterize ed with 

of quinolone. 

We 

erivatives enable e a 

inolone mo olecules, 

– . Thhe 

experim mentally 

otoinducedd 

reduction n of QN 

31+G(d) levvel 

of theo ory. The 

fine coupliing 

consta ants for 

densities iss 

presented d in Fig. 

cal anions aare 

analogo ous, and 

[QN(ox xo)] – 

Fig. .2 Plots of f B3LYP(IE EF-PCM=DMMSO)/6-31 

1+G(d) spin n densities of most probable p 

radical products monitorred 

upon UVA photoinduceed 

reduct tion of 

nitroquuinolones 

(Q QN, FQN) iin 

TiO2 susp pension 


Results oof 

calculations 

confirmmed 

for all l derivative es the domiinance 

of the t oxo- 

tauttomers 

in ppolar 

solven nts, contraryy 

to toluen ne and the hypothetica h al gas phase e, where 

the hydroxy-fforms 

are preferred p foor 

Q and FQ. 

The sim mulated TDD-DFT 

B3LY YP(IEF- 

PCMM=ACN)/6-31+G(d) 

UV/vis U speectra 

for oxo-tautom mers in AACN 

are in 

good 

accoordance 

wiith 

the expe erimental oones, 

despit te certain shifts 

of inddividual 

abs sorption 

bannds. 

Derivativves 

with ni itro substittution 

at 8-position 

ar re able to uundergo 

re eduction 

usinng 

photoexxcited 

TiO2 as a sourcee 

of electro ons to the correspondi c ding radical anions, 

monnitored 

by in situ EPR 

spectrosccopy. 

The correspond ding radicaal 

anions QN Q 

– and 

- 223 - 

[F FQN(oxo)] ] – 

Brno

XI. Workshhop 


E 

s´11 

FQN – were also studied th heoreticallyy. 

The hyp perfine cou upling consstants 

obtained 

from exxperimentaal 

EPR spec ctra are in ccorrelation 

with evalu uated quanttum 

theoretical 

data. 

5. AACKNOWLLEDGEME 

ENT 

This 

study wwas 

financia ally supportted 

by Scientific 

Gran nt Agency (VVEGA 

Proj jects 

1/0137/ /09, 1/10722/11, 

1/022 25/08 and 1/0018/09 9) and Res search andd 

Developm ment 

Agencyy 

of the Sloovak 

Repub blic (contraccts 

Nos. AP PVV-0055- 07, LPP 02230-09, 

SK-AT- 

0002-088 

and SK-AT-0016-0 

08) and byy 

the Aus strian Acad demic Excchange 

Ser rvice 

programm 

Wissennschaftlich- 

-Technischee 

Zusamm menarbeit Austria-Sllovakia 

un nder 

contracct 

No. ÖADD 

WTZ 11/2 2009. 

6. RREFERENCCES 

[1] Riimarčík, 

J., LLukeš, 

V., Klein, K E., Kellterer, 

A.-M. ,Milata, V., Vrecková, ZZ., 

Brezová, V.: V J. 

Phhotochem. 

Phhotobiol. 

A Chem., 

211 (20010), 

47. 

[2] Riimarčík, 

J., PPunyain, 

K., Lukeš, V., KKlein, 

E., Dv voranová, D. , Kelterer, AA.-M., 

Milata a, V., 

Liietava, 

J., Brezzová, 

V.: J. Mol. M Struc. (20011), 

doi:10.10 016/j.molstru uc.2011.02.0555. 

[3] Poople, 

J. A., et al., GAUSSIA AN 03, Revisiion 

A.1, Gaussian, 

Inc., Pit ttsburgh, PA, 2003. 

[4] Bööes, 

E.S., Livootto, 

P.R., Sta assen, H., Cheem. 

Phys. 331 (2006), 142. 

[5] Peetrenko, 

T., NNeese, 

F.: ORC CA ASA, Verrsion 

2.6.35, University U of Bonn, Germaany, 

2008 

- 224 - 

Brno


THIOPHENOLS: ENERGETICS OF S–H 

BOND CLEAVAGE 

Lenka ROTTMANNOVÁ 1 , Ján RIMARČÍK 1 , Adam VAGÁNEK 1 , Erik KLEIN 1 , 

Vladimír LUKEŠ 1 

1 Institute of Physical Chemistry and Chemical Physics, Slovak University of Technology, Radlinského 

9, SK-812 37 Bratislava, Slovak Republic 

Abstract 

In this contribution, we have investigated thermodynamics of S–H bonds cleavage in 

meta-substituted thiophenols in the gas-phase and in two solvents. Reaction 

enthalpies for each mechanism were studied using density functional theory (B3LYP 

functional) with 6-311++G** basis set. 


Sulfur-centered radicals play an important role in organic synthesis, 

biochemistry and atmospheric chemistry. Therefore, this study is devoted to S–H 

bond cleavage in thiophenols. Hydrogen atom transfer (HAT) represents the generally 

accepted mechanism of antioxidant action. The reaction enthalpy of this mechanism is 

known as bond dissociation enthalpy (BDE). 

ArSH ArS + H BDE (1) 

First step of single-electron transfer – proton transfer (SET-PT) mechanism is 

governed by ionization potential (IP) of molecule. The second step of this mechanism 

is proton dissociation enthalpy (PDE) 

ArSH ArS + + e – IP (2) 

ArSH + ArS + H + PDE (3) 

Sequential proton loss electron transfer (SPLET) mechanism is also two-step 

mechanism of ArS radical formation. The reaction enthalpy of the first step of SPLET 

is described by proton affinity (PA) of formed anion. In the second step, the reaction 

enthalpy represents electron transfer enthalpy (ETE) 

ArSH ArS – + H + PA (4) 

ArS – ArS + e – ETE (5) 

The main aim of this work was to compute reaction enthalpies for homolytic 

and heterolytic S–H bond cleavage for selected compounds in order to identify 

thermodynamically preferred mechanism in studied environments. We have studied 

S-H bond dissociation enthalpies (BDE) related to equation 1, ionization potentials 

(IP) related to equation 2 and proton affinities (PA) related to equation 4. Two 

solvents with various polarities: benzene (C6H6) and water, were chosen. All 

enthalpies were calculated for 298.15 K. 

We have studied mono-substituted thiophenols with various groups in meta 

position (Fig. 1). 

- 225 -


X 

Fig.1 Studied thiophenols, X = NMe2, NH2, OH, MeO, tBu, Me, Ph, F, Cl, Br, MeCO, 

CF3, CN, MeSO2, NO2. 


All calculations were performed using Gaussian 03 program package [1]. 

Geometries of each compound, radical or ionic structure was optimized using DFT 

method with B3LYP functional without any constraints using 6-311++G** basis set. 

The optimized structures were confirmed to be real minima by frequency analysis. 

Solvent contribution to the total enthalpies was calculated employing integral 

equation formalism IEF-PCM method. This approach was used for the parent 

molecules and radicals, radical cations and anions. All IEF-PCM calculations were 

performed using default settings of Gaussian 03 program. 


From the calculated total enthalpies we have determined following quantities 

BDE = H(ArS ) + H(H ) – H(ArSH) (6) 

IP = H(ArSH + ) + H(e – ) – H(ArSH) (7) 

PA = H(ArS – ) + H(H + ) – H(ArSH) (8) 

Tab. 1: S–H bond dissociation enthalpies, ionization potentials and proton affinities in 

kJ mol –1 

Substituent BDE IP PA 

Gas 

a 

C6H6 H2O b Gas 

c 

C6H6 H2O d Gas 

e 

C6H6 H2O f 

316 325 322 786 661 468 1412 402 174 

m-NH2 314 322 316 719 598 408 1420 409 178 

m-NMe2 313 322 316 683 578 402 1420 412 178 

m-tBu 315 323 319 758 648 462 1413 406 176 

m-Me 315 324 320 769 651 462 1415 405 175 

m-Ph 316 325 322 747 642 463 1401 399 173 

m-OH 315 324 321 771 647 456 1404 398 173 

m-MeO 314 323 320 751 639 456 1410 401 173 

m-F 319 329 327 809 681 484 1391 387 167 

m-Cl 319 329 327 803 679 484 1386 384 166 

m-MeCO 316 327 326 800 681 485 1382 382 167 

m-Br 319 329 327 799 677 483 1383 383 166 

m-CF3 321 331 329 833 697 493 1374 378 165 

m-CN 322 333 331 837 705 498 1362 370 162 

m-MeSO2 322 333 332 823 701 500 1362 370 160 

m-NO2 323 334 333 845 713 505 1357 366 159 

a 

solvH(H • ) = 6.4 kJ mol –1 [2], b 

kJ mol –1 [4], 

d 

hydrH(e – ) = –105 kJ mol –1 [4], e 

–1022 kJ mol –1 [4] 

- 226 - 

SH 

hydrH(H • ) = –4 kJ mol –1 [3], c 

solvH(H + ) = –894 kJ mol –1 [4], f 

solvH(e – ) = –7 

hydrH(H + ) =


In general, free energy is the criterion of the thermodynamically preferred 

mechanism. However, in the case of studied reactions the absolute values of the 

entropic term –TrS reach only few units or tens of kJ mol –1 and all free energies, rG 

= rH – TrS, are only slightly shifted in comparison to corresponding enthalpies 

[4,5,6]. Therefore, values of BDE, PA and IP (Tab. 1) are able to indicate 

thermodynamically preferred mechanism. Due to the large differences, exceeding 

several hundreds of kJ mol –1 , HAT mechanism is thermodynamically preferred in gasphase, 

where BDEs are significantly lower than ionization potentials. Proton transfer 

described by proton affinity is by ca one order higher than BDE. In benzene, 

hydrogen transfer mechanism remains preferred. In solution-phase, ionization 

potentials reached the highest values. In water, proton affinities are considerably 

lower than BDEs, therefore SPLET should be thermodynamically favored mechanism. 

Differences between BDEs and PAs in water grow with the increase in electronwithdrawing 

effect of substituents. 

4. CONCLUSION 

For studied mono-substituted thiophenols we found that solvents are able to 

change the thermodynamically favored pathway of ArS formation. We can conclude 

that in studied environments reaction enthalpies grow in this order: 

gas-phase: BDE < IP < PA 

benzene: BDE < PA < IP 

water: PA < BDE < IP 


This work has been supported by Scientific Grant Agency (VEGA Projects 

1/0137/09, 1/1072/11 and 1/0127/09). 

6. REFERENCES 

[1] Pople J. A. et al.: GAUSSIAN 03, Revision A.1, Gaussian, Inc., Pittsburgh, PA, 2003. 

[2] M. M. Bizarro, B. J. Costa Cabral, R.M.B dos Santos, J. A. Martinho Simões, Pure Appl. Chem. 71 

(1999) 1249. 

[3] W. D. Parker, J. Am. Chem. Soc. 114 (1992) 7458. 

[4] J. Rimarčík, V. Lukeš, E. Klein, M. Ilčin, J. Mol. Struct. (Theochem) 952 (2010) 25. 

[5] M.J.S. Dewar, The Molecular Orbital Theory of Organic Chemistry, McGraw-Hill, New York, 

1990. 

[6] J. Rimarčík, V. Lukeš, E. Klein, L. Rottmannová, Computational and Theoretical Chemistry, 

submitted. 

- 227 -


ANALYSIS OF METALLOTHIONEIN 

ISOFORMS BY CAPILLARY 

ELECTROPHORESIS 

Markéta RYVOLOVÁ 1 , Tereza HÁJKOVÁ 2 , Petr MAJZLÍK 1,2 , Jaromír HUBÁLEK 2,3 , 

Vojtěch ADAM 1,3 , Ivo PROVAZNÍK 4 , René KIZEK 1,3 


Zemedelska 1, CZ-613 00 Brno, Czech Republic 

2 Department of Microelectronics Faculty of Electrical Engineering and Communication, Brno 


3 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 




Abstract 

Prostate cancer (CaP) is the second most frequently diagnosed cancer in developed 

countries and the third most common cancer causing death in men. Number of 

compounds is tested as potential CaP markers to improve the reliability of commonly 

used tests. Metallothionein (MT), due to its properties including high cysteine 

content, high heavy metal affinity and thermostability, is extensively studied as a 

potential cancer marker. Two major isoforms (MT-1, MT-2) have been identified in 

mammals and diagnostic potential of their ratio is investigated. 

Capillary electrophoresis is a separation effective tool enabling separation and 

identification of MT isoforms in mixtures. However the complexity of real samples 

requires sample pre-treatment to obtain valuable results and minimize the 

interferences of other compounds. Thermal denaturation of the sample is one of the 

simplest and often used methods for elimination of interfering components taking 

advantage of the MT’s thermostability. The effect of thermal denaturation on the 

separation of MT isoforms was studied in this work. 


Metallothioneins belong to a class of low molecular mass proteins characterized 

by high cysteine content and the lack of aromatic amino acids. 1 MTs are single-chain 

proteins with amino acid number oscillating between app. 20 and more than 100 

residues according to organisms. Almost one third of this number is cysteine 

occurring in conserved sequences cys-x-cys, cys-x-y-cys a cys-cys where x and y 

represent other amino acid. 2 

MT binds metals such as zinc, cadmium, copper and mercury with high affinity 

and its functions include the homeostasis essential metals and detoxification of toxic 

metal ions and its compounds. Probably the most widely studied group of MTs is the 

mammalian subfamily. There are 4 mammalian MT isoforms (MT1 – MT4) known 

- 228 -


and 13 MT-like human proteins were identified. MT1 and MT2 are present in almost 

all soft tissues, MT3 is specific for brain, heart and kidney tissues and MT4 was found 

in epithelial cells. Differences of constituent forms come mainly from posttranslational 

modifications, small changes in primary structure, type of incorporated 

metal ion and speed of degradation. 

Capillary electrophoresis (CE) is nowadays well established and very powerful 

separation tool for analysis of complex biological samples. The extreme separation 

power combined with short time of analysis and low sample requirements are main 

advantages of this effective analytical technique. CE is suitable for the separation of 

low Mr molecules and therefore it is optimal for MT analysis as well as for 

identification of MT isoforms. 

The diagnostic potential of MT isoforms is also investigated. It was found in a 

work of Kawata et al. 3 that MT1/MT2 ratio may improve the diagnosis of chronic 

hepatic disorder. CE was used in this work as an efficient separation method. Sample 

of MT isoforms extracted from liver tissue was pre-treated by 2 minute thermal 

denturation (100°C). However due to the high concentration of MT in tissues, no 

problems with denaturation were observed. The aim of this work is to investigate the 

impact of the denaturation on the CE signal of MT1 and MT2 in various 

concentrations, especially in blood stream, where the concentration of MT is in the 

range of 0.01 mg/ml. 

2. EXPERIMENT 

MT1 and MT2 standards were purchased by Sigma Aldrich and subsequently 

dissolved in deionized water to the required concentration. Thermal denaturation was 

performed at 99°C for 20 minutes. 

Capillary electrophoresis system Backman PACE 5510 (USA) equipped with UV 

absorbance detector was used in this work. Signal of analytes was measured at 214 

nm. An un-coated fused silica capillary (Polymicro Technologies, USA) with total 

length of 47 cm, effective length of 40 cm and internal diameter of 50 μm was 

employed. Sample was injected hydrodynamicaly by pressure of 3.4 kPa applied for 20 

s. Separation voltage was set to 10 kV. Borate buffer (20 mM, pH 9.5) was used as a 

background electrolyte (BGE). 


Separation of MT isoforms (MT1 and MT2) by CE was optimized including 

electrolyte composition and pH, capillary length, and separation voltage. Typical 

electropherogram is shown in Fig. 1. Well resolved signals of MT1 and MT2 are 

present with migration times of 6.8 and 7.2 minutes. 

- 229 -

XI. Workshhop 


E 

s´11 

Fig.1: TTypical 

elecctropherogram 

of MTT1 

and MT T2 separatio on. Conditiions: 

BGE – 20 

mM boratee 

buffer pH 9.5, voltagge 

- +10 kV, 

injection - 20s, 3.4 kkPa, 

detection 

– 

214 nm 

CCalibration 

curves obt tained for each isofor 

(Fig. 2) ) and limitss 

of detecti ion were oof 

1×10 

and MTT2, 

respectiively. 

-7 rm exhibit 

M and 8×10- ted a very good linea arity 

-8 M determmined 

for MT1 M 

( (a) 

Fig. 2: Calibrationn 

curves for r (a) MT1 aand 

(b) MT T2 obtained d by CE. CConditions 

as a in 

Fig. 1. 

To 

analyse comple re eal samplee 

mixtures ussually sample prre-treatmen 

nt is 

requireed. 

One of ssuch 

pre-tre eatment meethods 

is th hermal dena aturation. IIn 

this case is it 

- 230 - 

(b) ( 

Brno

XI. WWorkshop 

of Phyysical 


takiing 

advantaage 

of therm mal stabilityy 

of MT in comparison 

to other pproteins. 

However H 

the influence oof 

this proc cedure on tthe 

MT ana alysis has to o be verifieed. 

Moreove er, even 

thouugh 

the CEE 

separation n is bassed mostly on the charge of the anaalyte 

the molecular 

struucture 

has somewhat impact as well. For this reason n, the impaact 

of the thermal 

dennaturation 

was invest tigated. It was foun nd that in concentraation 

highe er than 

1 mmg/ml 

the ddenaturatio 

on has no iinfluence 

and a signals s of MT1 aand 

MT2 are a well 

resoolved 

in denatured 

as a well as in not de enatured is soform mixxture 

(Fig. 3a, b). 

Howwever 

in concentratio 

ons lower than 1 mg g/ml, signifi icant changges 

in sign nal were 

obseerved. 

As sshown 

in Fig. F 3c, in nnon-denetu 

ured mixtur re with conncentration 

n of 0.08 

mg/ /ml a good separation of isoforms 

is present t. The samp ple of the saame 

concen ntration 

unddergoing 

deenaturation 

n (20 min, 999°C) 

exhib bits a major r peak withh 

migration time of 

about 

6 minuttes 

(Fig.3d) ) relating too 

the MT however h does 

not reppresent 

any y of the 

isofforms 

and aas 

well can not be useed 

for MT quantificati q ion. The na natre of this s peak is 

stilll 

not clear aand 

require es further innvestigation 

n. 

(a) 

(b) 

- 231 - 

Brno

XI. Workshhop 


E 

s´11 

(c) 

Fig. 3: CE of MTT1 

and MT2 2 mixture separated under u optim mized condditions. 

(a) not 

denatured, c = 2.5 mg g/ml; (b) deenatured, 

c = 2.5 mg/m ml; (c) not denatured, 

c = 

0.08 mg/mll; 

(d) denatured, 

c = 0. .08 mg/ml. 

4. CCONCLUSION 

MMT 

isoforms 

were effi iciently sepparated 

by CE with good 

sensitiivity 

as we ell as 

linearitty. 

Sample pre-treatm ment plays a significa ant role in analysis off 

real com mplex 

mixturees. 

Thermaal 

denaturat tion is one of the sim mplest pre-tr reatment mmethods 

and 

its 

influennce 

on the pprotein 

mix xture was iinvestigated 

d in this stu udy. Signifiicant 

impac ct of 

denaturration 

was observed in n MT sampples 

with co oncentration n below 1 mmg/ml. 


ENT 

The 

work has been supportedd 

by Nano oBioTECell GA CR 

NANIMMEL 

GA CRR 

102/08/15 546 and RECAMT 

GA A AV IAA40 01990701 


[1] HHamer 

D. H.: MMarine 

Environmental 

Ressearch, 

24, 17 71 (1988). 

[2] KKagi 

J. H. R. : Methods in i Enzymoology, 

205, 613 6 (1991). 

[3] KKawata 

T., Nakamura S., Nakayyama 

A., Fu ukuda H., Ebara M., Nagamine e T., 

MMinami 

T., SSakurai 

H.: Biological & Pharmac ceutical Bulletin, 

29, 4403 

(2006). 

- 232 - 

(d) ) 

Brno 

P102/11/1068,


ANALYSIS OF SARCOSINE BY CAPILLARY 

ELECTROPHORESIS 

Markéta RYVOLOVÁ 1 , Natalia CERNEI 1 , Michal MASAŘÍK 2 , René KIZEK 1,3 



2 Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Kamenice 5, CZ- 




Abstract 

Sarcosine (Src) is investigated as a potentially valuable molecule in diagnostics of 

prostate cancer. This potential induced an extensive research of its properties, 

biological functions and connections to certain diseases. In this work, capillary 

electrophoresis is used for determination of sarcosine as a complex with copper ions 

present in background electrolyte. Subsequently, the method was used for 

determination of sarcosine in lysates of prostate cancer cells treated with various 

concentrations of Zn ions. 


Src is an N-methyl derivative glycine. In some studies it was found that content 

of Src increases significantly in CaP patients 1-3 and therefore it is currently studied for 

the potential role in prostate cancer (CaP) diagnostics. This hypothesis induced an 

interest in the precise and sensitive determination of Src is various body fluids such as 

blood serum and/or urine. For this reason, a range of analytical methods were 

evaluated. Due to its basic properties capillary electrophoresis (CE) was also 

considered as a suitable analytical method. CE enables determination of a wide range 

of analytes including small molecules such as sarcosine and due to its high separation 

power it is optimal for analysis of complex matrices including body fluids. In this 

work, CE method for Src analysis employing the interaction of Cu ions with 

molecules having un-bonded electron pair was evaluated and applied for 

determination of Src in cell lysates of prostate tissue cells. 

2. EXPERIMENT 

All analyses were carried out using CE instrument by Beckman Coulter (P/ACE 

5500). Separations were performed in an uncoated fused silica capillary with an 

internal diameter of 50 m and external diameter of 375 m. The total length of the 

capillary was 47 cm and the effective length was 40 cm. In the case of the real samples 

the length was extended to 57 cm and 50 cm for the total and effective length, 

respectively. The capillary was flushed with 0.1 M NaOH for 5 minutes and with 

background electrolyte for 10 minutes prior to the first use. Conditioning with 0.1 M 

- 233 -


NaOH for 0.5 minute and 2 minutes with BGE was done before every run to obtain 

clean capillary surface and thus reproducible separation. 

The composition of the background electrolyte (BGE) was adopted from the 

work of Jiang et al. 4 where 50 mM CuSO4 in 0.05% HAc (v/v) was found to be an 

optimal BGE for the separation of amino acids in combination with the absorbance 

detection at 254 nm. The sample was injected hadrodynamicaly by applying of 3.4 kPa 

for period of 30 s and the separation voltage was set to 15 kV. 

Prostate cells were grown in media with 50, 150 and 300 M Zn 2+ and after 

harvesting mechanically manually homogenized in 50 μl of phosphate buffer (pH 7.4) 

for 3 minutes. Subsequently PBS up to 200 μl was added and thermal denaturation was 

carried out (99 °C for 15 minutes). The solution was centrifuged (14000 rpm for 30 

minutes at 4°C). These lysates were diluted by distilled water (1:1) and used for CE 

analysis. 


As discussed in the publication by Jiang et al. 4 during the separation Cu 2+ ions 

are able to create complexes based on coordination interactions with the analytes 

containing free electron pair. This method enables not only the direct absorbance 

detection of certain analytes including amino acids, but also online preconcentration 

of the analyte by coordination sweeping method. 

To optimize and characterize the method, separation of model mixture of six 

amino acids (His, Ser, Phe, Gln and Pro) as well as sarcosine (Src) as an isomer of 

alanine was performed (Fig. 1). 

A (AU) 

0,025 

0,02 

0,015 

0,01 

0,005 

his 

ser 

0 

10 15 20 

t (min) 

Fig.1: Electropherogram of standard amino acid mixture. Conditions: BGE – 50 mM 

CuSO4 in 0.05% HAc (v/v) pH 3.8, detection at 254 nm, voltage - +15 kV 

Also the applicability of the method for sarcosine analysis was verified and 

analytical figures of merit were determined (Tab. 1). It was proved that the method is 

suitable for analysis of Src in presence of other amino acids and the signal of Src 

- 234 - 

phe 

gln 

pro 

src


increases with the concentration. Calibration curve with a very good linearity (R 2 = 

0.9968) was obtained (Fig. 2). 

Tab. 1: Analytical figures of merit for CE analysis of Src 

LOD 

(M) 

3x10 - 

6 

A (AU) 

0,014 

0,012 

0,01 

0,008 

0,006 

0,004 

0,002 

RSD 

signal (n=3) 

11 % 

LOQ 

(M) 

8x10 - 

6 

- 235 - 

linear 

range 

LOQ - 6x10 - 

3 

RSD 

mig. time (n=3) 

0,2 % 

Fig. 2: Calibration curve of sarcosine obtained by CE. Conditions as in Fig. 1 

Subsequently the optimized method was utilized to analyze samples of prostate 

cell lysates. From the obtained results can be seen that the Src signal is well resolved 

and no directly interfering peaks are present. The identification of the Src peak was 

done by both, comparison with migration time of a standard solution as well as by 

standard addition method to take into account the matrix effect. The typical 

electropherogram of cell lysate sample is shown in Fig. 3. 

A (AU) 

0,0035 

0,003 

0,0025 

0,002 

0,0015 

0,001 

0,0005 

Fig. 3: Electropherogram of cell lysates. Conditions as in Fig. 1 

y = 2,1814x 

R² = 0,9968 

0 

0,000 0,001 0,002 0,003 0,004 0,005 0,006 0,007 

c (M) 

Src 

0 

10 15 20 

t (min) 

25 30

XI. Workshhop 


E 

s´11 

The 

influencce 

of conce entration oof 

Zn ions in i growing medium oon 

the Src level l 

was alsoo 

investigatted. 

For thi is reason, ceells 

culture ed in media a containingg 

different Zn 

concenntrations 

weere 

analyze ed. The commparison 

of o three dif fferent cell lysates sam 

shows tthe 

decreasse 

of the Src S signal (FFig. 

4). Flu uorescence microscopyy 

of intact 

proved that increasing 

con ncentrationn 

of Zn io ons decreas se the viabbility 

of c 

Therefoore 

the Src level is also o decreasinng. 

2+ 

mple 

cell 

cells. 

Fig. 4: Src level inn 

cell lysat te sample ttreated 

with h three con ncentrationns 

(50, 150 and 

300 μM) off 

Zn ions 


Itt 

was foundd 

that CE is s suitable foor 

analysis of Src. Sarc cosine amoount 

in pros state 

cell lysaates 

was deetermined 

by b the methhod 

employ ying compl lexation of Cu ions. It was 

also fouund 

that ZZn 

ions ha ave an infl fluence on the viabil lity of prosstate 

cells and 

therefoore 

on the aamount 

of sarcosine 

deetermined. 


ENT 

The 

work has been supported by GACR R 301/09/P P436, IGA 

NANOSSEMED 

GAA 

AV KAN2 208130801 and IGA MENDELU M 1/2011 


1. Kuuehn 

B. M.: JJama-Journal 

of the Ameriican 

Medical Association, A 301, 3 1008 (20009). 

2. Jeentzmik 

F., Sttephan 

C., Miller M K., Schrrader 

M., Erb bersdobler A., , Kristiansen G., Lein M., Jung 

K.: 

European UUrology, 

58, 12 

(2010). 

3. Coouzin 

J.: Sciennce, 

323, 865 (2009). 

4. Jiaang 

X. M., Xia 

Z. N., Wei W. W L., Gou QQ.: 

Journal of Separation S Sc cience, 32, 19227 

(2009). 

- 236 - 

Brno 

MZ 1020 00-3,


LAB-ON-CHIP: STATE OF THE ART 

Markéta RYVOLOVÁ 1 , Jaromír HUBÁLEK 2,3 , René KIZEK 1,3 



2 Department of Microelectronics, Faculty of Electrical Engineering and Communication, Brno 




Abstract 

Precise, sensitive and reliable determination of analytes in a wide range of natural 

matrixes is one of the main goals of natural sciences. In the real world, an analyte of 

interest (whether it is a small organic molecule or a much larger biopolymer) is 

usually present as a minor component in a complex mixture. This means that 

discrimination of the analyte from potential interferences is usually the critical step in 

the complete analysis process. 1 

Lab-on-chip systems, which are also known as micro total analysis systems (μTAS), 

can be defined as integrated micro electromechanical devices enable to carry out all 

stages of analytical process. They allow miniaturization and integration of complex 

functions that can automate repetitive laboratory tasks. The portability as well as 

compactness of these devices even support the rising trends of in situ analysis. 2 μTAS 

influences an extremely diverse range of analytical applications and on the other hand 

its own progress is enabled by numerous engineering disciplines. The lab-no-chip 

concept is closely connected to the manipulation with liquids at extremely low 

volumes – known as “microfluidics”. The history of microfluidics dates back to the 

early 1950s, when efforts to dispense small amounts of liquids in the nano and 

subnanolitre ranges were made for providing the basics of today’s ink-jet technology. 3 

μTAS concept itself was proposed in by Manz et al. 4 in the work from 1990, where a 

silicon chip analyze incorporating sample pretreatment, separation and detection was 

integrated. Since then the race for lower detection limits, lower sample and chemical 

consumption, higher throughput as well as lower costs is running. In last two decades 

numerous research groups invested their time and effort in developing new 

microfluidic components for liquid transport and low volume measurements, mixer, 

valves, concentrators and/or separators for analytes within miniaturized quantities of 

fluids. 3 

Moving the analysis to the micro-world brings specific obstacles that do not exist in 

macro-world and need to be effectively tackled. Therefore even closer connection 

between disciplines has developed. Currently, progress in nanotechnologies moves the 

miniaturization even further focusing on molecular machines and “nanobots”. This 

lecture is giving an overview of lab-on-chip state of art, highlighting some 

outstanding geometrical arrangements, manufacturing processes and/or applications. 

- 237 -



The work has been supported by CEITEC CZ.1.05/1.1.00/02.0068, 

NANOSEMED GA AV KAN208130801 

8. REFERENCES 

1. Jakeway S. C., de Mello A. J., Russell E. L.: Fresenius Journal of Analytical Chemistry, 366, 525 

(2000). 

2. Lim Y. C., Kouzani A. Z., Duan W.: Microsystem Technologies-Micro-and Nanosystems- 

Information Storage and Processing Systems, 16, 1995 (2010). 

3. Haeberle S., Zengerle R.: Lab on a Chip, 7, 1094 (2007). 

4. Manz A., Graber N., Widmer H. M.: Sensors and Actuators B-Chemical, 1, 244 (1990). 

- 238 -


THIN FILM HYDROGEN SENSOR BASED 

ON DIKETOPYRROLO-PYRROLES 

ANALOGUES 

Ota SALYK 1 , Jan VYŇUCHAL 2 

1 Brno University of Technology, Faculty of Chemistry, Purkyňova 118, 612 00 Brno, Czech Republic 

2 Synthesia a.s., Pardubice, Semtín 103, CZ-532 17 Pardubice, Czech Republic 

Abstract 

3,6-Diphenyl-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione (BPPB) and its analogues 

diketo-pyrrolo-pyrroles (DPPs) are industrially important organic pigments. DPPs 

have also attracted attention as materials useful for organic electronics because of 

conjugated chain inside the molecule enabling charge transport. Nitrogen atom in 

pyridyl substituent can create hydrogen bond and accept proton, which enhances 

molecule dipole momentum and N-doping of the substance resulting in conductivity 

increase. 3,6-bis-(4'-pyridyl)-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione 

(4PyPP4Py) and several other analogues of DPPs were investigated by IR and Raman 

spectroscopy, thermogravimetric analysis and other physical methods. X-ray 

difractometry was applied for elucidation of crystallographic polymorphism and 

hydrogen bridge bonds. 100 nm thin film structures with Pd interlayer were 

deposited by vacuum evaporation on substrates with platinum interdigital system of 

electrodes. Pd enabled hydrogen dissociation for DPPs doping. Hydrogen induced 

conductivity was measured; response and recovery time as well as temperature and 

oxygen free H2 mixture environment were tested. The conductivity increase of up to 5 

orders with hydrogen concentration up to 100 % was observed. Despite slow 

degradation in operation the system can be utilized in hydrogen sensor devices. 


With the advent of fuel cells based upon H2, H2 gas sensors have attracted 

attention, because hydrogen is the smallest atom and, thus, can leak easily. We have 

been so far involved in the research and development of a H2 gas sensor utilizing a 

high proton affinity of new materials with pyridyl substituents of phenyl in diketopyrrolo-pyrroles 

(DPPs). DPPs (Fig. 1) 0 are industrially important organic pigments 

used not only for paint industries but as in the imaging areas. DPPs have also attracted 

attention as a material useful for EL and colour filters for LCD applications and in 

photovoltaics. Recently an application in gas sensing devices was suggested by 

Mizuguchi et al. 0 who also explained the principle of proton bond effect on 

conductivity increase. 

- 239 -


X 2 

Y 2 

O 

O 

H N N H 

Y 1 

X 1 

BPPB X1=X2=Y1=Y2=CH 

2PyPPB X1=N, X2=Y1=Y2=CH 

2PyPP2Py X1= X2=N, Y1=Y2=CH 

4PyPPB 

4PyPP4Py 

(DPPP) 

X1=X2 = CH, 

Y1= N, 

Y2=CH 

X1= X2=CH, Y1=Y2=N 

Fig. 1 4PyPP4Py or 3,6-bis-(4´-pyridyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione 

(DPPP - DiPyridyldiketoPyrrolo-Pyrrole) 

and the family of some DPP 

analogues 0. 

- 240 - 

Pt 1000 thermometer 

interdigital electrode system 

heater 8±1 

Fig. 2 Commercial sensor 

platform KBI2 (Tesla Blatná) 

2. EXPERIMENT 

Corundum based sensor platform with interdigital system of electrodes (IDE) 

contains 10533 parallel squares between platinum contacts on a square flat 2x2 mm 

inside (Fig. 2). It is surrounded by heater of 8 and Pt 1000 thin film resistor for 

temperature measurement. It contains 79 contact strips 15 μm in width. 

The evaporated substance was available in powder form only poorly soluble in 

organic solvents, but they exhibit good thermal resistance against pyrolysis and can be 

sublimated as was before tested by thermogravimetric analysis 0. The deposition of 

the active DPP layer was carried out in the vacuum coating facility with ultimate 

pressure 1•10 -5 Pa. by irradiative heating of pellets 5.8 mm in diameter and of about 30 

mg of mass. The evaporator was designed in order to minimalize possible 

decomposition. The deposition rate was typically 0.2 to 0.5 nm/s. The hydrogen 

sensing device consisted of DPP(40 nm)–Pd(0,3 nm)–DPP(40 nm) three layer 

structure; Pd interlayer was evaporated from a graphite boat without vacuum 

breaking. 

Sensor testing facility was built up with respect to experience with operation of 

more complex facility presented in 0. The premixed 5 % hydrogen in nitrogen gas 

cylinder was used due to safety reasons, and for higher hydrogen concentrations an 

electrolyser followed by l-N2 cold trap with safety leakage fuse was used. The facility 

was controlled by a software virtual instrument programmed in LabView as well as 

the data acquisition. Keithley 500 electrometer with AD converter enabled to measure 

current down to 10 -14 A.

XI. WWorkshop 

of Phyysical 


3. RESULTTS 

AND DISCUSSI 

D ION 

The senssing 

structu ure accordinng 

to Fig. 2 was tested d at first in p 

for 24 hour peeriod 

load – Fig. 4. Thhe 

initial int trinsic cond ductance (w 

of tthe 

structurre 

depends s on Pd intterlayer 

thi ickness. Th he conducta 

wass 

round 10-13 

-1 , it correspondss 

to specifi ic conducti ivity 10 

periiod 

measurrement 

can be dividedd 

into 5 reg gions: 1 – b 

-12 

pure dry hy 

without hy 

ance of DP 

 

efore hydro 

-1cm-1 ydrogen 

ydrogen) 

PP layer 

. The 

long 

rogen inlet, voltage 

Fig. 3 Frresh 

sensor r response at a medium 

vvoltage 

and concentrat tion 

of 2 V was appplied 

and a relaxationn 

was obser rved for a few f minutees. 

Dry air flushed 

out the humiddity 

and it caused c fall ooff 

the init tial current. 

2 – 100% dry hydrog gen was 

appplied 

and ann 

increase of o the curreent 

over thr ree orders was w observeed, 

first rap pid, than 

conntinued 

slowwly 

for abou ut 1000 s. 33, 

4 – the sa aturation te endency waas 

replaced by slow 

degradation 

foor 

18 hours in hydrogeen. 

The cur rrent decrea ased by morre 

than an order. 5 

– fluush 

out witth 

dry air continued c tthe 

next day 

and drop the currennt 

by five orders o to 

2.100 

vari 

init 

13A. Oxygeen 

in air ca aused accelleration 

of recovery but b also irreeversible 

st tructure 

iation tookk 

effect in further cuurrent 

decr rease more than two orders bel low the 

ial current. . 

- 241 - 

Brno


Fig. 4 Fresh sensor response on pure dry hydrogen and recovery in dry air. Applied 

voltage was 2V. 

At the next sample (Fig. 3) 1 % H2 in N2 gas was switched on/off rapidly after 

500 s with dry air also for 500 s and this treatment was repeated for several times. The 

response time is faster and the recovery becomes slower using higher hydrogen 

concentration. Every air flushes out offered a dose of oxygen causing regular 

sensitivity decrease. So far also the intrinsic conductance falls down by the same slope 

and the ratio of hydrogen stimulated conductance to intrinsic conductance is 

constant, it can be explained as a decrement of sensitive component in the layer but it 

was not confirmed yet. The recovery continues in air for hours and in 24 hour the 

current fell down below the initial value. 

4. CONCLUSION 

Nitrogen in hetero-arenes can accept proton and shifts the charge dipole of the 

molecule. While the molecule contains a conjugated chain, a variation of conductivity 

occurs. A compound 4PyPP4Py was successfully applied for thin film hydrogen sensor 

fabrication and its characteristics were tested. The rapid response of several orders of 

magnitude on 100 % H2 was observed as well as in H2/N2 gas mixture, while in H2/air 

due to oxygen presence is the sensitivity about an order lower. But oxygen plays 

crucial role in recovery of initial state. Long period test cycling leads to sensor 

degradation, which principle is not explained yet. 


This work has been supported by the Czech Science Foundation in the project 

GACR 203/08/1594 and by the Ministry of Industry and Trade of the Czech Republic 

in the project FR-TI1/144. 

6. REFERENCES 

[1] Luňák, S., Vyňuchal, J. et al.: Journal of Molecular Structure, 983 (2010), 1-3, 39 

[2] Takahashi H., Mizuguchi J., J. Appl. Phys. 100(2006), 034908 

[3] Salyk, O., Castello, P. et al.: Measurement Science and Technology, 17(2006), 6, 3033 

- 242 -

XI. WWorkshop 

of Phyysical 


INNSTRUUMEN 

NT FOR 

FLU UORES SCENTT 

BIIOSENNSOR 

Jiří SEDLÁČEK 

HUUBÁLEK1 

K1 , Šárka BIDMANOVVÁ 

2 , Zbyně ěk PROKOP P2 , Břetislavv 

MIKEL3 , Jaromír 

Department oof 

Microelect tronics, Brno University of f Technology y, Technická 110, 

616 00 Brn no, Czech 

Republic 

2 Depart tment of Expeerimental 

Bio ology, Masary yk university, , Brno, Czech h Republic 

3 Institut ute of Scientifi fic Instrument ts of Academy y of Sciences s of the Czech h Republic 

1 D 

Abstract 

This 

device wwas 

design ned as a flluorescence 

e analyzer for the ddetection 

of o toxic 

subsstances 

due 

to (bio)c chemical reeaction 

tha at changes fluorescennce 

emissio on after 

lighht 

excitationn. 

Optical method m is aalso 

used fo or their nois se tolerancee 

and the ability 

to 

trannsfer 

signalls 

over long g distancess 

with only y minimal losses l of siggnal. 

Fluor rescence 

subsstance 

is appplied 

to th he end of ann 

optical fib bre which is connecteed 

to the de evice. It 

preddestines 

thee 

device to be used forr 

measurem ments in dee ep wells wiith 

drinking g water. 


N 

This insttrument 

wo orks as fluoorescence 

detector d for r biosensor. . Main part ts of the 

biossensor 

are bio-recog gnition parrt 

and ph hysical-chem mical trannsducer. 

Th he bio- 

recoognition 

paart 

must be in contact with the testing 

samp ple. The traansducer 

ge enerates 

optiical 

signal that is im mmediately converted d to electri ical signal for proces ssing by 

circcuitry. 

The basic prin nciple of itts 

function n is demonstrated 

in Fig. 1. An nalyte is 

carrried 

to bio-recognitio 

on part to rreact 

with the analyt te to detectt 

toxic sub bstances. 

Thee 

reaction of analyte with bio-recognition 

n part cha anges chemmical 

values s on its 

surfface 

causing 

fluoresce ence ampliffication/atte 

enuation. The T opticall 

signal is detected d 

by tthe 

light detector 

and d processedd. 

Acquired d information 

are anaalysed 

by so oftware. 

Thee 

sensing part prov vides non-stop 

elect tronic sign nal. The ssignal 

is directly 

propportional 

too 

concentra ation of onne 

or severa al chemical substances s in the ana alyte [1]. 

This 

device caan 

be used as analyzer 

of pestici ides for am mbient exammination 

(th he most 

ofteen 

in water). 

Fig. 11: 

Schematic 

of biose ensor. 

- 243 - 

Brno

XI. Workshhop 


E 

s´11 


The 

device cconsists 

of two main pparts: 

an el lectronic an nd an opticcal 

section. For 

controll 

and signall 

processing g the persoonal 

compu uter with so oftware is uused. 

The optic o 

part is iin 

Fig. 2 annd 

it is constructed 

wiith 

two cha annels with h one monoolithic 

dete ector 

of lightt. 

It consistts 

of two LE ED diodes as light sou urces about t wave lenggth 

of 590 nm. 

Part A is a dichroic 

mirror th hat reflectss 

light source 

and tran nsmits the ssignal 

from m the 

sensingg 

part. Part B is an optical 

lens. TThe 

lenses fo ocus the lig ght to the pphoton 

dete ector 

(PMT). Part C is aan 

optic filte er for speciified 

source e wave leng gth. 

TThe 

device has power supply fromm 

AC 230V V. Time div vision multi tiplex is use ed to 

scan thhe 

channelss. 

Light em mitted fromm 

LED diod de goes thr rough opticc 

filter (C) and 

reflectss 

on dichroiic 

mirror (A A) and goess 

to optic fib bre. The lig ght excites tthe 

fluorescent 

and creeates 

new optic radia ation. The new light goes throu ugh optic ffibre 

and optic o 

filter ( C) and thhrough 

dich hroic mirrror. 

The le ens (B) focuses 

the new light t on 

photodetector 

whhich 

convert ts the opticcal 

signal in nto an analo og electricaal 

signal. 

Fig. 2: Schhematic 

cu ut of optic part p 

AAn 

electric signal from m the phottodetector 

is processe ed by electtronic 

part. . All 

processsing 

is conntrolled 

by microconttroller. 

US SB bus is used u for coonnection 

and 

commuunication 

wwith 

PC. LE EDs inside oof 

the optic cal part are e controlledd 

with curr rent. 

Power supply is cconstructed 

inside of tthe 

device including transformer t r source of f 230 

Vac annd 

three monolithic 

voltage v reguulators. 

Tw wo of them m are used for symme etric 

source ±15 V for pphoto 

detec ctor and onee 

(+5 V) is used u for the 

electroniccs. 

3. RRESULTS 


Testing 

meaasurements 

were madee 

on the de eveloped fl luorescencee 

detector. The 

sample under testing 

was loc cated into tthe 

dark bo ox. Emitted d light, whiich 

was created 

by chemmical 

reaction, 

was fe ed via a plasstic 

optical fibre. The same opticcal 

fibre is used u 

for exciitation 

of bbio-chemica 

al transduccer. 

