Fluidic device and use thereof

Abstract

The fluid device of the present application has a substrate having a hole array of a plurality of identical holes regularly arranged with the holes opened on one surface, and a cover member arranged opposite the hole array, a space between the hole array and the cover member forming a flow path through which a fluid flows, a distance Dab between a center of gravity Ca of an opening of an arbitrary hole A of the hole array and a center of gravity Cb of an opening of a hole B closest to the hole A, and a diameter Da of a circle having the same area as that of the opening of the hole A satisfying the following formula (1), 0.8 ≤ Da / Dab < 1 (1).

Description

Technical Field

[0001] This invention relates to fluid devices and their applications. More specifically, this invention relates to fluid devices, methods for isolating aqueous media, and methods for detecting objects.

[0002] This application claims priority to Japanese Patent Application No. 2020-147507, filed on September 2, 2020 in Japan, the contents of which are incorporated herein by reference. Background Technology

[0003] Techniques for detecting biomolecules within fluidic devices are known. For example, in DNA microarray technology, biomolecules are detected by introducing them into tiny wells and conducting a reaction accompanied by heating. Furthermore, techniques for single-molecule detection of biomolecules are known. Examples of such techniques include digital measurement technologies such as digital ELISA (Digital Enzyme-Linked Immunosorbent Assay), digital PCR (Digital Polymerase Reaction), and digital Invasive Cleavaged Assay (Digital ICA).

[0004] These technologies require isolating the aqueous medium containing biomolecules within tiny reaction spaces. The inventors previously developed a method for isolating the aqueous medium by supplying the aqueous medium into the flow path of a reaction vessel having a flow path and multiple pores, filling the pores with the aqueous medium, and then supplying an oily sealing liquid into the flow path to seal the aqueous medium within the pores, thereby creating multiple independent reaction spaces for each pore (see, for example, Patent Document 1).

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: International Publication No. 2015 / 115635 Summary of the Invention

[0008] The technical problem that the invention aims to solve

[0009] However, the inventors discovered that when the volume of the pores in the pore array formed on the substrate decreases, when reagents are introduced into each pore, the air inside the pores is sometimes not replaced by the reagents and remains, which can hinder the detection of biomolecules.

[0010] Therefore, the object of the present invention is to provide a technique for suppressing the retention of air bubbles inside the pores when introducing an aqueous medium into the pores of a fluid device having a pore array.

[0011] Means for solving technical problems

[0012] The present invention includes the following methods.

[0013] [1] A fluid device comprising:

[0014] A substrate having at least a portion of a hole array consisting of a regular arrangement of multiple holes of the same shape opening on one surface; and

[0015] The cover member is arranged facing the aforementioned hole array.

[0016] The space between the aforementioned hole array and the aforementioned cover component forms a flow path for fluid.

[0017] The distance Dab between the centroid Ca of the opening of any hole A in the above hole array and the centroid Cb of the opening of the hole B closest to hole A, and the diameter Da of the circle with the same area as the opening of hole A, satisfy the following equation (1).

[0018] 0.8≤Da / Dab<1 (1).

[0019] [2] According to the fluid device described in [1] above, the diameter of the circle with the same area as the opening area of ​​the hole is 1 μm or more and 50 μm or less.

[0020] [3] According to the fluid device described in [1] or [2] above, the ratio of the sum of the areas of the openings of each hole in the hole array to the area of ​​the hole array on the one face is 30% or more and 90% or less.

[0021] [4] The fluid device according to any one of [1] to [3] above, wherein the volume of each of the above-mentioned holes is 10fL or more and 100pL or less.

[0022] [5] The fluid device according to any one of [1] to [4] above, wherein the sum of the volumes of each hole in the above-described hole array is 0.2 μL or more and 2.0 μL or less.

[0023] [6] The fluid device according to any one of [1] to [5] above, wherein the sum of the volumes of each hole in the above-described hole array is 5% or more and 40% or less relative to the volume of the above-described flow path.

[0024] [7] In any one of the above [1] to [6] fluid device, the diameter of the circle with the same area as the opening area of ​​the hole is 3% or more and 200% or less relative to the depth of the hole.

[0025] [8] The fluid device according to any one of [1] to [7] above, wherein the contact angle between the surface and the water is 70 degrees or more and 180 degrees or less.

[0026] [9] The fluid device according to any one of [1] to [8] above, wherein the contact angle between the side of the cover member facing the hole array and the water is 70 degrees or more and 180 degrees or less.

[0027]

[10] A method for isolating an aqueous medium, comprising the following steps:

[0028] The process of introducing the aqueous medium into the flow path of the fluid device described in any one of [1] to [9] above; and

[0029] The process of introducing the aqueous medium and then introducing a sealing liquid into the flow path to isolate the aqueous medium in each hole of the hole array.

[0030]

[11] A method for detecting an object, comprising the following steps:

[0031] After isolating the aqueous medium containing the detection object and the detection reagent using the method described above

[10] , the fluid device is heated to cause a reaction inside the orifice, generating a signal for detecting the detection object; and

[0032] The process of detecting the above signals.

[0033]

[12] According to the method described in

[11] above, the detection target is a biomolecule.

[0034]

[13] The method described in

[11] or

[12] above, wherein the above reaction is an isothermal reaction.

[0035]

[14] The method according to any one of

[11] to

[13] above, wherein the signal is fluorescence.

