Electrochemical hydrogen peroxide sensors fabricated using cytochrome immobilized on macroelectrodes

ColloidsandSurfacesB:Biointerfaces123(2014)866–869

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ColloidsandSurfacesB:Biointerfaces

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ShortCommunication

Electrochemicalhydrogenperoxidesensorsfabricatedusingcytochromecimmobilizedonmacroelectrodesandultramicroelectrodes

S.EhsanSalamifar,StephenLee,http://wendang.chazidian.comi?

DepartmentofChemistryUniversityofNebraska-Lincoln,651HamiltonHall,Lincoln,NE68588-0304,USA

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abstract

Articlehistory:

Received2August2014

Receivedinrevisedform4October2014Accepted15October2014

Availableonline28October2014

Keywords:Cytochromec

HydrogenperoxideUltramicroelectrodesRotatingdiskelectrodesMichaelis–MentenkineticsElectrontransferrate

Wereportthedesignandfabricationofhydrogenperoxide(H2O2)sensorsusinghemeproteinsimmo-bilizedonmacroelectrodesandultramicroelectrodes(UMEs).Inthissensordesign,thehemecentersaredirectly“wired”totheelectrodeviatheuseofanimidazole-terminatedself-assembledmonolayer.Wehavesystematicallyevaluatedtheeffectofelectrodetypeandsizeonsensorperformance.ThelimitofdetectionforH2O2determinedusinga10-?mgoldUMEissigni?cantlylowerthanthatobtainedusingastationarymacroelectrode.OurresultsalsohighlighttheadvantagesofusingUMEsforenzymekineticsanalysis;theKmdeterminedusinga10-?mUMEissimilartothatobtainedfromarotatingdiskelectrode.

©2014ElsevierB.V.Allrightsreserved.

Reactiveoxygenspecies(ROS)arecontinuouslyproducedinaer-obiccellsasby-productsofmetabolism.Someofthemarehighlytoxicandthusrapidlyscavengedbybothenzymaticandnonen-zymaticpathways[1,2].Owingtothesedetoxifyingmechanisms,intracellularROSlevelismaintainedataconstantlevel.Faultycon-trolofthesechemicalprocessescouldresultinROSaccumulationinsidecells,creatingaconditioncalled“oxidativestress”,whichhasbeenknowntocausecellgenomeinjury[3].Thecorrelationbetween“oxidativestress”andpathologicalconditionssuchasdif-ferenttypesofcancersiswellestablished[4,5].Thereisaneedfordevelopingrapidandsensitivemethodforinvivomeasure-mentofROSinbiomedicalresearch.Recently,ultramicroelectrodes(UMEs)andnanoelectrodes,duetotheiruniquepropertiessuchashigherratesofmasstransport,reducedcapacitanceandreducedohmicdrop,havefoundwidespreadapplicationsinrealtimeanal-ysisofROSatsinglecelllevel[6–8].WhileROScanbedetecteddirectlyusingPtUMEs,therearemeritsindevelopingbiosensorscapableofmeasuringROS.Hemeproteinssuchashemoglobin,myoglobin(Mb)andcytochromec(Cytc)areknowntobecapa-bleofcatalyzingreductionofROSsuchashydrogenperoxide(H2O2)[9].Biosensorshavesincebeenfabricatedusingthesepro-teins;however,fewhavebeenfabricateddirectlyonanimidazole

?Correspondingauthor.Tel.:+14024725340;fax:+1402472-9402.E-mailaddress:rlai2@unl.edu(http://wendang.chazidian.comi).

(Im)-terminatedself-assembledmonolayer(SAM)[10–12].HerewereportthedesignandfabricationofH2O2sensorsusinghemeproteinsthataredirectly“wired”totheelectrode.Thisstudyalsofocusesonthecomparisonofhemeprotein-basedH2O2sensorsfabricatedonUMEsandmacroelectrodes.

