Application of Fe3O4 mesoporous sphere modified carbon ionic liquid electrode as electrochemical

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ColloidsandSurfacesB:Biointerfaces

journalhomepage:www.elsevier.com/locate/colsurfb

ApplicationofFe3O4mesoporousspheremodifiedcarbonionicliquidelectrodeaselectrochemicalhemoglobinbiosensor

WeiSuna,b,∗,ZhaolanSunb,LiqiZhangc,XiaoweiQib,GuangjiuLib,JieWub,MeiWangc

a

CollegeofChemistryandChemicalEngineering,HainanNormalUniversity,Haikou,571158,PRChina

CollegeofChemistryandMolecularEngineering,QingdaoUniversityofScienceandTechnology,Qingdao266042,PRChinac

StateKeyLaboratoryofCoalCombustion,HuazhongUniversityofScienceandTechnology,Wuhan430074,PRChina

b

article

info

abstract

Articlehistory:

Received13January2012

Receivedinrevisedform26May2012Accepted10June2012

Available online 19 June 2012

Keywords:ChitosanHemoglobinIonicliquid

Fe3O4mesoporousspheresElectrochemicalbiosensor

Anewelectrochemicalhemoglobin(Hb)biosensorwasconstructedbasedonaFe3O4mesoporousspheresmodifiedcarbonionicliquidelectrode(CILE),whichshowedexcellentelectrocatalyticabilitytowardsthereductionoftrichloroaceticacid.CILEwaspreparedbyusingN-hexylpyridiniumhexafluorophosphate(HPPF6)asthemodifierandthebinderinthecarbonpaste.Ultraviolet-visibleandFouriertransforminfraredspectroscopicresultsindicatedthatHbmoleculesretainedthenativestructureinthechitosanandFe3O4mesoporousspherescompositefilm.Electrochemicalresultsindicatedthatapairofwell-󰀇

definedredoxpeaksappearedin0.1mol/Lphosphatebuffersolution(PBS)withtheformedpotential(E0)as−0.287V(vs.SCE),indicatingthatthedirectelectrontransferofHbinthecompositefilmwasrealized.ElectrochemicalbehaviorsofHbwerecarefullyinvestigatedwiththeelectrochemicalparameterssuchaselectrontransfercoefficient(˛),electrontransfernumber(n),andheterogeneouselectrontransferrateconstant(ks)calculated.DuetothespecificcharacteristicsofFe3O4mesoporousspherespresentontheelectrodesurface,theelectrontransferrateofHbwasgreatlypromoted.TheHbmodifiedelectrodeexhibitedexcellentelectrocatalyticpropertieswithwiderlinearrangeandlowerdetectionlimit.

© 2012 Elsevier B.V. All rights reserved.

1.Introduction

Directelectrochemistryofproteinscanbeusedfortheinvesti-gationontheelectrontransfermechanisminthebiologicalsystem[1,2].Toachievethedirectelectrontransferofredoxproteins,pro-teinfilmvoltammetryhasbeenwidelyused,whichcanprovideafavorablemicroenvironmentforkeepingthemolecularstructureandbiocatalyticabilityoftheproteins[3,4].Differentapproachessuchasdirectcasting,nanoparticle,layer-by-layer,polymers,etc.,havebeenusedforthefabricationofproteinfilmmodifiedelec-trodes[5–7].Proteinscanretaintheirnativestructuresandremainthebiocatalyticabilityinthefilms,andthenthedirectelectrontransferofproteinscanberealizedwithapairofwell-definedredoxpeaksappearedonthecyclicvoltammogram.Thefabricatedproteinmodifiedelectrodeshowpotentialapplicationsinbiomed-icaldevices,enzymaticbioreactorsandthirdgenerationbiosensors[8].

Asanewkindofgreensolvent,ionicliquids(ILs)haveattractedintensiveinterestbecauseoftheiruniquephysicochemicalprop-ertiessuchashigherionicconductivity,widerelectrochemical

∗Correspondingauthorat:HainanNormalUniversity,CollegeofChemistryandChemicalEngineering,Haikou571158,China.

E-mailaddress:swyy26@hotmail.com(W.Sun).

