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ASimpleandSensitiveAssayforMeasuringVerySmallVolumesofMicroprintedSolutions

AnalChemInsights.20116:6166.

PMCID:PMC3169343

Publishedonline2011Aug31.doi:10.4137/ACI.S7827

ASimpleandSensitiveAssayforMeasuringVerySmallVolumesofMicroprinted
Solutions
CharlesW.Sokolik, 1AnnieS.Walker, 1andGaryM.Nishioka2
1
DepartmentofChemistryandBiochemistry,DenisonUniversity,Granville,Ohio43023,USA
2
H&NInstrumentsInc.,POBox4338,Newark,Ohio43055,USA
Correspondingauthoremail:sokolik@denison.edu
Copyrighttheauthor(s),publisherandlicenseeLibertasAcademicaLtd.
Thisisanopenaccessarticle.Unrestrictednoncommercialuseispermittedprovidedtheoriginalworkisproperlycited.

Abstract

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Thisstudydescribesanextremelysensitiveandsimpleassaytomeasuresmallvolumesofsolutions,<1nL.The
assaytakesadvantageoftheSandellKolthoffreactioninwhichyellowcerium(IV)isreducedtocolorless
cerium(III)inthepresenceofarsenic(III)andcatalyticquantitiesofiodideion.Thereactionislinearwithrespectto
therateofCe(IV)reductionandthequantityofIpresent.Typicalassayscanmeasure10100picomolesofiodide
inasample.WhenIissubstitutedforchlorideioninstandardbiologicalbuffers,suchasTrisbufferedsaline,the
assaycanbeusedtodeterminethevolumeofsolutionprintedinamicroarray.
Keywords:iodideassay,smallvolumemeasurement,microarrays,siliconchips
Introduction

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Microfabricationtechniquesareplayinganincreasinglyimportantroleintheproductionofthelatestdiagnostic
devicesandsensors.Manyofthesedevicesrequirethefunctionalizationofpartofthesensorsurfacewitha
biologicallyactivecompound,suchasanantibodyorenzyme.Toachievethis,techniquessuchasmicrocontact
printing,quillpenspotting,andinkjetprintinghavebeendevelopedtoimpartasmallquantityofbiological
materialtosurfaces.16
Inmanyinstancesitisimportanttomeasurethequantityofbiologicalmaterialprintedbythesetechniques.The
quantityofactivematerialisreadilymeasuredusinganactivityassayforthebiologicalmaterial,forexample,an
enzymeassayifanenzymeisprinted,oranimmunoassayifanantibodyisprinted.However,thesemethodsdonot
necessarilymeasurethequantityofbiologicalmaterialactuallydeliveredtothesurface,sincedenaturingcanoccur
atvariouspointsintheprintingprocess.Denaturingcouldoccurduringprinting,duringadsorptiononthesurface,
orduringthestoragetimebetweenprintingsandassay.Evenifanexcessofmaterialisdeliveredtoasurface,the
resultingsurfaceconcentrationofactivematerialcouldbelowifsignificantdamageoccursduringprinting.
Werecentlymeasureddamagetotheenzymehorseradishperoxidase(HRP)causedbyinkjetprinting.7An
internalstandard,fluoresceinsodiumsalt,wasaddedtosolutionscontainingHRP,andthesesolutionswereprinted
usinganinkjet.Thesolutionswereprinteddirectlyintomicroplatewellscontainingabuffersolution.The
fluorescenceoftheinternalstandardmeasuresthetotalenzymeprinted,andastandardassaymeasuresthequantity
ofactiveenzyme.Comparingthesemeasurementsallowedustodeterminethefractionofperoxidasethatdenatured
duetoprinting.Wefoundthatdenaturingoftheperoxidasevariedlinearlywiththerateofcompressionofthe
piezoceramicduringprinting.
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Inthisreportwedescribeaninternalstandard,sodiumiodide,whichislessintrusive,moresensitive,andeasily
assayed.Theincreasedsensitivityofthisassaywhencomparedtothecommonlyusedfluorescentstandards,allows
forthemeasurementoflessthanonenanoliterintotalvolume.Thus,whenemployingmicroprintingtechniques
thatdeliververysmallvolumes,suchaselectrostaticprinting,whichcandeliverlessthanonepicoliterofvolume
perspot810(Nishioka,HollowayandSokolik,unpublishedresults),theuseofiodideasaninternalstandardis
required.Sodiumiodideissubstitutedforthesodiumchloridepresentincommonlyusedbiologicalbuffers,suchas
Trisbufferedsaline(TBS).Aftermicroprinting,thesmallquantitiesofiodidearemeasuredbyexploitingthe
reactionfirstdescribedbySandellandKolthoff.11Inthisreactioniodidecatalyzesthereductionofcerium(IV)to
cerium(III):