PriortousingagoldUMEasthesensorelectrode,weevalu-atedtheeffectofdifferentproteinimmobilizationproceduresontheelectrochemicalsignalofthesensorongoldmacroelectrodes.We?rstfabricatedsensorsusingtwoconventionalmethods,directadsorptionandcovalentattachmentvian-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(NHS-EDC)cou-pling[13,14].Forbothsystems,theeffectsofionicstrength,incubationtimeandconcentrationofreagentswerealsoinvesti-gated.Cyclicvoltammograms(CVs)ofthesensorsfabricatedusingtheoptimizedprotocolsareshowninFig.S1.Asetofredoxpeakswithahalf-wavepotential(E1/2)of??0.05Vwasobservedinadegassedphosphatebuffersolution(PBS),indicatingsuccess-fulimmobilizationofCytconthe11-mercaptoundecanoicacid(C11terminatedSAM(Fig.S1A).However,theelectro-chemicalsignalwasunstable,presumablybecauseofthegradualdesorptionofproteinsfromtheelectrodesurface.Incontrast,whiletheuseofNHS-EDCcouplingresultedinasensorwithastableelectrochemicalsignal(Fig.S1B),themulti-stepprocessrendersthisapproachlessattractive.Thisproteinimmobilizationmethodisalsolessreproducible(i.e.,largersensor-to-sensorvariationinprobecoverage),thusitisnotidealforsensorfabrication.Because

http://wendang.chazidian.com/10.1016/j.colsurfb.2014.10.0330927-7765/©2014ElsevierB.V.Allrightsreserved.

S.E.Salamifaretal./ColloidsandSurfacesB:Biointerfaces123(2014)866–869

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Scheme1.Schematicrepresentationofdirect“wiring”ofhemeproteinsontoagoldelectrodemodi?edwithC11-ImandC8-OH.

ofthesenegativeresults,immobilizationofMbwasnotattemptedusingthesetwoapproaches.

ToeffectivelyanchorCytcandMbforourcurrentapplica-tion,weemployedarecentlydevelopedimmobilizationmethodthatrelieson“direct-wiring”ofthehemecenterstotheelectrode(Scheme1andFig.S2)[10–12].Inbrief,agoldelectrodewas?rstmodi?edwith1-(11-mercaptoundecyl)imidazole(C11-Im)and8-mercapto-1-octanol(C8-OH),theSAM-modi?edelectrodewasthenexposedtoasolutioncontainingeitherCytcorMb.Theelectrochemicalsignaloftheimmobilizedproteinswasrecordedinaprotein-freePBSsolution.Otherpassivatingdiluents,including6-mercapto-1-hexanol(C6-OH)and4-mercapto-1-butanol(C4-OH),werealsoused;C8-OH,however,wasfoundtobebestsuitedforproteinimmobilization.SensorspassivatedwithC6-OHorC4-OHwerenoticeablylessstable,whichisnotunexpectedbasedonourpreviousstudiesonotherSAM-basedbiosensors[15].

Inadditiontothechoiceofpassivatingdiluent,theconcentra-tionratiobetweenthetwoalkanethiolsusedinSAMformationwasfoundtohaveaneffectontheresultantproteincoverage.Highestelectrochemicalsignal(i.e.,highestproteincoverage)wasachievedusingaC11-Im:C8-OHratioof1:1forCytcand7:3forMb(Fig.S3).Threehoursofexposuretimetotheproteinsolution(50?MforCytcand65?MforMb)resultedinthehighestelectrochemicalsignal.Thesensorsfabricatedusingtheoptimalprotocolshowedasetofredoxpeaks,verifyingsuccessfulimmobilizationofpro-teinsontotheC11-Im/C8-OHSAM.TheE1/2wasmorenegativethanthatrecordedfromsensorsfabricatedviadirectadsorptionorNHS-EDCcoupling;thiscouldbeduetothestronginteractionbetweenImandthehemecenter[10,11,16].Thelinearrelation-shipbetweenthepeakcurrent(Ip)andscanrate(??),aswellasthenon-linearcorrelationbetweenIpand??1/2,con?rmsthattheelectrontransferprocessissurface-con?nedinbothcases(Figs.S4andS5).Furthermore,thepeakseparation(??Ep=Epa?Epc)forbothsystemswasproportionaltolog??atscanrateshigherthan2Vs?1(Fig.S6).AccordingtotheLavirontheory[17],theelectrontransferrateconstant(ks)was443±36s?1and2136±353s?1forCytcandMb,respectively[18,19].Despitethelargerks,MbisnotidealforthisapplicationbecauseofitssensitivitytowarddissolvedO2(datanotshown).Eventhoughalltheexperimentswereperformedindegassedsolutions,thereareadvantagesinusingaproteinthatislesspronetointerferencesfromO2.Thus,Cytc,theproteinthatislesssensitivetoO2,wasusedfortherestofthestudy.