The fib bre spike wa as coated wwith 

the mi ix of 

fluoresccent 

indicaator, 

haloge en, halogennated 

analyt te and elem ments whichh 

degraded d the 

analytee. 

The 

measureement 

was made in aanalyte 

con ntaining pesticide. 

It wwas 

found that 

the signnal 

obtaineed 

is slight tly noised. Therefore, , averaging the samplles 

was car rried 

out. Thhe 

resulting waveform is in the Fiig 

3. 

- 244 - 

Brno

XI. WWorkshop 

of Phyysical 


Fig. .3: Graph of measur red curves, , after ave eraging filtering 

of mmeasured 

samples. s 

LED iintensity: 

100 %, signal was w recor rded in 4·10-9 M 5(6)- 

carboxyynaphthoflu 

uorescein 

dissolveed 

in 1 mM M HEPES buuffer 

with pH p 4.0 and pH 9.0. 


The deviice 

was dev veloped andd 

construct ted. The flu uorescent aanalyzer 

wa as tested 

in ddetail 

in Losschmidt 

lab boratories, MMasaryk 

University U of 

Brno 

Measurements 

show wed that thhe 

used fluo orescent giv ves an apprropriate 

sign nal. The 

reasson 

is the llow 

yield of o used fluoorescent 

an nd a small area a of the optical fiber 

spike 

thatt 

is coated wwith 

an enz zyme. 

Higher ssignals 

and lower deteection 

limi its will be obtained wwhen 

using g a fiber 

withh 

larger crross 

section n and anotther 

fluore escent, whi ich will haave 

a bette er yield 

emiission. 


EMENT 

This worrk 

has been n supported d by Grant Agency A of the t Czech RRepublic 

un nder the 

conntract 

GACRR 

P102/11/ 1068 and bby 

the Czec ch Ministry y of Educatiion 

in the frame f of 

Research 

Plan MSM 0021 1630503 (MMIKROSYN 

N). 

6. 

[1] 

[2] 

[3] 

REFEREENCES 

TRÖGL, JJosef. 

Biosenz zory. Automma 

[online]. 2004, 2 č. 4 [c cit. 2008-11--29]. 

On the e WWW: 

. ISSN 12210-9592. 

SEDLÁČEK, 

J. Biosenso or of Halogennated 

Compo ound as Instrument 

using fluorescence e method. 

Brno: Diploma 

thesis: Brno Univerrsity 

of Tech hnology, Facu ulty of Electrrical 

Enginee ering and 

Communiccations, 

2010. 

63 s. Supervvisor: 

doc. Ing g. Jaromír Hub bálek, Ph.D. 

BIDMANOOVÁ, 

Šárka. . Developmeent 

and Co onstruction of Biosensorrs 

for Dete ection of 

Halogenated 

Compoun nd in the EEnviroment. 

Brno, 2007. 75 s. Diplloma 

thesis. Masaryk 

Universityy, 

Faculty of Sciencies, Deept. 

of experimental 

micro obiology. Suppervisor 

Doc. Mgr. Jiří 

Damborskký, 

Dr. 

- 245 - 

Brno


NOVEL REACTIVITY – MAPPING 

TECHNIQUE FOR CHARACTERIZATION 

OF POLYELECTROLYTE BIOPOLYMERS 

Petr SEDLÁČEK 1 , Jiří SMILEK 2 , Martina KLUČÁKOVÁ 1 

1 Center for Materials’ Research, Brno University of Technology, Faculty of Chemistry, Purkyňova 118, 

Brno 

2 Institute of Physical and Applied Chemistry, Faculty of Chemistry, Purkyňova 118, Brno 

Abstract 

The contribution discusses possibilities of introducing simple diffusion techniques in 

automatized reactivity – mapping studies of biopolymers. As is illustrated on some 

preliminary results on humic acids, combination of a standard method of horizontal 

diffusion cells, optimized for required experimental conditions, with an appropriate 

hydrogel form of biopolymer under investigation provides interesting novel approach 

for simple macroscopic observation of interactions which take place on the molecules 

of the biopolymer. 


Polyelectrolyte biopolymers are found everywhere around us. On the one side, 

they are crucial constituents of living organisms, but in a form of humus, they also 

represent a key component of many non-living parts of nature – soils, waters and 

sediments. We routinely call these materials highly reactive but what exactly does it 

mean? And how such a property of wide comprehension can be measured and 

quantified? 

The most widespread approaches are based on adsorption experiments [1, 2] – 

biopolymers of various forms (solution, sol, suspension) are let in contact with a range 

of species to be sorbed and a variety of different sorption parameters is determined 

corresponding actual experimental condition. This approach brings several serious 

drawbacks. For example, the homogeneity of such media as colloidal sols or 

suspensions is always a question, as well as a corresponding size of biopolymer 

particles in them often resulting in the fact that a interaction of such a particle with a 

sorbate is limited just on the surface of the particle. The actual experimental 

conditions also play a crucial role: we should ask whether the medium is agitated or 

not and if yes, on what rate? Are we studying kinetics or equilibrium of the sorption? 

And finally, it is quite complicated to get the results from such diverse experiments in 

a universal form allowing a comparison in reactivity among various biopolymers. 

Diffusion techniques can provide an interesting and simple experimental 

alternative. If we prepare a homogeneous medium from studied polyelectrolyte and 

we let appropriate compound – probe – diffuse into or through the medium, we will 

be able to simultaneously explore the transport of the probe with its interactions with 

- 246 -


the surrounding media. Such diffusion processes are (under appropriate experimental 

conditions) easily observed; the mathematical apparatus for the data evaluation is well 

explained [3] and gives us some standard parameters – diffusion coefficients – in 

whose values all interactions in the system are involved. Just the first step – 

preparation of a homogeneous medium – could be a problem. 

For this purpose, preparation of a hydrogel form of a biopolymer could be the 

method of choice. The gel form can be considered a system allowing fixation of 

biopolymeric matrix in aqueous medium while enabling interactions in the whole 

volume. Consequently, not only physico-chemical interactions with the biopolymeric 

content but also simultaneous transport within the volume are observed 

experimentally. From the experimental point of view, gel media bring several 

advantages concerning a study of diffusion. It allows preparing sample in defined size 

and shape, which is necessary for mathematical description of a transport 

phenomenon. Besides, mechanical and thermal convection of a liquid is markedly 

suppressed by the gel matrix. For the measurement of diffusion coefficients, the 

majority of authors apply expensive and sophisticated spectroscopic (usually based on 

a light scattering) or nuclear methods [4-5]. In these cases, a homogeneous system is 

studied where no concentration gradient of the considered solute occurs and the value 

of diffusivity represents so called self - diffusion coefficients. On the other hand, there 

are simple laboratory methods that allow performing diffusion governed by a 

concentration gradient; usually, these techniques are not demanding on laboratory 

equipment and they provide a more reasonable sight on solute-release systems. A 

handlist of these methods is presented in [6]. For the automatized determination of 

diffusion coefficients, the method of the diffusion cells (often called the Stokes or 

Franz cells) represents the method of choice [7]. A probe is diffusing along initial 

concentration gradient between two solution compartments (cells) through a gel 

sample and a change in the concentration is monitored in both cells. A wide range of 

properties of these solutions can be easily adjusted and corresponding response in 

diffusivity can be determined. 

In previous works, several laboratory techniques were used in order to study 

diffusion of metal ions in a gel prepared from humic acids (HAs) [8-9]. This gel is 

prepared by the guided coagulation of alkaline solution of HAs by an addition of a 

strong acid. The humic gel was used as a model of natural environments which 

contains high amount of humic substances (soils and sediments). Humic acids are 

chemically reactive; they possess a wide range of functionalities that could change 

when structural modifications are made to acquire desired properties. This 

contribution represents the results of preliminary experiments concerning diffusion of 

metal ions in diffusion cells through various samples of hydrogels with content of 

humic acids. 

2. EXPERIMENT 

Exact method of isolation of lignitic HAs is published in details elsewhere [8-9]. 

Humic hydrogels were prepared by precipitation of dissolved HAs (concentration 8 

- 247 -


g.dm –3 ) by concentrated hydrochloric acids to pH ~ 1. After the suspension had been 

kept overnight at 4°C, the sedimented gel was separated by centrifugation at 4000 

RPM. Two gel samples used in diffusion experiments differed in the particular solvent 

utilized in dissolving original humic acids: 

sodium hydroxide was used in concentration 0.5 mol.dm –3 (resulting in gel A) 

and sodium triphosphate in concentration 0.1 mol.dm –3 (resulting in gel B). For 

determination of diffusion coefficient of cupric ions in both gels, the same diffusion 

cells apparatus with cell volume of 60 cm 3 was used. Initial concentrations of CuCl2 in 

“out” and “in” cells were 100 mol.m –3 and 0, respectively. In the apparatus, gel was 

fixed in a plastic insert as a cylinder 1 cm in length, 4 cm in diameter. Changes in 

concentration of cupric ions in the “in” cell were observed by means of UV-VIS 

spectroscopy – optical fibre probe was directly dipped in the cell and scanned UV-VIS 

spectra during the experiment. 


Fig. 1 shows the change in concentration of cupric ions in “in” cell (which had 

initially contained no cupric ions). As can be seen, there is not any pronounced 

difference between the two gels. Diffusion coefficients, calculated from regressions of 

the linear parts of these dependences are 3.58·10 –10 m 2 .s –1 (gel A) and 3.66·10 –10 m 2 .s –1 

(gel B). These results are well expected: the nature (morphological and chemical 

structure) of both gels was the same; there was no significant difference in solid 

content of the gel or in the inner pH. These values are lower as compared with 

diffusion of cupric ions in water (14.3·10 –10 m 2 .s –1 , see [10]). This is typically obtained 

for reactive hydrogels with high content of water (both gels contained about 85 % of 

water in weight). 

The diffusion cells apparatus is designed for measurement under various 

conditions. It allows us to change a parameters of inner solutions (pH, ionic strange) 

which gives us deeper knowledge about interactions which take place in the system. 

Besides, the temperature of the experiment can be easily altered. It was 

experimentally confirmed, that diffusion coefficient of cupric ions in gel A increases 

with increasing temperature as follows: 3.58·10 –10 m 2 .s –1 (30 °C), 6.43·10 –10 m 2 .s –1 (40 °C) 

and 9.08·10 –10 m 2 .s –1 (50°C). 

- 248 -


c Cu (mol.m –3 ) 

16 

14 

12 

10 

8 

6 

4 

2 

0 

gel A 

gel B 

0 20 40 60 80 

time (hrs.) 

Fig.1 Increase of the concentration of cupric ions in the “in” cell of the apparatus at 

30°C. 

4. CONCLUSION 

On the example of some preliminary results, the high potential of measurement 

of diffusion coefficient in biopolymer hydrogels using diffusion cells is presented. This 

provides an automatized, universal and experimentally non-demanding technique for 

some standardisable reactivity mapping studies on biopolymers. 


The work has been supported by government funding – Czech Science 

Foundation, project P106/11/P697. 

6. REFERENCES 

[1] Wan Ngah, W.S., Teong, L.C., Hanafiah, M.A.K.M.: Carbohydrate Polymers, 83 (2011), 4, 1446 

[2] Klučáková, M., Pekař, M.: Journal of Polymer Materials, 20 (2003), 2, 145 

[3] Crank J.: The Mathematics of Diffusion, Clarendon Press, 1956, Oxford 

[4] Manetti, C. et al.: Polymer, 43 (2001), 1, 87 

[5] Ray, S.S. et al.: Chemical Engineering Science, 53 (1998), 5, 869 

[6] García-Gutiérrez, M. et al.: Journal of Iberian Geology, 32 (2006), 1, 37 

[7] Falk, B., Garramone, S., Shivkumar, S.: Matterial Letters, 58 (2004), 3261 

[8] Sedláček, P., Klučáková, M.: Geoderma, 153 (2009), 1–2, 286 – 292 

[9] Sedláček, P., Klučáková, M.: Collect. Czech. Chem.C., 74 (2009), 9, 1323 – 1340 

[10] Lide D.R.: Handbook of Chemistry and Physics, 76th ed., CRC Press, 1995, New York 

- 249 -


DPPH AQUEOUS ANTIOXIDANT ASSAY 

SOLUTION 

Karel SEDLÁŘ 1 , Kristýna SMERKOVÁ 2 , Helena ŠKUTKOVÁ 1 , Jiří SOCHOR 2 , Vojtěch 

ADAM 2 , Ivo PROVAZNÍK 3 , René KIZEK 


University of Technology, Kolejni 4,CZ-61 200 Brno, Czech Republic 



3 International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic 

Abstract 

DPPH is a free radical commonly used in antioxidant assay. A purple coloured 

solution which becomes colourless after reaction with antioxidant is convenient to 

spectrofotometric analysis. Unfortunately, there are many protocols providing 

different results, so we are unable to compare them among laboratories. We tried to 

examine presumptions about stability of DPPH molecule as a common condition for 

many protocols. Using different temperatures and antioxidants (ascorbic acid, salicylic 

acid, homocysteine, etc.) we found out that some presumptions are wrong. 


Under normal conditions DPPH (2,2-diphenyl-1-picrylhydrazyl) is a dark 

crystalline substance [1] available as a powder. Free radical molecules are stable in this 

form. For antioxidant assay DPPH liquid solutions are used. Even in solution, 

molecules are considered as stable, unresponsive to molecules of solvent [2]. Purple 

colour solution is used for DPPH spectrofotometric assay, maximum absorbance is in 

the band about 520 nm [3]. Free radical molecules are neutralized in the reaction with 

antioxidants and solution becomes colourless. Due to stability and undemanding 

storage conditions DPPH is widely used. Except testing antioxidants DPPH is a 

scavenger for other free radicals. 

2. EXPERIMENT 

Because of many different protocols used in DPPH antioxidant assay we tried to 

test some factors that according to many protocols do not affect measurement results. 

These are mainly temperature influence and measuring time. However there are 

newer protocols that discovered some mistakes [4]. They do not use water as the 

solvent. 

We made samples with six different concentrations of DPPH (100 μM, 75 μM, 

50 μM, 20 μM, 10 μM, 5 μM). Solvent was distilled water. Then we watched the 

evolution of the spectrum, depending on the sample concentration at three different 

temperatures (15°C, 25°C, 35°C). For every sample at all temperatures we measured 

absorbance spectrum half an hour every 3 minutes for sample and for sample 

interfused with antioxidant separately. As antioxidants we took 100 μM solutions of 

- 250 -

XI. WWorkshop 

of Phyysical 


saliccylic 

acid, cysteine, acetylcystei a ine, homoc cysteine, tro olox, ascorbbic 

acid an nd gallic 

acidd. 

For interffusion 

we took t 1000 μμl 

of DPPH and 100 μl l of antioxiddant. 

3. 

RESULTTS 

AND DISCUSSI 

D ION 

Fig. .1 DPPH abbsorbance 

spectrum s deepending 

on 

concentr ration 

Firstly wwe 

took abso orbance speectrums 

for r every con ncentration and recons structed 

evolution 

of tthe 

spectru um dependiing 

on con ncentration of the sammple. 

We realized 

thatt 

concentraations 

lowe er than 20 μM are un nder resolut tion of specctrophotom 

meter so 

we released thhem 

from an nother anallysis. 

Becau use we had only 4 conncentrations 

on the 

inteerval 

betweeen 

20 and 100 μM wee 

interpose ed data with h cubic spliine. 

Thanks 

to this 

inteerpolation 

wwe 

got smo oother evollution 

that better repr resent real evolution. Results 

at 15°C 

and 355°C 

are simi ilar. 

From thee 

figure ab bove you caan 

also rea alize that ab bsorbance peak is hig gher for 

highher 

concenntrations. 

So S we toook 

absorba ance vector r for 100 μM DPPH H using 

funcction 

max tto 

get maxi imal absorbbance. 

Com mparing it with w vector we get its position p 

reprresenting 

wwavelength. 

The maxiimum 

absor rbance at 530 5 nm, wee 

set identic cally for 

all ttemperaturres. 

We use ed this wavvelength 

lat ter for getti ing decreasse 

of absorb bance of 

sammples 

with aantioxidant 

ts. 

Howeverr 

DPPH ra adical moleecules 

are considered c to be stablle 

in time even in 

soluution 

we decided 

to check c absoorbance 

spe ectrum evolution 

for one concen ntration 

deppending 

on time. 

- 251 - 

Brno

XI. Workshhop 


E 

s´11 

Fig.2 0. .75 μM DPPPH 

absorba ance spectrrum 

depend ding on tim me at 25°C (left) and 35°C 3 

(right) 

Frrom 

the figgures 

above e a decreasse 

of absorb bance in tim me is evideent. 

Unlike e the 

neutrallization 

reaaction 

with an antioxiidant, 

whic ch reduces only the hhighest 

pea ak in 

this casse 

the whoole 

spectrum m is reduceed. 

We thin nk it could mean thatt 

molecules s are 

not as sstable 

as theey 

are cons sidered. Thee 

explanation 

might be 

DPPH sppecific 

beha avior 

towards 

other raddicals 

when it acts as a radical tra ap [5]. The radiation ppassing 

thro ough 

the sammple 

in the spectropho otometer coould 

cause formation of free radi dicals in solv vent 

- waterr, 

which wwould 

react t with DPPPH. 

This reaction r could 

cause the absorp ption 

spectruum 

changinng. 

The 

point iis 

that it is s necessaryy 

to measu ure both DPPH D sampple 

and DP PPH 

sample with antiioxidant 

at t the same 

time. Th he result is then thhe 

decrease e of 

absorbaance 

(∆A) oobtained 

by y subtractinng 

the neutr ralized sam mple (An) byy 

DPPH sam mple 

itself As). (A Subscript 

t mean time. t Otherrwise 

the significant 

mistake m cann 

be brough ht to 

the anaalysis. 

∆ 

, 

- 252 - 

, 

(1) 

Brno

XI. WWorkshop 

of Phyysical 


Fig. .3 Decreasee 

of absorba ance using ssalicylic 

aci id 

You can see the results 

for saliicylic 

acid in i the figur re above. Itt 

shows absorbance 

decrreases 

for DPPH sample s intterfused 

with w salicy ylic acid. For the highest 


of DPPH the samplee 

at 15°C has h the high hest decreaases 

of abso orbance. 

Butt 

for the loower 

conce entrations the situatio on is chan nging. 75 μμM 

DPPH has the 

highhest 

decreaase 

at 25°C C and for oother 

conce entrations the maximmum 

decrea ase is at 

35°CC. 

If the ssamples 

we ere stable tthe 

decreas se would grow g in timme. 

But th here are 

seveeral 

situatioons 

where the decreaase 

is decre easing. Tha at shows innstability 

of f DPPH 

mollecules. 

Theese 

various s results shoow 

that the e antioxidant 

behavioour 

of salicy ylic acid 

is veery 

individdual 

and the ere is no general 

rule. 


Howeverr 

the DPPH H antioxidaant 

assay is commonly y used in mmany 

labora atories a 

lot of results can be wrong. 

DPPHH 

radical molecules m in 

solution are consid dered as 

stabble, 

but outt 

results sh hown someething 

diffe erent. Also antioxidannt 

behavior r highly 

deppends 

on twwo 

paramete ers: concenntration 

of DPPH D and sample temmperature. 


EMENT 

This woork 

was supported s by GAČR R 102/09/H H083, MSMM0021630513 

and 

Eurropean 

Regional Developpment 

Fund F - Project t FNUSA A-ICRC 

CZ. 1.05/1.1.000/02.0123. 

6. 

[1] 

REFEREENCES 

Kiers, C. T.; De Boe er, J. L.; Ollthof, 

R.; Sp pek, A. L. (1976). ( "Thhe 

crystal st tructure 

of a 2,2-diph henyl-1-piccrylhydrazy 

yl (DPPH H) moddification". 

Acta 

Crystalloographica 

Section S B SStructural 

Crystallogr C raphy and Crystal Ch hemistry 

32: 2297. . 

- 253 - 

Brno


[2] Szabo MR, Idit C, Chambre D, Lupea AX. Improved DPPH determination for antioxidant 

activity spectrophotometric assay. Chemical Papers. 2007;61(3):214-216. 

[3] Jasprica I, Bojic M, Mornar A, et al. Evaluation of antioxidative activity of Croatian propolis 

samples using DPPH* and ABTS*+ stable free radical assays. Molecules Basel Switzerland. 

2007;12(5):1006-1021. 

[4] Sharma O, Bhat T. DPPH antioxidant assay revisited. Food Chemistry. 2009;113(4):1202-1205 

[5] Price GJ, Garland L, Comina J, et al. Investigation of radical intermediates in polymer 

sonochemistry. Research on Chemical Intermediates. 2010;30(7):807 - 827 

- 254 -


QUANTUM CHEMICAL CALCULATIONS 

OF [LI(DMSO) N] + COMPLEXES 

Vladimír SLÁDEK 1 , Vladimír LUKEŠ 1 , Martin BREZA 1 , Michal ILČIN 1 

1 Institute of Physical Chemistry and Chemical Physics, Slovak University of Technology, SK-812 37 

Bratislava, Slovakia. 

Abstract 

DFT studies of optimal geometries, energetics, electron structure and electron spectra 

of [Li(DMSO)n] + complexes, n = 1 to 6, are presented. The coordination number 

increase causes Li-O bond elongation and decreasing electron density transfer from 

oxygen to lithium. TD-DFT calculations of vertical electronic transitions for IEFPCM 

polarizable continuum model in DMSO solutions and molecular orbital analysis 

indicate that the Li–DMSO bonding is responsible for the blue shift of the original 

DMSO excitation energy. These type complexes are important for the drug-transport 

through the cell membranes in biological systems. 


Dimethyl sulfoxide (DMSO), (CH3)2SO is remarkably versatile, theoretically 

interesting and technologically important aprotic solvent with relevance in various 

fields of chemistry. DMSO penetrates skin and other cell membranes without 

damaging and it could carry other compounds into biological systems [1, 2, 3]. This 

fact determines its use as a drug delivery system because it is less toxic than similar 

organic compounds of this class. In this context, the complex formation of DMSO 

molecules and metal ions receives an increasing attention in chemistry. Also the 

structure of solutions containing lithium cation has received unique attention in 

solution chemistry due to its special features in forming solvate shells. 

The main goal of our work is the DFT investigation of the optimal geometry, 

energetics, electron structure and electron spectra of [Li(DMSO)n] + complexes for n 

= 1 6. Vertical excited states will be calculated using Time-dependent DFT method 

(TD-DFT) [4, 5]. The computed characteristics may be helpful for the interpretation 

of spectroscopic measurements in solutions, as well as for the understanding of 

complex formation of organic molecules (e.g. potential drugs) with metal atom and 

DMSO molecules. 

2. QUANTUM CHEMICAL METHODS 

The electronic ground state geometries of the studied systems were optimized at 

DFT (B3LYP hybrid functional [6]) levels of theory. Based on the optimized B3LYP 

geometries, the vertical transition energies and oscillator strengths were computed by 

TD-B3LYP method. The DMSO solvent effects consequences on the vertical electron 

transitions of the studied systems were estimated using the Integral Equation 

Formalism Polarizable Continuum Model (IEFPCM) for vacuum geometries [7]. All 

- 255 -


calculations were performed using Gaussian 03 [8] program package. The calculations 

were performed in the basis set which consists of 6-31+G* for carbon, oxygen and 

hydrogen and 6-311+G* for sulphur and lithium atoms (B1 basis). 


In order to asses the differences between the complexes, all singlet electron 

transitions up to the 7.5 eV were evaluated using TD-B3LYP(IEFPCM) method. The 

obtained energies and relevant oscillator strengths are depicted in Figure 1. The 

lowest electron transitions with the first non-zero oscillator strengths are collected in 

Table 1. Free DMSO molecule gives six dominant transitions with the oscillator 

strength over 0.03. These results are in good agreement with the experimental 

absorption spectrum [9]. [Li(DMSO)n] + complexes formation causes the downshift of 

vertical excited states. These states are connected with the transitions within DMSO 

molecule (see molecular orbital analysis). On the other hand, the transitions reflecting 

the interaction between lithium atom and DMSO come into being. The next additions 

of DMSO up to n = 4 lead to the blue shift of the lowest excitation energy with nonzero 

oscillator strengths. The red shift of the transitions with dominant oscillator 

strength is indicated for the increasing coordination number (see the transition at 6.46 

eV for n = 3, 6.23 eV for n = 4, 6.13 eV for n = 4). The number of electron transitions 

with non-zero oscillator strengths is growing rapidly with increasing coordination 

number within the range from 5.4 to 6.5 eV. 

In order to understand the role of Li and DMSO interaction in the lowest 

vertical electronic transition, it is useful to examine the frontier molecular orbitals. 

The relevant B3LYP orbitals for the two lowest electronic transitions of [Li(DMSO)] + 

complex are presented in Figure 2. The HOMO orbital is regularly delocalized over 

DMSO molecule, i.e. mainly over sulphur and oxygen atoms. On the other hand, the 

LUMO lobe is spread over lithium atom. The HOMO to LUMO transition with 92 % 

contribution dominates in this optically allowed vertical transition. The shapes of the 

HOMO and LUMO+2 orbitals are identical with the HOMO and LUMO+1 orbitals of 

free DMSO molecule. The presence of Li atom is evidently responsible for the blue 

shift of the original DMSO energy transition from 5.59 eV to 6.97 eV. On the other 

hand, the consecutive catching of DMSO molecules by lithium cation has the 

tendency to shift the energy of this transition to 5.59 eV. 

Tab. 1: The lowest excitation energy with non-zero oscillator strength for studied 

systems. The order k of the transition is indicated by the second column and 

the values in parentheses stand for oscillator strengths. 

Complex k Energy [eV] Transitions 

DMSO 1 5.59 (0.058) HOMO LUMO+1 

[Li(DMSO)] + 

[Li(DMSO)2] + 

[Li(DMSO)3] + 

[Li(DMSO)4] + 

[Li(DMSO)5] + 

1 5.98 (0.019) 

HOMO LUMO 

6 

1 

9 

1 

5.99 (0.037) 

5.93 (0.014) 

5.89 (0.06) 

5.65 (0.006) 

HOMO LUMO+1 

HOMO LUMO 

HOMO LUMO; HOMO 

LUMO+1 

- 256 -

XI. WWorkshop 

of Phyysical 


Oscillator strenght 

[LLi(DMSO)66] 

+ 2 

0. 10 

0. 05 

0. 00 

180 190 200 210 220 230 

wavelenght (nm) 

Excitation energy (eVV) 

6.9 6.7 6.5 6.3 6.1 5.9 5.7 5.5 5.3 

0. 10 

0.005 

00.1 

0.005 

0. 10 

0.005 

0. 10 

0.005 

0. 10 

0.005 

0. 10 

0.005 

Excitation energy (eVV) 

6.9 6.7 6.5 6.3 6.1 5.9 5.7 5.5 5.3 

[Li(DMMSO)] 

italics s 

+ 200 210 220 230 Fig.2 Plo ots of the B3LYP/B1 B molecular orbitals 

wawelenght (nm) 

contribut ting to the e two lowwest 

transit tions of 

complex. 

The vvalues 

in parentheses p s are oscillaator 

streng gths and 

stand for pe ercentage exxcitation 

co ontribution ns to individdual 

transit tions. 

180 190 

5.31 ( (0.008) 

DMSO 

[Li(DMSO) 6 ] + 

[Li(DMSO) 5 ] + 

+ 

[Li(DMSO) 4 ] 

[Li(DMSO) 3 ] + 

[Li(DMSO) 2 ] + 

+ 

[Li(DMSO) 1 ] 

Fig. . 1 TD-B3LLYP/B1 

opt tical transittions 

(bar lines) l for fr ree DMSO and [Li(DM 

compleexes. 

The ex xperimentaal 

DMSO sp pectrum [9] (solid line) ) is normali ized. 


Our theeoretical 

DFT D studiees 

of [Li(D DMSO)n] 

elecctronic 

struucture 

and electron sspectroscop 

explain 

the opptimal 

coord dination nuumber 

of th 

quaantum-chemmical 

studie es, we havee 

found sta 

n = 5 and n = 66. 

The coor rdination nnumber 

inc 

weaakens 

the LLi-O 

bonds, , lowers thee 

DMSO st 

bluee 

shifts in eelectron 

spe ectra. Furthher 

experim 

soluutions 

of vaarious 

meta al salts are desirable t 

This 

may helpp 

us to und derstand thee 

DMSO-m 

trannsport 

throuugh 

the cel ll membrannes 

in biolog 

+ using geoometry, 

en nergetic, 

py argumen nts represeents 

an atte empt to 

he systems under studdy. 

Unlike previous p 

able structu ure for coorrdination 

numbers n 

rease cause es Li-O bonnd 

elongatio on. This 

tructure def formation aand 

corresp ponding 

mental and theoreticall 

studies on n DMSO 

to explain this t problemm 

in more details. 

metal-drug equilibrium e m during th he drug- 

gical system ms. 


EMENT 

This work 

has bee en supporteed 

by Slov vak Grant Agency A VEEGA 

(Proje ect Nos. 

1/01137/09, 

1/00127/09 

and d 1/1072/11) 

and by the Slovak k Research and Devel lopment 

Ageency 

(Projeect 

LPP-0230-09). 

TThis 

work k has benefited 

fromm 

the Ce entre of 

Exccellence 

Prrogramme 

of the Sloovak 

Acade emy of Scie ence in Brratislava, 

Slovakia 

S 

(COOMCHEM, 

Contract no. n II/1/20007). 

- 257 - 

HOMO H LLUMO+1 

HOMO H LLUMO+1 

5,71 1 eV 

(0.0133) 

HOMO → 

LUMO 

92 2 % 

6,50 0 eV 

(0.0078) 

HOMO 

→LUMO+1 

89 9 % 

Brno 

MSO)n] +


6. REFERENCES 

[1] Olver, I.N., Schwartz, M.A., Cancer Treat. Rep. 67 (1983) 407 

[2] Santhos, N.C., et al., Biochem. Pharmacol. 65 (2003) 1035 

[3] Alberts, D.S., Dorr, R.T., Oncol. Nurs. Forum 4 (1991) 693 

[4] Furche, F., Ahlrichs, R., J. Chem. Phys. 117 (2002) 7433 

[5] Bauernschmidt, R., Ahlrichs, R., Chem. Phys. Lett. 256 (1996) 454 

[6] Becke, A.D., J. Chem. Phys. 98 (1993) 5648 

[7] Cancès, M. T., et al., J. Chem. Phys. 107 (1997) 3032 

[8] Frisch, M.J., Pople, J.A., GAUSSIAN 03, Revision A.1, Gaussian, Inc., Pittsburgh, PA, 2003 

[9] K. Golnick, H.U. Stracke, Tetrahedron Lett. 12 (1973) 207 

- 258 -


ANALYSIS OF THIOL COMPOUNDS AND 

ANTIOXIDANT ACTIVITY IN PATIENTS 

SUFFERING FROM MALIGNANT DISEASE 

Jiří SOCHOR 1 , Ondřej ZÍTKA 1 , Petr BABULA 1 , Jaromír GUMULEC 2 , Michal 

Masařík 2 , Vojtěch Adam 1 , René Kizek 1* 

1 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University, Zemedelska 1, 

CZ-61300 Brno, Czech Republic 

2 Department of Pathological Physiology, Faculty of Medicine, Masaryk University, KAmenice 753/5, 

CZ-662 43 Brno, Czech Republic 

Abstract 

The aim of our work consisted in analysis of blood serum samples of patients suffering 

from the malignant disease – diagnosed prostate adenocarcinoma. Healthy persons 

served as a control group. We were focused on the monitoring of the rate of reduced 

(GSH) and oxidised (GSSG) glutathione and on the antioxidant capacity of blood 

serum. 


Glutathione is tripeptide composed of glutamic acid, cysteine and glycine. 

Glutathione is and essential compound produced practically in all eukaryotic cells. 

Glutathione can be in cells in two forms: free, or bound to protein. In this case, 

glutathione forms glutathionated proteins (Pastore, et al.). These proteins are in the 

focus of interest in different patophysiological processes, which is supported by some 

published studies (Cotgreave, et al.)(Klatt, et al.)(Fratelli, et al.). One of the most 

important glutathionated proteins is glutathionyl haemoglobin (S-glutathionated 

haemoglobin), especially due to its significance as a biochemical marker of oxidative 

stress in blood (Bursell, et al.)(Niwa, et al.). Free glutathione occurs in two forms – as 

reduced (GSH) or as oxidised (GSSG). GSH is in consequence of oxidative stress 

readily oxidised; however, due to activity of enzyme glutathione reductase is 

converted back to the reduced form. Under normal, “non-oxidative” conditions, the 

rate between GSH and GSSG is about 10:1. This rate may be significantly changed, 

especially as a result of oxidative stress, to the rates between 10:1 and 1:1 (Chai, et al.). 

Scavenging of hydrogen peroxide and free radicals in connection with ascorbic acid 

(glutathione-ascorbate cycle) are the most important mechanisms and functions of 

glutathione. Compounds that are able to generate reactive oxygen species (ROS) are 

for living organism very dangerous, ROS production is connected with many diseases, 

such as diabetes mellitus, atherosclerosis and cancer (Droge, et al.). Cytosolic, 

mitochondrial and microsomal enzymes glutathione S-transferase family (GST) are 

closely connected with GSH. Disruption in GST function is associated with cancer of 

urinary bladder, skin and possibly lungs (Hayes, et al.). 

- 259 -


Oxidative stress represents disruption of balance between formation and 

elimination of reactive oxygen species. Antioxidant activity is one of the most 

important markers of this balance (Sochor, et al.). Antioxidant activity is defined as 

the ability of compounds (or mixture of compounds) to eliminate oxidadative 

degradation of different compounds, for example lipids (inhibition of lipid 

peroxidation) (Singh, et al.). Methods of its determination are usually based on the 

direct reaction between studied compound and free radicals (their scavenging or 

quenching), or on the reaction with transient metals (Schlesier, et al.). 

2. EXPERIMENT 

Biological samples: 10 samples of blood serum of patients suffering from prostate 

adenocarcinoma were used in experiments. Staging of tumours ranged between 1c and 

4. Age of patients was from 48 to 78 years (average age 62.7 years). Control group was 

represented by 10 healthy persons of the age from 20 to 26 let, students of Masaryk 

University, Faculty of Sports Studies. 

Pretreatment of the biological samples for the GSH/GSG rate analysis: Samples 

of blood serum were diluted by the 10% TFA in the rate 1:1 (v/v) due to precipitation 

of proteins. Samples were subsequently vortexed (20 s) and centrifuged (Eppendorf 

centrifuge 5417R, 15 min., 16400 rpm, 4°C). Supernatant was used for analysis using 

HPLC with electrochemical detection. 

Analysis of thiol compounds: HPLC-ED consisted of two chromatographic 

pumps (Model 582 ESA, ESA Inc., Chelmsford, MA) (working range 0.001-9.999 

ml·min -1 ) and chromatographic column with reverse phase Zorbax eclipse AAA C18 

(150 × 4.6; 3,5 μm size of particles, Agilent Technologies, USA) and CoulArray 

electrochemical detector (Model 5600A, ESA, USA). Sample (20 μl) was automatically 

injected by autosampler (Model 542, ESA, USA), which contains thermostated space 

for column. Samples were during analysis stored in carousel at 8°C. Column was 

thermostated to 32 °C. Flow rate of mobile phase was 1 ml·min -1 . Mobile phase 

consisted of A: trifluoroacetic acid (80 mM) and B: 100% Met-OH. Time of one 

analysis was 20 minutes. 

Analysis of antioxidant activity: Automatic spectrophotometer BS-400 (Mindray, 

China) was used for measurement of antioxidant activity. This apparatus consists of 

cuvette space (thermostated to 37±0.1 °C), reagent space with carousel for reagents 

and for sample preparation (thermostated to 4±1 °C) and optic detector. Tungsten 

halogen lamp served as source of radiation. Transfer of samples was provided by 

robotic arm with dosage needle. Content of cuvettes is mixed by automatic stirrer 

immediately after addition of reagent of sample of volume of 2-45 μl. Contamination 

is minimised due to washing both dosage needle and stirrer by MilliQ water. For the 

detection, following wave lengths could be used: 340, 380, 412, 450, 505, 546, 570, 

605, 660, 700, 740, 800 nm. 

Volume of reagent (DPPH, ABTS, FRAP, DMPD) was incubated for 15 minutes 

with directly defined volume of blood serum; after it, absorbance of this mixture was 

- 260 -

XI. WWorkshop 

of Phyysical 


meaasured. 

Vallues 

of absorbance 

off 

reagent it tself and ab bsorbance oof 

sample after 15 

minnutes 

of inncubation 

were w used for determ mination of f antioxidannt 

activity – both 

valuues 

were subtracted. 

Resulting absorbance e was reca alculated inn 

accordance 

with 

calibration 

curve 

to the equivalentt 

of gallic acid. Proce edures of mmeasureme 

ents and 

prepparation 

of f reagents ar re describedd 

in the wo ork of Sochor 

et al. (Soochor, 

et al.). 

3. RESULTTS 

AND D 

All set of patients 

prosstate-speciffic 

antigen 

andd 

36,5 ng·mml 

histtologically 

appproaches 

we 

andd 

oxidised ( 

GSHH 

and GSS 

Valuues 

of the G 

(2,77 

at average 

valuues 

were be 

welll 

evident th 

the heavy hea 

antiitumour 

ag 

speccies. 

ROS a 

-1 DISCUSSI 

s suffering 

(PSA). Bef 

(9,7 ng·ml 

as acinar 

ere used for 

(GSSG) glu 

SG concent 

GSH:GSSG 

e) and relati 

etween 10, 

hat all patie 

alth troubl 

gents, whic 

are subseque 

-1 ION 

from mal lignant dis sease has iincreased 

level l of 

fore biopsy y, PSA leve el ranged beetween 

2,3 3 ng·ml 

at avverage). 

Ty ype of mal lignant dissease 

was 

r adenocarrcinoma 

of f the prostate. 

Diffferent 

met 

r the determmination 

of f oxidative stress. Ratee 

of reduced 

utathione wwas 

monitored. 

Rate was w determmined 

as a 

trations. TThese 

value es were determined 

bby 

the HP 

rate for pat atients (n=10) 

ranged between b 0,115 

and 7,6 ( 

ive standarrd 

deviation n RSD 86% . For the coontrol 

grou 

5 and 21,22 

(Fig. 1b) ( 13,69 at average); a RSSD 

was 24 

ents are expposed 

to ox xidative stre ess. These vvalues 

can 

les. Howevver, 

they can 

be conn nected witth 

the func 

ch effect iss 

connecte ed with generation 

oof 

reactive 

ently able tto 

damage tumour t tiss sue.. 

-1 

verified 

thodical 

d (GSH) 

ratio of 

PLC-ED. 

(Fig. 1a) 

up, these 

%. It is 

show to 

ction of 

oxygen 

Fig 1.: Values oof 

GSH/GSS SG in patieents 

(a) and in control group (b). 

Antioxiddant 

activity y was deterrmined 

usin ng four diff ferent methhods. 

DPPH H 

baseed 

on the aability 

of st table free rradicals 

of 2,2-dipheny 

2 yl-1-pikryllhydrazyle 

withh 

hydrogenn 

donors. ABTS A methhod 

is based d on the ne eutralisatioon 

of ABTS 

(2,22‘-azinobis( 

3-ethylben nzothiazolinne-6-supho 

onate). FRA AP methodd 

(Ferric R 

Anttioxidant 

Power) 

is ba ased on thee 

reduction of the ferr ric complexxes 

of TPTZ 

trippyridyl-S-trriazine) 

with ferrric 

chlo oride. DMPD D (NN,N-dimeth 

diamminobenzenne) 

is unde er activity of ferrous s salt transf formed intto 

relatively 

andd 

colour raadical 

form m. All resullts 

were re elated to th he equivaleents 

of gal 

(GAAE), 

whichh 

represen nts standarrd 

for antioxidant 

tests. 

This standard 

commparison 

noot 

only of obtained vvalues 

of pa atients and healthy peersons, 

but 

indiividual 

meethods, 

espe ecially witth 

respect of spectrum m of groupp 

of free r 

• test is 

to react 

• radical 

Reducing 

Z (2,4,6hyl-1,4y 

stable 

llic acid 

enables 

t also of 

radicals. 

- 261 - 

Brno


(Apak, et al.). Due to above-mentioned facts, it is suitable to use different methods for 

the determination of antioxidant activity. Obtained results are more comprehensive 

and more representable. 

Table 1.: Values of antioxidant activity of blood serum of patients suffering from 

acinar adenocarcinoma of prostate. 

Used method Range of values of 

mgGAE·ml -1 

- 262 - 

Average value of 

mgGAE·ml -1 

RSD [%] 

DPPH 0.01-0.15 0.06 19% 

ABTS 0.05-0.35 0.19 15% 

FRAP 0.09-0.53 0.22 18% 

DMPD 0.06-0.36 0.21 13% 

Number of patients n=10 

Number of repetitions of measurement n=3 

RSD – relative standard deviation related to the minimal and maximal values 

Table 2.: Values of antioxidant activity of blood serum of healthy persons – control. 

Used method Range of values of 

mgGAE·ml -1 

Average value of 

mgGAE·ml -1 

RSD [%] 

DPPH 0.09-0.45 0.30 27% 

ABTS 0.14-0.56 0.32 22% 

FRAP 0.38-0.86 0.51 24% 

DMPD 0.21-0.67 0.37 19% 

Number of healthy persons n=10 

Number of repetitions of measurement n=3 

RSD – relative standard deviation related to the minimal and maximal values 

Our study demonstrated significant differences in antioxidant activity of blood 

serum in group of patients suffering from acinar adenocarcinoma of prostate and 

healthy persons. This fact is connected with oxidative stress, which was verified by 

the GSH/GSSG rates. Our results agree with the results of exact studies focused on 

adenocarcinoma of prostate, which advert to the significant changes in protective 

antioxidant mechanisms in patients (Yilmaz, et al.). In these studies, values of lipid 

peroxidation index (Aydin, et al.), superoxid dismutase and catalase activities (Baker, 

et al.), which represent one of the most important protective mechanisms, were 

changed. These changes are responsible for reduction of antioxidant activity (Pace, et 

al.). 

4. CONCLUSION 

Evaluation of antioxidant state can serve as an important marker in the area of 

malignant diseases treatment. Results of our study verified significance of GSH/GSSG 

rate as a marker of oxidative stress in patients suffering from acinar adenocarcinoma 

of prostate.



Grants GAČR 301/09/P436 and CYTORES GA ČR P301/10/0356, Liga proti 

rakovině Praha 2011 are highly acknowledged. 

6. REFERENCES 

1. Pastore, A., et al., (2001): Determination of blood total, reduced, and oxidized glutathione in 

pediatric subjects, Clinical Chemistry, 47: 1467-1469. 

2. Cotgreave, I. A., et al., (1998): Recent trends in glutathione biochemistry - Glutathione-protein 

interactions: A molecular link between oxidative stress and cell proliferation?, Biochemical and 

Biophysical Research Communications, 242: 1-9. 

3. Klatt, P., et al., (2000): Regulation of protein function by S-glutathiolation in response to oxidative 

and nitrosative stress, European Journal of Biochemistry, 267: 4928-4944. 

4. Fratelli, M., et al., (2002): Identification by redox proteomics of glutathionylated proteins in 

oxidatively stressed human T lymphocytes, Proceedings of the National Academy of Sciences of 

the United States of America, 99: 3505-3510. 

