Suspended lipid bilayers, or black lipid membranes (BLMs), have been used to study the electrophysiological properties of ion channels (ICs); however, BLMs assembled from natural, non-polymerizable lipids are inherently unstable due to the non-covalent associations on which they are based. Lifetimes of several hours are commonly observed in BLMs until rupture due to mechanical, thermal, or chemical insults. One potential improvement is the use of polymerizable phospholipids (poly(lipids)). BLMs prepared using dienoyl functionalized poly(lipids) and binary mixtures of fluid, non-polymerizable lipids with poly(lipids) were investigated for IC recordings.poly(BLMs) exhibited enhanced lifetimes from several hours to upwards of 4 weeks while maintaining IC functionality for one week. Activity of ICs that require membrane fluidity was retained using binary phospholipid mixtures of fluid and polymeric phospholipids. IC activity was retained by inducing domain formation, wherein ICs incorporated into the fluid domains. The binary membranes exhibited marked enhancement in stability resulting from fractional poly(lipids) polymerization. Additionally, ICs can be reconstituted into the fluid domains following photopolymerization and subsequent domain formation, a key requirement when UV-sensitive ICs are utilized. Here, the electrical properties, stability, and incorporation of pore-forming ICs, including hemolysin, alamethicin, and gramicidin, into poly(lipid) membranes are reported. Potential applications developing ligand-gated IC based sensors for high throughput screening are being investigated.In parallel to the characterization of poly(lipids) for potential long-term IC membranes, a model ligand-gated IC was expressed, characterized, and reconstituted into non-polymerizable lipids. Mutant K_ATP channels were expressed in mammalian and yeast systems. The orientations of mutant K_ATP channels were studied using electrophysiological and immunohistochemical techniques. Large quantities were expressed and purified from <italic>Pichia pastoris</italic> and functionally reconstituted into BLMs. ATP and long-chaing coenzyme A ester sensitivity was maintained in reconstituted in BLMs. K_ATP channels will serve as a model system for testing the effect of poly(lipid) BLMs on IC function. Future utilization of poly(lipid) BLMs in combination with ligand-gated ICs offer major advancements to potential increased throughput for IC screening.

The work described here has focused on two types of supramolecular assemblies, supported lipid bilayers (SLBs) and giant vesicles (GVs) from polymerizable lipids. SLBs are explored extensively as structural models in biophysical studies of cell membranes and biosensor coatings. With regard to implementation as biocompatible scaffoldings for receptor-based molecular devices, fluid SLBs lack chemical, thermal and mechanical stability as lipids are self-organized by weak, noncovalent forces. One possible solution is to use synthetic lipid monomers that can be polymerized to form robust bilayers. A key question is how polymerization affects transmembrane protein structure and activity. Specifically it is unclear if lipid cross-linking can be achieved without adversely affecting the activity of incorporated proteins. In this work the effect of lipid polymerization on transmembrane protein activity was studied with rhodopsin. The protein was reconstituted into SLBs composed of polymerizable lipids, bis-SorbPC, bis-SorbPC:mono-SorbPC, bis-DenPC and bis-SorbPC:mono-SorbPE. Rhodopsin photoactivity was monitored using plasmon waveguide spectroscopy. The results show that reconstitution of rhodopsin into SLBs composed of phosphatidylcholine with the polymerizable moiety in the acyl chain terminus, followed by photoinduced cross-linking of the lipids, does not significantly perturb protein function. A possible explanation is that a bilayer with relatively low Xn retains sufficient elasticity to accommodate the membrane deformation that accompanies the conformational change associated with rhodopsin photoactivation when polymerized in the acyl chain terminus. GVs have diameters ranging from several to few hundred micrometers and thus can be observed by optical microscopic methods. This allows manipulation of individual vesicles and observation of their transformations in real time. GVs have attracted attention as microcontainers for enzymes and drugs, and as biosensors. With the aim of increasing stability for these types of applications, GVs were prepared from synthetic dienoyl lipids that can be polymerized to form robust vesicles. The stability of these vesicles after polymerization was investigated by surfactant treatment, drying and rehydration, and temperature variations. The structure of poly(GVs) was largely retained under these conditions which destroy unpolymerized vesicles. Permeability studies on poly(GVs) suggests that they could be potentially used in a variety of technological applications, including sensors, macromolecular carriers, and microreactors.

