Inspection Device and Inspection Method

Abstract

An inspection device (100) includes: a flow path (103) that is provided on one surface of a sheet substrate (101), and that transfers a liquid sample by means of capillary action; a sensing zone (105) that is provided in one portion of the flow path (103); and a fine uneven structure A that is provided at least in the sensing zone (105), and that has a plurality of protrusions formed integrally with the sheet substrate (101) in the flow path (103). In the sensing zone (105), a detector substance that specifically binds to a substance being detected in the liquid sample is immobilized on the surface of the fine uneven structure A, such that the substance being detected is detected according to the detection of a change in the optical properties of the sensing zone (105) produced by means of an enzymatic reaction.

Description

Technical Field

[0001] The present invention relates to an inspection device and an inspection method.

Background Art

[0002] As a technique for detecting a specific component in a sample, there is one described in Patent Document 1 (Japanese Patent Application Laid-Open No. 2005-502363). This document describes a method for determining the presence of an analyte in a sample. Specifically, such a method includes incorporating a lateral flow test strip having at least a starting region for receiving a sample and a reaction region having at least one immobilized enzyme, the starting region being capable of transporting a liquid by capillary action; causing a liquid phase to move through the test strip by contacting the sample with the starting region; and determining a detectable signal caused directly or indirectly in the reaction region by covalent modification of the analyte by the enzyme in the reaction region.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] When the present inventor studied the technique described in Patent Document 1, it became clear that there is room for improvement in terms of improving detection accuracy. The present invention provides an inspection device having excellent detection accuracy.

Means for Solving the Problems

[0005] According to the present invention, the following inspection device and inspection method are provided. [1] It is provided on one surface of a sheet-shaped resin substrate, and includes a flow path for transferring a liquid sample by capillary action, a detection zone provided in a part of the flow path, a fine uneven structure A having a plurality of convex portions provided at least in the detection zone and integrally formed with the resin substrate, and has, In the detection zone, a detection substance that specifically binds to the substance to be detected in the liquid sample is immobilized on the surface of the fine uneven structure A, An inspection device configured to detect the substance to be detected by detecting a change in the optical characteristics of the detection zone caused by an enzyme reaction. [2] The inspection device according to [1], wherein the detection substance is an antibody against the substance to be detected. [3] The inspection device according to [1] or [2], wherein the change in the optical characteristics is a color change in the detection zone. [4] The inspection device according to [3], wherein the color change can be visually confirmed. [5] The inspection device according to [1] or [2], wherein the change in the optical characteristics is chemiluminescence, bioluminescence or fluorescence in the detection zone. [6] The inspection device according to any one of [1] to [5], wherein in the detection zone, the shape of the convex portion is a columnar body, a cone or a frustum of a cone. [7] The inspection device according to any one of [1] to [6], wherein in the detection zone, the shape of the convex portion is a cone, and the detection substance is immobilized on the side surface of the cone. [8] The inspection device according to any one of [1] to [7], wherein in the detection zone, the plurality of convex portions are arranged in a grid pattern on a plane. [9] The inspection device according to any one of [1] to [8], wherein the diameter of the bottom surface of the convex portion is 10 μm or more and 1000 μm or less.

[10] The inspection device according to any one of [1] to [9], wherein the height of the convex portion is 10 μm or more and 500 μm or less.

[11] At least in the detection zone, a fine concavo-convex structure B is formed on the surface of the convex portion, The inspection device according to any one of [1] to

[10] , wherein the arithmetic mean roughness Ra of the roughness curve of the convex portion on which the fine concavo-convex structure B is formed is 0.005 μm or more and 1 μm or less.

[12] In one cross section, a plurality of the convex portions a first region located on one side of the convex portion from the center of the convex portion in the width direction of the convex portion; a second region located on the other side of the convex portion from the center of the convex portion in the width direction of the convex portion; and having In the one cross section, at least one of the outer edge of the first region and the outer edge of the second region of the convex portion has a concave portion. The inspection device according to any one of [1] to

[11] .

[13] The inspection device according to any one of [1] to

[12] , which is for an enzyme immunoassay method.

[14] An inspection method using the inspection device according to any one of [1] to

[13] , introducing the liquid sample upstream of the detection zone and guiding it to the detection zone by capillary action, and capturing the analyte in the fine concavo-convex structure A by the specific interaction between the analyte and the detection substance; After the capturing step, introducing a liquid containing a labeled antibody that specifically binds to the analyte into the detection zone, and capturing the labeled antibody in the fine concavo-convex structure A in which the analyte is captured; After the step of capturing the labeled antibody, introducing a compound that causes a change in optical properties in the detection zone by reacting with the labeled antibody into the detection zone; detecting or quantifying the analyte by detecting the change in optical properties in the detection zone; and including an inspection method.

Advantages of the Invention

[0006] According to the present invention, an inspection device with excellent detection accuracy can be provided.

Brief Description of the Drawings

[0007]

Figure 1

Figure 2

Figure 3

Figure 4

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Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Modes for Carrying Out the Invention

[0008] Hereinafter, embodiments of the present invention will be described with reference to the drawings. The drawings are schematic and do not match the actual dimensional ratios. In this embodiment, the composition can include each component either alone or in combination of two or more components. In this specification, "~" indicating a numerical range represents "or more" and "or less", and includes both end values.

[0009] Figure 1 is a top view showing a configuration example of the inspection device in this embodiment. The inspection device 100 shown in Figure 1 is provided on one surface of a sheet-shaped resin substrate (sheet substrate 101), and includes a flow path 103 for transferring a liquid sample by capillary action, a detection zone 105 provided in a part of the flow path 103, and a fine uneven structure A having a plurality of convex portions provided at least in the detection zone 105 and integrally formed with the sheet substrate 101 in the flow path 103. In the detection zone 105, a detection substance that specifically binds to the analyte in the liquid sample is immobilized on the surface of the fine uneven structure A. Further, the inspection device 100 is configured to detect the analyte by detecting a change in the optical characteristics of the detection zone 105 caused by an enzymatic reaction. Specifically, the inspection device 100 is a lateral flow immunoassay device. Further, the inspection device 100 is suitable for, for example, enzyme immunoassay.

[0010] (Sheet substrate) Specifically, the sheet substrate 101 is a substrate used as a base material of the inspection device 100. Examples of the shape of the sheet substrate 101 include a sheet shape and a plate shape. As in the example of Figure 1, the flow path 103 may be provided in a part of the region in a plan view on the sheet substrate 101, or the flow path 103 may be provided throughout the sheet substrate 101.

[0011] Specific examples of the planar shape of the sheet substrate 101 include polygons such as quadrilaterals, circles, and ellipses. When the sheet substrate 101 is a quadrilateral, the vertical width (length in the short side direction) of the sheet substrate 101 may be, for example, about 1 to 100 mm, and the horizontal width (length in the long side direction) of the sheet substrate 101 may be, for example, about 2 to 100 mm.

[0012] From the perspective of improving the strength of the inspection device 100, the thickness of the sheet substrate 101 is, for example, 0.05 mm or more, preferably 0.1 mm or more. Also, from the perspective of thinning the inspection device 100, the thickness of the sheet substrate 101 is, for example, 5 mm or less, preferably 3 mm or less.

[0013] Specific examples of the material of the sheet substrate 101 include one or more selected from the group consisting of resin materials such as poly (meth) acrylate such as polymethyl (meth) acrylate, polyester, polyolefin, polystyrene, polycarbonate, fluororesin, polyvinyl chloride, polyamide, and polyimide. Also, from the perspective of stably integrally molding the sheet substrate 101 and the plurality of convex portions, the material of the sheet substrate 101 is preferably a thermoplastic resin. Specific examples of the thermoplastic resin include one or more selected from the group consisting of polyester, polyolefin, polystyrene, polycarbonate, fluororesin, and (meth) acrylic resin. More specifically, one or more selected from the group consisting of polyethylene terephthalate (PET), cycloolefin polymer (COP), polypropylene (PP), polystyrene (PS), polycarbonate (PC), polyvinylidene fluoride (PVDF), polymethyl methacrylate (PMMA), and polyethylene (PE) are included.

