多光子激发荧光与毛细管电泳联用的实验研究

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 1.0×10-6 mol·L-1，FAD 7.4×10-７ mol·L-1，NADH 9.8×10-７ mol· 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。

Study on Multi-Photon Excited Fluorescence Combined with Capillary Electrophoresis

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 x L(-1) for 5HT, 7.4 x 10(-7) mol x L(-1) for FAD and 9.8 x 10(-7) mol x 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.