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SUPPLEMENTARYINFORMATIONChemicalEquilibrium-BasedMechanismfortheElectrochemicalReductionofDNA-BoundMethyleneBlueExplainsDoubleRedoxWavesinVoltammetryJ.D.Mahlumα,MiguelAllerPelliteroβandNetzahualcóyotlArroyo-Currásα,β,γ,δ,∗AffiliationsαChemistry-BiologyInterfaceProgram,ZanvylKriegerSchoolofArts&Sciences,JohnsHopkinsUniversity,Baltimore,MD21218.βDepartmentofPharmacologyandMolecularSciences,JohnsHopkinsUniversitySchoolofMedicine,Baltimore,MD21202.γDepartmentofChemicalandBiomolecularEngineering,WhitingSchoolofEngineering,JohnsHopkinsUniversity,Baltimore,MD21218.δInstituteforNanobiotechnology,JohnsHopkinsUniversity,Baltimore,MD21218.*Correspondenceto:NetzArroyo,Ph.D.JohnsHopkinsUniversitySchoolofMedicine316HunterianBuilding725NorthWolfeStreetBaltimore,MD21205netzarroyo@jhmi.edu443-287-4798S-1
1TableofContentsFigureS1.Averagingelectrodevoltammogramsresultsinartificialdecreaseinpeakcurrent....................3FigureS2.Diffusioncoefficientsdonotaffectvoltammetricfeaturesinournumericalmodel.....................4FigureS3.ComputedpercenterrorbetweenexperimentalvoltammogramsandthenumericalsimulationforFigure4....................................................................................................................................................5FigureS4.ChangingpKaofsimulatedmethylenebluevoltammogramsaffectspeaksplitting....................6FigureS5.Increasingtheprotonationrateofthemethyleneblueradicalincreasespeakcurrentmagnitudes......................................................................................................................................................................7FigureS6.ComputedpercenterrorbetweenexperimentalvoltammogramsandthenumericalsimulationforFigure5....................................................................................................................................................8FigureS7.ComputedpercenterrorbetweenexperimentalvoltammogramsandthenumericalsimulationforFigure6....................................................................................................................................................9FigureS8.ComputedpercenterrorbetweenexperimentalvoltammogramsandthenumericalsimulationforFigure7..................................................................................................................................................10FigureS9.ComputedpercenterrorbetweenexperimentalvoltammogramsandthenumericalsimulationforFigure8..................................................................................................................................................11FigureS10.Voltammogramsofelectrodesfunctionalizedwith6-Mercaptohexanolshowlargerchargingcurrentandlessfaradaiccurrentrelativeto1-hexanethiol-functionalizedelectrodes................................12FigureS11.6-mercaptohexanoicaciddisplaysextravoltammetricfeaturesthatmaskthesignalfrommethyleneblue............................................................................................................................................13TableS1.DNAconstructsusedforexperiments........................................................................................15TableS2.Homogeneousreactionconditionsforphosphate-bufferedsaline.............................................16TableS3.Figure4averagedparameters...................................................................................................17TableS4.Figure5averagedparameters...................................................................................................18TableS5.Figure6averagedparameters...................................................................................................19TableS6.Figure7averagedparameters...................................................................................................20SupplementaryReferences.........................................................................................................................21S-2
