Sample solution separation device, sample solution separation system, and sample solution separation method

Abstract

The present invention provides a sample solution separation device with a small dead volume. A sample solution separation device (101) comprises: a plurality of microchambers (105); a flow path (104) that connects the plurality of microchambers (105); a first opening (102) that is an inlet to the flow path (104) of a sample solution; a valve (103) that is provided between the first opening (102) and the plurality of microchambers (105); a second opening (107) that is an inlet to the flow path of a separation liquid separating between the plurality of microchambers (105) in which the sample solution is accommodated, and is provided on the opposite side of the first opening with the plurality of microchambers (105) in between; and a solid phase (106) provided between the second opening (107) and the plurality of microchambers (105). The solid phase (106) is air-permeable, hydrophobic to the sample solution, and permeable to the separation liquid.

Description

Technical Field

[0001] The present invention relates to a sample solution separation device, a sample solution separation system, and a sample solution separation method for separating a sample solution.

Background Art

[0002] Digital PCR (Polymerase Chain Reaction) is a technique for highly sensitive detection of nucleic acids, and can detect low-frequency gene mutations compared to conventional real-time quantitative PCR.

[0003] In digital PCR, after dividing a sample solution into minute volumes, PCR is performed within the divided solutions to amplify and measure the DNA to be measured. As a method of placing the sample solution in minute containers, Patent Document 1 discloses a method of sealing a port to evacuate the inside of the container and drawing the sample solution into the container. Further, Non-Patent Document 1 discloses a method of degassing the inside of the container, closing the valve on the outlet side, and opening the valve on the inlet side to introduce the solution. Patent Document 2 discloses a configuration for discharging the solution without waste using a gas-liquid separation filter.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

[0006] In digital PCR, a sample solution containing the target DNA is separated into multiple compartments, and PCR is performed on each compartment to identify the type of DNA present in each compartment. A key feature of digital PCR is that it allows for highly sensitive measurement of the target DNA by performing PCR after separation into each compartment. For example, liquid biopsy to detect cell-free DNA (cfDNA) present in trace amounts in blood is one suitable application. Cell-free DNA may contain ctDNA (circlating tumor DNA), which is tumor-derived DNA, and measuring the type and proportion of ctDNA mutations is expected to have applications in cancer diagnosis, treatment selection, and monitoring of treatment effectiveness.

[0007] Because cfDNA and ctDNA are present in trace amounts in the blood, they need to be measured with high sensitivity. In digital PCR, the sample solution is separated into multiple small volume chambers for measurement. However, not all of the sample solution can be used; some solution cannot be separated into the small volume chambers and therefore cannot be measured, resulting in a dead volume. To measure with high sensitivity, it is necessary to measure as much of the collected sample solution as possible. In other words, it is necessary to reduce the dead volume of the sample solution.

[0008] The present invention has been made in view of the above, and one of its objectives is to provide a sample solution separation device, a sample solution separation system, and a sample solution separation method that can reduce the dead volume of the sample solution. [Means for solving the problem]

[0009] To solve the above problems, the present invention provides a sample solution separation device for separating a sample solution, comprising: a plurality of microchambers; a flow path connecting the plurality of microchambers; a first opening which is an inlet for the sample solution into the flow path; a valve provided between the first opening and the plurality of microchambers; a second opening which is an inlet for the separation liquid into the flow path separating the plurality of microchambers containing the sample solution, provided on the opposite side of the first opening across the plurality of microchambers; and a solid phase provided between the second opening and the plurality of microchambers, wherein the solid phase is permeable, water-repellent to the sample solution, and permeable to the separation liquid.

[0010] Furthermore, the sample solution separation system of the present invention is a sample solution separation device for separating a sample solution, comprising: a plurality of microchambers; a flow path connecting the plurality of microchambers; a first opening which is an inlet for the sample solution into the flow path; a valve provided between the first opening and the plurality of microchambers; a second opening which is an inlet for the separation liquid into the flow path separating the plurality of microchambers containing the sample solution, provided on the opposite side of the first opening across the plurality of microchambers; and a solid phase provided between the second opening and the plurality of microchambers, wherein the solid phase is permeable, water-repellent to the sample solution, and permeable to the separation liquid; a pump for degassing air in the plurality of microchambers and flow path through the solid phase from the second opening; a valve control means for opening the valve; and a separation liquid introduction means for introducing the separation liquid from the second opening.

[0011] Furthermore, the present invention provides a sample solution separation device for separating a sample solution, comprising: preparing a sample solution separation device comprising: a plurality of microchambers; a flow path connecting the plurality of microchambers; a first opening which is an inlet for the sample solution into the flow path; a valve provided between the first opening and the plurality of microchambers; a second opening which is an inlet for the separation liquid into the flow path separating the plurality of microchambers containing the sample solution, provided on the opposite side of the first opening across the plurality of microchambers; and a solid phase provided between the second opening and the plurality of microchambers, wherein the solid phase is permeable, water-repellent to the sample solution, and permeable to the separation liquid; degassing the air in the plurality of microchambers and flow path via the solid phase; opening the valve and introducing the sample solution into the plurality of microchambers and flow path from the first opening; and introducing the separation liquid into the flow path via the solid phase from the second opening. [Effects of the Invention]

[0012] According to the sample solution separation device, sample solution separation system, and sample solution separation method of the present invention, it is possible to reduce the dead volume of the sample solution when separating the sample solution into multiple microchambers. Other challenges and novel features will become apparent from the description and accompanying drawings in this specification. [Brief explanation of the drawing]