windows,lowertoxicity,higherchemicalstabilityandnegligiblevaporpressure[9].InlastdecadeILshavebeenwidelyusedinthefieldsofelectrochemistryandelectroanalysis[10].Oneofitsapplicationsistoconstructnewkindsofmodifiedcarbonpasteelectrode.Bymixingcarbonpowder,ILand/orparaffintogether,carbonionicliquidelectrode(CILE)canbefabricatedwithmanyadvantagesincludingwiderelectrochemicalwindows,betteranti-foulingabilityandhigherdetectionsensitivity[11,12].Zhangetal.studiedtheelectrochemicalbehaviorofrutinonCILE[13].Sunetal.[14–16]applieddifferentCILEsforthedetectionofelectroactivesubstancessuchasadenosine,guanosineandhydroquinone.AlsoCILEcanbeusedasthesubstrateelectrodefortheimmobilizationofredoxproteins.Sunetal.[17,18]investigatedthedirectelec-trochemistryofredoxproteinsonthecarbonnanotubemodifiedCILE.Fe3O4nanoparticleisatypicalsuperparamagneticnanoma-terialwiththepropertiessuchasgoodbiocompatibility,strongsuperparamagnetism,lowtoxicityandeasypreparationprocess,whichhadbeenusedinthefabricationofelectrochemicalbiosen-sor.Reetzetal.[19]reportedmechanicallystablelipases/Fe3O4nanoparticle/sol–gelbiocatalystsbysimultaneousentrapmentoflipaseandnanostructuredFe3O4inhydrophobicsol–gelmaterials.Caoetal.[20]reportedanelectrochemicalbiosensorbasedonthehemeproteinsimmobilizedonFe3O4nanoparticles.Ontheotherhand,thebiopolymerchitosan(CTS)offersexcellentcharacteris-ticssuchasbiocompatibility,goodfilmformingability,nontoxicity,

0927-7765/$–seefrontmatter© 2012 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.colsurfb.2012.06.010

178W.Sunetal./ColloidsandSurfacesB:Biointerfaces101 (2013) 177–182

physiologicalinertnessandhighmechanicalstrength.Thus,ithasbeenextensivelyusedfortheimmobilizationofenzymesandtheconstructionofbiosensors[21,22].

Inthispaperthedirectelectrochemistryofhemoglobin(Hb)wasrealizedontheCTSandFe3O4mesoporousspherescom-positematerialmodifiedCILE.AnionicliquidN-hexylpyridiniumhexafluorophosphate(HPPF6)wasusedasthebinderforthepreparationofCILE,whichhadbeenusedfortheelectrochemicaldetection[14].Malekietal.[23]carefullyinvestigatedtheelec-trochemicalperformancesofCILE,andtheexperimentalresultsindicatedthatCILEcombinedmanygoodfeaturesofdifferenttypesofcarbonelectrodessuchasglassycarbonelectrode(GCE)andedgeplanepyrolyticgraphite.AlsoCILEprocessedtheadvan-tagesofcarbonpasteelectrode(CPE)suchaslowcost,simplicityofpreparation,surfacemodificationandrenewal.Thefavorableelectrochemicalresponse,higherconductivityandinherentelec-trocatalyticabilityobservedonCILEtowardbiomoleculesmakeitagoodcandidateforconstructionofbiosensors.Bycombinationoftheadvantagesofdifferentmaterialsused,directelectro-chemistryofHbwasrealizedonthecompositefilmmodifiedelectrode.Theelectrocatalyticabilityofthefabricatedelectrodetothereductionoftrichloroaceticacidwasfurtherinvestigatedcarefully.

2.Experimental

2.1.Apparatusandreagents

IonicliquidN-hexylpyridiniumhexafluorophosphate(HPPF6,>99%,LanzhouGreenchem.ILS.LICP.CAS.,China),bovinehemoglobin(Hb,MW64500,TianjinChuanyeBiochemicalLimitedCompany,China),chitosan(CTS,DalianXindieLimitedCompany,China),graphitepowder(averageparticlesize30␮m,ShanghaiColloidChemicalCompany,China)andtrichloroaceticacid(TCA,TianjinKemiouChemicalLimitedCompany,China)wereusedasreceived.0.1mol/LpH7.0phosphatebuffersolutions(PBS)wereusedasthesupportingelectrolyte.Alltheotherchemicalsusedwereofanalyticalreagentgradeanddoublydistilledwaterwasusedintheexperiments.

AlltheelectrochemicalexperimentswereexecutedonaCHI750Belectrochemicalanalyzer(ShanghaiCHInstrument,China).Astandardthree-electrodesystemwasusedwithanHbfilmmod-ifiedelectrodeasworkingelectrode,aplatinumwireasauxiliaryelectrodeandasaturatedcalomelelectrode(SCE)asreferenceelec-trode.Ultraviolet–visible(UV–vis)absorptionspectrawasrecordedonCary50probespectrophotometer(VarianCompany,Australia)andFouriertransforminfrared(FT-IR)spectrawasperformedonTensor27FT-IRspectrophotometer(BrukerCompany,Germany),respectively.AJSM-6700Fscanningelectronmicroscope(SEM,JapanElectronCompany,Japan)wasusedtorecordthescanningelectronmicroscopy.

2.2.PreparationofFe3O4mesoporousspheres

Fe3O4mesoporousspheresweresynthesizedbasedonthereportedprocedure[24].Inbrief,1.2gofFeCl3·6H2Oweredissolvedin75mLofglycolandthemixedsolutionwasstirringfor30min.Then3.6gofNaAcwasaddedtothesolutionfollowedwithstirringfor1h.Theresultingsolutionwastransferredtoa100mLTeflon-linedstainless-steelautoclaveandheatedat200◦Cfor16h,andthencoolednaturallytotheroomtemperature.Theproductwascollectedbyfiltration,washedwithdistilledwaterandabsoluteethylalcohol.Aftertheproductwasdriedat60◦Cfor6h,Fe3O4mesoporoussphereswereobtained.

