BiosensorsandBioelectronics51(2014)158–163ContentslistsavailableatScienceDirectBiosensorsandBioelectronicsjournalhomepage:www.elsevier.com/locate/biosCo-immobilizationofglucoamylaseandglucoseoxidaseforelectrochemicalsequentialenzymeelectrodeforstarchbiosensorandbiofuelcellQiaolinLanga,1,LongYina,c,1,JianguoShib,LiangLia,LinXiaa,AihuaLiua,c,nLaboratoryforBiosensing,QingdaoInstituteofBioenergy&BioprocessTechnology,KeyLaboratoryofBioenergy,ChineseAcademyofSciences,1SonglingRoad,Qingdao266101,ChinabKeyLaboratoryforBiosensorsofShangdongProvince,BiologyInstituteofShandongAcademyofSciences,19KeyuanRoad,Jinan250014,ChinacUniversityofChineseAcademyofSciences,19AYuquanRoad,Beijing100049,ChinaaarticleinfoArticlehistory:Received19April2013Receivedinrevisedform9July2013Accepted10July2013Availableonline30July2013Keywords:GlucoamylaseGlucoseoxidaseSequentialenzymebiosensorStarchbiosensorBiofuelcellabstractAnovelelectrochemicalsequentialbiosensorwasconstructedbyco-immobilizingglucoamylase(GA)andglucoseoxidase(GOD)onthemulti-walledcarbonnanotubes(MWNTs)-modifiedglassycarbonelectrode(GCE)bychemicalcrosslinkingmethod,whereglutaraldehydeandbovineserumalbuminwasusedascrosslinkingandblockingagent,respectively.Theproposedbiosensor(GA/GOD/MWNTs/GCE)iscapableofdeterminingstarchwithoutusingextrasensorssuchasClark-typeoxygensensororH2O2sensor.Thecurrentlinearlydecreasedwiththeincreasingconcentrationofstarchrangingfrom0.005%to0.7%(w/w)withthelimitofdetectionof0.003%(w/w)starch.Theas-fabricatedsequentialbiosensorcanbeapplicabletothedetectionofthecontentofstarchinrealsamples,whichareingoodaccordancewithtraditionalFehling'stitration.Finally,astablestarch/O2biofuelcellwasassembledusingtheGA/GOD/MWNTs/GCEasbioanodeandlaccase/MWNTs/GCEasbiocathode,whichexhibitedopencircuitvoltageofca.0.53Vandthemaximumpowerdensityof8.15μWcmÀ2at0.31V,comparablewiththeotherglucose/O2basedbiofuelcellsreportedrecently.Therefore,theproposedbiosensorexhibitedattractivefeaturessuchasgoodstabilityinweakacidicbuffer,goodoperationalstability,widelinearrangeandcapableofdeterminationofstarchinrealsamplesaswellasoptimalbioanodeforthebiofuelcell.&2013ElsevierB.V.Allrightsreserved.1.IntroductionAsoneofthemostgeneralcarbohydratesincrops,starchisusuallyusedasfoodprocessingauxiliarytoimprovethetasteandnutrition,andcanalsobeusedasfillerofcompositematerialsforthedegradationofcertainsyntheticpolymersduetoitscharacterofinnocuityandeasydegradation(Staretal.,2004).Duetoitscost-effectivity,starchisalsoconsideredasagoodfuelforbiofuelcells.Desirabletechnological,organoleptic,andnutritionalproper-tiesintheendproductsarealldependentontheadditionofstarchintheprocessessuchasthebakingofbread,theproductionofpastaproductsandstarch-basedsnackfoods,breakfastcereals,pregelatinizedflour,babyfoods,andparboiledcereals(OlkkuandRha,1978;LinebackandWongsrikasem,1980;LundandLorenz,1984).ThelevelofstarchcontentinfoodorpillisavitalparameterCorrespondingauthorat:LaboratoryforBiosensing,QingdaoInstituteofBioenergy&BioprocessTechnology,KeyLaboratoryofBioenergy,ChineseAcademyofSciences,1SonglingRoad,Qingdao266101,China.Tel.:+8653280662758.E-mailaddress:liuah@qibebt.ac.cn(A.Liu).1Theseauthorscontributedequallytothiswork.0956-5663/$-seefrontmatter&2013ElsevierB.V.Allrightsreserved.http://dx.doi.org/10.1016/j.bios.2013.07.021ninqualityinspectionofbrewing,foodindustryandpharmacy.Traditionalwaystodetectstarchincludepolarimetric(GarciaandWolf,1972)andtheFehlingtitrationmethod(Menyhert,1908),however,theyarecomplexandtime-consuminginsamplepre-treatment.Especially,theresultsofFehlingtitrationmethodaregreatlyaffectedbytheinterferencefromotherpossiblereducedsugarsco-existedinthesample.Ontheotherhand,theenzyme-basedelectrodesincombinationwithhydrogenperoxidesensors(Cordonnieretal.,1975;Mascinietal.,1983)oroxygensensors(CouletandBertrand,1979;BardelettiandCoulet,1987)werereportedforstarchmeasurement,whichwerebasedonthedeterminationofthereducedsaccharides,thehydrolyticproductsofstarch,nevertheless,itislaborious.Sequentialbiosensorscontainingtwoormoreenzymeswhichcatalyzesubstrateinsequence,aremostlyusedinthedeterminationofdisaccharides(ZhangandRechnitz,1994;Zhang,2000),starch(AbdulHamidetal.,1990)andcholesterol(MotonakaandFaulkner,1993).Theperformanceofthiskindofbiosensorsgreatlydependsontheamount,ratio,anddistributioncontroloftwoenzymesaswellastheimmobilizationmethods(Zhouetal.,2001).However,thesensitivityandoperationalstabilityareusuallynotsosatisfactoryQ.Langetal./BiosensorsandBioelectronics51(2014)158–163159comparedwithsingleenzymebiosensor,probablyarisingfromthecomplexityinenzymemembranepreparation(Zhouetal.,2001).Electrochemicalsequentialelectrodewasalsousedinthesurface-displayingenzymemicrobialfuelcell(Bahartanetal.,2012).Biofuelcell(BFC)whichemploysenzymesor/andmicroorgan-ismsasthebiocatalystsfortheproductionofelectricityfromrenewableorganicmatter,representsanewkindofgreenpowersourcesandhasattractedmuchattentioninrecentyears(Bullenetal.,2006;Duetal.,2007;Cracknelletal.,2008).Thereareintensivestudiesonthedesignandcharacterizationofenzyme-basedBFCs,however,mostofworkhasbeenfocusedonusingmonosaccharidessuchasglucoseorxyloseasfuels(Lietal.,2009;Gaoetal.,2010;Wenetal.,2011;Zebdaetal.,2011;Xiaetal.,2013).Incomparisonwithmonosaccharides,starchrepresentsanalternativeenergysourcewithlowercostandeasierprocessingprocedures.Byfar,starchhasbeenusedasenergyresourceinmicrobialfuelcells(Velasquez-Ortaetal.,2011;Herrero-Hernandezetal.,2013).However,therearenoreportsonBFCsbasedonsequentialenzymebioelectrocatalysisofstarch.Glucoamylase(GA,α-1,4-glucan-gluco-hydrolase,EC.3.2.1.3)isastarchhydrolyzingenzymewhichcatalyzesthehydrolysisofα-(1,4)glycosidicbondsatthenon-reducingendofstarchpolymertoreleasefreeglucose(Marin-NavarroandPolaina,2011).GAisanimportantenzymeextensivelyusedinbio-industryforproductionofstarchsugar,alcoholandsingle-cellprotein(Velasquez-Ortaetal.,2011;Yamakawaetal.,2012).Asanessentialpolysaccharidehydrolase,GAiswidelyusedinthehydrolysisofstarchintoglucosebeforetheirmeasurementwitheitherFehling'stitrationorelectrochemicalmethod(AbdulHamidetal.,1990;ZhangandRechnitz,1994;Zhang,2000).Inthepresentstudy,weconstructedasequentialbiosensorbasedontheco-immobilizationofGAandglucoseoxidase(GOD)forthedeterminationofstarch.Withtheincorporationofcarbonnanotubes,whichcouldfacilitatethedirectelectrontransferbetweenelectrodeandGOD,theredoxcenter(flavinadeninedinucleotide,FAD)ofGODpresenteddirectelectrochemistry.Thereductionpeakcurrentdecreasedwiththeincreasingofglucoseinsolutionbasedontheoxygenconsumption(Wangetal.,2009).TheproposedbiosensorenabledtodeterminestarchwithoutthemeasurementofH2O2,thussimplifiedstarchbiosensorandbiofuelcell.Tothebestofourknowledge,thisisthefirstreportontheconstructionofstarchbiosensorwithoutextrasensorssuchasClark-typeoxygensensororH2O2sensor.Finally,astarch/O2biofuelcellwasassembledusingtheGA/GOD/MWNTs/GCEelec-trodeasbioanodeandlaccase/MWNTs/GCEasbiocathode,whichexhibitedopencircuitvoltageuptoca.0.53Vandthemaximumpowerdensityof8.15μWcmÀ2at0.31V,comparablewiththeotherglucose/O2basedbiofuelcellsreportedrecently.2.MaterialsandMethods2.1.ChemicalsandreagentsGlucoseoxidase(GOD),laccaseandbovineserumalbumin(BSA)werepurchasedfromF.Hoffmann-LaRoche,Ltd.GA,starchandglutaraldehydewerepurchasedfromSinopharmChemicalReagentCo.,Ltd.Starchsolutionwaspreparedbysuspendingsuitableamountofstarchpowderinto0.1Mphosphatebufferunderheattoboilinginmicrowaveoven,whichwascooleddownatroomtemperaturebeforeuse.ThespecificenzymaticactivityofGAisdefinedastheamountofglucose(inμmol)generatedby1mgGAperminuteintheexcessofstarch,whilethespecificenzymaticactivityofGODisdefinedastheamountofglucose(inμmol)consumedby1mgGODperminuteintheexcessofglucose.Theiractivitiesweremeasuredseparatelybyspectrophotometrymethod,showingthat1mgGAcouldgenerate118μmolglucosefromstarchperminutewhile1mgGODcouldconsume297μmolglucoseperminuteatthesamecondition.2.2.PreparationofsequentialbiosensorThesequentialbiosensorwasfabricatedonaglassycarbonelectrode(GCE,diameterof3mm)whichwaspolishedtoamirrorfinishusing0.3and0.05μmaluminaslurry,followedbyrinsingthoroughlywithdeionizedwater.Afterultrasonicprocessinginanhydrousethanolandultrapurewater,respectively,theelectrodewasrinsedwithultrapurewateranddriedatroomtemperature.Inpreparationofsequentialbiosensor,5μLofmultiwalledcarbonnanotubes(MWNTs)suspension(2mgMWNTsdispersedin1mlultrapurewaterwithultrasonicprocessing)wasdrippedontheinvertedGCEsurfaceanddriedinair.Next,differentvolumesofGODsolution(3000U/ml)andGAsolution(1200U/ml),2μLofBSA(1%w/w)and5μLofglutaraldehyde(1%w/w)weremixedtogetherontheinvertedGCEtofabricatevariousmodifiedelectrodesanddriedovernightat41Cinrefrigerator.Aglucosebiosensorwasalsoconstructedwiththesimilarmethodinwhich6μLofGODsolution(3000U/ml),2μLofBSAand5μLofglutaraldehydewereapplied.2.3.ApparatusandelectrochemicalmeasurementsElectrochemicalmeasurementswereperformedusingaCHI660Dpotentiostat(CHInstruments,Chenhua,Shanghai,China).Theelectro-chemicalresponsewasmeasuredinaconventionalthree-electrodesystemusingachemicallymodifiedGCEasworkingelectrode,aPtwireauxiliaryelectrodeandasaturatedcalomelelectrode(SCE)asreferenceelectrode.Allpotentialswerereportedinthiscontextwithrespecttothisreference.Allmeasurementswereperformedatroomtemperature(∼231C).2.4.PreparationofbiofuelTheone-compartmentbiofuelcellcontainedGA/GOD/MWNTs/GCEemployedasthebioanodeandthelaccase/MWNTs/GCEasbiocathode,whichwereassembledtogetherin5mlof0.5%(w/w)starch(pH5.0)solution.Inthebiocathodefabrication,laccasewasusedasbiocatalysttocatalyzeoxygenreductiontowater.ToimprovethebioelectrocatalysisefficiencyofthelaccasebasedbiocathodetowardsO2reduction,2,2'-azinobis-(3-ethylbenzo-thiazoline-6-sulfonicacid)(ABTS)wasusedasaredoxmediator.3.Resultsanddiscussions3.1.ConstructionofsequentialbiosensorThesequentialbiosensorwasconstructedbyco-immobilizingGAandGODontheMWNTs-modifiedGCEbychemicalcross-linkingmethod,whereglutaraldehydeandBSAwasusedascrosslinkingandblockingagent,respectively(Fig.1A).Thethus-preparedbioelectrodeisdenotedasGA/GOD/MWNTs/GCE.Cyclicvoltammograms(CVs)ofdifferentmodifiedelectrodesareshowninFig.1B.NoredoxpeakscouldbefoundforbothGA/GCEandGA/GOD/GCEinthepresenceofstarch(Fig.1B,curvesa,b).OnlyincreasedbackgroundcurrentwasobservedatGA/MWNTs/GCEinthepresencestarchsolution(Fig.1B,curvec).Apairofwell-definedredoxpeakswereclearlyobservedatGA/GOD/MWNTs/GCEinbarephosphatebuffer(Fig.1B,curvef),whichmeantthedirectelectrontransferbetweenenzyme(GOD)andelectrodewasfacilitatedbyMWNTsthroughtheredoxcenterFAD/FADH2160Q.Langetal./BiosensorsandBioelectronics51(2014)158–163Fig.1.(A)Schematicconstructionofsequentialbiosensor.(B)CVsofGA/GCE(a)andGA/GOD/GCE(b)in0.05%(w/w)starchsolution;CVofGA/MWNTs/GCEinthepresenceof0.05%(w/w)starchsolution(c);CVsofGA/GOD/MWNTs/GCEinthepresenceof0.1%(w/w)glucosesolution(d),inthepresenceof0.05%(w/w)starchsolution(e)andinbare0.1Mphosphatebuffer(pH4.5)(f).Scanrate,50mV/s.embeddedintheGOD(Fig.1A)(Artesetal.,2011;Lietal.,2013).Moreover,thecathodicpeakcurrent(ipc)atÀ0.38VdecreasedatthesameGA/GOD/MWNTs/GCEinthepresenceofstarch(Fig.1B,curvee),suggestingthattheGAcatalyzedthehydrolysisofstarchtoglucose(reaction1),thelatterwasfurtherelectrocatalyticallyoxidizedbyGODimmobilizedontheelectrodesurfacetogluco-lactoneinthepresenceofO2,asschematicallyshowninFig.1Aandreactions2and3.Therefore,itispossibletodetectstarchusingasequentialbioelectrode.Ontheotherhand,GA/GOD/MWNTs/GCEexhibitedthesimilarCVsinthepresenceofglucoseorstarchsolution(Fig.1B,curvesdande),whichfurtherconfirmedtheredoxpeakswerecharacteristicpeaksofFAD/FADH2.However,thepeakpotentialsshouldbeshiftedalittlewithchangingthesubstrate(starchorglucose)concentration(Fig.1B,curvesdande).Additionally,theΔipcforGA/GOD/MWNTs/GCEinthepresenceofglucosehadthesimilartrendasthatvalueinthepresenceofstarch,thatis,forbothglucoseandstarchatthesamelowconcentration,boththeΔipcvalueswereroughlyequal.HeretheΔipcisdefinedasthedifferenceofthecathodicpeakcurrentfromCVsofthemodifiedelectrodeinthepresenceofsubstrateandinthepresenceofbarebuffersolution.Starch-GAGlucoseð1ÞGlucoseþGOxðFADÞ-GluconolactoneþGOxðFADH2Þð2ÞGOxðFADH2ÞþO2-GOxðFADÞþH2O2ð3Þ3.2.OptimizationofGAandGODloadingonresponseofthesequentialenzymeelectrodeThespecificenzymaticactivityofGAandGODweremeasuredtobe118U/mgand297U/mg,respectively.Inotherwords,thespecificenzymaticactivityratioofGOD/GAwas2.5,suggestingthatroughly,thecatalyticrateofGODwas1.5timesfasterthanthatofGAinthebeginningofreaction.Theloadingofenzymeonelectrodesurfaceisacrucialparameterintheconstructionofenzymebiosensor,especiallyforsequentialbiosensor,ofwhichtheperformancedependshighlyontheratiooftwoenzymes(Zhouetal.,2001).Fortheconvenientdesignofthesequentialbiosensor,theamountsofGAandGOD(inenzymaticactivityunit)appliedontheelectrodesurfaceshouldbeoptimized.Inthisstudy,theworkingelectrodeGA/GOD/MWNTs/GCEwaspreparedbyloadingbothenzymeswithdifferentGA/GODratios,andCVsweremeasuredinthepresenceof0.5%(w/w)starchsolutiontoinvestigatethechangeoftheipcataboutÀ0.38VfromtheCVs.AseriesofmodifiedelectrodeswereconstructedwithaconstantloadingofGOD(10U)andvaryingGAloadingrangingfrom8.0to40U.TheΔipcincreasedwiththeGAamountinthecastfilmontheelectroderangingfrom8to25U(Supplementarymaterial,Fig.S1A).Thereafter,thefurtherincreaseintheGAloadinginducedthecurrentdecrease.SoaGA(25U)wasloadedontheelectrodeforco-immobilizationinthesubsequentexperiments.Ontheotherhand,theGA/GOD/MWNTs/GCEbasedbiosensorwasfabricatedwithaconstant25UofGAandvaryingGODloading,andtheresponsesofthepreparedbiosensorstowardtheelectrocatalysisof0.5%(w/w)starchsolu-tionwererecordedseparately.ThedependenceofΔipcvalueasafunctionofGODloadingisshowninFig.S1B.Obviously,amaximalΔipcwasachievedwhen10UofGODwasloaded.However,whentheexcessamountoftwoenzymeswereloaded,theenzymemembranebecamethickenoughtoblockelectrontransferbetweenbuffersolutionandelectrodesurface(Lietal.,2013).Takentogether,aloadingof25UofGAand10UofGODwasappliedforthepreparationoftheGA/GOD/MWNTs/GCE.Thus,theoptimalGA/GODloadingratiowas2.5,whichiscloselycoincidentwiththespecificenzymaticactivityratioofGAandGOD.There-fore,itisclearthatthehydrolysisofstarchbyGA(reaction1)wasslower,whichwouldbecometherate-determiningstepinthesequentialenzymereaction,inagreementwiththeworkreportedbyVrbova'sgroup(Vrbováetal.,1993).3.3.OptimizationofbufferpHonresponseofthesequentialenzymeelectrodeConsideringthatGAusuallycatalyzesstarchinacidicsolution,thepH-dependentenzymaticactivityofGAwasinvestigatedwithinpH3–7.TheΔipcvalueincreasedsharplywhenbufferpHwaschangedfrom3to4.5,thereafterΔipcdecreasedwhenthepHwashigherthan5(Supplementarymaterial,Fig.S2).ThelargestΔipcvaluewasachievedwhenthepHwas4.5,whichwasinaccordancewithpreviousreport(MishraandDebnath,2002).Mostenzyme-basedbiosensorswerenotstableinextremepHcondition(Vrbováetal.,1993;Torresetal.,2013),however,ourbioelectrodecouldbestableinweakacidicbuffersolution(pH4.5)undercontinuousscans,suggestingtheattractivefeatureofoursequentialenzymesensor.3.4.CalibrationcurveofthesequentialenzymebiosensorTheCVsoftheGA/GOD/MWNTs/GCEin0.1Mphosphatebuffer(pH4.5)containingdifferentconcentrationsofstarchwereper-formedundertheambient-aircondition(Supplementarymaterial,Fig.S3).ItshouldbementionedherethatourmethodtodetectQ.Langetal./BiosensorsandBioelectronics51(2014)158–163161starchisbasedonthemeasurementofO2consumptionthroughthedecreaseofreductionpeak.Obviously,fromCVs,theΔipcatÀ0.38Vincreasedwiththeincreasingconcentrationofstarch(Supplementarymaterial,Fig.S3).However,thechangesinCVsaresominisculeatlowstarchconcentrationthatmighthardlyserveforanydifferentiation.Soamperometrywascarriedoutinpreparationofthecalibrationofthebiosensor.Thecurrent–timecurvewasobtainedwithGA/GOD/MWNTs/GCEbyusingampero-metryatanappliedpotentialofÀ0.4V(Fig