A multiplex PCR reaction chip based on thin film microfluidic technology

Abstract

The application belongs to the technical field of PCR reaction chip, and particularly relates to a multiplex PCR reaction chip based on thin film microfluidic technology, which is mainly a chip made of thin film technology and is composed of a non-welding area and a welding area; a channel is arranged in the welding area, a DNA sample pool is arranged on one side of the chip, a PCR enzyme Mix pool is arranged above the middle of the chip, and a PCR premix pool is arranged below the middle of the chip; the DNA sample pool is communicated with the PCR premix pool through the channel; a plurality of PCR reaction pools are arranged on the other side of the chip; a PCR pressure relief pool is arranged outside the PCR reaction pool and is communicated with the corresponding PCR reaction pool; the liquid inlet speed is adjusted by adjusting the width of the flow channel, so that the simultaneous injection is achieved; the multiplex PCR amplification detection is realized in the thin film microfluidic chip; and the actual use demand of simultaneously filling different PCR reaction chambers under the non-equal path condition is achieved.

Description

Technical Field

[0001] This invention relates to the field of PCR reaction chip technology, specifically a multiplex PCR reaction chip based on thin-film microfluidic technology. Background Technology

[0002] Multiplex PCR arrays work by adding multiple primer pairs to the same PCR reaction system. These primers specifically amplify different templates or different regions of the same template, thus obtaining multiple target fragments. Combined with certain detection methods, this allows for the diagnosis of multiple targets. Traditional microfluidic technology uses multiple reaction wells for multiplex PCR detection. However, when the liquid is distributed into the reaction chambers in the flow channel, the different distances between each reaction chamber and the liquid outlet cause uneven resistance during the flow process, resulting in different filling times for each reaction chamber and thus uneven distribution of liquid volume. Meanwhile, improper inlet and outlet settings can also lead to uneven distribution of fluid into each channel when entering the chip, resulting in an overall uneven distribution. If the fluid injection cannot be precisely controlled during the experiment, the fluid may stagnate in some areas, which will greatly reduce the experimental data. Therefore, we propose a multiplex PCR reaction chip based on thin-film microfluidic technology. Summary of the Invention

[0003] The purpose of this invention is to provide a multiplex PCR reaction chip based on thin-film microfluidic technology to solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a multiplex PCR reaction chip based on thin-film microfluidic technology, the main body of which is a chip made of thin-film technology, and the chip is composed of unbonded areas and bonded areas; The bonding area has a channel, a DNA sample pool is set on one side of the chip, a PCR enzyme mix pool is set above the middle of the chip, and a PCR premix pool is set below the middle of the chip. The DNA sample pool is connected to the PCR premix pool through the channel. A PCR reaction pool is set on the other side of the chip, and multiple PCR reaction pools are set. A PCR pressure relief pool is set outside the PCR reaction pool and is connected to the corresponding PCR reaction pool. The PCR enzyme Mix pool and the PCR premix pool are mixed by squeezing against each other, and then the mixture is guided into the PCR reaction pool through the channel. The channel connected to the PCR reaction pool is equipped with a flow rate regulating pool to control the flow rate. The PCR reaction pool has three distribution patterns: ring, linear symmetry, and asymmetry. The other side of the PCR reaction pool is connected to the PCR pressure relief pool. When liquid is injected into the PCR reaction pool, the air in the original cavity of the PCR reaction pool is squeezed into the PCR pressure relief pool. A valve is provided at the connection between the PCR pressure relief pool and the PCR reaction pool to control the opening and closing of the PCR pressure relief pool and the PCR reaction pool. The channel connecting the flow rate regulating tank and the PCR reaction tank is the main flow rate limiting channel, and the channel connecting the PCR reaction tank and the main flow rate limiting channel is the sub-flow rate limiting channel. The sub-flow rate limiting channels are distributed in three ways along the extension direction of the main flow rate limiting channel: annular, linearly symmetrical, and asymmetrical. The channel width of the sub-flow rate limiting channel is related to the liquid flow pressure. The liquid inlet speed is adjusted by the main flow rate limiting channel and the sub-flow rate limiting channel to achieve the purpose of simultaneous injection.

[0005] Preferably, the flow rate regulating pool is provided with a central regulating zone for regulating the flow rate, and a barrier is provided in the central regulating zone; the barrier is circular or conical with one end wider than the other; the central regulating zone is located in the main flow rate limiting channel, and the flow rate of the main flow rate limiting channel is controlled by the central regulating zone; PCR reaction pools are distributed on both sides of the main flow rate limiting channel, and the channel connecting the PCR reaction pools and the main flow rate limiting channel is a sub-flow rate limiting channel; when the barrier in the central regulating zone through which the liquid passes is conical, the flow rate of the main flow rate limiting channel is different due to the limitation of the shape of the central regulating zone, which in turn leads to different inlet pressures of each adjacent sub-flow rate limiting channel, and the width of the sub-flow rate limiting channel corresponds to the change in the width of the main flow rate limiting channel, so that the filling speed in each PCR reaction pool is the same.

[0006] Preferably, when the PCR reaction chamber, flow rate regulating chamber, main flow rate limiting channel, and sub-flow rate limiting channel are arranged in a ring, the barrier in the central regulating area through which the liquid passes is circular, and the width of the sub-flow rate limiting channels is the same. The liquid bypasses the circular barrier and is injected into the PCR reaction chamber to achieve synchronous liquid inflow. At the same time, the ring distribution of the reaction chamber includes three types: symmetrical ring, clockwise ring, and counterclockwise ring.

[0007] Preferably, the chip has multiple valves located at the connection points of the channels to control the opening and closing of the channels. The channels of the chip are formed by the longitudinal deformation of a flexible film. Under the same deformation force, the longitudinal deformation of the flexible film is constant. The PCR reaction pools have different path lengths but the same internal liquid pressure, which ensures that the longitudinal deformation within the PCR reaction pools remains consistent, and the capacity of the resulting cavity and channels also remains consistent.

[0008] The experimental steps for PCR reaction are as follows: S1, Liquid Pre-loading: Fill the DNA sample pool with the nucleic acid extraction solution of the sample to be tested, add PCR enzyme Mix to the PCR enzyme Mix pool, add primers and probes to the PCR reaction pool, place the chip into the matching detection fixture, and wait for the experiment. S2, Mixing Step 1: Adjust the valves in the fixture to ensure the channel from the DNA sample pool to the PCR premix pool remains open. Then squeeze the DNA sample pool to allow the sample to flow into the PCR premix pool. Next, adjust the valves in the fixture again to ensure the channel between the PCR enzyme mix pool and the PCR premix pool remains open. Then, circulate and squeeze the liquids in the PCR premix pool and the PCR enzyme mix pool to mix the sample with the PCR enzyme mix liquid. The mixed liquid flows along the main rate-limiting channel to the PCR reaction pool, and simultaneous injection is achieved by changing the flow of the secondary rate-limiting channel and the main rate-limiting channel. This allows the reaction solution to mix with the primers and probes in the PCR reaction pool, and the air in the PCR reaction pool chamber is expelled into the PCR depressurization pool. Finally, close all valves to completely fill the PCR reaction pool with liquid. S3, Mixing Step 2: Adjust the valves in the fixture to ensure the passage from the DNA sample pool to the PCR premix pool remains unobstructed. Then squeeze the DNA sample pool to allow the sample to flow into the PCR premix pool. Next, adjust the valves in the fixture again to ensure the passage between the PCR enzyme Mix pool and the PCR premix pool remains unobstructed. Then, circulate and squeeze the liquids in the PCR premix pool and the PCR enzyme Mix pool to mix the sample with the PCR enzyme Mix liquid. The mixed liquid flows along the main rate-limiting channel to the PCR reaction pool, and simultaneous injection is achieved through changes in the flow channels of the main rate-limiting channel and the sub-rate-limiting channels under the control of the central regulation area. This allows the reaction solution to mix with the primers and probes in the PCR reaction pool, and the air in the PCR reaction pool chamber is expelled into the PCR depressurization pool. Finally, close all valves to completely fill the PCR reaction pool with liquid. S4, PCR reaction: Based on the operation of Mix 1 or Mix 2, start the temperature control program of the detection process, control the temperature rise and fall of the PCR reaction chamber, collect fluorescence data, plot fluorescence curves, and output experimental results.

[0009] Compared with the prior art, the beneficial effects of the present invention are: This solution enables multiplex PCR amplification and detection within a thin-film microfluidic chip; it fulfills the practical need to simultaneously fill different PCR reaction chambers under non-isotropic conditions. The connection between the two reaction chambers on this chip is achieved through a narrow channel. Due to the longitudinal tension of the flexible film, this channel is tightly closed when no liquid is present. To allow liquid to pass through the channel, the longitudinal tension of the film must be overcome to open the channel. The magnitude of this opening force is related to the channel structure; the narrower and longer the channel, the greater the resistance to liquid passage and the longer the liquid takes to pass through that section of the channel; conversely, the wider and shorter the channel, the smaller the resistance to liquid passage and the shorter the time it takes to flow through that section of the channel. This principle is used to adjust the time it takes for liquid to reach different locations. This technology ensures that the liquid volume in three (or more) reaction chambers on the same chip is consistent, guaranteeing that each PCR reaction chamber has the same volume. The liquid chambers on the chip are formed by the longitudinal deformation of a flexible film. Under the same deformation force, the longitudinal deformation of the flexible film remains constant. The three PCR reaction chambers on the chip are interconnected through connecting channels. Since the pressure is the same everywhere in the liquid, the liquid pressure experienced by the three PCR reaction chambers is the same. The longitudinal deformation of the three chambers can be kept consistent, and the liquid volume that can be contained in the chambers can be kept consistent. By changing the shape and structure of the main velocity limiting channel and the sub-velocity limiting channel, the liquid flow rate in the channel can be limited, making the liquid distribution more uniform. Attached Figure Description

[0010] Figure 1 This is a front view of the chip structure of the present invention; Figure 2 This is a schematic diagram of the valve arrangement within the chip channel of the present invention; Figure 3 This invention relates to the first central island linear symmetric flow splitting method; Figure 4 For the present invention Figure 3 Enlarged view of the central island diversion method in the image; Figure 5 This invention relates to the symmetrical ring-shaped flow splitting method; Figure 6 This invention relates to a clockwise annular flow splitting method; Figure 7 This invention relates to the counterclockwise annular flow splitting method; Figure 8 This invention relates to the first central island asymmetric flow splitting method; Figure 9 This invention relates to the second central island asymmetric flow splitting method; Figure 10 This invention relates to the second central island linear symmetric flow splitting method; Figure 11 This is the first linear symmetric flow splitting method of the present invention; Figure 12 This is the second linear symmetric flow splitting method of the present invention; Figure 13 This is the first asymmetric flow splitting method of the present invention; Figure 14 This is the second asymmetric flow splitting method of the present invention.

