Biophysical aspects of agar-gel supported bilayer lipid nembranes: a new method for forming and studying planar bilayer lipid membranes

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

“…Note that, as discussed in Section S4, there are some optical effects due to the finite magnification of the CLSM system, and so to reduce these, data within 25 µm of the pipet on the trans side of the membrane were not analyzed. 25 As expected from visual inspection of the CLSM images, the permeation coefficients of each of the weak acids were found to increase monotonically with acyl tail length and water/octanol partition coefficient, K.…”

“…In order to verify the formation of a bilayer on 20 the pipet tip, i-V curves were recorded between the QRCEs in the pipet and the bulk solution for a gramicidin incorporated bilayer membrane (Supporting Information section S1). In a bilayer membrane, gramicidin acts as an ion-channel allowing transport of monovalent cations (K + in this case), hence decreasing the 25 bilayer membrane resistance. The membrane resistance was found to decrease from ~150 GΩ to ~13 GΩ after the incorporation of gramicidin into the bilayer, which is consistent with decrease in membrane resistances previously reported for supported bilayers confirming the assembly of a bilayer 30 membrane at the pipet tip.…”

“…[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.…”

“…32 Using this 20 technique, it is possible to study not only fluorescent permeants but also weak acids, by means of a pH-sensitive fluorophore, enabling visualization of local pH changes as the molecule permeates either into a liposome 16 or, as originally reported, across a planar lipid bilayer. 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.…”

“…For the measurement of permeation coefficients, the pipets are filled with the weak acid solution which is pH corrected to ensure the protonated form of the weak acid is the dominant species. Since charged species cannot permeate the bilayer to any 25 detectable extent, it is important to ensure the carboxylic acids are in the neutral form. 36 The bulk solution, into which the molecules permeate, contains the pH-sensitive fluorophore fluorescein, and is adjusted to pH 8 so that any permeating weak acid molecules tend to dissociate, changing the pH locally.…”