[0036] Invention Effects

[0037] According to the present invention, a technique can be provided to suppress the residual air bubbles inside the holes when introducing an aqueous medium into the holes of a fluid device having a hole array. Attached Figure Description

[0038] Figure 1(a) is a schematic cross-sectional view illustrating the structure of the fluid device; (b) is a top view of the fluid device; and (c) is a partially enlarged top view of the orifice array of the fluid device viewed from the orifice opening side.

[0039] Figure 2 (a) and (b) are photographs of the well arrays observed in bright field during the delivery of buffer solution to the fluid apparatus of Example 1 in Experimental Example 1.

[0040] Figure 3 (a) to (c) are photographs of the well arrays observed in bright field during the delivery of buffer solution to the fluid apparatus of Comparative Example 1 in Experimental Example 1.

[0041] Figure 4 (a) to (e) are photographs of the well arrays observed in bright field during the delivery of buffer solution to the fluid apparatus of Comparative Example 2 in Experimental Example 1.

[0042] Figure 5 (a) to (e) are cross-sectional views of the fluid apparatus representing the simulation results in Experiment Example 2.

[0043] Figure 6 (a) to (c) are cross-sectional views of the fluid apparatus representing the simulation results in Experiment Example 3.

[0044] Figure 7 (a) to (c) are cross-sectional views of the fluid apparatus representing the simulation results in Experiment Example 4.

[0045] Figure 8 (a) to (c) are cross-sectional views of the fluid apparatus representing the simulation results in Experiment Example 5. Detailed Implementation

[0046] The following is a reference to the appendix, depending on the situation. Figure 1 The embodiments of the present invention will be described in detail below. Furthermore, in the accompanying drawings, identical or equivalent portions are marked with the same or corresponding symbols, and repeated descriptions are omitted. Additionally, the dimensions in the figures are exaggerated for illustrative purposes and do not necessarily correspond to the actual dimensions.

[0047] [Fluid Equipment]

[0048] In one embodiment, the present invention provides a fluid device comprising: a substrate having at least a portion of a plurality of holes of the same shape regularly arranged to form an array of holes having openings on one surface; and a cover member disposed opposite to the hole array, wherein the space between the hole array and the cover member forms a flow path through which fluid flows, wherein the distance Dab between the centroid Ca of the opening of any hole A in the hole array and the centroid Cb of the opening of the hole B closest to hole A, and the diameter Da of a circle having the same area as the opening of hole A, satisfy the following formula (1).

[0049] 0.8≤Da / Dab<1 (1)

[0050] As described later in the embodiments, the fluid device according to this embodiment can suppress the residual air bubbles inside the holes when an aqueous medium is introduced into each hole of the hole array.

[0051] Figure 1 (a) A schematic cross-sectional view illustrating the structure of the fluid device in this embodiment. Figure 1 (b) is a top view of the fluid device in this embodiment. Figure 1 (c) is a partially enlarged top view (top view) of the orifice array of the fluid device of this embodiment, viewed from the opening side of the orifice.

[0052] like Figure 1 As shown in (a), the fluid device 100 of this embodiment includes a substrate 130 having at least a portion of a hole array 120 formed by regularly arranging a plurality of holes 110 of the same shape that open on one surface, and a cover member 140 disposed facing the hole array 120. The space 150 between the hole array 120 and the cover member 140 forms a flow path for fluid to flow through. The flow path 150 is a continuous space between the surface 131 of the substrate 130 and the surface 141 of the cover member 140.

[0053] like Figure 1 As shown in (b) and (c), the distance Dab between the centroid of the opening of any hole A in the hole array 120 (i.e., the centroid of the shape defined by the periphery of the opening) and the centroid Cb of the opening of the hole B closest to hole A, and the diameter Da assuming that the area of ​​the opening of hole A is the same as that of a circle, satisfy the following equation (1).

[0054] 0.8≤Da / Dab<1 (1)

[0055] In the fluid device of this embodiment, the lower limit of the Da / Dab value is 0.8, and it can also be 0.83 or higher. Furthermore, the upper limit of the Da / Dab value is less than 1, and can also be 0.92 or lower, or approximately 0.9. These lower and upper limits can also be combined arbitrarily.

[0056] like Figure 1 As shown in (a), the fluid device of this embodiment may further include an inlet 160 for introducing fluid into the flow path 150 and an outlet 170 for discharging fluid from the flow path 150. Additionally, Figure 1In example (a), the inlet 160 and outlet 170 are formed on the cover member 140, but the inlet 160 and outlet 170 can also be formed on the peripheral member 180 (described later) or on the substrate 130. The inlet 160 and outlet 170 are disposed at the position of the clamping hole array 120.

[0057] The shapes of the inlet 160 and outlet 170 are not particularly limited; they can be any shape that can transport fluid. For example, they can be circles, ellipses, triangles, quadrilaterals, pentagons, hexagons, heptagons, octagons, and other polygons.

[0058] like Figure 1 As shown in (a), the fluid device of this embodiment preferably includes a peripheral member 180. The peripheral member 180 functions as a separator separating the substrate 130 and the cover member 140 to form a flow path 150. The peripheral member 180 is also disposed around the hole array 120 between the substrate 130 and the cover member 140, forming part of the wall surface of the flow path 150. That is, the flow path 150 is preferably surrounded by the peripheral member 180 located between the surface 131 of the substrate 130 and the surface 141 of the cover member 140. The peripheral member 180 can also be provided as an integral member continuously connected to the cover member 140.

