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Elaboration of integrated microelectrodes for the
detection of antioxidant species
Céline Christophe, Fadhila Sekli-Belaidi, Jérôme Launay, Pierre Gros,
Emmanuel Questel, Pierre Temple-Boyer
To cite this version:
Céline Christophe, Fadhila Sekli-Belaidi, Jérôme Launay, Pierre Gros, Emmanuel Questel, et al..
Elab-oration of integrated microelectrodes for the detection of antioxidant species. Sensors and Actuators
B: Chemical, Elsevier, 2013, vol. 177, pp. 350-356. �10.1016/j.snb.2012.11.032�. �hal-00783050�
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: DOI:10.1016/j.snb.2012.11.032
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Eprints ID: 8030
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Christophe, Céline and Sekli-Belaidi, Fadhila and Launay, Jérôme and
Gros, Pierre and Questel, Emmanuel and Temple-Boyer, Pierre
Elaboration of integrated microelectrodes for the detection of antioxidant
species.
(2013) Sensors and Actuators B Chemical, vol. 177 . pp. 350-356.
ISSN 0925-4005
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Elaboration
of
integrated
microelectrodes
for
the
detection
of
antioxidant
species
C.
Christophe
a,b,d, F.
Sékli
Belaidi
a,b,c, J.
Launay
a,b, P.
Gros
c,
E.
Questel
d,
P.
Temple-Boyer
a,b,∗aCNRS,LAAS,7avenueducolonelRoche,F-31400Toulouse,France bUniversitédeToulouse,UPS,LAAS,F-31400Toulouse,France
cUniversitédeToulouse,LaboratoiredeGénieChimiqueUMRCNRS5503,UniversitéPaulSabatier,F-31062Toulouse,France dPierreFabreDermoCosmétique,CentredeRecherchesurlaPeau,31025Toulouse,France
Keywords: Thin-filmmicroelectrodes SU-8passivation Electrochemicalproperties Antioxidantspecies Oxidativestress
a
b
s
t
r
a
c
t
(Pt–Pt–Ag/AgCl)and (Au–Pt–Ag/AgCl)electrochemicalmicrocells(ElecCell) weredeveloped forthe detectionofredoxspeciesbycyclicvoltammetry.AspecialemphasiswasplacedontheSU-8 wafer-levelpassivationprocessinordertooptimizetheelectrochemicalpropertiesofthedifferent“thinfilm” metalliclayers,i.e.goldorplatinumfortheworkingelectrode,platinumforthecounterelectrodeand silver/silverchlorideforthereferenceelectrode.(Au–Pt–Ag/AgCl)microcellswereappliedforthe detec-tionofantioxidantspeciessuchasascorbicanduricacidsinphosphatebuffersolution,evidencinghigh sensitivitybutlowselectivity.Workswereextendedtoskinanalysis,demonstratingthatagood electri-calcontactwiththeskinhydrolipidicfilmallowedtheeffectiveevaluationoftheskinglobalantioxidant capacity.
1. Introduction
Electrochemistry provides an appropriate platform for the development of many analytical techniques in different fields including clinical biology, food industry or environment [1–5]. Indeed,electrochemicalproceduresprovidemanyadvantagessuch aseasyuse,lowcost,fastanalysis,aswellashighdetection sen-sitivityandselectivity[6].Researchworkswerethusdedicatedto developingtheelectrochemicaltheoryatamicroscalelevel, study-ingbioelectrochemicalsensitivelayerspropertiesandconstructing micro/nanoelectrodes. Due to theseworks, an improvement in electroanalyticalmethodswasachieves,consequentlyleadingto thedetectionofmanychemical,biochemicalandbiologicalredox species in complex media [7,8]. This is for instance the case for the evaluation of oxidative stress and the assay of specific antioxidantspeciesonskin.Oxidativestressisknowntobe respon-siblefortheproductionofreactiveoxygenspecies(ROS)[9–12]. Facetopro-oxidativeenvironments(atmosphere,pollutants,UV irradiations,pathogenicbacteria...),skinhasdevelopeddifferent strategies toprotect itself[9,13].Oneof them isrelated tothe productionofhydrophilicantioxidantspeciessuchasglutathione as well as ascorbic and uric acids. As a result, concerning the skinglobalantioxidantcapacity,thedetectionofthesedifferent moleculesbecameaveryimportantissuenotonlyforbiological
∗ Correspondingauthorat:CNRS,LAAS,7avenueducolonelRoche,F-31400 Toulouse,France.
