Formation Of Bilayer Lipid Membranes Research Articles

Detection of lipid bilayer membranes formed on silica fibers by double-long period fiber grating laser refractometry

The formation of lipid bilayer membranes on silica optical fiber surfaces is critical to the development of new optical biosensors based on supported lipid bilayer membrane technology. This paper reports on a new long period fiber grating (LPFG) method using double gratings, one serving as the sensor and another as the reference to correct for environmental factors, to study supported lipid membranes on a silica optical fiber. A method for correcting the effect of the environmental variation on sensor responses is described. The measurements show that formation of eggPC lipid bilayer membranes on the silica optical fiber surface takes place in about 3 min, which is accompanied with about a 500 picometer decrease in the resonance coupling wavelength of the sensor grating. The formation process of eggPC lipid bilayer membranes with and without Gramicidin was measured. It was found that while the presence of Gramicidin does not affect the rate of lipid bilayer membrane formation on the silica fiber, it causes a greater decrease in the resonance coupling wavelength than pure eggPC because of the increased film refractive index. Formation of a lipid bilayer membrane on the silica optical fiber surface was verified by confocal microscopy and through fluorescence recovery after photobleaching (FRAP) analysis, confirming the formation of a single lipid bilayer on the surface of the silica fiber with a diffusion coefficient of 1.2 μ m 2 s −1.

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.

Self-formation of bilayer lipid membranes on agarose-coated silicon surfaces studied by simultaneous electrophysiological and surface infrared spectroscopic measurements

Self-formation process of bilayer lipid membranes (BLMs) cushioned on agarose-coated Si surfaces was in situ monitored by simultaneous electrophysiological and infrared absorption spectroscopic (IRAS) measurements using IRAS with the multiple internal reflection geometry. IRAS signals corresponding to self-thinning of lipid solution to form BLMs were demonstrated. It was found that the appearance of IRAS bands due to C=O modes of phosphstidylcholine is related to formation of BLMs with a gigaohm seal. The functionality of the present BLM system was also demonstrated by incorporating gramicidin into the BLMs and recording its channel activities.

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 μF/cm2±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 <pH <4), while at higher pH the membranes could cover the aperture only partially. Thus, apertures in fluorosiliconized glass can be robustly used for formation of model lipid membranes under physiological conditions.

Formation of Bilayer Lipid Membranes with Microfluidic Devices

Membrane proteins play very important roles in cells. They are also useful in various industrial fields, including drug discovery, and highly sensitive ion-channel-based biosensors. Here, I describe our two approaches toward membrane protein chips: an array of membrane proteins on (1) planar lipid bilayers and (2) giant vesicles. First, a highly reproducible method was developed for planar lipid bilayer reconstitution. Planar lipid bilayers are formed at apertures by flowing lipid organic solution and buffer alternately into an integrated microfluidic channel. Using this technique, multiple lipid bilayers are formed simultaneously, and channel currents through peptide ion channels were recorded. Second, I describe a method for the preparation of lipid vesicles inspired by forming soap bubbles from a soap film. In our “blowing” method, lipid vesicles are blown out of the pre-formed bio-functional planar lipid membrane, directly encapsulating ejected materials. We believe that these devices are useful for an efficient and rapid analysis of singlespecies-specific membrane proteins.

Lipid Bilayer Microarray for Parallel Recording of Transmembrane Ion Currents

This paper describes a multiwell biochip for simultaneous parallel recording of ion current through transmembrane pores reconstituted in planar lipid bilayer arrays. Use of a thin poly(p-xylylene) (parylene) film having micrometer-sized apertures (phi=15-50 microm, t=20 microm) led to formation of highly stable bilayer lipid membranes (BLMs) for incorporation of transmembrane pores; thus, a large number of BLMs could be arrayed without any skillful technique. We optically confirmed the simultaneous formation of BLMs in a 5x5 matrix, and in our durability test, the BLM lasted more than 15 h. Simultaneous parallel recording of alamethicin and gramicidin transmembrane pores in multiple contiguous recording sites demonstrated the feasibility of high-throughput screening of transmembrane ion currents in artificial lipid bilayers.

Micromachined glass apertures for artificial lipid bilayer formation in a microfluidic system

The use of spark assisted chemical engraving (SACE) to produce glass apertures that are suitable for the formation of artificial bilayer lipid membranes is described. Prior to use, the glass apertures were rendered hydrophobic by a silanization process and were then incorporated into a simple microfluidic device. Successful bilayer lipid membrane (BLM) formation and the subsequent acquisition of single-channel recordings are demonstrated. Due to the simplicity and rapidity of the SACE process, these glass apertures could be easily integrated into an all-glass microfluidic system for BLM formation.

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.

