Fluid device

Abstract

This fluid device comprises: a base member having a plurality of wells formed on the upper surface thereof; and a lid member disposed over the wells. The lid member comprises a cover portion disposed over the wells, and a connection portion that connects the cover portion and the base member. The lower surface of the connection portion is welded to the upper surface of the base member. The lower surface of the connection portion and the upper edge portions of the wells are separated in a direction parallel to the upper surface of the base member.

Description

Fluidic Devices

[0001] This application claims priority to Japanese Patent Application No. 2023-185851, filed on October 30, 2023, the contents of which are incorporated herein by reference.

[0002] There are known techniques for detecting biomolecules in fluidic devices. For example, in DNA microarray technology, biomolecules are introduced into micropores and reacted with heat to detect the biomolecules.

[0003] Furthermore, techniques capable of detecting single biomolecules are known, such as digital measurement techniques such as digital enzyme-linked immunosorbent assay (ELISA), digital polymerase chain reaction (PCR), and digital invasive cleaved assay (ICA) described in Non-Patent Document 1.

[0004] For example, Patent Document 1 discloses a fluidic device comprising a substrate having a plurality of minute wells (depressions) formed on its upper surface and a lid material whose lower end is welded to the upper surface of the substrate.

[0005] In such a fluidic device, the lid material has a cover portion that covers the top of the multiple wells and a connection portion that connects the cover portion to the substrate, and an internal space that functions as a flow path is formed between the cover portion and the substrate.

[0006] To detect biomolecules, an aqueous medium containing a target substance is first pumped into the flow channel of the fluidic device, filling multiple wells on the flow channel with the aqueous medium. Next, an oily sealing liquid is pumped into the flow channel to seal the aqueous medium in the multiple wells. This creates multiple independent reaction spaces for each well. The fluidic device is then heated to heat the reaction solution, causing a detection reaction, thereby detecting the target substance.

[0007] International Publication No. 2019 / 098301

[0008] However, when a fluidic device is heated, bubbles may be generated inside the fluidic device, and the bubbles generated inside the device interfere with detection reactions and observations, so improvements were needed.

[0009] The present invention has been made in view of the above circumstances, and has an object to provide a fluidic device that can suppress the generation of bubbles and detect biomolecules with high accuracy.

[0010] The inventors observed a fluidic device in which bubbles were generated and found that bubbles were generated near the contact area between the upper edge of a well formed on the upper surface of a substrate and the underside of a connecting portion of a lid. Subsequently, the inventors discovered that the generation of bubbles could be suppressed by separating the upper edge of the well from the underside of the connecting portion of the lid in a direction parallel to the upper surface of the substrate (horizontal direction), and thus completed the present invention.

[0011] In order to solve the above problems, one aspect of the present invention includes the following aspects.

[0012] [1] A fluidic device comprising: a substrate having a plurality of wells formed on its upper surface; and a lid material covering the top of the wells, wherein the lid material comprises a cover portion covering the top of the wells and a connection portion connecting the cover portion to the substrate, the lower surface of the connection portion being welded to the upper surface of the substrate, and the lower surface of the connection portion and the upper edge of the wells being spaced apart in a direction parallel to the upper surface of the substrate.

[0013] [2] The fluidic device according to [1], wherein the separation distance between the upper edge of the well and the lower surface of the connection portion is 30 μm or more.

[0014] [3] The fluidic device according to [1] or [2], wherein the separation distance between the upper edge of the well and the lower surface of the connection portion is 155 μm or more. [4] The fluidic device according to any one of [1] to [3], wherein the volume of each well is 1 fL to 6 nL. [5] The fluidic device according to any one of [1] to [4], wherein the distance between the upper edge of the well and the lower surface of the connection portion is 500 μm or less. [6] The fluidic device according to any one of [1] to [5], wherein the connection portion has a through-hole that penetrates in the vertical direction of the stack of the substrate and the lid material, and the through-hole forms an internal space between the substrate and the lid material, and a well region in which the well is formed is provided inside the internal space as viewed from the vertical direction. [7] The fluidic device according to [6], wherein the wells are provided at equal intervals in the well region. [8] The fluidic device according to [6], wherein the wells are provided at irregular intervals in the well region. [9] The fluidic device according to any one of [6] to [8], wherein the lid member is provided with an inlet and an outlet communicating with the internal space, and when the direction in which the inlet and the outlet are aligned is defined as a front-rear direction and a left-right direction perpendicular to the front-rear direction and the up-down direction, the alignment direction of the wells in the well region differs from the front-rear direction and the left-right direction.

