Detection Device and Detection Method

Abstract

The inspection kit has: a flow path (2) provided on a substrate formed of resin for transporting a liquid sample from one end side to the other end side; a solid phase part (50) provided on the other end side of the flow path (2) and having an antibody immobilized thereon; a detection part (double electrode (20)) provided with an electrode part for detecting the reaction of the liquid sample with respect to the antibody; and a fine concavo-convex structure having a plurality of convex parts integrally formed on the flow path (2), the fine concavo-convex structure having: a first fine structure region (31) where the plurality of convex parts are provided relatively sparsely; and a second fine structure region (32) where the plurality of convex parts are provided relatively densely, and the first fine structure region (31) and the second fine structure region (32) are provided at positions closer to the one end side of the flow path (2) than the solid phase part (50).

Description

Technical Field

[0001] The present invention relates to a detection device and a detection method for detecting a substance to be detected in a liquid sample. Background Art

[0002] In recent years, point-of-care testing (POCT) reagents have received attention. POCT reagents are reagents for examining diseases, pregnancy, or measuring blood glucose levels, etc. by using antigen-antibody reactions, etc. In the detection and measurement using POCT reagents, results can be determined in a short time. In addition, the usage method of POCT reagents is relatively simple, and POCT reagents are relatively inexpensive. Since POCT reagents have these characteristics, they are often used for examinations at the stage of mild symptoms, regular examinations, etc. In addition, in home medical care, which is expected to increase in the future, POCT reagents will also become an important examination tool.

[0003] In the inspection or diagnosis using an inspection kit, which is one type of POCT reagent, a liquid sample such as blood is introduced into the inspection kit, and a specific substance to be detected contained in the liquid sample is detected. As a method for detecting a specific substance to be detected from a liquid sample, immunochromatography is often used. In immunochromatography, a liquid sample is dropped onto a membrane carrier provided in the inspection kit, and during the movement of the liquid sample on the membrane carrier, the substance to be detected in the liquid sample binds to a labeled substance. Further, the substance to be detected specifically and selectively binds to a substance (hereinafter referred to as a detection substance) fixed in the inspection kit. As a result, changes in color, weight, etc. generated in the inspection kit are detected. The detection substance can also be referred to as a reagent.

[0004] As a membrane carrier for moving a liquid sample, a nitrocellulose membrane is often used (refer to Patent Document 1 below). The nitrocellulose membrane has a plurality of fine pores with a diameter of about several μm, and the liquid sample moves in the pores by capillary force.

[0005] However, since the nitrocellulose membrane is derived from a natural product, the pore diameters in the membrane and the connection modes between the pores are not the same, so the flow rate of the liquid sample in the membrane varies depending on the membrane. If the flow rate varies, the time required for detecting the substance to be detected also changes. As a result, there may be an incorrect judgment that the substance to be detected is not detected before the substance to be detected binds to the labeled substance or the reagent.

[0006] To solve the above problems, a method of artificially creating a fine flow path for a liquid sample has been proposed (refer to Patent Documents 2 and 3 below). By using this method, a membrane carrier with a uniform structure can be fabricated. As a result, the possibility of an incorrect judgment that the substance to be detected is not detected before the substance to be detected binds to the labeled substance or the reagent can be reduced.

[0007] Prior art documents

[0008] Patent documents

[0009] Patent Document 1: Japanese Patent Laid-Open No. 2014-062820

[0010] Patent Document 2: Japanese Patent No. 4597664

[0011] Patent Document 3: Japanese Patent Publication No. 2012-524894 Summary of the invention

[0012] Problems to be solved by the invention

[0013] However, in immunochromatography, a liquid sample is dropped onto a membrane carrier provided in a test kit, and during the movement of the liquid sample on the membrane carrier, the analyte in the liquid sample binds to a labeling substance. Further, the analyte specifically and selectively binds to a detection substance immobilized in the test kit. As a result, changes in color, weight, etc. generated in the test kit are detected. As a method for detecting the analyte, a method (color change detection method) of detecting a color change in a detection area caused by the binding of the analyte bound to a labeling substance such as colored latex particles, fluorescent particles, or metal colloid particles to a reagent immobilized in the detection area using an optical measuring device such as an absorbance meter is known. In addition, there is also a method (electrochemical immunochromatography) of converting the concentration of a biomarker into the concentration of an electrochemically active substance for detection.

[0014] In electrochemical immunochromatography, it is necessary to develop a variety of solutions such as a reaction solution, a cleaning solution, and a secondary reaction solution, which poses problems of increased effort in use and longer detection time, and becomes an obstacle to the popularization of test kits using electrochemical immunochromatography. That is, in a test kit using electrochemical immunochromatography, there is a demand for a POCT reagent (test kit) that can determine the result in a short time, has a simple usage method, and is inexpensive. In addition, even in the case of using the color change detection method, the same problems exist in methods that require the development of a variety of solutions.

[0015] The present invention has been completed in view of the above circumstances, and an object thereof is to provide a technique that can save effort in use and shorten the detection time in a test kit using immunochromatography when developing a variety of solutions such as a reaction solution, a cleaning solution, and a secondary reaction solution.

[0016] Solutions for solving the problems

[0017] The inspection device (also referred to as "test kit") of the present invention has:

[0018] A flow path, which is provided on a substrate formed of resin and conveys a liquid sample from one end side to the other end side;

[0019] A solid phase part, which is provided on the other end side of the flow path and has an antibody immobilized thereon;

[0020] A detection part, which is provided with an electrode part and detects the reaction of the liquid sample with respect to the antibody; and

[0021] A fine uneven structure, which has a plurality of convex parts integrally formed on the flow path,

[0022] The fine uneven structure has:

[0023] A first uneven part, on which the plurality of convex parts are provided relatively sparsely; and

[0024] A second uneven part, on which the plurality of convex parts are provided relatively densely,

[0025] The first uneven part and the second uneven part are provided at a position closer to the one end side of the flow path than the solid phase part.

[0026] The inspection device of the present invention is a detection device for detecting a substance to be detected in a liquid sample, wherein,

[0027] The detection device has:

[0028] A flow path, which conveys the liquid sample from one end side to the other end side;

[0029] A solid phase part, which is provided on the other end side of the flow path and has an antibody immobilized thereon;

[0030] A detection part, which detects the reaction of the liquid sample with respect to the antibody; and

[0031] A fine uneven structure, which has a plurality of convex parts integrally formed on the flow path,

[0032] The fine uneven structure has:

[0033] A first uneven part, on which the plurality of convex parts are provided relatively sparsely; and

[0034] A second uneven part, on which the plurality of convex parts are provided relatively densely,

[0035] The first uneven part and the second uneven part are provided at a position closer to the one end side of the flow path than the solid phase part.

[0036] The detection method of the present invention uses the above detection device to detect the reaction of a liquid sample with respect to an antibody.

[0037] Effects of the Invention

[0038] According to the present invention, it is possible to provide a technique that can save the labor during use and shorten the detection time in an inspection kit using immunochromatography when various solutions such as a developing reaction solution, a cleaning solution, and a secondary reaction solution are used. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a top view of the inspection kit of the first embodiment.

[0040] Figure 2 It is a top view of the membrane carrier of the first embodiment.

[0041] Figure 3 It is a diagram showing the microstructure and convex portions of the first embodiment.

[0042] Figure 4 It is a perspective view of the convex portion of the first embodiment.

[0043] Figure 5 It is a diagram showing an enlarged view of the boundaries of the respective regions of the microstructure of the first embodiment.

[0044] Figure 6 It is a flowchart showing an example of an inspection method using the inspection kit of the first embodiment.

[0045] Figure 7 It is a top view of the membrane carrier of the second embodiment.

[0046] Figure 8 It is a diagram showing an enlarged view of the boundaries of the respective regions of the microstructure of the second embodiment.

[0047] Figure 9 It is a diagram showing an example of a structure for preventing backflow of a solution in the microstructure of the second embodiment.

[0048] Figure 10 It is a diagram showing a photograph of the test piece of Example 1.

[0049] Figure 11 It is a diagram of a test piece showing images 30 seconds, 140 seconds, and 310 seconds after the start of the test in Example 1.

[0050] Figure 12 It is a diagram showing a chart marking the elapsed time and the RGB component ratios of the measurement points in Example 1.

[0051] Figure 13 It is a diagram showing in Example 1 the analysis Figure 12 of the mixing ratios of the respective solutions calculated from the results obtained from the data.

[0052] Figure 14It is a diagram showing the results of the mixing ratios of the respective solutions calculated under the condition that nitrocellulose was used instead of the imprinting sheet in Comparative Example 2.

[0053] Figure 15 It is a diagram showing the structure of the test piece of Example 2.

[0054] Figure 16 It is a diagram showing the chart of the test results of Example 2.

[0055] Figure 17 It is a diagram showing the chart of the fluorescence intensity of the AB part - BG part in Example 2.

[0056] Figure 18 It is an example of a fluorescence photograph of Example 2.

[0057] Figure 19 It is a diagram showing an example of the arrangement of the electrode part in the two - electrode method of the first embodiment.

[0058] Figure 20 It is a diagram showing an example of the arrangement of the electrode part in the three - electrode method of the first embodiment.

