Digital immunodetection analysis method for accurate quantification of protein

Abstract

The invention relates to a digital immunodetection analysis method for accurate quantification of protein. Specifically, the invention provides a digital immunodetection analysis method for accurate quantification of protein, which comprises the following steps: (s1) providing a water phase mixture; (s2) mixing the aqueous phase mixture with a chromogenic substrate and an oil phase to form a first mixture; (s3) carrying out shearing treatment on the first mixture so as to form a sheared water-in-oil second mixture; wherein the second mixture contains a first micro-droplet and a second micro-droplet; the first micro-droplet is a micro-droplet containing a positive signal compound; the second micro-droplet is a micro-droplet containing a negative signal compound; and (s4) determining the first micro-droplets to obtain the number of the first micro-droplets so as to obtain a quantitative detection result of the protein.

Description

Technical Field

[0001] The invention belongs to the field of biological analysis and specifically relates to a digital immunoassay analysis method for accurate protein quantification. Background Art

[0002] For the determination of new biomarkers, enzyme-linked immunosorbent assay (ELISA) has been unable to meet the requirements so far. For example, for the quantitative analysis of proteins such as P-tau-181, 217, 231 antigens related to Alzheimer's disease, and neurofilament light chain (NfL), the sensitivity of this method can no longer meet the requirements. Although these have been written into clinical guidelines, traditional detection methods cannot achieve accurate determination because the content of these substances in the blood is too low. In addition, the response of the reporter molecule is limited due to the large reaction system. The concentration of the reporter molecule is too low, and the reaction system is too large, resulting in the effective signal concentration being diluted by the reaction solution.

[0003] In recent years, digital immunoassay methods have received increasing attention due to their high sensitivity and are regarded as the new gold standard. For example, Quanterix's simoa platform is a typical representative of this method. First, the simoa platform uses more than 200,000 micron-level micropores as detection sampling carriers, limiting the reaction system to the fL level, which is 1,000,000,000 times smaller than the uL level volume of the traditional ELISA method. In this way, the signal generated by a reporter molecule is confined to a smaller system, and its concentration can reach the range that the instrument's optical system can easily respond to (mg / mL level), and the number of observed molecules can be obtained by counting.

[0004] However, since digital immune signals cannot sample all molecules, due to technical constraints or sample concentration issues, it cannot be ensured that all molecules can be sampled and detected. In other words, it cannot be guaranteed that every molecule can be "captured" or "read". In this case, the sampling results will be affected by randomness, that is, only some molecules can be sampled and detected, and there is a certain uncertainty as to whether these molecules can represent all. At low concentrations, the sampling process conforms to the Poisson distribution, and 200,000 micropores can only meet the number of digital immune signal holes of about 99% under the conditions of about 1-6000 digital immune signals. In addition, due to the existence of Poisson distribution noise, the theoretical CV = (n is the number of positive molecules) is ≥10% (20% for 25 molecules) when the number of digital immune is less than 100, which determines that its linear range is narrow and can only be achieved under the state of 25-6000 molecules, which is difficult to meet the full digitalization requirements of practical applications.

[0005] In addition, the simoa platform uses magnetic beads as capture units, and then introduces the magnetic beads into the microwells for analysis. This process is essentially a secondary sampling, which undoubtedly increases the uncertainty of sampling. Since the efficiency of secondary sampling is not high, according to relevant literature reports, the sampling efficiency of the simoa platform HD-1 is only about 5%. This is mainly due to the limitation of the number of microwells by the chip area and the problem of well entry efficiency caused by the method of introducing magnetic beads into the microwells.

[0006] Therefore, to improve the performance of digital immunoassay, the key lies in increasing the number of micropores and improving the efficiency of secondary sampling. Although digital immunoassay has great potential value, the high cost of the simoa solution makes it difficult to increase the detection throughput and the linear range is too narrow, which makes it difficult to meet the needs of large-scale low-cost applications.

[0007] At present, the method of using microdroplets for digital detection has been widely used in digital PCR technology. The digital PCR systems on the market mainly use microdroplet generation technology, among which Bio-Rad's QX200 Droplet DigitalPCR System is a typical representative. This system generates microdroplets through microfluidic chips or microfluidic valves. Each microdroplet can be regarded as a tiny reaction chamber that can perform independent PCR reactions. In Bio-Rad's system, the generation of microdroplets is achieved through a technology called "oil-in-water", that is, surfactants are added to two immiscible liquids (such as water and oil) to form microdroplets through physical shear or pressure drive. The size of each microdroplet is about 1 nanoliter, and each PCR reaction can generate about 20,000 microdroplets. These microdroplets are evenly distributed in the reaction tube during the PCR reaction, so that the number of DNA molecules in each microdroplet conforms to the Poisson distribution, thereby realizing digital PCR detection.

[0008] This technology has been used in some fields. However, in contrast, digital immunoassays based on microdroplets have not yet been commercially applied.

[0009] Therefore, it is of great significance to develop a digital immunoassay method with high sensitivity, high accuracy and high efficiency in this field. Summary of the invention

[0010] The invention provides a digital immunoassay method with high sensitivity, high accuracy and high efficiency.

[0011] In a first aspect of the present invention, a digital immunoassay analysis method for accurate protein quantification is provided, comprising the steps of:

[0012] (s1) providing an aqueous phase mixture, wherein the aqueous phase mixture contains the following components:

[0013] (A) Positive signal complex: magnetic beads-antibody-antigen-detection antibody-enzyme marker, and / or magnetic beads-antibody

[0014] - antigen-detection antibody complex; and

[0015] (B) Negative signal complex: magnetic bead-antibody complex;

[0016] (s2) mixing the aqueous phase mixture with a chromogenic substrate and an oil phase to form a first mixture;

[0017] (s3) subjecting the first mixture to mechanical shearing treatment to form a sheared water-in-oil second mixture;

[0018] Wherein, the second mixture contains a first microdroplet and a second microdroplet; the first microdroplet is a microdroplet containing a positive signal complex; the second microdroplet is a microdroplet containing a negative signal complex; and

[0019] (s4) Counting the first micro-droplets to obtain the number of the first micro-droplets, thereby obtaining a quantitative detection result of the protein.

[0020] In another preferred embodiment, step (s1) further includes the following sub-steps:

[0021] (s1a) providing an aqueous monodisperse magnetic bead solution;

[0022] (s1b) adding a certain proportion of capture antibody (first antibody) to the magnetic bead solution to couple the magnetic beads with the capture antibody to form a magnetic bead-antibody complex, followed by washing;

[0023] (s1c) adding an antigen (a first antigen or a protein to be tested) to a solution containing the magnetic bead-antibody complex and reacting to form a magnetic bead-antibody-antigen complex, followed by washing;

[0024] (s1d) adding a detection antibody (secondary antibody) with biotin or horseradish peroxidase (HRP) to the solution containing the magnetic bead-antibody-antigen complex and reacting to form a magnetic bead-antibody-antigen-detection antibody complex, followed by washing;

[0025] When the detection antibody with biotin is added, step (s1e) is performed; when the detection antibody with horseradish peroxidase is added, step (s2) is directly performed;

[0026] (s1e) adding an enzyme label molecule to a solution containing the magnetic bead-antibody-antigen-detection antibody complex to form a magnetic bead-antibody-antigen-detection antibody-enzyme label, followed by washing; and

[0027] (s1f) Optionally, a fluorescent antibody (negative signal antibody) against the capture antibody is added to the solution containing the magnetic beads-antibody-antigen-detection antibody-enzyme marker, so that the magnetic beads are fluorescently labeled (negative signal marker), followed by washing.

[0028] In another preferred embodiment, the particle size of the magnetic beads is 0.03-100 μm.

[0029] In another preferred embodiment, the particle size of the magnetic beads is 1-5 μm.

[0030] In another preferred embodiment, step (s3) further includes the following sub-steps:

[0031] (s3a) subjecting the first mixture to mechanical shearing treatment, wherein the shearing treatment is to disperse the first mixture using mechanical segmentation (high-speed homogenizer) so that the magnetic beads are evenly dispersed in the microdroplets, thereby forming a second water-in-oil mixture; and

[0032] (s3b) allowing the second mixture to react for a period of time.

[0033] In another preferred embodiment, the following step is further included between steps (s3) and (s4): washing the sheared second water-in-oil mixture to remove empty droplets that do not contain any first microdroplets and second microdroplets.

[0034] In another preferred embodiment, step (s4) further includes the step of measuring the second microdroplets to obtain the number of the second microdroplets and the ratio of the number of the first microdroplets to the second microdroplets, thereby obtaining a quantitative detection result of the protein.

[0035] In another preferred embodiment, step (s2) further includes the following sub-steps:

[0036] (s2a) Evenly mixing the solution containing the magnetic beads-antibody-antigen-detection antibody-enzyme marker or magnetic beads-antibody-antigen-detection antibody complex with a colorimetric substrate, and immediately adding an oil phase reagent to form a first mixture.

[0037] In another preferred embodiment, the oil phase is selected from the group consisting of fluorinated oils: HFE7500, FC40, Fluo-Oil 7500, Fluo-Oil 40, Fluo-oil 135, Fluo-Oil 200, or a combination thereof.

[0038] In another preferred embodiment, a fluorinated surfactant is further added to the fluorinated oil to form a final oil phase.

[0039] In another preferred embodiment, the fluorinated surfactant is selected from the following group: fluoroalkyl polyether sulfate, fluoroalkyl polyethylene glycol ether sulfate, fluorocarbon-based alcohol and fluorocarbon-based acid, PFPE-PEG polymer, PFPE-b-PPO-PEO-PPO-b-PFPE, or a combination thereof.

[0040] In another preferred embodiment, the enzyme-labeled molecule is streptavidin-HRP.

[0041] In another preferred embodiment, the washing is performed by adsorbing and retaining droplets wrapped with single magnetic beads by magnetic force, thereby washing and enriching.

[0042] In another preferred embodiment, the magnetic beads are magnetic beads with fluorescent properties or magnetic beads without fluorescent properties.

[0043] In another preferred embodiment, the magnetic beads are magnetic beads with fluorescent properties.

[0044] In another preferred embodiment, the method uses multi-color magnetic beads with fluorescent properties to perform quantitative detection of multiple substances.

[0045] In another preferred embodiment, the method is used to measure n kinds of proteins, and the value of n is 1-200, preferably 1-20, and more preferably 1-10.

[0046] In another preferred embodiment, when n kinds of proteins are detected, the second mixture contains first microdroplets corresponding to each protein.

[0047] In another preferred embodiment, the total number of the first microdroplets and the second microdroplets in the second mixture effectively quantified by the method is greater than 50,000.

[0048] In another preferred embodiment, the total number of the first micro-droplets and the second micro-droplets in the second mixture effectively quantified by the method is 50,000 to 1,000,000.

[0049] In another preferred embodiment, the total number of the first micro-droplets and the second micro-droplets in the second mixture that can be effectively quantified by the method increases as the target surface of the optical camera increases.

[0050] In another preferred embodiment, the magnetic beads with fluorescent properties emit the following fluorescence: green (520nm), yellow (490nm), red (610nm), blue (450nm), and rare earth europium fluorescence and rare earth terbium fluorescence and other different wavelengths of light.

[0051] In another preferred embodiment, the magnetic beads also have different modifications.

[0052] In another preferred embodiment, the magnetic bead modifications include: carboxyl, amino, p-toluenesulfonic acid, NHS, biotin and streptavidin (AS).

[0053] In another preferred embodiment, the magnetic beads are magnetic beads without fluorescent properties.

[0054] In another preferred embodiment, when the magnetic beads are magnetic beads without fluorescent properties, step (s1f) is used to fluorescently label the magnetic beads.

[0055] In another preferred embodiment, in step (s1b), the antibody is coupled via a carboxyl group.

[0056] In another preferred embodiment, in step (s3), when the first mixture is subjected to mechanical shearing treatment, a second mixture containing more than 10,000 microdroplets is quickly formed.

[0057] In another preferred embodiment, in step (s3), when the first mixture is subjected to mechanical shearing treatment, a second mixture containing 50,000 to 10 billion microdroplets is quickly formed.

[0058] In another preferred embodiment, in step (s3), the volume of the second water-in-oil mixture is 10-1000 uL.

[0059] In another preferred embodiment, the total volume of the microdroplets in the second mixture is 10-1000 uL.

[0060] In another preferred embodiment, the concentration of the microdroplets in the second mixture is 0.5-90% (v / v).

[0061] In another preferred embodiment, the size of the microdroplets is 1-100 μm.

[0062] In another preferred embodiment, the mechanical shearing treatment is to use a high-speed homogenizer to perform high-speed homogenization on the first mixture.

[0063] In another preferred embodiment, the speed of the high-speed homogenizer is 2000-100000 RPM.

[0064] In another preferred embodiment, the speed of the high-speed homogenizer is 10000 RPM.

[0065] In another preferred embodiment, the ratio D1 / D2 of the diameter D1 of the first micro-droplet to the diameter D2 of the magnetic beads contained therein is 1.2-20, preferably 1.5-5, and more preferably 1.6-4.

[0066] In another preferred embodiment, the ratio D1 / D2 of the diameter D1 of the first micro-droplet to the diameter D2 of the magnetic beads contained therein is 1.8-3.

[0067] In another preferred embodiment, the total content of the first micro-droplets and the second micro-droplets in the second water-in-oil mixture is less than 30% relative to the content of the empty droplets, preferably less than 20%, and more preferably less than 10%.

[0068] In another preferred embodiment, the content of the first micro-droplets in the second water-in-oil mixture is greater than 30%.

[0069] In another preferred embodiment, the color developing substrate includes: ADHP (OxiRed probe), BBTP and FDG.

[0070] In another preferred embodiment, the color developing substrate is ADHP (OxiRed probe).

[0071] In another preferred embodiment, there is no cross-interference between the spectrum of the substance produced by the enzymatic reaction between the positive complex and the chromogenic substrate and the emission wavelength of the negative complex.

[0072] In another preferred embodiment, in step (s3b), the static reaction conditions are: reaction at 37°C for 5 minutes.

[0073] In another preferred embodiment, in step (s4), green and red are used. color The fluorescence signals of the two channels are used to accurately count the number of the first droplet and the second droplet after the reaction.

[0074] In another preferred embodiment, in step (s4), a droplet imaging instrument is used to perform fluorescence photography, and within one detection cycle, the following can be photographed: the number n of first micro-droplets and the number m of second micro-droplets.

[0075] In another preferred embodiment, in step (s4), the droplet imaging instrument uses a monochrome or color camera, which is a CCD camera or CMOS camera, monochrome or color camera with more than 5 million pixels;

[0076] In another preferred embodiment, in step (s4), a special low-imaging and low-distortion lens is selected so that a magnetic bead can be clearly distinguished.

[0077] In another preferred embodiment, in step (s4), the determination includes determining the second microdroplet in the following manner: using a fluorescent antibody to bind to a magnetic bead-antibody complex so that the magnetic beads are fluorescently labeled (negative signal labeled) to determine the second microdroplet; or selecting magnetic beads with fluorescent properties, based on the characteristic that there is no cross-interference between their emission wavelength and the spectrum of the substance generated by the enzymatic reaction in step (s3), thereby achieving the determination of the second microdroplet.

[0078] In another preferred embodiment, when the magnetic beads are magnetic beads with fluorescent properties, the fluorescence of the magnetic beads can produce different fluorescence intensities depending on the content of the fluorescent dye, thereby further identifying and distinguishing the magnetic beads.

