Agar-supported lipid bilayers — basic structures for biosensor design. Electrical and mechanical properties

The lipid bilayer is widely accepted as the basic structure of all biological membranes. Known as BLM (bilayer lipid membrane), it can be prepared artificially. Suitably modified, the BLM serves as a very appropriate model for biological membranes. Recent investigations have verified the high analytical potential of artificial lipid membranes. With a structure and composition almost identical to the lipid moiety of biomembranes, the BLM may serve as an ideal host for receptor molecules of biological origin, thus becoming a transducer which could “see” the environment the way the living cell does. For the construction of lipid bilayer based biosensors; however, stable, easy to prepare and long-lasting lipid membranes are required. With this aim in mind, we have prepared lipid bilayer membranes which use an agar gel as support. This as-BLM (agar-supported BLM) has been shown to possess the same electrical, mechanical and dynamic properties the conventional BLM is famous for, along with the benefits of long-term stability and considerably elevated breakdown voltages. Its preparation on the tip of an agar-filled Teflon tube of 0.5 mm diameter is easy and can be performed even by less-skilled personnel.

In an attempt of further miniaturization the concept of the as-BLM was applied to thin-film micro-systems manufactured by standard micro-electronic techniques. The result is a lipid bilayer system, which, while preserving all the essential properties of the bilayer lipid membrane, can serve as a basic building block for cheap, disposable biosensoric systems.     

自组装ITO/双层磷脂膜的制备及其光电行为研究

在ITO(Indium-tin-oxide)导电玻璃电极上制备上自组装双层磷脂膜和经C60修饰的双层磷脂膜,研究了这种自组装双层磷脂膜的光电行为,考察了偏压、溶液中的给体和受体的浓度对自组装膜光电流强度的影响,讨论了C60分子对光电子跨膜传递过程的促进作用。     

双层脂和膜生物能量学中的826-氧化还原反应

本文对超薄人造双分子层膜(BLM)和叶绿体及线粒体生物膜中的电子过程进行了研究,总结了由伏安技术所得的近期实验。还讨论了基础电化学在膜研究中的应用,尤其对Eyring方程、Butler-Volmer方程和Tafel方程,以及按照膜孔电积作用在膜中的电子过程的起源进行了论述。讨论了在缺少双层脂的情况下确定氧化还原蛋白组分的标准电位(U′_0,对双层脂膜中有关的电子转移和生物氧化还原提出了设想。     

Alkylated glass partition allows formation of solvent-free lipid bilayer by Montal-Mueller technique

Formation of bilayer lipid membrane (BLM) by Montal-Mueller technique across a small aperture in a partition film traditionally requires coating of the aperture with a hydrophobic substance, often just an organic solvent. However, we demonstrate here that the most effective coating is not strictly hydrophobic but rather provides water/oil repellent properties. BLM were formed from diphytanoylphosphatidylcholine (DPhPC) on small 0.1-0.8 mm apertures made in specially prepared alkylated glass coverslips. The coverslips were either fluorosiliconized by 3,3,3-Trifluoropropyl-trimethoxysilane, which reduces adsorption of DPhPC in addition to creation of hydrophobic surface, or silanized, which promote adsorption of DPhPC. At fluorosiliconized surfaces stable BLM were formed. Specific capacitance of these BLM was 0.86 microF/cm(2)+/-5%, while their lateral tension was estimated as 4.3+/-0.4 mN/m. BLM were stable for hours under moderate voltage applied. At silanized surfaces stable BLM were formed only in acidic medium (3 相似文献   

521—Light-induced redox reactions in pigmented BLM

This paper is concerned with light-mediated phenomena in membranes of photosynthesis and vision and their in vitro model bilayer lipid membranes (BLM) of planar configuration containing appropriate photoactive compounds. Chloroplast extract BLM, bovine rhodopsin BLM and bacteriorhodopsin BLM are used as examples. Particular emphasis is placed on those molecular mechanisms of photoelectrochemical energy transduction in these pigmented lipid bilayers, which are relevant for the elucidation of photosynthetic and visual processes. Additionally, a pigmented BLM separating two aqueous solutions containing redox couples has been likened to that of a double Schottky barrier cell.     

