Homogeneous multiple immunodetection method based on microscopic image analysis

Abstract

The invention discloses a homogeneous multiple immunodetection method based on microscopic image analysis. The method comprises the following steps: firstly, respectively preparing capture microspheres and detection microspheres; then preparing an immune complex containing'capture microsphere-target protein-detection microsphere '; uniformly spreading the immune complex on a detection slide, and collecting bright field images and dark field fluorescence images of different fluorescent microspheres by using a multi-channel fluorescence microscope; respectively recording center coordinates and radiuses of the capture microspheres and the detection microspheres; aligning the bright field image and the dark field fluorescence image through image registration, and calculating a space overlapping relationship between the capture microspheres and the detection microspheres by using a multi-modal microscopic co-localization calculation method to obtain the number of effective signals connected with the immune complex; and calculating through the standard curve to obtain the concentration of the target protein in the target sample solution to be detected. According to the method, co-localization analysis is adopted, signals are counted one by one, dependence on the overall signal strength is not needed, and simultaneous detection and quantitative detection of multiple targets can be achieved.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and particularly to a homogeneous multiplex immunoassay method and system based on microscopic image analysis for realizing high-sensitivity digital quantitative detection of multiple targets. Background Art

[0002] As an important part of biomolecular recognition technology, the immune reaction has experienced a development process from qualitative analysis to quantitative analysis since it was first used for disease detection, and has gradually become an indispensable tool in modern clinical and scientific research fields. Early immunoassay methods mainly relied on precipitation reactions and agglutination reactions, with low sensitivity and specificity, and could only meet the rough disease diagnosis needs. Subsequently, the advent of radioimmunoassay (RIA) marked the entry of immunoassay into the era of high-sensitivity quantitative detection. However, due to the safety issues associated with the use of radioactive substances, chemiluminescence immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) have gradually become the mainstream. However, despite the significant progress made by modern immunoassay techniques in terms of sensitivity, throughput, and accuracy, traditional methods still have many limitations. For example, CLIA and ELISA are usually limited to single-target detection or limited multi-target detection, and it is difficult to achieve efficient multiplex quantitative analysis.

[0003] Multiplex immune reactions play an increasingly important role in early disease screening, complex biological sample analysis, and new vaccine development. However, the complexity of multi-target detection poses higher requirements on existing technologies, mainly reflected in the following aspects: First, how to achieve efficient target discrimination and quantitative analysis in a limited detection system to avoid signal interference and misjudgment; Second, how to improve the applicability of the detection system to samples with complex backgrounds and reduce the interference of non-specific reactions; Third, how to balance low cost, high sensitivity, and convenient operation while performing multi-target detection. The liquid-phase chip (Luminex) technology, which has developed rapidly in recent years, has the ability to detect multiple targets, but it relies on complex instruments and sample liquid paths, with high operation difficulty and high cost, resulting in limited detection efficiency and popularization. The present invention provides an improved homogeneous multiplex immunoassay method and system by introducing microscopic image analysis and co-localization detection technology, achieving high-throughput and accurate quantitative analysis of multiple targets. Summary of the Invention

[0004] To solve at least one of the above problems, the present invention provides a homogeneous multiplex immunoassay method based on microscopic image analysis. By utilizing the immune complex formation mechanism of capture microspheres and detection microspheres, and through bright-field and dark-field fluorescence imaging and image processing techniques, efficient detection and statistics of target signals are completed without relying on the overall signal intensity, meeting the requirements of multi-target quantitative analysis.