[0014] (Detection zone) The detection zone 105 is provided with a fine uneven structure A. The fine uneven structure A has a plurality of convex portions. FIGS. 2(a) and 2(b) are diagrams showing a configuration example of the fine uneven structure A provided on the sheet substrate 101 and the convex portions constituting the fine uneven structure A. FIG. 2(a) is a top view showing an example of the arrangement of the convex portion 8 in the detection zone 105, and FIG. 2(b) is a perspective view showing an example of the shape of the convex portion 8. FIGS. 3(a) to 3(f) and FIG. 13 are perspective views (SEM images) showing configuration examples of the fine uneven structure A obtained in the examples described later.

[0015] In the detection zone 105, as shown in FIGS. 2(a) and 2(b), the sheet substrate 101 includes a flat portion 9 corresponding to the bottom surface of the flow channel 103 and a plurality of convex portions 8 protruding from the flat portion 9, and a fine concavo-convex structure A is formed by the totality of the plurality of convex portions 8. The liquid sample is transferred through the fine concavo-convex structure A from the sample introduction side on the left side of the drawing to the sample discharge side, specifically along the transfer direction d in FIGS. 1 and 2(a). In the example of FIG. 1, the transfer direction d is the longitudinal direction of the sheet substrate 101. The space between the plurality of convex portions 8 functions as a flow channel 103 for transferring the liquid sample along the surface of the sheet substrate 101. In other words, the voids in the fine concavo-convex structure A function as a flow channel 103 for transferring the liquid sample along the surface of the sheet substrate 101.

[0016] As shown in FIGS. 2(a), 3(a) to 3(f), the plurality of convex portions 8 are preferably arranged in a lattice pattern in a plane. More specifically, examples of the lattice arrangement include a square lattice and an oblique lattice such as a hexagonal lattice.

[0017] Specific examples of the shape of the convex portion 8 include columnar bodies such as cylinders and polygonal columns, cones such as cones and polygonal pyramids, and truncated cones such as truncated cones and truncated pyramids. These do not necessarily have to be geometrically precise shapes, and may be shapes with rounded corners or shapes with fine concavo-convexities on the surface. From the viewpoint of fabricating the convex portion 8 with better reproducibility and improving the measurement accuracy of the substance to be detected, the shape of the convex portion 8 is more preferably a cone or a truncated cone, and even more preferably a cone. From the same viewpoint, even more preferably, the shape of the convex portion 8 is a cone, and the detection substance is immobilized on the side surface of such a cone. FIGS. 2(b), 3(a) and 3(b) show examples in which the convex portion 8 is a cone. FIGS. 3(c) and 3(d) are examples of the convex portion 8 having a conical shape with concavo-convexities formed at the apex and a conical shape with a concave apex, respectively. Also, FIGS. 3(e) and (f) are examples of the convex portion 8 having a frustoconical shape. The shapes of the plurality of convex portions 8 may be the same or different. From the viewpoint of more reproducibly producing the desired fine concavo-convex structure A, it is preferable that the shapes of the plurality of convex portions 8 are the same.

[0018] When the planar shape of the bottom surface of the convex portion 8 (the bottom surface 10 in FIG. 2(b)) is circular, the diameter of the bottom surface of the convex portion 8 is preferably 10 μm or more, more preferably 20 μm or more, from the viewpoint of improving formability. Also, from the viewpoint of improving formability, the diameter of the bottom surface of the convex portion 8 (the diameter 4 in FIGS. 2(a) and 2(b)) is preferably 1000 μm or less, more preferably 500 μm or less.

[0019] The height of the convex portion 8 is preferably 10 μm or more, more preferably 20 μm or more, from the viewpoint of improving the measurement accuracy of the substance to be detected. From the same viewpoint, the height of the convex portion 8 (the height 6 in FIG. 2(b)) is preferably 500 μm or less, more preferably 200 μm or less, and even more preferably 100 μm or less.

[0020] The distance between adjacent convex portions 8, that is, the closest distance between the convex portions 8, is appropriately set according to the shape of the convex portion 8, and is, for example, about 0 to 500 μm. For example, when the convex portion 8 is a columnar body, the closest distance is preferably greater than 0 μm, and when the convex portion 8 is a conical body or a frustum of a cone, the closest distance is 0 μm or more. Also, the closest distance may be, for example, 0.1 μm or more, or may be, for example, 1 μm or more. The upper limit of the closest distance is preferably 500 μm or less, more preferably 100 μm or less, even more preferably 50 μm or less, and even more preferably 10 μm or less. Thereby, the contact area between the liquid sample and the flow path 103 increases and the capillary force increases, so that it becomes easy to move the liquid sample.

[0021] Here, the "distance between adjacent convex portions 8" is the distance defined by their peripheral surfaces on the line segment connecting the center points of the two convex portions 8 that are closest to each other in a top view. FIG. 4 is a top view showing an arrangement example of the convex portions 8 when the distance between adjacent convex portions 8 is 0. FIG. 4 shows an example in which the bottom surface shape of the convex portion 8 is circular, and at this time, the convex portion 8 is specifically a cone or a truncated cone. As shown in FIG. 4, for example, when the distance between adjacent convex portions 8 arranged in a hexagonal lattice is 0, the plurality of convex portions 8 have their bottom surfaces arranged without gaps, the number of convex portions 8 per unit area in a top view increases, and the capillary force further increases, making it easier to transfer the liquid sample.

[0022] Here, each numerical value regarding the size of the convex portion 8 is calculated, for example, by observing a cross-section perpendicular to the flat portion 9 of the convex portion 8 with a scanning electron microscope (SEM) or by observing a three-dimensional image of the convex portion 8 with an optical microscope.

[0023] Also, from the viewpoint of improving the measurement accuracy of the substance to be detected, it is also preferable that a fine uneven structure B is formed on the surface of the convex portion 8, at least in the detection zone 105. From the same viewpoint, at this time, the arithmetic mean roughness Ra of the roughness curve of the convex portion 8 on which the fine uneven structure B is formed is preferably 0.005 μm or more and 1 μm or less. Here, the arithmetic mean roughness Ra of the roughness curve is specifically measured in accordance with JIS B 0601:2013 using a three-dimensional roughness analysis scanning electron microscope.

[0024] From the viewpoint of more stably fixing the detected substance, the arithmetic mean roughness Ra of the roughness curve of the convex portion 8 on which the fine uneven structure B is formed is preferably 0.005 μm or more, more preferably 0.010 μm or more, still more preferably 0.050 μm or more, and even more preferably 0.080 μm or more. From the viewpoint of improving the formability of the fine uneven structure B, the arithmetic mean roughness Ra is preferably 1 μm or less, more preferably 0.5 μm or less, still more preferably 0.3 μm or less, and even more preferably 0.2 μm or less.

[0025] Hereinafter, taking Fig. 2(a) as an example, a method for measuring the arithmetic mean roughness Ra of the convex portion 8 in which the fine concavo-convex structure B is formed in the fine concavo-convex structure A will be described. Using a three-dimensional roughness analysis scanning electron microscope, a convex profile is measured along the surface of the convex portion 8 (along the straight line 20 when viewed from above) with the center of the vertex of the convex portion 8 (for example, the center 19 of the protrusion) as the center point. The straight line 20 is a single straight line with a length of 20d with the center of the vertex (for example, the center 19 of the convex portion 8) as the center point. The length 20d is the same length as the diameter of the bottom surface of the convex portion 8. When the straight line 20 is a straight line on the same plane (for example, when both ends and the center are on the same plane), that is, when the convex portion 8 has a shape such as a cylinder or a prism, the arithmetic mean roughness Ra of the roughness curve defined by JIS B 0601:2013 is calculated from the uneven profile. When the straight line 20 is not a straight line on the same plane, that is, when the convex portion 8 has a shape such as a cone, a pyramid, a hemisphere, a semi-ellipsoid, a frustum of a cone, or a frustum of a pyramid, slope correction is performed on the uneven profile, and the arithmetic mean roughness Ra of the roughness curve defined by JIS B 0601:2013 is calculated as a plane.