5. Bursell, S. E., et al., (2000): The potential use of glutathionyl hemoglobin as a clinical marker of 

oxidative stress, Clinical Chemistry, 46: 145-146. 

6. Niwa, T., et al., (2000): Increased glutathionyl hemoglobin in diabetes mellitus and hyperlipidemia 

demonstrated by liquid chromatography/electrospray ionization-mass spectrometry, Clinical 

Chemistry, 46: 82-88. 

7. Chai, Y. C., et al., (1994): S-THIOLATION OF INDIVIDUAL HUMAN NEUTROPHIL PROTEINS 

INCLUDING ACTIN BY STIMULATION OF THE RESPIRATORY BURST - EVIDENCE 

AGAINST A ROLE FOR GLUTATHIONE DISULFIDE, Archives of Biochemistry and 

Biophysics, 310: 273-281. 

8. Droge, W., et al., (1986): Glutathione augments the activation of cytotoxic lymphocytes-t invivo, 

Immunobiology, 172: 151-156. 

9. Hayes, J. D., et al., (1999): Glutathione and glutathione-dependent enzymes represent a Coordinately 

regulated defence against oxidative stress, Free Radical Research, 31: 273-300. 

10. Sochor, J., et al., (2010): An assay for spectrometric determination of antioxidant activity of a 

biological extract, Listy Cukrovarnicke a Reparske, 126: 416-417. 

11. Singh, S., et al., (2008): In vitro methods of assay of antioxidants: An overview, Food Reviews 

International, 24: 392-415. 

12. Schlesier, K., et al., (2002): Assessment of antioxidant activity by using different in vitro methods, 

Free Radical Research, 36: 177-187. 

13. Sochor, J., et al., (2010): Fully Automated Spectrometric Protocols for Determination of Antioxidant 

Activity: Advantages and Disadvantages, Molecules, 15: 8618-8641. 

14. Yilmaz, M. I., et al., (2004): Antioxidant system activation in prostate cancer, Biological Trace 

Element Research, 98: 13-19. 

15. Aydin, A., et al., (2006): Oxidative stress and antioxidant status in non-metastatic prostate cancer 

and benign prostatic hyperplasia, Clinical Biochemistry, 39: 176-179. 

16. Baker, A. M., et al., (1997): Expression of antioxidant enzymes in human prostatic adenocarcinoma, 

Prostate, 32: 229-233. 

17. Pace, G., et al., (2010): Oxidative Stress in Benign Prostatic Hyperplasia and Prostate Cancer, 

Urologia Internationalis, 85: 328-333. 

18. Apak, R., et al., (2007): Comparative evaluation of various total antioxidant capacity assays applied 

to phenolic compounds with the CUPRAC assay, Molecules, 12: 1496-1547. 

- 263 -


ANTIOXIDANT ACTIVITY OF TUMOUR 

CELL AND EFFECT OF CYTOSTATICS ON 

ANTIOXIDANT STATUS 

Jiří SOCHOR 1 , Tomáš ECKSCHLAGER 2 , Petr BABULA 1 , Vojtěch ADAM 1 , Marie 

STIBOROVÁ 3 , René KIZEK 1 

1 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University, Zemedelska 1, 

CZ-61300 Brno, Czech Republic 

2 Department of Paediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University in 

Prag, V Úvalu 84, CZ-150 06 Praha 5, Czech Republic 

3 Department of Biochemistry, Faculty of Science, Charles university in Prag, Albertov 2030, CZ-128 40 

Praha 2, Czech Republic 

Abstract 

Our work was focused to the implementation of methods for determination of 

antioxidant activity in tumour cells and in tumour cells treated by cytostatics. Level of 

oxidative stress was monitored as a change of antioxidant activity, or, alternatively as 

antioxidant state. Optimization of four different photometric methods represents the 

most important result of our work. 


Toxicity of cytostatics represents one of the most important problems in cancer 

treatment. Oxidative stress as a result of activity of cytostatics is still inadequately 

examined (Il'yasova, et al., Kansal, et al.). It is well known that oxidative stress in 

connected with misbalance between levels of pro-oxidants and protective antioxidant 

mechanisms (Singh, et al.). It seems that changes in these mechanisms are very 

important in the treatment of patients suffering from a malignant disease (Reuter, et 

al.). Direct measurement of reactive oxygen species (ROS) or markers of oxidative 

stress is in clinical medicine still very difficult (Franco, et al.). In addition, organisms 

is affected by simultaneous action of many factors (age of patients, nutrition, stress, 

next diseases, factors of the living environment), so, it is practically impossible to 

determine change of oxidation action due to malignant disease itself and cytostaticsbased 

therapy or radiotherapy (Il'yasova, et al., Omerovic, et al.). Therefore, it is 

necessary to understand processes on the cellular level in in vitro conditions without 

action of other factors. 

2. EXPERIMENT 

Antioxidant activity analysis: automatic spectrophotometer BS-400 (Mindray, 

China) was used for measurement of antioxidant activity. This apparatus consists of 

cuvette space (thermostated to 37±0.1 °C), reagent space with carousel for reagents 

and for sample preparation (thermostated to 4±1 °C) and optic detector. Tungsten 

halogen lamp served as source of radiation. Transfer of samples was provided by 

- 264 -

XI. WWorkshop 

of Phyysical 


robootic 

arm wwith 

dosage e needle. CContent 

of cuvettes is s mixed byy 

automatic c stirrer 

immmediately 

aafter 

additio on of reageent 

of samp ple of volum me of 2-45 μl. Contam mination 

is mminimised 

ddue 

to wash hing both ddosage 

need dle and stir rrer by MillliQ 

water. For the 

deteection, 

folllowing 

wav ve lengths could be used: u 340, 380, 3 412, 4450, 

505, 54 46, 570, 

605, 

660, 700, 740, and 80 00 nm. 

Volume of reagent (DPPH, ABBTS, 

FRAP P, DMPD) was w incubatted 

for 15 minutes m 

withh 

directly ddefined 

vol lume of bloood 

serum; after it, ab bsorbance oof 

this mixt ture was 

meaasured. 

Vallues 

of absorbance 

off 

reagent it tself and ab bsorbance oof 

sample after 15 

minnutes 

of inncubation 

were w used for determ mination of f antioxidannt 

activity – both 

valuues 

were subtracted. 

Resulting absorbance e was reca alculated inn 

accordance 

with 

calibration 

curve 

to the equivalentt 

of gallic acid. Proce edures of mmeasureme 

ents and 

prepparation 

of f reagents ar re describedd 

in the wo ork of Sochor 

et al. (Soochor, 

et al.). 

Biologicaal 

samples: s: cell line UKF-NB-4 

with am mplificationn 

of MYCN 

gene 

(sennsitive 

cell line) and derived d cell l line resista ant to the cisplatin, c wwhich 

was prepared p 

undder 

cisplatinn 

treatment t, were useed 

in experi iment. Cells 

were treaated 

by cisp platin in 


0.1 μM, 1 μM, and 100 

μM, control 

untreated 

cells weere 

also incl luded in 

experiment. 

Tiime 

of treat tment was 6 and 48 ho ours. 

Tabble 

1. Parammeters 

of ex xperimentall 

samples of f cells 

a) 

Sample 

designatio on 

1 

2 

3 

4 

5 

6 

7 

8 

Timee 

of Sensitivity 

treatmment 

to 

(h) ) cisp platin 

6/244 

sen nsitive 

6/244 

sen nsitive 

6/244 

sen nsitive 

6/244 

sen nsitive 

6/244 

resistant 

6/244 

resistant 

6/244 

resistant 

6/244 

resistant 

Figuure 

1. Cell lline 

UKF-N NB-4 withoout 

treatmen nt (a) and after a treatmment 

by cisp platin in 

concentration 

of 10μM 1 (b) 

- 265 - 

Concentrati 

C on 

of cisplatin (µ µM) 

0 

0.1 

1 

10 

0 

0.1 

1 

10 

Brno



Oxidative stress is caused by misbalance between formation and elimination of 

free radicals, especially reactive oxygen species. It is induced by formation of free 

radicals, or by depression of protective antioxidant mechanisms (Bulkley, Clutton). 

Antioxidant activity is one of the most important markers of oxidative stress 

(Antolovich, et al.). 

In the area of determination of antioxidant characteristics, many different 

methods have been proposed and verified (Schlesier, et al., Sochor, et al.). They are 

different in the view of principle of reaction. In addition, new and new modifications 

of these methods are still proposed and developed. Their importance consists in the 

characterization of antioxidant activity in conditions similar to physiological 

conditions. Methods used for antioxidant activity determination are based mostly on 

the direct reaction between studied compound or mixture of compounds with radicals 

(quenching or scavenging) or on reactions with transient metals. 

Four different methods for determination of antioxidant activity were used in 

our study – namely DPPH, ABTS, FRAP, and DMPD). These methods were optimised 

for the measurement by the automated analyser BS-400 Mindray. Calibration curves 

were designed for calculation of antioxidant activity to the equivalent of gallic acid. 

Table 2. Summary of parameters for individual methods and recalculation to the gallic 

acid equivalent. 

Method Wave 

length 

nm 

Range of 

measurement 

µg·ml -1 

Calibration 

equation 

- 266 - 

Confidence 

coefficient R 

Relative 

standard 

deviation 

% 

DPPH 505 1 – 10 y = -0.103ln(x) - 

0.093 

0,996 1,8 

ABTS 660 1 – 20 y = -0.011x - 

0.019 

0,996 2,1 

FRAP 570/605 1 – 300 y = 0.076x + 0.057 0,999 1,5 

DMPD 505 1 – 25 y = -0.060x + 

0.180 

0,999 2,3 

In the next step, cells of above-mentioned cell lines were analysed (Tab. 1). 

Values of antioxidant activity were different in accordance with cisplatin 

concentration and duration of treatment. Different values were also recorded by the 

use of different methods. This may be explained by the specificity of individual 

methods for individual types of free radicals. Using these methods, obtained data were 

more complex and comprehensive in comparison with use of only one method. Cells 

treated by cisplatin demonstrated lower values of antioxidant activity compared to 

control untreated cells.


4. CONCLUSION 

Looking for new markers of oxidative stress in they case of patients suffering 

from malignant disease is one of the main objects of research. There is only limited 

number of clinical tests, which are effective. However, they are usually invasive and 

limited to only some types of tumours. Determination of antioxidant activity 

represents important step in clinical medicine. Our aim consists in utilization of 

potential of these methods in malignant diseases and verification of importance of 

oxidative stress during treatment. 


Work was supported by grants GAČR 301/09/P436 and CYTORES GA ČR 

P301/10/0356. 

6. REFERENCES 

1. Il'yasova, D., et al., (2011): Individual responses to chemotherapy-induced oxidative stress, Breast 

Cancer Research and Treatment, 125: 583-589. 

2. Kansal, S., et al., (2011): Evaluation of the role of oxidative stress in chemopreventive action of fish 

oil and celecoxib in the initiation phase of 7,12-dimethyl benz(alpha)anthracene-induced 

mammary carcinogenesis, Tumor Biology, 32: 167-177. 

3. Singh, S., et al., (2008): In vitro methods of assay of antioxidants: An overview, Food Reviews 

International, 24: 392-415. 

4. Reuter, S., et al., (2010): Oxidative stress, inflammation, and cancer How are they linked?, Free 

Radical Biology and Medicine, 49: 1603-1616. 

5. Franco, R., et al., (2008): Oxidative stress, DNA methylation and carcinogenesis, Cancer Letters, 266: 

6-11. 

6. Il'yasova, D., et al., (2009): Markers of oxidative status in a clinical model of oxidative assault: a pilot 

study in human blood following doxorubicin administration, Biomarkers, 14: 321-325. 

7. Omerovic, E., et al., (2008): Aqueous fish extract increases survival in the mouse model of cytostatic 

toxicity, Journal of Experimental & Clinical Cancer Research, 27: 10. 

8. Sochor, J., et al., (2010): Fully Automated Spectrometric Protocols for Determination of Antioxidant 

Activity: Advantages and Disadvantages, Molecules, 15: 8618-8641. 

9. Bulkley, G. B., (1983): The role of oxygen free-radicals in human-disease processes, Surgery, 94: 407- 

411. 

10. Clutton, S., (1997): The importance of oxidative stress in apoptosis, British Medical Bulletin, 53: 

662-668. 

11. Antolovich, M., et al., (2002): Methods for testing antioxidant activity, Analyst, 127: 183-198. 

12. Schlesier, K., et al., (2002): Assessment of antioxidant activity by using different in vitro methods, 

Free Radical Research, 36: 177-187. 

13. Sochor, J., et al., (2010): An assay for spectrometric determination of antioxidant activity of a 

biological extract, Listy Cukrovarnicke a Reparske, 126: 416-417. 

- 267 -


ELECTROCHEMICAL ANODIZATION AS 

TOOL FOR NANOSTRUCTURES 

FORMATION 

Dmitry SOLOVEI 1 , Jaromíir HUBÁLEK 1 

1 Department of Microelectronics, Faculty of Electrical Engineering and Communication, Brno 

University of Technology, Technicka 3058/10, CZ-616 00, Brno, Czech Republic 

Abstract 

In this paper we describe the methods of electrochemical anodization of valve metals 

such as aluminum, titanium, niobium, for formation of nanoscale and nanostructured 

functional layers in the form of nanoporous matrix templates, nanopillars and 

nanodots with the basic geometric parameters of 10 to 100 nm, for future use in 

sensor, as well as micro-and nanoelectronics. 


The electrochemical anodization of valve metals has been known for more than 

80 years; oxalic acid anodizing was first patented in Japan in 1923 and later widely 

used in Germany [1]. But only through the invention of the electron microscope, 

scientists were able to understand that by using these methods can obtain nanoscale 

objects, such as nanoporous alumina matrix templates [2], metaloxide nanopillars [3] 

and metaloxide nanodots [4]. The main advantage of electrochemical anodization is a 

simple, reproducible and easily controllable formation of self-assembled nanosystems, 

which is very convenient for practical applications. In this paper, the methods of the 

electrochemical anodization of valve metals (Al, Ti, Nb) for creation of self-assembled 

nanostructured layers was describe. 

2. EXPERIMENT 

For electrochemical anodizing process are used metal foils and thin layers of 

valve metals sputtered in vacuum on dielectric or semiconducting substrate. 

Electrochemical anodization is carried out in aqueous solutions of organic and 

inorganic acids such as sulfuric, oxalic, phosphoric and citric with concentrations 

from 0.1 to 2 M. Anodization process was conducted in two modes: galvanic static 

mode with current densities from 2 to 10 mA/cm 2 and at the potential static mode 

with potential in electrochemical system from several to tens of volts. All process was 

held at temperature 20 °C with constant steering of electrolytes. For formation of 

nanoporous alumina templates was used known well two stage anodizing method: 

first porous anodizing on necessary thickness, removing of porous alumina matrixes 

and second porous anodizing for creation of nanoporous template with control high 

and porous diameter. Porous aluminum oxide was removed in a selective etchant 

based on chromium trioxide and phosphoric acid at 65 °C for 15 minutes. For 

formation of metaloxide nanopillars from different valve metals was used process of 

- 268 -


locally metal oxidization of underlying metal layers (Ti, Nb) through the porous 

alumina mask with additional reanodizing step when the potential smoothly 

increasing to value of 1,5 – 3 times higher than potential of porous alumina formation. 

For formation of metaloxide nanodots it was also used porous alumina oxide templates 

like for formation of metaloxide nanopillars but with low voltages below 10 volts. The 

process of underlying metal anodization lasted for 30 minutes and after reaching of 

constant minimum value of anode current was switched off. For revealing of 

metaloxide nanopillars and nanodots the last stage was completely removing of 

alumina templates by using chemical etching process. All electrochemical process was 

carried out by GPIB interfaces power supply and multimeters, scanning electron 

microscopy (SEM) techniques was used for investigation of uniformity and 

geometrical parameters of nanostructures layers. 

3. 3.RESULTS AND DISCUSSION 

On Fig. 1a is shown typical kinetic curve of anodizing 2 mkm aluminium layers 

in 0.9 M oxalic electrolyte at galvanic static mode with current of 5 mA/cm 2 . As see 

from Fig 1a the potential of porous alumina formation is 32.5 volts. By this potential 

we fabricated porous alumina matrix with pore diameter of about 20 nm located with 

step 80 – 85 nm (see Fig 1b). After selective removal of the porous matrix anodization 

process was repeated under initial conditions to obtain ordered porous alumina matrix 

with necessary thickness 500 – 700 nm (see Fig. 1c). So using this method on thin 

aluminum films we can fabricate low profile porous alumina matrix template with a 

pore diameter of 5 to 400 nm and a height of oxide from 300 to 2000 nm which can 

use for filing or coating by different materials. 

For formation of metaloxide nanopillars and nanodots firstly was made porous 

alumina matrixes with necessary pore diametres: for formation of niobium oxide 

nanopillars pore diametres was 30 nm and for formation of titanium oxide nanodots 

pore diametres was about 5 nm. During porous alumina formation when anodization 

front was reached of niobium or titanium underlayer anodization potential begins to 

increase with speed 1 V/s and its growth continued up to a certain limited value at 130 

volts for niobium layer and 11 volts for titanium layer. Then the anodizing mode was 

switched to the constant potential at which lasted electrochemical anodization of 

niobium and titanium underlayer. In this case, the current in the electrochemical 

system began to decline exponentially (see Fig. 2a), driven by the rising pillars of 

niobium and titanium oxide at the bottom of each pore of the matrix of anodic 

aluminum oxide (see Fig. 2b, c). During local oxidation of valve metals through the 

porous alumina matrixes there is a growth of oxides of these metals in the space of 

pores and by changing of the anodizing potential can be formed metaloxide 

nanopillars and nanodots of a necessary height and radius. Obtained using the method 

described above niobium oxide nanopillars has diameters of about 40 nm and height 

of about 250 nm and the titanium oxide nanodots has a diameter about 15 nm and a 

height of about 20 nm. 

- 269 -

XI. Workshhop 


E 

s´11 

Potential, V 

Fig.1 FFabrication 

of low pr rofile poroous 

alumin na matrixes s: a – anoddizing 

kin netic; 

b – SEM immage 

of the surface; c – SEM imag ge of the cr ross sectionn 

Current, A 

aa) 

0.000 07 

0.000 06 

0.000 05 

0.000 04 

0.000 03 

0.000 02 

0.000 01 

0.000 00 

0 250 5000 

750 1000 1250 

1500 1750 

a) 

35 

30 

25 

20 

15 

10 

5 

0 

0 100 200 3300 

400 500 600 

Time, s 

700 800 900 

Time, s 

b) ) 

Fig.2 Faabrication 

of nanopill lars and naanodots: 

a – anodizing g kinetic; b – SEM im mages 

of niobiumm 

oxide nanopillars; 

c – SEM imag ge of titaniu um oxide nnanodots 


Soo 

by usinng 

of described 

anoddizing 

tech hniques we w can forrm 

metalo oxide 

nanostrructured 

and 

nanosiz zed functiional 

layer rs of valve e metals wwith 

the main m 

geomettric 

dimenssions 

of 10 – 100 nm aat 

room tem mperatures and receivved 

layers have h 

good tiime 

stable of their fu unctional pproperties. 

This techn nique of thee 

formation 

of 

nanoscaale 

metaloxxide 

arrays s can be eeasily 

adap pted for a standard mmicroelectronic 

producttion 

process, 

which is one of the key technologic t cal advantaages. 

Obtained 

nanostrructured 

laayers 

of oxide 

valvee 

metals may m find im mmediate application n in 

- 270 - 

c) ) 

c) 

b) 

Brno


nanoelectronics and nanosensory for the development of new generation electronic 

devices. 


The work has been supported by the grants GAAV KAN208130801 and GACR 

102/08/1546 is highly acknowledged. 

6. REFERENCES 

[1] Sheasby, P. G., Pinner, R.: The Surface Treatment and Finishing of Aluminum 

and its Alloys, 2 (sixth ed.), ASM International & Finishing Publications, 2001, 

Materials Park, Ohio & Stevenage, UK, 427 

[2] Lei, Y., Cai, W., Wilde, G.: Progress in Materials Science, 52 (2007), 7, 465 

[3] Mozalev, A., Sakairi, M., Saeki, I., Takahashi, H.: Electrochimica Acta, 48 (2003), 20-22, 3155 

[4] Chen, Z., Lei, Y., Chew, H. G., Teo, L. W., Choi, W. K., Chim, W.K.: Journal of Crystal Growth, 

268 (2004), 3-4, 560 

- 271 -


THE IMPACT OF SELECTED PLATUM 

GROUP ELEMENTS ON ANTIOXIDANT 

ACTIVITY OF DUCKWEED (LEMNA 

MINO L.) 

Lenka STRAKOVÁ 1, 2 , Ivana SOUKUPOVÁ 1 , Jiří SOCHOR 2 , Vojtěch ADAM 2 

Miroslava BEKLOVÁ 1 , René KIZEK 2 

1 University of Veterinary and Pharmaceutical Sciences, Palackeho 1-3, 612 42 Brno, Czech Republic 

2 Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, 613 00 Brno, Czech Republic 

Abstract 

An actual problem in the environmental burden begins to be platinum group 

elements (PGEs), which come from automobile traffic. Increasing use of PGEs in 

exhaust catalysts and the runoff from surroundings of busy highways, lead to 

increased spread and accumulation of these metals in water ecosystem. To determine 

how these metals may affect the antioxidant activity of aquatic plants we chose 

duckweed (Lemna minor L.) as a bioindicator. In this experiment we used 

spectrophotometric methods for determination of the antioxidant activity. Plants 

were exposed by the solutions of SIS and selected metals from PGEs: platinum (PtCl4) 

and rhodium (RhCl3). Lemna minor was cultivated in various concentrations 

of platinum and rhodium (1, 10 and 100 μmol.l -1 ), respectively. The experiment lasted 

for seven days and sampling days were 2nd, 4th and 6th day. We chose two methods 

for this determination - FRAP and ABTS radical method. The influence of selected 

PGEs on Lemna minor was observed as a change of plant antioxidant activity. In the 

case of PtCl4 is an evident dependence between duration of the experiment and 

increasing concentrations of metals, but we do not observe such a big trend of 

increase of the antioxidant activity in the test with rhodium (RhCl3). 


Accumulation of platinum group elements (PGEs) in the environment has been 

increased over the time. Catalytic converters of modern vehicles are considered to be 

the main sources of PGEs pollution. The present literature survey shows that the 

concentration of these metals has increased significantly 

in the last decades [1]. Growing concentrations of PGEs in the culture medium 

correlates with the increasing concentrations in plants. Increased levels of PGEs may 

affect many cellular reactions. They affect the permeability of cell membranes, 

disrupting mitochondrial metabolism and interfere with protein synthesis. 

Duckweed (Lemna minor L.) is used in water quality studies to monitor heavy 

metals and other aquatic pollutants, because duckweed, like other water plants, may 

selectively accumulate certain chemicals. The plants possess physiological properties 

- 272 -


(small size, rapid growth in solutions of pH between 5 and 9, and vegetative 

propagation), which make them an ideal test system [2]. 

Protection against ROS is an intrinsic characteristic of any living cell. The 

photosynthetic electron transport system is the major source of ROS in plant tissues. 

ROS have the potential to generate singlet oxygen 1 O2 and superoxide O2 •− , which in 

turn, can be successively reduced to hydrogen peroxide (H2O2) and hydroxyl radical 

(•OH). ROS formation in plants stimulates defence mechanisms in which antioxidant 

systems are induced to detoxify different ROSs, and assist in acclimating plants to 

oxidative stress [4]. 

2. EXPERIMENT 

The experiment was performed using the CSN EN ISO 20079 - Water quality – 

Determination of the toxic effect of water constituents and waste water on duckweed 

(Lemna minor L.) – Duckweed growth inhibition test. We worked with two metal 

solutions of PGEs: PtCl4, RhCl3 with the concentrations of 1, 10 and 100 μmol·l -1 . For 

the growth of duckweed we used a micromethod (polystyrene macroplates of 10 ml 

sample volume). We took samples 2nd, 4th and 6th day to determine the dynamics of 

the effects of these metals. 

To determine the antioxidant activity, we used a biochemical analyzer Mindray 

BS 400 and choose two methods - a method of FRAP (Ferric Reducing Antioxidant 

Power) and ABTS radical method. The ABTS radical method is one of the most used 

assays for the determination of the concentration of free radicals. It is based on the 

neutralization of a radical-cation arising from the one-electron oxidation of the 

synthetic chromophore. The FRAP method (Ferric Reducing Antioxidant Power) is 

based on the reduction of complexes of 2,4,6-tripyridyl-s-triazine (TPTZ) with ferric 

chloride hexahydrate [3]. 


Increasing concentrations of PGEs negatively affected the growth of Lemna 

minor. During the experiment, we monitored the growth slowdown and changing the 

appearance of fronds. Plants started to turn yellow (chlorosis), and at the end of the 

experiment, we observed leaves with white or colourless areas (necrosis). We 

recalculated our results of the antioxidant activity on a basis of the total protein 

(reaction with red pyrogallol). 

Figures 1a and 1b show increasing antioxidant activities from 2nd day. The 

highest increase occurred at the end of the experiment. The increase in the 

antioxidant activity well corresponds with the increasing concentration 

of solution PtCl4. 

Figure 2a and 2b, the increase in the case of RhCl3 is not as obvious as in Figures 

1a and 1b. All values are almost at the same level all 3 days of testing. Only at the end 

of the experiment there was a large increase in the antioxidant activity in the 2nd 

concentration (1 μmol.l -1 ). 

- 273 -


FRAP [mgGAE·l -1 ] 

ABTS [mgGAE·l -1 ] 

20 

15 

10 

5 

0 

1400 

1200 

1000 

800 

600 

400 

200 

0 

control group 

1 µM 

10 µM 

100 µM 

PtCl4 RhCl3 

2nd day 4th day 6th day 

Sampling days 

control group 

1 µM 

10 µM 

100 µM 


Sampling days 

4. CONCLUSION 

The influence of selected PGEs on Lemna minor was observed as a change of 

plant antioxidant activity. Especially in the case of PtCl4 there is an evident 

dependence between duration of the experiment and increasing concentrations of 

metals. The experiment showed a good correlation with the increase of the 

antioxidant activity: the last sampling day leads to a very high increase of the 

antioxisdant activity. 

In contrast, we do not observe such a big trend of increase of the antioxidant 

activity in the test with rhodium (RhCl3). The values are virtually unchanged, only at 

the end of the experiment there is a blip at the lowest concentration. 

This experiment is only a part of a broader research of the effects of PGEs on 

Lemna minor. Within short time we are expecting more results from other methods 

from fully automated spectrometric estimation and from differential pulse 

voltammetry. And then we will be able to assess the influence do the PGEs on 

duckweed more suitable and accurately. 

- 274 - 

FRAP [mgGAE·l -1 ] 

ABTS [mgGAE·l -1 ] 

7 

6 

5 

4 

3 

2 

1 

0 

600 

500 

400 

300 

200 

100 

0 

control group 

1 µM 

10 µM 

100 µM 


Sampling days 

control group 

1 µM 

10 µM 

100 µM 


Sampling days



The work has been supported by MSMT 6215712402 and IGA 82/2001/FVHE. 

6. REFERENCES 

[1] Ravindra, K., Bencs, L., Van Grieken, R.: Science of the Total Environment, 318 (2004), 1-43. 

[2] Radic, S., Stipanicev, D., Cvjetko, P., Mikelic, I., L., Rajcic, M., M., Sirac, S., 

Pevalek-Kozlina, B., Pavlica, M.: Ecotoxicology, 19 (2010), 1,216-222. 

[3] Sochor, J., Ryvolova, M., Krystofova, O., Salas, P., Hubalek, J., Adam, V., Trnkova, L., Havel, L., 

Beklova, M., Zehnalek, J., Provaznik, I., Kizek, R.: Molecules. 15 (2010), 8618-8640. 

[4] Brain, R. A., Cedergreen, N: Biomarkers in Aquatic Plants: Selection and Utility, Springer, 2009, 

New York, 49 - 109. 

- 275 -


IMPORTANCE OF DIFFERENTIAL PULSE 

VOLTAMMOGRAMS FOR STUDYING OF 

BIOLOGICAL IMPORTANT 

PHENOMENONS 

Pavlína ŠOBROVÁ 1 , Lenka NOVÁKOVÁ 2 , Olga ŠTĚPÁNKOVÁ 2 , Miroslava 

BEKLOVÁ 2 , Vojtěch ADAM 1 , René KIZEK 1 

1Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, 


2 Department of Cybernetics, Faculty of Electrical Engineering, Czech Technical University, Technicka 

2, CZ-166 27 Prague 6, Czech Republic 

Abstract 

This work is consisted of observing of metallothionein level by Brdicka reaction. For 

experiment organs as liver, kidney, spleen, heart, muscle, brain, gonad and eye of 28 

days old rats were used and analysed electrochemically. The highest metallothionein 

level was observed in tissues as kidney (67 μg of MT/ g of tissue) and liver (48.75 μg of 

MT/ g of tissue) as organs response for organism detoxification. High level was also 

observed in brain (50.53 μg of MT/ g of tissue). Moreover, we compared the 

electrochemical curves of metallothionein of single tissues by our suggested software. 


Metallothioneins (MTs) are a group of low molecular mass, cysteine-rich 

proteins with a variety of functions including involvement in metal homeostasis, free 

radical scavenging, protection against heavy metal damage, and metabolic regulation 

via Zn donation. These proteins occur in whole animal kingdom with high degree of 

homology. MTs were discovered and originally located from horse kidney over five 

decades ago in 1957 by Margoshes and Valee [1,2]. Later, similar proteins from 

kidney, liver, and intestine tissues of other mammals, as well as from birds, fish, 

crustacean mussels, fungi [3] and plants [4] and from metal-resistant microorganisms 

[5,6] were characterized. The molecular weight of mammalian MT is from 6 to 10 kDa 

and is consisted of a chain of 61 or 62 amino acids with major representation of 

cystein (more than 30 % from all aminoacids). MT`s structure consists of two 

domains: more stable α (C-terminal), containing 4 divalent ion binding sites, and β 

(N-terminal) capable to incorporate 3 divalent ions [7]. MTs are expressed in four 

different isoforms. Despite the physical-chemical similarity of the forms, their roles 

and occurrence in tissues vary significantly. The most widely expressed isoforms are 

MT-1 and MT-2, which are present almost in all types of soft tissues. MT-2 appears to 

be expressed more in human tissues than MT-1. MT-3 is found mainly in the brain, 

and also is expressed in tongue, stomach, heart, kidney and reproductive tissues [8]. 

There have been few studies on MT-4, and the gene has been detected only in certain 

- 276 -

XI. WWorkshop 

of Phyysical 


squaamous 

epitthelia 

and the maternnal 

deciduu um [9]. Iso oforms mayy 

be distrib buted in 

variious 

ratios iin 

individu ual tissues and 

have dif ffering rate es of degraddation. 

The highest 


of MT in the t body is found in th he liver, kidney, 

intesstine 

and pa ancreas. 

Somme 

authors aalso 

describ bed increassing 

amount 

of MT in brain tissuees 

[10]. 

2. EXPERIIMENT 

Our worrk 

was aime ed at observving 

of met tallothionein 

level meeasured 

by Brdicka 

reacction 

in sinngle 

tissue as a kidney, lliver, 

spleen n, brain, he eart, musclee, 

gonad, ey ye of 28 

dayy 

old rats. Moreover, the Brdiccka 

curves were compared 

to eeach 

other by our 

sugggested 

PC pprogram. 

3. RESULTTS 

AND DISCUSSIO 

D ON 

We founnd 

that met tallothioneiin 

level diff fer in single 

tissue (Fiig. 

1A. The highest 


of MT was s found in tthe 

bodies providing detoxificatiion 

of xeno obiotics, 

suchh 

as kidneyy 

(67 μg of MT/ g of tiissue) 

and liver l (48.75 5 μg of MT/ / g of tissue e). High 

leveel 

was also observed in n brain (500.53 

μg of MT/ M g of tis ssue). Half concentrat tion was 

deteected 

in goonad, 

eye, heart h and mmuscle 

(in order o 30.32 2 μg, 27.88 μg, 25.97μ μg, 24.20 

μg oof 

MT/ g oof 

tissue.) Moreover, M we noticed d that sing gle electrocchemical 

re esponses 

difffer 

in singlee 

tissue. 

Fig. . 1: A) MTT 

levels in individual i rat. B) Typ pical voltam mmograms of electrol lyte and 

metalloothionein. 

The mecchanism 

of the reactioon 

is based on the cat talytic evollution 

of hy ydrogen 

on mercury ellectrodes 

fr rom solutioons 

of prot tein contain ning –SH ggroup 

in am mmonia 

bufffer 

and hexxaamminecobalt 

chlorride 

comple ex (Co(NH3)6Cl3) 

calledd 

Brdicka solution. 

Thee 

mechanisms 

of the reaction r is not detaile ed elucidate, 

but it is expected that t the 

cobalt(II) 

complex 

with protein, p pepptide 

or bas sic nitro compounds 

pplay 

import tant role 

in ccatalytic 

prrocess. 

Inter raction bettween 

coba alt(II) ion and a proteinn 

causes dec creasing 

of ccurrent 

maaximum 

of cobalt(II) and formation 

of two o other poolarographic 

c waves 

(volltammogramms 

peaks) at potentiial 

area from 

-1.2 to o -1.5 V. The reduc ction of 

commplex 

R(SHH)2 

and Co( (II) at poteential 

app. -1.35 V corresponds c s to first catalytic c 

signnal 

(RS2Co) ). Other tw wo signals Cat1 and Cat2 corr respond to the reduc ction of 

hyddrogen 

at thhe 

mercury y electrode and can be e used for quantificati q ion due to the fact 

thatt 

their heigght 

is propo ortional to cconcentrati 

ion of MT. In additionn, 

the signa al called 

- 277 - 

Brno


Co1 could occasionally result from reduction of [Co(H2O)6] 2+ . The mechanism of the 

hydrogen evolution at mercury electrode in Brdicka solution is shown in Fig. 1b. 

We focused our attention on shape of curves of different organs. We found out 

that all tissues gave voltammograms with different shape. The curves do not differ 

only in height of the peaks, but also in peaks appearance and their shape. Some 

voltammograms contained RS2Co, Cat1 and Cat2 only, however, in other curves also 

peak Co1 was detected at -0.7 V. Voltammograms with three peaks were obtained by 

measuring of spleen, gonad and muscle homogenates. Analyses of kidney, liver, brain, 

eye and heart homogenates gave four peaks. 

4. CONCLUSION 

Studying the impact of toxic substances in organisms is still very current and 

provides very interesting results. Toxic substances that are part of our diet, are the 

more objective of such studies because of the achievements we are able not only to 

define the level of toxicity, but also gain an idea of the biochemical effect of the 

studied substance, which can bring new opportunities in its detoxification. 


The financial support from the following projects GA AV IAA401990701, 

MSMT 6215712402 and IGAMZ 10200-3 is greatly acknowledged. The authors wish 

to express their thanks to Anna Vasatkova for excellent work with rat breeding. The 

first author is „Holder of Brno PhD Talent Financial Aid“. 

6. REFERENCES 

[1] Kagi, J.H.R.: Federation Proceedings 19 (1960) 340. 

[2] Margoshes, M., Vallee, B.L.: Journal of the American Chemical Society 79 (1957) 4813. 

[3] Lerch, K.: Nature 284 (1980) 368. 

[4] Rauser, W.E., Curvetto, N.R.: Nature 287 (1980) 563. 

[5] Bulman, R.A., Nicholson, J.K., Higham, D.P., Sadler, P.J.: Journal of the American Chemical 

Society 106 (1984) 1118. 

[6] Kojima, Y., Kagi, J.H.R., Trends in Biochemical Sciences 3 (1978) 90. 

[7] Petrlova, J., Potesil, D., Mikelova, R., Blastik, O., Adam, V., Trnkova, L., Jelen, F., Prusa, R., 

Kukacka, J., Kizek, R.: Electrochimica Acta 51 (2006) 5112. 

[8] Moffatt, P., Seguin, C.: DNA and Cell Biology 17 (1998) 501. 

[9] Liu, J., Cheng, M.L., Yang, Q., Shan, K.R., Shen, J., Zhou, Y.S., Zhang, X.J., Dill, A.L., Waalkes, 

M.P.: Environmental Health Perspectives 115 (2007) 1101. 

[10] Vasatkova, A., Krizova, S., Krystofova, O., Adam, V., Zeman, L., Beklova, M., Kizek, R.: 

Neuroendocrinology Letters 30 (2009) 163. 

- 278 -


ELECTROCHEMICAL BEHAVIUOR OF 

(PRP C ) AND CHANGED (PRP SC ) PRION 

PROTEIN 

Pavlína ŠOBROVÁ 1 , Dalibor HŮSKA 1 , Petr MAJZLÍK 1 , Vojtěch ADAM 1 , Jaromír 

HUBÁLEK 2 , Miroslava BEKLOVÁ 1 , René KIZEK 1* 

1 Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, CZ (E-mail: kizek@sci.muni.cz; phone: 

+420-5-4513-3350; fax: +420-5-4521-2044); 

2 Brno University of Technology, Technicka 10, CZ-616 00 Brno, CZ; 

Abstract 

Prion diseases are fatal neurodegenerative and infectious disorders of humans and 

animals, characterized by structural transition of the host-encoded cellular prion 

protein (PrP C ) into the aberrantly folded pathologic isoform PrP Sc . Prion protein is a 

biomolecule naturally occurring in the animal cells. This protein is present in all 

mammal cells and occurs primarily in neural cells and immune system cells. The main 

aim of this study was to optimize electrochemical methods for the detection of natural 

(PrP C ) and changed (PrPS C ) prion protein. To carry out the main objective a complex 

study of the electrochemical behaviour of both proteins was required. For this 

purpose fundamental electrochemical techniques were used. Both of the prions were 

characterized using different techniques, their limits of detection were found at pM 

levels and possible ability to change the structure of α-helix of natural prion (PrP C ) to 

β-sheet of the infectious prion (PrP Sc ) were monitored. 


Prion diseases are believed to propagate by the mechanism involving selfperpetuating 

conformational conversion of the normal form of the prion protein, 

PrP C , to the misfolded, pathogenic state, PrP Sc . The conformation change, from the αhelix 

in the natural protein form (PrP c ) to the β-sheet of the modified protein form 

(PrP Sc ), significantly influence the protein function. The mutated form (PrP Sc ) is 

extremely resistant to the cell degradation processes and may bind other PrP c 

molecules inducing the conformation change to the PrP Sc . The insufficiency of the 

physiological PrP c and toxic incidence of PrP Sc participate on the genesis of prion 

neurodegenerative diseases [1]. Prion diseases, also known transmissible spongiform 

encephalopathies (TSE) are a group of fatal neurodegenerative diseases. This group 

includes Creutzfeldt–Jakob disease, Gerstmann–Sträussler–Scheinker syndrome (GSS) 

and fatal familial insomnia (FFI) in humans [2]. Prion diseases can arise via infectious, 

hereditary or sporadic routes, and are among the most threatening diseases to humans 

[3,4]. In the animal kingdom, Bovine spongiform encephalopathy (BSE) is the most 

known, so called “the disease of mad cows”. With the outbreak of epidemics and 

discovery of BSE case almost everywhere in Europe, the question arises of how to 

improve screening methods and the possibility of detection of prions. 

- 279 -

XI. Workshhop 


E 

s´11 

2. EXPERIME 

OOur 

work w 

cellularr 

(PrP 

electroc 

techniq 

change 

prion (P 

C ) an 

chemical t 

ques, their 

the struct 

PrPSc ENT 

was aimed on 

study of 

nd changed d (PrP 

echniques. 

limits of d 

ture of α-h 

) weree 

monitored 

Sc the electro ochemical b 

). For this purpose p we 

Both of tthe 

prions were char 

detection wwere 

found at pM leve 

helix of nattural 

prion n (PrP 

d. 

C behaviour oof 

prion pro otein 

e optimizedd 

fundame ental 

racterized uusing 

diffe erent 

els and posssible 

abilit ty to 

) to β-sheet of f the infect tious 

3. RRESULTS 

AAND 

DISC 

Primarily, 

wwe 

attempt 

proteinn 

fragment 118 – 135; F 

using eelectrochemmistry 

and 

amountt 

of prion PrP 

AdTS iis 

based on 

electrodde 

circuit. 

electrodde 

in the b 

indifferrent 

electro 

accumuulation, 

bu 

responsse 

was obse 

(Fig 1B). 

Other co 

C CUSSION 

ed to char 

FW =11232 

optimized 

and PrP 

n the strong 

The exces 

buffer. The 

olyte (Fig 

uffer comp 

erved in pH 

onditions in 

SC racterize pr rion protein ns PrP 

2.3 and β – sheet break 

suitable method m for 

was measured using AdT 

g adsorbingg 

of prion on o the elec 

ss of prionn 

is rinsed d from the 

e adsorbed analyte is finally det 

1A). For optimizatio on we test 

position annd 

pH gradient. 

Th 

H 7.38 in phhosphate 

bu uffer and ti 

n different ccombination 

gave low 

C annd 

PrP 

ker peptide 

their deter 

TS DPV. Pr 

ctrode surfa 

surface o 

tected in th 

ted conditi 

he most e 

me of accu 

wer response 

SC (p prion 

e, FW = 159 97.9) 

rmination. The 

rinciple of f the 

face at an open o 

of the work king 

he presenc ce of 

ion as time 

of 

electrochem mical 

umulation 100 1 s 

es. 

Fig. 1: AdTS DPVV. 

A) Schem me of usingg 

of adsorpt tive transfe er strippingg 

technique e for 

study of prrions. 

Rene ewed surfaace 

of HMD DE (1) is placed p to dr drop contain ning 

prion standdard 

(2) wh here Prion is bond on nly. Other low l molecuular 

substances 

are washed 

out in the followwing 

step (3) HMDE E electrodee 

is placed d to 

supporting electrolyte e (4) and annalyzed 

by DPV. B) Dependence 

of peak he eight 

on time off 

accumulat tion. Curreent 

respons se enhanced d with incrreasing 

tim me of 

accumulatiion. 

The highest ppeak 

was determined 

under 100 s long 

accumulatiion. 

- 280 - 

Brno


4. CONCLUSION 

In conclusion, the electrochemical methods, particularly adsorptive transfer 

stripping technique coupled with differential pulse voltammetry appear to be suitable 

method for prion protein determination. In our work we optimised the technique for 

PrP C and PrP SC determination. The detection limit of the mentioned technique was at 

femtomole level. 

5. 5.ACKNOWLEDGEMENT 

The financial support from NANIMEL GA ČR 102/08/1546, NANOSEMED GA 

AV KAN208130801 and MSMT 6215712402 is highly acknowledged. The first author 

is „Holder of Brno PhD Talent Financial Aid“. 

6. REFERENCES 

[1] Ji, H.F., Zhang, H.Y.: Trends in Biochemical Sciences 35 (2010), 129. 

[2] Yokoyama, T., Mohri, S.: Current Medicinal Chemistry 15 (2008) 912. 

[3] Prusiner, S.B.: Archives of Neurology 50 (1993) 1129. 

[4] Prusiner, S.B., Hsiao, K.K.: Annals of Neurology 35 (1994) 385. 

