Protein adsorption on supported phospholipid bilayers

Quartz crystal microbalance with dissipation (QCM-D) measurements were used to investigate the adsorption of human fibrinogen, human serum albumin, bovine hemoglobin, horse heart cytochrome c, human immunoglobulin (hIgG), and 10% fetal bovine serum on supported bilayers of egg-phosphatidylcholine (eggPC) lipids. For comparison the adsorption of fibrinogen and hIgG to eggPC bilayers was also studied with surface plasmon resonance (SPR). The supported bilayers were formed in situ by vesicle adhesion and spontaneous fusion onto a SiO(2) surface. The supported lipid bilayer is highly protein resistant: The irreversible adsorption measured with the QCM-D technique was below the detection level, while reversible protein adsorption was detected for all the proteins in the range 0.3-4% of the saturation coverage on a hydrophobic thiol monolayer on gold. The adsorbed amounts were slightly higher for the SPR measurements. Possible mechanisms for the protein resistance of eggPC bilayers are briefly discussed.     

Hierarchical assembly of model cell surfaces: synthesis of mucin mimetic polymers and their display on supported bilayers

Molecular level analysis of cell-surface phenomena could benefit from model systems comprising structurally defined components. Here we present the first step toward bottom-up assembly of model cell surfaces-the synthesis of mucin mimetics and their incorporation into artificial membranes. Natural mucins are densely glycosylated O-linked glycoproteins that serve numerous functions on cell surfaces. Their large size and extensive glycosylation makes the synthesis of these biopolymers impractical. We designed synthetically tractable glycosylated polymers that possess rodlike extended conformations similar to natural mucins. The glycosylated polymers were end-functionalized with lipid groups and embedded into supported lipid bilayers where they interact with protein receptors in a structure-dependent manner. Furthermore, their dynamic behavior in synthetic membranes mirrored that of natural biomolecules. This system provides a unique framework with which to study the behavior of mucin-like macromolecules in a controlled, cell surface-mimetic environment.     

Arrays of lipid bilayers and liposomes on patterned polyelectrolyte templates

This paper presents novel methods to produce arrays of lipid bilayers and liposomes on patterned polyelectrolyte multilayers. We created the arrays by exposing patterns of poly(dimethyldiallylammonium chloride) (PDAC), polyethylene glycol (m-dPEG) acid, and poly(allylamine hydrochloride) (PAH) on polyelectrolyte multilayers (PEMs) to liposomes of various compositions. The resulting interfaces were characterized by total internal reflection fluorescence microscopy (TIRFM), fluorescence recovery after pattern photobleaching (FRAPP), quartz crystal microbalance (QCM), and fluorescence microscopy. Liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphate (monosodium salt) (DOPA) were found to preferentially adsorb on PDAC and PAH surfaces. On the other hand, liposome adsorption on sulfonated poly(styrene) (SPS) surfaces was minimal, due to electrostatic repulsion between the negatively charged liposomes and the SPS-coated surface. Surfaces coated with m-dPEG acid were also found to resist liposome adsorption. We exploited these results to create arrays of lipid bilayers by exposing PDAC, PAH and m-dPEG patterned substrates to DOPA/DOPC vesicles of various compositions. The patterned substrates were created by stamping PDAC (or PAH) on SPS-topped multilayers, and m-dPEG acid on PDAC-topped multilayers, respectively. This technique can be used to produce functional biomimetic interfaces for potential applications in biosensors and biocatalysis, for creating arrays that could be used for high-throughput screening of compounds that interact with cell membranes, and for probing, and possibly controlling, interactions between living cells and synthetic membranes.     

Arrays of mobile tethered vesicles on supported lipid bilayers

We report a new system of laterally mobile, arrayed vesicles that are encoded with DNA to control tethering to fluid-supported phospholipid bilayers. The motion of individual fluorescently labeled vesicles, specifically bound, are easily visualized by fluorescence video microscopy and observed to collide reversibly on the surface. This system is an ideal model for studying interactions involving membranes, in particular integral membrane proteins.     

