Microchannel device using self-spreading lipid bilayer as molecule carrier

We propose a microchannel device that employs a surface-supported self-spreading lipid bilayer membrane as a molecule carrying medium. The device has a micropattern structure fabricated on a SiO2 surface by photolithography, into which a self-spreading lipid bilayer membrane is introduced as the carrier medium. This system corresponds to a microchannel with a single lipid bilayer membrane height of approximately 5 nm, compared with conventional micro-fluidic channels that have a section height and width of at least several microm. The device is beneficial for detecting intermolecular interactions when molecules carried by the self-spreading lipid bilayer collide with each other in the microchannel. The validity of the device was confirmed by observing the fluorescence resonance energy transfer (FRET) between two dye molecules, coumarin and fluorescein.     

Tuning the dynamics and molecular distribution of the self-spreading lipid bilayer

The self-spreading dynamics of lipid bilayers were investigated at controlled electrolyte concentrations. The self-spreading velocity increased when the concentration of NaCl was increased from 1 to 100 mM. Comparing the experimentally determined spreading energy with that estimated from theoretical models, we found that the self-spreading dynamics were well explained by considering the van der Waals interaction, double layer interaction and hydration interaction energies between the self-spreading bilayer and the substrate. The characteristic behavior at high concentration is attributable to the increase in the density of the lipid layer, originating from the effective shielding of the molecular charges by the electrolyte ions in solution. The distribution of doped dye-labeled molecule within the spreading bilayer was also controllable by tuning the electrolyte concentration. All of these findings were explained by systematic changes in bilayer-substrate or inter-molecular interactions depending on the electrolyte concentration.     

Control of the structure of self-spreading lipid membrane by changing electrolyte concentration

We have controlled the structure of self-spreading lipid bilayer membranes prepared on surface-oxidized silicon substrates by changing electrolyte concentration. Analysis of the fluorescence intensity, considering the optical interference effect, clarified the stacking structure of the lipid membrane. By varying the electrolyte concentration, we can vary the number of single multilamellar lobes adsorbed on the underlying self-spreading bilayer. This dependence of the stacking ability on the electrolyte concentration was investigated on the basis of changes in the bilayer-lobe interaction energies, including van der Waals, electrostatic double layer, and hydration interaction energies. Theoretical estimation suggests that the observed electrolyte concentration dependence can be explained by the combination of the van der Waals attractive interaction energy and the repulsive double-layer interaction energy.     

Electrostatic control of lipid bilayer self-spreading using a nanogap gate on a solid support

We have demonstrated for the first time that the self-spreading of supported lipid bilayers can be controlled by the temporal switching of an electric field applied between nanogap electrodes. To account for this phenomenon, we propose an electrostatic trapping model in which an electric double layer plays an important role. The validity of this mechanism was verified by the dependence of self-spreading on the nanogap width and the ionic concentration of the electrolyte. Our results provide a promising tool for the temporal and spatial control of lipid bilayer formation for nanobio devices.     

Supported lipid bilayer self-spreading on a nanostructured silicon surface

We report on the self-spreading behavior of a supported lipid bilayer (SLB) on a silicon surface with various 100 nm nanostructures. SLBs have been successfully grown from a small spot of a lipid molecule source both on a flat surface and uneven surfaces with 100 nm up-and-down nanostructures. After an hour, the self-spreading SLB forms a large circle or an ellipse depending on the nanostructure pattern. The results are explained by a model that shows that a single-layer SLB grows along the nanostructured surfaces. The model is further supported by a quantitative analyses of our data. We also discuss the stability of the SLB on nanostructured surfaces in terms of the balance between its bending and adhesion energies.     

Control of dynamics and molecular distribution in a self-spreading lipid bilayer using surface-modified metal nanoarchitectures

The molecular distribution and spreading dynamics of self-spreading lipid bilayers can be tuned by surface-modified metallic nanoarchitectures. Interactions between lipids and molecules in the surface modification alter the self-spreading behavior at the gate regions between adjacent nanoarchitectures, leading to molecular filtering/concentrating effects and modification of the dynamics. The hydrophilic surface can tune the spreading velocity without changing the molecular distribution in the spreading bilayer, whereas the hydrophobic surface provides a molecular concentrating function to the nanogates. This indicates that a combination of unmodified/hydrophobic/hydrophilic nanoarchitectures has a wide range of potential applications since it can be used to independently control the self-spreading dynamics and the molecular distribution.     