2FigureS1.AveragingelectrodevoltammogramsresultsinartificialdecreaseinpeakcurrentWetooktheaverageofsixelectrodeswhichhad500nMofthetobramycinaptamerco-depositedwith1-hexanethiolforthreescanningrates:(A)1V∙s-1,(B)10V∙s-1,and(C)100V∙s-1.WecomparedthistotherawdatausedforFig.4A,C,andE.Weobservedthataveragingtheelectrodesresultedinanoveralldecreaseinpeakfaradaiccurrentandanartificialbroadeningofthepeaks.Thisledustouseonerepresentativereplicateforeachdatasettofitourmodel.S-3
3FigureS2.DiffusioncoefficientsdonotaffectvoltammetricfeaturesinournumericalmodelUsingexperimentaldata(solidlines)andsimulationparametersfromFig.4C,westudiedtheeffectdiffusioncoefficientsforspeciesO,R1,R1H,andR2Hhaveonthemagnitudeofvoltammetricfeaturesproducedbyourmodel.WeevaluatedsevenordersofmagnitudewhereDO=DR1=DR1H=DR2H=(A)10-2cm2·s-1,(B)10-3cm2·s-1,(C)10-4cm2·s-1,(D)10-5cm2·s-1,(E)10-6cm2·s-1,(F)10-7cm2·s-1,or(G)10-8cm2·s-1.(H)Additionally,weevaluatedonesetofdrasticallymodifieddiffusioncoefficientsforeachoftheelectroactivespeciesinourmodel:DO=10-3cm2·s-1,DR1=10-5cm2·s-1,DR1H=10-2cm2·s-1,andDR2H=10-8cm2·s-1.Doingthis,wedemonstratethat,becauseofthethin-layerelectrochemicalcellnatureofourmodel,diffusioncoefficientshavenoeffectonthevoltammetricfeaturesproducedbyoursimulations.S-4
4FigureS3.ComputedpercenterrorbetweenexperimentalvoltammogramsandthenumericalsimulationforFigure4Usingeq.22,wedeterminedtheaccuracyofournumericalmodelforeachofthesimulatedvoltammogramsinFig.4.(A)%errorvs.voltageplotsforboththereducingscan(toppanel)andoxidationscan(bottompanel)ofFig.4A.(B)SameforFig.4B,(C)sameforFig.4C,(D)SameforFig.4D,(E)SameforFig.4E,and(F)SameforFig.4F.S-5
5FigureS4.ChangingpKaofsimulatedmethylenebluevoltammogramsaffectspeaksplittingWesimulatedtheeffectsthatthreeaciddissociationconstantshadonpeakcurrentmagnitudeandsplitting:(A)pKa=6,(B)pKa=7,and(C)pKa=8.WeobservethatwithincreasingpKathereductionscan’speakcurrentincreasesconcomitantlywithanincreaseinsplittingoftheoxidationscan’speak.Thiscanbeexplainedbythechemicalequilibriumofprotonssimulatedbyourmodel(eq.2and20).Inthereductionwave,aspKaincreasesataconstantelectrolytepHandprotonationrate(kb,R1H),theleucomethyleneblueradical(LMB•+inFig.1)formedafterthefirstelectrontransferbecomesmorestablepereq.20(i.e.,ittendstobeprotonated).Thus,thesecondelectrontransferisnolongerlimitedbytheprotonationstep,andthemagnitudeofthereductionwaveincreasesbyvirtueofthenowoverlappingelectrontransfers.Atthesametime,higherpKa’sresultinmorepeaksplittingfortheoxidationscan.Thisisbecausethestabilityoftheleucomethyleneblueradicalcausesaresistancetowardsdeprotonation,whichinturncausesthesecondelectrontransfertooccuratincreasinglypositivepotentials.S-6
6FigureS5.IncreasingtheprotonationrateofthemethyleneblueradicalincreasespeakcurrentmagnitudesWesimulatedtheeffectsthatthreemethyleneblueradicalprotonationrates(eq.2)hadonpeakcurrentmagnitudeandsplitting:(A)kb,R1H=106m3∙s-1∙mol-1,(B)kb,R1H=107m3∙s-1∙mol-1,and(A)kb,R1H=108m3∙s-1∙mol-1.Weobservethatasprotonationrateincreases,thereductionscan’scurrentincreasesinmagnitude.Thisisbecause,afterthefirstelectrontransferstep(eq.1),thefasterthemethyleneblueradicalisprotonated,thequickerthenewlyprotonatedradicalisavailabletoundergoasecondelectrontransferstep(Fig.1).Thisleadstoanincreaseinpeakcurrent.Theincreaseincurrentfortheoxidationscanisduetotheincreaseinleucomethyleneblueformedduringthereductionprocess.S-7
