Micro fluid device, and method for manufacturing the same

Abstract

To provide a micro fluid device in which residual solvent is kept to an amount at which influence to a cultured cell can be suppressed.SOLUTION: A micro fluid device comprises: a first member made of resin, a second member made of resin, an adhesive layer which bonds an interface between the first member and the second member; and a flow channel which is formed in a part of the boundary between the first member and the second member. The adhesive layer includes a target substance being a component of organic solvent. In the case where components contained in a reference part positioned in a region separated from the flow channel and the adhesive layer and a flow channel part positioned in a region where the flow channel is formed are measured by thermal desorption mass spectrometry, a first temperature at which emission of a target substance is started from the reference part, and a second temperature at which emission of a target substance is started from the flow channel part are substantially the same.SELECTED DRAWING: Figure 11

Description

[Technical Field]

[0001] The present invention relates to a microfluidic device and a method for manufacturing the same. [Background technology]

[0002] In recent years, microfluidic devices (sometimes called "cell culture chips," "biochips," "microchips," or "microchannel chips") that can create three-dimensional (stereoscopic) cell culture and experimental environments have been proposed as devices used for cell and tissue culture.

[0003] Microfluidic devices are formed by assembling two or more components. Specifically, the formation of a microfluidic device requires a process of bonding two or more components. For this bonding process, a bonding method using an organic solvent (solvent bonding method) is commonly used.

[0004] However, due to the minute structural dimensions of microfluidic devices, microscopic gaps may be formed in the micro-channels during fabrication, and residual organic solvents (hereinafter referred to as "residual solvents") used for adhesion may penetrate into the micro-channels through the gaps and adversely affect cell culture and other assays.

[0005] In view of these circumstances, a technique has been proposed in the past in which members are heated for more than 30 minutes after being joined in order to remove the residual solvent (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2023-516548 Summary of the Invention [Problem to be solved by the invention]

[0007] At present, there is no established method for evaluating the amount of residual solvent contained in microfluidic devices manufactured using the solvent bonding method. Therefore, in order to suppress the effects on cultured cells using the technology of Patent Document 1, it is necessary to apply strong energy to the device by increasing the heating time or heating temperature. However, while such a method may remove the residual solvent, it also damages the components that make up the device, which may result in an effect on the cultured cells.

[0008] In view of the above-mentioned problems, the present invention aims to provide a determination index for determining whether the amount of residual solvent in a microfluidic device manufactured using a solvent bonding method is sufficient to suppress the effects on cultured cells. More specifically, the present invention aims to provide a method for manufacturing a microfluidic device that utilizes the determination index to limit the amount of residual solvent to a level sufficient to suppress the effects on cultured cells. Another object of the present invention is to provide a microfluidic device in which the amount of residual solvent is suppressed to a level sufficient to suppress the effects on cultured cells. [Means for solving the problem]

[0009] The microfluidic device according to the present invention comprises: a first member made of resin; a second member made of resin; an adhesive layer that bonds the interface between the first member and the second member; a flow path formed in a part of a boundary between the first member and the second member, the adhesive layer contains a target substance that is a component of an organic solvent; When the components contained in a reference portion located in an area spaced from the flow path and the adhesive layer and in a flow path portion located in the area where the flow path is formed are measured using thermal desorption mass spectrometry, the first temperature at which the target substance begins to be released from the reference portion and the second temperature at which the target substance begins to be released from the flow path portion are substantially identical.

[0010] In a microfluidic device manufactured by the solvent bonding method, a first member and a second member are bonded via an adhesive layer containing an organic solvent component.

[0011] Both the first member and the second member are made of resin. As a specific example, the main material of the first member may be one or more resin materials selected from the group consisting of COP (cycloolefin copolymer), COC (cycloolefin polymer), PS (polystyrene), PMMA (polymethyl methacrylate), and PC (polycarbonate). As a specific example, the main material of the second member may be one or more resin materials selected from the group consisting of COP, COC, PS, PMMA, and PC. In this specification, the term "main material" refers to a material that accounts for 50% or more by mass of the constituent materials.

[0012] Resins are produced by synthesizing (polymerizing) monomers using a solvent. For convenience, the solvent used in this synthesis is referred to as the "synthesis solvent." The first and second components may contain residues of the synthesis solvent before they are bonded together via the adhesive layer.

[0013] When the first and second members are bonded together via the adhesive layer to form a microfluidic device, the microfluidic device may contain residual solvent from the adhesive layer. Hereinafter, the solvent used for bonding will be referred to as the "adhesive solvent" for convenience.

[0014] According to the inventors' intensive research, it has been confirmed that residual adhesive solvents have adverse effects on cultured cells. In a microfluidic device, the space in which cells are cultured is within a flow channel (microchannel). Because the flow channel is formed at the boundary between the first and second members, the adhesive layer is disposed in close proximity to the flow channel, which makes it easy for adhesive solvents to remain within the flow channel, raising concerns about the effects on cells.