[10] The fluidic device according to any one of [6] to [9], wherein the connection portion is provided between a plurality of the well regions and has an auxiliary connection member that connects the cover portion and the substrate.

[11] The fluidic device according to any one of [6] to

[10] , wherein, when viewed from the up-down direction, the well region is surrounded by a welded portion formed by welding the lower surface of the connection portion to the upper surface of the substrate.

[0015] According to the present invention, it is possible to provide a fluidic device that can suppress the generation of bubbles and detect biomolecules with high accuracy.

[0016] 1 is a schematic perspective view of a fluidic device according to a preferred embodiment of the present invention; FIG. 2 is a schematic longitudinal sectional end view of the fluidic device taken along line X-X in FIG. 1; FIG. 3 is a schematic perspective view from below of a lid member; FIG. 4 is a schematic longitudinal sectional end view showing a state in which a reagent liquid is being supplied to an internal space of the fluidic device; FIG. 5 is a schematic longitudinal sectional end view showing a state in which a sealing liquid is being supplied to an internal space of the fluidic device; FIG. 6 is a schematic longitudinal sectional end view showing a state in which a sealing liquid is being supplied to an internal space of the fluidic device; FIG. 7 is a schematic longitudinal sectional end view of a conventional fluidic device in which bubbles are likely to be generated; FIG. 8 is a schematic longitudinal sectional end view showing a state in which a substrate and a connecting member are welded to each other according to the embodiment shown in FIG. 1; FIG. 9 is a schematic plan view showing a state in which a cover member has been removed from the fluidic device; FIG. 10 is a schematic longitudinal sectional end view of the fluidic device taken along line Y-Y in FIG. 1; FIG. 11 is a schematic plan view showing a state in which a large number of wells are irregularly arranged; FIG. 12 is a schematic plan view showing a state in which the alignment direction of the large number of wells is different from the extension direction of the connecting member; FIG. 13 is a schematic plan view showing a state in which a connecting member is welded to a position between wells on the surface of the substrate. FIG. 1 is a schematic side view showing the shape of each well of a substrate in an example. FIG. 2 is a planar image showing the vicinity of a well when a reagent liquid is heated with the upper edge of the well of the substrate and the lower surface of the connecting member in contact with each other. FIG. 3 is a planar image showing the vicinity of a well when a reagent liquid is heated with the upper edge of the well of the substrate and the lower surface of the connecting member separated by 30 μm in the horizontal direction. FIG. 4 is a planar image showing the vicinity of a well when a reagent liquid is heated with the upper edge of the well of the substrate and the lower surface of the connecting member separated by 40 μm in the horizontal direction. FIG. 5 is a planar image showing the vicinity of a well when a reagent liquid is heated with the upper edge of the well of the substrate and the lower surface of the connecting member separated by 100 μm in the horizontal direction. FIG. 6 is a planar image showing the vicinity of a well when a reagent liquid is heated with the upper edge of the well of the substrate and the lower surface of the connecting member separated by 200 μm in the horizontal direction.

[0017] Hereinafter, preferred embodiments of the present invention will be described in detail, with reference to the drawings as needed.

[0018] FIG. 1 is a schematic perspective view of a fluidic device 1 according to a preferred embodiment of the present invention, and FIG. 2 is a schematic longitudinal sectional end view of the fluidic device 1 taken along line XX shown in FIG.

[0019] As shown in FIGS. 1 and 2 , the fluidic device 1 is a hollow device including a substrate 11 and a lid member 12 covering one surface 11 a of the substrate 11, with an internal space S between the substrate 11 and the lid member 12. The fluidic device 1 is provided with an inlet 121 for injecting a reagent solution into the internal space S and an outlet 122 for discharging the reagent solution. Hereinafter, the side of the lid member 12 relative to the substrate 11 will be referred to as the “top,” the opposite side will be referred to as the “bottom,” and the direction in which the substrate 11 and the lid member 12 are stacked will be referred to as the up-down direction. The side of the outlet 122 relative to the inlet 121 will be referred to as the “front,” the opposite side will be referred to as the “rear,” and the direction in which the inlet 121 and the outlet 122 are aligned will be referred to as the front-to-back direction. The left side facing forward will be referred to as the “left,” the opposite side will be referred to as the “right,” and the direction perpendicular to the up-to-down direction and the front-to-back direction will be referred to as the left-to-right direction.

[0020] The fluidic device 1 is capable of detecting a target substance that may be contained in a reagent solution supplied to the internal space S.

