Stability Of Bilayer Lipid Membranes Research Articles

Ion transport across bilayer lipid membranes using a track-etched membrane filter

Bilayer lipid membranes (BLM) are widely employed to study the electrochemical behavior across cell membranes, particularly the ion transport in the presence of hydrophobic ions or ionophores. However, owing to the physical fragility of BLMs, they are readily broken down by external shocks, such as the applications of a potential difference, current, and vibration. Thus, in this study, a facile method for enhancing the physical stability of BLMs was developed using a track-etched membrane (TM). Physically stable BLMs were rapidly fabricated within the apertures of TM by dropping an n-decane solution containing lecithin and cholesterol on TM. Therefore, BLM formation was confirmed by capacitance measurement, and the estimated membrane thickness and mean capacitance were 3.5 ± 0.4 nm and 0.51 ± 0.05 μF cm−2, respectively. The applied membrane potential could be extended to ≥1.5 V, which is approximately 10 times higher than those of conventional BLMs, and the lifetime exceeded 40 min. The applicability of the fabricated BLM was verified by electrochemical measurements during ion transport.

STABILITY AND CONDUCTIVITY OF BILAYER LIPID MEMBRANE IN PRESENCE $\mathrm{Al_2O_3}$ NANOPARTICLES

The effect of aluminum oxide nanoparticles (Al2O3) on the stability and conductivity of BLM (the bilayer lipid membrane) in solution was studied. It has been shown that Al2O3 nanoparticles increase the stability of BLM in an electric field, and BLM becomes more stable with increasing their concentration. The experimental data are analyzed in terms of the well-known theory of BLM stability, which is based on the concept of spontaneous formation of microscopic pores on the BLM, the development of which leads to the loss of BLM stability. It is shown that aluminum oxide nanoparticles increase the value of the coefficient of linear tension of the pore edge, and with an increase in the concentration of nanoparticles, the linear tension also increases. It has been shown that the presence of nanoparticles in the solution leads to a decrease in BLM conductivity.

Effect of Sterols on the stability of bilayer lipid membranes

One of the main functions of biological membranes is a barrier function that regulates the penetration of solutes and solvents into and out of cells or their organelles. In other words, cellular membranes provide a selective, semipermeable barrier to the passive transport of molecules. From one side, the formation of conductive defects (pores) in the membrane disrupts this function leading to cell death. Contrastingly, this function finds application in various biomedical and biotechnological applications.

INFLUENCE OF ZINC OXIDE NANOPARTICLES ON BILAYER LIPID MEMBRANE PROPERTIES

The effect of zinc oxide nanoparticles ($ \mathrm{ZnO} $) on the stability and conductivity of the bilayer lipid membrane (BLM) in solution was studied. It has been shown that $ \mathrm{ZnO} $ nanoparticles increase the stability of BLM in an electric field, and BLM becomes more stable with increasing their concentration. The increase in the stability of BLM in an electrical field is mainly due to the increase in the coefficient of linear tension of the pore edge, which is forming in BLM. It is also shown, that the presence of $ \mathrm{ZnO} $ nanoparticles in the solution surrounding BLM leads to a decrease in the BLM conductivity.

Free-Standing Lipid Bilayers Based on Nanopore Array and Ion Channel Formation.

Integrated nanopores are novel and versatile single-molecule sensors for individual label-free biopolymer detection and characterization. However, their studies and application requires a stable lipid bilayer to maintain protein function. Herein, we describe a method for producing lipid bilayers across a nanopore array on a silicon nitride substrate. We used a painting technique commonly used with Teflon films to embed α-hemolysin (α-HL) into bilayer lipid membranes (BLMs) to form an ion channel. This was carried out in nanofluid developed in our lab. The membrane formation process, stability of BLMs and ion channel recordings were monitored by patch clamp in real-time. BLM formation was demonstrated by electrical recording (<10 pS conductance) of suspended lipid bilayers spanning a nanopore in the range of ±100 mV. Membrane resistance (Rm) and capacitance (Cm) of the device with the bilayer were assessed by membrane test as above 1.0 GΩ and ~20±2 pF, respectively. The silicon nitride surface and aperture edge were smooth at the nanometer lever leading to remarkable membrane stability. The membrane lifetime was 5-24 h. A single α-HL channel inserted in 30-60 min applied a potential of +100 mV. The α-HL channel currents were recorded at ~100±10 pA. Such integrated nanopores enable analysis of channel functions under various solution conditions from the same BLM. This will open up a variety of applications for ion channels including high-throughput medical screening and diagnosis.

