Nanopore unitary permeability measured by electrochemical and optical single transporter recording

Abstract

For the analysis of membrane transport processes two single molecule methods are available that differ profoundly in data acquisition principle, achievable information, and application range: the widely employed electrical single channel recording and the more recently established optical single transporter recording. In this study dense arrays of microscopic horizontal bilayer membranes between 0.8 mu m and 50 mu m in diameter were created in transparent foils containing either microholes or microcavities. Prototypic protein nanopores were formed in bilayer membranes by addition of Staphylococcus aureus alpha-hemolysin (alpha-HL). Microhole arrays were used to monitor the formation of bilayer membranes and single alpha-HL pores by confocal microscopy and electrical recording. Microcavity arrays were used to characterize the formation of bilayer membranes and the flux of fluorescent substrates and inorganic ions through single transporters by confocal microscopy. Thus, the unitary permeability of the alpha-HL pore was determined for calcein and Ca2+ ions. The study paves the way for an amalgamation of electrical and optical single transporter recording. Electro-optical single transporter recording could provide so far unresolved kinetic data of a large number of cellular transporters, leading to an extension of the nanopore sensor approach to the single molecule analysis of peptide transport by translocases.