Devices and test kits

Abstract

This device offers excellent sensitivity for detecting extracellular vesicles and is easy to manufacture. [Solution] The device 100 is a device for detecting exosomes 121 in a biological sample, comprising a sheet carrier 107 of a substrate 101 that constitutes the inner wall of a channel 103 through which the biological sample flows, and a detection zone 105 provided in the channel 103, wherein in the detection zone 105, an uneven structure A having a plurality of detection protrusions 8 is formed on the surface of the sheet carrier 107 that constitutes the channel 103, and antibodies 123 that specifically bind to exosomes 121 are provided on the surface of the plurality of detection protrusions 8, and the shape of the plurality of detection protrusions 8 is cone-shaped or frustum-shaped.

Description

Technical Field

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

Background Art

[0002] Exosomes are a type of extracellular vesicles with a diameter of about 50 to 150 nm released from cancer cells and the like, and their interior and surface contain specific DNA, RNA, and proteins depending on the type of cells that release the exosomes. Therefore, detecting specific exosomes contained in biological samples such as blood and urine is extremely important in the diagnosis of diseases such as cancer. So far, many detection methods for exosomes have been reported, which combine "separation" of exosomes by ultracentrifugation and "capture" using materials immobilized with capture molecules such as antibodies. However, there is room for improvement in that long-term operations and multi-step surface chemical treatments for antibody modification of materials are required.

[0003] As described above, exosomes have been used as an example. Among the techniques for detecting extracellular vesicles typified by exosomes, there are examples described in Patent Document 1 and Non-Patent Document 1 as techniques for more efficiently detecting extracellular vesicles in biological samples without using ultracentrifugation.

[0004] Patent Document 1 (Japanese Unexamined Patent Application Publication No. 2018-191636) describes a method of capturing exosomes in an extracellular vesicle capture section using a flow path structure provided with an immobilized carrier on which one or more high-mannose-type sugar chain-binding lectins capable of specifically binding to surface sugar chains possessed by extracellular vesicles derived from cancer cells are immobilized (Claims 1, 6). According to this document, by passing a surfactant through the above immobilized carrier after capture, microRNA extracted from exosomes can be recovered in the storage section, and it is said that cancer can be diagnosed (Paragraphs 0154, 0029).

[0005] Non-patent document 1 (Shailender Singh Kanwar et al., Lab on a Chip, 2014, Vol. 14, pp. 1891-1900) describes a method for immobilizing an antibody against the surface antigen of exosomes (anti-CD63) on the surface of a microfluidic structure consisting of arranged round chambers using 3-mercaptopropyltrimethoxysilane, GMBS, and neutraavidin, and a method for capturing exosomes using the microfluidic structure thus prepared. Results have been shown in capturing exosomes in the serum of cancer patients using a microfluidic structure with immobilized antibodies using this method. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2018-191636 [Patent Document 2] Shailender Singh Kanwar and 3 others, Lab on a Chip, 2014, Vol.14, pp.1891-1900 [Overview of the project] [Problems that the invention aims to solve]

[0007] When the inventors examined the techniques described in the aforementioned literature, they found that there was room for improvement in that multi-step chemical processing was required when immobilizing antibodies in the channel. Furthermore, there was room for improvement in that this made the procedure for creating structures to capture extracellular vesicles complicated and time-consuming, and could also lead to a decrease in the reproducibility of creating extracellular vesicle-capturing structures.

[0008] This invention provides a device that offers excellent sensitivity in detecting extracellular vesicles and ease of manufacture. [Means for solving the problem]

[0009] According to the present invention, the following devices and test kits are provided. [1] A device for detecting extracellular vesicles in a biological sample, A substrate constituting the inner wall of a channel through which the biological sample is flowed, A detection zone provided in the aforementioned flow path, Equipped with, In the detection zone, a surface of the substrate constituting the flow path has a surface with multiple detection protrusions A. Multiple detection protrusions are provided with a capture substance that specifically binds to the extracellular vesicles, A device in which the shape of the multiple detection protrusions is a cone or a frustum. [2] The device according to [1], wherein the average height of the detection protrusions is 5 μm or more and 1000 μm or less. [3] The device according to [1] or [2], wherein the average distance between adjacent detection protrusions in the detection zone is 500 μm or less. [4] The device according to any one of [1] to [3], wherein the bottom surface of the detection projection is circular and the average diameter of the bottom surface is 5 μm or more and 1000 μm or less. [5] The device according to any one of [1] to [4], wherein a fine uneven structure B is formed on the surface of the detection protrusion in at least the detection zone, and the arithmetic mean roughness Ra of the roughness curve of the detection protrusion on which the fine uneven structure B is formed is 0.005 μm or more and 1 μm or less. [6] A device according to any one of [1] to [5], wherein in one cross-section, a plurality of detection protrusions have a first region located on one side of the detection protrusion from the center of the detection protrusion in the width direction of the detection protrusion, and a second region located on the other side of the detection protrusion from the center of the detection protrusion in the width direction of the detection protrusion, and in one cross-section, at least one of the outer edge of the first region and the outer edge of the second region of the detection protrusion has a recess. [7] The device according to any one of [1] to [6], wherein, when the detection zone is viewed from above, a plurality of the detection protrusions are arranged in a grid pattern. [8] The device according to any one of [1] to [7], wherein the biological sample is a blood sample, urine, saliva, or breast milk. [9] The device according to any one of [1] to [8], wherein the extracellular vesicle is an exosome.

[10] The device according to any one of [1] to [9], wherein the capture substance is an antibody or an antigen-binding fragment thereof.

[11] The device according to any one of [1] to

[10] , wherein the capture substance is an antibody against the surface antigen of an exosome.

[12] The device according to

[11] , wherein the surface antigen of the exosome is one or more selected from the group consisting of CD9, CD63, CD81, and CD147.

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

[12] , wherein the material of the substrate is a thermoplastic resin.

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

[13] , wherein the material of the substrate comprises one or more selected from the group consisting of polyester, polyolefin, polystyrene, polycarbonate, fluororesin, poly(meth)acrylate, polyvinyl chloride, polyamide, and polyimide.

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

[14] , wherein a cover portion is provided to cover the flow path.

[16] The device according to

[15] , wherein the lid is made of one or more materials selected from the group consisting of thermoplastic resins, thermosetting resins, photocurable resins, and glass.

[17] A device according to any one of [1] to

[16] for use in the diagnosis, treatment, or prevention of cancer in a subject.

