Electrochemical properties of tethered lipid bilayers on thin film silver substrates

Abstract In this work, the assembly of functional tethered bilayer membranes (tBLMs) on silver surface was demonstrated for the first time. To avoid degradation of thin silver films the non-corrosive mixture of thiols composed of WC14 molecular anchor and 3-mercapto-1-propanol (3M1P) serving as a backfiller was used to self-assemble monolayers which allowed fusion of multilamellar vesicles and tBLM formation. tBLMs on silver films exhibited high stability and low residual defect densities as measured by the electrochemical impedance spectroscopy (EIS). Silver supported tBLMs demonstrated all features typical for interactions of pore-forming proteins and bilayer membranes. Specifically, the EIS spectral response was typical for the formation of water filled defects when tBLMs were exposed to the solution of vaginolysin (VLY), the cholesterol dependent cytolysin. Increase of defect density was observed only in case tBLMs contained significant amount, e.g., 50 %, of cholesterol, thus confirming functional reconstitution of vaginolysin into tBLMs. This observation is consistent with the biological nature of interaction between the bilayer and the cholesterol dependent cytolysins. The reconstitution of VLY was confirmed by the direct imaging of the protein entities in tBLMs by the atomic force microscopy (AFM). Notably, the quantitative estimate of the functional protein-pores in tBLMs measured by EIS, was found to be close to the number of VLY entities in tBLMs measured by the AFM. Our work demonstrates that silver can serve as less expensive alternative to gold in designing tBLM based devices, for example phospholipid biosensors, as well as the experimental platform for studying membrane protein interactions by electrochemical, and microscopy techniques.