多光子激发荧光与毛细管电泳联用的实验研究  

Study on Multi-Photon Excited Fluorescence Combined with Capillary Electrophoresis

作　　者：孙云旭[1] 朱峰[1] 马万云[1] 

机构地区：[1]清华大学物理系,原子分子纳米科学教育部重点实验室,北京100084

出　　处：《光谱学与光谱分析》2005年第4期502-505,共4页Spectroscopy and Spectral Analysis

基　　金：国家自然科学基金 (30 0 70 2 0 9;1 0 2 740 32 ) ;教育部博士点基金 (2 0 0 0 0 0 0 352 )资助项目

摘　　要：将多光子激发荧光探测与毛细管电泳技术相结合,研制了多光子激发荧光 毛细管电泳联用装置。这种方法可以高效快速的分离检测复杂样品中多种不同的荧光分子。作者对5HT ,FAD ,NADH这三种重要的生物分子,不用染料标记,分别用双光子激发和三光子激发,进行了直接的分离、识别和检测。得到的检测限分别是5HT 1. 0×10 -6mol·L-1,FAD 7. 4×10 -7mol·L-1,NADH 9. 8×10 -7mol·L-1。5HT的检测限比紫外吸收低2个数量级;FAD和NADH的检测限比紫外吸收低1个数量级。This paper describes a new method, multiphoton excitation fluorescence detection combined with capillary electrophoresis separation. The excitation source was a self mode-locked femtosecond titanium-sapphire laser (Spectra-physics Inc.), producing a stream of pulses with a pulse duration of about 100 fs at 82 MHz repetition rate. Its average power was about 200 mW at 750 nm. The laser beam was focused into a thin wall flow cell of capillary electrophoresis. A high numerical aperture objective (100 X NA 1.25) was chosen to focus the laser beam and to collect the fluorescence emitted by detecting molecules. Then the fluorescence was detected by a fast response PMT. All data were acquired and processed by a microcomputer. For three biological molecules, 5HT, FAD and NADH, it was demonstrated that they can be separated and detected efficiently by this method with three-photon and two-photon excitation respectively using only one 750 nm laser beam. The detection limits were 1.0 x 10(-6) mol (.) L-1 for 5HT, 7.4 x 10(-7) mol (.) L-1 for FAD and 9.8 x 10(-7) mol (.) L-1 for NADH using the criterion of three standard deviations above background. The results are much better than those with UV absorption detection by one or two magnitudes.

关 键 词：激发荧光 实验研究 毛细管电泳技术 NADH 紫外吸收 多光子激发 双光子激发 检测限 荧光探测 联用装置 荧光分子 复杂样品 分离检测 生物分子 FAD 数量级 

分 类 号：O623.624.2[理学—有机化学] O657.8[理学—化学]