WO2019208795A1 - Method for forming concentration gradient on microreactor chip, and microreactor chip - Google Patents

Abstract

A method for forming a concentration gradient on a microreactor chip comprises the steps of: introducing a first aqueous solution into a first liquid flow path arranged on the main surface of a hydrophobic layer of a microreactor chip provided with the hydrophobic layer, wherein the hydrophobic layer is made from a hydrophobic substance and has, formed on the main surface thereof, openings for multiple chambers, and the openings are arranged regularly on the main surface of the layer, whereby each of the chambers that face the first liquid flow path is filled with the first aqueous solution; introducing a second aqueous solution having a different concentration from that of the first aqueous solution through a first inlet port of the first liquid flow path to form a gradient in the concentration of an aqueous solution filled in each of the chambers, wherein the gradient corresponds to the distance from the first inlet port; and introducing an organic solvent containing a lipid and a third aqueous solution in this order into the first liquid flow path to form a lipid bilayer in such a manner that the opening for each of the chambers is sealed with an aqueous solution.

Description

Method for forming concentration gradient on microreactor chip and microreactor chip

The present disclosure relates to a concentration gradient forming method on a microreactor chip and a microreactor chip.

Japanese Patent Laying-Open No. 2015-040754 discloses a flat substrate and a plurality of minute capacitances of 4000 × 10 ⁻¹⁸ m ³ or less formed so as to be regularly and densely arranged on the surface of the substrate by a hydrophobic substance. Disclosed is a high-density microchamber array comprising a chamber and a lipid bilayer formed to seal the aqueous test solution at the openings of a plurality of microchambers filled with the aqueous test solution.

Based on the above-mentioned conventional high-density micro-chamber array, development of applied technology has been desired.

A concentration gradient forming method on a microreactor chip according to one aspect of the present disclosure includes:
A main layer of the hydrophobic layer of a microreactor chip having a hydrophobic layer, the layer comprising a hydrophobic substance, wherein the openings of the plurality of chambers are regularly arranged on the main surface of the layer. Introducing a first aqueous solution into a first liquid channel provided on a surface and filling each chamber facing the first liquid channel with the first aqueous solution;
A second aqueous solution having a concentration different from that of the first aqueous solution is introduced from the first introduction port of the first liquid flow path, and the gradient according to the distance from the first introduction port to the concentration of the aqueous solution filled in each chamber. Forming a step;
Introducing a lipid-containing organic solvent and a third aqueous solution into the first liquid channel in order, and forming a lipid bilayer so as to seal the aqueous solution at the opening of each chamber;
including.

FIG. 1 is a plan view illustrating an example of a schematic configuration of a microreactor chip according to an embodiment. FIG. 2 is a view showing a cross section of the microreactor chip shown in FIG. 1 taken along line AA. FIG. 3 is a flowchart illustrating an example of a method for manufacturing a microreactor chip according to an embodiment. FIG. 4A is a diagram for explaining a manufacturing method of a chip body of a microreactor chip according to an embodiment, and is a diagram illustrating a process of preparing a substrate. FIG. 4B is a diagram for explaining a manufacturing method of the chip body of the microreactor chip according to the embodiment, and is a diagram illustrating a process of forming a material film on the substrate. FIG. 4C is a diagram for explaining the manufacturing method of the chip body of the microreactor chip according to the embodiment, and is a diagram illustrating a process of forming a resist on the material film. FIG. 4D is a diagram for explaining the manufacturing method of the chip body of the microreactor chip according to the embodiment, and is a diagram illustrating a process of patterning a resist. FIG. 4E is a diagram for explaining a manufacturing method of the chip body of the microreactor chip according to the embodiment, and is a diagram illustrating a process of etching a material film using a patterned resist as a mask. FIG. 4F is a diagram for explaining the manufacturing method of the chip body of the microreactor chip according to the embodiment, and is a diagram illustrating a step of removing the resist. FIG. 5 is a plan view showing an apparatus configuration used in an example of a concentration gradient forming method on a microreactor chip according to an embodiment. FIG. 6 is a flowchart illustrating an example of a concentration gradient forming method on a microreactor chip according to an embodiment. FIG. 7A is a diagram for explaining an example of a concentration gradient forming method on a microreactor chip according to an embodiment, and shows a step (step S11) of introducing a first aqueous solution into a first liquid channel. It is. FIG. 7B is a diagram for explaining an example of the concentration gradient forming method on the microreactor chip according to the embodiment, and is a diagram showing a step of introducing the second aqueous solution into the first liquid channel (step S12). It is. FIG. 7C is a diagram for explaining an example of a concentration gradient forming method on the microreactor chip according to the embodiment, and an organic solvent containing lipid and a third aqueous solution are sequentially introduced into the first liquid channel. It is a figure which shows the process (step S13) to perform. FIG. 7D is a diagram for explaining an example of a concentration gradient forming method on the microreactor chip according to the embodiment, and an organic solvent containing lipid and a third aqueous solution are sequentially introduced into the first liquid channel. It is a figure which shows the process (step S13) to perform. FIG. 7E is a diagram for explaining an example of a concentration gradient forming method on a microreactor chip according to an embodiment, and an organic solvent containing lipid and a third aqueous solution are sequentially introduced into the first liquid channel. It is a figure which shows the process (step S13) to perform. FIG. 7F is a diagram for explaining an example of a concentration gradient forming method on the microreactor chip according to the embodiment, and an organic solvent containing lipid and a third aqueous solution are sequentially introduced into the first liquid channel. It is a figure which shows the process (step S13) to perform. FIG. 8 is a measurement result of the microreactor chip according to the first example, and is a diagram showing a fluorescence image of each chamber according to the distance from the first introduction port. FIG. 9A is a graph showing the measurement results of the microreactor chip according to the first example, and the relationship between the distance from the first inlet and the intensity of blue fluorescence when the second aqueous solution is introduced at different flow rates. It is. FIG. 9B is a graph showing the measurement results for the microreactor chip according to the first example, and the relationship between the distance from the first inlet and the intensity of green fluorescence when the second aqueous solution is introduced at different flow rates. It is. FIG. 10A is a graph showing the measurement results for the microreactor chip according to the first example, and the relationship between the distance from the first inlet and the intensity of blue fluorescence when the second aqueous solution is introduced with different capacities. It is. FIG. 10B is a graph showing the measurement results for the microreactor chip according to the first example, and the relationship between the distance from the first inlet and the intensity of green fluorescence when the second aqueous solution is introduced with different capacities. It is. FIG. 11 is a diagram for explaining the microreactor chip according to the second embodiment. FIG. 12A is a measurement result of the microreactor chip according to the second example and is a diagram illustrating a fluorescence image of the chamber at the position of the first introduction port. FIG. 12B is a measurement result of the microreactor chip according to the second example and is a diagram showing a fluorescence image of the chamber at a position 8.1 mm away from the first introduction port. FIG. 13 is a graph showing the measurement results for the microreactor chip according to the second example and showing the relationship between the elapsed time and the intensity of green fluorescence at a position where the distance from the first inlet is different. FIG. 14 is a graph showing the measurement results for the microreactor chip according to the second example and showing the relationship between the substrate concentration and the amount of change in fluorescence intensity per unit time. FIG. 15A is a diagram for explaining the microreactor chip according to the third embodiment. FIG. 15B is a measurement result of the microreactor chip according to the third example and is a diagram showing a fluorescence image of the chamber at a position 8.1 mm away from the first introduction port. FIG. 16 is a graph showing the measurement results for the microreactor chip according to the third example, and showing the relationship between the elapsed time and the fluorescence intensity of the chamber at a position 8.1 mm away from the first inlet. FIG. 17 is a graph showing the measurement results for the microreactor chip according to the third example and showing the relationship between the concentration of α-hemolysin and the ratio of the chamber in which nanopores are formed in the lipid bilayer membrane. FIG. 18 is a plan view showing a device configuration used in a modified example of the concentration gradient forming method on the microreactor chip according to the embodiment. FIG. 19 is a flowchart illustrating a modification of the concentration gradient forming method on the microreactor chip according to the embodiment.

In various biomolecular reactions that occur through lipid bilayer membranes, such as membrane transport processes, membrane permeation reactions, and enzyme reactions on the membrane surface, it takes a long time for the diffusion of reaction products, and substances associated with enzyme activity Since the change in concentration is extremely gradual, it is difficult to detect various biomolecular reactions that occur through the lipid bilayer with high sensitivity. When the capacity of the chamber is large, the concentration change in the chamber becomes small, and detection as a concentration change becomes difficult. When the number of chambers is small, the measurement throughput deteriorates. Therefore, there is a need for a high-density microchamber array in which a large number of microchambers with extremely small capacities sealed with a lipid bilayer membrane are formed at high density. Japanese Patent Laid-Open No. 2015-040754 discloses such a high-density microchamber array. However, there was an unexamined part about the applied technology.

The inventors diligently studied to find out the applied technology of the conventional high-density microchamber array. As a result, the following knowledge was obtained. It should be noted that the following knowledge is only a trigger for the present disclosure, and does not limit the present disclosure.

That is, the development of the above-described high-density micro-chamber array makes it possible to efficiently perform measurement such as transmembrane-type material transport using membrane proteins. However, in order to form a concentration gradient of a substrate, an inhibitor, etc. on a high-density microchamber array, an expensive and large-scale apparatus is required, and a technique for easily forming a substance concentration gradient has not been realized. Absent. Therefore, in the conventional high-density micro-chamber array, although a large number of micro-chambers are integrated, only one type of sample having a uniform concentration can be measured by one operation. This is a problem common to the entire high-density microchamber array, and remarkably limits the versatility of the analysis technique using the high-density microchamber array.

Based on this insight, the inventors have newly developed a method and mechanism for easily forming a concentration gradient of a substrate, an inhibitor, etc. on a high-density microchamber array, and specifically based on a fluid diffusion model. We have succeeded in developing a new protocol that forms a concentration gradient of a specific substance on a high-density microchamber array. In this technique, various concentration gradients can be formed by adjusting parameters that can be easily changed, such as flow rate and flow rate, and conventionally, it has been required to form concentration gradients. There is no need for expensive and large-scale equipment. In other words, it can be said that the development of this technology is an innovation in comprehensive functional analysis of membrane proteins.

The embodiment described below has been created based on such knowledge.

The concentration gradient forming method on the microreactor chip according to the first aspect of the embodiment includes:
A main layer of the hydrophobic layer of a microreactor chip having a hydrophobic layer, the layer comprising a hydrophobic substance, wherein the openings of the plurality of chambers are regularly arranged on the main surface of the layer. Introducing a first aqueous solution into a first liquid channel provided on a surface and filling each chamber facing the first liquid channel with the first aqueous solution;
A second aqueous solution having a concentration different from that of the first aqueous solution is introduced from the first introduction port of the first liquid flow path, and the gradient according to the distance from the first introduction port to the concentration of the aqueous solution filled in each chamber. Forming a step;
Introducing a lipid-containing organic solvent and a third aqueous solution into the first liquid channel in order, and forming a lipid bilayer so as to seal the aqueous solution at the opening of each chamber;
including.

According to such an aspect, after introducing the first aqueous solution into the first liquid flow path provided on the main surface of the hydrophobic layer and filling each chamber facing the first liquid flow path with the first aqueous solution, A second aqueous solution having a concentration different from that of the first aqueous solution is introduced from the first introduction port of the first liquid channel. At this time, since there is friction on the wall surface of the first liquid channel, the second aqueous solution is less likely to flow than the central portion of the first liquid channel, and due to dilution phenomenon at the interface between the first aqueous solution and the second aqueous solution, A concentration gradient corresponding to the distance from the first inlet is formed in the concentration of the aqueous solution filled in the chamber. That is, when the concentration of the second aqueous solution is lower than the concentration of the first aqueous solution, a concentration gradient is formed such that the concentration of the aqueous solution is lower in the chamber closer to the first inlet, and the concentration of the second aqueous solution is the first concentration. When the concentration is higher than the concentration of the aqueous solution, a concentration gradient is formed such that the concentration of the aqueous solution increases in the chamber closer to the first introduction port. Thereafter, an organic solvent containing lipid and a third aqueous solution are sequentially introduced into the first liquid channel to form a lipid bilayer membrane at the opening of each chamber. As a result, a substance concentration gradient can be easily formed in each chamber of the microreactor chip covered with the lipid bilayer membrane, and a comprehensive and highly efficient biological sample function analysis can be realized by one microreactor chip. .

The concentration gradient forming method on the microreactor chip according to the second aspect of the embodiment is the concentration gradient forming method according to the first aspect,
The first introduction port is formed at one end of the first liquid channel, and the other end of the first liquid channel is open to the atmosphere.

According to such an aspect, the liquid introduced from the first introduction port flows in one direction from one end to the other end of the first liquid flow path, that is, disturbance or stagnation occurs in the liquid flowing direction. Is reduced. Thereby, it is possible to reduce the occurrence of fluctuations in the substance concentration gradient formed on the microreactor chip.

The concentration gradient forming method on the microreactor chip according to the third aspect of the embodiment is the concentration gradient forming method according to the first or second aspect,
In the step of introducing the second aqueous solution into the first liquid flow path, a predetermined volume of the second aqueous solution is introduced at a predetermined flow rate using an electric pipette.

According to such an embodiment, the substance concentration gradient formed on the microreactor chip can be easily controlled, in other words, a desired substance concentration gradient can be easily formed on the microreactor chip.

A concentration gradient forming method on a microreactor chip according to a fourth aspect of the embodiment is a concentration gradient forming method according to any one of the first to third aspects,
The volume of the second aqueous solution introduced into the first liquid channel is 30% to 70% of the volume of the first liquid channel.

A concentration gradient forming method on a microreactor chip according to a fifth aspect of the embodiment is the concentration gradient forming method according to any one of the first to fourth aspects,
A fourth aqueous solution is introduced into a second liquid channel that is provided on the main surface of the hydrophobic tank and is different from the first liquid channel, and each chamber facing the second liquid channel is placed in the fourth aqueous solution. Step to fill with,
A fifth aqueous solution having a concentration different from that of the fourth aqueous solution is introduced from the second introduction port of the second liquid channel, and a gradient according to the distance from the second introduction port to the concentration of the aqueous solution filled in each chamber. Forming a step;
Introducing a lipid-containing organic solvent and a sixth aqueous solution into the second liquid channel in order, and forming a lipid bilayer so as to seal the aqueous solution at the opening of each chamber;
Further included.

According to such an embodiment, on one microreactor chip, a concentration gradient different from the concentration gradient formed in each chamber facing the first liquid flow path is given to each chamber facing the second liquid flow path. Can be formed. In other words, a plurality of kinds of substance concentration gradients can be easily formed on one microreactor chip. Thereby, comprehensive and highly efficient functional analysis of a biological sample by one microreactor chip can be further advanced.

A concentration gradient forming method on a microreactor chip according to a sixth aspect of the embodiment is a concentration gradient forming method according to the fifth aspect,
The second liquid channel is parallel to the first liquid channel.

According to such an aspect, the space between the first liquid channel and the second liquid channel can be narrowed, and the limited space of one microreactor chip can be effectively used.

A concentration gradient forming method on a microreactor chip according to a seventh aspect of the embodiment is the concentration gradient forming method according to any one of the first to sixth aspects,
The capacity of each chamber is 4000 × 10 ⁻¹⁸ m ³ or less.

The microreactor chip according to the eighth aspect of the embodiment is
A hydrophobic layer formed of a hydrophobic substance, wherein the openings of the plurality of chambers are regularly arranged on the main surface of the layer; and
A first liquid channel provided on a main surface of the hydrophobic layer;
With
Each chamber facing the first liquid channel is filled with an aqueous solution, and a lipid bilayer is formed at the opening of each chamber so as to seal the aqueous solution,
The concentration of the aqueous solution filled in each chamber has a gradient according to the distance from the reference position on the first liquid channel.

The microreactor chip according to the ninth aspect of the embodiment is the microreactor chip according to the eighth aspect,
The reference position is defined at one end of the first liquid channel, and the other end of the first liquid channel is open to the atmosphere.

The microreactor chip according to the tenth aspect of the embodiment is the microreactor chip according to the eighth or ninth aspect,
A second liquid channel different from the first liquid channel provided on the main surface of the hydrophobic layer;
Each chamber facing the second liquid channel is filled with an aqueous solution, and a lipid bilayer is formed at the opening of each chamber so as to seal the aqueous solution,
The concentration of the aqueous solution filled in each chamber has a gradient according to the distance from the reference position on the second liquid channel.

A microreactor chip according to an eleventh aspect of the embodiment is a microreactor chip according to the tenth aspect,
The second liquid channel is parallel to the first liquid channel.

A microreactor chip according to a twelfth aspect of the embodiment is the microreactor chip according to any of the eighth to eleventh aspects,
The capacity of each chamber is 4000 × 10 ⁻¹⁸ m ³ or less.

A microreactor chip according to a thirteenth aspect of the embodiment is the microreactor chip according to any one of the eighth to twelfth aspects,
The concentration gradient in the aqueous solution filled in each chamber facing the second liquid flow path is different from the concentration gradient in the aqueous solution filled in each chamber facing the first liquid flow path.

Hereinafter, specific examples of the embodiment will be described in detail with reference to the accompanying drawings. In addition, in each figure, the component which has an equivalent function is attached | subjected the same code | symbol, and detailed description of the component of the same code | symbol is not repeated.

[Configuration of microreactor chip]
FIG. 1 is a plan view illustrating an example of a schematic configuration of a microreactor chip 20 according to an embodiment. FIG. 2 is a view showing a cross section of the microreactor chip 20 shown in FIG. 1 cut along the line AA.

As shown in FIGS. 1 and 2, the microreactor chip 20 includes a substrate 22 and a hydrophobic layer 24 provided on the substrate 22.

The substrate 22 has translucency and is flat. The substrate 22 can be made of, for example, glass or acrylic resin. The material, thickness, shape, and the like of the substrate 22 are such that light incident on the substrate 22 from below the substrate 22 passes through the substrate 22 and enters the chamber 26, and from the chamber 26 to the substrate 22. The incident light is not particularly limited as long as the light can pass through the substrate 22 and escape to the lower side of the substrate 22. Specifically, for example, the thickness of the substrate 22 may be 0.1 mm or more and 5 mm or less, 0.3 mm or more and 3 mm or less, or 0.7 mm or more and 1.5 mm or less. Good. The size of the substrate 22 in plan view is not particularly limited.

The hydrophobic layer 24 is a layer made of a hydrophobic substance. Examples of the hydrophobic substance include a hydrophobic resin such as a fluororesin, and a substance other than a resin such as glass. The thickness of the hydrophobic layer 24 can be appropriately adjusted according to the capacity of the chamber 26 described later. Specifically, for example, it may be 10 nm or more and 100 μm or less, 100 nm or more and 5 μm or less, or 250 nm or more and 1 μm or less.

In the hydrophobic layer 24, openings of a plurality of minute chambers 26 are provided on the main surface of the hydrophobic layer 24 so as to be regularly and densely arranged. The capacity of the chamber 26 is 4000 × 10 ⁻¹⁸ m ³ or less (4000 μm ³ or less). The capacity of the chamber 26 may be, for example, 0.1 × 10 ⁻¹⁸ m ³ or more and 4000 × 10 ⁻¹⁸ m ³ or 0.5 × 10 ⁻¹⁸ m ³ or more and 400 × 10 ⁻¹⁸ m ^{3. Or} may be 1 × 10 ⁻¹⁸ m ³ or more and 40 × 10 ⁻¹⁸ m ³ or less.

The depth of the chamber 26 may be, for example, 10 nm or more and 100 μm or less, 100 nm or more and 5 μm or less, or 250 nm or more and 1 μm or less.

The opening of the chamber 26 can be circular, for example. The diameter of the circle in the case of a circle may be, for example, 0.1 μm or more and 100 μm or less, 0.5 μm or more and 5 μm or less, or 1 μm or more and 10 μm or less.

“Regular” means, for example, that the chambers are arranged on the substrate in a lattice shape, a matrix shape, a staggered shape, or the like as viewed from the thickness direction of the substrate. “Regular” may mean, for example, that the chambers are arranged at regular intervals in a plurality of rows.

“High density” means, for example, that the number of chambers per square mm (1 mm ² ) may be 0.1 × 10 ³ or more and 2000 × 10 ³ or less, or 1 × 10 ³ or more. It may be 1000 × 10 ³ or less, or 5 × 10 ³ or more and 100 × 10 ³ or less. When converted per 1 cm ² (1 × 10 ⁻⁴ m ² ), it may be 10 × 10 ³ or more and 200 × 10 ⁶ or less, or 100 × 10 ³ or more and 100 × 10 ⁶ or less. Alternatively, it may be 0.5 × 10 ⁶ or more and 10 × 10 ⁶ or less.

In the microreactor chip 20, the plurality of chambers 26 have a depth of 100 μm or less and are formed to have a diameter of 100 μm or less when converted into a circle, or have a depth of 2 μm or less and are converted into a circle. Sometimes, the diameter may be 10 μm or less, or the depth may be 1 μm or less, and the diameter may be 5 μm or less when converted into a circle. In this way, it is relatively easy to form the microreactor chip 20 before the formation of the lipid bilayer using a method of forming a thin film of a hydrophobic substance on the surface of the substrate 22 and forming a plurality of minute chambers 26 on the thin film. Can be manufactured. The “diameter” of “when converted to a circle” means a circular diameter having the same area as the shape of a cross section perpendicular to the depth direction. For example, when the cross section is a square of 1 μm square. The diameter when converted to a circle is 2 / √π≈1.1 μm.

The chamber 26 may be formed into a predetermined diameter range including a diameter of 1 μm when converted into a circular shape in a thin film made of a hydrophobic substance having a predetermined thickness range including a thickness of 500 nm. Considering the magnitude of the reaction rate of the biomolecule to be tested and the content of the biomolecule as well as the ease of production, it is considered that the depth and diameter of the chamber 26 are preferably several hundred nm to several μm. Here, the “predetermined thickness range” is, for example, a range of 50 nm, which is 0.1 times 500 nm, and 5 μm or less, which is 10 times 500 nm, or 1 μm, which is 250 nm or more, which is 0.5 times 500 nm, and twice 500 nm Or the following range. The “predetermined diameter range” is, for example, a range of 100 μm that is 0.1 times 1 μm and 10 μm or less that is 10 times that of 1 μm, or a range that is 500 nm or more that is 0.5 times that of 1 μm and 2 μm that is 2 times that of 1 μm. can do.

In one example, each chamber 26 is formed in the hydrophobic layer 24 having a thickness D of 1 μm so that the diameter R is 5 μm. Accordingly, the volume L of each chamber 26 is L = π (2.5 × 10 ⁻⁶ ) ² × 1 × 10 ⁻⁶ m ³ ≈19.6 × 10 ⁻¹⁸ m ³ . If the chambers 26 are arranged at intervals of 2 μm in length and width in plan view, the area S required for one chamber 26 is a square having a side of 7 μm, and S = (7 × 10 ⁻⁶ ) ² m ² = 49 × Calculated as 10 ⁻¹² m ² . Therefore, about 2 × 10 ⁶ chambers (20 × 10 ³ per square mm) per 1 cm ² (1 × 10 ⁻⁴ m ² ) are formed on the glass substrate 22.

Although illustration is omitted, an electrode may be provided in each chamber 26 (for example, the inner surface or the bottom surface of the chamber 26). Each electrode may be electrically connected to each other. The electrode may be made of a metal such as copper, silver, gold, aluminum, or chromium. The electrode is made of a material other than metal, for example, ITO (indium tin oxide), IZO (material made of indium tin oxide and zinc oxide), ZnO, IGZO (material made of indium, gallium, zinc, oxygen), etc. It may be configured.

The thickness of the electrode may be, for example, 10 nm or more and 100 μm or less, 100 nm or more and 5 μm or less, or 250 nm or more and 1 μm or less.

In this configuration, light incident on the substrate 22 from below the substrate 22 passes through the substrate 22 and enters the chamber 26, and light incident on the substrate 22 from the interior of the chamber 26 The light passes through 22 and escapes below the substrate 22.

[Microreactor chip manufacturing method]
Next, a method for manufacturing the microreactor chip 20 will be described with reference to FIG. 3 and FIGS. 4A to 4F. FIG. 3 is a flowchart showing an example of a method for manufacturing the microreactor chip 20. 4A to 4F are diagrams showing each step in the manufacturing method of the microreactor chip 20. FIG.

First, as shown in FIGS. 3 and 4A, as a cleaning process for cleaning the glass surface of the glass substrate 22, the glass substrate 22 is immersed in a 10M potassium hydroxide (KOH) solution for about 24 hours (step S111). Thereby, the surface of the glass substrate 22 is hydrophilic.

Next, as shown in FIG. 4B, a hydrophobic material (for example, fluororesin (CYTOP) manufactured by Asahi Glass Co., Ltd.) is spin coated on the surface of the glass substrate 22 to form a material film 24a. Is brought into close contact with the surface of the glass substrate 22 (step S112). As a condition for spin coating, for example, a condition of 2000 rps and 30 seconds can be used. In this case, the thickness of the material film 24a is about 1 μm. The adhesion of the material film 24a to the surface of the glass substrate 22 can be performed, for example, by baking for 1 hour on a hot plate at 180 ° C.

Next, as shown in FIG. 4C, a resist 25a is formed on the surface of the material film 24a by spin coating, and the resist 25a is brought into close contact with the surface of the material film 24a (step S113). As the resist 25a, AZ-4903 manufactured by AZ Electronic Materials can be used. As conditions for spin coating, for example, conditions of 4000 rps and 60 seconds can be used. The adhesion of the resist 25a to the surface of the material film 24a can be performed, for example, by baking for 5 minutes on a hot plate at 110 ° C. and evaporating the organic solvent in the resist 25a.

Next, as shown in FIG. 4D, the resist 25a is exposed using a mask of the pattern of the chamber 26, developed by immersing in a resist-dedicated developer, and the resist 25b from which a portion for forming the chamber 26 is removed is removed. Form (step S114). As the exposure condition, for example, a condition of irradiating with a UV power of 250 W for 7 seconds using an SAN-EI exposure machine can be used. As the development condition, for example, a condition of immersing in AZ developer made by AZ Electronic Materials for 5 minutes can be used.

Next, as shown in FIG. 4E, the material film 24a masked by the resist 25b is dry-etched to obtain a material film 24b from which the portion to become the chamber 26 is removed from the material film 24a (step S115). As shown in 5F, the resist 25b is removed (step S116). For dry etching, for example, a reactive ion etching apparatus manufactured by Samco can be used, and the conditions of O ₂ 50 sccm, Pressure 10 Pa, Power 50 W, and Time 30 min can be used as etching conditions. The resist 25b can be removed by immersing in acetone, washing with isopropanol, and then washing with pure water.

The plurality of chambers 26 may be formed in the thin film of the hydrophobic material by using a technique other than dry etching, for example, a technique such as nanoimprinting. In the case of dry etching, the inner surface of the chamber 26 is hydrophilic due to the action of O ₂ plasma, and it is preferable to fill the chamber 26 with an aqueous solution.

[Example of concentration gradient formation on a microreactor chip]
Next, an example of a concentration gradient forming method on the microreactor chip 20 according to an embodiment will be described with reference to FIGS. 5 to 7F. FIG. 5 is a plan view showing an apparatus configuration used in an example of a concentration gradient forming method on the microreactor chip 20 according to an embodiment. FIG. 6 is a flowchart illustrating an example of a concentration gradient forming method on the microreactor chip 20 according to an embodiment. 7A to 7F are diagrams showing each step in an example of the concentration gradient forming method on the microreactor chip 20 according to the embodiment.

First, as shown in FIG. 5, a glass plate 44 in which a first introduction port 45 a is formed while a spacer 42 having a “U” shape in plan view is interposed on the main surface of the hydrophobic layer 24 of the microreactor chip 20. Put it on. As a result, a first liquid channel 48a is formed in which the main surface of the hydrophobic layer 24 is a substantially horizontal bottom surface. In the illustrated example, the first liquid channel 48a has a length of 8.1 mm and a width of 2 mm. As shown in FIG. 5, the first introduction port 45a of the glass plate 44 may be positioned so as to be positioned at one end of the first liquid channel 48a, and the other end of the first liquid channel 48a may be open to the atmosphere. .

Next, as shown in FIGS. 6 and 7A, a first aqueous solution containing a surfactant is introduced into the first liquid channel 48a from the first inlet 45a, and the first liquid channel 48a and the first liquid channel 48a are introduced. Each chamber 26 facing the surface is filled with the first aqueous solution (step S11).

Next, as shown in FIGS. 6 and 7B, a second aqueous solution having a concentration different from that of the first aqueous solution is introduced from the first introduction port 45a into the first liquid channel 48a (step S12). At this time, a second aqueous solution having a predetermined volume may be introduced at a predetermined flow rate using an electric pipette. Further, the volume of the second aqueous solution introduced into the first liquid channel 48a may be 30% to 70% or 35% to 65% of the volume of the first liquid channel 48a. It may be 40% to 60%, or 45% to 55%.

When the second aqueous solution is introduced into the first liquid channel 48a from the first introduction port 45a, there is friction on the wall surface of the first liquid channel 48a, so that the second aqueous solution is compared with the central portion of the first liquid channel 48a. Is difficult to flow, and due to the dilution phenomenon at the interface between the first aqueous solution and the second aqueous solution, a concentration gradient corresponding to the distance from the first introduction port 45a is formed in the concentration of the aqueous solution filled in each chamber 26. That is, when the concentration of the second aqueous solution is lower than the concentration of the first aqueous solution, a concentration gradient is formed such that the concentration of the aqueous solution becomes lower in the chamber 26 closer to the first introduction port 45a. When the concentration is higher than the concentration of the first aqueous solution, a concentration gradient is formed such that the concentration of the aqueous solution becomes higher in the chamber 26 closer to the first introduction port 45a.

Next, as shown in FIGS. 6 and 7C to 7F, the organic solvent containing lipid and the third aqueous solution are sequentially introduced from the first introduction port 45a into the first liquid channel 48a (step S13). Here, natural lipids such as soybean and E. coli, and artificial lipids such as DOPE (dioleoylphosphatidylethanolamine) and DOPG (dioleoylphosphatidylglycerol) can be used as the lipid. As the organic solvent, hexadecane or chloroform can be used.

When an organic solvent containing lipid is introduced from the first introduction port 45a into the first liquid channel 48a, the hydrophilic group of the lipid faces the inside of the chamber 26 while the chamber 26 is filled with the aqueous solution. When the inner lipid monolayer film is formed so as to seal the opening of the chamber 26, and then the third aqueous solution is introduced into the first liquid channel 48a from the first introduction port 45a, the hydrophobic group of the lipid is changed. An outer lipid monolayer membrane facing the inner lipid monolayer membrane is formed so as to overlap the inner lipid monolayer membrane. As a result, a lipid bilayer is formed so that the aqueous solution is sealed in the opening of each chamber 26.

After the step of forming the lipid bilayer membrane, a step of reconstituting the membrane protein in the lipid bilayer membrane can also be provided. The process of reconstitution consists of cell membrane fragments containing membrane proteins, lipid bilayer membranes embedded with proteins, water-soluble proteins, liposomes incorporating proteins, and proteins solubilized with surfactants into lipid bilayer membranes. It may be a step of introducing a protein into a lipid bilayer membrane to form a membrane protein. As a method for incorporating a protein into the lipid bilayer membrane, membrane fusion or the like can be used in the case of liposomes, and thermal oscillation or the like can be used in the case of proteins solubilized with a surfactant.

By the method as described above, the concentration of the aqueous solution filled in each chamber 26 has a gradient according to the distance from the reference position (that is, the position of the first introduction port 45a) on the first liquid channel 48a. The chip 20 can be obtained.

Here, light incident on the substrate 22 from below the substrate 22 of the microreactor chip 20 passes through the substrate 22 and enters the chamber 26 and enters the substrate 22 from the inside of the chamber 26. Passes through the substrate 22 and escapes below the substrate 22. When the membrane protein is reconstituted in the lipid bilayer, the function of the membrane protein is to detect light emitted from the fluorescent substance contained in the aqueous solution contained in the chamber 26 using a confocal laser microscope. It can be analyzed by doing. An epi-focal confocal microscope may be used as the microscope.

Next, specific examples verified in connection with the above-described embodiment will be described.

[First embodiment]
As a first example according to the above-described embodiment, the inventors prepare microreactor chips A to E, and supply the first aqueous solution from the first introduction port 45a of each microreactor chip A to E to the first liquid channel 48a. Introduced, each chamber 26 facing the first liquid channel 48a was filled with the first aqueous solution. Here, as a first aqueous solution, Alexa 405 (blue fluorescent dye) and Alexa 488 (green fluorescent dye) are added to a liquid containing 100 mM TRIS and 1 mM magnesium chloride (hereinafter sometimes referred to as “buffer A”). What was added was used.

Next, the inventors introduced a second aqueous solution having a concentration different from that of the first aqueous solution into the first liquid channel 48a from the first introduction port 45a of each of the microreactor chips A to E. Here, as the second aqueous solution, a solution in which Alexa 405 was added to buffer A but Alexa 488 was not added was used. In addition, the inventors introduced a second aqueous solution having a predetermined volume as shown in Table 1 below into the first liquid channel 48a at a predetermined flow rate using an electric pipette.

Since Alexa 488 is not added to the second aqueous solution, the concentration of Alexa 488 in the second aqueous solution is lower than the concentration of Alexa 488 in the first aqueous solution. Therefore, when the second aqueous solution is introduced from the first inlet 45a into the first liquid channel 48a, the concentration of Alexa 488 is diluted in the chamber 26 closer to the first inlet 45a, that is, the aqueous solution filled in each chamber 26 is diluted. A concentration gradient is formed so that the concentration of Alexa 488 gradually increases according to the distance from the first introduction port 45a.

Next, the inventors sequentially introduce the organic solvent containing lipid and the third aqueous solution into the first liquid channel 48 a from the first introduction port 45 a of each microreactor chip A to E, and into the opening of each chamber 26. A lipid bilayer membrane was formed so as to seal the aqueous solution filled in the chamber 26. Here, chloroform containing 0.3 mg / ml POPC was used as the organic solvent containing lipid. Moreover, what diluted buffer solution A to 50% was used as 3rd aqueous solution.

Thereafter, the inventors detected the light emitted from the fluorescent substance contained in the aqueous solution in the chamber 26 for each of the microreactor chips A to E using a confocal laser microscope. FIG. 8 shows a fluorescence image of each chamber 26 according to the distance L from the first introduction port 45a for the microreactor chip C. In the fluorescence image shown in FIG. 8, the intensity of green fluorescence from Alexa 488 is smaller in the chamber 26 closer to the first introduction port 45a. From this, the aqueous solution in each chamber 26 is transferred from the first introduction port 45a. It can be confirmed that a concentration gradient is gradually formed so that the concentration of Alexa 488 gradually increases according to the distance.

FIG. 9A is a graph showing the relationship between the distance L from the first inlet 45a and the intensity of blue fluorescence for the microreactor chips A to C into which the second aqueous solution has been introduced at different flow rates, and FIG. 6 is a graph showing the relationship between the distance L from the first introduction port 45a and the intensity of green fluorescence for the microreactor chips A to C. As shown in FIG. 9B, the gradient of the intensity change of the green fluorescence (gradient of the graph) changes according to the flow rate when the second aqueous solution is introduced, that is, the flow rate when the second aqueous solution is introduced. Accordingly, it can be confirmed that the slope of the concentration gradient of Alexa 488 has changed.

FIG. 10A is a graph showing the relationship between the distance L from the first inlet 45a and the intensity of blue fluorescence for the microreactor chips C to E into which the second aqueous solution has been introduced with different capacities, and FIG. 6 is a graph showing the relationship between the distance L from the first introduction port 45a and the intensity of green fluorescence for the microreactor chips C to E. As shown in FIG. 10B, the gradient of the intensity change of the green fluorescence (gradient of the graph) changes according to the capacity when the second aqueous solution is introduced, that is, the capacity when the second aqueous solution is introduced. Accordingly, it can be confirmed that the slope of the concentration gradient of Alexa 488 has changed.

