雙光子敏化Eu3 高效發(fā)光活細(xì)胞成像納米生物探針

1、物理化學(xué)學(xué)報(bào)(WuliHuaxueXuebao)2480ArticleActaPhys.Chim.Sin.2012,28(10),2480-2486doi:10.3866/PKU.WHXB201208161O雙光子敏化Eu3+高效發(fā)光活細(xì)胞成像納米生物探針符小藝1,2,邵光勝1,韓榮成1,馬嚴(yán)1薛富民1付立民3張建平3王遠(yuǎn)1,*3楊帆3(1北京分子科學(xué)國家實(shí)驗(yàn)室,分子動(dòng)態(tài)與穩(wěn)態(tài)國家重點(diǎn)實(shí)驗(yàn)室,北京大學(xué)化學(xué)與分子工程學(xué)院,北京100871;2華南理工大學(xué)材料科學(xué)與工程學(xué)院,廣州510640;中國人民大學(xué)化學(xué)系,北京100872)摘要:將Eu(tta)3dpbt(dpbt:2-(N,N-dieth

2、ylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine;tta:thenoyltrifluoroacetonato)包埋在甲基丙烯酸甲酯-苯乙烯共聚物、正辛基三甲氧基硅及其水解縮合產(chǎn)物組成的雜化基質(zhì)中,制備了Eu(tta)3dpbt質(zhì)量分?jǐn)?shù)為40%的熒光納米粒子,其平均粒徑為45nm.所制備的發(fā)光納米粒子在水中分散穩(wěn)定性高、光穩(wěn)定性好、細(xì)胞毒性低、長波敏化Eu3+發(fā)光性能優(yōu)良,適宜作為生物分析的發(fā)光標(biāo)記物.所制備的發(fā)光納米粒子的可見區(qū)激發(fā)峰位于415nm,激發(fā)峰尾部延展至475nm,其發(fā)光量子產(chǎn)率為0.31(ex=415n

3、m,T=23C),最大雙光子激發(fā)作用截面為5.0105GM(ex=830nm,1GM=10-50cm4sphoto-1特異性標(biāo)記和雙光子激發(fā)Eu3+發(fā)光成像.關(guān)鍵詞:發(fā)光;銪配合物;雙光子激發(fā);納米生物探針;細(xì)胞成像O648particle-1).以轉(zhuǎn)鐵蛋白修飾上述發(fā)光納米粒子表面制備的納米生物探針被成功應(yīng)用于活的HeLa腫瘤細(xì)胞的中圖分類號(hào):NanoprobeswithEnhancedTwo-Photon-SensitizedEu3+LuminescencePropertiesforLiveCellImagingFUXiao-Yi1,2,SHAOGuang-Sheng1,YANGFan3FU

4、Li-Min3HANRong-Cheng1,ZHANGJian-Ping3MAYan1XUEFu-Min1WANGYuan1,*(1BeijingNationalLaboratoryforMolecularSciences,StateKeyLaboratoryforStructuralChemistryofUnstableandStableSpecies,CollegeofChemistryandMolecularEngineering,PekingUniversity,Beijing100871,P.R.China;2SchoolofMaterialsScienceandEngineerin

5、g,SouthChinaUniversityofTechnology,Guangzhou510640,P.R.China;3DepartmentofChemistry,RenminUniversityofChina,Beijing100872,P.R.China)Abstract:Luminescentnanospheres(EuPHS,dav=45nm)containing40%(w)ofEu(tta)3dpbt(tta=thenoyltrifluoroacetonato;dpbt=2-(N,N-diethylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-

6、1-yl)-1,3,5-triazine)werepreparedbyencapsulatingEu(tta)3dpbtinahybridmatrixformedinsitufrompoly(styrene-co-methylmethacrylate),octyltrimethoxysilane,andpoly(octylsiloxane).TheEuPHSarepromisingluminescentmarkersforbioanalysisbecauseoftheirgooddispersibilityinaqueoussolutions,highphotostability,lowcyt

7、otoxicity,andbrightEu3+luminescenceunderexcitationatlongwavelengths.EuPHSexhibitedexcellentvisiblelight-sensitizedandnear-infraredtwo-photon-sensitizedEu3+luminescenceproperties,withavisible-lightexcitationpeakat415nmandanexcitationwindowextendingupto475nm.ThequantumyieldforEu3+luminescencewas0.31(e

