Quartz Nanopore Membranes for Suspended Bilayer Ion Channel Recordings

Abstract

A method is described for fabricating 25-75 mum thick fused quartz membranes containing a single conical shaped nanopore (orifice radius ranging from 10 to 1000 nm). The quartz nanopore membrane (QNM) provides an excellent solid support structure for lipid bilayers in ion channel recordings due to the large electrical resistivity of fused quartz. Electrical measurements demonstrate that the leakage current through 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers suspended across a 500-1000 nm radius QNM orifice is immeasurably small, corresponding to a bilayer resistance greater than 10(12) ohms. Translocation of single-stranded DNA oligomers (poly dA 50-mer and poly dA 20-mer) through a protein ion channel (alpha-hemolysin) reconstituted in a DPhPC bilayer suspended across the QNM orifice is demonstrated.

Controllable Shrinking and Shaping of Glass Nanocapillaries under Electron Irradiation

“…Quartz nanopore membrane (QNM) require the incorporation of a sharp polished Pt wire into a glass membrane, which is etched away while monitoring the conductance of the opening nanopore. 22 A similar technique is used by Gao et al who corrode a nanopore into a sealed capillary pipette using an HF solution. 9 Again the pore opening is controlled nonoptically by measuring the conductance.…”

A new approach for the fabrication of microscale lipid bilayers at glass pipets: application to quantitative passive permeation visualization

“…[1][2][3][4][5][6][7][8] Passive permeation across the cell membrane is of particular interest since most drug compounds are transported via this mechanism, and, as such, planar lipid bilayers are used in 25 both high throughput drug screening [9][10][11] and more detailed studies to identify trends in permeation rates between molecules, 2,3,5,6 in addition to studies of charge transfer across the membrane. 12,13 Permeation coefficients of small molecules have commonly been analyzed using Overton's rule, 14 which considers the permeation 30 coefficient, P, of a molecule transported across a membrane between two aqueous solutions to be proportional to the product of the partition coefficient, K, and diffusion coefficient, D, of the molecule in the membrane. 15 There has, however, been some debate as to whether this simplistic view of the permeation 35 process is sufficient to describe the permeation of all small molecules, since it does not take into account the amphipathic nature of the phospholipids in a lipid bilayer.…”

“…28 The use 10 of small apertures, over which a bilayer is suspended, can also improve electrical and mechanical stability. 29,30 Liposomes have also been used, which do not contain any residual solvent molecules in the interior of the membrane, and are typically stable for considerably longer periods than planar 15 lipid bilayers. 31 However, since the interior of the liposome is inaccessible for sampling, measurement of permeation rates can be difficult.…”

“…1 25 Here, we report on the use of dual-barrel theta capillaries for the formation of stable, solvent-free, suspended BLMs. This could be viewed as a modification of the tip-dip method reported previously, 22, 33, 34 but herein, the monolayer is formed directly from a lipid solution rather than by moving the pipet out and into 30 a solution that contains a lipid monolayer at the air-water interface. Long lasting bilayers that are formed simply, quickly and reproducibly with high success rate (nearly 100%) (exhibit resistances up to 600 GΩ).…”

“…A buffered electrolyte 25 solution containing 0.1 M KCl, 50 µM HEPES (Fluka) and 5 µM of the sodium salt of fluorescein was also prepared and adjusted to pH 8 using NaOH (Fisher Scientific). Due to the relatively high pH in the bulk solution, the speciation of HEPES was such that the concentration of the neutral molecule was less than 0.1 30 nM and so did not need to be considered as a permeating species. For bilayer formation, DPPC or soy phosphatidylcholine (95%) (Avanti Polar Lipids) were dissolved in chloroform (BDH) to a concentration of 1 mg/ml.…”