PCR microfluidic chip and PCR analysis device

Abstract

To provide a technique for minimizing contamination into a liquid sample in allowing the liquid sample to flow in a flow channel.SOLUTION: A microfluid chip for PCR has: a flow channel through which a liquid sample used for PCR flows; introduction port which introduces the liquid sample into a flow channel; elastically stretchable two air chambers which are severally connected to both ends of the flow channel; and a surrounding member which surrounds the flow channel and the air chamber in an airtight manner, and in which a transparent part is provided for at least one side.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a microfluidic chip for PCR and a PCR analysis device. [Background technology]

[0002] PCR (polymerase chain reaction) is generally performed by placing a predetermined amount of the liquid sample to be used in a reaction vessel such as a PCR tube or a microplate (microwell) with multiple holes, but in recent years, it has become practical to perform PCR using a reaction vessel (also called a chip) with fine channels formed on a substrate. When PCR is performed using a chip with channels (microchannel chip), temperature zones such as high and low temperature zones are set in the channel, and the liquid sample is subjected to a thermal cycle by moving back and forth within the channel.

[0003] Conventional liquid transfer methods for microchannel chips include a method in which two microblowers or fans are connected to both ends of a channel and liquid is transferred by applying pressure through airflow, as disclosed in Patent Document 1. Also, a method of liquid transfer using a single pump and two switching valves has been proposed, as disclosed in Patent Document 2. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2019-47812 [Patent Document 2] International Publication No. 2019 / 139135 Summary of the Invention [Problem to be solved by the invention]

[0005] In PCR, it is important to minimize or completely prevent contamination of the liquid sample. In particular, when the liquid sample is flowing through a channel, it is desirable that the liquid sample does not come into contact with unwanted substances.

[0006] Therefore, the present invention provides a technique for minimizing contamination of a liquid sample when the liquid sample is caused to flow in a channel. [Means for solving the problem]

[0007] According to one aspect of the present invention, there is provided a microfluidic chip for PCR, which comprises a channel through which a liquid sample used in PCR flows, an inlet for introducing the liquid sample into the channel, two elastically expandable air chambers connected to both ends of the channel, and an enclosure member airtightly enclosing the channel and the air chambers and having a transparent portion on at least one side. 。

[0008] In this embodiment, when the inlet is closed, a completely closed flow channel system having a flow channel and two air chambers is formed. one When one of the air chambers is compressed, the liquid sample flows from the compressed air chamber to the other air chamber, causing the downstream air chamber to elastically expand. When the compressive force is removed, the expanded air chamber elastically contracts, causing the liquid sample to flow in the opposite direction toward the compressed air chamber, causing the compressed air chamber to elastically expand. In this way, the liquid sample can move back and forth through the flow channel. Both air chambers may be compressed alternately. Since the flow channel and the two air chambers are completely closed, contamination of the liquid sample can be minimized when the liquid sample is flowing through the flow channel. At least one side of the enclosing member is transparent, allowing the liquid sample in the flow channel to be observed. [Brief explanation of the drawings]

[0009] [Figure 1]FIG. 1 is a plan view of a PCR microfluidic chip according to an embodiment of the present invention. [Figure 2] FIG. 2 is an exploded perspective view of the PCR microfluidic chip of FIG. 1. [Figure 3] FIG. 1 is a front view of a PCR analysis device according to a first embodiment, showing a cross section of a PCR microfluidic chip. [Figure 4] FIG. 2 is a front view of the PCR analyzer according to the first embodiment when a liquid sample is introduced. [Figure 5] FIG. 2 is a front view of the PCR analyzer according to the first embodiment when the liquid sample is moving to the right. [Figure 6] FIG. 2 is a front view of the PCR analyzer according to the first embodiment when the liquid sample is moving leftward. [Figure 7] FIG. 10 is a front view of a PCR analysis device according to a second embodiment, showing a cross section of a PCR microfluidic chip. [Figure 8] FIG. 10 is a front view of a PCR analyzer according to a second embodiment when a liquid sample is introduced. [Figure 9] FIG. 10 is a front view of the PCR analyzer according to the second embodiment when the liquid sample is moving to the right. [Figure 10] FIG. 10 is a front view of the PCR analyzer according to the second embodiment when the liquid sample is moving leftward. [Figure 11] 10 is a table showing the results of examining the temperature settings of the heating device of a PCR analyzer and the state of a liquid sample. DETAILED DESCRIPTION OF THE INVENTION

[0010] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, several embodiments of the present invention will be described with reference to the accompanying drawings, in which the drawings are not necessarily drawn to scale and some features may be exaggerated or omitted.

[0011] As shown in FIG. 1, a PCR microfluidic chip 1 according to the embodiment has an axisymmetric shape, and includes a channel 2, ports 4 and 6, a first air chamber 10, and a second air chamber 12.

[0012] A liquid sample used in PCR flows through a flow channel 2. The flow channel 2 has a first curved flow channel section 7 that is bent, a second curved flow channel section 8 that is bent, and a straight flow channel section 9 that connects the first curved flow channel section 7 and the second curved flow channel section 8. The width of the flow channel 2 is preferably 0.5 to 1.0 mm. The length of each of the curved flow channel sections 7 and 8 is, for example, 80 mm, and the length of the straight flow channel section 9 is, for example, 15 mm.

[0013] The two air chambers 10, 12 are connected to both ends of the flow path 2, respectively, and are elastically expandable and contractible. The first air chamber 10 communicates with the first curved flow path section 7, and the second air chamber 12 communicates with the second curved flow path section 8.

[0014] Port 4 is connected to the section between the first curved flow path section 7 and the first air chamber 10, and port 6 is connected to the section between the second curved flow path section 8 and the second air chamber 12. One of ports 4 and 6 is used as an inlet for introducing a liquid sample into flow path 2, and the other is used as an air outlet for discharging air that is pushed out of flow path 2 by the liquid sample when the liquid sample is introduced into flow path 2. In the following description, port 4 is assumed to be an inlet, and port 6 is assumed to be an air outlet.

