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ElectrochemistryCommunications
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Novelelectrochemicalaptamerbiosensorbasedongoldnanoparticlessignalamplificationforthedetectionofcarcinoembryonicantigen
HuaweiShu,WeiWen,HuayuXiong,XiuhuaZhang,ShengfuWang⁎
HubeiCollaborativeInnovationCenterforAdvancedOrganicChemicalMaterials,MinistryofEducationKeyLaboratoryfortheSynthesisandApplicationofOrganicFunctionalMolecules&CollegeofChemistryandChemicalEngineering,HubeiUniversity,Wuhan430062,PRChina
articleinfoabstract
Anovelelectrochemicalaptamerbiosensorwasdesignedbasedonthesignalamplificationofgoldnanoparticles(AuNPs)forthedetectionofatumorbiomarker,carcinoembryonicantigen(CEA).TheelectrochemicalbiosensorwasconstructedbysandwichingtheCEAbetweenanAuelectrodemodifiedwiththiol-terminatedCEAaptamer-1(Apt1)andtheAuNPswiththiol-terminatedCEAaptamer-2(Apt2)and6-ferrocenylhexanethiol(Fc).Amper-ometricdetectionofFcbydifferentialpulsevoltammetry(DPV)ontheelectrochemicalbiosensorwasusedtoquantifytheconcentrationofCEA.Thebiosensorprovidedalinearrangefrom1to200ng/mLforCEAwithade-tectionlimitof0.5ng/mL.ItsperformancewassuccessfullyevaluatedwithhumanserumspikedwithCEA,indi-catingthattheaptasensorhasgreatpotentialforpracticalapplication.Inaddition,theelectrochemicalbiosensorexhibitedexcellentselectivityresponsesandgoodstabilitytowardthetargetanalyte.
©2013ElsevierB.V.Allrightsreserved.
Articlehistory:
Received15August2013
Receivedinrevisedform7September2013Accepted17September2013Availableonline5October2013Keywords:
CarcinoembryonicantigenAptamer
ElectrochemicalbiosensorGoldnanoparticles
1.Introduction
Carcinoembryonicantigen(CEA)isa180kDahighlyglycosylatedproteinover-expressedonbreast,colonandothercancercells[1].Argu-ably,itisthebeststudiedtumorepitopeandpresentonthelargestnum-beroftumors.These,andotherconditions,leadtoanincreaseinbloodCEA.Hence,clinically,serumCEAlevelsmaybeindicative(butnotdiag-nostic)ofthereturnofactivemetastaticdisease[2].Theusualtechniqueusedforthedeterminationoftumormarkersisimmunoassay.Immuno-assays,includingradioimmunoassays[3],enzymeimmunoassays[4],fluoroimmunoassays[5],andpiezoelectricimmunosensors[6],haveal-readybeenreportedforCEA.Butaptamers(Apts)havemanyadvantagesoverantibodiesintermsofrepeatablesynthesis,easymodification,long-termstability,lessimmunogenicityetc.,andareincreasinglyusedinbioanalysisandbiotechnology[7,8].
Aptsaresingle-strandedDNAorRNAmolecules.Theyhavebeense-lectedbySELEX(systematicevolutionofligandsbyexponentialenrich-ment)technologyfromacombinatoriallibrarybytheircapabilitytobindaspecifictargetsuchasanucleicacid,proteinortumorcell[9–11].Therearetwomainapproachestoconstructingelectrochemicalaptasensors.Thefirstmakesuseofthefactthataptamerscanreadilyundergosite-specificmodificationduringchemicalorenzymaticsyn-thesistoincorporateparticularreporters,linkers,orothermoieties.Al-ternatively,aptamersecondarystructurescanbeengineeredtoundergoanalyte-dependentconformationalchanges.This,combinedwiththe
⁎Correspondingauthor.Tel.:+862750865309;fax:+862788663043.E-mailaddress:wangsf@hubu.edu.cn(S.Wang).1388-2481/$–seefrontmatter©2013ElsevierB.V.Allrightsreserved.http://dx.doi.org/10.1016/j.elecom.2013.09.018
abilitytospecificallyplacechemicalagents,opensupawealthofpossi-blesignaltransductionschemas[12].Electrochemicalapproachesusingaptamersinanalyticalapplicationshavebecomeincreasinglypopularoverthepasttwodecades[13,14]becauseelectrochemicaltechniqueshavehighsensitivities,easyoperationandlowcost,andaresuitableforminiaturization.
Sofar,CEAAptshavebeenreported[15].However,CEAusuallybindstoonecopyofApt.Inthiswork,wereportanovelvoltammetricassayusingFccappedAuNPs–Apt2conjugatesfortheanalysisofCEAandApt1intheformofsandwichcomplexes.BecausetwodifferentAptsareusedsimultaneouslyforCEAidentification,theidentificationaccuracyishigherthanwithasingleaptamer,resultinginalowback-groundsignalandgoodselectivity.2.Experimentalsections2.1.Reagentsandapparatus
Hydrogentetrachloroaurate(III)trihydrate(HAuCl4·3H2O),tri-sodiumcitrate,6-ferrocenylhexanethiol(Fc),CEAfromhumanfluids(≥95%,SDS-PAGE),humanserumand6-mercapto-1-hexanol(MCH),tris(2-carboxyethy)phosphinehydrochloride(TCEP)werepurchasedfromSigma-Aldrich(USA).Allotherreagentswereofanalyticalgrade.OligonucleotidesweresynthesizedandpurifiedbySangon(Shanghai,China).Thesequencesoftheseoligonucleotidesusedinthisworkarelistedasfollows:
Apt1:3′-SH-ATACCAGCTTATTCAATT-5′;Apt2:3′-SH-AGGGGGTGAAGGGATACCC-5′.
16H.Shuetal./ElectrochemistryCommunications37(2013)15–19
TheelectrochemicalmeasurementswerecarriedoutonaCHI660Celectrochemicalworkingstation(CHInstruments,Inc.Shanghai)usingathree-electrodesystem.2.2.PreparationofAuNPs
AuNPswerepreparedaccordingtothemethodreportedpreviously[16].2.7mLof1%trisodiumcitratewasaddedto100mLofboiling0.01%HAuCl4solutionandstirredfor10minattheboilingpoint.ThepreparedcolloidAuNPswerestoredinbrownglassbottlesat4°C.ThefinalAuNPshadanaveragediameterofapproximately13nmasmeasuredbyaTransmissionElectronMicroscope(TEM)asshowninFig.1A.2.3.PreparationofFccappedAuNPs–Apt2conjugates
ThemodificationofAuNPswithFcandApt2waspreparedaccordingtothemethodreportedbyJ.Wangetal.[17].A500μLcolloidgoldsolu-tionwasmixedwith100μLofhexanecontaining5.0mMFcand200μLApt2for24honavortexstirrer.TheresultantmodifiedAuNPsremainedintheaqueousphase.Thesolutionwascentrifuged,andthehexanephasecontainingFcwasdecanted.TheAuNPscappedwithFcandApt2werethoroughlyrinsedwith500μLhexane,resuspendedin500μLof0.1mol/Lphosphatebufferedsaline(PBS,pH7.0).Followingcentrifugationofthemixturetoremovethesupernatant,theresultingFccappedAuNPswerewashedandredispersedin500μLPBSandstoredat4°Cforfurtheruse.
2.4.FabricationofthebiosensorandtheelectrochemicaldetectionThebareAuelectrodewaspolishedwith0.05mmaluminaslurriesandultrasonicallycleanedinethanolandultrapurewatertwicefor5min.TheAuelectrodeswerethenputin5%H2SO4solutionandscannedfrom0to1.6Vtomeasurethecyclicvoltammetry(CV)signalsuntilastandardcyclicvoltammetricpeakappeared.AfterwashingwithultrapurewateranddryingwithastreamofN2,thepre-cleanedAuelectrodewasincubatedfor1hatroomtemperaturein50μLof1μMApt1including10μLof5μMTCEPandwashedwithultrapurewater.Thentheelectrodewasimmersedin50μLof1mMMCHfor1htoobtainawell-alignedDNAmonolayer.Next,thiselectrodewasimmersedin50μLCEAsolution(solutionin0.1MPBS,pH7.4)for30min,rinsedwithPBStoremovethenonspecificallyboundCEA,andwasthenimme-diatelysoakedin50μLFccappedAuNPs–Apt2conjugatesforanother30min.TheelectrodeabovewasincontactwithPBSandscannedfrom0to0.6VtomeasuretheDPVsignals.Thewholepreparationpro-cessisoutlinedinFig.2.3.Resultsanddiscussion
3.1.Characterizationofbiosensorfabrication
Electrochemicalimpedancespectroscopy(EIS)andCVmeasure-mentswereusedtocharacterizetheelectrochemicalbiosensor[18].InthetermsofEIS,[Fe(CN)6]3−/[Fe(CN)6]4−wasutilizedastheredoxprobeandthesemicirclediameterwasequaltoelectron-transfer
A
18000160001400012000100008000600040002000
0
0
Babcd1000020000300004000050000
6420-2-4-6-8
Cad0.160.140.120.100.080.060.040.020.00
D
0.00.10.20.30.40.50.64:12:14:31:12:3
Potential / V
−/4−Fig.1.CharacteristicsofAuNPsTEM(A)andEIS(B)andCV(C)in0.5mMFe(CN)3atbareAu(a),Apt1/Au(b),CEA/Apt1/Au(c)andFc–AuNPs–Apt2/CEA/Apt1/Au(d)electrodes.Op-6timizationofexperimentalconditionforDPVresponsetothedifferentvolumeproportionsofAuNPsmodifiedFc(5mM)andApt2(1μM)(D).
