Detection kit, sealing liquid, method for storing sealing liquid, and method for detecting sample

Abstract

The detection kit is used for detecting a target substance contained in a liquid sample, and is provided with a fluid device that accommodates the sample, a detection reagent that reacts with the target substance, and an oily sealing liquid. The fluid device has an internal space in which a mixed aqueous solution of a sample and a detection reagent is accommodated, an injection port and a discharge port which communicate with the internal space, and a plurality of holes which open in the internal space and in which the mixed aqueous solution is accommodated, the dissolved oxygen content of the sealing liquid being 100 mg / L or less, and the dissolved nitrogen content of the sealing liquid being 260 mg / mL or less.

Description

Technical Field

[0001] The invention relates to a detection kit, a sealing liquid, a method for preserving the sealing liquid and a sample detection method.

[0002] This application claims priority based on Japanese Patent Application No. 2022-076057 filed in Japan on May 2, 2022, the contents of which are incorporated herein by reference. Background Art

[0003] There are known techniques for detecting biomolecules in fluidic devices. For example, in DNA microarray technology, biomolecules are sometimes detected by introducing biomolecules into microwells and performing a reaction accompanied by heating.

[0004] In addition, there are known technologies that can detect single molecules of biomolecules. Examples of such technologies include digital measurement technologies such as digital enzyme-linked immunosorbent assay (ELISA), digital polymerase chain reaction (PCR), and digital invasive cleavage assay (ICA) described in Non-Patent Document 1.

[0005] The inventors and others have previously developed a digital measurement technology using a reaction container (fluid device) with multiple wells (see patent document 1). In the previously developed method, first, an aqueous medium containing a target substance is fed to the flow path of the fluid device, and the aqueous medium is filled into multiple wells provided on the wall surface of the flow path. Next, an oily sealing liquid is fed to the flow path, and the aqueous medium in the multiple wells is sealed with the oily sealing liquid. Thus, multiple independent reaction spaces are formed in each well. In addition, the reaction solution is heated by heating the fluid device, and a detection reaction is performed, thereby detecting the target substance.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. 2015 / 115635

[0009] Non-patent literature

[0010] Non-patent literature 1: Large-scale femtoliter droplet array for digital counting of single biomolecules Soo Hyeon Kim, Lab Chip, 2012, 12, 4986-4991 Summary of the invention

[0011] Problems to be solved by the invention

[0012] However, in the method described in Patent Document 1, bubbles may be generated in the fluid device when the fluid device is heated. The generated bubbles may hinder the detection reaction and observation, and thus an improvement is required.

[0013] The present invention is completed in view of such a situation, and its purpose is to provide a detection kit that can suppress the generation of bubbles and perform biomolecule measurement with high precision. In addition, the purpose is to provide a sealing liquid for such a detection kit, a method for storing the sealing liquid, and a sample detection method using such a detection kit.

[0014] Means used to solve problems

[0015] [1] A detection kit for detecting a target substance contained in a liquid sample, the detection kit comprising: a fluid device for accommodating the sample, a detection reagent for reacting with the target substance, and an oily sealing liquid, the fluid device having: an internal space for accommodating a mixed aqueous solution of the sample and the detection reagent, and a plurality of holes for accommodating the mixed aqueous solution, the dissolved oxygen content of the sealing liquid being less than 100 mg / L, and the dissolved nitrogen content of the sealing liquid being less than 260 mg / mL.

[0016] [2] The detection kit according to [1], wherein no bubbles are visually confirmed in the sealing liquid by the following method:

[0017] (Confirmation method)

[0018] (1) Add 1.5 mL of the above sealing solution into a 1.5 mL microtube and seal it.

[0019] (2) Heating at 66°C for 25 minutes,

[0020] (3) Visually check the presence or absence of bubbles in the sealing liquid.

[0021] [3] A detection kit for detecting a target substance contained in a liquid sample, the detection kit comprising: a fluid device for accommodating the sample, a detection reagent for reacting with the target substance, and an oily sealing liquid, the fluid device comprising: an internal space for accommodating a mixed aqueous solution of the sample and the detection reagent, an injection port and an outlet connected to the internal space, and a plurality of holes opening in the internal space and accommodating the mixed aqueous solution, wherein no bubbles are visually confirmed in the sealing liquid by the following method:

[0022] (Confirmation method)

[0023] (1) Add 1.5 mL of the above sealing solution into a 1.5 mL microtube and seal it.

[0024] (2) Heating at 66°C for 25 minutes,

[0025] (3) Visually check the presence or absence of bubbles in the sealing liquid.

[0026] [4] The detection kit according to any one of [1] to [3], wherein the sealing liquid is sealed in a storage container, and the internal pressure of the storage container containing the sealing liquid is 2.0×10 2 Below Pa.

[0027] [5] The detection kit according to any one of [1] to [4], wherein the sealing liquid is fluorine-based oil.

[0028] [6] The detection kit according to any one of [1] to [5], wherein the material of the fluid device is a gas-impermeable resin.

[0029] [7] The detection kit according to any one of [1] to [6], wherein the contact angle of the sealing liquid with respect to a surface facing the internal space is 5° to 80°.

[0030] [8] The detection kit according to any one of [1] to [7], wherein the plurality of holes have the same shape when viewed from above and are arranged in a matrix, and any first hole and the second hole closest to the first hole satisfy the following formula (1):

[0031] 0.8≤Da / Dab<1 (1)

[0032] Da is an equivalent circle diameter of the opening of the first hole, and Dab is a distance between the center of gravity of the opening of the first hole and the center of gravity of the opening of the second hole.

[0033] [9] The detection kit according to [8], wherein the Da is 1 μm to 50 μm.

[0034]

[10] The detection kit according to any one of [1] to [9], wherein the average volume per well is 1 fL to 6 pL.

[0035]

[11] A sealing liquid, which is an oily sealing liquid used in the detection kit described in any one of [1] to

[10] , wherein the dissolved oxygen content of the sealing liquid is 100 mg / L or less, and the dissolved nitrogen content is 260 mg / mL or less.

[0036]

[12] The sealing liquid according to

[11] , wherein no bubbles are visually confirmed by the following method,

[0037] (Confirmation method)

[0038] (1) Add 1.5 mL of the above sealing solution into a 1.5 mL microtube and seal it.

[0039] (2) Heating at 66°C for 25 minutes,

[0040] (3) Visually check the presence or absence of bubbles in the sealing liquid.

[0041]

[13] The sealing liquid according to

[11] or

[12] , which is contained in a sealed container with an internal pressure of 2.0×10 2 Pa or less in a storage container.

[0042]

[14] A method for storing a sealing liquid, comprising storing the sealing liquid according to any one of

[11] to

[13] at a temperature of 15° C. or higher.

[0043]

[15] A sample detection method, which is a sample detection method for detecting a target substance contained in a liquid sample, the sample detection method comprising: a step of adjusting a mixed aqueous solution of a detection reagent that reacts with the target substance and the sample; a step of using a fluid device to introduce the mixed aqueous solution of the sample and the detection reagent into the internal space and fill the hole with the mixed aqueous solution, the fluid device having an internal space for accommodating the mixed aqueous solution, an injection port and a discharge port connected to the internal space, and a plurality of holes that are open in the internal space and accommodate the mixed aqueous solution; a step of introducing an oily sealing liquid into the fluid device and sealing the mixed aqueous solution in the hole with the sealing liquid; and a step of heating the fluid device to cause a reaction between the target substance and the detection reagent and detect the target substance; the dissolved oxygen content of the sealing liquid is less than 100 mg / L, and the dissolved nitrogen content of the sealing liquid is less than 260 mg / mL.