[0011] In the diagram: 1 Unwelded area; 2 Welded area; 3 Valve; 4 PCR pressure relief tank; 5 Flow rate regulating tank; 6 PCR enzyme mix tank; 7 DNA sample tank; 8 PCR premix tank; 9 PCR reaction tank; 10 Barrier body; 11 Central regulating area; 12 Rate limiting channel; 13 Main rate limiting channel. Detailed Implementation

[0012] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0013] In the description of this invention, it should be understood that the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0014] Please see Figure 1-14 The present invention provides the following technical solution: a multiplex PCR reaction chip based on thin film microfluidics technology, the main body of which is a chip made by thin film technology, and the chip is composed of an unbonded area 1 and a bonded area 2; The bonding area 2 has a channel. A DNA sample pool 7 is set on one side of the chip, a PCR enzyme mix pool 6 is set above the middle of the chip, and a PCR premix pool 8 is set below the middle of the chip. The DNA sample pool 7 is connected to the PCR premix pool 8 through the channel. A PCR reaction pool 9 is set on the other side of the chip, and multiple PCR reaction pools 9 are set. A PCR pressure relief pool 4 is set outside the PCR reaction pool 9, and the PCR pressure relief pool 4 is connected to the corresponding PCR reaction pool 9. After squeezing the PCR enzyme Mix tank 6 and the PCR premix tank 8 together, mix them. The mixed liquid can be stored in either the PCR enzyme Mix tank 6 or the PCR premix tank 8 (see attached). Figure 3As shown in the attached figure, the storage location of the liquid mixture is PCR enzyme Mix pool 6, and then it is guided to PCR reaction pool 9 through a channel. The channel connected to PCR reaction pool 9 is equipped with a flow rate regulating pool 5 to control the flow rate. PCR reaction pool 9 has three distribution patterns: annular, linear symmetrical, and asymmetrical. The other side of PCR reaction pool 9 is connected to PCR pressure relief pool 4. When liquid is injected into PCR reaction pool 9, the air in the original cavity of PCR reaction pool 9 is squeezed into PCR pressure relief pool 4. A valve 3 is provided at the connection between PCR pressure relief pool 4 and PCR reaction pool 9. The valve 3 controls the opening and closing of PCR pressure relief pool 4 and PCR reaction pool 9. The flow rate regulating tank 5 is connected to the central regulating zone 11 via a main flow rate limiting channel 13, and the PCR reaction tank 9 is connected to the main flow rate limiting channel 13 via a sub-flow rate limiting channel 12. The sub-flow rate limiting channels 12 are distributed in three ways along the extension direction of the main flow rate limiting channel 13: annular, linearly symmetrical, and asymmetrical. The channel width of the sub-flow rate limiting channels 12 is related to the liquid flow pressure. The liquid inlet speed is adjusted by the width of the main flow rate limiting channel 13 and the sub-flow rate limiting channels 12 to achieve simultaneous injection.

[0015] The flow rate regulating tank 5 is equipped with a central regulating zone 11 for regulating the flow rate. The central regulating zone 11 functions similarly to a flow island. A barrier 10 is installed in the central regulating zone 11. The barrier 10 in the central regulating zone 11 is circular or conical with one end wider than the other. The central regulating zone 11 is located within the main flow rate limiting channel 13 and controls the flow rate of the main flow rate limiting channel 13 through the central regulating zone 11. PCR reaction tanks 9 are distributed on both sides of the main flow rate limiting channel 13. The channel connecting the PCR reaction tank 9 and the main flow rate limiting channel 13 is a sub-flow rate limiting channel 12. When the barrier 10 in the central regulating zone 11 through which the liquid passes is conical, the shape of the central regulating zone 11 restricts the flow rate of the main flow rate limiting channel 13 to be different. Consequently, the inlet pressure of each adjacent sub-flow rate limiting channel 12 is also different. The width of the sub-flow rate limiting channel 12 corresponds to the width of the main flow rate limiting channel 13, ensuring that the filling speed in each PCR reaction tank 9 is the same.

[0016] When the PCR reaction chamber 9, flow rate regulating chamber 5, main flow rate limiting channel 13, and secondary flow rate limiting channel 12 are arranged in a ring, the barrier 10 in the central regulating area 11 through which the liquid passes is circular, and the width of the secondary flow rate limiting channels 12 is the same. The liquid bypasses the circular barrier 10 and is injected into the PCR reaction chamber 9, achieving synchronization of the liquid inflow. At the same time, the ring distribution of the reaction chamber 9 includes three types: symmetrical ring, clockwise ring, and counterclockwise ring.

[0017] Multiple valves 3 are installed in the channel of the chip. The valves 3 are distributed at the connection of the channel to control the opening and closing of the channel. The channel of the chip is formed by the longitudinal deformation of a flexible film. Under the same deformation force, the longitudinal deformation of the flexible film is constant. The path length of the PCR reaction cell 9 is different, but the liquid pressure is the same. The liquid pressure of the PCR reaction cell 9 is also the same, so that the longitudinal deformation in the PCR reaction cell 9 is consistent, and the capacity of the formed cavity and channel is also consistent.

