WO2016159068A1 - Réseau de micro-puits, son procédé de fabrication, dispositif micro-fluidique, procédé d'étanchéité de liquide aqueux dans un puits du réseau de micro-puits et procédé d'analyse de liquide aqueux - Google Patents

Réseau de micro-puits, son procédé de fabrication, dispositif micro-fluidique, procédé d'étanchéité de liquide aqueux dans un puits du réseau de micro-puits et procédé d'analyse de liquide aqueux Download PDF

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WO2016159068A1
WO2016159068A1 PCT/JP2016/060367 JP2016060367W WO2016159068A1 WO 2016159068 A1 WO2016159068 A1 WO 2016159068A1 JP 2016060367 W JP2016060367 W JP 2016060367W WO 2016159068 A1 WO2016159068 A1 WO 2016159068A1
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microwell array
well
aqueous liquid
wells
hydrophobic
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PCT/JP2016/060367
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English (en)
Japanese (ja)
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圭佑 後藤
牧野　洋一
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凸版印刷株式会社
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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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81B—MICROSTRUCTURAL DEVICES OR SYSTEMS, e.g. MICROMECHANICAL DEVICES
    • B81B1/00—Devices without movable or flexible elements, e.g. microcapillary devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81C—PROCESSES OR APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OR TREATMENT OF MICROSTRUCTURAL DEVICES OR SYSTEMS
    • B81C1/00—Manufacture or treatment of devices or systems in or on a substrate
    • 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
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass

Definitions

  • the present invention relates to a microwell array and a manufacturing method thereof, a microfluidic device, a method for enclosing an aqueous liquid in the wells of the microwell array, and a method for analyzing an aqueous liquid.
  • microwell arrays having various types of fine channel structures formed by using etching techniques, photolithography techniques, and the like used in semiconductor circuit manufacturing techniques have been studied.
  • the wells of these microwell arrays are used as chemical reaction vessels for causing various biochemical or chemical reactions in a small volume of fluid.
  • Patent Documents 1 to 4 describe the use of such a microfluidic system as various microchips and biochips.
  • digital PCR technology involves PCR amplification by dividing a mixture of a reagent and nucleic acid into countless microdroplets so that a signal such as fluorescence can be detected from the droplet containing nucleic acid. This is a technique for performing quantification by counting the number of detected droplets.
  • a method for producing a microdroplet As a method for producing a microdroplet, a method for producing a microdroplet by dividing with a sealing liquid, or a reagent is put into a hole formed on a substrate, and then a microdroplet is made by adding a sealing liquid.
  • a manufacturing method (see, for example, Patent Document 4) has been studied.
  • the microwell array according to the first aspect of the present invention includes a plurality of wells having a hydrophilic portion and a hydrophobic portion on the well surface.
  • the capacity of each of the plurality of wells may be 1 fL to 6 nL.
  • the density of the plurality of wells may be 100,000 to 10 million / cm 2 .
  • the plurality of wells may have the hydrophobic portion at the top of the well and have the hydrophilic portion at a position closer to the well bottom than the hydrophobic portion.
  • the plurality of wells may be provided on a substrate that is transparent to electromagnetic waves.
  • the electromagnetic waves may be X-rays, ultraviolet rays, visible rays, or infrared rays.
  • the substrate may be formed of a material that does not substantially have autofluorescence.
  • the contact angle of the hydrophilic portion measured in accordance with the sessile drop method defined in JIS R3257-1999 may be less than 70 degrees.
  • the hydrophilic portion may be formed of a siloxane polymer.
  • the contact angle of the hydrophobic part measured according to the sessile drop method defined in JIS R3257-1999 may be 70 degrees or more.
  • the hydrophobic portion may be formed of a novolac resin.
  • the microfluidic device includes a flow path and the microwell array according to the above aspect disposed in the flow path.
  • a method for enclosing an aqueous liquid in a well by preparing a microfluidic device including a flow path and the microwell array according to the above aspect disposed in the flow path.
  • An aqueous liquid is sent to the flow path of the fluidic device, the aqueous liquid is introduced to the well of the microwell array, a sealing liquid is sent to the flow path, and the aqueous liquid introduced into the well A sealing liquid layer is formed on the upper layer, and the aqueous liquid is sealed in the well.
  • the method for producing a microwell array according to the fourth aspect of the present invention includes forming a hydrophilic layer by laminating a hydrophilic material on a substrate having electromagnetic wave permeability, and laminating a hydrophobic material on the hydrophilic layer. Forming a layer and drilling the hydrophobic layer to form a plurality of wells;
  • the plurality of wells may be formed by excavating the hydrophobic layer and the hydrophilic layer.
