Issue 12, 2020

Double emulsion flow cytometry with high-throughput single droplet isolation and nucleic acid recovery

Abstract

Droplet microfluidics has made large impacts in diverse areas such as enzyme evolution, chemical product screening, polymer engineering, and single-cell analysis. However, while droplet reactions have become increasingly sophisticated, phenotyping droplets by a fluorescent signal and sorting them to isolate individual variants-of-interest at high-throughput remains challenging. Here, we present sdDE-FACS ([s with combining low line]ingle [d with combining low line]roplet [D with combining low line]ouble [E with combining low line]mulsion-FACS), a new method that uses a standard flow cytometer to phenotype, select, and isolate individual double emulsion droplets of interest. Using a 130 μm nozzle at high sort frequency (12–14 kHz), we demonstrate detection of droplet fluorescence signals with a dynamic range spanning 5 orders of magnitude and robust post-sort recovery of intact double emulsion (DE) droplets using 2 commercially-available FACS instruments. We report the first demonstration of single double emulsion droplet isolation with post-sort recovery efficiencies >70%, equivalent to the capabilities of single-cell FACS. Finally, we establish complete downstream recovery of nucleic acids from single, sorted double emulsion droplets via qPCR with little to no cross-contamination. sdDE-FACS marries the full power of droplet microfluidics with flow cytometry to enable a variety of new droplet assays, including rare variant isolation and multiparameter single-cell analysis.

Graphical abstract: Double emulsion flow cytometry with high-throughput single droplet isolation and nucleic acid recovery

Supplementary files

Article information

Article type
Paper
Submitted
12 Mar 2020
Accepted
20 Mar 2020
First published
17 May 2020
This article is Open Access
Creative Commons BY-NC license

Lab Chip, 2020,20, 2062-2074

Double emulsion flow cytometry with high-throughput single droplet isolation and nucleic acid recovery

K. K. Brower, C. Carswell-Crumpton, S. Klemm, B. Cruz, G. Kim, S. G. K. Calhoun, L. Nichols and P. M. Fordyce, Lab Chip, 2020, 20, 2062 DOI: 10.1039/D0LC00261E

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