Method for manufacturing an open microfluidic device

Abstract

To provide a method for efficiently producing an open microchannel device with an externally accessible microchannel that links supply and discharge ports.SOLUTION: A method for producing an open microchannel device includes: a joining step of joining a board to a hydrophobic resin substrate provided with an open microchannel groove whose top face is opened, so as to cover the opened face of the open microchannel groove; a hydrophilization step, followed by the joining step, of making the inside of the open microchannel groove hydrophilic; and a peeling step, followed by the hydrophilization step, of peeling the plate.SELECTED DRAWING: Figure 2

Description

【Technical Field】 【0001】 The present invention relates to a method for manufacturing an open microchannel device. 【Background Art】 【0002】 Microfluidic devices are widely used to separate a specific cell population from a cell mixture or ultimately to capture and separate single cells. As a conventional microfluidic device (microchannel), there are many devices that have a closed microchannel in which a sample supply port for a sample such as a cell mixture and a sample discharge port are formed and the supply port and the discharge port communicate with each other (Patent Document 1). 【Prior Art Documents】 【Patent Documents】 【0003】 【Patent Document 1】 International Publication No. 2009 / 016842 【Summary of the Invention】 【Problems to be Solved by the Invention】 【0004】 However, in a conventional microchannel, except for the supply port and the discharge port, the site for capturing and separating single cells and the channels before and after it are closed spaces from the outside, so there are problems such as being unable to collect the captured cells from the outside. Also, even when the channel is opened after capturing cells in a closed channel, there are problems such as difficulty in removing the lid and taking time, and the captured cells falling off during the removal operation. 【0005】 Therefore, an object of the present invention is to provide a method for efficiently manufacturing an open microchannel device in which the microchannel connecting the supply port and the discharge port has a structure accessible from the outside (open microchannel structure). 【Means for Solving the Problems】 【0006】 In other words, the present invention is as follows: [1] A bonding step involves bonding a plate to a hydrophobic resin substrate having open microfluidic grooves with an open surface at the top, such that the plate covers the open surface of the open microfluidic grooves. After the bonding step, a hydrophilization step is performed to make the inside of the open microchannel groove hydrophilic, After the hydrophilization step, a peeling step is performed to peel off the plates that were joined in the joining step, A method for manufacturing an open microfluidic device, including [the specified component]. [2] A method for manufacturing an open microfluidic apparatus according to [1], wherein in the hydrophilization step, a hydrophilization solution is flowed into the open microfluidic groove to make it hydrophilic. [3] The method for manufacturing an open microfluidic device according to [2], wherein the hydrophilic solution contains a nonionic surfactant. [4] A method for manufacturing an open microfluidic apparatus according to any one of [1] to [3], wherein the surface on which the open microfluidic grooves of the hydrophobic resin substrate are formed is subjected to surface modification treatment, and then the bonding step is performed. [5] A method for manufacturing an open microfluidic apparatus according to any one of [1] to [3], wherein, after peeling off the plate in the peeling step, the hydrophobic resin substrate is heat-treated at a temperature of 100 to 140°C for more than 0 minutes but no more than 60 minutes. [6] The method for manufacturing an open microfluidic apparatus according to [4], wherein, after peeling off the plate in the peeling step, the hydrophobic resin substrate having the grooves is heat-treated at a temperature of 100 to 140°C for more than 0 minutes but no more than 60 minutes. [Effects of the Invention] 【0007】 Because the method for manufacturing an open microfluidic device of the present invention has the above configuration, it is possible to efficiently manufacture an open microfluidic device. [Brief explanation of the drawing] 【0008】 [Figure 1]This is a schematic diagram showing an example of a hydrophobic resin substrate used in the bonding process. (A) is