Diffusive dynamics of vesicles tethered to a fluid supported bilayer by single-particle tracking

We recently introduced a method to tether intact phospholipid vesicles onto a fluid supported lipid bilayer using DNA hybridization (Yoshina-Ishii, C.; Miller, G. P.; Kraft, M. L; Kool, E. T.; Boxer, S. G. J. Am. Chem. Soc. 2005, 127, 1356-1357). Once tethered, the vesicles can diffuse in two dimensions parallel to the supported membrane surface. The average diffusion coefficient, D, is typically 0.2 microm(2)/s; this is 3-5 times smaller than for individual lipid or DNA-lipid conjugate diffusion in supported bilayers. In this article, we investigate the origin of this difference in the diffusive dynamics of tethered vesicles by single-particle tracking under collision-free conditions. D is insensitive to tethered vesicle size from 30 to 200 nm, as well as a 3-fold change in the viscosity of the bulk medium. The addition of macromolecules such as poly(ethylene glycol) reversibly stops the motion of tethered vesicles without causing the exchange of lipids between the tethered vesicle and supported bilayer. This is explained as a depletion effect at the interface between tethered vesicles and the supported bilayer. Ca ions lead to transient vesicle-vesicle interactions when tethered vesicles contain negatively charged lipids, and vesicle diffusion is greatly reduced upon Ca ion addition when negatively charged lipids are present both in the supported bilayer and tethered vesicles. Both effects are interesting in their own right, and they also suggest that tethered vesicle-supported bilayer interactions are possible; this may be the origin of the reduction in D for tethered vesicles. In addition, the effects of surface defects that reversibly trap diffusing vesicles are modeled by Monte Carlo simulations. This shows that a significant reduction in D can be observed while maintaining normal diffusion behavior on the time scale of our experiments.     

Ultra-high vacuum surface analysis study of rhodopsin incorporation into supported lipid bilayers

Planar supported lipid bilayers that are stable under ambient atmospheric and ultra-high-vacuum conditions were prepared by cross-linking polymerization of bis-sorbylphosphatidylcholine (bis-SorbPC). X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were employed to investigate bilayers that were cross-linked using either redox-initiated radical polymerization or ultraviolet photopolymerization. The redox method yields a more structurally intact bilayer; however, the UV method is more compatible with incorporation of transmembrane proteins. UV polymerization was therefore used to prepare cross-linked bilayers with incorporated bovine rhodopsin, a light-activated, G-protein-coupled receptor (GPCR). A previous study (Subramaniam, V.; Alves, I. D.; Salgado, G. F. J.; Lau, P. W.; Wysocki, R. J.; Salamon, Z.; Tollin, G.; Hruby, V. J.; Brown, M. F.; Saavedra, S. S. J. Am. Chem. Soc. 2005, 127, 5320-5321) showed that rhodopsin retains photoactivity after incorporation into UV-polymerized bis-SorbPC, but did not address how the protein is associated with the bilayer. In this study, we show that rhodopsin is retained in supported bilayers of poly(bis-SorbPC) under ultra-high-vacuum conditions, on the basis of the increase in the XPS nitrogen concentration and the presence of characteristic amino acid peaks in the ToF-SIMS data. Angle-resolved XPS data show that the protein is inserted into the bilayer, rather than adsorbed on the bilayer surface. This is the first study to demonstrate the use of ultra-high-vacuum techniques for structural studies of supported proteolipid bilayers.     

Bilayer lipid membrane formation on a chemically modified S-layer lattice

The present paper describes the generation of a biomimetic model lipid membrane on bacterial surface (S-)layer which covered the entire surface of various sensors. The S-layer lattice allows one to be independent from the underlying solid material and provides a biological surface and anchoring structure for lipid membranes. S-layer proteins were chemically modified via binding of two amine-terminated phospholipids. Subsequently, a bimolecular lipid membrane anchored to the previously generated viscoelastic lipid monolayer was generated by the rapid solvent exchange technique. Characterization of the intermediate (monolayer) and final membrane structures (bilayer) was performed by imaging, surface-sensitive, and electrochemical techniques. This bilayer lipid membrane generated on an S-layer lattice revealed a thickness of ～6 nm and constitutes a stable supported model membrane system with highly isolating properties showing a membrane resistance of 8.5 MΩ × cm(2).     

