Microfluidic multiplexed immunoassay system based on flow magnet-activated cell sorting, and method thereof

Abstract

The present invention relates to a microfluidic multiplexed immunoassay system based on flow magnet-activated cell sorting, and a method thereof. The system comprises a multiplexed immunoassay magnetic bead preparation module, a flow magnet-activated cell sorting microfluidic chip system, an optical detection module, and an image processing module, and achieves ultra-sensitive and rapid multiplexed protein quantitative detection by means of magnetic bead coding based on different magnetic contents and microfluidic chip decoding based on flow magnet-activated cell sorting. The microfluidic multiplexed immunoassay system of the present invention has the advantages of high detection sensitivity, short detection time, high signal-to-noise ratio, convenient and simple operation and low cost, is particularly suitable for multiplexed ultra-sensitive detection for monomolecular proteins, and has a potential economic value.

Description

A multiplex immune microfluidic detection system and method based on flow magnetic sorting Technical Field

[0001] The present invention mainly relates to the fields of microfluidics and biological detection, and proposes a multiplex immune microfluidics detection platform and method based on flow magnetic sorting. Background Art

[0002] Simultaneous detection of multiple targets can improve the accuracy of disease diagnosis and, on the other hand, increase analytical throughput, shorten detection time, and reduce detection costs. With the increasing demand for simultaneous and sensitive detection of multiple proteins in modern medicine, the development of advanced multiplex protein detection technologies is essential for improving the diagnostic capabilities of medical instruments. Traditional detection methods include immunochromatography, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), fluorescence, electrochemistry, chemiluminescence, and electrochemiluminescence. However, these technologies still suffer from low sensitivity, delayed time windows, high detection costs, high reagent consumption, and complex operations, making them unable to meet the growing demand for clinical diagnosis.

[0003] One of the major challenges in achieving immunoassay trace analysis of multiple protein markers in complex sample environments is the separation of target analytes and antibody complexes. Magnetic nanoparticles immobilized with antibodies have the inherent property of being attracted to magnets. This property can be exploited to directly capture, separate, and pre-concentrate target substances in complex environmental samples, providing a solution for immunoassay technology. Traditional large-scale immunoluminescence instrument immune reaction systems typically use traditional reagent tubes as reaction containers, requiring large amounts of reagents and high costs. Furthermore, since the entire reaction system is concentrated in the reagent tube, there are disadvantages such as insufficient incubation, insufficient mixing, and low magnetic separation efficiency.

[0004] Microfluidic technology can integrate basic operating units such as sample preparation, reaction, separation, and detection onto a micron-scale chip, automatically completing the entire analysis process. It has the characteristics of high mass and heat transfer efficiency, fast reaction rate, small reactor size, and high controllability. Microfluidic chips have become a popular research direction in the field of biological detection due to their high performance, miniaturization, integration, low cost, and rapid and instant detection. Although the flow pattern of fluids in microfluidic channels is usually laminar, the diffusion effect between fluids can be enhanced by designing serpentine or spiral curved channels to achieve rapid and thorough mixing and efficient mixing reactions. Flow magnetic separation in microchannels can achieve the movement of magnetic beads and non-magnetic materials with different magnetic contents in a magnetic field according to specific trajectories under different control boundaries. A high-throughput, high-magnetic-flux-density magnetic bead separation process is achieved during the magnetic field-flow field coupling process.

[0005] Combining multiple immunomagnetic beads with a flow magnetic sorting microfluidic chip can achieve magnetic bead encoding based on different magnetic contents and decoding based on a flow magnetic sorting microfluidic chip. Ultra-bright fluorescent microspheres are used as labels to improve fluorescence detection performance, and on-chip ultra-sensitive and rapid multiple protein quantitative detection can be developed. Compared with some current immunomagnetic microfluidic instant immunoassay products, it has overwhelming advantages in sensitivity, signal-to-noise ratio, convenience, simplicity, and reproducibility. For example, Chinese patents CN202111043290.4 and CN202211361964.X describe a microfluidic chip magnetic immunoassay system and analysis method. Although it can achieve highly sensitive optical immunoassay, it is still limited to the detection of a single protein. Chinese patents CN202210520133.6 and others disclose a multi-target quantitative detection method based on a microfluidic chip, which uses labeled microspheres of different sizes and colors and a machine vision algorithm to achieve multi-target detection on the same chip. Although the method is feasible, it has problems such as low signal-to-noise ratio and lack of sensitivity.

[0006] Most existing microfluidic detection chips focus on detecting a single biomarker and are unable to simultaneously detect multiple biomarkers in a sample. Currently available microfluidic platform technologies for quantitative multiplex protein detection do not utilize flow magnetic separation to achieve excess fluorescent marker removal and ultra-low background fluorescence. Furthermore, existing ultrasensitive detection methods for multiplex single-molecule proteins often utilize enzyme-linked immunosorbent assays to amplify the signal, which has the disadvantages of expensive reagents, difficult storage, and the need for isolation into independent small chambers.

[0007] Summary of the Invention

[0008] The present invention solves the problems of existing technology platforms such as high reagent consumption, insufficient mixing, complex and costly chips, need for improved sensitivity, and limited simultaneous detection of multiple targets, and provides a multiplex immune microfluidic detection platform and method based on flow magnetic sorting.

[0009] In one aspect, the present invention provides a flow magnetic sorting microfluidic chip, which includes an inlet, a mixing and incubation area, a washing and sorting area, a magnet, and an imaging and detection area; the inlet, the mixing and incubation area, the magnet, the washing and sorting area, and the imaging and detection area are integrated on the same microfluidic chip;

[0010] The injection port is used to inject the protein sample to be tested, the capture antibody-coated magnetic beads and the fluorescent-labeled detection antibody;

[0011] The mixing incubation zone is used to mix the protein sample to be tested, the capture antibody coated magnetic beads and the fluorescently labeled detection antibody to form sandwich immune complex magnetic beads containing the protein sample to be tested, the capture antibody coated magnetic beads and the fluorescently labeled detection antibody;

[0012] The washing and sorting zone is used to distribute the sandwich immune complex magnetic beads containing different protein samples to be tested to the sorting channel outlets in different imaging detection areas through dynamic magnetic sorting; the washing and sorting zone is connected to the mixing incubation zone through the magnetic bead inlet 4, and the washing and sorting zone includes a flow magnetic sorting channel 11 and a sandwich flow channel 10, the sandwich flow channel 10 connects the sandwich flow inlet 3 for injecting buffer and the flow magnetic sorting channel 11, the sandwich flow inlet 3 is used to inject buffer, the width of the sandwich flow channel 10 is greater than the width of the magnetic bead inlet 4, the sandwich flow channel 10 and the magnetic bead inlet 4 are connected to the flow magnetic sorting channel 11 in the same plane, the sandwich flow channel 10 is arranged on the side close to the permanent magnet 12, and the magnetic bead inlet 4 is arranged on the side away from the permanent magnet 12; when the sandwich immune complex magnetic beads enter through the magnetic bead inlet 4, the buffer injected by the sandwich flow inlet 3 causes the sandwich immune complex magnetic beads to focus near the side wall of the flow magnetic sorting channel, forming a focusing area with a width of 2-500 μm; the magnet is arranged on one side of the washing and sorting zone; and is arranged parallel to the flow magnetic sorting channel 11;

[0013] The imaging detection area is used to detect different samples obtained by sorting.

