Tethered Lipid Membranes Research Articles

The Effect of Benzyl Alcohol on the Voltage-Current Characteristics of Tethered Lipid Bilayers.

In this study a lipid bilayer membrane model was used in which the bilayer is tethered to a solid substrate with molecular tethers. Voltage-current (V-I) measurements of the tethered bilayer membranes (tBLM) and tBLM with benzyl alcohol (BZA) incorporated in their structures, were measured using triangular voltage ramps of 0-500mV. The temperature dependence of the conductance deduced from the V-I measurements are described. An evaluation of the activation energies for electrical conductance showed that BZA decreased the activation/ Born energies for ionic conduction of tethered lipid membranes. It is concluded that BZA increased the average pore radius of the tBLM.

Electrochemical assessment of dielectric damage to phospholipid bilayers by amyloid β-Oligomers

Amyloid beta (Aβ1-42) oligomers produced in vitro with and without the oligomerization inhibitor hexafluoroisopropanol (HFIP) were studied and compared as agents inflicting damage to the phospholipid bilayers. Tethered lipid membranes (tBLMs) of different compositions were used as model membranes. Dielectric damage of tBLMs by Aβ1-42 oligomers was monitored by the electrochemical impedance spectroscopy (EIS). Membranes containing sphingomyelin exhibited the highest susceptibility to Aβ1-42 oligomers when assembled in the absence of an inhibitor. The activation barrier of ion translocation through the Aβ1-42 oligomer entities in tBLMs was lowest in sphingomyelin membranes (<15 kJ/mol). This is consistent with the formation of water-filled, highly conductive (>50 pS) nanopores in tBLMs by Aβ1-42 oligomers assembled without HFIP. Conversely, HFIP-generated Aβ1- 42 oligomers exhibited conductance with high activation energies (>38 kJ/mol), suggesting the formation of assemblies with relatively narrow ion pores and the effective conductance in the range < 15 pS. Finally, the EIS data analysis revealed differences in the lateral distribution of Aβ1-42 oligomers in tBLMs. The inhibitor-free Aβ1-42 oligomers populate the tBLM surface in a random manner, whereas the HFIP-generated Aβ1-42 oligomers tend to cluster forming surface areas with markedly different densities of Aβ1-42 defects.

Tethered Lipid Membranes as a Nanoscale Arrangement towards Non-Invasive Analysis of Acute Pancreatitis.

Extracellular heat shock proteins (HSPs) mediate immunological functions and are involved in pathologies such as infection, stress, and cancer. Here, we demonstrated the dependence of an amount of HSP70 and HSP90 in serum vs. severity of acute pancreatitis (AP) on a cohort of 49 patients. Tethered bilayer lipid membranes (tBLMs) have been developed to investigate HSPs’ interactions with tBLMs that can be probed by electrochemical impedance spectroscopy (EIS). The results revealed that HSP70 and HSP90 interact via different mechanisms. HSP70 shows the damage of the membrane, while HSP90 increases the insulation properties of tBLM. These findings provide evidence that EIS offers a novel approach for the study of the changes in membrane integrity induced by HSPs proteins. Herein, we present an alternative electrochemical technique, without any immunoprobes, that allows for the monitoring of HSPs on nanoscaled tBLM arrangement in biologics samples such us human urine. This study demonstrates the great potential of tBLM to be used as a membrane based biosensor for novel, simple, and non-invasive label-free analytical system for the prediction of AP severity.

Design of tethered bilayer lipid membranes, using wet chemistry via aryldiazonium sulfonic acid spontaneous grafting on silicon and chrome