Thermotropic phase transition in soluble nanoscale lipid bilayers

The role of lipid domain size and protein-lipid interfaces in the thermotropic phase transition of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) bilayers in Nanodiscs was studied using small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and generalized polarization (GP) of the lipophilic probe Laurdan. Nanodiscs are water-soluble, monodisperse, self-assembled lipid bilayers encompassed by a helical membrane scaffold protein (MSP). MSPs of different lengths were used to define the diameter of the Nanodisc lipid bilayer from 76 to 108 A and the number of DPPC molecules from 164 to 335 per discoidal structure. In Nanodiscs of all sizes, the phase transitions were broader and shifted to higher temperatures relative to those observed in vesicle preparations. The size dependences of the transition enthalpies and structural parameters of Nanodiscs reveal the presence of a boundary lipid layer in contact with the scaffold protein encircling the perimeter of the disc. The thickness of this annular layer was estimated to be approximately 15 A, or two lipid molecules. SAXS was used to measure the lateral thermal expansion of Nanodiscs, and a steep decrease of bilayer thickness during the main lipid phase transition was observed. These results provide the basis for the quantitative understanding of cooperative phase transitions in membrane bilayers in confined geometries at the nanoscale.     

Supported lipid bilayer composition microarray fabricated by pattern-guided self-spreading

We report on the fabrication of a microarray of supported lipid bilayers (SLBs) with different chemical compositions and demonstrate its biosensing application. The technique utilizes the phenomenon of lipid self-spreading on a patterned surface, which offers complete positional selectivity for a supported lipid bilayer. We describe the fabrication of parallel 10-μm-wide lines, each filled with an SLB with a unique composition, at 5 μm intervals. Structures obtained with our new technique are finer and more highly integrated than previously reported structures that employ the vesicle fusion technique on patterned surfaces. We also detected specific binding between biotin and streptavidin with high contrast, indicating that the microarray is valuable for biosensing applications.     

Perturbation of phospholipid bilayer properties by ethanol at a high concentration

Atomistic molecular dynamics (MD) simulations have been carried out at 30 degrees C on a fully hydrated liquid crystalline lamellar phase of dimyrystoylphosphatidylcholine (DMPC) lipid bilayer with embedded ethanol molecules at 1:1 composition, as well as on the pure bilayer phase. The ethanol molecules are found to exhibit a preference to occupy regions near the upper part of the lipid acyl chains and the phosphocholine headgroups. The calculations revealed that the phosphocholine headgroup dipoles (P- --> N+) of the lipids prefer to orient more toward the aqueous layer in the presence of ethanol. It is noticed that the ethanol molecules modify the dynamic properties of both lipids as well as the water molecules in the hydration layer of the lipid headgroups. Both the in-plane "rattling" and out-of-plane "protrusion" motions of the lipids have been found to increase in the presence of ethanol. Most importantly, it is observed that the water molecules within the hydration layer of the lipid headgroups exhibit faster translational and rotational motions in the presence of ethanol. This arises due to faster dynamics of hydrogen bonds between lipid headgroups and water in the presence of ethanol.     

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.     

Interaction of the sugars trehalose, maltose and glucose with a phospholipid bilayer: a comparative molecular dynamics study

Molecular dynamics simulations are used to investigate the interaction of the sugars trehalose, maltose, and glucose with a phospholipid bilayer at atomic resolution. Simulations of the bilayer in the absence or in the presence of sugar (2 molal concentration for the disaccharides, 4 molal for the monosaccharide) are carried out at 325 and 475 K. At 325 K, the three sugars are found to interact directly with the lipid headgroups through hydrogen bonds, replacing water at about one-fifth to one-quarter of the hydrogen-bonding sites provided by the membrane. Because of its small size and of the reduced topological constraints imposed on the hydroxyl group locations and orientations, glucose interacts more tightly (at identical effective hydroxyl group concentration) with the lipid headgroups when compared to the disaccharides. At high temperature, the three sugars are able to prevent the thermal disruption of the bilayer. This protective effect is correlated with a significant increase in the number of sugar-headgroups hydrogen bonds. For the disaccharides, this change is predominantly due to an increase in the number of sugar molecules bridging three or more lipid molecules. For glucose, it is primarily due to an increase in the number of sugar molecules bound to one or bridging two lipid molecules.     