8、x=415nm,T=23C),andthetwo-photonexcitation(TPE)actioncrosssectionwas5.0105GM(1GM=10-50cm4sphoton-1particle-1)at830nm.BionanoprobespreparedReceived:June20,2012;Revised:August16,2012;PublishedonWeb:August16,2012.Correspondingauthor.Email:wangy;Tel:+86-10-62751491.Theseauthorscontributedequallytothiswor

9、k.TheprojectwassupportedbytheNationalNaturalScienceFoundationofChina(21073002,21133001,21227803,51121091)andNationalKeyBasicResearchSpecialFoundationofChina(2011CB808702).國家自然科學(xué)基金(21073002,21133001,21227803,51121091)及國家重點(diǎn)基礎(chǔ)研究專項(xiàng)經(jīng)費(fèi)(2011CB808702)資助EditorialofficeofActaPhysicoChimicaSinicaNo.10FUXiao-Yi

10、etal.:Two-Photon-SensitizedEu3+LuminescenceNanoprobesforLiveCellImagingbyadsorbingtransferrinonthesurfacesofEuPHSweresuccessfullyappliedintarget-specificlabelingandtwo-photon-excitationimagingofliveHeLacells.KeyWords:Luminescence;Europiumcomplex;Two-photonexcitation;Bio-nanoprobe;2481CellImaging1Int

11、roductionBioprobesbasedontwo-photon-sensitized(TPS)lumines-cenceofEu3+havebeenattractingintensiveattentioninthefieldsofmaterialscienceandbioanalysisbecauseoftheantici-pationthattwo-photonexcitationbioimagingbasedonsuchprobeswillcombinetheadvantagesofhighsensitivity,highsignal-to-noiseratio,deeppenet

12、ration,aswellaslowphoto-damagetolivingorganism.1-5Thesuperiorluminescentproper-tiesofeuropiumcomplexesarecharacterizedbythenar-row-lineemissionofEu3+inthered-lightregion,withaccept-abletransparenceformanybiosamples,largeStokesshifts,andlongluminescencelifetimes.3-5Remarkableprogresseshavebeenachieve

13、dinthedesignandsynthesisofEu3+complexeswithexcellenttwo-photonexcitation(TPE)luminescenceprop-erties,4,6-17andseveraloneshavebeensuccessfullyappliedinthemulti-photon-excitationbio-imagingofcells.8,9,18Incompar-isonwithfreeEu3+complexmolecules,bionanoprobespre-paredbyencapsulatingproperEu3+complexesi

14、ntowater-dis-persibleandbiocompatiblenanoparticlespossesstheaddition-albenefitsofmarkedlyenhancedluminescentbrightness,chemicalstabilityandphotostability,aswellaslowercytotox-icity.4,19,20ThepreviouslyreportedcomplexEu(tta)3dpbt21(dpbt:2-(N,N-diethylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3

15、,5-triazine;tta:thenoyltrifluoroacetonato,SchemeS1(seeSup-portingInformation)isoneofthemostproperdyesforprepar-ingbionanoprobeswithdesirabletwo-photon-sensitizedEu3+luminescencepropertiesinviewofitshighmolecularTPEac-tioncrosssection(,82GMat808nm,1GM=10-50cm4photon-1ofitsmolecule-1),andtheexcellentT

16、PEluminescentprop-sertiesmolecularaggregates.22,23However,theattemptstopreparedesirednanoprobesencapsulatingEu(tta)3dpbtoritsde-rivatives16,24byconventionalencapsulationmethodssuchasmi-croemulsionpolymerization25orStbermethod26failed,duetothedissociationofthecomplexesandlossoftheirintrinsiclu-minesc

17、encepropertiesduringtheencapsulationprocesses.Recently,wereportedaco-precipitation-assemblymethodforpreparingnanospheresencapsulating10%(w)ofEu(tta)3dpbt(EuLNPs)whichexhibitedfineTPEEu3+luminescenceproper-tieswithamaximalvalueforthenanospheresof1.2105GMat825nm.BionanoprobesusingEuLNPsasthelumines-ce

18、ntmarkerhavebeensuccessfullyappliedintheTPEcell-se-lective-imagingoflivecancercells.20Webelievethatthetwo-photonexcitationimagingqualitybasedonnanoprobesen-capsulatingEu(tta)3dpbtcouldbefurtherimprovedbyincreas-ingtheparticlesTPEactioncrosssection,whichcouldbereal-izedbyaugmentingtheEu(tta)3dpbtcont

