A novel nitromethane biosensor based on

BiosensorsandBioelectronics26 (2010) 991–995

ContentslistsavailableatScienceDirect

Biosensorsand

Bioelectronics

journalhomepage:http://wendang.chazidian.com/locate/bio

s

Anovelnitromethanebiosensorbasedonbiocompatibleconductiveredox

graphene-chitosan/hemoglobin/graphene/roomtemperatureionicliquidmatrix

LuWang,XiuhuaZhang,HuayuXiong,ShengfuWang?

Ministry-of-EducationKeyLaboratoryfortheSynthesisandApplicationofOrganicFunctionalMolecules,CollegeofChemistryandChemicalEngineering,HubeiUniversity,11XueyuanRoad,Wuhan430062,China

articleinfoabstract

Anovelamperometricbiosensorfornitromethane(CH3NO2)basedonimmobilizationofgraphene(GR),chitosan(CS),hemoglobin(Hb)androomtemperatureionicliquid(IL)onaglassycarbonelectrode(GCE)wasdevelopedforthe?rsttime.Thesurfacemorphologiesofasetofrepresentativemembraneswerecharacterizedbymeansofscanningelectronmicroscopy(SEM).Theelectrochemicalperformanceofthebiosensorwasevaluatedbycyclicvoltammetry(CV)andchronoamperometry.Apairofstableandwell-de?nedredoxpeaksofHbwithaformalpotentialof?0.240VwasobservedattheGR-CS/Hb/GR/IL/GCE.TheeffectsofphosphatebufferpH,scanrate,andtemperatureonthebiosensorwereinvestigatedtoprovideoptimumanalyticalperformance.Moreover,severalelectrochemicalparameters,e.g.,thehet-erogeneouselectrontransferrateconstant(ks),werecalculatedindetail.ThepresenceofbothGRandILnotonlydramaticallyfacilitatedtheelectrontransferofHb,butalsogreatlyenhancedelectrocatalyticactivitytowardsCH3NO2.TheapparentMichaelis–Mentenconstantwasdownto0.16?M,indicatingthatthebiosensorpossessedhighaf?nitytoCH3NO2.Besidesthis,theproposedbiosensorexhibitedfastamperometricresponse(<5s),lowdetectionlimit(6.0×10?10M),andexcellentlong-timestoragestabilityforthedeterminationofCH3NO2.

© 2010 Elsevier B.V. All rights reserved.

Articlehistory:

Received10May2010

Receivedinrevisedform21July2010Accepted12August2010

Available online 19 August 2010Keywords:HemoglobinGraphene

RoomtemperatureionicliquidNitromethaneElectrocatalysis

1.Introduction

Organicnitrocompoundshaveattractedagreatdealofinter-estoverthepastdecadesbecauseofthewidespreaduseofthesecompoundsasexplosivematerials(Fainberg,1992),fuels(Sabourinetal.,2009),specialsolvents(Subiratsetal.,2005),andevenaschemicalcoatings(Wangetal.,1998).Nitromethane(CH3NO2),thesimplestofthealiphaticnitrocompounds,isoneofthemostcommonpollutantswithrelativelyhighvaporpressure(JeanandMekki,2004;Singh,2007).Thechronicexposuretonitromethaneleadstomanycriticaleffectsonhumanhealthsuchasthyroidtoxicity,blooddyscrasiasandperipheralneuropathy(Pageetal.,2001).Hence,extensiveeffortshavebeendevotedtothedetectionofCH3NO2includingcolorimetry(JonesandRiddick,1956),solid-phasemicroextraction(SPME)-gaschromatography(GC)–highresolutionmassspectrometry(HRMS)(SPME-GC–HRMS)(Alwisetal.,2008),?uorimetry(MeaneyandMcGuf?n,2008).However,amajorityofthesemethodssufferedfromlimitations.Forinstance,?uorescencespectrometrydemandslargesamplesindespiteofthehighsensitivity.Nowadays,electrochemicalmethodappearsmuchmoreeleganttobeusedtodetectCH3NO2mainlyduetospeci?city,

?Correspondingauthor.Tel.:+862750865309;fax:+862788663043.E-mailaddress:wangsf6@http://wendang.chazidian.com(S.Wang).0956-5663/$–seefrontmatter© 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.bios.2010.08.027

fast,andeaseofmonitoring(Singh,2007).However,tothebestofourknowledge,verylittleworkhasbeenperformedonthedeter-minationofCH3NO2bythedirectelectrochemicalmeasurementwiththeenzymeelectrode.

