Name: automated lipid bilayer membrane formation using a polydimethylsiloxane thin film
Uploaded: 2016-07-10T13:00:00
Description: Watch this Scientific Journal Video about Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film at JoVE.com

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-pai...

<ol>
	<li>Hanke, W., Schulue, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Planar lipid bilayers: methods and applications. , (2012).</li><li>Mirzabekov, T. A., Silberstein, A. Y., Kagan, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+planar+lipid+bilayer+membranes+for+rapid+screening+of+membrane+active+compounds.">Use of planar lipid bilayer membranes for rapid screening of membrane active compounds.</a> Methods Enzymol. 294, 661-674 (1999).</li><li>Bayley, H., Cremer, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+sensors+inspired+by+biology.">Stochastic sensors inspired by biology.</a> Nature. 413 (6852), 226-230 (2001).</li><li>Fang, Y., Lahiri, J., Picard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein-coupled+receptor+microarrays+for+drug+discovery.">G protein-coupled receptor microarrays for drug discovery.</a> Drug. Discov. Today. 8 (16), 755-761 (2003).</li><li>Majd, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+biological+pores+in+nanomedicine,+sensing,+and+nanoelectronics.">Applications of biological pores in nanomedicine, sensing, and nanoelectronics.</a> Curr. Opin. Biotechnol. 21 (4), 439-476 (2010).</li><li>Kim, Y. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+Biomimetic+Membranes+and+Their+Sensor+Applications.">Synthetic Biomimetic Membranes and Their Sensor Applications.</a> Sensors (Basel). 12 (7), 9530-9550 (2012).</li><li>Ryu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+Ion+Channel+Activities+of+Gramicidin+A+in+the+Presence+of+Ionic+Liquids+Using+Model+Cell+Membranes.">Investigation of Ion Channel Activities of Gramicidin A in the Presence of Ionic Liquids Using Model Cell Membranes.</a> Sci Rep. 5, (2015).</li><li>Wood, C., Williams, C., Waldron, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamping+by+numbers.">Patch clamping by numbers.</a> Drug. Discov. Today. 9 (10), 434-441 (2004).</li><li>Mueller, P., Rudin, D. O., Tien, H. T., Wescott, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cell+membrane+structure+in+vitro+and+its+transformation+into+an+excitable+system.">Reconstitution of cell membrane structure in vitro and its transformation into an excitable system.</a> Nature. 194, 979-980 (1962).</li><li>Montal, M., Mueller, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+bimolecular+membranes+from+lipid+monolayers+and+a+study+of+their+electrical+properties.">Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties.</a> Proc. Natl. Acad. Sci. U. S. A. 69, 3561-3566 (1972).</li><li>Baaken, G., Sondermann, M., Schlemmer, C., Ruhe, J., Behrends, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+microelectrode-cavity+array+for+high-resolution+and+parallel+electrical+recording+of+membrane+ionic+currents.">Planar microelectrode-cavity array for high-resolution and parallel electrical recording of membrane ionic currents.</a> Lab Chip. 8 (6), 938-944 (2008).</li><li>Costello, R., Peterson, I., Heptinstall, J., Byrne, N., Miller, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+gel-bilayer+channel+biosensor.">A robust gel-bilayer channel biosensor.</a> Adv. Mater. Opt. Electron. 8 (2), 47-52 (1998).</li><li>Ide, T., Yanagida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+artificial+lipid+bilayer+formed+on+an+agarose-coated+glass+for+simultaneous+electrical+and+optical+measurement+of+single+ion+channels.">An artificial lipid bilayer formed on an agarose-coated glass for simultaneous electrical and optical measurement of single ion channels.</a> Biochem. Biophys. Res. Commun. 265 (2), 595-599 (1999).</li><li>Jeon, T. J., Malmstadt, N., Schmidt, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel-encapsulated+lipid+membranes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Lived+Planar+Lipid+Bilayer+Membranes+Anchored+to+an+In+Situ+Polymerized+Hydrogel.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+storable+and+shippable+lipid+bilayer+membrane+platform.">Long-term storable and shippable lipid bilayer membrane platform.</a> Lab. Chip. 8 (10), 1742-1744 (2008).</li><li>Ryu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+Lipid+Membrane+Formation+Using+a+Polydimethylsiloxane+Film+for+Ion+Channel+Measurements.">Automated Lipid Membrane Formation Using a Polydimethylsiloxane Film for Ion Channel Measurements.</a> Anal. Chem. 86 (18), 8910-8915 (2014).</li><li>Yaws, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemical Properties Handbooks: Physical, Thermodynamic, Environmental, Transport, Safety, and Health Related Properties for Organic and Inorganic Chemicals. , (1999).