This work was supported by the CREST program of the Japan Science and Technology Agency (JPMJCR14F3) and Grant in-Aids from Japan Society for the Promotion of Science (19H00846 and 18K14120). This work was partly carried out at the Laboratory for Nanoelectronics and Spintronics, Research Institute of Electrical Communication, Tohoku University.

1. Preparation of a hybrid solution
<ol>
	<li>Wash four 4 mL disposable glass vials and screw caps (with PTFE coated seals) in an ultrasonic bath for 10 min in distilled water (purified with a filtration system), followed by ethanol and chloroform, respectively. Dry the glass vials and caps in a stream of nitrogen gas.</li>
	<li>In an anaerobic glove box, prepare a CuPc stock solution (10 mg/mL) in a washed glass vial by dissolving powdered CuPc in chloroform.</li>
	<li>Filter the CuPc solution through a 0.2 &#181;m polytetrafluoroethylene (PTFE) membrane.</li>
	<li>Store the filtered solution in a washed glass vial filled with nitrogen, and seal the vial with parafilm.</li>
	<li>Take the purchased 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) chloroform solution (25 mg/mL) out of the -80 &#176;C refrigerator and allow it to warm to room temperature.</li>
	<li>Stir the DPhPC solution using a vortex mixer at 2300 rpm for 10 s.</li>
	<li>Rinse a glass micro-syringe with chloroform for 5 times.</li>
	<li>Transfer 200 &#181;L of the DPhPC chloroform solution into a pre-washed glass vial using the washed syringe. Evaporate the solvent in the vial with a gentle stream of nitrogen.</li>
	<li>Rinse a glass micro-syringe 5 times with chloroform.</li>
	<li>Add 202.6 &#181;L of chloroform to the glass vial with DPhPC using the cleaned syringe.</li>
	<li>Add 47.4 &#181;L of filtered 10 mg/mL CuPc solution into the DPhPC solution. The molar ratio of DPhPC to CuPc should be 10:1.</li>
	<li>Rinse the glass micro-syringe 5 times with chloroform.</li>
	<li>Add 250 &#181;L of hexane to the solution using the syringe. The final concentration of the solution should be 10 mg/mL.</li>
	<li>Mix the prepared solution using a vortex mixer at 2300 rpm for 10 s.</li>
	<li>Filter the CuPc solution through a 0.2 &#181;m polytetrafluoroethylene (PTFE) membrane.</li>
	<li>Seal the glass vial with parafilm. Put it in to a grip sealed bag filled with nitrogen and place the grip sealed bag in a freezer at -20 &#176;C. 
	NOTE: After step 1.13, the DPhPC and CuPc were dissolved in a mixed solvent, which was composed of chloroform and hexane (volume ratio of 1:1). In addition, the molar ratio of DPhPC to CuPc is not limited to 10:1. With a constant concentration of lipids (10 mg/mL), different molar ratios can be used. According to previous experimental results, a range from 10:1 to 3:1 is preferred for forming a high-quality hybrid lipid membrane.</li>
</ol>
2. Formation of a hybrid membrane at the water/air interface
<ol>
	<li>Cut Si substrates (3 cm x 3 cm) from a Si wafer.</li>
	<li>Clean the 3 cm x 3 cm Si substrates in an ultrasonic bath for 10 min in purified water, followed by ethanol and then chloroform. Treat the Si substrate with an O2 plasma for 5 min to remove adsorbed organic materials from the surface and to improve hydrophilicity.</li>
	<li>Wash a Teflon beaker with an inner diameter of 7.5 cm with flowing purified water for 3 min.</li>
	<li>Put the cleaned Si substrate in the washed PTFE beaker. The substrate is tilted at an angle of 30&#176; to the horizontal.</li>
	<li>Pour a sufficient amount of purified water into the Teflon beaker until the entire Si substrate is submerged.</li>
	<li>Take prepared hybrid solution out of freezer and allow it to warm to room temperature.</li>
	<li>Stir the hybrid solution using a vortex mixer at 2300 rpm for 15 s.</li>
	<li>Rinse a glass micro-syringe (50 &#181;L) 5 times with chloroform.</li>
	<li>Drop 3-5 &#181;L of the hybrid solution onto the water surface using the syringe to form a floating hybrid lipid membrane. 
	NOTE: When the solution is dropped, it is important to hold the droplet close to (less than 1 cm) the water surface. It should also be noted that a single-layer hybrid membrane is not visible to the naked eye, but the multi-layer hybrid membrane appears as thin, blue-colored film. In order to transfer the multi-layer hybrid membrane to the Si substrate, it is important to drop the mixed solution as close as possible to the Si substrate.</li>
</ol>
3. Transferring the membrane onto a Si substrate
<ol>
	<li>After the evaporation of organic solvent (it will take less than 2 seconds), lower the water level at a rate of 3 mm/min by pumping out the water through a rubber tube which is driven by a peristaltic pump, to transfer the floating hybrid membrane onto the Si substrate.</li>
	<li>After the transfer process is complete (about 5 min), place the Si substrate on a cleanroom wiper, and allow all of residual water to evaporate. 
	NOTE: Lowering the water level at such a low rate serves to minimize the turbulence of water and to protect the membrane.</li>
</ol>