19、entofthenano-spheres.However,withthepreviouslyreportedexperimentalconditions,thepreparationofnanospherescontainingmorethan15%(w)ofEu(tta)3dpbtproducedalargeamountofpre-cipitates.HereinwereportthepreparationandluminescentpropertiesofnewhybridnanosphereswithhighEu(tta)3dpbtloading(40%(w)andexcellentdi

20、spersion-stabilityinwater(EuPHS)whichwerepreparedbyaco-precipitation-condensationmeth-od.27,28Thetwo-photon-sensitizedEu3+luminescencepropertiesofEuPHSweremeasured.Andthetumorcelltargetingbehav-ioroftransferrin-modifiedEuPHS(Tf-EuPHS)wasobservedusinganon-invasivetwo-photonexcited(TPE)luminescenceima

21、ging.Receptor-mediatedtumorcelltargetingbehaviorofTf-EuPHSwasperformedinvitrobyinhibitionwithNaN3and2-deoxy-D-glucose.Inaddition,thebiocompatibilityofEuPHSwasalsoevaluatedwithcytotoxicitytests.2Materialsandmethods2.1MaterialsEu(tta)3dpbtwassynthesizedaccordingtothemethodreport-edpreviously.21Octyltr

22、imethoxysilane(OTS,97%)waspur-chasedfromAlfaAesar.Poly(styrene-co-methylmethacrylate)(P(ST-co-MMA),40%styrene,Mw100000-150000)waspur-chasedfromAldrich.Transferrin(98%)purchasedfromSig-ma,cetyltrimethylammoniumbromide(99%)obtainedfromAcrosOrganics,andtris(hydroxymethyl)aminomethanefromAmrescowereused

23、withoutfurtherpurification.Otherchemi-calsofARgradewereusedasreceived.2.2PreparationofEuPHSAcolloidalsolutionofEuPHSwaspreparedbyaco-precipi-tation-condensationmethod.27,28Inatypicalexperiment,2.5mLofacetonesolutioncontainingOTS(1.510-3molL-1),P(ST-co-MMA)(0.16gL-1),andEu(tta)3dpbt(1.310-4molL-1)was

24、dropwiseaddedintoanaqueoussolutionofcetyltri-methylammoniumbromide(CTAB)(7.0mL,1.410-3molL-1)understirringatroomtemperature.Themixturewasfur-therstirredforabout15mintoproduceayellowcolloidalsolu-tion.Toremovelargeparticles,theas-preparedcolloidalsolu-tionwascentrifugedat10000g.Thesupernatewasthentre

25、at-edbycentrifugingat40000g,andtheobtainedprecipitationwasre-dispersedinwaterof8mLtoproduceacolloidalsolu-tionofEuPHS.ThisprocesswasrepeatedtoremovemostofCTABandproduceastablecolloidsolutionofEuPHS(about52.5mgL-1,correspondingtoayieldofEuPHSof30%(w),seeSupportingInformationfordetails)withanaveragedi

26、ame-terof45nmasmeasuredbytransmissionelectronmicroscopy2482ActaPhys.Chim.Sin.2012Vol.28(TEM).Acolloidalsolutionofnanoparticles(EuHS)wasalsopreparedbytheaforementionedprocessesexceptfortheaddi-tionofP(ST-co-MMA).2.3PreparationofbionanoprobesTheobtainedcolloidalsolutionofEuPHS(5mL)wasmixedwithasolutio

27、noftransferrin(0.2mgin0.2mLwater),andthemixturewasincubatedat25Cfor2hinathermomixshakershakingat300rmin-1.Afterbeingseparatedbycentrifuging(40000g,5min)andwashedwithTris-HClbuffer(10mmolL-1,pH7.8)twice,theresultingEuPHSmodifiedwithtransfer-rin(Tf-EuPHS)weredispersedin5mLofTris-HClbuffertoobtainacoll

28、oidsolutionwhichwasstoredat4Cforuse.2.4CytotoxicitytestThecytotoxicitymeasurementswereperformedusingMTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbro-mide)assay.Briefly,HeLacellsweretrypsinizedandresus-pendedinDulbeccosmodifiedEaglesmedium(DMEM)con-taining10%(V/V)fetalbovineserum(FBS)and80

