Indium
tin
oxide
with
zwitterionic
interfacial
design
for
biosensing
applications
in
complex
matrices
Nadia
T.
Darwish,
Yatimah
Alias,
Sook
Mei
Khor
∗DepartmentofChemistry,FacultyofScienceUniversityofMalaya,50603KualaLumpur,Malaysia
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received5September2014
Receivedinrevisedform15October2014 Accepted28October2014
Availableonline5November2014 Keywords:
Biosensor Indiumtinoxide Anti-biofoulingcoating Zwitterionicmolecules
a
b
s
t
r
a
c
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Biosensinginterfacesconsistingoflinkermolecules(COOHorNH2)andcharged,antifoulingmoieties (( SO3−andN+(Me)3)forbiosensingapplicationswerepreparedforthefirsttimebytheinsitu depo-sitionofmixturesofaryldiazoniumcationsonindiumtinoxide(ITO)electrodes.Alinkermolecule isrequiredfortheattachmentofbiorecognitionmolecules(e.g.,antibodies,enzymes,DNAchains,and aptamers)closetothetransducersurface.Theattachedmoleculesimprovethebiosensingsensitivityand alsoprovideashortresponsetimeforanalytedetection.Thus,theincorporationofalinkerand antifoul-ingmoleculesisanimportantinterfacialdesignforbothaffinityandenzymaticbiosensors.Thereductive adsorptionbehaviorandelectrochemicalmeasurementwerestudiedfor(1)anindividualcompoundand (2)amixtureofantifoulingzwitterionicmoleculestogetherwithlinkermolecules[combination1: 4-sulfophenyl(SP),4-trimethylammoniophenyl(TMAP),and1,4-phenylenediamine(PPD);combination2: 4-sulfophenyl(SP),4-trimethylammoniophenyl(TMAP),and4-aminobenzoicacid(PABA)]ofaryl diazo-niumcationsgraftedontoanITOelectrode.ThemixtureratiosofSP:TMAP:PPDandSP:TMAP:PABAthat providedthegreatestresistancetonon-specificproteinadsorptionsofbovineserumalbuminlabeled withfluoresceinisothiocyanate(BSA–FITC)andcytochromeclabeledwithrhodamineBisothiocyanate (RBITC–Cytc)weredeterminedbyconfocallaserscanningmicroscopy(CLSM).Forthesurface antifoul-ingstudy,weused2-[2-(2-methoxyethoxy)ethoxy]aceticacid(OEG)asastandardcontrolbecauseof itsprominentantifoulingproperties.Surfacecompositionsofcombinations1and2werecharacterized usingX-rayphotoelectronspectroscopy(XPS).Field-emissionscanningelectronmicroscopy(FE-SEM) wasusedtocharacterizethemorphologyofthegraftedfilmstoconfirmtheevendistributionbetween linkerandantifoulingmoleculesgraftedontotheITOsurfaces.Combination1(SP:TMAP:PPD)witha ratioof0.5:1.5:0.37exhibitedthebestantifoulingcapabilitywithrespecttoresistingthenonspecific adsorptionofproteins.
1. Introduction
Biofoulingistheaccretionofproteins,cellsandotherbiological materialsontoabareelectrodesurface.Forfastclinicalscreening, immunosensorsaredesignedtodetectdiseaseanalytessuchas nucleicacids, proteinsorother diseasebiomarkersat very low concentrations from complicated sample media[1–5]. Such an accumulationof biofoulingsubstances leadstoreduced analyte diffusionandperfusiontothesensorinterface,whicheventually causesa decrease in sensor response and sensitivity. Polymers suchaspolyurethane,polyvinylchloride,poly(dimethylsiloxane) and poly(methacrylate) have been used as antifouling agents for biosensing surfaces,where theyprovide selective transport
∗Correspondingauthor.Tel.:+60379677022x2520;fax:+6039674193. E-mailaddress:[email protected](S.M.Khor).
of a targetedanalyte tothe biosensinginterface while preven-tingbiofoulingcomponentsfromreachingtheelectrodesurface
[6–10]. Although these polymers are exceptionally suitable in
clinical monitoring,their functional stability is greatly affected by the biocompatibility of the biosensor materials in contact with thebiological sample [11,12]. Poly(ethylene glycol)(PEG) andself-assembledmonolayerswitholigo(ethyleneglycol)chains (OEG-SAMs)havebeenusedtoreducethenon-specificadsorption ofnon-desiredproteins[13].
