DTIC ADA588241: Glucose Oxidase Catalyzed Self-Assembly of Bioelectroactive Gold Nanostructures

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Full Paper
Glucose Oxidase Catalyzed Self-Assembly of Bioelectroactive Gold
Nanostructures
Heather R. Luckarift,a,b* Dmitri Ivnitski,c Rosalba Rinco´n,c Plamen Atanassov,c* Glenn R. Johnsona
a MicrobiologyandAppliedBiochemistry(AFRL/RXQL),AirForceResearchLaboratory,139BarnesDrive,Suite#2,TyndallAir
ForceBase,FL32403,USA
*e-mail:[email protected];[email protected]
b UniversalTechnologyCorporation,1270N.FairfieldRoad,Dayton,OH45432,USA
c ChemicalandNuclearEngineeringDepartment,UniversityofNewMexico,Albuquerque,NM87131,USA
Received:June15,2009
Accepted:October1,2009
Abstract
Glucoseoxidasecatalyzestheformationofmetallicgoldparticlesinimmediateproximityoftheproteinfromgold(III)
chlorideintheabsenceofanyothercatalyticorreductivesubstrates.Theprotein-mediatedgoldreductionreactionleads
to size-controllable gold particle formation and concomitant association of the enzyme in an electrically conductive
metallictemplate.Such an enzymeimmobilization strategyprovides asimple and rapid method to createan intimate
interfacebetweenglucoseoxidaseandaconductivematrix,whichcanbejoinedtoanelectrodesurface.Modelelectrodes
were prepared by entraining the glucose oxidase/gold particles onto carbon paper. Voltammetry of the resulting
electrodesrevealedstableoxidationandreductionpeaksatapotentialclosetothatofthestandardvaluefortheFAD/
FADH cofactorofimmobilizedglucoseoxidase.Thegoldelectrodesexhibitcatalyticactivityinthepresenceofglucose
2
confirmingtheentrapmentofactiveglucoseoxidasewithinthegoldarchitecture.Theresultingcompositematerialcanbe
successfullyintegratedwithelectrodesofvariousdesignsforbiosensorandbiofuelcellapplications.
Keywords:Goldreduction,Glucoseoxidase,Electrontransfer,Nanocomposites,Self-assembly
DOI:10.1002/elan.200980003
1. Introduction 3s(cid:2)1 have been reported for immobilized GOx [7, 11, 12].
CreatingarchitecturescontainingCNT,however,hasproven
The ability to create stable enzyme/metal nanocomposites difficultandintegrationofenzymeswithCNTandwiththe
thatretainenzymeactivityathighsurface-to-volumeratios electrodeinterfacecontinuestopresentchallengesforscale-
providesexcellentopportunitiesincatalysis,biosensingand up and manufacturability. Similarly, gold nanoparticles,
biofuelcellapplications [1–4]. The synthesisofgoldnano- structured on the electrode interface can act as a scaffold
particleswithcontrollableshapesandstructuralproperties,in forGOximmobilizationandmediateDET[6,13,14].Inthe
particular, has provided significant advances in glucose presenceofgoldnanoparticlesofspecific size, the electron
sensing due to unique optical and electronic properties [5, transfer distance is significantly decreased, leading to an
6].Goldnanoparticleswiththeappropriatedimensionscan increaseintheelectrontunnelingrateofmorethan1000-fold
alsoactasabridgebetweentheredoxcenterofanenzyme insomecases[15].Therefore,thereisconsiderableinterestin
andthe bulkelectrode material tofacilitate direct electron methodsforcontrollingthepreparationofwell-definedgold
transfer(DET)[2,3,6].DETisadvantageousasitnegatesthe nanoparticles of different size and shape. It is ever more
useofmediatorsandelectrontransferoccursatapotential important to have those methods involve simultaneous
closetotheredoxpotentialoftheenzymeitself[7,8].Inthe enzymeimmobilizationwithaconductivephase,preferably
absenceofasuitable“connection”(electrochemicalmedia- forminginacontrolledsynergisticfashion.
tor, charge transfer relay, conductive polymer matrix), Awealthofchemicalapproacheshavebeendevelopedfor
however, electrons generated at the FAD/FADH active the synthesis of gold nanoparticles including the process
2
siteofglucoseoxidase(GOx)musttunnelca.15(cid:2)through reductionofmetalsaltsbyreagentssuchassodiumborohy-
the protein shell, severely limiting DET efficiency [9]. dride, hydroxylamine and polyvinyl pyrrolidone [1–3, 16].
