The lipid bilayer concept and its experimental realization: from soap bubbles,kitchen sink,to bilayer lipid membranes

The inspiration for lipid bilayer research, without question, comes from the biological world. Although self-assembled bilayer lipid membranes (BLMs) in vitro, were first reported in 1961, experimental scientists have been dealing with BLM-type interfacial adsorption phenomena since Robert Hooke’s time (1672). BLMs (of planar lipid bilayers) have been used in a number of applications ranging from basic membrane biophysics including transport, practical AIDS research, and ‘microchips’ studies, to the conversion of solar energy via water photolysis, to biosensor development using supported bilayer lipid membranes (s-BLMs), and to photobiology comprising apoptosis and photodynamic therapy. This paper presents an overview of the origin of the lipid bilayer concept and its experimental realization, as well as the studies of our laboratory and recent research of others on the use of BLMs as models of certain biomembranes. In addition, we describe briefly our present work on supported BLMs as biosensors and molecular devices; the experiments carried out in close collaboration with colleagues on s-BLMs are delineated.     

Polymerized planar suspended lipid bilayers for single ion channel recordings: comparison of several dienoyl lipids

The stabilization of suspended planar lipid membranes, or black lipid membranes (BLMs), through polymerization of mono- and bis-functionalized dienoyl lipids was investigated. Electrical properties, including capacitance, conductance, and dielectric breakdown voltage, were determined for BLMs composed of mono-DenPC, bis-DenPC, mono-SorbPC, and bis-SorbPC both prior to and following photopolymerization, with diphytanoyl phosphocholine (DPhPC) serving as a control. Poly(lipid) BLMs exhibited significantly longer lifetimes and increased the stability of air-water transfers. BLM stability followed the order bis-DenPC > mono-DenPC ≈ mono-SorbPC > bis-SorbPC. The conductance of bis-SorbPC BLMs was significantly higher than that of the other lipids, which is attributed to a high density of hydrophilic pores, resulting in relatively unstable membranes. The use of poly(lipid) BLMs as matrices for supporting the activity of an ion channel protein (IC) was explored using α-hemolysin (α-HL), a model IC. Characteristic i-V plots of α-HL were maintained following photopolymerization of bis-DenPC, mono-DenPC, and mono-SorbPC, demonstrating the utility of these materials for preparing more durable BLMs for single-channel recordings of reconstituted ICs.     

Biophysical aspects of agar-gel supported bilayer lipid nembranes: a new method for forming and studying planar bilayer lipid membranes

Conventional bilayer lipid membranes (BLMs), formed by either the painting method or the Langmuir-Blodgett technique, lack the desired stability. This paper presents a simple method for forming long-lived BLMs on agar-gel supports. The supported BLM reported has a greatly improved mechanical stability and also has desirable dynamic properties. These self-assembled BLMs are of significant interest, in view of their similarity of biological membranes, their molecular dimension and their spontaneous formation.     

Self-assembled BLMs: biomembrane models and biosensor applications

In the last few years, there have been a number of research papers on self-assemblies of molecules as ‘advanced’ or ‘smart’ materials. The inspiration for this exciting research, without question, comes from the biological world, where, for example, the lipid bilayer of the cell membrane is the most important self-assembling system. Although the first report on self-assembled bilayer lipid membranes (BLMs) in vitro was published in 1962, interface science, including surface and colloid science, has been dealing with these interfacial self-assemblies of amphiphilic molecules since Robert Hooke's time (1672). BLMs have been used in a number of applications, ranging from basic membrane biophysics studies to the conversion of solar energy via water photolysis, and to biosensor development using supported bilayer lipid membranes (s-BLMs and sb-BLMs). This paper briefly summarizes the past research on the use of BLMs as models of biological membranes and describes some details of our current work on supported BLMs as practical biosensors. Additionally, experiments carried out in close collaboration with others on s-BLMs and sb-BLMs are presented.     