Theactivityofanti-biofoulingcoatingsincomplexsamples,e.g., humansera,dependsonseveralfactors,includingthe concentra-tionsandchainlengthsofPEGandOEGandthetemperature.At 37◦C,thegraftedsurfacecanadsorbadditionalnon-specific pro-teins. Moreover,surfaces grafted withPEG and OEG are prone to auto-oxidation in most biochemically relevant solutions for invivostudies[6,14,15].Zwitterionicpolymerssuchas phospho-rylcholine,sulfobetaine,andcarboxybetaine,whichcontainboth
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Surface
Science
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(2015)
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Post-print version
anionicandcationicgroups,havebeenusedasnon-fouling mate-rials. Unfortunately, grafting of the long chains of zwitterionic polymerscanresult in theformation of high-impedancelayers andsubsequentlycauselossestoelectrodesensitivities.However, short-chainmoleculesexhibitpoorstability[15–17].Goodingand co-workersinvestigatedzwitterionicphenylderivativelayers bear-ingchargedmoieties,suchas SO3−and N+(Me)3,depositedonto
glassycarbonsurfacesasanalternativetoOEGphenyllayersand observedthatthegraftedzwitterionicphenylderivativelayersoffer betterchemicalstabilityandashorterelectrontransferpathway tothetransducersurface[15].Zwitterionicphenyllayers,suchas OEGalkanethiolSAMs,havebeenproposedaseffectivealternatives toconventionalantifoulingmoleculesbecauseoftheirantifouling properties.Suchcoatingsdonotcompletelypassivatetheelectrode surfaceandthereforeallowFaradaicelectrochemistry.Hence,they canbeutilizedinbiosensingapplications.
Indiumtinoxide(ITO)isabiosensingsubstrateofincreasing importanceinthefieldofbiomaterialsbecauseofitscombined propertiesoftransparency,conductivityandbiocompatibility[18]. Veryfewstudieshaveexaminedtheuseofantifoulingmolecules onITOforbioassaycontexts.Todate,onlyhydrophilicpolyacrylic acidbrushes[19]andzwitterionicpolymerbrushes[20]havebeen investigatedasantifoulingagents.However,inthesestudies,only BSA–FITCwasusedtoevaluatetheantifoulingcapability.Theuseof onlyasingleproteinmaynotberepresentativeofcomplexmedia, suchasbloodplasmaandserumthatcontainavarietyofproteins. Moreover,adifferentstudygraftedzwitterionicpolymersontothe ITOusing surface-initiatedatomtransferradicalpolymerization (SI-ATRP).Althoughtheobtainedorganiccoatingswereconsidered stableinaqueoussolution,thetechniquewastimeconsumingand hadmoderatethroughput[21].Theliteraturecontainsnoaccounts of research related to the capability of these antifouling moi-etiesaftertheyhavebeenmodifiedwithlinkermolecules,suchas 1,4-phenylenediamineor4-aminobenzoicacid.Alinkermolecule isrequiredfortheattachmentofbiorecognitionmolecules(e.g., antibodies,enzymes,DNA,or aptamers)closetothetransducer surface.
Theincorporation ofzwitterionicphenyllayerswithalinker moleculeisuseful for improvingtheselectivityof the biosens-inginterface,whichisresistanttothenonspecificadsorptionof proteins, andfor providing a shorter responsetime for specific analytedetection(e.g.,lowimpedanceduetoa shorterelectron pathway).Therefore,oneobjectiveofthisstudywastoanalyzethe electrochemicaldepositionbehaviorofOEGwhendepositedonto ITOelectrodescomparedwiththebehaviorsofasinglemolecule andtwomixedlayercoatingscomprising4-sulfophenyl(SP), 4-trimethylammoniophenyl(TMAP), 1,4-phenylenediamine (PPD), and4-aminobenzoicacid(BAPA):mixedlayers1(SP:TMAP:PPD) and mixed layers 2 (SP:TMAP:BAPA). The second objective of this study was to investigate the capability of an ITO surface modifiedwiththeaforementionedmoleculestorepelbotha nega-tivelychargedprotein(BSA–FITC)andapositivelychargedprotein (RBITC–Cytc).Theseexampleproteinswereusedtorepresent pro-teinscontainedinrealsamples.ITOwasmodifiedusingdifferent ratiosofantifoulingmolecules(SPorTMAP)tolinkermolecules (PPDorBAPA).Weexaminedtheantifoulingpropertiestowardthe nonspecificabsorptionoffluorescentlylabeledproteinsusing con-focallaserscanningmicroscopy(CLSM).Thethirdobjectiveofthis studywastocharacterizethemodifiedsurfacewiththelowest pro-teinadsorptionleveltobothBSA–FITCandRBITC–Cytcbyusing X-rayphotoelectronspectroscopy(XPS)toconfirmtheexistence ofSP,TMAP,PPDorPABAmoietiesonthegraftedITO.Thefinal objectiveofthisstudywastoexaminethesurfacemorphologyof thegraftedfilms,wherethesurfacedistributionofSP:TMAP:PPD couldbeindicated by NH-gold nanoparticlesand characterized usingfield-emissionscanningelectronmicroscopy(FE-SEM).
2. Experimentalmethods
2.1. Reagentsandmaterials
ITO coated glass slide (surface resistivity ≤7/sq, L×W× thickness 406mm×355mm×0.7mm, sheet) was purchased from (Sanyo, Japan). 4-sulfophenyl, 1,4-phenylenediamine, 4-aminobenzoic acid, sodiumnitrite (NaNO2), potassium chloride
(KCl), potassium dihydrogen phosphate (KH2PO4), potassium
phosphate dibasic (K2HPO4), hydrochloric acid (HCl),
potas-siumferricyanide(K3Fe(CN)6),hexamineruthenium(III)chloride
(Ru(NH3)6Cl3), sodium carbonate (Na2CO3) with 99% purity,
20nm gold nanoparticles, sodium bicarbonate (NaHCO3),
rho-damine B isothiocyanate, bovine serum albumin-fluorescein isothiocyanate, cytochrome c (from bovine heart) and 2-[2-(2-methoxyethoxy)ethoxy]acetic acid (OEG) were purchased from Sigma-Aldrich (USA). 4-Trimethylammoniophenyl (TMAP) was obtainedfromAcros(USA).Allsolutionswerepreparedusing Milli-Qwater(18M/cm)(Purelab,US).