E-mailaddress:temple@laas.fr(P.Temple-Boyer).
researchesbutalsoforroutineanalysis.Recently,non-integrated ultra-microelectrodes were developed and successfully applied to the skin analysis [14–16]. Nevertheless, in order to further improveskinelectrochemicalanalysis,microelectrodesfabrication processhad tobeimproved in ordertoensureminiaturization, massfabrication,reproducibility, reliability andeasiness ofuse. In order to obtain these specifications and develop integrated electrochemicalmicrocellscomprisedofathree-microelectrodes system,twodifferenttechnologieswereproposed.Firstly, screen-printing techniques were studied for the fabrication of “thick films”electrochemicalsensors[7,17].Unfortunately,theirspatial resolutionisnotefficientenoughtoreachmicroscalelevel. Sec-ondly,technologiesderivedfrommicroelectronicsweredeveloped [18]. By introducing photolithographic processes, silicon-based technologiesweresuccessfullyusedformassfabricationofthin metallic micro/nanoelectrodes and integration of electrochemi-calmicrocellsondifferentsubstrates(silicon,glass,polymers...) [19–23],dealingwithnumerousapplications:chemicaland bio-chemicaldetection[24–27],immunodetection[28],microfluidic electroanalysis [29–32], neurosciences [33,34],... Nevertheless, theso-fabricatedthinmetalliclayershavestilltobestudiedinorder tocontroltheirintrinsic electrochemicalcharacteristicsandthe relatedelectrochemicalmicrocellshavetobeoptimizedinorder tobefullycompatibleforskinelectrochemicalanalysis.
Thispaperdealswiththemassfabricationofintegrated, three-electrode (Pt–Pt–Ag/AgCl) and (Au–Pt–Ag/AgCl) electrochemical microcellsusingsilicon-basedmicrotechnologiesandtheir appli-cation in the detection of antioxidant species. To begin with, electrochemicalcharacteristicsofdifferentthinmetalliclayers,i.e.
Fig.1.Schematicsofthe(Au–Pt–Ag)electrochemicalmicrocell.
gold,platinum and silver, were studied.Then, (Au–Pt–Ag/AgCl) electrochemicalmicrocellsareusedforthedetectionofascorbic anduricacidsinvarioussolutions.Finally,theiruseisextendedto theskinanalysis,focusingonthewafer-levelpassivationprocess improvementandexaminingtheskinglobalantioxidantcapacity measurement.
2. Experimental
2.1. Electrochemicalmicrocellsfabrication
(Pt–Pt–Ag/AgCl)and (Au–Pt–Ag/AgCl)electrochemical micro-cells (ElecCell) were fabricated on oxidized (oxide thickness: ∼1mm) silicon wafers. The different thin metallic layers were depositedbyevaporationinconventionalphysicalvapour depo-sition (PVD) equipments, and patterned using a bilayer lift-off processinordertoimprovefabricationreproducibility.ThreePVD processeswereperformedinarow:firstly,a200nmplatinumlayer wasdepositedona20nmtitaniumunderlayerinordertoensure platinum adhesiononsilicon oxide,followed by a800nmgold anda400nmsilverlayer.Thesedifferentthicknessesvalueswere choseninordertoenhanceadhesionpropertieswhilelimiting tech-nologicaldefectsrelatedtomechanicalstressandinter-diffusion phenomenaofmetallicatoms(Ti,Pt,AuandAg).
Finally,abiocompatibleSU-8passivationlayer(thickness:1.6 or3mm)wasdeposited atthewaferlevelusing photolithogra-phytechniques(Fig.1).SU-83005photoresist(purchasedfrom MicroChemCorporation)wasspin-coatedandasoftbakewasthen performedat95◦C.Thepolymercrosslinkingwasachieveddue toanultraviolet(UV)exposurewhileactivezonesweredefined usingaspecificphotolithographicmask.Apost-exposurebakewas performed at95◦C. Finally, after revealingthe different metal-licareas (Pt,Au,Ag)bythedevelopmentoftheactivezonesin
PGMEA(propylene glycol monomethyl ether acetate),an addi-tionalUVexposureandafinalhard-bakewereconductedtoensure theSU-8layercompletepolymerization.Thiswafer-level passiv-ationprocesswasusedtoinsulateelectricallythedifferentmetallic layersanddefinepreciselythedifferentactivesurfaces.Thegold orplatinumworkingmicroelectrodesweredefinedasdisksand theirelectroactiveareawasapproximately4.9×10−4mm2 (diam-eter:25mm).Incontrast,verylargesilver/silverchloridereference microelectrode(0.02mm2)andplatinumcountermicroelectrode (1mm2)werefabricated.(Pt–Pt–Ag)and(Au–Pt–Ag) electrochem-icalmicrodevicesweremanufacturedonsiliconchip(Fig.2).The wholechipwasthenplacedandgluedbyanepoxyinsulatingglue ona specificallycoated printed circuit, wire bondedand pack-aged at thesystem level using a silicone glop-top in order to befullycompatiblewithliquidphasemeasurement(Fig.2).For eachmicrodevice,thesilver/silverchlorideAg/AgClreferencewas finallyobtainedbyoxidizingthesilvermetalliclayerina0.01M KClsolution.Thisoxidationwasperformedbylinearvoltammetry (potentialscanrate:1mVs−1 between0.1and0.25V/SCE)using astandardsaturatedcalomelelectrode(SCE)Hg/Hg2Cl2/KClsatas reference.
2.2. Electrochemicalmicrocellscharacterization
All the electrochemical characterizations were held using a multi-channelVMPpotentiostatfromBiologic.Surfacesofthe“thin film” platinumand goldworkingmicroelectrodeswere electro-chemically characterized by cyclic voltammetryin a deaerated 0.5M H2SO4 solution (potential scan rate: 50mVs−1 between −0.25and+1.2V/SCE,andbetween−0.3and+1.6V/SCE respec-tively). Their active surface was then evaluated by plotting a current–potentialcurve(potentialscanrate:50mVs−1 between −0.5and+0.3V/SCE)inadeaerated,0.1M,pH7.0phosphatebuffer
Fig.3. Useofthe(Au–Pt–Ag/AgCl)electrochemicalmicrocellfortheskinanalysis.
solution(PBS)containing0.005MofferricyanideionsFe(CN)63−. Consideringasteady-stateregime,themicroelectroderadiuswas calculatedaccordingtotheSaïtorelation[35]:
Ilim=4nFDCr (1)
whereFistheFaradayconstant(F=96500Cmol−1),nisthe num-ber of electrons exchanged per mole (n=1), and C and D are theFe(CN)63− ionconcentrationand diffusioncoefficient,being estimated to5×10−3molL−1 and 7×10−6cm2s−1 respectively [36,37].
The electrochemical characteristics of the “thin-film” silver microelectrodeswerestudiedbycyclicvoltammetryinadeaerated 0.1MKNO3acid(pH3.5)solution(potentialscanrate:50mVs−1 between−1and+0.5V/SCE).Finally,theefficiencyandstabilityof theresultingAg/AgClreferencemicroelectrodewereevaluatedby monitoringtheNernstpotentialatequilibriumina0.01KCl solu-tionandcomparedwithastandard saturatedcalomelelectrode (SCE).
AllthechemicalreagentswerepurchasedfromSigma. 2.3. Electrochemicalcharacterizationofantioxidantspeciesin solutionandontheskin
Thewhole(Au–Pt–Ag/AgCl)ElecCellmicrosensorsweretested in an autonomous way, i.e. using the “threeintegrated micro-electrodes” configuration, for the detection of two antioxidant molecules:ascorbic(AA)anduric(UA)acids.Thiswasperformed bycyclicvoltammetry(potentialscanrate:50mVs−1between−0.2 and+0.8V)inaPBSpH7.0containingascorbicacidand/oruricacid (concentration:10−3M,purchased fromSigma). Theiranalytical performanceswerecomparedwithstandard“non-integrated”gold microelectrodepresentedinpreviouspapers[14,16].Forabetter comparisonof thedifferentamperometricresponses,the resid-ualvoltammogrammsobtainedinPBSwerededucedfromthose obtainedwithAAand/orUAspecies.
