Improving electrophoretic resolution in microfabricated bioanalysis devices.

摘要

Microfabrication is a technology initially used by semiconductor manufacturing industries. With this technique, multiple components can be integrated on one cm²-sized chip and used as a micro-bioanalysis platform. Electrophoresis separation using polyacrylamide is one particular component of such a bioanalysis system that has been widely applied in gene analysis, mutation detection, disease diagnosis and forensic screening. A major concern for a micro-separation is the resolution loss due to shorter separation distances in comparison with conventional slab-gel or capillary systems. This thesis work focuses on improving separation resolution for a miniaturized device by both experimental measures and theoretical studies.; First, we have improved separation resolution for both ssDNA and dsDNA in a microfabricated DNA device by continuously flowing 1X TBE buffer over the electrodes at the cathodic end. Resolution for ssDNA primer separations has been increased about 4 times higher than that without a flow in a separation distance of 1.2cm. DNA electrophoretic behavior in microchannels has been studied with a transverse detection using confocal microscopy. A strong position-dependent distribution of DNA migration was found, and the distribution evolved with DNA migration distance from an initial center-biased distribution to a later top-and-bottom-biased distribution; indicating an unique interaction between a rigid rod-like molecule and a low-elastic fixed network, whose average pore size is much smaller than the axial dimension of the molecule.; A coarse-grained model was used to simulate ssDNA electrophoretic mobility and corresponding band broadening in a crosslinked or entangled network. Simulated mobility results showed a smooth transition from reptation to oriented reptation regime. With dimensionless numbers reflecting experimental conditions and physical properties, we were able to convert the simulated results into comparable experimental results for chains with 8 to 50 entanglements respectively (around 1500 to 8500 bases).; These studies suggest optimal process conditions for DNA separation in a micro-device, either by modifying the device design or understanding detailed mechanisms. In the future, an optimized micro-separation system can be designed for the applications of both genotyping and sequencing.