[0079] In a second aspect of the present invention, a digital immunoassay device for protein quantification is provided, the device comprising:

[0080] (a) a mixing module, wherein the mixing module is configured to form an aqueous phase mixture, wherein the aqueous phase mixture contains the following components:

[0081] (A) Positive signal complex: magnetic beads-antibody-antigen-detection antibody-enzyme marker, and / or magnetic beads-antibody

[0082] - antigen-detection antibody complex; and

[0083] (B) Negative signal complex: magnetic bead-antibody complex;

[0084] (b) a shearing module, wherein the shearing module is configured to add a color developing substrate and an oil phase to the aqueous phase mixture to form a first mixture; and perform a mechanical shearing treatment to form a sheared oil-in-water second mixture, wherein the second mixture contains a first microdroplet and a second microdroplet; the first microdroplet is a microdroplet containing a positive signal complex; and the second microdroplet is a microdroplet containing a negative signal complex;

[0085] (c) A digital detection module, wherein the digital detection module is configured to measure the first micro-droplets to obtain the number of the first micro-droplets, thereby obtaining a quantitative detection result of the protein.

[0086] In another preferred embodiment, the particle size of the magnetic beads is 0.03-100 μm.

[0087] In another preferred embodiment, the particle size of the magnetic beads is 1-5 μm.

[0088] In another preferred embodiment, the magnetic beads are magnetic beads with fluorescent properties or magnetic beads without fluorescent properties.

[0089] In another preferred embodiment, the oil phase is selected from the group consisting of fluorinated oils: HFE7500, FC40, Fluo-Oil 7500, Fluo-Oil 40, Fluo-oil 135, Fluo-Oil 200, or a combination thereof.

[0090] In another preferred embodiment, a fluorinated surfactant is further added to the fluorinated oil to form a final oil phase.

[0091] In another preferred embodiment, the fluorinated surfactant is selected from the following group: fluoroalkyl polyether sulfate, fluoroalkyl polyethylene glycol ether sulfate, fluorocarbon-based alcohol and fluorocarbon-based acid, PFPE-PEG polymer, PFPE-b-PPO-PEO-PPO-b-PFPE, or a combination thereof.

[0092] In another preferred embodiment, in step (s3), the volume of the second water-in-oil mixture is 10-1000 uL.

[0093] In another preferred embodiment, the concentration of the microdroplets in the second mixture is 0.5-90% (v / v).

[0094] In another preferred embodiment, the size of the microdroplets is 1-100 μm.

[0095] In another preferred embodiment, the mechanical shearing treatment is to use a high-speed homogenizer to perform high-speed homogenization on the first mixture.

[0096] In another preferred embodiment, the speed of the high-speed homogenizer is 2000-100000 RPM.

[0097] In another preferred embodiment, the speed of the high-speed homogenizer is 10000 RPM.

[0098] In another preferred embodiment, the ratio D1 / D2 of the diameter D1 of the first micro-droplet to the diameter D2 of the magnetic beads contained therein is 1.2-20, preferably 1.5-5, and more preferably 1.6-4.

[0099] In another preferred embodiment, the ratio D1 / D2 of the diameter D1 of the first micro-droplet to the diameter D2 of the magnetic beads contained therein is 1.8-3.

[0100] In another preferred embodiment, the total content of the first micro-droplets and the second micro-droplets in the second water-in-oil mixture is less than 30% relative to the content of the empty droplets, preferably less than 20%, and more preferably less than 10%.

[0101] In another preferred embodiment, the color developing substrate is ADHP (OxiRed probe).

[0102] In another preferred embodiment, in step (s4), a droplet imaging instrument is used to perform fluorescence photography, and within one detection cycle, the following can be photographed: the number n of first micro-droplets and the number m of second micro-droplets.

[0103] In another preferred embodiment, the method is used to measure n kinds of proteins, and the value of n is 1-200, preferably 1-20, and more preferably 1-10.

[0104] In another preferred embodiment, when n kinds of proteins are detected, the second mixture contains first microdroplets corresponding to each protein.

[0105] In another preferred embodiment, when n kinds of proteins are detected, the second mixture contains first microdroplets corresponding to each protein.

[0106] In another preferred embodiment, the total number of the first microdroplets and the second microdroplets in the second mixture effectively quantified by the method is greater than 50,000.

[0107] In another preferred embodiment, the total number of the first micro-droplets and the second micro-droplets in the second mixture effectively quantified by the method is 50,000 to 1,000,000.

[0108] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Figure 1 The digital immunoassay analysis method is a single-component, highly sensitive method.

[0110] Figure 2 Single molecule detection analysis method, multiplexed approach is shown.

[0111] Figure 3 A single molecule detection analysis method is shown in a rapid manner.

[0112] Figure 4 It shows: (A) after adding droplets to generate oil, no high-speed stirring or homogenization operation is used, the magnetic bead liquid phase floats on the upper layer, and the oil is in the lower layer; (B) after mechanical shearing is used to form immune microdroplets, it is in the state of oil-in-water magnetic bead solution, in which the oil phase is at the bottom and the droplets encapsulating the magnetic beads are in the upper layer.

[0113] Figure 5 The detection chip of the present invention is shown, which is composed of two layers of non-fluorescent material with a 100 um sandwich in the middle (unit: mm).

[0114] Figure 6 Shown are: (A) a fluorescence photograph taken under a 1X objective lens, a green channel fluorescence photograph (the oil-in-water fluorescent magnetic bead photograph includes negative and positive droplets); (B) a fluorescence photograph taken under a 1X objective lens, a red channel fluorescence photograph (positive droplets after HRP reaction); and (C) a fluorescence photograph taken under a 1X objective lens, the green and red channel fluorescence photographs are synthesized.

[0115] Figure 7A digitized immunoassay IL2 scatter plot is shown: the red population are positive droplets containing HRP, and the black population are negative droplets not containing HRP.

[0116] Figure 8 Digital immunoassay IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15 scatter plots are shown.

[0117] Fig. 9 Shown are (A) signal scatter plot and (B) red channel count histogram (50 fg / mL) for rapid analysis of SARS-CoV-2 antigen. DETAILED DESCRIPTION

[0118] After extensive and in-depth research, the inventors have developed for the first time a highly sensitive, highly accurate and highly efficient digital immunoassay method for accurate protein quantification. Specifically, the method comprises the following steps: (s1) providing an aqueous phase mixture, wherein the aqueous phase mixture contains the following complexes: (A) a positive signal complex: magnetic beads-antibody-antigen-detection antibody-enzyme marker, and / or a magnetic beads-antibody-antigen-detection antibody complex; and (B) a negative signal complex: a magnetic beads-antibody complex; (s2) mixing the aqueous phase mixture with a chromogenic substrate and an oil phase to form a first mixture; (s3) subjecting the first mixture to mechanical shearing treatment to form a sheared oil-in-water second mixture; wherein the second mixture contains a first microdroplet and a second microdroplet; the first microdroplet is a microdroplet containing a positive signal complex; the second microdroplet is a microdroplet containing a negative signal complex; and (s4) measuring the first microdroplet to obtain the number of the first microdroplet, thereby obtaining a quantitative detection result of the protein. The present invention is completed on this basis.

[0119] the term

[0120] As used herein, the abbreviation "HRP" refers to horseradish peroxidase, which is capable of binding to streptavidin to form a stable complex.

[0121] As used herein, the abbreviation "ADHP" is a highly sensitive and stable peroxidase substrate that selectively detects hydrogen peroxide (H 2 O 2 ). In the presence of horseradish peroxidase (HRP), this essentially colorless and non-fluorescent ADHP reacts with H 2 O 2 The reaction is carried out in a 1:1 chemical quantitative ratio to generate strong red fluorescent resorufin, which can be analyzed by fluorescence photometer and microplate reader. Many enzyme reactions can produce H 2 O 2Therefore, ADHP can be used in coupled enzymatic reactions to detect the biological activities of many different enzymes.

