固体支撑的自组装的双层类脂膜

具有通常BLM_s某些相似特性的固体支撑的双层类脂膜(S-BLM)能够通过两步自组装到新劈开的金属丝上面。如:(1)包有聚四氟乙烯的铂丝头部浸在类脂溶液里,用解剖刀把顶部切开;(2)包有类脂溶液的新铂丝末端转移到0.1mol/L KCl溶液里,电测定证实,数分钟后,在金属丝末端自动地形成了稳定的类脂双层。本文报道了这种固体支撑的BLM(S-BLM)在检测Pb²⁺离子中的应用。S-BLM为液晶结构,它可用于基础研究、生物传感器和分子电子器件等技术上的应用。     

A facile approach to immobilize protein for biosensor: self-assembled supported bilayer lipid membranes on glassy carbon electrode

A kind of solid substrate, glassy carbon (GC) electrode, was selected to support self-assembled lipid layer membranes. On the surface of GC electrode, we made layers of dimyristoylphosphatidylcholine (DMPG, a kind of lipid). From electrochemical impedance experiments, we demonstrated that the lipid layers on the GC electrode were bilayer lipid membranes. We immobilized horseradish peroxidase (HRP) into the supported bilayer lipid membranes (s-BLM) to develop a kind of mediator-free biosensor for H2O2. The biosensor exhibited fine electrochemical response, stability and reproducibility due to the presence of the s-BLM. As a model of biological membrane, s-BLM could supply a biological environment for enzyme and maintain its activity. So s-BLM is an ideal choice to immobilize enzyme for constructing the mediator-free biosensor based on GC electrode.     

Impedance spectroscopy of bilayer lipid membranes self-assembled on agar support - interaction with HDL

The electrical properties of the supported lipid bilayer membrane (s-BLM) of egg phosphatidylcholine (PC) self-assembled on agar surface were examined. To characterize the insulating properties of s-BLMs, electrochemical impedance spectroscopy (EIS) was used. The analysis of impedance spectra in terms of an equivalent circuit of agar/electrolyte and agar/s-BLM/electrolyte in the frequency range of 0.1 Hz-10 kHz was performed. The high-density lipoproteins (HDL)/s-BLM interaction in the concentration range from 20 microg/ml to 80 microg/ml of HDL was investigated. It is evident that treatment of s-BLM with HDL resulted in an increase of the lipid film resistance and a decrease of membrane capacitance.     

Supported bilayer lipid membranes modified with a phosphate ionophore

This article reports the electrical responses of a phosphate ionophore, the cyclic polyamine 3-decyl-1,5,8-triazacyclodecane-2,4-dione (N₃-cyclic amine) incorporated into metal supported bilayer lipid membranes (s-BLM). Teflon coated silver wire was used as a support. In a potentiometric mode, the ionophore had a response that was linearly related to the logarithm of HPO₄²⁻ concentration and was also dependant on pH. Selectivity coefficients for other anions compared to HPO₄²⁻ ions, determined by the separate solution method, fell within the range 1.73 × 10⁻⁴ to 6.38 × 10⁻².     

Conducting polymer polypyrrole supported bilayer lipid membranes

Electrochemically synthesized conducting polymer polypyrrole (PPy) film on gold electrode surface was used as a novel support for bilayer lipid membranes (BLMs). Investigations by surface plasmon resonance (SPR) suggest that dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and dimyristoyl-L-alpha-phosphatidyl-L-serine (DMPS) can form BLMs on PPy film surface but dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) and didodecyldimethylammonium bromide (DDAB) can not do so, indicating the formation of PPy supported bilayer lipid membranes (s-BLMs) is dependent on the chemical structure of the lipids used. The self-assembly of DMPC induces a smoother topography than the PPy layer with rms roughness decreasing from 4.484 to 2.914 nm convinced by atomic force microscopy (AFM). Impedance spectroscopy measurements confirm that the deposition of BLM substantially increases the resistance of the system indicating a very densely packed BLM structures. The little change of PPy film in capacitance shows that solvent and electrolyte ions still retain within the porous PPy film after BLM deposition. Therefore, the PPy supported BLM is to some extent comparable to conventional BLM with aqueous medium retaining at its two sides. As an example and preliminary application, horseradish peroxidase (HRP) reconstituted into the s-BLM shows the expected protein activity and can transfer electron from or to the underlying PPy support for its response to electrocatalytic reduction of hydrogen peroxide in solution. Thus the system maybe possesses potential applications to biomimetic membrane studies.     