4+

2Ce

(aq) + As

3+

[I

3+

(aq)
2Ce

(aq) + As

5+

(aq)

(yellow)(colourless)

Takingadvantageofthisreactionandmechanisticstudiesperformedbyotherinvestigators1216wehave
developedarapid,reliable,andconvenientassayfordetectingpicomolequantitiesofiodidepresentasaninternal
standardinsolution.TheiodideassayisconvenientlyperformedinpolystyreneUVtransparent96wellmicroplates
atroomtemperature.
Experimental

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Assayprocedure

Theassayisperformedbythesequentialadditionof100leachofsolutionscontainingarsenic(III),thesample
containingtheiodidestandard/sample,andcerium(IV).Theorderofadditionisextremelyimportantasismixing,
accomplishedbypipettingrapidlyupanddownthreetimes,aftereachcomponentisadded.Absorbanceat350nm
ismeasuredfollowingadditionofcerium(IV),andismeasuredagainafter2.0minutes.Therateofcerium(IV)
reductionisthencalculatedoverthistimeperiodthisratevarieslinearlywiththequantityofiodidepresentinthe
sample(seeFig.1).TheMolarabsorptivityofcerium(IV)is4282.5M1cm1,thepathlengthfor300Lof
solutioninatypical96wellUVtransparentmicroplateis0.88cm.Therateisconstantoverthefirst3minutesof
thereactionthereforeabsorbancechangecanbemeasuredoveranytimewithinthisperiod.
Figure1
Calibrationcurvefordeterminingpicomolesofiodidebymeasuringtherate
ofCe(IV)reduction.Thelineisthelinearbestfittotheaverageoffive
separateexperiments,y=0.929x+26.6.Errorbarsarethe95%confidence
intervals.Datawereobtained...
Assaysolutions

The15.0mMarsenic(III)solutionispreparedbydissolving0.297gofAs2O3in100mLof0.3MNaOHwith
heat.Afterdissolution,2.0mLofconcentratedsulfuricacidand6.0gNaClareadded,andthesolutionbroughtto
afinalvolumeof200mLwithwater.The100mMiodidestocksolutionispreparedbydissolving1.5gofNaIin
100mLwater.Aworkingstandardsolutionofiodideionof1M(1pmol/L)ispreparedbyserialdilutionofthe
stocksolutionwiththeappropriatebuffer.The2.4mMcerium(IV)solutionispreparedbydissolving0.194gof
Ce(SO4)24H2Oin200mLof0.9Msulfuricacid.
Buffers

Buffersusedwereasfollows:PBS:10mMphosphatebufferedsalinepH7.4,TBS:50mMTrisbufferedsalinepH
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7.6(2Amino2(hydroxymethyl)1,3propanediol),HBS:50mMHEPESbufferedsalinepH7.4(2[4(2
Hydroxyethyl)1piperazinyl]ethanesulfonicacid).
Chippreparation

<P100>siliconchipswereheatcleanedat500Cfor11hours.ForselfassembledmonolayeredC18(SAMC18)
coating,heatcleanedchipswereimmersedina1%(v/v)solutionofoctadecyltrichlorosilaneindicyclohexylfor5
minutes,thenwashed5timeswithtolueneandethanol,andairdriedinacleanhood.Theadvancingcontactangle
ofwateronthesesurfaceswas7578.Forpolyethyleneglycol(PEG)coating,heatcleanedchipswereimmersed
ina1%(v/v)solutionof2(methoxy(polyethyleneoxy)propyltrichlorosilaneindyclyclohexylfor5minutes,then
washed5timeswithtolueneandethanol,andairdriedinacleanhood.Waterspreadsonthesesurfaces.ThePEG
containsbetween6and9ethyleneoxideresidues.
Spottingsolutionandenzymeassay

Solutionswerepreparedconsistingof1.0mg/mLHRPand0.3Msodiumiodideinphosphatebuffer,Trisbuffer,
orHEPESbuffer(thesodiumiodidesubstitutesforsaline).Othersolutionscontainedinaddition10%w/v
trehalose.Thesesolutionswerediluted1:5,000withtheappropriatesalinebuffer(PBS,TBS,orHBS).Two
microlitersofeachsolutionwerethenspottedonchips,delivering0.4ngHRPand120picomolesiodide.To
produceHRPstandardactivitycurves,eachspottingsolutionwasdiluted3:1,1:1,and1:3intheappropriatebuffer.
Thesesolutionsandanegativecontrolwereassayedusing3,3,5,5tetramethylbenzidine(TMB)substrateina
microplatereader.Theriseinabsorbancewaslinearoverthefirst15minutes.Linearcalibrationplotsofabsorbance
versusconcentrationwereplottedforeachspottingsolution.Sampleswereassayedinasimilarfashion,andthe
calibrationplotswereusedtodetermineactiveHRPconcentration.Furtherdetailsaregiveninreference(1).
ResultsandDiscussion

