Adsorption Kinetics Dictate Mono layer Self-Assembly for Both Lipid-In and Lipid-Out Approaches to Droplet Interface Bilayer Formation

The droplet interface bilayer (DIB)a method to assemble planar lipid bilayer membranes between lipid-coated aqueous dropletshas gained popularity among researchers in many fields. Well-packed lipid monolayer on aqueous dropletoil interfaces is a prerequisite for successfully assembling DIBs. Such monolayers can be achieved by two different techniques: lipid-in, in which phospholipids in the form of liposomes are placed in water, and lipid-out, in which phospholipids are placed in oil as inverse micelles. While both approaches are capable of monolayer assembly needed for bilayer formation, droplet pairs assembled with these two techniques require significantly different incubation periods and exhibit different success rates for bilayer formation. In this study, we combine experimental interfacial tension measurements with molecular dynamics simulations of phospholipids (DPhPC and DOPC) assembled from water and oil origins to understand the differences in kinetics of monolayer formation. With the results from simulations and by using a simplified model to analyze dynamic interfacial tensions, we conclude that, at high lipid concentrations common to DIBs, monolayer formation is simple adsorption controlled for lipid-in technique, whereas it is predominantly adsorption-barrier controlled for the lipid-out technique due to the interaction of interface-bound lipids with lipid structures in the subsurface. The adsorption barrier established in lipid-out technique leads to a prolonged incubation time and lower bilayer formation success rate, proving a good correlation between interfacial tension measurements and bilayer formation. We also clarify that advective flow expedites monolayer formation and improves bilayer formation success rate by disrupting lipid structures, rather than enhancing diffusion, in the subsurface and at the interface for lipid-out technique. Additionally, electrical properties of DIBs formed with varying lipid placement and type are characterized.