Formation Of Bilayer Lipid Membranes Research Articles

Ohta–Kawasaki energy for amphiphiles: Asymptotics and phase-field simulations

We study the minimizers of a degenerate case of the Ohta–Kawasaki energy, defined as the sum of the perimeter and a Coulombic nonlocal term. We start by investigating radially symmetric candidates which give us insights into the asymptotic behaviors of energy minimizers in the large mass limit. In order to numerically study the problems that are analytically challenging, we propose a phase-field reformulation which is shown to Gamma-converge to the original sharp interface model. Our phase-field simulations and asymptotic results suggest that the energy minimizers exhibit behaviors similar to the self-assembly of amphiphiles, including the formation of lipid bilayer membranes.

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.

Formation of Lipid Bilayer Membranes and Domain Localization on Graphene Oxide

Formation of Lipid Bilayer Membranes and Domain Localization on Graphene Oxide

Bilayer lipid membrane formation on surface assemblies with sparsely distributed tethers.

A combined computational and experimental study of small unilamellar vesicle (SUV) fusion on mixed self-assembled monolayers (SAMs) terminated with different deuterated tether moieties (-(CD2)7CD3 or -(CD2)15CD3) is reported. Tethered bilayer lipid membrane (tBLM) formation of synthetic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine was initially probed on SAMs with controlled tether (d-alkyl tail) surface densities and lateral molecular packing using quartz crystal microbalance with dissipation monitoring (QCM-D). Long time-scale coarse-grained molecular dynamics (MD) simulations were then employed to elucidate the mechanisms behind the interaction between the SUVs and the different phases formed by the -(CD2)7CD3 and -(CD2)15CD3 tethers. Furthermore, a series of real time kinetics was recorded under different osmotic conditions using QCM-D to determine the accumulated lipid mass and for probing the fusion process. It is shown that the key factors driving the SUV fusion and tBLM formation on this type of surfaces involve tether insertion into the SUVs along with vesicle deformation. It is also evident that surface densities of the tethers as small as a few mol% are sufficient to obtain stable tBLMs with a high reproducibility. The described "sparsely tethered" tBLM system can be advantageous in studying different biophysical phenomena, such as membrane protein insertion, effects of receptor clustering, and raft formation.

Gapless Chip‐in‐Carrier Integration and Injectable Ag/AgCl‐Epoxy Reference Electrode for Bilayer Lipid Membrane Sensor

There are emerging needs for bilayer lipid membrane (BLM) sensors utilizing the vastly and highly developed molecular recognition ability of membrane proteins, especially those combined with CMOS chips. However, this type of BLM sensor has barely been studied so far mainly because of two problems: (i) the difficulty of their fabrication processes resulting from chip‐carrier discontinuity, (ii) a skill‐dependent and manual fashion to form BLMs. Here, we introduce a gapless chip‐in‐carrier integration process utilizing parylene as a gap filler and a microfluidic packaging method with an injectable Ag/AgCl‐epoxy reference electrode. These methods allow for subsequent photolithography processes and later microfluidic‐based BLM formation where the surface continuity of a microchannel is crucial. As a proof‐of‐concept device, we fabricated chip‐in‐carrier assemblies with a dummy chip coupled with a microfluidic system for BLM formation compatible with simultaneous optical observation. With this device, we validated the capability to form BLMs by microfluidic technique and characterized the configuration of a formed BLM through the observation of fluorescent localization. This chip integration and microfluidic packaging scheme can provide a solution for the full implementation of CMOS ICs with BLM sensors and broaden the possibilities of practical applications in the engineering and medical fields. © 2022 Institute of Electrical Engineers of Japan. Published by Wiley Periodicals LLC.

Direct fabrication of SU‐8 microchannel across an embedded chip for potentiometric bilayer lipid membrane sensor

AbstractMembrane proteins play a critically important role, especially in molecular and cellular transport processes, which makes them the most prevalent drug targets and also to be a good sensing element for biosensing applications. However, the skill‐dependent and manual fashion to form bilayer lipid membranes (BLMs) has limited their use for application purposes. Here, we introduce a fabrication technique of SU‐8 microchannel suitable for microfluidic‐based BLM formation and show a preliminary step of our ISFET‐based BLM sensor. Making notches on the corners of a rectangular through‐wafer hole etched in a silicon carrier substrate reduced the gap width between an embedded chip and the carrier as close as possible, leading to the gap depth of 4 μm. SU‐8 microchannel on the chip‐carrier assembly was directly fabricated by UV exposure through a bonded quartz lid and developing SU‐8 through the fluidic ports drilled in the lid, without needing a sealing process typically causes an alignment mismatch or void formation. Finally, the capability to form BLMs was tested and proven by the successfully encapsulated fluorescent dye in microwells fabricated on the embedded chip. This paper is the first report of the successful formation of BLMs on a chip‐in‐carrier assembly by the microfluidic method.

