Inspection device and inspection method

Abstract

This inspection device is provided with: a substrate (2); a microneedle (3) that sticks to the skin to collect body fluid; a body fluid flow path (4) that transfers the body fluid collected by the microneedle (3); and an immunoassay unit (5) that detects, through an antigen-antibody reaction, a biomarker that can be included in the body fluid that has been transferred through the body fluid flow path (4), wherein the microneedle (3), the body fluid flow path (4), and the immunoassay unit (5) are disposed on one surface (2a) of the substrate (2).

Description

Inspection device and inspection method

[0001] The present invention relates to a test device and a test method using an immunoassay method. This application claims priority based on Japanese Patent Application No. 2023-163247 filed in Japan on September 26, 2023, and Japanese Patent Application No. 2024-113244 filed in Japan on July 16, 2024, the contents of which are incorporated herein by reference.

[0002] BACKGROUND ART Testing devices have been known that collect body fluids from a living body that may contain biomarkers that indicate the presence or absence of disease, the stage of disease, pregnancy, etc., and perform diagnosis using an immunoassay method that utilizes an antigen-antibody reaction.

[0003] For example, Patent Document 1 describes a testing device that can collect body fluids by piercing the surface of a living body with microneedles arranged on one side of a substrate.

[0004] According to this testing device, after the collected body fluid is transferred to the other side, biomarkers in the body fluid can be detected by antigen-antibody reactions while the fluid is being spread radially.

[0005] International Publication No. 2019 / 118420

[0006] However, in the testing device of Patent Document 1, the microneedles are arranged on one side of the substrate, and the immunoassay section where the antigen-antibody reaction takes place is arranged on the other side of the substrate.

[0007] Therefore, during manufacturing, it is necessary to form microneedles or immunoassay portions on both the front and back surfaces of the substrate, which requires at least two special molds, resulting in high manufacturing costs for the testing device.

[0008] The present invention has been made in view of the above circumstances, and has an object to provide an inspection device that can reduce manufacturing costs, and an inspection method using the inspection device.

[0009] The present disclosure provides test devices for the detection of biomarkers.

[0010] In a specific embodiment, [1] an examination device comprising: a substrate; a microneedle that pierces the skin to collect a body fluid; a body fluid flow path that transports the body fluid collected by the microneedle; and an immunoassay unit that detects a biomarker that may be contained in the body fluid transported through the body fluid flow path by an antigen-antibody reaction, wherein the microneedle, the body fluid flow path, and the immunoassay unit are arranged on one side of the substrate.

[0011] [2] The test device according to [1], wherein a microstructure for improving the wettability of the body fluid flow path is formed on the surface of the body fluid flow path.

[0012] [3] The testing device according to [2], wherein the microstructure is a groove extending in the extension direction of the body fluid flow path.

[0013] [4] The testing device according to [2] or [3], wherein the plurality of grooves extend parallel to each other on the surface of the body fluid flow path.

[0014] [5] The testing device according to [3] or [4], wherein the number of the grooves increases toward the downstream side of the body fluid flow path.

[0015] [6] The testing device according to [4] or [5], wherein connecting grooves are formed to connect the plurality of grooves extending in the extension direction of the body fluid flow path.

[0016] [7] The inspection device according to any one of [3] to [6], wherein the width of the groove is in the range of 100 nm or more and less than 1000 nm.

[0017] [8] The testing device according to any one of [1] to [7], wherein at least a portion of the bodily fluid flow path has a shape in which the width increases toward the downstream side.

[0018] [9] The testing device according to any one of [1] to [8], wherein the body fluid flow path has a plurality of repeating units having the shape connected in the extending direction of the body fluid flow path.

[0019]

[10] The testing device according to any one of [1] to [9], wherein at least a portion of the bodily fluid flow path increases in depth toward downstream.

[0020]

[11] The testing device according to [2], wherein the microstructure is a plurality of microscopic protrusions provided in the body fluid flow path.

[0021]

[12] The inspection device according to

[11] , wherein the microprotrusions are substantially conical, and have a shape that satisfies the following formulas (1), (2), and (3), where a is the height of the microprotrusions, b is the width of the bottom surface of the microprotrusions, and c is the spacing between the microprotrusions: b≦600 (unit: nm) (1), a / b≧0.3 (2), and 200≦c≦400 (unit: nm) (3).

[0022]

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

[12] , wherein the surface of the microneedle is formed with irregularities.

[0023]

[14] The testing device according to any one of [1] to

[12] , wherein a protective film is provided on the tip side of the microneedle, and the thickness of the protective film is less than the height of the microneedle.

[0024]

[15] The test device according to any one of [1] to

[14] , wherein the portion of the substrate located on the other side of the immunoassay section is formed to be transparent.

[0025]

[16] The test device according to

[15] , wherein a magnifying optical system is provided on the other side of the portion located on the other side of the immunoassay section.

[0026]

[17] The testing device according to any one of [1] to

[16] , wherein the surface on one side of the substrate and the surface of the microneedle are formed of the same material, and the contact angle of water on the surface on one side is 45° or less.

[0027]

[18] The testing device according to any one of [1] to

[17] , wherein the microneedle is a solid, hollow, porous, or hydrogel structure.

[0028]

[19] The testing device according to any one of [1] to

[18] , wherein the body fluid flow path has a shape surrounding the microneedle.

[0029]

[20] The test device according to any one of [1] to

[18] , wherein the immunoassay unit includes a test unit that visualizes the presence of the biomarker in the body fluid, and the test unit includes a plurality of color-developing regions that visualize the presence of different biomarkers.

[0030]

[21] The testing device according to

[20] , wherein the color-producing region is formed in an inner region of a groove structure extending in a closed ring shape formed on the surface of the body fluid flow path, and the color-producing regions are each provided with antibodies that specifically capture different biomarkers.

[0031]

[22] A testing method comprising the steps of: puncturing the skin with a microneedle of a testing device having, on one side of a substrate, a microneedle, a body fluid flow path for transporting the body fluid, and an immunoassay unit for detecting a biomarker by an antigen-antibody reaction; transporting the collected body fluid through the body fluid flow path; and detecting a biomarker contained in the transported body fluid in the immunoassay unit.

[0032]

[23] The immunoassay unit includes a test unit that visualizes the presence of the biomarker in the body fluid, and the test unit includes a plurality of color-forming regions that visualize the presence of different biomarkers, the color-forming regions being formed in an inner region of a groove structure that extends in a closed ring on the surface of the body fluid flow path, and the plurality of color-forming regions are each provided with an antibody that specifically captures a different biomarker, and the antibody captures and detects a different biomarker in each color-forming region.

[0033] According to the present invention, since the microneedles and immunoassay units are concentrated on one surface of the substrate, the number of special molds required for the substrate can be reduced, thereby providing a testing device that can reduce manufacturing costs, and a testing method using the testing device.

[0034] 1. A schematic bottom view of a test device according to a preferred embodiment of the present invention. A longitudinal cross-sectional view of a test device taken along line Y-Y shown in FIG. 1. A schematic side view of the vicinity of a microneedle in the test device shown in FIG. 1. A cross-sectional end view of the vicinity of a microprotrusion according to a modified example, taken longitudinally on a plane passing through the center of the microprotrusion. A schematic bottom view of a second downstream section according to antibody immobilization method 2. A schematic cross-sectional view of the vicinity of the surface of the second downstream section according to antibody immobilization method 3. A schematic side view showing a method of forming microprotrusions according to antibody immobilization method 5. An enlarged side view of the second downstream section according to antibody immobilization method 6. An enlarged bottom view of the second downstream section according to antibody immobilization method 6. An enlarged side view of the second downstream section according to antibody immobilization method 7. An enlarged bottom view of the second downstream section according to antibody immobilization method 7. A schematic bottom view of a body fluid flow path according to another preferred embodiment of the present invention. A schematic diagram showing the function of a body fluid flow path according to yet another preferred embodiment of the present invention. A schematic perspective view of the vicinity of a body fluid flow path in a test device according to another preferred embodiment of the present invention. A schematic bottom view of the vicinity of the transfer groove shown in FIG. 14B is a schematic side view showing the up-and-down position of the substrate in the standard state of a test device according to yet another preferred embodiment of the present invention. FIG. 14C is a schematic side view showing the up-and-down position of the substrate in the puncture state of the test device shown in FIG. 14A. FIG. 14D is a schematic bottom view of the vicinity of the test section of a test device according to an embodiment of the present invention capable of detecting multiple items. FIG. 14E is a schematic side view showing a microneedle having circumferentially extending horizontal grooves formed therein. FIG. 14F is a schematic side view showing a microneedle formed in an arrowhead shape. FIG. 14G is a high-magnification image of a mold structure obtained by etching, with a pore period of 200 nm, a pore diameter of 150 nm, and a pore length of approximately 400 nm. FIG. 14H is a high-magnification image of a microstructure including pillars according to Example 1. FIG. 14I is a high-magnification image of a mold structure obtained by etching, with a pore period of 200 nm, a pore diameter of 150 nm, and a pore length of approximately 1 μm. FIG. 14J is a high-magnification image of a microstructure including pillars according to Example 2. FIG. 14J is a top view of a flow path prototype according to Example 3.

[0035] Hereinafter, preferred embodiments of the present invention will be described in detail, with reference to the drawings as needed.

[0036] [Test Device] FIG. 1 is a schematic bottom view of a test device 1 according to a preferred embodiment of the present invention, and FIG. 2 is a longitudinal sectional view of the test device 1 taken along line YY shown in FIG.

[0037] FIG. 3 is a schematic side view of the vicinity of the microneedle of the testing device 1.

[0038] The testing device 1 includes a substantially flat substrate 2, a large number of microneedles 3 and a body fluid flow path 4 formed on one surface 2a of the substrate 2, an immunoassay section 5 provided in part of the body fluid flow path 4, a fixed seal 6 disposed on the other side of the substrate 2, and an optical system 7 that improves the visibility of the detection results. In the following description, one side of the substrate 2 is referred to as the "bottom" ("L" in the drawings), and the opposite side (the other side) is referred to as the "top" ("U" in the drawings).

[0039] The testing device 1 is a device that punctures the skin with a microneedle 3 to collect body fluids and visualizes whether or not the body fluids contain biomarkers that indicate the presence or absence of disease, the degree of progression, pregnancy, etc.

[0040] The size of the substrate 2 is not particularly limited, but the larger of the depth and width of the substrate 2, width Z, is preferably 1 cm or more and 3 cm or less, and particularly preferably 1 cm or more and 2 cm or less.

[0041] The substrate 2 is preferably made of a biocompatible material. Examples of biocompatible materials include thermoplastic resins such as medical-grade silicone, polyglycolic acid, and cyclic olefin copolymer. The substrate 2 may be a laminate consisting of multiple layers made of different materials, but it is preferable to use a single material in order to reduce manufacturing costs.

[0042] The fixed seal 6 is formed to be wider than the substrate 2 in a bottom view. An adhesive layer is provided on the lower surface of the fixed seal 6, and no adhesive layer is provided on the upper surface. By adhering the lower surface of the portion of the fixed seal 6 that does not overlap with the substrate 2 in a bottom view to the skin, the microneedles 3 arranged on the lower surface 2a of the substrate 2 can be fixed in a state where they are punctured into the skin.

[0043] A through-hole 6a penetrating in the vertical direction is formed in the portion of the fixed seal 6 located above the optical system 7, so that the optical system 7 can be seen from above through the through-hole 6a. Each microneedle 3 is preferably conical or polygonal pyramidal, but is not limited to these. The microneedle 3 may have a shape in which a columnar (cylindrical, polygonal, etc.) base portion is joined to a conical or polygonal pyramidal tip portion.

[0044] The height of each microneedle 3 is preferably in the range of 100 μm to 2 mm, and more preferably in the range of 800 μm to 1 mm. By setting the height of the microneedle 3 within this range, the microneedle 3 can be inserted up to the boundary between the epidermis and the dermis, and mainly interstitial fluid (an example of a body fluid) can be collected, thereby realizing a testing device 1 that is less invasive than when blood is also collected.

[0045] However, the height of the microneedle 3 is not particularly limited, and the height of the microneedle 3 may be set to a height that reaches a depth in the body that allows collection of not only interstitial fluid but also blood. Hereinafter, the microneedle 3 may also be simply referred to as the "needle 3."

[0046] The angle of the tip of the microneedle 3 is not particularly limited, but is preferably in the range of 60°±40°. The microneedle 3 and the body fluid flow path 4 are preferably formed from the same material as the substrate 2. The materials for the microneedle 3, the body fluid flow path 4, and the entire substrate 2 are not particularly limited, but are preferably integrally and continuously molded from biocompatible materials such as polyglycolic acid, polycarbonate, cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polyether ether ketone (PEEK), alginate, chitin, chitosan, collagen, hydroxypropyl cellulose, and pullulan. Examples of molding methods include injection molding, thermal compression molding, and casting.

[0047] Hydroxyl groups and fine irregularities are formed on the surface of the needle 3 and the lower surface 2a of the substrate 2 by oxygen plasma treatment (hydrophilic treatment).

[0048] For this reason, these portions have extremely high hydrophilicity, and the contact angle of water on the lower surface 2a of the substrate 2 including the microneedles 3 and the body fluid flow path 4 is 45° or less. Therefore, the interstitial fluid collected by the microneedles 3 can be smoothly transferred to the body fluid flow path 4.

[0049] In addition, in order to make the contact angle of water on the lower surface 2a of the substrate 2 45° or less, in addition to plasma treatment using oxygen or the like, corona treatment, flame treatment, chemical treatment, UV treatment, etc. can be used, but are not limited to these.

[0050] Furthermore, the microneedles 3 and the body fluid flow paths 4 may be formed integrally with the substrate 2 as in this embodiment, but the substrate may be formed and then the microneedles and body fluid flow paths may be formed by processing the substrate itself, or the microneedles and body fluid flow path components formed separately from the substrate may be attached to one side of the substrate.

[0051] Although each microneedle 3 has a solid structure, the structure of the microneedle is not limited to a solid structure and may be a hollow structure, a porous structure, or a hydrogel structure. By making the microneedle a hollow structure, a porous structure, or a hydrogel structure, the wettability (hydrophilicity) of the microneedle can be improved.

[0052] Collection of body fluids by the microneedles 3 refers to attaching the body fluid to the surface of the microneedles 3, or storing the body fluid inside the hollow needles 3.

[0053] When the microneedle has a hollow structure, for example, by forming a through-hole that penetrates upward from the tip of the microneedle and connects to a body fluid flow path, body fluids such as interstitial fluid can be collected through the through-hole.

[0054] The microneedles 3 are arranged in an area surrounded by the bodily fluid flow path 4 on the lower surface 2 a of the substrate 2 .

[0055] After receiving interstitial fluid from the microneedles 3, the body fluid flow path 4 is capable of transporting the interstitial fluid from the upstream portion to the downstream portion of the body fluid flow path 4. The body fluid flow path 4 is provided with a needle peripheral portion 4a located on the upstream side and extending through the gaps between the microneedles 3, and a downstream portion 4b located downstream of the needle peripheral portion 4a.

[0056] The needle peripheral portion 4 a is a flow path whose upper portion is defined by the lower surface 2 a of the substrate 2 around the microneedle 3 .

[0057] The downstream portion 4b includes a first downstream portion 4b1 located approximately in the center of the area where a large number of microneedles 3 are arranged on the underside 2a of the substrate 2, a second downstream portion 4b2 extending horizontally in an approximately linear manner from the first downstream portion 4b1 to near the edge of the underside 2a of the substrate 2, and a third downstream portion 4b3 shaped to surround the microneedles 3 from the downstream end of the second downstream portion 4b2 (the end on the edge side of the substrate 2). The first downstream portion 4b1 serves to collect interstitial fluid collected from the living body by the microneedles 3 in the center of the substrate 2. The third downstream portion 4b3 serves solely to transport interstitial fluid downstream and corresponds to the water absorption portion of a testing device using a conventional immunoassay method.

[0058] Although the downstream portion 4b is formed by recessing the lower surface 2a of the substrate 2 upward, it is not necessarily required to form the downstream portion 4b in this manner. Furthermore, the height position of the surface of the downstream portion 4b may be higher or lower than the base of the microneedle 3 (the end on the substrate 2 side), or may be at the same height position.

