WO2019208795A1 - Procédé de formation d'un gradient de concentration sur une puce de microréacteur et puce de microréacteur - Google Patents

Procédé de formation d'un gradient de concentration sur une puce de microréacteur et puce de microréacteur Download PDF

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Publication number
WO2019208795A1
WO2019208795A1 PCT/JP2019/018008 JP2019018008W WO2019208795A1 WO 2019208795 A1 WO2019208795 A1 WO 2019208795A1 JP 2019018008 W JP2019018008 W JP 2019018008W WO 2019208795 A1 WO2019208795 A1 WO 2019208795A1
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aqueous solution
liquid channel
chamber
concentration
microreactor chip
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PCT/JP2019/018008
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English (en)
Japanese (ja)
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力也 渡邉
博行 野地
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国立大学法人東京大学
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations

Definitions

  • 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.
  • 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.
  • a concentration gradient forming method on a microreactor chip 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. 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 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. 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. 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. 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
  • 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.
  • 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. 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.
  • FIG. 10B is a graph showing the measurement
  • 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. 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
  • 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. 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. 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 ⁇ -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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • it can be said that the development of this technology is an innovation in comprehensive functional analysis of membrane proteins.
  • the concentration gradient forming method on the microreactor chip 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.
  • 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.
  • 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.
  • 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.
  • a concentration gradient is formed such that the concentration of the aqueous solution increases in the chamber closer to the first introduction port.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • a plurality of kinds of substance concentration gradients can be easily formed on one microreactor chip.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m or less, 100 nm or more and 5 ⁇ m or less, or 250 nm or more and 1 ⁇ m or less.
  • 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 ⁇ m 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 ⁇ m or less, 100 nm or more and 5 ⁇ m or less, or 250 nm or more and 1 ⁇ m 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 ⁇ m or more and 100 ⁇ m or less, 0.5 ⁇ m or more and 5 ⁇ m or less, or 1 ⁇ m or more and 10 ⁇ m 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.
  • the plurality of chambers 26 have a depth of 100 ⁇ m or less and are formed to have a diameter of 100 ⁇ m or less when converted into a circle, or have a depth of 2 ⁇ m or less and are converted into a circle.
  • the diameter may be 10 ⁇ m or less, or the depth may be 1 ⁇ m or less, and the diameter may be 5 ⁇ m 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 ⁇ m square. The diameter when converted to a circle is 2 / ⁇ 1.1 ⁇ m.
  • the chamber 26 may be formed into a predetermined diameter range including a diameter of 1 ⁇ m 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 ⁇ m.
  • the “predetermined thickness range” is, for example, a range of 50 nm, which is 0.1 times 500 nm, and 5 ⁇ m or less, which is 10 times 500 nm, or 1 ⁇ m, 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 ⁇ m that is 0.1 times 1 ⁇ m and 10 ⁇ m or less that is 10 times that of 1 ⁇ m, or a range that is 500 nm or more that is 0.5 times that of 1 ⁇ m and 2 ⁇ m that is 2 times that of 1 ⁇ m. can do.
  • 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 ⁇ m or less, 100 nm or more and 5 ⁇ m or less, or 250 nm or more and 1 ⁇ m or less.
  • 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. 3 is a flowchart showing an example of a method for manufacturing the microreactor chip 20.
  • 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.
  • KOH potassium hydroxide
  • a hydrophobic material for example, fluororesin (CYTOP) manufactured by Asahi Glass Co., Ltd.
  • CYTOP fluororesin
  • a condition for spin coating for example, a condition of 2000 rps and 30 seconds can be used.
  • the thickness of the material film 24a is about 1 ⁇ m.
  • 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.
  • 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).
  • the resist 25a AZ-4903 manufactured by AZ Electronic Materials can be used.
  • 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.
  • 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) 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.
  • 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).
  • the resist 25b is removed (step S116).
  • 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.
  • a technique other than dry etching for example, a technique such as nanoimprinting.
  • 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.
  • 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.
  • 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.
  • a first liquid channel 48a is formed in which the main surface of the hydrophobic layer 24 is a substantially horizontal bottom surface.
  • the first liquid channel 48a has a length of 8.1 mm and a width of 2 mm.
  • 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. .
  • 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).
  • 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).
  • a second aqueous solution having a predetermined volume may be introduced at a predetermined flow rate using an electric pipette.
  • 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%.
  • 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.
  • 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.
  • 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).
  • natural lipids such as soybean and E. coli
  • artificial lipids such as DOPE (dioleoylphosphatidylethanolamine) and DOPG (dioleoylphosphatidylglycerol) can be used as the lipid.
  • DOPE dioleoylphosphatidylethanolamine
