食品中的有害物质

10ColorimetricbiosensorforfoodchemicalhazardsdetectionJianlongWang1andH.SusanZhou21CollegeofFoodScienceandEngineering,NorthwestAgricultureandForestryUniversity,Yangling,Shaanxi,China2DepartmentofChemicalEngineering,WorcesterPolytechnicInstitute,Worcester,MA,USA10.1IntroductionNowadays,foodintakehasbeenidentifiedasthemajorsource(accountingfor>90%)forhumanstobeexposedtocertainchemicalcontaminantscomparedwithinhalationanddermalroutesofexposure(Fries,1995)andabout30%ofhumancancersarecausedbydietbecauseofcarcinogenicchemicalcontaminants(TrickerandPreussmann,1990).Thereasonisthatfoodiseasilycontaminatedbythousandsofbiological,chemical,andphysicalagentsinthelongandsophisticatedprocessingchain.Theterm“foodchemicalhazard”isrelatedtoaspecifictypeoffoodcontamination,whichmainlyresultsfromdifferenthazardouschemicalsubstancesinfoodproducts.Accordingtothecontaminationsourceandmechanismofactionofhazardouschemicals,foodchemicalhazardscanbeclassifiedintogroupssuchaspesticideresidues,veterinarydrugresidues(includingantibiotics,fluoroquinolones,hormonedrugs,nitrofuran,andantiparasiticagents),foodadditives,foodpackagingmaterials,persistentorganicpollutants,heavymetals,syntheticcompounds,toxins,andmycotoxins.Chemicalhazardsinfoodmatriceshavereceivedincreasingattentionowingtotheirgreaterharmfuleffectsonhumanhealthcomparedwithbiologicalandphysicalhazardousfactors.HencemonitoringandtestingofchemicalcontaminantsinfoodproductsareessentialforunderstandingandmanagingpotentialriskstohumanFoodChemicalHazardDetection:DevelopmentandApplicationofNewTechnologies,FirstEdition.EditedbyShuoWang.©2014JohnWileySons,Ltd.Published2014byJohnWileySons,Ltd.292CH10COLORIMETRICBIOSENSORhealthandtheenvironment.Forfoodchemicalhazarddetection,therearephysicochemicalconventionalmethodssuchashighperformanceliquidchromatography(HPLC),liquidchromatography–tandemmassspectrometry(LC–MSMS),andsoon.Althoughthesemethodshavebeenwidelyused,theyarecostlyandcomplicated,andcannotmeettheneedsofthefoodindustry.Colorimetricbiosensorsasanemergingandgrowingapplicationinfoodanalysishasbeenattractingincreasinginterestfromgovernmentregulatorsandfoodanalysts,andhavebeendevelopedforthescreeningofhazardouschemicals.Advantagesofferedbycolorimetricbiosensorsoverotherassays(suchasradioimmunoassay,enzymelinkedimmunosorbentassay,fluorescenceimmunoassay,andluminescenceimmunoassay)includesimplicity,onsiteapplication,lowcost,portability,automation,improvedreproducibility,andrapidexecution.Colorimetricbiosensorscurrentlyplayanimportantroleinthefieldoffoodsafety,andfurtheradvancesinnanotechnology,reagents,andsamplehandlingwillsurelyreinforcetheirposition.Theintentionofthischapteristopresenttheadvancesanddescribethetrendsincolorimetricbiosensorsforthedeterminationoffoodhazardouschemicalssuchasheavymetals,toxins,pesticides,andveterinarydrugresiduescoupledwithbioandnanotechnologies,namelyusinggoldnanoparticles(AuNPs)silvernanoparticles(AgNPs),aptamers,andGquadruplexbasedDNAzymesforsignalconversion(Table10.1).Themechanismoftheapplicationofdifferentcolorimetricbiosensorsisalsoelaborated.Table10.1TypesofcolorimetricbiosensorsfordetectionoffoodhazardouschemicalsTargetColorimetrictypeRealsampleReferencePesticidesAuNPsVegetablesXuetal.(2011)LAAuNPsFruitmatrixSunetal.(2011a)Ag@SiO2NH2NPs–Lietal.(2011c)VeterinarydrugsAuNPsHumanurineZhangetal.