Magnetic control liquid drop micro-fluidic chip, device and method for chemiluminescence immunoassay

Abstract

The invention discloses a magnetic control liquid drop micro-fluidic chip, device and method for chemiluminescence immunoassay, and belongs to the technical field of chemiluminescence immunoassay, the micro-fluidic chip comprises: a chip main body, which comprises a plurality of vertically through separation chambers for loading a reagent and serving as a reaction container, if the chip has the function of separating plasma from the whole blood sample, the first cavity adopts a bottom closed structure, and no gap is formed in the separation position between the first cavity and the second cavity; the top thin film and the bottom thin film are used for sealing the separated cavity of the chip main body; and the top gland and the bottom gland are matched with the chip main body in structural shape and are used for firmly attaching the two layers of films to the chip main body. According to the invention, the whole process integration of sample pretreatment, reagent mixing, reaction incubation and chemiluminescence detection can be realized, and a chemiluminescence immunoassay solution which is efficient, low in cost and easy to operate is provided through chip design, device integration and method optimization.

Description

Technical Field

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

[0002] Chemiluminescence Immunoassay (CLIA) is an immunoassay technology widely used in the field of in vitro diagnostics. 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 examinations, early disease screening, disease course monitoring, and prognosis evaluation, providing solid and reliable data support for clinical decision-making. Microfluidic technology achieves precise control and rapid response to small amounts of fluid, integrates multiple operations of chemiluminescence immunoassay into a single microchip, and can automatically complete the entire analysis process. Microfluidic chips are characterized by portability, low analysis cost, short reaction time, large-scale parallel processing, and automated operation, which makes them 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. The more mature technology in continuous flow microfluidics is centrifugal disc microfluidics. For example, the Chinese patent application with publication number CN105842468A provides a microfluidic chemiluminescent immunoassay device and its use method. A microfluidic detection unit is installed on the microfluidic disc, including various storage tanks, injection tanks, mixing tanks, waste liquid tanks, etc., and microfluidic pipelines connecting the tanks. Different angular velocities and angular accelerations are applied to the microfluidic disc to generate centrifugal force to control the transfer of samples and reagents in the microfluidic detection unit to achieve chemiluminescent immunoassay. The more mature technology in digital microfluidics is to use the dielectric wetting (Electrowetting-on-Dielectric, EWOD) technology to achieve discrete droplet manipulation. For example, the Chinese patent application with publication number CN114002447A provides a fully automatic digital microfluidic analysis platform, including an installation base and a magnetic separation control module, a chip support plate, a chip temperature control module, a chip drive electroacupuncture module and a multi-channel fluorescence detection system, wherein the chip drive electroacupuncture module provides voltage to the chip to cause the droplets to have a dielectric wetting effect and realize the drive of the droplets. The newer technology in digital microfluidics is to use magnetic excitation technology to achieve discrete droplet manipulation. For example, the Chinese patent application with publication number CN108479875A provides a digital microfluidic manipulation platform, the chip includes a chip upper plate and a chip lower plate, a microchannel is formed between the two plates, the movement of the microdroplets in the microchannel on the chip is controlled by setting a magnetic control module, and the magnetic control module is used to replace the microelectrodes in the EWOD drive chip. However, these technologies still face some challenges in practical applications. Although continuous flow microfluidics performs well in fluid control, its complex microchannel design and manufacturing process increase production costs and make it difficult to achieve high integration. Although digital microfluidics is flexible in droplet manipulation, its reliance on external drive devices increases the complexity and cost of the system, and the precise manipulation and stability of droplets still need to be further improved.

