Array of ultramicroelectrodes for the simultaneous detection of nitric oxide and peroxynitrite

ElectrochimicaActa140(2014)33–36

ContentslistsavailableatScienceDirect

ElectrochimicaActa

journalhomepage:http://wendang.chazidian.com/locate/electact

a

Arrayofultramicroelectrodesforthesimultaneousdetectionofnitricoxideandperoxynitriteinbiologicalsystems

LoanT.O.ThiKima,b,c,d,VirginieEscrioua,b,c,d,SophieGriveaua,b,c,d,AurélieGirarde,LaurentGriscome,FlorenceRazane,FethiBediouia,b,c,d,?

a

ChimieParisTech,EcoleNationaleSupérieuredeChimiedeParis,UnitédeTechnologiesChimiquesetBiologiquespourlaSanté,Paris,FranceCNRS,UnitédeTechnologiesChimiquesetBiologiquespourlaSantéUMR8258,Paris,Francec

UniversitéParisDescartes,SorbonneParisCité,UnitédeTechnologiesChimiquesetBiologiquespourlaSanté,Paris,Franced

INSERM,UnitédeTechnologiesChimiquesetBiologiquespourlaSanté(N?1022),Paris,Francee

SATIE-BIOMISCNRS8029,ENSCachan-Bretagne,Bruz,France

b

article

info

abstract

Articlehistory:

Received10December2013

Receivedinrevisedform8January2014Accepted12January2014

Availableonline23January2014

Keywords:NitricoxidePeroxynitriteArrayedsensors

HL60cells,Simultaneousdetection

Wereportheretheproofofconceptoftheuseofnewassembledarraysofultramicroelectrodes(UMEs)forthesimultaneousanddynamicsensingofreactivenitrogenspeciessuchasnitricoxide(NO)andperoxynitrite(ONOO?,so-calledPON)releasedbyculturedcells.Thesedevices?lltheneedtostudythekeyrolesofthesetworeactivenitrogenspecieswhereachemicalimbalanceisimpli-catedinseveralpathologiesandphysiologicaldisorders.Arrayscontainingover2400goldUMESweredesignedinsuchawaytoincreasesensitivityandallowdetectionoflowconcentrationsofthetargetanalytesinvivo.Thesedisposableplatformsarethenintegratedwithincellculturewellsandplates,resultinginaneasy-to-handletoolforcellularbiology.TheyweresuccessfullyusedtodetectinrealtimePONformedinsituuponthereactionofNOreleasedfromaNOdonormolecule((Z)-1-[N-(2-aminoepropyl)-N-(2-ammoniopropyl)amino]-diazen-1-ium-1,2-diolate)andsuperoxide(O2?)releasedbyPMA(phorbol-12-myristate-13-acetate)-inducedHL60cells.

©2014ElsevierLtd.Allrightsreserved.

1.Introduction

Thesimultaneousdetectionandmonitoringofmultiplechem-icalsubstancesreleasedbylivingsystemsandbiologicalsamplesisoftheutmostimportancefortheelucidationofsignaltransduc-tionpathwaysinvivoandalsofordrugtestingbycell-basedassays[1].Thedevelopmentofsensorsthatdisplayhighsensitivityandselectivitytowardstargetedmolecules,andatthesametimeareinsensitivetointerferingchemicalspresentincomplexbiologicalmedia,isthemajorhurdlethatmustbecrossedinordertoachievesimultaneousdetection.Inspiteoftheirinherentdif?culties,elec-trochemicalmethodsarerecognizedasbeingcurrentlytheonlylabel-freeanalyticaltoolsabletodeterminethelocal?uxofbio-logicallyactivecompoundswithoutdisturbingtheirmetabolismandassociatedregulatorypathways[2–4].ElectrochemicalSensorArrays(ESAs)provideeasyaccesstosimultaneousdetec-tionofmultiplespecies,wheneachindividualelectrodeofthearrayisspeci?callytailoredtosensitivelydetectaparticularanalyte.Inparticular,ESAsarewelladaptedtothestudyofreactiveoxygenandnitrogenspeciesincellularsignaltransduction[5–7].Among

?Correspondingauthor.

