A vertical microfluidic chip and method for PCR detection

Abstract

The present invention discloses a vertical microfluidic chip and method for PCR detection. The microfluidic chip includes: a chip body having a separation and purification chamber, a reagent chamber, and a reaction chamber; a first communication piston rotatably inserted into the chip body and having a first liquid flow groove capable of communicating the separation and purification chamber and the reagent chamber; a second communication piston rotatably inserted into the chip body and having a second liquid flow groove capable of communicating the separation and purification chamber and the reaction chamber; a driving piston movably disposed in a piston chamber of the chip body, the piston chamber being divided by the piston into a first piston chamber portion and a second piston chamber portion, the first piston chamber portion communicating with the separation and purification chamber; the first communication piston further having a first gas flow groove capable of communicating the reagent chamber and the second piston chamber portion; and the second communication piston further having a second gas flow groove capable of communicating the reaction chamber and the second piston chamber portion. The present invention has relatively accurate liquid quantification and no aerosol contamination.

Description

Technical Field

[0001] The present invention belongs to the technical field of PCR detection, and relates to a vertical microfluidic chip and method for PCR detection. Background Art

[0002] Currently, microfluidic chips are applied in the field of biological detection. They can be placed in a PCR instrument for reaction to achieve the purpose of detection. For example, nucleic acid extraction and amplification are carried out. To avoid contamination, the reagents required for the reaction are pre-loaded into the chambers in the microfluidic chip. During the reaction, the flow direction of the liquid is controlled according to the set reaction procedure, and the reagents, samples, reaction solutions, etc. are controlled to flow into the designated chambers. Therefore, a plurality of pistons for controlling the liquid flow direction or the connection between chambers are arranged on the microfluidic chip. By rotating or moving these pistons, the connection state of the flow channels between the chambers can be switched, or the driving force for controlling the liquid flow can be provided. Most of the microfluidic chips currently used for PCR nucleic acid detection are horizontal chips (the height is less than its size in the horizontal direction), that is, the chambers and the microchannels connecting the chambers are generally at the same horizontal height, and the inside of the chip needs to exchange air flow with the outside air, resulting in problems such as inaccurate liquid quantification and aerosol contamination. Pathogens may enter the outside air and cause contamination. Summary of the Invention

[0003] The object of the present invention is to provide a vertical microfluidic chip for PCR detection, which has relatively accurate liquid quantification and can reduce the risk of aerosol contamination or even eliminate aerosol contamination.

[0004] Another object of the present invention is to provide a method for PCR detection, which is easy to operate, has relatively accurate reagent quantification, and can reduce the risk of aerosol contamination or even eliminate aerosol contamination.

[0005] According to the first aspect of the present invention, a vertical microfluidic chip for PCR detection includes:

[0006] A chip body, the size of which in the up-down direction is greater than at least one of its sizes in the left-right direction or the front-back direction. The chip body has a separation and purification chamber for containing a sample, a reagent chamber for storing separation and purification reagents, and a reaction chamber for performing PCR amplification;

[0007] A first communication piston rotatably inserted into the chip body around a rotation axis extending in the left-right direction. The first communication piston has a first liquid flow groove capable of communicating the separation and purification chamber and the reagent chamber;

[0008] A second communication piston rotatably inserted into the chip body about a rotation axis extending in the left-right direction, the second communication piston having a second liquid flow groove capable of communicating the separation and purification chamber and the reaction chamber; and

[0009] A driving piston for driving the flow of liquid, a piston chamber provided on the chip body, the driving piston movably disposed in the piston chamber, the piston chamber being divided by the piston into a first piston chamber portion and a second piston chamber portion, the first piston chamber portion communicating with the separation and purification chamber;

[0010] Wherein, the first communication piston further has a first air flow groove capable of communicating the reagent chamber and the second piston chamber portion; the second communication piston further has a second air flow groove capable of communicating the reaction chamber and the second piston chamber portion.

[0011] In one embodiment, a sampling port communicating with the separation and purification chamber is provided on the chip body, and the chambers in the chip body can communicate with the outside only through the sampling port during sampling, and the chambers in the chip body are isolated from the outside after the sampling port is closed.

[0012] In one embodiment, the separation and purification chamber, the reagent chamber and the reaction chamber are arranged from top to bottom, the first communication piston is located between the separation and purification chamber and the reagent chamber, the second communication piston is located between the reagent chamber and the reaction chamber, the first communication piston further has a liquid flow transfer groove and an air flow transfer groove, the chip body further has a liquid flow transfer channel and an air flow transfer channel, the liquid flow transfer groove, the liquid flow transfer channel and the second liquid flow groove can be sequentially docked to communicate the separation and purification chamber and the reaction chamber, and the second air flow groove, the air flow transfer channel and the air flow transfer groove can be sequentially docked to communicate the reaction chamber and the second piston chamber portion.

[0013] More preferably, the vertical microfluidic chip has a first working state and a second working state. In the first working state, the separation and purification chamber communicates with the reagent chamber only through the first liquid flow groove, the reagent chamber communicating with the separation and purification chamber communicates with the second piston chamber portion through the corresponding first air flow groove, the liquid flow transfer groove and the liquid flow transfer channel are misaligned, and the air flow transfer groove and the air flow transfer channel are misaligned; in the second working state, the separation and purification chamber communicates with the reaction chamber sequentially through the liquid flow transfer groove, the liquid flow transfer channel and the second liquid flow groove, the reaction chamber communicates with the second piston chamber portion sequentially through the second air flow groove, the air flow transfer channel and the air flow transfer groove, the separation and purification chamber is misaligned with the first liquid flow groove, and the second piston chamber portion is misaligned with the first air flow groove.