[0018] The experimental steps for PCR reaction are as follows: S1, Liquid Pre-loading: Fill the DNA sample pool 7 with the nucleic acid extraction solution of the sample to be tested, add PCR enzyme Mix to the PCR enzyme Mix pool 6, add primers and probes to the PCR reaction pool 9, place the chip into the matching detection fixture, and wait for the experiment. S2, Mixing Step 1: Adjust valve 3 within the fixture to ensure unobstructed flow only from the DNA sample pool 7 to the PCR premix pool 8; then squeeze the DNA sample pool 7 to allow the sample to flow into the PCR premix pool 8. Next, adjust valve 3 within the fixture again to ensure unobstructed flow between the PCR enzyme Mix pool 6 and the PCR premix pool 8. Then, circulate and squeeze the liquids in the PCR premix pool 8 and the PCR enzyme Mix pool 6 to mix the sample with the PCR enzyme Mix liquid. The mixed liquid flows along the main rate-limiting channel 13 to the PCR reaction pool 9, achieving simultaneous injection through changes in the flow channels of the secondary rate-limiting channel 12 and the main rate-limiting channel 13. This allows the reaction solution to mix with the primers and probes in the PCR reaction pool 9, and the air in the chamber of the PCR reaction pool 9 is expelled into the PCR depressurization pool 4. Finally, close all valves 3 to completely fill the PCR reaction pool 9 with liquid. S3, Mixing Step 2: Adjust valve 3 within the fixture to ensure the passage from DNA sample pool 7 to PCR premix pool 8 remains unobstructed; then squeeze DNA sample pool 7 to allow the sample to flow into PCR premix pool 8. Next, adjust valve 3 again to ensure the passage between PCR enzyme Mix pool 6 and PCR premix pool 8 remains unobstructed. Then, circulate and squeeze the liquids in PCR premix pool 8 and PCR enzyme Mix pool 6 to mix the sample with the PCR enzyme Mix liquid. The mixed liquid flows along the main rate-limiting channel 13 to PCR reaction pool 9, achieving simultaneous injection through the flow changes of the main rate-limiting channel 13 and the sub-rate-limiting channel 12 under the control of the central regulation area 11. This allows the reaction solution to mix with the primers and probes in PCR reaction pool 9, and the air in the chamber of PCR reaction pool 9 is expelled into the PCR depressurization pool 4. Finally, close all valves 3 to completely fill PCR reaction pool 9 with liquid. S4, PCR reaction: Based on the operation of Mix 1 or Mix 2, start the temperature control program of the detection process, control the temperature rise and fall of PCR reaction chamber 9, collect fluorescence data, plot fluorescence curves, and output experimental results.

[0019] The function of the flow rate regulating tank 5: Liquid enters the flow rate regulating tank 5 and passes through the central regulating zone 11. The shape of the central regulating zone 11 can limit the flow rate of the liquid in the main velocity limiting channel 13. When the central regulating zone 11 has a conical barrier 10, the liquid has different velocities in the main velocity limiting channel 13. According to Bernoulli's principle, as the flow rate in the pipe increases, the pressure of the liquid on the inner wall of the pipe will decrease, which will result in different pressures at the inlet of each sub-velocity limiting channel 12. The pressure at the inlet of the velocity limiting channel 12 determines the liquid flow rate within the velocity limiting channel 12. By adjusting the shape of the central adjustment zone 11, the liquid flow rate within the velocity limiting channel 12 can be controlled, thereby achieving the goal of simultaneously filling the PCR reaction chamber 9 at different locations or with different flow paths. Multiplex PCR amplification detection is realized within the thin-film microfluidic chip, fulfilling the practical application requirement of simultaneously filling multiple PCR reaction chambers 9 under non-isotropic path conditions.

[0020] It should be noted that the appendix Figure 3-14 All are attached Figure 1 , 2 The preferred traffic acquisition methods derived from this are implemented as follows; Example 1: See attached document Figure 3 , 4 The experimental steps for the central island linear symmetric flow splitting method are as follows: S1, Liquid Pre-loading: Fill the DNA sample pool 7 with the nucleic acid extraction solution of the sample to be tested, add PCR enzyme Mix to the PCR enzyme Mix pool 6, add primers and probes to the PCR reaction pool 9, place the chip into the matching detection fixture, and wait for the experiment. S2, mix, adjust valve 3 in the fixture to keep only the channel from DNA sample pool 7 to PCR premix pool 8 unobstructed; then squeeze DNA sample pool 7 to let the sample flow into PCR premix pool 8, then adjust valve 3 in the fixture again to keep the channel between PCR enzyme Mix pool 6 and PCR premix pool 8 unobstructed, and then cycle the squeezing of PCR premix pool 8 and PCR enzyme Mix pool 6 to achieve mixing of sample and PCR enzyme Mix liquid; S3, Uniform flow splitting: The chip has multiple PCR reaction chambers 9, distributed below the PCR enzyme Mix chamber 6. The PCR reaction chambers 9 reach the PCR enzyme Mix chamber 6 via different paths. By adjusting the channel widths of the two PCR reaction chambers 9, the resistance to liquid flow in the multiple channels is made consistent, achieving synchronization of liquid reaching multiple PCR reaction chambers 9 simultaneously. Since the liquid pressure is the same, the liquid pressure experienced by the multiple PCR reaction chambers 9 is also the same, the longitudinal deformation within the chambers of the PCR reaction chambers 9 remains consistent, and the amount of liquid that can be contained within the chambers is also consistent. The central regulating zone 11 uses a conical barrier 10, and the PCR reaction chambers 9 on both sides are linearly symmetrically distributed. When the liquid passes through the central regulating zone 11, the liquid velocity in the main velocity limiting channel 13 is different. According to Bernoulli's principle, as the flow velocity in the channel increases, the pressure of the liquid on the inner wall of the channel will decrease. This results in different pressures at the inlet of each sub-velocity limiting channel 12. The liquid passes through different resistance channels to achieve the purpose of synchronous filling of the PCR reaction chambers 9. Excess liquid and air in the PCR reaction chambers 9 are discharged into the PCR depressurization chamber 4. Mix the reaction solution with the primers and probes in the PCR reaction chamber 9, and expel the air from the chamber into the PCR depressurization chamber 4; then close all valves 3 to completely fill the PCR reaction chamber 9 with liquid. S4. Start the temperature control program of the detection process, control the temperature rise and fall of PCR reaction chamber 9, collect fluorescence data, plot fluorescence curves, and output experimental results.

[0021] Example 2: See attached document Figure 5 The symmetrical ring-shaped flow splitting method was used, and the experimental steps were as follows: S1, Liquid premixed preloading: Fill the DNA sample pool 7 with the nucleic acid extraction solution of the sample to be tested, add PCR enzyme Mix to the PCR enzyme Mix pool 6, add primers and probes to the PCR reaction pool 9, place the chip into the matching detection fixture, and wait for the experiment. S2, mix, adjust valve 3 in the fixture to keep the channel from DNA sample pool 7 to PCR premix pool 8 unobstructed; then squeeze DNA sample pool 7 to let the sample flow into PCR premix pool 8, then adjust valve 3 in the fixture again to keep the channel between PCR enzyme Mix pool 6 and PCR premix pool 8 unobstructed, and then cycle the squeezing of PCR premix pool 8 and PCR enzyme Mix pool 6 to mix the sample with PCR enzyme Mix liquid; S3, uniform flow splitting, a ring-shaped central regulation zone 11 is set below the PCR enzyme Mix pool 6, and a circular barrier 10 is used in the central regulation zone 11. The PCR reaction pool 9 is distributed in a ring outside the central regulation zone 11. The PCR reaction pool 9 is connected to the central regulation zone 11 through a channel. The channel where the central regulation zone 11 is located is the main rate limiting channel 13. The channel connecting the PCR reaction pool 9 and the central regulation zone 11 is the secondary rate limiting channel 12. The secondary rate limiting channels 12 here have the same width. When liquid flows through the main velocity limiting channel 13, it is blocked and diverted by the annular central regulating zone 11, thus flowing towards the two side velocity limiting channels 12. Since the width of the two velocity limiting channels 12 is the same, the annular central regulating zone 11 can evenly divert the liquid to the surrounding area during operation. The liquid is forced into the two side velocity limiting channels 12 by pressure, achieving synchronous filling. Excess liquid in the PCR reaction tank 9 is discharged into the PCR pressure relief tank 4, and excess liquid and air in the PCR reaction tank 9 are discharged into the PCR pressure relief tank 4. S4. Start the temperature control program of the detection process, control the temperature rise and fall of PCR reaction chamber 9, collect fluorescence data, plot fluorescence curves, and output experimental results.

[0022] Example 3: See attached document Figure 6 , 7 The clockwise and counterclockwise annular flow splitting methods were used. The experimental steps were as follows: S1, Liquid premixed preloading: Fill the DNA sample pool 7 with the nucleic acid extraction solution of the sample to be tested, add PCR enzyme Mix to the PCR enzyme Mix pool 6, add primers and probes to the PCR reaction pool 9, place the chip into the matching detection fixture, and wait for the experiment. S2, mix, adjust valve 3 in the fixture to keep the channel from DNA sample pool 7 to PCR premix pool 8 unobstructed; then squeeze DNA sample pool 7 to let the sample flow into PCR premix pool 8, then adjust valve 3 in the fixture again to keep the channel between PCR enzyme Mix pool 6 and PCR premix pool 8 unobstructed, and then cycle the squeezing of PCR premix pool 8 and PCR enzyme Mix pool 6 to mix the sample with PCR enzyme Mix liquid; S3, uniform flow distribution: A central regulation zone 11 is set below the PCR enzyme Mix pool 6. PCR reaction pools 9 are spirally distributed outside the central regulation zone 11 and interconnected by channels. The channel connecting the central regulation zone 11 and the PCR enzyme Mix pool 6 is the main rate-limiting channel 13, and the channel connecting the central regulation zone 11 and each PCR reaction pool 9 is a sub-rate-limiting channel 12. The channel connecting the main rate-limiting channel 13 and the central regulation zone 11 is arc-shaped, and the sub-rate-limiting channels 12 connect the PCR reaction pools 9 to the main rate-limiting channel 13. The PCR reaction chambers 9, distributed around the central regulation zone 11, are tilted at a certain angle around the central regulation zone 11. Figure 6 and Figure 7The inclination directions are opposite, so that the PCR reaction chamber 9 and the main rate limiting channel 13 form a spiral shape around the central regulation area 11. When the liquid flows into the central regulation area 11 from the side along the main rate limiting channel 13, it flows into each sub-rate limiting channel 12 in the spiral direction and is injected into the surrounding distributed PCR reaction chambers 9 along the sub-rate limiting channels 12. The spiral provides a certain thrust so that the pressure of the liquid when it is injected into the sub-rate limiting channels 12 is the same, so as to achieve the purpose of synchronous filling. The excess liquid and air in the PCR reaction chamber 9 are discharged into the PCR depressurization chamber 4. S4. Start the temperature control program of the detection process, control the temperature rise and fall of PCR reaction chamber 9, collect fluorescence data, plot fluorescence curves, and output experimental results.

