Suspended Bilayers in Shaped Apertures Enable 24-Hour Ion Channel Recordings

Abstract

Electrophysiological characterization of reconstituted ion channels requires a stable and electrically addressable membrane, usually an aperture-suspended lipid bilayer in a septum that separates two aqueous compartments having polarizable electrodes for detection of picoampere channel gating events. Unfortunately, bilayers formed in conventional cylindrical apertures in septum materials such as Teflon and polystyrene are relatively fragile. Apertures with a low thickness/diameter ratio are expected to facilitate the formation of more stable bilayers, but thin septa are difficult to handle and also increase electrical noise in the ion channel current measurements due to an increased background capacitance. In this study we used 3D UV-lithography to create tapered apertures in layers of UV-curable polymers, which have similar electrical properties as Teflon or polystyrene sheets. In these beak- or triangle-shaped apertures, the septum thickness at the edge of the aperture, where the bilayer is formed, is substantially smaller than the bulk septum thickness. We established that >50 μm thick layers are sufficiently robust, following release from the substrate, to be used as septum sheets and also exhibit a low background capacitance. The moderate hydrophobicity of these photoresists was mitigated by vapor deposition of Parylene C, resulting in a contact angle of 110o, thus combining the favourable mechanical and electrical properties of traditional septum materials with lithography-enabled control of aperture diameter and shape. Bilayer formation with both the painting and the Montal-Mueller method was demonstrated for shaped apertures with an inner diameter of 60 μm. Significantly, these bilayers were stable for 24 hours or more at potentials >125 mV, and sustained continuous ion channel activity. This remarkable stability is suitable for automated bilayer array formation for high-throughput ion channel measurements.