Formation and stability of a suspended biomimetic lipid bilayer on silicon submicrometer-sized pores

We report the fabrication of a thin silicon membrane with an array of micrometer and submicrometer pores that acts as a scaffold for suspending a lipid bilayer. We successfully deposited a lipid bilayer by the Langmuir-Blodgett method on a synthetic silicon membrane bearing arrays of pores with sizes of 1000, 650, and 300 nm. Topographic images obtained by AFM showed a suspended lipid film spanning the pores, whatever the pore size. Higher stability of bilayers supported on smaller pores was shown by AFM characterization. These results represent an important first step to creating a biomimetic environment to study cell membrane dynamics and/or in developing a biosensor.     

Fluorescence microscopy of the pressure-dependent structure of lipid bilayers suspended across conical nanopores

Glass and fused-quartz nanopore membranes containing a single conically shaped pore are promising solid supports for lipid bilayer ion-channel recordings due to the high inherent stability of lipid bilayers suspended across the nanopore orifice, as well as the favorable electrical properties of glass and fused quartz. Fluorescence microscopy is used here to investigate the structure of the suspended lipid bilayer as a function of the pressure applied across a fused-quartz nanopore membrane. When a positive pressure is applied across the bilayer, from the nanopore interior relative to the exterior bulk solution, insertion or reconstitution of operative ion channels (e.g., α-hemolysin (α-HL) and gramicidin) in the bilayer is observed; conversely, reversing the direction of the applied pressure results in loss of all channel activity, although the bilayer remains intact. The dependence of the bilayer structure on pressure was explored by imaging the fluorescence intensity from Nile red dye doped into suspended 1,2-diphytanoyl-sn-glycero-3-phosphocholine bilayers, while simultaneously recording the activity of an α-HL channel. The fluorescence images suggest that a positive pressure results in compression of the bilayer leaflets and an increase in the bilayer curvature, making it suitable for ion-channel formation and activity. At negative pressure, the fluorescence images are consistent with separation of the lipid leaflets, resulting in the observed loss of the ion-channel activity. The fluorescence data indicate that the changes in the pressure-induced bilayer structure are reversible, consistent with the ability to repeatedly switch the ion-channel activity on and off by applying positive and negative pressures, respectively.     

Electrical measurements of bilayer membranes formed by Langmuir-Blodgett deposition on single-crystal silicon

Bilayer membranes on solid supports are used for fundamental studies of biophysical properties and for the development of biosensors and other devices. Here we report on electrically addressable bilayer membranes formed by Langmuir-Blodgett (LB)-based deposition on single-crystal silicon. The incorporation of a polymer cushion ensures high lipid mobility in both the lower and upper leaflet, allowing the potential for combined investigations of electrical, structural, and dynamic characteristics of membrane-associated proteins. Impedance spectroscopy is used to demonstrate that the lipid bilayers are robust and reproducible with an impedance of about 10(4) Omega cm2 and a capacitance of about 0.8 microF cm(-2). The ability to characterize ion channels is demonstrated using the model system gramicidin. These results demonstrate that artificial bilayers formed by LB deposition have many unique advantages for electrical measurements of membranes and their components.     

Tip penetration through lipid bilayers in atomic force microscopy

In studies of solid supported lipid bilayers with atomic force microscopes (AFM) the force between tip and bilayer is routinely measured. During the approach of the AFM tip in aqueous electrolyte first a short-range repulsive force is observed. For many solid-like and some liquid-like lipid bilayers a subsequent break-through is observed. We observe such a break-through also for dioleoyloxypropyl-trimethylammonium chloride (DOTAP) which is expected to be liquid-like. Here we describe a model which assumes that the jump reflects the penetration of the AFM tip through the lipid bilayer. The model predicts a logarithmical dependence of the break-through force on the approaching velocity of the AFM tip. Two parameters are introduced: The ratio A/αV, α being a geometric factor, A being the area over which pressure is exerted on the bilayer, V the activation volume, and k₀, the rate of spontaneous formation of a hole in the lipid bilayer that is big enough to allow the break-through of the tip. Experiments with bilayers consisting of DOTAP and dioleoylphosphatidylserine (DOPS) show that the break-through forces behave in the predicted way. For DOTAP we obtain ratios A/αV of about 58 nm⁻¹ and rates k₀ ranging from 1.9×10⁻⁸ to 2.5×10⁻⁴ s⁻¹. For DOPS the corresponding values are 162 nm⁻¹ and 2.0 s⁻¹.     

