Single molecule detection method and kit for detecting synaptic vesicle protein SV2A

Abstract

The present invention relates to the application of a single molecule detection method in detecting the concentration of synaptic vesicle protein SV2A, and to a counting-based single molecule detection method and a kit for detecting the concentration of synaptic vesicle protein SV2A. The present invention is the first to use a counting-based single molecule detection method in the detection of SV2A concentration. The method of the present invention can be implemented by a common fluorescence microscope, has low requirements for equipment, and has a low detection limit (high sensitivity), a wide detection dynamic range, and a low CV value.

Description

Technical Field

[0001] The present invention belongs to the field of single molecule detection, and specifically relates to a single molecule detection method for detecting SV2A protein (hereinafter, sometimes referred to as SV2A protein), a related kit, and the application of the single molecule detection method in detecting SV2A protein. Background Art

[0002] Synaptic vesicle protein 2 (SV2) is a glycoprotein with 12 transmembrane structures, present in all synaptic vesicles and endocrine vesicles. It was originally discovered in the electric organ of elasmobranch fish using anti-synaptic vesicle monoclonal antibodies. It is a small gene family with three subtypes: SV2A, SV2B, and SV2C. SV2A is the most widely distributed subtype, present in almost all neurons and widely distributed in the cerebral cortex. It is closely related to the release of neurotransmitters, endocrine vesicle exocytosis, and the maintenance of synaptic vesicle homeostasis. When the amount and function of SV2A protein change, it can induce epilepsy by causing a decrease in the amount of synaptotagmin 1 (Syt-1), leading to an imbalance in the release of inhibitory and excitatory neurotransmitters. Therefore, it is necessary to detect its concentration in cerebrospinal fluid and peripheral blood, especially the technology that can detect small changes in the concentration of SV2A protein. Based on this, technology that can detect trace amounts of SV2A protein is needed.

[0003] However, existing technologies mainly study the use of PET (positron emission tomography) and tracers to image SV2A protein for pathological research. No detection method for SV2A protein concentration has been reported, let alone how to detect trace amounts of SV2A protein in cerebrospinal fluid and peripheral blood.

[0004] Single molecule detection (SMD) is a method based on the analysis of a single molecule. So far, it has attracted much attention in the research of life sciences. In recent years, this method has been applied to cell imaging, protein interaction research, and quantitative detection of proteins and nucleic acids. In the above-mentioned applications, quantitative detection is achieved by counting the target molecules one by one, and this quantitative detection method represents the ultimate limit of detection. Traditional average measurement methods such as immunochromatography, chemiluminescence, and enzyme-linked immunosorbent assay are quantitative based on the relationship between signal intensity and target concentration. The higher the signal intensity, the higher the concentration of the target being measured. Unlike the average measurement method, in the SMD quantitative method, counting the molecules that can generate signals is more visible and digital, ensuring higher reproducibility. Since the SMD quantitative detection method has advantages that cannot be matched by average measurement, there are currently works that apply SMD to quantitative analysis (see patent documents 1~3).

[0005] Specifically, Patent Document 1 is a prior application of the inventor of the present application, which involves the use of in-situ signal-enhancing particles with a specific particle size (180-400 nm) to label the molecules to be tested, so that single-molecule signals can be captured and identified by optical imaging equipment, thereby achieving ultra-high sensitivity quantitative detection of the molecules to be tested. Among them, in-situ signal-enhancing particles and magnetic beads were used, and single-molecule detection of cTnI protein, IL-6 protein, DNA, etc. was performed based on the double antibody sandwich method, and a lower detection limit was obtained, but no mention was made of how to detect SV2A protein. It should be noted that the structure and molecular weight of SV2A protein are quite different from those of cTnI protein, IL-6 protein, etc. There is not even any literature in the prior art reporting how to measure SV2A protein, and how to achieve its quantitative detection requires a lot of creative work by those skilled in the art.

[0006] Patent document 2 is an application filed by Shenzhen Light and Biotechnology Co., Ltd., which involves a single-molecule detection method based on up-conversion fluorescent materials. Specifically, the sample to be detected is diluted and added to a detection substrate such as a glass sheet or a silicon wafer. The detected object is combined with the bioactive molecule B. After completion, the detection substrate is rinsed, and then the diluted immunofluorescent material is added to make the bioactive molecule A combine with the detected object that has been combined with the bioactive molecule B. After completion, the detection substrate is rinsed; the treated detection substrate is placed under a fluorescence microscope to count the number of immunofluorescent materials. This document uses high-concentration rare earth doping in up-conversion nanomaterial particles to make the up-conversion nanomaterial particles have a larger anti-Stokes shift. Compared with traditional organic dyes, the excitation light source and the emission band do not overlap, and the autofluorescence background noise can be effectively suppressed, which can significantly improve the signal-to-noise ratio of the detection signal. It uses a glass sheet or a silicon wafer as a carrier, and also does not involve how to detect SV2A protein.

[0007] Patent document 3 is an application filed by Caike (Suzhou) Biotechnology Co., Ltd., which involves a multiple immune molecule detection method, including the following steps: obtaining a coded microsphere with a capture molecule connected to the surface; capturing the target immune molecule through the capture molecule and adding an enzyme-labeled reagent to form an enzyme-labeled immune sandwich complex on the surface of the coded microsphere; driving the coded microsphere with the immune sandwich complex modified on the surface into the micropores of the microplate and sealing it; after a predetermined time, the microplate is detected by light excitation. Its principle is similar to the digital PCR technology of Quanterix Company in the United States, which is based on the micropores of the precision microplate to achieve the separation and detection of single molecules, and also does not mention how to detect SV2A protein.

[0008] Prior art literature

[0009] Patent document 1: CN111771126A;

[0010] Patent document 2: CN111735964A;

[0011] Patent document 3: CN111060683A. Summary of the invention

[0012] Problems to be solved by the invention

[0013] As mentioned above, no detection method for SV2A protein concentration has been reported in the prior art, and those skilled in the art are faced with a completely new field that has not yet been explored. In the field of immunodiagnosis, common methods include colloidal gold method, enzyme-linked immunosorbent assay, immunochromatography, fluorescent immunoassay, chemiluminescence, etc., and those skilled in the art are faced with a variety of choices. How to select a method that is well suited for a specific protein such as SV2A protein from a wide variety of detection methods is the problem to be solved by the present invention.

[0014] In view of the above-mentioned state of the prior art, the purpose of the present application is to provide a detection method and a related detection kit for SV2A protein that achieve excellent detection sensitivity (lower detection limit), a wider detection dynamic range and a better coefficient of variation at a low cost (without using a microfluidic chip or an unconventional fluorescence microscope).

[0015] Means for solving problems

[0016] The applicant has conducted in-depth research on the detection method of SV2A protein and surprisingly found that the single molecule detection method characterized by counting is particularly suitable for the detection of SV2A protein. The reason may be that SV2A proteins are not easy to bind to each other, which facilitates the detection and counting of each protein separately. It is further found that by applying the single molecule detection system characterized by in situ signal enhancement particles with specific particle size and double antibody sandwich method previously developed by the inventor to the detection of SV2A protein, satisfactory detection sensitivity, wide dynamic detection range and extremely low coefficient of variation can be obtained, which can meet the detection needs of trace SV2A protein.

[0017] A technical solution of the present application is as follows.

[0018] A counting-based single-molecule detection method for detecting the concentration of synaptic vesicle protein SV2A comprises the following steps:

[0019] (1) Immobilizing a capture antibody capable of binding to synaptic vesicle protein SV2A onto magnetic beads, adding a sample containing synaptic vesicle protein SV2A, allowing the first site of synaptic vesicle protein SV2A to bind to the capture antibody, thereby capturing the synaptic vesicle protein SV2A in the sample;

[0020] (2) adding a detection antibody that can bind to the second site of the synaptic vesicle protein SV2A, allowing the detection antibody to bind to the second site of the synaptic vesicle protein SV2A, and then adding an in situ signal enhancing particle that can directly or indirectly bind to the detection antibody; or first allowing the detection antibody to bind to the in situ signal enhancing particle to form a complex, and then adding the complex;

[0021] The in-situ signal enhancement particles have a particle size of 180nm to 350nm and contain fluorescent materials and carriers.

[0022] (3) imaging the fluorescent signal emitted by the in situ signal enhancing particle using an optical imaging device;

[0023] (4) The number of in situ signal-enhanced particles was counted, and the concentration of synaptic vesicle protein SV2A in the sample was further calculated.

[0024] Wherein, steps (1) and (2) can be replaced by the following steps (1') and (2'), respectively.

[0025] (1') mixing the detection antibody with the sample so that it binds to the second site of the synaptic vesicle protein SV2A in the sample, and then adding an in situ signal enhancing particle that can directly or indirectly bind to the detection antibody; or firstly allowing the in situ signal enhancing particle to bind to the detection antibody to form a composite material, and then adding the composite material to the sample so that the composite material binds to the second site of the synaptic vesicle protein SV2A in the sample; wherein the in situ signal enhancing particle is the same as the in situ signal enhancing particle in step (1);

[0026] (2') Immobilizing a capture antibody capable of binding to synaptic vesicle protein SV2A onto magnetic beads, and then adding the beads to the system of step (1'), so that the capture antibody binds to the first site of the synaptic vesicle protein SV2A, thereby capturing the synaptic vesicle protein SV2A.

[0027] Preferably, the particle size of the in situ signal enhancement particles is 200-300 nm, and the coefficient of variation (CV value) of the particle size is 1%-10%.

[0028] Preferably, the carrier is polyethylene glycol, styrene-(meth)acrylic acid copolymer, silicon dioxide, polystyrene, or polyacrylamide, preferably polyethylene glycol, polyacrylamide or silicon dioxide.

[0029] Preferably, the binding of the first site of the SV2A protein with the capture antibody in step (1) and step (2') is performed in the pH range of 8-10.

[0030] The present application also relates to the application of the single molecule detection method in detecting the concentration of synaptic vesicle protein SV2A.

[0031] The present application also relates to a single-molecule counting-based kit for detecting synaptic vesicle protein SV2A, which comprises in situ signal-enhanced particles and magnetic beads, characterized in that the in situ signal-enhanced particles comprise fluorescent materials and carriers, and have an average particle size of 180 to 350 nm, and a coefficient of variation (CV value) of the particle size of 1% to 10%.

[0032] Preferably, it further comprises a capture antibody that binds to the first site of synaptic vesicle protein SV2A and a detection antibody that binds to synaptic vesicle protein SV2A, respectively.

[0033] Preferably, it further comprises a buffer system, wherein the pH of the buffer system is 8-10, and the binding of the first site of the SV2A protein from the sample with the capture antibody can be performed within the pH range of 8-10.

[0034] The kit does not contain enzymes or chemiluminescent reagents.

[0035] The particle size in this application is measured using a scanning electron microscope (SEM) at a magnification that allows about 100 in-situ signal enhancement particles to be observed in one field of view, and the longest diameter of 50 randomly selected in-situ signal enhancement particles containing a fluorescent material and a carrier is measured with a caliper, and the number average of the values ​​is calculated and used as the average particle size. The coefficient of variation of the particle size of the in-situ signal enhancement particles is calculated by the following formula.

[0036] Coefficient of variation of particle size (CV value) = standard deviation of particle size / average particle size

[0037] Effects of the Invention

[0038] According to the present invention, a single-molecule detection method can be provided that achieves excellent detection sensitivity, a wide detection dynamic range, and an extremely low coefficient of variation at a low cost (without using a microfluidic chip or an unconventional fluorescence microscope). BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The standard curve obtained in Example 1 is shown (the vertical axis, namely CPN (copy number), is the number of single molecule signals). DETAILED DESCRIPTION

[0040] A single molecule detection method for detecting SV2A protein comprises the following steps:

[0041] (1) Immobilizing a capture antibody capable of binding to SV2A protein onto magnetic beads, adding a sample containing SV2A protein, allowing the first site of SV2A protein to bind to the capture antibody, thereby capturing the SV2A protein in the sample;

[0042] (2) adding a detection antibody that can bind to the second site of the SV2A protein, allowing the detection antibody to bind to the second site of the SV2A protein, and then adding an in situ signal enhancement particle that can directly or indirectly bind to the detection antibody; or first allowing the detection antibody to bind to the in situ signal enhancement particle to form a complex, and then adding the complex;

[0043] The particle size of the in-situ signal enhancement particle is 180nm~350nm, and contains fluorescent material and carrier.

[0044] (3) imaging the fluorescent signal emitted by the in situ signal enhancement particle using an optical imaging device;

[0045] (4) Count the number of in situ signal enhancement particles and further calculate the concentration of SV2A protein in the sample.

[0046] Wherein, steps (1) and (2) can be replaced by the following steps (1') and (2'), respectively.

[0047] (1') mixing the detection antibody with the sample to allow it to bind to the second site of the SV2A protein in the sample, and then adding an in situ signal enhancing particle that can directly or indirectly bind to the detection antibody; or first allowing the in situ signal enhancing particle to bind to the detection antibody to form a composite material, and then adding the composite material to the sample to allow the composite material to bind to the second site of the SV2A protein in the sample; wherein the in situ signal enhancing particle is the same as the in situ signal enhancing particle in step (1);

[0048] (2') Immobilizing a capture antibody capable of binding to the SV2A protein onto magnetic beads, and then adding the beads to the system of step (1'), so that the capture antibody binds to the first site of the SV2A protein, thereby capturing the SV2A protein.

[0049] The above detection method is based on double antibody sandwich immunoassay to detect the concentration of SV2A protein. Magnetic beads are used to separate and clean the detection sample and reagent. The above capture antibody is fixed on the surface of the magnetic bead by physical adsorption or chemical modification, and can bind to a binding site of the SV2A protein to separate the SV2A protein from the sample.

[0050] The above detection antibody can bind to another binding site of the SV2A protein of the molecule to be detected. The above in situ signal enhancement particles directly bind to the detection antibody, which means that the detection antibody is directly adsorbed or coupled to the in situ signal enhancement particles by physical adsorption or chemical modification, thereby realizing the functional modification of the in situ signal enhancement particles for recognition and labeling of the molecules to be detected. The above "indirect binding of the in situ signal enhancement particles to the detection antibody" means that the in situ signal enhancement particles are specifically labeled on the detection antibody through binding through anti-detection antibodies (i.e., secondary antibodies) or biotin-streptavidin systems.

[0051] The surface of the magnetic beads is modified with active functional groups that can be covalently coupled with antibodies, such as one or more of hydroxyl, carboxyl, amino, succinimidyl ester, sulfonyl (such as toluenesulfonyl) and their derivatives.

[0052] The capture antibodies can be classified according to antibody specificity, and can be one or both of polyclonal antibodies and monoclonal antibodies. The capture antibodies can be classified according to their sources, and can be one or more of mouse antibodies, rabbit antibodies, sheep antibodies, and alpaca antibodies.

[0053] The detection antibody can be classified according to antibody specificity, and can be one or both of polyclonal antibody and monoclonal antibody. The detection antibody can be classified according to source, and can be one or more of mouse antibody, rabbit antibody, sheep antibody and alpaca antibody.

[0054] The above-mentioned anti-detection antibodies can be classified according to the source of the detection antibodies, and can be one or more of anti-mouse antibodies, anti-rabbit antibodies, anti-sheep antibodies, and anti-alpaca antibodies. The anti-detection antibodies can be classified according to the source, and can be one or more of mouse-derived secondary antibodies, rabbit-derived secondary antibodies, sheep-derived secondary antibodies, and alpaca-derived secondary antibodies.

[0055] As used in this specification, the sensitivity of an assay, also known as the limit of detection (LoD), is the lowest concentration of the measured variable (i.e., the quantity planned to be measured) that can be detected at a specific confidence level. The confidence level is usually 95%, with a probability of a false negative measurement of 5%. Sensitivity is the lowest analyte concentration that can be reliably distinguished from the limit of blank (LoB) and can be detected.

[0056] The in-situ signal enhancement particle of the present invention is a material that enhances the fluorescence signal in-situ to a level that can be detected by conventional optical imaging equipment, and contains two parts: a fluorescent material and a carrier.

