基于罗丹明酰肼的铜离子荧光探针及其生物成像应用.pdf 12页

'中国科技论文在线http://www.paper.edu.cnAnewfluorogenicandcolorimetricchemosensorbasedonrhodaminehydrazideforcopper(II)ionsanditsapplication5tobioimaging1222**RENLiexiang,WANGPanfeng,LIUBin,YANGBinsheng(1.Lulianguniversity,Departmentofchemistryandchemicalengineering，Lishicity,033000;2.InstituteofMolecularScience,Shanxiuniversity,Taiyuan,030006)Abstract:AnewrhodaminehydrazidederivativeLcontainingalongchainschiffbasewithsalicylic2+10acidwassynthesizedandcharacterized.AdditionofCuinducedasignificantsolutioncolorchange(colorlesstopink)andanenhancementinfluorescenceoftheprobe,eveninthepresenceofother2+commonlycoexistentcationsinCH3CN/HEPESbuffersolvent(1:1,v/v,pH7.0).Cu-promotedring-openingandhydrolysisofrhodamineLinaqueousacetonitrilemedialedtothefluorescenceturn-onchangeswithhighselectivityandhighsensitivity(detectionlimit≤22.9nM)andarapid15responsetime(≤1min).Importantly,theprobewassuccessfullyusedforfluorescenceimagingincoloncancerSW480cells2+Keywords:Rhodaminederivative;Chemosensor;Cuion;Cellimaging0Introduction20Copperisanessentialtraceelementthatplaysacriticalroleinthephysiologyoflivingorganismsincludinggeneexpressionandthefunctioningandstructuralenhancementofproteins,[1]2+andisalsorequiredbythehumannervoussystem.However,excessiveaccumulationofCuinthehumanbodywillleadtoanoxidativestress,organtoxicityanddisordersassociatedwith[2][3]neurodegenerativediseasessuchasMenkesdisease,Wilson’sdisease,andAlzheimer’s[4]25disease.Copperisalsoknownasasignificantmetalpollutantduetoitswidespreaduse.Asa2+result,monitoringofCuisofconsiderableinterestandhasrecentlyattractedsignificantattention.Todate,thechemodosimeterhasattractedatremendousamountofattentionasanalternativetosensorsbasedonmetalionbindingmechanismduetoitshighsensitivityandarapidresponse.30Asaclassicdyes/fluorophores,rhodaminedyesarewidelyusedasfluorescentprobesinroutineopticalanalysisowingtotheirhighabsorptioncoefficient,fluorescenceemissioninvisibleregion,[5−8]andhighfluorescencequantumyield.Spirolactam-typerhodaminederivativesarenonfluorescentandcolorless,whereasspirolactamring-openinggivesrisetostrongfluorescenceemissionandapinkcolor.Somemetalcationscaninducespirolactamring-openingprocess,and35greatprogresshasachievedinthedevelopmentofnewluminescentchemodosimetersbasedon2+thisprocessinthepastseveralyears.Cucaninducethespirolactamring-openingprocessofrhodaminederivativesduetothechelationofmetalionswiththeprobes,whichhasbecomethe2+majorstrategyfordesigningCu-selectiveluminescentchemodosimeterswithaluminescece[9−31]2+turn-onresponse.Cu-chelation-inducedspirolactamring-openingprocessisreversible.In2+40addition,Cucanalsoinducethehydrolysisofactivatedesters,shift-bases,andhydrazone.The2+combinationofCu-promotedhydrolysisandspirolactamring-openingofrhodamineintoonesystemisausefulstrategytogeneratefluorescentturn-onchemodosimetersfortheselective2+[32−33]2+detectionofCuions.Cu-Inducedspirolactamring-openingandhydrolysisprocessesisirreversible.Interestingly,CH3CN/H2Osolutionisneededdefinitelyforallofthesehydrolysis45processes.Worksrelatedtothisareaareofgreatchallengeandincreasinginterest.Briefauthorintroduction:liexiangRen(1978),women,lecturer,bioinorganicchemstryCorrespondanceauthor:BinshengYang,1967.11.Professor,Biochemistry.E-mail:yangbs@sxu.edu.cn-1- 中国科技论文在线http://www.paper.edu.cn2+InanefforttoexpandourresearchondevelopingCufluorescentprobes,wewereinterestedinstudyingthepropertiesofrhodaminehydrazide.Rhodaminehydrazidecanforma5-membered2+ringcomplexwithCu,whichmightpromotetheredoxhydrolysisofanamidebond,andthuscausethefluorescencerecoveryofrhodamineB.Inaddition,Schiffbasesarecompoundswitha50functionalgroupcontainingaC=Nbond,whichisalwaysinvolvedinsomeenzymatictransformations.ThehydrolysisofSchiffbasescanbepromotedbyacids,aminesandmetalions.Itiswellknownthatrhodaminedyeisdifficulttoacrossthecellmembranes.Tosolvethisproblem,inthepresentwork,arhodaminehydrazidederivativeLcontainingalongaliphatic2+chainhasbeendemonstratedasachemodosimeterforCu.ItrevealsthatrhodaminehydrazideL2+55indeedexhibitsahighlyselectivefluorescenceonreactionwithCu,andmayfindausein2+detectingCuinbiosystems.1Materialandmethods1.1MaterialsandinstrumentsAllthereagentswereanalyticalgradeandusedasreceived.Allsolventswereusedafter3+3+3+2+2+2+2+2+60appropriatedistillationorpurification.SolutionsofFe,Cr,Al,Hg,Fe,Mg,Mn,Co,2+2+2+2+2+2++++Pb,Cu,Cd,Ni,Ca,Zn,K,Na,Agwerepreparedfromtheirchloridesalts.HEPESbuffersolutions(10mM,pH7.0)werepreparedinwater.Deionizedwaterwasusedthroughouttheexperiments.Astocksolutionofthechemodosimeter(1.0mM)waspreparedbydissolving0.01mMofthechemodosimeterin10mLofCH3CN.ColoncancercellSW480wereobtained65fromtheAmericanTypeCultureCollection.Themediumandheat-inactivatedfetalbovineserum(FBS)werefromGIBCO(GrandIsland,NY).113HNMRspectrawererecordedonBruker-300MHzspectrometersandCNMRspectrarecordedon75MHzspectrometers,thechemicalshifts(δ)werereportedasppminCHCl3.FluorescenceresponseswererecordedonaFluoromax-Xspectrofluorometer(HORIBA).ESI-MS70spectrawereconductedbyAgilent6520Accurate-MassQ-TOFLC/MSmassspectrometer.UV-visible(UV-Vis)spectraweremeasuredwithaVarian50BIOspectrophotometer.Thetemperatureofthesolutionswasmaintainedat37ºCbyajacketedcellholderconnectedtoanexternalcirculatingwaterbath(ShimadzuTB-85orHuber).ElementalanalysesweremeasuredonaVarioELIIIanalyzer.IRspectrawererecordedwithanBrukerTENSOR21FT-IR75spectrophotometer.AllpHmeasurementsweremadewithaModelpHS-3CpHmeter(Shanghai,China).BioimagingofthesensorswereperformedonanDaltaVision(AppliedPrecision,LLC,USA)invertedfluorescencemicroscopy.1.2SynthesisThegeneralsyntheticprocedureisgiveninScheme1.4-aminosalicylicacid(0.765g,5mM)80weredissolvedin20mLethanol,followedbyadditionofexcessiveglutaraldehyde(5mL,12.5mM)andthemixturewasrefluxedfor2h.Thesolutionwascooledtoroomtemperature.Theprecipitatewasfilteredandwashed3timeswith15mLcoldethanol.Afterdryingunderreducedpressure,thereactionaffordedayellowcrudeproduct1(0.9g,yield:76%),whichwasusedwithoutfurtherpurification.[34]85Therhodaminehydrazidewassynthesisaccordingtothereportedmethod.Thenrhodaminehydrazide(0.46g,1.0mM)and1(0.23g,1.0mM)weredissolvedin30mLethanol,andthemixturewasrefluxedfor3h.Aftercoolingtoroomtemperature,apinksolidwereprecipitated.Thesolidwaspurifiedbyrecrystallizationtoprovide0.32gLasapinksolid,yield,47.5%.-2- 中国科技论文在线http://www.paper.edu.cn90CompoundL.-1FT-IR(KBr):3078,2929,1699,1616,1514,1465,1265,1116cm.1H-NMR(CDCl3),δ(ppm,300MHz,TMS):δ7.93(d,2H),7.60(4H,Phen-H),7.35(1H,Phen-H),7.26(2H,Phen-H),7.06(2H,Xanthene-H),6.46(2H,Xanthene-H),3.33(8H,NCH2CH3),1.37(6H,CH2CH2CH2),0.88(12H,NCH2CH3)(Fig.S1).++95ESImassspectrometry:m/z=674.33[M+H],[M+H]calculated674.33(Fig.S2).Anal.CacldforC:71.30,H:6.43,N:10.39;Find:C:71.26,H:6.64,N:10.30.HOHOOglutaraldehydeONNH2OHOEtOHHO1OCHONNH2OONHONNEt2NONEt2Et2NONEt2EtOHLScheme1SynthesisofL.1002+1.3UV–visandfluorescencetitrationstudyofLwithCuTheinorganicsaltwasdissolvedindistilledwatertoafford1.0mMaqueoussolution.The1.0mMstocksolutionofLwaspreparedinabsoluteCH3CN.Allthemeasurementswerecarriedoutaccordingtothefollowingprocedure:To10mLvolumetricflaskcontaining100μLofthe105solutionofL,differentamounts(10–400μL)ofmetalionswereaddeddirectlywithmicropipette,thendilutedto2.0mLwithCH3CN–HEPESbuffersolvent(1:1,v/v,pH7.0,10mMHEPES)solution.Fluorescencemeasurementswerecarriedoutwithexcitationandemissionslitwidthof2.5and2.5nmandPMTVoltageandexcitationwavelengthwas400Vand540nm,respectively.2.4.Cellcultureandfluorescencemicroscopyimaging110SW480wasculturedinRPMI1640mediumsupplementedwith10%FBS,and1%penicillin-streptomycinat37ºCinahumidifiedatmospherewith5%CO2and95%air.CytotoxicityassaysshowthatLissafeenoughformicroscopyimagingatlowconcentrations.Beforeimagingexperiment,thecellswereseededin24-wellplates.Thecellswereincubatedwith15μMLat37ºCunder5%CO2for30min,washed3timestoremovetheremainingprobeand2+115bathedinDMEMcontainingnoFBSpriortoimaging.Then15μMCuwasaddedinthegrowthmediumfor0.5hat37ºC,washed3timeswithPBSbuffer.Then,cellswereimagedunderainvertedfluorescencemicroscopy(DaltaVision).-3- 中国科技论文在线http://www.paper.edu.cn2Resultsanddiscussion2.1UV-visblestudies120Fig.1(a)Absorptionspectraof50μMLinthepresenceof2.0equiv.ofdifferentmetalionsin1:1CH3CN–HEPESbuffersolvent(pH7.0,10.0mMHEPES).