Regeneration of tethered bilayer lipid membrane biosensors for repetitive use in toxin detection

The development of reusable biosensors based on tethered bilayer lipid membranes (tBLMs) is crucial to achieve cost-effective and sustainable pathogen detection. In this study, we investigated the regeneration of tBLMs assembled on fluorine-doped tin oxide (FTO) substrates using organic silane-based molecular anchors and a lipid mixture of dioleoylphosphatidylcholine and cholesterol. The sensors were exposed to α-hemolysin (αHL), a pore-forming toxin from Staphylococcus aureus, and regenerated by a two-step bilayer removal protocol. Electrochemical impedance spectroscopy (EIS) was used to assess the performance of the tBLM before and after each regeneration cycle. A reproducible but systematic shift in the EIS spectra was observed with each cycle, raising questions about the physical origin of this variability. Using an inverse modeling approach to EIS data, we determined that the observed spectral changes were not due to increasing membrane defect density, but rather to a significant decrease in the resistance of the submembrane layer separating the bilayer from the solid substrate, likely due to increased hydration of this layer. This finding was supported by stable mean defect densities and changes in membrane and Helmholtz capacitance. Our results demonstrate that tBLMs can be effectively regenerated even after exposure to membrane-disrupting toxins, but the electrochemical characteristics change due to submembrane physicochemical alterations. These insights highlight the importance of controlling submembrane reservoir properties to ensure analytical-grade reproducibility in reusable biosensor platforms. The findings inform future design strategies for robust, repeatable biosensing systems.