Amphiphobic Septa Enhance the Mechanical Stability of Free-Standing Bilayer Lipid Membranes.

Artificial bilayer lipid membranes (BLMs) provide well-defined systems for investigating the fundamental properties of membrane proteins, including ion channels, and for screening the effect of drugs that act on them. However, the application of this technique is limited due to the low stability and low reconstitution efficiency of the process. We previously reported on improving the stability of BLM based on the fabrication of microapertures having a tapered edge in SiO2/Si3N4 septa and efficient ion channel incorporation based on vesicle fusion accelerated by a centrifugal force. Although the BLM stability and incorporation probability were dramatically improved when these approaches were used, some BLMs were ruptured when subjected to a centrifugal force. To further improve the BLM stability, we investigated the effect of modifying the surface of the SiO2/Si3N4 septa on the stability of BLM suspended in the septa. The modified surfaces were characterized in terms of hydrophobicity, lipophobicity, and surface roughness. Diffusion coefficients of the lipid monolayers formed on the modified surfaces were also determined. Highly fluidic lipid monolayers were formed on the amphiphobic substrates that had been modified with long-chain perfluorocarbons. Free-standing BLMs formed in amphiphobic septa showed a much higher mechanical stability, including tolerance to water movement and applied centrifugal forces with and without proteoliposomes, than those formed in the septa that had been modified with a short alkyl chain. These results demonstrate that highly stable BLMs are formed when the surface of the septa has amphiphobic properties. Because highly fluidic lipid monolayers that are formed on the septa seamlessly connect with BLMs in a free-standing region, the high fluidity of the lipids contributes to decreasing potential damage to BLMs when mechanical stresses are applied. This approach to improve the BLM stability increases the experimental efficiency of the BLM systems and will contribute to the development of high-throughput platforms for functional assays of ion channel proteins.

INFLUENCE OF NANOPARTICLES OF $ \mathrm{CuO} $ ON THE STABILITY AND CONDUCTIVITY OF BLM

This study aims to provide arguments for an increase in the stability of bilayer lipid membranes (BLM) in an electrical field in presence of $ \mathrm{CuO} $ nanoparticles. Moreover, the sustainability of BLM becomes more stable with increasing of nanoparticles concentration. It is shown that an increase in the stability of BLM in an electrical field is mainly due to an increase in the coefficient of linear tension of the edge of a pore, which is forming in BLM. It is shown as well, that the presence of nanoparticles of $ \mathrm{CuO} $ in the solution surrounding BLM leads to a decrease in the conductivity of the BLM.

Real-Time Statistics on Ion-Channel Recordings using a Novel All-In-One BLM Educational Kit with Embedded Miniaturized Amplifier

Bilayer Lipid Membranes (BLM) are often used to perform single-channel recordings of different ion-channels. However, the success ratio and the BLM stability is strongly dependent on the specific electrophysiology setup, which is usually bulky and expensive. Moreover, the post-processing acquisition analysis is a time-consuming activity. These characteristics make BLM experiments difficult for educational purposes.Here we present a compact and complete BLM educational kit for accurate electrophysiological recordings allowing fast screening of ion-channels functional activity in lipid environments. A large number of standard experiments can be performed using the kit, which is particularly suitable for educational lab courses. Stable membranes can be easily formed to study ion-channels or toxins.The real-time statistic feature implemented in the amplifier control software enables quick and straightforward analysis of data about ion-channel properties, such as I/V curve for conductance analysis, FFT, dwell times, histograms and inter-events time statistics.The kit is contained into a 16x10x5cm Faraday cage and includes the recording chamber and the miniaturized amplifier. The chamber is based on two 1ml - 30x15x15mm Delrin cuvettes with individual electrodes. A laser drilled Teflon diaphragm, with holes varying between 50µm to 110µm, separates the two cuvettes.To prove the capability of the presented setup and of the real-time statistics, suspended lipid bilayer were easily formed by painting method and recordings of different proteins, such as Kcv, Gramicidin and alpha-hemolysin in presence of PEGs of different lengths, were analyzed. Thanks to the real-time statistics, scientists can perform a quick analysis on the ion-channel properties while experiments are running.The combination of the presented kit and the control software with real-time statistics feature allows quick and easy functional characterizations of many kinds of ion-channels for educational or research purposes.

