Tethered Bilayer Lipid Membranes Research Articles

Tethered Bilayer Lipid Membranes Based on Monolayers of Thiolipids Mixed with a Complementary Dilution Molecule. 1. Incorporation of Channel Peptides

Tethered bilayer lipid membranes (tBLMs) are described based on the self-assembly of a monolayer on template stripped gold of an archea analogue thiolipid, 2,3-di-o-phytanyl-sn-glycerol-1-tetraethylene glycol-d,l-alpha-lipoic acid ester lipid (DPTL), and a newly designed dilution molecule, tetraethylene glycol-d,l-alpha-lipoic acid ester (TEGL). The tBLM is completed by fusion of liposomes made from a mixture of diphytanoylphosphatidyl choline (DPhyPC), cholesterol, and 1,2-diphytanoyl-sn-glycero-3-phosphate (DPhyPG) in a molar ratio of 6:3:1. Melittin and gramicidin are incorporated into these tBLMs as shown by surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) studies. Ionic conductivity at 0 V vs Ag|AgCl, 3 M KCl, measured by EIS measurements are comparable to the results obtained by other research groups. Admittance plots as a function of potential are discussed on a qualitative basis in terms of the kinetics of ion transport through the channels.

Membrane on a Chip: A Functional Tethered Lipid Bilayer Membrane on Silicon Oxide Surfaces

Tethered membranes have been proven during recent years to be a powerful and flexible biomimetic platform. We reported in a previous article on the design of a new architecture based on the self-assembly of a thiolipid on ultrasmooth gold substrates, which shows extremely good electrical sealing properties as well as functionality of a bilayer membrane. Here, we describe the synthesis of lipids for a more modular design and the adaptation of the linker part to silane chemistry. We were able to form a functional tethered bilayer lipid membrane with good electrical sealing properties covering a silicon oxide surface. We demonstrate the functional incorporation of the ion carrier valinomycin and of the ion channel gramicidin.

Synthesis and Characterization of SAMs and Tethered Bilayer Membranes from Unsymmetrically Substituted 1,2-Dithianes

The synthesis of a series of new membrane-forming compounds, based on previously established components used in an ion-channel switch (ICS) biosensor (Nature 1997, 387, 580) is described. All new compounds are derived from 4,5-dihydroxy-1,2-dithiane, and contain a hydrophilic reservoir section (based on ethylene glycol units) and a lypophilic section (based on the natural lipid phytanol). Ellipsometry and contact angle measurements indicate that self-assembled monolayers (SAMs) formed from the new materials possess thicknesses and membrane packing comparable to previously reported biosensor components. Tethered bilayer lipid membranes (t-BLMs) incorporating the novel materials were studied using impedance spectroscopy with the ion-carrier valinomycin. The electrical behavior and calculated membrane thickness of the t-BLMs are comparable to those derived from the standard ICS components and this demonstrates that the novel membrane-forming compounds form stable membranes that are sufficiently fluid to transport potassium ions in the presence of valinomycin.

ＳｉＯ２表面へのアビジン単分子層作製と脂質膜形成への応用

Single molecular layers of avidin are fabricated on SiO2 surfaces, and characterized by atomic force microscopy (AFM) and infrared reflection absorption spectroscopy. Immobilization of avidin on the SiO2 surface is performed by; i) ester-group-modification of the surface using silane-coupling reagent, ii) carboxylation of the surface by hydrolyzation in HCl, and iii) amide bonding between the surface COOH and the-NH2 of the avidin. It is confirmed from the AFM observation that the immobilized avidin is dispersed and adsorbed as single molecules, not in aggregated state. Function-recognizing-AFM-observation shows that the binding ability of the avidin to biotin molecules is remained even after the covalent immobilization. Formation of tethered lipid bilayer membranes on the avidin single molecular layer bridged by avidin-biotin binding is also described.