Force measurements on myelin basic protein adsorbed to mica and lipid bilayer surfaces done with the atomic force microscope.

The mechanical and adhesion properties of myelin basic protein (MBP) are important for its function, namely the compaction of the myelin sheath. To get more information about these properties we used atomic force microscopy to study tip-sample interaction of mica and mixed dioleoylphosphatidylserine (DOPS) (20%)/egg phosphatidylcholine (EPC) (80%) lipid bilayer surfaces in the absence and presence of bovine MBP. On mica or DOPS/EPC bilayers a short-range repulsive force (decay length 1.0-1.3 nm) was observed during the approach. The presence of MBP always led to an attractive force between tip and sample. When retracting the tip again, force curves on mica and on lipid layers were different. While attached to the mica surface, the MBP molecules exhibited elastic stretching behavior that agreed with the worm-like chain model, yielding a persistence length of 0.5 +/- 0.25 nm and an average contour length of 53 +/- 19 nm. MBP attached to a lipid bilayer did not show elastic stretching behavior. This shows that the protein adopts a different conformation when in contact with lipids. The lipid bilayer is strongly modified by MBP attachment, indicating formation of MBP-lipid complexes and possibly disruption of the original bilayer structure.     

Probing membrane permeabilization by the antimicrobial peptide distinctin in mercury-supported biomimetic membranes

The mechanism of membrane permeabilization by the antimicrobial peptide distinctin was investigated by using two different mercury-supported biomimetic membranes, namely a lipid self-assembled monolayer and a lipid bilayer tethered to the mercury surface through a hydrophilic spacer (tethered bilayer lipid membrane: tBLM). Incorporation of distinctin into a lipid monolayer from its aqueous solution yields rapidly ion channels selective toward inorganic cations, such as Tl(+) and Cd(2+). Conversely, its incorporation in a tBLM allows the formation of ion channels permeable to potassium ions only at non-physiological transmembrane potentials, more negative than -340mV. These channels, once formed, are unstable at less negative transmembrane potentials. The kinetics of their formation is consistent with the disruption of distinctin clusters adsorbed on top of the lipid bilayer, incorporation of the resulting monomers and their aggregation into hydrophilic pores by a mechanism of nucleation and growth. Comparing the behavior of distinctin in tBLMs with that in conventional black lipid membranes strongly suggests that distinctin channel formation in lipid bilayer requires the partitioning of distinctin molecules between the two sides of the lipid bilayer. We can tentatively hypothesize that an ion channel is formed when one distinctin cluster on one side of the lipid bilayer matches another one on the opposite side.     

Dynamic force spectroscopy on supported lipid bilayers: effect of temperature and sample preparation

Biological membranes are constantly exposed to forces. The stress-strain relation in membranes determines the behavior of many integral membrane proteins or other membrane related-proteins that show a mechanosensitive behavior. Here, we studied by force spectroscopy the behavior of supported lipid bilayers (SLBs) subjected to forces perpendicular to their plane. We measured the lipid bilayer mechanical properties and the force required for the punch-through event characteristic of atomic force spectroscopy on SLBs as a function of the interleaflet coupling. We found that for an uncoupled bilayer, the overall tip penetration occurs sequentially through the two leaflets, giving rise to two penetration events. In the case of a bilayer with coupled leaflets, penetration of the atomic force microscope tip always occurred in a single step. Considering the dependence of the jump-through force value on the tip speed, we also studied the process in the context of dynamic force spectroscopy (DFS). We performed DFS experiments by changing the temperature and cantilever spring constant, and analyzed the results in the context of the developed theories for DFS. We found that experiments performed at different temperatures and with different cantilever spring constants enabled a more effective comparison of experimental data with theory in comparison with previously published data.     