We describe a bottom-up surface functionalization to design hybrid molecular coatings that tether biomembranes using wet chemistry. First, a monolayer was formed by immersion in a NH2-Ar-SO3H solution, allowing aryldiazonium salt radicals to spontaneously bind to it via strong C bonding. After formation of the air-stable and dense molecular monolayer (–Ar-SO3H), a subsequent activation was used to form highly reactive –Ar-SO2Cl groups nearly perpendicular to the monolayer. These can bind commercial surfactants, PEGylated oligomers and other inexpensive molecules via their -OH, -COOH, or -NH2 chain end-moieties, to build hybrid coatings. Metal and oxidized chromium, semi-conductor n-doped silicon (111), are the substrates tested for this protocol and the aromatic organic monolayers formed at their surface are characterized by X-ray photoelectron spectroscopy (XPS). XPS reveals unambiguously the presence of C–Cr and C–Si bonds, ensuring robustness of the coatings. Functional sulfur groups (–SO3H) cover up to 6.5×10−10 mol cm−2 of the silicon interface and 4.7×10−10 mol cm−2 of the oxidized chromium interface. These surface concentrations are comparable to the classic values obtained when the prefunctionalisation is driven by electrochemistry on conductors. Tethered lipid membranes formed on these coatings were analyzed by neutron reflectivity at the interface of functionalized n-doped silicon substrates after immersion in a solution of lipid vesicles and subsequent fusion. Results indicate a rather compact hybrid coating of Brij anchor-harpoon molecules that maintain a single lipid bilayer above the substrate, on top of a hydrated PEO cushion.

The Effect of Cholesterol on the Voltage-Current Characteristics of Tethered Lipid Membranes.

In this study, a lipid bilayer membrane model was used, in which the bilayer is tethered to a solid substrate with molecular tethers. The V-I characteristics of the lipid bilayers were found to be non-linear which suggests the presence of pores that are voltage-dependent. At high applied voltages, the conductance reached a limiting value, presumably indicating a limit on the maximum pore size. A decrease in the spacing between tethers (increasing tether density) caused a decrease in the membrane's conductance at high applied voltage, which is consistent with the maximum pore size being determined by the spacing between the tethers. The inclusion of 10M% cholesterol within the membrane lipid caused a decrease in the membrane conductance. However, the inclusion of higher levels of cholesterol increased the membrane conductance.

Model Biological Membranes and Possibilities of Application of Electrochemical Impedance Spectroscopy for their Characterization

AbstractBiological membranes are essential parts of living systems. They represent an interface between intracellular and extracellular space. Depending on their structure, they often perform very complex functions and play an important role in the transport of both charged and uncharged particles in any organism. Structure of the biological membranes, which play very important role in electrochemical processes inside living organisms, is very complicated and still not precisely defined and explained. Model lipid membranes are used to gain detail information about properties of real biological membranes and about associated electrochemical processes. Electrochemistry, especially electrochemical impedance spectroscopy (EIS), can play a useful role in the characterization of properties of model lipid membranes (planar and supported lipid bilayers, tethered lipid membranes, liposomes, etc.). This review is focused on model biological membranes and the possibilities and limitations of electrochemical methods and namely of EIS in this field.

Oriented Membrane Protein Reconstitution into Tethered Lipid Membranes for AFM Force Spectroscopy

Membrane proteins act as a central interface between the extracellular environment and the intracellular response and as such represent one of the most important classes of drug targets. The characterization of the molecular properties of integral membrane proteins, such as topology and interdomain interaction, is key to a fundamental understanding of their function. Atomic force microscopy (AFM) and force spectroscopy have the intrinsic capabilities of investigating these properties in a near-native setting. However, atomic force spectroscopy of membrane proteins is traditionally carried out in a crystalline setup. Alternatively, model membrane systems, such as tethered bilayer membranes, have been developed for surface-dependent techniques. While these setups can provide a more native environment, data analysis may be complicated by the normally found statistical orientation of the reconstituted protein in the model membrane. We have developed a model membrane system that enables the study of membrane proteins in a defined orientation by single-molecule force spectroscopy. Our approach is demonstrated using cell-free expressed bacteriorhodopsin coupled to a quartz glass surface in a defined orientation through a protein anchor and reconstituted inside an artificial membrane system. This approach offers an effective way to study membrane proteins in a planar lipid bilayer. It can be easily transferred to all membrane proteins that possess a suitable tag and can be reconstituted into a lipid bilayer. In this respect, we anticipate that this technique may contribute important information on structure, topology, and intra- and intermolecular interactions of other seven-transmembrane helical receptors.

Fluid and Resistive Tethered Lipid Membranes on Nanoporous Substrates.