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+dynamics+simulations+of+a+lipid+bilayer+and+of+hexadecane:+an+investigation+of+membrane+fluidity.">Molecular dynamics simulations of a lipid bilayer and of hexadecane: an investigation of membrane fluidity.</a> Science. 262 (5131), 223-226 (1993).</li><li>Ide, T., Ichikawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+artificial+lipid-bilayer+formation.">A novel method for artificial lipid-bilayer formation.</a> Biosensors and Bioelectronics. 21 (4), 672-677 (2005).</li><li>Funakoshi, K., Suzuki, H., Takeuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+bilayer+formation+by+contacting+monolayers+in+a+microfluidic+device+for+membrane+protein+analysis.">Lipid bilayer formation by contacting monolayers in a microfluidic device for membrane protein analysis.</a> Analytical Chemistry. 78 (24), 8169-8174 (2006).</li><li>Kongsuphol, P., Fang, K. 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The authors have nothing to disclose.

In the precursor solution of the hybrid membrane, a mixed organic solvent (chloroform and hexane) rather than pure chloroform is used to dissolve lipids and CuPc. If pure chloroform is used, the density of the precursor solution would be higher than water. Therefore, it is highly likely that the solution would sink to the bottom of water rather than spread on water surface. Adding hexane, a low density solvent, to the precursor solution, ensures that the solution will float on the water surface and form a uniform hybrid ...

As essential frameworks of cell membranes, the interior of cells are separated from the exterior by a lipid bilayer system. This system consists of amphiphilic phospholipids, which are composed of hydrophilic phosphoric ester &#8220;heads&#8221; and hydrophobic fatty acids &#8220;tails&#8221;. Due to remarkable fluidity and self-assembly ability of lipid bilayers in aqueous environment1,2, artificial lipid bilayers can be formed using simple methods3,4. Various types of membrane proteins, such as ion channels, membrane receptors and enzymes, have been incorporated into the artificial lipid bilayer to mimic and study the functions of cell membranes5,6. More recently, lipid bilayers have been doped with nanomaterials (e.g., metal nanoparticles, graphene, and carbon nanotubes) to form functional hybrid membranes7,8,9,10,11,12,13. A widely used method for forming such hybrid membranes involves the formation of doped lipid vesicles, which contain hydrophobic materials such as modified Au-nanoparticles7 or carbon nanotubes11, and the resulting vesicles are then fused into planar supported lipid bilayers. However, this approach is complex and time-consuming, which limits the potential uses of such hybrid membranes.
In this work, lipid membranes were doped with organic molecules to produce hybrid lipid membranes that formed at the water/air interface by self-assembly. This protocol involves three steps: preparation of the mixed solution, formation of a hybrid membrane at the water/air interface, and the transfer of the membrane onto a Si substrate. Compared with other previously reported methods, the method described here is simpler and does not require sophisticated instrumentation. Using this method, air-stable hybrid lipid membranes with a larger area can be formed in a shorter time. The nanomaterial used in this study is a semiconducting organic molecule, copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc), which is widely used in a number of applications, including solar cells, photodetectors, gas sensors and catalysis14,15. CuPc, a small organic molecule with a planar structure, has a high affinity for the &#8220;tails&#8221; of phospholipids duo to its hydrophobic characteristics. Other groups have reported that CuPc molecules can self-assemble on single-crystal surfaces with the formation of highly ordered structures16,17. Therefore, it is highly possible that the CuPc molecules could be incorporated into the lipid bilayers through self-assembly.
We provide a detailed description of the procedures used to form membranes and provide some suggestions for smoothly implementing this procedure. In addition, we present some presentative results of the hybrid lipid membranes, and discuss potential applications of this method.

