Characterization of supported membranes on topographically patterned polymeric elastomers and their applications to microcontact printing

This article describes the fluorescence microscopy and imaging ellipsometry-based characterization of supported phospholipid bilayer formation on elastomeric substrates and its application in microcontact printing of spatially patterned phospholipid bilayers. Elastomeric stamps, displaying a uniformly spaced array of square wells (20, 50, and 100 mum linear dimensions), are prepared using poly(dimethyl)siloxane from photolithographically derived silicon masters. Exposing elastomeric stamps, following UV/ozone-induced oxidation, to a solution of small unilamellar phospholipid vesicles results in the formation of a 2D contiguous, fluid phospholipid bilayers. The bilayer covers both the elevated and depressed regions of the stamp and exhibits a lateral connectivity allowing molecular transport across the topographic boundaries. Applications of these bilayer-coated elastomeric stamps in microcontact printing of lipid bilayers reveal a fluid-tearing process wherein the bilayer in contact regions selectively transfers with 75-90% efficiency, leaving behind unperturbed patches in the depressed regions of the stamp. Next, using cholera-toxin binding fluid POPC bilayers that have been asymmetrically doped with ganglioside Gm1 ligand in the outer leaflets, we examine whether the microcontact transfer of bilayers results in the inversion of the lipid leaflets. Our results suggest a complex transfer process involving at least partial bilayer reorganization and molecular re-equilibration during (or upon) substrate transfer. Taken together, the study sheds light on the structuring of lipid inks on PDMS elastomers and provides clues regarding the mechanism of bilayer transfer. It further highlights some important differences in stamping fluid bilayers from the more routine applications of stamping in the creation of patterned self-assembled monolayers.     

Atomic force microscopy of model lipid membranes

Supported lipid bilayers (SLBs) are biomimetic model systems that are now widely used to address the biophysical and biochemical properties of biological membranes. Two main methods are usually employed to form SLBs: the transfer of two successive monolayers by Langmuir–Blodgett or Langmuir–Schaefer techniques, and the fusion of preformed lipid vesicles. The transfer of lipid films on flat solid substrates offers the possibility to apply a wide range of surface analytical techniques that are very sensitive. Among them, atomic force microscopy (AFM) has opened new opportunities for determining the nanoscale organization of SLBs under physiological conditions. In this review, we first focus on the different protocols generally employed to prepare SLBs. Then, we describe AFM studies on the nanoscale lateral organization and mechanical properties of SLBs. Lastly, we survey recent developments in the AFM monitoring of bilayer alteration, remodeling, or digestion, by incubation with exogenous agents such as drugs, proteins, peptides, and nanoparticles.

Phospholipid morphologies on photochemically patterned silane monolayers

We have studied the spreading of phospholipid vesicles on photochemically patterned n-octadecylsiloxane monolayers using epifluorescence and imaging ellipsometry measurements. Self-assembled monolayers of n-octadecylsiloxanes were patterned using short-wavelength ultraviolet radiation and a photomask to produce periodic arrays of patterned hydrophilic domains separated from hydrophobic surroundings. Exposing these patterned surfaces to a solution of small unilamellar vesicles of phospholipids and their mixtures resulted in a complex lipid layer morphology epitaxially reflecting the underlying pattern of hydrophilicity. The hydrophilic square regions of the photopatterned OTS monolayer reflected lipid bilayer formation, and the hydrophobic OTS residues supported lipid monolayers. We further observed the existence of a boundary region composed of a nonfluid lipid phase and a lipid-free moat at the interface between the lipid monolayer and bilayer morphologies spontaneously corralling the fluid bilayers. The outer-edge of the boundary region was found to be accessible for subsequent adsorption by proteins (e.g., streptavidin and BSA), but the inner-edge closer to the bilayer remained resistant to adsorption by protein or vesicles. Mechanistic implications of our results in terms of the effects of substrate topochemical character are discussed. Furthermore, our results provide a basis for the construction of complex biomembrane models, which exhibit fluidity barriers and differentiate membrane properties based on correspondence between lipid leaflets. We also envisage the use of this construct where two-dimensionally fluid, low-defect lipid layers serve as sacrificial resists for the deposition of protein and other material patterns.     