[0044]

[16] The sample detection method according to

[15] , wherein the sealing liquid is free of bubbles that can be visually confirmed by the following method:

[0045] (Confirmation method)

[0046] (1) Add 1.5 mL of the above sealing solution into a 1.5 mL microtube and seal it.

[0047] (2) Heating at 66°C for 25 minutes,

[0048] (3) Visually check the presence or absence of bubbles in the sealing liquid.

[0049]

[17] The sample detection method according to

[15] or

[16] , wherein the sealing liquid is fluorine-based oil.

[0050]

[18] The sample detection method according to any one of

[15] to

[17] , comprising a step of degassing the sealing liquid before the sealing step.

[0051]

[19] The sample detection method according to any one of

[15] to

[18] , wherein in the detection step, the reaction temperature of the target substance and the detection reagent is 50°C to 99°C.

[0052] Effects of the Invention

[0053] According to the present invention, a detection kit capable of suppressing the generation of bubbles and performing biomolecule measurement with high precision can be provided. In addition, a sealing liquid for such a detection kit, a method for storing the sealing liquid, and a sample detection method using such a detection kit can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 It is a perspective schematic diagram showing a detection kit according to an embodiment.

[0055] Figure 2 is a cross-sectional view of the fluidic device contained in the test kit.

[0056] Figure 3 is a top view showing a portion of the plurality of microholes 110 .

[0057] Figure 4 It is an explanatory diagram of the sample detection method according to the present embodiment.

[0058] Figure 5 It is an explanatory diagram of the sample detection method according to the present embodiment.

[0059] Figure 6 It is an explanatory diagram of the sample detection method according to the present embodiment.

[0060] Figure 7 This is a schematic diagram illustrating an example of ICA reaction.

[0061] Figure 8 The following are photographs showing the evaluation results.

[0062] Fig. 9 The following are photographs showing the evaluation results. DETAILED DESCRIPTION

[0063] In this specification, when a numerical range is described as "1 to 10 μm", it means a range of 1 μm to 10 μm, and means a numerical range including 1 μm as a lower limit and 10 μm as an upper limit.

[0064] Below, refer to Figure 1 to Figure 6The detection kit, sealing liquid, method for storing the sealing liquid, and sample detection method of the present embodiment are described. In all the following drawings, the dimensions, ratios, etc. of the components are appropriately varied to facilitate viewing of the drawings.

[0065] <Test kit, sealing solution, and storage method of sealing solution>

[0066] Figure 1 It is a perspective schematic diagram showing the detection kit according to the present embodiment. Figure 2 is a cross-sectional view of the fluidic device contained in the test kit, Figure 1 Cross-sectional view along line segment II-II.

[0067] The detection kit 1 includes a fluid device 10, a detection reagent L1, and an oily sealing liquid L2. The detection reagent L1 and the sealing liquid L2 are stored in, for example, storage containers 91 and 92, respectively. The detection kit 1 is used for detecting a target substance contained in a liquid sample.

[0068] The sample is an aqueous solution containing a target substance, such as a biological sample or an environmental sample. The biological sample is not particularly limited, and examples thereof include serum, plasma, urine, and cell culture fluid. In addition, it may be a PCR reaction solution that uses a biological sample as a template and contains a staining reagent as a detection reagent. In addition, examples of environmental samples include river water and factory drainage.

[0069] As target substances, for example, DNA, RNA, protein, virus, cell, exosome, etc. can be mentioned. Here, as RNA, miRNA and mRNA, etc. can be mentioned. In addition, as cells, bacteria, yeast, animal cells, plant cells, insect cells, etc. can be mentioned.

[0070] The detection kit 1 detects the target substance by reacting the target substance contained in the sample as described above with the detection reagent L1 in the fluid device 10. At this time, in the detection kit 1, a reaction field suitable for the reaction between the target substance and the detection reagent L1 is formed using the sealing liquid L2, so that the target substance can be easily detected.

[0071] The following will explain them one by one.

[0072] [Fluid Equipment]

[0073] like Figure 1 and Figure 2 As shown, the fluid device 10 includes a well plate 11, a cover member 12, and a wall member 13. The fluid device 10 stores a sample in an internal space S and is used as a reaction container for performing a detection reaction of a target substance contained in the sample.

[0074] (Orifice Plate)

[0075] The orifice plate 11 is a plate-like member that is rectangular or long when viewed from above. "Viewed from above" refers to the field of view observed from the vertical direction of the upper surface 11a of the orifice plate. On the upper surface 11a of the orifice plate 11, a plurality of holes (also called micropores) 110 are provided in the center of the length direction of the orifice plate 11.

[0076] The micropore 110 is a recessed portion provided on the upper surface 11a of the orifice plate 11 and is open on the upper surface 11a. The micropore 110 is a space surrounded by the recessed portion and a virtual plane which is parallel to and in contact with the upper surface 11a.

[0077] The microwell 110 stores the sample stored in the internal space S, and functions as a reaction field between the target substance contained in the sample and the detection reagent L1.

[0078] The material of the orifice plate 11 is preferably hydrophobic. In detail, the contact angle between the material constituting the upper surface 11a of the orifice plate 11 and the sealing liquid L2 is preferably 5° to 80°. If the orifice plate 11 is formed by such a material, the contact angle between the upper surface 11a and the sealing liquid L2 becomes 5° to 80°. If the contact angle of the upper surface 11a is within the above range, when the sealing liquid is introduced into the internal space S by the method described later, there is a tendency to easily isolate the sample in the micropore 110.

[0079] The contact angle is determined by measuring using the sealing liquid L2 instead of water according to the sessile drop method specified in JIS R3257-1999.

[0080] Specifically, the following operation is performed: 4 μL or less of the sealing liquid L2 is dripped onto a test piece of a flat plate made of the same material as the well plate 11 placed horizontally to prepare a droplet. The following two values ​​are measured for this droplet.

[0081] r = radius of the surface of the test piece in contact with the droplet (mm)

[0082] h = height from the surface of the test piece to the top of the droplet (mm)

[0083] The contact angle θ was calculated from the following formula (1) using the obtained r and h. θ = 2tan -1 (h / r)

[0084] The material of the orifice plate 11 may or may not be permeable to electromagnetic waves. Here, as electromagnetic waves that are the object of permeability judgment, X-rays, ultraviolet rays, visible light, infrared rays, etc. can be cited. In the case where the orifice plate 11 has electromagnetic wave permeability, electromagnetic waves can be used to analyze the experimental results conducted in the fluid device 10 having the orifice plate 11. For example, fluorescence, phosphorescence, etc. generated as a result of irradiating electromagnetic waves can be measured from the orifice plate 11 side. In addition, "electromagnetic wave permeability" refers to the property of being able to transmit electromagnetic waves of various wavelengths, such as radio waves, light, X-rays, and gamma rays.

[0085] The details will be described later. For example, when fluorescence having a peak in the wavelength range of 400 to 700 nm, which is a visible light region, is generated in the micropore 110 and a sample is detected, a material having good transmittance to at least light in the visible light region may be used.

[0086] As materials having electromagnetic wave permeability, for example, glass and resin can be cited. As resins, for example, ABS resin, polycarbonate, COC (cycloolefin copolymer), COP (cycloolefin polymer), acrylic resin, polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyvinyl acetate, PET (polyethylene terephthalate) and PEN (polyethylene naphthalate) can be cited. These resins may contain various additives or may be polymer alloys formed by mixing multiple resins.

