Patent Publication Number: US-2021181194-A1

Title: Membrane carrier for liquid sample test kit, liquid sample test kit, method of manufacturing liquid sample test kit, test method of liquid sample, and membrane carrier

Description:
TECHNICAL FIELD 
     The present invention relates to a membrane carrier for a liquid sample test kit, a liquid sample test kit, a method of manufacturing a liquid sample test kit, a test method of a liquid sample, and a membrane carrier. 
     BACKGROUND ART 
     In recent years, a Point of Care Test (POCT) reagent, which measures an affliction of an infectious disease, pregnancy, a blood glucose level, and the like by using an antigen-antibody reaction and the like, has attracted attention. The POCT reagent has characteristics that a result can be determined in a short time, a use method is simple, and the cost is low. With these characteristics, the POCT reagent is frequently used for medical examinations and periodic medical examinations at a stage where the symptoms are mild, and is an important medical examination tool in home medical care, which is expected to increase in the future. 
     In many POCT reagents, a determination is made by introducing a liquid sample such as blood into a test kit and detecting a specific substance to be detected contained therein. As a method for detecting a specific substance to be detected from the liquid sample, an immunochromatography method is often used. An immunochromatography method refers to a technique in which a substance to be detected and a label are bound while a liquid dropped on a membrane carrier of a test kit moves on the membrane carrier, these specifically bind to a substance (hereinafter, referred to as detection substance) immobilized in the test kit, and a change in color or mass generated as a result is detected. The detection substance may be referred to as a reagent. 
     As a technique of detecting a substance to be detected, a method of detecting a change in color caused by using colored latex particles, fluorescent latex particles, metal colloidal particles, and the like as a label via an optical measurement device such as an absorbance measurement device is well known. 
     As a POCT reagent for optically determining the change in color, a lateral flow type kit using a nitrocellulose membrane is often used (Patent Document 1). The nitrocellulose membrane has a large number of fine holes having a diameter of about several μm, and the liquid sample moves in the holes by capillary force. 
     However, since the nitrocellulose membrane is derived from a natural product and a pore diameter and a way of connecting the pores are not uniform, there occurs a difference in a flow rate of the liquid sample flowing in each membrane. Patent Document 2 discloses a technique of controlling the flow rate. However, Patent Document 2 has a flow path which is porous body. The present invention has a flow path having a microstructure having a convex portion, and is different from Patent Document 2. Patent Document 2 uses a nitrocellulose membrane, and thus had a problem that the pore diameter and the way of connecting the pores are not uniform. In a case where a difference occurs in the flow rates, the time required to detect a substance to be detected is also changed, and as a result, the substance to be detected may be erroneously determined as non-detection before binding occurs. 
     In order to solve the above-mentioned problem, a technique of artificially preparing a fine flow path has been devised (Patent Documents 3 to 7). By using this technique, it is possible to prepare a membrane carrier having a uniform structure. Therefore, it is possible to decrease a possibility of erroneously determining a substance to be detected as non-detection before binding occurs. 
     In the above patent documents, the flow path structure in the system is uniform, and thus there is no limit in detection performance. Patent Document 8 discloses a technique for improving the detection performance at a time of using an artificial fine flow path, in which a groove-shaped flow path for the purpose of flow rate control is combined with a pillar-shaped flow path for the purpose of improving sensitivity. 
     RELATED DOCUMENT 
     Patent Document 
     [Patent Document 1] Japanese Unexamined Patent Publication No. 2014-062820 
     [Patent Document 2] International Publication No. WO 2016/051974 
     [Patent Document 3] Japanese Patent No. 4597664 
     [Patent Document 4] PCT Japanese Translation Patent Publication No. 2012-524894 
     [Patent Document 5] Japanese Patent No. 5609648 
     [Patent Document 6] Japanese Unexamined Patent Publication No. 2016-011943 
     [Patent Document 7] Japanese Unexamined Patent Publication No. 2013-113633 
     [Patent Document 8] Japanese Patent No. 5821430 
     [Patent Document 9] International Publication No. WO 2016/098740 
     SUMMARY OF THE INVENTION 
     Technical Problem 
     However, in the techniques disclosed in Patent Documents 1 to 8, attention is focused only on the detection substance and not on the flow of the substance to be detected or the flow of the label. In a system using an artificial fine flow path, the flow easily becomes a simple laminar flow. As a result, it becomes difficult to sufficiently stir the substance to be detected and the label, and this becomes a factor of deteriorating the detection performance. In particular, in a lateral flow type immunochromatography method, the detection system is simple, and the influence of the flow path structure is likely to be reflected in the test result. 
     Patent Document 9 discloses a membrane carrier for a liquid sample test kit that detects a substance to be detected in a liquid sample, in which at least one flow path capable of transporting the liquid sample is provided, and the microstructure that generates a capillary action for transporting the liquid sample is provided on a bottom surface of the flow path. However, Patent Document 9 does not disclose a level difference. 
     In view of the above problems, an object of the present invention is to provide a test kit capable of highly sensitive determination in an immunochromatography method capable of confirming that a substance to be detected has been detected by an optical technique, for example. 
     That is, the present invention is as follows. 
     (1) A membrane carrier for a liquid sample test kit that detects a substance to be detected in a liquid sample, the membrane carrier including: 
     at least one integrally-molded flow path capable of transporting the liquid sample, in which a microstructure that causes a capillary action for transporting the liquid sample is provided on a bottom surface of the flow path, in which at least one level difference, at which a height level of the bottom surface changes, is provided in the flow path, and in which the level difference is provided such that the height level of the bottom surface on a downstream side is higher than that on an upstream side in the transporting direction of the liquid sample. 
     (2) The membrane carrier for a liquid sample test kit according to (1), in which the microstructure has any one of a cone, a pyramid, a truncated cone, a truncated pyramid, a cylinder, a polygonal prism, a hemisphere, and a semi-ellipsoid. 
     (3) The membrane carrier for a liquid sample test kit according to (1) or (2), in which an amount of change in the height level of the bottom surface at the level difference is equal to or less than twice the height of the microstructure on the upstream side of the level difference. 
     (4) The membrane carrier for a liquid sample test kit according to any one of (1) to (3), in which an inclination is provided on the downstream side of the level difference in the flow path such that the height level of the bottom surface approaches the height level on the upstream side of the level difference. 
     (5) The membrane carrier for a liquid sample test kit according to any one of (1) to (4), in which the microstructure changes between the upstream side and the downstream side of the level difference with the level difference as a boundary. 
     (6) The membrane carrier fora liquid sample test kit according to any one of (1) to (5), in which the height of the microstructure on the downstream side is smaller than that on the upstream side with the level difference as a boundary. 
     (7) The membrane carrier fora liquid sample test kit according to any one of (1) to (6), in which a height of the microstructure is equal to or more than 10 μm and equal to or less than 500 μm in the flow path. 
     (8) A liquid sample test kit that detects a substance to be detected in a liquid sample, the liquid sample test kit including: 
     the membrane carrier for a liquid sample test kit according to any one of (1) to (7), in which the membrane carrier includes a detection zone that detects the substance to be detected in the liquid sample, and in which in the detection zone, a change in color occurs at a time when the substance to be detected is detected. 
     (9) The liquid sample test kit according to (8), in which the detection zone is provided on an inclined portion in the flow path. 
     (10) The liquid sample test kit according to (8) or (9), in which a label having an antibody specifically reacting with the substance to be detected in the liquid sample or an antigen-binding fragment thereof is provided in at least a part of the liquid sample test kit so as to react with the substance to be detected, and in which the change in color occurs due to the label that binds to the substance to be detected. 
     (11) The liquid sample test kit according to (10), in which the label is a particle in which the antibody or the antigen-binding fragment is bound to a colored latex particle or a fluorescent latex particle. 
     (12) The liquid sample test kit according to (10) or (11), in which a detection substance that detects the substance to be detected is immobilized in the detection zone, and the change in color occurs by the label being held in the detection zone by the detection substance and being colored. 
     (13) A method of manufacturing the liquid sample test kit according to any one of (8) to (12), the method including: immobilizing a detection substance in the detection zone that causes the change in color by holding the substance to be detected in the detection zone. 
     (14) A test method of a liquid sample using the liquid sample test kit according to any one of (8) to (12), the test method including: mixing the liquid sample with a label specifically binding to the substance to be detected in the liquid sample to prepare a mixture liquid sample, and binding the substance to be detected and the label to each other; dropping the mixture liquid sample in a dropping zone provided on the membrane carrier; transporting the mixture liquid sample from the dropping zone to the detection zone by the microstructure; and detecting a change in color in the detection zone. 
