Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 (< 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 (> 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 °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 °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.

Protokół

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 (v:v). Stir overnight using a rotator.
3. Preparation of lipid solution for membrane formation:
   1. 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.

2. Formation of a PDMS Thin Film

1. 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 µm). Spread PDMS pre-polymer using a spin coater at 800 rpm for 10 sec to form a thin film.
2. 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 °C.
3. In order to make a square PDMS thin film, cut the polymerized PDMS thin film into 2 x 2 cm² squares. Use a 500 µm 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.

3. Chamber Fabrication and Assembly

1. 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.
2. Craft the chamber using a PTFE block with a CNC machine and follow the manufacturer's instructions.

4. Chamber Assembly

1. 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.
2. 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.

5. Formation of Membrane Precursor with Expedited Self-assembly Formation (MPES)

1. Using a pipette, deposit 0.5 µl of 0.1% DPhPC lipid mixed in 2:8 n-decane:hexadecane onto the aperture of the PDMS thin film assembled with the chamber.
2. Prior to use, store the chamber in a freezer or a refrigerator below 10 °C.

6. Membrane Formation and Verification

1. 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 < 10 min until the frozen membrane precursor thaws.
2. 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.
3. On the other side, place a digital microscope vertically with respect to the light source to observe BLM formation (magnify by 200X).
4. To confirm BLM formation, observe the center of the aperture where the color becomes brighter than the annulus.

7. Electrical Recording

1. For electrical measurement, prepare Ag/Cl electrodes using a 208 µm-thick silver wire and bleach in sodium hypochlorite for > 1 min. Place the Ag/Cl electrodes into each side of the chamber deep enough to be dipped into the buffer solution.
2. Connect the electrodes to the microelectrode amplifier. Using electrophysiology software, apply a ±10 mV triangular waveform across the membrane to acquire a square wave. Set applying voltage by clicking the arrows indicated on V_clamp (mV).
3. 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.

8. Ion Channel Incorporation

NOTE: Gramicidin A (gA) incorporation occurs spontaneously upon formation of BLM, as gA is added directly to the lipid solution.

1. 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).
2. 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.
3. After electrical data acquisition, filter the data with a low-pass Bessel filter at 100 Hz using an electrophysiology software.
4. 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.

Wyniki

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...

Dyskusje

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...

Materiały

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)