Development of the elemental technologies for centrifugal microfluidic system and its applications in fully-automated molecular diagnostics

Abstract

This thesis describes elemental technologies and fully automated molecular diagnostics by utilizing a centrifugal microfluidic system. As the elemental technologies, a vertically-configured laser-irradiated ferrowax microvalve system was developed to have a compact structure and utilized it for the automated serial dilutions of the sample. Besides, a high-temperature ferrowax microvalve system was also developed for the valve-operation under the isothermal DNA amplification process. The optimal plano-convex collimating lens structure was designed by optical simulation and adapted to the injection-molded disc to amplify the fluorescence signal. A one-step centrifugal step emulsification method was developed that can be useful for digital quantitative analysis. Based on these elemental technologies, the centrifugal microfluidics-based standard quantitative analysis methods were automated. This allows the process of the standard addition analysis and external standard analysis to be carried out easily in a very short time while providing more accurate quantitative information. Finally, I integrate all the processes for molecular diagnostics, including DNA extraction and purification, isothermal DNA amplification, and fluorometric detection in a single disc. I targeted three kinds of major foodborne pathogens (Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes), and isothermal target and probe amplification (iTPA) was performed to amplify the specific sites of the target genes. Samples contaminated with foodborne pathogens could be automatically analyzed by the rotor system down to the dozens of CFU levels in 90 min. Besides, the presence of tuberculosis germs while also determining the resistance of major anti-tuberculosis drugs through single nucleotide polymorphism (SNP) analysis for the multi-drug resistant tuberculosis (MDR-TB) diagnostics. Loop-mediated isothermal amplification (LAMP) with cycling probe technology (CPT) was performed to discriminate the mutations of the target genes about drug-resistance of the rifampicin and isoniazid drugs. The simplicity and portability of the centrifugal microfluidics can provide an advanced platform for quantitative analysis and point-of-care testing, and can be effectively applied to the resource-limited environment.