[0015] In microfluidic devices, the synthesis solvent and adhesive solvent may contain the same organic substances, making it difficult to remove only the adhesive solvent. Furthermore, the affinity between resin and solvent is high. For these reasons, attempts have been made to remove residual solvent in microfluidic devices by applying higher energy than necessary. This is because applying low energy may not sufficiently remove the residual solvent in the microchannel, and there is no method to verify whether the residual solvent has been removed.

[0016] A microfluidic device for verification is prepared and separated into a reference portion located in an area away from the flow channel and the adhesive layer, and a flow channel portion located in an area where the flow channel is formed. As a detailed example, the reference portion can be obtained by cutting a portion of the microfluidic device away from the flow channel, i.e., a portion close to the surface, in a direction perpendicular to the main surface. The flow channel portion can be obtained by cutting a portion of the microfluidic device in an area away from the reference portion, including at least a portion of the area where the flow channel is formed.

[0017] The reference and flow channel sections obtained from the microfluidic device are analyzed by thermal desorption mass spectrometry (TDS-MS), which involves introducing a sample into the device, raising the temperature at a predetermined rate, and analyzing the relative amounts of substances contained in the resulting gas.

[0018] Comparing the solvent (adhesive solvent) remaining on the wall surface of the microfluidic device (more specifically, within the flow path) with the solvent (synthesis solvent) present in the components (first or second component) constituting the microfluidic device, the former is extracted at a lower temperature. This is because the bonding strength between the solvent present in the component constituting the microfluidic device and said component is thought to be stronger than the bonding strength between the solvent remaining on the wall surface of the flow path and said component. More specifically, this is thought to be because the solvent present in the component constituting the microfluidic device is located deeper than the solvent remaining on the wall surface, and because the glass transition temperature of the component constituting the microfluidic device is higher than that of the adhesive layer (the layer containing the solvent remaining on the wall surface).

[0019] In other words, when the reference portion and the channel portion obtained from the microfluidic device are measured by TDS-MS, if the temperature (first temperature) at which the substance (target substance) that is a constituent material of the solvent starts to be released from the reference portion and the temperature (second temperature) at which the target substance starts to be released from the channel portion are substantially the same, this indicates that there is substantially no residual solvent on the wall surface of the channel. Here, the phrase "the first temperature and the second temperature are substantially the same" may mean that the difference between the first temperature and the second temperature is 10% or less of the first temperature.

[0020] The temperature at which the release of the target substance begins can be the temperature at which a significant difference appears between the blank signal and the signal obtained from the gas extracted from the sample. The significant difference here may be defined as a P value of less than 0.05 in a two-sample equal variance t-test.

[0021] Various methods can be used to identify the target substance (target substance) contained in the gas obtained by TDS-MS analysis. First, if the components of the organic solvent used in the adhesive layer of the target microfluidic device are known, this known organic solvent can be used as the target substance. Then, the resulting mixed gas is subjected to a mass spectrometer while the temperature is increased, and the change in the intensity value of the m / z value corresponding to the target substance is observed over time. The temperature at which a significant difference in intensity value compared to the blank is observed can be determined as the temperature at which the release of the target substance begins.

[0022] When the components of the organic solvent used in the adhesive layer of a target microfluidic device are unknown, the same microfluidic device is measured using the TDS-MS method, and the substances contained in the gas when heated to a sufficiently high temperature are identified using a mass spectrometer. Among the identified substances, those corresponding to m / z values ​​with high signal intensity, excluding substances derived from air, can be identified as the target substance.

[0023] In other words, in the microfluidic device in which the first temperature at which the target substance starts to be released from the reference portion and the second temperature at which the target substance starts to be released from the flow path portion are substantially the same, it can be determined that there is virtually no adhesive solvent remaining in the microflow path, and therefore, by culturing cells using this microfluidic device, damage to the cells resulting from the adhesive solvent can be suppressed.

[0024] The target substance may be a substance selected from the group consisting of toluene, cyclohexane, dichloromethane, tetrahydrofuran, chloroform, ethyl acetate, methyl ethyl ketone, acetone, and dimethylformamide.

[0025] A typical example is: a main material of the first member and the second member is COP; The target substance is toluene The first temperature and the second temperature may both be greater than 75°C.

[0026] The method for producing a microfluidic device according to the present invention comprises the steps of: A step (a) of preparing a first member whose main material is a resin material and whose recessed groove is formed in a part thereof, and a second member whose main material is the resin material; a step (b) of bonding the first member and the second member together with an adhesive containing an organic solvent in a state where the side of the first member on which the grooves are formed faces the second member, thereby obtaining a device including a flow path formed by the grooves; (c) determining removal conditions for removing the organic solvent from the device; and (d) subjecting the device to removal of the organic solvent under the removal conditions; The step (c) a step (c1) of preparing a test member made of the resin material; a step (c2) of measuring a first temperature at which the target substance, which is a component of the organic solvent, starts to be released from the test member using thermal desorption mass spectrometry; (c3) removing the organic solvent from at least one of the test devices obtained in the step (b); After the step (c3), a step (c4) is performed on the test device, using thermal desorption mass spectrometry to measure a second temperature at which the target substance starts to be released; and a step (c5) of determining conditions for step (c3) under which the second temperature becomes substantially the same as the first temperature by performing step (c3) under different conditions when step (c3) is performed and setting the conditions as the removal conditions.

