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.     

Formation and diffusivity characterization of supported lipid bilayers with complex lipid compositions

The moving edge of a hydrodynamically manipulated supported lipid bilayer (SLB) can be used to catalyze SLB formation of adsorbed lipid vesicles that do not undergo spontaneous SLB formation upon adsorption on SiO(2). By removing the lipid reservoir of an initially formed SLB, we show how a hydrodynamically moved SLB patch composed of POPC can be used to form isolated SLBs with compositions that to at least 95% represent that of the adsorbed lipid vesicles. The concept is used to investigate the diffusivity of lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (rhodamine-DHPE) in SLBs made from complex lipid compositions, revealing a decrease in diffusivity by a factor of 2 when the cholesterol content was increased from 0% to 50%. We also demonstrate how the concept can be used to induce stationary domains in SLBs containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol (39:21:40 mol %, respectively). Because the method serves as a means to form SLBs with lipid compositions that hamper SLB formation via spontaneous rupture of adsorbed lipid vesicles, it opens up the possibility for new biophysical investigations of SLBs with more nativelike compositions.     

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.     

L‐Tryptophan‐Induced Electron Transport across Supported Lipid Bilayers: an Alkyl‐Chain Tilt‐Angle,and Bilayer‐Symmetry Dependence

Molecular orientation‐dependent electron transport across supported 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC) lipid bilayers (SLBs) on semiconducting indium tin oxide (ITO) is reported with an aim towards potential nanobiotechnological applications. A bifunctional strategy is adopted to form symmetric and asymmetric bilayers of DPPC that interact with L ‐tryptophan, and are analyzed by surface manometry and atomic force microscopy. Polarization‐dependent real‐time Fourier transform infrared reflection absorption spectroscopy (FT‐IRRAS) analysis of these SLBs reveals electrostatic, hydrogen‐bonding, and cation–π interactions between the polar head groups of the lipid and the indole side chains. Consequently, a molecular tilt arises from the effective interface dipole, facilitating electron transport across the ITO‐anchored SLBs in the presence of an internal Fe(CN)₆^4?/3? redox probe. The incorporation of tryptophan enhances the voltammetric features of the SLBs. The estimated electron‐transfer rate constants for symmetric and asymmetric bilayers (k_s=2.0×10^?2 and 2.8×10^?2 s^?1) across the two‐dimensional (2D) ordered DPPC/tryptophan SLBs are higher compared to pure DPPC SLBs (k_s=3.2×10^?3 and 3.9×10^?3 s^?1). In addition, they are molecular tilt‐dependent, as it is the case with the standard apparent rate constants ${k_{{\rm{app}}}^0 }$ , estimated from electrochemical impedance spectroscopy and bipotentiostatic experiments with a Pt ultramicroelectrode. Lower magnitudes of k_s and ${k_{{\rm{app}}}^0 }$ imply that electrochemical reactions across the ITO–SLB electrodes are kinetically limited and consequently governed by electron tunneling across the SLBs. Standard theoretical rate constants ${\left( {k_{{\rm{th}}}^0 } \right)}$ accrued upon electron tunneling comply with the potential‐independent electron‐tunneling coefficient β=0.15 Å^?1. Insulator–semiconductor transitions moving from a liquid‐expanded to a condensed 2D‐phase state of the SLBs are noted, adding a new dimension to their transport behavior. These results highlight the role of tryptophan in expediting electron transfer across lipid bilayer membranes in a cellular environment and can provide potential clues towards patterned lipid nanocomposites and devices.     

