碳纳米管的新型羧基化处理

MicrochimActa(2010)169:33–40DOI10.1007/s00604-010-0307-3

ORIGINALPAPER

Novelcarboxylationtreatmentandcharacterization

ofmultiwalledcarbonnanotubesforsimultaneoussensitivedeterminationofadenineandguanineinDNA

XinmanTu&XubiaoLuo&ShenglianLuo&LiushuiYan&FengZhang&QingjiXie

Received:2November2009/Accepted:14January2010/Publishedonline:23February2010#Springer-Verlag2010

AbstractAmethodispresentedforthecarboxylationofmultiwalledcarbonnanotubes(MWCNTs)viaatwo-stepprocess.ThehydroxygroupsofMWCNTswerefirstreactedwithepichlorohydrin,thenwithiminodiaceticacid.TheresultingMWCNTswerecharacterizedbymeansofFouriertransforminfraredspectroscopyandtransmissionelectronmicroscopy.TheglassycarbonelectrodemodifiedwiththeMWCNTsthuspreparedexhibitedenhancedelectrocatalyticactivityandgoodstabilityforthedetermi-nationofguanineandadenineinpH7.0phosphatebuffersolution.Theexperimentalparameterswereoptimized,andadirectelectrochemicalmethodwasdevelopedforthesimultaneousdeterminationofguanineandadenine.Thedetectionlimits(atS/N=3)forguanineandadenineare0.02and0.08μM,respectively.AsensitivemethodwasalsodevelopedforthedeterminationofguanineandadenineincalfthymusDNA.

KeywordsMultiwalledcarbonnanotubes.Carboxylation.Modifiedelectrode.Guanineandadenine.CalfthymusdsDNA

X.TuX.LuoS.Luo(*)L.YanF.ZhangSchoolofEnvironmentandChemicalEngineering,NanchangHangkongUniversity,Nanchang330063,Chinae-mail:[email protected]

X.Tu:Q.Xie

KeyLaboratoryofChemicalBiologyandTraditionalChineseMedicineResearch(MinistryofEducationofChina),HunanNormalUniversity,Changsha410081,China

Guanine(G)andadenine(A)areimportantcomponentsfoundindeoxyribonucleicacid.DeterminingindividualconcentrationsofguanineandadenineortheirratioinDNAisimportantwhenmeasuringthenucleicacidconcentrationitself.Indeed,nucleicacidcomponentsinphysiologicalfluids,tissuesandcellsarerelatedtothecatabolismofnucleicacids,enzymaticdegradationoftissuesanddietaryhabits.Therefore,detectionofelevatedlevelsofthesesubstancescouldbeindicativeofcertaindiseases[1].Manymethods,suchasthespectroscopicmethodscoupledwithchromatographyorelectrophoresis[1–6]andelectrophoresiswithelectrochemicaldetection[7–9],havebeendevelopedforthedetectionandquantifi-cationofpurinebasesinnucleicacids.Inaddition,voltammetrictechniquesarealsosuitablefortheanalysisofthesepurinesinnucleicacidsduetoadvantagesincludinghighsensitivityandselectivity,fastresponse,lowcostandultra-smallsamplevolumes.Butpurineandpyrimidinebasesusuallyprovidepoorresponsewithhighoxidativepotentialanddisturbeachotheronconventionalelectrodes.Todate,someelectrochemicaldetectionproto-colshavebeendeveloped,suchaselectrochemicallypretreatedglassycarbonelectrode[10],nafion-rutheniumoxidepyrochlorechemicallymodifiedelectrode[11],highlyboron-dopeddiamondelectrode[12],andcobalt(II)phthalocyaninemodifiedcarbonpasteelectrode[13],etc.However,thereareseveresetbacksforpurineelectro-chemicaldetermination,asthesepurinebasescouldirreversiblyadsorbontheelectrodesurfacethushamperingtheestimation.

