Simultaneous determination of catechol and hydroquinone by c(4)

3750

20

10

A

0μ / I-10-20

-30

E / V

Fig.2CVsofGOD/GCE(a),GOD/NiO/GCE(b),GOD/TiOGr/GCE(d)in0.1MPBS(pH7.0)

2–Gr/GCE(c),andGOD/NiO/TiO2–Electrochemicalimpedancespectroscopy(EIS)wasreportedasaneffectivemethodtomonitorthefeatureofsurface,allowingtheunderstandingofchemicaltransformationandprocessesassociatedwiththecon-ductiveelectrodesurface.Figure3showstheNyquistplotsofEISforthebareGCE,TiO2–Gr/GCE,NiO/GCE,NiO/TiO2–Gr/GCE,andGOD/NiO/TiO2–Gr/GCE.AtbareGCE,theredoxprocessofthe[Fe(CN)6]3 /4 probeshowedaveryweakelectrontransferresistance(curvea).TheEISincreasedwhenTiO2–Grwasmod-ifiedontheGCE(curveb).TheEISofNiO/GECobviouslyincreasedcomparedtobothoftheaforemen-tionedelectrodes(curvec).TheNiO/TiO2–Gr-modifiedGCEshowedamuchlowerresistancefortheredoxprobe(curved),implyingthatNiO/TiO2–Grwasanexcellentelectricconductingmaterialandacceleratedtheelectrontransfer.AfterGODwascoatedonNiO/TiO2–Gr/GCE,theresistanceincreaseddramatically

Ω

/ ''ZZ' / Ω

Fig.3EISspectraofbareGCE(a),TiO2–Gr/GCE(b),NiO/GCE(c),NiO/TiO2–Gr/GCE(d),andGOD/NiO/TiO2–Gr/GCE(e)in5mMFe(CN)63 /4 solutioncontaining0.1MKCl

JSolidStateElectrochem(2012)16:3747–3752

(curvee),suggestingthatthebulkyGODmoleculesblockedtheelectronexchangebetweentheredoxprobeandelectrodesurface.Differentscanratestudies

ThecyclicvoltammogramsofGOD/NiO/TiO2–Gr/GCEinPBSatdifferentscanrateswerestudied.BothIpaandIpcincreasedlinearlywithincreaseinscanratesfrom20to300mVs 1.ThisindicatedthattheelectrontransferprocessoccurringatGOD/NiO/TiO2–Gr/GCEwasasurface-confinedprocess.EffectofpH

TheeffectofpHonGODredoxcoupleatNiO/TiO2–Gr/GCEinvariousbuffersolutions(pH5.0to10.0)wasinves-tigated.TheredoxpeakcurrentincreasedwithincreaseofpHfrom5to6andthenremainedalmoststableinthepHrangeof6to8.ThepeakcurrentdecreasedwhenpHincreasedfrom8to10.So,pH7.0wasselectedastheoptimum.TheinfluenceofpHovertheanodicpeakpoten-tial(Epa)andcathodicpeakpotential(Epc)atNiO/TiO2–Gr/GCEwasstudiedanditshowedthatbothEpaandEpcexhibitedlineardependenceoverdifferentpHs.Thecorre-lationcoefficientwas0.998and0.995,respectively.TheslopevaluesofEpaandEpcwerefoundtobe 50.3and 50.5mVpH 1,respectively.Theslopeswereclosetothetheoreticalvalueof 58.6mVpH 1forareversiblereaction,whichindicatedanequalnumberofprotonandelectrontransferprocesses.

Amperometricresponseoftheglucosebiosensor

Theamperometriccurrent–timecurveofGOD/NiO/TiO2–Gr/GCEuponsuccessiveadditionofglucosetoacontinu-ouslystirredPBS(pH7.0)wasrecorded(Fig.4).Thebiosensorexhibitedarapidresponsefortheadditionofglucoseandachieved96%ofthesteady-statecurrentwithin3s.TheinsetofFig.4picturedthecalibrationcurveofGOD/NiO/TiO2–Gr/GCEforglucosedeterminationanditsequationwasI(μA)02.503+4.129Cglucose(mM)withacorrelationcoefficientof0.995.Agoodlinearrelationshipwasfoundbetweenthechronoamperometriccurrentandglucoseconcentrationfrom1to12mM.Meanwhile,thedetectionlimitof1.2μMwasestimatedatasignal-to-noiseratioof3.ThesensitivityofGOD/NiO/TiO2–Gr/GCEbio-sensor(4.129μAmM 1)wassuperiorthanreportedforbiosensorsofglucose,1suchasGOD/Chit-MWCNTs(0.45μAmM )[22],GrEC/Chit-CNT/GOD(1.38μAmM 1)[23],CS/glutaraldehyde/GOD(1.8μAmM 1)[24],andGOD/Au/CS-IL/MWNT(4.10μAmM 1)[25].ThehighsensitivityforGOD/NiO/