Calibration and Limits of Camera‐Based Fluorescence Correlation Spectroscopy: A Supported Lipid Bilayer Study

Camera‐based fluorescence correlation spectroscopy (FCS) approaches allow the measurement of thousands of contiguous points yielding excellent statistics and details of sample structure. Imaging total internal reflection FCS (ITIR‐FCS) provides these measurements on lipid membranes. Herein, we determine the influence of the point spread function (PSF) of the optical system, the laser power used, and the time resolution of the camera on the accuracy of diffusion coefficient and concentration measurements. We demonstrate that the PSF can be accurately determined by ITIR‐FCS and that the laser power and time resolution can be varied over a wide range with limited influence on the measurement of the diffusion coefficient whereas the concentration measurements are sensitive to changes in the measurement parameters. One advantage of ITIR‐FCS is that the measurement of the PSF has to be performed only once for a given optical setup, in contrast to confocal FCS in which calibrations have to be performed at least once per measurement day. Using optimized experimental conditions we provide diffusion coefficients for over ten different lipid membranes consisting of one, two and three constituents, measured in over 200000 individual correlation functions. Using software binning and thus the inherent advantage of ITIR‐FCS of providing multiple observation areas in a single measurement we test the FCS diffusion law and show how they can be complemented by the local information provided by the difference in cross‐correlation functions (ΔCCF). With the determination of the PSF by ITIR‐FCS and the optimization of measurement conditions ITIR‐FCS becomes a calibration‐free method. This allows us to provide measurements of absolute diffusion coefficients for bilayers with different compositions, which were stable over many different bilayer preparations over a time of at least one year, using a single PSF calibration.     

Continuous lipid bilayers derived from cell membranes for spatial molecular manipulation

Progress with respect to enrichment and separation of native membrane components in complex lipid environments, such as native cell membranes, has so far been very limited. The reason for the slow progress can be related to the lack of efficient means to generate continuous and laterally fluid supported lipid bilayers (SLBs) made from real cell membranes. We show in this work how the edge of a hydrodynamically driven SLB can be used to induce rupture of adsorbed lipid vesicles of compositions that typically prevent spontaneous SLB formation, such as vesicles made of complex lipid compositions, containing high cholesterol content or being derived from real cell membranes. In particular, upon fusion between the moving edge of a preformed SLB and adsorbed vesicles made directly from 3T3 fibroblast cell membranes, the membrane content of the vesicles was shown to be efficiently transferred to the SLB. The molecular transfer was verified using cholera toxin B subunit (CTB) binding to monosialoganglioside receptors (G(M1) and G(M3)), and the preserved lateral mobility was confirmed by spatial manipulation of the G(M1/M3)-CTB complex using a hydrodynamic flow. Two populations of CTB with markedly different drift velocity could be identified, which from dissociation kinetics data were attributed to CTB bound with different numbers of ganglioside anchors.     

Molecular Diffusion Measurement in Lipid Bilayers over Wide Concentration Ranges: A Comparative Study

Molecular diffusion in biological membranes is a determining factor in cell signaling and cell function. In the past few decades, three main fluorescence spectroscopy techniques have emerged that are capable of measuring molecular diffusion in artificial and biological membranes at very different concentration ranges and spatial resolutions. The widely used methods of fluorescence recovery after photobleaching (FRAP) and single‐particle tracking (SPT) can determine absolute diffusion coefficients at high (>100 μm^?2) and very low surface concentrations (single‐molecule level), respectively. Fluorescence correlation spectroscopy (FCS), on the other hand, is well‐suited for the intermediate concentration range of about 0.1–100 μm^?2. However, FCS in general requires calibration with a standard dye of known diffusion coefficient, and yields only relative measurements with respect to the calibration. A variant of FCS, z‐scan FCS, is calibration‐free for membrane measurements, but requires several experiments at different well‐controlled focusing positions. A recently established FCS method, electron‐multiplying charge‐coupled‐device‐based total internal reflection FCS (TIR‐FCS), referred to here as imaging TIR‐FCS (ITIR–FCS), is also independent of calibration standards, but to our knowledge no direct comparison between these different methods has been made. Herein, we seek to establish a comparison between FRAP, SPT, FCS, and ITIR–FCS by measuring the lateral diffusion coefficients in two model systems, namely, supported lipid bilayers and giant unilamellar vesicles.     

