Abstract
Artificial bilayer containing reconstituted ion channels, transporters and pumps serve as a well-defined model system for electrophysiological investigations of membrane protein structure–function relationship. Appropriately constructed microchips containing horizontally oriented bilayers with easy solution access to both sides provide, in addition, the possibility to investigate these model bilayer membranes and the membrane proteins therein with high resolution fluorescence techniques up to the single-molecule level. Here, we describe a bilayer microchip system in which long-term stable horizontal free-standing and hydrogel-supported bilayers can be formed and demonstrate its prospects particularly for single-molecule fluorescence spectroscopy and high resolution fluorescence microscopy in probing the physicochemical properties like phase behavior of the bilayer-forming lipids, as well as in functional studies of membrane proteins.
Highlights
Model membrane systems have proven to be useful tools for probing the molecular properties of lipid bilayers and transmembrane proteins
We describe construction details, required materials, and the procedure to set up a horizontal bilayer microchip system with long-term stable bilayers which optionally can be stabilized by cushions of natural or synthetic hydrogels as bilayer support
The most critical component within the sandwich architecture of the bilayer chip is the material of the septum/foil and the characteristics of the aperture hole which serves as bilayer scaffold
Summary
Model membrane systems have proven to be useful tools for probing the molecular properties of lipid bilayers and transmembrane proteins. We describe a universal usable microchip that allows formation of horizontal free standing and supported bilayer with long lifetime for combined or simultaneous electrical and high resolution fluorescence recordings. Bilayers are “painted” onto micropores (d ≈ 30–200 μm) within a Teflon® septum and the development of a bilayer is determined by measurement of the characteristic bilayer capacitance [16] These model bilayer systems can be used to study molecular properties of single ion channels in a chemically well-defined environment. To form bilayers on solid substrates, various spreading techniques where protein-containing vesicles are directly spread onto a hydrophilic surface were used This technique has been mainly used in combination with optical probes which allow for obtaining information on the physico-chemical properties of the bilayer membrane [17,18].
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