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Luminescence (In press)Published Calibration online analytical in Wiley applications InterScience studies (www.interscience.wiley.com) on PVP hydrogel-supported DOI: luminol 10.1002/bio.935CL ORIGINAL RESEARCH ORIGINAL RESEARCH1ORIGINAL RESEARCH<strong>Studies</strong> on PVP hydrogel-supported luminolchemiluminescence: 2. Luminometer calibration andpotential analytical applicationsErick Leite Bastos, 1 Luiz Francisco Monteiro Leite Ciscato, 1 Fernando Heering Bartoloni, 1 LuizHenrique Catalani, 1 Paulete Romoff 2 and Wilhelm Josef Baader 1 *1Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, 05508-900 São Paulo, SP, Brazil2 Faculdade de Ciências Biológicas, Exatas e Experimentais, Universidade Presbiteriana Mackenzie, Rua da Consolação 896, 01302-907 São Paulo, SP, BrazilReceived 27 March 2006; revised 16 May 2006; accepted 17 May 2006ABSTRACT: The use of poly(N-vinyl-2-pyrrolidone) (PVP) hydrogel-supported luminol chemiluminescence (CL) for the automaticdetermination of hydrogen peroxide and the quantification of the antiradical capacity of Trolox is described. The hydrogelcontaining luminol and hemin is prepared directly on a 96-well microplate and can be stored for up to 3 months without significantdecrease in CL quantum yields. Furthermore, this system can also be used as a secondary light standard for the calibration ofmicroplate luminometers. Copyright © 2006 John Wiley & Sons, Ltd.KEYWORDS: PVP; luminol; antiradical; hydrogen peroxide; luminometer calibrationINTRODUCTIONThe chemiluminescence (CL) of luminol (Scheme 1) canbe induced by free radical and/or redox processes andthe simplicity and sensitivity of this technique is used toadvantage in the development of analytical methods forthe monitoring of free radical production or consumption(1–5). In recent years, a series of luminol-basedanalytical methods on alternative supports has been proposedto detect hydrogen peroxide directly or substrateswhich can be converted to it. In addition, reductantshave also been quantified by these systems which, inmost cases, are used as remote chemical sensors (6–9).In CL assays, the oxidation of a specific substrateresults in the formation of a high-energy intermediatewhich is transformed into an electronically excitedproduct, responsible for light emission upon decay toits ground state. The CL reagents can be incorporatedinto flow-injection analysis systems, but this demandssignificant amounts of reagents and, consequently,increased residue production. The incorporation of CLreagents and/or catalysts (e.g. dehydrogenases,peroxidases, oxidases, hemin) within hydrogels andpolymeric thin films for the detection of inorganic and*Correspondence to: W. J. Baader, Instituto de Química, Universidadede São Paulo, Av. Prof. Lineu Prestes 748, 05508-900 São Paulo, SP,Brazil.E-mail: wjbaader@iq.usp.brContract/grant sponsor: FAPESP, Brazil; Contract/grant number:98/05445-9; 00/06652-0; 01/07477-0.Contract/grant sponsor: CNPq, Brazil.Contract/grant sponsor: CAPES, Brazil.Contract/grant sponsor: PADCT, Brazil.Copyright © 2006 John Wiley & Sons, Ltd.Scheme 1.CL oxidation of luminol in alkaline aqueous media.organic chemicals provides a simple alternative to suchsystems (8–10).Recently we have investigated the CL propertiesof hemin-catalysed luminol oxidation by hydrogen peroxidein a PVP hydrogel matrix. Multivariate factorialanalysis (MFA) was used to quantify the effect ofreagent concentrations on initial emission intensities(I 0 ), emission areas (S) and observed decay rate constants(k obs ). Using this approach, it is possible to accessoptimal experimental conditions for specific analyticalapplications (11).This paper describes the utilization of the PVPsupportedluminol–hemin system (PVP hydrogel CLsystem) for the calibration of commercial microplateluminometers and demonstrates that this automatedsystem can be used for the determination of hydrogenperoxide and Trolox, an antiradical standard.MATERIALS AND METHODSReagentsLuminol (5-amino-2,3-dihydro-phthalazine-1,4-dione)was obtained from Merck (Germany) and a stockLuminescence (In press)DOI: 10.1002/bio