Effect of cholesterol on diffusion in surfactant bilayers

Biological membranes consist of lipid bilayers with liquid-ordered and liquid-disordered phases. It is believed that cholesterol controls the size of the microdomains in the liquid-ordered phase and thereby affects the mobility as well as the permeability of the membrane. We study this process in a model system consisting of the nonionic surfactant C(12)E(5) and water in the lamellar phase. We measure the diffusion of fluorescent probe molecules (rhodamine B) by fluorescence correlation spectroscopy. For different surfactant to water ratios, we measure how the molecular mobility varies with the amount of cholesterol added. We find that a reduction of the diffusion coefficient is already detectable at a molar ratio of 8 mol % cholesterol.     

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.     

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.     

Quantifying lipid diffusion by fluorescence correlation spectroscopy: a critical treatise

Fluorescence correlation spectroscopy (FCS) measurements are widely used for determination of diffusion coefficients of lipids and proteins in biological membranes. In recent years, several variants of FCS have been introduced. However, a comprehensive comparison of these methods on identical systems has so far been lacking. In addition, there exist no consistent values of already determined diffusion coefficients for well-known or widely used membrane systems. This study aims to contribute to a better comparability of FCS experiments on membranes by determining the absolute diffusion coefficient of the fluorescent lipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD) in giant unilamellar vesicles (GUVs) made of dioleoylphosphatidylcholine (DOPC), which can in future studies be used as a reference value. For this purpose, five FCS variants, employing different calibration methods, were compared. Potential error sources for each particular FCS method and strategies to avoid them are discussed. The obtained absolute diffusion coefficients for DiD in DOPC were in good agreement for all investigated FCS variants. An average diffusion coefficient of D = 10.0 ± 0.4 μm(2) s(-1) at 23.5 ± 1.5 °C was obtained. The independent confirmation with different methods indicates that this value can be safely used for calibration purposes. Moreover, the comparability of the methods also in the case of slow diffusion was verified by measuring diffusion coefficients of DiD in GUVs consisting of DOPC and cholesterol.     

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.     

Drying and rehydration of DLPC/DSPC symmetric and asymmetric supported lipid bilayers: a combined AFM and fluorescence microscopy study

This work characterizes the impact of lipid symmetry/asymmetry on drying/rehydration reorganization in phase-separated dilauroylphosphatidylcholine (DLPC)/distearoylphosphatidylcholine (DSPC) supported lipid bilayers (SLBs) at the submicron and micron-scale. In addition the prevention of major drying/rehydration reorganization by the use of trehalose is demonstrated. Even though it was found using fluorescence microscopy that micrometer scale structure is preserved in the presence and absence of trehalose upon drying/rehydration, AFM and FRAP experiments successfully revealed major changes in the phase-separated structure such as defects, obstructions, lipid condensation, collapse structures, and complex incomplete DLPC-DSPC mixing/exchange in the absence of trehalose. In the presence of trehalose the membrane preserves its structure at the nanometer scale and mobility. We found that SLBs with asymmetric domain configurations underwent major rearrangements during drying and rehydration, whereas the symmetric domain configuration mainly rearranged during rehydration, that we hypothesize is related to lower transmembrane cohesiveness or lack of anchoring to the substrate in the case of the asymmetric domains.     

Separation and characterization of oxidized isomeric lipid–peptide adducts by ion mobility mass spectrometry

Phospholipids are major components of cell membranes and lipoprotein complexes. They are prone to oxidation by endogenous and exogenous reactive oxygen species yielding a large variety of modified lipids including small aliphatic and phospholipid bound aldehydes and ketones. These carbonyls are strong electrophiles that can modify proteins and, thereby, alter their structures and functions triggering various pathophysiological conditions. The analysis of lipid–protein adducts by liquid chromatography‐MS is challenged by their mixed chemical nature (polar peptide and hydrophobic lipid), low abundance in biological samples, and formation of multiple isomers. Thus, we investigated traveling wave ion mobility mass spectrometry (TWIMS) to analyze lipid–peptide adducts generated by incubating model peptides corresponding to the amphipathic β₁ sheet sequence of apolipoprotein B‐100 with 1‐palmitoyl‐2‐(oxo‐nonanoyl)‐sn‐glycerophosphatidylcholine (PONPC). The complex mixture of peptides, lipids, and peptide–lipid adducts was separated by TWIMS, which was especially important for the identification of two mono‐PONPC‐peptide isomers containing Schiff bases at different lysine residues. Moreover, TWIMS separated structural conformers of one peptide–lipid adduct possessing most likely different orientations of the hydrophobic sn‐1 fatty acyl residue and head group of PONPC, relative to the peptide backbone. Copyright © 2015 John Wiley & Sons, Ltd.     

