Chip-Based CE for Avidin Determination in Transgenic Tobacco and ...

Chip-Based CE for Avidin Determination in Transgenic Tobacco and ...

Chip-Based CE for Avidin Determinationin Transgenic Tobacco and Its Comparisonwith Square-Wave Voltammetry andStandard Gel Electrophoresis2008, 67, S75–S81Sona Krizkova 1 , Vendula Hrdinova 1 , Vojtech Adam 1,2 , Elisabeth P. J. Burgess 3 , Karl J. Kramer 4 ,Michal Masarik 5 , Rene Kizek 1,&1 Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry, Zemedelska 1, 613 00 Brno, Czech Republic;E-Mail: kizek@sci.muni.cz2 Department of Animal Nutrition and Forage Production, Faculty of Agronomy, Mendel University of Agriculture and Forestry,Zemedelska 1, 613 00 Brno, Czech Republic3 The Horticulture and Food Research Institute of New Zealand Limited, Mt Albert Research Centre, Private Bag, 92169 Auckland,New Zealand4 Grain Marketing and Production Research Center, Agricultural Research Service, US Department of Agriculture, Manhattan, KS 66502,USA5 Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Komenskeho namesti 2, 662 43 Brno, Czech RepublicReceived: 8 August 2007 / Revised: 13 February 2008 / Accepted: 18 February 2008Online publication: 18 April 2008IntroductionAbstractAvidin transgenic plants are a potential tool for providing resistance against various speciesof insect pests due to the sequestration of vitamin H (biotin) in the plant from the insect pests.In this project we compared three techniques for avidin determination in transgenic tobaccoplants, a novel chip-based capillary electrophoretic method (Experion), classical polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) and asquare wave voltammetric method using a carbon paste electrode. We determined that theautomated chip-based capillary electrophoretic method is rapid, sensitive and the resultsobtained are well repeatable. The avidin content measured in transgenic tobacco leavesusing chip-based capillary electrophoresis varied from 15 to 854 ng per mg of fresh massdepending on the individual plant.KeywordsChip-based capillary electrophoresisAvidin, transgenic tobacco plantsVoltammetrySDS-PAGEPresented at: Advances in Chromatographyand Electrophoresis 2007 and Chiranal 2007,Olomouc, Czech Republic, June 24–27, 2007.Protection of various agricultural cropsagainst insect pests that cause severeeconomic damage is an issue beingresearched worldwide [1–3]. One ofmany possibilities to achieve higherproductivity in agricultural crops is touse pesticides for crop protection. It is incommon knowledge that pesticide residuescan contaminate the environment,food and many of them are poisonous tohumans and other organisms [4].One way to reduce the use of harmfulpesticides is to exploit the methods ofgenetic engineering. One of the mostsuccessful applications of genetic engineeringis the utilization of recombinantendotoxins of Bacillus thuringiensis (Bt)[2, 5, 6]. Hood et al. in 1997 [5] andKramer et al. in 2000 [2] described thecommercial production and analysis ofavidin in maize. The susceptibility of thiscrop to stored-product insects had notbeen examined until the latter paper, buttoday it is clear that avidin maize ishighly resistant toward many species ofOriginal Chromatographia Supplement Vol. 67, 2008 S75DOI: 10.1365/s10337-008-0606-6Ó 2008 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH

stored-product insect pests like lessergrain borer (Rhyzopertha dominica),sawtoothed grain beetle (Oryzaephilussurinamensis), red flour beetle (Triboliumcastaneum), confused flour beetle(Tribolium confusum), flat grain beetle(Cryptolestes pusillus), Indianmeal moth(Plodia interpunctella), Mediterraneanflour moth (Anagasta kuehniella), warehousebeetle (Trogoderma variabile),which exhibit 95–100% mortality at>100 ppm avidin concentrations [2]. Sofar in addition to maize, transgenic avidinexpressing tobacco [7], rice [8], andapple [9] have been developed.Avidin is a glycosylated basic proteinwith isoelectric point of 10–10.5 composedof four subunits with a tetramericrelative molecular mass of about 67,000.Each subunit contains one binding sitewith high affinity for biotin (vitamin H,cis-hexahydro-2-oxo-1-H-thieno-[3,4]-imidazoline-4-valeric acid) with a dissociationconstant K d =10 15 M. Thisinteraction exhibits one of the highestknown affinities between a protein andits ligand, and can be utilized for variousfields of avidin-biotin technologyincluding immunohistochemistry, electronmicroscopy, DNA hybridizationand biosensors. In nature avidin occursas a minor component of bird, reptileand amphibian egg white and probablyplays a role in protecting embryos bybiotin sequestration [10]. Hereby it caninterfere with biotin-containing enzymes,which are involved in carboxylation,decarboxylation and transcarboxylationreactions [2].The most commonly used techniquesfor detection and evaluation of avidinbiotininteractions are ELISA, fluorimetry,and several electrochemicalmethods [11–16]. In our previous paperswe proposed alternative methods foravidin detection in transgenic plants.With a carbon paste electrode and suitableconditions for electrochemicalmeasurement, we were able to detectzeptomole levels of the protein in 5 lLsample volume [17]. In comparison withthe ELISA technique, the electrochemicaldetermination exhibited good agreement,but it was much more sensitive[17, 18].In the present work we evaluatedchip-based capillary electrophoresis foravidin analysis in transgenic tobaccoplants (Nicotiana tabacum) and comparedthis technique with two othermethods, standard gel electrophoresisand voltammetry. Tobacco plants areuseful model plant for genetic engineeringof plants and the methods for itstransformation via the used vectors areroutinely used and well handled. Thereforewe choose this model in our experiments.ExperimentalChemicalsAvidin, carbon powder, mineral oil andother chemicals were purchased fromSigma-Aldrich Chemical Corp. (St. Louis,USA). Solutions were prepared usingwater from Sigma Aldrich in ACS purity.Stock standard solutions of avidin at1 lg mL 1 were prepared and stored inthe dark at 4 °C. All solutions werefiltered through a 0.45 lm Teflon membranefilter (MetaChem, Torrance,CA, USA).Biological SamplesTransgenic tobacco plants (Nicotianatabacum cv. Samsun) were obtained fromthe Horticulture and Food ResearchInstitute of New Zealand. The constructionof plasmids used for transformationof maize with the chicken avidin gene,transformation, tissue culture, and generationof avidin-expressing transgenicplants were as described previously [19].As controls, nontransformed plants wereused. All plants were grown in a containmentglasshouse at approximately28 °C. Following harvest, leaves fromidentified plants were stored in 20 °C.Sample PreparationApproximately 0.1 g of the frozen planttissue (leaves) was homogenized usingmortar and liquid nitrogen, and extractedfor 4 h at 4 °C with constantstirring in buffer (5:1, w/v) containing50 mM sodium carbonate (pH 11),500 mM EDTA, and 0.05% (v/v)Tween-20. The homogenate was centrifugedat 16,000 · g for 15 min at 4 °C(Eppendorf, Type 16F6-38 rotor,Germany), supernatants were removedand centrifuged at 14,500 · g for 15 minat 4 °C, the pH was adjusted up to 10.5with 1 M NaOH, and then the mixturewas centrifuged at 11,000 rpm for30 min to remove precipitated proteins[5]. The protein concentration in plantsamples was determined according to themethod of Bradford [20]. The preparedsamples were stored at 20 °C.Electrochemical Determinationof AvidinElectrochemical measurements wereperformed using a CHI440A instrument(CH Instruments, Austin, USA). Thethree-electrode system consisted of acarbon-paste working electrode (CPE),an Ag/AgCl/3 M KCl reference electrode,and a platinum wire counterelectrode. Acetate buffer (0.2 MCH 3 COOH + 0.2 M CH 3 COONa inratio 9:2 (v/v), pH 