Droplet Contact Method (DCM) for Stable and Reproducible Lipid Bilayer Formation and its Application to Nanopore...

Droplet contact bilayers provide a versatile platform for reconstituting and measuring the activity of membrane proteins at the single-molecule level. This method enables stable lipid bilayer formation between aqueous droplets and allows the insertion of protein nanopores for electrophysiological analysis. Here, we describe the fabrication of a poly(methyl methacrylate)-based microdevice with a parylene membrane, the formation of bilayers by the droplet contact method, and the reconstitution of both protein nanopores and de novo designed peptide nanopores. We further outline procedures for detecting peptide fragments and performing nanopore-based sequencing using this system. This approach offers reproducible bilayer assembly and quantitative analysis of ion channel activity, supporting the development of nanopore technologies for diverse molecular sensing and sequencing applications.