[0021] The reagent solution may contain a biological sample or an environmental sample that may contain a target substance, as well as detection reagents such as enzymes, substrates, nucleic acids, antibodies, antibody fragments, fluorescent substances, and the like.

[0022] The type of biological sample is not particularly limited, and examples thereof include serum, plasma, urine, cell culture medium, PCR reaction solution, etc. Examples of environmental samples include river water, industrial wastewater, etc.

[0023] Examples of target substances include, but are not limited to, DNA, RNA, and proteins as biomolecules, as well as viruses, cells, exosomes, and the like.

[0024] The substrate 11 is a substantially flat plate-shaped member made of a light-transmitting resin, and has multiple wells (recesses) 110 formed on its upper surface. The wells 110 contain a reagent solution supplied to the internal space S and function as a reaction field between the target substance contained in the reagent solution and the detection reagent. Hereinafter, the rectangular region in which the wells 110 are formed will be referred to as the well region R. The well region R is not limited to a rectangle and may be circular, elliptical, or polygonal. As will be described in detail later, the multiple wells 110 may be arranged at equal intervals in the well region R or randomly. In the example of FIG. 1 , rows of three wells 110 arranged in the left-right direction and rows of two wells 110 arranged in the left-right direction are alternately arranged, so that the wells 110 are arranged at equal intervals from one another. The well region R is located in the center of the upper surface 11a in the left-right and front-back directions. When viewed from the top-bottom direction, the well region R is located in a position that does not overlap with the inlet 121 and the outlet 122. That is, the well region R is provided between the inlet 121 and the outlet 122. Note that the well region R is not limited to being provided between the inlet 121 and the outlet 122, as long as the well 110 is provided inside the internal space S when viewed from the top-bottom direction. For example, the well region R may be provided at a position overlapping the inlet 121 and the outlet 122 when viewed from the top-bottom direction.

[0025] The substrate 11 is preferably made of a resin that has little autofluorescence, such as cycloolefin polymer, cycloolefin copolymer, silicone, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluororesin, and amorphous fluororesin.

[0026] The method for manufacturing the substrate 11 is not particularly limited, and the substrate 11 can be manufactured using, for example, known injection molding, microimprinting technology, or nanoimprinting technology. The substrate 11 can also be manufactured by forming the wells 110 by etching using known photolithography technology.

[0027] The thickness of the substrate 11 can be determined appropriately. The thickness of the substrate 11 is greater than the depth of the well 110 and may be, for example, in the range of 0.6 mm to 5 mm. The thickness of the substrate 11 may also be in the range of 0.6 mm to 2 mm. When observing fluorescence from below the substrate 11 using a fluorescence microscope, the thickness of the substrate 11 may be, for example, 5 mm or less, 2 mm or less, or 1.6 mm or less. This is because if the thickness of the substrate 11 is excessively thick, the autofluorescence of the substrate 11 may increase, resulting in a decrease in detection sensitivity.

[0028] The shape of each well 110 is not particularly limited, but examples include a hemispherical shape, a cylindrical shape, a prismatic shape, and a frustum shape.

[0029] The size of each well 110 is not particularly limited, and it is preferable to design each well 110 to have a size suitable for introducing one target substance to be detected into each well 110 .

[0030] The volume of one well 110 is preferably from 1 fL to 6 nL, more preferably from 1 fL to 5 pL, even more preferably from 1 fL to 2 pL, and particularly preferably from 1 fL to 300 fL.

[0031] By designing the capacity of each well 110 within the above range, enzyme reactions performed in a microspace, such as digital PCR and invader reactions, can be suitably carried out. Digital PCR can be used to detect gene mutations, for example.

[0032] The bottom of the well 110 may be flat or curved (convex or concave).

[0033] The density of the wells 110 is, for example, 100,000 to 10,000,000 / cm ２ and preferably 100,000 to 5,000,000 particles / cm ２ and more preferably 100,000 to 1,000,000 particles / cm ２ Designing the density of the wells 110 to fall within the above range facilitates the operation of sealing the reagent solution in a predetermined number of wells 110. It also facilitates observation of the wells for analyzing the experimental results.

[0034] The lid member 12 includes a cover member (an example of a "cover portion") 12a that covers the top of the multiple wells 110, and a connecting member (an example of a "connecting portion") 12b that connects the cover member 12a to the base material 11. The cover member 12a and the connecting member 12b are fixed to each other by adhesive or welding. The cover member 12a and the connecting member 12b may be formed integrally.