2.3.Preparationofthemodifiedelectrode

CILEwaspreparedbyareportedprocedure[14],1.6gofHPPF6and3.2gofgraphitepowderweremixedcarefullyinamortar,heatedat60◦Cfor10minandgroundtoformahomogeneouscar-bonpaste.Thenthepreparedionicliquidmodifiedcarbonpastewastightlypackedintoacavityofglasstube(4.0mmdiameter)andtheelectricalcontactwasestablishedviaacopperwire.ThesurfaceofCILEwaspolishedtoasmoothsurfaceforthefurtheruse.

Themodifiedelectrodewaspreparedwiththefollowingprocedure.Typicallyasolutioncontaining20.0mg/mLHband0.33mg/mLFe3O4mesoporoussphereswaspreparedandmixedhomogeneously.Then10.0␮LofthepreparedHb–Fe3O4mixturewascastonthesurfaceofCILEandleftittodryatroomtem-peraturetogetanelectrodedenotedasHb–Fe3O4/CILE.Finally,5.0␮Lof1.0mg/mLCTSsolution(in1%HAcsolution)wasspreadontothesurfaceoftheHb–Fe3O4/CILEtogetthefinalmodifiedelectrode(CTS/Hb–Fe3O4/CILE).Forcomparison,differentmodifiedelectrodessuchasCTS/CILE,CTS/Fe3O4/CILEandCTS/Hb/CILE,etc.werefabricatedwithsimilardrop-castingprocedure.

2.4.Procedure

Electrochemicalmeasurementswereperformedina10mLelec-trochemicalcellcontaining0.1mol/LPBS,whichwaspurgedwithhighlypurifiednitrogenfor30minpriortoexperimentsandmain-tainedinanitrogenatmosphereduringtheexperiments.UV-Visspectroscopicexperimentswereperformedwithamixturesolu-tionofHb,CTS,andFe3O4mesoporoussphereswithpH7.0PBS.TheCTS/Hb–Fe3O4andHbfilmassembledonaglassslidewasusedforFT-IRmeasurements.

3.Resultsanddiscussion

3.1.SEMimages

SEMimageswererecordedtoidentifythesurfacemorpholo-gies.AsshowninFig.1A,thesynthesizedFe3O4materialexhibitedasmesoporousballwithanaveragediameterof400nm.Alsotheholesexistedontheroughsurfaceofmesoporousspherescouldbeobviouslyobserved.OnCTS/Hb–Fe3O4/CILE(Fig.1B)auniformsurfaceappearedwithroughinterface,whichwasduetothegoodfilmformingabilitityofCTSthatcouldincorporatethematerialsusedtightlyontheelectrodesurface.

3.2.Spectroscopicresults

InFT-IRspectroscopytheshapeandpositionofamideIandIIinfraredabsorbancebandsofHbcanprovideinformationonthesecondarystructureofthepolypeptidechain.TheamideIband(1700–1600cm−1)isattributedtotheCOstretchingvibrationofthepeptidelinkageinthebackboneofprotein.TheamideIIband(1600–1500cm−1)iscausedbythecombinationofNHinplanebendingandCNstretchingvibrationofthepeptidegroups[25].TheshapeofamideIandamideIIbandswilldiminishorevendisap-pearifHbmoleculeisdenatured[26,27].AsshowninFig.2Aa,thetwoamidebandsofpureHbwerelocatedat1649and1533cm−1.AftermixingwithCTSandFe3O4mesoporousspheres,thepositionofamideIandIIbandsappearedat1647and1533cm−1(Fig.2Ab).ThesimilaritiesoftheamidebandpositionssuggestedthatHbretaineditsnativestructureafterimmobilizedintheCTS–Fe3O4compositefilm.

InUV–visabsorptionspectrumtheSoretabsorptionbandfromthefourironhemegroupsofhemeproteinscanalsoprovide

W.Sunetal./ColloidsandSurfacesB:Biointerfaces101 (2013) 177–182

179

Fig.1.SEMimagesof(A)Fe3O4mesoporousspheresand(B)CTS/Hb–Fe3O4/CILE.

Fig.2.(A)FT-IRspectraof(a)Hbfilmand(b)CTS–Hb–Fe3O4film;(B)UV–visabsorptionspectraofHbinwater(a),CTS–Hb(b),Hb(c),Hb–Fe3O4(d)andCTS–Hb–Fe3O4(e)inpH7.0PBS.