Itiswellknownthatenzyme-basedmethodshowsfastspeed,highsensitivityandgoodselectivity.So,theelectrochemicalstudiesofenzymeandproteinsareofconsiderableimportanceinbiosen-sorsandbiocatalysts.Todate,avarietyofmaterialshavebeenemployedasmodi?erstorealizedirectelectrontransferofmet-alloproteins(e.g.,hemoglobin(Hb)),suchassurfactant(Liuetal.,2007),lipid(Tangetal.,2003),hydrogelpolymer(Liuetal.,2004)andnanomaterials(Xuetal.,2009).Graphene(GR)asa“risingstar”carbonnanomaterialhasbecomeahottopicinmyriadofresearchareasduringrecentyears(Sabourinetal.,2009;Chenetal.,2009).ItwasreportedthatGRexhibitshighsurfaceareaandexcellentelectricalconductivity(Stankovichetal.,2006).Fromthisitisclearthattheincreaseofeffectivesurfaceareaishelpingforintroducingalargernumberofactivesites.Thehighconductivitycanfacilitatetheelectrontransferofelectroactivespecies.Withthoseadvan-tages,anumberofelectrochemicalsensorsandbiosensorsbasedonGRweresuccessfullydevelopedforanalyticalpurposes(Wangetal.,2009;Shanetal.,2009;Kangetal.,2009).Morerecently,increasingattentionhasbeenpaidtothemodi?edelectrodeswithGRandroomtemperatureionicliquid(IL)inhopesofsynergizingtheirexceptionalproperties(Shanetal.,2010;Liuetal.,2010).

992L.Wangetal./BiosensorsandBioelectronics26 (2010) 991–995

ILisanioniccompoundcomposedofbulkyasymmetriccationsandsmallanions,whichpreservesliquidstateoverawidetemperaturerange(Moulthropetal.,2005).Itpossessesmanyfasci-natingpropertieslikelowtoxicity,negligiblevaporpressure,widepotentialwindow,highthermalstability,goodconductivityandelectrochemicalstability(Quinnetal.,2002).Therefore,ILsuggestsagreatpotentialapplicationinvariousareas.Inthe?eldofelectro-analysis,theapplicationsofILgenerallyfocusontwoaspects.OneisthatILisservedasanalternativesolventsorsupportingelec-trolyte(Wangetal.,2005).Forexample,ourgroupshadexploredthedirectelectrochemicalandelectrocatalysisofhemeproteinsimmobilizedbyagarosehydrogel?lmsinIL(Wangetal.,2005).Ontheotherhand,ILisusedasmodi?edmaterialinapplicationofbiosensorsandbiocatalysis(Chenetal.,2007).Thishasgainedmoreandmoreattentionsinrecentyears.Chitosan(CS)isanabundantnatural-bioploymerwithuniquestructure.Ithasbeenuniversallyusedtodispersenanomaterialsandimmobilizeenzymesowingtoitsgoodwaterpermeability,excellent?lmformingability,biocom-patibility,nontoxicity,andhighmechanicalstrength(Zhangetal.,2004).

Herein,anovelnitromethanebiosensorwithsandwichstructure(GR-CS/Hb/GR/IL)wasfabricatedbyintegratingtheadvantageousfeaturesofGR,IL,andCS.Theelectrochemicalprop-ertiesofthisbiosensorwereinvestigatedincludingthedirectelectrontransferofHbandelectrochemicaldeterminationofCH3NO2.Besides,thespecialsandwichstructureplayedacrucialroleinretainingthebioactivityofHbandavoidingtheleakageoftheimmobilizedprotein.2.Experimental