[0013] [Figure 1] This is a diagram showing the configuration of the sample solution separation device 101 according to Example 1. [Figure 2A] This figure shows how the sample solution 201 and the separation liquid 202 are introduced into the sample solution separation device 101 according to Example 1. [Figure 2B] This figure shows how the sample solution 201 and the separation liquid 202 are introduced into the sample solution separation device 101 according to Example 1. [Figure 2C]It is a diagram showing a state in which a sample solution 201 and a separation liquid 202 are introduced into a sample solution separation device 101 according to Example 1. [Figure 2D] It is a diagram showing a state in which a sample solution 201 and a separation liquid 202 are introduced into a sample solution separation device 101 according to Example 1. [Figure 3] It is a flow diagram when a sample solution 201 is separated and introduced into a microchamber 105 by introducing the sample solution 201 and the separation liquid 202 into the sample solution separation device 101 according to Example 1. [Figure 4] It is a configuration diagram of a sample solution separation system 400 that separates and introduces a sample solution 201 into a sample solution separation device 101 according to Example 1. [Figure 5] It is a configuration diagram of a sample solution separation device 501 according to Example 2. [Figure 6A] It is a top view of a sample solution separation device 601 according to Example 3. [Figure 6B] It is a side view of a sample solution separation device 601 according to Example 3. [Figure 7] It is a configuration diagram of a sample solution separation system 700 that separates and introduces a sample solution 201 into a sample solution separation device 601 according to Example 3. [Figure 8] It is a configuration diagram of a sample solution separation device 801 according to Example 4. [Figure 9] It is a configuration diagram of a digital PCR system 900 according to Example 5. [Figure 10] It is an operation flow diagram of a digital PCR system 900 according to Example 5.

Modes for Carrying Out the Invention

[0014] In the following embodiments, the description will be divided into multiple sections or embodiments where necessary for convenience. Unless otherwise specified, these are not unrelated, and one may be a modification, detail, or supplementary explanation of part or all of the other. Furthermore, in the following embodiments, when referring to the number of elements (including number, numerical value, quantity, range, etc.), unless otherwise specified or clearly limited to a specific number in principle, it is not limited to that specific number, and may be greater than or less than that number.

[0015] Furthermore, in the following embodiments, it goes without saying that the components (including element steps, etc.) are not necessarily essential unless specifically stated or considered to be clearly essential in principle. Similarly, in the following embodiments, when referring to the shape, positional relationship, etc., of the components, etc., it shall include those that substantially approximate or resemble their shape, etc., unless specifically stated or considered to be not in principle. The same applies to the numerical values ​​and ranges mentioned above.

[0016] In addition, in all the drawings used to explain the embodiments, the same reference numerals are generally used for identical components, and repeated explanations of them are omitted.

[0017] (Example 1) The sample solution separation device 101 according to Example 1 will be described with reference to Figures 1 to 4. Figure 1 is a configuration diagram of the sample solution separation device 101 according to Example 1. The sample solution separation device 101 is a device that separates and contains a sample solution in a plurality of microchambers 105. The sample solution separation device 101 comprises a first opening 102, a valve 103, a flow path 104, a plurality of microchambers 105, a solid phase 106, and a second opening 107.

[0018] The flow path 104 is connected to the first opening 102 via a valve 103. Multiple microchambers 105 are connected to the flow path 104. The flow path 104 is also connected to the second opening 107 via a solid phase 106. In other words, the flow path 104 connects the first opening 102, the multiple microchambers 105, and the second opening 107.

[0019] The first opening 102 is the inlet for the sample solution into the flow path 104.

[0020] The valve 103 is provided between the first opening 102 and the plurality of microchambers 105.

[0021] The second opening 107 is an inlet for the separation liquid flow path 104 that separates the multiple microchambers 105 containing the sample solution. This second opening 107 is located on the opposite side of the first opening 102, with the multiple microchambers 105 in between.

[0022] The solid phase 106 is provided between the second opening 107 and the plurality of microchambers 105. The solid phase 106 is permeable, water-repellent to the sample solution, and permeable to the separation solution.

[0023] Figures 2A to 2D show the process of introducing the sample solution 201 and the separation liquid 202 into the sample solution separation device 101 according to Example 1.

[0024] First, the sample solution 201 is introduced into the first opening 102. At the time the sample solution 201 is introduced into the first opening 102, the valve 103 is closed (Figure 2A). After closing the valve 103, the air inside the channel 104 and microchamber 105 is degassed through the solid phase 106 from the second opening 107. That is, the channel 104 and microchamber 105 are brought into a vacuum state.

[0025] By creating a vacuum in the flow path 104 and the microchamber 105 and opening the valve 103, the sample solution 201 is drawn into the flow path 104 and the microchamber 105 (Figure 2B).

[0026] Here, the solid phase 106 is a solid that is permeable, hydrophobic, and lipophilic. The permeability of the solid phase 106 allows air to pass through, and as described above, the flow path 104 and the microchamber 105 can be made into a vacuum. In addition, the hydrophobicity of the solid phase 106 repels aqueous solutions such as the sample solution 201, preventing the sample solution 201 from passing through.

[0027] Therefore, the sample solution 201 drawn into the channel 104 stops when it reaches the solid phase 106. Since the microchamber 105 is under vacuum, the sample solution 201 continues to be drawn in. The microchamber 105 is then filled with the sample solution 201 (Figure 2C).

[0028] The separation liquid 202 (see Figure 2D) used to separate the sample solution 201 from the multiple microchambers 105 is a substance (e.g., oil) that is immiscible with the sample solution 201. The solid phase 106 is lipophilic, allowing it to pass through this separation liquid 202.

[0029] Next, the sample solution 201 in the channel 104 is replaced with the separation liquid 202. The separation liquid 202 is introduced through the second opening 107. Since the solid phase 106 is lipophilic and allows the separation liquid 202 to pass through, the separation liquid 202 is introduced into the channel 104 from the second opening 107 through the solid phase 106. The sample solution 201 in the channel 104 is pushed back towards the first opening 102, and the channel 104 is filled with the separation liquid 202 (Figure 2D). At this time, the sample solution 201 remains in the microchamber 105, and the separation liquid 202 in the channel 104 separates the sample solution 201 in the multiple microchambers 105.

[0030] In this way, by using a solid phase 106 that is permeable, hydrophobic, and lipophilic, air can be removed from the flow channel 104 and the microchamber 105, and the sample solution 201 can be stopped at the position of the solid phase 106. Furthermore, the solid phase 106 can introduce the separation liquid 202 into the flow channel 104 and divide and seal the sample solution 201 into the microchamber 105.

[0031] Figure 3 is a flow chart showing the process of introducing the sample solution 201 and separation liquid 202 into the sample solution separation device 101 according to Example 1, and separating the sample solution 201 into the microchamber 105.

[0032] First, the sample solution 201 is placed in the first opening 102 (S301). At this time, the valve 103 closes the flow path 104.

[0033] Subsequently, the pump 403 (see Figure 4) connected to the second opening 107 is activated, causing the air in the flow path 104 and the multiple microchambers 105 to be discharged through the solid phase 106 and the second opening 107 (S302). As a result, the flow path 104 and the multiple microchambers 105 become a vacuum.

[0034] After the flow path 104 and the multiple microchambers 105 are under vacuum, the valve 103 opens the flow path 104, allowing the sample solution 201 to be introduced into the flow path 104 and the multiple microchambers 105 from the first opening 102 through the valve 103 (S303).

[0035] Subsequently, the pump 405 (see Figure 4) connected to the second opening 107 is driven, and the separation liquid 202 is introduced into the flow path 104 via the second opening 107 and the solid phase 106 (S304). The sample solution 201 in the flow path 104 is pushed back towards the first opening 102 by the separation liquid 202, and the flow path 104 is replaced from the sample solution 201 to the separation liquid 202, separating the multiple microchambers 105 by the separation liquid 202. After the separation liquid 202 is introduced into the flow path 104, the valve 103 may be closed to prevent the sample solution 201 in the multiple microchambers 105 and the separation liquid 202 in the flow path 104 from moving.

[0036] Figure 4 is a diagram of the sample solution separation system 400 that separates and introduces a sample solution 201 into a sample solution separation device 101 according to Example 1. The sample solution separation system 400 comprises a valve control mechanism 401, an opening contact part 402, a pump 403, an opening contact part 404, a pump 405, a moving mechanism 406, and a sample solution separation device 101.

[0037] The valve control mechanism 401 (valve control means) controls the opening and closing of the valve 103. When introducing the sample solution 201 into multiple microchambers 105, the valve control mechanism 401 opens the valve 103 from the closed state.

[0038] The opening contact portion 402 is connected to the second opening 107 of the sample solution separation device 101. The opening contact portion 402 is also connected to a pump 403 that performs vacuuming of the flow path 104 and the multiple microchambers 105. When vacuuming the flow path 104 and the multiple microchambers 105, the moving mechanism 406 moves the opening contact portion 402 to connect to the second opening 107.

[0039] The pump 403 degasses the air in the flow path 104 and the microchamber 105 through the solid phase 106 via the second opening 107, thereby creating a vacuum in the flow path 104 and the multiple microchambers 105.

[0040] The opening contact portion 404 is connected to the second opening 107 of the sample solution separation device 101. The opening contact portion 404 is also connected to the pump 405 that introduces the separation liquid 202 into the flow path 104.

[0041] The moving mechanism 406 (switching means) connects the opening contact portion 402 to the second opening 107 when degassing air from the flow path 104 and the multiple microchambers 105. Furthermore, when introducing the separation liquid 202 into the flow path 104, the moving mechanism 406 moves the opening contact portion 404 to connect it to the second opening 107. In other words, the moving mechanism 406 switches the connection destination of the second opening 107 to either pump 403 or pump 405.

[0042] The pump 405 (separation liquid introduction means) introduces the separation liquid 202 into the flow path 104 through the solid phase 106 via the second opening 107.

[0043] The pump 403 used for vacuuming is a pump capable of reducing the pressure from atmospheric pressure to, for example, 5 kPa to 0.01 kPa. A higher vacuum allows more air to be released from the microchamber 105, and more sample solution 201 to be added.

[0044] Furthermore, the pump 405 that introduces the separation liquid 202 may be, for example, a syringe pump. The syringe pump pushes out the pre-installed separation liquid 202 and introduces the separation liquid 202 into the flow path 104. Alternatively, the pump 405 may suck up the separation liquid 202 from a pre-installed container and push the sucked separation liquid 202 into the flow path 104. The pump 405 may also introduce the separation liquid 202 into the flow path under pressure, and may be, for example, a metering pump, a diaphragm pump, or a rotary pump. By pressurizing the separation liquid 202 when introducing it into the flow path 104, it becomes possible to push the separation liquid 202 in even when there is resistance from the solid phase 106 or flow path resistance.

[0045] Specifically, the solid phase 106 is a porous membrane. For example, PTFE (polytetrafluoroethylene) can be used for the solid phase 106. Because the solid phase 106 is porous and permeable, it can allow air to pass through; because it is water-repellent, it can stop the sample solution 201; and because it is lipophilic, it can allow the separation liquid 202 to pass through. Alternatively, the solid phase 106 may be PFA (perfluoroalkoxyalkylene), FEP (fluoroethylene propylene), ETFE (ethylene tetrafluoroethylene), or a fluorine-based water-repellent membrane, a silicone-based water-repellent membrane, a polymer-based water-repellent membrane, or a nanoporous membrane. Furthermore, the solid phase 106 does not have to be a membrane; it may be a channel 104 filled with hydrophobic beads. Because the beads are hydrophobic, they can repel the sample solution 201 and stop it. As the porous membrane, a hydrophobic membrane with a contact angle with water of, for example, 80 degrees or more may be used. Furthermore, the solid phase 106 is lipophilic and made of a material that allows the separation liquid 202 to permeate and pass through, so that the separation liquid 202 can pass through it.

[0046] Conventionally, instead of the solid phase 106 described above, a valve was used, for example, as disclosed in Non-Patent Document 1. In Non-Patent Document 1, closing the valve stops the flow of the sample solution, and opening it allows air to be removed or separation liquid to be introduced. However, if the valve is a pinch valve that crushes the tube, for example, a volume is required for the portion from the flow path to the tube and for the sample solution to pass through the tube. This sample solution remaining in the tube cannot be used for measurement and becomes a dead volume. This dead volume reduces the amount of target DNA that can be measured, leading to a decrease in detection sensitivity. On the other hand, by using the solid phase 106 of Example 1, the solid phase 106 can be placed close to the microchamber 105, and the dead volume of the sample solution 201 can be reduced.