ORIGINAL RESEARCHsolution (10 mmol/L) was prepared in 1 mol/L NaOH,kept at 4°C and used within 10 days. The workingstock solution was prepared by dilution in phosphatebuffer (0.1 mol/L, Na 3 PO 4 /Na 2 HPO 4 ), pH 11.6. Thefinal luminol concentration was determined spectrophotometricallyat 347 nm (ε = 7600 mol/L/cm).Hydrogen peroxide (Peróxidos do Brasil, Brazil)was obtained as a 60% w/w unstabilized aqueoussolution. The final concentration after dilution withdemineralized water (18 MΩ, Milli-Q, Millipore) wasdetermined spectrophotometrically, as described byCotton and Dunford (12). Hemin (ferriprotoporphyrinIX chloride) was purchased from Sigma (St. Louis,USA). A stock solution was prepared by dissolving2.5 mg hemin in 5 mL 1 mol/L aqueous NaOH. Theworking solution is a 1:100 dilution with 1 mol/L NaOH(8 µmol/L). The concentration was determined spectrophotometricallyusing ε = 58400 mol/L/cm at 382 nm(13). Poly(N-vinyl-2-pyrrolidone), known as PlasdoneK-90 (M w = 1.2· × 10 6 ; M n = 3.6· × 10 5 ), was obtained fromGAF Chem. Co. (USA). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was obtainedfrom Aldrich (Milwaukee, USA) and used as antiradicalstandard.InstrumentsThe CL emission experiments were performed on aBerthold EG&G 96V Lumimat microplate luminometer.Absorption spectra were recorded using a ShimadzuMultispec 1500 UV-visible spectrophotometer.Preparation of PVP hydrogel containing luminoland heminAliquots (200 µL) of a solution containing 120 mg/mLPVP and adequate concentrations of luminol and heminin phosphate buffer (pH 11.6, µ = 0.1 mol/L) were placedin a 96-well microplate, which was positioned 20 mm distantfrom a pen-type Heraeus (Hanau, Germany) 20 Wlow-pressure Hg lamp (λ em = 254 nm; 200 mm long). Thesolution was irradiated for 30 min under a slow flux ofinert gas. The resulting microplates can be used directlyor dried for storage and continuous use. For storage, themicroplates were submitted to a slow argon flux until athin film (ca. 2 mm) was formed, packed in PVC film andstored at 4°C.Hydrogen peroxide determinationThe freshly prepared 96-well microplate containing thePVP hydrogel-supported luminol–hemin system (PVPhydrogel CL system) was put into the cavity of aluminometer and thermostated to 25.0 ± 0.5°C. The reactionwas initiated by automatic jet injection of 100 µLaqueous hydrogen peroxide solution in adequateCopyright © 2006 John Wiley & Sons, Ltd.E. L. Bastos et al.concentration. To perform the experiments with storedmicroplates, 200 µL phosphate buffer (pH 11.6, µ =0.1 mol/L) were added to each well prior to placing itinto the luminometer cavity, followed by hydrogen peroxideaddition (100 µL) to initiate the measurement.The results obtained in both cases were very similar andfinal reagent concentrations were reported for a finalvolume of 300 µL (final concentrations: PVP, 80 mg/mL;luminol, 1.0 mmol/L; hemin, 7.7 nmol/L). Emission decaywas monitored for 1 h in cycles of 60 measurements/well/h.Quantification of the antiradical effect ofTroloxAliquots (60 µL) of a Trolox solution in adequateconcentration were added to each well of a microplatecontaining 200 µL PVP hydrogel CL system. Thereaction was initiated by addition of 40 µL aqueous0.75 mmol/L hydrogen peroxide solution to eachwell, using the automatic jet injection system presentin the Berthold luminometer (final concentrations:PVP, 80 mg/mL; luminol, 1.0 mmol/L; hemin, 7.7 nmol/L; H 2 O 2 , 0.10 mmol/L). Emission decay was monitoredfor 1 h in cycles of 60 measurements/well/h.Microplate luminometer calibrationAqueous media calibration. To a single well of a white96-well microplate thermostated to 25.0 ± 0.5°C, containing200 µL luminol solution (15.0 nmol/L) in phosphatebuffer (pH 11.6, µ = 0.1 mol/L), 40 µL 75.0 µmol/Laqueous hydrogen peroxide solution were added