TheelectrocatalyticcharacteristicofhemeproteinstowardH2O2reductioniswellestablished[20].ToverifytheactivityoftheimmobilizedCytc,weaddedatotalof800?MH2O2tothecell.Fig.S7showsCVsoftheCytc-modi?edelectrodebeforeandafterseveraladditionsofH2O2.AdditionofH2O2resultedinasimulta-neousincreaseanddecreaseinthecathodicpeakcurrent(Ipc)and

anodicpeakcurrent(Ipa),respectively.Theseresultsverifythatthehemecenteroftheprotein,evenafterbeingboundtoIm,retainsitselectrocatalyticpropertiestowardreductionofH2O2.AdditionofH2O2tosensorswithoutthehemeproteinonlyresultedinaminorchangeinthebackgroundcurrent,provingtheroleofCytcintheelectrocatalyticreductionofH2O2.Thelimitofdetection(LOD)ofthissensorwasfoundtobe200?M.DespitetherelativelyhighLOD,thesesensorsshowedgoodstability;weobserved<10%reductionintheCytcelectrochemicalsignalevenaftertheyhadbeenkeptindegassedPBSat4?Cfor5days.Theprobecoverageofsensorsfabricatedondifferentelectrodesvariedlessthan20%,verifyingreproducibilityandsensor-to-sensorconsistencyinthefabricationprocess.Regardingsensorselectivity,whilewedidnotperformanextensivestudyontheselectivityofthissensor,itisexpectedtobeselectivetowardH2O2reduction.Previousresearchhasdemon-stratedthatthesemediator-freeenzymebiosensors,alsoknownasthird-generationbiosensors,arehighlyselectivetowardtheirtargetsincetheyoperateatapotentialrangeclosetotheredoxpotentialoftheprotein[21].

Toimprovesensorsensitivity,wefabricatedthesamesensoronagoldrotatingdiskelectrode(RDE)andusedhydrodynamicamperometryasthedetectiontechnique.AsshowninFig.1A,successiveadditionsof10?MH2O2resultedinanincreaseintheelectrocatalyticcurrent.TheuseoftheRDEsystemenablesrapidsignalequilibration;steadystatecurrentwasobservedin<10saftertheadditionofH2O2.Thecatalyticcurrentwaspropor-tionaltotheconcentrationofH2O2,exhibitingalineardynamicrangebetween10and250?M(Fig.1B).TheLODwasestimatedtobe3?M(s/n=3),substantiallylowerthantheLOD(200?M)obtainedusingastationarymacroelectrode(Fig.S7).ItisworthnotingthattheresultantcurrentdeviatedfromlinearityatH2O2concentrationsbeyond250?M,suggestingthatthekineticsofthiselectrocatalyticprocessfollowstheMichaelis–Mentenmodel[22].TheapparentMichaelis–Mentenconstant(Km),whichindi-catestheenzyme–substratekinetics,wascalculatedusingtheLineweaver–Burkequation[23]:

11Km1=+·ssmaxmax(1)

where,Issisthesteady-statecurrentaftertheadditionofthesub-strate,CisthebulkconcentrationofsubstrateandImaxisthemaximumcurrentmeasuredundersaturatedsubstratesolution.Kmcanbecalculatedfromtheslopeandinterceptofthedoublereciprocalplot(1/Issvs.1/Cplot)(Fig.S8).AKmof1.38mMwasdeterminedforthissensor,suggestingthattheproteins,asimmo-bilized,haveahighaf?nitytowardthesubstrate,H2O2.ThisvalueiswithintherangeofpreviouslyreportedKmvalues[24–26].ForimmobilizedCytc,thereisagreatvariabilityintheKmvalues