[0087] The material having electromagnetic wave permeability preferably has substantially no autofluorescence. Substantially having no autofluorescence means that the material has no autofluorescence at the wavelength used for sample detection, or even if it has autofluorescence, it is so weak that it does not affect the sample detection. For example, if the autofluorescence is less than 1 / 2, more preferably less than 1 / 10 of the fluorescence of the detection object, it can be considered to be so weak that it does not affect the sample detection. If such a material is used to form the orifice plate 11, the detection sensitivity can be improved in the sample detection using electromagnetic waves.

[0088] As a material that is permeable to electromagnetic waves and does not emit autofluorescence, for example, quartz glass can be cited. As a raw material that has weak autofluorescence and does not cause obstacles to sample detection using electromagnetic waves, low-fluorescence glass, acrylic resin, COC (cycloolefin copolymer), COP (cycloolefin polymer) and CPP (cast polypropylene, unstretched polypropylene) can be cited.

[0089] The thickness of the well plate 11 can be appropriately determined. When fluorescence is observed from the well plate 11 side using a fluorescence microscope, the thickness of the well plate 11 may be, for example, 5 mm or less, 2 mm or less, or 1.6 mm or less.

[0090] In addition, the material of the orifice plate 11 is preferably a gas-impermeable resin. In the present embodiment, "gas impermeability" is evaluated using a gas permeability coefficient represented by the following definition. The gas permeability coefficient can be obtained based on the method described in JIS K 7126-1: 2006 "Plastics-Film and sheeting-Determination of gas-transmission rate-Part 1: Differential-pressure method" or ISO 15105-1: 2002, Plastics-Film and sheeting-Determination of gas-transmission rate-Part 1: Differential-pressure method.

[0091] Regarding the oxygen permeability coefficient, a material having an oxygen permeability coefficient not exceeding the following standard can be suitably used as a "gas impermeable resin".

[0092] Oxygen permeability coefficient ≤0.1(cm 3 (stp) 100μm / m 2 ·24hr·MPa)

[0093] If the orifice plate 11 is formed of such a resin, it is preferable because the dissolution of air (more specifically, oxygen) into the sealing liquid L2 can be suppressed during operation and measurement using the fluid device 10 , and measurement accuracy can be easily maintained.

[0094] The orifice plate 11 may be a single layer structure using only the above-mentioned material, or a laminate of multiple materials. When the laminate is processed to form the orifice plate 11, the layer having the micropores 110 and the layer supporting the layer may be made of different materials. In this case, the material of the layer having the micropores 110 is preferably hydrophilic (the contact angle with water is 70° to 180°) and is easily wetted by water.

[0095] (Micropores)

[0096] The micropore 110 may be in various shapes. Examples of the shape of the micropore 110 include cylindrical shapes such as a cylinder, an elliptical cylinder, and a polygonal cylinder, cone shapes such as a cone, a pyramid, and a truncated cone shape such as a truncated cone and a truncated cone. When the micropore 110 is a cone or a truncated cone, the opening diameter may be a shape that gradually decreases in the depth direction of the hole.

[0097] The bottom of the micropore 110 may be flat or curved (specifically, convex or concave).

[0098] When the micropore 110 is cylindrical, it is preferred that the maximum diameter of the micropore 110 in a plan view is, for example, 10 nm to 100 μm, more preferably 100 nm to 50 μm, and even more preferably 1 μm to 20 μm. In addition, it is preferred that the depth of the micropore 110 is, for example, 10 nm to 100 μm, more preferably 100 nm to 50 μm, and even more preferably 1 μm to 20 μm.

[0099] If the capacity of the microwell 110 is, for example, 1fL to 6nL, it is preferred, more preferably 1fL to 5pL, further preferably 1fL to 2pL, and particularly preferably 1fL to 300fL. If the average capacity of each microwell 110 is within such a range, enzyme reactions in microscopic spaces such as digital PCR and invasion reactions can be suitably performed. Digital PCR can be used to detect gene mutations, for example.

[0100] The volume of the micropore 110 can be calculated by measuring the size of the micropore 110 using a scanning electron microscope or a phase contrast microscope.

[0101] The orifice plate 11 has a plurality of micro-wells 110 of the same shape and size. The same shape and size only need to be the same shape and the same capacity to the extent required for digital measurement, and any deviation that is the degree of manufacturing error is acceptable.

[0102] The density of the micropores 110 is, for example, 100,000 to 10 million per cm 2 , preferably 100,000 to 5 million / cm 2 , more preferably 100,000 to 1,000,000 pieces / cm 2 When the density of the micropores 110 is within such a range, the operation of sealing the sample in a predetermined number of micropores 110 is easy. In addition, the observation of the pores for analyzing the experimental results is also easy.

[0103] For example, when measuring a mutation in cell-free DNA using the detection kit 1, when the presence ratio of the mutation to be detected relative to the wild type is about 0.01%, it is preferable to use, for example, 1 to 2 million microwells 110 .

[0104] Figure 3 FIG. 1 is a top view showing a portion of the plurality of micropores 110. Figure 3 As shown, in the well plate 11, the plurality of micro-holes 110 have the same shape in a plan view. In addition, a small number (eg, 1 to 4) of the plurality of micro-holes may have different shapes in order to be used as alignment marks in imaging or device manufacturing.

[0105] In addition, the plurality of micropores 110 are regularly arranged in a matrix. Here, the micropores 110 are "regularly arranged" means that the centers of gravity of the openings of the plurality of micropores 110 are arranged in a certain pattern.

[0106] For example, the centroids of the openings of the micropores 110 may be arranged in a quadrilateral lattice. In this case, a line connecting the centroids of the openings of four adjacent micropores 110 forms a rectangle, preferably a square.

[0107] Alternatively, the centroids of the openings of the micropores 110 may be arranged in a triangular lattice (also referred to as a hexagonal lattice). In this case, a line connecting the centroids of the openings of three adjacent micropores 110 forms an equilateral triangle.

[0108] An arbitrary hole (also referred to as a first hole) A among the plurality of microholes 110 and a hole (also referred to as a second hole) B closest to the hole A preferably satisfy the following formula (1).

[0109] 0.8≤Da / Dab<1 (1)

[0110] (Da is the equivalent circle diameter of the opening of hole A, and Dab is the distance between the center of gravity of the opening of hole A and the center of gravity of the opening of hole B.)

[0111] In the orifice plate 11, the lower limit of the value of Da / Dab is 0.8, and may be 0.83. In addition, the upper limit of the value of Da / Dab is less than 1, and may be 0.92, or may be about 0.9. These lower limits and upper limits may be combined arbitrarily. For example, Da / Dab may be greater than 0.8 and less than 1, or may be 0.83 to 0.92, or may be 0.83 to 0.9.

[0112] When the above formula (1) is satisfied and the plurality of micropores 110 are arranged in a triangular lattice, the distance between the centroid of the opening of any micropore 110 and the centroid of the opening of the micropore 110 closest to the micropore 110 is constant.

[0113] Figure 3 The micropores 110 shown are arranged in a triangular lattice. Figure 3 As shown, the centers of gravity Ca, Cb, and Cc of the openings of an arbitrary hole A, a hole B closest to the hole A, and a hole C closest to both the hole A and the hole B form an equilateral triangle with these centers of gravity as vertices.

[0114] Figure 3 In FIG. 1 , a line connecting the centroids Ca, Cb, and Cc of the openings of the holes A, B, and C, respectively, forms an equilateral triangle.

[0115] In the orifice plate 11 , the equivalent circle diameter of the opening of the micropore 110 is preferably 1 μm to 50 μm. In other words, the lower limit of the equivalent circle diameter of the opening of the micropore 110 is preferably 1 μm.

