Ultra-sensitive immunofluorescence detection method

Abstract

The invention belongs to the technical field of immunofluorescence detection, and particularly discloses an ultra-sensitive immunofluorescence detection method. Aiming at the problems of low sensitivity, poor repeatability and high cost of the existing method, the method comprises the following steps: capturing a target by adopting coated antibody-labeled magnetic microspheres, sequentially reacting with a biotin-labeled detection antibody and streptavidin-labeled horse radish peroxidase, and carrying out signal amplification dyeing by catalyzing casein fluorescent dye activation through peroxidase. And finally, percolating by using a microporous filter membrane or enriching by using a micro-fluidic chip, and detecting by using a fluorescence reading instrument or a microscope. According to the method, through multi-stage signal amplification and enrichment concentration, ultra-high-sensitivity detection below the picogram level is realized; the magnetic microspheres are used as reaction carriers, rapid separation, purification and washing are realized through magnetic field control, and the repeatability is remarkably improved; meanwhile, the antibody dosage is greatly reduced, the equipment requirement is lowered, and high sensitivity, high repeatability and economical efficiency are achieved.

Description

A highly sensitive immunofluorescence detection method Technical Field

[0001] This invention relates to the field of immunofluorescence detection technology, and more specifically to an ultrasensitive immunofluorescence detection method. Background Technology

[0002] Currently, methods for detecting low concentrations of proteins or small molecule compounds mainly include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), radioimmunoassay (RIA), and mass spectrometry (MS). Among these, CLIA and ELISA are more widely used, but each has its limitations.

[0003] Enzyme-linked immunosorbent assay (ELISA) involves many steps, and the incubation and washing conditions are prone to fluctuations, affecting the reproducibility of the results and resulting in poor precision. Moreover, the concentration level detected by this method is usually in the nanogram per milliliter range, and its sensitivity cannot meet the requirements for detecting ultra-low concentrations of proteins or small molecule compounds (such as less than 1 ng / ml).

[0004] Chemiluminescence assays are more sensitive than enzyme-linked immunosorbent assays (ELISA), but they are more expensive and the luminescent products are often unstable, which greatly affects the accuracy and repeatability of the test results. They also pose a significant challenge for detecting ultra-low concentrations of proteins or small molecule compounds at the picogram level per milliliter. Summary of the Invention

[0005] The present invention aims to improve the sensitivity and repeatability of detection results, and to achieve accurate detection of ultra-low concentration proteins or small molecule compounds. At the same time, the method adopted is economical and easy to popularize and apply.

[0006] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0007] A highly sensitive immunofluorescence detection method includes the following steps:

[0008] (1) Label magnetic microspheres with coated antibodies;

[0009] (2) The antibody-labeled magnetic microspheres are reacted with the protein sample to be tested to obtain a magnetic microsphere-antibody-target protein complex;

[0010] (3) Biotin-activated esters are reacted with the detection antibody to label the detection antibody with biotin;

[0011] (4) The magnetic microsphere-coated antibody-target protein complex is reacted with biotin-labeled detection antibody to obtain the magnetic microsphere-coated antibody-target protein-detection antibody-biotin complex;

[0012] (5) React the complex obtained in step (4) with streptavidin-labeled horseradish peroxidase (SA-HRP);

[0013] (6) Fluorescent staining: Add a solution containing hydrogen peroxide (H2O2) and tyrosine fluorescent dye to the reaction product. Under the catalysis of horseradish peroxidase, the tyrosine fluorescent dye is activated into free radicals and covalently binds to tyrosine residues on the protein, thus amplifying the fluorescent staining signal of the reaction product.

[0014] (7) Detection and analysis: After enriching the fluorescently stained reaction products by percolation through a microporous membrane or by centrifugation using a microfluidic chip, qualitative or quantitative detection and analysis are performed using a fluorescence reader or a fluorescence microscope. The fluorescence reader or fluorescence microscope are readily available and conventional equipment, and this invention does not impose specific limitations on them.

[0015] Preferably, in step (1), the magnetic microspheres are superparamagnetic microspheres with a particle size of 0.5-10 micrometers.