Attemptshavebeenmadetoreducetheelectrontunneling While chemical methods for gold reduction are well docu-
distance by using conductive nanostructures to shuttle mented,many biological materials including plant extracts,
electrons[10].Manipulationsofsuchnanoparticles,however, bacterialandfungalstrainswillcatalyzethereductionofgold
haveprovenextremelytimesensitiveandtheirpracticaluse saltstogoldnanoparticlesofdifferingsizeandmorphologies
dependsstronglyupontheprotocolsapplied.Carbonnano- [16–21]. As such, the interaction of biomolecules with
tubes (CNT) for example provide an excellent conduit for colloidal gold as well as the study of enzymatic activity of
electricalcommunicationandelectrontransferratesofca.1– bioconjugateshasalsoattractedattention[22–25].
784 (cid:3)2010Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim Electroanalysis2010,22,No.7-8,784–792
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1. REPORT DATE 3. DATES COVERED
2009 2. REPORT TYPE 00-00-2009 to 00-00-2009
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Glucose Oxidase Catalyzed Self-Assembly of Bioelectroactive Gold
5b. GRANT NUMBER
Nanostructures
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
Microbiology and Applied Biochemistry (AFRL/RXQL), Air Force REPORT NUMBER
Research Laboratory,139 Barnes Drive, Suite #2,Tyndall Air Force
Base,FL,32403
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13. SUPPLEMENTARY NOTES
Electroanalysis 2010, 22, No. 7-8, 784 - 792
14. ABSTRACT

15. SUBJECT TERMS
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
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a. REPORT b. ABSTRACT c. THIS PAGE Same as 10
unclassified unclassified unclassified Report (SAR)
Standard Form 298 (Rev. 8-98)
Prescribed by ANSI Std Z39-18
GlucoseOxidaseCatalyzedSelf-Assembly
GOxisknowntocatalyzegoldreductionbutonlyinthe molecular weight cutoff membrane (Ultrafree, Sigma-
presenceofglucose,duetothecatalyticformationofHO Aldrich,St.Louis,MO).100mLoftheGOx-Aucomposite
2 2
thatinturnactsasareducingagentforgold[10,13,26].We wasplacedontopoftheTPandpassedthroughthecolumn
reporthereinthatGOxcatalyzesthereductionofAu3þto bycentrifugation (5585(cid:3)g).Themolecular weightcutoff
metallicAuintheformofparticleswhosesizedependson membraneslowsthepassageofthegoldparticlestoaidin
thekineticsofthenucleationprocess.Thisprocessoccursin entrapment. A control electrode with GOx alone was
the absence of glucose or peroxide, formed as a result of preparedinthesamemannertoallowformeasurementof
enzymaticoxidationofglucoseoranyotherreducingagent. GOxactivitythatisretainedbysimplemolecularweightsize
The enzyme also acts as a self-assembly factor in the exclusionandphysicaladsorption.TheTPmodifiedGOx-
formation of gold particles and ultimately becomes en- Au electrodes were removed and washed with distilled
trained within the resulting gold nanostructures. The waterbeforeanalysis.
resultingglucoseoxidase/gold(GOx-Au)compositesdem-
onstrate DET between the FAD/FADH redox center of
2
GOxandthegoldparticles.Toourknowledge,thereduction
2.4. Characterization of Glucose Oxidase–Gold
ofAuCl(cid:2) byaredox-activeenzymethatinturnretainsits
4 Composites
catalyticactivityintheresultinggoldparticlehasnotbeen
reported previously. The direct encapsulation of enzymes ThespectroscopiccharacteristicsofGOx-Aunanoparticles
withingoldprovidesanopportunitytodevelopredox-active were measured using a Nanodrop 1000 (Thermo Fisher
conductive material for application in thedevelopment of Scientific,Waltham, MA). The surface morphology of the
micro-sizedenzyme-basedfuelcellsandlabel-freebiosens- GOx-Au electrodes were visualized using an Hitachi (S-
ingsystems. 2600)scanningelectronmicrscope(SEM)operatingunder
vacuumat20kVforimaging.Noconductivecoatingswere
added to the sample electrodes prior to SEM analysis.
