Micro-fluidic chip and detection method using micro-fluidic chip

Abstract

The invention relates to the technical field of in-vitro diagnosis, in particular to a micro-fluidic chip and a detection method applying the micro-fluidic chip, the micro-fluidic chip comprises a substrate, an air pressure control bin, an elastic film layer, a sample injection micro-plug group, a liquid outlet micro-plug group, a control plug and a driving source, and the detection method applying the micro-fluidic chip is used for detecting the sample injection micro-plug group, the liquid outlet micro-plug group, the control plug and the driving source. Comprising the following steps: construction of a detection platform, primary antibody incubation, secondary antibody incubation, color development, termination and quantitative analysis, and the micro-fluidic chip can provide pressure for sufficient flowing of liquid and is convenient to operate. According to the detection method applying the micro-fluidic chip, the experiment efficiency is improved, the detection result is averaged, the random error is reduced, and the reliability of the detection result is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to a microfluidic chip and a detection method using the microfluidic chip, in particular to the use and method of the microfluidic chip in immunomagnetic bead detection. Background Art

[0002] The biggest problem with the immunomagnetic bead method as a common method for detecting proteins and enriching cells is that it is complicated to operate, the washing process cannot be standardized, and it is time-consuming. Carrying out immune reactions in microfluidics will effectively solve the above problems. Therefore, in recent years, research on realizing ELISA reactions in microfluidics has gradually increased. For example, a Chinese invention patent "Microfluidic Chip and Its Use" with patent number CN202110484151.9 (announcement number CN113275047A) discloses a microfluidic chip that uses the immunomagnetic bead method to enrich cells. The liquid is driven to be injected through a power mechanism. The power mechanism is arranged between the injection port and the power source. The power mechanism can be a constant pressure pump, an air compressor, a piezoelectric driver or an ultrasonic driver.

[0003] However, the power mechanism involved in the above-mentioned microfluidic chip is relatively expensive, occupies a large space, and requires detection at a fixed position, which cannot be applied to rapid on-site detection, thereby increasing the cost. Another point is that the external equipment or the driving components integrated into the microfluidic system require regular maintenance, which increases the maintenance cost for the user. If the common method of manually pressing the pre-installed reagent in the liquid inlet chamber into the reaction chamber is used, there are certain problems. If the force is too light, it will result in incomplete injection. If the force is too strong, it may cause backflow due to excessive deformation and tilt of the chip, which is inconvenient for the experimenter to operate. Summary of the Invention

[0004] The first technical problem to be solved by the present invention is to provide a microfluidic chip with small size, low cost and convenient operation in response to the above-mentioned existing technical status.

[0005] The second technical problem to be solved by the present invention is to provide a detection method using the above-mentioned microfluidic chip in response to the above-mentioned existing technical status.

[0006] The first technical solution adopted by the present invention to solve the above technical problems is: the microfluidic chip includes:

[0007] A substrate, wherein the substrate is provided with an inlet area for adding a reaction liquid, and a reaction area and an outlet sequentially located downstream of the inlet area along the flow direction of the reaction liquid;

[0008] An air pressure control chamber is provided between the sample injection area and the reaction area, the air pressure control chamber and the sample injection area are connected via an injection flow channel, and the air pressure control chamber, the reaction area, and the outlet are connected via a liquid outlet flow channel;

[0009] It is characterized by further comprising:

[0010] an elastic film layer, disposed on the substrate and covering the air pressure control chamber, the sample inlet flow channel and the liquid outlet flow channel;

[0011] The sampling micro-plug group is provided on the elastic film layer, cooperates with the elastic film layer and acts on the sampling flow channel to open or close the sampling flow channel;

[0012] A liquid outlet micro-plug group is provided on the elastic film layer, cooperates with the elastic film layer and acts on the liquid outlet flow channel to open or close the liquid outlet flow channel;

[0013] A control pin is provided on the elastic film layer, wherein the bottom surface of the control pin is aligned with the top surface of the air pressure control chamber;

[0014] There are multiple driving sources, each providing power to the corresponding control plug, sample inlet micro-plug group and liquid outlet micro-plug group;

[0015] When the driving source drives the injection micro-plug group and the liquid outlet micro-plug group to press down and close the air pressure control chamber, the control plug can be lifted to increase the volume of the air pressure control chamber, and then the injection micro-plug group is lifted to open the injection flow channel, thereby allowing the reaction liquid to flow from the injection area into the air pressure control chamber;

[0016] When the driving source drives the sample injection micro-plug group and the liquid outlet micro-plug group to press down and close the air pressure control chamber, the volume of the air pressure control chamber can be reduced by pressing the control plug downward, and then the liquid outlet micro-plug group can be pulled up to open the liquid outlet flow channel, thereby realizing the reaction liquid flowing from the air pressure control chamber into the reaction area or out of the outlet.

[0017] In order to facilitate the user to control the force of the reaction liquid flowing into the reaction zone or out of the outlet, preferably, the air pressure control chamber is vertically distributed in the substrate, and the inner wall of the air pressure control chamber is provided with a partition membrane protruding inward along the circumferential direction, and the partition membrane divides the air pressure control chamber into:

[0018] an upper chamber, communicated with the sample injection area, and used to provide a movable space for the control pin;

[0019] The lower chamber is connected to the reaction zone and is used to provide a movable space for the control pin. The inner side of the separation membrane is surrounded by a flow area for connecting the upper chamber and the lower chamber. The cross-sectional size of the flow area is smaller than the outer diameter of the lower end of the control pin. The separation membrane is elastic.

[0020] When the driving source drives the sample inlet micro-plug group and the liquid outlet micro-plug group to press down and close the air pressure control chamber, the control plug can be pressed down from the top of the upper chamber to contact the separation membrane, and then the liquid outlet micro-plug group is pulled up to open the liquid outlet flow channel, thereby realizing the reaction liquid flowing from the air pressure control chamber into the reaction area;

[0021] When the driving source drives the sample inlet and outlet micro-plugs downward, sealing the air pressure control chamber, the downward pressure on the control plug drives the separator membrane into contact with the bottom of the lower chamber. The upward pressure on the liquid micro-plug assembly then opens the outlet channel, allowing the reaction liquid to flow out of the outlet. The user can selectively control the flow of the reaction liquid into the reaction zone or discharge the waste liquid through the outlet by pressing the control plug down until it releases the separator membrane or contacts the bottom of the lower chamber. The separator membrane also separates the upper and lower chambers when the control plug is pressed into the lower chamber, increasing the speed at which the pressure in the lower chamber increases and preventing any reaction liquid remaining in the reaction zone from flowing back into the sample inlet channel and contaminating the next reaction liquid after the control plug is lifted and reset, providing a dual-purpose advantage.

[0022] To ensure smooth depressing of the control pin, the substrate is preferably provided with a pressure relief channel connected to the upper chamber. This pressure relief channel, which communicates with the external environment, balances the air pressure within the upper chamber. This pressure relief channel provides a pressure relief channel when the upper chamber decreases in volume and increases in pressure, mitigating the pressure increase within the upper chamber and reducing resistance when the user depresses the control pin.