[0026] Also, it is also preferable that the convex portion 8 has the following configuration. That is, in one cross section, a plurality of convex portions 8 have a first region (RG1 in Fig. 5) located on one side of the convex portion 8 from the center of the convex portion 8 in the width direction of the convex portion 8, and a second region located on the other side of the convex portion 8 from the center of the convex portion 8 in the width direction of the convex portion 8. In the one cross section, it is also preferable that at least one of the outer edges of the first region and the second region of the convex portion 8 has a concave portion.

[0027] Fig. 5 is a diagram for explaining the concave portion R of the outer edge in the first region RG1 of the convex portion 8. Fig. 5 corresponds to an enlarged view of the outer edge of the convex portion 8 shown in Fig. 2(b). In Fig. 5, the X direction indicates a direction parallel to the flat portion 9 (Fig. 2(a)). The Y direction indicates a direction perpendicular to the flat portion 9.

[0028] The outer edge of the first region RG1 of the convex portion 8 has a concave portion R. The concave portion R is recessed by a distance D in the X direction with respect to the virtual straight line IL. The distance D is, for example, 50 nm or more and 500 nm or less. In FIG. 5, the virtual straight line IL extends in the Y direction. Specifically, the virtual straight line IL contacts the first position P1 of the outer edge of the convex portion 8 and passes through the second position P2. The first position P1 of the outer edge of the convex portion 8 is the position located most outside the convex portion 8 among the upper end portions of the concave portion R. Therefore, the virtual straight line IL contacts the first position P1. That is, at the first position P1 and its vicinity, the virtual straight line IL and the outer edge of the convex portion 8 geometrically share only the first position P1 (i.e., a single point). The second position P2 of the outer edge of the convex portion 8 is located below the first position P1 in the Y direction. FIG. 5 shows one concave portion R in a part of the outer edge in the first region RG1 of the convex portion 8. The number of concave portions R in the entire first region RG1 is, for example, 1 or more, preferably 2 or more, and, for example, 20 or less, preferably 10 or less, more preferably 5 or less. The concave portion of the outer edge in the second region of the convex portion 8 is also determined in the same manner as shown in FIG. 5, for example.

[0029] In the film carrier for the inspection kit according to the present embodiment, since the number of concave portions (for example, the concave portion R in the first region RG1) in the first region or the second region is a certain number or more, it is possible to further increase the amount of the detection substance in the detection zone 105, and the amounts of the reaction substances and coloring substances generated by the enzyme-substrate reaction, other chemical reactions, and biological reactions. As a result, the detection sensitivity of the substance to be detected in the liquid sample can be further improved. The reason why the amounts of the detection substance in the detection zone 105, and the reaction substances and coloring substances generated by the enzyme-substrate reaction, other chemical reactions, and biological reactions can be increased is not clear, but the following reasons are considered. First, the fact that the number of concave portions in the first region or the second region is a certain number or more serves as an index indicating that the surface area of the protrusion is increased due to the presence of the concave portions. Therefore, at least in the detection zone 105, it is considered that due to the number of recesses in the first region or the second region being equal to or greater than a certain number, a structure is formed that has a space suitable for carrying the detection substance, and reaction substances and coloring substances generated by enzyme-substrate reactions, other chemical reactions, and biological reactions. Thus, in the membrane carrier for the test kit according to the present embodiment, at least in the detection zone 105, due to the number of recesses in the first region or the second region being equal to or greater than a certain number, it is considered possible to increase the amount of the detection substance carried in the detection zone 105, as well as the reaction substances and coloring substances generated by enzyme-substrate reactions, other chemical reactions, and biological reactions. From the above, it is considered that the membrane carrier for the test kit according to the present embodiment can further improve the detection sensitivity of the substance to be detected in the liquid sample by the number of recesses in the first region or the second region being equal to or greater than a certain number.

[0030] FIG. 11(a) is a top view showing an example of the planar arrangement of the convex portions of the quadrangular prism, and FIG. 11(b) is a perspective view showing the shape of the convex portion 18 shown in FIG. 11(a). As shown in FIGS. 11(a) and 11(b), the shape of each convex portion 18 is a quadrangular prism, and a fine concavo-convex structure A of a line & space structure in which the convex portions 18 are linear can also be provided. When the planar shape of the convex portion 18 is rectangular, the diameter 14 of the bottom surface 11 of the convex portion 18 is the length of the shortest side of the bottom surface (rectangle) 11 (in FIG. 11(a), the length in the direction perpendicular to the transfer direction d of the liquid sample). Also, the height 16 of the convex portion 18 is the maximum length of the convex portion 18 in the direction perpendicular to the flat portion 9 (the height of the quadrangular prism).

[0031] In the detection zone 105, the detection substance is immobilized on the surface of the fine concavo-convex structure A. More specifically, the detection substance is selectively immobilized in the detection zone 105 of the flow path 103. The detection substance may be chemically immobilized or physically immobilized on the surface of the fine concavo-convex structure A. From the viewpoint of more stably holding the detection substance, the detection substance is preferably covalently bonded to the material of the sheet substrate 101 forming the fine concavo-convex structure A. At this time, the detection substance may be directly bonded to the material of the sheet substrate 101 in the fine concavo-convex structure A, or may be bonded to an intervening molecule such as a spacer molecule bonded to the material of the sheet substrate 101 in the fine concavo-convex structure A. The intervening molecule preferably contains glycol, ether, amine, ester, amide, alcohol, carboxylic acid, etc.

[0032] The detection substance is selected from substances that specifically bind to the substance to be detected. For example, when the substance to be detected is an antigen, the detection substance may be an antibody against the antigen or an antigen-binding fragment thereof, and is preferably an antibody against the substance to be detected. Here, the antigen may be a substance having immunogenicity alone or a hapten. Also, the substance to be detected may be a first antibody, and the detection substance may be a second antibody specific to the first antibody. When the detection substance is an antibody or an antigenic fragment thereof, the antibody may be a polyclonal antibody or a monoclonal antibody. When the substance to be detected is an antibody or its antigen-binding fragment, the substance to be detected may be a substance capable of reacting with the antibody in an antigen-antibody reaction, and examples thereof include various pathogens and various clinical markers. More specifically, examples of the substance to be detected include viral antigens such as influenza virus, norovirus, adenovirus, RSV, HAV, HBs, and HIV; bacterial antigens such as MRSA, group A streptococcus, group B streptococcus, and Legionella bacteria; toxins produced by bacteria, etc.; hormones such as mycoplasma, Chlamydia trachomatis, and human chorionic gonadotropin; C-reactive protein, myoglobin, cardiac troponin, various tumor markers, pesticides, and environmental hormones. When the substance to be detected is an item that requires urgent detection and treatment measures such as influenza virus, norovirus, C-reactive protein, myoglobin, and cardiac troponin, its usefulness is even greater. The substance to be detected is usually in a suspended or dissolved state in a liquid sample. The liquid sample may be, for example, a sample in which the substance to be detected is suspended or dissolved in a buffer solution.

[0033] The substance to be detected may be selected from the group consisting of proteins and peptides such as enzymes and antibodies; nucleic acids and nucleoproteins; polysaccharides; and glycoproteins. And the detection substance may be any substance having specificity for these substances to be detected. For example, the detection substance may be selected from the group consisting of proteins, nucleic acids, polysaccharides, and glycoproteins.

