Imaging functional bacterial pore-forming toxins in tethered bilayer lipid membranes using scanning electrochemical microscopy

This study demonstrates the straightforward application of scanning electrochemical microscopy (SECM) for characterizing pneumolysin-induced pores in tethered bilayer lipid membranes (tBLMs). Carbon-based nanoelectrodes with a tip radius of approximately 20 nm produced distinct feedback responses during approach curves to the sample. A positive feedback response was observed when approaching the self-assembled monolayer, while the few nanometers thick tBLMs exhibited characteristics of insulating layers, yielding a negative feedback response. Based on the computational calculations, the reconstitution of functional pneumolysin was further confirmed through electrochemical impedance spectroscopy, with a concentration of 5 nM pneumolysin resulting in an average pore density of 0.64 μm⁻². Finally, we demonstrated the practical utility of SECM for visualizing pneumolysin pores within the tBLM system. These experiments highlight the versatility and cost-effectiveness of electrochemical techniques for investigating membrane integrity, toxin activity, and biomolecular interactions at the nanoscale.