Recent progress in the development of near-infrared fluorescent probes for bioimaging applications

TUTORIAL REVIEW

Zhiqian Guo, Sookil Park, Juyoung Yoon and Injae ShinRecent progress in the development of near-infrared fl uorescent probes for bioimaging applications

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TUTORIALREVIEWView Article OnlineView Journal | View IssueCitethis:Chem.Soc.Rev.,2014,43,16

Recentprogressinthedevelopmentofnear-infraredfluorescentprobesforbioimagingapplications

ZhiqianGuo,abSookilPark,cJuyoungYoon*aandInjaeShin*cNear-infrared(NIR)fluorescentdyeshaveemergedaspromisingmodalitiesformonitoringthelevelsofvariousbiologicallyrelevantspeciesincellsandorganisms.TheuseofNIRprobesenablesdeepphotonpenetrationintissue,minimizesphoto-damagetobiologicalsamples,andproduceslowbackgroundauto-fluorescencefrombiomoleculespresentinlivingsystems.Thenumberofnewanalyte-responsiveNIRfluorescentprobeshasincreasedsubstantiallyinrecentyearsasaconsequenceofintenseresearchefforts.Inthistutorialreview,wehighlightrecentadvances(2010–2013)madeinthedevelopmentandapplicationsofNIRfluorescentprobes.ThereviewfocusesonNIRfluorescentprobesthathavebeen

Published on 19 September 2013. Downloaded on 08/12/2013 01:50:17. Received24thJuly2013DOI:10.1039/c3cs60271kwww.rsc.org/csr

devisedtosensevariousbiologicallyimportantspecies,includingROS/RNS,metalions,anions,enzymesandotherrelatedspecies,aswellasintracellularpHchanges.ThebasicprinciplesinvolvedinthedesignoffunctionalNIRfluorescentprobesandsuggestionsabouthowtoexpandapplicationsofNIRimagingagentsarealsodescribed.

Keylearningpoints(1)(2)(3)(4)(5)UsefulnessofNIRfluorescentprobesforinvitroandinvivostudiesNIRsensingsystemstodetectbiologicallyrelevantspeciesStrategyfordesignofNIRfluorescentprobesSensingmechanismsofNIRfluorescentprobesApplicationsofNIRfluorescentprobes1.Introduction

Fluorescenceimagingtechniqueshavebecomepowerfultoolsfornoninvasivevisualizationofbiologicalprocessesinrealtimewithhighspatialresolution.1–3Inparticular,near-infrared(NIR)emissiongreatlyfacilitatesinvivoimagingofmolecularprocesses.4,5Overthepastdecade,NIRfluorescentprobeshavebecomepromisingmodalitiesformonitoringinvitroandinvivolevelsofvariousbiologicallyimportantspecies.6–8Ingeneral,NIRfluorophoresaredefinedassubstancesthatemitfluorescenceintheNIRregion(650–900nm).

Comparedtomostotherconventionalfluorescentprobes,thosethatrelyonNIRfluorescencepossessuniqueadvantagesfortracingmolecularprocessesinvitroandinvivo.9Firstofall,

aDepartmentofChemistryandNanoScience,GlobalTop5ResearchProgram,EwhaWomansUniversity,Seoul120-750,Korea.E-mail:jyoon@ewha.ac.krbKeyLaboratoryforAdvancedMaterialsandInstituteofFineChemicals,EastChinaUniversityofScience&Technology,Shanghai200237,P.R.ChinacDepartmentofChemistry,YonseiUniversity,Seoul120-749,Korea.E-mail:injae@yonsei.ac.kr

hightissueauto-fluorescencetakingplacefromindigenousbiomoleculesinthelivingsystemsdoesnotinterferewithNIRemission.Moreover,NIRphotonscanpenetraterelativelydeeplyintotissuesandtheycauselessdamagetobiologicalsamples.Undoubtedly,NIRfluorescentprobeswillprovidechemistsandbiologistswithmanyopportunitiestoconductstudiesleadingtoagreaterunderstandingofbiologicalprocessesatthemolecularlevel.Actually,NIRprobeshavebeenemployedtosuccessfullyimagetumorsbothinvitroandinvivo.Inaddition,someotherapplicationsinclinicalpracticeshavealsobeendeveloped.

QuiteafewreviewshavebeencompileddescribingNIRfluorophores.4–8Forexample,in2010,Stronginandcoworkerspublishedanicereviewon‘‘NIRdyesforbioimagingapplica-tions’’,4whichcoversrecentprogressmadeupto2009inthesynthesisandevaluationofnewNIR-activeorganicdyes.Veryrecently,acriticalreviewoffar-redtoNIRfluorescentprobeswaspublishedbyLinandcoworkers.8However,forthemostpart,thesereviewsfocusedonimprovementsmadeinthephotophysicalandphotochemicalpropertiesofconventionalNIRorganicdyesusedforpotentialbioimagingapplications.

16|Chem.Soc.Rev.,2014,43,16--29Thisjournalis©TheRoyalSocietyofChemistry2014

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Published on 19 September 2013. Downloaded on 08/12/2013 01:50:17. Manyinterestingstudies,relatedtotheuseofNIRfluorescentprobesfortracingbiologicallyimportantspeciesinvitroandinvivo,havebeencarriedoutrecently.Inparticular,advanceshavebeenmadeinimprovinginvivoperformanceofNIRimagingagentsthroughencapsulationofNIRfluorescentprobesinnanoparticles.Inthistutorialreview,theresultsofrecentstudiesofNIRfluorescentprobesaredescribedusingaformatthatisbasedonsensingbiologicalspecies.Attentionisgiventocontributionsappearinginthe2010–2013timeperiodandtoNIRfluorescentprobesusedforsensingvariousimportantbiologicalspecies,includingreactiveoxygenspecies(ROS)andreactivenitrogenspecies(RNS),metalionsandanions,enzymesandotherrelatedspecies,aswellasintracellularpHchanges.TheprimaryfocusisgiventoprogressmadeintheapplicationoffunctionalNIRfluorescentprobesfortracingimportantintracellularspecies.

Inaddition,thedesignstrategies,sensingmechanisms,andinteractionmodesofrepresentativeNIRfluorescentprobesarealsodiscussed.Finally,suggestionsaregivenaboutthepossibledesignandapplicationofnewNIRfluorescentprobes.

2.ROSandRNSsensing

Freeradicalsandotherintracellularreactivespeciesserveasimportantsignalingagentsinphysiologicalandpathologicaleventsthatgoverncellularfunctions.Currently,itisclearthatROS/RNSareimportantmessengersthatmediatevariousbiologicalprocesses.However,agreatchallengestillexistsindetectingROS/RNSinvivoowingtotheirreactiveandshort-livednature.10Becauseoftheiradvantageousproperties,NIRfluorescent

ZhiqianGuo

ZhiqianGuoreceivedhisPhDdegreeinAppliedChemistryfromEastChinaUniversityofScienceandTechnology(ECUST,Shanghai)in2010underthesupervisionofProf.WeihongZhu.HethenservedasalectureratECUST.Aftercompletingpost-doctoralstudieswithProfessorJuyoungYoonatEwhaWomansUniversity(Korea)in2012,hereturnedtoECUSTandwasappointedAssociateProfessorin2013.Hisresearchinterestsfocusmainlyonfluorescentchemosensorsandmolecularlogicdevices.

SookilPark

SookilParkwasborninGyeongbuk,Korea,in1987.HeenteredYonseiUniversityinSeoul,Korea,asanundergraduatestudentin2007majoringinchemistryandphysics.HeiscurrentlyworkingwithProfessorInjaeShinoftheDepartmentofChemistryatYonseiUniversity.Hisresearchinterestsincludethesynthesisofbioactivesmallmoleculesandmolecularmodelingbasedoncomputationalscience.