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.

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).     

Characterization of Diblock Copolymer Order–Order Transitions in Semidilute Aqueous Solution Using Fluorescence Correlation Spectroscopy

The temperature and pH‐dependent diffusion of poly(glycerol monomethacrylate)‐block‐poly(2‐hydroxypropyl methacrylate) nanoparticles prepared via polymerization‐induced self‐assembly in water is characterized using fluorescence correlation spectroscopy (FCS). Lowering the solution temperature or raising the solution pH induces a worm‐to‐sphere transition and hence an increase in diffusion coefficient by a factor of between four and eight. FCS enables morphological transitions to be monitored at relatively high copolymer concentrations (10% w/w) compared to those required for dynamic light scattering (0.1% w/w). This is important because such transitions are reversible at the former concentration, whereas they are irreversible at the latter. Furthermore, the FCS data suggest that the thermal transition takes place over a very narrow temperature range (less than 2 °C). These results demon­strate the application of FCS to characterize order–order transitions, as opposed to order–disorder transitions.

     

Heterogeneous and anomalous diffusion inside lipid tubules

Self-assembled lipid tubules with crystalline bilayer walls are promising candidates for controlled drug delivery vehicles on the basis of their ability to release preloaded biological molecules in a sustained manner. While a previous study has shown that the release rate of protein molecules from lipid tubules depends on the associated molecular mass, suggesting that the pertinent diffusion follows the well-known Stokes-Einstein relationship, only a few attempts have been made toward investigating the details of molecular diffusion in the tubule interior. Herein, we have characterized the diffusion rates of several molecules encapsulated in lipid tubules formed by 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) using the techniques of fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). Our results show that the mobility of these molecules depends not only on their positions in the DC8,9PC tubules but also on their respective concentrations. While the former indicates that the interior of the DC8,9PC tubules is heterogeneous in terms of diffusion, the latter further highlights the possibility of engineering specific conditions for achieving sustained release of a "drug molecule" over a targeted period of time. In addition, our FCS results indicate that the molecular diffusions inside the crystalline bilayer walls of the DC8,9PC tubules strongly deviate from the normal, stochastic processes, with features characterizing not only anomalous subdiffusions but also motions that are superdiffusive in nature.     

A Monte Carlo simulation study of lipid bilayer formation on hydrophilic substrates from vesicle solutions

A general lattice Monte Carlo model is used for simulating the formation of supported lipid bilayers (SLBs) from vesicle solutions. The model, based on a previously published paper, consists of adsorption, decomposition, and lateral diffusion steps, and is derived from fundamental physical interactions and mass transport principles. The Monte Carlo simulation results are fit to experimental data at different vesicle bulk concentrations. A sensitivity analysis reveals that the process strongly depends on the bulk concentration C(0), adsorption rate constant K, and all vesicle radii parameters. A measure of "quality of coverage" is proposed. By this measure, the quality of the formed bilayers is found to increase with vesicle bulk concentration.     

Microcontact printing for creation of patterned lipid bilayers on tetraethylene glycol self-assembled monolayers

Supported lipid bilayers (SLBs) formed on many different substrates have been widely used in the study of lipid bilayers. However, most SLBs suffer from inhomogeneities due to interactions between the lipid bilayer and the substrate. In order to avoid this problem, we have used microcontact printing to create patterned SLBs on top of ethylene-glycol-terminated self-assembled monolayers (SAMs). Glycol-terminated SAMs have previously been shown to resist absorbance of biomolecules including lipid vesicles. In our system, patterned lipid bilayer regions are separated by lipid monolayers, which form over the patterned hexadecanethiol portions of the surface. Furthermore, we demonstrate that α-hemolysin, a large transmembrane protein, inserts preferentially into the lipid bilayer regions of the substrate.