Tethered bilayer lipid membranes self-assembled on mercury electrodes

In order to incorporate integral proteins in a functionally active state, metal-supported lipid bilayers must have a hydrophilic region interposed between the bilayer and the metal. This region is realized with a hydrophilic molecule terminating at one end with a sulfhydryl or disulfide group that anchors this "hydrophilic spacer" to the surface of a metal, such as gold or mercury. The other end of the hydrophilic spacer may be covalently linked to the polar head of a phospholipid molecule, giving rise to a supramolecule called "thiolipid" (TL). With respect to gold, mercury has the advantage of providing a defect-free and fluid surface to the self-assembling spacer. Hydrophilic spacers consisting of a polyethyleneoxy or a hexapeptide chain, as well as thiolipids derived from these spacers, were employed to fabricate mercury-supported lipid bilayers. The formation of a lipid bilayer on top of a self-assembled monolayer of a hydrophilic spacer, or of a single-lipid monolayer on top of a self-assembled monolayer of a thiolipid, was realized by simply immersing the coated mercury electrode into an aqueous solution across a lipid film previously spread on its surface at its spreading pressure. Particularly stable mercury-supported lipid bilayers were obtained by using thiolipids. The biomimetic properties of these lipid bilayers were tested by incorporating channel-forming polypeptides (gramicidin and melittin) and proteins (OmpF porin). The effect of the transmembrane potential on the function of these channels was estimated by using a simple electrostatic model of the mercury-solution interphase.     

Highly reproducible method of planar lipid bilayer reconstitution in polymethyl methacrylate microfluidic chip

We developed a highly reproducible method for planar lipid bilayer reconstitution using a microfluidic system made of a polymethyl methacrylate (PMMA) plastic substrate. Planar lipid bilayers are formed at apertures, 100 microm in diameter, by flowing lipid solution and buffer alternately into an integrated microfluidic channel. Since the amount and distribution of the lipid solution at the aperture determines the state of the lipid bilayer, controlling them precisely is crucial. We designed the geometry of the fluidic system so that a constant amount of lipid solution is distributed at the aperture. Then, the layer of lipid solution was thinned by applying an external pressure and finally became a bilayer when a pressure of 200-400 Pa was applied. The formation process can be simultaneously monitored with optical and electrical recordings. The maximum yield for bilayer formation was 90%. Using this technique, four lipid bilayers are formed simultaneously in a single chip. Finally, a channel current through gramicidin peptide ion channels was recorded to prove the compatibility of the chip with single molecule electrophysiology.     

Pore spanning lipid bilayers on mesoporous silica having varying pore size

Synthetic lipid bilayers have similar properties as cell membranes and have been shown to be of great use in the development of novel biomimicry devices. In this study, lipid bilayer formation on mesoporous silica of varying pore size, 2, 4, and 6 nm, has been investigated using quartz crystal microbalance with dissipation monitoring (QCM-D), fluorescent recovery after photo bleaching (FRAP), and atomic force microscopy (AFM). The results show that pore-spanning lipid bilayers were successfully formed regardless of pore size. However, the mechanism of the bilayer formation was dependent on the pore size, and lower surface coverages of adsorbed lipid vesicles were required on the surface having the smallest pores. A similar trend was observed for the lateral diffusion coefficient (D) of fluorescently labeled lipid molecules in the membrane, which was lowest on the surface having the smallest pores and increased with the pore size. All of the pore size dependent observations are suggested to be due to the hydrophilicity of the surface, which decreases with increased pore size.     

Single molecule measurements within individual membrane-bound ion channels using a polymer-based bilayer lipid membrane chip

The measurement of single poly(ethylene glycol) (PEG) molecules interacting with individual bilayer lipid membrane-bound ion channels is presented. Measurements were performed within a polymer microfluidic system including an open-well bilayer lipid membrane formation site, integrated Ag/AgCl reference electrodes for on-chip electrical measurements, and multiple microchannels for independent ion channel and analyte delivery. Details of chip fabrication, bilayer membrane formation, and alpha-hemolysin ion channel incorporation are discussed, and measurements of interactions between the membrane-bound ion channels and single PEG molecules are presented.     

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.     