Minimal F-actin cytoskeletal system for planar supported phospholipid bilayers

Preferential binding of F-actin to lipid bilayers containing ponticulin was investigated on both planar supported bilayers and on a cholesterol-based tethering system. The transmembrane protein ponticulin in Dictyostelium discoideum is known to provide a direct link between the actin cytoskeleton and the cell membrane ( Wuestehube, L. J. ; Luna, E. J. J. Cell Biol. 1987, 105, 1741- 1751 ). Purification of ponticulin has allowed an in vitro model of the F-actin cytoskeletal scaffold system to be formed and investigated by AFM, epi-fluorescence microscopy, surface plasmon resonance (SPR), and quartz crystal microbalance with dissipation (QCM-D). Single filament features of F-actin bound to the ponticulin containing lipid bilayer are shown by AFM to have a pitch of 37.3 +/- 1.1 nm and a filament height of 7.0 +/- 1.6 nm. The complementary techniques of QCM-D and SPR were used to obtain dissociation constants for the interaction of F-actin with ponticulin containing bilayers, giving 10.5 +/- 1.7 microM for a physisorbed bilayer and 10.8 +/- 3.6 microM for a tethered bilayer, respectively.     

Disruption of supported lipid bilayers by semihydrophobic nanoparticles

Understanding the interaction between functional nanoparticles and cell membranes is critical to use nanomaterials for broad biomedical applications with minimal cytotoxicity. In this work, we have investigated the effect of adsorbed semihydrophobic nanoparticles (NPs) on the dynamics and morphology of model cell membranes. We have systematically varied the degree of surface hydrophobicity of carboxyl end-functionalized polystyrene NPs of varied size in buffer solutions with varied ionic strength. It is observed that semihydrophobic NPs can readily adsorb on neutral SLBs and drag lipids from SLBs to NP surfaces. Above a critical NP concentration, the disruption of SLBs is observed, accompanied with the formation and rapid growth of lipid-poor regions on NP-adsorbed SLBs. In the study of the effect of solution ionic strength on NP surface hydrophobic degree and the growth of lipid-poor regions, we have concluded that the hydrophobic interaction enhanced by screened electrostatic interaction underlies the envelopment of NPs by lipids that are attracted from SLBs to the surface of NPs or their aggregates. Hence, the formation and growth of lipid-poor regions, or vaguely referred as "pores" or "holes" in the literature, can be controlled by NP concentration, size, and surface hydrophobicity, which is critical to design functional nanomaterials for effective nanomedicine while minimizing possible cytotoxicity.     

Interactions between titanium dioxide and phosphatidyl serine-containing liposomes: formation and patterning of supported phospholipid bilayers on the surface of a medically relevant material

Titanium is widely used in biomedical applications. Its mechanical properties and biocompatibility, conferred by a layer of oxide present on its surface, make titanium the material of choice for various implants (artificial hip and knee joints, dental prosthetics, vascular stents, heart valves). Furthermore, the high refractive index of titanium oxide is advantageous in biosensor applications based on optical detection methods. In both of the above fields of application, novel surface modification strategies leading to biointeractive interfaces (that trigger specific responses in biological systems) are continuously sought. In this report, we investigate the interactions between TiO2 and phosphatidyl serine-containing liposomes, present a novel approach for preparing supported phospholipid bilayers (SPBs) of various compositions on TiO2, and use the unique ability of liposomes to distinguish between different surfaces to create SPB corrals on SiO2/TiO2 structured substrates. These results represent an important first step toward the design of biointeractive interfaces on titanium oxide surfaces that are based on a cell membrane-like environment.     

Electrochemical biosensor based on supported planar lipid bilayers for fast detection of pathogenic bacteria

This paper presents a new ion-channel biosensor based on supported bilayer lipid membrane for direct and fast detection of Campylobacter species. The sensing element of a biosensor is composed of a stainless-steel working electrode, which is covered by artificial bilayer lipid membrane (BLM). Antibodies to bacteria embedded into the BLM are used as channel forming proteins. The biosensor has a strong signal amplification effect, which is defined as the total number of ions transported across the BLM. The total number of (univalent) ions flowing through the channels is 10¹⁰ ions s⁻¹. The biosensor showed a very good sensitivity and selectivity to Campylobacter species.     

Absence of ethanol-induced interdigitation in supported phospholipid bilayers on silica surfaces

Membranes prepared by the adsorption of phospholipid vesicles on solid supports are much-used model systems in biomedical research. However, there is accumulating evidence that such membranes may not always be equivalent to the free-standing cellular membranes that they are modeling. In the present study, sonicated DOPC/DOPS (80/20 mol %) vesicles were adsorbed on hydrophilic silica surfaces, a system that has been demonstrated to produce confluent bilayers. In addition, pure DOPC and DLPC membranes were studied. It is demonstrated that ethanol-induced membrane interdigitation, as demonstrated for free-standing bilayers, does not occur in these supported membranes.     