Carbonnanotubes(CNTs)representanovelcarbonnano-materialofgreatinterest,especiallyinelectroanalysis,duetoitsexcellentperformanceinenhancingtheelectrochemicalreactivity,promotingtheelectron-transferreactionsand

34alleviatingsurfacefouling[14,15].However,thecrucialobstacleofthenanotubesindiverseapplicationsistheirpoorsolubilityandprocessibility,whichiscausedbyinherentattractivevanderWaalsinteractionsbetweennanotubes[16].Asaresult,carbonnanotubesareaggregatedandexistasbundlesintheirnativestate.Theycanbedispersedinsomesolventsbysonication,butprecipitationimmediatelyoccurswhenthisprocessisinterrupted.Toovercomethisdifficulty,thebestmethodtospövetheseproblemsistointroducevariousfunctionalgroupsonthesurfaceofCNTs[17,18],whichcanimprovethedispersionofCNTsinsolutionorcompositematerials.Moreover,variousfunctionalgroupscanbeintroducedthroughfurtherchemicalmodificationonthesidewallofthecarboxylatedCNTs.Thewaytointroducecarboxylicacidgroups(COOH)toCNTswasusuallyacidtreatmentbyoxidation.Therearetwocommonacidtreat-mentsusedintheliterature.Onerefluxesthenanotubeswithasolutionofnitricacid[19–23],andtheotherexposesthesampletoamixtureofHNO3/H2SO4(1:3byvolume)underhighpowersonicationforamaximumof6h[20,21,24–26].Chenetal.[27]haveshownthatanoxidationprocessforsingle-wallcarbonnanotubes(SWCNTs)involvingextensiveultrasonictreatmentinamixtureofHNO3/H2SO4(1:3byvolume)canleadtotheopeningofthenanotubecapsaswellastheformationofholesinthesidewalls.Thefinalproductsarenanotubefragmentswithlengthsintherangeof100to300nm,whoseendsandsidewallsaredecoratedwithahighdensityofvariousoxygencontaininggroups(mainlycarboxylgroups).Nanotubesfunctionalizedinthismannerhaddamagedpristineelectronicandmechanicalproperties.Thus,werequirethedevelopmentofanovelwaytointroducecarboxylicacidgroups(COOH)toCNTsandretaintheirpristineelectronicandmechanicalproperties.

Thispaperpresentsasimpleapproachtotheintro-ductionofcarboxylicacidgroups(COOH)toCNTsandretaintheirpristineelectronicandmechanicalproperties.TheelectrocatalyticactivitytowardstheoxidationofguanineandadenineonthecarboxylatedCNTsmodifiedelectrodehasbeeninvestigatedutilizingcyclicvoltam-metry(CV),andthedetectionlimitandlinearrangeofadenineandguaninehavealsobeenstudiedbylinearsweepvoltammetry(LSV).Itwasfoundthatthismodifiedelectrodeshowedexcellentelectrocatalyticactivityintheoxidationofguanineandadenine,andbasedonthisadirectelectrochemicalmethodwasdevelopedtodeterminetracelevelsofDNA.Tothebestofourknowledge,thepresentstrategyforthepreparationofacarboxylatedCNTsmodifiedelectrodehassofarnotbeenreported,norhastheelectrocatalyticactivitytowardguanineandadenineoxidationbeenstudiedtodate.

X.Tuetal.

ExperimentalsectionInstrumentationandreagents

FTIRspectrawerecollectedonaNEXUS670FTIRspectrophotometer(Nicolet,USA,http://www.thermo.com/).TheCNTwaspressedintoKBrpelletsforFTIRmeasure-ments.Transmissionelectronmicroscopy(TEM)pictureswerecollectedonaJEOL-1230microscope(JEOL,Tokyo,Japan,http://www.jeol.com/)withalineresolutionof0.20nm,operatedatanaccelerationvoltageof100kV.Thesamplesweredispersedinwateranddrop-castontoacoppergridwithaholeycarbonfilm.

