荧光探针和分子传感器

1、XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYXIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYu全国高校化学教育研究中心第四届学术委员会副主任，全国高校化学教育研究中心第四届学术委员会副主任

2、，教育部高等学校本科教学工作水平评估专家，中国化教育部高等学校本科教学工作水平评估专家，中国化学会理事学会理事u 先后获得国家级奖先后获得国家级奖2项，省部级奖项，省部级奖4项，发表科研与项，发表科研与教学研究论文教学研究论文100100余篇。余篇。u研究兴趣：荧光光谱分析的原理与技术，在研项目涉研究兴趣：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。术研究，食品安全等。 u联系方式联系方式: :L), 2187288(O), 2182355(H)200507052

3、XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYuIntroductionuFluorescent Sensor 荧光传感荧光传感 uFluorescent Tracer 荧光示踪荧光示踪 uFRET 共振能量转移荧光共振能量转移荧光 uApplication 1. Radical probes 2. Ion probes 3. NIRF Fluorescence image 4. DNA s

4、equence5. Protein probes6. DNA probes7. Membrane probes8. pH probes200507053XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYuKey word: Fluorescen*uPeriod: Jan/2005-Jun/ 2005uData base: 美国化学会美国化学会 ( ACS )( ACS )数据库数据库 3566

5、3566 篇篇 WPSWPS电子期刊全文数据库电子期刊全文数据库 206 206 篇篇 SDOSSDOS全文数据库（上交全文数据库（上交 镜像）镜像） 1890 1890 篇篇 英国皇家化学会（英国皇家化学会（RSC) RSC) 数据库数据库 120 120 篇篇研究与应用领域涉及：生命研究与应用领域涉及：生命 环境环境 材料材料 等等讲座要求了解：讲座要求了解： 荧光荧光 探针探针 表征表征 应用应用 研究意义研究意义200507054XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLU

6、ORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY200507055XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYwww. probes. comwww. probes. com200507056T1h Ah AAbsorption10-15 sInternal conversion, IC, 10-1110-13 sIntersystem cro

7、ssing, ISC, 10-210-6 sFluorescencePhosphorescence ICISCS0S1S23 2 1 010-8 s10-3 100 sh Fh PHOMOLUMOXIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYExcitation wavelength 激发波长激发波长 exEmission wavelength 发射波长发射波长 emExtinction (

8、absorption) coefficient 吸光系数吸光系数 Stokes shift Fluorescence lifetime 荧光寿命荧光寿命 Fluorescence quantum yield 荧光量子产率荧光量子产率 Fluorescence Anisotropy 各项异性各项异性 r Fluorescence Polarization 荧光偏振荧光偏振 p200507058XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFL

9、UORESCENCE SPECTROSCOPY 1Excitation spectrumEmission spectraF emF ex em ex200507059nrk 1nrk+S RThvhvhv自由基传感自由基传感Z.-Y. Bian, X.-Q. Guo*, Analytical Science, 21 553-559, 2005XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYDD

10、hue -hvhvhvPET Photoinduced Electron TransferhvHg2+E. M. Nolan, et al., J. Am. Chem. Soc., 2003, 125: 14270-14271 SRT2005070513RShvhv FRET F D,ex D,em A,ex A,em F D,ex D,em A,ex A,emT+hvhvFluorescence Resonance Energy Transfer XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSC

11、OPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYADQFTarget DNAssDNA binding proteinDenaturing reagent QFFQ2005070515XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY+S-RThvhvAl3+hv2005070516XIAMEN UNIVERSITYXIAMEN UNIV

12、ERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY+S-RT+竞争型竞争型+S-RT催化剂中间体催化剂中间体+hvhv催化型催化型2005070517XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY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

13、C15C融合融合异核体异核体From Instant notes, Biochemistry, E2TracerTracer膜膜内内蛋蛋白白侧侧向向运运动动2005070518A 30 mthick section of a zebrafish embryo stained with CellTracker CM-DiI (C7000, C7001) prior to immunohistochemical analysis. A one-day-old zebrafish embryo was immobilized and impaled in the hindbrain with a m

14、icroelectrode filled with 1 mg/mL CellTracker CM-DiI in 95% ethanol. One day later, the brain was fixed, embedded, frozen in liquid nitrogen and sectioned on a cryostat. After blocking with 0.1% Triton X-100 in PBS containing 2% BSA and 2% normal goat serum, the 30 mthick section was incubated with

15、primary anti-glial antibody in conjunction with fluorescein goat antimouse IgG antibody. This section was then viewed sequentially through optical filter sets appropriate for rhodamine and fluorescein, and the resulting images were superimposed. The image was contributed by William Trevarrow, Beckma

16、n Institute, California Institute of Technology. 在免疫反应前后荧光探针自身的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荧光，荧光反比抗原量测固相洗脱，荧光反比抗原量测固相洗脱，荧光反比抗原量标记抗原标记抗原待测抗原待测抗原荧光分子荧光分子XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORES

17、CENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYJNrnkKDDT6422128)10(ln9000Rate of energy transfer D quantum yield of D D lifetime of Dn refrcative indexN Avogadros number F D,ex D,em A,ex A,em2005070522XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFL

18、UORESCENCE SPECTROSCOPYk = 2/3randomize by rotational diffusion prior to energy transfer2005070523XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY K. Kikuchi, et al., TrAC, 2004,23(6),407-4132005070524XIAMEN UNIVERSITYXIAM

19、EN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY环腺苷酸依赖蛋白激环腺苷酸依赖蛋白激酶支架结构四聚体酶支架结构四聚体表征表征cAMP2005070525Before hydrolysisAfter hydrolysis K. Kikuchi, et al., TrAC, 2004,23(6),407-413BeforeAfter 香豆素phosphodiesterasehvhvhvXIAMEN UNIVERSITYXIAMEN UNI