[0023] Example 4: See attached document Figure 8 The asymmetric flow splitting method using the first central island was employed, and the experimental steps were as follows: S1, Liquid premixed preloading: Fill the DNA sample pool 7 with the nucleic acid extraction solution of the sample to be tested, add PCR enzyme Mix to the PCR enzyme Mix pool 6, add primers and probes to the PCR reaction pool 9, place the chip into the matching detection fixture, and wait for the experiment. S2, mix, adjust valve 3 in the fixture to keep the channel from DNA sample pool 7 to PCR premix pool 8 unobstructed; then squeeze DNA sample pool 7 to let the sample flow into PCR premix pool 8, then adjust valve 3 in the fixture again to keep the channel between PCR enzyme Mix pool 6 and PCR premix pool 8 unobstructed, and then cycle the squeezing of PCR premix pool 8 and PCR enzyme Mix pool 6 to mix the sample with PCR enzyme Mix liquid; S3, Uniform Flow Distribution: A central regulation zone 11 is located below the PCR enzyme Mix chamber 6. The central regulation zone 11 uses a conical barrier 10. PCR reaction chambers 9 are staggered on the left and right sides of the central regulation zone 11, and are tilted downwards. The width of the main velocity-limiting channel 13 first narrows and then gradually widens due to the shape of the central regulation zone 11. The PCR reaction chambers 9 are connected to the main velocity-limiting channel 13 through sub-velocity-limiting channels 12, and the width of the sub-velocity-limiting channels 12 corresponds to the width of the main velocity-limiting channel 13. The central regulation zone 11 ensures consistent resistance to liquid flow in the multiple sub-velocity-limiting channels 12 in the vertical direction. Because the liquid pressure is the same, the liquid pressure experienced by the multiple PCR reaction chambers 9 is also the same. The longitudinal deformation within the chambers of the PCR reaction chambers 9 remains consistent, and the amount of liquid that can be contained within the chambers is also consistent; synchronous filling is achieved. Excess liquid and air in the PCR reaction chambers 9 are discharged into the PCR depressurization chamber 4. S4. Start the temperature control program of the detection process, control the temperature rise and fall of PCR reaction chamber 9, collect fluorescence data, plot fluorescence curves, and output experimental results.

[0024] Example 5: See attached document Figure 9 The asymmetric flow splitting method using the second central island was employed, and the experimental steps were as follows: S1, Liquid premixed preloading: Fill the DNA sample pool 7 with the nucleic acid extraction solution of the sample to be tested, add PCR enzyme Mix to the PCR enzyme Mix pool 6, add primers and probes to the PCR reaction pool 9, place the chip into the matching detection fixture, and wait for the experiment. S2, mix, adjust valve 3 in the fixture to keep the channel from DNA sample pool 7 to PCR premix pool 8 unobstructed; then squeeze DNA sample pool 7 to let the sample flow into PCR premix pool 8, then adjust valve 3 in the fixture again to keep the channel between PCR enzyme Mix pool 6 and PCR premix pool 8 unobstructed, and then cycle the squeezing of PCR premix pool 8 and PCR enzyme Mix pool 6 to mix the sample with PCR enzyme Mix liquid; S3, Uniform Flow Distribution: A central regulation zone 11 is located below the PCR enzyme Mix chamber 6. The central regulation zone 11 uses a conical barrier 10. PCR reaction chambers 9 are staggered on the left and right sides of the central regulation zone 11 and are tilted upwards. The width of the main velocity limiting channel 13 first narrows and then gradually widens due to the shape of the central regulation zone 11. The PCR reaction chambers 9 are connected to the main velocity limiting channel 13 through the sub-velocity limiting channel 12, and the width of the sub-velocity limiting channel 12 corresponds to the width change of the main velocity limiting channel 13. The central regulation zone 11 ensures that the resistance to liquid flow in multiple channels in the vertical direction is consistent. Because the liquid pressure is the same, the liquid pressure experienced by multiple PCR reaction chambers 9 is also the same. The longitudinal deformation within the chambers of the PCR reaction chambers 9 is consistent, and the amount of liquid that can be contained within the chambers is also consistent; synchronous filling is achieved. Excess liquid and air in the PCR reaction chambers 9 are discharged into the PCR depressurization chamber 4. S4. Start the temperature control program of the detection process, control the temperature rise and fall of PCR reaction chamber 9, collect fluorescence data, plot fluorescence curves, and output experimental results.