Particlesizewasmeasuredbydynamiclightscatteringusing
2. Experimental
aZetasizernanoCZ90(MalvernInstrumentsLtd.,Worch-
estershire,UK)usingarefractiveindexof0.47forgold[27].
2.1. Materials
Reported particle size measurements are an average of
TypeII-SGOxfromAspergillusniger(EC1.1.3.4)andgold threesamples(>12measurementspersample).Attenuated
(III) chloride solution (ca. 30wt% in dilute HCl) were Total Reflectance Fourier Transform-Infrared Spectrosco-
obtainedfromSigma-Aldrich(St.Louis,MO).Toraycarbon py(ATRFT-IR)wasperformedusingaNicoletFT-IR6700
paper (TP) TGPH-060, was obtained from E-TEK, New spectrophotometer equipped with a Smart Miracle single
Jersey,US(nowadivisionofBASF).Allotherreagentsand bouncediamondATRaccessory(ThermoFisherScientific,
chemicals were of analytical grade and obtained from Waltham, MA). The data collection was completed using
standardcommercialsources.Enzymestocksolutionswere OMNIC2.1software.ForFT-IR,5mLofGOx-Aucompo-
prepared in sodium acetate buffer (0.5M, pH6.5) and sitesweredepositedontoaglasscoverslipandallowedto
AuCl(cid:2) dilutions were prepared in distilled water.Glucose dry. The cover slip was placed face down on the diamond
4
solutionswerepreparedfromapuresubstrate,dissolvedin surfaceoftheFT-IRforanalysis.
distilledwaterandequilibratedatroomtemperaturebefore
theexperimentsformutarotation.
2.5. Treatment and Modification of Commercial Enzyme
Preparations
2.2. Preparation of the Glucose Oxidase–Gold
In order to confirm the involvement of GOx in gold
Composites
reduction, soluble contaminants were removed from the
GOx-Au composite particles were prepared as follows: commercialenzymepreparationbydialysis.GOx(10mLof
20mLofGOx(300mg/mL)wasaddedto175mLofsodium 300mg/mL) was dialyzed against three changes of 1L
acetate buffer (0.5M, pH6.5). To this was added a 5mL sodiumacetatebuffer(0.5M,pH6.5)over24hoursusinga
aliquotofAuCl(cid:2)atfivedilutions:30%,15%,7.5%,3.75% 10kDa molecular weight cut off dialysis cassette (Slide-a-
4
and0%togiveafinalconcentrationof35,18,9,4and0mM lyser;PierceInc.,Rockford,IL).