[0059] As shown in Figure 3, a large number of downwardly protruding microprotrusions 10 are formed on the needle peripheral portion 4a. The microprotrusions 10 can be formed by, for example, injection molding or nanoimprint lithography (NIL), but the method for forming the microprotrusions 10 is not limited to these. The microprotrusions 10 are an example of a "microstructure" for increasing the surface area of ​​the body fluid flow path 4 and improving the wettability of the needle peripheral portion 4a. Each microprotrusion 10 is approximately cylindrical.

[0060] The width b of the end of the microprotrusion 10 on the substrate 2 side is preferably 600 nm or less (more preferably 360 nm or less), and the dimensional ratio of the height a of the microprotrusion 10 to the width b is preferably 0.3 or more (more preferably 0.5 or more) (a / b≧0.3, a / b≧0.5). Furthermore, the spacing c between the microprotrusions 10 is preferably set in the range of 200 nm to 400 nm. Note that in the speech bubble in FIG. 3, "c" indicates the horizontal distance between the central axes of the microprotrusions 10.

[0061] By configuring the microprotrusions 10 in this manner, the surface area of ​​the needle peripheral portion 4a can be increased, and therefore the needle peripheral portion 4a can be maintained in a highly hydrophilic state with a wetted area ratio of 70% or more and a contact angle of 45° or less for a long period of time.

[0062] Similarly, a large number of microprotrusions 10 are also formed on the surface (lower surface) of the downstream portion 4b. By setting the width of the bottom surface, the ratio of height to width, and the spacing between the microprotrusions 10 of the downstream portion 4b within the above ranges, the downstream portion 4b can be maintained in a highly hydrophilic state with a wetted area ratio of 70% or more and a contact angle of 45° or less for a long period of time.

[0063] In measuring the wetted area ratio of the needle peripheral portion 4a or the downstream portion 4b, first, a plate-shaped test piece is prepared having a surface with the same surface condition as the needle peripheral portion 4a or the downstream portion 4b, such as material, surface treatment, and arrangement of microprotrusions.

[0064] Next, with the test piece held horizontally with the surface facing upward, water was poured into the test piece from a distance of 100 mm in several separate portions at a total rate of 0.016 cc / cm ２ For example, spray the surface with an amount of 100 cm ２ In this case, the amount of water sprayed is 1.6 cc.

[0065] Thereafter, the surface is held at a right angle for 5 seconds, and the wetted area ratio, which is the ratio of the area wetted by water (the area of ​​the portion wetted with water) to the entire area of ​​the surface of the test piece, is determined. The wetted area can be determined, for example, by photographing the surface of the test piece and analyzing the image obtained.

[0066] The shape of the microprotrusions formed in the body fluid flow path 4 is not particularly limited. For example, as shown in Figure 4, the microprotrusions 20 may be formed in a conical shape similar to the microneedles 3. In this case, too, in order to enhance the hydrophilicity of the body fluid flow path 4, it is preferable to set the width b of the bottom surface of the microprotrusions 20 (contacting the body fluid flow path 4) to a dimension of 600 nm or less (more preferably 360 nm or less), set the dimensional ratio of the height a of the microprotrusions 20 to the width b to 0.3 or more (more preferably 0.5 or more) (a / b ≥ 0.3, a / b ≥ 0.5), and set the arrangement interval c between the microprotrusions 10 in the range of 200 nm to 400 nm.

[0067] As described above, by making the body fluid flow path 4 highly hydrophilic and forming microprotrusions 10, 20 throughout the body fluid flow path 4, a portion of the interstitial fluid collected by the microneedle 3 can be passively and smoothly transported to the downstream side of the body fluid flow path 4.

[0068] At this time, due to the viscosity of the interstitial fluid, a flow of interstitial fluid is formed from the surface of the microneedle 3 and the needle periphery 4a toward the downstream side of the body fluid flow path 4 (in the direction of arrow X, which represents the extension direction of the body fluid flow path 4; see Figure 1).

[0069] In many conventional testing devices using immunochromatography, the body fluid flow path uses a fiber pad made of cellulose or the like.

[0070] In contrast, the entire body fluid flow path 4 of this embodiment has been subjected to surface treatment such as plasma treatment, corona treatment, flame treatment, chemical treatment, UV irradiation, etc. Therefore, compared to the body fluid flow paths of conventional testing devices, it is possible to prevent the transfer of body fluid from being stagnate, and to improve the controllability of the transfer of body fluid.

[0071] In the downstream section 4b of the body fluid flow path 4, a first downstream section 4b1 and a second downstream section 4b2 are formed with an immunoassay section 5 that detects biomarkers contained in the interstitial fluid by antigen-antibody reaction.

[0072] In this way, since the microneedles 3, the body fluid flow paths 4, and the immunoassay section 5 are all arranged on the lower surface 2a (one surface) of the substrate 2, it is possible to omit a special mold for the upper surface 2b of the substrate 2. Therefore, the manufacturing cost of the testing device 1 can be reduced.

[0073] 1, the third downstream section 4b3 is shaped to surround the microneedle 3, thereby ensuring a sufficient length of the body fluid flow path 4 even with a compact substrate 2. Therefore, even when detecting a biomarker that is contained in only trace amounts in interstitial fluid, a large amount of interstitial fluid can be supplied to the immunoassay section 5, and the color development of the test section 5b1, which will be described below, can be intensified.

[0074] The immunoassay section 5 includes a conjugate section 5a that labels the biomarker in the interstitial fluid, and a color-developing section 5b that visualizes the detection results.

[0075] The conjugate section 5a is formed in the first downstream section 4b1. The conjugate section 5a is provided with a labeling substance that specifically binds to and labels the biomarker to be detected.

[0076] The labeling substance has a binding moiety that specifically binds to the target biomarker and a labeling moiety, and the binding moiety can be an antibody, an antibody fragment, an aptamer, or the like.

[0077] Examples of the labeling moiety include, but are not limited to, metal colloid particles, fluorescent dye particles, colored latex particles, upconversion phosphor particles, quantum dot particles, etc. The particle diameter of the labeling moiety is preferably 20 nm or more and 1000 nm or less.

[0078] When the interstitial fluid is collected from the microneedle 3 to the conjugate portion 5a, the labeling substance of the conjugate portion 5a disperses into the interstitial fluid, resulting in a primary reaction in which biomarkers that may be contained in the interstitial fluid bind to the labeling substance at the conjugate portion 5a or a downstream position thereof and are labeled.

[0079] Thereafter, the biomarker bound to the labeling substance (hereinafter also referred to as "complex") flows downstream through the body fluid flow path 4 and is visualized by the color-developing portion 5b (secondary reaction).

[0080] The color-developing section 5b is formed in the second downstream section 4b2. The color-developing section 5b includes a test section 5b1 provided with a first antibody capable of specifically capturing a biomarker bound to a labeling substance, and a control section 5b2 disposed downstream of the test section 5b1.

[0081] Examples of the first antibody include, but are not limited to, antibodies, antibody fragments, and aptamers.

[0082] If the interstitial fluid contains a biomarker, the complex is successively captured by the first antibody in the test section 5b1 as the interstitial fluid reaches the immunoassay section 5 and flows further downstream in the direction of arrow X through the third downstream section 4b3.

[0083] As a result, the marker substance is concentrated in the test portion 5b1 along with the biomarker, and the presence or absence and intensity of reflected light, fluorescence, etc., derived from the marker substance can be confirmed visually or by using a device. If the target biomarker is not contained in the collected interstitial fluid, the test portion 5b1 will not develop a color. In this way, the test portion 5b1 can visualize the presence of the biomarker in the body fluid.

[0084] The control section 5b2 is provided with a second antibody that specifically captures the labeled substance. In this embodiment, the first and second antibodies are immobilized on beads and arranged among the numerous microprotrusions 10. However, as will be described in detail later with reference to Figures 5 to 9B, they may be immobilized on the surface (lower surface) of the second downstream section 4b2, or the antibody may be immobilized directly on the second downstream section 4b2 or the microprotrusions 10 without using beads. Examples of the second antibody include, but are not limited to, an antibody, an antibody fragment, an aptamer, etc.

[0085] When the interstitial fluid is transported through the body fluid flow path 4 and reaches the control section 5b2, the labeling substance dispersed in the interstitial fluid is captured by the second antibody.

[0086] As a result, the labeling substance is concentrated in the control section 5b2, and the presence or absence and intensity of reflected light, fluorescence, etc. derived from the labeling substance can be confirmed visually or by using a device.

