Single molecule immunodetection method based on enzymatic reaction

Abstract

The invention relates to the technical field of biology, in particular to a single-molecule immunodetection method based on enzymatic reaction. In the aspect of dispersing the magnetic particle carrier, the problem that the loading efficiency is unstable due to the fact that the magnetic particle carrier falls into holes in a microwell plate is solved, meanwhile, background noise and signal scattering can be reduced, the detection sensitivity can be improved, and very low-concentration protein can be detected; meanwhile, all the liquid drops can be counted at the same time by adopting optical imaging equipment, so that the accuracy is improved, and the CV value is reduced.

Description

[0001] This application claims the priority of the Chinese patent application filed with the China Patent Office on December 18, 2023, with application number 202311750171.1 and invention name “A single molecule immunoassay method based on enzymatic reaction”, the entire contents of which are incorporated by reference in this application. Technical Field

[0002] The invention relates to the field of biotechnology, and in particular to a single molecule immunoassay method based on enzymatic reaction. Background Art

[0003] Since its proposal, precision medicine has been considered the third medical revolution and the development direction of modern medicine. Its core consists of precision diagnosis and precision treatment. Precision diagnosis is the premise and foundation of precision treatment, and it is also the technical support and important guarantee of precision medicine. The precise measurement technology of protein content in blood is one of the important means indispensable for achieving precision diagnosis. Biomarkers refer to biochemical indicators that can mark changes or possible changes in the structure or function of systems, organs, tissues, cells and subcellular structures. They are widely used in the field of clinical diagnosis, among which protein biomarkers are the most important component. In the past half century, with the rapid development of immunoassay technology, protein biomarker detection systems have become more sensitive, more accurate and more user-friendly. At present, immunoassay is a widely used protein detection method in clinical practice. It can accurately detect specific antigens or antibody molecules in a short time. It has been widely used in tumor marker detection, prognosis monitoring, drug analysis and other fields.

[0004] Immunoassay is a chemical measurement method that uses the specific reaction between antigens and antibodies to identify the molecules to be tested, and then uses reagents to make the identified molecules to be tested generate signals for quantitative analysis. The molecules to be tested can be antigens or antibodies. Immunoassay was first invented by American scientists Berson and Yalow, and Yalow won the Nobel Prize in Medicine in 1977. So far, immunoassay has been developed for more than 50 years, with a variety of analysis modes and endless analysis types, and new methods are still being generated. Traditional immunoassays quantify by measuring the relationship between the intensity change of the macroscopic signal of the sample to be tested and the concentration of the target molecule. However, due to the limitations of the principle level, existing immunoassay technologies, such as chemiluminescence technology and enzyme-linked immunosorbent assay, can only achieve a detection limit of 1 pg / mL, and are helpless for many biomarkers in the pg / mL or sub-pg / mL concentration range, making a large number of biomarkers unable to be effectively used in clinical practice. It is difficult to meet the needs of early screening of major diseases, early diagnosis of neurological disorders, and early detection of cardiovascular diseases. In the early stages of major diseases, the concentration of antigens in the blood is 10 −15The mol / L level is even lower, and conventional immunoassays cannot measure it at all, let alone accurately measure it. For example, the mass concentration of prostate-specific antigen (PSA) in patients after prostate cancer surgery is 0.1×10 −12 ~100×10 −12 g / mL; the mass concentration of p24 antigen in early HIV patients is 0.01×10 −12 ~10×10 −12 g / mL; the minimum mass concentration of Alzheimer's-related β-amyloid protein is 0.3×10 −12 g / mL, such low concentrations are beyond the reach of traditional immunoassay technology.

[0005] Single-molecule immunoassay technology is considered to be the next generation of immunoassay technology because of its sensitivity performance that exceeds that of traditional immunoassay technology by 1,000 times. It is a key tool for developing clinical applications of these low-abundance new biomarkers. Single-molecule detection is a unique technology suitable for exploring the basic physical and chemical principles of molecules at the single-molecule level through conformational changes, dynamics, interactions between molecules, and manipulation of single molecules. It provides excellent sensitivity and high resolution that cannot be achieved by macroscopic measurements. The combination of single-molecule detection and immunoassay produces single-molecule immunoassay methods.

[0006] At present, the mainstream technical path for achieving single molecule immunoassay is:

[0007] Quanterix's Simoa technology is a method in which immune complexes are physically separated into separate compartments in a microwell array, and positive signals are observed only in microwells containing target complexes. An immune sandwich complex is formed on a 2.7 μm magnetic bead, labeled with an enzyme, and loaded into the array in the presence of a fluorescent enzyme substrate so that each bead is sealed in a reaction chamber of femtosecond volume. Microwells with enzyme-labeled immune complexes produce high concentrations of fluorescent products. By acquiring a time-lapse fluorescence image of the array using a standard microscope optical system, enzyme-labeled immune complex beads ("on") and unmodified beads ("off") can be distinguished, and the concentration of the analyte can be determined by counting the number of fluorescent wells. Although this method can achieve the detection limit of low-abundance proteins, it has high requirements for chip carriers, resulting in very high costs, low magnetic bead loading efficiency, and only partial areas are detected sequentially, which cannot achieve low detection errors, so it is difficult to meet the needs of clinical high-throughput detection.

