A Single‐Molecule Liposome Assay for Membrane Permeabilization

Abstract Cell membrane disruption is associated with numerous diseases and underlies the activity of various antimicrobial agents. The rapid screening of compounds capable of disrupting or permeabilizing biological membranes is essential to the search for new therapeutic drugs. Here, we present a single‐molecule confocal microscopy assay integrated with fast‐flow microfluidics to study membrane permeabilization in large unilamellar vesicles (LUVs) containing as few as seven dye molecules. This assay eliminates the need for liposome immobilization and achieves detection rates in the range of 1000 vesicles per minute, offering unparalleled sensitivity and detection limits as low as 135 pM, corresponding to just eight permeabilizing molecules per vesicle for active compounds such as ionomycin. It provides a robust platform for investigating membrane‐disrupting agents, including those with antimicrobial properties or implicated in neurodegenerative diseases.