E-mailaddress:fethi-bedioui@chimie-paristech.fr(F.Bedioui).

thosespecies,thebalancebetweennitricoxide(NO),superoxide(O2?)andtheirreactionproductperoxynitrite(ONOO?,so-calledPON)isknowntocontrolmanyessentialcellfunctionsincludingactivation,proliferationandapoptosis.ThisreactionbetweenNOandO2?israpidanddiffusion-limited.Theproductformed,PON,isaverypowerfuloxidantandcytotoxicagent.Indeed,PONunder-goeshomolyticorheterolyticcleavagethattriggersacascadeofhighlyreactivemoleculesincludingOH.andNO2.radicals[8].Thesemoleculesandtheirprogenitorsarethemajorcompoundsofoxida-tiveandnitroxidativestress[9,10].NOandPONareinvolvedinthedevelopmentofvarious(cardio)vascularandneurodegenera-tivepathologiesanddiseases.Forexample,inthecaseoftraumaticinjury,thede?ciencyofbioavailableNOandoverproductionofPONshouldbecloselymonitored[11].Amongalltheinvolvedspecies,andduetotheirveryshortlifetimes,onlyNOandPONcanbemoni-toredsimultaneously.ThedemonstrationthatNO/PONbalancecanbeusedasariskmarkeristightlylinkedtotheaccessibilityofanaccurateanalyticalconcept.Werecentlydevelopedthedesign,themicrofabricationprocessandanalyzedtheperformanceofanewfullyintegratedESAforthesimultaneousamperometricdetectionofNOandPON.

ThepresentworkaimedatassessingthefeasibilityofthemonitoringofPONformedbythereactionbetweenO2?producedbyDMSO-treatedHL60cell(toaquire

0013-4686/$–seefrontmatter©2014ElsevierLtd.Allrightsreserved.http://wendang.chazidian.com/10.1016/j.electacta.2014.01.053

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L.T.O.ThiKimetal./ElectrochimicaActa140(2014)33–36

Fig.1.(a)Layoutanddesignand(b)pictureoftheESAcontainedona50mmcircularwafer.Theelectricalconnectionsarepermittedthrough20PADsarrangedaroundtheperiphery.ThearrayedUMEsarecontainedwithinthecon?nesofthecounterelectrodes(CE,PADs1and2)whicharetwohalfcircleswitha7.5mmradius.Therearetworeferenceelectrodes,whicharecenteredverticalbars(Ref,PADs7&8).TheworkingUMEsarede?nedbytheetchingoftheparyleneinsulatinglayeronthegoldelectrodeareas.Theycanbeaddressedin4setsof617electrodesof20?mdiametereach(PADs3-6).(c)Photographyofthetwopartcell.(adaptedfrom[12]).

neurophilicphenotype)uponphorbol12-myristate13-acetate(PMA)stimulationthatactivatesNADPHoxidase,andNOreleasedfromNOdonorspecies,namely(Z)-1-[N-(2-aminoepropyl)-N-(2-ammoniopropyl)amino]-diazen-1-ium-1,2-diolate(so-calledPAPA-NONOate).Todosoweusedanewversionofarecentlydevelopedon-chipESAdeviceincludingfoursetsof617goldultramicroelectrodes(UMEs)groupedaroundtwonetworkswhereeachnetworkistailoredtodetectaspeci?canalyte[12].ThestructureoftheESAisshowninFig.1a.ThisnewversionallowsmakingsimultaneousmeasurementsofNOandPON?uxesinbiologicalsystems.

2.Experimental

2.1.DesignandmicrofabricationoftheESAs

TheESAsweregroupedintotwonetworksinviewtosimul-taneouslydetectNOandPON.Inthistwo-in-oneintegratedelectrochemicalsystem,allthegoldelectrodeswerearrangedinmatchingpairs:twosetsofarrayedworkinggoldUMEs,twogoldcounter-electrodesandtworeferenceelectrodes.Moreover,thepatternwasincludedina15mmdiametercircletosimulateacellculturewell.ThestructureoftheESAisshowninFig.1a.Thecounterelectrodesweredesignedintwopartsas2semicirclessurround-ingtheworkingUMEswitha14mmdiameter.Thewidthofthecounterelectrodesis500?mgivinga9.9mm2surfacearea.Thereferenceelectrodeswerearrangedinthecenterandrepresenttwo5×0.25mmbarswithasurfaceareaof1.25mm2each.EachdiscshapedworkingUMEis20micronsindiameterandisarrayed