Cell membranes perform important biological roles including compartmentalization, signaling, and transport of nutrients. Supported lipid membranes mimic the behavior of cell membranes and are an important model tool for studying membrane properties in a controlled laboratory environment. Lipid membranes may be supported on solid substrates; however, protein and lipid interactions with the substrate typically result in their denaturation. In this report, we demonstrate the formation of intact lipid membranes tethered on nanoporous metal thin films obtained via a dealloying process. Uniform lipid membranes were formed when the surface defect density of the nanoporous metal film was significantly reduced through a two-step dealloying process reported here. We show that the tethered lipid membranes on nanoporous metal substrates maintain both fluidity and electrical resistivity, which are key attributes to naturally occurring lipid membranes. The lipid assemblies supported on nanoporous metals provide a new platform for investigating lipid membrane properties, and potentially membrane proteins, for numerous applications including next generation biosensor platforms, targeted drug-delivery, and energy harvesting devices.

A coarse-Grained Solvent-Free Model for Tethered Lipid Membrane Simulation

In order to characterize biological lipid membranes and their constituents, a variety of experimental techniques, for instance fluorescence microscopy and X-ray/neutron reflection, require the bilayer systems to have a robust planar geometry. However, the adsorption onto a substrate can strongly affect some properties of the system, especially in the proximal leaflet. To alleviate artifacts such as the immobility of membrane-associated proteins, tethered bilayer systems have been developed. In these systems tethers consisting of short hydrophilic polymers elevate the bilayer from the substrate and also provide better control over the separation between the two.Different computational models for lipid memebranes with varying levels of resolution have been proposed in the past. However, only a few simulations have been dedicated to the supported systems, and none have studied the tethered bilayers. Here we present a mesoscopic coarse-grained model of tethered lipid membranes, which is based on an implicit-solvent three-bead lipid model [Cooke et. al., Phys. Rev. E 72, 011506 (2005)]. Each tether molecule consists of 1) a hydrophobic part that can anchor into lipid leaflets and share the properties of the lipids, 2) a hydrophilic chain mimicking polyethelene glycol, and 3) a headgroup bead which covalently attaches to the substrate. Under suitable conditions, these fixed tether molecules and free random lipids self-assemble into a flat tethered bilayer. Different physical observables, such as the fluctuation spectrum, the area density of lipids, and diffusion coefficients in proximal/distal leaflets, are measured. The results are compared to free and solid-supported membranes, providing important insights into the generic properties of this system.

Cationized albumin-biocoatings for the immobilization of lipid vesicles

Tethered lipid membranes or immobilized lipid vesicles are frequently used as biomimetic systems. In this article, the authors presented a suitable method for efficient immobilization of lipid vesicles onto a broad range of surfaces, enabling analysis by quantitative methods even under rigid, mechanical conditions-bare surfaces such as hydrophilic glass surfaces as well as hydrophobic polymer slides or metal surfaces such as gold. The immobilization of vesicles was based on the electrostatic interaction of zwitterionic or negatively charged lipid vesicles with two types of cationic chemically modified bovine serum albumin (cBSA) blood plasma proteins (cBSA-113 and cBSA-147). Quantitative analysis of protein adsorption was performed as the cBSA coatings were characterized by atomic force microscopy, surface zeta potential measurement, fluorescence microscopy, and surface plasmon spectroscopy, revealing a maximal surface coverage 270-280 ng/cm(2) for 0.02 mg/ml cBSA on gold. Small unilamellar vesicles as well as giant unilamellar vesicles (GUVs) were readily immobilized (∼15 min) on cBSA coated surfaces. GUVs with 5-10 mol% negatively charged 1,2,-dipalmitoyl-sn-glycero-3-phosphoglycerol remained stable in liquid for at least 5 weeks.

