Supported lipid bilayers on spacious and pH-responsive polymer cushions with varied hydrophilicity

We report the successful formation of supported multicomponent lipid bilayer membranes (sLBMs) on polymer cushions consisting of a set of alternating maleic acid copolymers. The formation of sLBMs was triggered by a transient reduction of the electrostatic repulsion between the polymer cushions and the lipid vesicles by lowering the solution's pH to 4. Upon formation, the stability of the sLBMs was not affected by subsequent variations of the environmental pH. The degree of hydrophilicity and swelling of the anionic polymer cushions was found to determine both the kinetics of the membrane formation and the mobility of the lipid bilayer with lipid diffusion coefficients in the range from 0.26 to 2.6 microm2s(-1). The introduced polymer cushion system is concluded to provide a versatile base for the integration of active transmembrane proteins in sLBMs.     

Lipid bilayers on polyacrylamide brushes for inclusion of membrane proteins

The ability of neutral polymer cushions to support neutral lipid bilayers for the incorporation of mobile transmembrane proteins was investigated. Polyacrylamide brush layers were grown on fused silica using atom-transfer radical polymerization to provide polymer layers of 2.5-, 5- and 10-nm thickness. Lipid bilayers composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) were formed by vesicle fusion onto bare fused silica and onto each of the polyacrylamide layers. Bilayer fluidity was assessed by the diffusion of a probe, NBD-labeled phosphatidylcholine, using fluorescence recovery after photobleaching. A transmembrane protein, the human delta-opioid receptor, was inserted into each lipid bilayer, and its ability to bind a synthetic ligand, DPDPE, cyclic[2-d-penicillamine, 5-d-penicillamine]enkephalin, was detected using single-molecule fluorescence spectroscopy by labeling this ligand with a rhodamine dye. The transmembrane protein was observed to bind the ligand for all bilayers tested. The protein's electrophoretic mobility was probed by monitoring the fluorescence from the bound ligand. The 5-nm polyacrylamide thickness gave the fastest diffusion for the fluorescent lipid probe (D(1) = 2.0(+/-1.2) x 10(-7) and D(2) = 1.2(+/-0.5) x 10(-6) cm(2)/s) and also the largest electrophoretic mobility for the transmembrane protein (3 x 10(-8) cm(2)/V.s). The optimum in polymer thickness is suggested to be a tradeoff between decoupling from the substrate and increasing roughness of the polymer surface.     

Supported lipid bilayers on biocompatible polysaccharide multilayers

Formation of supported lipid bilayers on soft polymer cushions is a useful approach to decouple the membrane from the substrate for applications involving membrane proteins. We prepared biocompatible polymer cushions by the layer-by-layer assembly of two polysaccharide polyelectrolytes, chitosan (CHI) and hyaluronic acid, on glass and silicon substrates. (CHI/HA)(5) films were characterized by atomic force microscopy, giving an average thickness of 57 nm and roughness of 25 nm in aqueous solution at pH 6.5. Formation of zwitterionic lipid bilayers by the vesicle fusion method was attempted using DOPC vesicles at pH 4 and 6.5 on (CHI/HA)(5) films. At higher pH adsorbed lipids had low mobility and large immobile lipid fractions; a combination of fluorescence and AFM indicated that this was attributable to formation of poor quality membranes with defects and pinned lipids rather than to a layer of surface-adsorbed vesicles. By contrast, more uniform bilayers with mobile lipids were produced at pH 4. Fluorescence recovery after photobleaching gave diffusion coefficients that were similar to those for bilayers on PEG cushions and considerably higher than those measured on other polyelectrolyte films. The results suggest that the polymer surface charge is more important than the surface roughness in controlling formation of mobile supported bilayers. These results demonstrate that polysaccharides provide a useful alternative to other polymer cushions, particularly for applications where biocompatibility is important.     

Lipid mobility controls the diffusion of small biopolymer adsorbates

Recent studies on the diffusion of adsorbed polymers such as DNA on supported lipid bilayers have suggested that such strongly adsorbed polymers can be treated similarly to a polymer "in" a 2D fluid, but this conjecture has not been experimentally verified. To test this hypothesis and also to gain a better understanding of polymer dynamics in two dimensions, we designed an experimental protocol-the lateral transport of a short, single-stranded DNA oligonucleotide adsorbed on a supported cationic lipid bilayer. Fluorescence recovery after photobleaching (FRAP) analysis reveals that the diffusivity of the adsorbed DNA quantitatively tracks that of the underlying lipid, even though the bilayer mobility changes by 2 orders of magnitude with changes in temperature. Interestingly, our results for short, extended, adsorbed biopolymers quantitatively track those for globular proteins in lipid bilayers. We thus conclude that short macromolecules that are strongly adsorbed on lipid bilayers can be treated similarly to macromolecules in the bilayer.     

