Microchannel chip and method for manufacturing same

Abstract

Provided are: a microchannel chip which suppresses optical noise from inside the channel, is capable of storing fluid leakage from an injection port and a discharge port, and is obtained by overlapping and integrally joining substrates with one another without using an adhesive; and a method for manufacturing the same. A microchannel chip 3 according to the present invention which is formed as an optically transparent plate and has a channel groove 12, the interior of which other than an injection port 22 and a discharge port 22 is sealed, said microchannel chip 3 being characterized by being equipped with a first substrate 1 and a second substrate 2 and in that: the first substrate has a channel groove formed therein, has a first joining surface 11 for overlapping and being integrally joined with the second substrate, and has a recess 13 formed therein which is not connected to the channel groove; the second substrate has a second joining surface 21 for overlapping and being integrally joined with the first substrate, and has an injection port which connects to the channel groove and / or a discharge port which connects to the channel groove which are formed therein; and an opening 23 which connects to the recess is formed in the first substrate and / or the second substrate.

Description

Microchannel chip and method of manufacturing the same

[0001] The present invention relates to a microchannel chip in which bonding surfaces of opposing substrates are bonded to form a microchannel therebetween, and to a method for manufacturing the same.

[0002] A microchannel chip is a molded product formed by adhesively bonding two or more substrates and having fine microchannel grooves with widths ranging from several hundred nanometers to 1 mm or less between the substrates. Reagents and specimens are injected through holes connected to the channel grooves, and the chip is used for separating, analyzing, detecting, and other applications of the samples.

[0003] Typical applications of microchannel chips include analysis in the fields of medicine and pharmacy, compound synthesis, environmental measurement, etc. When microchannel chips are used for these applications, it is possible to obtain effects such as shortening the analysis time compared to when conventional analytical devices suitable for similar applications are used.

[0004] In recent years, attempts have been made to improve the efficiency of measurement testing by placing multiple flow channels on a single chip and measuring different samples or reagents simultaneously, increasing the number of samples that can be measured at one time. There is also a demand for products that allow general users other than medical professionals to add liquids and obtain analysis results.

[0005] In manufacturing a microchannel chip, a method for bonding resin substrates together is important. For example, Patent Document 1 discloses a bonding method that can bond resin substrates together at a lower temperature than bonding by heat fusion.

[0006] Patent No. 4919474

[0007] However, when bonding is performed without using adhesive, there is a problem that air bubbles may get trapped between the bonded surfaces, causing liquid to leak from the bonded surfaces of the flow path portion.

[0008] In view of the above problems, an object of the present invention is to provide a microchannel chip that reduces the intrusion of air bubbles when bonding without using adhesive, and that makes it possible to collect liquid outside the channel when it leaks from the inlet and outlet, and a method for manufacturing the same.

[0009] A micro-channel chip according to one aspect of the present invention that solves the above-mentioned problems is a micro-channel chip formed in the shape of an optically transparent plate, having a channel whose interior is sealed except for an inlet and an outlet, the micro-channel chip comprising a first substrate and a second substrate, the first substrate having the channel formed therein, a first bonding surface for being overlaid on and integrally joined to the second substrate, and a recess not communicating with the channel, the second substrate having a second bonding surface for being overlaid on and integrally joined to the first substrate, and at least one of an inlet communicating with the channel or a outlet communicating with the channel, and an opening communicating with the recess is formed in at least one of the first substrate and the second substrate.

[0010] In addition, although not limited thereto, it is preferable that the openings are formed in the second substrate in the same direction as the recesses.

[0011] In addition, although not limited to this aspect, it is preferable that the opening be formed on at least one of the side surfaces of the first substrate and the second substrate.

[0012] In addition, in this respect, although not limited thereto, it is preferable that the area of ​​the recess is larger than the area of ​​the opening.

[0013] In addition, in this respect, although not limited thereto, it is preferable that at least a part of the side surface of the recessed portion is sloped.

[0014] In addition, in this respect, although not limited thereto, it is preferable that the depth of the recess from the first bonding surface is greater than the depth of the flow channel.

[0015] Furthermore, in this respect, although not limited thereto, it is preferable that the edge of the opening is formed lower than the height of the edge of either the inlet or the outlet, the difference in height between the edges is 0.1 μm or more, and a mouth recess is formed that is thinner than the thickness of the second substrate.