[0047] As described above, by using the solid phase 106 of Example 1, the solid phase 106 can be mounted on the flow channel 104, thereby suppressing dead volume such as that of a valve. Although the sample solution 201 remaining in the flow channel 104 may become dead volume, by designing and processing the cross-sectional area of ​​the flow channel 104 to be small, the volume of the sample solution 201 remaining in the flow channel 104 can be minimized, thereby reducing the dead volume of the sample solution 201.

[0048] In Example 1, a valve 103 is used before introducing the sample solution 201 into the flow path 104. The valve 103 is closed when vacuum is being applied and opens when the sample solution 201 is introduced. A one-time valve that opens only once when introducing the sample solution 201 into the flow path 104 can also be used for the valve 103. Specifically, for example, a resin film can be used as the valve, which is normally closed and opens by tearing the film when introducing the sample solution 201. This makes it possible to provide a low-cost and compact device. Note that a solenoid valve or a pinch valve that crushes the tube may also be used as the valve 103.

[0049] The separation solution 202 is a liquid that has properties that prevent it from being miscible with the sample solution 201. Specifically, for example, the separation solution 202 can be an oil such as silicone oil or mineral oil, paraffin wax, or a photocurable resin.

[0050] The separation liquid 202 may be a photocurable resin. When separating the sample solution 201 using a photocurable resin, the photocurable resin is introduced into the channel 104 in a liquid state. Then, by irradiation with light such as ultraviolet light, the photocurable resin is solidified, and the sample solution 201 can be separated and sealed in multiple microchambers 105 containing the sample solution 201. In addition, multiple types of separation liquids may be used instead of just one. Specifically, for example, the photocurable resin and oil are introduced in that order from the second opening 107. The photocurable resin reaches near the valve 103, and the channel 104 is filled with oil. Then, by curing the photocurable resin, the end of the channel 104 can be sealed with the photocurable resin, and the spaces between the microchambers 105 can be separated by oil.

[0051] Sample solution 201 is a solution containing, for example, the DNA to be measured, polymerase, buffer, primer, and probe. The volume of this solution is adjusted from approximately 10 μL to approximately 50 μL.

[0052] In Example 1, compared to the conventional configuration using valves on both sides of the flow path, the dead volume of the sample solution 201 can be reduced. As a result, most of the prepared sample solution 201 can be used for measurement, enabling highly sensitive detection.

[0053] Furthermore, when introducing the sample solution 201 into the first opening 102, another liquid (separation liquid) such as oil may be introduced continuously with the sample solution 201. By doing so, the oil can be used to push the sample solution 201 into the microchamber 105. This allows the flow path 104 to be filled with the separation liquid such as oil, further reducing the dead volume of the sample solution 201 in the flow path 104. In addition, when the separation liquid 202 is introduced, the liquid that is pushed back into the first opening 102 becomes the oil (separation liquid), which acts as a lid, thus preventing the nucleic acids in the sample solution 201 from diffusing into the atmosphere.

[0054] In this way, the sample solution separation device 101, into which the sample solution 201 is separated and introduced into multiple microchambers 105, undergoes a PCR thermal cycle. Through the thermal cycle, the target DNA is amplified by PCR in the multiple microchambers 105. The thermal cycle is set, for example, to a high temperature of 95°C for 20 seconds and a low temperature of 60°C for 40 seconds. At the high temperature, the double helix is ​​dissociated, and at the low temperature, annealing and extension occur, leading to PCR amplification. By measuring the fluorescence of the amplified product, the target DNA separated and introduced into the multiple microchambers 105 can be detected.

[0055] The volume of each microchamber 105 is, for example, several pL to several nL, and the number of microchambers 105 is several thousand to several million. In digital PCR, by dividing the target DNA into a large number of microchambers 105, the amount of background DNA is reduced, making it possible to detect the target DNA with high sensitivity.

[0056] In this embodiment, the opening contact portion 402 and the opening contact portion 404 were switched using the moving mechanism 406 (switching means), but the flow path may also be switched using a solenoid valve or the like.

[0057] The microchamber 105 and the channel 104 are connected by a narrow connecting channel that branches off from the main channel. This narrow connecting channel provides robust separation between multiple microchambers 105. When the separation liquid 202 is introduced into channel 104, the sample solution 201 introduced into the microchamber 105 flows through the main channel. The connecting channel has a smaller cross-sectional area than the main channel. Also, since the microchamber 105 beyond the connecting channel is filled with the sample solution 201, the separation liquid 202 does not enter the microchamber 105 from the connecting channel but flows through the main channel. Furthermore, because surface tension acts between the sample solution 201 and the separation liquid 202, the sample solution 201 becomes droplet-like within the microchamber 105, and the separation liquid 202 separates the multiple microchambers 105. In this way, by connecting multiple microchambers 105 with connecting channels having a smaller cross-sectional area than the main channel, the separation liquid 202 does not enter the microchambers 105 but passes through the channel 104, allowing the sample solution 201 to be separated.

[0058] (Example 2) In Example 1, an example was described in which the solid phase 106 is located in one place in the flow path 104, but the present invention is not limited to this. Figure 5 is a configuration diagram of the sample solution separation device 501 according to Example 2. The sample solution separation device 501 comprises a first opening 102, a valve 103, a flow path 104, a plurality of microchambers 105, a plurality of solid phases 506a to 506d, and a second opening 107. Descriptions similar to those in Example 1 will be omitted as appropriate. The flow path 104 is branched to connect to a plurality of microchambers 105. The plurality of branched flow paths 104a to 104d branched from the main flow path are connected to a plurality of microchambers 105, then merge again and are connected to the second opening 107. The solid phases 501a to 501d, which allow air to pass through, stop the sample solution 201, and allow the separation liquid 202 to pass through, can be installed at multiple locations between the microchambers 105 and the second opening 107. In the example shown in Figure 5, solid phases 501a to 501d are provided in each of the multiple branch channels 104a to 104d of the flow path 104. The number of multiple branch channels 104a to 104d and the number of solid phases 501a to 501d are not limited to four, but may be two to three, or five or more.

[0059] As in Example 2, compared to Example 1, by positioning the solid phases 501a to 501d closer to the microchamber 105, the amount of sample solution 201 remaining in the flow path 104 can be reduced, thereby reducing the dead volume of the sample solution 201.

[0060] (Example 3) The sample solution separation device 601 of Example 3 will be described with reference to Figures 6A, 6B, and 7. Figure 6A is a top view of the sample solution separation device 601 according to Example 3, and Figure 6B is a side view of the sample solution separation device 601 according to Example 3. As shown in Figures 6A and 6B, the sample solution separation device 601 comprises a first opening 602, a valve 603, a flow path 604, a plurality of microchambers 605, a solid phase 606, and a second opening 607. Descriptions similar to those of Examples 1 and 2 will be omitted as appropriate. The flow path 604 and the microchambers 605 are fabricated in the substrate 608. Through holes 610 and 611 are formed at one end and the other end of the flow path 604, penetrating the substrate 608. The through hole 610 is connected to the first opening 602 through the valve 603, and the through hole 611 is connected to the second opening 607 through the solid phase 606. Furthermore, the side of the substrate 608 where the multiple microchambers 605 and the flow channels 604 have been processed is sealed with a film 609.

[0061] In other words, the flow path 604 of the sample solution separation device 601 in Example 3 has a horizontal flow path 604a extending horizontally, to which a plurality of microchambers 605 are connected, and a vertical flow path 604b (through holes 610 and 611) connected to the horizontal flow path 604a and extending vertically. The solid phase 606 is provided at the upper end of the vertical flow path 604b (through hole 611).

[0062] The substrate 608 or film 609 is made of resin, but the present invention is not limited to this. For example, the substrate 608 or film 609 may be made of COP (cycloolefin polymer) or COC (cycloolefin copolymer) which have low autofluorescence. Alternatively, the substrate 608 or film 609 may be made of polycarbonate, polypropylene, PMMA (methacrylic resin), etc. Furthermore, a portion of the substrate 608 or film 609 may be made of a metal such as aluminum which has high thermal conductivity, or a material such as carbon which can suppress light reflection.

[0063] Valve 603 is a normally closed seal positioned to block the through-hole 610, and is a one-time valve that opens from a closed state when the hole is opened. A first opening 602 with a volume capable of receiving the sample solution 201 is positioned above valve 603. The solid phase 606 is in the form of a film and is positioned to block the through-hole 611. A second opening 607 with a volume capable of receiving the separation liquid 202 is positioned above the solid phase 606. This configuration allows for easy placement of the valve 603 and solid phase 606 on the substrate 608. Furthermore, since the solid phase 606 can be positioned close to the flow path 604, the dead volume of the sample solution 291 can be reduced.

[0064] Figure 7 is a diagram of the configuration of a sample solution separation system 700 that separates and introduces a sample solution 201 into a sample solution separation device 601 according to Example 3. The sample solution separation system 700 comprises a vacuum pump 701, a pressure sensor 702, a filter 703, a solenoid valve 704, a moving mechanism 705, an opening contact part 706, an opening contact part 707, a solenoid valve 708, a liquid pump 709, a container 710, a filter 711, a pressure sensor 712, a pressurizing pump 713, a valve control mechanism 714, an opening cover 715, a light source 717, a temperature controller 718, a controller 719, and a sample solution separation device 601.

[0065] The sample solution 201 is pre-filled into the first opening 602 and positioned so as to be in contact with the top of the valve 603. At this time, the valve 603 is in the closed state. The first opening 602 is provided with an opening cover 715. On the inside of the upper surface of the opening cover 715, there is a pointed part 716 that has a sharp tip and can break the seal (valve 603). The upper surface of the opening cover 715 is made of an elastic material, such as elastomer or rubber.

[0066] The second opening 607 of the sample solution separation device 601 and the opening contact portion 706 are connected by controlling the moving mechanism 705 (switching means). The solenoid valve 704 connects or disconnects the vacuum pump 701 and the opening contact portion 706. The vacuum pump 701 evacuates the flow path 604 and the multiple microchambers 605 of the sample solution separation device 601. After the flow path 604 and the multiple microchambers 605 are sufficiently depressurized, the valve control mechanism 714 (valve control means) pushes the top of the opening cover 715, causing the pointed component 716 to break the valve 603. This opens the valve 603. Here, as described above, the valve 603 is formed of a thin film seal such as aluminum or resin and can be broken by the pointed component 716. Because the flow path 604 and the multiple microchambers 605 are under reduced pressure, the sample solution 201 is drawn into the flow path 604 and the multiple microchambers 605, filling the flow path 604 and the multiple microchambers 605 with the sample solution 201. Because the solid phase 606 is water-repellent, the sample solution 201 passing through the flow path 604 is stopped at the solid phase 606. Furthermore, because the solid phase 606 is permeable, the flow path 604 and the microchambers 605 can be negatively pressurized by the vacuum pump 701 until the leading edge of the sample solution 201 reaches the solid phase 606, thereby increasing the vacuum level.

[0067] The solenoid valve 704 is used to seal the flow path 604, connect the vacuum pump 701 to the opening contact portion 706, and vent to the atmosphere. By opening the second opening 607 to the atmosphere, the flow path 604 of the sample solution separation device 601 is opened to atmospheric pressure. Subsequently, the moving mechanism 705 connects the opening contact portion 707 to the second opening 607. The solenoid valve 708 connects the opening contact portion 707 to the liquid pump 709. The liquid pump 709 draws in the separation liquid 202 that has been pre-installed in the container 710 and discharges it toward the opening contact portion 707. The separation liquid 202 is introduced into the flow path 604 through the second opening 607 and the solid phase 606.

[0068] Furthermore, the solenoid valve 708 switches the connection point with the second opening 607 from the liquid pump 709 to the pressure pump 713. The pressure pump 713 pressurizes the separation liquid 202 and pushes it into the flow path 604. The pressure pump 713 introduces the separation liquid 202 into the flow path 604 by pressurizing it to a pressure that exceeds the flow resistance of the flow path 604 and the resistance when passing through the solid phase 606. The pressurizing pressure is determined by the size of the separation liquid 202 and the flow path 604, the properties of the solid phase 606, etc., and is, for example, about 10 kPa to 200 kPa. The sample solution 201 in the flow path 604 is pushed back towards the first opening 602, and the flow path 604 is filled with the separation liquid 202. In this way, the sample solution 201 remains in each microchamber 605 and is separated by the separation liquid 202. Furthermore, by pressurizing the separation liquid 202 and introducing it into the flow path 604, the expansion of any remaining air bubbles for any reason can be suppressed, allowing the separation liquid 202 to be introduced into the flow path 604.

