Double Emulsion Flow Cytometry for Rapid Single Genome Detection

Established techniques in droplet microfluidics have utilized single emulsion (SE) drops to compartmentalize and analyze single cells achieving high-throughput, low input analysis. Building upon this foundation, double emulsion (DE) droplet microfluidics has emerged with distinct advantages in terms of stable compartmentalization, resistance to merging, and most importantly direct compatibility with flow cytometry. In this chapter, we describe a simple-to-fabricate, single-layer DE drop generation device that achieves spatial control over surface wetting with a plasma treatment step. This easy-to-operate device allows for the robust production of single-core DEs with excellent control over the monodispersity. We further explain the use of these DE drops for single-molecule and single-cell assays. Detailed protocols are described to perform single molecule detection using droplet digital PCR in DE drops and automated detection of DE drops on a fluorescence-activated cell sorter (FACS). Due to the wide availability of FACS instruments, DE methods can facilitate the broader adoption of drop-based screening. As the applications of FACS-compatible DE droplets are immensely varied and extend well beyond what can be explored here, this chapter should be seen as an introduction to DE microfluidics.