(2012c)AptamerAuNPsMilkSongetal.(2012)MPAAuNPsRawmilkSunetal.(2011b)HeavymetalsPAuNPsRiverwaterZhangetal.(2012b)QAAuNPsDrinkingwaterLiuetal.(2010)AptamerAuNPs–Lietal.(2009a)DNAzymePaintLiuandLu(2004)IDAAgNPsTapwaterQietal.(2012)FoodadditivesAgNPsRawmilkPingetal.(2012)AuNPsRawmilkLietal.(2010a)DNAAuNPsMilkHuangetal.(2011)4ATPGNRsDrinkingwaterXiaoandYu(2010)ToxinsAptamerAuNPs–Yangetal.(2011)DNAzymeWinesamplesYangetal.(2012)OtherhazardouschemicalsAptamerAuNPsWatersamplesMeietal.(2013)pSC6AuNPs–Hanetal.(2009)10.2DETECTIONOFHAZARDOUSCHEMICALSINFOODS29310.2DetectionofhazardouschemicalsinfoodsusingcolorimetricbiosensorsbasedonbionanomaterialsColorimetricbiosensorsbasedonbionanomaterialsaredesignedfortheanalysisarea,particularlywhenfunctionalgoldnanoparticles(AuNPs)silvernanoparticles(AgNPs)orfunctionalnucleicacids(aptamersandDNAzymes)areusedasnovelsensingprobes,andhavebecomeanadvantageousdetectiontechnologyforscreeningforchemicalcontaminantsinthefoodindustry.Inthisfield,theAuNPbasedcolorimetricbiosensorhasbeenextensivelyappliedforthedetectionofhazardouschemicalsinfoodsafetybecauseitexhibitssomeimportantfeaturessuchasrapidity,specificity,sensitivity,portability,selectivity,and,moreimportantly,simplicityinidentifyinganalytesofinterest.Specifically,comparedwithothermetalnanomaterials,AuNPspossessdistinctphysicalandchemicalattributesthatmakethemexcellentscaffoldsforthefabricationofnovelchemicalandbiologicalcolorimetricsensors(Haick,2007;Zhaoetal.,2008;BoisselierandAstruc,2009;RadwanandAzzazy,2009;BunzandRotello,2010;Zengetal.,2011;Sahaetal.,2012;Upadhyayula,2012).First,AuNPspossessstrongcompatibilitywithvarioustypesofchemically(includingorganicandmetallic)andbiologicallyactivemolecules.Thisfeaturemakesitpossibleforactivemoleculestomaintaintheirphysical,chemical,orbiologicalactivityunaffectedeventhoughtheyhavebeenimmobilizedonAuNPs;furthermore,thechanceofinteractionofactivemoleculeswithtargetchemicalsisincreased.Second,whensynthesizedusingtheclassicalcitratemethod,AuNPsholdchargeproperties,whichincreasestheopportunityforinteractionwithoppositelychargedmolecules.Third,whenAuNPsarecombinedwithappropriateligands,theycanprovidebiosensorswithahighsurfaceareatovolumeratioforbiorecognitionbetweendetectiontargets.Fourth,AuNPscanalsobetunedbyalteringtheirshape,size,andthesurroundingchemicalenvironmentaccordingtodifferentfabricationprinciples.Forexample,ameasurablesignalisgeneratedfromtothebindingeventbetweentherecognitionelementandtheanalyte,whichleadstoachangeinphysicochemicalpropertiessuchaselectricalconductivity,plasmonresonanceabsorption,orredoxbehavior.Additionally,byusingmorethanonedetectionmethodology,suchasspectroscopic(Julienetal.,2013),colorimetric(Duetal.,2012),fluorimetric(Luetal.,2013),andelectrochemical(Gaoetal.,2013)means,thesameanalytecanbedetectedusingmorethanonedetectionplatformsupportedbyAuNPs(Huetal.,2003;Simonianetal.,2005).Inrecentdecades,theuseofAuNPsasnovelcolorimetricprobestobuildcolorimetricbiosensorshasprovidedwidelyinnovativeapproachesforthedetectionofhazardouschemicalsinfoodsinarapidandefficientmanner.Thispartofthechaptersummarizesrecentadvancesincolorimetricbiosensorsforthedeterminationoffoodchemicalhazards.Italsoattemptstoprovidegeneralguidanceonhowtodesignandapplysuchdetectiontechnologyinthefoodindustry.InordertofabricatetheAuNPbasedcolorimetricsensingplatform,thekeyisthecontrolof294CH10COLORIMETRICBIOSENSORcolloidalAuNPdispersionandaggregationstagesbasedonbiologicalprocessesbetweenarecognitionelementandanalytesofinterest(interparticlebiorecognitionbasedonDNA–DNA,aptamer–target,antibody–antigen,streptavidin–biotin,lectin–sugar,metal–ligand,andotherchemicalinteractions).Consequently,theinterparticleattractiveandrepulsiveforcesbetweenAuNPs,whichdeterminewhetherAuNPsarestabilizedoraggregated,thuscausingthecolorofthesolution,arecentralinthedesignofsuchcolorimetricsensingsystems.Generally,therearetwomechanismsofaggregationofAuNPsincolorimetricbiosensors:,theinterparticlecrosslinkingmechanisminwhichtheinterparticlebondingformationovercomesinterparticlerepulsiveforces,andthenoncrosslinkingaggregationmechanismwherethecontrolledlossofstabilityofcolloidalAuNPsorthevanderWaalsattractiveforcesovercomeinterparticlerepulsiveforces(Zhaoetal.,2008).Inthiscase,thecolorofsolutionchangesfromredtopurple(orblue)whenAuNPaggregationoccurs,andfrompurpletorediftheAuNPaggregateisredispersed.Asanothersensingprobeincolorimetricbiosensors,thefunctionalnucleicacids(e.g.,aptamersandDNAzymes)haverecentlybeenemployedfortheanalysisoftargetsofinterest.Anaptamerisaspecificnucleicacidsequencethatwasfirstobtainedbythreedifferentlaboratoriesin1990usinganinvitroselectionprocessknownassystematicevolutionofligandsbyexponentialenrichment(SELEX)fromalibraryoflargerandomnucleicacids(EllingtonandSzostak,1990;RobertsonandJoyce,1990;TuerkandGold,1990).Sincethen,aptamerbasedbiosensorshavebeendevelopedfortargettestingsuchasproteins(Rotemetal.,2012),metalions(Kimetal.,2012),adenosine(Lietal.,2012b),explosives(Hoetal.,2012),EscherichiacoliO157:H7(Wuetal.,2012a),andcancerbiomarkers(Ilyasetal.,2012).Amongthesebiosensors,thecolorimetricbiosensorissimpleandconvenientaccordingtoitsdesignprinciple,andcanalsobeusedtodetecthazardouschemicalsinfoods.Further,theguaninerichsequences,whichcanformaGquadruplexstructure,areusedtobuildperoxidaselikeDNAzymeswhentheybindwithhemin.SuchDNAzymes,firstreportedinthe1990s(LiandSen,1997;Travascioetal.,1998),havemorerecentlyattractedgreatinterestinbiology,medicine,andbioanalyticalchemistryowingtotheirthermalstabilityandsimplerpreparation.TheoligonucleotidesmustadoptaGquadruplexstructuretoassuresuchcatalyticactivity,whichcanbindtheheminmoleculetoformGquadruplex–hemincomplexes.Thesecomplexesfacilitatearedoxreactionbetweenthetargetandhydrogenperoxide,whichresultsintheappearanceofcolorofanoxidizedtargetproduct.Suchapeculiarfeaturehasbeenexploitedbymanygroups,utilizingDNAzymesasprobesforchemiluminescenceorcolorimetricdetection,inwhichanintensesignalallowsobservationwiththenakedeyesuchasinvisualcatalysisusingasubstratelikeABTS[2,2′azinobis(3ethylbenzothiazoline6sulfonicacid)]thatcanconverttoagreensolubleproductuponoxidation(Jiaetal.,2012).DNAzymeshavebeenemployedtofabricatecolorimetricbiosensorsforsensingofmetalions(Yinetal.,2009;Qinetal.,2010;Zhuetal.,2011),smallmolecules(Sharonetal.,2010;Duetal.,2011;Yangetal.,2012),andmacromolecules(Pavlovetal.,2004).10.2DETECTIONOFHAZARDOUSCHEMICALSINFOODS29510.2.1ColorimetricdetectionofpesticidesPesticidesarewidelyutilizedinagriculturetocontroleliminateplantpestsanddiseasestoensuremaximumcropyields.Dependingonthepurposeofapplication,pesticidescanbeclassifiedasinsecticides,herbicides,fungicides,andothertypes.Accordingtotheirchemicalstructure,therearenumerousdifferenttypesofpesticidesonthemarket,themostcommoninagriculturalapplicationsbeingorganophosphates,carbamates,pyrethroids,arseniccompounds,andnitrophenolderivatives.Environmentalagencieshavesetmaximumlimitvaluesforthelevelsofpesticidecontaminationindrinkingandsurfacewaters(Richardson,2007;Thomaidisetal.,2012).Inaddition,thecompoundsproducedbydegradationofpesticidescanremaininvegetables,animals,andwatersources,andcanbecomemoreconcentratedastheymoveupthefoodchain(Atreyaetal.,2011).Manyseriousproblemswithfoodsafetyhaveresultedfromalackofsupervisionofgovernmentdepartmentsoftheuseofpesticides.AlthoughHPLCandgaschromatography–massspectrometry(GC–MS)offereffectivetraceanalysiswithhighreproducibilityandlowdetectionlimitsforpesticidedetection(Molinaetal.,1994;Fuerhackeretal.,2008;Lesueuretal.,2008;Fenolletal.,2009;MenezesFilhoetal.,2010),thesemethodsinvolveextensivepurification,includingderivatization,andrequiretrainedpersonnelandexpensiveequipment(Stan,2000).Therefore,forthedetectionofpesticidesandtheirderivatives,thedevelopmentofcosteffective,fast,anduserfriendlyassaymethodsisrequired.Recently,greatprogresshasbeenachievedinemployingdifferentkindsofnanomaterials(e.g.,AuNPs,AgNPs)tofabricatecolorimetricbiosensors(LiuandLin,2007;AragayandMerkoçi,2012).Comparedwithconventionaldetectiontechnologies,AuNPbasedcolorimetricbiosensorassayshaverecentlybecomeusefulforthedetectionofdifferenttypesofanalyteswithouttheneedforsophisticatedinstrumentsbecausemolecularinteractioneventscanbetransformedintovisiblecolorchanges(Cordrayetal.,2012;Derbyshireetal.,2012;Hombergeretal.,2012;Höppeneretal.,2012;Leeetal.,2012;Rosmanetal.,2012).Ingeneral,basedonthedistancedependentopticalpropertiesofAuNPs,thesolutionswilldisplayaredcolorwhenAuNPsarewelldispersed,whereasthosesolutionsthatcontaintargetsofinterestthatcanrapidlyinducetheaggregationofAuNPswillexhibitapurpleorbluecolor(Figure10.1).ThecolorchangeoftheAuNPsolutionisconfirmedbytheobviousshiftoftheabsorptionbandinthevisibleregionoftheelectromagneticspectrum.BasedonsuchcolorchangesofcolloidalAuNPsolutions,differenttypesofAuNPbasedcolorimetricbiosensorshavebeenappliedforscreeningofpesticidessuchasametryn(Zhaoetal.,2011a),cyromazine(Zhaoetal.,2011b),endosulfan(Nairetal.,2003),hexaconazole(Baietal.,2011),cartap(Liuetal.,2012b),andpymetrozine(Baietal.,2010).Forinstance,alabelfreeAuNPbasedcolorimetricbiosensorwasdevelopedforthedetectionofacetamiprid(Xuetal.,2011).TheAuNPsweresynthesizedbytheclassicalcitratereductionmethod(Doronetal.,1995),introducingnegativechargesthroughcitrateionsabsorbedtothesurfaceoftheAuNPs.Together296CH10COLORIMETRICBIOSENSORAuNPstargetFigure10.1SchemeofAuNPaggregationresultinginbindingofthetargetwiththecounterionsinthemedium,thenegativelychargedcitrateionsformedarepulsiveelectricdoublelayertostabilizetheAuNPswhenacetamipridwasnotaddedtotheAuNPsolution.Whenacetamipridwaspresent,itwasadsorbedonthesurfaceoftheAuNPsbecausethecyanogroupofacetamipridhasagoodaffinityforAuNPs.WiththebindingofacetamipridtotheAuNPs,theelectrostaticrepulsionforceoftheAuNPswasdiminishedandaggregationoftheAuNPsoccurred,whichresultedinacolorchangeofthesolutionfromredtopurple.Underoptimumassayconditions,thelinearrangeofacetamipriddetectionwasfrom0.66to6.6μM.Thismethodhasbeensuccessfullyemployedtodetectacetamipridresiduesinvegetables.Organophosphoruscompounds(OPs)arethemostcommonchemicalclassusedinagricultureandanimalhusbandry.ThereisaseriousconcernaboutfoodsafetycausedbyfarmproducecontaminatedwithOPsbecausesomemembersoftheOPpesticidegrouparehighlytoxictoanimalsandhumans.Forexample,theuseofthesecompoundsmayresultinthepoisoningof∼200000peopleperyeararoundtheworld(Kavalcietal.,2009).ForthedetectionofOPs,theuseofacetylcholinesterase(AChE)hydrolysisincolorimetricbiosensorsisawellknownmethod.Lietal.(2011b)employedpositivelychargedacetylthiocholineasasubstrateforAChE.AChEcatalyzestheacetylthiocholinehydrolysistoproducethiocholine,whichispositivelychargedandbearsanadditionalthiolgroup(–SH).Withthischaracteristic,thiocholinecanprefertosubstitutethecitrateandeasilybindtothesurfaceofAuNPs.Asaresult,crosslinkingandaggregationofAuNPswilloccur,andthecoloroftheAuNPsolutionchangesfromclaretredtopurpleorevengray.BecauseOPscanirreversiblyinhibitthecatalyticactivityofAChE,theaggregationofAuNPswillnotoccurinthepresenceofOPs.Similarly,anothergroup(Sunetal.,2011a)designedanAuNPwrappedcolorimetricbiosensorinspikedfruitmatricesforthedetectionoforganophosphatenerveagentsandpesticidesbasedonthecatalyticreactionofAChEandtheaggregationoflipoicacid(LA)cappedAuNPs,obtainingalimitofdetection(LOD)downtopicomolarlevels.Indetail,cationicthiocholine(TCh)wasproducedthroughAChEhydrolysisofacetylthiocholine(ATCh),andcouldinducetheaggregationofLAcappedAuNPsviatheTCh–LAinteractionalongwithadistinctcolorchangefromredtosteelblue.Asenzymeinhibitors,thepresenceofOPsinthissystemcouldsuppresstheproductionofTCh.AssociatedwithdifferentconcentrationsofOPs,theformoftheAuNPswouldbechangedfromanaggregatetowelldispersed,10.2DETECTIONOFHAZARDOUSCHEMICALSINFOODS297AChEAChEATChATChOPlipoicacid(LA)cappedAuNPsthiocholine(TCh)Figure10.2AggregationoftheLAcappedAuNPsinducedbythiocholinereflectingthechangesofAuNPsbothintheabsorbancespectraandinthechangeofcolorinsolution(Figure10.2).Morerecently,anovelcolorimetricbiosensorbasedonrhodamineBfunctionalizedgoldnanoparticles(RBAuNPs)wasdevisedwithdualreadouts(colorimetricandfluorimetric)forOPandcarbamatepesticidesinagriculturalproductsandriverwater(Liuetal.,2012a).Inthisstudy,RBwasappliedasanidealligandbecauseitpossessesinherentproperties(e.g.,photostability,watersolubility,andstrongfluorescence)andcouldreadilyadsorbonthesurfaceofAuNPsviaelectrostaticinteractionsresultinginfluorescencequenching.ATChandAChEwereaddedtoRBAuNPsolution,causingacolorchangeofthesolutionfromredtopurple,alongwiththesimultaneousemergingofthefluorescenceofRB(initiallyquenchedbyAuNPs).ThisphenomenonoccurredbecauseAChEhydrolyzedATCandgeneratedthiocholine,whichcanbindmorestronglythanRBtothesurfaceofAuNPs,thusremovingRBfromtheAusurfaceandrestoringtheRBfluorescence.Meanwhile,thiocholineandtheresidualRBattachedtodifferentAuNPsurfacescouldberecognizedbyelectrostaticinteractions,introducingtheaggregationofAuNPs,whichwasreflectedbytherapidcolorchangeoftheAuNPsolutionfromredtopurple.IfAChEwaspretreatedwithOPsorcarbamates,theAChEactivitywouldbeinhibitedbythesepesticides,thuspreventingthegenerationofthiocholinefromthehydrolysisofATCcatalyzedbyAChE.Asaconsequence,thecoloroftheRBAuNPsolutionremainsredandthefluorescenceofRBmoleculesremainsquenched.Bycontrast,othertypesofpesticidesinrealsamplesareunabletopreventtheAChEfrominducingtheredtopurplecolorchangeorfluorescencerecoveryowingtoitsultrasensitivitycomparedwiththosepreviouslyreported.SimilarlytoAuNPs,recentlyAgNPshavealsobeenusedtofabricatedifferenttypesofcolorimetricbiosensorsforthedetectionoffoodhazardouschemicalssuchaspesticides(XiongandLi,2008;Lietal.,2011c;Chenetal.,2012a).AgNPshavesomeadvantagesoverAuNPsbecausetheypossesshigherextinctioncoefficientsrelativetoAuNPsofthesamesize(Leeetal.,2007).TheinterfacefunctionalizationofAgNPsplaysacrucialroleinincreasingthestabilityandanalytical298CH10COLORIMETRICBIOSENSORapplicabilityofAgNPbasedbiosensorsbecauseofthesusceptibilityofthesilversurfacetooxidation(Yinetal.,2002).ThesurfaceofAgNPsiscommonlymodifiedwithappropriateligandstoenhancedoublelayerinteractionsoradsorbaprotectiveagenttopreventtheaggregationofNPs(Chenetal.,2000).Recently,acolorimetricbiosensorwasdesignedforthedetectionofthepyrethroidlambdacyhalothrin(LC)withaminemodifiedsilver–silicacore–shellnanoparticles(Ag@SiO2NH2NPs)(Lietal.,2011c).AsilicacoatinghaswidelybeenusedtosynthesizewatersolubleNPsbecauseoftheirreusability,stability,andgoodcatalyticproperties.Thesilicashellcanprotectthemetalcoreandintroducenewrecognitionsites.ThestrategywasbasedonthestronghydrogenbondinteractionsbetweenaminegroupsofAg@SiO2NH2NPsandthefluorineinthetrifluoromethylgroupofLC.ThecolorofAg@SiO2NH2NPschangedimmediatelyfromyellowtopinkonadditionofLC.Subsequently,aclearcolorprogressionfromyellowtoorangetoredwithincreasingLCconcentrationwasobserved,whichcanbeusedforthequantitativemeasurementoftheLC.Althoughthemechanismofdetectionseemslogicalintheory,thehighselectivityofthesystemforLCoverotherpyrethroidpesticidesneedsfurtherstudy.10.2.2ColorimetricdetectionofveterinarydrugsDetectionofantibioticsGenerally,antibioticsaredrugswithanantibacterialfunctioninhumansandanimals,andcanbesynthesized(orsemisynthesized)throughchemicalmethodsorbeproducedviamicrobialmetabolicactivity.Dependingonthediversityofthemolecularstructure,antibioticsaredividedintodifferenttypessuchasβlactams,quinolones,tetracyclines,macrolides,sulfonamides,andothers(Kümmerer,2009).Sincetheirfirstapplicationin1940,antibioticshavebeenusedincreasinglyforthetreatmentofbacterialdiseasesinhumansandanimalsowingtotheirexcellenttherapeuticeffects.However,thewidespreaduseinthetreatmentofanimaldiseaseshasledtothepresenceofresiduesofantibioticsinedibleanimalproducts,whichisofconcerntoregulatoryagenciesandconsumers.Reliableassaymethodsfortherapid,sensitive,andselectivedetectionoftheseresiduesareessentialtoensurefoodsafety.Afunctionalnucleicacidthatconsistsofanaptamer(DNAorRNA)representsanewanalyticalmethodforthedetectionofhazardoussubstancesinfoods.Inparticular,acolorimetricbiosensorcombiningAuNPsandtheaptamertechniquehasbeendevisedforthedetectionofantibioticswithhighselectivityandsensitivity.Heetal.(2012)employedacolorimetricbiosensorusingunmodifiedAuNPstodetectdaunomycininaqueoussolution.Intheabsenceofdaunomycin,freeantidaunomycinaptamer(Wochneretal.,2008)couldabsorbonthesurfaceoftheAuNPstopreventtheiraggregationinahighsaltsolutionowingtotherepulsiveinteractionbetweenthenegativelychargedaptamers.Inthepresenceofdaunomycin,theaptamerwasdisassociatedfromtheAuNPsurfacesduetotheformationofadaunomycin–aptamercomp