     (15) A membrane carrier for detecting a substance to be detected in a liquid sample, including: at least one flow path, in which a microstructure is provided on a bottom surface of the flow path, in which at least one level difference is provided in the flow path, and in which the level difference is provided such that a height level of the bottom surface on a downstream side is higher than that on an upstream side in a transporting direction of the liquid sample. 
     According to the present invention, in an immunochromatography method capable of confirming that a substance to be detected has been detected by an optical technique, it is possible to provide a test kit capable of highly sensitive determination. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The object, other objects, characteristics, and advantages will be further apparent from preferable embodiments to be described later and the following drawings accompanying thereof. 
         FIG. 1  is an example of an embodiment according to the present invention, and is a schematic top view of a test kit. 
         FIG. 2  is an example of an embodiment according to the present invention, and is a schematic top view of a membrane carrier. 
         FIG. 3( a )  is an example of an embodiment according to the present invention, and is a bird&#39;s-eye view (top view) of a microstructure, and  FIG. 3( b )  is a perspective view of a convex portion constituting the microstructure illustrated in  FIG. 3( a ) . 
         FIG. 4( a )  is an example of an embodiment according to the present invention, and is a bird&#39;s-eye view (top view) of the microstructure, and  FIG. 4( b )  is a perspective view of a convex portion constituting the microstructure illustrated in  FIG. 4( a ) . 
         FIG. 5( a )  is an example of an embodiment according to the present invention, and is a bird&#39;s-eye view (top view) of the microstructure, and  FIG. 5( b )  is a perspective view of a convex portion constituting the microstructure illustrated in  FIG. 5( a ) . 
         FIG. 6( a )  is an example of an embodiment according to the present invention, and is a bird&#39;s-eye view (top view) of the microstructure, and  FIG. 6( b )  is a perspective view of a convex portion constituting the microstructure illustrated in  FIG. 6( b ) . 
         FIG. 7  is an example of an embodiment according to the present invention, and is a sectional view of a membrane carrier having the microstructure. 
         FIG. 8  is an example of an embodiment according to the present invention, and is a sectional view of a level difference provided in a flow path. 
         FIG. 9  is an example of an embodiment according to the present invention, and is a sectional view of a level difference provided in a flow path. 
         FIG. 10  is an example of an embodiment according to the present invention, and is a schematic top view of a membrane carrier. 
         FIG. 11  is an example of an embodiment according to the present invention, and is a schematic view of a mold for forming a microstructure. 
         FIG. 12  is an example of an embodiment of the present invention, and is a schematic view (top view, sectional view) of a mold for forming a microstructure. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all the drawings, like components are denoted by common reference numerals, and description thereof will not be repeated. In addition, the drawing is a schematic view, and does not match the actual dimensional ratio. 
     The membrane carrier for a liquid sample test kit of the present embodiment refers to a membrane carrier for a liquid sample test kit that detects a substance to be detected in a liquid sample, for example. 
     Here, the substance to be detected is not limited at all, and may be any substance that can have an antigen-antibody reaction with an antibody, such as various pathogens and various clinical labels. Specific examples of the substance to be detected include virus antigens such as influenza virus, norovirus, adenovirus, RS virus, HAV, HBs, and HIV, bacterial antigens such as MRSA, group A  streptococcus , group B  streptococcus , and genus  Legionella  bacteria, toxins produced by viruses, hormones such as  mycoplasma, Chlamydia trachomatis , and human chorionic gonadotropin, C-reactive protein, myoglobin, cardiac troponin, various tumor labels, pesticides, and environmental hormones, but are not limited thereto. In particular, in a case where the substance to be detected is a substance that is urgently required in detection and therapeutic measures such as influenza virus, norovirus, C-reactive protein, myoglobin, and cardiac troponin, the liquid sample test kit and the membrane carrier according to the present embodiment are greatly useful. The substance to be detected may be an antigen capable of inducing an immune reaction alone, or may not be capable of inducing an immune reaction alone. However, the substance to be detected may be a hapten capable of inducing an immune reaction in a case being bound by an antigen-antibody reaction with an antibody. The substance to be detected is usually in a state of being floated or dissolved in the liquid sample. The liquid sample may be a sample in which the substance to be detected is floated or dissolved in a buffer solution, for example. 
     The liquid sample test kit (hereinafter, also referred to as test kit) according to the present embodiment detects a substance to be detected in a liquid sample.  FIG. 1  is a schematic top view of the test kit. For example, as illustrated in  FIG. 1 , the test kit  18  includes a membrane carrier  3  and a housing  18   a  that houses the membrane carrier  3 . The membrane carrier  3  has, on a surface thereof, a dropping zone  3   x  where a liquid sample is dropped, and a detection zone  3   y  for detecting the substance to be detected in the liquid sample. The dropping zone  3   x  is exposed at a first opening  18   b  of the housing  18   a . The detection zone  3   y  is exposed at a second opening  18   c  of the housing  18   a.    
       FIG. 2  is a schematic top view of the membrane carrier  3 . As illustrated in  FIG. 2 , the membrane carrier  3  includes at least one flow path  2  that transports a liquid sample. The flow path  2  is preferably provided by integral molding. A microstructure is provided on the bottom surface of the flow path  2  (not illustrated, details will be described later). The microstructure is positioned at least between the dropping zone  3   x  and the detection zone  3   y . The microstructure may be provided over the entire surface of the membrane carrier  3 . The entire surface of the membrane carrier  3  may be the flow path  2  of the liquid sample. The microstructure causes capillary action. Due to the capillary action of the microstructure, the liquid sample is transported to the detection zone  3   y  (along a transporting direction d) from the dropping zone  3   x  via the microstructure. In a case where the substance to be detected in the liquid sample is detected in the detection zone  3   y , color of the detection zone  3   y  changes. 
     The entire shape of the membrane carrier  3  is not particularly limited, but may be, for example, a polygon such as a square, a circle, or an ellipse. In a case where the membrane carrier  3  is a square, a vertical width (length in a short-side direction) L 1  of the membrane carrier  3  may be equal to or more than 2 mm and equal to or less than 100 mm, for example, and a horizontal width (length in a long-side direction) L 2  of the membrane carrier  3  may be equal to or more than 2 mm and equal to or less than 100 mm, for example. A thickness of the membrane carrier excluding a height of the microstructure may be equal to or more than 0.1 mm and equal to or less than 10 mm, for example. 
     The microstructure is preferably formed of any one of a cone, a pyramid, a truncated cone, a truncated pyramid, a cylinder, a polygonal prism, a hemisphere, and a semi-ellipsoid.  FIGS. 3( a ) to 6( b )  each illustrate an example of a microstructure provided on the bottom surface of the flow path and a convex portion constituting the microstructure in the present embodiment. In  FIGS. 3( a ) to 6( b ) ,  FIGS. 3( a ), 4( a ), 5( a ), and 6( a )  each are a bird&#39;s-eye view (top view) of the microstructure, and  FIGS. 3( b ), 4( b ), 5( b ), and 6( b )  each are a perspective view of a convex portion constituting the microstructure illustrated in  FIGS. 3( a ), 4( a ), 5( a ), and 6( a ) . As illustrated in  FIGS. 3( a ) to 6( b ) , a microstructure  7  is an entire body (aggregate) of a convex portion  8 . That is, the microstructure  7  includes a flat portion  9  corresponding to the bottom surface of the flow path  2  of the liquid sample, and a plurality of convex portions  8  protruding from the flat portion  9 . Due to the capillary action, a space between the plurality of convex portions  8  functions as the flow path  2  for transporting the liquid sample along a surface of the membrane carrier  3 . In other words, a void in the microstructure  7  functions as the flow path  2  for transporting the liquid sample along the surface of the membrane carrier  3  by the capillary action. The plurality of convex portions  8  may be regularly or translationally arranged in line on the surface of the membrane carrier  3 . 
     For example, as illustrated in  FIG. 3 , the shape of the convex portion  8   a  may be a cone. For example, as illustrated in  FIG. 4 , the shape of the convex portion  8   b  may be a quadrangular pyramid. For example, as illustrated in  FIG. 5 , the shape of the convex portion  8   c  may be a hexagonal pyramid. For example, as illustrated in  FIG. 6 , the shape of the convex portion  8   d  may be a horizontally disposed triangular prism (a triangular prism arranged such that one side surface (a square surface) of the triangular prism is in contact with the flat portion  9 ). From a viewpoint that the entire surface of the membrane carrier  3  can be visually recognized at a time when the microstructure  7  is viewed from a bird&#39;s-eye view (viewed from above), and a change in color at a time when the substance to be detected has been detected is easily confirmed by an optical technique, among these, a conical structure such as a cone or a pyramid is suitable as the shape of the convex portion  8 . In the conical structure, a cone is suitable as the shape of the convex portion  8 . 
     The shape of the convex portion  8  constituting the microstructure  7  does not need to be a geometrically accurate shape, and may be a shape with rounded corners or a shape with fine irregularities on the surface. 
     The membrane carrier  3  is provided with a level difference  11  in which the height level of the bottom surface of the flow path  2  changes.  FIG. 8  illustrates a level difference viewed from a direction perpendicular to a direction of a thickness of the membrane carrier and a transporting direction of the liquid sample. As illustrated in  FIG. 9 , a region B downstream of the level difference may be inclined such that the height level of the bottom surface approaches a region A upstream of the level difference. 