[0014] Further, the number of the reagent chambers is multiple and they are arranged side by side in the left - right direction. Each of the reagent chambers corresponds to and is respectively communicated with a first liquid flow groove and a first gas flow groove. The multiple first liquid flow grooves on the first communication piston are arranged at intervals along its circumferential direction, and the multiple first gas flow grooves on the first communication piston are arranged at intervals along its circumferential direction. The vertical microfluidic chip has multiple first working states. In any one of the first working states, only one of the reagent chambers is communicated with the separation and purification chamber through the corresponding first liquid flow groove and with the second piston chamber through the corresponding first gas flow groove.

[0015] More preferably, the number of the reaction chambers is multiple and they are arranged side by side in the left - right direction. The number of the second liquid flow grooves is one and at least a part of it extends in the left - right direction. Each of the reaction chambers corresponds to and is communicated with a second gas flow groove. The vertical microfluidic chip has a second working state. In the second working state, all the reaction chambers are communicated with the separation and purification chamber through the second liquid flow groove, and each reaction chamber is communicated with the gas flow transfer channel through the corresponding second gas flow groove.

[0016] More preferably, the microfluidic chip further includes a waterproof and breathable membrane covering the surface of the chip body. The waterproof and breathable membrane can allow air to pass through while blocking liquid. The waterproof and breathable membrane is located on the gas passage between the reaction chamber and the second piston chamber. Further, the waterproof and breathable membrane is provided on the gas passage between each reaction chamber and the second piston chamber, and the waterproof and breathable membrane is arranged before the gas flow transfer channel.

[0017] More preferably, the chip body further has liquid flow micro - channels and gas flow micro - channels corresponding to each chamber respectively.

[0018] The separation and purification chamber can be butt - connected and communicated with the inlet of the first liquid flow groove through a liquid flow micro - channel, and the separation and purification chamber is communicated with the first piston chamber through a gas flow micro - channel; and / or

[0019] Each reagent chamber can be butt - connected and communicated with the outlet of the first liquid flow groove through a liquid flow micro - channel, and each reagent chamber is communicated with the inlet of the first gas flow groove through a gas flow micro - channel;

[0020] and / or each reaction chamber can be butt - connected and communicated with the outlet of the second liquid flow groove through a liquid flow micro - channel, and each reaction chamber can be communicated with the inlet of the second gas flow groove through a gas flow micro - channel.

[0021] More preferably, the liquid flow transfer tank is located on the left side of the first liquid flow tank, the first gas flow tank is located on the right side of the corresponding first liquid flow tank, and the outlets of the gas flow transfer tank and the first gas flow tank are aligned; and / or, the second liquid flow tank has one inlet and a plurality of outlets spaced along the left-right direction, and a plurality of the second gas flow tanks are spaced along the left-right direction.

[0022] In one embodiment, the reagent chamber and the reaction chamber are formed on the surface of the chip body. A microchannel for chamber communication is provided inside and / or on the surface of the chip body. The vertical microfluidic chip further includes a sealing film covering the surface of the chip body; and / or, the thickness of the lower part of the chip body is smaller than that of the upper part, and the reaction chamber is provided on the lower part of the chip body; and / or, the vertical microfluidic chip further includes a magnet assembly capable of adsorbing or releasing nucleic acid by magnetic beads. The magnet assembly includes a rotatable mounting disk and a plurality of magnets, and the plurality of magnets are spaced along the circumferential direction of the mounting disk on the mounting disk.

[0023] According to the second aspect of the present invention, a method for PCR detection uses the vertical microfluidic chip as described above. The method includes the following steps:

[0024] A. Add the nucleic acid sample into the separation and purification chamber from the sample addition port, and seal the sample addition port;

[0025] B. Rotate the first communication piston to connect the first liquid flow tank with the separation and purification chamber and the reagent chamber, and connect the first gas flow tank with the reagent chamber and the second piston chamber part; move the driving piston to transfer the reagent into the separation and purification chamber;

[0026] C. Rotate the first communication piston and the second communication piston to connect the liquid flow transfer tank on the first communication piston, the liquid flow transfer channel on the chip body, and the second liquid flow tank on the second communication piston in sequence to connect the separation and purification chamber and the reaction chamber, and connect the gas flow transfer tank on the first communication piston, the gas flow transfer channel on the chip body, and the second gas flow tank on the second communication piston in sequence to connect the second piston chamber part and the reaction chamber; move the driving piston to distribute the liquid in the separation and purification chamber into the reaction chamber.

[0027] The present invention adopts the above solutions and has the following advantages compared with the prior art:

[0028] The vertical microfluidic chip for PCR detection of the present invention adopts a vertical structure, and during the amplification and detection process, a sealed space isolated from the outside air is formed inside the chip, and nucleic acid amplification and detection are carried out in this sealed space, effectively eliminating the influence of bubbles, enabling accurate liquid intake, realizing precise liquid quantification, avoiding the influence of outside air on the PCR reaction, reducing or even eliminating aerosol contamination, and preventing pathogens from escaping into the outside air. The method for PCR detection of the present invention is convenient and controllable in operation, capable of realizing automated detection, with accurate liquid intake, capable of realizing precise liquid quantification, avoiding the influence of outside air on the PCR reaction, reducing or even eliminating aerosol contamination, and preventing pathogens from escaping into the outside air. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings based on these drawings without creative efforts.

