In this study, we utilize a novel microfluidic device to perform simultaneous electrical interrogation of an array of droplet interface bilayers (DIB) that feature asymmetric phospholipid leaflet compositions. While asymmetry is vital to many cellular functions, it is has received very little attention in membrane-based engineered material systems for sensing, energy conversion, or actuation. This gap is due to challenges in constructing and interrogating networks of asymmetric membranes, limiting our understanding of how lipid asymmetry affects membrane active peptides, and vice versa. Our system overcomes these difficulties by enabling asymmetric membrane formation between many pairs of lipid-coated droplets in oil. We demonstrate its use in probing the interactions between alamethicin, a membrane-active peptide that forms voltage-induced ion channels, and asymmetric DPhPC:DOPhPC membranes, a choice that creates an intrinsic intramembrane potential of |137 mV| due to differences in their respective dipole potentials. Our experiments show that adding alamethicin peptides to one side of the membrane causes this inherent membrane potential to decrease over time, and it alters the value of external voltage that must be applied to drive alamethicin insertion for ion channel formation. These effects take place over the course of 1 to 5 hours after membrane formation, and both results are consistent with translocation and mixing of lipids across the leaflets of the membrane.

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