     The level difference  11  is preferably one in which the height level of the bottom surface of the flow path  2  changes discontinuously from a viewpoint of further promoting stirring in a height direction in the liquid sample. 
     In the level difference  11 , the height level of the bottom surface of the flow path  2  is changed, and a convex structure (for example, a protrusion) or a concave structure (for example, a groove) is removed. 
     The height of the bottom surface of the level difference  11  is higher on the downstream side than on the upstream side. In this case, the liquid sample is spread over the level difference  11  due to surface tension of the liquid sample, and at this time, stirring in the height direction is promoted in the liquid sample, and a reaction rate between the substance to be detected and the label is improved. Therefore, performance of the test kit is improved. 
     An amount of change in the height of the bottom surface level of the level difference  11  is preferably equal to or less than twice the height of the microstructure  8 A on the upstream side of the level difference  11 . In this case, advance of the liquid sample by the capillary force at the level difference  11  is performed more smoothly. Here, the height of the microstructure  8 A is a height of the convex portion  8 , for example. 
     The amount of change in the height of the bottom surface level of the level difference  11 , that is, the height of the level difference  11  is equal to or more than 5 μm and equal to or less than 1000 μm, and preferably equal to or more than 10 μm and equal to or less than 500 μm. 
     In the region B on the downstream side of the level difference  11 , an inclination may be provided such that the height level on the downstream side of the region B (for example, an opposite side  20 B of a specific side  20 A) approaches the height level on the upstream side (for example, the specific side  20 A). The inclination is preferably provided toward the downstream side. In this case, the liquid sample in the flow path is easily developed by the influence of gravity, the reaction time can be shortened, and coloring of the background due to the remaining label after the development of the liquid sample can be suppressed. 
     With the level difference  11  as a boundary, the shape of the convex portion  8  may change. At the level difference  11 , a change in a height of the liquid level of the developing liquid sample occurs. However, by changing the shape of the convex portion  8 , contact between the microstructure and the liquid sample can be promoted, and sensitivity can be improved. The height of the liquid level height mentioned herein refer to a height from the bottom surface of the flow path to the liquid level of the developing liquid sample in each region. The shape of the convex portion  8  mentioned herein includes a size of the convex portion  8 . 
     Specifically, the height of the liquid level immediately exceeding the level difference  11  is lower than the upstream side of the level difference. Therefore, although a flow amount of the liquid sample is reduced, the entire microstructure is brought into contact with the liquid sample with the level difference  11  as a boundary, contact between the microstructure and the substance to be detected in the liquid sample can be promoted, and sensitivity can be improved. With the level difference  11  as a boundary, in a case where the height of the microstructure on the downstream side of the level difference  11  is reduced, contact between the microstructure and the substance to be detected in the liquid sample can be further promoted, and sensitivity can be further improved. The flow rate mentioned herein refer to a volume of a liquid sample that has passed through a flow path section in a direction perpendicular to the developing direction of the liquid sample per unit time. 
     A height  6  of the convex portion  8  constituting the microstructure  7  is preferably equal to or more than 10 μm and equal to or less than 500 μm. The height  6  of the convex portion  8  may change within this range between a plurality of the convex portions  8  (may be different from one another). In a case where the height  6  of the convex portion  8  is equal to or more than 10 μm, the volume of the flow path  2  increases, and the liquid sample can be developed in a shorter time. In a case where the height  6  of the convex portion  8  is equal to or less than 500 μm, it is possible to reduce the time and the cost for preparing the microstructure  7 , and preparation of the microstructure  7  becomes easier. 
     The height  6  of the convex portion  8  is defined as a maximum length of the convex portion  8  in a direction orthogonal to the flat portion  9 . As illustrated in  FIGS. 3( a ), 3( b ) , and  7 , in a case where the shape of the convex portion  8   a  is a cone, the height  6   a  of the convex portion  8   a  is the maximum length of the convex portion  8   a  in the direction orthogonal to the flat portion  9  (the height of the cone). As illustrated in  FIGS. 4( a ) and 4( b ) , in a case where the shape of the convex portion  8   b  is a quadrangular pyramid, the height  6   b  of the convex portion  8   b  is the maximum length of the convex portion  8   b  in the direction orthogonal to the flat portion  9  (height of the quadrangular pyramid). As illustrated in  FIGS. 5( a ) and 5( b ) , in a case where the shape of the convex portion  8   c  is a hexagonal pyramid, the height  6   c  of the convex portion  8   c  is the maximum length of the convex portion  8   c  in the direction orthogonal to the flat portion  9  (height of the hexagonal pyramid). As illustrated in  FIGS. 6( a ) and 6( b ) , in a case where the shape of the convex portion  8   d  is a horizontally placed triangular prism, the height  6   d  of the convex portion  8   d  is the maximum length of the convex portion  8   d  in the direction orthogonal to the flat portion  9  (height of the horizontally placed triangular prism). 
     A diameter  4  of the bottom surface  10  of the convex portion  8  is defined as a representative length of the bottom surface  10  of the convex portion  8 . The representative length of the bottom surface  10  is a diameter in a case where the shape of the bottom surface  10  is a circle, a shortest side length in a case where the shape of the bottom surface  10  is a triangle or a quadrangle, a longest diagonal length in a case where the shape of the bottom surface  10  is a pentagon or more polygon, and a longest length on the bottom surface  10  in a case where the shape of the bottom surface  10  is another shape. 
       FIG. 7  is an arrow sectional view of the membrane carrier  3  having the microstructure  7   a  illustrated in  FIG. 3  taken along the line VII-VII. As illustrated in  FIGS. 3( a ), 3( b ) , and  7 , in a case where the shape of the convex portion  8   a  is a cone, the diameter  4   a  of the bottom surface  10   a  of the convex portion  8   a  is a diameter of the bottom surface (circle) of the cone. As illustrated in  FIGS. 4( a ) and 4( b ) , in a case where the shape of the convex portion  8   b  is a regular quadrangular pyramid, the diameter  4   b  of the bottom surface  10   b  of the convex portion  8   b  is a length of the side of the bottom surface (square)  10   b . As illustrated in  FIGS. 5( a ) and 5( b ) , in a case where the shape of the convex portion  8   c  is a regular hexagonal pyramid, the diameter  4   c  of the bottom surface  10   c  of the convex portion  8   c  is the length of a diagonal passing through the center of the bottom surface (regular hexagon)  10   c  (the length of the longest diagonal). As illustrated in  FIGS. 6( a ) and 6( b ) , in a case where the shape of the convex portion  8   d  is a horizontally disposed triangular prism, the diameter  4   d  of the bottom surface  10   d  of the convex portion  8   d  is a length of the shortest side of the bottom surface (rectangle)  10   d  (in  FIGS. 6( a ) and 6( b ) , the length in the direction orthogonal to the transporting direction d of the liquid sample). 
     A bottom area of the convex portion  8  (the area per one bottom surface  10  of the convex portion  8 ) constituting the microstructure  7  is preferably equal to or more than 75 μm 2  and equal to or less than 250,000 μm 2 . The bottom area of the convex portion  8  may change within this range between the plurality of convex portions  8  (may be different from one another). In a case where the bottom area of the convex portion  8  is equal to or more than 75 μm 2 , fine processing becomes easy, and the cost of preparing a microstructure is further reduced. In a case where the bottom area of the convex portion  8  is equal to or less than 250,000 μm 2 , the number of the convex portions  8  constituting the microstructure  7  in one test kit increases, and the development of the liquid sample becomes easier. 
     A closest distance  5  between the convex portions  8  constituting the microstructure  7  is preferably equal to or less than 500 μm, and more preferably equal to or more than 2 μm and equal to or less than 100 μm. The closest distance  5  between the convex portions  8  may change within this range between the plurality of convex portions  8  (may be different from one another). The closest distance  5  between the convex portions  8  cannot be less than 0 μm, and in a case where the closest distance  5  between the convex portions  8  is equal to or less than 500 μm, the contact area between the liquid sample and the flow path  2  increases. With this, the capillary force increases, and moving the liquid sample becomes easier. Here, “the closest distance between the convex portions  8 ” is the closest distance between a pair of adjacent convex portions  8  in the same region. 
     An aspect ratio of the convex portion  8  constituting the microstructure  7  is preferably equal to or more than 0.1 and equal to or less than 2.0. The aspect ratio mentioned herein is a value (Lh/Lv) obtained by dividing the height  6  (Lh) of the convex portion  8  by the representative length (diameter  4 ) (Lv) of the bottom surface  10  of the convex portion  8 . In a case where the aspect ratio is equal to or more than 0.1, the contact area between the liquid sample and the flow path  2  increases, and with this, the capillary force increases, thereby making it easier to move the liquid sample. In a case where the aspect ratio is equal to or less than 2.0, preparation of a microstructure becomes easier. 
     The microstructure  7  may be formed of convex portions  8  the same as one another in the same region. The microstructure  7  may be formed of convex portions  8  different from one another in the same region. In this case, the convex portions  8  different from one another may be arranged in the same region in accordance with a certain rule along the transporting direction d of the liquid sample. That is, in the same region, the convex portion  8  may be, for example, arranged in line such that at least one of the diameter  4  of the bottom surface  10  of the convex portion  8 , the height  6  of the convex portion  8 , the closest distance  5  between the convex portions  8 , and the aspect ratio of the convex portion  8  (height  6 /diameter  4 ) may change (increase or decrease) along the transporting direction d of the liquid sample in accordance with a certain rule. 