Preparation of an electrochemical biosensor based on lipid membranes in nanoporous alumina

Model lipid bilayers are versatile tools to investigate the molecular processes occurring at the membrane level. Among the model membranes, substrate supported bilayers have attracted much interest because they are robust and they can be investigated by powerful surface sensitive techniques such as electrochemical measurements. In a biosensor, lipid films can be used not only as a support for the biological sensing elements but also as sensing elements themselves to detect molecules that are able to alter the structure and the properties of biomembranes. In this work, we have prepared a tethered lipid membrane-based biosensor able to detect the alterations of membrane structure and fluidity. This tethered lipid membrane was prepared in a nanoporous aluminium oxide that provides a high surface area and a protective environment against dewetting. The membrane contained PEG-PE lipids as hydrating, protective and tethering agents and ubiquinone which is a redox lipophilic mediator embedded within the acyl chains of the lipid bilayer. The lipid membrane was prepared inside the pores of the nanoporous support by a PEG-triggered fusion of liposomes. This sensing system was efficient to detect the alterations of lipid membranes that are induced by the addition of a commonly used non-ionic detergent: Triton X-100.

In Situ PM-IRRAS Studies of an Archaea Analogue Thiolipid Assembled on a Au(111) Electrode Surface

Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) has been applied to determine the conformation, orientation, and hydration of a monolayer of 2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-dl-alpha-lipoic acid ester (DPTL) self-assembled at a gold electrode surface. This Archaea analogue thiolipid has been recently employed to build tethered lipid bilayers. By synthesizing DPT(d16)L, a DPTL molecule with a deuterium substituted tetraethylene glycol spacer, it was possible to differentiate the C-H stretch vibrations of the phytanyl chains from the tetraethylene glycol spacer and acquire the characteristic IR spectra for the chains, spacer, and lipoic acid headgroup separately. Our results show that the structure of the monolayer displays remarkable stability in a broad range of electrode potentials and that the phytanyl chains remain in a liquid crystalline state. The tetraethylene glycol chains are coiled, and the IR spectrum for this region shows that it is in the disordered state. The most significant result of this study is the information that in contrast to expectations the spacer region is poorly hydrated. Our results have implications for the design of a tethered lipid membrane based on this thiolipid.

Structural characterization of an elevated lipid bilayer obtained by stepwise functionalization of a self-assembled alkenyl silane film

This work reports a novel tethered lipid membrane supported on silicon oxide providing an improved model cell membrane. There is an increasing need for robust solid supported fluid model membranes that can be easily deposited on soft cushions. In such architecture the space between the membrane and the substrate should be tunable in the nanometer range. For this purpose a SiO(2) surface was functionalized with poly(ethylene glycol) (PEG)-lipid tethers and further modified with poly(ethylene glycol) making a biologically passivated substrate available for lipid bilayer deposition. First, a short chain self-assembled alkenyl silane film was oxidized to yield terminal COOH groups and then functionalized with amino-terminated PEG-lipids via N-hydroxysuccinimide chemistry. The functionalized silane film was then additionally passivated by functionalization of unreacted COOH groups with amino-terminated PEG of variable chain length. X-ray photoelectron spectroscopy (XPS) analysis of dry films, carried out near the C 1s ionization edge to characterize chemical groups formed in the near-surface region, confirmed binding of PEG-lipid tethers to the silane film. XPS further indicated that backfilling with PEG caused the lipid tails to stick up above the PEG layer which was confirmed by the x-ray reflectivity measurements. Lipid vesicle fusion on these surfaces in the presence of excess water resulted in the formation of supported membranes characterized by very high homogeneity and long range mobility, as confirmed by fluorescence bleaching experiments. Even after repeated drying-hydrating cycles, these robust surfaces provided good templates for high fluidity elevated membranes. X-ray reflectivity measurements of the tethered membranes, with a resolution of 0.6 nm in water, showed that these fluid membranes are elevated up to 8 nm above the silicon oxide surface.

Making lipid membranes even tougher

Biosensors based on lipid membranes promise an inexpensive and versatile platform for application in many fields of molecular sensing. An extensive review of the applications for tethered membranes was reported in the July 2006 MRS Bulletin [A.N. Parikh and J.T. Groves, Materials science of supported lipid membranes. MRS Bull.31(8), 507 (2006)]. The commercial use to which tethered lipid membranes have been applied has been limited by their stability under long-term storage. This report describes a novel membrane construct that is stable at room temperature for months, eliminates the mobile lipid phase present in lipid bilayers, and is robust against detergents under conditions that would destroy a lipid bilayer.