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Figure1plotstheeffectofiodideconcentrationontherateofcerium(IV)reduction.ThedatainFigure1areused
todeterminethequantityofiodideinsamplesandarereadilyreproduced.Becauseofroomtemperaturevariations
weroutinelymeasuredthecalibrationcurveforeachsetofsamplesbutonlyminordeviationsfromthecurvein
Figure1wereobserved.
Theapplicationofthisassayinmicroprintingisdemonstratedasfollows.Microprintingofproteins(using
electrospray)typicallyinvolvesthedepositionoflowvolumes(1nL)ofabufferedsalinesolution.Ifiodideis
substitutedforchlorideinbufferedsaline,then1nLofa0.14Miodidesolutionisdeposited,equaling140
picomolesofiodide.Inordertoquantitativelydeliverarepresentativeamountofiodideweusedastandard
micropipettetodepositarelativelylargevolume(2l)ofadiluteiodidesolution(100MNaI),thusdelivering200
picomolesofiodidetothesamplesurface.Wedepositedsolutionsconsistingof100Msodiumiodidedissolvedin
oneofthreebiologicalbuffers:phosphate,Tris,orHEPES.
Thesesolutionsweredepositedonsiliconchipsdicedfromasiliconwafer.Threesurfacesweretested:thebare
surface,whichconsistsofathinsilicalayer,asurfacecoatedwithaselfassembledmonolayerofoctadecylsilane
(SAMC18),andasurfacecoatedwithpolyethyleneglycol(PEG).Thesesurfacesrepresentahighenergyoxide
surface,alowenergyalkylsurface,andabiocompatiblelowadsorbingsurfacerespectively.
Threesamplesofeachsurfacewerespottedwith200picomolesofiodideandsubsequentlydriedovernightina
desiccator.Dryingwasperformedinordertodeterminewhetherthedehydrationwouldadverselyaffectiodide
recovery.Thedriedspotswereredissolvedin210loftheappropriatebuffer(withoutiodide).Eachrecovered
solutionwasassayedtwiceusing50loftherecoveredsolution+50loftheappropriatebuffer(PBS,TBS,or
HBS).Measuredrateswere70M/min,correspondingto50pmolesiodide(200pmoliodideintheentire
sample).
Thequantitiesofiodiderecoveredasapercentageofthequantitydelivered(200picomoles)areshowninTable1.
EachentryinTable1istheaverageofsixmeasurements.Thereportederroristhe95%confidenceintervalbased
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onthesemeasurements.Table1revealsthatthemeasuredrecoverediodideisingoodagreementwiththe
theoreticallydeliveredquantitywhenTrisandHEPESbufferswereused.Recoveryofiodideisnotinasgood
agreementwiththetheoreticallydeliveredquantityfromSAMandPEGsurfaceswhenphosphatebufferwasused.
Table1
Percentageiodiderecoveredfromsiliconchips.
Fromourdatawecanconcludethatsmallquantitiesofiodidecanbedeliveredandcompletelyrecoveredfroma
varietyofsurfacesusingTBSandHEPESbuffers.Iodidecanthusserveasaninternalstandardinthesebuffers.
RecoveryofiodidefromsurfacesusingPBSbuffermaypresentproblemsandshouldbeusedwithcaution.The
recoveryoftheiodidemaybeaffectedbythechargeofthemainbufferingspecies.UsingthezwitterionicHEPES
orpositivelychargedTrismayprovideforbetterrecoverywhencomparedtothenegativelychargedphosphate,but
additionstudieswillneedtobeperformedtoassessthebuffereffectofiodiderecovery.
Weinvestigatedtheuseoftheiodidestandardinthepresenceoftheenzymehorseradishperoxidase(HRP,type
12).WespottedsiliconchipscoatedwithPEGusingsolutionscontainingiodideandHRPindifferentbuffers.
Somesolutionsalsocontainedtrehalosetopreserveenzymeactivity.
Wespotted2lofeachsolutionontoPEGsiliconchips,delivering0.4ngHRPand120picomolesofiodideto
thesurface.Sampleswerespottedintriplicate.Samplesweredried4hoursinadesiccator,thenregeneratedin210
loftheappropriatebuffer.AliquotsfromeachsolutionwereassayedforHRPactivityandiodide.
Table2revealsthatunderallconditionstheiodiderecoveredwasclosetothetheoreticallydeliveredamount.In