Hydration properties of symmetric chain and asymmetric chain sphingomyelin bilayers

Hydration properties of lipid bilayer systems are compared for symmetric chain sphingomyelin (N-palmitoylsphingomyelin) and asymmetric chain sphingomyelin (N-lignoceroylsphingomyelin). These sphingomyelins were semisynthesized by a deacylation- reacylation process with a natural sphingomyelin used as a starting material. The number of differently bound water molecules was estimated by a deconvolution analysis of the ice-melting curves obtained by a differential scanning calorimetry (DSC) and was used to construct a water distribution diagram for these water molecules. Similarly to a natural sphingomyelin used for comparison, the asymmetric chain sphingomyelin was found to form small size vesicles having an internal cavity and incorporate 15 water molecules per molecule of lipid into its cavity, in contrast with 5 H₂O/lipid for freezable interlamellar water observed for large size multilamellar vesicles formed by the symmetric chain sphingomyelin.     

Sensitivity of hydrogen bond lifetime dynamics to the presence of ethanol at the interface of a phospholipid bilayer

Atomistic molecular dynamics simulations of a fully hydrated liquid crystalline lamellar phase of a dimyrystoylphosphatidylcholine lipid bilayer containing ethanol at 1:1 composition as well as of the pure lamellar phase of the bilayer have been performed. Detailed analyses have been carried out to investigate the effects of ethanol, if any, on the lifetime dynamics of lipid-water and water-water hydrogen bonds in the hydration layer of the lipid headgroups. The nonexponential hydrogen bond lifetime correlation functions have been analyzed by using the formalism of Luzar and Chandler, which allowed the identification of the bound states at the bilayer interface and the quantification of the dynamic equilibrium between the bound and the free water molecules, in terms of time-dependent relaxation rates. The calculations show that the overall relaxation of phosphate-water hydrogen bonds is faster in the presence of ethanol. Studies of the residence time and the number fluctuation of the hydration layer water molecules reveal that the presence of ethanol molecules decreases the rigidity of the lipid hydration layer.     

Micropatterned composite membranes of polymerized and fluid lipid bilayers

Micropatterned composite membranes of polymerized and fluid lipid bilayers were constructed on solid substrates. Lithographic photopolymerization of a diacetylene-containing phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC), and subsequent removal of nonreacted monomers by a detergent solution (0.1 M sodium dodecyl sulfate (SDS)) yielded a patterned polymeric bilayer matrix on the substrate. Fluid lipid bilayers of phosphatidylcholine from egg yolk (egg-PC) were incorporated into the lipid-free wells surrounded by the polymeric bilayers through the process of fusion and reorganization of suspended small unilamellar vesicles. Spatial distribution of the fluid bilayers in the patterned bilayer depended on the degree of photopolymerization that in turn could be modulated by varying the applied UV irradiation dose. The polymeric bilayer domains blocked lateral diffusion of the fluid lipid bilayers and confined them in the defined areas (corrals), if the polymerization was conducted with a sufficiently large UV dose. On the other hand, lipid molecules of the fluid bilayers penetrated into the polymeric bilayer domains, if the UV dose was relatively small. A direct correlation was observed between the applied UV dose and the lateral diffusion coefficient of fluorescent marker molecules in the fluid bilayers embedded within the polymeric bilayer domains. Artificial control of lateral diffusion by polymeric bilayers may lead to the creation of complex and versatile biomimetic model membrane arrays.     

