FACS-Sortable Triple Emulsion Picoreactors for Screening Reactions in Biphasic Environments

Biphasic environments can enable successful chemical reactions where any single solvent results in poor substrate solubility or poor catalyst reactivity. For screening biphasic reactions at high-throughput, a platform based on microfluidic double emulsions could use widely available FACS (Fluorescence Activated Cell Sorting) machines to screen millions of picoliter reactors in a few hours. However, encapsulating biphasic reactions within double emulsions to form FACS-sortable droplet picoreactors requires optimized solvent phases and surfactants to produce triple emulsion droplets that are stable over multi-hour assays and compatible with desired reaction conditions. This work demonstrates such FACS-sortable triple emulsion picoreactors with a fluorocarbon shell and biphasic octanol-in-water core. First, surfactants were screened to stabilize octanol-in-water emulsions for the picoreactor core. With these optimized conditions, stable triple emulsion picoreactors were produced (>70% of droplets survived to 24 hours), and the ability to produce protein in the biphasic core was demonstrated via cell-free protein synthesis. Finally, triple emulsion picoreactors were sorted based on fluorescence using commercial FACS sorters at >100 Hz with 75-80% of droplets recovered. These triple emulsion picoreactors have potential for future screening bead-encoded catalyst libraries, including enzymes such as lipases for biofuel production.