Gold nanocluster-based fluorescence biosensor for targeted imaging in cancer cells and ratiometr - 图文

BiosensorsandBioelectronics65(2015)183–190ContentslistsavailableatScienceDirectBiosensorsandBioelectronicsjournalhomepage:www.elsevier.com/locate/biosGoldnanocluster-based?uorescencebiosensorfortargetedimagingincancercellsandratiometricdeterminationofintracellularpHChangqinDinga,YangTiana,b,nabDepartmentofChemistry,TongjiUniversity,SipingRoad1239,Shanghai200092,People′sRepublicofChinaDepartmentofChemistry,EastChinaNormalUniversity,NorthZhongshanRoad3663,Shanghai200062,People′sRepublicofChinaarticleinfoArticlehistory:Received25July2014Receivedinrevisedform30September2014Accepted13October2014Availableonline18October2014Keywords:AunanoclustersRatiometric?uorescencebiosensorpHdeterminationTargetedimagingCancercellsFolatereceptorabstractThedysregulatedpHisworkingasamarkofcancer.ItisachallengefordevelopingabiosensorfortargetedimagingincancercellsandmonitoringofintracellularpH.Here,aratiometric?uorescencebiosensorforpHdeterminationwasdevelopedwithtargetedimagingintofolateacceptor(FR)-richcancercellsatthesametime.AuNCsprotectedbybovineserumalbumin(BSA)workedasreference?uorophoreand?uorescein-isothiocyanate(FITC)actedastheresponsesignalforpH.Fortargetedimagingofcancercells,theAuNCsweresimultaneouslyconjugatedwithfolicacid(FA).ThedevelopedratiometricbiosensorcanmonitorpHwithawidelinearrangefrom6.0–7.8withapKaat6.84.UndereverydifferentpHcondition,theprobeshowedhighselectivityovervariousmetalionsandaminoacidswithits?uorescenceratiostayedalmostconstant(o5%).ItalsoshowedgoodcyclicaccuracywhenpHswitchedbetween6.0and8.0,aswellaslowcytotoxicity.TheAuNC-basedinorganic–organicnanohy-bridbiosensorshowedgoodcell-permeability,lowcytotoxicity,andlong-termphotostability.Accord-ingly,thepHbiosensorwasemployedtogaintargetedimaginginFRtveHelacellswithFRàvelungcarcinomacellsA549ascomparison,andachievedtomonitorthepHchangesinHelacells.&2014ElsevierB.V.Allrightsreserved.1.IntroductionIntracellularpH(pHi)playsacriticalroleinthephysiologicalandpathologicalprocesses(RoosandBoron,1981).TheregulationofpHiisessentialformostcellularprocesses,includingreceptor-mediatedsignaltransduction,calciumregulation,iontransport,cellvolumeregulation,vesicletraf?cking,cellularmetabolism,cellmembranepolarityandsoon(GolovinaandBlaustein,1997;Loi-selleandCasey,2003).AbnormalpHishowsgreatrelationshipwithhumanphysiologyandpathophysiologydiseasessuchascancer,Alzheimer'sdisease,andcardiopulmonaryproblems(Izu-mietal.,2003;Lagadic-Gossmannetal.,1999;Tangetal.,2007).Recentyears,thedetectionofpHivalueincancercellsdrawsmoreattention,asthedysregulatedpHisworkingasamarkofcancer(Zhangetal.,2010).IthasbeenreportedthattheintracellularpHishigherthanextracellularpHincancerstumor,whichmayhelptopromotetheproliferation,migration,invasionofcancertumorsandsomeothercancerprocessions(Webbetal.,2011).Onthispoint,monitoringpHchangesincancercellsiscriticallyimportantCorrespondingauthorat:DepartmentofChemistry,TongjiUniversity,SipingRoad1239,Shanghai200092,People'sRepublicofChina.Fax:t862162237105.E-mailaddress:ytian@chem.ecnu.edu.cn(Y.Tian).http://dx.doi.org/10.1016/j.bios.2014.10.0340956-5663/&2014ElsevierB.V.Allrightsreserved.nforstudyingcellularfunctionsandgainingabetterunderstandingofphysiologicalprocesses.Uptonow,pHsensitivemicroelectrodes,nuclearmagneticresonance,UV–visabsorptionspectroscopy,and?uorescencespectroscopyhavebeenreportedforthedetectionofpHinvivo(Zhangetal.,2010).Amongthese,?uorescenceprobeprovidesapowerfultooltoassesspHinvivoandinvitrowithtechnicalandpracticaladvantagessuchashighsensitivity,subcellularresolutionandeasyoperation(Lietal.,2014;Pattersonetal.,1997).Inourpreviouswork,atwo-photon“turn-on”?uorescenceprobehasbeendesignedbasedoncarbondotsformonitoringpHchangesinlivecellsandtissues(Kongetal.,2012).Unfortunately,thiskindof“turn-on”?uorescenceprobe,aswellasthosereported“turn-off”?uorescentprobesforpHremainstwolimitations:oneislackofaccuracyinquantitativedetermination,becausethe?uorescenceintensitychangeswiththevariationofpHandthe?uorescentprobeunevenlydistributesinlivecells;anotheristheirlackse-lectivityinrecognitionofdifferentcells,becausetheycouldbetookupbyvariouskindsofcellswithoutspeci?crecognition.Tosolvethesetwoproblems,wedesignedaratiometric?uorescentbiosensorwithtargetedmarkerforimagingandbiosensingofpHincancercells.Ratiometric?uorescencemeasurementscanelim-inatethein?uenceofvariationsinthelocalprobeconcentrationanddistribution,thusenhancingtheaccuracyofmeasurements(Duetal.,2013;Fuetal.,2013;Gaoetal.,2014;Miaoetal.,2013;184C.Ding,Y.Tian/BiosensorsandBioelectronics65(2015)183–190Fig.1.(A)WorkingprincipleofthedevelopedFA-FITC@AuNC?uorescentbisensorforpHdetectionandcancercell-targetedimagingand(B)ReactionschemeofFITCwithHtandOHà.Zhuetal.,2012).Silicananoparticalsandpolymergelcoatingwithdifferentdyesandquantumdots(QDs)werewidelyusedasthereference?uorescencesforconstructionofraiometric?uorescenceprobes(Dennisetal.,2012;Pengetal.,2010;Tsouetal.,2014).Buttheseprobesaredif?culttoenterintolivecellsbecauseoftheirbigsizesandsomeofthemhavehighpotentialtoxicityduetotheirheavymetalcomponents.Morerecently,AuNCshavebeenre-portedasnewtypeofluminescentnanomaterialsforcatalysis,sensors,andbioimagingbecauseoftheirgoodbiocompatibility,highelectrocatalyticactivity,anduniqueopticalproperty(Kongetal.,2011;Liuetal.,2014;Sperlingetal.,2008;Xieetal.,2009;Xueetal.,2012;Zhuangetal.,2014).AuNCsprotectedbyBSAshowedgoodphotoluminescencepropertieswithalargeStokesshift(～150nm),goodbiocompatibility,andlowcytotoxicityduetoitssmallsize(centralcoreo5nm)andBSAshell.Inthisarticle,wedevelopedaAuNC-based?uorescencebio-sensorfortargetedimagingofcancercellsandratiometric?uor-escencedetectionofintracellularpHsimultaneously.AsshowninFig.1A,BSA-protectedAuNCswereemployedasreferencesignalandconjugatedwithFITCtoformaratiometric?uorescenceprobeFITC@AuNCforpHdetection.Here,FITCactedasthespeci?cre-cognitionelementforHtwith?uorescenceemissioncenteredat516.5nm.WhenpHincreases(OHàincreases),thelactoneringofFITCmolecularwillopentoformanion,causingstrong?uores-cenceemission;whenpHdecreased(Htincreases),thelactoneringwillclosedwithweak?uorescence(Fig.1B),whilethered?uorescenceascribedtoAuNCsat625nmremainedconstant.Ontheotherhand,FAwasalsohybridonFITC@AuNCsurfacetoformFA-FITC@AuNC?uorescentprobefortargetedimagingofcancercells.FAtransportsphysiologicallyacrosstheplasmamembranebyusingeitherreducedfolatecarrierorFR.Thelatterisfrequentlyoverexpressedincancercells,enablingFAbeusedasagoodtar-get-receptorforFRtvecancercells(LuandLow,2002;Wangetal.,2013).TheFA-FITC@AuNCbiosensorcanmonitorpHgradientsinapHrangeof6.0–7.8withhighselectivity,goodsensitivity,andhighcyclicaccuracy.Meanwhile,AuNC-based?uorescentbiosensorshowedlow-cytotoxicityandlong-termstabilityagainstlightil-lumination.Moreover,italsoexhibitedtheabilitytoachievetar-get-imagingofFRtvecancer,indicatinganacidi?cationprocessanditsrecoverywhenthesenanoparticlesenteringintocancercells.2.Materialandmethods2.1.ReagentsandchemicalsGold(III)chloridetrihydrate(HAuCl4