.2A).Thecurrentdecreasedafteradditionofstarchsolutionandreachedat95%steady-statevaluewithin10s(Fig.2A).TheplotofthedecreasedcurrentasafunctionofstarchconcentrationisshowninFig.2B,fromwhichthedecreasedcurrentwaslinearwithstarchconcen-trationwithin0.005–0.7%,andthereafter,thecurrentresponsewaslevelledoffwhenthestarchconcentrationwasfurtheradded.Sothelinearrangewas0.005–0.7%.Thelinearregressionequationisy¼0.0008+1.015xwiththecoefficientR¼0.999.Thelimitofdetec-tion(LOD)wasestimatedtobe0.003%starch(S/N¼3).Thelinearrangeinourworkwaswiderthanthosevaluesobtainedusingtri-enzymemodifiedClark-typeoxygensensorssuchasamyloglucosi-dase(AMG)/mutarotase(MUT)/GOD/catalase(CAT)-film/Clark-typeoxygensensor(0.01–0.4%)(Vrbováetal.,1993),AMG/MUT/GOD-film/Clark-typeoxygensensor(0.1–1%)(Watanabeetal.,1991)andAMG/MUT/GOD/Pd–Au/graphite(0.001–0.1%)(AbdulHamidetal.,1990).TheLODinourcasewaslittlehigherthan0.001%reportedfortheAMG/MUT/GOD/Pd–Au/graphite(AbdulHamidetal.,1990),Fig.2.(A)Current–timecurveobtainedattheGA/GOD/MWNTs/GCEonthesuccessiveadditionofstarchin0.1Mphosphate(pH4.5),onwhichthestarchconcentrationdenotedthestarchconcentrationinthebuffer.Appliedpotential,À0.4Vvs.SCE.(B)Typicalcalibrationgraphofthestarchbiosensor.whichwasprobablycausedbythelargecurrentnoiseofampero-metryduringvigorousstirring.3.5.SelectivityofthesequentialbiosensorTheselectivityofthebiosensorwasinvestigatedbycomparingthecurrentresponseofthebioelectrodeonthesuccessiveaddi-tionofstarchandvariousothersubstratesintothephosphatebufferwhenrecordingcurrent–timecurveatÀ0.4V.AsshowninFig.3,thesuccessiveadditionof0.1%starchresultedinobviouscurrentdecline(Fig.3,arrowsa,bandc).Thepresenceof0.04%D-glucosealsoexhibitedcurrentdecrease(Fig.3,arrowd),sug-gestingitsgoodresponsetoglucose.Thisisreasonable,becausethesequentialenzymebiosensorwasfabricatedwithGAandGOD,whereGODisspecifictoglucose.TheadditionofothersaccharidessuchasD-mannose,D-xylose,D-fructose,D-cellobioseandD-galac-toseaswellasD-xylitol(each0.2%)showedbaseline,suggestingthattheexistenceofthesespeciesdidnotaffectthedetectionofstarch.Theadditionofacetaminophen,ascorbicacid,anduricacid(each10mM)alsoshowednocurrentchange(Fig.3,arrowsk–m),suggestingthatthesecommoninterferingspecieshadnointer-ferencetothedetectionofstarch.Therefore,thisbiosensorcanbeusedtoselectivelydetectstarchwithoutinterferencefromcom-moninterferingspeciesandothersugarsexceptglucose.Actually,aGOD-modifiedelectrode,whichisconstructedsimilarlytostarchsequentialbiosensor,canbeusedtodetectglucosebeforethemeasurementofstarchusingthesequentialbiosensor.3.6.OperationalstabilityofthesequentialbiosensorThreedifferentelectrodeswerepreparedusingthesameprocedure,andtheirCVresponseswererecordedinthesamestarchsolution.AcontinuousmeasurementofCVwasperformedina0.1Mphosphatebuffercontaining0.5%starch(pH4.5).Itwasfoundthatthepeakcurrentsforstarchretainedover90%oftheinitialvalueafter200continuousscans(Supplementarymaterial,Fig.S4),whichshowedthatthemodifiedelectrodehadgoodoperationalstability(Chenetal.,2011).3.7.DeterminationofstarchinsamplesTheproposedbiosensorwasappliedforrealsampledetection.Beforemeasurement,suitablepretreatmentofthesamplesshouldbeperformed.Inageneralprocedure,anappropriateamountofFig.3.Current–timecurveobtainedfortheGA/GOD/MWNTs/GCEonthesucces-siveadditionof0.1%starch(a,bandc),0.04%D-glucose(d),0.5%D-mannose(e),0.5%D-xylose(f),0.5%D-xylitol(g),0.5%D-fructose(h),0.5%D-cellobiose(i),0.5%D-galactose(j),10mMacetaminophen(k),10mMascorbicacid(l),and10mMuricacid(m)in0.1Mphosphate(pH4.5).162Q.Langetal./BiosensorsandBioelectronics51(2014)158–163Table1Determinationofstarchcontentinrealsamples.SampleStarchcontent(%w/w)ThisworkFehling'stitrationLocalsnack46.670.450.070.1Kraft37.570.542.070.1Digestivepill49.070.351.070.1Localhamsausage8.370.19.070.2Banana0.7570.070.8070.2sampleisfreeze-driedandgroundintotinypowderwithmortar.Thenthepowderiswashedrepeatedlywithanhydrousethanoltoremoveanysolublesaccharides,theprecipitateiscollectedanddriedinovenat781Ctoremovethesolventethanol.Subse-quently,thedriedprecipitateisredissolvedin0.1Mphosphatebuffer(pH4.5)andfilteredtoremoveanyinsolubleparticlesthrougha0.22-μmmembrane,andthefiltrateiscollectedandaliquotedinto6equalvolumes.For3aliquots,theglucose+starchcontentinthesamplesolutionisdetectedbasedontheestab-lishedmethod.AGOD-modifiedelectrode,whichwasconstructedsimilarlytostarchsequentialbiosensor,isusedtodeterminethecontentofD-glucoseintheother3aliquots.ThecontributionfromtheinitialglucosecontainedinthesamplesbeforetheprocessofGAhydrolysisissubtracted.Theconcentrationsoftherealsampleswerecalculatedbasedonthecalibrationgraphmultiplyingthedilutionratios(Table1).Forcomparison,Fehling'stitrationwascarriedout.Resultsobtainedfromsequentialbiosensorcorre-spondedwelltotheresultsobtainedbyFehling'stitration(Table1).Apparently,ourmethodcanbeusedtodetectsamplewithlowerstarchcontent(suchasbanana),however,significanterrorwasobtainedwithFehling'stitration.ThestarchcontentsobtainedfromoursequentialsensorissystematicallylessthanthosevaluesmeasuredbyFehling'stitration(Table1).Itisreason-ablethatFehling'stitrationdetectedthetotalreducedsaccharidesinsamplesolution,whereasthesequentialbiosensorrespondedselectivelytoglucose.Further,Fehling'stitrationisusuallyinvolvedinalengthyhydrolysisprocessbeforetitration,whichistime-consuming.Takentogether,ourproposedmethodisadvan-tageousovertraditionalFehling'stitrationmethod.3.8.StarchbiofuelcellAsdemonstratedabove,sequentialbioelectrocatalyticoxida-tionofstarch(glucose)atGA/GOD/MWNTs/GCEmakesitpromis-ingasabioanode.Furthermore,directbioelectrocatalyticoxidationofglucoseatlowpotentialstartingatÀ0.4Vwillbefavorableforimprovingtheopencircuitvoltage(OCV)oftheBFC.Inthecurrentwork,wedevelopedstarch/O2biofuelcellbasedonourrecentworkwithmodification(Xiaetal.,2013).Theperformancesofthemodifiedbiocathodewereexaminedin0.1Mphosphatebuffer(pH5.0)containing0.5%w/wstarch.TheOCVvaluesvariedbetween0.45Vand0.52VdependingontheamountofenzymeimmobilizedontheMWNTs/GCE.Atthelaccase/MWNT/GCE,anincreasedcathodiccurrentappearedinthepresenceofO2,whilenocathodiccatalyticcurrentwasobservedunderN2atomosphere(Fig.4A).AsseenfromthepolarizationcurvesinFig.4A,theelectrocatalyticreductionofO2startedatabout0.52V.Toformamembrane-lessstarch/O2biofuelcell,thisnovelGA/GOD/MWNTs/GCEsequentialenzymebioanodewascombinedwiththeabovedescribedO2electroreducingcathode.Thedependenceofthepowerdensityontheoperatingvoltageoftheas-assembledbiofuelcellin0.5%(w/w)starchunderO2isshowninFig.4B.TheOCVoftheas-assembledBFCwasca.0.53Vandthemaximumpowerdensitywas8.15μWcmÀ2at0.31V.Fig.4.