[0116] In addition, the upper limit of the equivalent circular diameter of the opening of the micropore 110 can be less than 20 μm, or it can be less than 19 μm, or less than 18 μm, or less than 17 μm, or less than 16 μm, or less than 15 μm, or less than 14 μm, or less than 13 μm, or less than 12 μm, or less than 11 μm, or less than 10 μm.

[0117] Regarding the circle-equivalent diameter of the opening of the micropore 110 , the upper limit value and the lower limit value may be arbitrarily combined.

[0118] (Cover member)

[0119] The cover member 12 has the same outline shape (that is, a strip shape) as the orifice plate 11 in a plan view. The cover member 12 is disposed opposite to the upper surface 11 a of the orifice plate 11 with a gap therebetween.

[0120] The cover member 12 has two through holes penetrating in the thickness direction. The two through holes are respectively provided at one end side and the other end side in the length direction of the cover member 12. One through hole is an injection port 121 used when injecting a liquid into the internal space S of the fluid device 10, and the other through hole is an outlet port 122 used when discharging the liquid from the internal space S.

[0121] Here, the term "liquid substance" includes not only liquid samples but also detection reagents and sealing liquids.

[0122] The injection port 121, the internal space S and the discharge port 122 are connected in sequence to form a flow path FC as a whole. In the fluid device 10, the liquid is appropriately flowed in the flow path FC to perform a detection reaction of the target substance. In a plan view, a plurality of micropores 110 are arranged between the injection port 121 and the discharge port 122.

[0123] A cylindrical injection port 125 surrounding the injection port 121 is provided on the upper surface 12a of the cover member 12. The injection port 125 communicates with the injection port 121. The injection port 125 is used to connect a syringe filled with a liquid when the internal space is filled with a liquid, for example.

[0124] Likewise, a cylindrical discharge port 126 surrounding the discharge port 122 is provided on the upper surface 12a of the cover member 12. The discharge port 126 communicates with the discharge port 122. The discharge port 126 is used to connect a pipe through which liquid flows when the liquid is extracted from the internal space S, for example.

[0125] The material of the cover member 12 may be the same as or different from the material of the orifice plate 11 .

[0126] In addition, the material of the cover member 12 may or may not be electromagnetic wave transparent.

[0127] When the microwell 110 is observed in a bright field, at least one of the well plate 11 and the cover member 12 has light transmissivity.

[0128] The material of the cover member 12 is preferably hydrophobic. In detail, the contact angle between the material constituting the side facing the internal space S of the cover member 12 (that is, the lower surface 12b) and the sealing liquid L2 is preferably 5° to 80°. If the cover member 12 is formed by such a material, the contact angle between the lower surface 12b and the sealing liquid L2 becomes 5° to 80°. If the contact angle of the lower surface 12b is within the above range, when the sealing liquid is introduced into the internal space S by the method described later, there is a tendency to easily isolate the sample in the micropores 110.

[0129] (Wall component)

[0130] The wall member 13 is formed into a closed loop shape in a plan view and is arranged along the outer edge of the upper surface 11a of the orifice plate 11. Figure 1 In the embodiment, the wall surface facing the internal space S of the wall member 13 is substantially rectangular in a plan view, and the width thereof gradually decreases toward the injection port 121 side.

[0131] The wall member 13 is sandwiched by the orifice plate 11 and the cover member 12 to form a whole, thereby forming the fluid device 10. The space surrounded by the orifice plate 11, the cover member 12 and the wall member 13 is the internal space S for storing the liquid sample. The internal space S extends along the long orifice plate 11 in the longitudinal direction of the orifice plate 11.

[0132] The wall member 13 functions as a wall surface of the internal space S and also functions as a spacer between the orifice plate 11 and the cover member 12. The height of the wall member 13, that is, the height of the internal space S may be, for example, 100 μm or less.

[0133] The material of the wall member 13 is not particularly limited, and for example, a double-sided adhesive tape having an acrylic adhesive laminated on both sides of the core film can be suitably used. Examples of the material of the core film include silicone rubber and acrylic foam. By using such a material to form the wall member 13, the internal space S can be formed in a liquid-tight manner.

[0134] The wall member 13 may be made of the same material as the orifice plate 11. The wall member 13 made of such a material may be integrated with the orifice plate 11 and the cover member 12 by bonding with an adhesive or by welding such as heat welding, ultrasonic welding, or laser welding.

[0135] Furthermore, the wall member 13 may be formed integrally with the orifice plate 11 to constitute a part of the orifice plate 11. Likewise, the wall member 13 may be formed integrally with the cover member 12 to constitute a part of the cover member 12.

[0136] The above-mentioned orifice plate 11 can be manufactured by using known injection molding, micro-imprinting technology, or nano-imprinting technology. In addition, the orifice plate 11 can also be manufactured by using known photolithography technology and forming micro-holes 110 by etching.

[0137] The above-mentioned cover member 12 can be manufactured by known injection molding.

[0138] When the wall member 13 and the cover member 12 are formed integrally, the wall member 13 can be manufactured by known injection molding at the same time as the cover member 12. When the wall member 13 and the cover member 12 are separate bodies, the wall member 13 can be manufactured by cutting a double-sided adhesive tape formed by laminating an acrylic adhesive on both sides of the core film.

[0139] [Detection reagents]

[0140] The detection reagent L1 reacts with the target substance and is used for detecting the target substance. Examples of the detection reagent L1 include a buffer substance, an enzyme, a substrate, an antibody, and an antibody fragment.

[0141] For example, when the target substance is a nucleic acid, the enzyme is selected according to the content of the biochemical reaction in order to perform a biochemical reaction such as an enzyme reaction on a template nucleic acid related to the target substance. The biochemical reaction on the template nucleic acid is, for example, a reaction in which a signal amplification occurs under the condition of the presence of the template nucleic acid.

[0142] The detection reagent L1 is selected according to the detection reaction to be used. Specific detection reactions include ICA method, loop-mediated isothermal amplification (LAMP) method (registered trademark), 5'→3' nuclease method (TaqMan (registered trademark) method), fluorescent probe method, etc.

[0143] [Sealing liquid, storage method of sealing liquid]

[0144] The sealing liquid L2 is preferably oily and does not mix with the aqueous sample, or is not easily mixed with the aqueous sample. The sealing liquid L2 covers the opening of the microwell 110 containing the mixed aqueous solution of the sample and the detection reagent L1, and has the function of sealing the mixed aqueous solutions contained in the microwell 110 separately in a manner that prevents the mixed aqueous solutions contained in the microwell 110 from mixing with each other.

[0145] The sealing liquid L2 is preferably oil. As the oil, fluorine oil, silicone oil, hydrocarbon oil or a mixture thereof can be used. As specific sealing liquids, fluorine liquids such as FC-40, FC-43, FC-770, FC-72 and FC-3283 (all manufactured by 3M) and KF96 (manufactured by Shin-Etsu Chemical Co., Ltd.) can be cited.

[0146] Alternatively, mineral oil or the like used in PCR reactions or the like may be used as the sealing liquid L2.

[0147] According to the research of the inventors, when heating is performed for the detection of a target substance, if a sealing liquid containing a certain amount of microscopic bubbles (also called bubble precursors) that can become bubbles is injected into a fluid device before heating, bubbles that hinder the detection reaction and observation are easily generated. Specifically, it is believed that the reason is that when the sealing liquid is heated, the gas dissolved in the sealing liquid volatilizes to the bubble precursor, the bubble expands, and grows into a bubble of a size that can be visually recognized.

[0148] The amount of dissolved oxygen in the sealing liquid L2 is 100 mg / L or less, and the amount of dissolved nitrogen is 260 mg / mL or less.

