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.     

PHOTOELECTRIC EFFECTS IN DYE-SENSITIZED ARTIFICIAL LIPID MEMBRANES

Using dyes of known redox potentials the specific mechanisms of dark and light potential generation is analyzed in pigmented lipid membranes. The role of the ionic and electronic conductance, as well as the redox potential gradient is specifically related to the observed open circuit voltage developed across the membrane. The results can be most easily explained by the redox electrode model.     

ELECTRONIC PROCESSES AND PHOTOELECTRIC ASPECTS OF BILAYER LIPID MEMBRANES

Abstract— Owing to the complexity of biological membranes, many model systems have been studied in order to gain insight into the molecular mechanism of specific functions. One such model membrane extensively investigated in the past decade is the so-called bilayer lipid membrane (BLM). With suitable modifications, a BLM less than 100 A thick separating two aqueous solutions has been used as a model for a variety of biological membranes. This paper is devoted to a review of the properties and electronic processes of modified BLM.
Recent experiments using these membranes which contain photosynthetic pigments or dyes have demonstrated that, upon illumination, an EMF and a current can be generated. The connection between the photoelectric BLM and light-sensitive biological membranes and the rationale for this work are described.
Additionally, the effects of physical chemical parameters such as electric field, temperature, light intensity, duration of illumination and chemical agents (electron acceptors, donors, uncouplers, etc.) on the photoresponses of BLM are discussed. Other results indicate that BLM containing photoactive compounds behave similar to that of a silicon solar cell with one side of the membrane reducing and the other side oxidizing. The transverse pathway for the electron across the BLM could be provided by carotenoids such as β-carotene. Photoelectric BLM of this type represents a unique kind of energy transducing system and may well be useful in the conversion of solar energy into electricity and/or other forms of energy.     

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.     

Reconstitution of the voltage-gated K+ channel KAT1 in planar lipid bilayers

The single-channel current has been recorded for the voltage-gated K⁺ channel from Arabidopsis thaliana, KAT1, reconstituted in the planar bilayer lipid membrane (BLM). Channel-like current was observed between two aqueous phases after the addition of the proteoliposomes into one aqueous phase. In the potential range from 60 to 120 mV, the single-channel current was recorded and the conductance was calculated to 10.0–12.5 pS. The open channel probability increased with an increase of the applied membrane potential. These characteristics of the reconstituted channels are close to those of KAT1 reported by Hoshi et al. and Hedrich et al. with the patch clamp technique. This is the first work in which the isolated ion channel from higher plants was reconstituted in the planar BLM system.     

Characteristics of Single Ionic Channels Induced by Sulfonic Acid Derivatives of Dibenzo-18-Crown-6 in Bilayers

Sulfonic acid derivatives of dibenzo-18-crown-6 (DB18C6) have been shown to induce formation of ion channels permeable to monovalent cations in bilayer lipid membranes (BLM). Some electric characteristics of channels have been studied by means of the voltage clamp method. Channels displayed little interionic discrimination. The voltage dependence as well as the multiple state behaviors of the channels functioning was observed. The channels formation has been shown to be dependent on pH of bath solutions and on the presence of bivalent cations in them. The channels supposed to be formed from aggregates of complexes associates assembled in aqueous salt solutions.     

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。     

Interaction of pyridinium bis-retinoid (A2E) with bilayer lipid membranes

The accumulation of lipofuscin granules within the retinal pigment epithelium (RPE) cells is correlated with the progression of age-related macular degeneration. One of the fluorophores contained in lipofiscin granules is pyridinium bis-retinoid (A2E). To test its membrane-toxic effect, the interaction of A2E with bilayer lipid membranes (BLM) was studied. The incorporation of charged A2E molecules into the membranes has been detected as a change of either zeta-potential of multilayer liposomes or boundary potential of BLM. It was shown that the presence of up to 25mol% of A2E did not destabilize the bilayers made of saturated phosphatidylcholine (PC). However, the destabilizing effect became very significant when BLM contained negatively charged lipids such as cardiolipin or phosphatidylserine. The electrical breakdown measurements revealed that the A2E-induced decrease of BLM stability was primarily associated with the growing probability of lipid pore formation. It was found from the measurements of boundary potential of BLM that exposure of A2E to light initiates its transformation into at least two products. One of them is epoxy-A2E, which, being hydrophilic, moves from the membrane into water solution. The other product is a non-identified hydrophobic substance. Illumination of A2E-containing BLM made from unsaturated PC by visible light caused the membrane damage presumably due to oxidation of these lipids by singlet oxygen generated by excited A2E molecules. However, this effect was very weak compared to the effect of known photosensitizers. The illumination of BLM with A2E also leads to the damage of gramicidin incorporated into the membrane, as was detected by measuring the conductance of channels formed by this peptide.     