[0035] The material of the lid member 12 is not particularly limited, but it is preferable to select a resin that has little autofluorescence, and it is formed from a thermoplastic resin such as a cycloolefin polymer or a cycloolefin copolymer.

[0036] Furthermore, the lid member 12 is preferably formed from a material that does not transmit light of wavelengths close to the wavelength detected during fluorescence observation of the target substance, and may be formed from a thermoplastic resin to which carbon or metal particles, etc. are added in order to block such light. The method for manufacturing the lid member 12 is not particularly limited, but it can be manufactured by known injection molding, for example.

[0037] Two through holes are formed in the cover member 12a, penetrating in the thickness direction. The two through holes are provided at a first end side and a second end side in the longitudinal direction of the lid member 12, respectively. Of the two through holes, one is an inlet 121 used to inject a liquid material into the internal space S of the fluidic device 1, and the other is an outlet 122 used to discharge the liquid material from the internal space S. The liquid material may be a reagent liquid or a sealing liquid.

[0038] The inlet 121, the internal space S, and the outlet 122 are in communication with one another and together form a flow path FC. As will be described in detail later, various liquid substances are supplied to the flow path FC in a predetermined order, thereby enabling detection of a target substance.

[0039] A cylindrical injection port 125 is formed on the upper surface 12a1 of the cover member 12a so as to surround the injection port 121. The injection port 125 is in communication with the injection port 121. The injection port 125 is used to connect a syringe filled with a liquid to the injection port 121, for example, when the liquid is to be filled into the internal space S using the syringe.

[0040] Furthermore, a cylindrical discharge port 126 is formed on the upper surface 12a1 of the lid member 12 so as to surround the periphery of the discharge outlet 122. The discharge port 126 is in communication with the discharge outlet 122. The discharge port 126 is used, for example, to connect a tube to the discharge outlet 122 when extracting a liquid material from the internal space S.

[0041] FIG. 3 is a schematic perspective view of the lid member 12 from below.

[0042] As shown in Fig. 3, the connecting member 12b has a closed ring shape in a bottom view, and a rectangular parallelepiped through-hole 12b3 penetrating vertically is formed in the center in the front-rear and left-right directions. The connecting member 12b includes a first wall portion 12s1 and a second wall portion 12s2 extending in the front-rear direction and facing each other across the internal space S, and a third wall portion 12f1 and a fourth wall portion 12f2 extending in the left-right direction and facing each other across the internal space S. The first wall portion 12s1 is provided on the left side of the internal space S, and the second wall portion 12s2 is provided on the right side of the internal space S. The third wall portion 12f1 is provided in the front of the internal space S, and the fourth wall portion 12f2 is provided in the rear of the internal space S.

[0043] An upper surface 12b1 of the connecting member 12b is fixed to a lower surface 12a2 of the cover member 12a.

[0044] The outer edge of the connecting member 12b is aligned with the outer edges of the cover member 12a and the base material 11, and the lower surface (lower end) 12b2 of the connecting member 12b is welded and fixed to the upper surface 11a of the base material 11 by laser light irradiation. As a result, when viewed from the top and bottom, the well region R is surrounded by a welded portion formed by welding the lower surface 12b2 of the connecting member 12b to the upper surface 11a of the base material 11. Note that the method of fixing the connecting member 12b to the base material 11 is not limited to welding by laser light irradiation, and other welding methods using high frequency waves, ultrasonic waves, etc. may also be used.

[0045] As shown in Figure 3, the inlet 121 and outlet 122 of the cover member 12a are exposed due to the presence of the through hole 12b3, and liquid such as a reagent solution supplied through the inlet 121 is supplied to the inside of the through hole 12b3, i.e., the internal space S.

[0046] The internal space S is defined by the lower surface 12a2 of the cover member 12a, the upper surface 11a of the base material 11, and the inner surface 12b3a of the through-hole 12b3. The upper surface 11a of the base material 11 and the lower surface 12a2 of the cover member 12a are both horizontal.