withahexagonalspacingof500?m.Indeedthisarrangementofworkingelectrodesistoinsureanadditivecurrentmeasurementforeachadditionalworkingelectrode.ThegeometryoftheESAischosensothattheUMEsarespacedequidistantlyatagap(thatisatleasttentimestheelectrodediameter)toavoidoverlappingofthediffusionlayersduringtheelectrochemicalexperiments[13,14]andlossofsignalduetoanalytedepletion.Twosetsof617short-circuitedUMEscomposedanetworkoftheESAforNOandPONdetection.Thuseachnetworkcomprises1234UMEs,onebandshapedreferenceelectrodeandonesemi-circleshapedcounterelectrode(Fig.1a&b).

Standardcleanroommicrofabricationmethodsandequipmentwereusedtosatisfythestrictpurityrequirementsforsensitiveelectrochemicaldetection,aspreviouslyreported[12].Theclean-roommicrofabricationprocesscanbedescribedinthreephases.The?rstphaseinvolvesdepositingandpatterninggoldleadsfortheESAson1mmthickglasssubstrates.Theleads,inthesec-ondphase,arethenuniformlyinsulatedwithaone-micronthickparylene-Ccoating,whichispatternedandetchedbyareactiveionetching(RIE)processtode?nethemicroelectrodearrayaswellasthecounterandreferenceelectrodes.Inthe?nalphase,thesil-veriselectro-depositedandtransformedtoAgCltointegratethereferenceelectrodes.

2.2.Electrochemicaltechniquesandsensorfabrication

Foralltheelectrochemicalexperiments,theESAwasinsertedinatwopartcell(Fig.1c)consistingofanaluminumbaseplatesup-portingtheESAplatformandthesecondupperpartmadeofTe?oncontaining10sprunggoldplatedpins,whichassureelectricalcon-tactsanda15mmdiameterreservoirforthereceptionofliquidsabovetheelectrodes.Thewellwassealedusingano-ringthatwasclampedbetweenthetopplateandtheESAsubstrate[12].

ToachieveselectiveelectrochemicaloxidationofNOwithoutinterferencefromotherproducts,surfacemodi?cationoftheelec-trodearraydevotedtoNOisnecessary[12,15–17].TheUMEsoftheESAwerethencoatedbyelectropolymerizedeugenolandphenol?lms.Indeed,previousexperimentsshowedthatthecombinationofpoly(eugenol)withpoly(phenol)-amongseveralotherlay-eredmaterialsallowsobtainingthedesiredselectivitytowardsNO[12].TheelectrochemicaldeterminationofNOisthusachievedbyamperometryat+0.8VvsAg/AgCl.Theselectiveelectrochemi-caldetectionofPONisachievedthroughitsreductionat-0.1VvsAg/AgClatthearrayofUMEsdevotedtoPONwhichwereusedbarewithoutanychemicalmodi?cation[12,18].

Alltheelectrochemicalexperimentswereperformedatroomtemperature(25±3?C)inaeratedconditions.EitherAgwireelec-trochemicallycoatedbyAgClplacedintheelectrochemicalcellortheembeddedAg/AgClbandelectrodefabricatedwithintheESAwasusedasreferenceelectrode.Identicalresultswereobtainedusingeithertheexternalortheinternalpseudo-referenceelec-trode.ChronoamperometryexperimentswerecarriedoutusingaQuadStat(eDAQ).Chronoamperometricmeasurementsofsimulta-neousdetectionofNOandPONwereachievedbyusingcommonreferenceandcounterelectrodeforbothsensors.Eachexperimentwasrepeatedthreetimes.

2.3.Cellsandchemical

HL-60cells(CCL240)werefromATCC.TheywereculturedinRPMI1640withGlutamax,supplementedwith20%FBS(foetalbovineserum),penicillin(100U/mL),streptomycin(100?g/mL)andnon-essentialaminoacids.Toinducedifferentiationtogranulocyte-likecells,HL-60cellsuspensionswereincubatedwith1.3%(v/v)DMSOfor6days[19].Toinduceextracellularsuperoxideproduction,differentiatedcellswereincubatedwith

L.T.O.ThiKimetal./ElectrochimicaActa140(2014)33–36

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Fig.2.Simultaneousamperometricmeasurementsat+0.8VvsAg/AgClatthegoldUMEsarraycoatedbypoly(eugenol)/poly(phenol)(curvea)andat-0.1VvsAg/AgClatthebaregoldUMEs(curveb)uponinjectionofanaliquotof500?LofHEPESbufferedsolutioncontaining3millionsofnonactivatedHL60cellsandalka-linePAPA-NONOatestocksolution(100?M)totheelectrochemicalcellcontaining500?LofHEPESbuffer.