       FIG. 10  is a top view of a membrane carrier according to another embodiment. In the membrane carrier  3  illustrated in  FIG. 2 , the detection zone  3   y  is provided in the region B, but in the membrane carrier  13  illustrated in  FIG. 10 , the detection zone  13   y  is provided in the region B 1 . As illustrated in  FIG. 10 , the dropping zone  13   x  and the detection zone  13   y  may be formed over substantially the entirety of the membrane carrier  13  in a short-side direction. 
     The microstructure  7  and the membrane carrier  3  of the liquid sample test kit  18  of the present embodiment may be made of thermoplastic plastic. In other words, it is possible to prepare the membrane carrier  3  having the microstructure  7  by processing a membrane-shaped substrate formed of thermoplastic plastic. Examples of a processing method include thermal imprinting, UV imprinting, injection molding, etching, photolithography, mechanical cutting, and laser processing, for example. Among these, as a technique of performing accurate processing at a low cost, thermal imprinting on thermoplastic plastic is suitable. Examples of the thermoplastic plastic include a polyester resin, a polyolefin resin, a polystyrene resin, a polycarbonate resin, a fluorine resin, an acrylic resin, and the like. Specifically, it is possible to use various thermoplastic plastics such as polyethylene terephthalate (PET), cycloolefin polymer (COP), polypropylene (PP), polystyrene (PS), polycarbonate (PC), polyvinylidene fluoride (PVDF), and polymethyl methacrylate (PMMA). 
     In a case of a processing method using a mold such as imprinting or injection molding, the conical body has a smaller upper portion than the bottom surface. Therefore, a volume to be carved out is smaller at a time of preparing a mold than preparing a column with the same bottom surface, and the mold can be prepared at a preparation is smaller than production is smaller than that of a column with the same bottom. And the mold can be manufactured at a low cost. In this case, it is possible to perform detection of the substance to be detected in the liquid sample at a lower cost. 
     As described above, the membrane carrier  3  is a membrane carrier  3  for a liquid sample test kit  18  that detects the substance to be detected in the liquid sample, and the membrane carrier  3  includes the microstructure  7  that causes capillary action for transporting the liquid sample, provided on one surface of the membrane carrier  3 , the flow path  2  that transports the liquid sample, formed by the microstructure  7 , and the level difference  11  at which the height level of the bottom surface of the flow path  2  changes. 
     In the liquid sample test kit  18  according to the present embodiment, a change in color occurs at a time when the substance to be detected is detected in the detection zone  3   y  of the membrane carrier  3 . The change in color may be a change in color that can be confirmed by an optical technique. 
     Examples of the optical technique mainly include two techniques of visual determination and measurement of fluorescence intensity. In a case of visual determination, change in color preferably occurs in a case where color difference between two color stimuli at a time of measuring color before and after the detection by a color system of the CIE1976L*a*b* color space (A E described in JIS Z8781-4: 2013) is equal to or more than 0.5. In a case where the color difference is equal to or more than 0.5, it is easy to visually confirm the color difference. In a case where the fluorescence intensity is measured and determined, the change in color preferably occurs such that a ratio (FI 1 /FI 2 ) of fluorescence intensity (FI 1 ) in the detection zone  3   y  and fluorescence intensity (FI 2 ) in an upstream region and a downstream region adjacent to the detection zone  3   y= 10/1 or more. In a case where the ratio is equal to or more than 10/1, separation of signal and noise becomes easy. 
     In order to prepare the detection zone  3   y  in the liquid sample test kit  18  of the present embodiment, in an embodiment, a detection substance is immobilized on at least a part of the flow path  2 . That is, the detection substance that detects the substance to be detected is immobilized in the detection zone  3   y . The change in color in the detection zone  3   y  is caused by holding the substance to be detected in the detection zone  3   y  by the detection substance (reacting with the detection substance). 
     In other words, the method of manufacturing the liquid sample test kit  18  includes immobilizing the detection substance in the detection zone  3   y . As the detection substance, a detection substance that causes a change in color by holding the substance to be detected in the detection zone  3   y  is preferable. From a viewpoint that the detection substance (reagent) can be more efficiently immobilized in the detection zone  3   y , pre-surface treatment may be performed in a site provided with the detection zone  3   y  in the membrane carrier  3 . 
     The method of the surface treatment is not particularly limited, and various methods such as UV irradiation, UV/ozone treatment, various plasma treatments, and surface modification with 3-aminopropyl triethoxysilane or glutaraldehyde can be used. 
     In the present embodiment, examples of the detection substance (reagent) include an antibody, for example. The antibody is an antibody that reacts with the substance to be detected by an antigen-antibody reaction, and may be a polyclonal antibody or a monoclonal antibody. 
     The change in color in the detection zone  3   y  may be caused by a label having an antibody or an antigen-binding fragment thereof that specifically reacts with the substance to be detected in the liquid sample. The change in color occurs as the label is held in the detection zone  3   y  by the detection substance (reacts (binds) with the detection substance) and colored, for example. 
     The label may be one in which the antibody or antigen-binding fragment thereof is bound to particles such as colloidal particles and latex particles, for example. The antigen-binding fragment refers to a fragment that can specifically bind to a substance to be detected, for example, an antigen-binding fragment of an antibody. The label can bind to the substance to be detected via an antibody or an antigen-binding fragment thereof. The particles may have magnetism or fluorescent properties. Examples of the colloidal particles include metal colloidal particles such as gold colloidal particles and platinum colloidal particles. The particles are preferably latex particles in view of particle diameter control, dispersion stability, and binding easiness. Although the material of the latex particles is not particularly limited, polystyrene is preferable. 
     The particles are preferably colored particles or fluorescent particles, and more preferably colored particles in view of visibility. The colored particles may be any particles as long as the color can be detected with naked eye. The fluorescent particles may contain a fluorescent substance. The particles may be colored latex particles or fluorescent latex particles. In a case where the particles are colored latex particles, the change in color is suitably determined visually. In a case where the particles are fluorescent latex particles, the change in color is suitably determined by measuring the fluorescence intensity. 
     The label is provided in at least a part of the test kit  18  so as to react with the substance to be detected in the dropped liquid sample. For example, the label may be provided in a member in the test kit  18  or may be provided in at least a part (upstream side from the detection zone  3   y ) of the flow path  2  of the membrane carrier  3 . The label that has reacted (bound) with the substance to be detected is held in the detection zone  3   y  by the detection substance (by the detection substance reacting (binding) with the substance to be detected). With this, a change in color (coloring by the label) occurs in the detection zone  3   y.    
     The detection zone  3   y  may be provided on an inclined portion on the downstream side of the level difference  11 . In the inclined portion, the liquid sample in the flow path is easily developed due to the influence of gravity, and the suppression of the coloring of the background by the remaining label after the development of the liquid sample becomes particularly remarkable. Therefore, the change in color in the detection zone  3   y  is particularly easily recognized, and the detection sensitivity of the substance to be detected is improved. 
     The test method of the liquid sample according to an aspect of the present embodiment is a test method using the test kit  18 . 
     A test method of a liquid sample using the test kit  18  may include mixing a liquid sample and a label specifically binding to a substance to be detected in the liquid sample, preparing a mixture liquid sample (mixed liquid sample), and binding the substance to be detected and the label to each other; dropping the mixture liquid sample onto a dropping zone  3   x  provided in the membrane carrier  3 ; transporting the mixture liquid sample from the dropping zone  3   x  to the detection zone  3   y  by the microstructure  7 ; and detecting a change in color (development of color of the label) in the detection zone  3   y.    
     For example, the test method may include dropping the liquid sample onto the dropping zone  3   x  on the surface of the membrane carrier  3 ; transporting the liquid sample from the dropping zone  3   x  to the detection zone  3   y  through the microstructure  7  by the capillary action by the microstructure  7  (a plurality of convex portions  8 ) formed on the surface of the membrane carrier  3 ; and detecting a change in color in the detection zone  3   y  (optically determining the presence or absence of a change in color) by binding the substance to be detected in the liquid sample to a label via the antibody or antigen-binding fragment thereof, and binding the substance to be detected to a reagent immobilized on the detection zone  3   y , in the transporting process. 
     In binding the substance to be detected and the label to each other of the test method, the method of mixing the liquid sample with the label is not particularly limited. For example, the method may be a method of adding a liquid sample to a container containing the label, for example, a method of mixing a liquid containing the label with the liquid sample. For example, a filter may be interposed between dropping ports of the container containing the liquid sample, and the label may be immobilized in the filter. 
     EXAMPLES 
     Hereinafter, the present embodiment will be specifically described, but the present embodiment is not limited to these experimental examples. 
     Experimental Example 1 
     &lt;Preparation of Mold&gt; 
     A mold was prepared by laser processing and mechanical cutting.  FIG. 11  illustrates a mold  20  for preparing a microstructure. The mold  20  illustrated in  FIG. 11  has a plurality of regions (a first region A and a second region B), and a concave portion corresponding to the microstructure (a convex portion) illustrated in  FIG. 8  is formed on the surface (not illustrated). The mold  20  is made of an aluminum alloy A5052. At the center of the mold (die), fine processing is performed in a range of 30 mm×30 mm. Within a processing range of the mold  20 , a level difference  11  having a depth (height) of 100 μm is provided in parallel with a specific side ( 20 A) at a position of 5 mm in the processing range from the specific side ( 20 A). In a region (region A) between the level difference and the specific side ( 20 A) and a region (region B) other than the region A, a conical concave portion (a concave portion capable of forming a convex portion at a time of transferring the microstructure) having a diameter of 100 μm and a depth (also referred to as height in the table) of 100 μm is arranged in line in a triangular arrangement form of  FIGS. 3( a ), 3( b ) , and  8  with the closest distance between the microstructures (distance between the closest microstructures)  5  set to 5 μm. 
     A mold release treatment was performed on an irregular surface of the mold in order to easily and reliably peel off the mold at a time of transfer from thermoplastic plastic. The mold release treatment was performed by performing immersion in Optool HD-2100TH manufactured by Daikin Industries, Ltd. for about 1 minute, drying, and then allowing to stand overnight. 
     &lt;Transfer of Microstructure&gt; 
     The microstructure was transferred to a thermoplastic plastic using the mold obtained as described above. As the thermoplastic plastic, polystyrene (Denka Styrene sheet manufactured by Denka Corporation, film thickness 300 μm) was used. Thermal imprinting was used as a processing method, and X-300 manufactured by SCIVAX was used as an apparatus. At a molding temperature of 120° C. and an applied pressure of 5.5 MPa, transfer was performed for 10 minutes. After the transfer, the thermoplastic plastic and the mold were cooled to 80° C. while applying pressure, and then the pressure was removed to prepare a membrane carrier having a region A and a region B in order from one end side. 
     Experimental Example 2 
     Except that the depth of the level difference in Experimental Example 1 was 10 μm, and the microstructures in the region A and the region B were conical concave portions having a diameter of 10 μm and a depth of 10 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 1. 
     Experimental Example 3 
     Except that the depth of the level difference in Experimental Example 1 was 500 μm, and the microstructures in the region A and the region B were conical concave portions having a diameter of 500 μm and a depth of 500 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 1. 
     Experimental Example 4 
     Except that the depth of the level difference in Experimental Example 1 was 50 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 1. 
     Experimental Example 5 
     Except that the depth of the level difference in Experimental Example 1 was 200 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 1. 
     Experimental Example 6 
     Except that the microstructure of the region B in Experimental Example 1 was a conical concave portion having a diameter of 10 μm and a depth of 10 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 1. 
     Experimental Example 7 
     Except that the depth of the level difference in Experimental Example 1 was 500 μm and the microstructure of the region A was a conical concave portion having a diameter of 500 μm and a depth of 500 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 1. 
     Experimental Example 8 
     Except that the depth of the level difference in Experimental Example 1 was 500 μm, the microstructure of the region A was a conical concave portion having a diameter of 500 μm and a depth of 500 μm, and the microstructure of the region B was a conical concave portion having a diameter of 10 μm and a depth of 10 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 1. 
     Experimental Example 9 
       FIG. 12  illustrates a mold  20  for preparing the microstructure of the Experimental Example 9. Unless otherwise specified, it is the same as the mold  20  of Experimental Example 1.  FIG. 12  does not illustrate a concave portion corresponding to the microstructure (convex portion). In the region B, an inclination is provided in a region (region B 1 ) having a width of 5 mm in a direction opposite to the specific side  20 A from the level difference  11 , and the height level of the mold surface in a region (region B 2 ) other than the region B 1  in the region B was matched with the height level of the mold surface of the region A, a membrane carrier was prepared under the same conditions as in Experimental Example 1. However, the height level of the specific side  20 A, the height level of the opposite side  20 B of the specific side  20 A, and the height level of a boundary line  20 C between the region B 1  and the region B 2  were the same. 
     Experimental Example 10 
     Except that the depth of the level difference in Experimental Example 9 was 50 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 9. 
     Experimental Example 11 
     Except that the depth of the level difference in Experimental Example 9 was 200 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 9. 
     Experimental Example 12 
     Except that the microstructures of the region B 1  and the region B 2  in Experimental Example 9 were conical concave portions having a diameter of 10 μm and a depth of 10 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 9. 
     Experimental Example 13 
     Except that the depth of the level difference in Experimental Example 9 was 500 μm, and the microstructure of the region A was a conical concave portion having a diameter of 500 μm and a depth of 500 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 9. 
     Experimental Example 14 
     Except that the depth of the level difference in Experimental Example 9 was 500 μm, the microstructure of the region A was a conical concave portion having a diameter of 500 μm and a depth of 500 μm, and the microstructures of the region B 1  and the region B 2  were conical concave portions having a diameter of 10 μm and a depth of 10 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 9. 
     Experimental Example 15 
     Except that the microstructure of the region B 1  in Experimental Example 9 was a conical concave portion having a diameter of 10 μm and a depth of 10 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 9. 
     Experimental Example 16 
     Except that the depth of the level difference in Experimental Example 9 was 500 μm, and the microstructures of the region A and the region B 2  were conical concave portions having a diameter of 500 μm and a depth of 500 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 9. 
     Experimental Example 17 
     The depth of the level difference in Experimental Example 9 was 500 μm, the microstructures of the region A and the region B 2  were conical concave portions having a diameter of 500 μm and a depth of 500 μm, and the microstructure of the region B 1  was a conical concave portion having a diameter of 10 μm and a depth of 10 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 9. 
     Experimental Example 18 
     Except that a level difference was not provided in the fine processing range of the mold, and a conical concave portion having a diameter of 100 μm and a depth of 100 μm was provided over the entire range of 30 mm×30 mm, a membrane carrier was produced under the same conditions as in Experimental Example 1. 
     Experimental Example 19 
     Except that the height level of the bottom surface was lower by 100 μm on the downstream side than on the upstream side of the level difference, a membrane carrier was prepared under the same conditions as in Experimental Example 1. 
     Experimental Example 20 
     Except that the depth of the level difference was 300 μm, a membrane carrier was prepared under the same conditions as in Experimental Example 9. 
     &lt;Preparation of Detection Zone&gt; 
     In the region B of the membrane carrier prepared as described above, a region corresponding to a width of 5 mm (region B 1  in a structure having an inclination) in a direction opposite to a specific side ( 20 A) from the level difference  11  was subjected to UV treatment. An anti-type A influenza NP antibody suspension and an anti-type B influenza NP antibody suspension were each applied to the portion at a line width of 1 mm (application amount: 3 μL each), dried well in warm air, and the detection substance was immobilized. 
     &lt;Set of Labels&gt; 
     Purified anti-type A influenza virus NP antibody (another antibody as described above) and purified anti-type B influenza virus NP antibody (another antibody as described above) were used. The anti-type A influenza virus NP antibody was covalently labeled with blue latex particles (manufactured by CM/BL Seradyne) having a particle size of 0.394 μm, suspended in a tris buffer containing saccharide, surfactant, and protein such that a concentration of latex particles was 0.025 w/v % to prepare containing saccharide, surfactant and protein, and sonicated to prepare a sufficiently dispersed and floated anti-type A label. Similarly, an anti-type B label in which blue latex particles were labeled with an anti-type B influenza virus NP antibody was prepared. 
     The anti-type A label and the anti-type B label were mixed with each other. Regarding the mixture obtained by mixing, an amount of 50 μL per square centimeter was applied to glass fiber (33GLASS NO. 10539766 manufactured by Schleicher &amp; Schuell) having a size of 3 cm×1 cm, and dried well under warm air to prepare a label pad. Thereafter, the label pad was overlapped only at an end of 2 mm of the region A of the membrane carrier prepared as in Experimental Examples 1 to 20, and cut into strips having a width of 5 mm with a cutter to prepare an integrated liquid sample test kit. 
     &lt;Detection Evaluation&gt; 
     100 μL of the liquid sample was dropped on the label pad (dropping zone) at the end of the liquid sample test kit prepared as described above. As a liquid sample, using a specimen suspension attached to QuickNavi-Flu manufactured by Denka Seiken Co., Ltd. as a dilution solution, two kinds of a 4×10 4 -fold diluted type A influenza virus A/Beijing/32/92 (H3N2) (hereinafter, referred to as type Ain some cases) and a 4×10 3 -fold diluted type B influenza virus B/Shangdong/7/97 (hereinafter, referred to as type B in some cases) were used. 
     