[0015] 2, the microfluidic chip 1 has a resin lower substrate 20, a resin flow path substrate 21, a resin upper substrate 22, rubber elastic substrates 23 and 25, and rubber elastic membranes 24 and 26. The substrates 20, 21, and 22, the elastic substrates 23 and 25, and the elastic membranes 24 and 26 form an enclosing member that airtightly encloses the flow path 2 and the air chambers 10 and 12.

[0016] The substrates 20, 21, and 22 are made of a transparent resin such as acrylic or polypropylene, while the elastic substrates 23 and 25 and the elastic films 24 and 26 are made of silicone rubber, for example.

[0017] The lower substrate 20 is a rectangular flat plate with no grooves or holes formed therein, and has a thickness of, for example, 0.2 mm.

[0018] The flow path substrate 21 is a rectangular substrate in which through grooves 21a, 21b, and 21c and through holes 21d and 21e are formed, and has a thickness of, for example, 0.5 mm. When the flow path substrate 21 is joined to the lower substrate 20 and the upper substrate 22, the through groove 21a forms the flow path 2. Therefore, the height of the flow path 2 is, for example, 0.5 mm. The through groove 21b is connected to the through groove 21a and forms the lower part of port 4. The through groove 21c is connected to the through groove 21a and forms the lower part of port 6. The through hole 21d forms the lower part of the first air chamber 10. The through hole 21e forms the lower part of the second air chamber 12. The through grooves 21a, 21b, and 21c and the through holes 21d and 21e can be formed, for example, by laser processing.

[0019] Upper substrate 22 is a rectangular substrate in which through holes 22b, 22c, 22d, and 22e are formed, and has a thickness of, for example, 1.0 mm. Through hole 22b is located above port 4, and through hole 22c is located above port 6. Through hole 22d is located in part of first air chamber 10. Through hole 21e is located in part of second air chamber 12. Through holes 22b, 22c, 22d, and 22e can be formed by, for example, laser processing.

[0020] The substrates 20, 21, and 22 can be heated to, for example, 180° C. and bonded together by thermal welding.

[0021] The elastic substrates 23 and 25 are square flat plates, each having a thickness of, for example, 1.0 mm. A through-hole 23d is formed in the center of the elastic substrate 23, and the through-hole 23d becomes part of the first air chamber 10. A through-hole 25e is formed in the center of the elastic substrate 25, and the through-hole 25e becomes part of the second air chamber 12. The through-holes 23d and 25e can be formed, for example, by laser processing.

[0022] The elastic membranes 24 and 26 are square flat plates with no grooves or holes formed therein, and have a thickness of, for example, 0.3 mm.

[0023] The elastic substrate 23 and the elastic film 24 can be bonded together using vacuum ultraviolet light or oxygen plasma irradiation. The elastic substrate 25 and the elastic film 26 can also be bonded together using vacuum ultraviolet light or oxygen plasma irradiation. However, double-sided adhesive tape may be used to bond the elastic substrate 23 and the elastic film 24, and to bond the elastic substrate 25 and the elastic film 26.

[0024] 3, the rubber elastic substrates 23, 25 can be bonded to the resin upper substrate 22 with double-sided adhesive tape 27. However, to prevent or reduce the undesired inflow of organic matter into the flow channel 2 and the air chambers 10, 12, it is preferable to bond the elastic substrates 23, 25 to the upper substrate 22 using vacuum ultraviolet light or oxygen plasma irradiation.

[0025] As described above, by joining the substrates 20, 21, and 22, the elastic substrates 23 and 25, and the elastic membranes 24 and 26, a microfluidic chip 1 having a flow channel 2, ports 4 and 6, and air chambers 10 and 12 is completed. The flow channel 2 is formed by the through groove 21a in the flow channel substrate 21. The port 4 is formed by the through hole 22b in the upper substrate 22 and the through groove 21b in the flow channel substrate 21. The port 6 is formed by the through hole 22c in the upper substrate 22 and the through groove 21c in the flow channel substrate 21.

[0026] The first air chamber 10 is formed by the through-hole 21d of the flow path substrate 21, the through-hole 22d of the upper substrate 22, and the through-hole 23d of the elastic substrate 23. Because the elastic substrate 23 is formed of highly elastic rubber to have a large thickness, and the elastic membrane 24 is also formed of highly elastic rubber, the first air chamber 10 is elastically expandable and can be used as a diaphragm pump or an expandable accumulator.

[0027] The second air chamber 12 is formed by the through-hole 21e of the flow path substrate 21, the through-hole 22e of the upper substrate 22, and the through-hole 25e of the elastic substrate 25. Because the elastic substrate 25 is formed of highly elastic rubber to have a large thickness, and the elastic membrane 26 is also formed of highly elastic rubber, the second air chamber 12 is also elastically expandable and can be used as a diaphragm pump or an expandable accumulator.

[0028] In the microfluidic chip 1, when the ports 4 and 6 are closed, a completely closed channel system is formed, which includes the channel 2 and two air chambers 10 and 12. The two expandable air chambers 10 and 12 connected to both ends of the channel 2 are one When one of the air chambers 10 and 12 is compressed, the liquid sample flows from the compressed air chamber to the other, elastically expanding the downstream air chamber. When the compression force is removed, the expanded air chamber elastically contracts, causing the liquid sample to flow in the opposite direction toward the compressed air chamber, elastically expanding the compressed air chamber. In this way, the liquid sample can move back and forth through the flow channel 2. Both air chambers 10 and 12 may be compressed alternately. Since the flow channel 2 and the two air chambers 10 and 12 are completely closed, contamination of the liquid sample can be minimized when the liquid sample flows through the flow channel 2. The surrounding members, especially the thinnest lower substrate 20, are transparent, allowing the liquid sample in the flow channel 2 to be observed from below. The upper substrate 22 is also transparent, allowing the liquid sample in the flow channel 2 to be observed from above.

