DNA Amplification and Detection in Micro/nano-fluidic Chip.

摘要

In this thesis, several critical topics related to PCR-based micro/nano-fluidic chip were studied: from the chip integration and fabrication (three PCR chips and one DNA detection chip were introduced), PCR optimization to materials' PCR inhibition. A highly integrated PCR microfluidic chip combined giant electrorheological (GER) fluid actuated micromixer and micropump and a microheater array was demonstrated in chapter 2. Afterward a microheater and a thermal sensor were fabricated inside elastomeric PDMS micro channels by injecting silver paint (or other conductive materials) into the channels. With a high-precision control scheme, microheaters can be used for rapid heating, with precise temperature control and uniform thermal distribution. Using such a microheater and feedback system, the PCR experiment was carried out whereas the DNA was successfully amplified in 25 cycles, with 1 minute per cycle. As reported in chapter 4, we formulated a simplified PCR profile consisting of only two temperatures instead of the conventional three and found suitable PCR component concentrations for successful detection of genetic variation under optimized PCR conditions by utilizing raw saliva. We also tested the PCR-compatibility of 23 kinds of common materials in microfluidics with a simple and newly developed method. Our results provide an overview of which the materials are most PCR-friendly for microfluidic device fabrication. Based on the previous experimental results, a device consisting of an interchangeable PCR chamber, a temperature control component as well as an optical detection system was constructed. The DNA amplification happens on an interchangeable chip with the volumes as low as 1.25 microl and the total time of only 25 minutes to complete the 35 cycle PCR amplification. Within the PCR chip, the amplification of male-specific SRY gene marker by utilizing raw saliva was successfully achieved and the genetic identification was in-situ detected right after PCR by the optical detection system. Finally, initial experiments of DNA detection in nanoslot were reported. We have captured the impedance shift of the nanoslot for DNA is present in solution.