Demolish and Rebuild: Controlling Lipid Self-Assembly toward Triggered Release and Artificial Cells.

The ability to modulate the structures of lipid membranes, predicated on our nuanced understanding of the properties that drive and alter lipid self-assembly, has opened up many exciting biological applications. In this Perspective, we focus on two endeavors in which the same principles are invoked to achieve completely opposite results. On one hand, controlled liposome decomposition enables triggered release of encapsulated cargo through the development of synthetic lipid switches that perturb lipid packing in the presence of disease-associated stimuli. In particular, recent approaches have utilized artificial lipid switches designed to undergo major conformational changes in response to a range of target conditions. On the other end of the spectrum, the ability to drive the in situ formation of lipid bilayer membranes from soluble precursors is an important component in the establishment of artificial cells. This work has culminated in chemoenzymatic strategies that enable lipid manufacturing from simple components. Herein, we describe recent advancements in these two unique undertakings that are linked by their reliance on common principles of lipid self-assembly.

Impact of membrane protein-lipid interactions on formation of bilayer lipid membranes on SAM-modified gold electrode

In protein film electrochemistry for redox-active membrane proteins, the reconstitution of protein-tethered bilayer lipid membranes (ptBLMs) can maximize their functionality and stability at electrolyte/electrode interfaces. To understand the impact of protein–lipid interactions on the ptBLM formation, we tracked the reconstitution process of a lipid bilayer in the presence or the absence of a transmembrane enzyme of cytochrome c-dependent nitric oxide reductase (cNOR) on a mixed self-assembled monolayer (SAM)/Au electrode. Time-resolved surface-enhanced infrared absorption (SEIRA) spectroscopy and electrochemical measurements revealed that the protein–lipid interaction affected the formation kinetics for the phospholipid bilayer and the electrocatalytic stability of the cNOR-modified electrode but not the fluidity of the phospholipid bilayer.

Efficient Lipid Bilayer Formation by Dipping Lipid-Loaded Microperforated Sheet in Aqueous Solution

This paper describes a method for a bilayer lipid membrane (BLM) formation using a perforated sheet along with an open chamber. Microscopic observation of the formed membrane showed a typical droplet interface bilayer. We proved that the formed membrane was a BLM based on electrical measurements of the membrane protein α-hemolysin, which produces nanopores in BLMs. Unlike the conventional approach for BLM formation based on the droplet contact method, this method provides aqueous surfaces with no organic solvent coating layer. Hence, this method is suitable for producing BLMs that facilitate the direct addition of chemicals into the aqueous phase.

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.

Lipid Bilayer Membrane in a Silicon Based Micron Sized Cavity Accessed by Atomic Force Microscopy and Electrochemical Impedance Spectroscopy.

Supported lipid bilayers (SLBs) are widely used in biophysical research to probe the functionality of biological membranes and to provide diagnoses in high throughput drug screening. Formation of SLBs at below phase transition temperature (Tm) has applications in nano-medicine research where low temperature profiles are required. Herein, we report the successful production of SLBs at above—as well as below—the Tm of the lipids in an anisotropically etched, silicon-based micro-cavity. The Si-based cavity walls exhibit controlled temperature which assist in the quick and stable formation of lipid bilayer membranes. Fusion of large unilamellar vesicles was monitored in real time in an aqueous environment inside the Si cavity using atomic force microscopy (AFM), and the lateral organization of the lipid molecules was characterized until the formation of the SLBs. The stability of SLBs produced was also characterized by recording the electrical resistance and the capacitance using electrochemical impedance spectroscopy (EIS). Analysis was done in the frequency regime of 10−2–105 Hz at a signal voltage of 100 mV and giga-ohm sealed impedance was obtained continuously over four days. Finally, the cantilever tip in AFM was utilized to estimate the bilayer thickness and to calculate the rupture force at the interface of the tip and the SLB. We anticipate that a silicon-based, micron-sized cavity has the potential to produce highly-stable SLBs below their Tm. The membranes inside the Si cavity could last for several days and allow robust characterization using AFM or EIS. This could be an excellent platform for nanomedicine experiments that require low operating temperatures.

Formation of lipid bilayer membrane in a poly(dimethylsiloxane) microchip integrated with a stacked polycarbonate membrane support and an on-site nanoinjector.