Double cushions preserve transmembrane protein mobility in supported bilayer systems

Supported lipid bilayers (SLBs) have been widely used as model systems to study cell membrane processes because they preserve the same 2D membrane fluidity found in living cells. One of the most significant limitations of this platform, however, is its inability to incorporate mobile transmembrane species. It is often postulated that transmembrane proteins reconstituted in SLBs lose their mobility because of direct interactions between the protein and the underlying substrate. Herein, we demonstrate a highly mobile fraction for a transmembrane protein, annexin V. Our strategy involves supporting the lipid bilayer on a double cushion, where we not only create a large space to accommodate the transmembrane portion of the macromolecule but also passivate the underlying substrate to reduce nonspecific protein-substrate interactions. The thickness of the confined water layer can be tuned by fusing vesicles containing polyethyleneglycol (PEG)-conjugated lipids of various molecular weights to a glass substrate that has first been passivated with a sacrificial layer of bovine serum albumin (BSA). The 2D fluidity of these systems was characterized by fluorescence recovery after photobleaching (FRAP) measurements. Uniform, mobile phospholipid bilayers with lipid diffusion coefficients of around 3 x 10(-8) cm2/s and percent mobile fractions of over 95% were obtained. Moreover, we obtained annexin V diffusion coefficients that were also around 3 x 10(-8) cm2/s with mobile fractions of up to 75%. This represents a significant improvement over bilayer platforms fabricated directly on glass or using single cushion strategies.     

Selected-area sol-gel deposition of barium strontium titanate thin films on thermally oxidized silicon through mediation of self-assembled monolayers

Self-assembled monolayers (SAMs) of octadecyltrichlorosilane (OTS) and mercapto ethanol were used to modify the surface functionality of platinum/titanium or platinum/tantalum bilayer patterns on thermally oxidized silicon wafers. The attachment of OTS to the exposed oxide region made it hydrophobic, while the anchoring of mercapto ethanol to the bilayer pattern turned it hydrophilic. This patterned hydrophobicity and hydrophilicity was exploited to preferentially deposit barium strontium titanate (BST) thin films on the patterned bilayers from an aqueous sol-gel solution. The combination of the SAMs and the sol-gel film formation method allowed direct patterned deposition of BST thin films, which could be useful for on-chip electronic applications. Wet oxygen annealing at 50 °C was sufficient to stabilize the deposited sol-gel coating without adversely affecting the functionality of the OTS, thus permitting multiple dip-coatings to obtain patterned films of a desired thickness. Heat treatment at 750 °C allowed densification and conversion of the sol-gel coatings to perovskite BST films.     

Loosely packed self-assembled monolayer of N-hexadecyl-3,6-di(p-mercaptophenylacetylene)carbazole on gold and its application in biomimetic membrane research

Dithiols of N-hexadecyl-3,6-di(p-mercaptophenylacetylene)carbazole (HDMC) have been synthesized and employed to form self-assembled monolayers (SAMs) on gold. One characteristic of the HDMC molecule is its peculiar molecular structure consisting of a large and rigid headgroup and a small and flexible alkyl-chain tail. HDMC adsorbates can attach to gold substrates by a strong Au-S bond with weak van der Waals interactions between the alkyl-chain tails, leading to a loosely packed hydrophobic SAM. In this way we can couple hybrid bilayer membranes (HBMs) to gold surfaces with more likeness to a cell bilayer than the conventional HBMs based on densely packed long-chain alkanethiol SAMs. The insulating properties and stability of the HDMC monolayer as well as the HDMC/lipid bilayer on gold have been investigated by electrochemical techniques including cyclic voltammetry and impedance spectroscopy. To test whether the quality of the bilayer is sufficiently high for biomimetic research, we incorporated the pore-forming protein alpha-hemolysin) and the horseradish peroxidase into the bilayers, respectively. Experimental results demonstrated that this type of loosely packed hydrophobic SAM has great potential in biomimetic bilayer research and biosensor application.     

Sub-100 nm patterning of supported bilayers by nanoshaving lithography

Sub-100 nm wide supported phospholipid bilayers (SLBs) were patterned on a planar borosilicate substrate by AFM-based nanoshaving lithography. First, a bovine serum albumin monolayer was coated on the glass and then selectively removed in long strips by an AFM tip. The width of vacant strips could be controlled down to 15 nm. Bilayer lines could be formed within the vacant strips by vesicle fusion. It was found that stable bilayers formed by this method had a lower size limit of approximately 55 nm in width. This size limit stems from a balance between a favorable bilayer adhesion energy and an unfavorable bilayer edge energy.     

Supported lipid bilayer composition microarray fabricated by pattern-guided self-spreading

We report on the fabrication of a microarray of supported lipid bilayers (SLBs) with different chemical compositions and demonstrate its biosensing application. The technique utilizes the phenomenon of lipid self-spreading on a patterned surface, which offers complete positional selectivity for a supported lipid bilayer. We describe the fabrication of parallel 10-μm-wide lines, each filled with an SLB with a unique composition, at 5 μm intervals. Structures obtained with our new technique are finer and more highly integrated than previously reported structures that employ the vesicle fusion technique on patterned surfaces. We also detected specific binding between biotin and streptavidin with high contrast, indicating that the microarray is valuable for biosensing applications.     