<table><tbody><tr><td>Chloroform</td><td>Wako&amp;nbsp;Chemicals</td><td>033-08631</td><td></td></tr><tr><td>CuPc</td><td>Sigma-Aldrich</td><td>423165</td><td></td></tr><tr><td>DPhPc</td><td>Avanti Polar Lipids</td><td>850356C</td><td></td></tr><tr><td>Glass vials with screw cap</td><td>Nichiden-Rike Glass Co., Ltd</td><td>6-29801</td><td></td></tr><tr><td>Hexane</td><td>Wako&amp;nbsp;Chemicals</td><td>084-03421</td><td></td></tr><tr><td>Membrane filters</td><td>Merck Millipore Ltd.</td><td>R8CA42836</td><td></td></tr><tr><td>Micro-syringe</td><td>Hamilton</td><td>80530</td><td></td></tr><tr><td>Peristaltic pump</td><td>Tokyo Rikakikai Co., Ltd.</td><td>11914199</td><td></td></tr><tr><td>Vortex mixer</td><td>Scientific Industries, Inc.</td><td>SI-0286</td><td></td></tr></tbody></table>

self-assembly of hybrid lipid membranes doped with hydrophobic organic molecules at the water/air interface

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

The as-formed membrane has a uniform light blue color due to the presence of CuPc molecules. The area of the colored membrane is normally several square centimeters. In Figure 1A and Figure 1B, we show a microscopic image and an atomic force microscope (AFM) image (including a height profile) of the hybrid lipid membrane on a Si substrate. In the AFM image, the membrane in the upper left is thick, with a thickness of 79.4 nm and that at the lower right is thin, ...

Watch this Scientific Journal Video about Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface at JoVE.com

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers ...

self-assembly-hybrid-lipid-membranes-doped-with-hydrophobic-organic

Research

JoVE Journal

Chemistry

3.6K Views.  Tohoku University. Ons protocol toont een eenvoudige methode om organische molecule gedoped hybride lipide membranen te fabriceren. De lucht stabiele eigenschap van het membraan kan de toepassing van lipide bilayer structuur uit te breiden tot solid state apparaten. We gebruiken een zelfassemblageproces om hybride lipidemembranen te vormen met een dikte van enkele nanometers.Dit protocol is eenvoudig en gemakkelijk te volgen en vereist geen ingewikkelde apparatuur. Deze methode kan worden gebruikt om and...

Video: Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Biochemistry

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Bioengineering

Preparation of Hollow Polystyrene Particles and Microcapsules by Radical Polymerization of Janus Droplets Consisting of Hydrocarbon and Fluorocarbon Oils

In this article, we have demonstrated a method for producing hollow particles and microcapsules using oil droplets consisting of hydrocarbon oil (styrene) and fluorocarbon oil (perfluoro-n-octane, PFO) in aqueous surfactant (sodium dodecylsulfate, SDS) solutions. Since fluorocarbon oils are immiscible with hydrocarbon oils, the two oils are separated. Emulsions are prepared by stirring styrene/PFO/aqueous SDS solution mixtures at 80 &#176;C. The type of emulsions and the morphology of droplets in the emulsions are observed by light microscope and scanning confocal fluorescence microscope. It is found that oil droplets with Janus-type morphologies consisting of mutually immiscible styrene and PFO are formed in aqueous SDS solutions. Polystyrene particles are fabricated by radical polymerization of the ternary mixtures of styrene/PFO/aqueous SDS solution at 80 &#176;C. The morphologies of the polystyrene are confirmed by scanning electron microscopy and scanning transmission electron microscopy observations. These observations show the preparation of hollow polystyrene particles with a single hole on the surface. To our knowledge, this method is a novel strategy using the immiscibility of hydrocarbon and fluorocarbon oils. The hollow particles can also be applied to the preparation of microcapsules.

A protocol for the fabrication of hollow polymer particles and microcapsules by radical polymerization using emulsions consisting of styrene, perfluoro-n-octane, and aqueous SDS (sodium dodecylsulfate) solution is presented.

In this article, we have demonstrated a method for producing hollow particles and microcapsules using oil droplets consisting of hydrocarbon oil (styrene) ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular recognition within biosensors for diagnosis, functional membranes for artificial cells, and development of bioinspired micro/nano-reactors. In all of these applications, the characterization of their membrane properties is of fundamental importance. Among existing characterization techniques, micropipette aspiration, pioneered by E. Evans, allows the measurement of mechanical properties of the membrane such as area compressibility modulus, bending modulus and lysis stress and strain. Here, we present all the methodologies and detailed procedures to obtain giant vesicles from the thin film of a lipid or copolymer (or both), the manufacturing and surface treatment of micropipettes, and the aspiration procedure leading to the measurement of all the parameters previously mentioned.

The goal of the protocol is to reliably measure membrane mechanical properties of giant vesicles by micropipette aspiration.

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions&#8212;and perhaps the biological relevance&#8212;of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.

This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

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