4) was used as thesupporting electrolyte. Square wavevoltammetry (SWV) was performedusing the following parameters: initialpotential = 0.1 V, end potential = 1.5V, amplitude = 25 mV, step potential= 5 mV, and frequency = 260 Hz.All experiments were carried out at25 °C. The raw data were treated usingthe Savitzky and Golay filter, level 4(included in GPES software). GPESsoftware supplied by EcoChemie(Utrecht, Netherlands) was employedfor smoothing and baseline correction(peak width = 0.05). Other details canbe found in Kizek et al. [10].Preparation of Carbon PasteElectrodeThe carbon paste (about 0.5 g) wasmade of 70% graphite powder (Sigma-Aldrich) and 30% mineral oil (w/w)(Sigma-Aldrich; free of DNase, RNase,and protease). This paste was housed ina Teflon body having a 2.5-mm-diameterdisk surface. Prior to measurements,the electrode surface was renewed byS76 Chromatographia Supplement Vol. 67, 2008 Original

polishing with a soft filter paper inpreparation for measurement of a samplevolume of 5 lL.Electromigration MethodsSodium Dodecyl Sulphate-PolyacrylamideGel ElectrophoresisElectrophoresis was performed accordingto Laemmli [21] using a Biometramaxigel apparatus with gel dimension of17 · 18 cm (Biometra, Germany). First10% (w/v) running, then 5% (w/v)stacking gel was poured, the gels wereprepared from 30% (w/v) acrylamidestock solution with 1% (w/v) bisacrylamideconcentration; the polymerizationof the running gel was carried out atroom temperature for 1 h and 30 minfor the stacking gel. Prior to analysis thesamples were mixed with sample buffercontaining 5% (v/v) 2-mercaptoethanolin a 1:1 ratio. The samples were boiledfor 2 min, then loaded onto a gel in20 lL aliquots. For determination ofrelative molecular mass (M r ), the proteinladder ‘‘Precision plus protein standards’’from Bio-Rad was used. Theelectrophoresis was run at 150 V withcooling by tap water (approx. 7 °C) untilthe front dye reached the bottom of thegel. To compare the avidin amount,5,000 ng of avidin was run as a standardin one lane. Coomassie blue staining ofthe gels was performed according toDiezel et al. [22]. After staining, the gelwas scanned and analyzed using Biolightsoftware (Vilber-Lourmat, Germany).Experion SystemAnalyses on an automated microfluidicExperion electrophoresis system (Bio-Rad) were carried out according to themanufacturer’s instructions with suppliedchemicals (Experion Pro260 analysiskit, Bio-Rad). Each sample wasdiluted with water to the same proteinconcentration of 300 lg mL 1 , 4 lLaliquots were then mixed with 2 lL ofreducing sample buffer, and after 4 minof boiling, 84 lL of water was added.After priming of the chip with gel andgel-staining solution in the priming station,the mixture (6 lL) was loaded intoaPeak of avidin height (nA)14001200100080060040020000100 µg.mL -110 nA0.7 0.8 0.9 1.0Potential (V)2Ysample wells. The Pro260 Ladderincluded in the kit was used as a standard.Accuracy, Precision and RecoveryAccuracy, precision and recovery ofavidin were evaluated with homogenates(tobacco plant tissues) spiked with thestandard by Experion. Before samplepreparation, 100 lL avidin standardsand 100 lL water were added to tobaccotissue samples. Homogenates wereassayed blindly and avidin concentrationwas derived from the calibration curve.Accuracy was evaluated by comparingestimated concentrations with knownconcentrations of avidin. The spiking ofavidin was determined as a standardmeasured without presence of real sample.Calculation of accuracy (%Bias),precision (%C.V.) and recovery wascarried out as indicated by Causon [23],Bugianesi et al. [24], and Vanatta andColeman [25].Results and Discussion4y = 95.7151x - 48.4915R 2 = 0.9920Electrochemical Analysis ofAvidin and Its Determinationin Transgenic PlantsAs it has been shown in many previouslypublished papers [26, 27], tyrosine (Y)and tryptophan (W) residues areW6Concentration (µg.mL -1 )8101214bRelative avidin concentration (%)1201008060402001 2 3 4 5 6 7 8Plant sampleFig. 