JSolidStateElectrochem(2012)16:3747–37527060

50

A

40μ/ I3020100

Time / s

Fig.4TheamperometricresponseofGOD/NiO/TiO 0.3Vuponsuccessiveadditionsofglucose(1mM)2in–Gr/GCE0.1MpHat7.0PBS.Inset,plotofamperometriccurrentvs.glucoseconcentration

TiO2–Gr/GCEwasexpectedtooriginatepresentinfromthematrix.

thecombinedinfluenceofTiO2–GrandNiOAplateauincurrentresponsewasobservedforaglucoseconcentrationbeyond12mM.ThissignifiedtheoperationoftheMichaelis–Mentenkineticmechanismfortheenzyme-catalyzedprocess.TheapparentMichaelis–Mentenconstant(KM),aparameterofimportanceinenzyme–sub-stratekinetics,wasobtainedfromtheLineweaver–Burkequation[26]:1/Iss01/Imax+KM/ImaxC,whereIssisthesteady-statecurrentaftertheadditionofsubstrate,Cisthebulkconcentrationofsubstrate,andImaxisthemaximumcurrentmeasuredundersaturatedsubstratesolution.Analysisoftheslopeandinterceptfortheplotofthereciprocalofthesteady-statecurrentversusreciprocalofglucoseconcentra-tionallowsthedeterminationofKM.TheKMvaluefortheenzymeelectrodewasfoundtobe7.3mM.ThevalueofKMforGODatGOD/NiO/TiO2–Gr/GCEwaslowerincompar-isontootherglucosebiosensorsbasedonGOD-immobilizedPMMA-MWCNT(PDDA)-NFE(KM010.12mM)[27]andPrussianblue/MWCNTnanocomposites(KM018mM)[28].ThelowerKMvalueshowsabetteraffinitybetweenglucoseandenzymeelectrode.

Stability,repeatability,andinterferencedeterminationThestabilityoftheproposedbiosensorwasinvestigated.Whennotinuse,theelectrodewassuspendedabove0.1MPBSat4°C.Theresponseofthebiosensorto1.0mMglucosewastestedintermittently.Thebiosensorlostabout5.2%and10.3%ofitsoriginalresponseafter10and20days,respectively.Thebiosensoralsoshowedgoodreproducibilityforthedeterminationofglucoseconcentra-tioninitslinearrange.Therelativestandarddeviation(RSD)was1.9%forsixsuccessiveassaysataglucose

3751

concentrationof1.0mM.ThiscanbeduetothegoodbiocompatibilityofNiO/TiO2–Grcomposite,whichpro-videsafavorablemicroenvironmentforGOD.

Theabilityofthesensortodiscriminatetheinterfer-ingspecieshavingelectroactivitiessimilartothetargetanalyteisoneofthemostimportantanalyticalfactorsforanamperometricbiosensor.Easilyoxidizablecom-poundssuchasascorbicacid,dopamine,anduricacidnormallyco-existwithglucoseinnaturalsamples.Theinterferenceeffectof5.0mMl-cysteine,5.0mMglycin,2.0mMascorbicacid,2.0mMuricacid,and2.0mMdopamineontheamperometricresponseof1.0mMglucosewasinvestigated.Thecurrentresponseforsuchelectroactive-interferingspeciestothatofglucosebythesensorwasbelow5%.Therefore,goodselectivitycanbeobtainedwiththepreparedsensor.Samplesanalysis

Inanattempttoexplorethedevelopedsensorforpracticalapplications,GOD/NiO/TiO2–Gr/GCEwasappliedtodeter-mineglucoseinhumanbloodserumsamplesofhealthypeople.Arapidandstableamperometricresponsewasac-quiredat 0.3Vwiththedirect 1additionof20μLofsamplesinto20mLof0.1molLPBS.ThecontentofglucoseinthesampleswascalculatedfromthecalibrationcurveandtheobtainedresultsareshowninTable1.TherecoveryofglucosewasdeterminedbystandardadditionofpureglucosetothesolutionscontainingtheserumsamplesandthecorrespondingresultsaregiveninTable1.Onecanseethatthesensoralsogivesexactrecovery(96.3–103.4%).TheresultsdemonstratedhererevealthepotentialapplicationsofGOD/NiO/TiO2–Gr/GCEfordeterminationofglucoseinbiologicalfluids.