á3H2O,99%),dimethylsulfoxide(DMSO),nhydroxysuccinimide(NHS),andmethylthia-zolyltetrazolium(MTT)werepurchasedfromSigma-Aldrich.Fluorescein-isothiocyanate(FITC),N,N′-Dicyclohexylcarbodiimide(DCC),folicacid(FA),andouabainoctahydratewereobtainedfromAladdinChemistryCo.Ltd.Metalsalts,bovineserumalbumin(BSA),aminoacids,glucose,diethylether,andtriethylaminewereobtainedfromSinopharmChemicalReagentCo.Ltd.Solutionsofmetalionswereallpreparedfromtheirchloridesalts.Dialysistube(MWCO:3500)andSephadexG50wereobtainedfromEbioeasyCorporationandBiosharpCorporationseparately.CellculturemediaandsupplementsweresuppliedbyInvitrogenCor-poration.AllchemicalsfromcommercialsourceswereusedasC.Ding,Y.Tian/BiosensorsandBioelectronics65(2015)183–190185receivedwithoutfurtherpuri?cation.AllaqueoussolutionswerepreparedwithMilli-Qwater(18.2MΩcmà1).2.2.SynthesisofAuNCs,FITC@AuNC,andFA-FITC@AuNCprobesBSA-stabilizedAuNCsweresynthesizedaccordingtoagreenbio-mineralizationmethod(Xieetal.,2009)withalittlemod-ulation.ThecombinationofFITCandAuNCswasachievedthroughthereactionbetweentheisothiocyanategroupofFITCandtheaminogrouponthesurfaceofAuNCstoformFITC@AuNCandstoredat4°C.ForpreparationofFA-FITC@AuNCprobe,AuNCswere?rstlyconjugatedwithFAbyusingcouplingreagentsDCCandNHS(LeeandLow,1994).Inatypicalexperiment,FAwasreactedovernightwith500mgDCCand300mgNHSatroomtemperature.After?ltration,thereactionproductFA@AuNCwasprecipitatedandwashedthreetimeswithdiethylether,driedun-dervacuum,andstoredasyellowpowder.ThenFA@AuNCsolutionwasmixedwiththesolutionofNHS-folateinDMSOwithappro-priateproportionindarkforanother2h.Followed,FITCinEtOHwasaddedandthereactantwasstirredindarkatroomtem-peratureforanother12h.Finally,thenanohybridprobewasdialyzedinPBS(0.05M,pH?7.4)for8hbyusingdialysistube,puri?edthroughSephadexG50columnandstoredat4°C.3.Resultsanddiscussion3.1.CharacterizationofAuNCsandFA-FITC@AuNCTheas-preparedAuNCsshowedmono-dispersedwithaveragesizeof～5nm,asshowninFig.2A.AfurtherobservationrevealedthattheAuNCwasconsistentwith?111?spacingofAu(Fig.1B),whichwascon?rmedbyXRDdatawithadiffractionpeakat38.56°(JCPSDno.04-0784,Fig.2B).AftercombinedwithFAandFITC,suchdiffractionpeakshiftedto35.32°,indicatingthein-creaseofAulatticefrom～2.06?to～2.24?.ThechangewasalsoevidentbyXPSresults.AsdemonstratedinFig.2C,theAu4fXPSspectrumofAuNCsdisplayedadominantcomponentofAu°:83.5eV(Au4f7/2)and87.3eV(Au4f5/2),whileAu4fXPSspectrumofFA-FITC@AuNCshowedtwomainpeaksat84.1eV(Au4f7/2)and87.7eV(Au4f5/2),typicallyascribedtoAut(Guéveletal.,2011;Weietal.,2010).FT-IRspectrumofAuNCs(Fig.2D,curvea)ex-hibitedfourcharacteristicpeakslocatedat3450cmà1(VO–H),1260cmà1(VC–H),1662cmà1(VCQO),and1540cmà1(VN–H).Thepresenceofthesehydrophilicgroupsincluding?NH2,?COOHand/or?OHimpartsAuNCswater-solubility.Ontheotherhand,FT-IRspectrumforFITC(Fig.2D,curveb)showsfourcharacteristicpeakslocatedat3420cmà1,2040cmà1,1620cmà1,andAB

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Fig.2.Characterizationofthe?uorescentbiosensor.(A)TEMandHRTEM(inset)imagesofAuNCs;(B)X-raydiffractionpatternsof(a)AuNCsand(b)FA-FITC@AuNC;(C)XPSdataof(a)AuNCsand(b)FA-FITC@AuNC;(D)FT-IRspectraof(a)AuNCs,(b)FITC,(c)FAand(d)FA-FITC@AuNC.186C.Ding,Y.Tian/BiosensorsandBioelectronics65(2015)183–190A