1. Electrochemical behaviour of avidin on CPE surface. Calibration curve of avidinobtained by square wave voltammetry, in inset—electrochemical signals of avidin. Protein wasadsorbed from 5 lL drop of solution at the carbon paste electrode followed by electrode washingand transfer into the electrolytic cell with blank supporting electrolyte—sodium acetate, pH 4.0.SWV was performed under following conditions: frequency 260 Hz, initial potential 0.1 V, endpotential 1.5 V, step potential 5 mV, amplitude 25 mV, time of accumulation 60 s (a). Relativeavidin concentrations in analyzed samples were obtained by square wave voltammetry. The peakof 100% height corresponds to the highest avidin positive tobacco extract (b)responsible for the electroacitivity of aprotein at carbon electrodes. Squarewavevoltammetric analysis using solidcarbon electrodes is very sensitive andyields well-developed signals. However,using a CPE and base line correction ofthe data, we determined well-definedvoltammetric signals for both Y and Wat 0.78 and 0.92 V vs. Ag/AgCl/3 MKCl, respectively (Fig. 1a—inset). Thepeaks were measured at 100 lg mL 1of avidin by using the adsorptive transferstripping square-wave voltammetrytechnique. These electrochemical transfertechniques were described in detailpreviously [28–37]. Based on our experienceswith electrochemical detection ofavidin and streptavidin [10], we appliedadsorptive transfer stripping (AdTS)square wave voltammetry (SWV) in ourmeasurements. The dependence of peakY height on the frequency resulted inoptimal separation of the signal fromthe background at 260 Hz. The calibrationplot in expected concentration rangeof 0.75–12.5 lg mL 1 avidin is shownin Fig. 1. For t A = 60 s the height ofpeak Y increased linearly (equation:y = 95.7151x 48.4915, R 2 = 0.9920)with the avidin concentration up to1 lg mL 1 . Full coverage of the electrodesurface was not reached within theprotein concentration range studied. Therelative standard deviation (RSD) wasonly about 4%.Original Chromatographia Supplement Vol. 67, 2008 S77

aRelative peak area50y = 0.081x + 1.203740R 2 = 0.997930201000 100 200 300 400 500 600b20Avidin standard400 mAU250 ng.mL -1125 ng.mL -162.5 ng.mL -131.25 ng.mL -1marker10 20 25 37 50 75 100 150260 Mr × 1,00025 30 35 40 45 50Migration time (s)c260upper markerdAvidin200 mAUMr × 1,00015010075503720101.2Avidin formsmarker1,00050025012562.531.3Concentration of avidin (ng.mL -1 )tetramertrimerdimermonomerRelative avidin concentration (%)20120100806040200Avidin concentration ng.mL -1 1 2 3 4 5 6 7 810 20 25 37 50 75 100 15025 30 35 40 45 50Migration time (s)260Mr × 1,000Plant sampleFig. 2. Analysis of avidin in plant samples by Experion automated electrophoresis system. Calibration curve of avidin, the relative peak areas wererecounted by the Experion software according to the area of relative molecular mass standard 260 · 1,000 (a), different concentrations of avidinstandards in comparison with relative molecular mass standard (b), virtual gel output from Experion system, different concentrations of avidinstandards (c), relative avidin concentrations in analyzed samples obtained by Experion system. The peak of 100% height corresponds to the highestavidin positive tobacco extract. Inset—record of real plant sample (d)Under the optimized conditions theamount of avidin in various transgenictobacco plants in comparison to controlswas measured. The relative avidin concentrationsof avidin-positive tobaccoextracts are shown in Fig. 1b (n=3,RSD = 5%). For the avidin negativesamples, in which the content of electroactiveamino acids should be relativelylow, only very small signals of 5%compared to avidin positive sampleswere produced. The calculated contentof analyte in the control plants wassubtracted from the analyte content inavidin-positive plants as previously described.Using