Biological sample collection device

Abstract

To provide a biological sample collection device that includes a high-strength uneven structure on its surface and enhances extracellular vesicle capture efficiency and miRNA detection levels.SOLUTION: A biological sample collection device includes a polymer substrate with an uneven structure on its surface, and a covering layer that covers at least a part of the uneven structure, where the coating layer includes inorganic oxides or ionic polymers.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a device for collecting biological samples. [Background technology]

[0002] Disease testing using bodily fluids such as urine and blood is called liquid biopsy, and is attracting attention as a new testing method. Extracellular vesicles released by cells contain microRNA (microribonucleic acid; hereafter sometimes referred to as "miRNA"), which functions as an early diagnostic marker for cancer, and there is a demand for technology to capture extracellular vesicles.

[0003] Existing extracellular vesicle separation techniques include ultracentrifugation and agglutination reagent methods. The most commonly used ultracentrifugation method requires a sample volume of several tens of mL and a separation time of 4 to 5 hours, and the recovery rate is about 5 to 25%, making it difficult to efficiently separate extracellular vesicles. The agglutination reagent method is a simple method in which an agglutination reagent is dropped onto the target sample and allowed to stand, but it requires a long period of standing for separation, and has problems such as the use of a centrifuge, changes in particle size, a decrease in the number of particles, and a decrease in the amount of marker protein.

[0004] To solve the above problems, a method has been reported in which extracellular vesicles from small amounts of body fluid or culture supernatant are adsorbed onto nanowires formed in the microchannels of a chip, enabling highly efficient extraction of miRNA derived from extracellular vesicles (see Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6606786 Summary of the Invention [Problem to be solved by the invention]

[0006] While forming an uneven structure on a surface makes it easier to collect extracellular vesicles, there was room for improvement in the strength of the uneven structure in conventional devices with an uneven structure.

[0007] An object of the present invention is to provide a device for collecting biological samples that has a highly robust uneven surface structure and is capable of improving the efficiency of capturing extracellular vesicles and the amount of miRNA detected. [Means for solving the problem]

[0008] As a result of extensive research to achieve the above-mentioned object, the inventors discovered that the above-mentioned problems could be solved by a biological sample collection device having a concave-convex structure formed by nanoimprinting within a microchannel structure, and thus completed the present invention.

[0009] That is, the respective aspects of the present invention are [1] to [8] shown below. [1] A biological sample collection device comprising: a polymer substrate having a concave-convex structure on its surface; and a coating layer covering at least a portion of the concave-convex structure, the coating layer comprising an inorganic oxide or an ionic polymer. [2] The biological sample collection device according to [1], wherein the height of the uneven structure is 0.1 to 100 μm, the width of the uneven structure is 0.01 to 10 μm, and the ratio of the height of the uneven structure to the width of the uneven structure is 0.50 or more. [3] The biological sample collection device according to [1] or [2], wherein the uneven structure is formed by concave and / or convex portions, and the concave and convex portions have at least one structure selected from the group consisting of a cylindrical shape, a polygonal prism shape, a cone shape, a polygonal pyramid shape, and a dome shape. [4] The biological sample collection device according to any one of [1] to [3], wherein the coating layer contains one or more components selected from the group consisting of zinc oxide, titanium oxide, nickel oxide, yttria oxide, tin oxide, alumina oxide, silica, indium oxide, and indium tin oxide. [5] The biological sample collection device according to any one of [1] to [4], wherein when the uneven surface structure is viewed from the side where the coating layer is present, the area ratio of the area where the uneven surface structure is present to the entire polymer substrate is 10% or more. [6] The biological sample collecting device according to any one of [1] to [5], wherein the uneven structure is oriented in one direction. [7] The biological sample collection device according to any one of [1] to [6], further comprising a coating substrate having one or more through holes on the side of the coating layer opposite the polymer substrate, and at least one of the through holes forming a space for adding a biological sample. [8] The biological sample collection device described in [7], further comprising an intermediate layer between the coating layer and the coated substrate, the intermediate layer including an area for adhering the coating layer to the coated substrate and a space for forming a flow path for transporting the biological sample. Effect of the Invention