  • the method for analyzing an aqueous liquid according to the fifth aspect of the present invention detects the fluorescence or phosphorescence emitted from the well by irradiating electromagnetic waves to the wells of the microwell array according to the above aspect in which the aqueous liquid is enclosed.
  • the fluorescence or the phosphorescence may be detected on the substrate of the microwell array according to the above aspect.
  • a microwell array in which an aqueous liquid can be easily sealed in a well.
  • the manufacturing method of the microwell array which concerns on the said aspect, a microfluidic device, the method of enclosing aqueous liquid in the well of a microwell array, and the analysis method of aqueous liquid can be provided.
  • FIG. 1A is a perspective view showing a microfluidic device according to a second embodiment.
  • 1B is a cross-sectional view taken along line bb in the microfluidic device of FIG. 1A.
  • 1C is a cross-sectional view taken along line cc of the microfluidic device of FIG. 1A.
  • FIG. 2A is a perspective view showing a microfluidic device according to a third embodiment.
  • 2B is a cross-sectional view taken along line bb in the microfluidic device of FIG. 2A.
  • 2C is a cross-sectional view taken along line cc in the microfluidic device of FIG. 2A. It is sectional drawing which shows the structure of the microwell array concerning 5th Embodiment.
  • FIG. 6 is a cross-sectional view showing the structure of a microwell array of Comparative Example 1.
  • FIG. It is sectional drawing which shows the state which enclosed the aqueous liquid in the microwell array of the microfluidic device of Experimental example 1.
  • FIG. It is a photograph which shows the result of having observed the microwell array of the microfluidic device of Experimental example 1 from the board
  • FIG. It is sectional drawing which shows the state which enclosed the aqueous liquid in the microwell array of the microfluidic device of Experimental example 2.
  • FIG. It is a photograph which shows the result of having observed the microwell array of the microfluidic device of Experimental example 2 from the board
  • the first embodiment of the present invention provides a microwell array comprising a plurality of wells having a hydrophilic portion and a hydrophobic portion on a well surface.
  • enclosing the aqueous liquid means introducing the aqueous liquid into a plurality of wells constituting the microwell array, and further isolating the liquids introduced into the wells so as not to be mixed with each other.
  • the isolation method include forming an oil layer on the upper portion of the well after introducing an aqueous liquid into the well.
  • microwell array it is possible to easily enclose an aqueous liquid in 90% or more, for example, 95% or more, for example, 99% or more, for example, 100%, of the plurality of wells constituting the microwell array. .
  • the volume per well is, for example, 1 femtoliter (fL) to 6 nanoliters (nL), preferably 1 fL to 5 picoliters (pL), and more preferably Is 1 fL to 300 fL.
  • an enzymatic reaction such as digital PCR or invader reaction can be suitably performed.
  • gene mutation detection can be performed by the digital PCR.
  • the reaction volume is smaller even if the number of wells is the same.
  • the material of the microwell array is preferably a material that does not inhibit the enzyme reaction.
  • the shape and size of the wells of the microwell array are not particularly limited. For example, when various biochemical reactions are performed in microdroplets, one to several biomolecules or carriers can be accommodated. It preferably has a shape and size.
  • the carrier may be a bead, for example, and a biomolecule as a sample may be bound to the carrier.
  • the shape of the well is, for example, a cylindrical shape, a polyhedron composed of a plurality of surfaces (eg, rectangular parallelepiped, hexagonal prism, octagonal prism, etc.), inverted cone, inverted pyramid (inverted triangular pyramid, inverted quadrangular pyramid, inverted A pentagonal pyramid, an inverted hexagonal pyramid, an inverted polygonal pyramid with a heptagon or more), or a combination of two or more of these shapes.
  • a cylindrical shape a polyhedron composed of a plurality of surfaces (eg, rectangular parallelepiped, hexagonal prism, octagonal prism, etc.), inverted cone, inverted pyramid (inverted triangular pyramid, inverted quadrangular pyramid, inverted A pentagonal pyramid, an inverted hexagonal pyramid, an inverted polygonal pyramid with a heptagon or more), or a combination of two or more of these shapes.
  • a part may be cylindrical and the rest may be an inverted cone.
  • the bottom of the cone or pyramid is the entrance (opening) of the well, but the reverse cone shape or the inverted pyramid shape may be partially cut off from the top. .
  • the bottom of the microwell is flat.
  • the bottom of the microwell is usually flat, but may be a curved surface (convex surface or concave surface).
  • the bottom of the microwell can be curved as in the case of a shape obtained by cutting a part from the top of an inverted cone or inverted pyramid.