[0059] Furthermore, the cross-sectional shape of the surface of the flow path 150 perpendicular to the flow direction is not particularly limited; it can be any shape that can transport fluid. Examples include squares, rectangles, triangles, circles, and ellipses. Additionally, the cross-sectional shape of the flow path 150, from the inlet 160 to the outlet 170, can be constant or variable, but is preferably constant.

[0060] The fluid device of this embodiment can be a microfluidic path, commonly used in the processing of biomolecules. More specifically, the maximum cross-sectional area of ​​the flow path in the fluid device of this embodiment can be 0.01 to 1 mm. 2 .

[0061] The hole array 120 can be formed by forming a plurality of holes 110 on one surface 131 of the substrate 130. The substrate 130 can also be formed by stacking a first layer having a plurality of through holes and a second layer as a plate. The through holes form the holes 110, and the plurality of through holes form the hole array 120.

[0062] The material of the substrate 130 is not particularly limited, and examples include metals such as stainless steel, titanium, cobalt-chromium alloy, and magnesium alloy; glass; and resin materials such as common plastics, medical plastics, and cosmetic plastics. Furthermore, multiple materials formed from these materials can be laminated. Examples of resin materials include polyethylene, polypropylene, polystyrene, polyamide, polycarbonate, cyclic polyolefins, polylactic acid, polyglycolic acid, polycaprolactone, acrylic acid, urethane resins, silicone resins, fluoropolymers, aromatic polyetherketones, epoxy resins, and copolymers of these resins. More specific resin materials include, for example, ZEONEX (registered trademark) and CYTOP (registered trademark). The material of the cover member 140 is also not particularly limited, and examples include materials identical to those used for the substrate 130.

[0063] The shape of the opening of the orifice 110 (i.e., the shape of the orifice 110 in the top view of the fluid device 100 as viewed from the opening side) is not particularly limited. It can be a circle, an ellipse, or a polygon such as a triangle, quadrilateral, pentagon, hexagon, heptagon, or octagon. When the opening of the orifice 110 is circular, the centroid of the opening is the center of the circle.

[0064] The plurality of holes 110 constituting the hole array 120 are all of the same shape and are arranged regularly. However, for use as alignment marks in camera or equipment manufacturing, a few (e.g., 1 to 4) of the plurality of holes may be of different shapes. In this case, substantially all the holes (i.e., the holes other than the aforementioned few holes) are of the same shape and are arranged regularly.

[0065] Here, "regularly arranged holes" means that the centroids of the openings of the holes constituting the hole array are arranged in a constant pattern. For example, the centroids of the hole openings can be arranged in a square grid. In this case, the lines connecting the centroids of the openings of four adjacent holes form rectangles, preferably squares.

[0066] Alternatively, the centroids of the hole openings can be arranged in a triangular lattice (also called a hexagonal lattice). In this case, the lines connecting the centroids of the openings of three adjacent holes form an equilateral triangle. When the holes are arranged in a triangular lattice, the distance between the centroid of the opening of any hole and the centroid of the opening of the hole closest to it is constant for all holes.

[0067] Figure 1 The holes shown in (c) are configured in a triangular grid pattern. For example... Figure 1 As shown in (c), in the holes 110 constituting the hole array 120, the centroids Ca, Cb and Cc of the openings of any hole A, the hole B closest to hole A and the hole C closest to both hole A and hole B form equilateral triangles with themselves as vertices. Figure 1 In (c), the line connecting the centroids Ca, Cb, and Cc of the openings of holes A, B, and C is an equilateral triangle.

[0068] In the fluid device of this embodiment, the diameter of the orifice, assuming the opening area is a circle of the same area, is preferably 1 μm or more and 50 μm or less. That is, the lower limit of the diameter when the opening area of ​​the orifice is a circle of the same area is preferably 1 μm. Furthermore, the upper limit of the diameter when the opening area of ​​the orifice is a circle of the same area can be less than 20 μm, or less than 19 μm, or less than 18 μm, or less than 17 μm, or less than 16 μm, or less than 15 μm, or less than 14 μm, or less than 13 μm, or less than 12 μm, or less than 11 μm, or less than 10 μm. These lower and upper limits can be combined arbitrarily.

[0069] In the fluid device of this embodiment, the area of ​​the hole array 120 refers to the area of ​​the region on the surface 131 that is inscribed within the holes 110 present in the periphery of the hole array 120. The ratio of the sum of the areas of the openings of each hole 110 of the hole array 120 to the area of ​​the hole array 120 on one surface 131 (hereinafter sometimes referred to as the "opening area ratio") is preferably 30% or more and 90% or less.

[0070] The lower limit for the opening area ratio can be 60%, 45%, or 30%. The upper limit can be 90%, 80%, or 70%. These lower and upper limits can be combined arbitrarily. For example, the opening area ratio can be above 30% and below 90%, above 45% and below 80%, or above 60% and below 70%.

[0071] In the fluid device of this embodiment, the volume of each orifice 110 is preferably 10 fL or more and 100 pL or less, more preferably 100 fL or more and 30 pL or less. Furthermore, the sum of the volumes of all orifices 110 constituting the orifice array 120 is preferably 0.2 μL or more and 2.0 μL or less, more preferably 0.4 μL or more and 1.5 μL or less. Additionally, assuming the area of ​​the orifice 110 is the same as that of a circle with the same area as the opening, the ratio of the diameter to the depth of the orifice 110 is preferably 3% or more and 200% or less, more preferably 30% or more and 120% or less, and even more preferably 60% or more and 90% or less. When the size of the orifice 110 is within the above range, single-molecule detection of biomolecules can preferably be performed.

[0072] In the fluid device of this embodiment, the sum of the volumes of each hole 110 constituting the hole array 120 is preferably 5% or more and 40% or less relative to the volume of the flow path 150.

[0073] In the fluid device of this embodiment, the contact angle between one surface 131 and water is preferably 70 degrees or more and 180 degrees or less. Furthermore, the contact angle between the surface 141 of the cover member 140 facing the hole array 120 and water is preferably 70 degrees or more and 180 degrees or less. When the contact angles of surfaces 131 and 141 are within the above range, when the sealing fluid is introduced into the flow path 150, there is a tendency to easily isolate the aqueous medium within each hole of the hole array.

[0074] The fluid device of this embodiment can be manufactured, for example, in the following sequence. First, a substrate is prepared, and a resin layer is formed on one side of the substrate. A hole is formed on the substrate by forming a through hole in the resin layer. A second resin layer may also be provided between the substrate and the resin layer. Alternatively, an anchoring layer or the like may be provided to improve the adhesion between the substrate and the resin layer.

[0075] The resin layer can be formed from a material in which a colored component is mixed into the resin material. When the resin material is a photoresist, the content of the colored component can be, for example, 0.5% by mass (also called mass%) or more and 60 mass% or less. Regarding the content, it is preferable to be 5 mass% or more and 55 mass% or less, and more preferably 20 mass% or more and 50 mass% or less.

[0076] The content of the colored component can be appropriately set, taking into account the proportion of photosensitive components in the resist, to create the desired pattern. Furthermore, the colored component can be a pigment, and a dispersant can be appropriately added along with the pigment. When the resulting resin layer is formed from a material in which the colored component is mixed, the resin layer has a color based on the colored component.

[0077] Next, through holes are formed on the resin layer. Using photolithography, through holes can be formed simply and with good precision. When forming the resin layer using injection molding or the like, the resin layer formation and through hole formation can be performed in the same process. Alternatively, through holes can be formed using etching with a pattern mask or the like. When through holes are formed in the resin layer, a substrate 130 with a hole array 120 is obtained.

[0078] Alternatively, the hole array 120 can also be formed by forming a plurality of holes 110 on one surface 131 of the substrate 130. In this case, the substrate 130 having the hole array 120 can be manufactured by injection molding the resin material using a mold corresponding to the shape of the hole array 120.

[0079] Next, a peripheral member 180 is disposed around the hole array 120. Next, a cover member 140 is disposed on the peripheral member 180. Next, when the substrate 130, the peripheral member 180 and the cover member 140 are integrally joined, a fluid device 100 is obtained. A flow path 150 is formed between the cover member 140 and the substrate 130 using the peripheral member 180.

[0080] There is no particular limitation on the joining method of the substrate 130, the peripheral member 180 and the cover member 140. Known methods can be used, such as laser welding, bonding with double-sided adhesive, and bonding with adhesive.

[0081] In addition, in order to improve the adhesion of the substrate 130, the peripheral member 180 and the cover member 140, the surfaces of the substrate 130 and the cover member 140 may be subjected to surface treatment to enhance the adhesion effect before bonding. For example, as surface treatments, (1) chemical surface modification and (2) surface shape processing can be mentioned.

[0082] Alternatively, the peripheral member 180 and the cover member 140 can be integrally formed. In this case, the fluid device 100 can be manufactured by joining at least a portion of the substrate 130 having the hole array 120 and the cover member 140 integrally formed with the peripheral member 180. The flow path 150 is formed between the cover member 140 and the substrate 130 by the peripheral member 180 integrally formed on the cover member 140.

[0083] Fluid equipment can be manufactured using the methods described above. Furthermore, the methods for manufacturing fluid equipment are not limited to those described above; other known methods that can be applied analogously in various processes can also be used.

[0084] [Isolation methods for aqueous media]

[0085] In one embodiment, the present invention provides a method for isolating an aqueous medium, comprising the steps of: introducing an aqueous medium into the flow path of the fluid device; and, after introducing the aqueous medium, introducing a sealing liquid into the flow path to isolate the aqueous medium in each hole of the hole array.

[0086] According to the method of this embodiment, when an aqueous medium is filled into each hole of the pore array for isolation, residual air bubbles inside the pores can be suppressed.

[0087] Aqueous media and sealing fluids are preferably immiscible or difficult to mix. Specific examples of sealing fluids include fluorinated liquids such as FC-40, FC-43, FC-770, FC-72, and FC-3283 (all manufactured by 3M).

[0088] Aqueous media can contain both the analyte and the detection reagents. Examples of analytes include biomolecules such as nucleic acids, proteins, and lipids. Detection reagents are selected appropriately based on the detection method.

[0089] In this embodiment, the aqueous medium may also contain a surfactant. This allows for the suppression of residual air bubbles inside the pores and the suppression of non-specific adsorption on the pore-to-pore region of the surface 131 of the substrate 130.

[0090] The method of this embodiment may further include a step of introducing a filling liquid before introducing the aqueous medium, wherein the filling liquid and the aqueous medium are mixed with each other or easily mixed. This makes it easier to suppress residual air bubbles inside the pores. Specific examples of filling liquids include aqueous media containing surfactants, aqueous media without surfactants, and organic solvents that are easily mixed with aqueous media.

[0091] By including a surfactant in the filler, it is possible to achieve effects such as suppressing residual air bubbles inside the pores and suppressing non-specific adsorption on the pore-to-pore region on the surface 131 of the substrate 130. The surfactant may be included in both the aqueous medium and the filler, or only in either one.

[0092] In this embodiment, the aqueous medium and sealing fluid can be introduced through the inlet of a fluid device while pressure is applied. Introducing the aqueous medium and sealing fluid while pressure is applied can be done using a syringe or pipette. According to this embodiment, even when introducing the fluid while pressure is applied, it is easy to achieve the effect of suppressing residual air bubbles inside the orifice.

[0093] [Methods for detecting the target object]

[0094] In one embodiment, the present invention provides a method for detecting a target, comprising the following steps: after isolating an aqueous medium containing the target and a detection reagent within the pores of a pore array of a fluid device using the above-described aqueous medium isolation method, heating the fluid device to cause a reaction to occur inside the pores, thereby generating a signal for detecting the target; and detecting the signal.

[0095] According to the method of this embodiment, since the residual air bubbles inside the pore can be suppressed, it is easy to detect the object with high precision.

[0096] Aqueous media contain both the target molecules and the detection reagents. Target molecules can include, for example, biomolecules such as nucleic acids, proteins, and lipids. Detection reagents are selected appropriately based on the detection principle.

[0097] The reaction that generates the signal can be an isothermal reaction. Alternatively, the signal can be fluorescence. Examples of such reactions include Invasive Cleavaged Assay (ICA). In ICA, the target nucleic acid is detected, and detection reagents include flap probes, flanking endonucleases (FENs), and fluorescent substrates. Flap probes are nucleic acid fragments designed to hybridize with the nucleic acid of the target nucleic acid to form double-stranded nucleic acids with flanking structures. For ICA, the preferred reaction temperature for signal generation is above 55°C and below 75°C.

[0098] In the method of this embodiment, during the process of detecting signals, the signals can be detected by capturing images of the fluid equipment and analyzing the captured images.

[0099] In another aspect, the present invention includes the following methods.

[0100] [1] A fluid device comprising:

[0101] A substrate having at least a portion of a hole array consisting of a regular arrangement of multiple holes of the same shape opening on one surface; and

[0102] The cover member is arranged facing the aforementioned hole array.

[0103] The space between the aforementioned hole array and the aforementioned cover component forms a flow path for fluid.

[0104] The distance Dab between the centroid Ca of the opening of any hole A in the above hole array and the centroid Cb of the opening of the hole B closest to hole A, and the diameter Da of the circle with the same area as the opening of hole A, satisfy the following equation (1).

[0105] 0.8≤Da / Dab<0.92 (1).

[0106] [2] According to the fluid device described in [1] above, the diameter of the circle with the same area as the opening area of ​​the hole is 1 μm or more and 15 μm or less.

[0107] [3] According to the fluid device described in [1] or [2] above, the sum of the areas of the openings of each hole in the hole array is 60% or more and 80% or less relative to the area of ​​the hole array on the one surface.

[0108] [4] The fluid device according to any one of [1] to [3] above, wherein the volume of each of the above-mentioned holes is 100fL or more and 30pL or less.

[0109] [5] The fluid device according to any one of [1] to [4] above, wherein the sum of the volumes of each hole in the above-described hole array is 0.4 μL or more and 1.5 μL or less.

[0110] [6] The fluid device according to any one of [1] to [5] above, wherein the sum of the volumes of each hole in the above-described hole array is 5% or more and 100% or less relative to the volume of the above-described flow path.

[0111] [7] In any one of the above [1] to [6] fluid device, the diameter of the circle with the same area as the opening area of ​​the hole is 60% or more and 90% or less in proportion to the depth of the hole.

[0112] [8] The fluid device according to any one of [1] to [7] above, wherein the contact angle between the surface and the water is 70 degrees or more and 120 degrees or less.

[0113] [9] The fluid device according to any one of [1] to [8] above, wherein the contact angle between the side of the cover member facing the hole array and the water is 70 degrees or more and 120 degrees or less.

[0114]

[10] A method for isolating an aqueous medium, comprising the following steps:

[0115] The process of introducing the aqueous medium into the flow path of the fluid device described in any one of [1] to [9] above; and

[0116] The process of introducing the aqueous medium and then introducing a sealing liquid into the flow path to isolate the aqueous medium in each hole of the hole array.

[0117]

[11] A method for detecting an object, comprising the following steps:

[0118] After isolating the aqueous medium containing the detection object and the detection reagent using the method described above

[10] , the fluid device is heated to cause a reaction inside the orifice, generating a signal for detecting the detection object; and

[0119] The process of detecting the above signals.

[0120]

[12] According to the method described in

[11] above, the detection target is a biomolecule.

[0121]

[13] The method described in

[11] or

[12] above, wherein the above reaction is an isothermal reaction.

[0122]

[14] The method according to any one of

[11] to

[13] above, wherein the signal is fluorescence.

[0123] Example

[0124] [Manufacturing Example 1]

[0125] (Fabrication of the fluid device in Example 1)

[0126] The substrate and the cover component, both made of cyclic polyolefin (model "ZEONOR1020R", manufactured by ZEON Corporation of Japan), were respectively manufactured by injection molding.

[0127] The substrate thickness is 0.6 mm. An array of holes is formed on one surface of the substrate. The hole openings are circular. The hole dimensions are a diameter of 10 μm and a depth of 15 μm. Each hole has a volume of 824 fL, and the sum of the volumes of all holes is 0.76 μL. The ratio of the hole opening diameter to the hole depth is 66.7%. In a 6.0 mm × 30.0 mm area on the substrate, multiple holes are arranged in a triangular lattice pattern, with a distance of 12 μm between the center of each hole and the center of the nearest hole, forming a hole array. The contact angle between one surface of the substrate and water is 89 degrees.

[0128] The cover component and the height difference section (peripheral component) are integrally formed. The height of the flow path is 30 μm by adjusting the height of the height difference section to 30 μm. The actual height of the flow path is measured using a contact measuring instrument (model "TALYSURF PGI1240", manufactured by Taylor Hobson). The volume of the flow path is approximately 6 μL, and the sum of the volumes of all holes accounts for approximately 12.7% of the flow path volume. The contact angle between the face of the cover component facing the hole array and the water is 89 degrees.

[0129] Next, the height difference between the substrate and the cover component is joined by laser welding to fabricate the microfluidic device of Example 1.

[0130] [Manufacturing Example 2]

[0131] (The fabrication of fluid devices in Comparative Examples 1 and 2)

[0132] Except that the distance between the center of the pore and the center of the pore closest to it is 16 μm, the microfluidic device of Comparative Example 1 was fabricated in the same manner as in Example 1. The area of ​​the pore array was the same as in Example 1, and the sum of the volumes of the pores was 0.63 μL.

[0133] In addition, the microfluidic device of Comparative Example 2 was fabricated in the same manner as in Example 1, except that the distance between the center of the pore and the center of the pore closest to it was 20 μm. The area of ​​the pore array was the same as that of Example 1, and the sum of the volumes of each pore was 0.4 μL.

[0134] [Experimental Example 1]

[0135] An aqueous medium was introduced into each of the fluid devices of Example 1, Comparative Example 1, and Comparative Example 2 to evaluate the difficulty of bubble retention in the orifice.

[0136] First, an aqueous medium (i.e., buffer solution) with the composition shown in Table 1 below is injected into the flow path formed between the substrate and the cover member of each fluid device.

[0137] Table 1

[0138] Buffer composition

[0139] Element Final concentration <![CDATA[MgCl2]]> 20mM Tris pH 8.5 50mM Tween20 0.05％ distilled water Remaining

[0140] The buffer solution was injected while the fluid devices were observed from the substrate side using a bright-field microscope (model "BZ-710", manufactured by Keyence). A 10x objective lens was used, and the exposure time was 20 milliseconds. Then, the amount of buffer solution required to completely remove air bubbles from the wells was determined.

[0141] Figure 2 (a) and (b) are photographs showing bright-field observations of the pore array when delivering buffer solution to the fluid device of Example 1. Figure 2 (a) is a photograph showing the result of delivering 20 μL of buffer solution. Figure 2 (b) is a photograph showing the result of delivering 50 μL of buffer solution. Figure 2 The dimensions of the observed images (a) and (b) are 3,600 μm × 2,700 μm. The results show that the fluid apparatus of Example 1 effectively removed residual air bubbles from the orifices using a feed rate of only 50 μL of buffer solution.

[0142] in addition, Figure 3 (a) to (c) show bright-field observations of the pore array when delivering buffer solution to the fluid device of Comparative Example 1. Figure 3 (a) A photograph showing the result of delivering 100 μL of buffer solution. Figure 3 (b) A photograph showing the result of delivering 200 μL of buffer solution. Figure 3 (c) is a photograph showing the result of delivering 300 μL of buffer solution. Figure 3 The size of the observed images (a) to (c) is 3,600 μm × 2,700 μm. As a result, the fluid apparatus of Comparative Example 1 requires a delivery of 300 μL of buffer solution to adequately remove air bubbles remaining in the orifice.

[0143] in addition, Figure 4(a) to (e) are photographs showing the bright-field observation results of the pore array when delivering buffer solution to the fluid device of Comparative Example 2. Figure 4 (a) A photograph showing the result of delivering 100 μL of buffer solution. Figure 4 (b) A photograph showing the result of delivering 200 μL of buffer solution. Figure 4 (c) A photograph showing the result of delivering 300 μL of buffer solution. Figure 4 (d) is a photograph showing the result of delivering 400 μL of buffer solution. Figure 4 (e) is a photograph showing the result of delivering 500 μL of buffer solution. Figure 4 The size of the observed images (a) to (e) is 3,600 μm × 2,700 μm. As a result, the fluid apparatus of Comparative Example 2 requires a delivery of 500 μL of buffer solution to adequately remove air bubbles remaining in the orifice.

[0144] Table 2 below shows the diameter of the orifice, the depth of the orifice, the distance between the center of the orifice and the center of the orifice closest to it (center-to-center distance), the ratio of the orifice diameter to the center-to-center distance (diameter / center-to-center distance), the flow path height, the opening area ratio, the amount of buffer solution required to completely remove the air bubbles in the orifice, and the evaluation results of the poor retention of air bubbles in the orifice.

[0145] The opening area ratio is the ratio of the sum of the areas of the openings of the holes to the area of ​​the 6.0 mm × 30.0 mm region forming the hole array. Furthermore, the evaluation results for the poor retention of air bubbles within the holes are assessed using the following criteria.

[0146] (Evaluation Criteria)

[0147] Good: The required buffer solution delivery volume until all air bubbles in the well are completely removed is less than 50 μL.

[0148] Not good: The required buffer solution delivery volume to completely remove air bubbles from the well exceeds 50 μL.