[0016] In addition, in this respect, although not limited thereto, it is preferable that a plurality of openings are formed and that the mouth recess communicates with the plurality of openings.

[0017] In addition, in this respect, although not limited thereto, it is preferable that the first and second bonding surfaces do not have an adhesive layer or a pressure-sensitive adhesive layer.

[0018] In addition, in this respect, although not limited thereto, it is preferable that a plurality of flow channel grooves are independently arranged and recesses are arranged between the flow channel grooves.

[0019] In addition, in this respect, although not limited thereto, it is preferable that an optical measurement observation surface is formed on the second substrate located above the flow channel formed on the first substrate.

[0020] A method for manufacturing a micro-channel chip according to another aspect of the present invention is a micro-channel chip formed in the shape of an optically transparent plate and having a channel groove whose interior is sealed except for an inlet and an outlet, the micro-channel chip comprising a first substrate and a second substrate, the first substrate having the channel groove formed therein and a first bonding surface for being overlaid on and integrally joined to the second substrate, and a recess not communicating with the channel groove formed therein, the second substrate having a second bonding surface for being overlaid on and integrally joined to the first substrate, and at least one of an inlet communicating with the channel groove or a discharge port communicating with the channel groove formed therein, and at least one of the first substrate and the second substrate having an opening communicating with the recess.

[0021] As described above, according to the present invention, even if air bubbles remain between two substrates to be joined during the process of joining the joining surfaces, the openings can be used as degassing holes to remove the remaining air bubbles at the joining surfaces, and in addition, when liquid leaks from the inlet and outlet, it flows into the grooves of the recesses and can be stored, and a method for manufacturing the same can be provided.

[0022] In addition, in a preferred aspect of the present invention, by making the area of ​​the opening smaller than the area of ​​the recess, the opening acts as a burr portion for liquid that has accumulated in the groove of the recess, making it less likely for the liquid to spill out.

[0023] In addition, in a preferred aspect of the present invention, by angling the side surface of the recess, scattered light from the sample is refracted or totally reflected by the gradient portion, thereby suppressing light returning to the flow path, and also reducing noise light to other flow path portions.

[0024] FIG. 1 is an explanatory diagram showing an example of the configuration of a micro-channel chip as viewed from the second substrate side. FIG. 2 is a cross-sectional view showing an outline of the function of the micro-channel chip. FIG. 3 is a schematic diagram showing an example of an opening of a micro-channel chip. FIG. 4 is a schematic diagram showing an example of an opening of a micro-channel chip. FIG. 5 is a schematic diagram showing the function of the opening of a micro-channel chip. FIG. 6 is an explanatory diagram showing that a plurality of channel grooves and recesses are formed in a micro-channel chip. FIG. 7 is a cross-sectional view showing an outline of the side slope of a recess of a micro-channel chip. FIG. 8 is a diagram showing an example of the height difference of the edge surface of the opening. FIG. 9 is a cross-sectional view showing the function of the opening in a bonding step. FIG. 10 is a cross-sectional view showing an example of the arrangement and height difference of channel grooves and recesses according to an embodiment.

[0025] Preferred embodiments of the present invention will be described in detail below with reference to the drawings. However, the present invention can be embodied in many different forms and is not limited to the following examples. Dimensions, materials, and other specific numerical values ​​shown in the embodiments are merely examples to facilitate understanding of the invention and do not limit the present invention unless otherwise specified. In this specification and drawings, elements having substantially the same functions and configurations are designated by the same reference numerals to avoid redundant description, and elements not directly related to the present invention are not shown. Directions such as X, Y, and Z are indicated in the drawings to define the directions.

[0026] Fig. 1 is a diagram showing an outline of a micro-channel chip according to this embodiment (hereinafter also simply referred to as a "micro-channel chip") 3. Fig. 2 is a diagram showing a schematic cross-sectional view of the micro-channel chip 3. More specifically, the micro-channel chip 3 includes a first substrate 1 and a second substrate 2 that are stacked and integrally bonded together.

[0027] A flow channel 12, which is sealed except for an inlet 22 and an outlet 22, and a recess 13 that is not in communication with the flow channel 12 are formed on the bonding surface 11 of the first substrate 1. An opening 23 that opens upward, similar to the inlet 22 and the outlet 22, is formed on the bonding surface 21 of the second substrate 2.