respectively (i.e. GOx-Au:35). The reaction mixture was ThemodificationofthefreecysteinethiolgroupsofGOx
incubatedatroomtemperatureinthelightfor5hours. was adopted from a method reported previously [25]. A
stock solution of 6mM 2,2’-dithiobis(5-nitro-pyridine)
[DTNP] was prepared in DMSO and mixed with GOx in
potassiumphosphatebuffer(20mM,pH7.0)togiveafinal
2.3. Preparation of Carbon Paper Electrodes
concentrationof0.2mLDTNPin5mLofbuffercontaining
Carbon paper (TP) was cut into 4.5mm diameter disks, 15mg GOx. A control of GOx was prepared in the same
washedinethanolandrinsedwithwater.TheTPdiskwas manner with DMSO alone. The reaction mixture was
placed on top of a microcentrifuge filter with a 30kDa incubated at 48C overnight with stirring. The GOx was
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concentrated using an Amicon Ultra filter unit with a scanrateof5,10,20,40,60,80,100,200and500mV/sfor1
30kDamolecularweightcutoff(Sigma-Aldrich,St.Louis, cycleeach.Thecellwasthenspargedwithoxygenfor1hour
MO).Thefinalproteinconcentrationofthethiol-modifed andthecyclicvoltammetryrepeatedfrom(cid:2)0.8to(cid:2)0.2Vat
and untreated GOx was determined by bicinchoninic acid a scan rate of 20mV/s for 5 cycles. 25mM glucose (filter
(BCA) assay according to the manufacturers instructions sterilized)wasaddedandthecellwasspargedwithoxygen
(Pierce, Rockford, IL). The GOx concentration was nor- forafurther5minutestoaidmixingandthentheCVcycles
malizedandusedinthegoldreductionreactionasdescribed repeated. Peak maxima, heights and currents were deter-
above. mined from voltammograms using the V3 studio software
(PrincetonAppliedResearch).
2.6. Electrochemical Measurements
3. Results and Discussion
Electrochemical measurements were performed with a
potentiostat(PrincetonAppliedResearch,ModelVersastat
3.1. Synthesis and Characterization of Glucose Oxidase–
3,OakRidge,TN)inathree-electrodecellconsistingofthe
Gold Composites
TP GOx-Au working electrode, a glassy carbon counter
electrode (Metrohm, Switzerland) and an Ag/AgCl refer- Simple mixing of AuCl(cid:2) in a buffered solution of GOx at
4
ence electrode (CH Instruments Inc., Austin, TX) in a ambient conditions resulted in a significant color change
50mL working volume. The electrolyte solution used over5hoursofincubation.Thecolortransitionisindicative
throughoutconsistedofa1:1mixtureofphosphatebuffer ofachangeinthemetaloxidationstatefromAu3þtoAu0.No
(20mM,pH7.0)andKCl(0.1M).Electrochemicalexperi- goldformationwasobservedafteraperiodof7dayswith
ments were carried out at room temperature. Cyclic bufferandAuCl(cid:2)alone,confirmingthatGOxisessentialfor
4
voltammogramswereusedtocalculatetheelectrontransfer gold reduction. UV-visible spectroscopy of the resulting
rate constant using the method of Laviron [28, 29]. Each precipitatesshowstheappearanceofanabsorptionbandat
GOx-Au/TP disk was placed into a modified teflon cap 540–570nm, characteristic of the surface plasmon reso-
designedtofitsnugglyontoacommericalglassycarbondisk nanceforgoldnanoparticles(Fig.1).Glucoseoxidase–gold
electrode. The cell was sparged with nitrogen for 20min (GOx-Au) composites were prepared at a range of molar
before starting potentiostat measurements. Initial analysis concentrations that produced a shift in the absorption
was performed after subjecting the electrode to 10 cycles wavelength (and respective color of the reaction product)
withpotentialfrom(cid:2)0.8to(cid:2)0.2Vataconstantscanrate thatisdirectlydependentupontheratioofenzymetoAuCl(cid:2)
4
(20mV/s). The electrodes were then analyzed with varied andischaracteristicofavariationingoldnanoparticlesize
Fig.1. UV/VisabsorptionspectraofGOx-Aucomposites.
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GlucoseOxidaseCatalyzedSelf-Assembly
Fig.2. FT-IRspectraofGOx-Aucomposites.
[16]. At high concentrations of AuCl(cid:2) (GOx-Au:35), the to Au0 suggests that GOx acts as a reducing agent for the
4
formationofmacromoleculargoldparticlesisvisiblebyeye. metal and undergoes subsequent aggregation to form a
WithdecreasingconcentrationofAuCl(cid:2),theproductcolor planar microstructure. Gold synthesis in this way is there-
4
changes through the visible range from yellow to orange fore versatile as the size and morphology of the gold
(GOx-Au:18), brown (GOx-Au:9) and purple (GOx- compositescanbecontrolledbychangingtheratioofAuCl(cid:2)
4
Au:4). toGOx.