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Fig.3.FluorescentdeterminationofpH.(A)Fluorescencespectraof(a)AuNCs,(b)FITC,and(c)FA-FITC@AuNCunder488nmexcitation;(B)FluorescencespectraoftheratiometricbiosensortovariouspHtitration.Inset:PlotofFGreen/FRedasafunctionofthepH(5.0–9.0)(excitedat488nm,FGreen:510–550nm,FRed:580–680nm).1380cmà1,whichareascribedtothevibrationofO–H,NQCQS,CQOandC?O?Crespectively.Meanwhile,?vepeakslocatedat3420cmà1,1700cmà1,1600cmà1,1480cmà1,and1190cmà1wereclearlyobservedforFT-IRspectrumofFA,correspondingtothevibrationofVO–H,VCQO,δN–N,VC–NandVC–Orespectively.AfterFITCandFAwereconjugatedonAuNCssurface,theappearedpeaksat1650cmà1(AmideIVCQO)and1459cmà1(AmideIIIVC–N),andthedisappearedpeakat2040cmà1(VNQCQS)intheIRspectra(Fig.2D,curved)indicatedthesuccessfulattachmentofFAandFITConAuNCsbycovalentcombiningwithBSA.The?uorescenceemissionofAuNCswasobservedtobecen-teredat640nmunderexcitationat488nm,asshowninFig.3A(curvea).UsingrhodamineBasastandard,the?uorescencequantumyield(QY)ofAuNCswasestimatedtobe～9%(Fig.S1,SupplementaryInformation(SI)).However,aftercombinedwithFITCandFA,suchemissionshiftedto625nm.Meanwhile,theemissionpeakofFITCshiftedfrom515nmto516.5nmuponex-citationof488nm(Fig.3A,curveb).ThesuccessfulpreparationofFA-FITC@AuNCwasalsoprovedbyUV–Visspectrumwhentheas-preparedprobewaspuri?edthroughSephadexG50column,inwhichFA-FITC@AuNC?owedout?rstly(Fig.S2A,I,SI)andthenFA-FITC–BSAfollowed(Fig.S2A,II,SI).Thechromatogramwasobtainedbydetectingtheintensityof?uorescenceemission(λex?488nm)andUV–Visabsorbanceatthesametime.Thein-tensitiesof?uorescentemissionsat516.5nmand625nmpre-sentedtheexistencesofFITCandAuNCs,respectively.UV–Visabsorptionpeaksat280nmand350nmindicatedthecombina-tionofBSAandFA(Stellaetal.,2000).Furthermore,UV–Visab-sorptionspectrumshowedanewabsorptionshoulderaround355nmafterthecombinationofFA(Fig.S2B,SI),whichalsoin-dicatesthesuccessfulconjugationofFA,FITCandAuNCs.3.2.AnalyticalperformanceofFA-FITC@AuNCbiosensorforpHdeterminationThestandard?uorescencepHtitrationwasperformedinPBSattheexcitationof488nm,asshowninFig.3B.Inthisratiometricbiosensor,theorganicmoleculeFITCwasusedasresponsesignalfordeterminationofpH,whileAuNCservedasreferencesignalbecauseofitsgoodstabilityunderdifferentpH(Fig.S3,SI).WiththeincreasingpHofthebuffersolution,thegreenemissionfromFITCcontinuouslyincreased,whilethered?uorescenceascribedtoAuNCsstayedconstant.Asaresult,FGreen/FRed,theratiooftheintegratedintensitiesat510–550nm(FGreen)and580–680nm(FRed),graduallyincreasedwiththeincreasingpH.ThesignalratioshowedgoodlinearitywithpHintherangeof6.0–7.8withapKaat6.84,asplottedintheinsetofFig.3B.Asthe2δofthepHti-trationexperimentsis0.099,suggestingthattheFA-FITC@AuNCcoulddetectpHchangewithalevelof～0.1.Muchimportantly,thecomplexityofintracellularsystempre-sentsagreatchallengeforbiosensorsinrequirementofselectivityandstability.TheselectivityexperimentswerecarriedoutbymonitoringtheFGreen/FRedratioofthe?uorescentbiosensorinthepresenceofmetalionsandaminoacidswhichmaycoexistinlivingsystem.Variousmetalionssuchasaboundantcellularca-tions(1mMforKt,Nat,Ca2t,Mg2t)andtracemetalcationsinorganisms(10μMforCo2t,Cd2t,Cu2t,Fe2t,Fe3t,Mn2t,Ni2t,Zn2t)weretestedunderthreedifferentpHconditions(pH6.34,7.25,and8.09respectively).AsshowninFig.4A,underthesamepHconditions,thereisnoobviousdifferenceintheFGreen/FRedratiointhepresenceofvariouscations(o5%)comparedtothatindifferentpHsituations.Similarly,severaltypicalaminoacidsandglucose(10μM)wereexaminedunderthreedifferentpHcondi-tions.ItcouldbeseenthattheFGreen/FRedratioof?uorescentbiosensorcoexistedwithvariousaminoacidsandglucosestayednearlyunchangedunderthesamepHcondition,whileshiftedwiththechangesofpH(Fig.4B).TheresultsindicatethehighselectivityofthedevelopedratiometricprobefordeterminationofpHinthecomplicatedlivecells.ThephotostabilityofFA-FITC@AuNCbiosensorwasalsoin-vestigatedunderdifferentpHconditions(pH6.19,7.42,and8.06)andsummarizedinFig.4C.Afterbeingexposedtoa?uorescencespectrophotometer(λex?488nm)equippedwitha90WXenonlampfor2h,noobviouschanges(o5%)wereobservedfortheFGreen/FRedratioofthebiosensorundereverypHcondition,sug-gestingthegoodphotostabilityofthisAuNC-basedinorganic–or-ganic?uorescentbiosensor.Fig.4Dshowedthegood?uorescencereversibilityresponsesofFA-FITC@AuNCbiosensorintheratioofintegratedintensities(FGreen/FRed)inaPBSsolutionwhenpHwasswitchedbetween6.0and8.0forthreecycles.Besides,there-lationshipbetweenthe?uorescenceratio(FGreen/FRed)andreac-tiontimeshowedthatsuchprobecouldquicklyresponsetopHchangeswithin30s(Fig.S4,SI).Theseexperimentsindicatedthatthedual-emissionFA-FITC@AuNC?uorescentprobehadhighse-lectivity,long-termstability,goodreversibilityandquickresponseforpHdeterminationinbiologicalsystem.3.3.Cytotoxicity.ThepossibilityofFA-FITC@AuNCprobeformolecularreceptor-targetedopticaldetectionofcancerwastestedinthreetypesofcancercelllineswithdifferentlevelsofFRexpression:FRtveHelaC.Ding,Y.Tian/BiosensorsandBioelectronics65(2015)183–190187Fig.4.Selectivity,photostability,andreproducibilitytest.(AandB)FluorescenceresponsesofFA-FITC@AuNCunderdifferentpHof6.34,7.25,and8.09respectively,(A)towardvariousmetalions(1mMforNat,Kt,Ca2t,andMg2t;10μMforothercations);and(B)towardvariousaminoacids(10μMforcysteine,valine,histidine,isoleucine,leucine,lysine,methionine,serine,threonine,andglutamicacid)and10μMglucose.(C)PhotostabilityofFA-FITC@AuNCiunderdifferentpHof6.19,7.42,and8.06respectivelyfor2h.Errorbarsrepresentstandarderrormeasurements(S.E.M.)ofthreeparallelexperiments.(D)FluorescencereversibilityresponsesofFA-FITC@AuNCbetweenpH6.0and8.0.cells,FRàvelungcarcinomacellsA549,andFRàvehumankidneycells293T.Todevelopfurtherbiologicalimagingapplications,thelong-termcellulartoxicityofboth?uorescentprobeswerede-terminedbystandardMTTassays.InthepresenceofFITC@AuNCprobeswithconcentrationfrom5to100μgmLà1,thecellularviabilitiesofHelacellswereestimatedtobegreaterthan85%and80ˉterincubationfor24and48h(Fig.S5A,SI),whilethoseofA549cellsweregreaterthan80%and75%(Fig.S5B,SI).Mean-while,thecellularviabilitiesofcellstreatedwithFA-FITC@AuNCprobesfor24and48hwereupto80%and75%inHelacells,aswellas85%and90%inA549cells.Besides,non-cancerFRàvehumankidney293TsurvivedmuchmorewithFA-FITC@AuNCprobe(488%for24h,493%for48h)thanwithFITC@AuNCprobe(493%for24h,475%for48h)underthesametreatmentconcentration(Fig.S5C,SI).TheseresultsindicatedthatbothFIT-C@AuNCandFA-FITC@AuNCprobesisgenerallylow-toxicforcellularimaging,possiblybecauseofgoodbiocompatibilityofthesurroundedbiomoleculeBSA.3.4.FR-targetedimagingandcellularuptakeofFA-FITC@AuNCTheFR-targetedopticalimagesofFA-FITC@AuNCandFIT-C@AuNCwereobtainedbyusingFRtveHelacellsandFRàvelungcarcinomacellsA549.Fig.5presentstheoverlapimagesof?uor-escentchannelFRed(580nm–680nm)andbright?eldofFRàveA549cellsandFRtveHelacells,whichweretreatedwithFA-FIT-C@AuNCandFITC@AuNC.Hoechst33342stainingwascarriedouttodifferentiatethenucleusfromcytosol.ItdemonstratedthatAuNCsprobesweredispersedmainlynearthenucleusofcells(Fig.S6,SI).Therelativedifferenceintheuptakeofprobeswasmea-suredbycollectingthe?uorescenceofFRed(Fig.5C).ThereisnoobviousdifferencebetweentheconsumingamountofFITC@AuNCprobesintoFRtveHelacells(Fig.5A1)andFRàveA549cells(Fig.5B1)afterincubationof2h.Itappearedtobenosigni?cantstainingorcellularuptakerespondingtoFA-mediatedprocessintheintracellularuptakeprocessofFITC@AuNCprobes.However,FRtveHelacellstooknearlythreetimesofFA-FITC@AuNCprobes(Fig.5A2)asmuchasofFITC@AuNCprobes(Fig.5A1).Ascom-pared,FRàveA549cellsand239TcellstookuplessFA-FITC@AuNCthanHelacellsdidduringthesameculturetime(Fig.S8,SI),whichwascorrespondedwiththelackoffolatereceptoronitscyto-membrane.Furthermore,whenHelacellswerepretreatedwithFAfor2h,theuptakeofFA-FITC@AuNCintoHelacellssigni?cantlydecreased(Fig.5A3).TheseresultsclearlysuggestthatFA-FIT-C@AuNCwerespeci?callytakenupbyHelacellsviaaprocesscorrespondingtotheFRonthesurfaceofcells,andgatherednear