[0034] The inspection device 100 is configured to detect the substance to be detected by detecting a change in the optical properties of the detection zone 105 caused by an enzymatic reaction. For this reason, the inspection device 100 has excellent detection accuracy. Also, according to the present embodiment, it is possible to obtain, for example, an inspection device 100 having excellent detection accuracy and sensitivity. Specifically, the change in optical properties is a color change in the detection zone 105 or chemiluminescence, bioluminescence, or fluorescence in the detection zone 105. When the change in optical properties is a color change in the detection zone 105, the inspection device 100 is preferably configured to be able to visually confirm the above color change.

[0035] (Method for manufacturing inspection device) Next, a method for manufacturing the inspection device 100 will be described. The inspection device 100 can be obtained, for example, by forming a fine concavo-convex structure A in a predetermined region on one surface of the sheet substrate 101, and immobilizing a detection substance in a predetermined region within the formation region of the fine concavo-convex structure A to form a detection zone 105.

[0036] Examples of the method for forming the fine concavo-convex structure A include imprinting such as thermal imprinting and UV imprinting; injection molding; pattern formation on a UV-curable resin by photolithography, soft lithography using a UV-curable resin pattern-formed by photolithography as a mold, pattern formation by etching using a UV-curable resin pattern-formed by photolithography, etc.; mechanical cutting; and laser processing, etc. Among these, thermal imprinting on a thermoplastic resin and injection molding are suitable as methods for performing precise processing at low cost. Specific examples of the thermoplastic resin include those described above as the material of the sheet substrate 101. In the case where the shape of the convex portion 8 in the fine concavo-convex structure A is a cone, in a processing method using a mold such as imprinting or injection molding, since the cone is thinner at the top than at the bottom surface, the volume to be cut out during mold production is less than that for producing a columnar body with the same bottom surface, and the mold can be produced at low cost. In this case, it becomes possible to detect the substance to be detected in the liquid sample at a lower cost.

[0037] Also, the immobilization of the detection substance on the fine concavo-convex structure A can be performed, for example, by the following method. In the example where the detection substance is an antibody, for example, a method of applying an antibody solution on the fine concavo-convex structure A and incubating; a method of applying one or both of a silane coupling agent and a cross-linking agent on the surface of the fine concavo-convex structure A, reacting with the material of the sheet substrate 101, and then applying an antibody solution to the applied site and incubating, etc. can be mentioned.

[0038] Examples of the silane coupling agent in the latter method include one or more selected from the group consisting of a silane coupling agent having an amino group, a silane coupling agent having a mercapto group, a silane coupling agent having an epoxy group, a silane coupling agent having an acrylic group, a silane coupling agent having a methacrylic group, a silane coupling agent having a vinyl group, and a silane coupling agent having an isocyanate group. Examples of the silane coupling agent having an amino group include 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-(2-aminoethyl)aminopropyltriethoxysilane, and 3-(2-aminoethyl)aminopropyltrimethoxysilane. Examples of the silane coupling agent having a mercapto group include 3-mercaptopropyltrimethoxysilane, 3-mercaptopropyltriethoxysilane, 2-mercaptoethyltrimethoxysilane, and 2-mercaptoethyltriethoxysilane. Examples of the silane coupling agent having an epoxy group include 3-glycidoxypropyltrimethoxysilane, 5,6-epoxyhexyltriethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, and 3-glycidoxypropyltriethoxysilane. Examples of the silane coupling agent having an acrylic group include 3-acryloxypropyltrimethoxysilane, 3-acryloxypropylmethyldimethoxysilane, 3-acryloxypropylmethyldiethoxysilane, and 3-acryloxypropyltriethoxysilane. Examples of the silane coupling agent having a methacrylic group include 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, γ-(methacryloyloxypropyl)trimethoxysilane, and γ-(methacryloyloxypropylmethyl)dimethoxysilane. Examples of the silane coupling agent having a vinyl group include vinyltriethoxysilane and vinyltrimethoxysilane. Examples of the silane coupling agent having an isocyanate group include trimethoxysilylmethyl isocyanate, triethoxysilylmethyl isocyanate, tripropoxysilylmethyl isocyanate, 2-trimethoxysilylethyl isocyanate, 2-triethoxysilylethyl isocyanate, 2-tripropoxysilylethyl isocyanate, 3-trimethoxysilylpropyl isocyanate, 3-triethoxysilylpropyl isocyanate, 3-tripropoxysilylpropyl isocyanate, 4-trimethoxysilylbutyl isocyanate, 4-triethoxysilylbutyl isocyanate, and 4-tripropoxysilylbutyl isocyanate. From the viewpoint of increasing the amount of the detection substance supported on the fine concavo-convex structure A, the silane coupling agent is preferably at least one selected from the group consisting of a silane coupling agent having an amino group and a silane coupling agent having an epoxy group.

[0039] In addition, examples of the crosslinking agent include formaldehyde, glutaraldehyde, dextran, 1,4-phenyl diisocyanate, toluene-2,4 diisocyanate, polyethyleneimine, hexamethylene diisocyanate, hexamethylene diisothiocyanate, N,N'-polymethylene bisiodoacetamide, N,N'-ethylene bismaleimide, ethylene glycol bis succinimidyl succinate, bisdiazobenzidine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, succinimidyl 3-(2-pyridyldithio)propionate, N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, N-hydroxysuccinimide, N-sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, N-succinimidyl (4-iodoacetyl)aminobenzoate, N-succinimidyl 4-(1-maleimidophenyl)butyrate, N-(ε-maleimidocaproyl oxy)succinimide, iminothiolane, S-acetyl mercapto succinic anhydride, methyl-3-(4'-dithiopyridyl)propionimidate, methyl-4-mercapto butyrylimidate, methyl-3-mercaptopropionimidate, and N-succinimidyl-S-acetyl mercaptoacetate. One or more compounds selected from the group consisting of these are exemplified. From the viewpoint of increasing the amount of the detection substance supported on the fine concavo-convex structure A, the crosslinking agent is preferably one or more compounds selected from the group consisting of glutaraldehyde, dextran, 1,4-phenyl diisocyanate, toluene-2,4 diisocyanate, polyethyleneimine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and N-hydroxysuccinimide.

[0040] (Inspection method) In this embodiment, the inspection method using the inspection device 100 can be performed, for example, by the sandwich method. At this time, specifically, the inspection method is A step (step 11) of introducing a liquid sample containing a substance to be detected upstream of the detection zone 105, guiding it to the detection zone 105 by capillary action, and capturing the substance to be detected in the fine uneven structure A by specific interaction between the substance to be detected and the detection substance; After the capturing step, a step (step 12) of introducing a liquid containing a labeled antibody that specifically binds to the substance to be detected into the detection zone 105 and capturing the labeled antibody in the fine uneven structure A where the substance to be detected is captured; After the step of capturing the labeled antibody, a step (step 13) of introducing a compound that causes a change in optical properties in the detection zone 105 by reacting with the labeled antibody into the detection zone 105; and A step (step 14) of detecting or quantifying the substance to be detected in the liquid sample by detecting a change in optical properties in the detection zone 105 is included. Also, before step 11, between step 11 and step 12, between step 12 and step 13, and between step 13 and step 14, a step (step 15) of introducing a buffer solution or the like upstream of the detection zone 105 and flushing the detection zone 105 may be further performed.

[0041] For the labeled antibody in step 12, a labeled antibody used in immunoassays such as enzyme-linked immunosorbent assay (ELISA) or fluorescence immunoassay can be used. Examples of the labeled antibody include enzyme-labeled antibodies.

[0042] Specifically, step 13 is a step of introducing a substrate for causing color development or luminescence into the detection zone 105. As the compound in step 13, a compound that reacts with the labeled antibody to cause an optical change can be used. Examples of the above compound include chromogenic substrates and luminescent substrates that react with enzyme-labeled antibodies. Here, the luminescent substrate may be any of a chemiluminescent substrate, a bioluminescent substrate, and a fluorescent substrate. Also, in step 14, specifically, the labeled antibody captured in the fine concavo-convex structure A is detected. In step 14, a detection method corresponding to the type of labeled antibody used in step 12 can be used.