[0059] Figure 21 It is a diagram showing the structure of the test piece of Example 3.

[0060] Figure 22 It is a diagram showing the chart of the test results of Example 3.

[0061] Figure 23 It is a flowchart showing the solution dropping time of Example 4.

[0062] Figure 24 It is a diagram showing the chart of the test results of Example 4. Detailed implementation mode

[0063] <<First embodiment>>

[0064] Hereinafter, the embodiments of the present invention will be described.

[0065] <Summary of the inspection kit>

[0066] Figure 1 It is a top view of the inspection kit 18 of this embodiment. Figure 2 It is a schematic top view showing the membrane carrier 3. Figure 3 It shows the fine structure of the membrane carrier 3 (also called "fine concavo - convex structure") and the convex portion 8 constituting the fine structure. Figure 4 It shows a three - dimensional view (SEM image) of the convex portion 8.

[0067] The inspection kit 18 has the function of detecting the substance to be detected in the liquid sample.

[0068] The test kit 18 is one of the POCT reagents, which will be described in detail later. A liquid sample such as blood is introduced into the test kit 18, and a specific analyte contained in the liquid sample is detected. Immunochromatography is applied as a method for detecting a specific analyte from the liquid sample.

[0069] In the present embodiment, the test kit 18 having a structure using an electrochemical immunochromatography method for detecting by converting the concentration of a biomarker into the concentration of an electrochemically active substance will be described.

[0070] As described above, in the electrochemical immunochromatography method, it is necessary to develop a variety of solutions (liquid samples) such as a reaction solution, a cleaning solution, and a secondary reaction solution. Generally, the time required for use increases, resulting in a longer detection time. In the present embodiment, the flow path 2 of the membrane carrier 3 is divided into a plurality of regions (here, three regions: the first microstructural region 31 to the third microstructural region 33), and the flow rate of the solution in each region is controlled to be different. As a structure for making the flow rates different, a microstructure formed on the membrane carrier 3, that is, a structure having a capillary action that determines the speed of transporting the solution, is set in each region.

[0071] The following will be described in detail.

[0072] <Details of the test kit 18>

[0073] As Figure 1 shown, the test kit 18 includes a membrane carrier 3 and a housing 18a that houses the membrane carrier 3. In this figure, the direction from the upstream on the left side to the downstream on the right side in the illustration is described as the traveling direction d of the solution (also referred to as the "flow path direction").

[0074] On the surface of the membrane carrier 3, there are, in order from the left in the illustration, a cleaning solution area 3x for dropping a cleaning solution, a dropping area 3z for dropping a liquid sample, and a detection area 3y for detecting an analyte in the liquid sample. In addition, an absorption pad for absorbing excess solution is provided on the downstream side (the right side in the illustration) of the membrane carrier 3, but is not shown here.

[0075] The cleaning solution area 3x is exposed at the first opening 18b of the housing 18a. The dropping area 3z is exposed at the third opening 18d of the housing 18a. The detection area 3y is exposed at the second opening 18c of the housing 18a. In addition, a cleaning solution can be dropped in the dropping area 3z. In this case, the first opening 18b can be omitted. When there are multiple solutions, inlets (openings) are provided according to these solutions. That is, the inlets are provided according to which solution moves at what speed at what time. Multiple solutions can be dropped at a certain inlet, and the dropping times can be the same or different.

[0076] In order to perform detection using an electrochemical detection method, an electrode unit 20 is provided in the detection area 3y. The electrode unit 20 is, for example, a two-electrode (two-electrode method) including a working electrode 25 on the upstream side in the traveling direction d and a counter electrode 26 on the downstream side. In addition, as will be described later, the electrode unit 20 can also be a three-electrode method having a reference electrode 27. A measuring device 21 is connected to the electrode unit 20. The measuring device 21 can be a general measuring device or can be configured as a device in which a prescribed application program is introduced into a mobile terminal such as a smartphone.

[0077] <Details of the membrane carrier 3>

[0078] As Figure 2 shown, at least one flow path 2 for transporting a liquid sample is provided in the membrane carrier 3. As Figure 3 shown, a fine structure 7 is provided on the bottom surface of the flow path 2. In the present embodiment, the fine structure 7 is provided over the entire surface of the membrane carrier 3, and the entire surface of the membrane carrier 3 functions as the flow path 2 for the liquid sample.

[0079] Figure 3 (a) is a top view of the fine structure 7, Figure 3 (b) is a perspective view of the convex portion 8 constituting the fine structure. The fine structure 7 is the whole of the convex portions 8. That is, the membrane carrier 3 includes a flat portion 9 corresponding to the bottom surface of the flow path 2 for the liquid sample and a plurality of convex portions 8 protruding from the flat portion 9.

[0080] By capillary action, the space between the plurality of convex portions 8 functions as the flow path 2 for transporting the liquid sample along the surface of the membrane carrier 3. In other words, by capillary action, the voids in the fine structure 7 function as the flow path 2 for transporting the liquid sample along the surface of the membrane carrier 3. The plurality of convex portions 8 are regularly or translationally symmetrically arranged in a lattice configuration (for example, a rhombic lattice configuration, a square lattice configuration) and are regularly arranged in rows on the surface of the membrane carrier 3.

[0081] The convex portion 8 is, for example, in a conical shape. Here, as Figure 3 (b), Figure 4 shown, the convex portion 8 is in a conical shape. In addition to this, it can also be a pyramid, or can also be a shape obtained by cutting off the upper part of the cone (frustum of a cone). Whichever shape it is, as long as the fine structure 7 constituted by the convex portions 8 can generate capillary action to transport the liquid sample.

[0082] The fine structure 7 generates capillary action. By the capillary action of the fine structure 7, the liquid sample is transported via the fine structure 7 from the cleaning liquid area 3x or the dropping liquid area 3z on the left side in the drawing toward the detection area 3y (along Figure 2 the traveling direction d).

[0083] In this embodiment, as Figure 2 shown, the membrane carrier 3 is divided into three regions from the left side: a first microstructural region 31 (first uneven portion), a second microstructural region 32 (second uneven portion), and a third microstructural region 33 (third uneven portion). In the first microstructural region 31, the second microstructural region 32, and the third microstructural region 33, the microstructures 7 are different. As a result, the speed of transporting the solution is different in each region.

[0084] According to the Poiseuille equation used to explain the flow between parallel plates, the speed of transporting the solution can be understood. For example, in the microstructure 7 where capillary action occurs, for example, in a structure with a plurality of convex portions 8, the narrower the distance 5 between the convex portions 8, the greater the transport speed of the solution. That is, by appropriately setting the density of the microstructure ( Figure 3 the arrangement of the convex portions 8 shown), the speed of each region can be controlled.

[0085] In Figure 5 is a view showing the microstructure 7 observed from the upper surface. Figure 5 (a) of shows the region of the boundary (first boundary 41) between the first microstructural region 31 and the second microstructural region 32. Figure 5 (b) of shows the region of the boundary (second boundary 42) between the second microstructural region 32 and the third microstructural region 33. In all regions, the convex portions 8 are set to the same shape and size. In Figure 5 , the convex portion 8 is a cone, the diameter of the bottom surface is 30 μm, and the height is 30 μm. As shown in the attached drawings, regarding the arrangement (degree of density) of the convex portions 8, the first microstructural region 31 on the left side of the attached drawings is the sparsest, and the third microstructural region 33 on the right side is the densest. That is, the distance 5 between the convex portions 8 in the first microstructural region 31 is the widest, and the distance 5 between the convex portions 8 in the third microstructural region 33 is the narrowest. In the illustrated example, the distance 5 between the convex portions 8 in the first microstructural region 31 is 25 μm, the distance 5 between the convex portions 8 in the second microstructural region 32 is 15 μm, and the distance between the convex portions 8 in the third microstructural region 33 is 2 μm.

[0086] When the substance to be detected in the liquid sample reaches the detection area 3y, it is detected in the form of a current value by the measuring device 21 using the electrode portion 20 (working electrode 25, counter electrode 26) provided in the detection area 3y. That is, a potential difference is previously applied between the working electrode 25 and the counter electrode 26 of the electrode portion 20, and the oxidation current is measured by the measuring device 21. In addition, in the case of using a color change detection method, the substance to be detected is detected based on the change in the color of the detection area 3y.

[0087] <Material of the membrane carrier 3>

[0088] The film carrier 3 including the fine structure 7 (multiple convex portions 8) is formed of, for example, a thermoplastic. That is, by processing a film-shaped base material formed of a thermoplastic using hot embossing, the film carrier 3 having the fine structure 7 can be fabricated. The thermoplastic constituting the film carrier 3 can be, for example, at least one selected from the group including polyester-based resins, polyolefin-based resins, polystyrene-based resins, polycarbonate-based resins, fluorine-based resins, and acrylic-based resins. Specific thermoplastics can be, for example, at least one from the group including polyethylene terephthalate (PET), cycloolefin polymer (COP), polypropylene (PP), polystyrene (PS), polycarbonate (PC), polyvinylidene fluoride (PVDF), and polymethyl methacrylate (PMMA).