Lipid diffusion from single molecules of a labeled protein undergoing dynamic association with giant unilamellar vesicles and supported bilayers

It is demonstrated that single-molecule tracking of a fluorescently labeled protein undergoing transient binding to model membranes presents a useful method of obtaining fluid properties. The labeled ACBP protein was tracked during its binding to free-standing giant unilamellar vesicles (GUVs) and supported bilayers prepared from the GUVs in the same environment. The analysis of images that are blurred as a result of fast probe diffusion was discussed. An examination of the lateral diffusion trajectories revealed a homogeneous diffusion on the top segments of the GUVs with D = 6.9 +/- 0.3 microm(2)/s. The supported bilayer experiments revealed two diffusion processes, one with Df = 3.1 +/- 0.4 microm(2)/s and the other with Ds = 0.078 +/- 0.001 microm(2)/s. The 2-fold difference in the lipid bilayer mobility for the free-standing and fast components in the supported bilayers is attributed to the known effect of frictional coupling with the solid support. The slow mobile fraction in the bilayer is suggested to be associated with the migration of pore-like structures, originating from the interaction of the membrane with the glass support.     

Surface-supported bilayers with transmembrane proteins: role of the polymer cushion revisited

Protein lateral mobility in surface-supported bilayers is often much lower than the mobility of the lipids. In the present study we explore whether the incorporation of a PEG cushion between the bilayer and the substrate increases the lateral mobility of transmembrane proteins in bilayers produced via directed assembly, a method based on Langmuir-Blodgett deposition techniques. In our experiments, the PEG cushions were incorporated by adding PEG lipids to the protein/lipid monolayer at the air/water interface, at the first step of bilayer assembly. The protein and lipid mobilities in 160 different bilayers, with various PEG molecular weights and PEG lipid concentrations, were measured and compared. We found that the measured diffusion coefficients do not depend on the PEG molecular weight or the PEG lipid concentration and are very similar to the values measured in the absence of PEG. Therefore, contrary to our expectations, we found that a PEG cushion does not necessarily increase protein mobility, suggesting that the low protein mobility is not a consequence of protein-substrate interactions. Furthermore, we showed that the low protein mobility is not due to protein aggregation. The major determinant of protein mobility in surface-supported bilayer systems appears to be the method of bilayer assembly. While proteins were always mobile if the bilayers were prepared using the directed assembly method, in the presence and absence of a PEG cushion, other bilayer assembly protocols resulted in complete lack of protein mobility.     

Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions

The lateral diffusion coefficients of a BODIPY tail-labeled lipid in two model systems, namely, free-standing giant unilamellar vesicles (GUVs) and supported phospholipid bilayers (SPBs), were determined by fluorescence correlation spectroscopy (FCS) using the Z-scan approach. For the first time, the performed measurements on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers maintain exactly the same experimental conditions for both systems, which allows for a quantitative comparison of lipid diffusion in these two commonly used model membranes. The results obtained revealed that the lipid mobility in free-standing bilayers (D=7.8+/-0.8 microm2 s-1) is significantly higher than in the bilayer created on the solid support (mica) (D=3.1+/-0.3 microm2 s-1).     

Cholesterol slows down the lateral mobility of an oxidized phospholipid in a supported lipid bilayer

We investigated the mobility and phase-partitioning of the fluorescent oxidized phospholipid analogue 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N-Alexa647-ethanolamine (PGPE-Alexa647) in supported lipid bilayers. Compared to the conventional phospholipid dihexadecanoylphosphoethanolamine (DHPE)-Bodipy we found consistently higher diffusion constants. The effect became dramatic when immobile obstacles were inserted into the bilayer, which essentially blocked the diffusion of DHPE-Bodipy but hardly influenced the movements of PGPE-Alexa647. In a supported lipid bilayer made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the differences in probe mobility leveled off with increasing cholesterol content. Using coarse-grained molecular dynamics simulations, we could ascribe this effect to increased interactions between the oxidized phospholipid and the membrane matrix, concomitant with a translation in the headgroup position of the oxidized phospholipid: at zero cholesterol content, its headgroup is shifted to the outside of the DOPC headgroup region, whereas increasing cholesterol concentrations pulls the headgroup into the bilayer plane.     