AllelectrochemicalexperimentswereconductedonaCHI660Celectrochemicalworkstation(CHInstrumentCo.,USA,http://www.chinstruments.com/).Aconven-tionalthree-electrodecellwasused.Theglassycarbonelectrode(GCE)of3.0mmdiameterservedastheworkingelectrode.ThereferenceelectrodewasaKCl-saturatedcalomelelectrode(SCE),andallpotentialsinthispaperarereportedversusthisreference.Acarbonrodservedasthecounter-electrode.

Multiwalledcarbonnanotubeswithahydroxylgroup(95%purity,diameterof30–50nm,rateofsurfacecarbonatom:20–26mol%)werepurchasedfromtheChengduInstituteofOrganicChemistryoftheAcademyofSciences(http://www.timesnano.com).Adenine(A),guanine(G),andthecalfthymusdsDNAwerepurchasedfromSigma(http://www.sigmaaldrich.com/).Thestocksolutions(1.0mM)ofguanine(G)andadenine(A)werepreparedwithdilutedNaOHaqueoussolution.Theworkingsol-utionswerepreparedbydilutingthestocksolutionwithphosphatebuffersolution.0.10MK2SO4+0.10Mphos-phatebuffersolutionsconsistingofK2HPO4andKH2PO4wereemployedasthesupportingelectrolyte.ThedesiredsolutionpHwasadjustedbydifferentamountsof0.10MK2HPO4andKH2PO4solutions.Allchemicalswereofanalyticalgradeorbetterquality.Allsolutionswerepreparedusingredistilledwater.PreparationofDNAsamples

AgeneraltreatmentofDNAwith1.0mMHClleadstotheselectiveremovalofitspurinebasesbycleavageofpurineglycosidebonds[28].ThecalfthymusdsDNAwashydrolyzedasfollowsforquantificationofguanineandadenine:twomilligramsofdsDNAwasdigestedusing1.0mLof1.0MHClina10mLglasstube.Afterheatinginaboilingwaterbathfor80min,thepHofthesolutionwasadjustedwith1.0mLof1.0MNaOH.Aftercoolingtoroomtemperaturethesolutionwasdilutedto10mLusing0.10Mphosphatebuffersolution(pH7.0).

Novelcarboxylationtreatment35

CarboxylationofCNTs

Scheme1showsthecarboxylationreactionofCNTs.Theprocedurewasperformedasfollows:CNTs-OH(1.0g)wasdispersedin30mLof2.0MNaOHsolutionwhilestirring.110mgNaBH4and3.0mLepichlorohydrinwereaddedat37°C,andtheresultantmixturewasstirredfor10min.15mLof2.0MNaOHsolutionand9.0mLepichlorohy-drinwereaddeddropwiseundervigorousmagneticstirringoveraperiodof2h,andthemixturewasstirredfor24hat37°C.Theprecipitatewascollectedbyfiltrationunderreducedpressureandwashedwithdoublydistilledwater.Afterwards,theprecipitatewasdispersedin40mL2.0MNa2CO3solutionwhilestirring.2.5giminodiaceticacidwasadded,andtheresultantmixturewasstirredfor24hat37°C.Theprecipitatewascollectedbyfiltrationunderreducedpressureandwashedwithdoublydistilledwater.Theprecipitatewasdriedat50°C,andthecarboxylatedCNTswerefinallyobtained(DenotedasCNTs–COOH2).Forcomparison,theCNTs-OHwerefunctionalizedwithcarboxylicacidgroupsbysonicationina3:1sulfuric-acid/nitric-acidmixturefor8h[29].ThepretreatedMWCNTswereneutralizedwith0.10molL−1NaOH,washedwithwater,filtered,anddried(denotedasCNTs–COOH).Electrodemodifications

ThegeneralprocedureofGCEpretreatmentandmodifica-tionwasasfollows:priortouse,theworkingelectrodewaspolishedmechanicallywith0.05μmaluminapowdertoobtainamirror-likesurfaceandthenwashedwithdoublydistilledwaterandacetone.Electrochemicalactivationoftheelectrodewasperformedbycontinuouspotentialcyclingfrom−0.20to1.5Vatascanrateof100mVs−1in0.20molL−1HClO4solutionuntilastablevoltammo-gramwasobtained.Afterrinsingwithdoublydistilledwater,theactivatedelectrodewasmodifiedasfollows:TheCNTs-OHmodifiedGCEwaspreparedbydropping6.0μLofasolutionof2.0mgMWCNTs-OHdispersedin1.0mLofDMFonaGCE,andthenair-dried.TheCNTs-COOHfilmcoatedGCEwaspreparedbydropping6μLsolutionof2.0mgCNTs-COOHdispersedin1.0mLof

Scheme1SchematicdepictionofthecarboxylationofCNTs

OHOH

DMFontheGCE,andthenair-dried.TheCNTs-COOH2filmcoatedGCEwaspreparedbydropping6μLsolutionof2.0mgCNTs-COOH2dispersedin1.0mLofDMFontheGCE,andthenair-dried.

Resultsanddiscussion

CarboxylationofCNTsandcharacterization

Figure1showstheFTIRspectraofCNTs-OH(a)andcarboxylatedcarbonnanotube,CNTs-COOH(b)andCNTs-COOH2(c).ComparedwiththeFTIRspectrumofCNTs-OH,theCNTs-COOHhavesimilarcharacteristics.ForCNTs-COOH,thepeakat3,440cm−1isassignedtotheO-Hstretchingvibrations,thepeaksat1,710cm−1and1,210cm−1maybeassignedtotheC=OandC–Ostretchingvibrations,respectively.Thepeakat1,570cm−1canbeassociatedwiththestretchingofthecarbonnano-tubesbackbone.However,fortheCNTs-COOH2,peaksat2,920and2,850cm−1inthespectrum(c)aregreatlyenhancedbecauseoftheattachmentofadditionalmethy-lenegroups,andpeaksat1,100cm−1maybebecauseoftheexistenceofC-O-C[30].

Figure2showsTEMimagesofCNTs-OH(a),CNTs-COOH(b)andCNTs-COOH2(c).TheCNTs-OHwerehollowropeswithcleanwallsofca.40nmouterdiameter.AftertreatmentoftheCNTs-OHwithacidbyoxidation,theprocessleadstotheopeningofthenanotubecapsaswellastheformationofholesinthesidewalls,introducingdefectsonthewallsofthenanotubes.However,theimagescorrespondingtotheCNTs-OHsubjectedtothecovalentattachmentofCOOHtothesurfaceofCNTsshowthatthenanotubesidewallsdonotchangesignificantly.ThebiocompatiblesolubilizationofCNTsisbelievedtobesignificantforbiologicalapplicationsoftheCNTs.CNTs-OHhaveatendencytoaggregateinwaterduetothestrongvanderWaalsforces,asshownintheinsetofFig.2(a).However,theCNTsfunctionalizedwithCOOHcanformahomogeneousandblackdispersioninaqueoussolution,andthesuspensionwillkeepstableforatleast1week,asshownintheinsetsofFig.2bandc.

CH2COOHCH2COOH

ClHOOH

CH2COOHCH2COOH

36X.Tuetal.

1570

1710

3440

1570

28502920

1210

1710

2500

2000

1500

-1

3440

4000

3500

3000

1570

1000

500

wavelength /cm

Fig.1FTIRspectraofCNTs-OH(a),CNTs-COOH(b)andCNTs-COOH2(c)

