Microfluidic two-phase biochemical reaction systems for DNA analysis

The advance in microfluidics and microfabrication enables the miniaturization of the conventional biochemical reactions to achieve inexpensive and rapid assays. In this dissertation, biochemical reaction microsystems in two-phase microfluidic formats -- air-liquid and liquid-liquid (droplet-based), were developed and studied to perform single- and multi-step amplification-based DNA analyses.;Microfluidic valveless devices for single-liquid-phase-based polymerase chain reaction (PCR) were developed to reduce the evaporation loss relying on the principle of diffusion-limited evaporation. The evaporation can be suppressed using long narrow diffusion channels in conjunction with reducing the driving force for liquid evaporation by decreasing the interfacial temperature using thermal isolation and reducing the vapor concentration gradient through replenishing water vapor in the diffusion channels. With these techniques the evaporation loss can be limited to approximately 1% of the reaction content.;Performance of droplet-based PCR was also studied in a microdevice. Droplets with a volume range of 5-250nL can be formed on-chip reproducibly, and PCR in the droplets showed amplification efficiency comparable to benchtop reactions with no evaporation loss. Reaction parameters such as reagent concentration, hold time at each cycling step, template concentration and the droplet size were characterized. The droplet-based PCR also demonstrated amplification with a cycle threshold of ∼10 cycles earlier than the benchtop instruments.;Operation of the droplet-based microdevice was then automated using on-chip electronic sensing. The sensing signal was used to track the oil/aqueous interface and control the pneumatic supply for automatically generating and moving droplets. The actual droplet size was very close to the designed value with a standard deviation less than 3% of the droplet size. The droplets can be automatically confined in the reaction region with feedback pneumatic control. PCR has also been successfully performed in the automated generated and positioned droplets.;Droplet manipulations such as merging and mixing were also studied in a microdevice for applications in multi-step bioreactions. Mixing in the merged droplets was achieved with the droplet kept in the chamber or moved out of the chamber. A droplet-based two-step bioreaction, nested TaqManRTM PCR with enhanced sensitivity has been demonstrated by utilizing droplet generation, merging and mixing in the microdevice.