Influence of H2TOEtPyP4 porphyrin on the stability and conductivity of bilayer lipid membranes.

Many water-soluble cationic porphyrins are known to be prospective chemotherapeutics and photosensitizers for cancer treatment and diagnosis. The physicochemical properties of porphyrins, in particular their interactions with membranes, are important determining factors of their biological activity. The influence of cationic meso-tetra-[4-N-(2'-hydroxyethyl) pyridyl] porphyrin (H2TOEtPyP) on the stability and conductivity of bilayer lipid membranes (BLMs) was studied. H2TOEtPyP4 porphyrin was shown to decrease the stability of BLMs made of a mixture of DOPS and DPPE (1:1) in an electric field because of a reduction of line tension of spontaneously formed pore edges in the BLM. The presence of cationic porphyrin was found to reduce BLM surface tension. This effect was enhanced with increasing porphyrin concentration. H2TOEtPyP4 increased the probability of spontaneous pore formation. Further investigating the cyclic current-voltage characteristics of BLMs allowed determining the electrical capacity and conductivity of BLMs in the presence of H2TOEtPyP4 porphyrin. It was shown that in the presence of cationic porphyrin the electrical capacity as well as conductivity of the BLM increases.

Stability of bilayer lipid membrane in the presence of Co-containing metalloporphyrins

Stability of bilayer lipid membrane (BLM) in an electric field in the presence of Co-porphyrin in the solution surrounding BLM is investigated. We have shown that the presence of porphyrin in a solution reduces line tension of the pore edge and surface tension of BLM and increases the number of pores on the BLM. It is also shown that a decrease in the stability of BLM in the electric field in the presence of the porphyrin is a result of the reduction of line tension of the pore edge and increase in the number of pores.

Stable Lipid Bilayers Based on Micro- and Nano-Fabrication as a Platform for Recording Ion-Channel Activities

In this review, we will discuss our recent approaches for the formation of mechanically stable bilayer lipid membranes (BLMs) by combining with BLM formation and micro- and nano-fabrication techniques. BLMs were prepared across a microaperture fabricated in silicon (Si) chips or nanoporous alumina films using a minimized amount of organic solvent. Although BLMs spanned over the porous alumina film showed better electrical properties, such as background current noise and current transient, BLMs suspended in a thin Si(3)N(4) septum showed a much superior BLM stability. The BLMs showed tolerance to a high voltage of ±1 V, a membrane lifetime of >40 h, and tolerance to repetitive solution exchanges. Application to a drug screening system has been examined by using the human ether-a-go-go-related gene (hERG) potassium channel as an illustrative example. The potentiality of the present system as a platform of the high-throughput analysis for ion-channel protein is also discussed.

Mechanically Stable Free-Standing Bilayer Lipid Membranes in Microfabricated Silicon Chips

ABSTRACTIn this paper, we will discuss our recent approaches for improving the mechanical stability of free-standing bilayer lipid membranes (BLMs) by combining with BLM formation and microfabrication techniques. BLMs were prepared across a microaperture fabricated in a silicon (Si) chip and their mechanical stability and electric properties were investigated. BLMs suspended in a thin Si3N4 septum showed a dramatic improvement of BLM stability. The BLMs were resistant to voltage of ±1 V and the membrane lifetime was 15- ~40 h with and without incorporated channels. The membrane containing gramicidin channel exhibited tolerance to repetitive solution exchanges. At first, electric properties of the BLMs, such as noise level and current transient, were necessary to be improved. However, after coating the chip with insulator layers of Teflon and SiO2, total chip capacitance was reduced, leading to noise reduction (1-2 pA in peak-to-peak after low-pass filtering at 1 kHz) and elimination of current transients (&lt; 0.5 ms). Since the vicinity of the aperture edge was remained uncoated, the BLMs formed in the Si chips still showed high mechanical stability after the insulator coatings. The mechanically stable BLMs having electric properties suitable for recording activities of biological channels will open up a variety of applications including high-throughput analysis of ion-channel proteins.