[0004] In summary, there are technical bottlenecks in the existing technology in terms of the manufacturing process, manufacturing cost, control capability and complexity of microfluidic chips, which limits the widespread application of microfluidic technology in the field of small POCT (Point-of-Care Testing). Summary of the invention

[0005] In view of the above, the purpose of the present invention is to provide a magnetically controlled droplet microfluidic chip, device and method for chemiluminescent immunoassay. By designing a coating structure based on FEP material and a separation chamber with a micron-level notch at the bottom in the chip, there is no need to process the micron-level flow channel, thereby reducing the complexity and production cost of the chip manufacturing process, and further integrating the chip into the device to achieve efficient manipulation and separation of the immune magnetic bead complex through the magnetic field, thereby improving the detection efficiency and reducing the detection cost. According to the flexible design of the chip chamber, chemiluminescent immunoassay with the function of separating plasma from whole blood samples can be realized, and the full process integration of sample pretreatment, reagent mixing, reaction incubation and chemiluminescent detection is realized. The miniaturization and portability of the device make it particularly suitable for quickly completing high-quality biomarker detection in POCT scenarios.

[0006] In order to achieve the above-mentioned invention object, the technical solution provided by the present invention is as follows: In a first aspect, an embodiment of the present invention provides a magnetically controlled droplet microfluidic chip for chemiluminescent immunoassay, comprising: a top pressure cover, a top film, a chip body, a bottom film and a bottom pressure cover tightly connected from top to bottom; The chip body includes a plurality of vertically connected partition chambers for loading chemiluminescent immunoassay reagents and serving as chemiluminescent immunoreaction containers. A gap is left at the bottom of the adjacent partitions between the partition chambers. 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 used to seal the separated chambers of the chip body; The top pressure cover and the bottom pressure cover are adapted to the structural shape of the chip body and are used to firmly adhere the top film and the bottom film to the top surface and the bottom surface of the chip body respectively.

[0007] Preferably, the chip body is made of FEP material, the height of the bottom gap of the adjacent partitions between the separated chambers is 50-100 microns, and the separated chambers are made into the same size or different sizes according to the needs of chemiluminescent immunoassay.

[0008] Preferably, the top film and the bottom film are made of FEP films with a thickness of 100 to 200 microns, which are large enough to completely cover all the compartments of the chip body, and have folded edges for tensioning the film when the microfluidic chip is assembled as a whole.

[0009] Preferably, the top pressure cover and the bottom pressure cover are made of metal or plastic, and the chip body and the top film and the bottom film are connected into a whole through interference fit or snap-fit ​​design.

[0010] In a second aspect, an embodiment of the present invention further provides a magnetically controlled droplet microfluidic device for chemiluminescent immunoassay, which cooperates with the above-mentioned magnetically controlled droplet microfluidic chip for chemiluminescent immunoassay to realize chemiluminescent immunoassay, including: a spliced ​​structure permanent magnet, a magnet driving module, an incubation module, a mixing module and a 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.

[0011] Preferably, the incubation module comprises 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, 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 thermal resistor temperature sensor; the temperature control circuit board is used to collect the electrical signal of the temperature sensor and output the corresponding control signal via the temperature control algorithm to drive the heating component to work so as to achieve precise control of the temperature of the microfluidic chip.

[0012] Preferably, 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 the 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.

[0013] Preferably, the magnetically controlled droplet microfluidic device for chemiluminescent immunoassay with the function of separating plasma from whole blood samples also includes: an annular permanent magnet, which 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 a chip driving module is used instead of a 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 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.

[0014] In a third aspect, an embodiment of the present invention further provides a method for chemiluminescent immunoassay, which is implemented using the above-mentioned magnetically controlled droplet microfluidic device for chemiluminescent immunoassay, and includes the following steps: (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.

[0015] In a fourth aspect, an embodiment of the present invention further provides a method for chemiluminescent immunoassay, which has the function of separating plasma from whole blood samples, and is implemented using the above-mentioned magnetically controlled droplet microfluidic device for chemiluminescent immunoassay with the function of separating plasma from whole blood samples, comprising the following steps: (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.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The microfluidic chip provided by the present invention adopts a unique film structure. The gap between the film and the chip body forms a micron-level gap, which cleverly forms an immunomagnetic bead complex delivery microchannel, avoiding the complex process of traditional micron-level flow channel processing, and effectively reducing the difficulty and cost of chip production. At the same time, the film with a thickness of 100 to 200 microns greatly shortens the distance between the permanent magnet and the immunomagnetic beads, significantly enhances the magnetic field force acting on the immunomagnetic beads, improves the efficiency of magnetic bead manipulation, and provides more powerful support for subsequent immune reactions and detection processes. The design of the separated chamber supports the use of several to tens of microliters of droplets as reaction carriers, greatly shortening the distance of material diffusion and reaction. According to the principle of diffusion, the shortened distance greatly accelerates the reaction speed, and the detection can be completed in a shorter time. At the same time, the amount of sample and reagent used is also significantly reduced, which not only reduces the detection cost, but also improves the detection efficiency, providing a new idea for rapid and low-cost detection.