an overall perspective view, (B) is a cross-sectional view of (A) at XX, and (C) is a cross-sectional view of (A) at YY. [Figure 2] This diagram illustrates the manufacturing method of the open microfluidic device according to this embodiment. [Figure 3] This is a schematic diagram showing an enlarged view of the area around the trap section of a hydrophobic resin substrate or an open microfluidic device. [Figure 4] (A) is an example of an open microfluidic device. (B) and (C) are diagrams illustrating Example 1. [Figure 5] This graph shows the results of Example 3. [Figure 6] This is a photograph showing the results of Example 4. The black bar in Figure 6 is a 100 μm scale bar. [Modes for carrying out the invention] 【0009】 The following describes in detail embodiments for carrying out the present invention (hereinafter referred to as "this embodiment"). It should be noted that the present invention is not limited to the following embodiments, and can be implemented in various modifications within the scope of its gist. 【0010】 [Manufacturing method for open microfluidic devices] The method for manufacturing the open microfluidic device of this embodiment includes a bonding step of bonding a plate to a hydrophobic resin substrate having an open microfluidic groove with an open surface on the top, so as to cover the open surface of the open microfluidic groove; a hydrophilization step of making the inside of the open microfluidic groove hydrophilic after the bonding step; and a peeling step of peeling off the plate bonded in the bonding step after the hydrophilization step. The method for manufacturing the open microfluidic device of this embodiment may further include other steps. In this specification, the method for manufacturing an open microfluidic device may be simply referred to as the "manufacturing method." 【0011】 In the case of a device in which the microchannel is a closed space, it has been difficult to collect a single cell after capture or to perform different drug stimulations on each captured single cell. Since the open microchannel device manufactured by the manufacturing method of the present embodiment has an open microchannel accessible from the outside, what is captured in the open microchannel (for example, cells, etc.) can be easily collected and transferred to another container. Therefore, by using the above open microchannel device, culturing and analysis using the captured single cells become possible. In addition, since the outside of the channel is hydrophobic and the inside of the channel is hydrophilic, a solution containing what is to be captured (for example, cell fluid) can flow inside the channel due to the wettability of the channel surface. For example, it can flow inside the open microchannel by spontaneous capillary flows (SCF). Therefore, compared with the conventional method of pressurizing or sucking by a pump or the like, damage to what is to be captured (for example, cells) during capture can be significantly reduced. 【0012】 <Bonding step> (Hydrophobic resin substrate) The hydrophobic resin substrate will be described with reference to FIG. 1. For the sake of explanation, in FIG. 1, the open microchannel groove 21 is described as a straight groove, but it can be arbitrarily designed within the range where the effects of the present invention can be obtained, and the shape of an example of the open microchannel groove is as described later. The hydrophobic resin substrate 2 has an open microchannel groove 21. The open microchannel 21 communicates a supply port 26 and a discharge port 27 that penetrate in the thickness direction from the front to the back. In the example of FIG. 1, the portion between the supply port 26 and the discharge port 27 is the open microchannel groove 21. The open microchannel groove 21 has an open surface 22 upward (FIG. 1(B)). There may be a plurality of supply ports 26 and discharge ports 27. Also, the open microchannel groove may branch in the middle. 【0013】 Examples of materials constituting the above-mentioned hydrophobic resin substrate include hydrophobic resins such as PDMS, PMMA, PC, rigid polyethylene, polystyrene, polyvinyl chloride, polyurethane, epoxy resin, polyester, COP (cycloolefin polymer), and polylactic acid. Among these, PDMS is preferred from the viewpoint of ease of hydrophilization treatment. 