2.1.Reagentsandapparatus

GR(kindlyprovidedbyProfessorXianbaoWangfromFacultyofMaterialsScienceandEngineering,HubeiUniversity,China)wereusedasreceived.Theroomtemperatureionicliquid,1-butyl-3-methylimidazoliumtetra?uoroborate([bmim][BF4]),waspurchasedfromChengjieChemicalLtd.Corp.(Shanghai,China).BovineHbwasobtainedfromFluka(Switzerland).Theconcentra-tionofHbstocksolution,preparedbydissolvingHbin0.05MpH7.0phosphatebuffersolution(PBS),was20mg/ml.CH3NO2wasfromJingchunReagentLtd.Corp.(Shanghai,China).ThestocksolutionofCH3NO2waspreparedbydirectlydilutedwithPBS(0.1M,pH7.0)accordingtothedemands.CSoflowmolecularweightwasbroughtfromSigma(USA).CShydrogelwaspreparedbydissolving5mgCSin1mlHAc(0.1M).Supportingelectrolytesolutionforexperimentswas0.1MpH7.0PBS.Allotherchemicalswereofanalyticalreagentgrade.Deionizeddouble-distilledwaterwasusedthroughouttheexperiments.Unlessotherspeci?ed,allexperimentswereoperatedatroomtemperature.

AlltheelectrochemicalmeasurementswereperformedonaCHI660Aelectrochemicalworkstation.Astandardthree-electrodesystem,witha?lmmodi?edglassycarbonelectrode(GCE)astheworkingelectrode,asaturatedcalomelelectrode(SCE)asthereferenceelectrodeandaplatinumfoilastheauxiliaryelec-trode,wasusedinthemeasurements.Nitrogenatmospherewasmaintainedduringthemeasurements.ThepHvalueofelectrolytewasdeterminedbyusinga320-Saciditymeter(Mettler-Toledo,Switzerland).AJEOLJSM-5510LVscanningelectronmicroscopy(SEM,Japan)wasappliedforcharacterizingthepreparedsamples.2.2.PreparationoftheGR-CS/Hb/GR/IL/GCE

5?lof1%IL-ethanolsolutionwas?rstdroppedontothesur-faceofthepretreatedGCEtoformtheIL/GCE.Then,5?lofthe

homogeneousGR-DMFsolution(1mg/ml)wascoveredtheIL/GCE.Afterdrying,10?lHbsolutions(20mg/ml)werespreadontotheGR/IL/GCEsurface.Finally,5?loftheresultingCS-dispersedGRsolution,inwhichtheconcentrationofGRwas1mg/ml,wascoatedonthesurfaceofHb/GR/IL/GCE.TheelectrodereferredinthetextasGR-CS/Hb/GR/IL/GCEwasfabricated(seesupportinginformation,Scheme1).Forcomparison,GR-CS/Hb/GR/GCE,GR-CS/Hb/IL/GCE,GR-CS/GR/IL/GCEandCS/Hb/GR/IL/GCEwerealsopreparedbythissimpledrop-castingtechnique.3.Resultsanddiscussion

3.1.MorphologycharacterizationofthefabricatedbiosensorThesurfacemorphologiesofGR-CS/Hb/GR/ILmembraneandthreedifferentmembraneswereexaminedbySEMobservation.ThetopviewsofGR(Fig.1a)clearlyillustratedthetypically?ake-likewithslightlyscrollededgesshapes,whichwasconsistentwiththepreviousreports(Shanetal.,2009;Xuetal.,2010a).Fig.1bpresentedtheSEMimageofHb?lm.Thebrightglobu-larstructureswereassignabletothedepositionofHbmoleculeswithlargeaggregations.AscanbeseenfromFig.1c,theGR-CS/Hb/GRmembranelookedroughandincompact.However,forGR-CS/Hb/GR/ILmembrane(Fig.1d),atotallydifferentmorphol-ogywasobtained.ItseemedthattheGR-CS/Hb/GR/IL?lmwascharacterizedwithuniformandsmoothsurface.ThedegreeofevennesswasbetterwhenILwasadded,whichcouldbeattributedtothebindingandblanketingeffectofviscousIL(Xiaoetal.,2008).