[0016] Preferably, in step (3), the biotin-activated ester is a sulfonic acid group -NHS- (long chain). n -Biotin (SulfoNHS-(LC)) n -Biotin), where the number of long chains n depends on the steric hindrance of the antibody-protein binding structure and its corresponding immune activity. When the steric hindrance is large, a long chain with a larger n value can be selected, and vice versa, a long chain with a smaller n value or no long chain when the n value is zero can be selected.

[0017] Preferably, in step (6), the tyramide fluorescent dye is AF488 tyramide, AF546 tyramide, Cyanine 3 labeled tyramide, or other tyramide fluorescent dyes.

[0018] Preferably, in step (7), the microporous filter membrane is a polyethersulfone (PES) filter membrane, a hydrophilic polyvinylidene fluoride (PVDF) filter membrane, or a nitrocellulose (NC) membrane, and the pore size of the filter membrane is smaller than the particle size of the magnetic microspheres.

[0019] Preferably, in step (7), when using a microporous membrane to percolate and enrich the fluorescently stained reaction product, a magnet is first placed at the bottom of the microporous membrane before the reaction product is added dropwise to the microporous membrane, or a microporous membrane with a magnet at the bottom is selected for percolation and enrichment. The use of a magnet can accelerate the percolation speed of the magnetic balls, and when the sample volume is relatively large, the use of a magnet can focus the magnetic balls in a specific area, preventing the diffusion area from being too large and unfavorable for detection. The specific focusing area can be achieved by adjusting the size of the magnet and the placement area according to actual needs. In this invention, the microporous membrane can be used alone or in conjunction with a magnet.

[0020] Preferably, in step (7), the microfluidic chip is a disk-type microfluidic chip, which includes one or more microchannels, an array of chip holes with a diameter slightly larger than the magnetic microsphere particle size, a magnet below the microchannel, a sample inlet on one side of the microchannel, and a liquid storage tank on the other side; a cover plate is provided on the top of the microchannel, and an air hole is provided on one end of the liquid storage tank on the cover plate.

[0021] Preferably, in step (7), the qualitative analysis method using a fluorescence reader is as follows: the reaction product stained with fluorescence is first dropped onto a microporous filter membrane for percolation enrichment, the fluorescence signal intensity is detected using a fluorescence reader, and a threshold is set in conjunction with a negative control to complete the interpretation of positive and negative results.

[0022] Preferably, in step (7), the quantitative analysis method using a fluorescence reader is as follows: First, the target protein sample of known concentration is diluted according to a gradient concentration. Then, different concentrations of target protein fluorescent staining products are prepared using steps (1)-(6). Next, the different concentrations of target protein fluorescent staining products are dropped onto a microporous filter membrane for percolation enrichment. The fluorescence signal intensity at each concentration is detected using a fluorescence reader, a quantitative standard curve is established, and quantitative detection and analysis are performed (the concentration of subsequent protein samples can be determined by substituting the detected fluorescence signal intensity into the standard curve).

[0023] Preferably, in step (7), the qualitative analysis method using fluorescence microscopy is as follows: First, the reaction product stained with fluorescence is dropped onto the sample port of the disc-type microfluidic chip. By centrifugation, the liquid of the reaction product stained with fluorescence is moved from one end of the sample port of the chip to the storage tank. During the movement of the magnetic microspheres, they fall into the chip holes under the action of magnetic force. Then, the chip is placed under a fluorescence microscope for imaging, and the number of luminescent microspheres is collected. Combined with the negative control, the threshold is set, and the positive and negative interpretation of the detection results can be completed.

[0024] Preferably, in step (7), the quantitative analysis method using fluorescence microscopy is as follows: First, the target protein sample of known concentration is diluted according to a gradient concentration. Then, different concentrations of target protein fluorescent staining products are prepared using steps (1)-(6). Next, the liquid of different concentrations of target protein fluorescent staining products is moved from one end of the chip sample port to the storage tank. During the movement of the magnetic microspheres, they fall into the chip holes under the action of magnetic force. Then, the chip is placed under a fluorescence microscope for imaging. The number of luminescent microspheres corresponding to different concentrations of target protein fluorescent staining products is collected, a quantitative standard curve is established, and quantitative detection and analysis are performed (the concentration of subsequent protein samples can be determined by substituting the fluorescence signal intensity into the standard curve).