H.Shuetal./ElectrochemistryCommunications37(2013)15–1917
HSMCHAu0.400.350.30
a
Current/uA0.250.200.150.100.05
b
ab0.00
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Potential/V
SHAU NPsAU NPsSHCEAMCHSHSHSHSH-CEA aptamer-1SH-CEA aptamer-26-ferrocenylhexanethiolFig.2.SchematicdiagramabouttheelectrochemicalaptamerbiosensorbasedonAuNPssignalamplificationforthedetectionofCEA.
−/4−resistance.In0.5mMFe(CN)3,bareelectrodeexhibitedalmosta6straightline(Fig.1B,curvea),whichwascharacteristicofamassdiffu-sionlimitingstepoftheelectron-transferprocess.WhentheApt1wasself-assembledontothebareelectrodeviaAu\\Sbinding,theRetin-creased(Fig.1B,curveb),thiswasbecausethatthenegativelychargedphosphatebackboneoftheoligonucleotidesproducedanelectrostaticrepulsionforceto[Fe(CN)6]3−/[Fe(CN)6]4−.Itisalsofoundthattheas-semblyofCEAontheApt1modifiedelectrodeleadstoasignificantincreaseinRet(Fig.1B,curvec).AfterhybridizationwithApt2–AuNPs–Fc,thereisalargeincreaseinRet(Fig.1B,curved),implyingthattheintroductionofApt2–AuNPs–Fcgreatlyinhibitstheelectrontransferoftheredoxprobeontheelectrodesurface[19].TheseresultswereinagoodagreementwiththoseobtainedfromCVmeasurements(Fig.1C).Astheabovefigureshows,bothresultsofEISandCVdemon-stratethatthesensinginterfacehasbeenfabricatedsuccessfully.
3.3.Optimizationofincubationtime
ItwasfoundthatchangingtheincubationtimeoftheApt1modifiedelectrode(Fig.3B,curvea)andApt2–AuNPs–Fc(Fig.3B,curveb)inCEAsolution(50ng/mL)causedavisibledifferenceintheincreaseofpeakcurrent.Therefore,thedependenceofCEAincubationtimeonthein-creaseofthepeakcurrentwasstudiedtodeterminetheoptimumincu-bationtimeofCEA.AsshowninFig.3B,thecurrentresponseincreasedwiththeincreaseofincubationtimeandalmostleveledoffafter30min,whenthebuildingoftheApt1–CEA–Apt2–AuNPs–Fccomplexreachedsaturation.Therefore,30minwaschosenastheincubationtimeforthedetectionofCEA.3.4.Performanceofaptasensor
3.4.1.Calibrationcurveofaptasensor
TheaptasensorswereincubatedindifferentconcentrationsofCEAundertheoptimalconditionsandtheDPVresponsesoftheproposedaptasensorwererecorded.AsseeninFig.3C,whichdisplaysthecorre-spondingcalibrationplots,thecurrentwasproportionaltoCEAconcen-trationovertherangefrom1to200ng/mL.Thelimitofdetectionwas0.5ng/mL(S/N=3).ThelinearequationcouldbefittedasI(A)=1.68E−8+0.9373c(g/mL)(R=0.9978),whichislowerthanthede-tectionlimitof1.1ng/mL[20]obtainedusingimmunosensors.3.4.2.Specificity
ThespecificityoftheproposedaptasensorwasalsoexaminedbydetectingtheDPVchangeinthepresenceofCEAandthreeinterferingagents:myoglobin(MYO),mucoprotein(MUC),andbovineserumal-bumin(BSA).TheexperimentalresultsareshowninFig.3D:CEA(100ng/mL)producedamuchstrongercurrentresponse,whiletheotherinterferingagents(1μg/mL)causedalmostnegligibleelectro-chemicalchanges.
