Optical and Electrical Characterization of Nanopores in Freestanding Lipid Bilayers Formed on Microelectrode Cavity Arrays.

Single-channel electrical recording remains a fundamental technique for investigating biological nanopores embedded in lipid bilayer membranes. However, this method cannot reveal information from nanopores that fail to induce transmembrane ionic currents. Here, we present procedures that integrate a microelectrode cavity array with a single-molecule fluorescence microscope to probe labeled nanopores on freestanding lipid bilayers with light and electricity at the same time. Because oligomeric prepores are frequently employed in nanopore sensing applications, we explore the conversion of prepores into fully conducting pores. The protocol includes modified instructions for preparing and labeling alpha-hemolysin (αHL) heptamers, as well as a description of the instrument configuration and associated data analysis tools. We employ an Excel template (available as a supplemental file) to calculate a membrane-bound, but uninserted, heptameric nanopore diffusion coefficient of 8.1 ± 0.3 µm² s-¹ and an open-to-closed nanopore ratio of 10-³ to 10-⁴. The methods described here provide a general framework for extending orthogonal single-molecule optical and electrical measurements in microelectrode cavity arrays to a broad range of biological nanopores.