Electrical Impedance Analysis of Phospholipid Bilayer Membranes for Enabling Engineering Design of Bio-based Devices

ABSTRACTRecent research at Virginia Tech have shown that active transporter proteins reconstituted into suspended bilayer lipid membranes (BLMs) formed across an array of pores in synthetic substrates can convert chemical energy available in adenosine triphosphate (ATP) into electricity. Experimental results from this work show that this system—called BioCell—is capable of 1.7μW of electrical power per square centimeter of BLM area and per 15μL of ATPase enzyme. In support of such a system, the lipid membrane, as host to active biological proteins and channels, must be formed evenly across a porous substrate, remain stable and yet fluid-like for protein folding and activation, and provide sufficient electrical insulation. We report on the formation and characterization using electrical impedance spectroscopy (EIS) of BLMs formed across two types of porous substrates: polycarbonate filters and single-aperture silicon substrates. Equivalent electrical circuits describing the lipid membranes and their supporting substrates are approximated to fit the measured responses. The results show that BLMs formed in some but not all of the 400nm pores of the filters, while the formation of BLMs on the single-aperture silicon substrates was much more consistent.

An ionic polymer bead-supported lipid system

In this paper, we report on the preparation of an ionic polymer bead-supported lipid system several hundred micrometers in diameter. The electrostatic attractive interactions between anionic lipids and cationic polymer beads served as a “molecular glue” to immobilize the lipids on the beads, and then the immobilized lipids prompted the spontaneous formation of lipid bilayer membranes. Confocal fluorescence microscopic techniques revealed that the lipid bilayer membranes were located along the outline of the beads. In addition, the integrity of the lipid bilayer membranes was microscopically confirmed by a low-molecular-weight dye (trypan blue) exclusion test.

Deposition of lipid bilayers on OH-density-controlled silicon dioxide surfaces

We investigated the effect of the SiO2 surface hydrophilicity on the formation of lipid bilayer membranes using the vesicle fusion method with atomic force microscopy, and applied the results to constructing membrane arrays. We obtained SiO2 surfaces with different hydrophilicity by annealing chemically oxidized SiO2 surfaces at various temperatures under an N2 flow. The membrane formation rate is faster on less hydrophilic surfaces after depositing a 100-nm-filtered vesicle of dimyristoylphosphatidylcholine. Desorption of the surface hydroxyl groups causes a higher affinity between the lipid membrane and the substrate. We also describe a method to fabricate membrane arrays using “chemical patterning.” When the surface hydroxyl groups are locally removed using a focused ion beam (FIB), a bilayer membrane selectively forms on the FIB-patterned region.

Self-assembly of bilayer lipid membrane at multiwalled carbon nanotubes towards the development of photo-switched functional device

Bilayer lipid membrane (BLM) was self-assembled on a uniquely fabricated hydrophilic surface, containing N atoms from the carbon source of ethylene amine, of the multi-walled carbon nanotubes (MWNTs) to form the BLM/MWNTs nanocomposites. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and photoelectric experiments were taken to study the properties of the BLM/MWNTs nanocomposites. The thickness of the BLM, which was calculated from the CV data obtained at BLM/MWNTs electrode, turned out to be 4.38 nm, suggesting that the lipid self-assembled at the nanotubes surface was consistent with a bilayer structure. C 60-incorporated BLM could also be self-assembled at the nanotubes surface (C 60-BLM/MWNTs). The formation of BLM on the MWNTs surface blocked the diffusion of [Fe(CN) 6] 3/4− redox ions across BLM to the MWNTs electrode as no redox current was observed by CV measurement, whereas the incorporation of the electron mediator, C 60, resumed a pair of redox peaks at C 60-BLMs/MWNTs electrode. Moreover, the incorporation of C 60 led to a four order of magnitude reduction of the resistance of C 60-BLM/MWNTs (369.3 Ω) than that of BLM/MWNTs (3.238 × 10 6 Ω). MWNTs electrode exhibited an intrinsic cathodic photocurrent (166 μA cm −2) while BLM/MWNTs electrode blocked photocurrent response of the MWNTs. Interestingly, C 60-BLM/MWNTs electrode resumed partial photoelectric properties (photo current: 65 μA cm −2) due to the electron mediation effect of C 60 incorporated into the lipid membrane. As a result, the novel self-assembled BLM/MWNTs nanocomposites provided a simple yet useful model to study the C 60-mediated photoelectric properties of the BLM/MWNTs which may be applicable to develop new biosensors and molecular devices.

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.

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.

Single Ion Channel Sensitivity in Suspended Bilayers on Micromachined Supports

This paper describes the formation of bilayer lipid membranes on a micromachined support, consisting of a 128 μm (diameter) aperture on a gold/SU8 surface suspended over a small aqueous reservoir (∼25 nL). The bilayers are highly impermeable to ion transport with specific resistance values up to 107 Ω cm2. Single-channel activities of gramicidin and alamethicin were observed in these systems. This work demonstrates that micromachined supports for bilayers are promising for the development of a biosensor with single-channel sensitivity.