[18] A test kit having one of the devices described in [1] to

[17] . [Effects of the Invention]

[0010] According to the present invention, a device with excellent detection sensitivity for extracellular vesicles and ease of manufacture can be provided. [Brief explanation of the drawing]

[0011] [Figure 1] It is a perspective view showing an example of the configuration of the device in the embodiment. [Figure 2] It is a diagram showing an example of the configuration of the detection zone of the device in the embodiment. [Figure 3] It is a top view showing an example of the arrangement of the detection protrusions when the distance between adjacent detection protrusions is 0 in the embodiment. [Figure 4] It is a diagram for explaining the recess on the outer edge in the first region of the detection protrusion in the embodiment. [Figure 5] It is a perspective view showing an example of the configuration of the device in the embodiment. [Figure 6] It is a perspective view showing an example of the configuration of the device in the embodiment. [Figure 7] It is a perspective view showing a scanning electron microscope (SEM) image of the detection protrusions of the device in the example. [Figure 8] It is a diagram showing the capture result of the target substance in the example. [Figure 9] It is a diagram showing the capture result of the target substance in the example. [Figure 10] It is a diagram showing the capture result of the target substance in the example. [Figure 11] It is a diagram showing the capture result of the target substance in the example. [Figure 12] It is a diagram showing the capture result of the target substance in the example. [Figure 13] It is a diagram showing the capture result of the target substance in the example. [Figure 14] It is a diagram showing the capture result of the target substance in the example.

Mode for Carrying Out the Invention

[0012] Hereinafter, embodiments of the present invention will be described with reference to the drawings. The drawings are schematic and do not match the actual dimensional ratios. In this specification, "~" indicating a numerical range represents above and below, and includes both end values.

[0013] (device) Figure 1 is a perspective view showing an example of the device configuration in this embodiment. The device 100 shown in Figure 1 is a device for detecting a target substance (hereinafter also referred to as "detectable substance") in a biological sample containing the target substance. The target substance is specifically an extracellular vesicle, and the device is suitably used, for example, for detecting extracellular vesicles, preferably for detecting exosomes. The following explanation will primarily use the case where the target substance, an extracellular vesicle, is an exosome as an example.

[0014] The device 100 comprises a substrate 101 that constitutes the inner wall of a channel 103 through which a biological sample flows, and a detection zone 105 provided in the channel 103. In the detection zone 105, a bumpy structure A having a plurality of detection protrusions 8 is formed on the surface of the substrate 101 that constitutes the channel 103, and a capture substance (antibody 123) that specifically binds to extracellular vesicles (exosomes 121) is provided on the surface of the plurality of detection protrusions 8, and the shape of the plurality of detection protrusions 8 is cone-shaped or frustum-shaped.

[0015] Furthermore, in device 100, a partition wall 119 is provided on the substrate 101 along the direction of extension of the flow channel 103, that is, the flow direction of the biological sample. Furthermore, device 100 is provided with a lid portion 111 that covers the flow path 103 and faces the substrate 101. The bulkhead 119 and the cover 111 will be described later.

[0016] (base material) Specific examples of the planar shape of the base material 101 include polygons such as quadrilaterals, circles, and ellipses. If the base material 101 is quadrilateral, the vertical width (length in the shorter direction) of the base material 101 may be, for example, about 2 to 100 mm, and the horizontal width (length in the longer direction) of the base material 101 may be, for example, about 2 to 100 mm.

[0017] The base material 101 may be composed of a single component or of a combination of multiple components. In device 100, the base material 101 consists of a substrate 109 and a sheet carrier 107 attached to the surface of the substrate 109. Partition walls 119 are provided on both sides of the sheet carrier 107, and a lid portion 111 is positioned in contact with the upper surface of the partition walls 119. In the detection zone 105, an antibody 123, which is a substance that captures exosomes 121 (target substance), is provided on the surface of the detection projection 8 of the sheet carrier 107. The substrate 109 and the sheet carrier 107 will be described below.

[0018] (substrate) Specifically, the substrate 109 functions as a housing for the sheet carrier 107. Specifically, the material of the substrate 109 is one or more selected from the group consisting of glass such as quartz glass, soda-lime glass, and borosilicate glass; and resin materials such as poly(meth)acrylate such as polymethyl(meth)acrylate, polyester, polyolefin, polystyrene, polycarbonate, fluororesin, polyvinyl chloride, polyamide, and polyimide. The thickness of the substrate 109 is, for example, 0.2 mm or more, and preferably 0.5 mm or more, from the viewpoint of improving the strength of the device 100. Furthermore, from the viewpoint of making the device 100 thinner, the thickness of the substrate 109 is, for example, 5 mm or less, and preferably 4 mm or less.

[0019] (Sheet carrier) The sheet carrier 107 is provided with a channel 103 for transporting a biological sample. From the viewpoint of improving fluidity within the channel 103, the biological sample is preferably a liquid.

[0020] In the example shown in Figure 1, multiple detection protrusions 8 are formed on the sheet carrier 107. Specifically, the detection protrusions 8 are formed on the entire surface of the sheet carrier 107, extending from the upstream side (sample introduction side) to the downstream side (sample discharge side) of the detection zone 105, and the gaps between the detection protrusions 8 form the flow channels 103. At least one of the upstream side (sample introduction side) and downstream side (sample discharge side) of the detection zone 105, the flow path 103 may be provided with an uneven structure, and the uneven structure may include protrusions that are the same as or different in shape as, for example, the detection protrusions 8. In the example shown in Figure 1, the entire surface of the sheet carrier 107 is provided with protrusions that are the same shape and size as the detection protrusions 8, and the uneven structure (uneven structure A) is provided on the entire surface of the sheet carrier 107 except for the area where the partition wall 119 is provided.

[0021] The detection zone 105 only needs to be provided in at least a portion of the flow path 103. Specifically, the detection zone 105 is provided in the entire or a portion of the flow path 103. Figures 2(a) and 2(b) show examples of the configuration of a detection zone 105 provided with detection protrusions 8. Figure 2(a) is a top view showing an example of the arrangement of detection protrusions 8 in the detection zone 105, and Figure 2(b) is a perspective view showing an example of the shape of detection protrusions 8. As shown in Figure 2(a), in the detection zone 105, the sheet carrier 107 includes a flat portion 9 corresponding to the bottom surface of the flow path 103 and a plurality of detection protrusions 8 protruding from the flat portion 9.

[0022] In the detection zone 105, a ridged structure A is formed by the totality of multiple detection protrusions 8. The gaps between the multiple detection protrusions 8 form a channel 103 through which the biological sample moves. The biological sample is transported through the ridged structure A from the sample introduction side on the left side of the diagram to the sample discharge side (along the direction of travel d in Figure 2(a)). The spaces between the multiple detection protrusions 8 function as channels 103 for transporting the biological sample along the surface of the sheet carrier 107. In other words, the voids in the uneven structure A function as channels 103 for transporting the biological sample along the surface of the sheet carrier 107.

[0023] From the viewpoint of improving the capture efficiency of extracellular vesicles, when the detection zone 105 is viewed from above, the multiple detection protrusions 8 are preferably arranged in a grid pattern. More specifically, examples of grid patterns include oblique grids such as square grids and hexagonal grids.