Transmembrane domains of viral ion channel proteins: a molecular dynamics simulation study

Nanosecond molecular dynamics simulations in a fully solvated phospholipid bilayer have been performed on single transmembrane alpha-helices from three putative ion channel proteins encoded by viruses: NB (from influenza B), CM2 (from influenza C), and Vpu (from HIV-1). alpha-Helix stability is maintained within a core region of ca. 28 residues for each protein. Helix perturbations are due either to unfavorable interactions of hydrophobic residues with the lipid headgroups or to the need of the termini of short helices to extend into the surrounding interfacial environment in order to form H-bonds. The requirement of both ends of a helix to form favorable interactions with lipid headgroups and/or water may also lead to tilting and/or kinking of a transmembrane alpha-helix. Residues that are generally viewed as poor helix formers in aqueous solution (e.g., Gly, Ile, Val) do not destabilize helices, if located within a helix that spans a lipid bilayer. However, helix/bilayer mismatch such that a helix ends abruptly within the bilayer core destabilizes the end of the helix, especially in the presence of Gly and Ala residues. Hydrogen bonding of polar side-chains with the peptide backbone and with one another occurs when such residues are present within the bilayer core, thus minimizing the energetic cost of burying such side-chains.     

Nanomechanical recognition of sphingomyelin-rich membrane domains by atomic force microscopy

Sphingomyelin (SM) is a reservoir of signaling lipids and forms specific lipid domains in biomembranes together with cholesterol. In this study, atomic force microscopy (AFM) and force measurement were applied to investigate the interaction of SM-binding protein toxin, lysenin, with N-palmitoyl-D-erythro-sphingosylphosphorylcholine (palmitoyl sphingomyelin, PSM) bilayer spread over a mica substrate, in an aqueous buffer solution. Lysenin molecules were grafted on a silicon nitride tip for AFM by siloxane-thiol-amide coupling. The bilayers were prepared by the Langmuir-Blodgett (LB)/Langmuir-Schaefer (LS) method. By repeating cycles of tip approach/retraction motion, single-molecular adhesion motions were observed on the force curve, characterized as "fishing curves". The addition of cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) did not alter the peak force but increased the peak extension. Mixtures of PSM/DOPC/cholesterol exhibited 2-dimensional two-phase domain separation. The characteristic fishing curves were observed exclusively in one of the phases, indicating the selective interaction of the lysenin tip to PSM-rich membrane domains. Our results indicate that the AFM tips conjugated with lysenin are useful to detect the surface distribution of SM-rich membrane domains as well as the nanomechanical properties of the domains.     

Mechanism of the interaction of hydrophobically-modified poly-(N-isopropylacrylamides) with liposomes

Interactions of hydrophobically-modified poly-(N-isopropylacrylamides) (HM PNIPAM) with phospholipid liposomes were studied as a function of the lipid type, the lipid bilayer fluidity, and the polymer conformation. Fluorescence experiments monitoring non-radiative energy transfer (NRET), between naphthalene attached to the HM PNIPAM and 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into the lipid bilayer, confirmed the direct penetration of hydrophobic anchor groups linked to the polymer into the liposome hydrophobic core. Contraction of the polymer backbone above the lower critical solution temperature (LCST) resulted in a partial withdrawal of the anchor groups from the lipid bilayer. Analysis of polymer/lipid mixtures by centrifugation and quasi-elastic light scattering (QELS) revealed the polymer-induced fission of liposomes in the liquid-crystalline state, resulting in the formation of vesicles 150–230 nm in diameter. The process is reversible and upon transition of the bilayer into the gel state these vesicles are converted into larger aggregates. According to the results of gel-filtration experiments the HM PNIPAM is in dynamic exchange between the liquid-crystalline lipid bilayer and the water solution, while the binding to the bilayer in the gel state is more static in nature. The binding constant for mixture of HM PNIPAM with DMPC liposomes, evaluated from the centrifugation experiments, was found to be 120 M⁻¹.     