Soft lithographic patterning of supported lipid bilayers onto a surface and inside microfluidic channels

We present simple soft lithographic methods for patterning supported lipid bilayer (SLB) membranes onto a surface and inside microfluidic channels. Micropatterns of polyethylene glycol (PEG)-based polymers were fabricated on glass substrates by microcontact printing or capillary moulding. The patterned PEG surfaces have shown 97 +/- 0.5% reduction in lipid adsorption onto two dimensional surfaces and 95 +/- 1.2% reduction inside microfluidic channels in comparison to glass control. Atomic force microscopy measurements indicated that the deposition of lipid vesicles led to the formation of SLB membranes by vesicle fusion due to hydrophilic interactions with the exposed substrate. Furthermore, the functionality of the patterned SLBs was tested by measuring the binding interactions between biotin (ligand)-labeled lipid bilayer and streptavidin (receptor). SLB arrays were fabricated with spatial resolution down to approximately 500 nm on flat substrate and approximately 1 microm inside microfluidic channels, respectively.     

Surface Modification with Control over Ligand Density for the Study of Multivalent Biological Systems

In the study of multivalent interactions at interfaces, as occur for example at cell membranes, the density of the ligands or receptors displayed at the interface plays a pivotal role, affecting both the overall binding affinities and the valencies involved in the interactions. In order to control the ligand density at the interface, several approaches have been developed, and they concern the functionalization of a wide range of materials. Here, different methods employed in the modification of surfaces with controlled densities of ligands are being reviewed. Examples of such methods encompass the formation of self-assembled monolayers (SAMs), supported lipid bilayers (SLBs) and polymeric layers on surfaces. Particular emphasis is given to the methods employed in the study of different types of multivalent biological interactions occurring at the functionalized surfaces and their working principles.     

Model cell membranes: Techniques to form complex biomimetic supported lipid bilayers via vesicle fusion

Vesicle fusion has long provided an easy and reliable method to form supported lipid bilayers (SLBs) from simple, zwitterionic vesicles on siliceous substrates. However, for complex compositions, such as vesicles with high cholesterol content and multiple lipid types, the energy barrier for the vesicle-to-bilayer transition is increased or the required vesicle–vesicle and vesicle–substrate interactions are insufficient for vesicle fusion. Thus, for vesicle compositions that more accurately mimic native membranes, vesicle fusion often fails to form SLBs. In this paper, we review three approaches to overcome these barriers to form complex, biomimetic SLBs via vesicle fusion: (i) optimization of experimental conditions (e.g., temperature, buffer ionic strength, osmotic stress, cation valency, and buffer pH), (ii) α-helical (AH) peptide-induced vesicle fusion, and (iii) bilayer edge-induced vesicle fusion. AH peptide-induced vesicle fusion can form complex SLBs on multiple substrate types without the use of additional equipment. Bilayer edge-induced vesicle fusion uses microfluidics to form SLBs from vesicles with complex composition, including vesicles derived from native cell membranes. Collectively, this review introduces vesicle fusion techniques that can be generalized for many biomimetic vesicle compositions and many substrate types, and thus will aid efforts to reliably create complex SLB platforms on a range of substrates.     

Lipid bilayer deposition and patterning via air bubble collapse

We report a new method for forming patterned lipid bilayers on solid substrates. In bubble collapse deposition (BCD), an air bubble is first "inked" with a monolayer of phospholipid molecules and then touched to the surface of a thermally oxidized silicon wafer and the air is slowly withdrawn. As the bubble shrinks, the lipid monolayer pressure increases. Once the monolayer exceeds the collapse pressure, it folds back on itself, depositing a stable lipid bilayer on the surface. These bilayer disks have lateral diffusion coefficients consistent with high quality supported bilayers. By sequentially depositing bilayers in overlapping areas, fluid connections between bilayers of different compositions are formed. Performing vesicle rupture on the open substrate surrounding this bilayer patch results in a fluid but spatially isolated bilayer. Very little intermixing was observed between the vesicle rupture and bubble-deposited bilayers.     