From the above verification results, it is understood that the gradient of the concentration of the substance formed on the microreactor chip 20 can be easily controlled by controlling the volume and / or flow rate when the second aqueous solution is introduced, that is, the microreactor chip 20. It can be seen that the desired concentration gradient can be formed above.

[Second Embodiment]
As a second example according to the above-described embodiment, the inventors introduce a first aqueous solution into the first liquid channel 48a from the first inlet 45a as shown in FIG. Each chamber 26 facing the surface was filled with a first aqueous solution. Here, as the first aqueous solution, alkaline phosphatase (ALP) enzyme is added in advance to buffer A so as to have an average concentration of 1 or less per chamber 26, and Alexa 405 (blue fluorescent dye) and sTG- What added phos (green fluorescent dye) was used. Note that sTG-phos is a fluorescent substance that emits light when decomposed by ALP.

Next, the inventors introduced a second aqueous solution having a concentration different from that of the first aqueous solution into the first liquid channel 48a from the first introduction port 45a. Here, as the second aqueous solution, a solution in which Alexa 405 was added to buffer A but sTG-phos was not added was used. Since sTG-phos is not added to the second aqueous solution, the concentration of sTG-phos in the second aqueous solution is lower than the concentration of sTG-phos in the first aqueous solution. Therefore, when the second aqueous solution is introduced into the first liquid channel 48a from the first introduction port 45a, the concentration of sTG-phos is diluted in the chamber 26 closer to the first introduction port 45a, that is, each chamber 26 is filled. In the aqueous solution, a concentration gradient is formed so that the concentration of sTG-phos gradually increases according to the distance from the first inlet 45a (see FIG. 11).

Next, the inventors sequentially introduced the organic solvent containing lipid and the third aqueous solution into the first liquid channel 48a from the first inlet 45a, and the chambers 26 were filled in the openings of the chambers 26. A lipid bilayer was formed to seal the aqueous solution. Here, chloroform containing 0.3 mg / ml POPC was used as the organic solvent containing lipid. Moreover, what diluted buffer solution A to 50% was used as 3rd aqueous solution.

Thereafter, the inventors detected light emitted from the fluorescent material contained in the aqueous solution in the chamber 26 using a confocal laser microscope. FIG. 12A shows a green fluorescence image (right diagram), a blue fluorescence image (center diagram), and a green + blue fluorescence image (left diagram) of the chamber at the position of the first introduction port 45a. FIG. 12B shows a green fluorescence image (right diagram), a blue fluorescence image (center diagram), and a green + blue fluorescence image (left diagram) of the chamber at a position 8.1 mm away from the first introduction port 45a. .

As shown in FIG. 12A, green fluorescence indicating that sTG-phos was decomposed in the chamber 26 into which ALP was introduced was visually confirmed at a position 8.1 mm away from the first introduction port 45a. On the other hand, as shown in FIG. 12B, green fluorescence could not be visually confirmed in the chamber 26 into which ALP was introduced at the position of the first introduction port 45a. From this, the concentration of sTG-phos is smaller in the chamber 26 closer to the first introduction port 45a, that is, the aqueous solution in each chamber 26 gradually contains sTG in accordance with the distance from the first introduction port 45a. It can be confirmed that a concentration gradient is formed such that the concentration of -phos is high.

FIG. 13 shows the elapsed time at each position away from the first inlet 45a by 1.8 mm, 2.7 mm, 3.6 mm, 4.5 mm, 5.4 mm, 6.3 mm, 7.2 mm, and 8.1 mm. It is a graph which shows the relationship with the intensity | strength of green fluorescence. In the graph shown in FIG. 13, the gradient of the intensity change of green fluorescence (the gradient of the graph) increases with the distance from the first introduction port 45a, that is, the degradation rate of sTG-phos by the ALP enzyme increases. It can be confirmed that

FIG. 14 is a graph showing the relationship between the substrate concentration and the amount of change in fluorescence intensity per unit time. In FIG. 14, the horizontal axis indicates the concentration of the substrate (that is, sTG-phos) of the aqueous solution filled in each chamber, and the vertical axis indicates the amount of change in the intensity of green fluorescence per unit time, that is, the sTG by the ALP enzyme. -Shows the degradation rate of phos. From the graph shown in FIG. 14, it can be confirmed that the substrate (sTG-phos) concentration and the reaction rate (ie, activity) of the ALP enzyme are in a proportional relationship.

[Third embodiment]
As a third example according to the above-described embodiment, the inventors introduce a first aqueous solution into the first liquid channel 48a from the first inlet 45a of the microreactor chip 20 as shown in FIG. Each chamber 26 facing the liquid flow path 48a was filled with the first aqueous solution. Here, as the first aqueous solution, a solution obtained by adding Alexa 488 (green fluorescent dye) and α-hemolysin to buffer A was used.

Next, the inventors introduced a second aqueous solution having a concentration different from that of the first aqueous solution into the first liquid channel 48a from the first introduction port 45a. Here, as the second aqueous solution, a solution in which Alexa 488 was added to buffer A but α-hemolysin was not added was used. Since α-hemolysin is not added to the second aqueous solution, the concentration of α-hemolysin in the second aqueous solution is lower than the concentration of α-hemolysin in the first aqueous solution. Therefore, when the second aqueous solution is introduced into the first liquid channel 48a from the first introduction port 45a, the concentration of α-hemolysin is diluted in the chamber 26 closer to the first introduction port 45a, that is, each chamber 26 is filled. In the aqueous solution, a concentration gradient is formed such that the concentration of α-hemolysin gradually increases according to the distance from the first inlet 45a (see FIG. 15A).

Next, the inventors sequentially introduce the organic solvent containing lipid and the third aqueous solution into the first liquid channel 48a from the first introduction port 45a, and seal the aqueous solution at the opening of each chamber. A lipid bilayer was formed. Here, chloroform containing 0.3 mg / ml POPC was used as the organic solvent containing lipid. Moreover, what diluted buffer solution A to 50% was used as 3rd aqueous solution.

Thereafter, the inventors detected light emitted from the fluorescent material contained in the aqueous solution in the chamber 26 using a confocal laser microscope. FIG. 15B shows a green fluorescent image immediately after the start of measurement at a position away from the first introduction port 45a by 8.1 mm (left figure), a green fluorescent image after one hour has passed (center figure), and a difference image (right figure). ).

As shown in FIG. 15B, green fluorescence was visible in all the chambers 26 immediately after the start of measurement, but after 1 hour, green fluorescence was no longer visible in some chambers 26. This is because α-hemolysin in the chamber 26 is incorporated into the lipid bilayer during 1 hour, forming a micropore (nanopore) in the lipid bilayer, and Alexa 488 (green fluorescent dye) in the chamber 26 It is presumed that this leaked out of the chamber 26 through the nanopore.

FIG. 16 is a graph showing the relationship between the elapsed time and the fluorescence intensity of the chamber 26. In the graph shown in FIG. 16, the fluorescence intensity gradually decreases with the passage of time. From this, a lipid bilayer membrane in which nanopores due to α-hemolysin are formed is formed at the opening of the chamber 26. It can be confirmed that it exists.

FIG. 17 is a graph showing the relationship between the concentration of α-hemolysin and the ratio of the chamber 26 in which nanopores are formed on the lipid bilayer membrane. In FIG. 17, the horizontal axis indicates the concentration of α-hemolysin in the aqueous solution filled in each chamber 26, and the vertical axis indicates the ratio of the chamber 26 in which nanopores are formed in the lipid bilayer membrane, that is, a predetermined time elapsed. The ratio of the chambers 26 where green fluorescence can no longer be seen later is shown. From the graph shown in FIG. 17, it can be confirmed that when the concentration of α-hemolysin in the aqueous solution exceeds 1 μg / ml, the rate of formation of nanopores in the lipid bilayer increases in a seventh order function.

As described above, according to the above-described embodiment, the first aqueous solution is introduced into the first liquid channel 48 a provided on the main surface of the hydrophobic layer 24, and each chamber 26 facing the first liquid channel 48 a. Is filled with the first aqueous solution, and then a second aqueous solution having a concentration different from that of the first aqueous solution is introduced from the first introduction port 45a of the first liquid channel 48a. At this time, since there is friction on the wall surface of the first liquid channel 48a, the second aqueous solution is less likely to flow compared to the central portion of the first liquid channel 48a, and due to a dilution phenomenon at the interface between the first aqueous solution and the second aqueous solution. A concentration gradient corresponding to the distance from the first inlet 45a is formed in the concentration of the aqueous solution filled in each chamber 26. That is, when the concentration of the second aqueous solution is lower than the concentration of the first aqueous solution, a concentration gradient is formed such that the concentration of the aqueous solution becomes lower in the chamber 26 closer to the first introduction port 45a. When the concentration is higher than the concentration of the first aqueous solution, a concentration gradient is formed such that the concentration of the aqueous solution becomes higher in the chamber 26 closer to the first introduction port 45a. Thereafter, an organic solvent containing lipid and a third aqueous solution are sequentially introduced into the first liquid channel 48 a to form a lipid bilayer membrane at the opening of each chamber 26. As a result, a substance concentration gradient can be easily formed in each chamber of the microreactor chip covered with the lipid bilayer membrane, and a comprehensive and highly efficient biological sample function analysis can be realized by one microreactor chip. . For example, to contribute to drug discovery, in order to measure the affinity of a new drug for membrane proteins, which are the main target of marketed drugs, it is costly to measure a wide range of drug concentrations. An expensive and large-scale apparatus is necessary or complicated work is involved. In contrast, when the technology according to the above-described embodiment is used, a wide concentration gradient of the drug can be formed on one microreactor chip, so that the affinity of the drug to the membrane protein can be easily quantified with only one measurement. It becomes possible to do.

Further, according to the above-described embodiment, the first introduction port 45a is formed at one end of the first liquid channel 48a, and the other end of the first liquid channel 48a is open to the atmosphere. The liquid introduced from the introduction port 45a flows in one direction from one end to the other end of the first liquid channel 48a, that is, the occurrence of turbulence and stagnation in the liquid flowing direction is reduced. Thereby, it is possible to reduce the occurrence of fluctuations in the concentration gradient of the substance formed on the microreactor chip 20.

In addition, according to the above-described embodiment, the second aqueous solution having a predetermined volume is introduced into the first liquid channel 48a at a predetermined flow rate using the electric pipette, and thus formed on the microreactor chip 20. The substance concentration gradient can be easily controlled, in other words, a desired substance concentration gradient can be easily formed on the microreactor chip 20.

In the above-described embodiment, as shown in FIGS. 7A to 7F, each chamber 26 has an upward opening in a mode in which the first liquid channel 48a is provided above the hydrophobic layer 24 of the microreactor chip 20. Although the lipid bilayer membrane is formed, the present invention is not limited to this, and each chamber 26 is configured in a mode in which FIGS. 7A to 7F are turned upside down, that is, in a mode in which the first liquid channel 48a is provided below the hydrophobic layer 24. A lipid bilayer membrane may be formed in the downward opening. Alternatively, the lateral openings of the respective chambers 26 in a mode in which FIGS. 7A to 7F are rotated by 90 ° about a rotation axis extending in the left-right direction, that is, in a mode in which the first liquid channel 48a is provided on the side of the hydrophobic layer 24. A lipid bilayer membrane may be formed in the part.

Note that various modifications can be made to the above-described embodiment. Hereinafter, an example of modification will be described with reference to the drawings. In the following description and the drawings used in the following description, the same reference numerals as those used for the corresponding parts in the above-described embodiment are used for the parts that can be configured in the same manner as in the above-described embodiment, and overlapping descriptions are provided. Is omitted.

[Modified example of concentration gradient formation on microreactor chip]
FIG. 18 is a plan view showing an apparatus configuration used in a modified example of the concentration gradient forming method on the microreactor chip 20 according to the embodiment. FIG. 19 is a flowchart illustrating a modification of the concentration gradient forming method on the microreactor chip 20 according to the embodiment.

In the example shown in FIGS. 18 and 19, first, the first introduction port 45a and the second introduction port 45a are interposed on the main surface of the hydrophobic layer 24 of the microreactor chip 20 with a spacer 42 ′ having a “E” shape in plan view interposed therebetween. A glass plate 44 ′ having a mouth 45b is placed. Thus, the first liquid channel 48a and the second liquid channel 48b are formed in which the main surface of the hydrophobic layer 24 is a substantially horizontal bottom surface. The first liquid channel 48a and the second liquid channel 48b may have the same size, for example, a length of 8.1 mm and a width of 2 mm. As shown in FIG. 18, the first introduction port 45a of the glass plate 44 'is positioned so as to be positioned at one end of the first liquid channel 48a, and the other end of the first liquid channel 48a is opened to the atmosphere. Good. Similarly, the second introduction port 45b of the glass plate 44 'may be positioned so as to be positioned at one end of the second liquid channel 48b, and the other end of the second liquid channel 48b may be opened to the atmosphere.

As shown in FIG. 18, the second liquid channel 48b may be parallel to the first liquid channel 48a. In this case, the space between the first liquid channel 48a and the second liquid channel 48b can be narrowed, and the limited space of one microreactor chip 20 can be used effectively.

After the first liquid channel 48a and the second liquid channel 48b are provided on the main surface of the hydrophobic layer 24, referring to FIG. 19, the first liquid is supplied from the first inlet 45a in the same manner as in the above-described embodiment. The first aqueous solution is introduced into the flow channel 48a, and the first liquid flow channel 48a and the chamber 26 facing the first liquid flow channel 48a are filled with the first aqueous solution (step S11). A second aqueous solution having a concentration different from that of the first aqueous solution is introduced into the liquid channel 48a, and a concentration gradient corresponding to the distance from the first introduction port 45a is formed in the concentration of the aqueous solution filled in each chamber 26 (step S12), then, the lipid-containing organic solvent and the third aqueous solution are sequentially introduced from the first introduction port 45a into the first liquid channel 48a, and the aqueous solution is sealed in the opening of each chamber 26. Form a double membrane ( Step S13).

Next, after forming a lipid bilayer membrane at the opening of each chamber 26 facing the first liquid channel 48a, as shown in FIG. 19, using the electric pipette, the second liquid flow from the second inlet 45b. The fourth aqueous solution is introduced into the channel 48b, and the second liquid channel 48b and the chamber 26 facing the second liquid channel 48b are filled with the fourth aqueous solution (step S14). The fourth aqueous solution may have the same composition as the first aqueous solution or a different composition.

