Multi-immune microfluidic detection system based on flow magnetic separation and method thereof

Abstract

The invention relates to a multi-immune micro-fluidic detection system based on flow magnetic separation and a method thereof. The system comprises a multi-immune magnetic bead preparation module, a flow magnetic separation micro-fluidic chip system, an optical detection module and an image processing module, through coding based on magnetic beads with different magnetic contents and decoding based on a flow magnetic separation micro-fluidic chip, ultrasensitive and rapid multi-protein quantitative detection is realized. The multiple immune microfluidic detection system has the advantages of high detection sensitivity, short detection time, high signal-to-noise ratio, convenience and simplicity in operation and low cost, is particularly suitable for ultrasensitive detection of multiple single-molecule proteins, and has potential economic value.

Description

Technical Field

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

[0002] Simultaneous detection of multiple targets can improve the accuracy of disease diagnosis on the one hand. On the other hand, simultaneous detection of multiple targets improves the analysis throughput, shortens the detection time and reduces the detection cost. With the continuous improvement of the requirements for simultaneous sensitivity detection of multiple proteins in the modern medical field, developing advanced multiplex protein detection technology is the only way to improve the diagnostic ability of medical instruments. Traditional detection methods include immunochromatography, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), fluorescence, electrochemistry, chemiluminescence, and electrochemiluminescence, etc. However, traditional technologies still have problems such as low detection sensitivity, lagging time window, high detection cost, large reagent consumption, and complex operation, so they cannot meet the growing clinical diagnosis needs.

[0003] One of the major challenges in achieving immunotrace analysis of multiple protein markers in a complex sample environment lies in the separation of target analytes from antibody complexes. Magnetic nanoparticles immobilized with antibodies have the inherent property of being attracted to magnets. According to this property, they can be used to directly capture, separate, and preconcentrate targets in complex environmental samples and provide a solution for immunoassay technology. The immunoreaction system of traditional large-scale immunoluminescence instruments usually uses traditional reagent tubes as reaction containers, which requires a large amount of reagents, has a high cost, and due to the entire reaction system being 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 operation units such as sample preparation, reaction, separation, and detection onto a chip with a micron scale, automatically completing the entire analysis process, and 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 point-of-care testing. Although the fluid flow pattern in microfluidic channels is usually laminar flow, the diffusion effect between fluids can be enhanced by designing serpentine or spiral curved channels to achieve fast and thorough mixing and efficient mixing reactions. Flow magnetic sorting in microchannels can enable magnetic beads with different magnetic contents and non-magnetic materials in a magnetic field to move along specific trajectories under different regulation boundaries. A high-throughput and high magnetic flux density magnetic bead sorting process can be achieved during the coupling process of the magnetic field and the flow field.

[0005] Combining multiple immunomagnetic beads with flow magnetic sorting microfluidic chips can realize magnetic bead encoding based on different magnetic contents and decoding based on flow magnetic sorting microfluidic chips. Ultra-bright fluorescent microspheres are used as markers 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, CN202211361964.X, etc. 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, etc. disclose a multi-target quantitative detection method based on a microfluidic chip, which uses labeled microspheres of different sizes and colors and machine vision algorithms to achieve multi-target detection on the same chip. Although the method is feasible, it has problems of low signal-to-noise ratio and lack of sensitivity.

[0006] Most of the existing microfluidic detection chips detect a single biomarker and cannot detect multiple biomarkers in a sample at the same time. Among the currently disclosed microfluidic platform technologies for multiple protein quantitative detection, there is no method based on flow magnetic sorting to achieve excess fluorescent marker washing and ultra-low background fluorescence. In addition, the existing multiple single-molecule protein ultra-sensitive detection methods mostly use enzyme-linked immunosorbent amplification signals, which has the disadvantages of expensive reagents, difficult storage, and the need to be isolated into independent small chambers. Summary of the invention

[0007] The present invention solves the problems of high reagent consumption and insufficient mixing in the prior art platform, complex and costly chips, need for improved sensitivity and limited simultaneous detection of multiple targets, and provides a multiplex immune microfluidic detection platform based on flow magnetic sorting and a method thereof.

[0008] In one aspect, the present invention provides a flow magnetic sorting microfluidic chip, which comprises an injection port, a mixing incubation area, a washing and sorting area, a magnet, and an imaging detection area; the injection port, the mixing incubation area, the magnet, the washing and sorting area, and the imaging detection area are integrated on the same microfluidic chip;

[0009] 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;

[0010] The mixed 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;

[0011] The washing and sorting area 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 area is connected to the mixing and incubation area through the magnetic bead inlet 4. The washing and sorting area includes a flowing magnetic sorting channel 11 and a cross-flow channel 10. The cross-flow channel 10 connects the cross-flow inlet 3 for injecting buffer solution to the flowing magnetic sorting channel 11. The cross-flow inlet 3 is used to inject buffer solution. The width of the cross-flow channel 10 is greater than the width of the magnetic bead inlet 4. The cross-flow channel 10 and the magnetic bead inlet 4 are connected to the flowing magnetic sorting channel 11 in the same plane. The cross-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 far from the permanent magnet 12. When the sandwich immune complex magnetic beads enter through the magnetic bead inlet 4, the buffer solution injected from the cross-flow inlet 3 causes the sandwich immune complex magnetic beads to focus near the side wall of the flowing 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 area; and is arranged parallel to the flowing magnetic sorting channel 11;

[0012] The imaging detection area is used to detect different samples obtained by sorting.