[0010] According to the present invention, a device for collecting biological samples can be provided which has a highly robust uneven surface structure and which can improve the efficiency of capturing extracellular vesicles and the amount of miRNA detected. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows a schematic cross-sectional view of the device. [Diagram 2] FIG. 2 is a perspective view showing an example of a device in the form of a well plate. [Diagram 3] FIG. 3 is a perspective view showing an example of a flow channel configuration device. [Figure 4] FIG. 4 is a scanning electron microscope photograph of the surface of the device of Example 1. [Diagram 5] FIG. 5 is a scanning electron microscope photograph of the surface of the device of Example 2. [Figure 6]FIG. 6 is a scanning electron microscope photograph of the surface of the device of Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Hereinafter, the embodiment of the present invention will be described in detail with reference to the drawings. However, the present invention is not limited to the following embodiment. In this specification, "(meth)acryloyl group" refers to a methacryloyl group or an acryloyl group, and "(meth)acrylate" refers to a methacrylate or an acrylate. Unless otherwise specified, the materials exemplified below may be used alone or in combination of two or more.

[0013] Fig. 1 shows one embodiment of a biological sample collection device. The device 10 shown in Fig. 1 includes a polymer substrate 1 having a concave-convex structure on its surface, and a coating layer 2 that covers at least a part of the concave-convex structure. The coating layer 2 includes an inorganic oxide or an ionic polymer.

[0014] As used herein, a "biological sample" refers to a sample obtained from an animal (e.g., a human) or a plant. Examples of biological samples include cell culture supernatants, urine, blood, tissues, cells, and extracellular vesicles. These biological samples may contain DNA, RNA (e.g., miRNA), proteins, and the like.

[0015] Examples of materials for the polymer substrate 1 include cellulose-based resins such as triacetyl cellulose, diacetyl cellulose, and acetate butyrate cellulose; polyester resins such as polyethylene terephthalate; polycarbonate resins; acrylic resins such as polymethyl methacrylate; polyurethane resins; polyether resins; polysulfone resins; polyethersulfone; and polyether ketone.

[0016] The polymer substrate 1 has a concave-convex structure. The concave-convex structure is composed of concave and / or convex portions. In the device shown in FIG. 1, the concave-convex structure is formed by convex portions.

[0017] Examples of the shape of the concave and convex portions include a cylindrical shape, a polygonal prism shape (e.g., a triangular prism shape, a quadrangular prism shape, a hexagonal prism shape, etc.), a cone shape, a polygonal pyramid shape, a dome shape (hemisphere), etc. Since the concave and convex structure is suitable for increasing the capture rate of extracellular vesicles, it is preferable that the concave and convex portions have a cylindrical or polygonal prism shape, and it is more preferable that the concave and convex portions have a cylindrical or polygonal prism shape.

[0018] The concaves and convexities may be arranged regularly or irregularly. The concaves and convexities may be arranged in, for example, a dot-like, line-like, honeycomb-like, or lattice-like pattern. The polymer substrate 1 may include one of these patterns alone or in combination of two or more of them.

[0019] The height of the uneven structure is preferably 0.1 to 100 μm, since both the strength of the uneven structure and the performance of capturing a biological sample can be achieved. The height of the uneven structure is preferably 0.10 μm or more, 0.15 μm or more, or 0.20 μm or more, since it is suitable for improving the performance of capturing a biological sample, and may be 0.30 μm or more, 0.35 μm or more, 0.40 μm or more, 0.45 μm or more, 0.50 μm or more, 1.0 μm or more, 5.0 μm or more, or 10.0 μm or more. The height of the uneven structure is preferably 80.0 μm or less, 50.0 μm or less, 30.0 μm or less, 10.0 μm or less, 5.0 μm or less, 3.0 μm or less, 1.0 μm or less, 0.80 μm or less, or 0.60 μm or less, since it is suitable for improving the strength of the uneven structure.

