JP2019004839A - Micro-fluid device and reaction system - Google Patents

Abstract

To provide a micro-fluid device in which production of bubbles in a reaction chamber hardly occurs.SOLUTION: A micro-fluid device 1 in which a plurality of micro-channels are provided in a micro-channel chip 10, comprises: the micro-channel chip 10 and a temperature regulator 24 provided on a first principal surface 10a side of the micro-channel chip 10. The micro-channel has a main channel 12 and a plurality of branched channels 15 to 17 branched from the main channel 12. The main channel 12 has first and second valves 13, 14 provided on both ends of the main channel 12, a connection channel 20, an inlet-side channel 21, and a channel for waste liquid 22. At least one branched channel out of the plurality of branched channels 15 to 17 is a reaction chamber, and the inlet-side channel 21 and the reaction chamber are provided on the first principal surface 10a side of the micro-channel chip 10.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a microfluidic device provided with a microchannel through which a fluid is fed and a reaction system including the microfluidic device.

  Conventionally, various microfluidic devices provided with a microchannel through which a fluid is fed have been proposed for biochemical analysis and the like. For example, Patent Document 1 below discloses a microfluidic device provided with a microchannel. The micro channel has a main channel and a plurality of branch channels branched from the main channel. The plurality of branch channels are individually connected to the reaction chamber for each branch channel, and a valve is provided for each reaction chamber. In Patent Document 1, a gene amplification reaction or the like is performed in a reaction chamber.

Special table 2014-521306 gazette

  When a valve is individually provided for each of a plurality of reaction chambers as in Patent Document 1, there is a problem that the number of parts increases.

  In addition, the present inventors have found that the following problems arise when valves are provided at both ends of the main flow path and a plurality of reaction chambers are sealed together. Specifically, when a reaction that requires heating and cooling of the reaction solution in the reaction chamber, such as a gene amplification reaction, is performed, the amount of the reaction solution in the reaction chamber decreases or It has been found that bubbles may occur. In particular, when a gene amplification reaction or the like is detected from the outside by a detector, the presence of air bubbles makes it impossible to accurately detect the degree of reaction and the substance produced by the reaction.

  An object of the present invention is to provide a microfluidic device and a reaction system including the microfluidic device, in which bubbles are hardly generated in the reaction chamber.

  The microfluidic device according to the present invention is a microfluidic device in which a plurality of microchannels are provided in a microchannel chip, the microfluidic device having first and second main surfaces facing each other. A chip, and a temperature control device provided on the first main surface side of the microchannel chip, wherein the microchannel includes a main channel and a plurality of branches from the main channel A first and second valves provided at both ends of the main flow path, a connection flow path connected to the first valve, and the connection flow path. An inlet side channel connected to one end of the plurality of branch channels, and a waste liquid channel connecting the inlet side channel and the second valve, At least one branch channel among the plurality of branch channels is a reaction chamber, Fill port side flow passage and said reaction chamber is provided on the first main surface side of the micro-channel chip.

  On the specific situation with the microfluidic device which concerns on this invention, the said temperature control apparatus is provided on the said 1st main surface of the said microchannel chip.

  In another specific aspect of the microfluidic device according to the present invention, the connection channel and the waste liquid channel are provided on the first main surface side of the microchannel chip.

  In still another specific aspect of the microfluidic device according to the present invention, the microchannel chip includes a substrate and a cover layer provided on the substrate, the inlet-side channel and the reaction chamber, The temperature control device is separated by the cover layer.

  In still another specific aspect of the microfluidic device according to the present invention, all the branch channels among the plurality of branch channels are each a reaction chamber.

  In still another specific aspect of the microfluidic device according to the present invention, the reaction chamber is filled with a reaction solution.

  In still another specific aspect of the microfluidic device according to the present invention, the microchannel further includes an outlet-side channel connected to the other ends of the plurality of branch channels, and the outlet-side channel Is provided on the first main surface side of the microchip.

  The reaction system according to the present invention comprises a microfluidic device constructed according to the present invention.

  In a specific aspect of the reaction system according to the present invention, the reaction performed in the reaction chamber is a gene amplification reaction.

