Single molecule detection method

Abstract

The invention discloses a single molecule detection method, and relates to the field of single molecule immunodetection. The method comprises the steps that an imaging carrier is prepared, wherein the imaging carrier comprises a bottom plate and one or more grooves formed in the bottom plate; a sample, a capture antibody, a fluorescent dye labeled detection antibody and a solid-phase carrier are mixed and incubated, or the sample, a capture antibody coupled solid-phase carrier and the fluorescent dye labeled detection antibody are mixed and incubated, and a mixed and incubated product is centrifuged in a groove of the imaging carrier, and detecting and counting fluorescence points on the bottom surface of the groove by using a fluorescence microscope. The single molecule detection method has the advantages of short detection time and low detection cost, the detection sensitivity and accuracy are further improved, the detection limit is lower, and popularization of single molecule detection in scientific research and medical treatment is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of single molecule immunoassay, and in particular to a single molecule detection method. Background Art

[0002] Among traditional detection technologies, chemiluminescence and (or) immunoassays are the most widely used. However, they cannot reach the ideal measurement level in early screening of some major diseases, early diagnosis of neurological disorders, and early detection of cardiovascular diseases. Disadvantages of conventional immunoassays: 1. Slow detection speed. 2. Low sensitivity and high detection limit, which can only detect antigen molecules with nM-pM concentration in the blood. 3. Difficult method development.

[0003] Single-molecule detection technology is a new generation of detection technology that is different from traditional detection methods. In traditional detection methods (such as chemiluminescence and enzyme-linked immunosorbent assay), the overall signal in the solution is usually amplified to count the signal value of the sample. When the signal of the sample to be tested in the solution is low to a certain level, it is easily submerged by the noise in the solution, so it is difficult to perform accurate quantitative detection for low-concentration samples. In the single-molecule detection method, the signal to be tested is digitally segmented, and the single-molecule signal is directly observed and counted after signal amplification or fluorescent labeling. The quantitative detection at the single-molecule level is achieved through the quantitative relationship between the signal to be tested and the standard curve. The ability of single-molecule detection is determined by using different signal digital segmentation and detection methods. Including detection speed and detection sensitivity, there are currently several signal digitization strategies for single-molecule detection:

[0004] (1) Segmentation is performed through a microporous chip, and then the signal of each target unit is amplified for counting and observation. Represented by SIMOA of Quanterix in the United States, the enzymatic protein is coupled to the detection antibody, a special carrier with a microporous array is used, and the enzymatic substrate is added to the micropores. During the detection process, the microporous array digitizes the signal, amplifies the single-molecule signal through an enzymatic reaction, and realizes single-molecule quantitative detection. However, this scheme involves an enzymatic signal amplification process, which leads to a long detection time. At the same time, the special detection carrier will bring higher costs during use.

[0005] (2) Use magnetic beads to segment individual signals, use large-diameter fluorescent particles to label them, and count and observe the target signals. For example, Yuce Company (patent number CN 111771126) uses in situ enhanced fluorescent nanoparticles to label the detection antibody, and performs single-molecule detection by counting the fluorescent signal. The diameter of the fluorescent nanoparticles used in this method is 200-350nm. After coupling with the detection antibody, it will produce a large steric hindrance, which will cause the antigen-antibody immune reaction in the solution to have a low binding efficiency. At the same time, the magnetic beads further amplify the steric hindrance, and there is a problem of long reaction binding time. If the detection time is insufficient, it is difficult to meet the quantitative detection of low-concentration samples.

[0006] In summary, the problems of long detection time and high detection cost in the field of single-molecule immunoassay have greatly restricted the use and promotion of single-molecule detection in scientific research and medical treatment. Summary of the invention

[0007] Based on the technical problems existing in the background technology, the present invention proposes a single molecule detection method with short detection time, low detection cost, and further improved detection sensitivity and accuracy.

[0008] A single molecule detection method proposed by the present invention comprises the following steps:

[0009] Prepare an imaging carrier, the imaging carrier comprising a bottom plate and one or more grooves provided on the bottom plate, the groove having a flat bottom surface, the bottom surface of the groove and the bottom surface of the bottom plate are both horizontal surfaces, and the portion between the bottom surface of the groove and the bottom surface of the bottom plate is made of a transparent material;

[0010] First, the sample, the capture antibody and the detection antibody labeled with a fluorescent dye are mixed and incubated, and then a solid phase carrier is added and mixed and incubated, wherein the solid phase carrier and the capture antibody can be directly or indirectly coupled; or the solid phase carrier and the capture antibody are first directly or indirectly coupled to form a capture antibody-coupled solid phase carrier, and then the sample, the capture antibody-coupled solid phase carrier, and the detection antibody labeled with a fluorescent dye are mixed and incubated;

[0011] The mixed incubation product is centrifuged in the groove of the imaging carrier, and the number of fluorescent spots on the bottom surface of the groove is detected and counted using a fluorescence microscope;

[0012] The capture antibody can bind to the first binding site of the target molecule, the detection antibody can bind to the second binding site of the target molecule, and the first binding site and the second binding site are different binding sites;

[0013] The solid phase carrier is a microsphere with a diameter of 20nm to 500nm.

[0014] Preferably, the solid phase carrier is a microsphere with a diameter of 50 nm to 200 nm, more preferably a microsphere with a diameter of 100 nm to 200 nm.

[0015] Preferably, the target molecule is a protein (eg, an enzyme, a cytokine, a receptor protein), a nucleic acid (eg, DNA, RNA), a small molecule compound, a pathogen (eg, a bacterium, a virus), or a cell.

[0016] Preferably, the microspheres are surface-modified or non-surface-modified microspheres, and the surface modification includes at least one of amino modification, carboxyl modification, hydroxylation modification, sulfate modification, tosylation modification, epoxidation modification, streptavidin modification, biotin modification, and fluorescent dye modification.

[0017] Preferably, the microspheres are microspheres modified with surface fluorescent dyes, and the fluorescent dyes used to label the detection antibodies are different in color from the fluorescent dyes modified on the microsphere surfaces. In the present invention, by modifying the surface of the solid phase carrier and the detection antibody with fluorescent dyes emitting different colors, fluorescence co-localization can be achieved, further improving the sensitivity of detection.

[0018] Preferably, the fluorescent dye is at least one of fluorescein dyes, rhodamine dyes, coumarin dyes, quantum dot dyes, stilbene dyes, and naphthamide dyes.

[0019] More preferably, the fluorescent dye is selected from atto-647-NHS, atto-532-NHS or Alexa Fluor 647-NHS.

[0020] Preferably, the material of the microspheres is at least one of silicon, silica gel, silicon dioxide, polystyrene, iron, and iron oxide.

[0021] In the present invention, the coupling of the solid phase carrier and the capture antibody can adopt conventional methods in the art. Among them, the direct coupling of the solid phase carrier and the capture antibody means that the capture antibody is directly adsorbed or coupled to the solid phase carrier by physical adsorption or chemical modification; the indirect coupling of the solid phase carrier and the capture antibody means that the capture antibody is specifically coupled to the solid phase carrier by binding through an anti-capture antibody (i.e., a secondary antibody) or a biotin-streptavidin system. Preferably, the solid phase carrier and the capture antibody are coupled by at least one of glutaraldehyde cross-linking method, carbodiimide method, NHS ester and EDC cross-linking method, silanization method, affinity ligand mediation method, and physical adsorption method. In the detection, different coupling methods can be selected according to the properties of the target molecule, solid phase carrier, capture antibody, detection antibody and other materials used in the detection process.

