JP6964878B2 - Lipid bilayer property testing method, drug screening method, and lipid bilayer property analysis system - Google Patents

Description

The present invention relates to a method for examining the characteristics of a lipid bilayer membrane, a method for screening a drug, and a system for analyzing the characteristics of a lipid bilayer membrane.

A cell is generally covered with a cell membrane formed of a lipid bilayer membrane, which forms a boundary between the cell and the outside world.

The cell membrane not only has a function of preventing the substance from freely diffusing from the outside world into the cell, but also has a function of selectively taking in the substance from the outside world into the cell. Such functions of the cell membrane are expressed by lipids forming the cell membrane and various membrane proteins embedded in the cell membrane.

In recent years, with the development of cell membrane research, the development of a technique for artificially producing a lipid bilayer membrane and the development of a technique for inspecting and analyzing the characteristics of the lipid bilayer membrane have been required (for example,). See Patent Document 1 and Non-Patent Document 1).

Japanese Unexamined Patent Publication No. 2014-100672 (published on June 5, 2014)

Masayuki Iwamoto & Shigetoshi Oiki, Sci. Rep. 5, 9110, 2015

However, conventional techniques for inspecting and analyzing the properties of lipid bilayers (eg, physical properties such as membrane thickness and membrane tension) are from the perspective of simply inspecting or analyzing the properties of lipid bilayers. It was not enough from the beginning, and there was room for further improvement.

An object of an embodiment of the present invention is to provide an inspection method capable of inspecting the characteristics of a lipid bilayer membrane and an analysis system capable of analyzing the characteristics of the lipid bilayer membrane. Another object of the other embodiment of the present invention is to provide a method for screening a drug capable of screening a drug that changes the properties of a lipid bilayer membrane.

One embodiment of the present invention includes the following configurations.
[1] A method for inspecting the characteristics of a lipid bilayer membrane formed on a contact surface between a first bubble covered with a lipid monolayer and a second bubble covered with a lipid monolayer. Internal pressure detection step for detecting the internal pressure ΔP ₁ of the bubble and the internal pressure ΔP _{2 of the second bubble, the major axis R 11} and the minor _{axis R 21 of} the first bubble, and the major axis R ₁₂ and the minor axis of the second bubble. A major / minor axis detection step for detecting the diameter R _{22 , the contact angle θ 1} generated at the interface between the lipid double membrane and the lipid single membrane of the first bubble, and the lipid double membrane and the second bubble. _{A contact angle detection step for detecting the contact angle θ 2} generated at the interface with the lipid single membrane, and a tension calculation step for calculating the tension γ of the lipid double membrane from the following equations (1) to (5). A method for inspecting the properties of a lipid bilayer membrane, which comprises.

Equation (1) ΔP ₁ = γ ₁ × (1 / R ₁₁ + 1 / R ₂₁ )
Equation (2) ΔP ₂ = γ ₂ × (1 / R ₁₂ + 1 / R ₂₂ )
Equation (3) γ _b1 = γ ₁ × cos θ ₁
Equation (4) γ _b2 = γ ₂ × cos θ ₂
Equation (5) γ = γ _b1 + γ _b2
(Here, γ ₁ is the tension of the lipid single membrane of the first bubble, γ ₂ is the tension of the lipid single membrane of the second bubble, and γ _b1 is the tension of the first lipid bilayer membrane. The tension of the monolayer on the bubble side of the _{above, and γ b2} is the tension of the monolayer on the side of the second bubble in the lipid bilayer).
[2] Further, a capacitance detection step for detecting the capacitance C of the lipid bilayer membrane, an area detection step for detecting the area A of the lipid bilayer membrane, and the lipid bilayer from the following formula (6). The method for inspecting the characteristics of a lipid bilayer film according to [1], which comprises a film thickness calculation step of calculating the thickness T of the heavy film.

Equation (6) T = ε _m × ε ₀ × A / C
(Here, ε _m is the relative permittivity of the lipid bilayer film, and ε ₀ is the permittivity of the vacuum.)
[3] Characteristics of the above-mentioned lipid double membrane using the lipid double membrane formed on the contact surface between the first bubble covered with the lipid single membrane and the second bubble covered with the lipid single membrane. A method for screening a drug to be changed, which is a drug introduction step of introducing a drug into the lipid double membrane, a tension calculation step of calculating the tension γ of the lipid double membrane after the introduction of the drug, and a tension calculation step. The first drug determination step of determining a drug by comparing the tension γ with a reference value is included, and the tension calculation step includes the internal pressure ΔP _{1 of the first} bubble and the internal pressure ΔP _{2 of the second bubble.} The major / minor axis detection step for detecting the major axis R ₁₁ and the minor _{axis R 21 of} the first bubble, and the major axis R ₁₂ and the minor _{axis R 22 of} the second bubble, and the above-mentioned lipid. _{The contact angle θ 1} generated at the interface between the double membrane and the lipid monolayer of the first bubble, and the contact angle θ 1 generated at the interface between the lipid double membrane and the lipid monolayer of the second bubble. _{2. A} method for screening a drug, which comprises a step of detecting a contact angle for detecting 2, and is a step of calculating the tension γ of the lipid double membrane from the following formulas (1) to (5).

Equation (1) ΔP ₁ = γ ₁ × (1 / R ₁₁ + 1 / R ₂₁ )
Equation (2) ΔP ₂ = γ ₂ × (1 / R ₁₂ + 1 / R ₂₂ )
Equation (3) γ _b1 = γ ₁ × cos θ ₁
Equation (4) γ _b2 = γ ₂ × cos θ ₂
Equation (5) γ = γ _b1 + γ _b2
(Here, γ ₁ is the tension of the lipid single membrane of the first bubble, γ ₂ is the tension of the lipid single membrane of the second bubble, and γ _b1 is the tension of the first lipid bilayer membrane. The tension of the monolayer on the bubble side of the _{above, and γ b2} is the tension of the monolayer on the side of the second bubble in the lipid bilayer).
[4] Further, a film thickness calculation step of calculating the thickness T of the lipid bilayer membrane after the introduction of the drug, and a second drug for determining the drug by comparing the film thickness T with a reference value. The determination step is included, and the film thickness calculation step includes a capacitance detection step for detecting the capacitance C of the lipid bilayer membrane and an area detection step for detecting the area A of the lipid bilayer membrane. The method for screening a drug according to [3], which is a step of calculating the thickness T of the lipid bilayer membrane from the following formula (6).

Equation (6) T = ε _m × ε ₀ × A / C
(Here, ε _m is the relative permittivity of the lipid bilayer film, and ε ₀ is the permittivity of the vacuum.)
[5] This is an analysis system for the characteristics of the lipid bilayer formed on the contact surface between the first bubble covered with the lipid monolayer and the second bubble covered with the lipid monolayer. A first internal pressure detecting unit that detects the internal pressure ΔP _{1 of the bubble, a second internal pressure detecting unit that detects the internal pressure ΔP 2} of the second bubble, and a major axis R ₁₁ and a minor _{axis R 21 of the first bubble.} , And the major axis R ₁₂ and minor _{axis R 22 of the second bubble are detected, and the contact angle θ 1} generated at the interface between the lipid bilayer and the lipid single film of the first bubble. _{And the major / minor axis-contact angle detection unit that detects the contact angle θ 2} generated at the interface between the lipid bilayer and the lipid single film of the second bubble, and the following equations (1) to (5). A tension calculation unit for calculating the tension γ of the lipid bilayer from)), which is a characteristic analysis system for the lipid bilayer.

Equation (1) ΔP ₁ = γ ₁ × (1 / R ₁₁ + 1 / R ₂₁ )
Equation (2) ΔP ₂ = γ ₂ × (1 / R ₁₂ + 1 / R ₂₂ )
Equation (3) γ _b1 = γ ₁ × cos θ ₁
Equation (4) γ _b2 = γ ₂ × cos θ ₂
Equation (5) γ = γ _b1 + γ _b2
(Here, γ ₁ is the tension of the lipid single membrane of the first bubble, γ ₂ is the tension of the lipid single membrane of the second bubble, and γ _b1 is the tension of the first lipid bilayer membrane. The tension of the monolayer on the bubble side of the _{above, and γ b2} is the tension of the monolayer on the side of the second bubble in the lipid bilayer).
[6] Further, a capacitance detection unit that detects the capacitance C of the lipid bilayer membrane, an area detection unit that detects the area A of the lipid bilayer membrane, and the lipid bilayer from the following formula (6). The analysis system for the characteristics of a lipid bilayer film according to [5], which comprises a film thickness calculation unit for calculating the film thickness T of the heavy film.

Equation (6) T = ε _m × ε ₀ × A / C
(Here, ε _m is the relative permittivity of the lipid bilayer film, and ε ₀ is the permittivity of the vacuum.)