The Combined Effect of Electrostatic Field and Osmotic Pressure on the Stability of Bilayer Lipid Membranes

The issue of cell membrane stability is central in membranology. The extreme complexity of cell membranes urges to study this problem using a model, a bilayer lipid membrane (BLM). The membranes is often impacted both by electric forces and osmotic pressure. Therefore, it is of interest to investigate the joint activity of electric field and osmotic pressure on the stability of BLM. In the given work the joint activity of the osmotic pressure and transmembrane potential difference on the stability of BLM was experimentally investigated. As the capacity parameter a mean lifetime of BLM was taken at the given magnitudes of electric field and the osmotic pressure.At first the change of the mean lifetime of BLM with increasing of potential difference in absence of osmotic pressure was investigated. It was shown that the activity of electrostatic field brings decreasing of BLM mean lifetime. After the influence of potential difference on BLM mean lifetime was experimentally investigated at addition of osmotic pressure. It was shown that a dependence curve of BLM mean lifetime with potential for the second case is lower than in the first case. The loss of stability of BLM at addition of osmotic pressure is conditioned by two factors: either changing linear tension of BLM pore, changing the numbers of defects on BLM. The analysis theoretical formula and its confrontations with obtained data shows that the decreasing of mean lifetime of BLM at addition of osmotic pressure can be connected with the changing of linear tension. It is indicated that the difference in osmotic pressure of BLM does not bring to increasing of an area.

Simple and Stable Lipid Bilayer Formation: A Droplets Contacting Method using Parylene Micro-pores for Multiple Ion Channel Recordings

This paper describes multiple ion-channel recordings through membrane proteins reconstituted in bilayer lipid membranes (BLMs) array. The BLMs array can be prepared by “Droplets Contacting Method” which forms BLMs at the interface of two lipid monolayers. Since this method does not require skilled techniques, it is highly reproducible and can be applied to automated system. We used a double well chip (DWC) for the droplets contacting method. We attempted to confine the BLMs forming areas with parylene micro-pore (parylene double well chip, PDWC) to augment the mechanical stability of BLMs. Subsequently, we arrayed the PDWC with electrodes for multiple recordings of channel proteins. We successfully demonstrated 14 channels simultaneous ion channel recordings through α-hemolysin.

Stable lipid bilayers based on micro- and nano-fabrication

In this review, we will discuss our recent approaches for improving the mechanical stability of free-standing bilayer lipid membranes (BLMs) by combining with BLM formation and micro- and nano-fabrication techniques. BLMs were prepared across a microaperture fabricated in silicon (Si) chips or nanoporous alumina films, and their mechanical stability and electric properties were investigated. BLMs spanned over the porous alumina showed background noise currents small enough for recording activities of low-conductance channels, though further stability enhancement of porous alumina films was necessary. BLMs suspended in a thin Si3N4 septum showed a dramatic improvement of BLM stability. The BLMs were resistant to a voltage of ± 1 V and the membrane lifetime was 15–43 h with and without incorporated channels. The membrane containing gramicidin channel exhibited tolerance to repetitive solution exchanges, though the electric properties of the BLMs are necessary to be improved. The realisation of BLMs having both mechanical stability and proper electric properties will open up a variety of applications including highly sensitive biosensors and high-throughput drug screenings for ion channels.

Improved stability of free-standing lipid bilayers based on nanoporous alumina films

In this study, we propose a method for improving the stability of free-standing bilayer lipid membranes (BLMs) by preparing BLMs across nanoporous alumina films. The use of porous alumina reduced individual membrane size to improve the BLM stability. The BLMs were stable under an applied voltage of ±1 V. The lifetime of the BLMs was 16–30 h with and without incorporated gramicidin channels. Electric properties of the BLMs as a platform for channel-current recordings are also discussed. Since the total area of the BLMs is still large to facilitate protein incorporation, our approach is useful for designing highly sensitive biosensors based on channel proteins.