[0008] Merck's SMC single-molecule detection technology uses the classic double-antibody immune sandwich method, using two antibodies targeting different epitopes of the same antigen to complete the detection. The capture antibody is coated on a multi-well plate or magnetic beads, and the detection antibody is coupled to a fluorescent label. The experimental steps used are very similar to sandwich ELISA, but there is a fundamental difference from ELISA in terms of signal acquisition: before the detection step, an elution buffer is used to dissociate the immune complex, and the eluate is transferred to a new 384-well plate and sent to the Erenna system for detection. The confocal laser forms a focus, and when a single fluorescent-labeled secondary antibody molecule passes through the narrow detection space at the focus, a light flashing signal is generated, which can be captured by the instrument and digitally measured. The Airy disk focusing detection device used in this technology can identify the intensity of a single fluorescent molecule, so the equipment requires extremely high precision and is expensive, and cannot be widely promoted in scientific research and clinical medicine. In addition, the technical principle of the technology itself has limited sensitivity and poor repeatability. In summary:

[0009] 1. Traditional chemiluminescence analysis (CLIA) and enzyme linked immunosorbent assay (ELISA) have limited sensitivity, with the lowest detection limit only reaching the picogram level, and are unable to detect low-abundance proteins. An Elisa kit only has one specific indicator to detect antibodies, so a sample can only detect one indicator in one experiment, which cannot meet the needs of multiple tests for current clinical sample testing.

[0010] 2. Quanterix's single-molecule immunoassay technology Simoa has high requirements for chip carriers and uses microwell array plates, which results in very high costs. The magnetic bead loading efficiency is low, less than 20%, and the optical detection equipment only detects certain areas. Different areas are detected in a certain order and cannot be detected simultaneously, making it impossible to achieve a low detection error. This results in poor repeatability and increased CV values ​​of 10% to 20%, making it difficult to meet the needs of clinical high-throughput testing.

[0011] 3. Merck's single-molecule technology SMC has poor system stability, slow reaction speed, false positive problems, high precision requirements for detection instruments, and is expensive, making it unsuitable for scientific research and clinical projects. Summary of the invention

[0012] In view of this, the present invention provides a single molecule immunoassay method based on enzymatic reaction, which uses droplets for full encapsulation and counting, and counts all droplets at the same time, thereby improving accuracy and reducing CV value.

[0013] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0014] The present invention provides a single molecule immunoassay method based on an enzymatic reaction, comprising the following steps:

[0015] Step (1): mixing the sample to be tested with the capture antibody fixed on the solid phase carrier, incubating, washing, and obtaining a first complex, wherein the capture antibody binds to the first site of the target molecule in the sample to be tested;

[0016] Step (2): Mixing the enzyme bound to streptavidin and the detection antibody modified with biotin with the first complex described in step (1), incubating, and washing to obtain a second complex, wherein the detection antibody binds to the second site of the target molecule, and the biotin on the detection antibody binds to the streptavidin;

[0017] Step (3): encapsulating the second complex described in step (2) with droplets, dispersing, reacting, collecting optical signals with optical imaging equipment, analyzing, and obtaining detection results;

[0018] The droplets include a substrate and acceptable auxiliary materials or adjuvants.

[0019] In some specific embodiments of the present invention, the solid phase carrier in the above-mentioned single molecule immunoassay method based on enzymatic reaction includes a magnetic particle carrier;

[0020] The magnetic particle carrier is modified with a functional group capable of coupling with the capture antibody;

[0021] The functional group includes one or more of carboxyl, hydroxyl, amino, sulfonyl, succinimide ester and their derivatives;

[0022] The particle size of the magnetic particle carrier is 1 μm, 1.5 μm, 2 μm, 2.5 μm, 2.8 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm or 5 μm.

[0023] In some specific embodiments of the present invention, the enzyme in the above-mentioned single-molecule immunoassay method based on enzymatic reaction includes one or more of horseradish peroxidase, alkaline phosphatase, β-galactosidase, polymerized horseradish peroxidase, polymerized alkaline phosphatase or polymerized β-galactosidase;

[0024] The substrate comprises one or more of o-phenylenediamine, tetramethylbenzidine, 5-aminosalicylic acid, 4-methylumbelliferyl phosphate, sodium p-nitrophenylphosphate hexahydrate, 4-methylumbelliferyl-RD galactoside, resorufin β-D-galactopyranoside or fluorescein di-β-D-galactoside.

[0025] In some specific embodiments of the present invention, the capture antibody in the above-mentioned single-molecule immunoassay method based on enzymatic reaction includes polyclonal antibodies and monoclonal antibodies.

[0026] In some specific embodiments of the present invention, the capture antibody in the above-mentioned single-molecule immunoassay method based on enzymatic reaction includes one or more of mouse antibody, rabbit antibody, sheep antibody and alpaca antibody.

[0027] In some specific embodiments of the present invention, the capture antibody in the above-mentioned single-molecule immunoassay method based on enzymatic reaction is obtained from manufacturers including Medix, Hi-Tide, Ibaxin, Biospacific or Abcam.