FT-IR analysis of the GOx-Au composites showed uni- The material characterization using scanning electron
form bond stretching characteristic of the native enzyme, microscopy (SEM) revealed a mixture of spheroid nano-
irrespective of the gold particle size (Fig.2). The amide particles along with macromolecular gold triangles and
bonds;amide-I,amide-IIandamide-IIIofGOxarevisible plateletsdependinguponthegoldprecursorconcentration
at 1630–1670cm(cid:2)1, 1550cm(cid:2)1 and ca. 1340cm(cid:2)1 respec- of the preparation (Fig.3). The TP fibers are clearly
tively[30].AbroadbandattributedtoNHstretchwasalso distinguishable bySEM. Awellintegratedcoating ofgold
observed, centered around 3300cm(cid:2)1. The results are in particles is visible for the GOx-Au:18 composite whereas
agreementwithpreviousliteraturereportsshowingprotein- larger macromolecular platelets were found in the GOx-
mediated formation of gold particles [25]. The bond Au:35 composite and tended to collect on the TP surface
stretching associated with the native protein is retained duetosizerestriction(Fig.3).Theformationoftriangular
withintheGOx-Aucompositesindicatingtheretentionof and hexagonal particles at high gold concentrations is in
GOx within the gold particles with no apparent change in agreementwithpreviousreportsthatusedbiomoleculesas
protein conformation. A broad band from 930–960cm(cid:2)1 templatesforgoldformation[22,31].
was observed that increased relative to the apparent gold
concentrationandwasabsentwithGOxalone.
Dynamic light scatter measurements revealed detail in
the size distribution of nanoparticles in the GOx-Au Table1. Stoichiometry and particle size distribution of GOx-Au
composite materials that could not be ascertained from composites.
color and UV-visible spectroscopy observations (Table1).
Sample Ratio Particlesize
The particle size distribution of GOx-Au:35 was polydis- Gox:Au[a]
perseandindicatedthepresenceofgoldparticleaggregates Intensity(nm) Volume(nm)
withinthesample.Thelargeaggregatesscatterasignificant
GOx-Au:35 187:1 318.8(85.8%) 1040
portion of light due to their size but are present in low 71.4(11.6%)
numbers. Lower concentrations of AuCl(cid:2) in the reaction 5483.0(2.9%)
4
mixtureresultedinhomogeneousparticlesizedistributions GOx-Au:18 93:1 297.5 304.7
GOx-Au:9 47:1 255.0 252.6
in terms of intensity and volume size distribution. GOx-
GOx-Au:4 23:1 132.2 115.5
Au:18andGOx-Au:9formedsimilarlysizedgoldparticles
intheorderof250–300nm.TheobservedreductionofAu3þ [a]FinalconcentrationofGOxandAuassuming100%incorporation
Electroanalysis2010,22,No.7-8,784–792 (cid:3)2010Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim www.electroanalysis.wiley-vch.de 787
Full Paper H.R.Luckariftetal.
Fig.3. ScanningelectronmicrographsofGOx-Auelectrodesoncarbonpaper(TP).A)GOx-Au:35,B)GOx-Au:18,C)GOx-Au:9,
D)GOx-Au:4,E)GOx.Scalebar¼20mm
3.2. Direct Electrochemistry of Glucose Oxidase–Gold Au:18andGOx-Au:9indicatedthattheFADredoxcenter
Composites of GOx shows a quasi-reversible one-electron transfer
process on gold nanoparticles (Table2). At the highest
The electrochemical characteristics of the GOx-Au/TP (GOx-Au:35)andlowest(GOx-Au:4)goldconcentrations
electrodes were investigated by cyclic voltammetry (CV) investigated, the peak width increases 3–5 fold. GOx-
inthepresenceandabsenceofoxygen(Fig.4).GOx-Au:18 Au:35andGOx-Au:4electrodesshowmorevariabilityand
andGOx-Au:9electrodesshowreversibleelectrontransfer less stability in their redox characteristics, which may be
andapairofwell-definedredoxpeakswithaformalredox attributed to the gold particle size. For example, large
potentialofca.(cid:2)0.44V(atpH7.0vs.Ag/AgCl)(Table2). particleaggregatesareretainedontheelectrodesurfacedue
The standard redox potential of the FAD/FADH redox to size exclusion, but may not connect well with the TP
2
couple at pH7.0 is (cid:2)0.43V vs. Ag/AgCl [7]. The redox interface, whereas very small particles will likely pass
peaks are therefore attributed to the reversible reduction straightthroughtheTPcarbonfibermatrix.