[0029] 3 is a front view of a PCR analysis device 30 according to the first embodiment. 3 to 10 show a cross section of a PCR microfluidic chip 1.

[0030] As shown in FIG. 3, the PCR analysis device 30 includes a PCR microfluidic chip 1, two actuators 31 and 32, a heating device 33, an observation / analysis device 34, and a control device 35.

[0031] The actuators 31 and 32 are linear actuators having pistons 31a and 32a, respectively, and are driven by drive circuits 31b and 32b, respectively. The drive circuits 31b and 32b are controlled by a control device 35.

[0032] The actuator 31 is disposed above the first air chamber 10, and as shown in Figure 3, when the actuator 31 is not driven, the piston 31a is disposed at a distance from the elastic membrane 24 that defines the first air chamber 10. When the actuator 31 is driven, the piston 31a descends and presses down on the elastic membrane 24, thereby compressing the first air chamber 10.

[0033] The actuator 32 is disposed above the second air chamber 12, and as shown in Fig. 3, when the actuator 32 is not driven, the piston 32a is disposed at a distance from the elastic membrane 26 that defines the second air chamber 12. When the actuator 32 is driven, the piston 32a descends and pushes down the elastic membrane 26, compressing the second air chamber 12. Under the control of the control device 35, the actuators 31, 32 alternately compress the two air chambers 10, 12.

[0034] The actuators 31 and 32 are electric linear actuators, but may also be other actuators capable of compressing the air chambers 10 and 12, such as actuators having a mechanism for converting the rotation of a motor into linear motion, or hydraulic linear actuators.

[0035] The heating device 33 has a first heating section 37 that heats the first curved flow path section 7 and a second heating section 38 that heats the second curved flow path section 8. The first heating section 37 has a Peltier element 37a as a heating element and a metal plate 37b as a heat transfer element. The second heating section 38 has a Peltier element 38a as a heating element and a metal plate 38b as a heat transfer element.

[0036] Metal plate 37b is bonded to lower substrate 20 of microfluidic chip 1 at a position directly below first curved channel section 7, and Peltier element 37a is bonded to metal plate 37b. Metal plate 38b is bonded to lower substrate 20 of microfluidic chip 1 at a position directly below second curved channel section 8, and Peltier element 38a is bonded to metal plate 38b.

[0037] Peltier element 37a, 38a are driven by heating circuits 37c and 38c, respectively. The heating circuits 37c and 38c are controlled by the control device 35. In this embodiment, the Peltier elements 37a and 38b are used to perform local heating. 38a is used, but other heating elements may be used.

[0038] The observation / analysis device 34 observes the liquid sample in the straight channel section 9 of the channel 2 through the transparent lower substrate 20 of the microfluidic chip 1. The optical head 34a of the observation / analysis device 34 is disposed directly below the straight channel section 9 on the lower substrate 20. The observation / analysis device 34 may be a fluorescent probe that measures fluorescence emitted by an amplification product in the liquid sample in response to irradiation with excitation light (ultraviolet light). Alternatively, the observation / analysis device 34 may be an optical probe that detects turbidity caused by magnesium pyrophosphate, an amplification by-product in the liquid sample. The observation / analysis device 34 is connected to the control device 35, but it does not have to be connected to the control device 35.

[0039] The control device 35 is, for example, a PLC (Programmable Logic Controller), and controls the drive circuits 31b and 32b of the actuators 31 and 32 and the heating circuits 37c and 38c of the heating units 37 and 38. However, the control device 35 is configured by a PLC that controls the drive circuits 31b and 32b of the actuators 31 and 32, and a Peltier element 37a, 38a The heating element may have a temperature regulator that maintains the temperature constant.

[0040] The control device 35 may have at least one CPU (Central Processing Unit) and a storage device that stores an operation program and an observation program. In this case, the CPU may read the operation program from the storage device and control the drive circuits 31b and 32b of the actuators 31 and 32 and the heating circuits 37c and 38c of the heating units 37 and 38 in accordance with the operation program. The CPU may also read an observation program from the storage device and analyze the observation results of the observation analysis device 34 in accordance with the observation program. The observation program may be an artificial intelligence program.

[0041] Next, a method of using the PCR analyzer 30 will be described. First, as shown in FIG. 4, the liquid sample 40 is introduced, or injected, into the flow channel 2 through the port 4 using the introduction device 39. The introduction device 39 may be a micropipette or a syringe. It is not necessary to fill the flow channel 2 and the air chambers 10 and 12 with the liquid sample 40; the amount of the liquid sample 40 may be small, for example, 15 to 20 μl. The width and height of the flow channel 2 are small, and the liquid sample 40 aggregates into a rod-like shape due to interfacial tension, blocking the cross section of the flow channel 2. Assuming that the flow channel 2 has a rectangular cross section with a width of 0.5 mm and a height of 0.5 mm and the amount of the liquid sample 40 is 15 μl, the length of the liquid sample 40 in the flow channel 2 is 60 mm. Therefore, the liquid sample 40 divides the space within the flow channel 2 and the air chambers 10 and 12 into a left space and a right space. The left space includes the interior space of the first air chamber 10, and the right space includes the interior space of the second air chamber 12. Although not shown, a small amount of air may be discharged through port 6 to adjust the position of liquid sample 40 within flow channel 2 .

[0042] After this, the liquid sample 40 is allowed to release any air bubbles, and the ports 4 and 6 are closed and sealed with adhesive tape 41. The adhesive tape 41 may be cellophane tape or polyimide tape. Instead of the adhesive tape 41, resin or rubber plugs may be inserted into the ports 4 and 6 to close and seal the ports 4 and 6. In this way, a completely closed flow path system having the flow path 2 and the two air chambers 10 and 12 is formed.

[0043] Next, the control device 35 activates the drive circuit 31b to drive the actuator 31. Then, as shown in FIG. 5, the piston 31a of the actuator 31 descends, pushing down the elastic membrane 24 and compressing the first air chamber 10. Therefore, as shown by the arrow, the liquid sample 40 moves rightward within the flow channel 2. However, the piston 31a may have a larger lower end than shown in the figure so that it pushes down the elastic substrate 23 together with the elastic membrane 24. The liquid sample 40 passes through the first curved flow channel section 7 and the straight flow channel section 9, and then through the second curved flow channel section 8. Pressed by the liquid sample 40, the pressure in the right-hand space increases, and the second air chamber 12 elastically expands.

[0044] Next, the control device 35 deactivates the drive circuit 31b to raise the piston 31a of the actuator 31 to its initial position, and activates the drive circuit 32b to drive the actuator 32. Then, as shown in FIG. 6, the piston 32a of the actuator 32 descends, pushing down the elastic membrane 26 and compressing the second air chamber 12. Therefore, as shown by the arrow, the liquid sample 40 moves leftward within the flow channel 2. However, the piston 32a may have a larger lower end than shown in the figure so that it presses down the elastic substrate 25 together with the elastic membrane 26. The liquid sample 40 passes through the second curved flow channel section 8 and the straight flow channel section 9, and then through the first curved flow channel section 7. Pressed by the liquid sample 40, the pressure in the left space increases, and the first air chamber 10 elastically expands.

[0045] When the control device 35 deactivates the actuator 32 and activates the actuator 31, the liquid sample 40 moves to the right in the flow channel 2. When the control device 35 again deactivates the actuator 31 and activates the actuator 32, the liquid sample 40 moves to the left in the flow channel 2.

[0046] In this way, the control device 35 alternately drives the left and right actuators 31, 32 to alternately compress the left and right diaphragm pumps, i.e., the air chambers 10, 12. When the first air chamber 10 contracts and the pressure in the left space increases, the pressure in the right space also increases, but because the second air chamber 12 expands, the pressure in the right space does not increase abruptly. Conversely, when the second air chamber 12 contracts and the pressure in the right space increases, the pressure in the left space also increases, but because the first air chamber 10 expands, the pressure in the left space does not increase abruptly. In this way, since the pressure in the flow path 2 does not change abruptly, the generation of bubbles in the liquid sample 40 is reduced, and the rod-shaped liquid sample 40 is also prevented from breaking apart due to the generation of large bubbles.

[0047] The heating device 33 can perform thermal cycling during the reciprocating movement of the liquid sample 40 within the flow channel 2. In one example, while the liquid sample 40 reciprocates within the flow channel 2, the first heating element 37 on the left side may be fixed at a fairly high temperature (e.g., 94°C) for denaturing DNA (deoxyribonucleic acid) in the liquid sample 40, and the second heating element 38 on the right side may be fixed at a high temperature (e.g., 60°C) for annealing the primers. As the actuator 31 is driven to move the liquid sample 40 rightward within the flow channel 2, the liquid sample 40 reaches its denaturation temperature while passing through the first curved flow channel section 7 and reaches the primer annealing temperature while passing through the second curved flow channel section 8. DNA extension can also proceed at the annealing temperature. In this case, the annealing temperature can be referred to as the "extension-annealing temperature." Although the straight flow channel section 9 is not heated, the temperature drop of the liquid sample 40 during its passage through the straight flow channel section 9 is small due to its short length. On the other hand, while the actuator 32 is driven to move the liquid sample 40 leftward within the channel 2, the liquid sample 40 remains at the extension / annealing temperature while passing through the second curved channel section 8, and reaches the denaturation temperature while passing through the first curved channel section 7. Next, while the actuator 31 is driven to move the liquid sample 40 rightward within the channel 2, the liquid sample 40 remains at the denaturation temperature while passing through the first curved channel section 7, and reaches the extension / annealing temperature while passing through the second curved channel section 8. In this example, one round trip of the liquid sample 40 within the channel 2 corresponds to one cycle. The control device 35 alternately drives the left and right actuators 31 and 32 to match the cycle time.

[0048] In another example, while the liquid sample 40 moves back and forth within the channel 2, the first heating element 37 on the left side may be fixed at a high temperature (e.g., 60°C) for DNA extension and primer annealing within the liquid sample 40, and the second heating element 38 on the right side may be fixed at a significantly higher temperature (e.g., 94°C) for DNA denaturation. When the actuator 31 is driven to move the liquid sample 40 to the right within the channel 2, the liquid sample 40 reaches the extension / annealing temperature while passing through the first curved channel section 7 and reaches the denaturation temperature while passing through the second curved channel section 8. Because the straight channel section 9 is short, the temperature drop of the liquid sample 40 while passing through it is small. On the other hand, while the actuator 32 is driven to move the liquid sample 40 to the left within the channel 2, the liquid sample 40 remains at the denaturation temperature while passing through the second curved channel section 8 and reaches the extension / annealing temperature while passing through the first curved channel section 7. Next, the actuator 31 is driven to move the liquid sample 40 to the right within the channel 2; the liquid sample 40 remains at the extension / annealing temperature while passing through the first curved channel section 7, and reaches the denaturation temperature while passing through the second curved channel section 8. In this example, one round trip of the liquid sample 40 within the channel 2 corresponds to one cycle. The control device 35 alternately drives the left and right actuators 31 and 32 to match the cycle time.

[0049] In another example, the control device 35 may alternately change the temperatures of the left and right heating sections 37, 38. For example, while the actuator 31 is driven to move the liquid sample 40 to the right within the channel 2, the control device 35 may set the first heating section 37 on the left side to a fairly high temperature (e.g., 94°C) for denaturing DNA in the liquid sample 40, and the second heating section 38 on the right side to a high temperature (e.g., 60°C) for extending the DNA and annealing the primers. The liquid sample 40 reaches the denaturation temperature while passing through the first curved channel section 7, and reaches the extension / annealing temperature while passing through the second curved channel section 8.