JuyoungYoonwasborninPusan,Korea,in19.HereceivedhisPhD(1994)fromTheOhioStateUniversity.AftercompletingpostdoctoralstudiesatUCLAandScrippsResearchInstitute,hejoinedthefacultyatSillaUniversityin1998.In2002,hemovedtoEwhaWomansUniversity,whereheiscurrentlyaProfessorintheDepartmentofChemistryandNanoScienceandtheDepartmentofBioinspiredJuyoungYoon

Science.Hisresearchinterests

includeinvestigationoffluorescentchemosensors,molecularrecognitionandorganoELmaterials.

InjaeShinreceivedhisBS(1985)andMSdegrees(1987)inChemistryfromSeoulNationalUniversityinKorea.HisPhDstudieswerecarriedoutattheUniversityofMinnesotawithProfessorHung-wenLiu(1991–1995).Aftercarryingoutpostdoc-toralstudiesattheUniversityofCaliforniaatBerkeleywithPro-fessorPeterSchultz(1995–1998),hebeganhisindependentcareerasanAssistantProfessorofInjaeShin

ChemistryatYonseiUniversity

in1998wherehebecameanAssociateProfessorin2001andaProfessorin2006.Hisresearchinterestsincludethesynthesisofvariousbiologicallyandchemicallyinterestingcompounds,thedevelopmentofbioactivemoleculesthatcanbeusedforbiologicalandbiomedicalstudies,andfunctionalstudiesofglycansusingchemicaltoolsincludingglycanmicroarrays.

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probeswouldbehighlyusefulfortrackingROSandRNSincells,tissuesandorganismsatornearsitesoftheirproductionandaction.2.1

SensingROS

Published on 19 September 2013. Downloaded on 08/12/2013 01:50:17. Reactiveoxygenspecies(ROS),includinghydrogenperoxide(H2O2),hydroxylradicals(HO󰀂),superoxideanionradicals(O2󰀂À)andsingletoxygen(1O2),arehighlyreactivemolecules,whichareproducedasby-productsofcellularmetabolism.Areaction-basedstrategyisgenerallyemployedtodesignNIRfluorescentprobesforvariousROStargets.

AsamajorROSinlivingsystems,H2O2playsakeyroleasasecondmessengerinnormalcellularsignaltransduction.TheinvolvementofH2O2incellularsignalinganddiseasestateshasmotivatedthedevelopmentofchemicaltoolsthatcanbeemployedtounderstandhowcellsproduceandfunnelH2O2intospecificsignalingpathwaysinlivingsystems.FluorescentimagingwithH2O2-responsiveNIRfluorophorescreatesauniqueopportunityforreal-time,noninvasivedetectionofH2O2inbiologicalspecimens.Currently,boronate-basedfluorescentprobeshavebeenwidelyemployedtodetectintracellularH2O2.Theoperationofboronate-basedNIRprobestakesadvantageofthefactthatarylboronatesareselectivelyoxidativelytransformedtothecorrespondingphenolsbyH2O2.Recently,Satchi-Fainaro,ShabatandcoworkersexploitedNIRcyanine-basedprobe1,whichcontainsboronateasaspecificreactivemoiety,forsensingH2O2(Fig.1a).11Theboronate-maskedphenolgroupcauses1topossessareducedconjugatedp-electronsystem.Thus,intheabsenceofH2O2,probe1displaysalmostnofluorescenceinaqueoussolutions.Incontrast,H2O2-promotedtransformationoftheboronategroupin1toaphenolgroupresultsingenerationofanextensivelyconjugatedp-electronsystemthatemitsastrongNIRfluorescencesignal(lex=590nm,lem=720nm).Thisprobe

Fig.2Aproposedmechanismforsensingozoneby2.

Fig.1(a)Amechanismforsensinghydrogenperoxideby1;(b)detectionofendogenoushydrogenperoxideproducedinamouseduringalipopoly-saccharide(LPS)-inducedinflammatoryresponse,using1(reprintedfromref.11withpermission).

wasdemonstratedtobeeffectiveforimagingendogenousH2O2producedinanacuteinflammationmousemodel(Fig.1b).

Ithasbeensuggestedthatozone(O3)isproducedendogenouslyaspartofinflammatoryandantibacterialresponsesoftheimmunesystem.However,generationofO3incellsisdifficulttoprovemainlyasaconsequenceofthelackofsensitivedetectionmethods.Instudiesaimedatthisgoal,thetricarboncyanine2conjugatedL-tryptophan(Trp)asanO3-indicatorwasdevelopedbyTang’sgrouptoserveasaNIRozoneprobe(Fig.2).12Probe2displaysweakfluorescencebecauseitsexcitedstateisquenchedbyatwistedintramolecularchargetransfer(TICT)mechanism.UponadditionofO3,theL-tryptophanmoietyisoxidizedtoformaring-openedproduct,thenleadingtoconcurrentturn-onofaNIRfluorescencesignal(lem=770nm).Probe2wasfoundtohavealowdetectionlimitofca.17nMforO3andahighselectivityoverotherROS.ThisNIRprobewasemployedtomonitorthesubcellularlocationsofendogenousO3inmacro-phagesstimulatedbyphorbol12-myristate13-acetate(PMA).ThereactivitypatternsofcyaninedyeswithROShaveservedasthebasisfordesignofatwocyanine-conjugateddyeasanovelROS-sensitiveNIRfluorescentprobe.ItisknownthatthecentralpolymethinechainsofcyaninedyescanbeoxidativelycleavedbyROSspecies,suchassingletoxygenorsuperoxideanionradical.However,thereactivityofROSisgreatlyinfluencedbychemicalstructuresofthecyaninedyes.Inprobe3,wheretwodifferentcyaninedyes(Cy5SO3HandIR786S)areconjugatedtoeachotherviaashortlinker(Fig.3),13fluorescenceisquenchedowingtostackingoftwocyaninedyesbutnotbyaFRET(fluorescenceresonanceenergytransfer)mechanism.BecausetheCy5SO3Hmoietyin3ismorereactivewithROSthantheIR786Sgroup,reactionoftheCy5SO3HmoietyintheprobewithROSdisruptsthestacking.Asaconsequence,NIRfluorescenceoftheIR786Sfluorophoreat813nmisrecovered.Thisprobewassuccessfullyusedforinvivoimagingofoxidativestressinamousemodelofperitonitis.

CyaninedyesareconsideredtobethemostefficientNIRfluorophores,butdifficultiesinchemicalmodificationsofcyaninedyesserveasalimitationtotheirwidespreadapplication.Inordertoaddressthisproblem,alibraryofNIRfluorescentprobesbasedonthedistinctivedonor-two-acceptor(D2A)rationaldesignapproachhasbeenconstructedbyShabatandcoworkers.14Latentdonorsandmultipleacceptormolecules

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TutorialReviewFig.3AproposedmechanismforsensingROSby3.

wereincorporatedintothefluorescentprobes.Theseprobesare designedtoundergointramolecularchargetransfer(ICT)in.71:theirexcitedstatesviathelongerp-conjugatedsystems.For05:1example,theICTfromadonor(e.g.,phenolate)moietytooneof0 3thetwoacceptormoieties(e.g.,picoliniummoiety)leadsto10formationofanewfluorochromethatemitsNIRfluorescence/22/1(700nm).Arepresentativeexampleofthissystemisfoundin80 nNIRprobe4,whereaphenolgroupismaskedintheformofao dH2O2reactiveboronate(Fig.4).14Notably,membersofthisnewedafamilyofNIRprobesdisplaysignificantStokesshiftsthatlonwleadtoagreatersignal-to-noiseratio.AnalysisofconfocaloDimagesofH2O2inHeLacellsutilizing4indicatesthatthe .31probepenetratesintolysosomes.

02 Recently,anewfamilyofNIRfluorescentdyeshasbeenrebmdevisedfordetectionofH2O2byLin’sgroup(Fig.5).15TheseetpNIRfluorescentdyeshavelongerNIRemissionmaximaeS (>700nm)withhighfluorescencequantumyields.Significantly,91 nthesenewNIRdyeshavesimilarpropertiestothosecontainingo dthefluorescein/7-hydroxycoumarinplatform.Specifically,theyehsareapplicabletothedesignoffluorescentprobesbasedonilbuconversionofaboronatetoaphenolgroup.TakingadvantagePoftheirintegratedtunableopticalproperties,thenovelNIRfluorescentprobe5wasdesignedforH2O2detection.Thisprobeitselfexhibitsalmostnofluorescencewhenexcitedatca.690nm.However,followingadditionofH2O2,alargefluorescenceturn-onresponseoccurswithabouta180-foldenhancementinemissionat708nm.Probe5wasusedtoimageendogenousH2O2inmacrophagesandtheperitonealcavityofmiceduringLPS-inducedinflammatoryresponse.

Fig.4Aproposedmechanismforsensinghydrogenperoxideby4.

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Fig.5Aproposedmechanismforsensinghydrogenperoxideby5.

Fig.6AmechanismforsensingROSby6.

Rhodaminesarewidelyusedfluorescentprobesandmolecularmarkersbecauseoftheirexcellentphotophysicalproperties.How-ever,theabsorptionandemissionwavelengthsofmostrhodaminederivativesarebelow600nm.Thedesignofnewfar-redtoNIRrhodamineanaloguesthroughchemicalmodificationsoftheirxanthenecorehasattractedgreatinterest.Advanceshavebeenachievedbyreplacingtheoxygenatomatthe10positionofthexantheneringsystemwithSi(Si-rhodamine,SiR),Ge(GeR),Sn(SnR)andTe(TeR).Forinstance,NaganoandcoworkersdescribedthenewTe-rhodaminederivative6andshowedthatitisareversibleNIRfluorescenceprobefordetectingROS(Fig.6).16Theprobewasdesignedbyusinganoxidation–reductionstrategythattakesadvantageoftheredoxpropertiesofthetellurium(Te)atom.TheTe-rhodaminederivative6displaysveryweakfluorescencebutitsoxidizedformstronglyfluorescesat690nmowingtothereducedheavy-atomquenchingeffectofTeintheoxidizedform.Notably,theoxideformcanbereadilyreducedinthepresenceofglutathionetoregenerate6.ThisprobewasemployedtomonitorendogenousproductionofROSandsubsequenthomeostaticrecoveryoftheintracellularreductiveenvironmentinH2O2-stimulatedHL-60cells.TheredoxcharacteristicsandreversibleNIR-fluorescenceresponseof6suggestthattheprobehasthepotentialforcontinuouslymonitoringthedynamicsofROSproductioninvivo.2.2

SensingRNS

RNS,includingperoxynitrite(ONOOÀ),nitroxyl(HNO),nitro-soniumcation(NO+)andnitricoxide(NO),playcrucialrolesinmanyphysiologicalandpathologicalprocesses.IthasbeensuggestedthatoverproductionofRNScanresultindamageandinhibitionofthenormalfunctionsoflipids,proteinsandDNA.Forinstance,ONOOÀ,ahighlyreactiveRNSspecies,reactswithbiomolecules,causingcelldamageandtheonsetofnumerousdiseases.SeveralNIRprobeshavebeendevisedformonitoringONOOÀ,whicharebasedontheuniquereactivityoftheorganoseleniumreagentswithperoxynitrite.Inspiredbythespecificoxidationofdivalentselenium(Se2+)byONOOÀ,which

Chem.Soc.Rev.,2014,43,16--29|19

ChemSocRevFig.7AmechanismforsensingONOOÀby7.

.71:05:10 310Fig.8Amechanismofthereversibleredoxprocessof8and9promoted/22byONOOÀandGSH.

/180 no generatesquadrivalentselenium(Se4+),NIRprobe7wasdeddesignedtoincorporateabenzylselenidemoietyintothetri-aloncarbocyanineskeleton(Fig.7).17Theprobedisplaysanintensewofluorescencesignalat770nm.However,aftertheoxidationofD .3Se2+toSe4+oxidebyONOOÀ,almostnofluorescenceisdetectable102owingtotheoperationofphotoinducedelectrontransfer(PET) rebquenchingbetweenbenzylselenideoxideandthetricarbocyaninemegroup.InordertoimprovetheONOOÀselectivityof7,theprobetpeSwasencapsulatedintopolymericmicellestoproducenanoparticles. 91The7-encapsulatednanoprobewasappliedformonitoringONOOÀ no producedinlivemacrophages.

dehRedoxhomeostasisiscriticalformaintainingcellularfunctionsilbuandmetabolism.NIRfluorescentprobes,whichmonitorONOOÀPandenableimagingredoxcyclesinlivingcells,havebeendevisedtofollowthedynamicredoxhomeostasis.TheNIRcyanineprobe8operatesbyreversibleONOOÀ-promotedoxidation–reductionoftheseleniummoiety(Fig.8).18NIRfluorescenceofthisprobeisquenchedbyPETbetweenthesignaltransducercyanineunitandthemodulator4-(phenylselenyl)anilinegroup.OxidationoftheseleniummoietybyONOOÀinhibitsPET,aphenomenonthatresultsinanincreaseofafluorescencesignalat775nmina‘‘switchedon’’manner.Notably,asaconsequenceoftheopera-tionofanenzymaticcatalyticcycle,theselenoxidegroupintheoxidized9iseffectivelyandrapidlyreducedbyglutathione(GSH)toproducetheoriginalselenide8,accompaniedbya‘‘switchedoff’’decreaseoftheNIRfluorescence.Theprobe8wassuccessfullyusedforreal-timevisualizationoftheintracellularperoxynitriteconcentrationsduringreversibleendogenousONOOÀredoxcyclesinlivingcells.

3.Thiolsensing

3.1

Sensingcysteine,homocysteineandglutathione

Intracellularthiol-containingmolecules,suchascysteine(Cys),homocysteine(Hcy)andglutathione(GSH),playessentialrolesinmaintainingtheredoxhomeostasisofprotein,cellsandorganisms.

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Fig.9AproposedmechanismforsensingCysby10.

Abnormallevelsofthesemoleculeshavebeenshowntobeassociatedwithdetrimentaleffects,suchasslowgrowth,liverdamageandskinlesions,onhumanhealth.

Recently,Yoon,Parkandcoworkersdevelopedtheratio-metricNIRfluorescentprobeCyAC(10)forselectivedetectionofCysthatoperatesbasedonMichaeladditionofCysandsubsequentintramolecularcyclizationofathioetheradduct(Fig.9).19Inprobe10,anacrylategroupservesasthethiolspecifictriggermoiety.Oncetheacrylategroupisremovedfrom10byreactionwithCys,thep-conjugationsystemoftheprobeischanged.Theresultsofstudiesshowthatprobe10displaysacolorimetricandratiometricresponsetoCys.Inaddition,theprobehasanexcellentselectivityforCysoverHcyandGSHduetothegreatlydifferentratesoftheintra-molecularcyclizationprocessthatproducesthecyclohexanonecontainingproduct.Aratiometricfluorescentresponseof10toCysisenabledbythesignificantshiftintheemissionwave-lengthfrom780to625nminthepresenceofCys.TheprobewasutilizedforbioimagingCysinbreastcancercells.Notably,themodulationofapolymethinep-electronsystembyconjuga-tionandremovalofaspecifictriggermoietypavesthewayforanewstrategyforthegenerationofratiometriccyanine-basedNIRprobes.

In2013,AkkayaandcoworkerscreatedtheMichaelreaction-based,nitroethenyl-BODIPYconjugatedNIRfluorescentprobe11forselectivedetectionofbiologicalthiolsinaqueousmedia(Fig.10).20AdditionofCysresultsinblockingtheintra-molecularchargetransfer(ICT)processin11owingtodisrup-tionofthep-conjugationsystem.Inaddition,PETfromtheelectronrichtrimethoxyphenylmoietytotheelectrondeficient

Fig.10Amechanismforsensingthiolsby11.

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TutorialReviewFig.11Amechanismforsensingthiolsby12and13.