According to one embodiment of the present invention, the characteristics of the lipid bilayer membrane can be easily inspected or analyzed. Further, according to another embodiment of the present invention, a drug that changes the properties of the lipid bilayer membrane can be easily screened.

It is the schematic which shows the structure of the analysis system which concerns on one Embodiment of this invention. It is the schematic which shows the structure of the analysis system which concerns on one Embodiment of this invention. (A) It is a schematic diagram which shows the structure of the lipid bilayer membrane forming part in the analysis system which concerns on one Embodiment of this invention, and is the cross-sectional view of the cross section perpendicular to the lipid bilayer membrane. (B) is a cross-sectional view taken along the line AA of FIG. 3 (a). It is a block diagram which shows the analysis system which concerns on one Embodiment of this invention. It is a block diagram which shows the analysis system which concerns on one Embodiment of this invention. (A) It is a figure which shows the change of the shape of two bubbles and a lipid bilayer membrane with the reduction of the maintenance pressure of a bubble. (B) is a schematic view schematically showing (a) of FIG. It is a graph which shows the change of the ion current with the decompression of the maintenance pressure of a bubble.

An embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the configurations described below, and various modifications can be made within the scope of the claims. The present invention also includes embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples within the technical scope of the present invention. In addition, all the academic documents and patent documents described in the present specification are incorporated as references in the present specification. Further, unless otherwise specified in the present specification, "A to B" representing a numerical range is intended to be "A or more (including A and larger than A) and B or less (including B and smaller than B)".

[1. Features of the present invention]
In the inspection method according to the embodiment of the present invention, in order to measure the characteristics of the lipid bilayer membrane (specifically, the tension which is a physical characteristic), the internal pressure of the bubble, the major axis and the minor axis of the bubble, and the major axis and the minor axis of the bubble, and The contact angle between the lipid single membrane and the lipid bilayer membrane covering the bubble may be measured (detected) once.

Further, in the inspection method according to the embodiment of the present invention, in order to measure the characteristics of the lipid bilayer membrane (specifically, the film thickness which is a physical characteristic), the capacitance of the lipid bilayer membrane is used. The area and the area may be measured (detected) once.

Therefore, in the inspection method according to the embodiment of the present invention, the tension and the film thickness, which are the physical characteristics of the lipid bilayer membrane having a certain composition, are calculated extremely easily and in a short time (for example, calculated in real time). be able to.

Further, in the inspection method according to the embodiment of the present invention, not only the tension of the lipid bilayer membrane but also the tension of each of the two monolayers derived from the bubble constituting the lipid bilayer membrane can be calculated. .. The monolayer is a portion of the lipid monolayer of the bubble that constitutes the lipid bilayer.

[2. Method for inspecting the properties of lipid bilayer membranes]
One embodiment of the present invention provides a method for inspecting the characteristics of a lipid bilayer membrane, which includes an internal pressure detection step, a major / minor axis detection step, a contact angle detection step, and a tension calculation step.

The method for inspecting the characteristics of the lipid bilayer membrane according to the embodiment of the present invention can easily inspect the characteristics of the lipid bilayer membrane, particularly the tension of the lipid bilayer membrane, by having the above-mentioned configuration. It has the advantage of.

In the present specification, the "method for inspecting the properties of the lipid bilayer membrane" is also simply referred to as the "inspection method".

(Lipid bilayer formation step)
In the inspection method according to the embodiment of the present invention, the lipid bilayer membrane to be inspected is formed on the contact surface between the first bubble covered with the lipid monolayer and the second bubble covered with the lipid monolayer. It is a lipid bilayer membrane that is formed. Therefore, the present inspection method may include a lipid bilayer forming step of forming a lipid bilayer.

In the present lipid bilayer film forming step, the method for forming the lipid bilayer membrane is not particularly limited, and a conventionally known method can be used. For example, a method having the following (1) and (2) can be mentioned:
(1) At least two bubbles (also referred to as droplets) composed of an aqueous solution are formed in an oil (also referred to as an oil phase). Here, in the bubble composed of the aqueous solution formed in the oil, after the formation of the bubble, the desired lipid contained in the oil or contained in the aqueous solution in the bubble moves to the interface between the bubble and the oil. By doing so, a lipid monolayer containing the lipid is formed at the interface. As a result, the bubble becomes a bubble covered with a single lipid membrane;
(2) Next, by contacting at least two bubbles (that is, the first bubble and the second bubble) among the plurality of bubbles covered with the lipid monolayer, the lipid bilayer is brought to the contact surface of those bubbles. A film is formed.

Here, the aqueous solution constituting the bubble may be an electrolyte solution. When the aqueous solution is an electrolyte solution, the bubble can also be said to be an electrolyte solution bubble.

Examples of the method for forming a bubble composed of an aqueous solution in oil include, but are not limited to, the following methods (1) to (3): (1) Dropping an aqueous solution into oil using a micropipette or a syringe. (Hereinafter, also referred to as a dropping method); (2) A method in which the tip of a micropipette or a syringe is inserted into oil and an aqueous solution is injected into the oil (hereinafter, also referred to as an injection method); and (3). (I) At least two microchannels were immersed in oil so that the openings of the microchannels were close to each other and the openings of the microchannels were filled in the bubble forming channel. A method of arranging in a state, (ii) then discharging an aqueous solution from each opening to form a bubble composed of the aqueous solution in the oil, and then stopping the release of the aqueous solution from the opening.

As a method of bringing the two bubbles into contact with each other, for example, a method of manipulating the two bubbles with a manipulator, and a method of forming one bubble and then forming the other bubble on the bubble and using gravity 2 Examples thereof include, but are not limited to, a method of bringing two bubbles into contact with each other and a method of automatically bringing bubbles bulging from the two openings into contact with each other by bringing the openings of the two microchannel systems close to each other. ..

The lipid bilayer membrane forming step may be carried out using the technique described in Japanese Patent Application No. 2017-063796. The entire Japanese Patent Application No. 2017-063796 is incorporated herein by reference.

The oil used in this lipid bilayer forming step is not particularly limited, and examples thereof include phospholipids, hexadecanes, decanes, squalene, silicon oils, and mineral oils. Among these, hexadecane is preferable because it does not enter the gap between the two formed lipid monolayers and the formed lipid bilayer has a membrane structure close to that of a biological membrane and has low volatility.

The oil may contain lipids capable of forming a lipid monolayer. As another configuration, the aqueous solution may contain a lipid capable of forming a lipid monolayer. Alternatively, both the oil and the aqueous solution may contain lipids capable of forming a lipid monolayer. When the oil contains a lipid capable of forming a lipid monolayer, the bubble can be covered with the lipid monolayer even if the aqueous solution does not contain a lipid capable of forming a lipid monolayer. ..

The lipid capable of forming the lipid single membrane is not particularly limited as long as it can form a lipid bilayer membrane, and examples thereof include phospholipids and cholesterol. Examples of the phospholipid include, but are not limited to, phosphatidylcholine, difitanoylphosphatidylcholine, dipalmitoylphosphatidylcholine, palmitoyloleoil phosphatidylcholine, dioleoil phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, and phosphatidylglycerol. ..

When the oil contains a lipid capable of forming a lipid monolayer, the concentration of the lipid is not particularly limited, but is preferably 10 mg / ml to 200 mg / ml, and more preferably 15 mg / ml to 150 mg / ml. , 25 mg / ml to 100 mg / ml, more preferably 40 mg / ml to 60 mg / ml. With the above configuration, even when the aqueous solution does not contain a lipid capable of forming a lipid monolayer, a short-time (for example, less than 10 seconds) lipid monolayer formation time can be realized.

The aqueous solution used in this lipid bilayer forming step is not particularly limited as long as it is a solution containing water as a main component, and the aqueous solution contains various components (for example, salts, bases, sugars, lipids, proteins, etc.). You may be. The composition of the aqueous solution can be appropriately set, and may be a composition that imitates a specific environmental component in the living body such as a specific cytosol component, a blood component, or a lymph fluid component, for example.

The salt contained in the aqueous solution is not particularly limited, but is appropriately selected from salts contained in the living body, for example, metal salts such as sodium salt, potassium salt, magnesium salt, calcium salt, and phosphate, and amino acids. Examples include salt.

The base contained in the aqueous solution is not particularly limited, and examples thereof include guanine, adenine, thymine, and cytosine.

The lipid contained in the aqueous solution is not particularly limited, and examples thereof include cholesterol, glycolipid, phospholipid, and complex lipid. Some of these lipid components can move to the interface between the bubble and the oil in the formed bubble and become a component of the lipid monolayer. Such lipids that can be components of the lipid monolayer include cholesterol, glycolipids, phospholipids, and complex lipids. That is, in one embodiment of the present invention, the lipid monolayer covering the bubble may contain lipids such as cholesterol, glycolipids, phospholipids, and complex lipids.

The aqueous solution may contain lipids capable of forming a lipid monolayer. When the aqueous solution contains a lipid capable of forming a lipid monolayer, the bubble can be covered with the lipid monolayer even if the oil does not contain a lipid capable of forming a lipid monolayer. .. Examples of the lipid capable of forming the lipid single membrane contained in the aqueous solution include the same substances as the lipid capable of forming the lipid single membrane contained in the oil, as described above.

When the aqueous solution contains a lipid capable of forming a lipid monolayer, the concentration of the lipid is not particularly limited, but is preferably 10 mg / ml to 200 mg / ml, and more preferably 15 mg / ml to 150 mg / ml. , 25 mg / ml to 100 mg / ml, more preferably 40 mg / ml to 60 mg / ml. With the above configuration, even when the aqueous solution does not contain a lipid capable of forming a lipid monolayer, a short-time (for example, less than 10 seconds) lipid monolayer formation time can be realized.

The protein contained in the aqueous solution is not particularly limited, and examples thereof include an enzyme protein, a transport protein, and a membrane protein. These proteins are embedded within the lipid bilayer membrane, which allows the reproduction of cell membranes.

Membrane proteins include, but are not limited to, various ion channels, transmembrane adhesion proteins, transporters, transmembrane transport proteins, and the like. Further, the protein may be a protein whose function is unknown, a protein whose function is unknown even though it is a membrane protein, or a protein whose function is unknown.

The method for forming a lipid bilayer according to an embodiment of the present invention also forms a lipid bilayer, and at the same time or after the lipid bilayer is formed, for example, free diffusion and / or a micropipette, etc. By using, it is possible to embed a membrane protein in a lipid bilayer membrane.

The method for forming a lipid bilayer membrane according to an embodiment of the present invention may also include a method for expressing a membrane protein such as a channel protein. In this case, the expressed membrane protein can be embedded in the lipid bilayer membrane by free diffusion and / or by using a micropipette or the like.

As a method for expressing a membrane protein, for example, the aqueous solution in the bubble contains a gene encoding the membrane protein (DNA, RNA, etc.) and includes any protein necessary for transcribing and translating the gene in Invitro. By doing so, it can be achieved. Further, a fusion protein composed of a protein other than the membrane protein and a signal peptide for translocating to the membrane may be expressed.

The method for forming a lipid bilayer membrane according to an embodiment of the present invention may also include a method for measuring the function of a membrane protein. For example, when a channel protein is used as a membrane protein, the function of the channel protein is simply functioned by providing a current detection unit that detects a current in the bubble and preloading the membrane potential on the lipid bilayer membrane. It can be measured as a one-channel current.

By combining the method of expressing these membrane proteins and the method of measuring the function of the membrane protein, one embodiment of the present invention can provide a system capable of expressing the membrane protein and measuring the function of the membrane protein in real time. Specific examples of the system include the following systems. The DNA encoding the channel protein and any protein that can be transcribed and translated in Vitro are introduced into the first bubble of the system. Next, by maintaining the system at 37 degrees, the channel protein is synthesized in the bubble and the channel protein spontaneously translocates to the lipid bilayer membrane. By preloading the lipid bilayer with a membrane potential, the function of the channel protein can be measured as a single channel current.

In this lipid bilayer forming step, it is also possible to add any component into the bubble forming the lipid bilayer after the lipid bilayer is formed, or any component from the bubble. It is also possible to recover the components. In this case, for example, an arbitrary component may be injected into the bubble or an arbitrary component in the bubble may be sucked by using a micropipette or the like.

In this inspection method, the first bubble and the second bubble have the same size (major axis and minor axis), internal pressure, composition of the aqueous solution inside the bubble, and composition of the lipid single membrane covering the bubble. It may or may be different. Here, the "composition of the aqueous solution" refers to the types and contents of various components contained in the aqueous solution as described above. In addition, the "composition of a lipid monomembrane" refers to the type and content of various components contained in the lipid monomembrane (for example, lipids such as phospholipids and cholesterol, and proteins such as membrane proteins).
Further, the first bubble and the second bubble may have different contact angles generated at the interface between the lipid bilayer membrane and the lipid single membrane of the bubble. Further, as a result of the differences described above, the first bubble and the second bubble may have the same or different tension and / or film thickness of the lipid single membrane covering the bubbles. good.

(Internal pressure detection process)
The internal pressure detection step is a step of detecting the internal pressure ΔP ₁ of the first bubble and the internal pressure ΔP ₂ of the second bubble. Further, the detected _{information on the internal pressure ΔP 1} and the internal pressure ΔP ₂ can be used as an internal pressure signal in the tension calculation step described later.

Here, the internal pressure ΔP ₁ and the internal pressure ΔP ₂ are collectively referred to as an internal pressure ΔP. The internal pressure ΔP is also the pressure applied to the lipid single membrane of the bubble, and is the difference between the pressure applied from the oil phase to the lipid single membrane of the bubble and the pressure applied from the inside of the bubble to the lipid single membrane of the bubble.

The internal pressure detection step only needs to be able to detect the internal pressure ΔP of the bubble, and the specific method for detecting the internal pressure ΔP is not particularly limited. Some examples of methods for detecting internal pressure ΔP are described below, but the present invention is not limited to these methods.

For example, when the method of forming a bubble composed of an aqueous solution in oil is the above-mentioned injection method, the pressure applied to the aqueous solution when the aqueous solution is injected into the oil can be set to the internal pressure ΔP. In this case, the pressure applied to the aqueous solution (the pressure to be applied) can be detected by a micropipette for injecting the aqueous solution or a sensor provided in the space inside the syringe. Further, an internal pressure ΔP is obtained based on the pressure, and the obtained value of the internal pressure ΔP can be used in the tension calculation step described later.

Further, for example, the pressure in the bubble is directly detected by a sensor inserted in the bubble, the internal pressure ΔP is obtained based on the pressure, and the obtained value of the internal pressure ΔP is used in the tension calculation step described later. Can be done.

That is, the main internal pressure detecting step may be a step of directly detecting the pressure in the bubble, or may be a step of indirectly detecting the pressure in the bubble from the force applied to the bubble.

The sensor described above is not particularly limited as long as each detection target can be detected, and a well-known sensor can be used.

Here, for the two bubbles forming the lipid bilayer, the first bubble and the second bubble, when the composition of the aqueous solution inside the bubble and the composition of the lipid single membrane covering the bubble are both the same. think of. In this case, it may be assumed that the _{internal pressure ΔP 1} and the internal pressure ΔP _{2 are equal.} Therefore, the internal pressure ΔP of one of the two bubbles forming the lipid bilayer membrane may be detected and the internal pressure ΔP may be set _{to the internal pressure ΔP 1 and the internal pressure ΔP 2.}

(Long and short diameter detection process)
The major / minor axis detection step is a step of detecting the major axis R ₁₁ and the minor _{axis R 21 of} the first bubble, and the major axis R ₁₂ and the minor _{axis R 22 of the second bubble. Here, the major axis R 11} of the first bubble and the major axis R ₁₂ of the second bubble are collectively referred to as the major axis R _1, and the minor axis R ₂₁ of the first bubble and the minor axis R ₂₂ of the second bubble are referred to. Together, it is also referred to as minor diameter R _2. The detected information on the major axis R ₁ and the minor _{axis R 2} can be used in the tension calculation step described later, respectively.

The present long and short diameter detecting step as long as it can detect the diameter R ₁ and bubble minor R ₂ bubble, a specific method for detecting diameter R ₁ and minor axis R ₂ of the bubble, particularly limited Not done. Note that the major axis R ₁ of the bubble, refers to the longest diameter of the diameter of the bubble, and the minor axis R ₂ of the bubble, refers to the shortest diameter of the diameter of the bubble. The diameter of the bubble is the length of a line segment cut by the lipid single membrane of the bubble in the straight line passing through the center of the bubble. An example of this long / short diameter detection process will be described below.

In this major / minor axis detection step, for example, the bubble may be imaged using an imaging unit, and the major _{axis R 1 and the minor axis R 2} of the bubble may be detected from the imaging data.

In this major / minor axis detection step, it is preferable to acquire one imaging data in which two bubbles are projected together. Further, when acquiring one imaging data in which two bubbles are projected together, it is preferable that these two bubbles do not overlap each other in the imaging data. By imaging two bubbles from a direction parallel to the interface between the lipid bilayer membrane and the lipid single membrane of the bubble using the imaging unit, one imaging data including two bubbles that do not overlap each other can be obtained. It becomes possible to acquire. The direction parallel to the interface between the lipid bilayer membrane and the lipid single membrane of the bubble can be said to be the direction parallel to the lipid bilayer membrane. The interface between the lipid bilayer membrane and the lipid single membrane of the bubble is also the contact surface between the two bubbles.

The shape of the bubble in this inspection method is not particularly limited, but may be a substantially circular shape such as an elliptical shape or a substantially perfect circle.

Here, for the two bubbles forming the lipid bilayer (first bubble and second bubble), the composition of the aqueous solution inside the bubble and the composition of the lipid single membrane covering the bubble are both the same. think of. In this case, it can be assumed that the _{major axis R 11} and the major axis R ₁₂ are equal, _{and the minor axis R 21} and the minor axis R _{22 are equal.} Therefore, the major axis and the minor axis of one of the two bubbles forming the lipid bilayer membrane are detected, and the major axis and the minor axis are set to the major axis R ₁₁ and the minor _{axis R 21 of the first bubble.} , And the major axis R ₁₂ and the minor _{axis R 22 of} the second bubble.

(Contact angle detection process)
_{The contact angle detection step included in this inspection method includes a contact angle θ 1} generated at the interface between the lipid bilayer membrane and the lipid single membrane of the first bubble, and the lipid bilayer membrane and the lipid single membrane of the second bubble. This is a step of detecting the _{contact angle θ 2} generated on the boundary surface of the above. Further, the detected information on the contact angle θ ₁ and the contact angle θ ₂ can be used in the tension calculation step described later.

In the present specification, the "boundary surface between the lipid bilayer membrane and the lipid single membrane of the bubble" may be referred to as the "boundary surface between the lipid bilayer membrane and the bubble" or simply the "boundary surface". Further, as described above, the boundary surface between the lipid bilayer membrane and the lipid single membrane of the bubble is also the contact surface of the two bubbles.

In this contact angle detection step, it is sufficient that the contact angle θ generated at the interface between the lipid bilayer membrane and the bubble can be detected, and the specific method for detecting the contact angle θ is not particularly limited. An example of this contact angle detection step will be described below.

In this contact angle detection step, for example, the boundary surface may be imaged using an imaging unit, and the contact angle θ may be detected from the imaged data.

In the present contact angle detection step, when the boundary surface is imaged, it is preferable to image the contact angle from a direction parallel to the boundary surface. With the above configuration, since the boundary surface and the bubble do not overlap, the contact angle θ can be detected accurately.

Here, for the two bubbles forming the lipid bilayer (first bubble and second bubble), the composition of the aqueous solution inside the bubble and the composition of the lipid single membrane covering the bubble are both the same. think of. In this case, it can be assumed that _{the contact angle θ 1} and the contact angle θ _{2 are equal.} Therefore, the contact angle θ of one of the two bubbles forming the lipid bilayer membrane may be detected, and the contact angle θ may be set as _{the contact angle θ 1 and the contact angle θ 2.}

(Tension calculation process)
The tension calculation step included in this inspection method is a step of calculating the tension γ of the lipid bilayer membrane from the following formulas (1) to (5).

Equation (1) ΔP ₁ = γ ₁ × (1 / R ₁₁ + 1 / R ₂₁ )
Equation (2) ΔP ₂ = γ ₂ × (1 / R ₁₂ + 1 / R ₂₂ )
Equation (3) γ _b1 = γ ₁ × cos θ ₁
Equation (4) γ _b2 = γ ₂ × cos θ ₂
Equation (5) γ = γ _b1 + γ _b2
(Here, γ ₁ is the tension of the lipid single membrane of the first bubble _{, γ 2} is the tension of the lipid single membrane of _{the second bubble, and γ b 1} is the side of the first bubble in the lipid bilayer membrane. Γ _b2 is the tension of the monolayer on the side of the second bubble in the lipid bilayer).

Specifically, this tension calculation step is performed from (i) the internal pressure ΔP ₁ of the first bubble, the major axis R ₁₁ and the minor _{axis R 21 of} the first bubble, based on the equation (1), of the first bubble. _{The tension γ 1} of the lipid monolayer is calculated, and from (ii) the internal pressure ΔP ₂ of the second bubble, the major axis R ₁₂ and the minor _{axis R 22 of} the second bubble, the tension of the second bubble is calculated based on the formula (2). _{The tension γ 2} of the lipid monolayer was calculated, and (iii) _{from the tension γ 1} obtained in (i) above, based on the formula (3), the monolayer on the side of the first bubble in the lipid bilayer. The tension γ _b1 is calculated, and (iv) _{from the tension γ 2 obtained in (ii) above, the tension γ b2} of the monolayer on the side of the second bubble in the lipid duplex film is calculated based on the formula (4). Calculate and calculate the tension γ of the lipid double membrane based on the formula (5) from _{(v) the tension γ b1} obtained in the above (iii) and _{the tension γ b2} obtained in the above (iv). It is a process.

Here, the internal pressure ΔP ₁ and the internal pressure ΔP ₂ can be the internal pressure ΔP detected in the internal pressure detection step, and the major axis R ₁ and the minor _{axis R 2} are the major axis R _{1 detected in the major / minor axis detection step.} and be a minor axis R _2, the contact angle theta, it can be a contact angle theta detected by the contact angle detecting step.

In this tension calculation step, as long as the tension γ of the lipid bilayer membrane can be calculated, the specific method for calculating the tension γ is not particularly limited, and a well-known method can be used. In this tension calculation step, for example, a computer including a storage unit and a calculation unit can be used.

Here, for the two bubbles forming the lipid bilayer (first bubble and second bubble), the composition of the aqueous solution inside the bubble and the composition of the lipid single membrane covering the bubble are both the same. think of. In this case, it can be assumed that _{the tension γ 1} and the tension γ ₂ are equal, and the tension γ _b1 and the tension γ _{b2 are equal.} Therefore, the tension of the lipid single membrane and the tension of the monolayer are calculated for one of the two bubbles forming the lipid bilayer, and the tension of the monolayer is set to the tension γ _b1 and the tension. It may be _{γ b2.}

The method for inspecting the characteristics of the lipid bilayer according to the embodiment of the present invention further includes a capacitance detection step, an area detection step, and a film thickness calculation step, and calculates the thickness T of the lipid bilayer film. Is preferable.

The inspection method according to the embodiment of the present invention, that is, the present inspection method has the above-mentioned configuration, and as a characteristic of the lipid bilayer membrane, the thickness of the lipid bilayer membrane is determined in addition to the tension of the lipid bilayer membrane. It has the advantage that it can be easily inspected.

(Capacitance detection process)
The capacitance detection step is a step of detecting the capacitance C of the lipid bilayer membrane. Further, the detected capacitance C information can be used in the film thickness calculation step described later.

In this capacitance detection step, it is sufficient that the capacitance C of the lipid bilayer membrane can be detected, and the specific method for detecting the capacitance C is not particularly limited, and a well-known method is used. Can be done. An example of this capacitance detection step will be described below.

For example, using electrodes arranged in the first bubble and in the second bubble, the capacitive current value of the lipid bilayer film is obtained by generating a ramp wave between the electrodes. Capacitance C can be detected from the capacitive current value. In this case, each electrode is arranged so that the lipid bilayer membrane exists between the electrodes. From the viewpoint of more accurately detecting the capacitance C of the lipid bilayer membrane, it is preferable that each of the above electrodes is arranged at a position close to the lipid bilayer membrane. The device for generating the lamp wave is not particularly limited, and a well-known device can be used.

(Area detection process)
The area detection step is a step of detecting the area A of the lipid bilayer membrane. Further, the detected area A information can be used in the film thickness calculation step described later.

In this area detection step, it is sufficient that the area A of the lipid bilayer membrane can be detected, and the specific method for detecting the area A is not particularly limited. An example of this area detection step will be described below.

In this area detection step, the area A may be detected by, for example, imaging a lipid bilayer membrane using an imaging unit and acquiring imaging data.

When imaging the lipid bilayer membrane in this area detection step, it is preferable to image the lipid bilayer membrane from a direction perpendicular to the lipid bilayer membrane. With the above configuration, it is possible to acquire imaging data on which the entire surface of the lipid bilayer membrane is projected, so that the area A can be detected accurately. The direction perpendicular to the lipid bilayer can be said to be the direction perpendicular to the interface between the lipid bilayer and the lipid monolayer of the bubble.

If the lipid bilayer can be approximated as a perfect circle, the diameter of the lipid bilayer can be determined from the obtained imaging data by imaging the lipid bilayer from a direction parallel to the lipid bilayer. It can be obtained, and the area A can be detected from such a diameter. The direction parallel to the lipid bilayer is the direction parallel to the interface. Therefore, when the lipid bilayer membrane can be approximated as a perfect circle, the area A can be detected from the imaging data obtained in the contact angle detection step. Here, the case where the lipid bilayer membrane can be approximated as a perfect circle is, for example, the case where the diameter of the bubble is 50 μm or less.

(Film thickness calculation process)
The film thickness calculation step included in this inspection method is a step of calculating the film thickness T of the lipid bilayer membrane from the following formula (6).

Equation (6) T = ε _m × ε ₀ × A / C
(Here, ε _m is the relative permittivity of the lipid bilayer film, and ε ₀ is the permittivity of the vacuum.)
Specifically, this film thickness calculation step is performed by the formula (6) from the capacitance C and area A of the lipid bilayer, the relative permittivity ε _m of the lipid bilayer, and the permittivity ε _{0 of the vacuum.} ), The thickness T of the lipid bilayer film is calculated. Further, the calculation of the film thickness T of the lipid bilayer membrane may be performed using the analysis unit.

Here, the capacitance C may be the capacitance C detected in the capacitance detection step, and the area A may be the area A detected in the area detection step.

In the present specification, the "relative permittivity of the lipid bilayer" also means an oil sandwiched between a phospholipid hydrocarbon chain layer existing in the hydrophobic core region of the lipid bilayer and the hydrocarbon chain layer. It is the relative permittivity of the composite layer when the composite layer composed of is regarded as a uniform oil phase. When the oil is hexadecane, the relative permittivity of the lipid bilayer membrane is, for example, 2.2.

The above-mentioned "permittivity of vacuum" is a basic physical constant, and in this specification, the recommended value provided by the Committee on Data for Science and Technology (CODATA) is adopted. For example, if the permittivity of the vacuum is rounded off to the third decimal place based on the recommended value in 2014, it is ^{8.854 × 10-12} Fm ^-1.

[3. Drug screening method]
Another embodiment of the present invention provides a method for screening a drug that changes the properties of a lipid bilayer membrane, including a drug introduction step, a tension calculation step, and a first drug determination step.

According to the method for screening a drug according to an embodiment of the present invention, a drug that changes the characteristics of a lipid bilayer membrane, particularly the tension of the lipid bilayer membrane, can be easily screened by having the above-mentioned configuration. Has advantages.

In the present specification, the "drug screening method" is also referred to as a "drug screening method".

(Lipid bilayer formation step)
The drug screening method according to the embodiment of the present invention uses a lipid bilayer membrane formed on the contact surface between the first bubble covered with the lipid single membrane and the second bubble covered with the lipid single membrane. .. Therefore, the drug screening method may include a lipid bilayer forming step of forming a lipid bilayer.

The mode of the lipid bilayer membrane forming step in the drug screening method is described in the above [1. Methods for testing the properties of lipid bilayer membranes] section can be incorporated.

(Drug introduction process)
The drug introduction step is a step of introducing a drug into the lipid bilayer membrane.

In the drug introduction step, it is sufficient that the drug can be introduced into the lipid bilayer membrane, and the method for introducing the drug is not particularly limited. For example, a method of adding a drug to oil and / or into a bubble using a micropipette or the like can be mentioned. The oil and the drug added in the bubble can be introduced into the lipid bilayer by diffusion or interaction with any component in the lipid bilayer.

In the present drug introduction step, in order to introduce the drug more easily, it is preferable that the drug is introduced into the lipid bilayer membrane by the perfusion method. The perfusion method will be described below.

The perfusion method is a method of introducing an arbitrary component into the lipid bilayer membrane by injecting an arbitrary component in the vicinity of the interface between the lipid bilayer membrane and the bubble in oil (that is, the lipid bilayer membrane). .. Any injected component is rapidly incorporated into the lipid bilayer membrane.

(Tension calculation process)
The tension calculation step is a step of calculating the tension γ of the lipid bilayer after the introduction of the drug, and includes an internal pressure detection step, a major / minor axis detection step, and a contact angle detection step, and includes the following formulas (1) to (5). ) To calculate the tension γ of the lipid bilayer.

Equation (1) ΔP ₁ = γ ₁ × (1 / R ₁₁ + 1 / R ₂₁ )
Equation (2) ΔP ₂ = γ ₂ × (1 / R ₁₂ + 1 / R ₂₂ )
Equation (3) γ _b1 = γ ₁ × cos θ ₁
Equation (4) γ _b2 = γ ₂ × cos θ ₂
Equation (5) γ = γ _b1 + γ _b2
(Here, γ ₁ is the tension of the lipid single membrane of the first bubble _{, γ 2} is the tension of the lipid single membrane of _{the second bubble, and γ b 1} is the side of the first bubble in the lipid bilayer membrane. Γ _b2 is the tension of the monolayer on the side of the second bubble in the lipid bilayer)
The embodiments of the internal pressure detection step, the major / minor axis detection step, and the contact angle detection step in the tension calculation step included in the drug screening method are described in the above [1. Methods for testing the properties of lipid bilayer membranes] section can be incorporated.

Further, the tension calculation step of the drug screening method comprises the pressure [Delta] P, diameter R _1, and calculates the tension of the minor axis R ₂ of a lipid single film, further lipids from the tension and contact angle θ of the lipid single layer two The tension γ of the bilayer film is calculated. Therefore, the substantial aspect of the tension calculation step included in the present drug screening method is described in the above [1. It is the same as the mode of the tension calculation step described in the section of [Method for inspecting the characteristics of lipid bilayer membrane], and is the same as the above [1. Methods for testing the properties of lipid bilayer membranes] section can be incorporated.

(First drug determination step)
The first drug determination step is a step of determining a drug by comparing the tension γ obtained through the tension calculation step with a reference value.

The above reference value may be the value of the tension γ obtained through the tension calculation step before the introduction of the drug, or may be the value of the tension γ obtained through the tension calculation step after the introduction of the other drug. , Or it may be a predetermined value set in advance.

Changes in the value of tension γ can change the function of membrane proteins present in the lipid bilayer. That is, an agent capable of altering the tension γ can regulate the function of the membrane protein (eg, increase the enzymatic activity of the membrane protein or decrease the enzymatic activity of the membrane protein). Therefore, a substance that can be a candidate for a drug that targets a membrane protein can be selected by the first drug determination step.

The above-mentioned "determining a drug by comparing the tension γ obtained through the tension calculation step with a reference value" is, for example, based on the absolute value of the difference between the tension γ obtained through the tension calculation step and the reference value. , It is intended to determine whether the administered drug is a drug capable of changing the tension γ of the lipid bilayer membrane.

The drug screening method according to the embodiment of the present invention preferably further includes a film thickness calculation step and a second drug determination step.

The drug screening method according to the embodiment of the present invention, that is, the drug screening method has the above-mentioned configuration, and as a characteristic of the lipid bilayer membrane, in addition to the tension of the lipid bilayer membrane, the membrane of the lipid bilayer membrane It has the advantage that drugs that change the thickness can be easily screened.

(Film thickness calculation process)
The thickness calculation step is a step of calculating the thickness T of the lipid bilayer membrane after the introduction of the drug, and includes a capacitance detection step and an area detection step, and the lipid bilayer membrane is calculated from the following formula (6). This is a step of calculating the film thickness T of the above.

Equation (6) T = ε _m × ε ₀ × A / C
(Here, ε _m is the relative permittivity of the lipid bilayer film, and ε ₀ is the permittivity of the vacuum.)
The aspects of the capacitance detection step and the area detection step in the film thickness calculation step included in the drug screening method are described in the above [1. Methods for testing the properties of lipid bilayer membranes] section can be incorporated.

Further, in the film thickness calculation step included in this drug screening method, the thickness T of the lipid bilayer film is calculated from the capacitance C, the area A, the relative permittivity ε _m of the lipid bilayer film, and the dielectric constant ε _{0 of the vacuum.} It is to be calculated. Therefore, the substantial aspect of the film thickness calculation step included in the present drug screening method is described in the above [1. It is the same as the aspect of the film thickness calculation step described in the section of [Method for inspecting the characteristics of lipid bilayer membrane], and is the same as the above [1. Methods for testing the properties of lipid bilayer membranes] section can be incorporated.

(Second drug determination step)
The second drug determination step is a step of determining a drug by comparing the film thickness T obtained through the film thickness calculation step with a reference value.

The above reference value may be the value of the film thickness T obtained through the film thickness calculation step before the introduction of the drug, or the value of the film thickness T obtained through the film thickness calculation step after the introduction of the other drug. It may be, or it may be a predetermined value set in advance.

When the value of the film thickness T changes, the function of the membrane protein present in the lipid bilayer can change. That is, an agent capable of changing the film thickness T can regulate the function of the membrane protein (for example, increase the enzymatic activity of the membrane protein or decrease the enzymatic activity of the membrane protein). Therefore, a substance that can be a candidate for a drug can be selected by the second drug determination step.

The above-mentioned "determining a drug by comparing the film thickness T obtained through the film thickness calculation step with a reference value" means, for example, the absolute difference between the film thickness T obtained through the film thickness calculation step and the reference value. Based on the value, it is intended to determine whether or not the administered drug is a drug capable of changing the thickness T of the lipid bilayer membrane.

[4. Analysis system for lipid bilayer properties]
Another embodiment of the present invention comprises a lipid bilayer characteristic analysis system comprising a first internal pressure detection unit, a second internal pressure detection unit, a major / minor axis-contact angle detection unit, and a tension calculation unit. offer.

The lipid bilayer characteristic analysis system according to the embodiment of the present invention can easily analyze the characteristics of the lipid bilayer membrane, particularly the tension of the lipid bilayer membrane, by having the above-described configuration. It has the advantage of.

An analysis system according to an embodiment of the present invention, that is, the present analysis system will be described with reference to FIGS. 1 to 5.

FIG. 1 is a schematic view showing a configuration of an analysis system 100 according to an embodiment of the present invention. The analysis system 100 includes a lipid bilayer film forming unit 1, a first internal pressure detecting unit 10, a second internal pressure detecting unit 20, a major / minor axis-contact angle detecting unit 30, and a tension calculating unit 40. Here, the analysis system 100 includes the lipid bilayer film forming unit 1, but an analysis system that does not include the lipid bilayer film forming unit 1 is also within the scope of the present invention.

In the lipid bilayer film forming section 1, a lipid bilayer film formed on the contact surface between the first bubble covered with the lipid monolayer and the second bubble covered with the lipid monolayer is produced. Next, the internal pressure ΔP ₁ of the first bubble is detected by the first internal pressure detecting unit 10, the internal pressure ΔP ₂ of the second bubble is detected by the second internal pressure detecting unit 20, and the major axis R of the first bubble is detected. ₁₁ , the minor axis R ₂₁ and the contact angle θ ₁ , and the major axis R ₁₂ and the minor axis R _{22 of} the second bubble, and the contact angle θ ₂ are detected by the major axis-contact angle detecting unit 30. After that, from the internal pressure ΔP ₁ , internal pressure ΔP ₂ , major axis R ₁₁ , minor axis R ₂₁ , major axis R ₁₂ , minor axis R ₂₂ , contact angle θ ₁ , and contact angle θ ₂ , the tension γ of the lipid bilayer film is the tension calculation unit. Calculated by 40.

(Lipid bilayer membrane forming part 1)
The lipid bilayer membrane forming portion 1 will be further described with reference to FIG. FIG. 3A is a schematic view showing the configuration of a lipid bilayer film forming portion in the analysis system according to the embodiment of the present invention, and is a cross-sectional view of a cross section perpendicular to the lipid bilayer film.

The lipid bilayer film forming portion 1 is composed of a container 2, an oil phase 3, and a bubble 4. The container 2 is configured to accommodate the oil phase 3 and the bubble 4. A well-known structure is used for the shape and material of the container 2. The oil phase 3 covers the bubble 4, that is, the bubble 4 is in contact with the oil phase 3 and another bubble 4.

The oil that can be used as the oil phase 3 is described in [1. The description in the section (Step of forming a lipid bilayer membrane) in [Method for examining the properties of a lipid bilayer membrane] can be incorporated. The bubble 4 is composed of an aqueous solution, and is composed of a first bubble M1 and a second bubble M2. The number of bubbles 4 in the analysis system 100 is two, but in another aspect of the present invention, the number of bubbles 4 is not particularly limited as long as it is two or more. Examples of the aqueous solution used for the bubble 4 include the above [1. The description in the section (Step of forming a lipid bilayer membrane) in [Method for examining the properties of a lipid bilayer membrane] can be incorporated.

The first bubble M1 is laminated vertically above the second bubble M2, and a lipid bilayer film 7 is formed on the contact surface 6 of the first bubble M1 and the second bubble M2. Since the contact surface 6 is also a boundary surface between the lipid bilayer membrane and the lipid single membrane of the bubble, the contact surface 6 has the same configuration as the boundary surface 6 described later. Further, in the analysis system 100, the first bubble M1 and the second bubble M2 are stacked in the vertical direction, but in another aspect of the present invention, the first bubble and the second bubble are arranged in the horizontal direction. It may be arranged. When the first bubble and the second bubble are stacked vertically, the lipid bilayer film is formed horizontally, and when the first bubble and the second bubble are arranged horizontally. The lipid bilayer is formed vertically. FIG. 3B is a cross-sectional view taken along the line AA of FIG. 3A.

Other aspects of the lipid bilayer membrane forming portion in this analysis system include the above [1. Methods for testing the properties of lipid bilayer membranes] section may be incorporated as appropriate.

(First internal pressure detection unit 10 and second internal pressure detection unit 20)
In the analysis system 100, the first internal pressure detecting unit 10 and the second internal pressure detecting unit 20 are provided inside the micropipette 5, respectively. Each of the micropipettes 5 is arranged to exert a force on the first bubble M1 or the second bubble M2. Here, the first internal pressure detecting unit 10 and the second internal pressure detecting unit 20 are also simply referred to as "internal pressure detecting units 10 and 20".

The internal pressure detecting units 10 and 20 each include sensors capable of detecting the force applied to the micropipette 5 (that is, the repulsive force due to the bubble, which can be said to be the pressure of the bubble). The internal pressure detecting units 10 and 20 may further detect the internal pressure ΔP from the force (or pressure) detected by the sensor, respectively.

As shown in FIG. 4, the internal pressure ΔP detected by the internal pressure detecting units 10 and 20 is output to the tension calculating unit 40.

The sensor is not particularly limited as long as it can detect the force applied to the micropipette 5, and a well-known sensor can be used.

In this analysis system, the first internal pressure detecting unit and the second internal pressure detecting unit are not particularly limited as long as they can detect the internal pressure ΔP of the bubble, respectively.

Other aspects of the first internal pressure detection unit and the second internal pressure detection unit in this analysis system include the above [1. Method for Examining the Characteristics of Lipid Bilayer Membrane] section may be appropriately incorporated. That is, the first internal pressure detecting unit and the second internal pressure detecting unit may include a sensor provided in the bubble, and the sensor may be configured to be able to detect the pressure in the bubble.

(Long and short diameter-contact angle detection unit 30)
The major / minor diameter-contact angle detecting unit 30 images the bubble 4 from the horizontal direction, and acquires the imaged data on which the bubble 4 is projected. Therefore, in the analysis system 100, the major / minor axis-contact angle detection unit 30 can be said to be a major / minor axis-contact angle imaging unit. The horizontal direction is a direction parallel to the contact surface (boundary surface) 6 and the lipid bilayer membrane 7. Therefore, the major / minor axis-contact angle detection unit 30 can simultaneously image the first bubble and the second bubble. From the imaging data, the major axis and minor axis of the first bubble, the major axis and minor axis of the second bubble, the contact angle generated at the interface between the lipid single membrane and the lipid bilayer membrane of the first bubble, and The contact angle generated at the interface between the lipid single membrane and the lipid bilayer membrane of the second bubble can be detected at the same time.

_{The major / minor axis-contact angle detection unit 30 detects the major axis R 1} and the minor _{axis R 2} of the bubble, and the contact angle θ by analyzing the imaging data obtained by imaging the bubble 4 as described above. It may be something that can be done.

As shown in FIG. 4, the imaging data, the major axis R ₁ , the minor axis R ₂ , and / or the contact angle θ are output to the tension calculation unit 40.

The major / minor axis-contact angle imaging unit may be any as long as it can acquire imaging data having a resolution capable of analyzing _{the major axis R 1} and the minor _{axis R 2 of the bubble and the contact angle θ, and a well-known configuration is used.} Can be (eg, the lens of a microscope, or the microscope itself). The lens may be, for example, an objective lens used in a microscope. Therefore, the major / minor axis-contact angle image pickup unit may be composed of an objective lens and an image pickup element that receives light incident on the objective lens.

In this analysis system, the major / minor axis-contact angle detection unit is not particularly limited to the above configuration as long as it can detect _{the major axis R 1} and the minor _{axis R 2 of the bubble and the contact angle θ.}

(Tension calculation unit 40)
The tension calculation unit 40 includes internal pressure ΔP obtained from the first internal pressure detection unit 10 and the second internal pressure detection unit 20, major and minor _{axis R 1 and minor axis R 2} obtained from the major / minor axis-contact angle detection unit 30, and contact. From the angle θ, the tension γ of the lipid bilayer membrane is calculated by applying the following equations (1) to (5).

Equation (1) ΔP ₁ = γ ₁ × (1 / R ₁₁ + 1 / R ₂₁ )
Equation (2) ΔP ₂ = γ ₂ × (1 / R ₁₂ + 1 / R ₂₂ )
Equation (3) γ _b1 = γ ₁ × cos θ ₁
Equation (4) γ _b2 = γ ₂ × cos θ ₂
Equation (5) γ = γ _b1 + γ _b2
(Here, γ ₁ is the tension of the lipid single membrane of the first bubble M1, γ ₂ is the tension of the lipid single membrane of the second bubble M2, and γ _b1 is the tension of the first bubble in the lipid bilayer membrane. Is the tension of the monolayer on the side of, and γ _b2 is the tension of the monolayer on the side of the second bubble in the lipid bilayer).

The tension calculation unit 40 is not particularly limited, but for example, a computer including a storage unit and a calculation unit can be used.

The analysis system according to the embodiment of the present invention preferably further includes a capacitance detection unit, an area detection unit, and a film thickness calculation unit. FIG. 2 is a schematic view showing the configuration of the analysis system 200 according to the embodiment of the present invention. The analysis system 200 includes a capacitance detection unit 50, an area detection unit 60, and a film thickness calculation unit 70, in addition to the configuration of the analysis system 100 described above.

In the analysis system 200, the thickness T of the lipid bilayer membrane is calculated in addition to the tension γ of the lipid bilayer membrane. Specifically, the capacitance C of the lipid bilayer membrane is detected by the capacitance detection unit 50, and the area A of the lipid bilayer membrane is detected by the area detection unit 60. After that, the thickness T of the lipid bilayer is calculated by the film thickness calculation unit 70 from the capacitance C, the area A, the relative permittivity of the lipid bilayer, and the permittivity of the vacuum.

(Capacitance detection unit 50)
The capacitance detection unit 50 includes an electrode and a lamp wave generator. The electrodes are respectively arranged inside the first bubble and the second bubble, and the electric resistance value of the lipid bilayer membrane is obtained by generating a lamp wave between the electrodes. In FIG. 2, the electrodes are arranged inside the micropipette 5, and an electric current is transmitted to the inside of each bubble through the electrolyte solution filled inside the micropipette 5.

The capacitance detection unit 50 detects the capacitance C of the lipid bilayer membrane 7 by analyzing the electric resistance value of the lipid bilayer membrane 7 acquired as described above.

As shown in FIG. 5, the capacitance C can be output to the film thickness calculation unit 70.

In this analysis system, the capacitance detection unit is not particularly limited to the above configuration as long as it can detect the capacitance C of the lipid bilayer membrane.

(Area detection unit 60)
The area detection unit 60 images the lipid bilayer film 7 from vertically below, and acquires the imaging data on which the lipid bilayer film 7 is projected. The vertical lower direction is a direction perpendicular to the contact surface (boundary surface) 6 and the lipid bilayer membrane 7. Therefore, the area detection unit 60 can image the entire surface of the lipid bilayer membrane 7.

The area detection unit 60 detects the area A of the lipid bilayer membrane by analyzing the imaging data obtained by imaging the lipid bilayer membrane 7 as described above.

As shown in FIG. 5, the area A of the lipid bilayer membrane can be output to the film thickness calculation unit 70.

In this analysis system, the area detection unit is not particularly limited to the above configuration as long as the area A of the lipid bilayer membrane can be detected.

(Film thickness calculation unit 70)
The film thickness calculation unit 70 uses the following formula from the capacitance C obtained from the capacitance detection unit 50, the area A obtained from the area detection unit 60, the relative permittivity of the lipid double film, and the permittivity of the vacuum. (6) is applied to calculate the thickness T of the lipid double film.

Equation (6) T = ε _m × ε ₀ × A / C
(Here, ε _m is the relative permittivity of the lipid bilayer film, and ε ₀ is the permittivity of the vacuum.)
The film thickness calculation unit 70 is not particularly limited, but for example, a computer including a storage unit and a calculation unit can be used. The same device may be used for the tension calculation unit 40 and the film thickness calculation unit 70.

(Use of this analysis system)
This analysis system can be used as a system for carrying out the above-mentioned method for inspecting the characteristics of a lipid bilayer membrane. In addition, this analysis system can be suitably used as a system for carrying out the above-mentioned drug screening method.

In each part and each processing step of the present invention, a calculation means such as a CPU executes a program stored in a storage means such as a ROM (Read Only Memory) or a RAM, and an input means such as a keyboard or an output such as a display. It can be realized by controlling a means or a communication means such as an interface circuit.

Therefore, the present invention can be realized only by a computer having these means reading the recording medium on which the program is recorded and executing the program. Further, by recording the program on a removable recording medium, the above-mentioned various functions and various processes can be realized on an arbitrary computer.

As the recording medium, a memory (not shown) such as a ROM for processing by a microcomputer may be a program medium, or a program reading device (not shown) is provided as an external storage device. It may be a program medium that can be read by inserting a recording medium therein.

Further, in any case, the stored program is preferably configured to be accessed and executed by the microprocessor. Further, it is preferable that the program is read, the read program is downloaded to the program storage area of the microcomputer, and the program is executed. It is preferable that the download program is stored in the main unit in advance.

The program media is a recording medium that is separable from the main body, and is a tape system such as a magnetic tape or a cassette tape, a magnetic disk such as a flexible disk or a hard disk, or a disk such as a CD / MO / MD / DVD. Fixed including disk system, card system such as IC card (including memory card), or semiconductor memory such as mask ROM, EPROM (Erasable Programmable Read Only Memory), EEPROM (Electrically Erasable Programmable Read Only Memory), flash ROM, etc. There may be a recording medium or the like that carries the program.

Further, if the system configuration is such that a communication network including the Internet can be connected, it is preferable that the recording medium carries the program in a fluid manner so as to download the program from the communication network.

Further, when the program is downloaded from the communication network in this way, it is preferable that the program for download is stored in the main unit in advance or installed from another recording medium.

The present inventors have found for the first time that the function of the membrane is changed by changing the characteristics of the lipid bilayer membrane. Hereinafter, in the examples, it will be demonstrated that the activity of the membrane protein embedded in the membrane changes when the tension of the lipid bilayer membrane is changed.
(operation)
(1. Preparation of lipid bilayer membrane)
First, a liposome solution (ie, a proteoliposomal solution) containing a membrane protein at a concentration of 2 mg / mL was prepared. Here, the proteoliposomal solution is a solution in which liposomes and membrane proteins are dissolved in an electrolytic solution. As the membrane protein, KcsA, which is a potassium channel, was used. The prepared proteoliposome solution was filled in two micropipettes, and then the micropipettes were inserted into a hexadecane (oil phase) filled in a container. Here, the container was placed on the stage of an inverted microscope (IX73, manufactured by Olympus Corporation). The tip of the micropipette was about 30 μm.

Next, the proteoliposome solution was extruded into hexadecane from each of the two micropipettes to create two bubbles covered with a single lipid membrane. The diameter of the bubble was about 100 μm. Next, the two created bubbles were brought into contact with each other by operating a micropipette to form a lipid bilayer membrane on the contact surface of the two bubbles. Here, the operation of the micropipette was performed using a motor-driven micromanipulator (EMM-3NV, manufactured by Narikige Co., Ltd.). In addition, the internal pressure of the micropipette was controlled using a pneumatic micropipette (IM-11-2). The internal pressure of the micropipette is also the maintenance pressure of the bubble.

Here, in order to measure the change in ionic current due to the action of KcsA (that is, the activity of KcsA), the pH of the proteoliposomal solution in the two bubbles was made asymmetric (pH 7 and pH 4). The reference electrode was connected to a micropipette that produced a bubble containing a proteoliposomal solution of pH 7.5.

(2. Measurement of ion current)
Electrodes for measuring ionic current were placed inside the micropipette. As the electrode, an Ag / AgCl metal wire electrode (E255, manufactured by Warner Instruments) was used. The ion current was measured by voltage immobilization using a patch clamp amplifier (EPC800USB, manufactured by HEKA). The current signal was filtered at 1 kHz for frequency cutoff and sampled at 5 kHz by an A / D converter (Digitata 1550A, Molecular Devices). The sampled current signal was stored in a computer using software (pCLAMP, manufactured by Molecular Devices).

(3. Changes in lipid bilayer membrane due to decompression of bubble maintenance pressure)
By reducing the internal pressure of the micropipette, the maintenance pressure of the bubble is reduced (ie, decompressed). FIG. 6 shows the changes in the two bubbles covered with the lipid single membrane and the lipid bilayer membrane formed on the contact surface of the two bubbles when the maintenance pressure of the bubbles was reduced. FIG. 6A is a diagram showing changes in the shapes of the two bubbles and the lipid bilayer membrane as the maintenance pressure of the bubbles is reduced. FIG. 6B is a schematic view schematically showing FIG. 6A.

From FIG. 6, it was found that the area of the lipid bilayer increases and the contact angle θ generated at the interface between the lipid bilayer and the lipid monolayer of the bubble increases as the maintenance pressure of the bubble is reduced. ..

A decrease in the maintenance pressure of the bubble also causes a decrease in the internal pressure of the bubble. Therefore, referring to the formulas (1) and (2) of the present invention, it can be seen that the tension of the lipid monolayer of the bubble decreases as the internal pressure of the bubble decreases. Further, referring to the formulas (3) to (5) of the present invention, it can be seen that the tension of the lipid bilayer membrane is reduced by the reduction of the tension of the lipid single membrane of the bubble.

(4. Changes in membrane protein activity due to decompression of bubble maintenance pressure)
Next, the change in the activity of the membrane protein (KcsA) with the decrease in the maintenance pressure of the bubble was evaluated by measuring the change in the ion current with the decrease in the maintenance pressure of the bubble.

While continuing to measure the ion current over time, the maintenance pressure of the bubble was reduced at some point and the change in the ion current was evaluated. The results are shown in FIG. FIG. 7 is a graph showing the change in the ion current with the decompression of the maintenance pressure of the bubble.

From FIG. 7, it was shown that the ion current decreased as the maintenance pressure of the bubble was reduced. A decrease in ion current indicates a decrease in the activity of the potassium channel KcsA. Therefore, it was shown that the activity of the potassium channel KcsA decreased with the decrease in the maintenance pressure of the bubble, that is, the decrease in the tension of the lipid bilayer membrane.

The present invention can be used in the field relating to lipid bilayer membranes.

1 Lipid bilayer membrane forming part 2 Container 3 Oil phase 4 Bubble 5 Micropipette 6 Contact surface 7 Lipid bilayer membrane 10 First internal pressure detection part 20 Second internal pressure detection part 30 Long and short diameter-contact angle detection part 40 Tension calculation Part 50 Capacitance detection part 60 Area detection part 70 Film thickness calculation part 100, 200 Analysis system

Claims (4)

This is a method for inspecting the tension γ of the lipid bilayer membrane formed on the contact surface between the first bubble covered with the lipid monolayer and the second bubble covered with the lipid monolayer.
Internal pressure detection step of detecting the internal _{pressure ΔP 1} of the first bubble and the internal _{pressure ΔP 2} of the second bubble,
A major / minor axis detection step for detecting _{the major axis R 11} and the minor _{axis R 21 of} the first bubble and the major _{axis R 12} and the minor _{axis R 22 of} the second bubble.
_{The contact angle θ 1} generated at the interface between the lipid bilayer membrane and the lipid monolayer of the first bubble, and the contact generated at the interface between the lipid bilayer membrane and the lipid monolayer of the second bubble. A contact angle detection step for detecting an angle θ ₂ , and a contact angle detection step, and
Tension calculating step of following formulas (1) to (5) for calculating the tension γ of the lipid bilayer membrane, characterized in that it comprises a method for inspecting a tension γ of the lipid bilayer.
Equation (1) ΔP ₁ = γ ₁ × (1 / R ₁₁ + 1 / R ₂₁ )
Equation (2) ΔP ₂ = γ ₂ × (1 / R ₁₂ + 1 / R ₂₂ )
Equation (3) γ _b1 = γ ₁ × cos θ ₁
Equation (4) γ _b2 = γ ₂ × cos θ ₂
Equation (5) γ = γ _b1 + γ _b2
(Here, γ ₁ is the tension of the lipid single membrane of the first bubble, γ ₂ is the tension of the lipid single membrane of the second bubble, and γ _b1 is the tension of the first lipid bilayer membrane. The tension of the monolayer on the bubble side of the _{above, and γ b2} is the tension of the monolayer on the side of the second bubble in the lipid bilayer). This is a method for inspecting the tension γ and the thickness T of the lipid bilayer membrane formed on the contact surface between the first bubble covered with the lipid monolayer and the second bubble covered with the lipid monolayer.
Internal pressure detection step of detecting the internal _{pressure ΔP 1} of the first bubble and the internal _{pressure ΔP 2} of the second bubble,
A major / minor axis detection step for detecting _{the major axis R 11} and the minor _{axis R 21 of} the first bubble and the major _{axis R 12} and the minor _{axis R 22 of} the second bubble.
_{The contact angle θ 1} generated at the interface between the lipid bilayer membrane and the lipid monolayer of the first bubble, and the contact generated at the interface between the lipid bilayer membrane and the lipid monolayer of the second bubble. Contact angle detection step, which detects the angle θ _2,
A tension calculation step of calculating the tension γ of the lipid bilayer membrane from the following formulas (1) to (5).
Capacitance detection step for detecting the capacitance C of the lipid bilayer membrane,
An area detection step for detecting the area A of the lipid bilayer membrane, and
Thickness calculation step of the following formulas (6) to calculate the thickness T of the lipid bilayer membrane, characterized in that it comprises a method for inspecting a tension γ and thickness T of the lipid bilayer.
Equation (1) ΔP ₁ = γ ₁ × (1 / R ₁₁ + 1 / R ₂₁ )
Equation (2) ΔP ₂ = γ ₂ × (1 / R ₁₂ + 1 / R ₂₂ )
Equation (3) γ _b1 = γ ₁ × cos θ ₁
Equation (4) γ _b2 = γ ₂ × cos θ ₂
Equation (5) γ = γ _b1 + γ _b2
(Here, γ ₁ is the tension of the lipid single membrane of the first bubble, γ ₂ is the tension of the lipid single membrane of the second bubble, and γ _b1 is the tension of the first lipid bilayer membrane. The tension of the monolayer on the bubble side of the _{above, and γ b2} is the tension of the monolayer on the side of the second bubble in the lipid bilayer).
Equation (6) T = ε _m × ε ₀ × A / C
(Here, ε _m is the relative permittivity of the lipid bilayer film, and ε ₀ is the permittivity of the vacuum). It is an analysis system of the tension γ of the lipid bilayer membrane formed on the contact surface between the first bubble covered with the lipid monolayer and the second bubble covered with the lipid monolayer.
A first internal pressure detecting unit that detects the internal pressure ΔP ₁ of the first bubble, and a second internal pressure detecting unit that detects _{the internal pressure ΔP 2 of the second bubble.
The major axis R 11} and minor _{axis R 21 of} the first bubble and the major _{axis R 12} and minor _{axis R 22 of} the second bubble are detected, and the lipid bilayer membrane and the first bubble detecting the _2, contact angle theta generated at the interface between the contact angle theta ₁ and the lipid bilayer and the second bubble lipid single film occurs at the interface between the lipid single film, long and short diameters - contact Angle detector, as well as
Tension calculating unit formula (1) to (5) for calculating the tension γ of the lipid bilayer membrane, characterized in that it comprises a lipid bilayer analysis system tension γ of.
Equation (1) ΔP ₁ = γ ₁ × (1 / R ₁₁ + 1 / R ₂₁ )
Equation (2) ΔP ₂ = γ ₂ × (1 / R ₁₂ + 1 / R ₂₂ )
Equation (3) γ _b1 = γ ₁ × cos θ ₁
Equation (4) γ _b2 = γ ₂ × cos θ ₂
Equation (5) γ = γ _b1 + γ _b2
(Here, γ ₁ is the tension of the lipid single membrane of the first bubble, γ ₂ is the tension of the lipid single membrane of the second bubble, and γ _b1 is the tension of the first lipid bilayer membrane. The tension of the monolayer on the bubble side of the _{above, and γ b2} is the tension of the monolayer on the side of the second bubble in the lipid bilayer). This is an analysis system for the tension γ and the thickness T of the lipid bilayer membrane formed on the contact surface between the first bubble covered with the lipid monolayer and the second bubble covered with the lipid monolayer.
A first internal pressure detecting unit that detects the internal pressure ΔP ₁ of the first bubble, and a second internal pressure detecting unit that detects _{the internal pressure ΔP 2 of the second bubble.
The major axis R 11} and minor _{axis R 21 of} the first bubble and the major _{axis R 12} and minor _{axis R 22 of} the second bubble are detected, and the lipid bilayer membrane and the first bubble detecting the _2, contact angle theta generated at the interface between the contact angle theta ₁ and the lipid bilayer and the second bubble lipid single film occurs at the interface between the lipid single film, long and short diameters - contact Corner detector,
A tension calculation unit that calculates the tension γ of the lipid bilayer membrane from the following formulas (1) to (5).
Capacitance detector for detecting the capacitance C of the lipid bilayer membrane,
An area detection unit that detects the area A of the lipid bilayer membrane, and
Thickness calculating unit that the following formulas (6) to calculate the thickness T of the lipid bilayer membrane, characterized in that it comprises the analysis system tension γ and thickness T of the lipid bilayer.
Equation (1) ΔP ₁ = γ ₁ × (1 / R ₁₁ + 1 / R ₂₁ )
Equation (2) ΔP ₂ = γ ₂ × (1 / R ₁₂ + 1 / R ₂₂ )
Equation (3) γ _b1 = γ ₁ × cos θ ₁
Equation (4) γ _b2 = γ ₂ × cos θ ₂
Equation (5) γ = γ _b1 + γ _b2
(Here, γ ₁ is the tension of the lipid single membrane of the first bubble, γ ₂ is the tension of the lipid single membrane of the second bubble, and γ _b1 is the tension of the first lipid bilayer membrane. The tension of the monolayer on the bubble side of the _{above, and γ b2} is the tension of the monolayer on the side of the second bubble in the lipid bilayer).
Equation (6) T = ε _m × ε ₀ × A / C
(Here, ε _m is the relative permittivity of the lipid bilayer film, and ε ₀ is the permittivity of the vacuum).

Images