[0122] As used herein, "microspheres" and "magnetic beads" are used interchangeably to refer to magnetic beads with or without fluorescent properties of the present invention.

[0123] As used herein, "the first antigen of the present invention", "the antigen of the present invention" and "the protein to be tested of the present invention" are used interchangeably and refer to the protein to be accurately quantified. As used herein, the term "comprising" can be open, semi-closed or closed. In other words, the term includes the following definitions: "comprising", "essentially consisting of", "consisting of".

[0124] ELISA

[0125] Enzyme-linked immunosorbent assay (ELISA) is a commonly used protein quantitative analysis method. Since this technology (enzyme-linked immunosorbent assay) was proposed in the 1970s, it has been widely used in various scientific researches and has become an indispensable classic technology in the field of biomedical research and development. ELISA is a basic immunoassay method that uses enzyme reaction catalysis to perform quantitative analysis of antigen-antibody binding. It is used to identify peptides, proteins, antibodies, hormones, etc. In the past few decades, due to its high sensitivity and high throughput, it has always been the preferred technology for protein quantitative analysis and is also recognized as the "gold standard" in the industry. Its basic reaction process is to specifically bind the well plate coated with antibodies to the antigen in the sample, then bind the detection antibody labeled with the reporter molecule to the antigen, and finally use the signal generated by the reporter molecule, such as color reaction, light reaction, magnetic reaction, and electrochemical reaction, to indirectly quantitatively analyze the antigen. Although this method has certain advantages, it also has some unavoidable defects. Its biggest problem is that it is essentially still an analog quantitative test technology and can no longer meet the needs of new biomarker determination.

[0126] Immune responses and droplet-based digital immunoassays

[0127] Unlike molecular hybridization reactions which involve only the interaction of two molecules (probe and target), immune reactions usually involve multifaceted interactions of antigen, antibody, and accessory molecules.

[0128] Therefore, in order to effectively carry out immune response, it is necessary to use a carrier containing antibodies to capture and enrich the target protein. This method has been widely used in various immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA).

[0129] Secondly, regarding the need for signal amplification. In immunoassays, since the number of target molecules is extremely small, additional reporter molecules need to be introduced to amplify the detection signal. This is usually achieved by introducing fluorescently labeled or enzyme-labeled reporter molecules, which can bind to target molecules or antibodies to produce detectable signals.

[0130] Finally, the characteristics of Poisson distribution need to be taken into account. In droplet microfluidics systems, each droplet can be regarded as an independent reaction chamber, so the number of molecules in each droplet should conform to Poisson distribution.

[0131] In the digital immunoassay of droplets, the Poisson distribution formula can be used to describe the number distribution of target molecules in the droplets. The Poisson distribution formula is as follows:

[0132]

[0133] Where P(x) is the probability that the droplet contains x target molecules, and λ is the average number of target molecules. According to the characteristics of Poisson distribution, when the average frequency of an event (λ) increases, the probability of the event (P) will also increase accordingly.

[0134] However, it is necessary to control the number of droplets containing more than two target molecules to be detected, that is, the number when k>1, such as λ=0.1, k=1, P 1 ≈0.090, the cumulative probability P of k>1 1-∞ ≈0.0046, P 1 / P 1-∞ ≈20, that is, when λ=0.1, the ratio of the number of droplets containing only one molecule to the number of droplets containing more than two molecules is 20, and the probability of droplets when K=0 is 0.90, which can be understood as 90% of the droplets are empty droplets, 9% of the droplets contain only one molecule, and 0.47% of the molecules contain more than two droplets. The present invention sets the above conditions as the minimum λ standard. The existence of empty droplets greatly reduces the number of droplets that can be observed to effectively contain magnetic beads. To improve the efficiency of observing magnetic beads, effective means should be used to eliminate empty droplets.

[0135] λ is the average number of target molecules, which is the ratio of the number of detected molecules (n) to the detection unit (m). Therefore, when λ≤0.1, effectively increasing the number of m is the key technology to achieve digital detection.

[0136] In addition to the characteristics of Poisson distribution, other factors need to be considered in digital immunoassays of droplets, such as antibody capture and enrichment, signal amplification, etc.

[0137] By comprehensively considering these factors, the inventors designed an efficient droplet digital immunoassay method to achieve sensitive detection of target molecules.

[0138] These are the key factors that need to be considered in droplet-based digital immunoassays. Only when these conditions are met can effective digital immunoassays be achieved.

[0139] Digital immunoassay analysis for accurate protein quantification

[0140] The present invention utilizes micro-droplet technology and disperses magnetic beads into the form of water-in-oil micro-droplets within 1-10 seconds by mechanical emulsification dispersion. The magnetic beads are randomly wrapped in the micro-droplets to form water-in-oil droplets. The number of magnetic beads in the micro-droplets conforms to the Poisson distribution, and then the micro-droplets containing magnetic beads are washed and intercepted by magnetic force, and the empty droplets are separated, thereby significantly increasing the micro-droplets containing magnetic beads that can be used for observation.

[0141] The present invention selects to use monodisperse magnetic beads with a particle size of 1-5 μm. These magnetic beads can be widely used in the field of life sciences, including products of companies such as Dynabeads, MagSi, MyOne, M-270, Dynal Biotech, and Sera-Mag. The particle size of these magnetic beads ranges from 0.03 μm to 100 μm. The magnetic beads used in this patent have or do not have fluorescent properties, and can emit light of different wavelengths such as green (520 nm), yellow (490 nm), red (610 nm), blue (450 nm), and rare earth europium fluorescence and rare earth terbium fluorescence. These magnetic beads also have different modifications, including carboxyl, amino, p-toluenesulfonic acid, NHS, biotin (Biotin) and streptavidin (AS), etc.

[0142] The present invention uses fluorinated oil as the oil phase, which is widely used in the field of life sciences. Common fluorinated oils include HFE7500, FC40, Fluo-Oil 7500, Fluo-Oil 40, Fluo-oil 135 and Fluo-Oil 200, etc. Fluorine surfactants are added to fluorinated oils to form an oil phase. Common surfactants include fluoroalkyl polyether sulfates, fluoroalkyl polyethylene glycol ether sulfates, fluorocarbon-based alcohols and fluorocarbon-based acids, PFPE-PEG high molecular polymers, PFPE-b-PPO-PEO-PPO-b-PFPE, etc. After the oil phase is mixed with the water phase, a high-speed homogenizer can be used to quickly form oil-in-water microdroplets.

[0143] The aqueous phase of the present invention is a magnetic bead solution, which is added to the oil phase solution. The magnetic beads can be broken up into monodisperse water-in-oil microdroplets using a high-speed homogenizer, and the number of magnetic beads is controlled to be 500,000. Using 100uL of aqueous phase, it is estimated that about millions to billions of microdroplets can be generated each time, and under this condition, the magnetic beads exist in the form of a single droplet encapsulating a single magnetic bead. Due to the excellent properties of the surfactant in the fluorinated water-in-oil, the microdroplets can stably exist in the oil phase without leaking fluorescent substances to each other.

[0144] After being attracted by the magnet, the water-in-oil magnetic beads can be temporarily and stably adsorbed on the glass slide, and the remaining droplets that do not contain magnetic beads are washed away by the oil phase. At this time, the water-in-oil magnetic beads form a monodisperse magnetic bead layer on the glass slide, which can be observed by fluorescence photography. One magnetic bead can be clearly distinguished.