Ion Channel Behavior of a Supported Bilayer Lipid Membrane Composed of 5,5－Ditetradecyl－2－ （2－trimethyl－ammonioethyl）－l, 3－dioxane Bromide Modified Glassy Carbon Electrode

A synthetic cationic surfactant, 5,5-ditetradecyl-2-(2-trimethyl-ammonioethyl)-l,3-dioxane bromide (DTDB), was used to construct a supported bilayer lipid membrane (s-BLM) coatedon an underlying glassy carbon electrode (GCE). Electrochemical impedance spectroscopy (EIS), small-angle X-ray diffraction (SAXD) and cyclic voltammetry were used to characterize the s-BLM. Both EIS and SAXD data indicated that the synthetic lipid exists as a well-oriented bilayer in the membrane.The voltammetric study showed that the lipid membrane can open ion channels in the presence of ClO4^- stimulant with Ru(bpy)3^2 as marker ions and give distinct channel currents.The channels can be dosed and open up again many times by removing or introducing ClO4^- anions.     

Voltammetric Elucidation of Ion Transfer Through an Extremely Thin Membrane

Digital simulation of the cyclic voltammogram for the ion transfer through a liquid membrane of thickness from 1 mm to 10 nm was performed. The magnitude of current and the shape of the voltammogram simulated for extremely thin membrane (10 nm thick) were similar to those observed experimentally with a bilayer lipid membrane, BLM, of about 10 nm in thick, when the diffusion coefficient of an ion in the BLM was assumed to be extraordinary small (10^?13 to 10^?14 cm² s^?1).     

Transport of Cesium Ion Across a Bilayer Lipid Membrane and Its Facilitation in the Presence of Iodide Ion

The ion transport current was clearly observed across a bilayer lipid membrane (BLM) between two aqueous phases (W1, W2) containing 0.1 M CsCl. This indicates that Cs⁺ can spontaneously penetrate the BLM in the absence of any kind of transporter. In addition, the current density at a given potential between W1 and W2 increased using I^? instead of Cl^? as the counter ion. Our results strongly suggest that the cell membrane transport is partially responsible for the internal exposure of ¹³⁴Cs and ¹³⁷Cs.     

细胞色素C在阴离子修饰的盐桥支撑双层类脂膜上的电化学行为及其应用

采用循环伏安法研究了细胞色素C在月桂酸阴离子修饰的盐桥支撑双层类脂膜上的氧化还原反应;对盐桥支撑双层类脂膜的特性、细胞色素C的电化学反应动力学以及有关影响细胞色素C循环伏安行为各种因素进行了详细探讨;并对用该双层类脂膜体系电化学测定细胞色素C进行了初步尝试。     

OPTICAL SPECTROSCOPY OF BILAYER MEMBRANES

Abstract— The absorption and fluorescence spectroscopy of natural and model bilayer lipid membranes is reviewed. Basic structural features of biological membranes and the relative advantages of black lipid membranes (BLM) and of liposomes are discussed. Theoretical considerations show that the wavelengths of absorption maxima are affected by the refractive index and dielectric constant of the medium surrounding the chromophore. Techniques of obtaining photoelectric action spectra, direct absorption spectra, and reflection spectra of BLM are described. Polarized spectra can give information about the orientation of membrane constituents and show, for example, that the porphyrin ring of chlorophyll in BLM is tilted at 45 ± 5° to the membrane surface. Absorption maxima of chlorophyll in BLM are compared with solution spectra of various chlorophyll adducts and aggregates. It is concluded that chlorophyll in BLM exists largely as solvated monomer and dimer (or oligomer), depending on concentration, and is not coordinated with water. From the theory of fluorescence spectroscopy it follows that aggregation and the polarity of the environment affect the fluorescence yield and lifetime of a membrane component, and also the wavelength of its emission maximum. The microviscosity of the membrane matrix affects the anisotropy of fluorescence. Techniques of steady-state fluorescence spectroscopy and of fluorescence lifetime measurements are reviewed. Examples of the use of fluorescent probes in membrane studies are given. Certain probes such as anilinonaphthalene sulfonate (ANS) preferentially bind to membrane proteins. The location of a probe in a particular membrane region can be pinpointed from its fluorescence yield and emission maximum. The orientation of the hydrocarbon chains of membrane lipids has been found, from fluorescence polarization of certain probes, to be normal to the membrane surface as postulated a priori on the basis of the lipid bilayer model. Anisotropy of fluorescence shows that elongated probe molecules rotate rapidly about their long axes when surrounded by phospholipids but become immobilized when bound to proteins. Changes in intensity and anisotropy of fluorescence as function of temperature have demonstrated the existence of phase transitions and phase equilibria of membrane lipids. Excimer fluorescence has been used as a measure of the available lipid core volume of membranes. Mechanisms of energy transfer between membrane components are reviewed. The theoretical dependence of energy transfer on distance and orientation for several rigid and fluid membrane models is discussed in terms of the structural information it can provide. Fluorescence sensitization resulting from energy transfer within and across bilayer membranes has been demonstrated in various systems. Quantitative measurement of energy transfer efficiency in BLM has shown that such transfer is about five times more efficient than in solutions at comparable donor-acceptor distances. Lipid membranes can be viewed as structures which maintain their components at high concentrations, in a reactive state, and at favourable orientations.     