【0014】 Examples of cross-sectional shapes in the thickness direction of the open microfluidic groove 21 include U-shape (Figure 1(B)), V-shape, polygonal shapes such as squares, and semicircular shapes. From the viewpoint of facilitating natural capillary flow, a shape with a large aspect ratio of the fluid channel cross-section (a shape in which the depth is large relative to the width of the fluid channel cross-section) is preferred. The cross-sectional shape in the thickness direction may be the same or different in the direction of extension of the open microfluidic groove, but it is preferable that it be the same from the viewpoint of facilitating natural capillary flow. In the trap section described later, the cross-sectional shape in the thickness direction may be different. 【0015】 In this specification, the side of the open microfluidic channel groove 21 toward the supply port 26 may be referred to as the "upstream," the side toward the discharge port 27 as the "downstream," and the direction from the supply port 26 toward the discharge port 27 as the "flow direction." The open microfluidic channel groove 21 has a liquid supply section 23, a trap section 24, and a drain section 25 in that order from upstream to downstream in the flow direction between the supply port 26 and the discharge port 27 (Figure 3). There may be one or more trap sections 24. 【0016】 Preferably, the open microfluidic channel groove 21 extends continuously from the supply port 26 to the discharge port 27 in the extending direction and has an open surface 22 facing upward. 【0017】 (board) The size of the plate 3 is not particularly limited as long as it is large enough to cover the open microfluidic grooves 21 of the hydrophobic resin substrate 2. However, from the viewpoint of ease of manufacturing and ease of joining, it is preferable that the plate 3 be the same size as the hydrophobic resin substrate, except in the thickness direction. The shape of the plate 3 is not particularly limited as long as it is large enough to cover the open microfluidic grooves 21 of the hydrophobic resin substrate 2, but it is preferably a flat plate. Here, covering the open microfluidic channel groove 21 means joining the hydrophobic resin substrate 2 and the plate 3 to temporarily seal the open surface 22 (i.e., sealing the open microfluidic channel groove 21 except for the supply port 26 and the discharge port 27). 【0018】 The plate 3 may be made of resin, glass, metal, etc., but it is preferably made of resin, and more preferably made of the same material as the hydrophobic resin substrate 2. In particular, from the viewpoint of excellent adhesion during temporary bonding, it is preferable that both the hydrophobic resin substrate and the plate be made of PDMS. 【0019】 (Joining) The above bonding process forms a composite structure in which the hydrophobic resin substrate 2 and the plate 3 are joined (Figure 2(b)-(d)). Because the open microfluidic channels 21 of the composite structure are sealed, even if a solution is added from the supply port 26, the solution does not flow into any area other than the open microfluidic channels 21. The above-mentioned bonding refers to the process of temporarily bonding the hydrophobic resin substrate 2 and the plate 3 in the bonding step, and then peeling off the temporarily bonded hydrophobic resin substrate 2 and plate 3 in the peeling step. 【0020】 In the above bonding process, the surface of the hydrophobic resin substrate 2 on which the open microfluidic grooves 21 are provided is placed opposite one surface of the plate 3, and the two surfaces are bonded together. 【0021】 The method for joining the hydrophobic resin substrate 2 and the plate 3 is not particularly limited, as long as it is a method that allows for joining to a degree that the inside of the open microchannel grooves can be made hydrophilic in a later hydrophilization step. Examples include methods of pressing them together under pressure, joining using a removable adhesive, joining using removable adhesive tape, and tying the composite with rubber or the like. 【0022】 The composite of the hydrophobic resin substrate 2 and the plate 3 after bonding is preferably bonded at least around the open surface 22 of the open microfluidic groove 21, but it may also be bonded over the entire surface other than the open microfluidic groove 21. 【0023】 Figures 1 and 2 show an example in which a hydrophobic resin substrate 2 is provided with a supply port 26 and an outlet port 27. However, for example, a hydrophobic resin substrate 2 without a supply port and an outlet port, having only open microchannel grooves 21 on one surface, and a plate 3 having through holes at a position opposite to the open microchannel grooves 21 when joined may also be used. 【0024】 <Hydrophilization process> Methods for hydrophilizing the open microchannel groove 21 in the above hydrophilization step include flowing a hydrophilization solution into the open microchannel groove. In the above hydrophilization step, it is preferable to hydrophilize only the open microchannel groove 21. One method for flowing the hydrophilic solution is to pour the hydrophilic solution 4 into the composite of the hydrophobic resin substrate 2 and the plate 3 after bonding, leave it for a certain period of time with the microfluidic groove filled with the hydrophilic solution 4, and then suction and discharge the hydrophilic solution 4 from the discharge port 27 (Figure 2(b)-(d)). 【0025】 The hydrophilic solution 4 described above preferably contains a nonionic surfactant. Examples of nonionic surfactants include block polymer ethers, polyoxyethylene hydrogenated castor oil, sucrose fatty acid esters (sugar esters), polyoxyethylene sorbitan fatty acid esters, and sucrose fatty acid esters. Examples of the above-mentioned block polymer type ethers include polyoxyethylene (196) polyoxypropylene (67) glycol (Pluronic F127), polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68), polyoxyethylene (42) polyoxypropylene (67) glycol (Pluronic P123), and polyoxyethylene oxypropylene cetyl ether (20E.O4P.O). Examples of the above-mentioned hydrogenated castor oil include hydrogenated castor oil polyoxyethylene ether and hydrogenated polyoxyethylene castor oil. Examples of the above-mentioned polyoxyethylene sorbitan fatty acid esters include polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 65, polysorbate 80 (Tween 80), and polyoxyethylene sorbitan monolaurate (20E.O). In particular, from the viewpoint of suppressing the adhesion of cells and other organisms to open microfluidic grooves and further facilitating natural capillary flow, block polymer type ethers are preferred, more preferably polyoxyethylene polyoxypropylene glycol, and even more preferably Pluronic F68. 【0026】 The mass ratio of the nonionic surfactant to 100% by mass of the hydrophilic solution is preferably 0.1 to 10% by mass, more preferably 0.3 to 5% by mass, and even more preferably 0.5 to 3% by mass. If the mass ratio is less than 0.1% by mass, the hydrophilic effect in the open microfluidic channels is small, which is undesirable. If it is greater than 10% by mass, the viscosity of the hydrophilic solution becomes high, making it difficult for the hydrophilic solution to flow into the open microfluidic channels, or components in the hydrophilic solution accumulate in the open microfluidic channels, preventing uniform hydrophilicity, which is also undesirable. 【0027】 The hydrophilic solution described above contains the nonionic surfactant described above and may also contain other components. In particular, it is preferable that the hydrophilic solution consists only of water and the nonionic surfactant described above. 【0028】 The time for leaving the open microfluidic groove 21 filled with the hydrophilic solution 4 to stand may be 10 to 200 minutes, or 20 to 100 minutes. Furthermore, the temperature during the standing period may be 5 to 40°C or 15 to 30°C. It is preferable that the temperature of the hydrophilic solution 4 when it is poured into the open microfluidic channel is the same as the temperature during the standing period. When the open microfluidic channel groove 21 is left filled with the hydrophilic solution 4, it is preferable to block the supply port 26 and / or discharge port 27 in order to reduce the evaporation of the hydrophilic solution. Methods for blocking the supply port, etc. include, for example, blocking the supply port, etc. with removable adhesive tape. 【0029】 One method for discharging the hydrophilic solution 4 from the open microfluidic channel groove is to remove it from the outlet 27 by applying negative pressure using a vacuum or the like (Figure 2(d)). 【0030】 <Removal process> After the hydrophilization step described above, the hydrophobic resin substrate 2 and the plate 3 that were joined in the joining step described above are peeled apart to obtain an open microfluidic device 1 in which the inside of the open microfluidic groove 21 is hydrophilized. The hydrophobic resin substrate 2 after peeling may be heat-treated (Figure 2(e)). This heat treatment can dry the hydrophilic solution remaining in the open microchannels and can also more firmly fix the hydrophilic components in the open microchannel grooves. The temperature for the above heat treatment is such that the hydrophobic resin substrate does not deform or change, and may be, for example, 100 to 140°C. The duration of the above heat treatment is the time during which the hydrophobic resin substrate does not deform or change, and may be, for example, more than 0 minutes and within 60 minutes. 【0031】 <Other processes> (Surface modification process) In the manufacturing method of the open microfluidic device of this embodiment, it is preferable to include a surface modification step before the joining step, since the hydrophilization step allows only the inside of the open microfluidic groove to be hydrophilized separately from the surface outside the groove. In the above surface modification process, the surface of the hydrophobic resin substrate 2 on which the open microchannel grooves 21 are formed is subjected to surface modification treatment. The surface modification treatment may be performed on the entire surface of the hydrophobic resin substrate 2 on which the open microchannel grooves 21 are formed, or it may be performed only on the open microchannel grooves 21. The surface can be made hydrophilic in the surface modification process described above. However, with the hydrophilicization in the surface modification process, the hydrophilicity of the surface is lost after a certain period of time, and wettability that causes natural capillary flow with high capture efficiency may not be obtained. Therefore, from the viewpoint of obtaining an open microfluidic device with high capture efficiency, it is preferable to perform the bonding process and the hydrophilization process consecutively after the surface modification process. Examples of the surface modification treatment process include ultraviolet irradiation, corona discharge, and plasma treatment such as oxygen plasma treatment. Among these, ultraviolet irradiation is preferred from the viewpoint of ease of treatment. The ultraviolet irradiation treatment is preferably performed by irradiating with light in the vacuum ultraviolet region with a wavelength of 10 to 200 nm (preferably light with a wavelength of 150 to 180 nm) from a distance of 10 to 30 mm for 5 to 20 minutes. 【0032】 [Open Microfluidic Device] The manufacturing method for the open microfluidic device of this embodiment described above allows for the production of an open microfluidic device 1 in which the inside of the open microfluidic groove 21 is hydrophilic. Preferably, all parts of the open microfluidic device other than the open microfluidic groove 21 are hydrophobic. The supply port 26 and discharge port 27 are used when flowing the hydrophilic solution during the hydrophilization process, but they do not need to be used when flowing the solution by natural capillary flow, so they may be hydrophilic or hydrophobic. The open microfluidic device 1 described above is a hydrophobic resin substrate 2 in which the inside of the open microfluidic grooves is hydrophilic. 【0033】 When the open microfluidic device 1 has a through hole on the upstream side (for example, when it has a supply port 26), it is preferable to use it with a cover 6 on the vertically lower side of the through hole (Figure 2(f)). The cover 6 may be hydrophilic or hydrophobic, and it is preferable that it be made of the same material as the hydrophobic resin substrate 2. 【0034】 Because the upper surface of the channel in the open microfluidic device 1 described above is open, it is not possible to aspirate the solution using a vacuum or the like. The open microfluidic device 1 can have a solution containing something to be captured (e.g., cell sap) dropped into the open microfluidic channel, and the fluid can be made to flow by the wettability of the channel surface. For example, the solution can be made to flow by natural capillary flow. The above-described open microfluidic device 1 is a device having an open microfluidic channel that can flow a solution by natural capillary flow. 【0035】 The open microfluidic channel 21 of the open microfluidic device 1 described above has a liquid supply section 23, a trap section 24, and a drain section 25 in that order from the upstream side to the downstream side in the flow direction. A solution containing the material to be captured (e.g., cell sap) is dropped upstream of the liquid supply section 23, and the solution flows to the liquid supply section 23. Here, the flow velocity of the solution near the trap section is faster in the trap section 24 than in the flow velocity Q2 that bypasses the trap section due to the narrow width of the trap section 24 (Figure 3, Q1>Q2). Therefore, the first material to be captured (e.g., cells) that flows in is captured