[0028] In some specific embodiments of the present invention, the detection antibody in the above-mentioned single-molecule immunoassay method based on enzymatic reaction comprises one of a polyclonal antibody and a monoclonal antibody.

[0029] In some specific embodiments of the present invention, the detection antibody in the above-mentioned single-molecule immunoassay method based on enzymatic reaction includes one or more of mouse antibody, rabbit antibody, sheep antibody and alpaca antibody.

[0030] In some specific embodiments of the present invention, the detection antibodies in the above-mentioned single-molecule immunoassay method based on enzymatic reaction are from manufacturers including Medix, Hi-Tide, Ibaxin, Biospacific or Abcam.

[0031] In some specific embodiments of the present invention, the target molecule in the above-mentioned single-molecule immunoassay method based on enzymatic reaction includes one or more of proteins, polysaccharides, small molecules with biological activity or complexes of small molecules and proteins.

[0032] In some specific embodiments of the present invention, the target molecule in the above-mentioned single-molecule immunoassay method based on enzymatic reaction includes one or more of cellular inflammatory factors, blood biomarkers of Alzheimer's disease or myocardial markers;

[0033] The cellular inflammatory factors include IL-6 and / or IL-17;

[0034] The Alzheimer's disease blood biomarkers include one or more of p-tau 181, p-tau 217, p-tau 231, NFL, Aβ-42 or Aβ-40;

[0035] The myocardial markers include cTnI;

[0036] The tumor markers include PSA.

[0037] In some specific embodiments of the present invention, the optical imaging device in the above-mentioned single-molecule immunoassay method based on enzymatic reaction includes one or more of a CCD camera, a CMOS camera or a CIS camera.

[0038] In some specific embodiments of the present invention, the optical imaging device in the above-mentioned single-molecule immunoassay method based on enzymatic reaction does not include a microscopic imaging device.

[0039] In some specific embodiments of the present invention, the acceptable excipients or auxiliary agents in the above-mentioned single-molecule immunoassay method based on enzymatic reaction include a fluorescence enhancer;

[0040] The fluorescence enhancer includes one or more of sodium hydroxide, calcium chloride, 4-methyl-2,6-dimethoxyaniline or prodigiosin.

[0041] In some specific embodiments of the present invention, in the above-mentioned single molecule immunoassay method based on enzymatic reaction:

[0042] The incubation temperature in step (1) is 20°C, 25°C, 30°C, 35°C, 40°C, 45°C or 50°C; and / or

[0043] The incubation time in step (1) is 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min, 85 min or 90 min.

[0044] In some specific embodiments of the present invention, in the above-mentioned single molecule immunoassay method based on enzymatic reaction:

[0045] The incubation temperature in step (2) is 20°C, 25°C, 30°C, 35°C, 40°C, 45°C or 50°C; and / or

[0046] The incubation time in step (2) is 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min.

[0047] In some specific embodiments of the present invention, in the above-mentioned single molecule immunoassay method based on enzymatic reaction:

[0048] The reaction time is 5 min, 10 min, 15 min, 20 min, 25 min or 30 min;

[0049] The reaction temperature is 20°C, 25°C, 30°C, 35°C, 40°C, 45°C or 50°C.

[0050] In some specific embodiments of the present invention, the incubation temperature range of the detection antibody and the enzyme-bound streptavidin in the above-mentioned single-molecule immunoassay method based on enzymatic reaction is 20°C, 25°C, 30°C, 35°C, 40°C, 45°C or 50°C, and the incubation time is 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45min, 50 min, 55 min or 60 min.

[0051] The detection method of the present invention has the following effects:

[0052] In terms of counting magnetic particle carriers, the present invention uses droplets for full encapsulation and counting, and the magnetic particle carriers are fully loaded, thereby ensuring the stability of the statistical quantity between different samples or different batches, improving the repeatability of sample data, and reducing the CV value;

[0053] The present invention improves the stability, injection volume and rate of generated droplets. The size of the generated droplets remains uniform, and the injection volume is less than 5 μL, which can save precious samples and reduce matrix effects. The time from droplet generation to completion does not exceed 5 minutes, which reduces the detection time;

[0054] In terms of dispersing magnetic particle carriers, the present invention solves the problem of unstable loading efficiency caused by magnetic particle carriers falling into the microporous plate, and can reduce background noise and signal scattering, improve detection sensitivity, and detect very low concentration proteins;

[0055] In terms of optimizing the detection of fluorescent signals, the present invention ensures the stability of the fluorescent signals and improves the intensity, shortens the detection time and improves the detection sensitivity by adding a fluorescent enhancer.

