Microfluidic devices and sample analysis methods

Abstract

The present invention provides a microfluidic device that can suppress the generation of bubbles and improve detection efficiency when forming tiny droplets, heating them, and performing optical detection. [Solution] The system has a flow channel and a plurality of droplet holding parts connected to the flow channel, wherein the height of the flow channel is greater than 0 μm and less than or equal to 30 μm, and 1 cm 2 A microfluidic device in which the number of droplet-holding sections per unit is between 100,000 and 10,000,000, the ratio of the total volume of the droplet-holding sections to the volume of the flow path is between 5% and 40%, and the total volume of the droplet-holding sections is between 0.2 μL and 2.0 μL.

Description

Technical Field

[0001] The present invention relates to a microfluidic device and a sample analysis method using the microfluidic device. This application claims priority to Japanese Patent Application No. 2019-034228, filed on February 27, 2019, and incorporates its content herein by reference.

Background Art

[0002] In recent years, micro-well arrays having various forms of fine channel structures formed by using etching technology, photolithography technology used in the manufacturing technology of semiconductor circuits, or molding methods of fine plastics have been studied. The wells of these micro-well arrays are used as chemical reaction vessels for causing various biochemical or chemical reactions in a fluid of a minute volume.

[0003] As materials for manufacturing a microfluidic system, hard substances such as silicon and glass, various polymer resins such as PDMS (polydimethylsiloxane), and soft substances such as silicone rubber are used. For example, Patent Documents 1 to 3 and Non-Patent Document 1 describe using such a microfluidic system as various microchips or biochips.

[0004] Techniques for detecting biomolecules in a channel device are known. For example, in DNA microarray technology, biomolecules are introduced into minute pores and reactions accompanied by heating are performed to detect the biomolecules. Further, techniques capable of detecting biomolecules at the single molecule level are known. Examples of the techniques capable of detecting single molecules include digital measurement techniques such as digital ELISA (Digital Enzyme-Linked ImmunoSorbent Assay), digital PCR (Digital Polymerase Reaction), and digital Invasive Cleavaged Assay (Digital ICA). Digital PCR technology is a technique that divides a mixture of reagents and nucleic acids into countless tiny droplets, performs PCR amplification, and ensures that a signal such as fluorescence is detected from the droplets containing nucleic acids. Quantification is then performed by counting the droplets from which a signal is detected.

[0005] Methods for forming microdroplets include forming microdroplets by cleaving a mixture of reagent and nucleic acid with a sealing solution, and forming microdroplets by placing a mixture of reagent and nucleic acid into pores formed on a substrate, followed by adding a sealing solution. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Patent No. 6183471 [Patent Document 2] Special Publication No. 2014-503831 [Patent Document 3] International Publication No. 2013-151135 [Non-patent literature]

[0007] [Non-Patent Document 1] Kim SH, et al., Large-scale femtoliter droplet array for digital counting of single biomolecules., Lab on a Chip, 12 (23), 4986-4991, 2012. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] However, when forming tiny droplets and heating them for optical detection using fluorescence or other methods, bubbles may form inside the channel, which can interfere with detection.

[0009] Therefore, the present invention aims to provide a microfluidic device that can suppress the generation of bubbles and improve detection efficiency when forming minute droplets, heating them, and performing optical detection. Furthermore, the present invention aims to provide a sample analysis method that can suppress the generation of bubbles when forming and heating minute droplets, thereby improving the detection efficiency of the sample, when the sample is optically detected after heating the minute droplets. [Means for solving the problem]

[0010] To achieve the above objectives, the present invention employs the following configuration. [1] A microfluidic device having a channel and a droplet holding portion formed in the channel, wherein the height of the channel is greater than 0 μm and less than or equal to 30 μm. [2] The microfluidic device according to [1], wherein the flow channel has a planar substrate and the droplet holding portion is a hole in the substrate. [3] The microfluidic device according to [1] or [2], wherein a plurality of droplet-holding sections are present. [4] A microfluidic device according to any one of [1] to [3], wherein the device has a lid member and the flow path is a space sandwiched between the lid member and the substrate. [5] The microfluidic device according to any one of items [1] to [4], wherein the volume of each droplet-holding section is 10 fL or more and 100 pL or less. [6] The microfluidic device according to any one of [1] to [5], wherein the total volume of the droplet holding portion is 0.2 μL or more and 2.0 μL or less. [7] The microfluidic device according to any one of [1] to [6], wherein the ratio of the total volume of the droplet holding portion to the volume of the flow path is 5% or more and 40% or less. [8] The microfluidic device according to any one of [2] to [7], wherein the ratio of the depth of the droplet holding portion to the height of the flow channel is 3% or more and 150% or less. [9] The microfluidic device according to any one of [2] to [8], wherein the ratio of the total area of ​​the openings of the droplet holding portion to the unit area of ​​the region on the substrate in which the droplet holding portion is formed is 23% or more and 90% or less. A sample analysis method using a microfluidic device as described in any one of

[10] [1] to [9], comprising: a sample supply step of supplying an aqueous liquid containing a sample into the channel and holding the aqueous liquid in the droplet holding section; a sealing step of introducing a sealing liquid into the channel and replacing the aqueous liquid present in the channel, thereby sealing the aqueous liquid in the droplet holding section; a reaction step of causing a reaction in the droplet holding section to generate a signal for detection; and a detection step of detecting the signal.

[11] The sample analysis method according to

[10] , wherein the sample is a biomolecule.

[12] The sample analysis method according to

[10] or

[11] , wherein in the reaction step, the microfluidic device is heated to cause the reaction, and the temperature at which it is heated is 60°C or higher.

[13] A sample analysis method according to any one of

[10] to

[12] , wherein the signal is detected by image capture.

[14] The sample analysis method according to any one of

[10] to

[13] , wherein the signal is fluorescent.

[15] The method for analyzing a sample according to any one of

[10] to

[14] , wherein the reaction is an isothermal reaction.

[0011] Another aspect of the present invention includes the following embodiments.

[16] A microfluidic device having a channel and one or more droplet holding parts connected to the channel, wherein the height of the channel is greater than 0 μm and less than or equal to 30 μm.

[17] The microfluidic device according to

[16] , further comprising a flat substrate, wherein the flow path is located on the flat substrate and the droplet holding portion is a hole in the substrate.

[18] The microfluidic device according to

[17] , further comprising a lid member, wherein the flow path is a space sandwiched between the lid member and the substrate.

[19] The ratio of the total area of the openings of the droplet holding portions per unit area of the region where the droplet holding portions are formed on the substrate is 23% or more and 90% or less, the microfluidic device according to

[17] or

[18] .

[20] The microfluidic device according to any one of

[16] to

[19] , wherein the droplet holding portions are a plurality of droplet holding portions.

[21] The microfluidic device according to any one of

[16] to

[20] , wherein the volume per one of the droplet holding portions is 10 fL or more and 100 pL or less.

[22] The microfluidic device according to any one of

[16] to

[21] , wherein the total volume of the droplet holding portions is 0.2 μL or more and 2.0 μL or less.

[23] The microfluidic device according to any one of

[16] to

[22] , wherein the ratio of the total volume of the droplet holding portions to the volume of the flow path is 5% or more and 40% or less.

[24] The microfluidic device according to any one of

[16] to

[23] , wherein the ratio of the depth of the droplet holding portion to the height of the flow path is 3% or more and 150% or less.