- 281 -


ELECTROCHEMICAL BIOSENSOR FOR 

DETECTION OF BIOAGENTS 

Eva ŠVÁBENSKÁ 1, 2 , Petr SKLÁDAL 1 , Jan PŘIBYL 1 

1 National Centre for Biomolecular Research, Masaryk University,Brno, Czech Republic 

2 VOP-026 Šternberk,s.p.,VTÚO Brno Division, Czech Republic 

Abstract 

Simple and rapid detection and identification of dangerous agents is one of the most 

important ways preventing illness or even death of people due to infectious diseases 

and bioterroristic threats. An electrochemical immunosensor system is developed in 

our group, it employs specific capture of microbes in the sensitive area by formation 

of an immunocomplex and subsequent binding of the tracer, secondary antibody 

conjugated to peroxidase. Thus, the presence of the target microorganisms is indicated 

using amperometric measurement. 


In the last two decades, the expansion of biosensor technologies for detection 

and identification of chemical and biological species followed the requirements for 

simple, robust, fast and cost-effective analytical systems. Several types of biosensors 

working on electrochemical, optical, magnetic, piezoelectric and thermometric 

principle are available. Nanomaterials and nanoparticles come into the focus of the 

scientists as advantageous tools for preparation of enhanced biosensors layers. 

2. EXPERIMENT 

The biosensor working on amperometric principle was chosen. Before the actual 

detection of microorganisms, the detailed study of immunospecific layers and 

optimization of responses was realized. Thick film based sensors produced by screenprinting 

were produced at BVT Technologies, various types of working electrodes 

based on Au, Pt, carbon were evaluated. These sensors were modified with different 

gold nanoparticles and carbon nanotubes in order to enhance signal from peroxidase. 

Methods used for characterization of the sensing surfaces included amperometry, 

cyclic voltammetry (CV) with using redox probes, atomic force microscopy (AFM) 

and near field scanning optical microscopy (SNOM). 


The sensors were modified by different combinations of peroxidase, albumin 

and/or gelatine (inert proteins), glutaraldehyde (cross-linker) and nanoparticles / 

nanotubes. The mixture was applied either on a pure surface of electrode or attached 

by means of a cystamine-based self-assambled monolayer. Evaluation of the activity of 

thus immobilized peroxidase was measured by amperometry (activity of the enzyme) 

and cyclic voltammetrywith a redox probe (porosity of the biolayer). 

- 282 -

XI. WWorkshop 

of Phyysical 


For detecction 

of microbes, m sandwich assay fo ormats weere 

applie ed with 

ampperometricc 

evaluatio on, antiboodies 

prov viding high h specificitty 

and im mproved 

deteection 

limits 

were se elected. Seeveral 

test ts for the rapid r detecction 

of bio ological 

ageents 

(Escherichia 

col li DH5α, BBacillus 

subtilis 

var. niger, n Franncisella 

tul larensis 

LVSS) 

were foocused 

on n model ssamples 

in n water. Chemical C ssubstances 

were 

deteected 

on a similar r principlee 

using in nhibition of o the enzyme 

act tivity of 

choolinesterasee. 

At prese ent, detecttion 

of biological 

sub bstances inn 

bioaeroso ols was 

initiated 

usingg 

the biosensor 

tecchnology 

coupled to 

cyclonee-based 

sa ampling 

sysstem. 



det tection of bbiological 

agents a has a large poteential 

for ra apid and 

speccific 

responnse 

to a targ get species oof 

microorg ganisms. 


EMENT 

The worrk 

has been n supported 

by the Ministry M of f Defense oof 


(proojects 

no. OOVVTUO20 

008001 andd 

OSVTUO2 2006003). 

6. 

[1] 

[2] 

[3] 

[4] 

[5] 

REFEREENCES 

Skládal, P., Přibyl, J., Šafář, B.: Coommercial 

and Pre-Commerciaal 

Cell De etection 

Technoloogies 

for Defence D agaiinst 

Bioterr ror. Technol logy, Market and Society 39, 2008, 

Amsterdamm, 

ISBN 978- -1-58603-8588-8, 

p. 21-29. 

Skládal, P. , Pohanka, M., M Kupská, E. ., Šafář, B.: Bi iosensors, Ser rra, P. A. (Edd.), 

2010, Zagr reb, ISBN 

978-953-77619-99-2, 

p. 115-125. 

Pedrosa, VV.A. 

et all: Jou urnal of Electrroanalytical 

Chemistry, C 60 02 (2007) 149– 9–155 

Krejčí, J., Grosmanová, , Z., Krejčovvá, 

D., Skláda al, P.: Comm mercial andd 

Pre-Com mmercial 

Cell Deteection 

Tech hnologies ffor 

Defence e against Bi ioterror. NAATO 

Science for Peace 

and Securiity 

Series: 39, 2008, Amsteerdam, 

ISBN 978-1-58603- 

9 -858-8, p. 1500-165. 

Skládal, P. , Symerská, Y., Pohankaa, 

M., Šafář, B., B Macele, A.: A Defense e against bi 

NATO Seccurity 

through h science seriies 

B, 2005, Dordrecht, D ISB BN 1-4020-33385-0, 

p. 221 1-232. 

- 283 - 

Brno 

bioterror.


ELECTROANALYSIS WITH NON-MERCURY 

METAL-MODIFIED CARBON PASTE 

ELECTRODES IN THE NEW MILLENNIUM 

Ivan ŠVANCARA 1 

1 Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, 

Studentská 573, HB/C, 532 10 Pardubice, Czech Republic 

Abstract 

In this contribution, an overview on non-mercury metal-modified carbon paste 

electrodes is given focused on the research activities in the period of 2001-2011 and 

concerning scientific collaboration between the electroanalytical group at the 

University of Pardubice and similarly orientated partners across Europe (namely: 

Austria, Poland, Slovakia, Lithuania, Greece, Slovenia, and Serbia). 


In the new millennium, under the increasing significance of the 

environmentally friendly ("green") chemistry [1], a new family of non-mercury 

metal-based electrodes (MeEs) has occupied a prominent position in the modern 

electroanalysis and, especially, in electrochemical stripping analysis (ESA). All this 

movement had been propelled by introducing of bismuth film-coated glassy carbon 

electrode (BiF-GCE) in 2000 [2], followed soon by numerous alternate configurations 

(see [3-5] and refs. therein), amongst which carbon paste-based substrates were 

actually the second electrode material, recommended and more widely tested in 

combination with bismuth films and related arrangements [6-11]. All the 

configurations were more or less successfully used in ESA for the determination of 

various heavy metals (e.g., Zn, Cd, Pb, Tl, Sn, and In), offering a comparable 

electroanalytical performance to that of traditional mercury electrodes (i.e.: HMDE 

and MFE) that as still more controversial within the green chemistry concept 

are now undergoing a general decline of interest. 

2. THE STATE OF THE ART 

Initially, a configuration of the (i) BiF-CPE [7,9] type was presented, where the 

bismuth film had been deposited electrolytically (ether in situ, or externally, from 

special plating solutions), followed soon by further two bismuth-based variants 

enabled by easy-to-make bulk-modification of the carbon paste. These configurations, 

(ii) Bi2O3-CPE [6,8] and (iii) Bi-CPE [10,11] containing solid oxide or finely pulverised 

metal, have then contributed to the continuing popularity of carbon paste-based and 

bismuth-modified electrodes, sensors, and detectors in the up-coming years. There has 

been yet another variant (iv) BiF4-CPE [12], whereas the original BiF-CPE could be 

miniaturised [13] or combined with a new, electroactive carbon paste substrate [14]. 

Qualitatively new contributions in employing the carbon paste-based substrates have 

- 284 -


then come in the mid and late 2000s in the form of other types of MeEs; namely, as 

antimony- [15,16] and lead- [17] film plated CPEs. 

The latter configurations represent, in fact, a new concurrence to the already 

established bismuth-based variants although the use of both antimony and lead 

and, especially, of their Sb III and Pb II salts (for preparation of the respective films) 

means a certain step back with respect to environmentally friendly procedures. Thus, 

of interest are now some novel configurations that contain less harmful Sb- (or Pb-) 

based precursors like carbon pastes bulk-modified with Sb2O3, SbOCl [18], SbF3, and 

Sb-powder [19]. 

3. FUTURE PROSPECTS 

As documented in a retrospective review a year ago [20], the young field of ESA 

with MeEs has already spread throughout the world. Together with some types of 

screen-printed electrodes, the family of MeEs is the first successful alternative of 

mercury-based electrodes dominating in the electrochemistry and electroanalysis for 

lengthy decades. And, in some respect, MeEs are also the real aspirants for the full 

replacement of both HMDE and MFE. Although the newest activities correspond to 

such predictions, it is wise to wait a little bit with so conclusive statements. As known 

in science, only time will show... 


Financial grants from the Ministry of Education, Youth, and Sports of the Czech 

Republic (projects № MSM0021627502 and LC 06035) are gratefully acknowledged 

5. REFERENCES 

[1] Wang, J.: Ass. Chem. Res. 35 (2002) 811-816. 

[2] Wang, J., Lu, J.-M., Hočevar, S. B., Farias, P. A. M., Ogorevc, B.: Anal. Chem. 72 (2000) 3218- 

3222. 

[3] Economou, A.: Trends Anal. Chem. 24 (2005) 334-340. 

[4] Wang, J.: Electroanalysis 17 (2005) 1341-1346. 

[5] Švancara, I., Vytřas, K.: Chem. Listy 100 (2006) 90-113. 

[6] Pauliukaitė, R., Kalcher, K.: (2001) YISAC '01: 8 th Young Investigators’ Seminar on Analytical 

Chemistry, Book of Abstracts), University of Pardubice, Pardubice, 2001, pp. 10-11. 

[7] Królicka, A., Pauliukaitė, R., Švancara, I., Metelka, R., Norkus, E., Bobrowski, A., Kalcher, K., 

Vytřas K.: Electrochem. Commun. 4 (2002) 193-196. 

[8] Pauliukaitė, R., Metelka, R., Švancara, I., Królicka, A., Bobrowski, A., Vytřas, K., Norkus, E., 

Kalcher, K.: Anal. Bioanal. Chem. 374 (2002) 1155-1158. 

[9] Vytřas, K., Švancara, I., Metelka, R.: Electroanalysis 14 (2002) 1359-1364. 

[10] Švancara, I., Baldrianová, L., Tesařová, E., Hočevar, S.B., Elsuccary, S.A.A., Economou, A., 

Sotiropoulos, S., Ogorevc, B., Vytřas, K.: Electroanalysis 18 (2006) 177-185. 

[11] Hočevar, S. B., Švancara, I., Ogorevc, B., Vytřas, K.: Electrochim. Acta 51 (2005) 706-710. 

[11] Sopha, H., Baldrianová, L., Tesařová, E., Grincienė, G., Weidlich, T., Švancara, I., Hočevar, S.B.: 

Electroanalysis 22 (2010) 1489-1493. 

[12] Baldrianová, L., Švancara, I., Sotiropoulos, S.: Anal. Chim. Acta 599 (2007) 249- 

255. Papp, Zs., Guzsvány, V., Švancara, I., Vytřas, K., Gaál, F., Bjelica, L., 

Abramović, B.; in: Sensing in Electroanalysis, Vol. 4 (K. Vytřas, K. Kalcher, I. 

Švancara; eds.), Univ. Pardubice Press Centre, 2009, pp. 47-58. 

- 285 -


[13] Švancara, I., Hočevar, S.B., Baldrianová, L., Tesařová, E., Vytřas, K.: Sci. Pap. 

Univ. Pardubice, Ser. A 13 (2007) 5-19. 

[14] Tesařová, E., Baldrianová, L., Hočevar, S.B., Švancara, I., Vytřas, K., Ogorevc, B.: 

Electrochim. Acta 54 (2009) 1506-1510. 

[15] Bobrowski, A., Królicka, A., Łyczkowska, E.: Electroanalysis 20 (2008) 61-67. 

[16] Švancara, I., Florescu, M., Stočes, M., Baldrianová, L., Svobodová, E., Badea, M.; 

in: Sensing in Electroanalysis, Vol. 5 (K. Vytřas, K. Kalcher, I. Švancara; eds.), 

Univ. Pardubice Press Centre, 2010, pp. 109-125. 

[17] Švancara, I., Svobodová, E., Stočes, M.: article(s) in preparation. 

[18] Švancara, I., Prior, C., Hočevar, S.B., Wang, J.: Electroanalysis 22 (2010) 1405- 

1420. 

- 286 -


ELECTROCHEMISTRY OF OSMIUM(VI)- 

MODIFIED CARBOHYDRATES 

Mojmír TREFULKA 1 , Martin BARTOŠÍK 1 , Emil PALEČEK 1 

1 Institute of Biophysics, AS CR, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic 

Abstract 

Oligo- and polysaccharides (Sachs) were modified with complexes of six-valent 

osmium with nitrogen ligands [Os(VI)L]. The most tested ligands were N,N,N´,N´- 

tetramethylethylenediamine (temed) and 2,2´-bipyridine (bipy). At HMDE, cyclic 

voltammograms (CVs) of Sachs-Os(VI)L adducts showed three cathodic peaks Ic-IIIc 

and their anodic counterparts Ia-IIIa. Potential (EP) of these peaks were in the range 

from 0 V to -1.1 V. In addition, peak IVc and electrocatalytic peaks Vc and Va (EP - 

1.2 V against Ag/AgCl/3 M KCl electrode) were observed, using some ligands, e.g 

bipy. With square wave voltammetry, peak Ic of dextran-Os(VI)temed was detectable 

down to 3.2 ng/ml directly in the reaction mixture. Using peak Vc, by differential 

pulse voltammetry, purified dextran-Os(VI)bipy was detectable down to 40 pg/ml. 

CVs of Sachs-Os(VI)L at PGE differed from those at HMDE by absence of an 

equivalent of sharp peak Ic and catalytic peaks Vc and Va. 


Compounds containing 1,2-diol groups, including mono-, oligo- and 

polysaccharides (Sachs) can be selectively modified with complexes of sixvalent 

osmium with nitrogen ligands [Os(VI)L] (Fig. 1), yielding electroactive adducts 

producing redox couples at carbon and Hg electrodes and electrocatalytic peaks at Hg 

electrodes. 

2. EXPERIMENT 

Voltammetric measurements were performed with an Autolab analyzer (Eco 

Chemie, Utrecht, The Netherlands) in connection with VA-Stand 663 (Metrohm, 

Herisau, Switzerland). Three-electrode system was used consisting of Ag/AgCl/3M 

KCl electrode as a reference and platinum wire as an auxiliary electrode. The hanging 

mercury drop electrode (HMDE) with an area of 0.4 mm 2 or basal plane pyrolytic 

graphite electrodes (PGE), 9 mm 2 were the working electrodes. All measurements 

were performed at room temperature. The samples were freed from oxygen by 

bubbling 180 s with argon before each measurement. 


At HMDE, cyclic voltammograms (CVs) of Sachs-Os(VI)L showed three 

cathodic peaks Ic-IIIc and their anodic counterparts Ia-IIIa (Fig.2A). Potential (EP) of 

these peaks depending on the type of ligand [1, 2] were within range from 0 V to -1.1 

V. In addition to these redox couples, peak IVc and peaks Vc and Va were produced 

by Sachs-Os(VI)L, using some ligands, e.g. bipy [3] (Fig. 2B). Both peaks V (EP -1.2 V 

- 287 -


against Ag/AgCl/3 M KCl electrode) were assigned to the catalytic hydrogen 

evolution. These peaks were much larger than less negative peaks. CVs of Sachs- 

Os(VI)L at PGE differed from those at HMDE by absence of an equivalent sharp peak 

Ic and catalytic peaks V [2, 4]. 

We have found that the electrochemical behavior of Sachs-Os(VI)L adducts at 

carbon and mercury electrodes is in some features similar to the electrochemical 

behavior of Os(VIII)L-modified nucleic acids [5] and proteins. Using the Adsorptive 

Transfer Stripping (AdTS, ex situ) voltammetry with carbon electrode, Sachs can be 

analyzed directly in the reaction mixture. Similar analysis of DNA-Os(VIII)bipy 

adducts at HMDE was not possible because the reagent interfered with the analysis. 

We have found that in difference to Os(VI)bipy, the Os(VI)temed as a free reagent 

produced only poorly developed peaks at HMDE that could be easily resolved from 

the peaks of the Sachs-Os(VI)temed adduct. Moreover, free Os(VI)temed could be 

removed from the electrode surface by simple washing with water. These findings 

enabled us to analyze Os(VI)temed-modified dextran at HMDE directly in the 

reaction mixture and determine dextran in presence of an excess of monomeric 

glucose or sucrose. In difference to previous analyses of Os(VIII)L-modified DNA and 

proteins [6], we have found that Sachs-Os(VI)temed can be determined at HMDE in 

presence of the reaction mixture not only by AdTS but also by conventional AdS (in 

situ) methods; such determination was earlier not possible with other Os(VI)L 

complexes, regardless of the type of the electrode used. Using square wave 

voltammetry, nanomolar concentrations of Sachs can be determined. For example, 

dextran was detectable down to 20 nM concentration (related to polysaccharide 

monomer content; this concentration corresponded to about 3.2 ng/ml). Our 

preliminary results suggest that using catalytic peak Vc, detection limits of Sachs can 

be decreased at least by two orders of magnitude as compared to detection limits 

obtained with any of the redox couples. Dextran was detectable with peak Vc down to 

about 40 pg/ml. Os(VI)L-modified carbohydrates were also suitable for the analysis at 

carbon electrodes. 

4. CONCLUSION 

We developed a new, highly sensitive electrochemical method of carbohydrate 

analysis allowing their determination at nM and pM level. Using a transfer (ex situ) 

method, volumes of the analyte sample may be reduced to μl. To our knowledge this 

method represents the most sensitive way of determination of polysaccharides and 

oligosaccharides. For instance, improved phenol-sulfuric method [7] requires 1–150 

nmol of sugars as compared to fmols necessary for our methods. New electrochemical 

methods for Sachs analysis may become very important in biomedicine, including 

cancer and neurodegenerative diseases. Alternations in protein glycosylation are 

associated with a variety of tumors. For example, prostate-specific antigen (PSA), 

considered as the best tumor marker for diagnosing early prostate carcinoma [8], is a 

28,400 Da glycoprotein containing ~8% carbohydrate in the form of an N-linked 

oligosaccharide side chain. PSA concentration in human sera is very low (in the order 

- 288 -

XI. WWorkshop 

of Phyysical 


of nng/ml), 

makking 

the stu udies of glyycan 

structu ures of patie ent serum PPSA 

difficu ult. High 

senssitivity 

of f the new w electrocaatalytic 

sig gnal of Os(VI)L-mo 

O odified pol ly- and 

oliggosacchariddes 

holds a great g promiise 

for a fas st, inexpens sive and simmple 

technique 

for 

thesse 

studies. 

Fig. . 1 Chemiccal 

modific cation of poly- 

andd 

oligosacccharides 

with w Os(VVI)L 

commplexes. 

A part of α-1,4-gluucan 

(ammylose, 

dexttrin) 

molecu ule formingg 

an 

addduct 

with aan 

Os(VI) L complex 

is 

showwn. 

As lligands 

(L) 

nitrogennous 

commpounds, 

ssuch 

as te emed or bbipy, 

werre 

used. 


EMENT 

This worrk 

was supported 

by ggrant 

of th he Grant Agency Ag of thhe 


301/10/P548. 

6. 

[1] 

[2] 

[3] 

[4] 

[5] 

[6] 

[7] 

[8] 

REFEREENCES 

Fojta M., KKostecka 

P., Trefulka T M., HHavran 

L., an nd Palecek E.: Anal. Chem. ., 79 (2007), 1022. 1 

Trefulka MM. 

and Palecek 

E.: Electroaanalysis, 

22 (2 2010), 1837. 

Palecek E. and Trefulka a M.: Analyst, 

136 (2011), 321. 

Trefulka MM. 

and Palecek 

E.: Electroaanalysis, 

21 (2 2009), 1763. 

Palecek E. . and Jelen F., F in Perspecctives 

in Bioa analysis, Elect trochemistry of Nucleic Acids A and 

Proteins. TTowards 

Elec ctrochemical Sensors for Genomics an nd Proteomiccs., 

Vol. 1 (Pa alecek E., 

Scheller F. ., and Wang J., J eds.), Elsevvier, 

Amsterdam, 

2005, p. 73. 7 

Palecek E. : Methods En nzymol., 212 ( (1992), 139. 

Masuko T., 

Minami A. , Iwasaki N., Majima T., Nishimura N S. I., and Lee YY. 

C.: Anal. Biochem., B 

339 (2005) ), 69. 

Tabares G., 

Radcliffe C. 

M., Barrabees 

S., Ramirez z M., Aleixan ndre R. N., Hooesel 

W., Dw wek R. A., 

Rudd P. MM., 

Peracaula R., R and de Lloorens 

R.: Glyc cobiology, 16 (2006), 132. 

- 289 - 

I [µA] 

I [µA] 

1 

0 

-1 

0 

-4 

-8 

A 

B 

Va 

Vc 

-1.5 

IIIa 

IIIc 

IIa 

IIc 

-1.0 -0.5 

E [V] 

IIc 

0.0 

Fig. 2 Cyclic C vooltammogra 

ams of 

dextran modified m either A, A with 

Os(VI)tem med (red) or B, with 

Os(VI)bipy y (blue). AA, 

30 μM dextran- d 

Os(VI)tem med, accumuulation 

tim me, tA 60 

s, scan rat te, v 2V/s; B, 5 μM dextran- d 

Os(VI)bipy y, tA 600 

s, v 0.1 0 V/s. 

AUTOLAB B, HMDE, AA) 

80 mM Britton- B 

Robinson buffer, pHH 

7.0, B) 100 

mM 

Britton-Ro obinson bufffer, 

pH 4.7 75. 

Ia 

Brno

XI. Workshop of Physical Chemists and Electrochemists ´11 Brno 

ADSORPTIVE STRIPPING ELIMINATION 

VOLTAMMETRY 

Libuše TRNKOVÁ 1 

1 Laboratory of Biophysical Chemistry and Electrochemistry, Department of Chemistry, Faculty of 

Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic 

LABIFEL: http://labifel.byethost24.com/, e-mail: libuse@chemi.muni.cz; litrn@seznam.cz 

Abstract 

The contribution is devoted to the transformation of linear sweep voltammetric (LSV) 

or cyclic voltammetric (CV) results into elimination voltammetry with linear scan 

(EVLS) for the case of adsorbed electroactive species. The approach can be named 

adsorptive stripping elimination voltammetry (AdS EVLS) or adsorptive transfer 

stripping elimination voltammetry (AdTS EVLS). In connection with mercury or 

graphite electrode it is illustrated advantages of this electroanalytical approach. Using 

AdS EVLS the differences in the reduction and oxidation processes of biologically 

important molecules such as nucleic acids, oligonucleotides and nucleobases were 

presented. 


Adsorptive stripping voltammetry (AdSV) and adsorptive transfer stripping 

voltammetry (AdTSV) 1-4 belong to the family of stripping voltammetric procedures in 

which the pre-concentration step plays a key role. While in cathodic or anodic 

stripping technique the pre-concentration step is controlled by electrolysis, in 

adsorptive stripping this step is controlled by adsorption. The adsorption step proceeds 

on the working electrode surface and it is realized by adsorption of reactants, 

intermediates or products of electrode processes. It is also one of the method of the 

modified electrodes preparation, often useful for electrochemical sensors. AdSV can 

be employed in the trace analysis of organic compounds exhibiting surface active 

properties. When the compounds studied contains an electrochemically reducible or 

oxidizable group, then we can observe current peaks on the voltammetric curves and 

can determine very low concentration of the electroactive species. Stripping 

techniques are usually combined with conventional voltammetric methods such as 

pulse, differential pulse, square wave and linear sweep voltammetry. Recently it has 

been found that elimination voltammetry with linear scan (EVLS) is capable to 

improve voltammetric signals not only from the point of view of current sensitivity 

but also of the resolution of overlapped signals. It was found that EVLS in 

combination with adsorptive stripping procedure (AdS EVLS) is a promising tool for 

achieving very good resolution of electrode processes, for qualitative and quantitative 

analysis, as well as for identification of the structures of compounds studied 5-11 . 

The EVLS can be considered as a mathematical model of the transformation of 

current-potential curves capable of eliminating some selected current components, 

290


while conserving others by means of elimination functions 5-7 . For the calculation of 

the elimination functions three voltammetric curves at different scan rates should be 

recorded under identical experimental conditions. One scan rate is chosen as a 

reference scan rate and the total voltammetric current I is affected by the ratio 

ref 

ref and 

the ratio has exponent according to the dependence of current component 

on the scan rate: 

1 2 I I I ..... 

I ref 

ref d ref c k 

where I d , I c , Ik is the diffusion (exponent ½), charging (exponent 1), and 

kinetic (exponent 0) current component. The equation (1) can be regarded as a 

normalization equation where the reference state is characterized by the total current 

measured at reference scan rate ref . Three equations with three different ratios 

ref provide the set of total currents measured at different scan rates. From 

experimental point of view the integer 2 is suitable. 

These total currents were converted into a current elimination function which is 

expressed as a linear combination of them. The best results are obtained using the 

function E4 eliminating kinetic and charging current components and conserving the 

diffusion current component: 

f ( I ) 11. 

657I1 

2 17. 

485 I 5. 

8284I2 

(2) 

I1 

2 

1 2 

ref ref 

Where , I , and I 2 are total voltammetric currents measured at , 

2 

ref , and , respectively. 

The ELSV has several advantages: (a) expanded available electrode potential 

range, (b) increased current sensitivity, and (c) improved signal resolution. The two 

latter advantages result from the fact that the elimination of charging and kinetic 

currents reduces the irreversible current width and increases the peak height. This 

effect is particularly pronounced in the case of an adsorbed substance yielding a welldeveloped 

elimination signal (peak-counterpeak). As a practical application, the AdS 

EVLS of thermally denatured DNA, ODNs and nucleobases on hanging mercury and 

carbon electrodes has been performed. 

2. EXPERIMENT 

Voltammetric measurements were performed using the analyzer Autolab 20 

EcoChemie connected with the VA Stand 663 and controlled by GPES manager 

software. The main part of this apparatus was electrochemical vessel equipped with 

three electrodes: the hanging mercury drop electrode or carbon electrode as a 

working electrode, the argent chloride electrode (Ag/AgCl/3MKCl) as a reference 

electrode and the platinum wire as a counter electrode. The samples were analyzed 

usually in the phosphate – acetate buffer. Experimental conditions of LSV were 

following: the time of accumulation was 120s, conditioning of electrode was 2 

seconds, the potential step was 2 mV. The LSV curves were measured at different scan 

291 

(1)


rates from 100 to 800 mV/s. All voltammetric measurements were performed in inert 

atmosphere (argon) and at room temperature. Three of these curves with integer 2 

(1/2, 1, and 2 multiple of reference scan rate) were taken for EVLS procedure. 


Theoretical transition of EVLS of an irreversible adsorbed electroactive particle 

predicted the specific signal in which a sharp positive peak immediately followed by a 

negative peak (peak-counterpeak). The verification of EVLS theory for adsorbed 

depolarizer was carried out on model compounds - denatured calf thymus DNA, 

which provides a common reduction on HMDE signal of cytosine and adenine 

residues 7 . It was found that the EVLS yields significantly higher sensitivity in the 

detection of voltammetric signals not only from the current increase (7-15 times 

higher), but also by their separation when the peak potentials are closer than 40 mV8- 

9. Therefore EVLS (E4 function) is applied to the joint reduction peak of adenine and 

cytosine in oligonucleotides (ODNs) 10-13 . At the beginning of ODN research homo- 

ODNs (nonamers) were studied. EVLS E4 function was applied to the separation 

adenine and cytosine signals in the mixtures of dA9 and dC9. In the case of hetero- 

ODNs (nonamers) the different base sequences were studied. In comparison to 

common voltammetric methods only EVLS was able to separate overlapping signals 

(two ODN structure forms). Our research continues to trend higher voltammetric 

signal amplification which is accomplished by two techniques, adsorptive stripping 

voltammetry (AdSV - adsorptive stripping voltammetry) or AdTS EVLS (AdTSV - 

adsorptive transfer stripping voltammetry). Both methods are able not only to 

determine the ratio of adenine and cytosine in ODN molecules but also to 

distinguish the relative positions of these nucleobases in the ODN chain. It should be 

noted that an important parameter influencing the signals ODNs EVLS the pH, ionic 

strength, accumulation time and accumulation potential 10-13 . 

4. CONCLUSION 

Despite persistent prejudices against elimination voltammetry it was showed 

that EVLS has a chance to win in the field of electroanalysis. Each method has 

advantages and disadvantages, but EVLS has numerous advantages overcoming its 

disadvantages. If the experiment is performed correctly (i.e., the same number of I - E 

pairs = the same potential step for all measured curves, good working potentiostat), 

then EVLS: 

is fast, inexpensive and not time-consuming method; 

is able to eliminate the currents of different types (not limited to capacitive 

current); 

guarantees the simultaneous elimination of the multiple current components; 

is not limited only by reducing process on a mercury electrode; 

helps to increase the sensitivity of irreversible current signals; 

expands potential window (eliminating Ik); 

distinguishes overlapped signals; 

reveals minor (hidden) processes in the major ones; 

292


confirms quickly the electron transfer in an adsorbed state (peak-counterpeak) 

is able to detect possible interactions, changes in the structure on charged 

interfaces; 

does not need the fundamental line current (baseline correction); 

is able to help in the study of reaction mechanism; 

allows calculation of from different potential peak values of two functions 

(preferably E1 and E4). 

EVLS disadvantages can be considered: 

results of the elimination procedure does not exactly correspond to the theoretical 

conclusions (the currents do not affect each other), then EVLS curves are 

distorted. One of these distortions is a peak-counterpeak signal; 

EVLS errors on solid electrodes are higher than of LSV or CV errors. 

However, the theoretical and practical development of EVLS and AdS EVLS 

continues 14 . We can conclude that the combination of both techniques AdS or AdTS 

and EVLS is a suitable tool for the study of absorbable molecules such as nucleic acids 

and their constituents. 


This work has been supported by projects INCHEMBIOL MSM0021622412, 

BIO-ANAL-MED LC06035 and MUNI/A/0992/2009 by the Ministry of Education, 

Youth and Sports of the Czech Republic. 

6. REFERENCES 

[1] Bard A.J.; Faulkner L.R.: Electrochemical Methods, Fundamentals and Applications, 

2nd edition, John Wiley & Sons, Inc., NewYork, 2000. 

[2] Kissinger P.T.; Heineman W.R.: Laboratory Techniques in Electroanalytical Chemistry, Marcel 

Dekker, New York, 1984. 

[3] Bond A.: Modern Polarographic Methods in Analytical Chemistry, Marcel Dekker, New York 

and Basel, 1980. 

[4] Paleček E.; Scheller F.: Electrochemistry of Nucleic Acids and Proteins, Towards Electrochemical 

Sensors for Genomics and Proteomics, Perspectives in Bioanalysis, Vol. 1, 1st ed., Wang J. (ed.), 

Elsevier, Chapter 3: Electrochemistry of Nucleic Acids (E. Paleček, F. Jelen) 2006. 

[5] Dračka O.: J.Electroanal. Chem., 402 (1996)19. 

[6] Trnkova, L.; Dracka, O.: J. Electroanal. Chem. 413 (1996) 123. 

[7] Trnkova, L.; Kizek, R.; Dracka, O.: Electroanalysis 12 (2000) 905. 

[8] Trnkova L.: J. Electroanal. Chem., 582 (2005)258. 

[9] Trnkova, L.: Chem. Listy 95 (2001) 518. 

[10] Trnkova, L.; Jelen, F.; Petrlova, J.; Adam, V.; Potesil, D.; Kizek, R.: Sensors 5 (2005) 448. 

[11] Trnkova, L.; Jelen, F.; Postbieglova, I.: Electroanalysis 15 (2003) 1529. 

[12] Trnková L., Jelen F., Postbieglova I.: Electroanalysis 18 (2006) 662. 

[13] Trnkova, L.; Postbieglova, I.; Holik, M.: Bioelectrochemistry 63 (2004) 25. 

[14] Serrano, N.; Klosova, K.; Trnkova, L.: Electroanalysis 22 (2010) 1873. 

293


THEORETICAL STUDY OF N–H BOND 

DISSOCIATION ENTHALPIES IN PARA 

SUBSTITUTED ANILINES 

Adam VAGÁNEK 1 , Ján RIMARČÍK 1 , Lenka ROTTMANNOVÁ 1 , Erik KLEIN 1 , 

Vladimír LUKEŠ 1 

1 Institute of Physical Chemistry and Chemical Physics, Slovak University of Technology, Radlinského 

9, SK-812 37 Bratislava, Slovak Republic 

Abstract 

In this work, we have calculated bond dissociation enthalpies of N–H bonds in twelve 

para-substituted anilines using density functional theory (DFT) with PBE functional 

and 6-311++G** basis set, and density functional tight binding method, DFTB+, in 

order to assess the applicability of employed methods for the study of substituent 

induced changes in N–H BDE. 


Bond dissociation enthalpy, BDE, is defined, as BDE = H(R ) + H(H ) – H(R–H) 

where H(R ) is the total enthalpy of the radical, H(H ) is the total enthalpy of the 

abstracted hydrogen atom, and H(R-H) is the total enthalpy of the molecule. Hence, 

bond dissociation enthalpy is the reaction enthalpy of homolytic abstraction of a 

hydrogen atom from a molecule. Hydrogen atom transfer, HAT, represents common 

mechanism in organic chemistry. 

The main aim of this work is to assess the reliability of DFTB+ semiempirical 

computational approach for the description of the substituent effect in terms of N–H 

bond BDEs in para-substituted anilines (Fig. 1). 

X 

Fig.1 Para-substituted anilines X = OMe, Me, COMe, CF3, CN, NO2, COtBU, CCH, F, 

COOMe, Cl, Br 


For the geometry of each compound and radical and their total electronic 

energy calculations, DFT method with PBE functional and 6-311++G** basis set in 

Gaussian 03 [1] program package was employed. In the case of DFTB+ method, 

Release 1.1 of DFTB+ program [2] was employed. DFTB+ method is based on PBE 

functional. 

294 

NH 2



Because DFTB+ program does not provide total enthalpies, all BDE values were 

approximated from the total electronic energies. For the sake of exact comparison of 

substituent effect description for DFT and DFTB+ approaches, we have chosen the 

PBE functional in DFT calculations. Although anilines are widely studied compounds, 

only several experimental BDEs are available. 

To verify the reliability of employed computational approach for substituent 

effect description, it is inevitable to compare calculated and experimental ΔBDE 

values, where ΔBDE = BDE(X-PhNH2) – BDE(PhNH2) represents the difference 

between substituted and non-substituted aniline BDEs. Table 1 summarizes computed 

and available experimental ΔBDEs together with the Hammett p constants [3]. 

Calculated ΔBDEs are presented in the DFT and DFTB+ columns. Experimental 

ΔBDEs estimated on the basis of the equilibrium acidities and the oxidation potentials 

of conjugated anions of involved anilines are given in the EC column [4]. 

Table 1. BDE* values in kJ mol –1 , and Hammett constants σp. 

Substituent DFT DFTB+ EC σp 

p-OMe –16 –21 –8 –0,27 

p-Me –6 –6 –1 –0,17 

p-F –5 –8 – 0,06 

p-CCH –2 –5 – 0,23 

p-Cl –1 – 1 0,23 

p-Br 0 – 0 0,23 

p-COtBu 8 –1 – 0,32 

p-COOMe 9 1 – 0,45 

p-COMe 10 2 8 0,50 

p-CF3 11 9 18 0,54 

p-CN 12 3 12 0,66 

p-NO2 18 12 19 0,78 

*BDE of non-substituted aniline: 399 kJ mol –1 (DFT), 406 kJ mol –1 (DFTB+), 386 

kJ mol –1 (EC) 

Comparison of experimental BDE of non-substituted aniline with the two 

calculated values indicates that employed approaches overestimate its N–H BDE. 

Calculation results show that DFT/PBE/6-311++G** method describes substituent 

effect in good agreement with experiment in terms of BDE values. Average deviation 

between 8 experimental and calculated BDEs reached 3.4 kJ mol –1 . This indicates 

that DFT/PBE/6-311++G** approach provides reliable BDEs for para-substituted 

anilines. From Table 1 it can be seen that DFTB+ method provides less reliable BDEs, 

differences between DFTB+ and experimental BDE values are in 5–13 kJ mol –1 range. 

4. CONCLUSION 

We have found that DFTB+ approach is not very suitable for substituent effect 

description in studied compounds, because there is no good agreement between 

DFTB+ BDE values with the experimental ones. Both employed approaches 

295


overestimate individual BDEs of studied anilines. DFT/PBE/6-311++G** method 

describes substituent effect in accordance to the experimental BDE values. 


The work has been supported by Scientific Grant Agency (VEGA Project 

1/0137/09). 

6. REFERENCES 

[1] Pople, J. A., et al.: GAUSSIAN 03, Revision A.1, Gaussian, Inc., Pittsburgh, PA, 2003. 

[2] Koskinen, P., Mäkinen, V.: Computational Materials Science, 47 (2009), 237–253. 

[3] Hansch, C., Leo, A., Taft, R.W.: Chem. Rev. 91 (1991), 165–195. 

[4] Bordwell, F. G., Zhang, X.-M., Cheng, J.-P.: J. Org. Chem. 58 (1993), 6410–6416. 

296


SOFTWARE FOR PROCESSING OF 

CATALYTIC METALLOTHIONEIN SIGNALS 

Martin VALLA 1 , Martin KLIMEK 1 , Jiří DVOŘÁČEK 1 , Ivo PROVAZNÍK 1 , Petr 

MAJZLÍK 2 , René KIZEK 2 


University of Technology, 

2 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Brno, 


e-mail: martin.valla@phd.feec.vutbr.cz 

Abstract 

The goal of this topic deal with processing and analysis catalytic metalothionein 

signals. In the analysis is used Fourier transform for noise filtering and cyclic loop for 

detect peaks on the signal. Results are displaying in user friendly graphic interface. 


Electrochemical analysis of proteins and nucleic acids is at the beginning of 21 st 

century actual and may broaden our knowledge or may be used in routine diagnostics 

[1,2]. More than eighty years ago, catalytic signals of proteins in ammonium medium 

with presence of cobalt ions were described. Free sulfhydryl groups of proteins are 

responsible for these catalytic signals [3,4] . Even if principle of this catalytic reaction 

is still unknown, this way of electrochemical analysis is very suitable for proteins with 

high content of –SH groups [5]. Thermostabile metallothionein is one of these 

proteins [6,7]. Metallothionein (MT) is a small protein [8] of which primary function 

is in keeping of metals homoeostasis in living organisms. 

Synthesis of human metallothionein may be induced by increasing metals 

concentrations. Researches associate significant relation of MT concentration with 

carcinogenesis, spontaneous mutagenesis, and participation in mechanisms of action 

of anti-tumour drugs and pharmaceutical products [9-11]. Overexpression of 

metallothionein is under investigation as a new prognostic marker in many types of 

malignant tumours [12,13]. The main aim of this paper is processing of 

electrochemical catalytical signals of metallothionein. 

2. METHODS AND EXPERIMENT 

For operation with measured data, it is necessary to adjust these data using 

suitable mathematical method. Pre-treatment of these data concludes elimination of 

undesirable components of signal (noise). In the case of repeated measurement of one 

sample, its ergodicity can be used. First, noise filtering was performed by averaging. 

Then, noise suppression was done in frequency domain. After application of discrete 

Fourier transform, selected Fourier coefficients were zeroed and filtered signal was 

restored by inverse discrete Fourier transform. Sampling period of the measured 

signals was 2mV, then sampling "frequency" fs=1/0.002=500 V -1 . Cut-off frequency of 

297

XI. Workshhop 


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th 

types oof 

filters, ba 

signals to main fig 

of nummber 

measur 

-1 (0.0 05 fs). Indivvidual 

peak ks within signals 

were detected using u 

f compariso on three nneighborgin 

ng signal sa amples. Thhe 

method was 

y sample-b by-sample while a sa ample with h one preceeding 

and one 

of lower value wass 

searched. 

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software was w developped 

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MATL LAB, 

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llows loadin ng of RAWW 

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means ove er all measuuring 

chan nnels. Abou ut -1,5V, peeak 

is used d for 

bration line. 

Extendedd 

function of this inte erface consiists 

in prop perty 

d and automatic 

dettection 

aft ter comput ting. Theree 

are specially 

and plottin ng (Fig. 1) bbellow. 

Nex xt option is s zoom regiion 

of inter rests 

reshold det tection, ploot 

actual fil le path for work, chooosing 

diffe erent 

aseline dete ection withh 

options ch hanging ran nge of proccessing, 

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signa al from dataset, 

dynammical 

detec ction 

ring (maxim mum 9) andd 

button for r ending pr rogramme. 

Fig 1. AAnalyzing 

software fo or signals oof 

metalloth hionein wi ith graphicc 

user inter rface 

GUI develooped 

in env vironment oof 

MATrix LABorator ry MATLABB. 

3. RRESULTS 


Inn 

additionn, 

we ac ccentuated automatiz zation of detectionn 

because of 

approxiimation 

of this metho od to needss 

of clinica al practice to t rapid annd 

standard dized 

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n of 

biologiccal 

materiaal. 

Current t responsess 

are result ts of electr rochemical analysis using 

u 

298 

Brno


Brdicka reaction. Principle of this reaction is very difficult and its character is 

partially of catalytic origin. In obtained signals, following phenomenons are described 

(without protein presence): at potential of -0.25 V, cobalt(III) ions are reduced to 

cobalt(II) ions and at potential of -1.1 V, cobalt(II) ions are reduced to cobalt. In 

presence of proteins, dramatic changes are well evident. Signal of reduction of 

cobalt(II) ions is significantly reduced and at potential of -1 V, signal of originating 

complex of cobalt with–SH groups (RS2Co) appears. After signal of this complex, in 

dependence on concentration and amount of free –SH groups, catalytic signals Cat 1 

at potential about -1.25 V, Cat 2 at potential of -1.35 V, and Cat 3 at potential about - 

1.45 V appear. In addition, at very negative potential (about -1.7 V), catalytic signal 

marked as H peak appears. From our experimental results, it is well evident that Cat2 

signal is directly proportional to level of MT in sample. Individual maxima of signals 

were characterized by their position (potential) and height (current). This method 

simplified evaluation of very complicated electrochemical records. As the registered 

signals are noise-free with well recognizable peaks, reliability of the peak detector was 

high. 

4. CONCLUSION 

Precise and rapid evaluation of biological signals enables enhance of efficiency 

of bioanalytical analysis and above all leads to reduction of random mistakes caused 

by human factor. For MT detection, we used in clinical practice uncommon 

electrochemical methods, which demonstrate high sensitivity, selectivity, and low 

costs. Software for processing is used on MEDELU university. 


This work was supported from: GAČR 301/09/P436, GAČR 102/07/1473 GAČR 

102/08/H083, IGAMZ10200-3, GA AV IAA401990701, GA AV KAN208130801 and 

MSM0021630513. 

6. REFERENCES 

[1] J. Petrlova, D. Potesil, R. Mikelova, O. Blastik, V. Adam, L. Trnkova, F. Jelen, R. Prusa, J. 

Kukacka and R. Kizek Attomole voltammetric determination of metallothionein, Electrochimica 

Acta 51 (2006) 5112-5119. 

[2] I. Fabrik, J. Kukacka, V. Adam, R. Prusa, T. Eckschlager and R. Kizek Metallothionein and its 

relation to anticancer treatment by platinum complexes, Prakticky Lekar 88 (2008) 90-93. 

[3] S. Krizkova, V. Adam, T. Eckschlager and R. Kizek Using of chicken antibodies for 

metallothionein detection in human blood serum and cadmium-treated tumour cell lines after 

dot- and electroblotting, Electrophoresis 30 (2009) 3726-3735. 

