A Multiplexed Cell‐Free Assay to Screen for Antimicrobial Peptides in Double Emulsion Droplets

The global surge in bacterial resistance against traditional antibiotics triggered intensive research for novel compounds, with antimicrobial peptides (AMPs) identified as a promising candidate. Automated methods to systematically generate and screen AMPs according to their membrane preference, however, are still lacking. We introduce a novel microfluidic system for the simultaneous cell‐free production and screening of AMPs for their membrane specificity. On our device, AMPs are cell‐free produced within water‐in‐oil‐in‐water double emulsion droplets, generated at high frequency. Within each droplet, the peptides can interact with different classes of co‐encapsulated liposomes, generating a membrane‐specific fluorescent signal. The double emulsions can be incubated and observed in a hydrodynamic trapping array or analyzed via flow cytometry. Our approach provides a valuable tool for the discovery and development of membrane‐active antimicrobials.