[0056] The method adopted by the present invention has low requirements on optical imaging equipment, and the results can be counted and analyzed without using expensive microscopic imaging technology;

[0057] The present invention uses optical imaging equipment to count all droplets at the same time. The statistical method is full statistics, which is performed on all generated droplets to improve accuracy and reduce CV value to within 5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0059] Figure 1 The standard curve of IL-6 detected by single molecule immunoassay in Example 1 is shown;

[0060] Figure 2 The standard curve of single molecule immunoassay p-Tau 181 in Example 2 is shown;

[0061] Figure 3 The standard curve of IL-6 detected by single molecule immunoassay in Comparative Example 1 is shown;

[0062] Figure 4 The standard curve of IL-6 detected by single molecule immunoassay in comparative example 2 is shown. DETAILED DESCRIPTION

[0063] The present invention discloses a single molecule immunoassay method based on an enzymatic reaction. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0064] A single molecule immunoassay method based on an enzymatic reaction, the specific steps are as follows:

[0065] 1. Immobilize antibody 1, i.e., capture antibody, onto a magnetic particle carrier, and utilize the capture antibody to bind to the first site of the target molecule, thereby capturing the target molecule in the sample;

[0066] 2. Add biotin-modified antibody 2, i.e., detection antibody, and let the detection antibody bind to the second site of the target molecule. Add enzyme-bound streptavidin to bind to the biotin-modified detection antibody to form a complex; or pre-bind enzyme-bound streptavidin to the biotin-modified detection antibody and then add it to bind to the second site of the target molecule;

[0067] 3. The combined complex and the substrate containing the fluorescence enhancer required for the enzymatic reaction are simultaneously introduced into the droplet generation chip device, and the generated droplets are introduced into the observation area;

[0068] 4. After standing for a period of time, use an optical imaging device to detect all optical signals emitted by the reaction products in the droplets;

[0069] 5. The number of magnetic particle carriers and the optical signals emitted by the reaction products in the droplets are counted, and the concentration information of the target molecules in the sample is further calculated.

[0070] Statistics and analysis:

[0071] 1. Detect the optical signals emitted by the reaction products in the droplets through optical imaging equipment, which can be divided into two statistical calculation methods;

[0072] 2. When the percentage of positive magnetic particle carriers in the droplet is less than 70% of the total magnetic particle carriers, the digital model AEB (Average Enzyme per Bead, the average number of enzyme molecules per magnetic particle carrier) is calculated by magnetic particle carrier counting and Poisson statistics, as shown in Formula I:

[0073] (Formula I)

[0074] where f on Indicates the percentage of magnetic particle carriers in the "on" state or active magnetic particle carriers;

[0075] 3. When the percentage of positive magnetic particle carriers in the droplets is higher than 70% of the total magnetic particle carriers, it is necessary to calculate the simulated digital model AEB by measuring the average fluorescence intensity of the positive magnetic particle carriers in the droplets, as shown in Formula II and Formula III:

[0076] (Formula II)

[0077] (Formula III)

[0078] in represents the average fluorescence intensity produced by a single enzyme in a droplet, It represents the average fluorescence intensity of positive magnetic particle carriers in the droplets.

[0079] 4. Use the concentration of the calibrator as the X-axis of the calibration curve, and the AEB value calculated by optical signal detection as the Y-axis of the calibration curve. The calibration curve is obtained by parameter simulation fitting. The AEB value of the sample to be tested detected and calculated by the optical imaging device is substituted into the calibration curve to calculate the concentration of the sample to be tested.

[0080] The key to the present invention lies in the selection of the particle size of the magnetic particle carrier, which is between 1 μm and 5 μm; including magnetic microspheres of conventional specifications such as 1 μm, 2 μm, 2.8 μm, 3 μm, and 4 μm; and the surface of the magnetic particle carrier is modified with active functional groups that can be covalently coupled with antibodies, specifically one or more of carboxyl, hydroxyl, amino, sulfonyl, succinimidyl ester and their derivative groups;

[0081] The key to the present invention lies in the selection of enzymes in the enzymatic reaction, and the enzyme can be one of horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-Gal), polymerized horseradish peroxidase (poly-HRP), polymerized alkaline phosphatase (poly-ALP) or polymerized β-galactosidase (poly-β-Gal);

[0082] The key to the present invention lies in the selection of substrates in the enzymatic reaction, and the substrates can be one or more of o-phenylenediamine (OPD), tetramethylbenzidine (TMB), 5-aminosalicylic acid (5-ASA), 4-methylumbelliferone phosphate (4-MUP), sodium p-nitrophenylphosphate hexahydrate (p-NPP), 4-methylumbelliferone-RD galactoside (4-MUU), resorufin β-D-galactopyranoside (RGP) and fluorescein di-β-D-galactoside (FDG);

[0083] The key to the present invention lies in the selection of capture antibodies. According to the antibody specificity, it can be one of polyclonal antibodies and monoclonal antibodies; according to the antibody source, it can be one or more of mouse antibodies, rabbit antibodies, sheep antibodies and alpaca antibodies. The antibody manufacturers include Medix, Haitide, Yibaixin, Biospacific, Abcam, etc.

[0084] The key to the present invention lies in the selection of detection antibodies. According to the antibody specificity, it can be one of polyclonal antibodies and monoclonal antibodies; according to the antibody source, it can be one or more of mouse antibodies, rabbit antibodies, sheep antibodies and alpaca antibodies. The antibody manufacturers include Medix, Haitide, Yibaixin, Biospacific, Abcam, etc.

[0085] The key to the present invention lies in the selection of target molecules, including one or more of proteins, polysaccharides, small molecules with biological activity, or complexes of small molecules and proteins. It is more suitable for low-abundance target molecules such as inflammatory factors IL-6 and IL-17, blood biomarkers of Alzheimer's disease p-Tau 181, p-Tau 217, p-Tau 231, NFL, Aβ-42, Aβ-40, myocardial marker cTnI, tumor marker PSA, etc.