[0022] Preferably, the temperature of the mixed incubation is 25 to 45° C., and the time is 3 to 30 minutes.

[0023] Preferably, the imaging carrier is a well plate or a chip or glass slide with grooves.

[0024] Preferably, the transparent material is glass or plastic, and the distance between the bottom surface of the groove and the bottom surface of the bottom plate is 0.08-0.25 mm.

[0025] In the present invention, the mixed incubation of the sample can be carried out in the groove of the imaging carrier. At this time, in order to ensure the accuracy and stability of the detection results, the inside of the groove needs to be sealed; the mixed incubation of the sample can also be carried out in other containers. After the reaction in other containers is completed, the product is added to the groove of the imaging carrier. At this time, the inside of the groove can be sealed or not. If the sealing treatment is performed, the product can be directly added to the groove. If the sealing treatment is not performed, in order to reduce the interference of free fluorescent substances on the detection results, the mixed incubation product needs to be washed with a solvent and then added to the groove. The washing can be one or more times, and the solvent used for washing can be a buffer commonly used in the art, such as Tris-HCl buffer, DPBS buffer, sodium acetate buffer, and the solvent used for washing can also include a surfactant with a concentration of 0.01-1%, and the surfactant is preferably at least one of Tween20, tritonX-100, and NP40.

[0026] In the present invention, the mixed incubation product can be first evenly dispersed in a solvent, and then centrifuged and detected and counted; the solvent used for dispersion can be a buffer commonly used in the art, such as Tris-HCl buffer, DPBS buffer, sodium acetate buffer, and the solvent used for dispersion can also include a surfactant with a concentration of 0.01 to 1%, and the surfactant is preferably at least one of Tween20, tritonX-100, and NP40.

[0027] In the present invention, the inside of the groove of the imaging carrier can be sealed with a conventional sealing agent in the art, for example, one or more of skim milk powder, bovine serum albumin (BSA), serum, casein, formaldehyde, gelatin, and polyethylene glycol. The specific method of the sealing treatment can be a conventional method in the art.

[0028] Preferably, in S3, the centrifugal rotation speed is 500-2500 rpm.

[0029] The beneficial effects of the present invention are as follows:

[0030] The present invention uses small-size microspheres as solid phase carriers to connect capture antibodies, so that the capture antibodies are combined with fluorescent dye-labeled detection antibodies and target molecules, and centrifugal sedimentation is performed in an imaging carrier with a groove with a flat bottom surface, so that the microspheres can be uniformly and highly dispersedly deposited at the bottom of the groove of the imaging carrier, and then the number of fluorescent points on the bottom of the groove is detected and counted using a fluorescence microscope, thereby achieving rapid and accurate single-molecule detection. The detection cost of this method is low, the detection time is short, and under the premise of high-sensitivity detection, the entire detection process can be completed within 30 minutes, and its detection sensitivity is greatly improved, and the detection limit can reach less than 10fg / ml, which is more conducive to the promotion of single-molecule detection in scientific research and medical fields, and can be widely used in biomedicine (including clinical diagnosis, biopharmaceutical quality control), chemistry, materials science, environmental science and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the standard curve diagram in Example 1.

[0032] Figure 2 The fluorescence imaging results of single molecule detection of samples of different concentrations in Example 1 are shown.

[0033] Figures 3 to 7 It is the standard curve diagram in Examples 2 to 6 of the present invention.

[0034] Figures 8 to 10 It is the standard curve diagram in comparative examples 1 to 3 of the present invention.

[0035] Figures 11 to 21 It is the standard curve diagram in Examples 7 to 17 of the present invention. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is described in detail below through specific embodiments.

[0037] Example 1

[0038] 1. Experimental materials: carboxyl magnetic beads (particle size 200nm), IL-6 capture antibody (Abcam ab233706), N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), IL-6 detection antibody (Abcam ab9324), sodium acetate buffer (1.8mL glacial acetic acid and 82.0g sodium acetate (CH3COONa·3H2O) added to deionized water to 1000mL, pH 5.0), ethanolamine, fluorescent marker (atto-647-NHS), Tris buffer (12.114g Tris was added to deionized water to make up to 1000 ml, pH 7.5), DPBS buffer (8.0 g sodium chloride, 0.2 g potassium chloride, 1.15 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate were added to deionized water to make up to 1000 ml, pH 7.4), magnetic bead storage solution (0.1% tween-20, 0.5% BSA dissolved in DPBS buffer), coupling buffer (0.5% BSA dissolved in Tris buffer, pH 7.5), antibody diluent (0.5% BSA dissolved in DPBS buffer), washing buffer (0.5% tween 20 dissolved in DPBS buffer, pH 7.4), imaging buffer (0.1% tween 20 dissolved in DPBS buffer at pH 7.4), IL-6 antigen (Thermo Fisher Scientific Human IL-6 Recombinant Protein, 200-06-20UG), transparent glass-bottom 96-well plate (imaging carrier, model Cellvis P96-1.5HN).

[0039] 2. Experimental steps:

[0040] 2.1 Preparation of IL-6 capture antibody-coupled magnetic beads:

[0041] (1) Take 2 mg of 200 nm carboxyl magnetic beads and wash them four times with 200 μL DPBS buffer;

[0042] (2) Dissolve 0.5 mg NHS and 0.5 mg EDC in 200 μL DPBS buffer and mix well;

[0043] (3) Add the mixture obtained in (2) to the magnetic beads washed in (1), mix well, and incubate on a shaker at room temperature for 30 min;

[0044] (4) Wash the magnetic beads after incubation in (3) once with 200 μL DPBS buffer, add 0.5 mg IL-6 capture antibody and mix well, place in a shaker at room temperature for 4 h;

[0045] (5) The reaction product obtained in (4) was washed once with 200 μL DPBS buffer, mixed with 200 μL ethanolamine, and placed in a shaker for reaction at room temperature for 1 h;

[0046] (6) The reaction product obtained in (5) was washed four times with 200 μL DPBS buffer to obtain IL-6 capture antibody-coupled magnetic beads, which were suspended and stored in magnetic bead storage solution for later use.

[0047] 2.2 Preparation of fluorescent dye-labeled IL-6 detection antibody:

[0048] (1) Dissolve 1 mg of atto-647-NHS in 1 mL of DMSO to obtain a fluorescent dye solution with a concentration of 1 mg / ml;

[0049] (2) Mix 50 μL of fluorescent dye solution with 1 mg of IL-6 detection antibody, add coupling buffer to make up the volume to 200 μL, and place on a shaker at room temperature for 1 h;

[0050] (3) After the reaction is completed, the mixture is placed in a refrigerator at 4°C for overnight dialysis to obtain a fluorescent dye-labeled IL-6 detection antibody.