  • DOPG dioleoylphosphatidylglycerol
  • the organic solvent hexadecane or chloroform can be used.
  • the hydrophilic group of the lipid faces the inside of the chamber 26 while the chamber 26 is filled with the aqueous solution.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Alexa 405 blue fluorescent dye
  • Alexa 488 green fluorescent dye
  • buffer A a liquid containing 100 mM TRIS and 1 mM magnesium chloride
  • 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.
  • the second aqueous solution a solution in which Alexa 405 was added to buffer A but Alexa 488 was not added was used.
  • 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.
  • Alexa 488 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.
  • 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.
  • chloroform containing 0.3 mg / ml POPC was used as the organic solvent containing lipid.
  • what diluted buffer solution A to 50% was used as 3rd aqueous solution.
  • 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.
  • 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
  • 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.
  • the gradient of the intensity change of the green fluorescence 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
  • 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.
  • the gradient of the intensity change of the green fluorescence 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.
  • 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.
  • 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.
  • 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.
  • Alexa 405 blue fluorescent dye
  • sTG- What added phos green fluorescent dye
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • chloroform containing 0.3 mg / ml POPC was used as the organic solvent containing lipid.
  • what diluted buffer solution A to 50% was used as 3rd aqueous solution.
  • 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. .
  • 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
  • the gradient of the intensity change of green fluorescence 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.
  • 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.
  • 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.
  • the first aqueous solution a solution obtained by adding Alexa 488 (green fluorescent dye) and ⁇ -hemolysin to buffer A was used.
  • 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.
  • the second aqueous solution a solution in which Alexa 488 was added to buffer A but ⁇ -hemolysin was not added was used. Since ⁇ -hemolysin is not added to the second aqueous solution, the concentration of ⁇ -hemolysin in the second aqueous solution is lower than the concentration of ⁇ -hemolysin in the first aqueous solution.
  • the concentration of ⁇ -hemolysin is diluted in the chamber 26 closer to the first introduction port 45a, that is, each chamber 26 is filled.
  • a concentration gradient is formed such that the concentration of ⁇ -hemolysin gradually increases according to the distance from the first inlet 45a (see FIG. 15A).
  • 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.
  • chloroform containing 0.3 mg / ml POPC was used as the organic solvent containing lipid.
  • what diluted buffer solution A to 50% was used as 3rd aqueous solution.
  • 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). ).
  • FIG. 16 is a graph showing the relationship between the elapsed time and the fluorescence intensity of the chamber 26.
  • the fluorescence intensity gradually decreases with the passage of time. From this, a lipid bilayer membrane in which nanopores due to ⁇ -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 ⁇ -hemolysin and the ratio of the chamber 26 in which nanopores are formed on the lipid bilayer membrane.
  • the horizontal axis indicates the concentration of ⁇ -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 ⁇ -hemolysin in the aqueous solution exceeds 1 ⁇ g / ml, the rate of formation of nanopores in the lipid bilayer increases in a seventh order function.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lateral openings of the respective chambers 26 in a mode in which FIGS.
  • a lipid bilayer membrane may be formed in the part.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the second liquid channel 48b may be parallel to the first liquid channel 48a.
  • 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.
  • 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.
  • Step S13 Form a double membrane
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne un procédé de formation d'un gradient de concentration sur une puce de microréacteur comprenant les étapes consistant à : introduire une première solution aqueuse dans un premier trajet d'écoulement de liquide disposé sur la surface principale d'une couche hydrophobe d'une puce de microréacteur pourvue de la couche hydrophobe, la couche hydrophobe étant constituée d'une substance hydrophobe et ayant, formée sur sa surface principale, des ouvertures pour de multiples chambres, et les ouvertures étant disposées régulièrement sur la surface principale de la couche, moyennant quoi chacune des chambres qui font face au premier trajet d'écoulement de liquide étant remplie avec la première solution aqueuse ; introduire une deuxième solution aqueuse ayant une concentration différente de celle de la première solution aqueuse par un premier orifice d'entrée du premier trajet d'écoulement de liquide pour former un gradient dans la concentration d'une solution aqueuse remplissant chacune des chambres, le gradient correspondant à la distance par rapport au premier orifice d'entrée ; et introduire un solvant organique contenant un lipide et une troisième solution aqueuse dans cet ordre dans le premier trajet d'écoulement de liquide pour former une bicouche lipidique de telle sorte que l'ouverture pour chacune des chambres est scellée avec une solution aqueuse.
PCT/JP2019/018008 2018-04-26 2019-04-26 Procédé de formation d'un gradient de concentration sur une puce de microréacteur et puce de microréacteur WO2019208795A1 (fr)

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Citations (2)

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JP2015040754A (ja) * 2013-08-21 2015-03-02 国立大学法人 東京大学 高密度微小チャンバーアレイおよびその製造方法
WO2018003856A1 (fr) * 2016-06-28 2018-01-04 学校法人 慶應義塾 Plaque de micropuits pour former un réseau de gouttelettes et procédé de fabrication d'un réseau de gouttelettes

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JP2015040754A (ja) * 2013-08-21 2015-03-02 国立大学法人 東京大学 高密度微小チャンバーアレイおよびその製造方法
WO2018003856A1 (fr) * 2016-06-28 2018-01-04 学校法人 慶應義塾 Plaque de micropuits pour former un réseau de gouttelettes et procédé de fabrication d'un réseau de gouttelettes

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