Stabilized filter‐supported bilayer lipid membranes (BLMs) for automated flow monitoring of compounds of clinical, pharmaceutical, environmental and industrial interest

This paper describes the results of analytical applications of electrochemical biosensors based on bilayer lipid membranes (BLMs) for the automated rapid and sensitive flow monitoring of substrates of hydrolytic enzymes, antigens and triazine herbicides. BLMs, composed of mixtures of egg phosphatidylcholine (egg PC) and dipalmitoylphosphatidic acid (DPPA), were supported on ultrafiltration membranes (glass microfibre or polycarbonate filters) which were found to enhance their stability for flow experiments. The proteins (enzymes, antibodies) were incorporated into a floating lipid matrix at an air-electrolyte interface, and then a casting procedure was used to deliver the lipid onto the filter supports for BLM formation. Injections of the analyte were made into flowing streams of the carrier electrolyte solution and a current transient signal was obtained with a magnitude related to the analyte concentration. Substrates of hydrolytic enzyme reactions (acetylcholine, urea and penicillin) could be determined at the micromolar level with a maximum rate of 220 samples/h, whereas antigens (thyroxin) and triazine herbicides (simazine, atrazine and propazine) could be monitored at the nanomolar level in less than 2 min. The time of appearance of the transient response obtained for herbicides was increased to the order of simazine, atrazine and propazine which has permitted analysis of these triazines in mixtures.

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: 1. formation of self-assembled BLMs over ss DNA adsorbed on the electrode surface; 2. direct adsorption of ss DNA into a BLM modified GC electrode; 3. formation of a BLM with incorporated ss DNA at the electrode surface using the monolayer folding technique. 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.

Electrochemical transduction of interactions of atrazine with bilayer lipid membranes

AbstractThis work investigates the interactions of atrazine with bilayer lipid membranes (BLMs) that can be used for the direct electrochemical sensing of this herbicide. Egg phosphatidylcholine (PC) and dipalmitoylphosphatidic acid (DPPA) were used for the formation of solventless BLMs. The interactions of atrazine with these membranes were found to be electrochemically transduced by BLMs in the form of a transient current signal with a duration of seconds, which reproducibly appeared within 1 min after exposure of the membranes to atrazine. The sensitivity of the response was maximized by use of BLMs composed of 35% (w./w.) DPPA, and by alteration of the phase distribution within membranes by the introduction of calcium ions in bulk solution. The mechanism of signal generation was related to the adsorption of atrazine with a consequent rapid reorganization of the membrane electrostatics due to atrazine aggregation at the surface of BLMs. Hydrogen bonding between atrazine and the carbonyl group of the lipid was explored by addition of platelet‐activating factor (PAF; an ether analog of PC) in BLMs composed of PC. Differential scanning calorimetry of vesicles composed of 15% DPPA was used to study the aggregation of atrazine in membrane domains enriched in the charged lipid. The magnitude of the transient current signal was linearly related to the concentration of atrazine in bulk solution with sub‐micromolar detection limits. This electrochemical transduction of atrazine interactions with BLMs holds prospects for flow injection monitoring of triazine herbicides.

Bilayer lipid membranes as electrochemical detectors for flow injection immunoanalysis

AbstractThis work describes the use of filter‐supported stabilized bilayer lipid membranes (BLMs) for the rapid electrochemical monitoring of an immunological reaction in flowing solution streams. BLMs were prepared from egg phosphatidylcholine (egg PC) and dipalmitoyl phosphatidic acid (DPPA) and the ultrafiltration membranes used were composed of glass microfibers. Thyroxin (T4)/anti‐rabbit T4 was used as a representative immunological reaction for these studies. Antibody was incorporated into a floating lipid matrix at an air–electrolyte interface, and then a casting procedure was used to deliver the lipid onto the filter supports for BLM formation. Injections of antigen were made into flowing streams of a carrier electrolyte solution. Experiments were done in a stopped‐flow mode using lipid mixtures containing 15% (w/w) DPPA to provide only a single transient current signal with a magnitude related to the antigen concentration. Differential scanning calorimetric experiments provided evidence that the antibody‐lipid interactions at the BLMs occurred through electrostatic interactions. BLMs containing 35% DPPA were used to examine regeneration of the active sites of antibody after complex formation by washing with the carrier electrolyte solution. Repetitive cycles of injection of antigen followed by regeneration of antibody binding activity have shown that the maximum number of cycles is about 5, followed by a degradation of signal for a larger number of injections. However, the sensor can also be easily regenerated by recasting of the existing lipid/antibody film at the air–electrolyte interface to form fresh BLMs.

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.