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Lowering the potential of electroenzymatic glucose
oxidation on redox hydrogel-modified porous carbon
electrode
Aimi Suzuki, Nicolas Mano, Seiya Tsujimura
To cite this version:
Aimi Suzuki, Nicolas Mano, Seiya Tsujimura. Lowering the potential of electroenzymatic glucose
oxidation on redox hydrogel-modified porous carbon electrode. Electrochimica Acta, Elsevier, 2017,
232, pp. 581-585. �10.1016/j.electacta.2017.03.007�. �hal-01512668�
Lowering
the
potential
of
electroenzymatic
glucose
oxidation
on
redox
hydrogel-modi
fied
porous
carbon
electrode
Aimi
Suzuki
a,
Nicolas
Mano
b,
Seiya
Tsujimura
a,*
aDivisionofMaterialsScience,FacultyofPureandAppliedSciences,UniversityofTsukuba,1-1-1Tennodai,Tsukuba,Ibaraki305-8573,Japan
b
CentredeRecherchePaulPascal,CRPP-UPR8641-CNRS,Univ.Bordeaux,AvenueAlbertSchweitzer,Pessac,France
Keywords: Glucoseoxidase redoxhydrogel porouscarbon biofuelcell Herein,we demonstratehighcurrentdensitiesper projectedsurface areafortheelectroenzymatic
oxidationofglucoseatlowpotentialusingahierarchicallystructuredelectrodebasedonMgO-templated carbon.Themodifiedelectrodewaspreparedbyassemblingglucoseoxidase(GOx)withan osmium-basedredoxpolymerwithalowformalpotential(0.03Vvs.Ag|AgCl),inwhichOscomplexesare tetheredtothepolymerbackboneviaa13-atomalkylchainandacrosslinker.Aglucoseoxidationcurrent densityof15mAcm2wasmeasuredat0Vvs.Ag|AgClat37CandpH7,withaplateauvalueof 50mAcm2at0.3V.ThehydrogelelectrodescomposedofthesameredoxpolymerandFAD-dependent glucosedehydrogenasedeliveredonly10%ofthecurrentdensitiesobtainedwiththeGOxelectrode.
1.Introduction
Werecentlyreportedthedevelopmentofaglucose-oxidizing electrodeusingMgO-templatedmesoporouscarbon(MgOC),with anaverageporediameterof38nm,asanelectrodematerial.The MgOCwas coatedwitha hydrogelcontaininga redoxpolymer, glucose oxidase (GOx), as well as FAD-dependent glucose dehydrogenase(FADGDH)[1,2].Withtheobjectiveofimproving themasstransferoffuelandelectrolyteionsthroughtheporous carbonlayer,porouscarbonparticlesweredepositedonaglassy carbonsubstratebyanelectrophoretictechnique,toform10
m
m-scale macropores. Thehydrogel, composed of a redoxpolymer, partiallyquaternizedpoly(1-vinylimidazole)complexedwith[Os (bipyridine)2Cl](polymerI,E’=0.22Vvs.Ag|AgCl)[3,4],GOx,andacrosslinker,producedglucoseoxidationcurrentdensitiesashigh as60mAcm2at0.7Vat37C. Thiscurrentdensityis12times higher than that obtained with thesame polymer on a glassy carbonelectrode(GC-E).Forcomparisonwithother GOx-hydrogel-based electrodes,theeffect of thehierarchical structure of the MgOC-basedelectrodecanbeelucidatedusingpolymerI,which hasbeenused onotherporouscarbon materials [4–6]. Similar hydrogel-modified porous carbon electrodes, composed of the sameredoxpolymeranddeglycosylatedFADGDH,haveachieved currentdensities of 180mAcm2at 0.7V and 37C, whichis 3
timeshigherthancurrentdensitiesforGOx-basedelectrodes[2]. However,thiselectrodesystemwasfarfromoptimum,becausethe redox mediator potential was too high for use in efficient biosensorsandbiofuelcells(BFCs)[7].Aredoxmediatorwitha lowerpotentialisrequiredtoavoidtheelectrochemicaloxidation ofinterferingsubstances,toachievehighaccuracysensors,andto increase the operational voltage of BFCs [8–10]. Mano et al. developedredoxpolymerswithlowerredoxpotentials[11].The rateofelectrontransferfromtheactivecenterofGOx,FAD,tothe Os complextetheredtothepolymer backbonewasreportedto increase as the potential difference between the two species increased.ThispaperalsoreportedthatO2canbereducedonthese
polymerstoproduceH2O2,withanE’valuelowerthan+0.07Vvs.
