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首页> 外文会议>Conference on Laser Applications to Chemical and Environmental Analysis, Feb 11-13, 2000, Santa Fe, New Mexico >Single molecule DNA sequencing in microcapillaries
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Single molecule DNA sequencing in microcapillaries

机译：微毛细管中的单分子DNA测序

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摘要

Since the fust measurements of single molecule fluorescence decay times in flowing sample streams several groups have improved the ability to distinguish between differently labeled single molecules due to their characteristic fluorescence decay times using time-correlated single-photon counting (TCSPC). Using confocal excitation/detection techniques detection volumes in the femtoliter region can be easily attained which permit signal-to-background ratios (SBRs) > 100. For efficient detection of all analyte molecules a technique has to be developed which constrains all analyte molecules to flow through the detection volume. If a detection volume in the picoliter (pl) range is used, hydrodynamic focusing of the sample stream in a sheath flow cuvette has been applied successfully. Recently, efficient detection of single fluorescent molecules eluting off a polystyrene microsphere optically trapped in a flowing sheath stream has been demonstrated. Unfortunately, for the application of femtoliter detection volumes microcapillaries or microchannels with inner diameters smaller than the detection area (< 1μm) have to be used. Recently, we were able to demonstrate the time-resolved identification of individual fluorescent dyes as they flow through a microcapillary with an inner diameter of 500+-200 nm applying an electrical tension of a few volts. Addition of a nonionic detergent (Tween 20) efficiently suppressed adsorption of molecules to the glass surface of the capillary and reduced the electroosmotic flow.
机译：由于在流动的样品流中对单分子荧光衰减时间进行了简便的测量，由于使用时间相关的单光子计数（TCSPC）的特征性荧光衰减时间，几个组已经提高了区分不同标记的单分子的能力。使用共聚焦激发/检测技术，可以轻松实现飞升区域中的检测体积，允许信噪比（SBR）>100。要有效检测所有分析物分子，必须开发一种技术来限制所有分析物分子流动通过检测量。如果使用的检测体积在皮升（pl）范围内，则已经成功应用了鞘流比色皿中样品流的流体动力学聚焦。近来，已经证明有效检测从光学捕获在流动的鞘流中的聚苯乙烯微球洗脱的单个荧光分子。不幸的是，对于飞升检测体积的应用，必须使用内径小于检测面积（<1μm）的微毛细管或微通道。最近，我们能够证明在单个荧光染料流过微毛细管时的时间分辨鉴定，该微毛细管的内径为500 + -200 nm，施加的电压为几伏。加入非离子型去污剂（吐温20）可有效抑制分子吸附到毛细管玻璃表面，并减少电渗流。

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