Incorporation of Na+,K+-ATP-ase into the Thiolipid Biomimetic Assemblies via the Fusion of Proteoliposomes

The black lipid membranes (BLMs) are artificial membrane systems that have been widely used in the study of different biological processes. In this paper the planar bilayer lipid membranes have been used to study the behavior of thiolipid molecules-dipalmitoyl-phosphatidyl-ethanolamine-mercaptopropionamide (DPPE-MPA) and cholesteryl 3-mercaptopropionate (Chs-MPA)-as compared to classical BLM made of natural lipids. We present our experiments on black thiolipid bilayer (BTM) formation from a thiolipid solution and basic results of pump currents generated by sodium-potassium pump-Na(+),K(+)-ATP-ase-introduced to such bilayer systems via proteoliposome adsorption with subsequent fusion. Our results imply that no substantial difference exists between BLMs formed from classical lipids and those made from thiolipids used in this study. The same thiolipid molecules were subsequently used for the formation of covalently bound, tethered bilayer lipid membranes (t-BLMs) on polycrystalline gold electrodes. Similarly, as in the case of BLMs, we took advantage of proteoliposome adsorption/fusion to obtain a t-BLM system with reconstituted enzyme. The vesicle fusion on hydrophobic or hydrophilic substrates is one of the main ways to obtain a bilayer system with incorporated biological species. In this paper we present also our preliminary results of electrochemical experiments using rapid solution exchange technique on such t-BLMs systems and their comparison with painted solid supported membranes (SSMs) and BLMs. We have also followed the process of vesicles fusion onto thiolipid monolayer by means of in situ atomic force microscopy in tapping mode (TM-AFM). On the basis of these experiments, we conclude that DPPE-MPA and Chs-MPA molecules used in our experiments preserve lipid properties, allowing for at least partial reconstitution of Na(+),K(+)-ATP-ase into such t-BLMs. On the other hand, the relatively compact organization on polycrystalline gold and the hydrophobic nature of the first monolayer of tethered thiolipids slows down the proteoliposome fusion onto such monolayers and consequently hinders the protein insertion. However, this effect can be overcome by mechanical stimulus that facilitates proteoliposome delamination onto the self-assembled monolayer.

The Protein-Tethered Lipid Bilayer: A Novel Mimic of the Biological Membrane

A new concept of solid-supported tethered bilayer lipid membrane (tBLM) for the functional incorporation of membrane proteins is introduced. The incorporated protein itself acts as the tethering molecule resulting in a versatile system in which the protein determines the characteristics of the submembraneous space. This architecture is achieved through a metal chelating surface, to which histidine-tagged (His-tagged) membrane proteins are able to bind in a reversible manner. The tethered bilayer lipid membrane is generated by substitution of protein-bound detergent molecules with lipids using in-situ dialysis or adsorption. The system is characterized by surface plasmon resonance, quartz crystal microbalance, and electrochemical impedance spectroscopy. His-tagged cytochrome c oxidase (C cO) is used as a model protein in this study. However, the new system should be applicable to all recombinant membrane proteins bearing a terminal His-tag. In particular, combination of surface immobilization and membrane reconstitution opens new prospects for the investigation of functional membrane proteins by various surface-sensitive techniques under a defined electric field.

The study of the interaction of a model α-helical peptide with lipid bilayers and monolayers

We studied the interaction of the α-helical peptide acetyl–Lys 2–Leu 24–Lys 2–amide (L 24) with tethered bilayer lipid membranes (tBLM) and lipid monolayers formed at an air–water interface. The interaction of L 24 with tBLM resulted in adsorption of the peptide to the surface of the bilayer, characterized by a binding constant K c=2.4±0.6 μM −1. The peptide L 24 an induced decrease of the elasticity modulus of the tBLM in a direction perpendicular to the membrane surface, E ⊥. The decrease of E ⊥ with increasing peptide concentration can be connected with a disordering effect of the peptide to the tBLM structure. The pure peptide formed a stable monolayer at the air/water interface. The pressure–area isotherms were characterized by a transition of the peptide monolayer, which probably corresponds of the partial intercalation of the α-helixes at higher surface pressure. Interaction of the peptide molecules with lipid monolayers resulted in an increase of the mean molecular area of phospholipids both in the gel and liquid crystalline states. With increasing peptide concentration, the temperature of the phase transition of the monolayer shifted toward lower temperatures. The analysis showed that the peptide–lipid monolayer is not an ideally miscible system and that the peptide molecules form aggregates in the monolayer.