[4] S. Krizkova, V. Adam and R. Kizek Study of metallothionein oxidation by using of chip CE, 

Electrophoresis 30 (2009) 4029-4033. 

[5] V. Adam, O. Blastik, S. Krizkova, P. Lubal, J. Kukacka, R. Prusa and R. Kizek Application of the 

Brdicka reaction in determination of metallothionein in patients with tumours, Chemicke Listy 

102 (2008) 51-58. 

[6] M. Beklova, I. Fabrik, P. Sobrova, V. Adam, J. Pikula and R. Kizek Electrochemical 

determination of metallothionein in the domestic fowl, Natura Croatica 17 (2008) 283-292. 

299


[7] O. Blastik, J. Hubalek, V. Adam, J. Prasek, M. Beklova, C. Singer, B. Sures, L. Trnkova, J. 

Zehnalek and R. Kizek Electrochemical sensor for determination of metallothionein as 

biomarker, Proceedings of IEEE Sensors, Daegu, 2006, pp. 1171-1174. 

[8] T. Eckschlager, V. Adam, J. Hrabeta, K. Figova and R. Kizek Metallothioneins and cancer, 

Current Protein and Peptide Science 10 (2009) 360-375. 

[9] I. Fabrik, S. Krizkova, D. Huska, V. Adam, J. Hubalek, L. Trnkova, T. Eckschlager, J. Kukacka, R. 

Prusa and R. Kizek Employment of electrochemical techniques for metallothionein 

determination in tumor cell lines and patients with a tumor disease, Electroanalysis 20 (2008) 

1521-1532. 

[10] I. Fabrik, J. Kukacka, J. Baloun, I. Sotornik, V. Adam, R. Prusa, D. Vajtr, P. Babula and R. Kizek 

Electrochemical investigation of strontium - metallothionein interactions - analysis of serum and 

urine of patients with osteoporosis, Electroanalysis 21 (2009) 650-656. 

[11] S. Krizkova, P. Blahova, J. Nakielna, I. Fabrik, V. Adam, T. Eckschlager, M. Beklova, Z. 

Svobodova, V. Horak and R. Kizek Comparison of metallothionein detection by using Brdicka 

reaction and enzyme-linked immunosorbent assay employing chicken yolk antibodies, 

Electroanalysis 21 (2009) 2575-2583. 

[12] S. Krizkova, I. Fabrik, V. Adam, J. Hrabeta, T. Eckschlager and R. Kizek Metallothionein - a 

promising tool for cancer diagnostics, Bratislava Medical Journal 110 (2009) 93-97. 

[13] J. Petrlova, O. Blastik, R. Prusa, J. Kukacka, R. Mikelova, M. Stiborova, B. Vojtesek, V. Adam, O. 

Zitka, T. Eckschlager and R. Kizek Determination of metallothionein content in patients with 

breast cancer, colon cancer, and malignant melanoma, Klinicka Onkologie 19 (2006) 138-142. 

300


MATERIALS FOR ORGANIC ELECTRONICS 

Martin VALA 1 , Martin WEITER 1 

1 Brno University of Technology, Faculty of Chemistry, Purkyňova 464/118, Brno 61200, Czech 

Republic 

Abstract 

Model materials for organic electronics were modified in order to alter their electron 

properties. Diketo-pyrrolo-pyrroles substituted with electron donating groups showed 

bathochromic and hyperchromic shift, giving a good chance to be used in solar cell 

applications. Derivatives with larger Stokes shift obtained by N-alkylation were used 

in OLED applications. We were able to achieve good charge balance that led to a low 

turn-on voltage. 


The material basis for organic electronics is very broad. It covers low molecular 

weight structures, polymers, nanoparticles, biomolecules etc. The common feature is 

the possibility to modify their properties via chemical synthesis and therefore to 

prepare virtually unlimited number of modifications with precisely tuned properties. 

It becomes to be common practise to design molecules and predict their 

characteristics prior to the synthesis utilising quantum chemical modelling. We used 

this goal-targeted approach to modify diketo-pyrrolo-pyrroles (DPP), Figure 1, to 

prepare derivatives for organic electronics, namely for solar cells (SC), light emitting 

diodes (OLEDS), and field effect transistors (FET). 

The DPPs as high-performance pigments have attracted researches and 

companies also from the field of organic electronics because of their exceptional 

stability, good scalability of the chemical synthesis, and other properties such as high 

absorption coefficients, fluorescence quantum yields etc. In this contribution we 

present general strategies involved to tune the electron properties of this class of 

materials. The impacts on relevant parameters important in organic electronics are 

discussed rather than the chemical synthetic procedures. 

2. EXPERIMENT 

The devices were prepared in the form of thin films by either spin coating or 

vacuum deposition techniques. The electrical devices use indium tin oxide (ITO) as 

transparent electrode and Al as the counter electrode. The organic layers were 

sandwiched in between. 

To characterise the materials we used various electrical methods (currentvoltage 

characteristics, admittance spectroscopy), optoelectrical methods 

(electroluminescence measurement, steady state and time resolved photoconductivity 

measurement) and optical characterization (absorption and steady state and time 

resolved photoluminescence spectroscopy). 

301



To alter the electronic properties mainly groups with electron donating or 

withdrawing ability are employed. Introduction of polar substituents into organic 

chromophores causes a redistribution of electronic density in both the ground state 

and the excited state, which can strongly modify their absorption and fluorescence 

properties [1]. The important parameters such as positions of the electron and/or hole 

transport levels, the absorption coefficients and fluorescence quantum yields, etc. are 

modified. Furthermore, the push–pull substituted organic compounds are at the 

centre of interest of physicists, because they can produce strong second-order 

nonlinear optical effects 0. 

The effects of electron-donor (piperidino) and electron-acceptor (cyano) groups 

on the electronic spectra were investigated both, experimentally and theoretically. It 

was found, that in general, cyano group stabilizes both phenyl molecular orbitals, 

while piperidino group destabilizes them (and even to a greater extent). An electrondonor 

substituent increases the electron density on the phenyl group to which it is 

attached, and on acceptor C=O group of the second pyrrolinone ring in HOMO, while 

in LUMO further CT is observed to the opposite phenyl group. This indicates the 

electron-acceptor character of the whole central dipyrrolinone mainly localized on 

keto groups. 

The absorption spectra show poor resolution of vibronic structure in the case of 

unsymmetrically piperidino substituted and push-pull piperidino–cynao substituted 

compounds. We ascribed this to the significantly higher dipole moment interacting 

with the polar solvent (dimethylsulfoxide) by dipole–dipole interaction. 

The fluorescence spectra of DPPs usually show small Stokes shifts, which are 

significantly increased by N-substitution (e.g. alkylation) inducing higher degree of 

nonplanarity [3]. Thus the N-substituted derivatives are promising with respect to 

applications like OLED, laser, etc. The Stokes shift between 0-0 vibronic bands in 

absorption and fluorescence spectra is higher for all derivatives with electon donating 

or withdrawing substituents than that for parent compound. Its significant increase is 

observed the asymmetrically piperidino substituted (47 nm) and push-pull piperidino– 

cynao substituted (78 nm), as a result of much stronger excited state solvent 

(dimethylsulfoxide) relaxation of these polar compounds. The dipolar character of 

these compounds gives them also some chance to produce second-order nonlinear 

optical phenomena. This behaviour has been already confirmed using two photon 

excited fluorescence. 

Introduction of electron-donating groups increased the molar absorption 

coefficient and was accompanied with strong bathochromic shift. This behaviour 

implies that charge separation occurs via electron delocalization leading to creation of 

permanent dipole moment. Blurring of vibration structure in absorption spectra of 

302


mono substituted derivatives imply interaction with polar dimethylsulfoxide (DMSO) 

and shows polar character of the substituted. 

Introduction of the N-alkylation led to the decrease of the and hypsochromic 

shift. First N-alkylation causes only small change, whereas second alkylation lead to 

the value of almost similar to the parent, non N-substituted, DPP. This decrease is 

accompanied by the hypsochromic shift and loss of vibrational structure. We 

proposed the same mechanism as for the N-alkylated only derivatives: the Nalkylation 

causes rotation of the phenyls and consequently breaks the molecule 

symmetry, and hence, the effective conjugation and increases the polarity. 

On the basis of the findings described above symmetrically N-alkylated 

derivatives resulted as suitable for electroluminescence characterization. The prepared 

organic diode from the phenyl di-piperidino substituted N,N-alkylated DPP showed 

that the turn-on voltage for this diode is ~3V. At this voltage the previously ohmic 

nature of the I-V characteristic changes to the space-charge-limited, where the 

current flow is bulk-limited. The low value of the turn-on voltage implies that 

reasonable charge balancing was achieved due to the barrier reducing pre-contact 

layers (PEDOT:PSS and Alq3). This allowed us to measure also the spectrally resolved 

electroluminescence of several selected derivatives. 

R 1 

 

R 4 

N 

O 

 

Fig.1 General formula of the basic diketo-pyrrolo-pyrrole used as parent molecule in 

this study. Substitution on nitrogens (R1 and R2) referred as N-alkylation was 

used to alter solubility. Substitution on phenyls (R3 and R4) were used to 

introduce electron donating or withdrawing groups, and therefore, to 

influence the electron spectra. 

4. CONCLUSION 

In this contribution, we showed the influence of various chemical modifications 

of basic structure on the electron properties of organic molecules. The diketo-pyrrolopyrroles 

served only as a model class of materials suitable for production of organicbased 

electronic devices. Derivatives suitable for solar cell applications were obtained 

303 

O 

N 

R 3 

R 2


by substitution with electron withdrawing groups. Derivatives for OLED applications 

were obtained using N-alkylation leading to the higher Stokes shift. 


The work has been supported by MŠMT (OP VaVpI) via project Centres for 

materials research at FCH BUT No. CZ.1.05/2.1.00/01.0012. 

6. REFERENCES 

[1] Griffiths, J.: Colour and constitution of organic molecules. 1976 London: Academic Press 

[2] Marder, S.R.: Chemical Communications (2006), 2, 131. 

[3] Vala, M., Weiter, M., Vyňuchal, J., Toman, P., Luňák, S.: Journal of Fluorescence. (2008), 18(6), 

1181. 

304


DEVELOPMENT OF OPTICAL SENSORS 

BASED ON COMPLEXES OF 

MACROCYCLIC COMPOUNDS 

Jakub VANĚK 1 , Přemysl LUBAL 1 


Abstract 

The Ln(DO3A) structural motif was utilized for proposal of sensitive fluorosensor for 

determination of hydrogencarbonates. The thermodynamic study of Eu(DO3A) 

complex with bidentate ligands using luminescence spectroscopy demonstrate the 

order: CO3 2- > oxalate 2- > picolinate - > phthalate 2- citrate 3- as consequence of 

increasing chelate ring size. The ternary [EuL(Picolinate)] - and [TbL(Picolinate)] - 

complexes show good photophysical properties due to antenna effect. The high 

quenching effect of carbonate and less of oxalate enables to construct linear 

calibration plot under optimized experimental conditions (e.g. concentration of 

reagents, pH, wavelength, etc.). The Tb(III) sensor shows better sensitivity while the 

limit of detection about 0.4 mM was found for both sensors. In addition, the analysis 

of real samples shows no systematic error of proposed analytical procedure and the 

results are comparable with values giving by CITP. 


The lanthanide complexes of macrocyclic ligands (mostly DOTA derivatives) are 

often used as MRI agents (Gd), luminescent probes (Eu, Tb in VIS, Yb, Nd in NIR 

range) or for radioimunotherapy (Y, Sm, Ho, Lu). DO2A (1,4,7,10tetraazacyclododecane-1,7-diacetate) 

and DO3A (1,4,7,10-tetraazacyclododecane- 

1,4,7-triacetate) are hexa- and heptadentate macrocyclic ligands capable of formation 

very stable complex with europium(III) ion 1-3 . Both complexes are able to form 

ternary lanthanide(III) species with mono- and bidentate ligands (DO2A-fluoride, 

acetate, hydrogenphosphate, DO3A-hydrogenphosphate, hydrogencarbonate) 1-3 . 

Different stability of these ternary complex systems can be used as a sensor for 

selective determination of different anions. The use of chromophore, such as picolinic 

acid and its analogues, capable of forming ternary complex [Fig.1] with lanthanide(III) 

macrocyclic complexes leads to fluorescence enhancement due to antenna-effect and 

the determination itself can be more sensitive. The ternary complex with picolinic 

acid is less stabile than the ternary complex of analyzed anion and this leads to 

fluorescence decrease which can be used for sensitive determination. This 

contribution enlarges the study of the formation of ternary complex europium(III) 

species with important bioanalytes (e.g. hydrogencarbonate, oxalate, citrate, etc.) by 

means of molecular luminescence spectroscopy. The results are used for proposal of 

selective anion sensor. 

305


N 

O - H 

O - 

O O - 

N N 

Eu 

N N 

3+ 

O H VIS rad 

2 

H2O Fig.1 Ternary Eu(DO3A)(picolinate) complex 

O 

2. EXPERIMENT 

All luminescent spectra including were recorded on spectrofluorimeter 

Aminco-Bowman Series 2 (Thermo-Spectronic, USA) – operating with continuous 

and flash Xe lamp in wavelength range 200 - 800nm. The compounds were purchased 

from SIGMA-ALDRICH (all of analytical grade) and used as received. 


We have focused on study of formation of ternary species with bidentate 

ligands having potential analytical application. The thermodynamic study of 

formation of ternary Eu(III) species was followed by fluorescence spectroscopy. As it 

can be seen on example (Fig. 2), the formation of ternary Eu(III) complex is 

accompanied by increase of fluorescence intensity since two water molecules are 

excluded from the first coordination shell after bidentate ligand complexation 4-5 . 

Antenna effect of picolinate can be utilized for higher luminescence enhancement. 

The radiation of wavelength at maximum of absorption band of picolinate, 286 

nm, improved the luminescence of ternary complex to factor 170 (Fig. 2) however the 

quenching effect of ligand on formed complex is observed at higher picolinate 

concentration. 

O - 

306 

O 

UV rad 

O


I f (a.u.) 

300 

200 

100 

I f (a.u.) 

400 

300 

200 

100 

0.0 2.0x10 -3 

4.0x10 -3 

6.0x10 -3 

8.0x10 -3 

1.0x10 -2 

0 

c picolinate / M 

0 

580 600 620 640 

Wavelength / nm 

Fig. 2 The emission spectra of EuL complex after addition of picolinic acid (cEuL = 0.1 

mM, pH = 7.4, exc = 286 nm). In offset there is plot of luminescence intensity 

of Eu(III) complex at 616 nm as function of picolinic acid. 

Adding hydrogencarbonate in solution, the new ternary [EuL(Carb)] 2- complex 

is formed which does not show excellent luminescence properties as [EuL(Pic)] - 

complex and therefore it should be accompanied by decrease of luminescence in 

concentration region from 10 -4 M to 10 -2 M at pH 7.4. Increasing pH to 10.0, the 

substitution reaction is taking place in lower concentration region. 

4. CONCLUSION 

Eu(DO3A) complex forms stabile complex having longer luminiscence life-time 

as the consequence of two coordinated water molecules eliminated from Eu(III) 

coordination sphere. Ternary complexes with bidentate ligands and their stability 

follows the order: CO3 2- > oxalate 2- > picolinate - > phthalate 2- citrate 3- . 

This order is reflected by the size of chelate ring and acidobasic properties of 

ligand. The ternary complex Eu(DO3A)(picolinate) or Tb(DO3A)(picolinate) can be 

applied for sensitive analytical determination of carbonate (hydrogencarbonate) and 

oxalate in mM scale in alkaline pH under optimal experimental conditions (cEuL = 0.1 

mM, cpicolinate = 5.0 mM) in presence of other anions (phosphate, citrate) which are not 

interfering. 


This work was supported by Ministry of Education of the Czech Republic 

(ME09065, LC06035, MUNI/A/0992/2009) and performed in framework of COST D38 

EU program. 

307


6. REFERENCES 

[1] Wu S.-Li, DeW. Horrocks W., Anal. Chem. (1996), 68, 394. 

[2] Kimpe K., D’Olieslager W., Görller-Wahrland C., Figurienha A., Kovács Z., C.F.G.C. Geraldes, J. 

Alloys Comp. (2001), 323-324, 828 

[3] Supkowski R., DeW. Horrocks W., Inorg. Chem. (1999), 38, 5616 

[4] Táborský P., Svobodová I., Hnatejko Z., Lubal P., Lis S., Försterová M., Hermann P., Lukeš I., Havel 

J., J. Fluorescence (2005) 15, 507. 

[5] Táborský P., Svobodová I., Lubal P., Hnatejko Z., Lis S., Hermann P., Polyhedron (2007), 26, 4119. 

308


MONITORING DNA MODIFICATION BY 

PLATINUM COMPLEXES USING 

CATALYTIC HYDROGEN EVOLUTION AT 

MERCURY ELECTRODES 

Pavlína VIDLÁKOVÁ 1 , Petra HORÁKOVÁ 1 , Hana PIVOŇKOVÁ 1 , Luděk HAVRAN 1 , 

Miroslav FOJTA 1 

1 Pavlína Vidláková, Petra Horáková, Hana Pivoňková, Luděk Havran, Miroslav Fojta Institute of 

Biophysics ASCR,v.v.i., Královopolská 135, CZ-612 65 Brno, Czech Republic 

Abstract 

The voltammetry at the HMDE was used for the monitoring of DNA modification 

with cis-diamminedichloroplatinum (cisplatin), [(1R,2R)-cyclohexane-1,2diamine](ethanedioato-O,O')platinum(II) 

(oxaliplatin) and cisdiammine(cyclobutane-1,1-dicarboxylate-O,O')platinum(II) 

(carboplatin). These 

complexes binds covalently to DNA, forming several kinds of adducts. Using cyclic 

voltametry at HMDE was observed remarkable enhancement of cathodic currents in 

the presence of platinated DNA. These effects have been ascribed to catalytic 

hydrogen evolution accompanying electrochemical reduction of the platinated DNA 

adducts. In square-wave voltammetry the catalytic currents gave rise to well 

developed and analytically useful peak. This signal was used for monitoring of DNA 

modification. 


Electroanalytical methods are used for analysis of natural as well as chemically 

modified nucleic acids since the second half of 50s years of 20th century [1,2]. In 

cyclic voltammetry at mercury electrodes adenine and cytosine produce a cathodic 

peak CA, guanine produce an anodic peak G (due to oxidation of reduction product of 

guanine that is reduced at very negative potentials) [2]. When the DNA is chemically 

modified, its electrochemical responses can be changed. In some cases, attachment of 

a new electrochemically active group to DNA can give rise to a new signal which can 

be used for monitoring of DNA modification. 

Cisplatin, oxaliplatin and carboplatin are used as cytotoxic and antineoplastic 

metallodrugs by treatment of various malignancies [3]. The drugs binds covalently to 

DNA, forming several kinds of adducts, where the primary site the DNA modification 

is guanine. The most frequent of them being intrastrand cross-links in sequence 

motifs GG, AG and GNG (where N stands for any nucleotide). Previously we proposed 

a simple electrochemical technique for the monitoring of DNA modification with 

cisplatin [4]. Here, application of the technique is extended towards analysis of DNA 

modification with other platinum complexes. 

309


2. EXPERIMENTAL 

DNA (usually single strand calf thymus DNA) was incubated with cisplatin 

(oxaliplatin, carboplatin) in 0.1 M NaClO4 at 37 °C overnight in the dark. DNA 

concentration in the reaction mixture was 20 g.ml -1 . Concentracion of platinum 

complexes complied with rb values (rb is the number of platinum atoms bound per 

DNA nucleotide). Platinated DNA was purified using magnetoseparation procedure. 

Voltammetric responses of the modified DNA were measured using the adsorptive 

transfer stripping (AdTS) procedure at HMDE. All measurements were performed at 

room temperature with an Autolab analyzer connected to VA-Stand 663 in threeelectrode 

setup (Ag/AgCl/3 M KCl electrode as a reference and platinum wire as an 

auxiliary electrode), under argon. 


We used CV at HMDE for analysis of unmodified and platinated DNA (Fig.1). 

The unmodified DNA yielded two signals - catodic peak CA at -1.51 V and anodic 

peak G at -0.25 V. In the cathodic part of voltammogram of DNA modified by 

cisplatin, the negative current started to increase sharply around -1.2 V and reached 

its maximum at -1.75 V, forming a wide peak. In the anodic part of the 

voltammogram were three waves (around -1.75, -1.45 and -1.3 V), and between -1.53 

and -1.18 V it was going through higher negative current values than the cathodic 

part in the same region. Such behavior suggested a kinetic process coupled to 

reversible electron transfer reactions, most likely catalytic hydrogen evolution 

accompanying redox processes of the platinum moieties. Peak G produced by the 

cisplatinated DNA was lower than the same signal produced by the unmodified DNA 

and was shifted to more negative potentials. 

In square wave voltammograms of DNA modified by cisplatin, oxaliplatin or 

carboplatin we observed two signals, peak G at -0.26 V (corresponding to signal of 

guanine moieties) and peak P at -1.25 V (characteristic for the platinum-DNA adduct) 

(Fig. 2). Intensity of this signal increased linearly up to rb=0.12 within the region 

relevant for the biochemical studies. Differences in the intensity of the peak P for 

DNAs modified with various complexes under the same conditions, suggesting 

different feasibilities of the adducts formation. 

310


I/A 

0 

-10 

-20 

-30 

-40 

-50 

-60 

I/mA 

0.36 

0.18 

0.00 

-0.18 

-0.36 

G 

-0.40 -0.32 -0.24 -0.16 

311 

E/V 

DNA + cisplatin (rb 1) 

unmodified DNA 

-2.0 -1.5 -1.0 -0.5 0.0 

Fig. 1 CV of DNA modified by cisplatin: background electrolyte 0.3 M ammonium 

formate, 0.05 M sodium phosphate, pH 6.9 (AFP), initial potential 0.0 V, 

switching potential -1.85 V, scan rate 1 Vs -1 

I/A 

0.98 

0.84 

0.70 

0.56 

0.42 

0.28 

0.14 

0.00 

P 

P 

E/V 

DNA + oxaliplatin (rb 0.1) 

DNA + carboplatin (rb 0.1) 

unmodified DNA 

DNA + cisplatin (rb 0.1) 

-1.5 -1.0 -0.5 0.0 

E/V 

Fig. 2 AdTS SWV of DNA modified by platinum complexes: background electrolyte 

AFP, initial potential -1.85 V, end potential 0.0 V, frequency 200 Hz, 

amplitude 50 mV, potential step 5 mV 

4. CONCLUSION 

Using square-wave voltammetry at HMDE the catalytic currents gave rise to 

well developed and analytically useful catalytic peak (peak P). Intensity of this peak 

responded to the extent of DNA modification at levels relevant for biochemical 

studies (rb=0.01 – 0.10, where rb is the number of platinum atoms bound per DNA 

nucleotide). We show that intensity of the peak P reflects different reactivity of 

individual platinum complexes towards the DNA. 


This work was supported by grants of the GA ASCR (IAA400040903, 

IAA500040701), Czech Science Foundation (P206/11/1638), MEYS CR (LC06035) and 

institutional research plans Nos. AV0Z50040507 and AV0Z50040702. 

G


6. REFERENCES 

[1] Palecek, E., Jelen, F: In Electrochemistry of nucleic acids and proteins Towards electrochemical 

sensors for genomics and proteomics Palecek, E., Scheller, F., Wang, J., Elsevier, 2005, 

Amsterodam 

[2] Fojta, M: Collect Czech. Chem. Commun. (2004), 69, 715 

[3] Kasparkova, J., Fojta, M., Farrell, N., Brabec, V.: Nucleic Acids Res. (2004), 32, 5546 

[4] Horakova, P., Tesnohlidkova, L., Havran, L., Vidlakova, P., Pivonkova, H., Fojta, M.: Anal. 

Chem. (2010), 82, 2969. 

312


SYNTHESIS AND CHARACTERIZATION OF 

NANOPARTICLES OF SN-3,5AG-0,5ZN 

Vít VYKOUKAL 1 , Klára SUCHÁNKOVÁ 1 , David ŠKODA 1 , Jíří SOPOUŠEK 1 


Abstract 

Sn-3,5Ag-0,5Zn alloy nanoparticles were synthesized from NaBH4, 

Polyvinylpyrrolidone, SnSO4, AgNO3, Zn(NO3)2 precursors by using chemical 

reduction wet synthese at 50°C ubder pernament 12 h stirring. Transmission electron 

microscopy (TEM) was used to investigate the size and shape of the nanoparticle alloy 

product. TEM observation revealed that smaller aggregations were formed from small 

primary nanoparticles with size varied from 3 to 5 nm. Aggregation of small 

nanoparticle was spontaneous. It was found that aggregates with a greater proportion 

of nanoparticles are heated by passing of electron beam in TEM in such a way that 

nanoparticles melt and subsequently redeposit on carbon membrane. 


Until recently lead-tin alloys were mostly used for soldering. The advantage of 

these alloys is the price, ductility and low surface tension. Considerable disadvantage 

is toxicity of lead and its negative effects on the environment. Today's modern and 

progressive trend for soldering is using solders without addition of lead. These solder 

alloys exhibit eutectic behavior and a sufficient reduction in melting point. However 

nanoparticles are considered for using. 

Physical, electrical and thermodynamic properties of nanoparticles are different 

from the compact particles. The melting point of nanoparticles depends on their size 

and can be much lower than the melting point of compact materials. On the other 

hand, reduction of the melting point can indicate reduction of nanoparticles size. The 

reason for this phenomenon is the higher surface energy. Nanoparticles of metals and 

their alloys can easily be aggregated and may interact with other materials 

(substrates). This phenomenon is a possible alternative to soldering, which is a 

common manufacturing process in electrical engineering. 

Preparation of nanoparticles is also possible by chemical synthesis. Well usable 

are reduction methods, which are cheap and can provide nanoparticles of metals and 

their alloys. Reaction conditions can control the size and the shape of synthesized 

nanoparticles. However, the choice of suitable reducing agent is also important. 

To characterize the obtained nanoparticles of metals and alloys the methods of 

electron microscopy and thermal analysis can also be used. The shape and size 

of particles can be detected by the methods of electron microscopy. 

By means of transmission electron microscopy (TEM) the size distribution curve 

of nanoparticle array and the effect of aggregation processes can be determined. 

313


Electron microanalysis of the sample (EDX) we can then determine the composition 

of the sample. Thermal analysis – differential thermal analysis (DTA) and differential 

scanning calorimetry (DSC) – can be used for monitoring depression of the melting 

point, which is given by the difference of melting point of nano-objects and compact 

materials of the same chemical composition. Melting point depression is a function of 

nanoparticle size. 

2. EXPERIMENT 

Sn-3,5Ag-0,5Zn alloy nanoparticles were synthesized by precipitation with 

2,405 g NaBH4 and 2 g PVP in 200 ml aqueous solutions at 50°C. This solution was 

prepared in the first beaker. At first we dissolved the PVP in water. PVP is not well 

soluble in water. We adjusted water pH to about 9 and then we added NaBH4. By 

increasing of pH we wanted to relieve reaction boisterousness. NaBH4 is releasing 

hydrogen in acidic environment and reaction could be very turbulent. In the 2nd 

beaker we simultaneously dissolved 0,138 g AgNO3, 3,471 g SnSO4 a 0,030 g 

Zn(NO3)2.The salts were dissolving very badly, pH of solution was about 2. Yellow 

suspension was formed, which looked like a thick porridge. For better solubility of the 

suspension we heated it. From resulted color we concluded that SnSO4 is very badly 

soluble. Suspension during heating became darker and the black dots began to 

explore. After heating we filtered suspension at atmospheric pressure. The filtrate was 

clear, filter paper, beige with black dots. Afterwards we very carefully and dropwise 

got together both solutions. We added filtrate to the solution of NaBH4 a PVP. The 

solution immediately began to darken. First it was brown, but progressively it was 

getting dark until black. A large amount of gas released and formed in the center of 

beaker large bubble. During permanent stirring both solutions were mixed at 

laboratory temperature. After mixing we started to heat the final solution. We heated 

for 12h with permanent stirring. 


The final product was a suspension containing solid phase containing the 

nanoparticles and liquid water phase. Solid phase could be separated by decantation, 

like black powder. The obtained solid samples were sent for analysis by TEM. Before 

TEM analysis the samples of the suspensions were briefly shaken in the ultrasound 

and then deposited onto copper grid with carbon membrane. As follows from the 

micrographs the particles form clusters (aggregates). The high degree of aggregation 

can be explained by a large part of unoxidized ( metallic) nanoparticle surface by 

means of which the nanoparticles reduce their energy. For smaller aggregates we can 

conclude that primary size of nanoparticles was 3-5 nm (see Fig. 1-2). Unfortunately 

aggregation is not controlled, it is spontaneous. Therefore this process was not 

monitored by the thermal analysis. 

314

XI. WWorkshop 

of Phyysical 

Chemists and Electrocheemists 

´11 

Fig. . 1: Agregattes 

of nanop particles (TTEM) 

Fig. 2: Detail of o agregatees 

of nanop particles 

(TEM) 

It was ffound 

that aggregatess 

with a greater g prop portion of nanoparticles 

are 

heaated 

by passsing 

of electron 

beam in TEM in such a way y that nanooparticles 

melt m and 

subssequently 

rredeposite 

(small ( ballss) 

on carbon n membran ne. The ball lls are form med with 

dimmensions 

of 10 nm to 100 1 nm (seee 

Fig. 3 to 4). 4 Accordin ng to EDX micro-anal lysis the 

Sn wwas 

confirmed. 

The silver and zinc conte ents were on o the deteection 

limit 

of the 

usedd 

micro-annalysis. 

Fig. 3: AAgregates 

of o nanopartticles 

Fig. 4: 4 Detail of rede eponted 

nanopaarticles 

after r interactioon 

with elec ctron beam m TEM. Interraction 

electronn 

beam TEM M. 

with 


Alloy naanoparticles 

s of Sn-3,5AAg-0,5Sn 

al lloy with si ize 3-5 nmm 

were prep pared by 

reduuction 

of pprecursors 

by wet syynthesis 

method. m The e particles showed a simple 

aggrregation, 

caaused 

by a low degreee 

of adsorp ption of foreign 

materrials 

on the surface 

of thhe 

nanoparrticles. 

A hi igh proporttion 

of met tallic surface 

led to agggregation, 

which w is 

diffficult 

to control. 

Inter raction witth 

electron n beam cau uses furtherr 

instability y of the 

nannoparticles. 

Agregates with high concentration 

of nan noparticles easily capt ture the 

energy 

of thee 

electron beam. Naanoparticle 

es melt an nd then leead 

to sub bsequent 

315 

Brno


redeposition on carbon membrane. It was proved that tin is the main component of 

redeposited particles. 


The authors acknowledge the project support through grant GACR 106/09/0700 

(Thermodynamics and microstructure of environmentally friendly nanoparticle 

solders). 

6. REFERENCES 

[1] C. Y. Lin, U.S. Mohany, J. H. Chou: Journal of Alloys and Compounds 501 (2010) 204-210 

[2] Hongjin Jiang, Kyoung-sik Moon, Fay Hua, and C. P. Wong: Chem. Mater. (2007), 19, 4482-4485 

316



BIOLOGICALLY ACTIVE COMPOUNDS 

Jiří ZIMA 1 , Jiří BAREK 1 , Hana DEJMKOVÁ 1 

1 Charles University in Prague, Faculty of Science, Department of Analytical Chemistry, UNESCO 

Laboratory of Environmental Electrochemistry, Albertov 6, 128 43 Prague, Czech Republic 

Abstract 

This contribution deals with recent studies based on electrochemical determination of 

submicromolar and nanomolar concentrations of various biologically active 

compounds including carcinogens, environmental pollutants, or pharmaceuticals 

(nitrated polycyclic aromatic hydrocarbons, heterocyclic compounds, azo compounds, 

aromatic amino compounds, etc.) employing both traditional electrodes as mercury 

electrodes and non-traditional types of electrodes such as solid amalgam electrodes, 

carbon paste electrodes, boron doped diamond film electrodes, platinum tubular 

electrodes in batch or flow arrangement. 


Modern electrochemical methods possess high sensitivity and could be utilized 

for monitoring purposes of biologically active compounds [1,2]. The strength of 

electrochemical methods is in their versatility, possibility to determine substances 

which are both reducible and oxidizable. Electrochemical methods are especially 

suited for large scale monitoring of chemical carcinogens or environmental pollutants 

in matrices of drinking and surface waters, biological fluids matrices or 

pharmaceutical products [3,4]. When used in a form of electrochemical detectors in 

HPLC or electromigration methods, they could be utilized even for more complicated 

environmental matrices where preliminary separation is inevitable for successful 

analyte determination. The most important factor influencing the performance of the 

method is the type and quality of the electrode material. For determination of 

electrochemically reducible organic compounds such as azodyes, aromatic nitroso or 

nitro compounds, mercury-based electrodes could be utilized with limits of 

determination (LOD) being in the range from 10 -9 to 10 -10 mol/L [5], or their 

environmentally friendly alternatives - amalgam based electrodes with LODs down to 

10 -7 mol/L, which are also suitable for both batch methods of analysis and for HPLC. 

For determination of electrochemically oxidizable organic compounds such as 

phenols, thiols or aromatic amines, classical bare or chemically or biologically 

modified carbon paste electrodes are well suited with with LOD down to 10 -7 mol/L, 

pastes based on glassy carbon spherical microparticles which are compatible with high 

content of organic solvents and thus applicable for HPLC [6] with LOD down to 10 -7 

mol/L [7]. The main disadvantage of electrochemical methods, which is lower 

selectivity, could be overcome by their combination with a suitable preliminary 

separation method such as liquid-liquid extraction or solid phase extraction, the 

317


passivation of the electrode surface could be eliminated with periodical 

electrochemical or mechanical renewal of working surface of the electrode, sensor or 

detector [8]. 

2. 2.EXPERIMENTAL 

Studied substances were purchased from Sigma or Merck. Their stock solutions 

(usually c = 1 mmol/L) were prepared by dissolving the exact amount of the respective 

substance in methanol or water and were kept at a laboratory temperature. 

Spectrophotometric measurements were used for checking their stability for 

prolonged periods of time. Britton-Robinson (B-R) buffers served as supporting 

electrolytes for voltammetric measurements. The same 10 times diluted buffers or 

phosphate buffers in mixtures with methanol or acetonitrile of various ratios served as 

mobile phases in HPLC or FIA measurements. The details for carbon paste 

composition, amalgam electrode composition or other details will be described. Other 

used chemicals were of commercial origin and of p.a. or of HPLC purity. Deionized 

water was prepared by Millipore system. 

Voltammetric batch measurements were performed using Eco-Tribo- 

Polarograph, controlled by software Polar Pro 5.1 (both PolaroSensors, Prague, Czech 

Republic) and they included cyclic voltammetry (CV), differential pulse voltammetry 

(DPV), direct current voltammetry (DCV) and their adsorptive stripping variants in 

three electrode arrangement with a chosen working electrode, a platinum wire 

auxiliary electrode, and Ag/AgCl (3 M KCl) reference electrode RAE 113 

(Monokrystaly Turnov, Czech Republic), to which all the potential values are 

referred. The same electrode set was used for electrochemical detection in a flow 

arrangement. HPLC measurements were performed using high pressure pump Beta 

10, injector valve with 20 L loop, UV/VIS detector Sapphire 800 (all Ecom, Czech 

Republic) and amperometric detector ADLC 2 (Laboratorní přístroje, Czech Republic) 

if not specified otherwise. The HPLC system was controlled via Clarity 2.3 software 

(DataApex, Czech Republic). 

3. 3.RESULTS AND DISCUSSION 

Electroanalytical chemistry has to deal with the growing requirements and 

demands for solving practical tasks in more and more complicated matrices and in 

lower and lower concentration ranges. So that new types of electrodes or 

arrangements are sought, together with new potential programs and workup of 

current signals including preliminary electrochemical pre-treatment of the working 

electrode. Preliminary separation and pre-concentration of analytes is helping to 

further increase the sensitivity, robust methods deal with the most serious problem of 

electrochemistry which is the passivation. Nevertheless, electrochemical methods 

could be successfully used for large scale monitoring of electroactive compounds in 

the environment as the absence of proper signal is often enough for claiming the 

absence of particular analyte of interest, while in positive case more expensive and 

demanding separation methods could be used. Electroanalytical methods thus present 

independent alternative to separation or optical methods of analysis. 

318


In this contribution, examples of successful use of carbon paste electrodes, 

carbon film electrodes, mercury electrodes, amalgam electrodes, amalgam crystal 

electrodes, boron doped diamond film electrodes, in batch and flow methods, in walljet, 

tubular and thin layer arrangement in HPLC or FIA will be described. 

The illustration of the importance of proper regeneration of electrode surface is 

depicted in Figure 1 showing the repeatability of DPV signal of 2,4-dinitrophenol 

using meniscus modified silver solid amalgam electrode (m-AgSAE). Under the 

chosen potential program up to 25 curves could be measured with standard deviations 

not exceeding 5 %. 

I p [nA] 

-600 

-400 

-200 

0 

0 5 10 15 20 25 

N 

Fig. 1 Peak heights of DP voltammograms of 1.10 -4 M 2,4-dinitrophenol using m- 

AgSAE in BR buffer pH 4. 

Electrochemical electrode regeneration: 1 Eini = 100 mV; Efin = -1100 mV; 2 Eini = 100 

mV; Efin = -1400 mV; 3 Eini = 0 mV; Efin = -1400 mV. 

In case of carbon paste electrode quite nice illustration of the persistence of bare 

carbon paste based on microspheres of glassy carbon in a mobile phase with high 

content of methanol is depicted in Figure 2. The chromatograms of consecutive 30 

injections of 20 μL of 1.10 -4 M aminoglutethimide for HPLC with electrochemical 

detection (Edet = 1.3 V) using carbon paste electrode in a mobile phase composed of 

phosphate buffer and methanol (1:1, v/v) had the relative standard deviation (RSD) of 

only 1.4 % compared with RSD 1.9 % for UV detection at 242 nm. Carbon pastes 

based on spherical microparticles of glassy carbon could be used the whole day 

without mechanical or electrochemical renewal of working area during single 

measurements, the paste does not decompose, the noise being the same and very low. 

319 

2 

3 

1

XI. Workshhop 


E 

s ´11 

Fig. 2 

Chromatogram[9] 

of o 30 reppeated 

injections 

of 

aminogluteethimide, 

mobile m phasse 

0.01M ph hosphate bu 

(v/v), ED at 

CPE +1,3 V. flow ratte 

1 ml·min n−1 f 20 μl 1·10 

uffer pH 4: 

. 

−4 mo ol·l 

:MeOH = 5 

−1 

0:50 

OOther 

intereesting 

electrode 

materrials 

which is at present 

studied intensively y are 

boron ddoped 

diammond 

film electrodes 

(BBDBFE). 

BD DDFEs hav ve usually hhave 

very br road 

potentiial 

window spanning for f more thhan 

3 V, low w noise, low w passivatioon, 

mechan nical 

and eleectrochemiical 

stability, 

they aare 

biocom mpatible an nd already commercially 

availablle. 

BDDFEE 

could be used for bboth 

batch methods and 

flow mmethods 

in thin 

layer orr 

wall jet aarrangemen 

nt. On the oother 

hand d, the inertness 

of its surface usu ually 

preventts 

utilizatioon 

of adsor rptive accummulation 

of o analytes on their suurface 

and thus 

furtherr 

decreasingg 

limits of f determinaation. 

Exam mples of th he use of BBDDFE 

for r the 

determmination 

off 

carcinoge ens and otther 

organ nic analyte es in moddel 

samples 

of 

environnmental 

and 

biologica al matrices wwill 

be give en. 

Fiinally, 

exammples 

of a single silveer 

amalgam m crystal as detector inn 

voltamme etric 

analysiss 

will be deescribed. 


Both 

traditiional 

and non-tradittional 

elec ctrode materials 

conttribute 

to the 

renaissaance 

of eleectrochemi 

ical methoods 

for mo onitoring purposes 

wh where they can 

compette 

with seeparation 

methods m oor 

can be used as electrochem 

e mical sensi itive 

detectoors 

for separration 

meth hods. The ffact 

that th he analyte molecule m orr 

ion is a di irect 

source of signal, high sensi itivity, brooad 

linear dynamic ranges, r neww 

strategie es in 

minimiizing 

probllems 

with the passivvation, 

poss sibility of analysing ooxidizable 

and 

reducibble 

compouunds 

even in the preesence 

of large l amou unts of nonn-electroac 

ctive 

substannces, 

new mmaterials 

or r componennts 

of sensor 

materials, 

high speed, 

low runn ning 

and invvestment 

ccosts, 

comp patibility wwith 

green chemistry, 

support tthis 

trend. We 

have shhown 

that either new w electrodee 

materials s or classic cal electrodde 

material ls in 

combinnation 

withh 

new pre- -concentrattion 

approa aches or electrode 

arrrangement 

can 

320 

Brno


provide us with sensitive and selective methods of determination of biologically active 

compounds including carcinogenics, environmental pollutants, toxic compound etc. 

Performance of electrochemical methods of analysis, especially the utilization of 

micro electrodes is of special importance in in vivo measurements, and in 

voltammetric immunoanalysis. Also membrane electrodes, fast measurement 

techniques and dual or multi-channel detection are the future of electrochemistry. 


The work has been supported by project No. SVV 261204 and by the Czech 

Ministry of Education, Youth and Sports (projects No. MSM 0021620857, RP14/63 

and LC 06035) and KONTAKT (AMVIS) project ME10004. 

6. REFERENCES 

[1] Barek J., Mejstřík V., Muck A., Zima J.: Polarographic and Voltammetric Determination of 

Chemical Carcinogens. Crit. Rev. Anal. Chem. 30 (2000), 35. 

[2] Zima J., Barek J., Muck A.: Monitoring of Environmentally and Biologically Important Organic 

Substance at Carbon Paste Electrodes. Rev. Chim. (Bucuresti) 55 (2004), 657. 

[3] Barek, J., Cvačka J., Muck A., Quaiserová V., Zima, J.: Electrochemical methods for monitoring 

of environmental carcinogens. Fresenius J. Anal. Chem. 369 (2001), 556. 

[4] Švancara I., Vytřas K., Barek J., Zima J.: Carbon paste electrodes in modern electroanalysis. Crit. 

Rev. Anal. Chem. 31 (2001), 311. 

[5] Barek, J.; Fogg, A.G.; Muck, A.; Zima, J. Polarography and voltammetry at mercury electrodes. 

Crit. Rev. Anal. Chem. 31(2001), 291. 

[6] Zima J., Cienciala M., Barek J., Moreira J.C.: Determination of thymol using HPLC-ED with 

glassy carbon paste electrode, Chem. Anal. (Warsaw) 52 (2007) 1049. 

[7] Yosypchuk B., Novotný L.: Nontoxic electrodes of solid amalgams. Crit. Rev. Anal. Chem. 32 

(2002), 141. 

[8] Zima J.,Svancara I., Barek J., Vytras K.: Recent Advances in Electroanalysis of Organic 

Compounds at Carbon Paste Electrodes, Crit. Rev. Anal. Chem. 39 (2001), 204. 

[9] Vlachova K.: Diploma Thesis, Charles University in Prague, Faculty of Science, 2010. 

