The comparative study of two types of tethered bilayer membranes (tBLMs), one formed on atomically smooth gold films and the other on fluorine-doped tin oxide (FTO) films on glass is reported. These solid substrates exhibited different surface morphology and roughness. After tailoring with thiol or silane compounds, both formed intact tBLMs as demonstrated by electrochemical impedance spectroscopy (EIS). In contrast to the flat Au surfaces, the FTO substrates showed the presence of local water pockets, which caused a lower electrical resistance of the submembrane space separating the bilayers and the solid substrate. The EIS data analysis showed that the residual lateral defects, with an average radius between 10 and 20 nm, are heterogeneously distributed over the surface of the tBLMs. The concentration of defects on the surface is very low, ranging from 10 to 30 defects per 1000 mu m2. Both types of tBLMs responded to the pore-forming toxin, the alpha-hemolysin (alpha HL) from Staphylococcus aureus in a concentration dependent manner. The EIS response and the sensitivity to the toxin were stronger for tBLMs on FTO than on Au. We show that, independent of the solid substrate, the property that affects the sensitivity of tBLM sensors to membrane-damaging toxins is related to the resistance of the submembrane reservoir. The lower the resistance, the higher the sensitivity. Although we cannot exclude other factors such as membrane composition, buffer salts, and pH, we conclude that, in general, sub-membrane resistance is the fundamental factor determining the sensitivity of tBLM sensors.

Tethered bilayer lipid membranes (tBLMs) with two different molecular architectures were investigated in the context of their utility for biosensing of pore-forming toxins (PFTs). The different architectures were achieved by using two back-filler molecules of different chain length to act as surface diluents for the molecular anchors [20tetradecyloxy-3,6,9,12,15,18,22-heptaoxahexatricontane-1-thiol (WC14)] that ensure immobilization of the phospholipid bilayers on a solid conductive (metal, such as gold) support. The back-filler molecules were beta-mercaptoethanol (BME) and HS(CH2)3(CH2CH2O)5H thiol (C3EO5H). In the case of BME, the submembrane space separating solid surface from attached bilayer (typically 1-2 nm thick) is populated with the ice-like, presumably low entropy and low dielectric constant water, however, the water fraction in the submembrane is high. Much bulkier C3EO5H back-filler renders less space for water in the submembrane, however, it is populated with more mobile water molecules resulting in higher dielectric constant. These two opposing effects result in similar submembrane specific conductances, i.e., the specific electrochemical impedance spectral (EIS) characteristics of the tBLMs and their variation, upon exposure to and reorganization with, the pore-forming toxin (PFT), alpha-hemolysin, occur in the same frequency range for both back-fillers. Significantly, the sensitivity of the C3EO5H-containing tBLMs to PFT is approximately 7-8 times higher than that of the BME-containing surface constructs. At least two physical phenomena contribute to this difference. The bulkier C3EO5H backfillers facilitate the formation of nanoclusters of the molecular anchors, WC14, with cluster sizes ranging from 20 to 120 nm. These highly anchor-clustered, "patchy" tBLMs, consequently exhibit other domains devoid, or nearly so, of anchors, resulting in lowering the tBLM global 2-D viscosity by a factor of 3. Thus, a major conclusion of this study is that lower tBLM 2D fluidity increases the sensitivity of tBLMs to PFTs.