Finally, electrochemical characterization of antioxidant molecules was extended to skin analysis by applying directly the(Au–Pt–Ag/AgCl) ElecCellmicrosensors ontheforearmskin surface(Fig.3).
3. Resultsanddiscussion
3.1. Electrochemicalcharacteristicsoftheplatinumthinfilm (Pt–Pt–Ag/AgCl) electrochemical microcells were first char-acterized by cyclic voltammetry. Fig. 4 shows the cyclic voltammogramtypicallyobtainedwiththe“thinfilm”platinum working microelectrode (SU-8 thickness: 1.6mm) in a 0.5M deaeratedH2SO4 solution.Thecurve shape isrepresentative of polycrystalline platinum [38–40]: the oxidation and reduction
Fig.4. Cyclicvoltammogramobtainedwiththe“thinfilm”platinumworking elec-trode(SU-8thickness:1.6mm)inadeaerated0.5MH2SO4solution(potentialscan
rate:50mVs−1).
peaksrelatedtothePtH2andPtHspeciesappearedinapotential rangebetween−0.25and0V.Theplatinumoxidationbeganaround 0.6Vandthesolventoxidationwasaround1.2V.Thepeakpotential correspondingtothereductionoftheplatinumoxidespreviously obtainedattheelectrodesurfacewasfoundaround0.45V.
Cyclicvoltammogramsrecordedwiththe“thinfilm”platinum workingmicroelectrodeina deaerated0.005MFe(CN)63− solu-tionyieldedasteady-statediffusion-limitedcurrentevenwithout stirring the solution and with a relatively high potential scan rateof 50mVs−1 (figure not shown). Accordingto Eq. (1), the Ilim experimental value was approximately34nA current. This valuecorrespondedtoanelectroactivesurfaceofapproximately 5×10−4mm2,i.e.toaradiusaround25mm,whichwasagreement withtheworkingmicroelectrodedimension.
Inconclusion,the“thinfilm”platinumlayerexhibited electro-chemicalpropertiessimilartothoseusuallydemonstratedonsolid platinumaswellaswell-definedelectroactivearea.
3.2. Electrochemicalcharacteristicsofthegoldthinfilm
Similarexperimentswereperformedforthe(Au–Pt–Ag/AgCl) electrochemical microcells and the associated “thin film” gold microelectrode. Fig. 5 shows successive cyclic voltammograms recordedinadeaerated0.5MH2SO4 solutionfora1.6mm-thick SU-8 passivationlayer. The curves demonstrated that the gold
Fig.5.Cyclicvoltammogramsobtainedwiththe“thinfilm”goldworkingelectrode (SU-8thickness:1.6mm)inadeaerated0.5MH2SO4solution(potentialscanrate:
Fig.6. Cyclicvoltammogramsobtainedwiththe“thinfilm”goldworkingelectrode (SU-8thickness:3mm)inadeaerated0.5MH2SO4solution(potentialscanrate:
50mVs−1).
oxidationwavebeganat1Vandthatpeakcorrespondingtogold oxidesreduction appearedcloseto0.85V [41,42].Nevertheless, someotheramperometricsignalsarealsoobservedat−0.2and 1.3Vintheanodicpartofthecurveaswellasat0.3and−0.2V duringthecathodicscan.Allthesesignalsincreasedwiththe num-berofpotentialcycles(seeevolutionarrowsonFig.5).Sincethese unexpectedcurves werenotevidenced forthe“thin-film” plat-inummicroelectrode(seeSection3.1),theycanbeattributedto theAu/SU-8interfacephenomena.Ontheonehand,non-adhesion oftheSU-8filmongoldaswellaspoorstepcoverageduetothe Ti/Pt/Austackinghigherthickness(∼1000nmratherthan∼200nm for the Ti/Pt one) is responsible to passivation defects, allow-ingthetitanium/platinumunderlayertobeincontact withthe electrolyticsolution[43].Ontheotherhand,thesulphur-based tri-arisulfoniumsaltsoftheSU-8resinareelectrochemicallyactive onthegold surface[44].Thiswas confirmedbycyclic voltam-metryperformed onSU-8/cyclopentanone solutions (figure not shown).