in the trap section 24. After that, the flow velocity in the trap section 24 decreases, and Q2>Q1, so the solution flows around the trap section 24. Therefore, the trap section 24 can capture one material to be captured at a time (e.g., a single cell). By providing multiple trap units 24 directly and / or in parallel, single cells, etc., can be captured at multiple locations. When multiple trap units 24 are provided in series, the drainage section 25 of one trap unit 24 corresponds to the fluid supply section 23 of the subsequent trap unit 24. Furthermore, since the open microfluidic channel groove 21 has an open surface 22 at the top, the cells captured in the trap section 24 can be collected with a pipette or the like. For example, a captured single cell can be aspirated with a pipette and transferred to a dish containing culture medium for cell culture. Because the cells captured in the open microfluidic channel groove 21 are captured at the flow rate of natural capillary flow, there is significantly less damage, and cells can be captured while maintaining structures such as dendrites and cell surface structures, thus capturing cells in a state closer to that of a living organism. 【0036】 By adjusting the width, depth, and contact angle of the groove surface of the open microfluidic groove 21 described above, the efficiency of natural capillary flow can be increased, further improving capture efficiency and reducing damage to cells. 【0037】 The width of the open microfluidic groove 21 is preferably 500 μm or less, more preferably 100 μm or less, and even more preferably 50 μm or less, from the viewpoint of facilitating the flow of the solution through natural capillary flow. The above width may also be 1 μm or more. Here, the width of the open microfluidic groove may be the average width of the flow channel 50 μm upstream of the trap section 24. Furthermore, the width of the flow channel refers to the width in a cross-section perpendicular to the flow direction, and measurement should be taken excluding branching points where the liquid flows in multiple directions. 【0038】 The depth of the open microfluidic groove 21 is preferably 500 μm or less, more preferably 100 μm or less, and even more preferably 50 μm or less, from the viewpoint of facilitating the flow of the solution through natural capillary flow. Here, the depth of the open microfluidic groove may be the average depth of the flow channel 50 μm upstream of the trap section 24. Furthermore, the depth of the flow channel refers to the depth in a cross-section perpendicular to the flow direction. 【0039】 The surface of the open microchannel groove is preferably wettable. The contact angle θ of the surface of the open microchannel groove is preferably less than 90°, more preferably 50° or less, and even more preferably 30° or less. The contact angle θ refers to the value measured by the tangent method at any location in the channel 50 μm upstream of the trap portion 24. 【0040】 From the viewpoint of facilitating the occurrence of SCF, the relationship among the width W (μm), depth H (μm), and contact angle θ of the open microchannel groove 21 preferably satisfies W / (2H + W) < cosθ, more preferably satisfies W / (2H + W) < 0.8, and even more preferably satisfies W / (2H + W) < 0.6. 【0041】 Examples of what is captured by the trap portion 24 include cells such as mammalian cells, insect cells, and plant cells, bacteria, beads, mixtures thereof, etc., and cells are preferred. 【0042】 The width of the trap portion 24 is not particularly limited as long as it is narrow enough to capture what is to be captured, and can be appropriately changed according to the type of what is to be captured. For example, it is preferably 0.1 to 20 μm, more preferably 0.5 to 15 μm, and even more preferably 1 to 10 μm. 【0043】 From the viewpoint of the capture efficiency in the trap portion, the width of the trap portion 24 is preferably narrower than the width of the open microchannel groove 21, and is preferably 10 to 80% of the diameter of what is to be captured, more preferably 20 to 60%, and even more preferably 30 to 50%. 【0044】 The open microchannel groove 21 may have a plurality of branches and a plurality of trap portions (Fig. 4). 【Examples】 【0045】 Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited by these examples. 