[0023] In order to further ensure that the mixed liquid in the reaction zone does not contaminate the reaction liquid during the operation of the reaction liquid flowing from the injection zone into the pressure control chamber, preferably, the substrate includes:

[0024] A reaction liquid sampling layer is provided below the elastic film layer, wherein the sampling area and the upper cavity are provided in the reaction liquid sampling layer, and the sampling area is used for storing the reaction liquid;

[0025] The reaction layer is disposed below the reaction liquid injection layer, and the reaction zone is disposed in a lower chamber in the reaction layer. The separator membrane is disposed at the junction of the reaction liquid injection layer and the reaction layer. The upper chamber vertically disposed within the reaction liquid injection layer and the lower chamber disposed within the reaction layer prevent the mixed liquid in the reaction zone from flowing back into the injection channel despite gravity, thereby reducing the risk of the mixed liquid in the reaction zone flowing into the injection channel.

[0026] To facilitate cleaning of the reaction zone, the reaction layer is preferably provided with a wash liquid injection chamber, which communicates with the lower chamber via a wash liquid injection channel for injecting wash liquid. A wash liquid injection micro-plug is provided on the elastic film layer, which cooperates with the elastic film layer and acts on the wash liquid injection channel to open or close the wash liquid injection channel. By lifting the control plug, wash liquid can enter the lower chamber directly, eliminating the need to wait for the reaction liquid to flow from the upper chamber to the lower chamber as is required during reaction liquid injection, simplifying the process of cleaning the reaction zone with the wash liquid.

[0027] To facilitate user control of the flow of liquid within the reaction zone, the liquid outlet channel preferably includes a reaction zone inlet channel and a control channel connecting the reaction zone and the lower chamber. The liquid outlet micro-plug assembly cooperates with the elastic film layer and acts on the control channel to open or close the control channel. The liquid outlet micro-plug assembly manipulates the open and closed state of the control channel to control the flow of liquid into or out of the reaction zone.

[0028] To ensure high efficiency of chip detection, preferably, the reaction zone includes independently arranged first, second, and third reaction chambers, and the reaction zone liquid inlet flow channels include a first reaction zone liquid inlet flow channel connected to the first reaction chamber, a second reaction zone liquid inlet flow channel connected to the second reaction chamber, and a third reaction zone liquid inlet flow channel connected to the third reaction chamber, and the first reaction zone liquid inlet flow channel, the second reaction zone liquid inlet flow channel, and the third reaction zone liquid inlet flow channel are of the same size. The arrangement of the first, second, and third reaction chambers within the chip enables the detection process to be carried out independently in the three reaction chambers, and the identical size of the first, second, and third reaction zone liquid inlet flow channels ensures that the reaction liquid can enter the three reaction chambers at the same time, ensuring that the detection processes in different reaction chambers are carried out synchronously.

[0029] In order to increase the experimental efficiency, preferably, the control flow channel includes a first control flow channel communicating with the first reaction chamber, a second control flow channel communicating with the second reaction chamber, and a third control flow channel communicating with the third reaction chamber;

[0030] Correspondingly, the liquid outlet micro-plug group includes a first liquid outlet micro-plug located directly above the first control flow channel for controlling the on / off state of the first control flow channel, a second liquid outlet micro-plug located directly above the second control flow channel for controlling the on / off state of the second control flow channel, and a third liquid outlet micro-plug located directly above the third control flow channel for controlling the on / off state of the third control flow channel. The first, second, and third liquid outlet micro-plugs are connected by a liquid outlet micro-plug connector. The provision of the liquid outlet micro-plug connector ensures that the first, second, and third reaction chambers are simultaneously sealed or open, facilitating the synchronous detection process.

[0031] The second technical solution adopted by the present invention to solve the above technical problem is: a detection method using the above microfluidic chip comprises the following steps:

[0032] S1 detection platform construction, reagent presetting and state initialization: a magnetic field generating device is placed under the chip body, which acts on the reaction area and is used to control whether the magnetic beads in the reaction chamber are in a magnetic attraction state; the sampling area includes a first sampling chamber, a second sampling chamber, a third sampling chamber, and a fourth sampling chamber; the sampling flow channel includes a first sampling flow channel connected to the first sampling chamber, a second sampling flow channel connected to the second sampling chamber, a third sampling flow channel connected to the third sampling chamber, and a fourth sampling flow channel connected to the fourth sampling chamber; the sampling micro-plug group includes a first sampling micro-plug for controlling the on-off state of the first sampling flow channel, a second sampling micro-plug for controlling the on-off state of the second sampling flow channel, a third sampling micro-plug for controlling the on-off state of the third sampling flow channel, and a fourth sampling micro-plug for controlling the on-off state of the fourth sampling flow channel.

[0033] Add the magnetic bead primary antibody solution, the test sample solution, the enzyme-labeled secondary antibody solution, the color developing solution, and the washing solution into the first, second, third, fourth, and washing solution injection chambers, respectively; press down the control plug, the injection micro-plug group, and the liquid outlet micro-plug group; seal the air pressure control chamber, the injection flow channel, and the liquid outlet flow channel; and turn off the magnetic field generating device;

[0034] S2: primary antibody incubation: introducing the magnetic bead primary antibody solution and the sample solution to be tested in step S1 into the reaction zone; after mixing and incubating, turning on the magnetic field generating device for magnetic separation, and washing to obtain the sample-primary antibody magnetic beads magnetically attracted to the reaction zone;

[0035] S3 secondary antibody incubation: introducing the enzyme-labeled secondary antibody solution in step S1 into the reaction zone, turning off the magnetic field generator, mixing with the sample-primary antibody magnetic beads obtained in step S2, incubating, and then turning on the magnetic field generator for magnetic separation. After washing again, the enzyme-labeled secondary antibody-sample-primary antibody magnetic beads magnetically attracted to the reaction zone are obtained;

[0036] S4 color development, termination, and quantitative analysis: the color development solution in step S1 is introduced into the reaction area, the magnetic field generating device is turned off, and the color development solution is mixed and incubated with the enzyme-labeled secondary antibody-sample-primary antibody magnetic beads obtained in step S3, and then the stop solution is added to obtain the solution to be tested; a detection module for performing absorbance analysis on the reaction area is also provided above the reaction area. The detection module is turned on, and the absorbance values ​​obtained by absorbance analysis of the solution to be tested in the first reaction chamber, the second reaction chamber, and the third reaction chamber are averaged and converted into the content of the marker in the solution to be tested.