[0051] 2.3 Make a standard curve:

[0052] (1) using fetal bovine serum to dilute the IL-6 antigen concentration to obtain samples with concentrations of 0 pg / mL, 0.0098 pg / mL, 0.039 pg / mL, 0.156 pg / mL, 0.625 pg / mL, 2.5 pg / mL, 10 pg / mL, and 40 pg / mL;

[0053] (2) Dilute the IL-6 capture antibody-coupled magnetic beads to 1 mg / mL using magnetic bead storage solution, and dilute the fluorescent dye-labeled IL-6 detection antibody to 10 μg / mL using antibody diluent;

[0054] (3) Take 50 μL of the sample of each concentration obtained in (1), add 50 μL of the IL-6 capture antibody-coupled magnetic bead dilution obtained in (2), then add 10 uL of the fluorescent dye-labeled IL-6 detection antibody dilution obtained in (2), mix well, and incubate at 37°C for 10 min;

[0055] (4) The incubation product obtained in (3) was washed three times with 100 μL of washing buffer, resuspended in 100 μL of imaging buffer, added to the groove of a transparent glass-bottomed 96-well plate, centrifuged at 2200 rpm for 3 min on a centrifuge (Hangzhou Aosheng MPC-P25), and then single-molecule imaging was performed using a fluorescence microscope (Nikon Eclipse Ti) to count the number of fluorescent spots on the bottom of the groove; each concentration was tested three times, and a standard curve was drawn based on the test results to calculate the detection limit. The standard curve was drawn as shown in FIG. Figure 1 The calculated lower limit of detection is shown in Table 1. The fluorescence imaging results of single molecule detection of samples with different concentrations are shown in Figure 2 As shown, Figure 2 Medium, A: 40pg / mL; B: 10pg / mL; C: 2.5pg / mL; D: 0.625pg / mL; E: 0.156pg / mL; F: 0.039pg / mL; G: 0.0098pg / mL; H: 0pg / mL. from Figure 2 It can be seen that the fluorescence signals at different concentrations are significantly different. The more fluorescence signal points, the higher the concentration of the target molecule. Figure 2 It can also be seen that the detection method of the present invention has good dispersion of the fluorescence signal of the detected sample, which is beneficial to improving the sensitivity and efficiency of the detection.

[0056] Example 2

[0057] The only difference between Example 2 and Example 1 is that the carboxyl magnetic beads used have a particle size of 50 nm; the rest of the experimental materials and operations are the same as those of Example 1. The standard curve obtained in Example 2 is as follows Figure 3 The calculated lower limit of detection is shown in Table 1.

[0058] Example 3

[0059] The only difference between Example 3 and Example 1 is that the carboxyl magnetic beads used have a particle size of 100 nm; the rest of the experimental materials and operations are the same as those of Example 1. The standard curve obtained in Example 3 is as follows Figure 4 The calculated lower limit of detection is shown in Table 1.

[0060] Example 4

[0061] The only difference between Example 4 and Example 1 is that the carboxyl magnetic beads used have a particle size of 150 nm; the remaining experimental materials and operations are the same as those of Example 1. The standard curve obtained in Example 4 is as follows Figure 5 The calculated lower limit of detection is shown in Table 1.

[0062] Example 5

[0063] The only difference between Example 5 and Example 1 is that the carboxyl magnetic beads used have a particle size of 300 nm; the rest of the experimental materials and operations are the same as those of Example 1. The standard curve obtained in Example 5 is as follows Figure 6 The calculated lower limit of detection is shown in Table 1.

[0064] Example 6

[0065] The only difference between Example 6 and Example 1 is that the carboxyl magnetic beads used have a particle size of 500 nm, and when preparing the standard curve, the IL-6 antigen concentration is diluted with fetal bovine serum to obtain samples with concentrations of 0 pg / mL, 0.156 pg / mL, 0.625 pg / mL, 2.5 pg / mL, 10 pg / mL, 40 pg / mL, and 160 pg / mL; the remaining experimental materials and operations are the same as in Example 1. The standard curve obtained in Example 6 is as follows Figure 7 The calculated lower limit of detection is shown in Table 1.

[0066] Comparative Example 1

[0067] The difference between Comparative Example 1 and Example 1 is that the particle size of the carboxyl magnetic beads used is 1 μm, and when preparing the standard curve, the IL-6 antigen concentration is diluted with fetal bovine serum to obtain samples with concentrations of 0 pg / mL, 0.156 pg / mL, 0.625 pg / mL, 2.5 pg / mL, 10 pg / mL, 40 pg / mL, and 160 pg / mL; the remaining experimental materials and operations are the same as those in Example 1. The standard curve obtained in Comparative Example 1 is as follows Figure 8 The calculated lower limit of detection is shown in Table 1.

[0068] Comparative Example 2

[0069] The difference between Comparative Example 2 and Example 1 is that the particle size of the carboxyl magnetic beads used is 2.7 μm, and when preparing the standard curve, the IL-6 antigen concentration is diluted with fetal bovine serum to obtain samples with concentrations of 0 pg / mL, 0.156 pg / mL, 0.625 pg / mL, 2.5 pg / mL, 10 pg / mL, 40 pg / mL, and 160 pg / mL; the remaining experimental materials and operations are the same as those in Example 1. The standard curve obtained in Comparative Example 2 is as follows Fig. 9 The calculated lower limit of detection is shown in Table 1.

[0070] Comparative Example 3

[0071] The difference between Comparative Example 3 and Example 1 is that in step (4), the centrifugation at 2200 rpm for 3 min on a centrifuge (Hangzhou Aosheng MPC-P25) is replaced by using a magnet for 3 min. The standard curve obtained in Comparative Example 3 is as follows: Fig.10 The calculated lower limit of detection is shown in Table 1.

[0072] Example 7

[0073] 1. Experimental materials: SA magnetic beads (particle size 200nm), IL-6 capture antibody (Abcam ab233706), IL-6 detection antibody (Abcam ab9324), fluorescent marker (atto-647-NHS), biotin-NHS (thermo), dimethyl sulfoxide (DMSO), DPBS buffer (8.0 g sodium chloride, 0.2 g potassium chloride, 1.15 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate, added to deionized water to make up to 1000 mL, pH 7.4), magnetic bead storage solution (0.1% tween-20, 0.5% BSA dissolved in DPBS buffer), coupling buffer (0.5% BSA dissolved in Tris buffer, pH 7.5), antibody diluent (0.5% BSA dissolved in DPBS buffer), washing buffer (0.5% tween20 dissolved in DPBS buffer, pH 7.4), imaging buffer (0.1% tween20 dissolved in DPBS buffer, pH 7.4), IL-6 antigen (Thermo Fisher Scientific Human IL-6 Recombinant Protein, 200-06-20UG), transparent glass-bottom 96-well plate (imaging carrier, model Cellvis P96-1.5HN).

