37. Focused Single Molecule DNA Detection in Microfabricated Capillary Electrophoresis Chips 

Brian B. Haab and Richard A. Mathies 
Department of Chemistry, University of California, Berkeley, CA 94720 
rich@zinc.cchem.berkeley.edu 

Single-molecule fluorescence burst counting is a highly sensitive method for detecting electrophoretic separations of ds-DNA fragments¹ with applications in environmental monitoring and health care diagnostics. We previously presented methods for optimizing dye labeling, laser power and data analysis, and conventional CE separations of ds DNA fragments in the 100-1000 bp range were detectable when only 50-100 molecules passed through the probe volume.² We have now performed single DNA molecule detection in glass capillary electrophoresis (CE) chips which offer improved optics, faster separations, and increased molecular detection efficiency compared to conventional capillaries.³ Chips were fabricated with a 145 mm thick top plate that was matched to the design specifications of the 100X, 1.3 NA objective, yielding a two-fold increase in light collection efficiency. The channels were designed to focus a greater number of molecules through the laser beam to achieve enhanced detection sensitivity. The sample was constricted in the region of the 1 mm diameter focused laser beam by physical narrowing of the separation channel and by electrokinetic focusing caused by additional side channels in the detection region. The sample stream width decreased and the single molecule count rate increased linearly with the focusing current density. A four-fold improvement in molecular detection efficiency was achieved while maintaining single molecule sensitivity. The CE separation of a 500 bp PCR product was then detected using molecular focusing, which showed a two-fold increase in signal compared with conventional detection. A 300 fM sample was easily detectable with a signal-to-noise ratio of eight. These developments will enhance our ability to use CE separations to detect trace pathogen contamination or DNA mutation. 

¹ B. B. Haab and R. A. Mathies, Anal. Chem. 34, 3253-3260 (1995) 

² B. B. Haab and R. A. Mathies, Appl. Spec. 51, 1579-1584 (1997) 

³ B. B. Haab and R. A. Mathies, Proc. SPIE 3259, 104-112 (1998)