This work was supported by the Pioneer Research Center Program (NRF-2012-0009575) and National Research Foundation Grants (NRF-2012R1A1B4002413, NRF-2014R1A1A2059341) from the National Research Foundation of Korea. This work was also partially supported by the Inha University Research Grant.

1. Solution Preparation
<ol>
	<li>Preparation of buffer solution:
	<ol>
		<li>To formulate buffer solution, dissolve 1 M KCl (Potassium chloride), 10 mM Tris-HCl (Tris-hydrochloride), and 1 mM EDTA (Ethylenediaminetetraacetic acid) in distilled water and adjust pH to 8.0.</li>
		<li>Filter the solution using a 0.20 &#181;m filter. To sterilize, autoclave the solution at 121 &#176;C for 15 min.</li>
	</ol>
	</li>
	<li>Preparation of lipid solution for pre-painting:
	<ol>
		<li>To formulate the lipid solution for pre-painting, dissolve 3% DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine) lipid (w:v) in a mixture of 2:8 n-decane and hexadecane (v:v). Stir overnight using a rotator.</li>
	</ol>
	</li>
	<li>Preparation of lipid solution for membrane formation:
	<ol>
		<li>To formulate the lipid solution for membrane formation, dissolve 0.1% DPhPC (1, 2-diphytanoyl-sn-glycero-3-phosphatidylcholine) lipid (w:v) in a mixture of 2:8 squalene and hexadecane (v:v). Stir overnight using a rotator.</li>
	</ol>
	</li>
</ol>
2. Formation of a PDMS Thin Film
<ol>
	<li>Mix PDMS and curing agent in a 9:1 (w/w) ratio in a mixing cup to form the PDMS prepolymer. Add 5 g of PDMS prepolymer to a Petri dish to form the PDMS thin film (thickness 200 - 250 &#181;m). Spread PDMS pre-polymer using a spin coater at 800 rpm for 10 sec to form a thin film.</li>
	<li>Place the Petri dish into a vacuum desiccator at a pressure of 100 mTorr for 2 hr to remove air bubbles. To polymerize the pre-polymer thin film, bake in an oven for 5 hr at 70 &#176;C.</li>
	<li>In order to make a square PDMS thin film, cut the polymerized PDMS thin film into 2 x 2 cm2 squares. Use a 500 &#181;m micro punch to make an aperture in the center of the PDMS thin film. Pre-paint apertures with 3% DPhPC lipid solution mixed in 2:8 n-decane and hexadecane.</li>
</ol>
3. Chamber Fabrication and Assembly
<ol>
	<li>To fabricate the BLM chamber, design two symmetric blocks of the chamber using 3D drawing software with outer dimensions of 4 cm x 1.5 cm x 1 cm and inner-well dimensions of 1.5 cm x 1.3 cm x 0.8 cm17.</li>
	<li>Craft the chamber using a PTFE block with a CNC machine and follow the manufacturer&#39;s instructions.</li>
</ol>
4. Chamber Assembly
<ol>
	<li>To assemble the chamber, place the pre-painted-PDMS thin film between the two PTFE blocks such that the aperture on the PDMS thin film is aligned with the hole in the chamber.</li>
	<li>Seal the outer edges of the chamber using a cover glass with grease (facilitating optical observation). Immobilize the assembled chamber using nuts and bolts. 
	NOTE: Make sure the chamber is well-sealed so that there is no liquid leakage.</li>
</ol>
5. Formation of Membrane Precursor with Expedited Self-assembly Formation (MPES)
<ol>
	<li>Using a pipette, deposit 0.5 &#181;l of 0.1% DPhPC lipid mixed in 2:8 n-decane:hexadecane onto the aperture of the PDMS thin film assembled with the chamber.</li>
	<li>Prior to use, store the chamber in a freezer or a refrigerator below 10 &#176;C.</li>
</ol>
6. Membrane Formation and Verification
<ol>
	<li>To form a BLM with MPES, withdraw the chamber from the refrigerator and suspend 2 ml of buffer solution in each side of the chamber. Set the chamber aside for &#60; 10 min until the frozen membrane precursor thaws.</li>
	<li>Place the chamber onto a micromanipulator to precisely control the elevation with respect to the light source and the microscope. Illuminate one side of the chamber as a light source using a halogen fiber optic illuminator to brighten the aperture of the PDMS thin film for optical observation of BLM formation process.</li>
	<li>On the other side, place a digital microscope vertically with respect to the light source to observe BLM formation (magnify by 200X).</li>
	<li>To confirm BLM formation, observe the center of the aperture where the color becomes brighter than the annulus.</li>
</ol>
7. Electrical Recording
<ol>
	<li>For electrical measurement, prepare Ag/Cl electrodes using a 208 &#181;m-thick silver wire and bleach in sodium hypochlorite for &#62; 1 min. Place the Ag/Cl electrodes into each side of the chamber deep enough to be dipped into the buffer solution.</li>
	<li>Connect the electrodes to the microelectrode amplifier. Using electrophysiology software, apply a &#177;10 mV triangular waveform across the membrane to acquire a square wave. Set applying voltage by clicking the arrows indicated on V_clamp (mV).</li>
	<li>Record the electrical properties of the membrane by clicking the record button (red dot icon). Proceed with recording until a uniform square wave is observed. Quit the recording by clicking the black square icon.</li>
</ol>
8. Ion Channel Incorporation
NOTE: Gramicidin A (gA) incorporation occurs spontaneously upon formation of BLM, as gA is added directly to the lipid solution.
<ol>
	<li>To observe gA channel activities, apply 100 mV across the membrane at a sample rate of 5 kHz to measure holding potential of the membrane. Set applying voltage by clicking the arrows indicated on V_clamp (mV).</li>
	<li>Record the electrical properties of the gA incorporation by clicking the record button (red dot icon). Proceed the recording until current jumps is observed. Quit the recording by clicking the black square icon.</li>
	<li>After electrical data acquisition, filter the data with a low-pass Bessel filter at 100 Hz using an electrophysiology software.</li>
	<li>Observe current jumps in the filtered holding potential data (each current jump, ~0.15 nS, represents dimerization of a gA ion channel) to verify gA incorporation.</li>
</ol>

