Micropatterning of Phase-Segregated Supported Lipid Bilayers and Binary Lipid Phases through Polymer Stencil Lift-Off

Supported lipid bilayers (SLBs) provide an excellent model system for studying structural and functional characteristics of biomembranes. Patterning model membranes on solid supports has elicited much interest because lipid bilayer arrays at cellular or subcellular scales provide attractive platforms for reconstituting tissue-like conditions for cell culture, and for creating simplified physiological environments to study biological processes. Phase-segregated SLB patterns can be especially useful for such studies, as the selective functionalization of the lipid phases with different lipids, receptors, or proteins can be achieved to mimic the key features of plasma membrane. However, it remains challenging to pattern phase-segregated lipid bilayers and to spatially control the lipid phases at the micron scale. Current methods to achieve this involve multiple surface modification and patterning steps, elaborate techniques such as microfluidic, microcontact printing, or electrochemical control, among others. To overcome the complexity in producing phase-segregated patterns, we have developed simple and rapid strategies to pattern SLBs with phase separation utilizing the polymer stencil lift-off (PSLO) technique. PSLO is a powerful technique for SLB patterning, since it allows the faithful pattern transfer of micron-sized lipid domains onto solid surfaces under aqueous conditions, which eliminates the need for controlled humidity and reduces the risk of bilayer disruption through drying. By integrating postetching substrate cleaning and a blocking treatment, well-defined homogeneous and phase-segregated SLB patterns were achieved with lipid mobility that matches that of SLBs formed on clean SiO₂ wafer substrates. A two-step incubation method was also developed for patterning binary lipid phases, which allowed precise control of their position and geometries. The created phase-segregated SLB patterns were used to study lipid phase behavior within confined areas, and quantitative analysis showed that smaller pattern sizes resulted in smaller gel phase domains, which also covered a smaller fraction of the total patterned SLB area. This was attributed to the decreased mobility of the bottom leaflet of the SLB, which lies in close proximity to the substrate, and the resulting hindered exchange of lipid molecules between the bottom and upper leaflets through the SLB boundary. By further integration with functional groups, the phase-segregated lipid bilayer patterns might find relevant application in tissue engineering, biophysical studies of biomolecular and cellular interactions, and biosensing platforms.