3.2.Cyclicvoltammetrycharacterizationofthebiosensor

Todiscerntheroleofeachcomponentandpossiblesynergybetweenthem,cyclicvoltammograms(CVs)ofdifferentelectrodeswererecordedinFig.2.NovoltammetricresponsewasobservedforGR-CS/GR/IL/GCEexceptforalargebackgroundcurrent(Fig.2a).Whereasapairofwell-de?nedandnearlyreversiblepeakswiththeformalpotentialof?0.240VappearedonGR-CS/Hb/GR/IL/GCE(Fig.2b),indicatingthatthecoupleofpeaksarisesfromtheredoxreactionofHb–Fe(III)/Fe(II)(Wangetal.,2005).Incomparison,thecurrentresponsesofGR-CS/Hb/GRmatrixwithoutILwerefaintandfeeble(Fig.2c),meaningthatILplayedanimportantroleinfacilitatingthedirectelectrontransferofHb.Althoughthepeakcur-rentsdecreasedapparentlyandthepeak-to-peakseparation(??Ep)increasedsynchronouslyatbothGR-CS/Hb/IL/GCE(Fig.2d)andCS/Hb/GR/IL/GCE(Fig.2e),thepeakcurrentofHbdecreasedmoresharplyatGR-CS/Hb/IL/GCE.ThephenomenondemonstratedthatGR,especiallyclosetothesurfaceofelectrode,greatlypromotedtheelectrontransferbetweenredoxenzymeandtheunderlyingelec-trode(Xuetal.,2010a).Thus,thecombinationofILandGRshowedanextraordinarysynergisiticenhancementfortheelectrontransferrateofHbenzyme.

3.3.Selectionoftheoptimumexperimentalconditions

3.3.1.In?uenceofpHonelectrochemicalbehaviorsofthebiosensor

ThepHeffectofsupportingelectrolyteontheelectrochemi-calbehaviorsofHbwasexploredovertherangeof4.0–9.0(seesupportinginformation,Fig.S1).Boththeanodic(Epa)andcathodic(Epc)potentialshiftednegativelywiththeincreaseofpH,implyingthattheelectrochemicalprocessinvolvedprotontransfer.Intion,theEpa,EpcandE0??

addi-(formalpotential)werelinearlydependentonthepHvalueofPBSwiththeslopevaluesof45,42and44mV/pH,respectively.Allthoseslopevaluesweresmallerthanthetheo-reticalvalueof57.6mV/pHat18?Cforasingle-protoncoupled

L.Wangetal./BiosensorsandBioelectronics26 (2010) 991–995

993

Fig.1.SEMimagesof(a)GR,(b)Hb,(c)GR-CS/Hb/GRand(d)GR-CS/Hb/GR/ILontheglassycarbon.

reversibleone-electrontransfer(Bond,1980;Meites,1965).Thereasonforthisdifferencemightresultfromthein?uenceoftheprotonationoftransandresidueligandsaroundheme,orthepro-tonationofthewatermoleculecoordinatedtothecentraliron(Luetal.,2007).Theanodicpeakcurrentschangedsigni?cantlyinthepHrange.ThemaximumvalueofpeakcurrentswasachievedatpH7.0.SopH7.0wasdeemedastheoptimumpHandusedinthefurther

experiments.

Fig.2.CVsof(a)GR-CS/GR/IL/GCE,(b)GR-CS/Hb/GR/IL/GCE,(c)GR-CS/Hb/GR/GCE,(d)GR-CS/Hb/IL/GCEand(e)CS/Hb/GR/IL/GCEin0.1MpH7.0PBSatascanrateof0.1V/s.