[0043] For example, in step 14, when the change in optical properties is a color change or a luminescence change due to the reaction of an enzyme and a substrate in the detection zone, in step 14, the presence or absence of color development, absorbance, fluorescence, chemiluminescence, bioluminescence, etc. based on the labeled antibody substrate is measured in the detection zone 105, whereby the substance to be detected can be detected or quantified. Specific examples of the combination of the enzyme-labeled antibody and the chromogenic substrate include, for example, a mixture of an alkaline phosphatase (ALP)-labeled antibody, 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (BCIP-NBT); a mixture of a horseradish peroxidase (HRP)-labeled antibody, 3,3'-diaminobenzidine (DAB) and hydrogen peroxide (H2O2); a mixture of a horseradish peroxidase (HRP)-labeled antibody, 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2), etc.

[0044] Also, in step 14, when the change in optical properties is fluorescence, chemiluminescence or bioluminescence in the detection zone 105, in step 14, a substrate for the enzyme immobilized on the labeled antibody is introduced into the detection zone 105, and the presence or absence of fluorescence or chemiluminescence based on the substrate, or the fluorescence intensity, chemiluminescence intensity or bioluminescence intensity is measured, whereby the substance to be detected can be detected or quantified.

[0045] Specific examples of the combination of an enzyme-labeled antibody and a fluorescent substrate include the combination of an alkaline phosphatase (ALP)-labeled antibody and 1,3-dichloro-9,9'-dimethyl-acridine-2-one-7-yl phosphate (DDAO phosphate), the combination of an ALP-labeled antibody and ELF (registered trademark, Thermo Fischer Scientific), the combination of an ALP-labeled antibody and AttoPhos (registered trademark, JBL Scientific), the combination of an ALP-labeled antibody and 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), the combination of an ALP-labeled antibody and 4-methylumbelliferyl phosphate (MUP), and the combination of an ALP-labeled antibody and fluorescein diphosphate tetraammonium salt (FDP).

[0046] Specific examples of the combination of an enzyme-labeled antibody and a chemiluminescent substrate include the combination of a horseradish peroxidase (HRP)-labeled antibody and a mixture of luminol, hydrogen peroxide (H2O2), and an iodophenol compound, the combination of an ALP-labeled antibody and AMPPD (registered trademark, Tropix), the combination of an ALP-labeled antibody and CDP-Star (trademark, C 18 H 19 Cl2O7PNa2), the combination of an ALP-labeled antibody and CSPD (registered trademark, Tropix)C 18 H 20 ClO7PNa2), and the combination of an ALP-labeled antibody and Lumigen (registered trademark) PPD (C 18 H 21 O7PNa2).

[0047] Specific examples of the combination of an enzyme-labeled antibody and a bioluminescent substrate include the combination of a luciferase-labeled antibody and luciferin.

[0048] (Chip) In the present embodiment, the inspection device 100 can also be used for a chip. Further, the chip has the inspection device 100 in the above-described present embodiment. The chip may be composed of the inspection device 100, or may further include other members. Specific examples of the other members include a housing that houses or holds the inspection device 100, and a member that absorbs the liquid that has flowed through the inspection device 100.

[0049] As described above, the embodiments of the present invention have been described with reference to the drawings. These are examples of the present invention, and various configurations other than the above can also be adopted.

Example

[0050] Hereinafter, the present embodiment will be specifically described with reference to examples and comparative examples, but the present embodiment is not limited to these examples.

[0051] (Production Examples 1 to 7) In this example, a sheet substrate 101 having the schematic structure shown in FIG. 1 was manufactured by the following procedure.

[0052] (Production Example 1) Production of Sheet Substrate 1 Thermal imprinting was performed on a polycarbonate sheet (manufactured by Teijin Limited, PC-2151, 50 mm × 50 mm, sheet thickness 200 μm) to form a conical convex portion 8 having a diameter of 30 μm at the bottom surface of the fine concavo-convex structure A (convex portion 8) (hereinafter, also referred to as "diameter of the convex portion" or "diameter") and a height of 30 μm (hereinafter, also referred to as "height"). A sheet substrate 1 (Production Example 1: FIG. 3(a)) in which the convex portions 8 were arranged in a hexagonal lattice pattern with a distance of 2 μm between the convex portions 8 was produced. The planar shape of the formation region of the convex portion 8 in the sheet substrate 1 was 30 mm × 30 mm. Here, when performing thermal imprinting, by using a laser-processed mold, a fine concavo-convex structure B was formed on the surface of the fine concavo-convex structure A (convex portion 8), and the number of recesses R on the outer edge in the first region RG1 of the convex portion 8 on which the fine concavo-convex structure B was formed and the number of recesses R on the outer edge in the second region RG2 were set to the values shown in Table 1.

[0053] The processing method of the mold used for manufacturing the sheet substrate 1 is as follows. A plurality of pulses of light were irradiated from a laser processing apparatus (Ultra-short Pulse Laser Processing Machine R-200 manufactured by Dongcheng Electro Beam Co., Ltd., laser wavelength: 1552 nm, rated output: 10 W, pulse: femtosecond) onto an aluminum alloy flat plate to obtain a mold having a conical concave portion corresponding to the convex portion 8.

[0054] (Production Example 2) Production of Sheet Substrate 2 A sheet substrate 2 (Production Example 2: FIG. 3(b)) was produced in accordance with the sheet substrate in Production Example 1, except that the distance between the convex portions 8 was 15 μm (distance between the centers of the cones: 45 μm) and they were arranged in a square lattice.

[0055] (Production Example 3) Production of Sheet Substrate 3 A plurality of pulses of light were irradiated from a laser processing apparatus (Ultra-short Pulse Laser Processing Machine R-200 manufactured by Dongcheng Electro Beam Co., Ltd., laser wavelength: 1552 nm, rated output: 10 W, pulse: femtosecond) onto a nickel flat plate to obtain a mold having a conical concave portion corresponding to the convex portion 8, and a sheet substrate 3 (Production Example 3: FIG. 3(c)) was produced in accordance with the sheet substrate in Production Example 1, except that the obtained mold was used for imprint processing.

[0056] (Production Example 4) Production of Sheet Substrate 4 After subjecting an acrylic flat plate to mechanical cutting, a mold having a conical concave portion corresponding to the convex portion 8 was obtained by Ni electroforming twice, and a sheet substrate 4 (Production Example 4: FIG. 3(d)) was produced in accordance with the sheet substrate in Production Example 1, except that the obtained mold was used for imprint processing.

[0057] (Production Example 5) Production of Sheet Substrate 5 The diameter of the bottom surface of the fine concavo-convex structure A (protrusion 8) was set to 60 μm, the height of the fine concavo-convex structure A (protrusion 8) was set to 90 μm, and the distance between the protrusions 8 was set to 2 μm. After subjecting an acrylic flat plate to mechanical cutting, Ni electroforming was performed twice to obtain a mold having a conical recess corresponding to the protrusion 8. Except for using the obtained mold for imprinting, a sheet substrate 5 (Production Example 5: FIG. 3(e)) was produced according to the sheet substrate in Production Example 1.

[0058] (Production Example 6) Production of sheet substrate 6 After subjecting an acrylic flat plate to mechanical cutting, Ni electroforming was performed twice, and further etching was performed with an iron chloride solution to obtain a mold having a conical recess corresponding to the protrusion 8. Except for using the obtained mold for imprinting, a sheet substrate 6 (Production Example 6: FIG. 3(f)) was produced according to the sheet substrate in Production Example 5.