[0013] Furthermore, the sample inlet is connected to the mixing and incubation area. The sample inlet includes at least one protein sample inlet 1 to be tested and at least one inlet 2 for magnetic beads coated with capture antibodies and fluorescently labeled detection antibodies;

[0014] Furthermore, the mixing and 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 shape. The magnetic beads coated with capture antibodies, the fluorescently labeled detection antibodies, and the protein sample to be tested can form sandwich immune magnetic beads in the mixing flow channel; the serpentine shape is a serpentine detour, the detour angle is 180 degrees, and at least 8 - 10 detours are provided.

[0015] Furthermore, the width of the magnetic bead inlet 4 is lower than the width of the mixing flow channel 9.

[0016] Furthermore, the flowing magnetic sorting channel 11 is a single channel, and the magnet is a permanent magnet. The length of the permanent magnet is the same as the length of the flowing magnetic sorting channel 11. Furthermore, the flowing magnetic sorting channel 11 is rectangular or trapezoidal. When it is trapezoidal, the width at the starting end close to the cross-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 cross-flow channel is the same as the width at the sorting channel outlet.

[0017] Furthermore, 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. 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 far from the permanent magnet.

[0018] Another aspect of the present invention provides a multiplex immunodetection microfluidic system based on flow magnetic sorting, and the multiplex immunodetection microfluidic system includes a multiplex immunomagnetic bead preparation module, a flow magnetic sorting microfluidic chip system, an optical detection module, and an image processing module;

[0019] The multiplex immunomagnetic bead preparation module is used to prepare capture antibody-coated magnetic beads and fluorescently labeled detection antibodies;

[0020] The flow magnetic sorting microfluidic chip system includes the above-mentioned flow magnetic sorting microfluidic chip and auxiliary support components;

[0021] The optical detection module is used to detect and record the sample conditions at the sorting channel outlet of the flow magnetic sorting microfluidic chip;

[0022] The image processing module is used to process the images of the sample conditions at the sorting channel outlet obtained by the optical detection module, and analyze and calculate the concentrations of the samples at different sorting channel outlets.

[0023] Further, the auxiliary support components include three programmable injection pumps, a microtube, and a sampling head, and the programmable injection pumps can provide a flow rate of 0.01-100 μL / min.

[0024] Further, 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 passes through the emission light filter and the optical path will transmit the light to a charge-coupled device (CCD) camera; 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.

[0025] Further, 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.

[0026] Another solution of the present invention provides a multiplex protein detection method, and the multiplex protein detection method is to perform detection using the above-mentioned multiplex immunodetection microfluidic system.

[0027] Or the multiplex protein detection method includes the following steps:

[0028] S001) Prepare capture antibody-coated magnetic beads and fluorescently labeled detection antibodies: Prepare multiple groups of capture antibody-coated magnetic beads and fluorescently labeled detection antibodies according to the multiplex proteins to be detected. Among them, the capture antibody and the detection antibody in each group can specifically bind to one protein in the multiplex proteins respectively. The magnetic beads and the fluorescent labels in each group are the same, and the magnetic beads and the fluorescent labels in different groups are different; the different magnetic beads have different particle sizes or different magnetic contents or both different particle sizes and magnetic contents, and the emission wavelengths of the fluorescent labels in different groups are different;

[0029] S002) Prepare the capture antibody-coated magnetic beads and the fluorescently labeled detection antibodies into a mixed solution of the capture antibody-coated magnetic beads and the fluorescently labeled detection antibodies. In the mixed solution, based on 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, the concentration of the fluorescently labeled detection antibody is higher than the concentration of the capture antibody-coated magnetic beads;

[0030] S003) Obtain the above-mentioned flow-through magnetic sorting microfluidic chip;

[0031] S004) Inject the multiplex protein sample to be detected into the flow-through magnetic sorting microfluidic chip through the sample injection port 1 for the protein to be detected. The mixed solution obtained in step S002) is injected into the flow-through magnetic sorting microfluidic chip through the injection port 2 for the capture antibody-coated magnetic beads and the fluorescently labeled detection antibodies, and the multiplex protein sample to be detected and the mixed solution are mixed in the mixing channel 9 of the flow-through magnetic sorting microfluidic chip to form sandwich immune complex magnetic beads, and enter the cross-flow channel 10 through the magnetic bead inlet 4; the buffer solution is injected into the flow-through magnetic sorting microfluidic chip through the cross-flow inlet 3, and is mixed with the fluid injected through the magnetic bead inlet 4 through the cross-flow channel 10, and a sheath flow is formed outside the fluid injected through 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 through the magnetic bead inlet 4 are both restricted near the wall on the side far from the permanent magnet in the flow-through magnetic sorting channel 11; with the continuous injection of the buffer solution, the magnetic beads in the sandwich immune complex magnetic beads will also be deflected to different degrees in the direction of the permanent magnet 12 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 on the side farthest from the permanent magnet;

[0032] 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;

[0033] Optionally, in S006), an image processing module is used to analyze the image collected in step S005) to obtain the concentration value of the object under test.

[0034] Furthermore, the multiplex protein detection method can be qualitative detection or quantitative detection.