321


SPECTROFOTOMETRIC ANALYSIS OF 

CYSTEINE-CADMIUM COMPLEXES USING 

MUREXIDE INDICATOR 

Ondřej ZÍTKA 1 , Jiří SOCHOR 1 , Vojtěch ADAM 1 , René KIZEK 1, * 



Abstract 

Question of interaction of biomolecules with heavy metals is not sufficiently 

determined area of the research. We can principally divide the metals into two groups 

by physiological point of view. Essential metals are beneficial for organism in nature 

physiological biochemical proceses. Toxic metals or essential metals in higher 

concentration could cause death of organism. Metal ions are not occurring in the 

organism in the free state because they are bond to the biomolecules eg 

metalloproteins. The possibility of the bonding on the smaller molecules such as 

aminoacids is partially remaining unclear. Aminoacid analysis could be done by many 

various approaches. Study of complexes of free amino acids with toxic metals is not so 

well known area but it has its importance in bioscience and biochemical research. In 

vitro studies of aminoacids and heavy metals could be done for obtaining basic 

knowledge about the complex creation of particular group of compounds of interest. 

In our work we presented using of UV-VIS spectrophotometric detection as easy to 

use method for these purposes. Aim of this work was to observe an interaction 

between free aminoacids Cysteine, Homocysteine and N-acetyl cysteine with 

cadmium ions. 


Free amino acids are these which are not in the time or along of its whole 

existence bounded neither in any peptide chain which is representing proteins or 

enzymes nor in other biomolecules. Next group is biogenic amines or their precursors 

during their biosynthesis or group of biogenic amines which originates by 

modification of other common aminoacids. If the metal is free occurring in the 

organism it can induce a number of reactive oxygen species (ROS) which are danger 

for the organism [1]. Free metals like a copper and iron could have a negative role 

because could acting like an catalizators for creation of hydroxyl radicals [2] . These 

radicals can cause damaging of genetic information of cytoplasmatic membranes and 

thus to disturbing of the homeostasis of the organism. In the front defend line against 

these influences stays the metal binding proteins or aminoacid which often contains 

thiol groups like cysteine, homocysteine or N-acetyl-cysteine. Cysteine is part of well 

known tripeptide glutathione (GSH) which is very important and has many benefitial 

and crucial functions. Other aminoacids which could have the similar qualities and 

322


thus could bind some toxic metal are methionine, histidine or tryptophan. All of these 

has a nucleophilic qualities because of their side chains. In this work we studied 

interaction between free aminoacids cysteine, homocysteine and N-acetyl-cysteine 

with cadmium ions. Cadmium is one of most dangerous metal in the environment 

thanks to anthropogenic influence. Various methods for determination of specific 

amino acid could be used. Specifity and selectivity of the reaction is critical and 

depends on the concrete method according the reagent and structure of the 

determined amino acid. In the stationary systems which are common like a reaction 

in the cuvette are the products of reactions of aminoacids with specific reagent 

monitored to obtain informations about change of absorption maximum or intensity 

in one wavelength. These changes could be monitored in the ultraviolet or visible part 

of spectra. Methods like a Sakaguchi reaction are based on by eye visible color change 

and it is specific on the side chain of the amoniacid. Determination of aminoacids like 

tyrosine, tryptophan and Phenylalanin, is possible by Xantoprotein reaction. Pauly´s 

reaction is suitable for detection of tyrosine a histidin. For the cystein and its dimer 

cystin most suitable method is reaction with Ellman reagent 5,5´-dithiobis-2nitrobenzooic 

acid (DNTB) called as Ellman´s method. Thiol group is reacting under 

simultanoueous cleaving of DTNB on two monomers. One monomer is immedietly 

connected to the free thiol group of Cysteine and second monomer (5-merkapto-2nitrobenzoic 

anino) is monitored under 412 nm [3]. There is many other methods 

which could be mentioned but we interested about studying of complexes with metals 

and thus we decided to apply rather different approach. We used a metalochromic 

indicator murexide and we supposed that it will indicate the cadmium ions in free 

state. On the other side when metal will be bounded by the aminoacid there should 

be a change of absorbance or absorption spectrum occuring. 

2. EXPERIMENT 

Murexide was achieved from Lachema (Brno, Czech Republic). All other used 

chemicals includind cadmium chloride were purchased from Sigma Aldrich in ACS 

purity, if there is´t noted otherwise. Stock solutions of all standards (concentrations 1 

mg.ml -1 ) were prepared in ACS water (Aldrich, USA) and stored in dark and 

temperature -20 °C. New working solutions were prepared every day. The pH value 

was measured using inoLab Level 3 with terminal Level 3 (Wissenschaftlich- 

Technische Werkstätten - WTW, Weilheim, Germany), controlled by the personal 

computer program (MultiLab Pilot; WTW). The pH-electrode (SenTix-H, pH 0– 

14/3M KCl) was calibrated by set of buffers (WTW). The pH value and conductivity 

was measured using inoLab Level 3 (Wissenschaftlich-Technische Werkstatten 

GmbH; Weilheim, Germany). Deionised water underwent demineralization by 

reverse osmosis using the instruments Aqua Osmotic 02 (Aqua Osmotic, Tisnov, 

Czech Republic) and then it was subsequently purified using Millipore RG (Millipore 

Corp., USA, 18 MΏ) – MiliQ water. For spectrophotometric analysis UV-VIS 

spectrophotometer Specord - 210 (Jena Analytic, Germany) was used. 

Spectrophotometer was equipped by moving carousel for eight samples. Plastic 

cuvettes (Kartell, Italy) with optical length 1cm and total volume 1,5ml were used. 

323

XI. Workshhop 


E 

s ´11 

Carouseel 

were teermostated 

on speciffic 

temper rature by flow aggreegate 

JULA ABO 

F12/EDD 

(Labortecchnik 

Gmb bH, Germmany), 

whe ere a distilled 

waterr 

was used d as 

mediumm. 

All anallysis 

was done 

under temperatu ure 25°C. In n the scannning 

mode the 

range wwas 

400-7000 

nm. 

3. RRESULTS 


WWe 

used a Murexide as a colorr 

change in ndicator w 

functioon 

which iss 

bounding to heavy mmetals 

Cu, Cd, C Co, Pb 

[4]. Wee 

supposedd 

that by in nteraction of Murexi ide with he 

createdd 

a colored complex which w absorrption 

max ximum cou 

400-7000 

nm. We aalso 

assume ed that afteer 

an addition 

of amin 

compleex 

with thee 

metal we will be abble 

to moni itor some c 

the commplex 

and thus to cha ange of thee 

height an nd position 

The preeparation 

oof 

reaction mixture wwas 

done pa artially acco 

one diffference. 

WWe 

used fr ree cysteinn 

instead of o glutathio 

solutionn 

of the reagent 

consists 

of 50 mmM 

KCl a 50 mM Tri 

every rrepetitions 

we used same 

conceentration 

of f the Mure 

repetitiions 

differdd 

in applied d concentrat ations of Cd d 

0,16; 0, ,32; 0,48 a 0,64 mM. For each vvariant 

of 

four cooncentratioon 

repetitions 

of cyssteine 

10, 

Homoccysteine 

and 

N-acetyl l cysteine wwere 

studie 

and fastt 

mixing off 

all parts of 

solution wwere 

8 cuve 

row off 

the cadmmium 

and d in one independe 

concenntration 

of each amin noacid anaalyzed. 

For 

dependdent 

measurrement 

was s done. 

+2 with a commpetitive 

lig gand 

, Zn, Ag a Hg in ratio o 1:1 

eavy metall 

there wil ll be 

ld be moniitored 

in ra ange 

no acid whic ich can crea ate a 

hange of thhe 

structur re of 

of absorpttion 

maxim mum. 

ording literrature 

[5] with w 

one reuducced. 

The basic b 

is-Mes undder 

pH 7,0. For 

exide 100 μμM. 

Individual 

, which were w 0,01; 00,02; 

0,04; 0,08; 0 

Murexide with w cadmmium 

there was 

25, 50 a 100 μM. All Cyste eine, 

ed by this approach. After addi ition 

ettes which h containedd 

concentra ation 

nt analysis 

every tiime 

only one 

r these sam mples discoontinual 

ti ime- 

Fig.1 SStructure 

oof 

murexide 

(A) and d record of f spectra of o murexidd 

complex (B). 

Complexess 

are ma arked: Muurexide+Cysteine+Cad 

dmium (mmodrá 

křiv vka), 

Murexid+KKadmium 

(b blue curve) ), Murexide+Cysteine 

(green currve) 

and single 

Murexid (ppurple 

curv ve). 

The 

changess 

of height of o absorptioon 

maximu um of murex xide whichh 

testified by b its 

decreasse 

and shift ft respective ely about ccreation 

of f complex between b mmetal, 

murexide 

and amminoacid. 

Thhe 

change of the absoorption 

ma aximum of murexide wwas 

influen nced 

324 

Brno


by two factors actually by concentration of metal and concentration of amino acid. 

Intensity was influenced by time. Firstly we analyzed individual combinations: single 

murexide 100 μM, murexide 100 μM + cysteine 100 μM, murexide 100 μM + cadmium 

0,64 mM a murexide + cysteine 100 μM + cadmium 0,64 mM. After comparing 

obtained spectra we determined the addition of cysteine as decreases absorption of 

maximum of murexide. Addition of cadmium moves the absorption maximum to 

longest wavelengths. But if all three components are in mixture, the absorption 

maximum moved to shorter wavelengths and decreased slightly (Fig. 1). For quantify 

of rate of complex formation which is based on the change of absorption maximum, 

we must proceed to normalize output values. Normalization was performed on the 

basis of presumption that the highest value of absorbation is getting from interaction 

with all three components. We subtracted from highest signal murexide + amino acid 

+ cadmium the absorbance values of mixtures murexide + aminoacid and murexide + 

cadmium. To realize our computation we had to do same time dependent analysis for 

concentration repetitions of murexide + cadmium and murexide + amino acid. Values 

of differences for amino acid cystein in appropriated concentration repetitions are 

showed in graphs. For all dependences for changes of absorbance based on change of 

concentrations of metal and amino acid on various terms which are mentioned in 

graphs in this chapter was performed correlation through power law trendline. This 

curve is very objective expressing behavior for all trends and it’s characterized by 

quadratics Y=a.Xb. It is seem differences of absorbance for all concentrations of 

cysteine are uniformly decreased in time. This fact could induct of competitive 

character of interactions of murexide with metal, where cysteine molecules are 

bonded to metal ions in time and because of this the proportion of free metal ions for 

bond with murexide is decreasing. By that are decreasing value of absorbance in 

absorption maximum, this was for complex of murexide + aminoacid + metal in 

485nm. This assumption confirms trend, where is the lowest dispersion between 

highest and lowest values of absorbance of time dependence at the highest applied 

concentrations. In coincidence with this thesis is change, which could see between 

particular applied concentrations. After comparison of scale of particular axis 

(absorbance) is seen, that with highest concentration of cystein occurs for penetrative 

decreases of absorbance. In the case of amino acid homocysteine, the creation of 

complex wasn´t so intensive. For middle applied concentrations was decrease of 

absorbance in time hardly 25% compared to decrease observing in the case of cystein. 

Increased creation of complex was noted for amino acid N-acetyl-cystein. The 

different of the highest and the lowest values of absorbance isn´t change slightly at 

time dependent measurement, but the change of absorbencies between particular 

applied concentrations of metal was relatively very high. The highest differencies of 

values of complex absorbance was again determined by the highest applied 

concentration of amino acid, like both previous cases. 

4. CONCLUSION 

We proved that the spectrophotometric method could be useful as easy to use 

method for characterizing of complex creation between amino acids and free metal 

325


ions like cadmium. More options like time dependent measurements, temperature or 

concentrations of both reagents could be studied. We observed the best interaction 

between cadmium and cysteine 50 μM after 60 minutes of incubation. Interactions of 

homocysteine and N-acetyl-cysteine had a smaller efficiency. Moreover homocysteine 

best conditions for interaction were recorded in concentration 50 μM after 10-15 

minutes and N-acetyl-cysteine interaction were slightly increasing to 30 minutes but 

then were negligible. 


The work has been supported by NANIMEL GA ČR 102/08/1546, NANOSEMED 

GA AV and KAN208130801 and NanoBioTECell P102/11/1068 GA ČR. 

6. REFERENCES 

[1] W. Droge, Physiological Reviews 82 (2002) 47. 

[2] B. Halliwell, Journal of Neurochemistry 59 (1992) 1609. 

[3] J.Z. Karasova, K. Kuca, D. Jun, J. Bajgar, Chemicke Listy 104 46. 

[4] J. Zolgharnein, H. Tahmasebi, S. Amani, Russian Journal of Coordination Chemistry 35 (2009) 

512. 

[5] P. Leverrier, C. Montigny, M. Garrigos, P. Champeil, Analytical Biochemistry 371 (2007) 215. 

326


DETERMINATION OF LACTOFERRINE IN 

HUMAN SALIVA 

Ondřej ZÍTKA 1 , Jiří SOCHOR 1 , Sylvie SKALÍČKOVA 1 , Jiří LITZMAN 2 ,Marcela 

VLKOVA 2 , Ester MEJSTRIKOVÁ 3 ,Vojtěch ADAM 1 , René KIZEK 1 

1 Mendel University in Brno, Brno, Czech Republic 

2 Department of Clinical immunology and allergology, University Hospital, Pekarska 53, CZ-656 91 


3 Department of Paediatric Haematology and Oncology, 2 nd Faculty of Medicine, Charles University, 

V Uvalu 84, CZ-150 06 Prague 5, Czech Republic 

Abstract 

Salivary is body fluid, which consists of a lot of substances as enzymes, hormones, 

proteins, glykoproteins, electrolytes and perform lot of tasks for body functions. 

Lactoferrin is a glycoprotein of about 77000 Da and into of two its domains, C- and Nterminal, 

can bind one iron ion to each domain. Aims of this work were to optimize 

FPLC separation for purification of lactoferrin from whole saliva and to determine 

concentration of lactoferrin in real samples of whole saliva by spectrophotometry. We 

analyzed of whole saliva by four healthy volunteers. After isolation of fraction which 

contained a purified lactoferrin this fraction was analyzed by Pyrogallol red method 

which was conducted by automatic spectrophotometer. Sample of whole saliva was 

also analyzed for determination of albumin concentrations. Then the amount of 

Lactoferrin was corrected on amount of albumin concentrations (μg of lactoferrin / mg 

of albumin) because of different physiological dilution of real sample. Obtained resuts 

for 4 volunteers were: 78,2 μg/mg, 82 μg/mg, 100,1 μg/mg, 89,5 μg/mg. Verification of 

purity of from FPLC yielded fractions was performed by capillary chip 

electrophoresis. 


Saliva is a fluid secretion of human salivary glands, specifically parotid glands, 

submaxillary glands, sublingual gland and other small glands which are interspersed 

in oral cavity. Production of saliva is controlled by autonomic vegetative system based 

on conditioned and unconditioned reflexes. Daily production of saliva is 1,5 – 2 l per 

day in humans depending on type of ingested food and frequency of eating. Rapid 

decrease of salivary production is in sleep or in deficiency of liquids [1]. Saliva is 

composed of 99% water. Remaining components are mucin, electrolytes (Na, Ca, K, 

Mg, F), hormones, proteins – immunoglobulin A, lysozyme, lactoferrin, digestive 

enzymes which enable digestion of some compounds as a starch or fats [2]. The main 

functions of saliva are antimicrobial activity, ability to reduce acidity, disinfection and 

protection against formation of dental decay [3]. Lactoferrin (LF) is a glycoprotein 

consisted from 703 amino acid residues. Hololactoferrin is formed from one linear 

polypeptide chain forming two spherical domains (C- and N-terminal), each domain 

contains one iron binding site (see in the picture). Its molecular weight is of about 

327

XI. Workshhop 


E 

s ´11 

77000 D 

place, b 

Co3+ Da. Lactofe 

but with lo 

, CCu 

iron ho 

It is 

antiinfl 

tumour 

2+ , Zn2+ errin is abl le to bind different heavy h meta als into the 

ower affinit ty. For exaample 

it is able to bin nd Ga 

, aand 

trivale ent lanthannoids. 

The biological 

omeostasis rregulation. 

Iron captuuring 

occurs s primary in 

related too 

cell gro owth reguulation, 

cel ll differen 

lammatory activity [ 4], [5]. Baased 

on th hese functi 

r diseases annd 

metastas ses has beenn 

published d [6]. 

3+ e same bind 

, All 

function o 

n the intest 

ntiation an 

ions possib 

3+ , VO2+ ding 

, Mn M 

of lactoferri 

tine from f 

nd it has 

ble relation 

3+ , 

in is 

food. 

an 

n to 

2. EXPERIME 

The 

instrum 

deliveryy 

pumps, c 

Sloveniia), 

auto-inj 

collectoor. 

Volume 

(25mMM, 

pH = 7), B 

linear iincreasing 

g 

(100% B) -> 10.40 

was 1 mmLmin-1 

ENT 

ment for FPLC 

(Bio-Raad, 

USA) (F Fig. 1) was composed of two solv vent 

chromatogr raphic monnolithic 

colu umn (CIM SO3 disk, BBia 

separati ions, 

jection valv ve with 2 mml 

injection n loop, UV- -VIS detecttor 

and frac ction 

e of collecte ed fraction was 1ml. Mobile M pha ase consist of A: Tris-HCl 

B: NaCl in mobile m phaase 

A (2M). Lactoferrin n was eluteed 

by follow wing 

gradient: 0 – 1 min (1100% 

A) -> > 1 – 10 min 

(100% BB) 

-> 10 – 10.40 

0 – 13.40 min m (100% A) -> 13.4 40 – 13.80 min (100% % A). Flow rate 

. 

Fig. 3: : FPLC syst tem 

Testing 

grouup 

was 4 healthy 

subjjects 

22 – 28 2 years old. 

Saliva wwas 

collecte ed in 

the moorning 

1 hoour 

before eating. Sammples 

were collected using u Salivvette 

centrifuge 

vessel ( (Sarstedt, GGermany) 

by b chewingg 

swab for 2 minutes. After thatt, 

samples were w 

centrifuugated 

(30000 

rcf 5 min), m (Eppenndorf 

centr rifuge). The 

preparedd 

samples were w 

diluted 1:1 with wwater 

and filtered usiing 

micro filter f (micr roStar 0,45μμm 

CA, Co ostar 

Cambriidge). 

Apprroximate 

ac cquired voluume 

of sam mple was 1 ml. m Before purification n on 

FPLC ssamples 

weere 

analyzed 

for conteent 

of albu umin by spectrophotoometry 

analysis 

(BS 2000, 

Mindray, 

China). Prepared 

sammples 

were e fractionali ized on FPLLC. 

Conten nt of 

LF in ssaliva 

was iinvestigated 

d by spectrrophotometry 

(BS 200 0, Mindrayy, 

China) using u 

Pyrogalllol 

red reaagent. 

Used d wavelengtth 

was 605 5 nm. Obtained 

fractioons 

of LF were w 

analyzeed 

by capilllary 

chip electrophoreesis 

(Exper rion, Bio-Ra ad, USA). AAnalyses 

on n an 

328 

Brno


automated microfluidic Experion electrophoresis system (Bio-Rad, USA) were carried 

out according to the manufacturer’s instructions with supplied chemicals (Experion 

Pro260 analysis kit, Bio-Rad). Each sample was diluted with water to the same protein 

concentration of 300 μg.mL-1, 4 μl aliquots were then mixed with 2 μl of reducing 

sample buffer, and after 4 min of boiling, 84 μl of water was added. After priming of 

the chip with gel and gel-staining solution in the diluted priming station sample, the 

mixture (6 μl) was loaded into sample wells. The Pro260 Ladder included in the kit 

was used as a standard. 


There are many interfering compounds in saliva. Therefore isoelectric point 

of lactoferrin (IP~7.8) could be separated using ion exchange chromatography from 

other protein substances which have lower IP. Content of LF in human whole saliva 

was determined using purification by FPLC and subsequent quantitative analysis by 

spectrophotometer. At first, we optimized separation of LF on FPLC. Firstly we 

observed dependence of signal height on flow rate. We tested following flow rates: 1 

ml/min, 2 ml/min, 3 ml/min, 4 ml/min, 5 ml/min. According to results obtained (Fig. 

2A.) the best flow rate was 4 ml/min. We investigated influence of different 

concentration of NaCL as an eluting mobile phase. We tested: 0,5M, 0,75M, 1M, 1, 

25M, 1,5M and 2M solutions of NaCl (mobile phase B). With increasing concentration 

of NaCl there was increased separation resolution. The 2M NaCl was the most 

effective for elution of LF from chromatographic column (Fig 2B.). 

Fig. 4: Comparison of eluting conditions; (a): Dependence of 

signal height on flow rate (b): Dependence of signal height on salt 

concentration in eluent 

After optimizing of separation conditions, we fractionalized LF from saliva 

samples. After that we also analyzed some obtained fractions from saliva by capillary 

chip electrophoresis to ensure about the purity of the fraction. We analyzed LF alone 

(Fig.3A) and then LF fractionalized (Fig. 3B). The migration time was almost same 

nevertheless there were not any other peaks on the electroforeogram detected. 

329


Obtained fractions were analyzed by automatic spectrophotometer with Pyrogallol 

red method employed. To determination of the reliability of the whole approach we 

prepared two types of calibration curves. One was pure LF standard disolved in the 

mobile phase B (2M NaCl). Second we calibration was LF standard in the water and 

then we separated fractions by optimized FPLC method. These fractions were then 

used as second calibration curve. Both standard curves showed good linearity but they 

were slightly different. We assumed that it was thanks to little differed structure of LF 

which could be caused by ionic strength of the 2M NaCl. With regard to optimized 

process we definitely used this second calibration for determinination of LF 

concentration. Limit of detection was 15μg/ml. Other step which was necessary to be 

done was to determine level of albumin protein in the whole saliva and then 

recalculate the amount of the LF on Albumin because of correction of physiological 

dilution of real sample of saliva. Limit of detection for Albumin method was 60 μg/ml. 

Results were reached as μg of LF on mg of albumin in whole saliva. As shown in Tab. 

1, the content of LF in saliva was between 78,2 – 100,1 μg/mg of albumin. Other 

authors were found 10 – 25 μg/ml LF in whole saliva by spectrophotometric analysis 

[7], [8] 

Fig. 5: (a): Peak of standard LF (500μg/ml); (b): Peak of fractionalized saliva 

Tab. 1 Concentration of LF in whole saliva 

Sample of Concentration of LF Concentration of total concentration of LF on 

saliva 

(pyrogal red) proteins (albumin) total proteins 

(µg/ml) (µg/ml) (µg/mg) 

S 1 75,1 960,3 78,2 

S 2 74,0 902,4 82,0 

S_3 76,4 763,3 100,1 

S_4 67,4 752,4 89,5 

A. B. 

Fluorescence (mFU) 

120 

100 

80 

60 

40 

20 

77 kDa 

0 

25 

‐20 

30 35 40 45 50 

Migration time (s) 

330 

Fluorescence (mFU) 

35 

30 

25 

20 

15 

10 

5 

0 

35 37 39 41 

‐5 

Migration time (s)


4. CONCLUSION 

Lactoferrin is related to antimicrobial activity, cell grow regulation and 

differentiation. It could also play a role as marker for various diseases associated with 

immune system. In this study we optimized purification of LF by FPLC and 

subsequent analysis by spectrophotometer with possible clarification of purity of 

obtained fractions. The most effective conditions were flow rate 4 ml/min and elution 

by 2M NaCl. To determine of level of LF in whole saliva the spectrophotometric 

analysis was used. The concentrations of LF of healthy humans were 78,2 μg/mg 

albumin, 82 μg/mg albumin, 100,1 μg/mg albumin, 89,5 μg/mg albumin. 


The work has been supported by NANOSEMED GA AV KAN208130801, 

NANIMEL GA ČR 102/08/1546 and NanoBioTECell GA ČR P102/11/1068. 

6. REFERENCES 

[1] I.D. Mandel, Journal of Dental Research 66 (1987) 623. 

[2] N.N. Rehak, S.A. Cecco, G. Csako, Clinical Chemistry and Laboratory Medicine 38 (2000) 335. 

[3] W.M. Edgar, British Dental Journal 172 (1992) 305. 

[4] D. Legrand, E. Elass, M. Carpentier, J. Mazurier, Cellular and Molecular Life Sciences 62 (2005) 

2549. 

[5] Y. Pan, A. Lee, J. Wan, M.J. Coventry, W.P. Michalski, B. Shiell, H. Roginski, International 

Dairy Journal 16 (2006) 1252. 

[6] P.P. Ward, E. Paz, O.M. Conneely, Cellular and Molecular Life Sciences 62 (2005) 2540. 

[7] K. Komine, T. Kuroishi, A. Ozawa, Y. Komine, T. Minami, H. Shimauchi, S. Sugawara, 

Molecular Immunology 44 (2007) 1498. 

[8] J. Tenovuo, O.P.J. Lehtonen, A.S. Aaltonen, P. Vilja, P. Tuohimaa, Infection and Immunity 51 

(1986) 49. 

331


EMPLOYMENT OF HPLC WITH 

COULOMETRIC DETECTION FOR 

ANALYSIS OF PHYTOCHELATIN 

SYNTHASE ACTIVITY 

Ondřej ZÍTKA 1 , Olga KRYŠTOFOVÁ 1 , Pavlína ŠOBROVÁ 1 , Josef ZEHNÁLEK 1 , 

Miroslava BEKLOVÁ 1 , Vojtěch ADAM 1 , René KIZEK 1 


Zemedelska1, CZ-613 00 Brno, Czech Republic 

Abstract 

Phytochlatins are physiologically active peptides which have to be synthesized by 

phytochelatin synthase (PCS). The main aim of this study was to optimize high 

performance liquid chromatography coupled with electrochemical detector (HPLC- 

ED) as suitable tool for determination of the phytochelatin synthase activity. 

Coulometric detector with porous graphite working electrode was used. Firstly we 

optimized a separation of two compounds which are important for monitoring of PCS 

activity. It was glutathione reduced as substrate for the reaction and fytochelatin-2 as 

a product. PCS is the best activated by cadmium ions. We used a model with BY-2 

tobacco cells. We conducted the in vivo 3 day cultivation experiment where BY-2 

cells were treated by various concentrations of cadmium. We then homogenized the 

cells and immediately analyzed the extracts by optimized HPLC-ED method. 

Moreover we observed that with higher concentrations of applied cadmium there was 

increasing of amount of PC-2. The lowest concentration of the toxic metal ions caused 

almost three times enhancing PCS activity compared to control samples. 


Heavy metals are occurring in the environment partially due to increasing 

anthropogenic activities. The heavy metals are toxic for both plants and animals [1-3]. 

But plants has an advantage against animals to be more resistant to them because are 

able to synthesize plant stress peptides as phytochelatins (PC; a basic formula (γ-Glu- 

Cys)n-Gly (n = 2 to 11)) [4-7]. Phytochelatins can bind heavy metal ions via –SH 

groups of cysteine units and consequently transport them to vacuole [5-9], thereby 

toxicity of the metal is decreased. Biosynthesis of Phytochelatins is catalyzed by γ- 

Glu-Cys dipeptidyl transpeptidase (EC 2.3.2.15), which has been named as 

phytochelatin synthase (PCS) [10-11]. In the most preferred reactions of PCS it takes 

two molecules of reduced glutathion (γ-Glu-Cys-Gly) as an substrate and produces of 

one molecule of PC-2 and one molecule of Glycine. The same mechanism is applied in 

case of biosynthesis of PC-3,4 or 5 but every time a GSH is donor and PC is an 

acceptor of γ-Glu-Cys dipeptide Fig. 1. We decided to optimize an HPLC method with 

coulometric detection for analysis of GSH and PC-2 simultaneously in Cell BY-2 

332


Tobacco extract. Electrochemical techniques as differential pulse and cyclic 

voltammetry are suitable methods for detection of thiols [12-17]. The electrochemical 

methods are sensitive but for our purposes it need to be connected to separation 

method such as high performance liquid chromatography. The coulometric detector is 

one of the most suitable because of its sensitivity, low noise background and 

possibility in baseline correction application which is needed if gradient elution is 

applied. Moreover the higher area of the working electrode which is made from 

porous graphite is capable to oxidise or reduce more then 90% of the analyzed 

substance. And this is more than classic graphite planar electrodes in flow 

arrangement [18]. 

2. EXPERIMENT 

Reduced (GSH) and oxidized (GSSG) glutathione, and trifluoroacetic acid (TFA) 

were purchased from Sigma-Aldrich (St. Louis, USA). Phytochelatin2 (PC2) (γ-Glu- 

Cys)2-Gly was synthesized in Clonestar Biotech (Brno, Czech Republic) with a purity 

above 90 %. HPLC-grade methanol (>99.9%; v/v) was from Merck (Dortmund, 

Germany) were used. Other chemicals were purchased from Sigma-Aldrich (St. Louis, 

USA) unless noted otherwise. Stock standard solutions of the thiols (1 mg.ml -1 ) were 

prepared with ACS water (Sigma-Aldrich, USA) and stored in dark at -20 °C. Working 

standard solutions were prepared daily by dilution of the stock solutions. All solutions 

were filtered through 0.45 μm Nylon filter discs (Millipore, Billerica, Mass., USA) 

prior to HPLC analysis. The pH value was measured using WTW inoLab Level 3 with 

terminal Level 3 (Weilheim, Germany), controlled by software MultiLab Pilot; 

Weilheim, Germany. The pH-electrode (SenTix H, pH 0..14/0..100°C/3mol.l -1 KCl) 

was regularly calibrated by set of WTW buffers (Weilheim, Germany). HPLC-ED 

system consisted of two solvent delivery pumps operating in the range of 0.001-9.999 

ml.min -1 (Model 582 ESA Inc., Chelmsford, MA), Zorbax eclipse AAA C18 (150 × 4.6; 

3,5 μm particles, Agilent Technologies, USA) and a CoulArray electrochemical 

detector (Model 5600A, ESA, USA). The electrochemical detector includes one flow 

cells (Model 6210, ESA, USA). The cell consists of four analytical cells containing 

working carbon porous electrode, two auxiliary and two reference electrodes. The 

sample (20 μl) was injected using autosampler (Model 542, ESA, USA). Other 

experimental parameters were optimized. 


Firstly we optimized the chromatographic method for separation of glutathiones 

reduced (GSH) and oxidized (GSSG) and PC-2. We were able to observe clearly 

separated peaks of all compounds of interest. The PC-2 has a retention time about 10,7 

minutes. Than BY-2 Tobacco cells in liquid medium were treated by Cd(NO3)2 in 

various concentrations 0, 5, 10, 25, 50 and 100 μM. Cells were harvested after 3 day of 

cultivation and in same time centrifuged and then immediately homogenized in liquid 

nitrogen and phosphate buffer with 1mM TCEP added. After centrifugation we 

obtained supernatant which was initial solution for further tests of activity. We took 

100 μl of the supernatant from cell extract and added a various concentrations of 

333


reduced glutathione (0, 0.05, 0.1, 0.25, 0.5, 1, 2 and 5 mM) as a substrate for the PCS 

reaction. 

GSH……..…. 

PC‐2…. 

γ‐glu‐cys‐gly 

PC‐3…. 

γ‐glu‐cys‐gly 

γ‐glu‐cys‐gly + γ‐glu‐cys‐gly 

(γ‐glu‐cys) n=2 ‐gly + gly 

(γ‐glu‐cys) n=3 ‐gly + 2gly 

PC‐4….…………. 

PC‐5….…………. 

n = 4 

n = 5 

Fig.1 Scheme of Fytochelatin synthase functions. 

Than cadmium(II) ions (50 μM Cd(NO3)2) were added for initializing of PCS 

activity. We optimized that mixtures should be incubated at 35 °C for 30 min for 

obtaining the highest yield of PC-2. Using HPLC-ED, PC2 was determined. The signal 

of PC-2 were increasing with increase of applied concentration of GSH. The highest 

activity of PCS as 278 fkat was determined in cells treated with 100 Cd(II) ions. 

4. CONCLUSION 

We optimized HPLC-ED method for detection of PC-2 in femtomole 

concentrations and we used this method for analysis of the series of cells extracts 

treated by different concentrations of cadmium. With partially optimized method we 

processed the cells extracts and we analyzed the PCS activity. The cells treated with 

100 μM Cd(II) ions had more than seven times active PCS compared to control ones. 

These results are in well agreement with those published by Nakazawa et al. [19] and 

334 

PHYTOCHELATIN 

SYNTHASE 

EC 2.3.2.15


Ogawa et al. [20]. We proved that HPLC-ED method like this we developed can be 

useful for these kinds of biochemical purposes. 


The work has been supported by REMEDTECH GACR 522/07/0692, GACR 

204/09/H002 and IGA MENDELU 2/2011. 

6. REFERENCES 

[1] C.S. Cobbett, Plant Physiol. 123 (2000) 825. 

[2] J.L. Hall, J. Exp. Bot. 53 (2002) 1. 

[3] M.H. Zenk, Gene 179 (1996) 21. 

[4] W. Bae, W. Chen, A. Mulchandani, R.K. Mehra, Biotechnology and Bioengineering 70 (2000) 

518. 

[5] C.S. Cobbett, Curr. Opin. Plant Biol. 3 (2000) 211. 

[6] M.H. Zenk, Gene 179 (1996) 21. 

[7] L.S. di Toppi, R. Gabbrielli, Environmental and Experimental Botany 41 (1999) 105. 

[8] C.S. Cobbett, P.B. Goldsbrough, Annu. Rev. Plant. Biol. 53 (2002) 159. 

[9] E. Grill, E.-L. Winnacker, M.H. Zenk, Science 320 (1985) 674. 

[10] C.S. Cobbett, Trends Plant Sci. 4 (1999) 335. 

[11] O.K. Vatamaniuk, E.A. Bucher, J.T. Ward, P.A. Rea, J. Biol. Chem. 276 (2001) 20817. 

[12] R. Kizek, J. Vacek, L. Trnkova, F. Jelen, Bioelectrochemistry 63 (2004) 19. 

[13] J. Vacek, J. Petrek, R. Kizek, L. Havel, B. Klejdus, L. Trnková, F. Jelen, Bioelectrochemistry 63 

(2004) 347. 

[14] B. Yosypchuk, I. Sestakova, L. Novotny, Talanta 59 (2003) 1253. 

[15] N.S. Lawrence, J. Davis, L. Jiang, T.G.J. Jones, Analyst 125 (2000) 661. 

[16] I. Sestakova, P. Mader, Cell. Mol. Biol. 46 (2000) 257. 

[17] J. Vitecek, J. Petrlova, J. Petrek, V. Adam, D. Potesil, L. Havel, R. Mikelova, L. Trnkova, R. 

Kizek, Electrochim. Acta 51 (2006) 5087. 

[18] C.N. Svendsen, Analyst 118 (1993) 123. 

[19] R. Nakazawa, H. Kato, Y. Kemeda, H. Takenaga, Biol. Plantarum 45 (2002) 311. 

[20] S. Ogawa, T. Yoshidomi, T. Shirabe, E. Yoshimura, Journal of Inorganic Biochemistry 104 (2010) 

442. 

335


FAST AND ROBUST METHOD FOR 

DETECTION COPPER AND CADMIUM 

IONS BY FLOW INJECTION METHOD 

WITH AMPEROMTRIC DETECTION 

Ondřej ZÍTKA 1 , Miguel-Ángel MERLOS 2 , Nuria FERROL 2 , Vojtěch ADAM 1 , René 

KIZEK 1 



2 Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, 

CSIC, Profesor Albareda 1, Granada 18008, Spain 

Abstract 

The problem of heavy metal contamination is obvious in the less developed countries. 

Besides this problem is often connected to mining which has a devastating impact on 

the landscape, agriculture and whole environment. The contamination of water and 

farmland has a direct influence on local inhabitants. We developed a method which is 

suitable, fast and robust for determination of metals as cadmium and copper in the 

water. Cadmium is one of most environmental occurring toxic heavy metal but copper 

is intensively mined. Copper even it is essential element it can be in some case 

generating of ROS which are dangerous for organism. Our method based on 

amperometric detection using glassy carbon planar electrode as working is due to 

connection with flow injection analysis (FIA) method robust and fast enough for this 

purposes. We optimized a parameters like flow rate, pH of the working buffer of most 

effective potential from hydrodynamic voltammograms which has been constructed. 


The toxic metals contamination in some less developed countries is world-wide 

problem which has only partial scientific of public importance. The hardest impact 

has contamination in the pour countries such like in Africa or South America where 

the ore and precious metal mining is in motion. And even if the mining stopped many 

years after that there is still danger of toxic metals which is mostly occurring in the 

water and soil. Soil is then used for growing of the plants for local inhabitants and this 

causes the most risk which is hard predictable. But extraordinary dangerous still be 

the water which is used in these localities. The monitoring of the contamination level 

of the water is most important step to have an initial solution of these kinds of 

problems. Problem of metal contamination is not black and white because there are 

many aspects as what metal and in which concentration is occurring. In case there are 

many of metal there could be very completive dangerous for the human organism. 

Physiologicaly we can take cobalt, iron, manganese, molybdenum, nickel, selenium, 

vanadium, calcium, tungsten, zinc and copper like essential metals. These are very 

336


important by its occurring in proteins. Functionality up to 70% of all described 

proteins is dependent on the metals which are bond in their structures [1-3]. The most 

toxic metals for human are arsenic, lead, mercury and cadmium [4-5]. Toxicity of the 

metals is widely studied but it is important to say that even essential metals could be 

toxic if are occurring in organism in high concentrations. There are many ways of 

toxic affection for various metals. Arsenic, chromium and platinum are cancerotoxic. 

Gold, cobalt, chromic, nickel and platinum are immunotoxic. Mercury has 

terratogenic and embryotoxic affecting. Cadmium, lead and thalium are spermiotoxic. 

Cadmium and uranium are nefrotoxic and copper, iron, selen and zinc are neurotoxic 

[6]. There are many reasons why we should study all of these metals because their 

biological danger. In this study we oriented to cadmium which is one of the most 

environmental occurring toxic metal nex to lead and mercury and copper which is 

essential but in some cases it can play some negative roles. If the copper ions are free 

occurring in the organism it can induce a number of reactive oxygen species (ROS) 

which are danger for the organism [7]. We used an electrochemical amperometric 

detector with glassy carbon planar electrode. We have chosen a flow injection 

analysis arrangement. The methods like a flow injection analysis or sequential 

injection analysis were founded for easy to use method for mixing of any reagents and 

immediate analysis by simple detectors as UV-VIS or than more difficult like ICP-MS. 

The next generation of this method is sequential injection analysis SIA which 

complementary to Lab on Valve area which is analogous to Lab on Chip area. 

2. EXPERIMENT 

HPLC-grade methanol (>99.9%; v/v) was from Merck (Dortmund, Germany) 

were used. Other chemicals were purchased from Sigma-Aldrich (St. Louis, USA) 

unless noted otherwise. Stock standard solutions of the thiols (1 mg.ml -1 ) were 

prepared with ACS water (Sigma-Aldrich, USA) and stored in dark at -20 °C. Working 

standard solutions were prepared daily by dilution of the stock solutions. All solutions 

were filtered through 0.45 μm Nylon filter discs (Millipore, Billerica, Mass., USA) 

prior to HPLC analysis. The pH value was measured using WTW inoLab Level 3 with 

terminal Level 3 (Weilheim, Germany), controlled by software MultiLab Pilot; 

Weilheim, Germany. The pH-electrode (SenTix H, pH 0..14/0..100°C/3mol.l -1 KCl) 

was regularly calibrated by set of WTW buffers (Weilheim, Germany). The 

instrument for flow injection analysis with electrochemical detection (FIA-ED) 

consisted of solvent delivery pump operating in range of 0.001-9.999 ml.min-1 (Model 

582 ESA Inc., Chelmsford, MA, USA), a reaction coil (1 m) and an electrochemical 

detector. The electrochemical detector includes one low volume flow-through 

analytical cell (Model 5040, ESA, USA), which is consisted of glassy carbon working 

electrode, hydrogen-palladium electrode as reference electrode and auxiliary 

electrode, and Coulochem III as a control module. The sample (5 μl) was injected 

manually using 6-way injection valve. The data obtained were treated by Clarity 

software (Version 3.0.04.444, Data Apex, Czech Republic). The measuring 

experiments were carried out at room temperature. A glassy carbon electrode was 

polished mechanically by 0.1 μm of alumina (ESA Inc., USA) and sonicated at room 

337

XI. Workshhop 


E 

s ´11 

temperrature 

for 5 min usin ng a Sonorrex 

Digital l 10 P Son nicator (Ban andelin, Berlin, 

Germanny) 

at 40 WW. 

Other ex xperimentall 

parameter rs were opti imized. 

3. RRESULTS 


AAim 

of this work was s to optimiize 

fast and d high thro oughput mmethod 

for easy 

determmination 

of f these tw wo metals in the wa ater. We used u an eelectrochem 

mical 

amperoometric 

dettector 

with h glassy carrbon 

planar r electrode. 

We have chosen a flow f 

injectioon 

analysiss 

arrangem ment for hhigh 

throu ughput ana alsis. We cconnected 

the 

electrocchemical 

ddetector 

to automated a autosample er wich con ntained a 66-way 

injec ction 

valve aand 

1m lonng 

reaction n coul betwween 

valve and detect tor. Thankks 

this fact one 

analysiss 

of the saample 

took k about 15 second wh hen the fastest 

injecttion 

mode was 

appliedd. 

We optimmized 

the parameters p like influen nce of flow w rate on thhe 

peak hei ight, 

influennce 

of pH oon 

the peak k height annd 

we analyzed 

all of o these parrameters 

like 

a 

compleex 

hydroodynamic 

voltammmogram 

(HDV) ( records. 

HHydrodyna 

amic 

voltammmograms 

wwere 

built from numbber 

of injections 

and each injecction 

was done d 

under cconcrete 

coontrolled 

po otential. WWe 

measured d HDV´s in n different sscales 

like from f 

0mV too 

1000mV. The upper r range is deeterminate 

by durabil lity and staability 

of gl lassy 

carbon electrodee. 

After selecting the best conditions 

for meeasurement 

t of 

hydroddynamic 

volltammograms 

which wwas 

flow ra ate 1 ml/ml and injectiion 

about 20uL 2 

we anaalyzed 

the different pH p of workking 

buffer r Britton-Robinson. 

TThis 

buffer was 

very usseful 

for widde 

pH rang ging from 2 to 8. 

Fig.1 (AA) 

Hydrodyynamic 

vol ltammogramms 

for various 

concen ntration of copper(II) ions 

(100,200 annd 

400 μg/m ml). (B) Eleectrochemic 

cal cell ESA A 5040 withh 

glassy car rbon 

electrode. 

338 

Brno


4. CONCLUSION 

The most simplification of the analysis approach is very important in 

development of the method which should be complementary to be applied in extreme 

conditions. The FIA method is fast and robust for these purposes. If we combine the 

automatic injection system which can by easy replaced by manual valve with an 

electrochemical detector we obtain fast and robust instrument. 


The work has been supported by REMEDTECH GA ČR (522/07/0692) 

INCHEMBIOL MSM0021622412 and IGA MENDELU 2/2011.This work is dedicated 

to UNEP Lead and Cadmium activities. 

6. REFERENCES 

[1] W. Shi, M.R. Chance, Cellular and Molecular Life Sciences 65 (2008) 3040. 

[2] J.A. Tainer, V.A. Roberts, E.D. Getzoff, Current Opinion in Biotechnology 2 (1991) 582. 

[3] K. Degtyarenko, Bioinformatics 16 (2000) 851. 

[4] N.N. Greenwood, Earnshaw, Chemie prvku I., II., Informatorium, Praha, 1993. 

[5] D. Voet, J.G. Voet, Biochemie, Victoria Publishing, Praha, 1995. 

[6] J. Szpunar, Analytical and Bioanalytical Chemistry 378 (2004) 54. 

