<title>Single-molecule DNA detection in microfabricated capillary electrophoresis chips</title>

Abstract

Single-molecule fluorescence burst counting is a highly sensitive method for detecting electrophoretic separations of ds-DNA fragments. In previous work we demonstrated the detection of single DNA fragments as small as 100 bp in capillary electrophoresis separations. To further enhance the applicability of this method to low level pathogen and mutation detection, we have now successfully performed single molecule detection of DNA separations in microfabricated glass capillary electrophoresis (CE) chips. By fabricating CE chips with a 200 micrometers thick top cover plate and by using a 40X, 1.3 NA immersion microscope objective, the S/N ratio for single molecule detection is enhanced by more than two-fold over conventional capillaries. By constricting the sample in the detection region to an approximately 10 micrometers wide by approximately 10 micrometers deep cross section, approximately 10% of the molecules passing through the channel are probed by the approximately 2 micrometers wide focused laser beam. This is a 1000-fold improvement over our previous work. We have now achieved an on-column detection limit of approximately 600 fM or 500 molecules for 500 bp DNA fragments (S/N equals 3).© (1998) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.