The self-assembled monolayers (SAMs) of the new lipidic anchor molecule HC18 [Z 20-(Z-octadec-9-enyloxy)-3,6,9,12,15,18,22-heptaoxatetracont-31-ene-1-thiol], and mixed HC18/β-mercaptoethanol (βME) SAMs were studied by spectroscopic ellipsometry, contact angle measurements, reflection adsorption infrared spectroscopy (RAIRS), electrochemical impedance spectroscopy (EIS), and evaluated in tethered bilayer lipid membranes (tBLMs). Our data indicate that HC18, containing a double bond in the alkyl segments, forms highly disordered SAMs up to anchor/βME molar fraction ratios of 80/20 and result in tBLMs that exhibit higher lipid diffusion coefficients, relative to previous anchor compounds with saturated alkyl chains, as determined by fluorescence correlation spectroscopy. EIS data shows the HC18 tBLMs, completed by rapid solvent exchange (RSE) or vesicle fusion, form more easily than with saturated lipidic anchors, exhibit excellent electrical insulating properties indicating low defect densities, and readily incorporate the pore forming toxin, α-hemolysin. Neutron reflectivity measurements on HC18 tBLMs confirm the formation of complete tBLMs, even at low tether compositions and high ionic lipid compositions. Our data indicates HC18 results in tBLMs with improved physical properties for incorporation of integral membrane proteins (IMPs) and that 80% HC18 tBLMs appear to be optimal for practical applications such as biosensors where high electrical insulation and IMP/peptide reconstitution is imperative.

Structure of the self-assembled monolayers (SAMs) used to anchor phospholipid bilayers to surfaces affects the functional properties of the tethered bilayer membranes (tBLMs). SAMs of the same surface composition differing in the lateral distribution of the anchor molecule give rise to tBLMs of profoundly different defectiveness with residual conductance spanning 3 orders of magnitude. SAMs composed of anchors containing saturated alkyl chains, upon exposure to water (72 h), reconstruct to tightly packed clusters as deduced from reflection absorption infrared spectroscopy data and directly visualized by atomic force microscopy. The rearrangement into clusters results in an inability to establish highly insulating tBLMs on the same anchor layer. Unexpectedly, we also found that nanometer scale smooth gold film surfaces, populated predominantly with (111) facets, exhibit poor performance from the standpoint of the defectiveness of the anchored phospholipid bilayers, while corrugated (110) dominant surfaces produced SAMs with superior tethering quality. Although the detailed mechanism of cluster formation remains to be clarified, it appears that smooth surfaces favor lateral translocation of the molecular anchors, resulting in changes in functional properties of the SAMs. This work unequivocally establishes that conditions that favor cluster formation of the anchoring molecules in tBLM formation must be identified and avoided for the functional use of tBLMs in biomedical and diagnostic applications.

Phosphatidylcholine and cholesterol exchange between vesicles and planar tethered bilayer lipid membranes (tBLMs) was demonstrated from electrochemical impedance spectroscopy (EIS), fluorescence microscopy (FM), and neutron reflectometry (NR) data. Cholesterol is incorporated into the tBLMs, as determined by the functional reconstitution of the pore forming toxin α-hemolysin (EIS data), attaining cholesterol concentrations nearly equal to that in the donor vesicles. Using fluorescently labeled lipids and cholesterol, FM indicates that the vesicle-tBLM exchange is homogeneous for the lipids but not for cholesterol. NR data with perdeuterated lipids indicates lipid exchange asymmetry with two lipids exchanged in the outer leaflet for every lipid in the inner leaflet. NR and EIS data further show different exchange rates for cholesterol (t1/2 < 60 min) and phosphatidylcholine (t1/2 > 4 h). This work lays the foundation for the preparation of robust, lower defect, more biologically relevant tBLMs by essentially combining the two methods of tBLM formation-rapid solvent exchange and vesicle fusion.

A facile and reproducible preparation of surface-supported lipid bilayers is essential for fundamental membrane research and biotechnological applications. We demonstrate that multilamellar vesicles fuse to molecular-anchor-grafted surfaces yielding low-defect-density, tethered bilayer membranes. Continuous bilayers are formed within 10min, while the electrically insulating bilayers with <0.1μm defect density can be accomplished within 60min. Surface plasmon resonance spectroscopy indicates that an amount of lipid material transferred from vesicles to a surface is inversely proportional to the density of an anchor, while the total amount of lipid that includes tethered and transferred lipid remains constant within 5% standard error. This attests for the formation of intact bilayers independent of the tethering agent density. Neutron reflectometry (NR) revealed the atomic level structural details of the tethered bilayer showing, among other things, that the total thickness of the hydrophobic slab of the construct was 3.2nm and that the molar fraction of cholesterol in lipid content is essentially the same as the molar fraction of cholesterol in the multilamellar liposomes. NR also indicated the formation of an overlayer with an effective thickness of 1.9nm. These overlayers may be easily removed by a single rinse of the tethered construct with 30% ethanol solution. Fast assembly and low residual defect density achievable within an hour of fusion makes our tethered bilayer methodology an attractive platform for biosensing of membrane damaging agents, such as pore forming toxins.

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