Material Properties of Lipid Microdomains: Force-Volume Imaging Study of the Effect of Cholesterol on Lipid Microdomain Rigidity

The effect of cholesterol (CHOL) on the material properties of supported lipid bilayers composed of lipid mixtures that mimic the composition of lipid microdomains was studied by force-volume (FV) imaging under near-physiological conditions. These studies were carried out with lipid mixtures of dioleoylphosphatidylcholine, dioleoylphosphatidylserine, and sphingomyelin. FV imaging enabled simultaneous topology and force measurements of sphingomyelin-rich domains (higher domain (HD)) and phospholipid-rich domains (lower domain (LD)), which allowed quantitative measurement of the force needed to puncture the lipid bilayer with or without CHOL. The force required to penetrate the various domains of the bilayer was probed using high- and low-ionic-strength buffers as a function of increasing amounts of CHOL in the bilayer. The progressive addition of CHOL also led to a decreasing height difference between HD and LD. FV imaging further demonstrated a lack of adhesion between the atomic force microscope tip and the HD or LD at loads below the breakthrough force. These results can lead to a better understanding of the role that CHOL plays in the mechanical properties of cellular membranes in modulating membrane rigidity, which has important implications for cellular mechanotransduction.     

Direct imaging of individual intrinsic hydration layers on lipid bilayers at Angstrom resolution

The interactions between water and biological molecules have the potential to influence the structure, dynamics, and function of biological systems, hence the importance of revealing the nature of these interactions in relation to the local biochemical environment. We have investigated the structuring of water at the interface of supported dipalmitoylphosphatidylcholine bilayers in the gel phase in phosphate buffer solution using frequency modulation atomic force microscopy (FM-AFM). We present experimental results supporting the existence of intrinsic (i.e., surface-induced) hydration layers adjacent to the bilayer. The force versus distance curves measured between the bilayer and the AFM tip show oscillatory force profiles with a peak spacing of 0.28 nm, indicative of the existence of up to two hydration layers next to the membrane surface. These oscillatory force profiles reveal the molecular-scale origin of the hydration force that has been observed between two apposing lipid bilayers. Furthermore, FM-AFM imaging at the water/lipid interface visualizes individual hydration layers in three dimensions, with molecular-scale corrugations corresponding to the lipid headgroups. The results demonstrate that the intrinsic hydration layers are stable enough to present multiple energy barriers to approaching nanoscale objects, such as proteins and solvated ions, and are expected to affect membrane permeability and transport.     

Effect of ion-binding and chemical phospholipid structure on the nanomechanics of lipid bilayers studied by force spectroscopy

The nanomechanical response of supported lipid bilayers has been studied by force spectroscopy with atomic force microscopy. We have experimentally proved that the amount of ions present in the measuring system has a strong effect on the force needed to puncture a 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer with an atomic force microscope tip, thus highlighting the role that monovalent cations (so far underestimated, e.g., Na(+)) play upon membrane stability. The increase in the yield threshold force has been related to the increase in lateral interactions (higher phospholipid-phospholipid interaction, decrease in area per lipid) promoted by ions bound into the membrane. The same tendency has also been observed for other phosphatidylcholine bilayers, namely, 2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dioleoyl-sn-3-phosphocholine, and also for phosphatidylethanolamine bilayers such as 1-palmitoyl-2-oleoyl-sn-3-phosphoethanolamine. Finally, this effect has been also tested on a natural lipid bilayer (Escherichia coli lipid extract), showing the same overall tendency. The kinetics of the process has also been studied, together with the role of water upon membrane stability and its effect on membrane nanomechanics. Finally, the effect of the chemical structure of the phospholipid molecule on the nanomechanical response of the membrane has also been discussed.     

