Molecular engineering of tethered bilayer lipid membranes for impedimetric detection of pore-forming toxins

Tethered bilayer lipid membranes (tBLMs) with two different molecular architectures were investigated in the context of their utility for biosensing of pore-forming toxins (PFTs). The different architectures were achieved by using two back-filler molecules of different chain length to act as surface diluents for the molecular anchors [20-tetradecyloxy-3,6,9,12,15,18,22-heptaoxahexatricontane-1-thiol (WC14)] that ensure immobilization of the phospholipid bilayers on a solid conductive (metal, such as gold) support. The back-filler molecules were β-mercaptoethanol (BME) and HS(CH₂)₃(CH₂CH₂O)₅H thiol (C₃EO₅H). In the case of BME, the submembrane space separating solid surface from attached bilayer (typically 1-2 nm thick) is populated with the ice-like, presumably low entropy and low dielectric constant water, however, the water fraction in the submembrane is high. Much bulkier C₃EO₅H back-filler renders less space for water in the submembrane, however, it is populated with more mobile water molecules resulting in higher dielectric constant. These two opposing effects result in similar submembrane specific conductances, i.e., the specific electrochemical impedance spectral (EIS) characteristics of the tBLMs and their variation, upon exposure to and reorganization with, the pore-forming toxin (PFT), alpha-hemolysin, occur in the same frequency range for both back-fillers. Significantly, the sensitivity of the C₃EO₅H-containing tBLMs to PFT is approximately 7–8 times higher than that of the BME-containing surface constructs. At least two physical phenomena contribute to this difference. The bulkier C₃EO₅H back-fillers facilitate the formation of nanoclusters of the molecular anchors, WC14, with cluster sizes ranging from 20–120 nm. These highly anchor-clustered, “patchy” tBLMs, consequently exhibit other domains devoid, or nearly so, of anchors, resulting in lowering the tBLM global 2-D viscosity by a factor of 3. Thus, a major conclusion of this study is that lower tBLM 2D fluidity increases the sensitivity of tBLMs to PFTs.