[0057] In the in-situ signal enhancement particles, the carrier plays a very important role. For example, it can bind more fluorescent materials to make the luminescent signal stronger; it provides sites for functional modification, can bind a large number of antibodies, and improves the reaction activity; it provides the possibility of single molecule detection by conventional fluorescence microscope. If there is no carrier, single molecule detection cannot be achieved. The carrier can be classified according to the material, and can be one or more of polyethylene glycol, styrene-(meth)acrylic acid copolymer, silica, polystyrene, or polyacrylamide. The carrier can be classified according to the structure, and can be one or more of a hollow structure, a core-shell structure, a porous structure, an alloy structure, and a hydrogel structure. Among them, from the viewpoint of making the fluorescent material evenly distributed and making the brightness of the fluorescent material high, the carrier is preferably polyethylene glycol, silica and polyacrylamide.

[0058] The fluorescent material in the in-situ signal enhancement particle is also necessary to achieve single molecule detection. The fluorescent material can be one or more of fluorescent dye molecules, rare earth elements, rare earth chelates, fluorescent proteins, quantum dots, aggregation-induced luminescence materials, semiconductor conjugated polymer fluorescent materials and up-conversion nanoparticles. The fluorescent material is preferably fluorescein (such as fluorescein isothiocyanate), rhodamine (such as rhodamine green, rhodamine B, etc.), coumarin, quantum dots (such as ZnO), rare earth elements (such as Eu, Ce) and their complexes. The fluorescent material is adsorbed or wrapped on the surface or inside of the carrier by one or more of covalent modification, chelation, spatial encapsulation, hydrophobic effect and electrostatic adsorption. It should be noted that from the perspective of facilitating optical imaging recognition and improving sensitivity, it is preferred that the fluorescent material is uniformly wrapped inside the carrier.

[0059] In the present application, the fluorescent dye is preferably PEG-encapsulated fluorescent quantum dots ZnO, fluorescent particles formed by silica-encapsulated fluorescent dye molecules (such as fluorescein), fluorescent particles formed by polyacrylamide-encapsulated fluorescent dye molecules (such as fluorescein), fluorescent particles formed by polystyrene-encapsulated quantum dots, fluorescent particles formed by polystyrene-encapsulated rare earth elements or rare earth chelates, etc.

[0060] In the present invention, the particle size of the in situ signal enhancement particles needs to be controlled within the range of 180 to 350 nm, for example, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm. If the particle size of the fluorescent dye is less than 180 nm, for example, 150 nm, no signal can be detected in conventional optical imaging equipment. If the particle size is greater than 350 nm, for example, 360 nm, the detection sensitivity cannot meet the requirements and it is difficult to achieve the sensitivity required by the clinic. It should be noted that the particle size can be a primary particle size or a secondary particle size. The secondary particle size refers to the particle size formed after the primary particles and the secondary particles are agglomerated. The particle size of the in-situ signal enhancement particles can be controlled by adjusting parameters such as the mass ratio of the fluorescent material to the carrier, the reaction temperature, the reaction time, the stirring speed, and the amount of the solvent.

[0061] In the present application, the surface of the in situ signal enhancement particle is modified with active functional groups that can be covalently coupled to antibodies, including one or more of hydroxyl, carboxyl, amino, thiol, alkene, alkynyl, succinimide ester groups and their derivative groups.

[0062] In the present application, the surface of the in situ signal enhancing particle can be modified with a connecting arm of a certain length, and the connecting arm includes a multi-carbon straight chain, a multi-carbon branched chain, a polymer chain, a peptide chain, and a protein. The length of the connecting arm is preferably 180 to 350 nm, more preferably 2 to 20 nm, and most preferably 5 to 10 nm.

[0063] In step (1) of the present application, the incubation temperature of the sample and the capture antibody is 10-50°C, preferably 20-40°C, and particularly preferably 37°C, and the incubation time is 1 min to 60 min, preferably 3 min to 30 min, and particularly preferably 10 min to 30 min. In step (2) of the present application, the incubation temperature of the detection antibody (or the in situ signal enhancement particle combined with the detection antibody) and the sample is 10-50°C, preferably 20-40°C, and particularly preferably 37°C, and the incubation time is 1 min to 60 min, preferably 2 min to 30 min, and particularly preferably 15 min to 25 min.

[0064] After in-depth research, the inventors of the present application unexpectedly discovered that the binding conditions of the first site of the SV2A protein and the capture antibody in step (1) and step (2'), especially pH, have a significant effect on sensitivity, dynamic range and coefficient of variation. Specifically, in the double antibody sandwich immunoassay, the binding of the protein to be tested and the capture antibody is usually carried out under neutral conditions (pH is about 7.4). The applicant also followed the conventional pH conditions in the art in the detection of cTnI protein, IL-6 protein, p-Tau181 protein, p-Tau217 protein, Aβ40 protein, Aβ42 protein, SV2A protein and NF-L protein. However, in actual experiments, it was found that for SV2A protein, although conventional neutral conditions can also perform single-molecule detection on it, there is still room for improvement in the effect. After further in-depth research, the applicant unexpectedly found that when the pH at which the first site of the SV2A protein binds to the capture antibody in step (1) and step (2') is controlled under alkaline conditions, for example, 8 to 10 (particularly around 9), the sensitivity, dynamic range and coefficient of variation can be significantly improved. Therefore, it is preferred that the pH at which the first site of the SV2A protein binds to the capture antibody in step (1) and step (2') is controlled within the range of 8 to 10 (particularly around 9).