Cluster formation is a widely-observed phenomenon, which results in unique sets of properties both in physical, chemical and biological domains of nature. Here, we present a qualitative description of clustering of membrane defects, specifically ion-conducting protein pores in tethered phospholipid bilayers, a versatile biomimetic model of biological membranes accomplished on chemically modified gold films. By invoking the Voronoi tessellation concept we demonstrate the possibility to distinguish between random and sparsely clustered patterns both in computer generated and real-world systems using one single parameter sigma, the standard deviation of the normalized Voronoi sector areas distribution. For random systems, sigma approximate to 0.54, for sparsely clustered patterns sigma > 0.54. Because of a specific structure and dielectric properties of tethered bilayers, they can be characterized by an alternating current technique, electrochemical impedance spectroscopy (EIS). EIS measures macroscopic parameters of dielectric systems and it is not a structural technique per se. However, we found the EIS spectra-derived quantitative metric zeta to be a diagnostic parameter that allows assessment of the distribution type (homogeneous, random or clustered) of defects at nanometer level. One of the most interesting findings of the current study is the fact that the EIS derived zeta parameter is sensitive to an average size of the defects thus enabling a purely electrochemical methodology to access fine structural information such as size of incomplete protein pores in phospholipid bilayers. Overall, our results demonstrate a fundamental property of the macroscopic technique, electrochemical impedance spectroscopy, to probe structural arrangement of defects with sizes between 0.5 nm to 25.5 nm located in a thin, 2 nm thick phospholipid dielectric layer. Our findings can be utilized in designing precision electrochemical biosensors, and/or solving specific physical, biochemical or electrochemical problems related to other types of nanometer thick dielectric films on conducting surfaces. (C) 2020 Elsevier Ltd. All rights reserved.

Protein corona formed on nanomaterial surfaces play an important role in the bioavailability and cellular uptake of nanomaterials. Modification of surfaces with oligoethylene glycols (OEG) are a common way to improve the resistivity of nanomaterials to protein adsorption. Short-chain ethylene oxide (EO) oligomers have been shown to improve the protein resistance of planar Au surfaces. We describe the application of these EO oligomers for improved protein resistance of 30 nm spherical gold nanoparticles (AuNPs). Functionalized AuNPs were characterized using UV-Vis spectroscopy, dynamic light scattering (DLS), and zeta potential measurements. Capillary electrophoresis (CE) was used for separation and quantitation of AuNPs and AuNP-protein mixtures. Specifically, nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was employed for the determination of equilibrium and rate constants for binding between citrate-stabilized AuNPs and two model proteins, lysozyme and fibrinogen. Semi-quantitative CE analysis was carried out for mixtures of EO-functionalized AuNPs and proteins, and results demonstrated a 2.5-fold to 10-fold increase in protein binding resistance to lysozyme depending on the AuNP surface functionalization and a 15-fold increase in protein binding resistance to fibrinogen for both EO oligomers examined in this study.

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 10 min, while the electrically insulating bilayers with < 0.1 μm− 2 defect density can be accomplished within 60 min. 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 3.2 nm 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 1.9 nm. 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.

We present a facile strategy to modify poly(dopamine) (PDA)-coated substrates. Using thiol-terminated short chain ethylene oxide oligomers (OEG) under aqueous conditions, we explore the creation of a model surface exhibiting resistance to nonspecific protein absorption (RPA) by engineering the surface properties of a PDA adlayer. Surprisingly, dithiol-terminated OEG molecules demonstrated significantly greater coverage on PDA surfaces than analogous monothiol molecules. Successful RPA is only achieved with dithiol-terminated OEGs.

Nanoporous platinum membranes can be straightforwardly fabricated by forming a Pt coating inside the nanopores of anodic alumina membranes (AAO) using atomic layer deposition (ALD). However, the high-aspect-ratio of AAO makes Pt ALD very challenging. By tuning the process deposition temperature and precursor exposure time, enhanced infiltration depth along with conformal coating was achieved for Pt ALD inside the AAO templates. Cross-sectional scanning electron microscopy/energy dispersive x-ray spectroscopy and small angle neutron scattering were employed to analyze the Pt coverage and thickness inside the AAO nanopores. Additionally, one application of platinum-coated membrane was demonstrated by creating a high-density protein-functionalized interface.

