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.     

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.     

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.     

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.     

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.     

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.     

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.     

Study on selectivity of permeating chiral complexes in bilayer lipid membranes (BLMs)

Electrical properties such as membrane potential (E_m) of planar bilayer lipid membranes (BLMs) are readily measured. Planar BLMs have been extensively used as models of biomembranes. In this paper we report BLMs formed in the solutions containing chiral complexes: d-K[Co(EDTA)], l-K[Co(EDTA)]; d-[Co(C₂O₄)(en)₂]I, and l-[Co(C₂O₄)(en)₂]I, whose E_m values display great differences, implying strong chiral selectivity. The permeability ratios of different chiral complexes calculated from E_m are the same as those obtained from human erythrocyte experiments. These results showed that chiral selectivity of cell uptake was mainly caused by the chirality of the membrane phospholipid itself. As a rapid and sensitive analytic tool, the BLM may be used to study permeating pathways and drug–membrane interactions. With further research, the BLM system may be developed into a useful method for drug screening.     

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.     

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.     

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.     

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.     

933 — Chemical modification of the bilayer lipid membrane biosensor dipolar potential

The bilayer lipid membrane (BLM) is an interesting model for a transducer, based on transmembrane ion current modulation by selective complexation of a membrane-embedded receptor with a suitable stimulant. The dipolar potential, which originates from the lipid bilayer headgroup zone, partially controls transmembrane ion current. Dipolar potentials associated with phosphatidyl choline/steroid lipid monolayers on a Langmuir-Blodgett thin-film trough have been measured with a non-contacting electrostatic voltmeter, and have been correlated with lipid chemistry. Lipid molecular interactions established by monolayer compression and BLM Arrhenius energy barriers determined from thermal dependence of ion conductivity of BLM have provided an evaluation of the dipolar potential with respect to its role in controlling ion current, surface ion adsorption and BLM structural integrity. Additionally, this work deals with the characteristics required for an optimum receptor-membrane system, which would best operate by dramatically influencing membrane fluidity/packing parameters as well as dipolar potential.     

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。     

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.     

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.     

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.     

Imaging of voltage-gated alamethicin pores in a reconstituted bilayer lipid membrane via scanning electrochemical microscopy

Voltage-gated biological ion channels were simulated by insertion of the peptaibol antibiotic alamethicin into reconstituted phosphatidylcholine bilayer lipid membranes (BLMs). Scanning electrochemical microscopy (SECM) was utilized to probe initial BLM resistivity, the insertion of alamethicin pores, and mass transport across the membrane. Acquired SECM images show the spatial location of inserted pore bundles, the verification of voltage control over the pore conformational state (open/closed), and variations in passive mass transport corresponding to different topographical areas of the BLM. SECM images were also used to evaluate overall BLM integrity prior to insertion as well as transport (flux in open state) and leakage (flux in closed state) currents following insertion.     

468—the study of the BLM reversible electrical breakdown mechanism in the presence of UO22+

The nature of the reversible decrease (by more than 7 orders of magnitude) of the resistance for bilayer lipid membranes (BLM), modified by UO₂²⁺ ions, in the electrical field is studied. It is shown that the process of BLM current growth during a reversible breakdown is accompanied by not more than a 2% change in the membrane capacitance. This result, the reproduced character of current oscillograms and the linear increase of breakdown current point to the appearance in the BLM, modified by UO₂²⁺, of a considerable number of local defects developed due to the effect of the electrical field. The nature of such defects is discussed.