[0145] The present invention selects a low-deformation, low-dispersion fluorescence microscope lens, giving priority to 1x, 2x, 4x, 10x, 20x, 40x, 60x, and 100x objective lenses. By adjusting the optical configuration, it is ensured that the formed phase field can observe the fluorescence signals generated by the magnetic beads and the substrate. The present invention uses a dual-channel fluorescence filter, which includes at least the following two channels: a DAPI channel, with an emission wavelength of about 450-500nm; a FITC channel, with an emission wavelength of about 515-530nm; a GFP channel, with an emission wavelength of about 509-525nm; an AlexaFluor 488 channel, with an emission wavelength of about 519-545nm; a Cy3 channel, with an emission wavelength of about 565-615nm; a Rhodamine Red-X channel, with an emission wavelength of about 600-680nm; a Texas Red channel, with an emission wavelength of about 600-680nm; and a Cy5 channel, with an emission wavelength of about 665-725nm. The present invention uses a CCD or CMOS camera to capture fluorescent images, and the target size of the selected camera matches the lens. For example, cameras of sizes such as 4 / 3 inch, 1.2 inch, 1.1 inch, 1 inch, 1 / 1.1 inch, 1 / 1.2 inch, 2 / 3 inch, 1 / 1.8 inch, 1 / 2 inch, 1 / 3 inch, 36 / 24 mm, 45 / 60 mm, 60 / 60 mm, etc. can be used. The number of pixels of the camera can vary from 1 million to 100 million pixels. The pixel size should match the numerical aperture (NA) of the selected objective lens. For example, when using a 1x objective lens with an NA of 0.045, according to the Rayleigh criterion (0.61λ / NA), its resolution at a wavelength of 500nm is about 4.35um. Therefore, the present invention can select a matching 3.45um (2 / 3 inch camera) pixel size. In the case of 2-5um magnetic beads, a pixel bright spot on the phase field can represent a magnetic bead signal.

[0146] After the immune reaction, the magnetic beads and the enzyme substrate are mixed evenly at low temperature (2-8°C), and then the oil phase reagent is immediately added and dispersed using a high-speed homogenizer for 1s-60s. In this way, the magnetic beads can be evenly dispersed into microdroplets. Since the number of empty droplets is much larger than the number of magnetic beads, according to the principle of Poisson distribution, the magnetic beads will be evenly dispersed into a single droplet. The size of the magnetic bead droplets will be larger than the diameter of the magnetic beads. After measurement, the droplet size is about 1-10 times the size of the magnetic beads. The size of the droplets is mainly affected by the speed of the high-speed homogenizer, the content of the oil phase surfactant, and the action time of the high-speed homogenizer. The typical droplet size is between 1-2 times the size of the magnetic beads. Due to the effect of shear force, the droplet size conforms to the normal distribution. In order to control the uniform distribution of the droplets as much as possible, the reaction system parameters can be adjusted, including the speed of the high-speed homogenizer, the content of the oil phase surfactant, and the length of the high-speed homogenizer action time. In the color development process after the reaction, enzyme fluorescent substrates are used for color development, and commonly used ones include ADHP (OxiRed probe) of horseradish peroxidase, BBTP of alkaline phosphatase, FDG substrate of galactosidase, etc.

[0147] The first micro-droplets finally formed have the characteristics of fluorescent magnetic beads and fluorescent substrate color development, and meet the following conditions: 1) droplets containing only fluorescent magnetic beads; 2) droplets containing fluorescent magnetic beads and containing fluorescence for enzyme substrate color development; 3) droplets containing only fluorescent substrate color development. These three conditions exist at the same time.

[0148] Magnetic beads are coupled with a certain proportion of capture antibodies. After cleaning the magnetic beads, the magnetic beads are washed and blocked with bovine serum albumin solution. After washing, a magnetic bead-antibody complex is formed for use. The antibody-coated magnetic beads formed are counted to determine that the concentration of the magnetic beads is 500,000. The number of magnetic beads is controlled, and the reaction system is controlled to 50uL. The above magnetic beads are reacted with the antigen for 15 minutes, and then washed three times with a magnetic stand PBST 200uL, followed by adding the biotinylated detection antibody solution, reacting for 15 minutes, and then using a magnetic stand PBST 200uL to wash three times, adding streptavidin HRP, reacting for 15 minutes, and then using a magnetic stand PBST 200uL to wash three times, adding anti-mouse IgG-FITC 10uL to shake and react for 10 minutes, and then using a magnetic stand plus PBST200uL to wash three times, using pre-cooled PBS (2-8°C) to configure the above magnetic beads into a 100uL solution (50uL pre-cooled PBS and 50uL pre-cooled ADHP), and the reaction is as follows Figure 1 shown.

[0149] After the above magnetic bead reaction solution is fully prepared, the liquid mixture is quickly poured into 100uL of droplet generation oil at one time, and a high-speed homogenizer is used to disperse it for 1s-60s to form an oil-in-water droplet mixture. The droplet mixture is reacted at 37°C for 5min, and the above droplets are attracted and washed with a magnetic rack to remove unreacted empty droplets.

[0150] Add the above-mentioned micro-droplet solution to the detection pool for detection and counting, and record the total number of green channel bright spots n, and the total number of red channel bright spots m. The static detection pool is composed of a fluorescence counting plate with a 100μm sandwich in the middle. The green and red channels have wavelengths of 570 excitation, 585nm emission and 470 excitation and 490 emission. The two detection bands do not interfere with each other. The detection method is as follows Figure 1 The detection chip used in conjunction with this technology is shown in Figure 5 As shown. After the solution is added, due to the magnetic force, the magnetic beads will form a monodisperse magnetic bead layer on the surface corresponding to the 8*6mm magnet. Under the 2 / 3-inch CMOS camera, adjust the algorithm threshold to calculate the number of magnetic beads and the number of signal droplets.

[0151] You can also use multicolor fluorescent microspheres for multiple marker detection, adjust the instrument settings, use one color and multiple fluorescent microspheres of different intensities as carriers, couple magnetic beads of each color intensity with a certain proportion of different capture antibodies, wash the magnetic beads after washing, and use bovine serum albumin solution to block them. After washing, magnetic bead antibody complexes with different fluorescence intensities will be formed for use. Count the antibody-coated magnetic beads formed, determine that the concentration of magnetic beads is greater than 50,000, and then mix the magnetic beads. The total volume of the magnetic beads was controlled to control the reaction system to 50uL, the magnetic beads were reacted with the antigen for 15min, then washed three times with 200uL of PBST on a magnetic stand, then added with a biotinylated detection antibody solution, reacted for 15min, then washed three times with 200uL of PBST on a magnetic stand, added with streptavidin-HRP, reacted for 15min, then washed three times with 200uL of PBST on a magnetic stand, and the magnetic beads were configured into a 100uL solution (50uL of pre-cooled PBS and 50uL of pre-cooled ADHP) using pre-cooled PBS (2-8°C), and the reaction was as follows: Figure 2 shown.

[0152] You can also use a fluorescent microsphere for rapid marker detection, adjust the instrument settings, use a color of fluorescent microspheres as a carrier, couple the magnetic beads with a certain ratio of different capture antibodies, wash the magnetic beads after cleaning, block them with bovine serum albumin solution, and form a magnetic bead antibody complex after washing for use. Count the antibody-coated magnetic beads formed, determine the concentration of the magnetic beads to be greater than 50,000, and then mix the magnetic beads. Control the total volume of the magnetic beads, control the reaction system to 50uL, react the above magnetic beads with the antigen for 5 minutes, then wash three times with 200uL of PBST on a magnetic stand, then add the HRP detection antibody solution, react for 5 minutes, then wash three times with 200uL of PBST on a magnetic stand, use pre-cooled PBS (2-8°C) to configure the above magnetic beads into a 100uL solution (50uL pre-cooled PBS and 50uL pre-cooled ADHP), and react as Figure 3 shown.

[0153] The micro-droplet magnetic bead algorithm process of the present invention is as follows:

[0154] 1) Read grayscale image;

[0155] 2) Apply adaptive thresholding to segment the image into highlights and background;

[0156] 3) Find connected components and separate bright spots into different areas;

[0157] 4) Count the number of connected components (minus background).