29、UmL-1gentamycinsulfate.Thecellswereseededatadensityof0.2-1.0millioncellsperwellina96-wellplate.After24hofincubationat37Cin5%CO2,thecellswerewashed3timeswithphosphatebufferedsolution(PBS,pH7.4).Colloidalsolu-tionsofEuPHSorTf-EuPHSwithdifferentconcentrations(100L)wereaddedtothewells.After20hofincubati

30、onat37Cin5%CO2,asolutionofMTT(20L,5.0mgmL-1)wasaddedtoeachwell.After4hofincubationat37Cin5%CO2,themediumwasdiscarded,andtheintracellularprecipi-tateofformazanwascollectedbydimethylsulfoxide(DMSO)(100L).Theabsorbanceat570nmwasmeasuredonaBio-RadModel550microplatereader.Eachdatapointwascollect-edbyaver

31、agingtheabsorbancevaluesofsixwells,andtheun-treatedcellswereusedascontrols.Statisticalsignificancewasassessedbythetwo-sampleStudentst-testusingSPSS13.0;Pvalues(levelofsignificance)(0.01)wereconsideredstatisti-callysignificant.2.5CellularuptakeTheeffectsofNaN3and2-deoxy-D-glucoseonthecellularuptakeof

32、Tf-EuPHSwereexamined.Thecellswerepre-incu-batedinPBSbuffersolutionandsupplementedwith10mmolL-1NaN3and50mmolL-12-deoxy-D-glucosefor30minat37CfollowedbyincubationinasolutionofTf-EuPHS.2.6CellcultureandimagingHeLacellswerepropagatedinDulbeccosmodifiedEaglesmedium(DMEM)supplementedwith10%(V/V)fetalbovin

33、eserumand80UmL-1gentamycinsulfate.Thentheculturedcellsweretrypsinizedandre-suspendedinthisDMEMataconcentrationofabout7.5105mL-1.Thecellsuspension(100L)wastransferredtoaconfocaldish(35mm).Afterincuba-tionfor24hat37Cin5%CO2,thecellswerecarefullyrinsedwithPBSsolution(pH7.4).ThenacolloidalsolutionofTf-E

34、uPHS(100L,0.2mgmL-1)wasadded.After3hincuba-tionat37Cin5%CO2,thedishwasrinsedthreetimeswithPBSsolution(pH7.4)andthen1mLoffreshserum-freemedi-umwasadded.Theplateswereincubatedforanother10minat37Candthendirectlyimagedonanuprightconfocalmicro-scope(LeicaTCSSP5)equippedwithafemtosecondTi:sap-phirelaseran

35、da20waterimmersionobjective(CarlZeiss).Thetwo-photon-excitationluminescenceimagesweretakenuponfemtosecond800nmirradiation(800nm=2.5105GM)withanaveragelaserpowerof10mW,andintegrationofEu3+luminescenceinthespectralrangefrom590to620nm.Forcomparison,alloftheconfocalimagesweretakenatthesamesetting.Transm

36、ittedlightdifferentialinterferencecontrast(DIC)imagesweretakenonthesameinstrument.2.7CharacterizationsTransmissionelectronmicroscopy(TEM)andhigh-resolu-tiontransmissionelectronmicroscopy(HRTEM)imagesweretakenonatransmissionelectronmicroscope(JEM2000FX,Hi-tachi,Japan)andafieldemissionmicroscope(Tecna

37、iF30,FEI,Netherlands),respectively.EnergydispersiveX-rayspec-troscopy(EDX)measurementsandelementalmappingwerecarriedoutonafieldemissiontransmissionmicroscope(Tec-naiG2F20U-TWIN,Netherlands)withenergydispersiveX-rayspectroscope(EDX,EPMA-1600,Shimadzu,Japan).UV-Visabsorptionandphotoluminescencemeasure

38、mentswerecarriedoutonanabsorptionspectrometer(CARY1E,Varian,German)andafluorescencespectrophotometer(F-4500,Hita-chi,Japan).ThephotoluminescencedecaykineticsofEuPHScolloidalnanoparticleswasmeasuredbyFLS920(EdinburghInstruments,German).Luminescencequantumyield()ofthepreparednanoparticleswasdetermined