[0086] The key to the present invention lies in the incubation temperature and incubation time of the sample and the capture antibody, the temperature range is 20-50°C, the best is room temperature 25°C, the time range is 30 min-90 min, the best is 30 min;

[0087] The key to the present invention lies in the incubation temperature and incubation time of the sample and the detection antibody, the temperature range is 20-50°C, the best is room temperature 25°C, the time range is 15 min-60 min, the best is 30 min;

[0088] The key to the present invention lies in the incubation temperature and incubation time of the detection antibody and the streptavidin combined with the enzyme, the temperature range is 20-50°C, the best is room temperature 25°C, the time range is 5 min-60 min, the best is 15 min;

[0089] The key to the present invention lies in the incubation temperature and time of the enzyme and substrate, the temperature range is 20-50°C, the best is room temperature 25°C, the time range is 5 min-30 min, the best is 15 min;

[0090] The key to the present invention is to add a fluorescence enhancer to the enzymatic reaction substrate, wherein the enhancer includes one or more of sodium hydroxide, calcium chloride, 4-methyl-2,6-dimethoxyaniline (4-MBN), prodigiosin, etc., which can improve the generation efficiency of the fluorescent product and reduce the quenching rate of the fluorescent product;

[0091] The key to the present invention lies in the optical imaging device, which uses a relatively low-cost optical imaging device, including one or more of a CCD, CMOS or CIS camera;

[0092] The key to the present invention is to perform a full inspection on all generated droplets at the same time, count the fluorescence intensities of all generated droplets at the same time, and perform analysis.

[0093] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0094] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0095] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0096] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0097] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0098] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0099] The present invention will be further described below in conjunction with embodiments:

[0100] Example 1: An example of detecting IL-6 samples using a single molecule immunoassay method based on an enzymatic reaction

[0101] 1. Coupling of magnetic beads to capture antibodies

[0102] 1) Transfer 16.7 μL of 30 mg / mL Dynabeads M-270 carboxylic acid magnetic beads (Thermo M270) to a reaction tube, wash three times with 300 μL of magnetic bead washing solution (10 mM PBS buffer), and remove the washing solution;

[0103] 2) Add 300 μL of magnetic bead diluent (10 mM PBS containing 0.1% TW20, pH=7.4) and wash three times, and add 291 μL of pre-cooled magnetic bead coupling buffer (50 mM MES, pH 5.0) for the last wash;

[0104] 3) Add 9 μL of 10 mg / mL pre-cooled EDC to the reaction tube and shake at 2-8°C for 30 min to activate the magnetic beads;

[0105] 4) Wash three times with 300 μL magnetic bead coupling buffer and remove the washing solution;

[0106] 5) Dilute the antibody to a final concentration of 0.2 mg / mL with 300 μL pre-cooled magnetic bead diluent, add to the reaction tube, shake at 2-8°C for 2 h, and couple the capture antibody (Abcam Anti-IL-6 antibody, ab9324) to the magnetic beads;

[0107] 6) Wash three times with 300 μL magnetic bead washing solution, add 300 μL magnetic bead blocking solution (purchased from Biotech, catalog number: P0220) to the reaction tube, and shake at room temperature for 45 min;

[0108] 7) Wash three times with 300 μL magnetic bead washing solution and remove the washing solution;

[0109] 8) Add 300 μL of magnetic bead diluent to wash once. After removing the washing solution, add 300 μL of magnetic bead storage solution (10 mM PBS containing 0.1% BSA and 0.1% TW20, pH=7.4) and store at 2-8°C.

[0110] 2. Standard curve

[0111] 1) Dilute the IL-6 antigen to 0, 0.001, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 and 50 pg / mL with sample diluent (10 mM PBS);

[0112] 2) Take 100 μL of the above samples of various concentrations, add 1 μL of 1.6 mg / mL magnetic bead antibody complex, mix and shake at room temperature for 30 min;

[0113] 3) Add 10 μL of biotin-labeled detection antibody (Abcam Biotin Anti-IL-6 antibody, ab84251), mix and shake at room temperature for 30 min, wash three times with washing solution, and remove the supernatant;

[0114] 4) Add 10 μL of streptavidin-coupled β-galactosidase (purchased from Thermo, S931), mix and shake at room temperature for 15 min, wash three times with cleaning solution, remove the supernatant, and add 10 μL of 10 mM PBS containing 0.1% TW20, pH = 7.4 to make up to volume.

[0115] 5) The complex generated by the immune reaction and the reaction substrate required for enzyme catalysis (fluorescein di-β-D-galactoside (FDG), purchased from Thermo, F1179) and fluorescence enhancer (0.1% NaOH) were simultaneously introduced into the droplet generation chip (purchased from Zhongxin Qiheng, ZX-LD-100Y) device, and the droplets generated at room temperature were allowed to react for 15 minutes;

[0116] 6) Use an optical imaging device CCD camera to detect the optical signal intensity in the generated droplets, and calculate through a mathematical model to obtain the concentration of the sample to be tested.

