Rapid Systems for Detection of Pathogen and Human Nucleic Acids via Microfluidic Amplification
DuVall, Jacquelyn, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Landers, James, Department of Chemistry, University of Virginia
Nucleic acid amplification serves an important role in many applications, including, but not limited to: clinical diagnostics, biomedical analyses, and forensics. Traditional amplification, either via polymerase chain reaction (PCR) or isothermal methods, is time-consuming and dependent upon expensive instrumentation and equipment. Microfluidics has emerged as a powerful tool to address these limitations, and extensive research has been focused on the application of micro total analysis systems (μTAS) to nucleic acid amplification. This dissertation presents two specific applications of microfluidic amplification: pathogen detection and forensic human identification.
The goal of the work presented in Chapters 2 and 3 was an inexpensive and simplified method for pathogen detection. Building upon a previously described technique, chaotrope-induced aggregation (CIA), a novel magnetic bead assay was developed, and coupled to an isothermal amplification reaction, for rapid detection of multiple microorganisms, including: Rift Valley fever virus, Escherichia coli, Salmonella, Influenza H1N1, and Clostridium difficile. The magnetic bead assay, referred to as product-inhibited bead aggregation (PiBA), as well as the isothermal amplification, were both successfully demonstrated on a polyester microdevice. Functionality, including spinning, heating, and application of a rotating magnetic field (RMF), was achieved with a custom-built system, and image analysis allowed for detection of targeted pathogens with an inexpensive 15-megapixel cell phone camera.
Chapters 4 and 5 present work aimed at developing an inexpensive and rapid platform for human identification using short tandem repeat (STR) analysis. Multiplexed PCR was demonstrated for the first time on a polyester microdevice, and the amplification was further integrated with upstream liquid extraction (LE) and downstream microchip electrophoresis (ME). A custom-built system was designed for rapid Peltier-mediated thermocycling, and allowed for the successful amplification of 10 genetic loci in only 15 minutes. The print, cut, laminate (PCL) method was utilized for rapid prototyping, and this approach allowed for the fabrication of a fully-integrated microdevice using inexpensive, commercially available substrates. Fluid flow control was achieved through the use of centrifugal force and laser-actuated valves, thereby eliminating the need for cumbersome external pumps and valves. Overall, a fully- integrated microdevice was demonstrated, capable of DNA extraction, multiplexed STR- based PCR amplification, and fragment separation, for rapid human identification via generation of a unique STR profile.
PHD (Doctor of Philosophy)
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