Hydrophilic/Omniphobic Droplet Arrays for High-throughput and Quantitative Enzymology.

Engineered enzymes with enhanced or novel functions are specific catalysts with wide-ranging applications in industry and medicine. Here, we introduce droplet array microfluidic enzyme kinetics (DA-MEK), a high-throughput enzyme screening platform that combines water-in-air droplet microarrays formed on patterned superhydrophilic/omniphobic surfaces with cell-free protein synthesis to enable cost-effective expression and quantitative kinetic characterization of enzyme variants. By printing DNA templates encoding enzyme variants onto hydrophilic spots, stamping slides to add cell-free expression mix, and imaging the resulting arrays, we demonstrate reproducible expression of enzyme variants across hundreds of microwells per slide, with line of sight toward replicating this across larger libraries. By specifically patterning slides with antibodies, we further demonstrate parallel immobilization, purification, and iterative characterization of the expressed variants. Subsequent stamping of fluorogenic substrates and time-lapse imaging allows determination of Michaelis-Menten parameters for each variant, with measured catalytic efficiencies spanning 5 orders of magnitude and agreeing well with values obtained via traditional microtiter plate assays. DA-MEK consumes orders of magnitude less reagents than plate-based assays, while providing accurate and detailed kinetic information for both beneficial and deleterious mutations. In future work, we anticipate that DA-MEK will provide a powerful and versatile platform to accelerate enzyme engineering and enable screening of large variant libraries under diverse conditions.