Supported membrane nanodevices.

Abstract

Supported membrane nanodevices are based on natural or artificial ion channels embedded in a lipid membrane deposited on a chip wafer. Membrane conductance is modulated by biorecognitive events, with the use of intrinsic binding sites of the ion channel or via artificial sites fused to the channel protein. Artificial ion gates are constructed by coupling a specific ligand for the analyte near the channel entrance or a site important to triggering channel conformation. The binding event leads to the closure of the ion channel or induces a conformational change of the channel, reducing the ion flux. The signal transduced from the device is the decrease in the ion flux-induced electron current at a silver-silver chloride electrode at ultimate single-molecule sensitivity. Among the natural ion channels, gramicidin A, a transport antibiotic, was found to be most suitable, and thus was used by AMBRI, Australia, to set up prototypes of membrane biochips, using self-association of the dimer. Covalent dimerization-based devices, developed by the Vienna group, make use of the down-regulation of the permanently open membrane-spanning bisgramicidine ion channel. The reactive group at the C-terminus, a hydroxy group, allows precise coupling of the analyte-binding moiety in gramicidin as well as bisgramicidin. The device is set up with bilayer membranes deposited on apertures of a hydrophobic frame structure produced via microlithography, facing an aqueous or hydro-gel micro-environment on both sides, constructing black lipid membranes or patch-clamp devices "on chip." The setup of the device needs gel membrane supports that allow membrane formation and contribute to the stability of the bilayer by exposure of functional groups that promote electrostatic interaction and formation of hydrogen bridges and enable the introduction of covalent spacers and anchors. Photo-cross-linked polyvinylpyrrolidone and polyacrylamide, electropolymerized polydiaminobenzene and coated agarose, as well as various chemical modifications of these polymers, were employed as membrane supports. With optimized assemblies, the membrane support did allow the formation of stable bilayer membranes, proved by "gigaseal" (electrical sealing with giga-ohm resistance) to be free of any point defects in the lipid assembly. Supports with and without hydrophilic and hydrophobic anchors were studied with reference to promoting the formation of a self-assembled membrane, to their electric resistance, and to the capability to insert functional ionophores. All components, including novel chemically engineered ion channels, novel amphiphilic lipids, a microlithographically designed chip, isolating polymer frames, and a hydrogel membrane support, are combined in the new bionanodevice. Sensitivity and specificity were proved, for example, with the use of an antibody-antigen couple down-regulating the ion flux through the membrane channel. Single ion channels incorporated in the supported lipid bilayer gave stable signals at an operational stability of several hours, which is already sufficient to test and screen for membrane receptors but still insufficient to use this device as a sensor for off-site application. Further optimization to increase operational and storage stability is done by a number of groups to allow a broad application of these devices.