3.3.2.In?uenceofscanrateonelectrochemicalbehaviorsofthebiosensor

Withtheincreaseofscanratefrom10to500mV/s,theanodicandcathodicpeakcurrentsofHbgrewlinearly,demonstrat-ingthattheredoxprocessofHbwassurface-con?ned(Zhangetal.,2007)(seesupportinginformation,Fig.S2).What’smore,thechargeconsumed(Q),obtainedbytheintegrationofreduc-tionpeaks,keepalmostunchangedregardlessofthescanrate.AccordingtoFaraday’slaw,Q=nFA??*(wherenisthenumberofelectrontransferred,FandAstandfortheFaradayconstantandthegeometricareaofthemodi?edelectrodesurface,respec-tively).Therefore,thesurfaceconcentrationsofelectroactiveHbatGR-CS/Hb/GR/IL/GCEwasestimatedtobe7.70×10?10molcm?2,whichwasabout41timesaslargeasthetheoreticalmonolayercoverage(1.89×10?11molcm?2)(Liuetal.,2004)and1.8timeshigherthanthat(4.25×10?10molcm?2)atHb/RTIL/PDDA-G/GCE(Liuetal.,2010).ThisrevealedthatmultilayersofHbparticipatedintheelectron-transferprocess.ThepercentageofelectroactivityHbontheelectrodesurfacewas2.19%,illuminatingthatonlytheproteinmoleculesclosetotheelectrodesurfaceandwithsuitableorientationcouldexchangeelectronswiththeelectrodesurface(Wangetal.,2005).

Ontheotherhand,thepeakpotentialsshiftslightlywithincreasingscanratefrom0.01to1V/s.However,thevaluesofEpcandEpawereproportionaltothelogarithmofscanrateswithslopesof2.3RT/(1??)nFand2.3RT/?nFathighscanrates.Sotheelectrontransfercoef?cient(?)wascalculatedtobe0.42.BasedontheLavirontheory(Laviron,1979),theheterogeneouselec-trontransferrateconstant(ks)ofHbcouldbedeterminedandestimatedtobe57.3s?1,whichwassuperiortothatincorpo-ratedinDDAB-HIMIMPF6(11.6s?1)(Xuetal.,2010b),SA-MWCNTs(9.54±0.883s?1)(Zhaoetal.,2009),andPVA/MWCNTs/CILE(1.05s?1)(Sunetal.,2009).Thepromotiontoelectrontransferof

994L.Wangetal./BiosensorsandBioelectronics26 (2010) 991–995

HbmaybeduetothesynergeticfunctionofGRandILwithgoodbiocompatibilityandhighconductivity.

3.3.3.In?uenceoftemperatureonthedetectionofCH3NO2bythebiosensor

Undertheoptimalconditionsestablishedabove,theeffectoftemperatureonthecurrentresponseofthebiosensorto2.12mMCH3NO2wasinvestigatedattherangefrom10to60?C(datanotshown).Itcanbenotedthatthecurrentincreasedwiththetemper-atureupto30?C,andthengraduallydecreasedasthetemperaturefurtherincreases.TheresultsmaybeattributedtothepartialdenaturationofHbathightemperatures.Consideringboththelife-timeandcurrentresponseofthebiosensor,theroomtemperature(25?C)waschosenastheoptimumtemperatureindetectionofCH3NO2.

3.4.AmperometricresponseoftheCH3NO2biosensor

AsshowninFig.3A,therewasnoelectrochemicalresponseatGR-CS/GR/IL/GCEintheabsenceofCH3NO2(curvea).However,acathodicpeakcorrespondingtothereductiveofCH3NO2wasobtainedat?1.01VwhenCH3NO2existed(curveb).Inaddition,itwasnecessarytomentionthatanewpeak,appearedat?0.790Vwithconsiderablyhighercurrentresponse,wasclearlyobservedontheGR-CS/Hb/GR/IL/GCEwhenCH3NO2wasaddedintothedetec-tionsolution(curved).ThepresenceofHbledtothegreataugmentinthecurrentresponseofCH3NO2andtherapiddeclineoftheover-potential,whichwereallcharacteristicofelectrocatalyticreductionofCH3NO2byHb.Furthermore,thedecreaseoftheoverpotentialwasfavorabletoavoidpossibleinterferencesfromtheelectroactivesubstancesinbiologicalmatrixes(Duetal.,2009).