Voltammetricstudyofadenineandguanineonthemodifiedelectrode

Inthepresentwork,weexaminetheenhancedelectro-chemicalresponseofadenineandguanineataCNTs-COOH2modifiedelectrode.Figure3illustratestheCVresponseoftheanalytemixtureonthemodifiedelectrodesandbareGCE.Itcanbeseenthat,inthecaseofbareGCE,thevoltammogramsofbasesAandGexhibitjusttwosmallhumppeaks.Comparedwiththeelectrochemicalresponseonthebareelectrode,inthecaseofCNTs-OH/GCE,CNTs-COOH/GCEandCNTs-COOH2/GCE,thepeakcurrentsignalsofAandGwereenhancedsignificantly.Further-more,itisevidentthattheelectrochemicalresponseonthe

CNTs-COOH2/GCEwasmuchstrongerthanthatontheCNTs-OH/GCEandCNTs-COOH/GCE.Nocathodicpeakswereobservedonthereversescanwithintheinvestigatedpotentialrange,indicatingthattheoxidationofGandAiselectrochemicallyirreversible.

WhenimmersingtheCNTs-COOH2/GCEelectrodeintoaquietphosphatebuffer(pH7.0)containingguanineandadeninefor5min,thecyclicvoltammogramshowsverylowresponses.After5minofstirringatopencircuit,however,theresponsesgreatlyimprove.AfterimmersionoftheCNTs-COOH2/GCEelectrodeinthestirringsolutioncontainingguanineandadeninefor5minandtransferringtoapH7.0phosphatebuffersolution,peaksofguanineandadeninecanbeobserved.ThissuggeststhatguanineandadeninecanbeadsorbedontheCNTs-COOH2/GCEelectrodesurface.Thepeaksofguanineandadeninealmostdisappearedinthesecondcycle.Thisphenomenonmaybepartlyattributedtotheconsumptionofadsorbedguanineandadenineandtheadsorptionofelectrochemicaloxida-tionproductsattheCNTs-COOH2/GCEsurface.

ToinfermoreaboutthereactionprocessofCNTs-COOH2/GCE,westudiedthescanrateeffectontheguanineelectrochemicalresponse(seeFig.4).ThepeakcurrentsofCNTs-COOH2/GCEinthepresenceof20μMAandGinphosphatebuffersolutionaredirectlyproportionaltothescanrateovertherangeof25–400mVs−1,furtherconfirmingthatAandGwereadsorbedonthesurfaceoftheelectrode.Inaddition,withincreasingscanrate,theoxidationpeakpotential(Ep)shiftstomorepositivevaluesandthereisalinearcorrelationbetweenthepeakpotentialandthelogarithmofthescanrate,logv.Thelogarithmplotpeakcurrent(logipa)vs.logarithmofscanrates(logv)ofGandAhavealinearrelationship,withaslopeof0.732(logipavs.logv,R=0.998)and0.731(logipavs.logv,R=0.999),respectively.Thesevaluesarebetween0.5and1.Theseresultsindicatethatthe

electrode

Fig.2TransmissionelectronmicrographsofCNTs-OH(a),CNTs-COOH(b)andCNTs-COOH2(c).Insetsshowthephotographsof2mgmL−1CNTsdispersionsintowaterfor1week

Novelcarboxylationtreatment

0.2

0.4

0.6

0.8

1.0

1.2

1.4

E /V vs SCE

Fig.3Cyclicvoltammetriccurvesin0.10MpH7.0phosphatebuffersolutioncontaining20μMguanineand20μMadenineatbareGCE(a),CNTs-OH/GCE(b),CNTs-COOH/GCE(c)andCNTs-COOH2/GCE(d).Accumulatedatanopencircuitpotentialfor240s,scanratewas100mVs−1