theinformationontheconformationalintegrityandthepossi-bledenaturationortheconformationalchangeaboutthehemeregion[3].AsshowninFig.2B,theSoretbandofHbappearedat405.0nminwater(curvea)andpH7.0PBS(curvec).AftermixingHbwithCTS,Fe3O4andCTS–Fe3O4,respectively,theabsorptionvaluealsoappearedat405.0nmwithoutchanges(curvesb,dande),whichsuggestedthatHbinthecompositefilmretaineditsnativestructure.TheresultsalsoprovedthebiocompatibilityofFe3O4mesoporoussphereandCTSwithprotein.Fe3O4nanopar-ticleisatypicalnanomaterialwiththepropertiessuchasgoodbiocompatibility,strongsuperparamagnetismandsoon[20].CTSisanabundantnaturalbiopolymeroriginatedfromtheexoskele-tonofcrustaceans,whichisbiocompatible,biodegradablewithouttoxicity[28].SotheCTS–Fe3O4compositefilmwasasuitableimmobilizationmatrixforredoxproteins.

c),whichmaybeduetothepresenceofsemiconductorFe3O4mesoporousspherewithhighsurfaceareainthecompositefilmacceleratedtheelectrontransfer.Thedifferencesofcyclicvoltam-mogramsindicateddifferentimmobilizationstepsontheelectrodesurfaceandthesuccessfulfixationofHbontheelectrodesurface.

3.4.Directelectrochemistryofthemodifiedelectrodes

DirectelectrochemistryoftheimmobilizedHbwasfurthercare-fullyinvestigatedbycyclicvoltammetry.Fig.4showedthetypicalcyclicvoltammogramsofdifferentmodifiedelectrodesinadeaer-ated0.1mol/LPBS.OnbareCILE(curvea)andCTS/CILE(curveb)noelectrochemicalresponseswereobservedattheselectedpotentialrange,whichindicatedthatnoelectroactivesubstancespresentontheelectrodesurface.Theincreaseofthebackgroundcurrentcould

3.3.Electrochemicalcharacteristicsofthemodifiedelectrodes

Potassiumferricyanideiscommonlyusedasaprobetoeval-uatetheperformanceofdifferentmodifiedelectrodeswiththetypicalcyclicvoltammogramsshowninFig.3.OnCILEapairofwell-definedredoxpeaksappeared(curveb)withthepeak-to-peakseparation(󰀅Ep)as0.079V(vs.SCE),indicatingthegoodreversibilityonCILE.OnCTS/CILEtheredoxpeakcurrentsincreasedapparently(curvea).CTSisapositivelychargedbiopolymerandthenegativelycharged[Fe(CN)6]3−/4−couldbeeasilyadsorbedontheelectrodesurface,sothevoltammetricresponseincreasedcor-respondingly.OnCTS–Hb/CILEtheredoxpeakcurrentdecreasedgreatly(curved),indicatingthepresenceofHbmoleculesinthecompositefilmhinderedtheelectrontransferrate.WhileonCTS/Hb–Fe3O4/CILEtheredoxpeakcurrentsincreased(curve

Fig.3.Cyclicvoltammogramsof(a)CTS/CILE,(b)CILE,(c)CTS/Hb–Fe3O4/CILEand(d)CTS/Hb/CILEinamixturesolutionof1.0mmol/LK3[Fe(CN)6]and0.5mol/LKClwiththescanrateas100mV/s.

180W.Sunetal./ColloidsandSurfacesB:Biointerfaces101 (2013) 177–182

Fig.4.Cyclicvoltammogramsof(a)CILE,(b)CTS/CILE,(c)CTS/Hb/CILEand(d)CTS/Hb–Fe3O4/CILEinpH7.0PBSwiththescanrateas100mV/s.

beascribedtothepresenceofILinthecarbonpaste.ByusingILasthebinderinCPE,alayerofILfilmwaspresentonthesurfaceofgraphitepower,whichcouldactasthedoublelayerwithincreaseofthecapacitance,sothebackgroundcurrentwasincreasedgreatly.OnCTS/Hb/CILEapairofunsymmetricredoxpeaksappeared(curvec),whichindicatedthatthedirectelectrontransferofHbwithCILEhadtakenplace.CILEhadbeenelucidatedwiththeadvan-tagessuchashigherconductivityandbetterreversibility.SothedirectelectrontransferofHbcanberealizedontheCILE.WhileonCTS/Hb–Fe3O4/CILE(curved),apairofwell-definedredoxpeaksappearedwithincreasedelectrochemicalresponse,indicatingthatthepresenceofFe3O4mesoporoussphereontheelectrodesur-faceplayedimportantrolesinfacilitatingtheelectrontransferofHb.Fe3O4mesoporousspherehaveexhibitedgoodbiocompatibil-ity,betterconductivityandbiggersurfaceareawithmanysmallholesonthesurface,whichcouldadsorbmoreHbmoleculeswiththeelectroactivecenterofHbmoleculesmoreclosertothesphereandathree-dimensionalstructurecouldbeformed.SothedirectelectrontransferofHbwaseasilyrealizedonthemodifiedelec-trode.Theformalpotential(E0󰀇)wascalculatedas−0.287V,whichwasthecharacteristicofthehemeFe(III)/Fe(II)redoxcouplesoftheprotein[29].

