Regulated attachment method for reconstituting lipid bilayers of prescribed size within flexible substrates

A new method called the regulated attachment method (RAM) for reproducibly forming lipid bilayers within flexible substrates has been developed that enables precise control over the size of the bilayer. This technique uses a deformable flexible substrate to open and close an aperture that subdivides aqueous volumes submersed in an organic solvent. Phospholipids incorporated as vesicles in the aqueous phase self-assemble at the oil/water interface to form lipid monolayers that encapsulate each aqueous volume. Controlled attachment of opposing lipid monolayers is achieved by regulating the dimensions of the aperture in the substrate that separates the adjacent aqueous volumes. In this manner, the size of a lipid bilayer formed within a flexible substrate is a function of the substrate and aperture dimensions, and not determined by the sizes or shapes of the aqueous volumes. Lipid bilayers formed within the prototype flexible substrate exhibit DC resistances consistently higher than 10 GOmega and can survive 20-30x changes in area without rupture. Furthermore, RAM permits lipid bilayers to be completely unzipped after thinning by applying sufficient force to fully close the dividing aperture and even allows the introduction of species, such as alamethicin channels, into preformed lipid bilayers via controlled injection through an intersecting channel within the substrate. Controlling the size of the interface through indirect interactions with the supporting substrate offers a new platform for assembling durable lipid bilayers. We envision that this technology can be scaled to higher dimensions consisting of multiple apertures required for creating aqueous networks partitioned by functional lipid bilayers and to smaller length scales to produce very small lipid bilayers capable of hosting single proteins.