  • the diameter When the well is cylindrical, the diameter may be 3 to 10 ⁇ m for the purpose of enclosing an aqueous liquid containing biomolecules.
  • the depth may be 3 to 10 ⁇ m.
  • the size of a carrier such as a bead to which a biomolecule is attached, and a suitable ratio between the dimensions of the well, etc., the size of the well is one per microwell, or It is determined appropriately so that several biomolecules are accommodated.
  • the detection target to be detected using the microwell array according to the present embodiment may be, for example, a sample collected from a living body such as blood, a PCR product, or the like, or an artificially synthesized compound or the like.
  • the well may have a shape and size that allow one molecule of DNA to enter.
  • biomolecules include proteins, RNA (including RNA such as miRNA, mtRNA, tRNA), sugars, and amino acids (including peptides).
  • the density of a plurality of wells is, for example, 100,000 to 10 million pieces / cm 2 , preferably 100,000 to 5 million pieces / cm 2 , and more preferably 100,000. ⁇ 1 million pieces / cm 2 .
  • the density of the well is in such a range (100,000 to 10,000,000 / cm 2 )
  • the presence ratio of the mutation to be detected to the wild type is about 0.01%, about 100,000 to 5 million pieces / cm 2 , more preferably 100,000 to 1 million.
  • the number of cells / cm 2 is present, rapid measurement is possible even if all wells are detected without impairing the detection accuracy.
  • each well (out of a plurality of wells) has the hydrophobic portion at the top of the well, and the hydrophilic portion is positioned closer to the well bottom (bottom side) than the hydrophobic portion. It is preferable to have a sex part. As will be described later, the inventors have found that such a configuration makes it very easy to enclose an aqueous liquid in the wells of the microwell array.
  • the above-mentioned hydrophilic part (material of the hydrophilic part) may have a contact angle of less than 70 degrees measured according to the sessile drop method defined in JIS R3257-1999.
  • being hydrophilic means that the contact angle measured according to the sessile drop method defined in JIS R3257-1999 is less than 70 degrees.
  • the hydrophobic part (the material of the hydrophobic part) may have a contact angle of 70 degrees or more measured according to the sessile drop method defined in JIS R3257-1999.
  • being hydrophobic means that the contact angle measured according to the sessile drop method specified in JIS R3257-1999 is 70 degrees or more.
  • hydrophilic resin The resin forming the hydrophilic portion (hereinafter sometimes referred to as “hydrophilic resin”) is not particularly limited as long as the effect of the present invention is exerted. Examples thereof include resins having a group. Examples of the hydrophilic group include a hydroxyl group, a carboxyl group, a sulfone group, a sulfonyl group, an amino group, an amide group, an ether group, and an ester group.
  • a siloxane polymer an epoxy resin; a polyethylene resin; a polyester resin; a polyurethane resin; a polyacrylamide resin; a polyvinylpyrrolidone resin; an acrylic resin such as a polyacrylic acid copolymer; a cationized polyvinyl alcohol, a silanolated polyvinyl alcohol, Polyvinyl alcohol resins such as sulfonated polyvinyl alcohol; polyvinyl acetal resins; polyvinyl butyral resins; polyethylene polyamide resins; polyamide polyamine resins; cellulose derivatives such as hydroxymethyl cellulose and methyl cellulose; polyalkylenes such as polyethylene oxide and polyethylene oxide-polypropylene oxide copolymers Oxide derivatives; maleic anhydride copolymer; ethylene-vinyl acetate copolymer; At least one resin having a contact angle of less than 70 degrees measured according to the sessile drop method specified in JIS R3257-1999 is
  • the hydrophilic resin may be a thermoplastic resin, a thermosetting resin, a resin curable by an active energy ray such as an electron beam or UV light, or an elastomer. .
  • the hydrophilic layer may also have a function of bringing the substrate and the hydrophobic layer into close contact when a substrate described later is present.
  • the hydrophilic layer may be formed by applying a thermosetting silane coupling agent or the like to the substrate and thermally curing to form a siloxane polymer.
  • hydrophobic resin The resin that forms the hydrophobic portion (hereinafter sometimes referred to as “hydrophobic resin”) is not particularly limited as long as the effects of the present invention are exhibited.
  • novolak resin acrylic resin; methacrylic resin; styrene resin; Polyvinyl resin; Polyamide resin; Polyacetal resin; Polycarbonate resin; Polyphenylene sulfide resin; Polysulfone resin; Fluorine resin; Silicone resin; Yuria resin; Melamine resin; Any of the above resin combinations can be appropriately selected from at least one resin having a contact angle of 70 degrees or more measured according to the sessile drop method defined in JIS R3257-1999. .
  • the hydrophobic resin may be a thermoplastic resin, a thermosetting resin, a resin curable by an active energy ray such as an electron beam or UV light, or an elastomer. .
  • the hydrophobic portion may be formed of a resist, for example.
  • the resist may be a photoresist from the viewpoint of easily forming a fine structure.
  • the photoresist may be, for example, a photosensitive novolac resin.
  • the microwell array according to this embodiment may be formed on a substrate.
  • the substrate may be a substrate having electromagnetic wave permeability or a substrate having no electromagnetic wave permeability.
  • examples of the electromagnetic wave include X-rays, ultraviolet rays, visible rays, and infrared rays.
  • electromagnetic waves can be used to analyze the results of experiments performed on the microwell array. For example, fluorescence, phosphorescence, and the like generated as a result of irradiation with electromagnetic waves can be measured from the substrate side (via the substrate and on the substrate).
  • the fluorescence and phosphorescence include light having different wavelengths at the time of irradiation and detection (wavelengths are converted).
  • wavelength conversion include a case where fluorescence or phosphorescence in the visible light region is generated as a result of irradiation with ultraviolet light, and a case where fluorescence or phosphorescence in the infrared region is generated as a result of irradiation with visible light.
  • a fluorescence microscope can be used.
  • the electromagnetic wave may be irradiated, for example, from the substrate side (to the substrate), or may be irradiated, for example, from the entrance side of the well (to the entrance of the well).
  • Examples of the substrate having electromagnetic wave permeability include glass and resin.
  • Examples of the resin substrate include ABS resin, polycarbonate resin, COC (cycloolefin copolymer), COP (cycloolefin polymer), acrylic resin, polyvinyl chloride, polystyrene resin, polyethylene resin, polypropylene resin, polyvinyl acetate, and PET (polyethylene). Terephthalate), PEN (polyethylene naphthalate) and the like. These resins may contain various additives, and a plurality of resins may be mixed.
  • the substrate does not substantially have autofluorescence.
  • substantially having no autofluorescence means that the substrate has no autofluorescence of the wavelength used for detection of the experimental results, or even if it has autofluorescence, the detection of the experimental results is affected. It means that it is so weak as not. For example, if the autofluorescence is about 1/10 or less and 1/100 or less as compared with the fluorescence to be detected, it can be said that it is so weak that it does not affect the detection of the experimental result.
  • quartz glass As a material that has electromagnetic wave transparency and does not emit autofluorescence at all, for example, quartz glass can be cited.
  • materials that have weak autofluorescence and that do not interfere with detection of experimental results using electromagnetic waves include low fluorescent glass, acrylic resin, COC (cycloolefin copolymer), and COP (cycloolefin polymer).
  • fluorescent substances such as fluorescent dyes, FRET (fluorescence resonance energy transfer), reaction of fluorescently labeled avidin and streptavidin, enzyme reaction using horseradish peroxidase, FRET such as Invader (registered trademark) method
  • FRET fluorescent resonance energy transfer
  • Invader registered trademark
  • the observation of the fluorescence emitted from the biomolecule accommodated in the well can be performed from the substrate side of the microwell array (via the substrate and from the substrate) using, for example, an inverted microscope.
  • the number of target molecules can be specified by counting the number of microwells emitting predetermined fluorescence.
  • the biomolecule may be treated with a fluorescent label that specifically labels the target molecule before being accommodated in the well.
  • the bead is accommodated in the well and brought into contact with a fluorescent label capable of specifically labeling the target molecule, and the target molecule is placed in the well. May be fluorescently labeled.
  • the thickness of the substrate can be appropriately determined.
  • the fluorescence is observed from the substrate side (on the substrate) using a fluorescence microscope, it is, for example, 5 mm or less, for example, 2 mm or less, for example, 1.6 mm or less. Is preferred.
  • Turbidity can be used in addition to fluorescence. Turbidity can be measured, for example, by the transmittance of light having a wavelength of about 400 to 1000 nm in the detection target.
  • Microfluidic device One embodiment of the present invention provides a microfluidic device comprising a flow path and the above-described microwell array disposed in the flow path. By using such a microfluidic device, it becomes easy to enclose an aqueous liquid in the wells of the microwell array.
  • FIG. 1A is a perspective view showing a microfluidic device according to a second embodiment.
  • 1B is a cross-sectional view taken along line bb in the microfluidic device of FIG. 1A.
  • 1C is a cross-sectional view taken along line cc of the microfluidic device of FIG. 1A.
  • the microfluidic device 1000 includes a bottom member 1100, a lid member 1200, a flow channel 1300, a flow channel inlet 1210 provided at one end (first end) of the flow channel 1300, and the flow channel 1300.