andoxidationreactionoftheFAD/FADH cofactorinthe TheabilityofthegoldencapsulatedGOxtoundergoDET
2
active site of GOx. Control experiments of TP with GOx withtheelectrodesurfaceandretainitsbiocatalyticactivity
alone (in the absence of gold nanoparticles) showed no was investigated using cyclic voltammetry of solutions
discernible redox characteristics confirming that the gold containing and lacking the substrates of the enzymatic
particlesprovidethesoleelectricalconnectionbetweenthe recation:d-glucoseanddi-oxygen(Fig.4).Theoxidationof
enzymeandtheelectrodesurface. d-glucose by GOx involves a redox change of the FAD
Thetheoreticalhalf-heightpeakwidthforaoneelectron- cofactoroftheenzyme.Glucoseiscatalyticallyconvertedto
transfer process is 91mV at 298K [32]. The redox peak gluconolactonewiththeconcomitantconversionofoxygen
separation (DE ) and theoretical half peak heights mea- to hydrogen peroxide. In the oxygenated electrochemical
p
sured for the GOx-Au:18 and GOx-Au:9 electrodes cell,theadditionofglucosecausesadecreaseincurrentdue
showedclosestmatchtotheoreticalvaluesfortheelectrodes tothecatalyticremovalofoxygenattheelectrodesurface.
tested (Table2). The experimental peak width for GOx- The addition of glucose to the GOx-Au electrodes in all
788 www.electroanalysis.wiley-vch.de (cid:3)2010Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim Electroanalysis2010,22,No.7-8,784–792
GlucoseOxidaseCatalyzedSelf-Assembly
Table2. DirectelectrochemistrycharacteristicsofGOx-Aucomposites.
Scanrate20mV/sunderaN atmospherewith25mMglucosevs.Ag/AgCl;ND:notdetermined–notmeasurableat20mV/sscanrate.
2
Sample Anodicpeak Cathodicpeak Formal DE (mV) Half-heightpeak
p
potential(V) width(mV)
(V) (mA) (V) (mA)
GOx-Au:35 (cid:2)0.417 1.38 (cid:2)0.548 (cid:2)3.47 (cid:2)0.482 131 71–72
GOx-Au:18 (cid:2)0.435 4.02 (cid:2)0.460 (cid:2)5.30 (cid:2)0.447 25 82–93
GOx-Au:9 (cid:2)0.420 2.30 (cid:2)0.460 (cid:2)3.14 (cid:2)0.440 40 92–95
GOx-Au:4 (cid:2)0.392 1.33 (cid:2)0.507 (cid:2)2.77 (cid:2)0.449 115 ND
casesresultedinadecreaseincurrentdensitybuttovarying 3.3. Characterization of Bioelectrocatalytic Activity of
degrees.Thecurrentresponseconfirmedtheencapsulation Glucose Oxidase–Gold Composite Electrodes
ofactiveGOxwithinthegoldcompositesandisconsistent
withinfluencefromenzymethatisinanintimateassociation Frompreliminarystudies,thecompositioncorrespondingto
with the electrode. The bioelectrocatalytic activity was thesynthesisparametersofGOx-Au:18compositesexhib-
greatestforGOx-Au:18andisagainattributedtothegold ited optimal direct electrochemical activity. Evidence of
particlesizewhichallowsforeffectivecommunicationwith surface-confined redox centers was obtained from the
theelectrode. observation of a correlation of anodic and cathodic peak
Fig.4. ElectrochemicalanalysisofGOx-Auelectrodes:A)GOx-Au:35,B)GOx-Au:18,C)GOx-Au:9,D)GOx-Au:4,E)GOxonTP
electrodes.Scanrate;20mV/sin20mMphosphatebuffer/0.1MKCl,(pH7.0).N saturatedelectrolyte(blackline,#10of10scans),O
2 2
saturatedelectrolyteþ25mMglucose(greyline,#5of5scans),O saturatedelectrolyte(dashedline,#5of5scans).