[0028] In the micro-channel chip 3 according to this embodiment, the substrate 1 on which the flow channel 12 and the recess 13 are formed is referred to as the "first substrate," and the other substrate that is overlaid on the first substrate is referred to as the "second substrate." However, "first" and "second" are counters used to easily distinguish between the two substrates when describing the micro-channel chip according to this embodiment, and have no other technical meaning. Of course, it is not prohibited to provide a recess in the second substrate as well as the first substrate. That is, the flow channel 12 and the recess 13 may be formed at the bonding interface 31 between the bonding surface 11 of the first substrate 1 and the bonding surface 21 of the second substrate 2. In the micro-channel chip 3 according to this embodiment, a plurality of recesses 13 may be formed in the first substrate 1.

[0029] In the micro-channel chip 3 according to this embodiment, an inlet 22 and an outlet 22 are formed in the second substrate 2. The inlet 22 and the outlet 22 are connected to the channel 12, and it is possible to inject a liquid from the inlet 22 and discharge the liquid from the outlet 22 via the channel 12. It is preferable that both the inlet 22 and the outlet 22 are provided on the bonding surface 21 of the second substrate, but this is not a limitation, and it is sufficient that at least one of these is formed in the second substrate 2.

[0030] It is also important that the micro-channel chip 3 has optical transparency. If there are no particular problems with the required heat resistance and durability, a resin that is suitable for mass production by a manufacturing method is selected as the material, but glass that has excellent transparency is also a preferred example.

[0031] The resin, which is the main component of the microchannel chip 3, is preferably a polymeric material that can be processed and molded into a desired shape. As will be described later, the resin that can be used for the first substrate 1 is not limited as long as it can obtain the desired refractive index, but examples include thermoplastic resins such as polycarbonate, polyethylene, polypropylene, polyvinyl chloride, polyester, acrylic, cycloolefin polymer (COP), and cyclic olefin copolymer (COC), thermosetting resins such as phenolic resin, polyurethane, and thermosetting polyimide, and photocurable resins.

[0032] The resin is not limited as long as it has optical transparency, and may be colorless transparent, colorless translucent, colored transparent, or colored translucent.

[0033] Each substrate before bonding can be produced by various molding methods such as injection molding using a mold in which a microchannel and a convex portion are formed. The thickness of the substrate does not matter, and it may be a film.

[0034] In the micro-channel chip 3 according to this embodiment, the size of the channel 12 is not limited and various widths and shapes can be adopted as long as the function of the micro-channel chip can be exhibited. However, a width of 0.1 μm to 1 mm and a depth of 0.1 μm to 1 mm are assumed to be typical preferred ranges.

[0035] Commonly known methods for producing fine irregularities in grooves such as flow channels include metal cutting dies, a method that combines electron beam lithography and plasma etching, and optical lithography. The formed microstructure can also be formed using an electroformed die. The formed fine irregularities in the flow channel grooves can be transferred to a resin or the like as a die, enabling mass production.

[0036] Furthermore, the penetration portions of the injection port 22, the discharge port 22, and the opening 23 into the second substrate 2 can be arbitrarily selected from molding using a mold structure, additional machining by cutting, pressing, laser processing, and the like.

[0037] Furthermore, in order to prevent the liquid from spilling outside the desired flow channel 12, a cover is provided on the flow channel 12, and the substrates are bonded together in a bonding process.

[0038] Furthermore, in this embodiment, an example is shown in which the recess 13 and the opening 23 connected thereto are in the same direction, and this configuration has the advantage that even if liquid leaks from the inlet and outlet 22 formed in the second substrate 2, the liquid can be stored in the recess 13 from the opening 23, but they do not necessarily have to be in the same direction, as long as openings 23 connected to the recess 13 are formed in the first substrate 1 and the second substrate 2. Note that "the same direction" here means that they are formed on the same surface side when viewing the micro-channel chip 3 according to this embodiment.

[0039] 3, 4, and 5 show an example of another embodiment of the present invention in which openings 23 are formed in a perpendicular direction instead of in the same direction. In the example shown in these figures, openings 23 are formed on the side surface of micro-channel chip 3. This has the advantages of facilitating molding, reducing the area required for bonding, and increasing bonding strength.

