Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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-painting, dissolve 3% DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine) lipid (w:v) in a mixture of 2:8 n-decane and hexadecane ....

Results

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 bilay.......

Discussion

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.......

Materials

List of materials used in this article

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)