Measuring Thousands of Single-Vesicle Leakage Events Reveals the Mode of Action of Antimicrobial Peptides

[Image: see text] Host defense or antimicrobial peptides hold promise for providing new pipelines of effective antimicrobial agents. Their activity quantified against model phospholipid membranes is fundamental to a detailed understanding of their structure–activity relationships. However, classical characterization assays often lack the ability to achieve this insight. Leveraging a highly parallelized microfluidic platform for trapping and studying thousands of giant unilamellar vesicles, we conducted quantitative long-term microscopy studies to monitor the membrane-disruptive activity of archetypal antimicrobial peptides with a high spatiotemporal resolution. We described the modes of action of these peptides via measurements of the disruption of the vesicle population under the conditions of continuous peptide dosing using a range of concentrations and related the observed modes to the molecular activity mechanisms of these peptides. The study offers an effective approach for characterizing membrane-targeting antimicrobial agents in a standardized manner and for assigning specific modes of action to the corresponding antimicrobial mechanisms.