BODIPYcoreisalsosuppressed.Bycontrollingthereactiontime,NIRprobe11displaysselectiveandsensitiveresponsetoCysoverHcyandGSH.

Chen’sgroupdesignedtwoNIRfluorescentprobesCy–NiSe .71(12)andCy–TfSe(13)thatarebasedonaSe–Nbondcleavage:05reactionbythiolstoproducetheselenenylsulfide(Fig.11).21:10 Twostrongelectron-withdrawinggroups,2-nitrophenylselane310and3-(trifluoromethyl)phenylselane,arepresentin12and13,/22respectively.ThesegroupsacttomodulatetheNIRcyanine/180fluorescencesignalthroughadonor-excitedphotoinduced no electrontransfer(d-PET)process.UponadditionofGSHtodedeitherprobe,aNIRfluorescenceenhancement(lem=750nm)alonoccurs.Probe12and13exhibithighlysensitiveandselectiveworesponsestosulfhydryl-containingmoleculescomparedtoD .3otherbiologicallyrelevantanalytes.Thesetwoprobeswere102appliedtomonitoringthiollevelsinmacrophagesandto rebimagingthiolsinratlivertissues.

meItisimportanttovisualizestatesofredoxstressbyusingatpeSprobethatcanrespondtochangesinbothoxidationandreduction 91events.Guidedbytheoxidationofthecatecholaminergicneuro- no transmitterdopamine,thedopaminemoietywasincorporatedasadehredoxmodulatorintoaNIRcyaninescaffoldtoproduceDA–Cy(14,silbFig.12).22Thisprobeexhibitson–off–onfluorescencechangesforuPdetectionofbothH2O2oxidativestressandGSHreducingrepairprocesses.Specifically,reactionof14withH2O2triggersformationofanon-fluorescentproductO–DA–Cy(switch-off)thatisaccompaniedbyacolorchangefrombluetopurple.Subse-quently,O–DA–CyundergoesMichaeladditionreactionwithGSHtoproduceGSH–DA–Cy,whichdisplaysNIRfluorescenceat755nm(switch-on).Probe14wasusedtodetectintracellularredoxenvironmentsinlivingcells.Studiesusingfreshrathippocampustissuesshowthat14issuitableformonitoringH2O2oxidativestressandGSHreducingrepairinvivo.

Becausealterationsintheredoxstatusofsubstratesarecloselyassociatedwithphysiologicalandpathologicalprocesses,

Fig.12AmechanismforsensingH2O2andGSHby14.

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Fig.13SchematicillustrationofGSHandH2O2promotedinterconver-sionbetween15and16.

itisimportanttobeabletotracethesechangesinlivingsystems.Forexample,theredoxcouplecomposedofGSHandhydrogenperoxideinlivingcellsplayskeyrolesinredoxhomeostasisandsignaling.Inordertoestablishareal-timeinvivoimagingmethodtomonitorchangesintheredoxstatusofthiscouple,Tang’sgroupdevelopedtheNIRfluorescentprobeCy–O–Eb(15,Fig.13).23ThisprobecanbeusedtodeterminechangesinGSH–H2O2levelsbasedonanon–offfluorescencechangeassociatedwithopeningofthefive-memberedringpromotedbySe–Nbondcleavage.InthepresenceofGSH,theSe–Nbondof15iscleavedtoformCy–O–SeH(16),inwhichNIRfluorescenceat768nmisefficientlyquenchedbyaPETprocess.AdditionofH2O2to16inducesitsoxidationthatleadstorecoveryofthefive-memberedringin15concomitantwithanincreaseinthefluorescenceintensity.Notably,thisreversibleredoxcyclecanberepeatedseveraltimes.Probe15hasbeenappliedtoreal-timeimagingofchangesintheredoxstatusofGSH/H2O2duringapoptosisandusedformonitoringH2O2concentrationchangesatthewoundmargininzebrafishlarvae.3.2

SensingH2S

Hydrogensulfideisproducedthroughreductionofsulfate,sulfiteandthiosulfatebyanaerobicbacteria.Thismoleculefreelydiffusesintocellswhereitreachestoxiclevels,leadingtosuppressionofcellularrespirationandproliferation.Endogen-ouslyproducedH2Sisinvolvedinanumberofbiologicalprocesses.AdvancesmadeinstudiesofH2Shaveprovidedagreaterunderstandingofthepropertiesandfunctionsofthissubstanceinbiologicalsystems.24ThestrongreducingornucleophilicpropertiesofH2Shavebeenusedadvantageouslyinthedesignoffluorescentprobes.Forexample,reductionofthefluorophore-tetheredazideintheprobeCy–N3(17)byH2S,leadingtogenerationofthecorrespondingamineCy–NH2,isaccompaniedbyachangeinthefluorescencesignal(Fig.14a).25Thischangeisaconsequenceoftheelectron-donatingabilityoftheaminesubstituentpresentinCy–NH2.Recently,FanandPengetal.describedanewNIRdicyanomethylene-4H-pyranchromophore(18)forinvivomonitoringofH2Sthatreliesonthesamestrategy(Fig.14b).26InthepresenceofH2S,theazidegroupof18isconvertedtoanNH2group,aprocessthatbringsabouta65-foldenhancementofthefluorescencesignalat670nm.Probe18hasbeenutilizedtotrackH2Sinlivingcells.Notably,theprobewasalsoappliedtoH2Simaginginmice.

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ChemSocRevFig.14AmechanismforsensingH2Sby(a)17and(b)18.

.71:05:10 310/22/180 no dedalonwFig.15AmechanismforsensingH2Sby19.

oD .3102Lin’sgroupdevelopedthefluorescentturn-onH2Sprobe reb(19),whichwasdesignedusingastrategythatisbasedonmetdinitrophenyletherchemistry(Fig.15).27Probe19displayspeSalmostnofluorescencesignalowingtoefficientPETquenching. 91However,whenthedinitrobenzenemoietyin19isremovedby no Hd2Stogenerate20,afluorescencesignalappearsat708nmehs(turn-on).Analysisofbioimagingdatashowsthatprobe19ilbusensesH2Sinlivingcellsinadosedependentmanner.

PInspiredbythenucleophilicadditionofaphenolaniontotheindoleniumC-2atominthespiropyran,GuoandcoworkersrecentlydesignedaratiometricH2SsensorCouMC(21,Fig.16),28whichhasaquickresponseratetoH2Sand,asaresult,canbeutilizedforreal-timeintracellularimaging.Asahybridofcoumarinandmerocyaninefluorophores,probe21exhibitsratiometricsensingbehaviorderivedfromreactionwithH2SattheindoleniumC-2center,whichleadstosuppres-sionofmerocyaninefluorescence,whileretainingcoumarinemission.Ithasbeenshownthatprobe21displaysahigherselectivityforH2SthanotherbiologicalthiolsincludingCys,HcyandGSH,whicharethemaininterferingspeciesinintracellularH2Sdetection.Moreover,probe21hasafasterresponse(about30s)thanmostotherreportedH2Sprobes.Thus,thisprobehasgreatbenefitswhenaccountistakenofthe

Fig.16AproposedmechanismforsensingH2Sby21.22|Chem.Soc.Rev.,2014,43,16--29View Article Online

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variablenatureandquickmetabolismofendogenousH2Sinbiologicalsystems.Theresultsoflivingcellimagingexperi-mentsshowthatprobe21isusefulforratiometrictrackingofH2Sinmitochondria.

4.Ionsensing

4.1

Sensingmetalions

4.1.1Zn2+.Zincion,thesecondmostabundanttransitionmetalioninthehumanbody,playskeyrolesinthecatalyticfunctionofmanyenzymesandingenetranscription.Mostofthezincionsinbiologicalsystemsaretightlyboundtoproteinsforstructuralandcatalyticfunctions.However,looselyboundzincionsarealsofoundinvarioushumantissuesincludingthebrain.FailureinhomeostasisoffreezincionsiscloselyassociatedwithneurologicaldiseasessuchasAlzheimer’sdisease,amyotrophiclateralsclerosis,Parkinson’sdisease,ischemicstrokeandepilepsy.Inaddition,freezincionsarealsoinvolvedinapoptosis(orprogrammedcelldeath).