E-cadherin tethered to micropatterned supported lipid bilayers as a model for cell adhesion

Cell-cell adhesion is a dynamic process requiring recruitment, binding, and reorganization of signaling proteins in the plane of the plasma membrane. Here, we describe a new system for investigating how this lateral mobility influences cadherin-based cell signaling. This model is based on tethering of a GPI-modified E-cadherin protein (hEFG) to a supported lipid bilayer. In this report, membrane microfluidics and micropatterning techniques are used to adopt this tethered protein system for studies with the anchorage-dependent cells. As directly formed from proteoliposomes, hEFG exhibits a diffusion coefficient of 0.6 +/- 0.3 microm(2)/s and mobile fraction of 30-60%. Lateral structuring of the supported lipid bilayer is used to isolate mobile proteins from this mixed mobile/immobile population, and should be widely applicable to other proteins. MCF-7 cells seeded onto hEFG-containing bilayers recognize and cluster this protein, but do not exhibit cell spreading required for survival. By micropatterning small anchors into the supported lipid bilayer, we have achieved cell spreading across the bilayer surface and concurrent interaction with mobile hEFG protein. Together, these techniques will allow more detailed analysis of the cellular dynamics involved in cadherin-dependent adhesion events.     

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.     

In situ formation and characterization of poly(ethylene glycol)-supported lipid bilayers on gold surfaces

Inclusion of a polymer cushion between a lipid bilayer membrane and a solid surface has been suggested as a means to provide a soft, deformable layer that will allow for transmembrane protein insertion and mobility. In this study, mobile, tethered lipid bilayers were formed on a poly(ethylene glycol) (PEG) support via a two-step adsorption process. The PEG films were prepared by coadsorbing a heterofunctional, telechelic PEG lipopolymer (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000-N-[3-(2-(pyridyldithio)propionate]) (DSPE-PEG-PDP) and a nonlipid functionalized PEG-PDP from an ethanol/water mixture, as described in a previous paper (Munro, J. C.; Frank, C. W. Langmuir 2004, 20, 3339-3349). Then a two-step lipid adsorption strategy was used. First, lipids were adsorbed onto the PEG support from a hexane solution. Second, vesicles were adsorbed and fused on the surface to create a bilayer in an aqueous environment. Fluorescence recovery after photobleaching experiments show that this process results in mobile bilayers with diffusion coefficients on the order of 2 microm2/s. The mobility of the bilayers is decreased slightly by increasing the density of tethered lipids. The formation of bilayers, and not multilayer structures, is also confirmed by surface plasmon resonance, which was used to determine in situ film thickness, and by fluorimetry, which was used to determine quantitatively the fluorescence intensity for each 18 by 18 mm sample. Unfortunately, fluorescence microscopy also shows that there are large defects on the samples, which limits the utility of this system.     

Single Lipid Bilayers Constructed on Polymer Cushion Studied by Sum Frequency Generation Vibrational Spectroscopy

Planar solid supported single lipid bilayers on mica, glass, or other inorganic surfaces have been widely used as models for cell membranes. To more closely mimic the cell membrane environment, soft hydrophilic polymer cushions were introduced between the hard inorganic substrate and the lipid bilayer to completely avoid the possible substrate-lipid interactions. In this article, sum frequency generation (SFG) vibrational spectroscopy was used to examine and compare single lipid bilayers assembled on the CaF(2) prism surface and on poly (L-lactic acid) (PLLA) cushion. By using asymmetric lipid bilayers composed of a hydrogenated 1,2-dipalmitoyl-sn-glycerol-3-phosphoglycerol (DPPG) leaflet and a deuterated 1,2-dipalmitoyl-(d62)-sn-glycerol-3-phosphoglycerol (d-DPPG) leaflet, it was shown that the DPPG lipid bilayers deposited on the CaF(2) and PLLA surfaces have similar structures. SFG has also been applied to investigate molecular interactions between an antimicrobial peptide Cecropin P(1) (CP1) and the lipid bilayers on the above two different surfaces. Similar results were again obtained. This research demonstrated that the hydrophilic PLLA cushion can serve as an excellent substrate to support single lipid bilayers. We believe that it can be an important cell membrane model for future studies on transmembrane proteins, for which the possible inorganic substrate-bilayer interactions may affect the protein structure or function.     