[0020] The "height of the uneven structure" refers to the maximum length of the uneven structure in the out-of-plane direction of the polymer substrate 1. In the device 10 having protrusions shown in FIG. 1, the height of the uneven structure is the height 3A of the protrusions. In a device having an uneven structure formed by recesses on the surface, the height of the uneven structure is the height of the recesses. The height of the uneven structure can be calculated by measuring the length on a scanning electron microscope photograph.

[0021] The width of the uneven structure is preferably 0.01 to 10 μm because it is possible to improve the strength of the uneven structure while increasing the surface area of ​​the uneven structure and thereby improving the performance of capturing a biological sample. The width of the uneven structure is preferably 0.05 μm or more, 0.10 μm or more, 0.15 μm or more, or 0.20 μm or more because it is suitable for increasing the strength of the uneven structure, and may be 0.30 μm or more, 0.40 μm or more, 0.50 μm or more, 0.80 μm or more, 1.0 μm or more, 3.0 μm or more, or 5.0 μm or more. The width of the uneven structure is preferably 8.0 μm or less, 4.0 μm or less, 2.0 μm or less, 1.50 μm or less, 1.00 μm or less, 0.80 μm or less, 0.50 μm or less, 0.40 μm or less, or 0.30 μm or less, as this is suitable for improving the performance of capturing biological samples.

[0022] The "width of the uneven structure" refers to the maximum diameter of the uneven structure in the in-plane direction of the polymer substrate 1. In the device 10 having protrusions shown in FIG. 1, the width of the uneven structure is the width 3B of the protrusions. In a device having an uneven structure formed by recesses on the surface, the width of the uneven structure is the width of the recesses. The width of the uneven structure can be calculated by measuring these lengths on a scanning electron microscope photograph.

[0023] The ratio of the height of the uneven structure to the width of the uneven structure (height / width) is preferably 0.50 or more, since it is possible to balance the strength of the uneven structure and the performance of capturing a biological sample. Since it is suitable for improving the strength of the uneven structure and the performance of capturing a biological sample, the ratio of the height of the uneven structure to the width of the uneven structure is preferably 0.60 or more, 0.70 or more, 0.80 or more, or 0.85 or more, and may be 1.0 or more, 1.5 or more, 2.0 or more, or 10 or more. The ratio of the height of the uneven structure to the width of the uneven structure may be, for example, 5.0 or less, 3.0 or less, 2.0 or less, 1.5 or less, or 1.0 or less.

[0024] The pitch of the uneven structure may be, for example, 0.10 μm or more, 0.20 μm or more, 0.30 μm or more, 0.40 μm or more, 0.50 μm or more, 0.60 μm or more, 0.70 μm or more, 0.80 μm or more, or 0.90 μm or more, and may be 5.0 μm or less, 4.0 μm or less, 3.0 μm or less, 2.0 μm or less, 1.0 μm or less, 0.80 μm or less, 0.60 μm or less, or 0.50 μm or less.

[0025] The pitch of the uneven structure is the distance between the centers of the bottom surfaces of the recesses of the uneven structure, or the distance between the apexes of the protrusions. In the device 10 having protrusions shown in Figure 1, the pitch of the uneven structure is the distance 3C between the apexes of the protrusions. The pitch of the uneven structure can be calculated by measuring these lengths on a scanning electron microscope photograph.

[0026] The ratio of the pitch of the uneven structure to the width of the uneven structure (pitch / width) is preferably 10.0 or less, 9.0 or less, 8.0 or less, 7.0 or less, 6.0 or less, 5.0 or less, 4.0 or less, 3.0 or less, 2.5 or less, or 2.0 or less, and may be 1.5 or less. The ratio of the pitch of the uneven structure to the width of the uneven structure (pitch / width) may be 1.1 or more, 1.3 or more, 1.5 or more, 1.7 or more, or 1.9 or more.

[0027] The uneven structure may be provided on at least a part of the main surface of the polymer substrate 1 on the side where the coating layer is provided. When viewed from the side where the uneven structure is present, the ratio of the area of ​​the region where the uneven structure is present to the entire polymer substrate 1 (hereinafter also referred to as the "uneven structure presence ratio") is preferably 10% or more. When the uneven structure presence ratio is 10% or more, the performance of capturing a biological sample can be improved. Since it is suitable for improving the performance of capturing a biological sample, the uneven structure presence ratio is preferably 15% or more, 20% or more, 25% or more, or 30% or more, and may be 50% or more, or 80% or more. The uneven structure presence ratio may be, for example, 100% or less, 80% or less, 60% or less, 50% or less, or 40% or less.

[0028] The proportion of the uneven structure can be determined by measuring the area of ​​the region where the uneven structure is present in an image of the surface of the biological sample collection device, which is measured with a scanning electron microscope from a direction perpendicular to the surface, and dividing the area by the area of ​​the measurement range, which is equivalent to the area of ​​a square with one side 100 times or more the width of the uneven structure.

[0029] In the case of a cell collection application, it is preferable that the concave-convex structure is oriented in one direction. In the case of a biological sample collection device having a flow channel structure, the concave-convex structure is oriented in one direction, and by arranging the concave-convex structure against the flow of the biological sample in the flow channel, the collection efficiency of the biological sample of the concave-convex structure can be increased. In addition, in the case of a biological sample such as a cell, the components inside the biological sample are easily extracted by crushing with the concave-convex structure. The orientation of the concave-convex structure in one direction can be determined by the presence or absence of bright spots in a Fourier transform image obtained by measuring a scanning electron microscope image of the surface of the biological sample collection device and Fourier transforming the image. By the Fourier transform, the shading of the original image can be converted into a two-dimensional image representing the power (the square of the absolute value of the brightness) relative to the wave number of each sine wave when the shading of the original image is approximated by superimposing sine waves, and if there is regularity in the shading of the original image, bright spots corresponding to each sine wave will appear in the Fourier transform image.

[0030] In the application of collecting extracellular vesicles, it is preferable that the concave-convex structure is irregularly arranged. By having the concave-convex structure be irregularly arranged rather than oriented in one direction, in the case of a biological sample collection device having a flow channel structure, the reproducibility of the biological sample collection efficiency can be stabilized without depending on the direction of the concave-convex structure in the flow channel.

[0031] The method for forming the concave-convex structure is preferably nanoimprinting, which can be a thermal imprinting method in which a concave-convex pattern is transferred by heat using a thermoplastic resin, or a photoimprinting method in which a concave-convex pattern is transferred by ultraviolet light using a photocurable resin.

[0032] The coating layer 2 includes an inorganic oxide or an ionic polymer. When the coating layer 2 includes an inorganic oxide or an ionic polymer, it becomes possible to further increase the capture rate of extracellular vesicles.

[0033] The inorganic oxide may contain one or more components selected from the group consisting of zinc oxide (ZnO), titanium oxide (TiO2), nickel oxide (NiO), alumina oxide (Al2O3), silica (SiO2), yttria oxide (Y2O3), tin oxide (e.g., SnO), indium oxide, and indium tin oxide (ITO). Since it is suitable for increasing the capture rate of extracellular vesicles, the inorganic oxide is more preferably zinc oxide, titanium oxide, nickel oxide, alumina oxide, silica, yttria oxide, tin oxide, indium oxide, and indium tin oxide, and particularly preferably zinc oxide, titanium oxide, tin oxide, indium oxide, and indium tin oxide, and most preferably zinc oxide or indium tin oxide.

[0034] There are no particular limitations on the method for forming an inorganic oxide on the surface of the uneven structure, but examples of the method include a method in which the desired inorganic oxide is directly coated by sputtering, chemical vapor deposition, deposition, etc., and a method in which an inorganic oxide precursor is wet-coated and then grown into the inorganic oxide by a sol-gel method.

[0035] The ionic polymer may contain a cationic polymer and / or an anionic polymer. The cationic polymer is preferably a polymer having a secondary amino group, a tertiary amino group, or a quaternary ammonium group. Examples of the cationic polymer include polyelectrolytes such as polyethyleneimine and its quaternary derivatives, polydiallyldimethylammonium chloride, poly(N,N'-dimethyl-3,5-dimethylene-piperidinium chloride) polyallylamine and its quaternary derivatives, polydimethylaminoethyl(meth)acrylate and its quaternary derivatives, and polydimethylaminopropyl(meth)acrylamide and its quaternary derivatives.

