IMPEDANCE ANALYSIS OF SUPPORTED LIPID BILAYER-MEMBRANES - A SCRUTINY OF DIFFERENT PREPARATION TECHNIQUES

One topic of this study is the comparison of different preparation tec hniques to build up solid supported lipid bilayers onto gold substrate s. The deposited lipid bilayers were investigated by a.c. impedance sp ectroscopy. Three different strategies were applied: (1) The gold surf ace was initially covered with a chemisorbed monolayer of octadecaneth iol or 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). The se cond monolayer consisting of phospholipids was then deposited onto thi s hydrophobic surface by (i) the Langmuir-Schaefer-technique, (ii) fro m lipid solution in n-decane/isobutanol, (iii) by the lipid/detergent dilution technique or (iv) by fusion of vesicles. (2) Charged molecule s carrying thiol-anchors for attachment to the gold surface by chemiso rption were used. Negatively charged surfaces of 3-mercaptopropionic a cid were found to be excellent substrates that allow the attachment of planar lipid bilayers by applying positively charged dimethyldioctade cylammoniumbromide (DODAB) vesicles or negatively charged 1,2-dipalmit oyl-sn-glycero-3-phosphoglycerol vesicles in the presence of chelating Ca2+-ions. If positively charged first monolayers of mercaptoethylamm oniumhydrochloride were used we were able to attach mixed l/1,2-dimyri stoyl-sn-glycero-3-phosphoethanolamine vesicles to form planar lipid b ilayers via electrostatic interaction. (3) Direct deposition of lipid bilayers is possible from vesicles containing 1,2-dimyristoyl-sn-glyce ro-3-phosphothioethanol (DMPTE). A critical amount of more than 50 mol % of DMPTE was found to be necessary to form a solid supported lipid b ilayer. Bilayers obtained with these different preparation techniques were scrutinized with respect to their capacitances, kinetics of forma tion and their long-term stabilities by impedance spectroscopy. The se cond feature of this paper is the application of the supported bilayer s to study ion transport through channel-forming peptides. We used a D ODAB-bilayer for the reconstitution of gramicidin D channels. By circu lar dichroism measurements we verified that the peptide is in its chan nel conformation. The ion transport of Cs+-ions through the channels w as recorded by impedance analysis.