Lateral mobility of tethered vesicle-DNA assemblies

Supported lipid membranes are particularly attractive for use in biochemical assays because of their resistance to nonspecific adsorption and their unique ability to host transmembrane proteins. Although ideal for use in many surface-based detection techniques, supported bilayers can make the incorporation of proteins problematic due to the steric constraints of the underlying substrate. A recently developed strategy overcomes this obstacle by tethering liposomes to supported lipid bilayers via cholesterol-tagged DNA. Due to the fluidity of the bilayer, the vesicle assemblies exhibited significant lateral mobility. The corresponding diffusion coefficients were then investigated using fluorescence recovery after photobleaching (FRAP). The diffusivity was neither sensitive to the size of the vesicles nor to the length of the DNA tether. However, changing from single cholesterol tethers to double cholesterol tethers caused a decrease in the diffusivity of the assemblies by a factor of 3. Perhaps even more notable was the fact that single cholesterol-DNA without vesicles diffused 6 times faster than the corresponding assemblies. Double cholesterol-DNA diffused 11 times faster. This discrepancy is believed to arise from the fact that each vesicle is tethered to the bilayer by multiple DNA pairs.     

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.     

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.     

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。     

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.     

Surface tension effect on transmembrane channel stability in a model membrane

The effect of surface tension on the lipid bilayer membrane is a question that has drawn considerable research effort. This interest has been driven both by the desire to determine the surface tension effects on the lipid bilayer and from the suggestion that adding finite surface tension to a small membrane system may provide more realistic lipid properties in molecular dynamics simulations. Here, the effect of surface tension on a palmitololelylphosphatidylcholine (POPC) bilayer membrane containing a four-helix transmembrane alamethicin peptide bundle is investigated. Simulations of 10 ns were undertaken for two different ensembles, NPT and NP(z)gammaT with a surface tension, gamma, of 20 mN m(-1) per interface, which is near the pore-forming region. The significance of differences between the tension-free and surface tension simulations was determined using nonparametric statistical analysis on replicate simulations with different initial conditions. The results suggest that, when the membrane is under surface tension, the peptide helical structure is perturbed from that in the tension-free state but that the bundle conformation is more stable than that in the tension-free state, with hydrogen bonding playing an important stabilizing role. Surface tension counteracts the influence of the transmembrane helix bundle on nearby lipid order, making the lipid order more uniform throughout the membrane in the tension state. Conversely, the lipid mobility was less uniform in the tension state, with lipids far from the bundle being significantly more mobile than those near the bundle. One general implication of the results is that surface tension can affect the membrane nonuniformly, in that the properties of lipids near the peptide are different from those further away.     

Lipid bilayer deposition and patterning via air bubble collapse

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

Controlling two-dimensional tethered vesicle motion using an electric field: interplay of electrophoresis and electro-osmosis

We recently introduced methods to tether phospholipid vesicles or proteoliposomes onto a fluid-supported lipid bilayer using DNA hybridization (Yoshina-Ishii, C.; Miller, G. P.; Kraft, M. L.; Kool, E. T.; Boxer, S. G. J. Am. Chem. Soc. 2005, 127, 1356-1357). These intact tethered vesicles diffuse in two dimensions parallel to the supporting membrane surface. In this article, we report the dynamic response of individual tethered vesicles to an electric field applied parallel to the bilayer surface. Vesicles respond to the field by moving in the direction of electro-osmotic flow, and this can be used to reversibly concentrate tethered vesicles against a barrier. By adding increasing amounts of negatively charged phosphatidylserine to the supporting bilayer to increase electro-osmosis, the electrophoretic mobility of the tethered vesicles can be increased. The electro-osmotic contribution can be modeled well by a sphere connected to a cylindrical anchor in a viscous membrane with charged headgroups. The electrophoretic force on the negatively charged tethered vesicles opposes the electro-osmotic force. By increasing the amount of negative charge on the tethered vesicle, drift in the direction of electro-osmotic flow can be slowed; at high negative charge on the tethered vesicle, motion can be forced in the direction of electrophoresis. The balance between these forces can be visualized on a patterned supporting bilayer containing negatively charged lipids that reorganize in an externally applied electric field to create a gradient of charge within a corralled region. The charge gradient at the surface creates a gradient of electro-osmotic flow, and vesicles carrying similar amounts of negative charge can be focused to a region perpendicular to the applied field where electrophoresis is balanced by electro-osmosis, away from the corral boundary. Electric fields are effective tools to direct tethered vesicles and concentrate them and to measure the tethered vesicle's electrostatic properties.     