electrochemical methodswe determined that avidin levels intransgenic tobacco plants varied from 42to 503 ng avidin per mg of fresh mass ofthe plant tissue.Determination of Avidinby Experion: AutomatedElectrophoresis SystemExperion is an automated microfluidicelectrophoresis system that uses a CaliperLife Sciences innovative LabChipmicrofluidic separation technology withsensitive fluorescent sample detection. Itperforms rapid and repeatable analysesof protein, DNA and RNA samples,which allows the analysis of protein ornucleic acids samples within 30 min. Theseparation, detection and data analysisare performed within a single platform,so the time-consuming steps in classicelectrophoretic methods are minimized.Many types of samples, such as bacteriallysates, protein extracts, and chromatographyfractions, can be analyzed. Inaddition to the significant shortening oftime required, the chip-based methodallows both repeatable and accuratesizing and quantification of the proteins.After application of different concentrationsof an avidin standard on the chip,the highest responses were observed at(25.8 ± 0.6) · 1,000 (migration time =27.3 ± 0.1 s), which corresponds to thesubunit dimer. In the case of the fourhighest concentrations (125, 250, 500 and1,000 ng mL 1 ) analyzed, the signalsS78 Chromatographia Supplement Vol. 67, 2008 Original

corresponding to monomer ((16.4 ± 0.4)· 1,000, migration time = 24.9 ± 0.1s), trimer ((44.7 ± 0.7) · 1,000, migrationtime = 30.7 ± 0.1 s) and tetramerof subunits ((66.7 ± 0.3) · 1,000, migrationtime = 34.0 ± 0.1 s), exhibited aslight signal shift with decreasing concentrationof the protein to a higherrelative molecular mass of about 0.5(Fig. 2b, c). Considering the fact that thesignals for monomer, trimer and tetramerwere observed only at the fourhighest avidin concentrations tested,with each representing approximately9.5, 7.3, 4.5 and 1.5% of the total signalintensity, respectively, it can be assumedthat the absolute majority of the avidinpresent in plant extracts exists in theform of a subunit dimer. Within therange from 31.25 to 500 ng mL 1 concentration,a linear dependence of signalintensity on avidin concentration wasobtained as defined by the equation ofy = 0.0810x + 1.2037 with an R 2 =0.9979 (Fig. 2). The RSD of a measurementwas 5.5% and the detection limitwas 15.5 ng mL 1 with analysis time nothigher than 1 min.Based on the previous results, thepresence and intensity of the signalcorresponding to the subunit dimer(peak of relative molecular mass of(26 ± 2) · 1,000) was measured in theplant protein extracts. The resultsobtained with plant samples are shownin Fig. 2d. With all of the analyzedplants, an increased intensity of thedimer peak signal compared to nontransformedcontrol plants was observed(Fig. 2d). The average intensity of thispeak measured in extracts from thetransgenic plants was 7 ± 2 times higherthan in control extracts. To determinethe amount of avidin in the transgenicplants, the concentration of proteinswith the same mobility determined incontrol plant extracts was subtractedfrom the concentration determined fromthe calibration curve. The avidinconcentration in the transgenic plantsranged from 15 to 854 ng mg 1 of freshmass of the plant tissue. This largevariability can be attributed to differentavidin expression levels in individualtransgenic plants. It clearly follows fromthe results obtained that Experion can beused for analysis of avidin in extractsTable 1. Recovery of avidin spiked in a tobacco tissue sample (n =5)Homogenate(lM) afrom transgenic tobacco plants.However, the influence of the biologicalmatrix on the avidin responses is considerablewith recovery of 90%(Table 1).Sodium Dodecyl Sulphate-Polyacrylamide GelElectrophoresisSpiking(lM) aIn the case of sodium dodecyl sulphate—polyacrylamide gel electrophoresis (SDS-PAGE) using reducing conditions, themost intense band corresponding to