[0149] The dissolved oxygen content is more preferably 80 mg / L or less, and further preferably 60 mg / L or less. The less the dissolved oxygen content, the better. Theoretically, it can be as low as below the detection limit, but for ease of adjustment, it can be 20 mg / L or more. The upper limit and lower limit of the dissolved oxygen content can be arbitrarily combined. In other words, the dissolved oxygen content can be 20 mg / L to 80 mg / L, or 20 mg / L to 60 mg / L.

[0150] The dissolved nitrogen content is more preferably 200 mg / L or less, and further preferably 150 mg / L or less. The lower the dissolved nitrogen content, the better. Theoretically, it can be as low as below the detection limit, but for ease of adjustment, it can be 50 mg / L or more.

[0151] The upper limit and lower limit of the dissolved nitrogen content may be arbitrarily combined. That is, the dissolved nitrogen content may be 50 mg / L to 200 mg / L, or 50 mg / L to 150 mg / L.

[0152] The dissolved oxygen content and dissolved nitrogen content of the sealing liquid L2 can be measured using a gas chromatograph (for example, GC-2014AT, manufactured by Shimadzu Corporation) under the conditions described in the examples.

[0153] The sealing liquid L2 included in the detection kit 1 is preferably adjusted so that no bubbles are visually observed when heated under the following conditions.

[0154] (condition)

[0155] (1) Add 1.5 mL of the above sealing solution into a 1.5 mL microtube and seal it.

[0156] (2) Heating at 66°C for 25 minutes,

[0157] (3) Visually check the presence or absence of bubbles in the sealing liquid.

[0158] The sealing liquid L2 adjusted in this way is sealed and stored in the storage container 92. The internal pressure of the storage container 92 storing the sealing liquid L2 is 2.0×10 2 That is, the sealing liquid is contained in a sealed container with an internal pressure of 2.0×10 2 Pa or less. The internal pressure can be measured using a known internal pressure measuring instrument (e.g., KDM30 (manufactured by KRONE Co., Ltd.)). The lower limit of the internal pressure depends on the pressure resistance of the storage container 92, and various values ​​can be adopted. For example, the lower limit of the internal pressure can be 1×10 4 Pa or more, or 1×10 3 Pa or more. The upper and lower limits of the internal pressure can be arbitrarily combined, for example, 1×10 4 Pa~2.0×10 2 Pa, or 1×10 3 Pa~2.0×10 2 Pa.

[0159] When storing the sealing liquid L2, it is sealed in the storage container 92 under the above-mentioned internal pressure state and stored at a temperature above 15°C. The storage temperature of the sealing liquid L2 can be above 20°C, or above 30°C. In addition, the storage temperature of the sealing liquid L2 can be below 70°C, or below 40°C. The upper limit and lower limit of the storage temperature can be arbitrarily combined. For example, the storage temperature of the sealing liquid L2 can be 20°C to 70°C, or 30°C to 40°C. By storing under such conditions, the redissolution of oxygen and nitrogen in the air into the sealing liquid L2 can be suppressed, and the detection of the target substance can be appropriately performed.

[0160] When stored in the storage container 92, in order to prevent the air in the container from dissolving into the sealing liquid, the volume of the sealing liquid is preferably larger than the volume of the air in the container. The volume of the sealing liquid may be 1.01 to 10 times the volume of the air in the container, preferably 1.01 to 5 times, more preferably 1.01 to 2 times, and more preferably 2 times or less. In addition, when stored in the storage container 92, the surface area in the storage container may be 1 times or more relative to the area where the air in the container contacts the sealing liquid.

[0161] The material of the storage container 92 is selected from resins such as PE (polyethylene), PVC (polyvinyl chloride), PP or PET, metal, and glass. When the material of the storage container 92 is a resin, a gas-impermeable resin is more preferable.

[0162] <Sample testing method>

[0163] Figures 4 to 6 : is an explanatory diagram of the sample detection method of the present embodiment. The sample detection method of the present embodiment is a sample detection method for detecting a target substance contained in a liquid sample, and includes the following steps.

[0164] (1) Step of preparing a mixed aqueous solution of the detection reagent L1 and the sample that reacts with the target substance

[0165] (2) A step of introducing a mixed aqueous solution into the internal space S and filling the mixed aqueous solution into the micropores 110 using a fluid device 10, wherein the fluid device 10 comprises an internal space S for storing the mixed aqueous solution, an injection port 121 and an outlet communicated with the internal space S, and a plurality of micropores 110 opened in the internal space S and storing the mixed aqueous solution.

[0166] (3) Step of introducing the sealing liquid L2 into the fluid device 10 and sealing the mixed aqueous solution in the micropore 110 with the sealing liquid L2

[0167] (4) Step of heating the fluid device 10 to cause a reaction between the target substance and the detection reagent L1 and detecting the target substance

[0168] The sample detection method was carried out using the detection kit 1 described above.

[0169] (1) Step of adjusting the mixed aqueous solution

[0170] First, a liquid sample is mixed with the detection reagent L1 to prepare a mixed aqueous solution. The concentration of the detection reagent L1 in the mixed aqueous solution is appropriately adjusted according to the type of detection reagent L1 used and the type of detection reaction.

[0171] The detection reagent L1 used may contain an anti-adsorption agent. The anti-adsorption agent has a function of inhibiting the adsorption of the target substance, enzyme, etc. contained in the mixed aqueous solution to the member. Examples of the anti-adsorption agent include surfactants and proteins.

[0172] In order to facilitate the detection of the target substance, the sample can be pretreated before the adjustment of the mixed aqueous solution. As pretreatment, concentration adjustment (dilution or concentration), loading using a carrier, binding reaction of two or more target substances, filtration sterilization using filtration, pH adjustment, and modification (annealing) of the target substance using heat treatment can be cited.

[0173] (2) Step of filling the micropores with the mixed aqueous solution

[0174] Next, if Figure 4 As shown, the mixed aqueous solution L containing the detection reagent L1 is injected from the injection port 121 into the internal space S. When the mixed aqueous solution L flows in the internal space S (ie, the flow path FC), the micropores 110 opened in the internal space S are also filled with the mixed aqueous solution L.

[0175] After the internal space S and all the micropores 110 are filled with the mixed aqueous solution L, the mixed aqueous solution L passing through the flow path FC is discharged from the discharge port 122 .

[0176] At this time, it is preferable to pre-adjust the concentration of the mixed aqueous solution L so that one target substance is filled inside one micropore 110. For example, after the sample detection method described later is implemented, if the mixed aqueous solution L has a high concentration and the quantitative determination of the target substance is difficult, the result is used as a preliminary experimental result and the mixed aqueous solution L is diluted.

[0177] Through the above operation, one or less, that is, 0 or 1, target substance is filled in one microwell 110. Thus, it is confirmed that the number of microwells 110 after the detection reaction described later corresponds to the number of target substances, and the target substance can be detected in units of 1, that is, digital measurement can be performed. In addition, it is not necessary to introduce the target substance into all microwells 110.

[0178] There is no particular limitation on the method for introducing the target substance into the microwell 110, and an appropriate method can be selected according to the target substance to be detected. For example, the target substance can be allowed to settle in the fluid device 10 (specifically, in the flow channel FC) by its own weight and distributed to the microwell.

[0179] In addition, a substance that captures the target substance (also referred to as a capture substance) can be used to combine the capture substance with the target substance that is not easy to settle due to its own weight to carry out liquid delivery. In addition, by pre-immobilizing the capture substance in the micropore, the capture substance is made to capture the target substance that is carried out with the mixed aqueous solution L, thereby also being able to improve the introduction efficiency of the target substance into the micropore 110.