[0047] The vertical length of the connecting member 12b, i.e., the height of the internal space S, is not particularly limited. The height of the internal space S may be, for example, a height that allows the reagent solution to pass through the flow path FC. The height of the internal space S may be, for example, 100 μm or less, 50 μm or less, or approximately 30 μm. The height of the internal space S may be in the range of 20 to 30 μm. In this embodiment, an example has been described in which the fluidic device 1 is constructed by welding the lid member 12 including the cover member 12a and the connecting member 12b to the substrate 11, but this example is not limiting. For example, the connecting member 12b and the substrate 11 may be fixed by welding, and then the cover member 12a may be fixed to the connecting member 12b. Alternatively, the substrate 11 and the connecting member 12b may be integrally formed, and the cover member 12a may be fixed to a member including the substrate 11 and the connecting member 12b. In this way, even when the substrate 11 and the connecting member 12b are integrally formed, the portion of the integrated member that is provided on a plane including the upper surface 11a of the substrate 11 is the lower surface 12b2 of the connecting member 12b. Alternatively, the cover member 12a, the connecting member 12b, and the substrate 11 may each be separate members, and the fluidic device 1 may be formed by stacking these three members and simultaneously fixing the cover member 12a and the connecting member 12b, and the connecting member 12b and the substrate 11 together.

[0048] Fig. 4 is a schematic longitudinal sectional end view showing a state in which a reagent liquid is supplied to the internal space S of the fluidic device 1. Fig. 5 is a schematic longitudinal sectional end view showing a state in which a sealing liquid is supplied to the internal space S of the fluidic device 1. Fig. 6 is a schematic longitudinal sectional end view showing a state of the internal space S after the sealing liquid L2 has been supplied.

[0049] In the fluidic device 1 configured as described above, a target substance that may be contained in the reagent liquid L1 can be detected by, for example, performing the following operation.

[0050] First, a reagent solution L1 containing a sample diluted to a concentration such that one target substance (one molecule in the case of molecules) is contained per well 110 is supplied to the internal space S through the injection port 125 and the injection inlet 121.

[0051] As a result, as shown in FIG. 4, the reagent liquid L1 is transported forward within the internal space S, and each well 110 is filled with the reagent liquid L1.

[0052] Next, the sealing liquid L2 is supplied into the internal space S through the injection port 125 and the injection port 121. The sealing liquid L2 is preferably oil.

[0053] As a result, as shown in FIG. 5, each well 110 is sealed with sealing liquid L2 while containing reagent liquid L1 inside.

[0054] Examples of oils used for the sealing liquid L2 include fluorine-based oils, silicone-based oils, hydrocarbon-based oils, and mixtures thereof. Specific examples of the sealing liquid include fluorine-based oils such as FC-40, FC-43, FC-770, FC-72, and FC-3283 (all manufactured by 3M), and silicone oils such as KF96 (manufactured by Shin-Etsu Chemical Co., Ltd.), NOVEC (model HFE-7500, manufactured by 3M), and FLUO-OIL7500 (manufactured by Emulseo).

[0055] As shown in Figure 6, after the sealing liquid L2 is supplied until the internal space S is filled with the sealing liquid L2, the wells 110 are left to stand until various reactions corresponding to the target substances in the reagent liquid L1 are completed. At this time, it is preferable to heat the reagent liquid L1 in each well 110. This can promote various reactions. Examples of methods for heating the reagent liquid L1 include, but are not limited to, a method in which a heater is brought into contact with the substrate 11 from below and heat is applied to the reagent liquid L1 through the substrate 11.

[0056] Finally, the target substance can be detected by measuring the fluorescence or the like in each well 110 from below through the substrate 11.

[0057] In conventional fluidic devices, bubbles are generated in the internal space during the above-mentioned process of heating the reagent solution, which can interfere with the detection reaction and post-reaction observation. The generation of bubbles in conventional fluidic devices is described in detail below.

[0058] FIG. 7 is a schematic longitudinal sectional end view of a conventional fluidic device 2 in which bubbles are likely to occur.

[0059] The inventor observed a conventional fluidic device 2 that was prone to generating bubbles and found that, as shown at position α in Figure 7, the upper edge 210a of the well 210 formed on the upper surface of the substrate 21 was welded to the lower surface 22b2 of the connecting member 22b in a contacting state (in other words, the connecting member 22b was overlapping the upper edge 210a of the well 210), and that bubbles were generated near the contact point.

[0060] Without being bound by any particular theory, it is believed that if the upper edge 210a of the well 210 is in contact with the connecting member 22b, the shape of the well 210 changes during welding. As a result, it is presumed that when a reagent solution is supplied to the internal space 2S of the fluidic device 2, air remains inside the well 210, and bubbles are generated when the reagent solution is heated.

[0061] Fig. 8 is a schematic longitudinal sectional end view showing the welding of the base material 11 and the connecting member 12b according to the embodiment shown in Fig. 1. Fig. 9 is a schematic plan view showing the fluidic device 1 with the cover member 12a removed, and Fig. 10 is a schematic longitudinal sectional end view of the fluidic device 1 taken along line Y-Y shown in Fig. 1.