Inordertoreducebothredoxphenomena,twodevelopments wereproposed.Firstly,theSU-8polymerizationprocesswas opti-mized in terms of exposure dose and annealing duration to counteractanyelectrochemicalinterference.Secondly, theSU-8 thicknesswasincreasedfrom1.6to3mmtoimprovestep cover-ageandavoidanycontactbetweenthemetallicunderlayersand theelectrolyticsolution.Fig.6showstheresultingcyclic voltam-mograminthesamedeaerated0.5MH2SO4solution.Thecurvewas verysimilartothattypicallyrecordedwithsolidgoldwhilethe cur-rentpeakscorrespondingtotheoxidationofgoldandthereduction ofgoldoxideswereonlyobservedinthiscase.ComparedtoFig.5, lowercurrentlevelswereevidenced,demonstratingthatabetter controlofthegoldmicroelectrodeelectrochemicalactiveareawas obtained.Thiswasconfirmedbycharacterizingbycyclic voltam-metrytheseoptimised“thinfilm”goldworkingmicroelectrodesin adeaerated0.005MFe(CN)63− solution(resultnotshown). Sim-ilarlytothe“thinfilm”platinummicroelectrode(Section3.1), a steady-statediffusion-limitedcurrentof32nAwasrecorded.As expected,accordingtoEq.(1),theworkingelectrodeelectroactive surfaceandradiuswereestimatedto4.3×10−4mm2and23.5
mm respectively.
So, by improving the SU-8 polymerisation process and by increasing the SU-8 passivation thickness from 1.6 to 3mm, “thinfilm”goldworkingmicroelectrodeswithgood electrochem-ical properties and well-defined active area, were successfully obtained.Finally,itshouldbementionedthattheincreaseinSU-8 thicknessfrom1.6to3micrometersdidnotinduceanydegradation
Fig.7.Cyclicvoltammogramofthe“thinfilm”silverelectrode(SU-8thickness: 3mm)inadeaerated0.1MKNO3solution(potentialscanrate:50mVs−1).
ofthe“thinfilm”platinumworkingmicroelectrodes(cf.Section 3.1).
3.3. StudyoftheAg/AgClreferencemicroelectrode
Cyclicvoltammogramstypicallyobtainedwiththe“thin-film” silver microelectrodes (SU-8 passivation thickness: 3mm)were recordedinadeaerated0.1MKNO3acid(pH3.5)solution(Fig.7). Typicalcurveswereobtained:thesilveroxidation startsaround 0.3V,theassociatedreductionwasclearlyshownaround0.4Vand thefinalprotonreductionisevidencedunder−0.5V.
Finally,thetemporalstabilityofthe“thinfilm”Ag/AgCl refer-encemicroelectrodewasstudiedbymeasuringitspotentialusing theSCE referenceelectrode in a 0.01 KClsolutionfor a 10min duration.TheAg/AgClmicroelectrodepotentialatequilibriumwas foundtobequitestablearound0.1225V(resultnotshown).This valueishigherthanthetheoreticalonegivenbytheNernstlaw,i.e. 0.098V.SuchdiscrepancywasrelatedtoAgCllayerdefectsandto chemicalactivitiesofchlorideionsCl−and/orAg-relatedspecies [45,46].Nevertheless,afteraninitialdecreasearound0.5mV dur-ing1min,thepotentialdriftoftheAg/AgClintegratedelectrode wasverylow(around1mVs−1),inagreementwithliterature[46]. 3.4. Detectionofantioxidantspecies
(Au–Pt–Ag/AgCl)electrochemicalmicrocells(SU-8passivation layerthickness:3mm)wereappliedforthedetectionof antioxi-dantmolecules,andmorepreciselyascorbicanduricacids(AAand UA),inpH7.0phosphatebuffersolution(PBS).Allthecyclic voltam-mogramswereobtainedinanautonomousway,i.e.usingthe“three integratedmicroelectrodes”configuration.Inallcases,no signifi-cantsignalwasobservedinPBSpH7.0exceptforthegoldoxidation starting around 0.8V. This PBS-related residual voltammogram wassystematicallydeducedfromthatobtainedwithantioxidant moleculesinordertoimproveresultsanalysis.