[0030] Figure 1 FIG. 9 is a three-dimensional schematic diagram of a vertical microfluidic chip according to an embodiment of the present invention;

[0031] Figure 2 FIG. 13 is a side view of the microfluidic chip according to an embodiment of the present invention;

[0032] Figure 3 FIG. 17 is a schematic diagram of one side surface of the chip body;

[0033] Figure 4 FIG. 21 is a schematic diagram of the other side surface of the chip body;

[0034] Figure 5a 、 Figure 5b FIGS. 27(a) and 27(b) are schematic diagrams of chamber communication of the microfluidic chip in two different perspectives in the first working state;

[0035] Figure 6a 、 Figure 6b FIGS. 33(a) and 33(b) are schematic diagrams of chamber communication of the microfluidic chip in two different perspectives in the second working state;

[0036] Figure 7a 、 Figure 7b 、 Figure 7c 、 Figure 7d FIGS. 43(a), 43(b), 43(c), and 43(d) are schematic diagrams of the first connecting piston in four different perspectives;

[0037] Figure 8a 、 Figure 8b FIGS. 49(a) and 49(b) are schematic diagrams of the second connecting piston in two different perspectives.

[0038] Among them,

[0039] 1. Chip body; 11. Separation and purification chamber; 11a. Sampling port; 111. Reserved chamber; 112. Freeze-drying ball chamber; 113. Waste liquid chamber; 12. Reagent chamber; 13. Reaction chamber; 14. Piston chamber; 141. First piston chamber part; 142. Second piston chamber part; 101a. Liquid flow microchannel; 101b. Liquid flow microchannel; 101c. Liquid flow microchannel; 102a. Gas flow microchannel; 102b. Gas flow microchannel; 102c. Gas flow microchannel; 102d. Gas flow microchannel; 103. Liquid flow transfer channel; 104. Gas flow transfer channel; 104a. Inlet; 104b. Outlet;

[0040] 2. First connecting piston; 21. First liquid flow groove; 22. First gas flow groove; 23. Liquid flow transfer groove; 24. Gas flow transfer groove;

[0041] 3. Second connecting piston; 31. Second liquid flow groove; 31a. Inlet; 31b. Outlet; 32. Second gas flow groove;

[0042] 4. Driving piston; 41. Piston rod;

[0043] 5. Magnet assembly; 51. Mounting plate; 52. Magnet; 53. Motor;

[0044] 6. Waterproof and breathable membrane. Detailed implementation manners

[0045] The following elaborates on the preferred embodiments of the present invention in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art. It should be noted here that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation to the present invention.

[0046] As shown in this specification and the claims, the terms "including" and "comprising" only indicate the inclusion of the steps and elements that have been clearly identified, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements. The term "and / or" used herein includes any combination of one or more of the related listed items.

[0047] It should be noted that unless otherwise specified, when a feature is referred to as "fixed" or "connected" to another feature, it can be directly fixed or connected to another feature, or indirectly fixed or connected to another feature. In addition, the up, down, left, right, etc. descriptions used in the present invention are only relative to the mutual positional relationship of the components of the present invention in the accompanying drawings. Specifically, based on the Figure 3 、 5a And 6a, with the upper side of the paper surface as the top, the lower side as the bottom, the left side as the left, and the right side as the right.

[0048] It can be further understood that in the present disclosure, "a plurality of" means two or more, and other quantifiers are similar thereto. "And / or" describes the association relationship of associated objects and indicates that three relationships can exist. For example, A and / or B can represent three situations: A exists alone, A and B exist simultaneously, and B exists alone.

[0049] It can be further understood that the terms "first", "second", etc. are used to describe various information, but this information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other and do not represent a specific order or importance. In fact, expressions such as "first" and "second" can be used interchangeably. For example, without departing from the scope of the present disclosure, the first information can also be referred to as the second information, and similarly, the second information can also be referred to as the first information.

[0050] According to an embodiment of the present invention, a vertical microfluidic chip for PCR detection is as Figures 1 to 8b shown. Referring to Figures 1 to 8b , the vertical microfluidic chip mainly includes a chip body 1, a first communication piston 2, a second communication piston 3, a driving piston 4 and a magnet assembly 5. Chambers and microchannels are provided inside and / or on the surface of the chip body 1. The first communication piston 2 and the second communication piston 3 are used to switch the connection or blockage of the chambers and microchannels. The driving piston 4 is used to provide the power for liquid flow, and the magnet assembly 5 is used to provide a magnetic field for adsorbing samples with magnetic beads.