Ep=1.130–0.058pH(peakG,r=0.9945)andEp=1.451–0.060pH(peakA,r=0.9975),respectively.Theoxidationofadenineandguaninefollowsatwo-stepmechanisminvolvingthetotallossof4e-andthefirst2e-oxidationistherate-determiningstep.Theslopesof58and60mV/pHshowsthattwoprotonstakepartintherate-determiningstep[33].TheinfluencesoftheamountofCNTs-COOH2onthepeakcurrentwasexaminedbyCV.However,theoxidationpeakcurrentsofadenineandguaninearecloselyrelatedtotheamountofCNTs-COOH2.TheoxidationpeakcurrentincreasesgreatlywhentheCNTs-COOH2amountimprovesfrom0to9μL,andfinallydecreaseswhentheCNTs-COOH2amountishigherthan9μL,beingdueprobablytothelimitedmasstransportofadenineandguanineinsideathickerfilm.Meanwhile,thebackgroundcurrentgraduallyincreaseswhiletheCNTs-COOH2amountincreases.Inthiswork,theamountofCNTs-COOH2waschosentobe9μLforahigherpeakcurrentandalowerbackgroundcurrent.Voltammetricseparationdeterminationofadenineandguanine

Figure5showstheLSVwhen2.0μMguanineandadenineinconcentrationsfrom0.05to10.0μMcoexistinthesolution.Ascanbeseen,thecompetitiveadsorptionphenomenononCNTs-COOH2isnegligible,andthelinearrelationbetweenadenineconcentrationandthepeakcurrent(Ipa∼CA)isavailableintheinvestigatingcondition,whichilluminatesthatthepresenceofcertainconcentrationsofguaninedonotinterferewithadeninedetermination.Itisthereforepossibletodeterminequantitativelywithoutanyconsiderablerecip-rocalinfluenceoveralargerrangeofconcentrations.

Similarly,undertheexperimentalconditions,andinthepresenceof2.0μMadenine,theLSVpeaksofaseriesofguanine(withdifferentconcentrations)arespikyandhavea

1.10.90.8

-3.0

processwassimultaneouslyinfluencedbydiffusionandadsorption[31].Inthiscase,thechargetransfercoefficient(a)canbeworkedoutfromthefollowingequations[32],whichis0.66and0.68forAandG,respectively.Epa¼E0þ

2:3RTð1ÀaÞnFv

log

0ð1ÀaÞnFRTKs

ð1Þ

TheeffectofpHontheelectrochemicalresponseofthe

modifiedelectrodetowardsthesingledeterminationofadenineandguaninewasstudiedovertherangeof4.0–9.0.TheoxidationpeakcurrentofadenineandguanineatthemodifiedelectrodedecreasesslightlywithanincreaseofthepHvalue.ThepHdependenceofoxidationpeakpotentialsofguanineandadenineobeystheequations

900

[**************]0-150

0.70.6

logIpa

-3.6

-4.2

0.300.450.600.750.901.051.201.35

-4.8

E /V vs SCE

Fig.4aScanratedependenceofthecyclicvoltammetricresponseofCNTs-COOH2/GCEin0.10MpH7.0phosphatebuffersolutioncontaining20μMguanineand20μMadenine.Scanratefrominnertoouter:25,50,75,100,200,300,400mVs−1.Insetshowsplotof

logv

anodicpeakcurrentvs.scanrate.bPlotsofanodicpeakpotentialandthelogarithmofanodicpeakcurrentsasfunctionsofthelogarithmofthescanrate

Epa /V

1.0

200175

[1**********]

50250

0.300.450.600.750.901.051.201.35E/V vs SCE

Fig.5Linearsweepstrippingvoltammogramsofguanineatvariousconcentrations(0.2–10.0μM)in2.0μMadeninesolution.Inset:plotofpeakcurrentsvs.guanineconcentrations.OtherconditionswerethesameasinFig.3

favorablelinearrelationship(seeFig.6).Comparedwiththereportedresultsforguanineandadenine,thehighlinearlimitisimprovedupto10μM.Theseobservationsclearlydemonstratethatthetwopurinebasescanbeestimatedfromamixtureintheconcentrationrangestudied.Here,thedetectionlimitsofGandAforindividualanalysisarecalculatedtobe0.02and0.08μM(withS/N=3criterion),respectively.Thus,thismethodallowssimultaneousandsensitivedeterminationofguanineandadenine.Analyticalapplication

Theacid-denaturedDNAproducestwowell-definedpeaksattheCNTs-COOH2/GCEduetotheoxidationofguanine

225

[***********]0250

0.300.450.600.750.901.051.201.35

E /V vs SCE

Fig.6Linearsweepstrippingvoltammogramsofvariousconcen-trationsofadeninefrom0.2to10.0μMin2.0μMguaninesolution.Inset:plotofpeakcurrentsvs.adenineconcentrations.OtherconditionswerethesameasinFig.3

X.Tuetal.

