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>
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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. 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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 ...

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