Supported lipid bilayer nanosystems: stabilization by undulatory-protrusion forces and destabilization by lipid bridging

Control of the stabilization/destabilization of supported lipid bilayers (SLBs) on nanoparticles is important for promotion of their organized assembly and for their use as delivery vehicles. At the same time, understanding the mechanism of these processes can yield insight into nanoparticle-cell interactions and nanoparticle toxicity. In this study, the suspension/precipitation process of zwitterionic lipid/SiO(2) nanosystems was analyzed as a function of ionic strength and as a function of the ratio of lipid/SiO(2) surface areas, at pH = 7.6. Salt is necessary to induce supported lipid bilayer (SLB) formation for zwitterionic lipids on silica (SiO(2)) (Seantier, B.; Kasemo, B., Influence of Mono- and Divalent Ions on the Formation of Supported Phospholipid Bilayers via Vesicle Adsorption. Langmuir 2009, 25 (10), 5767-5772). However, for zwitterionic SLBs on SiO(2) nanoparticles, addition of salt can cause precipitation of the SLBs, due to electrostatic shielding by both the lipid and the salt and to the suppression of thermal undulation/protrusion repulsive forces for lipids on solid surfaces. At ionic strengths that cause precipitation of SLBs, it was found that addition of excess SUVs, at ratios where there were equal populations of SUVs and SLBs, restored the undulation/protrusion repulsive forces and restabilized the suspensions. We suggest that SUVs separate SLBs in the suspension, as observed by TEM, and that SLB-SLB interactions are replaced by SLB-SUV interactions. Decreasing the relative amount of lipid, to the extent that there was less lipid available than the amount required for complete bilayer coverage of the SiO(2), resulted in precipitation of the nanosystem by a process of nanoparticle lipid bridging. For this case, we postulate a process in which lipid bilayer patches on one nanoparticle collide with bare silica patches on another SiO(2) nanoparticle, forming a single bilayer bridge between them. TEM data confirmed these findings, thus indicating that lipid bridges are composed of half bilayers on adjoining SiO(2) nanoparticles.     

Micropatterned composite membranes of polymerized and fluid lipid bilayers

Micropatterned composite membranes of polymerized and fluid lipid bilayers were constructed on solid substrates. Lithographic photopolymerization of a diacetylene-containing phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC), and subsequent removal of nonreacted monomers by a detergent solution (0.1 M sodium dodecyl sulfate (SDS)) yielded a patterned polymeric bilayer matrix on the substrate. Fluid lipid bilayers of phosphatidylcholine from egg yolk (egg-PC) were incorporated into the lipid-free wells surrounded by the polymeric bilayers through the process of fusion and reorganization of suspended small unilamellar vesicles. Spatial distribution of the fluid bilayers in the patterned bilayer depended on the degree of photopolymerization that in turn could be modulated by varying the applied UV irradiation dose. The polymeric bilayer domains blocked lateral diffusion of the fluid lipid bilayers and confined them in the defined areas (corrals), if the polymerization was conducted with a sufficiently large UV dose. On the other hand, lipid molecules of the fluid bilayers penetrated into the polymeric bilayer domains, if the UV dose was relatively small. A direct correlation was observed between the applied UV dose and the lateral diffusion coefficient of fluorescent marker molecules in the fluid bilayers embedded within the polymeric bilayer domains. Artificial control of lateral diffusion by polymeric bilayers may lead to the creation of complex and versatile biomimetic model membrane arrays.     

Immobilization of acetylcholinesterase in lipid membranes deposited on self-assembled monolayers

Human red blood cell acetylcholinesterase was incorporated into planar lipid membranes deposited on alkanethiol self-assembled monolayers (SAMs) on gold substrates. Activity of the protein in the membrane was detected with a standard photometric assay and was determined to be similar to the protein in detergent solution or incorporated in lipid vesicles. Monolayer and bilayer lipid membranes were generated by fusing liposomes to hydrophobic and hydrophilic SAMs, respectively. Liposomes were formed by the injection method using the lipid dimyristoylphosphatidylcholine (DMPC). The formation of alkanethiol SAMs and lipid monolayers on SAMs was confirmed by sessile drop goniometry, ellipsometry, and electrochemical impedance spectroscopy. In this work, we report acetylcholinesterase immobilization in lipid membranes deposited on SAMs formed on the gold surface and compare its activity to enzyme in solution.     