[0037] In order to facilitate the user's operation during the reaction solution injection process, preferably, the operation steps of introducing the magnetic bead primary antibody solution and the test sample solution in step S1 into the reaction zone in step S2 include:

[0038] S21 Magnetic bead injection: lift up the first injection micro-plug and the control plug, and suck the magnetic bead primary antibody solution of step S1 from the first injection chamber into the air pressure control chamber, then press down the first injection micro-plug to close the first injection flow channel; press down the control plug until it contacts the separation membrane, and lift up the liquid outlet micro-plug connector, and the magnetic bead primary antibody solution flows from the air pressure control chamber into the reaction zone; then turn on the magnetic field generating device to perform magnetic separation on the magnetic bead primary antibody solution, obtain the primary antibody magnetic beads adsorbed on the bottom of the reaction zone and the separated waste liquid, and discharge the waste liquid;

[0039] S22: Injecting the sample to be tested: Lift the second injection micro-plug upward and the control plug upward to draw the sample solution to be tested from step S1 into the air pressure control chamber, then press down the second injection micro-plug to seal the second injection channel; press down the control plug until it contacts the separator membrane, and lift up the liquid outlet micro-plug connector, allowing the sample solution to be tested to enter the reaction zone from the air pressure control chamber. Step S22 is completed after the sample solution to be tested has completely entered the reaction zone and contacted the antimagnetic beads obtained in step S21. By pressing down the control plug until it contacts the separator membrane, the reaction solution flows into the reaction zone rather than flowing directly out of the outlet, making it easier for the user to control the force of the downward pressure.

[0040] In order to facilitate the user to perform the mixing and incubation operation, preferably, the mixing and incubation steps in step S2 and step S3 include: turning off the magnetic field generating device, controlling the control plug to reciprocate in the upper chamber, and completing the mixing and incubation operation after the magnetic beads are evenly distributed in the reaction zone. The reciprocating movement of the control plug in the upper chamber ensures that the reaction liquid in the reaction zone is always mixed at the outlet of the reaction zone liquid inlet flow channel and in the reaction zone, and does not flow back into the lower chamber. At the same time, because the upper chamber is connected to the pressure relief channel, the suction and thrust of the fluid during the mixing and incubation operation are relatively small, thereby preventing the mixed liquid from flowing back into the upper chamber due to excessive suction.

[0041] In order to facilitate the user to fully clean the reaction area, preferably, the washing operation in steps S2 and S3 is to open the magnetic field generating device, lift up the washing liquid sampling micro-plug and the control plug, and the washing liquid in step S1 flows into the lower cavity from the washing liquid sampling chamber; after the washing liquid has completely flowed into the lower cavity, press down the washing liquid sampling micro-plug and then press down the control plug until it contacts the separation membrane, lift up the liquid outlet micro-plug connector, and the washing liquid flows into the reaction area; after the washing liquid has completely entered the reaction area, continue to press down the control plug until the separation membrane contacts the bottom surface of the lower cavity, and the liquid in the reaction area flows to the outlet. After the washing liquid has completely flowed out of the reaction area, lift up the control plug until the separation membrane recovers its deformation, press down the liquid outlet micro-plug connector, the control flow channel is blocked, and the washing operation is completed. During the entire washing operation, the control plug only needs to complete the action of lifting and pressing down. The operation is simple and easy to implement. Pressing down the washing liquid injection micro-plug has pressed the control plug down until it contacts the separation membrane, which prevents the washing liquid from flowing back into the upper chamber and the washing liquid injection chamber, and provides sufficient power for the washing liquid to enter the reaction area or flow out from the outlet. The cleaning is fast and the operation is simple.

[0042] To facilitate the user in draining the reaction zone, preferably, the operation of draining the waste liquid in step S21 is to press the control plug downward until the separator contacts the bottom surface of the lower chamber, causing the waste liquid to flow from the reaction zone to the outlet, thereby draining the waste liquid. The user can drain the waste liquid by pressing the control plug downward to the bottom surface of the lower chamber.

[0043] Compared with the prior art, the advantages of the present invention are:

[0044] 1. This microfluidic chip achieves the flow of the reaction liquid from the injection zone to the reaction zone or outlet through the coordinated use of the injection micro-plug group, the liquid outlet micro-plug group, the control plug, and the elastic film layer. Compared with the power mechanism in the existing technology, it is low-cost and small in size.

[0045] 2. The setting of the air pressure control chamber and the sealing of the air pressure control chamber by the sample inlet micro-plug group and the liquid outlet micro-plug group enable the movement of the control plug to quickly change the pressure in the air pressure control chamber, thereby providing sufficient power for the flow of the reaction liquid and speeding up the detection process;

[0046] 3. Limiting the range of motion of the control bolt by pressing it to the bottom of the air pressure control chamber or lifting the control bolt out of the air pressure control chamber can facilitate operation;

[0047] 4. In the detection method using the above-mentioned microfluidic chip, only the change in air pressure is used to drive the reaction liquid to flow in the chip, which is low in cost. The pressure generated by the movement of the control plug allows the reaction liquid to fully flow into the multiple reaction chambers in the reaction area. The size setting of the control flow channel and the setting of the liquid outlet microplug connector enable the reactions in multiple reaction chambers to start simultaneously, thereby improving the experimental efficiency. The average of the detection results reduces random errors and increases the reliability of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a schematic structural diagram of Example 1 of the present invention;

[0049] Figure 2 This is an exploded view of the structure of Example 1 of the present invention;

[0050] Figure 3 This is a schematic structural diagram of the reaction layer in Example 1 of the present invention;

[0051] Figure 4 This is a schematic structural diagram of the reaction liquid injection layer of Example 1 of the present invention;

[0052] Figure 5 Schematic diagram of the internal structure of the microfluidic chip after longitudinal section according to Example 2 of the present invention;

[0053] Figure 6 A longitudinal cross-sectional view of the reaction liquid flowing from the injection zone into the upper chamber during the injection process of Example 2 of the present invention (the arrows in the flow channel represent the flow direction of the reaction liquid, and the arrows on the control plug, injection micro-plug group, and discharge micro-plug group represent the movement directions of the corresponding micro-plugs);

[0054] Figure 7 A longitudinal cross-sectional view of the reaction liquid flowing from the upper chamber into the lower chamber during the injection process of Example 2 of the present invention (the arrows in the flow channel represent the flow direction of the reaction liquid, and the arrows on the control plug, injection micro-plug group, and discharge micro-plug group represent the movement directions of the corresponding micro-plugs);

[0055] Figure 8 A longitudinal cross-sectional view of the reaction liquid flowing from the lower chamber into the reaction zone during the injection process of Example 2 of the present invention; (the arrows within the flow channel represent the flow direction of the reaction liquid, and the arrows on the control plug, injection micro-plug assembly, and discharge micro-plug assembly represent the movement directions of the corresponding micro-plugs);

[0056] Figure 9 A longitudinal cross-sectional view of the reaction solution in the reaction zone during mixing and incubation during the injection process of Example 2 of the present invention; (the arrows in the flow channel represent the flow direction of the reaction solution, and the arrows on the control plug, injection micro-plug group, and discharge micro-plug group represent the movement directions of the corresponding micro-plugs);

[0057] Figure 10A longitudinal cross-sectional view of the reaction liquid during the injection process of Example 2 of the present invention during waste liquid discharge; (the arrows within the flow channel represent the flow direction of the reaction liquid, and the arrows on the control plug, injection micro-plug assembly, and discharge micro-plug assembly represent the movement directions of the corresponding micro-plugs);

[0058] Figure 11 This is a schematic diagram of the internal structure of the microfluidic chip according to Example 2 of the present invention after longitudinal sectioning in another manner;