[0065] In the present application, the measurement object is not particularly limited, and various biological samples can be cited, for example, body fluids such as blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, nasal mucus, nasal swab fluid, throat swab fluid, and tears, preferably blood, serum, plasma or cerebrospinal fluid.

[0066] In the present application, the optical imaging device mainly includes the following components: an excitation light source, an objective lens, a filter, a photosensitive element, a data acquisition module, a data processing module and a dichroic mirror (if it is an upright microscope, there may be no dichroic mirror). Among them, the excitation light source is an optical emitting device used to excite the reacted sample to emit an optical signal. The objective lens is used for signal acquisition and amplification of the sample to be tested. The dichroic mirror is used for reflection of the excitation light path and acquisition of sample optical signals. The filter is used for filtering the excitation light band and filtering the sample emission light signal. The photosensitive element is used for acquisition of sample optical signals. The data acquisition module is configured to receive the optical signal captured by the photosensitive element and convert it into a digital signal. The data processing module is configured for conversion of digital signals and formation and processing of optical images.

[0067] In some embodiments of the device, the excitation light source includes one or more of a gas laser, a solid laser, a semiconductor laser, a liquid laser, and a free electron laser. In some embodiments of the device, the objective lens is classified according to the magnification, including one or more of 1X, 2X, 4X, 5X, 10X, 20X, 40X, 50X, and 100X; the objective lens is classified according to the field curvature correction, including a flat objective lens or a curved objective lens. In some embodiments of the device, the photosensitive element includes one or both of CCD (Charge Coupled Device) or CMOS (Complementary Metal-Oxide Semiconductor).

[0068] By adopting a specific detection system, the present application has low requirements for optical imaging equipment, and a conventional optical imaging equipment (i.e., an optical imaging equipment that has not broken the optical diffraction limit) can be used. There is no need for expensive imaging equipment that has broken the optical diffraction limit, such as a total internal reflection fluorescence microscope, an epifluorescence microscope, a scanning near-field optical microscope, or a confocal fluorescence microscope.

[0069] In the present application, there are two ways to calculate the concentration of SV2A protein: single molecule counting mode and fluorescence intensity integration mode. Among them, in terms of the single molecule counting mode, the number of bright spots formed by the in-situ signal enhancement particles in the generated image is directly analyzed and counted, and the number of bright spots is directly or indirectly converted to the concentration information of SV2A protein in the sample. The so-called "direct conversion to the concentration information of SV2A protein in the sample" refers to absolute quantification, that is, it can be converted to concentration information without standard curve correction. The so-called "indirect conversion to the concentration information of SV2A protein in the sample" refers to the conversion to concentration information by the number of bright spots and the standard curve (or correction parameter). In terms of the fluorescence intensity integration mode, the bright spot area formed by the in-situ signal enhancement particles in the generated image is counted and integrated, and the integral result is divided by a specific parameter, such as the average bright spot area or bright spot area related variables (such as power square, square root, and polynomial, etc.) formed by each in-situ signal nanoparticle, thereby converting the approximate number of in-situ signal enhancement particles, and then converting the value to the concentration information of SV2A protein in the sample. Among them, the average bright spot area is obtained by counting the bright spot areas of single molecules at lower concentrations and taking the average value. From the perspective of obtaining a larger detection dynamic range, it is important to use the single molecule counting mode in the low concentration range and the fluorescence intensity integration mode in the high concentration range, and then merge the standard curves drawn in these two modes to draw a complete standard curve. It should be noted that the dividing line between the above low concentration and high concentration is generally the concentration when more than one molecule to be tested is bound to the surface of a magnetic bead. It can also be preferably the concentration when an average of 0.5 molecules to be tested are bound to the surface of a magnetic bead or the concentration when 2 molecules to be tested are bound according to the standard curve fitting results. Example

[0070] Hereinafter, the present application will be described in further detail with reference to Examples and Comparative Examples, but the present application is not limited thereto.

[0071] 1. Determination of particle size and coefficient of variation of in situ signal-enhanced particles

[0072] Taking polyethylene glycol-loaded fluorescent particles, styrene-(meth)acrylic acid copolymer-loaded fluorescent particles, and silica-loaded fluorescent particles as examples, each in situ signal enhancement particle was diluted 1000 times with water, and then 100 μL was dropped on the surface of a clean silicon wafer and dried. A small sputtering device was used to sputter-deposit 5 nm of platinum on the surface, and imaging analysis was performed using SEM (SU3900 manufactured by Hitachi High-Tech Corporation, Japan) to determine the particle size.

[0073] The coefficient of variation of the particle size was calculated by the following formula.

[0074] Coefficient of variation of particle size (CV value) = standard deviation of particle size / average particle size

[0075] Taking polyacrylamide-loaded fluorescent particles as an example, the obtained polyacrylamide fluorescent nanoparticles were diluted 1000 times with pure water, and the particle size of the particles was measured using a Malvern particle size analyzer (Zetasizer Nano S90).

[0076] 2. Single-molecule imaging

[0077] Single-molecule imaging can be performed using a conventional fluorescence microscope such as the Nikon Eclipse Ti-U fluorescence microscope. In addition, other fluorescence microscopes in the Nikon Eclipse Ti series, the Leica DMi8 fluorescence microscope, etc. can also be used.

[0078] 3. Standard curve drawing method

[0079] In this application, the single molecule counting mode and the fluorescence intensity integration mode are used in combination, and the specific implementation method is as follows:

[0080] When the concentration of the molecule to be tested is low, the number of magnetic beads is greater than the number of the molecule to be tested bound to the magnetic beads, so the single molecule counting mode is used to draw a standard curve for samples of the molecule to be tested with different concentrations;

[0081] When the concentration of the molecule to be tested exceeds a certain threshold, more than one molecule to be tested may be bound to the surface of a magnetic bead, and single-molecule signals are easily superimposed, resulting in deviations in the detection results. Therefore, it is more appropriate to use the fluorescence intensity integration mode.

[0082] Specifically, when the number of single molecules in an imaging picture does not exceed the set threshold, the single molecule counting mode is used to draw the standard curve; when the number of single molecules in an imaging picture exceeds the set threshold, the fluorescence intensity integration mode is used, and the total fluorescence intensity area is divided by the average fluorescence intensity area of ​​each molecule to convert it into the "approximate number of single molecules" for drawing the standard curve.

[0083] Finally, the standard curve obtained using the single molecule counting mode was combined with the standard curve obtained using the fluorescence intensity integration mode, and the curves were fitted using the fitting formula to draw a complete standard curve.

[0084] 4. Preparation of in situ signal enhancement particles

[0085] (a) Taking the preparation of in situ signal-enhancing particles with polyethylene glycol as a carrier as an example, the steps are as follows.

[0086] Polyethylene glycol and fluorescent material are dissolved in an organic solvent in a specified ratio, stirred and slightly heated, and the solution is dropped into an aqueous solution containing a surfactant such as Pluronic F-127, ultrasonicated to form an emulsion, and mixed with the aqueous solution containing the surfactant again. The organic solvent is evaporated after magnetic stirring, and washed after centrifugation to obtain in situ signal enhancement particles with polyethylene glycol as a carrier and embedded with the above fluorescent material.

[0087] (b) In-situ signal enhancement particles using styrene-(meth)acrylic acid copolymer as a carrier can be prepared by the following method:

[0088] (1) A step of preparing a seed particle dispersion containing a styrene-(meth)acrylic acid copolymer, specifically, adding 400 g of pure water, 1 g of methacrylic acid, 10 g of styrene, and 0.20 g of potassium persulfate (initiator) to a reactor equipped with a stirrer, a reflux condenser, a temperature sensor, and a nitrogen inlet tube, and then stirring the container at 65°C at a speed of 200 rpm for polymerization for 24 hours. After the polymerization is completed, the solution is filtered with filter paper to remove the latex particles, and then dialyzed with a dialysis membrane for 48 hours to obtain latex particles as seed particles;

[0089] (2) a step of preparing an emulsion containing the fluorescent material and an organic solvent. Specifically, 0.4 g of the fluorescent material as the light-emitting material is dissolved in 31 g of ethyl acetate to prepare a solution, and the solution is added to an aqueous solution (150 g of water) in which 0.1 g of sodium styrene sulfonate is dissolved, followed by mixing to prepare an emulsion;

[0090] (3) adding the emulsion to the seed particle dispersion so that the mass of the fluorescent material becomes 0.11 times the mass of the seed particles, and causing the seed particles to absorb the fluorescent material and the organic solvent to obtain a swollen particle droplet dispersion;

[0091] and (4) drying the organic solvent while stirring the dispersion of the swollen particle droplets at 65° C. and 200 rpm for 24 hours to obtain in-situ signal enhancement particles containing the fluorescent material and using styrene-(meth)acrylic acid copolymer as a carrier, wherein the mass fraction of the fluorescent material is 10%.

[0092] When the carrier is styrene, the in situ signal enhancement particles can be synthesized by a similar method to that when styrene-(meth)acrylic acid copolymer is used as the carrier (omitting the (meth)acrylic acid). When the carrier is silica or polyacrylamide, the method for manufacturing the in situ signal enhancement particles can refer to the description of the examples in Patent Document 1.

[0093] Example 1: Used for the detection of SV2A protein in human serum (using PEG-encapsulated quantum dot ZnO as in-situ signal enhancement particles with a particle size of 200 nm).

[0094] 1. Experimental components

[0095] Tosyl-activated M280 magnetic beads (Thermo), capture antibody (self-developed), detection antibody (self-developed), silane coupling agent (APTES), ammonia water, tetraethyl orthosilicate (TEOS), in situ signal enhancement particles containing fluorescein isothiocyanate and silica carrier (average particle size of 200 nm, particle size CV value of 5%, purchased from Qiyue Biotechnology), succinic anhydride, serum samples to be tested, PBS buffer, Buffer C (3 mM (NH4)2SO4 dissolved in 10 mM PBS buffer, pH = 7.4), Buffer D (0.01% NaCl, 0.5% BSA dissolved in 10 mM PBS, pH = 7.4), Buffer E (0.0088% NaCl, 0.1% BSA dissolved in 10 mM PBS, pH=7.4), N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), enhanced particle storage solution, sample diluent and PBS washing solution.

[0096] 2.1 Covalent coupling of magnetic beads and capture antibodies

[0097] (1) Take 166.6 μL of 25 mg / mL tosyl-activated M280 magnetic beads, wash three times with 10 mM PBS buffer, and remove the buffer.

[0098] (2) Take 120 μg of capture antibody, dilute it with 120 μL of 10 mM PBS buffer, and add it to the magnetic beads in (1). Mix well. Add 100 μL of Buffer C, rotate and mix well at 37°C, and react for 45 minutes.