Therewasnodoubtthatthecurrentresponseofelectrochem-icalbiosensorswasstronglydependentontheappliedpotentials.Ontheotherhand,theselectivityofelectrochemicalbiosensorsisalsopotential-dependant.Tothebestofourknowledge,thecoex-istingbiologicalcompound,suchasO2andNO2?,mayinterferewiththeelectrochemicaldeterminationofCH3NO2atthemorenegativelypotentials(Zhuetal.,2009;Weietal.,2009).There-fore,amperometricresponsesofthebiosensortoCH3NO2wereexaminedatdifferentpotentialsrangingfrom?0.6to0V.Amongthesepotentials,theoptimizedworkingpotentialof?0.35Vwasselectedtoensurearelativelyhighsensitivity,suf?cientcur-rentresponse,aswellasrelativelylowbackgroundcurrentofthebiosensor.InordertodemonstratetheperformanceoftheproposedCH3NO2biosensor,acomparisonoftheamperomet-ricresponseoftheGR-CS/Hb/GR/IL/GCEwiththatoftheothermodi?edelectrodesattheappliedpotentialof?0.35Vwasdis-playedinFig.3B.ForboththeGR-CS/GR/IL/GCE(curveb)andCS/Hb/IL/GCE(curvec),thevisionofsmallcurrentresponsewascaught.Incontrast,thelargeststeppedgrowthofreductioncur-rentcouldbeseenatGR-CS/Hb/GR/IL/GCE(curvea).ItwasinferredthatthesynergisticeffectoftheelectrocatalyticactivityofHbandGRresultedinthebettercatalyticpropertyofGR-CS/Hb/GR/IL/GCEtowardCH3NO2thanthatofothercases.ItwasknownfromtheinsetofFig.3BthattheGR-CS/Hb/GR/IL/GCEhadanexcel-lentlinearresponsetoCH3NO2intheconcentrationrangefrom2.0×10?9to2.3×10?7Mwithacorrelationcoef?cientof0.9991,whichwasmuchwiderthanthatof2.0×10?9–7.5×10?8MatCS/Hb/IL/GCE.Alowdetectionlimitof6.0×10?10M(S/N=3)wasobtainedbythepreparedbiosensor,whichwasevidentlylowerthanthevalueof3.3×10?5MbyMb/ddab/PGelectrode(JeanandMekki,2004)andwasalsocomparabletothatof1.6×10?10MbySPME-GC–HRMSmethod(Alwisetal.,2008).Thesensitivityofthebiosensor(788?AmM?1)wasimprovedapparentlycomparedwiththatof329?AmM?1atGR-CS/GR/IL/GCEand211?AmM?1atCS/Hb/IL/GCE.Moreover,95%ofthesteady-statecurrent

was

Fig.3.(A)CVsat0.1V/sin0.1MpH7.0PBSfor(a)GR-CS/GR/IL/GCEand(c)GR-CS/Hb/GR/IL/GCEwithoutCH3NO2;(b)GR-CS/GR/IL/GCEand(d)GR-CS/Hb/GR/IL/GCEinbuffercontaining2.12mMCH3NO2.(B)Amperometricresponseof(a)GR-CS/Hb/GR/IL/GCE,(b)GR-CS/GR/IL/GCEand(c)CS/Hb/IL/GCEuponsucces-siveadditionsof7.92?MCH3NO2intostirring0.1MpH7.0PBS.Appliedpotential:?0.35V.Inset:thecorrectedcurvesofcatalyticcurrent(Icat)againstCH3NO2con-centrationfor(a)GR-CS/Hb/GR/IL/GCE,(b)GR-CS/GR/IL/GCEand(c)CS/Hb/IL/GCEin0.1MpH7.0PBS.Therelativestandarddeviation(RSD)islessthan7%.

reachedwithin5swhenaliquotsofCH3NO2wereadded,indicat-ingtherapidresponseofthebiosensor.SuchfastresponsemaybeonaccountoftheintroductionofGRandtheeasydiffusionofsub-strateintheGR-CS/Hb/GR/ILmatrix.ThecalibrationcurvetendedtoleveloffwhentheconcentrationofCH3NO2becamelarger,manifestingthecharacteristicsoftheMichaelis–Mentenmechanism.TheapparentMichaelis–Mentenconstant(Kapp

kinetic

m)forthebiosensortoCH3NO2wascalculatedtobe0.16?MaccordingtotheLineweaver–Burkequation(KaminandWilson,1980),whichwasremarkablylowerthanthevaluereportedtrode(JeanandMekki,2004).ThelowKapp

byMb/ddab/PGelec-mvaluesuggestedthatHbinGR-CS/Hb/GR/ILmembraneretaineditsbioelectrocatalyticactivityandexhibitedahighaf?nitytoCH3NO2.3.5.Reproducibilityandstabilityofthebiosensor