[0025] The present invention has the following beneficial effects:

[0026] 1. Significantly Improved Detection Sensitivity. In conventional immunoassay methods using microspheres as coating carriers, small-diameter microspheres have a large specific surface area but relatively low detection sensitivity, while large-diameter microspheres are prone to sedimentation, reducing reaction efficiency and causing poor reagent stability. The magnetic microspheres used in this method have a particle size between 0.5-10 μm, ensuring not only high detection sensitivity but also good reagent stability and high reaction efficiency. Furthermore, this method employs a multi-stage signal amplification system, including a biotin-streptavidin signal amplification system (BAS), superimposed with horseradish peroxidase (HRP)-catalyzed TSA (tyramine signal amplification) technology. The fluorescently stained reaction products, after multi-stage signal amplification, are then filtered and enriched through a microporous membrane, further concentrating the fluorescent products and enhancing the fluorescence signal intensity per unit scale, thereby further improving detection sensitivity.

[0027] 2. Improved repeatability. This method uses magnetic microspheres as the core reaction carrier. By adding or subtracting magnetic fields, product purification and washing can be completed simply and quickly, reducing the fluctuations in detection results introduced by multiple steps in traditional immunoassay, such as manual processing or centrifugation, thus improving the repeatability of the results. Furthermore, the fluorescent products obtained after enrichment and separation in this method are more stable (especially the dry fluorescent products obtained by microporous membrane filtration), and the fluorescence signal remains stable over a long period, which also contributes to improved repeatability. Using the microporous membrane-based detection method of this invention to test a 5 pg / ml IL-6 protein sample, the intra-day precision (n=10) CV was 5.2%, and the inter-day precision (n=40) CV was 8.7%, showing that the microfluidic chip-based detection method is superior. In contrast, the same sample detected using chemiluminescence immunoassay showed an intra-day precision (n=10) CV of 11.5% and an inter-day precision (n=40) CV ≤ 18.2%.

[0028] 3. Reduced detection costs and improved economic efficiency of detection applications. In immunoassay methods, antibodies, as the core active raw materials, are usually expensive and account for a major portion of the cost of the detection reagents. Thanks to the multi-stage signal amplification system used in this method, the amount of antibody raw material used in the reaction system is less than that used in conventional methods, greatly reducing the cost of raw materials. At the fluorescence signal acquisition end, the reaction product only needs to be filtered and enriched on the filter membrane, and the fluorescence signal can be read using a fluorescence reader; or the reaction product can be dropped onto the microfluidic chip, and the microspheres can fall into the chip wells, allowing fluorescence imaging analysis of individual magnetic microspheres using a fluorescence microscope, greatly reducing the need for expensive equipment or complex liquid circuit design and control. Attached Figure Description

[0029] Figure 1 shows the process flow of the detection method of the present invention.

[0030] Figure 2 is a photograph of the reaction product after fluorescent staining in Example 1 being trapped and enriched on the surface of the filter membrane.

[0031] Figure 3 shows the fluorescence chromatograms (partial) of the negative control and positive sample in Example 1.

[0032] Figure 4 is the standard curve of protein concentration-fluorescence intensity fitting in Example 2.

[0033] Figure 5 is a schematic diagram of the structure of the disc-type microfluidic chip. In the figure, (a) is a top view of the disc-type microfluidic chip, and (b) and (c) are the top view and side sectional view of the microchannel in the disc-type microfluidic system, respectively. The numbers refer to the component names as follows: 1-microchannel, 11-sample loading port, 12-chip aperture, 13-magnet, 14-liquid reservoir, 15-vent.

[0034] Figure 6 is the standard curve of protein concentration-fluorescence intensity fitting in Example 4. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. All the following embodiments use human interleukin-6 (IL-6) as the target protein for verification, and the process flow is shown in Figure 1.

[0036] Unless otherwise specified, all reagents used in the following embodiments are commercially available and conventional reagents, and all experimental procedures used are conventional procedures in the field unless otherwise specified.

[0037] The main equipment and raw material information is as follows:

[0038]

[0039] Example 1

[0040] This embodiment provides a qualitative detection method based on a microporous filter membrane, the specific steps of which are as follows:

[0041] 1. Superparamagnetic microspheres with a particle size of 1.5 μm were labeled with a coating antibody.

[0042] 1.1 Weigh 1 mg of magnetic beads into a 1.5 ml centrifuge tube using an electronic balance, add 500 μl of DMF, and vortex to mix well so that the magnetic beads are evenly suspended in DMF;

[0043] 1.2 Place the magnetic ball on the magnetic rack, add 1 ml of PBST washing buffer (10 mM phosphate, 0.1% Tween 20, pH 7.4) and wash twice, discarding the supernatant on the last wash;

[0044] 1.3 Dilute the mouse anti-human IL-6 monoclonal antibody to 50 μg / ml using PBST;

[0045] 1.4 Take 100 μl of the diluted antibody solution and add it to the centrifuge tube containing the magnetic beads from step 1.2. Resuspend and mix well, then place in a mixer at room temperature and shake overnight.

[0046] 1.5 Transfer the magnetic ball to the magnetic rack, apply a magnetic field, and discard the supernatant;

[0047] 1.6 Add 1 ml of PBST washing buffer and wash twice, discarding the supernatant on the last wash;

[0048] 1.7 Add 1 ml of blocking solution (10 mM phosphate, 0.1% Tween 20, 1% bovine serum albumin, pH 7.4), and then place in a mixer and shake at room temperature for 2 hours.

[0049] 1.8 Add 1 ml of PBST washing buffer and wash twice, discarding the supernatant on the last wash.

[0050] 2. The antibody-labeled magnetic microspheres are reacted with the sample to obtain a magnetic microsphere-antibody-target protein complex.

[0051] 2.1 Dilute the IL-6 protein sample to 0.1 ng / ml using PBST solution; the negative control is a PBST solution without IL-6 protein.

[0052] 2.2 Take 100 μl of IL-6 protein dilution solution and add it to the centrifuge tube containing magnetic beads from step 1.8. Resuspend and mix well, then place it in a mixer and shake at room temperature for 30 minutes.

[0053] 3. Biotin is labeled onto the detection antibody by reacting it with sulfonic acid-NHS-Biotin (Sulfo NHS-Biotin).

[0054] 3.1 Dilute the IL-6 detection antibody to 1 mg / ml using PBST solution;

[0055] 3.2 Take 5 μl of 1 mg / ml Sulfo NHS-Biotin solution, add 1 μl of the above-diluted IL-6 detection antibody diluent, and then add 14 μl of purified water to a total volume of 20 μl. Incubate at room temperature for 2 hours.

[0056] 4. React the magnetic microsphere-coated antibody-target protein complex with biotin-labeled detection antibody to obtain the magnetic microsphere-coated antibody-target protein-detection antibody-biotin complex.

[0057] 4.1 Dilute the reaction product from step 3.2 above with PBST to a final antibody concentration of 20 μl / ml;

[0058] 4.2 Add 10 μl of biotin-labeled detection antibody dilution solution to the reaction centrifuge tube in step 2.2 above, vortex to mix, and place in a mixer to continue vortexing for 30 minutes.

[0059] 5. React the complex obtained in step 4 above with streptavidin-labeled horseradish peroxidase (SA-HRP).

[0060] 5.1 Add 10 μl of 5 μg / ml streptavidin-labeled horseradish peroxidase (SA-HRP) to the reaction solution in step 4.2, mix well, and continue to shake the mixture for 10 minutes.

[0061] 5.2 Transfer the magnetic ball to the magnetic rack, apply a magnetic field, and discard the supernatant;

[0062] 5.3 Add 1 ml of PBST washing buffer and wash twice, discarding the supernatant on the last wash.

[0063] 6. Fluorescent staining.

[0064] Add 1 ml of staining solution (10 mM tris(hydroxymethyl)aminomethane (pH 8.5), 0.1% Tween 20, 0.03% hydrogen peroxide (H2O2), 1 μl AF488 Tyramide fluorescent dye) to the reaction product of step 5.3 above, and let it stand at room temperature in the dark for 20 minutes.

[0065] 7. Microporous membrane percolation enrichment.

[0066] The fluorescently stained reaction product was dropped onto a 0.8 μm PVDF filter membrane and filtered. The fluorescently stained reaction product was retained and enriched on the surface of the filter membrane, as shown in Figure 2.

[0067] 8. Qualitative analysis using a fluorescence reader.

[0068] The intercepted and enriched fluorescent staining products were read using a fluorescence reader with excitation / emission wavelengths of 495 / 519 nm. A threshold was set in conjunction with a negative control (stained sample without IL-6 protein) to perform qualitative interpretation of the positive and negative results. The fluorescence images are shown in Figure 3, and the fluorescence signal reading results are shown in the table below.

[0069]

[0070] Results analysis: The 100 pg / ml protein sample showed obvious fluorescence signal compared with the negative control (Figure 3). The fluorescence signal intensity values ​​in the table above show that the fluorescence signal intensity of the positive sample is 430 times that of the background signal intensity of the negative sample, which is significantly different and can be clearly distinguished. This indicates that the method has an effective qualitative detection capability for low concentration target proteins, and the detection limit can reach at least 100 pg / ml.

[0071] Example 2

[0072] This embodiment provides a quantitative detection method based on a microporous filter membrane, the specific steps of which are as follows:

[0073] 1. Superparamagnetic microspheres with a particle size of 2.8 μm were labeled with a coating antibody.

[0074] The operation steps are basically the same as steps 1.1-1.8 of Example 1, except that the magnetic ball particle size used is 2.8μm.

[0075] 2. The antibody-labeled magnetic microspheres are reacted with the sample to obtain a magnetic microsphere-antibody-target protein complex.

[0076] 2.1 Use PBST solution to serially dilute IL-6 protein samples to 500 pg / ml, 250 pg / ml, 125 pg / ml, 25 pg / ml, 5 pg / ml, and 0 pg / ml (PBST solution without IL-6 protein);

[0077] 2.2 Take 100 μl of IL-6 protein dilution solution and add it to the centrifuge tube containing magnetic beads from step 1.8. Resuspend and mix well, then place it in a mixer and shake at room temperature for 30 minutes.

[0078] 3. Biotin is labeled onto the detection antibody by reacting it with sulfonic acid-NHS-Biotin (Sulfo NHS-Biotin).

[0079] 3.1 Dilute the IL-6 detection antibody to 1 mg / ml using PBST solution;

[0080] 3.2 Take 6 μl of 1 mg / ml Sulfo NHS-Biotin solution, add 1 μl of the above-diluted IL-6 detection antibody diluent, and then add 13 μl of purified water to a total volume of 20 μl. Incubate at room temperature for 2 hours.

[0081] 4. React the magnetic microsphere-coated antibody-target protein complex with biotin-labeled detection antibody to obtain the magnetic microsphere-coated antibody-target protein-detection antibody-biotin complex.

[0082] 5. React the complex obtained in step 4 above with streptavidin-labeled horseradish peroxidase (SA-HRP).

[0083] 6. Fluorescent staining.

[0084] Steps 4-6 are the same as in Example 1.

[0085] 7. Microporous membrane percolation enrichment.

[0086] The fluorescently stained reaction products were dropped onto a 1.2 μm PVDF filter membrane and filtered. The fluorescently stained reaction products were retained and enriched on the surface of the filter membrane.

[0087] 8. Quantitative analysis using a fluorescence reader.

[0088] The intercepted and enriched fluorescent staining products were read using a fluorescence reader with excitation / emission wavelengths of 495 / 519 nm. Different concentrations of target proteins were detected, and the corresponding fluorescence signal values ​​were collected to establish a quantitative standard curve for quantitative detection and analysis.

[0089] The test values ​​are shown in the table below, and the standard curve is shown in Figure 4.