2.2. ITOelectrodesurfacemodification
ITOsubstratesweresoakedand cleanedin anultrasonicator usingdichloromethaneandthen99%methanolfor10mineach, followedbytreatmentwith0.5MK2CO3ina3:1methanol:Milli-Q
watermixturefor30minundersonicationtoremoveanyresidual organiccontaminants.TheITOsubstrateswerethenrinsedwith copiousamountsofMilli-Qwater,driedandstoredinan nitrogen-filledcontainer[22]. Inthefirst experiment,theITOelectrodes weremodifiedwithonlya singlecompound:SP,PPD,BAPA,or TMAP.Inthesecondexperiment,theITOelectrodeswere modi-fiedwithacombinationofSP:TMAP:PPDatdifferentmolarratios: 1:1:0.37(mix1),1.5:0.5:0.37(mix2)and0.5:1.5:0.37(mix3).Inthe thirdexperiment,theITOelectrodesweremodifiedwitha combi-nationofSP:TMAP:PABAatdifferentmolarratios:0.8:1:0.2(mix1), 1:1:0.1(mix2)and0.5:1:0.2(mix3).Forthecontrolexperiment, OEGwasusedastheantifoulingmoleculeontheITOslides.The OEGwaspreparedbytheelectrodepositionofamixedlayerof 4-aminobenzoicacid(8mM)and1,4-phenyldiamine(2mM)onITO slides.Theelectrodeinterfacewasfabricatedusingcarbodiimide chemistry:welinkedtheOEGtotheCOOHmoietiesofPPDamine groupsbyincubatingthemodifiedsurfaceinanethanolsolution containing10mMOEGand40mMDCCfor6hatroom temper-ature.Inalloftheaforementionedexperiments,insitumethods wereusedinwhicharyldiazoniumcationswereelectrochemically reducedwithtwotimesthemolaramountofNaNO2in0.05MHCl.
Thesolutionwaspurgedwithnitrogengasfor20minbeforethe surfacemodification.Theelectrodepositionwasperformedusing cyclicvoltammetryat−0.6Vto0.2V;theelectrodeswerescanned for5cyclesat100mVs−1[23–25].
2.3. Surfacepassivationstudyviaelectrochemicalmeasurements
All voltammetry measurements were performed using an AutoLabIII potentiostat (Metrohm AutoLab, Netherlands) and a conventionalthree-electrodesystemcomprisinganITOworking electrode,aplatinumwireastheauxiliaryelectrode,andAg/AgCl (3.0MNaCl)asthereferenceelectrode.Allpotentialswerereported relativetotheAg/AgClreferenceelectrodeatroomtemperature. Thesurfaceblockingstudiesonthemodifiedsensinginterfacewere performedin two aqueoussolutions containing differentredox probes.[Fe(CN)6]4−/3−,[Ru(NH3)6]3+andCVwereusedtoprobethe
integrityofthefunctionalizedlayersontheelectrodesurfaces.The respectiveredox-activesolution(1mM)wascomposedof0.05M KCl,0.05MK2HPO4,and0.05MKH2PO4andwasadjustedtopH7.0.
3)werecharacterizedusingXPS.TheXPSspectrawereanalyzed usingtheCasaXPSsoftware(version2.3.15).
2.4. ConjugationofcytochromectorhodamineBisothiocyanate
ThefluorescentdyerhodamineBwasconjugatedwithCytc accordingtothemethodreportedbyGuietal.[26].Tenmilligrams of Cyt c was dissolved in 1mL of 0.1M sodium carbonate-bicarbonatebuffer(pH9.05),and0.25mLof10mg/mL RBITCin DMSOwassubsequentlyaddedtoobtainafivefoldmolarexcess ofCytcrelativetoRBITC.Thetubecontainingthemixture solu-tionwaswrappedwithaluminumfoilandincubatedinthedark for1hundercontinuousmagneticstirring.Themixturewas fil-teredthrougha0.22mporesizesyringefilter(non-sterile,13mm diameter; MerckMillipore,Germany), andtheclear conjugated solutionwassubsequentlytransferredtoanAmiconUltra-15 cen-trifugalfilterdevice(3kDaMWcut-off,EMDMillipore,USA).The cappedfilterdevicewasplacedintheswingingbucketrotorofa centrifugeandspunfor10minat4000rpm.Theseparated solu-tionintheupperreservoirofthefilterdevicecontainedRBITC–Cyt c,whichwasthencharacterizedbyUV–visandfluorescence spec-troscopy.TheRBITC–Cytcsolutionwasstoredat−20◦Cuntiluse. The functionalized ITOslides were stored in clean Petri dishes beforetheantifoulingstudy.