[0059] (Production Example 7) Production of sheet substrate 7 In this example, a sheet substrate having a schematic structure shown in FIGS. 12(a) and 12(b) of the fine concavo-convex structure A was produced. FIG. 12(a) is a top view showing the arrangement of the protrusions 18 in the fine concavo-convex structure A, and FIG. 12(b) is a perspective view showing the shape of the protrusions 18. In this example, a sheet substrate 7 (Production Example 7: FIG. 13) was produced in which rectangular parallelepiped protrusions 18 of 10 μm (diameter of the bottom surface 14) × 30 μm × 30 μm (height) were arranged as shown in FIG. 12(a) with a distance of 30 μm between the protrusions 18.

[0060] The processing method of the mold used for the production of the sheet substrate 7 is as follows. After patterning recesses on a glass substrate by photolithography, Ni electroforming was performed twice to obtain a mold having a rectangular parallelepiped-shaped recess corresponding to the protrusion 18.

[0061] FIGS. 3(a) to 3(f) and FIG. 13 are diagrams showing SEM images of the fine concavo-convex structure A of the sheet substrates obtained in Production Examples 1 to 7, respectively. Table 1 shows the measured values regarding the sizes of the protrusions 8 of the sheet substrates obtained in each example.

[0062]

Table 1

[0063] (Experimental Example 1: Example 1 and Comparative Example 1) In Example 1 and Comparative Example 1, on the sheet substrate 1 and the film substrate 1 made of nitrocellulose membrane without processing (having no fine concavo-convex structure A) (manufactured by Merck Millipore, thickness about 0.25 mm), respectively, an anti-CRP (C-Reactive Protein) antibody was immobilized as a detection substance to manufacture devices (Example 1: Device 1, Comparative Example 1: Device 2). Using the obtained devices, CRP, an enzyme-labeled anti-CRP antibody, and a substrate were sequentially dropped, and an immunoassay by the sandwich method was performed. The specific procedure is shown below.

[0064] (Immobilization of detection substance) An antibody solution with an antibody concentration of about 0.1 μg / mL was prepared by adding an anti-CRP antibody (manufactured by Betyl laboratories) to the prepared buffer solution (composition: 50 mM tris(hydroxymethyl)aminomethane-hydrochloric acid buffer, pH 7.5, trehalose 2 (w / v)%). In Example 1, the formation region of the fine concavo-convex structure A on the sheet substrate 1 was cut into a size of 5 mm in width × 30 mm in length, and in Comparative Example 1, the film substrate 1 was cut into the same size and used for the devices in each example. The prepared antibody solution was applied dropwise, 1 μL at a time, using a micropipette at the center in the width direction of the substrate at a position 1.2 mm from the upstream end of each substrate, and then each substrate was dried at 42°C for 1 hour to prepare a detection zone (sheet substrate 1: circular shape with a diameter of about 1 mm, film substrate 1: circular shape with a diameter of about 2 mm).

[0065] (Fabrication of device) Each of the sheet substrate 1 and the film substrate 1 on which the antibody was immobilized was overlapped with the end portion (position 5 mm from the downstream end of the substrate) of an absorption pad (PVA sponge, A-3150HP, manufactured by Ion) cut into a size of 5 mm in width × 50 mm in length, and fixed using tape.

[0066] (Immunoassay) Using the devices obtained in each example, an immunoassay was performed according to the following procedure. For Example 1 and Comparative Example 1, the immunoassay was performed with n = 3 for each condition using a sample containing an antigen or a sample not containing an antigen. In the following procedure, unless otherwise specified, each aqueous solution was dropped at the end of the substrate of each example (at a position 5 mm from the end on the opposite side to the side overlapped with the absorption pad). 1. 20 μL of a washing aqueous solution (0.05 (v / v)% Triton X-100 / tris-buffered saline (TBS)) was dropped to wash the excess antibodies on the substrate of each example. 2. An aqueous solution (CRP concentration 10 μg / mL) obtained by diluting CRP (manufactured by BBI Solutions) with 0.05 (v / v)% Triton X-100 / TBS, or an aqueous solution not containing CRP (0.05 (v / v)% Triton X-100 / TBS) was dropped, and waited for 3 minutes. 3. 20 μL of an alkaline phosphatase-labeled anti-CRP antibody solution (antibody concentration of about 20 μg / mL) was dropped, and waited for 2 minutes. This operation was repeated one more time. 4. 20 μL of a BCIP-NBT solution prepared by mixing a BCIP-NBT color development stock solution (manufactured by Nacalai Tesque) and a tris-hydrochloric acid buffer solution (manufactured by Nacalai Tesque) at a volume ratio of 1:100 was dropped, and waited for 3 minutes. 5. 20 μL of a washing aqueous solution (0.05 (v / v)% Triton X-100 / TBS) was dropped to wash the excess antigen, substrate, and product on the substrate. 6. The substrate of each example after washing was imaged using a digital camera (manufactured by Casio, EXILIM EX-100F).

[0067] Images of each substrate in the above 6 are shown in FIGS. 6(a) to 6(d). FIGS. 6(a) and 6(b) are diagrams showing the results of dropping CRP concentrations of 10 μg / mL and 0.05 (v / v)% Triton X-100 / TBS in the above procedure 2 in Example 1, respectively. FIGS. 6(c) and 6(d) are diagrams showing the results of dropping CRP concentrations of 10 μg / mL and 0.05 (v / v)% Triton X-100 / TBS in the above procedure 2 in Comparative Example 1, respectively.

[0068] From FIGS. 6(a) to 6(d), a purple color development was confirmed on each substrate by the reaction between the enzyme (alkaline phosphatase) introduced onto the substrate in the above procedure 3 and the substrate (BCIP-NBT) introduced onto the substrate in the above procedure 4. For Device 1 using the sheet substrate having the fine concavo-convex structure A of Example 1, a clear color development was observed from the detection zone (the region coated with the antibody) of the substrate on which the sample containing the antigen was measured. Also, almost no color development was confirmed in the region other than the detection zone of the substrate on which the sample containing the antigen was measured (the region not coated with the antibody) and in the substrate on which the sample not containing the antigen was measured.

[0069] On the other hand, in Device 2 using the film substrate of Comparative Example 1, color development was observed not only in the region coated with the antibody of the substrate on which the sample containing the antigen was measured, but also in the region not coated with the antibody and in the substrate on which the sample not containing the antigen was measured.

[0070] From the above results, Device 1 using the sheet substrate having the fine concavo-convex structure A of Example 1 can efficiently immobilize the detection substance and the coloring substance on the convex portion 8 as compared with Device 2 using the film substrate of Comparative Example 1, so that in the detection zone 105, it becomes easier to recognize the detection signal (color development), which is a change in optical characteristics caused by the enzyme reaction, that is, the S / N ratio becomes higher, and it is shown that highly sensitive and highly accurate measurement is possible.

[0071] (Calculation of ΔRGB in the region coated with the antibody) For Example 1 and Comparative Example 1, images of the substrates of each example after immunoassay were analyzed using image analysis software Image J (manufactured by the National Institutes of Health, USA), and the RGB values in the area coated with the antibody were calculated. Specifically, the captured color image was separated into an RGB image, and the R value, G value, and B value at the center of the antibody-coated part (circular shape with a diameter of 800 μm) were calculated respectively. For the substrate of each example, ΔRGB was calculated according to the following formula using the RGB value of the substrate measured with the sample containing the antigen and the RGB value of the substrate measured with the sample not containing the antigen.

[0072]

Equation

[0073] In the above formula, R n , G n , B n represent the R value, G value, and B value of the substrate measured with the sample containing the antigen respectively, and R0, G0, B0 represent the R value, G value, and B value of the substrate measured with the sample not containing the antigen respectively. The calculation results of ΔRGB for each substrate are shown in Table 2.