[7] W. Droge, Physiological Reviews 82 (2002) 47. 

339


IMPLEMENTATION OF WATERRING 

MEASUREMENT 

TO ENVIRONMENT MONITOR SYSTEM 

Jaromír ŽÁK 1 , Jaromír HUBÁLEK 1 , René KIZEK 2 

1 Department of microelectronics, Brno University of Technology, Brno, Czech Republic 

2 Department of chemistry and biochemistry, Mendel University, Brno, Czech Republic 

Abstract 

Environment monitoring is these days one of the most important areas of 

environmental measurements. In this work new modules for various values 

measurements are being developed (temperature, conductivity and pH of waterring). 

These modules are constructed for being connected to Environment Monitor – 

universal measuring device which has been developed in our laboratories. In 

connection to the later developed device for basic meteorological values 

measurements, the new complex device becomes a strong tool for ecology applications 

too. 


New modules that measure humidity, acidity, conductivity, and temperature of 

precipitation are being developed for interconnection to the actual device called 

Environment Monitor using the previously developed communication protocols and 

standard links. General description of the device is in the [1]. Communication 

protocols were developed for simple and efficient data transferring between main 

device and one of the sensor modules. We can connect up to 16 various sensor 

modules with internal calibration and automatic type of module detection. 

Communication can be realized by one wire (and ground) or by wireless connection 

via IQRF [2] modules. Communication has implemented Master Slave serial protocol 

with 9bits word length and three types of packets (Command, Data and 

Acknowledgement packets are used). 

2. EXPERIMENT 

There is a design of multipurpose sensor made first, with integrated three sensor 

types. Sensors are designed with respect to thick film technology, there are five 

sensors on standard ceramic substrate (a square substrate with side length 2”). We 

have created a small thick-film sensor for impedance, temperature and pH 

measurements (see Figure 1A). The sensor topology has small dimensions: 15 mm × 10 

mm for sensing area. There are six layers of materials used on the sensor. The first 

layer is conductive layer (grey colour) realized by Ag/Pd paste. Next four layers are 

function layers. Meander for temperature sensing (on the left side) is realized by PTC 

thermo resistive paste. Electrodes for conductivity measurement are made by Au 

paste. Dimensions and number of electrodes are selected with relation to expected 

340

XI. WWorkshop 

of Phyysical 


´11 

preccipitation 

cconductivit 

ty, which wwill 

be rela atively high h (water iss 

contamin nated by 

ferttilizers; 

connductivity 

is s similar to drinking water). w 

pH sensoor 

is compo osed of twoo 

electrodes s. MnO2 [4] 

or IrOx+TTiO2 

[5] is used u for 

senssing 

electroode 

(greater r circle elecctrode 

on the t right sid de in the mmiddle, 

red colour). 

Thee 

powders wwere 

synth hesized, mixxed 

with 3w wt% of glas ss frit and hhomogeniz 

zed with 

terppineol 

based 

vehicle to t obtain thhixotropic 

paste print ted circle aand 

fired at t 520°C. 

Ag/ /AgCl pastee 

is used fo or referencce 

pH elec ctrode (rect tangle electtrode 

in th he right 

botttom 

cornerr, 

light blue e colour). 

The last layer is a standard dielectric layer l with low absorrption 

of moisture m 

(greeen 

colour) ). This laye er covers noon 

sensing parts of th he sensor, sseparates 

analyzed a 

elecctrolyte 

frrom 

critic cal parts of sensor r and im mproves ellectrical 

isolating i 

characteristicss. 

Sensor will 

be connnected 

to th he electron nic circuits by 1.27 mm 

pitch 

connnector. 

Thiis 

connecto or must be iisolated 

and d waterproo of too. 

Fig.1 Me easuring sennsor 

and electronic 

module 

desiggn 

Input mmodules 

were 

designeed 

for auto onomic ser rvice withoout 

externa al cable 

connnections. 

PPower 

supp ply can be realized by y standard slim 3.3 V batteries for one 

monnth 

measurement, 

bu ut it can bbe 

connect ted to exte ernal poweer 

supplies s too, if 

prefferable. 

Daata 

connection 

to thhe 

main module 

is re ealized wirreless 

using 

IQRF 

moddules 

[2], bbut 

they can c be connnected 

dir rectly by wire w conneection 

as standard s 

(earrlier 

develooped) 

modu ules. Measuurement 

of conductivi ity (or imppedance, 

if needed) 

is reealized 

on one fixed frequency generated by genera ator in powwer 

supply circuits 

(seee 

Figure 1B) ). Temperature 

is meaasured 

by si imple resist tance to volltage 

conve erter. 

Output oof 

the pH sensor is vvoltage 

with h negative offset (aboout 

250 mV V). This 

offsset 

is comppensated 

by y adding thhis 

voltage e and the signal s is ammplified. 

All A three 

signnals 

are coonverted 

to o digital siggnals 

in microcontro 

m oller and thhey 

are pr rocessed 

digiitally. 

341 

Brno

XI. Workshhop 


E 

s ´11 

3. 3.RESULTSS 

AND DISCUSSIO 

D ON 

Fiifty 

waferss 

with prot totypes of sensors were w created d first (1255 

sensors with w 

MnO2 [ [4] and 1255 

sensors with w IrOx+TTiO2 

[5] ion n selective layer l of pHH 

sensor). After A 

that, baasic 

characcteristics 

of f sensors wwere 

measu ured. First measuremment 

was made m 

with thhe 

temperatture 

sensor r. Measuredd 

resistance of created sensor wass 

about 300 00 Ω 

and aveerage 

tempperature 

coe efficient wwas 

13 Ω/K. Temperatu ure dependdency 

was very v 

linear. The secondd, 

pH senso or didn’t haave 

strictly linear characteristics 

s (see Figure 

2). 

Each of 

measuredd 

sensors had h a differrent 

output t voltage to o pH. The most diffe erent 

value wwas 

offset vvoltage. 

The e offset volltage 

was varied v betw ween 150 mmV 

and 300 mV 

(measured 

for miinimal 

volt tage with pH 14). This T offset will be coompensated 

d by 

hardwaare. 

The nnext 

comp pensation step will be realize ed in thee 

firmware e of 

microcoontroller. 

OOpposite 

to t temperaature 

senso or, calibrati ion of pH sensor is not 

linear, but it is rrealized 

by polynomiaal 

equation n. MnO2 la ayer has smmaller 

abra asion 

resistannce 

and layeer 

cohesion n was unstaable. 

RReal 

cell connstant 

for sensor 

of coonductivity 

y was determ 

with thhe 

standard calibration n conductivve 

solution (1413 μS/c 

μS/cm). . We obtainned 

average e cell consttant 

KREAL = 299.6 m 

= 0.9933. 

-1 mined fromm 

measurem ment 

m, 5000 μSS/cm 

and 12 2880 

, the correcction 

factor r is α 

Fig.2 3-point 

calibrration 

of pH H measurem ment 


There 

are soome 

device es which wwere 

design ned for thi is project. It is the main m 

device and its sofftware 

for collecting c iinformation 

n from slav ve measuremment 

modu ules. 

There aare 

two typpes 

of meas surement mmodules. 

There 

is the e measuremment 

modul le of 

temperrature, 

hummidity, 

atmo ospheric preessure, 

illumination 

and 

CO2 gass 

concentra ation 

(develooped 

in the first part of o this projeect). 

The other o measu urement moodule 

has been b 

developped 

and it allows us s to measuure 

values of pollutio on, temperrature, 

pH and 

conducctivity. 

WWe 

have created sm mall thick film sens sors for measuring m nneeded 

va alues 

altogethher 

as welll. 

Measurem ment for teesting 

and describing d newly n creaated 

sensors s for 

pollutioon 

was madde 

after com mpleting thhe 

prototyp pe of sensor rs. The nexxt 

work on this 

project will be to iimplement 

new technniques 

to th he existing device. d 

342 

Brno



This work has been supported by Grant Agency of the Czech Republic under 

the contract GACR 102/08/1546 (NANIMEL) and by the Czech Ministry of Education 

in the frame of Research Plan MSM 0021630503 (MIKROSYN). 

6. REFERENCES 

[1] Zak, J.: "Pristroj pro monitorovani prostredi pri kultivaci rostlin – Bakalarska prace", Brno, 2008, 

65p. 

[2] MICRORISC, IQRF solution specification, Jicin, 2011, Available from the web: 

. 

[3] Hubalek, J., Adamek, M.: "Mikrosenzory a mikroelektromechanicke systemy", Brno, 1999, 122p. 

[4] Qingwen, L., Yiming, W., Guoan, L.: "pH Response of nanosized MnO2 prepared with solid state 

reaction route at room temperature", Sensors and Actuators B 59, 1999, page 42 – 47, China 

[5] G. M. da Silva, S. G. Lemos, L. A. Pocrifka, P. D. Marreto, A. V. Rosario, E. C. Pereira: 

"Development of low cost metal oxide pH electrodes based on the polymeric precursor method", 

Analytica Chimica Acta 616, 2008, page 36 – 41, Available from the web: 

 

343


TOXIC METALS IN BRNO URBAN SOILS 

AND THE PLANTS OF COMMON 

DANDELION (TARAXACUM OFFICINALE) 

Andrea KLECKEROVÁ 1 , Michaela ŠEBKOVÁ 2 , Hana DOČEKALOVÁ 1 

1 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Brno, 


2 Institute of Chemistry and Technology of Environmental Protection, Faculty of Chemistry, Brno 

University of Technology, Brno, Czech Republic 

Abstract 

To examine the metal content of dandelion leaves and roots in relation to 

environmental metal levels, the concentrations of cadmium, mercury and lead were 

analyzed in plant part and soil samples collected at five sites in the Brno city 

differentially impacted by pollution. The sampling place Opuštěná represented the 

heavily polluted locality with heavy traffic density situated in the city centre. 

Sampling places Vídeňská and Podstránská belonged to medium polluted localities 

that were situated close to frequented roads. Relatively clean localities were 

represented by Musorgského and Šrámkova Street, which were situated in peripheral 

city district with lower traffic density. Soils and plants were collected twice in 

November 2008 and March 2009. In the studied soil samples average amounts of 

cadmium, mercury and lead did not exceed the limits based on the Regulation No. 

13/1994 of the Ministry of Environment Czech Republic. The highest value of metals content 

was found in the soil sampled at Opuštěná site in accordance with contamination 

loading. The content of lead and mercury in leaves of common dandelion was higher 

than the content in roots that indicated preferred atmospheric pollution. On the other 

hand the higher cadmium content was measured in underground part of the plant 

that indicated soil contamination. The content of toxic metals in soils and common 

dandelion plants corresponded well with the contamination load of the sampling 

place. 


The accumulation of high levels of toxic metals in urban soils is caused by many 

antropogenic activities. These toxic metals can present an environmental risk, 

therefore searching to find reliable, low cost methods of assessing the extent of metal 

contamination at a locality and the exposure risk to the biota is high desirable. Toxic 

metal deposition in plants from antropogenic sources has increased the attention on 

inorganic pollution and established plants as passive biological monitors. A good 

biological monitor should be a species that is represented by large numbers of 

individuals over a wide geographic area, has a broad toxitolerance, and accumulates 

metals at levels reflecting those in the environment so that their chemical 

composition will provide a quantitative measure of the magnitude of contamination 

344


[1-3]. The widespread distribution of the common dandelion (Taraxacum officinale) 

make this ‘species’ an attractive candidate for biological monitoring at the global level 

[4]. A number of studies have begun to investigate the suitability of dandelions for 

monitoring metal pollution at sites in Europe and Canada [5] Common dandelion is 

characterized by a high relative accumulation factor of some contaminants. It uses 

include evaluating environmental pollution with SO2, polycyclic aromatic 

hydrocarbons and heavy metals [6]. 

The aim of this work was to investigate the suitability of the common dandelion 

for monitoring environmental metal pollution by analyzing the metal content of 

dandelion leaves and roots from plants growing at sites in Brno city with a range of 

pollution contamination. While previous studies indicated that dandelions could 

accumulate metals from the atmosphere as well as from the soil [7] the contents of 

studied metals (cadmium, mercury and lead) in the soil were measured 

simultaneously. 

2. EXPERIMENT 

Brno is the second largest city of Czech Republic with wide range of industrial 

activities including smelting operations and automotive exhaust. The soil and plant 

samples were collected from the topsoil of five sampling sites of the Brno city twice at 

November 2008 and March 2009. Location of sampling sites is shown in Figure No 1. 

The sampling site Opuštěná represents the heavily polluted locality with high traffic 

density situated in the city centre. Sampling sites Vídeňská and Podstránská belong to 

medium polluted localities that are situated close to frequented roads. Relatively clean 

localities are represented by Musorgského and Šrámkova Street, which are situated in 

peripheral city district with smaller traffic density. The soils were sampled from a 

depth horizon of 0-10 cm, ten samples at every sampling place, represented an area of 

3x3m. Ten plants of common dandelion grown on the sampling place were extracted 

from the soil, washed with distilled water and air-dried. For characterization of soils, 

a fine soil fraction of particle size below 2 mm was obtained by sieving the air-dried 

raw sample, from which large components were separated (stones, plant parts). Single 

leaching procedure with 2 mol.l -1 HNO3 was used for cadmium and lead determination 

in soils samples. 

345

XI. Workshhop 


E 

s ´11 

Fig. 1 LLocation 

off 

sampling sites: A – OOpuštěná, 

B – Vídeňs ská, C – Poodstránská, 

D – 

Musorgského, 

E – Šrámkova 

Portion 

of 7 g dried and d sieved soil was shake 

temperrature 

(25ºCC) 

with 70 ml of 2 mmol.l 

immediiately 

filterred 

and the e filtrate w 

homogeenized 

plannt 

samples were w minera 

1200, Ittaly) 

using 6 ml of conc centrated HN 

Mercury in ddried 

and sie eved soil an 

absorption 

spectrommetry 

using g the AMA 

concenntrations 

wwere 

determ mined in b 

Electroothermal 

attomic-abso 

orption spe 

Germanny) 

was useed 

under th he recomme 

-1 en in an ext traction boottle 

at amb bient 

nitric c acid for 16 

hours. TThe 

extract was 

was collected 

in polyet thylene botttle. 

Dried and 

alized in th he microwa ave oven (MMilestone, 

MLS M 

NO3 and 1 ml l of 30% H2O O2. 

nd in dried plant p samples 

was deterrmined 

by at tomic 

A 254 (Altec, 

s.r.o., ČR). Č Cadmiium 

and lead 

both the HNO3 H leac chates and plant dig gests. 

ectrometer (AAS ZEEnit 

60, AAnalytik 

Jena, J 

ended cond ditions specified 

by thee 

producer. . 

3. RRESULTS 


Thhe 

highest mmercury 

co ontent in soiil 

was found 

Novemmber 

2008 aand 

March 2009. The average co 

0.607 ± 0.101mg.kg-1 

. At the sam mpling site PPodstránská 

w 

of merccury 

in the soil, 0.212 ± 0.022 mgg.kg 

collecteed 

from othher 

three sampling 

sit 

dandelioon 

roots corrresponded 

with w the merc 

green pplant 

partss 

was high her than i 

atmosphheric 

deposittion 

of mercu ury species. 

-1 d at the sam mpling site OOpuštěná 

both 

at 

ncentration of mercury wwas 

at this site 

was recorded 

the secondd 

highest content 

. The concentra ations of mmercury 

in soil 

tes were much m lower. 

Contents oof 

mercury in n the 

cury content ts in soil. Th he content of mercur ry in 

in roots at t all samp pling sites. That indic cates 

The low contamination 

of soil as weell 

as of plan nt by 

346 

Brno


mercury, comparable with Brno clean areas, was found at Vídeňská Street. Obtained results are 

summarized at Fig. 2. 

mHg (g.kg -1 DW) 

650 

600 

550 

500 

450 

400 

350 

300 

250 

200 

150 

100 

50 

0 

607 

66 

84 

58 

9 

37 

212 

43 

74 

Fig. 2. Average content of mercury in soil, roots and leaves of dandelion. 

The highest content of lead in soil was found at the sampling site Opuštěná. The 

average amount of lead in soil was 51.6 ± 9.3 mg.kg-1. Approximately half the levels 

of lead in soil were detected at sampling locations Vídeňská, Podstránská and 

Musorgského. The lowest lead concentration, 9 ± 1.1 mg.kg-1, was measured in soil 

samples collected at sampling site Šrámkova. The contents of lead in the dandelion 

roots fluctuated around 2 mg.kg-1 at all sampling sites. The contents of lead in 

dandelion leaves corresponded to contamination load, the highest lead content, 10.04 

± 3.1 mg.kg-1, was found at the sampling site Opuštěná with heavy traffic density 

(Fig. 3.). Similarly to mercury higher contents of lead were found in dandelion leaves. 

The content of mercury and lead in dandelion leaves could predict atmospheric 

pollution of these two toxic elements. 

mPb (mg.kg -1 DW) 

60 

55 

50 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

Fig. 3. Average content of lead in soil, roots and leaves of dandelion 

The highest content of cadmium in soil was found at the sampling site 

Opuštěná, average concentration 0.645 ± 0.022 mg.kg-1. Significantly lower amounts 

of cadmium than other collector sites were detected at the sampling site Šrámkova, 

the average content of cadmium in soil was 0.114 ± 0.026 mg.kg-1. Obtained results 

were summarized in (Fig. 4.). Compared to lead and mercury higher contents of 

cadmium were found in dandelion roots collected at all studied localities. The main 

source of cadmium was in this case in the soil. Conversely to mercury and lead 

347 

48 

5 

34 

41 

8 

25 

Opuštěná Vídeňská Podstránská Mosurgského Šrámkova 

51,6 

2,39 

10,04 

25,7 

2,09 

6,49 

Sampling sites 

25,9 

2,14 

6,15 

18,7 

2,02 

4,77 

9 

1,82 

soil 

root 

leaves 

Opuštěná Vídeňská Podstránská 


Mosurgského Šrámkova 

soil 

root 

leaves 

2,67


cadmium was found in soils at the mobile and labile forms and enters the plant 

primarily through the root system. 

Fig. 4. Average content of cadmium in soil, roots and leaves of dandelion 

4. CONCLUSION 

Levels of cadmium, mercury and lead in studied Brno urban soil samples based 

on the Regulation No. 13/1994 of the Ministry of Environment Czech Republic were 

not exceed. The amount of metals measured in soils and in leaves and roots of 

common dandelion corresponded with the contamination load of the sampling place. 

It was demonstrated, that the common dandelion could be used as biological monitor. 

The higher content of lead and mercury in dandelion leaves than in roots indicated 

predominantly atmospheric pollution of sampling place. Cadmium was preferentially 

accumulated in dandelion roots and thus determination of cadmium in dandelion 

roots could be used for prediction of soil contamination. 


The work has been supported by Grant Agency of the Czech Republic, Project 

No. P503/10/2002). 

6. REFERENCES 

mCd (g.kg -1 DW) 

700 

650 

600 

550 

500 

450 

400 

350 

300 

250 

200 

150 

100 

50 

0 

645 

217 

118 

427 

[1]. Bargagli, R., Trace elements in terrestrial plants : an ecophysiological approach to biomonitoring 

and biorecovery. Environmental intelligence unit. 1998: Berlin [etc.] : Springer. 

[2]. Martin, M.H. and P.J. Coughtrey, Biological monitoring of heavy metal pollution: land and air. 

Biological monitoring of heavy metal pollution: land and air., 1982. 

[3]. Wittig, R., General aspects of biomonitoring heavy metals by plants. Plants as biomonitors, ed. 

B. Markert. 1993: Weinheim, Germany: VCH Verlagsgesellschaft. 

[4]. Djingova, R. and I. Kuleff, Monitoring of heavy metal pollution by Taraxacum officinale. . Plant 

as biomonitors, ed. B. Markert. 1993: Wiley-VCH Verlag GmbH. 435-460. 

[5]. Marr, K., H. Fyles, and W. Hendershot, Trace metals in montreal urban soils and the leaves of 

Taraxacum officinale. Canadian Journal of Soil Science, 1999. 79(2): p. 385-387. 

149 

348 

54 

330 

130 

Opuštěná Vídeňská Podstránská Mosurgského Šrámkova 

48 


317 

82 

35 

114 

59 

soil 

root 

leaves 

32


[6]. Krolak, E., Accumulation of Zn, Cu, Pb and Cd by dandelion (Taraxacum officinale Web.) in 

environments with various degrees of metallic contamination. Polish Journal of Environmental 

Studies, 2003. 12(6): p. 713-721. 

[7]. Kabata-Pendias, A. and S. Dudka, Trace metal contents;Taraxacum officinale (dandelion) as a 

convenient environmental indicator. Environmental Geochemistry and Health, 1991. 13(2): p. 

108-113. 

349


LANTHANUM 3+ - A NEW PHOSPHATE 

CHELATOR USED IN CHRONIC KIDNEY 

DISEASE - POTENTS APOPTOSIS IN PC-3 

AND 22RV1 PROSTATE CANCER CELL 

LINES AND IN HEALTHY PROSTATE CELLS 

PNT1A 

Marian HLAVNA 1 , Michal MASAŘÍK 1 , Petr BABULA 2 , Jaromír GUMULEC 1 , Markéta 

SZTALMACHOVÁ 1 

1 Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Kamenice 5, CZ- 


2 Department of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical 

Sciences, Palackeho 1-3, CZ-612 42 Brno, Czech Republic, 


Lanthanum 3+ belongs to group of lanthanides – a so called rare Earth’s elements. 

Lanthanides are widely used in agriculture, chemistry and industry as well as 

medicine; so, they are able to enter the living environment and food chains. 

Ecological risk, as well as effect of lanthanides on living organisms on different levels 

– molecular, cytological, histological – are still almost unknown. Lanthanum 3+ 

carbonate is used as a relatively new drug for binding an inorganic phosphate in 

patients suffering with renal functional impairment [1-3]. Nevertheless, the fact that 

lanthanum 3+ is considered to be only low available, increase of lanthanum 3+ plasmatic 

levels was detected in pharmacological studies. Incorporation of lanthanum 3+ ions into 

bone structures was shown [4]. In animals, interference of lanthanum 3+ ions with 

calcium channels as well as inhibition of different enzymes was demonstrated. 

Recently, in in vitro experiments, affinity of lanthanides ions to possible binding sites 

for zinc 2+ ions has been demonstrated [5]. As prostate cancer cells are characteristic by 

decreased ability to uptake, accumulate and metabolize zinc ions and lanthanum 3+ 

shows some similar properties, we decided to investigate effect of lanthanum 3+ 

treatment on PC-3 and 22Rv1 prostate cancer cell lines and PNT1A cell line 

representing healthy prostate epithelial cells. 

2. EXPERIMENT 

We treated PC-3 and 22Rv1 prostate cancer cell lines and PNT1A healthy 

prostate epithelium cell line with increasing concentration of lanthanum 3+ . 22Rv1 and 

PNT1A cell lines were treated with 100 μM, 300 μM, 600 μM and 1200 μM 

concentration of lanthanum 3+ in medium, and 50 μM, 150 μM, 500 μM and 1000 μM 

for Firstly, after lanthanum 3+ treatment we observed morphological changes in nuclei 

350


of prostate cell lines. Therefore we decided to investigate gene expression after 

lanthanum 3+ treatment on mRNA level using RT-PCR combined with real time-PCR 

quantification. We concerned especially on genes conducted with zinc uptake (ZIP1) 

and its extracellular exclusion (ZnT-1), proliferation (FOS, JUN) apoptosis (p53, NFkB), 

genes involved in metabolism, transport and storage of heavy metals and 

protection against oxidative stress (MT1A, MT2A). Furthermore, we looked in detail 

on morphological changes on cellular level using immunohistochemistry and 

fluorescent microscopy. 


Morphological changes in cells observed after lanthanum 3+ treatment are shown 

in Fig. 1. In case of PNT1A and PC-3 cells is clear evidence of chromatin 

condensation, fragmentation of nuclei and apoptosis. A bit different morphological 

changes have been found in 22Rv1 cell line. We observed forming of micronuclei 

after lanthanum 3+ treatment rather than nuclei segmentation and apoptosis (Fig.1 C 

and F.) showing that this cell line is sensitive for lanthanum 3+ treatment in different 

manner. 

Interestingly, lanthanum 3+ treatment has different effect on transcriptional level 

of some investigated genes across used prostate cancer/healthy epithelium cell lines 

(shown in Fig. 2). 

We found that increasing lanthanum 3+ concentration caused increasing 

expression of MT1A in 22Rv1 cancer cell line but rapid decrement of MT1A 

expression in PC-3 cancer cell line. Moreover, in healthy prostate epithelium cell line 

PNT1AA caused lanthanum 3+ treatment, except 100 μM concentration (>8-fold 

increase), no significant change in MT1A expression (Fig 2). Similarly, we found that 

only 100 μM lanthanum 3+ treatment caused significant change in MT2A expression 

and only in PNT1AA cell line (>5-fold increase). Surprisingly, we observed in 22Rv1 

and PC-3 cell line opposite lanthanum 3+ effect than in MT1A. Lanthanum 3+ caused 

slightly decreasing trend in MT2A expression in 22Rv1 and increasing in PC-3, 

respectively. 

351

XI. Workshhop 


E 

s ´11 

A. 

B. 

C. 

Fiig. 

1: Propiidium 

iodid de stained p 

(A) PNNT1AA 

proostate 

cell line l withou 

prostatee 

cancer ceell 

line con ntrol and (C 

(D) PNNT1AA 

cellls 

after 48 hour lanth 

Upper arrow shoows 

nucleu us fragmen 

chromaatin 

condennsation 

and d lower arr 

cells aft fter 48h 10000 

μM lant thanum 

late apooptosis. 

(F) ) 22RV1 ce 

show mmicronucleii. 

3+ prostate ce 

ut lanthan 

C) are 22R 

hanum 

tr 

ell line afte 

3+ ll lines afte 

num 

tre 

ntation an 

row shows 

reatment. A 

er 48h 1200 

3+ er lanthanu 

treatm ment (cont 

v1 prostate e cancer ce 

eatment in 1200 μM 

nd apoptosis, 

middle 

formation of micronu 

Arrow show ws cell frag 

0 μM lanth hanum3+ um 

tre 

3+ treatm ment. 

trol), (B) PC-3 P 

ell line con ntrol. 

concentrat tion. 

arrow sh hows 

uclei. (E) PC-3 P 

gmentation and 

eatment, ar rrow 

AAs 

we obserrved 

by fluo 

investiggation 

the llanthanum3 

orescent miicroscopy 

apoptosis a we w have beeen 

intereste ed in 

3+ treatmennt 

effect on cell cycle regulatory r ggenes 

FOS/ /JUN 

352 

D. 

E. . 

F. . 

A 

Brno

XI. WWorkshop 

of Phyysical 


´11 

andd 

also antiaapoptotic 

ge ene NF-kB. 

No signif ficant chan nge on mRNNA 

level has h been 

obseerved 

in thhese 

genes. 

As prosta 

andd 

our prev 

trannsporters 

fo 

leveels 

of Zip1 

humman 

cells. I 

ZnTT-1 

transpo 

lantthanum3+ 

ate cancer cells are chharacteristi 

ic by zinc ion metaboolism 

dereg gulation 

vious exper riments shhow 

that ZIP1/ZnT-1 

Z 1 transport rters can serve s as 

or other me etal ions suuch 

as cadm mium or copper, 

we innvestigated 

d mRNA 

– the main n zinc ion iimporter 

an nd ZnT1 – main expoorter 

of zinc 

ion in 

In our experiment 

wee 

did not observe o sig gnificant chhange 

in ZI IP1 and 

orter expres ssion. It is likely, that t these tran nsporters arre 

not essen ntial for 

immport 

and export e fromm 

the cell. 

In case oof 

22Rv1 an nd PNT1A c 

p533 

expressionn 

after lanthanum 

expression 

in PPC-3 

cell l 

thesse 

cell liness 

are likely 

3+ cell lines we w also foun nd non-signnificant 

cha anges in 

trreatment 

an nd only mo odest decreeasing 

trend d in p53 

ine (Fig 2). . Our resul lts suggestin ng that apooptosis 

obse erved in 

p53 indepeendent. 

Fig. 2: RRelative 

exp pression of 

22RRv1 

and PNNT1AA 

lan nthanum 

decrrease 

(p=0, ,05) in mRN 

alsoo 

found stattistically 

sig 

ZIPP1 

in 22RRv1 

cell li 

3+ 

f investigated 

genes after 

prostat 

treatment t. We obse erved statis 

NA level oof 

MT2A af fter lanthan num 

gnificant inncrease 

of MT1A M mRN 

ine (p=0,055). 

PNT1A A cells sh 

3+ te cell line es PC-3, 

stically sig gnificant 

treattment 

in PC3. P We 

NA and decrrease 

of Zn nT-1 and 

how signifi ficant increase 

in 

353 

Brno


MT1A/MT2A expression only in relatively low concentrations of lanthanum 3+ 

(100μM). 

4. CONCLUSION 

Our results show, that lanthanum 3+ causes chromatin condensation, forming of 

micronuclei and nuclei segmentation in prostate cancer cell lines and healthy prostate 

epithelial cells. This nuclei segmentation and apoptosis are most probably NF-kB and 

p53 independent. Even that lanthanum 3+ shows similar properties as zinc ions, main 

zinc ion cell transporters ZIP1 (influx) and ZnT-1 (efflux) are likely not involved in 

lanthanum 3+ transport and lanthanum 3+ ions are coming through membranes by other 

mechanism. To reveal apoptotic pathway activated after lanthanum 3+ treatment is 

desirable to investigate also other possible pathways e.g. Ca 2+ dependent or Fas 

receptor mediated apoptotic pathways. 


The work has been supported by grants GACR 301/09/P436 and NSI0200-3 

6. REFERENCES 

[1] Scaria, P.T., Gangadhar, R., Pisharody, R.: Indian J. Pharmacol. 41, (2009), 187-191. 

[2] Arenas, M.D., Rebollo, P., Malek, T., Moledous, A., Gil, M.T., Alvarez-Ude, F., Morales, A., 

Cotilla, E.: J. Nephrol. 23, (2010), 683-692 

[3] Samy, R., Faustino, P.J., Adams, W., Yu, L., Khan, M.A., Yang, Y.S.: J. Pharm. Biomed. Anal. 51 

(2010), 1108-1112. 

[4] Bronner, F., Slepchenko, B.M., Pennick, M., Damment, S.J.P.: Clin. Pharmacokinet. 47, (2008), 

543-552. 

[5] Huidrom, B., Devi, N.R., Ch, S., Singh, T.D., Yaiphaba, N., Singh, N.R.: J. Indian Chem. Soc. 87, 

(2010), 1391-1394. 

354


OPTIMIZATION OF PLANAR THREE- 

ELECTRODES SYSTEMS 

FOR ELECTROCHEMICAL APPLICATIONS 

Jan PRÁŠEK 1 


Abstract 

This paper studies the optimization possibilities of planar thick film three-electrode 

electrochemical systems for detection of species in aqueous solutions. The aim of this 

work was to find suitable material for reference electrodes fabrication and determine 

how the shape and size of the electrodes affect the output signal. Various commercial 

and own materials were used for Ag/AgCl reference electrodes fabrication and 

compared with standard reference electrode. The best results were achieved with 

commercial materials for reference electrodes fabrication. Moreover several 

differently sized and shaped electrodes were fabricated for their electrochemical 

behaviour evaluation. Finally some conclusions and recommendations for electrodes 

sizes and shapes come out too. 


Commercial solid electrodes are usually created from a big glass body with 

attached metal wire or plate, but the necessities of today’s analyses are small-size 

electrodes. One possibility of miniaturization of solid electrodes is their integration 

onto a small substrate. Such electrochemical system forming a small sensor could be 

fabricated using thick film technology (TFT). The advantages of this solution are low 

dimensions, good electrical and mechanical properties of electrodes, good 

reproducibility and well accessible and ecological fabrication process. Unconventional 

applications of TFT also open wide possibilities of creation of electrodes of sensors and 

biosensors using chemical active electrode materials. 

In last few years, various commercial electrochemical sensors have been 

developed and presented. Besides them, many scientific works reported very good 

results using different electrochemical thick-film sensors, but nowhere is explained, 

why the selected topography, electrode materials, etc. was used. Also their influence 

to output current response of electrochemical sensors is not reported. The aim of 

presented paper is to check the behaviour of output current response of thick film 

planar reference electrodes (RE) depending on material used for their fabrication and 

to check, how the size and shape of the electrodes influence the output current 

response of the three-electrode electrochemical sensor. The behaviour was studied in 

a three-electrode system using standard electrochemical solution of potassium ferroferricyanide. 

355

XI. Workshhop 


E 

s ´11 


The 

design of new thr ree-electrodde 

systems for all exp periments ccame 

out from f 

the stanndard 

plannar 

voltamm metric elecctrochemica 

al three-ele ectrode thiick-film 

sensor 

topograaphy 

commmonly 

used d in our labboratory 

[1 1]. The sta andard TFTT 

voltamme etric 

sensor ttopographyy 

used in ou ur laboratorry 

is shown n in the figu ure 1. For aall 

experim ments 

the samme 

sized aluumina 

subst trate (25.4× ×7.2 mm) an nd the same 

size of plaanar 

electro odes 

area (7× ×7 mm) weere 

used for r sensors deesign 

in this s work. 

Figg.1 

Standard TFT voltamm metric 

sensor ttopography 

OOwn 

referennce 

Ag/AgC Cl electroddes 

were ele ectrodeposi ited accordding 

to [2] over o 

standarrd 

Ag based 

TFT elec ctrode (ESLL 

9912-K). . Another two DuPoont 

commercial 

polymeer 

pastes wwere 

used fo or RE fabriication: 

pas ste type 5870 

(Ag:AgCCl 

ratio: 80 0:20) 

and 58774 

(Ag:AgCCl 

ratio: 65: 35). Pastes were scree en-printed and a cured aat 

120 °C fo or 10 

minutees. 

Foor 

electroddes 

size expe eriment sevven 

differen nt sensors with w strip eelectrodes 

were w 

designeed 

(Figure 22). 

The size e of workinng 

electrod des (WE) was 

fixed annd 

the auxil liary 

electroddes 

(AE) annd 

RE wer re varied. FFor 

electrode 

shape experiment, 

, seven sen nsors 

with diifferent 

shaape 

of electr rodes and tthe 

same ge eometrical electrodes area sizes were w 

designeed 

(Figure 33). 

Soolution 

of f a 0.05 mol/L m pottassium 

fer rrocyanide, , 0.05 mool/L 

potass sium 

ferricyaanide 

and 00.2 

mol/L KOH K was pprepared 

us sing 18MΩ redistilled d and deion nized 

water and used for all experiments 

e s. All the e measurem ments werre 

done using u 

potentiiostat 

Voltalab 

PST050 

(Radiommeter 

analytical, 

De enmark). TThe 

measu uring 

methodd 

was cyclicc 

voltamme etry (CV) iin 

range of f the potent tial from -3300 

to 600 mV. 

The scaan 

rate wass 

set to 20 mV/sec. m Thhe 

measurement 

setup p and respoonse 

evalua ation 

were ddone 

usingg 

a stand dard personnal 

compu uter. In RE R experimment 

stand dard 

commeercial 

WE aand 

SE were e used. 

3. RRESULTS 


Fiirst 

experimment 

was devoted d to the materi ial of RE fo or electrochhemical 

planar 

sensorss. 

For thhe 

first experimennt 

severa al Ag/AgC Cl screenn-printed 

and 

electrocchemically 

prepared planar p referrence 

electr rodes were fabricated and compa ared. 

356 

Brno 

Fig. 2 Designed D stri rip 

electrodes 

for electtrode 

size 

experim ment 

Fig. 3 Designed D sennsors 

for 

the electrode 

shapee 

experim ment

XI. WWorkshop 

of Phyysical 


´11 

Thee 

results (see 

Figure 4) 4 showed that the results r obta ained with h both com mmercial 

DuPPont 

pastees 

are very 

similar although the Ag/AgCl 

ratio is differen nt. The 

commparison 

wwith 

classic cal Ag/AgCCl 

referenc ce electrod de showed that the current 

respponse 

is commparable. 

Different D siituation 

wa as obtained d in compar arison of ha alf-wave 

poteentials, 

whhere 

was ob bserved thee 

potential shift in ra ange of ~ 1100 

mV. Th he half- 

wavve 

potentiaals 

of the el lectrochemmically 

fabri icated refer rence electrrodes 

were e similar 

to hhalf-wave 

ppotentials 

of 

the DuPoont 

polymer 

pastes. Th he current ddiffered 

jus st in the 

peakk 

current oof 

the redu uction proccess. 

Mentio oned result ts led us too 

decision that t the 

DuPPont 

pastess 

are the be est solutionn 

for constr ruction of planar p referrence 

electr rodes of 

TFTT 

sensors beecause 

of ve ery good annd 

reproduc cible respon nse. 

Second eexperiment 

t was devooted 

to the influence investigatiion 

of geom metrical 

sizee 

of reference 

and auxiliary 

electrrodes 

to the 

sensor an nodic outpuut 

current response r 

andd 

wave poteential. 

It wa as found thaat 

the depe endence of output currrent 

respon nse with 

the change off 

RE area size s had neegative 

grad dient in co omparison wwith 

our previous p 

resuults. 

The ccurrent 

res sponse deppendence 

with w the change 

of AAE 

area size 

was 

incrreasing. 

Thhe 

size of AE A was probbably 

chose en badly an nd it needs to be meas sured in 

widder 

range oof 

AE area sizes in thee 

next experiments, 

but b it couldd 

be expect ted that 

withh 

next incrrement 

of the 

AE size, 

the curren nt will be stabilized s at a fixed va alue due 

to mmaximum 

ccurrent 

den nsity passing 

through WE. W The in nfluences oof 

RE and AE A areas 

sizees 

to anodicc 

wave pote ential was aalmost 

negli igible in bo oth cases. 

The thirrd 

experim ment was ddevoted 

to the influe ence invest stigation of f sensor 

elecctrode 

area shape with h the fixed geometrica al area size of all three 

electrode es to the 

senssor 

anodicc 

output cu urrent respponse 

and wave pot tential. Thhe 

highest current 

respponse 

and rreproducibi 

ility was acchieved 

with 

the sens sor numberr 

S4 (see Fi igure 5). 

Thee 

lowest cuurrent 

respo onse with a relative good g reprod ducibility wwas 

achieved 

with 

the sensor S1 that repre esents commmonly 

used d rounded shape of tthe 

electrod de area. 

Connsidering 

soome 

kind of o error for the first se et of electro odes, the chhange 

of ha alf wave 

poteential 

was aalmost 

inde ependent onn 

the shape e of electrod de area. 

Fig. 4 All measured 

refe erence electrrodes 

mmaterials 

commparison 

with h standard AAg/AgCl 

electrod de 

357 

I [µA] 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0 

1 

S 1 

S 5 

S 2 S 3 

S 6 S 7 

2 3 4 

S 4 

Set # [-] 

Brno 

Fig. F 5 Depend dence of outp tput current response r 

on the shap pe of the sennsor 

electrod de area 

5


4. CONCLUSION 

The optimization of planar three-electrode system was studied in this work 

including material for reference electrodes fabrication, shape and geometrical size of 

the electrodes. All obtained results, conclusions and recommendations are mentioned 

in previous section. 


Funding for this work was provided by the Grant agency of the Czech Republic 

under the contracts GACR 102/08/1546, and Czech Ministry of Education in the 

frame of Research Plan MSM 0021630503 MIKROSYN 

6. REFERENCES 

[1] Prasek, J., et al.: 33rd International Spring Seminar on Electronics Technology, 2010, pp. 1-4 

[2] Lanz, M., et al.: Journal of Photochemistry and Photobiology A: Chemistry, 1999, 120, pp. 105- 

117 

358


OBSAH 

ELECTROCHEMISTRY OF METALLOTHIONEINS ........................................................................ 7 

ELECTROCHEMISTRY IN SPACE ................................................................................................ 12 

ANATOMICAL STUDY OF VEGETATIVE ORGANS OF RHUS HIRTA (L.) SUDW. 