For integral membrane proteins (IMP), an assessment of their structures and interactions with other membrane proteins within a bio-mimetic lipid bilayer environment is critical for determining their cellular function. Hydrophobic sequences prevalent within the transmembrane domain(s) of IMPs, however, make these proteins susceptible to aggregation, and thus create difficulties in examining their structural and functional properties via canonical techniques. Working exclusively with transmembrane (TM) segments of polytopic membrane proteins - in the form of soluble peptides - bypasses many of the pitfalls of full-length protein preparations, while allowing for the opportunity to examine the properties of TM domains within bio-mimetic membrane environments. In this study, peptides mimicking the TM domains of the epidermal growth factor (EGFR) and CD4 receptors, both cell-signaling membrane proteins, have been reconstituted into POPC lipid bilayers. The formation of their native alpha-helical structure within vesicle membranes was observed from CD spectra, and proper orientation of the peptides passing through the membrane was demonstrated by tryptophan fluorescence using brominated lipids. Functionalized with an N-terminal biotin tag, and utilizing an engineered planar lipid bilayer system ideally set up for surface plasmon resonance measurements, the TM peptides demonstrated capabilities of “activating” a membrane surface by the capture of streptavidin. Prospectively, these peptides reconstituted as near-native membrane proteins within artificial bilayer systems could be utilized in formulating bioactive surfaces for powerful biomedical and biosensor applications. Novel techniques for re-forming the full-length IMP construct will be discussed.

In human cancer cells, hMGL is the principal regulator of fatty acids, controlling the hydrolysis of cellular lipids and the production of free fatty acid-derived oncogenic lipid messengers that promote cancer cell migration and invasiveness. hMGL is a membrane proximal protein that belongs to the serine hydrolase family involved in the endocannabinoid signaling system (ECBSS). As a major part of the ECBSS that is composed of two main cannabinoid receptors CB1 and CB2 and endogenous ligands, hMGL exclusively hydrolyzes 2-arachidonoylglycerol (2-AG), making it a particularly critical modulator for the endocannabinoid transmission. hMGL also plays important roles in the metabolism of lipid storage. The key roles of hMGL in tuning homeostatic ECBSS and in supporting aggressive tumorogenesis make this protein a promising therapeutic target for treating cancer as well as managing pain and treating inflammatory, neurodegenerative, and immunological disorders. Using a custom built device that allows simultaneous surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) measurements in real-time, we report for the first time, that hMGL introduces significant defects into pre-formed, intact, tethered phospholipid bilayer membranes (tBLMs) composed of phosphatidylcholines and other lipids. To probe the mechanism of hMGL's ‘destructive activity’ to tBLMs we investigated wild-type and mutant hMGLs with respect to concentration, inhibitors, and pH. Our data shows that MGL's lid domain, lytic activity center, and hydrophobic pocket all play important roles in this process. This work supported by NIDA grant DA-3801

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.

As part of an effort to develop biointerfaces for structure-function studies of integral membrane proteins (IMPs) a series of oligo(ethylene oxide) self-assembled monolayers (OEO-SAMs) were evaluated for their resistance to protein adsorption (RPA) of IMPs on Au and Pt. Spectroscopic ellipsometry (SE) was used to determine SAM thicknesses and compare the RPA of HS(CH2)3O(CH2CH2O)6CH3 (1), HS(CH2)3O(CH2CH2O)6H (2), [HS(CH2)3]2CHO(CH2CH2O)6CH3 (3) and [HS(CH2)3]2CHO(CH2CH2O)6H (4), assembled from water. For both substrates, SAM thicknesses for 1 to 4 were found to be comparable indicating SAMs with similar surface coverages and OEO chain order and packing densities. Fibrinogen (Fb), a soluble plasma protein, and rhodopsin (Rd), an integral membrane G-protein coupled receptor, adsorbed to the SAMs of 1, as expected from previous reports, but not to the hydroxy-terminated SAMs of 2 and 4. The methoxy-terminated SAMs of 3 were resistant to Fb but, surprisingly, not to Rd. The stark difference between the adsorption of Rd to the SAMs of 3 and 4 clearly indicate that a hydroxy-terminus of the OEO chain is essential for high RPA of IMPs. The similar thicknesses and high RPA of the SAMs of 2 and 4 show the conditions of protein resistance (screening the underlying substrate, packing densities, SAM order, and conformational mobility of the OEO chains) defined from previous studies on Au are applicable to Pt. In addition, the SAMs of 4, exhibiting the highest resistance to Fb and Rd, were placed in contact with undiluted fetal bovine serum for 2h. Low protein adsorption (≈12.4ng/cm(2)), obtained under these more challenging conditions, denote a high potential of the SAMs of 4 for various applications requiring the suppression of non-specific protein adsorption.

