Fluorescence-based analysis of single membrane protein transport kinetics on nanopore arrays

We present a nanopore-based system for optical analysis of membrane transport processes. The system is designed as a general platform for the kinetic study of proteins that facilitate membrane transport. It is especially suited to study membrane proteins involved in non-electrogenic transport. Arrays of 45x45 nanopores (20-120 nm) in Si₃N₄ are sealed by suspended lipid membranes which are formed from lipid vesicles or proteoliposomes in physiological conditions. This implies the potential to study medically relevant transport proteins that are often sensitive to changes in pH, ion strength and the presence of organic solvents. Each nanopore addresses an individual 30 fl-compartment that is used for independent fluorescence readout through the transparent 50 nm Si₃N₄ top layer. Transported substances are monitored by time-lapse fluorescence microscopy. The capacities of the system are exemplified by measurements of transport through single alpha-hemolysin protein complexes. Several types of transported species can be monitored simultaneously by the use of different fluorescent labels.