[25] A sample analysis method using the microfluidic device according to any one of

[16] to

[24] , comprising introducing an aqueous liquid containing a sample into the flow path, holding the aqueous liquid in the droplet holding portion, introducing a sealing liquid into the flow path to replace the aqueous liquid present in the flow path, enclosing the aqueous liquid in the droplet holding portion, causing a reaction in the droplet holding portion to generate a signal for detection, and detecting the signal.

[26] The sample analysis method according to

[25] , wherein the sample is a biomolecule.

[27] Generating the signal for detection includes causing the reaction by heating the microfluidic device, and the temperature when the microfluidic device is heated is 60°C or more, the sample analysis method according to

[25] or

[26] .

[28] The sample analysis method according to any one of

[25] to

[27] , wherein the signal is detected by imaging the microfluidic device. The sample analysis method according to any one of

[25] to

[28] , wherein the signal is fluorescence. The sample analysis method according to any one of

[26] to

[29] , wherein the reaction is an isothermal reaction.

Advantages of the Invention

[0012] According to the microfluidic device of the present invention, it is possible to form minute droplets and suppress the generation of bubbles when heating the minute droplets. Further, according to the sample analysis method of the present invention, when forming minute droplets, heating the minute droplets, and optically detecting a sample, the generation of bubbles when heating the minute droplets is suppressed, and the detection efficiency of the sample can be improved.

Brief Description of the Drawings

[0013] [Figure 1] It is a perspective view showing a microfluidic device according to an embodiment of the present invention. [Figure 2] It is a cross-sectional view taken along line b-b of FIG. 1. [Figure 3] It is a cross-sectional view taken along line b-b of FIG. 1. [Figure 4] It is a top view of a microchannel device according to an embodiment of the present invention, in which there is a region where a droplet holding portion is formed only in a part of the substrate. [Figure 5] It is a cross-sectional view showing a microfluidic device according to an embodiment of the present invention. [Figure 6] It is a view showing a state during use of a microfluidic device according to an embodiment of the present invention. [Figure 7] It is a view showing a state during use of a microfluidic device according to an embodiment of the present invention. [Figure 8] It is a view showing a state during use of a microfluidic device according to a comparative example. [Figure 9A] It is a view showing a fluorescence image observed using a microfluidic device according to an embodiment of the present invention. [Figure 9B]This figure shows fluorescence images observed using a microfluidic device related to a comparative example. [Modes for carrying out the invention]

[0014] One embodiment of the present invention will be described with reference to Figures 1 to 5. In this specification, the dimensional ratios in each drawing are exaggerated for illustrative purposes and do not necessarily correspond to the actual dimensional ratios.

[0015] Figure 1 is a perspective view showing a microfluidic device 1 according to this embodiment. Figures 2 and 3 are cross-sectional views along the line bb in Figure 1. As shown in Figures 2 and 3, the microfluidic device 1 includes a flow channel 35 and a droplet holding section 11. The flow channel 35 may be formed by arranging a lid member 20 and a substrate 10 opposite each other at a certain distance. In this case, the flow channel 35 is the space sandwiched between the lid member 20 and the substrate 10. A peripheral member 34 is located between the substrate 10 and the lid member 20. The region sandwiched between the substrate 10 and the lid member 20 and surrounded by the peripheral member 34 is the flow channel 35. The peripheral member 34 may be formed integrally with the lid member 20.

[0016] If the substrate 10 forming the flow path 35 is planar, i.e., a flat plate, the droplet holding portion 11 is preferably a hole, i.e., a well, present in the substrate 10. The droplet holding portion 11 is preferably a microwell 33 having multiple holes (wells) on the substrate 10, as shown in Figure 3. In other words, it is preferable that the substrate 10 has multiple microwells 33. Hereinafter, the microfluidic device 1 according to this embodiment will be described using as an example a microfluidic device 1 in which the droplet holding portion 11 is a microwell 33 having multiple holes (wells) on the substrate 10.

[0017] The microwell array 30 may have a bottom layer 31, a wall layer 32 (which may also be described as a partition wall 32), and a plurality of microwells 33. The bottom layer 31 is provided on the substrate 10. The wall layer 32 is formed on the bottom layer 31. The plurality of microwells 33 are composed of a plurality of through holes 32a formed in the thickness direction of the bottom layer 31 and the wall layer 32. The plurality of microwells 33 are formed in an array shape in the wall layer 32. In the internal space S between the substrate 10 and the lid member 20, there is a gap between the microwell array 30 and the lid member 20, in other words, between the upper surface of the wall layer 32 and the lid member 20. This gap functions as a flow path 35 that communicates with the plurality of microwells 33, as well as the first hole 21 and the second hole 22.

[0018] The substrate 10 may be permeable to electromagnetic waves. Examples of electromagnetic waves include X-rays, ultraviolet rays, visible light, and infrared rays. Because the substrate 10 is permeable to electromagnetic waves, fluorescence or phosphorescence produced by the reaction between the sample enclosed in the microfluidic device 1 and the reagent can be observed from the substrate 10 side. The substrate 10 may be capable of transmitting only electromagnetic waves within a predetermined wavelength range. For example, if the presence of a sample in a microwell is determined by detecting fluorescence having a peak in the visible light region, specifically in the wavelength range of 350 to 700 nm, then the substrate 10 should be capable of transmitting visible light within at least the above wavelength range.

[0019] Examples of materials for forming the substrate 10 include glass and resin. Examples of resin substrate materials include ABS resin, polycarbonate resin, COC (cycloolefin copolymer), COP (cycloolefin polymer), acrylic resin, polyvinyl chloride, polystyrene resin, polyethylene resin, polypropylene resin, polyvinyl acetate, PET (polyethylene terephthalate), and PEN (polyethylene naphthalate). These resins may contain various additives. Examples of additives include antioxidants, water-repellent additives, and hydrophilic additives. The resin substrate may contain only one of the above resins, or it may be a mixture of multiple resins.

[0020] Since the sample analysis methods described later may utilize fluorescence or phosphorescence, it is preferable to use a material that is substantially free of autofluorescence as the substrate 10. Here, "substantially free of autofluorescence" means that the substrate either has no autofluorescence at all at the wavelength used to detect the experimental results, or if it does have autofluorescence, it is so weak that it does not affect the detection of the experimental results. For example, if the autofluorescence is 1 / 2 or less, preferably 1 / 10 or less, compared to the fluorescence of the target to be detected, it can be said to be weak enough not to affect the detection of the experimental results.

[0021] The thickness of the substrate 10 can be determined as appropriate, but to facilitate transmission of fluorescence and phosphorescence emitted during sample analysis, it is preferably 5 millimeters (mm) or less, more preferably 2 mm or less, and even more preferably 1.6 mm or less. The lower limit of the thickness of the substrate 10 can be determined as appropriate, but it is best to set the thickness so that no distortion occurs even when the internal pressure of the microfluidic device 1 increases. For example, 0.1 mm or more is preferred, 0.2 mm or more is more preferred, and 0.4 mm or more is even more preferred. The upper and lower limits of the thickness of the substrate 10 can be arbitrarily combined. For example, the thickness of the substrate 10 is preferably 0.1 mm or more and 5 mm or less, more preferably 0.2 mm or more and 2 mm or less, and even more preferably 0.4 mm or more and 1.6 mm or less.

[0022] The bottom layer 31 constitutes the bottom surface of the microwell 33. Therefore, if hydrophilicity is to be imparted to the bottom surface, the bottom layer 31 should be formed from a hydrophilic material. It is preferable that the bottom layer 31 be formed so as to be able to transmit electromagnetic waves and not interfere with the observation of the sample in the microwell 33 from the substrate 10 side. Also, if hydrophobicity is to be to be to the bottom surface, the bottom layer 31 should be formed from a hydrophobic material. It is preferable that the bottom layer 31 not interfere with the observation of the sample in the microwell 33 from the substrate 10 side. Furthermore, it is preferable to use a material that substantially does not exhibit autofluorescence for the bottom layer 31. Here, the substrate 10 and the bottom layer 31 integrated together can also be simply referred to as the substrate.

[0023] Furthermore, if the characteristics of the bottom surface of the microwell 33 are the same as those of the substrate 10 and there is no problem, the bottom layer 31 may be omitted, and the wall layer 32 may be formed directly on the substrate 10. In that case, the microwell 33 is formed by the surface of the substrate 10 and the through holes 32a of the wall layer 32.

[0024] The wall layer 32 has a plurality of through holes 32a arranged in an array when viewed in the thickness direction. The inner surface of each through hole 32a constitutes the inner wall surface of each microwell 33.

[0025] As the material for forming the wall layer 32, the same resin as the material for forming the substrate 10 can be used, but a material may also be used in which a colored component that absorbs electromagnetic waves of a predetermined wavelength is mixed into the resin. As for the resin material, considering the properties required for the microwells 33, either a hydrophilic resin in which the molecules of the resin components have hydrophilic groups, or a hydrophobic resin in which the molecules of the resin components have hydrophobic groups, can be used.

[0026] Examples of hydrophilic groups include hydroxyl groups, carboxyl groups, sulfone groups, sulfonyl groups, amino groups, amide groups, ether groups, and ester groups. Examples of hydrophilic resins include siloxane polymers, epoxy resins, polyethylene resins, polyester resins, polyurethane resins, polyacrylamide resins, polyvinylpyrrolidone resins, acrylic resins such as polyacrylic acid copolymers, polyvinyl alcohol resins such as cationized polyvinyl alcohol, silanolized polyvinyl alcohol, and sulfonated polyvinyl alcohol, polyvinyl acetal resins, polyvinyl butyral resins, polyethylene polyamide resins, polyamide polyamine resins, cellulose derivatives such as hydroxymethylcellulose and methylcellulose, polyalkylene oxide derivatives such as polyethylene oxide and polyethylene oxide-polypropylene oxide copolymers, maleic anhydride copolymers, ethylene-vinyl acetate copolymers, styrene-butadiene copolymers, and combinations of the above resins, which can be appropriately selected and used. Examples of hydrophobic resins include, for example, novolac resin; acrylic resin; methacrylic resin; styrene resin; vinyl chloride resin; vinylidene chloride resin; polyolefin resin; polyamide resin; polyimide resin; polyacetal resin; polycarbonate resin; polyphenylene sulfide resin; polysulfone resin; fluororesin; silicone resin; urea resin; melamine resin; guanamine resin; phenol resin; cellulose resin; and combinations of the above resins. Materials with a contact angle of 70 degrees or more, measured according to the static droplet method specified in JIS R3257-1999, can be appropriately selected and used. In other words, hydrophobicity in this specification means a contact angle of 70 degrees or more, measured according to the static droplet method specified in JIS R3257-1999. Alternatively, the contact angle can be measured using a method compliant with ASTM D5725-1997 instead of the static droplet method specified in JIS R3257-1999.

[0027] Both the hydrophilic and hydrophobic resins may be thermoplastic or thermosetting resins. Furthermore, they may be resins that harden with active energy rays such as electron beams or UV light, or they may be elastomers. By using photoresist as the resin material, numerous fine through-holes 32a can be precisely formed in the wall layer 32 by photolithography. When using photolithography, known methods can be appropriately selected for the selection of the type of photoresist to be used, its application, exposure (photosensitization), and the removal of unwanted photoresist. If a resist is not used, the wall layer 32 can be formed, for example, by injection molding.

[0028] Examples of colored components include organic or inorganic pigments. Specifically, black pigments include carbon black, acetylene black, and iron black. Yellow pigments include chromium yellow, zinc yellow, ochre, Hansa yellow, permanent yellow, and benzine yellow. Orange pigments include orange lake, molybdenum orange, and benzine orange. Red pigments include red iron oxide, cadmium red, antimony vermilion, permanent red, risole red, lake red, brilliant scarlet, and thioindigo red. Blue pigments include ultramarine, cobalt blue, phthalocyanine blue, ferrocyanine blue, and indigo. Green pigments include chromium green, viridian naphthol green, and phthalocyanine green.

[0029] Furthermore, when the wall layer 32 is formed by injection molding or the like, not only pigments dispersed in the resin but also various dyes dissolved in the resin can be used as coloring components. Examples of dyes can be found in various dyeing methods. Specifically, these include direct dyes, basic dyes, cationic dyes, acid dyes, mordant dyes, acid mordant dyes, sulfur dyes, vat dyes, naphthol dyes, disperse dyes, and reactive dyes. Disperse dyes are often selected, especially when dyeing resin.

[0030] The lid member 20 (which may also be simply referred to as lid 20) is a plate-shaped or sheet-shaped member. The lid member 20 faces the substrate 10 at a distance from it. In other words, the lid member 20 covers the multiple microwells 33. The flow path 35 is the region surrounded by the lid member 20, the microwell array 30, and the peripheral member 34. The flow path 35 is connected to the openings of the multiple microwells 33 and is located above the multiple microwells 33.

[0031] The lid member 20 has a first hole 21 and a second hole 22 that penetrate in the thickness direction. In a plan view of the lid member 20, the first hole 21 and the second hole 22 are positioned to sandwich one or more of the liquid-holding portions. The first hole 21 and the second hole 22 communicate with the internal space S, which includes the microwell array 30 and the flow path 35, in the completed microfluidic device 1. The first hole 21 and the second hole 22 function as an inlet for supplying fluid to the internal space S and an outlet for discharging fluid, respectively. The material used to form the lid member 20 and the thickness of the lid member 20 can be the same as those used for the substrate 10. If the lid member 20 is electromagnetic wave permeable, the electromagnetic wave permeability can be set as appropriate. For example, if the electromagnetic wave irradiation process described later is not performed from the lid member 20 side, the lid member 20 does not need to be able to transmit electromagnetic waves.

[0032] As shown in Figure 2, the height h of the channel 35 is the height from the uppermost surface of the substrate 10 on the channel 35 side (the surface of the substrate 10 on the channel 35 side in the portion where no holes are formed) to the surface of the lid member 20 on the channel 35 side. If the microfluidic device 1 includes a wall layer 32, as shown in Figure 3, the height h is the height from the surface 32b of the wall layer 32 on the channel 35 side to the surface 20a of the lid member 20 on the channel side.

[0033] The height h of the channel 35 is greater than 0 μm and less than 50 μm, preferably greater than 0 μm and 30 μm or less, preferably 2 μm or more and 30 μm or less, preferably 3 μm or more and 25 μm or less, more preferably 5 μm or more and 20 μm or less, and particularly preferably 8 μm or more and 18 μm or less. By having a channel height greater than 0 μm and less than 50 μm, the generation of bubbles is suppressed when the droplets formed in the droplet holding section 11 (microwell 33) are heated. Therefore, the sample can be detected by fluorescence or the like without the detection of fluorescence or the like being hindered by bubbles.

[0034] From the viewpoint of suppressing bubble generation, a smaller channel height h is preferable, preferably less than 50 μm. However, if the channel height h is less than 2 μm, it may become difficult to easily fill the channel with the sample. When the channel height is 2 μm or more, the pressure does not rise too much when filling the channel with the aqueous liquid containing the sample, and the aqueous liquid can be easily filled. When the channel height h is 50 μm or more, bubbles are more likely to be generated when the droplets formed in the droplet holding section 11 (microwell 33) are heated, and this bubble generation makes it difficult to detect the sample by fluorescence or the like. In other words, when the channel height h is less than 50 μm, bubbles are less likely to be generated even when the droplets formed in the droplet holding section 11 (microwell 33) are heated, and the sample can be accurately detected by fluorescence or the like. As a result, when the channel height h is less than 50 μm, it leads to an improvement in the yield rate of microfluidic devices. In addition, when the channel height h is less than 50 μm, the sample detection efficiency is improved, and the success rate of the detection operation is improved.

[0035] The height of the channel 35 may be the average of the heights at multiple points on the uppermost surface of the substrate 10 on the channel 35 side (the surface on the channel 35 side in the portion of the substrate 10 where no holes are formed), or it may be the height at a representative point. The representative point can be any position on the substrate 10. For example, it may be the center of the substrate 10 forming the channel 35, or it may be 10% to 50% from the leading edge of the line connecting the leading edge of the substrate 10 forming the channel 35 and the center. Considering ease of measurement, it is preferable to use the position at which a virtual orthogonal line at the center of a virtual line connecting the center of the first hole 21 and the center of the second hole 22, when viewed from above the lid member 20, intersects with the peripheral member 34 as the representative point. Furthermore, if the microfluidic device 1 includes a wall layer 32, the representative location may be the average height of multiple points on the surface 32a of the wall layer 32 facing the flow channel 35, or it may be the height of a representative location. The representative location can be any position on the wall layer 32. For example, the representative location may be the center of the wall layer 32. Alternatively, the representative location may be 10% to 50% from the leading edge of the line connecting the leading edge of the wall layer 32 facing the flow channel 35 to the center. Considering ease of measurement, it is preferable to use the position at which a virtual orthogonal line at the center of a virtual line connecting the first hole 21 and the second hole 22 intersects with the peripheral member 34 as the representative location. Here, the "tip" refers to the end of the microfluidic device 1 in a direction parallel to the imaginary line connecting the first hole 21 and the second hole 22 (referred to as the longitudinal direction). The "end" refers to the portion that contacts the peripheral member 34, which will be described later.

[0036] Furthermore, it is preferable that the surface of the lid member 20 that forms the flow channel 35 is smooth. By making the surface of the lid member 20 that forms the flow channel 35 smooth, the generation of bubbles can be further suppressed when the droplets in the droplet holding section 11 (microwell 33) are heated. In this specification, smooth means that there are few irregularities when observed with an optical microscope, that is, there are few microscopic irregularities. In this specification, smooth does not mean that the lid member 20 is not curved on the inside of the peripheral member 34. In this embodiment, the lid member 20 may be curved on the inside of the peripheral member 34. For example, the lid member 20 may be curved near the center of the main surface of the lid member 20 so as to protrude toward the substrate 10. In other words, the flow channel 35 in the part that is in contact with the peripheral member 34 may be higher than the flow channel 35 near the center of the main surface of the lid member 20. Here, the center of the main surface of the lid member 20 is the geometric center of the main surface of the lid member 20.

[0037] In this specification, a microwell refers to a well with a volume of 10 nanoliters (nL) or less. By setting the volume of the droplet holding section 11 to the volume of the microwell, reactions performed in a minute space, such as digital PCR and digital ICA reactions, can be suitably carried out. Using the method described above, for example, gene mutation detection can be performed. The volume of the droplet holding section 11 (microwell 33) is not particularly limited, but is preferably 10 femtoliters (fL) or more and 100 picoliters (pL) or less, more preferably 10 fL or more and 5 pL or less, and most preferably 10 fL or more and 2 pL or less. Setting the volume within this range is suitable for accommodating only one to several biomolecules or carriers in a single droplet holding section 11 (microwell 33) during the sample analysis described later.

[0038] The shape of the microwell 33 is not particularly limited as long as its volume is within the range described above. Therefore, it may be cylindrical, a polyhedron composed of multiple faces (e.g., a rectangular prism, a hexagonal prism, and an octagonal prism), an inverted cone, or an inverted pyramidal shape (inverted triangular pyramidal shape, inverted square pyramidal shape, inverted pentagonal pyramidal shape, inverted hexagonal pyramidal shape, and inverted polygonal pyramidal shape with seven or more sides). Furthermore, the shapes of the multiple macrowells 33 may be combinations of two or more of the shapes described above. For example, some of the multiple macrowells 33 may be cylindrical and the rest inverted cone shape. Also, if the microwell 33 is inverted cone or inverted pyramidal, the bottom surface of the cone or pyramidal form an opening that connects the flow channel 35 and the microwell 33. In this case, the bottom of the microwell 33 may be made flat by using a shape obtained by cutting off a part from the top of the inverted cone or inverted pyramidal. As another example, the bottom of the microwell 33 may be a curved shape that protrudes toward the opening, or the bottom of the macrowell 33 may be a curved shape that is recessed.

[0039] The unit area (usually 1 mm²) of the region where the droplet holding portion of the substrate 10 is formed. 2 The ratio of the total area of ​​the openings of the droplet holding section 11 (microwell 33) per unit area of ​​the region where the droplet holding section is formed on the substrate 10 is preferably 23% to 90%, more preferably 25% to 90%, more preferably 30% to 90%, more preferably 35% to 80%, more preferably 39% to 76%, and more preferably 39% to 64%. When the ratio of the total area of ​​the openings of the droplet holding section 11 (microwell 33) per unit area of ​​the region where the droplet holding section is formed on the substrate 10 is within the above preferred range, the generation of bubbles in the flow path can be effectively suppressed. This is because, when the aqueous liquid in the droplet holding section 11 (microwell 33) is heated, the total area of ​​the openings is not too large, which suppresses the pressure applied to the sealing liquid in the flow path 35 to some extent.

[0040] Hereinafter, the ratio of the total area of ​​the openings of the droplet holding portion 11 (microwell 33) per unit area of ​​the region where the droplet holding portion is formed on the substrate 10 will also be called the "opening area ratio".

[0041] If the region where the droplet-holding portion 11 (microwell 33) is formed extends across the entire surface of the substrate 10, the total area of ​​the openings of the droplet-holding portion 11 (microwell 33) on the substrate 10 can be approximated to be the same as the area of ​​the region demarcated by the peripheral member of the substrate 10. The region where the droplet-holding portion 11 (microwell 33) is formed may exist only in a part of the substrate 10, as shown in Figure 4. Here, the region where the droplet-holding portion 11 (microwell 33) is formed refers to the region where, when the substrate 10 is viewed from a vertical direction, the distance between the droplet-holding portions 11 (microwell 33) remains constant, as shown in Figure 4. In this case, the total area of ​​the openings of the droplet-holding portion 11 (microwell 33) on the substrate 10 can be approximated to the area of ​​the region enclosed by imaginary lines passing through the centers of the openings of the multiple droplet-holding portions 11 (microwell 33) located at the outermost edge of the region where the droplet-holding portion 11 (microwell 33) is formed. The term "constant interval" does not mean that the intervals must be strictly constant; naturally, manufacturing tolerances are acceptable.

[0042] The larger the area of ​​the region where the droplet holding portion 11 is formed relative to the area of ​​the region demarcated by the peripheral member of the substrate 10, the easier it is for the aqueous liquid to evaporate and for bubbles to be generated. However, by setting the height h of the channel 35 to less than 50 μm, as in this embodiment, the generation of bubbles in the channel 35 can be effectively suppressed.

[0043] If a wall layer 32 is present, the thickness of the wall layer 32 defines the depth of the droplet holding section 11 (microwell 33). When the microwell is cylindrical, for the purpose of encapsulating an aqueous liquid (sample) containing biomolecules, the thickness of the wall layer 32 can be, for example, within the range of 10 nm to 100 μm, preferably 100 nm to 50 μm, more preferably 1 μm to 30 μm, even more preferably 2 μm to 15 μm, and even more preferably 3 μm to 10 μm. The dimensions of each part of the microwell 33 should be determined appropriately, taking into consideration the amount of aqueous liquid to be contained and the size of the carrier such as beads to which biomolecules are attached, so that one or several biomolecules can be contained in each microwell.

[0044] The ratio of the depth of the droplet holding section 11 (microwell 33) to the height h of the flow path 35 is preferably 3% to 150%, more preferably 10% to 100%, even more preferably 12% to 75%, even more preferably 15% to 75%, even more preferably 33% to 75%, and even more preferably 33% to 50%. Being within this range allows for a better balance between suppressing the generation of bubbles and facilitating the introduction of the target droplet into the droplet holding section 11 (microwell 33).

[0045] The number and density of microwells 33 in the microwell array 30 can be set as appropriate, but it is preferable to set the number and density of microwells 33 so that the total volume of the microwells 33 is, for example, 0.2 μL or more and 2.0 μL or less, preferably 0.5 μL or more and 1.5 μL or less. Furthermore, the total volume of the microwells 33 is preferably 5% to 40%, more preferably 8% to 30%, and most preferably 10% to 20% of the volume of the flow path 35. By having the ratio of the total volume of the microwells 33 to the volume of the flow path 35 within a preferred range, the generation of bubbles when the droplets in the microwells 33 are heated can be further suppressed.

[0046] 1cm 2 The number of microwells 33 per unit area is, for example, between 10,000 and 10,000,000, preferably between 100,000 and 5,000,000, and more preferably between 100,000 and 1,000,000. In this specification, 1 cm 2The number of microwells per unit area is sometimes referred to as the microwell density. When the microwell density is within this range, the operation of sealing the aqueous liquid sample into a predetermined number of wells is easy. Furthermore, when the microwell density is within this range, observation of the wells for analyzing experimental results is also easy. For example, when detecting mutations in cell-free DNA, if the proportion of the target mutant DNA relative to wild-type DNA is about 0.01%, it is preferable to use, for example, about 1 to 2 million microwells. Figure 1 shows an example of a one-dimensional array in which multiple microwells 33 are arranged in a row. However, as mentioned above, when providing a large number of microwells, a two-dimensional array in which multiple microwells are arranged in two dimensions may also be used.

[0047] A peripheral member 34, shaped like a frame in plan view, is positioned around the microwell array. The dimensions of the peripheral member 34 in the thickness direction of the microfluidic device 1 are larger than those of the wall layer 32. The peripheral member 34 supports the lid member 20, thereby ensuring a gap between the lid member 20 and the microwell array and maintaining the flow path 35. In other words, the flow path 35 is the region sandwiched between the microwell array 30 and the lid member 20 and surrounded by the peripheral member 34. There are no particular restrictions on the material of the peripheral member 34, but examples include a double-sided adhesive tape in which an acrylic adhesive is laminated on both sides of a core material film made of silicone rubber or acrylic foam. The peripheral member 34 may be integrally molded with the lid member 20. In that case, the peripheral member 34 becomes a stepped portion of the lid member 20, and this stepped portion secures a gap between the lid member 20 and the microwell array, maintaining the flow path 35.

[0048] The microfluidic device 1 configured as described above can be manufactured, for example, by the following procedure. First, prepare the substrate 10 and form a wall resin layer on the surface of the substrate 10, which will become the wall layer 32. If a bottom layer 31 is to be provided, form the bottom layer 31 before forming the wall resin layer. Even if a bottom layer 31 is not provided, an anchor layer or the like may be provided on the surface of the substrate 10 as needed to improve the adhesion between the substrate 10 and the wall resin layer.

[0049] The wall resin layer may be formed from a material obtained by mixing a colored component with a resin material. When the resin material is a resist, the content of the colored component relative to the total mass of the resin material and the colored component can be, for example, 0.5% by mass (mass%) or more and 60% by mass or less. Preferably, the content is 5% by mass or more and 55% by mass or less, and more preferably 20% by mass or more and 50% by mass or less. The content of the colored component relative to the total mass of the resin material and the colored component can be appropriately set to enable the construction of a desired pattern, taking into account the proportion of photosensitive components, etc., contained in the resist. Furthermore, when the colored component is a pigment, the particle size of the pigment is set and prepared to satisfy the predetermined conditions described above for the microwells to be formed. A dispersant may be appropriately added to the resin material along with the pigment. If the formed wall resin layer is made from a material in which a colored component is mixed with the resin material, the wall resin layer will have a color based on the colored component contained in the wall resin layer.

[0050] Next, through-holes 32a are formed in the formed wall resin layer. As described above, photolithography allows for the simple and accurate formation of through-holes 32a. When the wall resin layer is formed by injection molding or the like, the formation of the wall resin layer and the formation of the through-holes can be carried out in the same process. In addition, through-holes 32a can also be formed by etching using a pattern mask or the like. Once the through-hole 32a is formed, the wall resin layer becomes the wall layer 32, and the microwell array 30 is completed.

[0051] Subsequently, the peripheral member 34 is placed around the microwell array 30, and then the lid member 20 is placed on the peripheral member 34. When the substrate 10, the peripheral member 34, and the lid member 20 are joined together, the microfluidic device 1 is completed. The flow path 35 is formed between the lid member 20 and the substrate 10 by the peripheral member 34. The joining method is not particularly limited, but examples include joining with adhesive, joining with double-sided tape, joining by laser welding, and joining by heat welding. When the sample analysis method using the microfluidic device 1 includes a heating reaction, joining with adhesive, joining with double-sided tape, and joining by laser welding are preferred because they can sufficiently withstand the pressure increase in the internal space S due to heating.

[0052] Furthermore, the microfluidic device 1 may have a substrate 10 and a wall layer 32 integrally molded, and a peripheral member 34 and a lid member 20 integrally molded. Figure 5 shows a microfluidic device 2 in which the substrate 10 and the wall layer 32 are integrally molded, and the peripheral member 34 and the lid member 20 are integrally molded. The microfluidic device 2 can be manufactured by placing the substrate 10, which is integrally molded with the wall layer 32, on the lid member 20, which is integrally molded with the peripheral member 34, and joining the stepped portion formed by the integral molding of the peripheral member 34 and the lid member 20 to the substrate 10, which is integrally molded with the wall layer 32. The flow path 35 is formed between the lid member 20 and the substrate 10 by the stepped portion formed on the lid member 20. The configuration of the microfluidic device 2 is the same as that of the microfluidic device 1 described above, except that the substrate 10 and the wall layer 32 are integrally molded, and the peripheral member 34 and the lid member 20 are integrally molded.

[0053] In another embodiment, the microfluidic device may have a substrate 10 and a wall layer 32 as separate elements, and the peripheral member 34 and the lid member 20 may be integrally formed. In this case as well, the configuration of the microfluidic device other than the integral formation of the peripheral member 34 and the lid member 20 is the same as that of the microfluidic device 1 described above.

[0054] Next, the sample analysis method of this embodiment using the microfluidic device 1 according to this embodiment will be described with reference to Figures 6 and 7. The sample analysis method of this embodiment is a sample analysis method using the microfluidic device 1 according to this embodiment, The aqueous liquid containing the sample is introduced into the flow path 35, and the aqueous liquid is held in the droplet holding unit 11, The sealing liquid is introduced into the channel 35 and replaced with the aqueous liquid present in the channel 35, thereby sealing the aqueous liquid in the droplet holding section 11. A reaction occurs in the droplet holding unit 11, generating a signal for detection. This is a sample analysis method comprising detecting the aforementioned signal.

[0055] Here, the aqueous liquid may include water, buffer solution, and detection reaction reagents in addition to the sample. Enzymes may also be included in the aqueous liquid. For example, if the sample is nucleic acid, PCR, ICA, LAMP (trademarked Loop-Mediated Isothermal Amplification), TaqMan (registered trademark), or fluorescent probe methods can be used. For example, if the sample is protein, ELISA (registered trademark) can be used. Furthermore, additives such as surfactants may be included in the aqueous liquid.

[0056] Examples of buffer solutions include Tris-HCl buffer, acetate buffer, and phosphate buffer. Examples of enzymes include DNA polymerase, RNA polymerase, reverse transcriptase, and flap endonuclease. Examples of surfactants include Tween 20 (also known as polyoxyethylene sorbitan monolaurate), Triton-X100 (also known as polyethylene glycol mono-4-octylphenyl ether (n=approximately 10)), glycerol, octylphenol ethoxylate, and alkyl glycosides.

[0057] The microfluidic device of this embodiment can suitably maintain the aqueous liquid in the wells even when the temperature of the enclosed aqueous liquid is changed, for example, in the detection of gene mutations. The range of temperature to be changed, that is, the range from the lower limit to the upper limit of the temperature change, is, for example, 0°C to 100°C, preferably 20°C to 100°C, more preferably 20°C to 90°C, even more preferably 20°C to 80°C, and still more preferably 20°C to 70°C. When the aqueous solution enclosed in the wells is within this range, reactions performed in a microspace, such as PCR reactions and ICA reactions, can be suitably carried out. In this embodiment, since the channel height h of the microfluidic device is less than 50 μm, the generation of bubbles can be suppressed even when the droplet holding section 11 (microwell 33) is heated within the above temperature range.

[0058] Examples of samples to be analyzed using the microfluidic device 1 according to this embodiment include samples taken from living organisms, such as blood. The target to be detected by sample analysis may be a PCR product using DNA contained in the sample as a template, or an artificially synthesized compound (for example, an artificially synthesized nucleic acid that mimics the DNA in the sample). For example, when DNA, a biomolecule, is the target to be detected, the well may have a shape and size that can accommodate one DNA molecule.

[0059] The sample may include biomolecules such as DNA, RNA, miRNA, mRNA, proteins, or lipids. Lipids may include lipid bilayer structures. Biomolecules may also include cells collected from humans for non-therapeutic purposes, cells collected from animals, microorganisms, or bacteria. When the target of detection is a biomolecule, the sample analysis method in one aspect of the present invention can be described as a biomolecule detection method, and the microfluidic device in one aspect of the present invention used in the biomolecule detection method can be described as a biomolecule detection device.

[0060] The following describes the details of the sample analysis method. As a preparation step, an aqueous liquid containing the sample to be sealed in the microwells is prepared. The aqueous liquid containing the sample is a liquid in which water containing the target to be detected is the main solvent. Examples include a PCR reaction solution using a biological sample as a template and containing SYBR Green as the detection reagent, or an ICA reaction solution containing an allergen probe, ICA oligo, FEN-1, and a fluorescent substrate. During preparation, a surfactant may be added to make it easier for the sample to enter the microwells. Alternatively, beads that specifically recognize the target to be detected may be added to capture the target. The target to be detected may not be directly or indirectly bound to a carrier such as beads, but may be suspended in the aqueous liquid.

[0061] Next, using a syringe or the like, the aqueous liquid 100 containing the prepared sample is introduced into the flow path 35 through the first hole 21, and the droplet holding unit 11 is made to hold the aqueous liquid containing the sample (also called the sample supply step). The supplied aqueous liquid 100 containing the sample is filled into each microwell 33 and the flow path 35, as shown in Figure 6. The gas in the flow path 35 is removed in advance by a degassing operation before the sample supply step. This degassing operation may be performed by filling the flow path 35 with a buffer. Examples of buffers include water, water containing a buffer, water containing a surfactant, and water containing both a buffer and a surfactant.

[0062] Next, a sealing step is performed in which the aqueous liquid 100 containing the sample is sealed into the droplet holding section 11 (microwell 33). Before the sealing step, the target to be detected in the sample contained in the aqueous liquid may be labeled with a fluorescent or other label. The fluorescent labeling process may be performed before the sample supply step, for example, during sample preparation, or after the sample supply step, by introducing the fluorescent label into the flow path 35. In the sealing process, the sealing solution 110 is supplied from the first hole 21 to the flow path 35 using a syringe or the like. The supplied sealing solution 110 flows through the flow path 35 and, as shown in Figure 7, pushes the aqueous liquid 100 containing the sample present in the flow path 35 toward the second hole 22. The sealing solution 110 then replaces the aqueous medium 100 that was filling the flow path 35, and the flow path 35 is filled with the sealing solution 110. As a result, the aqueous liquid 100 containing the sample is placed only in each microwell 33, independently of each other, and the sealing of the sample is completed.

[0063] In this specification, the sealing liquid 110 refers to a liquid used to isolate the aqueous liquids introduced into each microwell 33 of the microwell array 30 so that they do not mix with each other, and examples include oils. Examples of oils that can be used include Sigma's product name "FC40", 3M's product name "HFE-7500", and mineral oil used in PCR reactions, etc. Preferably, the sealing solution 110 has a contact angle of 5 degrees or more and 30 degrees or less with respect to the material of the wall layer 32. When the contact angle of the sealing solution 110 is within this range, the sample can be suitably sealed in each microwell 33. The contact angle of the sealing solution 110 can be measured, for example, by using the sealing solution 110 instead of water, in accordance with the static droplet method specified in JIS R3257-1999. Alternatively, the contact angle may be measured by a method in accordance with ASTM D5725-1997 instead of the static droplet method specified in JIS R3257-1999.

[0064] Next, a reaction step is performed in which a reaction occurs in the droplet holding section 11 (microwell 33) of the microfluidic device 1 to generate a signal for detection. Examples of signals for detection include fluorescence, chemiluminescence, color development, potential changes, or pH changes, but fluorescence is preferred. Before the reaction step, the microfluidic device 1 may be subjected to a thermal cycler to perform enzymatic reactions such as PCR or ICA reactions, as needed. The reaction may be, for example, a biochemical reaction, or more specifically, an enzymatic reaction. Alternatively, the reaction may be initiated by heating the microfluidic device 1. The heating temperature is determined appropriately depending on the reaction, but is, for example, between 60°C and 100°C. Preferably, the heating temperature is between 60°C and 90°C, more preferably between 60°C and 80°C, and even more preferably between 60°C and 70°C. The heating temperature refers not to the actual temperature of the reagent solution in the droplet holding section 11 (microwell 33), but to the heating temperature of the microfluidic device set by a thermal cycler or incubator. Furthermore, the statement that the heating temperature is, for example, between 60°C and 100°C means that the maximum temperature reaches between 60°C and 100°C, and it is not necessary for it to always be between 60°C and 100°C. That is, the temperature of the microfluidic device 1 may change within the range of temperature variation described above. An example of a reaction is a signal amplification reaction. The signal amplification reaction is an isothermal reaction in which the microfluidic device 1 is maintained for a predetermined time, for example, at least 10 minutes, preferably about 15 minutes, under constant temperature conditions, for example, between 60°C and 100°C, where the desired enzyme activity can be obtained, with a reagent solution containing an enzyme for signal amplification contained in the droplet holding section 11 (microwell 33).

[0065] Next, the signal generated from the droplet holding section 11 (microwell 33) by the above reaction is detected (also called the detection step). For example, if the signal is fluorescence, the microfluidic device 1 is set in a fluorescence microscope and excitation light (electromagnetic waves) is irradiated onto it. The wavelength of the excitation light is set appropriately according to the fluorescent label being used. Electromagnetic wave irradiation may be performed from the substrate 10 side of the microfluidic device 1, from the lid member 20 side, i.e., from the upper side of the microwell 33, or from any other direction. Furthermore, detection of fluorescence or phosphorescence generated as a result of electromagnetic wave irradiation may be performed from the substrate side of the microwell array, from the well side, or from any other direction. However, when detecting fluorescence or phosphorescence using a fluorescence microscope, for example, it is convenient to perform the detection from the substrate 10 side of the microfluidic device 1.

[0066] Next, the number of microwells 33 that make up the microwell array 30 that emit fluorescence or phosphorescence is measured. The measurement may also be performed by taking a fluorescence image of the microwell array 30 and using the fluorescence image. For example, by performing a PCR reaction in a microwell array 30 and detecting the fluorescence of SYBR Green in the microwells 33 where PCR amplification is observed, the proportion of microwells 33 that show amplification relative to the total number of microwells 33 can be calculated. If the target of detection is, for example, a single nucleotide polymorphism (SNP), the expression frequency of the SNP can be analyzed by counting the number of fluorescent microwells 33.

[0067] Another aspect of the present invention includes the following embodiments.

[31] A microfluidic device comprising a substrate, a lid member located on the substrate, a peripheral member connecting the substrate and the lid member, a flow channel located between the substrate and the lid member and partitioned by the peripheral member, and one or more droplet holding portions located on the substrate and connected to the flow channel, wherein the height of the flow channel is greater than 0 μm and less than or equal to 30 μm.

[32] The microfluidic device according to

[31] , wherein the droplet holding portion is a hole provided on the surface of the substrate.

[33] The microfluidic device according to

[31] , having at least one first hole for introducing liquid into the flow path and at least one second hole for discharging liquid from the flow path, wherein the first hole and the second hole are positioned to sandwich one or more liquid-holding portions.

[34] The microfluidic device according to

[31] to

[33] , wherein the bottom of the droplet holding portion is on the substrate side, the droplet holding portion has an opening on the lid member side, and the flow path is located on the opening.

[35] The microfluidic device according to any one of

[31] to

[34] , wherein the volume of each droplet-holding portion is 10 fL or more and 100 pL or less.

[36] The microfluidic device according to any one of

[31] to

[35] , wherein the total volume of the droplet holding portion is 0.2 μL or more and 2.0 μL or less.

[37] The microfluidic device according to any one of

[31] to

[36] , wherein the ratio of the total volume of the droplet holding portion to the volume of the flow path is 5% or more and 40% or less.

[38] The microfluidic device according to any one of

[31] to

[37] , wherein the ratio of the depth of the droplet holding portion to the height of the flow channel is 3% or more and 150% or less.

[39] The microfluidic device according to any one of

[31] to

[38] , wherein the ratio of the total area of ​​the openings of the droplet holding portion to the unit area of ​​the region in which the droplet holding portion is formed on the substrate is 23% or more and 90% or less.

[40] The microfluidic device according to any one of

[31] to

[39] , wherein the peripheral member is a stepped portion formed integrally with the lid member. A method for analyzing a sample using a microfluidic device as described in any one of

[41] ,

[31] , to

[40] , comprising: introducing an aqueous liquid containing a sample into the channel and holding the aqueous liquid in the droplet holding portion; introducing a sealing liquid into the channel to replace the aqueous liquid present in the channel with the sealing liquid and sealing the aqueous liquid in the droplet holding portion; causing a reaction in the droplet holding portion to generate a signal for detection; and detecting the signal.

[42] The sample analysis method according to

[41] , wherein the sample is a biomolecule.

[43] The sample analysis method according to

[41] or

[42] , wherein generating the signal for detection includes heating the microfluidic device to cause the reaction, the temperature at which the microfluidic device is heated is 60°C or higher.

[44] A sample analysis method according to any one of

[41] to

[43] , wherein the signal is detected by imaging of the microfluidic device.

[45] The method for analyzing a sample according to any one of

[41] to

[44] , wherein the signal is fluorescent.

[46] The method for analyzing a sample according to any one of

[41] to

[45] , wherein the reaction is an isothermal reaction. [Examples]

[0068] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0069] (Example 1) Two resin components were prepared: a substrate made of COP (ZEONOR1010R, manufactured by Nippon Zeon Co., Ltd.) formed by injection molding, and a lid component also made of COP (ZEONOR1010R, manufactured by Nippon Zeon Co., Ltd.). The total volume of micropores (i.e., microwells) was adjusted by changing the number of micropores molded into the COP substrate. The lid component and the stepped portion were formed integrally, and the height of the stepped portion was adjusted to 30 μm, thereby setting the height of the flow channel to 30 μm. The micropores, with a diameter of 10 μm and a depth of 15 μm, were arranged in a 9000 mm × 30000 mm area within the flow channel surface. The aperture area ratio was defined as the ratio of the total area of ​​the micropore openings per 6.5 mm × 9.0 mm region where micropores are formed. The substrate used was a 0.6 mm thick, injection-molded substrate with micro-perforations across its entire surface. A contact-type measuring instrument (TALYSURF PGI1240, Taylor Hobson) was used to measure the channel height. A microfluidic device was fabricated by laser welding the stepped portion of the substrate and the lid member together. A fluorescent reagent (Fluorescein, manufactured by Tokyo Chemical Industry Co., Ltd.) having the composition shown in Table 1 was injected into the channel formed between the substrate and the lid member. Furthermore, multiple microwells were individually sealed with fluorocarbon oil (FC40, manufactured by Sigma Corporation). In this example, a fluorescence reaction was not performed, but an enzyme was added to the fluorescent reagent to achieve a state similar to that of a fluorescence reaction.

[0070] [Table 1]

[0071] The above microfluidic device was heated at 66°C for 30 minutes. After being left at room temperature, the presence or absence of bubbles was checked from the top surface, i.e., from the lid side, and photographed using a digital camera (CX-4, Ricoh). In addition, micropore fluorescence images were observed using a fluorescence microscope (BZ-710, KEYENCE) with a 4x objective lens. The exposure time was 20 msec in brightfield, and 3000 msec using a GFP (Green Fluorescent Protein) fluorescence filter.

[0072] Table 2 shows the bubble generation rate for the above microfluidic devices. The bubble generation rate was calculated by preparing multiple microfluidic devices of the same design and dividing the number of devices with bubbles in each device by the total number of microfluidic devices analyzed. The presence or absence of bubbles was determined if bubbles of a size visible to the naked eye were present in the microfluidic device, and if no bubbles were present under the same conditions, it was determined that there were no bubbles. The maximum size of bubbles visible under the above conditions was approximately 500 μm or larger. Even if small bubbles were present, if the size of the bubbles was smaller than the distance between the edges of the nearest adjacent micropores (i.e., microwells) when the microfluidic device was observed with a microscope using a 4x objective lens, it was determined that there were no bubbles. The reason for this determination is that if bubbles smaller than the above size are present, they do not substantially hinder detection, and it is considered that bubble generation is suppressed. As a result, as shown in Table 2, the microfluidic device manufactured in Example 1 had a low rate of bubble generation. Figure 8 shows the observation results of typical bubbles in the comparative microfluidic device (channel height: 100 μm). In Figure 8, the areas indicated by arrows show typical bubble generation areas. Figure 8 shows that bubbles of several millimeters were present, as well as multiple small bubbles of about 500 μm. The microfluidic device of Example 1, with a channel height of 30 μm, showed a reduced rate of such bubble generation.

[0073] Next, the fluorescence observation results are shown. As shown in Figure 9A, in the microfluidic device of Example 1 with a channel height of 30 μm, the droplets did not collapse, and it was possible to observe the micropores (i.e., microwells).

[0074] (Example 2) A microfluidic device was manufactured in the same manner as in Example 1, except that the channel height was set to 20 μm, and the bubble generation rate was measured. The results are shown in Table 2. As shown in Table 2, the microfluidic device manufactured in Example 2 had a low bubble generation rate.

[0075] (Comparative Example 1) A microfluidic device was manufactured in the same manner as in Example 1, except that the channel height was set to 100 μm, and the bubble generation rate was measured. The results are shown in Table 2. As shown in Table 2, the microfluidic device manufactured in Comparative Example 1 had a high bubble generation rate. Figure 9B shows the fluorescence observation results. In the microfluidic device with a channel height of 100 μm, multiple bubbles were generated, making it difficult to observe the micropores. In Figure 9B, the areas indicated by arrows show typical bubble generation areas.

[0076] (Comparative Example 2) A microfluidic device was manufactured in the same manner as in Example 1, except that the well depth was set to 3.5 μm and the channel height to 100 μm, and the bubble generation rate was measured. The results are shown in Table 2. As shown in Table 2, the microfluidic device manufactured in Comparative Example 2 had a high bubble generation rate.

[0077] [Table 2]

[0078] (Example 3) A substrate made of COP (ZEONOR1010R, manufactured by Nippon Zeon Co., Ltd.) formed by injection molding was used. Micropores with a diameter of 5 μm and a depth of 3.5 μm were arranged in a 6.5 mm × 9.0 mm area within the flow channel surface. As the lid component, a lid component made of COP (ZEONOR1010R, manufactured by Nippon Zeon Co., Ltd.) formed by injection molding (with liquid supply and waste liquid ports) was used, and as the peripheral component, a 30 μm thick PET (polyethylene terephthalate) base double-sided tape (No. 5603 BN, manufactured by Nitto Denko Co., Ltd.) was used.

[0079] Aside from these, the microfluidic devices were manufactured in the same manner as in Example 1, and the bubble generation rate was measured. The results are shown in Table 3. In Example 3, four microfluidic devices were linked together to form a single device group. Three of these device groups were created, resulting in a total of 12 microfluidic devices. Furthermore, the "ratio of the area where the well is formed" was defined as the ratio of the area where micropores are formed to the area of ​​the region surrounded by the peripheral member, i.e., the area where the flow channel is formed.

[0080] (Comparative Example 3) A microfluidic device was manufactured in the same manner as in Example 1, except that a 50 μm thick PET (polyethylene terephthalate) base double-sided tape (product number: No. 5603 BN, manufactured by Nitto Denko) was used as the peripheral material, and the bubble generation rate was measured. The results are shown in Table 3.

[0081] (Comparative Example 4) A microfluidic device was manufactured in the same manner as in Example 1, except that a 100 μm thick PET (polyethylene terephthalate) base double-sided tape (product number: No. 5603 BN, manufactured by Nitto Denko) was used as the peripheral material, and the bubble generation rate was measured. The results are shown in Table 3.

[0082] [Table 3]

[0083] The channel height of the microfluidic device in Example 4 corresponds to the thickness of the peripheral member, and can therefore be said to be 30 μm. No bubbles were generated in the microfluidic device of Example 4.

[0084] On the other hand, in the microfluidic devices of Comparative Example 3, where the channel height was 50 μm, and Comparative Example 4, where the channel height was 100 μm, the bubble generation rates were high, at 50% and 100%, respectively. Based on the above, it was confirmed that the bubble generation rate increases when the channel height of the microfluidic device is 50 μm or more, but decreases when the channel height is 30 μm. [Industrial applicability]

[0085] The present invention provides a microfluidic device that can suppress the generation of bubbles when forming and heating minute droplets. Furthermore, it provides a sample analysis method that can suppress the generation of bubbles when forming and heating minute droplets and optically detecting the sample, thereby improving the detection efficiency of the sample. When biomolecules are used as the sample, it is necessary to heat them to a high temperature for a certain period of time or longer, and the present invention can effectively suppress the generation of bubbles even in such cases. [Explanation of Symbols]

[0086] 1, 2 Microfluidic Devices 10 circuit boards 11 Droplet holding part 20 Lid member 30 microwell array 32 Wall layer 33 microwells 35 channels 100 aqueous liquid 110 Sealing liquid

Claims

1. It has a flow channel and a plurality of droplet holding parts connected to the flow channel, and the height of the flow channel is greater than 0 μm and less than or equal to 30 μm. 1 cm ２ The number of droplet-holding parts per unit is between 100,000 and 10,000,000. The ratio of the total volume of the droplet holding section to the volume of the flow path is 5% or more and 40% or less. A microfluidic device in which the total volume of the droplet-holding portion is 0.2 μL or more and 2.0 μL or less.

2. Furthermore, the microfluidic device according to claim 1, having a flat substrate, the flow path being located on the flat substrate, and the droplet holding portion being a hole present on the substrate.

3. Furthermore, the microfluidic device according to claim 2, further comprising a lid member, wherein the flow path is a space sandwiched between the lid member and the substrate.

4. The microfluidic device according to claim 2 or 3, wherein the ratio of the total area of ​​the openings of the droplet-holding portion to the unit area of ​​the region in which the droplet-holding portion is formed on the substrate is 23% or more and 90% or less.

5. The microfluidic device according to any one of claims 1 to 4, wherein the ratio of the depth of the droplet holding portion to the height of the flow channel is 3% or more and 150% or less.

6. A method for analyzing a sample using a microfluidic device according to any one of claims 1 to 5, The aqueous liquid containing the sample is introduced into the flow path, and the aqueous liquid is held in the droplet holding section. The sealing liquid is introduced into the channel to replace the aqueous liquid present in the channel, and the aqueous liquid is sealed in the droplet holding portion. A reaction occurs in the droplet holding section, generating a signal for detection. To detect the aforementioned signal, A sample analysis method comprising the following features.

7. The sample analysis method according to claim 6, wherein the sample is a biomolecule.

8. The sample analysis method according to claim 6 or 7, wherein generating the signal for detection includes heating the microfluidic device to cause the reaction, and the temperature at which the microfluidic device is heated is 60°C or higher.

9. The sample analysis method according to any one of claims 6 to 8, wherein the signal is detected by imaging the microfluidic device.

10. The sample analysis method according to any one of claims 6 to 9, wherein the signal is fluorescence.

11. The method for analyzing a sample according to any one of claims 6 to 10, wherein the reaction is an isothermal reaction.

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