[0158] The main advantages of the present invention include:

[0159] (a) A digital immunoassay system is constructed using a multi-channel micro-droplet generation chip. The previous magnetic bead immunoreaction can be simply converted into a digital immunoassay using micro-droplets generated by a droplet generation device, which greatly improves the sensitivity and the detection capacity can reach 1fg / mL.

[0160] (b) This patent uses HRP enzyme to amplify digital immune signals. Beta-galactosidase, alkaline phosphatase, metal catalysis, and fluorescent particles can also be used to greatly expand the detection range and reduce the adaptability of the system.

[0161] (c) Using high-speed homogenization to generate micro-droplet magnetic bead complexes can quickly generate hundreds of millions of picoliter-level micro-droplets. At the same time, due to the use of magnetic bead separation technology, micro-droplets containing magnetic beads can be effectively enriched, and empty droplets without magnetic beads can be effectively separated, making the sampling efficiency extremely high, which can effectively avoid the waste of secondary sampling and greatly improve the detection efficiency.

[0162] (d) The microdroplet fluorescence used can produce two different wavelengths of fluorescence, one channel is used to count the number of negative magnetic beads, and the other channel is used to count the number of positive magnetic beads. The concentration of the solution is then calculated using Poisson distribution statistics.

[0163] (e) The present invention can use colored magnetic beads instead, label different types of antibodies on the magnetic beads, so as to provide multiple detection capabilities by utilizing the massive generation capacity of microdroplets. For example, a series of colored microspheres with different color ratios formed by mixing different dyes in different proportions can be used as analysis carriers, thereby achieving theoretically 100 multi-component ultra-high sensitivity analysis capabilities.

[0164] (f) Since the microdroplets used in the present invention are at the picoliter level, a short incubation process such as 5 minutes can be used to achieve rapid detection. The sensitivity calculation method is converted from a digital immune Poisson distribution counting method to a microdroplet fluorescence intensity method, thereby achieving rapid detection and the ability to quantify protein within 15 minutes.

[0165] (g) Using digital immunoassay technology, the droplet number measurement range can reach more than 4 log range, that is, 100,000 to 1 million. At the same time, as the target surface of the optical camera is expanded, the droplet recognition capability has the potential to be further improved. For example, using a 60*60mm CMOS camera, such as the GSENSE6060CMOS, and equipped with a 1X lens, it is possible to achieve a droplet recognition capability of about hundreds of millions on the target surface.

[0166] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0167] Example 1

[0168] Digital immunoassay for high sensitivity analysis of IL2

[0169] 1. Experimental Materials

[0170] The experimental materials used in this embodiment are shown in Table 1.

[0171] Table 1

[0172]

[0173]

[0174] 2. Coating method

[0175] (1) Take a 2 mL centrifuge tube, add 30 uL of sulfonated magnetic beads (MS300), wash three times with 0.3 mL of 20 mM PBS, and then dry the washing solution on a magnetic rack.

[0176] (2) Add the required raw materials, CB buffer, and 3 M ammonium sulfate in proportion as shown in Table 2.

[0177] Table 2

[0178] Ingredients / Magnetic beads 1mg Coating antibody material + CB buffer 200uL 3M Ammonium Sulfate 100uL Total volume 300uL

[0179] (3) Place the centrifuge tube in step 2 on a multi-purpose rotary shaker at 2-8°C for 20 hours.

[0180] (4) Place the centrifuge tube in step 3 on the magnetic rack for 5 minutes, remove the supernatant, add 0.3 mL of magnetic bead blocking pre-wash solution, and wash three times.

[0181] (5) Add 0.3 mL of blocking solution to the magnetic beads washed in step 4, mix well, and react on a multipurpose rotary shaker at 37°C for 6 hours.

[0182] (6) Repeat step 4.

[0183] (7) Resuspend and add 0.3 mL of magnetic bead working solution to each tube. Place on a multi-purpose rotary shaker and incubate at 37°C for 20 hours.

[0184] (8) Place the centrifuge tube in step 3 on the magnetic rack for 5 minutes, remove the supernatant, add 0.3 mL of magnetic bead blocking pre-wash solution, and wash three times, each wash for 5 minutes.

[0185] (9) Add the washed magnetic beads to the magnetic bead working solution to prepare a 120 ug / mL magnetic bead working solution and store at 2-8°C.

[0186] (10) Prepare the solution concentration to 10 million / mL.

[0187] 3. Detection Methods

[0188] (1) Take a series of sample solutions (50uL), including 0, 1, 5, 10, 100, 400, and 1000fg / mL, 10 5fg / mL calibrators and 100fg / mL calibrators, 13 normal human serum samples, and 10 gastric cancer patient serum samples.

[0189] (2) Mix 50uL of magnetic beads and 50uL of sample, react at 37°C for 15min, and then wash the magnetic beads three times with 200uL of PBST using a magnetic stand.

[0190] (3) Add 100uL of biotin-coated detection antibody (0.1ug), react at 37°C for 15min, and then wash the magnetic beads three times using a magnetic stand with 200uL of PBST.

[0191] (4) Add 100uL streptavidin-labeled HRP (1:5000 dilution), react at 37°C for 15 min, and then wash the magnetic beads three times using a magnetic stand with 200uL PBST.

[0192] (5) Add 100uL anti-mouse IgG-FITC (0.1ug), react at 37°C for 15min, and then wash the magnetic beads three times with 200uL PBST using a magnetic stand.

[0193] (6) After the above reaction is completed, disperse the magnetic beads into 50uL of PBS solution and place it in pre-cooled condition.

[0194] 4 Droplet generation

[0195] (1) Immediately add 50uL of pre-cooled ADHP fluorescent substrate and mix well, then immediately add 200uL of droplet generation oil.

[0196] (2) Insert the head of a high-speed homogenizer into the above mixed solution, adjust the speed to high speed mode (10000 RPM), and homogenize for 10 seconds.

[0197] The mixed liquid is layered and the final homogenate is shown in the figure below. Figure 4 As shown in AB.

[0198] (3) The above water-in-oil mixture was placed at 37°C for reaction for 5 minutes.

[0199] Here, if the reaction is performed first and then mechanically cut, the fluorescence of the empty droplets will increase, which is not a good effect.

[0200] 5. Detection

[0201] (1) Place the above reaction solution on a magnetic rack, use droplet formation oil to absorb and wash 3 times, and finally resuspend in 20uL droplet formation oil.

[0202] (2) To the detection chip ( Figure 5 ) to form a monolayer distribution, and then observe and detect under a microscope.

[0203] (3) Take photos of the green and red channels, such as Figure 6 As shown in AC.

[0204] (4) Analyze the fluorescence photos under two excitation wavelengths: 570 nm excitation, 585 nm emission and 470 nm excitation, 490 nm emission. Calculate the fluorescence intensity of each bright spot and make a scatter plot of all data points. The droplets that do not contain magnetic beads are blank droplets.

[0205] (5) Count the number of positive droplets containing magnetic beads and the number of negative droplets containing magnetic beads, such as Figure 7 As shown, the corresponding test data is listed in Table 3 below:

[0206] Table 3 Digital immunoassay IL2 standard curve data

[0207]

[0208]

[0209] Regression analysis of the calibrator concentration and the percentage of positive magnetic beads in Table 3 showed that the calibrator concentration was linear in the range of 1-1000 fg / mL, and had a good correlation compared with the value calculated by Poisson theory.

[0210] (6) Using the above method, a set of IL2 standard solutions with concentrations of 5 fg / mL and 100 fg / mL were simultaneously measured 10 times each. The calculated CV (digital immunoassay IL2 repeatability data) are shown in Tables 4-5:

[0211] Table 4

[0212]

[0213] Table 5

[0214]

[0215]

[0216] Table 4-5 shows that the CV of the 5fg / mL sample is 7.54%, and the CV of the 100fg / mL sample is 0.01%

[0217] (7) The above method was used to measure the corresponding serum of 10 normal samples and 13 patient samples, as shown in Table 6:

[0218] Table 6 IL2 determination data of normal subjects and patients

[0219]

[0220] The results in Table 6 show that the method of the present invention can effectively detect IL6 in serum.

