Immunomagnetic bead detection system and detection method based on micro-fluidic chip

Abstract

The invention relates to the technical field of micro-fluidic detection equipment, and provides an immunomagnetic bead detection system based on a micro-fluidic chip, the immunomagnetic bead detection system comprises a support and the micro-fluidic chip, and a magnetic field device comprises a magnet; the device is characterized in that the magnet is a cylinder magnetized in the diameter direction, the central axis of the magnet is perpendicular to the bottom face of the reaction bin, the diameter of the magnet is larger than the width of the reaction bin, the diameter of the magnet is smaller than the length of the reaction bin, and the diameter of the magnet is larger than or equal to two times of the width of the reaction bin. The magnetic field intensity of the surface of the magnet ranges from 20 mT to 150 mT; meanwhile, the invention also provides a detection method applying the immunomagnetic bead detection system based on the micro-fluidic chip. The magnetic bead detection system has the beneficial effects that the magnetic beads left in the reaction bin after a user washes the magnetic beads are few, the cleaning effect is good, and the detection limit is reduced; the detection method has the advantages of low detection limit and high detection speed.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic detection devices, and particularly to an immunomagnetic bead detection system and detection method based on a microfluidic chip. Background Art

[0002] Microfluidic chip technology is a technology that can integrate various biochemical reactions onto a microchip. The miniaturization of large instruments can reduce the occupied space of experimental instruments, and the integration of each experimental step on the chip can also simplify and shorten the experimental process and time.

[0003] The integration of biological reactions can greatly improve the experimental efficiency. For example, the Chinese invention patent "Chemiluminescence Detection Microfluidic Chip and Chemiluminescence Detection Microfluidic Chip System and Their Applications" with the patent number CN201711206029.5 (publication number CN107930710A) discloses a chemiluminescence microfluidic chip that integrates sample injection, reaction, and detection. The chip includes a sample injection unit, a liquid storage unit, a reaction unit, and a waste liquid unit. The sample to be tested flows from the sample injection unit and reacts with the reaction reagents (enzyme-labeled antibody reagent, microbead-coupled antibody reagent, cleaning solution) quantitatively released from the liquid storage unit in the reaction chamber. After magnetic beads capture the target, the magnetic field is turned on and the cleaning solution is added to wash away the excess enzyme-labeled antibody. The chromogenic solution and the terminating solution are added, and the absorbance is detected by the light detection unit. Finally, the magnetic beads and the reaction solution are rinsed into the waste liquid unit together. The detection process of this chip is simple, can quantify three reaction samples, has high sensitivity and strong repeatability. However, during the reaction process, when the magnetic beads adsorbed on the bottom surface of the reaction unit and already coupled with the sample to be tested are mixed with the enzyme-labeled secondary antibody, due to the uneven magnetic field distribution in the reaction chamber, the magnetic beads tend to concentrate in the strong magnetic field area corresponding to the bottom of the reaction chamber by the magnet, resulting in uneven distribution of magnetic beads in the reaction chamber. Most magnetic beads stack and gather at the edge of the strong magnetic field area. When the enzyme-labeled secondary antibody solution enters the reaction chamber, the magnetic field is turned off, and the stacked magnetic beads will reduce the collision probability between the secondary antibody sites coupled with the sample to be tested on the surface of the magnetic beads and the enzyme-labeled secondary antibody, resulting in a significant decrease in the coupling efficiency, increasing the experimental time and affecting the experimental results, and easily leading to false negatives. At the same time, in the final rinsing step, due to the uneven magnetic field distribution, some reactants will remain near the side wall area of the reaction chamber with a slower flow rate, making it difficult to clean and affecting the next reaction in this reaction chamber, thus reducing the experimental efficiency. At the same time, it takes a long time to prepare the magnetic bead-primary antibody reagent in advance before the experiment, increasing the experimental cycle. If purchased directly, the cost will increase. Summary of the Invention

[0004] The first technical problem to be solved by the present invention is to provide an immunomagnetic bead detection system based on a microfluidic chip with high coupling efficiency and easy to clean in view of the above-mentioned prior art status.

[0005] The second technical problem to be solved by the present invention is to provide a detection method that uses the above-mentioned immunomagnetic bead detection system and can efficiently prepare a primary antibody probe on a chip in response to the above-mentioned existing technical status.

[0006] The technical solution adopted by the present invention to solve the first technical problem is: the immunomagnetic bead detection system based on the microfluidic chip includes a support,

[0007] A microfluidic chip is mounted on the support. A reaction chamber is provided inside the microfluidic chip. The reaction chamber contains a plurality of magnetic beads for coupling reaction with labeled antibodies and test proteins. The reaction chamber has a reaction chamber inlet and a reaction chamber outlet for liquid circulation.

[0008] a magnetic field device, disposed in the support and below the microfluidic chip, the magnetic field device acting on the reaction chamber, comprising a magnet for generating a magnetic field in the reaction chamber;

[0009] Its characteristics are:

[0010] The magnet is a cylinder magnetized along the diameter direction, the central axis of the magnet is perpendicular to the bottom surface of the reaction chamber, the diameter of the magnet is greater than the width of the reaction chamber and the diameter of the magnet is less than the length of the reaction chamber, the diameter of the magnet is greater than or equal to twice the width of the reaction chamber, and the magnetic field strength on the surface of the magnet ranges from 20 to 150 mT.

[0011] In order to facilitate the user to control the intensity of the magnetic field in the reaction chamber, preferably, the magnetic field device further includes a driving member capable of driving the magnet (21) to move closer to or further away from the reaction chamber. When the driving member drives the magnet (21) to be in close contact with the bottom of the reaction chamber, the number of magnetic beads adsorbed and gathered on the flow path formed from the reaction chamber inlet to the reaction chamber outlet in the reaction chamber is greater than the number of magnetic beads gathered on both sides perpendicular to the flow path. The lifting and lowering movement of the driving member controls the intensity of the magnetic field in the reaction chamber. The characteristics of the magnet itself and its relative position to the reaction chamber determine that when the magnet is in close contact with the bottom of the reaction chamber, the number of magnetic beads gathered on both sides of the reaction chamber is less than the number of magnetic beads gathered near the reaction chamber inlet and the reaction chamber outlet.

[0012] To improve coupling efficiency during use and facilitate remixing of the magnetic beads after magnetic attraction, the diameter of the magnet is preferably twice the width of the reaction chamber. With this size relationship, when the magnet fits against the bottom of the reaction chamber, the magnetic field strength within the chamber is evenly distributed across the width. This evenly distributes the magnetic beads in the central area between the chamber's sidewalls, reducing the amount of beads stacked together. This allows for easier dispersion during remixing and improves coupling efficiency with the reactants.

[0013] To improve the accuracy of using the above-mentioned immunomagnetic bead detection system, preferably, the magnetic field strength range on the surface of the magnet is 100-130 mT. This technical solution can achieve that when the magnet fits against the bottom of the reaction chamber, the magnetic beads can be firmly magnetically attracted to the bottom of the reaction chamber and will not be washed out of the reaction chamber by the washing liquid and flowing air, increasing the number of magnetic beads during detection and reducing the risk of false negatives in detection.

[0014] Furthermore, the thickness of the magnet: the diameter of the magnet is 1:8.

[0015] The present invention provides a detection method to solve the second technical problem, which is characterized by applying the above-mentioned immunomagnetic bead detection system based on a microfluidic chip, and includes the following steps:

[0016] S1. Construction of the microfluidic detection platform: The microfluidic chip is also provided with an inlet communicating with the inlet of the reaction chamber, a detection chamber communicating downstream of the reaction chamber, a mixing channel communicating downstream of the detection chamber and an outlet communicating downstream of the mixing channel; controlling the driving member to adjust the magnetic field device to the initial state where the magnet is farthest from the reaction chamber, and a detection device for performing chromaticity analysis on the liquid in the detection chamber is also provided above the microfluidic chip; the inlet is connected to a washing device for pumping washing liquid and air into the reaction chamber; a mixing device is provided above the microfluidic chip, and the mixing device acts on the reaction chamber to accelerate the coupling reaction;

[0017] S2. Preparation of magnetic bead primary antibody probe: Add avidin-coated magnetic beads and biotin-labeled antibody from the inlet in step S1 to the reaction chamber of the microfluidic chip, and after opening the mixing device and vibrating and mixing, the coupling is completed; adjust the driving member to move the magnet from the initial state in step S1 to the magnetic attraction state where it fits against the bottom of the reaction chamber to complete magnetic separation; open the washing device to empty, wash, and empty the reaction chamber to obtain the primary antibody probe;

[0018] S3. Preparation of immunomagnetic beads with a double antibody sandwich structure: Add the protein solution to be detected and the enzyme-labeled antibody into the reaction chamber in sequence; adjust the driving member, and the magnetic field device is restored from the magnetic attraction state in step S2 to the initial state, open the mixing device and vibrate and mix, adjust the driving member to make the magnetic field device in the magnetic attraction state, and after the magnetic beads are completely separated, wash and empty to obtain immunomagnetic beads with a double antibody sandwich structure;

[0019] S4. Color development and termination: Add the color developing solution into the reaction chamber, adjust the magnetic field device from the magnetic attraction state in step S3 to the initial state, open the mixing device and vibrate and mix; adjust the magnetic field device to the magnetic attraction state, add the termination solution and let it stand for 1-3 min, and the solution changes color to obtain the reaction solution to be detected;

[0020] S5, Absorbance Detection: Transfer the reaction solution to be measured in Step 4 from the reaction chamber to the detection chamber for absorbance detection, and record the experimental data;

[0021] S6, Chip Cleaning: Adjust the magnet to its initial state, wash and empty it.

[0022] To shorten the coupling time between the reactants and magnetic beads in the reaction chamber, preferably, the mixing device includes a vibration head and a vibration motor that is controllably connected to the vibration head. The parameters set for the vibration motor in the vibration mixing operations of Steps S2, S3, and S4 are: voltage greater than or equal to 3.7V, current 0.1A - 0.23A, vibration time 1 - 3 min, and vibration frequency 20000 - 35000 times / min. This technical solution realizes the full mixing of magnetic beads and reactants in a short time, further improves the coupling efficiency, and reduces the detection limit of the sample to be measured.

[0023] To be able to wash the excess reactant solution in the reaction chamber and the immunomagnetic beads in the reaction chamber after detection, preferably, the washing device includes a washing pump for pumping washing liquid into the reaction chamber and an air pump for pumping air into the reaction chamber. The washing operation in Steps S2, S3, and S6 is to turn on the washing pump to pump in the washing liquid, and the pumping flow rate of the washing pump is set to 300 μl / min, and the pumping time is set to 30 s; the emptying operation is to turn on the air pump to pump in air, and the pumping flow rate of the air pump is set to 300 μl / min, and the pumping time is set to 30 - 60 s. This technical solution can wash the magnetic beads in the center of the reaction chamber clean.

[0024] Further, the washing operation in Steps S2, S3, and S6 is performed 1 - 2 times.

[0025] Compared with the prior art, the advantages of the present invention are:

[0026] 1. In the immunomagnetic bead detection system based on a microfluidic chip, the technical features that the magnet is a cylinder magnetized along the diameter direction, the central axis of the magnet is perpendicular to the bottom surface of the reaction chamber, and the relationship between the diameter of the magnet and the size of the reaction chamber make the magnetic induction line density on both sides of the reaction chamber significantly less than that near the inlet and outlet of the reaction chamber. The range parameters of the magnetic field intensity in the reaction chamber reduce the magnetic induction line density on both sides of the reaction chamber to such an extent that the magnetic beads do not significantly accumulate on both sides of the reaction chamber. The synergistic effect of the magnet diameter and the magnetic field intensity makes it difficult for the magnetic beads to remain on both sides of the reaction chamber when the user cleans the reaction chamber through the washing device. After the user washes the magnetic beads, there are fewer magnetic beads remaining in the reaction chamber, and the cleaning effect is good;

[0027] 2. The technical feature that the diameter of the magnet is smaller than the length of the reaction chamber enables the magnetic beads to always be magnetically attracted to the bottom of the reaction chamber under the influence of the magnet's magnetic field rather than on the wall surface or outside the reaction chamber, ensuring that the reaction process between the magnetic beads and other solutions can always occur within the reaction chamber, reducing the risk of false negatives and lowering the detection limit.

[0028] 3. The detection method of this application's immunomagnetic bead detection system based on a microfluidic chip can prepare magnetic bead primary antibodies within the chip without the need to purchase magnetic bead primary antibody reagents, reducing costs. Moreover, the preparation time is controlled within 5 minutes, improving experimental efficiency. The high-frequency vibration of the vibration motor on the reaction chamber fully mixes the magnetic beads and reactants in a short time, further enhancing experimental efficiency. At the same time, the magnet controls the movement of the magnetic beads only within the reaction chamber to prevent the magnetic beads from running out of the reaction chamber prematurely, reducing the detection limit. After applying the above immunomagnetic bead detection system, the cleaning time is shortened and the cleaning effect is good, significantly shortening the experimental cycle compared to the prior art. In the case of a short cleaning time, the subsequent experiment will not be affected by the previous experiment, featuring a low detection limit and a fast detection speed. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic structural diagram of Embodiment 1 of the present invention;

[0030] Figure 2 It is an exploded view of the structure of the microfluidic chip of Embodiment 1 of the present invention;

[0031] Figure 3 It is a schematic structural diagram of the magnetic field device of Embodiment 1 of the present invention;

[0032] Figure 4 It is a schematic structural diagram of the magnet of Embodiment 1 of the present invention;

[0033] Figure 5 It is a schematic diagram of the positional relationship between the magnet and the microfluidic chip of Embodiment 1 of the present invention;

[0034] Figure 6 It is a partial longitudinal sectional view of the immunomagnetic bead detection system based on a microfluidic chip in the magnetically attracted state of Embodiment 2 of the present invention;

[0035] Figure 7 It is a distribution diagram of magnetic induction lines in the reaction chamber in the magnetically attracted state of Embodiment 2 of the present invention;

[0036] Figure 8 It is a partial longitudinal sectional view of the immunomagnetic bead detection system based on a microfluidic chip in the initial state of Embodiment 2 of the present invention;

[0037] Figure 9Top view of the magnetic field intensity simulation in the reaction chamber when the magnet is at different distances from the bottom of the reaction chamber in Embodiment 1 of the present invention (the first row of pictures represents the simulation diagram of the magnetic field intensity in the reaction chamber when the magnet is in contact with the bottom of the reaction chamber; the second row of pictures represents the simulation diagram of the magnetic field intensity in the reaction chamber when the upper surface of the magnet is 0.4 cm away from the bottom surface of the reaction chamber; the third row of pictures represents the simulation diagram of the magnetic field intensity in the reaction chamber when the upper surface of the magnet is 0.8 cm away from the bottom surface of the reaction chamber; the fourth row of pictures represents the simulation diagram of the magnetic field intensity in the reaction chamber when the upper surface of the magnet is 1.2 cm away from the bottom surface of the reaction chamber);

[0038] Figure 10 Longitudinal sectional view of the magnetic field intensity simulation in the reaction chamber when the magnet is at different distances from the bottom of the reaction chamber in Embodiment 1 of the present invention (the first row of pictures represents the simulation diagram of the magnetic field intensity in the reaction chamber when the magnet is in contact with the bottom of the reaction chamber; the second row of pictures represents the simulation diagram of the magnetic field intensity in the reaction chamber when the upper surface of the magnet is 0.4 cm away from the bottom surface of the reaction chamber; the third row of pictures represents the simulation diagram of the magnetic field intensity in the reaction chamber when the upper surface of the magnet is 0.8 cm away from the bottom surface of the reaction chamber; the fourth row of pictures represents the simulation diagram of the magnetic field intensity in the reaction chamber when the upper surface of the magnet is 1.2 cm away from the bottom surface of the reaction chamber, and the color from blue to red represents the magnetic field intensity from small to large);

[0039] Figure 11 Standard curve graph of the concentration of interleukin-6 and absorbance in Embodiment 4 of the present invention;

[0040] Figure 12 Standard curve graph of the concentration of procalcitonin and absorbance in Embodiment 5 of the present invention;

[0041] Figure 13 Standard curve graph of the concentration of C-reactive protein and absorbance in Embodiment 6 of the present invention. Detailed implementation mode

[0042] The present invention will be further described in detail below in conjunction with the embodiments with reference to the accompanying drawings.

[0043] As Figures 1 to 6 shown, it is a preferred embodiment of the present invention. The immunomagnetic bead detection system based on a microfluidic chip in this embodiment is mainly used for the detection of the antigen to be detected. When this detection is used for on-site detection, the system must shorten the detection time and for the detection of some trace substances, it requires a lower detection limit. The main part affecting the detection time is the coupling time of the magnetic beads and the reactants. At the same time, on-site detection also requires that the system has a strong self-cleaning ability so that the results of the previous experiment do not affect the results of the next experiment. Therefore, it will be beneficial to provide an immunomagnetic bead detection system and detection method based on a microfluidic chip with high coupling efficiency and easy cleaning. The following will elaborate on the detailed description of this immunomagnetic bead detection system and its usage method:

[0044] Embodiment 1

[0045] Please refer to Figures 1 to 5 , this immunomagnetic bead detection system based on a microfluidic chip includes a support 10, a microfluidic chip 1 disposed on the support 10. Inside the microfluidic chip 1, there is a reaction chamber 11. The reaction chamber 11 contains a plurality of magnetic beads for the conjugation reaction with labeled antibodies and proteins to be detected. The reaction chamber 11 has a reaction chamber inlet 111 and a reaction chamber outlet 112 for liquid circulation; inside the support 10, there is a magnetic field device 2 for controlling the magnetic field strength inside the reaction chamber 11. The magnetic field device 2 is located below the microfluidic chip 1 and acts on the reaction chamber 11. The magnetic field device 2 includes a magnet 21 for generating a magnetic field inside the reaction chamber. The magnet 21 is a cylinder magnetized along the diameter direction. The central axis of the magnet 21 is perpendicular to the bottom surface of the reaction chamber 11. The diameter of the magnet 21 is greater than the width of the reaction chamber 11 and the diameter of the magnet 21 is less than the length of the reaction chamber 11. The diameter of the magnet 21 is greater than or equal to twice the width of the reaction chamber 11. The magnetic field strength range on the surface of the magnet 21 is 20 - 150 mT. The magnetic field generated by the magnet 21 can drive the magnetic beads to gather on the flow path formed from the reaction chamber inlet 111 to the reaction chamber outlet 112 in the reaction chamber. Among them, the width of the reaction chamber 11 is 4 mm and the length is 14.75 mm; the magnet 21 is a permanent magnet.

[0046] In this embodiment, the magnetic field device 2 further includes a driving member 22 that can drive the magnet 21 to approach or move away from the reaction chamber 11. In the state where the driving member 22 drives the magnet 21 to be in close contact with the bottom of the reaction chamber 11, the number of magnetic beads adsorbed and gathered on the flow path formed from the reaction chamber inlet 111 to the reaction chamber outlet 112 in the reaction chamber 11 is greater than the number of magnetic beads gathered on the two sides perpendicular to the flow path. The movement of the driving member 22 approaching or moving away from the reaction chamber 11 controls the magnitude of the magnetic field strength inside the reaction chamber 22. The characteristics of the magnet 21 itself and its relative position with the reaction chamber 11 determine that when the magnet 21 is in close contact with the bottom of the reaction chamber 11, the number of magnetic beads gathered on both sides of the reaction chamber 11 is less than the number of magnetic beads gathered near the reaction chamber inlet 111 and the reaction chamber outlet 112. For the influence of the distance between the magnet 21 and the bottom of the reaction chamber 11 on the magnetic force of the magnetic beads, please refer to Figure 10 , from Figure 10It can be obtained that when the magnet 21 is in contact with the bottom of the reaction chamber 11, the magnetic adsorption settlement distribution of the magnetic beads in the reaction chamber 11 is mainly in a "semicircular" distribution near the edge of the magnet 21 in the reaction chamber 11, and the distribution range is relatively wide, which is beneficial to the uniform distribution of the magnetic beads at the bottom of the reaction chamber 11; as the distance between the magnet 21 and the bottom of the reaction chamber 11 gradually increases from 0 cm to 0.4 cm, 0.8 cm, and 1.2 cm, the magnetic field intensity in the reaction chamber 11 gradually weakens; the range parameter of the magnetic field intensity in the reaction chamber 11 makes the magnetic beads on both sides of the reaction chamber 11 not accumulate significantly. The selection of the magnet 21 and the synergistic effect of the limited range of the magnetic field intensity make it difficult for the magnetic beads to remain on both sides of the reaction chamber when the user cleans the reaction chamber through the washing device, improving the cleaning efficiency of the user during use.

[0047] In this embodiment, the diameter of the magnet 21 is twice the width of the reaction chamber 11. As Figure 9 shown in the top view of the simulated magnetic field distribution in the reaction chamber 11 when the distance between the magnet 21 and the bottom surface of the reaction chamber 11 changes, the relationship between the diameter of the magnet 21 and the size of the reaction chamber 11 makes the magnetic induction line density on both sides of the reaction chamber 11 significantly smaller than that near the inlet 111 and the outlet 112 of the reaction chamber; therefore, when the magnet 21 in this size relationship is in contact with the bottom of the reaction chamber 11, the magnetic field intensity in the reaction chamber 11 is evenly distributed in the width direction, and the uniform distribution of the magnetic beads in the central area between the side walls of the reaction chamber 11 reduces the number of magnetic beads stacked together. When remixed, the magnetic beads are relatively easy to disperse and the coupling efficiency with the reactants is improved, enhancing the coupling efficiency of the user during use, that is, the magnetic beads are easy to remix after magnetic adsorption.

[0048] In this embodiment, the magnetic field intensity range on the surface of the magnet 21 is 100 - 130 mT. Under this magnetic field intensity range, the magnetic beads can be firmly magnetically adsorbed at the bottom of the reaction chamber and are not washed out of the reaction chamber by the washing liquid and flowing air, increasing the number of magnetic beads during detection and reducing the risk of false negatives in detection. To improve the accuracy of using the above-mentioned immunomagnetic bead detection system. In this embodiment, the thickness: diameter of the magnet 21 is 1:8, where the thickness of the magnet 21 is 1 mm and the diameter is 8 mm.

[0049] The working process and principle of this microfluidic chip-based immunomagnetic bead detection system are as follows: Control the driving member 22 to move the magnet 21 away from the bottom surface of the reaction chamber 11. Then, add magnetic beads, primary antibody solution, protein to be detected, and secondary antibody solution into the reaction chamber 11 in sequence from the inlet 12. Turn on the mixing device 4 to accelerate the coupling reaction. After the reaction is completed, turn off the mixing device 4. Control the driving member 22 to make the magnet 21 fit the bottom surface of the reaction chamber 11. The magnetic beads in the reaction chamber 11 are adsorbed to the bottom of the reaction chamber 11. Turn on the washing device 2 to pump in the washing liquid and drain the waste liquid. Add the chromogenic solution and termination solution into the reaction chamber 11 in sequence from the inlet 12. Control the driving member 22 to move the magnet 21 away from the bottom surface of the reaction chamber 11. Turn on the mixing device 4 to accelerate the chromogenic and termination reactions to obtain the solution to be detected. After the reaction is completed, turn off the mixing device 4. Turn on the washing device 3 to pump in air. The solution to be detected enters the detection chamber 13 from the reaction chamber 11 for absorbance detection. After the detection is completed, turn on the washing device 3 to pump in the washing liquid to clean the reaction chamber 11 and the detection chamber 13. Then, turn on the washing device 3 to pump in air to drain the washing liquid in the reaction chamber 11 and the detection chamber 13, and the experiment ends.

[0050] Example 2 Microfluidic Immunomagnetic Bead Detection Method

[0051] Please refer to Figure 6 and Figure 8 , the detection method of the microfluidic chip-based immunomagnetic bead detection system in Example 1 of this application includes the following steps:

[0052] S1. Microfluidic detection platform construction: The microfluidic chip 1 is also provided with an inlet 12 communicating with the reaction chamber inlet 111, a detection chamber 13 communicating downstream of the reaction chamber 11, a mixing channel 14 communicating downstream of the detection chamber 13, and an outlet 15 communicating downstream of the mixing channel 14. Control the driving member 22 to adjust the magnetic field device 2 to the initial state where the magnet 21 is farthest from the reaction chamber 11. Above the microfluidic chip 1, there is also a detection device for performing chromaticity analysis on the liquid in the detection chamber 13. The inlet 12 is connected to a washing device 3 for pumping washing liquid and air into the reaction chamber 11. Above the microfluidic chip 1, there is a mixing device 4, and the mixing device 4 acts on the reaction chamber 11 to accelerate the coupling reaction. The detection device includes an LED light source located above the detection chamber 13, a photoelectric sensor located below the detection chamber 13 for receiving the LED light source, and a control board connected to the photoelectric sensor for receiving sensor data. A display screen for displaying the absorbance value is provided on this control board. The magnetic adsorption state is as Figure 6 shown, and the magnetic induction line distribution in the reaction chamber 11 in the magnetic adsorption state is as Figure 7 shown, and the initial state is as Figure 8 shown;

[0053] S2. Preparation of magnetic bead primary antibody probe: Add streptavidin-coated magnetic beads and biotin-labeled antibody into reaction chamber 11 of microfluidic chip 1 from inlet 12 in step S1. After coupling is completed by turning on mixing device 4 to vibrate and mix evenly; adjust driving member 22 to move magnet 21 from the initial state in step S1 to the magnetic adsorption state where it fits against the bottom of reaction chamber 11 to complete magnetic adsorption separation; turn on washing device 3 to empty, wash, and then empty reaction chamber 11 to obtain the primary antibody probe;

[0054] S3. Preparation of immunomagnetic beads with double antibody sandwich structure: Add the protein solution to be detected and enzyme-labeled antibody into reaction chamber 11 in sequence; adjust driving member 22 to restore magnetic field device 2 from the magnetic adsorption state in step S2 to the initial state, turn on mixing device 4 to vibrate and mix evenly, adjust driving member 22 to make magnetic field device 2 in the magnetic adsorption state. After the magnetic beads are completely separated, wash and empty to obtain immunomagnetic beads with double antibody sandwich structure;

[0055] S4. Color development and termination: Add color developing solution into reaction chamber 11, adjust magnetic field device 2 from the magnetic adsorption state in step S3 to the initial state, turn on mixing device 4 to vibrate and mix evenly; adjust magnetic field device 2 to the magnetic adsorption state, add termination solution and let it stand for 1 - 3 min. The solution changes color to obtain the reaction solution to be detected;

[0056] S5. Absorbance detection: Transfer the reaction solution to be detected in step 4 from reaction chamber 11 to detection chamber 13, turn on detection device to perform absorbance detection, and record experimental data;

[0057] S6. Chip cleaning: Adjust magnet 21 to the initial state, wash and empty.

[0058] In this embodiment, mixing device 4 includes vibration head 41 and vibration motor 42 that is controlledly connected to vibration head 41. The parameters set for vibration motor 42 in the operations of vibrating and mixing evenly in steps S2, S3, and S4 are: voltage is greater than or equal to 3.7 V, current is 0.1 A - 0.23 A, vibration time is 1 - 3 min, and vibration frequency is 20000 - 35000 times / min. Thus, sufficient mixing of magnetic beads and reactants is achieved in a short time, further improving the coupling efficiency, reducing the detection limit of the sample to be detected, and shortening the coupling time between reactants and magnetic beads in the reaction chamber.

[0059] In this embodiment, the washing device 3 includes a washing pump 31 for pumping washing liquid into the reaction chamber 11 and an air pump 32 for pumping air into the reaction chamber 11. In steps S2, S3, and S6, the operation of washing is to turn on the washing pump 31 to pump in the washing liquid. The pumping flow rate of the washing pump 31 is set to 300 μl / min, and the pumping time is set to 30 s. The operation of emptying is to turn on the air pump 32 to pump in air. The pumping flow rate of the air pump 32 is set to 300 μl / min, and the pumping time is set to 30 - 60 s, so as to wash the magnetic beads in the center of the reaction chamber clean with the washing liquid. Among them, the washing pump 31 is an injection pump.

[0060] Example 3 Magnet Selection and Magnetic Attraction Effect Verification Experiment

[0061] 1. Experimental materials: Select magnets 21 of a series of sizes;

[0062] 2. Experimental device: Use the microfluidic detection platform built in step S1 of Example 2 for the experiment;

[0063] 3. Experimental operations:

[0064] 1) Magnetic attraction: Control the driving member 22 to move the magnet 21 away from the bottom surface of the reaction chamber 11. Replace the magnet 21 in Example 1 with the magnets 21 of a series of sizes in this embodiment. Add magnetic beads into the reaction chamber 11 through the inlet 12. Turn on the mixing device 4 to mix the magnetic beads evenly in the reaction chamber 11. Record the aggregated state of the evenly mixed magnetic beads. Control the driving member 22 to make the magnet 21 fit the bottom surface of the reaction chamber 11. Observe the aggregation of the magnetic beads in the reaction chamber 11, judge whether they aggregate at the two side edges of the reaction chamber 11 and record;

[0065] 2) Washing: Control the driving member 22 to make the magnet 21 fit the bottom surface of the reaction chamber 11. Turn on the washing device 3 to pump in the washing liquid. The pumping flow rate is set to 300 μl / min, and the pumping time is set to 30 s. Flush once. Observe the aggregation of the magnetic beads in the reaction chamber 11, and compare whether the density has changed significantly compared with the aggregation of the magnetic beads before washing (that is, whether there is loss after washing) and record;

[0066] 3) Mixing: Control the driving member 22 to move the magnet 21 away from the bottom surface of the reaction chamber 11. Turn on the mixing device 4 to work for 30 s. Observe the aggregation of the magnetic beads in the reaction chamber 11, and compare with the aggregated state of the evenly mixed magnetic beads in step 1) to judge whether the magnetic beads are easy to remix and record.

[0067] 4. Experimental results and discussion

[0068] Table 1 Comparison of Magnet 21 Selection and Magnetic Bead Aggregation

[0069]

[0070] (Note: where φ represents the diameter of the magnet 21, and d represents the thickness of the magnet 21; when the dimensions are expressed in terms of φ and d, the magnet 21 is in the shape of a cylinder; the dimensions of the magnet 21 numbered 1, "14.5×4.75×1.75", represent a rectangular magnet with a length of 14.5, a width of 4.75, and a thickness of 1.75)

[0071] It can be obtained from Table 1 that when the magnet 21 is a cylinder with a diameter of 8 mm and a thickness of 1 mm, after the magnetic field is turned on, the magnetic beads are easily adsorbed evenly on the bottom of the reaction chamber 11, and basically no magnetic beads are lost in the reaction chamber 11 after one washing, so that the magnetic beads are retained as much as possible, reducing the detection limit; when the magnetic field is turned off and the mixing device 4 is turned on, the magnetic beads are easily mixed with the liquid again in the reaction chamber 11, accelerating the coupling reaction speed with the secondary antibody solution during the re-mixing, and improving the experimental efficiency.

[0072] Example 4 Experiment on Detection of Interleukin-6 Concentration

[0073] Example 4 applies the microfluidic immuno-magnetic bead detection method in Example 2 to the detection of interleukin-6 concentration. The detection steps of interleukin-6 concentration and the corresponding results are as follows:

[0074] Step 1: Preparation of Immuno-Magnetic Beads (IMBs) in the Microfluidic Chip 1

[0075] ① Add 2.5 μl of streptavidin-coated magnetic beads (SA-MBs) with a concentration of 10 mg / mL and 100 μl of biotin (Biotin)-labeled interleukin-6 (IL-6) monoclonal antibody with a concentration of 10 μg / mL into the reaction chamber 11 of the microfluidic chip 1 through the inlet 12; then turn on the vibration motor 42 and vibrate and mix for 3 min under the drive of a 3.7 v voltage;

[0076] ② After the vibration ends, the magnet 21 is raised to a position in contact with the bottom of the reaction chamber 11. After magnetic adsorption for 10 s, turn on the air pump 32 to empty the Biotin-IL6 recognition antibody in the reaction chamber 11 within 30 s;

[0077] ③ Turn on the washing pump 31 containing TBST washing solution with a concentration factor of 1.5, set the flow rate to 300 μl / min, and the running time to 30 s. At this time, the washing solution just completely covers the magnetic beads in the reaction chamber 11, thereby removing the Biotin-labeled IL6 monoclonal antibody that has not bound to the SA-MBs. Turn on the air pump 32 to work for 30 - 60 s to empty the washing solution in the reaction chamber 11, and the preparation of immuno-magnetic beads (IMBs) is completed.

[0078] Step 2: Preparation of the Double-Antibody Sandwich Structure on the Immuno-Magnetic Beads (IMBs)

[0079] ① Add 25 μl of IL-6 protein solution with a concentration of 100 ng / mL and 25 μl of IL-6 monoclonal antibody labeled with horseradish peroxidase (HRP) with a concentration of 0.125 μg / mL to the reaction chamber 11 in sequence; then move the magnet 21 down to a position about 10 mm away from the bottom of the reaction chamber 11. At this time, the magnet 21 has no magnetic adsorption on the IMBs in the reaction chamber 11; then turn on the vibration motor 42 and vibrate and mix for 3 min under the drive of a 3.7 V voltage.

[0080] ② After the vibration ends, the magnet 21 is raised to a position in contact with the bottom of the reaction chamber 11. After magnetic adsorption for 10 s, turn on the air pump 32 and empty the HRP-IL6 detection antibody in the reaction chamber 11 within 30 s.

[0081] ③ Turn on the washing pump 31 containing TBST washing solution with a concentration factor of 1.5, set the flow rate to 300 μl / min, and the running time to 30 s. At this time, the washing solution just completely covers the magnetic beads in the reaction chamber 11, thereby removing the HRP-IL6 detection antibody that has not bound to the IMMBs. Then turn on the air pump 32 to work for 30 - 60 s to empty the washing solution in the reaction chamber 11. Thus, the double antibody sandwich structure based on the magnetic beads is formed.

[0082] Step 3: Color reaction on the immunomagnetic beads (IMBs): Add 50 μl of the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) to the reaction chamber 11 of the chip, and then move the magnet 21 down to a position about 10 mm away from the bottom of the reaction chamber 11. At this time, the magnet 21 has no magnetic adsorption on the IMBs in the reaction chamber 11; then turn on the vibration motor 42 and vibrate and mix for 5 min under the drive of a 3.7 V voltage. After the vibration ends, the solution in the reaction chamber 11 of the chip turns blue, and a blue solution is obtained.

[0083] Step 4: Terminate the reaction: Raise the magnet 21 to a position in contact with the bottom of the reaction chamber 11 for magnetic adsorption, and then add 25 μl of the termination solution and let it stand for a period of time until the blue solution is mixed with the colorless termination solution to form a yellow solution.

[0084] Step 5: Absorbance detection: Use the air pump 32 to push the yellow solution to completely fill the detection chamber 13 of the chip, and turn on the detection device. Among them, the LED light source at 450 nm passes through the yellow solution in the detection chamber 13. Part of the light source is absorbed by the yellow solution, and the other part passes through the yellow solution in the detection chamber 13 of the chip and is transmitted to the photoelectric sensor. Finally, read the absorbance on the display screen of the control board of the detection device. After parallel determination three times, the average value of the absorbance of the IL-6 protein solution with a concentration of 100 ng / mL is 1.9046, the standard deviation is 0.0731, and the coefficient of variation is 3.83%; for 8 concentrations, after each is tested 3 times, please refer to Figure 11, the concentration and absorbance value are linearly related.

[0085] Step 6: Calculation of detection limit: The detection limit was calculated using the formula LOD = 3*δ / s, where δ is the standard deviation of the 11 blank absorbance values and s is the slope of the fitted linear equation. The calculated minimum detection limit of IL-6 was 0.43 ng / mL.

[0086] Example 5 Procalcitonin concentration detection experiment

[0087] Example 5 applies the microfluidic immunomagnetic bead detection method in Example 2 to the detection of procalcitonin (PCT) concentration. The detection steps of PCT concentration and the corresponding results are as follows:

[0088] Step 1: Preparation of immunomagnetic beads (IMBs) in microfluidic chip 1;

[0089] ① 2.5 μl of 10 mg / mL streptavidin-coated magnetic beads (SA-MBs) and 100 μl of 6.66 μg / mL biotin-labeled PCT monoclonal antibody were also added to the reaction chamber 11 of the microfluidic chip 1 through the inlet 12; then, the vibration motor 42 was turned on and driven by a voltage of 3.7 V to vibrate and mix for 3 minutes;

[0090] ② After the vibration is completed, the magnet 21 is raised to a position where it is in contact with the bottom of the reaction chamber 11. After 10 seconds of magnetic attraction, the air pump 32 is turned on to empty the Biotin-PCT recognition antibody in the reaction chamber within 30 seconds;

[0091] ③ Turn on the washing pump 31 filled with TBST washing solution with a concentration factor of 1.5, set the flow rate to 300 μl / min, and the running time to 30 seconds. At this time, the washing solution just completely covers the magnetic beads in the chip reaction chamber 11, thereby removing the biotin-labeled PCT monoclonal antibodies that are not bound to the SA-MBs. Turn on the air pump 32 and work for 30-60 seconds to empty the washing solution in the reaction chamber 11. The preparation of immunomagnetic beads (IMBs) is completed.

[0092] Step 2: Preparation of double antibody sandwich structure on immunomagnetic beads (IMBs)

[0093] ① 25 μl of 100 ng / mL PCT protein solution and 25 μl of 0.25 μg / mL horseradish peroxidase (HRP)-labeled PCT monoclonal antibody were added to the reaction chamber 11 in sequence; the magnet 21 was then lowered to a distance of approximately 10 mm from the bottom of the reaction chamber 11, at which point the magnet 21 had no magnetic attraction to the IMBs in the reaction chamber 11; the vibration motor 42 was then turned on and vibrated at 3.7 V for 3 minutes to mix the mixture;

[0094] ② After the vibration is completed, the magnet 21 is raised to a position flush with the bottom of the reaction chamber 21. After magnetic attraction for 10 seconds, the air pump 32 is turned on to empty the HRP-PCT detection antibody in the reaction chamber 11 within 30 seconds;

[0095] ③ Turn on the washing pump 31 filled with TBST washing solution with a concentration factor of 1.5, set the flow rate to 300 μl / min, and the running time to 30 s. At this time, the washing solution just completely covers the magnetic beads in the reaction chamber 11, thereby removing the HRP-PCT detection antibodies that are not bound to the IMMBs. Then turn on the air pump 32 and work for 30-60 s to empty the washing solution in the reaction chamber 11. At this point, the double antibody sandwich structure based on the magnetic beads is formed.

[0096] Step 3: Color reaction on immunomagnetic beads (IMBs): Add 50 μl of the color development substrate 3,3',5,5'-tetramethylbenzidine (TMB) to the reaction chamber 11 of the chip, and then move the magnet 21 down to a distance of about 10 mm from the bottom of the chip. At this time, the magnet 21 has no magnetic adsorption on the IMBs in the chip reaction chamber 11; then turn on the vibration motor 42, and vibrate and mix for 5 minutes under the drive of 3.7V voltage. After the vibration ends, the solution in the chip reaction chamber 11 turns blue, and a blue solution is obtained.

[0097] Step 4: Stop the reaction: Raise the magnet 21 to the same level as the bottom of the reaction chamber 11 to perform the magnetic attraction process, then add 25 μl of stop solution and let it stand for a while until the blue solution and the colorless stop solution are mixed into a yellow solution.

[0098] Step 5: Absorbance detection: Use an air pump to push the yellow solution to completely fill the detection chamber 13 of the chip, and turn on the detection device. The 450nm LED light source passes through the yellow solution in the detection chamber 13. Part of the light source is absorbed by the yellow solution, and the other part passes through the yellow solution in the detection chamber 13 and is transmitted to the photoelectric sensor. Finally, the absorbance is read on the display screen on the control panel of the detection device. After three parallel measurements, the average absorbance of the PCT protein solution with a concentration of 100ng / mL is 2.2997, the standard deviation is 0.082, and the coefficient of variation is 3.58%. Among them, 8 concentrations were tested 3 times respectively. Please refer to Figure 12 , the concentration and absorbance value are linearly related.

[0099] Step 6: Calculation of detection limit: Same as step 6 in Example 4, the minimum detection limit of PCT is 0.93 ng / mL.

[0100] Example 6 C-reactive protein concentration detection experiment

[0101] Example 6 applies the microfluidic immunomagnetic bead detection method in the examples to the detection of C-reactive protein concentration. The detection steps of C-reactive protein concentration and the corresponding results are as follows:

[0102] Step 1: Preparation of immunomagnetic beads (IMBs) in the microfluidic chip 1;

[0103] ① Add 2.5 μl of streptavidin-coated magnetic beads (SA-MBs) with a concentration of 10 mg / mL and 100 μl of biotin (Biotin)-labeled C-reactive protein (CRP) monoclonal antibody with a concentration of 6.66 μg / mL into the reaction chamber 11 of the microfluidic chip 1 through the inlet 12; then turn on the vibration motor 42 and vibrate and mix for 3 min under the drive of a 3.7 v voltage;

[0104] ② After the vibration ends, the magnet 21 is raised to a position where it fits the bottom of the reaction chamber 11. After magnetic adsorption for 10 s, turn on the air pump 32 and empty the Biotin-CRP recognition antibody in the reaction chamber within 30 s;

[0105] ③ Turn on the washing pump 31 containing TBST washing solution with a concentration factor of 1.5, set the flow rate to 300 μl / min, and the running time to 30 s. At this time, the washing solution just completely covers the magnetic beads in the reaction chamber 11, thereby removing the Biotin-labeled CRP monoclonal antibody that has not bound to the SA-MBs. Turn on the air pump 32 to work for 30 - 60 s to empty the washing solution in the reaction chamber 11. Thus, the immunomagnetic beads (IMBs) are prepared.

[0106] Step 2: Preparation of the double-antibody sandwich structure on the immunomagnetic beads (IMBs)

[0107] ① Add 25 μl of CRP protein solution with a concentration of 100 ng / mL and 25 μl of horseradish peroxidase (HRP)-labeled CRP monoclonal antibody with a concentration of 0.25 μg / mL into the reaction chamber 11 in sequence; then move the magnet 21 down to a position about 10 mm away from the bottom of the reaction chamber 11. At this time, the magnet 21 has no magnetic adsorption on the IMBs in the chip reaction chamber 11; then turn on the vibration motor 42 and vibrate and mix for 3 min under the drive of a 3.7 v voltage;

[0108] ② After the vibration ends, the permanent magnet 21 is raised to a position flush with the bottom of the reaction chamber 21. After magnetic adsorption for 10 s, turn on the air pump 32 and empty the HRP-CRP detection antibody in the reaction chamber within 30 s;

[0109] ③ Turn on the washing pump 31 filled with TBST washing solution with a concentration factor of 1.5, set the flow rate to 300 μl / min, and the running time to 30 s. At this time, the washing solution just completely covers the magnetic beads in the reaction chamber 11, thereby removing the HRP-CRP detection antibody that is not bound to the IMMBS. Then turn on the air pump 32 and work for 30-60 s to empty the washing solution in the reaction chamber 11. At this point, the double antibody sandwich structure based on the magnetic beads is formed.

[0110] Step 3: Color development reaction on immunomagnetic beads (IMBs): Add 50 μl of the color development substrate 3,3',5,5'-tetramethylbenzidine (TMB) to the reaction chamber 11, and then move the magnet 21 down to a distance of about 10 mm from the bottom of the reaction chamber 11. At this time, the magnet 21 has no magnetic attraction to the IMBs in the reaction chamber 11; then turn on the vibration motor 42 and vibrate and mix for 5 minutes under the drive of 3.7V voltage.

[0111] Step 4: Termination of the reaction: After the vibration is completed, the solution in the chip reaction chamber 11 turns blue. Then, the magnet 21 is raised to be flush with the bottom of the chip reaction chamber 11 for magnetic attraction. Then, 25 μl of stop solution is added and the mixture is allowed to stand for a period of time until the blue solution and the colorless stop solution are mixed into a yellow solution.

[0112] Step 5: Absorbance detection: Use the air pump 32 to push the yellow solution to completely fill the detection chamber 13 of the chip, and turn on the detection device. The 450nm LED light source passes through the yellow solution in the detection chamber 13. Part of the light source is absorbed by the yellow solution, and the other part passes through the yellow solution in the detection chamber 13 and is transmitted to the photoelectric sensor. Finally, the absorbance is read on the display screen on the control panel of the detection device. After three parallel measurements, the average absorbance of the CRP protein solution with a concentration of 28ng / mL was 2.055, the standard deviation was 0.007, and the coefficient of variation was 0.34%. Among them, 8 concentrations were tested 3 times respectively. Please refer to Figure 13 , the concentration and absorbance value are linearly related.