[0035] Furthermore, 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.

[0036] Beneficial effects

[0037] The present invention provides a multiplex immunomicrofluidic detection platform based on flow magnetic sorting and its method. The platform mainly includes a multiplex immunomagnetic bead reagent system, a modular microfluidic chip and an imaging detection system, which are used for the injection, mixing, capture, sorting and detection of the biological sample to be detected with specific immunomagnetic beads and multiplex fluorescent labels. This method develops an on-chip ultrasensitive and rapid multiplex protein quantitative detection through magnetic bead encoding based on different magnetic contents and decoding based on a flow magnetic sorting microfluidic chip. During the coupling process of the magnetic field and the flow field, flow magnetic sorting can achieve high-throughput sorting of magnetic beads with different magnetic contents and non-magnetic labels in the microchannel.

[0038] Second, the multiplex immunomicrofluidic detection platform of the present invention can achieve single molecule separation and detection. When injecting the capture antibody-coated magnetic beads and the fluorescently labeled detection antibody into the microfluidic chip, when the capture antibody-coated magnetic beads used are more than 10 times the predicted concentration of the protein to be detected, each magnetic bead can capture only one protein to be detected. Furthermore, when observing at the outlet of the sorting channel with a fluorescence imaging system, single protein detection can be achieved, and the concentration of the protein to be detected can be calculated by the recognition and analysis of the image processing module.

[0039] Secondly, in the solution of the present invention, ultra-bright fluorescent microspheres are used as fluorescent labels, greatly improving the fluorescence detection performance.

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

[0041] This platform has the advantages of high detection sensitivity, short detection time, high signal-to-noise ratio, convenient and simple operation, and low cost. It is especially suitable for multiplex single molecule protein ultrasensitive detection and has potential economic value. Description of the drawings

[0042] Figure 1 It is a schematic diagram for constructing a multiplex sandwich immunomagnetic bead.

[0043] Figure 2 It is a schematic diagram of a multiplex immunomicrofluidic chip based on flow magnetic sorting.

[0044] Figure 3 Schematic diagram of the mask structure of a rectangular microfluidic chip for hybrid and flow magnetic separation. Among them, 1 - sample injection port for proteins to be detected containing multiple targets, 2 - sample injection port for magnetic beads coated with capture antibodies and detection antibodies labeled with fluorescence, 3 - cross-flow inlet, 4 - multiple immunomagnetic bead inlet, 5 - outlet of the first sorting channel, 6 - outlet of the second sorting channel, 7 - outlet of the third sorting channel, 8 - outlet of the fourth sorting channel, 9 - mixing channel, 10 - cross-flow channel, 11 - flow magnetic separation channel, 12 - permanent magnet.

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

[0046] Figure 5 Schematic diagram of the fluorescence imaging system of the microfluidic chip.

[0047] Figure 6 Physical pictures of the effects at each stage of flow magnetic separation.

[0048] Figure 7 Washing effect diagram of flow magnetic separation of blank samples.

[0049] Figure 8 Fluorescence imaging diagrams after processing by algorithms of immunomagnetic beads with different concentrations.

[0050] Figure 9 Schematic diagram of the mask structure of a trapezoidal microfluidic chip for hybrid and flow magnetic separation. Among them, 1 - sample injection port for proteins to be detected containing multiple targets, 2 - sample injection port for magnetic beads coated with capture antibodies and detection antibodies labeled with fluorescence, 3 - cross-flow inlet, 4 - multiple immunomagnetic bead inlet, 5 - outlet of the first sorting channel, 6 - outlet of the second sorting channel, 7 - outlet of the third sorting channel, 8 - outlet of the fourth sorting channel, 9 - mixing channel, 10 - cross-flow channel, 11 - flow magnetic separation channel, 12 - permanent magnet. Detailed implementation manners

[0051] In order to make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description of the specific implementation manners of the present invention is provided, but it should not be construed as a limitation on the implementable scope of the present invention.

[0052] The present invention discloses a multiplex immunodetection microfluidic platform based on flow magnetic separation and its method. The platform mainly includes multiplex immunomagnetic bead preparation, a magnetic separation microfluidic chip, and an imaging detection system, and is used for the injection, mixing, incubation, washing, sorting, and detection of a biological sample to be detected with specific immunomagnetic beads and multiplex fluorescent markers.

[0053] The following will provide a detailed description of the design method of the multiplex immunodetection microfluidic platform based on flow magnetic separation, specifically as follows:

[0054] S1) Preparation of multiple immunomagnetic beads: As Figure 1 shown, 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 at most one single-molecule protein and improve the luminescence intensity of the fluorescent label. Generally, when the ratio of the number of magnetic beads to the number of protein molecules is greater than 10, it follows the Poisson distribution. It mainly includes carboxylated fluorescent microspheres activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide (EDC-NHS) to conjugate detection antibodies and streptavidin magnetic beads conjugated with biotinylated capture antibodies. After bovine serum albumin (BSA) blocks the immunologically active sites, they bind to the target protein in the sample to be tested to form immunocomplex magnetic beads, as follows:

[0055] S11) Design and preparation of capture antibody-coated magnetic beads: Antibody-coated magnetic beads are prepared outside the magnetic sorting microfluidic chip. Estimate the number of moles of the protein to be tested in the sample, and use at least 10 times the number of streptavidin magnetic beads conjugated with biotinylated capture antibodies to obtain capture antibody-coated magnetic beads. According to the Poisson distribution, at this ratio, each microbead carries at most a single protein. The magnetic beads described above include at least two or more kinds, such as 3 kinds, 4 kinds or 5 kinds. At the same time, each kind of magnetic bead is a magnetic bead with different magnetic contents or the same magnetic content but different particle sizes. The capture antibodies contained on each kind of magnetic bead are the same, while the capture antibodies contained on different kinds of magnetic beads are different. The capture antibody can specifically bind to the target protein to be tested.

[0056] S12) Preparation of fluorescently labeled detection antibodies: Ultra-bright fluorescent microspheres such as quantum dots are used as fluorescent labels to conjugate detection antibodies to form fluorescently labeled detection antibodies. The fluorescent microspheres have different detection wavelengths, and there are at least two or more kinds of fluorescent microspheres with different wavelengths. Each kind of fluorescent microsphere with the same wavelength is conjugated with the same kind of detection antibody, and different kinds of fluorescent microspheres are conjugated with different kinds of detection antibodies. The detection antibody described above is an antibody that can specifically bind to the target protein to be tested. Figure 4 is the fluorescence imaging diagram of a single ultra-bright fluorescent microsphere with a particle size of 120 nm. Under the microscope, a single bright microsphere can be clearly seen. Based on the high brightness of a single fluorescent microsphere, it provides the possibility for discrete immunomagnetic beads and accurate recognition of single-molecule protein signals. The method of conjugating ultra-bright fluorescent microspheres with detection antibodies can be achieved by any conventional means in the art. For example, carboxylated ultra-bright fluorescent microspheres are conjugated with detection antibodies after activation by EDC-NHS.

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

[0058] S2) Design and preparation of a magnetic sorting microfluidic chip.

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

[0060] The sample inlet is connected to the mixing and incubation area. The sample inlet includes at least one sample inlet 1 for the protein to be detected and at least one sample inlet 2 for magnetic beads coated with capture antibodies and fluorescently labeled detection antibodies, where the fluorescently labeled detection antibodies are in excess relative to the magnetic beads coated with capture antibodies.

[0061] The mixing and incubation area includes a mixing channel 9 and a magnetic bead inlet 4. The mixing channel 9 is arranged in a serpentine or spiral shape. The magnetic beads coated with capture antibodies, the fluorescently labeled detection antibodies, and the protein to be detected can form a sandwich immunomagnetic bead in the mixing channel. The serpentine arrangement is a serpentine detour arrangement with a detour angle of 180 degrees and at least 8 - 10 detours. The serpentine detour arrangement can enhance the diffusion effect between fluids, realizing the rapid and thorough mixing and efficient mixing reaction of the specific capture antibody magnetic beads, the sample to be detected, and the multiple fluorescently labeled detection antibodies to form a sandwich immunomagnetic bead. The magnetic bead inlet 4 is used to connect to the washing and separation area and is arranged on one side of the starting end of the flow magnetic separation channel 11 far from the permanent magnet 12. The width of the magnetic bead inlet 4 is lower than the width of the mixing channel.

[0062] The washing and separation area is connected to the mixing and incubation area through the magnetic bead inlet 4. The washing and separation area also includes a pinch channel 10. The pinch channel 10 connects the pinch inlet 3 for injecting buffer solution to the flow magnetic separation channel 11. The width of the pinch channel 10 is greater than the width of the magnetic bead inlet 4. The pinch channel 10 and the magnetic bead inlet 4 are connected to the flow magnetic separation channel 11 in the same plane. The pinch channel 10 is arranged on the side close to the permanent magnet 12, while the magnetic bead inlet 4 is arranged on the side far from the permanent magnet 12. When the sandwich immunocomplex magnetic beads enter through the magnetic bead inlet 4, the buffer solution injected from the pinch inlet 3 focuses the sandwich immunocomplex magnetic beads near the side wall of the flow magnetic separation channel, forming a focused area with a width of 2 - 500 μm.

[0063] The flow magnetic separation channel 11 is used to sort different magnetic beads and non-magnetic signal probes that have not participated in the reaction, so that they form different displacements in the width direction of the flow magnetic separation channel to enter different sorting channel outlets. The flow magnetic separation channel 11 is a single channel. A permanent magnet 12 is arranged on one side of the flow magnetic separation channel 11. The permanent magnet 12 is arranged parallel to the flow magnetic separation channel 11, and the length of the permanent magnet 12 is the same as the length of the flow magnetic separation channel 11.

[0064] As the magnetic beads flow in the magnetic separation channel 11, they will 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. Larger-sized magnetic beads are subject to a greater magnetic force, so their lateral displacement is more significant; while smaller-sized magnetic beads are subject to a smaller force and have a smaller lateral displacement; non-magnetic materials (excessive multiple fluorescent markers) are not affected by the magnetic force and have no lateral displacement. This displacement difference enables magnetic beads of different sizes to move along specific trajectories under different regulation boundaries within the channel and be diverted to different sorting outlets.

[0065] The imaging detection area is located at the end of the flowing magnetic separation channel 11. The imaging detection area includes 4 sorting channel outlets 5, 6, 7, 8, and can also be set to 3, 5, or 6 according to actual needs. The sorting channel outlets are connected to the flowing magnetic separation channel 11 in the same plane and are arranged in sequence from the side close to the permanent magnet 12 to the side far from the permanent magnet.

