Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Summary

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

Abstract

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

Introduction

As essential frameworks of cell membranes, the interior of cells are separated from the exterior by a lipid bilayer system. This system consists of amphiphilic phospholipids, which are composed of hydrophilic phosphoric ester “heads” and hydrophobic fatty acids “tails”. Due to remarkable fluidity and self-assembly ability of lipid bilayers in aqueous environment^1,2, artificial lipid bilayers can be formed using simple methods^3,4. Various types of membrane proteins, such as ion channels, membrane receptors and enzymes, have been incorporated into the artificial lipid bilayer to mimic and study the functions of cell membranes^5,6. More recently, lipid bilayers have been doped with nanomaterials (e.g., metal nanoparticles, graphene, and carbon nanotubes) to form functional hybrid membranes^{7,8,9,10,11,12,13}. A widely used method for forming such hybrid membranes involves the formation of doped lipid vesicles, which contain hydrophobic materials such as modified Au-nanoparticles⁷ or carbon nanotubes¹¹, and the resulting vesicles are then fused into planar supported lipid bilayers. However, this approach is complex and time-consuming, which limits the potential uses of such hybrid membranes.

In this work, lipid membranes were doped with organic molecules to produce hybrid lipid membranes that formed at the water/air interface by self-assembly. This protocol involves three steps: preparation of the mixed solution, formation of a hybrid membrane at the water/air interface, and the transfer of the membrane onto a Si substrate. Compared with other previously reported methods, the method described here is simpler and does not require sophisticated instrumentation. Using this method, air-stable hybrid lipid membranes with a larger area can be formed in a shorter time. The nanomaterial used in this study is a semiconducting organic molecule, copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc), which is widely used in a number of applications, including solar cells, photodetectors, gas sensors and catalysis^14,15. CuPc, a small organic molecule with a planar structure, has a high affinity for the “tails” of phospholipids duo to its hydrophobic characteristics. Other groups have reported that CuPc molecules can self-assemble on single-crystal surfaces with the formation of highly ordered structures^16,17. Therefore, it is highly possible that the CuPc molecules could be incorporated into the lipid bilayers through self-assembly.

We provide a detailed description of the procedures used to form membranes and provide some suggestions for smoothly implementing this procedure. In addition, we present some presentative results of the hybrid lipid membranes, and discuss potential applications of this method.

Protocol

1. Preparation of a hybrid solution

1. Wash four 4 mL disposable glass vials and screw caps (with PTFE coated seals) in an ultrasonic bath for 10 min in distilled water (purified with a filtration system), followed by ethanol and chloroform, respectively. Dry the glass vials and caps in a stream of nitrogen gas.
2. In an anaerobic glove box, prepare a CuPc stock solution (10 mg/mL) in a washed glass vial by dissolving powdered CuPc in chloroform.
3. Filter the CuPc solution through a 0.2 µm polytetrafluoroethylene (PTFE) membrane.
4. Store the filtered solution in a washed glass vial filled with nitrogen, and seal the vial with parafilm.
5. Take the purchased 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) chloroform solution (25 mg/mL) out of the -80 °C refrigerator and allow it to warm to room temperature.
6. Stir the DPhPC solution using a vortex mixer at 2300 rpm for 10 s.
7. Rinse a glass micro-syringe with chloroform for 5 times.
8. Transfer 200 µL of the DPhPC chloroform solution into a pre-washed glass vial using the washed syringe. Evaporate the solvent in the vial with a gentle stream of nitrogen.
9. Rinse a glass micro-syringe 5 times with chloroform.
10. Add 202.6 µL of chloroform to the glass vial with DPhPC using the cleaned syringe.
11. Add 47.4 µL of filtered 10 mg/mL CuPc solution into the DPhPC solution. The molar ratio of DPhPC to CuPc should be 10:1.
12. Rinse the glass micro-syringe 5 times with chloroform.
13. Add 250 µL of hexane to the solution using the syringe. The final concentration of the solution should be 10 mg/mL.
14. Mix the prepared solution using a vortex mixer at 2300 rpm for 10 s.
15. Filter the CuPc solution through a 0.2 µm polytetrafluoroethylene (PTFE) membrane.
16. Seal the glass vial with parafilm. Put it in to a grip sealed bag filled with nitrogen and place the grip sealed bag in a freezer at -20 °C.
    NOTE: After step 1.13, the DPhPC and CuPc were dissolved in a mixed solvent, which was composed of chloroform and hexane (volume ratio of 1:1). In addition, the molar ratio of DPhPC to CuPc is not limited to 10:1. With a constant concentration of lipids (10 mg/mL), different molar ratios can be used. According to previous experimental results, a range from 10:1 to 3:1 is preferred for forming a high-quality hybrid lipid membrane.