[0024] The shape of the detection projection 8 can be, specifically, a cone, a pyramidal pyramid, or other pyramidal shape; or a frustum, a pyramidal pyramid, or other frustum-shaped shape. These do not need to be geometrically precise shapes; they may have rounded corners or surfaces with fine irregularities. The shapes of the multiple detection protrusions 8 may be the same or different. From the viewpoint of producing the desired uneven structure A with better reproducibility, it is preferable that the shapes of the multiple detection protrusions 8 are the same.

[0025] In this embodiment, since the shape of the detection projection 8 is a cone or a frustum, the gap distance of the detection projection 8 can be made narrower at the lower part (bottom side) of the flow channel 103. This effectively improves the capture rate of extracellular vesicles compared to the case where a columnar body such as a cylinder is used. Furthermore, in processing methods using molds such as imprinting and injection molding, if the shape of the detection projection 8 is a cone or a frustum, the upper part is narrower than the bottom, so less volume needs to be removed during mold manufacturing compared to manufacturing a columnar body with the same bottom, and molds can be manufactured at a lower cost, thus improving the productivity of the molds. In this case, it becomes possible to detect extracellular vesicles more efficiently. From the viewpoint of producing the uneven structure A with better reproducibility, the shape of the detection projection 8 is more preferably a cone or a frustocone. Figure 2(b) shows an example in which the detection projection 8 is conical.

[0026] When the bottom surface of the detection projection 8 is circular, the average diameter of the bottom surface (diameter 4 in Figures 2(a) and 2(b)) is preferably 5 μm or more, more preferably 10 μm or more, and even more preferably 20 μm or more, from the viewpoint of efficient capture of extracellular vesicles. Furthermore, from the viewpoint of efficient capture of extracellular vesicles, the average diameter of the base of the detection projection 8 is preferably 1000 μm or less, more preferably 200 μm or less, and even more preferably 100 μm or less. From a similar viewpoint, when the extracellular vesicles are exosomes, the average diameter of the base of the detection projection 8 is even more preferably 50 μm or less. Here, as the average diameter of the bottom surface of the detection protrusions 8, for example, five arbitrary detection protrusions 8 can be selected from the detection zone 105, and the average value of the diameters of the bottom surfaces of the five selected detection protrusions 8 (diameter 4 in Figures 2(a) and 2(b)) can be adopted.

[0027] The average height of the detection projection 8 is preferably 5 μm or more, more preferably 10 μm or more, and even more preferably 20 μm or more, from the viewpoint of efficient capture of extracellular vesicles. Furthermore, from the viewpoint of efficient capture of extracellular vesicles, the average height of the detection protrusion 8 is preferably 1000 μm or less, more preferably 200 μm or less, and even more preferably 100 μm or less. From a similar viewpoint, when the extracellular vesicles are exosomes, the average height of the detection protrusion 8 is even more preferably 50 μm or less. For example, the height of the detection protrusion 8 can be determined by selecting five arbitrary detection protrusions 8 from the detection zone 105 and adopting the average value of the heights of the five selected detection protrusions 8 (height 6 in Figure 2(b)).

[0028] The average distance between adjacent detection protrusions 8 in the detection zone 105, i.e., the average nearest distance between detection protrusions 8, is preferably 500 μm or less, more preferably 100 μm or less, and even more preferably 20 μm or less, from the viewpoint of efficient capture of extracellular vesicles. From a similar viewpoint, when the extracellular vesicle is an exosome, the average nearest distance is even more preferably 10 μm or less. Also, the average nearest distance is 0 μm or more. Furthermore, the average nearest distance may be, for example, 0.1 μm or more, or for example, 1 μm or more. Here, "average distance between adjacent detection protrusions 8" refers to the closest proximity distance between adjacent pairs of detection protrusions 8. Specifically, five distances between any two adjacent detection protrusions 8 (distance 5 in Figure 2(a)) can be selected from the detection zone 105, and the average value of these five selected distances can be adopted.

[0029] Figure 3 is a top view showing an example of the arrangement of detection protrusions 8 when the distance between adjacent detection protrusions 8 is 0. Figure 3 shows an example where the base shape of the detection protrusions 8 is circular, in which case the detection protrusions 8 are specifically cones or frustocones. As shown in Figure 3, for example, when the distance between adjacent detection protrusions 8 arranged in a hexagonal grid is 0, the bases of the multiple detection protrusions 8 are lined up without gaps, increasing the number of detection protrusions 8 per unit area in a top view, and further increasing the capillary force, making it easier to transport biological samples.

[0030] Here, the numerical values ​​for the size of the detection projection 8 are calculated, for example, by observing a cross-section perpendicular to the flat portion 9 of the detection projection 8 using a scanning electron microscope (SEM), or by observing a three-dimensional image of the detection projection 8 using an optical microscope.

[0031] Furthermore, from the viewpoint of improving the detection accuracy of extracellular vesicles, it is also preferable that a fine uneven structure B is formed on the surface of the detection projection 8 in at least the detection zone 105. From a similar viewpoint, the arithmetic mean roughness Ra of the roughness curve of the detection projection 8 on which the fine uneven structure B is formed is preferably 0.005 μm or more and 1 μm or less. Here, the arithmetic mean roughness Ra of the roughness curve is specifically measured using a three-dimensional roughness analysis scanning electron microscope in accordance with JIS B 0601:2013.

[0032] From the viewpoint of more stably fixing the captured substance, the arithmetic mean roughness Ra of the roughness curve of the detection protrusion 8 on which the fine uneven structure B is formed is preferably 0.005 μm or more, more preferably 0.010 μm or more, even more preferably 0.050 μm or more, and even more preferably 0.080 μm or more. From the viewpoint of improving the moldability of the fine uneven structure B, the above arithmetic mean roughness Ra is preferably 1 μm or less, more preferably 0.5 μm or less, even more preferably 0.3 μm or less, and even more preferably 0.2 μm or less.

[0033] The following describes how to measure the arithmetic mean roughness Ra of the detection protrusion 8 on which the fine uneven structure B is formed, using Figure 2(a) as an example. Using a three-dimensional roughness analysis scanning electron microscope, a convex profile is measured along the surface of the detection protrusion 8 (along a straight line 20 when viewed from above), with the center of the vertex of the detection protrusion 8 (for example, the center 19 of the detection protrusion 8) as the center point. The straight line 20 is a single straight line 20d with a length of 20d, with the center of the vertex (for example, the center 19 of the detection protrusion 8) as the center point. The length 20d is the same as the diameter of the bottom surface of the detection protrusion 8. If the straight line 20 is a straight line on the same plane (for example, both ends and the center are on the same plane), i.e., if the detection protrusion 8 has the shape of a cylinder, polygonal prism, etc., the arithmetic mean roughness Ra of the roughness curve specified in JIS B 0601:2013 is calculated from the convex-convex profile. If the straight line 20 is not a straight line on the same plane, that is, if the detected projection 8 is a shape such as a cone, pyramidal polygon, hemisphere, semiellipsoid, frustum of a cone, or frustum of a polygon, the slope correction is applied from the unevenness profile and the arithmetic mean roughness Ra of the roughness curve specified in JIS B 0601:2013 is calculated as a plane.