188C.Ding,Y.Tian/BiosensorsandBioelectronics65(2015)183–190Fig.5.FluorescentmicroscopicimagesshowinginteractionofFA-FITC@AuNCandFITC@AuNCwith(A)Helacellsand(B)A549cells:(A1)FRtveHelaand(B1)FRàveA549cellswithFITC@AuNC,(A2)FRtveHelaand(B2)FRàveA549withFA-FITC@AuNCofincubationfor2hat37°C,andFRtveHelacellsincubatedwithFA-FITC@AuNCfor2haftertreated(A3)withFAfor2h,(A4)at4°C,and(A5)10mMNaN3for2h.(C)CellularinternalizationamountofFA-FITC@AuNCandFITC@AuNCindifferenttypesofcelllinesundervarioustreatments.Errorbar:standarderrormeasurements(S.E.M.).Scalebar:50μm.nucleuswiththe?uorescenceintensityremainingintact.Whencellswereculturedwithprobesat4°C(Fig.5A4)orpretreatedwithNaN3(Fig.5A5)for2htoblocktheenergy-dependenten-docytosis,theconsumingamountofprobeswereidenticallyde-creased(Fig.5C).TheseresultsindicatethattheuptakeofFA-FITC@AuNCprobesweretaken-upintoHelacellsmainlyviaapathwaywithfolic-receptorinvolvedandenergydependent.ItalsoindicatedthemaintenanceoftheprobesanditspotentialapplicationforpHdetectionintheintracellularregions.Next,theprocessofcellularuptakeofFA-FITC@AuNCprobesintoHelacellsweremonitoredbyculturingHelacellsinserum-freemediawith50μgmLà1FA-FITC@AuNCprobesat37°Cfor0.5h,1h,2h,4h,and24h,respectively.The?uorescencein-tensitiesfrom510to550nm(FGreen)andfrom580to680nm(FRed)werecollectedtoindicatepHvaluesinthecellularuptakeprocessaswellastheconsumingamountofprobesincells.Fig.S7(SI)showedthatafter2hincubation,theconsumingofprobesintocellsreachedmaximum.Then,theamountofprobesincellsdecreasedrapidlyandalmostdisappearedafter24hincubation(Fig.S7A-E,SI).Itindicatesaquickmetabolismaswellasthelowcytotoxicityofsuchprobesincells.Besides,itcouldbenoticedthattheprobeswere?rstlygatherednearcytomembrane(Fig.S7B,SI),thentransferrednearnuclear(Fig.S7D,SI).Inthisprocess,theemissionofFGreenfromFITCincreasedgraduallywiththecolorofthe?uorescentmergedimagesturnedfromfalsered(Fig.S7B,SI)toyellow(Fig.S7D,SI).ItsuggeststhatthepHoftheen-vironmentofprobesincreasedamongtheprobemovingfromnear-cytomembranetonear-nuclearregion.ThoughtheprecisemechanismofFRtransportofFAintocellsremainedunsolved,itisclearthatfolateconjugatesweretakenupnondestructivelybymammaliancellsviareceptor-mediateden-docytosis(LuandLow,2002;Rothbergetal.,1990a,1990b;Tureketal.,1993).SuchfolateconjugateswereobservedtointernalizeendosomesaftertheybondtoFRonthecancercellsurface(Tureketal.,1993;VarmaandMayor,1998;Wuetal.,1997).Ithasalsobeenreportedthatfolateconjugate-containingendosomeshaveC.Ding,Y.Tian/BiosensorsandBioelectronics65(2015)183–190189ABCDEFGHIJKLFig.6.(A–C)Confocal?uorescenceimagesof(A)FGreen(510–550nm),(B)FRed(580–680nm),and(C)overlapped?uorescenceandbright?eldimageofHelacellsincubatedwithFA-FITC@AuNCfor1hexcitedby488nm;(D)FluorescenceemissionscanfromHelacellswithFA-FITC@AuNCprobes.(E–G)Nat–Htexchangedependentpseudocolorimages(FGreen/FRed)ofHelacellswithFA-FITC@AuNCprobesstimulatedbyouabain.(E)HelacellsincubatedinClà-freeRinger'ssolutionscontaining0.1mMouabainfor45min,followedby(F)treatedinNat-freeRinger'ssolutionsforanother5min,and(G)adding100mMNatforanother5min.