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

DNA separations have been performed using microfabricated capillary electro phoresis chips and detected using a single-molecule fluorescence burst coun ting technique. We enhanced the percentage of electromigrating DNA molecule s that were detected by focusing the sample through the 1-mu m-diameter foc used laser beam. The sample was focused by introducing a taper in the separ ation channel and by a sheath now 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 las er power for each focusing condition was investigated using dilute solution s of pBluescript DNA. Although fluorescence burst heights and background va ried with laser power, the signal-to-noise ratio was only weakly dependent on this parameter using our single-molecule counting technique. Focused sin gle-molecule counting was used to detect separations of a 100-1000-bp DNA s izing ladder. The increase in molecular detection efficiency was quantified by applying a focusing current midway through the ladder separation and co mparing observed count rates to the known molecular concentration in the ba nds. The molecular detection efficiency varied linearly with the applied cu rrent density ratio, and greater than 3-fold enhancements in detection effi ciency were achieved.