Formation Of Bilayer Lipid Membranes Research Articles

Stabilized bilayer lipid membranes for flow‐through experiments

AbstractThis work reports a technique for the stabilization of solventless bilayer lipid membranes (BLMs), and the use of stabilized BLMS as flow detectors. Microporous filters composed of glass fibers, polytetrafluoroethylene (PTFE) and polycarbonate (nominal pore sizes from 1 to 5 μM) can serve as interfaces that separate two solution compartments. The micropores in the filter media can act as supports for formation and stabilization of BLMs. One of the solution compartments is used to cast lipid films on the filters, while a carrier electrolyte solution concurrently flows through the opposing compartment. Optimization of the flow cell design, and the chemical composition and methods for preparation of stabilized BLMs, are described. Lipid membranes composed of mixtures of phosphatidyl choline and phosphatidic acid could respond rapidly to pH alterations of the carrier electrolyte solution. Signals would reproducibly appear within a few seconds following the injection of an electrolyte of different pH than the carrier. Signals took the form of a single ion current transient with magnitude of tens of picoamperes (pA) and a duration of seconds. The mechanism of signal generation is explored by differential scanning calorimetry. The results show that a phase transition within a lipid membrane can be triggered by pH alterations of the electrolyte solution. Stabilized BLMs which provide artificial ion gating events hold prospects for chemical sensing of process streams.

Bilayer lipid membranes for flow injection monitoring of acetylcholine, urea, and penicillin.

This work describes a technique for the rapid and sensitive determination of acetylcholine, urea, and penicillin in flowing solution streams using stabilized systems of solventless bilayer lipid membranes (BLMs). This method of monitoring substrates of hydrolytic enzyme reactions made use of BLMs which were supported on ultrafiltration membranes such as polycarbonate and glass microfiber; these filter membranes were found to enhance the stability of BLMs for uses in flow injection experiments. The enzymes were immobilized on BLMs by incorporating the protein solution into the lipid matrix at the air/electrolyte interface before the BLM formation, followed by injections of the substrates into flowing streams of a carrier electrolyte solution. Hydronium ions produced by the enzymatic reaction at the BLM surface caused dynamic alterations of the electrostatic fields and phase structure of BLMs, and as a result ion current transients were obtained; the magnitude of these signals was correlated to the substrate concentration, which could be determined at the micromolar level. The response times were ca. 10 s, and acetylcholine, urea, and penicillin could be determined in continuous flowing systems with a maximum rate of 220 samples/h. It is expected that this analytical utility of stabilized BLMs for flow stream uses will provide new opportunities in this strategy of chemical sensing.

Direct electrochemical sensing of insecticides by bilayer lipid membranes

This work describes the use of bilayer lipid membranes (BLMs) as sensitive detectors for the direct electrochemical monitoring of the organophosphate and carbamate insecticides monocrotofos and carbofuran, respectively. Egg phosphatidylcholine (PC) and dipalmitoylphosphatidic acid (DPPA) were used for the formation of BLMs. The interactions of monocrotofos and carbofuran with BLMs produced a transient current signal with a duration of seconds, which reproducibly appeared within 3 to 5 min after exposure of the membranes to the insecticides. The sensitivity of response was maximized by use of high concentrations of the charged lipid, and by alteration of the phase distribution within membranes by the introduction of calcium ions in bulk solution. The mechanism of signal generation is related to the absorption of the lipophilic insecticide molecules with a consequent rapid reorganization of the membrane electrostatics. The magnitude of the transient current signal was linearly related to the concentration of monocrotofos or carbofuran in bulk solution with sub-micromolar detection limits.

Comparative Study on the Potentiometric Responses between a Valinomycin-Based Bilayer Lipid Membrane and a Solvent Polymeric Membrane