For the antifouling study, the modified ITO electrode was immersedinanadequateamountofeither1mg/mLBSA–FITC(an exampleofanegativelychargedprotein)or2mg/mLRBITC–Cyt c(anexampleofa positivelychargedprotein);both immersion solutionswerepreparedusingPBSbuffer,pH7.4.ThePetridishes werekeptindarknessfor1h, andtheslidesweresubsequently transferredtoanotherPetridishthatcontainedonlyPBS.TheITO slides were soaked for 7minto remove weakly adsorbed pro-teinsfromthesurfacesoftheslides.Afterward,theslideswere washedwithMilli-Qwatertoremoveanyremainingresidues.The fluorescent-labeled-protein-adsorbedsurfacesweredried under flowingnitrogen.Thesamplesweremountedwith50%aqueous glycerolsolutionandcoveredwith22×60mm2microscopecover
slides (SuperiorMarienfeld,Germany). Thedried sampleswere mountedface-downontoacoverglass(22×60mm2,No.1,Paul
Marienfeld,GmbH&Co.KG,Germany) with50% aqueous glyc-erol solutionasthemounting agent.For eachtype of modified surface,threereplicatesweretested.Fiveimageswerecaptured under10×magnificationforsurveystudies.Fifteenimageswere capturedunder 63× magnification, and theirmeangray values werecalculatedtodeterminethefluorescenceoftheadsorbed pro-teins.Themeangrayvalueswerecomparedwithdifferentsurface types.
2.5. Surfaceantifoulingstudyviaconfocallaserfluorescent microscopeimaging
BSA–FITCand RBITC–Cyt cproteinsadsorbedonto function-alized electrode surfacesweremeasured usinga confocal laser fluorescence microscope (Leica Microsystems Pty, Ltd., USA) equipped withan argon laser (at 29% power) and operated in thexyzscanmode(stepsize0.04m).Images(1024×1024px2)
werecapturedat8-bitresolutionusingtheLasAFsoftware (Ori-gin).Theimagingwascarriedoutusingtwoobjectivelenses:an HCXPLAPOCS10.0×0.40 DryUV andanHCXPLAPOlambda blue63.0×1.40OILUV.Thegrayvaluerepresentsthefluorescence intensity.CapturedTIF-formatimageswerefurtheranalyzedusing ImageJ(IJ-1.48g.jar)softwaretodeterminethemeangrayvalue afterbackgroundsubtraction.Forreference,theemission band-widthofBSA–FITCrangesfrom506nmto575nm.Inthecaseof RBITC–Cytc,thebandwidthrangesfrom524nmto594nm.
2.6. ITOsurfacecharacterizationviaXPSanalysis
SpectrawereobtainedusingXPS(AXISUltraDLD spectrome-terwithahemisphericalanalyzer,amultichanneldetectoranda monochromaticAl-K␣source;1486.6eV).Spectrawere accumu-latedatatake-offangleof90◦witha0.9mm2spotsizeatapressure
oflessthan1×10−8mbar.Surveyscans(0–1000eV)werecarried
outata1.0eVstepsize,100msdwelltime,and100eVanalyzer passenergy.High-resolutionscans(S2p,C1s,N1s)wereperformed usinga0.1eVstepsize,100msdwelltime,and20eVpassenergy. Bindingenergiesofelementswerecorrectedwithreferencetothe C1speakofgraphiticcarbon(284.4eV).
2.7. ITOsurfacecharacterizationviaFE-SEManalysis
Fourdifferenttypesofsurfacesweremorphologically charac-terizedusingaHitachi SU8000field-emissionscanningelectron microscope(Tokyo,Japan):(1)bareITO,(2)ITO/goldnanoparticles (GNPs),(3)ITO/SP:TMAP:PPD/GNPs,and (4)ITO/SP:TMAP:PABA. Theslidesweretestedatslowscanspeedslessthan2kVandat 50,000×magnification. Theworkdestinationwas2.1mmatan acceleratingvoltageof2kV.GNPswereusedtoindicatethe pres-enceof PPDterminalaminegroups.GNPswereimmobilizedby immersingITO/SP:TMAP:PPDandbareITOinaGNPsolutionfor 3hatroomtemperature[24].Energy-dispersiveX-ray(EDX)was usedtocharacterizethegraftedGNPs.
3. Resultsanddiscussion
3.1. Reductiveadsorptionandelectrochemicalmeasurementof individualaryldiazoniumcationsandtheirmixtures
The reductive adsorption peaks of SP (10mM) and PABA (10mM)appearedat−0.473Vand−0.10V,respectively.Acyclic voltammogram of SP diazonium cation solution exhibited an irreversiblecathodicpeaklocatedat−0.47V(Fig.1(1a)),similar totheCVresultspreviouslyreportedforglassycarbon surfaces
[27,28].AtypicalcyclicvoltammogramofPABAdiazoniumcation
solution showed a single irreversible cathodic peak at −0.10V (Fig. 1(1d)). The cyclic voltammograms for bare ITO in 1mM [Fe(CN)6]4−/3− showeda pairof well-definedredoxpeaks.
