Horizontal Bilayer Research Articles

Regulation of Interfacial Anchoring Orientation of Anisotropic Nanodumbbells.

Nanoparticles exhibiting geometrical and chemical anisotropies hold promise for environmentally responsive materials with tunable mechanical properties. However, a comprehensive understanding of their interfacial behaviors remains elusive. In this paper, we control the interfacial anchoring orientation of polystyrene nanodumbbells by adjusting interparticle forces. The film nanostructure is characterized by the orientation angle analysis of individual dumbbells from cross-sectional EM data: dumbbells undergo orientation transitions from a distinctive horizontal bilayer to an isotropic anchoring when electrostatic repulsion is suppressed by either an ionic strength increase or surface amine-modification. This anchoring orientation influences the film's mechanical properties and foam stability, as investigated by a 2D isotherm and dark/bright-field microscopy measurements. Our findings highlight the potential for precise control of supra-colloidal structures by modulating particle alignment, paving the way for smart delivery systems.

Membrane binding and pore forming insertion of PEX5 into horizontal lipid bilayer.

The assembly of the peroxisomal translocon involves the transition of a soluble form of the peroxisomal targeting receptor PEX5 into a membrane-bound form, which becomes an integral membrane component of the import pore for peroxisomal matrix proteins. How this transition occurs is still a mystery. We addressed this question using a artificial horizontal bilayer in combination with fluorescence time-correlated single photon counting (TCSPC) and electrophysiological channel recording. Purified human isoform PEX5L and truncated PEX5L(1-335) lacking the cargo binding domain were selectively labeled with thiol-reactive Atto-dyes. Diffusion coefficients of labeled protein in solution show that PEX5L is monomeric with a rather compact spherical conformation, while the truncated protein appeared in a more extended conformation. Labeled PEX5L and the truncated PEX5L(1-335) bind stably to horizontal bilayer thereby accumulating around 100-fold. The diffusion coefficients of the membrane-bound PEX5L forms are 3-4 times lower than in solution, indicating the formation of larger complexes. Electrophysiological single channel recording shows that membrane-bound labeled and non-labeled PEX5L, but not the truncated PEX5L(1-335), can form ion conducting membrane channels. The data suggest that PEX5L is the pore-forming component of the oligomeric peroxisomal translocon and that spontaneous PEX5L membrane surface binding might be an important step in its assembly.

Heat Transfer Performance of Micro-Porous Copper Foams with Homogeneous and Hybrid Structures Manufactured by Lost Carbonate Sintering

ABSTRACTThe heat transfer coefficients of homogeneous and hybrid micro-porous copper foams, produced by the Lost Carbonate Sintering (LCS) process, were measured under one-dimensional forced convection conditions using water coolant. In general, increasing the water flow rate led to an increase in the heat transfer coefficients. For homogeneous samples, the optimum heat transfer performance was observed for samples with 60% porosity. Different trends in the heat transfer coefficients were found in samples with hybrid structures. Firstly, for horizontal bilayer structures, placing the high porosity layer by the heater gave a higher heat transfer coefficient than the other way round. Secondly, for integrated vertical bilayer structures, having the high porosity layer by the water inlet gave a better heat transfer performance. Lastly, for segmented vertical bilayer samples, having the low porosity layer by the water inlet offered the greatest heat transfer coefficient overall, which is five times higher than its homogeneous counterpart.

Intercalation and configurations of organic dye acridine orange in a high-charge montmorillonite as influenced by dye loading

Cationic dyes have strong affinity for negatively charged particle surfaces. The uptake of cationic dyes by clay minerals was attributed to cation exchange. In this study, the mechanism of acridine orange (AO) interaction with a high-charge swelling clay and its interlayer configuration were studied by batch studies, Fourier transform infrared, derivative thermal gravity, and X-ray diffraction (XRD) analyses and assisted by molecular dynamic simulation. At lower loading levels up to 0.8 mmol/g of the clay mineral, the uptake of AO was mainly attributed to cation exchange. At higher AO uptake levels, AO molecular association into a horizontal bilayer would be anticipated in the interlayer of montmorillonite as deduced by the d-spacing expansion on XRD patterns and supported by molecular dynamic simulation. Both the external and internal sorption sites and higher AO adsorption capacity made montmorillonite a superior candidate for the removal of cationic dyes.

