Regulated Attachment Method for Reconstituting Lipid Bilayers of Prescribed Size within Flexible Substrates

摘要

A new method called the regulated attachment methodn(RAM) for reproducibly forming lipid bilayers withinnflexible substrates has been developed that enablesnprecise control over the size of the bilayer. This techniquenuses a deformable flexible substrate to open and close annaperture that subdivides aqueous volumes submersed innan organic solvent. Phospholipids incorporated as vesiclesnin the aqueous phase self-assemble at the oil/waterninterface to form lipid monolayers that encapsulate eachnaqueous volume. Controlled attachment of opposing lipidnmonolayers is achieved by regulating the dimensions ofnthe aperture in the substrate that separates the adjacentnaqueous volumes. In this manner, the size of a lipidnbilayer formed within a flexible substrate is a function ofnthe substrate and aperture dimensions, and not determinednby the sizes or shapes of the aqueous volumes.nLipid bilayers formed within the prototype flexible substratenexhibit DC resistances consistently higher than 10nGΩ and can survive 20-30× changes in area withoutnrupture. Furthermore, RAM permits lipid bilayers to bencompletely unzipped after thinning by applying sufficientnforce to fully close the dividing aperture and even allowsnthe introduction of species, such as alamethicin channels,ninto preformed lipid bilayers via controlled injectionnthrough an intersecting channel within the substrate.nControlling the size of the interface through indirectninteractions with the supporting substrate offers a newnplatform for assembling durable lipid bilayers. We envisionnthat this technology can be scaled to higher dimensionsnconsisting of multiple apertures required for creatingnaqueous networks partitioned by functional lipidnbilayers and to smaller length scales to produce very smallnlipid bilayers capable of hosting single proteins.