High-throughput piezoelectric-actuated micro-fluorescence-activated cell sorter (muFACS).

摘要

In this thesis, I have developed a piezoelectric-actuated micro-fluorescence-activated cell sorter (muFACS) and demonstrate its performances using various biological samples including mammalian cell and bacteria. Three major developments in this work included a high-sensitivity detection system, fast-response on-chip piezoelectric cell sorting module, and system integration. In my early work, optical arrayed waveguides combined with cross-correlation signal processing algorithm are implemented to achieve high-sensitivity scattering detection. The insight gained from the algorithm further allowed me to design and implement a spatial-filter based (space-time coding) fluorescent detection system. The system enables not only signal amplification (∼ 18 dB SNR enhancement) but also sorting even verification, allowing real-time optimization of sorting parameters. The first generation on-chip cell-sorting module involves flow-redirection using the principle of nozzle-diffuser, but due to the periodic flow and high fluid disturbance resulted from high-voltage piezoelectric actuation, the sorting module was redesigned, resulting in piezoelectric-actuated cell sorting module. The inexpensive module was able to manipulate single cells at high rate (> 1000 cells/s) under low powered actuation ( 10 mW and 10 Vp-p).;Integration of detection and sorting systems is achieved through the implementation of the preprogrammed FPGA-embedded external driver enables closed-loop control for triggering fluorescence-activated cell sorting. With the sorting event verification capability, sorting efficiency was found to be > 80%. Sample enriching experiments were done using beads and human mammalian cells, showing an enrichment factor > 200 fold (comparable to commercial FACS), which is the highest among muFACS systems.;The developed integrated muFACS was also applied to address the challenges (detection sensitivity and cell-free DNA contamination) commonly encountered in single-cell genome sequencing. Flow cytometry-modified Tyramide Signal Amplification Fluorescence in situ Hybridization (TSA-FISH) and two-step optofluidic light confinement were implemented to enhance sensitivity. Also, dual-round cell-free DNA purification was performed and compared to commercial FACS, showing comparable results. Sorting of rare bacteria was achieved, showing 223-fold enrichment.;I hope the work sets the benchmark for the future development of muFACS systems. I believe the realization of a truly hand-held muFACS that can be afforded by every research labs and clinics is not far off.