CHARGE TRANSFER ACROSS PIGMENTED BILAYER LIPID MEMBRANE AND ITS INTERFACES

Abstract— The technique of forming bilayer lipid membranes (BLM) has made it possible to study photoreactions of pigments in an environment that is much closer to those in photosynthetic and visual membranes. A pigmented BLM system with Mg²⁺-porphyrins as membrane-bound pigments and with ferricyanide and ferrocyanide as the aqueous electron acceptor and donor, respectively, was used to illustrate the photoelectric effects due to coupled interfacial charge transfer reactions.
The steady-state continuous photoresponse was studied by means of the voltage clamp method and a null current method. The independence of the pigment conductance channel and the ionic conductance channel was demonstrated. A tunable voltage clamp method was used to study the transient pulsed photoresponses. Such a measurement permits us to characterize the photosystem in terms of an equivalent circuit model which contains a novel chemical capacitance. Molecular interpretation of this equivalent circuit model was given.
A microscopic model based on the Gouy–Chapman theory and chemical kinetics calculation leads to an equivalent circuit which is also equivalent to the previous one. Generalization of this microscopic model further leads to a physical mechanism of the generation of the early receptor potential (ERP) in visual membranes. Relevance of pigmented BLM research to photosynthesis and other disciplines was also discussed.     

Electrochemical biosensor based on supported planar lipid bilayers for fast detection of pathogenic bacteria

This paper presents a new ion-channel biosensor based on supported bilayer lipid membrane for direct and fast detection of Campylobacter species. The sensing element of a biosensor is composed of a stainless-steel working electrode, which is covered by artificial bilayer lipid membrane (BLM). Antibodies to bacteria embedded into the BLM are used as channel forming proteins. The biosensor has a strong signal amplification effect, which is defined as the total number of ions transported across the BLM. The total number of (univalent) ions flowing through the channels is 10¹⁰ ions s⁻¹. The biosensor showed a very good sensitivity and selectivity to Campylobacter species.     

Redox reactions in lipid bilayers and membrane bioenergetics

This paper begins with a consideration of electronic processes in ultrathin artificial bilayer lipid membranes (BLM) and biomembranes of chloroplasts and mitochondria. Methods and materials used in the study of BLMs are described next. The results of recent experiments, as obtained by voltammetric techniques, are summarized. The remaining sections of the paper deal with the application of basic electrochemistry to membrane research, in particular, the Eyring equation, the Butler-Volmer equation and the Tafel equation, and the origin of electronic processes in membranes in terms of electrostenolysis. The paper concludes with a discussion on the determination of standard potentials (U_o′) of redox protein components in the absence of the lipid bilayer, and finally a suggestion of the future experiments in relation to electron-transfer and bioredox reactions in bilayer lipid membranes.     

Facilitated Transport of Ions and Glucose by Amphotericin B Across Lipid Bilayers in the Presence or Absence of Cholesterol

The transport of ions and glucose across bilayer lipid membranes (BLM) facilitated by amphotericin B (AmB) is studied by use of planar BLMs and liposomal membranes. The transport characteristics change with time in the presence of cholesterol, while it is independent of time in the absence of cholesterol. The carrier‐type transport is observed immediately after the addition of AmB. In the presence of cholesterol, AmB forms a 1 : 1 complex with cholesterol and the channel is formed by aggregation of AmB‐cholesterol complexes. It is concluded that the number of the channels increases with time and that the carrier‐type transport decreases instead.     

