Droplet interface bilayers are versatile model membranes useful for synthetic biology and biosensing; however, to date they have always been confined to fluid reservoirs. Here, we demonstrate that when two or more water droplets collide on an oil-infused substrate, they exhibit noncoalescence due to the formation of a thin oil film that gets squeezed between the droplets from the bottom up. We show that when phospholipids are included in the water droplets, a stable droplet interface bilayer forms between the noncoalescing water droplets. As with traditional oil-submerged droplet interface bilayers, we were able to characterize ion channel transport by incorporating peptides into each droplet. Our findings reveal that droplet interface bilayers can function in ambient environments, which could potentially enable biosensing of airborne matter.
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