[0180] The reaction of the capture substance binding to the target substance can be performed at any time, for example, by bringing the target substance into contact with the capture substance in a sample tube before adjusting the mixed aqueous solution.

[0181] Alternatively, after the capture substance is introduced into the microwell 110 , the target substance may be introduced into the microwell 110 , and the capture substance and the target substance may be brought into contact with each other in the microwell 110 .

[0182] The capture substance is a substance that can capture the target substance. For example, the capture substance may be a combination of a solid phase and a substance that specifically binds to the target substance.

[0183] As the solid phase constituting the captured object, particles, membranes, substrates, etc. can be cited. In addition, the specific binding substance for the target substance can be one, or two or more. For example, it can be three, or four, or five or more.

[0184] As particles, there are no particular restrictions, and polymer particles, magnetic particles and glass particles can be cited. Particles are preferably particles that have been surface treated to avoid nonspecific adsorption. In addition, in order to immobilize the specific binding substance, particles having functional groups such as carboxyl groups on the surface are preferred. More specifically, the trade name "Magnosphere LC300" manufactured by JSR Corporation can be used.

[0185] Furthermore, for example, when a virus is used as a target substance, cells to which the virus can attach (ie, cells having a virus receptor) can be used as captured objects.

[0186] (3) Step of sealing the mixed aqueous solution in the micropores 110

[0187] Next, if Figure 5 As shown, the sealing liquid L2 is supplied to the flow path FC from the injection port 121. The sealing liquid L2 supplied to the flow path FC flows along the surface direction of the upper surface 11a in the internal space S, and washes and replaces the mixed aqueous solution L not contained in the micropores 110 in the mixed aqueous solution L supplied to the flow path FC.

[0188] Thus, the sealing liquid L2 seals the plurality of microwells 110 containing the mixed aqueous solution L containing the target substance, and the microwells 110 become independent reaction spaces. When the flow path FC is filled with the sealing liquid L2 , excess sealing liquid L2 is discharged from the discharge port 122 . Figure 6 The state in which all the plurality of micropores 110 are sealed with the sealing liquid L2 and all the flow paths FC are replaced with the sealing liquid L2 is shown.

[0189] When injecting the sealing liquid L2, the sealing liquid L2 may be injected slowly so that no bubbles are contained in the sealing liquid L2 in the internal space S. After injecting the sealing liquid L2, it is visually confirmed that no bubbles are contained in the sealing liquid L2 due to the injection operation.

[0190] At this time, the sealing liquid L2 used is preferably the sealing liquid L2 attached to the above-mentioned detection kit 1, especially fluorine-based oil. The sealing liquid L2 is preferably adjusted so that no bubbles can be visually observed when heated under the following conditions.

[0191] (condition)

[0192] (1) Add 1.5 mL of the above sealing solution into a 1.5 mL microtube and seal it.

[0193] (2) Heating at 66°C for 25 minutes,

[0194] (3) Visually check the presence or absence of bubbles in the sealing liquid.

[0195] As described above, the amount of dissolved oxygen in the sealing liquid L2 is 100 mg / L or less, and the amount of dissolved nitrogen is 260 mg / mL or less.

[0196] (4) Step of detecting target substance

[0197] Next, the fluid device 10 is heated to cause a reaction between the target substance and the detection reagent L1. At this time, the sealing liquid L2 that seals the micropore 110 suppresses the generation of bubbles caused by heating as described above. Therefore, in the process of detecting the target substance, the generation of bubbles in the sealing liquid L2 can be suppressed, and the target substance can be detected with high precision.

[0198] The method for detecting the target substance can use any known detection method according to the characteristics of the target substance to be detected. For example, it can be performed as follows: first, a reaction (also referred to as a signal amplification reaction) is performed to amplify the signal from the target substance to a detectable level as required, and then the amplified signal is detected using an appropriate method.

[0199] Examples of the signal that can be used in the detection method of the present embodiment include fluorescence, chemiluminescence, color development, potential change, and pH change.

[0200] The signal amplification reaction may be, for example, a biochemical reaction, more specifically an enzyme reaction. As an example, the signal amplification reaction is an isothermal reaction in which a reagent solution containing an enzyme for signal amplification is contained in a well and the fluid device is kept at a certain temperature condition at which the desired enzyme activity can be obtained, for example, above 60° C., preferably about 66° C., for a predetermined time, for example, at least 10 minutes, preferably about 15 minutes.

[0201] Specifically, when a reaction for detecting nucleic acid is used, examples of the signal amplification reaction include ICA reactions such as the Invader (registered trademark) method, the LAMP method (registered trademark), the TaqMan (registered trademark) method, and the fluorescent probe method.

[0202] As a signal amplification reaction, the ICA reaction is particularly preferably used. In the ICA reaction, signal amplification is performed by (1) complementary binding of nucleic acids to each other, and (2) recognition and cutting of the triple chain structure of the enzyme through the cycle of these two reactions. In such a signal amplification reaction, the influence of the reaction cycle obstruction caused by inclusions other than the target substance is small. Therefore, in the case where various components other than the target substance (that is, inclusions) are present in the micropore 110, the target substance can also be detected with good accuracy by using the ICA reaction.

[0203] For example, when an ICA reaction is used in the signal amplification reaction, the mixed aqueous solution L contains a reaction reagent and a template nucleic acid required for the ICA reaction. When the biochemical reaction in the reaction step is an ICA reaction, by an enzyme reaction utilizing an isothermal reaction, when a target substance exists in the well, the fluorescent substance is released from the quenching substance, thereby emitting a predetermined fluorescent signal corresponding to the excitation light.

[0204] When the ICA reaction is used in the signal amplification reaction, the reaction temperature of the target substance and the detection reagent is preferably 50°C to 99°C.

[0205] The ICA reaction will be described in more detail below.

[0206] Figure 7 This is a schematic diagram illustrating an example of the ICA method. Figure 7 Detection of DNA as a target substance by the ICA method is shown in FIG.

[0207] Examples of the reaction reagents required for the ICA reaction include ICA reaction reagents such as the FLAP probe 810 , the FLAP endonuclease FEN, the fluorescent substrate 820 , and the invader probe (invader oligonucleotide) 830 .

[0208] The FLAP probe 810 and the invader probe 830 are nucleic acid fragments (oligonucleotides) designed to hybridize with DNA (target DNA) as a target substance and form a FLAP structure with the double-stranded nucleic acid 140 .

[0209] The fluorescent substrate 820 is a nucleic acid fragment formed by combining a fluorescent substance F with a hairpin structure and a light-extinguishing substance Q. Figure 7In the fluorescent substrate 820 shown, a fluorescent substance F is bonded to the 5' end of the nucleic acid fragment, and an extinguishing substance Q is bonded to the 3' side several bases from the 5' end. The extinguishing substance Q suppresses the emission of the fluorescent substance F.

[0210] First, the FLAP probe 810 and the invader probe 830 are hybridized with the target DNA. The FLAP probe 810 and the invader probe 830 overlap by one base at the SNP site of the target DNA, respectively, to form an unstable three-base-like structure. As a result, a first FLAP site 811 is formed. The first FLAP site 811 is a portion of the FLAP probe 810 that does not hybridize with the target DNA.

[0211] Next, FEN recognizes the three-base-like structure and reacts, thereby cleaving the first FLAP site 811 to generate a nucleic acid fragment 811, which is released in the mixed aqueous solution L.

[0212] The generated nucleic acid fragment 811 hybridizes with the fluorescent substrate 820. The nucleic acid fragment 811 invades the hairpin structure of the fluorescent substrate 820, overlaps one base at the SNP site, and forms an unstable three-base-like structure. As a result, a second FLAP site 821 is formed. The second FLAP site 821 is a portion of the fluorescent substrate 820 that will not hybridize due to the invasion of the nucleic acid fragment 811.