[0005] In order to achieve the above object, the present invention adopts the following technical means:

[0006] The first aspect of the present invention discloses a homogeneous multiple immunoassay method based on microscopic image analysis, comprising the following steps:

[0007] S1. Preparation of capture microspheres: coating the capture antibody on the surface of microsphere C1 to form capture microspheres;

[0008] S2. Preparation of detection microspheres: coating the detection antibody on the surface of microsphere D1 to form detection microspheres;

[0009] S3. Preparation of immune complexes:

[0010] Add capture microspheres and detection microspheres to a solution containing the target protein, mix and fully incubate to obtain an immune complex containing "capture microspheres-target protein-detection microspheres";

[0011] The target protein solution includes a target sample solution to be detected and a target protein calibrator solution with known gradient concentrations;

[0012] S4, image acquisition: the immune complex prepared in S3 is evenly spread on a detection slide, and a multi-channel fluorescence microscope is used to collect bright field images of different fluorescent microspheres in a bright field detection mode and dark field images in a dark field fluorescence detection mode under different channels;

[0013] S5. Signal processing and statistics: Use bright field images to record the center coordinates and radius of the captured microspheres, and use dark field fluorescence images to record the center coordinates and radius of different fluorescence signals of the detected microspheres through multi-channel detection; align the bright field image and the dark field fluorescence image through image registration, and use the multimodal microscopic co-localization calculation method to calculate the spatial overlap relationship between the captured microspheres and the detected microspheres to obtain the number of effective signals of immune complex connection;

[0014] S6. Quantitative analysis: Use target proteins with known gradient concentrations as calibrators to establish a standard curve between the number of effective signals and the concentration of the target protein. Then, based on the number of signals in the sample solution to be tested, the concentration of the target protein in the target sample solution to be tested is calculated using the standard curve.

[0015] In some embodiments of the present invention, the microscope can switch between bright-field detection and dark-field fluorescence detection modes; in the bright-field detection mode, the sample is scanned and imaged, and the image is saved; in the dark-field fluorescence detection mode, the sample is scanned and imaged, and multi-channel detection is achieved through different excitation light and detection light filters. For example, channels 1, 2, and 3 can respectively collect the signals of fluorescent microspheres D1a, D1b, and D1e, and output three photos taken in different channels; the signal acquisitions of fluorescent microspheres D1a, D1b, and D1e do not interfere with each other.

[0016] In some embodiments of the present invention, the multi-modal microscopic co-localization calculation method in step S5 is as follows:

[0017] S1. Bright-field image processing: The background noise is removed by using existing noise reduction techniques, the spheres are segmented by an algorithm, the contours and center coordinates of the captured microspheres are extracted, and the center coordinates (x A , y A , and the radius r A ) of each captured microsphere C A are recorded, where A = 1, 2, 3... n, representing the number of captured microspheres;

[0018] S2. Dark-field image processing: The image signals of each fluorescence channel are enhanced respectively, the detected microspheres are segmented separately for each fluorescence channel, and the center coordinates (x 1a , y D1a , and the radius r D1a ) of the detected microsphere D D1a are extracted, where a = 1, 2, 3... n; representing the number of detected microspheres;

[0019] S3. Image registration: The feature points of the bright-field image and the dark-field image are extracted respectively, and the image alignment registration is completed based on the affine transformation or perspective transformation of feature point matching;

[0020] S4. Co-localization analysis: For the center coordinates (x A , y A ) of each captured microsphere, a neighborhood query radius d A = r A + Δ is constructed, where Δ is the redundant width for connection detection;

[0021] For the neighborhood of each captured microsphere, it is checked whether the center of the detected microsphere falls within the neighborhood range. If there is a detected microsphere in the neighborhood of the captured microsphere and the distance between the two satisfies , it is determined that there is a co-localization relationship between the captured microsphere and the detected microsphere, and it is recorded as a valid signal.

[0022] In some embodiments of the present invention, the redundant width Δ for connection detection is 0.15 - 0.3 μm.

[0023] In some embodiments of the present invention, when counting the number of detection microspheres in the neighborhood of each capture microsphere, if a capture microsphere is connected to multiple detection microspheres, each connection is counted as an independent valid signal number. If a detection microsphere is simultaneously connected to multiple capture microspheres, each pair of connections is counted as an independent valid signal number.