Kinetics of valinomycin-mediated K + ion transport through tethered bilayer lipid membranes

Bilayer lipid membranes tethered to planar gold electrodes were prepared, based on a self assembled monolayer (SAM) of 2,3-di- O-phytanyl-sn-glycerol-1-tetraethylene glycol-DL-α-lipoic acid ester lipid (DPTL). When the SAM's were exposed to a suspension of liposomes made from diphytanoylphosphodatidyl choline (DPhyPC), tethered lipid bilayers (tBLMs) were formed with good sealing properties. The preformed tBLMs were doped with valinomycin, and the K + ion concentration in the bathing solution was increased stepwise by adding KCl. Electrical impedance spectra were recorded following every addition. These data were modelled by means of the network simulation program spice, using parameter values of the undoped membrane and a kinetic scheme for the K +/valinomycin system, whose rate constants were determined previously in independent measurements. Experimental and simulated data are in reasonable agreement despite some simplifying assumptions used in the spice simulations. Experimental data were also fitted to a conventional equivalent circuit, which reveals that the representation of the K +/valinomycin system by an ohmic resistor can be considered as no more than an approximation.

Tethered Bilayer Membranes Containing Ionic Reservoirs: Selectivity and Conductance

Ion channels, such as gramicidin A, selectively facilitate the transport of ions across biological and synthetic membranes. The conductance properties of ion channels are frequently characterized in synthetic bilayer lipid membranes (BLMs). The instability of BLMs has seriously limited the range of applications for these structures, and tethered bilayer lipid membranes (tBLMs) have addressed the problem through tethering many of the membrane components to a solid surface. In the present study, thin gold substrates have been used to tether thiol- and disulfide-terminated membrane components to form a tBLM electrode to provide a reservoir for ions. This study reports on the ion selectivity and apparent permeability of gramicidin channels in such tethered bilayer membranes. The investigations using electrical impedance spectroscopy indicated that the magnitude of ionic conductance varies substantially in reservoirs with different chemical structures. This study addressed the effect of changing ionic concentr...

Archaea analogue thiolipids for tethered bilayer lipid membranes on ultrasmooth gold surfaces.

Simple vesicle fusion allowed the formation of a bilayer lipid membrane from the self-assembled monolayer of lipoic acid ester 1 (see formula). Film thicknesses determined by surface plasmon resonance (SPR) spectroscopic measurements agree with the theoretical thicknesses of the stretched conformation. Electrochemical impedance spectroscopy (EIS) of the model system shows that the latter exhibits similar electrical properties to biological membranes.

Supramolecular functional interfacial architectures for biosensor applications

This short review describes some of our efforts in generating bio-functional supramolecular interfacial architectures for their use as affinity coatings in biosensor development based on a recently introduced novel optical recording principle combining the resonant excitation of surface plasmon modes and fluorescence detection schemes. Examples are given for multilayer assemblies designed for surface hybridization reactions between a grafted oligonucleotide catcher probe and target strands from solution. By describing the binding behavior of fluorophore-labeled rabbit–anti-mouse IgG to surface-attached mouse IgG, the limit of detection of the surface plasmon fluorescence spectroscopy will be shown to be in the lower femtomolar concentration range. These DNA-and protein binding studies will be complemented by examples for membrane-based biosensor platforms. We will document that tethered lipid bilayer membranes can be assembled with specific capacities of Cm = 0.5μF cm−2 and specific resistivities in excess of Rm > 20 MΩ cm2. The incorporation of the synthetic ionophore valinomycin can mediate the K+-ion translocation across these tethered membranes, thus reducing the resistivity selectively and reversibly by more than four orders of magnitude.

Tethered-bilayer lipid membranes as a support for membrane-active peptides.