[0117] 3. Experimental Results

[0118] The results showed that the linear range of the IL-6 detection project of the present invention was 0-50 pg / mL, with the lowest detection limit of 0.001 pg / mL, and the CV value of the sample detection was less than 5%, which met the requirements of single molecule immunoassay. Figure 1 shown.

[0119] Example 2: A single molecule immunoassay method based on enzymatic reaction to detect p-Tau 181 sample example

[0120] 1. Coupling of magnetic beads to capture antibodies

[0121] 1) Transfer 21.9 μL of 25.4 mg / mL Bangs 3.06 μm carboxylated (Bangs 11653) magnetic beads to a reaction tube, wash three times with 300 μL of magnetic bead washing solution (10 mM PBS buffer), and remove the washing solution;

[0122] 2) Add 300 μL of magnetic bead diluent (10 mM PBS containing 0.1% TW20, pH=7.4) and wash three times, and add 291 μL of pre-cooled magnetic bead coupling buffer (10 mM MES, pH 5.0) for the last wash;

[0123] 3) Add 9 μL of 10 mg / mL pre-cooled EDC to the reaction tube and shake at 2-8°C for 30 min to activate the magnetic beads;

[0124] 4) Wash three times with 300 μL magnetic bead diluent and remove the washing solution;

[0125] 5) Dilute the antibody to a final concentration of 0.5 mg / mL with 300 μL pre-cooled magnetic bead diluent, add to the reaction tube, shake at 2-8°C for 2 h, and couple the capture antibody (Medix, Anti-h p-Tau 181 R13321 SPTN-5) to the magnetic beads;

[0126] 6) Wash three times with 300 μL magnetic bead washing solution, add 300 μL magnetic bead blocking solution (purchased from Biotech, catalog number: P0220) to the reaction tube, and shake at room temperature for 45 min;

[0127] 7) Wash three times with 300 μL magnetic bead washing solution and remove the washing solution;

[0128] 8) Add 300 μL of magnetic bead diluent to wash once. After removing the washing solution, add 300 μL of magnetic bead storage solution (10 mM PBS containing 0.1% BSA and 0.1% TW20, pH=7.4) and store at 2-8°C.

[0129] 2. Standard curve

[0130] 1) Dilute the p-Tau 181 antigen to 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50 and 100 pg / mL with sample diluent (10 mM PBS).

[0131] 2) Take 100 μL of the above samples of various concentrations, add 1 μL of 1.6 mg / mL magnetic bead antibody complex, mix and shake at room temperature for 30 min;

[0132] 3) Add 10 μL of biotin-labeled detection antibody (Medix, Anti-h Tau R13301 SPTN-5), mix and shake at room temperature for 45 min, wash three times with washing solution, and remove the supernatant;

[0133] 4) Add 10 μL of streptavidin-coupled polymerized horseradish peroxidase (Streptavidin Poly-HRP80Conjugate, purchased from Fitzgerald, 65R-S105PHRP), mix and shake at room temperature for 15 min, wash three times with cleaning solution, remove the supernatant, and add 10 μL of 10 mM PBS containing 0.1% TW20, pH=7.4 to make up to volume.

[0134] 5) The complex generated by the immune reaction and the reaction substrate required for enzyme catalysis (tetramethylbenzidine (TMB), purchased from Thermo, 002023) and the fluorescence enhancer (0.1% NaOH) were simultaneously introduced into the droplet generation chip device (purchased from Zhongxin Qiheng, ZX-LD-100Y), and the droplets generated at room temperature were allowed to react for 15 minutes;

[0135] 6) Use an optical imaging device CCD camera to detect the optical signal intensity in the generated droplets, and calculate through a mathematical model to obtain the concentration of the sample to be tested.

[0136] 3. Experimental Results

[0137] The results show that the linear range of the p-Tau 181 detection project of the present invention is 0-100 pg / mL, the lowest detection limit is 0.01 pg / mL, and the CV value of the sample detection is less than 5%, which meets the requirements of single molecule immunoassay. The standard curve is as follows Figure 2 shown.

[0138] Comparative Example 1: A single molecule immunoassay based on an enzymatic reaction to detect IL-6 samples (without using a droplet method)

[0139] 1. Coupling of magnetic beads to capture antibodies

[0140] 1) Transfer 16.7 μL of 30 mg / mL Dynabeads M-270 carboxylic acid magnetic beads (Thermo M270) to a reaction tube, wash three times with 300 μL of magnetic bead washing solution (10 mM PBS buffer), and remove the washing solution;

[0141] 2) Add 300 μL of magnetic bead diluent (10 mM PBS containing 0.1% TW20, pH=7.4) and wash three times, and add 291 μL of pre-cooled magnetic bead coupling buffer (50 mM MES, pH 5.0) for the last wash;

[0142] 3) Add 9 μL of 10 mg / mL pre-cooled EDC to the reaction tube and shake at 2-8°C for 30 min to activate the magnetic beads;

[0143] 4) Wash three times with 300 μL magnetic bead coupling buffer and remove the washing solution;

[0144] 5) Dilute the antibody to a final concentration of 0.2 mg / mL with 300 μL pre-cooled magnetic bead diluent, add to the reaction tube, shake at 2-8°C for 2 h, and couple the capture antibody (Abcam Anti-IL-6 antibody, ab9324) to the magnetic beads;

[0145] 6) Wash three times with 300 μL magnetic bead washing solution, add 300 μL magnetic bead blocking solution (purchased from Biotech, catalog number: P0220) to the reaction tube, and shake at room temperature for 45 min;

[0146] 7) Wash three times with 300 μL magnetic bead washing solution and remove the washing solution;

[0147] 8) Add 300 μL of magnetic bead diluent to wash once. After removing the washing solution, add 300 μL of magnetic bead storage solution (10 mM PBS containing 0.1% BSA and 0.1% TW20, pH=7.4) and store at 2-8°C.