Ag|AgCl; the rate of this reaction increasedexponentially with decreasingE’.
Therefore,inthisstudyweselectedaredoxpolymer(polymer II),whichconsistsofpoly(vinylpyridine)complexedwithOs(1,10 -dimethyl-2,20-biimidazole)2-2-[6-methylpyrid-2-yl]imidazole)2+/3 +[11–13].Thispolymerhasaredoxpotentialof0.03Vvs.Ag|AgCl
andallowsGOxtoproduceanefficientglucoseoxidationcurrent (2.5mAcm2) on a GC-E at 37C, with a hydrogel loading of
200
m
gcm2 [12]. To achieve further enhancement in current density,KetjenBlack(KB,atypeofcarbonblackwith800m2g1ofhighspecificsurfacearea)[12]andathree-dimensional carbona-ceousfoam[13]wereusedaspotentialporouselectrodematerials inordertoincreasetheefficiencyoftheenzymaticanode.The KB-modified electrode, with 200
m
gcm2 of hydrogel loading, produced 8mAcm2 of catalytic current density at 37C and* Correspondingauthor.
6000rpm,whilethelatterporouscarbonaceouselectrode, with 1734
m
gcm2ofhydrogelloading,produced18mAcm2at37C and 2000rpm. However, the enhancement in catalytic current achieved using these porous structured carbon electrodes was quitelimited.Thepaststudiesofglucoseanodebasedon hydrogel-porouscarbonaresummarizedinTable1.To circumvent this limitation, in this study, hydrogels with polymerIIandGOxorFADGDHwerepreparedonhierarchically structured MgOC-based porous electrodes. The present work showsthathighcatalyticcurrentsatlowredoxpotentialscanbe obtainedforGOximmobilizedwithapolymerII-basedhydrogel matrix,withoutasignificantdecreaseinelectroenzymaticcurrent for glucose oxidation. Surprisingly, although FADGDH has a superior electron exchangeability over GOx toward polymer I
[14],polymerIIwasfoundnottobeabletoconnectefficientlyto FADGDH.
2. Experimental
TheMgO-templatedcarbon-modifiedelectrode(MgOC-E)was fabricatedaccordingtoourpreviouspapers[1,2].MgO-templated carbon, with an average pore diameter of 38nm, was kindly donated by Toyo Tanso (CNovel1, Osaka, Japan). The mixed biocatalyst solution was composed of enzyme, GOx (from Aspergillusniger, Wako PureChemical,40mgmL1)orFADGDH (fromAspergillusterreus,IkedaThoka,40mgmL1),redoxpolymer II(8mgmL1),andacrosslinker,poly(ethyleneglycol)diglycidyl ether(PEGDGE,molecularweight500,Sigma-Aldrich,8mgmL1). Thetotalhydrogelloadingontheelectrodesurfacewasfixedat 1000
m
gcm2.Thepercentageweightofthecrosslinkerfortotal hydrogelloadingwasvaried(10,15, 20,30%relativetothetotal hydrogel),whilemaintainingaconstant(1:1)ratioofenzymeto polymerII.ThisbiocatalystsolutionwaspipettedontotheMgOC-E surface,whichwasconferredwithhydrophilicitybypriorplasma oxidation(10min).Aftermodification,theelectrodewasdriedat 4Cfor18h.Rotatingdisccyclicvoltammetrywasperformedonan electrochemicalanalyzer(BSA50W).Theelectrodewasrotatedat 8000rpm,usingarotator(RDE-2, BAS),toobservethecatalytic activity-limitedcurrent.Platinumwirecounter andAg|AgCl|sat. KCl reference electrodes were used. All measurements were performed at 37C in a thermostated water jacket-equippedelectrolysiscell.Theelectrolysissolutionwascomprisedof0.1M phosphatebuffer(pH7)inatotalvolumeof20mL.