themonomer of avidin subunit with a relativemolecular mass of 15.8 · 1,000 wasmeasured. Bands corresponding to otheravidin forms were not detected probablydue to the denaturing conditions and thehigh concentration of reducing agent(2.5% in the sample mixture). The opticaldensity of the band with the samemobility as standard avidin monomerwas approximately evaluated by Biolightsoftware. In comparison to the control,the intensity of this band was of about 10times higher in all transgenic plants. Inone sample (plant #2) this intensity wasmarkedly higher and in three plants (#5,6 and 8) lower (Fig. 3a).Comparison of the TechniquesUsed for Avidin Determinationin Transgenic Tobacco PlantsHomogenate Recovery+ spiking (lM) a (%) bAvidin 380 ± 30 (7.8) 250 ± 11 (4.4) 570 ± 51 (8.9) 90 ± 8a Avidin concentration; expressed as mean ± SD (C.V.%)b Recovery; expressed as mean ± C.V.%Avidin-expressing transgenic plantscould be useful in insect control technologydue to the toxicity of avidin tomany species of insect pests. All threedetection techniques used in this studyare nonspecific because both electrochemicaland electromigration methodsdemonstrate the presence of any protein.The electrochemical method (AdTSSWV at CPE) utilizes the electroactivityof two amino acids, tyrosine and tryptophan[26, 38, 39], while electromigrationmethods utilize mobilities ofcharged proteins after a subsequentcolour reaction. Particularly the novelExperion system is based on binding ofthe specific fluorescent dye to a proteinand subsequent detection of fluorescenceintensity. The Experion analysis kit canseparate and quantify protein samplesranging from relative molecular massfrom 10 to 260 · 1,000. The result ofeach analysis provides a logging ofindividual peaks of proteins and thecreation of virtual electrophoreograms.The sensitivity of this kit is comparableto colloidal Coomassie Blue staining ofproteins in SDS-PAGE gels, which hasbeen also used.As we mentioned above, the threetechniques employed for avidin determinationin transgenic plants are quitedifferent. In the case of electromigrationmethods, it was necessary to denaturethe sample thermally and chemicallyprior to its loading into the Experionchip or SDS-PAGE gel. Therefore, thenative avidin was converted to monomeric(SDS-PAGE) and dimeric units(Experion). The reason why avidin wasdetected as a dimer in the case of theautomated electrophoretic system is dueto an incomplete denaturation and/orpartial renaturation during the dilutionand loading of samples into the Experioninstrument. The 5–6 unit differences ofthe expected and obtained molecularmass of dimeric and trimeric avidinforms can be explained by used chiptechnology. Although the proteins canbe quickly separated on the Experionsystem, sizing results may vary fromthose obtained using conventional SDS-PAGE (5,299 and 5,423 Bio-Rad bulletin).Electrochemical measurements werecarried out with native avidin because itwas not necessary to denature it. Theelectrochemical method well correlateswith Experion with correlation coeffi-Original Chromatographia Supplement Vol. 67, 2008 S79

a7550markerstandardPlant sample1 2 3 4 5 6 7 8controlAcknowledgmentThe financial support from MSM6215712402 is gratefully acknowledged.bExperion ( -1 )Mr × 1,000100080060040020037252015Correlationy = 1.7114x + 19.071R 2 = 0.9305cient higher than 0.9300 (Fig. 3b).However, compared to electrophoreticmethods, the electrochemical methodyields lower amounts (*40%), Fig. 3b.This difference can be explained byconsidering features of the differentmethods of detection. In both of theelectromigration methods, the peptidicbond present in all proteins is detected,but the electrochemical determination isbased on the detection of only theelectroactive amino acids, Tyr and Trp,whose content is markedly increased inavidin relative to other proteins.In comparison with the literaturewhere the values of avidin amount