[0036] Anionic polymers are -COOH, -COO -, -SO3H, or -SO3 - Examples of the negatively charged polymer include poly(meth)acrylic acid and its ionized product, poly(sodium polystyrenesulfonate), poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyamic acid, and poly(potassium vinylsulfonate).

[0037] The ionic polymer preferably contains a cationic polymer, since it is possible to further increase the extracellular vesicle capture rate. The ionic polymer preferably contains a cationic polymer and does not contain an anionic polymer (cationic polymer), or contains a cationic polymer and an anionic polymer, since it is possible to further increase the extracellular vesicle capture rate.

[0038] The method of forming the ionic polymer on the surface of the uneven structure is not particularly limited, and can be appropriately selected depending on the material of the uneven structure and the types of cationic polymer and anionic polymer. For example, a method including surface-treating the surface of the uneven structure, and attaching a liquid containing a cationic polymer or anionic polymer to the surface-treated surface of the uneven structure, washing with water or the like as necessary, and then drying can be used. For example, plasma treatment can be used for the surface treatment. For a method of forming an ionic polymer layer containing a cationic polymer and an anionic polymer on the surface of the uneven structure, a method can be used in which the surface of the uneven structure is coated with one of the cationic polymer and the anionic polymer, and then coated with the other polymer.

[0039] The thickness of the coating layer 2 may be 10 nm or more, 50 nm or more, 80 nm or more, or 90 nm or more, and may be 500 nm or less, 200 nm or less, 160 nm or less, 120 nm or less, or 110 nm or less. The thickness of the coating layer 2 can be measured by the following method. The polymer substrate 1 on which the coating layer 2 is formed is cut with a microtome to prepare a thin film slice. The slice is observed with a scanning electron microscope to measure the length from the interface between the coating layer 2 and the polymer substrate 1 to the surface of the coating layer 2. The thickness of the coating layer 2 is measured after elemental mapping is performed, if necessary, to clarify the interface of the coating layer 2.

[0040] The width of the uneven structure coated with the coating layer 2 (coated uneven structure) may be 0.05 μm or more, 0.10 μm or more, or 0.12 μm or more, since the strength of the uneven structure can be improved. The width of the coated uneven structure may be 8.0 μm or less, 4.0 μm or less, 2.0 μm or less, 1.5 μm or less, 1.0 μm or less, 0.80 μm or less, or 0.60 μm or less, since it is suitable for improving the performance of capturing a biological sample. The width of the coated uneven structure is the width of the convex portion coated with the coating layer 2 or the concave portion coated with the coating layer 2. The width of the coated uneven structure can be calculated by measuring these lengths on a scanning electron microscope photograph of the surface on the side where the uneven structure coated with the coating layer 2 exists.

[0041] The pitch of the coated uneven structure may be the same as the numerical range exemplified as the pitch of the uneven structure (uncoated uneven structure) described above. The pitch of the coated uneven structure may be, for example, 0.10 μm or more, 0.25 μm or more, or 0.40 μm or more, and may be 3.0 μm or less, 2.0 μm or less, or 1.5 μm or less. The pitch of the coated uneven structure is the distance between the centers of the bottom surfaces of the recesses coated with the coating layer 2, or the distance between the apexes of the protrusions coated with the coating layer 2. The pitch of the coated uneven structure can be calculated by measuring these lengths on a scanning electron microscope photograph of the surface on the side where the uneven structure coated with the coating layer 2 exists.

[0042] The ratio of the pitch of the coated concave-convex structure to the width of the coated concave-convex structure (pitch / width) is suitable for enhancing the performance of capturing a biological sample, and may be 10.0 or less, 9.0 or less, 8.0 or less, 7.0 or less, 6.0 or less, 5.0 or less, 4.0 or less, or 3.5 or less. The ratio of the pitch of the coated concave-convex structure to the width of the coated concave-convex structure (pitch / width) is 1.1 or more, 1.2 or more, 1.3 or more, or 1.5 or more, and may be 1.1 or more, 1.2 or more, 1.3 or more, or 1.5 or more, and may be 1.0 or less, ...

[0043] The device 10 may further include a coated substrate having one or more through-holes on the surface of the coating layer 2 opposite to the polymer substrate 1. The device may have a space for adding a biological sample formed by at least one of the through-holes.

[0044] FIG. 2 shows an example of a device further comprising a coating substrate, which is a well plate type device. The device 10A shown in FIG. 2 comprises a polymer substrate 1, a coating layer 2, and a coating substrate 6. The coating substrate 6 has a plurality of spaces 7. In the device 10A, the spaces 7 are formed for adding biological samples. Since the spaces 7 are separated from the other spaces, different biological samples can be added to each space in one device without mixing the biological samples. Therefore, the device 10A can handle multiple types of biological samples at the same time. The spaces 7 may be through-holes, since this promotes the reaction of the biological samples.

[0045] The number of spaces 7 in the coated substrate 6 may be 2 or more. The number of spaces 7 in the coated substrate 6 is preferably, for example, 6, 24, 48, 96, 384, or 1536. The spaces 7 may also be 2 or more through holes.

[0046] Fig. 3 shows an example of a device further comprising a coated substrate 6, which further comprises an intermediate layer 8 between the coating layer 2 and the coated substrate 6, and the intermediate layer 8 has a flow path structure including a region for bonding the coating layer 2 and the coated substrate 6, and a space for forming a flow path 9 for transporting a biological sample. The device 10B shown in Fig. 3 has a polymer substrate 1, a coating layer 2, an intermediate layer 8, and a coated substrate 6 laminated in this order.

[0047] Two spaces 7a, 7b are formed in the coated substrate 6. The spaces 7a, 7b are through holes. One space 7a is a space for adding a liquid containing a biological sample, and the other space 7b is a space for recovering the liquid. Since the liquid added to one space 7a flows out into the other space 7b after passing through the flow path, the liquid from which at least some of the components in the biological sample have been removed can be recovered.

[0048] The intermediate layer 8 is disposed between the covering layer 2 and the covering substrate 6, and includes a region for bonding the covering layer 2 and the covering substrate 6, and a space (internal space) that forms a flow path 9 for transporting a biological sample.

[0049] The flow path 9 has a central region and two end regions, and the two end regions are arranged so as to be located vertically below the spaces 7a and 7b, respectively. The intermediate layer 8 can be formed, for example, from an adhesive tape. In this case, the coating layer 2 and the coating substrate 6 are bonded together with the adhesive tape, and the flow path 9 can be formed by the region where the adhesive tape is not present.

[0050] The biological sample is added onto the coating layer 2 in the end region of the flow channel 9 through the space 7a. The added biological sample is transported to the space 7b along the direction d while in contact with the uneven structure covered by the coating layer 2. According to the device 10B, the biological sample can be brought into contact with the uneven structure covered by the coating layer 2 in a minute space, and components can be efficiently extracted even from a small amount of biological sample.

[0051] The device according to this embodiment has an improved extracellular vesicle capture rate. The extracellular vesicle capture rate is preferably 50% or more, more preferably 60% or more, particularly preferably 80% or more, and may be 90% or more. The extracellular vesicle capture rate is calculated by the following formula 1. Details of the measurement method are as described in the Examples below. Formula 1: ([Amount of extracellular vesicles in the original extracellular vesicle sample]-[Amount of untrapped extracellular vesicles]) x 100 / [Amount of extracellular vesicles in the original extracellular vesicle sample] (%)

[0052] The device according to the present embodiment has a high extracellular vesicle capture rate, and can be suitably used for recovering miRNA contained in extracellular vesicles. The amount of recovered miRNA is preferably 0.10 ng / μL or more, more preferably 0.15 ng / μL or more, and may be 0.20 ng / μL or 0.25 ng / μL. The amount of recovered miRNA is measured by the method described in the Examples below.

[0053] When forming a concave-convex structure (nanowires) by utilizing crystal growth (see, for example, Patent Document 1), the material of the concave-convex structure is limited to certain materials such as zinc oxide, and it is difficult to flexibly change the material depending on the type of biological sample to be captured. In contrast, in the device according to the present embodiment, a concave-convex structure coated with an inorganic oxide or an ionic polymer is formed by forming a coating layer 2 on a polymer substrate 1 having a concave-convex structure, so that the material of the surface of the concave-convex structure can be easily changed depending on the type of biological sample to be captured, and high capture efficiency can be achieved for various biological samples. EXAMPLES

[0054] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited thereto. In the examples and comparative examples, the extracellular vesicle capture rate and the amount of miRNA were measured by the following methods.

[0055] [Measurement of extracellular vesicle capture rate and miRNA amount] The medium in which the cells were cultured was used as the extracellular vesicle sample. The extracellular vesicle sample was dropped at 250 μL / well and left to stand overnight. The supernatant was collected and the extracellular vesicle concentration was measured to determine the amount of extracellular vesicles that were not captured. Lysis buffer was dropped at 250 μL / well and left to stand for 5 minutes. The lysis buffer was collected and purified using a nucleic acid purification kit, after which the miRNA concentration was measured. The extracellular vesicle capture rate was calculated using the following formula. ([Amount of extracellular vesicles in the original extracellular vesicle sample] - [Amount of untrapped extracellular vesicles]) x 100 / [Amount of extracellular vesicles in the original extracellular vesicle sample] (%)

[0056] [Strength evaluation] The strength of the protrusions was evaluated by scratching the film surface having the protrusions five times with a Bemcot (manufactured by Asahi Kasei Corporation) under a load of 50 g, and then checking for damage to the protrusions using a scanning electron microscope. ○: No damage to the protrusions. ×: Protrusions are damaged.

[0057] [Proportion of area with uneven structure] The film surface with protrusions was measured using a scanning electron microscope (Keyence VE-9800). The obtained micrograph of the film surface with protrusions was binarized so that the areas with protrusions were white and the areas without protrusions were black, and the proportion of the white area was calculated. This was taken as the proportion of the area where the uneven structure was present.

[0058] [Example 1] A nanoimprinted film (manufactured by SCIVAX; product name FLP230 / 200 / 460-120) having a pillar structure with a concave-convex structure having a height of 0.2 μm, a width of 0.23 μm, and a pitch of 0.46 μm was used, and the surface was coated with zinc oxide (ZnO) to a thickness of 100 nm by sputtering.

[0059] The substrate having the above-mentioned zinc oxide sputtering layer was attached to the bottom surface of a bottomless 24-well plate to produce a device of Example 1 having an uneven structure on the bottom surface of the well. The device of Example 1 is a device in which an uneven structure is formed by a plurality of cylindrical protrusions. The device of Example 1 was packaged in a sterilization bag and sterilized with ethylene oxide gas. After aeration for 24 hours, the device was taken out and used for evaluation.

[0060] The fabricated device had an extracellular vesicle capture rate of 87.8%, and was excellent in extracellular vesicle capture performance. In addition, the amount of recoverable miRNA was 0.196 ng / μL, and the amount of recoverable miRNA was large. FIG. 4 is a scanning electron microscope photograph of the surface on the coating layer side of the device of Example 1. The width of the convex parts covered with the coating layer (coated convex parts) was 0.28 μm, the pitch of the coated convex parts was 0.45 μm, and the ratio of the pitch to the width of the coated convex parts was 1.61.

[0061] [Example 2] A nanoimprinted film (manufactured by SCIVAX Corp.; product name FLP500 / 500 / 1000-50×50) having a pillar structure with a height of 0.5 μm, width of 0.5 μm, and pitch of 1 μm was used to fabricate a device of Example 2 in the same manner as in Example 1. The device of Example 2 is a device in which a projection-recess structure is formed by a plurality of cylindrical projections.

[0062] The fabricated device had an extracellular vesicle capture rate of 87.1%, and was excellent in extracellular vesicle capture performance. In addition, the amount of recoverable miRNA was 0.104 ng / μL, and the amount of recoverable miRNA was large. Figure 5 is a scanning electron microscope photograph of the surface on the coating layer side of the device of Example 2. The width of the coated convex portions was 0.59 μm, the pitch of the coated convex portions was 1.0 μm, and the ratio of the pitch to the width of the coated convex portions was 1.69.