[0040] Although not limited thereto, a pretreatment step is preferably performed before bonding the first substrate 1 and the second substrate 2. The pretreatment step is a treatment step for facilitating bonding of the substrates. A specific example of the pretreatment step is surface modification of the first bonding surface 11 and the second bonding surface 21 of each substrate to make them hydrophilic. The modification method may be either vacuum plasma treatment or atmospheric pressure plasma treatment. Furthermore, the surface modification of the bonding surfaces of the two substrates may be performed by plasma treatment, vacuum ultraviolet (VUV) treatment in which vacuum ultraviolet (VUV) rays are irradiated onto the bonding surfaces from an excimer lamp, corona discharge treatment, or the like. Because heat generation may distort the fine flow path, vacuum plasma treatment or vacuum ultraviolet (VUV) treatment, which suppresses heat generation, is preferred. However, vacuum ultraviolet (VUV) treatment is more preferred for production processes under atmospheric pressure.

[0041] Furthermore, the pretreated substrates are preferably bonded together at their bonding surfaces by a thermal pressing process. Heating the substrates to the upper limit of their glass transition temperature allows for efficient bonding by applying pressure from both sides. The heating time is preferably adjusted appropriately between several seconds and several minutes, depending on the heat resistance of the resin and the thickness of the substrate, as it takes longer for heat to be transferred to the bonding surfaces. Depending on the desired bonding strength, bonding can also be performed at room temperature. While bonding can be performed with a pressure of several kgf, the pressure must be increased depending on the size of the microchannel chip 3 to be bonded and the presence of air bubbles on the bonding surfaces. After the bonding pretreatment, the thermal pressing process should be performed promptly within a few minutes, but thermal pressing bonding can also be performed over several days.

[0042] The above bonding method does not use any adhesive or glue, so bonding is possible without blocking or closing the cross section of the microchannel groove. In this case, there is no adhesive or sticky layer between the first bonding surface 11 and the second bonding surface 21. Needless to say, bonding is also possible in a similar manner for a microchannel chip 3 having three or more stacked substrates. Furthermore, the presence of the recess 13 and opening 23 reduces the area of ​​the bonding surface to be pressed, resulting in bonding with less pressure and reducing deformation of the channel groove 12, etc.

[0043] Furthermore, the outer surface of the micro-channel chip 3 according to this embodiment may be covered with a coating, vapor deposition, or sputtering protective layer, or a seal, film, or paint such as ink, as long as the characteristics of the liquid injection and discharge sections, optical observation section, and openings are maintained, allowing for greater freedom in product design.

[0044] 6 , in the configuration of the present invention, in the unlikely event that liquid leaks from inlet 22 and outlet 22 during use, the liquid can be stored in recess 13 from opening 23. Alternatively, recess 13 can serve as a receptacle to store liquid that leaks from bonding interface 31 due to unintended liquid pressure, thereby preventing it from leaking out of micro-channel chip 3. Furthermore, the presence of opening 23 prevents the pressure in recess 13 from increasing due to the leaked liquid, thereby preventing further peeling of bonding interface 31 and preventing unnecessary liquid from leaking back onto the surface of micro-channel chip 3.

[0045] 7, in the micro-channel chip 3 according to this embodiment, it is preferable from the viewpoint of performing a plurality of processes that a plurality of independent flow channels 12 are formed on one substrate, and when a plurality of independent flow channels 12 are formed, it is preferable that the recess 13 is disposed between the plurality of independent flow channels 12. In this way, even if an unexpected situation occurs, such as when one flow channel 12 is filled with liquid and the substrates are not sufficiently tightly attached, the liquid will flow into the recess 13, thereby reducing the risk of the liquid leaking into the other flow channel 12.

[0046] Furthermore, in the micro-channel chip 3 according to this embodiment, it is preferable that the area of ​​the opening 23, as viewed from the Z direction (top and bottom sides) of the chip surface, is smaller than the area of ​​the recess 13. From another perspective, it is preferable that the area of ​​the recess 13, as viewed from the Z direction of the micro-channel chip 3 surface, is larger than the area of ​​the opening 23. In this case, the bonding surface of the second substrate 2 covers the recess 13, forming a burr surface 15, making it difficult for accumulated liquid to return to the surface. Note that there may be multiple openings 23 for one recess 13. The shape may also be designed arbitrarily as long as it can perform its function. In the case of multiple openings 23, it is similarly preferable that the bonding surface covers the recess 13. Furthermore, the cross-sectional shape of the recess 13 may have a step.