7FigureS6.ComputedpercenterrorbetweenexperimentalvoltammogramsandthenumericalsimulationforFigure5Usingeq.22,wedeterminedtheaccuracyofournumericalmodelforeachofthesimulatedvoltammogramsinFig.5.(A)%errorvs.voltageplotsforboththereducingscan(toppanel)andoxidationscan(bottompanel)ofFig.5A.(B)SameforFig.5B,(C)sameforFig.5C,and(D)sameforFig.5D.S-8
8FigureS7.ComputedpercenterrorbetweenexperimentalvoltammogramsandthenumericalsimulationforFigure6Usingeq.22,wedeterminedtheaccuracyofournumericalmodelforeachofthesimulatedvoltammogramsinFig.5.(A)%errorvs.voltageplotsforboththereducingscan(toppanel)andoxidationscan(bottompanel)ofFig.6A.(B)SameforFig.6B,(C)sameforFig.6C,(D)sameforFig.6D,(E)sameforFig.6E,and(F)sameforFig.6F.S-9
9FigureS8.ComputedpercenterrorbetweenexperimentalvoltammogramsandthenumericalsimulationforFigure7Usingeq.22,wedeterminedtheaccuracyofournumericalmodelforeachofthesimulatedvoltammogramsinFig.7.(A)%errorvs.voltageplotsforboththereducingscan(toppanel)andoxidationscan(bottompanel)ofFig.7A.(B)SameforFig.7Band(C)sameforFig.7C.S-10
10FigureS9.ComputedpercenterrorbetweenexperimentalvoltammogramsandthenumericalsimulationforFigure8Usingeq.22,wedeterminedtheaccuracyofournumericalmodelforeachofthesimulatedvoltammogramsinFig.8.(A)%errorvs.voltageplotsforboththereducingscan(toppanel)andoxidationscan(bottompanel)ofFig.8A(leftpanel).(B)SameforFig.8A(middlepanel),(C)sameforFig.8A(rightpanel),(D)sameforFig.8B(leftpanel),(E)sameforFig.8B(middlepanel),(F)sameforFig.8B(rightpanel),(G)sameforFig.8C(leftpanel),(H)sameforFig.8C(middlepanel),and(I)sameforFig.8C(rightpanel).S-11
11FigureS10.Voltammogramsofelectrodesfunctionalizedwith6-Mercaptohexanolshowlargerchargingcurrentandlessfaradaiccurrentrelativeto1-hexanethiol-functionalizedelectrodes.Thisdifferenceinmeasuredsignal-to-noiseratiosresultinnoisierbackground-subtractedvoltammogramsforelectrodesfunctionalizedwith6-mercaptohexanolmonolayers(redtrace)relativeto1-hexanethiolmonolayers(blacktrace)andafluorinatedmonolayer(bluetrace).S-12
12FigureS11.6-mercaptohexanoicaciddisplaysextravoltammetricfeaturesthatmaskthesignalfrommethyleneblueWeco-deposited6-mercaptohexanoicacidwith100nMoftobramycinaptamer(Table1)andcollectedcyclicvoltammogramsat(A)0.5V∙s-1,(B)1V∙s-1,(C)10V∙s-1,(D)50V∙s-1,and(E)100V∙s-1before(leftpanels)andafter(middlepanels)aguanidiniumchloridetreatment.Anelectrodewithonly6-S-13
13mercaptohexanoicaciddepositedonitssurfacewasusedtodeterminethecontributionofthemonolayertotheFaradaiccurrent(rightpanels,blacktrace).Weseethat6-mercaptohexanoicacidhasvoltammetricfeaturesthatoverlapwithmethyleneblue’speakcurrents(A-E,leftpanels).Becauseofthisbackgroundcurrent,wechosenottouse6-mercaptohexanoicacidinourstudy.S-14
14TableS1.DNAconstructsusedforexperiments§Allsequenceswererunthroughthemfoldwebservertopredictsecondarystructure.1*Nopredictedsecondarystructure**ΔGcalculatedtobe-5.27kcal∙mol-1.S-15
15TableS2.Homogeneousreactionconditionsforphosphate-bufferedsalineAlldeprotonationrateconstants,equilibriumconstants,anddiffusioncoefficientswerefoundinref.2.S-16
16TableS3.Figure4averagedparametersS-17
17TableS4.Figure5averagedparametersS-18
18TableS5.Figure6averagedparametersS-19
19TableS6.Figure7averagedparametersS-20
20SupplementaryReferences1.Zuker,M.,Mfoldwebserverfornucleicacidfoldingandhybridizationprediction.NucleicAcidsRes2003,31,3406-3415.DOI:10.1093/nar/gkg5952.Eden,A.;Scida,K.;Arroyo-Curras,N.;Eijkel,J.C.T.;Meinhart,C.D.;Pennathur,S.,Modelingfaradaicreactionsandelectrokineticphenomenaatananochannel-confinedbipolarelectrode.JPhysChemC2019,123,5353-5364.DOI:10.1021/acs.jpcc.8b10473S-21