[0148] 2. Standard curve

[0149] 1) Dilute the IL-6 antigen to 0, 0.001, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 and 50 pg / mL with sample diluent (10 mM PBS);

[0150] 2) Take 100 μL of the above samples of various concentrations, add 1 μL of 1.6 mg / mL magnetic bead antibody complex, mix and shake at room temperature for 30 min;

[0151] 3) Add 10 μL of biotin-labeled detection antibody (Abcam Biotin Anti-IL-6 antibody, ab84251), mix and shake at room temperature for 30 min, wash three times with washing solution, and remove the supernatant;

[0152] 4) Add 10 μL of streptavidin-coupled β-galactosidase (purchased from Thermo, S931), mix and shake at room temperature for 15 min, wash three times with cleaning solution, remove the supernatant, and add 10 μL of 10 mM PBS containing 0.1% TW20, pH = 7.4 to make up to volume.

[0153] 5) The complex generated by the immune reaction, the reaction substrate required for enzyme catalysis (fluorescein di-β-D-galactoside (FDG), purchased from Thermo, F1179) and the fluorescence enhancer (0.1% NaOH) were simultaneously introduced into the detection well, and the droplets generated at room temperature were allowed to react for 15 min;

[0154] 6) Use the optical imaging device CCD camera to detect the optical signal intensity in the hole, and calculate it through the mathematical model to obtain the concentration of the sample to be tested.

[0155] 3. Experimental Results

[0156] The results showed that the linear range of the IL-6 detection project of the present invention was 0-50 pg / mL, with the lowest detection limit of 0.01 pg / mL, and the CV value of the sample detection was between 10% and 20%, which was higher than that of the droplet method. Figure 3 shown.

[0157] Comparative Example 2: A single molecule immunoassay method based on enzymatic reaction to detect IL-6 samples (using a microscopic imaging system):

[0158] 1. Coupling of magnetic beads to capture antibodies

[0159] 1) Transfer 16.7 μL of 30 mg / mL Dynabeads M-270 carboxylic acid magnetic beads (Thermo M270) to a reaction tube, wash three times with 300 μL of magnetic bead washing solution (10 mM PBS buffer), and remove the washing solution;

[0160] 2) Add 300 μL of magnetic bead diluent (10 mM PBS containing 0.1% TW20, pH=7.4) and wash three times, and add 291 μL of pre-cooled magnetic bead coupling buffer (50 mM MES, pH 5.0) for the last wash;

[0161] 3) Add 9 μL of 10 mg / mL pre-cooled EDC to the reaction tube and shake at 2-8°C for 30 min to activate the magnetic beads;

[0162] 4) Wash three times with 300 μL magnetic bead coupling buffer and remove the washing solution;

[0163] 5) Dilute the antibody to a final concentration of 0.2 mg / mL with 300 μL pre-cooled magnetic bead diluent, add to the reaction tube, shake at 2-8°C for 2 h, and couple the capture antibody (Abcam Anti-IL-6 antibody, ab9324) to the magnetic beads;

[0164] 6) Wash three times with 300 μL magnetic bead washing solution, add 300 μL magnetic bead blocking solution (purchased from Biotech, catalog number: P0220) to the reaction tube, and shake at room temperature for 45 min;

[0165] 7) Wash three times with 300 μL magnetic bead washing solution and remove the washing solution;

[0166] 8) Add 300 μL of magnetic bead diluent to wash once. After removing the washing solution, add 300 μL of magnetic bead storage solution (10 mM PBS containing 0.1% BSA and 0.1% TW20, pH=7.4) and store at 2-8°C.

[0167] 2. Standard curve

[0168] 1) Dilute the IL-6 antigen to 0, 0.001, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 and 50 pg / mL with sample diluent (10 mM PBS);

[0169] 2) Take 100 μL of the above samples of various concentrations, add 1 μL of 1.6 mg / mL magnetic bead antibody complex, mix and shake at room temperature for 30 min;

[0170] 3) Add 10 μL of biotin-labeled detection antibody (Abcam Biotin Anti-IL-6 antibody, ab84251), mix and shake at room temperature for 30 min, wash three times with washing solution, and remove the supernatant;

[0171] 4) Add 10 μL of streptavidin-coupled β-galactosidase (purchased from Thermo, S931), mix and shake at room temperature for 15 min, wash three times with cleaning solution, remove the supernatant, and add 10 μL of 10 mM PBS containing 0.1% TW20, pH = 7.4 to make up to volume.