[0014] Furthermore, the injection port is connected to the mixing incubation zone, and the injection port includes at least one injection port 1 for a protein to be tested and at least one injection port 2 for magnetic beads coated with a capture antibody and a fluorescently labeled detection antibody;

[0015] Furthermore, the mixed incubation area includes a mixing flow channel 9 and a magnetic bead inlet 4, and the mixing flow channel 9 is arranged in a serpentine or spiral bending configuration, and the capture antibody-coated magnetic beads, the fluorescently labeled detection antibody and the protein to be tested can form sandwiched immunomagnetic beads in the mixing flow channel; the serpentine configuration is a serpentine winding configuration, the winding angle is 180 degrees, and at least 8-10 windings are set.

[0016] Furthermore, the width of the magnetic bead inlet 4 is smaller than the width of the mixing channel 9 .

[0017] Furthermore, the flowing magnetic separation channel 11 is a single channel, and the magnet is a permanent magnet, and the length of the permanent magnet is the same as the length of the flowing magnetic separation channel 11. Furthermore, the flowing magnetic separation channel 11 is rectangular or trapezoidal. When it is trapezoidal, the width near the starting end of the sandwich flow channel is greater than the width near the separation channel outlet. When it is rectangular, the width near the starting end of the sandwich flow channel is the same as the width near the separation channel outlet.

[0018] Furthermore, the imaging detection area is located at the end of the flowing magnetic sorting channel 11, and the imaging detection area includes 3-6 sorting channel outlets, which are connected to the flowing magnetic sorting channel 11 in the same plane and arranged in sequence from the side close to the permanent magnet 12 to the side away from the permanent magnet.

[0019] Another aspect of the present invention provides a multiplex immunoassay microfluidic system based on flow magnetic sorting, the multiplex immunoassay microfluidic system comprising a multiplex immunomagnetic bead preparation module, a flow magnetic sorting microfluidic chip system, an optical detection module and an image processing module;

[0020] The multiple immunomagnetic bead preparation module is used to prepare capture antibody-coated magnetic beads and fluorescent-labeled detection antibodies;

[0021] The flow magnetic separation microfluidic chip system comprises the above-mentioned flow magnetic separation microfluidic chip and auxiliary support components;

[0022] The optical detection module is used to detect and record the sample conditions at the outlet of the sorting channel of the flow magnetic sorting microfluidic chip;

[0023] The image processing module is used to process the image of the sample conditions at the outlet of the sorting channel obtained by the optical detection module, and to analyze and calculate the concentrations of the samples at the outlets of different sorting channels.

[0024] Furthermore, the auxiliary support component includes three programmable syringe pumps, a soft tubing, and an injection head, and the programmable syringe pump can provide a flow rate of 0.01-100 μL / min.

[0025] Furthermore, the optical detection module includes a fluorescence imaging system, which includes an excitation light source, an excitation light filter, an emission light filter, and a dichroic mirror. The fluorescence imaging system includes a fluorescence excitation light path and a fluorescence detection light path. Along the fluorescence excitation light path, the excitation light emitted by the light source device is incident on the imaging detection area of ​​the microfluidic chip in sequence through the convex mirror and the excitation light filter; along the fluorescence detection light path, the fluorescence generated by the excitation light in the imaging detection area of ​​the microfluidic chip is transmitted in sequence through the emission light filter and the path to the charge-coupled device (CCD) camera; the fluorescence excitation light path is combined with the emission light path after passing through the excitation light filter by the dichroic mirror and introduced into the fluorescence excitation light path.

[0026] Furthermore, the image processing module includes a memory, a processor, and a computer program stored in the memory and executable on the processor; when executing the computer program, the processor utilizes a machine learning algorithm to splice and analyze the charge-coupled device image, provides an image and protein concentration analysis model, and outputs a recognition result; and simultaneously calculates the probability density of different luminescent microbeads according to the Poisson distribution probability formula = -ln(1-number of luminescent microbeads / total number of microbeads), thereby calculating the concentration value of the object being measured.

[0027] Another embodiment of the present invention provides a multiplex protein detection method, wherein the multiplex protein detection method is to use the above-mentioned multiplex immunoassay microfluidic system for detection.

[0028] Alternatively, the multiple protein detection method comprises the following steps:

[0029] S001) Preparing capture antibody-coated magnetic beads and fluorescently labeled detection antibodies: preparing multiple groups of capture antibody-coated magnetic beads and fluorescently labeled detection antibodies according to the multiple proteins to be detected, wherein the capture antibody and detection antibody in each group can respectively specifically bind to one protein in the multiple proteins, the magnetic beads and fluorescent labels in each group are the same, and the magnetic beads and fluorescent labels in different groups are different; the different magnetic beads have different magnetic bead particle sizes or different magnetic contents, or different magnetic bead particle sizes and magnetic contents, and the emission wavelengths of the fluorescent labels in different groups are different;

[0030] S002) preparing a mixed solution of capture antibody-coated magnetic beads and fluorescently labeled detection antibody with capture antibody-coated magnetic beads and fluorescently labeled detection antibody, wherein the concentration of the fluorescently labeled detection antibody in the mixed solution is higher than the concentration of the capture antibody-coated magnetic beads, as measured by the molar concentration of the detection antibody in the fluorescently labeled detection antibody and the molar concentration of the capture antibody in the capture antibody-coated magnetic beads;

[0031] S003) obtaining the above-mentioned flow magnetic separation microfluidic chip;

[0032] S004) injecting the multiple protein samples to be detected into the flow magnetic sorting microfluidic chip through the protein injection port 1 to be detected, and injecting the mixed solution obtained in step S002) into the flow magnetic sorting microfluidic chip through the capture antibody coated magnetic beads and the fluorescent labeled detection antibody injection port 2, and the multiple protein samples to be detected and the mixed solution are mixed in the mixing flow channel 9 of the flow magnetic sorting microfluidic chip to form sandwich immune complex magnetic beads, and enter the sandwich flow channel 10 through the magnetic bead inlet 4; the buffer solution is injected into the flow magnetic sorting microfluidic chip through the sandwich flow inlet 3, and mixed with the fluid injected by the magnetic bead inlet 4 through the sandwich flow channel 10, At the initial stage of the washing and sorting zone, a sheath flow is formed outside the fluid injected into the magnetic bead inlet 4, so that the sandwich immune complex magnetic beads and excess fluorescent-labeled detection antibodies in the fluid injected into the magnetic bead inlet 4 are confined to the wall of the flowing magnetic sorting channel 11 away from the permanent magnet. With the continuous injection of the buffer solution, the magnetic beads in the sandwich immune complex magnetic beads are also affected by the magnetic field force. While moving toward the sorting channel outlet, they will produce different degrees of deviation toward the permanent magnet 12 and enter different sorting channel outlets, while the non-magnetic particles continue to move horizontally along the wall to the sorting channel outlet farthest from the permanent magnet.

[0033] S005) using a fluorescence imaging system to detect at the exit of the sorting channel, observing the fluorescent markers at the exits of different sorting channels, and collecting images to achieve qualitative detection;

[0034] Optionally, S006) uses an image processing module to analyze the image collected in step S005) to obtain the concentration value of the object being measured.

[0035] Furthermore, the multiplex protein detection method can be a qualitative detection or a quantitative detection.

[0036] Furthermore, the fluorescent label is selected from any one or more of fluorescein, quantum dots, rare earth elements, rare earth chelates, fluorescent proteins or upconversion nanoparticles. Beneficial effects

[0037] This invention proposes a multiplexed immunomicrofluidic detection platform and method based on flow magnetic sorting. The platform primarily comprises a multiplexed immunomagnetic bead reagent system, a modular microfluidic chip, and an imaging detection system. The platform is used for the injection, mixing, capture, sorting, and detection of biological samples, specific immunomagnetic beads, and multiple fluorescent markers. This method utilizes encoding of magnetic beads based on varying magnetic content and decoding using a flow magnetic sorting microfluidic chip to develop on-chip, ultrasensitive, and rapid multiplexed protein quantitative detection. Through magnetic field-flow coupling, flow magnetic sorting enables high-throughput sorting of magnetic beads of varying magnetic content and non-magnetic markers within a microfluidic channel.