The potentiometric behavior of valinomycin incorporated bilayer lipid membranes (BLMs) was studied in view of a comparison with its solvent polymeric membrane counterpart. The preparation of membranes has greatly relied on the Takagi Montal method as well as synthetic lipid chemistry. The prepared membrane was First characterized regarding its electrical resistance, electrical capacitance and the effect of gramicidin to reveal its bilayer nature. The membrane potential was systematically studied in terms of i) the valinomycin concentration in the lipid membrane and ii) the effect of the lipid charge. The observed membrane potentials were compared with those of valinomycin-incorporated solvent polymeric membranes in terms of i) the detection limit, ii) the dynamic range and iii) the selectivity for alkali metal ions. It was found that the detection limit, dynamic range and optimum valinomycin concentration for the K+ ion-induced Nernstian response for the BLM electrode were comparable to those of the solvent polymeric membranes. The selectivity coefficients (Ki,jpot) for alkali metal ions determined by the separate solution method were also comparable to those of liquid membranes. However, a marked difference between both the membranes was found with respect to the effect of the membranous charge. The potentiometric response of the BLMs was found to depend on the charges of lipids. With negatively charged BLMs, the detection limit and dynamic range for K+ ions were improved. On the contrary, the response with positively charged BLMs was significantly reduced without any change in the Nernstian slope. This effect of lipid charge was compared with the anion effect for a solvent polymeric membrane. Finally, the first successful formation of planar BLMs using totally synthetic lipids was also demonstrated.

Relationships between monolayer and BLM properties of biomembrane components

Monolayers of galactocerebrosides and their mixtures with 1-monooleoyl-glycerol at the air/ water interface were studied as a function of temperature. Information on the bidimensional phases of the pure components and on their miscibility in mixtures was obtained from the spreading isotherms. The conductance and capacitance of bilayer lipid membranes (BLMs) containing 1-monooleoyl-glycerol, 1-monostearoyl-glycerol, galactocerebrosides, 1-monooleoyl-glycerol/1-monostearoyl-glycerol and galactocerebrosides/1-monooleoyl-glycerol in squalene were studied. The results obtained showed that the interfacial orientation was the most important factor for the monolayer miscibility and that it influenced the BLM formation. In fact, at a constant temperature stable BLMs were obtained only from substances giving fluid noncondensed films at the air/water interface and from mixtures whose components were miscible in monolayers at the air/ water interface.

Permeation of water through bilayer lipid membranes

This paper, the fourth of a series, is concerned with the movement of water across the bilayer lipid membrane (BLM). The water permeability coefficients, Po, were determined for BLM formed from oxidized cholesterol, cholesterol-DAP (dodecyl acid phosphate), and cholesterol-HDTAB (hexadecyltrimethylammonium bromide). When NaCl was used as the solute, the value of Po for oxidized cholesterol membranes was found to be 8.4 ± 0.5 × 10−4 cm/sec at 22.5°C. In either NaI or CsCl solution, however, the value of Po was found to be about three times larger. The temperature dependence of Po for BLM generated from oxidized cholesterol in n-octane was also measured in the range of 22.5–44.0°C. Experimental activation energy was calculated to be 6.8 ± 0.5 kcal/mole. From the temperature-permeability coefficient data, various derived quantities from absolute reaction rate theory have been evaluated. The nature of pathway for water permeation and the factors governing the rate of water transport across the BLM are discussed in terms of the transition state theory. The results of this study suggest that the bifacial region of the membrane plays an important role in water permeability. During the last few years, a new experimental structure of less than 90 A thick has been extensively investigated. This membranous structure in aqueous solution known variously as bimolecular, bilayer, or black lipid membrane (abbreviated as BLM), possesses some of the properties of the biological membrane. These properties include thickness, certain electrical characteristics, bifacial tension, ionic selectivity, and water permeability. A detailed summary of the studies on BLM together with the relevant literature references may be found in a recent publication (1). In this paper we report the results of measurements of the osmotic permeability coefficient of water across BLM formed from various lipid solutions. Previously, the permeability coefficients of BLM have been measured by several groups of investigators (2–7). The values reported vary from 4.9–104 μ/sec. It is not certain from these studies whether this large discrepancy is due to the difference in the experimental techniques or the composition of the lipid solutions used for BLM formation or both. Our measurements with the BLM generated from various lipid solutions should therefore be of some interest. Regarding the mechanism of water permeation through the BLM, it is generally recognized that only a very limited amount of information can be derived from measurements of water permeability coefficient at a single temperature. Hence there is an obvious need to carry out studies at various temperatures. Further, one would like to raise the question of the nature of the interfacial region (i.e., the zone near the solution/membrane interface) upon the water permeability. Therefore, we have measured the permeability coefficients of water through the BLM as a function of (a) the temperature and (b) the ionic composition of the bathing medium. The permeability-temperature data permit an evaluation of the various derived quantities from the absolute reaction rate theory, from which certain speculations concerning the mechanism of water permeation through the BLM are now possible. The permeability coefficient data determined as a function of solute used provide an opportunity to examine the effect of ions on water permeation and possibly on the structure of the BLM/solution interface.