Biosensor for dopamine based on stabilized lipid films with incorporated resorcin[4]arene receptor

This work reports a technique for the stabilization after storage in air of a lipid film with incorporated resorcin[4]arene receptor based biosensor for dopamine. Microporous filters composed of glass fibers (nominal pore sizes, 0.7 and 1.0 microm) were used as supports for the formation and stabilization of these devices and the lipid film is formed on the filter by polymerization prior its use. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2'-azobis-(2-methylpropionitrile) was the initiator. The stability of the lipid films by incorporation of a receptor for the preparation of stabilized lipid film biosensor is studied throughout this work. The response towards dopamine of the present stabilized for repetitive uses lipid membrane biosensor composed of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidic acid was compared with planar freely suspended bilayer lipid membranes (BLMs). The stabilized lipid membranes provided similar artificial ion gating events as BLMs in the form of transient signals and can function for repetitive uses after storage in air. However, the response of the stabilized lipid films was slower than that of the freely suspended BLMs. This will allow the practical use of the techniques for chemical sensing based on lipid films and commercialization of these devices, because it is now possible to prepare stabilized lipid film based biosensors and store them in the air.     

Formation of artificial lipid bilayers using droplet dielectrophoresis

We describe the formation of artificial bilayer lipid membranes (BLMs) by the controlled, electrical manipulation of aqueous droplets immersed in a lipid-alkane solution. Droplet movement was generated using dielectrophoresis on planar microelectrodes covered in a thin insulator. Droplets, surrounded by lipid monolayers, were brought into contact and spontaneously formed a BLM. The method produced BLMs suitable for single-channel recording of membrane protein activity and the technique can be extended to create programmable BLM arrays and networks.     

双层磷脂膜的电化学性质及其在生物传感器中的应用

由于双层磷脂膜（BLM）可模仿自然界的生物细胞膜的生物相容性，成为物分子的天然固定化材料，因此生物传感器的研制领域显示出广泛的应用前景，本文介绍了BLM、s-BLM的电化学性质，制备技术，并评述了其在生物传感器的应用研究进展。     

Photosensitized damage of bilayer lipid membrane in the presence of haematoporphyrin dimethylether

The variations in electrical conductivity and surface tension of planar bilayer lipid membranes (BLMs) sensitized by a haematoporphyrin dimethylether (HpDME) on visible light irradiation are reported. The irradiation of BLMs immediately leads to a decrease in membrane surface tension. On irradiation the conductivity of BLMs remains constant for a certain period of time (induction time), followed by an increase, terminated by membrane breakage. The induction time is not dependent on stirring of the solution, the addition of azide or ferricyanide to the solution, the addition of antioxidant to the lipid or substitution of air for argon in the cell. The induction time decreases for repeated irradiations or for any new BLM formed in the same cell immediately after the previous membrane has been broken. The conductivity shift consists of reversible and irreversible components. These results suggest that the irradiation of BLMs sensitized by HpDME leads to an accumulation of photoproducts in the membrane which induce pore formation and to a decrease in BLM stability when the concentration of the photoproducts exceeds a critical level.     

Planar artificial biomembranes optimized for biochemical assay

Langmuir-Blodgett monolayer and planar bilayer lipid membrane (BLM) experiments are used to study the relative significance of dipolar potential and packing/fluidity in the control of the permeability of phospholipid/steroid BLMs to potassium ion. Practical chemical construction of BLMs designed to achieve particular dipolar potential and packing/fluidity characteristics are described. The ability to modify selectively the salient physical properties of BLMs allows optimization of the ion permeability and receptor activity of the membrane. The use of BLMs to quantify drug response and receptor activity is illustrated by examples involving valinomycin, phloretin, concanavalin A and auxin receptor.     

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。     

Miniaturized biochemical sensing devices based on planar bilayer lipid membranes

Methods of in-vitro artificial formation of bilayer lipid membranes (BLM) and their analytical applications are reviewed, on the basis of 122 literature references. Different techniques for preparation of free-suspended planar BLMs, and gel-, filter-, and solid-supported systems are presented. The analytical applications developed are based on direct interaction of analytes with chemically unmodified BLMs, and with systems modified by use of redox mediators, ionophores, ion-channel forming species, enzymes, antibodies, or DNA.     