[0024] In some embodiments of the present invention, for the detection of target protein a, the principle of "colocalization counting" and the method for realizing the detection are as follows: Collect all the signal positions of C1 in bright field, and collect all the signal positions of D1a in dark field channel 1. Only the signals where the positions of C1 and D1a overlap will be counted as a valid signal; the number of valid signals depends on the number of fluorescent microspheres D1a and magnetic beads C1 that meet the colocalization counting rules. In case one, there is 1 fluorescent microsphere D1a and 1 magnetic bead C1 at the same coordinate position, which is counted as 1 valid signal; in case two, there are n fluorescent microspheres D1a and 1 magnetic bead C1 at the same coordinate position, which is counted as n valid signals; in case three, there is 1 fluorescent microsphere D1a and m magnetic beads C1 at the same coordinate position, which is counted as m valid signals.

[0025] In some embodiments of the present invention, the capture microspheres described in step S1 are magnetic microspheres. For different detection target samples, the specifications of the magnetic beads can be the same or different, and the capture antibodies coated are different due to different detection targets. In some embodiments of the present invention, the particle size of the magnetic beads is 1500 nm.

[0026] In some embodiments of the present invention, the detection microspheres described in step S2 are fluorescent microspheres. For different detection target samples, fluorescent microspheres with different excitation lights are selected. In some embodiments of the present invention, the particle size of the fluorescent microspheres is 200 nm.

[0027] In some embodiments of the present invention, a uniform magnetic field is applied below the detection slide in step S4 to make the immune complexes evenly spread on the surface of the slide.

[0028] In some embodiments of the present invention, according to the number of detection targets in the solution of the target sample to be detected, the types of capture microspheres and detection microspheres are determined. In some embodiments of the present invention, if the detection targets are two proteins a and b, there are two types of capture microspheres, namely capture microspheres C1a coated with anti-a antibody and capture microspheres C1b coated with anti-b antibody. At the same time, there are also 2 types of detection microspheres, namely detection microspheres D1a coated with anti-a antibody and detection microspheres D1b coated with anti-b antibody. Similarly, if the detection targets in addition to proteins a and b also contain protein e, the capture microspheres in addition to C1a and C1a also contain C1e. At the same time, there are also 3 types of detection microspheres, namely D1a, D1b and D1e.

[0029] In some embodiments of the present invention, the mixing and incubation in the preparation of the immune complex in step S3 are as follows: vortex mix for 25 - 35 s, and then place it on a mixer and rotate and incubate it in a constant temperature incubator at 250 - 300 r and 37 °C for 0.5 - 1 h.

[0030] In some embodiments of the present invention, the method for preparing the capture microspheres is as follows: mix microspheres C1 and an activator and react for a period of time. After the surface of microspheres C1 is activated, wash away the excess unreacted activator adhered to microspheres C1 with a washing buffer; add a certain amount of the sample of the capture antibody to the container where the activated microspheres C1 are located. After sufficient incubation, part of the capture antibody binds to the surface of microspheres C1; wash away the excess unbound capture antibody with a washing buffer, and the collected microspheres C1 with the capture antibody bound to the surface are the capture microspheres.

[0031] In some embodiments of the present invention, the method for preparing the detection microspheres is as follows: mix fluorescent microspheres D1 and an activator and react for a period of time. After the surface of the fluorescent microspheres D1 is activated, wash away the excess unreacted activator adhered to the fluorescent microspheres D1 with a washing buffer; add a certain amount of the sample of the detection antibody to the container where the activated fluorescent microspheres D1 are located. After sufficient incubation, part of the detection antibody binds to the surface of the fluorescent microspheres D1; wash away the excess unbound detection antibody with a washing buffer, and after blocking, the collected fluorescent microspheres D1 with the detection antibody bound to the surface are the detection microspheres; the preparation process of the detection microspheres is strictly carried out under light - proof conditions.