Inordertodetectmobilezincions,Tianandcoworkersdevelopedthenaphthalenediimide-basedNIRfluorescentprobesPND(22)andPNT(23)(Fig.17),29inwhichanaphthal-enediimide(NDI)fluorophoreisconjugatedtothezincbindingreceptors,di(pyridin-2-ylmethyl)ethane-1,2-diamine(DPEA)andtri(pyridin-2-ylmethyl)ethane-1,2-diamine(TPEA),respectively.Thetwoprobescontainthestronglyelectrondonatingpiperidinemoiety,whichcausesalongwavelengthshift(orbathochromicshift)oftheabsorptionandemissionmaximaincomparisonwiththeanalogousbromine-substitutedprobe.Interestingly,probes22and23withrespectiveDPEAandTPEAmoietiesdisplaydifferentbindingbehaviorstowardZn2+.PND(22)showsasignificantincreaseinfluorescentintensityuponzincionbindingasaconsequenceofthereliefofPETquenchingbythelonepairofelectronsonthetertiarynitrogenatomintheDPEAmoiety.

However,thefluorescenceandabsorptionmaximaofPNT(23)undergodistinctblueshiftswithconcomitantfluorescenceenhancementuponbindingtoZn2+.TheblueshiftsuggeststhatthenitrogenattachedtotheNDImoietyparticipatesinbindingtoZn2+,forminga5-coordinatecomplexandresultinginadecreaseinitselectrondonatingability.Theadditionalpyridylnitrogenisalsoaligand,forminga5-coordinateZn2+complex.

Fig.17

AproposedmechanismforsensingZn2+by22and23.

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TutorialReviewFig.18

AproposedmechanismforsensingZn2+by24.

Thesynergisticchelatingeffectcauses23tohavealowerselectivitytowardZn2+than22,reflectedbythefactthattheresponseof23tothezincionisinterferedwithbyCo2+,Ni2+andCd2+.BothprobeshavebeenappliedtoimagingZn2+inlivingcellswhereanincreaseinthefluorescenceisobservedinzinc .71ion-treatedcells.

:05:Zhuandcoworkersdevelopedthe6-hydroxyindoleBODIPY-10 3basedNIRfluorescentprobe24forZn2+detection(Fig.18).3010Probe24containsaBODIPYscaffoldwithsalicylaldehyde/22benzoylhydrazoneasatridentatechelatingmoietyforZn2+./180 Thesensingmechanismof24isbasedonachelation-enhancedno dfluorescence(CHEF)effectinducedbytarget-triggereddepro-edatonation.Ingeneral,dyesthatcontainacyclicCQNbondsarelonweaklyfluorescentbecauseisomerizationabouttheCQNwoDbondsefficientlydeactivatestheexcitedstates.However,upon .3bindingofZn2+to24,thephenolgroupisdeprotonatedandthe102CQNbondbecomesincorporatedinasmallchelatering rebsystem.Asaconsequence,theresultingcomplex,Zn(1-O)2,metdisplaysalongwavelengthemissionat680nmwithaZn2+peSdetectionlimitof9.7Â10À7M.Importantly,probe24shows 91 greatselectivityforZn2+inaqueoussolutionswithlittleinter-no dferencefromCd2+.ThissensorhasbeenusedtomonitorZn2+ehslocatedintheperinuclearareaofcytosolinlivingcells.

ilbuWedevelopedthecyanine-basedNIRfluorescentprobe25Pfordetectingendogenouszincionsinlivingcellsandorganisms(Fig.19a).31Thisprobeiscomposedofacyanine-basedfluoro-phoreconjugatedtotris(2-pyridylmethyl)amine(TMPA)asareceptorforZn2+.WhentheTMPAmoietyin25bindstoZn2+,theamineattachedtothecenterofthepolymethinechainoftricarbocyanineisreadilydeprotonatedtoformaniminethatiscross-conjugatedwiththelessdelocalizeddiamino-tetraenegroup.Reduceddelocalizationinducesalargehypsochromicshiftinbothabsorptionandemissionmaximaof25.Whilefree25hasanabsorptionmaximaat670nm,Zn2+-bound25showsanabsorptionmaximaat510nmandtheratiooftheabsorbanceisproportionaltoZn2+concentrations.Inaddition,25hasalmostnoemissionat590nmuponexcitationat510nm.However,theemissionmaximumoftheprobeundergoesalargehypsochromicshiftfrom730nmto590nmuponbindingtoZn2+.Moreover,probe25hasaverystrongbindingaffinity(Kd=1.2nM)andagoodselectivityforZn2+overothermetalions.Thepropertiesof25enableittoserveasanexcellentprobetomonitorZn2+inthenanomolarrange.Importantly,thisprobewasusedtodetectZn2+releasedduringapoptosisandintactZn2+intheneuromastsofzebrafish(Fig.19b),whichisnotobservedforotherzincprobes,suchasNBD-TPEAandZTRS.

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Fig.19(a)AproposedmechanismforsensingZn2+by25;(b)fluorescentdetectionofzincionsinneuromastsofzebrafishusing25(reprintedfromref.31withpermission).

4.1.2Copperions.Copperisanessentialtransitionmetalinlivingorganismsbecauseitisinvolvedinvariousphysiologicalprocessessuchasenzymecatalysis,developmentandgrowth.LossofcopperhomeostasisinanorganismresultsinneurodegenerativediseasesincludingAlzheimer’sdisease,Menkesdisease(copperdeficiency),Wilson’sdisease(copperoverload)andamyotropiclateralsclerosis.Despitethebiologicalsignificanceofcopperions,onlyafewNIRfluorescentprobeshavebeenexploitedfortheirdetection.

TangandcoworkersdevelopedtheNIRfluorescentprobe26,composedoftricarbocyanineasafluorophoreand2,20-azanediylbis(N-hydroxyacetamide)asaCu2+receptor,fordetectionofCu2+(Fig.20).32Whilefree26displaysweakfluorescencebecauseofaPETquenchingmechanism,itsfluorescenceisstronglyenhanceduponbindingtoCu2+,whichblocksPETquenching.Probe26hasanabsorptionmaximumat750nmandemissionmaximumat800nm.However,uponbindingtoCu2+,a30nmblue-shiftoftheemissionbandoccurswithasix-foldincreaseinthefluorescencequantumyield.Probe26,whichhasgoodselectivitytowardCu2+overotherbiologicallyrelevantcations,hasbeenemployedtoimageexogenousCu2+inlivecellsandzebrafish.Itshouldbenotedthatbecause

Fig.20AproposedmechanismforsensingCu2+by26.

Chem.Soc.Rev.,2014,43,16--29|23

ChemSocRevFig.21

AproposedmechanismforsensingCu+by27and28.

probe26displaysamorestrongfluorescenceinanacidicthaninaneutralenvironmentafterbindingtoCu2+,itcanbeusedto imageCu2+inthelysosome(pHB5).