[0027] According to the above method, conditions (removal conditions) that allow the adhesive solvent to be effectively removed are set in step (c), and the organic solvent is removed under the set removal conditions in step (d), which makes it possible to remove the adhesive solvent remaining in the flow path without causing undue damage to the device.

[0028] In detail, steps (c2) and (c4) involve placing a sample in a reaction tube, heating it at a predetermined heating rate, sending the gas (mixed gas) in the reaction tube to a mass spectrometer, and performing mass analysis in the mass spectrometer.The temperature at which the intensity value of the signal derived from the target substance is significantly different from the blank can be determined to be the temperature at which the release of the target substance begins.

[0029] More specifically, the steps (c3) and (d) include a step of heating the device in a state where the device is placed in a predetermined space, The removal condition may be specified by information including the temperature of the space in which the device is installed and the time for which the device is installed in the space.

[0030] The step (c) may further include, prior to the step (c2), a step (c1a) of setting the substance having the highest signal intensity, excluding substances derived from air, obtained when the test member is heated to a verification temperature near the glass transition temperature using thermal desorption mass spectrometry as the target substance.

[0031] In this specification, the term "near the glass transition temperature" refers to a temperature range that includes an error of ±20% from the glass transition temperature.

[0032] The resin material may be one or more selected from the group consisting of COP, COC, PS, PMMA, and PC.

[0033] The target substance may be a substance selected from the group consisting of toluene, cyclohexane, dichloromethane, tetrahydrofuran, chloroform, ethyl acetate, methyl ethyl ketone, acetone, and dimethylformamide.

[0034] In a typical example, the resin material is COP, the target substance is toluene, In the step (c5), the removal conditions may be set to the conditions in the step (c3) where both the first temperature and the second temperature are higher than 75°C. [Effects of the Invention]

[0035] According to the present invention, in a microfluidic device manufactured using a solvent bonding method, it is possible to reduce the amount of solvent remaining in the microchannel while suppressing damage to the device. [Brief explanation of the drawings]

[0036] [Figure 1] FIG. 1 is a perspective view schematically illustrating the structure of an embodiment of a microfluidic device. [Figure 2] FIG. 2 is a schematic plan view of the microfluidic device as viewed from the first member side. [Figure 3] 3 is a schematic cross-sectional view of the microfluidic device taken along line X1-X1 in FIG. 2. FIG. [Figure 4] 1 is a schematic diagram illustrating a reference portion and a channel portion, which are parts of a microfluidic device. [Figure 5] FIG. 1 is a block diagram schematically illustrating an example of an apparatus configuration for performing a TDS-MS method. [Figure 6] 1 is a flowchart schematically illustrating an example of the steps of a method for manufacturing a microfluidic device. [Figure 7] FIG. 1 is a conceptual diagram schematically illustrating an example of a procedure for manufacturing a microfluidic device. [Figure 8]1 is a flowchart schematically illustrating an example of a procedure for setting conditions for removing an organic solvent remaining in a microfluidic device. [Figure 9] 1 shows an example of the results of a mass spectrometry spectrum obtained when a COP resin is analyzed using a TDS-MS method. [Figure 10] 1 is a graph showing the change in the signal intensity of m / z=91, which is a signal derived from toluene, when mass spectrometry was performed on the gas in a reaction tube while raising the temperature of a test member made of COP resin. [Figure 11] A graph showing the change in the signal intensity of m / z=91, which is a signal derived from toluene, when mass spectrometry was performed on the gas in the reaction tube while the temperature of the test device was being increased, is superimposed on the graph in Figure 10. [Figure 12] 10 is a graph showing the results of verifying the influence of organic solvents remaining in a microfluidic device on cultured cells. [Figure 13] 1 is a graph showing the change in the signal intensity of m / z=56, which is a signal derived from cyclohexane, when mass spectrometry was performed on the gas in the reaction tube while the temperature of the test member and the test device was being increased. DETAILED DESCRIPTION OF THE INVENTION

[0037] A microfluidic device and a method for manufacturing the same according to the present invention will be described with reference to the drawings. Note that the drawings are merely schematic illustrations. That is, the dimensional ratios in the drawings do not necessarily match the actual dimensional ratios, and the dimensional ratios between the drawings do not necessarily match.

[0038] Fig. 1 is a perspective view schematically illustrating the structure of one embodiment of a microfluidic device. As shown in Fig. 1, the microfluidic device 1 includes a first member 11 and a second member 12, which are bonded together via an adhesive layer 3 (see Fig. 3). Fig. 2 is a schematic plan view of the microfluidic device 1 as viewed from the first member 11 side. Fig. 3 is a schematic cross-sectional view of the microfluidic device 1 taken along line X1-X1 in Fig. 2.

[0039] As shown in Fig. 3, the microfluidic device 1 includes a channel 20 formed in a part of the boundary between the first member 11 and the second member 12. The channel 20 forms a space for culturing cells or the like. In the example shown in Figs. 1 to 3, the microfluidic device 1 includes wells 21 and 22 connected to the channel 20. The wells 21 and 22 form an inlet or outlet for the culture medium or the like.