(b)UV–vistitrationprofileofLupongradualaddition2+2+2+ofCu(0–1.5equiv.),(c)Job’splotoftheLwithCu,totalconcentrationofL+Cuwaskeptconstantat50.02+μM(d)AbsorptionofLat554nm(50.0μM)to2.0equiv.ofCuin1:1CH3CN–HEPESbuffersolution(10.0125mMHEPES,pH7.0)containing20equiv.ofvariousmetalions.2+2+2+2+Toexplorethesensitivityofthesensortowardsvariouscations(e.g.Ni,Hg,Fe,Co,2+2+2+2+2+3+3+3++++Ca,Cd,Mn,Cu,Zn,Cr,Fe,Al,K,Na,Ag),chloridesaltsofeachoftheguestmetalionswereemployedinCH3CN–HEPESbuffer(1:1,v/v,25ºC)atpH7.0(10mMHEPES130buffer).AsshowninFig.1a,theUV–visabsorptionspectrumofprobeLinCH3CN–HEPESbuffer(1:1,v/v)hasnoabsorptionbandabove400nm.Theabsorptionspectrumdoesnotchange2+significantlyinthepresenceofdifferentmetalionsexceptCu,suggestingthespirolactamringof2+rhodamineBunitprefersitsring-closedstateatthiscondition.WhileuponinteractionwithCuions,anewstrongabsorptionpeakforprobeLin500–590nmrangeissimultaneouslyobserved,135andthecolorofthesolutionchangesfromacolorlesstopink(Fig.2),clearlysuggestingtheformationofthering-openedamideformofL.Thecolor-onreactionshowsahighselectivity2+towardCuonly,ratherthanotherions.Fig.1bshowsthechangeintheabsorptionspectrum2+uponadditionofCutoasolutionoftheprobe(50μM/L)in1:1CH3CN–HEPESbuffersolution2+(10.0mMHEPES,pH7.0).WithincreasingCuconcentration,thenewabsorptionpeakat554140nmcomesout,indicatingtheformationofanewcompound.Theabsorbanceintensityenhances2+almost12-folduponadditionof1.0equiv.ofCuin1:1CH3CN–HEPESbuffersolution.-4- 中国科技论文在线http://www.paper.edu.cnFig.2PhotosofchangesincolorandfluorescentemissionoftheL(50μM)uponadditionof2.0equiv.variousmetalionsin1:1CH3CN–HEPESbuffersolution(10.0mMHEPES,pH7.0).145Bindinganalysisusingthemethodofcontinuousvariations(Job’splot)wasmeasured.AsillustratedinFig.1c,amaximumabsorbanceat554nmisobservedwhenthemolecularfraction2+ofLiscloseto0.5,whichestablishesthe1:1complexformationbetweenLandCu.The[35,36]stoichiometryissupported(Fig.S4,SupportingInformation)bytheHillplotmethod.As150wellas,thebindingconstant(logKa)isfurtherfoundtobe5.0,whichisalsodeterminedaccording2+totheHillplotmethod(Fig.S4).ThedetectionlimitofprobeforCuisdeterminedtobe1.0μM(Fig.S5)usingtheequationDL=K×Sb1/S,whereK=3,Sb1isthestandarddeviationoftheblank[37]solution(10times)andSistheslopeofthecalibrationcurve.Achievinghighselectivityfortheanalyteofinterestoverotherpotentiallycompetingspecies155isanecessityforbioimagingprobes.Thus,theabsorbancechangesofLweremeasuredbythe2+treatmentof2.0equiv.Cuionsinthepresenceof10equiv.otherinterferingmetalions2+2+2+2+2+2+2+2+3+3+3++++includingNi,Hg,Fe,Co,Ca,Cd,Mn,Zn,Cr,Fe,Al,K,NaandAg.The2+resultsdemonstratesthattheabsorbanceofprobeLisenhancedeffectivelybyCuionswiththeseionsasbackground(Fig.1d).Thisobservationconfirmsthatthecoexistentionshave2+160negligibleinterferingeffectonCusensingbyprobeL.2.2Fluoresencestudies2+Fig.3(a)FluorescencespectraofL(50μM))uponadditionof2.0equiv.Cuandothermetalionswiththe165excitationat540nmin1:1CH3CN–HEPESbuffersolution(10.0mMHEPES,pH7.0).