(H)BargraphrepresentingFGreen/FRed.(I–L)Nat–Htexchangedependentpseudocolorimages(FGreen/FRed)ofHelacellswithFA-FITC@AuNCprobesstimulatedbyNH4Cl:(I)HelacellsareincubatedinClà-containingRinger'ssolutionswith30mMNH4Clfor4min,followedby(J)washingNH4Clfromthesolution,and(K)addingNat-containingRinger'ssolutionsforanother2min;(L)BargraphrepresentingFGreen/FRed.Valuesarethemeanratiosgeneratedfromtheintensitiesfrom?verandomlyselected?elds.Errorbarsrepresentstandarderrormeasurements(S.E.M.).Scalebar:50μm.beenshowntohavepHvaluesbetween4.3and6.9duetoaprocesscalledendosomeacidi?cation(Leeetal.,1996).Asatar-geted-introducer,FAactedasimilarroleintheuptakeofFA-FIT-C@AuNCprobeforthedetectionofintracellularpH.Asaresult,FA-FITC@AuNCprobeenteredFRtveHelacellsspeci?callyviaapathwaycorrespondingtoitsrichfolatereceptorsonthesurface.Suchprocesswasenergy-depended,andcouldbeblockedbylowtemperatureorthepresenceofNaN3.Afterbeingtakenintothecells,thetwoemissions(FGreenandFRed)ofprobeacteddifferentlywhenittransferredfromtheregionsnearcytomembranetogathernearthenuclear.Inthistransmission,theaverageratiooftwoemissionchannelincreasedgradually,indicatinganincreaseinthepHoftheintracellularenvironment.3.5.BioimagingandbiosensingofpHincancercellsAccordingtothe?uorescenceimages(Fig.6AandB)andtheiroverlayimageswithbright-?eldimage(Fig.6C),itwasclearthattheprobehadgoodcell-permeability.The?uorescencescaninHelacellstreatedwithFA-FITC@AuNCprobes(Fig.6D)con?rmedthepresentprobesmaintainedthedual-emissionincellularen-vironment,withtwoemissionscenteredat520nmand640nm.Theemissionsincellshadalittlered-shiftcomparedtothatinPBSduetothevariouscellularenvironmentanddeviationsfromdif-ferentdetectors(Zhuangetal.,2014).Next,FA-FITC@AuNCbiosensorwasusedintheapplicationofintracellularpHdetection.TheNat–HtexchangehasbeenacommonmechanismforregulatingcytosolicpH(Paradisoetal.,1984).Uponexcitationat488nm,theratioimagesofHelacellstreatedwithFA-FITC@AuNCwerecollectedfromtwoseparatedchannels.Byusinga“pseudocolorimage”technology,theratiovalueofthegrayvalueofgreenchannelandredchannelwascodedintodifferentcolors:redfortheratioof0.1andbluefortheratioof1.5.AfterHelacellswereincubatedinClà-freeRinger'ssolutionscontaining0.1mMouabainfor45min,theaverageemissionratioFGreen/FRedis1.06170.191(Fig.6E),similartothatinHelacellsincubatedwithoutouabaintreatment(Fig.6H).Then,theratioslightlydecreasedto1.00870.265whenthecellswerecontinuouslyincubatedwithaNat-freeRinger'ssolutionsforanother5min(Fig.6H).Thepseudocolorclearlychangedfrombluetored(Fig.6F).Suchchangeindicatedarapidlyacidi?cationofcellsunderstimulateoftheNat-freeRinger'ssolutions.Afteraddinganother100mMNatsolution,suchratioincreasedbackto1.05770.149(Fig.6Gand6H),suggestingarecoverofpHintheintracellularenvironment.Inanotherset,HelacellswithFA-FITC@AuNCwereincubatedinaClà-containingRinger'ssolutionfor30min,thentreatedwith30mMNH4Clfor4min(Fig.6I).TheacidloadwasachievedbywashingtheNH4Clfromthesolutions(Fig.6J).Afteranothersti-mulatebyNat,cellswouldcomealkalinizedtoahigherpH,with190C.Ding,Y.Tian/BiosensorsandBioelectronics65(2015)183–190thepseudo-colorimageturnedintoblue(Fig.6K).Suchchangeswasmonitoredbyusingamodeoftimescan(Fig.6L).ItcouldbenoticedthataftertheremoveofNH4Cl,theratiovalueofFGreenandFRedchannelsdecreasedto0.97570.040,indicatethepHincellsdecreasedto～6.5.AstheadditionofNat,theratiovalueoftwochannelsincreasedto1.62270.066?rstly,thenrecoveredto1.49570.059.ItshowedthatsuchstimulatewillcauseasuddenincreaseofpHincells,thenrecoveredafter～4min.TheseinitialexperimentsinHelacellsdemonstratedthatFA-FITC@AuNCprobecouldbeusedtoobservethechangesofpHinlivecellsthroughthe?uorescenceratioofdifferentemissionchannels,suggestingthegreatpotentialofthisdual-emissionprobeforfurtherfunda-mentalbiologyresearches.4.ConclusionsToconclude,aratiometric?uorescencebiosensorforpHde-tection,FA-FITC@AuNC,hasbeendevelopedsuccessfullyforspe-ci?cbioimagingandbiosensingincancercellsatthesametime.Inthisself-calibrationbiosensor,?uorescentAuNCsworkasre-ferencesignal,FITCplaystheroleforpHresponseandFAactsasspeci?crecognitionelementforcancercellstarget.ThedevelopedbiosensorhasbeenusedtomonitorpHgradientsinapHrangeof6.0–7.8withgoodsensitivity,highcyclicaccuracy,andshortre-sponsetime.Italsoexhibitshighselectivityovervariousmetalionsandbiologicalspecies.Furthermore,theAuNC-basedin-organic-organicbiosensorshowsgoodbiocompatibilityandlong-termstabilityagainstlightillumination.Asaresult,thetargetimaginghavebeentesti?edbyusingFRtveHelacells,FRàveA549cellsandFRàve293Tcells,andachievedthebiosensingofpHinHelacells.Ithasbeenalsocon?rmedanacidi?cationprocessanditsrecoveryinthetransversionofFAconjugatesenteringintocancercells.Thisinvestigationhasprovidedamethodologytodesigningtheratiometric?uorescentbiosensorwithtargetedmolecules,byconjugatingdifferentfunctionunits,suchastar-getedanddetectormoleculesononekindofspeci?cnanomaterial.Inaddition,thisworkcanbeextendedtoconstructfutureratio-metric?uorescentbiosensorsfortargetedimaging,drugdeliver,orthedetectionofotherbiomolecules,suchasmetalions,proteins,andotherbiologicalspecies.AcknowledgmentsThisworkwas?nanciallysupportedbytheNSFC(21175098,and21175044)andNationalNatureScienceFundfordistinguishedyoungscholars(21325521).AppendixA.SupplementarymaterialSupplementarydataassociatedwiththisarticlecanbefoundintheonlineversionatdoi:http://dx.doi.org/10.1016/j.bios.2014.10.034ReferencesDennis,A.M.,Rhee,W.J.,Sotto,D.,Dublin,S.N.,Bao,G.,2012.ACSNano6,2917–2924.Du,F.,Ming,Y.,Zeng,F.,Yu,C.,Wu,S.,2013.Nanotechnology24,365101.Fu,Y.,Ding,C.,Zhu,A.,Deng,Z.,Tian,Y.,Jin,M.,2013.Anal.Chem.85,11936–11943.Gao,X.,Ding,C.,Zhu,A.,Tian,Y.,2014.Anal.Chem.86,7071–7078.Golovina,V.A.,Blaustein,M.P.,1997.Science275,1643–1648.Guével,X.L.,H?tzer,B.,Jung,G.,Hollemeyer,K.,Trouillet,V.,Schneider,M.,2011.J.Phys.Chem.C115,10955–10963.Izumi,H.,Torigoe,T.,Ishiguchi,H.,Uramoto,H.,Yoshida,Y.,Tanabe,M.,Ise,T.,Murakami,T.,Yoshida,T.,Nomoto,M.,Kohno,K.,2003.CancerTreat.Rev.29,541–549.Kong,B.,Zhu,A.,Ding,C.,Zhao,X.,Li,B.,Tian,Y.,2012.Adv.Mater.24,5844–5848.Kong,B.,Zhu,A.,Luo,Y.,Tian,Y.,Yu,Y.,Shi,G.,2011.Angew.Chem.Int.Ed.50,1837–1840.Lagadic-Gossmann,D.,Rissel,M.,Galisteo,M.,Guillouzo,A.B.,1999.J.Pharmacol128,1673–1682.Lee,R.J.,Low,P.S.,1994.J.Biol.Chem.269,3198–3204.Lee,R.J.,Wang,S.,Low,P.S.,1996.Biochim.Biophys.Acta1312,237–242.Li,P.,Xiao,H.,Cheng,Y.,Zhang,Wen,Huang,F.,Zhang,W.,Wang,H.,Tang,B.,2014.Chem.Commun.50,7184–7187.Liu,J.,Guan,Z.,Lv,Z.,Jiang,X.,Yang,S.,Chen,A.,2014.Biosens.Bioelectron52,265–270.Loiselle,F.B.,Casey,J.R.,2003.Methods.Mol.Biol.227,259–280.Lu,Y.,Low,P.S.,2002.Adv.Drug.Deliv.Rev.54,675–693.Miao,F.,Song,G.,Sun,Y.,Liu,Y.,Guo,F.,Zhang,W.,Tian,M.,Yu,X.,2013.Biosens.Bioelectron50,42–49.Paradiso,A.M.,Tsien,R.Y.,Machen,T.E.,1984.Proc.Natl.Acad.Sci.81,7436–7440.Patterson,G.H.,Knobel,S.M.,Sharif,W.D.,Kain,S.R.,Piston,D.W.,1997.Biophys.J.73,2782–2790.Peng,H.,Stolwijk,J.A.,Sun,L.,Wegener,J.,Wolfbeis,O.S.,2010.Angew.Chem.Int.Ed.49,4246–4249.Roos,A.,Boron,W.F.,198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