The ElecCellmicrosensorwasfirst usedfor thedetection of ascorbicacid(AA–0.001M)anduricacid(UA–0.001M) sepa-rately(Figs.8and9).Inbothcases,cyclicvoltammogramsevidence an oxidation wave starting around0V for theAA solution and around0.3VfortheUAone[47,48].Comparedtothesolidgold microelectrodeand whatever theantioxidant species, the Elec-Cellmicrosensorresponsesshowedsimilarhalf-wavepotentialE1/2 andhigherdiffusioncurrentdensityilim,i.e.thusprovidinghigher sensitivity(Table1).Suchenhancementshouldberelatedtothe electrocatalyticpropertiesofsolidgoldelectrodeandthinmetallic layerdepositedbyPVD,respectivelyandcouldresultfromchange
Fig.8. Cyclicvoltammogramofthe(Au–Pt–Ag/AgCl)electrochemicalmicrocell (solidline)andofthestandard“nonintegrated”goldelectrode(dottedline)ina PBSpH7.0solutioncontainingAA0.001M(potentialscanrate:50mVs−1).
Fig.9. Cyclicvoltammogramofthe(Au–Pt–Ag/AgCl)electrochemicalmicrocell (solidline)andofthestandard“nonintegrated”goldelectrode(dottedline)ina PBSpH7.0solutioncontainingUA0.001M(potentialscanrate:50mVs−1).
incrystallographicorientationorsurfaceroughness.Anyway,the useof(Au–Pt–Ag/AgCl)ElecCellmicrosensorsforthedetectionof ascorbicand/oruricacidsinliquidphasewassuccessfully demon-strated.
Finally, the ElecCell microsensor was studied in phosphate buffersolutioncontainingbothascorbicand uricacids(AA+UA – 0.001M(Fig.10).Asexpected and similarlyto thatobtained withthestandard“nonintegrated”goldelectrode,cyclic voltam-mograms evidenced two oxidation waves related to the two antioxidant species. Asbefore, slightly higher diffusion current Ilim,andthereforehigherdetectionsensitivity,wereobtainedwith theElecCellmicrosensor,resultinginhighersensitivity(Table1). Nevertheless,thesewavesarenotclearlydefinedandare there-foredifficulttoseparate.Half-wavepotentialE1/2 wereroughly defined,leadingtolowdetectionselectivitybetweenascorbicand uric acids[49,50].This problemcouldbesolved by developing
Fig.10.Cyclicvoltammogramofthe(Au–Pt–Ag/AgCl)electrochemicalmicrocell (solidline)andofthestandard“nonintegrated”goldelectrode(dottedline)ina PBSpH7.0solutioncontainingAAandUA0.001M(potentialscanrate:50mVs−1).
specifiedfunctionalizationbasedonelectroactivepolymerssuch aspoly(3,4-ethylenedioxythiophene)(PEDOT)[16].Worksarein progressinthisway.
3.5. Skinanalysisfortheantioxidantcapacitymeasurement The(Au–Pt–Ag/AgCl) electrochemicalmicrocells werefinally testedforskinanalysisinordertodeterminetheglobal antioxi-dantcapacitydetectionfeasibility.Theexperimentalprotocolwas quitesimplesincetheElecCellmicrodeviceswereapplieddirectly ontheforearmwithoutanypre-treatment(Fig.3).Themain prin-ciplewastousethe“stratumcorneum”naturalhydrolipidicfilmto guaranteetheelectricalcontactbetweentheworking,counterand referencemicroelectrodes[51].