2
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Full Paper H.R.Luckariftetal.
Fig.5. Electrochemical analysis of GOx-Au:18 electrode. A) Effect of increasing scan rate from 5 to 500mV/s (from inner scan
outwards)in20mMphosphatebuffer/0.1MKCl(pH7.0)andthecorrespondingLavironplot(B).Effectofglucoseconcentrationon
current;cyclicvoltammetryat0,5,10,15,20and25mMglucose(C)andthecorrespondingcalibrationcurve(D).
currents against scan rate (Fig.5A). The peak separation TheCVofGOx-Au:18undergoesasignificantchangein
from10to100mV/sforelectrodeGOx-Au:18rangesfrom currentdensityinthepresenceofoxygenattributedtothe
27.64 ((cid:4)3.08) minimum to 45.08 ((cid:4)4.36) maximum, indi- catalyticconversionofO toHO (Fig.5C).Itisconfirmed
2 2 2
cating that the electron-transfer process is rapid and that in oxygen-saturated buffer, the addition of glucose
reversible.Theseparationbetweenreductionandoxidation causes a decrease in current due to catalytic removal of
peakmaximaasafunctionofscanratewasusedtocalculate oxygen at the electrode surface, confirming an intimate
an electron transfer rate (K) of 2.59s(cid:2)1 (n¼3) for a association between GOx and the gold composite. The
s
triplicate ofGOx-Au:18electrodes[28].The fastelectron bioelectrooxidation of glucose by the GOx-Au:18/TP
transferindicatesthatthegoldmatrixprovidesaneffective electrodeincreaseswithglucoseconcentrationuptoavalue
electrical communication between the FAD redox center of 15mM at which point the kinetics of catalytic activity
andtheTPelectrode.ThesymmetryevidentintheLaviron plateau as expected as a result of enzymatic Michaelis-
plot is indicative of a quazi-reversible charge-transfer Menten kinetics (Fig.5D). Interestingly, the linear detec-
process associated with the redox couple FAD/FADH in tionrangeforthissystemcoversthephysiologicallevelofca.
2
theactivesiteoftheenzyme(Fig.5B).Ininstanceswhere 4–6mM present in human blood, lending itself to a
theprotein(GOx)isdenatured,thecathodicpeakpositions potentialapplicationinreagentlessglucosedetection.
shift more significantly than the anodic ones, resulting in
asymmetrical Laviron plots. This allows us to hypothesize
that, the voltammetry observations of redox activity at
3.4. Catalytic Mechanism of Glucose Oxidase–Gold
cathodicpotentialsarenotaproductofFADcofactorthat
Composites
mayhavebeenreleasedfromtheenzymeduringdenatura-
tion and subsequently bound to the electrode composite Althoughacompleteunderstandingofthemechanismfor
matrix. biocatalytic goldreduction remainsunclear,itis generally
believedthatthenatureoftheaminoacidfunctionalgroups
790 www.electroanalysis.wiley-vch.de (cid:3)2010Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim Electroanalysis2010,22,No.7-8,784–792
GlucoseOxidaseCatalyzedSelf-Assembly
((cid:2)SH,(cid:2)NH,etc.)playanimportantroleingoldreduction particle formation (Fig.6). We must emphasize, however,
2
andaggregation.Asstabilizingagentsingoldnanoparticles that no seed particles, additional glucose or exogenous
synthesis,themostcommongroupsarealkanethiols[25,33]. reaction components were added to the commercial GOx
The thiol-gold bond is most commonly described as a preparation during gold reduction. Results from prelimi-
surfaceboundthiolate[33].Recently,thepresenceoffree naryexperimentsinwhichGOxwastreatedwithDTNPto
thiol groups has been proposed as a mechanism for gold modifythefreethiolgroups[25],remainedinconclusive,as
reductioninpureenzymes[25].Thethiolgroup((cid:2)SH)inthe thereactionwascomplicatedbytherequirementforDMSO
sidechainofcysteineresiduesareknowntoreactwithgold inthereactionmixture.Amoredetailedinvestigationofthe
to form Au-S bonds and is implicated as a mechanism for kinetics and reaction mechanism of gold reduction from
gold reduction in a-amylase and other enzymes with highlypurifiedGOxisthereforecurrentlyunderway.
