| I have developed the microchip technology needed to make a miniaturized and highly-integrated multichannel polymerase chain reaction---capillary electrophoresis (PCR-CE) microprocessor for high-throughput DNA analysis. The challenges of realizing this PCR-CE processor are to develop scalable microfluidic valves and pumps for sample introduction and immobilization during thermal cycling, scalable heaters and temperature sensors for parallel thermal cycling of multiple reaction chambers, and an improved injector for sensitive and quantitative analysis of PCR amplicons.; To this end, a normally-closed, pneumatically actuated silicone elastomer based polydimethylsiloxane (PDMS) membrane valve was developed. The PDMS microvalves can seal reliably against pressure as high as 75 kPa with an applied valve pressure of 45 kPa, and when opened they allow a flow rate of 370 nL/s with a fluidic pressure of 30 kPa. Moreover, the fabrication of the PDMS valves is monolithic and dense arrays of valves can be fabricated in a single operation to allow parallel actuation with an integrated manifold.; Different heater and temperature sensor configurations have been explored, and characterized. The optimized integrated heaters with serpentine Ti/Pt heating elements together with Ti/Pt RTD's enable heating rates of > 15°C s-1, cooling rates of > 10°C s-1, and 30 PCR cycles are completed in < 27 min. With proper fluidic and temperature control, we then developed a multichannel PCR-CE microdevice that demonstrates good amplification uniformity and sensitivity down to 10 initial template copies in the 380 nL reactor (∼ 43 attomolar concentration) with signal-to-noise ratio (S/N) greater than 10. However, further performance advancement was hampered by the inefficient cross-injector typically used in microfabricated CE systems.; To provide improved quantitative analysis capability, a novel inline injection method was developed that utilizes a thermally switchable oligonucleotide affinity capture gel to mediate the concentration, purification and injection of dsDNA. Unlike the traditional cross-injector, this inline injection method enables the efficient capture and injection of dsDNA amplicons which facilitates the quantitative analysis of products from integrated nL-scale PCR reactors arrays. The work present here provides a strong foundation that will enable the development of massively parallel genetic analysis microdevices. |
