FACS-Sortable Picoreactors for Ultra High-throughput Screening of Catalysts in Biphasic Environments

Biphasic environments can enable successful chemical reactions in systems where any single solvent would be ineffective due to poor substrate solubility or poor catalyst reactivity. The broader application of biphasic reactions to screening small molecule and enzyme catalysts would be greatly enhanced by platforms for high-throughput library screening. Microfluidic droplets use pL of reagents per condition, are compatible with widely available commercial FACS machines, and are capable of processing libraries with millions of candidate catalysts. However, developing FACS-sortable droplet picoreactors compatible with biphasic screening requires optimizing solvent phases and surfactants to produce triple emulsion droplets that are < 50 µm, stable over multi-hour assays, compatible with reaction conditions, and have a shell that encapsulates the biphasic reaction core. Here, we demonstrate the generation of FACS-sortable triple emulsion picoreactors with a fluorocarbon shell and biphasic octanol-in-water core. To accomplish this, we first developed a plate reader assay and screened for surfactants that could stabilize octanol-in-water emulsions for the picoreactor core. We then demonstrated that PDMS droplet generators could produce triple emulsion droplets with the desired architecture and stability (>70% of droplets survived to 24 hours), and that proteins could be expressed via cell-free protein synthesis in the biphasic inner core. Finally, we demonstrated that these triple emulsion picoreactors can be screened, sorted, and recovered using commercial FACS sorters. These triple emulsion picoreactors have potential for screening bead-encoded catalyst libraries, including enzymes. Additionally, our droplet optimization approach is extensible to other water-immiscible organic solvents.