PMA(100ng.mL?1)for15minat37?C.

DMSO-differentiatedcellswerewashedwithcellculturemediumandre-suspendedinthesamemedium.

NOdonorcompound,PAPA-NONOate((Z)-1-[N-(2-aminoep-ropyl)-N-(2-ammoniopropyl)amino]-diazen-1-ium-1,2-diolate)wasfromCaymanChemical(USA)andkeptat-20?C.Silver(99.9%)wireswerefromGoodfellow(UK).Allotherchemicalswerereagentgradeandwereusedwithoutfurtherpuri?cation.Allaqueoussolutionsweremadeusingultrapurewaterwitharesistivityof18.2M??.cm.Allexperimentswereachievedat37±1?CinHEPESbuffer.

TheNOdonorstocksolutionswerepreparedbydissolv-ingPAPA-NONOateinNaOH(0.01M)solution(10mMPAPA-NONOate).Theobtainedsolutionswerealiquotedandkeptat?20?Cinthedarkfortwomonthsmaximum.AlkalinestocksolutionswerethawedimmediatelybeforeuseandreleaseofNOwasinitiatedbyaddingaliquotstoaerobicHEPESbuffersolution(pH7.4)atroomtemperature.

3.Results

ThewellcontainingtheelectrochemicalESAwas?rst?lledwith500?LofHEPESbufferedsolution.Afterstabilizationofthebackgroundcurrentforca.15minutesatbothsensornetworks,analiquotof500?LofHEPESbufferedsolutioncontaining3millionsofnonactivatedHL60cells(noproductionofsuperoxide)andalka-linePAPA-NONOatestocksolution(100?M)wasrapidlymixedandinjectedtothewell.TheexpectedproductionofNOfromPAPA-NONOatedecompositioninthebufferedHEPESsolutionwasfollowedbymeasuringthetemporalevolutionoftheoxidationcurrentattheNO-sensor.Fig.2ashowstheobtainedampero-grams.Assoonasthemixtureisinjectedandmixed,afast

increase

Fig.3.Simultaneousamperometricmeasurementsat+0.8VvsAg/AgClatthegoldUMEsarraycoatedbypoly(eugenol)/poly(phenol)(curvea)andat-0.1VvsAg/AgClatthebaregoldUMEs(curvesb)uponinjectionofanaliquotof500?LofHEPESbufferedsolutioncontaining3millionsofPMA-activatedHL60cellsandalka-linePAPA-NONOatestocksolution(100?M)totheelectrochemicalcellcontaining500?LofHEPESbuffer.Curvecisobtainedat-0.1VvsAg/AgClatthebaregoldUMEsuponinjectionofanaliquotof500?LofHEPESbufferedsolutioncontain-ing3millionsofPMA-activatedHL60cells,alkalinePAPA-NONOatestocksolution(100?M)andSOD(600U/mL)totheelectrochemicalcellcontaining500?LofHEPESbuffer.

oftheoxidationcurrentisobserved,duetotheoxidationofthespontaneouslyreleasedNOfromtheNO-donor,uptoamaxi-mum.Then,arelativelyslowdecreaseofthecurrentisobservedassoonastheoxidationofNObymolecularoxygenpresentintheaerobicelectrolyticsolutiondominatestheoverallreaction.Suchbehaviorisverysimilartothepreviouslyreportedandwell-knownoneinphosphatebuffersolutionatpH=7.4[20].Fig.2bshowstheamperometricsignalrecordedsimultaneouslyatthePON-sensor.Asexpected,nospecialfeaturesrelatedtotheelectrochemicalreductionofPONareobserved[12,18],con?rmingtheabsenceofPON,sincetheHL60werenonactivatedandnotabletoproducethesuperoxideanionresponsiblefortheformationofPON.ThisalsoshowsthatnointerferingsignalfromNOoccursatthePON-sensornetwork.Wehaveclearlyshowedinapreviousstudythatnointerferencesfromoxygen,hydrogenperoxideandotheranalytesareobservableat-0.1Vvs.Ag/AgClongoldUMEs[18].However,itshouldbenotedthatalargepositiveonsetofthecurrentmeasuredatthePON-sensorisobservedupontheadditionoftheinactivatedcells.Thismightbeexplainedbyanimmediatechangeofthechargedistribution,convectionandsurfacecompositionofthebaregoldelectrodesduetothepresenceofthecellsandbythereactivitybetweengoldandextracellularproteinthiolgroups.