[0050] In this example, while the actuator 32 is driven to move the liquid sample 40 leftward within the flow channel 2, the control device 35 may set the second heating element 38 on the right side to a fairly high temperature (e.g., 94°C) for denaturing the DNA in the liquid sample 40, and the first heating element 37 on the left side to a high temperature (e.g., 60°C) for extending the DNA and annealing the primers. The liquid sample 40 reaches its denaturation temperature while passing through the second curved flow channel section 8, and reaches its extension / annealing temperature while passing through the first curved flow channel section 7. In this example, one round trip of the liquid sample 40 within the flow channel 2 corresponds to two cycles. The control device 35 alternately drives the left and right actuators 31 and 32 to match the cycle time.

[0051] After the driving of both actuators 31 and 32 is stopped, the liquid sample 40 stops moving when the pressures in the left and right spaces (the pressures in the two air chambers 10 and 12) are balanced. The stopping position of the liquid sample 40 remains constant regardless of the number of times the actuators 31 and 32 are driven.

[0052] The liquid sample 40 that has stopped moving can be observed by the observation and analysis device 34 through the transparent lower substrate 20. The liquid sample 40 can be observed by the observation and analysis device 34 even while it is moving.

[0053] The applicant repeatedly alternately drove the actuators 31 and 32, causing the liquid sample 40 to move back and forth 50 times within the flow channel 2. No bubbles were observed in the liquid sample 40, and no breakage of the rod-shaped liquid sample 40 was observed.

[0054] In this embodiment, the flow channel 2 and the two air chambers 10, 12 are completely closed by the resin substrates 20, 21, 22, the rubber elastic substrates 23, 25, and the rubber elastic membranes 24, 26, which minimizes contamination of the liquid sample when the liquid sample is caused to flow through the flow channel 2. The microfluidic chip 1 is made of rubber and resin and does not contain any metal, ceramic, or electronic components, so it is inexpensive and can be easily disposed of.

[0055] The transport mechanism for the liquid sample 40, which has the left and right actuators 31, 32 and the left and right diaphragm pumps, is simple yet capable of stable and continuous liquid transfer.

[0056] 7 is a front view of a PCR analysis device 50 according to the second embodiment. As shown in FIG. 7, the PCR analysis device 50 includes a PCR microfluidic chip 1, one actuator 31, a heating device 33, an observation / analysis device 34, and a control device 35.

[0057] The microfluidic chip 1 may be exactly the same as the microfluidic chip 1 used in the first embodiment. In this embodiment, the first air chamber 10 compressed by the actuator 31 is used as a diaphragm pump, and the second air chamber 12 not compressed by the actuator is used as an expandable and contractible accumulator.

[0058] The actuator 31, the heating device 33, the observation and analysis device 34, and the control device 35 are the same as those in the first embodiment, and will not be described in detail.

[0059] The method of using the PCR analyzer 50 will now be described. First, as shown in FIG. 8, a liquid sample 40 is introduced, or injected, into the flow channel 2 through the port 4 using an introduction device 39 (e.g., a micropipette or syringe). It is not necessary to fill the flow channel 2 and the air chambers 10 and 12 with the liquid sample 40; the amount of the liquid sample 40 may be small, for example, 15 to 20 μl. The liquid sample 40 divides the space within the flow channel 2 and the air chambers 10 and 12 into a left space and a right space. The left space includes the internal space of the first air chamber 10, and the right space includes the internal space of the second air chamber 12. Although not shown, the position of the liquid sample 40 within the flow channel 2 may be adjusted by slightly venting air through the port 6.

[0060] After this, the liquid sample 40 is allowed to release air bubbles, and the ports 4 and 6 are closed and sealed with adhesive tape 41. The adhesive tape 41 may be cellophane tape or polyimide tape. Instead of the adhesive tape 41, resin or rubber plugs may be inserted into the ports 4 and 6 to close and seal the ports 4 and 6.

[0061] Next, the control device 35 activates the drive circuit 31b to drive the actuator 31. As a result, as shown in FIG. 9, the piston 31a of the actuator 31 descends, pushing down the elastic membrane 24 and compressing the first air chamber 10. Therefore, as shown by the arrow, the liquid sample 40 moves rightward within the flow channel 2. However, the piston 31a may have a larger lower end than shown in the figure so that it pushes down the elastic substrate 23 together with the elastic membrane 24. The liquid sample 40 passes through the first curved flow channel section 7 and the straight flow channel section 9, and then through the second curved flow channel section 8. Pressed by the liquid sample 40, the pressure in the right-hand space increases, and the second air chamber 12 elastically expands.

[0062] Next, as shown in FIG. 10, the control device 35 deactivates the drive circuit 31b, causing the piston 31a of the actuator 31 to rise to its initial position. By removing the compressive force from the first air chamber 10, the expanded second air chamber 12 elastically contracts. Therefore, as shown by the arrow, the liquid sample 40 moves leftward within the flow path 2, toward the compressed first air chamber 10. The liquid sample 40 passes through the second curved flow path section 8 and the straight flow path section 9, and then through the first curved flow path section 7. Pressed by the liquid sample 40, the pressure in the left-hand space increases, causing the first air chamber 10 to elastically expand.

[0063] When the control device 35 drives the actuator 31 again, the liquid sample 40 moves to the right within the flow channel 2. When the control device 35 deactivates the actuator 31 again, the liquid sample 40 moves to the left within the flow channel 2.

[0064] In this way, the control device 35 intermittently drives the left and right actuators 31 to intermittently compress the diaphragm pump, i.e., the first air chamber 10. When the first air chamber 10 contracts and the pressure in the left space increases, the pressure in the right space also increases, but because the second air chamber 12 expands, the pressure in the right space does not increase abruptly. Conversely, when the second air chamber 12 contracts and the pressure in the right space increases, the pressure in the left space also increases, but because the first air chamber 10 expands, the pressure in the left space does not increase abruptly. In this way, since the pressure in the flow path 2 does not change abruptly, the generation of bubbles in the liquid sample 40 is reduced, and the rod-shaped liquid sample 40 is also prevented from breaking apart due to the generation of large bubbles.