       
         
           
               
               
               
               
               
               
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
               
               
                   
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Microstructure height 
                 100 
                 10 
                 500 
                 100 
                 100 
                 100 
                 500 
                 500 
               
               
                 of region A (μm) 
               
               
                 Microstructure diameter 
                 100 
                 10 
                 500 
                 100 
                 100 
                 100 
                 500 
                 500 
               
               
                 of region A (μm) 
               
               
                 Microstructure height 
                 100 
                 10 
                 500 
                 100 
                 100 
                 10 
                 100 
                 10 
               
               
                 of region B (μm) 
               
               
                 Microstructure diameter 
                 100 
                 10 
                 500 
                 100 
                 100 
                 10 
                 100 
                 10 
               
               
                 of region B (μm) 
               
               
                 Height of level difference 
                 100 
                 10 
                 500 
                  50 
                 200 
                 100 
                 500 
                 500 
               
               
                 (μm) 
               
               
                 Limit magnification at 
                 7 × 10 4   
                 7 × 10 4   
                 6 × 10 4   
                 6 × 10 4   
                 7 × 10 4   
                 8 × 10 4   
                 7 × 10 4   
                 7 × 10 4   
               
               
                 which type A visual 
               
               
                 determination is possible 
               
               
                 Limit magnification at 
                 7 × 10 3   
                 7 × 10 3   
                 6 × 10 3   
                 6 × 10 3   
                 7 × 10 3   
                 8 × 10 3   
                 7 × 10 3   
                 7 × 10 3   
               
               
                 which type B visual 
               
               
                 determination is possible 
               
               
                 Time until density 
                  6 
                  7 
                  5 
                  5 
                  7 
                 7 
                  6 
                 7 
               
               
                 is stabilized (minute) 
               
               
                 Comprehensive evaluation 
                 A 
                 B 
                 B 
                 B 
                 B 
                 A 
                 A 
                 B 
               
               
                 Notes 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
               
               
                   
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
             
            
               
                   
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
               
               
                   
                 9 
                 10 
                 11 
                 12 
                 13 
                 14 
                 15 
               
               
                   
               
               
                 Microstructure height 
                 100 
                 100 
                 100 
                 100 
                 500 
                 500 
                 100 
               