Size-dependent properties of small unilamellar vesicles formed by model lipids

The size-dependent behavior of small unilamellar vesicles is explored by dissipative particle dynamics, including the membrane characteristics and mechanical properties. The spontaneously formed vesicles are in the metastable state and the vesicle size is controlled by the concentration of model lipids. As the vesicle size decreases, the bilayer gets thinner and the area density of heads declines. Nonetheless, the area density in the inner leaflet is higher than that in the outer. The packing parameters are calculated for both leaflets. The result indicates that the shape of lipid in the outer leaflet is like a truncated cone but that in the inner leaflet resembles an inverted truncated cone. Based on a local order parameter, our simulations indication that the orientation order of lipid molecules decreases as the size of the vesicle reduces and this fact reveals that the bilayer becoming thinner for smaller vesicle is mainly attributed to the orientation disorder of the lipids. The membrane tension can be obtained through the Young-Laplace equation. The tension is found to grow with reducing vesicle size. Therefore, small vesicles are less stable against fusion. Using the inflation method, the area stretching and bending moduli can be determined and those moduli are found to grow with reducing size. Nonetheless, a general equation with a single numerical constant can relate bending modulus, area stretching modulus, and bilayer thickness irrespective of the vesicle size. Finally, a simple metastable model is proposed to explain the size-dependent behavior of bilayer thickness, orientation, and tension.     

A Halogen-Bond-Driven Artificial Chloride-Selective Channel Constructed from 5-Iodoisophthalamide-based Molecules

Despite considerable emphasis on advancing artificial ion channels, progress is constrained by the limited availability of small molecules with the necessary attributes of self-assembly and ion selectivity. In this study, a library of small molecules based on 5-haloisophthalamide and a non-halogenated isophthalamide were examined for their ion transport properties across the lipid bilayer membranes, and the finding demonstrates that the di-hexyl-substituted 5-iodoisophthalamide derivative exhibits the highest level of activity. Furthermore, it was established that the highest active compound facilitates the selective chloride transport that occurs via an antiport-mediated mechanism. The crystal structure of the compound unveils a distinctive self-assembly of molecules, forming a zig-zag channel pore that is well-suited for the permeation of anions. Planar bilayer conductance measurements proved the formation of chloride selective channels. A molecular dynamics simulation study, relying on the self-assembled component derived from the crystal structure, affirmed the paramount significance of intermolecular hydrogen bonding in the formation of supramolecular barrel-rosette structures that span the bilayer. Furthermore, it was demonstrated that the transport of chloride across the lipid bilayer membrane is facilitated by the synergistic effects of halogen bonding and hydrogen bonding within the channel.     

Structure and dynamics of crystalline protein layers bound to supported lipid bilayers

We study proteins at the surface of bilayer membranes using streptavidin and avidin bound to biotinylated lipids in a supported lipid bilayer (SLB) at the solid-liquid interface. Using X-ray reflectivity and simultaneous fluorescence microscopy, we characterize the structure and fluidity of protein layers with varied relative surface coverages of crystalline and noncrystalline protein. With continuous bleaching, we measure a 10-15% decrease in the fluidity of the SLB after the full protein layer is formed. We propose that this reduction in lipid mobility is due to a small fraction (0.04) of immobilized lipids bound to the protein layer that create obstacles to membrane diffusion. Our X-ray reflectivity data show a 40 A thick layer of protein, and we resolve an 8 A layer separating the protein layer from the bilayer. We suggest that the separation provided by this water layer allows the underlying lipid bilayer to retain its fluidity and stability.     

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.     

A silicate gel for promoting deposition of lipid bilayers

A simple method for modifying a polymer surface to induce lipid bilayer formation by vesicle fusion is described. A silicate gel was prepared by condensation of tetraethyl orthosilicate (TEOS) in the presence of acid. When applied to a poly(methylmethacrylate) substrate, either a rough or a smooth layer could be produced, depending on the method used for the application. The smooth surface induced formation of a supported lipid bilayer by fusion of lipid vesicles; the rough silicate surface induced adsorption of a vesicle layer. A high-frequency acoustic waveguide device was used to follow the initial adsorption of vesicles, the transition from a vesicle layer to a bilayer, and the formation of a complete bilayer; the time required to form a bilayer was determined as a function of lipid concentration in suspension. The presence of a bilayer on the smooth silicate surface was confirmed by fluorescence recovery after photobleaching. An additional procedure is described to modify a gold surface to induce bilayer formation.