39、accordingtothemeth-oddescribedbyDemasandGrosby,29usingDCM(4-dicyano-methylene-2-methyl-6-p-dimethylaminostyryl-4H-pyran)inn-propanol(=0.570.02)asthereference.Thephotobleachingexperimentswerecarriedoutonafluorescencespectrophotom-eterusinga150Wxenonlampasanexcitationsource.Colloi-dalsolutionsofthenan

40、oparticleswerecontinuouslyirradiated,andtheemissionintensitywasrecordedat8sintervals.ThemeasurementsofTPEactioncrosssectionsonthecolloidalso-lutionofEuPHSwereconductedaccordingtothemethodre-portedpreviously,22usingRhodamineB(RhB)asastandardwithknowntwo-photo-absorptioncrosssections.3Resultsanddiscus

41、sion3.1PreparationandcharacterizationofEuPHSAcolloidalsolutionofluminescentnanoparticlesencapsulat-ingEu(tta)3dpbtwaspreparedbyaddingdropwiseofanace-tonesolutioncontainingP(ST-co-MMA),OTS,andEu(tta)3dpbttoanaqueoussolutionofCTABunderstirring.Thepreparedcolloidalsolutionwascentrifugedtogetaprecipitat

42、eofEu-PHSwhichwasre-dispersedinwateroraTris-HClbuffersolu-tion(10mmolL-1,pH7.8)toformcolloidalsolutions.Thefi-nallyobtainedcolloidalsolutionswereverystableandnopre-cipitatewasobservedafterstandingformonths(seeExperi-mentalSection).ThepreparedEuPHSwerecomposedof40%(w)ofEu(tta)3dpbt,10%(w)ofOTS,35%(w)

43、ofpoly(octylsi-No.10(a)FUXiao-Yietal.:Two-Photon-SensitizedEu3+LuminescenceNanoprobesforLiveCellImaging2483.1RepresentativeTEMimage(a)andthesizedistribution(b)ofEuPHS(N=334)TheinsetshowsahigherresolutionimageofoneofEuPHS.loxane),and15%(w)ofP(ST-co-MMA)asestimatedfromtheresultsofelementalanalysis,gas

44、chromatography,andinduc-tivelycoupledplasmaatomicemissionspectroscopy(ICP-AES)(seeSupportingInformationfordetails).TheEu(tta)3dpbtcontentofEuPHSis4timesashighasthatinthepreviouslyreportedEuLNPsnanospheres.20Fig.1showstheTEM,HRTEMimages(inset),andthesizedistributionofEuPHSwhicharesphericaland45nmindi

45、ame-ter.EDXmeasurementsonindividualEuPHSrevealedthattheywerecomposedofC,Si,Eu,andS(Fig.S1(seeSupport-ingInformation).ElementalmappingconductedbyEDXinascanningtransmissionelectronmicroscope(STEM)indicatedthattheCandEudistributionsamongthenanosphereswereho-mogeneous,whileSiwasenrichedinthesurfacelayer

46、ofthenanoparticles(Fig.2).TheseresultssuggestthatEu(tta)3dpbtmoleculesorsmallmolecularaggregatesarewelldispersedinthematrixoftheencapsulationmaterials,andhydrolysisprod-uctsofoctyltrimethoxysilane(poly(octylsiloxane)areen-richedinthesurfacesofthenanoshperes.SincetheprecipitationofP(ST-co-MMA)intheso

47、lutionwasaquickprocess,whilethehydrolysisofOTSinthegivenconditionswasaslowone,webelievethattheformationpro-cessoftheEuPHSconsistsoftwosteps.Whentheacetoneso-lutionwasaddedintotheaqueoussolutionofCTAB,nanoparti-clesofthehydrophobicpolymerP(ST-co-MMA)containinghydrophobicEu(tta)3dpbtandOTSquicklyforme

48、dbyprecipi-tation.ThenthehydrolysisofOTSaroundthesurfacesofthepreformednanoparticlesoccurred,whichwasaccompaniedbyacondensationreactionofthehydrolysisproductstoformpoly(octylsiloxane)shellscoveredonthecores.AsmeasuredbygaschromatographyandICP-AES(seeSupportingInforma-tionfordetails),themolarratioofO

49、TStothehydrolyzedOTSinEuPHSwasabout1:4.Therefore,itisreasonabletodeducethatthecoresofEuPHSnanoparticlesaremainlycomposedofP(ST-co-MMA),Eu(tta)3dpbt,andOTS.ItwasfoundthatOTSplayedakeyroleintheformationofthesphericalnanoparti-cles.ThenanoparticlespreparedintheabsenceofOTSwererandominshapeasshowninthei