[0062] In consideration of the generation of bubbles near the contact portion, in this embodiment, no well 110 is formed in the portion of the upper surface 11a of the substrate 11 that contacts the connecting member 12b of the lid member 12. In the manufacturing process of the fluidic device 1, as shown in Fig. 8, the substrate 11 is placed upside down on the lower surface 12b2 of the lid member 12 that is also inverted. Thereafter, the substrate 11 and the lid member 12 are welded together by laser light, with the upper edge 110a of the well 110 not in contact with the lower surface 12b2 of the connecting member 12b.

[0063] As a result, as shown in Fig. 9, in a top view, none of the wells 110 located near any of the front, rear, left, or right edges of the base material 11 overlap the connecting member 12b of the lid material 12. Also, as shown in Fig. 10, for each well 110, the entire upper edge portion 110a, which is circular in a top view, is horizontally separated from the lower end (lower surface 12b2) of the connecting member 12b. Note that the "horizontal direction" refers to a direction perpendicular to the up-down direction, or in other words, a direction parallel to the upper surface 11a of the base material 11.

[0064] By horizontally separating the lower surface 12b2 of the connecting member 12b from the upper edge 110a of the well 110, air can be prevented from remaining inside the well 110 when the reagent liquid L1 is supplied, and the generation of bubbles during heating can be effectively suppressed.

[0065] The horizontal separation distance W between the upper edge 110a of the well 110 and the lower surface 12b2 of the connecting member 12b is preferably 30 μm or more, more preferably 155 μm or more, as will be shown in the examples below.

[0066] Since the upper edge 110a of the well 110 is spaced 30 μm from the lower surface 12b2 of the connecting member 12b, excessive heat transfer to the well 110 can be prevented during welding, and the generation of bubbles can be effectively suppressed during the above-mentioned process of heating the reagent solution.

[0067] Furthermore, by providing a separation of 155 μm or more between the upper edge 110a of the well 110 and the lower surface 12b2 of the connecting member 12b, even if one or both of the substrate 11 and the connecting member 12b move horizontally due to vibration or other factors during welding, or if the substrate 11 or the lid member 12 is produced with dimensions slightly different from the design values ​​due to processing accuracy, the separation between the upper edge 110a of the well 110 and the lower surface 12b2 of the connecting member 12b can be ensured for all products, including those. Furthermore, the separation distance W is preferably 500 μm or less. If the separation distance W is greater than 500 μm, the well region R, which is the area in which wells 110 can be placed, becomes smaller, reducing the number of wells that can be placed, which may result in reduced detection accuracy. While the separation distance W may satisfy the above-mentioned condition throughout the entire periphery of the well region R, it is particularly preferable that the separation distance W in the left-right direction be 30 μm or more and 500 μm or less. Only the left-right separation distance W between the upper edge 110a of the well 110 and the lower surfaces 12b2 of the first wall 12s1 and the second wall 12s2 of the connecting member 12b is 30 μm or more and 500 μm or less, and the front-rear distance between the upper edge 110a of the well 110 and the lower surfaces 12b2 of the third wall 12f1 and the fourth wall 12f2 of the connecting member 12b may be greater than the separation distance W. This is because, for example, there are cases where the well 110 is not formed at a position that overlaps with the inlet 121 and the outlet 122 when viewed from the top-bottom direction.

[0068] 11 to 13 are schematic plan views showing variations in the arrangement of wells 110, 115 on the substrate 11. In detail, Fig. 11 is a schematic plan view showing a state in which a plurality of wells 110, 115 are irregularly arranged, Fig. 12 is a schematic plan view showing a state in which the alignment direction of the plurality of wells 110, 115 differs from the extension direction of the connecting members 12b, and Fig. 13 is a schematic plan view showing a state in which connecting members 12b are welded to positions between the wells 110 on the upper surface (11a) of the substrate.

[0069] As shown in Figure 11, even if multiple wells 110, 115 are arranged irregularly, the upper edge 110a of the well 115 closest to the lower surface of the connecting member 12b is horizontally separated from the lower surface of the connecting member 12b, thereby suppressing the generation of bubbles.

[0070] Furthermore, as shown in Figure 12, even if the alignment direction of multiple wells 110, 115 is different from the extension direction of connecting member 12b (front-to-back and / or left-to-right), the upper edge 110a of well 115 closest to the underside of connecting member 12b is horizontally separated from the underside of connecting member 12b, thereby suppressing the generation of bubbles.