3.2.Optimizationofexperimentalconditions
AsshowninFig.1D,whentheratiowaslessthanthebestpropor-tion,becausetheamountofFcwasless,theDPVsignalwasrelativelylow;whentheratiowasgreaterthanthebest,becausethequantityofApt2wasless,affectingtheamountofAuNPscompoundscombinedwiththeCEA,thereisadecreaseintheelectrochemicalsignal.There-fore,theoptimizedvolumeproportionofAuNPsmodifiedFc(5mM)andApt2(1μM)wasdeterminedtobe1:2.
IntheoptimizedvolumeproportionofAuNPsmodifiedFc(5mM)andApt2(1μM),thedosageoftheApt2determinedtheefficiencywithwhichtheApt2bindstoCEA,andwouldaffecttheelectrochemicalsignal.AsshowninFig.3A,withanincreaseintheamountofApt2,thesignalintensitygraduallyincreasedandthenwasnearlybalancedwhentheamountofApt2was200μL.Therefore,theoptimaldosageoftheApt2was200μL.
18H.Shuetal./ElectrochemistryCommunications37(2013)15–19
0.070.120.100.080.06A0.060.050.040.03B
ab0.040.020.020.010.000.00501001502002503000102030405060VCEA-apt 2/µL 0.25Time/min0.200.160.120.080.040.000.0R=0.997840.200.150.100.050.00y=1.68E-8+0.9373x200ngC0.16D
1ng0.120501001502000.080.040.000.10.20.30.40.50.6CEAMYOMUCBSAFig.3.OptimizationofexperimentalconditionforDPVresponseto1μMApt2ofdifferentvolumes(A)andDPVpeakcurrentsplottedagainstincubationtimeoftheApt1modifiedelec-trode(a)andApt2–AuNPs–Fccompounds(b)inCEA(50ng/mL)solutionat37°C(B).DPVof1,5,10,25,50,75,100,150,and200ngCEAinthe0.1MPBS(pH7.0).Insert:linearrela-tionshipbetweentheincreasepeakcurrentandtheconcentrationofCEA.Theselectivityoftheaptasensor:CEA(100ng/mL),MYO(1μg/mL),MUC(1μg/mL),BSA(1μg/mL)(D).
3.5.Analyticalapplicationoftheproposedaptasensor
Toevaluatetheapplicabilityoftheproposedaptasensor,theconcen-trationofCEAinhumanbloodserumsamplewasdeterminedbythestandardadditionmethod.Inthistest,30μLoforiginalhumanserumwasdilutedto3.0mLwithPBS.ThendifferentamountsofCEA(100,120,140ng)wereaddedin1mLdilutedhumanserumsamplestopre-parethreeserumspecimenscontainingCEA.Eachsamplewasdetectedthreetimes.Thedetectionvaluewastheaverageofthreeresults.TheanalyticalresultsareshowninTable1.Therecoverywasintherange95.6–101.0%,indicatingthattheaptasensorhadgoodaccuracyandhadgreatpotentialfortheanalysisofCEAinrealclinicalsamples.4.Conclusion
Insummary,anovelelectrochemicalaptasensorforthedetectionofCEAhasbeendeveloped.Unlikemostexistingelectrochemicalaptasensors,twodifferentAptswereusedsimultaneouslyforCEAiden-tificationbyusingApt2–AuNPs–FcconjugatesfortheanalysisofCEA
andApt1intheformofsandwichcomplexes.Thisproducedbetteriden-tificationaccuracythanwhenusingasingleaptamer,resultinginalowbackgroundsignalandgoodselectivity.Thiselectrochemicalaptasensorwaseasytoproduce,andmightprovepromisingforapplicationsinbi-ologicalorclinicaltargetanalysis.Acknowledgments
ThisworkwassupportedbytheNationalNaturalScienceFounda-tionofChina(No.21175032)andtheNaturalScienceFundforCreativeResearchGroupsofHubeiProvinceofChina(No.2011CDA111).References
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Table1
DeterminationsofCEAinhumanserumsamples.Samples123
Added(ng/mL)100.0120.0140.0
Found(ng/mL)95.6117.8141.4
Recovery(%)95.698.2101.0
RSD(%)4.91.73.1
H.Shuetal./ElectrochemistryCommunications37(2013)15–19
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19
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