3. ResultsandDiscussion
Fig.1A depicts the cyclic voltammograms for the hydrogel electrodespreparedwithGOxandpolymerIIonanMgOC-E(blue curve(a))oraGC-E(redcurve(b)),inthepresenceandabsence (graycurve(c))of0.5Mglucose,withatotalhydrogelloadingof 1000
m
gcm2at37C.Thesymmetricvoltammogramobservedin theabsenceofglucose(graydashed-curve(c))indicatesthatthe formalpotentialis0.022Vvs.Ag|AgClandthatthetotalsurface density of theOs complex is 2107molcm2,indicating that mostoftheOscomplexappliedtotheelectrodeisinvolvedinthe surfaceredoxreaction.Theonsetpotentialoftheglucoseoxidation currentwasobservedtobe0.12Vvs.Ag|AgCl,andthecurrent reachedasteadystateat0.3V.Thesteady-stateglucoseoxidation current on theMgOC-E was determined tobe 457mAcm2, whichisalmost20timeshigherthanthatfortheGC-E(redcurve (b),2.30.1mAcm2).Thecurrent densityat0Vvs. Ag|AgClis 15mAcm2,whileatthesamepotential,thehydrogelelectrode comprising conventional polymer I is not able toproduce any current(Table1)[1,2,4–6].Thesteady-statecatalyticglucoseoxidationprocessdependson thehydrogelcomposition,asshowninFig.1,panelB.Ataconstant total hydrogel loading of 1000
m
gcm2, the highest current densities,508and 506mAcm2,are observedforhydrogel GOx:polymer:crosslinker compositions of 40:40:20 and 42.5:42.5:15respectively.Asthepercentageweightofthe cross-linkerincreasesfrom10%to15or20%,althoughthecontentofthe biocatalyst decreases, the efficiency of current production increases. The stability of the continuous current response improves astheweightratioofcrosslinkerincreases[15,16].In presenceof10wt%crosslinker,thecurrentdensitywasobservedto decreaseby40%aftertheelectrodehasrotatedat8000rpm,at 37C,for5minutes.Underthesameoperatingconditions,almost 90%oftheinitialcurrentremainsfor theelectrodesmadewith hydrogelsamplescontaining15%or20%crosslinker.Fig.2(panelA)showsthedependenceoftheglucoseoxidation current on hydrogel loading. The catalytic current increases linearly,toaloadingof1000
m
gcm2,owingtothehighsurfaceTable1
Comparisonoftheconstructionandperformanceofsomerecenthydrogel-porouscarbon-basedglucoseelectrodes. Ref.
No.
Enzyme Polymer Hydrogelloading Electrode material(carbon) Buffer(conc.,pH, temp.) Temp. Glucose conc. Electrode rotationrate Onsetpotentialvs. Ag|AgCl/V Currentdensity [1] GOx Polymer I 1000mgcm2 MgOConGC 0.1MPB,pH7, 37C 500mM 8000rpm 0.05 60mAcm2 @0.7V [2] d-FADGDH Polymer I 1600mgcm2 MgOConGC 1MPB,pH7, 37C 500mM 9000rpm 0.05 180mAcm2 @0.7V [4] GOx Polymer I 800mgcm2 CNTonCP PB,pH7.1, 37.5C 50mM 4000rpm 0.10 22mAcm2 @0.6V [6] GOx Polymer I 131010molcm2of Oscomplex
CNTforest PB,pH7 37.5C 200mM Stirringthe solution 0.10 25mAcm2 @0.4V [12] GOx Polymer II 200mgcm2 Ketjenblackon GC 20mMPB+140mM NaCl,pH7, 37C 100mM 6000rpm 0.20 8mAcm2@ 0.3V [13] GOx Polymer II 1734mgcm2 Carbonaceous form 100mMPB,pH7.2 37C 50mM 2000rpm 0.10 18mAcm2 @0.3V [14] d-GOx Polymer I 200mgcm2 GC 100mMPB,pH7 37C 200mM 5000rpm 0.10 5.4mAcm2 @0.4V [14] d-FADGDH Polymer I 200mgcm2 GC 100mMPB,pH7 37C 200mM 5000rpm 0.10 7.5mAcm2 @0.4V This work GOx Polymer II 1000mgcm2 MgOConGC 100mMPB,pH7 37C 500mM 8000rpm 0.12 50mAcm2 @0.3V d-FADGDH Polymer II 1000mgcm2 MgOConGC 100mMPB,pH7 37C 500mM 8000rpm 0.05 8mAcm2@ 0.3V PB:phosphatebuffer.
