Specific Sorting of Single Bacterial Cells with Microfabricated Fluorescence-Activated Cell Sorting and Tyramide Signal Amplification Fluorescence in Situ Hybridization

Abstract

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.

A microfluidic platform for profiling biomechanical properties of bacteria

“…The current work therefore adds the ability to detect biomechanical phenotypes of bacteria to the list of other bacterial phenotypes currently detectable using microchip-based methods such as ratcheting microchannels to fractionate a population of bacteria by cell length 31 , positive dielectrophoretic measurements 32 , surface enhanced Raman spectroscopy 33 , and microfabricated fluorescence-activated cell sorting 34 . Cell sorting/separation and selection applications related to biomechanical phenotypes have potential advantages over selection based on other phenotypes in that biomechanical phenotypes are naturally present and could potentially be performed on environmental samples including uncultivable organisms.…”

Automated, Miniaturized, and Integrated Quality Control-on-Chip (QC-on-a-Chip) for Cell-Based Cancer Therapy Applications

“…The on-chip integration of piezoelectric zirconate titanate films (PZT) actuators (Chen et al, 2009) in μFACS devices allows hydrodynamic focusing of single cells in the sub-nanoliter volume (Figure 4A). When a targeted cell enters the sorting junction, the PZT actuator is activated using a voltage pulse to deflect the cell-containing fluid from the center position toward a side collection channel (Chen et al, 2011), thus enabling an operating limit of >1000 cells/s. Currently, commercially available is the miniaturized flow cytometer tabletop-box (NanoCellect, San Diego, CA, USA), which is based on integrated optical system, the microdevice is combined with PZT actuators (http: //nanocellect.com/).…”

Exploring Symbioses by Single-Cell Genomics

“…This method uses reaction volumes of only 60 nl, which reduces the likelihood of contaminating the sample. Microfluidic devices have also been recently developed for FISH and tyramide-signalamplification FISH (tsa-FISH) followed by cell sorting via flow cytometry directly on the device (Chen et al, 2011;Liu, P., et al, 2011). This approach holds great promise for 16S rRNA gene-based identification of single cells, while bearing low risks of contamination.…”