[0051] The chip body 1 has a dimension in the vertical direction greater than at least one of its dimensions in the left-right direction or the front-back direction. Further, the chip body 1 is vertical, and its height is greater than its length and width. The chip body 1 has a separation and purification chamber 11 for containing a sample, a reagent chamber 12 for storing separation and purification reagents, and a reaction chamber 13 for performing PCR amplification. Specifically, the number of separation and purification chambers 11 is one, which is provided inside the chip body 1; a sample addition port 11a communicating with the separation and purification chamber 11 is provided on the chip body 1. The chambers inside the chip body 1 can only communicate with the outside through the sample addition port 11a during sample addition. After the sample addition port 11a is closed, the chambers inside the chip body 1 are isolated from the outside. That is, only during sample addition, the sample addition port 11a is opened to add a nucleic acid sample into the separation and purification chamber 11; after sample addition is completed, the sample addition port 11a is sealed with a cover or a film to isolate the chambers inside the microfluidic chip from the outside air, and nucleic acid extraction, purification, amplification, etc. are performed in a closed environment. The reagent chamber 12 is located below the separation and purification chamber 11 and is used to store reagents for nucleic acid extraction and purification. Further, the number of reagent chambers 12 is multiple and they are arranged at intervals in the left-right direction. Specifically in this embodiment, the number of reagent chambers 12 is five, which are used to store eluent, magnetic bead-containing reagent, lysis solution / isopropanol, washing solution A, and washing solution B in sequence. The reaction chamber 13 is located below the reagent chamber 12 and is used to store reagents such as primers for PCR amplification and provide the reaction chamber 13 for performing PCR amplification. Further, the number of reaction chambers 13 is multiple and they are arranged at intervals in the left-right direction.

[0052] The first connecting piston 2 is rotatably inserted into the chip body 1 about a rotation axis line extending in the left-right direction. The first connecting piston 2 has a first liquid flow groove 21 capable of communicating the separation and purification chamber 11 and the reagent chamber 12. The second connecting piston 3 is rotatably inserted into the chip body 1 about a rotation axis line extending in the left-right direction. The second connecting piston 3 has a second liquid flow groove 31 capable of communicating the separation and purification chamber 11 and the reaction chamber 13. A piston chamber 14 is provided inside the upper part of the chip body 1. The driving piston 4 is arranged in the piston chamber 14 so as to be movable in the left-right direction. The piston chamber 14 is divided into a first piston chamber part 141 and a second piston chamber part 142 by the piston. The first piston chamber part 141 is communicated with the separation and purification chamber 11. The first connecting piston 2 further has a first air flow groove 22 capable of communicating the reagent chamber 12 and the second piston chamber part 142; the second connecting piston 3 further has a second air flow groove 32 capable of communicating the reaction chamber 13 and the second piston chamber part 142. Specifically, the overall bodies of the first connecting piston 2 and the second connecting piston 3 extend in the left-right direction respectively. Their rotation axis lines extend in the left-right direction and are parallel to each other. The major-minor axis ratio of the two connecting pistons is greater than 1, preferably greater than 5; the driving piston 4 is driven by a piston rod 41 extending in the left-right direction to move left and right in the piston chamber 14. A first piston chamber part 141 is formed on the left side of the driving piston 4, and a second piston chamber part 142 is formed on the right side of the driving piston 4. The driving piston 4, the first connecting piston 2 and the second connecting piston 3 are arranged from top to bottom.

[0053] Specifically in this embodiment, the separation and purification chamber 11, the reagent chamber 12 and the reaction chamber 13 are arranged from top to bottom. The first connecting piston 2 is located between the separation and purification chamber 11 and the reagent chamber 12. The second connecting piston 3 is located between the reagent chamber 12 and the reaction chamber 13. The first connecting piston 2 further has a liquid flow transfer groove 23 and an air flow transfer groove 24. The chip body 1 further has a liquid flow transfer channel 103 and an air flow transfer channel 104. The liquid flow transfer groove 23, the liquid flow transfer channel 103 and the second liquid flow groove 31 can be sequentially docked to communicate the separation and purification chamber 11 and the reaction chamber 13. The second air flow groove 32, the air flow transfer channel 104 and the air flow transfer groove 24 can be sequentially docked to communicate the reaction chamber 13 and the second piston chamber part 142. The vertical microfluidic chip has a first working state and a second working state. Combining Figure 5a and Figure 5b As shown, in the first working state, the separation and purification chamber 11 is only communicated with a reagent chamber 12 through a first liquid flow groove 21. The reagent chamber 12 communicated with the separation and purification chamber 11 is communicated with the second piston chamber part 142 through a corresponding first air flow groove 22. The liquid flow transfer groove 23 and the liquid flow transfer channel 103 are misaligned, and the air flow transfer groove 24 and the air flow transfer channel 104 are misaligned. Combining Figure 6a and Figure 6bAs shown, in the second working state, the separation and purification chamber 11 is sequentially connected to the reaction chamber 13 through the liquid flow transfer tank 23, the liquid flow transfer channel 103, and the second liquid flow tank 31. The reaction chamber 13 is sequentially connected to the second piston chamber part 142 through the second gas flow tank 32, the gas flow transfer channel 104, and the gas flow transfer tank 24. The separation and purification chamber 11 is misaligned with the first liquid flow tank 21, and the second piston chamber part 142 is misaligned with the first gas flow tank 22.