(b)Emissionprofile-5- 中国科技论文在线http://www.paper.edu.cn2+of50μMLuponconcomitantadditionsofCu.Insert:Changesofemissionintensityat575nmwith2+2+incrementaladditionofCu(c)HillplotofthefluorescencetitrationdataofLat575nmwithCuinCH3CN–HEPESsolution.(d)Fluorescenceintensitychangesof50μMLupontheadditionofvariousmetalionsinCH3CN/HEPES(pH7.0,1:1,v/v)solution.λex=540nm,slit:2.5nm/2.5nm.1702+ThehighselectivityofLforCuwasfurtherobservedinthefluorescentspectra.The2+fluorescenceresponseofLtovariouscationsanditsselectivityforCuareillustratedinFig.3.ItisclearthatLshowsaveryweakfluorescenceintheabsenceofmetalionsinCH3CN–HEPES2+buffer(1:1,v/v).However,theadditionofCu(2.0equiv.)resultsinaremarkablyenhanced175fluorescenceintensity(λex=540nm).Wealsonotethatitisvisualwithasolutioncolorchangefromcolorlesstoyellowunderilluminationwitha365nmUVlamp(Fig.2).Noobvious2+2+2+2+2+2+2+2+responsescanbeobservedupontheadditionofNi,Hg,Fe,Co,Ca,Cd,Mn,Zn,3+3+3++++Cr,Fe,Al,K,Na,Ag,respectively(Fig.3a),whichisconsistantwiththeresultofUV–visspectra.TheseobservationsindicatesthatLhasahighsensitivityandexcellentselectivityfor2+2+180CuinCH3CN–HEPESbuffer(1:1,v/v).UpongradualadditionofCu,thefluorescenceintensityofthesolutionat575nmincreases(Fig.3b).Thefluorescenceintensityenhancesalmost2+2+8-folduponadditionof1.0equiv.ofCuinCH3CN–HEPESbuffer(1:1,v/v).WhenmoreCuistitrated,thefluorescenceintensityshowsnegligiblechanges.FromtheJob’splot(Fig.S6),amaximumfluorescencechangeisobservedwhenthemolar2+185fractionofthesensor[L]versus[Cu]+[L]is0.5,indicatinga1:1bindingstoichiometrybetween2+LandCu.Basedona1:1bindingmode,thebindingconstantiscalculatedtobelogKa=5.28fromtheHillplotofthefluorescencetitration(Fig.3c)withsatisfactorycorrelationcoefficientvaluesR=0.990.Thecalculateddetectionlimitis22.9nMfromthefluorescencespectra(Fig.S7),whichismuchlowerthanthetypicalconcentrationofbloodcopper(15.7–23.6μM)innormal[38]190individualsandthelimitofcopperindrinkingwater(~20μM)setbytheU.S.Environmental[39]ProtectionAgency.Competitionexperimentswerealsocarriedoutbymonitoringthechangeinfluorescence2+intensityat575nmuponadditionof2.0equiv.CuiontoasolutionofLanddifferentmetalions(10equiv.)inthebufferedsolution(Fig.3d).Itcanbeobservedthatnoneofthemetalionsexert195virtuallyanyeffectonthefluorescenceintensity.Therefore,theprobeiscapableoffluorescent2+recognitionofCuwithhighselectivityandsensitivity.2.3EffectofpHandkineticcharacteristicoftheswitch2+200Fig.4Thechangesofemissionintensityat575nmofL(50μM)intheabsenceandpresenceof2.0equiv.CuinH2O/CH3CN(v/v,1/1)atdifferentpH.