[0069] Furthermore, the separation liquid 202 may be a photocurable resin. By using a photocurable resin that does not mix with the sample solution 201, the sample solution 201 can be separated into each microchamber 605. In this case, the photocurable resin in liquid form is introduced into the channel 604, and then the photocurable resin is cured using the light source 717. This allows the sample solution 201 to be sealed inside the microchamber 605.

[0070] Furthermore, when introducing the sample solution 201 and the separation liquid 202, it is possible to control the temperature of the sample solution separation device 601 using the temperature controller 718. Generally, the viscosity of the sample solution 201 and the separation liquid 202 decreases at higher temperatures. Therefore, by heating them compared to room temperature, the viscosity of the sample solution 201 and the separation liquid 202 decreases, making it easier for the sample solution 201 and the separation liquid 202 to enter the flow path 604 and the microchamber 605. This makes it possible to shorten the introduction time of the solution (sample solution 201 and separation liquid 202) and to robustly introduce the sample solution 201 into all of the microchambers 605. For example, it is preferable to heat the temperature using the temperature controller 718 to about 35°C to 70°C.

[0071] Furthermore, the pressure during vacuuming with the vacuum pump 701 is monitored and controlled by the pressure sensor 702. The pressure during pressurization with the pressurizing pump 713 is monitored and controlled by the pressure sensor 712. In addition, filters 703 and 711 prevent debris from contaminating the sample solution 201 and the separation liquid 202.

[0072] The control of the above-described series of operations is performed by the controller 719. The controller 719 has a processor 720, a memory 721, and an interface 722. The processor 720 executes various programs on the memory 721 and outputs control signals via the interface 722 to control the above-described operations.

[0073] The valve control mechanism 714 is, for example, a solenoid, which moves when current flows through it and opens the valve 603. The moving mechanism 705 is, for example, a two-axis motor drive mechanism with horizontal and vertical axes.

[0074] By using a solid phase 606 that is permeable, hydrophobic, and lipophilic, it becomes possible to control the vacuum of the flow path 604, stop the flow of the sample solution 201, and control the passage of the separation liquid 202 without using valves. Furthermore, it becomes possible to place the solid phase 606 near the microchamber 605, allowing the sample solution 201 to be divided and placed into the microchamber 605 without wasting any of it.

[0075] Furthermore, by using a one-time valve with a structure that creates a hole in a thin film as valve 603, a valve with a simple structure can be implemented. Since the sample solution 201 comes into contact with valve 603, it is necessary to make it disposable to prevent carryover, making a one-time valve useful. Also, because valve 603 is a valve with a simple structure, it can be provided at a low cost. In addition, by using a photocurable resin as the separation liquid 202 and solidifying it after introducing it in liquid form into the channel 604, the microchamber 605 can be sealed. This eliminates the need to close the first opening 602 and the second opening 607, making it possible to simplify the structure.

[0076] Valve 603 may also be punctured with a pipette tip. The sample solution separation device 601 according to Example 3 can also be applied to automated systems that connect to sample pretreatment such as nucleic acid extraction, reagent mixing, and dispensing. For example, a sample collected in a blood collection tube is centrifuged to extract plasma, and the plasma is purified to extract nucleic acids from the plasma. The nucleic acids are mixed with a reagent, and the target nucleic acid is detected by digital PCR. In this case, a dispensing system is used to aspirate and dispense the mixed sample solution with a pipette tip, and the sample solution 201 is injected into the first opening 602. At this time, the valve may be punctured with the tip of the pipette tip so that the sample solution 201 is introduced into the flow path.

[0077] Furthermore, by introducing oil (separation solution) continuously with the sample solution 201 through the first opening 602, the dead volume of the sample solution 201 can be further reduced. Specifically, after the sample solution 201 is introduced into the microchamber 605 by negative pressure, oil (separation solution) continuously enters the flow path 604, thereby reducing the amount of sample solution 201 remaining in the flow path 604. In this state, by introducing the separation solution 202 from the second opening 607 in the opposite direction to the sample solution 201, the sample solution 201 in the flow path 604 is completely replaced with the separation solution 202, allowing for separation between multiple microchambers 605. In addition, the oil (separation solution) introduced continuously with the sample solution 201 acts as a lid, preventing the nucleic acids in the sample solution 201 from becoming aerosols and being released into the atmosphere. In this way, the dead volume of the sample solution 201 can be reduced even more compared to the case where only the sample solution 201 is introduced through the first opening 602.

[0078] In this embodiment, the separation liquid 202 in the container 710 is introduced into the flow path 604 through the second opening 607, but the present invention is not limited to this. For example, the separation liquid 202 may be pre-filled in the sample solution separation device 601, and the separation liquid 202 filled in this sample solution separation device 601 may be introduced into the flow path 604.

[0079] (Example 4) In the above-described Example 3, a thin film solid phase 606 was installed at the upper end of the through hole 611 of the substrate 608, but the present invention is not limited to this. Figure 8 is a configuration diagram of the sample solution separation device 801 according to Example 4. The solid phase 806 of the sample solution separation device 801 according to Example 4 is a plurality of microparticles having permeability, hydrophobicity, and lipophilicity. The plurality of microparticles are packed into the through hole 611 (vertical channel) to form the solid phase 806. The microparticles are, for example, beads with a size of 0.1 μm to 10 μm. The microparticles are easier to introduce into the channel 604 than a film. Also, since air can pass between the microparticles, vacuuming is possible. In addition, since the microparticles are hydrophobic, the sample solution 201 can be stopped. Also, since the microparticles are lipophilic, they can pass through the separation liquid 202.