[0047] It is also preferable that the bottom of the recess 13 is set to be the same as or lower than the depth of the flow channel 12. In this way, the volume of the recess 13 can be made larger than the overall volume of the flow channel 12, allowing for a margin of liquid to be stored.

[0048] Furthermore, when a plurality of recesses 13 are formed, these recesses 13 may be connected to each other via grooves that do not communicate with the flow path groove 12, or may be arranged so as to surround the flow path groove 12. Since liquid may similarly flow in directions other than those indicated by the arrows in Figure 6, it is preferable to form openings 23 and recesses 13 as appropriate depending on the design of the flow path groove 12.

[0049] In the microchannel chip 3, the substrate is optically transparent. Here, "optically transparent" refers to the property of transmitting at least a portion of incident light. While not limited to this, specifically, the transmittance of light (633 nm) in the flat portion and the finely textured portion of the substrate is preferably more than 10% and not more than 60%, more preferably 20% or more and not more than 50%. A transmittance of less than 10% is not preferred because the light for detection will not be transmitted.

[0050] 8, a suitable slope may be provided on the recess side surface 14 of the recess 13 of the first substrate 1. In the production process, when the molded product is made of resin, this not only facilitates release from the mold, but also reduces optical noise by making it difficult for unwanted light 4 generated in the flow channel 12 to return to the flow channel side due to refraction and total reflection of the light on the recess side surface 14.

[0051] Furthermore, if a slope is provided on the recess side surface 14 of the recess 13, when the substrate is colored and transparent, there is a drawback in that the light transmittance decreases, but there is an effect of absorbing scattered light and reducing noise light, and the optical path length and the thickness of the molded part can be designed appropriately.

[0052] Furthermore, the plate-like portions of the first substrate 1 and the second substrate 2 may be made of a resin that transmits only specific wavelength ranges such as ultraviolet and infrared rays, thereby eliminating the need for filters and other components on the detection device side.

[0053] Furthermore, as shown in the schematic cross-sectional view of Figure 9, by positioning the height of the opening 23 lower in the direction of gravity (-Z side direction) than the height of the mouths of the inlet 22 and outlet 22, the leaked liquid can be more easily guided into the recess 13 due to the height difference 33 between the edge 24 of the opening and the edges of the inlet 22 and outlet 22.

[0054] The mouth recess 25 of the opening 23 can be shaped to have a flow path shape when viewed from the Z direction or a shape that surrounds the inlet 22 and the outlet 22, thereby improving the function by guiding the leaked liquid 32 to the opening 23. In other words, the "mouth recess" refers to a recess formed on at least one of the first substrate and the second substrate, which connects multiple openings.

[0055] 10, by providing a degassing through-hole 53 in the heat-pressure mold 5 used for applying pressure in accordance with the positions of the opening 23, injection port 22, and discharge port 22 during the heat-pressure bonding process, air bubbles remaining on the bonding surface can be removed like a degassing flow 51, thereby increasing the strength of the bond. Furthermore, depending on the area of ​​the recess 13, the height of the edge of the opening 23 can be formed lower than the height of the edges of the injection port 22 and discharge port 22 so that distortion occurs during heat-pressure bonding. By providing a mouth recess 25 larger than the edge 24 of the opening in advance when producing the second substrate 2, it is also possible to easily align the hole positions of the second substrate 2 and the heat-pressure mold 5.

[0056] As shown in the schematic cross-sectional view of FIG. 11 , the closer the optical measurement observation surface 26 formed on the second substrate 2 is to the first substrate 1 (the thinner the second substrate is), the easier it is to observe the flow channel 12. In addition, shortening the optical path length through the substrate has the advantages of improving the S / N ratio and suppressing autofluorescence.

[0057] Furthermore, since the heat-pressure mold 5 does not come into contact with the optical measurement observation surface 26 during heat-pressure application in the process, scratches on the observation surface can be prevented. Furthermore, the configuration can prevent scratches when the microchannel chips are stacked on top of each other or placed during the process, transportation, or use.

[0058] As described above, the microchannel chip according to this embodiment can contain any liquid leakage that occurs during use by being given a unique design, and can be used not only as a microchannel chip for medical use, but also as a molded product that reduces optical noise, such as a chip for a chemical analysis device.