[0066] The channel heights of the mixing incubation area, the washing and sorting area, and the imaging detection area are the same.

[0067] The magnetic separation microfluidic chip designs its specific characteristic structure according to the washing and sorting function requirements of the flowing magnetic separation unit, combining a permanent magnet and magnetic beads with different magnetic contents. In the microchannel, flowing magnetic separation can achieve the sorting of multiple immunomagnetic beads with different magnetic contents in a magnetic field, and during the magnetic field-fluid field coupling process, 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) Prepare the magnetic separation microfluidic chip: According to the design in step S21), the microfluidic chip can be fabricated by microelectromechanical system (MEMS) technology, soft lithography method, 3D printing, injection molding or imprinting.

[0069] Taking the soft lithography method as an example to introduce the preparation process: The lithography technology includes steps such as pretreatment, spin-coating photoresist, soft baking, exposure, post-baking, and development.

[0070] S3) Set up a fluorescence imaging system for the microfluidic chip. The fluorescence imaging system is as shown in Figure 5. The fluorescence imaging system can achieve on-chip ultrasensitive detection, especially immunoassay at the single-molecule level. 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 passes through the emission light filter and the optical path where the light will occur and is transmitted to a charge-coupled device (CCD) camera; the dichroic mirror combines the emission optical path after the excitation light filter in the fluorescence excitation optical path and imports it into the fluorescence excitation optical path. The dichroic mirror, the excitation light filter, and the emission light filter are adapted to the fluorescent microsphere labels in the fluorescently labeled detection antibody. Using the above fluorescence imaging system, bright-field magnetic bead and dark-field fluorescence imaging diagrams of immunomagnetic beads in the detection imaging area of the sorted microfluidic chip can be obtained.

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

[0072] In some specific embodiments, the fluorescence imaging system uses an objective lens with a magnification of more than 40 times.

[0073] S4) Further, to achieve adaptive protein detection and analysis, combined with the charge-coupled device image, a machine learning algorithm is used to splice and analyze the charge-coupled device image, provide an image and a protein concentration analysis model, and intuitively output the recognition result. And the probability density of the 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 obtain the concentration value of the object to be measured.

[0074] The present invention also provides an alternative improvement scheme for the magnetic sorting microfluidic chip, as Figure 9 shown. Different from the above chip, in this alternative improvement scheme, a trapezoidal flow magnetic sorting channel 11 and four outlets 5, 6, 7, 8 are provided. The trapezoidal structure design can narrow the channel width. The purpose of doing this is to make the flowing magnetic beads approach 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 towards the permanent magnet 12 and be subjected to a stronger magnetic force, resulting in significant lateral displacement of them. This innovative design improves the sorting resolution and efficiency of magnetic beads with lower magnetism.

[0075] During the sorting process, the buffer solution injected through the cross-flow inlet 3 focuses the sandwich immuno-complex magnetic beads near the sidewall of the flow-through magnetic sorting channel, with the focusing width controlled within 2 - 500 μm. As the magnetic beads flow in the channel 11, even small magnetic beads with a low yield can be subjected to sufficient magnetic force for effective sorting. Since the magnetic force is proportional to the volume of the magnetic beads, larger-sized magnetic beads are subjected to a greater magnetic force, resulting in a larger lateral displacement and rapidly changing their motion trajectory; while smaller-sized magnetic beads are less affected by the force initially and have a small lateral displacement, but as they approach the trapezoidal structure, they gradually move to the high magnetic field region, and their motion trajectory begins to change, and eventually they will also have a lateral shift and deviate from the original trajectory. This design enables the diversion of magnetic beads of different sizes to corresponding sorting outlets according to the displacement differences of the magnetic beads at the channel outlet. For example, as shown in Figure 9 the figure, larger-sized magnetic beads will enter the first sorting outlet 5, while medium-sized and smaller-sized magnetic beads will be respectively guided to the second and third sorting outlets 6 and 7. For those signal probes without magnetic labels, since they are not affected by the magnetic field, they will go straight to the fourth sorting outlet 8 and enter the waste liquid channel. This integration process realizes the effective magnetic sorting of magnetic beads while washing away the background signal in the same channel, ensuring the precise distinction of magnetic beads of different sizes.

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

[0077] the sample inlet is connected to the mixing and incubation area, and the sample inlet includes at least one sample inlet 1 for the protein to be tested and at least one sample inlet 2 for the capture antibody-coated magnetic beads and the fluorescence-labeled detection antibody;

[0078] the mixing and 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 shape, and the capture antibody-coated magnetic beads, the fluorescence-labeled detection antibody, and the protein to be tested can form sandwich immuno-magnetic beads in the mixing flow channel; the serpentine setting is a serpentine detour setting, the detour angle is 180 degrees, and at least 8 - 10 detours are provided. The width of the magnetic bead inlet 4 is lower than the width of the mixing flow channel 9.

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

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

[0081] The flow magnetic separation channel 11 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.

[0082] The imaging detection area is located at the end of the flow magnetic separation channel 11. The imaging detection area contains 3 - 6 sorting channel outlets. The sorting channel outlets are connected to the flow magnetic separation channel 11 in the same plane and are arranged in sequence from the side close to the permanent magnet 12 to the side far 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 separation, which comprises a multiplex immunomagnetic bead preparation module, a flow magnetic separation microfluidic chip system, an optical detection module, and an image processing module.

[0084] The multiplex 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 an auxiliary support component.

[0086] Among them, the flow magnetic separation microfluidic chip includes a sample inlet, a mixing and incubation area, a washing and sorting area, a magnet, and an imaging detection area; the sample inlet, the mixing and incubation area, the magnet, the washing and sorting area, and the imaging detection area are integrated on the same microfluidic chip.

[0087] The sample inlet is connected to the mixing and incubation area. The sample inlet includes at least one test protein sample inlet 1 and at least one capture antibody-coated magnetic bead and fluorescent-labeled detection antibody sample inlet 2;

[0088] The mixing and 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 shape. The capture antibody-coated magnetic beads, fluorescent-labeled detection antibodies, and test proteins can form a sandwich immunomagnetic bead in the mixing flow channel. The serpentine setting is a serpentine detour setting with a detour angle of 180 degrees and at least 8 - 10 detours. The width of the magnetic bead inlet 4 is lower than the width of the mixing flow channel 9.

[0089] The washing and sorting area is connected to the mixing and incubation area through the magnetic bead inlet 4. The washing and sorting area includes a flow magnetic separation channel 11 and a pinch flow channel 10. The pinch flow channel 10 connects the pinch flow inlet 3 for injecting buffer solution to the flow magnetic separation channel 11. The pinch flow inlet 3 is used to inject buffer solution. The width of the pinch flow channel 10 is greater than the width of the magnetic bead inlet 4. The pinch flow channel 10 and the magnetic bead inlet 4 are connected to the flow magnetic separation channel 11 in the same plane. The pinch 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 far from the permanent magnet 12. When the sandwich immune complex magnetic beads enter through the magnetic bead inlet 4, the buffer solution injected by the pinch flow inlet 3 focuses the magnetic beads near the channel sidewall, forming a focusing area with a width of 2 - 500 microns.

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

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

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

[0093] The auxiliary support component includes three programmable injection pumps and a microfluidic tube. The programmable injection pumps 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 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 passes through the emission light filter and the optical path where the light will occur and is transmitted to a charge-coupled device (CCD) camera; 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.

[0094] 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, it uses machine learning algorithms to splice and analyze the charge-coupled device images, provides an image and a protein concentration analysis model, and outputs the recognition result; at the same time, the probability density of different luminescent beads is calculated according to the Poisson distribution probability formula = -ln(1 - the number of luminescent beads / the total number of beads), so as to calculate the concentration value of the object to be measured.

[0095] Based on the above design scheme, the present invention provides a method for detecting multiple proteins using the above-mentioned multiple immunodetection microfluidic system.

[0096] The method for detecting multiple proteins includes the following steps:

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

[0098] S002) Prepare a mixed solution of capture antibody-coated magnetic beads and fluorescence-labeled detection antibodies. In the mixed solution, based on the molar concentration of the detection antibody in the fluorescence-labeled detection antibody and the molar concentration of the capture antibody in the capture antibody-coated magnetic beads, the concentration of the fluorescence-labeled detection antibody is higher than the concentration of the capture antibody-coated magnetic beads;

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

[0100] S004) Inject the multi-protein sample to be detected into the flow-through magnetic separation microfluidic chip through the protein sample injection port 1 for testing. Inject the mixed solution obtained in step S002) into the flow-through magnetic separation microfluidic chip through the capture antibody-coated magnetic beads and fluorescent-labeled detection antibody injection port 2, and mix the multi-protein sample to be detected with the mixed solution in the mixing channel 9 of the flow-through magnetic separation microfluidic chip to form sandwich immuno-complex magnetic beads, and enter the cross-flow channel 10 through the magnetic bead inlet 4; Inject the buffer solution into the flow-through magnetic separation microfluidic chip through the cross-flow inlet 3, mix it with the fluid injected from the magnetic bead inlet 4 through the cross-flow channel 10, and form a sheath flow outside the fluid injected from the magnetic bead inlet 4 at the starting stage of the washing and separation area, so that the sandwich immuno-complex magnetic beads and the excess fluorescent-labeled detection antibody in the fluid injected from the magnetic bead inlet 4 are both restricted near the wall on the side far from the permanent magnet in the flow-through magnetic separation channel 11; With the continuous injection of the buffer solution, the magnetic beads in the sandwich immuno-complex magnetic beads will also move towards the sorting channel outlet while being affected by the magnetic field force, and will shift to different degrees towards 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 on the side farthest from the permanent magnet.

[0101] In some specific technical solutions, in step S004), after injecting the protein to be tested, the capture antibody-coated magnetic beads and the fluorescent-labeled detection antibody, air is injected through the protein sample injection port 1 for testing and the capture antibody-coated magnetic beads and the fluorescent-labeled detection antibody injection port 2 to ensure that all the proteins to be tested, the capture antibody-coated magnetic beads and the fluorescent-labeled detection antibody enter the flow-through magnetic separation channel 11 through the magnetic bead inlet 4.

[0102] S005) Use a fluorescence imaging system to detect at the sorting channel outlet, observe the fluorescence labels at different sorting channel outlets, and collect images to achieve qualitative detection;

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

[0104] In the solution of the present invention, the multi-protein detection method can be qualitative detection or quantitative detection.

