Specific sorting of single bacterial cells with microfabricated fluorescence-activated cell sorting and tyramide signal amplification fluorescence in situ hybridization.

When attempting to probe the genetic makeup of diverse bacterial communities that elude cell culturing, researchers face two primary challenges: isolation of rare bacteria from microbial samples and removal of contaminating cell-free DNA. We report a compact, low-cost, and high-performance microfabricated fluorescence-activated cell sorting (µFACS) technology in combination with a tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) to address these two challenges. The TSA-FISH protocol that was adapted for flow cytometry yields a 10-30-fold enhancement in fluorescence intensity over standard FISH methods. The µFACS technology, capable of enhancing its sensitivity by ~18 dB through signal processing, was able to enrich TSA-FISH-labeled E. coli cells by 223-fold. The µFACS technology was also used to remove contaminating cell-free DNA. After two rounds of sorting on E. coli mixed with λ-phage DNA (10 ng/µL), we demonstrated over 100,000-fold reduction in λ-DNA concentration. The integrated µFACS and TSA-FISH technologies provide a highly effective and low-cost solution for research on the genomic complexity of bacteria as well as single-cell genomic analysis of other sample types.

HIGH-THROUGHPUT CELL SORTER WITH PIEZOELECTRIC ACTUATION.

Currently, most of the integrated sorting modules in the microfabricated DEP-based and fluorescent-activated cell sorters (µFACS) still suffer from low-throughput operation and require complex fabrication process (e.g. embedded electrodes) and high power consumption (e.g. electrokinetically-driven sorters). In this paper, we demonstrate an easy-to-fabricate, low-powered and high-speed sorting module (at a single cell level) using an on-chip integrated piezoelectric (PZT) actuator. By controlling the bending motion of the PZT actuator, we have investigated and verified the high-speed flow-switching and sorting capabilities both theoretically (dynamic simulation) and experimentally using beads and biological agents.