Elec-trodesurfacesmodifiedwitharyldiazoniumcations,SPandPABA showed good passivation toward the penetration of negatively chargedredox-activespecieswhenthemodifiedsurfacewastested in [Fe(CN)6]4−/3−. However, these surfaces showed weak
pas-sivationpropertiestowardthepenetrationofpositivelycharged redox active species when the surfaces were tested in 1mM [Ru(NH3)]63+.Thisweakpassivationwaslikelyaconsequenceof
thenegativelychargedlayerthatformedontheITOelectrode sur-face,whichrepelled[Fe(CN)6]4−/3−butallowedthepenetrationof
[Ru(NH3)]63+fromthesolutiontotheITOelectrode surface.An
opposite effect wasobservedwhen theITO wasmodified with TMAP(Fig.1(1b)).Thispositivelychargedaryldiazoniumcation withareductiveadsorptionpeakat−0.26Vallowed[Fe(CN)6]4−/3−
topenetratethroughthelayerbutprevented[Ru(NH3)6]3+from
Fig.1.Cyclicvoltammogramsof(1)reductiveadsorption;(2)surfacepassivationstudytestedin1mM[Fe(CN)6]3−/4−before(blue)andafter(red)surfacemodification;
(3)surfacepassivationstudytestedin1mM[Ru(NH3)6]3+beforeandaftersurfacemodificationfor(a)SP;(b)TMAP;(c)PPD;(d)BAPA;(e)mixedlayerofSP:TMAP:PPD; (f)mixedlayerofSP:TMAP:PABA;(g)mixedlayerof4-aminobenzoicacidand2-[2-(2-methoxyethoxy)ethoxy)aceticacid(OEG).(Note:theconditionusedforITOsurface modificationwas10mMaryldiazoniumcation,20mMNaNO2,0.5MHCl,10cyclescansofCVat100mV/s).(Forinterpretationofthereferencestocolorinthisfigurelegend, thereaderisreferredtothewebversionofthisarticle.)
PPDattachmentontheglassycarbonsurfacehasalsobeenreported
[30].ThisobservationmaybeduetoPPDhavingthefastest diffu-sionrateandhavingthelowestmolecularweightamongthearyl diazoniumcationsusedinthisstudy[31].
Thereductive-adsorption cyclic voltammogramsof the mix-tureofSP,TMAPandPPDshowedreductiveadsorptionpeaksat approximately−0.35Vand−0.1V.Thepeakpotentialforthe mix-ture(SP:TMAP:PPDis more positive thanthat required for the adsorptionofanindividualaryl diazoniumcation:SP(−0.47V), TMAP(−0.26V)andPPD(−0.36V).Thisshiftinthepeakpotential suggests that, in mixed layers, the energy barriers for reduc-ing diazonium cations may have decreased. A decrease in the energybarrierlikelyoccurredbetweenTMAPandPPDreductive peakstoyielda singlepeak at−0.1V. Thefirstpeakat−0.35V maybe attributedtoSP diazoniumwith a smallpositive shift. Smallerreductive peak sizeswithbetterpassivation properties wereobservedforthismixtureontheITOsurfacecomparedwith surfacesmodified witheitherSPorTMAPalone. Similarresults werealsoreportedbyauthorsofotherstudiesofglassycarbon surfaces[26,27](Fig.1(1e)).
Thereductive-adsorption cyclic voltammogramsof the mix-tureofSP:TMAP:PABAshowedasinglereductiveadsorptionpeak atapproximately−0.13V,which waslikely associatedwiththe reduction of PABA (Fig. 1(1f)). The dominance of PABA onthe ITOsurfaceisattributedtoitslowernegativereductivepotential (−0.1V)comparedtothoseofSP(−0.47V)andTMAP(−0.26V). Thepassivationofthemodifiedelectrodesurface(fromthe sec-ondcycleofmodifications)mayindicatethattherateofradical depositionontheITOsurfacewasmuch higherinthis mixture comparedwiththatonITOsurfacesmodifiedwithsinglespecies ofradicals[27].Thereductive-adsorptionvoltammogramsofthe mixtureofPABAandPPDshowedsinglereductiveadsorptionpeaks atapproximately−0.5V.ThepassivationoftheITOelectrode sur-facewasachievedafter5cyclesofsurfacemodification.Themixed layerofthePABA-andPPD-modifiedITOsurfacewasfurtherlinked
withtheOEGaryldiazoniumcation(Fig.1(1g)).ThePABA-and PPD/OEG-modifiedITOsurfacewastestedinboth[Fe(CN)6]4−/3−
and [Ru(NH3)6]3+ redox-active solutions (1mM,pH 7).A
mod-erate reduction in the cathodicand anodic peaks, respectively, wasobserved.Thisreductionmaybeduetothepassivationeffect ofOEG,which is usedinbiomedical applicationstoprovidean inertsurface[32](Fig.1((2g)and(3g)).Weexpectedthatgrafted ITOsurfaceswithSP:TMAP:PPDwouldrepelboth[Fe(CN)6]4−/3−
and[Ru(NH3)6]3+becauseoftheoverallneutralchargeofthese
mixturesonITOsurfaces.InthecaseofSP:TMAP:PABAmixtures, moderatepassivationoftheITOelectrodeandreducedintensities ofcathodicandanodicpeakswereobservedinthevoltammograms ofthe[Fe(CN)6]4−/3−testsolutions.However,nosucheffectswere
observedin thevoltammograms of[Ru(NH3)6]3+ test solutions.