An immunosensing device, comprising a lipid membrane incorporating ion channels tethered to the surface of a gold electrode, has been reported [Cornell, Braach-Maksvytis, King, Osman, Raguse, Wieczorek and Pace (1997) Nature (London) 387, 580-583]. The present article describes key steps in the assembly of the device and provides further evidence for its proposed sensing mechanism.

Tethered bilayer lipid membranes as a support for membrane active peptides

Tethered bilayer lipid membranes as a support for membrane active peptides

Kinetics of the competitive response of receptors immobilised to ion-channels which have been incorporated into a tethered bilayer.

A competitive ion channel switch (ICS) biosensor has been modelled yielding ligand mediated monomer-dimer reaction kinetics of gramicidin (gA) ion-channels within a tethered bilayer lipid membrane. Through employing gramicidin A, functionalized with the water-soluble hapten digoxigenin, it is possible to cross-link gramicidin to antibody fragments tethered at the membrane/aqueous interface. The change in ionic conductivity of the channel dimers may then be used to measure the binding kinetics of hapten-protein interactions at the membrane surface. The approach involves measuring the time dependence of the increase in impedance following the addition of a biotinylated antibody fragment (b-Fab'), which cross-links the functionalized gramicidin monomers in the outer layer of the lipid bilayer to tethered membrane spanning lipid. The subsequent addition of the small molecule digoxin, (M(r) 781 Da), competes with and reverses this interaction. The model provides a quantitative description of the response to both the cross-linking following the addition of the b-Fab' and the competitive displacement of the hapten by a water-soluble small analyte. Good agreement is obtained with independent measures of the cross-linking reaction rates of the gramicidin monomer-dimer and the b-Fab: hapten complex. The rate and amplitude of the competitive response is dependent on concentration and provides a fast and sensitive detection technique. Estimates are made of the concentration of gramicidin monomers in both the inner and outer monolayer leaflets of the membrane. This is used in the calculation of the gramicidin monomer/dimer equilibrium constant, K2D3. Other considerations include the membrane impedance limit set by the membrane leakage which is also a function of the concentration of the gA monomer concentration, and the two-dimensional kinetic association constant k2D2, of the hapten: b-Fab' complex. The gA dimer concentration is dependent on both the concentration of gA-dig and of the tethered streptavidin: b-Fab' complexes. The model shows that the 2D dissociation constant k2D3(-1), must be at least 10 times faster than the 3D dissociation constant k3D2(-1) for digoxin to completely reverse the cross-linked hapten-receptor interaction at the membrane interface.

Tethered Lipid Bilayer Membranes: Formation and Ionic Reservoir Characterization

Using novel synthetic lipids, a tethered bilayer membrane (tBLM) was formed onto a gold electrode such that a well-defined ionic reservoir exists between the gold surface and the bilayer membrane. Self-assembled monolayers of reservoir-forming lipids were first adsorbed onto the gold surface using gold−sulfur interactions, followed by the formation of the tBLM using the self-assembly properties of phosphatidylcholine-based lipids in aqueous solution. The properties of the tBLM were investigated by impedance spectroscopy. The capacitance of the tBLM indicated the formation of bilayer membranes of comparable thickness to solvent-free black (or bilayer) lipid membranes (BLM). The ionic sealing ability was comparable to those of classical BLMs. The function of the ionic reservoir was investigated using the potassium-specific ionophore valinomycin. Increasing the size of the reservoir by increasing the length of the hydrophilic region of the reservoir lipid or laterally spacing the reservoir lipid results in a...

Type VI membrane proteins?

Phospholipid vesicles containing ponticulin have been used to form solid supported and tethered bilayer lipid membranes. The ponticulin serves as both a nucleation site for actin polymerization as well as a binding site for F-actin. Studies of F-actin binding to such bilayers have demonstrated the formation of an in vitro actin scaffold. The dissociation constant for the binding of F-actin filaments to a ponticulin-containing tethered bilayer was found to be 11 ± 5 nM, indicative of high affinity binding.