areawitheffectivemorphologyforhydrogelloading.Thecatalytic currentdensity for theKB-basedhydrogelelectrode was deter-minedtobe8mAcm2at200
m
gcm2ofhydrogelloading,which isinagreementwithearlierresults[12].Theresultssuggestthat thebioelectrochemicalreactionsontheKBandMgOCelectrodes proceedinthesamemanneratthelowhydrogelloading;inthis situationhydrogelcanbespreadoverthesurfaceofporouscarbon electrode(Fig.3(a)and(c)).Ontheotherhand,it isdifficultto increasethecatalyticcurrentfortheKB-basedelectrodebysimply increasingthehydrogelloading[12];thecatalyticcurrentforthis electrode reacheda maximumvalue at lowerhydrogel loading comparedtotheMgOC-E.Similarresultswerealsoobservedona carbon nanotube-modified carbon paper electrode; 22mA at 800m
gcm2ofhydrogelloading[4].Infact,theKBelectrodehasa largespecificsurfaceareathatisbuiltbytheaggregationof40 nm-diameterKBparticles.Despite this,someareas(spaces)arenot utilized to support the hydrogel because the porous carbonstructure created by the KB particles is uniform and not interconnected in a manner that permits the penetration of polymerandenzymeintotheentirecarbonlayer(Fig.3(b)).Onthe otherhand,electrophoreticallydepositedMgOClayershavemuch moreeffectivesurfacesforhydrogelloadingowingtothepresence of10
m
m-scaleinterconnectedmacroporesand 38nm-diameter mesopores,sothatthehydrogellayercanbewidelydistributedon the carbon layer, while mass transfer of fuel are not limited (Fig.3(d))[2].Fig.2(panelB)showsthedependenceoftheglucoseoxidation current, at 0.3V, on the electrode rotation rate. The catalytic current showed a significant increase as the rotation rate is increased to 500rpm that plateaued asymptotically beyond 1000rpm.Atlowrotationrates(i.e.500rpm),60%ofthemaximum glucoseoxidationcurrentisobserved,whichindicatesthatmass transfer through theMgOC-E layeris effective [12]. The three-dimensional macro-cellular carbonaceous foam electrode,
Fig.1.(A)RotatingdiskcyclicvoltammogramsforglucoseoxidationcatalyzedbythepolymerIIGOxhydrogel,modifiedontoMgOC(bluecurve(a))andGC(redcurve(b)) electrodes,inaphosphatebuffer(0.1M,pH7.0,37C)andinthepresenceof0.5Mglucose.Thegraydashedcurve(c)correspondstoGOxandpolymerII-modifiedMgOC-Ein
theabsenceofglucose.Thetotalhydrogelloadingwas1000mgcm2andthepolymer:enzyme:crosslinkerratiowas45:45:10.Thescanratewas20mVs1.(B)Dependenceof thecatalyticcurrentdensityat0.6V(opencircle,leftaxis)andresidualcurrent(percentageofcatalyticcurrentafter5minconstant-potentialelectrolysisat0.6Vinthe presenceofglucosetotheinitialcurrent,closecircle,rightaxis)oncrosslinkercontent.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferred tothewebversionofthisarticle.)
