Ultra high-throughput fluorescence detection for single cell applications in drop microfluidics
Schaefer, Robert Willman
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Conventional methods in microbiology can be limited by assay execution and analysis times, phenotypic dominance within bulk communities, reagent volumes, and single-use supply costs. These limitations can be overcome using drop-based microfluidics. In this discipline, pico-liter sized, water-in-oil emulsions serve as independent 'test tubes,' allowing for the compartmentalization of community constituents and interrogation at the single cell level. Furthermore, two-phase, continuous flow microfluidic devices enable drop populations to be manipulated and analyzed at kilohertz rates according to experimental needs. In this research, a fluorescence-based method for drop analysis and sorting was developed and applied, in conjunction with other microfluidic techniques, to perform assays in microbiology. The applications explored include cell dormancy within P. aeruginosa subpopulations, microalgae lipid accumulation for the production of biofuels, optimization of microbially-induced calcite precipitation (MICP), and human norovirus infectivity. Results from each application include: 1. The hibernation promoting factor (Hpf) was found to play a key role in the maintenance of P. aeruginosa viability during planktonic starvation. 2. Progress was made on a Nile Red based, ultra high-throughput, single cell algal lipid detection platform. 3. MICP was demonstrated at the single cell level. 4. A drop based human norovirus infection platform was attempted using human B cells as the viral host.