Therelativestandarddeviation(RSD)was5.41%for10suc-cessivedeterminationsof1.36×10?7MCH3NO2usingonesingleGR-CS/Hb/GR/IL/GCE.Theresultilluminatedthatthebiosensor

L.Wangetal./BiosensorsandBioelectronics26 (2010) 991–995995

showedtheexcellentreproducibilityforthedeterminationofCH3NO2.Thestabilityofthebiosensorwasalsoinvestigated.Thebiosensorcouldmaintain90%ofitsinitialresponseafterbeingstoredfor50daysintherefrigerator.Asexpected,thebiosen-sorposedoutstandinglong-timestoragestability,whichmightbeduetothefollowingaspects.Firstly,theuniquesandwich-likestructuresofGR-CS/Hb/GR/ILmatrixcouldprovideafavorablemicroenvironmentforHbtoremainitsconformationandbioactiv-ity.Additionally,theoutlayerofGR-CShybrid?lmcouldef?cientlypreventtheleakageoftheboundHb(Xuetal.,2010a;Zhangetal.,2007).Owingtotheaboveadvantages,itcanbeseenthatthismethodwasreliableforthedeterminationofCH3NO2.3.6.Selectivityandapplicationofthebiosensor

Thein?uenceoftheinterferingspecieson7.84×10?8MCH3NO2determinationwasexamined.Itwasfoundthat1000-foldNO3?,C7H8,C6H5NO2didnotinterferewiththetestofCH3NO2.Thegoodselectivityofthebiosensormightberelatedtothelowwork-ingpotentialof?0.35V.Todemonstratetheapplicationpotentialofthebiosensorinenvironmentalanalysis,realfreshwatersamplesdetectionwasperformed.ItwasfoundthattheCH3NO2contentinthetestedsampleswastoolowtobedetectedbythebiosen-sor(datanotshown).However,thereisanevidentincreaseinthecurrentresponseifdifferentconcentrationsofCH3NO2wereaddedintothesamples.ThissuggestedthattheproposedbiosensorcouldbeappliedtothedeterminationofCH3NO2inenvironmentsamples,withalinearrangefrom9.9×10?9to4.0×10?7Mandasensitivityof353?AmM?1.AlthoughtheresultsofCH3NO2anal-ysiswerenotsosatis?edinrealsamples,itisstillanareathatwewillbeworkinghardtoimprove.4.Conclusion

Inthispaper,theemploymentofGRnanosheets,ILandbiopolymer(CS)associatedwiththeinherentcatalyticactivityofHbtowardCH3NO2,provideustofabricateanewCH3NO2biosen-sor.Thisbiosensorofferedseveralfascinatingadvantages:?rst,theespecialsandwichstructureofthebiosensorcouldprovideafriendlymicroenvironmentforHbtopreserveitsnativestructureandbioactivity.Second,theappearanceofGRandILwithhighconductivityandprominentbiocompatibilitygreatlypromotedthedirectelectrontransferbetweenHbandtheunderlyingelec-trode.Therefore,thebiosensorachievedalimitofdetectiondownto6.0×10?10MforquantitativeCH3NO2analysis.Inaddition,theconstructedbiosensordisplayedhighsensitivity,enhancedaf?nity,andaprolongedgoodstabilizationtothedetectionofCH3NO2.AlthoughthedeterminationofCH3NO2inrealsamplesbythebiosensorisintheimprovementstages,westillbelievethisapproachcanpaveanewandcost-effectivewayforbiomon-itoringofCH3NO2bothinmethodologicalstudyandinclinicallaboratories.Acknowledgements

ThisworkwassupportedbytheNationalNaturalScienceFoun-dationofChina(No.20875023).

AppendixA.Supplementarydata

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