[0040] The first member 11 and the second member 12 are both made primarily of resin. Specifically, the primary material of the first member 11 and the second member 12 is one or more selected from the group consisting of COP, COC, PS, PMMA, and PC.

[0041] The first member 11 and the second member 12 are bonded together via an adhesive layer 3. This adhesive layer 3 contains a target substance α, which is a component of an organic solvent. As a specific example, the target substance α is a substance selected from the group consisting of toluene, cyclohexane, dichloromethane, tetrahydrofuran, chloroform, ethyl acetate, methyl ethyl ketone, acetone, and dimethylformamide.

[0042] FIG. 4 is a schematic diagram illustrating a reference portion 31 and a channel portion 32, which are parts of the microfluidic device 1, and is a schematic diagram of a cutaway portion of the microfluidic device 1 shown in FIG. 3. The reference portion 31 is a portion of the microfluidic device 1 located in a region away from the channel 20 and the adhesive layer 3. The channel portion 32 is a portion of the microfluidic device 1 located in a region where the channel 20 is formed. Note that, since the channel 20 no longer exists when the reference portion 31 and the channel portion 32 are separated from the microfluidic device 1, in FIG. 4, the wall surface of the microfluidic device 1 corresponding to the location where the channel 20 existed before separation is indicated by the reference symbol 20a.

[0043] When the microfluidic device 1 is separated into the reference portion 31 and the flow path portion 32 and each is measured using temperature programmed desorption mass spectrometry (TDS-MS), the temperature at which the target substance α begins to be released from the reference portion 31 (hereinafter, for convenience, referred to as the "first temperature T1") and the temperature at which the target substance α begins to be released from the flow path portion 32 (hereinafter, for convenience, referred to as the "second temperature T2") are substantially the same.

[0044] An example of a specific method for performing the TDS-MS method will be described with reference to Fig. 5. Fig. 5 is a block diagram showing a schematic example of an apparatus configuration for performing the TDS-MS method. Note that this method is preferably performed using the same apparatus as that used for steps #12 and #14 described below.

[0045] The analytical apparatus 40 illustrated in FIG. 5 includes a reaction tube 41, a heating furnace 42, a vacuum chamber 43, a nude gauge 44, a molecular pump 45, a mass spectrometer 46, and a processing unit 47. A sample consisting of the reference portion 31 or the flow path portion 32 is placed in the reaction tube 41. Then, the temperature inside the reaction tube 41 is increased by the heating furnace 42 while the pressure inside the reaction tube 41 is reduced through the vacuum chamber 43. The gas obtained inside the reaction tube 41 is sent to the mass spectrometer 46, where mass analysis is performed. As a more detailed example, the mass spectrometer 46 can be a quadrupole mass spectrometer. The nude gauge 44 monitors the pressure inside the reaction tube 41. Information related to the analysis results from the mass spectrometer 46, as well as temperature and pressure information inside the reaction tube 41, are input to the processing unit 47. The molecular pump 45 removes background gas (unnecessary gas from the surroundings) in the reaction tube 41 and efficiently supplies gases and volatile components released from the sample to the mass spectrometer .

[0046] The fact that the first temperature T1 and the second temperature T2 are substantially the same means that almost no solvent (target substance α), which is a component of the adhesive layer 3, remains in the flow path portion 32. This point will be described below in conjunction with a method for manufacturing the microfluidic device 1.

[0047] Fig. 6 is a flowchart that schematically illustrates an example of the steps of a method for manufacturing the microfluidic device 1. Fig. 7 is a conceptual diagram that schematically illustrates a part of the example of the steps of a method for manufacturing the microfluidic device 1. In the following description, the step numbers in Fig. 6 will be referenced as appropriate.

[0048] (Step #1) First, a first member 11 and a second member 12 made of resin are prepared. In the example shown in FIG. 7, a groove 20b is formed in a part of the first member 11, and this groove 20b will later be bonded to the second member 12 to form the flow path 20. As described above, the resin material can be one or more selected from the group consisting of COP, COC, PS, PMMA, and PC. A typical example of the resin material is COP. Note that the groove 20b in this specification is not limited to the structure shown in FIG. 7, as long as the member having the groove 20b (the first member 11) can be bonded to the second member 12 to form the flow path 20. As another example, the first member 11 having the groove 20b may be a member obtained by bonding another member to a member having a through hole.

[0049] The first member 11 and the second member 12 can be obtained by, for example, injection molding using a metal mold. This makes it possible to obtain the first member 11 with the grooves 20b formed therein. As another example, the grooves 20b may be formed by cutting a plate-shaped resin member to obtain the first member 11. Although the wells 21 and 22 shown in FIG. 1 are not shown in FIG. 7, the wells 21 and 22 may also be formed by injection molding or by cutting.

[0050] This step #1 corresponds to process (a).