-6- 中国科技论文在线http://www.paper.edu.cn2+InordertoinvestigatetheeffectoftheenvironmentalfactorstothedetectionofCu,the2+spectrumresponsewithorwithoutCuinthebuffersolutionsatdifferentpHvalueswasfirstlyevaluatedatroomtemperature.Fig.4showsthatnoobviousfluorescenceemissionofLis205observedbetweenpH4and12,suggestingthatthecompoundisinsensitivetopHandthatthespirolactamformisstillpreferredinthiscondition.IntherangepH5–7.5,amarkedfluorescence2+enhancementisobserveduponadditionofCu.Themoststrongfluorescenceintensityis2+observedaroundpH7.0forCu,whichisclosetophysiologicalconditions.Therefore,furtherstudiesarecarriedoutinH2O/CH3CN(v/v,1/1)atpH7.0.2102+Fig.5ChangesofemissionintensityofLat575nminthepresenceandabsenceof2.0equiv.ofCuandasthefunctionoftime(0–150s).Thetimecourseforthefluorescenceresponseof50μMprobeLupontheadditionof2.02+215equiv.CuinbufferedsolutionatroomtemperatureisshowninFig.5.Itisfoundthatthe2+obviousspectralchangeisobservedwithin1minuponadditionof2.0equiv.Cu.Thesolutionisstabletolightandairatroomtemperature.Therefore,thissystemcanbeusedforreal-time2+trackingofCuincellsandorganisms.Inthispaper,2minisusedforalltitrations.2.4Stoichiometricratioandmechanism2202+Fig.6IRspectraofL(a)andL-Cu(b)inKBrdisks.InordertoinvestigatewhythesignificantabsorbanceandfluorescencechangesofL,theIR2+225spectraexperimentsweremeasured.IRspectraofLandL-Cu(gotfromevaporationof-7- 中国科技论文在线http://www.paper.edu.cnacetonitrilesolution)weretakeninKBrdiscs,respectively,andtheresultswereshowninFig.6−1andFig.S8-S9.AsclearlyshowninFig.6,thepeakat1616cm,whichcorrespondstothe−1characteristicabsorptionofamidecarbonylgroup,isshiftedtolowerfrequency(near1589cm)2+uponchelatingwithCu.Thissamephenomenonhasalsobeenfoundinotherliteratures,during−1[28]−1[40]230whichthepeakcanshiftfrom1619to1595cmorfrom1616to1590cm.HydrazidesarealsoknowntobindCu(II)thuslywithresultingenhancedtransacylationreactivity.ThissupportsthenotionthatthecarbonylOatomandamidoNatominthehydrazideisinvolvedinthe2+coordinationofCuforminga5-memberedringcomplex.2352+2+2+Fig.7PhotosofchangesincolorofL(a)1:L,2:L+Cu,3:L+Cu+DETAinCH3CN;(b)1:L,2:L+Cu,3:2+2+L+Cu+DETA,4:L+Cu+EDTAinH2O/CH3CN(1:1,v/v).2+ToconfirmCumediatedhydrolysis,reversibilityexperimentswerecarriedoutinboth240acetonitrileandacetonitrile/watersystems.Theadditionofdiethylenetriamine(DETA)tothe2+L-CucomplexinpureacetonitrilerestoresthefluorescencesignalofLtoitsoriginallevel,a2+showninFig.7a.FurtheradditionofCuionstothesamesolutiongivestheoutcomeofthe2+L-CucomplexindicatingthereversiblebehaviorofLinacetonitrile.Ontheotherhand,addition2+ofCuchelatingagents,suchasEDTAorDETAtoacetonitrile/watersystems,doesnotdecrease245theintensityofthefluorescencesignals(Fig.7b),confirmingtheirreversiblecharacterofthisprocess.ThechangesofabsorbancespectraarealsoshowninFig.S10–S11.Tofurtherclarifythemechanism,thehydrolyticproductwassubjectedtotheESImassspectralanalysis.Theresultsshowthatthepeakatm/z=443.23(Fig.S3)correspondstorhodamineB(Calc.m/z=443.23)astheresultinghydrolyticproduct.Notably,thepeakat674.3disappearswhenLisfullyhydrolyzed2+250(Fig.S3).Takentogether,alikelysensingmechanismbasedontheCu-triggeredspirocyclicring-openingprocessisproposedinScheme2.-8- 中国科技论文在线http://www.paper.edu.cnOHNOHOOHOCu2+OCu2+ONNNHONNDETANONNONweakfluorescencestrongfluorescenceLL-Cu2+DH2OsisElyTroAdOhyHONONRing-openedRhodamine2+Scheme2ProposedbindingmechanismbetweenLandCu2552.5FluorescentimaginginSW480cellsFig.8BioimagingapplicationofchemodosimeterLinSW480cells.Representativefluorescenceimagesof2+SW480cellstreatedwithL(5μM)ineithertheabsence(a)orthepresence(b)of5equiv.Cu(30minat37ºC).(c)Bright-fieldimageofcellsshowninpanelb.(d)Overlayimageof(b)and(c).260Tofurtherevaluatethepracticalapplicabilityofthedesignedchemodosimeter-functionalizedprobe,fluorescentimagingexperimentswerecarriedouttodemonstrateitsvalueinbioimaging2+intracellularCuinSW480celllines.SW480cellwereincubatedwithreceptorL(5μM)inanRPMI-1640mediumfor30minat37ºCandwashedwithphosphatebufferedsaline(PBS)buffer265(pH7.4)toremoveexcessreceptorL.Asdeterminedbyfluorescentimagingmicroscopy,stainingcellstreatedwith5μMsolutionofLfor30mingivenointracellularfluorescence(Fig.8a).-9- 中国科技论文在线http://www.paper.edu.cn2+However,aftertreatmentwithCuions(25μM)underthesameconditions,thecellspretreatedwithreceptorL(5μM)showasignificantfluorescenceincreasefromtheintracellularregion(Fig.8b).Theappearanceofredfluorescenceisattributedtothehydrazidederivativeandring-opened2+270formofrhodamineproducedbyCuionmediatedhydrolysisofL.Theseresultssuggestthat2+receptorLiscellpermeableandaneffectiveintracellularCuionimagingagentwithturn-onredcoloredfluorescenceemissions.3ConclusionInsummary,awatersolublerhodaminefluorescentprobeLcontainingalongchainschiff275basewassynthesizedanditschemosensingpropertieswereinvestigated.Leasilyundergoes2+Cu-inducedhydrolysiscleavageoftheamidebondandspirocyclicringopeningoftherhodaminefluorophoretoproducepinkcolorationalongwith‘off–on’fluorescencechange.AlltheseselectiveandsensitiveresultsindicatethatchemosensorLcouldmeettheselectiverequirementsforenvironmentalapplication.Inaddition,thesensorcanalsobeappliedasan2+280excellentfluorescenceprobeforanalyticaluseinintracellularimagingofCuinlivingcellwithtunableredemission,whichwillfacilitateunderstandingofthebiologicalprocessesatthemolecularlevel.Acknowledgements285ThisworkwassupportedbyPh.D.ProgramsFoundationoftheMinistryofEducationofChina(20131401110011).References[1]G.Muthaup,A.Schlicksupp,L.Hess,D.Beher,T.Ruppert,C.L.Masters,K.Beyreuther,Theamyloid290precursorproteinofAlzheimer"sdiseaseinthereductionofcopper(II)tocopper(I),Science,1996,271,1406-1409.