[0034] Furthermore, it is also preferable that the detection projection 8 has the following configuration. That is, in one cross-section, a plurality of detection projections 8 have a first region (RG1 in Figure 4) located on one side of the detection projection 8 from the center of the detection projection 8 in the width direction of the detection projection 8, and a second region located on the other side of the detection projection 8 from the center of the detection projection 8 in the width direction of the detection projection 8, and it is also preferable that at least one of the outer edges of the first region and the second region of the detection projection 8 has a recess in one cross-section.

[0035] Figure 4 is a diagram illustrating the recess R at the outer edge of the first region RG1 of the detection projection 8. Figure 4 corresponds to an enlarged view of the outer edge of the detection projection 8 shown in Figure 2(b). In Figure 4, the X direction is parallel to the flat portion 9 (Figure 2(a)). The Y direction is perpendicular to the flat portion 9.

[0036] The outer edge of the first region RG1 of the detection projection 8 has a recess R. The recess R is recessed by a distance D in the X direction relative to a virtual line IL. The distance D is, for example, between 50 nm and 500 nm. In Figure 4, the virtual line IL extends in the Y direction. In detail, the virtual line IL is tangent to a first position P1 on the outer edge of the detection projection 8 and passes through a second position P2. The first position P1 on the outer edge of the detection projection 8 is the outermost position of the detection projection 8 at the upper end of the recess R. Therefore, the virtual line IL is tangent to the first position P1. That is, at and near the first position P1, the virtual line IL and the outer edge of the detection projection 8 geometrically share only the first position P1 (i.e., a single point). The second position P2 on the outer edge of the detection projection 8 is located below the first position P1 in the Y direction. Figure 4 shows a single recess R in a portion of the outer edge of the first region RG1 of the detection projection 8. The total number of recesses R in the first region RG1 is, for example, 1 or more, preferably 2 or more, and also, for example, 20 or less, preferably 10 or less, and more preferably 5 or less. The recesses on the outer edge of the second region of the detection projection 8 are also determined, for example, in a similar manner to that shown in Figure 4. A certain number of recesses in the first or second region indicates that the surface area of ​​the detection projection 8 is increased due to the presence of these recesses. Therefore, a certain number of recesses in the first or second region suggests that the structure has a more suitable space for carrying the captured substance, making it possible to increase the amount of captured substance that can be carried. From the above, it is thought that a certain number of recesses in the first or second region can further improve the capture efficiency of extracellular vesicles.

[0037] A capture substance is provided on the detection projection 8. In the example shown in Figure 1, the antibody 123 is directly immobilized on the surface of the detection projection 8 without the need for other structures such as microparticles. More specifically, the capture substance is adsorbed or bound to the surface of the detection projection 8. The capture substance may be physically adsorbed or chemically adsorbed or bound to the detection projection 8. Furthermore, the captured substance may be directly adsorbed or bound to the surface of the detection projection 8, or it may be adsorbed or bound to an intervening substance bound to or adsorbed on the detection projection 8. Specific examples of intervening substances include crosslinking agents and spacer molecules. Alternatively, a coupling agent such as a silane coupling agent may be attached to the surface of the detection projection 8, and the capture substance may be bound to the coupling agent.

[0038] From the viewpoint of improving the detection sensitivity of extracellular vesicles, the immobilized concentration (surface concentration) of the captured substance at the detection projection 8 is preferably 0.1 ng / mm³. 2 The above is true, and more preferably 0.2 ng / mm 2 More preferably 0.5 ng / mm 2 That's all. Furthermore, from the viewpoint of noise reduction, the above immobilization concentration is preferably 10 μg / mm³. 2 The following is more preferable: 1 μg / mm³ 2 More preferably 0.5 μg / mm³ 2 The following applies:

[0039] Next, the material of the sheet carrier 107 and the method for forming the flow channel 103 will be described. Specifically, the material for the sheet carrier 107 may be one or more selected from the group consisting of glass such as quartz glass, soda-lime glass, and borosilicate glass; and resin materials such as poly(meth)acrylate such as polymethyl(meth)acrylate, polyester, polyolefin, polystyrene, polycarbonate, fluororesin, polyvinyl chloride, polyamide, and polyimide. Here, (meth)acrylate is at least one of acrylate and methacrylate.

[0040] Furthermore, in terms of enabling the sheet carrier 107 and the detection projection 8 to be integrally molded, it is preferable that the material of the sheet carrier 107 be a thermoplastic resin. Specifically, thermoplastic resins include one or more selected from the group consisting of polyester, polyolefin, polystyrene, polycarbonate, fluororesin, poly(meth)acrylate, polyvinyl chloride, polyamide, and polyimide, and more specifically, one or more selected from the group consisting of polyethylene terephthalate (PET), cycloolefin polymer (COP), polypropylene (PP), polystyrene (PS), polycarbonate (PC), polyvinylidene fluoride (PVDF), polymethyl methacrylate (PMMA), and polyethylene (PE).

[0041] The thickness of the sheet carrier 107 is, for example, 50 μm or more, preferably 100 μm or more, and more preferably 150 μm or more, from the viewpoint of improving the strength of the device 100. Furthermore, from the viewpoint of making the entire device 100 thinner, the thickness of the substrate 101 is, for example, 2000 μm or less, and preferably 1000 μm or less.

[0042] Methods for manufacturing the sheet carrier 107 provided with the channel 103 include thermal imprinting, UV imprinting, injection molding, pattern formation on UV-curable resin using photolithography, soft lithography in which a UV-curable resin pattern formed using photolithography is used as a template to transfer to a resin, etching, mechanical cutting, and laser processing of silicone resin or glass patterned using photolithography. Among these, thermal imprinting or injection molding of thermoplastic resin is preferred as an inexpensive method for precise processing. For example, a sheet carrier 107 having an uneven structure A can be obtained by processing a sheet material made of thermoplastic plastic by thermal imprinting.

[0043] Furthermore, when the detection projection 8 is provided with the aforementioned fine uneven structure B, the sheet carrier 107 may be prepared by the following method. In other words, when manufacturing the sheet carrier 107 by thermal imprinting, the Ra value at the detection protrusion 8 on which the fine uneven structure B is formed can be adjusted to within the above numerical range by, for example, etching, photolithography, mechanical cutting, laser processing, etc. In particular, it is preferable to adjust the Ra value at the detection protrusion 8 on which the fine uneven structure B is formed by setting the Ra value of the surface of the mold used for thermal imprinting to a predetermined value. For example, it is preferable to adjust the Ra value at the detection protrusion 8 on which the fine uneven structure B is formed by etching, photolithography, mechanical cutting, polishing, laser processing, etc. Polishing methods include cutting by dicing, sandblasting, etc. In laser processing, Ra can be adjusted by controlling the output of the laser.

[0044] Furthermore, the immobilization of the captured substance onto the sheet carrier 107 can be performed, for example, by applying a liquid containing the captured substance to the sheet carrier 107 or by introducing and delivering it using a pump such as a syringe pump, and then incubating it.

[0045] (bulkhead) The material of the partition wall 119 may be the same as that of the base material 101 or the lid portion 111, or it may be a different material. In device 100, since the partition wall 119 is placed on the sheet carrier 107 that constitutes the base material 101, the partition wall may be made of the same material as the sheet carrier 107. Also, the partition wall 119 may be formed integrally with the base material 101 or the lid portion 111. Furthermore, the partition wall 119 may be formed by applying a water-repellent solution to the surface of the sheet carrier 107 and drying it.

[0046] The height of the septum 119 is preferably 5 μm or more, more preferably 10 μm or more, and even more preferably 20 μm or more, from the viewpoint of efficient capture of extracellular vesicles. Furthermore, from the viewpoint of more stably capturing extracellular vesicles, the height of the septum 119 may be, for example, 1200 μm or less, preferably 1000 μm or less, more preferably 200 μm or less, and even more preferably 100 μm or less. From a similar viewpoint, it is preferable that the difference between the height of the septum 119 and the height of the detection projection 8 is not too large, more preferably the difference between these heights is 10 μm or less, and even more preferably the height of the septum 119 is the same as the height of the detection projection 8.

[0047] (Lid) The device 100 is preferably provided with a lid that covers the flow path 103. In Figure 1, a configuration in which the device 100 has a lid 111 that covers the entire substrate 101 is illustrated, but the lid 111 may cover the entire substrate 101 or a part of the substrate 101. By providing the lid 111, drying of the biological sample in the flow path 103 can be suppressed. In addition, the flow of the biological sample in the detection zone 105 can be made more stable.

[0048] The thickness of the lid portion 111 is, for example, 50 μm or more, and preferably 100 μm or more, from the viewpoint of improving the strength of the device 100. Furthermore, from the viewpoint of making the device 100 thinner, the thickness of the lid portion 111 is, for example, 3 mm or less, and preferably 1.5 mm or less.

[0049] The lid 111 is made of one or more materials selected from the group consisting of, for example, thermoplastic resin, thermosetting resin, photocurable resin, and glass. From the viewpoint of improving visibility from the top of the device 100, the lid 111 is preferably made of a transparent material. Specific examples of thermoplastic resins include poly(meth)acrylate, polyester, polyolefin, polystyrene, polycarbonate, fluororesin, polyvinyl chloride, polyamide, and polyimide. Specific examples of thermosetting resins include silicone resins such as polydimethylsiloxane, epoxy resins, melamine resins, urethane resins, and phenolic resins. Examples of photocurable resins include acrylate-based resins. Specific examples of glass include quartz glass, soda-lime glass, and borosilicate glass.

[0050] (Biological samples, extracellular vesicles) The biological sample contains extracellular vesicles (exosome 121 in Figure 1). From the viewpoint of more stable detection of extracellular vesicles, the biological sample is preferably a blood sample, urine, saliva, or breast milk. Specific examples of blood samples include blood and blood-derived samples.

[0051] The International Society for Extracellular Vesicles (ISEV) defines extracellular vesicles as "particles that are released from cells, are delimited by a lipid bilayer, and cannot replicate on their own" (Joshua A. Welsh et al., "Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches," Journal of Extracellular Vesicles, 2024, Volume 13, Issue 2, e12404). Extracellular vesicles are specifically classified based on differences in their production mechanisms. Examples of extracellular vesicles include exosomes (diameter 50-150 nm), microvesicles (diameter 100-1000 nm), and apoptotic vesicles (diameter 5 μm). From the viewpoint of suitability for cancer diagnosis, etc., extracellular vesicles are preferably exosomes.

[0052] (captured substance) The capture substance is selected from substances that specifically bind to extracellular vesicles, which are the target substances, and may preferably be an antibody or an antigen-binding fragment that specifically binds to an antigen present on the surface of the extracellular vesicle, and is preferably antibody 123. When the capture substance is antibody 123, antibody 123 may be a polyclonal antibody or a monoclonal antibody. Other examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain antibodies.

[0053] When the extracellular vesicle is an exosome 121, the antibody 123 is preferably an antibody against the surface antigen of the exosome, from the viewpoint of more stably capturing the exosome 121. From a similar viewpoint, the surface antigen of exosomes is preferably one or more selected from the group consisting of CD9, CD63, CD81, and CD147.

[0054] In this embodiment, a plurality of cone-shaped or frustoconical detection protrusions 8 are arranged in the detection zone 105 of the device 100, and a capture substance is provided on the surface of the detection protrusions 8. This allows for favorable fluidity of extracellular vesicles in the biological sample in the detection zone 105, enabling efficient and stable capture of extracellular vesicles by the detection protrusions 8, resulting in excellent detection sensitivity for extracellular vesicles. Furthermore, the device 100 has a simple structure with a ridged structure A having a plurality of detection protrusions 8 formed on the surface of the sheet carrier 107, and antibodies 123 are provided on the surface of the plurality of detection protrusions 8. This allows for easy manufacturing with good reproducibility.

[0055] Furthermore, in device 100, the internal space surrounded by the sheet carrier 107, the partition wall 119, and the lid portion 111 serves as a channel 103 for the biological sample containing extracellular vesicles. Since the height of the detection projection 8 is equal to the height from the bottom surface of the sheet carrier 107 to the lid portion, the contact efficiency between the extracellular vesicles and the detection projection 8 is increased, making detection more sensitive and accurate.

[0056] Because device 100 can detect extracellular vesicles with high accuracy, it is suitable for use, for example, in the diagnosis, treatment, or prevention of cancer in a subject. Examples of animals that can be studied include vertebrates such as humans, monkeys, rabbits, mice, rats, camels, cattle, goats, sheep, and guinea pigs.

[0057] In device 100, preferred combinations of materials for the substrate 109, sheet carrier 107, and lid 111 include a combination where the substrate 109 and sheet carrier 107 are polycarbonate and the lid 111 is silicone resin; and a combination where the substrate 109 is glass, the sheet carrier 107 is polycarbonate, and the lid 111 is silicone resin. Device 100 may further include components other than those described above.

[0058] The following describes variations of the device. The explanation will focus on the differences from device 100 shown in Figure 1. Multiple devices with the same or different structures can also be used in combination.

[0059] In device 100, the entire sheet carrier 107 is shown as a flow channel 103, but the flow channel 103 may be provided in only a part of the sheet carrier 107. Furthermore, although the device 100 is exemplified as having a substrate 101 comprising a substrate 109 and a sheet carrier 107, the configuration of the substrate 101 is not limited. For example, the base material 101 may consist only of a sheet carrier 107 having a plurality of detection protrusions.

[0060] Furthermore, although device 100 (Figure 1) was provided with a partition wall 119, the device does not necessarily need to have a partition wall. Examples of devices without partition walls are shown in Figure 5 and Figure 6, which will be described later. Figure 5 is a perspective view showing an example of the device configuration in this embodiment. The device 120 shown in Figure 5 comprises a substrate 101, a channel 103 provided on one surface of the substrate 101 for flowing a biological sample, and a detection zone 105 provided in the channel 103. In the device 120 as well, the channel 103 in the detection zone 105 is formed by a plurality of detection protrusions (not shown), and a capture substance that specifically binds to target cells is provided on the surface of the plurality of detection protrusions.

[0061] Furthermore, while device 100 (Figure 1) has a flat plate-shaped lid portion 111, device 120 shown in Figure 5 has a lid portion 111 with a recess 113. The lid portion 111 has a recess 113 in the region facing the flow path 103, as well as extending upstream and downstream thereof. By providing a recess 113 in the lid portion 111, the depth of the flow path 103 can be stably controlled to a desired size.

[0062] Furthermore, the outer surface of the lid portion 111 is provided with an inlet 115 and an outlet 117 that communicate with the flow path 103. The inlet 115 is an opening provided on the upstream side of the flow path 103, and a biological sample is introduced into the device 100 from the inlet 115. The outlet 117 is an opening provided on the downstream side of the flow path 103, and the biological sample that has passed through the flow path 103 is discharged from the outlet 117.

[0063] Furthermore, although the above description has used a device 100 in which the base material 101 is provided with a flow channel 103 in a sheet carrier 107 attached to the surface of a substrate 109, the flow channel 103 may also be provided in the substrate 109, as shown in Figure 6.

[0064] Figure 6 is a perspective view showing the configuration of device 110. The basic configuration of device 110 is the same as that of the device shown in Figure 5, but it differs in that the base material 101 is made up of a substrate 109.

[0065] In the device 110, as a method for forming detection protrusions by microfabrication of the substrate 109, for example, the method described above for manufacturing the sheet carrier 107 provided with the flow channel 103 can be used.

[0066] (Detection method) In this embodiment, device 100 can be suitably used for detecting extracellular vesicles such as exosomes 121. A detection method using a device is, for example, Step 11 involves introducing a biological sample containing extracellular vesicles, which are the substances to be detected, to the upstream side of the detection zone 105 and guiding it into the detection zone 105, and capturing the extracellular vesicles on the detection projection 8 through specific interactions between the extracellular vesicles and the capture substance; and, Step 12: Detect extracellular vesicles captured by the detection projection 8. Includes. Furthermore, a step (step 13) may be performed in which a buffer solution or the like is introduced upstream of the detection zone 105 to wash away the detection zone 105 at least one stage before step 11, between step 11 and step 12, and after step 12.

[0067] In the example of device 100 shown in Figure 1, in step 11, for example, a biological sample is introduced from an inlet (not shown), and it is allowed to flow through the channel 103 and guided to the detection zone 105.

[0068] Various types of pumps may be used for transporting biological samples. Examples of pump types include syringe pumps, diaphragm pumps, and peristaltic pumps. For example, in step 11, a liquid delivery tube communicating with the inlet may be connected, and the biological sample may be pumped into the flow path 103 via the liquid delivery tube using a liquid delivery pump such as a syringe pump.

[0069] The flow rate of the biological sample in detection zone 105 is preferably 1 μL / min or more, more preferably 2 μL / min or more, and even more preferably 4 μL / min or more, from the viewpoint of improving the capture rate of extracellular vesicles. From a similar viewpoint, the flow rate of the biological sample in detection zone 105 is preferably 300 μL / min or less, more preferably 150 μL / min or less, and even more preferably 100 μL / min or less.

[0070] Furthermore, the detection method in step 12 can be selected, for example, depending on the type of extracellular vesicle, and the extracellular vesicles may be stained with a fluorescent substance that specifically acts on extracellular vesicles. The fluorescent substance may be modified, for example, with a secondary antibody specific to the extracellular vesicle.

[0071] (Test kit) In this embodiment, the test kit has the device described in this embodiment. For example, the test kit may include the device in this embodiment and a reagent to be applied to the device. Examples of reagents include antibodies and solvents. An antibody is an antibody that specifically binds to the substance to be detected (extracellular vesicle), and may be a polyclonal antibody or a monoclonal antibody. It may also be a fragment antibody. The test kit may also include instructions for use, which may be for the purpose of using the kit for the diagnosis, treatment, or prevention of cancer in the subject.

[0072] The embodiments of the present invention have been described above with reference to the drawings, but these are merely examples of the present invention, and various other configurations can also be adopted. [Examples]

[0073] This embodiment will be described in detail below with reference to examples and comparative examples, but this embodiment is not limited to these examples. In the following, the detection projection 8 will also be referred to as the "protrusion" as appropriate.

[0074] (Manufacturing Example 1) In this example, a sheet carrier 107 equipped with a cone-shaped detection projection 8 was manufactured using the following procedure. Figure 7 shows a scanning electron microscope (SEM) image of the detection projection of the sheet carrier obtained in Manufacturing Example 1. Table 1 shows the measured values ​​for the size of the detection projection 8 of the sheet carrier obtained in each example.

[0075] (Manufacturing Example 1) Manufacturing of Sheet Carrier 1 A sheet carrier 1 (Figure 7) was fabricated by applying thermal imprinting to a polycarbonate sheet (Teijin Corporation, PC-2151, 50 mm x 50 mm, sheet thickness 200 μm) to create a sheet carrier 1 having a hexagonal lattice structure A in which conical detection protrusions 8, each with a bottom diameter (hereinafter sometimes referred to as "diameter of detection protrusion") of 30 μm and a height (hereinafter sometimes referred to as "height") of 30 μm, are arranged at a distance of 2 μm between detection protrusions 8 (distance between cone centers 32 μm). The planar shape of the area where the detection protrusions 8 are formed on the sheet carrier 1 was set to 30 mm x 30 mm. The measured values ​​regarding the size of the detection protrusions 8 on the obtained sheet carrier are shown in Table 1. Here, when applying thermal imprinting, the detection projection 8 and the fine uneven structure B on its surface were formed by using a laser-processed mold.