50、rTEMimages(Fig.S2(seeSupportingInformation).3.2LuminescencepropertiesofEuPHSFig.3showstheexcitationandluminescencespectraofEu(tta)3dpbtintolueneandinEuPHSnanospheresdispersedinanaqueoussolution.Theexcitationpeak(em=614nm)ofEu(tta)3dpbtintoluene,relatedtothesensitizationeffectofco-ordinateddpbt,locat

51、esat402nm,whilethatinEuPHSred-shiftsto415nm,withanedgeofthisbandextendingupto475nm.TheluminescencespectrumofthetoluenesolutionofEu(tta)3dpbtdisplaysasmallpeakcenteredat440nmwhichistheemittingbandoffreedpbtderivedfromthedissociationofEu(tta)3dpbt.21Incontrast,thisfluorescencesignalofdpbtcouldnotbeobs

52、ervedintheluminescencespectrumofEuPHS,indicatingtheintactnessofcoordinationstructurebetweenEu3+anddpbtinEuPHS.Thephotoluminescencedecaykineticsattheprobingwave-lengthof614nmcouldbeaccountedbyatwo-exponentialde-caymodelfunction,whichyieldedtheapparentdecaytimeconstantsof51s(8%)and508s(92%)(Fig.S3(see

53、Sup-portingInformation).Thephenomenaofred-shiftintheexci-tationpeakandthemulti-exponentialdecaywerealsoob-servedinthepreviouslyreportednanosizedEu(tta)3dpbtconge-riespreparedbyaprecipitationmethod,22whichcouldbeex-plainedbytheformationofJ-typeaggregatesofthepolarEu(tta)3dpbtmolecules.AsshowninFig.4,

54、theUV-VisabsorptionspectrumofEuPHSinthecolloidalsolutionshowsamaximalabsorptionpeakcenteredat415nmwithamolarextinctioncoefficient()of2.4104mol-1Lcm-1.Theluminescencequantumyield()fortheEu3+emissionofthepreparednanoparticleswasmea-Fig.2STEMimage(a)andtheelementalmappingofC(b),Si(c),andEu(d)ofoneofEuP

55、HS2484ActaPhys.Chim.Sin.2012Vol.28.3Normalizedexcitation(dashedline,em=614nm)andemission(solidline,ex=415nm)spectraofEu(tta)3dpbtinacolloidalsolutionofEuPHS(upper)andintoluene(lower)suredtobe0.310.03upontheexcitationat415nmat23C.Assumingthatthenanoparticleshaveadiameterof45nmandthedensityofthenanopa

56、rticlesisequalto1.0gcm-3(thedensitiesofP(ST-co-MMA),OTS,andEu(tta)3dpbtare1.05,0.91,and0.92gcm-3,respectively),itisestimatedthateachnanoparticlecontainsabout10000Eu(tta)3dpbtmolecules(seeSupportingInformationfordetails).Theluminescentbright-nessofEuPHS(definedas)isestimatedtobe7.4107mol-1Lcm-1undere

57、xcitationat415nm,suggestingaremark-ablesignalamplificationcapabilityofEuPHSinbioanalysis.ThemaximalTPEactioncrosssection()oftheeuropi-umcomplexmoleculesinEuPHSwasmeasuredtobe50GMat23Cat830nm(Fig.5).Thisvaluewasabout62%ofthatofEu(tta)3dpbtmoleculesintoluene,6indicatingthattheexcellenttwo-photon-sensi

58、tizedluminescencepropertiesofEu(tta)3dpbtmoleculesweremaintainedmostlyinEuPHS.Consideringthenumberofeuropiumcomplexmolecules(10000)ineachnanoparticlehavingtheaveragediameter,themaximalTPEactioncrosssectionofEuPHS(dav=45nm)wasroughlyestimatedtobe5105GM,whichwasabout10timeshigherthanthehighestvaluereportedforCdSe/ZnScore-shellquantumdots(dav4.5nm)determinedbyasimilarmethod,30andwasabout4timeshigherthanthatofourpreviouslyreport-edEuLNPsnanoparticles(52nminaveragediameter).20.4UV-VisabsorptionspectrumofEuPHSinwater.5Two-photonexc