KSEF RDE: Development of plasmonic nanostructures for simultaneous single channel electrophysiology and single molecule fluorescence of ion channels in live cells

Ion channels play a crucial role in neuronal function by converting chemical signals, in the form of ligand binding, to the electrical signals that pass between neurons. A series of complex biological processes, beginning with ligand recognition, orchestrate the spatial rearrangement and activation of the channel. Despite their importance as drug targets, little is known about the fundamental mechanisms that regulate ion channel activity including how agonist binding initiates the conformational changes that drive signal transduction. Studies have primarily been limited by the absence of techniques capable of resolving functional and conformational dynamics of individual proteins in live cells. We propose to develop a novel method that will allow us to simultaneously study the functional and structural dynamics of individual receptors within complex biological systems. We will fabricate nanostructured plasmonic devices containing apertures (150-200 nm) capable of isolating small portions of the cell's plasma membrane containing single ion channels. Utilizing these devices to create nanoscale observation volumes will allow us to simultaneously measure single molecule fluorescence (SMF) and single channel electrophysiology (SCE) of membrane proteins (ion channels, membrane receptors, bacterial toxins) on live cell membranes. Recently we have demonstrated that nanoscale wells in metal films can be used for low background single molecule fluorescence measurements at high molecular concentrations (mM or uM) to explore molecular mechanisms inside a cell. To integrate electrophysiological measurements, we will design devices that allow an electrode to be placed within a thin layer of agarose film incorporated below the fused silica substrate supporting the thin (150 nm) metal film. While this is a high-risk proposal, we believe that it provides the basis for a novel and innovative methodology capable of resolving ion channel dynamics and drug-receptor interactions. These advancements would provide critical insight necessary for the development of new and more effective therapeutics.