[0017] (2) The microfluidic chip provided by the present invention, the chip body and the two layers of film are 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 chemiluminescent immunoassay reactions. At the same time, it has excellent high and low temperature resistance, can adapt to different experimental environment temperature requirements, and broaden the application scenarios of the chip. Moreover, good mechanical properties ensure the structural stability of the chip during the production and use process. Most importantly, there is no need for surface treatment, which greatly reduces the complexity of the chip manufacturing process, reduces the production process and cost, and provides convenience for large-scale production.

[0018] (3) The microfluidic device provided by the present invention uses a spliced ​​structure permanent magnet and utilizes the magnetic field superposition effect to maximize the magnetic field force at the joint. Under this design, the immunomagnetic bead complex can quickly gather at the joint of the spliced ​​structure permanent magnet. The strong magnetic field force enables a very small amount of magnetic beads to overcome the interfacial tension of the droplets to achieve separation, which not only improves the magnetic bead separation efficiency, but also reduces the demand for the number of magnetic beads, further optimizes the detection process, and improves the sensitivity and reliability of the detection.

[0019] (4) The method for chemiluminescent immunoassay provided by the present invention integrates a magnetic bead lyophilized reagent coupled to an RBC antibody on a microfluidic chip, thereby achieving direct separation of plasma from a whole blood sample on the chip and using it for chemiluminescent immunoassay. This eliminates the need for complex pretreatment operations such as centrifugation of whole blood samples in traditional methods, simplifies the detection process, reduces errors and contamination that may be caused by sample processing, improves the accuracy and convenience of the detection, and makes the detection process more efficient and rapid, making it more suitable for POCT scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 is a schematic diagram of the structure of a magnetically controlled droplet microfluidic chip for chemiluminescent immunoassay provided in an embodiment of the present invention, wherein A is an exploded view and B is a longitudinal cross-sectional view; Figure 2 Schematic diagram of the structure of a magnetically controlled droplet microfluidic device for chemiluminescent immunoassay provided in an embodiment of the present invention; Figure 3 is a schematic structural diagram of a spliced ​​structure permanent magnet provided by an embodiment of the present invention; Figure 4It is a schematic structural diagram of a magnetically controlled droplet microfluidic device for chemiluminescent immunoassay with the function of separating plasma from whole blood samples provided by an embodiment of the present invention; Figure 5 is a schematic diagram of a chemiluminescent immunoassay method based on a microfluidic chip and device provided in an embodiment of the present invention; Figure 6 It is a comparison diagram of the magnetic field force and magnetic bead aggregation effect of the spliced ​​structure permanent magnet and the conventional permanent magnet, wherein A and D are schematic diagrams of the magnetic field force and magnetic bead aggregation effect of the spliced ​​structure permanent magnet, B and E, as well as C and F are schematic diagrams of the magnetic field force and magnetic bead aggregation effect of conventional permanent magnets of different sizes; Figure 7 It is a diagram showing the relationship between magnetic field force and interfacial tension and a diagram showing the theoretical value of the minimum amount of magnetic beads used; Figure 8 This is a schematic diagram of the magnetic bead loss rate and cleaning effect under different cleaning times; Fig. 9 It is a schematic diagram of the correlation analysis and Brand-Altman analysis between the detection results of the method of the present invention and the detection results of the traditional tube-based commercial CLIA method. DETAILED DESCRIPTION

[0022] To make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific implementation methods described herein are only used to explain the present invention and do not limit the scope of protection of the present invention.

[0023] The inventive concept of the present invention is: in view of the technical bottlenecks in the manufacturing process, manufacturing cost, control ability and complexity of the microfluidic chip in the prior art, the embodiments of the present invention provide a magnetically controlled droplet microfluidic chip, device and method for chemiluminescent immunoassay, through the chip FEP material and coating structure and the separation chamber design including the bottom notch, and combined with the splicing structure permanent magnet and magnet driving module of the corresponding device to achieve precise control and effective separation of magnetic beads under the action of the magnetic field, finally based on the chip and the device to achieve chemiluminescent immunoassay and chemiluminescent immunoassay with the function of separating plasma from whole blood samples, through innovative chip design, device integration and method optimization, provide an efficient, low-cost and easy-to-operate chemiluminescent immunoassay solution, which can meet the needs of multiple scenarios such as clinical diagnosis, disease screening and health monitoring.

[0024] Figure 1 FIG. 1 is a schematic diagram of the structure of a magnetically controlled droplet microfluidic chip for chemiluminescent immunoassay provided in an embodiment of the present invention. Figure 1As shown in A in the figure, the embodiment provides a magnetically controlled droplet microfluidic chip for chemiluminescent immunoassay, including: a top pressure cover, a top film, a chip body, a bottom film and a bottom pressure cover tightly connected from top to bottom.

[0025] The chip body is used to load chemiluminescent immunoassay reagents and serve as a chemiluminescent immunoassay reaction container, including several vertically connected compartments. The compartments are made into the same or different sizes according to the needs of chemiluminescent immunoassay, and each compartment can hold tens to hundreds of microliters of liquid. Figure 1 As shown in B, there is a gap at the bottom of the adjacent partition between the separated chambers, and the height of the bottom gap is 50~100 microns. 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 main body of the chip is made of FEP (Fluorinated Ethylene Propylene, fluorinated ethylene propylene copolymer) material. FEP has excellent chemical stability, high and low temperature resistance, and good mechanical properties, and does not require surface treatment.

[0026] The top film and the bottom film are used to seal the compartments of the chip body. The top film and the bottom film are made of 100-200 micron thick FEP film, which is large enough to completely cover all compartments of the chip body, and have folded edges for tensioning the film when the microfluidic chip is assembled as a whole.

[0027] The top and bottom glands are adapted to the shape of the chip body structure, and the middle is hollowed out to expose the top film at the top of the separation chamber and the bottom film at the bottom. The top and bottom films are firmly attached to the top and bottom surfaces of the chip body respectively through the edges of the top and bottom glands. The top and bottom glands are made of metal or plastic, and the chip body and the top and bottom films are connected as a whole through interference fit or snap-on design.

[0028] Specifically, a magnetically controlled droplet microfluidic chip for chemiluminescent immunoassay, and its manufacturing method is as follows: (1) Use the bottom pressure cover to firmly attach the bottom film to the bottom surface of the chip body; (2) adding paraffin oil into each compartment, the paraffin oil is used to coat the droplets in each compartment to prevent the droplets from evaporating and to maintain the droplet shape; (3) adding chemiluminescent immunoassay reagents into each compartment in turn, and the reagents form droplets in paraffin oil; (4) Cover the top surface of the chip with a top film. This process should ensure that no gas is trapped in each chamber. (5) Use the top cover to firmly attach the top film to the top surface of the chip body.

[0029] Specifically, a magnetically controlled droplet microfluidic chip for chemiluminescent immunoassay having the function of separating plasma from whole blood samples is prepared as follows: (1) Use the bottom pressure cover to firmly attach the bottom film to the bottom surface of the chip body; (2) adding a magnetic bead reagent labeled with RBC antibody into the first chamber with a closed bottom and performing a freeze-drying treatment; (3) adding paraffin oil into other compartments, the paraffin oil is used to coat the droplets in each compartment to prevent the droplets from evaporating and to maintain the droplet shape; (4) adding chemiluminescent immunoassay reagents into other compartments in sequence, and the reagents form droplets in paraffin oil; (5) Cover the top surface of the chip with a top film. This process should ensure that no gas is trapped in each chamber. (6) Use the top cover to firmly attach the top film to the top surface of the chip body.

