Integrated capillary electrophoresis system for DNA analysis.

Abstract

Since the early 1990's researchers have applied the principles of capillary electrophoresis to microfabricated devices. The majority of these devices have been fabricated using bulk micromachining of glass substrates and employ costly optical systems to detect fluorescent analytes. The real power of miniaturization is lost, however, when these devices rely on a room full of equipment. The realization of on-chip detection with these devices paves the way to practical and reliable hand held DNA assay systems. Surface micromachining is an attractive method not only for the integration of electrophoresis devices with fluorescence on-chip detection but also for the fabrication of complex DNA analysis chips. Such a technology must be able to include not only separation and detection systems but also heaters, temperature sensors, reaction chambers, pumps, valves and possibly even CMOS circuitry. This proposal concludes former research on capillary electrophoresis based DNA analysis, explores capability for higher DNA resolution and proposes a new design of surface micromachined integrated capillary electrophoresis systems for DNA analysis. This technology uses photoresist sacrificial layers in conjunction with vacuum deposited parylene-C. Channels up to 20mum in height and nearly 2 cm in length have been fabricated. The low temperature fabrication process allows these channels to be fabricated on top of heaters, electrodes, photodiodes, temperature sensors, etc. without affecting device performance. Channels can be constructed on top of virtually any topography due to the conformal deposition of parylene-C. Cross-linked sieve matrix materials such as cross-linked polyacrylamide(UV initiated polymerized gel) are commercially used for high resolution DNA sequencing. However acrylamide or similar gels cannot polymerize in the presence of O₂. Due to the porous characteristics of parylene films, UV gels cannot polymerize in parylene channel: In order to overcome this problem, we explore the use of UV gel in parylene channels with an aluminum oxide layer overcoat. The alumina layer serves as an O₂ barrier. Results show alumina can dramatically decrease parylene film permeability. Both dsDNA and ssDNA separation have been tested up to 5bp resolution. In order to achieve maximum fluorescence and minimum noise for ssDNA detection, a newly designed optical detection system has been presented. It includes an on-chip high sensitivity photodiode and high power excitation scheme. The high sensitivity photodiode has dark current as low as 30pA biased at -5V. DNA single base pair resolution has been achieved through 1bp ladder separation.