A biological membrane not only forms a protective outer boundary for cells and organelles but also houses ion channels that are attractive drug targets. The characterisation of membrane-embedded ion channels hence is of prime importance, but in vivo studies have been hindered by the complexity of the natural membranes. Lipid bilayers suspended in apertures have provided a simple and controlled model membrane system for ion channel studies, but short lifetimes and poor mechanical stability of suspended bilayers have limited the experimental throughput of bilayer electrophysiology experiments. Although suspended bilayers are more stable when smaller apertures are used, ion channel incorporation through vesicle fusion with the suspended bilayer becomes increasingly difficult. In this project, in an alternative bilayer stabilization approach, shaped apertures with tapered sidewalls have been fabricated with serial two-photon laser lithography and high-throughput grayscale lithography in photoresist. Bilayers formed at the 2µm thin tip of the shaped apertures, either with the painting or the folding method, displayed drastically increased lifetimes, typically >20 hours, and mechanical stability, being able to withstand extensive perturbation of the buffer solution, as compared to the control shapes. Single-channel electrical recordings of the peptide Alamethicin, water soluble protein α-Hemolysin and of the proteoliposome-delivered potassium and sodium channels KcsA, hERG and NavSp pore domains demonstrate channel conductance with low noise, made possible by the small capacitance of the 50µm thick resist septum, which is only thinned around the aperture, and unimpeded proteoliposome fusion, enabled by the large aperture diameter of 80µm. Optically accessible horizontal bilayers in shaped apertures were developed to visualize suspended bilayers and incorporated ion channels. It is anticipated that these shaped apertures with micrometer edge thickness can substantially enhance the throughput of channel characterisation by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:628778 |
| Date | January 2014 |
| Creators | Kalsi, Sumit |
| Contributors | De Planque, Maurits |
| Publisher | University of Southampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://eprints.soton.ac.uk/368792/ |
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