Atomic force microscopy imaging and electrical recording of lipid bilayers supported over microfabricated silicon chip nanopores: lab-on-a-chip system for lipid membranes and ion channels

We describe a silicon chip-based supported bilayer system to detect the presence of ion channels and their electrical conductance in lipid bilayers. Nanopores were produced in microfabricated silicon membranes by electron beam lithography as well as by using a finely focused ion beam. Thermal oxide was used to shrink pore sizes, if necessary, and to create an insulating surface. The chips with well-defined pores were easily mounted on a double-chamber plastic cell recording system, allowing for controlling the buffer conditions both above and below the window. The double-chamber system allowed using an atomic force microscopy (AFM) tip as one electrode and inserting a platinum wire as the second electrode under the membrane window, to measure electrical current across lipid bilayers that are suspended over the pores. Atomic force imaging, stiffness measurement, and electrical capacitance measurement show the feasibility of supporting lipid bilayers over defined nanopores: a key requirement to use any such technique for structure-function study of ion channels. Online addition of gramicidin, an ion-channel-forming peptide, resulted in electrical current flow across the bilayer, and the I-V curve that was measured using the conducting AFM tip indicates the presence of many conducting gramicidin ion channels.     

脂双层膜表面结构与稳定性的原子力显微镜研究

用原子力显微镜研究了1,2－二油酸甘油－3－磷酸－1甘油（DOPG）脂双层膜 的表面结构与稳定性。实验结果表明,原子力显微镜的探针与脂双层膜的相互作用 导致脂双层膜表面产生一个永久的损伤。静电相互作用对脂双层膜结构和稳定性的 影响表明,在NaCl溶液中制成的脂质体,随着NaCl浓度的增加,它们的双层膜更稳 定。在低的NaCl浓度则经常被损伤,在1 mol/L NaCl溶液中制备的指双层变得更稳 定。在KCl溶液中结果恰好相反。在高的KCl浓度中经常被损伤,随着KCl浓度的降 低,它们的双层膜更稳定。葡萄糖和蔗糖对脂双层膜结构有稳定作用。     

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.     

Creation of lipid partitions by deposition of amphipathic viral peptides

Phospholipid vesicles exhibit a natural characteristic to fuse and reform into a continuous single bilayer membrane on hydrophilic solid substrates such as glass, mica, and silica. The resulting solid-supported bilayer mimics physiological tendencies such as lipid flip-flop and lateral mobility. The lateral mobility of fluorescently labeled lipids fused into solid-supported bilayers is found to change upon deposition on the membrane surface of an amphipathic alpha-helical peptide (AH) derived from the hepatitis C virus (HCV) NS5A protein. The binding of the AH peptide to a phospholipid bilayer, with the helical axis parallel to the bilayer, leads to immobilization of the bilayer. We used AFM to better understand the mechanistic details of this specific interaction, and determined that the diminished fluidity of the bilayer is due to membrane thinning. Utilizing this specific interaction between AH peptides and lipid molecules, we demonstrate a novel process for the creation of lipid partition by employing AH peptides as agents to immobilize lipid molecules, thus creating a patterned solid support with partition-defined areas of freely mobile lipid bilayers. This architecture could have a wide range of applications in novel sensing, biotechnology, high-throughput screening, and biomimetic strategies.     

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.     

Groove-spanning behavior of lipid membranes on microfabricated silicon substrates

We report on a spreading behavior of phospholipid membranes that arise from a lump of phospholipid (a lipid source) on topographically patterned substrates immersed in an aqueous solution. Microgrooves with well-defined shapes were prepared on Si111 surfaces by anisotropic etching in an alkaline solution. A spreading front that consists of membrane lobes and a single lipid bilayer was observed on the patterned silicon substrates by utilizing fluorescence interference contrast (FLIC) microscopy. FLIC images indicate that the membrane lobes span the microgrooves, while the underlying single lipid bilayer spread along the surface of the microgrooves. In fact, fluorescent polystyrene nanoparticles could be encapsulated in the microgrooves that were completely covered with the membrane lobes. The groove-spanning behavior of membrane lobes is discussed in terms of a balance between adhesion and bending energies of lipid 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.     

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.     

Pulling nanotubes from supported bilayers

The force required to form a nanoscale tube from a supported lipid bilayer (SLB) by pulling was measured using an atomic force microscope (AFM). The equilibrium membrane shape during an AFM pulling experiment was calculated and used to derive a general force-distance relationship for pulling a tube from an SLB. We compare these theoretical results with our experimental data and determine the tube radius, the force required to elongate the tube, and, consequently, the surface tension. For a dioleoylphosphatidylcholine (DOPC) SLB, the tension was found to be close to membrane rupture during the pulling experiment.     

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.     