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   

LIGHT-INDUCED ELECTRICAL EFFECTS IN A LIQUID CRYSTAL BLM CONTAINING TCNQ

Abstract— The light-induced capacitance changes and also both photovoltage and photocurrent under continuous illumination have been investigated in pigmented liquid crystal bilayer membranes (PBLM)‡ containing TCNQ as photosensitizer with Na₂SO₃ electron donor on one side and methylene blue electron acceptor on the other side. The results have shown that TCNQ in cyanobiphenyl membrane produces a unique photoactive BLM system in which all three main parameters (conductivity, capacity and voltage across the membrane) are in a wide range altered by the light. It is shown that a TCNQ-cyanobiphenyl charge transfer complex is responsible for the observed photochanges. The possible mechanism of photoinduced electrical effects in this type of PBLM is discussed.     

Synthetic ion channel activity documented by electrophysiological methods in living cells

Hydraphiles are synthetic ion channels that use crown ethers as entry portals and that span phospholipid bilayer membranes. Proton and sodium cation transport by these compounds has been demonstrated in liposomes and planar bilayers. In the present work, whole cell patch clamp experiments show that hydraphiles integrate into the membranes of human embryonic kidney (HEK 293) cells and significantly increase membrane conductance. The altered membrane permeability is reversible, and the cells under study remain vital during the experiment. Control compounds that are too short (C(8)-benzyl channel) to span the bilayer or are inactive owing to a deficiency in the central relay do not induce similar conductance increases. Control experiments confirm that the inactive channel analogues do not show nonspecific effects such as activation of native channels. These studies show that the combination of structural features that have been designed into the hydraphiles afford true, albeit simple, channel function in live cells.     

Bilayer lipid membranes from falling droplets

We describe a system that provides a rapid and simple way of forming suspended lipid bilayers within a microfluidic platform from an aqueous droplet. Bilayer lipid membranes are created in a polymeric device by contacting monolayers formed at a two-phase liquid–liquid interface. Microdroplets, containing membrane proteins, are injected onto an electrode positioned above an aperture machined through a conical cavity that is filled with a lipid–alkane solution. The formation of the BLM depends solely on the device geometry and leads to spontaneous formation of lipid bilayers simply by dispensing droplets of buffer. When an aqueous droplet containing transmembrane proteins or proteoliposomes is injected, straightforward electrophysiology measurements are possible. This method is suitable for incorporation into lab-on-a-chip devices and allows for buffer exchange and electrical measurements.

Zeta potential of ion-conductive membranes by streaming current measurements

Surface charge properties have a significant influence on membrane retention and fouling performance. As a key parameter describing the surface charge of membranes used in aqueous applications, zeta potential measurements on membranes of various types have attracted great attention. During the zeta potential characterization of a series of ion-conductive sulfonated poly(sulfone) membranes, it was found that the measured streaming current varied with the thickness of the sample, which is not predicted by the classical Smoluchowski equation. Moreover, for higher conductivity membranes with an increased concentration of sulfonate groups, the zeta potential tended toward zero. It was determined that the influence of membrane bulk conductance on the measured streaming current must be taken into account in order to correctly interpret the streaming current data for ion-conductive polymers and understand the relationship between membrane chemical composition and zeta potential. Extrapolating the measured streaming current to a membrane thickness of zero has proven to be a feasible method of eliminating the error associated with measuring the zeta potential on ion conductive polymer membranes. A linear resistance model is proposed to account for the observed streaming currents where the electrolyte channel is in parallel with the ion-conductive membranes.     

Study of the Ion Channel Behavior of Didodecyldimethylammonium Bromide Formed Bilayer Lipid Membrane Stimulated by PF6

Bilayer lipid membranes(BLM) formed from didodecyldimethylammonium bromide were made on the freshly exposed surface of a galssy carbon(GC) and were demonstrated by the acimpedance spectroscopy.The ion channels of membrane properties induced by PF6^- were studied by the cyclic voltammetric methods.Experimental results indicated that the ion channel of BLM was open in the presence of the PF6^- due to the interaction of PF6^- with the BLM,while it was switched off in the absence of PF6^-.Because the ion channel behavior was affected by the concentration of PF6^-,a sersor for PF6^- can be developed.     