[0221] Example 2

[0222] Digital immune sensitivity analysis Multiple cytokines (IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15) Human adaptive immunity 7-fold quantitative analysis

[0223] 1. Experimental Materials

[0224] The experimental materials of this example are shown in Table 7.

[0225] Table 7

[0226]

[0227] 2. Magnetic Bead Coating Method

[0228] The magnetic bead coating method of this embodiment is the EDC coupling method, and the specific operation steps are as follows:

[0229] (1) Thoroughly mix the homemade carboxyl magnetic beads with different fluorescence intensities in advance.

[0230] (2) Washing: Take one Eppendorf tube and add 20uL of 100mg / mL carboxyl (2mg) magnetic beads, add 1.8mL 100mM pH 5.0 MES, wash by inversion for 3min, adsorb on magnetic rack for 3min, remove supernatant, and repeat twice.

[0231] (3) Activation:

[0232] Activation Solution 1: Prepare 100 mg / mL EDC: weigh 10 mg EDC, add 100 uL, and dissolve in pre-cooled pH 5.0 100 mM MES.

[0233] Activation Solution 2: Prepare 100 mg / mL NHS: weigh 20 mg NHS, add 200 uL, and dissolve in pre-cooled pH 5.0 100 mM MES.

[0234] Prepare according to demand.

[0235] (4) Quickly add the liquids to the prepared magnetic beads in the order listed in Table 8. Rotate at room temperature for 60 minutes. Add in a ratio of 1:1:2.

[0236] Table 8

[0237] 100mM pH 5.0 pre-chilled MES 5uL 100mg / mL EDC (first) mix well and then add NHS 5uL 100mg / mL NHS 50uL

[0238] (5) Washing: Place on magnetic rack for 4 min, remove supernatant, add 1.8 mL of ice-cold 100 mM MES pH 5.0, and place on magnetic rack for 3 min. Remove supernatant, and repeat 4 times.

[0239] (6) Binding Antibody:

[0240] For example, for coupling, magnetic beads: antibody = 50:1, the total volume should be no less than 250uL according to Table 9. (The concentration of IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, and IL-15 coated antibodies is calculated according to the instructions provided by the corresponding manufacturers, and the preservatives, glycerol and other components are removed by dialysis.) Mix well and combine at room temperature for 60 minutes.

[0241] Table 9

[0242] Magnetic beads to antibody ratio 50:1 Magnetic beads 2mg Antibody quality 0.04mg Antibody volume (Add according to antibody concentration) 0.02M pH 8.0 boric acid buffer (Add according to antibody volume) Total volume 250uL

[0243] (7) Blocking: Add 1.8 mL of 50 mM pH 7.4 Tris + 0.1% Tween-20 + 1% BSA buffer and wash three times. Add 1.8 mL of 50 mM pH 7.4 Tris + 0.1% Tween-20 + 1% BSA 0.5% Proclin buffer again and incubate at room temperature for 90 min.

[0244] (8) Washing: adsorb on a magnetic rack for 3 minutes, remove the supernatant, add 1.8 mL of 0.02 M pH 7.3 PB + 0.1% BSA + 0.5% ProcLin buffer, adsorb on a magnetic rack for 3 minutes, and repeat 3 times.

[0245] (9) Prepare working solution: add 16.67 mL 0.02 M pH 7.3 PB + 0.1% BSA + 0.5% Proclin buffer to 120 ug / mL magnetic beads and store at 2-8°C.

[0246] (10) Prepare the solution concentration to 10 million beads / mL and mix the above IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15 and other magnetic bead solutions before use.

[0247] 3. Detection Methods

[0248] (1) Take a series of samples of IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, and IL-15 solution (50uL), including a mixture of calibrators with different concentrations including 0, 5, 10, 100, and 500fg / mL.

[0249] (2) Mix 50uL of magnetic beads and 50uL of sample, react at 37°C for 15min, and then wash the magnetic beads three times with 200uL of PBST using a magnetic stand.

[0250] (3) Add 100uL of biotin-coated detection antibody, react at 37°C for 15 min, and then wash the magnetic beads three times using a magnetic stand with 200uL of PBST.

[0251] (4) Add 100uL streptavidin-labeled HRP (1:5000 dilution), react at 37°C for 15 min, and then wash the magnetic beads three times using a magnetic stand with 200uL PBST.

[0252] (5) After the above reaction is completed, disperse the magnetic beads into 50uL of PBS solution.

[0253] 4. Droplet Generation

[0254] (1) Immediately add 50uL of pre-cooled ADHP fluorescent substrate and mix well, then immediately add 200uL of droplet generation oil.

[0255] (2) Insert the head of a high-speed homogenizer into the above mixed solution, adjust the speed to high speed mode (10000 RPM), and homogenize for 10 seconds.

[0256] (3) The above water-in-oil mixture was placed at 37°C for reaction for 5 minutes.

[0257] 5. Detection

[0258] (1) Place the above reaction solution on a magnetic rack, use droplet formation oil to absorb and wash 3 times, and finally resuspend in 20uL droplet formation oil.

[0259] (2) Add the above magnetic bead droplet mixture into the detection chip to form a monolayer distribution, and place it under a microscope for observation and detection.

[0260] (3) Take photos of the green and red channels.

[0261] (4) Analyze the fluorescence photos under two excitation wavelengths: 570 nm excitation, 585 nm emission and 470 nm excitation, 490 nm emission. Calculate the fluorescence intensity of each bright spot and make a scatter plot of all data points. The droplets that do not contain magnetic beads are blank droplets.

[0262] (5) Count the number of positive droplets containing magnetic beads and the number of negative droplets containing magnetic beads, such as Figure 8 As shown, the corresponding test data is listed in Table 10 below:

[0263] Table 10 Digital immunoassay data for IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15

[0264]

[0265]

[0266] Table 10 shows that the use of seven fluorescent magnetic beads with different green fluorescence intensities can effectively perform quantitative analysis of multiple cytokines (IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15), with a detection range of 0-500 fg / mL.

[0267] Example 3

[0268] Rapid analysis of SARS-CoV-2 antigens

[0269] 1. Experimental Materials

[0270] The experimental materials of this example are shown in Table 11.

[0271] Table 11

[0272]

[0273] 2. Coating method

[0274] (1) Take a 2 mL centrifuge tube, add 30 uL (3 mg) Tosylactivated green fluorescent magnetic beads, wash three times with 0.3 mL 20 mM PBS, at a speed of 30 times / min, each adsorption for 5 min, and after washing, place the washing solution on a magnetic rack to dry.

[0275] (2) Add the required raw materials, CB buffer, and 3M ammonium sulfate in proportion as shown in Table 12.

[0276] Table 12

[0277] Ingredients / Magnetic beads 1mg Coating antibody material + CB buffer 200uL 3M Ammonium Sulfate 100uL Total volume 300uL

[0278] Note: The final concentration of ammonium sulfate in the coating solution is 1M / L, and the minimum coating volume must not be less than 300uL.

[0279] (3) Place the centrifuge tube in step 2 on a multi-purpose rotary shaker at 2-8°C for 20 hours.

[0280] (4) Place the centrifuge tube in step 3 on the magnetic rack for 5 minutes, remove the supernatant, add 0.3 mL of magnetic bead blocking pre-wash solution, and wash three times.

[0281] (5) Add 0.3 mL of blocking solution to the magnetic beads washed in step 4, mix well, and react on a multipurpose rotary shaker at 37°C for 6 hours.

[0282] (6) Repeat step 4.