ThecompositionofthemodifierontheelectrodewasoptimizedtogetthestablestandhighestelectrochemicalresponsesofHb.ByvaryingtheratioofHb,Fe3O4mesoporousspheresandCTS,directelectrochemistryofHbmodifiedelectrodewasinvestigatedandcompared.ExperimentalresultsshowedthattheamountofFe3O4mesoporoussphereshadgreatinfluencesontheredoxresponsesofthemodifiedelectrode.ThepresenceofFe3O4mesoporousspheresprovidedaspecificinterfacewithhighsurfacearea,butexcessiveamountsofFe3O4mesoporousspheresontheelectrodesurfacewouldincreasethethicknessofthefilm.Duetothegoodfilmform-ingabilityofCTS,thecompositewithoutCTSfilmwasnotstableandthemodifiercouldbeeasilyleakedouttothebuffersolution.Theexperimentalresultsindicatedthatthecompositefilmthatcon-taining20.0mg/mLHb,0.33mg/mLFe3O4mesoporousspheresand1.0mg/mLCTSwastheoptimalratioforthefabricationofmodifiedelectrode.

equalheightofredoxpeakcurrents,indicatingaquasi-reversibleelectrodeprocess.TheresultsindicatedthatalltheelectroactiveHbFe(III)inthecompositefilmwasreducedtoHbFe(II)onthefor-wardscanandthenreoxidizedtoHbFe(III)onthereversescan.Boththeredoxpeakcurrentsincreasedlinearlywithscanrate(Fig.5B),demonstratingthattheelectrodeprocessofHbwasasurface-controlledthin-layerelectrochemicalreaction.Withtheincreaseofscanratethevalueof󰀅Epwasalsoincreasedgradually,sotheelectrochemicalparametersoftheHbmodifiedelectrodereactioncanbecalculatedaccordingtotheLaviron’sequation[30].

Epc=E0−

󰀇

2.3RT

log󰀄˛nF(1)(2)

(1−˛)˛Fn󰀅EpRT−nFv2.3RT

(3)

Epa=E0+

󰀇

2.3RT

log󰀄

(1−˛)nF

logks=˛log(1−˛)+(1−˛)log˛−log

3.5.ElectrochemicalbehaviorsoftheCTS/Hb–Fe3O4/CILE

TheinfluenceofscanrateontheelectrochemicalresponsesofCTS/Hb–Fe3O4/CILEwasfurtherinvestigatedintherangefrom50.0to500.0mV/swiththeresultshowninFig.5A.Withtheincreaseofscanrateapairofsymmetricredoxpeaksappearedwiththealmost

TwostraightlinesweregotbetweentheEpvaluewiththeloga-rithmofscanrate(Fig.5C)withtheequationsasEpa(V)=0.057log󰀄(V/s)−0.326(n=15,󰀂=0.998)andEpc(V)=−0.142log󰀄(V/s)−0.071(n=15,󰀂=0.996).Thenthevaluesoftheelec-trontransfercoefficient(˛)andtheheterogeneouselectrontransferrateconstant(ks)werecalculatedas0.29and0.478s−1,respectively.Itiswell-knownthatksreflectsthelocalmicroenvi-ronmentoftheproteinimmobilizedontheelectrode.Thevalueobtainedhereisintherangeofksfortypicalsurface-controlledquasi-reversibleelectrontransferprocesses.ThisksishigherthanthatofHbimmobilizedonananocrystalinetitaniumoxidemodifiedelectrode(0.137s−1)[31],sodiumalginateandSiO2nanoparticlebionanocompositefilm(0.077s−1)[32]andcarbonnanotube(0.062s−1)[33].TheincreaseoftheelectrontransferrateofHbwasduetothespecificcharacteristicsofFe3O4mesoporoussphereswithhighsurfaceandgoodconductivity.

Byintegrationofthecyclicvoltammetriccurve,analmostunchangedchargevalue(Q)wasgotregardlessofthechangeofscanrate.Basedontheequationof󰀔*=Q/nAF,thesur-faceconcentration(󰀔*)ofelectroactiveHbwascalculatedas9.75×10−9mol/cm2.WhiletheelectroactivesurfaceconcentrationofHbonCTS/Hb/CILEwascalculatedbythesamemethodwiththeresultsas1.88×10−9mol/cm2,whichwasmuchsmallerthanthatofCTS/Hb–Fe3O4/CILE.TheresultsindicatedthatthepresenceofFe3O4mesoporousspherecouldprovideathree-dimensional

W.Sunetal./ColloidsandSurfacesB:Biointerfaces101 (2013) 177–182

181

Fig.5.(A)InfluenceofscanrateonthecyclicvoltammogramsofCTS/Hb–Fe3O4/CILEinpH7.0PBS;(B)therelationshipofthecathodicandanodicpeakcurrentwithscanrate(󰀄);(C)therelationshipofthecathodicandanodicpeakpotentialagainstlog󰀄.

structurewithmorechannelsfortheHbmoleculestoexchangeelectrons.WhilethetotalamountofHbcastedontheelectrodesurfacewas2.47×10−8mol/cm2,so39.47%oftheHbmoleculesonCTS/Hb–Fe3O4/CILEtookpartintheelectrochemicalreaction,whichwaslargerthansomereportedvalues[34,35].TheresultalsodemonstratedthatthemultilayersofHbinthecompositefilmparticipatedintotheelectrontransferprocess,whichcouldbeattributedtothespecificspherestructureofFe3O4mesoporouswithlargesurfacearea.

