JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

Published: July 10th, 2016

DOI:

10.3791/54258

1Department of Biological Engineering, Inha University, 2Department of Mechanical Engineering, Inha University, 3Biohybrid Systems Research Center (BSRC), Inha University, 4Convergent Research Center for Metabolism and Immunoregulation (CRCMI), Inha University

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 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 °C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of < 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

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 suscept....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Solution Preparation

  1. Preparation of buffer solution:
    1. 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.
    2. Filter the solution using a 0.20 µm filter. To sterilize, autoclave the solution at 121 °C for 15 min.
  2. Preparation of lipid solution for pre-painting:
    1. To formulate the lipid solution for pre-pai.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

  1. Hanke, W., Schulue, W. . Planar lipid bilayers: methods and applications. , (2012).
  2. Mirzabekov, T. A., Silberstein, A. Y., Kagan, B. L. Use of planar lipid bilayer membranes for rapid screening of membrane active compounds. Methods Enzymol. 294, 661-674 (1999).
  3. Bayley, H., Cremer, P. S. Stochastic sensors inspired by biology. Nature. 413 (6852), 226-230 (2001).
  4. Fang, Y., Lahiri, J., Picard, L. G protein-coupled receptor microarrays for drug discovery. Drug. Discov. Today. 8 (16), 755-761 (2003).
  5. Majd, S., et al. Applications of biological pores in nanomedicine, sensing, and nanoelectronics. Curr. Opin. Biotechnol. 21 (4), 439-476 (2010).
  6. Kim, Y. R., et al. Synthetic Biomimetic Membranes and Their Sensor Applications. Sensors (Basel). 12 (7), 9530-9550 (2012).
  7. Ryu, H., et al. Investigation of Ion Channel Activities of Gramicidin A in the Presence of Ionic Liquids Using Model Cell Membranes. Sci Rep. 5, (2015).
  8. Wood, C., Williams, C., Waldron, G. J. Patch clamping by numbers. Drug. Discov. Today. 9 (10), 434-441 (2004).
  9. Mueller, P., Rudin, D. O., Tien, H. T., Wescott, W. C. Reconstitution of cell membrane structure in vitro and its transformation into an excitable system. Nature. 194, 979-980 (1962).
  10. Montal, M., Mueller, P. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc. Natl. Acad. Sci. U. S. A. 69, 3561-3566 (1972).
  11. Baaken, G., Sondermann, M., Schlemmer, C., Ruhe, J., Behrends, J. C. Planar microelectrode-cavity array for high-resolution and parallel electrical recording of membrane ionic currents. Lab Chip. 8 (6), 938-944 (2008).
  12. Costello, R., Peterson, I., Heptinstall, J., Byrne, N., Miller, L. A robust gel-bilayer channel biosensor. Adv. Mater. Opt. Electron. 8 (2), 47-52 (1998).
  13. Ide, T., Yanagida, T. An artificial lipid bilayer formed on an agarose-coated glass for simultaneous electrical and optical measurement of single ion channels. Biochem. Biophys. Res. Commun. 265 (2), 595-599 (1999).
  14. Jeon, T. J., Malmstadt, N., Schmidt, J. J. Hydrogel-encapsulated lipid membranes. J Am Chem Soc. 128 (1), 42-43 (2006).
  15. Malmstadt, N., Jeon, T. J., Schmidt, J. J. Long-Lived Planar Lipid Bilayer Membranes Anchored to an In Situ Polymerized Hydrogel. Adv. Mater. 20 (1), 84-89 (2008).
  16. Jeon, T. J., Poulos, J. L., Schmidt, J. J. Long-term storable and shippable lipid bilayer membrane platform. Lab. Chip. 8 (10), 1742-1744 (2008).
  17. Ryu, H., et al. Automated Lipid Membrane Formation Using a Polydimethylsiloxane Film for Ion Channel Measurements. Anal. Chem. 86 (18), 8910-8915 (2014).
  18. Yaws, C. . Chemical Properties Handbooks: Physical, Thermodynamic, Environmental, Transport, Safety, and Health Related Properties for Organic and Inorganic Chemicals. , (1999).
  19. Windholz, M., Budavari, S., Stroumtsos, L. Y., Fertig, M. N. . The Merck index. An encyclopedia of chemicals and drugs. , (1976).
  20. Miller, C. . Ion Channel Reconstitution. , (1986).
  21. Miller, C. Open-state substructure of single chloride channels from Torpedo electroplax. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 299 (1097), 401-411 (1982).
  22. Benz, R., Frohlich, O., Lauger, P., Montal, M. Electrical capacity of black lipid films and of lipid bilayers made from monolayers. Biochim. Biophys. Acta. 394 (3), 323-334 (1975).
  23. Priel, A., Gil, Z., Moy, V. T., Magleby, K. L., Silberberg, S. D. Ionic requirements for membrane-glass adhesion and giga seal formation in patch-clamp recording. Biophys. J. 92 (11), 3893-3900 (2007).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved