Magnetically Controlled Droplet Microfluidic Chip, Device and Method for Chemiluminescence Immunoassay

Abstract

The present invention discloses a magnetically controlled droplet microfluidic chip, device and method for chemiluminescence immunoassay, belonging to the technical field of chemiluminescence immunoassay. The microfluidic chip includes: a chip body, which includes a plurality of partition chambers that penetrate up and down, and is used for loading reagents and serving as a reaction container. There are gaps at the bottom of the adjacent partitions between the partition chambers. If the chip has the function of separating plasma from whole blood samples, the first chamber adopts a bottom-closed structure and there is no gap at the partition between the first and second chambers; a top film and a bottom film, which are used to seal the partition chambers of the chip body; a top cover and a bottom cover, which are adapted to the structure and shape of the chip body and are used to firmly attach the two films to the chip body. The present invention can realize the full-process integration of sample pretreatment, reagent mixing, reaction incubation and chemiluminescence detection, and provides an efficient, low-cost and easy-to-operate chemiluminescence immunoassay solution through chip design, device integration and method optimization.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemiluminescence immunoassay, and particularly relates to a magnetically controlled droplet microfluidic chip, device and method for chemiluminescence immunoassay. Background Art

[0002] Chemiluminescence Immunoassay (CLIA) is a widely used immunoassay technology in the field of in vitro diagnosis. It has extremely high sensitivity and specificity, and can accurately detect human biomarkers quantitatively. It has played an indispensable core role in many medical scenarios such as health check-ups, early disease screening, disease course monitoring and prognosis evaluation, providing solid and reliable data support for clinical decision-making. Microfluidic technology realizes precise control and rapid response to a small amount of fluid, integrates various operations of chemiluminescence immunoassay onto a single microchip, and can automatically complete the entire analysis process. The microfluidic chip has the characteristics of portability, low analysis cost, short reaction time, large-scale parallel processing and automated operation, making it have great prospects in the integration and miniaturization of CLIA systems.

[0003] Microfluidic chips can be divided into continuous-flow microfluidics based on microchannels and digital microfluidics based on discrete droplets. A relatively mature technology in continuous-flow microfluidics is centrifugal disk microfluidics. For example, the Chinese patent application with the publication number CN105842468A provides a microfluidic chemiluminescence immunoassay device and its usage method. A microfluidic detection unit is installed on the microfluidic disk, including various storage tanks, injection tanks, mixing tanks, waste liquid tanks, etc., as well as microfluidic pipeline channels connecting these tanks. By applying different angular velocities and angular accelerations to the microfluidic disk, centrifugal force is generated to control the transfer of samples and reagents within the microfluidic detection unit, thereby realizing chemiluminescence immunoassay. A relatively mature technology in digital microfluidics is to use the electrowetting-on-dielectric (EWOD) technology to achieve discrete droplet manipulation. For example, the Chinese patent application with the publication number CN114002447A provides a fully automatic digital microfluidic analysis platform, including an installation base, a magnetic separation control module, a chip carrier plate, a chip temperature control module, a chip driving electro-pin module, and a multi-channel fluorescence detection system. Among them, the chip driving electro-pin module provides voltage for the chip to cause the dielectric wetting effect of the droplets, thus realizing the driving of the droplets. A relatively new technology in digital microfluidics is to use magnetic excitation technology to achieve discrete droplet manipulation. For example, the Chinese patent application with the publication number CN108479875A provides a digital microfluidic manipulation platform. The chip includes an upper chip plate and a lower chip plate, and a microchannel is formed between the two plates. By setting a magnetic control module, the movement of the micro-droplets in the microchannel on the chip is controlled, and the magnetic control module is used to replace the microelectrodes in the EWOD-driven chip. However, these technologies still face some challenges in practical applications. Although continuous-flow microfluidic technology performs well in fluid control, its complex microchannel design and manufacturing process increase the production cost and it is difficult to achieve high integration. Although digital microfluidic technology has flexibility in droplet manipulation, its reliance on external driving devices increases the complexity and cost of the system, and at the same time, the precise manipulation and stability of the droplets still need to be further improved.

[0004] In summary, there are technical bottlenecks in aspects such as the manufacturing process, manufacturing cost of microfluidic chips, and the manipulation ability and complexity of microfluidic devices in the prior art, which limit the wide application of microfluidic technology in the field of small-scale POCT (Point-of-Care Testing) detection. Summary of the Invention

[0005] In view of the above, the object of the present invention is to provide a magnetically controlled droplet microfluidic chip, device and method for chemiluminescence immunoassay. By designing a film covering structure based on FEP material and a partition chamber with a micron-level notch at the bottom in the chip, there is no need to process micron-level flow channels, reducing the complexity and manufacturing cost of the chip manufacturing process. Further, the chip is integrated into the device to achieve efficient manipulation and separation of immunomagnetic bead complexes through a magnetic field, improving the detection efficiency and reducing the detection cost. It can realize chemiluminescence immunoassay with the function of separating plasma from whole blood samples according to the flexible design of the chip chamber, achieving the full-process integration of sample pretreatment, reagent mixing, reaction incubation and chemiluminescence detection. The miniaturization and portability of the device make it particularly suitable for quickly completing high-quality biomarker detection in the POCT scenario.

[0006] To achieve the above object of the invention, the technical solutions provided by the present invention are as follows:

[0007] In the first aspect, a magnetically controlled droplet microfluidic chip for chemiluminescence immunoassay provided by an embodiment of the present invention includes: a top cover, a top film, a chip body, a bottom film and a bottom cover that are tightly connected from top to bottom;

[0008] The chip body includes a number of partition chambers that penetrate up and down, which are used to load chemiluminescence immunoassay reagents and serve as chemiluminescence immunoassay reaction containers. There are notches at the bottom of the adjacent partitions between the partition chambers. If the microfluidic chip has the function of separating plasma from whole blood samples, the first chamber adopts a bottom-closed structure and there is no notch at the adjacent partition between the first chamber and the second chamber;

[0009] The top film and the bottom film are used to seal the partition chambers of the chip body;

[0010] The top cover and the bottom cover are adapted to the structure and shape of the chip body, and are used to firmly attach the top film and the bottom film to the top surface and the bottom surface of the chip body respectively.

[0011] Preferably, the chip body is made of FEP material, and the height of the bottom notch at the adjacent partition between the partition chambers is 50 - 100 microns. The partition chambers are made into the same or different sizes according to the needs of chemiluminescence immunoassay.

[0012] Preferably, the top film and the bottom film are made of FEP films with a thickness of 100 - 200 microns, and their sizes completely cover all the partition chambers of the chip body, and there are flanges for the tensioning of the films during the overall assembly of the microfluidic chip.

[0013] Preferably, the top cover and the bottom cover are made of metal or plastic materials, and the chip body, the top film and the bottom film are connected into a whole through interference fit or snap design.

[0014] In a second aspect, an embodiment of the present invention further provides a magnetically controlled droplet microfluidic device for chemiluminescence immunoassay, which is used in conjunction with the above-mentioned magnetically controlled droplet microfluidic chip for chemiluminescence immunoassay to achieve chemiluminescence immunoassay, including: a spliced structure permanent magnet, a magnet driving module, an incubation module, a mixing module, and a PMT photoelectric detection module;

[0015] The incubation module is used to accommodate the microfluidic chip and maintain a constant temperature in the chamber of the microfluidic chip;

[0016] The mixing module is used to oscillate and mix the reagent droplets and the immunomagnetic bead complexes generated by the reaction in the chamber of the microfluidic chip;

[0017] The spliced structure permanent magnet is composed of two rectangular permanent magnets or semi-cylindrical permanent magnets with their N and S poles attracted to each other, and is used to control the aggregation and separation operations of the immunomagnetic bead complexes;

[0018] The magnet driving module is used to achieve the movement control of the spliced structure permanent magnet along the arrangement direction of the chamber of the microfluidic chip and the movement control of the spliced structure permanent magnet approaching or departing from the bottom of the microfluidic chip;

[0019] The PMT photoelectric detection module is used to measure the light signal intensity when the immunomagnetic bead complexes enter the chamber containing the substrate and undergo chemiluminescence reaction to quantitatively analyze the content of the target analyte.

[0020] Preferably, the incubation module includes a microfluidic chip mounting seat, a heating component, a temperature sensor, and a temperature control circuit board; wherein, the microfluidic chip mounting seat is used to accommodate and fix the microfluidic chip; the heating component is arranged outside the microfluidic chip mounting seat to indirectly adjust the temperature of the microfluidic chip, preferably a polyimide heating film or a Peltier element; the temperature sensing element of the temperature sensor is in close contact with the microfluidic chip mounting seat to measure and feedback the actual temperature of the microfluidic chip mounting seat, preferably a PT1000 platinum resistance temperature sensor; the temperature control circuit board is used to collect the electrical signal of the temperature sensor and output a corresponding control signal through a temperature control algorithm to drive the heating component to work to achieve precise control of the temperature of the microfluidic chip.

[0021] Preferably, the PMT photoelectric detection module includes a single photon counter, a detection window adapter, and a shutter; wherein, the single photon counter uses a PMT as the core detection element and is equipped with a corresponding signal processing circuit and counting system; the detection window adapter is used to connect the detection window in the single photon counter and the chamber in the microfluidic chip that accommodates the substrate, and isolates ambient light during the detection process to avoid interference from external light; the shutter is used to control the exposure time of the photosensitive window in the single photon counter, and opens or closes the shutter in coordination with the chemiluminescence reaction process to protect the single photon counter.

[0022] Preferably, a magnetically controlled droplet microfluidic device for chemiluminescence immunoassay with the function of separating plasma from whole blood samples further includes: an annular permanent magnet, which is controlled by a magnet driving module to move vertically upward and close to the bottom of the microfluidic chip with the function of separating plasma from whole blood samples, or a chip driving module is used instead of the magnet driving module, and the microfluidic chip with the function of separating plasma from whole blood samples is controlled by the chip driving module to move and close to the upper part of the annular permanent magnet, and the aggregation and annular arrangement of the immunomagnetic bead complex combined with red blood cells are controlled under the action of the magnetic field to achieve plasma separation at the center of the ring.

[0023] Thirdly, an embodiment of the present invention further provides a method for chemiluminescence immunoassay, which is implemented by using the above-mentioned magnetically controlled droplet microfluidic device for chemiluminescence immunoassay, and includes the following steps:

[0024] (1) Embedding reagents: The chemiluminescence immunoassay reagents are pre-added to the first chamber of the microfluidic chip, the washing buffer is pre-added to multiple intermediate chambers, and the substrate solution is pre-added to the last substrate chamber;

[0025] (2) Adding samples: The sample to be tested is added to the first chamber in which the reagents are embedded;

[0026] (3) Loading the chip: The microfluidic chip is loaded into the microfluidic chip mounting seat of the magnetically controlled droplet microfluidic device for chemiluminescence immunoassay;

[0027] (4) Incubation and binding: The mixing module performs a mixing operation on the microfluidic chip, and the incubation module performs a heat preservation operation on the microfluidic chip. In the first chamber, the antigen to be tested in the sample to be tested specifically binds to the antibody in the chemiluminescence immunoassay reagent to form an immunomagnetic bead complex;

[0028] (5) Magnetic bead separation: The magnet driving module controls the spliced structure permanent magnet to move vertically upward to close to the bottom film at the bottom of the microfluidic chip. Under the action of the magnetic field, the immunomagnetic bead complex quickly aggregates to the bottom of the droplet and arranges in a line along the joint of the spliced structure permanent magnet. The magnet driving module continues to control the horizontal movement of the spliced structure permanent magnet. During the movement, the immunomagnetic bead complex further aggregates into dots, and finally the immunomagnetic bead complex separates from the droplet under the action of the magnetic field force to overcome the interfacial tension;

[0029] (6) Multi-round washing: The magnet driving module continues to horizontally move the spliced permanent magnet. The separated immunomagnetic bead complexes enter the next chamber through the tiny gap formed by the bottom notch between the partition chambers and the bottom film under the action of the magnetic field force and fuse with the washing buffer droplets therein. After that, the spliced permanent magnet is vertically moved downward away from the bottom of the microfluidic chip, the magnetic field action is withdrawn, and the mixing module performs a mixing operation to redisperse the aggregated immunomagnetic bead complexes. Repeat the above steps (5) and (6) for multi-round washing to remove interfering substances;

[0030] (7) Substrate reaction: After multi-round washing is completed, the immunomagnetic bead complexes enter the substrate chamber and fuse and mix with the substrate liquid therein. The substrate liquid reacts with the immunomagnetic bead complexes to produce a chemiluminescence reaction;

[0031] (8) Luminescence detection: The photons released in the chemiluminescence reaction are captured by the PMT photoelectric detection module and converted into electrical signals, and then the content of the target analyte in the sample is calculated.

[0032] Fourthly, the embodiment of the present invention also provides a method for chemiluminescence immunoassay, which has the function of separating plasma from whole blood samples and is realized by using the above-mentioned magnetically controlled droplet microfluidic device for chemiluminescence immunoassay with the function of separating plasma from whole blood samples, including the following steps:

[0033] (1) Embedding reagents: The magnetic bead reagent labeled with RBC antibody is pre-added to the first chamber at the bottom of the microfluidic chip with the function of separating plasma from whole blood samples and freeze-dried. The chemiluminescence immunoassay reagent is pre-added to the second chamber. The washing buffer is pre-added to multiple intermediate chambers after the second chamber. The substrate liquid is pre-added to the last substrate chamber;

[0034] (2) Adding samples: Add the whole blood sample to be tested to the first chamber where the reagents are embedded;

[0035] (3) Loading the chip: Install the microfluidic chip with the function of separating plasma from whole blood samples into the microfluidic chip mounting seat of the magnetically controlled droplet microfluidic device for chemiluminescence immunoassay with the function of separating plasma from whole blood samples;

[0036] (4)Plasma extraction: The mixing module performs a mixing operation on the microfluidic chip with the function of separating plasma from whole blood samples. During this process, red blood cells in the whole blood sample to be tested react with the magnetic bead reagent labeled with RBC antibody to form an immunomagnetic bead complex combined with red blood cells. The magnet driving module or the chip driving module controls the upper part of the ring permanent magnet to be close to the bottom of the microfluidic chip with the function of separating plasma from whole blood samples. Under the action of the magnetic field, the immunomagnetic bead complex combined with red blood cells quickly aggregates to the bottom of the chamber and arranges in a ring along the edge of the ring permanent magnet, and the separated plasma is obtained in the center of the ring. The separated plasma is extracted and added to the second chamber pre-embedded with reagents;

[0037] (5)Incubation and binding: The mixing module performs a mixing operation on the microfluidic chip with the function of separating plasma from whole blood samples, and the incubation module performs a heat preservation operation. In the second chamber, the antigen to be tested in the plasma specifically binds to the antibody of the chemiluminescent immunoassay reagent to form an immunomagnetic bead complex;

[0038] (6)Magnetic bead separation: The magnet driving module or the chip driving module controls the splicing structure permanent magnet to be close to the bottom film of the microfluidic chip with the function of separating plasma from whole blood samples. Under the action of the magnetic field, the immunomagnetic bead complex quickly aggregates to the bottom of the droplet and arranges in a line along the joint of the splicing structure permanent magnet. The magnet driving module controls the horizontal movement of the splicing structure permanent magnet or the chip driving module controls the horizontal movement of the microfluidic chip with the function of separating plasma from whole blood samples. During the movement, the immunomagnetic bead complex further aggregates into dots, and finally the immunomagnetic bead complex separates from the droplet under the action of the magnetic field force to overcome the interfacial tension;

[0039] (7)Multiple rounds of washing: The magnet driving module continues to control the horizontal movement of the splicing structure permanent magnet or the chip driving module continues to control the horizontal movement of the microfluidic chip with the function of separating plasma from whole blood samples. The separated immunomagnetic bead complex enters the next chamber through the tiny gap formed by the bottom notch and the bottom film between the partition chambers under the action of the magnetic field force and fuses with the washing buffer droplet therein. After that, the splicing structure permanent magnet is moved away from the bottom of the microfluidic chip, the magnetic field action is cancelled, and the mixing module performs a mixing operation to disperse the aggregated immunomagnetic bead complex again. Repeat the above steps (6) and (7) for multiple rounds of washing to remove interfering substances;

[0040] (8)Substrate reaction: After multiple rounds of washing, the immunomagnetic bead complex enters the substrate chamber and fuses and mixes with the substrate liquid therein. The substrate liquid reacts with the immunomagnetic bead complex to produce a chemiluminescent reaction;

[0041] (9)Luminescence detection: The photons released in the chemiluminescent reaction are captured by the PMT photoelectric detection module and converted into electrical signals, and then the content of the target analyte in the sample is calculated.

[0042] Compared with the prior art, the beneficial effects of the present invention at least include:

[0043] (1) The microfluidic chip provided by the present invention adopts a unique film covering structure, where a micron-level gap is formed between the film and the notch of the chip body, ingeniously constituting a microchannel for transporting immunomagnetic bead complexes, avoiding the complex process of traditional micron-level flow channel processing, effectively reducing the chip manufacturing difficulty and cost. At the same time, the film with a thickness of 100 - 200 microns significantly shortens the distance between the permanent magnet and the immunomagnetic beads, remarkably enhancing the magnetic field force acting on the immunomagnetic beads, improving the magnetic bead manipulation efficiency, and providing stronger support for subsequent immune reactions and detection processes. Through the design of the separation chamber, droplets with a volume of several to dozens of microliters are supported as reaction carriers, greatly shortening the distance of substance diffusion and reaction. According to the diffusion principle, the shortening of the distance significantly accelerates the reaction rate, enabling detection to be completed in a shorter time. At the same time, the sample and reagent consumption are also significantly reduced, not only reducing the detection cost but also improving the detection efficiency, providing new ideas for rapid and low-cost detection.

[0044] (2) For the microfluidic chip provided by the present invention, the chip body and the two layers of films are both made of FEP material, which has excellent chemical stability and can withstand the erosion of various chemical reagents, ensuring that the chip performance is not affected in complex chemiluminescence immunoassay reactions. At the same time, it has excellent high-temperature and low-temperature resistance, can adapt to different experimental environmental temperature requirements, and broadens the application scenarios of the chip. Moreover, the good mechanical properties ensure the structural stability of the chip during manufacturing and use. Most importantly, no surface treatment is required, which greatly reduces the complexity of the chip manufacturing process, reduces the manufacturing processes and costs, and provides convenience for large-scale production.

[0045] (3) For the microfluidic device provided by the present invention, a spliced structure permanent magnet is adopted, and by using the magnetic field superposition effect, the magnetic field force at the joint reaches the maximum. Under this design, the immunomagnetic bead complex can quickly gather at the joint of the spliced structure permanent magnet, and the strong magnetic field force enables a very small amount of magnetic beads to overcome the droplet interfacial tension for separation, not only improving the magnetic bead separation efficiency but also reducing the requirement for the number of magnetic beads, further optimizing the detection process, and enhancing the sensitivity and reliability of the detection.

[0046] (4) For the method for chemiluminescence immunoassay provided by the present invention, by integrating a magnetic bead freeze-dried reagent conjugated with RBC antibody on the microfluidic chip, direct separation of plasma from whole blood samples on the chip and its use for chemiluminescence immunoassay are realized, thus eliminating the need to rely on complex pretreatment operations such as centrifugation of whole blood samples in traditional methods, simplifying the detection process, reducing errors and contamination that may be brought about during sample processing, improving the accuracy and convenience of detection, making the detection process more efficient and rapid, and being more suitable for POCT scenarios. Brief Description of the Drawings

[0047] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0048] Figure 1 It is a schematic structural diagram of a magnetically controlled droplet microfluidic chip for chemiluminescence immunoassay provided by an embodiment of the present invention, where A is an exploded view and B is a longitudinal sectional view;

[0049] Figure 2 It is a schematic structural diagram of a magnetically controlled droplet microfluidic device for chemiluminescence immunoassay provided by an embodiment of the present invention;

[0050] Figure 3 It is a schematic structural diagram of a spliced - structure permanent magnet provided by an embodiment of the present invention;

[0051] Figure 4 It is a schematic structural diagram of a magnetically controlled droplet microfluidic device for chemiluminescence immunoassay with the function of separating plasma from whole - blood samples provided by an embodiment of the present invention;

[0052] Figure 5 It is a schematic diagram of a chemiluminescence immunoassay method based on a microfluidic chip and device provided by an embodiment of the present invention;

[0053] Figure 6 It is a comparison diagram of the magnetic field force and the magnetic bead aggregation effect between the spliced - structure permanent magnet and the conventional permanent magnet. Among them, A and D are respectively the schematic diagrams of the magnetic field force and the magnetic bead aggregation effect of the spliced - structure permanent magnet, and B and E, as well as C and F are respectively the schematic diagrams of the magnetic field force and the magnetic bead aggregation effect of conventional permanent magnets of different sizes;

[0054] Figure 7 It is a diagram of the relationship between the magnetic field force and the interfacial tension and a schematic diagram of the theoretical value of the minimum magnetic bead dosage;

[0055] Figure 8 It is a schematic diagram of the magnetic bead loss rate and the cleaning effect under different cleaning times;

[0056] Figure 9 It is a schematic diagram of the correlation analysis and the Bland - Altman analysis of the detection results of the method of the present invention and the detection results of the traditional tube - type commercial CLIA method. Detailed Embodiments

[0057] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not limit the protection scope of the present invention.

[0058] The inventive concept of the present invention is as follows: Aiming at the technical bottlenecks in aspects such as the manufacturing process, manufacturing cost of microfluidic chips, and the control ability and complexity of microfluidic devices in the prior art, the embodiments of the present invention provide a magnetically controlled droplet microfluidic chip, device, and method for chemiluminescence immunoassay. Through the chip FEP material, the film covering structure, and the design of the partition chamber including a bottom notch, and in combination with the splicing structure of the permanent magnet and the magnet driving module of the corresponding device, precise control and effective separation of magnetic beads under the magnetic field are realized. Finally, based on the chip and the device, chemiluminescence immunoassay and chemiluminescence immunoassay with the function of separating plasma from whole blood samples are realized. Through innovative chip design, device integration, and method optimization, an efficient, low-cost, and easy-to-operate chemiluminescence immunoassay solution is provided, which can meet the requirements of multiple scenarios such as clinical diagnosis, disease screening, and health monitoring.

[0059] Figure 1 It is a schematic structural diagram of a magnetically controlled droplet microfluidic chip for chemiluminescence immunoassay provided by an embodiment of the present invention. As Figure 1 shown in A of, the embodiment provides a magnetically controlled droplet microfluidic chip for chemiluminescence immunoassay, including: a top cover, a top film, a chip body, a bottom film, and a bottom cover that are tightly connected from top to bottom.

[0060] The chip body is used to load chemiluminescence immunoassay reagents and serve as a chemiluminescence immunoassay reaction container, and includes a number of partition chambers that penetrate up and down. According to the needs of chemiluminescence immunoassay, the partition chambers are made into the same or different specifications and sizes, and each chamber can accommodate dozens to hundreds of microliters of liquid. As Figure 1 shown in B of, there is a notch at the bottom of the adjacent partition between the partition chambers, and the height of the bottom notch is 50 - 100 micrometers. If the microfluidic chip has the function of separating plasma from whole blood samples, the first chamber adopts a bottom-closed structure and there is no notch at the adjacent partition between the first chamber and the second chamber. The chip body is made of FEP (Fluorinated Ethylene Propylene) material, and FEP has excellent chemical stability, high and low temperature resistance, and good mechanical properties, and no surface treatment is required.

[0061] The top film and the bottom film are used to enclose the separated chambers of the chip body. The top film and the bottom film are made of FEP film with a thickness of 100 - 200 microns, which completely cover all the separated chambers of the chip body and have flanges for the tensioning of the film during the overall assembly of the microfluidic chip.

[0062] The top cover and the bottom cover are adapted to the structural shape of the chip body, with a hollow in the middle to expose the top film on the upper part of the separated chamber and the bottom film on the lower part. The top film and the bottom film are firmly attached to the top surface and the bottom surface of the chip body respectively through the edges of the top cover and the bottom cover. The top cover and the bottom cover are made of metal or plastic materials, and the chip body, the top film and the bottom film are connected into a whole through interference fit or snap design.

[0063] Specifically, a magnetically controlled droplet microfluidic chip for chemiluminescence immunoassay is fabricated as follows:

[0064] (1) Use the bottom cover to firmly attach the bottom film to the bottom surface of the chip body;

[0065] (2) Add paraffin oil into each separated chamber. The paraffin oil is used to coat the droplets in each chamber to prevent droplet evaporation and to maintain the droplet shape;

[0066] (3) Add the chemiluminescence immunoassay reagents into each separated chamber in sequence. The reagents form droplets in the paraffin oil;

[0067] (4) Cover the top film on the top surface of the chip body. During this process, it should be ensured that no gas is trapped in each chamber;

[0068] (5) Use the top cover to firmly attach the top film to the top surface of the chip body.

[0069] Specifically, a magnetically controlled droplet microfluidic chip for chemiluminescence immunoassay that also has the function of separating plasma from whole blood samples is fabricated as follows:

[0070] (1) Use the bottom cover to firmly attach the bottom film to the bottom surface of the chip body;

[0071] (2) Add the magnetic bead reagent labeled with RBC antibody into the first chamber closed at the bottom and perform freeze-drying treatment;

[0072] (3) Add paraffin oil into the other separated chambers. The paraffin oil is used to coat the droplets in each chamber to prevent droplet evaporation and to maintain the droplet shape;

[0073] (4) Add the chemiluminescence immunoassay reagents into the other separated chambers in sequence. The reagents form droplets in the paraffin oil;

[0074] (5) Cover the top film on the top surface of the chip body, ensuring that no gas is trapped in each chamber during this process;

[0075] (6) Use the top gland to firmly attach the top film to the top surface of the chip body.

[0076] Figure 2 It is a schematic structural diagram of a magnetically controlled droplet microfluidic device for chemiluminescence immunoassay provided by an embodiment of the present invention. The device includes: a spliced - structure permanent magnet, a magnet driving module, an incubation module, a mixing module, and a PMT photoelectric detection module.

[0077] The spliced - structure permanent magnet is used to achieve the aggregation and separation operations of immunomagnetic bead complexes during the chemiluminescence immunoassay process. The spliced - structure permanent magnet adopts two cuboid permanent magnets or semi - cylindrical permanent magnets and attracts their N - poles and S - poles together, as Figure 3 shown. The spliced - structure permanent magnet can significantly enhance the magnetic field force acting on chemiluminescence immunomagnetic beads, which is beneficial to the precise control of the chemiluminescence immunoassay process. The material of the spliced - structure permanent magnet is preferably N56 neodymium - iron - boron magnet.

[0078] The magnet driving module includes a mechanical driving component for controlling the horizontal movement of the spliced - structure permanent magnet and a mechanical driving component for controlling the vertical movement of the spliced - structure permanent magnet. The combination of the two realizes the movement control of the spliced - structure permanent magnet along the arrangement direction of the microfluidic chip chambers and the movement control of the spliced - structure permanent magnet approaching or departing from the bottom of the microfluidic chip.

[0079] The incubation module is used to accommodate the microfluidic chip and maintain the temperature of the chemiluminescence immunoassay reaction in the microfluidic chip chamber constant, including a microfluidic chip mounting seat, a heating component, a temperature sensor, and a temperature control circuit board. Among them, the microfluidic chip mounting seat is used to accommodate and fix the microfluidic chip. The heating component is arranged outside the microfluidic chip mounting seat and is used to increase or decrease the temperature of the microfluidic chip mounting seat, thereby indirectly adjusting the temperature of the microfluidic chip. Preferred are polyimide heating film sheets, Peltier elements, etc. The temperature - sensing element of the temperature sensor is in close contact with the microfluidic chip mounting seat and is used to measure and feedback the actual temperature of the microfluidic chip mounting seat. Preferred is a PT1000 platinum resistance temperature sensor. The temperature control circuit board is used to collect the electrical signal of the temperature sensor and output a corresponding control signal through a temperature control algorithm to drive the heating component to work, thereby realizing the precise control of the temperature of the microfluidic chip.

[0080] The mixing module is used to oscillate and mix the reagent droplets and the immunomagnetic bead complexes generated by the reaction in the chamber of the microfluidic chip. Specifically, it is used to disperse the immunomagnetic bead complexes during the chemiluminescence immunoassay process through the oscillating mixing operation, and promote the specific binding reaction between antigens and antibodies and the redox reaction between the substrate and the luminescent substance. The mixing module adopts an eccentric wheel connecting rod mechanism to drive the microfluidic chip mounting seat to perform reciprocating linear motion, causing the reagent droplets in the microfluidic chip chamber to shake. By setting the eccentricity of the eccentric wheel, the oscillation amplitude can be adjusted, and by setting the rotation speed of the eccentric wheel, the oscillation frequency can be adjusted. The mixing module can also use an ultrasonic motor or a vibration motor to trigger the vibration of the bottom film of the microfluidic chip, causing the high-frequency vibration of the reagent droplets in the microfluidic chip chamber to achieve the purpose of reagent mixing.

[0081] The PMT (Photomultiplier Tube) photoelectric detection module is used to measure the light signal intensity when the immunomagnetic bead complex enters the chamber containing the substrate and undergoes a chemiluminescence reaction to quantitatively analyze the content of the target analyte. During the chemiluminescence reaction, the intensity of the luminescence signal is usually proportional to the concentration of the target analyte. By measuring the luminescence intensity, the content of the target analyte can be quantitatively analyzed. The PMT photoelectric detection module includes a single photon counter, a detection window adapter, and a shutter. Among them, the single photon counter uses a PMT as the core detection element and is equipped with corresponding signal processing circuits and counting systems. The detection window adapter is used to connect the detection window in the single photon counter and the chamber in the microfluidic chip that accommodates the substrate, and isolates ambient light during the detection process to avoid interference from external light. The shutter is used to control the exposure time of the photosensitive window in the single photon counter, and opens or closes the shutter in coordination with the chemiluminescence reaction process to protect the single photon counter.

[0082] Figure 4 It is a schematic structural diagram of a magnetically controlled droplet microfluidic device for chemiluminescence immunoassay with the function of separating plasma from whole blood samples provided by an embodiment of the present invention. The device includes: an annular permanent magnet, a spliced structure permanent magnet, a chip driving module, an incubation module, a mixing module, and a PMT photoelectric detection module. Among them, the annular permanent magnet acts on the first chamber of the microfluidic device with the function of separating plasma from whole blood samples to achieve plasma separation. Specifically, the microfluidic chip with the function of separating plasma from whole blood samples is controlled by the chip driving module to move vertically downward to the upper surface of the annular permanent magnet and close to the bottom of the chip. Under the action of the magnetic field, the aggregation and annular arrangement of the immunomagnetic bead complexes combined with red blood cells are controlled to achieve plasma separation at the center of the ring.

[0083] Based on the magnetically controlled droplet microfluidic chip and device for chemiluminescence immunoassay provided in the above embodiments, an embodiment of the present invention also provides a method for chemiluminescence immunoassay, including the following steps:

[0084] (1)Pre-embedded reagents: For example, Figure 5 As shown in Figure 5 , taking the one-step reaction as an example, the chemiluminescent immunoassay reagents (alkaline phosphatase-labeled antibody (ALP-Abs) and magnetic bead-coated antibody (MB-Abs)) are pre-added to the first chamber of the microfluidic chip, the washing buffer is pre-added to multiple intermediate chambers (the second to the fourth chambers), and the substrate solution (APS-5) is pre-added to the last substrate chamber (the fifth chamber). If it is a multi-step reaction, the number and order of the chambers are rearranged according to the specific reaction sequence;

[0085] (2)Sample addition: Add the test sample containing the target antigen to the first chamber pre-embedded with reagents;

[0086] (3)Chip loading: Load the microfluidic chip into the microfluidic chip mounting seat of the magnetically controlled droplet microfluidic device for chemiluminescent immunoassay;

[0087] (4)Incubation and binding: The mixing module performs a mixing operation on the microfluidic chip for 20 - 30 seconds, and the incubation module performs a heat preservation operation at 37 °C on the microfluidic chip for 5 - 10 minutes. In the first chamber, the target antigen in the test sample specifically binds to the antibody in the chemiluminescent immunoassay reagent to form an immunomagnetic bead complex;

[0088] (5)Magnetic bead separation: The magnet drive module controls the vertical upward movement of the spliced structure permanent magnet to the bottom of the microfluidic chip close to the bottom film. Under the action of the magnetic field, the immunomagnetic bead complex rapidly aggregates to the bottom of the droplet and arranges in a line along the seam of the spliced structure permanent magnet. The magnet drive module continues to control the horizontal movement of the spliced structure permanent magnet. During the movement, the immunomagnetic bead complex further aggregates into dots. Finally, the immunomagnetic bead complex overcomes the interfacial tension and separates from the droplet under the action of the magnetic field force;

[0089] (6)Multiple rounds of washing: The magnet drive module continues to move the spliced structure permanent magnet horizontally. The separated immunomagnetic bead complex enters the next chamber through the small gap formed by the 50 - 100 - micron bottom notch between the partition chambers and the bottom film under the action of the magnetic field force and fuses with the washing buffer droplet therein. Then, the spliced structure permanent magnet is moved vertically downward away from the bottom of the microfluidic chip to cancel the magnetic field action. The mixing module performs a mixing operation for 20 - 30 seconds to redisperse the aggregated immunomagnetic bead complex. Repeat the above steps (5) and (6) for multiple rounds of washing to remove interfering substances such as unbound free enzyme-labeled antibodies and impurities in the sample;

[0090] (7)Substrate reaction: After multiple rounds of washing are completed, the immunomagnetic bead complex enters the substrate chamber and fuses with the substrate solution therein, and is mixed for 20 - 30 seconds. The substrate solution reacts with the alkaline phosphatase in the immunomagnetic bead complex to produce a chemiluminescent reaction;

[0091] (8) Luminescence detection: The photons released in the chemiluminescence reaction are captured by the PMT photoelectric detection module and converted into electrical signals. The intensity of the optical signal is related to the amount of alkaline phosphatase in the reaction system, and thus there is a certain proportional relationship with the amount of the antigen to be detected. The content of the target analyte in the sample can be calculated by methods such as a standard curve.

[0092] Furthermore, based on the magnetically controlled droplet microfluidic chip and device for chemiluminescence immunoassay with the function of separating plasma from whole blood samples provided in the above embodiments, the embodiments also provide a chemiluminescence immunoassay method with the function of separating plasma from whole blood samples, including the following steps:

[0093] (1) Embedding reagents: Taking the one-step reaction as an example, the magnetic bead reagent labeled with RBC antibody is pre-added to the first chamber at the bottom of the microfluidic chip with the function of separating plasma from whole blood samples and freeze-dried. The chemiluminescence immunoassay reagents (alkaline phosphatase-labeled antibody (ALP-Abs) and magnetic bead-coated antibody (MB-Abs)) are pre-added to the second chamber. The washing buffer is pre-added to multiple intermediate chambers after the second chamber (if it is a three-step washing, the third to fifth chambers are set accordingly). The substrate solution (APS-5) is pre-added to the last substrate chamber (the sixth chamber). If it is a multi-step reaction, the number and order of the chambers are rearranged according to the specific reaction sequence;

[0094] (2) Adding samples: The whole blood sample to be tested containing the target antigen is added to the first chamber where the freeze-dried magnetic bead reagent is embedded.

[0095] (3) Loading the chip: The microfluidic chip with the function of separating plasma from whole blood samples is loaded into the microfluidic chip mounting seat of the magnetically controlled droplet microfluidic device for chemiluminescence immunoassay with the function of separating plasma from whole blood samples.

[0096] (4) Extracting plasma: The mixing module performs a mixing operation on the microfluidic chip with the function of separating plasma from whole blood samples for 20 to 30 seconds. During this process, the red blood cells in the whole blood sample to be tested react with the magnetic bead reagent labeled with RBC antibody to form an immunomagnetic bead complex binding red blood cells. The chip driving module moves the microfluidic chip with the function of separating plasma from whole blood samples vertically downward to the upper surface of the annular permanent magnet and close to the bottom of the chip. Under the action of the magnetic field, the immunomagnetic bead complex binding red blood cells quickly aggregates to the bottom of the chamber and arranges in a ring along the edge of the annular permanent magnet, and the separated plasma is obtained in the center of the ring. The separated plasma is extracted using a Tip head or a sampling needle and added to the second chamber where the reagents are embedded.

[0097] (5) Incubation and binding: The mixing module performs a mixing operation on the microfluidic chip with the function of separating plasma from whole blood samples for 20 - 30 seconds, and the incubation module performs a heat preservation operation at 37°C for 5 - 10 minutes. In the second chamber, the antigen to be detected in the plasma specifically binds to the antibody of the chemiluminescence immunoassay reagent to form an immunomagnetic bead complex;

[0098] (6) Magnetic bead separation: The chip driving module controls the microfluidic chip with the function of separating plasma from whole blood samples to move vertically downward until the upper surface of the permanent magnet of the splicing structure is close to the bottom film of the chip. Under the action of the magnetic field, the immunomagnetic bead complex quickly aggregates to the bottom of the droplet and arranges in a line along the seam of the splicing structure permanent magnet. The chip driving module continues to control the microfluidic chip with the function of separating plasma from whole blood samples to move horizontally. During the movement, the immunomagnetic bead complex further aggregates into dots, and finally the immunomagnetic bead complex separates from the droplet under the action of the magnetic force to overcome the interfacial tension;

[0099] (7) Multiple rounds of washing: The chip driving module continues to move the microfluidic chip with the function of separating plasma from whole blood samples horizontally. The separated immunomagnetic bead complex enters the next chamber through the small gap formed by the 50 - 100 - micron bottom notch between the partition chambers and the bottom film under the action of the magnetic force and merges with the washing buffer droplet therein. Then, the microfluidic chip with the function of separating plasma from whole blood samples is moved vertically upward away from the splicing structure permanent magnet to cancel the magnetic field action. The mixing module performs a mixing operation for 20 - 30 seconds to redisperse the aggregated immunomagnetic bead complex. Repeat the above steps (6) and (7) for multiple rounds of washing to remove interfering substances such as unbound free enzyme - labeled antibodies and impurities in the sample;

[0100] (8) Substrate reaction: After multiple rounds of washing, the immunomagnetic bead complex enters the substrate chamber and merges with the substrate liquid therein, and is mixed for 20 - 30 seconds. The substrate liquid reacts with the immunomagnetic bead complex to produce a chemiluminescence reaction;

[0101] (9) Luminescence detection: The photons released in the chemiluminescence reaction are captured by the PMT photoelectric detection module and converted into electrical signals. The intensity of the optical signal is related to the amount of alkaline phosphatase in the reaction system, and thus there is a certain proportional relationship with the amount of the antigen to be detected. The content of the target analyte in the sample can be calculated by methods such as the standard curve.

[0102] The following experimentally verifies the magnetic control effect and detection effect of the immunomagnetic bead complex (referred to as magnetic beads) in the chip, device, and method involved in the embodiments of the present invention.

[0103] As Figure 6 shown, the magnetic force of the conventional permanent magnet points to the edge of the permanent magnet (as shown by B and C in Figure 6 ), and the magnetic force of the splicing structure permanent magnet points to the seam and is significantly enhanced (as shown inFigure 6 as shown in A of Figure 6 D of Figure 6 as shown in E and F of

[0104] As Figure 7 shown, the magnetic force on the magnetic beads is proportional to the mass of the magnetic beads, and the interfacial tension is proportional to the cube root of the mass of the magnetic beads. The mass of the magnetic beads at the intersection point of the two is the minimum amount of magnetic beads that can be separated from the droplet. The minimum amount of magnetic beads used for the spliced permanent magnet in the embodiment of the present invention is 0.225 micrograms, which is about 1 / 3 of that of the conventional permanent magnet.

[0105] As Figure 8 shown, in the chemiluminescent immunoassay method provided in the embodiment of the present invention, the loss rates of magnetic beads after the first round, the second round, and the third round of washing are 0.5%, 1.31%, and 2.71% respectively. And after three rounds of washing, the CL (Chemiluminescence) intensity of the negative sample is close to the background noise, indicating that the free enzyme-labeled antibody and impurities in the sample have been removed after three rounds of washing and will not interfere with the detection results.

[0106] As Figure 9 shown, the present invention is compared with a traditional tube-type CLIA commercial instrument (large). Figure 9 The correlation analysis results shown in A of Figure 9 show a strong correlation between the two (R² = 0.9977). The Bland-Altman analysis results shown in B of

[0107] The specific embodiments described above have detailed the technical solutions and beneficial effects of the present invention. It should be understood that the above are only the most preferred embodiments of the present invention and are not used to limit the present invention. Any modifications, supplements, equivalent replacements, etc. made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A magnetically controlled droplet microfluidic chip for chemiluminescent immunoassay, characterized in that: include: A top cover, a top film, a chip body, a bottom film and a bottom cover tightly connected from top to bottom; The chip body is made of FEP material, including a plurality of vertically connected partition chambers for loading chemiluminescent immunoassay reagents and serving as chemiluminescent immunoreaction containers. A gap with a height of 50 to 100 microns is left at the bottom of the adjacent partitions between the partition chambers, so that the separated immunomagnetic bead complex can enter the next chamber from the tiny gap formed by the bottom gap between the partition chambers and the bottom film under the action of the magnetic field force. If the microfluidic chip also has the function of separating plasma from whole blood samples, the first chamber adopts a bottom-closed structure and there is no gap at the adjacent partition between the first chamber and the second chamber; The top film and the bottom film are made of FEP film with a thickness of 100 to 200 microns, which are used to seal the separated chambers of the chip body; The top pressure cover and the bottom pressure cover are adapted to the chip body structure shape, and the chip body and the top film and the bottom film are connected into a whole through interference fit or snap-fit ​​design, so as to firmly fit the top film and the bottom film to the top surface and the bottom surface of the chip body respectively.

2. The magnetically controlled droplet microfluidic chip for chemiluminescent immunoassay according to claim 1, characterized in that: The separated chambers are made into the same size or different sizes according to the needs of chemiluminescent immunoassay.

3. The magnetically controlled droplet microfluidic chip for chemiluminescent immunoassay according to claim 1, characterized in that: The size of the top film and the bottom film completely covers all the partition chambers of the chip body, and a folded edge is left for tensioning the film when the microfluidic chip is assembled as a whole.

4. The magnetically controlled droplet microfluidic chip for chemiluminescent immunoassay according to claim 1, characterized in that: The top pressure cover and the bottom pressure cover are made of metal or plastic.

5. A magnetically controlled droplet microfluidic device for chemiluminescent immunoassay, which cooperates with the magnetically controlled droplet microfluidic chip for chemiluminescent immunoassay according to any one of claims 1 to 4 to realize chemiluminescent immunoassay, characterized in that: include: Splicing structure permanent magnet, magnet driving module, incubation module, mixing module and PMT photoelectric detection module; The incubation module is used to accommodate the microfluidic chip and maintain a constant temperature in the chamber of the microfluidic chip; The mixing module is used for oscillating and mixing the reagent droplets in the chamber of the microfluidic chip and the immunomagnetic bead complex produced by the reaction; The spliced ​​structure permanent magnet uses two rectangular permanent magnets or semi-cylindrical permanent magnets and attracts the N poles and S poles thereof to each other, and is used to control the aggregation and separation operation of the immunomagnetic bead complex; The magnet driving module is used to realize the motion control of the splicing structure permanent magnet along the arrangement direction of the microfluidic chip chamber and the motion control of the splicing structure permanent magnet approaching or moving away from the bottom of the microfluidic chip; The PMT photoelectric detection module is used to measure the intensity of the light signal when the immunomagnetic bead complex enters the chamber containing the substrate and undergoes a chemiluminescent reaction to quantitatively analyze the content of the target analyte.

6. The magnetically controlled droplet microfluidic device for chemiluminescent immunoassay according to claim 5, characterized in that: The incubation module includes a microfluidic chip mounting seat, a heating component, a temperature sensor and a temperature control circuit board; wherein the microfluidic chip mounting seat is used to accommodate and fix the microfluidic chip; the heating component is arranged on the outside of the microfluidic chip mounting seat to indirectly adjust the temperature of the microfluidic chip; the temperature sensing element of the temperature sensor is in close contact with the microfluidic chip mounting seat to measure and feedback the actual temperature of the microfluidic chip mounting seat; the temperature control circuit board is used to collect the electrical signal of the temperature sensor and output the corresponding control signal through the temperature control algorithm to drive the heating component to work so as to achieve precise control of the temperature of the microfluidic chip.

7. The magnetically controlled droplet microfluidic device for chemiluminescent immunoassay according to claim 5, characterized in that: The PMT photoelectric detection module includes a single-photon counter, a detection window adapter and a shutter; wherein the single-photon counter uses PMT as a core detection element and is equipped with a corresponding signal processing circuit and a counting system; the detection window adapter is used to connect the detection window in the single-photon counter and the chamber containing the substrate in the microfluidic chip, and isolate the ambient light during the detection process to avoid interference from external light; the shutter is used to control the exposure time of the photosensitive window in the single-photon counter, and to open or close the shutter in conjunction with the chemiluminescence reaction process to protect the single-photon counter.

8. The magnetically controlled droplet microfluidic device for chemiluminescent immunoassay according to any one of claims 5 to 7, which has the function of separating plasma from whole blood samples, is characterized in that: Also includes: The annular permanent magnet is controlled by a magnet driving module to move vertically upward and close to the bottom of a microfluidic chip with the function of separating plasma from whole blood samples, or the chip driving module is used instead of the magnet driving module. The chip driving module is used to control the microfluidic chip with the function of separating plasma from whole blood samples to move and close to the upper part of the annular permanent magnet, and the aggregation and annular arrangement of the immune magnetic bead complex bound to red blood cells are controlled under the action of the magnetic field to achieve plasma separation in the center of the ring.

9. A method for chemiluminescent immunoassay for non-disease diagnosis and treatment purposes, implemented using the magnetically controlled droplet microfluidic device for chemiluminescent immunoassay according to any one of claims 5 to 7, characterized in that: The following steps are involved: (1) Pre-embedded reagents: pre-add the chemiluminescent immunoassay reagent into the first chamber of the microfluidic chip, pre-add the washing buffer into the middle chambers, and pre-add the substrate solution into the last substrate chamber; (2) Adding samples: adding the sample to be tested into the first chamber pre-embedded with reagents; (3) Loading the chip: loading the microfluidic chip into the microfluidic chip mounting seat of the magnetically controlled droplet microfluidic device for chemiluminescent immunoassay; (4) Incubation and binding: the mixing module performs a mixing operation on the microfluidic chip, and the incubation module performs a heat preservation operation on the microfluidic chip. In the first chamber, the antigen to be tested in the sample to be tested specifically binds to the antibody in the chemiluminescent immunoassay reagent to form an immunomagnetic bead complex; (5) Magnetic bead separation: The magnet drive module controls the splicing structure permanent magnet to move vertically upward to the bottom of the microfluidic chip close to the bottom film. Under the action of the magnetic field, the immunomagnetic bead complex quickly gathers at the bottom of the droplet and is arranged in a line along the joint of the splicing structure permanent magnet. The magnet drive module controls the splicing structure permanent magnet to move horizontally. During the movement, the immunomagnetic bead complex further gathers into a point shape. Finally, the immunomagnetic bead complex overcomes the interfacial tension under the action of the magnetic field force and is separated from the droplet; (6) Multiple rounds of washing: The magnet driving module continues to move the splicing structure permanent magnet horizontally. Under the action of the magnetic field force, the separated immunomagnetic bead complex enters the next chamber from the tiny gap formed by the bottom notch and the bottom film between the chambers and merges with the washing buffer droplet therein. Thereafter, the splicing structure permanent magnet is moved vertically downward away from the bottom of the microfluidic chip, the magnetic field is canceled, and the mixing module performs a mixing operation to redisperse the aggregated immunomagnetic bead complex. The above steps (5) and (6) are repeated to perform multiple rounds of washing to remove interfering substances. (7) Substrate reaction: After multiple rounds of washing, the immunomagnetic bead complex enters the substrate chamber and merges with the substrate solution therein, and the substrate solution reacts with the immunomagnetic bead complex to produce a chemiluminescent reaction; (8) Luminescence detection: The photons released in the chemiluminescence reaction are captured by the PMT photoelectric detection module and converted into electrical signals, which are then used to calculate the content of the target analyte in the sample.

10. A method for chemiluminescent immunoassay for non-disease diagnosis and treatment purposes, implemented by using the magnetically controlled droplet microfluidic device for chemiluminescent immunoassay according to claim 8, which has the function of separating plasma from whole blood samples, characterized in that: The following steps are involved: (1) Pre-embedded reagents: A magnetic bead reagent labeled with RBC antibodies is pre-added to the first chamber with a bottom seal in a microfluidic chip that has the function of separating whole blood samples from plasma and freeze-dried, a chemiluminescent immunoassay reagent is pre-added to the second chamber, a cleaning buffer is pre-added to multiple chambers in the middle after the second chamber, and a substrate solution is pre-added to the last substrate chamber; (2) Adding samples: adding the whole blood sample to be tested into the first chamber pre-embedded with reagents; (3) Loading the chip: loading the microfluidic chip with the function of separating plasma from whole blood samples into the microfluidic chip mounting seat of the magnetically controlled droplet microfluidic device for chemiluminescent immunoassay with the function of separating plasma from whole blood samples; (4) Extracting plasma: The mixing module performs a mixing operation on the microfluidic chip that has the function of separating plasma from whole blood samples. During this process, the red blood cells in the whole blood sample to be tested react with the magnetic bead reagent labeled with RBC antibodies to form an immune magnetic bead complex that binds to the red blood cells. The magnet driving module or the chip driving module controls the upper part of the annular permanent magnet to be close to the bottom of the microfluidic chip that has the function of separating plasma from whole blood samples. Under the action of the magnetic field, the immune magnetic bead complex that binds to the red blood cells quickly gathers to the bottom of the chamber and is arranged in a ring along the edge of the annular permanent magnet to obtain separated plasma in the center of the ring. The separated plasma is extracted and added to the second chamber pre-embedded with the reagent; (5) Incubation and combination: The mixing module performs a mixing operation on the microfluidic chip that has the function of separating whole blood samples from plasma, and the incubation module performs a heat preservation operation. In the second chamber, the antigen to be tested in the plasma specifically binds to the antibody in the chemiluminescent immunoassay reagent to form an immunomagnetic bead complex; (6) Magnetic bead separation: The magnet driving module or the chip driving module controls the splicing structure permanent magnet to be close to the bottom film of the microfluidic chip with the function of separating whole blood sample plasma. Under the action of the magnetic field, the immunomagnetic bead complex quickly gathers at the bottom of the droplet and is arranged in a line along the seam of the splicing structure permanent magnet. The magnet driving module controls the horizontal movement of the splicing structure permanent magnet or the chip driving module controls the horizontal movement of the microfluidic chip with the function of separating whole blood sample plasma. During the movement, the immunomagnetic bead complex further gathers into a point shape. Finally, the immunomagnetic bead complex overcomes the interfacial tension under the action of the magnetic field force and is separated from the droplet; (7) Multiple rounds of washing: the magnet driving module continues to control the horizontal movement of the splicing structure permanent magnet or the chip driving module continues to control the horizontal movement of the microfluidic chip that has the function of separating whole blood samples from plasma. Under the action of the magnetic field force, the separated immunomagnetic bead complex enters the next chamber from the tiny gap formed by the bottom notch and the bottom film between the separation chambers and merges with the washing buffer droplet therein. Thereafter, the splicing structure permanent magnet is moved away from the bottom of the microfluidic chip, the magnetic field is canceled, and the mixing module performs a mixing operation to redisperse the aggregated immunomagnetic bead complex. The above steps (6) and (7) are repeated to perform multiple rounds of washing to remove interfering substances. (8) Substrate reaction: After multiple rounds of washing, the immunomagnetic bead complex enters the substrate chamber and merges with the substrate solution therein, and the substrate solution reacts with the immunomagnetic bead complex to produce a chemiluminescent reaction; (9) Luminescence detection: The photons released in the chemiluminescence reaction are captured by the PMT photoelectric detection module and converted into electrical signals, which are then used to calculate the content of the target analyte in the sample.

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