[0030] Figure 2 It is a structural schematic diagram of a magnetically controlled droplet microfluidic device for chemiluminescent immunoassay provided in an embodiment of the present invention, the device comprising: a spliced ​​structure permanent magnet, a magnet driving module, an incubation module, a mixing module and a PMT photoelectric detection module.

[0031] The spliced ​​structure permanent magnet is used to realize the aggregation and separation of the immunomagnetic bead complex in the chemiluminescent immunoassay process. The spliced ​​structure permanent magnet uses two rectangular permanent magnets or semi-cylindrical permanent magnets and attracts their N poles and S poles together. Figure 3 As shown. The spliced ​​structure permanent magnet can significantly enhance the magnetic field force acting on the chemiluminescent immunomagnetic beads, which is beneficial to the precise control of the chemiluminescent immunoassay process. The spliced ​​structure permanent magnet material is preferably N56 needle iron boron magnet.

[0032] The magnet drive module includes a mechanical drive component for controlling the horizontal movement of the splicing structure permanent magnet and a mechanical drive component for controlling the vertical movement of the splicing structure permanent magnet. The combination of the two realizes the movement control of the splicing structure permanent magnet along the arrangement direction of the microfluidic chip chamber and the movement control of the splicing structure permanent magnet close to or away from the bottom of the microfluidic chip.

[0033] The incubation module is used to accommodate the microfluidic chip and maintain the constant temperature of the chemiluminescent immunoreaction in the chamber of the microfluidic chip, 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 on the outside of the microfluidic chip mounting seat, which is used to realize the heating or cooling of the microfluidic chip mounting seat, thereby indirectly adjusting the temperature of the microfluidic chip, preferably a polyimide heating film, a Peltier element, 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, preferably a PT1000 platinum thermal resistor temperature sensor. 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, thereby realizing the precise control of the temperature of the microfluidic chip.

[0034] The mixing module is used to oscillate and mix the reagent droplets in the chamber of the microfluidic chip and the immunomagnetic bead complex produced by the reaction. It is specifically used to achieve the dispersion operation of the immunomagnetic bead complex in the chemiluminescent immunoassay process through oscillation and mixing operations, and promote the specific binding reaction between the antigen and the antibody 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. The oscillation amplitude can be adjusted by setting the eccentric distance of the eccentric wheel, and the oscillation frequency can be adjusted by setting the rotation speed of the eccentric wheel. The mixing module can also use an ultrasonic motor or a vibration motor to trigger the vibration of the thin film at the bottom of the microfluidic chip, causing high-frequency vibration of the reagent droplets in the microfluidic chip chamber to achieve the purpose of reagent mixing.

[0035] The PMT (Photomultiplier Tube) 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. The intensity of the luminescent signal during the chemiluminescent reaction is usually proportional to the concentration of the target analyte. By measuring the luminescent 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; the single-photon counter uses 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 containing the substrate in the microfluidic chip, isolating 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 the shutter is opened or closed in conjunction with the chemiluminescent reaction process to protect the single-photon counter.

[0036] Figure 4It is a schematic diagram of the structure of a magnetically controlled droplet microfluidic device for chemiluminescent immunoassay with the function of separating plasma from whole blood samples provided by an embodiment of the present invention, and 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 with the function of separating plasma from whole blood samples to achieve plasma separation, specifically, the chip driving module controls the microfluidic chip with the function of separating plasma from whole blood samples to move vertically downward to the upper surface of the annular permanent magnet and close to the bottom of the chip, and controls the aggregation and annular arrangement of the immune magnetic bead complex bound to red blood cells under the action of the magnetic field to achieve plasma separation in the center of the ring.

[0037] Based on the magnetically controlled droplet microfluidic chip and device for chemiluminescent immunoassay provided in the above embodiment, the embodiment of the present invention further provides a method for chemiluminescent immunoassay, comprising the following steps: (1) Pre-embedded reagents: Figure 5 As shown, taking a one-step reaction as an example, chemiluminescent immunoassay reagents (alkaline phosphatase labeled antibodies (ALP-Abs) and magnetic bead-coated antibodies (MB-Abs)) are pre-added to the first chamber of the microfluidic chip, the washing buffer is pre-added to the middle chambers (the second to 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; (2) Adding sample: adding the sample to be tested containing the target antigen into the first chamber pre-embedded with the reagent; (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 for 20 to 30 seconds, and the incubation module performs a 37°C incubation operation on the microfluidic chip for 5 to 10 minutes. 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 drive 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 gap of 50-100 microns between the chambers and the bottom film and merges with the washing buffer droplet therein. After that, 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 for 20-30 seconds to redisperse the aggregated immunomagnetic bead complex. Repeat the above steps (5) and (6) and perform multiple rounds of washing to remove interfering substances such as unbound free enzyme-labeled antibodies and impurities in the sample; (7) Substrate reaction: After multiple rounds of washing, the immunomagnetic bead complex enters the substrate chamber and merges with the substrate solution therein. After mixing for 20 to 30 seconds, the substrate solution reacts with the alkaline phosphatase in 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. The intensity of the light signal is related to the amount of alkaline phosphatase in the reaction system, and in turn has a certain proportional relationship with the amount of the antigen to be tested. The content of the target analyte in the sample can be calculated using methods such as the standard curve.

[0038] Further, based on the above embodiment, a magnetically controlled droplet microfluidic chip and device having the function of separating plasma from whole blood samples for chemiluminescent immunoassay is provided, the embodiment also provides a chemiluminescent immunoassay method having the function of separating plasma from whole blood samples, comprising the following steps: (1) Pre-embedded reagents: Taking a one-step reaction as an example, 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 then freeze-dried. A chemiluminescent immunoassay reagent (alkaline phosphatase labeled antibody (ALP-Abs) and magnetic bead coated antibody (MB-Abs)) is pre-added to the second chamber. A cleaning buffer is pre-added to multiple intermediate chambers after the second chamber (if a three-step cleaning is performed, the third to fifth chambers are set accordingly). A substrate solution (APS-5) is pre-added to the last substrate chamber (the sixth chamber). If a multi-step reaction is performed, the number and order of the chambers are rearranged according to the specific reaction sequence. (2) Adding samples: Adding the whole blood sample containing the target antigen to be tested into the first chamber pre-embedded with freeze-dried magnetic bead reagent; (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 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 antibodies to form an immune magnetic bead complex bound to the 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 immune magnetic bead complex bound 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. The separated plasma is obtained in the center of the ring. The separated plasma is extracted using a tip head or a sample suction needle and added to the second chamber pre-buried with the reagent. (5) Incubation and binding: The mixing module performs a mixing operation on the microfluidic chip that has the function of separating whole blood samples from plasma for 20 to 30 seconds, and the incubation module performs a 37°C insulation operation for 5 to 10 minutes. 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 chip driving module controls the microfluidic chip with the function of separating whole blood sample plasma to move vertically downward until the upper surface of the splicing structure permanent magnet is close to the bottom film of the chip. 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 chip driving module continues to control the microfluidic chip with the function of separating whole blood sample plasma to move horizontally. During the movement, the immunomagnetic bead complex further gathers into dots. 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 chip driving module continues to move the microfluidic chip with the function of separating whole blood samples from plasma 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 of 50 to 100 microns between the chambers and the bottom film and merges with the washing buffer droplet therein. Thereafter, the microfluidic chip with the function of separating whole blood samples from plasma is moved vertically upward away from the permanent magnet of the splicing structure, the magnetic field is canceled, and the mixing module performs a mixing operation for 20 to 30 seconds 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 such as unbound free enzyme-labeled antibodies and impurities in the sample; (8) Substrate reaction: After multiple rounds of washing, the immunomagnetic bead complex enters the substrate chamber and merges with the substrate solution therein. After mixing for 20 to 30 seconds, the substrate solution and the immunomagnetic bead complex react 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. The intensity of the light signal is related to the amount of alkaline phosphatase in the reaction system, and in turn has a certain proportional relationship with the amount of the antigen to be tested. The content of the target analyte in the sample can be calculated using methods such as the standard curve.

