Ion channels are one of the main drug targets and are in the focus of various membrane biosensor applications and drug screening assays. The aim of this thesis was to develop and characterize a novel membrane system suspending highly ordered porous substrates. This hybrid membrane system was supposed to combine the advantages of freestanding and solid supported lipid membranes. While part of the lipid bilayer anchored to the surface of the porous matrix resembles a solid supported membrane, the pore-suspending parts can be viewed as freestanding lipid membranes.
Hexagonally ordered porous alumina and macroporous silicon substrates with pore diameters in the nano- and micrometer range were fabricated by different etching procedures and characterized in detail by impedance spectroscopy and scanning electron microscopy. These highly ordered sieve-like pore arrays of billions of pores per square centimeter were used as supports for lipid bilayer immobilization.
Two different methods were successfully developed to obtain pore-suspending lipid bilayers based on these porous substrates: (1) a painting technique and (2) a technique based on vesicle spreading and fusion:
(1) Painting technique: In order to ensure that the prepared bilayers suspend the pores and do not cover the inner pore walls, the top of the pore columns was selectively functionalized by coating with a thin gold layer followed by chemisorption of either
1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol or 1-octadecanethiol. Lipid bilayers suspending the pores were obtained by painting 1,2-diphytanoyl-sn-glycero-3-phosphocholine dissolved in n-decane across the porous matrix. The membrane formation process was followed by means of electrical impedance spectroscopy, and membrane specific parameters were extracted from the impedance data by modeling the electrical behavior of these membrane systems by an adequate equivalent circuit consisting of a simple parallel RmCm-element. Specific membrane capacitances of
CmA = 0.7 µF/cm2 were calculated indicating the formation of single lipid bilayers. Suspended lipid bilayers on porous alumina, which we termed nano-black lipid membranes (nano-BLMs) and those suspending macroporous silicon substrates, termed micro-BLMs, both exhibited membrane resistances in the gigaohm regime allowing for single ion channel recordings and an extraordinary high long-term stability. In contrast to classical BLMs, which rupture in one single step, the membrane resistance of nano- and micro-BLMs decreases continuously, which was attributed to the fact that each membrane suspending a single pore can rupture individually due to the separation of the freestanding bilayer parts by the chemisorbed hydrophobic submonolayer. To prove this hypothesis porous matrix-supported BLMs were formed by painting 1,2-diphytanoyl-sn-glycero-3-phosphocholine across macroporous silicon substrates without pre-functionalization. Indeed, without the chemisorbed submonolayer, these matrix-supported lipid bilayers resemble classical BLMs. Though they exhibit typical membrane specific parameters, they rupture in one single event.
(2) Vesicle spreading and fusion: The formation of solvent-free pore-suspending lipid bilayers was achieved by spreading and fusion of thiolipid-containing large unilamellar vesicles on porous alumina substrates, which were covered on top of the pore columns with a thin gold layer. Impedance analysis revealed that these membranes were, however, not defect-free and thus, were as yet not suited for single channel recordings.
Pore-suspending lipid bilayers formed by the painting technique were proven to be ideally suited as membrane biosensors with fully functional transmembrane ion channels. The peptide antibiotics gramicidin and alamethicin as well as the transmembrane domain of the HIV-1 accessory peptide Vpu were successfully inserted into these novel chip-based membrane systems and peptide-characteristic conductance states were recorded. For Vpu, different amiloride derivates were elucidated as potential drugs to inhibit its channel activity. These measurements confirm the potential of nano- and micro-BLMs as membrane biosensors.
Pore-suspending lipid bilayers based on hexagonally ordered pore arrays will allow for automation and parallelization of ion channel recordings and will thus enable the development of high-throughput screening assays. Furthermore, the highly ordered porous structure serving as membrane support will allow adressing each substrate pore by space-resolved electrochemical techniques. This will enable one to perform several measurements on one support quasi-simultaneously