[0032] Advantages of the present invention

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. Strong multi - target detection ability: When there are multiple detection targets in the target solution to be detected, multi - channel fluorescence and image processing technologies can be used to simultaneously detect and quantitatively detect multiple targets.

[0035] 2. High digitization and precision: Based on the co - localization analysis method in this application, signal counting is realized one by one, without relying on the overall signal intensity.

[0036] 3. Simple operation and wide applicability: It supports the microscope platform, does not require complex instruments, and is suitable for a variety of detection scenarios.

[0037] 4. High specificity and resolution: Through microscopic image analysis technology, accurate detection with a high signal - to - noise ratio is realized.

[0038] 5. Excellent cost - effectiveness: Compared with flow - through fluorescence technology and chemiluminescence technology, the instrument requirements are low and the experimental operation has strong flexibility. Brief Description of the Drawings

[0039] Figure 1 It shows a schematic diagram of the detection method of the present invention;

[0040] Figure 2 It shows the images of the immune complexes detected under the microscope in each channel in the embodiment;

[0041] Figure 3 It shows the standard curve obtained according to the IL-6 standard product in the embodiment;

[0042] Figure 4 It shows the standard curve obtained according to the IL-10 standard product in the embodiment. Detailed Description of the Invention

[0043] The following examples are used herein to demonstrate the preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention, and thus can be regarded as preferred embodiments for implementing the present invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, and still obtain the same or similar results, without departing from the spirit or scope of the present invention.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The materials cited herein and their citations will be incorporated by reference. Those skilled in the art will recognize or be able to learn many equivalent techniques to many of the specific embodiments of the invention described herein through routine experimentation. These equivalents will be included in the claims.

[0045] The technical solutions of the present application will be further described in detail below in conjunction with the specific embodiments.

[0046] Example 1: Preparation Method of Immune Microspheres

[0047] This example provides a preparation method of immune microspheres coated with anti-human IL-6 antibody and anti-human IL-10 antibody, which specifically includes the following contents:

[0048] 1. Preparation of Capture Microspheres

[0049] Take magnetic microspheres (JSR MS160) with a diameter of 1.5 μm and surface carboxyl modification and coat them with anti-human IL-6 antibody and anti-human IL-10 antibody respectively.

[0050] First, take 1 mg of magnetic beads and place them in a 2 mL centrifuge tube. Add 200 μL of MES buffer (25 mM, pH = 5), vortex to mix evenly, separate the magnetic beads on a magnetic stand, and use a pipette to remove the supernatant. Repeat this step a total of 2 times to wash the magnetic beads.

[0051] To the washed magnetic beads, add 200 μL of MES buffer (25 mM, pH = 5), 5 μL of 50 mg / mL NHS, and 5 μL of 50 mg / mL EDC respectively, vortex to mix evenly, and activate at room temperature for 1 h on a shaker at 250 r;

[0052] Wash the activated magnetic beads 2 times with 200 μL of MES buffer (25 mM, pH = 5), and collect the magnetic beads by magnetic absorption;

[0053] Resuspend the magnetic beads in 200 μL of MES buffer (25 mM, pH = 5), add 15 μg of antibody, vortex to mix evenly, and activate at room temperature for 2.5 h on a shaker at 250 r;

[0054] After the reaction, remove the supernatant by magnetic separation, collect the magnetic beads, add 200 μL of blocking solution (50 mM Tris buffer pH = 7.4, containing 0.5% BSA, 0.05% TW - 20), and incubate at room temperature for 1 h on a shaker at 500 r for the blocking reaction;

[0055] After blocking, wash the magnetic beads 3 times with the washing solution (10 mM PBS, 0.2 wt% Tween 20), and store them in 200 μL of magnetic bead storage solution (50 mM Tris buffer pH = 7.4, containing 0.5% BSA, 0.1% TW - 20)

[0056] 2. Preparation of detection microspheres

[0057] Select orange fluorescent microspheres with a particle size of 200 nm to coat with anti - human IL - 6 antibody, and select red fluorescent microspheres with a particle size of 200 nm to coat with anti - human IL - 10 antibody.