Investigation of finite system-size effects in molecular dynamics simulations of lipid bilayers

In the absence of external stress, the surface tension of a lipid membrane vanishes at equilibrium, and the membrane exhibits long wavelength undulations that can be described as elastic (as opposed to tension-dominated) deformations. These long wavelength fluctuations are generally suppressed in molecular dynamics simulations of membranes, which have typically been carried out on membrane patches with areas <100 nm2 that are replicated by periodic boundary conditions. As a result, finite system-size effects in molecular dynamics simulations of lipid bilayers have been subject to much discussion in the membrane simulation community for several years, and it has been argued that it is necessary to simulate small membrane patches under tension to properly model the tension-free state of macroscopic membranes. Recent hardware and software advances have made it possible to simulate larger, all-atom systems allowing us to directly address the question of whether the relatively small size of current membrane simulations affects their physical characteristics compared to real macroscopic bilayer systems. In this work, system-size effects on the structure of a DOPC bilayer at 5.4 H2O/lipid are investigated by performing molecular dynamics simulations at constant temperature and isotropic pressure (i.e., vanishing surface tension) of small and large single bilayer patches (72 and 288 lipids, respectively), as well as an explicitly multilamellar system consisting of a stack of five 72-lipid bilayers, all replicated in three dimensions by using periodic boundary conditions. The simulation results are compared to X-ray and neutron diffraction data by using a model-free, reciprocal space approach developed recently in our laboratories. Our analysis demonstrates that finite-size effects are negligible in simulations of DOPC bilayers at low hydration, and suggests that refinements are needed in the simulation force fields.     

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.     

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.     

Surface response methodology for the study of supported membrane formation

We report on the investigations of the formation of the tethered lipid bilayer by vesicle deposition on amine-functionalized surfaces. The tethered bilayer was created by the deposition of egg-PC vesicles containing 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly-(ethyleneglycol)-N-hydroxysuccinimide as anchoring molecules on an amine-coated surface. This approach is an easy route for the formation of a biomimetic-supported membrane. A Doelhert experimental design was applied to determine the conditions leading to the formation of a continuous and defect-free tethered bilayer on different surfaces (gold and glass). Doehlert designs allow modeling of the experimental responses by second-order polynomial equations as a function of experimental factors. Four factors expected to influence bilayer formation were studied: the lipid concentration in the vesicle suspension, the mass percentage of anchoring molecules in the vesicles, the contact time between the vesicles and the surface, and the resting time of the membrane after buffer rinse. The optimization of the membrane preparation parameters was achieved by monitoring lipid assembly formation using surface plasmon resonance spectroscopy on gold and by fluorescence recovery after photobleaching on glass. Three characteristic responses were systematically measured: the bilayer thickness, the lipid diffusion coefficient, and the lipid mobile fraction. The simultaneous inspection of the three characteristics revealed that a restricted experimental domain leads to properties that are in accordance with a bilayer presence. The factors of this domain are a lipid concentration from 0.1 to 1 mg/mL, 4-8% of anchoring molecules in the vesicles, 1-4 h of contact time between vesicles and surface, and 21-24 h of resting time after buffer rinse. Under these conditions, a membrane having a lipid mass per surface between 545 +/- 5 and 590 +/- 10 ng/cm2, a diffusion coefficient of between 2.5 +/- 0.3 x 10(-8) and 3.60 +/- 0.5 x 10(-8) cm2/s, and a mobile fraction between 94 +/- 2 and 99 +/- 1% was formed. These findings were confirmed by atomic force microscopy observations, which showed the presence of a continuous and homogeneous bilayer in the determined experimental domain. This formation procedure presents many advantages; it provides an easily obtainable biomimetic membrane model for proteins studies and offers a versatile tethered bilayer because it can be adapted easily to various types of supports.     

Steady-state oxidation of cholesterol catalyzed by cholesterol oxidase in lipid bilayer membranes on platinum electrodes

Cholesterol oxidase is immobilized in electrode-supported lipid bilayer membranes. Platinum electrodes are initially modified with a self-assembled monolayer of thiolipid. A vesicle fusion method is used to deposit an outer leaflet of phospholipids onto the thiolipid monolayer forming a thiolipid/lipid bilayer membrane on the electrode surface. Cholesterol oxidase spontaneously inserts into the electrode-supported lipid bilayer membrane from solution and is consequently immobilized to the electrode surface. Cholesterol partitions into the membrane from buffer solutions containing cyclodextrin. Cholesterol oxidase catalyzes the oxidation of cholesterol by molecular oxygen, forming hydrogen peroxide as a product. Amperometric detection of hydrogen peroxide for continuous solution flow experiments are presented, where flow was alternated between cholesterol solution and buffer containing no cholesterol. Steady-state anodic currents were observed during exposures of cholesterol solutions ranging in concentration from 10 to 1000 μM. These data are consistent with the Michaelis-Menten kinetic model for oxidation of cholesterol as catalyzed by cholesterol oxidase immobilized in the lipid bilayer membrane. The cholesterol detection limit is below 1 μM for cholesterol solution prepared in buffered cyclodextrin. The response of the electrodes to low density lipoprotein solutions is increased upon addition of cyclodextrin. Evidence for adsorption of low density lipoprotein to the electrode surface is presented.     

