Continuous FACS Sorting of Double Emulsion Picoreactors with a 3D-Printed Vertical Mixer

Area of Science:

• Biotechnology
• Biochemistry
• Synthetic Biology

Background:

• Microfluidic picoreactors enable high-throughput screening for enzyme directed evolution.
• Double emulsion picoreactors (DEs) simplify sorting using commercial fluorescence-activated cell sorting (FACS).
• Previous DE methods faced challenges with loading variability and low yields.

Purpose of the Study:

• To optimize double emulsion (DE) stability and loading for efficient FACS sorting.
• To enhance the accessibility and throughput of directed evolution using microfluidic picoreactors.
• To overcome barriers in DE loading rates and yields for improved enzyme discovery.

Main Methods:

• Optimized surfactant formulations to improve DE stability.
• Developed a 3D-printed corkscrew mixer for continuous sample line loading.
• Utilized commercial FACS for sorting and analyzed sorting accuracy and recovery rates.

Main Results:

• Achieved stable DEs with enhanced loading efficiency.
• Analyzed 1.17 million DEs across four 10-minute sorting rounds at high frequencies (up to 480 Hz).
• Demonstrated high sorting accuracy (89 ± 1%) and recovery (78.0 ± 0.9%) in mock sorts.

Conclusions:

• Optimized DE loading conditions significantly improve FACS-sortable picoreactor throughput and ease of use.
• Enhanced DE methods expand accessibility of directed evolution for creating high-activity enzymes.
• This approach facilitates controlled in vitro evolution for enzyme engineering applications.