[0058] Take 25 μL of fluorescent microspheres (1% solid content) and add them to 1 mL of MES buffer solution (50 mM, pH = 5.5), centrifuge at 12000 rpm for 8 min to collect the microspheres, and then redissolve them in MES and sonicate for 2 min.

[0059] Add 4 μL of 5 mg / mL EDC and 40 μL of 5 mg / mL NHS in sequence, first vortex for 3 s to mix evenly, then hold the tube and sonicate for 1 min to disperse the microspheres, and then place them on a shaker at 200 r in the dark at 37 °C for 25 min for activation.

[0060] After activation, centrifuge at 12000 rpm for 8 min to collect the microspheres.

[0061] Wash once with 1 mL of PBST buffer solution (10 mM PBS, pH = 7.4, 0.05% Tween-20), then centrifuge at 12,000 rpm for 8 min to collect the microspheres.

[0062] The microspheres were resuspended in 1 mL of PBST buffer solution and dispersed by sonication for 1 min. Add 10 μg of antibody, vortex to mix well, and incubate in the dark at 250 r on a shaker at 37 °C for 2 h.

[0063] After incubation, add 20 μL of 20% BSA, vortex to mix well, and block in the dark at 250 r on a shaker at 37 °C for 0.5 h.

[0064] After blocking, wash three times with 1 mL of PBST buffer solution (10 mM PBS, pH = 7.4, 0.05% Tween-20) and redissolve in 200 μL of PBS buffer (10 mM, pH = 7.4).

[0065] Example 2: Drawing of the standard curve for the homogeneous multiplex immunoassay method based on microscopic image analysis

[0066] This example provides a standard curve establishment scheme for dual-target detection based on IL-6 and IL-10, including the following steps:

[0067] Step 1: Preparation of standards.

[0068] Dilute IL-6 and IL-10 standard proteins with PBS buffer solution (10 mM, pH = 7.4) respectively to prepare solutions with concentrations of IL-6 and IL-10 both being 100 ng / mL. Then, use the serial dilution method to sequentially dilute the concentration of the standard protein by 10 times to obtain solutions with concentrations of IL-6 and IL-10 both being 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, 1 pg / mL, and 0 pg / mL. Prepare 3 portions of each concentration solution, with each solution volume being 200 μL, and label them as S1, S2, S3, S4, S5, and S6 respectively.

[0069] Step 2: Establishment of the standard curve.

[0070] Detect and record the values of the above S1 - S6 samples, and take the average of the three detections as the final signal response value of the sample. Take the Log values respectively with the sample concentration as the abscissa and the final signal response value of the sample as the ordinate, draw a scatter plot, perform linear fitting on the scatter plot to establish the standard curve, and calculate the goodness of fit R 2 , The standard curve obtained from the IL-6 standard is as Figure 3 shown; The standard curve obtained from the IL-10 standard is as Figure 4as shown

[0071] Among them, the summary of the test data of the IL-6 standard product is shown in Table 1 as follows

[0072] Table 1 Summary Table of Test Data of IL-6 Standard Product

[0073]

[0074] The summary of the test data of the IL-10 standard product is shown in Table 2 as follows

[0075] Table 2 Summary Table of Test Data of IL-10 Standard Product

[0076]

[0077] Fitting curves from the above data, the fitting curve for the IL-6 standard product test is y = 0.535x + 0.8456, and the linear correlation coefficient R 2 = 0.9952. The fitting curve for the IL-10 standard product test is Y = 0.4637x + 1.0904, and the linear correlation coefficient R 2 = 0.9821, indicating that within the detection range of 1 - 10000 pg / mL, the linear correlation is very good