Functional reconstitution of the cholesterol-dependent cytolysin vaginolysin (VLY) from Gardnerella vaginalis into artificial tethered bilayer membranes (tBLMs) has been accomplished. The reconstitution of VLY was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to VLY solutions were consistent with the formation of water-filled pores in the membranes. It was found that reconstitution of VLY is a strictly cholesterol-dependent, irreversible process. At a constant cholesterol concentration reconstitution of VLY occurred in a concentration-dependent manner, thus allowing the monitoring of VLY concentration and activity in vitro and opening possibilities for tBLM utilization in bioanalysis. EIS methodology allowed us to detect VLY down to 0.5 nM (28 ng/mL) concentration. Inactivation of VLY by certain amino acid substitutions led to noticeably lesser tBLM damage. Pre-incubation of VLY with the neutralizing monoclonal antibody 9B4 inactivated the VLY membrane damage in a concentration-dependent manner, while the non-neutralizing antibody 21A5 exhibited no effect. These findings demonstrate the biological relevance of the interaction between VLY and the tBLM. The membrane-damaging interaction between VLY and tBLM was observed in the absence of the human CD59 receptor, known to strongly facilitate the hemolytic activity of VLY. Taken together, our study demonstrates the applicability of tBLMs as a bioanalytical platform for the detection of the activity of VLY and possibly other cholesterol-dependent cytolysins.

The self-assembled monolayers (SAMs) of 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-absorption infrared spectroscopy, and electrochemical impedance spectroscopy (EIS) and were 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 those of previous anchor compounds with saturated alkyl chains, as determined by fluorescence correlation spectroscopy. EIS data shows the HC18 tBLMs, completed by rapid solvent exchange 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 indicate that HC18 results in tBLMs with improved physical properties for the 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 are imperative.

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.

We have developed a method based on self-assembly of thiols on Au substrates to immobilize membrane proteins at interfaces. Using water soluble nitrilotriacetic acid (NTA)-terminated oligo(ethylene glycol) thiols, a histidine-tagged G protein-coupled membrane receptor (GPCR) was captured in a defined orientation with little nonspecific binding.

Tethered bilayer lipid membranes (tBLMs) were prepared by the self-assembly of thiolated lipidic anchor molecules on gold, followed by phospholipid precipitation via rapid solvent exchange. They were characterized by their in-plane structure, dynamics and dielectric properties. We find that the in-plane homogeneity and resistivity of the tBLMs depend critically on a well-controlled sample environment during the rapid solvent-exchange procedure. The in-plane dynamics of the systems, assessed by fluorescence correlation spectroscopy (FCS) as the diffusivity of free, labeled phospholipid dissolved in the membrane, depend on the density of the lipidic anchors in the bilayer leaflet proximal to the substrate as well as on details of the molecular structure of the anchor lipid. In DOPC tBLMs in which tethers are laterally dilute (sparsely tethered bilayer lipid membranes, stBLMs), measured diffusivities, D ≈ 4 μm(2) s(-1), are only slightly greater than those reported in physisorbed bilayers (M. Przybylo, J. Sykora, J. Humpolíckova, A. Benda, A. Zan and M. Hof, Langmuir, 2006, 22, 9096-9099). However, when we distinguish label diffusion in the proximal and in the distal bilayer leaflets, we observe distinct diffusivities, D ≈ 2 μm(2) s(-1) and 7 μm(2) s(-1), respectively. The value observed in the distal leaflet is identical to that in free membranes. stBLMs completed with phytanoyl lipids (DPhyPC) show consistently lower label diffusivity than those completed with unsaturated chains (DOPC). As the length of the tether chain increases, a reduction in the apparent diffusivity is observed, which we interpret as an increased propensity of the proximal bilayer leaflet to host free lipid. We also investigated preparation conditions that control whether the tBLMs are laterally homogeneous, as assessed by optical microscopy. In laterally heterogeneous bilayers, the label diffusivity varies only by a factor of ~2 to 4, indicating that the regions in the bilayers with different label solubilities do not correspond to distinct phases, such as a fluid phase coexisting with a gel phase.