  In another specific aspect of the reaction system according to the present invention, the reaction system further includes a detector for detecting the amplification reaction of the gene.

  According to the present invention, it is possible to provide a microfluidic device in which bubbles are hardly generated in the reaction chamber.

1 is a perspective view showing an appearance of a microfluidic device according to a first embodiment of the present invention. It is a schematic plan view for demonstrating the microchannel of the microfluidic device which concerns on the 1st Embodiment of this invention. It is typical sectional drawing of the part which follows the AA line in FIG. It is typical sectional drawing of the part which follows the BB line in FIG. It is typical sectional drawing for demonstrating the reaction system as the 2nd Embodiment of this invention. 3 is a schematic cross-sectional view for explaining a reaction system of Comparative Example 1. FIG.

  Hereinafter, the present invention will be clarified by describing specific embodiments of the present invention with reference to the drawings.

  It should be pointed out that each embodiment described in this specification is an exemplification, and a partial replacement or combination of configurations is possible between different embodiments.

  FIG. 1 is a perspective view showing the appearance of the microfluidic device according to the first embodiment of the present invention. FIG. 2 is a schematic plan view for explaining a microchannel of the microfluidic device according to the first embodiment of the present invention. 3 is a schematic cross-sectional view of a portion along the line AA in FIG. 2, and FIG. 4 is a schematic cross-sectional view of a portion along the line BB in FIG.

  As shown in FIG. 3, the microfluidic device 1 includes a microchannel chip 10 and a temperature control device 24. The microchannel chip 10 has first and second main surfaces 10a and 10b facing each other. A temperature control device 24 is provided on the first main surface 10 a of the microchannel chip 10.

  As shown in FIG. 1, the microchannel chip 10 is not particularly limited, but has a plate-like substrate 2 in the present embodiment. The plate-like substrate 2 includes a substrate body 3, a first cover layer 4 provided so as to cover the upper surface of the substrate body 3, and a second cover layer 5 stacked on the lower surface side of the substrate body 3. . The first cover layer 4 is provided on the second main surface 10b side. The second cover layer 5 is provided on the first main surface 10a side. The laminated structure is not particularly limited.

  In the plate-like substrate 2, a micro flow path 11 shown in a plan view in FIG. 2 is provided. The micro flow path 11 refers to a fine flow path that causes a micro effect when a fluid is conveyed. In such a microchannel 11, the fluid is strongly influenced by the surface tension and behaves differently from the fluid flowing through a normal large-sized channel.

  The cross-sectional shape and size of the microchannel 11 are not particularly limited as long as the microchannel 11 generates the above micro effect. For example, when using a pump or gravity when flowing a fluid through the microchannel 11, the cross-sectional shape of the microchannel 11 is generally rectangular (including a square) from the viewpoint of further reducing the channel resistance. The dimension of the smaller side is preferably 20 μm or more, more preferably 50 μm or more, and even more preferably 100 μm or more. Further, from the viewpoint of further downsizing the microfluidic device 1, it is preferably 5 mm or less, more preferably 1 mm or less, and further preferably 500 μm or less.

  Moreover, when the cross-sectional shape of the microchannel 11 is almost circular, the diameter (in the case of an ellipse, the short diameter) is preferably 20 μm or more, more preferably 50 μm or more, and even more preferably 100 μm or more. From the viewpoint of further miniaturizing the microfluidic device 1, the diameter (in the case of an ellipse, the short diameter) is preferably 5 mm or less, more preferably 1 mm or less, and even more preferably 500 μm or less.

  On the other hand, for example, when flowing a fluid through the microchannel 11, when the capillary phenomenon is used more effectively, if the cross-sectional shape of the microchannel 11 is generally rectangular (including a square), the smaller one is used. The side dimension is preferably 5 μm or more, more preferably 10 μm or more, and further preferably 20 μm or more. The smaller side dimension is preferably 200 μm or less, and more preferably 100 μm or less.