.71:Owingtothepotentredoxactivityofthecopperions,05:1detectionofCu+inbiologicalsystemsisalsoimportant.Chang0 3andcoworkersdevelopedtheNIRfluorescentprobe27for10invivoimagingoflabileCu+(Fig.21).33Theprobeconsistsof/22/1acyaninedyeasafluorophoreandelectron-rich9-aza-2,6,13-80 ntrithiapentadecaneasaCu+-selectivereceptor.Theprobedoesnoto dexperienceaspectralshiftuponbindingtoCu+,buta15-foldedaenhancementinfluorescenceintensityat790nmisobservedinlonwresponsetoCu+asaconsequenceofsuppressionofthePEToDquenchingmechanism.Probe27hasa1:1bindingstoichiometry .31andadissociationconstant(Kd)of3.0Â10À11MwithCu+.For02 biologicalapplications,amodifiedformof27wasproduced.Therebmnewprobe28hasenhancedcell-membranepermeability.Onceetpinsidecells,theacetoxymethylgroupin28isremovedbyintra-eS cellularesterasestogenerate27whosecarboxylateshelptrapthe91 nprobewithincells.Theresultofinvitroandinvivoexperimentso dshowthatprobe28detectslabileCu+inlivingcellsandmice.Inehsiaddition,theprobewasemployedtomonitorthepathologicallbudevelopmentofWilson’sdiseaseinmicebydetectingcopperlevels.P4.1.3Mercuryions.Amongtoxicmetalions,mercuryisoneofthemostprevalentonearth.Thismetalionreadilypassesthroughbiologicalmembranesanditisconvertedtoorganomercuryspeciesthatbecomeaccumulated,asmethyl-mercuryionsforthemostpart,inlivingorganisms.Mercuryspeciesinthebodycausedamagemainlytothecentralnervoussystem.Concernoverthetoxicityofmercuryhasstimulatedthedevelopmentofselectiveandefficientmethodstosensemer-curyionsincellsandorganisms.Althoughmanyfluorescentprobeshavebeendevelopedforthispurpose,mercury-selectiveNIRfluorescentprobeshavebeenonlyrecentlydescribed.Tianandcoworkersdevelopedthetricarbocyanine-basedNIRfluorescentprobes29and30tomonitorHg2+andCH3Hg+(Fig.22).34Theresponseofthesedyestomercuryspeciesis

Fig.22

AproposedmechanismforsensingHg2+orMeHg+by29and30.

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basedonthestrongthiophilicaffinityofmercury.Bindingofmercuryspeciestotheprobespromotesdesulfurizationofthethioureamoietyandcyclizationtogeneratethecorrespondingcyclicguanidines.Inthecyclizationprocess,thecentralbridgingsecondaryamineofthetricarbocyanineisconvertedtotheimidazolinetertiaryamine.Asaconsequence,theelectron-donatingabilityofthisnitrogenisattenuatedand,thus,anICTprocessoperatingintheprobeissharplyreduced.Thischangeleadstoalargeredshiftinabsorptionmaximafor29(from6nmto840nm)and30(from668nmto830nm),whichallowsdetectionofthecolorchangebythenakedeye.Influorescentproperties,probe30displaysanemissionmaximumat780nm,theintensityofwhichisremarkablydecreasedinthepresenceofHg2+,uponexcitationat670nm.However,mercuryion-inducedturn-onfluorescenceisobservedat830nmuponexcitationat810nm.AsimilarfluorescenceresponsetoHg2+isobservedin30.TheseprobesshowagreatselectivitytoHg2+overothermetalions.Theresultsofcellexperimentsrevealthatprobes29and30canbeappliedtothemonitoringofmercuryionsincells.Jiangandcoworkersalsodevelopedacyanine-basedNIRfluorescentprobeforHg2+,35whichiscomposedofatricarbocyaninefluorophoreandathymine(T)moietythatisknowntoassociatewithHg2+toformT–Hg2+–T.ThebindingstoichiometryoftheprobewithHg2+wasdeterminedtobe2:1,suggestingtheformationofT–Hg2+–T.However,theprobewasnotappliedforbioimagingHg2+.

Theturn-onNIRfluorescentprobe31forHg2+,whichcontainsaBODIPYfluorophore,wasdevelopedbyGuoandcoworkers(Fig.23).36Despitetheirmanyadvantageousfeatures,NIRfluorescentdyesgenerallysufferfromlowfluores-cenceefficiencies.Toovercomethisproblem,anewstrategywasemployedwhichreliedonindirectexcitationfromtheS0totheS2stateoftheprobe.Probe31containsdi(methoxystyryl)-BODIPYasafluorophoreandaHg2+bindingmoiety.Intheabsorptionspectraof31,astrongS0-S2transitionexistsat370nmalthoughthetypicalS0-S1transitionisalsoobservedat0nm.Ingeneral,thephotonnumberabsorbedisdepen-dentonthewavelengthanditincreaseswithadecreaseinthewavelength.Inprobe31,morephotonswithhigh-energyareabsorbedbytheindirectS0-S2excitationthanbythedirectS0-S1excitation,leadingtoa2.5-foldemissionenhancement.UponadditionofHg2+,thelong-wavelengthbandof31under-goesaslightredshift,whilearemarkable30-foldenhancement

Fig.23

AproposedmechanismforsensingHg2+by31.

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TutorialReviewFig.24

AmechanismforsensingHg2+by32.

ofthefluorescencesignalat655nmtakesplaceasaresultofthesuppressionofPETfromtheaniline-containingreceptortoBODIPY.AlthoughAg+,Ni2+andCd2+causeaslightincreaseinfluorescenceof31at655nm,theprobehasagoodselectivity forHg2+overothermetalions.Thisprobewassuccessfully.71:employedtomonitorexogenousHg2+inlivingcells.Akkaya05:1andcoworkersalsoexploitedaBODIPY-basedHg2+-selective0 3probe,37whichconsistsofthedithiaazacrownligandasaHg2+10binderandtwoBODIPYfluorophoresthatcanfunctionasan/22excitationenergydonorandacceptor.Theexcitedstateenergy/180 ofthedonorBODIPYistransferredtothereceptorBODIPY.no dThisprobehasagoodselectivityforHg2+overothermetalions,edabutithasnotbeenappliedforbioimagingHg2+duetoitspoorlonwwatersolubility.

oDWuandcoworkersdevelopedasilicone-incorporated .31rhodamine-basedNIRfluorescentprobe32fordetectionof02 Hg2+(Fig.24).38Unliketypicalrhodaminedyes,Si-rhodaminerebmhasabsorptionandemissionintheNIRregionbecauseoftheetplow-lyingLUMOcausedbys*-p*conjugation.OwingtotheeS red-shiftedabsorptionband,Si-rhodaminehasabluecolor91 nwhenitsspirolactoneorspirolactamringisopened.Upono dbindingtoHg2+,32undergoesdesulfurizationofthethioureaehsmoietyandcyclizationtoformthecorrespondingoxadiazole.ilbuDuringthisprocess,theprobechangesfrombeingcolorlesstoPiconicblue.Thespirolactamring-openedformoftheprobehastheabsorptionbandcenteredat6nmandaturn-onfluorescencemaximumcenteredat680nm.Probe32showsamarkedenhance-ment(ca.500-fold)influorescenceintensityinresponsetoHg2+.ThisprobehasahighselectivityforHg2+overotherionsandhasbeenutilizedtoimageexogenousHg2+inlivingcells.4.2

Anions

Liketheirmetalcationcounterparts,anionsoftenplaycrucialrolesinbiologicalprocesses.Therefore,detectionofthebiologicallyrelevantanionswithhighselectivityandsensitivityinbiologicalsystemsisanimportantgoal.ThekeypathwayfollowedbymostNIRfluorescentprobes,whicharedesignedtodetectanions,reliesontheaffinityofanionstometalcations,especiallycopperions,thatareincorporatedintoprobes.ThisprocessleadstoreleaseoftheNIRfluorophore(displacementapproach)andconcomitantrestorationofitsfluorescence.SeveralNIRfluorescentprobesfordetectingspecificanions,whichutilizethisapproach,havebeendescribedrecently.