[0051] (Step #2) Next, the first member 11 and the second member 12 are bonded together via the adhesive layer 3. As a specific example of this method, as shown in Fig. 7, after exposing the surface of the second member 12 to the vapor of an organic solvent, the side of the first member 11 on which the recessed grooves 20b are formed is brought into contact with the surface of the second member 12, and pressure is applied while heating as necessary. In Fig. 7, the application of an external force by pressure to the first member 11 and the second member 12 is schematically indicated by the symbol PF.

[0052] By carrying out this step, a flow path 20 is formed at the boundary between the side of the first member 11 where the recessed groove 20b is formed and the second member 12. However, at this point, the target substance α, which is a component of the organic solvent that constitutes the adhesive layer 3, remains on the wall surface of the flow path 20.

[0053] This step #2 corresponds to process (b).

[0054] (Step #3) Next, a removal step is performed to remove the remaining target substance α. This removal step is performed under a preset removal condition Ef. This step #3 corresponds to step (d).

[0055] This removal condition Ef is set according to the procedure shown in the flowchart of Fig. 8. A method for setting the removal condition Ef will be described below. In the following description, the step numbers in Fig. 8 will be referred to as appropriate.

[0056] (Step #11) First, a test member Y1 is prepared. The test member Y1 is made of the same resin material as the first member 11 and the second member 12, and is processed to a size that allows analysis using the TDS-MS method.

[0057] This step #11 corresponds to process (c1).

[0058] (Step #12) Next, the test member Y1 is analyzed using the TDS-MS method. More specifically, using the analysis device 40 described above with reference to Fig. 5, the test member Y1 is placed in the reaction tube 41, and mass spectrometry is performed on the gas in the reaction tube 41 while increasing the temperature inside the reaction tube 41.

[0059] Specifically, first, the temperature inside the reaction tube 41 is raised to a verification temperature, which is a temperature near the glass transition temperature of the test member Y1. Then, mass analysis is performed on the gas inside the reaction tube 41 obtained at this time, and substances derived from the organic solvent used in synthesizing the test member Y1 are identified from substances other than air-derived substances (N2, O2, HO, CO2, etc.). The substance identified in this manner corresponds to the "target substance α." As a more specific example of this method, mass analysis is performed on the gas inside the reaction tube 41 obtained by heating at the verification temperature, and the substance with the highest signal intensity, excluding air-derived substances, may be identified as the target substance α. This step of identifying the target substance α corresponds to process (c1a).

[0060] FIG. 9 shows an example of a mass spectrum obtained by analyzing the gas in the reaction tube 41 using TDS-MS when the test member Y1 is made of a COP resin. In FIG. 9, m / z values ​​corresponding to the toluene molecular ion peak, toluene fragment peak, or toluene isotope peak are indicated by black dots. The results in FIG. 9 indicate that a strong signal derived from toluene was detected in the gas in the reaction tube 41. This suggests that the toluene contained in the organic solvent used to synthesize the test member Y1 separated from the resin and evaporated.

[0061] The following formulas (1) and (2) show typical synthesis routes for COP and COC, which are examples of the main materials for the first component 11 and the second component 12. COP is prepared by ring-opening metathesis polymerization of cycloolefins followed by hydrogenation. In this case, toluene may be used as a solvent or co-solvent for the polymerization reaction. Furthermore, cyclohexane may be used as a solvent for the dehydrogenation reaction of COP. COC is obtained by copolymerization of cycloolefins with ethylene or α-olefins, and in this case, toluene may also be used as a solvent or co-solvent for the polymerization reaction.

[0062] [ka]

[0063] After the target substance α is identified, another test member Y1 is analyzed using the TDS-MS method. That is, the test member Y1 is placed in the reaction tube 41, and mass analysis is performed on the gas inside the reaction tube 41 while the temperature inside the reaction tube 41 is increased at a predetermined temperature increase rate. Then, the temperature at which a signal derived from the target substance α begins to be observed above the noise level in the obtained mass spectrum is identified. This temperature corresponds to the "first temperature T1."

[0064] Fig. 10 is a graph showing the change in the signal intensity of m / z=91, which is a signal derived from toluene, when mass spectrometry is performed on the gas in the reaction tube 41 while increasing the temperature of the test member Y1 made of COP resin. In detail, Fig. 10 shows the change in the signal intensity of m / z=91, which is a signal derived from toluene, when 0.1 g of the test member Y1 is placed in a quartz reaction tube 41 having a diameter of 35 mm and a length of 600 mm, and heated under vacuum (1 x 10 -6 10 is a graph obtained when the reaction tube 41 was heated from 24°C to 115°C at a heating rate of 2°C / min under a pressure of 100 Pa. In Fig. 10, the data group labeled "COP" shows the results obtained from the test member Y1 made of a COP resin.

[0065] 10, the horizontal axis represents the elapsed time, the left vertical axis represents the signal intensity of m / z=91, and the right vertical axis represents the temperature inside the reaction tube 41. The plots in FIG. 10 correspond to the average values ​​obtained when three measurements were performed at approximately the same timing.