  As shown in FIG. 2, the microchannel 11 has a main channel 12. A plurality of branch portions 12 a to 12 c are provided in the main channel 12. Branch flow paths 15 to 17 are connected to the branch portions 12a to 12c, respectively. The branch flow channels 15 to 17 have branch flow channel main body portions 15a to 17a connected to the branch portions 12a to 12c. Branch flow channel narrowing portions 15b to 17b are connected to downstream end portions of the branch flow channel main body portions 15a to 17a. Branch flow channel expanding portions 15c to 17c are connected to downstream end portions of the branch flow channel narrowing portions 15b to 17b. The downstream end portions of the branch flow channel expanding portions 15 c to 17 c are connected to the outlet side flow channel 18. A bypass channel 19 is provided so as to connect the main channel 12 and the outlet side channel 18.

  The branch flow path 15 constitutes the reaction chamber 23 shown in FIG. Similarly, the branch channels 16 and 17 also constitute a reaction chamber 23 (not shown). In the present invention, it is only necessary that at least one branch channel among the plurality of branch channels 15 to 17 constitutes the reaction chamber 23. Each reaction chamber 23 is filled with a reaction solution. The amount of the reaction solution in one reaction chamber 23 can be set to 1 μL or more and 100 μL or less, for example.

  The microfluidic device 1 is used to detect a reaction by a gene amplification reaction (PCR) in the reaction chamber 23. However, the use of the microfluidic device 1 is not particularly limited.

  Next, the structure of the main channel 12 will be described.

  As shown in FIG. 3, the main flow path 12 includes a connection flow path 20, an inlet-side flow path 21, and a waste liquid flow path 22. The connection flow path 20 is a flow path provided between the first valve 13 and the inlet-side flow path 21. The inlet channel 21 is a channel that connects the connection channel 20 and the waste fluid channel 22. The inlet side channel 21 is connected to the branch channels 15 to 17. Further, the waste liquid channel 22 is a channel provided between the inlet-side channel 21 and the second valve 14. In FIG. 3, the boundary lines of the inlet-side flow path 21, the connection flow path 20, and the waste liquid flow path 22 are indicated by broken lines. The width and depth of the inlet-side channel 21 can be set to, for example, 0.1 mm or more and 1.0 mm or less, respectively.

  In the present embodiment, the first and second valves 13 and 14 are provided so as to collectively seal the reaction solutions filled in the reaction chambers 23 provided in the branch flow paths 15 to 17. ing. The first and second valves 13 and 14 are valves that open and close the flow path by pressing the first cover layer 4 with the first and second pressing members 13a and 14a, respectively. Therefore, in the present embodiment, the first cover layer 4 is desirably made of a flexible material. Examples of the material having flexibility include synthetic resin films and elastomers. In the present invention, the first and second valves 13 and 14 are not particularly limited, and may be valves having a valve structure that can be optically switched between an open state and a closed state.

  In this embodiment, since it has the above valve structure, the 1st and 2nd valves 13 and 14 are provided in the 1st cover layer 4 side. On the other hand, the inlet-side flow path 21 is provided on the second cover layer 5 side. In particular, in the present embodiment, the inlet-side flow path 21 is provided so as to be in contact with the second cover layer 5. Therefore, the connection flow path 20 and the waste liquid flow path 22 that connect the first and second valves 13 and 14 and the inlet-side flow path 21 respectively extend in the thickness direction of the substrate 2.

  In the present embodiment, since the PCR reaction is performed in the reaction chamber 23, the temperature adjustment device 24 is disposed on the first main surface 10a side of the microchannel chip 10. The temperature adjusting device 24 can be configured by an appropriate temperature adjusting device that can repeat heating and cooling. Further, the temperature adjusting device 24 does not necessarily need to be in contact with the first main surface 10a of the microchannel chip 10 and may be separated. However, preferably, the temperature control device 24 is provided so as to be in contact with the first main surface 10 a of the microchannel chip 10. In that case, generation | occurrence | production of the bubble mentioned later can be made still more difficult to produce.

  The material constituting the microfluidic device 1 is not particularly limited. For example, the substrate body 3, the first cover layer 4 and the second cover layer 5 constituting the substrate 2 may have a laminated structure of synthetic resin moldings. Alternatively, the substrate body 3 may be an injection molded product, and the first cover layer 4 and the second cover layer 5 may be a synthetic resin film attached to the upper surface or the lower surface of the injection molded product.