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<li>Costello, R., Peterson, I., Heptinstall, J., Byrne, N., Miller, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+robust+gel-bilayer+channel+biosensor%5BTitle%5D)+AND+%22Adv.+Mater.+Opt.+Electron%22%5BJournal%5D)">A robust gel-bilayer channel biosensor.</a> Adv. Mater. Opt. Electron. 8 (2), 47-52 (1998).</li>
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<li>Jeon, T. J., Malmstadt, N., Schmidt, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrogel-encapsulated+lipid+membranes%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">Hydrogel-encapsulated lipid membranes.</a> J Am Chem Soc. 128 (1), 42-43 (2006).</li>
<li>Malmstadt, N., Jeon, T. J., Schmidt, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-Lived+Planar+Lipid+Bilayer+Membranes+Anchored+to+an+In+Situ+Polymerized+Hydrogel%5BTitle%5D)+AND+%22Adv.+Mater%22%5BJournal%5D)">Long-Lived Planar Lipid Bilayer Membranes Anchored to an In Situ Polymerized Hydrogel.</a> Adv. Mater. 20 (1), 84-89 (2008).</li>
<li>Jeon, T. J., Poulos, J. L., Schmidt, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+storable+and+shippable+lipid+bilayer+membrane+platform%5BTitle%5D)+AND+%22Lab.+Chip%22%5BJournal%5D)">Long-term storable and shippable lipid bilayer membrane platform.</a> Lab. Chip. 8 (10), 1742-1744 (2008).</li>
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The authors have nothing to disclose.

Our BLM formation technique provides a powerful tool for cell membrane and ion channel studies, in contrast to conventional techniques that have limited potential for industrial use. We developed a membrane precursor using a PDMS thin film, and devised a frozen membrane precursor with expedited self-assembly.
As opposed to conventional membrane formation methods with hydrophobic films, where membrane formation only occurs via surface interactions between the film and the lipid solution,20...