[0089] The glass transition point Tg or melting point Tm of the above thermoplastic can be 80 to 180 °C. The storage modulus of the thermoplastic at a temperature 20 °C higher than the glass transition point Tg can be 1.0 Pa or more and 1.0×10 7 Pa or less. The storage modulus of the thermoplastic at a temperature 20 °C higher than the melting point Tm can be 1.0 Pa or more and 1.0×10 7 Pa or less.

[0090] When the thermoplastic undergoes glass transition or melting at a temperature below 80 °C, and the storage modulus of the thermoplastic at a temperature 20 °C higher than the glass transition point or melting point is 1.0×10 7 Pa or less, it is difficult to use the thermoplastic in a solid form at room temperature in practical applications, and it is difficult to fabricate the film carrier using hot embossing.

[0091] When the thermoplastic undergoes glass transition or melting at a temperature higher than 180 °C, the molding temperature during hot embossing becomes high, and the productivity of the film carrier decreases. That is, when the temperature required to soften the thermoplastic during hot embossing is higher than 180 °C, the productivity of the film carrier decreases.

[0092] When the storage modulus of the thermoplastic at a temperature 20 °C higher than the glass transition point or melting point is 1.0×10 7 Pa or less, the molding pressure required for fabricating the fine structure can be suppressed to a small value, and fabrication can be carried out under relatively mild conditions, thus improving the production efficiency.

[0093] The convex portion 8 of the cone (here a circular cone) can be formed by hot embossing using a mold. When forming a cone using a mold, compared with the case of forming a groove-shaped flow path (line and space structure) using a mold, the volume of the metal shaved off from the surface of the metal member when manufacturing the mold is significantly reduced, and the processing cost of the mold is reduced. In contrast, when manufacturing a mold for forming a line and space structure, a large amount of metal must be shaved off from the metal member.

[0094] In addition, the upper part of the cone is thinner than the bottom surface of the cone. Therefore, when forming a cone using a mold, compared with the case of forming a cylinder having the same bottom surface as the cone using a mold, the volume of the metal shaved off from the surface of the metal member when manufacturing the mold is significantly reduced, and the processing cost of the mold is reduced.

[0095] Furthermore, the porosity of the fine structure formed by regularly arranging cones in rows is greater than the porosity of the line and gap structure. In addition, the porosity of the fine structure formed by regularly arranging cones in rows is greater than the porosity of the structure formed by regularly arranging a plurality of cylinders having the same bottom surface as the cone in rows. Therefore, according to the fine structure formed by regularly arranging cones in rows, the flow rate of the liquid sample can be increased, which is beneficial to the detection of the substance to be detected.

[0096] <Shape and Dimensions of Membrane Carrier 3>

[0097] As described above, the shape of the bottom surface 10 of the cone (convex portion 8) can be freely selected, and can be, for example, as shown in (b) of Figure 3 or Figure 4 a circular cone, or can also be a pyramid (a quadrangular pyramid, a hexagonal pyramid, etc.). In order to facilitate the processing of the mold and suppress the processing cost, the bottom surface 10 of the cone (convex portion 8) is preferably circular or polygonal (such as a square, a rhombus, a rectangle, a triangle, or a hexagon, etc.).

[0098] The diameter 4 of the bottom surface 10 of the convex portion 8 is, for example, 10 to 1000 μm. When the diameter 4 of the bottom surface 10 of the convex portion 8 is less than 10 μm, the microfabrication cost of the mold increases. In addition, it is difficult to uniformly fabricate numerous microstructures 7 on the surface of the relatively large film carrier 3. Therefore, the too-small microstructures 7 are not suitable for practical applications. In addition, when the diameter 4 of the bottom surface 10 of the microstructures 7 is less than 10 μm, there is a tendency for the capillary force required to move the liquid sample to weaken. When the diameter 4 of the bottom surface 10 of the microstructures 7 is greater than 1000 μm, the volume of the metal cut out from the metal member when fabricating the mold becomes larger, resulting in an increase in the fabrication costs of the mold and the film carrier 3. In addition, when the diameter 4 of the bottom surface 10 of the microstructures 7 is greater than 1000 μm, the area of the flow path 2 in the film carrier 3 must also be increased, and the inspection kit 18 becomes larger, which is not conducive to the transportation of the inspection kit 18 itself. When the convex portion 8 (microstructure 7) is a cone, the diameter 4 of the bottom surface 10 of the convex portion 8 can be the diameter 4 of the bottom surface 10 (circle) of the cone.

[0099] The height 6 of the convex portion 8 is, for example, 10 to 500 μm. When the height 6 of the convex portion 8 is less than 10 μm, there is a tendency for the capillary force required to move the liquid sample to weaken. When the height 6 of the convex portion 8 is greater than 500 μm, it is difficult to completely fill the thermoplastic into the concave portion (the pit corresponding to the shape of the convex portion 8 of the microstructure 7) of the mold during hot embossing.

[0100] The overall shape of the film carrier 3 is not particularly limited. For example, it can be a polygon such as a quadrilateral, a circle, or an ellipse. When the film carrier 3 is a quadrilateral, the longitudinal width L1 of the film carrier 3 can be, for example, 2 to 100 mm, and the transverse width L2 of the film carrier 3 can be, for example, 3 to 100 mm. In addition, the transverse widths L21 to L23 of the first to third microstructure regions 31 to 33 can also be, for example, 1 to 50 mm respectively. The thickness of the film carrier 3 other than the height 6 of the microstructure 7 (i.e., the convex portion 8) can be, for example, 0.1 to 10 mm.

[0101] The aspect ratio Lv / Lh of the convex portion 8 can be 1 / 10 or more and 2 / 1 or less. When the aspect ratio Lv / Lh is less than 1 / 10, the contact area between the liquid sample and the flow path 2 is small, and the capillary force decreases. Therefore, there is a tendency for it to be difficult to move the liquid sample. When the aspect ratio Lv / Lh is greater than 2 / 1, the productivity of the film carrier 3 formed by hot embossing decreases. As in the present embodiment, when the convex portion 8 is a cone (more specifically, a cone), the length Lh in the horizontal direction of the convex portion 8 can be the diameter 4 of the bottom surface 10 of the convex portion 8. In addition, the length Lv in the vertical direction of the convex portion 8 can be the height 6 of the convex portion 8 with respect to the flat portion 9 of the film carrier 3.

[0102] The ratio D2 / D1 of the diameter 4 (D1) of the bottom surface of the convex portion 8 to the distance D2 between the closest centers of the convex portions 8 to each other can be greater than 1 and 5 or less. The ratio D2 / D1 cannot be 1 or less. When the ratio D2 / D1 is greater than 5, the contact area between the liquid sample and the flow path 2 decreases, the capillary force decreases, and there is a tendency that it is difficult to move the liquid sample. In the case where the convex portion 8 is a cone as in the present embodiment, the diameter 4 (D1) of the bottom surface 10 of the convex portion 8 can be the diameter of the bottom surface of the cone, and the distance D2 between the closest centers can be the distance between the vertices of a pair of adjacent convex portions 8 (cones). The diameter 4 (D1) of the bottom surface 10 of the convex portion 8 can be made to coincide with the length Lh in the horizontal direction of the convex portion 8 described above. Therefore, the aspect ratio Lv / Lh can also be expressed as Lv / D1.

[0103] In addition, regarding the pitch (distance between vertices) between the convex portions 8 of the fine concavo-convex structure, when comparing between adjacent regions, the ratio (P1 / P2) of the pitch P1 of the region where the fine concavo-convex structure is formed relatively sparsely to the pitch P2 of the region where the fine concavo-convex structure is formed relatively densely is 1.1 or more and 5 or less. Here, when the pitch between the convex portions 8 in the first fine structure region 31 is P11, the pitch between the convex portions 8 in the second fine structure region 32 is P21, and the pitch between the convex portions 8 in the third fine structure region 33 is P23, the ratio (P11 / P21) is 1.1 or more and 5 or less, and in addition, the ratio (P21 / P23) is 1.1 or more and 5 or less. The ratio (P1 / P2) is set according to the speed at which the solution is desired to move. In addition, regarding the lower limit of the ratio (P1 / P2), when it is small, the speed difference between regions disappears, and the significance of setting a difference in the density of the fine concavo-convex structure decreases. From this point of view, the ratio (P1 / P2) is preferably 1.2 or more, more preferably 1.3 or more. Regarding the upper limit, if it becomes too large, the difference in the moving speed of the solution between regions becomes too large, and it becomes difficult to adjust the speed in the entire inspection kit 18. From this point of view, it is preferably 4 or less, more preferably 3 or less.

[0104] <Configuration of Electrode Portion>

[0105] Refer to Figure 19 and Figure 20 , and an example of the configuration of the electrode portion 20 will be described.

[0106] Figure 19 in (a) to Figure 19 in (c) show an example of the arrangement of the working electrode 25 and the counter electrode 26 in the case where the electrode portion 20 is of a two-electrode type. The working electrode 25 and the counter electrode 26 are provided separately. Here, the working electrode 25 is provided at the same position as the counter electrode 26 or at a position upstream of the counter electrode 26 in the traveling direction d. The working electrode 25 can also be configured as a comb-shaped electrode, for example.