TheeffectofbufferpHonthecyclicvoltammetricresponsesofthemodifiedelectrodeswasfurtherinvestigated.InthepHrangefrom4.0to9.0,astableandwell-definedcyclicvoltammetriccurveswereobtained.WiththeincreaseofbufferpHtheredoxpeakpoten-tialshiftedtothenegativedirection,implyingthatprotonswereinvolvedintheelectrodeprocess.Agoodlinearregressiontionshipwasgotbetweentheformalpotential(E0󰀇

rela-)andpHwith

theequationasE0󰀇

(V)=−47.7pH+3.15(n=7,␥=0.997).Theslopevalueof−47.7mV/pHwassmallerthanthetheoreticalvalueof−56.0mV/pHat20◦Cforasingle-protoncoupledreversibleone-electrontransferprocess.Thereasonmightbeoriginatedfromtheinfluenceoftheprotonationstatesoftransligandstothehemeironandaminoacidsaroundtheheme,ortheprotonationofthewatermoleculecoordinatedtothecentraliron.However,italsocouldindicatethatasingleprotonationaccompanieswithoneelectrontransferofHbFe(III)toelectrode.Sotheelectrochemicalreactionequationcouldbeexpressedas:HbhemeFe(III)+H++e−→HbhemeFe(II).

3.6.Electrocatalyticactivity

Itiswell-knownthatredoxproteinwhichcontainsapros-theticgrouphavegoodelectrocatalyticactivitytowardsdifferentsubstratesincludingTCA,NaNO2,H2O2,etc.,whichareimportantdetectiontargetsinthefieldssuchasbiochemistryandenvi-ronmentalanalysis.TCAisametabolicbyproductofCCl4underreducedoxygentension.Alsoitisananalogueofaceticacidwiththreehydrogenatomsofmethylgroupreplacedbychlorineatoms.TCAhasbeenwidelyusedinthefieldofbiochemistryforthepre-cipitationofbiomacromoleculessuchasproteins,DNAandRNA.ItcanalsobepresentinthedrinkingwaterastheresultofChlorinedisinfectionwiththepotentialtokillnormalcells.SoitisnecessarytoestablishsensitivemethodforTCAdetectionandtheelectrocat-alyticreductionofTCAonCTS/Hb–Fe3O4/CILEwereexaminedwiththeresultsshowninFig.6.Itcanbeseenthatthecathodicpeakcur-rentincreaseddramaticallyat−0.362VwiththedisappearanceoftheanodicpeakaftertheadditionofdifferentamountsofTCAintoPBS(curvea–l),whichwasthetypicalelectrocatalyticbehaviorsofHbimmobilizedontheelectrode.TheresultsindicatedthattheactivationenergyofTCAreductionwasdecreasedgreatlyduetothepresenceofHbinthemodifiedelectrode.Inaddition,another

newreductionpeakappearedat−0.63VwiththefurtherincreaseoftheTCAconcentration.Basedonthereference[36],thesecondreductionpeakcouldbetentativelyassignedtothehighlyreducedformofHbFe(I),whichwasanactivereductantthatcoulddechlori-natedi-andmonochloroaceticacidafterthedechlorinationofTCAbyHbFe(II).TheoverallelectrocatalyticTCAreductionmechanismcanbeexpressedwiththefollowingequations:

HbFe(III)+e→HbFe(II)

(1)

2HbFe(II)+Cl3CCOOH+H+→2HbFe(III)+Cl2CHCOOH+Cl−

(2)

HbFe(II)+e→HbFe(I)

(3)

2HbFe(I)+Cl2CHCOOH+H+→2HbFe(II)+ClCH2COOH+Cl−

(4)

2HbFe(I)+ClCH2COOH+H+→2HbFe(II)+CH3COOH+Cl−

(5)

ThecatalyticreductivepeakcurrentincreasedlinearlywiththeTCAconcentrationintherangefrom2.4to20.0mmol/LwiththelinearregressionequationasIp(mA)=0.089c(mmol/L)+0.015(n=21,󰀂=0.998)andthedetectionlimitas0.033mmol/L(3󰀃).ThedetectionlimitwassmallerthanthatofNafion-CdS-HbmodifiedCILE(10.0mmol/L)[37]andcarbonnanochips(CNCs))/Hb/Nafion

Fig.6.CyclicvoltammogramsofCTS/Hb–Fe3O4/CILEinpH7.0PBScontaining2.4,3.0,4.2,4.8,6.0,7.2,8.0,10.0,11.0,12.0,15.0,16.0mmo/LTCA(curvea–l),respec-tively,withthescanrateas100mV/s(insetwasthelinearrelationshipofcatalyticreductionpeakcurrentsandtheTCAconcentration.

