荧光探针和分子传感器

XIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY200507051郭祥群博士，博士生导师全国高校化学教育研究中心第四届学术委员会副主任，教育部高等学校本科教学工作水平评估专家，中国化学会理事先后获得国家级奖2项，省部级奖4项，发表科研与教学研究论文100余篇。研究兴趣：荧光光谱分析的原理与技术，在研项目涉及生物活性自由基的荧光表征，海洋污染生物监测技术研究，食品安全等。联系方式:[e-mail]0592-2181637(L),2187288(O),2182355(H)XIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY200507052FluorescentProbesIntroductionFluorescentSensor荧光传感FluorescentTracer荧光示踪FRET共振能量转移荧光Application1.Radicalprobes2.Ionprobes3.NIRFFluorescenceimage4.DNAsequence5.Proteinprobes6.DNAprobes7.Membraneprobes8.pHprobesXIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY200507053ThankyouThankyouXIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY200507054FluorescenceresearchKeyword:Fluorescen*Period:Jan/2005-Jun/2005Database:美国化学会(ACS)数据库3566篇WPS电子期刊全文数据库206篇SDOS全文数据库（上交镜像）1890篇英国皇家化学会（RSC)数据库120篇研究与应用领域涉及：生命环境材料等讲座要求了解：荧光探针表征应用研究意义XIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY200507055MolecularProbeMolecularreporterFromJ.FluorescenceXIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY200507056homepageAhAAbsorption10-15sVibrationalrelax,VR,10-1210-14sInternalconversion,IC,10-1110-13sIntersystemcrossing,ISC,10-210-6sFluorescencePhosphorescenceICISCS0S1S2321010-8s10-3100shFhPHOMOLUMOXIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY200507058CharacteristicsofFluorophoreExcitationwavelength激发波长exEmissionwavelength发射波长emExtinction(absorption)coefficient吸光系数Stoke’sshiftFluorescencelifetime荧光寿命Fluorescencequantumyield荧光量子产率FluorescenceAnisotropy各项异性rFluorescencePolarization荧光偏振pXIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY200507059Excitationandemissionspectra1ExcitationspectrumEmissionspectraFemFexemexFluorescencelifetimenrkΓ1relaxation(10-12s)S0S1S1hvAhvFknrSimplifiedJablonskidiagram:EmissionrateKnr:nonradiativedecayrateDefinitionAveragelifetime:theaveragetimethemoleculespendsintheexcitedstatepriortoreturntothegroundstate.ExpressionFluorescenceQuantumYieldnrkΓΓΦDefinitionThefluorescencequantumyieldistheratioofthenumberofphotonsemittedtothenumberabsorbedExpressionSensor(1)+SRTS信号单元，R识别单元，T靶分子自由基传感Z.-Y.Bian,X.-Q.Guo*,AnalyticalScience,21553-559,2005XIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY2005070513Sensor(2)DDhue-hvhvhvPETPhotoinducedElectronTransferhvHg2+E.M.Nolan,etal.,J.Am.Chem.Soc.,2003,125:14270-14271SRTSensor(3)RShvhvFRETFD,exD,emA,exA,emFD,exD,emA,exA,emT+hvhv’FluorescenceResonanceEnergyTransferXIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY2005070515ProbingDNAMolecularbeacons分子信标ADQFTargetDNAssDNAbindingproteinDenaturingreagentQFQXIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY2005070516Sensor(4)+S-RThvhvAl3+hvXIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY2005070517Sensor(5)+S-RT+竞争型+S-RT催化剂中间体+hv催化型XIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY2005070518TheFusionofIntegalMembraneProtein荧光素标记鼠蛋白质罗丹明标记人蛋白质鼠细胞人细胞37°C15°C融合异核体FromInstantnotes,Biochemistry,E2Tracer膜内蛋白侧向运动CelltracerA30µm–thicksectionofazebrafishembryostainedwithCellTrackerCM-DiI(C7000,C7001)priortoimmunohistochemicalanalysis.Aone-day-oldzebrafishembryowasimmobilizedandimpaledinthehindbrainwithamicroelectrodefilledwith1mg/mLCellTrackerCM-DiIin95%ethanol.Onedaylater,thebrainwasfixed,embedded,frozeninliquidnitrogenandsectionedonacryostat.Afterblockingwith0.1%TritonX-100inPBScontaining2%BSAand2%normalgoatserum,the30µm–thicksectionwasincubatedwithprimaryanti-glialantibodyinconjunctionwithfluoresceingoatanti–mouseIgGantibody.Thissectionwasthenviewedsequentiallythroughopticalfiltersetsappropriateforrhodamineandfluorescein,andtheresultingimagesweresuperimposed.TheimagewascontributedbyWilliamTrevarrow,BeckmanInstitute,CaliforniaInstituteofTechnology.C7000C68H105Cl2N3O抗体在免疫反应前后荧光探针自身的荧光量子产率不发生变化的情况测反应剩余的标记抗原，荧光正比抗原量测固相荧光，荧光反比抗原量测固相洗脱，荧光反比抗原量F标记抗原待测抗原∆—F荧光分子F∆∆∆∆FRETFluorescenceResonanceEnergyTransferXIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY2005070522PrincipleofFRETJNrnkKDDT6422128)10(ln9000RateofenergytransferDquantumyieldofDDlifetimeofDrdistancebetweenAandDnrefrcativeindexNAvogadro’snumberkorientationfactorJoverlapintegralFD,exD,emA,exA,emXIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY2005070523Orientationfactork=2/3randomizebyrotationaldiffusionpriortoenergytransferXIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY2005070524FRETsensordesignbasedondonorandacceptordistancechangesK.Kikuchi,etal.,TrAC,2004,23(6),407-413XIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY2005070525FRETprobingcAMPK.Kikuchi,etal.,TrAC,2004,23(6),407-413cAMPcyclicadenosinemonophosphate环腺苷酸环腺苷酸依赖蛋白激酶支架结构四聚体表征cAMPFRETprobingPDE磷酸二酯酶BeforehydrolysisAfterhydrolysisK.Kikuchi,etal.,TrAC,2004,23(6),407-413BeforeAfter香豆素phosphodiesterasehvhvhvXIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY2005070527EmissionspectraPDEXIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY2005070528FRETsensordesignbasedonmodulationofspectraloverlapFD,exD,emA,exA,emFD,exD,emA,exA,em有利于能量转移不利于能量转移Probingphosphatase磷酸酶LFabsQFabsDonorEmiLF(内酯）QF(醌型）K.Kikuchi,etal.,TrAC,2004,23(6),407-413PTP蛋白质酪氨酸磷酸酶香豆素荧光素XIAMENUNIVERSITYFLUORESCENCESPECTROSCOPY2005070530生物活性自由基研究的重要性•OHO2-•RO•ROO•