Viral potassium channels as a robust model system for studies of membrane–protein interaction

The viral channel KcvNTS belongs to the smallest K+ channels known so far. A monomer of a functional homotetramer contains only 82 amino acids. As a consequence of the small size the protein is almost fully submerged into the membrane. This suggests that the channel is presumably sensitive to its lipid environment. Here we perform a comparative analysis for the function of the channel protein embedded in three different membrane environments. 1. Single-channel currents of KcvNTS were recorded with the patch clamp method on the plasma membrane of HEK293 cells. 2. They were also measured after reconstitution of recombinant channel protein into classical planar lipid bilayers and 3. into horizontal bilayers derived from giant unilamellar vesicles (GUVs). The recombinant channel protein was either expressed and purified from Pichia pastoris or from a cell-free expression system; for the latter a new approach with nanolipoprotein particles was used. The data show that single-channel activity can be recorded under all experimental conditions. The main functional features of the channel like a large single-channel conductance (80pS), high open-probability (>50%) and the approximate duration of open and closed dwell times are maintained in all experimental systems. An apparent difference between the approaches was only observed with respect to the unitary conductance, which was ca. 35% lower in HEK293 cells than in the other systems. The reason for this might be explained by the fact that the channel is tagged by GFP when expressed in HEK293 cells. Collectively the data demonstrate that the small viral channel exhibits a robust function in different experimental systems. This justifies an extrapolation of functional data from these systems to the potential performance of the channel in the virus/host interaction. This article is part of a Special Issue entitled: Viral Membrane Proteins—Channels for Cellular Networking.

Horizontal Bilayer for Electrical and Optical Recordings

Artificial bilayer containing reconstituted ion channels, transporters and pumps serve as a well-defined model system for electrophysiological investigations of membrane protein structure–function relationship. Appropriately constructed microchips containing horizontally oriented bilayers with easy solution access to both sides provide, in addition, the possibility to investigate these model bilayer membranes and the membrane proteins therein with high resolution fluorescence techniques up to the single-molecule level. Here, we describe a bilayer microchip system in which long-term stable horizontal free-standing and hydrogel-supported bilayers can be formed and demonstrate its prospects particularly for single-molecule fluorescence spectroscopy and high resolution fluorescence microscopy in probing the physicochemical properties like phase behavior of the bilayer-forming lipids, as well as in functional studies of membrane proteins.

Controlled-release formulation of antihistamine based on cetirizine zinc-layered hydroxide nanocomposites and its effect on histamine release from basophilic leukemia (RBL-2H3) cells

A controlled-release formulation of an antihistamine, cetirizine, was synthesized using zinc-layered hydroxide as the host and cetirizine as the guest. The resulting well-ordered nanolayered structure, a cetirizine nanocomposite “CETN,” had a basal spacing of 33.9 Å, averaged from six harmonics observed from X-ray diffraction. The guest, cetirizine, was arranged in a horizontal bilayer between the zinc-layered hydroxide (ZLH) inorganic interlayers. Fourier transform infrared spectroscopy studies indicated that the intercalation takes place without major change in the structure of the guest and that the thermal stability of the guest in the nanocomposites is markedly enhanced. The loading of the guest in the nanocomposites was estimated to be about 49.4% (w/w). The release study showed that about 96% of the guest could be released in 80 hours by phosphate buffer solution at pH 7.4 compared with about 97% in 73 hours at pH 4.8. It was found that release was governed by pseudo-second order kinetics. Release of histamine from rat basophilic leukemia cells was found to be more sensitive to the intercalated cetirizine in the CETN compared with its free counterpart, with inhibition of 56% and 29%, respectively, at 62.5 ng/mL. The cytotoxicity assay toward Chang liver cells line show the IC50 for CETN and ZLH are 617 and 670 μg/mL, respectively.