[0066] 10, when the temperature of the reaction tube 41 was 86° C., a deviation (significant difference) exceeding the noise level was observed between the signal intensity of m / z=91 of the test member Y1 and the blank. Note that a two-sample equal variance t-test was performed between the blank and the test member Y1, and the first confirmation of a P value of <0.05 may be regarded as indicating the presence of a significant difference.

[0067] That is, when the results shown in FIG. 10 are obtained, the first temperature T1 of the test member Y1 is set to 86°C.

[0068] This step #12 corresponds to process (c2).

[0069] (Step #13) Next, the residual organic solvent is removed from one of the devices obtained through step #2 (referred to as "test device Y2"), with removal condition Ei set as a predetermined initial condition E0. One example of a method for removing the residual organic solvent is to place test device Y2 in a predetermined space and heat it. In this case, the removal conditions can be defined by the heating temperature and heating time. In this case, the initial condition E0 is a condition in which the heating temperature and heating time are defined as predetermined initial values.

[0070] This step #13 corresponds to process (c3).

[0071] (Step #14) Next, the test device Y2 that has undergone step #13 is subjected to analysis using the TDS-MS method in the same manner as step #12. Specifically, the test device Y2 or a small piece of the test device Y2 is placed in the reaction tube 41, and mass analysis is performed on the gas in the reaction tube 41 while the temperature inside the reaction tube 41 is increased at a predetermined heating rate. Then, the temperature at which the signal from the target substance begins to exceed the noise level in the obtained mass spectrum is identified. This temperature corresponds to the "second temperature T2." The method for determining that the noise level has been exceeded for the first time is the same as that for determining the first temperature T1.

[0072] In step #14, when a small piece of test device Y2 is used, the small piece preferably includes the flow path portion 32 described above with reference to Fig. 4. In the latter case, the small piece may be obtained by scraping 100 µm or more from the surface of test device Y2 at a position directly above the flow path portion 32.

[0073] This step #14 corresponds to process (c4).

[0074] (Steps #15, #16, #17) Next, the first temperature T1 obtained in step #12 is compared with the second temperature T2 obtained in step #14. If the second temperature T2 is significantly different from the first temperature T1, that is, if the first temperature T1 and the second temperature T2 are not found to be substantially the same (No in step #15), the removal condition Ei is changed (step #16). In this example, the removal condition Ei executed immediately before was E0, so the removal condition Ei is changed from E0 to E1. Specifically, the heating temperature, heating time, or both for the test device Y2 are changed. Then, steps #13 to #15 are repeatedly executed.

[0075] Then, when it is confirmed that the first temperature T1 obtained in step #12 and the second temperature T2 obtained in step #14 are substantially the same (Yes in step #15), the removal condition Ei executed in the immediately preceding step #13 is set to the removal condition Ef (step #17).

[0076] Whether the first temperature T1 and the second temperature T2 are substantially the same may be determined based on whether the difference between the first temperature T1 and the second temperature T2 is 10% or less of the first temperature T1.

[0077] This step #17 corresponds to process (c5).

[0078] The second temperature T2 being substantially the same as the first temperature T1 means that the amount of target substance α generated by heating the test device Y2 including the adhesive layer 3 is substantially the same as the amount of target substance α generated by heating the test member Y1 before bonding. In other words, the second temperature T2 being substantially the same as the first temperature T1 means that the target substance α remaining in the flow path 20 of the test device Y2 including the adhesive layer 3 has been removed.

[0079] Therefore, by setting the removal condition Ei as the removal condition Ef, under which the second temperature T2 is substantially the same as the first temperature T1, and then removing the residual solvent under this removal condition Ef in step #3 after step #2, it is possible to obtain a microfluidic device 1 in which the organic solvent remaining on the walls of the flow path 20 has been removed as much as possible.

[0080] 4, when microfluidic device 1 is separated into reference portion 31 and channel portion 32 and each is measured by TDS-MS, the temperature at which target substance α starts to be released from reference portion 31 corresponds to first temperature T1, and the temperature at which target substance α starts to be released from channel portion 32 corresponds to second temperature T2. The fact that the temperature at which target substance α starts to be released from reference portion 31 and the temperature at which target substance α starts to be released from channel portion 32 are substantially the same indicates that the organic solvent remaining on the wall surface of channel 20 of microfluidic device 1 has been successfully removed as much as possible.

[0081] FIG. 11 is a graph superimposed on FIG. 10, showing the change in signal intensity of m / z=91, which is a signal derived from toluene, when mass spectrometry was performed on the gas in the reaction tube 41 while increasing the temperature of the test device Y2. In FIG. 11, the data group labeled COP-T shows the results obtained from the test device Y2. Note that COP-T shown in FIG. 11 corresponds to the results obtained when mass spectrometry was performed on the test device Y2 before the removal process in step #13 was performed. Note that, like the data group labeled COP shown in FIG. 10, the data group labeled COP-T also corresponds to the average value of three measurements taken at approximately the same time.