[0099] (3) Wash five times with 10 mM PBS buffer and add 1 mL of Buffer D for blocking reaction. Rotate and mix at 37°C for 45 min.

[0100] (4) Wash five times with 10 mM PBS buffer and store in 250 μL Buffer E for later use.

[0101] 2.3 Covalent coupling of detection antibodies to in situ signal enhancement particles

[0102] (1) Take 10 μL of the in situ signal enhancement particles stored in the enhancement particle storage solution, add 50 μL of PBS buffer and sonicate for 1 min.

[0103] (2) Take 0.005 g of EDC and dissolve it in 60 μL of PBS buffer. Take 0.0135 g of NHS and dissolve it in 100 μL of PBS buffer.

[0104] (3) The in situ signal enhancement particles were centrifuged at 10,000 rpm, the supernatant was removed, and 50 μL of PBS buffer was added to resuspend the particles. After sonication for 1 min, 2.5 μL of EDC solution was added. After sonication for 1 min, 7.5 μL of NHS solution was added. The mixture was rotated at 37 °C for 15 min, and the mixture was centrifuged at 12,000 rpm for 15 min. The supernatant was removed and the nanoparticles were resuspended in 50 μL of PBS buffer.

[0105] (4) Add 20 μg of detection antibody (self-developed) and rotate to mix at 37°C for 2 h.

[0106] (5) Add 25 μL of Buffer D, incubate the reaction at 37°C for 45 min, centrifuge at 12,000 rpm for 15 min, and resuspend in 100 μL of Buffer E.

[0107] 3. Experimental methods

[0108] Preparation of standard curve

[0109] (1) Use fetal bovine serum to dilute the concentration of SV2A protein antigen to 0, 1.715, 5.144, 15.43, 46.30, 138.89, 416.67, 1666.67, 3333.33, and 5000 pg / mL, respectively.

[0110] (2) Dilute the magnetic beads labeled with capture antibody to 1 mg / mL, take 50 μL, add 50 μL of the sample of each concentration obtained in (1) at pH 9, and incubate at 37°C for 45 min. Wash three times with 100 μL of washing buffer to remove residual sample, and aspirate the supernatant.

[0111] (3) Add 10 μL of in situ signal enhancement particles bound to detection antibodies, incubate at 37°C for 45 min, wash four times with washing buffer to wash away residual in situ signal enhancement particles, and remove the supernatant.

[0112] (4) Add 5 μL of detection solution to resuspend the magnetic beads and transfer them to the detection well. Use a magnet to attract the magnetic beads to the bottom of the detection well. Use a fluorescence microscope (Nikon Eclipse Ti-U) for single-molecule imaging. Use the single-molecule counting mode and fluorescence intensity integration mode together to complete subsequent single-molecule counting statistics and analysis.

[0113] (5) Complete a series of concentration tests, repeat each concentration point 6 times, draw a standard curve based on the test results, and calculate the CV% value of each point.

[0114] 4. Experimental results

[0115] The results of the standard curve are as follows Figure 1 As shown in FIG. 1 , it can be seen that in this embodiment, the detection range of SV2A protein is 1 pg / mL to 100 ng / mL, and within this range, the single molecule signal number (i.e., CPN) has a good linear relationship with the sample concentration (R 2 =0.994, very close to 1), its detection limit can reach 1pg / mL, and the detection sensitivity is high. Moreover, the detection dynamic range is also wide, and the coefficient of variation CV value is only 3%. Example

[0116] The same operation as in Example 1 was performed except that the pH when adding the sample to the magnetic beads labeled with the capture antibody was adjusted to 7.4. As a result, the detection range of SV2A protein was 12pg / mL~100ng / mL. In this range, the single molecule signal number (i.e., CPN) showed a good linear relationship with the sample concentration, and the detection limit was 12pg / mL, and the detection sensitivity was acceptable. The coefficient of variation CV value was 9%, which met the requirement of less than 10%. Example

[0117] The same operation as in Example 1 was performed except that the pH when adding the sample to the magnetic beads labeled with the capture antibody was adjusted to 8.0. As a result, the detection range of SV2A protein was 3pg / mL~100ng / mL. In this range, the single molecule signal number (i.e., CPN) showed a good linear relationship with the sample concentration, and the detection limit was 3pg / mL, indicating excellent detection sensitivity. The coefficient of variation CV value was 4%. Example

[0118] The same operation as in Example 1 was performed except that the pH when adding the sample to the magnetic beads labeled with the capture antibody was adjusted to 10.0. As a result, the detection range of SV2A protein was 4 pg / mL to 100 ng / mL. In this range, the single molecule signal number (i.e., CPN) showed a good linear relationship with the sample concentration, and the detection limit was 4 pg / mL, indicating excellent detection sensitivity. The coefficient of variation CV value was 5%. Example

[0119] The same operation as in Example 1 was performed except that the pH value when adding the sample to the magnetic beads labeled with the capture antibody was adjusted to 10.5. As a result, the detection range of SV2A protein was 20pg / mL~100ng / mL. In this range, the single molecule signal number (i.e., CPN) showed a good linear relationship with the sample concentration, and the detection limit was 20pg / mL, and the detection sensitivity was acceptable. The coefficient of variation CV value was 9%, which met the requirement of less than 10%.