[0074] 2. Experimental steps:

[0075] 2.1 Preparation of IL-6 capture antibody-coupled SA magnetic beads:

[0076] (1) Weigh 5.677 mg of biotin-NHS and dissolve it in DMSO to obtain a biotin solution with a concentration of 10 uM;

[0077] (2) Take 0.5 mg of IL-6 capture antibody, add 6.5 uL of the biotin solution obtained in (1), and then add coupling buffer to make up the volume to 500 μl, place in a shaker and incubate at room temperature for 1 hour to obtain a biotinylated capture antibody solution;

[0078] (3) Take 1 mg of 200 nm SA magnetic beads, wash them four times with 200 μL DPBS buffer, add 20 μL of the biotinylated capture antibody solution obtained in (2), mix well, and place on a shaker at room temperature for 30 min;

[0079] (4) The magnetic beads after the reaction in (3) were washed three times with 200 μL DPBS buffer to obtain IL-6 capture antibody-coupled magnetic beads, which were suspended and stored in magnetic bead storage solution for later use.

[0080] 2.2 Preparation of fluorescent dye-labeled IL-6 detection antibody:

[0081] (1) Dissolve 1 mg of atto-647-NHS in 1 mL of DMSO to obtain a fluorescent dye solution with a concentration of 1 mg / ml;

[0082] (2) Mix 50 μL of fluorescent dye solution with 1 mg of IL-6 detection antibody, add coupling buffer to make up the volume to 200 μL, and place on a shaker at room temperature for 1 h;

[0083] (3) After the reaction is completed, the mixture is placed in a refrigerator at 4°C for overnight dialysis to obtain a fluorescent dye-labeled IL-6 detection antibody.

[0084] 2.3 Make a standard curve:

[0085] (1) using fetal bovine serum to dilute the IL-6 antigen concentration to obtain samples with concentrations of 0 pg / mL, 0.0098 pg / mL, 0.039 pg / mL, 0.156 pg / mL, 0.625 pg / mL, 2.5 pg / mL, 10 pg / mL, and 40 pg / mL;

[0086] (2) Dilute the IL-6 capture antibody-coupled magnetic beads to 1 mg / mL using magnetic bead storage solution, and dilute the fluorescent dye-labeled IL-6 detection antibody to 10 μg / mL using antibody diluent;

[0087] (3) Take 50 μL of the sample of each concentration obtained in (1), add 50 μL of the IL-6 capture antibody-coupled magnetic bead dilution obtained in (2), then add 10 uL of the fluorescent dye-labeled IL-6 detection antibody dilution obtained in (2), mix well, and incubate at 37°C for 10 min;

[0088] (4) The incubation product obtained in (3) was washed three times with 100 μL of washing buffer, resuspended in 100 μL of imaging buffer, added to the groove of a transparent glass-bottomed 96-well plate, centrifuged at 2200 rpm for 3 min on a centrifuge (Hangzhou Aosheng MPC-P25), and then single-molecule imaging was performed using a fluorescence microscope (Nikon Eclipse Ti) to count the number of fluorescent spots on the bottom of the groove; each concentration was tested three times, and a standard curve was drawn based on the test results to calculate the detection limit. The standard curve was drawn as shown in FIG. Fig.11 The calculated lower limit of detection is shown in Table 1.

[0089] Example 8

[0090] 1. Experimental materials: Tosyl magnetic beads (particle size 200 nm), IL-6 capture antibody (Abcam ab233706), IL-6 detection antibody (Abcam ab9324), fluorescent marker (atto-647-NHS), biotin-NHS (thermo), dimethyl sulfoxide (DMSO), DPBS buffer (8.0 g sodium chloride, 0.2 g potassium chloride, 1.15 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate, added to deionized water to make up to 1000 mL, pH 7.4), magnetic bead storage solution (0.1% tween-20, 0.5% BSA dissolved in DPBS buffer), coupling buffer (0.5% BSA dissolved in Tris buffer, pH 7.5), antibody diluent (0.5% BSA dissolved in DPBS buffer), washing buffer (0.5% tween20 dissolved in DPBS buffer, pH 7.4), imaging buffer (0.1% tween20 dissolved in DPBS buffer, pH 7.4), IL-6 antigen (Thermo Fisher Scientific Human IL-6 Recombinant Protein, 200-06-20UG), transparent glass-bottom 96-well plate (imaging carrier, model: Cellvis P96-1.5HN).

[0091] 2. Experimental steps:

[0092] 2.1 Preparation of IL-6 capture antibody-coupled magnetic beads:

[0093] (1) Take 10 mg of 200 nm tosyl magnetic beads and wash them three times with 200 uL DPBS buffer;

[0094] (2) Take 0.2 mg of IL-6 capture antibody and add DPBS buffer to make the volume up to 200 μL to obtain IL-6 capture antibody dilution solution;

[0095] (3) adding the IL-6 capture antibody dilution obtained in (2) to the washed magnetic beads in (1), mixing well, and incubating on a shaker at room temperature for 4 h;

[0096] (4) The incubation product obtained in (3) was washed three times with 200 μL DPBS buffer to obtain IL-6 capture antibody-coupled magnetic beads, which were suspended and stored in magnetic bead storage solution for later use.

[0097] 2.2 Preparation of fluorescent dye-labeled IL-6 detection antibody:

[0098] (1) Dissolve 1 mg of atto-647-NHS in 1 mL of DMSO to obtain a fluorescent dye solution with a concentration of 1 mg / ml;

[0099] (2) Mix 50 μL of fluorescent dye solution with 1 mg of IL-6 detection antibody, add coupling buffer to make up the volume to 200 μL, and place on a shaker at room temperature for 1 h;

[0100] (3) After the reaction is completed, the mixture is placed in a refrigerator at 4°C for overnight dialysis to obtain a fluorescent dye-labeled IL-6 detection antibody.

[0101] 2.3 Make a standard curve:

[0102] (1) using fetal bovine serum to dilute the IL-6 antigen concentration to obtain samples with concentrations of 0 pg / mL, 0.0098 pg / mL, 0.039 pg / mL, 0.156 pg / mL, 0.625 pg / mL, 2.5 pg / mL, 10 pg / mL, and 40 pg / mL;

[0103] (2) Dilute the IL-6 capture antibody-coupled magnetic beads to 1 mg / mL using magnetic bead storage solution, and dilute the fluorescent dye-labeled IL-6 detection antibody to 10 μg / mL using antibody diluent;

[0104] (3) Take 50 μL of the sample of each concentration obtained in (1), add 50 μL of the IL-6 capture antibody-coupled magnetic bead dilution obtained in (2), then add 10 uL of the fluorescent dye-labeled IL-6 detection antibody dilution obtained in (2), mix well, and incubate at 37°C for 10 min;

[0105] (4) The incubation product obtained in (3) was washed three times with 100 μL of washing buffer, resuspended in 100 μL of imaging buffer, added to the groove of a transparent glass-bottomed 96-well plate, centrifuged at 2200 rpm for 3 min on a centrifuge (Hangzhou Aosheng MPC-P25), and then single-molecule imaging was performed using a fluorescence microscope (Nikon Eclipse Ti) to count the number of fluorescent spots on the bottom of the groove; each concentration was tested three times, and a standard curve was drawn based on the test results to calculate the detection limit. The standard curve was drawn as shown in FIG. Fig.12 The calculated lower limit of detection is shown in Table 1.