[0076] The processing method for the mold used in the manufacture of sheet carrier 1 is as follows: A mold having a cone-shaped recess corresponding to the detection projection 8 was obtained by irradiating an aluminum alloy plate with pulsed light multiple times from a laser processing device (Tosei Electrobeam Co., Ltd. ultrashort pulse laser processing machine R-200, laser wavelength: 1552nm, rated output: 10W, pulse: femtosecond).

[0077] [Table 1]

[0078] (Example 1, Comparative Examples 1 and 2) In this example, devices were fabricated using the sheet carrier obtained in Manufacturing Example 1. Culture supernatant of human breast cancer cell line MCF-7 cells and serum-free medium (a control sample without exosomes) were introduced into each fabricated device using a syringe pump. After introduction, the capture of exosomes in the culture supernatant was verified by staining the lipid bilayer of the exosomes with a fluorescent dye. Furthermore, specific exosome capture by the antibody was confirmed by comparing the results with the case where MCF-7 cell (breast cancer cell line) culture supernatant was flowed through the sheet carrier without antibody immobilization.

[0079] (Method of manufacturing a device) Device 1, in which an antibody was immobilized on the surface of a sheet carrier, and Device 2, in which an antibody was not immobilized on the surface of a sheet carrier, were fabricated using the following procedure. A sheet carrier was prepared by applying a water-repellent solution (A-PAP PEN, manufactured by Daido Sangyo Co., Ltd.) to its surface and drying it, thereby forming a detection protrusion in a 6mm x 6mm area near the center, and surrounding the outer periphery with an area coated with the water-repellent solution. An aqueous solution of anti-CD63 antibody (BD Biosciences, Purified Mouse Anti-Human CD63, model number 556019, antibody concentration: 50 μg / mL) was dropped onto the surface of the prepared sheet carrier, and the sheet carrier was left to stand at room temperature for 1 hour, after which the solution was dried in a 40°C oven (manufactured by ESPEC). After that, a glass plate (76mm x 26mm, thickness 0.8~1.0mm) was placed on the underside of the sheet carrier. Meanwhile, a polydimethylsiloxane sheet with a planar shape similar to that of the glass plate was prepared as a lid. Two holes, a liquid introduction port and a liquid discharge port, were pre-made at the position where the polydimethylsiloxane sheet would be placed on the sheet carrier. A polydimethylsiloxane sheet was placed on a sheet carrier, and the polydimethylsiloxane sheet and the glass plate were bonded together by plasma bonding at the outer periphery of the sheet carrier. By connecting Teflon® tubes to the liquid introduction and liquid discharge sections, a device 1 was fabricated that could pump the sample. Furthermore, in the preparation of device 1 described above, device 2 was obtained in the same manner as the preparation of device 1, except that the anti-CD63 antibody solution was not dropped onto the surface of the sheet carrier, allowed to stand, and dried.

[0080] (Experimental method) In this example and the following examples, the method for confirming exosome capture involved fluorescently labeling the exosomes with a fluorescent dye or antibody after capture, and then taking fluorescence microscope images of the detection protrusions. The amount of captured exosomes was then calculated from the fluorescence images using the image analysis software ImageJ (National Institutes of Health, USA), and the relative fluorescence intensity was evaluated.

[0081] (Method for preparing the sample) As a model sample for exosomes, we used the culture supernatant containing exosomes released by MCF-7 cells (human breast cancer cell line, provided by RIKEN BioResource Center). We used 3.6 × 10⁶ MCF-7 cells. 5 Cells were seeded on a dish at a concentration of cells / mL and cultured for 72 hours (±1 hour) in a serum-containing culture medium. At this point, the culture medium was replaced with serum-free medium (Advanced DMEM, Thermo Fisher Scientific) and cultured for 48 hours to release exosomes into the culture medium. The culture medium was collected and centrifuged at 4°C, 3000 g, and 30 minutes, and 1000 μL of the supernatant was collected. The collected supernatant was mixed with 25 μL of a cell adhesion inhibitor (Through Path Plus 40x (OnChip Biotechnologies)) and used as the introduction sample. (conditions) • Sample introduction volume: 500 μL • Sample flow rate: 10 μL / min • Combination of sample and device: The following combinations were used in each example. Example 1: Culture supernatant containing exosomes, device 1 (using antibody-immobilized sheet) Comparative Example 1: Serum-free culture medium (exosome-free solution), Device 1 (using antibody-immobilized sheet) Comparative Example 2: Culture supernatant containing exosomes, Device 2 (using an antibody-unimmobilized sheet) • Number of measurements N=1

[0082] (Methods for capturing exosomes) In each case, after introducing the sample described above, washing was performed by introducing 500 μL of washing solution (2.5% Through Path Plus 40x (OnChip Biotechnologies) / PBS, which does not contain cells; the same applies hereafter unless otherwise specified). Subsequently, PBS containing Vybrant DiO (Thermo Fisher Scientific) was introduced and allowed to stand at 37°C for 10 minutes, after which washing was performed again by introducing another 500 μL of washing solution. Fluorescence microscopy images of green-stained exosomes were taken to confirm exosome capture. Furthermore, the amount of captured exosomes was quantitatively evaluated by image analysis. The results are shown in Figures 8 and 9.

[0083] (Evaluation results) Figure 8 shows the capture results of the target substance (exosomes), and is a fluorescence microscope image of the detection protrusion after sample introduction, staining, and washing. Figure 9 also shows the capture results of the target substance, and is a graph showing the brightness values ​​of multiple fluorescence microscope images, including the fluorescence microscope image shown in Figure 8, as relative fluorescence intensity. These results indicate that when the culture supernatant containing exosomes was introduced onto an antibody-immobilized sheet (Example 1), strong fluorescence intensity was observed, indicating that the exosomes were captured. On the other hand, when serum-free culture medium was introduced onto an antibody-immobilized sheet (Comparative Example 1) and when the culture supernatant containing exosomes was introduced onto an antibody-unimmobilized sheet (Comparative Example 2), the fluorescence intensity was low in both cases. This result indicates that in Example 1, the exosomes were specifically captured by the antibody.

[0084] (Examples 2-4, Comparative Example 3) In this example, device 1 was fabricated using the sheet carrier obtained in manufacturing example 1, and a sample of MCF-7 cell culture supernatant diluted to an appropriate concentration was introduced. The fluorescence intensity was then evaluated to see if it changed in accordance with the exosome concentration.

[0085] (Methods for capturing exosomes) The relative concentration of the culture supernatant containing exosomes was changed as follows. Aside from this change, the experiment was conducted in the same manner as described in Example 1. The conditions are shown below. (conditions) • Relative concentration of culture supernatant containing exosomes: 0% (Comparative Example 3), 10% (Example 2), 50% (Example 3), 100% (Example 4) • Number of measurements N=1

[0086] Figure 10 shows the capture results of the target substance, and is a graph showing the relative values ​​of fluorescence intensity at each relative concentration. From Figure 10, it can be seen that the fluorescence intensity tends to increase with increasing relative concentration. This result indicates that the capture of exosomes is quantitative.

