title: Bilayer lipid membranes supported on Teflon filters: a functional environment for ion channels.
abstract: Many ion channel proteins have binding sites for toxins and pharmaceutical drugs and therefore have much promise as the sensing entity in high throughput technologies and biosensor devices. Measurement of ionic conductance changes through ion channels requires a robust biological membrane with sufficient longevity for practical applications. The conventional planar BLM is 100-300 μm in diameter and typically contains fewer than a dozen channels whereas pharmaceutical screening methods in cells use current recordings for many ion channels. We present a new, simple method for the fabrication of a disposable porous-supported bilayer lipid membrane (BLM) ion channel biosensor using hydrated Teflon (polytetrafluoroethylene, PTFE) filter material (pore size 5 μm, filter diameter=1 mm). The lipid layer was monitored for its thickness and mechanical stability by electrical impedance spectroscopy. The results showed membrane capacitances of 1.8±0.2 nF and membrane resistances of 25.9±4.1 GΩ, indicating the formation of lipid bilayers. The current level increased upon addition of the pore-forming peptide gramicidin. Following addition of liposomes containing voltage-gated sodium channels, small macroscopic sodium currents (1-80 pA) could be recorded. By preloading the porous Teflon with sodium channel proteoliposomes, prior to BLM formation, currents of 1-10 nA could be recorded in the presence of the activator veratridine that increased with time, and were inhibited by tetrodotoxin. A lack of rectification suggests that the channels incorporated in both orientations. This work demonstrates that PTFE filters can support BLMs that provide an environment in which ion channels can maintain their functional activity relevant for applications in drug discovery, toxin detection, and odour sensing.
authors.affiliation: AgResearch, Grasslands Research Centre, Palmerston North, New Zealand.
authors.forename: Thai
authors.initials: T
authors.lastname: Phung
authors.forename: Yanli
authors.initials: Y
authors.lastname: Zhang
authors.forename: James
authors.initials: J
authors.lastname: Dunlop
authors.forename: Julie
authors.initials: J
authors.lastname: Dalziel
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D058266:Dielectric Spectroscopy / Q000379:methods; D004867:Equipment Design; D005374:Filtration / Q000295:instrumentation; D006096:Gramicidin / Q000494:pharmacology; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry*; D011138:Polytetrafluoroethylene / Q000737:chemistry*; D015222:Sodium Channels / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D055767:Immobilized Proteins; D008051:Lipid Bilayers; D015222:Sodium Channels; D006096:Gramicidin; D011138:Polytetrafluoroethylene