Linking of sensor molecules with amino groups to amino-functionalized AFM tips

The measuring tip of an atomic force microscope (AFM) can be upgraded to a specific biosensor by attaching one or a few biomolecules to the apex of the tip. The biofunctionalized tip is then used to map cognate target molecules on a sample surface or to study biophysical parameters of interaction with the target molecules. The functionality of tip-bound sensor molecules is greatly enhanced if they are linked via a thin, flexible polymer chain. In a typical scheme of tip functionalization, reactive groups are first generated on the tip surface, a bifunctional cross-linker is then attached with one of its two reactive ends, and finally the probe molecule of interest is coupled to the free end of the cross-linker. Unfortunately, the most popular functional group generated on the tip surface is the amino group, while at the same time, the only useful coupling functions of many biomolecules (such as antibodies) are also NH(2) groups. In the past, various tricks or detours were applied to minimize the undesired bivalent reaction of bifunctional linkers with adjacent NH(2) groups on the tip surface. In the present study, an uncompromising solution to this problem was found with the help of a new cross-linker ("acetal-PEG-NHS") which possesses one activated carboxyl group and one acetal-protected benzaldehyde function. The activated carboxyl ensures rapid unilateral attachment to the amino-functionalized tip, and only then is the terminal acetal group converted into the amino-reactive benzaldehyde function by mild treatment (1% citric acid, 1-10 min) which does not harm the AFM tip. As an exception, AFM tips with magnetic coating become demagnetized in 1% citric acid. This problem was solved by deprotecting the acetal group before coupling the PEG linker to the AFM tip. Bivalent binding of the corresponding linker ("aldehyde-PEG-NHS") to adjacent NH(2) groups on the tip was largely suppressed by high linker concentrations. In this way, magnetic AFM tips could be functionalized with an ethylene diamine derivative of ATP which showed specific interaction with mitochondrial uncoupling protein 1 (UCP1) that had been purified and reconstituted in a mica-supported planar lipid bilayer.     

Growth of cationic lipid toward bilayer lipid membrane by solution spreading: scanning probe microscopy study

The growth of cationic lipid dioctadecyldimethylammonium bromide (DODAB) toward bilayer lipid membrane (BLM) by solution spreading on cleaved mica surface was studied by atomic force microscopy (AFM). Bilayer of DODAB was formed by exposing mica to a solution of DODAB in chloroform and subsequently immersing into potassium chloride solution for film developing. AFM studies showed that at the initial stage of the growth, the adsorbed molecules exhibited the small fractal-like aggregates. These aggregates grew up and expanded laterally into larger patches with time and experienced from monolayer to bilayer, finally a close-packed bilayer film (5.4+/-0.2 nm) was approached. AFM results of the film growth process indicated a growth mechanism of nucleation, growth and coalescence of dense submonolayer, it revealed the direct information about the film morphology and confirmed that solution spreading was an effective technique to prepare a cationic bilayer in a short time.     

Theory of electrostatic effects in soft biological interfaces using atomic force microscopy.

We calculated the electrostatic force between a planar interface, such as a planar-supported lipid bilayer membrane, and the tip of a stylus on which another lipid bilayer or some other biomacromolecular system might be deposited. We considered styli with rounded tips as well as conical tips. To take into account the effect of dynamical hydrogen-bonded structures in the aqueous phase, we used a theory of nonlocal electrostatics. We used the Derjaguin approximation and identified the systems for which its use is valid. We pointed out where our approach differs from previous calculations and to what extent the latter are inadequate. We found that 1) the nonlocal interactions have significant effects over distances of 10-15 A from the polar zone and that, at the surface of this zone, the effect on the calculated force can be some orders of magnitude; 2) the lipid dipoles and charges are located a distance L from the hydrophobic layer in the aqueous medium and this can have consequences that may not be appreciated if it is ignored; 3) dipoles, located in the aqueous region, can give rise to forces even though the polar layer is unchanged, and if this is ignored the interpretation of force data can be erroneous if an attempt is made to rationalize an observed force with a knowledge of an uncharged surface; 4) the shape of the stylus tip can be very important, and a failure to take this into account can result in incorrect conclusions, a point made by other workers; and 5) when L is nonzero, the presence of charges and dipoles can yield a force that can be nonmonotonic as a function of ionic concentration.     