  When the reaction in the reaction chamber 23 is optically detected from the outside by a detector, it is desirable that the upper part and / or the lower part of the reaction chamber 23 is translucent.

  As shown in FIGS. 3 and 4, in this embodiment, an inlet-side flow path 21 and a reaction chamber 23 are provided on the upper surface of the second cover layer 5, and temperature adjustment is performed on the lower surface of the second cover layer 5. A device 24 is provided. That is, the inlet-side flow path 21 and the reaction chamber 23 are separated from the temperature adjustment device 24 only through the second cover layer 5. In the present embodiment, all portions of the inlet-side flow path 21 and the reaction chamber 23 are provided on the temperature adjusting device 24 side.

  In the present embodiment, since the inlet side flow path 21 and the reaction chamber 23 are provided on the temperature adjusting device 24 side, bubbles are hardly generated in the reaction chamber 23. This can be explained as follows.

  In the case where the first valve 13 and the second valve 14 are provided at both ends of the main flow path 12 and the reaction liquid is sealed together in the plurality of reaction chambers 23, the inlet-side flow path connecting the plurality of reaction chambers 23. An empty space may occur in 21. Therefore, when a reaction that requires heating and cooling of the reaction solution in the reaction chamber 23 is performed, such as a gene amplification reaction, the reaction solution expands when heated, and the inlet-side flow path that has the empty space when cooled. The inner wall of 21 may condense. As a result, the amount of the reaction solution in the reaction chamber 23 may decrease or bubbles may be generated in the reaction chamber 23.

  However, in this embodiment, in addition to the reaction chamber 23, the inlet-side flow path 21 is provided on the temperature adjusting device 24 side. Therefore, since the inlet-side flow path 21 can also be heated, even if the reaction liquid expands during heating, the reaction liquid can be returned to the reaction chamber 23 during cooling. As a result, the inner wall of the inlet-side flow path 21 in which the reaction solution has the empty space is hardly condensed during cooling, and the generation of bubbles can be suppressed. In addition, it is possible to make it difficult for contamination to occur in the reaction chamber 23. Therefore, in the microfluidic device 1, when a gene amplification reaction or the like is detected from the outside by a detector, the detection accuracy can be increased.

  In the present embodiment, as described above, the first and second valves 13 and 14 are provided at both ends of the main flow path 12, and the reaction liquid is sealed together in the plurality of reaction chambers 23. Generation of bubbles can be suppressed. Therefore, it is not necessary to provide a valve individually for each of the plurality of reaction chambers 23, and the number of parts can be reduced.

  In the present invention, the inlet side flow path 21 and the reaction chamber 23 may be provided on the temperature adjusting device 24 side, and between the inlet side flow path 21 and the reaction chamber 23 and the temperature adjusting device 24, Other layers other than the second cover layer 5 may be provided. Further, the inlet-side flow path 21 and the reaction chamber 23 do not have to be in contact with the second cover layer 5. However, it is preferable that the inlet-side flow path 21, the reaction chamber 23, and the temperature control device 24 are separated only by the second cover layer 5 from the viewpoint of more reliably suppressing the generation of bubbles. In this case, the thickness of the second cover layer 5 is preferably 300 μm or less. Thereby, generation of bubbles can be made even more difficult.

  In the present invention, it is preferable that the outlet side channel 18 and the bypass channel 19 are also provided on the temperature control device 24 side. In that case, generation | occurrence | production of a bubble can be suppressed still more reliably. But the exit side flow path 18 and the bypass flow path 19 do not need to be provided in the temperature control apparatus 24 side.

  In the present invention, the connection flow path 20 and the waste liquid flow path 22 may not be provided. That is, the first and second valves 13 and 14 and the inlet-side flow path 21 may be directly connected on the same plane. Even in that case, the inlet side flow path 21 and the reaction chamber 23 should just be provided in the temperature control apparatus 24 side.