[0120] Comparative Example 1

[0121] The concentration of SV2A protein was determined using an enzyme-linked immunosorbent assay (ELISA), a common method in the field of immunoassays. The kit was purchased from Shanghai Yaji Biotechnology Co., Ltd. The detection limit was 300 pg / mL, the dynamic range was 300 pg / mL to 100 ng / mL, and the coefficient of variation was 20%.

[0122] Comparative Example 2

[0123] The applicant tried to use chemiluminescence to determine the concentration of SV2A protein. Since there was no commercially available kit, the common chemiluminescent agent acridinium ester and self-developed antibody pairs were used for determination, and Beckman Coulter's fully automatic chemiluminescent immunoassay instrument UniCel DxI 800 Access was used for the determination. As a result, the detection limit was 200 pg / mL and the coefficient of variation was 15%.

[0124] Comparative Example 3

[0125] The test was conducted using the Quanterix HD-1 device based on the single-molecule detection technology of the American company Quanterix. Since the company did not have a matching kit for the SV2A protein, the matching reagents (antibodies were changed) of its mature kit for cTnI were used for reference. As a result, the detection limit was 100 pg / mL and the coefficient of variation was 13%.

[0126] Examples 6-8 (Experiments with different carriers)

[0127] The same operation as in Example 1 was carried out except that the carrier in the in situ signal enhancing particle was replaced with polyacrylamide, silica, and polystyrene, respectively, to obtain the lower limit of detection of each example, which is shown in Table 1 below.

[0128] Table 1

[0129] Example 1 Example 6 Example 7 Example 8 Sensitivity 1pg / mL 2fg / mL 4pg / mL 7pg / mL

Claims

1. A counting-based single-molecule detection method for detecting the concentration of synaptic vesicle protein SV2A, comprising the following steps: (1) fixing a capture antibody capable of binding to synaptic vesicle protein SV2A onto magnetic beads, adding a sample containing synaptic vesicle protein SV2A, allowing the first site of synaptic vesicle protein SV2A to bind to the capture antibody, thereby capturing synaptic vesicle protein SV2A in the sample; (2) adding a detection antibody that can bind to the second site of the synaptic vesicle protein SV2A, allowing the detection antibody to bind to the second site of the synaptic vesicle protein SV2A, and then adding an in situ signal enhancement particle that can directly or indirectly bind to the detection antibody; or first allowing the detection antibody to bind to the in situ signal enhancement particle to form a complex, and then adding the complex; The in-situ signal enhancement particles have a particle size of 180nm to 350nm and contain fluorescent materials and carriers. (3) imaging the fluorescent signal emitted by the in situ signal enhancement particle using an optical imaging device; (4) Counting the number of in situ signal enhancement particles, and further calculating the concentration of synaptic vesicle protein SV2A in the sample, Wherein, steps (1) and (2) can be replaced by the following steps (1') and (2'), respectively, (1') mixing the detection antibody with the sample to make it bind to the second site of the synaptic vesicle protein SV2A in the sample, and then adding the in situ signal enhancing particle that can directly or indirectly bind to the detection antibody; or firstly making the in situ signal enhancing particle bind to the detection antibody to form a composite material, and then adding the composite material to the sample to make the composite material bind to the second site of the synaptic vesicle protein SV2A in the sample; wherein the in situ signal enhancing particle is the same as the in situ signal enhancing particle in step (1); (2') fixing a capture antibody capable of binding to synaptic vesicle protein SV2A onto magnetic beads, and then adding the magnetic beads to the system of step (1'), so that the capture antibody binds to the first site of the synaptic vesicle protein SV2A, thereby capturing the synaptic vesicle protein SV2A, The binding of the first site of the synaptic vesicle protein SV2A to the capture antibody in step (1) and step (2') is carried out in the pH range of 8-10.

2. The single molecule detection method according to claim 1, wherein: The particle size of the in-situ signal enhancement particle is 200-300 nm, and the coefficient of variation (CV value) of the particle size is 1%-10%.

3. The single molecule detection method according to claim 1 or 2, wherein: The carrier is polyethylene glycol, styrene-(methyl)acrylic acid copolymer, silicon dioxide, polystyrene, or polyacrylamide, and the fluorescent material is fluorescent dye, quantum dots, aggregation-induced luminescence material, or semiconductor conjugated polymer fluorescent material. 4 . Use of the single molecule detection method according to claim 1 in detecting the concentration of synaptic vesicle protein SV2A.

5. A single-molecule counting kit for detecting synaptic vesicle protein SV2A, comprising in situ signal enhancement particles and magnetic beads, wherein the in situ signal enhancement particles comprise a fluorescent material and a carrier, and have an average particle size of 180 to 350 nm, and a coefficient of variation (CV value) of the particle size of 1% to 10%. The invention also comprises a buffer system, wherein the pH value of the buffer system is 8-10, and the first site of the synaptic vesicle protein SV2A from the sample can be combined with the capture antibody within the pH range of 8-10.

6. The kit according to claim 5, wherein The carrier is polyethylene glycol, styrene-(methyl)acrylic acid copolymer, silicon dioxide, polystyrene, or polyacrylamide, and the fluorescent material is fluorescent dye, quantum dots, aggregation-induced luminescence material, or semiconductor conjugated polymer fluorescent material. 7 . The kit according to claim 5 , further comprising a capture antibody that binds to the first site of synaptic vesicle protein SV2A and a detection antibody that binds to synaptic vesicle protein SV2A, respectively.

8. The kit according to claim 5 or 6, which does not contain enzyme or chemiluminescent reagent.

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