[0213] Next, FEN recognizes the three-base-like structure and reacts. As a result, the second FLAP site 821 is cut off, generating a nucleic acid fragment 821. Figure 7 In FIG. 8 , the remaining portion after the nucleic acid fragment 821 is cut from the fluorescent substrate 820 is indicated by symbol 820′.

[0214] As a result, the fluorescent substance F is separated from the quenching substance Q, and fluorescence FL is generated. By detecting the fluorescence FL, the target DNA can be detected.

[0215] In addition, the detection of the target substance can also be performed by binding a substance that specifically binds to the target substance (also referred to as a specific binding substance) to the target substance and detecting the specific binding substance after binding. For example, when the target substance is a protein, ELISA can be used for detection. More specifically, detection can be performed, for example, by sandwich ELISA using the principle of FRET.

[0216] In the case of a sandwich method utilizing the FRET principle, first, a first specific binding substance (e.g., an antibody) labeled with a first fluorescent substance (also called a donor) and a second specific binding substance labeled with a second fluorescent substance (also called an acceptor) having an absorption wavelength that overlaps the fluorescence wavelength of the first fluorescent substance are prepared.

[0217] Next, the target substance (e.g., antigen) is brought into contact with both the first specific binding substance and the second specific binding substance to form a complex. If a complex is formed, the distance between the donor and the acceptor becomes closer, and the fluorescence wavelength of the acceptor can be detected by irradiating the excitation wavelength of the donor. Alternatively, the specific binding substance can be labeled with a nucleic acid fragment, and the nucleic acid fragment can be detected by an ICA reaction.

[0218] As specific binding substances, substances similar to the specific binding molecules for structures described later, such as antibodies, antibody fragments, nucleic acid aptamers, etc., can be used. In order to detect the specific binding substances bound to the target substance, the specific binding substances can be directly or indirectly labeled by enzymes such as horseradish peroxidase (HRP). When using more than two specific binding substances, each specific binding molecule can be identified in a mutually recognizable manner.

[0219] The signal observation method can select a known appropriate method according to the type of signal to be observed. For example, in the case of bright field observation, white light is irradiated in the vertical direction to the substrate provided with the hole array. In the case of observing the fluorescent signal, excitation light corresponding to the fluorescent substance is irradiated into the hole from the bottom side of the hole, and the fluorescence emitted by the fluorescent substance is observed. The image of all or part of the observed hole array is photographed and saved, and image processing is performed using a computer system.

[0220] In addition, if the sealing liquid L2 cannot suppress the generation of bubbles due to the preheating as described above, the sealing liquid L2 may be degassed before the sealing step using the sealing liquid L2. The sealing liquid L2 may be degassed by reducing pressure, heating, ultrasonic irradiation, or a combination thereof.

[0221] According to the detection kit 1 having the above-described configuration, the generation of bubbles can be suppressed, and biomolecule measurement can be performed with high accuracy.

[0222] Furthermore, the sealing liquid having the above-described structure can be used in the detection kit 1 to perform biomolecule measurement with high accuracy in order to suppress the generation of bubbles due to heating.

[0223] Furthermore, according to the sealing liquid storage method having the above-described configuration, the sealing liquid with generation of bubbles suppressed can be appropriately stored.

[0224] Furthermore, according to the sample detection method having the above-described configuration, biomolecule measurement can be performed with high accuracy.

[0225] The preferred embodiments of the present invention are described above with reference to the accompanying drawings, but the present invention is not limited to the above-mentioned embodiments. The various shapes and combinations of the components shown in the above-mentioned embodiments are examples, and various changes can be made based on design, method, etc. within the scope of the main purpose of the present invention.

[0226] Example

[0227] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples.

[0228] [Manufacture of fluid equipment]

[0229] An orifice plate made of cyclic polyolefin (product number "ZEONOR1020R", manufactured by Zeon Corporation) and a cover member made of cyclic polyolefin (product number "ZEONOR1020R", manufactured by Zeon Corporation) were respectively produced by injection molding. The thickness of the orifice plate was 0.6 mm.

[0230] A plurality of micropores were formed on one side of the orifice plate by injection molding. The opening shape of the micropores was set to be circular. The size of the micropores was 10 μm in diameter and 15 μm in depth. In an area of ​​6.0 mm × 30.0 mm on the substrate, a plurality of micropores were arranged in a triangular lattice so that the distance between the center of a micropore and the center of the micropore closest to the micropore was 12 μm, forming a hole array.

[0231] The cover member is formed integrally with the wall member. By adjusting the height of the wall member to 30 μm, the height of the flow path is set to 30 μm.

[0232] Next, the orifice plate and the cover member (wall member) were joined by laser welding to produce the fluid devices of Examples 1 and 2.

[0233] [Buffer adjustment]

[0234] The aqueous medium (ie, buffer) for delivering the fluid to the fluidic device was adjusted according to the composition shown in Table 1.

[0235] [Table 1]

[0236] Buffer composition

[0237] Element Final concentration <![CDATA[MgCl2]]> 20mM Tris pH 8.5 50mM Tween20 0.05％ Distilled water Remaining

[0238] [Determination of dissolved gas concentration]

[0239] The sealing liquid was measured by gas chromatography, and the dissolved oxygen concentration and dissolved nitrogen concentration of the sealing liquid after degassing were obtained by the calibration curve method. During the measurement, the GC injection port was replaced with He to prevent contamination by the atmosphere.

[0240] The measurement sample was collected using a syringe at a depth of about 10 mm from the liquid surface, and the collection position was adjusted visually and manually so that the collection position was substantially the same.

[0241] (Measurement conditions)

[0242] Device used: GC-2014AT (Shimadzu Corporation)

[0243] Pre-column: Porapak Q

[0244] Column: Molecular Sieve 5A

[0245] Carrier gas: He

[0246] Inlet temperature: 170℃

[0247] Column temperature: 50°C

[0248] Detector: Thermal conductivity detector (100mA)

[0249] Detector temperature: 150°C

[0250] Measurement sample volume: 20 μL

[0251] As a standard product of the component, the following standard gas (mother gas) was diluted (2 / 201) with He gas using a vacuum bottle, and the measurement was performed in the same manner as the sample to prepare a calibration curve.

[0252] (Standard gas)

[0253] Nitrogen and oxygen mixed gas: O2 21%,

[0254] N2 79%, (manufactured by Kansai Gas First Co., Ltd.)

[0255] The injection amount of each standard gas is as follows.

[0256] Mother gas: 20, 100, 200 μL

[0257] Dilution gas: 20, 100, 200 μL

[0258] The component masses of the standard gas are calculated using the following formula.

[0259] Component mass (g) = component molecular weight (g / mol) × standard gas concentration (%) / 100 × injection volume (L) / 24.47 (L)

[0260] In addition, "24.47" in the above formula is the volume (unit: L) of 1 mol of ideal gas at 25°C.

[0261] The dissolved gas concentrations of the untreated sealing liquid after storage at room temperature were 120 mg / L for oxygen and 260 mg / L for nitrogen.

[0262] [Example 1]

[0263] Fluorine oil (Fluorinert FC-40, manufactured by SIGMA-ALDRICH) was used as the sealing liquid.

[0264] The sealing liquid was degassed by the following method.

[0265] (Degassing method)

[0266] 50 ml of the sealing liquid was placed in a screw-cap bottle (SV-50A, manufactured by Nippon Rika Glass Co., Ltd.), and heated at 70° C. for 2 hours using a hot plate.

