Microcavity PDMS and gold substrates for supported lipid udbilayers

摘要

Cell membranes surround all living cells and are comprised of a complex matrix of phospholipids and proteins. The proteins embedded in or bound to the exterior of the membrane are responsible for a wide range of processes, for example cell signalling, and transport of material in and out of the cell. Understanding how transmembrane proteins behave within the lipid membrane system will allow for a better understanding of molecular mechanisms of diseases as well as the development more targeted therapeutics. However, due to the complex nature of the cell membrane environment, it is difficult to selectively study single protein species within the whole cell system. This has driven the development of model membrane systems, which allow for the sub-division of these complex systems into simpler forms and allow for the study of individual membrane proteins. Solid supported lipid bilayers have been widely used as model systems, however they have multiple limitations, the most important being the influence of the underlying substrate on the bilayer. This can impede lipid fluidity and is particularly detrimental to mobility of reconstituted proteins as substrate-protein interactions can impede motion and even cause protein to denature. ududThis thesis attempts to address this by developing substrates for studying membrane proteins in a biomimetic environment where such interactions are minimized. The initial substrates, designed for optical measurements, comprise of a microcavity array substrate formed in Polydimethylsiloxane (PDMS). A method for spanning bilayers over these PDMS microcavity arrays was developed and lipid diffusion dynamics over the cavities was assessed using Fluorescence Lifetime Correlation Spectroscopy (FLCS). Importantly, diffusion coefficients for lipids over these cavities are 2 to 3 times faster than on flat PDMS, and are more akin to diffusion rates normally observed in liposomes, indicating that the bilayer is minimally influenced by the underlying substrate.udIn the second part of this thesis an analogous substrate and bilayer deposition method was developed using gold substrates with the objective of using electrochemical methods to address the bilayer or trigger events within the cavity. ududFirstly lipid bilayers are spanned in a similar manner as developed for PDMS and the bilayer modified gold was characterised by electrochemical impedance spectroscopy (EIS). Incorporation of ion transporting molecules into the supported bilayers is also investigated by EIS. Finally a novel means of inducing electrically controlled release of reagent from inside the gold cavities to a lipid bilayer suspended across the cavity was developed using a ferrocene/cyclodextrin complex. To demonstrate this Streptavidin was released to a biotinylated lipid bilayer and its interaction with the bilayer was monitored using electrochemical impedance spectroscopy.