[0090]

[0091] Results Analysis: This embodiment used 2.8 μm magnetic microspheres and established a good linear relationship within the concentration range of 0-500 pg / ml, with a detection limit of up to 5 pg / ml, far lower than the nanogram-level detection limit of conventional enzyme-linked immunosorbent assays (ELISA). The fluorescence signal value increased gradient with increasing target protein concentration, with a correlation coefficient R² > 0.99, indicating that this method has excellent quantitative detection performance and a wide linear range.

[0092] Example 3

[0093] This embodiment provides a qualitative detection method based on a microfluidic chip, the specific steps of which are as follows:

[0094] 1. Superparamagnetic microspheres with a particle size of 2.8 μm were labeled with a coating antibody.

[0095] The operation steps are basically the same as steps 1.1-1.8 of Example 1, except that the magnetic ball particle size used is 2.8μm.

[0096] 2. React the antibody-labeled magnetic microspheres with the sample to obtain a magnetic microsphere-antibody-target protein complex. The procedure is the same as in Example 1.

[0097] 3. Biotin is labeled onto the detection antibody by reacting it with sulfonic acid-NHS-Biotin (Sulfo NHS-Biotin).

[0098] 3.1 Dilute the IL-6 detection antibody to 1 mg / ml using PBST solution;

[0099] 3.2 Take 7 μl of 1 mg / ml Sulfo NHS-Biotin solution, add 1 μl of the above-diluted IL-6 detection antibody diluent, and then add 12 μl of purified water to a total volume of 20 μl. Incubate at room temperature for 2 hours.

[0100] 4. React the magnetic microsphere-coated antibody-target protein complex with biotin-labeled detection antibody to obtain the magnetic microsphere-coated antibody-target protein-detection antibody-biotin complex.

[0101] 5. React the complex obtained in step 4 above with streptavidin-labeled horseradish peroxidase (SA-HRP).

[0102] 6. Fluorescent staining.

[0103] Steps 4-6 are the same as in Example 1.

[0104] 7. Microfluidic chip enrichment.

[0105] The disc-type microfluidic chip was placed on a centrifuge platform, and 100 μl of the fluorescently stained reaction product was added to the chip's sample well. Centrifugal force drove the liquid to move to the reservoir, and the magnetic microspheres fell into the chip's small wells (pore diameter 3 μm).

[0106] Specifically, the structure of the disc-type microfluidic system is shown in Figure 5. It includes a disc body and multiple microchannels 1 disposed on the disc body. Each microchannel 1 is provided with a sample inlet 11, a chip aperture 12, a magnet 13, a reservoir 14, and a top cover. The sample inlet 11 is located on one side of the microchannel 1, the reservoir 14 is located at the bottom of the other side, the chip aperture 12 and the magnet 13 are located between the two, and the magnet 13 is located below the chip aperture 12 area. The chip aperture 12 is arranged in an array within the microchannel 1. The top of the microchannel 1 is provided with the top plate, and the top plate has an air hole 15 for venting and stabilizing pressure at one end of the reservoir 14. After the sample liquid is added through the sample inlet 11, it flows through the chip aperture 12 area. Due to the magnetic attraction of the magnet 13, the magnetic microspheres are adsorbed onto the chip aperture 12, and excess liquid flows to the reservoir 14.

[0107] 8. Qualitative analysis using fluorescence microscopy

[0108] The chip containing the magnetic microspheres was imaged under a fluorescence microscope (excitation / emission wavelengths 495 / 519 nm), and the number of fluorescent microspheres was counted. Qualitative analysis was then performed using a negative control (a stained sample without IL-6 protein). The counting results are shown in the table below:

[0109]

[0110] Results Analysis: In this embodiment, microfluidic chip centrifugation enrichment was used to detect 100 pg / ml IL-6 protein. The number of fluorescent magnetic beads in the positive sample was 494 times that in the negative control, which was significantly different and could be clearly distinguished. This shows that the microfluidic chip enrichment method combined with fluorescence microscopy counting can effectively achieve sensitive qualitative detection of ultra-low concentration targets.