This paper describes a new and facile approach for the formation of pore-spanning bilayer lipid membranes (BLMs) within a poly(dimethylsiloxane) (PDMS) microfluidic device. Commercially, readily available polycarbonate (PC) membranes are employed for the support of BLMs. PC sheets with 5 μm, 2 μm, and 0.4 μm pore diameters, respectively, are thermally bonded into a multilayer-stack, reducing the pore density of 0.4 μm-pore PC by a factor of 200. The BLMs on this support are considerably stable (a mean lifetime: 17 h). This multilayer-stack PC (MSPC) membrane is integrated into the PDMS chip by an epoxy bonding method developed to secure durable bonding under the use of organic solvents. The microchip has a special channel for guiding a micropipette in the proximity of the MSPC support. With this on-site injection technique, tens to hundreds of nanoliters of solutions can be directly dispensed to the support. Incorporating gramicidin ion channels into BLMs on the MSPC support has confirmed the formation of single BLMs, which is based on the observation from current signals of 20 pS conductance that is typical to single channel opening. Based on the bilayer capacitance (1.4 pF), about 15% of through pores across the MSPC membrane are estimated to be covered with BLMs.

Multiplexed Microfluidic Device for Parallel Electrophysiological Measurements on Independent Planar Lipid Bilayers

Bilayer lipid membranes (BLMs) are excellent platforms to study ion channels in a functional environment, and for drug screening assays. However, conventional electrophysiological BLM approaches do not meet the requirements for high throughput experiments due to the large volumes utilized, and the extensive manual procedures involved. In that context, microfluidics is a promising format to develop new platforms for high throughput electrophysiological measurements on ion channels incorporated in BLMs. Previously, we reported a simple microfluidic device for experimentation on BLMs (Stimberg, Small, 2013), and here we describe a multiplexed device along the same line, which contains 3 BLMs, each being optically accessible and independently electrically addressable. Due to the larger footprint, bonding of the device turned out to be a challenge and required thorough optimization. Furthermore, a novel procedure was developed for BLM formation: it consists of 3 pipetting steps, and is therefore easily amenable to automation using pipetting robots. BLMs were prepared using DPhPC (25 mg/mL) in n-decane, and measurements were conducted in 1 M KCl 10 mM HEPES buffer (pH 7.0). First, BLMs were formed successfully in the different apertures of the device. Next, they exhibit good stability over time upon continuous application of a 100 mV voltage, as well as excellent electrical properties (RSEAL= 27.9±4.9 GΩ; CMspec = 0.77±0.02 μF/cm2 & IRMS = 500±50 fA for 100-μm diameter apertures (n=6)), enabling low noise single channel recordings. Finally, first experiments conducted on pore-forming Gramicidin demonstrated a well-defined single channel behavior with a chord conductance of ca. 33 pS at 100 mV. We will report the fabrication of the multiplexed device, together with its thorough characterization, membrane preparation, as well as multiplexed measurements on various pore-forming peptides and proteins (e.g., gramicidin, alpha-hemolysin, OmpF).

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.

Lipid bilayer array for simultaneous recording of ion channel activities

This paper describes an array of stable and reduced-solvent bilayer lipid membranes (BLMs) formed in microfabricated silicon chips. BLMs were first vertically formed simultaneously and then turned 90° in order to realize a horizontal BLM array. Since the present BLMs are mechanically stable and robust, the BLMs survive this relatively tough process. Typically, a ∼60% yield in simultaneous BLM formation over 9 sites was obtained. Parallel recordings of gramicidin channel activities from different BLMs were demonstrated. The present system has great potential as a platform of BLM-based high throughput drug screening for ion channel proteins.

The Role of Hydrodynamic Interactions in Self-Organization of Biological Molecules

Self-organization is one of the most fundamental phenomena in biology. Elucidating the mechanisms of the self-assembly of biological molecules, such as protein folding/binding and formation of lipid bilayer membranes, have been a fascinating topic over decades in biological field. Hydrodynamic interactions (HIs) give rise to collective motions between molecules. Therefore, they may play an important role on dynamics of self-assembly. Although there are significant advances in theoretical and computational studies on thermodynamics and kinetics of self-assembling systems recent years, analysis on effects of HIs to the reactions are limited due to their high complexity. Here, by using a Brownian dynamics (BD) simulation technique, we evaluate the importance of HIs on kinetic of self-assembly in a set of examples, formation of lipid membrane and actin polymerization reaction. We first built coarse-grained models of lipid and actin monomer. A cluster of lipid molecules was represented by two particles, one is hydrophilic head group and the other is hydrophobic tail group, and attractive interaction between tail groups was applied. For actin polymerization system, each monomer was represented by two particles and an angle-dependent attractive interaction was applied to form long filaments. In BD simulations, the Rotne-Prager-Yamakawa tensor was used to account for HIs between particles. In the early stage of self-assembly, HIs between inter-monomer particles decelerated the association rate of monomers to form oligomers compared with that in the simulations without inter-monomer HIs in both systems. On the other hand, the HIs accelerate the reaction of oligomers to form a fully-assembled state or much longer polymers. This phenomenon could be explained by the Kirkwood-Riseman theory. Those results clearly suggest that HIs greatly affect kinetics of self-assembly reactions and considering the interactions is crucial for studying dynamics of biological systems.