[0063] [Example 3] A nanoimprinted film (manufactured by SCIVAX Corp.; product name FLP500 / 500 / 1000-50×50) having a hole structure with a height of 0.2 μm, width of 0.23 μm, and pitch of 0.46 μm was used to fabricate the device of Example 3 in the same manner as in Example 1. The device of Example 3 is a device in which a concave-convex structure is formed by a plurality of cylindrical concaves. The width of the concaves covered with the coating layer (coated concaves) was 0.14 μm, the pitch of the coated concaves was 0.43 μm, and the ratio of the pitch to the width of the coated concaves was 3.07.

[0064] The fabricated device had an extracellular vesicle capture rate of 87.5%, and was excellent in extracellular vesicle capture performance. In addition, the amount of miRNA that could be recovered was 0.108 ng / μL, and the amount of miRNA that could be recovered was large. Figure 6 is a scanning electron microscope photograph of the surface of the coating layer side of the device of Example 3.

[0065] The devices obtained in Examples 1 to 3 have excellent extracellular vesicle trapping performance and were able to recover large amounts of miRNA, and are therefore suitable devices for collecting biological samples.

[0066] [Comparative Example 1] A smooth polycarbonate film was used as is, and without performing sputtering, it was attached to a bottomless 24-well plate in the same manner as in Example 1 to prepare a device of Comparative Example 1.

[0067] The device prepared in Comparative Example 1 had an extracellular vesicle capture rate of 46.0%, which was poor.

[0068] [Table 1]

[0069] The device of the embodiment had a highly robust uneven structure on the surface and a high extracellular vesicle capture rate. It was shown that the device of the embodiment had a high miRNA recovery rate. [Explanation of symbols]

[0070] 1...polymer substrate, 2...coating layer, 3A...height of convex portion, 3B...width of convex portion, 3C...distance between apexes of convex portions, 6...coating substrate, 7, 7a, 7b...space, 8...intermediate layer, 9...flow path, 10, 10A, 10B...device.

Claims

1. The present invention includes a polymer substrate having a concave-convex structure on a surface thereof, and a coating layer that covers at least a portion of the concave-convex structure, The biological sample collection device, wherein the coating layer comprises an inorganic oxide or an ionic polymer.

2. The height of the uneven structure is 0.1 to 100 μm, The width of the uneven structure is 0.01 to 10 μm, 2. The biological sample collection device according to claim 1, wherein a ratio of the height of the concave-convex structure to the width of the concave-convex structure is 0.50 or more.

3. The uneven structure is formed by recesses and / or protrusions, 3. The biological sample collecting device according to claim 1, wherein the recessed portion and the protruding portion have at least one structure selected from the group consisting of a cylindrical shape, a polygonal prism shape, a cone shape, a polygonal pyramid shape, and a dome shape.

4. 3. The biological sample collection device according to claim 1 or 2, wherein the coating layer contains one or more components selected from the group consisting of zinc oxide, titanium oxide, nickel oxide, yttria oxide, tin oxide, alumina oxide, silica, indium oxide, and indium tin oxide.

5. The biological sample collection device according to claim 1 or 2, wherein when the uneven structure is viewed from the side where the coating layer is present, the area ratio of the area where the uneven structure is present to the entire polymer substrate is 10% or more.

6. The biological sample collecting device according to claim 1 or 2, wherein the uneven structure is oriented in one direction.

7. The device further comprises a coated substrate having one or more through holes on a surface of the coating layer opposite the polymer substrate; 3. The biological sample collecting device according to claim 1, wherein a space for adding a biological sample is formed by at least one of the through-holes.

8. the device further comprising an intermediate layer between the coating layer and the coated substrate; The biological sample collection device according to claim 7 , wherein the intermediate layer includes a region for bonding the covering layer and the covering substrate, and a space for forming a flow path for transporting the biological sample.

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