contrast,thereisalargevariationinrecoveredHRPactivity.Thisisnotsurprising,sinceHRPactivityisreadilylost
duringdryingorthroughadsorption.TrehaloseisseentopreserveenzymeactivityinthephosphateandTris
buffers,butnotintheHEPESbuffer.Thisresultissomewhatperplexinggiventhatintheabsenceoftrehalosethe
HRPactivityisgreatlydiminishedwithTBSandPBS,whileitiscompletelyrecoveredwhenHEPESaloneis
used.AdditionalstudiesareneededtobetterunderstandtheeffectofpreservativessuchastrehaloseonHRP
activitywhendepositedandstoredonvariouscoatedsiliconsurfaces.
Table2
IodideandHRPrecoveredfromPEGsiliconchips.
Whenaddingnonnativechemicalstobiologicalsamples,careshouldbetakentoensurethatthereisnoadverse
affectonthebiomolecules.Theadditionofiodideiontobiologicalsamplesseemstoberelativelybenign.Wehave
analyzedtheactivityofHRP,alkalinephosphataseandgalactosidaseinthepresenceofiodideionwithno
measuredlossofactivity(unpublishedresults).Althoughthereactionofiodinewithsulfhydralgroupsandaromatic
ringsoftyrosineandhistidineiswelldescribed,17itisunlikelyundertheconditionsoftheassaythattheiodideion
wouldbeoxidizedtoiodinewhichcouldthenreactwithproteinsinthesample.
Therearealsosomecompoundsthatareknowntointerferewiththeiodidecatalyzedreaction.Ru(IV)and
Os(VIII)canalsocatalyzethereductionofCe(IV),whileAg(I)andHg(II)ionssignificantlyinhibitthereactionat
lowconcentrations.11,16However,theseionscanbecontrolledforinthebufferedsolutionstypicallyusedin
microprintingapplicationsandthereforeareoflittleconcern.Othermorecommonions,suchasPO43,SO42,
ClO,Cl,Br,Fe3+,Al3+,Mg2+,Cu2+,andZn2+havenoaffectontheassayalthoughanyspeciesthatabsorbs
intheregionof350nmwillinterfere.11,16
TheresultssummarizedinTables1and2indicatethatiodidecanbeusedasaninternalstandardtoprovidea
measureofthevolumeandquantityofmaterialprinted.Werecommenditsuseinmicroprintingapplicationsthat
depositsubnanolitervolumesofsolutionasfollows.(1)Substitutesodiumiodideforthesalinesaltusedinthe
biologicalbufferintheprintsolution.(2)Assaythebiologicalactivityoftheprintsolutionanddeterminetheratio
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ofactivitytoiodideconcentration.(3)Printthesolution.(4)Removetheprintediodidefromthesubstrateand
assay.(5)Assaythebiologicalactivityoftheprintedsample.(6)Comparethebiologicalactivity/iodideratioafter
printingtotheratiooftheoriginalsolution.
Insummary,theuseofthisiodideassaycouldprovidevaluableinsightsonthemicroprintingprocess,specifically
thelossofactivityofaprintedbiologicalmaterial.Aproperlydesignedassaycoulddistinguishlossduetothe
actualprintingprocess,denaturingfromdrying,ordenaturingduetoadsorption.
Acknowledgments

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ThisworkwassupportedbyaDenisonUniversityRobertC.GoodFacultyFellowship,theDenisonUniversity
ResearchFoundation,DenisonUniversityAndersonResearchAssistantship,andtheNationalInstitutesofHealth,
AREAgrant1R15GM6278101andSBIRgrant1R43GM08080301.
Footnotes

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Disclosures
Author(s)haveprovidedsignedconfirmationstothepublisheroftheircompliancewithallapplicablelegalandethicalobligations
inrespecttodeclarationofconflictsofinterest,funding,authorshipandcontributorship,andcompliancewithethical
requirementsinrespecttotreatmentofhumanandanimaltestsubjects.Ifthisarticlecontainsidentifiablehumansubject(s)
author(s)wererequiredtosupplysignedpatientconsentpriortopublication.Author(s)haveconfirmedthatthepublishedarticle
isuniqueandnotunderconsiderationnorpublishedbyanyotherpublicationandthattheyhaveconsenttoreproduceany
copyrightedmaterial.Thepeerreviewersdeclarednoconflictsofinterest.

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