byautomatic injection. The reaction was initiated byaddition of 20 µL hemin solution (0.8 nmol/L). Aftercomplete emission decay in about 20 s, 20 µL hemin(2.4 nmol/L) solution were added and a low-intensityemission was observed. Finally, an additional injectionof the concentrated hemin solution was made (20 µLhemin 2.4 nmol/L), normally not leading to measurablelight emission, to assure complete luminol consumption.The final reaction mixture contains 0.37 nmol/L hemin,10.0 nmol/L luminol and 10.0 µmol/L hydrogen peroxidein 300 µL final volume.Hydrogel calibration. To a single well of a microplatefilled with 240 µL of PVP hydrogel CL system([luminol], 12.5 nmol/L; [hemin], 0.26 nmol/L), 40 µL75.0 µmol/L hydrogen peroxide solution were addedby automatic injection. After total emission depletion,an additional volume of 20 µL hemin (2.4 nmol/L)solution was added to the reaction mixture. In theexperimental conditions utilized, the last hemin additionleads to very low-intensity light emission and theemission area was, in all cases tested, less than 5% ofthe initial emission area. The final reaction mixtureLuminescence (In press)DOI: 10.1002/bio

Calibration analytical applications studies on PVP hydrogel-supported luminol CL ORIGINAL RESEARCH ORIGINAL RESEARCH3contains 80 mg/mL PVP, 0.37 nmol/L hemin, 10.0 nmol/L luminol and 10.0 µmol/L hydrogen peroxide in 300 µLfinal volume.Calculation method. The area of emission can be convertedfrom arbitrary units (a.u.) to Einstein (E, ‘mols’of photons) using the luminol conversion factor ( f lum ),which is calculated by equation 1:flumCopyright © 2006 John Wiley & Sons, Ltd.lumnlum= φ [ Eau / . .](1)Slumwhere φ lum is the luminol quantum yield, n lum is thenumber of mols of luminol and S lum is the area under thecurve of emission, which is obtained by integration ofthe light emission intensity as a function of the reactiontime. The quantum yield for a certain CL orbioluminescence (BL) reaction (φ sample ) is obtained byequation 2:sample lum photoφ samplensample= S f f(2)where S sample is the area under the curve of the BL/CLreaction to be calibrated, f lum is the luminol factorobtained from equation 1, n sample is the number of molsof the limiting reagent, and f photo is the photomultipliertube (PMT) wavelength sensitivity factor. The latterfactor is required if light emission from the samplesystem occurs in a wavelength region different fromluminol emission (420 nm), since commercial PMTsshow wavelength-dependent sensibility. The relativesensibility of a specific PMT with the emission wavelengthis normally available from the equipment manufacturer.For the PVP hydrogel CL system this factor isf photo = 1, as its emission spectrum is identical to that ofthe aqueous system (11).The emission intensity (I, a.u./s) can also be calibratedand thus expressed as calibrated intensity (I c ) in E/susing f lum and f photo (equation 3). In this way, light emissionintensities can be reported in absolute values andtherefore experimental results obtained by differentacquisition equipments (i.e. luminometers, photoncounters and fluorimeters) in different research laboratoriesbecome comparable:I C [E/s] = I[a.u./s] × f lum [E/a.u.] × f photo (3)Statistical and mathematical analysisAll values were expressed as mean ± standard deviation(SD) of at least three independent experiments. All calculationswere performed using Origin 6.1 (OriginLabCorp., 2000).RESULTS AND DISCUSSIONLuminometer calibration using thePVP-supported luminol–hemin systemPrimary calibration of photomultipliers can be accessedby the use of a source of light radiance calibrated withreference to black body radiation (14). However, severalissues concerning the light emission intensity, refractiveindex discrepancies and, most importantly, geometricfactors make the use of this methodology difficult (14–17). To overcome these technical difficulties, secondarycalibration with reference to the CL oxidation of luminoleliminates the necessity