  • a flow path outlet 1220 provided at the other end (second end), a sealing member 1400 that forms part of the inner wall of the flow path 1300, and a microwell array m disposed inside the flow path 1300 are provided.
  • the fluid may be introduced from the channel inlet 1210 and discharged from the channel outlet 1220 using a syringe or the like.
  • the microwell array m is provided inside the flow path 1300.
  • the structure of the microwell array m will be described later.
  • the substrate 1100 of the microwell array m and the bottom member 1100 of the microfluidic device 1000 may be integrated. Further, the hydrophilic layer 1120 and the hydrophobic layer 1130 of the microwell array m may be integrally formed on the bottom member 1100 of the microfluidic device 1000. That is, the microwell array m may be formed on the bottom member 1100 of the microfluidic device 1000.
  • the same material as that of the substrate of the microwell array described above can be used.
  • the material of the sealing member 1400 is not particularly limited.
  • a double-sided pressure-sensitive adhesive tape in which an acrylic pressure-sensitive adhesive is laminated on both sides of a core material film formed of silicone rubber or acrylic foam, or an adhesive is laminated.
  • spacer members resin, metal, paper, inorganic materials such as glass
  • these sealing members those which do not easily react with the liquid to be fed can be appropriately selected.
  • what coated the surface of the sealing member so that it may become difficult to react with the liquid sent can also be employ
  • a double-sided adhesive tape was used.
  • FIG. 2A is a perspective view showing a microfluidic device according to a third embodiment of the present invention.
  • 2B is a cross-sectional view taken along line bb in the microfluidic device of FIG. 2A.
  • 2C is a cross-sectional view taken along line cc in the microfluidic device of FIG. 2A.
  • the microfluidic device 2000 has a configuration in which a part of the flow channel 1300 is enlarged in a plan view of the microfluidic device 1000 according to the first embodiment described above.
  • the microwell array m (second microwell array m-2) in the microfluidic device 2000 has a larger area in plan view than the microwell array m in the microfluidic device 1000.
  • Other configurations of the microfluidic device 2000 are the same as those of the microfluidic device 1000.
  • plan view means a state in which the microfluidic device or the microwell array is viewed from a direction perpendicular to the bottom member of the microfluidic device or the substrate of the microwell array.
  • a microfluidic device including a flow path and the microwell array according to the above-described embodiment disposed in the flow path is prepared, and the flow path of the microfluidic device is aqueous. Liquid is fed, the aqueous liquid is introduced into the wells of the microwell array, the sealing liquid is fed into the flow path, and the sealing liquid layer is formed on the upper layer of the aqueous liquid introduced into the well.
  • a method is provided for encapsulating an aqueous liquid within a well of a microwell array, wherein the aqueous liquid is encapsulated within the well.
  • the aqueous liquid means water, a biological sample such as a buffer solution containing a biomolecule to be detected, an enzyme reaction solution, or the like.
  • the biological sample is generally an aqueous liquid.
  • the aqueous liquid may be, for example, a PCR reaction solution containing a biological sample as a template and SYBR Green as a detection reagent.
  • an additive such as a surfactant may be contained in the aqueous liquid. By adding a surfactant to the aqueous liquid, the aqueous liquid can be easily enclosed in the wells of the microwell array.
  • the sealing liquid means a liquid used for isolating the liquid introduced into each well (among a plurality of wells) of the microwell array so that they are not mixed with each other.
  • oils are used. Can do.
  • the oil for example, trade name “FC40” manufactured by Sigma, trade name “HFE-7500” manufactured by 3M, mineral oil used for PCR reaction, or the like can be used.
  • FC40 trade name “FC40” manufactured by Sigma
  • HFE-7500” manufactured by 3M mineral oil used for PCR reaction, or the like
  • mineral oil used for PCR reaction, or the like can be used.
  • mineral oil used for PCR reaction, or the like can be used in a conventional microwell array.
  • mineral oil In a conventional microwell array, it may be difficult to use mineral oil as a sealing liquid.
  • an aqueous liquid is added to the well. Can be enclosed.
  • the sealing liquid preferably has a contact angle of 5 to 80 degrees with respect to the material of the hydrophobic layer of the microwell array.
  • the contact angle of the sealing liquid is within this range (5 to 80 degrees)
  • the aqueous liquid can be sealed in the wells of the microwell array.
  • the contact angle of the sealing liquid may be measured using a sealing liquid instead of water, for example, according to the sessile drop method defined in JIS R3257-1999.
  • a liquid other than the specific oil described above can also be used as the sealing liquid by appropriately selecting the combination of the material forming the hydrophobic layer of the microwell array and the sealing liquid.