膜片电极支撑自组装双层脂膜中短杆菌肽离子通道的形成

通过一个两步程序在膜片电极尖端形成自组装双层脂膜：（1）膜片电极尖端沾取成膜液；（2）将吸附成膜液的尖端浸入电解液中，排除尖端多余的成膜液，通过电学方法监测双层脂膜的形成。将短杆菌肽通道蛋白分散在成膜液和电解质溶液中，在制备膜片电极支撑双层脂膜过程中，短杆菌肽重组到双层脂膜中形成离子通道，对通道的一般特性进行了研究，并观察到通道开放和关闭的现象。     

Formation of individual protein channels in lipid bilayers suspended in nanopores

Free-standing lipid bilayers are formed in regularly arranged nanopores of 200, 400 and 800 nm in a 300 nm thin hydrophobic silicon nitride membrane separating two fluid compartments. The extraordinary stability of the lipid bilayers allows us to monitor channel formation of the model peptide melittin and α-hemolysin from Staphylococcus aureus using electrochemical impedance spectroscopy and chronoamperometry. We observed that melittin channel formation is voltage-dependent and transient, whereas transmembrane heptameric α-hemolysin channels in nano-BLMs persist for hours. The onset of α-hemolysin-mediated conduction depends on the applied protein concentration and strongly on the diameter of the nanopores. Heptameric channel formation from adsorbed α-hemolysin monomers needs more time in bilayers suspended in 200 nm pores compared to bilayers in pores of 400 and 800 nm diameters. Diffusion of sodium ions across α-hemolysin channels present in a sufficiently high number in the bilayers was quantitatively and specifically determined using ion selective electrodes. The results demonstrate that relatively small variations of nano-dimensions have a tremendous effect on observable dynamic biomolecular processes. Such nanopore chips are potentially useful as supports for stable lipid bilayers to establish functional assays of membrane proteins needed in basic research and drug discovery.     

Direct introduction of single protein channels and pores into lipid bilayers

We have developed a mechanical method for inserting single pores and channels into lipid bilayers. A hand-operated hydrogel probe, coated with a layer of proteins, is mechanically engaged with the lipid bilayer. The two major classes of membrane proteins (beta barrels and alpha-helix bundles) that can be inserted, thereby demonstrating the wide applicability of the approach. Recordings from the proteins show that they retain electrical properties that are the same as those of proteins inserted from solution. Protein-loaded probes can be used repeatedly, allowing individual pores to be rapidly screened one at a time. The method has implications for fundamental studies of cell membranes, array fabrication, and chemical screening.     

Role of nanomechanical properties in the tribological performance of phospholipid biomimetic surfaces

The role of phospholipid bilayers in controlling and reducing frictional forces between biological surfaces is investigated by three complementary experiments: friction forces are measured using a homemade tribometer, mechanical resistance to indentation is measured by AFM, and lipid bilayer degradation is controlled in situ during friction testing using fluorescence microscopy. DPPC lipid bilayers in the solid phase generate friction coefficients as low as 0.002 (comparable to that found for cartilage) that are stable through time. DOPC bilayers formed by the vesicle fusion method or the adsorption of mixed micelles generate higher friction coefficients. These coefficients increased through time, during which the bilayers degraded. The friction coefficient is correlated with the force needed to penetrate the bilayer with the AFM tip. With only one bilayer in the contact region, the friction increased to a similar value of about 0.08 for the DPPC and DOPC. Our study therefore shows that good mechanical stability of the bilayers is essential and suggests that the low friction coefficient is ensured by the hydration layers between adjacent lipid bilayers.     

Incorporation of alpha-hemolysin in different tethered bilayer lipid membrane architectures

Tethered bilayer lipid membranes are stable solid supported model membrane systems. They can be used to investigate the incorporation and function of membrane proteins. In order to study ion translocation mediated via incorporated proteins, insulating membranes are necessary. The architecture of the membrane can have an important effect on both the electrical properties of the lipid bilayer as well as on the possibility to functionally host proteins. Alpha-hemolysin pores have been functionally incorporated into a tethered bilayer lipid membrane coupled to a gold electrode. The protein incorporation has been monitored optically and electrically and the influence of the molecular structure of the anchor lipids on the insertion properties has been investigated.     

Asymmetric droplet interface bilayers

In cell membranes, the lipid compositions of the inner and outer leaflets differ. Therefore, a robust model system that enables single-channel electrical recording with asymmetric bilayers would be very useful. We and others recently developed the droplet interface bilayer (DIB), which is formed by connecting lipid monolayer-encased aqueous droplets submerged in an oil-lipid mixture. Here, we incorporate lipid vesicles of different compositions into aqueous droplets and immerse them in an oil bath to form asymmetric DIBs (a-DIBs). Both alpha-helical and beta-barrel membrane proteins insert readily into a-DIBs, and their activity can be measured by single-channel electrical recording. We show that the gating behavior of outer membrane protein G (OmpG) from Escherichia coli differs depending on the side of insertion in an asymmetric DIB with a positively charged leaflet opposing a negatively charged leaflet. The a-DIB system provides a general platform for studying the effects of bilayer leaflet composition on the behavior of ion channels and pores.     