Fig.2. (A)Dependenceoftheglucoseoxidationcurrentat0.6Vonthehydrogelloadinginaphosphatebuffer(0.1M,pH7.0,37C)containing0.5Mglucose.Theelectrode rotationratewas8000rpm.(B)Dependenceoftheglucoseoxidationcurrentat0.3Vontheelectroderotationrateinaphosphatebuffer(0.1M,pH7.0,37C)containing0.5M
modified with 1734
m
gcm2 of hydrogel, generated a current densityof18mAcm2at37Cand2000rpm[13].Incomparison, theMgOC-Egenerateddoublethecurrentdensity(35mAcm2)at 2000rpmandlowerhydrogelloading(1000m
gcm2).Thehigher current density can be ascribed to highly efficient electron transportthroughthethinhydrogellayer[17,18],aswellasthe hierarchicalelectrode morphology. Macro-structuresamongthe unevenlydeposited carbonparticlesallow forthesmoothmass transfer of glucose through the carbon layer, while the 38nm mesoporous structure allows for the formation of very thin hydrogelstructures,reducingmasstransferresistancethroughthe hydrogelasglucosepassesfromsolutiontotheenzyme(Fig.3(d)). Themaximalcatalyticcurrentdensity,atinfiniterotationrate,that can be reached when glucose is sufficiently delivered to the catalyticsite ofthemodifiedhydrogel, wasestimatedfromthe intercept of the Koutecky-Levich plot (1/currentdensity vs. 1/ (radians1)1/2).ThemaximalcurrentdensityforMgOC-E,with1000
m
gcm2 of hydrogel, was determined to be 60mAcm2, which is 6-fold higher than that for the KBelectrode with a loading 200m
gcm2 [12]. The diffusion coefficient of glucose, estimatedfromtheslopeoftheplot,is2.8107cm2s1,whichisofthesame orderof magnitudethan thatfor theKBelectrode (3.7107cm2s1on100mMglucose)[12].Theseresultssuggest thatthereare nodistinct differencesbetweenMgOCandKBin currentproduction efficiency,but MgOCis thebetterplatform, havingalargercapacityforhydrogelloading.
Fig.4presentsacomparisonofthecyclicvoltammogramsfor the various hydrogel electrodes on MgOC-E, using the high-potential polymerI (0.22V),and the low-potentialpolymers II (0.03V,withlongspacerchain).Althoughtheredoxpotentialof thehydrogelwithpolymerIIismorereducingthanthatofpolymer I,andclosetotheredoxpotentialofGOx,thedifferenceintheir steady-statecatalytic currentswas quitesmall.Thelongcarbon chainlinkingtheOscomplexwiththebackbonepolymerallows theredoxcentertocollectelectronsmoreefficientlyfromtheGOx active center [17,18]. In addition, the macro-meso-hierarchal structureoftheMgOC-Ewasalsoveryeffectiveforproducinga hydrogelelectroderedoxpolymerwithlowpotential.Inprevious papers we demonstrated that polymer I-based hydrogels
incorporatingdeglycosylatedFAD-dependentglucose dehydroge-nase(d-FADGDH,fromA.terreus,IkedaTohka,Fukuyama,Japan), produced catalytic currents of 100 and 4mAcm2 at 25C on MgOC-EandGC-E,respectively[2,14].Fig.5(A) showsthecyclic voltammograms for glucose-oxidation using the GOx-hydrogel-andd-FADGDH-hydrogel-MgOC-EwithpolymerII.Thecatalytic currentof theGOx-hydrogelelectrodeis almost6timeshigher than that of the d-FADGDH-hydrogel electrode (8mAcm2). Notably, when glycosylated (or non-deglycosylated) FADGDH wasusedasanalternativetod-FADGDH,noincreaseincatalytic currentisobserved.Fig.5(B)showsthecyclicvoltammogramsfor glucose-oxidation using the GOx-hydrogel- and d-FADGDH-hydrogel on GC electrode with polymer II. The ratio of the catalyticcurrentonGOxelectrodetothatonFADGDHelectrode wassameincase ofMgOC-EandcaseofGC-E.Thecomparison indicatesthat the low catalytic current onFADGDH-polymer II electrode was not due to the electrode material, but to the combination of enzyme and redox polymer. Considering the differences in glucose oxidation current between GOx and FADGDH,it isclearthat thelongspacerdoesnotlead tomore efficientelectroncapturefromtheactivecenterintheFADGDH system. Some interactions between the spacer chain and the enzymesurfaceorenzymeactivesitemightleadtothedecreased flexibilityoftheOscomplexinwithinpolymerIIinthehydrogel, which is important for the efficient collection of electrons. Althoughthe3DcrystalstructureofFADGDHfromA.terreusused inthisstudyhasnotbeenreported,thestructureofthesimilar FADGDH from A. flavus, which shares 60% of its amino acid sequence homology withFADGDHfromA.terreus, hasrecently beenreported[19].BycomparisonwiththatfromA.terreus,the surfaceofFADGDHwould beexpectedtobemorehydrophobic than GOx, and therefore, long hydrophobic spacers may get entangledatthehydrophobicsurfaceofFADGDHbyvanderWaals forces, orhydrophobic interactions,therebylosing flexibility.In ourpreviouspaper,wereportedthat catalyticcurrentincreases withincreasesinphosphatebufferconcentration(0.1to1M)fora d-FADGDHhydrogelelectrodefrompolymerI[2].However,the cyclic voltammogramof the hydrogel modified electrode made
Fig.3.Proposedschematicillustrationofhydrogel distributionwithin porous carbonelectrode.Porouscarbonwithrandomporesize(likeKB)withlowhydrogel loading(A)andhighloading(B),andporesize-controlledporouscarbonelectrode (MgO-templatedcarbon)withlowhydrogelloading(C)andhighloading(D).