Supported lipid bilayers lifted from the substrate by layer-by-layer polyion cushions on self-assembled monolayers

The formation of lipid bilayers, lifted from the solid substrate by layer-by-layer polyion cushions, on self-assembled monolayers (SAMs) on gold was investigated by surface plasmon resonance (SPR) and fluorescence recovery after photobleaching (FRAP). The polyions poly(diallyldimethylammonium chloride) (PDDA) and polystyrene sulfonate (PSS) sodium salt were used for the layer-by-layer polyion macromolecular assembly. The cushion was formed by electrostatic interaction of PDDA/PSS/PDDA layers with a negatively charged surface of an SAM of 11-mercaptoundecanoic acid (MUA) on gold. The lipid bilayer membranes were deposited by vesicle fusion with different compositions of SOPS (an anionic lipid, 1-stearoyl-2-oleoyl-phosphatidylserine) and POPC (a zwitterionic lipid, 1-palmitoyl-2-oleoylphosphatidylcholine). In the case of pure SOPS and for lipid mixtures with a POPC composition up to 25%, single bilayers were deposited. FRAP experiments showed that single bilayers supported on PDDA/PSS/PDDA/MUA were mobile at room temperature, with lateral coefficients of approximately (1.2–2.1)×10⁻⁹ cm²/s. The kinetics of the addition of the ion-channel-forming peptide protegrin-1 to the supported bilayers was detected by SPR. A two-step interaction was observed, similar to the association behavior of protegrin-1 with bilayers supported on PDDA/MUA. The results are similar to that of supported lipid bilayers without a layer-by-layer cushion. The model membrane system in this work is a potential biosensor for mimicking the natural activities of biomolecules and is a possible tool to investigate the fundamental properties of biomembranes.     

Effects of surface pressure and internal friction on the dynamics of shear-driven supported lipid bilayers

Supported lipid bilayers (SLBs) are one of the most common model systems for cell membrane studies. We have previously found that when applying a bulk flow of liquid above an SLB the lipid bilayer and its constituents move in the direction of the bulk flow in a rolling type of motion, with the lower monolayer being essentially stationary. In this study, a theoretical platform is developed to model the dynamic behavior of a shear-driven SLB. In most regions of the moving SLB, the dynamics of the lipid bilayer is well explained by a balance between the hydrodynamic shear force arising from the bulk flow above the lipid bilayer and the friction between the upper and lower monolayers of the SLB. These two forces result in a drift velocity profile for the lipids in the upper monolayer of the SLB that is highest at the center of the channel and decreases to almost zero at the corners of the channel. However, near the front of an advancing SLB a very different flow behavior is observed, showing an almost constant drift velocity of the lipids over the entire bilayer front. In this region, the motion of the SLB is significantly influenced by gradients in the surface pressure as well as internal friction due to molecules that have accumulated at the front of the SLB. It is shown that even a modest surface fraction of accumulated molecules (～1%) can drastically affect the behavior of the SLB near the bilayer front, forcing the advancing lipids in the SLB away from the center of the channel out toward the sides.     

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.     

Using bicellar mixtures to form supported and suspended lipid bilayers on silicon chips

Bicellar mixtures, planar lipid bilayer assemblies comprising long- and short-chain phosphatidylcholine lipids in suspension, were used to form supported lipid bilayers on flat silicon substrate and on nanotextured silicon substrates containing arrays of parallel troughs (170 nm wide, 380 nm deep, and 300 nm apart). Confocal fluorescence and atomic force microscopies were used to characterize the resulting lipid bilayer. Formation of a continuous biphasic undulating lipid bilayer membrane, where the crests and troughs corresponded to supported and suspended lipid bilayer regions, is demonstrated. The use of interferometric lithography to fabricate nanotexured substrates provides an advantage over other nanotextured substrates such as nanoporous alumina by offering flexibility in designing different geometries for suspending lipid bilayers.     

Supported lipid bilayer microarrays created by non-contact printing

Arrays of supported lipid bilayers (SLBs) provide great potential for future drug development and multiplexed biological research, but are difficult to prepare due to the sensitivity of both the lipid and protein structural arrangement to air exposure. A novel way to produce arrays of SLBs is presented based on non-contact dispensing of vesicles to a substrate through a thin surface confined water film. The approach presents many degrees of freedom since it is not limited to a specific substrate, lipid composition, linker or controlled environment. The method allows adjustment of spot size (180-360 μm) by repeated dispensing as well as control over the composition of the spots and subsequent analytes. SLB formation by vesicle adsorption and rupture allows for incorporation of membrane proteins through pre-formed proteoliposomes. Dispensing through a dip-and-rinse water film avoids contamination, disruptive drying and the need for complex buffer compositions. Furthermore, no humidity control is necessary which simplifies the production step and prolongs the life-time of the spotting system. We characterize the method with respect to control over spot size, bilayer mobility and the formation process as well as demonstrate the possibility to fuse bilayer spots with subsequently added vesicles. Since complex lipid compositions and multiple spotting nozzles can be used, this novel technique is expected to be a promising platform for future applications, e.g. patterning to monitor peptide/protein-lipid interactions, for glycomics using glycolipids or lipopolysaccharides, and to study mixing of spatially confined lipid membranes.