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

Abstract

We have developed a novel workflow (sdDE-FACS, s̲ingle d̲roplet D̲ouble E̲mulsion FACS) that allows robust production, screening, and sorting of single double emulsion droplets with complete nucleic acid recovery.

Droplet flow cytometry for single-cell analysis

“…[45][46][47] By selectively controlling the wettability of microchannels, microuidic devices enabled a hierarchical generation of monodisperse W/O/W DEs encapsulated with individual cells. [47][48][49][50] There are two main approaches to generate W/O/W DEs: the two-step batch method and the one-step continuous ow method using microuidic devices. 51 In the rst approach, water phase and oil phase are mixed by stirring to form W/O droplets, which can then be encapsulated by another water phase via constantly adding more water under the stirring condition.…”

“…In the DE, the aqueous internal phase can be composed of a reaction mixture (e.g., substrates, buffers, uorescent dyes and antibodies), cell suspensions or a gene library, providing a suitable environment for different cellular assays. 49,53,54 The oil shell is bioinert and non-adhesive to hydrophilic activities and cells. The usage of uorinated oil (e.g., HFE-7500 and FC-40) acting as a selective barrier can reduce hydrophilic mass transfer and communication between DEs.…”

“…The usage of uorinated oil (e.g., HFE-7500 and FC-40) acting as a selective barrier can reduce hydrophilic mass transfer and communication between DEs. 49,55,56 The aqueous external phase allows the suspension of DEs in nutrient medium for long-term cell culture and the compatibility with commercial cytometers for high-throughput assays 18,49,57,58 (with the sorting speed >10 7 events per hour 31,32,56 ).…”

“…Moreover, the isolated cells from heterogeneous populations allow downstream bioinformatics analysis (e.g., tandem genomic, epigenomic and transcriptomic analyses) of the same target, revealing the biological functions of massive genetic pathways. [48][49][50]…”

Counting of Enzymatically Amplified Affinity Reactions in Hydrogel Particle-Templated Drops

“…These cytometers are often equipped with more sensitive detectors like photomultiplier tubes that will also help further distinguish positive and negative signals. While others have analyzed double emulsions in flow cytometers 48,49 since dropicles are supported by a hydrogel particle core they may be more likely to sustain fluid shear stresses in a flow cytometer that can operate with an oil continuous phase, for example the On-Chip Sort 50 . Ultimately, we envision the hydrogel particles can be produced at Please do not adjust margins Please do not adjust margins centralized sites and conveniently distributed to enable highly sensitive immunoassays for various biomarkers.…”