[0113] Step 6: Calculation of detection limit: Same as step 6 in Example 3, the minimum detection limit of CRP is 0.36 ng / mL.

Claims

1. An immunomagnetic bead detection system based on a microfluidic chip, comprising a support (10), a microfluidic chip (1) disposed on the support (10). The microfluidic chip (1) is internally provided with a reaction chamber (11), and a plurality of magnetic beads for the coupling reaction with labeled antibodies and proteins to be detected are provided in the reaction chamber (11). The reaction chamber (11) has a reaction chamber inlet (111) and a reaction chamber outlet (112) for liquid circulation; an inlet (12) communicating with the reaction chamber inlet (111) and a detection chamber (13) communicating and located downstream of the reaction chamber (11) are further provided on the microfluidic chip (1). A magnetic field device (2) is disposed in the support (10) and below the microfluidic chip (1). The magnetic field device (2) acts on the reaction chamber (11) and includes a magnet (21) for generating a magnetic field in the reaction chamber (11). It is characterized in that: The magnet (21) is a cylinder magnetized along the diameter direction. The central axis of the magnet (21) is perpendicular to the bottom surface of the reaction chamber (11). The diameter of the magnet (21) is larger than the width of the reaction chamber (11) and the diameter of the magnet (21) is smaller than the length of the reaction chamber (11). The diameter of the magnet (21) is greater than or equal to twice the width of the reaction chamber (11). The magnetic field intensity range on the surface of the magnet (21) is 20 - 150 mT.

2. The immunomagnetic bead detection system based on a microfluidic chip according to claim 1, characterized in that: The magnetic field device (2) further includes a driving member (22) capable of driving the magnet (21) to approach or move away from the reaction chamber (11). In a state where the driving member (22) drives the magnet (21) to be in close contact with the bottom of the reaction chamber (11), the number of magnetic beads adsorbed and aggregated on the flow path formed by the reaction chamber inlet (111) to the reaction chamber outlet (112) in the reaction chamber (11) is greater than the number of those aggregated on both sides perpendicular to the flow path.

3. The immunomagnetic bead detection system based on a microfluidic chip according to claim 1 or 2, characterized in that: The diameter of the magnet (21) is twice the width of the reaction chamber (11).

4. The immunomagnetic bead detection system based on a microfluidic chip according to claim 3, wherein: The magnetic field intensity range on the surface of the magnet (21) is 100 - 130 mT.

5. The immunomagnetic bead detection system based on a microfluidic chip according to claim 4, wherein: The thickness of the magnet (21): the diameter of the magnet (21) is 1:

8.

6. A detection method, characterized in that Applying the immunomagnetic bead detection system based on a microfluidic chip according to any one of claims 1 - 5, includes the following steps: S1. Construction of the microfluidic detection platform: An inlet (12) communicating with the inlet (111) of the reaction chamber is further provided on the microfluidic chip (1), a detection chamber (13) located downstream of the reaction chamber (11) and in communication therewith, a mixing channel (14) located downstream of the detection chamber (13) and in communication therewith, and an outlet (15) located downstream of the mixing channel (14) and in communication therewith; the driving member (22) is controlled to adjust the magnetic field device (2) to the initial state where the magnet (21) is at the farthest distance from the reaction chamber (11), and a detection device for performing chromaticity analysis on the liquid in the detection chamber (13) is further provided above the microfluidic chip (1); the inlet (12) is communicated with a washing device (3) for pumping washing liquid and air into the reaction chamber (11); a mixing device (4) is provided above the microfluidic chip (1), and the mixing device (4) acts on the reaction chamber (11) to accelerate the coupling reaction; S2. Preparation of magnetic bead primary antibody probe: Add avidin-coated magnetic beads and biotin-labeled antibody from the inlet (12) in step S1 into the reaction chamber (11) of the microfluidic chip (1), turn on the mixing device (4) to vibrate and mix, and the coupling is completed after mixing; adjust the driving member (22) so that the magnet (21) moves from the initial state in step S1 to the magnetic adsorption state where it fits against the bottom of the reaction chamber (11) to complete magnetic adsorption separation; turn on the washing device (3) to empty, wash, and empty the reaction chamber (11) to obtain the primary antibody probe; S3. Preparation of immunomagnetic beads with a double-antibody sandwich structure: Add the protein solution to be detected and the enzyme-labeled antibody into the reaction chamber (11) in sequence; adjust the driving member (22), the magnetic field device (2) is restored from the magnetic adsorption state in step S2 to the initial state, turn on the mixing device (4) to vibrate and mix, adjust the driving member (22) so that the magnetic field device (2) is in the magnetic adsorption state, and after the magnetic beads are completely separated, wash and empty to obtain immunomagnetic beads with a double-antibody sandwich structure; S4. Color development and termination: Add the color-developing solution into the reaction chamber (11), adjust the magnetic field device (2) from the magnetic adsorption state in step S3 to the initial state, turn on the mixing device (4) to vibrate and mix; adjust the magnetic field device (2) to the magnetic adsorption state, add the termination solution and let it stand for 1 - 3 min, and the solution changes color to obtain the reaction solution to be detected; S5. Absorbance detection: Transfer the reaction solution to be detected in step (4) from the reaction chamber (11) to the detection chamber (13), perform absorbance detection, and record the experimental data; S6. Clean the chip: Adjust the magnet (21) to the initial state, wash and empty.

7. The immunomagnetic bead detection system based on a microfluidic chip according to claim 6, wherein: The mixing device (4) includes a vibration head (41) and a vibration motor (42) that is controllably connected to the vibration head (41). The parameters set for the vibration motor (42) in the vibration mixing operations in steps S2, S3, and S4 are: voltage greater than or equal to 3.7 V, current 0.1 A - 0.23 A, vibration time 1 - 3 min, and vibration frequency 20000 - 35000 times / min.

8. The immunomagnetic bead detection system based on a microfluidic chip according to claim 7, wherein: The washing device (3) includes a washing pump (31) for pumping washing liquid into the reaction chamber (11) and an air pump (32) for pumping air into the reaction chamber (11). In steps S2, S3, and S6, the washing operation is to turn on the washing pump (31) to pump in the washing liquid. The pumping flow rate of the washing pump (31) is set to 300 μl / min, and the pumping time is set to 30 s. The emptying operation is to turn on the air pump (32) to pump in air. The pumping flow rate of the air pump (32) is set to 300 μl / min, and the pumping time is set to 30 - 60 s.

9. The immunomagnetic bead detection system based on a microfluidic chip according to claim 8, wherein: The washing operations in steps S2, S3, and S6 are carried out 1 to 2 times.

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