[0038] Second, the multiplexed immune microfluidic detection platform of the present invention enables single-molecule separation and detection. When injecting capture antibody-coated magnetic beads and fluorescently labeled detection antibodies into the microfluidic chip, if the capture antibody-coated magnetic beads are at least 10 times the predicted concentration of the protein to be detected, each magnetic bead can capture only one protein to be detected. This enables single-protein detection when observed at the exit of the sorting channel using a fluorescent imaging system. The concentration of the protein to be detected can then be calculated using the image processing module's recognition and analysis.

[0039] Secondly, the solution of the present invention uses ultra-bright fluorescent microspheres as fluorescent markers, which greatly improves the fluorescence detection performance.

[0040] In addition, optical microscopy can be well integrated with the microfluidic platform, allowing microscopic images to be analyzed by machine learning algorithms and intuitively output recognition results.

[0041] This platform has the advantages of high detection sensitivity, short detection time, high signal-to-noise ratio, simple and convenient operation, and low cost. It is particularly suitable for ultra-sensitive detection of multiple single-molecule proteins and has potential economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is a schematic diagram of the construction of multiple sandwich immunomagnetic beads.

[0043] Figure 2 is a schematic diagram of a multiplexed immune microfluidic chip based on flow magnetic sorting.

[0044] Figure 3 is a schematic diagram of the mask structure of a rectangular microfluidic chip for mixing and flow magnetic sorting. Among them, 1 is the inlet for the multi-target protein to be tested, 2 is the inlet for the capture antibody-coated magnetic beads and the fluorescent-labeled detection antibody, 3 is the clamp inlet, 4 is the inlet for the multiple immunomagnetic beads, 5 is the outlet for the first sorting channel, 6 is the outlet for the second sorting channel, 7 is the outlet for the third sorting channel, 8 is the outlet for the fourth sorting channel, 9 is the mixing channel, 10 is the clamp channel, 11 is the flow magnetic sorting channel, and 12 is the permanent magnet.

[0045] Figure 4 shows fluorescence imaging of a single fluorescent microsphere.

[0046] Figure 5 is a schematic diagram of the microfluidic chip fluorescence imaging system.

[0047] Figure 6 is a physical picture of the effects of each stage of flow magnetic separation.

[0048] Figure 7 is a diagram showing the washing effect of flow magnetic separation of blank samples.

[0049] FIG8 is a fluorescence imaging diagram of different concentrations of immunomagnetic beads after algorithm processing.

[0050] Figure 9 is a schematic diagram of the mask structure of a trapezoidal microfluidic chip for mixing and flow magnetic sorting. Among them, 1 is the inlet for the multi-target protein to be tested, 2 is the inlet for capture antibody-coated magnetic beads and fluorescently labeled detection antibodies, 3 is the clamp inlet, 4 is the inlet for multiple immunomagnetic beads, 5 is the outlet for the first sorting channel, 6 is the outlet for the second sorting channel, 7 is the outlet for the third sorting channel, 8 is the outlet for the fourth sorting channel, 9 is the mixing channel, 10 is the clamp channel, 11 is the flow magnetic sorting channel, and 12 is the permanent magnet. DETAILED DESCRIPTION

[0051] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0052] The present invention discloses a multiplex immunoassay microfluidic platform based on flow magnetic sorting and a method thereof. The platform mainly includes multiplex immunomagnetic bead preparation, magnetic sorting microfluidic chip and imaging detection system, which are used for the injection, mixing, incubation, washing, sorting and detection of biological samples to be detected, specific immunomagnetic beads and multiple fluorescent markers.

[0053] The following is a detailed description of the design method of a microfluidic platform for multiplex immunoassays based on flow magnetic sorting, as follows:

[0054] S1) Preparation of multiple immunomagnetic beads: As shown in Figure 1, capture antibody-coated magnetic beads and fluorescently labeled detection antibodies are designed and prepared. For single-molecule protein detection, it is necessary to ensure that each magnetic bead binds to at most one single-molecule protein and to increase the luminescence intensity of the fluorescent marker. This is generally achieved by following a Poisson distribution when the ratio of the number of magnetic beads to the number of protein molecules is greater than 10. It mainly includes carboxylated fluorescent microspheres activated by carbodiimide-N-hydroxysuccinimide (EDC-NHS) to couple the detection antibody and streptavidin magnetic beads to couple the biotinylated capture antibody. After bovine serum albumin (BSA) blocks the immune active site, it combines with the target protein in the sample to be tested to form an immune complex magnetic bead, as follows:

[0055] S11) Design and prepare capture antibody-coated magnetic beads: prepare antibody-coated magnetic beads outside the magnetic sorting microfluidic chip. Estimate the molar number of the protein to be tested in the sample, use at least 10 times the number of streptavidin magnetic beads coupled with biotinylated capture antibodies, and obtain capture antibody-coated magnetic beads. According to the Poisson distribution, a single microbead carries at most a single protein at this ratio. The magnetic beads contain at least two or more types, for example, 3, 4 or 5 types. At the same time, each magnetic bead has a different magnetic content or a different particle size with the same magnetic content. The capture antibody contained on each magnetic bead is the same, while the capture antibodies contained on different types of magnetic beads are different. The capture antibody can specifically bind to the target protein to be tested.

[0056] S12) Prepare fluorescently labeled detection antibodies: Use ultra-bright fluorescent microspheres such as quantum dot balls as fluorescent labels, couple detection antibodies, and form fluorescently labeled detection antibodies. Fluorescent microspheres have different detection wavelengths, and there are at least two or more fluorescent microspheres with different wavelengths. Fluorescent microspheres of each wavelength are coupled with the same type of detection antibody, and different types of fluorescent microspheres are coupled with different types of detection antibodies. The detection antibody is an antibody that can specifically bind to the target protein to be detected. Figure 4 is a fluorescent imaging image of a single ultra-bright fluorescent microsphere with a particle size of 120nm. A single bright microsphere can be clearly seen under a microscope. Based on the high brightness of a single fluorescent microsphere, it is possible to discrete immunomagnetic beads and realize accurate identification of single-molecule protein signals. The method of coupling ultra-bright fluorescent microspheres to detection antibodies can be achieved by any conventional means in the art, for example, using carboxylated ultra-bright fluorescent microspheres and coupling them with detection antibodies after activation with EDC-NHS.

[0057] The number of types of the antibody-coated magnetic beads is the same as the number of types of the fluorescent-labeled detection antibodies.

[0058] S2) Design and fabricate a magnetic separation microfluidic chip.

[0059] S21) Design a magnetic separation microfluidic chip: The magnetic separation microfluidic chip includes an inlet, a mixing and incubation area, a washing and sorting area, a magnet, and an imaging detection area. The inlet, mixing and incubation area, magnet, washing and sorting area, and imaging detection area are integrated on a single microfluidic chip.

[0060] The injection port is connected to the mixing incubation area, and includes at least one protein injection port 1 to be tested and at least one capture antibody coated magnetic beads and fluorescent labeled detection antibody injection port 2, wherein the fluorescent labeled detection antibody is in excess relative to the capture antibody coated magnetic beads.