Dynamics and state of lipid bilayer-internal water unraveled with solution state 1H dynamic nuclear polarization

The dynamics and state of lipid bilayer-internal hydration water of unilamellar lipid vesicles dispersed in solutions is characterized. This study was enabled by a recently developed technique based on Overhauser dynamic nuclear polarization (DNP)-driven amplification of (1)H nuclear magnetic resonance (NMR) signal of hydration water. This technique can, in the full presence of bulk water, selectively quantify the translational dynamics of hydration water within ～10 ? around spin labels that are specifically introduced to the local volume of interest within the lipid bilayer. With this approach, the local apparent diffusion coefficients of internal water at different depths of the lipid bilayer were determined. The modulation of these values as a response to external stimuli, such as the addition of sodium chloride or ethanol and the lipid phase transitions, that alter the fluctuations of bilayer interfaces together with the activation energy values of water diffusivity shows that water is not individually and homogeneously solvating lipid's hydrocarbon tails in the lipid bilayer. We provide experimental evidence that instead, water and the lipid membrane comprise a heterogeneous system whose constituents include transient hydrophobic water pores or water structures traversing the lipid bilayer. We show how these transient pore structures, as key vehicles for passive water transport can better reconcile our experimental data with existing literature data on lipid bilayer hydration and dynamics.     

Interactions of membrane active peptides with planar supported bilayers: an impedance spectroscopy study

Membrane active peptides exert their biological effects by interacting directly with a cell's lipid bilayer membrane. These therapeutically promising peptides have demonstrated a variety of activities including antimicrobial, cytolytic, membrane translocating, and cell penetrating activities. Here, we use electrochemical impedance spectroscopy (EIS) on polymer-cushioned supported lipid bilayers constructed on single crystal silicon to study two pairs of closely related membrane active peptides selected from rationally designed, combinatorial libraries to have different activities in lipid bilayers: translocation, permeabilization, or no activity. Using EIS, we observed that binding of a membrane translocating peptide to the lipid bilayer resulted in a small decrease in membrane resistance followed by a recovery back to the original value. The recovery may be directly attributable to peptide translocation. A nontranslocating peptide did not decrease the resistance. The other pair, two membrane permeabilizing peptides, caused an exponential decrease of membrane resistance in a concentration-dependent manner. This permeabilization of the supported bilayer occurs at peptide to lipid ratios as much as 1000-fold lower than that needed to observe effects in vesicle leakage assays and gives new insights into the fundamental peptide-bilayer interactions involved in membrane permeabilization.     

Mechanisms of Membrane Pore Formation by Amyloidogenic Peptides in Amyotrophic Lateral Sclerosis

Using unbiased atomic‐detailed molecular dynamics simulations, the C‐terminal fragments of TDP‐43 are observed to aggregate and form disordered‐toroidal pores in a lipid bilayer. Cytotoxicity of TDP‐43 may be inferred from the observation that the membrane pores catalyze lipid flip‐flop between bilayer leaflets and conduct water at high rates.     

Supported bilayer lipid membrane arrays on photopatterned self-assembled monolayers

This work demonstrates the use of photocleavable cholesterol derivatives to create supported bilayer lipid membrane arrays on silica. The photocleavable cholesteryl tether is attached to the surface by using the reaction of an amine-functionalized self-assembled monolayer (SAM) and the N-hydroxysuccinimide-based reagent 9. The resultant SAM contains an ortho-nitrobenzyl residue that can be cleaved by photolysis by using soft (365 nm) UV light regenerating the original amine surface, and which can be patterned using a mask. The photoreaction yield was approximately 75 % which was significantly higher than previously found for related ortho-nitrobenzyl photochemistry on gold substrates. The SAMs were characterized by means of contact angle measurements, ellipsometry and X-ray photoelectron spectroscopy. Patterned surfaces were characterized with SEM and AFM. After immersing the patterned surface into a solution containing small unilamellar vesicles of egg phosphatidylcholine (PC), supported lipid membranes were formed comprised of lipid bilayer over the amine functionalized "hydrophilic" regions and lipid monolayer over the cholesteryl "hydrophobic" regions. This was confirmed by fluorescence microscopy and AFM. FRAP studies yielded a lateral diffusion coefficient for the probe molecule of 0.14+/-0.05 microm(2) s(-1) in the bilayer regions and approximately 0.01 microm(2) s(-1) in the monolayer regions. This order of magnitude difference in diffusion coefficients effectively serves to isolate the bilayer regions from one another, thus creating a bilayer array.     