  • an aqueous liquid such as a PCR reaction solution or an invader reaction solution is fed from the flow channel inlet 1210 of the microfluidic device 1000 using a syringe, a micropipette, or the like.
  • an aqueous liquid is introduced into each well of the microwell array m arranged inside the flow path 1300.
  • the sealing liquid is fed into the microfluidic device 1000 from the flow path inlet 1210 using a syringe or the like.
  • a sealing liquid layer is formed in the vicinity of the well entrance of each well of the microwell array m arranged in the flow path 1300, and the aqueous liquid is sealed in the well. Since the microwell array m has the above-described configuration, an aqueous liquid can be easily enclosed in each well of the microwell array m by the method of the present embodiment.
  • an enzyme reaction such as a PCR reaction or an invader reaction can be performed using a thermal cycler or the like.
  • a hydrophilic material is formed on a substrate having electromagnetic wave permeability to form a hydrophilic layer, and a hydrophobic material is formed on the hydrophilic layer to form a hydrophobic layer.
  • a method of manufacturing a microwell array is provided in which a layer is drilled to form a plurality of wells.
  • FIG. 3 is a cross-sectional view showing the structure of the microwell array 100 according to the fifth embodiment.
  • the hydrophilic layer 120 is formed on the surface 115 of the substrate 110.
  • the substrate 110 may be a glass substrate, for example.
  • the hydrophilic layer 120 may be, for example, a siloxane polymer formed from a silane coupling agent or a curable resin.
  • the hydrophilic layer 120 is formed by applying the curable resin onto the substrate 110 and curing it by heat treatment or the like.
  • a silane coupling agent may be added to the curable resin.
  • the adhesion of the hydrophilic layer 120 to the substrate 110 is improved.
  • a hydrophobic resin as a material for the hydrophobic layer 130 is applied on the hydrophilic layer 120.
  • a photoresist may be used as the hydrophobic resin.
  • a photoresist is applied on the hydrophilic layer 120 by spin coating or the like. After the spin coating, the photoresist is prebaked by applying heat and solidified to generate the hydrophobic layer 130.
  • the hydrophobic layer 130 is excavated to form a well.
  • the excavation can be performed by developing a photoresist, for example.
  • a pattern mask is attached on the hydrophobic layer 130 and exposed to ultraviolet rays or the like to be exposed.
  • the photoresist for example, a positive type photoresist is used.
  • a positive type photoresist is decomposed when exposed to light and dissolved in a developer.
  • a pattern mask having a design in which a hole of a target pattern is opened at a position where a microwell is to be formed is used as the pattern mask.
  • the exposure time and conditions may be appropriately determined with reference to the photoresist data sheet to be used.
  • the microwell array 100 is formed.
  • the development time is appropriately determined with reference to the data sheet of the photoresist to be used.
  • stop the development with a rinse solution and wash. For example, pure water or the like may be used as the rinse liquid.
  • post-baking is performed to stabilize the photoresist.
  • the microwell array 100 according to this embodiment can be manufactured.
  • the bottom of the well of the microwell array is formed by the hydrophilic layer 120.
  • a plurality of wells may be formed by excavating the hydrophobic layer 130 and the hydrophilic layer 120.
  • FIG. 4 is a cross-sectional view showing the structure of a microwell array 200 according to the sixth embodiment.
  • the microwell array 200 having wells where the surface 115 of the substrate 110 is exposed can be manufactured.
  • the seventh embodiment of the present invention is a method for analyzing an aqueous liquid in which an aqueous liquid is enclosed, the wells of the microwell array according to the above embodiment are irradiated with electromagnetic waves, and fluorescence or phosphorescence emitted from the wells is detected. provide.
  • the analysis method according to the present embodiment can measure how many of the wells constituting the microwell array emit fluorescence or phosphorescence. For example, by performing a PCR reaction in a microwell array and detecting fluorescence of SYBR Green in wells in which PCR amplification was observed, the proportion of wells in which amplification was observed can be calculated.
  • the analysis method according to the present embodiment may be performed using, for example, a fluorescence microscope.
  • the electromagnetic wave irradiation may be performed from the substrate side of the microwell array, from the well side, or from any other direction.
  • Detection of fluorescence or phosphorescence generated as a result of irradiation with electromagnetic waves may be performed from the substrate side of the microwell array, may be performed from the well side, or may be performed from any other direction.
  • detecting fluorescence or phosphorescence using a fluorescence microscope it is convenient to detect fluorescence or phosphorescence from the substrate side of the microwell array.
  • Example 1 The microwell array of Example 1 was manufactured according to the manufacturing method of the fifth embodiment described above.