Fig.3showsthesimultaneousamperometricmeasurementsattheNO-sensor(curvea)andatthePON-sensor(curveb)upontheadditionofanaliquotof500?LofHEPESbufferedsolutioncontaining3millionsofPMA-activatedHL60cellsandalkaline

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PAPA-NONOatestocksolution(100?M)totheelectrochemicalcellcontaining500?LofHEPESbuffersolution.TheshapeoftheamperometricsignalmeasuredattheNOsensorissimilartothatpreviouslyreportedforareleaseofNOfromtheNO-donor,exceptthatitfeaturesafasterandamoreintensedecreasecom-paredtothatobtainedinpresenceofthenonactivatedcells.TheamperometricsignalrecordedatthePON-sensorhasamuchdif-ferentshapethantheonerecordedinpresenceofthenonactivatedcells.Indeed,itshowstheapparitionofasigni?cantreductioncur-rent?veminutesaftertheadditionofthemixturethatcanbeattributedtothechemicalreductionofPONatthegoldUMEs[12]uponthereactionbetweenNOreleasedfromPAPA-NONOateandsuperoxideanionproducedbythePMA-activatedHL60cells.Itwascon?rmedinpreliminaryexperimentsthatthePMA-activatedcellsproducesuperoxideaspreviouslyreportedintheliterature[19].Itshouldbenotedthattheelectrochemicalresponsesdonotre?ecttherealproductionofNOandPONandtheirtotalquan-titiesduetotheirchemicalunstability(decomposition)andhighreactivity.Also,itcanbeeasilyunderstandablenottoconverttheamperometriccurrentsmeasuredinthecellculturemediumtoNOandPONconcentrations.HowevercomparisonbetweenthechargesdevelopedundertheNOcurvesinabsenceandinpresenceofthesimultaneouslyproducedsuperoxideshowsthat≈40%ofNOisconsumedwhenitisreleasedfromPAPA-NONOateinpres-enceoftheactivatedHL60cells.ThisevaluationisroughlydoneduethefactthattheNObaselineamperometriccurrentchangessigni?cantly.

Inordertoensurethespeci?cityoftheamperometricsignalatthePON-sensor,anotherexperimentwasperformedbyaddinganaliquotof500?LHEPESbufferedsolutioncontaining3millionPMA-activatedHL60cells,alkalinePAPA-NONOatestocksolu-tion(100?M)andSOD(600UM/mL)totheelectrochemicalcell(SOD:superoxidedismutatsecatalysesthedismutationofO2?).TheamperometricsignalmeasuredatthePONsensorisshowninFig.3,curvec.ThesedataindicatethatthereductioncurrentattributedtoPONissuppressedinpresenceofSODduetothecom-petitivescavengingofO2?decreasinglargelytheamountofPONformed.Indeed,aquitelargeamountofSOD,comparedtothatoftheNOdonormoleculewasusedhereandthisexplainsthesup-pressioneffectalthoughthereactionofNOandsuperoxideis3timesgreaterthanforthereactionofsuperoxidewithSOD[21,22].

4.Conclusion

Thisstudyshowstheproofofconceptoftheuseofnewassembledarraysofultramicrosensorsforthesimultaneousand

dynamicelectrochemicalsensingofNOandPONformeduponthedecompositionofNO-donorandtheactivationandHL60cells.Thisconceptisnowfurtherdevelopedtoanalyzethebiologicalfunc-tionsofdesignatedlivingsystems.

Acknowledgements

Financialsupportfromthe“AgenceNationaledelaRecherche”(ANRFrance)intheframeworkoftheprojectMECANOANR-08-PCVI-0018isacknowledged.Theauthorsarethank-fultoDrDamienQuintonforhisscienti?ccontributionsanddiscussions.

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