182W.Sunetal./ColloidsandSurfacesB:Biointerfaces101 (2013) 177–182

modifiedGCE(0.06mmol/L)[38],indicatingthehighersensitiv-ityofthemodifiedelectrode.WhentheTCAconcentrationwasmorethan20.0mmol/L,thecurrentresponseturnedtoleveloff,whichindicatedasaturationofHbcatalyticentMichalies–Mentenconstant(Kapp

reaction.Sotheappar-M),whichisanindicatoroftheenzyme–substratereactionkinetics,iscalculatedtoevalu-atethebiologicalactivityoftheimmobilizedHb.ByusingtheLineweaver–Burkequation:

1app

=1

+KMI(4)

ssImaxImaxcwhereIssisthesteady-statecurrentaftertheadditionofsub-strate,cisthebulkconcentrationofsubstrateandImaxmaximumcurrentundersaturatedsubstratesolution.Kapp

isthe

bytheanalysisofslopeandinterceptoftheplotMcanbeobtainedofthereciprocalsofthesteady-statecurrentversusTCAconcentration.

TheKapp

MofCTS/Hb–Fe3O4/CILEwasestimatedtobe14.9mmol/L,whichwassmallerthanthatofHbimmobilizedinagarosehydro-gelfilmsin1-butyl-3-methylimidazoliumhexafluorophosphateonGCE(47.0mmol/L)[34],Nafion/CdSnanorodsfilmmodifiedCILE(98.5mmol/L)[37]andCTS-MWCNTs-HbfilmmodifiedCILE(18.7mmol/L)[18].

3.7.Anti-interferingactivity

ThepotentialinterferencesonCTS/Hb–Fe3O4/CILEtothedeter-minationof4.0mmol/LTCAwereinvestigated.Undertheselectedconditions,theelectrocatalyticreductionpeakcurrentofTCAwasindividuallymeasuredinthepresenceofdifferentamountofinterferentswiththechangesofthepeakcurrentrecorded.Exper-imentalresultsindicatedthat100-foldconcentrationofZn2+,Ni2+,Cu2+,Mg2+,Ca2+,Cl−,SO42−,N03−didnotinterferewiththecat-alyticreductionpeakwiththepeakcurrentchangesbelow±5%.SoCTS/Hb–Fe3O4/CILEexhibitedgoodselectivitytotheTCAdetection.

3.8.Reproducibilityandstabilityofthemodifiedelectrode

ThestabilityofCTS/Hb–Fe3O4/CILEwasinvestigatedbyexam-iningtheredoxpeakcurrentsafter50successivelyscanningandthevoltammetricresponseswerenotchanged,indicatingthatCTS/Hb–Fe3O4/CILEwasstableinbuffersolution.Thelong-termstoragestabilityoftheHbmodifiedelectrodewasinvestigatedbykeepingtheelectrodeat4◦Cwhennotuse.93.2%oftheinitialcur-rentresponsewasretainedafter2weeksstorage.After30days,theredoxcurrentresponsedecreasedabout9%.Theresultsindi-catingthatCTS/Fe3O4nanocompositematerialwasveryefficientforpreventingtheleakageoftheproteinsandretainingelectro-catalyticactivityofHb.FiveHbmodifiedelectrodeswerepreparedbythesameprocedureindependentlyandtherelativestandarddeviation(RSD)forthedeterminationof7.0mmol/LTCAwascal-culatedas2.6%,whichindicatedthemodifiedelectrodehadgoodrepeatability.ThereproducibilityoftheHbelectrodewascheckedbysuccessivelydetecting7.0mmol/LTCAfor6timesandtheRSDvaluewasgotas3.1%,demonstratingagoodreproducibilityofthepreparedelectrode.

4.Conclusions

InthispaperaFe3O4mesoporousspherewasusedasthemodifierwithaHPPF6basedCILEasthesubstrateelectrode,

whichprovidedaspecificmicroenvironmentforthefurtherimmo-bilizationofHb.DirectelectrochemistryofHbwasrealizedin

theCTS–Fe3O4nanocompositefilmmodifiedelectrodewiththeelectrochemicalparameterscalculated.Duetothespecificchar-acteristicsofFe3O4mesoporoussphere,thesecondarystructureandcatalyticactivityofHbwasremained.TheCTS/Hb–Fe3O4/CILEshowedgoodelectrocatalyticabilitytothewiderdynamicrange,smallKapp

reductionofTCAwith

SothemodifiedMvalue,lowdetectionlimitandlongtermstability.electrodehasthepotentialapplicationinthethird-generationelectrochemicalbiosensor.

Acknowledgements

WeacknowledgethefinancialsupportoftheNationalNatu-ralScienceFoundationofChina(No.21075071,51076056),theShandongProvinceNaturalScienceFoundation(ZR2011BQ023),theFoundationofStateKeyLaboratoryofCoalCombustionofHuazhongUniversityofScienceandTechnology(FSKLCC1010)andtheDoctoralFoundationofQUST(0022424).