[0061] The mixed incubation area includes a mixing flow channel 9 and a magnetic bead inlet 4. The mixing flow channel 9 is arranged in a serpentine or spiral bending configuration. The capture antibody-coated magnetic beads, the fluorescently labeled detection antibody and the protein to be tested can form sandwiched immunomagnetic beads in the mixing flow channel. The serpentine configuration is a serpentine circuitous configuration with a circuitous angle of 180 degrees, and at least 8-10 circuitous configurations are provided. The serpentine circuitous configuration can enhance the diffusion effect between the fluids, thereby achieving rapid and thorough mixing and efficient mixing reactions of specific capture antibody magnetic beads, test samples and multiple fluorescently labeled detection antibodies to form sandwiched immunomagnetic beads. The magnetic bead inlet 4 is used to connect the washing and sorting zone and is arranged on the side of the starting end of the flow magnetic sorting channel 11 away from the permanent magnet 12. The width of the magnetic bead inlet 4 is lower than the width of the mixing flow channel.

[0062] The washing and sorting zone is connected to the mixing and incubation zone through the magnetic bead inlet 4. The washing and sorting zone also includes a sandwich flow channel 10, which connects the sandwich flow inlet 3 for injecting buffer and the flow magnetic sorting channel 11. The width of the sandwich flow channel 10 is greater than the width of the magnetic bead inlet 4. The sandwich flow channel 10 and the magnetic bead inlet 4 are connected to the flow magnetic sorting channel 11 in the same plane. The sandwich flow channel 10 is arranged on the side close to the permanent magnet 12, while the magnetic bead inlet 4 is arranged on the side away from the permanent magnet 12. When the sandwich immune complex magnetic beads enter through the magnetic bead inlet 4, the buffer injected from the sandwich flow inlet 3 causes the sandwich immune complex magnetic beads to focus near the side wall of the flow magnetic sorting channel, forming a focusing area with a width of 2-500 μm.

[0063] The flowing magnetic separation channel 11 is used to separate different magnetic beads and non-magnetic signal probes that are not involved in the reaction, causing them to form different displacements across the width of the channel and enter different separation channel exits. The flowing magnetic separation channel 11 is a single channel, with a permanent magnet 12 positioned on one side of the channel 11, parallel to the channel 11, and having the same length as the channel 11.

[0064] As the magnetic beads flow through the magnetic separation channel 11, they experience a magnetic field perpendicular to the flow direction and a gradient magnetic field. The magnetic force is proportional to the volume of the beads. Larger beads experience a greater magnetic force, resulting in more significant lateral displacement. Smaller beads experience less force and therefore less lateral displacement. Non-magnetic materials (excessive amounts of multiple fluorescent markers) are not affected by the magnetic force and do not experience lateral displacement. This displacement difference allows beads of different sizes to move along specific trajectories within the channel under different control boundaries and be diverted to different separation outlets.

[0065] The imaging detection area is located at the end of the flowing magnetic sorting channel 11. The imaging detection area includes four sorting channel outlets 5, 6, 7, and 8. According to actual needs, 3, 5, or 6 can also be set. The sorting channel outlets are connected to the flowing magnetic sorting channel 11 in the same plane and are arranged in sequence from the side close to the permanent magnet 12 to the side away from the permanent magnet.

[0066] The channels of the mixed incubation area, the washing and sorting area, and the imaging and detection area have the same height.

[0067] The magnetic sorting microfluidic chip is designed to meet the washing and sorting function requirements of the flow magnetic sorting unit by combining permanent magnets and magnetic beads with different magnetic contents. The flow magnetic sorting in the microchannel can realize multiple immunomagnetic beads with different magnetic contents in the magnetic field. In the process of magnetic field-flow field coupling, the magnetic beads in the microchannel can be sorted out in the vertical direction, and the magnetic beads can be prevented from adhering to the wall in the horizontal direction, and the microchannel will not be blocked, thereby realizing a high-throughput multiple immunomagnetic bead sorting process.

[0068] S22) Preparation of magnetic separation microfluidic chip: According to the design of step S21), the microfluidic chip can be made by using microelectromechanical system (MEMS) technology, soft lithography method, 3D printing, injection molding or embossing.

[0069] Taking soft lithography as an example, the preparation process is introduced: lithography technology includes the steps of pretreatment, spin coating of photoresist, soft baking, exposure, post-baking and development.

[0070] S3) Build a microfluidic chip fluorescence imaging system. The fluorescence imaging system is shown in Figure 5. The fluorescence imaging system can achieve on-chip ultra-sensitive detection, especially single-molecule level immunoassay. The fluorescence imaging system includes an excitation light source, an excitation light filter, an emission light filter, and a dichroic mirror. The fluorescence imaging system includes a fluorescence excitation light path and a fluorescence detection light path. Along the fluorescence excitation light path, the excitation light emitted by the light source device is incident on the imaging detection area placed on the microfluidic chip in sequence through a convex mirror and an excitation light filter; along the fluorescence detection light path, the fluorescence generated by the excitation light in the imaging detection area of ​​the microfluidic chip is sequentially transmitted through an emission light filter and a path to a charge-coupled device (CCD) camera; the fluorescence excitation light path is combined with the emission light path after passing through the excitation light filter by a dichroic mirror and introduced into the fluorescence excitation light path. The dichroic mirror, excitation light filter, and emission light filter are adapted to the fluorescent microsphere label in the fluorescent-labeled detection antibody. The above-mentioned fluorescence imaging system can obtain bright-field magnetic beads and dark-field fluorescence imaging images of the immune magnetic beads in the detection imaging area of ​​the microfluidic chip after sorting.

[0071] In some specific embodiments, a cooled high-sensitivity CCD is used to achieve ultrasensitive detection.

[0072] In some specific embodiments, the fluorescence imaging system uses a 40x or greater objective lens.

[0073] S4) Further, to achieve adaptive protein detection and analysis, the CCD images are combined and analyzed using a machine learning algorithm, providing an image and protein concentration analysis model and intuitively outputting the recognition results. The probability density of the luminescent beads is calculated according to the Poisson distribution probability formula = -ln(1-number of luminescent beads / total number of beads), thereby obtaining the concentration value of the measured object.

[0074] The present invention also discloses an alternative improvement scheme for the magnetic sorting microfluidic chip, as shown in Figure 9. Unlike the above-mentioned chip, this alternative improvement scheme is provided with a trapezoidal flow magnetic sorting channel 11 and four outlets 5, 6, 7, and 8. The trapezoidal structure design can reduce the channel width. The purpose of this is to allow the magnetic beads flowing through to be closer to a stronger magnetic field. When the magnetic beads enter the chip from the magnetic bead inlet 4, as the trapezoidal channel gradually narrows, even the magnetic beads with weaker magnetism will be forced to move toward the permanent magnet 12 and be subjected to a stronger magnetic force, causing them to have a significant lateral displacement. This innovative design improves the sorting resolution and efficiency of magnetic beads with lower magnetism.

[0075] During the sorting process, the buffer injected through the sandwich flow inlet 3 focuses the sandwich immune complex magnetic beads near the sidewalls of the flow magnetic sorting channel, with a focus width controlled between 2 and 500 μm. As the beads flow through channel 11, even small beads with low yields are subjected to sufficient magnetic force for effective sorting. Because the magnetic force is proportional to the volume of the beads, large beads experience greater magnetic force, resulting in greater lateral displacement and a rapid change in their trajectory. Small beads, on the other hand, initially experience less force and smaller lateral displacement. However, as the trapezoidal structure approaches, they gradually move into the high magnetic field region, where their trajectory begins to change, eventually shifting laterally and deviating from their original trajectory. This design allows beads of different sizes to be diverted to corresponding sorting outlets at the channel outlet based on their displacement differences. For example, as shown in FIG9 , large beads will enter the first sorting outlet 5, while intermediate and small beads will be directed to the second and third sorting outlets 6 and 7, respectively. Unlabeled magnetic signal probes, unaffected by the magnetic field, travel directly to the fourth sorting outlet 8 and into the waste channel. This integrated process achieves both washing to remove background signals and effective magnetic separation of magnetic beads in the same channel, ensuring precise differentiation of magnetic beads of different sizes.