</li><li>Windholz, M., Budavari, S., Stroumtsos, L. Y., Fertig, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Merck index. An encyclopedia of chemicals and drugs. , (1976).</li><li>Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion Channel Reconstitution. , (1986).</li><li>Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-state+substructure+of+single+chloride+channels+from+Torpedo+electroplax.">Open-state substructure of single chloride channels from Torpedo electroplax.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 299 (1097), 401-411 (1982).</li><li>Benz, R., Frohlich, O., Lauger, P., Montal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+capacity+of+black+lipid+films+and+of+lipid+bilayers+made+from+monolayers.">Electrical capacity of black lipid films and of lipid bilayers made from monolayers.</a> Biochim. Biophys. Acta. 394 (3), 323-334 (1975).</li><li>Priel, A., Gil, Z., Moy, V. T., Magleby, K. L., Silberberg, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+requirements+for+membrane-glass+adhesion+and+giga+seal+formation+in+patch-clamp+recording.">Ionic requirements for membrane-glass adhesion and giga seal formation in patch-clamp recording.</a> Biophys. J. 92 (11), 3893-3900 (2007).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="1448">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Potassium Chloride</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">P9333</td>
			<td style="width:743px;">For buffer solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Tris-hydrochloride</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">1185-53-1</td>
			<td>For buffer solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">60-00-4</td>
			<td>For buffer solution</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:335px;">n-decane</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">44074-U</td>
			<td>For lipid solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Hexadecane</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">544-76-3</td>
			<td>For lipid solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Squalene</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">S3626</td>
			<td>For lipid solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Gramicidin A</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">11029-61-1</td>
			<td>Membrane protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">1,2-diphytanoyl-sn-glycero-3-phosphocholine</td>
			<td style="width:172px;">Avanti Polar Lipids, Inc.</td>
			<td style="width:199px;">850356</td>
			<td>For membrae formation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Sylgard 184a and 184b elastromer kit</td>
			<td style="width:172px;">Dow Corning Asia</td>
			<td style="width:199px;"></td>
			<td>To produce PDMS thin film</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">0.2 &#956;m filter</td>
			<td style="width:172px;">Satorius stedim</td>
			<td style="width:199px;">16534----------K</td>
			<td>To filter buffer solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Rotator</td>
			<td style="width:172px;">FinePCR</td>
			<td style="width:199px;">AG</td>
			<td>To dissolve lipid homogeneously</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Autoclave</td>
			<td style="width:172px;">Biofree</td>
			<td style="width:199px;">BF-60AC</td>
			<td>To sterilize buffer solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Spin coater</td>
			<td style="width:172px;">Shinu Mst</td>
			<td style="width:199px;">SP-60P</td>
			<td>To spread PDMS prepolymer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Vaccum dessiccator</td>
			<td style="width:172px;">Welch</td>
			<td style="width:199px;">2042-22</td>
			<td>To remove air bubble in PDMS prepolymer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">500 &#956;m&#160; punch</td>
			<td style="width:172px;">Harris Uni-Core</td>
			<td style="width:199px;">0.5</td>
			<td>To create an aperture on the PDMS thin film</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">CNC machine</td>
			<td style="width:172px;">SME trading</td>
			<td style="width:199px;">SME 2518</td>
			<td>To fabricate membrane formation chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Halogen fiber optic illuminator</td>
			<td style="width:172px;">Motic</td>
			<td style="width:199px;">MLC-150C</td>
			<td>To illuminate the aperture of PDMS thin film for optical observation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Digital microscope</td>
			<td style="width:172px;">Digital blue</td>
			<td style="width:199px;">QX-5</td>
			<td>To optically observe lipid bilayer membrane formation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Electrode</td>
			<td style="width:172px;">A-M Systems</td>
			<td style="width:199px;"></td>
			<td>To electrically observe membrane formation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Microelectrode amplifier (Axopatch amplifier)</td>
			<td style="width:172px;">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 ...