Storable droplet interface lipid bilayers for cell-free ion channel studies

An artificially created lipid bilayer is an important platform in studying ion channels and engineered biosensor applications. However, a lipid bilayer created using conventional techniques is fragile and short-lived, and the measurement of ion channels requires expertise and laborious procedures, precluding practical applications. Here, we demonstrate a storable droplet lipid bilayer precursor frozen with ion channels, resulting in a droplet interface bilayer upon thawing. A small vial with an aqueous droplet in organic solution was flash frozen in -80 °C methanol immediately after an aqueous droplet was introduced into the organic solution and gravity draws the droplet down to the interface upon thawing. A lipid bilayer created along the interface using this method had giga-ohm resistance and typical specific capacitance values. The noise level of this system is favorably comparable to the conventional system. The subsequent incorporation of ion channels, alpha-hemolysin and gramicidin A, showed typical conductance values consistent with those in previous literatures. This novel system to create a lipid bilayer as a whole can be automated from its manufacture to use and indefinitely stored when frozen. As a result, ion channel measurements can be carried out in any place, increasing the accessibility of ion channel studies as well as a number of applications, such as biosensors, ion channel drug screening, and biophysical studies.     

Bending and puncturing the influenza lipid envelope

Lysosomes, enveloped viruses, as well as synaptic and secretory vesicles are all examples of natural nanocontainers (diameter ≈ 100 nm) which specifically rely on their lipid bilayer to protect and exchange their contents with the cell. We have applied methods primarily based on atomic force microscopy and finite element modeling that allow precise investigation of the mechanical properties of the influenza virus lipid envelope. The mechanical properties of small, spherical vesicles made from PR8 influenza lipids were probed by an atomic force microscopy tip applying forces up to 0.2 nN, which led to an elastic deformation up to 20%, on average. The liposome deformation was modeled using finite element methods to extract the lipid bilayer elastic properties. We found that influenza liposomes were softer than what would be expected for a gel phase bilayer and highly deformable: Consistent with previous suggestion that influenza lipids do not undergo a major phase transition, we observe that the stiffness of influenza liposomes increases gradually and weakly (within one order of magnitude) with temperature. Surprisingly, influenza liposomes were, in most cases, able to withstand wall-to-wall deformation, and forces >1 nN were generally required to puncture the influenza envelope, which is similar to viral protein shells. Hence, the choice of a highly flexible lipid envelope may provide as efficient a protection for a viral genome as a stiff protein shell.     

Melittin-induced bilayer leakage depends on lipid material properties: evidence for toroidal pores

The membrane-lytic peptide melittin has previously been shown to form pores in lipid bilayers that have been described in terms of two different structural models. In the "barrel stave" model the bilayer remains more or less flat, with the peptides penetrating across the bilayer hydrocarbon region and aggregating to form a pore, whereas in the "toroidal pore" melittin induces defects in the bilayer such that the bilayer bends sharply inward to form a pore lined by both peptides and lipid headgroups. Here we test these models by measuring both the free energy of melittin transfer (DeltaG degrees ) and melittin-induced leakage as a function of bilayer elastic (material) properties that determine the energetics of bilayer bending, including the area compressibility modulus (K(a)), bilayer bending modulus (k(c)), and monolayer spontaneous curvature (R(o)). The addition of cholesterol to phosphatidylcholine (PC) bilayers, which increases K(a) and k(c), decreases both DeltaG degrees and the melittin-induced vesicle leakage. In contrast, the addition to PC bilayers of molecules with either positive R(o), such as lysoPC, or negative R(o), such as dioleoylglycerol, has little effect on DeltaG degrees , but produces large changes in melittin-induced leakage, from 86% for 8:2 PC/lysoPC to 18% for 8:2 PC/dioleoylglycerol. We observe linear relationships between melittin-induced leakage and both K(a) and 1/R(o)(2). However, in contrast to what would be expected for a barrel stave model, there is no correlation between observed leakage and bilayer hydrocarbon thickness. All of these results demonstrate the importance of bilayer material properties on melittin-induced leakage and indicate that the melittin-induced pores are defects in the bilayer lined in part by lipid molecules.