[0107] In Figure 19 In the configuration example shown in (a), the working electrode 25 is disposed across the entire width direction of the flow path 2. The counter electrode 26 is disposed across the entire width direction of the flow path 2 in a region that is separated from the working electrode 25 by a predetermined distance toward the downstream side. In addition, although the working electrode 25 and the counter electrode 26 are rectangular in a plan view, they are not limited to this shape and can be various shapes such as an ellipse or a semi-circle. Further, although the working electrode 25 and the counter electrode 26 are disposed so as to block the width direction of the flow path 2, they are not limited thereto, and as shown in Figure 1 , Figure 2 , they may be disposed only in a partial region with respect to the width direction. Further, the widths of both or either one of the working electrode 25 and the counter electrode 26 may be shortened.

[0108] In Figure 19 In the configuration example shown in (b), the counter electrode 26 is disposed in a shape of a Japanese syllable "コ" that is concave on the upstream side in a plan view. Further, the working electrode 25 is disposed in a rectangular shape in the concave region of the counter electrode 26. The position of the most upstream side of the working electrode 25 and the position of the upstream side of the counter electrode 26 are the same position.

[0109] In Figure 19 In the configuration example shown in (c), the rectangular working electrode 25 and the counter electrode 26 are symmetrically disposed in the width direction, respectively.

[0110] Figure 20 (a) to Figure 12 (f) of

[0111] In Figure 20 In the configuration example shown in (a), the working electrode 25, the reference electrode 27, and the counter electrode 26 are arranged from the upstream side to the downstream side and are disposed across the entire width direction of the flow path 2. It can also be said that in the configuration shown in (a) of Figure 19 , there is a structure in which the reference electrode 27 is disposed between the working electrode 25 and the counter electrode 26.

[0112] In Figure 20 In the configuration example shown in (b), in the configuration shown in (a) of Figure 20 , the width of the reference electrode 27 is shortened and the reference electrode 27 is disposed in a rectangular shape at the center in the width direction of the flow path 2.

[0113] In Figure 20In the configuration example shown in (c), a working electrode 25 and a counter electrode 26 are provided on the left side of the flow path 2 from the upstream side, and a reference electrode 27 is provided on the right side of the flow path 2. The reference electrode 27 is provided elongated along the travel direction d from the upstream side of the working electrode 25 to the downstream side of the counter electrode 26.

[0114] exist Figure 20 In the configuration example shown in (d), the configuration of the working electrode 25 and the counter electrode 26 is the same as Figure 19 The configuration is the same as that shown in (b), and a reference electrode 27 is provided at a position upstream of the working electrode 25 on the left side in the figure.

[0115] exist Figure 20 In the configuration example shown in (e), Figure 19 In the arrangement shown in (b), the concave right end portion of the counter electrode 26 becomes shorter toward the downstream side, and the reference electrode 27 is provided in the shortened region.

[0116] exist Figure 20 In the configuration example shown in (f), the counter electrode 26 is provided across the entire width direction of the flow channel 2. Furthermore, the working electrode 25 is provided on the left side of the flow channel 2, and the reference electrode 27 is provided on the right side. The working electrode 25 and the reference electrode 27 are bilaterally symmetrical.

[0117] <Structure of the electrode section>

[0118] The electrode portion 20 (a working electrode 25 and a counter electrode 26 in the case of a two-electrode system, and a reference electrode 27 in the case of a tripole electrode) can also be formed by providing a conductive material on the convex portion 8 of the fine structure 7. The conductive material is not particularly limited, and examples thereof include gold, silver, platinum, palladium, carbon, graphene, carbon nanotubes (CNTs), and composite materials thereof. The reference electrode 27 is not particularly limited, and examples thereof include an Ag / AgCl electrode.

[0119] The conductor material of the protrusion 8 can be, for example, a conductor film formed by at least one of sputtering, vacuum evaporation, laser ablation and CVD (chemical vapor deposition), or a printed layer of a paste (ink) containing conductor particles formed by inkjet printing, screen printing, etc. For antibody immobilization, the working electrode 25 can be surface-modified with thiol or the like.

[0120] In this case, the maximum peak height Rp of the roughness curve of the electrode portion 20 is 0.005 μm or more and 10 μm or less, and the average length RSm of the roughness curve elements is 0.01 μm or more and 15 μm or less. By setting the surface roughness in this way, good capillary force can be generated. In addition, since the surface area of the electrode portion 20 increases, the amount of signal that can be obtained can be increased. In addition, these surface roughnesses can be calculated by analyzing the SEM image as shown in Figure 4 shown below.

[0121] <Method for manufacturing the test kit 18>

[0122] The method for manufacturing the test kit 18 can be obtained through the following processes.

[0123] Process 1: Hot embossing process

[0124] It includes the following processes: bringing the surface of a mold (die) having a plurality of recesses into contact with a film-like substrate made of a thermoplastic resin, and heating the substrate, thereby manufacturing a film carrier 3 having a fine structure (a plurality of protrusions 8) corresponding to the shape of the recesses.

[0125] The method for manufacturing the test kit 18 further includes the following process: fixing a reagent or a labeling substance to the detection area 3y on the surface of the film carrier 3 having the fine structure 7, more specifically, to the solid phase portion 50 of the third fine structure region 33.

[0126] The microfabrication method of the mold used in the hot embossing process can be, for example, etching, photolithography, mechanical cutting, or laser processing, etc. A microfabrication method suitable for the processing size and processing range can be selected.

[0127] Before performing hot embossing, it is desirable to perform a mold release treatment. In the mold release treatment, for example, a self-assembled monolayer can be formed on the mold surface to reduce the surface energy. As a result, after hot embossing, it is easy to peel the film carrier 3 made of a thermoplastic resin from the surface of the mold 1.

[0128] The hot embossing method can be either a flat pressing type or a roll type. In the flat pressing type, between the upper and lower worktables facing each other in parallel, the mold and the substrate made of a thermoplastic resin are overlapped and sandwiched between the worktables. Then, the mold and the substrate are heated and pressed via the worktables. Such a flat pressing method is excellent in terms of good forming accuracy. The roll type is a method of forming using a heated roll-type mold and the clamping pressure between the rolls. The productivity of the roll type is excellent.

[0129] When performing hot embossing, conditions such as the forming temperature, forming pressure, and transfer time can be selected according to the size of the microfabrication, the shape of the microstructures (protrusions 8), the size of the processing range, etc. For example, in the case of flat pressing, the forming temperature can be a temperature 20 to 50 °C higher than the glass transition point Tg, or a temperature 20 to 50 °C higher than the melting point Tm. The forming pressure can be 1 to 10 MPa. The transfer time (the time maintained while pressing the mold and the substrate) can be 3 to 10 minutes. By performing hot embossing under the above conditions, it is easy to accurately transfer the microstructures of the mold to the surface of the substrate.

[0130] Depending on the type of thermoplastic resin constituting the film carrier 3 and the type of reagent (test substance), it may be difficult to fix the reagent (test substance) to the solid phase portion 50 of the film carrier 3. In this case, by performing an appropriate surface treatment only on the detection area 3y in advance, the reagent (test substance) can be easily fixed to the detection area 3y (i.e., the solid phase portion 50) of the film carrier 3.

[0131] The surface treatment method of the detection area 3y is not limited in any way. For example, it can be various plasma treatments, UV treatments, UV / ozone treatments, or various methods such as surface modification using 3-aminopropyltriethoxysilane or glutaraldehyde.

[0132] The reagent (test substance) fixed to the detection area 3y can be an antibody, for example. For example, in Figure 2 the antibody is fixed to the solid phase portion 50 of the third microstructural region 33. The solid phase portion 50 is provided at a position upstream of the electrode portion 20 in the solution traveling direction d.

[0133] An antibody is a substance that causes an antigen-antibody reaction with the substance to be detected. The antibody can be a polyclonal antibody or a monoclonal antibody. The substance to be detected is not limited in any way and can be any substance that can cause an antigen-antibody reaction with the antibody, such as various pathogens, various clinical markers, etc. Specific examples of the substance to be detected can be viral antigens such as influenza virus, norovirus, adenovirus, RSV, HAV, HBs, HIV, etc. The substance to be detected can also be bacterial antigens such as MRSA, group A hemolytic streptococcus, group B hemolytic streptococcus, Legionella bacteria, toxins produced by bacteria, etc. The substance to be detected can also be hormones such as mycoplasma, Chlamydia trachomatis, human chorionic gonadotropin, etc. The substance to be detected can also be C-reactive protein, myoglobin, cardiac troponin, various tumor markers, pesticides, environmental hormones, etc. In particular, in cases where detection of substances to be detected such as influenza virus, norovirus, C-reactive protein, myoglobin, and cardiac troponin is urgently needed and treatment measures for diseases caused by them are required, the usefulness of the test kit 18 of the present embodiment is particularly great. In addition, the substance to be detected can also be an antigen that can individually induce an immune response. The substance to be detected can also be a hapten that cannot individually induce an immune response but can bind to an antibody through an antigen-antibody reaction with the antibody.