20、VERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYPDE2005070527XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY F D,ex D,em A,ex A,em F D,ex D,em A,ex A,em2005070528LF absQF

21、 absDonor EmiLF (内酯）QF (醌型） K. Kikuchi, et al., TrAC, 2004,23(6),407-413PTP 蛋白质酪氨酸磷酸酶XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY2005070530XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLU

22、ORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY2005070531XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYGSHROSGSGS-GSSGO2O2蛋白质巯基细胞膜磷酯-2005070532XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYF

23、LUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYNOFluorophoreNOFluorophore.+R.RIIIParamagnetic(Low fluorescence)Diamagnetic(High fluorescence)2005070533XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYNHOCOO-ONON HNN HH NO

24、OO M eF eNOHCOO-ONONONHNNHHNOOOMeFeNO2005070534XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFura-Ca2+FuraExcitation Emission2005070536XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCE

25、NCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYIndo-1-Ca2+2005070537XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYICT principle2005070538XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE

26、SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFalse-color image（假色成像）（假色成像） of free Ca2+ concentration in a Purkinje neuron （梨状（梨状神经元）神经元）from embryonic mouse cerebellum（胚鼠小（胚鼠小脑）脑） The composite image, which represents the ratio of images obtained with excitation at 340 nm and 380 nm, reveals the mobilizati

27、on of internal Ca2+ stores without contribution from Ca2+ influx.Fura-2-Ca2+胚鼠小脑梨状神经元中游离胚鼠小脑梨状神经元中游离Ca2+浓度荧光成像浓度荧光成像2005070539XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFluo-3 (F1240) confocal image of a spiral Ca2+ w

28、ave initiated by injection of a nonhydrolyzable analog of inositol 1,4,5-triphosphate（肌醇三磷酸非水解模拟物） in a Xenopus laevis oocyte（瓜蟾卵母细胞）. The image was contributed by David Clapham, Harvard University, and reproduced with permission from Cell 69, 283 (1992). 瓜蟾卵母细胞中螺旋瓜蟾卵母细胞中螺旋Ca2+波共焦荧光成像波共焦荧光成像Fluo-320

29、05070540XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYSBFISBFISBFI-Na+海马切片海马切片CA1锥形锥形神经元神经元Na+成像成像2005070541XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENC

30、E SPECTROSCOPYFluorescence emission spectra of a mixture of 1 M BTC-5N (B-6845) and 1 M TCPP in solutions containing zero to 1.0 M Cd2+. BTC-5N, tetrapotassium salt2005070542XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY

31、APTRA-BTC, tripotassium salt Fluorescence excitation spectra of APTRA-BTC (A-6895) in solutions containing zero to 25 M Zn2+. 2005070543XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY5-sulfofluorescein diacetate, sodium s

32、alt (SFDA)2005070544XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYApoptosis2005070546XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY通过静脉注射或腹腔

33、注射到生物体内的近红外通过静脉注射或腹腔注射到生物体内的近红外荧光探针吸着并聚集于肿瘤等组织中，肿瘤中荧光探针吸着并聚集于肿瘤等组织中，肿瘤中存在特异的细胞、蛋白质、酶等，这些物质可存在特异的细胞、蛋白质、酶等，这些物质可与荧光探针结合或反应而发出荧光，可根据荧与荧光探针结合或反应而发出荧光，可根据荧光的位置、强度得知肿瘤的位置、大小，是否光的位置、强度得知肿瘤的位置、大小，是否转移、扩散及肿瘤的阴性、阳性等一系列信息转移、扩散及肿瘤的阴性、阳性等一系列信息. .2005070547XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUOR

34、ESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY2005070548GFP 绿色绿色荧光蛋白荧光蛋白Endostatin内生抑制素内生抑制素Cy5.5 花菁花菁荧光染料荧光染料NIR1, NIR2, GFP 不同滤光片不同滤光片XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY2005070550XIAM

35、EN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY2005070551XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY酶活化酶活化酶作用位点酶作用位点C-PGC肿瘤伴生溶酶体蛋白酶特异识别肿瘤伴生溶酶体

36、蛋白酶特异识别自猝灭荧光探针（或细胞成像）自猝灭荧光探针（或细胞成像）2005070552XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY2005070553XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECT

37、ROSCOPYLysosomal compartment 溶酶体分隔溶酶体分隔2005070554XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPY2005070555Template 3-CCGGTAGCAACT-5Primer 5-GG-3GGCCAGGCCATCGTTGAGGCGGCCGGCCATCGGCCATCGGGCCATCGTTGGGCCATGGCCATCGTGGCCATCGTTdA

38、TPdCTPdGTPdTTPddATPdATPdCTPdGTPdTTPddCTPdATPdCTPdGTPdTTPddGTPdATPdCTPdGTPdTTPddTTPF1-AF2-CF3-GF4-TDNA polymerasedATP 脱氧腺苷dCTP 脱氧胞苷dGTP 脱氧鸟苷dTTP 脱氧胸苷ddATP 双脱氧腺苷ddCTP 双脱氧胞苷ddGTP 双脱氧鸟苷ddTTP 双脱氧胸苷FXIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORE

39、SCENCE SPECTROSCOPY七次甲基花菁素类七次甲基花菁素类(图图2，a) 2005070557WWWXIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYAbsorption and fluorescence emission spectra of ethidium bromde bound to DNA Ethidium bromide 溴化乙锭溴化乙锭2005070559XIAMEN UNIVERSITYXIAMEN UNIVERSITYXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCOPYFLUORESCENCE SPECTROSCO