Microfluidic device and sample analysis method

Abstract

To provide a microfluidic device reducing non-specific adsorption of reagents, and a sample analysis method capable of, when detecting a signal generating from a microwell, correctly detecting the signal by preventing fluorescence, etc. generated by non-specific adsorption from becoming noise.SOLUTION: There are provided a microfluidic device 1 and a sample analysis method using the microfluidic device 1. The microfluidic device comprises a microwell array 30 including a plurality of microwells, a lid member 20 facing the microwell array 30 with a space therebetween, and a hydrophobic coating layer applied to a surface of the lid member on the microwell array side, where a flow path is provided between the microwell array 30 and the lid member 20. An arithmetic mean roughness Ra of a surface 20a of the lid member 20 on the microwell array 30 side is 50 nm or less, and the thickness of the coating layer is 0.01 μm or more and 3 μm or less.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a microfluidic device and a sample analysis method. This application claims priority from Japanese Patent Application No. 2019-037543, filed on March 1, 2019, the contents of which are incorporated herein by reference. [Background technology]

[0002] In recent years, microwell arrays with various types of fine channel structures have been investigated, formed using etching and photolithography techniques used in semiconductor circuit manufacturing or microplastic molding methods. The wells of these microwell arrays are used as chemical reaction vessels for various biochemical or chemical reactions in microvolumes of fluid.

[0003] Materials used to fabricate microfluidic systems include hard substances such as silicon and glass, various polymer resins such as PDMS (polydimethylsiloxane), and soft substances such as silicone rubber. For example, Patent Documents 1 to 3 and Non-Patent Document 1 describe the use of such microfluidic systems as various microchips and biochips.

[0004] Recently, attention has been focused on technologies for testing biological materials by carrying out reactions in a microscopic space with a very small volume. One example of such a technology is digital measurement technology. One example of a new approach to nucleic acid detection and quantification is digital PCR (Digital Polymerase Reaction). Digital PCR is a technology in which a mixture of reagents and nucleic acid is divided into countless microdroplets for PCR amplification, and a signal such as fluorescence is detected from the droplets containing the nucleic acid, and the droplets from which the signal is detected are counted to perform quantification.

[0005] Methods under consideration for producing microdroplets include a method in which a mixture of reagent and nucleic acid is fragmented using a sealing liquid to form microdroplets, and a method in which a mixture of reagent and nucleic acid is poured into a hole formed on a substrate, followed by pouring in a sealing liquid to form microdroplets. [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. Summary of the Invention [Problem to be solved by the invention]

[0008] However, when analyzing samples using a microfluidic device, reagents may be nonspecifically adsorbed to the cover member. In conventional microfluidic devices, when detecting fluorescent signals in minute holes, the fluorescence emitted from nonspecific adsorption of reagents, which was not a problem when using non-digital measurement techniques, can become noise when counting the number of wells containing fluorescent substances using digital measurement. Although it is possible to suppress adsorption of reagents onto the lid member by using a material that is generally considered to be hydrophobic, such as PDMS, for the lid member, this limits the materials that can be used.

[0009] The present inventors have discovered that it is possible to reduce nonspecific adsorption of reagents to a lid member and improve detection efficiency without using a material generally considered to be hydrophobic, and have completed the present invention. The present invention aims to provide a microfluidic device that can reduce nonspecific adsorption of reagents to a lid member and improve detection efficiency. Another aim of the present invention is to provide a sample analysis method that can accurately detect signals by suppressing the fluorescence and other emissions caused by nonspecific adsorption from becoming noise when detecting signals generated from microwells. [Means for solving the problem]

[0010] In order to achieve the above object, the present invention includes the following aspects. [1] A microwell array having a plurality of microwells, a cover member facing the microwell array while being spaced apart from the microwell array, and a hydrophobic coating layer applied to the surface of the cover member facing the microwell array, wherein a flow path is provided between the microwell array and the cover member, the arithmetic mean roughness Ra of the surface of the cover member facing the microwell array is 70 nm or less, and the Ra / Rz of the surface of the cover member facing the microwell array is 0.10 or more and 0.24 or less, Rz is the surface roughness (ten-point average roughness) of the surface of the cover member facing the microwell array, A microfluidic device, wherein the coating layer has a thickness of 0.01 μm or more and 3 μm or less. [2] The microfluidic device according to [1], wherein the coating layer contains at least one of a fluorine-based coating agent, a fluorine-containing polymer, and a silicone resin. [3] A sample analysis method using the microfluidic device described in [1] or [2], comprising: supplying an aqueous liquid containing a sample to the flow path; introducing a sealing liquid into the flow path to replace the aqueous liquid present in the flow path, sealing the aqueous liquid in the microwell; causing a reaction in the microwell to generate a signal for detection; and detecting the signal. [4] The sample analysis method according to [3], wherein the sample is DNA, RNA, protein, lipid, cell, or bacterium. [Effects of the Invention]

[0011] The present invention can provide a microfluidic device that reduces nonspecific adsorption of reagents, and can also provide a sample analysis method that can accurately detect signals by suppressing the fluorescence and other emissions caused by nonspecific adsorption from becoming noise when detecting signals generated from microwells. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a perspective view showing a microfluidic device according to an embodiment of the present invention. [Figure 2] FIG. 2 is a cross-sectional view taken along line bb in FIG. [Figure 3] 1 is a cross-sectional view showing a microfluidic device according to one embodiment of the present invention. [Figure 4] 1A and 1B are diagrams showing a state in which a microfluidic device according to an embodiment of the present invention is in use. [Figure 5] 1A and 1B are diagrams showing a state in which a microfluidic device according to an embodiment of the present invention is in use. [Figure 6A] FIG. 1 shows a fluorescent image obtained using a microfluidic device according to one embodiment of the present invention. [Figure 6B] FIG. 10 is a diagram showing a fluorescent image obtained using the microfluidic device according to Comparative Example 1. [Figure 7] FIG. 10 is a diagram showing measurement data of the surface roughness of a cover member of a microfluidic device according to one embodiment of the present invention. [Figure 8] FIG. 10 is a diagram showing measurement data of the surface roughness of a cover member of a microfluidic device according to one embodiment of the present invention. [Figure 9] FIG. 10 is a diagram showing measurement data of the surface roughness of the cover member of the microfluidic device according to Comparative Example 2. [Figure 10]FIG. 10 is a diagram showing measurement data of the surface roughness of the cover member of the microfluidic device according to Comparative Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0013] An embodiment of the present invention will be described with reference to Figures 1 to 5. In this specification, the dimensional ratios in the drawings are exaggerated for the purpose of explanation and do not necessarily correspond to the actual dimensional ratios.

[0014] FIG. 1 is a perspective view showing a microfluidic device 1 according to this embodiment. FIG. 2 is a cross-sectional view taken along line bb in FIG. 1. As shown in FIGS. 1 and 2, the microfluidic device 1 comprises a microwell array 30 having a plurality of wells, and a cover member 20 facing the microwell array 30 at a distance, with a channel 35 between the microwell array 30 and the cover member 20. The microwell array 30 may comprise only a substrate 10, or may comprise a bottom layer 31 and a wall layer 32 in addition to the substrate 10. A peripheral member 34 is located between the microwell array 30 and the cover member 20. The region sandwiched between the microwell array 30 and the cover member 20 and surrounded by the peripheral member 34 constitutes the channel 35. The peripheral member 34 may be formed integrally with the cover member 20.

[0015] The substrate 10 may be capable of transmitting electromagnetic waves. Here, examples of electromagnetic waves include X-rays, ultraviolet rays, visible light, and infrared rays. By allowing the substrate 10 to transmit electromagnetic waves, fluorescence, phosphorescence, and the like generated by a reaction between a sample and a reagent enclosed in the microfluidic device 1 can be observed from the substrate 10 side. Substrate 10 may be capable of transmitting only electromagnetic waves in a predetermined wavelength range. For example, when the presence of a sample in a microwell is determined by detecting fluorescence having a peak in the wavelength range of 350 to 700 nm, which is the visible light region, a substrate capable of transmitting at least visible light in the above wavelength range may be used as substrate 10.

[0016] Examples of materials for forming the substrate 10 include glass and resin. Examples of materials for resin substrates 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, additives that impart water repellency, and additives that impart hydrophilicity. The resin substrate may contain only one of the above resins, or may be a mixture of multiple resins.

[0017] Since the sample analysis method described below utilizes fluorescence or phosphorescence, a material that is substantially free of autofluorescence is used for the substrate 10. Here, "substantially free of autofluorescence" means that the substrate has no autofluorescence at the wavelength used to detect the experimental results, or that even if it does have autofluorescence, it is so weak that it does not affect the detection of the experimental results. For example, autofluorescence that is less than half, preferably less than one-tenth, of the fluorescence to be detected can be said to be so weak that it does not affect the detection of the experimental results.

[0018] 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. Furthermore, to facilitate processing, it is preferably 0.1 mm or more, more preferably 0.2 mm or more. 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 even more preferably 0.4 mm or more and 1.6 mm or less.

[0019] The lid member 20 (which may be simply referred to as the lid 20) is a member formed in the shape of a plate or sheet. The lid member 20 faces the microwell array 30 at a distance. In other words, the lid member 20 covers the multiple microwells 33. The flow path 35 is an area 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.

[0020] The cover member 20 has a first hole 21 and a second hole 22 that penetrate through the thickness direction. In a plan view of the cover member 20, the first hole 21 and the second hole 22 are positioned so as to sandwich one or more of the liquid retaining portions. In the completed microfluidic device 1, the first hole 21 and the second hole 22 communicate with the internal space S that includes the microwell array 30 and the flow channel 35. 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 the fluid, respectively. The material and thickness of the lid member 20 can be the same as those of the substrate 10. When the lid member 20 has electromagnetic wave transparency, the electromagnetic wave transparency can be set appropriately. For example, when the electromagnetic wave irradiation step 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.

[0021] The arithmetic mean roughness (Ra) of the surface 20a of the cover member 20 facing the microwell array 30 is 70 nm or less, preferably 60 nm or less, more preferably 50 nm or less, even more preferably 40 nm or less, and particularly preferably 35 nm or less. Furthermore, the arithmetic mean roughness (Ra) of the surface 20a of the cover member 20 facing the microwell array 30 may be 30 nm or less, 25 nm or less, 20 nm or less, or 15 nm or less. By having the arithmetic mean roughness (Ra) of the surface 20a of the cover member 20 facing the microwell array 30 be 50 nm or less, nonspecific adsorption of reagents to the surface 20a of the cover member 20 facing the microwell array 30 can be suppressed.

[0022] The lower limit of the arithmetic mean roughness (Ra) of the surface 20a of the cover member 20 on the side of the microwell array 30 is not particularly limited, but is, for example, 5 nm.

[0023] The upper and lower limits of the arithmetic mean roughness (Ra) of surface 20a of cover member 20 facing microwell array 30 can be arbitrarily combined. For example, the arithmetic mean roughness (Ra) of surface 20a of cover member 20 facing microwell array 30 may be 5 nm to 70 nm, 5 nm to 60 nm, 6 nm to 50 nm, 7 nm to 40 nm, 7 nm to 35 nm, 8 nm to 30 nm, 8 nm to 25 nm, 9 nm to 20 nm, or 10 nm to 15 nm.

[0024] The arithmetic mean roughness (Ra) can be measured, for example, according to the measurement method specified in JIS B 0601-2001. The measurement range may be the entire surface of the lid member or a representative area. In this embodiment, the measurement range is defined as a straight line having the center of the lid member 20 as the center of the measurement range, and the length of the straight line is 800 μm to 1.5 mm.

[0025] There are no particular limitations on the method for making the arithmetic mean roughness (Ra) of surface 20a of cover member 20 facing microwell array 30 50 nm or less, but examples include mirror polishing of cover member 20. When cover member 20 is manufactured by injection molding, the mold for cover member 20 may also be mirror-polished using diamond paste or the like.

[0026] The contact angle with water of surface 20a of cover member 20 on the microwell array 30 side may be 70 degrees or more. The contact angle between water and the surface 20a of the cover member 20 facing the microwell array 30 is 180 degrees or less, so the contact angle between water and the surface of the cover member 20 facing the microwell array 30 is, for example, 70 degrees or more and 180 degrees or less. The contact angle with water can be measured, for example, according to the sessile drop method specified in JIS R 3257-1999. Instead of the sessile drop method specified in JIS R 3257-1999, the contact angle may be measured by a method in accordance with ASTM D5725-1997.

[0027] The surface roughness (ten-point average roughness) (Rz) of the surface 20a of the cover member 20 facing the microwell array 30 is preferably 350 nm or less, more preferably 300 nm or less, and particularly preferably 250 nm or less. Furthermore, the surface roughness (ten-point average roughness) (Rz) of the surface 20a of the cover member 20 facing the microwell array 30 may be 200 nm or less, 150 nm or less, 120 nm or less, 110 nm or less, 100 nm or less, or 90 nm or less. By having the surface roughness (Rz) of the surface 20a of the cover member 20 facing the microwell array 30 be 350 nm or less, nonspecific adsorption of reagents to the surface 20a of the cover member 20 facing the microwell array 30 can be further suppressed.

[0028] The lower limit of the surface roughness (ten-point average roughness) (Rz) of the surface 20a of the cover member 20 on the side of the microwell array 30 is not particularly limited, but is, for example, 50 nm. The upper and lower limit values ​​can be arbitrarily combined for the surface roughness (ten-point average roughness) (Rz) of surface 20a of cover member 20 facing microwell array 30. For example, the surface roughness (ten-point average roughness) (Rz) of surface 20a of cover member 20 facing microwell array 30 may be 50 nm to 350 nm, 55 nm to 300 nm, 60 nm to 250 nm, 60 nm to 200 nm, 60 nm to 150 nm, 65 nm to 120 nm, 70 nm to 110 nm, 70 nm to 100 nm, or 80 nm to 90 nm.

[0029] The surface roughness (Rz) can be measured in accordance with the measurement method specified in JIS B 0601-2001. The measurement range may be the entire surface of the lid member or a representative area. In this embodiment, the measurement range is defined as a straight line having the center of the lid member 20 as the center of the measurement range, and the length of the straight line is 800 μm to 1.5 mm.

[0030] There are no particular limitations on the method for making the surface roughness (Rz) of surface 20a of cover member 20 facing microwell array 30 350 nm or less, but examples include mirror polishing of cover member 20. When cover member 20 is manufactured by injection molding, the mold for cover member 20 may be mirror polished with diamond paste.

[0031] The Ra / Rz of the surface 20a of the cover member 20 facing the microwell array 30 is preferably 0.10 or more. The Ra / Rz of the surface 20a of the cover member 20 facing the microwell array 30 is preferably 0.24 or less, more preferably 0.23 or less, and even more preferably 0.225 or less. Furthermore, the Ra / Rz of the surface 20a of the cover member 20 facing the microwell array 30 may be 0.16 or less, 0.15 or less, 0.14 or less, or 0.13 or less.

[0032] The upper and lower limit values ​​of Ra / Rz of surface 20a of cover member 20 facing microwell array 30 can be combined arbitrarily. For example, Ra / Rz of surface 20a of cover member 20 facing microwell array 30 may be 0.10 or more and 0.24 or less, 0.10 or more and 0.23 or less, 0.10 or more and 0.225 or less, 0.10 or more and 0.16 or less, 0.10 or more and 0.15 or less, 0.10 or more and 0.14 or less, or 0.10 or more and 0.13 or less.

[0033] When the Ra / Rz of the surface 20a of the lid member 20 facing the microwell array 30 is 0.10 or more and 0.24 or less, nonspecific adsorption of reagents to the surface 20a of the lid member 20 facing the microwell array 30 can be further suppressed, even if the material of the lid member 20 is hydrophilic. This is thought to be because not only the electrical and chemical interactions between the surface 20a of the lid member 20 facing the microwell array 30 and the reagent are suppressed, but also the physical interactions can be suppressed due to the small micro-irregularities on the surface 20a of the lid member 20 facing the microwell array 30. When the Ra / Rz is less than 0.10, this indicates that the surface 20a of the lid member 20 facing the microwell array 30 has partially protruding portions, which are likely to cause nonspecific adsorption of reagents. When the Ra / Rz is greater than 0.24, this indicates that the surface 20a of the lid member 20 facing the microwell array 30 is generally rough, which is likely to cause nonspecific adsorption of reagents.

[0034] In the microfluidic device of this embodiment, the arithmetic mean roughness (Ra) within the above range can be achieved by applying a hydrophobic coating to surface 20a of cover member 20 facing microwell array 30. Therefore, by applying a hydrophobic coating to surface 20a of cover member 20 facing microwell array 30, nonspecific adsorption of reagents to surface 20a of cover member 20 facing microwell array 30 can be suppressed.

[0035] Examples of methods for applying a hydrophobic coating to the surface 20a of the lid member 20 facing the microwell array 30 include applying a hydrophobic coating agent to the surface 20a of the lid member 20 facing the microwell array 30 and then drying it. Examples of the coating agent include fluorine-based coating agents, fluorine-containing polymers, silicone resins, etc. Examples of the coating method include dry coating and wet coating.

[0036] When the surface 20a of the cover member 20 facing the microwell array 30 is coated with a hydrophobic coating agent, the thickness of the coating layer is preferably 0.01 μm or more and 3 μm or less, and more preferably 0.05 μm or more and 1 μm or less.

[0037] The microwell array 30 may have a bottom layer 31, a wall layer 32 (which may also be referred to as partitions 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 is composed of the bottom layer 31 and a plurality of through-holes 32a formed in the thickness direction of the wall layer 32. The plurality of microwells 33 are formed in an array in the wall layer 32. In the internal space S between the substrate 10 and the cover member 20, there is a gap between the microwell array 30 and the cover member 20, in other words, between the upper surface of the wall layer 32 and the cover member 20. This gap functions as a flow path communicating with the plurality of microwells 33 and the first and second holes 21 and 22.

[0038] The bottom layer 31 constitutes the bottom surface of the microwell 33. Therefore, if it is desired to impart hydrophilicity to the bottom surface, the bottom layer 31 may be formed from a hydrophilic material. The bottom layer 31 is preferably formed to be permeable to electromagnetic waves and not to interfere with observation of the sample in the microwell 33 from the substrate 10 side. Furthermore, if it is desired to impart hydrophobicity to the bottom surface, the bottom layer 31 may be formed from a hydrophobic material. It is preferable that the bottom layer 31 not interfere with observation of the sample in the microwell 33 from the substrate 10 side. Furthermore, it is preferable to use a material that is substantially free of autofluorescence for the bottom layer 31.

[0039] If there is no problem as long as the characteristics of the bottom surface of the microwell 33 are the same as those of the substrate 10, the wall layer 32 may be formed directly on the substrate 10 without providing the bottom layer 31. In this case, therefore, the microwell 33 is formed by the surface of the substrate 10 and the through-hole 32a of the wall layer 32.

[0040] 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 forms the inner wall surface of each microwell 33. The wall layer 32 may be integrally formed with the substrate 10. In this case, the microwells 33 are formed on the surface of the substrate 10.

[0041] The material for forming the wall layer 32 can be a resin similar to the material for forming the substrate 10, but it is also possible to use a material in which a colored component that absorbs electromagnetic waves of a predetermined wavelength is mixed into the resin. As the resin material, either a hydrophilic resin in which the molecules of the resin constituents have hydrophilic groups, or a hydrophobic resin in which the molecules of the resin constituents have hydrophobic groups can be used, taking into consideration the properties desired for the microwells 33.

[0042] 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, silanolated polyvinyl alcohol, and sulfonated polyvinyl alcohol, polyvinyl acetal resins, polyvinyl butyral resins, polyethylene polyamide resins, polyamide polyamine resins, cellulose derivatives such as hydroxymethyl cellulose and methyl cellulose, 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. Examples of hydrophobic resins include novolac resins, acrylic resins, methacrylic resins, styrene resins, vinyl chloride resins, vinylidene chloride resins, polyolefin resins, polyamide resins, polyimide resins, polyacetal resins, polycarbonate resins, polyphenylene sulfide resins, polysulfone resins, fluororesins, silicone resins, urea resins, melamine resins, guanamine resins, phenolic resins, cellulose resins, and combinations of the above resins. Materials having a contact angle of 70 degrees or greater measured according to the sessile drop method specified in JIS R3257-1999 can be appropriately selected and used. In other words, the term "hydrophobic" as used herein means that the contact angle measured according to the sessile drop method specified in JIS R3257-1999 is 70 degrees or greater. The contact angle may be measured according to ASTM D5725-1997 instead of the sessile drop method specified in JIS R3257-1999.

[0043] Both the hydrophilic resin and the hydrophobic resin may be a thermoplastic resin or a thermosetting resin, and may further be a resin that is cured by active energy rays such as electron beams or UV light, or an elastomer. When photoresist is used as the resin material, a large number of minute through holes 32a can be formed in the wall layer 32 with high precision by photolithography. When photolithography is used, known methods can be appropriately selected for the selection of the type of photoresist to be used, the application, exposure (photoexposure), and removal of unnecessary photoresist. If no resist is used, the wall layer 32 can be formed by, for example, injection molding.

[0044] Examples of colored components include organic and inorganic pigments. Specific examples of black pigments include carbon black, acetylene black, and iron black. Yellow pigments include chrome 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, lithol red, lake red, brilliant scarlet, and thioindigo red. Blue pigments include ultramarine blue, cobalt blue, phthalocyanine blue, ferrocyanine blue, and indigo. Green pigments include chrome green, viridian naphthol green, and phthalocyanine green.

[0045] 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 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 used when dyeing resins.

[0046] As used herein, a microwell refers to a well with a volume of 10 nanoliters (nL) or less. By minimizing the volume of the microwell 33 to this extent, enzyme reactions such as PCR and ICA (Invasive Cleavaged Assay) reactions can be carried out in a small space. Digital PCR can be used to detect gene mutations, for example. The volume of the microwell 33 is not particularly limited, but is preferably 10 femtoliters (fL) to 100 picoliters (pL), more preferably 10 fL to 5 pL, and most preferably 10 fL to 2 pL. Setting the volume within this range is suitable for accommodating one to several biomolecules or carriers in each microwell 33 during sample analysis, which will be described later.

[0047] The shape of the microwell 33 is not particularly limited as long as the volume is within the above-mentioned range, and may be, for example, a cylinder, a polyhedron composed of multiple faces (e.g., a rectangular parallelepiped, a hexagonal prism, an octagonal prism, etc.), an inverted cone, or an inverted pyramid (an inverted triangular pyramid, an inverted square pyramid, an inverted pentagonal pyramid, an inverted hexagonal pyramid, or an inverted polygonal pyramid with seven or more sides). Furthermore, the shape of the multiple macrowells 33 may be a combination of two or more of the above shapes. For example, some of the multiple macrowells 33 may be cylindrical, and the rest may be inverted cone-shaped. When the multiple macrowells 33 are inverted cone-shaped or inverted pyramid-shaped, the bottom of the cone or pyramid serves as an opening connecting the flow path 35 and the microwells 33. In this case, the bottom of the microwells 33 may be flattened by using a shape in which a portion of the apex of the inverted cone or inverted pyramid is cut off. As another example, the bottom of the macrowell 33 may have a curved shape that protrudes toward the opening, or the bottom of the macrowell 33 may have a curved shape that is recessed.

[0048] The thickness of the wall layer 32 defines the depth of the microwell 33. When the microwell is cylindrical, for the purpose of enclosing 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 can be appropriately determined so that one or several biomolecules can be accommodated in one microwell, taking into consideration the amount of aqueous liquid to be accommodated and the size of the carrier such as beads to which the biomolecules are attached.

[0049] The number and density of microwells 33 provided in microwell array 30 can be set as appropriate. 1cm 2 The number of microwells 33 per cm is, for example, 10,000 to 10,000,000, preferably 100,000 to 5,000,000, and more preferably 100,000 to 1,000,000. 2 The number of microwells 33 per well is sometimes referred to as the microwell density. When the microwell density is within this range, it is easy to seal the aqueous liquid sample into a predetermined number of wells. Furthermore, when the microwell density is within this range, it is easy to observe the wells to analyze the experimental results. For example, when detecting mutations in cell-free DNA, if the proportion of mutant DNA to be detected relative to wild-type DNA is about 0.01%, it is preferable to use, for example, about 1 to 2 million microwells. 1 shows an example of a one-dimensional array in which a plurality of microwells 33 are arranged in a line. However, when providing a large number of microwells as described above, a two-dimensional array in which a plurality of microwells are arranged two-dimensionally may also be used.

[0050] A peripheral member 34, which is frame-shaped in plan view, is arranged around the microwell array. The dimension of peripheral member 34 in the thickness direction of microfluidic device 1 is larger than that of wall layer 32. Peripheral member 34 supports cover member 20, thereby ensuring a gap between cover member 20 and the microwell array and maintaining flow channel 35. In other words, flow channel 35 is the region sandwiched between microwell array 30 and cover member 20 and surrounded by peripheral member 34. There are no particular restrictions on the material of the peripheral member 34, but examples include double-sided adhesive tape in which an acrylic adhesive is laminated on both sides of a core film made of silicone rubber or acrylic foam. It should be noted that peripheral member 34 may be molded integrally with cover member 20. In this case, peripheral member 34 serves as a step portion of cover member 20, and this step portion ensures a gap between cover member 20 and the microwell array, thereby maintaining flow path 35.

[0051] The microfluidic device 1 configured as above can be manufactured, for example, by the following procedure. First, the substrate 10 is prepared, and a wall resin layer that will become the wall layer 32 is formed on the surface of the substrate 10. If a bottom layer 31 is to be provided, the bottom layer 31 is formed before the formation of the wall resin layer. Even if the bottom layer 31 is not provided, an anchor layer or the like that improves adhesion between the substrate 10 and the wall resin layer may be provided on the surface of the substrate 10 as needed.

[0052] 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 mass% or more and 60 mass% or less. The content is preferably 5 mass% or more and 55 mass% or less, and even more preferably 20 mass% or more and 50 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 so that the desired pattern can be constructed, taking into account the proportion of the photosensitive component contained in the resist. Furthermore, when the colored component is a pigment, the particle diameter of the pigment is set and prepared so as to satisfy the above-mentioned predetermined conditions for the microwells to be formed. A dispersant may be added to the resin material together with the pigment. When the formed wall resin layer is made of a material in which a colored component is mixed into a resin material, the wall resin layer has a color based on the colored component contained in the wall resin layer.

[0053] Next, through holes 32a are formed in the formed wall resin layer. As described above, the through holes 32a can be formed easily and accurately by using photolithography. 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 performed in the same process. Alternatively, the through holes 32a can be formed by etching using a pattern mask or the like. When the through-holes 32a are formed, the wall resin layer becomes the wall layer 32, and the microwell array 30 is completed.

[0054] Thereafter, a peripheral member 34 is placed around the microwell array 30, and then the cover member 20 is placed on the peripheral member 34. At this time, the cover member 20 is placed so that the surface 20a facing the microwell array 30 has an Ra of 50 nm or less. Next, the substrate 10, peripheral member 34, and cover member 20 are bonded together to complete the microfluidic device 1. A flow path is formed between the cover member 20 and the substrate 10 by the peripheral member 34. The bonding method is not particularly limited, but examples include bonding with an adhesive, bonding using double-sided tape, and bonding by laser welding.

[0055] Furthermore, in the microfluidic device 1, the substrate 10 and the wall layer 32 may be integrally molded, or the peripheral member 34 and the lid member 20 may be integrally molded. FIG. 3 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 integrally molded with the wall layer 32 on the lid member 20 integrally molded with the peripheral member 34, and bonding the step formed by the integral molding of the peripheral member 34 and the lid member 20 to the substrate 10 integrally molded with the wall layer 32. The flow channel 35 is formed between the lid member 20 and the substrate 10 by the step formed in 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 cover member 20 are integrally molded.

[0056] In another embodiment, the microfluidic device may have the substrate 10 and the wall layer 32 as separate elements, and the peripheral member 34 and the lid member 20 are integrally formed. In this case, the configuration of the microfluidic device is the same as that of the microfluidic device 1 described above, except for the peripheral member 34 and the lid member 20 being integrally formed.

[0057] Next, a sample analysis method according to this embodiment using the microfluidic device 1 according to this embodiment will be described with reference to FIGS. The sample analysis method of this embodiment is a method for analyzing a sample using the microfluidic device 1 according to this embodiment, Supplying an aqueous liquid containing a sample to the flow channel 35; introducing a sealing liquid into the flow channel 35 to replace the aqueous liquid present in the flow channel 35, and sealing the aqueous liquid in the microwell 33; heating the microfluidic device to cause a reaction in the microwells 33 to generate a signal for detection; detecting the signal; The sample analysis method comprises:

[0058] Here, the aqueous liquid may contain, in addition to the sample, water, a buffer solution, a detection reaction reagent, and the like. The aqueous liquid may also contain an enzyme. For example, if the sample is a nucleic acid, PCR, ICA, LAMP (registered trademark, Loop-Mediated Isothermal Amplification), TaqMan (registered trademark), or a fluorescent probe method may be used. For example, if the sample is a protein, ELISA (registered trademark), or the like may be used. Furthermore, the aqueous liquid may contain additives such as surfactants.

[0059] Examples of the buffer solution include Tris-HCl buffer, acetate buffer, and phosphate buffer. Examples of the enzyme 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 glycoside.

[0060] The microfluidic device of this embodiment can suitably retain the aqueous liquid in the wells even when the temperature of the enclosed aqueous liquid is changed, for example, in detecting gene mutations, etc. The temperature range to be changed, i.e., the range from the lower limit to the upper limit of the temperature change, is, for example, 0°C to 100°C, preferably 0°C to 80°C, and more preferably 20°C to 70°C. When the aqueous solution enclosed in the wells is within this range, reactions that are carried out in a microspace, such as PCR and ICA reactions, can be carried out suitably.

[0061] Examples of samples that can be analyzed using the microfluidic device 1 according to this embodiment include DNA, RNA, miRNA, mRNA, proteins, lipids, cells, and bacteria. The sample may be, for example, a sample collected from a living organism, such as blood. Furthermore, the detection target detected by sample analysis may be a PCR product using DNA contained in the sample as a template, or an artificially synthesized compound (e.g., an artificially synthesized nucleic acid that mimics the DNA of the sample). For example, when DNA, a biological molecule, is the detection target, the well may have a shape and size that allows one DNA molecule to be placed inside.

[0062] The sample analysis method is described in detail below. In the preparation step, an aqueous liquid containing a sample to be sealed in the microwell is prepared. The aqueous liquid containing the sample is a liquid containing the target substance and in which water is the main solvent. Examples include a PCR reaction solution using a biological sample as a template and containing SYBR Green as a detection reagent, or an ICA reaction solution containing an allele probe, ICA oligo, FEN-1, and a fluorescent substrate. During preparation, a surfactant may be added to facilitate the sample's entry into the microwell. Alternatively, beads that specifically recognize the target substance may be added to capture the target substance. The target substance may be suspended in the aqueous liquid without being directly or indirectly bound to a carrier such as beads.

[0063] Next, aqueous liquid 100 containing the prepared sample is supplied from first hole 21 to flow channel 35 using a syringe or the like (this is also referred to as a sample supplying step). As shown in FIG. 4, the supplied aqueous liquid 100 containing the sample fills each microwell 33 and flow channel 35. Gas within flow channel 35 is removed in advance by a degassing operation prior to the sample supplying step. This degassing operation may be performed by filling flow channel 35 with a buffer. Examples of buffers include water, water containing a buffer solution, water containing a surfactant, and water containing a buffer solution and a surfactant.

[0064] Next, an encapsulation step is performed in which an aqueous liquid containing sample 100 is encapsulated in microwell 33. Before the encapsulation step, the target substance to be detected in the sample contained in the aqueous liquid may be labeled with a fluorescent label or the like. 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 a fluorescent label into flow channel 35. In the sealing step, a syringe or the like is used to supply sealing liquid 110 from first hole 21 to flow channel 35. The supplied sealing liquid 110 flows through the flow channel and, as shown in FIG. 5 , pushes aqueous liquid 100 containing the sample present in flow channel 35 toward second hole 22. Then, sealing liquid 110 replaces aqueous liquid 100 that had filled flow channel 35, and flow channel 35 is filled with sealing liquid 110. As a result, aqueous liquid 100 containing the sample is placed only in each microwell 33, independently of one another, and sealing of the sample is completed.

[0065] As used herein, sealing liquid 110 refers to a liquid used to isolate the aqueous liquids introduced into each microwell 33 of microwell array 30 so that they do not mix with each other, and examples of such liquids include oils. Examples of oils that can be used include Sigma's product name "FC40" and 3M's product name "HFE-7500," as well as mineral oils used in PCR reactions and the like. The contact angle of the sealing liquid 110 with respect to the material of the wall layer 32 is preferably 5 degrees or more and 30 degrees or less. When the contact angle of the sealing liquid 110 is in this range, the sealing liquid 110 easily pushes the aqueous liquid 100, and the aqueous liquid 100 is less likely to remain on the surface of the cover member 20. As a result, the sample can be suitably sealed in each microwell 33. The contact angle of the sealing liquid may be measured, for example, according to the sessile drop method specified in JIS R3257-1999, using the sealing liquid instead of water. Instead of the sessile drop method specified in JIS R3257-1999, the contact angle may also be measured according to a method in accordance with ASTM D5725-1997.

[0066] Subsequently, a reaction step is carried out in which the microfluidic device 1 is heated to cause a reaction in the microwells 33 and generate a signal for detection. Examples of signals for detection include fluorescence, chemiluminescence, color development, potential change, and pH change, with fluorescence being preferred. Before the reaction step, the microfluidic device 1 may be subjected to a thermal cycler to carry out an enzymatic reaction such as a PCR reaction or an ICA reaction, if necessary. The reaction may be, for example, a biochemical reaction, more specifically, an enzymatic reaction. The heating temperature is determined appropriately depending on the reaction, but is, for example, between 60°C and 100°C. The heating temperature does not refer to the actual temperature of the reagent solution in the microwells 33, but refers to the heating temperature of the microfluidic device set by a thermal cycler, incubator, or the like. Furthermore, a heating temperature of, for example, between 60°C and 100°C means that the maximum temperature reaches between 60°C and 100°C, but does not necessarily have to be between 60°C and 100°C. In other words, the temperature of the microfluidic device 1 may change within the above-mentioned temperature range. An example of a reaction is a signal amplification reaction. The signal amplification reaction is an isothermal reaction in which a reagent solution containing an enzyme for signal amplification is contained in the microwells 33, and the microfluidic device 1 is maintained under constant temperature conditions, for example, between 60°C and 100°C, at which the desired enzymatic activity is obtained, for a predetermined time, for example, at least 10 minutes, preferably about 15 minutes.

[0067] Next, a signal generated from the microwell 33 by the above reaction is detected (detection step). For example, if the signal is fluorescence, the microwell 33 is set in a fluorescence microscope and irradiated with excitation light (electromagnetic waves). The wavelength of the excitation light is appropriately set depending on the fluorescent label used. Electromagnetic waves may be applied from the substrate 10 side of the microfluidic device 1, from the lid member 20 side, i.e., from above the microwells 33, or from any other direction. Fluorescence or phosphorescence generated as a result of the application of electromagnetic waves may be detected 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 detect it from the substrate 10 side of the microfluidic device 1.

[0068] Next, it is measured how many of the microwells 33 that make up the microwell array 30 are emitting fluorescence or phosphorescence. The measurement may be performed by taking a fluorescent image of the microwell array 30 and using the fluorescent image. For example, by performing a PCR reaction within the microwell array 30 and detecting SYBR Green fluorescence in microwells 33 where PCR amplification is observed, it is possible to calculate the ratio of the number of microwells 33 where amplification is observed to the total number of microwells 33. When the detection target is, for example, a single nucleotide polymorphism (SNP), the frequency of SNP expression, etc. can be analyzed by counting the number of microwells 33 that emit fluorescence.

[0069] In this measurement process, the aqueous liquid may contain proteins or enzymes as a sample or may contain enzymes as a reagent. When these proteins or enzymes are not adsorbed to beads but are suspended in the aqueous liquid, they are particularly likely to be adsorbed to the surface of the lid member 20. If proteins or enzymes are nonspecifically adsorbed to the lid member 20 and emit fluorescence or phosphorescence, this fluorescence or phosphorescence will be detected as noise. According to the microfluidic device of the present invention, adsorption of proteins or enzymes contained in the sample or reagent to the lid member 20 is reduced, thereby suppressing the generation of the noise.

[0070] Another aspect of the present invention includes the following embodiments. [8] A microfluidic device comprising a microwell array having a plurality of microwells and a cover member facing the microwell array at a distance, with a flow path between the microwell array and the cover member, wherein the arithmetic mean roughness Ra of the surface of the cover member facing the microwell array is 5 nm or more and 50 nm or less, and the ten-point mean roughness (Rz) is 50 nm or more and 250 nm or less. [9] The microfluidic device according to [8], further comprising a peripheral member positioned between the microwell array and the cover member and surrounding the flow channel.

[10] The microfluidic device according to [9], wherein the peripheral member is a stepped portion formed integrally with the lid member.

[11] The microfluidic device according to any one of [8] to

[10] , wherein the surface of the cover member facing the microwell array has a contact angle with water of 70 degrees or more.

[12] The microfluidic device according to any one of [8] to

[11] , wherein the surface of the cover member facing the microwell array has an Ra / Rz of 0.10 or more and 0.23 or less.

[13] The microfluidic device according to any one of [8] to

[12] , wherein the surface of the cover member facing the microwell array is hydrophobically coated.

[14] The microfluidic device according to any one of [8] to

[13] , wherein the hydrophobic coating is one of a fluorine-based coating agent, a fluorine-containing polymer, and a silicone resin.

[15] A sample analysis method using the microfluidic device described in any one of [8] to

[14] , comprising: supplying an aqueous liquid containing a sample to the flow path; introducing a sealing liquid into the flow path to replace the aqueous liquid present in the flow path, sealing the aqueous liquid in the microwell; causing a reaction in the microwell to generate a signal for detection; and detecting the signal.

[16] The sample analysis method according to

[15] , wherein the sample is DNA, RNA, protein, lipid, cell, or bacterium. [Example]

[0071] 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.

[0072] Example 1 Two resin components were prepared: a rectangular substrate made of COP (ZEONOR1010R, manufactured by Zeon Corporation) formed by injection molding, and a rectangular COP lid member. The COP substrate had cylindrical microholes, 10 μm in diameter and 15 μm deep, arranged across the entire surface of the substrate, and was injection molded. The lid member had a step with a height of 100 μm, i.e., a peripheral member, an inlet, and an outlet. The inlet and outlet were positioned inside the peripheral member along the longitudinal direction of the lid member. The surface of the die for the steel lid member that corresponds to the bottom surface of the lid member (the surface on which the step is formed) was polished with #3000 diamond paste to give the die a mirror finish. The contact angle of the bottom surface of the molded lid member with water was measured using a contact angle measuring instrument SA-20 (manufactured by Kyowa Interface Science Co., Ltd.) in accordance with the sessile drop method specified in JIS R 3257-1999. The contact angle of the bottom surface of the lid member of Example 1 with water was 85 degrees. The surface roughness of the molded lid member was measured using a contact surface roughness measuring instrument (TALYSURF PGI1240, manufactured by Taylor Hobson). In this measurement, the height difference from the starting point (height 0 nm) was obtained within a scanning range of 800 μm. Table 1 shows the measurement results of Ra and Rz of the bottom surface of the lid member.

[0073] [Table 1]

[0074] The above-mentioned COP substrate and lid member were bonded together by applying mineral oil to the stepped portion of the lid member so that the mirror-finished surface of the lid member faced the substrate, thereby producing a microfluidic device. 200 μL of a buffer having the composition shown in Table 2 below was pumped into the flow path between the substrate and the cover member, filling each well of the micropore chip with the buffer.

[0075] [Table 2]

[0076] Next, 10 μL of a fluorescent reagent (Fluorescein, manufactured by Tokyo Chemical Industry Co., Ltd.) having the composition shown in Table 3 below was pumped into the flow path, replacing the buffer with the fluorescent reagent. Furthermore, 150 μL of fluorocarbon oil (FC40, manufactured by Sigma) was pumped to individually seal each well of the micropore chip. Note that, although a fluorescent reaction was not performed in this example, an enzyme was added to the fluorescent reagent to create conditions similar to those in which a fluorescent reaction was performed.

[0077] [Table 3]

[0078] The microfluidic device was placed on a hot plate and heated at 66°C for 15 minutes. Fluorescent images of the micropore chip were then observed using a 4x objective lens under a fluorescence microscope (BZ-710, Keyence Corporation). The exposure time was 20 msec in bright field and 3000 msec using a GFP (Green Fluorescent Protein) fluorescent filter.

[0079] Figure 6A shows the results of fluorescence observation of the micropore chip after droplet formation in the microfluidic device of Example 1. The size of the observed image was 580 μm × 580 μm. The microfluidic device of Example 1 was able to accurately count the number of droplets without nonspecific adsorption of the reagent to the bottom surface of the lid member.

[0080] (Comparative Example 1) A microfluidic device was fabricated in the same manner as in Example 1, except that the surface of the mold corresponding to the bottom surface of the lid member was not mirror-finished. The measurement results of Ra and Rz of the bottom surface of the lid member are shown in Table 1. The contact angle of the bottom surface of the lid member of Comparative Example 1 with water was 85 degrees.

[0081] In the same manner as in Example 1, a buffer and a fluorescent reagent were delivered to the microfluidic device fabricated above to form droplets, and a fluorescent image of the micropore chip was observed. Figure 6B shows the results of fluorescence observation of the micropore chip after droplet formation in the microfluidic device of Comparative Example 1. The arrows in the figure indicate typical reagent adhesion. In addition to the arrowed areas, numerous reagent adhesions were observed throughout the entire field of view. As shown in Figure 6B, in the microfluidic device of Comparative Example 1, which used a cover member that was not mirror-finished, the reagent adhered nonspecifically to the bottom surface of the cover member, and the correct number of droplets could not be measured due to the adhesion points. These results revealed that even if the contact angle of the bottom surface of the lid member with water is the same, non-specific adsorption of the reagent to the bottom surface of the lid member can be reduced by having an Ra of 50 nm or less.

[0082] Example 2 A COP substrate was manufactured using the same method as in Example 1. A COP lid member was manufactured using the same method as in Example 1, except that the mirror-finishing time for the surface of the lid member mold corresponding to the bottom surface was extended. The surface roughness of the molded lid member was measured using a surface roughness measuring instrument (SJ-210, manufactured by Mitutoyo). The scanning range for this measurement was set to the center of the lid member and 1.5 mm in the direction perpendicular to the longitudinal direction of the lid member, and the height difference relative to the starting point (height 0 nm) was obtained. Table 4 shows the measurement results for Ra and Rz of the bottom surface of the lid member in Example 2. Figure 7 shows the measurement data for the surface roughness of the lid member of the microfluidic device in Example 2.

[0083] [Table 4]

[0084] The base material and the lid member were bonded together by applying mineral oil to the stepped portion of the lid member so that the mirror-finished surface of the lid member faced the substrate, thereby producing a microfluidic device. The buffer, fluorescent reagent, and mineral oil were delivered to the microfluidic device under the same conditions as in Example 1, and the fluorescent reagent was individually sealed in each well of the micropore chip. Note that in this example, as in Example 1, no fluorescent reaction was performed, but an enzyme was added to the fluorescent reagent to create a condition similar to that in which a fluorescent reaction was performed.

[0085] The microfluidic device was placed on a hot plate and heated at 66°C for 15 minutes. Fluorescent images of the micropore chip were then observed using a 4x objective lens under a fluorescence microscope (BZ-710, Keyence Corporation). The exposure time was 20 msec for bright field and 3000 msec for GFP (Green Fluorescent Protein) fluorescence filter. The observation field was a 3.6 mm x 2.7 mm area.

[0086] In the fluorescent image, the intensity of the fluorescence emitted by the enzyme reaction when the target molecule is present in the well was used as a reference, and areas emitting fluorescence intensity above the reference were determined to be areas where the fluorescent reagent had been adsorbed, and the area of ​​the areas where the fluorescent reagent had been adsorbed was calculated. The area of ​​the portion where the fluorescent reagent was adsorbed relative to the area of ​​the entire fluorescent image was calculated as the ratio (%) of the adsorption area. When the ratio of the adsorption area was less than 10%, adsorption suppression was evaluated as good, and when it was 10% or more, adsorption suppression was evaluated as poor. The results are shown in Table 4.

[0087] Example 3 The reagent adsorption area ratio of Example 3 was determined using the same method as in Example 2, except that the time for mirror-finishing the surface of the mold for the lid member corresponding to the bottom surface of the lid member was shortened. The results are shown in Table 4. Figure 8 shows measurement data for the surface roughness of the lid member of the microfluidic device of Example 3.

[0088] (Comparative Examples 2 and 3) The reagent adsorption area ratios of Comparative Examples 2 and 3 were determined using the same method as in Example 2, except that the mirror finish was not performed on the surface of the mold for the lid member corresponding to the bottom surface of the lid member. The results are shown in Table 4. Figures 9 and 10 show measurement data for the surface roughness of the lid members of the microfluidic devices of Comparative Examples 2 and 3, respectively.

[0089] In Examples 2 and 3, where the Ra of the bottom surface of the lid member, that is, the surface of the lid member on the microwell array side, was 70 nm or less, the adsorption area ratios were good values ​​of 1.8% and 3.6%, respectively.

[0090] On the other hand, in Comparative Examples 2 and 3, where the surface of the lid member facing the microwell array had an Ra of greater than 70 nm, the adsorption area ratios were 22.3% and 21.0%, respectively. In Comparative Examples 2 and 3, the surface of the lid member mold corresponding to the bottom surface was not mirror-finished, so the surface roughness of the bottom surface of the lid member could not be controlled and variations in surface roughness occurred.

[0091] The reason why the adsorption suppression was evaluated as being good when the adsorption area ratio was less than 10% is as follows. If the adsorption area ratio exceeds 10%, for example, when measuring a low-concentration sample in which only one target molecule can fit in each well of a microfluidic device, there is a probability of more than 10% that the target molecule will be trapped in the well that overlaps with the area where the reagent is adsorbed. Even if the well where the reagent is adsorbed emits light due to an enzyme reaction, the luminescence cannot be detected, resulting in a false negative result (false negative). Even if the same measurement is performed twice, there is a probability of more than 1% (more than 1 in 100) that a false negative will result. On the other hand, when the adsorption area ratio is less than 10%, for example, when the adsorption area ratio is 5% or less as in Examples 2 and 3, the probability of a false negative is 5% or less, and by performing the same measurement twice, the probability of a false negative can be reduced to 0.25% or less. [Industrial Applicability]

[0092] The present invention provides a microfluidic device and a sample analysis method that can detect fluorescence, phosphorescence, and the like of aqueous liquid in wells as accurately as possible. For example, when performing diagnosis by detecting biological DNA, RNA, and the like, it becomes possible to introduce nucleic acids together with reagents into a microscopic space. [Explanation of symbols]

[0093] 1. Microfluidic Device 10 Substrate 20 Cover member 30 Microwell Array 32 Wall layer 33 microwells 100 aqueous liquid 110 Sealing liquid

Claims

1. a microwell array having a plurality of microwells; a cover member facing the microwell array at a distance; a hydrophobic coating layer applied to the surface of the cover member facing the microwell array, a flow path between the microwell array and the cover member; the arithmetic mean roughness Ra of the surface of the cover member on the microwell array side is 70 nm or less; the Ra / Rz of the surface of the cover member facing the microwell array is 0.10 or more and 0.24 or less; Rz is the surface roughness (ten-point average roughness) of the surface of the cover member facing the microwell array, A microfluidic device, wherein the coating layer has a thickness of 0.01 μm or more and 3 μm or less.

2. The microfluidic device according to claim 1 , wherein the coating layer comprises at least one of a fluorine-based coating agent, a fluorine-containing polymer, and a silicone resin.

3. A sample analysis method using the microfluidic device according to claim 1 or 2, comprising: supplying an aqueous liquid containing a sample to the flow channel; introducing a sealing liquid into the flow channel to replace the aqueous liquid present in the flow channel, and sealing the aqueous liquid in the microwell; heating the microfluidic device to cause a reaction in the microwell to generate a signal for detection; detecting the signal; A sample analysis method comprising:

4. 4. The sample analysis method according to claim 3, wherein the sample is DNA, RNA, protein, lipid, cell, or bacterium.

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