In-situ spectroscopic ellipsometry (SE) was utilized to examine the formation of the self-assembled monolayers (SAMs) of the water-soluble oligo(ethylene oxide) [OEO] disulfide [S(CH2CH2O)6CH3]2 {[S(EO)6]2} and two analogous thiols – HS(CH2CH2O)6CH3 {(EO)6} and HS(CH2)3O(CH2CH2O)5CH3 {C3(EO)5} – on Au from aqueous solutions. Kinetic data for all compounds follow simple Langmuirian models with the disulfide reaching a self-limiting final state (d=1.2nm) more rapidly than the full coverage final states of the thiol analogs (d=2.0nm). The in-situ ellipsometric thicknesses of all compounds were found to be nearly identical to earlier ex-situ ellipsometric measurements suggesting similar surface coverages and structural models in air and under water. Exposure to bovine serum albumin (BSA) shows the self-limiting (d=1.2nm) [S(EO)6]2 SAMs to be the most highly protein resistant surfaces relative to bare Au and completely-formed SAMs of the two analogous thiols and octadecanethiol (ODT). When challenged with up to near physiological levels of BSA (2.5mg/mL), protein adsorption on the final state [S(EO)6]2 SAM was only 3% of that which adsorbed to the bare Au and ODT SAMs.

The complexity of cells makes molecular-scale characterizations of structure and interactions of biomembranes in vivo extremely difficult, thus driving the development of synthetic membrane models. tBLMs are resilient biomimetic systems stabilized by the proximity of an inorganic interface.1,2 We characterize their in-plane structure, dynamics and dielectric properties using fluorescence microscopy, fluorescence correlation spectroscopy (FCS) and electrochemical impedance spectroscopy (EIS). The in-plane dynamics of tBLMs depend on structural details of the anchor lipid and its lateral density in the bilayer leaflet proximal to the substrate.3 In tBLMs with homogeneous lateral label distributions, the fluidity of the distal leaflet is comparable to that in vesicle membranes (2D diffusion constant, D ∼ 7 μm2/s) while that in the proximal leaflet is moderately reduced (D ∼ 2-3 μm2/s). tBLMs completed with phytanoyl lipids (DPhyPC) show lower label diffusivity than those completed with unsaturated chains (DOPC). In laterally heterogeneous bilayers, the label diffusivity varies only slightly, indicating that distinct regions in the bilayers do not correspond to distinct phases. Concurrently, we investigate the effect of charged lipids (DOPS) and cholesterol on lipid diffusivity. This aims at a characterization of changes in membrane dynamics as amyloid-β (Aβ) oligomers associate with tBLMs where they have been shown to affect the resistance of the bilayer to ion conduction.4,5 Supported by the NIH (1P01AG032131) and the AHAF (A2008-307). 1McGillivray, D.J., et al. 2007. Biointerphases 2:21-33. 2Vockenroth, I.K., et al. 2008. Biointerphases 3:FA68-73. 3Shenoy, S., et al., 2009. Soft Matter, submitted. 4Arispe, N., et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90:567-571. 5Valincius, G., et al., 2008. Biophys. J. 95:4845-4861.