Theseresultsare attributabletothecoating of ITOwitha thin SP:TMAP:PABAlayer,whichhasamoderateresistanceto inner-sphereelectron-transferringredoxprobes(e.g.,resistancetothe transferofanelectronthroughtheformationofsignificant cova-lentbondingbetweenthetwospeciesinaredoxchemicalreaction), suchas[Fe(CN)6]4−/3−butaminimalbarriereffecttoredoxprobes
thatperformouter-sphereelectrontransfers(e.g.,withoutcovalent bonding),suchas[Ru(NH3)6]3+.Therefore,outer-sphereelectron
transfersarenotaffected.
3.2. AntifoulingstudybyCLSM
3.2.1. ITOsurfacemodifiedwithamixtureofSP:TMAP:PPD
Fig.2. Confocallaserscanningmicroscopy(CLSM)imageunder63×magnificationfrom(a)FITC–BSAand(b)RBITC–Cytcadsorbedondifferentsurfaces.(c)Comparisonof intensityofFITC–BSAadsorbedondifferentsurfaces.(d)ComparisonofintensityofRBITC–Cytcadsorbedondifferentsurfaces.
studywasthattheITOsurfacemodifiedwithSP:TMAP:PPDwas capableofrepellingbothBSA–FITCandRBITC–Cytc,asindicated bythelowfluorescenceintensities.(Notethatpercentagesof flu-orescentadsorptionsfordifferentITOsurfaceswerecalculatedon thebasisofthemeangrayvalue,whereallvalueswerenormalized totheITOelectrodemodifiedwithPPD:PABA/OEG.)
Thefluorescenceintensitypercentagefor adsorbedBSA–FITC onSP:TMAP:PPD-modifiedITOsurfacesfor mixtures 1,2and 3 werelowerthanthatforITOfunctionalizedwithPPD:PABA/OEG. However, mix 3 exhibited the greatest resistance to BSA–FITC adsorption at 63% (a mean gray value of 1.4, s=0.7 and n=3) compared with the 100% absorption of BSA–FITC in the con-trol experiment (a mean gray value of 2.2, s=0.3 and n=3), whichinvolvedanITOsurfacefunctionalizedwithPPD:PABA/OEG
(Table1).TheITOmodifiedwithSP:TMAP:PPDmixtures1,2or3
alsoexhibitedexcellentresistancetononspecificprotein adsorp-tion.Inthisstudy,mixture3exhibitedthebestresistancebecause thefluorescenceintensitypercentagefortheamountofadsorbed RBITC–Cytconmix3wasonly6.9%(ameangrayvalueof0.15, s=0.05andn=3)comparedwiththe100%absorptionofRBITC–Cyt cofthecontrolexperiment(ameangrayvalue2.1,s=0.4andn=3), whichinvolvedfunctionalizedITOmodifiedwithPPD:PABA/OEG (Fig.2).
Interestingly,thebareITOexhibitedthebestcapabilitytorepel BSA–FITC,withafluorescenceintensitypercentageof35.6%anda meangrayvalueofonly0.81(s=0.2,n=3).Incontrast,thebare ITOexhibited a highfluorescenceintensity of1.9 (s=0.3,n=3)
Table1
ThepercentageofBSA–FITCandRBITC–Cytcadsorptionatdifferentsurfaces repre-sentedbythenormalizedfluorescenceintensitiesrelativetoITOelectrodemodified withPPD:PABA/OEG.
Surfacetype Thepercentageofthe fluorescentintensity foradsorbedBSA–FITC
Thepercentageofthe fluorescentintensityfor adsorbedRBITC–Cytc
BareITO 35.68 87.89
ITO/PPD 109.54 16.45
ITO/SP:TMAP:PPDwith molarityratioof 1:1:0.37(mix1)
75.94 16.10
ITO/SP:TMAP:PPDwith molarityratioof 1.5:0.5:0.37(mix2)
95.62 15.86
ITO/SP:TMAP:PPDwith molarityratioof 0.5:1.5:0.37(mix3)
63.33 6.99
ITO/PABA 175.64 42.68
ITO/SP:TMAP:PABA withmolarityratioof 0.8:1:0.2(mix1)
132.14 64.82
ITO/SP:TMAP:PABA withmolarityratioof 1:1:0.1(mix2)
104.58 68.45
ITO/SP:TMAP:PABA withmolarityratioof 0.5:1:0.2(mix3)
86.72 50.52
Table2
XPSatomicratiocomparisonofS,C,NbetweenSP:TMAP:PPD–ITOandSP:TMAP:PABA–ITO.
Corelevelpeaks S2p C1s N1s
S1 C1 C2 C3 N1 N2
SP:TMAP:PPD 3.43 12.1 25.7 – 0.64 2.75
SP:MAP:PABA 0.41 18.9 8.5 2.9 1.0 0.6
Fig.3. X-rayphotoelectronspectra(XPS)ofSP:TMAP:PPDmodifiedITOsurfaceswithmolarityratioof0.5:1.5:0.37(mix3).Thesurveyspectrawithmolecularstructureof S2p,C1sandN1score-levelspectra.
afteradsorbingRBITC–Cytc.Thisobservationisexplainedbythe chemicalcompositionoftheITOsurfaces,whichconsistof neg-ativelycharged complexes of stoichiometricoxide, hydroxides, oxyhydroxides,andphysisorbedindiumhydroxides.Hence,these negativechargesmayrepelBSA–FITCbutincreasetheadsorption ofpositivelychargeproteinssuchasRBITC–Cytc[22].