The overall goal of this procedure is to describe a method that can be used to study automated lipid bilayer formation using a polydimethylsiloxane thin film. This method can help answer key questions in the nanobiosensor field regarding membrane-protein interactions. The main advantage of this technique is that the lipid bilayer formation is automated and reproducible.To start, prepare the buffer solution by combining potassium chloride, tris hydrochloride, and EDTA in distilled water. Then, adjust the PH of the solution to 8.0. Then, filter the solution using a 0.2 micron filter, and autoclave the solution at 121 degrees celsius for 15 minutes.Next, prepare the lipid solution for pre-painting by dissolving 3%of the lipid in a mixture consisting of two parts undecane and eight parts hexadecane by volume. Stir the solution overnight using a rotator. Also, prepare the lipid solution for membrane formation by dissolving 0.1%of the lipid in the same mixture of two parts undecane and eight parts hexadecane by volume.Stir this solution overnight on a rotator as well. In order to prepare a PDMS thin film, mix nine parts prepolymer with one part curing agent in a mixing cup. Place five grams of this mixture into a petri dish, and spin coat the dish at 800 rpm for 10 seconds to form a film that is 200 to 250 microns thick.Next, place the petri dish into a vacuum desiccator and pull a vaccum of 100 millitorr for two hours to remove the residual air bubbles. Then, bake the thin film in an oven for five hours at 70 degrees celsius to polymerize the PDMS. Once cooled, cut the polymerized thin film into two centimeter by two centimeter squares, and use a 500 micron diameter punch to make a small aperture in the center of the square.Then, use a micropipette to prepaint the apertures with one microliter of the 3%lipid solution. To create the black lipid membrane chamber, design two symmetric blocks for the chamber using 3D drawing software. Then, fabricate the chamber using a block of PTFE and a CNC machine.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 centered with the hole in the chamber. Use cover glass and vacuum grease to seal the outer edges of the chamber. Once sealed, immobilize the assembled chamber using nuts and bolts.It is important to make sure the chamber's well sealed so that there is no liquid leakage. Using a pipette, deposit 0.5 microliters of the 0.1%lipid solution onto the aperture of the PDMS thin film, assembled with the chamber. Then, place the chamber in a cooled environment below 10 degrees celsius and store it until it is needed.To form a black lipid membrane with expedited self-assembly, withdraw the chamber from the refrigerator and add two milliliters of the buffer solution into each side of the chamber. Set the chamber aside for 10 minutes until the frozen membrane precursor thaws. Then, place the chamber onto a micro manipulator to precisely control the elevation with respect to the light source and the microscope.Using a halogen fiber optic illuminator, illuminate one side of the chamber to brighten the aperture of the PDMS thin film. On the other side, place a 20x objective in line with the aperture and observe the center where the color becomes brighter than the annulus. For electrical measurement, prepare silver chloride electrodes by placing a 208 micrometer thick silver wire in a solution of sodium hypochlorite for at least one minute.Next, connect the electrodes to a microelectrode amplifier. Then, place the silver chloride electrodes into each side of the chamber, so that they are in the buffer solution. Using electrophysiology software, apply a 10 millivolt triangular wave form across the membrane to acquire a square wave.Record the electrical properties of the membrane by clicking the record button. Continue recording until a uniform square wave is observed. Then, quit recording by clicking on the black square icon.Two methods can be employed to incorporate gramicidin A into the lipid bilayer. First, gramicidin A can be added directly to the lipid solution before spreading the lipid solution onto the aperture of the PDMS thin film. Second, gramicidin A solution can be added to the chamber when a lipid bilayer forms.To observe gramicidin A channel activities, apply 100 millivolts across the membrane at a sample rate of 5 kilohertz to measure the holding potential of the membrane. Record the electrical properties as previously done, by clicking the record button. As ion channel activities are shown, keep recording until various current jumps are observed to confirm ion channel incorporation.Once confirmed, quit the recording by clicking the black square icon. When the recording is finished, filter the data with a low pass bessel filter at 100 hertz using electrophysiology software. Observe the current jumps in the filtered holding potential data.These jumps represent the dimerization of a gramicidin A ion channel. When the frozen membrane precursor was brought in to room temperature to thaw, the lipid bilayer formation is facilitated due to extraction of hydrophobic solvent into the PDMS thin film. This sequence of images shows the formation of a lipid bilayer, which self assembles when warmed from its frozen form.Here, the electrical conductance illustrates the incorporation and dimerization of gramicidin A.Upon dimerization, the gramicidin A forms an ion channel and the conductance levels jump to 28 picosiemens, which is consistent with the results of previous reports and shows immediate incorporation of this antibiotic into the bilayer. Our membrane formation technique provides powerful tool for cell membrane and nanochannel studies in contrast to conventional techniques that have limited potentials for practical application. Our system requires no expertise for either membrane formation or ion channeling incorporation making it well suited for drug screening and bio sensing applications.

Research

JoVE Journal

Bioengineering

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

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 ...

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

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 ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

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 ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...