In a recent paper, we hypothesized that the continuous increase in membrane conductance observed for nano-BLMs is the result of an independent rupturing of single membranes or membrane patches covering the pores of the porous material. To prove this hypothesis, we prepared micro-BLMs on porous silicon substrates with a pore size of 7 mum. The upper surface of the silicon substrate was coated with a gold layer, followed by the chemisorption of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) and subsequent addition of a droplet of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) dissolved in n-decane. The lipid membranes were fluorescently labeled and investigated by means of fluorescence microscopy and impedance spectroscopy. Impedance spectroscopy revealed the formation of pore-suspending bilayers with high membrane resistance. Increases in membrane capacitance and membrane conductance were observed. This increase in membrane conductance could be unambiguously related to the individual rupturing of membranes suspending the pores of the porous material as visualized by means of fluorescence microscopy. Moreover, by fluorescence recovery after photobleaching experiments, we investigated the lateral mobility of the lipids within the micro-BLMs leading to a mean effective diffusion coefficient of Deff = (14 +/- 1) microm2/s.

The phase transition of individually addressable microstructured lipid bilayers was investigated by means of imaging ellipsometry. Microstructured bilayers were created on silicon substrates by micromolding in capillaries, and the thermotropic behavior of various saturated diacyl phosphatidylcholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipentadecoyl-sn-glycero-3-phosphocholine, and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) bilayers as well as DMPC/cholesterol membranes was determined by measuring the area expansion and thickness of the bilayer as a function of temperature. We found an increase in the main phase transition temperature T(M) of 2-6 degrees C and a substantially reduced cooperativity compared to multilamellar vesicles. Measurements of lateral diffusion constants D employing fluorescence recovery after photobleaching revealed, however, only a marginal decrease in D compared to those found for vesicles and multibilayers. The known dependencies of T(M) both on the chain length of diacyl PC membranes and on the cholesterol content were reproduced on a solid support. Microstructured bilayers offer the unique advantage of integrating an internal standard of known thermotropic properties, which turned out to be important for reducing the measurement error and for ruling out the slightly changing impact of the surface on the phase transition behavior due to the surface pretreatment.

The aim of this study was to investigate the long term stability and lateral mobility of micro-BLMs (black lipid membrane) by means of impedance spectroscopy and fluorescence microscopy. Micro-BLMs are a novel hybrid system based on porous silicon that combines the advantages of solid supported membranes and freestanding lipid bilayers. By means of fluorescence microscopy and impedance spectroscopy, it was shown that micro-BLMs rupture independently from each other, resulting in a continuous decrease in membrane resistance. To determine the lateral mobility of the lipids in micro-BLMs, fluorescence recovery after photobleaching (FRAP) was used. The diffusion coefficient of micro-BLMs, composed of a submonolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE), on which 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was painted, was determined to be (9 ± 5) µm 2 s -1 with an immobile fraction of (24 ± 5) %. Small amounts of solvent remain in the micro-BLMs, which contributes to the fluidity of the membrane system. This was deduced from the fact that pore suspending membranes prepared by spreading of giant unilamellar vesicles on porous silicon with the same pore diameter and also functionalized with DPPTE exhibit a diffusion coefficient that is by a factor of two smaller compared to the solvent-containing micro-BLMs. Diffusion measurements of the upper lipid monolayer demonstrated that only this monolayer contributes significantly to the lateral mobility. Finite-element-simualtions elucidated that the measured diffusion coefficient is the sum of two individual diffusion constants, one that can be attribed to the free diffusion of the lipids in the pore suspending membrane area, while the other one is due to diffusion of the phospholipids on the pore rims. The lateral diffusion of lipids in solvent free micro-BLMs prepared on the one hand on DPPTE and on the other hand on the spacer lipid (cholesterylpolyethylenoxy)thiol (CPEO3) was compared. Compared to solvent-free micro-BLMs prepared on DPPTE functionalized substrates, the diffusion coefficient on CPEO3 functionalized substrates was by a factor of 1.8 larger, while the immobile fraction was by a factor 2 lower. This is caused by a lower surface coverage of CPEO3, which provides free space for the formation of mobile non immobilized lipid areas in the bottom lipid monolayer leading to an increased mobility of the lipids. The insertion of diacetylene lipids (23:2 PC diyene) in the membrane and its polymerization by irradiation with UV-light resulted in a reduction of the lateral mobility of the membrane lipids. In summary, the results demonstrate the strong influence of the boundary conditions (pore size, surface functionalization) on the lateral mobility of the lipids and the long term stability of the pore suspending membrane. It has been proven that micro-BLMs can serve as an adequate model system for biological membranes.