Ion Channel Behavior of a Supported Bilayer Lipid Membrane Composed of 5,5-Ditetradecyl-2-(2-trimethyl-ammonioethyl)-1,3-dioxane Bromide Modified Glassy Carbon Electrode

A synthetic cationic surfactant, 5,5-ditetradecyl-2-(2-trimethyl-ammonioethyl)-1,3-dioxane bromide (DTDB), was used to construct a supported bilayer lipid membrane (s-BLM) coated on an underlying glassy carbon electrode (GCE). Electrochemical impedance spectroscopy (EIS), small-angle X-ray diffraction (SAXD) and cyclic voltammetry were used to characterize the s-BLM. Both EIS and SAXD data indicated that the synthetic lipid exists as a well-oriented bilayer in the membrane. The voltammetric study showed that the lipid membrane can open ion channels in the presence of C1O4- stimulant with Ru(bpy)32+ as marker ions and give distinct channel currents. The channels can be closed and open up again many times by removing or introducing ClO4- anions.

Biphasic Nano-Domains of Planar Lipid Bilayer Complexed with Fluorogenic Polymer Reporter Tailored for Antimicrobial Detection

We have fabricated an unexpected type of supported planar bilayer composed of receptor phospholipids and single-chained diacetylenes as fluorogenic reporters using protruded anchor moieties with a positive terminal charge.Nanoscale topographical and surface thermodynamic analyses,as well as molecular dynamics simulations,revealed the coexistence of well-dispersed liquid-condensed(L_(c))domains forming nano-islands and liquid-expanded(L_(e))region in the planar bilayer,enhancing sensitivity against a prototype of ubiquitous membrane-associated antimicrobial peptides,melittin.The L_(e)regions,acting as target receptors,enabled sensitive detection as the melittin adsorbed and inserted into these regions due to strong hydrophobic interactions between phospholipids and melittin.The L_(c)domains,serving as signal reporters,enabled diacetylenes to assemble,polymerize,and fluoresce in response to the insertion of melittin into the L_(e)regions.Thus,biphasic nanodomains of the planar lipid bilayer finally endowed this sensor system with a detection range of 100μMto 50 nM and a limit of detection(LOD)of∼37 nM for melittin.This exceeded the operational performance of the colorimetric polydiacetylene vesicle solution 45 times,which reportedly ranged from 100 to 4μM with an LOD of∼1.7μM.

Novel Experimental Strategy for High Resolution AFM Imaging of Membrane-Associated Bacterial Toxins

Bacterial pore-forming toxins(PFTs) are essential virulence factors of many human pathogens. Knowledge of their structure within the membrane is critical for an understanding of their function in pathogenesis and for the development of useful therapy. Atomic force microscopy(AFM) has often been employed to structurally interrogate many membrane proteins, including PFTs, owing to its ability to produce sub-nanometer resolution images of samples under aqueous solution. However, an absolute prerequisite for AFM studies is that the samples are single-layered and closely-packed, which is frequently challenging with PFTs. Here, using the prototypical member of the cholesterol-dependent cytolysin family of PFTs, perfringolysin O(PFO), as a test sample, we have developed a simple, highly robust method that routinely produces clean, closely-packed samples across the entire specimen surface. In this approach, we first use a small Teflon well to prepare the supported lipid bilayer, remove the sample from the well, and then directly apply the proteins to the bilayer. For reasons that are not clear,bilayer preparation in the Teflon well is essential. We anticipate that this simple method will prove widely useful for the preparation of similar samples, and thereby enable AFM imaging of the greatest range of bacterial PFTs to the highest possible resolution.