The influence of different membrane components on the electrical stability of bilayer lipid membranes

A good understanding of cell membrane properties is crucial for better controlled and reproducible experiments, particularly for cell electroporation where the mechanism of pore formation is not fully elucidated. In this article we study the influence on that process of several constituents found in natural membranes using bilayer lipid membranes. This is achieved by measuring the electroporation threshold ( V th) defined as the potential at which pores appear in the membrane. We start from highly stable 1,2-diphytanoyl- sn-glycero-3-phosphocholine (DPhPC) membranes ( V th ∼ 200 mV), and subsequently add therein other phospholipids, cholesterol and a channel protein. While the phospholipid composition has a slight effect (100 mV ≤ V th ≤ 290 mV), cholesterol gives a concentration-dependent effect: a slight stabilization until 5% weight ( V th ∼ 250 mV) followed by a noticeable destabilization ( V th ∼ 100 mV at 20%). Interestingly, the presence of a model protein, α-hemolysin, dramatically disfavours membrane poration and V th shows a 4-fold increase (∼ 800 mV) from a protein density in the membrane of 24 × 10 − 3 proteins/μm 2. In general, we find that pore formation is affected by the molecular organization (packing and ordering) in the membrane and by its thickness. We correlate the resulting changes in molecular interactions to theories on pore formation.

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 ζ-potential of multilayer liposomes or boundary potential of BLM. It was shown that the presence of up to 25 mol% 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.

Amphiphile bilayer films from DPPC: bilayer lipid membranes and Newton black films

Amphiphile bilayer films are obtained from 1,2 dipalmitoyl-glycero-3-phosphocholine (DPPC): bilayer lipid membranes (BLM) and Newton black films (NBF), through thinning of the respective thin liquid films, thus allowing for a very precise determination of the moment of their formation. Stability (or rupture) and formation of BLM and NBF are considered from a unified point of view with the microscopic theory of Kashchiev–Exerowa [J. Colloid Interface Sci., 77 (1980) 501–511], based on the formation of nanoscopic holes in them. BLM and NBF are obtained and studied with the microinterferometric method of Scheludko–Exerowa in its contemporary version. The equivalent thickness of both BLM (in benzene solution between two water phases with 0.1 M NaCl) and NBF in aqueous DPPC solution (in the presence of 0.1 M NaCl) is determined as being h w = 7.0 nm for BLM and h w = 7.8 nm for NBF. By means of the dependences: BLM lifetime versus DPPC concentration and probability for BLM formation versus DPPC concentration, it is established that there exist metastable BLM and stable NBF. The good fit between the experimental results of τ( C) dependence and theory in the case of BLM allow to determine the three constants: pre-exponential factor A = 1.5 × 10 −3 s, related to the process kinetics; constant B = 20.2 ± 0.2, related to the specific hole energy γ = 1.7 × 10 −11 J/m and the equilibrium concentration C e = 6 × 10 −4 ± 7.2 × 10 −6 m/l. The specific hole linear energy γ = 1.7 × 10 −11 J/m determined as well as the binding energy Q between first neighbor molecules in the bilayers Q = 1.48 × 10 −19 J (36 kT) are lower than the ones determined for DPPC foam bilayer in gel state γ = 9.1 × 10 −11 J/m and Q = 55 kT. This means that interaction is weaker in the case of BLM. The critical concentration C c at which bilayer formation starts is: for BLM C c = 30 μg/ml and for NBF C c = 70 μg/ml. This concentration characterizes quantitatively the formation of the amphiphile bilayer and is a very useful parameter that can be used for various purposes.

Stability of bilayer lipid membranes on different metallic supports

In order to arrive at an optimized s-BLM protocol, the influence of various metallic supports (silver, iridium and stainless steel) of different diameters (from 0·14 to 0·33 mm) on the process of forming and stability of supported phospholipid bilayer membranes was studied. In addition, experiments were made using different membrane-forming solutions, ionic strength and pH. The transmembrane resistance and the current ratio between bare and lipid-coated wire at +670 mV were measured. Tefloncoated stainless steel support (0·33 mm in diameter) covered with 2% crude ox brain fraction of phospholipids was most convenient for the formation of supported bilayer lipid membranes on solid support. The well-known stabilizing effect of cholesterol was confirmed for the s-BLM system. Both the pH in the range 5·0–8·0 (silver and stainless steel supports) and the ionic strength (for all types of supports) in the range from 0·025 to 0·1 mol/l at pH 7·0 were found not to be crucial.