[0283] (7) Resuspend and add 0.3 mL of magnetic bead working solution to each tube. Place on a multipurpose rotary shaker at 37°C for 24 hours at a speed of 30 times / min.

[0284] (8) Place the centrifuge tube in step 3 on the magnetic rack for 5 minutes, remove the supernatant, add 0.3 mL of magnetic bead blocking pre-wash solution, wash three times, each wash for 5 minutes, adsorb on the magnetic rack for 5 minutes, and aspirate the wash solution.

[0285] (9) Add the washed magnetic beads to the magnetic bead working solution to prepare a 120 ug / mL magnetic bead working solution and store at 2-8°C.

[0286] (10) Prepare the solution concentration to 10 million / mL.

[0287] 3. Detection Methods

[0288] (1) Take 50uL of a series of antigen standard solutions, including 0, 50, 100, 1000, 5000, and 10000 fg / mL.

[0289] (2) Mix 50uL of magnetic beads and 50uL of sample, react at 37°C for 5 min, and then wash the magnetic beads three times using a magnetic stand with 200uL of PBST.

[0290] (3) Add 100uL HRP-labeled detection antibody (0.1ug), react at 37°C for 5 min, and then wash the magnetic beads three times using a magnetic stand with 200uL PBST.

[0291] (4) After the above reaction is completed, disperse the magnetic beads into 50uL of PBS solution.

[0292] 4. Droplet Generation

[0293] (1) Immediately add 50uL of pre-cooled ADHP fluorescent substrate and mix well, then immediately add 200uL of droplet generation oil.

[0294] (2) Insert the head of a high-speed homogenizer into the above mixed solution, adjust the speed to high speed mode (10000 RPM), and homogenize for 10 seconds.

[0295] (3) The above water-in-oil mixture was placed at 37°C for reaction for 5 minutes.

[0296] 5. Detection

[0297] (1) Place the above reaction solution on a magnetic rack, use droplet formation oil to absorb and wash 3 times, and finally resuspend in 20uL droplet formation oil.

[0298] (2) Add the above magnetic bead droplet mixture into the detection chip to form a monolayer distribution, and place it under a microscope for observation and detection.

[0299] (3) Take photos of the green and red channels.

[0300] 6. Analysis

[0301] Analyze the fluorescence photos under two wavelength excitation conditions: 570 excitation, 585nm emission and 470 excitation, 490 emission. Calculate the fluorescence intensity of each bright spot and make a scatter plot of all data points. The droplets without magnetic beads are blank droplets.

[0302] The results are as follows Fig. 9 As shown in A, 4000 is used as the threshold, and the signals above 4000 are retained. The following is the empty droplet signal containing magnetic beads. ADHP and frequency are fitted, and the fitting end point is taken as the final calculation signal output. The corresponding concentration calculation values ​​are shown in Table 13 below. The red channel histogram of the rapid analysis of SARS-CoV-2 antigen is shown in Fig. 9 As shown in B.

[0303] Table 13 Rapid analysis of SARS-CoV-2 antigen results

[0304] Calibrator concentration fg / mL Positive beads percentage Number of positive magnetic beads 0 0.3313％ 1424 50 0.5217％ 2463 100 1.0888％ 4367 500 3.0510％ 14256 5000 12.8219％ 53562 10000 14.0522％ 63423

[0305] Table 13 shows that the SARS-CoV-2 antigen standard can be detected quantitatively at 0-10000 fg / mL using the method of this example.

[0306] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A digital immunoassay analysis method for protein quantification, characterized in that: Includes steps: (s1) providing an aqueous phase mixture, wherein the aqueous phase mixture contains the following complex: (A) positive signal complex: magnetic beads-antibody-antigen-detection antibody-enzyme marker, and / or magnetic beads-antibody-antigen-detection antibody complex; and (B) Negative signal complex: magnetic bead-antibody complex; (s2) mixing the aqueous phase mixture with a chromogenic substrate and an oil phase to form a first mixture; (s3) subjecting the first mixture to mechanical shearing treatment to form a sheared water-in-oil second mixture; Wherein, the second mixture contains a first microdroplet and a second microdroplet; the first microdroplet is a microdroplet containing a positive signal complex; the second microdroplet is a microdroplet containing a negative signal complex; and (s4) measuring the first microdroplets to obtain the number of the first microdroplets, thereby obtaining a quantitative detection result of the protein.

2. The method according to claim 1, characterized in that Step (s1) also includes the following sub-steps: (s1a) providing an aqueous monodisperse magnetic bead solution; (s1b) adding a certain proportion of capture antibody to the magnetic bead solution to couple the magnetic beads with the capture antibody to form a magnetic bead-antibody complex, followed by washing; (s1c) adding an antigen to a solution containing the magnetic bead-antibody complex and reacting to form a magnetic bead-antibody-antigen complex, followed by washing; (s1d) adding a detection antibody with biotin or horseradish peroxidase (HRP) to a solution containing the magnetic bead-antibody-antigen complex and reacting to form a magnetic bead-antibody-antigen-detection antibody complex, followed by washing; Wherein, when the detection antibody with biotin is added, step (s1e) is performed, and when the detection antibody with horseradish peroxidase is added, step (s2) is directly performed; (s1e) adding an enzyme label molecule to a solution containing the magnetic bead-antibody-antigen-detection antibody complex to form a magnetic bead-antibody-antigen-detection antibody-enzyme label, followed by washing; and (s1f) Optionally, a negative signal antibody against the capture antibody is added to the solution containing the magnetic beads-antibody-antigen-detection antibody-enzyme label, so that the magnetic beads are labeled with a negative signal, followed by washing.

3. The method according to claim 1, characterized in that The mechanical shearing treatment is to use a high-speed homogenizer to homogenize the first mixture at high speed, and the speed of the high-speed homogenizer is 2000-100000RPM.

4. The method according to claim 1, characterized in that The total content of the first microdroplets and the second microdroplets in the second water-in-oil mixture is less than 30% relative to the content of the empty droplets, preferably less than 20%, and more preferably less than 10%.

5. The method according to claim 1, characterized in that The method further includes the following step between steps (s3) and (s4): washing the sheared second water-in-oil mixture to remove empty droplets that do not contain any first micro-droplets and second micro-droplets.

6. The method according to claim 5, characterized in that The washing is to adsorb and retain the droplets wrapped with single magnetic beads by magnetic force, so as to wash and enrich the magnetic beads.

7. The method according to claim 1, characterized in that The magnetic beads are magnetic beads with fluorescent properties or magnetic beads without fluorescent properties.

8. The method according to claim 1, characterized in that In step (s3), the ratio D1 / D2 of the diameter D1 of the first microdroplet to the diameter D2 of the magnetic beads contained therein is 1.2-20, preferably 1.5-5, more preferably 1.6-4, and even more preferably 1.8-3.

9. The method according to claim 1, characterized in that The method is used to measure n kinds of proteins, and the value of n is 1-200, preferably 1-20, and more preferably 1-10.

10. A digital immunoassay device for protein quantification, characterized in that: The device comprises: (a) a mixing module, wherein the mixing module is configured to form an aqueous phase mixture, wherein the aqueous phase mixture contains the following components: (A) positive signal complex: magnetic beads-antibody-antigen-detection antibody-enzyme marker, and / or magnetic beads-antibody-antigen-detection antibody complex; and (B) Negative signal complex: magnetic bead-antibody complex; (b) a shearing module, wherein the shearing module is configured to add a color developing substrate and an oil phase to the aqueous phase mixture to form a first mixture; and perform a mechanical shearing treatment to form a sheared oil-in-water second mixture, wherein the second mixture contains a first microdroplet and a second microdroplet; the first microdroplet is a microdroplet containing a positive signal complex; and the second microdroplet is a microdroplet containing a negative signal complex; (c) A digital detection module, wherein the digital detection module is configured to measure the first micro-droplets to obtain the number of the first micro-droplets, thereby obtaining a quantitative detection result of the protein.

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