[0087] The operator of the testing device 1 can confirm whether or not the interstitial fluid, which is the sample, has been successfully supplied to the color-producing section 5b by checking whether or not the control section 5b2 has developed a color. If the control section 5b2 has developed a color, the operator can determine that the interstitial fluid has been successfully supplied to the color-producing section 5b.

[0088] Here, the portion of the substrate 2 located above the color-producing portion 5b is formed to be transparent.

[0089] In addition, an optical system 7 is disposed above the transparent portion of the substrate and the color-producing portion 5b to enhance the visibility of the color-producing portion 5b that indicates the detection result.

[0090] Therefore, the operator of the inspection device 1 can easily check the reflected light from the color-producing portion 5b from above the through hole 6a of the fixed seal 6 through the optical system 7 with the naked eye or using an imaging device such as a smartphone.

[0091] Examples of optical systems 7 that enhance visibility include, but are not limited to, magnifying optical systems such as magnifying lenses that magnify the light reflected from the color-producing portion 5b, optical systems that prevent scattered and / or reflected light, and optical systems that enhance contrast. Examples of optical systems that prevent scattered light include optical systems that reduce the reflection and scattering of light by applying a coating to the surface of an optical material such as glass. For example, forming an anti-reflection film (AR coating) on ​​the surface of the optical material can reduce light of specific wavelengths and improve the characteristics of the optical material. In this case, improved contrast can also be expected.

[0092] Below, a detailed description is given of a method for immobilizing antibodies (first and second antibodies) on the hydrophilically treated surface (lower surface) of the second downstream section 4b2 or on any position on the microprotrusions 10. The antibodies can be immobilized directly on the surface of the second downstream section 4b2 or on the microprotrusions 10, or they can be immobilized via a carrier. The material of the carrier is not particularly limited, but it can be composed of, for example, polymer particles such as beads or glass particles. Note that the method for immobilizing the antibodies is not limited to the following method.

[0093] <Immobilization on the surface of the second downstream section 4b2> (Immobilization method 1) An antibody or a carrier on which the antibody is immobilized is embedded in a highly viscous gel and attached to and adsorbed in a desired region of the second downstream section 4b2. In detail, the antibody or the carrier on which the antibody is immobilized is mixed with a gel monomer, and a desired amount is added to the surface of the second downstream section 4b2 and allowed to stand until gelation occurs, thereby immobilizing the antibody on the second downstream section 4b2.

[0094] (Immobilization method 2) Fig. 5 is a schematic bottom view of the second downstream section 4b2 according to antibody immobilization method 2. As shown in Fig. 5, carrier 13 on which an antibody is immobilized is added by inkjet or the like to the inside of frame 14, which has been subjected to a hydrophobic treatment on the surface of second downstream section 4b2, and then dried and immobilized. Note that it is also possible to add only the antibody without using carrier 13.

[0095] The hydrophobic frame 14 may be formed, for example, as a fine groove carved into a part of the hydrophilic second downstream portion 4b2 by laser processing, or may be formed by applying a hydrophobic composition to the second downstream portion 4b2.

[0096] For example, if a hydrophobic frame 14 is formed in the test section 5b1 and a carrier 13 to which a first antibody is immobilized is immobilized in the region inside the frame 14, when the interstitial fluid initially flows into the test section 5b1 on the second downstream section 4b2, the interstitial fluid will pass around the frame 14, bypassing it. However, as the amount of interstitial fluid flowing through the test section 5b1 per unit time increases, at some point the interstitial fluid will exceed the frame 14 and invade the hydrophilic region within the frame 14. As a result, the complex in the interstitial fluid is captured and concentrated by the first antibody, causing the inside of the frame 14 to develop a color.

[0097] By placing the antibody or the carrier 13 to which the antibody is attached within the hydrophobic frame 14, it is possible to prevent the antibody from being washed downstream together with the interstitial fluid.

[0098] The diameter of the frame 14 may be approximately 150 μm, but the size of the frame 14 is not particularly limited. Furthermore, in Fig. 5, the frame 14 is circular when viewed from the bottom, but the shape of the hydrophobic frame surrounding the first antibody is not particularly limited, and it may be formed into another closed ring shape, such as a rectangle. Furthermore, multiple circular frames 14 may be arranged in a direction substantially perpendicular to the extension direction X of the body fluid flow path 4 so that the multiple circular frames 14 appear as a single line as a whole.

[0099] (Immobilization Method 3) Fig. 6 is a schematic cross-sectional view of the surface of the second downstream section 4b2 according to antibody immobilization method 3. As shown in Fig. 6, a mask 17 is attached to at least the area of ​​the surface of the second downstream section 4b2 where the antibody is not to be immobilized, and the antibody can be immobilized by adding a carrier 13 onto which the antibody is immobilized to the second downstream section 4b2 and drying it. The mask 17 is removed after the carrier 13 is immobilized. Note that it is also possible to add only the antibody without using the carrier 13.

[0100] (Immobilization Method 4) First, as in Immobilization Method 3, a region of the surface of the second downstream section 4b2 to which no antibody is immobilized is masked, and then a binding molecule is added to the second downstream section 4b2 and dried. The binding molecule is a molecule capable of binding to an antibody or a carrier 13. After drying, the mask is removed, and an antibody or a carrier 13 to which an antibody is immobilized is added to the second downstream section 4b2 and dried, thereby immobilizing the antibody to the second downstream section 4b2. After drying the antibody, the second downstream section 4b2 may be washed by supplying a buffer solution or the like to remove the antibody that is not bound to the binding molecule.

[0101] <Immobilization onto the Microprotrusions 10> As a method for immobilizing antibodies onto the microprotrusions 10, the above-mentioned immobilization methods 1 to 4 can be used, and also, for example, the following immobilization methods 5 to 7 can be used.

[0102] (Immobilization Method 5) FIG. 7 is a schematic side view showing a method for forming microprotrusions 10 according to antibody immobilization method 5. As shown in FIG. 7 , first, a photocurable resin 16 containing dispersed antibodies or carriers 13 with antibodies immobilized thereon is added to the second downstream section 4b2. A mask 17 used in photolithography is then attached to the surface of the resin 16 to cover the areas where the microprotrusions 10 will not be formed, and the photocurable resin 16 is partially cured by irradiating it with light (e.g., ultraviolet light) of a wavelength appropriate for the type of resin. Finally, the uncured photocurable resin 16 is removed by washing and dried, yielding microprotrusions 10 with antibodies immobilized on their surfaces.

[0103] (Immobilization Method 6) Figure 8A is an enlarged side view of the second downstream section 4b2 according to antibody immobilization method 6, and Figure 8B is an enlarged bottom view of the second downstream section 4b2 according to antibody immobilization method 6. First, with the microprotrusions 10 formed by microfabrication such as injection molding or NIL facing upward, carrier 13 to which an antibody is immobilized is added near the microprotrusions 10 and allowed to stand for a certain period of time. Thereafter, the second downstream section 4b2 is washed to remove carrier 13 located outside the spaces between the microprotrusions 10, and then dried.

[0104] (Immobilization Method 7) Figure 9A is an enlarged side view of the second downstream section 4b2 according to antibody immobilization method 7, and Figure 9B is an enlarged bottom view of the second downstream section 4b2 according to antibody immobilization method 7. As shown in Figure 9A, first, a well structure (recess) 15 is formed at a desired position in the second downstream section 4b2 by microfabrication such as injection molding or NIL. Next, a carrier 13 on which an antibody is immobilized is added near the well structure 15 and allowed to stand for a certain period of time. Thereafter, the carrier 13 located outside the well structure 15 is removed by washing and dried, thereby allowing the antibody to be immobilized at a desired position in the second downstream section 4b2.

[0105] 1, the second downstream section 4b2 of the body fluid flow path 4, where the color-developing section 5b is formed, has a shape that widens in width as it extends downstream. By forming the second downstream section 4b2 of the body fluid flow path 4 in this manner, the flow rate of the interstitial fluid that passively flows through the immunoassay section 5 can be increased compared to when the second downstream section 4b2 is formed with a constant width.

[0106] 10, the bodily fluid flow path 4 may be formed in a shape in which a plurality of repeating units U, each of which has a shape that increases in width as it proceeds downstream, are connected in the direction in which the bodily fluid flow path extends (the direction X in which the bodily fluid flows). By forming the repeating units U in this way, the speed at which the interstitial fluid passively flows through the bodily fluid flow path 4 can be further increased compared to when the width simply continues to increase as it proceeds downstream.

[0107] Furthermore, in this embodiment, a needle peripheral portion 4a is arranged around the microneedle 3, and a downstream portion 4b is arranged to surround the needle peripheral portion 4a, but the arrangement and shape of the microneedle and the body fluid flow path are not particularly limited.

[0108] FIG. 11 is a schematic diagram showing the function of the body fluid flow path 45 according to yet another preferred embodiment. As shown in FIG. 11, the body fluid flow path 45 may be formed linearly on the underside of the substrate, and the needle peripheral portion 45a, the first downstream portion 45b1, the second downstream portion 45b2, and the third downstream portion 45b3 may be arranged in a straight line, in that order from the microneedle 3 side. In addition, in this case, the entire linear body fluid flow path may be formed so that its width increases toward the downstream side. Note that in FIG. 11, the same reference numerals are used to designate the same components as those in the testing device 1 according to the embodiment shown in FIG. 1.

[0109] As shown in FIG. 11 , a conjugate portion 55a provided with a labeling substance may be formed in the second downstream portion 45b2. If the conjugate portion 55a is formed in the second downstream portion 45b2 and the distance between the conjugate portion 55a where the primary reaction occurs and the color-developing portion 5b where the secondary reaction occurs becomes excessively short, it is preferable to form a step 8 between the conjugate portion 55a and the color-developing portion 5b on the body fluid flow path 45 to ensure sufficient time for labeling the biomarker contained in the interstitial fluid. By forming the step 8 in the body fluid flow path 45, the body fluid passing through the conjugate portion 55a can come into contact with the step 8, thereby slowing the flow rate of the body fluid and lengthening the time it takes for the body fluid to reach the color-developing portion 5b. The step 8 is formed so that the downstream portion of the second downstream portion 45b2 where the color-developing portion 55b is formed is located lower than the portion where the conjugate portion 55a is formed. However, the portion of the second downstream section 45b2 where the color-developing section 55b is formed may be positioned higher than the portion where the conjugate section 55a is formed.

[0110] Furthermore, fibers such as cellulose, glass, cotton, etc. may be placed in the body fluid flow path, as in conventional testing devices using immunoassay methods. The location in the body fluid flow path where the fibers are placed is not particularly limited, and the fibers may be placed in part or all of the body fluid flow path.

[0111] 11, fibers may be disposed in place of or together with the microprotrusions 10 in the linearly formed body fluid flow path 45. The fibers may be disposed in the first downstream section 45b1 that collects interstitial fluid. The fibers may also be disposed in the second downstream section 45b2, in a section where the conjugate section 55a where the primary reaction occurs is formed, or in a section where the color-developing section 5b where the secondary reaction occurs is formed. The fibers may also be disposed in the third downstream section 45b3 that primarily transports interstitial fluid downstream.

[0112] FIG. 12 is a schematic perspective view of the vicinity of the body fluid flow path 4 of a testing device 100 according to another preferred embodiment of the present invention, and FIG. 13 is a schematic bottom view of the vicinity of the transfer groove 40 shown in FIG.

[0113] FIG. 12 shows a downstream portion 4b, which is a part of the body fluid flow path 4, including a longitudinal cross section.

[0114] Note that Figures 12 and 13 show different portions of the transport groove 40 in the extension direction of the downstream portion 4b, and the transport groove 40 shown in Figure 12 is the portion immediately downstream of the transport groove 40 shown in Figure 13.

[0115] The testing device 100 according to this embodiment has the same configuration as the testing device 1 according to the embodiment shown in FIGS. 1 to 3, except for the following points.

[0116] A plurality of transport grooves 40, which are an example of a "microstructure" for increasing the surface area of ​​the downstream portion 4b and improving the wettability of the downstream portion 4b, are formed on the surface of the downstream portion 4b.

[0117] 12 and 13, the bottom of each transfer groove 40 is shown as 40a.

[0118] Each transport groove 40 is formed in the downstream portion 4b in the desired direction of interstitial fluid flow, i.e., toward the downstream side. The transport grooves 40 extend parallel to each other on the surface of the downstream portion 4b. By forming the transport grooves 40 on the surface of the downstream portion 4b in this way, the surface area of ​​the downstream portion 4b can be increased, thereby improving the transport speed of interstitial fluid.

[0119] The width of each transport groove 40 is preferably in the range of 100 nm or more and less than 1000 nm.

[0120] 13, the transport grooves 40 branch off at a branch point B1 in the downstream section 4b. Increasing the number of transport grooves 40 toward the downstream side of the downstream section 4b in this way increases the surface volume of the downstream section 4b, increases the passive transport force of the interstitial fluid by the downstream section 4b, and increases the flow rate of the interstitial fluid, thereby suppressing stalling of the interstitial fluid downstream.

[0121] In the downstream portion 4b, two adjacent transport grooves 40 are connected to each other by connecting grooves 41, 42 extending in a direction perpendicular to the body fluid flow direction X. This allows for stable transport of interstitial fluid.

[0122] Although the connecting groove 41 connects two adjacent transport grooves 40 and the connecting groove 42 connects three adjacent transport grooves 40, the number of transport grooves 40 connected by a single connecting groove is not limited to two or three. Therefore, a single connecting groove may be configured to connect four or more adjacent transport grooves 40.

[0123] Figure 14A is a schematic side view showing the up and down positions of the substrate 2000 in the standard state of the testing device 1000 in yet another preferred embodiment of the present invention, and Figure 14B is a schematic side view showing the up and down positions of the substrate 2000 in the puncture state of the testing device 1000.

[0124] The testing device 1000 according to this embodiment has the same configuration as the testing device 1 according to the embodiment shown in FIGS. 1 to 3, except for the following points.

[0125] The testing device 1000 does not have a fixed seal 6 , and is provided with a pair of substrate support parts 9 that support a substrate 2000 , and a connecting part 12 that connects the pair of substrate support parts 9 .

[0126] A through hole (not shown) that penetrates in the vertical direction is formed in the part of the connecting portion 12 that is located above the optical system 7, and the color-producing portion 5b can be seen through this through hole and the optical system 7.

[0127] Each substrate support part 9 has a slide part 9a that is slidable in the vertical direction. The substrate 2000 is attached to the slide parts 9a of the pair of substrate support parts 9.

[0128] A protective film 11 for protecting the microneedles 3 is provided on the tip side, i.e., below, of the microneedles 3. The left and right ends of the protective film 11 on the paper surface (drawing) are fixed to the lower surfaces of a pair of substrate support parts 9.

[0129] The protective film 11 includes a first layer 11a fixed to the pair of substrate support members 9 and a second layer 11b attached to the underside of the first layer 11a. An adhesive layer is formed on the underside of the second layer 11b, but the presence or absence of the adhesive layer is not particularly limited. Furthermore, the protective film 11 does not necessarily need to include both the first layer 11a and the second layer 11b; it may include only one of the first layer 11a and the second layer 11b. Furthermore, the protective film 11 may include one or more layers in addition to the first layer 11a and the second layer 11b. In other words, the number of layers included in the protective film 11 is not particularly limited. Possible functions of additional layers to the protective film 11 include, for example, improving permeability to bodily fluids and providing adhesive properties, but are not limited thereto.

[0130] The first layer 11a serves to stretch the second layer 11b. In the first layer 11a, a through-hole 11a1 is formed in a portion located below each microneedle 3, penetrating in the vertical direction.

[0131] The second layer 11b is designed to be pierceable by the microneedles 3, and serves to stretch the skin and protect the microneedles 3 when the skin is punctured.

[0132] In the standard state before puncturing, the substrate 2000 is located at the up-down position shown in FIG. 14A.

[0133] With the pair of substrate support members 9 and the underside of the protective film 11 in contact with the skin, the substrate 2000 is pressed downward and slid to the up or down position shown in Figure 14B, allowing the microneedles 3 to puncture the skin (puncture state).

[0134] As shown in FIG. 14B, in the puncturing state in which the microneedles 3 are puncturing the skin, each microneedle 3 penetrates the second layer 11b through the through-hole 11a1 and pierces the skin.

[0135] In this way, by piercing the skin through the protective film 11 while the underside of the protective film 11 is placed against the skin, the skin can be punctured while minimizing bending, and interstitial fluid can be collected minimally invasively.

[0136] The thickness of the protective film 11 is not particularly limited as long as it is less than the height dimension of the microneedles 3, and can be set appropriately. For example, when the height of the microneedles 3 is set in the range of 100 μm to 2 mm as described above, the thickness is preferably 50 μm to 1950 μm depending on the height of the microneedles 3. The materials of the first layer 11a and the second layer 11b are not particularly limited, and examples that can be used include titanium, stainless steel, polyglycolic acid, polycarbonate, cyclic olefin copolymer (COC), cyclic olefin polymer, polyether ether ketone (PEEK), alginate, chitin, chitosan, collagen, hydroxypropyl cellulose, and pullulan.

[0137] The protective film 11 preferably includes a layer permeable to interstitial fluid or other bodily fluids as the first layer 11a, the second layer 11b, or another layer. This increases the amount of bodily fluid that can be collected from the skin. Examples of materials permeable to interstitial fluid or other bodily fluids include nitrocellulose, polyvinylidene fluoride, nylon, and polyethersulfone.

[0138] FIG. 15 is a schematic bottom view of the vicinity of the test portion 50b1 of the testing device according to the embodiment of the present invention capable of detecting multiple items.

[0139] In this embodiment, the second downstream portion configured in the same manner as in the embodiment shown in FIG. 1 is designated by the same reference numerals and description thereof will be omitted.

[0140] In this embodiment, the test section 50b1 includes four coloring regions 50b1a-50b1d arranged in the second downstream section 4b2 along the direction X of bodily fluid flow. Each of the four coloring regions 50b1a-50b1d is provided with a type of first antibody that specifically captures a different antigen, making it possible to detect a total of four biomarkers. The following describes the process from collecting interstitial fluid to visualizing the presence of the four biomarkers.

[0141] The conjugate portion formed in the first downstream portion 4b1 contains a mixture of four types of labeling substances that bind to the four types of biomarkers in a one-to-one relationship. The colors of the four types of labeling substances may be the same or different from one another.

[0142] When interstitial fluid is collected by puncturing with the microneedle 3 and transported through the needle peripheral portion 4a to the first downstream portion 4b1, the four types of labeled substances in the conjugate portion disperse in the interstitial fluid. The four types of labeled substances then bind to biomarkers that may be contained in the interstitial fluid while mixing with the interstitial fluid and flowing through the first downstream portion 4b1 and the second downstream portion 4b2 (the portion upstream of the position where the test portion 50b1 is formed). As a result, up to four types of complexes are formed.

[0143] Each of the four color-producing regions 50b1a to 50b1d is provided with a first antibody that specifically captures one of the four types of complexes, and up to four types of complexes are captured in the color-producing regions 50b1a to 50b1d. As a result, the biomarkers bound to the labeling substances are concentrated in up to four color-producing regions 50b1a to 50b1d, and develop a color intensity that can be seen through the optical system 7.

[0144] In this way, the four color-forming regions 50b1a-50b1d can visualize the presence of different biomarkers. The color-forming regions 50b1a-50b1d in the second downstream section 4b2 are preferably arranged such that the color-forming regions that visualize biomarkers present in small amounts in the body fluid are located downstream, and the color-forming regions that visualize biomarkers present in large amounts in the body fluid are located upstream. This increases the transport distance of the biomarkers present in small amounts from the conjugate section to the corresponding color-forming regions, ensuring sufficient binding time (binding opportunity) between the biomarkers present in small amounts and the labeling substance, thereby increasing the color development of the biomarkers present in small amounts.

[0145] Furthermore, each color-producing region of the test section may be formed in an inner region (not inside the groove) of a groove extending in a closed ring shape (e.g., circular, rectangular, etc.) formed on the surface (bottom surface) of a body fluid flow path such as the second downstream section 4b2, as will be described in detail later in Example 3.

[0146] When the amount of body fluid transported through the body fluid flow path increases and the body fluid passes over the groove structure that extends around the color-producing region so as to surround the color-producing region and reaches the inner color-producing region, the biomarker that may be contained in the body fluid and the labeled substance bound to the biomarker are captured in the color-producing region, and the biomarker is detected by coloring.

[0147] Furthermore, the number of types of antibodies and the number of color-developing regions are not limited to four, but may be three or less or five or more.

[0148] In addition, in this embodiment, as shown in Figure 15, four coloring regions 50b1a to 50b1d are arranged along the extension direction of the body fluid flow path (the direction of arrow X), but multiple coloring regions may also be arranged, for example, in the width direction of the body fluid flow path (the direction approximately perpendicular to arrow X).

[0149] Furthermore, in an embodiment capable of detecting multiple items, the transport grooves 40 and connecting grooves 41 shown in Figure 13 may be formed in the downstream portion 4b instead of the microprotrusions 10. Even in the case of detecting multiple items, it is desirable that the number of transport grooves 40 increases toward the downstream side of the body fluid flow path 4.

[0150] Additionally, in this embodiment, the microneedle 3, the body fluid flow path 4, and the immunoassay section including the test section 50b1 are arranged on the underside 2a (one side) of the substrate 2, but a through-hole may be formed that penetrates the substrate in the vertical direction, and the through-hole may be used as a body fluid flow path to transfer body fluid collected by the microneedle 3 on the underside of the substrate to the upper surface of the substrate, and an immunoassay section including a test section capable of detecting multiple items may be provided on the upper surface.

[0151] Also in this embodiment, fibers such as cellulose, glass, cotton, etc. may be disposed in part or all of the body fluid flow path 4 .

[0152] [Testing method] The testing method of this embodiment includes the steps of puncturing the skin with the microneedle of a testing device having a microneedle, a body fluid flow path for transporting body fluid, and an immunoassay section for detecting biomarkers by antigen-antibody reaction on one side of a substrate to collect body fluid, transporting the collected body fluid through the body fluid flow path (in other words, on the body fluid flow path), and detecting the biomarkers contained in the body fluid transported through the body fluid flow path using the immunoassay section.

[0153] The testing method of this embodiment is a method for testing for the presence or absence of a biomarker using the testing device according to each embodiment shown in Figures 1 to 15 or a testing device described below. Therefore, in the testing method of this embodiment, as described above, a microneedle is punctured into the skin, and while the body fluid that has flowed along the needle surface or passed through the hollow part inside the needle is transported through the body fluid flow path, a biomarker that may be contained in the body fluid is detected in an immunoassay unit located in the body fluid flow path.

[0154] The immunoassay section of the test device used in this test method may have a color-developing section as described above, or may have a test section that visualizes the presence of a biomarker in a body fluid. The test section may have multiple color-developing regions that visualize different biomarkers (in other words, the types of biomarkers visualized may differ among the multiple color-developing regions).

[0155] In this case, the color-forming region may be formed in an area inside a groove structure that extends in a closed loop (e.g., a circle or a rectangle) formed on the surface of the body fluid flow path, as in Example 3 described below. Multiple color-forming regions may be located inside a single groove structure, or each color-forming region may have its own groove structure outside it.

[0156] Each of the multiple color-producing regions may be provided with an antibody that specifically captures a different biomarker (in other words, each color-producing region may have a different type of antibody), allowing each color-producing region to capture and visualize (detect color with the naked eye or an image sensor) a different biomarker and detect it.

[0157] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these modifications are also included within the scope of the present invention.

[0158] For example, in the embodiment shown in Figures 1 to 3, fine irregularities are formed on the surface of the needle 3 by oxygen plasma treatment, but as shown in Figure 16A, a circumferentially extending horizontal groove 3a may be formed on the surface of the approximately conical microneedle 3, or as shown in Figure 16B, a portion of the height of the microneedle 3 may be formed in an arrowhead shape that protrudes radially outward.

[0159] Forming unevenness such as the lateral grooves 3a and protrusions 3b on the microneedles 3 improves the wettability of the microneedles 3 and also forms a partial gap between the microneedles 3 and the skin when the microneedles 3 are punctured into the skin, making it easier to supply interstitial fluid exuding from the skin to the body fluid flow path. Note that in microneedles 3 having the lateral grooves 3a formed therein, when viewed from inside the lateral grooves 3a, the corners at the entrances of the lateral grooves 3a are convex, and in arrowhead-shaped microneedles 3, there are concaves above the protrusions 3b.

[0160] When forming the lateral grooves 3a in the microneedles 3, a trapezoidal tool having protrusions on the left and right sides corresponding to the lateral grooves 3a is inserted into the space between adjacent microneedles 3, which is approximately trapezoidal in side view, and the lateral grooves 3a can be formed by moving the tool in and out in the depth direction of the drawing. Similarly, when forming the microneedles 3 into an arrowhead shape, the microneedles 3 can be formed by moving in and out a trapezoidal tool having protrusions on the left and right sides that cut the base portion of the needles 3. Note that instead of using a tool, it is also possible to form the lateral grooves 3a and protrusions using, for example, semiconductor lithography technology.

[0161] 12 and 13, transport grooves 40 are formed in the body fluid flow path 4 as an example of a microstructure, but the body fluid flow path 4 may also be formed so that its width increases downstream. Alternatively, as shown in Fig. 10, the body fluid flow path may be formed so that multiple repeating units U, each of which increases in width downstream, are connected in the direction of extension of the body fluid flow path. Such a configuration can increase the flow rate of interstitial fluid.

[0162] Furthermore, the configuration relating to the protective film 11 and the pair of substrate support parts 9 shown in FIG. 14 may be applied to the embodiment shown in FIGS.

[0163] 1 to 3 and the embodiment shown in FIG. 10, the downstream portion 4b has a shape in which its width increases downstream. However, at least a portion of the downstream portion 4b in its extending direction may be configured so that its depth increases downstream. This means that the vertical position of the upper surface of the downstream portion 4b shifts upward (the amount of upward recession of the substrate 2 increases), and the height of the walls defining both sides of the downstream portion 4b increases downstream. This configuration increases the surface area of ​​the downstream portion 4b and increases the flow rate of interstitial fluid flowing through the downstream portion 4b.

[0164] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0165] Example 1: Fabrication of a microstructure for improving wettability (1) (Fabrication of a microstructure) First, a fine pre-relief pattern was transferred onto the surface of an aluminum plate (2 x 2 x 0.014 mm, purity 99.99%) using a 200 nm periodic protrusion structure mold, and then anodized in the following electrolytic solution to obtain a nanopore structure with a pore period of 200 nm. The electrolysis conditions were as follows: Electrolytic solution composition: phosphoric acid 5.0 g / L Electrolysis voltage: 100 V constant voltage Electrolysis temperature: 0°C Electrolysis time: 10 minutes

[0166] Next, the pore structure was adjusted by etching under the following conditions: Etching solution composition: 50 g / L phosphoric acid, Etching solution temperature: 30°C, Etching time: 10 minutes. Figure 17 shows a high-magnification image of the mold structure obtained by this etching process, with a pore period of 200 nm, a pore diameter of 150 nm, and a pore length of approximately 400 nm. The scale bar in Figure 17 is 200 nm.

[0167] After washing and drying the resulting simple mold, a nanopore structure-forming substrate was attached. Methyl methacrylate (MMA) containing 10 wt% polyethylene glycol and 5 wt% benzoyl peroxide was dripped onto the nanopore structure under vacuum to fill the pores and coat the pore structure substrate. The mixture was then heated at 40°C for 48 hours for polymerization to produce PMMA. The PMMA-coated nanopore structure-forming substrate was removed from the simple mold and immersed in a phosphoric acid-chromic acid mixed solution (35 mL of 85% phosphoric acid + 20 g / L chromium trioxide solution) at 30°C for 3 hours, selectively dissolving and removing the alumina. A structure with a pillar diameter of 150 nm, a pillar height of 400 nm, and a pillar spacing of 200 nm was produced. A high-magnification image of the resulting microstructure (channel structure) is shown in Figure 18. The scale bar in Figure 18 is 200 nm.

[0168] (Evaluation of wettability) To evaluate wettability, a contact angle meter (CA-X manufactured by Kyowa Interface Science Co., Ltd.) was used to measure the contact angle of 1 μL of water dispensed from a syringe onto the obtained microstructure. Measurements were performed three times, and the average value was used as the contact angle for each sample. As a comparative example, measurements were also performed on PMMA containing 10% PEG without a microstructure. The contact angle measurement results are shown in Table 1 below.

[0169]

[0170] As shown in Table 1, the flow channel structure having pillars on the surface had a significantly smaller water contact angle than the comparative example having no pillars.

[0171] Example 2: Fabrication of a microstructure for improving wettability (2) (Fabrication of a microstructure) First, a fine pre-relief pattern was transferred onto the surface of a titanium plate (2 x 2 x 0.014 mm, purity 99.99%) using a 200 nm protrusion periodic structure mold, and then anodized in the following electrolytic solution to obtain a nanopore structure with a pore period of 200 nm. The electrolysis conditions were as follows: Electrolytic solution composition: 0.5 M phosphoric acid + 0.14 M sodium fluoride Electrolysis voltage: The anodization voltage was continuously ramped from the open circuit potential to a higher value, typically 10 to 30 V, at a rate of 0.1 V / sec, and then increased to 40 V. Electrolysis temperature: 40°C Electrolysis time: 45 minutes

[0172] Next, the pore structure was adjusted by etching under the following conditions: Etching solution composition: 50 g / L phosphoric acid, Etching solution temperature: 30°C, Etching time: 10 minutes. Figure 19 shows a high-magnification image of the mold structure obtained by this etching process, with a pore period of 200 nm, a pore diameter of 150 nm, and a pore length of approximately 1 μm. The scale bar in Figure 19 is 200 nm.

[0173] The resulting simple mold was washed with water and dried, after which a nanopore structure-forming substrate was attached. Methyl methacrylate (MMA) containing 10 wt% polyethylene glycol and 5 wt% benzoyl peroxide was dripped onto the nanopore structure under vacuum to fill the pores and coat the pore structure substrate. The mixture was then heated at 40°C for 48 hours for polymerization to produce PMMA. The PMMA-coated nanopore structure-forming substrate was removed from the simple mold and immersed in a phosphoric acid-chromic acid mixed solution (35 mL of 85% phosphoric acid + 20 g / L chromium trioxide solution) at 30°C for 3 hours, selectively dissolving and removing the alumina. A microstructure (channel structure) with a pillar diameter of 150 nm, a pillar height of 1 μm, and a pillar-to-pillar spacing of 200 nm was fabricated. Figure 20 shows a high-magnification image of the resulting microstructure. The scale bar in Figure 20 is 200 nm.

[0174] (Evaluation of wettability) To evaluate wettability, a contact angle meter (CA-X manufactured by Kyowa Interface Science Co., Ltd.) was used to measure the contact angle of 1 μL of water dispensed from a syringe onto the obtained microstructure. Measurements were performed three times, and the average value was used as the contact angle for each sample. As a comparative example, measurements were also performed on PMMA containing 10% PEG without a microstructure.

[0175]

[0176] As shown in Table 2, the flow channel structure having pillars on the surface had a significantly smaller water contact angle than the comparative example having no pillars.

[0177] Example 3: Detection of multiple biomarkers using the immunoassay section Materials and methods Reagents The reagents shown in Table 3 below were used.

[0178]

[0179] (Materials) The materials used were those shown in Table 4 below.

[0180]

[0181] (Preparation of antibody-modified colored beads for detection) 20 μL of colored bead C solution and 1 mg of anti-human IgG antibody [KT131], 20 μL of colored bead M solution and 1 mg of anti-human serum albumin antibody [15C7], and 20 μL of colored bead Y solution and 1 mg of anti-CRP-1 antibody [EPR14345-43] were added to each of the dedicated buffers so that the total volume was 200 μL, and the mixture was mixed by inversion at room temperature for 1 hour to prepare three types of antibody-modified beads for detection.

[0182] (Direct immobilization of capture antibody to immunochromatography flow channel or immobilization via beads) Anti-human IgG antibody [RM116] and biotinylated human C reactive protein antibody [PTX-1] (1 mg each) were added to a dedicated buffer to a total volume of 500 μL to prepare a capture antibody solution. Next, an immunochromatographic flow channel prototype 30 was constructed, which had two circular groove structures 31, 32 (circular diameter 7 mm, groove diameter 0.75 mm, groove depth 0.25 mm) formed on the bottom (surface) 33b of a body fluid flow channel structure (a recess structure formed in a substrate) capable of autonomous fluid transfer. The color-developing regions 31a, 32a were located inside the groove structures. To one of the color-developing regions 31a, 10 μL of the prepared anti-human IgG antibody [RM116] solution was added, and to the other color-developing region 32a, 10 μL of the prepared biotinylated human C reactive protein antibody [PTX-1] solution was added. The resulting channel prototype 30 was then left to stand overnight at 4°C and dried. A top view of the resulting channel prototype 30 is shown in FIG. 21.