[0111] Example 4

[0112] This embodiment provides a quantitative detection method based on a microfluidic chip, the specific steps of which are as follows:

[0113] 1. Label 2.8 μm superparamagnetic microspheres with coated antibodies. The procedure is the same as in Example 3.

[0114] 2. The antibody-labeled magnetic microspheres are reacted with the sample to obtain a magnetic microsphere-antibody-target protein complex.

[0115] 2.1 Use PBST solution to serially dilute IL-6 protein samples to 100 pg / ml, 50 pg / ml, 10 pg / ml, 1 pg / ml, 0.1 pg / ml, and 0 pg / ml (PBST solution without IL-6 protein);

[0116] 2.2 Take 100 μl of IL-6 protein dilution solution and add it to the centrifuge tube containing magnetic beads from step 1.8. Resuspend and mix well, then place it in a mixer and shake at room temperature for 30 minutes.

[0117] 3. Biotin is labeled onto the detection antibody by reacting it with sulfonic acid-NHS-Biotin (Sulfo NHS-Biotin).

[0118] 4. React the magnetic microsphere-coated antibody-target protein complex with biotin-labeled detection antibody to obtain the magnetic microsphere-coated antibody-target protein-detection antibody-biotin complex.

[0119] 5. React the complex obtained in step 4 above with streptavidin-labeled horseradish peroxidase (SA-HRP).

[0120] 6. Fluorescent staining.

[0121] Steps 3-6 are the same as those in Example 3.

[0122] 7. Microfluidic chip enrichment.

[0123] The disc-type microfluidic chip was placed on a centrifuge platform. 100 μl of the reaction product of the target protein sample after fluorescent staining was added to the chip sample well. Centrifugal force drove the liquid to move to the storage tank, and the magnetic microspheres fell into the small well (pore diameter 3 μm).

[0124] 8. Quantitative analysis using fluorescence microscopy

[0125] The chip containing magnetic microspheres was imaged under a fluorescence microscope (excitation / emission wavelengths 495 / 519 nm). The number of fluorescent microspheres in the reaction products of target protein samples of different concentrations after fluorescence staining was counted, a standard curve was established, and quantitative analysis was performed. The counting results are shown in the table below, and the standard curve is shown in Figure 6.

[0126]

[0127] Results Analysis: This embodiment achieved quantitative detection of IL-6 protein in the concentration range of 0-100 pg / ml, with a detection limit of 0.1 pg / ml. The number of fluorescent magnetic beads showed a good linear relationship with the target protein concentration, with a correlation coefficient R² > 0.99. These results indicate that microfluidic chip enrichment combined with fluorescence microscopy counting can achieve highly sensitive and accurate quantitative detection of ultra-low concentration targets at the picogram level.

[0128] Example 5

[0129] This embodiment is basically the same as embodiment 1, except that in step 7, a magnet is placed at the bottom of the PVDF filter membrane before the fluorescently stained reaction product is dropped onto the filter membrane for percolation.

[0130] Example 6

[0131] This embodiment is basically the same as embodiment 2, except that in step 7, a magnet is placed at the bottom of the PVDF filter membrane before the fluorescently stained reaction product is dropped onto the filter membrane for percolation.

[0132] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.

Claims

1. A highly sensitive immunofluorescence detection method, characterized in that, Includes the following steps: (1) Label magnetic microspheres with coated antibodies; (2) React the coated antibody-labeled magnetic microspheres with the protein sample to be tested to obtain a magnetic microsphere-coated antibody-target protein complex; (3) React biotin-activated ester with the detection antibody to label the detection antibody with biotin; (4) React the magnetic microsphere-coated antibody-target protein complex with the biotin-labeled detection antibody to obtain a magnetic microsphere-coated antibody-target protein-detection antibody-biotin complex; (5) React the complex obtained in step (4) with horseradish peroxidase labeled with streptavidin; (6) Fluorescent staining: Add a solution containing hydrogen peroxide and tyrosine fluorescent dye to the reaction product; (7) Detection and analysis: After enriching the fluorescently stained reaction product by percolation through a microporous filter membrane or centrifugation of a microfluidic chip, perform qualitative or quantitative detection and analysis using a fluorescence reader or fluorescence microscope.