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Fig.1.ChronoamperometricscanofaRDEmodi?edwithC11-Im/C8-OHandCytcbeforeandaftersuccessiveadditionofH2O2indegassedPBS(A).AlsoshownisthecalibrationplotshowingalinearincreaseincurrentwithincreasingH2O2concentrationbetween10and250?M(B).Thesensorelectrodewasbiasedat?0.4V(vs.Ag/AgCl)andtherotationspeedwas1200rpm.

reportedintheliterature,presumablybecauseoftheuseofdiffer-entproteinimmobilizationmethodsandexperimentalconditions[16].AlthoughtheKmisnotextremelylow,thisproteinimmobi-lizationstrategyhasdemonstratedtobeeffective,versatile,andtheresultantsensorisstableenoughtobeusedinthefabricationofanultramicro-biosensor.

Toevaluatetheeffectofelectrodesizeonsensorperformance,wefabricatedthesameCytc-basedsensoronsmallgolddiskelec-trodeswiththreedifferentdiameters,100?m,25?mand10?m.Fig.2Ashowstheeffectofelectrodesizeontheelectrochemi-calsignaloftheimmobilizedCytc.Asexpected,theredoxpeaksdecreasedinsizewithdecreasingelectrodesize,giventhatfewerproteinscanbeimmobilizedonanelectrodewithasmallersur-facearea.However,despitebeingdwarfedbytheCVsobtainedfromthelargerelectrodes,theredoxpeaksintheCVofthe10-?mUMEwerestilldistinguishable.Inadditiontothemainredoxpeaks,weobservedasmallpeakat?0.09V,whichcouldbeattributedtothenonspeci?cally-adsorbedCytconthemonolayer.UMEshaveseveraladvantages,forexample,theratiooffaradaic/non-faradaiccurrentisoftenimprovedbecauseofthesmallIRdrop.Thus,inCVthecurrentreachesasteadystatevalueathighoverpotentials,whichcanbedescribedusingthefollowingequation:

iss=4nFDCa

(2)

whereissisthediffusion-limitedsteady-statecurrent,aistheradiusoftheelectrode,nisthenumberofelectronstransferredpermolecule,FistheFaraday’sconstant,andDandCarethedif-fusioncoef?cientandbulkconcentrationoftheredoxmolecule,respectively[27].Becauseoftheseuniqueproperties,UMEsarewell-suitedforuseassensorelectrodes,inparticular,forimprov-ingsensorsensitivity.AscanbeseeninFig.2B,achangeintheCVcurrentwasevidentaftertheadditionof10?MH2O2forthesen-sorfabricatedona10-?mUME.ThisLODisslightlyhigherthan