(A),CVsofthelaccase/MWNTs/GCEbiocathodein0.1Mphosphatebufferwith0.5%(w/w)starch(pH5.0)underN2-saturatedatmospherewithoutABTS(dashedline),andinpresenceof0.5mMABTSunderN2-saturated(dottedline)andunderoxygen-saturatedatmosphere(solidline).(B)Dependenceofthepowerdensityonthecelloperatingvoltageofthestarch/O2BFC.BecausetheMWNTcanfacilitatetheGOxcatalysis(Lietal.,2013;Wangetal.,2013),theperformanceofthisBFCwasquitecomparabletothoseofglucose/oxygenBFCreportedrecently(Lietal.,2009;Gaoetal.,2011;Wenetal.,2011).Totesttheoperationalstabilityoftheas-assembledBFC,thecellwasoper-atedcontinuouslyina0.5%w/wstarchsolutionunderambientair.After12hoperation,itretained%ofitsmaximalpower,suggestingafavorablystablepoweroutputprocess.4.ConclusionsAnovelelectrochemicalsequentialbiosensorGA/GOD/MWNTs/GCEwassuccessfullyconstructedforthedetectionofstarch.Theproposedbiosensorwasbasedonthemeasurementofthedecreaseinthepresenceofstarch,enablingtodeterminestarchwithoutthemeasurementofH2O2,thussimplifiedstarchbiosen-sor.Thecurrentlinearlydeclinedwiththeincreasingconcentra-tionofstarchrangingfrom0.005%to0.7%(w/w).Theas-fabricatedsequentialbiosensorcanbeapplicabletothedetectionofthecontentofstarchinsnacks,pillsandfruits,whichwereingoodaccordancewithtraditionalFehling'stitration.Therefore,theproposedbiosensorexhibitedattractivefeaturessuchasgoodoperationalstability,widelinearrangeandcapableofdetermina-tionofstarchinrealsamples.Finally,thestarch/O2biofuelcellwasassembledusingtheGA/GOD/MWNTs/GCEelectrodeasbioanodeQ.Langetal./BiosensorsandBioelectronics51(2014)158–163163andlaccase/MWNTs/GCEasbiocathode,whichexhibitedopencircuitvoltageofca.0.53Vandthemaximumpowerdensityof8.15μWcmÀ2at0.31V,comparablewiththeotherglucose/O2basedbiofuelcellsreportedrecently.Thisresearchprovidesanewparadigmfortheinvestigationofothersequentialenzymesystems.AcknowledgmentsThisworkwasfinanciallysupportedbytheNationalNaturalScienceFoundationofChina(Nos.91227116and21275152)andtheHundred-Talent-Project(No.KSCX2-YW-BR-7)andtheKnowl-edgeInnovationProjectinBiotechnology(No.KSCX2-EW-J-10-6),ChineseAcademyofSciences.WeareindebtedtoProf.Dr.GeboPaninSuzhouInstituteofNan-TechandNano-Biomics,ChineseAcademyofSciencesforgiftingmulti-walledcarbonnanotubes.AppendixA.SupportinginformationSupplementarydataassociatedwiththisarticlecanbefoundintheonlineversionathttp://dx.doi.org/10.1016/j.bios.2013.07.021.ReferencesAbdulHamid,J.,Moody,G.J.,Thomas,J.D.,1990.Analyst115,12–1295.Artes,J.M.,Diez-Perez,I.,Sanz,F.,Gorostiza,P.,2011.ACSNano5,2060–2066.Bahartan,K.,Amir,L.,Israel,A.,Lichtenstein,R.G.,Alfonta,L.,2012.ChemSusChem5,1820–1825.Bardeletti,G.,Coulet,P.R.,1987.EnzymeandMicrobialTechnology9,652–657.Bullen,R.A.,Arnot,T.C.,Lakeman,J.B.,Walsh,F.C.,2006.BiosensorsandBioelec-tronics21,2015–2045.Chen,H.,Guo,L.,Ferhan,A.R.,Kim,D.-H.,2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