  • the microwell array of Example 1 had a cylindrical well having a diameter of about 5 ⁇ m and a height of about 3 ⁇ m, and the bottom surface of the well was formed of a hydrophilic resin (hydrophilic layer).
  • the well density was 1 million / cm 2 .
  • a glass substrate was used as the substrate.
  • a silane coupling agent model number “Z6094”, manufactured by Toray Dow Corning Co., Ltd., contact angle measured according to the sessile drop method defined in JIS R3257-1999: 55.3 degrees
  • hydrophobic resin a positive photoresist (model number “S1818”, manufactured by Dow, contact angle measured according to the sessile drop method defined in JIS R3257-1999: 88.4 degrees) was used. Therefore, the wells of the microwell array of Example 1 have a hydrophobic portion near the well entrance on the well surface, and the hydrophilic portion is located closer to the bottom of the well (position closer to the bottom of the well) than the hydrophobic portion. And the well bottom was also hydrophilic.
  • Example 2 The microwell array of Example 2 was produced according to the manufacturing method of the sixth embodiment described above.
  • the microwell array of Example 2 had a cylindrical well having a diameter of about 5 ⁇ m and a height of about 3 ⁇ m.
  • the well density was 1 million / cm 2 .
  • a glass substrate contact angle measured according to the sessile drop method defined in JIS R3257-1999: 65.0 degrees
  • hydrophilic resin a silane coupling agent (model number “Z6094”, manufactured by Toray Dow Corning Co., Ltd., contact angle measured according to the sessile drop method defined in JIS R3257-1999: 55.3 degrees) was used. .
  • hydrophobic resin a positive photoresist (model number “S1818”, manufactured by Dow, contact angle measured according to the sessile drop method defined in JIS R3257-1999: 88.4 degrees) was used. Therefore, the wells of the microwell array of Example 2 have a hydrophobic portion near the well entrance on the well surface, and the hydrophilic portion is located closer to the bottom of the well (position closer to the bottom of the well) than the hydrophobic portion. Had.
  • FIG. 5 is a cross-sectional view showing the structure of the microwell array 300 of the first comparative example.
  • the microwell array of Comparative Example 1 had a cylindrical well having a diameter of about 5 ⁇ m and a height of about 3 ⁇ m.
  • a glass substrate was used as the substrate.
  • a positive photoresist (model number “S1818”, manufactured by Dow, contact angle measured according to the sessile drop method defined in JIS R3257-1999: 88.4 degrees) was used. As shown in FIG.
  • the microwell array 300 has a structure in which the hydrophobic layer 130 is directly laminated on the substrate 110, and the well is formed inside the hydrophobic layer 130. Therefore, the wells of the microwell array of Comparative Example 1 all had well surfaces formed from hydrophobic portions.
  • Example 1 A microfluidic device comprising the microwell array of Example 1 was assembled. Subsequently, an aqueous solution in which a proper amount of FITC (fluorescein isothiocyanate), which is a fluorescent substance, and a surfactant were dissolved was sent to the microfluidic device using a syringe. Thereafter, oil (model number “FC40”, manufactured by Sigma, contact angle of 7.0 ° with respect to positive photoresist (model number “S1818”)) was sent to the microfluidic device using a syringe.
  • FITC fluorescein isothiocyanate
  • S1818 contact angle of 7.0 ° with respect to positive photoresist
  • FIG. 6 is a cross-sectional view showing a state after oil is fed to the microfluidic device of this experimental example.
  • the microfluidic device 400 was provided with a bottom member 410 that holds a microwell array, a lid member 420, and a microchannel 430.
  • the bottom member 410 was integrated with the substrate 110 of the microwell array 100 of Example 1. That is, the microwell array 100 of Example 1 was formed using the bottom member 410 as the substrate 110.
  • the aqueous liquid 440 (an aqueous solution in which an appropriate amount of FITC and a surfactant was dissolved) was sent to the microfluidic device 400 from the channel inlet (not shown), the aqueous liquid 440 was introduced into the wells of the microwell array 100. Subsequently, when the oil 450 is supplied to the microfluidic device 400 from a channel inlet (not shown), a layer of the oil 450 is formed near the well inlet of the well while the aqueous liquid 440 is held in the well of the microwell array 100. Been formed. That is, the aqueous liquid 440 was sealed in the wells of the microwell array 100. Each well functions as a chemical reaction vessel that performs various biochemical or chemical reactions in a small volume of aqueous liquid 440.
  • FIG. 7 is a photograph showing the result of fluorescence microscopic observation of the microwell array of Example 1 from the substrate side (on the substrate, the bottom of the microfluidic device) after the oil was fed.
