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).

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