[0059] First, a resist pattern for the channel recesses and protrusions was formed using photolithography. A nickel mold was then fabricated by electroforming to transfer the fine recesses and protrusions from the resist pattern to a mold for injection molding. The nickel mold was then attached to the mold and transferred to the resin by injection molding.

[0060] The molded article thus produced had a plurality of flow channels each having a pair of flat surfaces, a thickness of 1 mm, and a depth of 400 μm.

[0061] It was confirmed that thermocompression bonding can be achieved without using adhesives for resin materials such as acrylic, polystyrene, polycarbonate, cycloolefin polymer, and COC.

[0062] When molded products with openings and those without openings were observed during the joining process, it was confirmed that the openings had the effect of degassing.

[0063] As described above, it has been confirmed that the effects of the present invention can be fully achieved in actual resin molded products.

[0064] The present invention has industrial applicability as a micro-channel chip and a manufacturing method thereof. Specifically, the present invention is suitable for a micro-channel chip in which substrates are stacked and bonded together to form a micro-channel between the bonded surfaces, and a manufacturing method thereof.

[0065] 1: First substrate 2: Second substrate 3: Microchannel chip 4: Unwanted light 5: Heat and pressure mold 11: Bonding surface of first substrate 12: Flow channel 13: Recess 14: Recess side 15: Burr surface 21: Bonding surface of second substrate 22: Inlet / outlet 23: Opening 24: Edge of opening 25: Recess at opening 26: Optical measurement observation surface 31: Bonding boundary surface 32: Leaking liquid 33: Height difference at edge 51: Degassing flow 53: Through hole

Claims

1. A microchannel chip formed in a plate shape with light transmissivity and having a flow channel groove sealed inside except for the injection port and the discharge port, The microchannel chip includes a first substrate and a second substrate, The first substrate has the flow channel groove formed therein, has a first bonding surface for bonding integrally with the second substrate, and has a recess formed that does not communicate with the flow channel groove, The second substrate has a second bonding surface for bonding integrally with the first substrate, and at least one of the injection port communicating with the flow channel groove or the discharge port communicating with the flow channel groove is formed, An opening is formed in at least one of the first substrate and the second substrate that is connected to the recess, A microchannel chip, wherein at least a part of the side surface of the recess has a gradient formed thereon.

2. The microchannel chip according to claim 1, The microchannel chip, wherein the opening is formed in the second substrate in the same direction as the recess.

3. The microchannel chip according to claim 1, The microchannel chip, wherein the opening is formed on at least one side surface of the first substrate and the second substrate.

4. The microchannel chip according to claim 1, The microchannel chip, wherein the area of the recess is larger than the area of the opening.

5. The microchannel chip according to claim 1, The microchannel chip, wherein the depth of the recess from the first bonding surface is deeper than the depth of the flow channel groove.

6. The microchannel chip according to any one of claims 1 to 5, The edge of the opening is formed lower than the height of the edge of either the injection port or the discharge port, the height difference of the edge is 0.1 μm or more, and a mouth recess thinner than the thickness of the second substrate is formed. A microchannel chip characterized by this.

7. The microchannel chip according to claim 6, The microchannel chip, wherein a plurality of the openings are formed, and the mouth recess communicates with the plurality of openings.

8. The microchannel chip according to any one of claims 1 to 5, The microchannel chip, wherein there is no adhesive layer or adhesive layer on the first bonding surface and the second bonding surface.

9. A microchannel chip according to any one of Claims 1 to 5, wherein a plurality of the channel grooves are arranged independently, and the recess is arranged between the channel grooves.

10. A microchannel chip according to any one of Claims 1 to 5, wherein an optical measurement observation surface is formed on the second substrate located above the channel groove formed on the first substrate.

11. A method for manufacturing a microchannel chip formed in a light-transmissive plate shape and having a channel groove sealed inside except for an inlet and an outlet, the method comprising: producing a first substrate having the channel groove and a recess not communicating with the channel groove formed therein; producing a second substrate having at least one of the inlet communicating with the channel groove or the outlet communicating with the channel groove formed therein; superposing and integrally joining the first substrate and the second substrate; wherein an opening connecting to the recess is formed in at least one of the first substrate and the second substrate; and a gradient is formed on at least a part of a side surface of the recess.