[0078] Example 3: Implementation Scheme of Homogeneous Multiplex Immunoassay Method Based on Microscopic Image Analysis

[0079] Select 10 serum samples with known IL-6 and IL-10 concentrations assigned by a chemiluminescence kit, perform dual-target testing using the homogeneous multiplex immunoassay method based on microscopic image analysis of the present invention, and compare the test results with the sample concentration values to evaluate its accuracy and reliability

[0080] Step 1: Prepare immune microsphere complexes

[0081] Respectively take 5 μL of the capture microspheres prepared in Example 1 and 2 μL of the detection microspheres prepared in Example 1 and add them to 100 μL of the specimen solution to be tested. Mix well with a vortex oscillator for 30 s, and then place it in an incubator at 250 r and 37 °C on a mixer and rotate and incubate for 0.5 h to obtain the prepared immune complexes

[0082] Step 2: Image extraction and analysis

[0083] Vortex the above-prepared immune complexes evenly, take 5 μL and spread it evenly on a glass slide, and then place the slide on a stage with a uniform magnetic field applied at the bottom and let it stand for 2 min

[0084] Image acquisition is performed using a microscope with a fluorescence module. When the microscope is switched to bright field, the focal length is adjusted until the image is clear, and the CMOS camera's photographing function is used to save the image of the sample under bright field; when switched to the orange fluorescence channel, the focal length is adjusted until the image is clear, and the CMOS camera's photographing function is used to save the image of the sample under the orange fluorescence channel; when switched to the red fluorescence channel, the focal length is adjusted until the image is clear, and the CMOS camera's photographing function is used to save the image of the sample under the red fluorescence channel.

[0085] Extract the images, process the images using computer software, and record the number of effective signals of the captured microsphere - orange fluorescence detection microsphere complex and the captured microsphere - red fluorescence detection microsphere complex. Substitute the effective signal data into the standard product fitting curves corresponding to IL-6 and IL-10 in Example 2 to obtain the corresponding concentration values of IL-6 and IL-10. The detection comparison results of the clinical assignments and test values of different sample concentrations of IL-6 and IL-10 are shown in Table 3.

[0086] Table 3 Comparison of the results of different sample concentrations of IL-6 and IL-10

[0087]

[0088] The results show that the test results are basically consistent with the clinical assignments, and the error range is within ±8%. Through linear regression analysis, the correlation coefficients (R 2 ) of the test results and clinical assignments of 10 samples are all greater than 0.99, indicating that the linear correlation between the test results of the method of the present invention and the chemiluminescence method is good, and the difference is small.

[0089] All the documents mentioned in the present invention are cited in this application as references, just as if each document is cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by this application.

Claims

1. A homogeneous multiple immunoassay method based on microscopic image analysis, characterized in that: The steps include: S1, preparation of capture microspheres: coating the capture antibody on the surface of microsphere C1 to form capture microspheres; S2. Preparation of detection microspheres: coating the detection antibody on the surface of microsphere D1 to form detection microspheres; S3. Preparation of immune complexes: Add capture microspheres and detection microspheres to a solution containing the target protein, mix and fully incubate to obtain an immune complex containing "capture microspheres-target protein-detection microspheres"; The target protein solution includes a target sample solution to be detected and a target protein calibrator solution with known gradient concentrations; S4, image acquisition: the immune complex prepared in S3 is evenly spread on a detection slide, and a multi-channel fluorescence microscope is used to collect bright field images of different fluorescent microspheres in a bright field detection mode and dark field images in a dark field fluorescence detection mode under different channels; S5. Signal processing and statistics: Use bright field images to record the center coordinates and radius of the captured microspheres, and use dark field fluorescence images to record the center coordinates and radius of different fluorescence signals of the detected microspheres through multi-channel detection; align the bright field image and the dark field fluorescence image through image registration, and use the multimodal microscopic co-localization calculation method to calculate the spatial overlap relationship between the captured microspheres and the detected microspheres to obtain the number of effective signals of immune complex connection; S6. Quantitative analysis: Use target proteins with known gradient concentrations as calibrators to establish a standard curve between the number of effective signals and the concentration of the target protein. Then, based on the number of signals in the sample solution to be tested, the concentration of the target protein in the target sample solution to be tested is calculated using the standard curve.