  In addition, fluid feeding in the microchannel 11 can be performed by a liquid feeding means provided inside or outside the microchannel chip 10. The liquid feeding means is not particularly limited, and examples thereof include a micropump. Specifically, there is a means for sending liquid by sending liquid, air, or a predetermined gas into the micro flow path 11 using a micro pump. In this case, the micropump may be provided inside the microchannel chip 10 or may be provided outside the microchannel chip 10.

  Further, as another liquid feeding means, a gas generating member disposed in a space connected to the upstream side from the first valve 13 may be mentioned. The gas generating member is a member that generates gas by an external force such as light or heat. A gas can be generated by applying an external force to the gas generating member at a predetermined timing, and the gas can be sent into the microchannel 11. Thereby, the fluid can be fed. Examples of the gas generating member include a gas generating tape.

[Reaction system]
FIG. 5 is a cross-sectional view showing a reaction system as a second embodiment having the microfluidic device 1. The reaction system 31 includes the microfluidic device 1, the temperature adjustment device 24, and a detector 32.

  The detector 32 is provided for detecting the PCR reaction in the reaction chamber 23 described above. The detector 32 can be constituted by a detection device based on various measurement principles such as an optical detection device.

  The detector 32 is preferably one that can measure the PCR reaction in the reaction chamber 23 in a non-contact manner. Therefore, in this embodiment, the detector 32 is provided separately from the microfluidic device 1.

  However, the detector 32 may be in contact with the surface on the first cover layer 4 side or the surface on the second cover layer 5 side of the microfluidic device 1.

  In addition, the use of the microfluidic device 1 and the reaction system 31 is not limited to the use of detecting nucleic acid or the like using a PCR reaction. However, in the PCR reaction, the gene is amplified by repeating the steps of heating and cooling the reaction solution. Therefore, by repeating such heating and cooling, dissolved oxygen or the like in the reaction solution may be generated as bubbles. Therefore, the present invention is effective when used for reactions in which such bubbles are likely to occur.

  Next, the operation of the detection method using the reaction system 31 will be described.

  First, a micropump (not shown) provided on the upstream side of the first valve 13 is driven as liquid feeding means in the microfluidic device 1. Then, the reaction liquid as the fluid supplied from the upstream side of the main flow path 12 is sent from the main flow path 12 to the branch flow paths 15 to 17. In this case, the first and second valves 13 and 14 are open. In this case, the branch flow paths 15 to 17 are filled with the reaction liquid at substantially the same timing. In this state, the first and second valves 13 and 14 are closed. Thereby, the reaction solution is confined in the reaction chamber 23. Preferably, the volumes of the first and second valves 13 and 14 and the reaction chamber 23 are determined so that the reaction solution occupies 97% of the volume of the reaction chamber 23 in this state. In that case, bubbles are less likely to be generated. Therefore, even if heating and cooling are repeated, the detection accuracy is more unlikely to deteriorate.

  Preferably, a push-type liquid feeding means for pressing the reaction liquid is desirable as the liquid feeding means. Thereby, bubbles are less likely to be generated in the reaction chamber 23.

  As described above, the first and second valves 13 and 14 are closed, and in this state, heating and cooling are repeated using the temperature controller 24 to advance the PCR reaction. The set temperature is not particularly limited, and a high temperature state and a low temperature state necessary for the PCR reaction may be repeated.

  Next, the concentration of the nucleic acid amplified in each reaction chamber 23 is optically detected using the detector 32. In this case, since it is difficult for bubbles to be generated in each reaction chamber 23, nucleic acid, DNA, and the like can be detected with high accuracy.

  Hereinafter, the present invention will be clarified by giving specific examples and comparative examples of the present invention. In addition, this invention is not limited to a following example.

Example 1
In Example 1, the reaction system 31 shown in FIG. 5 was produced.

  Specifically, the micro flow path is made of a synthetic resin film in which the substrate body 3 is made of an injection molded product, and the first cover layer 4 and the second cover layer 5 are attached to the upper surface or the lower surface of the injection molded product. The chip 10 was manufactured, and the temperature adjusting device 24 was provided on the lower surface of the second cover layer 5. Further, the detector 32 is disposed above the first cover layer 4 so as to be separated from the first cover layer 4.

