| Fluorescence detection has established itself as one of the main techniques of interrogation of biological systems. Extending those techniques to decrease the sample size to single molecules provides an absolute standard for bulk sample calibrations, as well as better insights since individual behavior is observed instead of population averages. We observed a number of fluorophores, including GFP, at the single-molecule level at room temperature. Calibrations gave a correct estimate of bulk surface densities over four orders of magnitude, through an optical, non-invasive, non-destructive means. Novel surface chemistry enabled visualization of single tagged nucleotide incorporations inside DNA immobilized on a glass surface at the single-molecule level. This technology was later extended to successful single-molecule DNA sequencing.; At the same time, PDMS microfluidics was developed to provide the plumbing control, speed, and economy of scale for a broad range of applications. Novel surface chemistry anchored DNA to the PDMS microchannels, which allowed sequencing-by-synthesis to be conducted in the microfluidic environment using optical techniques. Materials, device, and architecture problems were also solved. Finally, all technology was put together and successful microfluidic bulk-fluorescence DNA sequencing was demonstrated. The same technology is applicable to any DNA studies in microfluidic environments and can eventually be extended to close the circle to single-molecule detection. |