[0074]

Table 2

[0075] From Table 2, the device using the sheet substrate having the fine concavo-convex structure A of Example 1 had a higher ΔRGB value compared to the device using the film substrate without the fine concavo-convex structure A of Comparative Example 1. Also, regarding the coefficient of variation of the measurement results of ΔRGB in the three tests, the coefficient of variation of the device using the sheet substrate having the fine concavo-convex structure A of Example 1 became smaller compared to the device using the film substrate of Comparative Example 1. Therefore, it was shown that the device using the sheet substrate having the fine concavo-convex structure A of Example 1 could efficiently immobilize the detection substance and the coloring substance at the convex portions 8, and could detect the antigen with higher sensitivity and higher precision as compared with the device using the film substrate of Comparative Example 1.

[0076] (Experimental Example 2: Examples 2 to 8) In Examples 2 to 8, anti-CRP (C-Reactive Protein) antibody was immobilized as a detection substance on each of the aforementioned sheet substrates 1 to 7, respectively, to produce devices (Example 2: Device 3, Example 3: Device 4, Example 4: Device 5, Example 5: Device 6, Example 6: Device 7, Example 7: Device 8, Example 8: Device 9). Using the obtained devices, CRP, enzyme-labeled anti-CRP antibody, and substrate were sequentially dropped, and immunoassay by the sandwich method was performed. The specific procedure is shown below.

[0077] (Immobilization of detection substance) According to the method described above in Experimental Example 1, anti-CRP antibody was immobilized on each substrate.

[0078] (Fabrication of device) According to the method described above in Experimental Example 1, each substrate was combined with an absorption pad. By the above procedure, Device 3 using the sheet substrate 1 obtained in Production Example 1, Device 4 using the sheet substrate 2 obtained in Production Example 2, Device 5 using the sheet substrate 3 obtained in Production Example 3, Device 6 using the sheet substrate 4 obtained in Production Example 4, Device 7 using the sheet substrate 5 obtained in Production Example 5, Device 8 using the sheet substrate 6 obtained in Production Example 6, and Device 9 using the sheet substrate 7 obtained in Production Example 7 were obtained.

[0079] (Immunoassay) Using Devices 3 to 9, immunoassay was performed by the following procedure, respectively. Unless otherwise specified, each aqueous solution was dropped at the end of the substrate of each example (at a position 5 mm from the end on the side opposite to the side overlapped with the absorption pad). 1. 10 μL of a washing aqueous solution (0.05 (v / v)% Triton X-100 / tris-buffered saline (TBS)) was dropped to wash the excess antibodies on the substrate of each example. 2. An aqueous solution (CRP concentration 10 μg / mL) in which CRP (manufactured by BBI Solutions) was diluted with 0.05 (v / v)% Triton X-100 / TBS, or an aqueous solution containing no CRP (0.05 (v / v)% Triton X-100 / TBS) was dropped in an amount of 10 μL, and the mixture was left standing for 3 minutes. 3. 10 μL of an alkaline phosphatase-labeled anti-CRP antibody solution (antibody concentration approximately 20 μg / mL) was dropped, and the mixture was left standing for 1 minute. This operation was repeated two more times. 4. 10 μL of a washing aqueous solution (0.05 (v / v)% Triton X-100 / tris-buffered saline (TBS)) was dropped to wash the excess labeled antibodies on the substrate of each example. 5. 10 μL of a BCIP-NBT solution prepared by mixing BCIP-NBT chromogenic stock solution (manufactured by Nacalai Tesque) and tris-hydrochloride buffer solution (manufactured by Nacalai Tesque) at a volume ratio of 1:100 was dropped, and the mixture was left standing for 3 minutes. 6. 10 μL of a washing aqueous solution (0.05 (v / v)% Triton X-100 / TBS) was dropped to wash the excess antigen, substrate, and product on the substrate. 7. The substrate of each example after washing was imaged using a digital camera (EXILIM EX-100F, manufactured by Casio).

[0080] Among the above 7., the images of Device 3 (Example 2) and Device 4 (Example 3) are shown in FIGS. 7(a) and 7(b), respectively. From FIGS. 7(a) and 7(b), purple color development was confirmed on each substrate by the reaction between the enzyme (alkaline phosphatase) introduced onto the substrate in the above-mentioned procedure 3 and the substrate (BCIP-NBT) introduced onto the substrate in the above-mentioned procedure 5. In the devices of both Example 2 and Example 3, distinct color development was observed from the detection zone (the region where the antibody was applied) of the substrate on which the sample containing the antigen was measured. Also, almost no color development was confirmed from the regions other than the detection zone of the substrate on which the sample containing the antigen was measured (the regions where the antibody was not applied), and from the substrates on which the samples not containing the antigen were measured.

[0081] (Observation of SEM image in the antibody-coated part) FIGS. 8(a), 8(b), 9(a) and 9(b) are diagrams showing SEM images of the detection zone 105 (the region where the antibody was applied in the figure) and the regions other than the detection zone 105 (the regions where the antibody was not applied in the figure) on each substrate after performing the immunoassay by the aforementioned procedure in Example 2 (FIGS. 8(a) and 8(b)) and Example 3 (FIGS. 9(a) and 9(b)). From FIGS. 8(a) and 9(a), it was confirmed that coloring substances with sizes ranging from several tens of nm to several hundreds of μm were deposited on the fine concavo-convex structure A without gaps in the detection zone 105 (the region where the antibody was applied) of each example. On the other hand, almost no deposition of coloring substances with sizes ranging from several tens of nm to several hundreds of μm was confirmed in the regions other than the detection zone 105 (the regions where the antibody was not applied).

[0082] (Calculation of ΔRGB in the region where the antibody was applied) ΔRGB was calculated according to the method described above in Experimental Example 1. The calculation results of ΔRGB for each substrate are shown in Table 3.

[0083] [Table 3]

[0084] From Table 3, in any device using the sheet substrate having the fine concavo-convex structure A, ΔRGB was higher as compared with the device using the film substrate of Comparative Example 1 (Table 2). Among them, in the device using the sheet substrate having the fine concavo-convex structure A of Example 2, ΔRGB was even higher.

[0085] (Experimental Example 3: Examples 9 and 10, Comparative Example 2) In Examples 9 and 10 and Comparative Example 2, detection substances were immobilized on the aforementioned sheet substrates 1, 2, and film substrate 1, respectively, to manufacture devices (Example 9: Device 10, Example 10: Device 11, Comparative Example 2: Device 12), and an immunoassay using chemiluminescence by the luminol reaction was performed using the obtained devices.

[0086] (Immobilization of detection substance) According to the method described above in Experimental Example 1, anti-CRP antibody was immobilized on each substrate.

[0087] (Fabrication of device) According to the method described above in Experimental Example 1, each substrate and an absorption pad were combined. By the above procedure, Device 10 using the sheet substrate 1 obtained in Production Example 1 and Device 11 using the sheet substrate 2 obtained in Production Example 2 were obtained. On the other hand, a detection substance was immobilized on the film substrate 1 made of nitrocellulose membrane to obtain Device 12.

[0088] (Immunoassay) Using Devices 10 to 12, immunoassays were performed according to the following procedure, respectively. Unless otherwise specified, each aqueous solution was dropped at the end of the substrate of each example (at a position 5 mm from the end on the side opposite to the side where the absorption pad was overlapped). 20 μL of a washing aqueous solution (0.05 (v / v)% Triton X-100 / TBS) was dropped to wash the excess antibody on the substrate of each example. 2. An aqueous solution (CRP concentration 10 μg / mL) obtained by diluting CRP (manufactured by BBI Solutions) with 0.05 (v / v)% Triton X-100 / TBS, or an aqueous solution not containing CRP (0.05 (v / v)% Triton X-100 / TBS) was dropped in an amount of 20 μL, and left for 3 minutes. 3. 20 μL of an anti-CRP antibody labeled with horseradish peroxidase (HRP) (antibody concentration approximately 0.2 μg / mL) was dropped and left for 2 minutes. This operation was repeated one more time. 4. 20 μL of a washing aqueous solution (0.05 (v / v)% Triton X-100 / TBS) was dropped to wash the excess labeled antibody on the substrate of each example. 5. A substrate solution was prepared by mixing equal amounts of Stable Peroxide Solution and Luminol / Enhancer Solution in SuperSignal West Femto Maximum Sensitivity Substrate (trade name, manufactured by Thermo Fischer SCIENTIFIC), and 20 μL was dropped. 6. After dropping, the chemiluminescence image 1 minute and 45 seconds later was captured by a chemiluminescence measuring device (ChemiDoc XRS Plus system, manufactured by BIORAD).