[0065] The heating device 33 can perform thermal cycling during the reciprocating movement of the liquid sample 40 within the flow channel 2. In one example, while the liquid sample 40 reciprocates within the flow channel 2, the first heating section 37 on the left side may be fixed at a fairly high temperature (e.g., 94°C) for denaturing DNA within the liquid sample 40, and the second heating section 38 on the right side may be fixed at a high temperature (e.g., 60°C) for DNA extension and primer annealing. As the actuator 31 is driven to move the liquid sample 40 to the right within the flow channel 2, the liquid sample 40 reaches its denaturation temperature while passing through the first curved flow channel section 7 and its extension and annealing temperature while passing through the second curved flow channel section 8. The straight flow channel section 9 is not heated, but because it is short, the temperature drop of the liquid sample 40 during its passage therethrough is small. On the other hand, while the actuator 31 is deactivated and the liquid sample 40 moves leftward within the channel 2, the liquid sample 40 remains at the extension / annealing temperature while passing through the second curved channel section 8, and reaches the denaturation temperature while passing through the first curved channel section 7. Next, while the actuator 31 is activated and the liquid sample 40 moves rightward within the channel 2, the liquid sample 40 remains at the denaturation temperature while passing through the first curved channel section 7, and reaches the extension / annealing temperature while passing through the second curved channel section 8. In this example, one round trip of the liquid sample 40 within the channel 2 corresponds to one cycle. The control device 35 intermittently drives the actuator 31 to match the cycle time.

[0066] In another example, while the liquid sample 40 moves back and forth within the channel 2, the first heating element 37 on the left side may be fixed at a high temperature (e.g., 60°C) for DNA extension and primer annealing within the liquid sample 40, and the second heating element 38 on the right side may be fixed at a much higher temperature (e.g., 94°C) for DNA denaturation. While the actuator 31 is driven and the liquid sample 40 moves rightward within the channel 2, the liquid sample 40 reaches the extension / annealing temperature while passing through the first curved channel section 7, and reaches the denaturation temperature while passing through the second curved channel section 8. On the other hand, while the actuator 31 is not driven and the liquid sample 40 moves leftward within the channel 2, the liquid sample 40 remains at the denaturation temperature while passing through the second curved channel section 8, and reaches the extension / annealing temperature while passing through the first curved channel section 7. Next, the actuator 31 is driven to move the liquid sample 40 to the right within the channel 2; the liquid sample 40 remains at the extension / annealing temperature while passing through the first curved channel section 7, and reaches the denaturation temperature while passing through the second curved channel section 8. In this example, one round trip of the liquid sample 40 within the channel 2 corresponds to one cycle. The control device 35 intermittently drives the actuator 31 to match the cycle time.

[0067] In another example, the control device 35 may alternately change the temperatures of the left and right heating sections 37, 38. For example, while the actuator 31 is driven to move the liquid sample 40 to the right within the channel 2, the control device 35 may set the first heating section 37 on the left side to a fairly high temperature (e.g., 94°C) for denaturing DNA in the liquid sample 40, and the second heating section 38 on the right side to a high temperature (e.g., 60°C) for extending the DNA and annealing the primers. The liquid sample 40 reaches the denaturation temperature while passing through the first curved channel section 7, and reaches the extension / annealing temperature while passing through the second curved channel section 8.

[0068] In this example, conversely, while the actuator 31 is deactivated and the liquid sample 40 moves leftward within the flow channel 2, the control device 35 may set the second heating element 38 on the right side to a fairly high temperature (e.g., 94°C) for denaturing the DNA in the liquid sample 40, and the first heating element 37 on the left side to a high temperature (e.g., 60°C) for extending the DNA and annealing the primers. The liquid sample 40 reaches its denaturation temperature while passing through the second curved flow channel section 8, and reaches its extension / annealing temperature while passing through the first curved flow channel section 7. In this example, one round trip of the liquid sample 40 within the flow channel 2 corresponds to two cycles. The control device 35 intermittently drives the actuator 31 to match the cycle time.

[0069] After the actuator 31 is stopped, the liquid sample 40 stops moving when the pressures in the left and right spaces (the pressures in the two air chambers 10, 12) are balanced. The stopping position of the liquid sample 40 remains constant regardless of the number of times the actuator 31 is driven.

[0070] The liquid sample 40 that has stopped moving can be observed by the observation and analysis device 34 through the transparent lower substrate 20. The liquid sample 40 can be observed by the observation and analysis device 34 even while it is moving.

[0071] The applicant repeatedly intermittently drove the actuator 31, causing the liquid sample 40 to move back and forth 50 times within the flow channel 2. No bubbles were observed in the liquid sample 40, and no breakage of the rod-shaped liquid sample 40 was observed.

[0072] In this embodiment, the flow channel 2 and the two air chambers 10, 12 are completely closed by the resin substrates 20, 21, 22, the rubber elastic substrates 23, 25, and the rubber elastic membranes 24, 26, which minimizes contamination of the liquid sample when the liquid sample is caused to flow through the flow channel 2. The microfluidic chip 1 is made of rubber and resin and does not contain any metal, ceramic, or electronic components, so it is inexpensive and can be easily disposed of.

[0073] The transport mechanism for the liquid sample 40, which includes the actuator 31 and the left and right diaphragm pumps, is simple yet capable of stable and continuous liquid transfer. Moreover, this embodiment does not require the actuator 32 of the first embodiment, allowing for a reduction in the number of parts.

[0074] The applicant conducted an experiment on the temperature settings of the heating sections 37 and 38 to perform a more suitable thermal cycle.