Logically, the ElecCellmicrosensors with a 3mm-thick SU-8 passivationlayerwerefirststudied.Inthiscase,no amperomet-ric signal was evidenced on the skin while performing cyclic voltammetryexperiment.However,assoonasfewpH7.0 phos-phatebuffersolution(PBS)droplets weredepositedontheskin surface, thetypicalgold voltammogram wasfoundagain.Such resultdemonstratesthatthehydrolipidicfilmwasnotsufficient toprovidetheelectricalcontactbetweenthedifferent microelec-trodessinceitsthicknessisknowntobearoundonemicron[51,52]. Inordertotackleoffthisbottleneckandtoimprovetheelectrical contactontheskinsurface,themicroelectrodesrecessand there-foretheSU-8layerthicknessmustbedecreased.So,eveniftheir electrochemicalperformanceswerelower,theElecCell microsen-sorswitha 1.6mm-thickSU-8passivationlayerwerealsoused for theskinanalysis usingcyclic voltammetry(Fig.11). In this case,thankstothepassivationthicknessdecrease,agood electri-calcontactwasobtainedwiththehydrolipidicfilm.Furthermore, noelectrochemicalinterferencewasevidenced(cf.Section3.2). This improved electrical behaviour was related to the hydroli-pidicfilmphysicalproperties.Comparedtowater-basedsolutions, thisemulsion-like,two-dimensionalmediumischaracterizedby ahigherviscosity,lowerdiffusivitiesandahigherelectrical resis-tivity[51,52].Thus,obtainedvoltammogramsweretypicalofthe Table1
Characteristicsofthe(Au–Pt–Ag/AgCl)electrochemicalmicrosensorresponsesforthedetectionofascorbicand/oruricacids.
Ascorbicacid(AA) Uricacid(UA) Ascorbicanduricacids(AA+UA) ElecCellmicrosensor E1/2(V/SCE) 0.47±0.08 0.51±0.03 0.31±0.03/0.53±0.01
ilim(mAcm−2) 0.9±0.1 1.1±0.3 0.69±0.04/0.91±0.07
Standardgoldelectrode E1/2(V/SCE) 0.35±0.02 0.48±0.02 0.31±0.03/0.52±0.02 ilim(mAcm−2) 0.73±0.08 0.67±0.05 0.6±0.05/0.81±0.07
Fig.11.Cyclicvoltammogramofthe(Au–Pt–Ag/AgCl)electrochemicalmicrocellon theskinsurface(potentialscanrate:50mVs−1).
electrochemicalskinproperties[14,53].Theascorbicacidoxidation wasresponsibleforthefirstcurrentwavearound0.6V/SCE.Then, thesecondwavearound0.8V/SCEwasrelatedtouricacid, nicotin-amideadeninedinucleotidephosphate(NADPH)andcystein.The lastwavearound1.2V/SCEwasassociatedtotheglutathione oxi-dation.Finally,thereductionpeakobservedaround0.4V/SCEwas duetothegoldoxides.
Allinall,bydecreasingtheSU-8layerthicknessandtherefore themicroelectrodesrecessto1.6mm,theskinhydrolipidicfilmwas sufficienttoobtainagoodelectricalcontactbetweenthedifferent microelectrodesandtodetectelectrochemicallydifferent antioxi-dantspeciespresentontheskinsurface.
4. Conclusion
(Pt–Pt–Ag/AgCl)and (Au–Pt–Ag/AgCl)electrochemical micro-cells (ElecCell)werefabricatedusing silicon-based technologies derivedfrommicroelectronicsinordertodetectredoxspecies,and morespecificallyantioxidantmoleculessuch asascorbicand/or uricacidsinliquidphase.Whatismore,electrochemicalanalysisfor theskinglobalantioxidantcapacitymeasurementwasperformed. SpecificemphasiswasplacedontheSU-8wafer-levelpassivation process. In order tooptimize theelectrochemical properties of thedifferent“thinfilm”metalliclayers,i.e.goldfortheworking electrode,platinumforthecounterelectrodeandsilver/silver chlo-rideforthereferenceelectrode,itwasnecessarytoincreasethe SU-8layerthicknessupto3mm.Thus,theElecCellmicrosensors werestudiedforthedetectionofascorbicanduricacidsin phos-phatebuffersolution.Comparedtostandard“nonintegrated”gold electrodes,similarresponseswereshownbycyclicvoltammetry, evidencinghigherdetectionsensitivitybutlowdetection selectiv-ity.
Workswereextendedtoskinanalysis.Inthiscase,the3 mm-thickSU-8layerwasresponsibleforaveryhighmicroelectrode recesstoprovideagoodelectricalcontactwiththeskin hydroli-pidic film. However, using a non-optimized 1.6mm-thick SU-8 layer,oxidationwavesrelatedtodifferentantioxidantspecieswere successfullyobtainedontheskin,demonstratingthatthestratum corneumglobalantioxidantcapacitycanbeeffectivelymeasured.