Artificial lipid bilayer membrane, or black lipid membrane (BLM), is an important tool for elucidating mechanisms of cell membranes and ion channels, as well as for understanding interactions between ion channels and ions/molecules.1-7 Although the patch-clamp method is often considered the gold standard for cell membrane studies, it is laborious and requires highly skilled operators for ion channel measurements.8 While artificially reconstituted lipid bilayer membranes have emerged as alternative tools for ion channel studies,9,10 they are also associated with laborious processes and specific expertise. Moreover, membranes are susceptible to mechanical perturbations. Hence, lipid bilayer technologies introduced to date have limited practical applications.11
In order to enhance robustness and longevity of lipid bilayer membranes, Costello et al.12, and Ide and Yanagida13 have devised a free-standing lipid bilayer supported by hydrogels. Despite enhanced longevity however (&#60; 24 hr), bilayer robustness was not improved. Jeon et al.14 devised a hydrogel encapsulated membrane (HEM) with intimate hydrogel-lipid bilayer contact, resulting in enhanced longevity (up to several days). To further increase the lifetime of the HEM, Malmstadt and Jeon et al. created a hydrogel-encapsulated membrane with hydrogel-lipid binding via in-situ covalent conjugation (cgHEM).15 In both systems, membrane lifetimes increased substantially (&#62; 10 days). However, the membrane formation systems were not sufficiently robust, and could not be stored or delivered where required to liberate expertise for use of the lipid bilayers.
The development of a lipid bilayer platform has primarily revolved around increasing robustness and longevity of BLMs. Although the longevity of BLMs has been substantially enhanced recently, their applications have been limited due to a lack of transportability and storability. To overcome these issues, Jeon et al. created a storable membrane system and introduced a membrane precursor (MP).16 To construct an MP, they prepared a mixture of n-decane and hexadecane containing 3% DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine) to control the freezing point of the lipid solution such that it would freeze at ~14 &#176;C (below room temperature, above typical refrigerator temperature). In this experiment, the MP was spread over a small aperture on a polytetrafluoroethylene (PTFE) film and subsequently frozen in a refrigerator at 4 &#176;C. When the MP was brought to room temperature, the MP thawed and a lipid bilayer was automatically formed, eliminating the expertise typically associated with membrane formation. However, the success rate of BLM made from the MP was as low as ~27%, and membrane formation time was inconsistent (30 min to 24 hr), limiting its practical applications.
In this study, a polydimethylsiloxane (PDMS) thin film is used instead of a conventional hydrophobic thin films (PTFE, polyoxymethylene, polystyrene) to (a) control fabrication time and (b) increase the success rate of BLM formation as previously reported by Ryu et al.17 Herein, membrane formation was facilitated by extraction of solvents due to the porous nature of PDMS, and the time required for membrane formation was successfully controlled in this study. In this system, as the lipid solution was absorbed into the PDMS thin film, a consistent membrane formation time was achieved. Moreover, membrane lifetime was prolonged due to slow absorption of solvents into the PDMS thin film, a result of the addition of squalene to the lipid solution. We conducted optical and electrical measurements to verify that membranes formed using this technique are suitable for ion channels studies.