References

[1]F.A.Armstrong,H.A.O.Hill,N.J.Walton,Acc.Chem.Res.21(1988)407–413.[2]F.W.Scheller,N.Bistolas,S.Q.Liu,M.Janchem,M.Katterle,U.Wollenberger,

AdvColloidInterfaceSci.116(2005)111–120.

[3]J.F.Rusling,A.E.F.Nassar,J.Am.Chem.Soc.115(1993)11891–11897.[4]Z.Lu,Q.Huang,J.F.Rusling,J.Electroanal.Chem.423(1997)59–66.[5]G.Decher,Science277(1997)1232–1237.

[6]G.Decher,J.D.Hong,J.Schmitt,ThinSolidFilms831(1992)210–211.

[7]Z.H.Zhu,L.N.Qu,X.Li,Y.Zeng,W.Sun,X.T.Huang,Electrochim.Acta55(2010)

5959–5965.

[8]P.Bianco,Rev.Mol.Biotechnol.82(2002)393–409.

[9]P.Sun,D.W.Armstrong,Anal.Chim.Acta661(2010)1–16.

[10]M.C.Buzzeo,R.G.Evans,R.G.Compton,Chem.Phys.Chem.5(2004)1106–1120.[11]H.T.Liu,P.He,Z.Y.Li,C.Y.Sun,L.H.Shi,Y.Liu,G.Y.Zhu,J.H.Li,Electrochem.

Commun.7(2005)1357–1363.

[12]N.Maleki,A.Safavi,F.Sedaghati,F.Tajabadi,Anal.Biochem.369(2007)

149–153.

[13]Y.Zhang,J.B.Zheng,Talanta77(2008)325–330.

[14]W.Sun,Y.Y.Duan,Y.Z.Li,T.R.Zhan,K.Jiao,Electroanalysis21(2009)

2667–2673.

[15]W.Sun,Y.Z.Li,Y.Y.Duan,K.Jiao,Electrochim.Acta54(2009)4105–4110.

[16]W.Sun,Q.Jiang,M.X.Yang,K.Jiao,Bull.KoreanChem.Soc.29(2008)915–920.[17]W.Sun,X.Q.Li,Z.Q.Zhai,K.Jiao,Electroanalysis20(2008)2649–2654.

[18]W.Sun,Z.Q.Zhai,X.Q.Li,L.N.Qu,T.R.Zhan,K.Jiao,Anal.Lett.42(2009)

2460–2473.

[19]M.T.Reetz,A.Zonta,V.Vijayakrishnan,K.Schimossek,J.Mol.Catal.A:Chem.

134(1998)251–258.

[20]D.Cao,P.He,N.Hu,Analyst128(2003)1268–1274.[21]L.Chen,W.Gorski,Anal.Chem.73(2001)2862–2868.

[22]J.J.Feng,G.Zhao,J.J.Xu,H.Y.Chen,Anal.Biochem.342(2005)280–286.[23]N.Maleki,A.Safavi,F.Tajabadi,Anal.Chem.78(2006)3820–3826.

[24]D.W.Qi,J.Lu,C.H.Deng,X.M.Zhang,J.Phys.Chem.C113(2009)15854–15861.[25]J.K.Kauppinen,D.J.Moffatt,H.H.Mantsch,D.G.Cameron,Appl.Spectrosc.35

(1981)271–276.

[26]Y.P.Song,M.C.Petty,J.Yarwood,W.J.Feast,J.Tsibouklis,S.Mukherjee,Langmuir

8(1992)257–261.

[27]A.E.Nassar,W.S.Willis,J.F.Rusling,Anal.Chem.67(1995)2386–2392.[28]M.S.Lin,H.J.Leu,Electroanalysis17(2005)2068–2073.[29]J.F.Rusling,Acc.Chem.Res.31(1998)363–369.[30]E.Laviron,J.Electroanal.Chem.101(1979)19–28.

[31]Q.Li,G.Luo,J.Feng,Electroanalysis13(2001)359–363.

[32]W.Sun,D.Wang,Z.Zhai,R.Gao,K.Jiao,J.Iran.Chem.Soc.6(2009)412–419.[33]Y.D.Zhao,Y.H.Bi,W.D.Zhang,Q.M.Luo,Talanta65(2005)489–494.

[34]S.F.Wang,T.Chen,Z.L.Zhang,X.C.Shen,Z.X.Lu,D.W.Pang,K.Y.Wong,Langmuir

21(2005)9260–9266.

[35]X.B.Lu,J.Q.Hu,X.Yao,Z.P.Wang,J.H.Li,Biomacromolecules7(2006)975–980.[36]X.Ma,X.J.Liu,H.Xiao,G.X.Li,Biosens.Bioelectron.20(2005)1836–1842.

[37]W.Sun,D.D.Wang,G.C.Li,Z.Q.Zhai,R.J.Zhao,K.Jiao,Electrochim.Acta53

(2008)8217–8221.

[38]S.George,H.K.Lee,J.Phys.Chem.B113(2009)15445–15454.