[0039] The magnetic control effect and detection effect of the immunomagnetic bead complex (magnetic beads for short) in the chip, device and method involved in the embodiments of the present invention are experimentally verified as follows.

[0040] like Figure 6 As shown, the magnetic field force of a conventional permanent magnet points to the edge of the permanent magnet (such as Figure 6 As shown in B and C in the figure, the magnetic field force of the permanent magnet in the spliced ​​structure is directed to the joint and is significantly enhanced (as shown in Figure 6 As shown in A in the figure). Under the action of magnetic field force, magnetic beads quickly gather at the joints of permanent magnets in the spliced ​​structure (as shown in Figure 6 As shown in D in the figure, the magnetic beads aggregated within 2 seconds without any residue. The magnetic beads slowly aggregated to the edge of the conventional permanent magnet (as shown in Figure 6 As shown in E and F in Figure 3, some beads still did not aggregate after 10 seconds.

[0041] like Figure 7 As shown, the magnetic field force on the magnetic beads is proportional to the mass of the magnetic beads, 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 of the two is the minimum amount of magnetic beads that can be separated from the droplets, and the minimum amount of magnetic beads in the spliced ​​structure permanent magnet used in the embodiment of the present invention is 0.225 micrograms, which is about 1 / 3 of that of a conventional permanent magnet.

[0042] like Figure 8 As shown, in the chemiluminescent immunoassay method provided in an embodiment of the present invention, the magnetic bead loss rates after one, two, and three rounds of washing are 0.5%, 1.31%, and 2.71%, respectively, and the CL (Chemiluminescence) intensity of the negative sample after three rounds of washing is close to the background noise, indicating that the free enzyme-labeled antibodies and impurities in the sample have been removed after three rounds of washing and will not interfere with the test results.

[0043] like Fig. 9 As shown, the present invention is compared with the traditional tube-type CLIA commercial instrument (large-scale), Fig. 9 The correlation analysis results shown in A show that the two are strongly correlated (R²=0.9977). Fig. 9The Bland-Altman analysis results shown in B show that the average deviation between the two is -0.0586 ng / mL (95% CI, -0.80 to 0.68), and 92.86% of the data are distributed in this range. The results show that the two methods have good consistency, indicating that the detection method provided in the embodiment of the present invention is comparable to the traditional tube-type CLIA commercial instrument in accuracy and reliability, and even performs better in some aspects. This further verifies the feasibility and superiority of the present invention in practical applications, and provides a more convenient and efficient solution for clinical testing.

[0044] The specific implementation methods described above provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above is only the most preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, supplements and equivalent substitutions 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 includes a plurality of vertically connected partition chambers for loading chemiluminescent immunoassay reagents and serving as chemiluminescent immunoreaction containers. A gap is left at the bottom of the adjacent partitions between the partition chambers. 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 used to seal the separated chambers of the chip body; The top pressure cover and the bottom pressure cover are adapted to the structural shape of the chip body and are used to firmly adhere 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 chip body is made of FEP material, the height of the bottom gap of the adjacent partitions between the separated chambers is 50-100 microns, and 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 top film and the bottom film are made of FEP films with a thickness of 100 to 200 microns, which are large enough to completely cover all the compartments of the chip body, and have folded edges 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, and the chip body and the top film and the bottom film are connected into a whole through interference fit or snap-fit ​​design.

5. A magnetically controlled droplet microfluidic device for chemiluminescent immunoassay, which is used in conjunction with the magnetically controlled droplet microfluidic chip for chemiluminescent immunoassay according to any one of claims 1 to 4 to implement 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, 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 thermal resistor temperature sensor; the temperature control circuit board is used to collect the electrical signal of the temperature sensor and output a corresponding control signal via a 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, implemented by 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, implemented by using the magnetically controlled droplet microfluidic device for chemiluminescent immunoassay according to claim 8, having 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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