2. The ultrasensitive immunofluorescence detection method according to claim 1, characterized in that, In step (1), the magnetic microspheres are superparamagnetic microspheres with a particle size of 0.5-10 micrometers.

3. The ultrasensitive immunofluorescence detection method according to claim 1, characterized in that, In step (3), the biotin-activated ester is a sulfonic acid group -NHS- (long chain). n -Biotin.

4. The ultrasensitive immunofluorescence detection method according to claim 1, characterized in that, In step (6), the tyrosine fluorescent dye is AF488 tyrosine, AF546 tyrosine, Cyanine 3-labeled tyrosine, or other tyrosine fluorescent dyes.

5. The ultrasensitive immunofluorescence detection method according to claim 1, characterized in that, In step (7), the microporous filter membrane is a polyethersulfone filter membrane, a hydrophilic polyvinylidene fluoride filter membrane, or a nitrocellulose membrane, and the pore size of the filter membrane is smaller than the particle size of the magnetic microspheres.

6. The ultrasensitive immunofluorescence detection method according to claim 1 or 5, characterized in that, In step (7), the microporous filter membrane is used alone or in conjunction with a magnet.

7. The ultrasensitive immunofluorescence detection method according to claim 6, characterized in that, The method of using the magnet includes: adding the fluorescently stained reaction product to a microporous filter membrane with a magnet at the bottom, or placing a magnet at the bottom of the microporous filter membrane before adding the reaction product to the microporous filter membrane.

8. The ultrasensitive immunofluorescence detection method according to claim 1, characterized in that, In step (7), the microfluidic chip is a disk-type microfluidic chip, which includes one or more microchannels. The microchannels have an array of chip holes with a diameter slightly larger than that of the magnetic microspheres. There is a magnet below the microchannels, a sample inlet on one side of the microchannels, and a liquid storage tank on the other side.

9. The ultrasensitive immunofluorescence detection method according to claim 1, characterized in that, In step (7), the qualitative analysis method using a fluorescence reader is as follows: First, the reaction product stained with fluorescence is dropped onto a microporous filter membrane for percolation enrichment. The fluorescence signal intensity is detected using a fluorescence reader. The threshold is set in conjunction with a negative control, and the positive and negative results can be interpreted. The quantitative analysis method using a fluorescence reader is as follows: First, the target protein sample of known concentration is diluted according to the gradient concentration. Then, the fluorescent staining products of different concentrations of target protein are prepared by steps (1)-(6). Next, the fluorescent staining products of different concentrations of target protein are dropped onto the microporous filter membrane for percolation enrichment. The fluorescence signal intensity at each concentration is detected by the fluorescence reader, a quantitative standard curve is established, and quantitative detection and analysis are performed.

10. The ultrasensitive immunofluorescence detection method according to claim 1, characterized in that, In step (7), the qualitative analysis method using fluorescence microscopy is as follows: First, the reaction product stained with fluorescence is dropped onto the sample port of the disc-type microfluidic chip. By centrifugation, the liquid of the reaction product stained with fluorescence is moved from one end of the sample port of the chip to the storage tank. During the movement, the magnetic microspheres fall into the chip holes under the action of magnetic force. Then, the chip is placed under a fluorescence microscope for imaging, and the number of luminescent microspheres is collected. Combined with the negative control to set the threshold, the positive and negative interpretation of the detection results can be completed. The quantitative analysis method using fluorescence microscopy is as follows: First, the target protein sample of known concentration is diluted according to the gradient concentration. Then, different concentrations of target protein fluorescent staining products are prepared using steps (1)-(6). Then, the liquid of different concentrations of target protein fluorescent staining products is moved from one end of the chip sample port to the storage tank. During the movement of the magnetic microspheres, they fall into the chip hole under the action of magnetic force. Then, the chip is placed under the fluorescence microscope for imaging. The number of luminescent microspheres corresponding to different concentrations of target protein fluorescent staining products is collected, a quantitative standard curve is established, and quantitative detection and analysis are performed.

Images