[0076] Based on the above design, the present invention provides a flow magnetic sorting microfluidic chip, which includes an inlet, a mixing and incubation area, a washing and sorting area, a magnet, and an imaging detection area; the inlet, mixing and incubation area, the magnet, washing and sorting area, and the imaging detection area are integrated on the same microfluidic chip;

[0077] The injection port is connected to the mixing incubation area, and the injection port includes at least one protein injection port 1 to be tested and at least one capture antibody coated magnetic beads and fluorescent labeled detection antibody injection port 2;

[0078] The mixing and incubation zone includes a mixing channel 9 and a magnetic bead inlet 4. The mixing channel 9 is arranged in a serpentine or spiral shape. The capture antibody-coated magnetic beads, the fluorescently labeled detection antibody, and the test protein can form a sandwich of immunomagnetic beads within the mixing channel. The serpentine shape is a serpentine arrangement with a winding angle of 180 degrees and at least 8-10 windings. The width of the magnetic bead inlet 4 is less than the width of the mixing channel 9.

[0079] The washing and sorting zone is connected to the mixing and incubation zone through the magnetic bead inlet 4. The washing and sorting zone includes a flow magnetic sorting channel 11 and a sandwich flow channel 10. The sandwich flow channel 10 connects the sandwich flow inlet 3 for injecting buffer and the flow magnetic sorting channel 11. The sandwich flow inlet 3 is used to inject buffer. The width of the sandwich flow channel 10 is greater than the width of the magnetic bead inlet 4. The sandwich flow channel 10 and the magnetic bead inlet 4 are connected to the flow magnetic sorting channel 11 in the same plane. The sandwich flow channel 10 is arranged on the side close to the permanent magnet 12, while the magnetic bead inlet 4 is arranged on the side away from the permanent magnet 12. When the sandwich immune complex magnetic beads enter through the magnetic bead inlet 4, the buffer injected from the sandwich flow inlet 3 causes the sandwich immune complex magnetic beads to focus near the side wall of the flow magnetic sorting channel, forming a focusing area 2-500 μm wide.

[0080] The flowing magnetic separation channel 11 is a single channel. A permanent magnet 12 is provided on one side of the flowing magnetic separation channel 11 . The permanent magnet 12 is provided parallel to the flowing magnetic separation channel 11 , and the length of the permanent magnet 12 is the same as that of the flowing magnetic separation channel 11 .

[0081] The flow magnetic separation channel 11 is rectangular or trapezoidal. When it is trapezoidal, the width near the starting end of the clamp flow channel is greater than the width near the outlet of the separation channel. When it is rectangular, the width near the starting end of the clamp flow channel is the same as the width near the outlet of the separation channel.

[0082] The imaging detection area is located at the end of the flowing magnetic sorting channel 11. The imaging detection area includes 3-6 sorting channel outlets, which are connected to the flowing magnetic sorting channel 11 in the same plane and are arranged in sequence from the side close to the permanent magnet 12 to the side away from the permanent magnet.

[0083] Based on the above design scheme, the present invention also provides a multiplex immunoassay microfluidic system based on flow magnetic sorting, which includes a multiplex immunomagnetic bead preparation module, a flow magnetic sorting microfluidic chip system, an optical detection module, and an image processing module.

[0084] The multiple immunomagnetic bead preparation module is used to prepare capture antibody-coated magnetic beads and fluorescently labeled detection antibodies.

[0085] The flow magnetic separation microfluidic chip system includes a flow magnetic separation microfluidic chip and auxiliary support components.

[0086] The flow magnetic sorting microfluidic chip includes an injection port, a mixing and incubation area, a washing and sorting area, a magnet, and an imaging and detection area; the injection port, the mixing and incubation area, the magnet, the washing and sorting area, and the imaging and detection area are integrated on the same microfluidic chip;

[0087] The injection port is connected to the mixing incubation area, and the injection port includes at least one protein injection port 1 to be tested and at least one capture antibody coated magnetic beads and fluorescent labeled detection antibody injection port 2;

[0088] The mixing and incubation zone includes a mixing channel 9 and a magnetic bead inlet 4. The mixing channel 9 is arranged in a serpentine or spiral shape. The capture antibody-coated magnetic beads, the fluorescently labeled detection antibody, and the test protein can form a sandwich of immunomagnetic beads within the mixing channel. The serpentine shape is a serpentine arrangement with a winding angle of 180 degrees and at least 8-10 windings. The width of the magnetic bead inlet 4 is less than the width of the mixing channel 9.

[0089] The washing and sorting zone is connected to the mixing and incubation zone via a magnetic bead inlet 4. The washing and sorting zone includes a flow magnetic sorting channel 11 and a sandwich flow channel 10. The sandwich flow channel 10 connects the sandwich flow inlet 3 for injecting buffer and the flow magnetic sorting channel 11. The sandwich flow inlet 3 is used to inject buffer. The width of the sandwich flow channel 10 is greater than the width of the magnetic bead inlet 4. The sandwich flow channel 10 and the magnetic bead inlet 4 are connected to the flow magnetic sorting channel 11 in the same plane. The sandwich flow channel 10 is located near the permanent magnet 12, while the magnetic bead inlet 4 is located away from the permanent magnet 12. When the sandwich immune complex magnetic beads enter through the magnetic bead inlet 4, the buffer injected into the sandwich flow inlet 3 causes the magnetic beads to focus near the channel sidewalls, forming a focusing area 2-500 microns wide.

[0090] The flowing magnetic separation channel 11 is a single channel. A permanent magnet 12 is provided on one side of the flowing magnetic separation channel 11 . The permanent magnet 12 is provided parallel to the flowing magnetic separation channel 11 , and the length of the permanent magnet 12 is the same as that of the flowing magnetic separation channel 11 .

[0091] The flow magnetic separation channel 11 is rectangular or trapezoidal. When it is trapezoidal, the width near the starting end of the clamp flow channel is greater than the width near the outlet of the separation channel. When it is rectangular, the width near the starting end of the clamp flow channel is the same as the width near the outlet of the separation channel.

[0092] The imaging detection area is located at the end of the flowing magnetic sorting channel 11. The imaging detection area includes 3-6 sorting channel outlets, which are connected to the flowing magnetic sorting channel 11 in the same plane and are arranged in sequence from the side close to the permanent magnet 12 to the side away from the permanent magnet.

[0093] The auxiliary support components include three programmable syringe pumps and a microtubule. The programmable syringe pump can provide a flow rate of 0.01-100 μL / min. The optical detection module includes a fluorescence imaging system, which includes an excitation light source, an excitation light filter, an emission light filter and a dichroic mirror. The fluorescence imaging system includes a fluorescence excitation light path and a fluorescence detection light path. Along the fluorescence excitation light path, the excitation light emitted by the light source device is incident on the imaging detection area placed on the microfluidic chip through the convex mirror and the excitation light filter in turn; along the fluorescence detection light path, the fluorescence generated by the excitation light in the imaging detection area of ​​the microfluidic chip is transmitted to the charge-coupled device CCD camera in turn through the emission light filter and the path; the fluorescence excitation light path is combined with the emission light path after passing through the excitation light filter by a dichroic mirror and introduced into the fluorescence excitation light path.

[0094] The image processing module includes a memory, a processor, and a computer program stored in the memory and executable on the processor. When executing the computer program, the processor utilizes a machine learning algorithm to stitch and analyze charge-coupled device images, provides an image and protein concentration analysis model, and outputs a recognition result. Simultaneously, the probability density of different luminescent microbeads is calculated according to the Poisson distribution probability formula = -ln(1-number of luminescent microbeads / total number of microbeads), thereby calculating the concentration value of the measured object.

[0095] Based on the above design scheme, the present invention provides a method for performing multiple protein detection using the above multiple immunoassay microfluidic system.

[0096] The multiplex protein detection method comprises the following steps:

[0097] S001) Preparing capture antibody-coated magnetic beads and fluorescently labeled detection antibodies: preparing multiple groups of capture antibody-coated magnetic beads and fluorescently labeled detection antibodies according to the multiple proteins to be detected, wherein the capture antibody and detection antibody in each group can respectively specifically bind to one protein in the multiple proteins, the magnetic beads and fluorescent labels in each group are the same, and the magnetic beads and fluorescent labels in different groups are different; different magnetic beads have different magnetic bead particle sizes or different magnetic contents or different magnetic bead particle sizes and magnetic contents, and the emission wavelengths of the fluorescent labels in different groups are different; the fluorescent labels are selected from any one or more of fluorescein, quantum dots, rare earth elements, rare earth chelates, fluorescent proteins or upconversion nanoparticles.

[0098] S002) preparing a mixed solution of capture antibody-coated magnetic beads and fluorescently labeled detection antibody with capture antibody-coated magnetic beads and fluorescently labeled detection antibody, wherein the concentration of the fluorescently labeled detection antibody in the mixed solution is higher than the concentration of the capture antibody-coated magnetic beads, as measured by the molar concentration of the detection antibody in the fluorescently labeled detection antibody and the molar concentration of the capture antibody in the capture antibody-coated magnetic beads;

[0099] S003) obtaining the above-mentioned flow magnetic separation microfluidic chip;

[0100] S004) injecting the multiple protein samples to be detected into the flow magnetic sorting microfluidic chip through the protein injection port 1 to be detected, and injecting the mixed solution obtained in step S002) into the flow magnetic sorting microfluidic chip through the capture antibody coated magnetic beads and the fluorescent labeled detection antibody injection port 2, and the multiple protein samples to be detected and the mixed solution are mixed in the mixing flow channel 9 of the flow magnetic sorting microfluidic chip to form sandwich immune complex magnetic beads, and enter the sandwich flow channel 10 through the magnetic bead inlet 4; the buffer solution is injected into the flow magnetic sorting microfluidic chip through the sandwich flow inlet 3, and mixed with the fluid injected by the magnetic bead inlet 4 through the sandwich flow channel 10, At the initial stage of the washing and sorting zone, a sheath flow is formed outside the fluid injected into the magnetic bead inlet 4, so that the sandwich immune complex magnetic beads and excess fluorescent-labeled detection antibodies in the fluid injected into the magnetic bead inlet 4 are confined to the wall of the flowing magnetic sorting channel 11 away from the permanent magnet. With the continuous injection of the buffer solution, the magnetic beads in the sandwich immune complex magnetic beads are also affected by the magnetic field force. While moving toward the sorting channel outlet, they will produce different degrees of deviation toward the permanent magnet 12 and enter different sorting channel outlets, while the non-magnetic particles continue to move horizontally along the wall to the sorting channel outlet farthest from the permanent magnet.

[0101] In some specific technical solutions, step S004) further includes injecting air through the test protein inlet 1 and the capture antibody coated magnetic beads and fluorescently labeled detection antibody inlet 2 after injecting the test protein, capture antibody coated magnetic beads and fluorescently labeled detection antibody to ensure that all the test protein, capture antibody coated magnetic beads and fluorescently labeled detection antibody enter the flow magnetic separation channel 11 through the magnetic bead inlet 4.

[0102] S005) using a fluorescence imaging system to detect at the exit of the sorting channel, observing the fluorescent markers at the exits of different sorting channels, and collecting images to achieve qualitative detection;

[0103] Optionally, S006) uses an image processing module to analyze the image collected in step S005) to obtain the concentration value of the object being measured.

[0104] In the scheme of the present invention, the multiple protein detection method can be a qualitative detection or a quantitative detection.

[0105] In some specific embodiments of the present invention, as shown in Figures 1 and 2, a schematic diagram of the construction of multiple sandwich immunomagnetic beads is shown, which mainly includes the following steps: 1) Estimate the molar number of the protein to be detected in the sample, use at least 10 times (based on Poisson distribution, at this ratio, a single magnetic bead can carry at most a single protein) a number of streptavidin-modified magnetic beads as carriers to fully react and incubate with the capture antibody outside the chip, and the particle size of the magnetic beads is 1-5μm; obtain capture antibody-coated magnetic beads. 2) Prepare N-hydroxysuccinimide (NHS) with phosphate buffer (0.01M, pH 7.4), and activate the carboxyl group with a mixed solution of EDC-NHS (40mM EDC, 10mM NHS) with carbodiimide (EDC), use fluorescent microspheres as luminescent reporter molecules, bind to the detection antibody, and incubate at 37°C for 15 minutes; obtain fluorescent-labeled detection antibody. 3) Using 3-5% bovine serum albumin (BSA) solution to block the immune active sites of the capture antibody-coated magnetic beads and the fluorescently labeled detection antibody; 4) Preparing the fluorescently labeled detection antibody and the capture antibody-coated magnetic beads for injection, and constructing sandwich immunomagnetic beads with the protein to be tested in the sample.

[0106] In some specific embodiments of the present invention, a photolithography method is used to prepare a microfluidic chip, specifically including the steps of pretreatment, spin coating of photoresist, soft baking, exposure, post-baking, and development. Pretreatment is used to change the surface properties of the silicon wafer so that it can adhere firmly to the photoresist. The main method is to heat the silicon wafer to 120°C in a closed oven and then perform plasma treatment for 5 minutes to remove moisture and form a hydrophilic bond surface. SU-8 3025 photoresist is spin-coated at a speed of 4000 rpm and a spin coating time of 35 seconds to obtain a 20μm thick, uniform and stable photoresist. After the spin coating is completed, the photoresist is first dried and fixed to remove the solvent in the photoresist, pre-baked at 65°C for 5 minutes, and then adjusted to 95°C for 15 minutes; exposure is performed, the exposure time is 60 seconds, and the exposure dose is: 150-215mJ / cm 2; After exposure, the sample is placed on a drying table for post-baking treatment: first, the temperature is adjusted to 65°C for 1 minute, and then adjusted to 95°C and maintained for 7 minutes. Finally, the sample is developed by immersing it in SU-8 developer, developing it at room temperature, and then washing away the developer with ethanol to complete the production of the complementary master. The microfluidic channel pattern on the master is copied to the PDMS by molding. After obtaining the PDMS microchannel substrate, the PDMS microchannel substrate and the SU-8 microwell substrate are first cleaned with isopropyl alcohol for 40 to 50 seconds to remove any organic matter and impurity particles that may exist on the surface of the SU-8 substrate and the PDMS substrate, and then washed with deionized water for 1 to 2 minutes to remove any residual isopropyl alcohol and impurity particles; blow dry with clean air or nitrogen. Next, seven holes are drilled: inlet 1 for the multi-target protein sample, inlet 2 for capture antibody-coated magnetic beads and fluorescently labeled detection antibodies, inlet 3 for the clamp flow, and outlets 5, 6, 7, and 8 for the first, second, third, and fourth sorting channels. The microfluidic chip is then treated with oxygen plasma for 40 to 60 seconds at a power of 200 to 400 W to modify the PDMS bonding surface from hydrophobic to hydrophilic. Finally, a clean thin glass sheet is press-bonded to the PDMS and baked to form a complete microfluidic chip.