[0054] Referring to Figure 5b and Figure 6b As shown, a reserved chamber 111 is also provided on the chip body 1. The reserved chamber 111 is located on the right side of the separation and purification chamber 11 and is reserved for other uses. Referring to Figure 5b and Figure 6b As shown, a freeze-dried ball chamber 112 is also provided on the chip body 1. The freeze-dried ball chamber 112 is located on the right side of the separation and purification chamber 11, specifically on the right side of the reserved chamber 111. The freeze-dried ball chamber 112 is used to store or stores freeze-dried balls, and the freeze-dried balls contain reagents for amplification. The freeze-dried ball chamber 112 and the separation and purification chamber 11 are connected through a liquid flow channel, and the freeze-dried ball chamber 112 and the second piston chamber part 142 are connected through a gas flow channel. The eluent in the reagent chamber 12 can enter the freeze-dried ball chamber 112 to dissolve the freeze-dried balls and then be transferred into the separation and purification chamber 11 and then injected into the reaction chamber 13. Referring to Figure 5b and Figure 6b As shown, a waste liquid chamber 113 is also provided on the chip body 1. The waste liquid chamber 113 is used to store the waste liquid generated during the separation and purification process. The waste liquid chamber 113 is located on the right side of the separation and purification chamber 11, specifically on the right side of the freeze-dried ball chamber 112. The waste liquid chamber 113 and the separation and purification chamber 11 are connected through a liquid flow channel, and the waste liquid chamber 113 and the second piston chamber part 142 are connected through a gas flow channel so that the waste liquid in the separation and purification chamber 11 can be controlled to flow into the waste liquid chamber 113 by moving the driving piston 4.

[0055] As Figure 3 shown, each reagent chamber 12 corresponds to and is respectively connected to a first liquid flow tank 21 and a first gas flow tank 22. As Figures 7a to 7dAs shown, a plurality of first liquid flow grooves 21 on the first communication piston 2 are arranged at intervals along its circumferential direction, and a plurality of first gas flow grooves 22 on the first communication piston 2 are arranged at intervals along its circumferential direction. The vertical microfluidic chip has a plurality of first working states. In any one of the first working states, only one of the reagent chambers 12 and the separation and purification chamber 11 are connected through the corresponding first liquid flow grooves 21 and are connected to the second piston chamber portion 142 through the corresponding first gas flow grooves 22. The first communication piston 2 can selectively connect one of the separation and purification chamber 11 and the reagent chambers 12, while disconnecting the other reagent chambers 12; at the same time, the first communication piston 2 can also selectively connect only the reagent chamber 12 connected to the separation and purification chamber 11 and the second piston chamber portion 142. As the first communication piston 2 rotates by a certain angle, the first communication piston 2 connects the other one of the separation and purification chamber 11 and the reagent chambers 12, while disconnecting the other reagent chambers 12; the first communication piston 2 also only connects the other reagent chamber 12 and the second piston chamber portion 142. Specifically, it is connected through the first liquid flow grooves 21 and the first gas flow grooves 22 provided on the outer surface of the first communication piston 2.

[0056] As Figure 4 , Figure 8a and Figure 8b shown, the number of the second liquid flow grooves 31 is one and at least a part of it extends in the left-right direction, and each reaction chamber 13 corresponds to and is connected to a second gas flow groove 32. The vertical microfluidic chip has a second working state. In the second working state, all the reaction chambers 13 are connected to the separation and purification chamber 11 through the second liquid flow grooves 31, and each reaction chamber 13 is connected to the gas flow transfer channel 104 through the corresponding second gas flow grooves 32.

[0057] The chip body 1 also has liquid flow microchannels 101a, 101b, 101c and gas flow microchannels 102a, 102b, 102c, 102d corresponding to each chamber respectively. The separation and purification chamber 11 can be connected to the inlet of the first liquid flow groove 21 through a liquid flow microchannel 101a, the separation and purification chamber 11 is connected to the first piston chamber portion 141 through a gas flow microchannel 102a, and the second piston chamber portion 142 can be connected to the outlet of the first gas flow groove 22 or the outlet of the gas flow transfer groove 24 on the first communication piston 2 through a gas flow microchannel 102b. Each reagent chamber 12 can be connected to the outlet of the first liquid flow groove 21 through a liquid flow microchannel 101b, and each reagent chamber 12 is connected to the inlet of the first gas flow groove 22 through a gas flow microchannel 102c. Each reaction chamber 13 can be connected to the outlet of the second liquid flow groove 31 through a liquid flow microchannel 101c, and each reaction chamber 13 is connected to the inlet of the second gas flow groove 32 through a gas flow microchannel 102d.

[0058] The reagent chamber 12 and the reaction chamber 13 are opened on the surface of the chip body 1. The liquid flow microchannels 101a, 101b, 101c, the gas flow microchannels 102a, 102b, 102c, 102d, the liquid flow transfer channel 103, and part of the gas flow transfer channel 104 are located on the surface of the chip body 1 while part is inside the chip body 1. The vertical microfluidic chip further includes a sealing film (not shown in the figure) covering the surface of the chip body 1 to seal the reagent chamber 12, the reaction chamber 13, and the microchannels connected thereto.

[0059] For the layout of the upper grooves of the first communication piston 2, refer to Figures 7a to 7d As shown, the inlets of the first liquid flow grooves 21 and the inlet of the liquid flow transfer groove 23 are aligned with each other, so that they can all be aligned and connected to the liquid flow microchannel 101a of the separation and purification chamber 11 to achieve communication with the separation and purification chamber 11; thus, the inlets of the first liquid flow grooves 21 and the inlet of the liquid flow transfer groove 23 are all located at the same circumferential position of the first communication piston 2, that is, their axial distance is zero. The outlets of the first gas flow grooves 22 and the outlet of the gas flow transfer groove 24 are aligned with each other, so that they can all be aligned and connected to the gas flow microchannel 102b of the second piston cavity 142 to achieve communication with the second piston cavity 142; thus, the outlets of the first gas flow grooves 22 and the outlet of the gas flow transfer groove 24 are all located at the same circumferential position of the first communication piston 2, that is, their axial distance is zero. Combining Figures 7a to 7d As shown, the liquid flow transfer groove 23 is located on the left side of the first liquid flow groove 21, the first gas flow groove 22 is located on the right side of the corresponding first liquid flow groove 21, and the outlet of the gas flow transfer groove 24 is aligned with the outlet of the first gas flow groove 22.

[0060] For the layout of the upper grooves of the second communication piston 3, refer to Figure 8a and Figure 8b As shown, the second liquid flow groove 31 has an inlet 31a and a plurality of outlets 31b arranged at intervals in the left-right direction. After the second communication piston 3 rotates to a certain position, the inlet 31a of the second liquid flow groove 31 is aligned and connected to the outlet of the liquid flow transfer channel 103, and each outlet 31b is respectively aligned and connected to the inlet of the liquid flow microchannel 101c of a corresponding reaction chamber 13. A plurality of second gas flow grooves 32 are arranged at intervals in the left-right direction, and their inlets are aligned and connected to the outlets of the gas flow microchannels 102d of a corresponding reaction chamber 13. The outlets of the second gas flow grooves 32 converge inside the chip body 1 or converge through the gap between the surface of the chip body 1 and the sealing film thereon and then communicate with the inlet of the gas flow transfer channel 104. For example, refer to Figure 4As shown, the microfluidic chip further includes a waterproof and breathable membrane 6 covering the surface of the chip body 1, which can allow air to pass through while blocking liquids. The waterproof and breathable membrane 6 is located on the gas passage between each reaction chamber 13 and the second piston chamber 142, so as to serve as an obstacle to block liquids on the gas passage, thereby blocking the reaction liquid distributed to each reaction chamber 13 and achieving precise quantitative liquid separation. When performing quantitative liquid separation, the reaction liquid can be injected into all the reaction chambers 13 at one time (such as Figure 6b the 12 reaction chambers shown), and there is no need to inject liquid into each reaction chamber 13 separately. Specifically, in this embodiment, the waterproof and breathable membrane 6 is covered on the surface of the chip body 1 at a position opposite to the second communication piston 3, and an air flow gap is formed between the waterproof and breathable membrane 6 and the sealing membrane covered on the chip body 1. When the second communication piston 3 rotates to make the microfluidic chip in the second working state, this air flow gap can communicate with the inlet 104a of the air flow transfer channel 104 (the inlet 104a can be multiple, such as Figure 4 the six shown in), the outlet 104b of the air flow transfer channel 104 is connected and communicated with the first air flow groove 22, and the first air flow groove 22 is connected and communicated with the second piston chamber 142 through the air flow microchannel 102b. That is to say, the gas passage between the reaction chamber 13 and the second piston chamber 142 is composed of the air flow microchannel 102d, the second air flow groove 32, the above-mentioned air flow gap, the air flow transfer channel 104, the first air flow groove 22 and the air flow microchannel 102b. The waterproof and breathable membrane 6 can block the reaction liquid in the reaction chamber no matter where it is set in the air flow channel. Preferably, it is located before the air flow transfer channel 104, that is, before the air flow discharged from each reaction chamber 13 converges.

[0061] The first communication piston 2 and the second communication piston 3 respectively include a main body integrally in a cylindrical shape and a flexible sealing layer covering the main body. Each communication groove or transfer groove is opened on the flexible sealing layer; or, each communication groove or transfer groove is opened on the main body and penetrates the flexible sealing layer, and the periphery of each communication groove or transfer groove is surrounded by the flexible sealing layer. The flexible sealing layer is made of a flexible material, such as rubber.

[0062] The first communication piston 2 and the second communication piston 3 can be respectively driven to rotate by a power source, and the power source can specifically be a motor. The first communication piston 2 has a driving end for engaging with the power source, and this driving end has a connecting groove in a polygonal or special-shaped shape for the output shaft of the motor to be inserted and connected. The second communication piston 3 has a driving end for engaging with the power source, and this driving end has a connecting groove in a polygonal hole or special-shaped hole for the output shaft of the motor to be inserted and connected.

[0063] The thickness of the lower part of the chip body 1 is less than that of the upper part, and the reaction chambers 13 are arranged on the lower part of the chip body 1 to facilitate cooperation with the structure of the PCR instrument for laser irradiation and fluorescence collection.

[0064] The vertical microfluidic chip further includes a magnet assembly 5 capable of adsorbing or releasing nucleic acids by magnetic beads. The magnet assembly 5 includes a rotatable mounting disc 51 and a plurality of magnets 52. The plurality of magnets 52 are arranged on the mounting disc 51 at intervals along the circumferential direction of the mounting disc 51. The mounting disc 51 is driven to rotate by a motor 53. The magnet 52 can apply a magnetic field to the magnetic beads in the separation and purification chamber 11 so that the magnetic beads can adsorb nucleic acids and separate them; when the magnetic field leaves the magnetic beads, the nucleic acids on the magnetic beads are released and separated from the magnetic beads.

[0065] This embodiment also provides a method for PCR detection, using the above vertical microfluidic chip. The method includes the following steps:

[0066] A. Add the nucleic acid sample into the separation and purification chamber 11 from the sample inlet 11a, and seal the sample inlet 11a.

[0067] B. Rotate the first communication piston 2 to connect the separation and purification chamber 11 and the reagent chamber 12 through the first liquid flow groove 21, and connect the reagent chamber 12 and the second piston chamber part 142 through the first gas flow groove 22; move the driving piston 4 to the right to transfer the reagent into the separation and purification chamber 11.