The effect of pigments on the crystallization and properties of polypropylene

The effect of pigments of different chemical compositions on the crystellization process of PP was studied by means of differential scanning calorimetry in isothermal mode. The Avrami equation was applied to obtain the crystallization parameters of polypropylene with different pigments. Lipatov's equations were applied for evaluation of the thickness and volume content of the transition layers. Results show that pigments affect not only the degree of crystallinity but also the structure of the amorphous phase and the integrity of the crystalline/amorphous interphase. Finally, the morphology and mechanical properties of pigmented polypropylene were studied in view of the different chemical compositions of the various pigments.     

Activity monitoring of functional OprM using a biomimetic microfluidic device

This paper describes the fabrication and use of a biomimetic microfluidic device for the monitoring of a functional porin reconstituted within a miniaturized suspended artificial bilayer lipid membrane (BLM). Such a microfluidic device allows for (1) fluidic and electrical access to both sides of the BLM and (2) reproducible membrane protein insertion and long-term electrical monitoring of its conductance (G(i)), thanks to the miniaturization of the BLM. We demonstrate here for the first time the feasibility to insert a large trans-membrane protein through its β-barrel, and monitor its functional activity for more than 1 hour (limited by buffer evaporation). In this paper, we specifically used our device for the monitoring of OprM, a bacterial efflux channel involved in the multidrug resistance of the bacteria Pseudomonas aeruginosa. Sub-steps of the OprM channel conductance were detected during the electrical recordings within our device, which might be due to oscillations between several structural conformations (sub-states) adopted by the protein, as part of its opening mechanism. This work is a first step towards the establishment of a genuine platform dedicated to the investigation of bacterial proteins under reconstituted conditions, a very promising tool for the screening of new inhibitors against bacterial channels involved in drug resistance.     

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.     

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.     

Supra-physiological membrane potential induced conformational changes in K+ channel conducting system of skeletal muscle fibers

The effects of a supra-physiological membrane potential shock on the conducting system of the delayed rectifier K(+) channels in the skeletal muscle fibers of frogs were studied. An improved double Vaseline gap voltage clamp technique was used to deliver stimulation pulses and to measure changes in the channel currents. Our results showed that a single 4 ms, -400 mV pulsed shock can cause a reduction in the K(+) channel conductance and a negative-shift of the channel open-threshold. Following the Boltzmann theory of channel voltage-dependence, we analyzed the shock-induced changes in the channel open-probability by employing both two-state and multi-state models. The results indicate a reduction in the number of channel gating particles after the electric shock, which imply possible conformational changes at domains that gate the channels proteins. This study provides further evidence supporting our hypothesis that high intensity electric fields can cause conformational changes in membrane proteins, most likely in the channel gating system. These structural changes in membrane proteins, and therefore their dysfunctions, may be involved in the mechanisms underlying electrical injury.     

Simultaneous impedance measurements of two one-face sealed resonating piezoelectric quartz crystals for in situ monitoring of electrochemical processes and solution properties

Through admittance measurements of two piezoelectric quartz crystals in parallel on one impedance analyzer and then non-linear fitting according to an equivalent circuit of two parallel Butterworth-Van Dyke circuits, we have simultaneously obtained accurate and precise impedance responses of two one-face sealed crystals to changes in solution density and viscosity, temperature, conductance, and/or electrode mass. A series of sucrose aqueous solutions, ferri-/ferrocyanide redox switching, hot water cooling, a series of NaClO₄ aqueous solutions, bovine serum albumin adsorption and silver electrodeposition/stripping were selected as model systems for such purposes. Galvanostatic charging/discharging reactions at positive and negative poles in a Ni-Zn battery were synchronously monitored, with some quartz crystal microbalance (QCM) insights into the second reduction process of nickel hydroxide film. In all cases, the crystal immersion angle effect was found to be negligible. The present method as a versatile one is highly recommended for informative two-electrode monitoring of two concurrent chemical or biological events, or for check and/or compensation of effects due to solution density, viscosity, temperature and/or conductance during QCM researches.