thatdeterminedusingtheRDEsystem(3?M).DespitetheslightdifferenceintheLOD,UMEshaveclearlydemonstratedtobeuse-fulinthefabricationofthisclassofSAM-basedbioelectrocatalyticsensors,albeitwithouttheneedofamoresophisticatedapparatus.Intheory,duetotheenhanceddiffusion,anUMEcouldbeusedinplaceofaRDE,whichmainattributeistocircumventmasstrans-ferlimitations.Tosupporttheabovetheory,wedeterminedKmusingissrecordedwiththe10-?mUMEafterseveraladditionsofH2O2(Fig.2B).Thedoublereciprocalplotderivedfromissrecordedat?0.4VwasusedtodetermineKmaccordingtoEq(1)(Fig.S9).ThecalculatedKmwas720?M,muchlowerthanthevalueobtainedusingtheRDE.ThisdifferencecouldbeattributedtotheenhanceddiffusionattheUME,therebyprovidingahigher?uxofH2O2totheelectrodesurface.Whileamorecomprehensivestudy,whichincludestheuseofdifferentrotationspeed,Cytcsurfacecoverage,andionicstrength,isnecessarytoelucidatethereasonsbehindthisobservation,ourresultshighlighttheadvantagesofusingUMEsinbioelectrocatalyticsensingapplications.Furthermore,itisversatileandcanpotentiallybeusedforanalysisofenzymekineticsofawiderangeofhemeproteins.TheanalyticalperformanceofthecurrentH2O2sensorcanbecomparedtopreviouslydevelopedenzymaticandnon-enzymaticH2O2sensors[28,29].Ingeneral,formediator-freeenzymaticbiosensors,inwhichthesignalingmechanismisbasedonthedirectelectrontransfer(DET)betweentheimmobi-lizedredoxproteinsandtheelectrode,theLODcanreachaslowas10?8M.However,formostH2O2sensorsthatarefabricatedusingCytcimmobilizedonSAMs,suchasthatreportedbyJietal.[30]whichutilizesabinarySAMofthiocticacidandthiocticamide,theLODishigher(e.g.,10?M).DespitedifferencesintheSAMusedinsensorfabrication,theperformanceofmanyotherCytc-basedsensorsisquitesimilar.Ofnote,themainchallengeinconstruc-tingthesemediator-freeenzymaticbiosensorsistheoptimizationofDETbetweenthehemeproteinsandtheunderlyingelectrode.

Fig.2.CVsof10-?m(gray),25-?m(dashed)and100-?m(black)goldelectrodesmodi?edwithC11-Im/C8-OHandCytc(A).CVsofa10-?mUMEmodi?edwithC11-Im/C8-OHandCytcbeforeandaftertheadditionof10,20,30,40,50,and60?MH2O2.AllCVswerecollectedatascanrateof4Vs?1inPBS.

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Itisgenerallydif?culttoachieveDETofredoxproteinsowingtothefactthatactivesitesaredeeplyembeddedintheproteinshells.Moreover,adsorptionorentrapmentofenzymesontothedifferentmaterialsorelectrodesurfaceusuallyresultsinthedenaturationandlossofbioactivityoftheimmobilizedenzymes[31].Thus,inthedesignofthisclassofsensors,?ndingasimplemethodtofacili-tateDETinprede?nedpathwaysinterconnectingtheactivecenterandtheelectrodesurfaceisextremelycrucial[32].Inthiscase,theIm-terminatedSAMservesasanidealplatformforimmobilizationofCytc,therebypreventingtheproteinfromdenaturation,aswellasenabling“directwiring”betweentheredoxcenterofthepro-teinandtheunderlyingelectrode.ItisasimpleyeteffectivewaytofacilitateDET,circumventingthemainchallengeinthedesignandfabricationofthisclassofbiosensors.

Inconclusion,wehavesuccessfullydesignedandfabricatedH2O2sensorsthatarebasedondirectimmobilizationofhemeproteinssuchasCytcongoldelectrodesofdifferentsizes.OurresultsshowthatUMEsarecompatiblewiththissensordesignandarecapableofachievingaLODthatisnotpossiblewithsta-tionarymacroelectrodes.Moreimportantly,UMEscanbeusedtocalculateKmforimmobilizedenzymes,animportantparameterinbioelectrocatalysisthatisoftendeterminedusingRDEs.Owingtothesimplicityofthissensordesign,potentialapplicationsarenumerous;itiswellsuitedfordirectdetectionofextracellularandintracellularROSinlivecellsandthesestudiesarecurrentlyunderwayinthelaboratory[8,31,33].

Acknowledgments

ThisresearchwassupportedbyNSF(CHE-0955439andDMR-0851703),NEEPSCoR(EPS-1004094).TheauthorswouldliketothankAnitaJ.Zaitounaforthehelpfuldiscussions.

AppendixA.Supplementarydata

Supplementarymaterialrelatedtothisarticlecanbefound,intheonlineversion,athttp://wendang.chazidian.com/10.1016/j.colsurfb.2014.10.033.

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