for complex geometrical factorcalculations and is widely used (15–18). The typicalprocedures consist of the reaction of luminol with hydrogenperoxide in the presence of hemin, which has anabsolute CL emission quantum yield (φ CL ) of 0.0124in alkaline aqueous media (17–19). This value is independentfrom luminol concentration over a wide range(10 −9 –2.0 mmol/L) and the methods feasibility requiresan at least 100 times molar excess of hydrogen peroxideover luminol and successive additions of catalyst to guaranteecomplete luminol consumption (17–19).The amount of light produced by the conventionalaqueous calibration system is compared with that of thePVP hydrogel CL system obtained in the same experimentalconditions (i.e. identical concentrations ofreagents). The obtained results allow us to determine theabsolute light emission of the luminol–hemin–hydrogenperoxide reaction in the cross-linked polymer. Theluminol conversion factor is determined from theaqueous calibration system as f lum = 6.87 ± 0.21 × 10 −18E/a.u. (equation 1), and the emission quantum yield inthe PVP hydrogel system is φ CL = 2.39 ± 0.07 × 10 −3 E/mol(equation 2).The amount of luminol used in calibration (3.0× 10 −12 mol) is able to generate a total emission of7.16 ± 0.22 × 10 −15 E in the PVP hydrogel system.The calibrated initial emission intensity (I C ) in thestandard PVP hydrogel system is determined to beI C = 9.99 × 10 −14 E/s; absolute emission intensities are alsoreported here for the analytical applications of thesystem.Stability of the hydrogel CL systemTo access the stability of the PVP-supported luminol–hemin system, the amount of light produced in the presenceof hydrogen peroxide has been monitored for 3months. Microplates were stored at 4°C under argonatmosphere and nine experiments (three independentreplicates in three microplates) were performed every 10days (Fig. 1).The emission intensity and φ CL proved to beconstant for about 30 days. After this period, aLuminescence (In press)DOI: 10.1002/bio

Calibration analytical applications studies on PVP hydrogel-supported luminol CL ORIGINAL RESEARCH ORIGINAL RESEARCH5of 6.8 × 10 −7 mol/L. The regression equation for thelinear dependence is S C = 7.78 × 10 −11 + 1.05 × 10 −5 [H 2 O 2 ]with correlation coefficient (R) = 0.9977 (n = 6). Athigher peroxide concentrations the total light emissionshows a significant decrease (Fig. 3A, insert), a factwhich may indicate the destruction of hemin by excessof peroxide and incomplete conversion of hydrogenperoxide. Similar results are obtained if the initial emissionintensities are correlated with [H 2 O 2 ]; in this casethe regression equation for the linear dependence isI C = 9.04 × 10 −16 + 5.83 × 10 −10 [H 2 O 2 ] with R = 0.9998(n = 6). Also the light intensities show a sharp decreaseat higher peroxide concentrations (Fig. 3B).As can be seen from Table 1, the observed detectionlimit (6.8 × 10 −7 mol/L) is compatible with several otherdetection methods based on luminol CL, immobilized orin solution (21–29).We also can predict from the MFA that the linearrange of the peroxide dependence can be extended tohigher concentrations by using higher hemin concentrations,without significant changes in the detection limit(11). The results obtained for the detection of hydrogenperoxide are very promising in terms of application automation.For example, oxidases could be inserted intothe polymeric matrix together with luminol and hemin,allowing the automatic detection of specific substrates,such as glucose or lactose, in the µmol/L concentrationrange by quantification of hydrogen peroxide generatedby enzyme catalysed substrate oxidation (30, 31).Figure 3. Correlation of total light emission (A) and emissionintensity (B) with the hydrogen peroxide concentration.