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 mu m-2. Finally, we demonstrated the practical utility of SECM for visualizing pneumolysin pores within the tBLM system. These experiments highlight the versatility and costeffectiveness of electrochemical techniques for investigating membrane integrity, toxin activity, and biomolecular interactions at the nanoscale.