[0068] C. Rotate the first communication piston 2 and the second communication piston 3 to connect the liquid flow transfer groove 23 on the first communication piston 2, the liquid flow transfer channel 103 on the chip body 1, and the second liquid flow groove 31 on the second communication piston 3 in sequence to connect the separation and purification chamber 11 and the reaction chamber 13, and connect the gas flow transfer groove 24 on the first communication piston 2, the gas flow transfer channel 104 on the chip body 1, and the second gas flow groove 32 on the second communication piston 3 in sequence to connect the second piston chamber part 142 and the reaction chamber 13; move the driving piston 4 to distribute the liquid in the separation and purification chamber 11 into the reaction chamber 13.

[0069] In step B, step B is repeated multiple times so that the separation and purification chamber 11 is connected to each reagent chamber 12 in sequence, and magnetic beads, lysis solution / isopropanol, washing solution A, washing solution B, and elution solution are transferred into the separation and purification chamber 11 to extract, purify, and elute nucleic acid molecules in the sample. After each treatment is completed, the waste liquid is discharged into the waste liquid chamber; for example, after lysing the sample with the lysis solution, a variable magnetic field is applied to the separation and purification chamber 11 to mix the magnetic beads, so that the magnetic beads adsorb the lysed nucleic acid molecules, and the driving piston 4 is moved to discharge impurities such as unadsorbed cell debris into the waste liquid chamber 113.

[0070] This embodiment adopts a vertical structure, integrating nucleic acid extraction, amplification and detection; it can simultaneously perform real-time fluorescence detection of 1 to 12 gene loci, and during the amplification and detection process, a closed space isolated from the internal structure of the chip and the outside air is formed, and nucleic acid amplification and detection are carried out in this closed space, effectively eliminating the influence of bubbles, making the liquid intake accurate, enabling precise liquid quantification, without the need for an additional gas path, avoiding the influence of outside air on the PCR reaction, and reducing or even eliminating aerosol contamination and preventing pathogens from escaping into the outside air; the structure is compact and simple, facilitating batch production; the liquid path of the microfluidic chip is switched through a connecting piston, with convenient and controllable operation and easy automation; this chip uses a waterproof and breathable membrane to achieve precise quantification, and when quantitatively separating and dispensing the liquid, it can be injected into 12 reaction chambers at one time, without the need to inject the liquid into each reaction chamber separately.

[0071] The above embodiments are only for illustrating the technical concept and characteristics of the present invention. They are a preferred embodiment, and their purpose is to enable those familiar with this technology to understand the content of the present invention and implement it accordingly. However, the protection scope of the present invention cannot be limited thereby. Any equivalent transformation or modification made according to the principle of the present invention should be covered within the protection scope of the present invention.

Claims

1. A vertical microfluidic chip for PCR detection, characterized in that, it includes: a chip body, the size of which in the up-down direction is greater than at least one of its sizes in the left-right direction or the front-back direction, and the chip body has a separation and purification chamber for containing a sample, a reagent chamber for storing separation and purification reagents, and a reaction chamber for performing PCR amplification; a first communication piston, which is rotatably inserted into the chip body around a rotation axis extending in the left-right direction, and the first communication piston has a first liquid flow groove capable of communicating the separation and purification chamber and the reagent chamber; a second communication piston, which is rotatably inserted into the chip body around a rotation axis extending in the left-right direction, and the second communication piston has a second liquid flow groove capable of communicating the separation and purification chamber and the reaction chamber; and a driving piston for driving the flow of liquid, a piston chamber is provided on the chip body, the driving piston is movably arranged in the piston chamber, and the piston chamber is divided into a first piston chamber part and a second piston chamber part by the piston, and the first piston chamber part is communicated with the separation and purification chamber; wherein, the first communication piston further has a first air flow groove capable of communicating the reagent chamber and the second piston chamber part; the second communication piston further has a second air flow groove capable of communicating the reaction chamber and the second piston chamber part; the first communication piston has an air flow transfer groove, the chip body has an air flow transfer channel, and the second air flow groove, the air flow transfer channel and the air flow transfer groove can be sequentially docked to communicate the reaction chamber and the second piston chamber part; the reaction chamber corresponds to and communicates with the second air flow groove, the reaction chamber can be communicated with the separation and purification chamber through the second liquid flow groove, and the reaction chamber is communicated with the air flow transfer channel through the second air flow groove; the separation and purification chamber, the reagent chamber and the reaction chamber are arranged from top to bottom, the first communication piston is located between the separation and purification chamber and the reagent chamber, the second communication piston is located between the reagent chamber and the reaction chamber, the first communication piston further has a liquid flow transfer groove, the chip body further has a liquid flow transfer channel, and the liquid flow transfer groove, the liquid flow transfer channel and the second liquid flow groove can be sequentially docked to communicate the separation and purification chamber and the reaction chamber; the separation and purification chamber can be docked and communicated with the inlet of the first liquid flow groove through a first liquid flow microchannel, and the separation and purification chamber is communicated with the first piston chamber part through a first air flow microchannel; each reagent chamber can be docked and communicated with the outlet of the first liquid flow groove through a second liquid flow microchannel, and each reagent chamber is communicated with the inlet of the first air flow groove through a second air flow microchannel; each reaction chamber can be docked and communicated with the outlet of the second liquid flow groove through a third liquid flow microchannel, and each reaction chamber is communicated with the inlet of the second air flow groove through a third air flow microchannel; The microfluidic chip further includes a waterproof and breathable membrane covering the surface of the chip body. The waterproof and breathable membrane can allow air to pass through while blocking liquid. The waterproof and breathable membrane is located on the gas passage between the reaction chamber and the second piston chamber. Wherein, the waterproof and breathable membrane covers the position on the surface of the chip body opposite to the second communication piston, and an air flow gap is formed between the waterproof and breathable membrane and the sealing membrane covered on the chip body. The air flow gap can communicate with the inlet of the air flow transfer channel, the outlet of the air flow transfer channel is butt-connected and communicated with the first air flow groove, the first air flow groove is communicated with the second piston chamber through a fourth air flow microchannel, and the gas passage between the reaction chamber and the second piston chamber is composed of the third air flow microchannel, the second air flow groove, the air flow gap, the air flow transfer channel, the first air flow groove and the fourth air flow microchannel.

2. The vertical microfluidic chip according to claim 1, wherein, a sampling port communicating with the separation and purification chamber is opened on the chip body. The chamber in the chip body can communicate with the outside only through the sampling port during sampling, and the chamber in the chip body is isolated from the outside after the sampling port is closed.

3. The vertical microfluidic chip according to claim 1, wherein, the vertical microfluidic chip has a first working state and a second working state. In the first working state, the separation and purification chamber is only communicated with the reagent chamber through the first liquid flow groove, the reagent chamber communicated with the separation and purification chamber is communicated with the second piston chamber through the corresponding first air flow groove, the liquid flow transfer groove and the liquid flow transfer channel are misaligned, and the air flow transfer groove and the air flow transfer channel are misaligned. In the second working state, the separation and purification chamber is sequentially communicated with the reaction chamber through the liquid flow transfer groove, the liquid flow transfer channel and the second liquid flow groove, the reaction chamber is sequentially communicated with the second piston chamber through the second air flow groove, the air flow transfer channel and the air flow transfer groove, the separation and purification chamber is misaligned with the first liquid flow groove, and the second piston chamber is misaligned with the first air flow groove.

4. The vertical microfluidic chip according to claim 3, wherein, the number of the reagent chambers is multiple and they are arranged side by side in the left-right direction. Each reagent chamber corresponds to and is respectively communicated with a first liquid flow groove and a first air flow groove. The multiple first liquid flow grooves on the first communication piston are arranged at intervals along its circumferential direction, and the multiple first air flow grooves on the first communication piston are arranged at intervals along its circumferential direction. The vertical microfluidic chip has multiple first working states. In any one of the first working states, only one of the reagent chambers is communicated with the separation and purification chamber through the corresponding first liquid flow groove and with the second piston chamber through the corresponding first air flow groove.

5. The vertical microfluidic chip according to claim 3, wherein, The number of the reaction chambers is multiple and they are arranged side by side in the left - right direction. The number of the second liquid flow grooves is one and at least a part of it extends in the left - right direction. Each reaction chamber corresponds to and communicates with one of the second air flow grooves. The vertical microfluidic chip has one second working state. In the second working state, all the reaction chambers are communicated with the separation and purification chamber through the second liquid flow groove, and each reaction chamber is communicated with the air flow transfer channel through the corresponding second air flow groove.

6. The vertical microfluidic chip according to claim 1, characterized in that, the liquid flow transfer groove is located on the left side of the first liquid flow groove, the first air flow groove is located on the right side of the corresponding first liquid flow groove, and the outlet of the air flow transfer groove is aligned with the outlet of the first air flow groove; and / or, the second liquid flow groove has one inlet and multiple outlets arranged at intervals in the left - right direction, and multiple second air flow grooves are arranged at intervals in the left - right direction.

7. The vertical microfluidic chip according to claim 1, characterized in that, the reagent chamber and the reaction chamber are opened on the surface of the chip body. Micro - channels for chamber communication are provided inside and / or on the surface of the chip body. The vertical microfluidic chip further includes a sealing film covering the surface of the chip body; and / or, the thickness of the lower part of the chip body is less than that of the upper part, and the reaction chamber is arranged on the lower part of the chip body; and / or, the vertical microfluidic chip further includes a magnet assembly capable of adsorbing or releasing nucleic acid by magnetic beads. The magnet assembly includes a rotatable mounting disk and multiple magnets, and the multiple magnets are arranged at intervals along the circumferential direction of the mounting disk on the mounting disk.

8. A method for PCR detection, characterized in that, using the vertical microfluidic chip according to any one of claims 1 to 7, the method includes the following steps: A. Add the nucleic acid sample into the separation and purification chamber from the sample loading port and seal the sample loading port; B. Rotate the first communication piston to make the first liquid flow groove communicate the separation and purification chamber with the reagent chamber, and make the first air flow groove communicate the reagent chamber with the second piston chamber part; Move the driving piston to transfer the reagent into the separation and purification chamber; C. Rotate the first communication piston and the second communication piston to make the liquid flow transfer groove on the first communication piston, the liquid flow transfer channel on the chip body and the second liquid flow groove on the second communication piston be docked in sequence to communicate the separation and purification chamber with the reaction chamber, and make the air flow transfer groove on the first communication piston, the air flow transfer channel on the chip body and the second air flow groove on the second communication piston be docked in sequence to communicate the second piston chamber part with the reaction chamber; Move the driving piston to distribute the liquid in the separation and purification chamber into the reaction chambers.

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