Single Ion-Channel Recordings Using Glass Nanopore Membranes

Abstract

Protein ion-channel recordings using a glass nanopore (GNP) membrane as the support structure for lipid bilayer membranes are presented. The GNP membrane is composed of a single conical-shaped nanopore embedded in a ∼50 μm-thick glass membrane chemically modified with a 3-cyanopropyldimethylchlorosilane monolayer to produce a surface of intermediate hydrophobicity. This surface modification results in lipid monolayer formation on the glass surface and a lipid bilayer suspended across the small orifice (100−400 nm-radius) of the GNP membrane, while allowing aqueous solutions to fully wet the glass nanopore. The GNP membrane/bilayer structures, which exhibit ohmic seal resistances of ∼70 GΩ and electrical breakdown voltages of ∼0.8 V, are exceptionally stable to mechanical disturbances and have lifetimes of at least 2 weeks. These favorable characteristics result from the very small area of bilayer (10^-10−10^-8 cm²) that is suspended across the GNP membrane orifice. Fluorescence microscopy and vibrational sum frequency spectroscopy demonstrate that a lipid monolayer forms on the 3-cyanopropyl-dimethylchlorosilane modified glass surface with the lipid tails oriented toward the glass. The GNP membrane/bilayer structure is well suited for single ion-channel recordings. Reproducible insertion of the protein ion channel, wild-type α-hemolysin (WTαHL), and stochastic detection of a small molecule, heptakis(6-O-sulfo)-β-cyclodextrin, are demonstrated. In addition, the insertion and removal of WTαHL channels are reproducibly controlled by applying small pressures (−100 to 350 mmHg) across the lipid bilayer. The electrical and mechanical stability of the bilayer, the ease of which bilayer formation is achieved, and the ability to control ion-channel insertion, coupled with the small bilayer capacitance of the GNP membrane-based system, provide a new and nearly optimal system for single ion-channel recordings.