2. Formation of a hybrid membrane at the water/air interface

1. Cut Si substrates (3 cm x 3 cm) from a Si wafer.
2. Clean the 3 cm x 3 cm Si substrates in an ultrasonic bath for 10 min in purified water, followed by ethanol and then chloroform. Treat the Si substrate with an O₂ plasma for 5 min to remove adsorbed organic materials from the surface and to improve hydrophilicity.
3. Wash a Teflon beaker with an inner diameter of 7.5 cm with flowing purified water for 3 min.
4. Put the cleaned Si substrate in the washed PTFE beaker. The substrate is tilted at an angle of 30° to the horizontal.
5. Pour a sufficient amount of purified water into the Teflon beaker until the entire Si substrate is submerged.
6. Take prepared hybrid solution out of freezer and allow it to warm to room temperature.
7. Stir the hybrid solution using a vortex mixer at 2300 rpm for 15 s.
8. Rinse a glass micro-syringe (50 µL) 5 times with chloroform.
9. Drop 3-5 µL of the hybrid solution onto the water surface using the syringe to form a floating hybrid lipid membrane.
   NOTE: When the solution is dropped, it is important to hold the droplet close to (less than 1 cm) the water surface. It should also be noted that a single-layer hybrid membrane is not visible to the naked eye, but the multi-layer hybrid membrane appears as thin, blue-colored film. In order to transfer the multi-layer hybrid membrane to the Si substrate, it is important to drop the mixed solution as close as possible to the Si substrate.

3. Transferring the membrane onto a Si substrate

1. After the evaporation of organic solvent (it will take less than 2 seconds), lower the water level at a rate of 3 mm/min by pumping out the water through a rubber tube which is driven by a peristaltic pump, to transfer the floating hybrid membrane onto the Si substrate.
2. After the transfer process is complete (about 5 min), place the Si substrate on a cleanroom wiper, and allow all of residual water to evaporate.
   NOTE: Lowering the water level at such a low rate serves to minimize the turbulence of water and to protect the membrane.

Results

The as-formed membrane has a uniform light blue color due to the presence of CuPc molecules. The area of the colored membrane is normally several square centimeters. In Figure 1A and Figure 1B, we show a microscopic image and an atomic force microscope (AFM) image (including a height profile) of the hybrid lipid membrane on a Si substrate. In the AFM image, the membrane in the upper left is thick, with a thickness of 79.4 nm and that at the lower right is thin, ...

Discussion

In the precursor solution of the hybrid membrane, a mixed organic solvent (chloroform and hexane) rather than pure chloroform is used to dissolve lipids and CuPc. If pure chloroform is used, the density of the precursor solution would be higher than water. Therefore, it is highly likely that the solution would sink to the bottom of water rather than spread on water surface. Adding hexane, a low density solvent, to the precursor solution, ensures that the solution will float on the water surface and form a uniform hybrid ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chloroform	Wako Chemicals	033-08631
CuPc	Sigma-Aldrich	423165
DPhPc	Avanti Polar Lipids	850356C
Glass vials with screw cap	Nichiden-Rike Glass Co., Ltd	6-29801
Hexane	Wako Chemicals	084-03421
Membrane filters	Merck Millipore Ltd.	R8CA42836
Micro-syringe	Hamilton	80530
Peristaltic pump	Tokyo Rikakikai Co., Ltd.	11914199
Vortex mixer	Scientific Industries, Inc.	SI-0286