[0106] Example 9 Capture antibody-solid phase carrier affinity ligand method coupled single molecule detection of IL-6 antigen

[0107] 1. Experimental materials: SA magnetic beads (particle size 200nm), IL-6 capture antibody (Abcam ab233706), IL-6 detection antibody (Abcam ab9324), fluorescent marker (atto-647-NHS), biotin-NHS (thermo), dimethyl sulfoxide (DMSO), DPBS buffer (8.0 g sodium chloride, 0.2 g potassium chloride, 1.15 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate, added to deionized water to make up to 1000 mL, pH 7.4), magnetic bead storage solution (0.1% tween-20, 0.5% BSA dissolved in DPBS buffer), coupling buffer (0.5% BSA dissolved in Tris buffer, pH 7.5), antibody diluent (0.5% BSA dissolved in DPBS buffer), washing buffer (0.5% tween20 dissolved in DPBS buffer, pH 7.4), imaging buffer (0.1% tween20 dissolved in DPBS buffer, pH 7.4), IL-6 antigen (Thermo Fisher Scientific Human IL-6 Recombinant Protein, 200-06-20UG), transparent glass-bottom 96-well plate (imaging carrier, model Cellvis P96-1.5HN).

[0108] 2. Experimental steps:

[0109] 2.1 Preparation of biotin-coupled IL-6 capture antibody:

[0110] (1) Weigh 5.677 mg of biotin-NHS and dissolve it in DMSO to obtain a biotin solution with a concentration of 10 uM;

[0111] (2) Take 0.5 mg of IL-6 capture antibody, add 6.5 μL of the biotin solution obtained in (1), and then add coupling buffer to make up the volume to 500 μL. Incubate at room temperature for 1 hour on a shaker, and then dialyze overnight at 4°C to obtain biotin-coupled IL-6 capture antibody.

[0112] 2.2 Preparation of fluorescent dye-labeled IL-6 detection antibody:

[0113] (1) Dissolve 1 mg of atto-647-NHS in 1 mL of DMSO to obtain a fluorescent dye solution with a concentration of 1 mg / ml;

[0114] (2) Mix 50uL of fluorescent dye solution with 1mg of IL-6 detection antibody, add coupling buffer to make up the volume to 200uL, and place on a shaker at room temperature for 1h;

[0115] (3) After the reaction is completed, the mixture is placed in a refrigerator at 4°C for overnight dialysis to obtain a fluorescent dye-labeled IL-6 detection antibody.

[0116] 2.3 Make a standard curve:

[0117] (1) using fetal bovine serum to dilute the IL-6 antigen concentration to obtain samples with concentrations of 0 pg / mL, 0.0098 pg / mL, 0.039 pg / mL, 0.156 pg / mL, 0.625 pg / mL, 2.5 pg / mL, 10 pg / mL, and 40 pg / mL;

[0118] (2) diluting the biotin-conjugated IL-6 capture antibody to 2 μg / mL and diluting the fluorescent dye-labeled IL-6 detection antibody to 10 μg / mL using antibody diluent;

[0119] (3) Take 50 μL of the biotin-coupled IL-6 capture antibody dilution obtained in (2), add 50 μL of the sample of each concentration obtained in (1), then add 10 μL of the fluorescent dye-labeled IL-6 detection antibody dilution obtained in (2), mix well, incubate at 37°C for 10 min, and then add 0.1 mg of SA magnetic beads and incubate for 10 min;

[0120] (4) The incubation product obtained in (3) was washed three times with 100 μL of washing buffer, resuspended in 100 μL of imaging buffer, added to the groove of a transparent glass-bottomed 96-well plate, centrifuged at 2200 rpm for 3 min on a centrifuge (Hangzhou Aosheng MPC-P25), and then single-molecule imaging was performed using a fluorescence microscope (Nikon Eclipse Ti) to count the number of fluorescent spots on the bottom of the groove; each concentration was tested three times, and a standard curve was drawn based on the test results to calculate the detection limit. The standard curve was drawn as shown in FIG. Fig.13 The calculated lower limit of detection is shown in Table 1.

[0121] Example 10

[0122] The only difference between Example 10 and Example 9 is that the SA magnetic beads used have a particle size of 100 nm; the rest of the experimental materials and operations are the same as those of Example 9. The standard curve obtained in Example 10 is as follows Fig.14 The calculated lower limit of detection is shown in Table 1.

[0123] Embodiment 11

[0124] The only difference between Example 11 and Example 1 is that the fluorescent marker used is cy5-NHS; the rest of the experimental materials and operations are the same as those of Example 1. The standard curve obtained in Example 11 is as follows Fig.15 The calculated lower limit of detection is shown in Table 1.

[0125] Example 12

[0126] The only difference between Example 12 and Example 1 is that the fluorescent marker used is Alexa Fluor 647-NHS; the rest of the experimental materials and operations are the same as those of Example 1. The standard curve obtained in Example 12 is as follows Fig.16 The calculated lower limit of detection is shown in Table 1.

[0127] Embodiment 13

[0128] The only difference between Example 13 and Example 1 is that the imaging carrier used is a transparent glass-bottom 384-well plate (model: Cellvis P384-1.5HN); the rest of the experimental materials and operations are the same as those in Example 1. The standard curve obtained in Example 13 is as follows Fig.17 The calculated lower limit of detection is shown in Table 1.

[0129] Example 14 One-step, no-cleaning, single-molecule detection of IL-6 antigen (imaging carrier blocking method)

[0130] 1. Experimental materials: carboxyl magnetic beads (particle size 200nm), IL-6 capture antibody (Abcam ab233706), N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), IL-6 detection antibody (Abcam ab9324), sodium acetate buffer (1.8mL glacial acetic acid and 82.0g sodium acetate (CH3COONa·3H2O) added to deionized water to 1000mL, pH 5.0), ethanolamine, fluorescent marker (atto-647-NHS), Tris buffer (12.114g Tris was added to deionized water to make up to 1000 ml, pH 7.5), DPBS buffer (8.0 g sodium chloride, 0.2 g potassium chloride, 1.15 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate were added to deionized water to make up to 1000 ml, pH 7.4), magnetic bead storage solution (0.1% tween-20, 0.5% BSA dissolved in DPBS buffer), coupling buffer (0.5% BSA dissolved in Tris buffer, pH 7.5), antibody diluent (0.5% BSA dissolved in DPBS buffer), washing buffer (0.5% tween20 dissolved in DPBS buffer, pH 7.4), blocking buffer (2.5% BSA dissolved in DPBS buffer), imaging buffer (0.1% tween20 dissolved in DPBS buffer, pH 7.4), IL-6 antigen (Thermo Fisher Scientific Human IL-6 Recombinant Protein, 200-06-20UG), transparent glass-bottom 96-well plate (imaging carrier, model: Cellvis P96-1.5HN).