[0080] Furthermore, although the valve 603 in Example 3 is a one-time valve, the present invention is not limited thereto. For example, the valve 803 of the sample solution separation device 800 according to Example 4 may be an expandable tube. The tube is extended to connect to the through hole 610 of the substrate 608, forming the valve 803. For example, the valve 803 is a silicone tube. The silicone tube, when compressed, forms a pinch valve that stops the flow inside the tube. By using a pinch valve, it can be opened and closed multiple times. The valve 803 is closed when vacuuming and opened when adding the sample solution 201. After introducing the separation liquid 202, the valve 803 is closed to seal the sample solution 201 and the separation liquid 202.

[0081] (Example 5) The sample solution separation device of the present invention can be used in digital PCR. Figure 9 is a configuration diagram of the digital PCR system 900 according to Example 5. The digital PCR system 900 (sample solution separation system) comprises a sample solution separation device 601, a valve control mechanism 901, a pump 902, a liquid pump 903, a temperature controller 913, an optical system 914 (measurement means), and an analysis unit 912. The optical system 914 includes a light source 904, lenses 905, 908, 910, a bandpass filter 906, a bandpass filter 909, a dichroic mirror 907, and a CMOS sensor 911.

[0082] The sample solution 201 is set into the sample solution separation device 601 through the first opening 602. The pump 902 drives the air in the flow path 604 and microchamber 605 through the second opening 607, creating negative pressure. The valve control mechanism 901 opens the valve 603, introducing the sample solution 201 into the flow path 604 and microchamber 605. Subsequently, the liquid pump 903 drives the separation liquid 202 into the flow path 604 via the second opening 607. In this way, the sample solution 201 can be separated and introduced into multiple microchambers 605.

[0083] Here, sample solution 201 is a PCR reaction solution containing the DNA to be detected, polymerase, buffer, primer, and probe. A thermal cycling treatment is performed on a device in which the sample solution 201, which is the PCR reaction solution, is separated and placed in multiple microchambers 605. During the thermal cycling treatment, the temperature controller 913 is controlled to the temperature range in which denature, annealing, and extension occur. The temperature controller 913 is composed of a heater, a Peltier element, etc., but the present invention is not limited to this. During the thermal cycling treatment, if the DNA to be measured is present in the microchamber 605, the target DNA is amplified.

[0084] The optical system 914 (measurement means) measures the target nucleic acid in the PCR-amplified sample solution 201. Light emitted from the light source 904 is collimated by the lens 905, transmitted to a predetermined wavelength by the bandpass filter 906, reflected by the dichroic mirror 907, and passed through the lens 908 to irradiate the sample solution separation device 601. Fluorescence from the microchamber 605 of the sample solution separation device 601 passes through the lens 908, the dichroic mirror 907, the bandpass filter 909, and the lens 910, and is imaged by the CMOS sensor 911. The analysis unit 912 performs analysis based on the image captured by the CMOS sensor 911 and detects the target DNA in each microchamber.

[0085] Figure 10 is an operational flowchart of the digital PCR system 900 according to Example 5.

[0086] The valve control mechanism 901 opens the valve 603 and introduces the sample solution 201 set in the first opening 602 into the sample solution separation device 601 (S1001).

[0087] The liquid pump 903 introduces the separation liquid 202 into the sample solution separation device 601 through the second opening 607 (S1002). As a result, the sample solution 201 is separated and introduced into multiple microchambers 605.

[0088] Next, the temperature controller 913 puts the sample solution separation device 601 into a thermal cycle (S1003).

[0089] The optical system 914 irradiates the sample solution separation device 601 with light and measures the fluorescence intensity of each microchamber 605 (S1004).

[0090] Then, the analysis unit 915 analyzes the fluorescence intensity measured by the optical system 914 to detect the target DNA in the sample solution 201 (S1005).

[0091] Furthermore, when introducing the sample solution 201 and the separation liquid 202, the sample solution 201 and the separation liquid 202 may be heated. This changes the viscosity of the sample solution 201 and the separation liquid 202, making it easier to introduce them into the flow path 604. It also shortens the time required to introduce the sample solution 201 and the separation liquid 202. The temperature controller used to heat the sample solution 201 and the separation liquid 202 may be the temperature controller 913 that performs the thermal cycle, or it may be a temperature controller separate from the temperature controller 913.

[0092] Asymmetric PCR may be performed by varying the concentrations of the forward and reverse primers in the PCR reaction solution, so that one of the single-stranded DNA molecules is amplified more than the other. In this way, single-stranded DNA is detected by molecular beacons. Furthermore, the temperature of the sample solution separation device is controlled during fluorescence measurement, and melting curve analysis is performed. Fluorescence measurement is performed using multiple filters and multiple colors. By identifying the target DNA using the fluorescence color, fluorescence intensity, and melting temperature of each microchamber, high multiplex and high-sensitivity measurement can be achieved.

[0093] (Method for separating sample solutions) Here, we will describe a method for separating the sample solution 201 into multiple microchambers 105. The sample solution separation method is as follows: Prepare the sample solution separation device 101 described above. Degassing of air from multiple microchambers 105 and flow channels 104 via solid phase 106, Open the valve 103 and introduce the sample solution 201 from the first opening 102 into the multiple microchambers 105 and the flow path 104, and The method involves introducing the separation liquid 202 into the flow path 104 via the solid phase 106 through the second opening 107. The sample solution separation device 101 to be prepared may be one of the sample solution separation devices 501, 601, or 801.

[0094] Furthermore, the method for separating the sample solution is: A thermal cycle of PCR (polymerase chain reaction) is performed on the sample solution separation device 101 in which the sample solution 201 is separated into multiple microchambers 105. To measure the fluorescence intensity of multiple microchambers 105, and The method further includes analyzing fluorescence intensity to detect target DNA in sample solution 201.

[0095] Furthermore, introducing the sample solution 201 from the first opening 102 into the multiple microchambers 105 and flow channels 104 includes introducing the sample solution 201 and the oil (separation liquid) following the sample solution 201 from the first opening 102.

[0096] (modified version) This disclosure is not limited to the embodiments described above, but includes various modifications. For example, the embodiments described above are described in detail for the purpose of explaining this disclosure and are not necessarily limited to those having all the configurations described. Furthermore, it is possible to replace parts of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add configurations from other embodiments to the configuration of one embodiment. In addition, it is possible to add, delete, or replace parts of the configuration of each embodiment with other configurations.