[0172] 5) The complex generated by the immune reaction and the reaction substrate required for enzyme catalysis (fluorescein di-β-D-galactoside (FDG), purchased from Thermo, F1179) and the fluorescence enhancer (0.1% NaOH) were simultaneously introduced into the droplet generation chip device (purchased from Zhongxin Qiheng, ZX-LD-100Y), and the droplets generated at room temperature were allowed to react for 15 minutes;

[0173] 6) Use a Leica DMi8 fluorescence microscope to detect the intensity of the optical signal in the generated droplets, and calculate it through a mathematical model to obtain the concentration of the sample to be tested.

[0174] 3. Experimental Results

[0175] The results showed that the linear range of the IL-6 detection item of the present invention was 0-50 pg / mL, with the lowest detection limit of 0.05 pg / mL, and the CV value of the sample detection was between 10% and 20%, which was higher than that of the non-microscopic optical imaging system. The standard curve is shown in Figure 4 shown.

[0176] Comparison of data between embodiment and comparative example:

[0177]

[0178] 1) Detection of CV value is an important indicator for ensuring data accuracy and stability of immunological detection methods. The lower the value, the higher the accuracy and stability. Example 1 and Example 2 use a droplet dispersion method and a fully observed optical imaging system, and the accuracy and stability can be maintained within 5%. Comparative Example 1 that does not use a droplet dispersion method or Comparative Example 2 that does not use a fully observed optical imaging system will have a reduced accuracy and stability of between 10% and 20%;

[0179] 2) The minimum detection limit is an important indicator for ensuring the sensitivity of immunological detection methods. The lower the value, the higher the sensitivity. Example 1 uses a droplet dispersion method and a fully observed optical imaging system, and the sensitivity can reach 0.001 pg / mL. Comparative Example 1 that does not use a droplet dispersion method or Comparative Example 2 that does not use a fully observed optical imaging system has a lower sensitivity.

[0180] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A single molecule immunoassay method based on enzymatic reaction, characterized in that: The steps include: Step (1): mixing the sample to be tested with the capture antibody fixed on the solid phase carrier, incubating, washing, and obtaining a first complex, wherein the capture antibody binds to the first site of the target molecule in the sample to be tested; Step (2): Mixing the enzyme bound to streptavidin and the detection antibody modified with biotin with the first complex described in step (1), incubating, and washing to obtain a second complex, wherein the detection antibody binds to the second site of the target molecule, and the biotin on the detection antibody binds to the streptavidin; Step (3): encapsulating the second complex described in step (2) with droplets, dispersing, reacting, collecting optical signals with optical imaging equipment, analyzing, and obtaining detection results; The droplets include a substrate and acceptable auxiliary materials or adjuvants.

2. The single molecule immunoassay method based on enzymatic reaction according to claim 1, characterized in that: The solid phase carrier comprises a magnetic particle carrier; The magnetic particle carrier is modified with a functional group capable of coupling with the capture antibody; The particle size of the magnetic particle carrier is 1 μm to 5 μm.

3. The single molecule immunoassay method based on enzymatic reaction according to claim 1 or 2, characterized in that: The enzyme includes one or more of horseradish peroxidase, alkaline phosphatase, β-galactosidase, polymerized horseradish peroxidase, polymerized alkaline phosphatase or polymerized β-galactosidase; The substrate comprises one or more of o-phenylenediamine, tetramethylbenzidine, 5-aminosalicylic acid, 4-methylumbelliferyl phosphate, sodium p-nitrophenylphosphate hexahydrate, 4-methylumbelliferyl-RD galactoside, resorufin β-D-galactopyranoside or fluorescein di-β-D-galactoside.

4. The single molecule immunoassay method based on enzymatic reaction according to any one of claims 1 to 3, characterized in that: The target molecule includes one or more of a protein, a polysaccharide, a small molecule with biological activity, or a complex of a small molecule and a protein.

5. The single molecule immunoassay method based on enzymatic reaction according to any one of claims 1 to 4, characterized in that: The optical imaging device includes one or more of a CCD camera, a CMOS camera or a CIS camera.

6. The single molecule immunoassay method based on enzymatic reaction according to any one of claims 1 to 5, characterized in that: The optical imaging device does not include a microscopic imaging device.

7. The single molecule immunoassay method based on enzymatic reaction according to any one of claims 1 to 6, characterized in that: The acceptable excipients or auxiliary agents include fluorescence enhancers; The fluorescence enhancer includes one or more of sodium hydroxide, calcium chloride, 4-methyl-2,6-dimethoxyaniline or prodigiosin.

8. The single molecule immunoassay method based on enzymatic reaction according to any one of claims 1 to 7, characterized in that: include: The incubation temperature in step (1) is 20 to 50°C; and / or The incubation time in step (1) is 30 min to 90 min.

9. The single molecule immunoassay method based on enzymatic reaction according to any one of claims 1 to 8, characterized in that: include: The incubation temperature in step (2) is 20 to 50°C; and / or The incubation time in step (2) is 15 min to 60 min.

10. The single molecule immunoassay method based on enzymatic reaction according to any one of claims 1 to 9, characterized in that: include: The reaction time is 5 min to 30 min; The reaction temperature is 20-50°C.

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