Kinetic control of histidine-tagged protein surface density on supported lipid bilayers

Nickel-chelating lipids are general tools for anchoring polyhistidine-tagged proteins to supported lipid bilayers (SLBs), but controversy exists over the stability of the protein-lipid attachment. Here, we show that chelator lipids are suitable anchors for building stable, biologically active surfaces but that a simple Langmuirian model is insufficient to describe their behavior. Desorption kinetics from chelator lipids are governed by the valency of surface binding: monovalently bound proteins desorb within minutes (t1/2 approximately 6 min), whereas polyvalently bound species remain bound for hours (t1/2 approximately 12 h). Evolution between surface states is slow, so equilibrium is unlikely to be reached on experimental timescales. However, by tuning incubation conditions, the populations of each species can be kinetically controlled, providing a wide range of protein densities on SLBs with a single concentration of chelator lipid. We propose guidelines for the assembly of SLB surfaces functionalized with specific protein densities and demonstrate their utility in the formation of hybrid immunological synapses.     

Parallel-oriented fibrogenesis of a beta-sheet forming peptide on supported lipid bilayers

Peptide self-assembly on substrates is currently an intensively studied topic that provides a promising strategy for fabrication of soft materials and is also important for revealing the surface chemistry of amyloidogenic proteins that aggregate on cell membranes. We investigated the fibrogenesis of a beta-sheet forming peptide Abeta(26-35) on supported lipid bilayers (SLBs) by in situ atomic force microscopy (AFM), circular dichroism (CD), and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The results show that the Abeta(26-35) nanofilaments' growth is oriented to a specific direction and formed a highly ordered, large-scale, parallel-oriented surface pattern on membranes. The parallel-oriented fibrogenesis of Abeta(26-35) was able to occur on different lipid membranes rather than on solid substrates. It implies that the parallel-oriented fibrogenesis was associated with the distinct properties of lipid membranes, such as the fluid nature of lipid molecules on membranes. The membrane fluidity may allow the peptide assemblies to float at the water-membrane interface and easily orient to an energetically favorable state. These results provide an insight into the surface chemistry of peptide self-assembly on lipid membranes and highlight a possible way to fabricate supramolecular architectures on the surface of soft materials.     

Directed assembly of surface-supported bilayers with transmembrane helices

The lateral assembly of transmembrane (TM) helices gives rise to membrane proteins with complex folds, which play important roles in biochemical processes. Therefore, the assembly of surface-supported bilayers containing TM helices is the first step toward the development of functional biomembrane mimetics. Here we report novel directed assembly of surface-supported lipid bilayers with laterally mobile TM helices. The TM helices were incorporated into lipid monolayers at the air/water interface, and the monolayers were then transferred onto glass substrates using Langmuir-Blodgett (LB) deposition. Finally, bilayers were assembled using lipid vesicle fusion on top of the LB monolayers. The novelty is the incorporation of the peptides into the monolayer at the first step of bilayer assembly, which allows control over the peptide concentration and orientation. The transmembrane orientation of the peptides was confirmed using oriented circular dichroism (OCD), lateral mobility was assessed using fluorescence recovery after photobleaching (FRAP), and diffusion coefficients were determined using a novel boundary profile evolution (BPE) method. The described directed-assembly approach can be used to develop versatile bilayer platforms for studying membrane proteins interactions in native bilayer environments.     

Asymmetric distribution of anionic phospholipids in supported lipid bilayers

Lipid bilayers with a controlled content of anionic lipids are a prerequisite for the quantitative study of hydrophobic-electrostatic interactions of proteins with lipid bilayers. Here, the asymmetric distribution of zwitterionic and anionic lipids in supported lipid bilayers is studied by neutron reflectometry. We prepare POPC/POPS (3:1) unilamellar vesicles in a high-salt-concentration buffer. Initially, no fusion of the vesicles to a SiO(2) surface is observed over hours and days. Once the isotonic buffer is exchanged with hypotonic buffer, vesicle fusion and bilayer formation occur by osmotic shock. Neutron reflectivity on the bilayers formed this way reveals the presence of anionic lipids (d(31)-POPS) in the outer bilayer leaflet only, and no POPS is observed in the leaflet facing the SiO(2) substrate. We argue that this asymmetric distribution of POPS is induced by the electrostatic repulsion of the phosphatidylserines from the negatively charged hydroxy surface groups of the silicon block. Such bilayers with controlled and high contents of anionic lipids in the outer leaflet are versatile platforms for studying anionic lipid protein interactions that are key elements in signal transduction pathways in the cytoplasmic leaflet of eukaryotic cells.     