[0089] The chemiluminescence images of each substrate after measurement are shown in FIGS. 10(a) to 10(f). In each of these figures, the rectangular frame indicates the frame of the image. In Example 9 (FIGS. 10(a) and 10(b)) and Example 10 (FIGS. 10(c) and 10(d)), chemiluminescence was confirmed in the detection zone 105 (the region where the antibody was applied) of the sheet substrate (FIGS. 10(a) and 10(c)). Also, almost no luminescence was confirmed from the regions other than the detection zone 105 of the sheet substrate (the regions where the antibody was not applied) and the substrates on which samples not containing the antigen were measured (FIGS. 10(b) and 10(d)).

[0090] On the other hand, in Comparative Example 2 (Figs. 10(e) and 10(f)), for the device 12 using the film substrate 1, luminescence was observed not only in the detection zone 105 (the region coated with the antibody) of the substrate for measuring the sample containing the antigen, but also in the regions other than the detection zone 105 (the regions not coated with the antibody) and on the substrate on which the sample not containing the antigen was not measured.

[0091] From the above results, compared with the device 12 using the film substrate of Comparative Example 2, the devices 10 and 11 using the sheet substrate having the fine concavo-convex structure A of Examples 9 and 10 can efficiently immobilize the detection substance and the coloring substance on the convex portions 8, so that in the detection zone 105, it becomes easier to recognize the detection signal (chemiluminescence), which is a change in optical properties caused by the enzymatic reaction, that is, the S / N ratio becomes higher, and it has been shown that highly sensitive and highly accurate measurement is possible.

[0092] This application claims priority based on Japanese Patent Application No. 2021-052926 filed on March 26, 2021, and incorporates all of its disclosures herein.

Explanation of Reference Numerals

[0093] 4 Diameter 5 Distance 6 Height 8 Convex Portion 9 Flat Portion 10 Bottom Surface 11 Bottom Surface 14 Diameter 16 Height 18 Convex Portion 19 Center 20 Straight Line 100 Inspection Device 101 Sheet Substrate 103 Flow Path 105 Detection Zone d Transfer Direction R Concave Portion

Claims

1. An inspection method using an inspection device, comprising: the inspection device includes: a flow path provided on one surface of a sheet-shaped resin substrate for transferring a liquid sample by capillary action; a detection zone provided in a part of the flow path; a fine concavo-convex structure A provided at least in the detection zone and having a plurality of convex portions formed integrally with the resin substrate; and,[ in the detection zone, a detection substance that specifically binds to the substance to be detected in the liquid sample is immobilized on the surface of the fine concavo-convex structure A; at least in the detection zone, a fine concavo-convex structure B is formed on the surface of the convex portion; the arithmetic mean roughness Ra of the roughness curve of the convex portion on which the fine concavo-convex structure B is formed is 0.005 μm or more and 1 μm or less; configured to detect the substance to be detected by detecting a change in the optical characteristics of the detection zone caused by an enzyme reaction, wherein the change in the optical characteristics is a color change, chemiluminescence, bioluminescence or fluorescence in the detection zone; the inspection method includes: introducing the liquid sample upstream of the detection zone and guiding it to the detection zone by capillary action, and capturing the substance to be detected in the fine concavo-convex structure A by a specific interaction between the substance to be detected and the detection substance; after the capturing step, introducing a liquid containing an enzyme-labeled antibody that specifically binds to the substance to be detected into the detection zone, and capturing the enzyme-labeled antibody in the fine concavo-convex structure A in which the substance to be detected is captured; after the step of capturing the enzyme-labeled antibody, introducing a substrate that causes a change in optical characteristics in the detection zone by reacting with the enzyme-labeled antibody into the detection zone, wherein the change in the optical characteristics is a color change, chemiluminescence, bioluminescence or fluorescence in the detection zone; detecting or quantifying the substance to be detected by detecting the change in the optical characteristics in the detection zone, wherein the change in the optical characteristics is a color change, chemiluminescence, bioluminescence or fluorescence in the detection zone; An inspection method comprising the above steps.

2. An inspection method using an inspection device, comprising: the inspection device includes: a flow path provided on one surface of a sheet-shaped resin substrate for transferring a liquid sample by capillary action; a detection zone provided in a part of the flow path; At least a fine concavo-convex structure A provided in the detection zone and having a plurality of convex portions formed integrally with the resin substrate, having, In the detection zone, a detection substance that specifically binds to the substance to be detected in the liquid sample is immobilized on the surface of the fine concavo-convex structure A, In a cross-section, a plurality of the convex portions, A first region located on one side of the convex portion from the center of the convex portion in the width direction of the convex portion, A second region located on the other side of the convex portion from the center of the convex portion in the width direction of the convex portion, having, In the cross-section, at least one of the outer edges of the first region and the second region of the convex portion has a concave portion, configured to detect the substance to be detected by detecting a change in the optical characteristics of the detection zone caused by an enzymatic reaction, and the change in the optical characteristics is a color change, chemiluminescence, bioluminescence, or fluorescence in the detection zone, The inspection method is, introducing the liquid sample upstream of the detection zone and guiding it to the detection zone by capillary action, and capturing the substance to be detected in the fine concavo-convex structure A by specific interaction between the substance to be detected and the detection substance, After the capturing step, introducing a liquid containing an enzyme-labeled antibody that specifically binds to the substance to be detected into the detection zone, and capturing the enzyme-labeled antibody in the fine concavo-convex structure A where the substance to be detected is captured, After the step of capturing the enzyme-labeled antibody, introducing a substrate that causes a change in optical characteristics in the detection zone by reacting with the enzyme-labeled antibody into the detection zone, and the change in the optical characteristics is a color change, chemiluminescence, bioluminescence, or fluorescence in the detection zone, detecting or quantifying the substance to be detected by detecting the change in the optical characteristics in the detection zone, and the change in the optical characteristics is a color change, chemiluminescence, bioluminescence, or fluorescence in the detection zone, An inspection method including.

3. The inspection method according to claim 1 or 2, wherein the detection substance is an antibody against the substance to be detected.

4. The inspection method according to any one of claims 1 to 3, wherein the change in the optical characteristics is a color change in the detection zone.

5. The inspection method according to claim 4, wherein the color change can be visually confirmed.

6. The inspection method according to any one of claims 1 to 3, wherein the change in the optical property is chemiluminescence, bioluminescence or fluorescence in the detection zone.

7. The inspection method according to any one of claims 1 to 6, wherein in the detection zone, the shape of the convex portion is a columnar body, a cone or a frustum.

8. The inspection method according to any one of claims 1 to 7, wherein in the detection zone, the shape of the convex portion is a cone, and the detection substance is immobilized on the side surface of the cone.

9. In the detection zone, the plurality of convex portions are arranged in a grid pattern on a plane. The inspection method according to any one of claims 1 to 8.

10. The inspection method according to any one of claims 1 to 9, wherein the diameter of the bottom surface of the convex portion is 10 μm or more and 1000 μm or less.

11. The inspection method according to any one of claims 1 to 10, wherein the height of the convex portion is 10 μm or more and 500 μm or less.

12. The inspection method according to any one of claims 1 to 11, which is for an enzyme immunoassay method.

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