[0134] <Detection method of test kit 18>

[0135] Refer to Figure 6 the flowchart shown and the above Figures 1 - 5 , the test method based on the test kit 18 will be described. In Figure 6 , the third microstructure region 33 of the microstructure 30 is shown with a focus.

[0136] S1: Device preparation process

[0137] First, prepare the test kit 18 and the solutions used (reaction solution, cleaning solution, secondary reaction solution). As described above, the antibody 51 is fixed to the solid phase portion 50 of the third microstructure region 33.

[0138] S2: Reaction solution spreading process

[0139] When the reaction solution is dropped from the droplet region 3z to the second microstructure region 32, the reaction solution moves to the third microstructure region 33 under the capillary action of the microstructure 7.

[0140] The detection target 91 and the detection target (marker) 92 in the reaction solution react with the antibody 51 and are fixed. The excess reaction solution is absorbed by the water-absorbing pad, but a part of the detection target 91a and the detection target (marker) 92a that are not fixed remain on the third microstructure region 33.

[0141] S3: Cleaning solution spreading process

[0142] Next, cleaning liquid 93 is dropped from the self-cleaning liquid area 3x to clean the detection target 91a and the detection target (marker) 92a that are not fixed to the solid phase part 50 and remain in the third microstructural region 33. The detection target (marker) 92a has an alkaline phosphatase (ALP) marker.

[0143] S4: Secondary reaction liquid development step

[0144] After cleaning, the secondary reaction liquid (for example, p-aminophenylphosphoric acid 94) dropped onto the second microstructural region 32 moves to the third microstructural region 33 by the capillary action of the microstructures 7. p-aminophenylphosphoric acid 94 reacts with the detection target (marker) 92 fixed to the antibody 51 to generate an electroactive substance (here, p-aminophenol 95). This substance is related (proportional) to the amount (concentration) of the detection target (marker) 92 fixed to the antibody 51. Therefore, the concentration of the measurement target can be accurately and stably measured using the value of the oxidation current measured by the electrode part 20.

[0145] As described above, according to the present embodiment, in the inspection kit 18 applying immunochromatography, it is possible to set multiple regions with different speeds (speeds based on capillary action) for the solution to move in the flow path 2 of the microstructures 7 of the membrane carrier 3. As a result, even when multiple solutions such as a reaction liquid, a cleaning liquid, and a secondary reaction liquid need to be developed, the timing of developing these solutions can be adjusted according to the usage method. Therefore, the labor such as the original timing adjustment can be omitted, and an appropriate inspection can be stably performed. Specifically, a prescribed jig or the like can be used to drop the solutions simultaneously at multiple positions considering the difference in the timing of developing each solution. That is, each solution can be developed with only one operation.

[0146] <<Second Embodiment>>

[0147] Reference Figure 7 and Figure 8 to explain the inspection kit of the present embodiment. The difference from the first embodiment lies in the structure of the membrane carrier 103. The differences will be mainly explained, and the explanations of the same structures and functions will be appropriately omitted.

[0148] Figure 7 is a schematic top view showing the membrane carrier 103. In Figure 8 shows an enlarged image of the boundary between adjacent regions. Figure 8 The (a) of

[0149] As shown in the figure, the film carrier 103 has a rectangular shape with a specified longitudinal width L10 and a transverse width L20. The film carrier 103 includes a first microstructural region 131 (transverse width L201), a second microstructural region 132 (transverse width L202), a third microstructural region 133 (transverse width L203), and a fourth microstructural region 134 (transverse width L204) from the left side. In these regions, similar to the first embodiment, the density of the convex portions in the microstructure is different, and as a result, the speed based on capillary action is different.

[0150] Specifically, the first microstructural region 131 is set to be the sparsest (region A11), then the third microstructural region 133 is set to be the second sparsest (region A13), the second microstructural region 132 is set to be the third sparsest (region A12), and the fourth microstructural region 134 is set to be the densest (region A14). In addition, a solid phase portion 150 is provided in the fourth microstructural region 134.

[0151] In addition, a buffer region with a specified width L31 is provided at the second boundary 142 between the second microstructural region 132 and the third microstructural region 133. No microstructure (i.e., convex portion) is provided in the buffer region. Similarly, a buffer region with a specified width L32 is also provided at the third boundary 143 between the third microstructural region 133 and the fourth microstructural region 134. By providing such buffer regions, the difference in the transport amount of the solution in each region can be absorbed to prevent the occurrence of backflow and the like. For example, in the second microstructural region 132 and the third microstructural region 133, the microstructure of the downstream third microstructural region 133 is sparser. Therefore, the moving speed of the solution in the second microstructural region 132 is larger. As a result, if there is no buffer region at the second boundary 142, backflow may occur depending on the amount of the developed solution. However, by providing a buffer region that does not generate capillary force as in the present embodiment, the occurrence of backflow caused by the moving speed and the developed amount of the solution can be prevented.

[0152] <<Third Embodiment>>

[0153] In the present embodiment, the anti-backflow structure of the solution is described with six examples. In addition, a cross-sectional view of a partial region corresponding to the structures of the above-mentioned film carriers 3 and 103 is extracted for description here, but it can also be applied to other regions.

[0154] Figure 9 In the film carrier 203 shown in (a), a stepped portion 241 is provided at the boundary between the first microstructural region 231 and the second microstructural region 232 so that the second microstructural region 232 side is lower.

[0155] Figure 9In the film carrier 303 shown in FIG. (b), an inclined portion 341 that decreases as it goes downstream is provided at the boundary between the first microstructural region 331 and the second microstructural region 332. The inclined portion 341 may be a buffer region without protrusions, or may be a micro uneven structure with protrusions.

[0156] In Figure 9 In the film carrier 403 shown in FIG. (c), an inclined portion 441 that decreases as it goes upstream is provided at the boundary between the first microstructural region 431 and the second microstructural region 432. The boundary between the inclined portion 441 and the second microstructural region 432 becomes a stepped portion 442.

[0157] In Figure 9 In the film carrier 503 shown in FIG. (d), a concave portion 541 is provided at the boundary between the first microstructural region 531 and the second microstructural region 532.

[0158] In Figure 9 In the film carrier 603 shown in FIG. (e), the first microstructural region 631 and the third microstructural region 633 are formed horizontally, but the second microstructural region 632 has a slope that decreases more towards the downstream side.

[0159] In Figure 9 In the film carrier 703 shown in FIG. (f), the first microstructural region 731, the second microstructural region 732, and the third microstructural region 733 all have slopes that decrease more towards the downstream side. Here, a structure with the same inclination angle for all is shown, but each region may have a different inclination angle.

[0160] It is possible to appropriately combine the structures of FIGS. (a) to Figure 9 of the above Figure 9 of (f) to set a desired film carrier, and it is possible to achieve an optimal flow path and a solution movement speed corresponding to the type and amount of the developed solution.

[0161] As described above, the embodiments of the present invention have been described with reference to the drawings, but these are examples of the present invention, and various structures (modification examples) other than the above can also be adopted. For example, the flow path 2 is the micro structure (micro uneven structure) of the film carrier 3 on the substrate formed of resin, but as long as it is possible to provide micro structures (micro uneven structures) with different densities according to the region of the flow path 2, various structures and materials can be adopted.

[0162] <Summary of the Embodiment>

[0163] As described above, if the features of the present invention are simply summarized, they are as follows.

[0164] (1) An inspection device (inspection kit), wherein,

[0165] The inspection device (inspection kit) has:

[0166] A flow path provided on a substrate formed of resin for transporting a liquid sample from one end side to the other end side;

[0167] A solid phase part provided on the other end side of the flow path, having an antibody on the solid phase;

[0168] A detection part provided with an electrode part for detecting the reaction of the liquid sample with respect to the antibody; and

[0169] A fine uneven structure having a plurality of convex parts integrally formed on the flow path,

[0170] The fine uneven structure has:

[0171] A first uneven part on which the plurality of convex parts are relatively sparsely provided; and

[0172] A second uneven part on which the plurality of convex parts are relatively densely provided,

[0173] The first uneven part and the second uneven part are provided at a position closer to the one end side of the flow path than the solid phase part.

[0174] (2) A detection device for detecting a substance to be detected in a liquid sample, wherein,

[0175] The detection device has:

[0176] A flow path for transporting the liquid sample from one end side to the other end side;

[0177] A solid phase part provided on the other end side of the flow path, having an antibody on the solid phase;

[0178] A detection part for detecting the reaction of the liquid sample with respect to the antibody; and

[0179] A fine uneven structure having a plurality of convex parts integrally formed on the flow path,

[0180] The fine uneven structure has:

[0181] A first uneven part on which the plurality of convex parts are relatively sparsely provided; and

[0182] A second uneven part on which the plurality of convex parts are relatively densely provided,

[0183] The first uneven part and the second uneven part are provided at a position closer to the one end side of the flow path than the solid phase part.

[0184] (3) Alternatively, the detection part may be provided at a position closer to the other end side of the flow path than the solid phase part.

[0185] (4) Alternatively, the first concavo-convex portion may be provided at a position closer to one end side of the flow path than the second concavo-convex portion.

[0186] (5) Alternatively, the detection device may have a buffer region where the convex portion is not provided at the boundary between the first concavo-convex portion and the second concavo-convex portion.

[0187] (6) Alternatively, a step or a slope may be provided at the boundary between the first concavo-convex portion and the second concavo-convex portion.

[0188] The region of the step or the slope closer to the first concavo-convex portion is higher than the region of the step or the slope closer to the second concavo-convex portion.

[0189] (7) Alternatively, a concave portion region having a concave portion may be provided at the boundary between the first concavo-convex portion and the second concavo-convex portion.

[0190] (8) Alternatively, the convex portion may have a region provided in a rhombic lattice shape.

[0191] (9) Alternatively, the convex portion may have a region provided in a regular lattice shape.

[0192] (10) Alternatively, when the first concavo-convex portion and the second concavo-convex portion are adjacent, the ratio (P1 / P2) of the pitch (P1) between the convex portions in the first concavo-convex portion to the pitch (P2) between the convex portions in the second concavo-convex portion is 1.1 or more and 5 or less.

[0193] (11) Alternatively, the convex portion may be formed as a cone.

[0194] (12) Alternatively, the detection device may further include an introduction portion that introduces the liquid sample into the flow path.

[0195] The liquid sample includes a plurality of solutions.

[0196] The introduction portion is provided at a plurality of positions according to the plurality of solutions.

[0197] (13) Alternatively, the electrode portion is formed on the convex portion of the micro concavo-convex structure, the maximum peak height Rp of the roughness curve of the electrode portion is 0.005 μm or more and 10 μm or less, and the average length RSm of the roughness curve elements is 0.01 μm or more and 15 μm or less.

[0198] (14) Alternatively, the electrode portion has a conductor film layer formed of a conductor material by at least one of sputtering, vacuum evaporation, laser ablation, and CVD on the convex portion of the micro concavo-convex structure.

[0199] (15) Alternatively, the electrode part may have a printed layer of a paste containing particles of a conductor on the convex part of the fine uneven structure.

[0200] (16) Alternatively, the electrode part may have a working electrode and a counter electrode separated from the working electrode.

[0201] The working electrode is disposed at the same position as the counter electrode or at a position upstream of the counter electrode in the flow path direction.

[0202] (17) Alternatively, the counter electrode may be disposed across the entire width direction of the flow path.

[0203] (18) Alternatively, the working electrode may be disposed across the entire width direction of the flow path.

[0204] (19) Alternatively, the working electrode may be configured as a comb-shaped electrode.

[0205] (20) Alternatively, the electrode part may further have a reference electrode.

[0206] (21) The detection method uses the above detection device to detect the reaction of the liquid sample with respect to the antibody.

[0207] Examples

[0208] Hereinafter, Examples (Example 1 and Example 2) will be given to specifically illustrate the present invention, but the present invention is not limited to these Examples. In addition, in the following Examples, an evaluation experiment of the flow rate control of the solution was conducted when the fine structure was configured to have a structure with a plurality of regions having different densities in the membrane carrier.

[0209] [Example 1]

[0210] In this Example, an experiment for quantitatively evaluating the case where the solution is replaced when three-color aqueous solutions are developed on the membrane carrier will be described.

[0211] 1. Experiment

[0212] (1) In order to confirm the technique of controlling the solution development by using the change of the fine structure, for the membrane carrier 3 having the structure shown in the first embodiment, a flow path 2 formed by continuously connecting three regions with the distances between the convex parts 8 in the first fine structure region 31, the second fine structure region 32, and the third fine structure region 33 being 25 μm, 15 μm, and 2 μm respectively was fabricated from polycarbonate (PC-2151 manufactured by Teijin Limited). The manufacturing conditions (hot embossing process) are as described below. Figure 2 The manufacturing conditions (hot embossing process) are as follows.

[0213] <Hot embossing process (transfer of the fine structure)>

[0214] Using the following hot embossing process, the fine structure on the surface of the mold is transferred to the surface of a film-like substrate formed of a thermoplastic plastic. In the hot embossing process, an X-300 manufactured by SCIVAX is used. In the hot embossing process, the surface of the mold having the fine structure (a plurality of recesses) is brought into contact with the film-like substrate formed of a thermoplastic plastic, and while heating the mold and the substrate, pressure is applied. The molding temperature is 180 °C. The applied pressure is 5.5 MPa. The transfer time is 5 minutes. After the transfer of the fine structure, while pressure is applied to the mold and the substrate, the mold and the substrate are cooled to 140 °C. After cooling, the pressure is removed. Using the above hot embossing process, the film carrier of Example 1 is obtained. The film carrier has a surface including a plurality of cones (fine structures) and flat portions. The shape and size of the convex portions (cones) on the surface of the film carrier are identical to the shape and size of the recesses (inverted cones) formed in the mold.

[0215] The convex portion 8 is a conical structure with a diameter of 4 and a height of 6, both being 30 μm.

[0216] The longitudinal width L1 of the film carrier 3 is 5 mm, and the respective transverse widths L21 to L23 of the first fine structure region 31 to the third fine structure region 33 are 20 mm.

[0217] (2) The water-absorbing pad used in Navi-Flu is pasted in such a manner that it overlaps only 5 mm at the end of the region of the third fine structure region 33. Moreover, at the points where the distance between the fine structures changes (the positions corresponding to the first boundary 41 and the second boundary 42 of Figure 2 ), the conjugate pad used in Navi-Flu is fixed to produce a test piece. Figure 10 The photograph of the produced test piece is shown in

[0218] (3) The conjugate pads are numbered "1", "2", and "3" in the order approaching the water-absorbing pad, and an aqueous solution having the composition shown in Table 1 is dropped onto each pad. The amount of the dropped liquid is determined in consideration of the distance from the water-absorbing pad and the amount captured by the intermediate conjugate pads during spreading. In addition, for simplifying the operation and result verification of this experiment, it is dropped onto the pads in the order of "1", "2", and "3" every 10 seconds.

[0219] [Table 1]

[0220] Table 1: Composition and dropping amount of the aqueous solution

[0221]

[0222] (4) The spreading of the solution of each color is video-recorded, and the color change at the midpoint between the dropping portion 1 and the water-absorbing pad is analyzed by image analysis.

[0223] 2. Results

[0224] The captured video is visualized every 10 seconds and imported into an image analysis software (software name "Image J"). The images after 30 seconds, 140 seconds, and 310 seconds from the start of the experiment are shown in Figure 11 . Figure 11 (a) in is the image after 30 seconds, Figure 11 (b) in is the image after 140 seconds, Figure 11 (c) in is the image after 310 seconds.

[0225] Determine the measurement point at the midpoint between the dripping site 1 and the absorbent pad, and record the RGB display data at this point. Then, calculate the component ratio of each RGB color according to Equation 1. As an example, the conversion data of the aqueous solutions of red (R), green (G), and blue (B) are shown in Table 2.

[0226] (Component ratio of R or G or B) = (Value of R or G or B) / (Value of R + Value of G + Value of B)

[0227] … Equation 1

[0228] [Table 2]

[0229] Table 2: Conversion from RGB values to component ratios

[0230]

[0231] In Figure 12 , a graph showing the elapsed time and the RGB component ratios of the measurement points is shown. According to Figure 12 the results shown, it can be confirmed that the numerical values of the situation where it gradually changes from green → red → blue over time can be obtained.

[0232] Moreover, in order to quantitatively evaluate the inspection results and the flow situation, as a method for quantitatively evaluating the mixing ratio, the following Equation 2 was studied. This Equation 2 is an equation for obtaining to what extent the component ratios of the single colors in Table 2 should be mixed to achieve the RGB component ratios at each measurement point. In order to minimize the error ε between the measured value and the calculated value, the solver function of the spreadsheet software Excel was used.

[0233] ε = |R_r x + G_r y + B_r z - r| + |R_g x + G_g y + B_g z - g| + |R_b x + G_b y + B_b z - b|

[0234] … Equation 2

[0235] Find x, y, and z that minimize ε expressed by Equation 2.

[0236] Here, R_r, R_g, and R_b respectively represent the R, G, and B component ratios of the red solution. G and B have the same meaning for the green solution and the blue solution respectively. r, g, and b are the measured values of the R, G, and B component ratios at the measurement point. x, y, and z are the mixing ratios of the red, green, and blue solutions at the measurement point, which are restricted in the form of x + y + z = 1.

[0237] The result obtained by analyzing the Figure 12 data using Equation 2 can represent the mixing ratio of each solution as Figure 13 such.

[0238] Based on the above results, the time change of the mixing ratio of the solution is quantitatively evaluated using image analysis. That is, it can be confirmed that by appropriately setting the density state (interval of convex portions) of the fine structure of each region of the membrane carrier, the moving speed of the solution in the flow path of the membrane carrier can be adjusted, and multiple solutions can be developed.

[0239] The results (comparative example) of conducting the same test and evaluation using nitrocellulose instead of the imprinting sheet are shown in Figure 14 . In the case of the imprinting sheet, since the structure (convex portion) is conical, the solution in the flow path can be observed from directly above. However, since nitrocellulose is in the form of a non-woven fabric, only the color change on the outermost surface can be observed. Pay attention to this point when examining the results.