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.     

Biosensors for the Rapid Repetitive Detection of Adrenaline Using Stabilized Bilayer Lipid Membranes (BLMs) with Incorporated Calix[4]resorcinarene Receptor

This work uses lipid film based biosensors with incorporated calix[4]resorcinarene receptor (lipophilic macrocyclic host molecule) for the rapid electrochemical detection of adrenaline. Freely‐suspended and metal supported BLMs (composed of egg phosphatidylcholine (PC) and 35% (w/w) dipalmitoyl phosphatidic acid) modified with the resorcin[4]arene receptor were used as one shot sensors to rapidly detect this catecholamine. The interactions of this compound with freely‐suspended BLMs were found to be electrochemically transduced in the form of a transient current signal with duration of seconds, which reproducibly appeared about 14 s after exposure of the membranes to adrenaline. The response time for these BLMs without incorporated receptor for adrenaline was about 1.5 min. The magnitude of the transient current signal was related to the concentration of adrenaline in bulk solution in the micromolar range. Differential scanning calorimetric (DSC) experiments were performed to explore the mechanism of interactions of BLMs with incorporated receptor with adrenaline. The interactions of adrenaline with surface‐stabilized bilayer lipid membranes (sBLMs) with incorporated receptor produced electrochemical ion current increases, which reproducibly appeared within a few seconds after exposure of the membranes to the stimulant. The use of the receptor in sBLMs increased the sensitivity of the method 6‐fold. The current signal increases were related to the concentration of adrenaline in bulk solution in the micromolar range. Stabilized lipid membranes formed by polymerization on glass fiber microfilters were used as practical chemical biosensors for the rapid detection of adrenaline. The interactions of polymerized lipid films with adrenaline were also found to provide transient current signals similar to those of freely‐suspended BLMs. The magnitude of the transient current signal was also related to the concentration of the stimulating agent in bulk solution in the micromolar range and these stabilized lipid films can be used again after storage in air. No interferences from ascorbic acid were noticed because of the negatively charged lipids in membranes. The effect of other compounds such as proteins and other compounds closely related to adrenaline was also investigated. Results of recovery experiments using human urine have shown recoveries ranged between 94 to 105%, which shows no interferences from matrix effects due to the presence of urine constituents. The present sensor based on stabilized lipid films can be used for the rapid repetitive detection of this pharmaceutical substance and keep prospects for the selective determination of catecholamines in biofluids.     

Ion permeability through bilayer lipid membranes for biosensor development: control by chemical modification of interfacial regions between phase domains.

Based on the results of studies on cystic fibrosis, which implicated hydroxystearic acid (HSA) as a contributing factor in altered biomembrane function, solvent-free bilayer lipid membranes (BLMs) and monolayer films were prepared from a lipid mixture containing (by mass) 34% phosphatidylcholine, 19% dipalmitoylphosphatidyl serine, 47% cholesterol and variable amounts of 10- and 12-HSA (0-50%). Ion currents, resulting from K+ permeation through BLMs that were supported in 0.1 mol dm-3 KCl solutions buffered to pH 7.4, were monitored with use of a d.c. circuit. The structures of monolayer films at the air-water interface of a Langmuir-Blodgett trough were studied by pressure-area correlations and by further correlation with microscopic phase separation as revealed by fluorescence microscopy. In order to elucidate the role of the hydroxyl moieties in ion permeability, the transmembrane ion current was corrected for the effect of the negative surface charge of the carboxylic acid by replacement of the HSA component with stearic acid. The ion current was found to increase with the molar proportion of the HSAs. Two models for ion conduction through BLMs were considered: 'hopping' via hydrophilic sites within the hydrophobic zone of the BLMs, introduced by the hydroxyl moiety of 10- or 12-HSA; and transport through interfacial regions between phase domains that represent areas of low steric density and low structural order within monolayers. Although the two mechanisms are not distinct, the ion permeability results indicate a change in the response of ion current to HSA concentration at 35 mol-%, suggesting a change in the relative proportion of the mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Planar bilayer lipid memebranes