Mechanism of acridine orange removal from water by low-charge swelling clays

Swelling clays have been extensively studied to remove contaminants from aqueous solution. This research aimed at elucidating the mechanism of adsorption of acridine orange (AO), a cationic dye, on low charge montmorillonite in order to better understand the principles behind AO removal using swelling clays. Proportional desorption of exchangeable cations from the clays accompanying AO adsorption confirmed cation exchange as the most dominant mechanism for AO removal. FTIR analyses further confirmed the presence of cationic dye AOH + on the surface and in the interlayer of the swelling clays. XRD and TG-DTG analyses revealed interlayer adsorption, thus, intercalation of AO molecules with different conformations at low and higher AO adsorption levels. Molecular simulation showed dimer and horizontal bilayer formation of intercalated AO at the low and high adsorption levels. At the intermediate AO adsorption levels, the dimer intercalated and bilayer intercalated swelling clays formed mixtures at the single crystal scale, resulting in a significant broadening of (0 0 1) peak. The results showed that the uptake of AO could far exceed the cation exchange capacity, an advantage of using swelling clays to treat wastewater containing cationic dyes.

Macromol. Symp. 301

Cover: The diagrams show possible accommodation of TCS within C15A featuring; (a) the Százdi and Pukánszky arrangement of alkyl chains, and (b) a horizontal bi‐layer with both alkyl chains in contact with the MMT platelet surface. More details can be found in the article by Christopher M. Liauw et. al. p 96 ft

Onset of convection in two layers of a binary liquid

We perform linear stability calculations for horizontal bilayers of a two-component fluid that can undergo a phase transformation, taking into account both buoyancy effects and thermocapillary effects in the presence of a vertical temperature gradient. Critical values for the applied temperature difference across the system that is necessary to produce instability are obtained by a linear stability analysis, using both numerical computations and small wavenumber approximations. Thermophysical properties are taken from the aluminum–indium monotectic system, which includes a liquid–liquid miscibility gap. In addition to buoyant and thermocapillary modes of instability, we find an oscillatory phase-change instability due to the combined effects of solute diffusion and fluid flow that persists at small wavenumbers. This mode is sensitive to the ratio of the layer depths, and for certain layer depths can occur for heating from either above or below.

Towards Simultaneous Single Channel Current and Fluorescence Recordings in Planar Lipid Bilayer

The ion conducting pore of K+ channels has two gates -- one at the selectivity filter and another at the helical bundle crossing. Both gates have to open for ions to pass through the pore. Opening of the helical bundle crossing, which is linked to the voltage sensor movement in voltage-dependent channels, is thought to occur in one cooperative step of all four subunits. On the other hand, subconductance levels, which are states of lower conductance than the normal conducting state, are suggested to be caused by a partial (i.e. not all four subunits) opening of the channel. It remains unknown whether full opening is necessary or whether partial opening is sufficient to get ion conduction. Here, we present our development towards studying the correlation between the subunits' opening and the associated current of the channel. We are investigating purified KcsA channels fluorescently labeled at the helical bundle crossing. The channels are reconstituted at low concentration in lipid vesicles and inserted into horizontal planar lipid bilayer until single channel current is observed. In the horizontal bilayer configuration, we have optical access and electrical control simultaneously. Using fluorescence spectroscopy techniques, we monitor the opening of single subunits while single channel current is recorded. Fluorescence intensity changes of FRET pairs or environment-sensitive dyes allow us to distinguish the movement of the four subunits (Blunck et al., 2008). We successfully imaged the diffusion of single channels in the bilayer using an EMCCD camera and now seek to correlate their fluorescence intensity, which is associated to a partial or a full opening, with the occurrence of subconductance levels.This work is supported by CIHR MOP-81351, CRC 202965 & NSERC 327201DG; H.M. holds a student fellowship of FQRNT.