exposedthiolgroups[25].Similarly,glutathionereductase
catalyzes the NADPH-dependent reduction of AuCl(cid:2) to
4
form gold nanoparticles via catalytic cysteines within the 4. Conclusions
enzymeactivesite[34].Theinvolvementofdisulfidebridges
in gold reduction by bovine serum albumin has recently The method describes a simple and benign method to
beenproposed,althoughthepurity(andpotentialpresence associate GOx in metallic gold nanoparticles via GOx-
ofcontaminants)ofthecommercialproteinpreparationwas catalyzedgoldreduction.Thegoldparticlesforminsucha
notreported[22].Recentstudiesalsoproposeanalternative waythattheyfacilitateelectrontransferbetweentheredox
mechanism in which aromatic amines can be used as center of GOx and an electrode surface. The electron
reducing and capping agents for the synthesis of gold transfer system requires no external mediator and the
nanoparticles [35–38]. Selvakannan etal., for example, effectivecommunicationofelectronsbetweenGOxandthe
reported a water-soluble gold nanoparticle, functionalized electrode occurs at a potential of approximately (cid:2)0.44V.
with the amino acid lysine [39]. Similarly, Subramaniam Such a negative potential provides an effective anode
etal.statedthatthereductionofauricionsandtheoxidation configuration for enzyme-based fuel cell applications or
of the aromatic amine occur simultaneously [38]. The efficientinterference-freebiosensingelectrodes.TheGOx-
oxidative polymerization of the amines proceeds simulta- Au composites demonstrate an electron transfer rate
neouslywiththeformationofgoldnanoparticlessuchthat comparable to immobilization utilizing carbon nanotubes
thepolymerizedamineencapsulatesthegoldnanoparticle. but with the advantage of simplicity of preparation that
Assuch,theinteractionoftheamineswithgoldisstrongand potentiallymayresultinhighertechnologyadaptabilityand
similar to thiolate bonds [37]. GOx contains two disulfide bettermanufacturabilityandtechnologydevelopment.The
bridges and two free sulfhydryl groups as well as amino bio-conjugatesofGOx-Auformnanoparticlesunderbenign
groups presented on the surface [40]. GOx possesses physiological conditions in an aqueous solvent and the
thirteenlysineresiduesonitssurfaceandasaresult,carries reaction can be tuned to control size and shape of metal
a net negative charge. The thiol group ((cid:2)SH) in the side nanoparticleswithvariedopticalproperties.
chain of cysteine residues and amine groups of GOx may
thereforebeproposedtoberesponsibleforthesynthesisof
goldnanoparticles. Acknowledgements
Additional experiments were pursued to examine the
GOx-catalyzed gold reduction in light of other literature TheUNMportionofthisworkwassupportedinpartbya
reports. After GOx was dialyzed against acetate buffer to grantfromDOD/AFOSRMURIAwardNumber:FA9550-
limitpotentialcontaminantsfromthecommercialprepara- 06-1-0264,FundamentalsandBioengineeringofEnzymatic
tion,thegoldreductionactivitywasretainedinthedialyzed FuelCells.AFRLresearchwasfundedbytheUSAirForce
GOx,confirmingthatGOxisindeedtheactivecomponent Research Laboratory, Materials Science Directorate and
withinthemixture.Therateofgoldreductionwashigher, theAirForceOfficeofScientificResearch.
however, in the crude commercial preparation suggesting
that a component in the mixture may accelerate gold
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