Biomimetic tethered lipid membranes designed for membrane-protein interaction studies

The complexity of the biological membranes restricts their direct investigation at the nanoscale. Lipid bilayer membranes have been developed as a model of biological membranes in order to allow the interaction and insertion of peptides and membrane proteins in a functional manner. Promising models have been developed in the past two decades and tethered bilayer design traduces constant improvement of membrane models. The formation of protein free solid tethered membranes can be achieved by direct vesicle fusion, Langmuir-Blodgett, Langmuir-Schaffer transfers, self assembly of various building blocks such as thiol on gold, silane on quartz, grafting of polymers, as well as ligand receptor recognition. In this review, the current state of different tethered bilayer membrane will be described. We will focus on critical analysis of the main advantages/drawbacks of each kind of model construction and their ability to allow protein incorporation in non-denaturing conditions. Some of the current drawbacks encountered in these biomimetic models can be overcome using an innovative tethered bilayer design based on a reliable and fast formation method. The successful protein incorporation of the Adenylate Cyclase produced by Bordetella pertussis and the voltage dependent anion channel (VDAC) was demonstrated on this model.

AFM characterization of gramicidin-A in tethered lipid membrane on silicon surface

Tethered lipid bilayers were formed on oxidized Si surfaces using the avidin–biotin interaction to investigate the lipid–membrane protein interactions by using gramicidin-A (g-A) as a model membrane protein. The morphology of the tethered lipid bilayer, observed by in situ atomic force microscopy (AFM), changed drastically by the reconstruction of g-A. The aggregation behavior of g-A was clearly different in the tethered membrane from those in simple supported membranes on mica and SiO 2 surfaces.

S1e2-5 Immunosensors based on Tethered Lipid Membranes(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS &amp; BSJ 2006)

S1e2-5 Immunosensors based on Tethered Lipid Membranes(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS &amp; BSJ 2006)

Investigating the Function of Ion Channels in Tethered Lipid Membranes by Impedance Spectroscopy

AbstractThe function of biologically important ion channels can be measured in supported lipid membranes by impedance spectroscopy. This approach offers substantial advantages over traditional electrophysiological measurements. In this article, we present an overview of the field, with a special emphasis on the reconstitution of ion channels in lipid bilayers tethered to gold electrodes and the modulation of their channel activity by specific ligand binding.

Formation of Tethered and Streptavidin-Supported Lipid Bilayers on a Microporous Electrode for the Reconstitution of Membranes of Large Surface Area

A new approach for the self-assembly of supported and tethered lipid membranes of large surface area is proposed. The template is a microporous electrode made by anodic etching of aluminum and covered with a monolayer of streptavidin. We show that spontaneous fusion of biotinylated lipid vesicles on the affinity layer is a slow process despite abundant accumulation of lipid material at the template surface. To increase dramatically the efficiency of the self-assembly, fast fusion is provoked with the help of a fusogen solution of poly(ethylene glycol). The extent of fusion is assessed by electrochemical monitoring of the long-range lateral mobility of ubiquinone (coenzyme Q10) in the supported bilayer. Finally, the geometrical characterization of the honeycomb structure at key steps of the self-assembly procedure is performed by electrochemical measurement of the porosity. As expected, the formation of the supported bilayer causes a decrease in the apparent inner diameter of the pores. It is expected that the type of supported lipid membrane built according to the present approach can be adequate for the incorporation of transmembrane proteins in structures that would mimic the membrane stacking found in chloroplasts or mitochondria.

Electrochemical studies of blocking properties of solid supported tethered lipid membranes on gold

The insulating properties of self-assembled thiolipid monolayers and tethered lipid bilayers on polycrystalline gold electrodes were studied by means of cyclic voltammetry (CV). These films were formed by two-step self-assembly processes. Electrochemical measurements of the heterogeneous electron transfer rate constant of different redox couples such as potassium ferrocyanide (K 4[Fe(CN) 6]) and dopamine (DP) were used to examine the molecular integrity and structural defects and pinholes within the monolayers. We demonstrate by means of cyclic voltammetry that the bilayer lipid membranes tethered to the gold surface are blocking, stable, yet retaining their dynamic properties and can be used as a model of the cell membrane.