[0059] Figure 12 A longitudinal cross-sectional view of the reaction liquid flowing from the wash liquid sample inlet chamber into the lower chamber during the wash process of Example 2 of the present invention; (the arrows within the flow channel represent the flow direction of the reaction liquid, and the arrows on the control plug, the sample inlet micro-plug assembly, and the liquid outlet micro-plug assembly represent the movement directions of the corresponding micro-plugs);

[0060] Figure 13 A longitudinal cross-sectional view of the reaction liquid flowing into the reaction zone during the washing process of Example 2 of the present invention; (the arrows in the flow channel represent the flow direction of the reaction liquid, and the arrows on the control plug, the sample inlet micro-plug assembly, and the liquid outlet micro-plug assembly represent the movement directions of the corresponding micro-plugs);

[0061] Figure 14 This is a longitudinal cross-sectional view of the reaction liquid in the waste liquid discharge state during the washing process of Example 2 of the present invention; (the direction of the arrow in the flow channel represents the flow direction of the reaction liquid, and the arrows on the control plug, the injection micro-plug group, and the liquid discharge micro-plug group represent the movement direction of the corresponding micro-plug). DETAILED DESCRIPTION

[0062] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0063] Example 1

[0064] like Figures 1 to 4 The figure shows a preferred embodiment of the present invention. The microfluidic chip of the present invention is mainly used to detect samples to be tested through biochemical reactions. When the microfluidic chip is used in the field of rapid detection, users need to be able to detect samples to be tested through convenient operation, and the cost of the microfluidic chip itself needs to be reduced for mass production. Therefore, it would be beneficial to provide a microfluidic chip that is easy to operate, has a fast detection speed, and is low in cost. The microfluidic chip is described in detail below:

[0065] See Figures 1 to 2, the microfluidic chip includes a substrate 1, an air pressure control chamber 3, an elastic film layer 4, an injection micro-plug group 5, a liquid outlet micro-plug group 6, a control plug 7 and a driving source, wherein the substrate 1 is provided with an injection area 11 for adding a reaction liquid and a reaction area 12 and an outlet 2 located downstream of the injection area 11 along the flow direction of the reaction liquid; the air pressure control chamber 3 is arranged between the injection area 11 and the reaction area 12, the air pressure control chamber 3 is connected to the injection area 11 through an injection flow channel 13, and the air pressure control chamber 3, the reaction area 12, and the outlet 2 are connected through a liquid outlet flow channel 14; the elastic film layer 4 is arranged on the substrate 1 and covers the air pressure control chamber 3, the injection flow channel 13 and the liquid outlet flow channel 14; the injection micro-plug group 5 is arranged on the elastic film layer 4, and is used in conjunction with the elastic film layer 4 to act on the injection flow channel 13, so as to open or close the injection flow channel 13; the liquid outlet micro-plug group 6 is arranged on the elastic film layer 4, and is used in conjunction with the elastic film layer 4 to act on The liquid outlet channel 14 is used to open or close the liquid outlet channel 14; the control plug 7 is provided on the elastic film layer 4, and the bottom surface of the control plug 7 is aligned with the top surface of the air pressure control chamber 3; there are multiple driving sources, which provide power to the corresponding control plug 7, sampling micro-plug group 5 and liquid outlet micro-plug group 6 respectively; when the driving source drives the sampling micro-plug group 5 and the liquid outlet micro-plug group 6 to press down and close the air pressure control chamber 3, the control plug 7 can be lifted to increase the volume of the air pressure control chamber 3, and then the sampling micro-plug group 5 is lifted to open the sampling flow channel 13, thereby realizing the reaction liquid from the sampling area 11 to flow into the air pressure control chamber 3; when the driving source drives the sampling micro-plug group 5 and the liquid outlet micro-plug group 6 to press down and close the air pressure control chamber 3, the control plug 7 can be pressed down to reduce the volume of the air pressure control chamber 3, and then the liquid outlet micro-plug group 6 is lifted to open the liquid outlet channel 14, thereby realizing the reaction liquid from the air pressure control chamber 3 to flow into the reaction area 12 or out of the outlet 2. In addition, a micro-plug mounting layer is provided on the elastic film layer 4, and the micro-plug mounting layer is provided with corresponding micro-plug mounting holes for mounting the sample injection micro-plug group 5, the liquid discharge micro-plug group 6, and the control plug 7. The sample injection micro-plug group 5, the liquid discharge micro-plug group 6, and the control plug 7 can be lifted up and pressed down along the hole walls of their corresponding micro-plug mounting holes, and the driving source can be manual drive or mechanical drive.

[0066] The air pressure control chamber 3 is vertically distributed in the substrate 1. The inner wall of the air pressure control chamber 3 is provided with a partition membrane 33 that protrudes inward along the circumferential direction. The partition membrane 33 divides the air pressure control chamber 3 into an upper chamber 31 and a lower chamber 32, wherein the upper chamber 31 is connected to the sample injection area 11 to provide a movable space for the control plug 7; the lower chamber 32 is connected to the reaction area 12 to provide a movable space for the control plug 7; the inner side of the partition membrane 33 is surrounded by a flow area 331 for connecting the upper chamber 31 and the lower chamber 32, and the cross-sectional size of the flow area 331 is smaller than the outer diameter of the lower end of the control plug 7; the partition membrane 33 is elastic; when driving When the driving source drives the sample injection micro-plug group 5 and the liquid outlet micro-plug group 6 to press down and close the air pressure control chamber 3, the downward pressure control plug 7 can be used to contact the separation membrane 33 from the top of the upper chamber 31, and then the liquid micro-plug group 6 is raised to open the liquid outlet flow channel 14, thereby realizing the reaction liquid flowing from the air pressure control chamber 3 into the reaction area 12; when the driving source drives the sample injection micro-plug group 5 and the liquid outlet micro-plug group 6 to press down and close the air pressure control chamber 3, the downward pressure control plug 7 can be used to drive the separation membrane 33 to contact the bottom of the lower chamber 32, and then the liquid micro-plug group 6 is raised to open the liquid outlet flow channel 14, thereby realizing the reaction liquid flowing out from the outlet 2. The user can selectively control the reaction liquid to enter the reaction area 12 or discharge the waste liquid from the outlet 2 by choosing to press the control plug 7 down until it is released from the separation membrane 33 or contacts the bottom surface of the lower chamber 32. The setting of the separation membrane 33 also allows the control plug 7 to separate the upper chamber 31 and the lower chamber 32 when the control plug 7 is pressed down into the lower chamber 32, thereby increasing the speed at which the pressure in the lower chamber 32 increases and preventing the reaction liquid remaining in the reaction area 12 from flowing back into the injection channel 13 and contaminating the next reaction liquid after the control plug 7 is reset. It has the advantage of "one thing for two uses" and facilitates the user to control the force with which the reaction liquid flows into the reaction area 12 or flows out of the outlet 2.

[0067] Substrate 1 is also provided with a pressure relief channel 15 connected to upper chamber 31. This channel is connected to the external environment and serves to balance the air pressure within upper chamber 31. The provision of pressure relief channel 15 provides a pressure-dividing channel when the volume of upper chamber 31 decreases and the air pressure increases, thereby mitigating the increasing pressure within upper chamber 31. This reduces resistance when the user depresses control pin 7, allowing for smooth depressing of control pin 7. Furthermore, because the connection between pressure relief channel 15 and upper chamber 31 is relatively small, the pressure exerted by control pin 7 at the connection within upper chamber 31 is insufficient to push the reaction solution out of upper chamber 31 through pressure relief channel 15.

[0068] See Figures 3 and 4The substrate 1 of this embodiment includes a reaction liquid injection layer 8 and a reaction layer 9. The reaction liquid injection layer 8 is disposed below the elastic film layer 4, with an injection area 11 and an upper chamber 31 disposed within the reaction liquid injection layer 8. The injection area 11 is used to store the reaction liquid. The reaction layer 9 is disposed below the reaction liquid injection layer 8, with a reaction area 12 and a lower chamber 32 disposed within the reaction layer 9. A separation membrane 33 is disposed at the junction of the reaction liquid injection layer 8 and the reaction layer 9. The upper chamber 31 vertically disposed within the reaction liquid injection layer 8 and the lower chamber 32 disposed within the reaction layer 9 prevent the mixed liquid within the reaction zone 12 from flowing back against gravity, thereby reducing the risk of the mixed liquid within the reaction zone 12 flowing into the injection channel 13. This further ensures that the mixed liquid already within the reaction zone 12 does not contaminate the reaction liquid during the process of the reaction liquid flowing from the injection area 11 into the air pressure control chamber 3.

[0069] Reaction layer 9 also includes a wash liquid sampling chamber 91, which communicates with lower chamber 32 via wash liquid sampling channel 16 for sampling wash liquid. Elastic film layer 4 includes a wash liquid sampling micro-plug 41, which cooperates with elastic film layer 4 and acts on wash liquid sampling channel 16 to open or close wash liquid sampling channel 16. By lifting control plug 7, wash liquid can enter lower chamber 32 directly, eliminating the need to wait for the reaction liquid to flow from upper chamber 31 to lower chamber 32, as is the case with reaction liquid sampling. This simplifies the cleaning of reaction zone 12 with wash liquid and facilitates user cleaning of reaction zone 12.

[0070] The liquid outlet channel 14 includes a reaction zone inlet channel 141 and a control channel 142, connecting the reaction zone 12 and the lower chamber 32. The liquid outlet micro-plug assembly 6 cooperates with the elastic film layer 4 and acts on the control channel 142 to open or close it. A waste liquid reservoir is also connected between the reaction zone 12 and the outlet 2. This waste liquid reservoir is connected to the reaction zone 12 via the control channel 142. The liquid outlet micro-plug assembly 6 controls the open and closed state of the control channel 142 to allow liquid to flow into or out of the reaction zone. The user can manipulate the open and closed state of the control channel 142 via the liquid outlet micro-plug assembly 6 to control liquid flow into or out of the reaction zone.

[0071] The reaction zone 12 includes a first reaction chamber 121, a second reaction chamber 122 and a third reaction chamber 123 which are independently arranged. The reaction zone liquid inlet channel 141 includes a first reaction zone liquid inlet channel 1411 connected to the first reaction chamber 121, a second reaction zone liquid inlet channel 1412 connected to the second reaction chamber 122 and a third reaction zone liquid inlet channel 1413 connected to the third reaction chamber 123. The first reaction zone liquid inlet channel 1411, the second reaction zone liquid inlet channel 1412 and the third reaction zone liquid inlet channel 1413 have the same size. The arrangement of the first reaction chamber 121, the second reaction chamber 122 and the third reaction chamber 123 in the chip enables the detection process to be carried out independently in the three reaction chambers, and the same size of the first reaction area liquid inlet channel 1411, the second reaction area liquid inlet channel 1412 and the third reaction area liquid inlet channel 1413 ensures that the reaction liquid can enter the three reaction chambers at the same time, ensuring the synchronization of the detection process in different reaction chambers and ensuring the high efficiency of chip detection.

[0072] The control channel 142 includes a first control channel 1421 connected to the first reaction chamber 121, a second control channel 1422 connected to the second reaction chamber 122, and a third control channel 1423 connected to the third reaction chamber 123; the liquid outlet micro-plug group 6 includes a first liquid outlet micro-plug 61 located directly above the first control channel 1421 for controlling the on-off state of the first control channel 1421, a second liquid outlet micro-plug 62 located directly above the second control channel 1422 for controlling the on-off state of the second control channel 1422, and a third liquid outlet micro-plug 63 located directly above the third control channel 1423 for controlling the on-off state of the third control channel 1423. The first liquid outlet micro-plug 61, the second liquid outlet micro-plug 62 and the third liquid outlet micro-plug 63 are connected by a liquid outlet micro-plug connector 64. The provision of the liquid outlet micro-plug connector 64 ensures that the first reaction chamber 121, the second reaction chamber 122 and the third reaction chamber 123 are closed or circulated at the same time, which is beneficial to the synchronous progress of the detection process and increases the experimental efficiency.

[0073] The microfluidic chip can mainly realize the injection and discharge of reaction liquid. The operation process is that the user drives the injection micro-plug group 5 and the liquid outlet micro-plug group 6 to press down to close the air pressure control chamber, lifts the control plug 7 to increase the volume of the air pressure control chamber 3, and then lifts the injection micro-plug group 5 to open the injection flow channel 13, and the reaction liquid flows from the injection area 11 into the upper chamber 31; after waiting for the reaction liquid to flow from the upper chamber 31 into the lower chamber 32 by gravity, the driving source drives the injection micro-plug group 5 and the liquid outlet micro-plug connector 64 to press down to close the air pressure control chamber 3, and then The control plug 7 is pressed down until it contacts the separation membrane 33 to reduce the volume of the air pressure control chamber 3, and then the liquid micro-plug connector 64 is pulled up to open the liquid outlet channel 14, and the reaction liquid flows into the reaction area 12 from the lower chamber 32, completing the injection operation; after the reaction liquid in the reaction area 12 is completed, a mixed liquid is obtained, and the liquid outlet micro-plug connector 64 is pressed down to close the liquid outlet channel 14, and the control plug 7 is pressed down until it contacts the bottom surface of the lower chamber 32, and then the liquid outlet micro-plug connector 64 is pulled up to open the liquid outlet channel 14, and the mixed liquid flows out from the outlet 2, completing the discharge operation.

[0074] Example 2 Detection method using the above-mentioned microfluidic chip

[0075] This embodiment is a detection method using the microfluidic chip in Example 1, please refer to Figures 5 to 14 The following steps are involved:

[0076] S1 detection platform construction, reagent presetting and state initialization: a magnetic field generating device is placed under the chip body, which acts on the reaction area 12 and is used to control whether the magnetic beads in the reaction chamber are in a magnetic attraction state; the sample injection area 11 includes a first sample injection chamber 111, a second sample injection chamber 112, a third sample injection chamber 113, and a fourth sample injection chamber 114; the sample injection channel 13 includes a first sample injection channel 131 connected to the first sample injection chamber 111, a second sample injection channel 13 connected to the second sample injection chamber 112, and a fourth sample injection chamber 114. 2. A third sample injection channel 133 connected to the third sample injection chamber 113, a fourth sample injection channel 134 connected to the fourth sample injection chamber 114, and a sample injection micro-plug group 5 including a first sample injection micro-plug 51 for controlling the on / off state of the first sample injection channel 131, a second sample injection micro-plug 52 for controlling the on / off state of the second sample injection channel 132, a third sample injection micro-plug 53 for controlling the on / off state of the third sample injection channel 133, and a fourth sample injection micro-plug 54 for controlling the on / off state of the fourth sample injection channel 134.

[0077] Add the magnetic bead primary antibody solution, the test sample solution, the enzyme-labeled secondary antibody solution, the color developing solution, and the washing solution into the first sample injection chamber 111, the second sample injection chamber 112, the third sample injection chamber 113, the fourth sample injection chamber 114, and the washing solution injection chamber 91, respectively. Press down the control plug 7, the sample injection micro-plug group 5, and the liquid outlet micro-plug group 6. The air pressure control chamber 3, the sample injection channel 13, and the liquid outlet channel 14 are blocked, and the magnetic field generating device is turned off.

[0078] S2: Primary antibody incubation: The magnetic bead primary antibody solution and the sample solution to be tested in step S1 are introduced into the reaction zone 12; after mixing and incubation, a magnetic field generating device is turned on for magnetic separation, and after washing, the sample-primary antibody magnetic beads magnetically attracted to the reaction zone 12 are obtained;

[0079] S3 secondary antibody incubation: The enzyme-labeled secondary antibody solution in step S1 is introduced into the reaction zone 12, the magnetic field generator is turned off, and the solution is mixed with the sample-primary antibody magnetic beads obtained in step S2 and incubated, and then the magnetic field generator is turned on for magnetic separation. After washing again, the enzyme-labeled secondary antibody-sample-primary antibody magnetic beads are magnetically attracted to the reaction zone 12;

[0080] S4 color development, termination, and quantitative analysis: The color development solution in step S1 is introduced into the reaction area 12, the magnetic field generating device is turned off, and the solution is mixed and incubated with the enzyme-labeled secondary antibody-sample-primary antibody magnetic beads obtained in step S3, and then the stop solution is added to obtain the solution to be tested; a detection module for performing absorbance analysis on the reaction area 12 is also provided above the reaction area 12. The detection module is turned on, and the absorbance values ​​obtained by absorbance analysis of the solution to be tested in the first reaction chamber 121, the second reaction chamber 122, and the third reaction chamber 123 are averaged and converted into the content of the marker in the solution to be tested. Among them, the first reaction chamber 121, the second reaction chamber 122 and the third reaction chamber 123 are each provided with a stop solution injection port for adding the stop solution, and each stop solution injection port is independently connected to the one-way valve 10. The flow direction of the one-way valve 10 is from the outside of the stop solution injection port to the inside of the reaction zone 12, which is used to prevent the reaction solution from leaking out of the stop solution injection port during mixing and incubation. Therefore, the operation of adding the stop solution in step S4 is to inject the stop solution into the stop solution injection port corresponding to the first reaction chamber 121, the second reaction chamber 122 and the third reaction chamber 123 respectively. The stop solution is added through the one-way valve 10. At the same time, the first sampling chamber 111, the second sampling chamber 112, the third sampling chamber 113, and the fourth sampling chamber 114 are all provided with sampling ports for injecting the reaction liquid. Each sampling port is independently provided with a one-way valve 10. The magnetic field generating device is a permanent magnet. When the permanent magnet is placed under the reaction zone 12, the magnetic field generating device is turned on and a magnetic field is generated in the reaction zone 12; when the permanent magnet is removed from under the reaction zone 12, the magnetic field generating device is turned off and the magnetic field in the reaction zone 12 disappears.

[0081] See Figures 5 to 8 In step S2 of this embodiment, the operation steps of introducing the magnetic bead primary antibody solution and the test sample solution in step S1 into the reaction zone 12 include:

[0082] S21 Magnetic bead injection: lift up the first injection micro-plug 51 and the control plug 7, and suck the magnetic bead primary antibody solution in step S1 from the first injection chamber 111 into the air pressure control chamber 3, then press down the first injection micro-plug 51 to close the first injection flow channel 131; press down the control plug 7 until it contacts the separation membrane 33, and lift up the liquid outlet micro-plug connector 64, so that the magnetic bead primary antibody solution flows from the air pressure control chamber 3 into the reaction area 12; then turn on the magnetic field generating device to perform magnetic separation on the magnetic bead primary antibody solution, obtain the primary antibody magnetic beads adsorbed on the bottom of the reaction area 12 and the separated waste liquid, and discharge the waste liquid;

[0083] S22: Sample injection: Lift the second injection micro-plug 52 and the control plug 7 upward to draw the sample solution from step S1 into the air pressure control chamber 3. Then, press down the second injection micro-plug 52 to seal the second injection channel 132. Press down the control plug 7 until it contacts the separator 33, and lift up the outlet micro-plug connector 64. The sample solution enters the reaction zone 12 from the air pressure control chamber 3. Once the sample solution has completely entered the reaction zone 12 and contacts the antimagnetic beads obtained in step S21, step S22 is complete. By pressing down the control plug 7 until it contacts the separator 33, the reaction solution flows into the reaction zone 12 rather than flowing directly out of the outlet 2. This allows the user to control the force of the downward pressure and facilitates operation during the injection of the reaction solution.

[0084] See Figure 9 The mixing and incubation steps in step S2 and step S3 of this embodiment include: turning off the magnetic field generating device, controlling the control plug 7 to perform reciprocating motion in the upper chamber 31, and completing the mixing and incubation operation after the magnetic beads are evenly distributed in the reaction zone 12. The reciprocating motion of the control plug 7 in the upper chamber 31 ensures that the reaction liquid in the reaction zone is always mixed at the outlet of the reaction zone liquid inlet channel 141 and the reaction zone 12, and does not flow back into the lower chamber 32. At the same time, since the upper chamber 31 is connected to the pressure relief channel 15, the suction and thrust of the fluid during the mixing and incubation operation are relatively small, thereby preventing the mixed liquid from flowing back into the upper chamber due to excessive suction, and facilitating the user's mixing and incubation operation.

[0085] Please see Figures 10 to 14The washing operation in steps S2 and S3 of this embodiment is to turn on the magnetic field generating device, lift up the washing liquid sampling micro-plug 41 and the control plug 7, and the washing liquid in step S1 flows into the lower chamber 32 from the washing liquid sampling chamber 91; after the washing liquid has completely flowed into the lower chamber 32, press down the washing liquid sampling micro-plug 41 and then press down the control plug 7 until it contacts the separation membrane 33, lift up the liquid outlet micro-plug connector 64, and the washing liquid flows into the reaction zone 12; after the washing liquid has completely entered the reaction zone 12, continue to press down the control plug 7 until the separation membrane 33 contacts the bottom surface of the lower chamber 32, and the liquid in the reaction zone 12 flows to the outlet 2. After the washing liquid has completely flowed out of the reaction zone 12, lift up the control plug 7 until the separation membrane 33 recovers its deformation, press down the liquid outlet micro-plug connector 64, control the flow channel 142 to be blocked, and the washing operation is completed. During the entire washing operation, the control plug 7 only needs to complete the lifting and pressing actions, which is simple and easy to implement. Pressing down the washing liquid injection micro-plug 41 has pressed the control plug 7 down until it contacts the separation membrane 33, which prevents the washing liquid from flowing back into the upper chamber 31 and the washing liquid injection chamber 91, and provides sufficient power for the washing liquid to enter the reaction area 12 or flow out from the outlet 2. The cleaning is fast and easy to operate, which is convenient for the user to fully clean the reaction area 12.