(ANACARDIACEAE) ...................................................................................................................... 17 

ANATOMICAL STUDY OF VEGETATIVE ORGANS OF PHARMACEUTICALLY AND 

TOXICOLOGICALLY IMPORTANT PLANT ACONITUM NAPELLUS L. EM. SKALICKY 

(RANUNCULACEAE) ..................................................................................................................... 24 

ANATOMICAL STUDY OF PHARMACEUTICALLY IMPORTANT PLANT MACLURA POMIFERA 

(RAF.) C.K. SCHNEID. (MORACEAE) ........................................................................................... 31 

VOLTAMMETRIC MONITORING OF RIBOFLAVIN REDUCTION ON MERCURY MENISCUS 

MODIFIED SILVER SOLID AMALGAM ELECTRODE ................................................................... 38 

PHOTOINDUCED OXYGEN ACTIVATION IN THE PRESENCE OF 4-ANILINOQUINAZOLINES 

........................................................................................................................................................ 42 

ELECTROCHEMICAL DETECTION OF RNA USING A COMPLEX OF SIX-VALENT OSMIUM .. 46 

THE SPECTROSCOPIC STUDY OF PHOTOINDUCED REACTIONS OF N-HETEROCYCLIC 

COMPOUNDS ................................................................................................................................ 49 

TECHNICAL SOLUTION OF PLANET EXPLORATION ................................................................ 53 

ELECTROCHEMICAL DATA FROM SPACE .................................................................................. 56 

SPECTROPHOTOMETRIC ACID-BASE CHARACTERIZATION OF ADENINE ANALOGUES ... 59 

ROBOTIC MODULE EURYDICE ................................................................................................... 62 

CHEMORESITANCE OF CANCER CELLS- MODELS FOR STUDY IN VITRO AND IN VIVO .... 67 

PROCESSING OF ELECTROCHEMICAL SIGNALS FOR DETERMINATION OF 

METALLOTHIONEIN CONCENTRATION ...................................................................................... 70 

STRUCTURE AND MAGNETISM OF CLEAN AND IMPURITY-DECORATED GRAIN 

BOUNDARIES IN NICKEL FROM FIRST PRINCIPLES ................................................................ 74 

USE OF LIGAND STEP GRADIENT FOCUSING IN COMBINATION WITH 

ISOTACHOPHORESIS (LSGF-ITP) FOR THE EFFECTIVE PRE-CONCENTRATION AND 

ANALYSIS OF HEAVY METALS .................................................................................................... 78 

LIGNITE – A LOW QUALITY SOLID FUEL WITH ATTRACTIVE SORPTION ABILITIES ............. 81 

APPLICATIONS OF IRON OXIDE NANOPARTICLES .................................................................. 85 

EXPRESSING OF BACTERIAL DIHYDRODIPICOLINATE SYNTHASE IN GRAIN OF 

TRANSGENIC BARLEY ................................................................................................................. 89 

DETERMINATION PROSTATE SPECIFIC ANTIGEN AND TESTOSTERONE IN TUMOUR CELL 

LINES WITH ENCORE ZINC AND SARCOSINE AS WELL AS IN PROSTATE CANCER 

PATIENTS ....................................................................................................................................... 95 

FABRICATION AND CHARACTERIZATION OF TIO2 QUANTUM DOTS ARRAY ...................... 101 

DETERMINATION OF SARCOSINE AS POSSIBLE TUMOUR MARKER OF PROSTATE 

TUMOURS AND ROUTINE BIOCHEMICAL TESTS IN URINE SAMPLES ................................ 105 

PHOTOCURRENT GENERATION IN INKJET PRINTED TIO2 LAYERS .................................... 111 

OPTICAL SPECTROSCOPY IN MODERN BIOPHYSICAL RESEARCH ................................... 115 

AN ELECTROCHEMICAL SPM UNDER FULL ENVIRONMENTAL CONTROL ......................... 118 

359


HEAVY METALS IN PROSTATE CANCER CELL LINES ............................................................. 119 

NEW POSSIBILITIES IN FUNCTIONALIZATION AND LABELING OF DNA FOR 

ELECTROCHEMICAL ANALYSIS ............................................................................................... 125 

REVIEW OF ELECTROREDUCTION OF HYDROGEN IONS ON MERCURY .......................... 128 

QUANTUM DOTS WITH FUNCTIONALIZED SHELL LAYERS FOR ENHANCED 

BIOCONJUGATION AND FLUOROIMMUNOASSAYS ............................................................... 129 

USING OF TEMPLATES BASE METHOD FOR FARICATION OF NANORODS STRUCTURES 

FOR ELECTROCHEMICALSENZING ELEMENTS .................................................................... 133 

TRENDS IN CHEMICAL SENSORS ............................................................................................ 137 

NANOTECHNOLOGY FOR SENSORS AND DIAGNOSIS ......................................................... 140 

ELECTROCHEMICAL ANALYSIS OF DNA AND CADMIUM IONS INTERACTION ................... 144 

FABRICATION OF COPPER MICROPARTICLES BASED WORKING ELECTRODES FOR 

ELECTROCHEMICAL DETECTION OF ADENINE ..................................................................... 148 

PREPARATION OF WATER SOLUBLE GLUTATHIONE-COATED CDTE QUANTUM DOTS ... 152 

APPLICATION OF CITP FOR BIOMINERAL ANALYSIS ............................................................ 156 

UTILIZATION OF FRACTIONAL EXTRACTION FOR CHARACTERIZATION OF THE 

INTERACTIONS BETWEEN HUMIC ACIDS AND METALS ....................................................... 158 

MONITORING OF STRESS MARKERS IN MAIZE (ZEA MAYS L.) EXPOSED TO CADMIUM 

AND ZINC IONS .......................................................................................................................... 162 

SYNDROM OF NEWLY FILLED RESERVOIR FROM THE MERCURY POINT OF VIEW ........ 168 

UTILIZATION OF TOTAL ANTIOXIDANT CAPACITY TO EVALUATE THE EFFECT OF 

MULTIWALL CARBON NANOTUBES AND MAGNETIC NANOPARTICLES ON SUSPENSION 

TOBACCO CULTURE .................................................................................................................. 171 

EPR AND UV-VIS SPECTROELECTROCHEMICAL STUDIES OF NOVEL SYNTHESIZED 

COPPER, NICKEL AND COBALT-BASED COMPLEX DERIVATIVES ....................................... 174 

VARIOUS MICROWAVE DIGESTION PROCEDURE OF SAMPLES FOR HEAVY METALS 

ELECTROCHEMICAL DETERMINATION ................................................................................... 177 

ELECTROCHEMICAL DETERMINATION OF HEAVY METALS IN RAINWATER ...................... 182 

ELECTROCHEMICAL DETERMINATION OF MT1 AND MT2 ISOFORMS ................................ 185 

INFLUENCE OF PTCL4 ON MAIZE AND PEA ............................................................................ 190 

PLATINUM GROUPS ELEMENTS AND THEIR INFLUENCE ON PEA SEEDLINGS ................ 193 

UTILIZATION OF ELECTROCHEMICAL METHODS IN STUDIES OF TRANSPORTING 

PROCESSES ACROSS THE PHOSPHOLIPID BILAYERS ........................................................ 196 

OPTICAL CHARACTERIZATION OF ORGANIC SEMI-CONDUCTORS .................................... 200 

ELECTROCHEMISTRY OF BIOMACROMOLECULES. TRENDS IN PROTEIN ANALYSIS ..... 203 

THE STUDY OF 6 – BENZYLAMINOPURINE AND ITS DERIVATIVES BY ELECTROCHEMICAL 

AND SPECTRAL METHODS ....................................................................................................... 207 

SPRAY-COATED WORKING ELECTRODES FOR ELECTROCHEMICAL SENSORS ............. 213 

IMPROVEMENT OF SENSING PROPERIES OF THE THICK-FILM ELECTROCHEMICAL 

SENSORS BY SPECIFICATION MEASUREMENT WITH THE ROTATING VESSEL CELL ...... 217 

FLUORO/NITRO DERIVATIVES OF QUINOLONES: THEORETICAL AND SPECTROSCOPIC 

STUDY ......................................................................................................................................... 221 

360


THIOPHENOLS: ENERGETICS OF S–H BOND CLEAVAGE ..................................................... 225 

ANALYSIS OF METALLOTHIONEIN ISOFORMS BY CAPILLARY ELECTROPHORESIS ........ 228 

ANALYSIS OF SARCOSINE BY CAPILLARY ELECTROPHORESIS ......................................... 233 

LAB-ON-CHIP: STATE OF THE ART ........................................................................................... 237 

THIN FILM HYDROGEN SENSOR BASED ON DIKETOPYRROLO-PYRROLES ANALOGUES 

...................................................................................................................................................... 239 

INSTRUMENT FOR FLUORESCENT BIOSENSOR ................................................................... 243 

NOVEL REACTIVITY – MAPPING TECHNIQUE FOR CHARACTERIZATION OF 

POLYELECTROLYTE BIOPOLYMERS ....................................................................................... 246 

DPPH AQUEOUS ANTIOXIDANT ASSAY SOLUTION ................................................................ 250 

QUANTUM CHEMICAL CALCULATIONS OF [LI(DMSO) N] + COMPLEXES ............................... 255 

ANALYSIS OF THIOL COMPOUNDS AND ANTIOXIDANT ACTIVITY IN PATIENTS SUFFERING 

FROM MALIGNANT DISEASE .................................................................................................... 259 

ANTIOXIDANT ACTIVITY OF TUMOUR CELL AND EFFECT OF CYTOSTATICS ON 

ANTIOXIDANT STATUS ............................................................................................................... 264 

ELECTROCHEMICAL ANODIZATION AS TOOL FOR NANOSTRUCTURES FORMATION ..... 268 

THE IMPACT OF SELECTED PLATUM GROUP ELEMENTS ON ANTIOXIDANT ACTIVITY OF 

DUCKWEED (LEMNA MINO L.) ................................................................................................. 272 

IMPORTANCE OF DIFFERENTIAL PULSE VOLTAMMOGRAMS FOR STUDYING OF 

BIOLOGICAL IMPORTANT PHENOMENONS ............................................................................ 276 

ELECTROCHEMICAL BEHAVIUOR OF (PRP C ) AND CHANGED (PRP SC ) PRION PROTEIN .. 279 

ELECTROCHEMICAL BIOSENSOR FOR DETECTION OF BIOAGENTS ................................. 282 

ELECTROANALYSIS WITH NON-MERCURY METAL-MODIFIED CARBON PASTE 

ELECTRODES IN THE NEW MILLENNIUM ................................................................................ 284 

ELECTROCHEMISTRY OF OSMIUM(VI)-MODIFIED CARBOHYDRATES ................................ 287 

ADSORPTIVE STRIPPING ELIMINATION VOLTAMMETRY ...................................................... 290 

THEORETICAL STUDY OF N–H BOND DISSOCIATION ENTHALPIES IN PARA SUBSTITUTED 

ANILINES ..................................................................................................................................... 294 

SOFTWARE FOR PROCESSING OF CATALYTIC METALLOTHIONEIN SIGNALS .................. 297 

MATERIALS FOR ORGANIC ELECTRONICS ............................................................................ 301 

DEVELOPMENT OF OPTICAL SENSORS BASED ON COMPLEXES OF MACROCYCLIC 

COMPOUNDS .............................................................................................................................. 305 

MONITORING DNA MODIFICATION BY PLATINUM COMPLEXES USING CATALYTIC 

HYDROGEN EVOLUTION AT MERCURY ELECTRODES ......................................................... 309 

SYNTHESIS AND CHARACTERIZATION OF NANOPARTICLES OF SN-3,5AG-0,5ZN ........... 313 

ELECTROCHEMICAL DETERMINATION OF BIOLOGICALLY ACTIVE COMPOUNDS ............ 317 

SPECTROFOTOMETRIC ANALYSIS OF CYSTEINE-CADMIUM COMPLEXES USING 

MUREXIDE INDICATOR .............................................................................................................. 322 

DETERMINATION OF LACTOFERRINE IN HUMAN SALIVA ..................................................... 327 

EMPLOYMENT OF HPLC WITH COULOMETRIC DETECTION FOR ANALYSIS OF 

PHYTOCHELATIN SYNTHASE ACTIVITY .................................................................................. 332 

361


FAST AND ROBUST METHOD FOR DETECTION COPPER AND CADMIUM IONS BY FLOW 

INJECTION METHOD WITH AMPEROMTRIC DETECTION ..................................................... 336 

IMPLEMENTATION OF WATERRING MEASUREMENT TO ENVIRONMENT MONITOR 

SYSTEM ....................................................................................................................................... 340 

TOXIC METALS IN BRNO URBAN SOILS AND THE PLANTS OF COMMON DANDELION 

(TARAXACUM OFFICINALE) ...................................................................................................... 344 

LANTHANUM 3+ - A NEW PHOSPHATE CHELATOR USED IN CHRONIC KIDNEY DISEASE - 

POTENTS APOPTOSIS IN PC-3 AND 22RV1 PROSTATE CANCER CELL LINES AND IN 

HEALTHY PROSTATE CELLS PNT1A ........................................................................................ 350 

OPTIMIZATION OF PLANAR THREE-ELECTRODES SYSTEMS FOR ELECTROCHEMICAL 

APPLICATIONS ........................................................................................................................... 355 

OBSAH ......................................................................................................................................... 359 

AUTORSKÝ REJSTŘÍK ............................................................................................................... 363 

POZNÁMKY ................................................................................................................................. 366 

SEZNAM ÚČASTNÍKŮ XI. PRACOVNÍHO SETKÁNÍ FYZIKÁLNÍCH CHEMIKŮ A 

ELEKTROCHEMIKŮ .................................................................................................................... 367 

PODĚKOVÁNÍ OBCHODNÍM SPOLEČNOSTEM ZA SPONZOROVÁNÍ XI. SETKÁNÍ 

FYZIKÁLNÍCH CHEMIKŮ A ELEKTROCHEMIKŮ:...................................................................... 370 

362


AUTORSKÝ REJSTŘÍK 

Vojtěch ADAM 7, 12, 17, 24, 32, 57, 63, 

120, 145, 153, 163, 172, 191, 194, 229, 

251, 260, 265, 273, 277, 280, 323, 328, 

333, 337 

Petr BABULA ..... 17, 24, 32, 90, 96, 106, 

120, 260, 265, 350 

Jana BALÁŽIKOVÁ ............................ 43 

Zdeňka BALCAROVÁ ...................... 208 

Lenka BANDŽUCHOVÁ ................... 39 

Zuzana BARBIERIKOVÁ ................... 43 

Jiří BAREK ........................................ 318 

Martin BARTOŠÍK ............. 47, 204, 288 

Miroslava BEKLOVÁ ....... 191, 194, 273, 

277, 280, 333 

Šárka BIDMANOVÁ ........................ 244 

Ondřej BLASTIK ................................... 7 

Miroslava BOBENIČOVÁ .................. 50 

Martin BREZA .................................. 256 

Vlasta BREZOVÁ ....................... 43, 222 

Pavel BUČEK ...................................... 60 

Petra BUŠÍNOVÁ . 82, 86, 134, 149, 214 

Natalia CERNEI ... 90, 96, 106, 163, 194, 

234 

Hana ČERNOCKÁ ............................ 204 

Hana DEJMKOVÁ ............................ 318 

Hana DOČEKALOVÁ ...................... 344 

Jana DRBOHLAVOVÁ ..... 102, 134, 153 

Dana DVORANOVÁ .................. 50, 222 

Jiří DVOŘÁČEK ............................... 298 

Petr DZIK .......................................... 112 

Tomáš ECKSCHLAGER ............. 68, 265 

Ivo FABRIK .......................................... 7 

363 

Tomáš FESSL ..................................... 116 

Nuria FERROL .................................. 337 

Miroslav FOJTA ........................ 126, 310 

Fiona FREHILL ................................. 119 

Lenka GAJDOVÁ ............................. 169 

Petr GALUSZKA ................................ 90 

Eliška GLOVINOVÁ .......................... 79 

Jaromír GUMULEC ... 96, 106, 120, 260, 

350 

Tereza HÁJKOVÁ ............................ 229 

Wendy A. HARWOOD ..................... 90 

Luděk HAVRAN ....................... 126, 310 

Patricie HEINRICHOVÁ ................. 201 

Michael HEYROVSKÝ ..................... 129 

Antonín HLAVÁČEK ....................... 130 

Marian HLAVNA ..................... 120, 350 

Michal HOCEK ................................. 126 

Sylvie HOLUBOVÁ .......................... 208 

Petra HORÁKOVÁ .................. 126, 310 

Roman Hrabec .................................. 120 

Jan Hraběta.......................................... 68 

Zuzana HRDINOVÁ ........................ 172 

Radim HRDÝ .................................... 134 

Jaromír HUBÁLEK .... 12, 54, 57, 63, 82, 

86, 102, 134, 138, 141, 149, 153, 229, 

238, 244, 269, 280, 341 

Dalibor Hůska ........................... 145, 280 

David HYNEK .. 163, 178, 183, 186, 191, 

194 

Jana CHOMOUCKÁ .. 86, 134, 149, 153, 

214 

Michal Ilčin ....................................... 256


Soňa JANTOVÁ .................................. 43 

Libor JANU ....................................... 153 

Zdeňka Jarolímová ............................ 157 

Ondřej JAŠEK ................................... 172 

Michal KALINA ................................ 159 

Anne-Marie KELTERER .................. 222 

Renata KENSOVÁ ............................ 169 

René KIZEK 7, 12, 17, 24, 32, 57, 63, 71, 

90, 96, 106, 120, 141, 145, 153, 163, 

172, 178, 183, 186, 191, 194, 229, 234, 

238, 251, 260, 265, 273, 277, 280, 298, 

323, 328, 333, 337, 341 

Andrea KLECKEROVÁ .................... 163 

Andrea KLECKEROVÁ .................... 344 

Erik KLEIN ........................ 222, 226, 295 

Martin KLIMEK ................................ 298 

Martina KLUČÁKOVÁ ............ 159, 247 

Anna KORVASOVÁ ........................... 17 

Kamila KRUŽÍKOVÁ ....................... 169 

Olga KRYŠTOFOVÁ 163, 172, 191, 194, 

333 

Sona KRÍŽKOVA ...................... 120, 172 

Vladimíra KUBICOVÁ ....................... 71 

Vít KUDRLE ..................................... 172 

Šárka KUCHTICKOVÁ .................... 120 

Simona KVAŠŇÁKOVÁ ..................... 24 

Jiří LITZMAN ................................... 328 

Přemysl LUBAL ................................ 157 

Přemysl LUBAL .................. 60, 157, 306 

Vladimír LUKEŠ ....... 222, 226, 256, 295 

Hana MACÍČKOVÁ-CAHOVÁ ...... 126 

Petr MAJZLÍK 7, 71, 178, 183, 186, 229, 

280, 298 

364 

Vladimír MAREČEK ........................ 197 

Michal MASAŘÍK ...... 96, 106, 120, 234, 

260, 350 

Ester MEJSTRIKOVÁ ....................... 328 

Miguel-Ángel MERLOS ................... 337 

Břetislav MIKEL ............................... 244 

Hana Mikulášková .................... 191, 194 

Magdalena Morozová ....................... 112 

Katarina MRIZOVÁ ........................... 90 

Tomáš NAVRÁTIL ........................... 197 

Petra NETROUFALOVÁ ................... 60 

Lenka NOVÁKOVÁ ......................... 277 

Ludmila OHNOUTKOVÁ .................. 90 

Veronika OSTNATÁ ........................ 204 

Imad OUZZANE ............................... 201 

Emil PALEČEK ................... 47, 204, 288 

Miloslav PEKAŘ ................................. 82 

Jitka PETRLOVÁ .................................. 7 

Iveta PILAŘOVÁ .............................. 208 

Hana PIVOŇKOVÁ ................. 126, 310 

Kristýna PLEVOVÁ ............................ 50 

Jitka POLJAKOVÁ ............................. 68 

Jan POSPÍCHAL ................................. 79 

Jan PRÁŠEK ...... 134, 149, 214, 218, 355 

Zbyněk PROKOP ............................. 244 

Ivo PROVAZNÍK ........ 71, 229, 251, 298 

Jan PŘIBYL ....................................... 283 

Kraiwan PUNYAIN .......................... 222 

Zdenek PYTLÍČEK ........................... 218 

M.A. RAHMAN .................................. 68 

Tereza REICHLOVÁ ........................ 186 

Ján RIMARČÍK ................. 222, 226, 295


Lenka ROTTMANNOVÁ ......... 226, 295 

Arne ROVNÝ .................................... 120 

Markéta RYVOLOVÁ ..... 153, 229, 234, 

238 

Ota SALYK ........................................ 240 

Jiří SedlÁČek ..................................... 244 

Petr SEDLÁČEK ....................... 159, 247 

Karel SEDLÁŘ ................................... 251 

Núria SERRANO ............................... 208 

Eliška SISPEROVA ............................. 79 

Sylvie SKALÍČKOVA ....................... 328 

Petr SKLÁDAL .......................... 130, 283 

Vladimír SLÁDEK ............................ 256 

Jan SLAVÍK ....................................... 145 

Mark A. SMEDLEY ............................ 90 

Kristýna SMERKOVÁ ...................... 251 

Jiří SMILEK ....................................... 247 

Jiří SOCHOR ...... 96, 106, 172, 251, 260, 

265, 273, 323, 328 

Dmitry SOLOVEI ............................. 102 

Dmitry SOLOVEI ..................... 102, 269 

Jíří SOPOUŠEK ................................. 314 

Ivana SOUKUPOVÁ ......................... 273 

Marie STIBOROVÁ .................... 68, 265 

Lenka STRAKOVÁ ........................... 273 

Klára SUCHÁNKOVÁ ...................... 314 

Zdeňka SVOBODOVÁ ..................... 169 

Markéta SZTALMACHOVÁ .... 120, 350 

Michaela ŠEBKOVÁ ......................... 344 

Martin ŠEDINA ................................ 201 

Renáta ŠELEŠOVSKÁ ......................... 39 

Ivana ŠESTÁKOVÁ .......................... 197 

365 

David Škoda ...................................... 314 

Helena ŠKUTKOVÁ ......................... 251 

Mojmír ŠOB ........................................ 75 

Pavlína ŠOBROVÁ ...... 7, 163, 191, 194, 

277, 280, 333 

Olga ŠTĚPÁNKOVÁ ........................ 277 

Eva ŠVÁBENSKÁ ............................. 283 

Ivan Švancara .................................... 285 

Jana TABAČIAROVÁ ........................ 50 

Jana TKADLEČKOVÁ ........................ 32 

Mojmír TREFULKA ........... 47, 204, 288 

Libuše TRNKOVÁ . 7, 60, 145, 149, 208, 

291 

Adam VAGÁNEK ..................... 226, 295 

Martin Vala ....................................... 201 

Martin VALLA .................... 71, 298, 302 

Jakub VANĚK ................................... 306 

Jana VAŠKOVÁ .................................. 90 

Michal VESELÝ ................................ 112 

Pavlína VIDLÁKOVÁ ...................... 310 

Marcela VLKOVA ............................ 328 

Marina VOROZHTSOVÁ ................ 134 

Milan VRÁBEL ................................. 126 

Monika VŠIANSKÁ ............................ 75 

Vít VYKOUKAL ............................... 314 

Jan VYŇUCHAL ............................... 240 

Libor VYSLOUŽIL ............................ 172 

Martin WEITER ....................... 201, 302 

Josef ZEHNÁLEK ............................. 333 

Jiří ZIMA ........................................... 318 

Ondřej ZÍTKA ...... 90, 96, 106, 260, 323, 

328, 333, 337 

Jaromír ŽÁK ...................................... 341


POZNÁMKY 

366


SEZNAM ÚČASTNÍKŮ 

XI. PRACOVNÍHO SETKÁNÍ FYZIKÁLNÍCH CHEMIKŮ A 

ELEKTROCHEMIKŮ 

Adam Vojtěch 

Ústav chemie a biochemie, Fakulta agronomická, MENDELU Brno, Zemědělská 1, 613 00 Brno 

Bandžuchová Lenka 

Ústav environmentálního a chemického inženýrství, Fakulta chemicko-technologická, Univerzita 

Pardubice, Studentská 95, 532 10 Pardubice 

Barbieriková Zuzana 

Institute of Physical Chemistry and Chemical Physics, Slovak University of Technology in Bratislava, 

Radlinského 9, 812 37 Bratislava, Slovakia 

Bartošík Martin 

Biofyzikální ústav AV ČR, v.v.i., Královopolská 135, 612 65 Brno 

Bittová Miroslava 

Ústav chemie, PřF MU v Brně, Kotlářská 2, 611 37 Brno 

Bobeničová Miroslava 



Buček Pavel 


Cernei Natalia 


Dzik Petr 

Ústav fyzikální a spotřební chemie, Fakulta chemická VUT v Brně, Purkyňova 118, 612 00 Brno 

Tomáš Fessl 

Institute of Physical Biology, University of South Bohemia, Zamek 136, Nove Hrady 

Frehill Fiona 

Agilent Technologies, 610 Wharfedale Road, IQ Winnersh, Wokingham, Berkshire, RG41 5TP, UK 

Gumulec Jaromír 


Hasoň Stanislav 


Havran Luděk 


Heyrovský Michael 

Ústav fyzikální chemie J. Heyrovského AV ČR, Dolejškova 3, 182 23 Praha 8 

Hlaváček Antonín 

Ústav biochemie, PřF MU v Brně, Kotlářská 2, 611 37 Brno 

Húska Dalibor 


Janů Libor 


Jarolímová Zdeňka 


Kalina Michal 

Fakulta chemická VUT v Brně, Ústav fyzikální a spotřební chemie, Purkyňova 118, 612 00 Brno 

Kizek René 


Klein Erik 

367




Koudelka Petr 


Kryštofová Olga 


Křížková Soňa 


Lubal Přemysl 


Lušpai Karol 



Navrátil Tomáš 

Ústav fyzikální chemie J. Heyrovského AV ČR, Dolejškova 3, 182 23 Praha 8 

Ouzzane Imad 

Ústav fyzikální a spotřební chemie, Fakulta chemická, VUT Brno, Purkyňova 118, 612 00 Brno 

Paleček Emil 


Pilařová Iveta 


Pouch Milan 


Rimarčík Ján 



Rottmannová Lenka 



Ruttkay - Nedecký Branislav 


Ryvolová Markéta 


Salyk Ota 

Ústav fyzikální a spotřební chemie, Fakulta chemická, VUT Brno, Purkyňova 118, 612 00 Brno 

Sedláček Petr 


Sládek Vladimír 



Sochor Jiří 

Ústav šlechtění a množení zahradnických rostlin, Fakulta zahradnická, MENDELU Brno, Valtická 337, 691 

44 Lednice 

Sztalmachová Martina 


Šob Mojmír 


Šobrová Pavlína 


Švábenská Eva 

VOP-026 Šternberk,s.p.,VTÚO Brno Division, Czech Republic 

368


Švancara Ivan 

Katedra analytické chemie, Fakulta chemicko-technologická, Univerzita Pardubice, Studentská 95, 532 10 

Pardubice 

Novotná Kamila 

Ústav veřejného veterinárního lékařství a toxikologie, FVHE, VFU Brno, Palackého 1/3, 612 42 

Trefulka Mojmír 


Trnková Libuše 


Vagánek Adam 



Vala Martin 


Vaněk Jakub 


Vidláková Pavlína 


Vykoukal Vít 

Zima Jiří 

Katedra analytické chemie, PřF, UK v Praze, Hlavova 8,128 43 Praha 2 

Zítka Ondřej 


369


PODĚKOVÁNÍ OBCHODNÍM SPOLEČNOSTEM ZA SPONZOROVÁNÍ 

XI. SETKÁNÍ FYZIKÁLNÍCH CHEMIKŮ A 

ELEKTROCHEMIKŮ: 

ANTON PAAR, organizační složka, Bělohorská 

85, 169 00 Praha 6 - Břevnov 

http://www.anton-paar.cz/ 

Výroba přístrojů pro aplikace ve výzkumných, 

vývojových, provozních a průmyslových 

pracovištích 

BECKMAN COULTER, Radiová 1, 102 27 

Praha, 

http://www.immunotech.cz/services.htm 

BIO-RAD, Nad ostrovem 1119/7, 147 00 

Praha 4 

www.qcnet.com/cz 

Prodej laboratorních přístrojů 

BVT TECHNOLOGIES, Hudcova 78c, 612 00 

BRNO 

www.bvt.cz 

Vývoj a výroba biosenzorů 

CLONESTAR PEPTIDE SERVICES, s. r. o. 

Křižíkova 70, 612 00 Brno 

http://www.clonestar.cz/ 

Zakázková syntéza peptidů 

ENVINET a.s. Modřínová 1094 674 01 Třebíč 

www.envinet.cz 

Výrobce a dodavatel měřicích a informačních 

systémů pro energetiku a průmyslovou 

automatizaci. 

EPPENDORF Czech&Slovakia, Kolovratská 

1476, 251 01 Říčany u Prahy 

http://www.eppendorf.cz/ 

Prodej laboratorní techniky 

H Test Šafránkova 1243/3, 155 00 Praha- 

Stodůlky 

www.htest.cz 

Autorizovaný dovozce měřicí techniky Agilent 

do ČR a SR 

CHROMSERVIS s. r. o. Jakobiho 327, 109 00 

Praha 10 – Petrovice http://chromservis.cz/cz 

Prodej měřicí a regulační techniky se 

specializací na chromatografii a techniku pro 

sledování kvality životního a pracovního 

prostředí. 

370 

ChromSpec Jindřicha Plachty 28, 150 00 Praha 

www.chromspec.cz 

Přístrojová technika pro laboratoře 

LABOSERV Hudcova 532/78b, 612 00 Brno 

www.laboserv.cz 

Partner klinických laboratoří 

MANEKO, spol. s.r.o. Na Pískách 71, 16000 

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Laboratorní přístroje a technika 

MEDESA s.r.o., Na Vyšehradě 1092, 572 01 

Polička http://www.medesa.cz/ 

Nabízíme kompletní vybavení klinických 

laboratoří. 

METROHM ČESKÁ REPUBLIKA S.R.O Na 

harfě 935/5c 

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www.metrohm.cz 

PRAGOLAB s.r.o. Nad Krocínkou 55, 19000 

Praha 9 

www.pragolab.cz 

Vybavení laboratoří – chemické, biologické, 

fyzikální 

RADANAL s. r. o., Okružní 613, 530 03 

Pardubice http://www.radanal.cz 

Vědecký výzkum a vzdělávání v oblasti 

aplikace přírodních věd Lifescience; výživa a 

zdravotní diagnostika 

SIGMA ALDRICH Sokolovská100/94, 186 00 

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http://www.sigmaaldrich.com/czechrepublic.html 

SCHOELLER INSTRUMENTS Vídeňská 

1398/124, 148 00 Praha 

www.schoeller.cz 

Prodej a servis laboratorní techniky a 

zdravotnických přístrojů 

TRIGON-PLUS Západní 93, 251 70 Čestlice 

www.trigon-plus.cz 

Řešení pro vaši laboratoř


Sborník příspěvků 

XI. Pracovní setkání fyzikálních chemiků a elektrochemiků 

Uspořádala: Libuše Trnková 

Technická úprava: Sylvie Holubová, Petr Koudelka 

Vydala: Mendelova univerzita v Brně 

Tisk: Ediční středisko MENDELU v Brně 

První vydání, 2011¨ 

Náklad 

ISBN 978‐80‐7375‐514‐0 

371

➤ Týmová spolupráce 

4 Měření hustoty & koncentrace 

4 Reometry a viskozimetry 

4 Příprava vzorků/mikrovlnné pece 

4 Měření CO 2 a O 2 v nápojích 

4 Mikrovlnná syntéza 

4 Elektrokinetická analýza 

4 X-ray strukturální analýza 

4 Vysoce přesné měření teploty 

4 Refraktometry a polarimetry 

Anton Paar GmbH 

organizační složka 

Česká republika/ 

Slovenská republika 

Bělohorská 238/85 

169 00 Praha 6 - Břevnov 

CZECH REPUBLIC 

Tel.: +420 233 356 634 

Fax: +420 233 356 636 

info.cz@anton-paar.com 

info.sk@anton-paar.com 

www.anton-paar.cz 

www.anton-paar.sk 

210x297_basketball.indd 1 18.04.11 14:17

Pr ˇ ístroje a reagencie 

pro klinickou diagnostiku 

a biomedicínský výzkum 

Genetické 

analyzátory 

Automatizace 

Spektrofotometry 

a Readery 

Beckman Coulter Česká republika s.r.o. 

Murmanská 1475/4, 100 05 Praha 10 - Vršovice, www.beckman.cz 

Průtokové 

cytometry 

Centrifugy 

Analyzátory 

buněk

Electrochemical tools 

BVT Technologies, a.s., 

Hudcova 533/78c 

612 00 Brno 

Czech Repulic 

info@bvt.cz 

www.bvt.cz 

We offer a wide range of Electrochemical Sensors 

TFT Sensor substrates 

destinated for amperometric and conductometric measurements and also biosensors with immobilized enzymes. 

The standard sensor formats are formed on alumina ceramics with different combinations of electrode materials. 

The sensor response can be measured using our Biosensor analyzer or any other suitable potentiostat. Our facility 

offer includes design and development of sensor formats depending on user specific needs. Long term R&D projects 

co-operations are welcomed also. 

AC1 The most often used three-electrode sensor. It has 

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used as a substrate for glucose oxidase or acetylcholine 

esterase biosensor preparation (AC1.GOD, AC1.AChE). 

AC3 Narrow single working electrode sensor designed 

as replacement of metal electrodes from expensive materials. 

AC5 Array of eight working electrodes, each electrode has its own reference electrode. 

It is possible to measure eight signals at once. One electrode can be a standard, other 

electrodes can be modified by antibodies, enzymes, DNA, etc. to get an assay for more 

target substances in one measurement. 

AC6 Electrochemical sensor with one working, one 

reference and two auxiliary electrodes. 

AC8 Electrochemical sensor with 4 working electrodes; 

some of them may be modified by enzyme, DNA, antibody, 

etc. for multianalyte detection. 

AC10 Electrochemical sensor with array of 20 working 

electrodes and one common reference electrode; it was 

designed for complicated complex analysis of 20 analytes 

in one step. 

AC2 Electrochemical sensor with two working electrodes 

and one reference electrode serves for differential 

measurement of two signals at once. 

AC4 Single working electrode sensor with bigger metal 

surface compared to AC3. It may serve for metal coating. 

AC7 This sensor is shorter version of AC1 for different 

connector – soldered pins. 

AC9 Electrochemical sensor with array of 8 working 

electrodes and one common reference electrode; it was 

designed as a substrate for biosensor for multianalyte 

detection. 

CC1 Conductivity measurement sensor with finger 

structure of electrodes 

CC2 Conductivity measurement sensor designed for 

differential measurements of conductivity on the background 

of another effect.

Electrochemical tools 

Pump 

Accessories for Electrochemical Sensors 

BVT produces a wide variety of electrochemical sensors using 

screen printing technology. 

MicroFlow System (MFS) 

Our Microflow System is designed to 

remove the conventional problems 

rising within the standard electrochemical 

measurement setup. The 

system allows noiseless stirring and regulated 

liquid flow onto the electrode surface, control of 

temperature and atmosphere. The sensor chamber 

design is suitable to our AC1 sensor formats 

and permits easy replacement of used substrates. 

The Microflow system is predominantly used for 

Batch Injection analysis. 

Flow Cell 

The flow cell enables the use of AC1, CC1 sensor in a flow 

through arrangement. The cell ensures the wall-jet flow 

around the working electrode. The cell contains also the 

contact and output cable. 

A range of micro-flow pumps designed and developed to provide you with low 

order flow rates (0,01 – 200 µl per minute). The pumps are extremely small in size 

and come with pre-defined as well as user changeable flow rates. 

Potentiostats 

BVT Bioanalyzer 

The Biosensor analyzer BA1 is a small 

compact amperometric analyzer designed 

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analysis. The device is equipped with 

sophisticated program for biosensors 

signals evaluation. 

Connector 

Cell 

Pump 

Connector 

Sensor 

Flow 

Potentiostats 

Micro Flow 

Systems 

The connector enables the use of the electrochemical 

sensors AC1, AC4, CC1, CC2 in classical electrochemical 

arrangement with 

stirred vessel. 

Cell 

Simple glass cell serves for 

electrochemical measurements 

employing our TFT sensors. Cell openings are 

designed for sensor connector and stirrer. 

PalmSens potentiostat 

the most compact portable potentiostat 

for all kinds of electrochemical sensors 

and cells. It can be configured for a single 

application but can also be used as a generic electrochemical 

instrument. PalmSens can be extended to a multichannel potentiostat. 

eDAQ potentiostat 

a four-channel potentiostat that can operate each channel 

independently in three electrode mode, or operate 

two (bipotentiostat), three or four working electrodes with a common 

reference and auxiliary electrode.

ZAKÁZKOVÁ SYNTÉZA PEPTIDŮ 

• Produkt v závislosti na sekvenci a množství v čistotě od 70 do 98 % 

• Kvalitativní analýza (MALDI-TOF,ESI) 

• Kvantitativní analýza (HPLC) 

• Purifi kace peptidu pomocí HPLC 

• Možnost modifi kace peptidu (fosforylace, biotinylace, acetylace, 

značení izotopem, FAM) 

• Možnost konjugace peptidu na proteiny KLH (BSA) přes glutaraldehyd 

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• Dodací lhůty do 3 týdnů 

ZAKÁZKOVÁ VÝROBA 

PROTILÁTEK 

• Syntéza peptidu a následná konjugace na nosič (KLH, BSA) 

• Produkce polyklonálních protilátek v králících 

• Testování protilátek 

• Afi nitní purifi kace až 50 ml séra 

• Vzorek 5 -10 mg peptidů zdarma přiloženého k zásilce protilátky 

• Standardní dodání za 10-12 týdnů 

Clonestar 

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www.clonestar.cz

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Eppendorf Czech & Slovakia s.r.o. · Kolovratská 1476 · 251 01 Říčany u Prahy · Česká republika 

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Zastoupení na Slovensku: 

Eppendorf Czech & Slovakia s.r.o. - organizačná zložka · Prírodovedecká fakulta UK v Bratislave · Mlynská dolina 

842 15 Bratislava · Slovenská republika · Mobil: +421 911 181 474 · E-mail: eppendorf@eppendorf.sk · www.eppendorf.sk

Technologie AFM/SPM mikroskopů 

umožňuje zobrazování v atomárním 

měřítku s rozlišením až na desetiny 

nanometrů. Zásadní výhodou těchto 

mikroskopů je, že vzorky lze zobrazovat v 

libovolných podmínkách. Je možné měřit 

při různých teplotách dle potřeb aplikace 

nebo dokonce i přímo v kapalinách. 

AFM mikroskopie 

Agilent Technologies 

Společnost H TEST a.s. je výhradním distributorem Nanomeasurement Division Agilent 

Technologies, která zahrnuje AFM mikroskopii a nanoindentory. 

H TEST a.s. 

Šafránkova 3 

155 00 Praha 5 

Tel: 235 365 207 

info@htest.cz 

www.agilent.com/find/nano 

5500 AFM/SPM 

AFM mikroskop Agilent 5500 je vrcholný 

víceúčelový výzkumný mikroskopický systém 

pro AFM a SPM. Modulární koncepce 

této série dovoluje jednoduchou integraci 

stojanu pro velké vzorky až 150x200mm 

(LS), invertovaného mikroskopu (ILM), 

skenerů pro malé i velké zobrazované 

plochy, adaptérů pro uchycení vzorků, 

soupravy pro elektrochemii nebo videomikroskopu.

110415_chromservis_letak_Dani_SHS_CZ_fin.indd 1 21.4.2011 12:23:21

110415_chromservis_letak_Dani_SHS_CZ_fin.indd 2 21.4.2011 12:23:28

• 

• 

• 

• 

• 

• 

AKCE KINETEX 

2011 

Výběr z fází C18, XB-C18, PFP, C8 a HILIC 

Částice 2,6 µm pro systémy do 600 barů, 

částice 1,7 µm pro systémy do 1000 barů. 

Separační účinnost až až 300 000 teoretických pater. 

Nové Nové rozměry. 

Až 14× rychlejší analýzy, vyšší citlivost a a rozlišení 

Výrazné zvýšení výkonu výkonu a produktivity v laboratoři 

PLATNOST NABÍDKY 

DO 30. 6. 2011 

PHENOMENEX GUARANTEE 

SLEVA 20% 

PŘI ZAKOUPENÍ JEDNÉ KOLONY KINETEX 

SLEVA 30% 

PŘI ZAKOUPENÍ DVOU KOLON KINETEX 

Pokud kolony Kinetex s pevným jádrem a porézním 

povrchem neposkytnou nejlepší výsledky v LC 

technologiích, které jste kdy 

používali, zašlete nám data 

porovnávající srovnatelný 

VYŽÁDEJTE SI 

produkt do 40 dnů 

CENOVOU NABÍDKU NABÍDKU 

a PONECHEJTE SI KOLONU 

ZDARMA. 

Chromservis s.r.o., Jakobiho 327, 109 00 Praha 10 - Petrovice, www.chromservis.cz, tel: +420 274 021 211 

Chromservis SK s.r.o., Nobelova 34, 831 02 Bratislava, www.chromservis-sk.sk, tel: +421 911 181 098

ORGANICKÁ ROZPOUŠTĚDLA A KINETEX 

2.6 µm 

 

Rozpouštědlo 

RADY A TIPY 

Existuje několik kritických parametrů (viz tabulka dole), které je potřeba vzít v úvahu při výběru vhodného organického 

rozpouštědla použitého jako mobilní fáze pro kolony Kinetex. Jednou z nejdůležitějších vlastností je viskozita rozpouštědla, 

protože solventy s vysokou viskozitou mohou vyvolat v HPLC systému příliš velký protitlak. Dalšími důležitými vlastnostmi rozpouštědel 

jsou UV cuttoff, náklady a index polarity. Rozpouštědlo s vysokým UV cutoff bude mít za následek nízkou citlivost 

při použití UV/VIS detekce. Rozpouštědla s nízkým indexem polarity mají zpravidla rychlejší eluci organických sloučenin a jsou 

běžně používány pro čištění kolon. 

• Acetonitril je pravděpodobně nejlepším organickým rozpouštědlem, protože ve směsi s vodou dává nejnižší protitlak systému 

a má také velmi nízký UV cutoff a tím lepší UV/Vis citlivost detekce. 

• Metanol je další populární organické rozpouštědlo, protože má srovnatelnou eluční sílu s acetonitrilem, má relativně nízkou 

UV absorbanci, a je levnější než acetonitril. Hlavní nevýhodou metanolu, a to zejména při použití malých zrn fáze, je to, 

že jeho používání může vést k protitlaku, který překračuje mnohé limity HPLC systémů. 

• Aceton je méně běžně používané rozpouštědlo, protože má vysokou UV absorbanci, ale může být úspěšně použit, pokud 

analyty absorbují při vyšších vlnových délkách nebo v případě použití jiných typů detektorů jako např. MS. Má podobné 

eluční vlastnosti jako acetonitril. 

• Etanol se obecně nedoporučuje, protože vede k velmi vysokému protitlaku ve směsi s vodou. 

• Iso-, n-propanol mají poměrně velkou eluční sílu a jsou nejběžněji používány k čištění kolon při nízkých průtocích, protože 

rovněž dávají v systému vysoký protitlak. 

• Tetrahydrofuran má podobnou eluční sílu jako n-propanol. 

*Přibližná hodnota tlaku směsi s vodou (50/50) na koloně Kinetex 150 mm × 4,6 mm při 

průtoku 1,2 ml/min (20 °C) 

1.7 µm 

Bližší informace o výrobcích najdete na našich webových stránkách www.chromservis.cz nebo se informujte u našich regionálních zástupců. 

Tato nabídka platí do 30. 6. 2011 a nemůže být kombinována s jinými slevami. Cenovou nabídku a bližší informace o nabízených produktech si prosím 

vyžádejte prostřednictvím e-mailu prodej@chromservis.cz, nebo predaj@chromservis-sk.sk. Obrázky jsou pouze ilustrativní. 

 

Viskozita 

[cP] 

Zpětný tlak 

[bar]* 

Index 

polarity 

UV cutoff 

[nm] 

Acetonitril 0,37 200 5,8 190 

Metanol 0,60 390 5,1 205 

Aceton 0,32 325 5,1 330 

Etanol 1,20 630 5,2 210 

n-Propanol 2,27 650 3,9 210 

Tetrahydrofuran 0,55 430 4,0 215 

Chromservis s.r.o., Jakobiho 327, 109 00 Praha 10 - Petrovice, www.chromservis.cz, tel: +420 274 021 211 

Chromservis SK s.r.o., Nobelova 34, 831 02 Bratislava, www.chromservis-sk.sk, tel: +421 911 181 098

NABÍDKA SPEKTROFOTOMETRŮ 

Dvoupaprskové UV/Vis spektrofotometry s chlazenými 

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• monochromátor s vysokým rozlišením (štěrbina 1,4 nm 

nebo nastavitelná. Optické prvky Zeiss 

• zabudovaný holmiový filtr pro kontrolu vlnových délek 

• rychlost měření spekter až 6000 nm/min 

• rychlé kinetiky 3D 

CHROMSPEC spol. s r.o. 

PURELAB flex 

AstraGene Life Sciences Spectrophotometer je 

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vzorků o objemu od 2 µl přímo ve špičce automatické 

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Se spektrofotometrem AstraGene je dodávána i 

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Mníšek pod Brdy, 252 10, Lhotecká 594 

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analytikjena 

Jednotka pro přípravu ultračisté vody je určena pro 

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Pro zajištění nízké úrovně bakteriálního znečištění a 

nízkých hodnot TOC je model PURELAB flex vybaven 

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SPOL. S R.O. 

SPECORD Plus 

AstraGene 

email: info@chromspec.cz 

www.chromspec.cz

Maneko, spol. s r. o. 

Na Pískách 71 

160 00 Praha 6 

www.maneko.cz 

Katalogy zasíláme zdarma 

Fax: 

233 335 638-9 

233 336 579 

233 332 656

Metrohm Česká republika s.r.o. 

Na Harfě 935/5c Telefon: 00420 246 063 433 

190 00 Praha 9 Fax: 00420 246 063 468 

Web: www.metrohm.cz 

Titrace, iontová chromatografie, elektrochemie… 

Kompletní řada iontových analýz pod jednou střechou 

Naše firma se specializuje v oblastech přístrojového vybavení 

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Naše přístroje jsou široce 

využívány v oblastech chemického 

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Jen to nejlepší řešení je dost dobré pro Vaše 

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Spolehlivé výsledky měření po celou dobu životnosti Vašeho přístroje 

Bez ohledu na to zda provádíte analýzu vody s 

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VÝROBKY NEJVYŠŠÍ KVALITY 

Mrazící boxy - 40°C, - 86°C, -152°C 

Lednice laboratorní, farmaceutické, transfúzní 

CO 2 inkubátory 

Chlazené inkubátory 

Autoklávy 

Sušárny 


mail@schoeller.cz Tel.: 261 009 111 Fax: 244 470 745

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