  Next, a micropump (not shown) provided on the upstream side of the first valve 13 as the liquid feeding means in the microfluidic device 1 was driven. And the reaction liquid as a fluid supplied from the upstream of the main flow path 12 was sent from the main flow path 12 to the branch flow paths 15-17. Thereby, the branch flow paths 15 to 17 were filled with the reaction liquid at substantially the same timing. In this state, the first and second valves 13 and 14 were closed.

  The first and second valves 13 and 14 were closed, and in this state, heating and cooling were repeated using the temperature control device 24 to perform a PCR reaction. As a result, in Example 1, almost no bubbles were generated in the reaction chamber 23.

(Comparative Example 1)
In Comparative Example 1, the reaction system 101 shown in FIG. 6 was produced.

  Specifically, in Example 1, the inlet-side flow path 21 and the reaction chamber 23 were provided on the temperature adjusting device 24 side, whereas in Comparative Example 1, a part of the inlet-side flow path 21 was It is provided not on the adjusting device 24 side (first main surface 10a side) but on the second main surface 10b side. In other respects, a reaction system 101 was prepared in the same manner as in Example 1, and a PCR reaction was performed. As a result, in Comparative Example 1, many bubbles were generated in the reaction chamber 23.

DESCRIPTION OF SYMBOLS 1 ... Microfluidic device 2 ... Board | substrate 3 ... Board | substrate main body 4,5 ... 1st, 2nd cover layer 10 ... Microchannel chip 10a, 10b ... 1st, 2nd main surface 11 ... Microchannel 12 ... Mainstream Paths 12a to 12c ... Branch portions 13, 14 ... First and second valves 13a, 14a ... First and second pressing members 15-17 ... Branch channels 15a, 16a, 17a ... Branch channel main body 15b, 16b, 17b ... Branch channel narrowing portions 15c, 16c, 17c ... Branch channel enlarged portion 18 ... Outlet channel 19 ... Bypass channel 20 ... Connection channel 21 ... Inlet channel 22 ... Waste liquid channel 23 ... Reaction chamber 24 ... Temperature control device 31 ... Reaction system 32 ... Detector

Claims (10)

A microfluidic device in which a plurality of microchannels are provided in a microchannel chip,
The microchannel chip having first and second main surfaces facing each other;
A temperature control device provided on the first main surface side of the microchannel chip;
With
The microchannel has a main channel and a plurality of branch channels branched from the main channel,
The main flow path is connected to the first and second valves provided at both ends of the main flow path, a connection flow path connected to the first valve, and the plurality of the connection flow paths. An inlet side channel connected to one end of the branch channel, and a waste liquid channel connecting the inlet side channel and the second valve;
At least one branch channel among the plurality of branch channels is a reaction chamber,
The microfluidic device, wherein the inlet channel and the reaction chamber are provided on a first main surface side of the microchannel chip.   The microfluidic device according to claim 1, wherein the temperature adjusting device is provided on the first main surface of the microchannel chip.   3. The microfluidic device according to claim 1, wherein the connection flow path and the waste liquid flow path are provided on the first main surface side of the micro flow path chip. The microchannel chip comprises a substrate and a cover layer provided on the substrate,
The microfluidic device according to any one of claims 1 to 3, wherein the inlet-side channel and the reaction chamber are separated from the temperature adjusting device via the cover layer.   The microfluidic device according to any one of claims 1 to 4, wherein all of the plurality of branch channels are reaction chambers.   The microfluidic device according to claim 1, wherein the reaction chamber is filled with a reaction solution.   The micro-channel further includes an outlet-side channel connected to the other ends of the plurality of branch channels, and the outlet-side channel is provided on the first main surface side of the microchip. The microfluidic device according to any one of claims 1 to 6.   A reaction system comprising the microfluidic device according to claim 1.   The reaction system according to claim 8, wherein the reaction performed in the reaction chamber is a gene amplification reaction.   The reaction system according to claim 9, further comprising a detector that detects an amplification reaction of the gene.

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