[0131] 2. Experimental steps:

[0132] 2.1 Preparation of IL-6 capture antibody-coupled magnetic beads:

[0133] (1) Take 2 mg of 200 nm carboxyl magnetic beads and wash them four times with 200 μL DPBS buffer;

[0134] (2) Dissolve 0.5 mg NHS and 0.5 mg EDC in 200 μL DPBS buffer and mix well;

[0135] (3) Add the mixture obtained in (2) to the magnetic beads washed in (1), mix well, and incubate on a shaker at room temperature for 30 min;

[0136] (4) Wash the magnetic beads after incubation in (3) once with 200 μL DPBS buffer, add 0.5 mg IL-6 capture antibody and mix well, place in a shaker at room temperature for 4 h;

[0137] (5) The reaction product obtained in (4) was washed once with 200 μL DPBS buffer, mixed with 200 μL ethanolamine, and placed in a shaker for reaction at room temperature for 1 h;

[0138] (6) The reaction product obtained in (5) was washed four times with 200 μL DPBS buffer to obtain IL-6 capture antibody-coupled magnetic beads, which were suspended and stored in magnetic bead storage solution for later use.

[0139] 2.2 Preparation of fluorescent dye-labeled IL-6 detection antibody:

[0140] (1) Dissolve 1 mg of atto-647-NHS in 1 mL of DMSO to obtain a fluorescent dye solution with a concentration of 1 mg / ml;

[0141] (2) Mix 50 μL of fluorescent dye solution with 1 mg of IL-6 detection antibody, add coupling buffer to make up the volume to 200 μL, and place on a shaker at room temperature for 1 h;

[0142] (3) After the reaction is completed, the mixture is placed in a refrigerator at 4°C for overnight dialysis to obtain a fluorescent dye-labeled IL-6 detection antibody.

[0143] 2.3 Sealing of imaging carrier

[0144] (1) Wash the transparent glass-bottomed 96-well plate three times with washing buffer;

[0145] (2) Add 200 μL of blocking buffer to each groove and react at 37°C for 1 h;

[0146] (3) After the reaction is completed, aspirate the blocking solution and wash with washing buffer 5 times, soaking for 30 seconds each time;

[0147] (4) After cleaning is completed, the remaining cleaning solution is aspirated to obtain a sealed glass-bottomed 96-well plate.

[0148] 2.4 Make a standard curve:

[0149] (1) using fetal bovine serum to dilute the IL-6 antigen concentration to obtain samples with concentrations of 0 pg / mL, 0.0098 pg / mL, 0.039 pg / mL, 0.156 pg / mL, 0.625 pg / mL, 2.5 pg / mL, 10 pg / mL, and 40 pg / mL;

[0150] (2) Dilute the IL-6 capture antibody-coupled magnetic beads to 1 mg / mL using magnetic bead storage solution, and dilute the fluorescent dye-labeled IL-6 detection antibody to 10 μg / mL using antibody diluent;

[0151] (3) Take 50 μL of the sample of each concentration obtained in (1), add 50 μL of the IL-6 capture antibody-coupled magnetic bead dilution obtained in (2), then add 10 uL of the fluorescent dye-labeled IL-6 detection antibody dilution obtained in (2), add to the groove of the blocked glass-bottom 96-well plate, mix well, incubate at 37°C for 10 min, centrifuge at 2200 rpm for 3 min on a centrifuge (Hangzhou Aosheng MPC-P25), and then use a fluorescence microscope (Nikon Eclipse Ti) for single-molecule imaging to count the number of fluorescent spots on the bottom of the groove; repeat the test 3 times for each concentration, draw a standard curve based on the test results, calculate the detection limit, and draw the obtained standard curve as shown in Figure 2. Fig.18 As shown, the calculated lower limit of detection is shown in Table 1. Example 15 Fluorescence co-localization single molecule detection of IL-6 antigen (magnetic bead fluorescence labeling method)

[0152] 1. Experimental materials: carboxyl magnetic beads (particle size 200nm), IL-6 capture antibody (Abcam ab233706), N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), IL-6 detection antibody (Abcam ab9324), sodium acetate buffer (1.8mL glacial acetic acid and 82.0g sodium acetate (CH3COONa·3H2O) added to deionized water to 1000mL, pH 5.0), ethanolamine, fluorescent marker 1 (atto-532-NHS), fluorescent marker 2 (atto-647-NHS), Tris buffer (12.114g Tris was added to deionized water to make up to 1000 ml, pH 7.5), DPBS buffer (8.0 g sodium chloride, 0.2 g potassium chloride, 1.15 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate were added to deionized water to make up to 1000 ml, pH 7.4), magnetic bead storage solution (0.1% tween-20, 0.5% BSA dissolved in DPBS buffer), coupling buffer (0.5% BSA dissolved in Tris buffer, pH 7.5), antibody diluent (0.5% BSA dissolved in DPBS buffer), washing buffer (0.5% tween20 dissolved in DPBS buffer, pH 7.4), imaging buffer (0.1% tween20 dissolved in DPBS buffer at pH 7.4), IL-6 antigen (Thermo Fisher Scientific Human IL-6 Recombinant Protein, 200-06-20UG), transparent glass-bottom 96-well plate (imaging carrier, model Cellvis P96-1.5HN).

[0153] 2. Experimental steps:

[0154] 2.1 Preparation of atto-532 coupled magnetic beads:

[0155] (1) Dissolve 1 mg of atto-532-NHS in 1 mL of DMSO to obtain an atto-532-NHS solution with a concentration of 1 mg / ml;

[0156] (2) Take 2 mg of 200 nm carboxyl magnetic beads and wash them four times with 200 μL DPBS buffer;

[0157] (3) 0.1 μL of the atto-532-NHS solution obtained in (1) was mixed with 200 μL of the washed carboxyl magnetic beads in (2), and the mixture was placed on a shaker and reacted at room temperature for 1 h;

[0158] (4) The magnetic beads obtained in reaction (3) were washed four times with a washing buffer to obtain atto-532-coupled magnetic beads, which were resuspended in 200 μL of magnetic bead storage solution for later use;

[0159] 2.2 Preparation of IL-6 capture antibody and atto-532 coupled magnetic beads:

[0160] (1) Take 200 μL of the atto-532-coupled magnetic bead suspension obtained in 2.1 and wash it four times with 200 μL DPBS buffer;

[0161] (2) Dissolve 0.5 mg NHS and 0.5 mg EDC in 200 μL DPBS buffer and mix well;

[0162] (3) Add the mixture obtained in (2) to the magnetic beads washed in (1), mix well, and incubate on a shaker at room temperature for 30 min;

[0163] (4) Wash the magnetic beads after incubation in (3) once with 200 μL DPBS buffer, add 0.5 mg IL-6 capture antibody and mix well, place in a shaker at room temperature for 4 h;

[0164] (5) The reaction product obtained in (4) was washed once with 200 μL DPBS buffer, mixed with 200 μL ethanolamine, and placed in a shaker for reaction at room temperature for 1 h;

[0165] (6) The reaction product obtained in (5) was washed four times with 200 μL DPBS buffer to obtain IL-6 capture antibody and atto-532 coupled magnetic beads, which were suspended and stored in magnetic bead storage solution for later use.

[0166] 2.2 Preparation of atto-647 labeled IL-6 detection antibody:

[0167] (1) Dissolve 1 mg of atto-647-NHS in 1 mL of DMSO to obtain an atto-647-NHS solution with a concentration of 1 mg / ml;

[0168] (2) Mix 50uL of atto-647-NHS solution with 1mg of IL-6 detection antibody, add coupling buffer to make up the volume to 200uL, and place on a shaker at room temperature for 1h;

[0169] (3) After the reaction is completed, the mixture is placed in a refrigerator and dialyzed overnight at 4°C to obtain atto-647-labeled IL-6 detection antibody.

[0170] 2.3 Make a standard curve:

[0171] (1) using fetal bovine serum to dilute the IL-6 antigen concentration to obtain samples with concentrations of 0 pg / mL, 0.0098 pg / mL, 0.039 pg / mL, 0.156 pg / mL, 0.625 pg / mL, 2.5 pg / mL, 10 pg / mL, and 40 pg / mL;

[0172] (2) Dilute the IL-6 capture antibody and atto-532-coupled magnetic beads to 1 mg / mL using magnetic bead storage solution, and dilute the atto-647-labeled IL-6 detection antibody to 10 μg / mL using antibody diluent;

[0173] (3) Take 50 μL of the sample of each concentration obtained in (1), add 50 μL of the IL-6 capture antibody and atto-532-coupled magnetic beads dilution obtained in (2), then add 10 uL of the atto-647-labeled IL-6 detection antibody dilution obtained in (2), mix well, and incubate at 37°C for 10 min;

[0174] (4) The incubation product obtained in (3) was washed three times with 100 μL of washing buffer, and then resuspended in 100 μL of imaging buffer, added to the groove of a glass-bottomed 96-well plate, and centrifuged at 2200 rpm for 3 min on a centrifuge (Hangzhou Aosheng MPC-P25). Then, single-molecule imaging was performed using a fluorescence microscope (Nikon Eclipse Ti) to count the number of fluorescent spots on the bottom of the groove; each concentration was tested three times, and a standard curve was drawn based on the test results to calculate the detection limit. The standard curve was drawn as shown in FIG. Fig.19 The calculated lower limit of detection is shown in Table 1.

[0175] Example 16

[0176] 1. Experimental materials: carboxyl magnetic beads (particle size 200nm), TNF-α capture antibody (ab1793), N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), TNF-α detection antibody (ab307164), sodium acetate buffer (1.8mL glacial acetic acid and 82.0g sodium acetate (CH3COONa·3H2O) added to deionized water to 1000mL, pH 5.0), ethanolamine, fluorescent marker (atto-647-NHS), Tris buffer (12.114g Tris was added to deionized water to make up to 1000 ml, pH 7.5), DPBS buffer (8.0 g sodium chloride, 0.2 g potassium chloride, 1.15 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate were added to deionized water to make up to 1000 ml, pH 7.4), magnetic bead storage solution (0.1% tween-20, 0.5% BSA dissolved in DPBS buffer), coupling buffer (0.5% BSA dissolved in Tris buffer, pH 7.5), antibody diluent (0.5% BSA dissolved in DPBS buffer), washing buffer (0.5% tween20 dissolved in DPBS buffer, pH 7.4), imaging buffer (0.1% tween20 dissolved in DPBS buffer at pH 7.4), TNF-α antigen (Ibotek Recombinant Human TNF-alpha Protein RP00993), and a transparent glass-bottom 96-well plate (imaging carrier, model Cellvis P96-1.5HN).

[0177] 2. Experimental steps:

[0178] 2.1 Preparation of TNF-α capture antibody-coupled magnetic beads:

[0179] (1) Take 2 mg of 200 nm carboxyl magnetic beads and wash them four times with 200 μL DPBS buffer;

[0180] (2) Dissolve 0.5 mg NHS and 0.5 mg EDC in 200 μL DPBS buffer and mix well;

[0181] (3) Add the mixture obtained in (2) to the magnetic beads washed in (1), mix well, and incubate on a shaker at room temperature for 30 min;

[0182] (4) Wash the magnetic beads after incubation in (3) once with 200 μL DPBS buffer, add 0.5 mg TNF-α capture antibody and mix well, place in a shaker at room temperature for 4 h;

[0183] (5) The reaction product obtained in (4) was washed once with 200 μL DPBS buffer, mixed with 200 μL ethanolamine, and placed in a shaker for reaction at room temperature for 1 h;

[0184] (6) The reaction product obtained in (5) was washed four times with 200 μL DPBS buffer to obtain TNF-α capture antibody-coupled magnetic beads, which were suspended and stored in magnetic bead storage solution for later use.

[0185] 2.2 Preparation of fluorescent dye-labeled TNF-α detection antibody:

[0186] (1) Dissolve 1 mg of atto-647-NHS in 1 mL of DMSO to obtain a fluorescent dye solution with a concentration of 1 mg / ml;

[0187] (2) Mix 50uL of fluorescent dye solution with 1mg of TNF-α detection antibody, add coupling buffer to make up the volume to 200uL, and place on a shaker at room temperature for 1h;

[0188] (3) After the reaction is completed, the mixture is placed in a refrigerator at 4°C for overnight dialysis to obtain a fluorescent dye-labeled IL-6 detection antibody.

[0189] 2.3 Make a standard curve:

[0190] (1) using fetal bovine serum to dilute the TNF-α antigen concentration to obtain samples with concentrations of 0 pg / mL, 0.0098 pg / mL, 0.039 pg / mL, 0.156 pg / mL, 0.625 pg / mL, 2.5 pg / mL, 10 pg / mL, and 40 pg / mL;

[0191] (2) diluting the magnetic beads coupled to the TNF-α capture antibody to 1 mg / mL using magnetic bead storage solution, and diluting the TNF-α detection antibody labeled with a fluorescent dye to 10 μg / mL using antibody diluent;

[0192] (3) Take 50 μL of the sample of each concentration obtained in (1), add 50 μL of the TNF-α capture antibody-coupled magnetic bead dilution obtained in (2), then add 10 uL of the fluorescent dye-labeled TNF-α detection antibody dilution obtained in (2), mix well, and incubate at 37°C for 10 min;

[0193] (4) The incubation product obtained in (3) was washed three times with 100 μL of washing buffer, resuspended in 100 μL of imaging buffer, added to the groove of a transparent glass-bottomed 96-well plate, centrifuged at 2200 rpm for 3 min on a centrifuge (Hangzhou Aosheng MPC-P25), and then single-molecule imaging was performed using a fluorescence microscope (Nikon Eclipse Ti) to count the number of fluorescent spots on the bottom of the groove; each concentration was tested three times, and a standard curve was drawn based on the test results to calculate the detection limit. The standard curve was drawn as shown in FIG. Fig. 20 The calculated lower limit of detection is shown in Table 1.

[0194] Example 17 Capture antibody-solid phase carrier affinity ligand method coupled single molecule detection of TNF-α antigen

[0195] 1. Experimental materials: SA magnetic beads (particle size 200nm), TNF-α capture antibody (ab1793), TNF-α detection antibody (ab307164), fluorescent marker (atto-647-NHS), biotin-NHS (thermo), dimethyl sulfoxide (DMSO), DPBS buffer (8.0g sodium chloride, 0.2g potassium chloride, 1.15g disodium hydrogen phosphate, 0.2g potassium dihydrogen phosphate added to deionized water to 1000mL, pH7.4), magnetic bead storage solution ( 0.1% tween-20, 0.5% BSA dissolved in DPBS buffer), coupling buffer (0.5% BSA dissolved in Tris buffer, pH7.5), antibody diluent (0.5% BSA dissolved in DPBS buffer), washing buffer (0.5% tween20 dissolved in DPBS buffer, pH7.4), imaging buffer (0.1% tween20 dissolved in DPBS buffer, pH7.4), TNF-α antigen (Ibotek Recombinant Human TNF-alpha Protein RP00993), transparent glass-bottom 96-well plate (imaging carrier, model Cellvis P96-1.5HN).