[0071] 13, a plurality of wells 110 are arranged in a row in the longitudinal direction (front-rear direction) of the upper surface 11a of the substrate, and a plurality of rows of wells 110 are formed in the left-right direction. A gap is formed in the short-side direction (left-right direction) between the rows of wells 110. In the example of FIG. 13, two well regions R, each having one or more rows of wells 110, are provided, and a gap is formed between the two well regions R.

[0072] The lid member 12 further includes an auxiliary connecting member 125b in the left-right center that connects the cover portion 12a and the substrate 11. The lower surface of the auxiliary connecting member 125b, located in the center of the short side of the connecting member 12b, is welded to a gap located in the center of the left-right (short side) direction of the substrate upper surface 11a. The upper surface of the auxiliary connecting member 125b is fixed to the center of the left-right direction of the lower surface of the cover member 12a. The lower surface 12b2 of the auxiliary connecting member 125b and the upper edge portion 110a of the well 110 are spaced apart in a direction parallel to the upper surface 11a of the substrate 11. It is preferable that the auxiliary connecting member 125b extend parallel to the flow path FC. Therefore, although the auxiliary connecting member 125b extends in the front-rear direction in the example of FIG. 13 , this example is not limiting, and the auxiliary connecting member 125b may extend in a direction intersecting the front-rear direction.

[0073] In this way, even if the auxiliary connecting member 125b is positioned at a position other than the horizontal end of the fluidic device, the generation of bubbles can be suppressed by separating the lower surface of the auxiliary connecting member 125b from the upper edge 110a of each well 110.

[0074] Although the preferred embodiments have been described in detail above, the present invention is not limited to the above embodiments, and various modifications are possible within the scope of the invention described in the claims.

[0075] 1 to 13, the cover member 12a and the connecting member 12b of the lid member 12 are separate bodies, but they may be formed integrally. In this case, the portion of the lid member that covers the multiple wells is the cover portion, and the portion that connects the cover portion to the base material is the connecting portion.

[0076] In addition, in the embodiment shown in Figures 1 to 10, the liquid discharged from the discharge port 126 is configured to spread around the fluid device 1, but a wall may be provided at the edge of the upper surface 12a1 of the cover member 12a so that the liquid can be contained on the cover member 12a.

[0077] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0078] In this experiment, we confirmed whether the generation of bubbles when the reagent solution is heated can be suppressed by horizontally separating the upper edge of the well 110 of the substrate 11 shown in Figure 2 from the lower surface of the connecting member 12b of the lid material 12.

[0079] In this experiment, a fluidic device was used in which the base material 11 and the connecting member 12b were welded by laser light.

[0080] The substrate 11 and the lid member 12 were each made of cycloolefin polymer ZEONOR® 1020R manufactured by Zeon Corporation. The lid member 12 was colored black by mixing it with carbon black.

[0081] The reagent solution contains 20 mM (mol / L) NaCl and 25 mM MgCl ２An enzyme solution consisting of 50 mM Tris-HCl buffer (pH 8.5), 0.05% surfactant, 0.2 mg / mL enzyme, and water was used. This reagent solution was heated at 66°C for 25 minutes through the substrate 11. Tween™ 20, for example, can be used as the surfactant.

[0082] Silicone oil KF-96 manufactured by Shin-Etsu Chemical Co., Ltd. was used as the sealing liquid.

[0083] 14 is a schematic side view showing the shape of each well 110 in the substrate 11 in the example. The wells 110 were cylindrical, with the diameter gradually decreasing from top to bottom. Each well 110 had a diameter S1 of 12.5 μm at its upper edge, a diameter S2 of 11.0 μm at its bottom, and a depth S3 of 10 μm. The spacing between the wells 110 (the distance between the upper edges of the wells 110) was 2.5 μm. Approximately 1 million wells 110 of this shape and dimensions were formed in the substrate 11.

[0084] 15 is a planar image of the vicinity of the well 110 when the reagent solution is heated with the upper edge of the well 110 of the substrate 11 in contact with the underside of the connecting member 12b, and is an image according to a comparative example. The horizontal overlap between the well 110 and the connecting member 12b was approximately 5 μm to 6 μm.