In conclusion, a surface modified withzwitterionicor simi-larmoleculesexhibitedbetterantifoulingpropertiesinrepelling BSA–FITC and RBITC–Cyt c compared with ITO/PPD:PABA/OEG. This better antifouling behavior may be due to good packing
and charge balancing of zwitterionic groups on the ITO modi-fiedwithSP:TMAP:PPDmixtures.TheITOsurfacemodifiedwith SP:TMAP:PPD(mix3)showedthebestresistancetononspecific adsorptionsofBSA–FITCandRBITC–Cytccomparedwithmix1and mix2.Weattributethedifferencesintheantifoulingcapabilityof SP:TMAP:PPDmixturestotheelectrostaticinteractionbetweenthe threemoleculesusedtofabricatetheinertsurfaces.Atthisratio,the electrostaticinteractionbetweenthepositivelychargedtailgroup (TMAP:PPD)andthenegativelychargedtailgroup(SP)appearto providethebestelectricallyneutralelectronicstructure.
Fig.5.E-SEMimageof(a)ITO/GNP.(b)ITO/SP:TMAP:PPD/GNPwithmolarityratio(0.5:1.5:0.37).(c)ITO/SP:TMAP:PABAwithmolarityratio(0.5:1:0.2)undermagnification 50K.(d)ITO/SP:TMAP:PABAandbareITOsurfaceundermagnification1.5K.(e)EDXspectraITO/SP:TMAP:PPD/GNP.
3.2.2. ITOsurfacemodifiedwithamixtureofSP:TMAP:PABA ITOsurfacesweremodified withSP:TMAP:PABA atdifferent molarityratiosof0.8:1:0.2(mix1),1:1:0.1(mix2)and0.5:1:0.2 (mix3).Mix3exhibitedthebestantifoulingcapabilitiesamong thethreecombinationsagainstbothBSA–FITC(86.7%fluorescent intensity,ameangrayvalueof1.96;s=0.5,n=3)andRBITC–Cyt c(50.5%fluorescentintensity,a mean grayvalueof 1.1;s=0.4, n=3) and compared with the ITO surface functionalized with PPD:PABA/OEG(100%BSA–FITCfluorescentintensity,ameangray valueof2.2;s=0.3,n=3;100%RBITC–Cytcfluorescentintensity,a meangrayvalueof2.1;s=0.4,n=3;Fig.2;Table1).
TheITOelectrodemodifiedwithPPD:PABA/OEGaccumulated asignificantamountofRBITC–Cytc.Itexhibitedthehighest fluo-rescenceintensityamongtheinvestigatedsurfacetypes(Fig.2(d)). RBITC–Cytchastheabilitytochangeitsconformationupon adsorp-tionontoawiderangeofinterfaces[33,34].Furthermore,thefailure ofOEGmoietiestoresistthenonspecificadsorptionofundesired proteinsmaybeduetotheauto-oxidationofOEGmoieties[15,35], whichmakesthePPD:PABA/OEGlayerunabletorepelBSA–FITC andRBITC–Cytc.Antifouling coatingalternativesthat aremore effectivethanOEGshouldthereforebeidentified.
ITO modified with SP:TMAP:PPD mixtures exhibited better resistancetothenonspecificadsorptionofBSA–FITCandRBITC–Cyt ccomparedwithSP:TMAP:PABA.Thisbetterresistancemaybedue tothedominationofPABAmoleculesinSP:TMAP:PABAmixtures (Fig.1(1f)).Theadsorptionofaryldiazoniumcationmixtureswas dominatedbythearyldiazoniumcation,whichwaseasiertoreduce (e.g.,havingalessnegativepotential)[36];suchbehavior subse-quentlyreducedadsorptionsoftheothertwomolecules(e.g.,SP andTMAP,whichwerenecessaryforaneutralchargeinterface.In theSP:TMAP:PPDmixture,nosuchdominationwasobservedfor thePPDmolecule.XPSanalysisshowedthattheatomicratiofor SPandTMAPwashigherinSP:TMAP:PPDthaninSP:TMAP:PABA mixtures(Table2).
IncontrasttoPABA,PPDcouldbeusedasalinkermoleculeand beincorporatedwithzwitterionicmolecules(e.g.,SPandTMAP) toresistthenonspecific adsorptionofBSA–FITC and RBITC–Cyt c.PPDwasmorereadilydepositedontotheITOsurface,andthe completepassivationoftheelectrodesurfacerequiredonlytwo cycles(Fig.1(1c)).WithPABA,thepassivationwassignificantlyless
efficient,requiring10cycles(Fig.1(1d)).PPDalonecanresistthe nonspecificadsorptionofBSA–FITCandRBITC–Cytc(Fig.1,Table1). Suchbehaviorenhancedtheantifoulingefficiencyofzwitterionic molecules(e.g.,SPandTMAP).Moreover,thereductiveadsorption potentialforPPDwas−0.36V,which,incomparisontothe poten-tialfor PABA(−0.1V),wasclosertoreductiveadsorptionsofSP (−0.473V)andTMAP(−0.26V).Therefore,aryldiazoniumcations withsimilarreductiveadsorptionpotentialspreventedthe domi-nationofonearyldiazoniumcationovertheother,whichaidedthe fabricationofaneutrallychargedantifoulingcoating.