               
                 of region A (μm) 
               
               
                 Microstructure diameter 
                 100 
                 100 
                 100 
                 100 
                 500 
                 500 
                 100 
               
               
                 of region A (μm) 
               
               
                 Microstructure height 
                 100 
                 100 
                 100 
                 10 
                 100 
                 10 
                 10 
               
               
                 of region B1 (μm) 
               
               
                 Microstructure diameter 
                 100 
                 100 
                 100 
                 10 
                 100 
                 10 
                 10 
               
               
                 of region B1 (μm) 
               
               
                 Microstructure height 
                 100 
                 100 
                 100 
                 10 
                 100 
                 10 
                 100 
               
               
                 of region B2 (μm) 
               
               
                 Microstructure diameter 
                 100 
                 100 
                 100 
                 10 
                 100 
                 10 
                 100 
               
               
                 of region B2 (μm) 
               
               
                 Height of level 
                 100 
                  50 
                 200 
                 100 
                 500 
                 500 
                 100 
               
               
                 difference (μm) 
               
               
                 Limit magnification at 
                 7 × 10 4   
                 6 × 10 4   
                 7 × 10 4   
                 8 × 10 4   
                 7 × 10 4   
                 7 × 10 4   
                 8 × 10 4   
               
               
                 which type A visual 
               
               
                 determination is possible 
               
               
                 Limit magnification at 
                 7 × 10 3   
                 6 × 10 3   
                 7 × 10 3   
                 8 × 10 3   
                 7 × 10 3   
                 7 × 10 3   
                 8 × 10 3   
               
               
                 which type B visual 
               
               
                 determination is possible 
               
               
                 Time until density is 
                  5 
                  4 
                  6 
                 6 
                  5 
                 7 
                 6 
               
               
                 stabilized (minute) 
               
               
                 Comprehensive evaluation 
                 A 
                 B 
                 A 
                 A 
                 A 
                 B 
                 A 
               
               
                 Notes 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
               
               
                   
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
               
               
                   
                   
                 16 
                 17 
                 18 
                 19 
                 20 
               
               
                   
                   
               
               
                   
                 Microstructure height 
                 500 
                 500 
                 100 
                 100 
                 100 
               
               
                   
                 of region A (μm) 
               
               
                   
                 Microstructure diameter 
                 500 
                 500 
                 100 
                 100 
                 100 
               
               
                   
                 of region A (μm) 
               
               
                   
                 Microstructure height 
                 100 
                 10 
                 100 
                 100 
                 100 
               
               
                   
                 of region B1 (μm) 
               
               
                   
                 Microstructure diameter 
                 100 
                 10 
                 100 
                 100 
                 100 
               
               
                   
                 of region B1 (μm) 
               
               
                   
                 Microstructure height 
                 500 
                 500 
                 100 
                 100 
                 100 
               
               
                   
                 of region B2 (μm) 
               
               
                   
                 Microstructure diameter 
                 500 
                 500 
                 100 
                 100 
                 100 
               
               
                   
                 of region B2 (μm) 
               
               
                   
                 Height of level 
                 500 
                 500 
                  0 
                 −100 
                 300 
               
               
                   
                 difference (μm) 
               
               
                   
                 Limit magnification at 
                 7 × 10 4   
                 7 × 10 4   
                 4 × 10 4   
                 Liquid remains on 
                 7 × 10 4   
               
               
                   
                 which type A visual 
                   
                   
                   
                 upstream side of 
               
               
                   
                 determination is possible 
                   
                   
                   
                 level difference 
               
               
                   
                 Limit magnification at 
                 7 × 10 3   
                 7 × 10 3   
                 4 × 10 3   
                 Liquid remains on 
                 7 × 10 3   
               
               
                   
                 which type B visual 
                   
                   
                   
                 upstream side of 
               
               
                   
                 determination is possible 
                   
                   
                   
                 level difference 
               
               
                   
                 Time until density is 
                  4 
                 7 
                  5 
                 Liquid does not 
                  10 
               
               
                   
                 stabilized (minute) 
                   
                   
                   
                 flow to 
               
               
                   
                   
                   
                   
                   
                 downstream side 
               
               
                   
                   
                   
                   
                   
                 and measurement 
               
               
                   
                   
                   
                   
                   
                 is not possible 
               
               
                   
                 Comprehensive evaluation 
                 A 
                 B 
                 D 
                 D 
                 C 
               
               
                   
                 Notes 
                 Working 
                 Working 
                 Comparative 
                 Comparative 
                 Working 
               
               
                   
                   
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
               
               
                   
                   
               
            
           
         
       
     
     In Example 19, the height level of the bottom surface was lower by 100 μm on the downstream side than on the upstream side of the level difference. 
     The determination of the detection was performed by visually observing the presence or absence of a colored line in the detection zone (the type A influenza virus detection portion and the type B influenza virus detection portion) after 15 minutes. 
     As a result of the determination, in a case where a solution obtained by diluting A/Beijing/32/92 (H3N2) by 4×10 4  was used, a change in color was confirmed only in the type A detection zone, and in a case where a solution obtained by diluting B/Shangdong/7/97 by 4×10 3  was used, a change in color was confirmed only in the type B detection zone. 
     From the membrane carrier prepared as in Experimental Examples 1 to 20, a liquid sample test kit was prepared as described above. Subsequently, in a case where a dilution rate of the type A influenza virus A/Beijing/32/92 (H3N2) was increased from 4×10 4 , a dilution rate (type A limit magnification at which visual determination is possible), at which the presence or absence of a colored line could not be visually observed 15 minutes after the start of the test, was obtained. In a case of performing test at a dilution rate of 1/2 of the dilution rate, a time from the start of the test to the color density of the colored line was stabilized (time until the type A density was stabilized) was obtained. The result is shown in Tables 1 and 2. 
     From the membrane carrier prepared as in Experimental Examples 1 to 20, a liquid sample test kit was prepared as described above. Subsequently, in a case where a dilution rate of the type B influenza virus B/Shangdong/7/97 was increased from 4×10 3 , a dilution rate (type B limit magnification at which visual determination is possible), at which the presence or absence of a colored line could not be visually observed, was obtained. In a case of performing test at a dilution rate of 1/2 of the dilution rate, a time from the start of the test to the color density of the colored line was stabilized (time until the type B density was stabilized) was obtained. The result is shown in Tables 1 and 2. 
     As for the time until the density is stabilized, an average value of a time until the type A density is stabilized and a time until the type B density is stabilized was used as a time until the density is stabilized. 
     Tables 1 and 2 also show the results of the comprehensive evaluation based on the following criteria for each experimental example. 
     A: Those that can be determined at a dilution rate of equal to or more than 7×10 4  for type A and at a dilution rate of equal to or more than 7×10 3  for type B, or those that can be determined at a dilution rate of equal to or more than 8×10 4  for type A and at a dilution rate of equal to or more than 8×10 3  for type B, within 6 minutes of determination time (time until the density is stabilized). 
     B: Those for which comprehensive evaluation does not apply to either of A and C. 
     C: Those for which determination time is equal to or more than 8 minutes and equal to or less than 10 minutes. 
     D: Those for which determination time exceeds 10 minutes or those of which dilution rate capable of determination is equal to or less than 4×10 4  for type A and equal to or less than 4×10 3  for type B. 
     Experimental Examples 21 to 37 
     The preparation of the membrane carrier in Experimental Examples 21 to 37 was performed in the regions A, B 1 , and B 2 . 
     The experiment was performed in the same manner as in Experimental Example 1 except that the depth of the level difference, the diameter of the microstructure (convex portion), and the height of the microstructure (convex portion) were set as shown in Tables 3 and 4. 
     Subsequently, preparation of the detection zone, setting of the label, and evaluation of the detection were performed in the same manner as in Experimental Examples 1 to 17, except that the particles to be used were changed from colored latex particles to fluorescent latex particles (micromer-F fluorescent latex particles material polystyrene manufactured by Corefront Corporation), and 4 minutes after the start of the test, a magnification (limit magnification at which fluorescence can be determined), at which the presence or absence of a colored line could not be read by an immunochromatography reader (C11787 manufactured by Hamamatsu Photonics), was obtained. The results are shown in Tables 3 and 4. 
     Tables 3 and 4 also show the results of the comprehensive evaluation based on the following criteria for each experimental example. 
     A: Those of which limit magnification at which fluorescence can be determined 4 minutes after the start of the test is equal to or more than 1×10 6  for type A and equal to or more than 1×10 5  for type B. 
     B: Those for which comprehensive evaluation does not apply to either of A and C. 
     C: Those of which limit magnification at which fluorescence can be determined at 4 minutes after the start of the test is less than 7×10 5  for type A and less than 7×10 4  for type B. 
     