It would be beneficial to incorporate transmembrane proteins (TMPs) into biosensors, because TMPs are important for cell function in healthy and diseased states. These devices would employ an artificial cell membrane to maintain TMP function since cell membranes, which are mostly lipids, are necessary for the TMPs to function. These artificial lipid membranes must be robust for sensor applications. The ruggedness of these artificial membranes can be increased by using polymerizable lipids. Some polymerized lipid membranes exhibit increased stability, while successfully incorporating TMPs.Some polymerized membranes do not support the activity of certain TMPs, while maintaining the function of others. It is believed the physical properties of the membranes are important for TMP function. Some important physical properties of polymerizable lipid membranes have not yet been measured. Here, fluidity and mechanical properties of polymerizable dienoylPC lipid membranes were investigated.Fluorescence Recovery After Photobleaching was used to measure the fluidity of polymerizable dienoylPC membranes. Unpolymerized, UV-polymerized, and redox-polymerized membranes were investigated. Three types of membranes were found: fluid, partially fluid, and immobile. Unpolymerized and some polymerized membranes were fluid, while only polymerized membranes were partially fluid or immobile. Polymer size is believed to cause the differences in fluidity. This study highlights how polymerization parameters can influence membrane fluidity.Micropipette Aspiration was used to measure the mechanical properties of Giant Unilamellar Vesicles (GUVs) composed of dienolyPC lipids. Unpolymerized and UV-polymerized GUVs were investigated. Strength measurements showed that denoylPC GUVs were stronger than sorbylPC GUVs. Area expansion moduli of denoylPCs and mono-SorbPC GUVs were slightly lower than SOPC GUVs, while bis-SorbPC GUVs were substantially easier to stretch. The bending moduli of all GUVs was similar. UV-polymerization had no significant effect on the parameters. The difference in strength between denoylPCs and sorbylPCs is hypothesized to be due to the porous nature of sorbylPCs. It is thought UV-polymerization of these GUVs created polymers too small to significantly alter mechanical properties.It was demonstrated that some stable membranes are also fluid, which is important for the function of certain TMPs. A correlation cannot be made between the bending and stretching moduli of polymerizable membranes and function of TMPs.

Cellular energy transduction processes are often driven by transmembrane ion gradients, and a number of artificial membrane systems have been developed that allow for chemically or light-induced transport of ions across lipid bilayers. These liposomal architectures, however, are not readily interfaced to a solid-state transducer. A significant step toward this goal is described here by assessing the possibility of coupling a lipid bilayer directly to a transducer to form a stable uniform film using hybrid bilayer membranes (HBMs).Although the surface attachment of self-assembled monolayer increases the robustness of the lipid assembly, HBMs cannot maintain film uniformity under harsher conditions due to the absence of strong lipid-lipid interactions. Therefore, HBMs were prepared and characterized using a cross-linking polymerizable lipid, bis-SorbPC. Several parameters relating to lipid deposition and film stabilization through polymerization were examined. Film characterization strongly suggests that polymerization of bis-SorbPC stabilizes the HBM such that its structure is largely preserved even after the dehydration process. This work suggests that network formation in the upper monolayer is not enough to prevent oligomer desorption, intermonolayer covalent linking is also a prerequisite in making uniform, defect-free planar supported lipid assemblies.Some of the challenges associated with the application of lipids involve the creation of supported bilayers that are stable to chemical and physical disruptions, yet retain their ion barrier properties, and allow transmembrane ion transport by lipid-soluble shuttles. Polymerized lipid films provide the stability required for these structures, but permeability properties of cations across poly(lipid) membranes are not known. Therefore, convenient liposome-based proton and calcium permeability assays were developed. These assays were applied to various poly(lipid) compositions.In addition, three novel sorbyl-substituted head group polymerizable lipids, which have been synthesized based on a strategy that head group polymerization would minimally perturb the characteristic ion impermeability of the membrane, were evaluated for their lipid characteristics and ability to form polymers. None of these compounds forms vesicles by itself. Therefore, attempts were made to form mixed vesicles with other fluid lipids. The miscibility of the mixed monolayers was assessed using Langmuir isotherms.