Single-Molecule Detection of DNA Separations in Microfabricated Capillary Electrophoresis Chips Employing Focused Molecular Streams
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- 作者：Brian B. Haab and Richard A. Mathies
- 刊名：Analytical Chemistry
- 出版年：1999
- 出版时间：November 15, 1999
- 年：1999
- 卷：71
- 期：22
- 页码：5137 - 5145
- 全文大小：214K
- 年卷期：v.71,no.22(November 15, 1999)
- ISSN：1520-6882
文摘
DNA separations have been performed using microfabricated capillary electrophoresis chips and detected usinga single-molecule fluorescence burst counting technique.We enhanced the percentage of electromigrating DNAmolecules that were detected by focusing the samplethrough the 1-
m-diameter focused laser beam. Thesample was focused by introducing a taper in the separation channel and by a sheath flow delivered from crosschannels. The sample stream width, single-moleculevelocities, and single-molecule count rates varied linearlywith the current density ratio as expected. The optimallaser power for each focusing condition was investigatedusing dilute solutions of pBluescript DNA. Althoughfluorescence burst heights and background varied withlaser power, the signal-to-noise ratio was only weaklydependent on this parameter using our single-moleculecounting technique. Focused single-molecule countingwas used to detect separations of a 100-1000-bp DNAsizing ladder. The increase in molecular detection efficiency was quantified by applying a focusing currentmidway through the ladder separation and comparingobserved count rates to the known molecular concentration in the bands. The molecular detection efficiencyvaried linearly with the applied current density ratio, andgreater than 3-fold enhancements in detection efficiencywere achieved.
m-diameter focused laser beam. Thesample was focused by introducing a taper in the separation channel and by a sheath flow delivered from crosschannels. The sample stream width, single-moleculevelocities, and single-molecule count rates varied linearlywith the current density ratio as expected. The optimallaser power for each focusing condition was investigatedusing dilute solutions of pBluescript DNA. Althoughfluorescence burst heights and background varied withlaser power, the signal-to-noise ratio was only weaklydependent on this parameter using our single-moleculecounting technique. Focused single-molecule countingwas used to detect separations of a 100-1000-bp DNAsizing ladder. The increase in molecular detection efficiency was quantified by applying a focusing currentmidway through the ladder separation and comparingobserved count rates to the known molecular concentration in the bands. The molecular detection efficiencyvaried linearly with the applied current density ratio, andgreater than 3-fold enhancements in detection efficiencywere achieved.