Rapid and Controlled Single-Nanopore Insertion via Hydrogel-Confined Localized Delivery

Abstract Controlled insertion of a single protein pore, also referred to as a nanopore, into a free-standing lipid bilayer underpins high-resolution single-channel electrophysiology. Hitherto, conventional bulk reconstitution is stochastic, slow, and prone to multiple insertions. To overcome this, we adapted a hydrogel-confined delivery strategy in which an agarose-filled glass capillary delivers nanopores directly to the bilayer. An optimal window of agarose concentration, aerolysin concentration, and membrane potential raised the single-pore yield to >70% and shortened the waiting time to the first insertion from tens of minutes to a few minutes, without altering native pore conductance. The approach generalizes across the β-barrel toxin aerolysin, the mitochondrial TOM core complex, and a DNA nanopore, making nanopore insertion rapid, controlled, and reproducible.