Peter Osman

A tethered bilayer sensor containing alamethicin channels and its detection of amiloride based inhibitors.

Alamethicin, a small transmembrane peptide, inserts into a tethered bilayer membrane (tBLM) to form ion channels, which we have investigated using electrical impedance spectroscopy. The number of channels formed is dependent on the incubation time, concentration of the alamethicin and the application of DC voltage. The properties of the ion channels when formed in tethered bilayers are similar to those for such channels assembled into black lipid membranes (BLMs). Furthermore, amiloride and certain analogs can inhibit the channel pores, formed in the tBLMs. The potency and concentration of the inhibitors can be determined by measuring the change of impedance. Our work illustrates the possibility of using a synthetic tBLM for the study of small peptide voltage dependent ion channels. A potential application of such a device is as a screening tool in drug discovery processes.

The effect of pulsed electric fields on the phosphorus-31 spectra of lipid bilayers

A technique is described for measuring the effect of electric fields on the conformation of lipid bilayer membranes by solid state nuclear magnetic resonance. An apparatus was devised to obtain spectra from samples of aligned phospholipid dispersions at varying electric field strengths up to 100 MV/m. Measurements were carried out on membranes made from dioleoylphosphatidylethanolamine and dioleoylphosphatidylcholine, which resulted in electric field induced phase changes. Calibration experiments were performed using bilayers formed from dimyristoylphosphatidylcholine with glycerol and with a nematic liquid crystal. An electric field induced change, from L alpha to HII, was also seen in a dimyristoylphosphatidylcholine/alamethicin bilayer.

AMBRI biosensor: stabilizing artificial membranes and receptor attachment

The AMBRI Ion Channel Switch biosensor is a novel scanning technology with broad application in a variety of fields. The technology is based on an artificial bilayer membrane attached to gold through hydrophilic tethers. The lamellar bilayer membrane possesses electrical characteristics similar to black (bilayer) lipid membranes being sealed with a capacitance of approximately 0.6 (mu) F/cm2, is fluid, and is stable to a variety of media including plasma and whole blood and to challenges with solvent solutions. Receptors/antibodies can be attached to the membrane through biotin-streptavidin linkages, and use of caged biotin species allows optical patterning of the membrane surface.

Gated ion channel biosensor: a functioning nanomachine

Biosensors combine a biological recognition mechanisms with a physical transduction technique. In nature, the transduction mechanism for high sensitivity molecular detection is modulation of cell membrane ionic conductivity, through specific ligand - receptor binding induced switching of ion channels. This effects an inherent signal amplification of 6-8 orders of magnitude, corresponding to the total ion flow arising from the single channel gating event. Here we describe the first reduction of this principle to a practical sensing device, which is a planar impedance element composed of a macroscopically supported synthetic bilayer membrane incorporating ion channels. The membrane and ionic reservoir are covalently attached to an evaporated gold surface. The channels have specific receptor groups attached which permit switching of the channels by analyte binding to the receptors. The device may then be made specific for the detection of a very wide range of analytes, including proteins, drugs, hormones, antibodies, DNA, etc., currently in the 10-7-10-12 M range. It also lends itself readily to microelectronic fabrication, the optimum sensitivity range of the device may be tuned over several orders of magnitude.

The phosphorus-31 spectra of dielectrophoretically reoriented tubules in the HII phase of DOPE

31P electric field nuclear magnetic resonance measurements are described which assess the effect of electric field on the orientation of tubules comprising the HII phase of dioeleoylphosphatidylethanolamine. A model, based on dielectrophoretic effects, was used to predict that a field of 4 MV/m would change the orientation of the lipid tubules in a HII phase. The excitation pulse was biphasic to help discriminate electric field interactions with free ions or permanent dipoles from interactions with induced dipoles, as well as to control the problems of ohmic heating, electrolysis and polarisation associated with dc or unbalanced ac excitation voltages. Spectra consistent with irreversible electrorotation and with reversible and transient electrorotation were observed. No response to the electric field was seen in certain cases. The conditions for irreversible and reversible electrorotation and failure to rotate have been tabulated and are discussed. Finally, some simple models are considered, in order to calculate the energies involved, if the observed NMR spectra are interpreted as arising from lipid HII phase reorientations.