[0149] Table 2

[0150] Example 1 Comparative Example 1 Comparative Example 2 Diameter (μm) 10 10 10 Hole depth (μm) 15 15 15 Intercenter distance (μm) 12 16 20 Diameter / Center-to-center distance 0.833 0.625 0.5 Flow path height (μm) 30 30 30 Opening area ratio 63 35 23 Liquid delivery volume (μL) 50 300 500 Evaluation results good not good not good

[0151] The results above indicate the following tendency: when the ratio of the diameter of the hole to the distance between the center of the hole and the center of the hole closest to it (center-to-center distance) is greater than 0.8, air bubbles are less likely to remain in the hole.

[0152] [Experimental Example 2]

[0153] (Simulation 1)

[0154] This study investigates the persistent presence of air bubbles within the orifices of a fluid processing device through simulation. The simulation was conducted using software (AnsysFluent, manufactured by ANSYS).

[0155] Simulations were performed on the flow path of a fluid device having an array of orifices of the shapes shown in Table 3, to which a liquid with the properties shown in Table 4 was supplied. Table 3 shows the diameter of each orifice, the depth of the orifice, the distance between the center of the orifice and the center of the orifice closest to it (center-to-center distance), the ratio of the orifice diameter to the center-to-center distance (diameter / center-to-center distance), the flow path height, the taper, and the presence or absence of burrs and fillets. In Table 4, wettability is expressed as a value reflecting the properties of the fluid device. Furthermore, a taper refers to a shape where the area of ​​the orifice opening differs from the area of ​​the bottom of the orifice, tapering towards the front end of the bottom. The taper angle represents the angle between the side of the substrate containing the orifice opening and the side of the orifice; when the taper angle is 0°, the angle between the side of the substrate containing the orifice opening and the side of the orifice is 90°.

[0156] The fluid device simulated in this experimental example is equivalent to the fluid device in Example 1.

[0157] Table 3

[0158] Simulation 1 Diameter (μm) 10 Hole depth (μm) 15 Intercenter distance (μm) 12 Diameter / Center-to-center distance 0.833 Flow path height (μm) 30 Conical (°) 0 Burrs and rounded corners none

[0159] Table 4

[0160] Physical properties of liquids

[0161] Viscosity (Pa·s) 0.0012 <![CDATA[Density (Kg / m 3 )]]> 789.24 Surface tension (N / m) 12 Diameter / Center-to-center distance 0.022 Wettability (°) 10

[0162] The simulation was conducted for the following scenarios: liquid was delivered at a flow rate of 33 mm / s from the start of delivery until 0.027 seconds later, and liquid was delivered at a flow rate of 330 mm / s from 0.027 seconds later until 0.047 seconds later.

[0163] Figure 5 (a) to (e) are cross-sectional views of the fluid equipment representing the simulation results. Figure 5 (a) shows the simulation results before the liquid delivery started. Figure 5 (b) shows the simulation results 0.02 seconds after the start of liquid delivery. Figure 5 (c) shows the simulation results 0.03 seconds after the start of liquid delivery. Figure 5 (d) shows the simulation results 0.04 seconds after the start of liquid delivery. Figure 5 (e) shows the simulation results 0.047 seconds after the start of liquid delivery. Figure 5 In (a) to (e), the scale represents the outline of the bubbles and the liquid.

[0164] The results show that after the liquid delivery begins, the bubbles in the orifice gather and merge, and the merged bubbles flow while entraining other bubbles.

[0165] [Experiment Example 3]

[0166] (Simulation 2)

[0167] This study investigates the persistent presence of air bubbles within the orifices of a fluid processing device through simulation. The simulation was conducted using software (AnsysFluent, manufactured by ANSYS).

[0168] A simulation was performed on the case of delivering a liquid with the properties shown in Table 4 to a flow path of a fluid device having an array of orifices with the shapes shown in Table 5 below.

[0169] The simulated fluid device in this experimental example is equivalent to the fluid device in Comparative Example 1.

[0170] Table 5

[0171] Simulation 2 Diameter (μm) 10 Hole depth (μm) 15 Intercenter distance (μm) 16 Diameter / Center-to-center distance 0.625 Flow path height (μm) 30 Conical (°) 0 Burrs and rounded corners none

[0172] The simulation was conducted for the following scenarios: liquid was delivered at a flow rate of 33 mm / s from the start of delivery until 0.02 seconds later, and at a flow rate of 330 mm / s from 0.02 seconds later.

[0173] Figure 6 (a) to (c) are cross-sectional views of the fluid equipment representing the simulation results. Figure 6 (a) shows the simulation results before the liquid delivery started. Figure 6 (b) shows the simulation results 0.02 seconds after the start of liquid delivery. Figure 6 (c) Simulation results 0.03 seconds after the start of liquid delivery. Figure 6 In (a) to (c), the scale represents the outline of the bubbles and the liquid.

[0174] The results show that even when liquid is delivered, the air bubbles inside the hole do not gather and merge, leaving residual air bubbles.

[0175] [Experiment Example 4]

[0176] (Simulation 3)

[0177] This study investigates the persistent presence of air bubbles within the orifices of a fluid processing device through simulation. The simulation was conducted using software (AnsysFluent, manufactured by ANSYS).

[0178] A simulation was performed on the case of conveying a liquid with the properties shown in Table 4 into the flow path of a fluid device having an array of orifices with the shapes shown in Table 6 below.

