Single-molecule detection of DNA separations in microfabricated capillary electrophoresis chips employing focused...

DNA separations have been performed using microfabricated capillary electrophoresis chips and detected using a single-molecule fluorescence burst counting technique. We enhanced the percentage of electromigrating DNA molecules that were detected by focusing the sample through the 1-microm-diameter focused laser beam. The sample was focused by introducing a taper in the separation channel and by a sheath flow delivered from cross channels. The sample stream width, single-molecule velocities, and single-molecule count rates varied linearly with the current density ratio as expected. The optimal laser power for each focusing condition was investigated using dilute solutions of pBluescript DNA. Although fluorescence burst heights and background varied with laser power, the signal-to-noise ratio was only weakly dependent on this parameter using our single-molecule counting technique. Focused single-molecule counting was used to detect separations of a 100-1000-bp DNA sizing ladder. The increase in molecular detection efficiency was quantified by applying a focusing current midway through the ladder separation and comparing observed count rates to the known molecular concentration in the bands. The molecular detection efficiency varied linearly with the applied current density ratio, and greater than 3-fold enhancements in detection efficiency were achieved.