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.

Multiplexed Microfluidic Device for Bilayer Experimentation and Drug Screening Assays on Membrane Proteins

Membrane proteins represent >50% of the targets for the development of new drugs. However, to date no appropriate platform is available for high throughput drug screening assays on these proteins. In that context, we developed a 1-plex microfluidic device for experimentation on BLMs (bilayer lipid membrane) and membrane proteins.1 Furthermore, we have proposed a novel methodology to prepare membranes in a closed environment, the “lipid-plug thinning” technique, and we have validated our device for the detection of single pore-forming species.Here, we report the multiplexing of the platform for parallel experimentation on a series of independent BLMs. The same 3-layer glass-Teflon-glass structure is employed, where the glass substrates house microchannels and the Teflon layer microfabricated apertures (15-40 μm O) for BLM formation. Two potential designs are investigated. In the first design, a series of independent 1-plex devices is placed in parallel. The second design resembles the structure of a fishbone, with one common channel in one glass substrate and a series of independent channels in the second glass substrate to reduce the number of reservoirs. Bilayers are formed in the multiplexed devices with a slightly modified approach where the lipid solution is introduced in one channel only, while buffer is present in the other one. Bilayer formation is successful (∼75% yield) for both designs, and BLMs exhibit good properties in terms of seal resistance (>10 GΩ), and capacitance (1.35-5.01 pF for 30-μm apertures).We are currently comparing the two designs for both automation of the membrane formation and simultaneous electrical measurements on independent BLMs. Thereafter, we will conduct experiments on pore-forming species and membrane properties using the in-house developed gramicidin-based assay.21. Stimberg et al., Proceedings MicroTAS 2010 & 2011.2. Stimberg et al., Small, submitted.

Bilayer Lipid Membrane Formation on a Chemically Modified S-Layer Lattice

The present paper describes the generation of a biomimetic model lipid membrane on bacterial surface (S-)layer which covered the entire surface of various sensors. The S-layer lattice allows one to be independent from the underlying solid material and provides a biological surface and anchoring structure for lipid membranes. S-layer proteins were chemically modified via binding of two amine-terminated phospholipids. Subsequently, a bimolecular lipid membrane anchored to the previously generated viscoelastic lipid monolayer was generated by the rapid solvent exchange technique. Characterization of the intermediate (monolayer) and final membrane structures (bilayer) was performed by imaging, surface-sensitive, and electrochemical techniques. This bilayer lipid membrane generated on an S-layer lattice revealed a thickness of ∼6 nm and constitutes a stable supported model membrane system with highly isolating properties showing a membrane resistance of 8.5 MΩ × cm(2).

Integrated Microfluidic Platform for Studies on Membrane Proteins and Drug Screening Assays

The combination of microfluidics with BLM (bilayer lipid membrane) experimentation provides a promising route towards high-throughput screening platforms for studies and drug screening on membrane proteins. The microfluidic format is ideal for multiplexed and automated assays, the miniaturization of the device goes together with enhanced electrical capabilities and more stable bilayers, and dual optical and electrical measurements are possible with a horizontal configuration of the membrane. In that context, we have developed a sandwich device consisting of two glass substrates separated by a Teflon membrane. The glass substrates contain two independent and orthogonal microfluidic channels being the two fluidic reservoirs for BLM experimentation; the Teflon film presents a micrometer-size aperture, located at the intersection of the microchannels, and across which BLMs are made. Leakage-free assembly of the three layers is demonstrated using an optical adhesive. The closed configuration of the device prompted us to develop a novel methodology for BLM preparation. Lipid solution and buffer are successively flushed in both channels, so that the lipid plug deposited in the aperture spontaneously thins into a bilayer. BLM formation is monitored electrically (patch-amplifier) and optically ((fluorescence) microscopy). This lipid-plug-thinning technique gives highly stable BLMs (> 7 hrs lifetime) with an almost 100% success yield. The resulting membranes exhibit reproducible characteristics in terms of sealing quality (14 ± 4 giga-ohm) and surface area (∼80% aperture coverage, measured optically and electrically (10 ± 3.4 pF capacitance)). Insertion of alpha-hemolysin (in the BLM) confirms the formation of a bilayer structure and demonstrates the potential of the platform for single protein measurements. We will present the detailed fabrication of the microfluidic platform, BLM formation and characterization in the closed environment, and on-chip single proteins studies using optical and electrical techniques.