JP5544186B2 - Method for forming planar lipid bilayer membrane - Google Patents

Description

  The present invention relates to a technique for forming a planar lipid bilayer membrane used in fields such as electrophysiology, biotechnology, biochip, membrane protein analysis, drug discovery screening, biosensor, and DNA sequencing.

  The patch clamp method developed by Neher and Sakmann in 1976 has made it possible to understand the function of channel proteins. In this method, the channel protein existing on the living cell membrane is patched with a glass pipette, and the flow of ions accompanying the structural change is detected by amplifying the current. The behavior of the channel “one molecule” is captured in real time. It is a pioneer in single molecule measurement methods.

  On the other hand, a research technique called an artificial planar membrane method has recently been developed in which an artificial lipid bilayer membrane is developed in a hole of 100 μm to several tens of μm opened in a hydrophobic plastic film, and the current of an ion channel to be measured is measured. It is done actively. The feature of this method is that, unlike the patch clamp method, the experiment can be performed with a simple reconstruction system that artificially reproduces the cell membrane. As a result, the channel can be reconfigured in a system in which external conditions such as lipid type, solution salt concentration, composition, pH and the like are freely selected, so that experimental conditions can be clearly limited. Thus, the artificial planar membrane method is superior to the patch clamp method in that the basic mechanism of the ion channel can be studied more deeply by conducting experiments in a system that is as simple and controlled as possible. At present, many attempts have been made to apply the artificial planar membrane method to various devices and biosensors such as highly accurate single molecule detection, drug diagnosis, and DNA base identification.

  When the artificial planar membrane method is extended to the above measurement system, the instability of the planar membrane is a big problem. On the other hand, it has already been reported that the stability of the double membrane is improved by reducing the diameter of the hole that expands the membrane. It has also been reported that the reduction of the hole diameter reduces the membrane capacity and reduces current noise. Therefore, the reduction of the pore diameter is considered to be important in applying the artificial planar membrane method.

  For example, in the method for forming a planar lipid bilayer described in Patent Document 1, an oxide film is formed on the upper and lower surfaces of a silicon substrate, the oxide film is patterned, and micropores having a width of 50 to 100 μm are formed by reactive ion etching. Is forming. In addition, as described in Patent Document 2, a method is also known in which a stainless steel rod is pressed against a resin to form irregularities, and the convex portions are scraped off with a razor or the like to form minute holes. In Non-Patent Document 1, when a micropore is formed on a silicon wafer with a micropore having a diameter of 20 to 30 μm, and a lipid bilayer membrane is formed after performing a hydrophobic treatment, it is stable even when a voltage of 1 V is applied. It is disclosed that the obtained film was obtained.

International Publication No. 2005/071405 JP 2005-91305 A

Langmuir 2010, 26 (3), pp. 1949-1952

  However, as in Patent Document 1 and Non-Patent Document 1, an expensive and large-scale apparatus is required to form micropores using a microfabrication technique. In addition, if a silicon substrate is selected as the material for forming the micropores, there is a risk of cracking during the processing. When a resin is used as in Patent Document 2, there is no risk of such cracking, but in the method of forming irregularities as described in the same document and scraping with a razor, 0.1 to It is the limit to make a hole with a size of about 1 mm, and it is difficult to make a microhole of the order of several μm. Therefore, there is a demand for a method for forming a stable planar lipid bilayer membrane by a simple operation.

The present inventors have succeeded in providing micropores in a resin film by a very simple and inexpensive method. Then, it was found that when a planar lipid bilayer membrane was formed in the micropores, a very stable membrane was obtained and channel current recording was possible. The gist of the present invention is as follows.
(1) A plane including heating a sharpened metal needle and pressing it against the resin film to form micropores in the resin film, and forming a lipid bilayer in the micropores provided in the resin film Formation method of lipid bilayer membrane.
(2) The method according to (1), wherein the micropore has a width of less than 50 μm.

(3) The resin is selected from fluorine resin, polyethylene, polypropylene, polyvinyl chloride, polystyrene, polyvinyl acetate, ABS resin, AS resin, acrylic resin, polyethylene terephthalate and polybutylene terephthalate, (1) or ( The method according to 2).
(4) The method according to any one of (1) to (3), wherein the metal needle is a needle made of a metal selected from tungsten, platinum, a platinum alloy, and nickel sharpened by electrolytic polishing.

  According to the method for forming a planar lipid bilayer membrane of the present invention, a stable planar lipid bilayer membrane can be formed at a low cost by a simple operation. Since the lipid bilayer membrane that can be formed by the method of the present invention is developed into very small pores, it is excellent in mechanical properties (shock resistance). The method for forming a planar lipid bilayer membrane of the present invention is very useful in experimental studies and applications using lipid bilayer membranes.

It is the figure which showed an example of the method of this invention which forms a micropore in a resin film. It is the microscope image observed when forming a micropore by the method shown in FIG. The shadow of the needle is reflected through the resin film. A to C, E, and G to H are SEM images of micropores formed in the resin film. D and F are SEM images of the needle tip. I is a diagram for explaining a cross-sectional view of an assumed minute hole. It is a figure explaining the structure of the cell for lipid bilayer membrane formation. It is a figure explaining the state of the apparatus at the time of an electric current measurement. It is a result of channel current recording. A to C are SEM images of micropores formed in a polyethylene film having a thickness of 15 μm, and D to E are formed in a polypropylene film having a thickness of 15 μm. A and B are SEM images of micropores formed in a PET film having a thickness of 13 μm, and C to E are formed in a PTFE film having a thickness of 13 μm. A to C are SEM images of micropores formed in an FEP film having a thickness of 25 μm, D and E of 50 μm and F and G of 125 μm.

  The method of the present invention includes forming a lipid bilayer membrane in micropores provided in a resin film. Formation of the lipid bilayer membrane can be performed by any known method. Examples of the method for forming the lipid bilayer membrane include a painting method, a bonding method, a vesicle fusion method, and a method for directly capturing cells.

  In the painting method, lipid molecules are dispersed in a decane solvent, and the solution is applied with a brush or pipette so as to close the micropores. If it does so, it will be in the state where the lipid solution became the state pinched | interposed into the aqueous solution from the both sides of a micropore, and it is a principle that a lipid molecule spontaneously forms a bilayer by hydrophobic interaction. The laminating method applies a monomolecular film forming technique called a Langmuir-Blodgette method to planar film formation. First, a monolayer of lipid molecules is formed at the gas-liquid interface between two solutions sandwiching micropores. Next, the interface of the monomolecular film is raised so that the monomolecular films on both sides are bonded together in the micropores. This is the principle by which the intended planar lipid bilayer is formed. In the vesicle fusion method, lipid molecules are added and dispersed in a solution to produce a spherical endoplasmic reticulum (called a vesicle) in which the lipid bilayer is rounded in the solution. In this method, the lipid bilayer is transferred to the substrate surface by the interaction between the vesicle and the substrate surface.

  The resin film that can be used in the method of the present invention is not particularly limited, but is preferably a film made of a thermoplastic resin. Examples of such thermoplastic resins include fluororesin, polyethylene, polypropylene, polyvinyl chloride, polystyrene, polyvinyl acetate, ABS resin, AS resin, acrylic resin, polyethylene terephthalate and polybutylene terephthalate. Here, the fluororesin is a group of resins known by the general name of Teflon (registered trademark). In this specification, the fluororesin includes PTFE (polytetrafluoroethylene) and PFA (tetrafluoroethylene / perfluoroalkyl vinyl ether). Polymer), FEP (tetrafluoroethylene / hexafluoropropylene copolymer), ETFE (tetrafluoroethylene / ethylene copolymer), PVDF (polyvinylidene fluoride) PCTFE (polychlorotrifluoroethylene) and ECTFE (chlorotrifluoro) Ethylene / ethylene copolymer). As the thermoplastic resin, various resins can be used from those having a melting point of 100 ° C. or lower to those exceeding 400 ° C. Among the above resins, fluorine resin, polyethylene, polypropylene, and polyethylene terephthalate are particularly preferable, and PTFE, FEP, ETFE, polypropylene, and polyethylene terephthalate are particularly preferable. The thickness of the resin film is preferably in the range of 1 to 500 μm, more preferably in the range of 5 to 200 μm, and particularly preferably in the range of 10 to 150 μm.

  The width of the micropores provided in the resin film is preferably less than 50 μm, particularly less than 20 μm, particularly less than 5 μm. The smaller the micropore size, the greater the stability of the formed planar lipid bilayer. In addition, when the size of the micropore is very small, there is a possibility that when the lipid bilayer membrane is formed by the painting method, the solvent may block the hole and the membrane may not be formed. In such a case, the painting method is used. What is necessary is just to form a lipid bilayer membrane by methods other than.

  In the method of the present invention, the micropores on the resin film are formed by heating a sharpened metal needle and pressing it against the resin film. In this specification, “pressing” means bringing at least a part of a metal needle into contact with the resin film, or penetrating the resin film with the metal needle. The micropores on the resin film obtained by the method of the present invention have a very sharp edge, which is advantageous for the development of the lipid bilayer membrane.

  The material of the metal needle is not particularly limited as long as it does not melt when heated, and examples thereof include platinum alloys such as tungsten, platinum, platinum-iridium, and nickel. The sharpening of the metal needle can be performed by any polishing method such as electrolytic polishing, chemical polishing, and mechanical polishing, and electrolytic polishing is particularly preferable. Techniques for sharpening the needle by electropolishing are known to those skilled in the art, for example, in the field of STM probes. Sharpening a metal needle by electrolytic polishing is generally performed by immersing the tip of the needle base material in an electrolytic solution and etching the probe by an electrochemical reaction to control the shape and sharpen the most advanced part. That means.

  The heating of the metal needle can be performed by any method such as bringing a heating element such as a nichrome wire into contact therewith. The heating is preferably performed so that the temperature of the metal needle is a temperature near the melting point of the resin, for example, the melting point of the resin is ± 10 ° C., ± 5 ° C., ± 3 ° C., or ± 1 ° C. When the nichrome wire is brought into contact with the needle and heated, the temperature of the needle can be adjusted by the amount of current passed through the nichrome wire. The amount of current may be appropriately adjusted according to the desired hole size. For example, when the thickness of the nichrome wire is 0.2 mm and the thickness of the metal needle is 0.5 mm, and the nichrome wire is wound around the sharp portion of the metal needle several times (for example, twice), the current amount is about several A For example, about 0.5 to 2.5 A is appropriate.

  When the heated needle is pressed against the resin film, it is preferable that there is a support such as a glass plate on the side opposite to the side on which the needle of the resin film hits. When there is a glass plate, the effect of preventing the needle from being stuck too much and the hole from becoming too large, and the effect of rounding the edge of the hole are produced. When the pore edge is smooth, the formed lipid membrane is more stable. Further, the fact that the cross section of the edge of the pore is sharp also contributes to the stability of the formed lipid membrane. Furthermore, for example, when a glass plate provided with a transparent conductive film (ITO) is used, the contact of the needle with the glass plate can be detected electrically, and the control of the needle becomes easier. However, even without a support such as a glass plate, it is possible to provide micropores having a width of less than 50 μm, less than 20 μm, or less than 5 μm. In addition, when the support is made of a material having a lower hardness than the glass plate, for example, a resin plate, the needle can be prevented from being damaged by contact with the support.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to these Examples.
[Example 1]
(1) Formation of micropores in fluororesin (ETFE) film A silver plate (25 mm square, thickness 0.5 mm) having a 0.4 mm hole in the center was prepared. The silver plate was preliminarily soaked overnight in a 1M KClO ₂ aqueous solution and then subjected to Ag-AgCl treatment. An ETFE film (20 mm square, thickness 12 μm) was attached to this silver plate with an epoxy adhesive, and it was turned over and placed on a glass plate. This was set on a stage fixed on the objective lens of an inverted microscope (Olympus IX-50). A tungsten wire (φ = 0.5 mm) was sharpened by electrolytic polishing (10% KOH, 60 Hz, 20 Vp-p) to obtain a needle for making a hole. A nichrome wire (thickness 0.2 mm) was wound around the needle several times, and electricity was passed through the nichrome wire so that the needle could be heated. The degree of heating of the needle was adjusted by the magnitude of the current passed through the nichrome wire. . While the needle was fixed and the position of the needle was confirmed with a microscope (FIG. 2), the needle was pressed against the ETFE film using a microscope aiming device to make a hole. FIG. 1 schematically shows the method for forming the micropores described above.

  FIG. 3 shows SEM images of micropores created under various conditions. (A) is a SEM image of ETFE in which an unheated needle is pressed. It can be seen that the deformation of ETFE is insufficient and micropores are not formed. On the other hand, when a heated needle was used (B), a microhole having a diameter of 3 μm was formed, and the periphery thereof had a beautiful conical shape. This was thought to be due to the fact that ETFE was heat-melted to reflect the shape of the needle tip. However, when the needle was heated too much, no beautiful conical micropores were formed (C).

  The opening angle of the microhole (E) created using the needle (D) having a high degree of sharpness was smaller than that of the microhole (G) processed by the needle (F) having a low degree of sharpness. From this, it is considered that the shape of the microhole changes depending on not only the temperature of the needle tip but also the shape of the needle tip used.

  From the above, it is considered that the shape of the micropores can be freely controlled to some extent by adjusting the temperature of the needle tip and the sharpness thereof. It is considered that a conical minute hole having a large opening angle can be easily processed by using a needle having a low sharpness at an optimum temperature. Such microholes have low stray capacitance and access resistance and are considered ideal for channel current measurement.

  Further, the shape of the microhole on the glass plate side was flat unlike the side to which the needle was applied (HEM: SEM image of the G microhole on the glass plate side). Therefore, it is thought that the edge part of the created microhole is very sharp like the microhole obtained by the conventional method of scraping with a razor (I: sectional view of assumed microhole). If the edges of the micropores are sharp, lipid thinning is promoted, so it is considered possible to develop a lipid bilayer membrane using a painting method even in micropores with a diameter of 3 μm.

(2) Cell for forming a lipid bilayer membrane Two silicon O-rings (inner diameter 11.0 mm, thickness 2.6 mm) and a new silver plate (25 mm square, thickness 0.8 mm) were prepared. The O-ring, the silver plate on which the ETFE film of (1) above was pasted, and the O-ring were stacked in this order on the silver plate. Holes (φ2 mm) were made in advance on the four sides of the two silver plates, and each was fixed with M2 screws and screws. FIG. 4 schematically shows the above-described cell creation method. Chambers are provided on each of the cis side (upper side) and the transformer side (lower side) by a silicon O-ring.

(3) Formation of artificial lipid bilayer A planar lipid bilayer was formed using the cell of (2) above. The trans and cis chambers of the cell were pre-filled with base solution (2M KCl, Hepes 10 mM at pH 7.4). Formation of the lipid bilayer membrane was performed by a painting method. As a lipid solution, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DphPC) (Avanti polar lipid) is dispersed in n-decane (Wako Pure Chemical Industries), and the final concentration is DphPC 20 mg / ml n- What was decanted was used. A very small amount of lipid solution was attached to the tip of a micropipette (for 20 μL), and excess solvent was blown off in pure water. Thereafter, bubbles were formed at the tip of the pipette in the base solution on the cis side, and the lipid solution was applied by passing through micropores provided in the ETFE film. When a large amount of lipid solution was applied to the micropores and the film formation did not progress, air bubbles were blown with a new pipette to remove the excess lipid solution and promote thinning. After the formation of lipid duplex was confirmed, the cis side was replaced with 2M KCl solution.

(4) Current measurement Gramicidin channels were introduced into the planar lipid bilayer provided in the ETEF micropores with a diameter of 3 μm in (3) above, and current measurement was performed with +100 mV applied using a silver plate as a ground electrode. The final concentration of gramicidin was 0.1 nM. For the measurement of current, a patch clamp amplifier (CEZ-2400, Nihon Kohden) was used and digitized at a sampling frequency of 1 kHz. Data were analyzed using commercial software (pClamp 10.2, Axon). FIG. 5 schematically shows the state of the apparatus during current measurement. FIG. 6A shows the result of current measurement.

  From the result of the current measurement shown in FIG. 6A, a conductance step of 3 pA changing every few seconds was observed, and the behavior of the gramicidin channel was captured. This indicates that a lipid bilayer could be formed in 3 μm ETFE micropores. When the micropore diameter is as small as several μm, it is considered difficult to form a lipid bilayer using the painting method without draining the solvent and thinning the lipid. It became clear that a double membrane could be formed. Further, the obtained current measurement result clearly shows a change in current even when compared with the non-patent document 1 cited above, for example.

(5) Membrane stability test A lipid bilayer membrane was formed in 3 μm ETFE micropores, and what changes occurred when a membrane potential of 1000 mV was applied was examined. The result is shown in FIG. 6B.

  When +1000 mV was applied with +100 mV initially applied, the membrane current reached the measured saturation value. However, when the membrane potential was returned to +100 mV, the current recovered from the saturation value. Since the change in conductance derived from the gramicidin channel was recorded before applying a membrane potential of +1000 mV, it was clear that a lipid bilayer was formed. In addition, when the double membrane is irreversibly destroyed due to the application of +1000 mV, the current value recovery phenomenon cannot occur. Therefore, even if the double membrane is applied with a membrane potential of 1000 mV, it is not destroyed. It has been found that it continues to exist stably. It is known that a double membrane formed in a micropore of about several tens of μm is irreversibly destroyed when 300 to 400 mV is applied. That is, this result shows that the double membrane formed in the micropores on the order of several μm is overwhelmingly stable.

[Example 2]
It was verified by the method of Example 1 (1) whether micropores could be formed in resin films of other materials. 7A to 7C are SEM images of micropores formed in a polyethylene film having a thickness of 15 μm, and D to E being formed in a polypropylene film having a thickness of 15 μm. 8A and 8B are SEM images of micropores formed on a 13 μm thick PET film, and C to E on a 13 μm thick PTFE film. In the figure, the value of the current passed through the nichrome wire for heating the needle is shown. “Using ITO glass” means using a glass plate with ITO (transparent conductive film) on the surface as a glass plate to support the resin film, and opening a hole while electrically detecting contact between the glass plate and the needle. It means that. “Glass not used” means that a hole is formed without using a glass plate that supports the resin film.

  As can be seen from these SEM images, micropores having a width of several μm to 100 μm could be formed by the method of the present invention in any film. 9A to 9C are SEM images of micropores formed in an FEP film having a thickness of 25 μm, D and E of 50 μm, and F and G of 125 μm. It was found that micropores having a width of several μm to several tens of μm can be formed by the method of the present invention in any thickness of FEP film. It is expected that a stable planar lipid bilayer is formed by utilizing the micropores formed in these resin films.

Claims (4)

In a state where a sharpened metal needle is heated by bringing a heating element into contact with the resin film, the resin film is pressed against the resin film to provide micropores with a width of less than 5 μm , and provided in the resin film. A method for forming a planar lipid bilayer, comprising forming a lipid bilayer in a micropore. The method according to claim 1, wherein the heating element is a nichrome wire carrying a current. The method according to claim 1 or 2 , wherein the metal needle is a needle made of a metal selected from tungsten, platinum, a platinum alloy and nickel sharpened by electropolishing. The resin according to any one of claims 1 to 3, wherein the resin is selected from fluororesin, polyethylene, polypropylene, polyvinyl chloride, polystyrene, polyvinyl acetate, ABS resin, AS resin, acrylic resin, polyethylene terephthalate and polybutylene terephthalate . 2. The method according to item 1 .

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