Creating fluid and air-stable solid supported lipid bilayers

Solid supported lipid bilayers are rapidly delaminated when drawn through the air/water interface. We have discovered that a close packed monolayer of specifically bound protein prevents this process. The protection mechanism worked in two ways. First, when protein-protected bilayers were drawn through the air/water interface, a thin bulk water layer was visible over the entire bilayer region, thereby preventing air from contacting the surface. Second, a stream of nitrogen was used to remove all bulk water from a protected bilayer, which remained fully intact as determined by fluorescence microscopy. The condition of this dried bilayer was further probed by fluorescence recovery after photobleaching. It was found that lipids were not two-dimensionally mobile in dry air. However, when the bilayer was placed in a humid environment, 91% of the bleached fluorescence signal was recovered, indicating long-range two-dimensional mobility. The diffusion coefficient of lipids under humid conditions was an order of magnitude slower than the same bilayer under water. Protected bilayers could be rehydrated after drying, and their characteristic diffusion coefficient was reestablished. Insights into the mechanism of bilayer preservation were suggested.     

DNA concentration modulation on supported lipid bilayers switched by surface acoustic waves

Spatially addressable arrays of molecules embedded in or anchored to supported lipid bilayers are important for on-chip screening and binding assays; however, methods to sort or accumulate components in a fluid membrane on demand are still limited. Here we apply in-plane surface acoustic shear waves (SAWs) to laterally accumulate double-stranded DNA segments electrostatically bound to a cationic supported lipid bilayer. The fluorescently labeled DNA segments are found to segregate into stripe patterns with a spatial frequency corresponding to the periodicity of the standing SAW wave (~10 μm). The DNA molecules are accumulated 10-fold in the regions of SAW antinodes. The superposition of two orthogonal sets of SAW sources creates checkerboard like arrays of DNA demonstrating the potential to generate arrayed fields dynamically. The pattern relaxation time of 0.58 s, which is independent of the segment length, indicates a sorting and relaxation mechanism dominated by lipid diffusion rather than DNA self-diffusion.     

Measuring lipid asymmetry in planar supported bilayers by fluorescence interference contrast microscopy

There is substantial scientific and practical interest in engineering supported lipid bilayers with asymmetric lipid distributions as models for biological cell membranes. In principle, it should be possible to make asymmetric supported lipid bilayers by either the Langmuir-Blodgett/Schafer (LB/LS) or Langmuir-Blodgett/vesicle fusion (LB/VF) techniques (Kalb et al. Biochim. Biophys. Acta 1992, 1103, 307-316). However, the retention of asymmetry in biologically relevant lipid bilayers has never been experimentally examined in any of these systems. In the present work, we developed a technique that is based on fluorescence interference contrast (FLIC) microscopy to measure lipid asymmetry in supported bilayers. We compared the final degree of lipid asymmetry in LB/LS and LB/VF bilayers with and without cholesterol in liquid-ordered (l(o)) and liquid-disordered (l(d)) phases. Of five different fluorescent lipid probes that were examined, 1,2-dipalmitoyl-phosphatidylethanolamine-N-[lissamine rhodamine B] was the best for studying supported bilayers of complex composition and phase by FLIC microscopy. An asymmetrically labeled bilayer made by the LB/LS method was found to be at best 70-80% asymmetric once completed. In LB/LS bilayers of either l(o) or l(d) phase, cholesterol increased the degree of lipid mixing between the opposing monolayers. The use of a tethered polymer support for the initial monolayer did not improve lipid asymmetry in the resulting bilayer. However, asymmetric LB/VF bilayers retained nearly 100% asymmetric label, with or without the use of a tethered polymer support. Finally, lipid mixing across the center of LB/LS bilayers was found to have drastic effects on the appearance of l(d)-l(o) phase coexistence as shown by epifluorescence microscopy.     

Fluid biomembranes supported on nanoporous aerogel/xerogel substrates

Planar supported lipid bilayers have attracted immense interest for their properties as model cell membranes and for potential applications in biosensors and lab-on-a-chip devices. We report the formation of fluid planar biomembranes on hydrophilic silica aerogels and xerogels. Scanning electron microscopy results showed the presence of interconnected silica beads of approximately 10-25 nm in diameter and nanoscale open pores of comparable size for the aerogel and grain size of approximately 36-104 nm with approximately 9-24 nm diameter pores for the xerogel. When the aerogel/xerogel was prehydrated and then allowed to incubate in l-alpha-phosphatidylcholine (egg yolk PC) unilamellar vesicle (approximately 30 nm diameter) solution, lipid bilayers were formed due to the favorable interaction of vesicles with the hydroxyl-abundant silica surface. Lateral mobility of labeled lipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine was retained in the membranes. A diffusion coefficient of 0.61 +/- 0.22 microm(2)/s was determined from fluorescence recovery after photobleaching analysis for membranes on aerogels, compared to 2.46 +/- 0.35 microm(2)/s on flat glass. Quartz crystal microbalance-dissipation was utilized to monitor the kinetics of the irreversible adsorption and fusion of vesicles into bilayers on xerogel thin films.     