[0025] Example 6 Reference Figure 10 The second central island linear symmetric flow splitting method was adopted, and the experimental steps were as follows: S1, Liquid premixed preloading: Fill the DNA sample pool 7 with the nucleic acid extraction solution of the sample to be tested, add PCR enzyme Mix to the PCR enzyme Mix pool 6, add primers and probes to the PCR reaction pool 9, place the chip into the matching detection fixture, and wait for the experiment. S2, mix, adjust valve 3 in the fixture to keep the channel from DNA sample pool 7 to PCR premix pool 8 unobstructed; then squeeze DNA sample pool 7 to let the sample flow into PCR premix pool 8, then adjust valve 3 in the fixture again to keep the channel between PCR enzyme Mix pool 6 and PCR premix pool 8 unobstructed, and then cycle the squeezing of PCR premix pool 8 and PCR enzyme Mix pool 6 to mix the sample with PCR enzyme Mix liquid; S3, Uniform Flow Distribution: A central regulation zone 11 is located below the PCR enzyme Mix chamber 6. The central regulation zone 11 uses a conical barrier 10. PCR reaction chambers 9 are symmetrically distributed on the left and right sides of the central regulation zone 11, and are tilted upwards. The width of the main velocity limiting channel 13 first narrows and then gradually widens due to the shape of the central regulation zone 11. The PCR reaction chambers 9 are connected to the main velocity limiting channel 13 through the sub-velocity limiting channel 12, and the width of the sub-velocity limiting channel 12 corresponds to the width change of the main velocity limiting channel 13. The central regulation zone 11 ensures that the resistance to liquid flow in multiple channels in the vertical direction is consistent. Because the liquid pressure is the same, the liquid pressure experienced by multiple PCR reaction chambers 9 is also the same. The longitudinal deformation within the chamber of the PCR reaction chamber 9 is consistent, and the amount of liquid that can be contained in the chamber is also consistent; synchronous filling is achieved. Excess liquid and air in the PCR reaction chamber 9 are discharged into the PCR depressurization chamber 4. S4. Start the temperature control program of the detection process, control the temperature rise and fall of PCR reaction chamber 9, collect fluorescence data, plot fluorescence curves, and output experimental results.

[0026] Example 7 Reference Figure 11-12 The first linear symmetric flow splitting method and the second linear symmetric flow splitting method are adopted; refer to Figure 13-14 The first asymmetric flow splitting method and the second asymmetric flow splitting method are used. All four attached figures use the same principle for flow splitting. The experimental steps are as follows: S1, Liquid premixed preloading: Fill the DNA sample pool 7 with the nucleic acid extraction solution of the sample to be tested, add PCR enzyme Mix to the PCR enzyme Mix pool 6, add primers and probes to the PCR reaction pool 9, place the chip into the matching detection fixture, and wait for the experiment. S2, mix, adjust valve 3 in the fixture to keep the channel from DNA sample pool 7 to PCR premix pool 8 unobstructed; then squeeze DNA sample pool 7 to let the sample flow into PCR premix pool 8, then adjust valve 3 in the fixture again to keep the channel between PCR enzyme Mix pool 6 and PCR premix pool 8 unobstructed, and then cycle the squeezing of PCR premix pool 8 and PCR enzyme Mix pool 6 to mix the sample with PCR enzyme Mix liquid; S3, uniform flow distribution: No central regulation zone 11 is provided below the PCR enzyme Mix chamber 6; direct current is used for distribution. PCR reaction chambers 9 are symmetrically or staggered on the left and right sides of the main velocity-limiting channel 13. PCR reaction chambers 9 are tilted upwards or downwards. The main velocity-limiting channel 13 has the same width. PCR reaction chambers 9 are directly connected to the main velocity-limiting channel 13 through sub-velocity-limiting channels 12, but the widths of the sub-velocity-limiting channels 12 are different. This ensures consistent resistance to liquid flow in multiple channels in the vertical direction. Due to the consistent internal liquid pressure, the liquid pressure experienced by multiple PCR reaction chambers 9 is also the same. The longitudinal deformation within the chambers of PCR reaction chambers 9 remains consistent, resulting in a consistent liquid capacity within the chambers. This achieves synchronous filling. Excess liquid and air in the PCR reaction chambers 9 are discharged into the PCR depressurization chamber 4. S4. Start the temperature control program of the detection process, control the temperature rise and fall of PCR reaction chamber 9, collect fluorescence data, plot fluorescence curves, and output experimental results.

[0027] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description. Therefore, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.

[0028] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A multiplex PCR reaction chip based on thin-film microfluidic technology, the main body of which is a chip fabricated using thin-film technology, and the chip is composed of an unbonded area (1) and a bonded area (2), characterized in that: The welding area (2) has a channel, a DNA sample pool (7) is provided on one side of the chip, a PCR enzyme mix pool (6) is provided above the middle of the chip, and a PCR premix pool (8) is provided below the middle of the chip. The DNA sample pool (7) is connected to the PCR premix pool (8) through the channel. A PCR reaction pool (9) is provided on the other side of the chip. Multiple PCR reaction pools (9) are provided. A PCR pressure relief pool (4) is provided outside the PCR reaction pool (9). The PCR pressure relief pool (4) is connected to the corresponding PCR reaction pool (9). The PCR enzyme Mix pool (6) and the PCR premix pool (8) are mixed by squeezing each other, and then the mixture is guided into the PCR reaction pool (9) through the channel. The channel connected to the PCR reaction pool (9) is equipped with a flow rate regulating pool (5) to control the flow rate. The PCR reaction pool (9) has three distribution patterns: ring, linear symmetry, and asymmetry. The other side of the PCR reaction pool (9) is connected to the PCR pressure relief pool (4). When liquid is injected into the PCR reaction pool (9), the air in the cavity of the PCR reaction pool (9) is squeezed into the PCR pressure relief pool (4). A valve (3) is provided at the connection between the PCR pressure relief pool (4) and the PCR reaction pool (9). The valve (3) controls the opening and closing of the PCR pressure relief pool (4) and the PCR reaction pool (9). The channel connecting the flow rate regulating pool (5) and the PCR reaction pool (9) is the main flow rate limiting channel (13), and the channel connecting the PCR reaction pool (9) and the main flow rate limiting channel (13) is the sub-flow rate limiting channel (12). The sub-flow rate limiting channels (12) are distributed in three ways along the extension direction of the main flow rate limiting channel (13): annular, linearly symmetrical, and asymmetrical. The channel width of the sub-flow rate limiting channel (12) is related to the pressure of the liquid flow. The liquid inlet speed is adjusted by the main flow rate limiting channel (13) and the sub-flow rate limiting channel (12) to achieve the purpose of simultaneous injection.

