Microfluidic microarray for pathogenic DNA Analysis: single-base-pair-mismatch discrimination, and modeling/simulation of centrifugal flows and dynamic hybridization

In the development of surface-based biosensors, the combination of microfluidic technology with the DNA microarray chip has been realized with the intersection method. The method shows the advantages of less sample usage, multiple sample capability, enhanced hybridization signals, and reduced assay time. In this thesis, line arrays of DNA probes were printed on a glass chip through the microfluidic method. Target microchannels orthogonally intersected with these line arrays, and complementary DNA molecules, upon hybridization, were retained at the intersections as rectangular spots. Detection was achieved through the read-out of the fluorescent labels on the targets. The high surface-to-volume ratio in microchannels of nanolitre volume enhanced the detection sensitivity as compared to that obtained with the bulk solution method. The spot shape is more regular than that from the pin-spotted microarray, which is an advantage for subsequent image processing. For diagnostic purposes, PCR products amplified from the genomic DNA of fungal pathogens were detected with this microfluidic intersection method. Moreover, with the aid of gold nanoparticles, two 260-bp DNA sequences with single base-pair difference were discriminated from each other at room temperature for the first time. In addition to the pressure-driven flows used in rectangular chips, a centrifugal-pumping method was employed to drive liquid movement in a CD-like microchip. Connections from electrodes or tubing were avoided, resulting in the ease of conducting parallel hybridizations in multi-channels. For example, up to 100 samples can be analysed simultaneously with the CD-like microchip. The centrifugal-pumping flow in the spiral channel was modeled mathematically and simulated numerically by the computational flow dynamics (CFD) method. It was found that the mathematical results and simulation results are consistent with the experimental findings. Other than the flow study, the kinetics of microfluidic DNA hybridization was also studied and simulated. The effects of probe coverage, channel depths, assay conditions, and sample delivery rate on DNA hybridization were investigated. The variation of signal intensity inside a hybridization spot and from spots to spots was also discussed and compared with the experimental findings. The proposed method provides a way to optimize both chip design and experimental conditions during surface-based biosensor assays.