| This dissertation presents the development of a molecular diagnostics microfluidic instrument. This device, identified herein as the "Continuous-Flow Thermal Gradient" system, is capable of performing PCR (polymerase chain reaction) and spatial DNA melting analysis. The amplification and detection of nucleic acids have been performed simultaneously, as well as individually. This device represents a significant departure from the architecture and methodology of other continuous-flow PCR microfluidic systems, and is the first known embodiment of a device capable of spatial DNA melting analysis. From the original concept of this system, the device, supporting instrumentation, and analytical software were all created to successfully demonstrate this envisioned system. Initial prototypes have been successfully tested, characterized, and improved. Notable experimental achievements include: 40 PCR cycles in less than 9 minutes with an efficiency and specificity comparable to macroscale commercial systems; in situ melting analysis of the PCR product during its amplification; and spatial resolution of DNA melting capable of single polymorphism (SNP) detection with a signal to noise ratio above 80. Numerical models have been developed to examine the thermal behavior of the device, specifically with regards to the effects of liquid flow. The parametric dependencies of the flow-induced temperature changes were investigated. It was found that the mean fluid velocity and the corresponding cross-sectional area of the microfluidic channel have little effect on the thermal stability of the device, while the volumetric flow rate, geometric channel spacing, and especially the thermal conductivity of the substrate govern the local thermal behavior. |