[0105] In some specific implementation manners of the present invention, such as Figure 1 and 2As shown, a schematic diagram of the construction of a multiple sandwich immunomagnetic bead is presented, mainly including the following steps: 1) Estimate the number of moles of the protein to be detected in the sample. Use at least 10 times (according to the Poisson distribution, at this ratio, a single magnetic bead can carry at most a single protein) the number of magnetic beads modified with streptavidin as a carrier to fully react and incubate with the capture antibody outside the chip. The particle size of the magnetic beads is 1 - 5 μm; obtain magnetic beads coated with the capture antibody. 2) Prepare a mixed solution of N-hydroxysuccinimide (NHS) and carbodiimide (EDC) (EDC-NHS, 40 mM EDC, 10 mM NHS) by mixing NHS with phosphoric acid buffer (0.01 M, pH 7.4) to activate carboxyl groups. Use fluorescent microspheres as luminescent reporter molecules, incubate at 37 °C for 15 min after binding the detection antibody; obtain fluorescently labeled detection antibody. 3) Block the immunologically active sites of the magnetic beads coated with the capture antibody and the fluorescently labeled detection antibody with a 3 - 5% bovine serum albumin (BSA) solution. 4) Prepare the fluorescently labeled detection antibody and the magnetic beads coated with the capture antibody for injection, and construct a sandwich immunomagnetic bead with the protein to be detected in the sample.

[0106] In some specific embodiments of the present invention, a lithography method is used for the preparation of the microfluidic chip, specifically including steps such as pretreatment, spin-coating photoresist, soft baking, exposure, post-baking, and development. Among them, 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 sealed oven and then perform plasma treatment for 5 min to remove water vapor and form a hydrophilic bond surface. Spin-coat the su-8 3025 photoresist, use the spin-coating method at a speed of 4000 rpm, set the spin-coating time to 35 s to obtain a photoresist with a thickness of 20 μm, which is uniform and stable. After spin-coating is completed, first dry and fix the photoresist to remove the solvent in the photoresist, pre-bake at 65 °C for 5 min, and then adjust the baking table to 95 °C and maintain it for 15 min; perform exposure, the exposure time is 60 s, and the exposure dose is 150 - 215 mJ / cm 2After exposure, the sample is placed on a baking table for post-baking treatment: First, the temperature is adjusted to 65 °C for 1 min, and then it is adjusted to 95 °C and maintained for 7 min. Finally, development is carried out. The sample is immersed in SU-8 developer, and after development at room temperature, the developer is washed off with ethanol to complete the production of the complementary master plate. The microfluidic channel pattern on the master plate is replicated onto PDMS by casting. After obtaining the PDMS microchannel substrate, the PDMS microchannel substrate and the SU-8 micro-well substrate are first cleaned with isopropanol for 40 - 50 s to remove possible organic substances and impurity particles on the surfaces of the SU-8 substrate and the PDMS substrate, and then washed with deionized water for 1 - 2 min to remove the residual isopropanol and impurity particles; they are dried with clean air or nitrogen. Then, holes are drilled at seven positions: the sample injection port 1 for the protein to be detected containing multiple targets, the sample injection port 2 for the capture antibody-coated magnetic beads and the fluorescence-labeled detection antibody, the cross-flow inlet 3, and the outlets of the first sorting channel 5, the second sorting channel 6, the third sorting channel 7, and the fourth sorting channel 8. Then, oxygen plasma treatment is carried out for 40 - 60 s at a power of 200 - 400 W to hydrophilically modify the bonding surface of PDMS from hydrophobic. Finally, a clean thin glass is taken and pressed and bonded with the PDMS and baked to form a complete microfluidic chip.

[0107] In some specific embodiments of the present invention, as Figure 3 shown, a schematic structural diagram of a mask plate of a rectangular microfluidic chip for hybrid and flow magnetic sorting is presented, including a mixing channel, a magnetic sorting channel, two sample injection ports (including a cross-flow inlet and a magnetic bead inlet), and four outlets. When the sandwich immuno-complex magnetic beads enter through the magnetic bead inlet 4, the buffer solution injected through the cross-flow inlet 3 causes the magnetic beads to focus near the sidewall of the channel, forming a focusing region with a width of 2 - 500 microns. As the magnetic beads flow in the magnetic sorting channel 11, they will 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. Larger-sized magnetic beads are subjected to a greater magnetic force, so the lateral displacement is more significant; while smaller-sized magnetic beads are subjected to a smaller force and have a smaller lateral displacement. 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 the figure, large-sized magnetic beads with high magnetic content (such as 5 microns) enter the first sorting outlet 5, medium-sized magnetic beads with medium magnetic content (such as 2.8 microns) enter the second sorting outlet 6, and small-sized magnetic beads with low magnetic content (such as 1 micron) enter the third sorting outlet 7. The non-magnetic signal probes that do not participate in the reaction are directed to the fourth sorting outlet 8 and enter the waste liquid channel due to the unchanged direction of movement. This design realizes 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 Figure 6As shown, these are the physical pictures of the effects of each stage of microfluidic chip flow magnetic separation. Taking 2.8μm magnetic beads as an example, at the initial stage of the washing and separation area, due to the sheath flow, magnetic particles and excessive fluorescently labeled detection antibodies that are non-magnetic are both restricted near the wall ( Figure 6 A in); as the particles move in the horizontal direction, they are affected by the magnetic force in the vertical direction, and the magnetic particles form pearl-shaped chains ( Figure 6 B in); at the same time, due to the shear force of the flow, the micron-sized magnetic particles gradually become single discrete particles distributed ( Figure 6 C in), while the non-magnetic particles continue to move horizontally along the wall to the Figure 3 No. 8 chamber in, 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 deflected to different vertical positions due to different magnitudes of the magnetic force, and finally enter the three chambers 5-7 respectively.

