| Despite huge strides made towards the development of microfabricated DNA separation systems, there are significant hurdles to the realization of small, portable DNA analysis systems. The design of microfabricated separation systems have been based more on heuristics, and less on a scientific understanding of the migration parameters and the factors influencing the resolution of separation. Most of the miniaturized separation systems developed, so far, use high electric fields to overcome the diffusion/dispersion of DNA fragments in the sieving medium, which hinders the use of battery operation. In addition, most of these devices require external optical readers thereby increasing the cost substantially, and diminishing the benefits of microfabrication. Finally, high-resolution separations tend to require long separation lengths that result in devices with a relatively large footprint. These limitations that have restricted the applicability of microfabricated devices to laboratory based settings.; Upon establishing the feasibility of DNA separations in microfabricated devices, a detailed study of the migration of single stranded DNA under typical DNA sequencing conditions was performed. The implications of this study on the design of future separation systems was also explored. Based upon these studies, it appeared that polyacrylamide gels offered the possibility of realizing high-resolution separations in short separation lengths at low electric fields. Subsequently, techniques for the efficient loading of the sieving medium into the separation channels, and for injecting a defined amount of sample were developed. The use of these technologies for double stranded DNA separations in microfabricated separation systems was also demonstrated. Finally, an on-chip fluorescent detection system was developed, and used, to detect individual DNA bands migrating in a microfabricated electrophoretic device. Fluorescence-based detection of DNA bands was achieved by incorporating a highly sensitive photodiode beneath the electrophoresis channel along with a thin film optical filter deposited above the diode.; The development of these techniques for loading the sieving medium, injecting the sample, and detecting the presence of fluorescently labeled moities, could significantly help accelerate the realization of integrated, portable ‘lab-on-a-chip’ systems. |