[0183] (Preparation of developing sample solution) A dedicated buffer was mixed with a solution containing the above-mentioned antibody-modified colored beads for detection to prepare a total volume of 200 μL of developing sample solution containing IgG protein, Alb protein, and / or CRP protein in the composition shown in Table 5 below. Sample A was a negative control containing no proteins.

[0184]

[0185] (Immunochromatographic assay) 50 μL of developing sample solution was added to the closed end 33 a, which is one end of the body fluid flow path structure 33 of the immunochromatographic flow path prototype 30, and the developing sample solution was passively transported to the other end while the sample was left standing for 15 minutes.

[0186] (Biomarker detection evaluation) The detection of biomarkers in each color-developing region 31a, 32a was evaluated visually and by imaging using a tabletop stereo microscope (Sanwa Supply, LPE-07W) and image analysis using ImageJ. For visual evaluation, the presence or absence of coloration and the color were recorded. For imaging using a tabletop stereo microscope (Sanwa Supply, LPE-07W) and image analysis using ImageJ, a profile was created as a CMYK image from the captured data using ImageJ, and the presence or absence of C, M, and Y signal detection in the color-developing regions 31a, 32a was evaluated from the average signal values ​​of any five points in the image.

[0187] The evaluation results are shown in Table 6 below. For the visual evaluation, if coloration was present, it was rated as "present (color)", and if not, it was rated as "absent". For signal detection, if a signal was detected, it was rated as "all signals detected (CMY)", and if none was detected, it was rated as "absent".

[0188]

[0189] As a result, in sample A, which did not contain any of the protein components of sample A, no coloration was observed visually or by signal detection, whereas in sample B, which contained all of the protein components, blue coloration was observed visually in color region 31a and yellow coloration in color region 32a, and signal detection detected a C signal in color region 31a and a Y signal in color region 32a. Furthermore, in sample C, which contained different protein components, blue coloration was observed visually in color region 31a and no coloration in color region 32a, and signal detection confirmed a C signal in color region 31a and no signal in color region 32a. In sample D, no coloration was observed visually in color region 31a and yellow coloration in color region 32a, and signal detection confirmed no signal in color region 31a and a Y signal in color region 32a. Furthermore, no M signal was detected in either color-producing region 31a or 32a, and no red coloration was observed visually, so there was no false detection of Alb contained in samples B, C, and D.

[0190] From the above, it was demonstrated that the immunoassay part of the present invention can simultaneously detect and measure multiple biomarkers.

[0191] According to the present invention, the microneedles, body fluid channels, and immunoassay units are concentrated on one surface of the substrate, which reduces the number of special molds required for the substrate, thereby reducing manufacturing costs and making the invention industrially applicable.

[0192] 1 Test device 2 Substrate 2a One side surface of substrate (lower surface) 2b Other side surface of substrate (upper surface) 3 Microneedle 3a Lateral groove 3b Protrusion 4 Body fluid flow path 4a Needle peripheral portion 4b Downstream portion 5 Immunoassay portion 5a Conjugate portion 5b Color-developing portion 5b1 Test portion 5b2 Control portion 6 Fixed seal 7 Optical system 9 Substrate support portion 10 Microprotrusion 11 Protective film 11a First layer 11b Second layer 11a1 Through-hole 13 Carrier 14 Frame 15 Well structure 20 Microprotrusion 30 Immunochromatography flow path prototype 31 Groove structure 31a Color-developing region 32 Groove structure 32a Color-developing region 33 Body fluid flow path structure 33a Closed end 33b Bottom surface 40 Transport groove 40a Bottom 41 Connecting groove U Repeating unit

Claims

1. A testing device comprising: a substrate; a microneedle that pricks the skin to collect a bodily fluid; a bodily fluid flow path that transports the bodily fluid collected by the microneedle; and an immunoassay unit that detects biomarkers that may be contained in the bodily fluid transported through the bodily fluid flow path through an antigen-antibody reaction, wherein the microneedle, the bodily fluid flow path and the immunoassay unit are arranged on one side of the substrate.

2. The testing device according to claim 1, wherein a microstructure is formed on the surface of the body fluid flow path to improve the wettability of the body fluid flow path.

3. The testing device according to claim 2, wherein the microstructure is a groove extending in the extension direction of the body fluid flow path.

4. The testing device of claim 3, wherein a plurality of said grooves extend parallel to one another on the surface of said body fluid flow path.

5. The testing device according to claim 4, wherein the number of said grooves increases downstream in said body fluid flow path.

6. The testing device according to claim 5, further comprising connecting grooves formed to connect the plurality of grooves extending in the extension direction of the body fluid flow path.

7. The inspection device of claim 6, wherein the groove has a width in the range of 100 nm to less than 1000 nm.

8. The testing device according to any one of claims 1 to 7, wherein at least a portion of the body fluid flow path has a shape that widens in the downstream direction.

9. The testing device according to claim 8, wherein the body fluid flow path has a plurality of repeating units having the shape linked together in the extending direction of the body fluid flow path.

10. The testing device according to any one of claims 1 to 7, wherein at least a portion of the body fluid flow path becomes deeper in the downstream direction.

11. The testing device according to claim 2, wherein the microstructure is a plurality of microprotrusions provided in the body fluid flow path.

12. The inspection device according to claim 11, wherein the microprotrusions are substantially conical, and when the height of the microprotrusions is a, the width of the bottom surface of the microprotrusions is b, and the arrangement interval of the microprotrusions is c, the microprotrusions have a shape that satisfies the following formulas (1), (2), and (3): b≦600 (unit: nm) (1) a / b≧0.3 (2) 200≦c≦400 (unit: nm) (3) 13. An inspection device described in any one of claims 1 to 7, wherein the surface of the microneedle is formed with projections and recesses.

14. An inspection device described in any one of claims 1 to 7, wherein a protective film is provided on the tip side of the microneedle, and the thickness of the protective film is less than the height of the microneedle.

15. The test device according to any one of claims 1 to 7, wherein the portion of the substrate located on the other side of the immunoassay section is formed to be transparent.

16. The testing device according to claim 15, further comprising a magnifying optical system provided on the other side of said portion located on the other side of said immunoassay portion.

17. An inspection device described in any one of claims 1 to 7, wherein the surface on one side of the substrate and the surface of the microneedle are formed from the same material, and the contact angle of water on the surface on one side is 45° or less.

18. The testing device of any one of claims 1 to 7, wherein the microneedle is a solid, hollow, porous, or hydrogel structure.

19. An testing device according to any one of claims 1 to 7, wherein the body fluid flow path is shaped to surround the microneedle.

20. An inspection device described in any one of claims 1 to 7, wherein the immunoassay portion includes a test portion for visualizing the presence of the biomarker in the bodily fluid, and the test portion includes a plurality of color-developing regions for visualizing the presence of different biomarkers.

21. The testing device described in claim 20, wherein the color-producing region is formed in an inner region of a groove structure extending in a closed ring shape formed on the surface of the body fluid flow path, and each of the multiple color-producing regions is provided with an antibody that specifically captures a different biomarker from the others.

22. A testing method comprising the steps of: puncturing the skin with a microneedle of a testing device having, on one side of a substrate, a bodily fluid flow path for transporting bodily fluid, and an immunoassay section for detecting a biomarker by antigen-antibody reaction, transporting the collected bodily fluid through the bodily fluid flow path; and detecting a biomarker contained in the transported bodily fluid in the immunoassay section.

23. The testing method described in claim 22, wherein the immunoassay section comprises a test section which visualizes the presence of the biomarker in the body fluid, the test section comprises a plurality of color-producing regions which visualize the presence of different biomarkers, the color-producing regions being formed in an inner region of a groove structure extending in a closed ring formed on the surface of the body fluid flow path, and each of the plurality of color-producing regions is provided with an antibody which specifically captures a different biomarker, and the antibody captures and detects a different biomarker in each color-producing region.

Images