2. The multiplex PCR reaction chip based on thin-film microfluidic technology according to claim 1, characterized in that: The flow rate regulating pool (5) is provided with a central regulating zone (11) for regulating the flow rate, and a barrier (10) is provided in the central regulating zone (11); the barrier (10) is circular or conical; the central regulating zone (11) is located in the main flow rate limiting channel (13), and the flow rate of the main flow rate limiting channel (13) is controlled by the central regulating zone (11); PCR reaction pools (9) are distributed on both sides of the main flow rate limiting channel (13), and the PCR reaction pools (9) are connected to the main flow rate limiting channel (13). The channel is a sub-rate limiting channel (12); when the barrier (10) in the central regulation zone (11) through which the liquid passes is conical, the central regulation zone (11) restricts the flow rate of the main rate limiting channel (13) by the shape, which in turn leads to different inlet pressures of each adjacent sub-rate limiting channel (12). The width of the sub-rate limiting channel (12) corresponds to the width of the main rate limiting channel (13), so that the filling speed in each PCR reaction pool (9) is the same.

3. The multiplex PCR reaction chip based on thin-film microfluidic technology according to claim 2, characterized in that: When the PCR reaction pool (9) is arranged in a ring with the flow rate regulating pool (5), the main flow rate limiting channel (13), and the sub-flow rate limiting channel (12), the barrier (10) in the central regulating area (11) through which the liquid passes is circular, and the width of the sub-flow rate limiting channel (12) is the same. The liquid bypasses the circular barrier (10) and is injected into the PCR reaction pool (9), thus achieving synchronization of the liquid inflow. At the same time, the ring distribution of the reaction pool (9) includes three types: symmetrical ring, clockwise ring, and counterclockwise ring.

4. The multiplex PCR reaction chip based on thin-film microfluidic technology according to claim 1, characterized in that: The chip has multiple valves (3) in its channel. The valves (3) are distributed at the connection of the channel to control the opening and closing of the channel. The channel of the chip is formed by the longitudinal deformation of a flexible film. Under the same deformation force, the longitudinal deformation of the flexible film is fixed. The PCR reaction pool (9) has different path lengths, but the liquid pressure is the same. The liquid pressure of the PCR reaction pool (9) is also the same, so that the longitudinal deformation in the PCR reaction pool (9) is consistent, and the capacity of the formed cavity and channel is also consistent.

5. An experimental method for a multiplex PCR reaction chip based on thin-film microfluidic technology according to any one of claims 1, 2, or 3, characterized in that: The experimental steps for PCR reaction are as follows: S1, Liquid preloading: Fill the DNA sample pool (7) with the nucleic acid extract of the sample to be tested, add PCR enzyme Mix to the PCR enzyme Mix pool (6), add primers and probes to the PCR reaction pool (9), put the chip into the matching detection fixture, and wait for the experiment; S2, Mixing 1: Adjust the valve (3) in the fixture to keep the channel from DNA sample pool (7) to PCR premix pool (8) unobstructed; then squeeze the DNA sample pool (7) to let the sample flow into the PCR premix pool (8), and then adjust the valve (3) in the fixture again to keep the channel between PCR enzyme Mix pool (6) and PCR premix pool (8) unobstructed, and then circulate the squeezing of the liquid in PCR premix pool (8) and PCR enzyme Mix pool (6) to achieve mixing of sample and PCR enzyme Mix liquid. The mixed liquid flows along the main rate limiting channel (13) to PCR reaction pool (9). Simultaneous injection is achieved by changing the flow channels of the sub-rate limiting channel (12) and the main rate limiting channel (13); let the reaction solution mix with the primer probe in PCR reaction pool (9), and the air in the chamber of PCR reaction pool (9) is discharged into PCR depressurization pool (4); then close all valves (3) to completely fill PCR reaction pool (9) with liquid. S3, Mixing 2: Adjust the valve (3) in the fixture to keep the channel from DNA sample pool (7) to PCR premix pool (8) unobstructed; then squeeze the DNA sample pool (7) to let the sample flow into the PCR premix pool (8), and then adjust the valve (3) in the fixture again to keep the channel between PCR enzyme Mix pool (6) and PCR premix pool (8) unobstructed, and then squeeze the liquid in PCR premix pool (8) and PCR enzyme Mix pool (6) in a cycle to achieve mixing of sample and PCR enzyme Mix liquid. The mixed liquid flows along the main rate limiting channel (13) to PCR reaction pool (9). Simultaneous injection is achieved by changing the flow channels of the main rate limiting channel (13) and the sub-rate limiting channel (12) under the restriction of the central regulation area (11); let the reaction solution mix with the primer probe in PCR reaction pool (9), and the air in the chamber of PCR reaction pool (9) is discharged into PCR depressurization pool (4); then close all valves (3) to completely fill PCR reaction pool (9) with liquid. S4, PCR reaction: Based on the operation of Mix 1 or Mix 2, start the temperature control program of the detection process, control the temperature rise and fall of the PCR reaction pool (9), collect fluorescence data, draw fluorescence curves, and output experimental results.

Images