[0196] 2. Experimental steps:

[0197] 2.1 Preparation of biotin-coupled TNF-α capture antibody:

[0198] (1) Weigh 5.677 mg of biotin-NHS and dissolve it in DMSO to obtain a biotin solution with a concentration of 10 μM;

[0199] (2) Take 0.5 mg of TNF-α capture antibody, add 6.5 μL of the biotin solution obtained in (1), and then add coupling buffer to make up the volume to 500 μL. Incubate at room temperature for 1 hour on a shaker, and then dialyze overnight at 4°C to obtain biotin-coupled TNF-α capture antibody.

[0200] 2.2 Preparation of fluorescent dye-labeled TNF-α detection antibody:

[0201] (1) Dissolve 1 mg of atto-647-NHS in 1 mL of DMSO to obtain a fluorescent dye solution with a concentration of 1 mg / ml;

[0202] (2) Mix 50 μL of fluorescent dye solution with 1 mg of TNF-α detection antibody, add coupling buffer to make up the volume to 200 μL, and place on a shaker at room temperature for 1 h;

[0203] (3) After the reaction is completed, the mixture is placed in a refrigerator at 4°C for overnight dialysis to obtain a fluorescent dye-labeled TNF-α detection antibody.

[0204] 2.3 Make a standard curve:

[0205] (1) using fetal bovine serum to dilute the TNF-α antigen concentration to obtain samples with concentrations of 0 pg / mL, 0.0098 pg / mL, 0.039 pg / mL, 0.156 pg / mL, 0.625 pg / mL, 2.5 pg / mL, 10 pg / mL, and 40 pg / mL;

[0206] (2) diluting the biotin-conjugated TNF-α capture antibody to 2 μg / mL and diluting the fluorescent dye-labeled TNF-α detection antibody to 10 μg / mL using antibody diluent;

[0207] (3) Take 50 μL of the biotin-coupled TNF-α capture antibody dilution obtained in (2), add 50 μL of the sample of each concentration obtained in (1), then add 10 μL of the fluorescent dye-labeled TNF-α detection antibody dilution obtained in (2), mix well, incubate at 37°C for 10 min, and then add 0.1 mg of SA magnetic beads and incubate for 10 min;

[0208] (4) The incubation product obtained in (3) was washed three times with 100 μL of washing buffer, resuspended in 100 μL of imaging buffer, added to the groove of a transparent glass-bottomed 96-well plate, centrifuged at 2200 rpm for 3 min on a centrifuge (Hangzhou Aosheng MPC-P25), and then single-molecule imaging was performed using a fluorescence microscope (Nikon Eclipse Ti) to count the number of fluorescent spots on the bottom of the groove; each concentration was tested three times, and a standard curve was drawn based on the test results to calculate the detection limit. The standard curve was drawn as shown in FIG. Fig.21 The calculated lower limit of detection is shown in Table 1.

[0209] Table 1

[0210]

[0211]

[0212] Combination Figure 1 , Figures 3 to 21 From the data in Table 1, it can be seen that the single molecule detection method of the present invention selects small-size microspheres in a specific size range as solid phase carriers, combined with the collection method of centrifugal sedimentation, which can significantly improve the sensitivity of detection, and the detection limit can reach below 10fg / ml.

[0213] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A single molecule detection method, characterized in that: The following steps are involved: Prepare an imaging carrier, the imaging carrier comprising a bottom plate and one or more grooves provided on the bottom plate, the groove having a flat bottom surface, the bottom surface of the groove and the bottom surface of the bottom plate are both horizontal surfaces, and the portion between the bottom surface of the groove and the bottom surface of the bottom plate is made of a transparent material; First, the sample, the capture antibody and the detection antibody labeled with a fluorescent dye are mixed and incubated, and then a solid phase carrier is added and mixed and incubated, wherein the solid phase carrier and the capture antibody can be directly or indirectly coupled; or the solid phase carrier and the capture antibody are first directly or indirectly coupled to form a capture antibody-coupled solid phase carrier, and then the sample, the capture antibody-coupled solid phase carrier, and the detection antibody labeled with a fluorescent dye are mixed and incubated; The mixed incubation product is centrifuged in the groove of the imaging carrier, and the number of fluorescent spots on the bottom surface of the groove is detected and counted using a fluorescence microscope; The capture antibody can bind to the first binding site of the target molecule, the detection antibody can bind to the second binding site of the target molecule, and the first binding site and the second binding site are different binding sites; The solid phase carrier is a microsphere with a diameter of 20nm to 500nm.

2. The single molecule detection method according to claim 1, characterized in that: The solid phase carrier is a microsphere with a diameter of 50nm to 200nm.

3. The single molecule detection method according to claim 1, characterized in that: The microspheres are surface-modified or non-surface-modified microspheres, and the surface modification includes at least one of amino modification, carboxyl modification, hydroxylation modification, sulfate modification, tosylation modification, epoxidation modification, streptavidin modification, biotin modification, and fluorescent dye modification.

4. The single molecule detection method according to claim 1, characterized in that: The microspheres are microspheres modified with a fluorescent dye on the surface, and the fluorescent dye used to mark the detection antibody has a different color from the fluorescent dye modified on the surface of the microspheres.

5. The single molecule detection method according to any one of claims 1 to 4, characterized in that: The fluorescent dye is at least one of fluorescein dyes, rhodamine dyes, coumarin dyes, quantum dot dyes, stilbene dyes, and naphthamide dyes.

6. The single molecule detection method according to any one of claims 1 to 4, characterized in that: The fluorescent dye is selected from atto-647-NHS, atto-532-NHS or AlexaFluor 647-NHS.

7. The single molecule detection method according to claim 1, characterized in that: The material of the microspheres is at least one of silicon, silica gel, silicon dioxide, polystyrene, iron, and iron oxide.

8. The single molecule detection method according to claim 1, characterized in that: The solid phase carrier is coupled with the capture antibody by at least one of NHS ester and EDC cross-linking method, glutaraldehyde cross-linking method, carbodiimide method, silanization method, affinity ligand mediation method and physical adsorption method.

9. The single molecule detection method according to claim 1, characterized in that: The temperature of the mixed incubation is 25-45° C., and the time is 3-30 minutes.

10. The single molecule detection method according to claim 1, characterized in that: The imaging carrier is a well plate or a chip or glass slide with grooves.

11. The single molecule detection method according to claim 1, characterized in that: The transparent material is glass or plastic, and the distance between the bottom surface of the groove and the bottom surface of the bottom plate is 0.08-0.25 mm.

12. The single molecule detection method according to claim 1, characterized in that: In S3, the centrifugal rotation speed is 500-2500 rpm.

Images