[0075] In the first experiment, while the liquid sample 40 was moving back and forth within the channel 2, the first heating element 37 on the left side was fixed at a fairly high denaturation temperature (104°C) to denature the DNA in the liquid sample 40, and the second heating element 38 on the right side was fixed at a high extension / annealing temperature (60°C) to elongate the DNA and anneal the primers. The denaturation temperature was set at 104°C, which is higher than the normal denaturation temperature, to facilitate boiling of the liquid sample 40 (and thus the generation of bubbles in the liquid sample 40). While the actuator 31 was driven to move the liquid sample 40 to the right within the channel 2, the liquid sample 40 reached its denaturation temperature while passing through the first curved channel section 7 and its extension / annealing temperature while passing through the second curved channel section 8. While the actuator 31 is deactivated and the liquid sample 40 moves leftward within the channel 2, the liquid sample 40 remains at the extension / annealing temperature while passing through the second curved channel section 8, and reaches the denaturation temperature while passing through the first curved channel section 7. Next, while the actuator 31 is activated and the liquid sample 40 moves rightward within the channel 2, the liquid sample 40 remains at the denaturation temperature while passing through the first curved channel section 7, and reaches the extension / annealing temperature while passing through the second curved channel section 8.

[0076] In the second experiment, while the liquid sample 40 was moving back and forth within the channel 2, the first heating element 37 on the left side was fixed at a high extension / annealing temperature (60°C) for DNA extension and primer annealing within the liquid sample 40, and the second heating element 38 on the right side was fixed at a much higher denaturation temperature (104°C) for DNA denaturation. While the actuator 31 was driven and the liquid sample 40 was moving rightward within the channel 2, the liquid sample 40 reached the extension / annealing temperature while passing through the first curved channel section 7 and reached the denaturation temperature while passing through the second curved channel section 8. While the actuator 31 was not driven and the liquid sample 40 was moving leftward within the channel 2, the liquid sample 40 remained at the denaturation temperature while passing through the second curved channel section 8 and reached the extension / annealing temperature while passing through the first curved channel section 7. Next, the actuator 31 is driven to move the liquid sample 40 to the right within the channel 2, while the liquid sample 40 remains at the extension / annealing temperature while passing through the first curved channel section 7 and reaches the denaturation temperature while passing through the second curved channel section 8.

[0077] In the experiments, the liquid sample 40 was stopped in the denaturation temperature range (first curved flow path section 7 in the first experiment, and second curved flow path section 8 in the second experiment), and the state of the liquid sample 40 (whether bubbles were generated, and whether the liquid sample 40 was broken up due to the generation of large bubbles) was examined 15 seconds, 30 seconds, and 60 seconds after the stoppage. The results are shown in FIG. 11. In the first experiment, the liquid sample 40 broke up 30 seconds after the liquid sample 40 stopped in the first curved flow path section 7. On the other hand, in the second experiment, although bubbles were generated after the liquid sample 40 stopped in the second curved flow path section 8, the liquid sample 40 did not break up even after 60 seconds had passed.

[0078] Therefore, the liquid sample 40 is less likely to boil when the second curved channel section 8, which is connected to the second air chamber 12 that is not compressed by the actuator, is controlled to the denaturation temperature than the first curved channel section 7, which is connected to the first air chamber 10 that is compressed by the actuator. This is thought to be because the second curved channel section 8 is farther from the first air chamber 10, in which the liquid sample 40 is compressed by the actuator, than the first curved channel section 7. In other words, regardless of the direction of movement of the liquid sample 40, when the liquid sample 40 is located in the second curved channel section 8, a higher pressure is applied to the liquid sample 40 than when the liquid sample 40 is located in the first curved channel section 7. The boiling point is higher under higher pressure.

[0079] In PCR thermal cycles, the DNA denaturation time is often 30 seconds or less. Therefore, it is preferable to set the second curved channel section 8 to a higher temperature than the first curved channel section 7. That is, it is preferable that the first heating section 37 heats the liquid sample in the first curved channel section 7 to the extension temperature, and the second heating section 38 heats the liquid sample in the second curved channel section 8 to a denaturation temperature higher than the extension temperature.

[0080] Although the present invention has been shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that changes may be made in form and detail without departing from the scope of the invention as set forth in the appended claims. Such changes, modifications and alterations are intended to be included within the scope of the invention.

[0081] For example, in the above embodiment, elastic substrates 23 and 25 are provided to define air chambers 10 and 12. However, bellows may be used instead of elastic substrates 23 and 25. In this case, the bellows airtightly connects elastic membrane 24 to upper substrate 22, and airtightly connects elastic membrane 26 to upper substrate 22.

[0082] In the first embodiment, the material and structure of the members defining the first air chamber 10 are preferably the same as the material and structure of the members defining the second air chamber 12. However, in the second embodiment, the material and structure of the members defining the first air chamber 10 may be the same as or different from the material and structure of the members defining the second air chamber 12.

[0083] In the above embodiment, the PCR analysis device 30 or the PCR analysis device 50 has the observation analysis device 34. However, after the thermal cycle is completed, the liquid sample 40 may be extracted from the microfluidic chip 1 using a micropipette or syringe, and the extracted liquid sample 40 may be filtered by electrophoresis to measure the size of the amplified product in the liquid sample 40. Therefore, the observation analysis device 34 is not essential.

[0084] Aspects of the invention are also described in the following numbered clauses.

[0085] Clause 1. A flow path through which a liquid sample used in PCR flows; an inlet for introducing the liquid sample into the flow channel; Two elastically expandable air chambers connected to both ends of the flow path, respectively; an enclosing member that encloses the flow path and the air chamber in an airtight manner and has a transparent portion on at least one side; A microfluidic chip for PCR.

[0086] Clause 2. A PCR microfluidic chip according to clause 1; two actuators that alternately compress the two air chambers; a heating device for heating the flow path; PCR analysis equipment.

[0087] According to this provision, two actuators alternately compress two air chambers connected to both ends of the flow path, thereby promoting the reciprocating movement of the liquid sample. A heating device heats the flow path during the reciprocating movement, subjecting the liquid sample to thermal cycling. After the actuators are stopped, the liquid sample stops moving when the pressures in the two air chambers are equilibrated.

