Integration of biological ion channels onto optically addressable micro-fluidic electrode arrays for single molecule characterization
The challenge of modeling the organization and function of biological membranes on a solid support has received considerable attention in recent years, primarily driven by potential applications in biosensor design. Affinity-based biosensors show great promise for extremely sensitive detection of BW agents and toxins. Receptor molecules have been successfully incorporated into phospholipid bilayers supported on sensing platforms. However, a collective body of data detailing a mechanistic understanding of membrane processes involved in receptor-substrate interactions and the competition between localized perturbations and delocalized responses resulting in reorganization of transmembrane protein structure, has yet to be produced. This report describes a systematic procedure to develop detailed correlation between (recognition-induced) protein restructuring and function of a ligand gated ion channel by combining single molecule fluorescence spectroscopy and single channel current recordings. This document is divided into three sections: (1) reported are the thermodynamics and diffusion properties of gramicidin using single molecule fluorescence imaging and (2) preliminary work on the 5HT{sub 3} serotonin receptor. Thirdly, we describe the design and fabrication of a miniaturized platform using the concepts of these two technologies (spectroscopic and single channel electrochemical techniques) for single molecule analysis, with a longer term goal of using the physical and electronic changes caused by a specific molecular recognition event as a transduction pathway in affinity based biosensors for biotoxin detection.