[0086] In step S21, the waste liquid is discharged by pressing the control plug 7 downward until the separator 33 contacts the bottom surface of the lower chamber 32. The waste liquid flows from the reaction zone 12 to the outlet 2, thereby being discharged. By pressing the control plug 7 downward to the bottom surface of the lower chamber 32, the user can empty the waste liquid, making it convenient for the user to empty the reaction zone 12.

Claims

1. A microfluidic chip comprising: A substrate (1) is provided with an inlet area (11) for adding a reaction liquid, and a reaction area (12) and an outlet (2) sequentially located downstream of the inlet area (11) along the flow direction of the reaction liquid; An air pressure control chamber (3) is provided between the sample inlet region (11) and the reaction region (12); the air pressure control chamber (3) and the sample inlet region (11) are connected via an inlet flow channel (13); and the air pressure control chamber (3), the reaction region (12), and the outlet (2) are connected via a liquid outlet flow channel (14); It is characterized in that Also included are: An elastic film layer (4) is provided on the substrate (1) and covers the air pressure control chamber (3), the sample inlet flow channel (13) and the liquid outlet flow channel (14); The sampling micro-plug group (5) is provided on the elastic film layer (4), cooperates with the elastic film layer (4) and acts on the sampling flow channel (13) to open or close the sampling flow channel (13); A liquid outlet micro-plug group (6) is provided on the elastic film layer (4), cooperates with the elastic film layer (4) and acts on the liquid outlet flow channel (14) to open or close the liquid outlet flow channel (14); A control pin (7) is provided on the elastic film layer (4), and the bottom surface of the control pin (7) is aligned with the top surface of the air pressure control chamber (3); A plurality of driving sources are provided, each providing power to the corresponding control plug (7), the sample inlet micro plug group (5) and the liquid outlet micro plug group (6); When the driving source drives the sample injection micro-plug group (5) and the liquid discharge micro-plug group (6) to press down and close the air pressure control chamber (3), the volume of the air pressure control chamber (3) can be increased by lifting the control plug (7), and then the sample injection micro-plug group (5) is lifted to open the injection flow channel (13), thereby realizing that the reaction liquid flows from the sample injection area (11) into the air pressure control chamber (3); When the driving source drives the sample inlet micro-plug group (5) and the liquid outlet micro-plug group (6) to press down and close the air pressure control chamber (3), the volume of the air pressure control chamber (3) can be reduced by pressing down the control plug (7), and then the liquid outlet micro-plug group (6) can be raised to open the liquid outlet channel (14), thereby realizing that the reaction liquid flows from the air pressure control chamber (3) into the reaction area (12) or flows out from the outlet (2).

2. The microfluidic chip according to claim 1, wherein: The air pressure control chamber (3) is vertically distributed in the substrate (1), and the inner wall of the air pressure control chamber (3) is provided with a separation membrane (33) protruding inwardly along the circumferential direction, and the separation membrane (33) divides the air pressure control chamber (3) into: An upper chamber (31) is communicated with the sample injection area (11) and is used to provide a movable space for the control pin (7); The lower chamber (32) is connected to the reaction zone (12) and is used to provide a movable space for the control plug (7); the inner side of the separation membrane (33) is surrounded by a flow area (331) for connecting the upper chamber (31) and the lower chamber (32); the cross-sectional size of the flow area (331) is smaller than the outer diameter of the lower end of the control plug (7); the separation membrane (33) is elastic; When the driving source drives the sample inlet micro-plug group (5) and the liquid outlet micro-plug group (6) to press down and close the air pressure control chamber (3), the control plug (7) can be pressed down from the top of the upper chamber (31) to contact the separation membrane (33), and then the liquid outlet micro-plug group (6) can be raised to open the liquid outlet flow channel (14), thereby realizing that the reaction liquid flows from the air pressure control chamber (3) into the reaction area (12); When the driving source drives the sample inlet micro-plug group (5) and the liquid outlet micro-plug group (6) to press down and close the air pressure control chamber (3), the separation membrane (33) can be driven to contact the bottom of the lower chamber (32) by pressing down the control plug (7), and then the liquid outlet micro-plug group (6) is pulled up to open the liquid outlet channel (14), thereby realizing the reaction liquid flowing out of the outlet (2).

3. The microfluidic chip according to claim 2, wherein: The substrate (1) is further provided with a pressure relief channel (15) communicating with the upper cavity (31); the pressure relief channel (15) is in communication with the external environment and is used to balance the air pressure in the upper cavity (31).

4. The microfluidic chip according to claim 2, wherein: The substrate (1) comprises: A reaction liquid sampling layer (8) is provided below the elastic film layer (4), the sampling area (11) and the upper cavity (31) are provided in the reaction liquid sampling layer (8), and the sampling area (11) is used for storing the reaction liquid; The reaction layer (9) is arranged below the reaction liquid injection layer (8), and the reaction zone (12) is arranged in the lower cavity (32) of the reaction layer (9); the separation membrane (33) is arranged at the junction of the reaction liquid injection layer (8) and the reaction layer (9).

5. The microfluidic chip according to claim 4, characterized in that: The reaction layer (9) is further provided with a washing liquid sampling chamber (91), which is connected to the lower chamber (32) via a washing liquid sampling flow channel (16) and is used for sampling the washing liquid; the elastic film layer (4) is provided with a washing liquid sampling micro-plug (41), which cooperates with the elastic film layer (4) and acts on the washing liquid sampling flow channel (16) to open or close the washing liquid sampling flow channel (16).

6. The microfluidic chip according to claim 5, characterized in that: The liquid outlet channel (14) comprises a reaction zone liquid inlet channel (141) and a control channel (142) connecting the reaction zone (12) and the lower chamber (32); the liquid outlet micro-plug group (6) cooperates with the elastic film layer (4) and acts on the control channel (142) to open or close the control channel (142).

7. The microfluidic chip according to claim 6, characterized in that: The reaction zone (12) includes a first reaction chamber (121), a second reaction chamber (122) and a third reaction chamber (123) which are independently arranged; the reaction zone liquid inlet flow channel (141) includes a first reaction zone liquid inlet flow channel (1411) connected to the first reaction chamber (121), a second reaction zone liquid inlet flow channel (1412) connected to the second reaction chamber (122) and a third reaction zone liquid inlet flow channel (1413) connected to the third reaction chamber (123); the first reaction zone liquid inlet flow channel (1411), the second reaction zone liquid inlet flow channel (1412) and the third reaction zone liquid inlet flow channel (1413) have the same size.