[2]C.Vulpe,B.Levinson,S.Whitney,S.Packman,J.Gitschier,IsolationofacandidategeneforMenkesdiseaseandevidencethatitencodesacopper-transportingATPase,Nat.Genet.3(1993)7-13.[3]P.C.Bull,G.R.Thomas,J.M.Rommens,J.R.Forbes,D.W.Cox,TheWilsondiseasegeneisaputativecoppertransportingP-typeATPasesimilartotheMenkesgene,Nat.Genet.5(1993)327-337.295[4]Y.H.Hung,A.I.Bush,R.A.Cherny,CopperinthebrainandAlzheimer"sdisease,J.Biol.Inorg.Chem.15(2010)61-76[5]H.N.Kim,M.H.Lee,H.J.Kim,J.S.Kim,J.Yoon,Anewtrendinrhodamine-basedChemsensors:applicationofspirolactamring-openingtosensingions.Chem.Soc.Rev.37(2008)1465-1472.[6]M.Beija,C.A.M.Afonso,J.M.G.Martinho,Synthesisandapplicationsofrhodaminederivativesasfluorescent300probes,Chem.Soc.Rev.38(2009)2410-2433.[7]X.Q.Chen,T.Pradhan,;F.Wang,J.S.Kim,J.Yoon,Fluorescentchemosensorsbasedonspiroring-openingofxanthenesandrelatedderivatives,Chem.Rev.112(2012)1910−1956.[8]Y.M.Yang,Q.Zhao,W.Feng,F.Y.Li,Luminescentchemodosimetersforbioimaging,Chem.Rev.113(2013)192−270305[9]L.Mei,Y.Xiang,N.Li,A.J.Tong,AnewfluorescentprobeofrhodamineBderivativeforthedetectionofcopperion,Talanta72(2007)1717-1722[10]X.Zhang,Y.Shiraishi,T.Hirai,Cu(II)-selectivegreenfluorescenceofarhodamine−diaceticacidconjugate,Org.Lett.9(2007)5039-5042[11]Y.Xiang,Z.F.Li,X.T.Chen,A.Tong,Highlysensitiveandselectiveopticalchemosensorfordetermination310ofCu2+inaqueoussolution,Talanta74(2008)1148-1153.[12]K.Swamy,S.K.Ko,S.K.Kwon,H.N.Lee,C.Mao,J.M.Kim,K.H.Lee,J.H.Kim,I.Shin,J.Y.Yoon,Boronicacid-linkedfluorescentandcolorimetricprobesforcopperions,Chem.Commun.44(2008)5915-5917.[13]X.Zhang,S.Sumiya,Y.Shiraishi,T.Hirai,EffectsofalkylchainlengthonCu(II)-selectivegreenfluorescenceofrhodamine-diaceticacidconjugates,J.Photochem.Photobiol.A205(2009)215-220.315[14]Y.Zhao,X.B.Zhang,Z.X.Han,L.Qiao,C.Y.Li,L.X.Jian,G.L.Shen,R.Q.Yu,Highlysensitiveandselectivecolorimetricandoff-OnfluorescentchemosensorforCu2+inaqueoussolutionandlivingcells,Anal.Chem.81(2009)7022-7030.[15]M.L.Zhao,X.F.Yang,S.H.He,L.P.AWang,Rhodamine-basedchromogenicandfluorescentchemosensorforcopperioninaqueousmedia,Sens.ActuatorB:Chem.135(2009)625-631.-10- 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中国科技论文在线http://www.paper.edu.cn基于罗丹明酰肼的铜离子荧光探针及385其生物成像应用1222任列香，王攀峰，刘斌，杨斌盛（1.吕梁学院，化学化工系,离石,033000；2.分子科学研究所，山西大学，太原，030006）摘要：本文利用罗丹明酰肼合成了一个长链的希夫碱化合物L，并利用多种方法进行了表2+390征。研究表明，在CH3CN/HEPES缓冲液中（1:1,pH=7.0）,Cu离子的滴加引起该化合物2+颜色有无色变为粉色，同时荧光大大增强。其他共存离子不会对Cu产生干扰。机理研究2+表明，Cu离子的加入引起了罗丹明螺环的开环过程，并在水分子的做一下，酰胺键断裂。2+探针对Cu离子具有较高的选择性和灵敏度，反应时间短，检出限为22.9nM。另外，该检测过程在SW480细胞内得以实现。395关键词：罗丹明；铜离子；传感器；细胞成像中图分类号：O621.3-12-'