intransgenic plants were between 70 and2,500 ng per mg of fresh mass of thetissue, our results are in relatively goodagreement with those amounts. In general,insect pests cannot develop andsurvive feeding on transgenic plantsexpressing 47 ppm (e.g. ng per mg offresh mass) avidin or more [2, 7, 9].Therefore, the data demonstrate that thetransgenic tobacco plants expressed sufficientavidin such that they would beresistant to insect attack.Avidin00 100 200 300 400 500 600SWV ( -1 )Fig. 3. SDS-PAGE analysis of avidin in plant samples. SDS-PAGE gel, standards of relativemolecular mass, 5,000 ng of avidin standard, 1–8—samples of transgenic plants 1–8 respectively,control plant (a). Correlation of square wave voltammetry (SWV) and Experion method used todetermine avidin in tobacco transgenic tissue sample (b)ConclusionsWe have proposed the use of a novelautomated electrophoresis system(Experion) for avidin determination intransgenic plants. The technique is basedon mobility of charged proteins in anelectric field, the binding of fluorescentdye to the protein and subsequent analysisof the fluorescence intensity. Webelieve that this technology offers usefuland promising possibilities in the field ofbiosensor development. We have alsodemonstrated the application of Experionfor detection of avidin extracted fromtransgenic tobacco. In the case of avidindetermination, we also compared thechip-based capillary electrophoreticmethod with the SDS-PAGE techniqueand square wave voltammetric method.Based on the results obtained here, it canbe concluded that the square-wave voltammetryand Experion are sensitive,rapid and repeatable tools for quantitativeprotein analysis. These new approachesare likely to be a suitable toolin detection and quantification of arange of proteins and other biologicallyimportant compounds.References1. Flinn PW, Kramer KJ, Throne JE,Morgan TD (2006) J Stored Prod Res42:218–2252. Kramer KJ, Morgan TD, Throne JE,Dowell FE, Bailey M, Howard JA (2000)Nat Biotechnol 18:670–6743. Hilder VA, Boulter D (1999) Crop Prot18:177–1914. Snedeker SM (2001) Environ HealthPerspect 109:35–475. Hood EE, Witcher DR, Maddock S,Meyer T, Baszczynski C, Bailey M, FlynnP, Register J, Marshall L, Bond D,Kulisek E, Kusnadi A, Evangelista R,Nikolov Z, Wooge C, Mehigh RJ, HernanR, Kappel WK, Ritland D, Li CP,Howard JA (1997) Mol Breed 3:291–3066. Zhu YC, Adamczyk JJ, West S (2005)J Econ Entomol 98:1566–15717. Burgess EPJ, Malone LA, Christeller JT,Lester MT, Murray C, Philip BA, PhungMM, Tregidga EL (2002) Transgenic Res11:185–1988. Yoza K, Imamura T, Kramer KJ,Morgan TD, Nakamura S, Akiyama K,Kawasaki S, Takaiwa F, Ohtsubo K(2005) Biosci Biotechnol Biochem69:966–9719. Markwick NP, Docherty LC, Phung MM,Lester MT, Murray C, Yao JL, Mitra DS,Cohen D, Beuning LL, Kutty-Amma S,Christeller JT (2003) Transgenic Res12:671–68110. Kizek R, Masarik M, Kramer KJ, PotesilD, Bailey M, Howard JA, Klejdus B,Mikelova R, Adam V, Trnkova L, Jelen F(2005) Anal Bioanal Chem 381:1167–117811. Nardone E, Rosano C, Santambrogio P,Curnis F, Corti A, Magni F, Siccardi AG,Paganelli G, Losso R, Apreda B, BolognesiM, Sidoli A, Arosio P (1998) Eur JBiochem 256:453–46012. Lahely S, Ndaw S, Arella F, HasselmannC (1999) Food Chem 65:253–25813. Kuramitz H, Natsui J, Tanaka S, HasebeK (2000) Electroanalysis 12:588–59214. Kuramitz H, Sugawara K, Tanaka S(2000) Electroanalysis 12:1299–130315. Sugawara K, Tanaka S, Nakamura H(1994) Bioelectrochem Bioenerg 33:205–20716. Sugawara K, Yamauchi Y, Hoshi S,Akatsuka K, Yamamoto F, Tanaka S,Nakamura H (1996) Bioelectrochem Bioenerg41:167–17217. Petrlova J, Masarik M, Potesil D, AdamV, Trnkova L, Kizek R (2007) Electroanalysis19:1177–118218. Masarik M, Kizek R, Kramer KJ, BillovaS, Brazdova M, Vacek J, Bailey M, JelenF, Howard JA (2003) Anal Chem75:2663–2669S80 Chromatographia Supplement Vol. 67, 2008 Original

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