Development of a tethered biomembrane biosensing platform for the incorporation of ion channels

摘要

Solid-supported membranes present a biomimetic platform that can be adapted for bio-physical, biochemical and electrophysiological studies. In addition to this they offer an environment to host membrane proteins for the purposes of biosensing. This thesis examines the use of such a system and the possibilities it presents for the studies of ion channels and their potential applications in biosensing. Electrochemical impedance spectroscopy (EIS) is a powerful technique in the study of solid-supported membranes giving access to capacitance and resistance data, and as such was employed as the main method of characterisation. Electrodes were designed for this purpose in conjunction with Philips Research, and the suitability of the surface for the formation of insulating tethered bilayer lipid membranes (tBLMs). The development of these electrodes led to the incorporation of a SiO2 insulating layer, however its addition resulted in diculties with the formation of self-assembled monolayers (SAMs). However, further refinement of the manufacturing process should resolve these issues. Despite these diffculties, studies were performed using first generation electrodes (P1). Two ionophores, valinomycin and gramicidin, were employed in the characterisation of the ion transport properties of the tBLM system. These studies yielded important information about the structure of the tBLM system under investigation, as well as the ways ion transport can be presented in EIS. Using the work on ionophores as a foundation, an investigation into the incorporation and characterisation of ion channels in tBLM was conducted. Three channels were studied - a ligand-gated eukaryotic Ca2+-permeant channel (TRPC5), a voltage-gated prokaryotic Na+-permeant channel (NavCbt), and a pH-gated K+-permeant channel (KcsA). The success of these studies varied, but provided strong evidence that ion channel incorporation is possible. Further investigation of channel function in the tBLM is required as measured activity is lower than that suggested by literature.