[0082] 11, when mass spectrometry was performed on the test device Y2 before the removal step in step #13, the signal intensity of m / z=91, a signal derived from toluene, was recognized as a significant value when the temperature exceeded 24°C. Comparing this result with the result obtained from the test member Y1 made of COP resin, labeled COP, it can be seen that when the bonding step using the adhesive layer 3 is performed without performing the organic solvent removal step, the organic solvent remaining on the wall surfaces of the flow channel 20 and the like is easily extracted. In other words, when the second temperature T2 is sufficiently lower than the first temperature T1, it can be seen that the organic solvent derived from the adhesive layer 3 remains on the wall surfaces of the flow channel 20 and the like of the microfluidic device 1.

[0083] When the test device Y2 was placed in an air atmosphere under removal conditions Ef, which included a heating temperature of 60°C and a heating time of 24 hours, and then step #14 was performed, the signal intensity of m / z=91, a signal derived from toluene, was recognized as a significant value when the temperature exceeded 84°C. This temperature is substantially the same as the first temperature T1 of the test member Y1, 86°C, described above with reference to FIG. 10. In other words, it can be determined that the microfluidic device 1 obtained after performing step #3 under these removal conditions Ef has had the organic solvent derived from the adhesive layer 3 removed as much as possible from the wall surfaces of the flow channel 20, etc.

[0084] 12 is a graph showing the results of verifying the influence of the organic solvent remaining in the microfluidic device 1 on the cultured cells. Specifically, the results are as follows.

[0085] (Reference example) SH-SY5Y human neuroblastoma cells stained with Annexin V apoptosis marker labeled with Hoechst 33258 fluorescent dye and Alexa 594 fluorescent dye (Annexin V-Alexa 594) were seeded on the surface of a 1 cm x 1 cm COP plate placed in a petri dish and cultured for 3 days in a humidified incubator at 37°C containing 5% (v / v) CO2. The percentage of apoptotic cells was determined from microscopic photographs of cells stained with Annexin V-Alexa 594.

[0086] (Comparative Example) The percentage of apoptotic cells was determined under the same conditions as in the Reference Example, except that the surface of the COP member in the Reference Example was coated with toluene by exposing it to vapor containing toluene, and then cells were seeded after drying. The Comparative Example simulated a situation in which organic solvent derived from the adhesive layer 3 remained on the wall surface of the flow channel 20 in the microfluidic device 1.

[0087] (Example) The percentage of apoptotic cells was determined under the same conditions as in the Reference Example, except that the surface of the COP member of the Reference Example was similarly coated with toluene-containing vapor, dried, and then heated at a high temperature to evaporate the toluene before seeding with cells. The Example simulates the situation in which the organic solvent originating from the adhesive layer 3 remaining on the wall surface of the flow channel 20 is removed from the microfluidic device 1 of the Comparative Example.

[0088] 12, it was confirmed that the Example had an apoptosis rate equivalent to that of the Reference Example. On the other hand, it was confirmed that the Comparative Example had a higher apoptosis rate than the Reference Example and the Example. From these results, it was confirmed that if organic solvent derived from adhesive layer 3 remains on the wall surface of flow channel 20, it affects the cultured cells, and that by performing the removal process related to step #3, it is possible to suppress the effect on the cultured cells.

[0089] In the above embodiment, the case where the target substance is toluene has been described as an example. However, it is understood that when the substance contained in the resin constituting the first member 11 and the second member 12 is the same as the component substance of the organic solvent constituting the adhesive layer 3, the organic solvent derived from the adhesive layer 3 can be removed as much as possible from the wall surface of the flow channel 20, etc., by using a similar manufacturing method.

[0090] FIG. 13 is a graph showing the change in signal intensity at m / z=56, which is a signal derived from cyclohexane, when mass spectrometry was performed on the gas in the reaction tube 41 while increasing the temperature of a test member Y1 made of COP resin and a test device Y2 including a cyclohexane-based adhesive layer 3. In FIG. 13, the data group labeled COP-C shows the results obtained from the test device Y2 including the cyclohexane-based adhesive layer 3. Note that this test device Y2 corresponds to the test device Y2 before the removal process in step #13 was performed. The plots in FIG. 13 correspond to the average values ​​obtained when three measurements were performed at approximately the same time.

[0091] 13, when the temperature of the reaction tube 41 was 84°C, the signal intensity of m / z=56 of the test member Y1 was found to deviate (significantly differ) from the blank by more than the noise level. On the other hand, when mass spectrometry was performed on the test device Y2 before the removal process in step #13, the signal intensity of m / z=56, which is a signal derived from cyclohexane, was found to be significant when the temperature exceeded 45°C. In other words, this result shows that, similar to the case of toluene described above with reference to FIG. 11, if the organic solvent removal process is not performed after the bonding process using the adhesive layer 3, the organic solvent remaining on the wall surface of the flow channel 20, etc., is easily extracted.

[0092] Then, in step #3, the organic solvent is removed under removal condition Ef, thereby removing the target substance α (here, cyclohexane) derived from the organic solvent. Therefore, the temperature (second temperature T2) at which the target substance α starts to be released from the channel portion 32 of the microfluidic device 1 obtained after this step is substantially equal to 84°C, and this temperature corresponds to the temperature at which the target substance α starts to be released from the reference portion 31 of the microfluidic device 1.