[0240] Compared with Figure 13 (Example of the imprinting sheet), it can be seen that the test time is significantly different. This shows the difference in the developing flow rate between the imprinting sheet and nitrocellulose, and it can be confirmed that for quickly replacing the developing solution, the imprinting sheet with a larger flow rate is advantageous. By adjusting the total length of the nitrocellulose, the shortening of the test time can be achieved, but in this case, the risk of mixing between the simultaneously dropped solutions increases. In an actual test where the total length and the dropped liquid volume are halved, the solutions are mixed. By designing the structure, it may be possible to fabricate a device with a short judgment time and no solution mixing, but due to the inability to adjust the flow rate, etc., the design freedom is low. Regarding this point, when the membrane carrier is composed of the imprinting sheet as shown in Example 1, the freedom in adjusting the flow rate, etc., is high.

[0241] 3. Summary

[0242] According to Example 1, in the case of the membrane carrier of the imprinting sheet, the flow rate can be controlled by adjusting the fine structure and the material, and it can flexibly meet the market demands.

[0243] [Example 2]

[0244] 1. Experiment

[0245] (1) In order to confirm the technique of controlling the spreading of a solution through changes in the microstructures, for the membrane carrier 103 with the structure shown in Figure 7 , a flow path with four regions was fabricated from polycarbonate (PC-2151 manufactured by Teijin Limited) such that the vertex-to-vertex distances of the protrusions 8 in the first microstructural region 131, the second microstructural region 132, the third microstructural region 133, and the fourth microstructural region 134 were 105 μm, 60 μm, 80 μm, and 30 μm, respectively. The fabrication conditions were the same as those in Example 1. In Figure 15 , (a) shows a schematic view of the flow path observed from the side, and in Figure 15 , (b) shows a top view (photo) of the actually fabricated test piece.

[0246] The protrusion 8 has a conical structure with a diameter of 4 and a height of 6, both being 32 μm.

[0247] The longitudinal width L1 of the membrane carrier 103 is 5 mm, the transverse width L201 of the first microstructural region 131 is 15 mm, the transverse width L202 of the second microstructural region 132 is 30 mm, the transverse width L203 of the third microstructural region 133 is 45 mm, and the transverse width L204 of the fourth microstructural region 134 is 40 mm.

[0248] When viewed from the side, the second microstructural region 132, the third microstructural region 133, and the fourth microstructural region 134 are inclined at an inclination angle of 2.2°.

[0249] The first boundary 141 is the boundary where the first microstructural region 131 without a buffer region is continuous with the second microstructural region 132.

[0250] The width L31 of the buffer region (unprocessed region) of the second boundary 142 is 0.15 mm.

[0251] The width L32 of the buffer region (unprocessed region) of the third boundary 143 is 0.20 mm.

[0252] (2) Experiment 1 (RGB image analysis):

[0253] In Experiment 1, using the same experimental and analysis methods as in Example 1, the color change of a specified point was analyzed according to the RGB components to quantitatively confirm the liquid replacement situation.

[0254] Specifically, the color change of a point 5 mm away from the most downstream (the right end of the fourth microstructural region 134 shown in the figure) was analyzed according to the RGB components. That is, based on the RGB component ratios of each solution, the mixing ratio of the solutions during spreading was evaluated. Figure 16 is a chart showing the evaluation results. This chart corresponds to that of Example 1 in Figure 13, it is possible to confirm that as time passes, a relatively large proportion of the components gradually change into green component (G), red component (R), and blue component (B).

[0255] (3) Experiment 2 (CRP detection performance evaluation):

[0256] In this experiment, as Figure 15 shown in (a) of

[0257] , as the solution developing process, the following process is carried out: 10 μL of cleaning solution is dropped onto the fourth microstructural region 134; 10 seconds later, 10 μL of CRP solution is dropped onto the third microstructural region 133; 1 minute later, 15 μL of fluorescent labeling solution is dropped onto the second microstructural region 132; finally, 30 μL of cleaning solution is dropped onto the first microstructural region 131 after 2 minutes. The fluorescence intensity is measured 10 minutes after dropping the last solution (cleaning solution).

[0258] Cleaning solution: PBS added with 2 wt% Triton X-100;

[0259] CRP solution: A solution formed by mixing the cleaning solution and the CRP solution at a specified concentration;

[0260] Fluorescent labeling solution: A solution formed by mixing the cleaning solution and the fluorescent-labeled anti-CRP antibody solution such that the antibody concentration becomes 30 μg / mL.

[0261] In Figure 17 , the phosphor intensity for each CRP concentration is shown. In addition, in Figure 18 , the image at the time of observing the fluorescence intensity is shown. Figure 18 (a) of Figure 18 is the graph when the CRP concentration is 0 ng / mL, Figure 17 and Figure 18 (b) of

[0262] [Example 3]

[0263] 1. Experiment

[0264] (1) Test piece (membrane carrier 103)

[0265] In Example 3, for the purpose of making the test system closer to the actual use state, part of the test conditions of Experiment 2 in Example 2 were changed, and a CRP detection test in which human serum was mixed in the sample solution was carried out.

[0266] For the structure of the test piece (membrane carrier 103) corresponding to Figure 7 a flow path having four regions was fabricated from polycarbonate (PC-2151 manufactured by Teijin Limited) such that the vertex-to-vertex distances of the convex portions 8 in the first microstructural region 131, the second microstructural region 132, the third microstructural region 133, and the fourth microstructural region 134 were 100 μm, 60 μm, 95 μm, and 30 μm, respectively. The fabrication conditions were the same as those in Example 1 and Example 2.

[0267] In Figure 21 a schematic view of the test piece (flow path) observed from the side is shown. As the test piece, 36 samples were prepared. The convex portion 8 is a conical structure having a diameter of 4 and a height of 6, both of 32 μm.

[0268] The longitudinal width L1 of the membrane carrier 103 is 5 mm, the transverse width L201 of the first microstructural region 131 is 36.95 mm, the transverse width L202 of the second microstructural region 132 is 5 mm, the transverse width L203 of the third microstructural region 133 is 40 mm, and the transverse width L204 of the fourth microstructural region 134 is 40 mm.

[0269] Slopes inclined at an inclination angle of 2.1° in a side view were given to the second microstructural region 132, the third microstructural region 133, and the fourth microstructural region 134.

[0270] The first boundary 141 is the boundary where the first microstructural region 131 without a buffer region and the second microstructural region 132 are continuous.

[0271] The width L31 of the buffer region (unprocessed region) of the second boundary 142 is 0.15 mm.

[0272] The width L32 of the buffer region (unprocessed region) of the third boundary 143 is 0.15 mm.

[0273] (2) Antibody solid phase

[0274] 1 μL of an anti-CRP antibody solid phase solution was dropped at a position 17.5 mm from the most downstream end of the test piece and dried for 1 hour in an environment of 45°C, thereby immobilizing 25 ng of the anti-CRP antibody.

[0275] As Figure 21As shown, a 2.1° slope is set in the flow path, and the development is carried out in the order of CRP solution, fluorescently labeled anti-CRP antibody solution (in the developing solution, the concentration of the fluorescently labeled anti-CRP antibody is 45 μg / mL), and developing solution (PBS containing 2 wt% Triton X-100) from different dropping sites. The composition of the CRP solution used is shown in Table 3. The volume of the developing solution and the interval of solution dropping are shown in Table 4. In addition, each test is carried out under the condition of n = 3.

[0276] After developing all the solutions for 10 minutes, the blotting pad is separated from the imprinting sheet (membrane carrier 103) to prevent the backflow of the solution, and then the fluorescence intensity of the antibody solid phase part is measured.

[0277] 2. Results

[0278] The fluorescence intensity measurement results of each test are shown in Figure 22 . The fluorescence intensity shown here is the value obtained by subtracting the background fluorescence intensity around it from the fluorescence intensity of the antibody solid phase part. In addition, in Figure 22 (a), the exposure time during fluorescence intensity measurement is set to 1 second, and in Figure 22 (b), the exposure time during fluorescence intensity measurement is set to 1 / 6 second.

[0279] Regardless of the presence or absence of serum, the same minimum detection sensitivity and a measurement range of three digits or more (detection range) can be achieved.

[0280] In addition, due to the presence of serum, there is a tendency for the fluorescence intensity to decrease. It is speculated that the protein in the serum (~80 mg / mL) inhibits the reaction between the antibody and CRP.

[0281] [Table 3]

[0282] Table 3: Composition of CRP Solution

[0283]

[0284] [Table 4]

[0285] Table 4: Test Scheme

[0286]

[0287] [Example 4]

[0288] In Example 4, based on the results of Examples 1 to 3, an electrochemical detection test on the imprinting sheet is carried out. In addition, in this example, from the viewpoint of confirming whether electrochemical detection can be appropriately carried out, all the solutions are dropped to the same position in the fourth microstructural region 134 in a manner of changing the dropping time.

