Stabilität und laterale Mobilität von porenüberspannenden Membranen auf porösen Siliziumsubstraten

Abstract

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) µm2 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.