[0107] In some specific embodiments of the present invention, as shown in FIG3 , a schematic diagram of the structure of a rectangular microfluidic chip mask for mixing and flow magnetic sorting is shown, including a mixing channel, a magnetic sorting channel, two sample inlets (including a sandwich inlet and a magnetic bead inlet), and four outlets. When the sandwich immune complex magnetic beads enter through the magnetic bead inlet 4, the buffer injected into the sandwich inlet 3 causes the magnetic beads to focus near the side wall of the channel, forming a focusing area 2-500 microns wide. As the magnetic beads flow in the magnetic sorting channel 11, they experience a magnetic field and a gradient magnetic field perpendicular to the flow direction. The magnetic force is proportional to the volume of the magnetic beads. Large-sized magnetic beads are subjected to a larger magnetic force, so the lateral displacement is more significant; while small-sized magnetic beads are subjected to less force and the lateral displacement is also small. This displacement difference causes magnetic beads of different sizes to be diverted to different sorting outlets at the channel outlet. As shown in Figure 3, large-sized magnetic beads with high magnetic content (e.g., 5 microns) enter the first sorting outlet 5, intermediate-sized magnetic beads with medium magnetic content (e.g., 2.8 microns) enter the second sorting outlet 6, and small-sized micron-sized magnetic beads with low magnetic content (e.g., 1 micron) enter the third sorting outlet 7. Non-magnetic signal probes that do not participate in the reaction, due to their unchanged direction of movement, are directed to the fourth sorting outlet 8 and into the waste channel. This design enables simultaneous washing to remove background signals and effective magnetic sorting of magnetic beads of different sizes in the same channel.

[0108] In some specific embodiments of the present invention, as shown in Figure 6, it is a physical picture of the effects of each stage of flow magnetic sorting in a microfluidic chip. Taking 2.8μm magnetic beads as an example, at the initial stage of the washing and sorting zone, due to the sheath flow, the magnetic particles and the non-magnetic excess fluorescent-labeled detection antibodies are all confined near the wall (A in Figure 6); as the particles move in the horizontal direction, they are affected by the magnetic field force in the vertical direction, and the magnetic particles form pearl-like chains (B in Figure 6); at the same time, due to the shear force of the flow, the micron-sized magnetic particles gradually become a single discrete particle distribution (C in Figure 6), while the non-magnetic particles continue to move horizontally along the wall to chamber 8 in Figure 3, so the immunomagnetic beads are fully washed and the background signal is controlled at an extremely low level. Immunomagnetic beads of different sizes are also offset to different vertical positions due to the different magnitudes of the magnetic field force, and finally enter chambers 5-7 respectively.

[0109] In some specific embodiments of the present invention, a blank sample without the protein to be tested, such as a PBS solution, is used for testing, and the magnetic separation microfluidic chip and the magnetic rack of the present invention are used for separation, respectively. The experimental results show that the magnetic separation microfluidic chip has a lower background signal than the magnetic rack, and the low noise brings a higher signal-to-noise ratio. As shown in Figure 7, B in Figure 7 is an exemplary detection image of the sorting channel outlet of the blank sample flow magnetic separation microfluidic chip close to the permanent magnet. A in Figure 7 is the effect of washing the same sample with a magnetic rack. Among them, the blue dots represent the capture antibody-coated magnetic beads that are not bound to the protein to be tested, the green dots represent the sandwich immune complex magnetic beads (the image recognition algorithm of the present invention marks the position where the bright field magnetic beads and the dark field fluorescent label detection antibody overlap as sandwich immune complex magnetic beads), and the red dots represent the unbound free fluorescent label detection antibody. Although there is no protein in the sample and sandwich immune complexes cannot be formed, it can be seen from the image that when the magnetic frame is used for sorting, the fluorescently labeled detection antibody and the capture antibody coated magnetic beads cannot be effectively separated. As a result, during observation, the capture antibody coated magnetic beads and the fluorescently labeled detection antibody overlap and are labeled as sandwich immune complex magnetic beads. If this is in actual detection, it will cause false positives and high detection background problems. The experimental results of the present invention are shown in Figure 7 B. It can be seen that after sorting by the magnetic sorting chip, the sorting channel outlet only contains magnetic beads. The excess fluorescently labeled detection antibodies are not affected by the magnet and have not been deflected and have been washed to the sorting channel outlet farthest from the magnet. The other outlets only contain magnetic beads of uniform size. It can be seen that the method and chip of the present invention can improve the detection signal-to-noise ratio and show obvious advantages.

[0110] In some specific embodiments of the present invention, different concentrations of high-sensitivity C-reactive protein are used for sorting, and the number of capture antibody-coated magnetic beads used (10^6 magnetic beads) is more than 10 times the molar number of high-sensitivity C-reactive protein. Under this condition, each magnetic bead binds at most one high-sensitivity C-reactive protein molecule, and the detection results are shown in Figure 8. Figure 8 is a fluorescence imaging image of three different concentrations of high-sensitivity C-reactive protein after being subjected to a flow magnetic sorting microfluidic detection platform. Based on ultra-bright fluorescent nanospheres and image recognition algorithms, it can be seen that under the condition that the number of magnetic beads is more than tens of times the molar number of the protein to be detected, the number of sandwich immune complex magnetic beads represented by green dots in the field of view increases with the increase of high-sensitivity C-reactive protein concentration, and single molecule counting detection as low as 35fg / mL protein can be achieved.

Claims

1. A flow-through magnetic separation microfluidic chip, which comprises a sample inlet, a mixing and incubation zone, a washing and sorting zone, a magnet, and an imaging and detection zone; Characterized in that, The sample inlet, the mixing and incubation zone, the magnet, the washing and sorting zone, and the imaging and detection zone are integrated on the same microfluidic chip; The sample inlet is used for injecting a protein sample to be tested, magnetic beads coated with capture antibodies, and fluorescently labeled detection antibodies; The mixing and incubation zone is used for mixing the protein sample to be tested, magnetic beads coated with capture antibodies, and fluorescently labeled detection antibodies to form sandwich immunocomplex magnetic beads containing the protein sample to be tested, magnetic beads coated with capture antibodies, and fluorescently labeled detection antibodies; The washing and sorting zone is used for distributing the sandwich immunocomplex magnetic beads containing different protein samples to be tested to the sorting channel outlets in different imaging and detection zones through dynamic magnetic separation; the washing and sorting zone is connected to the mixing and incubation zone through a magnetic bead inlet. The washing and sorting zone includes a flow-through magnetic separation channel and a sheath flow channel. The sheath flow channel connects a sheath flow inlet for injecting buffer solution to the flow-through magnetic separation channel. The sheath flow inlet is used for injecting buffer solution. The width of the sheath flow channel is greater than the width of the magnetic bead inlet. The sheath flow channel and the magnetic bead inlet are co-injected into the flow-through magnetic separation channel on the same plane. The sheath flow channel is arranged on the side close to the permanent magnet, while the magnetic bead inlet is arranged on the side far from the permanent magnet; when the sandwich immunocomplex magnetic beads enter through the magnetic bead inlet, the buffer solution injected by the sheath flow inlet causes the sandwich immunocomplex magnetic beads to focus near the side wall of the flow-through magnetic separation channel, forming a focusing region with a width of 2 - 500 μm; The magnet is arranged on one side of the washing and sorting zone and is arranged parallel to the flow-through magnetic separation channel; The imaging and detection zone is used for detecting different immunomagnetic beads of the protein to be tested obtained by sorting.

2. The flow-through magnetic separation microfluidic chip according to claim 1, Characterized in that, The sample inlet is connected to the mixing and incubation zone. The sample inlet includes at least one protein sample inlet to be tested and at least one inlet for magnetic beads coated with capture antibodies and fluorescently labeled detection antibodies.