[0109] In some specific embodiments of the present invention, a blank sample without the protein to be detected, such as PBS solution, is used for testing, and separation is carried out using the magnetic separation microfluidic chip and the magnetic force rack of the present invention respectively. The experimental results show that compared with the magnetic force rack, the magnetic separation microfluidic chip has a lower background signal, and the low noise brings a higher signal-to-noise ratio. As Figure 7 shown, Figure 7 B in is an exemplary detection image of the sorting channel outlet of the blank sample flow magnetic separation microfluidic chip close to the permanent magnet. Figure 7 A in is the effect of washing the same sample with a magnetic force rack. Among them, the blue dots represent the capture antibody-coated magnetic beads that have not bound to the protein to be detected, the green dots represent the sandwich immunocomplex magnetic beads (the image recognition algorithm of the present invention marks the position where the bright-field magnetic beads overlap with the dark-field fluorescently labeled detection antibody as the sandwich immunocomplex magnetic beads), and the red dots represent the unbound free fluorescently labeled detection antibodies. Although there is no protein in the sample and no sandwich immunocomplex can be formed, it can be seen from the image that when sorting with a magnetic force rack, since the fluorescently labeled detection antibody cannot be effectively separated from the capture antibody-coated magnetic beads, during observation, due to the overlap of the capture antibody-coated magnetic beads and the fluorescently labeled detection antibody, they are marked as sandwich immunocomplex magnetic beads, which will bring problems of false positives and high detection background in actual detection. And the experimental result of the present invention is Figure 7 B in, it can be seen that after sorting by the magnetic separation chip, only magnetic beads are included at the sorting channel outlet. The excess fluorescently labeled detection antibody has not been deflected because it is not affected by the magnet and has been washed to the sorting channel outlet farthest from the magnet. Only magnetic beads of uniform size are included at other outlets. Thus, it can be known 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, sorting is performed with high-sensitivity C-reactive protein at different concentrations, 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 as Figure 8 shown. Figure 8 0 fg / mL ( Figure 8 A in), 35 fg / mL ( Figure 8 B in), 350 fg / mL ( Figure 8 C in) are fluorescence imaging diagrams of three different concentrations of high-sensitivity C-reactive protein after passing through the flow magnetic sorting microfluidic detection platform. Based on the ultra-bright fluorescent nanospheres and the image recognition algorithm, it can be seen that under the condition that the number of magnetic beads is more than 10 times the molar number of the protein to be measured, as the concentration of high-sensitivity C-reactive protein increases, the number of sandwich immuno-complex magnetic beads represented by the green dots in the field of view also increases, and single-molecule counting detection of proteins as low as 35 fg / mL can be achieved.

Claims

1. A flow-through magnetic separation microfluidic chip, which includes a sample injection port, a mixing and incubation area, a washing and sorting area, a magnet, and an imaging and detection area; Characterized in that, The sample 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; The sample injection port is used to inject a protein sample to be tested, magnetic beads coated with capture antibodies, and fluorescently labeled detection antibodies; The mixing and incubation area is used to mix 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 area is used to distribute the sandwich immunocomplex magnetic beads containing different protein samples to be tested to the sorting channel outlets in different imaging and detection areas through dynamic magnetic separation; the washing and sorting area is connected to the mixing and incubation area through a magnetic bead inlet. The washing and sorting area 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 to inject 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 in 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 focused area with a width of 2 - 500 μm; The magnet is arranged on one side of the washing and sorting area; and is arranged parallel to the flow-through magnetic separation channel; The imaging and detection area is used to detect 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 injection port is connected to the mixing and incubation area, and the sample injection port includes at least one sample injection port for the protein to be tested and at least one sample injection port 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 area includes a mixing flow channel and a magnetic bead inlet. The mixing flow 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 flow channel; Preferably, the serpentine shape is a serpentine detour shape, the detour angle is 180 degrees, and at least 8 - 10 detours are arranged; Preferably, the width of the magnetic bead inlet is lower than the width of the mixing flow 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 fluorescently 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 syringe pumps, a microfluidic tube and a sampling head; Preferably, the programmable syringe 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 sequentially incident on the imaging detection area of the microfluidic chip through a convex lens and an excitation light filter; along the fluorescence detection optical path, the fluorescence generated by the imaging detection area of the microfluidic chip under the excitation light is sequentially incident on the charge-coupled device CCD camera through an emission light filter and a light path that will transmit the light; 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 or 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 the magnetic beads coated with capture antibodies and the 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, and inject the mixed solution obtained in step S002) into the flow-through magnetic sorting microfluidic chip through the injection port for the magnetic beads coated with capture antibodies and the fluorescently labeled detection antibodies, and mix the multiplex protein sample to be detected and 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 field 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.

Images