Anomalous diffusion in supported lipid bilayers induced by oxide surface nanostructures

Hierarchic structure and anomalous diffusion on submicrometer scale were introduced into an artificial cell membrane, and the spatiotemporal dependence of lipid diffusion was visualized on nanostructured oxide surfaces. We observed the lipid diffusion in supported lipid bilayers (SLBs) on step-and-terrace TiO(2)(100) and amorphous SiO(2)/Si surfaces by single molecule tracking (SMT) method. The SMT at the time resolution of 500 μs to 30 ms achieved observation of the lipid diffusion over the spatial and temporal ranges of 100 nm/millisecond to 1 μm/second. The temporal dependence of the diffusion coefficient in the SLB on TiO(2)(100) showed that the crossover from anomalous diffusion to random diffusion occurred around 10 ms. The surface fine architecture on substrates will be applicable to induce hierarchic structures on the order of 100 nm or less, which correspond to the microcompartment size in vivo.     

Characterization of phospholipid bilayer formation on a thin film of porous SiO2 by reflective interferometric Fourier transform spectroscopy (RIFTS)

Classical methods for characterizing supported artificial phospholipid bilayers include imaging techniques such as atomic force microscopy and fluorescence microscopy. The use in the past decade of surface-sensitive methods such as surface plasmon resonance and ellipsometry, and acoustic sensors such as the quartz crystal microbalance, coupled to the imaging methods, have expanded our understanding of the formation mechanisms of phospholipid bilayers. In the present work, reflective interferometric Fourier transform spectrocopy (RIFTS) is employed to monitor the formation of a planar phospholipid bilayer on an oxidized mesoporous Si (pSiO(2)) thin film. The pSiO(2) substrates are prepared as thin films (3 μm thick) with pore dimensions of a few nanometers in diameter by the electrochemical etching of crystalline silicon, and they are passivated with a thin thermal oxide layer. A thin film of mica is used as a control. Interferometric optical measurements are used to quantify the behavior of the phospholipids at the internal (pores) and external surfaces of the substrates. The optical measurements indicate that vesicles initially adsorb to the pSiO(2) surface as a monolayer, followed by vesicle fusion and conversion to a surface-adsorbed lipid bilayer. The timescale of the process is consistent with prior measurements of vesicle fusion onto mica surfaces. Reflectance spectra calculated using a simple double-layer Fabry-Perot interference model verify the experimental results. The method provides a simple, real-time, nondestructive approach to characterizing the growth and evolution of lipid vesicle layers on the surface of an optical thin film.     

Engineered lipids that cross-link the inner and outer leaflets of lipid bilayers

The application of supported lipid bilayer systems as molecular sensors, diagnostic devices, and medical implants is limited by their lack of stability. In an effort to enhance the stability of supported lipid bilayers, three pairs of phosphatidylcholine lipids were designed to cross-link at the termini of their 2-position acyl chain upon the formation of lipid bilayers. The cross-linked lipids span the lipid bilayer, resembling naturally occurring bolaamphiphiles that stabilize archaebacterial membranes against high temperatures. The three reactions investigated here include the acyl chain cross-linking between thiol and bromine groups, thiol and acryloyl groups, and cyclopentadiene and acryloyl groups. All three reactive lipid pairs were found to cross-link in liposomal membranes, as determined by thin-layer chromatography, ion-spray mass spectrometry, and 1H NMR. The monolayer film properties of the reactive amphiphiles were characterized by surface pressure-area isotherms and showed that stable monolayers formed at the air-water interface with limiting molecular areas comparable to that of pure saturated phosphatidylcholine lipids. Langmuir-Blodgett bilayers of dimyristoylphosphatidylcholine incorporating 15 mol % of the reactive thiol and acryloyl lipids had diffusion coefficients comparable with pure dimyristoylphosphatidylcholine, while bilayers with more than 25 mol % of the reactive lipids were immobile, suggesting that interleaflet cross-linking of the lipids inhibited membrane diffusion. Our results show that the reactive lipids can cross-link within a lipid bilayer and are suitable for assembling supported lipid bilayers using Langmuir-Blodgett deposition. By using terminally reactive amphiphiles to build up supported lipid bilayers with cross-linked leaflets, bolaamphiphiles can be incorporated into asymmetric solid supported membranes to increase their stability in biosensor and medical implant applications.     

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.