[Luminol], 1.0 mmol/L; [hemin], 7.7 nmol/L; [PVP], 80 mg/mL.concentrations), since we are interested here in detectingH 2 O 2 at the low concentration range. A lineardependence of the calibrated total light emission (S C )with the concentration of H 2 O 2 is observed up to 5 ×10 −4 mol/L (Fig. 3A), with an estimated detection limitAntiradical assay: effect of Trolox on emissionintensities and emission areasIn order to use the luminol CL as the basis ofan antiradical assay, the kinetics should consist in slowlight emission decay (32). This behaviour indicates aconstant, or nearly constant, production of free radicalintermediates derived from luminol, hemin and hydrogenperoxide. From our MFA we chose intermediateconcentrations of all the three reagents to access slowemission decay and a relatively slow consumption of theTable 1. Detection limits for hydrogen peroxide using several chemiluminescencemethodsDetection limitSystem (mol/L) ReferenceLuminol–KIO 4 3.0 × 10 −8 (21)Luminol–K 3 Fe(CN) 6 6.0 × 10 −6 (22)Luminol–HRP 8.0 × 10 −6 (24)Luminol–peroxidase 1.0 × 10 −8 (25)Luminol–Co(II) 5.0 × 10 −9 (26)Luminol–calixarene/Cu(II) 3.0 × 10 −6 (27)DTMC 4.0 × 10 −8 (28)Luminol–hemin 3.0 × 10 −9 (23)Luminol–hemin–PVP 6.8 × 10 −7 This workDTCM, 7-(4,6-Dichloro-1,3,5-triazinylamino)-4-methylcoumarin.Copyright © 2006 John Wiley & Sons, Ltd.Luminescence (In press)DOI: 10.1002/bio

ORIGINAL RESEARCHantiradical compound (Fig. 2). This setting allows thequantitative determination of the antiradical capacityof compounds in the range of µmol/L and sub-µmol/Lconcentrations (33–36).The response of the hydrogel system to antiradicalcompounds is investigated by using Trolox, a stablewater-soluble analogue of α-tocopherol. This compoundis widely used as a reference in such assays and it isassumed that it is able to scavenge two radical species,resulting in the formation of quinoidal analogues (37–44). The area under the CL decay curve, indicating thetotal light emitted, also represents the total amount ofthe reactive species present in the reaction media which,ultimately, lead to light emission. Radical scavengingcompounds will consume these reactive species and suppressthe light emission until all antiradical compoundsin the system are completely depleted. The emissionsuppression area (S SUP = total emission in the absenceof antiradical—total emission in the presence of antiradical)is therefore directly proportional to the number ofradical species deactivated by the antiradical and can beutilized as a measure for the antiradical capacity of thecompound (20).Addition of the antiradical to the hydrogel containingluminol and hemin before adding hydrogen peroxideleads to a drastic change in the emission intensitiesand only slight variations in the kinetic emission profile(Fig. 4A). A linear correlation between Trolox concentrationand the total light inhibition is observed (Fig. 4B)with a regression equation of total light inhibition =0.01 + 0.20 [Trolox] (R = 0.9995, n = 5), demonstratingthat this system can be utilized for antiradical capacitydetermination in analogy to the luminol CL antiradicalassay in aqueous medium (20).An antiradical added to the system will compete withluminol for the hemin-derived oxidizing intermediatesand any other oxidant species present in the reactionmixture, as well as intercepting luminol-derived freeradical species and oxidants. In this way, a delay periodin CL emission would be expected until all antiradicalspresent have been consumed. This lag-time should beproportional to the antiradical concentration and itscapacity, as it results from the oxidation of a non-CLsubstrate instead of luminol (45). However, the additionof antiradicals prior to initiation of the radical reactionmay interfere in the initial radical formation step, whichcould lead to less reliable results than those obtainedwith addition of antiradicals during the reaction course,where a constant steady-state concentration of radicalsis already established (46). A delay phase is notobserved in the PVP hydrogel system; however, theemission intensities are considerably lower in the presence(I AOH ) than in the absence (I) of the antiradical. Alinear