[0267] The heated sealing liquid was cooled to room temperature, and the dissolved gas concentration was measured by the above-mentioned method. The results were: oxygen: 100.0 mg / L, nitrogen: 260.0 mg / L.

[0268] (Confirmation method)

[0269] 20 μL of a buffer solution having a composition shown in Table 1 was injected into the flow path of the fluidic device. Next, 200 μL of a degassed sealing liquid was injected into the fluidic device into which the buffer solution was injected, and the buffer solution in the flow path was replaced with the sealing liquid.

[0270] Then, the fluid device into which the sealing liquid had been injected was placed on a transparent hot plate (model: MP-10DMFH-PG, manufactured by Kitazato Corporation) heated to 66° C. and allowed to stand for 25 minutes.

[0271] The condition in the flow path of the heated fluid device was observed in the heated state using the observation tool shown below, and the presence or absence of bubble generation was visually confirmed.

[0272] (Observation equipment)

[0273] USB camera: CMOS camera (Template: DFK22AUC03, manufactured by Imaging Source) Lens: Ai Nikkor 105mm f2.5mm (manufactured by Nikon)

[0274] Lighting: Metal halide lamp (Model: LSM210, manufactured by Sumita Optical Co., Ltd.)

[0275] [Comparative Example 1]

[0276] The presence or absence of bubble generation was confirmed in the same manner as in Example 1 except that an untreated sealing liquid stored at room temperature was used. Figure 8 The following are photographs showing the evaluation results.

[0277] As a result of the evaluation, bubbles were generated in the fluid device using the sealing liquid of Comparative Example 1, but bubbles were not generated in the fluid device using the sealing liquid of Example 1.

[0278] [Example 2]

[0279] The sealing liquid was degassed by the following method.

[0280] (Degassing method)

[0281] The screw-capped vial with 50 ml of sealing liquid was placed in a vacuum desiccator while keeping it open. The desiccator was depressurized using a rotary pump (model: DW-120, manufactured by Sato Vacuum Co., Ltd.) at an initial pressure of 1.6 × 10 2 Keep it for 6 days.

[0282] The pressure in the desiccator was returned to normal pressure, and the dissolved gas concentration of the depressurized sealing liquid was measured by the above-mentioned method. The results were: oxygen: 5.0 mg / L, nitrogen: 8.6 mg / L.

[0283] The presence or absence of bubble generation was confirmed in the same manner as in Example 1 except that a degassed sealing liquid was used. Fig. 9 2 is a photograph showing the evaluation results. As a result of the evaluation, no bubbles were generated in the fluid device using the sealing liquid of Example 2.

[0284] From the above results, it can be seen that the present invention is useful.

[0285] Industrial Applicability

[0286] According to the present invention, a detection kit capable of suppressing the generation of bubbles and performing biomolecule measurement with high precision can be provided. In addition, a sealing liquid for such a detection kit, a method for storing the sealing liquid, and a sample detection method using such a detection kit can be provided.

[0287] Explanation of symbols

[0288] 1…detection kit, 10…fluid device, 91, 92…storage container, 110…well (microwell), 120…well array, 121…injection port, 122…discharge port, A…well (first well), B…well (second well), Ca…center of gravity, L…mixed aqueous solution, L1…detection reagent, L2…sealing liquid, S…internal space

Claims

1. A detection kit for detecting a target substance contained in a liquid sample, the detection kit comprising: a fluid device for storing the sample, a detection reagent that reacts with the target substance, and Oily sealing fluid, The fluid device comprises an internal space for accommodating a mixed aqueous solution of the sample and the detection reagent, and a plurality of holes for accommodating the mixed aqueous solution. The amount of dissolved oxygen in the sealing liquid is 100 mg / L or less, and the amount of dissolved nitrogen in the sealing liquid is 260 mg / mL or less.

2. The detection kit according to claim 1, wherein The sealing liquid has no bubbles visually confirmed by the following method: (Confirmation method) (1) Add 1.5 mL of the sealing solution into a 1.5 mL microtube and seal it. (2) Heating at 66°C for 25 minutes, (3) The presence or absence of bubbles in the sealing liquid is visually confirmed.

3. The detection kit according to claim 1 or 2, wherein: The sealing liquid is sealed and stored in a storage container. The internal pressure of the storage container containing the sealing liquid is 2.0×10 2 Below Pa.

4. The detection kit according to claim 1, wherein The sealing liquid is fluorine oil.

5. The detection kit according to claim 1, wherein The material of the fluid device is a gas-impermeable resin.

6. The detection kit according to claim 1, wherein The contact angle of the sealing liquid with respect to the surface facing the internal space is 5° to 80°.

7. The detection kit according to claim 1, wherein The plurality of holes have the same shape when viewed from above and are arranged in a matrix. Any first hole and the second hole closest to the first hole satisfy the following formula (1): 0.8≤Da / Dab<1 (1) Da is an equivalent circle diameter of the opening of the first hole, and Dab is a distance between the center of gravity of the opening of the first hole and the center of gravity of the opening of the second hole.

8. The detection kit according to claim 7, wherein The Da is 1 μm to 50 μm.

9. The detection kit according to claim 1, wherein The average volume of each of the pores is 1 fL to 6 pL.

10. A sealing liquid, which is an oily sealing liquid used in the detection kit according to claim 1, in, The sealing liquid has a dissolved oxygen content of 100 mg / L or less and a dissolved nitrogen content of 260 mg / mL or less.

11. The sealing liquid according to claim 10, wherein no bubbles can be visually confirmed by the following method: (Confirmation method) (1) Add 1.5 mL of the sealing solution into a 1.5 mL microtube and seal it. (2) Heating at 66°C for 25 minutes, (3) The presence or absence of bubbles in the sealing liquid is visually confirmed.

12. The sealing liquid according to claim 10 or 11, which is contained in a sealed container with an internal pressure of 2.0×10 2 Pa or less in a storage container.

13. A method for storing a sealing liquid, comprising storing the sealing liquid according to claim 10 at a temperature of 15°C or higher.

14. A sample detection method, which is a sample detection method for detecting a target substance contained in a liquid sample, the sample detection method comprising: A step of preparing a mixed aqueous solution of a detection reagent that reacts with the target substance and the sample; A step of introducing a mixed aqueous solution of the sample and the detection reagent into the internal space and filling the hole with the mixed aqueous solution using a fluid device, wherein the fluid device has an internal space for accommodating the mixed aqueous solution, an injection port and a discharge port communicating with the internal space, and a plurality of holes opening in the internal space and accommodating the mixed aqueous solution; A step of introducing an oily sealing liquid into the fluid device to seal the mixed aqueous solution in the hole by the sealing liquid; as well as The step of heating the fluid device to cause a reaction between the target substance and the detection reagent, and detecting the target substance; The amount of dissolved oxygen in the sealing liquid is 100 mg / L or less, and the amount of dissolved nitrogen in the sealing liquid is 260 mg / mL or less.

15. The sample detection method according to claim 14, wherein: The sealing liquid has no bubbles visually confirmed by the following method: (Confirmation method) (1) Add 1.5 mL of the sealing solution into a 1.5 mL microtube and seal it. (2) Heating at 66°C for 25 minutes, (3) Visually confirm the presence or absence of bubbles in the sealing liquid.

16. The sample detection method according to claim 14 or 15, wherein: The sealing liquid is fluorine oil.

17. The sample detection method according to claim 14, wherein: Prior to the step of sealing, a step of degassing the sealing liquid is included.

18. The sample detection method according to claim 14, wherein: In the detection step, the reaction temperature of the target substance and the detection reagent is 50°C to 99°C.

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