Hydrogencyanide(HCN),producedbyPseudomonasaeruginosa,issuggestedtobeinvolvedinthepathogenesisofcysticfibrosislungdisease.Therefore,monitoringcyanideinvivoisanimportant

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Fig.25Aproposedmechanismforsensing(a)CNÀby33-Cu2+,(b)sulfideanionsby34-Cu2+,and(c)PPiby35-Cu2+.

componentofstudiesaimedatgaininganunderstandingoftheprocessofCNÀproductionbybacteriaandthedetrimentaleffectofHCNoncysticfibrosispatients.Towardthisend,wedevelopedanewNIRfluorescentprobefordetectingcyanideanions.39Theprecursorprobe(33)iscomposedofatricarbocyaninedyeandaCu2+receptormoiety(Fig.25a).When33bindstoCu2+toformthecomplex33-Cu2+,theabsorptionmaximumat718nmshiftsto743nmandthefluorescenceat748nm(excitationat680nm)isquenched.WhenCNÀisaddedtoasolutionof33-Cu2+,thefluorescenceintensityat748nmincreasesasaresultofthereactionofCNÀwithCu2+thatformsthestable[Cu(CN)x]nÀanditiscompletelyrecoveredafteradditionofexcessCNÀ.Otheranionsdonottriggerthissensingsystem,indicatingthat33-Cu2+selectivelyrespondstoCNÀ.ThisprobehasbeensuccessfullyemployedtodetectbiogenicCNÀproducedbyP.aeruginosainC.elegans(Fig.26).Asimilarapproachwasusedtodesignprobestodetectsulfideandpyrophosphate(PPi)anions.Tomonitorsulfideanions,LinandcoworkersdevelopedaNIRfluorescentprobe

Fig.26NIRimagingofCNÀinC.elegans.ThenematodeswereexposedtovariousconcentrationsofNaCNfor4h.(a)Nocyanide;(b)1mMCNÀ;(c)100mMCNÀ;(d)theenlargedimageinthevicinityoftheintestineinthenematodeexposedto100mMNaCN(P=pharynx;I=intestine;n=nucleus;c=cytosol)(reprintedfromref.39).

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Published on 19 September 2013. Downloaded on 08/12/2013 01:50:17. 34-Cu2+,whichiscomposedofatricarbocyaninedye,an8-amino-quinolinebinderandcopper(Fig.25b).40Theprecursorprobe(34)exhibitsfluorescenceemissionat794nm,whichisalmostcompletelyquenchedbyadditionofCu2+.Theprobehasa1:1bindingmodeandtheassociationconstantof3.0Â105MÀ1withCu2+.Whensulfideanionsareaddedto34-Cu2+,turn-onfluorescenceat794nmtakesplacebringingaboutareturnofitsintensitytoessentiallythatofthefreeprobe34.ThisphenomenoniscausedbycoordinationofCu2+tosulfidetoformthestableCuS(Ksp=1.27Â10À36).Probe34-Cu2+hasahighselectivityforsulfideoverotheranions,however,ithasnotbeenappliedtomonitoringsulfideincells.

ZhuandcoworkersdevelopedaNIRfluorescentprobe35-Cu2+,whichishighlyselectivetoPPiinaqueoussolutions(Fig.25c).41Theprecursorprobe(35),whichconsistsofdicyano-methylene-4H-chromeneasafluorophoreandaniminodiacetategroupasareceptor,exhibitsNIRfluorescenceat675nmthatisquenchedbychelationofCu2+tothediacetatemoietyoftheprobe.ThisquenchingphenomenonisobservedonlyuponadditionofCu2+butnotothermetalions.Probe35formsa2:1complexwithCu2+.WhenPPiisaddedto35-Cu2+,fluores-cenceat675nmincreases,butitsintensitydoesnotcompletelyreturntothatoffree35,suggestingthatcoordinationofPPiisnotstrongenoughtocausecompleteremovalofCu2+from35-Cu2+.ThisprobedisplaysagoodselectivityforPPioverthevariousanions,butH2PO4À,HPO42ÀandPO43Àslightlyrespondtotheprobe.Incubationofcellswith35-Cu2+promotesturn-onfluorescenceintheperinuclearareaofthecytosol.

Fig.27AmechanismforsensingpHchangesby36and37.

Fig.28AmechanismforsensingpHchangesby38.

5.SensingpHchanges

KnowledgeofintracellularorextracellularpHchangesprovidesimportantinformationaboutthenatureofphysiologicalandpathologicalprocesses.42Becauseacidicenvironmentsaregenerallyassociatedwithinflammationandtumor,sensingslightdecreasesinpHintumormicroenvironmentshasbecomeanimportantissueinthedesignofanti-tumortherapy.TherequirementsforimagingtumormicroenvironmentshavedriventhedevelopmentofNIRpH-activatableindicatorsforvisualizationofpHchanges.

Cyanine-basedpHprobeshaveattractedgreatattentionasaresultoftheiruniqueNIRspectraproperties.Ingeneral,twostrategieshavebeendevisedforthedesignofpH-sensitiveprobes.ThefirstreliesonreversibleprotonationanddeprotonationoftheindoliumringnitrogenofpH-sensitivecyaninedyes,whichleadstoalternationsofpelectronicchargedensities.Forinstance,Achilefuandcoworkersconstructedthenon-N-alkylatedindoliumcyanine36asapHindicator(Fig.27).43Probe36isalmostnon-fluorescentwhenthenitrogenatomisnotprotonated,butitbecomesstronglyfluorescentat800nmuponprotonationinacidicenvironments.Thecharacteristicalternationofpelectronicchargedensitiesinprotonated36resultsinthehypsochromicshiftoftheabsorptionmaxima.Toimprovethetumortargetingsensitivityandspecificityoftheprobe,acyclicarginine-glycine-asparticacid(cRGD)groupwasconjugatedtoapH-sensitiveNIRdyetoformprobe37.43Theresultsofinvitroandinvivostudies

showthat37issuitableforthedetectionofprimaryandmetastaticbreasttumorsinbothsubcutaneousandorthotopicmousemodelswithhighsensitivityandspecificity.

ThesecondstrategyforconstructionofpH-sensitivecyaninedyesreliesonthesuppressionorenhancementofPETquenchingthroughprotonation/deprotonationoftheelectrondonormoiety.ThesetypesofpH-sensitivedyes,exemplifiedby38(Fig.28),consistofacyaninefluorophoreandanitrogen-containingelectrondonor.Forexample,anethylenediamineorpiperazinemoietyisconjugatedtothecyanineringsystemtoprepareratiometricNIRfluorescentpHprobesthathavevariouspKavalues.Intheseprobes,theexcitationwavelengthsofamine-substitutedtricarbocyaninecanbemodulatedbycontrollingtheelectron-donatingabilitiesoftheaminesubstituent.Inthisway,thepH-sensitiveprobe38exhibitsobservableshiftsofitsabsorptionmaximum.44ThedrasticchangesintheabsorptionspectralmaximapermititsuseasareversibleratiometricpHprobe.ThestrategyinvolvingtuningthepKavalueofthediaminemoietyprovidesaneffectivemethodtodesignandpredictratiometricmeasurementsofpHchangesoverawiderangebothinvitroandinvivo.

AlthoughBODIPYdyeshavegoodbrightnessandexcellentphoto-stabilitycharacteristicsascomparedtoNIRcyaninedyes,theysufferfromthefactthattheirabsorptionmaximalieinthevisibleregionand,thus,theyarenotoptimalfornoninvasiveinvivoimaging.AspartofeffortsmadetoimprovephotophysicalpropertiesofBODIPY,Borisovandcoworkers

Fig.29AproposedmechanismforpH-sensitiveprobe39.

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TutorialReviewexploredaBF2-chelatedtetraarylazadipyrromethanedye39asaNIRfluorescentpHprobe,whichisamenabletostructuralmodifications.45Upondeprotonationofphenolin39,theprobeexhibitslargeemissionintensitychangesinthe700nmregion(Fig.29)asaresultofefficientPETfromatwistedphenolatetotheaza-BODIPYsubunit.ThispH-sensitiveprobehasbeenemployedtomonitorthepHincoralswithafiber-opticpHoptode.