Detection of supported lipid bilayers using their electric charge

We describe an electronic detection method for charged lipid bilayers supported on a Si 3N 4/SiO 2/Si substrate. The flat-band voltage was used to monitor the charge of the bilayers. We show that the flat-band voltage varies with lipid adsorption depending on the polarity and mole ratio of the charged lipids, the salt concentration, and the surface coverage. Cationic and anionic bilayers produced a decrease and an increase in the flat-band voltage, respectively. The voltage change increased as the percentage of charged lipid components was elevated in the planar bilayers with full surface coverage. In addition, the voltage variation increased when the salt concentration was decreased or when the surface coverage of planar bilayer patches was increased. These results demonstrate that charged bilayers can be detected from the field effect that they exert on a solid support.     

Direct photochemical patterning and refunctionalization of supported phospholipid bilayers

A wet photolithographic route for micropatterning fluid phospholipid bilayers is demonstrated in which spatially directed illumination by short-wavelength ultraviolet radiation results in highly localized photochemical degradation of the exposed lipids. Using this method, we can directly engineer patterns of hydrophilic voids within a fluid membrane as well as isolated membrane corrals over large substrate areas. We show that the lipid-free regions can be refilled by the same or other lipids and lipid mixtures which establish contiguity with the existing membrane, thereby providing a synthetic means for manipulating membrane compositions, engineering metastable membrane microdomains, probing 2D lipid-lipid mixing, and designing membrane-embedded arrays of soluble proteins. Following this route, new constructs can be envisaged for high-throughput membrane proteomic, biosensor array, and spatially directed, aqueous-phase material synthesis.     

Patterned supported lipid bilayers and monolayers on poly(dimethylsiloxane)

A simple and practical method for patterning supported lipid bilayers on poly(dimethylsiloxane) is presented. By using electron microscopy grids to laterally control the extent of plasma oxidation, the substrate is partitioned into regions of different hydrophilicities. Addition of vesicles then results in the spontaneous formation of lipid bilayers and monolayers side-by-side on the surface, separated by regions that contain no lipid and/or a region with adhering vesicles. By using millimeter-sized plastic masks we are able to control the formation of these lipid structures on macroscopic patches by simply varying the plasma-cleaning time. For the first time, we are able to influence, in a controlled fashion, the chemical composition of a substrate in such a way that it supports fluid lipid monolayers, rejects lipid adhesion, adsorbs intact lipid vesicles, or supports fluid bilayers.     

Asymmetric distribution of phosphatidyl serine in supported phospholipid bilayers on titanium dioxide

Supported phospholipid bilayers (SPBs) are useful for studying cell adhesion, cell-cell interactions, protein-lipid interactions, protein crystallization, and applications in biosensor and biomaterial areas. We have recently reported that SPBs could be formed on titanium dioxide, an important biomaterial, from vesicles containing anionic phospholipid phosphatidyl serine (PS) in the presence of calcium. Here, we show that the mobility of the fluorescently labeled PS present in these bilayers is severely restricted, whereas that of the zwitterionic phosphatidyl choline is not affected. Removal of calcium alleviated the restriction on the mobility of PS. Both components were found to be mobile in SPBs of identical compositions prepared in the presence of calcium on silica. To explain these results, we propose that, on TiO2, PS is trapped in the proximal leaflet of the bilayers. This proposal is supported by the results of protein adsorption experiments carried out on bilayers containing various amounts of PS prepared on silica and titania.     

Structuring of supported hybrid phospholipid bilayers on electrodes with phospholipase A2

The effect of a lipolytic enzyme, pork pancreatic phospholipase A(2), on hybrid bilayer membranes was monitored using voltammetry, impedance spectroscopy and surface plasmon resonance. The hybrid bilayers were prepared by Langmuir-Schaefer transfer of lipid monolayers onto gold electrodes modified with self-assembled alkanethiol monolayers, or by liposome spreading. The electrodes were immersed in the phospholipase aqueous solution to allow adsorption of the enzyme and cleavage of the ester bond in the sn-2 position of phospholipids in the outer leaflet of the hybrid layers. The action of phospholipase A(2) led to perforation of the lipid films. Impedance spectroscopy and surface plasmon resonance were used for monitoring enzyme adsorption, phospholipid hydrolysis and product desorption. The results obtained show that transport efficiency of an electroactive probe, ferrocyanate, and of an electroactive drug, doxorubicin, through the bilayer depends on the action of the enzyme; the state of the lipid layer covering the electrode surface depends on the latter as well. Cyclic voltammetry and electrochemical impedance spectroscopy were used to study this effect. The doxorubicin reduction/oxidation signals appearing at potentials close to those observed using a bare gold electrode indicated facilitated penetration of the drug into the layer. The results obtained were interpreted in terms of pore formation in the lipid matrix; phospholipase A(2) can be considered as a nano-device for high precision perforation of the lipid layer.