8. The microfluidic chip according to claim 7, characterized in that: The control flow channel (142) includes a first control flow channel (1421) communicating with the first reaction chamber (121), a second control flow channel (1422) communicating with the second reaction chamber (122), and a third control flow channel (1423) communicating with the third reaction chamber (123); Correspondingly, the liquid outlet micro-plug group (6) includes a first liquid outlet micro-plug (61) located directly above the first control flow channel (1421) and used to control the on-off state of the first control flow channel (1421), a second liquid outlet micro-plug (62) located directly above the second control flow channel (1422) and used to control the on-off state of the second control flow channel (1422), and a third liquid outlet micro-plug (63) located directly above the third control flow channel (1423) and used to control the on-off state of the third control flow channel (1423). The first liquid outlet micro-plug (61), the second liquid outlet micro-plug (62) and the third liquid outlet micro-plug (63) are connected via a liquid outlet micro-plug connector (64).

9. A detection method, characterized in that The microfluidic chip according to claim 8 is applied, comprising the following steps: S1 detection platform construction, reagent presetting and state initialization: a magnetic field generating device is placed below the chip body, which acts on the reaction area (12) and is used to control whether the magnetic beads in the reaction chamber are in a magnetic attraction state; the sample injection area (11) includes a first sample injection chamber (111), a second sample injection chamber (112), a third sample injection chamber (113), and a fourth sample injection chamber (114); the sample injection channel (13) includes a first sample injection channel (131) connected to the first sample injection chamber (111), a second sample injection channel (132) connected to the second sample injection chamber (112), and a fourth sample injection chamber (114). ), a third sample injection channel (133) communicating with the third sample injection chamber (113), a fourth sample injection channel (134) communicating with the fourth sample injection chamber (114), the sample injection micro-plug group (5) comprising a first sample injection micro-plug (51) for controlling the on-off state of the first sample injection channel (131), a second sample injection micro-plug (52) for controlling the on-off state of the second sample injection channel (132), a third sample injection micro-plug (53) for controlling the on-off state of the third sample injection channel (133), and a fourth sample injection micro-plug (54) for controlling the on-off state of the fourth sample injection channel (134). The magnetic bead primary antibody solution, the sample solution to be tested, the enzyme-labeled secondary antibody solution, the color developing solution, and the washing solution are respectively added to the first sample injection chamber (111), the second sample injection chamber (112), the third sample injection chamber (113), the fourth sample injection chamber (114), and the washing solution injection chamber (91); the control plug (7), the sample injection micro-plug group (5), and the liquid outlet micro-plug group (6) are pressed down; the air pressure control chamber (3), the sample injection flow channel (13), and the liquid outlet flow channel (14) are blocked; and the magnetic field generating device is turned off; S2 primary antibody incubation: introducing the magnetic bead primary antibody solution and the sample solution to be tested in step S1 into the reaction zone (12); after mixing and incubating, turning on the magnetic field generating device for magnetic separation, and obtaining the sample-primary antibody magnetic beads magnetically attracted to the reaction zone (12) after washing; S3 secondary antibody incubation: introducing the enzyme-labeled secondary antibody solution in step S1 into the reaction zone (12), turning off the magnetic field generating device, mixing and incubating with the sample-primary antibody magnetic beads obtained in step S2, and then turning on the magnetic field generating device for magnetic separation, and washing again to obtain the enzyme-labeled secondary antibody-sample-primary antibody magnetic beads magnetically attracted to the reaction zone (12); S4 color development, termination, and quantitative analysis: the color development solution in step S1 is introduced into the reaction zone (12), the magnetic field generating device is turned off, and the solution to be tested is mixed and incubated with the enzyme-labeled secondary antibody-sample-primary antibody magnetic beads obtained in step S3, and then a termination solution is added to obtain a solution to be tested; a detection module for performing absorbance analysis on the reaction zone (12) is also provided above the reaction zone (12), the detection module is turned on, and the absorbance values ​​obtained by performing absorbance analysis on the solution to be tested in the first reaction chamber (121), the second reaction chamber (122), and the third reaction chamber (123) are averaged and converted into the content of the marker in the solution to be tested.

10. The detection method according to claim 9, characterized in that The operation steps of introducing the magnetic bead primary antibody solution and the sample solution to be tested in step S1 into the reaction zone (12) in step S2 include: S21 Magnetic bead injection: lift up the first injection micro-plug (51) and the control plug (7), and suck the magnetic bead and antibody solution of step S1 from the first injection chamber (111) into the air pressure control chamber (3), and then press down the first injection micro-plug (51) to close the first injection flow channel (131); press down the control plug (7) until it contacts the separation membrane (33), and lift up the liquid outlet micro-plug connector (64), and the magnetic bead and antibody solution flows from the air pressure control chamber (3) into the reaction zone (12); then turn on the magnetic field generating device to perform magnetic separation on the magnetic bead and antibody solution, and obtain the antibody magnetic beads adsorbed on the bottom of the reaction zone (12) and the waste liquid after separation, and discharge the waste liquid; S22: Injection of the sample to be tested: lift up the second injection micro-plug (52) and the control plug (7), and then press down the second injection micro-plug (52) after the sample solution to be tested in step S1 is sucked into the air pressure control chamber (3), and the second injection flow channel (132) is blocked; press down the control plug (7) until it contacts the separation membrane (33), and lift up the liquid outlet micro-plug connector (64), and the sample solution to be tested enters the reaction zone (12) from the air pressure control chamber (3). After the sample solution to be tested completely enters the reaction zone (12) and contacts the antimagnetic beads obtained in step S21, step S22 is completed.

11. The detection method according to claim 10, characterized in that The mixing and incubation steps in step S2 and step S3 include: turning off the magnetic field generating device, controlling the control pin (7) to reciprocate in the upper chamber (31), and completing the mixing and incubation operation after the magnetic beads are evenly distributed in the reaction area (12).

12. The detection method according to claim 10, characterized in that The washing operation in steps S2 and S3 is to open the magnetic field generating device, lift up the washing liquid sampling micro-plug (41) and the control plug (7), and the washing liquid in step S1 flows into the lower chamber (32) from the washing liquid sampling chamber (91); after the washing liquid completely flows into the lower chamber (32), press down the washing liquid sampling micro-plug (41) and then press down the control plug (7) until it contacts the separation membrane (33), lift up the liquid outlet micro-plug connector (64), and the washing liquid flows out. into the reaction zone (12); after the washing liquid completely enters the reaction zone (12), continue to press the control plug (7) downward until the separation membrane (33) contacts the bottom surface of the lower chamber (32), and the liquid in the reaction zone (12) flows to the outlet (2); after the washing liquid completely flows out of the reaction zone (12), lift the control plug (7) upward until the separation membrane (33) recovers its deformation, press down the liquid outlet micro-plug connector (64), and the control flow channel (142) is blocked, and the washing operation is completed.

13. The detection method according to claim 11, characterized in that The operation of discharging the waste liquid in step S21 is to press the control bolt (7) downward until the separation membrane (33) contacts the bottom surface of the lower chamber (32), and the waste liquid flows from the reaction zone (12) to the outlet (2) to be discharged.

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