<table><tbody><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P9333</td><td>For buffer solution</td></tr><tr><td>Tris-hydrochloride</td><td>Sigma-Aldrich</td><td>1185-53-1</td><td>For buffer solution</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma-Aldrich</td><td>60-00-4</td><td>For buffer solution</td></tr><tr><td>&lt;em&gt;n&lt;/em&gt;-decane</td><td>Sigma-Aldrich</td><td>44074-U</td><td>For lipid solution</td></tr><tr><td>Hexadecane</td><td>Sigma-Aldrich</td><td>544-76-3</td><td>For lipid solution</td></tr><tr><td>Squalene</td><td>Sigma-Aldrich</td><td>S3626</td><td>For lipid solution</td></tr><tr><td>Gramicidin A</td><td>Sigma-Aldrich</td><td>11029-61-1</td><td>Membrane protein</td></tr><tr><td>1,2-diphytanoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine</td><td>Avanti Polar Lipids, Inc.</td><td>850356</td><td>For membrae formation</td></tr><tr><td>Sylgard 184a and 184b elastromer kit</td><td>Dow Corning Asia</td><td></td><td>To produce PDMS thin film</td></tr><tr><td>0.2 &amp;mu;m filter</td><td>Satorius stedim</td><td>16534----------K</td><td>To filter buffer solution</td></tr><tr><td>Rotator</td><td>FinePCR</td><td>AG</td><td>To dissolve lipid homogeneously</td></tr><tr><td>Autoclave</td><td>Biofree</td><td>BF-60AC</td><td>To sterilize buffer solution</td></tr><tr><td>Spin coater</td><td>Shinu Mst</td><td>SP-60P</td><td>To spread PDMS prepolymer</td></tr><tr><td>Vaccum dessiccator</td><td>Welch</td><td>2042-22</td><td>To remove air bubble in PDMS prepolymer</td></tr><tr><td>500 &amp;mu;m&amp;nbsp; punch</td><td>Harris Uni-Core</td><td>0.5</td><td>To create an aperture on the PDMS thin film</td></tr><tr><td>CNC machine</td><td>SME trading</td><td>SME 2518</td><td>To fabricate membrane formation chamber</td></tr><tr><td>Halogen fiber optic illuminator</td><td>Motic</td><td>MLC-150C</td><td>To illuminate the aperture of PDMS thin film for optical observation</td></tr><tr><td>Digital microscope</td><td>Digital blue</td><td>QX-5</td><td>To optically observe lipid bilayer membrane formation</td></tr><tr><td>Electrode</td><td>A-M Systems</td><td></td><td>To electrically observe membrane formation</td></tr><tr><td>Microelectrode amplifier (Axopatch amplifier)</td><td>Axon Instruments</td><td>Axopatch 200B Amplifier</td><td>To measure capacitance of the membrane (described as microelectrode amplifier in the manuscript)</td></tr></tbody></table>

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.

Optimization of MPES Solution Composition 
Different compositions of lipids and solvents were tested to successfully reconstitute lipid bilayer membranes from MPES. The MP system with a mixture of n-decane and hexadecane containing 3% DPhPC14 exhibited a low success rate of membrane formation (~27%). In addition, as the PDMS film continuously extracted lipid solution, it was necessary to optimize solvent composition to maintain an intact lipid bil...

Watch this Scientific Journal Video about Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film at JoVE.com

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

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 ...

automated-lipid-bilayer-membrane-formation-using-polydimethylsiloxane

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JoVE Journal

Bioengineering

18.2K Views.  Inha University.  We tonen een opgeslagen of vervoerd lipidedubbellaag formatie systeem. Een lipide dubbellaag membraan kan worden gevormd binnen 1 uur meer dan 80% succes als een bevroren membraan precursor tot omgevingstemperatuur wordt gebracht. Dit systeem tijdrovend proces en deskundigheid geassocieerd met ionkanalen verminderen. 

Video: Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

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 ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

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 ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

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.

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 ...

Chemistry

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 ...

Biochemistry

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It is not only passively generated by cellular activities, but also actively regulates protein interactions and is involved in many intracellular signaling. Thus, it is of great value to examine the role of membrane curvature in regulating the distribution and dynamics of proteins and lipids. Recently, many techniques have been developed to study the relationship between the curved membrane and protein in vitro. Compared to traditional techniques, the newly developed nanobar-supported lipid bilayer (SLB) offers both high-throughput and better accuracy in membrane curvature generation by forming a continuous lipid bilayer on patterned arrays of nanobars with a pre-defined membrane curvature and local flat control. Both the lipid fluidity and protein sensitivity to curved membranes can be quantitatively characterized using fluorescence microscopy imaging. Here, a detailed procedure on how to form a SLB on fabricated glass surfaces containing nanobar arrays&#160;and the characterization of curvature-sensitive proteins on such SLB are introduced. In addition, protocols for nanochip reusing and image processing are covered. Beyond the nanobar-SLB, this protocol is readily applicable to all types of nanostructured glass chips for curvature sensing studies.

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Here, a nanobar-supported lipid bilayer system is developed to provide a synthetic membrane with a defined curvature that enables the characterization of proteins with curvature sensing ability in vitro.

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

Neuroscience

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

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 ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

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