correlation between the light intensity inhibition(I − I AOH /I) and the concentration of Trolox is observed(Fig. 4C), showing the existence of a competitionE. L. Bastos et al.Figure 4. (A) Effect of Trolox addition on luminol CL, usingthe PVP hydrogel CL system. (B) Correlation of emissionsuppression areas (S SUP ) with the concentration of addedTrolox. (C) Correlation of the suppression of light intensity(I − I AOH /I) with the concentration of added Trolox. [Luminol],1.0 mmol/L; [hemin], 7.7 nmol/L; [H 2 O 2 ], 0.1 mmol/L; [PVP],80 mg/mL.Copyright © 2006 John Wiley & Sons, Ltd.Luminescence (In press)DOI: 10.1002/bio

Calibration analytical applications studies on PVP hydrogel-supported luminol CL ORIGINAL RESEARCH ORIGINAL RESEARCH7between the antiradical compound and luminol for theoxidizing species, e.g. Hem–Fe(IV) in the polymermatrix system (light intensity inhibition = 0.02 + 0.21[Trolox]; R = 0.9971, n = 5). This competition leads tolower emission intensities in steady-state conditions inthe presence of the antiradical and also to lower totalemission areas. Depending on the specific application,both parameters (suppression area and intensity inhibition)can be utilized to quantify antiradical capacity, asshown by the linear correlation between the two parametersand the Trolox concentration (Fig. 4B, C).The different behaviour of the PVP-supportedluminol CL system, as compared to analogous aqueousassays (20), is supposed to be related to diffusion factorsin the polymer system. One possible explanationcould be that the reaction rate might be determinedmuch more by the diffusion of the species than by itsreactivity, thereby ‘masking’ the much higher reactivityof the antiradical Trolox towards the oxidizing speciesand allowing the competition by luminol anion, even ifboth species are present in comparable concentrations.In fact, in the system studied the luminol concentrationis three orders of magnitude higher than the Troloxconcentration; however, in the analogous aqueoussystem Trolox is sufficiently more reactive and ableto scavenge all the oxidizing species present until itseffective complete depletion (20). Whatever may bethe exact reason for the different behaviour of theaqueous and PVP-supported luminol CL system in thepresence of an antiradical compound, it is shown in thiswork that the polymer-supported system can beperfectly utilized for antiradical capacity determinationand opens up the possibility of automation usingmicroplate luminometers.CONCLUSIONSIn conclusion, we have shown that the PVP hydrogel CLsystem can be utilized up to 90 days after preparation forthe determination of hydrogen peroxide concentrationand as an antiradical capacity assay, since optimizedconditions are used to maximize its sensibility and reproducibility.Furthermore, it can be conveniently utilizedfor the calibration of microplate luminometer PMTsand, consequently, the determination of CL quantumyields. This approach allows the direct comparison of theexperimental results (emission intensities and total lightemission) obtained in different research laboratories.AcknowledgementsA donation of 60% hydrogen peroxide by Solvay–Peróxidos do Brasil LTDA Co. is gratefully acknowledged.This work was supported by Fundação deAmparo à Pesquisa do Estado de São Paulo (FAPESP;Copyright © 2006 John Wiley & Sons, Ltd.98/05445-9, 00/06652-0, 01/07477-0), Conselho Nacionalde Desenvolvimento Científico e Tecnológico (CNPq),Coordenação de Aperfeiçoamento de Pessoal de NívelSuperior (CAPES) and Programa de Apoio ao DesenvolvimentoCientífico e Tecnológico (PADCT).REFERENCES1. Merényi G, Lind J, Eriksen TE. Luminol chemiluminescence—chemistry, excitation, emitter. J Biolumin. Chemilumin. 1990; 5:53–56.2. Rao PS, Luber JM Jr, Milonowicz J, Lalezari P, Mueller HS.Specificity of oxygen radical scavengers and assessment of thefree radical scavenger efficiency using luminol enhancedchemiluminescence. Biochem. 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