【0046】 (Example 1) A hydrophobic resin substrate with open microchannel grooves, as shown in Figure 4(A), was manufactured by pouring PDMS into a mold and curing it. The thickness of the hydrophobic resin substrate was 16 μm, the width of the open microchannel grooves was 30 μm, the depth was 16 μm, and the width of the trap section was 8 μm. The substrate was placed with the side where the open microchannel grooves were formed facing upwards in a Samco compact etcher FA-1a (manufactured by Samco Corporation) and subjected to oxygen plasma treatment for 5 seconds (air supply rate: 20 ml / min, LEV: 75 W, FWD: 75, REV: 0). In addition, a separate PDMS plate was fabricated. The surface of the hydrophobic resin substrate with open microchannel grooves formed after oxygen plasma treatment was placed opposite one surface of a flat plate, and the two surfaces were pressed together by hand. Then, a 1% PluronicF68 aqueous solution was poured into the open microchannel grooves from the supply port on the upstream side, filling the grooves, and left to stand at room temperature for 30 minutes. After that, a tube was inserted into the outlet, and negative pressure was applied to remove the 1% PluronicF68 aqueous solution from the open microchannel grooves. Subsequently, the flat plate was peeled off, and the surface with the open microchannel grooves formed was placed face down on a hot plate, and the hydrophobic resin substrate was heated at 120°C for 20 minutes. The water contact angle θ measured by the tangential method on a PDMS flat plate that had undergone a similar hydrophilization treatment was approximately 20°. Then, one of the holes in the supply port was covered with a PDMS plate to manufacture an open microfluidic device. From the upstream of the obtained open microchannel, 10.2 μm diameter beads (number concentration 5 × 10) 5 A solution (beads / ml) was added dropwise and allowed to flow by natural capillary flow. One bead was captured in all 10 trap locations. Two more similar tests were conducted, and in each test, one bead was captured in all 10 trap locations. Figure 4(B) is a magnified view of the area around the trap section before the beads 5 are captured, and Figure 4(C) is a magnified view of the area around the trap section after the beads 5 have been captured and the beads flow to the drain section. 【0047】 (Example 2) An open microfluidic device was manufactured in the same manner as in Example 1. An open microfluidic device is placed in a 2-inch diameter dish, and Jurkat cell solution (at a concentration of 1 × 10⁻¹⁶) is introduced from the upstream of the open microfluidic device. 5 ~1 × 10 7 A culture medium (RPMI1640 + 10% FBS) containing ) cells / ml was added dropwise and allowed to flow by natural capillary flow. One cell was trapped in the trap section, and the trapped cell was aspirated with a pipette and dispensed into a test tube containing RPMI1640 + 10% FBS. 【0048】 (Comparative Example 1) A hydrophobic resin substrate was manufactured in the same manner as in Example 1, and the entire hydrophobic resin substrate was subjected to oxygen plasma treatment. Then, one of the holes in the supply port was covered with a PDMS plate to manufacture an open microfluidic device. From the upstream of the obtained open microchannel, 10.2 μm diameter beads (number concentration 5 × 10) 5 When a beads / ml aqueous solution was dropped onto the groove, the solution did not flow only within the groove but spread across the entire surface. Furthermore, some of the bead solution flowed into the groove, allowing the beads to be captured in the trap section, but the efficiency was extremely poor. 【0049】 (Example 3) Hydrophobic resin substrates with open microfluidic channels having a rectangular cross-sectional shape in the thickness direction were manufactured by pouring PDMS into a mold and allowing it to cure. The open microfluidic channels were long channels with the same cross-sectional shape in the thickness direction along their extension. The rectangular cross-section in the thickness direction had a width of 50 μm, and three different depths of 16, 42, and 78 μm. A total of six hydrophobic resin substrates were prepared, two of each type. In all of the hydrophobic resin substrates, the open microfluidic channels were long channels with the same cross-sectional shape in the thickness direction along their extension. In addition, six flat PDMS plates were fabricated separately. The surfaces of each hydrophobic resin substrate with open microfluidic grooves were placed opposite one surface of each flat plate, and they were joined together by pressing them with fingers. Then, a 1% Pluronic F68 aqueous solution or a 1% PVA aqueous solution was poured into the open microfluidic grooves from the supply port on the upstream side, filling the grooves, and