       
         
           
               
               
               
               
               
               
               
               
               
             
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
               
               
                   
                 21 
                 22 
                 23 
                 24 
                 25 
                 26 
                 27 
                 28 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Microstructure height of region A (μm) 
                 100 
                 10 
                 500 
                 100 
                 100 
                 100 
                 500 
                 500 
               
               
                 Microstructure diameter of region A (μm) 
                 100 
                 10 
                 500 
                 100 
                 100 
                 100 
                 500 
                 500 
               
               
                 Microstructure height of region B (μm) 
                 100 
                 10 
                 500 
                 100 
                 100 
                 10 
                 100 
                 10 
               
               
                 Microstructure diameter of region B (pm) 
                 100 
                 10 
                 500 
                 100 
                 100 
                 10 
                 100 
                 10 
               
               
                 Height of level difference (μm) 
                 100 
                 10 
                 500 
                 50 
                 200 
                 100 
                 500 
                 500 
               
               
                 Limit magnification at which type A fluorescence 
                 1 × 10 6   
                 9 × 10 5   
                 1 × 10 6   
                 1 × 10 6   
                 9 × 10 5   
                 2 × 10 6   
                 2 × 10 6   
                 1 × 10 6   
               
               
                 determination is possible 4 minutes after start of test 
               
               
                 Limit magnification at which type B fluorescence 
                 1 × 10 5   
                 9 × 10 4   
                 1 × 10 5   
                 1 × 10 5   
                 9 × 10 4   
                 2 × 10 5   
                 2 × 10 5   
                 1 × 10 5   
               
               
                 determination is possible 4 minutes after start of test 
               
               
                 Comprehensive evaluation 
                 A 
                 B 
                 A 
                 A 
                 B 
                 A 
                 A 
                 A 
               
               
                 Notes 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
               
               
                   
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
               
               
             
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
               
               
                   
                 29 
                 30 
                 31 
                 32 
                 33 
                 34 
                 35 
                 36 
                 37 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Microstructure height of region A (μm) 
                 100 
                 100 
                 100 
                 100 
                 500 
                 500 
                 100 
                 500 
                 500 
               
               
                 Microstructure diameter of region A (μm) 
                 100 
                 100 
                 100 
                 100 
                 500 
                 500 
                 100 
                 500 
                 500 
               
               
                 Microstructure height of region B1 (μm) 
                 100 
                 100 
                 100 
                 10 
                 100 
                 10 
                 10 
                 100 
                 10 
               
               
                 Microstructure diameter of region B1 (μm) 
                 100 
                 100 
                 100 
                 10 
                 100 
                 10 
                 10 
                 100 
                 10 
               
               
                 Microstructure height of region B2 (μm) 
                 100 
                 100 
                 100 
                 10 
                 100 
                 10 
                 100 
                 500 
                 500 
               
               
                 Microstructure diameter of region B2 (μm) 
                 100 
                 100 
                 100 
                 10 
                 100 
                 10 
                 100 
                 500 
                 500 
               
               
                 Height of level difference (μm) 
                 100 
                 50 
                 200 
                 100 
                 500 
                 500 
                 100 
                 500 
                 500 
               
               
                 Limit magnification at which type A 
                 2 × 10 6   
                 2 × 10 6   
                 1 × 10 6   
                 2 × 10 6   
                 1 × 10 6   
                 1 × 10 6   
                 2 × 10 6   
                 2 × 10 6   
                 1 × 10 6   
               
               
                 fluorescence determination is possible 4 
               
               
                 minutes after start of test 
               
               
                 Limit magnification at which type B 
                 2 × 10 5   
                 2 × 10 5   
                 1 × 10 5   
                 2 × 10 5   
                 1 × 10 5   
                 1 × 10 5   
                 2 × 10 5   
                 2 × 10 5   
                 1 × 10 5   
               
               
                 fluorescence determination is possible 4 
               
               
                 minutes after start of test 
               
               
                 Comprehensive evaluation 
                 A 
                 A 
                 A 
                 A 
                 A 
                 A 
                 A 
                 A 
                 A 
               
               
                 Notes 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
                 Working 
               
               
                   
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
               
               
                   
               
            
           
         
       
     
     From the results of Tables 1 to 4, it was shown that the liquid sample test kit according to the present embodiment can promote stirring of the substance to be detected and the label by providing a level difference in the flow path, and can perform a highly sensitive test. It was shown that it was possible to suppress coloring of the background by preparing the detection zone on an inclined portion on a downstream side of the level difference, and thus the sensitivity was further improved. In addition, from the results of Tables 3 and 4, it was confirmed that highly sensitive test could be performed even in a case where the particles were fluorescent latex particles in the liquid sample test kit. 
     In a case where the level difference was not provided, high sensitivity was not obtained (Experimental Example 18). In a case where the height level of the bottom surface was lower on the downstream side than on the upstream side of the level difference, the liquid did not flow to the downstream side, and measurement was not possible (Experimental Example 19). 
     In a case where the amount of change in the height level of the bottom surface at the level difference exceeds twice the height of the microstructure on the upstream side of the level difference, the determination time was long (Experimental Example 20). 
     Since the liquid sample test kit of the present embodiment can perform a highly sensitive test at a low cost, it is useful as a disposable POCT reagent. 
     According to the present embodiment, by using a flow path provided with an artificial fine convex portions having high design flexibility, it is possible to control the flow in the liquid sample test kit more easily compared to a technique of adjusting a pore diameter or thickness of a porous body having a non-uniform structure as in Patent Document 2. By providing a level difference in the flow path, stirring of a substance to be detected or a label is promoted, and it is possible to improve sensitivity in the detection zone compared to cases of Patent Documents 3 to 8 using a smooth artificial flow path. 
     In this application, priority is claimed on Japanese Patent Application No. 2017-236604, filed on Dec. 11, 2017, the content of which is incorporated herein by reference. 
     REFERENCE SIGNS LIST 
     
         
         
           
               2 : flow path 
               3 ,  13 : membrane carrier 
               3   x ,  13   x : dropping zone 
               3   y ,  13   y : detection zone 
               4 : representative length at the bottom surface of the convex portion (diameter of the bottom surface of the convex portion) 
               5 : distance between closest microstructures 
               6 ,  6   a ,  6   b ,  6   c ,  6   d : height of convex portion 
               8 ,  8   a ,  8   b ,  8   c ,  8   d : convex portion 
               7 ,  7   a ,  7   b ,  7   c ,  7   d : microstructure 
               9 : flat portion 
               10 ,  10 ,  10   b ,  10   c ,  10   d : bottom surface of convex portion 
               18 : liquid sample test kit 
               18   a : housing 
               18   b : first opening 
               18   c : second opening 
               20 : mold 
               20 A: specific side 
               20 B: opposite side of specific side  20 A 
               20 C: boundary between region B 1  and region B 2   
             A: first region 
             B: second region 
             B 1 : region 
             B 2 : region other than B 1   
             d: flow direction of liquid sample (transporting direction)