[0093] [Another embodiment] Another embodiment of the microfluidic device 1 will now be described.

[0094] <1> In the microfluidic device 1, the shapes and arrangements of the wells 21 and 22 and the number of the channels 20 are not limited.

[0095] <2> In the above embodiment, the microfluidic device 1 is configured by bonding together the first member 11 and the second member 12. However, in the microfluidic device 1, the number of members bonded together is not limited to two, and the microfluidic device 1 may be configured by bonding together three or more members.

[0096] <3> In the above embodiment, the groove 20b is formed in the first member 11, and the flat plate-shaped second member 12 and the first member 11 are bonded together to obtain the microfluidic device 1. However, the groove 20b may be formed in both the first member 11 and the second member 12.

[0097] <4> The present invention is not limited to the above-described embodiments and includes various modifications. For example, the above-described embodiments have been described in detail to provide a better understanding of the present invention, and the present invention is not necessarily limited to those having all of the configurations described. The scope of the present invention is defined by the claims, and it is intended to include all modifications within the meaning and scope of the claims. [Explanation of symbols]

[0098] 1: Microfluidic device 3: Adhesive layer 11: First member 12: Second member 20: Flow path 20a: Wall of the flow channel 20b: Concave groove 21: Well 22: Well 31:Reference part 32: Flow path part 40: Analyzer 41: Reaction tube 42:Heating furnace 43: Vacuum chamber 44: Nude Gauge 45: Molecular pump 46:Mass spectrometer 47: Processing unit

Claims

1. a first member made of resin; a second member made of resin; an adhesive layer that bonds the interface between the first member and the second member; a flow path formed in a part of a boundary between the first member and the second member, the adhesive layer contains a target substance that is a component of an organic solvent; A microfluidic device characterized in that, when components contained in a reference portion located in an area spaced from the flow path and the adhesive layer, and a flow path portion located in an area where the flow path is formed, are measured by thermal desorption mass spectrometry, a first temperature at which the target substance begins to be released from the reference portion and a second temperature at which the target substance begins to be released from the flow path portion are substantially identical.

2. The microfluidic device according to claim 1, wherein the main material of the first member and the second member is one or more resin materials selected from the group consisting of COP, COC, PS, PMMA, and PC.

3. 2. The microfluidic device according to claim 1, wherein the target substance is a substance selected from the group consisting of toluene, cyclohexane, dichloromethane, tetrahydrofuran, chloroform, ethyl acetate, methyl ethyl ketone, acetone, and dimethylformamide.

4. a main material of the first member and the second member is COP; the target substance is toluene, The microfluidic device of claim 1 , wherein the first temperature and the second temperature are both higher than 75° C.

5. 1. A method for manufacturing a microfluidic device, comprising: A step (a) of preparing a first member whose main material is a resin material and whose groove is formed in a part thereof, and a second member whose main material is the resin material; a step (b) of bonding the first member and the second member together with an adhesive containing an organic solvent in a state where the side of the first member on which the grooves are formed faces the second member, thereby obtaining a device including a flow path formed by the grooves; (c) determining removal conditions for removing the organic solvent from the device; and (d) subjecting the device to removal of the organic solvent under the removal conditions; The step (c) a step (c1) of preparing a test member made of the resin material; a step (c2) of measuring a first temperature at which the target substance, which is a component of the organic solvent, starts to be released from the test member using thermal desorption mass spectrometry; (c3) removing the organic solvent from at least one test device obtained in the step (b); After the step (c3), a step (c4) is performed on the test device, using thermal desorption mass spectrometry to measure a second temperature at which the release of the target substance begins; and performing steps (c3) and (c4) under different conditions when performing step (c3), thereby determining conditions for step (c3) under which the second temperature is substantially the same as the first temperature, and setting the conditions as the removal conditions.

6. the step (c3) and the step (d) include a step of heating the device in a state where the device is placed in a predetermined space, The method for manufacturing a microfluidic device according to claim 5 , wherein the removal conditions are defined by information including the temperature of the space in which the device is placed and the time for which the device is placed in the space.

7. 6. The method for manufacturing a microfluidic device according to claim 5, wherein the step (c) further comprises, prior to the step (c2), a step (c1a) of setting the substance that has the highest signal intensity, excluding substances derived from air, as the target substance when the test member is heated to a verification temperature near the glass transition temperature using thermal desorption mass spectrometry.

8. 8. The method for manufacturing a microfluidic device according to claim 5, wherein the resin material is at least one selected from the group consisting of COP, COC, PS, PMMA, and PC.

9. 8. The method for manufacturing a microfluidic device according to claim 5, wherein the target substance is a substance selected from the group consisting of toluene, cyclohexane, dichloromethane, tetrahydrofuran, chloroform, ethyl acetate, methyl ethyl ketone, acetone, and dimethylformamide.

10. the resin material is COP, the target substance is toluene, The method for manufacturing a microfluidic device according to any one of claims 5 to 7, wherein the step (c5) is characterized in that the removal conditions are set to be the conditions of the step (c3) in which the first temperature and the second temperature are both higher than 75°C.

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