left to stand at room temperature for 30 minutes. After that, a tube was inserted into the outlet, and negative pressure was applied to remove the 1% Pluronic F68 aqueous solution or 1% PVA aqueous solution from the open microfluidic grooves. Then, the plate was peeled off, and one of the holes in the supply port was covered with a PDMS plate to fabricate an open microfluidic device with a rectangular cross-section depth of 16, 42, or 78 μm, which was hydrophilized with a 1% Pluronic F68 aqueous solution or a 1% PVA aqueous solution. Water was dropped from the upstream end of the resulting open microchannel and allowed to flow by natural capillary flow. Figure 5 is a graph with time (seconds) to the power of 1 / 2 on the horizontal axis and the distance (mm) traveled by natural capillary flow on the vertical axis. In all flow channel devices with groove depths of 16, 42, and 78 μm, the water flow was faster in the devices hydrophilized with 1% Pluronic F68 aqueous solution than in the devices hydrophilized with 1% PVA aqueous solution. Furthermore, natural capillary flow did not occur in the flow channel device with a groove depth of 16 μm that was hydrophilized with 1% PVA aqueous solution. 【0050】 (Example 4) An open microchannel manufactured in the same manner as in Example 1, and an open microchannel apparatus manufactured in the same manner as in Example 1, except that heat treatment was not performed after peeling off the flat plate, were prepared. The open microfluidic grooves of the flow channel device in Example 4 were of the shape shown in Figure 6. Water was dropped into two types of open microfluidic devices that were manufactured, and the spread of the water droplets was observed. In the heat-treated open microfluidic device (Figure 6, right), it was possible to control the hydrophilicity appropriately, preventing the raised area of ​​the dropped water droplet from spreading too much, and controlling the range of the region where natural capillary flow occurs only within the open microfluidic groove. On the other hand, in the open microfluidic device that did not undergo heat treatment (Figure 6 left), perhaps due to its high hydrophilicity, the dropped water spread over a wide area, making it difficult to control the range of the region where natural capillary flow occurred only within the open microfluidic groove. [Industrial applicability] 【0051】 The manufacturing method of the open microfluidic device of this embodiment allows for the individual separation and capture of target substances contained in a mixture by flowing the solution through natural capillary flow. [Explanation of Symbols] 【0052】 1. Open microfluidic device 2. Hydrophobic resin substrate 21 Open microfluidic grooves 22 Open surface 23 Liquid supply section 24 Trap section 25 Drainage section 26 supply ports 27 Outlet 3 boards 4 Hydrophilization solution 5 beads 6 Lid

Claims

[Claim 1] A bonding step involves bonding a plate to a hydrophobic resin substrate having open microfluidic grooves with an open surface at the top, such that the plate covers the open surface of the open microfluidic grooves. After the bonding step, a hydrophilization step is performed to make the inside of the open microchannel groove hydrophilic, After the hydrophilization step, a peeling step is performed to peel off the plates that were joined in the joining step, Includes, A method for manufacturing an open microfluidic device, comprising the step of hydrophilizing the open microfluidic grooves by flowing a hydrophilizing solution through them. [Claim 2] The method for manufacturing an open microfluidic device according to claim 1, wherein the hydrophilic solution contains a nonionic surfactant. [Claim 3] A method for manufacturing an open microfluidic apparatus according to claim 1 or 2, wherein the surface on which the open microfluidic grooves of the hydrophobic resin substrate are formed is surface modified before performing the bonding step. [Claim 4] A method for manufacturing an open microfluidic device according to claim 1 or 2, wherein, after peeling off the plate in the peeling step, the hydrophobic resin substrate is heat-treated at a temperature of 100 to 140°C for more than 0 minutes but not exceeding 60 minutes. [Claim 5] The method for manufacturing an open microfluidic device according to claim 3, wherein, after peeling off the plate in the peeling step, the hydrophobic resin substrate is heat-treated at a temperature of 100 to 140°C for more than 0 minutes but no more than 60 minutes.

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