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.     

The 40th anniversary of bilayer lipid membrane research

This paper presents a historic perspective on the origin of the lipid bilayer concept and its experimental realization. Additionally, current studies in close collaboration with our colleagues on the use of supported BLMs as biosensors and molecular devices are delineated. Further, recent research of others on BLMs (planar lipid bilayers) is referenced.     

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.     

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.     

The design of molecular sensing interfaces with lipid-bilayer assemblies

We describe the current status of techniques for preparing planar lipid bilayers, the fusion of sensory elements and the design of bilayer interfaces for analytical applications. Advances in bilayer fabrication have allowed preparation of lipid bilayers with membrane resistance of >1 GΩ (“gigaseal”) in flow, microfluidic and array formats, which have enabled single-channel and multi-channel recording. Not only biological but also engineered channels have been adopted as sensory elements for lipid-bilayer-based biosensors. Synthetic and inorganic channels are also emerging for designing membranes for lipid-bilayer sensors. We discuss the potential of lipid bilayers containing biological, engineered or synthetic channels for the design of biosensors, including drug-screening biosensors.     

Bilayer lipid membranes formed from thermotropic liquid crystals

This paper reports a new type of bilayer lipid membrane (BLM) with aqueous interfaces which are formed from thermotropic liquid crystals (nematic-6CB, smectic-8CB, and cholesteric-cholesteryl palmitate, ChP). The electrical properties of these unmodified membranes have been investigated. We suggest that BLMs of this type in their 'black' states consist of two molecular layers with a smectic-like structure.     

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.     

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.     

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.     

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.     

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。     

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.     

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.     

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.     

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.     

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。     

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.     

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.     

Evaluation of a glassy carbon electrode modified by a bilayer lipid membrane with incorporated DNA

The objective of the present work was the evaluation and characterization of a glassy carbon (GC) electrode modified by a bilayer lipid membrane (BLM) with incorporated single-stranded deoxyribonucleic acid fss DNA). Various procedures were developed and tested for the incorporation of ss DNA at the electrode modified by the lipidic membrane: Differential pulse voltammetry (i.e. oxidation of guanine and adenine residues) was used to monitor the incorporation of ss DNA at the GC electrode modified by the BLM. The results have shown that the lipid membrane enhances the stability of ss DNA during a "medium-exchange" of the electrode and prohibits its diffusion from the electrode surface. The third scheme was proven to be the most appropriate as both electrode modification by the BLM and DNA adsorption occur in one stage and much faster (as no BLM thinning process is required) as compared to the former two techniques; furthermore, maximized loading of DNA in BLMs is achieved which reduces by ca. 10-fold the DNA amounts that can be detected electrochemically. Conventional planar "free-suspended" and self-assembled metal supported BLMs were used to monitor in situ the incorporation of ss DNA in these membranes. The results have shown that the adsorption of ss DNA at lipid membranes (as a medium for DNA incorporation on an electrode surface) can occur much faster, using milder conditions and smaller amounts of DNA than by previously described techniques.     

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。