[0085] 16 is a planar image showing the vicinity of the well 110 when the reagent liquid is heated with the upper edge of the well 110 of the substrate 11 and the lower surface of the connecting member 12b spaced apart horizontally by 30 μm. FIG. 17 is a planar image showing the vicinity of the well 110 when the reagent liquid is heated with the upper edge of the well 110 of the substrate 11 and the lower surface of the connecting member 12b spaced apart horizontally by 40 μm. FIG. 18 is a planar image showing the vicinity of the well 110 when the reagent liquid is heated with the upper edge of the well 110 of the substrate 11 and the lower surface of the connecting member 12b spaced apart horizontally by 100 μm. FIG. 19 is a planar image showing the vicinity of the well 110 when the reagent liquid is heated with the upper edge of the well 110 of the substrate 11 and the lower surface of the connecting member 12b spaced apart horizontally by 200 μm.

[0086] 16 to 19, the distance between the upper edge of the well 110 and the lower surface of the connecting member 12b is indicated as W.

[0087] 15 to 19 were acquired using an all-in-one fluorescence microscope BZ-X810 manufactured by Keyence Corporation. Images of the inside of the fluidic device were taken from above through the cover member 12a of the lid member 12 (see FIG. 2, etc.).

[0088] When the reagent solution was heated while the upper edge of the well 110 of the substrate 11 was in contact with the lower surface of the connecting member 12b, multiple bubbles K were generated near the welded portion Y between the substrate 11 and the connecting member 12b, as shown in Figure 15.

[0089] In contrast, when the reagent liquid was heated with the upper edge of the well 110 of the substrate 11 and the lower surface of the connecting member 12b separated horizontally, no bubbles K were generated, as shown in Figures 16 to 19.

[0090] From the above, it was confirmed that the generation of bubbles K when the reagent is heated can be effectively suppressed by welding the substrate 11 and the connecting member (connecting portion) while separating the upper edge of the well 110 of the substrate 11 from the lower surface of the connecting member 12b in the horizontal direction (a direction parallel to the upper surface of the substrate 11).

[0091] 1...fluidic device, 11...substrate, 12...lid material, 12a...cover member (an example of a cover portion), 12b...connecting member (an example of a connecting portion), 12b2...lower surface (lower end portion), 125b...auxiliary connecting member, 110...well (microwell), 110a...upper edge portion, 121...inlet, K...air bubble, 122...outlet, L1...reagent solution, L2...sealing liquid, S...internal space, S1...diameter of upper edge portion of well, S2...diameter of bottom surface of well, S3...depth of well, W...separation distance between upper edge portion of well and lower surface of connecting member (an example of a connecting portion)

Claims

1. A fluidic device comprising: a substrate having a plurality of wells formed on an upper surface thereof; and a lid material covering above the wells, the lid material comprising a cover portion covering above the wells; and a connection portion connecting the cover portion to the substrate, the lower surface of the connection portion being welded to the upper surface of the substrate, and the lower surface of the connection portion and an upper edge of the wells being spaced apart in a direction parallel to the upper surface of the substrate.

2. The fluidic device according to claim 1, wherein the separation distance between the upper edge of the well and the lower surface of the connection portion is 30 μm or more.

3. The fluidic device according to claim 2, wherein the separation distance between the upper edge of the well and the lower surface of the connection portion is 155 μm or more.

4. A fluidic device according to any one of claims 1 to 3, wherein the volume of each of the wells is between 1 fL and 6 nL.

5. A fluidic device according to any one of claims 1 to 3, wherein the distance between the upper edge of the well and the lower surface of the connection portion is 500 μm or less.

6. A fluidic device as described in claim 1, wherein the connection portion has a through hole formed therein that penetrates in the vertical direction between the substrate and the lid material that are stacked, the through hole forms an internal space between the substrate and the lid material, and a well region in which the well is formed is provided inside the internal space when viewed from the vertical direction.

7. The fluidic device according to claim 6, wherein the wells are provided at equal intervals in the well region.

8. The fluidic device according to claim 6, wherein the wells are irregularly spaced in the well region.

9. A fluidic device as described in any one of claims 6 to 8, wherein the lid material is provided with an inlet and an outlet that communicate with the internal space, and when the direction in which the inlet and the outlet are aligned is defined as a front-to-rear direction and a left-to-right direction perpendicular to the front-to-rear direction and the up-down direction, the alignment direction of the wells in the well region is different from the front-to-rear direction and the left-to-right direction.

10. A fluid device according to any one of claims 6 to 8, wherein the connection portion has an auxiliary connection member provided between a plurality of the well regions and connecting the cover portion and the substrate.

11. A fluidic device according to any one of claims 6 to 8, wherein when viewed from the top-bottom direction, the well region is surrounded by a welded portion formed by welding the lower surface of the connection portion and the upper surface of the substrate.

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