2. The method according to claim 1, characterized in that: The multimodal microscopic colocalization calculation method in step S5 is as follows: S1. Bright field image processing: Use existing noise reduction technology to remove background noise, segment the sphere through the algorithm, extract the outline and center coordinates of the captured microsphere, and record the C of each captured microsphere. A The center coordinates (x A ,y A ) and radius r A , A=1.2.3...n; S2. Dark field image processing: Enhance the image signal of each fluorescent channel separately, segment the detection microspheres for each fluorescent channel, and extract the detection microspheres D 1a The center coordinates (x D1a ,y D1a ) and radius r S1a , a=1.2.3...n; S3, image registration: extract feature points of bright field image and dark field image respectively, and complete image alignment and registration based on affine transformation or perspective transformation of feature point matching; S4. Co-localization analysis: The center coordinates (x A ,y A ), construct the neighborhood query radius d A =r A +Δ, Δ is the redundant width of connection detection; For each captured microsphere neighborhood, check whether the center of the detection microsphere falls within the neighborhood. If there is a detection microsphere in the captured microsphere neighborhood and the distance between the two satisfies It is determined that the capture microspheres and the detection microspheres are co-localized and recorded as valid signals.

3. The method according to claim 2, characterized in that: The redundant width Δ of the connection detection is 0.15-0.3 μm.

4. The method according to claim 2, characterized in that: For each capture microsphere, when counting the number of detection microspheres in its neighborhood, if a capture microsphere is connected to multiple detection microspheres, each connection is counted as an independent valid signal number; if a detection microsphere is connected to multiple capture microspheres at the same time, each pair of connections is counted as an independent valid signal number.

5. The method according to claim 1, characterized in that: The capture microspheres described in step S1 are magnetic microspheres. For different detection target samples, the specifications of the magnetic beads are the same or different, and the coated capture antibodies are different depending on the detection target.

6. The method according to claim 1, characterized in that: The detection microspheres in step S2 are microspheres with fluorescence, and fluorescent microspheres with different excitation lights are selected for different detection target samples.

7. The method according to claim 1, characterized in that: In step S4, a uniform magnetic field is applied under the detection slide to make the immune complex spread evenly on the surface of the slide.

8. The method according to claim 1, characterized in that: The types of capture microspheres and detection microspheres are determined according to the amount of the target to be detected in the target sample solution.

9. The method according to claim 1, characterized in that: In step S3, the mixing and incubation in the preparation of the immune complex is as follows: vortex mixing for 25-35 seconds, and then rotating and incubating in a constant temperature box at 250-300r and 37°C on a mixer for 0.5-1h.

10. The method according to claim 1, characterized in that: The preparation method of the capture microsphere / detection microsphere is as follows: the capture microsphere / detection microsphere and the activator are mixed and reacted for a period of time, and after the surface of the capture microsphere / detection microsphere is activated, the excess activator adhering to the capture microsphere / detection microsphere that has not reacted is washed away with a washing buffer; a certain amount of capture / detection antibody sample is added to the container where the activated capture microsphere / detection microsphere is located, and after sufficient incubation, part of the capture antibody / detection antibody is bound to the surface of the capture microsphere / detection microsphere; the excess unbound capture antibody / detection antibody is washed away with a washing buffer, and the microspheres with the capture antibody / detection antibody bound to the surface obtained are the capture microsphere / detection microsphere; During the preparation of the detection microspheres, the operation was strictly protected from light.

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