Fig.4. RotatingdiskcyclicvoltammogramforglucoseoxidationbyaGOx-hydrogel modified MgOC-E inphosphatebuffer(0.1M,pH 7.0, 37C)containing0.5M
glucose,usingpolymerI(bluecurve(a)),andpolymerII(redcurve(b)).Total hydrogelloadingswere1000mgcm2andthepolymer:enzyme:crosslinkerratio was45:45:10forpolymerIandpolymerII.(Forinterpretationofthereferencesto colorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
from polymer II remained unchanged when the electrolyte solution was replaced with a 1M potassium phosphate buffer solution. FADGDH from A. terreus can be used with organic mediators with relatively low redox potentials (below 0.1V), suchasnaphthoquinonesorotherredoxmolecules,atacceptable rates.Forexample,thebimolecularrateconstantforitsusewith 1,2-naphthoquionone (140mV) is 2–3 orders of magnitude higherthanthatofGOx(unpublished). Rossetal.alsoreported thata1,2-naphthoquinone-tetheredpolymerfacilitatedelectron transferfromFADGDHtotheelectrode,butthiswasnotthecase forGOx[20].Thelowcatalyticcurrentdensitydemonstratedin thisstudyisascribedtothelowaffinityofthepolymer-tetheredOs complex toward the active site of FADGDH. This is still under investigation.
Inconclusion,aglucoseoxidationcurrentdensityof50mAcm2 wasrecordedat0.3VusingMgO-templatedporouscarbon,with 38nmaveragediameterpores,modifiedwithaGOxhydrogel,anda redoxpolymerwithaformalpotentialof0.03Vvs.Ag|AgCl.The onsetpotentialwasmeasuredtobe0.12V,whichis0.2Vlower thanthatreportedearlier[1].Contraryto expectation[14],GOx producesahighercatalyticcurrentthanFADGDHordeglycosylated FADGDH. WhileFADGDHs gathersconsiderableattentions asan electrochemical biocatalyst for bioelectrochemical application because they don’t use O2 as anelectron acceptor, FADGDH-Os
complex hydrogel systems having both acceptable potentials at whichreactionsproceedandcurrentdensityfromtheviewpointof applicationforbioelectrochemicaldevices,especiallyforbiofuelcell, havenotbeenreported(Table1)sofar,norachievedinthisstudy. WhenGOxisusedasanelectrocatalystinthepresenceofO2inthe
system,hydrogenperoxidecanbegenerated,whichmayleadan instability of the enzyme electrode and prevent the long-term operationofthebioelectrochemicaldevices.However,recentlywe reportedthatGOx-basedhydrogelanodecanbestabilizedbyadding catalaseintothehydrogeltoeliminatetheH2O2[1].
Acknowledgements
This work was partially supported by a grant from JSPS KAKENHI (Grant Number 15K14684). The author (NM) thanks fundingfromlaRégionAquitaineand theANRRATIOCELLS (12-BS08-0011-01).
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Fig.5.Rotatingdiskcyclicvoltammogramsin0.1Mphosphatebuffercontaining0.5Mglucose,atpH7.0,37C,8000rpm,and20mVs1forpolymerII-basedelectrodes.
GlucoseoxidationbyGOx(bluecurve(a))andd-FADGDH(redcurve(b)).Thetotalhydrogelloadingwas1000mgcm2andthepolymer:enzyme:crosslinkerratiowas 45:45:10forpolymerIandpolymerII.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)