Advances in Artificial Cell Membrane Systems as a Platform for Reconstituting Ion Channels

An artificial cell membrane that is composed of bilayer lipid membranes (BLMs) with transmembrane proteins incorporated within them represents a well‐defined system for the analysis of membrane proteins, especially ion channel proteins that are major targets for drug design. Because the BLM system has a high compatibility with recently developed cell‐free expression systems, it has attracted attention as a next‐generation drug screening system. However, three issues associated with BLM systems, i. e., their instability, the need for non‐volatile organic solvents and a low efficiency of ion channel incorporation, have limited their use as a drug screening platform. In this personal account, we discuss our recent approaches to address these issues based on microfabrication. We also discuss the potential for using the BLM system combined with cell‐free expression systems as a drug screening system for future personalized medicine.     

Receptor–ligand interactions in a reconstituted bilayer lipid membrane

A simple method is described to reconstitute membrane receptors into bilayer lipid membranes (BLMs). After reconstitution, the receptor still retains its ligand activity. Furthermore, the relationship between receptor–ligand interactions and electrical properties of reconstituted BLMs such as membrane capacitance (C_m) and membrane resistance (R_m) was studied. When glycophorin in erythrocyte and asialoglycoprotein in hepatocyte were taken as examples, it was found that the resistance of reconstituted BLM decreased when adding blood type monoclonal antibody or the solutions of galactose, respectively, and the decrease is ligand-concentration dependent; however, the membrane capacitance was not influenced. This provides a simple, practical approach to determining the interactions between the receptor and its ligand.     

The analytical potential of chemoreception at bilayer lipid membranes

The potential use of the bilayer lipid membrane as an electrochemical sensor is discussed through a study of model systems known to cause increased membrane conductance. The limit of detection for amphotericin B, a molecule capable of forming membrane pores, is in the region of 1O^-9 M. The current—time profile is discussed in terms of a mechanism which involves micelle formation in the aqueous and lipid phases. Unlike previous experiments, two current maxima with time are observed for valinomycin response (limit of detection 1O^-11 M). The first transient signal is attributed to increased membrane permeability caused by a conformational change in valinomycin in the “surface” volume of the bilayer. Selective interactions at membranes and the nature of membrane responses are discussed in terms of analytical parameters.     

Bilayer lipid membranes: An experimental system for biomolecular electronic devices development

The lipid bilayer postulated as the basic structural matrix of biological membranes is widely accepted. At present, the planar bilayer lipid membrane (BLM) together with spherical lipid bilayers (liposomes), upon suitable modification, serves as a most appropriate model for biological membranes. In recent years, advances in microelectronics and interest in ultrathin organic films, including BLMs and Langmuir-Blodgett (L-B) films, have resulted in a unique fusion of ideas toward the development of biosensors and transducers. Furthermore, recent trends in interdisciplinary studies in chemistry, electronics, and biology have led to a new field of research: biomolecular electronics. This exciting new field of scientific-technological endeavor is part of a more general approach toward the development of a new, post-semiconductor electronic technology, namely, molecular electronics with a long-term goal of molecular computers.

Recently, it has been demonstrated that BLMs, after suitable modification, can function as electrodes and exhibit nonlinear electronic properties. These and other experimental findings relevant to sensor development and to “biomolecular electronic devices” (BED) will be described in more details in the present review article. Also the potential use of the BLM system together with its modifications in the development of a new class of organic diodes, switches, biosensors, electrochemical photocells, and biofuel cells will be discussed. Additionally, this paper reports also a novel technique for obtaining BLMs (or lipid bilayers) on solid supports. The presence of solid support on one side of the BLM greatly enhances its mechanical stability, while retaining the dynamic properties of the lipid bilayer. Advantages of the new techniques for self-assembling amphiphilic molecules on rigid substrates are discussed in terms of their possible uses. It is evident that the new BLM system (s-BLMs) is potentially useful for technological applications in the area of biosensors and enzyme electrodes as well as molecular electronics.