The planar bilayer lipid membrane, also known as lipid bilayer membrane, black lipid membrane or simply BLM(s), for short, has been investigated since its inception in 1960, the details of which have been described in a monograph published in 1974. This review is a report on the advances in the BLM research since that time.After a brief introduction, the first five sections consider various aspects of experimental methods, optical properties, thermodynamics of lipid bilayers, permeability, and electrical properties of BLMs. Section 7 deals with the use of BLM as energy transducer, particularly the transduction of light into electrical energy. Section 8, the longest portion of the paper, is devoted to modelling of biomembranes, such as the plasma membrane of cells, the thylakoid membrane of chloroplasts, the cristae membrane of mitochondria, the visual receptor membrane of the eye, and the nerve membrane. The concluding section points out that studies of BLMs facilitate the initial testing of working hypothses and may lead to a better choice of appropriate $\text{in vivo}$ and reconstituted membrane experiments.     

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.     

Air-exposure technique for the formation of artificial lipid bilayers in microsystems

To develop a reliable method for on-chip bilayer lipid membrane (BLM) formation, which could be employed for use in a biosensor array platform, a polymer microfluidic device has been constructed, and the formation of suspended BLMs within it has been investigated. A simple, yet reproducible BLM formation protocol has been developed, in which a brief air-exposure period is employed to induce the rapid thinning of an initially thick lipid-solvent layer. The technique is rapid, reproducible, and amenable to the simple injection of proteins or analytes, as well as to buffer exchange on both sides of the membrane. Scaling up the technique for use in an array platform is also straightforward, the simultaneous formation of three individually addressable BLMs being demonstrated.     

双层类脂膜的修饰及其在分析化学中的应用

双层类脂膜（ＢＬＭｓ）是公认的与众多生命过程直接相关的生物膜模拟体系。作者评述了大环化合物超分子试剂、药物和抗原抗体对ＢＬＭｓ的镶嵌修饰以及被修饰的ＢＬＭｓ体系和分析化学中的应用研究进展,其中包括基于ＢＬＭｓ的生物传感器和模拟酶;展望了发展趋势。引用文献６６篇。     

Micro‐ and Nano‐Technologies for Lipid Bilayer‐Based Ion‐Channel Functional Assays

Ion channel proteins provide gated pores that allow ions to passively flow across cell membranes. Owing to their crucial roles in regulating transmembrane ion flow, ion channel proteins have attracted the attention of pharmaceutical investigators as drug targets for use in the studies of both therapeutics and side effects. In this review, we discuss the current technologies that are used in the formation of ion channel‐integrated bilayer lipid membranes (BLMs) in microfabricated devices as a potential platform for next‐generation drug screening systems. Advances in BLM fabrication methodology have allowed the preparation of BLMs in sophisticated formats, such as microfluidic, automated, and/or array systems, which can be combined with channel current recordings. A much more critical step is the integration of the target channels into BLMs. Current technologies for the functional reconstitution of ion channel proteins are presented and discussed. Finally, the remaining issues of the BLM‐based methods for recording ion channel activities and their potential applications as drug screening systems are discussed.     

膜片电极支撑自组装双层脂膜中短杆菌肽离子通道的形成

通过一个两步程序在膜片电极尖端形成自组装双层脂膜：（1）膜片电极尖端沾取成膜液；（2）将吸附成膜液的尖端浸入电解液中，排除尖端多余的成膜液，通过电学方法监测双层脂膜的形成。将短杆菌肽通道蛋白分散在成膜液和电解质溶液中，在制备膜片电极支撑双层脂膜过程中，短杆菌肽重组到双层脂膜中形成离子通道，对通道的一般特性进行了研究，并观察到通道开放和关闭的现象。