[0088] Clause 3. A PCR microfluidic chip according to clause 1; an actuator that intermittently compresses a first air chamber of the two air chambers; a heating device for heating the flow path; PCR analysis equipment.

[0089] According to this provision, when the first air chamber is compressed by the actuator, the liquid sample flows from the compressed first air chamber toward the other air chamber, causing the downstream air chamber to elastically expand. When the compression force is removed, the expanded air chamber elastically contracts, causing the liquid sample to flow in the opposite direction toward the compressed first air chamber, causing the compressed first air chamber to elastically expand. In this way, the liquid sample can move back and forth through the flow path. A heating device heats the flow path during the reciprocating movement, causing the liquid sample to be thermally cycled. After the actuator is stopped, the liquid sample stops moving when the pressures in the two air chambers are equilibrated.

[0090] Clause 4. The flow path has a first curved flow path portion communicating with the first air chamber compressed by the actuator, a second curved flow path portion communicating with a second air chamber of the two air chambers that is not compressed by the actuator, and a straight flow path portion connecting the first curved flow path portion and the second curved flow path portion; the heating device has a first heating section that heats the first curved flow path section and a second heating section that heats the second curved flow path section, The first heating section heats the liquid sample in the first curved channel section to an extension / annealing temperature, and the second heating section heats the liquid sample in the second curved channel section to a denaturation temperature higher than the extension / annealing temperature. 4. A PCR analysis device according to clause 3, characterized in that

[0091] This provision prevents the generation of bubbles in the liquid sample or the disruption of the liquid sample.

[0092] Clause 5. The PCR microfluidic chip further includes an observation and analysis device for observing and analyzing the liquid sample in the flow channel through the transparent portion of the PCR microfluidic chip. 5. A PCR analysis device according to any one of clauses 2 to 4, characterized in that

[0093] This provision allows for easy observation and analysis of liquid samples during or after movement has stopped. [Explanation of symbols]

[0094] 1. Microfluidic chip for PCR 2 Flow path 4 ports (inlet) 6 ports 10 First air chamber 12 Second air chamber 7 First curved flow path section 8 Second curved flow path section 9 Straight flow path section 20 Lower substrate (enclosure member) 21 Flow path substrate (enclosure member) 22 Upper substrate (enclosure member) 23, 25 Elastic substrate (enclosure member) 24, 26 Elastic membrane (enclosure member) 30,50 PCR analyzer 31,32 Actuator 33 Heating device 34 Observation and analysis equipment 35 Control device 37 First heating section 38 Second heating section 37a, 38a Peltier element 37b,38b metal plate 40 liquid samples

Claims

1. a flow channel through which a liquid sample used in PCR flows; an inlet for introducing the liquid sample into the flow channel; a first air chamber and a second air chamber, each connected to both ends of the flow path and capable of elastically expanding and contracting; an enclosing member that airtightly encloses the flow path, the first air chamber, and the second air chamber and has a transparent portion on at least one side; Has, the surrounding member has a flow path substrate made of resin in which a through groove forming the flow path is formed, a lower substrate joined to a lower surface of the flow path substrate, and an upper substrate made of resin joined to an upper surface of the flow path substrate, a first air chamber is defined by the lower substrate, a through-hole formed in the flow path substrate and connected to the through groove, a through-hole formed in the upper substrate, a through-hole formed in a first elastic substrate made of rubber and bonded to the upper substrate, and a first elastic film made of rubber that is a flat plate bonded to the first elastic substrate; The second air chamber is defined by the lower substrate, a through-hole formed in the flow path substrate and connected to the through groove, a through-hole formed in the upper substrate, a through-hole formed in a second elastic substrate made of rubber and bonded to the upper substrate, and a second elastic film made of rubber that is a flat plate bonded to the second elastic substrate. Microfluidic chip for PCR.

2. The PCR microfluidic chip according to claim 1; two actuators that alternately compress the first air chamber and the second air chamber; a heating device for heating the flow path; A PCR analysis device having:

3. The PCR microfluidic chip according to claim 1; an actuator that intermittently compresses a first air chamber of the two air chambers; a heating device for heating the flow path; A PCR analysis device having:

4. the flow path includes a first curved flow path portion communicating with the first air chamber compressed by the actuator, a second curved flow path portion communicating with a second air chamber of the two air chambers that is not compressed by the actuator, and a straight flow path portion connecting the first curved flow path portion and the second curved flow path portion, the heating device has a first heating section that heats the first curved flow path section and a second heating section that heats the second curved flow path section, The first heating section heats the liquid sample in the first curved channel section to an extension / annealing temperature, and the second heating section heats the liquid sample in the second curved channel section to a denaturation temperature higher than the extension / annealing temperature. The PCR analysis device according to claim 3 .

5. The PCR microfluidic chip further includes an observation and analysis device for observing and analyzing the liquid sample in the flow channel through the transparent portion of the PCR microfluidic chip.

5. The PCR analysis device according to claim 2, wherein the PCR analysis device is a PCR analyzer.

6. A flow path through which a liquid sample used in PCR flows; an inlet for introducing the liquid sample into the flow channel; a first air chamber and a second air chamber, each connected to both ends of the flow path and capable of elastically expanding and contracting; an enclosing member that encloses the flow path and the air chamber in an airtight manner and has a transparent portion on at least one side; a PCR microfluidic chip having the an actuator that intermittently compresses the first air chamber; a heating device that heats the flow path, When the actuator compresses the first air chamber, the liquid sample in the flow path passes through a portion of the flow path that is heated by the heating device and a portion of the flow path that is not heated, causing the second air chamber to elastically expand; When the actuator removes the compressive force on the first air chamber, the second air chamber elastically contracts, forcing the liquid sample in the flow path back through a portion of the flow path that is not heated by the heating device and a portion of the flow path that is heated, causing the first air chamber to elastically expand. PCR analyzer.

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