3.3. ITOsurfacecharacterizationviaXPSanalysis
ForITOsurfacesmodifiedwithSP:TMAP:PPD(mix3),the pres-enceofSPwasconfirmedbyasignalpeakindicativeofsulfur(S2p) at167.9eV[27,28].Thissignalpeakwasabsentinthenarrowscan oftheS2pregionforbareITO(Fig.3).TMAPwasdetectedbythe presenceofaC-Nlinkagepeak,whichrepresentsthelinkbetween nitrogenand4-methylgroups,atabindingenergyof285.0eV(C2). Anotherpeak(C1)centeredat284.4eVinanarrowscan ofthe carbonregionrepresentedaromaticcarbonsinSP,PPDandTMAP moieties[27,30].PPDwascharacterizedbytheN1399.5eVpeak observedinthenarrowscanoftheN1sregion,whichisattributed
toH2N C[23–25,37].Incontrast,XPSsurveyspectraforthemixed
SP:TMAP:PABAlayer(Fig.4)revealed a peakat 167.8eV corre-spondingtoS2p,whichindicatedanSPgraft[23,27,28].Anarrow scan ofthecarbon C1sregionrevealedthree peakscenteredat 284.7eV(C1),286.1eV(C2),and288.5eV(C3).TheC1andC2peaks wereattributedtoaromaticcarbonatomsandtoN (CH3)3inthe
trimethylammoniogroup,respectively.TheC3peakcorresponded toCOO-;thepresenceofthispeakindicatesthesuccessfulgrafting
ofPABAmoleculesontotheITOsubstrate[23,38,39].
3.3.1. ITOsurfacecharacterizationviaFE-SEManalysis
onlyformedwiththepresentofPPD.TheEDXresultsconfirmed thedepositionofGNPsontomodifiedITOelectrodesurfacesatan atomicratioof2.11(Fig.5(e)).Thelinkageofaminesinthemixture ofSP:TMAP:PPDtoGNPswasmorelikelytooccurby dehydrogena-tionfromNH-GNPbecauseofitsstrongercovalentbond[24,40]. Suchobservationsconfirmedthehomogenousgraftingofthelinker molecule(PPD) ontotheITOsurface, unliketheSP:TMAP:PABA mixture,whichwasdominatedwithPABAmolecule(XPSresults areshowninTable2).TheimageofanSP:TMAP:PABA-modified ITOelectrodeshowedathin,smoothandfeaturelessmorphology indicatesthatnoGNPscouldbedepositedontothistypeofsurface (Fig.5(c)).Anobviousdifferencewasnoticedinimagesbetween thebareandSP:TMAP:PABAmodifiedITOsurfaceswhichcaptured at1500×magnification(Fig.5(d)).
4. Conclusions
A biosensing interface consisting of antifouling and linker moleculesbasedonin-situ-generatedaryldiazoniumcationswas described.Thedifferencesinelectrochemicalbehaviorsamongall typesofmodifiedsurfaces(eitherwiththeindividualcompound ora mixed layer of aryl diazonium cations) were investigated and evaluated. An antifouling study using confocal laser scan-ningmicroscopy showed that ITO surfaces coated with mixed layers of SP:TMAP:PPD 0.5:1.5:0.37 (mix 3) or SP:TMAP:PABA 0.5:1:0.2(mix3)exhibitedbetterantifoulingcapabilitiesagainst bothBSA–FITCandRBITC–CytccomparedtoITOsurfacescoated with PPD:PABA/OEG. However, PPD:PABA/OEG-coated ITO sur-facesshowedloweramountsofBSA–FITCadsorbedcomparedto SP:TMAP:PABA-coatedITOsurfaces.ThemixtureofSP:TMAP:PPD showedbetterresistancetononspecificadsorptionsofBSA–FITC and RBITC–Cyt ccompared to SP:TMAP:PABA.Analyses of XPS surveyspectrafurtherconfirmedthedepositionofSP:TMAP:PPD andSP:TMAP:PABAontoITOsurfaces.FE-SEMandEDXanalyses showeddenseandhomogenousdistributionsofGNPs,which indi-catedagooddistributionoflinkerandantifoulingmoleculeson theITOsensinginterfaces.Thisstudyhighlightstheprospective useofaryl-diazonium-cation-derivedzwitterioniccompoundsand linkermoleculesfortheeasyfabricationofITObiosensing inter-faces.Theseinterfacescanrepelundesirableanionicandcationic proteinsanddetecttargetanalytesinhighlycomplicatedbiological matrices,suchasblood,serum,andurine.
Acknowledgments
ThisworkwasfinanciallysupportedbytheUniversityofMalaya ResearchGrant(UMRG)(RG159-12SUS,RP012C-14SUS),the Fun-damental Research Grant Scheme (FRGS) from the Ministry of Higher Education of Malaysia (MOHE) (FP014-2013A), a High ImpactResearchGrantfromtheMinistryofHigherEducationof Malaysia(HIR-MoHE F000004-21001),andUniversityofMalaya PostgraduateResearchGrant(PG120-2012B).
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