3. The flow-through magnetic separation microfluidic chip according to claim 1, Characterized in that, The mixing and incubation zone includes a mixing channel and a magnetic bead inlet. The mixing channel is arranged in a serpentine or spiral shape. Magnetic beads coated with capture antibodies, fluorescently labeled detection antibodies, and the protein to be tested can form sandwich immunomagnetic beads in the mixing channel; Preferably, the serpentine shape is a serpentine detour shape, the detour angle is 180 degrees, and at least 8 - 10 detours are provided; Preferably, the width of the magnetic bead inlet is lower than the width of the mixing channel.

4. The flow-through magnetic separation microfluidic chip according to claim 1, Characterized in that, The flow-through magnetic separation channel is a single channel, and the magnet is a permanent magnet; Preferably, the length of the permanent magnet is the same as the length of the flow-through magnetic separation channel.

5. The flow-through magnetic separation microfluidic chip according to claim 1, Characterized in that, The flow-through magnetic separation channel is rectangular or trapezoidal. When it is trapezoidal, the width at the starting end close to the sheath flow channel is greater than the width at the sorting channel outlet. When it is rectangular, the width at the starting end close to the sheath flow channel is the same as the width at the sorting channel outlet.

6. The flow-through magnetic separation microfluidic chip according to claim 1, characterized in that, the imaging detection area is located at the end of the flow-through magnetic separation channel. The imaging detection area includes 3-6 sorting channel outlets, and the sorting channel outlets are connected to the flow-through magnetic separation channel in the same plane and are arranged in sequence from the side close to the permanent magnet to the side far from the permanent magnet.

7. A multiplex immunoassay microfluidic system based on flow-through magnetic separation, characterized in that, the multiplex immunoassay microfluidic system includes a multiplex immunomagnetic bead preparation module, a flow-through magnetic separation microfluidic chip system, an optical detection module and an image processing module; the multiplex immunomagnetic bead preparation module is used to prepare capture antibody-coated magnetic beads and fluorescence-labeled detection antibodies; the flow-through magnetic separation microfluidic chip system includes the flow-through magnetic separation microfluidic chip according to any one of claims 1-6 and auxiliary support components; the optical detection module is used to detect and record the sample conditions at the sorting channel outlets of the flow-through magnetic separation microfluidic chip; the image processing module is used to process the images of the sample conditions at the sorting channel outlets obtained by the optical detection module, and analyze and calculate the concentrations of the samples at different sorting channel outlets; Preferably, the auxiliary support components include three programmable injection pumps, a micro-soft tube and a sampling head; Preferably, the programmable injection pump can provide a flow rate of 0.01-100 μL / min.

8. The multiplex immunoassay microfluidic system based on flow-through magnetic separation according to claim 7, characterized in that, the optical detection module includes a fluorescence imaging system, and the fluorescence imaging system includes an excitation light source, an excitation light filter, an emission light filter and a dichroic mirror; The fluorescence imaging system includes a fluorescence excitation optical path and a fluorescence detection optical path. Along the fluorescence excitation optical path, the excitation light emitted by the light source device is incident on the imaging detection area of the microfluidic chip through a convex lens and an excitation light filter in sequence; along the fluorescence detection optical path, the fluorescence generated by the imaging detection area of the microfluidic chip under the excitation light is incident on the charge-coupled device CCD camera through an emission light filter and a path where light transmission occurs in sequence; the emission optical path after the excitation light filter in the fluorescence excitation optical path is combined by a dichroic mirror and introduced into the fluorescence excitation optical path.

9. The multiplex immunoassay microfluidic system based on flow-through magnetic separation according to claim 7, characterized in that, the image processing module includes a memory, a processor and a computer program stored on the memory and executable on the processor; when the processor executes the computer program, machine learning algorithms are used to splice and analyze the charge-coupled device images, provide an image and a protein concentration analysis model, and output an identification result; at the same time, the probability density of different luminescent microbeads is calculated according to the Poisson distribution probability formula = -ln(1 - the number of luminescent microbeads / the total number of microbeads), so as to calculate the concentration value of the object to be measured.

10. A method for detecting multiplex proteins, characterized in that, the method for detecting multiplex proteins is to use the multiplex immunoassay microfluidic system according to any one of claims 7-9 for detection, or the method for detecting multiplex proteins includes the following steps: S001) Prepare magnetic beads coated with capture antibodies and fluorescently labeled detection antibodies: Prepare multiple sets of magnetic beads coated with capture antibodies and fluorescently labeled detection antibodies according to the multiplex proteins to be detected. Among them, the capture antibodies and detection antibodies in each set can specifically bind to one protein in the multiplex proteins respectively. The magnetic beads and fluorescent labels in each set are the same, while the magnetic beads and fluorescent labels in different sets are different; the different magnetic beads have different particle sizes, different magnetic contents, or both different particle sizes and magnetic contents, and the emission wavelengths of the fluorescent labels in different sets are different; S002) Prepare the magnetic beads coated with capture antibodies and the fluorescently labeled detection antibodies into a mixed solution of magnetic beads coated with capture antibodies and fluorescently labeled detection antibodies. In the mixed solution, based on the molar concentration of the detection antibodies in the fluorescently labeled detection antibodies and the molar concentration of the capture antibodies in the magnetic beads coated with capture antibodies, the concentration of the fluorescently labeled detection antibodies is higher than the concentration of the magnetic beads coated with capture antibodies; S003) Obtain the flow-through magnetic sorting microfluidic chip according to any one of claims 1-6; S004) Inject the multiplex protein sample to be detected into the flow-through magnetic sorting microfluidic chip through the sample injection port for the protein to be detected. Inject the mixed solution obtained in step S002) into the flow-through magnetic sorting microfluidic chip through the injection port for magnetic beads coated with capture antibodies and fluorescently labeled detection antibodies, and mix the multiplex protein sample to be detected with the mixed solution in the mixing channel of the flow-through magnetic sorting microfluidic chip to form sandwich immune complex magnetic beads, and enter the cross-flow channel through the magnetic bead inlet; Inject the buffer solution into the flow-through magnetic sorting microfluidic chip through the cross-flow inlet, and mix it with the fluid injected from the magnetic bead inlet through the cross-flow channel, and form a sheath flow outside the fluid injected at the magnetic bead inlet 4 at the starting stage of the washing and sorting area, so that the sandwich immune complex magnetic beads and the excess fluorescently labeled detection antibodies in the fluid injected from the magnetic bead inlet are both restricted near the wall on the side far from the permanent magnet in the flow-through magnetic sorting channel; With the continuous injection of the buffer solution, the magnetic beads in the sandwich immune complex magnetic beads will also produce different degrees of deviation towards the permanent magnet while moving towards the sorting channel outlet due to the magnitude of the magnetic force, and enter different sorting channel outlets, while the non-magnetic particles continue to move horizontally along the wall to the sorting channel outlet farthest from the permanent magnet; S005) Detect at the sorting channel outlet using a fluorescence imaging system, observe the fluorescent labels at different sorting channel outlets, and collect images to achieve qualitative detection; Optionally, S006) Analyze the images collected in step S005) using an image processing module to obtain the concentration value of the object to be measured; Preferably, the multiplex protein detection method is qualitative detection or quantitative detection; Preferably, the multiplex protein detection is particularly suitable for ultrasensitive low-concentration single-molecule protein counting immunoassay; Preferably, the fluorescent label is selected from any one or more of fluorescein, quantum dots, rare earth elements, rare earth chelates, fluorescent proteins, or upconverting nanoparticles; Preferably, the particle size of the fluorescent label is 100-500 nm.

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