[0097] For example, in the above-described embodiment, a separation liquid was used to separate the multiple microchambers 105, but the present invention is not limited to this, and a separation gas may also be used. In this case, the solid phase only needs to have physical properties that allow the separation gas to pass through. [Explanation of symbols]

[0098] 101, 501, 601, 801 Sample Solution Separation Device 102 First opening 103 Valve 104 Flow Channel 105 Microchamber 106 Solid phase 107 Second opening 201 Sample Solution 202 Separation liquid 400,700 Sample Solution Separation System 401 Valve control mechanism 402,404 Opening Contact Section 403,405 pumps 406 Moving mechanism 506a,506b,506c,506d solid phase 602 First opening 603 Valve 604 Flow channel 604a Horizontal channel 604b Vertical channel 605 Microchamber 606 Solid phase 607 Second opening 608 circuit board 609 film 610, 611 Through holes 701 Vacuum pump 702,712 Pressure Sensors 703,711 filters 704,708 Solenoid valves 705 Moving mechanism 706, 707 Opening Contact Section 709 Liquid pump 710 Container 713 Pressure pump 714 Valve control mechanism 715 Opening lid 716 Pointed parts 717 Light source 718 Temperature controller 719 Controller 720 Processor 721 memory 722 Interface 803 Valve 806 Solid phase 900 Digital PCR System 901 Valve control mechanism 902 Pump 903 Liquid Pump 904 Light source 905, 908, 910 lenses 906,909 Bandpass Filter 907 Dichroic Mirror 911 CMOS sensor 912 Analysis Department 913 Temperature controller 914 Optical system

Claims

1. A sample solution separation device for separating sample solutions, Multiple microchambers and A channel connecting the aforementioned multiple microchambers, A first opening which is the inlet for the sample solution into the flow path, A valve provided between the first opening and the plurality of microchambers, An inlet to a channel for a separation liquid that separates the plurality of microchambers containing the sample solution, comprising a second opening provided on the opposite side of the first opening across the plurality of microchambers, The system comprises a solid phase provided between the second opening and the plurality of microchambers, The solid phase is permeable, water-repellent to the sample solution, and permeable to the separation liquid. A sample solution separation device characterized by the following features.

2. The flow path has a plurality of branched flow paths to which each of the microchambers is connected. The solid phase is provided in each of the plurality of branched channels. The sample solution separation device according to claim 1.

3. The aforementioned flow path is The aforementioned multiple microchambers are connected, and a horizontal channel extends horizontally, It has a vertical channel connected to the horizontal channel and extending vertically, The solid phase is provided at the upper end of the vertical flow channel. The sample solution separation device according to claim 1.

4. The aforementioned flow path is The aforementioned multiple microchambers are connected, and a horizontal channel extends horizontally, It has a vertical channel connected to the horizontal channel and extending vertically, The solid phase is a plurality of fine particles provided within the vertical channel. The sample solution separation device according to claim 1.

5. The valve is a one-time valve that is opened only once when the sample solution is introduced into the flow path. The sample solution separation device according to claim 1.

6. The solid phase is a porous membrane that is water-repellent to the sample solution and permeable to the separation solution. A sample solution separation device according to feature 1.

7. The material of the porous membrane is PTFE (polytetrafluoroethylene). The sample solution separation device according to feature 6.

8. The separating liquid is a photocurable resin, The separated liquid hardens upon light irradiation within the flow path. The sample solution separation device according to claim 1.

9. The separation liquid is an oil having properties that make it incompatible with the sample solution. A sample solution separation device according to claim 1, characterized in that...

10. A sample solution separation device for separating a sample solution, comprising: a plurality of microchambers; a flow path connecting the plurality of microchambers; a first opening which is an inlet for the sample solution into the flow path; a valve provided between the first opening and the plurality of microchambers; a second opening which is an inlet for a separation liquid separating the plurality of microchambers containing the sample solution, provided on the opposite side of the first opening across the plurality of microchambers; and a solid phase provided between the second opening and the plurality of microchambers, wherein the solid phase is permeable, water-repellent to the sample solution, and permeable to the separation liquid. A pump that degassses the air in the plurality of microchambers and the flow path through the solid phase from the second opening, Valve control means for opening the valve, and It has a separation liquid introduction means for introducing the separation liquid through the second opening. A sample solution separation system characterized by the following features.

11. The system further includes a switching means for switching the connection destination of the second opening to the pump or the separation liquid introduction means. The sample solution separation system according to claim 10.

12. The system further includes a pressurizing pump that pressurizes the separated liquid and introduces it into the flow path. The sample solution separation system according to claim 10.

13. The system further comprises a measurement means for measuring the target nucleic acid in the sample solution from which PCR (polymerase chain reaction) has been performed within the microchamber. The sample solution separation system according to claim 10.

14. A sample solution separation device for separating a sample solution, comprising: a plurality of microchambers; a flow path connecting the plurality of microchambers; a first opening which is an inlet for the sample solution into the flow path; a valve provided between the first opening and the plurality of microchambers; a second opening which is an inlet for a separation liquid separating the plurality of microchambers containing the sample solution, provided on the opposite side of the first opening across the plurality of microchambers; and a solid phase provided between the second opening and the plurality of microchambers, wherein the solid phase is permeable, water-repellent to the sample solution, and permeable to the separation liquid, is provided. Degassing the air in the plurality of microchambers and the flow path via the solid phase, Open the valve and introduce the sample solution into the plurality of microchambers and the flow path through the first opening, and The method involves introducing the separated liquid into the flow path through the solid phase from the second opening. A method for separating sample solutions characterized by the following features.

15. A thermal cycle of PCR (polymerase chain reaction) is performed on the sample solution separation device in which the sample solution is separated into the plurality of microchambers. To measure the fluorescence intensity of the aforementioned plurality of microchambers, and The method further comprises analyzing the fluorescence intensity and detecting the target DNA in the sample solution. The method for separating sample solutions according to feature 14.

16. Introducing the sample solution into the plurality of microchambers and the flow path through the first opening includes introducing the sample solution and the separation solution following the sample solution through the first opening. The method for separating sample solutions according to feature 14.

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