[0289] 1. Experiment

[0290] (1) Test piece (membrane carrier 103)

[0291] A membrane carrier 103 having the same structure as the test piece prepared in Example 3 was prepared. The vertical width L1 of the membrane carrier 103 was 5 mm, the horizontal width L201 of the first microstructure region 131 was 36.95 mm, the horizontal width L202 of the second microstructure region 132 was 5 mm, the horizontal width L203 of the third microstructure region 133 was 40 mm, and the horizontal width L204 of the fourth microstructure region 134 was 40 mm.

[0292] (2) Electrode part (working electrode, counter electrode)

[0293] An imprint sheet was attached to a substrate formed by attaching a polyimide tape to a SUS plate, and gold was vacuum-deposited through a mask processed into an electrode shape to form the electrode portion 20. The electrode shape is as follows:

[0294] Working electrode: 1mm×5mm (flow path width);

[0295] Counter electrode: 3mm×5mm (flow path width);

[0296] Gap between electrodes: 0.5mm;

[0297] Electrode position: The downstream end of the counter electrode was located 15.5 mm from the most downstream end of the flow path.

[0298] (3) Antibody solid phase

[0299] At a position 5 mm upstream from the working electrode, 1 μL of the anti-CRP antibody solid phase solution was dropped in the same manner as in Example 3, and dried at 45° C. for 1 hour to solidify 25 ng of the anti-CRP antibody.

[0300] (4) Measuring device

[0301] The conduction between the electrode part 20 (working electrode, counter electrode) and the substrate was obtained using silver paste (DOTITE D-550), and the substrate was clamped with an alligator clip of an electrochemical measuring device (Solartron 1252A) and measured. A potential of +50mV was applied between the working electrode and the counter electrode to develop the solution, and the current value was marked with time (refer to Figure 24 ).

[0302] (5) Solution addition time and solution addition

[0303] Figure 23 This is a flow chart showing the timing of solution dropwise addition in Example 4.

[0304] First, 10 μL of cleaning solution is added (the first step S11). Two minutes after the first step S11, 10 μL of the CRP and ALP-labeled CRP mixed solution is added (the second step S12). Further, two minutes after the second step S12, 10 μL of the cleaning solution (4%) is added (the third step S13). Finally, three minutes after the third step S13, 10 μL of the sodium 4-aminophenyl phosphate solution is added (the fourth step S14).

[0305] The added solutions are as follows:

[0306] Cleaning solution: PBS containing 2 wt% of Triton X-100;

[0307] ALP-labeled CRP: Used for labeling commercially available CRP with a labeling kit (LK13 manufactured by Dojindo Laboratories);

[0308] CRP and ALP-labeled CRP mixed solution: CRP and ALP-labeled CRP are suspended in the cleaning solution to a specified concentration;

[0309] Cleaning solution (4%): PBS containing 4 wt% of Triton X-100;

[0310] Sodium 4-aminophenyl phosphate solution: Sodium 4-aminophenyl phosphate is dissolved in the cleaning solution to a concentration of 5 mM.

[0311] 2. Results

[0312] The measurement results are as Figure 24 shown. Figure 24 (a) is the result when the CRP concentration of the CRP and ALP-labeled CRP mixed solution is set to 0 μg / mL and the ALP-CRP concentration is set to 1.25 μg / mL, Figure 24 (b) is the result when the CRP concentration of the above mixed solution is set to 12.5 μg / mL and the ALP-CRP concentration is set to 1.25 μg / mL. As shown in the figure, current values reflecting the dropping time of the solution and the CRP concentration are detected.

[0313] This application claims priority based on Japanese Patent Application No. 2020-079367 filed on April 28, 2020, and incorporates the entire contents disclosed therein into this specification.

[0314] Explanation of reference numerals

[0315] 2. Flow path; 3, 103, 203, 303, 403, 503, 603, 703, Membrane carrier; 3x, Cleaning liquid area; 3y, Detection area; 3z, Dropping area; 8, Protrusion; 18, Test kit; 18a, Housing; 18b, First opening; 18c, Second opening; 18d, Third opening; 20, Electrode part; 21, Measuring device; 25, Working electrode; 26, Counter electrode; 27, Reference electrode; 31, 131, 231, 331, 431, 531, 631, 731, First microstructural region; 32, 132, 232, 332, 432, 532, 632, 732, Second microstructural region; 33, 133, Third microstructural region; 134, Fourth microstructural region; 41, 141, 241, First boundary; 42, 142, Second boundary; 143, Third boundary; 50, 150, Solid phase part; 51, Antibody; 341, 441, Inclined part; 541, Recess.

Claims

1. A detection device, wherein, the detection device has: a flow path provided on a substrate formed of resin, for transporting a liquid sample from one end side to the other end side; a solid phase part provided on the other end side of the flow path, having an antibody immobilized thereon; a detection part provided with an electrode part, for detecting the reaction of the liquid sample with respect to the antibody; and a fine concavo-convex structure having a plurality of convex parts integrally formed on the flow path, the fine concavo-convex structure has: a first concavo-convex part, on which the plurality of convex parts are relatively sparsely provided; and a second concavo-convex part, on which the plurality of convex parts are relatively densely provided, the first concavo-convex part and the second concavo-convex part are provided at a position closer to the one end side of the flow path than the solid phase part, the detection device further has an introduction part for introducing the liquid sample into the flow path, the liquid sample includes a plurality of solutions, the introduction part is provided at a plurality of positions according to the plurality of solutions, the detection part is provided at a position closer to the other end side of the flow path than the solid phase part, when the first concavo-convex part and the second concavo-convex part are adjacent to each other, the ratio (P1 / P2) of the pitch (P1) between the convex parts in the first concavo-convex part to the pitch (P2) between the convex parts in the second concavo-convex part is 1.1 or more and 5 or less.

2. A detection device for detecting a substance to be detected in a liquid sample, wherein, the detection device has: a flow path for transporting the liquid sample from one end side to the other end side; a solid phase part provided on the other end side of the flow path, having an antibody immobilized thereon; a detection part provided with an electrode part, for detecting the reaction of the liquid sample with respect to the antibody; and a fine concavo-convex structure having a plurality of convex parts integrally formed on the flow path, the fine concavo-convex structure has: a first concavo-convex part, on which the plurality of convex parts are relatively sparsely provided; and a second concavo-convex part, on which the plurality of convex parts are relatively densely provided, the first concavo-convex part and the second concavo-convex part are provided at a position closer to the one end side of the flow path than the solid phase part, the detection part is provided at a position closer to the other end side of the flow path than the solid phase part, when the first concavo-convex part and the second concavo-convex part are adjacent to each other, the ratio (P1 / P2) of the pitch (P1) between the convex parts in the first concavo-convex part to the pitch (P2) between the convex parts in the second concavo-convex part is 1.1 or more and 5 or less, the detection device further has an introduction part for introducing the liquid sample into the flow path, the liquid sample includes a plurality of solutions, the introduction part is provided at a plurality of positions according to the plurality of solutions.

3. The detection device according to claim 1 or 2, wherein, the first concavo-convex part is provided at a position closer to the one end side of the flow path than the second concavo-convex part.

4. The detection device according to claim 1 or 2, wherein, the detection device has a buffer region where no convex part is provided at the boundary between the first concavo-convex part and the second concavo-convex part.

5. The detection device according to claim 1 or 2, wherein, a step or a slope is provided at the boundary between the first concavo-convex part and the second concavo-convex part. The area of the step or the slope on the side of the first uneven portion is higher than the area of the step or the slope on the side of the second uneven portion.

6. The detection device according to claim 1 or 2, wherein A recessed area with a recess is provided at the boundary between the first uneven portion and the second uneven portion.

7. The detection device according to claim 1 or 2, wherein The convex portion has a region formed in a diamond lattice pattern.

8. The detection device according to claim 1 or 2, wherein The convex portion has a region formed in a square lattice pattern.

9. The detection device according to claim 1 or 2, wherein The convex portion is formed as a cone.

10. The detection device according to claim 1 or 2, wherein The electrode portion is formed on the convex portion of the fine uneven structure. The maximum peak height Rp of the roughness curve of the electrode portion is 0.005 μm or more and 10 μm or less, and the average length RSm of the roughness curve elements is 0.01 μm or more and 15 μm or less.

11. The detection device according to claim 1 or 2, wherein The electrode portion has a conductor film layer formed of a conductor substance by at least one of sputtering, vacuum evaporation, laser ablation, and CVD on the convex portion of the fine uneven structure.

12. The detection device according to claim 1 or 2, wherein The electrode portion has a printed layer of a paste containing conductor particles on the convex portion of the fine uneven structure.

13. The detection device according to claim 1 or 2, wherein The electrode portion has a working electrode and a counter electrode separated from the working electrode. The working electrode is provided at the same position as the counter electrode or at a position upstream of the counter electrode in the flow path direction.

14. The detection device according to claim 13, wherein The counter electrode is provided across the entire width direction of the flow path.

15. The detection device according to claim 13, wherein The working electrode is provided across the entire width direction of the flow path.

16. The detection device according to claim 13, wherein The working electrode is configured as a comb-shaped electrode.

17. The detection device according to claim 13, wherein The electrode portion further has a reference electrode.

18. A detection method, wherein This detection method uses the detection device according to any one of claims 1 to 17 to detect the reaction of a liquid sample with respect to an antibody.

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