Self-assembly of supported lipid multi-bilayers investigated by time-resolved X-ray diffraction

Supported lipid multi-bilayers or bilayer stacks are an important model membrane system, particularly suitable for surface-sensitive characterization methods like X-ray and neutron diffraction. Spreading organic solution (sOS) is one of the most widely used protocols for the preparation of lipid multi-bilayers. Despite its great popularity, the self-assembly mechanism of the bilayers is not yet fully elucidated, limiting further improvements of this protocol. In order to solve this problem, we investigated the formation process of lipid bilayers in the sOS protocol, using in-situ time-resolved X-ray diffraction, complemented by X-ray reflectivity and molecular dynamics simulation. Results reveal a simultaneous self-assembly scheme for both cholesterol-free and cholesterol-containing bilayers, with one bilayer phase forming at the surface and the other forming in the solution. The solution phase gradually transforms into the surface phase, yielding clean single phase in the end.