Next, as shown in FIG. 19, a fifth aqueous solution having a concentration different from that of the fourth aqueous solution is introduced from the second introduction port 45b into the second liquid channel 48b using an electric pipette (step S15). The fifth aqueous solution may have the same composition as the second aqueous solution or a different composition. When the fifth aqueous solution is introduced into the second liquid channel 48b from the second introduction port 45b, the fifth aqueous solution flows compared to the central portion of the second liquid channel 48b because there is friction on the wall surface of the second liquid channel 48b. It is difficult, and due to the dilution phenomenon at the interface between the fourth aqueous solution and the fifth aqueous solution, a concentration gradient corresponding to the distance from the second introduction port 45b is formed in the concentration of the aqueous solution filled in each chamber 26. That is, when the concentration of the fifth aqueous solution is lower than the concentration of the fourth aqueous solution, a concentration gradient is formed such that the concentration of the aqueous solution becomes lower in the chamber 26 closer to the second introduction port 45b. When the concentration is higher than the concentration of the fourth aqueous solution, a concentration gradient is formed such that the concentration of the aqueous solution becomes higher in the chamber 26 closer to the second introduction port 45b.

Next, as shown in FIG. 19, an organic solvent containing lipid and a sixth aqueous solution are sequentially introduced from the second introduction port 45 b into the second liquid channel 48 b, and the aqueous solution is introduced into the opening of each chamber 26. A lipid bilayer is formed so as to be sealed (step S16). The sixth aqueous solution may have the same composition as the third aqueous solution or a different composition.

After the step of forming a lipid bilayer membrane (steps S13 and S16), a step of reconstituting membrane proteins in the lipid bilayer membrane may be provided. The process of reconstitution consists of cell membrane fragments containing membrane proteins, lipid bilayer membranes embedded with proteins, water-soluble proteins, liposomes incorporating proteins, and proteins solubilized with surfactants into lipid bilayer membranes. It may be a step of introducing a protein into a lipid bilayer membrane to form a membrane protein. As a method for incorporating a protein into the lipid bilayer membrane, membrane fusion or the like can be used in the case of liposomes, and thermal oscillation or the like can be used in the case of proteins solubilized with a surfactant.

According to such an embodiment, on one microreactor chip 20, a concentration gradient different from the concentration gradient formed in each chamber 26 facing the first liquid channel 48a is caused to face the second liquid channel 48b. Can be formed in each chamber 26. In other words, a plurality of types of substance concentration gradients can be easily formed on one microreactor chip 20. As a result, it is possible to further proceed with comprehensive and highly efficient biological sample function analysis using one microreactor chip 20.

In the example shown in FIGS. 18 and 19, a spacer 42 ′ having three extending portions parallel to each other on the main surface of the hydrophobic layer 24 of the microreactor chip 20 (a spacer 42 ′ having a “E” shape in plan view). The two liquid flow paths 48a and 48b that are parallel to each other are formed on the main surface of the hydrophobic layer 24 by placing the glass plate 44 'on which two inlets 45a and 45b are formed. However, the present invention is not limited to this, while interposing spacers 42 ′ having (N + 1) (N is a natural number of 3 or more) extending portions parallel to each other on the main surface of the hydrophobic layer 24 of the microreactor chip 20, N liquid flow paths parallel to each other may be formed on the main surface of the hydrophobic layer 24 by placing a glass plate 44 ′ having N introduction ports.

The description of the above-described embodiments and individual modifications and the disclosure of the drawings are merely examples for explaining the invention described in the claims, and the description of the above-described embodiments and individual modifications. The invention described in the scope of claims is not limited by the description or the disclosure of the drawings. The components of the above-described embodiments and individual modifications can be arbitrarily combined without departing from the gist of the invention.

Claims (13)

A main layer of the hydrophobic layer of a microreactor chip having a hydrophobic layer, the layer comprising a hydrophobic substance, wherein the openings of the plurality of chambers are regularly arranged on the main surface of the layer. Introducing a first aqueous solution into a first liquid channel provided on a surface and filling each chamber facing the first liquid channel with the first aqueous solution;
A second aqueous solution having a concentration different from that of the first aqueous solution is introduced from the first introduction port of the first liquid flow path, and the gradient according to the distance from the first introduction port to the concentration of the aqueous solution filled in each chamber. Forming a step;
Introducing a lipid-containing organic solvent and a third aqueous solution into the first liquid channel in order, and forming a lipid bilayer so as to seal the aqueous solution at the opening of each chamber;
A method for forming a concentration gradient on a microreactor chip. The first inlet is formed at one end of the first liquid channel, and the other end of the first liquid channel is open to the atmosphere.
The concentration gradient forming method according to claim 1. In the step of introducing the second aqueous solution into the first liquid channel, the second aqueous solution having a predetermined volume is introduced at a predetermined flow rate using an electric pipette.
The concentration gradient forming method according to claim 1 or 2. The volume of the second aqueous solution introduced into the first liquid channel is 30% to 70% of the volume of the first liquid channel.
The concentration gradient forming method according to any one of claims 1 to 3. A fourth aqueous solution is introduced into a second liquid channel that is provided on the main surface of the hydrophobic tank and is different from the first liquid channel, and each chamber facing the second liquid channel is placed in the fourth aqueous solution. Step to fill with,
A fifth aqueous solution having a concentration different from that of the fourth aqueous solution is introduced from the second introduction port of the second liquid channel, and a gradient according to the distance from the second introduction port to the concentration of the aqueous solution filled in each chamber. Forming a step;
Introducing a lipid-containing organic solvent and a sixth aqueous solution into the second liquid channel in order, and forming a lipid bilayer so as to seal the aqueous solution at the opening of each chamber;
The method for forming a concentration gradient according to any one of claims 1 to 4, further comprising: The second liquid channel is parallel to the first liquid channel;
The concentration gradient forming method according to claim 5. The capacity of each chamber is 4000 × 10 ⁻¹⁸ m ³ or less,
The concentration gradient forming method according to any one of claims 1 to 6. A hydrophobic layer formed of a hydrophobic substance, wherein the openings of the plurality of chambers are regularly arranged on the main surface of the layer; and
A first liquid channel provided on a main surface of the hydrophobic layer;
With
Each chamber facing the first liquid channel is filled with an aqueous solution, and a lipid bilayer is formed at the opening of each chamber so as to seal the aqueous solution,
The microreactor chip, wherein the concentration of the aqueous solution filled in each chamber has a gradient corresponding to the distance from the reference position on the first liquid channel. The reference position is defined at one end of the first liquid channel, and the other end of the first liquid channel is open to the atmosphere.
The microreactor chip according to claim 8. A second liquid channel different from the first liquid channel provided on the main surface of the hydrophobic layer;
Each chamber facing the second liquid channel is filled with an aqueous solution, and a lipid bilayer is formed at the opening of each chamber so as to seal the aqueous solution,
The concentration of the aqueous solution filled in each chamber has a gradient according to the distance from the reference position on the second liquid channel.
The microreactor chip according to claim 8 or 9. The second liquid channel is parallel to the first liquid channel;
The microreactor chip according to claim 10. The capacity of each chamber is 4000 × 10 ⁻¹⁸ m ³ or less,
The microreactor chip according to any one of claims 8 to 11. The concentration gradient in the aqueous solution filled in each chamber facing the second liquid flow path is different from the concentration gradient in the aqueous solution filled in each chamber facing the first liquid flow path.
The microreactor chip according to any one of claims 8 to 12.

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