Directional Potassium Transport through a Unimolecular Peptide Channel

Three unimolecular peptide channels have been designed and prepared by using the β‐helical conformation of gramicidin A (gA). The new peptides bear one to three NH₃⁺ groups at the N‐end and one to three CO₂^? groups at the C‐end. These zwitterionic peptides were inserted into lipid bilayers in an orientation‐selective manner. Conductance experiments on planar lipid bilayers showed that this orientation bias could lead to observable directional K⁺ transport under multi‐channel conditions. This directional transport behavior can further cause the generation of a current across a planar bilayer without applying a voltage. More importantly, in vesicles with identical external and internal KCl concentrations, the channels can pump K⁺ across the lipid bilayer and cause a membrane potential.     

非支撑磷脂双层膜制备及温度对其力学性质的影响

结合聚苯乙烯球刻蚀和微机电系统技术加工氮化硅纳米多孔膜, 并在其上用囊泡法制备非支撑磷脂双层膜, 通过温控原子力显微术(AFM)的成像模式和力曲线模式对非支撑磷脂双层膜的形貌和力学性质进行研究. 实验结果表明, 该方法制备的非支撑磷脂双层膜具有流动性, 能进行自我修复, 该特点有利于提供足够的非支撑磷脂双层膜区域用于其性质研究; 非支撑磷脂双层膜的膜破力和粘滞力均随着温度的升高而减小, 即膜的机械稳定性随着温度的升高而降低. 非支撑磷脂双层膜膜破力小于支撑磷脂双层膜的膜破力, 并且非支撑磷脂双层膜粘滞力随温度的变化趋势与支撑磷脂双层膜的变化趋势相反.     

Ultrathin spin-coated dioleoylphosphatidylcholine lipid layers in dry conditions: a combined atomic force microscopy and nanomechanical study

Atomic force microscopy (AFM) has been used to study the structural and mechanical properties of low concentrated spin-coated dioleoylphosphatidylcholine (DOPC) layers in dry environment (RH ≈ 0%) at the nanoscale. It is shown that for concentrations in the 0.1-1 mM range the structure of the DOPC spin-coated samples consists of an homogeneous lipid monolayer ～1.3 nm thick covering the whole substrate on top of which lipid bilayer (or multilayer) micro- and nanometric patches and rims are formed. The thickness of the bilayer structures is found to be ～4.5 nm (or multiples of this value for multilayer structures), while the lateral dimensions range from micrometers to tens of nanometer depending on the lipid concentration. The force required to break a bilayer (breakthrough force) is found to be ～0.24 nN. No dependence of the mechanical values on the lateral dimensions of the bilayer structures is evidenced. Remarkably, the thickness and breakthrough force values of the bilayers measured in dry environment are very similar to values reported in the literature for supported DOPC bilayers in pure water.     

AFM-based force-clamp monitors lipid bilayer failure kinetics

The lipid bilayer rupture phenomenon is here explored by means of atomic force microscopy (AFM)-based force clamp, for the first time to our knowledge, to evaluate how lipid membranes respond when compressed under an external constant force, in the range of nanonewtons. Using this method, we were able to directly quantify the kinetics of the membrane rupture event and the associated energy barriers, for both single supported bilayers and multibilayers, in contradistinction to the classic studies performed at constant velocity. Moreover, the affected area of the membrane during the rupture process was calculated using an elastic deformation model. The elucidated information not only contributes to a better understanding of such relevant process, but also proves the suitability of AFM-based force clamp to study model structures as lipid bilayers. These findings on the kinetics of lipid bilayers rupture could be extended and applied to the study of other molecular thin films. Furthermore, systems of higher complexity such as models mimicking cell membranes could be studied by means of AFM-based force-clamp technique.     

Atomic force microscopy assisted immobilization of lipid vesicles

We report on a new approach to direct the immobilization of unilamellar lipid vesicles on substrate-supported lipid bilayers in a spatially confined manner. The adsorption of vesicles from solution is limited to areas of disorder in the bilayers, which is induced by scanning a pattern in situ with an atomic force microscopy (AFM) tip using high imaging forces. Lines of vesicles with a length exceeding 25 microm and a width corresponding to that of a single surface-immobilized vesicle have been fabricated. The adsorbed vesicles are effectively immobilized and do not desorb spontaneously. However, AFM with forces of several nanoNewtons allows one to displace vesicles selectively. The novel methodology described, which may serve as a platform for research on proteins incorporated in the lipid bilayers comprising the vesicles, does not require chemical labeling of the vesicles to guide their deposition.