Conclusions

Theconstructionofanelectrochemicalbiosensorbymodi-ficationofaglassycarbonelectrodewithafilmcontainingTiO2–GrandNiOwasreported.TheGOD/NiO/TiO2–Gr/GCEbiosensorwaspreparedtodetectglucoseusingNiO/

Table1Determinationofglucoseinhumanserumsamples(n04)SampleConcentration(nM)

RSD(%)Added(nM)Recovery(%)

13.451.5598.222.682.5596.334.561.95102.544.283.45103.45

2.15

4.1

5

97.6

3752TiO2–GrnanocompositetoimmobilizeGODasamodelenzyme.TheevaluationofGOD/NiO/TiO2–Grnanocomposite–Gr/GCEhadadem-onstratedthatNiO/TiO2goodabilitytoretainthebioactivityofGOD.Cyclicvoltammetryshowedapairofwell-definedredoxpeaks,correspondingtothedirectelectrontransferofGOD.ThepresenceoftheredoxpeaksindicatedthattheNiO/TiO2–GrnanocompositefacilitatedthedirectelectrontransferofGOD.Themethodpresentedcanbeusedfortheimmobilizationandevaluationofthedirectelectrontransferofotherenzymesorproteins.

AcknowledgmentsThisworkwassupportedbytheNationalNatu-ralScienceFoundationofChina(20805040),ProgramforScienceandTechnologyInnovationTalentsinUniversitiesofHenanProvince(2010HASTIT025),andExcellentYouthFoundationofHe’nanScientificCommittee(104100510020).

References

1.BayachouM,BoutrosJA(2004)JAmChemSoc126:12722–12723

2.DengC,ChenJ,NieZ,SiS(2010)BiosensBioelectron26:213–2193.LiGX(2006)Protein-basedvoltammetricsensors.In:Encyclope-diaofsensors.AmSci8:301–313

4.LiGX(2007)Protein-basedbiosensorsusingnanomaterials.Nanotechnologiesforlifesciences.Wiley,NanomaterBiosens8:278–310

5.ZhangHJ,ToshimaN(2011)ApplCatalA400:9–13

6.WuH,WangJ,KangXH,WangCM,WangDH,LiuJ,AksayIA,LinYH(2009)Talanta80:403–406

7.WangCY,ChenSH,XiangY,LiWJ,ZhongX,CheX,LiJJ(2011)JMolCatalB69:1–7

8.CarnesCL,KlabundeKJ(2003)JMolCatalA194:227–236

JSolidStateElectrochem(2012)16:3747–3752

9.IchiyanagiY,WakabayashiN,YamazakiJ,YamadaS,KimishimaY,KomatsuE,TajimaH(2003)PhysicaB862:329–333

10.SalimiA,SharifiE,NoorbakhashA,SoltanianS(2007)Biophys

Chem125:540–548

11.SalimiA,SharifiE,NoorbakhashA,SoltanianS(2007)Biosens

Bioelectron22:3146–3153

12.GuoSJ,WenD,ZhaiY,DongSJ,WangEK(2010)ACSNano

4:3959–3968

13.ZhouM,ZhaiYM,DongSJ(2009)AnalChem81:5603–561314.ZhaoJ,ChenGF,ZhuL,LiGX(2011)ElectrochemCommun

13:31–33

15.ZhouH,GanX,WangJn,ZhuXL,LiGX(2005)AnalChem

77:6102–6104

16.ZhouH,LiuL,YinK,LiuSL,LiGX(2006)ElectrochemComm

8:1168–1172

17.FanY,LuHT,LiuJH,YangCP,JingQS,ZhangYX,YangXK,

HuangKJ(2011)ColloidsSurfB83:78–82

18.FanY,HuangKJ,NiuDJ,YangCP,QingJS(2011)Electrochim

Acta56:4685–4690

19.SunJY,HuangKJ,ZhaoSF,FanY,WuZW(2011)Biochem

82:125–130

20.KovtyukhovaNI,OllivierPJ,MartinBR,MalloukTE,Chizhik

SA,BuzanevaEV,GorchinskiyAD(1999)ChemMater11:771–778

21.ZhangH,LvX,LiY,WangY,LiJ(2010)ACSNano4:380–38622.WuBY,HouSH,YuM,QinX,LiS,ChenQ(2009)MaterSci

EngC29:346–349

23.GhicaME,PauliukaiteR,FilhoOF,BrettCMA(2009)Sens

ActuatorsB142:308–315

24.LiangRP,FanLX,WangR,QiuJD(2009)Electroanalysis

21:1685–1691

25.RagupathyD,GopalanAI,LeeKP(2009)ElectrochemCommun

11:397–401

26.LiJ,TanSN,GeH(1996)AnalChimActa335:137–145

27.ManeshKM,KimHT,SanthoshP,GopalanAI,LeeKP(2008)

BiosensBioelectron23:771–779

28.ZhuL,ZhaiJ,GuoY,TianC,YangR(2006)Electroanalysis

18:1842–1846