[0087] (Example 5) In this example, two devices 1, fabricated using the sheet carrier obtained in Manufacturing Example 1, were connected in series, and the same experiment as in Example 1 was performed to verify whether the amount of exosomes introduced and the area of ​​the detection protrusions were appropriate.

[0088] (Methods for capturing exosomes) The experiment was conducted in the same manner as described above in Example 1, except that the following conditions were changed. The conditions are as follows. (conditions) • Create two identical devices (Device 1). • Connect the outlet of the upstream device and the inlet of the downstream device using a Teflon tube. • Sample to introduce: Culture supernatant containing exosomes • Number of measurements N=1

[0089] Figure 11 shows the capture results of the target substance, and is a fluorescence microscope image of the detection protrusions in the upstream and downstream devices after sample introduction, staining, and washing. Figure 12 shows the capture results of the target substance, and is a graph showing the brightness values ​​of multiple microscope images, including the fluorescence microscope shown in Figure 12, as relative fluorescence intensity for the upstream and downstream devices, respectively. From these figures, it was confirmed that the relative fluorescence intensity of the detection protrusions in the downstream device was lower compared to the upstream device. This result indicates that exosomes in the sample were captured more efficiently in the upstream device.

[0090] (Example 6, Comparative Example 4) In this example, a device was fabricated using the sheet carrier obtained in Manufacturing Example 1. The culture supernatant of MCF-7 cells, a human breast cancer cell line, was introduced into the fabricated device using a syringe pump. After introduction, the membrane protein (CD63) present in the lipid bilayer of exosomes was specifically stained using an antibody to verify whether the exosomes contained in the culture supernatant were specifically captured.

[0091] (Method of capturing cells) The experiments in Comparative Example 1 and Example 1 were conducted in the same manner as described above, except that the following conditions were changed. The conditions are shown below. (conditions) • Combination of sample and device: The following combinations were used in each example. Comparative Example 4: Serum-free culture medium (exosome-free solution), Device 1 (using antibody-immobilized sheet) Example 6: Culture supernatant containing exosomes, device 1 (using antibody-immobilized sheet) • Staining method after sample introduction: 500 μL of 0.1% PBST (PBS containing 0.1% Tween20, prepared in-house) containing 2 μg / mL of rabbit-derived anti-human CD63 antibody (Sigma-Aldrich) as the primary antibody was introduced, followed by the introduction of 500 μL of washing solution (0.1% PBST). Next, 500 μL of 0.1% PBST containing 4 μg / mL of Alexa Fluor594-labeled donkey anti-rabbit antibody (Thermo Fisher Scientific) was introduced as the secondary antibody, followed by washing with 500 μL of washing solution (0.1% PBST). • Number of measurements N=1

[0092] Figure 13 shows the capture results of the target substance, and is a fluorescence microscope image of the detection protrusions on the device after sample introduction, staining, and washing. Figure 14 shows the capture results of the target substance, and is a graph showing the brightness values ​​of the fluorescence microscope images of the detection protrusions as relative fluorescence intensity in each example. When the culture supernatant was introduced, strong fluorescence intensity was observed, indicating that CD63-expressing exosomes were captured (Example 6). On the other hand, the fluorescence intensity was weak when serum-free culture medium without exosomes was introduced (Comparative Example 4). This result indicates that specific capture of exosomes actually occurred. [Explanation of symbols]

[0093] 4 diameter 5 distance 6 Height 8 Detection protrusion 9 Flat area 19 center 20 straight line 20d length 100 devices 101 Base material 103 Flow channel 105 detection zones 107 Sheet carrier 109 circuit boards 110 devices 111 Lid 113 Recess 115 Inlet 117 Outlet 119 Bulkhead 120 devices 121 Exosomes 123 Antibodies d Direction of travel

Claims

1. A device for detecting extracellular vesicles in biological samples, A substrate constituting the inner wall of a channel through which the biological sample is flowed, A detection zone provided in the aforementioned flow path, Equipped with, In the detection zone, a surface of the substrate constituting the flow path has a surface with multiple detection protrusions A. Multiple detection protrusions are provided with a capture substance that specifically binds to the extracellular vesicles, A device in which the shape of the multiple detection protrusions is a cone or a frustum.

2. The device according to claim 1, wherein the average height of the detection protrusions is 5 μm or more and 1000 μm or less.

3. The device according to claim 1 or 2, wherein the average distance between adjacent detection protrusions in the detection zone is 500 μm or less.

4. The device according to claim 1 or 2, wherein the shape of the bottom surface of the detection projection is circular, and the average diameter of the bottom surface is 5 μm or more and 1000 μm or less.

5. The device according to claim 1 or 2, wherein a fine uneven structure B is formed on the surface of the detection projection in at least the detection zone, and the arithmetic mean roughness Ra of the roughness curve of the detection projection on which the fine uneven structure B is formed is 0.005 μm or more and 1 μm or less.

6. The device according to claim 1 or 2, wherein in one cross-section, a plurality of detection protrusions have a first region located on one side of the detection protrusion from the center of the detection protrusion in the width direction of the detection protrusion, and a second region located on the other side of the detection protrusion from the center of the detection protrusion in the width direction of the detection protrusion, and in one cross-section, at least one of the outer edge of the first region and the outer edge of the second region of the detection protrusion has a recess.

7. The device according to claim 1 or 2, wherein, when the detection zone is viewed from above, a plurality of the detection protrusions are arranged in a grid pattern.

8. The device according to claim 1 or 2, wherein the biological sample is a blood sample, urine, saliva, or breast milk.

9. The device according to claim 1 or 2, wherein the extracellular vesicle is an exosome.

10. The device according to claim 9, wherein the capture substance is an antibody or an antigen-binding fragment thereof.

11. The device according to claim 10, wherein the capture substance is an antibody against the surface antigen of an exosome.

12. The device according to claim 11, wherein the surface antigen of the exosome is one or more selected from the group consisting of CD9, CD63, CD81, and CD147.

13. The device according to claim 1 or 2, wherein the material of the substrate is a thermoplastic resin.

14. The device according to claim 1 or 2, wherein the material of the substrate includes one or more selected from the group consisting of polyester, polyolefin, polystyrene, polycarbonate, fluororesin, poly(meth)acrylate, polyvinyl chloride, polyamide, and polyimide.

15. The device according to claim 1 or 2, wherein a cover portion is provided to cover the aforementioned flow path.

16. The device according to claim 15, wherein the lid portion is made of one or more materials selected from the group consisting of thermoplastic resins, thermosetting resins, photocurable resins, and glass.

17. The device according to claim 1 or 2, for use in the diagnosis, treatment, or prevention of cancer in a subject.

18. A test kit having the device according to claim 1 or 2.

Images