杂多酸K7Fe^3^+P2W17O62H2对硫醇-磷脂杂化双层膜通透性的影响

利用涂抹冷冻法制备了硫醇-磷脂杂化双层膜,采用循环伏安和交流阻抗方法,研究了硫醇-磷脂杂化双层膜与杂多酸K7Fe^3^+P2W17O62H2作用前后通透性的变化,发现该种杂多酸能够诱导硫醇-磷脂杂化双层膜产生一些孔洞,降低了膜电阻,增加了膜电容,也增加了探针Fe(CN)^3-/4-~6与电极的电子传递。同时对产生该现象的机理进行了初步的探讨。     

Determination of the lipid bilayer breakdown voltage by means of linear rising signal

Electroporation is characterized by formation of structural changes within the cell membrane, which are caused by the presence of electrical field. It is believed that "pores" are mostly formed in lipid bilayer structure; if so, planar lipid bilayer represents a suitable model for experimental and theoretical studies of cell membrane electroporation. The breakdown voltage of the lipid bilayer is usually determined by repeatedly applying a rectangular voltage pulse. The amplitude of the voltage pulse is incremented in small steps until the breakdown of the bilayer is obtained. Using such a protocol each bilayer is exposed to a voltage pulse many times and the number of applied voltage pulses is not known in advance. Such a pre-treatment of the lipid bilayer affects its stability and consequently the breakdown voltage of the lipid bilayer. The aim of this study is to examine an alternative approach for determination of the lipid bilayer breakdown voltage by linear rising voltage signal. Different slopes of linear rising signal have been used in our experiments (POPC lipids; folding method for forming in the salt solution of 100 mM KCl). The breakdown voltage depends on the slope of the linear rising signal. Results show that gently sloping voltage signal electroporates the lipid bilayer at a lower voltage then steep voltage signal. Linear rising signal with gentle slope can be considered as having longer pre-treatment of the lipid bilayer; thus, the corresponding breakdown voltage is lower. With decreasing the slope of linear rising signal, minimal breakdown voltage for specific lipid bilayer can be determined. Based on our results, we suggest determination of lipid bilayer breakdown voltage by linear rising signal. Better reproducibility and lower scattering are obtained due to the fact that each bilayer is exposed to electroporation treatment only once. Moreover, minimal breakdown voltage for specific lipid bilayer can be determined.     

Minimal F-actin cytoskeletal system for planar supported phospholipid bilayers

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

Using wavelets to analyze AFM images of thin films: surface micelles and supported lipid bilayers

This paper presents micro- and nanoanalysis of thin films based on images obtained by atomic force microscopy (AFM). The analysis exploits the discrete wavelet transform and the resulting wavelet spectrum to study surface features. It is demonstrated that the wavelet technique can characterize micro- and nanosurface features and distinguish between similar surface structures. The use of a feature extraction method is shown. The method involves the separation of certain frequency content from the original AFM images and analyzing the data independently to gain quantitative information about the images. By using the feature extraction method, soft surfaces in water are analyzed and nanofeatures are measured. The packing of surface micelles of sodium dodecyl sulfate on a self-assembled monolayer is analyzed. The characteristics of pore formation, due to penetration of the antibacterial peptide protegrin, into a solid-supported lipid bilayer are quantified. The sizes of the pores are obtained, and it is observed that the line tension of the pores reduces the fluctuations of the lipid bilayer.     

Forming Lipid Bilayer Membrane Arrays on Micropatterned Polyelectrolyte Film Surfaces

A novel method of forming lipid bilayer membrane arrays on micropatterned polyelectrolyte film surfaces is introduced. Polyelectrolyte films were fabricated by the layer‐by‐layer technique on a silicon oxide surface modified with a 3‐aminopropyltriethoxysilane (APTES) monolayer. The surface pK_a value of the APTES monolayer was determined by cyclic voltammetry to be approximately 5.61, on the basis of which a pH value of 2.0 was chosen for layer‐by‐layer assembly. Micropatterned polyelectrolyte films were obtained by deep‐UV (254 nm) photolysis though a mask. Absorbed fluorescent latex beads were used to visualize the patterned surfaces. Lipid bilayer arrays were fabricated on the micropatterned surfaces by immersing the patterned substrates into a solution containing egg phosphatidylcholine vesicles. Fluorescence recovery after photobleaching studies yielded a lateral diffusion coefficient for probe molecules of 1.31±0.17 μm² s^?1 in the bilayer region, and migration of the lipid NBD PE in bilayer lipid membrane arrays was observed in an electric field.     

The analytical potential of chemoreception at bilayer lipid membranes

The potential use of the bilayer lipid membrane as an electrochemical sensor is discussed through a study of model systems known to cause increased membrane conductance. The limit of detection for amphotericin B, a molecule capable of forming membrane pores, is in the region of 1O^-9 M. The current—time profile is discussed in terms of a mechanism which involves micelle formation in the aqueous and lipid phases. Unlike previous experiments, two current maxima with time are observed for valinomycin response (limit of detection 1O^-11 M). The first transient signal is attributed to increased membrane permeability caused by a conformational change in valinomycin in the “surface” volume of the bilayer. Selective interactions at membranes and the nature of membrane responses are discussed in terms of analytical parameters.