Nanopore Unitary Permeability Measured by Electrochemical and Optical Single Transporter Recording

For the analysis of membrane transport processes two single molecule methods are available that differ profoundly in data acquisition principle, achievable information, and application range: the widely employed electrical single channel recording and the more recently established optical single transporter recording. In this study dense arrays of microscopic horizontal bilayer membranes between 0.8 microm and 50 microm in diameter were created in transparent foils containing either microholes or microcavities. Prototypic protein nanopores were formed in bilayer membranes by addition of Staphylococcus aureus alpha-hemolysin (alpha-HL). Microhole arrays were used to monitor the formation of bilayer membranes and single alpha-HL pores by confocal microscopy and electrical recording. Microcavity arrays were used to characterize the formation of bilayer membranes and the flux of fluorescent substrates and inorganic ions through single transporters by confocal microscopy. Thus, the unitary permeability of the alpha-HL pore was determined for calcein and Ca(2+) ions. The study paves the way for an amalgamation of electrical and optical single transporter recording. Electro-optical single transporter recording could provide so far unresolved kinetic data of a large number of cellular transporters, leading to an extension of the nanopore sensor approach to the single molecule analysis of peptide transport by translocases.

Scanning electrochemical microscopy. 40. Voltammetric ion-selective micropipet electrodes for probing ion transfer at bilayer lipid membranes.

Voltammetric ion-selective micropipet electrodes for use in scanning electrochemical microscopy (SECM) for detection of potassium ion were fabricated. These used pulled borosilicate capillaries with tip orifice radii of 0.7-20 microm with silanized inner walls filled with a solution of 10 mM valinomycin and 10 mM ETH 500 in dichloroethane. The electrodes were characterized by determining the steady-state tip current for K+ concentrations of 0.05-0.3 mM. The tips were used in the SECM feedback and generation-collection modes to study K+ transfer through gramicidin channels in a horizontal bilayer lipid membrane (glycerol monooleate).

Horizontal ‘solvent-free’ lipid bimolecular membranes with two-sided access can be formed and facilitate ion channel reconstitution

We present here an easily used method and apparatus for formation of horizontal ‘solvent-free’ lipid bilayer membranes affording two-sided access. These horizontal bilayers allow direct delivery of submicroliter volumes of samples onto the membrane upper surface increasing the efficacy of reconstitution by several orders of magnitude, as demonstrated using Staphylococcus aureus α-toxin. Also, they permit creation of locally high and transient transbilayer osmotic gradients to initiate fusion of ion-channel containing liposomes with planar membrane, which, following fusion, leaves the membrane and channel in essentially symmetric bathing solutions. This method is especially advantageous for cases where thickness of the membrane, absence of hydrocarbon solvent, or presence of differing lipid compositions in the two monolayers is critical.

Fusion of synaptic vesicle membranes with planar bilayer membranes

The interaction of synaptic vesicles with horizontal bilayer lipid membranes (BLMs) was investigated as a model system for neurotransmitter release. High concentrations (200 mM) of the fluorescent dye, calcein, were trapped within synaptic vesicles by freezing and thawing. In the presence of divalent ions (usually 15 mM CaCl2), these frozen and thawed synaptic vesicles (FTSVs) adhere to squalene-based phosphatidylserine-phosphatidylethanolamine BLMs whereupon they spontaneously release their contents which is visible by fluorescence microscopy as bright flashes. The highest rate of release was obtained in KCl solutions. Release was virtually eliminated in isotonic glucose, but could be elicited by perfusion with KCl or by addition of urea. The fusion and lysis of adhering FTSVs appears to be the consequence of stress resulting from entry of permeable external solute (KCl, urea) and accompanying water. An analysis of flash diameters in experiments where Co+2, which quenches calcein fluorescence, was present on one or both sides of the BLM, indicates that more than half of the flashes represent fusion events, i.e., release of vesicle contents on the trans side of the BLM. A population of small, barely visible FTSVs bind to BLMs at calcium ion concentrations of 100 microM. Although fusion of these small FTSVs to BLMs could not be demonstrated, fusion with giant lipid vesicles was obvious and dramatic, albeit infrequent. Addition of FTSVs or synaptic vesicles to BLMs in the presence of 100 microM-15 mM Ca2+ produced large increases in BLM conductance. The results presented demonstrate that synaptic vesicles are capable of fusing with model lipid membranes in the presence of Ca+2 ion which, at the lower limit, may begin to approach physiological concentrations.