Inordertouse(Au–Pt–Ag/AgCl)ElecCellmicrosensorsforthe skinanalysis, a compromise hasto befoundon theSU-8 pas-sivation process.On theone handandas demonstratedforthe liquidphaseanalysis,ahighSU-8thickness,i.e.3mmandmore, isresponsibleforacorrectstepcoverageofthedifferentmetallic layersand,consequently,forgoodelectrochemicalpropertiesand
well-controlledactiveareaoftherelatedmicroelectrodes.Onthe otherhand,alowSU-8thickness,i.e.1.6mmorless,isstillrequired forobtainingalowmicroelectroderecessand,finally,toprovide thenecessaryelectricalcontactwiththeskinhydrolipidicfilm.This hastobefurtherstudiedintheframeofwafer-levelpassivation processes(basedonSU-8polymersand/orothersbiocompatible materials).
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Biographies
C.ChristophewasbornonDecember16,1981.ShereceivedherEngineer’sDegree inmaterialssciencefromthe“InstitutNationalPolytechniquedeToulouse”(France) in2006.Shejoinedthe“Laboratoired’Architectureetd’AnalysedesSystèmes”from theFrench“CentreNationaldelaRechercheScientifique”(LAAS-CNRS)in2007.She isworkingonthedevelopmentofelectrochemicalmicrosensorsforchemicaland biochemicaldetection.
F.SékliBelaidiwasbornonFebruary22,1980.ShereceivedherMaster’sDegree inprocessandenvironmentalengineeringfromthe“InstitutNationaldesSciences AppliquéesdeToulouse”(France)in2006.Shejoinedthe“LaboratoiredeGénie Chimique”(LGC)fromtheUniversityofToulouse(France)in2007.Sheisworking onthedevelopmentofelectrochemicalmicrosensorsforchemicalandbiochemical detection.
J.LaunaywasbornonMarch11,1975.Hereceivedthedegreeinelectronic engi-neeringfromtheInstitutNationaldesSciencesAppliquéesdeToulouse”(France)in 1998.Hejoinedthe“Laboratoired’Architectureetd’AnalysedesSystèmes”from theFrench“CentreNationaldelaRechercheScientifique”(LAAS-CNRS)in1998 andreceivedthePhDdegreefromthe“InstitutNationaldesSciencesAppliquées deToulouse”(France)in2001.In2002,hebecamelecturerattheUniversityof Toulouse(France).Hisresearchactivitiesincludethedevelopmentof electrochem-icalmicrosensorsforthedetectioninliquidphase.
P.Groswasbornin1970.HegraduatedinPhysicalChemistryin1992andreceived hisPhDdegreeinChemicalEngineeringin1996attheUniversityPaulSabatierin Toulouse.HeisnowProfessorinElectroanalyticalEngineeringintheChemical Engi-neeringLaboratory(Toulouse-France).Heiscurrentlyworkingonthedevelopment ofelectrochemical(bio)sensors.
E.QuestelwasbornonOctober16,1965.HereceivedhisEngineer’sDegreein molecularandsupramolecularchemistryfromtheUniversityofToulouse(France)in 1992.HejoinedtheFrench“GroupedeRecherchesurl’Oncogénèse,lesUltraviolets etlaPigmentationCutanée”in1994.In1997,hejoinedtheFrench“Laboratoire PierreFABREDermocosmetique”companyinToulouse.Since2004,heisincharge ofthe“SkinPhotobiologydepartment”.Heisworkingonclinicalstudiesonhealthy volunteers.
P.Temple-Boyer was born onOctober 25, 1966. Hereceived his Engineer’s Degreeinelectronicengineeringfromthe“EcoleSupérieured’Electricité”(Paris– France)in1990andhisMaster’sDegreeinmicroelectronicsfromtheUniversityof Toulouse(France)in1992.Hejoinedthe“Laboratoired’Architectureetd’Analyse desSystèmes”(LAAS)fromtheFrench“CentreNationaldelaRecherche Scien-tifique”(CNRS)in1992andreceivedthePhDdegreefromthe“InstitutNational desSciencesAppliquéesdeToulouse”(France)in1995.Sincethen,asaCNRS researcher,hehasworkedatLAASonthedevelopmentofphysicalandchemical microsensors.
