Array of Freestanding Planar Lipid Bilayers for Parallel Optical and Electrical Recordings

Abstract

Here we report on the design and validation of an array platform for parallel high-resolution electrophysiological and optical recordings. The recording chip is based on a SU-8 coated glass slide (ca. 200 μm thickness) containing 4 cavities with individual ring-shaped Ag/AgCl-microelectrodes as well as a common ground electrode. Four suspended lipid bilayers can be self-assembled on the chip surface over the cavities. The electrodes are connected to a multichannel amplifier capable of simultaneous electrical recording with high-bandwidth and low noise (<1pA [email protected]). The ring shape of the electrodes creates an optical window allowing access with a high-NA oil or water-immersion objective on an inverted microscope. This allows for e.g. single-molecule fluorescence measurements from one of the four bilayers using time-correlated-single-photon-counting (TCSPC). Fluorescently labeled alpha-hemolysin for optical and electrical measurements was expressed in-vitro and purified. BODIPY at position 131 was incorporated during the translation in a site-directed manner via stop or four-base codon suppression using pre-charged tRNAs. A series of characterization and validation experiments performed with the system include: (1)Studies of diffusion rates of fluorescently labelled Hemolysin and lipid molecules in the bilayer using fluorescence correlation spectroscopy (FCS); (2)Monitoring of alpha-hemolysin membrane insertion using fluorescence lifetime imaging (FLIM) and (3)Visualization of fusion of labeled proteoliposomes resulting in membrane channel insertion. In the two latter cases, changes in fluorescence intensity were monitored simultaneously with alterations in membrane conductance. This system allows for rapid membrane array formation and by providing ready combination of fluorescence microscopy with high-resolution electrophysiology, enables correlated optical and electrical characterization and monitoring of the interactions of channel forming proteins in artificial lipid bilayers on a single-molecule level. The technology can be further implemented for studies of membrane protein folding and mobility as well as of membrane transport.