Amphiphobic Septa Enhance the Mechanical Stability of Free-Standing Bilayer Lipid Membranes

Artificial bilayer lipid membranes (BLMs) provide well-defined systems for investigating the fundamental properties of membrane proteins, including ion channels, and for screening the effect of drugs that act on them. However, the application of this technique is limited due to the low stability and low reconstitution efficiency of the process. We previously reported on improving the stability of BLM based on the fabrication of microapertures having a tapered edge in SiO₂/Si₃N₄ septa and efficient ion channel incorporation based on vesicle fusion accelerated by a centrifugal force. Although the BLM stability and incorporation probability were dramatically improved when these approaches were used, some BLMs were ruptured when subjected to a centrifugal force. To further improve the BLM stability, we investigated the effect of modifying the surface of the SiO₂/Si₃N₄ septa on the stability of BLM suspended in the septa. The modified surfaces were characterized in terms of hydrophobicity, lipophobicity, and surface roughness. Diffusion coefficients of the lipid monolayers formed on the modified surfaces were also determined. Highly fluidic lipid monolayers were formed on the amphiphobic substrates that had been modified with long-chain perfluorocarbons. Free-standing BLMs formed in amphiphobic septa showed a much higher mechanical stability, including tolerance to water movement and applied centrifugal forces with and without proteoliposomes, than those formed in the septa that had been modified with a short alkyl chain. These results demonstrate that highly stable BLMs are formed when the surface of the septa has amphiphobic properties. Because highly fluidic lipid monolayers that are formed on the septa seamlessly connect with BLMs in a free-standing region, the high fluidity of the lipids contributes to decreasing potential damage to BLMs when mechanical stresses are applied. This approach to improve the BLM stability increases the experimental efficiency of the BLM systems and will contribute to the development of high-throughput platforms for functional assays of ion channel proteins.