E /V vs SCE

Fig.7Linearsweepstrippingvoltammogramsforthesimultaneousdeterminationofguanineandadenineinacid-denatured−1DNAwithincreasingconcentrationfrom0.50to25μgmL.Inset:plotofthepeakcurrentsvs.acid-denaturedDNAconcentrations.Othercon-ditionswerethesameasinFig.3

andadenineresidues.Figure7showsthetypicalLSVresponsesofguanineandadenineresidueswithincreasingDNAconcentrationsrangingfrom0.50to25μgmL−1.TherelationshipbetweenthepeakcurrentandDNAconcentra-tionislinearintherangeof0.50–15μgmL−1.

Thedeterminationofguanineandadenineconcentra-tionswasperformedbythestandardadditionmethodasfollows.TwentymicrolitersofdenaturedDNAwasaddedtoacellcontaining10mLpH7.0phosphatebuffersolution,thenthepeakcurrentsofguanineandadenineresiduesweremeasured.Subsequentlyacertainquantityofguanineoradeninesolutionwasadded,andthepeakcurrentofguanineoradeninewasmeasuredagain.TheconcentrationsofguanineandadenineinDNAcanbecalculatedfromthepeakcurrentusingthecalibrationgraphobtainedpreviouslyforsimultaneousdeterminationofguanineandadenine.TheresultsaregiveninTable1.Whenusingthemethodpresented,avalue(G+C)/(A+T)of0.81wasobtainedforacalfthymusDNAsampledigestedwithHCl,whichcoincidedwiththestandardvalueof0.77[34].Thedeterminationlimitof0.08μg·mL−1wasalso

Table1ContentofguanineandadenineinHCl-digestedcalfthymusDNAsimultaneouslydeterminedwiththeCNTs-COOH2/GCE

CalfthymusDNAtreatedwithHClGuanine(mol%)22.41Adenine(mol%)

27.67Molarratio(G+C)/(A+T)

0.81

Novelcarboxylationtreatmentobtainedforthecalf−1thymusDNA.Tencontinuousmeasure-mentsof1μg·mLcalfthymusshowedagoodreproduc-ibilitywitha2.6%R.S.D.

Conclusions

Insummary,wehavedevelopedanovelrouteforthecarboxylationofmultiwalledcarbonnanotubes.Ourobserva-tionsindicatethatthecarboxylatedCNTshaveexcellentfunctionalpropertieswithgoodelectrochemicalactivityonguanineandadeninebiomolecules.ItappearsthattheexcellentelectrochemicalbehavioroftherelevantbasesmaycomefromtheselectiveaccumulationonthecarboxylatedCNTselectrodesurface.Therefore,thissimpleandreliablestrategybasedonanelectrochemicaltechniqueatCNTs-COOH2/GCEcouldbeusedforthedevelopmentofsensitivevoltammetricbiosensorsforsimultaneousdeterminationofguanineandadenineintherelatedbiologicalprocess.

AcknowledgementsThisworkwassupportedbytheNationalOutstandingYouthFoundationsofChina(No.50725825),NationalNaturalScienceFoundationofChina(No.50908113,20765003),theNaturalScienceFoundationofJiangxiProvince(No.2008GZH0008),theYouthFoundationofJiangxiProvincialDepartmentofEducation(No.GJJ09483),andtheOpeningFundoftheKeyLaboratoryofChemicalBiologyandTraditionalChineseMedicineResearch(MinistryofEducationofChina),HunanNormalUniversity(No.KLCBTCMR2008-08).

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