Development of Microfluidic Devices for Genetic Analysis: Continuous Flow Infrared-mediated PCR (cfIR-PCR) and Microwave Thermal Cycling Systems
Oh, Kyudam, Chemical Engineering - School of Engineering and Applied Science, University of Virginia
Landers, James, Chemistry, University of Virginia
The work in this dissertation constitutes four research efforts focused on the development of microfluidic instrumentation and methodology for advanced genetic analysis in polymeric microdevices. Laser ablation has been mainly employed to construct polymeric devices as a result of rapid prototyping and ease of microfabrication. As part of a genetic analyzer, two types of heating systems were investigated to drive thermocycling in these polymeric microdevices: infrared and microwave, ultimately capable of multiple and high-throughput reactions with portability in DNA analysis. Chapter 1 introduced the general purpose in the development of microfluidic devices for genetic analysis, especially describing details of benefits in “Lab on a chip” technologies and microfluidic Polymerase Chain Reaction (PCR) that are directly associated with this work. In Chapter 2, the development of continuous flow infrared-mediated PCR (cfIR-PCR) on a single lamp is outlined. An IR-mediated heating system has been utilized in our group to facilitate non-contact PCR inside glass and polymeric microfluidic devices for clinical and forensic applications. However, the number of PCR reactions was limited due to the configuration of our heating system. In present work, the cfIR-PCR system has shown the capability of multi-segmented DNA amplifications from multiple DNA targets that were encapsulated inside emulsion solutions without any cross-contamination, which further proves the feasibility of high-throughput genetic analysis on a single lamp.
As an alternative heating system, a microwave-mediated thermal cycling system is presented in Chapter 3. Due to the characteristics of dielectric heating, microwave offers ultrafast thermal cycling of micro and nano-scale reaction solutions in microfluidic devices. Three types of microwave-mediated heating control (on/off, open-loop, and proportional voltage controls) concepts were assessed using polymeric materials to optimize our microwave heating setup. As two major approaches in temperature sensing, a thermocouple (contact sensing) and an IR-pyrometer (non-contact sensing) were utilized to measure temperature in the PCR solution to control thermocycling and to accomplish successful DNA amplifications under a microwave field. Additionally, the potential for PCR applications to be used in miniaturized genetic analyzers for Point-of-Care (POC) DNA analysis was demonstrated. In Chapter 4, IR-mediated PCR in the single chamber of the polymeric microdevice was shown to achieve the rapid detection of exogenous and evolved genetic materials (DNA/RNA) using degenerate primers. Furthermore, the novel concept of rapid and reliable drug susceptibility testing (DST) for M. tuberculosis is then introduced in Chapter 5. Standard PCR coupled with hybridization-induced bead aggregation was successfully introduced for the detection of PCR amplicons as an advanced alternative of microchip electrophoresis. Finally, summaries and future directions were outlined in Chapter 6.
PHD (Doctor of Philosophy)
All rights reserved (no additional license for public reuse)