6.Enzymesensing

Protease-cleavablepeptideprobes,whichcontainaNIRfluoro-phorelinkedtoaquencherviaapeptidesubstrate,havebeen .7developedtodetecttheactivityofmatrixmetalloproteases1:0(MMPs),whichcatalyzethecleavageoftheextracellularmatrix5:10andareknowntobeabiomarkeroftumors,cardiovascular 31diseasesandinflammation.TheseFRET-basedprobesare0/22typicallynon-orveryweaklyfluorescent.However,whena/18peptidemoietyintheprobeiscleavedbyactionofaprotease,0 nothefluorophoreandquencherbecomeseparated,which deinducesanenhancementinthefluorescenceintensity.

daAnexampleofthisdesignisfoundintheMMPNIRprobe40lonwdevelopedbyLinderandcoworkers,whichpossessestheazo-bondoD containingBlackHoleQuencher3(BHQ-3)asaquencherand.310IRDye800CWasaNIRfluorophore,linkedviaapeptidemoiety2 re(Fig.30a).46AlthoughthespectraloverlapbetweenfluorescenceofbmIRDye800CWandabsorptionofBHQ-3isnotoptimalforFRETetpeapplications,thefluorescenceofIRDye800CWisalmostcompletelyS 9quenchedbythepresenceofBHQ-3.Treatmentoftheprobe1 nowithMMPsresultsina56-foldincreaseinfluorescence,caused debyaprocessthatgeneratestheseparatedfragments41and42hsilbetweenwhichFRETquenchingcannotoccur.Whenprobe40isbuPinjectedintomice,theexpectedfragment41isformedtogetherwithseveralmetabolitesincludingadearginatedmetabolite

Fig.30AmechanismforsensingMMPactivitiesusingFRETprobes(a)40and(b)43.

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IRDye800CW-PLGLK(BHQ-3)-Aandanazobondcleavedmeta-bolite.ThisfindingshowsthatalthoughtheIRDye800CW/BHQ-3pairisusefulforFRETapplications,instabilityoftheazobondinBHQ-3needstobeconsideredforinvivostudies.Inalatereffort,NaganoandcoworkersemployedasimilarapproachindevelopingFRETprobe43todetectMMPactivityinvitroandinvivo(Fig.30b).47ItshouldbenotedthatthisstrategycanbeusedtoexploitFRET-basedNIRfluorescentprobesfordetectingtheactivitiesofotherenzymes.

7.Nanomaterial-basedNIRfluorescentprobes

Fluorescentnanomaterialshavebeenextensivelyexploredinthecontextofbiologicalandbiomedicalapplications.Inparticular,owingtotheirenhancedpermeabilityandretentioneffect,thesematerialshavefoundgreatuseinmonitoringtumor.ZhuandcoworkerspreparedtheNIRfluorescentnano-particleCyN-12@NHswithasizeofca.35nmforinvivobioimagingoftumors(Fig.31).48Thenanocompositeiscon-structedbyencapsulationofatricarbocyaninedyeintothehydrophobiccore,throughself-assemblyofamphiphilicblockcopolymerPS-b-PAA(polystyrene-b-polyacrylamide),andsubse-quentcrosslinkingofthemicellarassemblieswithsilane.CyN-12@NHshassomeadvantageouscharacteristicscomparedtothoseofthefreedye,inthatfluorescentdyesencapsulatedinthenanoparticledisplayenhancedchemicalandphoto-stability,betterresistancetoROS,andgoodwatersolubility.CyN-12@NHshasanabsorptionmaximumat690nmandemitsstrongfluores-cenceat800nm.Thisnanoparticlewasusedtoimagecancercellsandtumorsinxenograftedmice.Liandcoworkersdevelopedbiodegradablecore-crosslinkedpolymericmicelles,whichcontainbothNIRfluorophoresand111indium,fordualmodal,NIRfluorescentandnucleartumorimaging.49Thisnanomaterialhasbeenappliedforinvivodetectionoftumorsinmicebyg-scintigraphyandNIRfluorescence.

8.NIRchemiluminescentdyes

Chemiluminescentprobeshavesomeadvantagesoverconven-tionalfluorescentprobesthatarisebecauseoftheirinherenthighsignal-to-noiseratios.However,mostchemiluminescent

Fig.31PreparationofaNIRfluorophore-encapsulatedCyN-12@NHs.

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ChemSocRevFig.32Chemiluminescentmechanismof44thatreleases1O2and

concomitantlyemitsNIRlight.

.71:05:1dyesareshort-livedasaconsequenceoftheirspontaneous0 3degradationwhenexposedtolightand,asaresult,theycannot10bestoredforlongperiods,andtheynormallyexhibitvisible/22/1lightemission.RecentlyaNIRfluorescentandchemilumines-80 ncentdye(44)wasdevelopedbySmithandcoworkers(Fig.32).50o dThisdyepossessesaninterlockedrotaxaneskeleton,whichisedacomposedofdumb-bell-shapedsquaraineencapsulatedinatetra-lonwlactammacrocyclewithathermallyunstable9,10-anthraceneoDmonoendoperoxidegroup.Probe44isproducedbyirradiationof .31theparentrotaxanewithredlightinthepresenceofairandcanbe02 storedforalongperiodoftimeatÀ201C.However,uponwarmingrebm44to371C,thedyeundergoesaunimolecularcycloreversionetpreactiontoreleasesingletoxygenandconcomitantlyemitNIRlighteS withmaximaat733nmfromtheencapsulatedsquarainechromo-91 nphore.Itwassuggestedthat,inthisprocess,energytransfertakeso dplacefromsingletoxygentotheencapsulatedsquaraine.When44,ehsiadsorbedontomicroparticles,wasinjectedintomice,stronglbuchemiluminescencewasobservedinrelativelydeeptissuesowingPtoverylowbackgroundemissionfromtheanimal.

9.Concludingremarks

Inthisreview,recentadvances(2010–2013)madeinthestudyofNIRfluorescentprobesweredescribed.ParticularattentionwasgiventotheapplicationofNIRfluorescentprobesforbioimagingvariousbiologicallyimportantspecies.Thedesignstrategies,sensingmechanisms,andinteractionmodesofrepresentativeNIRfluorescentprobeswerealsodiscussedinthecontextofstrategiestodesignandapplyNIRfluorescentprobestoimportantbiologicalproblems.

Despitethegreatadvancesthathavebeenmadeinthisarea,largechallengesremaininthedevelopmentofNIRfluorescentprobesforinvivobiologicalimaging.Forexample,todate,theonlyclinicallyFDAapprovedmaterialforthispurposeisindocyaninegreen(ICG).ThemajorobstacleindevisingsuitablefluorophoresasNIRplatformsistheneedforthesesystemstohaveexcellentchemicalandphotochemicalpropertiessuchashighquantumyields,largeStokesshifts,greaterphoto-stability,andreadymodification.FutureeffortsaimedatdesigningnewNIRfluorescentprobeswillneedtofocusonhowtheNIR

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fluorescencesignalswillbeintegratedwithmoreversatilefunctionalities.ThenextgenerationfunctionalfluorescentprobeswillberequiredtonotmerelyrespondtothepresenceofreactivesmallbiologicalspeciesbyNIRfluorescentsignals.Inaddition,NIRfluorescentprobescanbeutilizedasacomponentofthedeliverysystemtocarryandmonitorthedrugatthespecifictargetsiteinanaccuratemannerwithoutcausingphoto-damagetotissues.

SignificantopportunitiesandgreatchallengesremaininthedevelopmentofidealNIRfluorescenceprobesthatcanbeemployedasimagingandtherapeuticagentsinvivo.Itshouldbenotedthatnanoparticle-basedNIRtheranosticagents,whichovercomeseveraldrawbacksofsmallmoleculecancerdrugsandimagingagents,havealreadybeendesignedandconstructed.Clearly,devisinggeneralandeffectiveapproachestoversatileNIRfluorescentprobeswillbetheimpetusfordevelopingexcitingnewmethodsforopticalimaginginvivo.

Acknowledgements

ThisworkwassupportedbytheNationalCreativeResearchInitiativeprogram(grantno.2010-0018272toI.S.and2012R1A3A2048814toJ.Y.).Z.G.acknowledgesNSFC/China(Grant61177034)andtheShanghaiPujiangProgram.

Notesandreferences

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