Templated assembly of biomembranes on silica microspheres using bacteriorhodopsin conjugates as structural anchors

Membrane proteins are some of the most sophisticated molecules found in nature. These molecules have extraordinary recognition properties; hence, they represent a vast source of specialized materials with potential uses in sensing and screening applications. However, the strict requirement of the native lipid environment to preserve their structure and functionality presents an impediment in building biofunctional materials from these molecules. In general, the purification protocols remove the native lipid support structures found in the cellular environment that stabilize the membrane proteins. Furthermore, the membrane protein structure is often highly complex, typified by large, multisubunit complexes that not only span the lipid bilayer but also contain large (>2 nm) cytoplasmic and extracellular domains that protrude from the membrane. The present study is focused on using a biomimetic approach to build a stable, fluid microenvironment to be used to incorporate larger membrane proteins of interest into a tether-supported lipid bilayer membrane adequately spaced above a substrate passivated to liposome fusion and nonspecific adsorption. Our aim is to reintroduce the supporting structures of the native cell membrane using self-assembled supramolecular complexes constructed on microspheres in an artificial cytoskeleton motif. Central to our architecture is to utilize bacteriorhodopsin (bR), a transmembrane protein, as a biomembrane anchoring molecule to be tethered to surfaces of interest as a sparse structural element in the design. Compared to a typical lipid tether, which inserts into one leaflet of the lipid bilayer, bR anchoring provides an over 8-fold greater hydrophobic surface area in contact with the bilayer. In the work presented here, the silica microsphere surface was biofunctionalized with streptavidin to make it a suitable supporting interface. This was achieved by self-assembly of (p-aminophenyl)trimethoxysilane on the silica surface followed by subsequent conjugation of biotin-PEG3400 (PEG = poly(ethylene glycol) and PEG2000 for further passivation and the binding of streptavidin. We have conjugated bR with biotin-PEG3400 through amine-based coupling to use it as a tether. The biotin-PEG-bR conjugate was further labeled with Texas Red to facilitate localization via fluorescence imaging. Confocal microscopy was utilized to analyze the microsphere surface at different stages of surface modification by employing fluorescent staining techniques. Sparely tethered supported lipid bilayer membranes were constructed successfully on streptavidin-functionalized silica particles (5 mum) using a detergent-based method in which tethered bR nucleates self-assembly of the bilayer membrane. The fluidity of the supported membranes was analyzed using fluorescence recovery after photobleaching in confocal imaging detection mode. The phospholipid diffusion coefficients obtained from these studies indicated that nativelike fluidity was achieved in the tether-supported membranes, thus providing a prospective microenvironment for insertion of membrane proteins of interest.     

双层类脂膜及其在电化学生物传感器中的应用

详细评述了各种双层类脂膜包括传统的双层类脂膜（ＢＬＭ）、固体载体支撑的自组双层类脂膜（ｓ－ＢＬＭ）、固体载体支撑的混合双层类脂膜（ｅ－ＢＬＭ）的制备方法和特性,比较了其优缺点。介绍了双层类脂膜在电化学生物传感器中的应用,并展望了发展前景。     

Amphipols can support the activity of a membrane enzyme

Amphipathic polymers ("amphipols") were introduced several years ago (Tribet, C.; Audebert, R.; Popot, J.-L. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15047-15050) as an alternative method for solubilizing integral membrane proteins in stable, nativelike conformations. However, direct maintenance of full membrane protein functionality in amphipol solutions has not previously been demonstrated in the absence of added lipid or detergent. In this contribution, the first zwitterionic amphipol "PMAL-B-100" is introduced. PMAL-B-100 not only maintains membrane protein structure and solubility, but also supports the full catalytic activity of an integral membrane enzyme, diacylglycerol kinase, in the complete absence of additional lipid or detergent. All of the roles which a lipid bilayer normally plays in maintaining diacylglycerol kinase's structure and in facilitating catalysis are satisfied by the environment and interactions supplied by PMAL-B-100.     

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.