[0179] Table 6

[0180] Simulation 3 Diameter (μm) 20 Hole depth (μm) 15 Intercenter distance (μm) 22 Diameter / Center-to-center distance 0.909 Flow path height (μm) 30 Conical (°) 0 Burrs and rounded corners none

[0181] The simulation was conducted on the case where liquid was delivered at a flow rate of 33 mm / s from the start of delivery until 0.02 seconds later.

[0182] Figure 7 (a) to (c) are cross-sectional views of the fluid equipment representing the simulation results. Figure 7 (a) shows the simulation results before the liquid delivery started. Figure 7 (b) shows the simulation results 0.01 seconds after the start of liquid delivery. Figure 7 (c) shows the simulation results 0.02 seconds after the start of liquid delivery. Figure 7 In (a) to (c), the scale represents the outline of the bubbles and the liquid.

[0183] The results show that when the diameter of the hole is large, air bubbles are less likely to remain inside the hole.

[0184] [Experiment Example 5]

[0185] (Simulation 4)

[0186] This study investigates the persistent presence of air bubbles within the orifices of a fluid processing device through simulation. The simulation was conducted using software (AnsysFluent, manufactured by ANSYS).

[0187] A simulation was performed on the case of conveying a liquid with the properties shown in Table 4 into the flow path of a fluid device having an array of orifices with the shapes shown in Table 7 below.

[0188] Table 7

[0189] Simulation 4 Diameter (μm) 30 Hole depth (μm) 15 Intercenter distance (μm) 32 Diameter / Center-to-center distance 0.938 Flow path height (μm) 30 Conical (°) 0 Burrs and rounded corners none

[0190] The simulation was conducted on the case where liquid was delivered at a flow rate of 33 mm / s from the start of delivery until 0.02 seconds later.

[0191] Figure 8 (a) to (c) are cross-sectional views of the fluid equipment representing the simulation results. Figure 8 (a) shows the simulation results before the liquid delivery started. Figure 8 (b) shows the simulation results 0.01 seconds after the start of liquid delivery. Figure 8 (c) shows the simulation results 0.02 seconds after the start of liquid delivery. Figure 8 In (a) to (c), the scale represents the outline of the bubbles and the liquid.

[0192] The results show that when the diameter of the hole is large, air bubbles are less likely to remain inside the hole.

[0193] Industrial availability

[0194] According to the present invention, a technique can be provided to suppress the retention of air bubbles inside the holes when an aqueous medium is introduced into each hole of a fluid device having a hole array. Furthermore, according to the method for detecting a target according to the present invention, when an aqueous medium is introduced into each hole of a fluid device having a hole array for isolation and signal generation for detection, the retention of air bubbles inside the holes can be suppressed, thereby improving the detection efficiency of the target.

[0195] Symbol Explanation

[0196] 100 fluid device, 110 holes, 120 hole array, 130 substrate, 140 cover component, 131 and 141 surfaces, 150 flow path, 160 inlet, 170 outlet, 180 peripheral component, A, B, and C holes, Da diameter, Dab distance, Ca, Cb, and Cc centroid.

Claims

1. A fluid apparatus comprising: A substrate having at least a portion of a hole array consisting of a regular arrangement of multiple holes of the same shape opening on one surface; and A cover member arranged facing the hole array, The space between the hole array and the cover member forms a flow path for fluid. The distance Dab between the centroid Ca of the opening of any hole A in the hole array and the centroid Cb of the opening of the hole B closest to hole A, and the diameter Da of the circle with the same area as the opening of hole A, satisfy the following equation (1). 0.8≤Da / Dab<1 (1) Furthermore, the diameter of the circle with the same area as the opening of the hole is 1 μm or more and 20 μm or less.

2. The fluid device according to claim 1, wherein, The sum of the areas of the openings of each hole in the hole array is more than 30% and less than 90% of the area of ​​the hole array on the surface.

3. The fluid apparatus according to claim 1 or 2, wherein, The volume of each of the holes is more than 10 fL and less than 100 pL.

4. The fluid apparatus according to claim 1 or 2, wherein, The sum of the volumes of the pores in the pore array is greater than 0.2 μL and less than 2.0 μL.

5. The fluid apparatus according to claim 1 or 2, wherein, The sum of the volumes of the holes in the hole array is more than 5% and less than 40% of the volume of the flow path.

6. The fluid apparatus according to claim 1 or 2, wherein, The ratio of the diameter of a circle with the same area as the opening of the hole to the depth of the hole is more than 3% and less than 200%.

7. The fluid apparatus according to claim 1 or 2, wherein, The contact angle between the surface and the water is greater than 70 degrees and less than 180 degrees.

8. The fluid apparatus according to claim 1 or 2, wherein, The contact angle between the cover member and the water on the side facing the hole array is greater than 70 degrees and less than 180 degrees.

9. A method for isolating an aqueous medium, comprising the following steps: The step of introducing an aqueous medium into the flow path of the fluid device according to any one of claims 1 to 8; and The process of introducing the aqueous medium and then introducing a sealing liquid into the flow path to isolate the aqueous medium in each hole of the hole array.

10. A method for performing non-diagnostic testing on a test object, comprising the following steps: After isolating the aqueous medium containing the detection object and the detection reagent using the method of claim 9, the fluid device is heated to cause a reaction inside the orifice, generating a signal for detecting the detection object; and The process of detecting the signal.

11. The method according to claim 10, wherein, The detection target is biomolecules.

12. The method according to claim 10 or 11, wherein, The reaction is an isothermal reaction.

13. The method according to claim 10 or 11, wherein, The signal is fluorescence.

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