Giga-seal solvent-free bilayer lipid membranes: from single nanopores to nanopore arrays

Abstract

A robust platform providing a fluid lipid bilayer is in great demand not only for specific basic research on membrane proteins, but also for related applications. Here we present electrically sealing solvent-free bilayer lipid membranes spanned over arrays of cylindrical nanopores. The nanopores are milled through thin Si₃N₄ diaphragms using a focused ion beam (FIB). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) reveal pores with regular shapes and inter-pore spacing. Nanopore-spanning bilayer lipid membranes (npsBLMs) are formed reproducibly by directed fusion of giant unilamellar vesicles (GUVs) to the pore-containing diaphragms. The arrays of npsBLMs exhibit electrical resistances in the GΩ range, lifetimes of up to several days, and breakdown voltages above 250 mV. Perfusion robustness of the npsBLMs and low aspect ratio of the nanopores allow easy access to both sides of the bilayers. npsBLM conductance in the presence of the pore-forming toxin gramicidin D increases depending on the concentration levels. Peptide-to-lipid molar ratios can reach as high as 1 : 23. Recordings of ionic currents through alamethicin channels are possible with single-channel resolution after dielectric passivation of the substrates. This demonstrates the applicability of the platform to biophysical research of membrane proteins as well as pharmaceutical drug screening assays.