  • FITC-derived fluorescence was regularly observed in the well pattern of the microwell array of Example 1.
  • the white portion is a fluorescent well
  • the black portion is a substrate. This result shows that the FITC solution could be encapsulated in each well of the microwell array of Example 1.
  • Example 2 As a microwell array, FITC and a surfactant were dissolved in appropriate amounts in the microwell array in the same manner as in Experimental Example 1, except that the microwell array of Example 2 was used instead of the microwell array of Example 1. An aqueous solution was enclosed.
  • FIG. 8 is a cross-sectional view showing a state after oil is fed to the microfluidic device of this experimental example.
  • the microfluidic device 600 includes a bottom member 610 that holds a microwell array, a lid member 620, and a microchannel 630.
  • the bottom member 610 was integrated with the substrate 110 of the microwell array 200 of Example 2. That is, the microwell array 200 of Example 2 was formed using the bottom member 610 as the substrate 110.
  • aqueous liquid 440 an aqueous solution in which an appropriate amount of FITC and a surfactant was dissolved
  • the aqueous liquid 440 was introduced into the wells of the microwell array 200.
  • the oil 450 is supplied to the microfluidic device 600 from a channel inlet (not shown)
  • a layer of the oil 450 is formed near the well inlet of the well while the aqueous liquid 440 is held in the well of the microwell array 200. Been formed. That is, the aqueous liquid 440 was sealed in the wells of the microwell array 200.
  • FIG. 9 is a photograph showing a result of observing the microwell array of Example 2 from the substrate side with a fluorescence microscope after feeding the oil. As a result, as shown in FIG. 9, FITC fluorescence was regularly observed in the well pattern of the microwell array of Example 2. In FIG. 9, the white portion is a well emitting fluorescence, and the black portion is a substrate. This result shows that the FITC solution could be encapsulated in each well of the microwell array of Example 2.
  • the novolak resin used as the hydrophobic resin has autofluorescence, but the fluorescence value of the novolac resin is sufficiently low, and is the detection value and the fluorescence value of the novolac resin. Since there was a sufficient difference from the fluorescence value of FITC, there was no problem in the detection of FITC fluorescence.
  • Example 3 As a microwell array, FITC and a surfactant were dissolved in appropriate amounts in the same manner as in Experimental Example 1, except that the microwell array of Comparative Example 1 was used instead of the microwell array of Example 1. An aqueous solution was enclosed.
  • FIG. 10 is a cross-sectional view showing a state after oil is fed to the microfluidic device of this experimental example.
  • the microfluidic device 700 was provided with a bottom member 710 for holding a microwell array, a lid member 720, and a microchannel 730.
  • the bottom member 710 was formed integrally with the substrate 110 of the microwell array 300 of Comparative Example 1. That is, the microwell array 300 of Comparative Example 1 was formed using the bottom member 710 as the substrate 110.
  • aqueous liquid 440 an aqueous solution in which an appropriate amount of FITC and a surfactant is dissolved
  • the oil 450 is fed from an unillustrated channel inlet
  • Liquid 440 could not be retained in the wells of microwell array 300. That is, in the microwell array 300 of Comparative Example 1, it was difficult to enclose the aqueous liquid 440 in the well.
  • FIG. 11 is a photograph showing a result of observing the microwell array of Comparative Example 1 from the substrate side with a fluorescence microscope after feeding the oil.
  • fluorescence was observed across a plurality of wells, and regular FITC fluorescence along the well pattern was not observed.
  • This result shows that the FITC solution could not be encapsulated in the microwell array of Comparative Example 1. That is, when the microwell is formed only by the hydrophobic layer, it becomes difficult to hold the aqueous solution, and when the oil is fed, it becomes clear that all the aqueous solution in the well is replaced with the oil. It was.
  • a microwell array in which an aqueous liquid can be easily enclosed in a well.
  • the manufacturing method of said microwell array, a microfluidic device, the method of enclosing aqueous liquid in the well of a microwell array, and the analysis method of aqueous liquid can be provided.
  • diagnosis is performed by detecting DNA or RNA derived from a living body, a nucleic acid can be put together with a reagent into a minute space.

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Abstract

L'invention concerne un réseau de micro-puits doté d'une pluralité de puits ayant des portions hydrophiles et des portions hydrophobes sur les surfaces de puits.
PCT/JP2016/060367 2015-03-30 2016-03-30 Réseau de micro-puits, son procédé de fabrication, dispositif micro-fluidique, procédé d'étanchéité de liquide aqueux dans un puits du réseau de micro-puits et procédé d'analyse de liquide aqueux WO2016159068A1 (fr)

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