Single molecule detection method and kit for detecting high-sensitivity cardiac troponin I

Abstract

The invention relates to a counting-based single molecule detection method and kit for high-sensitivity cardiac troponin I. According to the invention, a single molecule detection method based on a specific fluorescent material is applied to the detection of the concentration of the high-sensitivity cardiac troponin I, and the CV value can be obviously reduced while excellent sensitivity is obtained.

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 high-sensitivity cardiac troponin I (hereinafter referred to as cTnI), a related kit and the application of the single molecule detection method in detecting cTnI. Background Art

[0002] Cardiac troponin (cTn) is a myocardial-specific protein. When the myocardium is damaged, it is released into the blood and can be used as a specific marker of myocardial damage. The cardiac troponin subunits used for clinical detection are cTn I and cTn T, and cTn I is more commonly used. According to the sensitivity of the detection technology, it is divided into conventional cardiac troponin detection technology (Contemporary cTn, con-cTn) and high-sensitivity cardiac troponin detection technology (hs-cTn). Among them, the molecular mass of cTnI is 23.9kD, containing 210 amino acid residues. Elevated cTnI content can be used as an important serological indicator for diagnosing acute myocardial infarction (AMI). When acute myocardial infarction occurs, cTnI is released into the blood within 4-8 hours due to myocardial obstruction, so its concentration is out of the concentration range of healthy people.

[0003] Since cTnI has the advantages of rapid dynamic release after myocardial injury, complete curve, obvious peak, strong tissue specificity, long window diagnosis period, fast determination method, and early appearance in blood, the diagnosis of AMI based on cTnI is better than the diagnosis of AMI based on CK-MB, and has been widely accepted in clinic. cTnI has not only become the "gold standard" for diagnosing acute myocardial infarction, but also has become the most suitable marker for monitoring the condition, observing the efficacy, stratifying the risk, and evaluating the prognosis of myocardial diseases.

[0004] There are many methods for measuring cTnI, mainly radioisotope immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography (ICA), chemiluminescence assay (CLIA), etc. 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, the molecules that can generate signals are counted, which is more visible and digital, ensuring higher reproducibility. Since the SMD quantitative detection method has advantages that cannot be matched by average determination, there are studies that have applied SMD to quantitative analysis (see Patent Document 1). In previous studies, single-molecule quantitative detection methods were also used to detect cTnI (see Patent Document 2).

[0005] Prior art literature

[0006] Patent document 1: CN111771126B;

[0007] Patent document 2: CN115932248B Summary of the invention

[0008] Problems to be solved by the invention

[0009] In previous studies, single-molecule quantitative detection of high-sensitivity cTnI was able to achieve excellent sensitivity, with the best result being 1 fg / mL (see Patent Document 2). However, after repeated experiments, it was found that at this concentration level, its CV value was too high (about 15%) and could not be well applied in clinical scenarios and large-scale testing.

[0010] Means for solving problems

[0011] In response to the above problems, the applicant has repeatedly conducted in-depth research on single-molecule detection systems suitable for cTnI, such as luminescent materials, carriers, particle sizes, magnetic bead systems, buffer systems, etc. It was found that this may be due to the characteristics of the aggregation-induced luminescent materials used. The fluorescence intensity of such luminescent materials is only excellent enough when they are well aggregated, but due to the instability of aggregation, it is easy to cause poor repeatability, which in turn leads to a high CV value. Based on this, the applicant accidentally discovered that when coronene is used as the luminescent material in the single-molecule detection system of cTnI, the CV value can be significantly reduced while ensuring excellent detection sensitivity (even better than before), ensuring the stability of the detection system, and can be well applied to large-scale detection.

[0012] A technical solution of the present invention is as follows.

[0013] A counting-based single-molecule detection method for high-sensitivity cardiac troponin I, comprising the following steps:

[0014] (1) Immobilizing a capture antibody capable of binding to cTnI onto magnetic beads, adding a sample containing cTnI, allowing the first site of cTnI to bind to the capture antibody, thereby capturing cTnI in the sample;

[0015] (2) adding a detection antibody capable of binding to the second site of cTnI, allowing the detection antibody to bind to the second site of cTnI, and then adding in situ signal enhancement particles capable of directly or indirectly binding to the detection antibody; or firstly combining the detection antibody with the in situ signal enhancement particles to form a complex, and then adding the complex; the in situ signal enhancement particles have a particle size of 180 nm to 350 nm, and contain a fluorescent material and a carrier, wherein the fluorescent material is coronene

[0016] (3) imaging the fluorescent signal emitted by the in situ signal enhancement particle using an optical imaging device;

[0017] (4) The number of in situ signal-enhanced particles is counted and the concentration of cTnI in the sample is further calculated.

[0018] Preferably, the particle size of the in situ signal enhancing particles is 200 to 300 nm.

[0019] Preferably, the carrier is polyethylene glycol, styrene-(meth)acrylic acid copolymer, silica, polystyrene, or polyacrylamide.

[0020] Preferably, the binding of the first site of cTnI to the capture antibody in step (1) is performed in the pH range of 8-10.

[0021] Another technical solution of the present invention relates to the application of a single molecule detection method in detecting the concentration of cTnI.

[0022] Another technical solution of the present invention relates to a single molecule counting-based kit for detecting cTnI, which comprises in situ signal enhancement particles and magnetic beads. The in situ signal enhancement particles comprise a fluorescent material and a carrier, and have an average particle size of 180 to 350 nm. The fluorescent material is coronene.

[0023] Effects of the Invention

[0024] According to the present invention, a single-molecule detection method for cTnI can be provided that can significantly reduce the CV value while maintaining excellent detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The standard curve obtained in Example 1 is shown (the ordinate represents the number of single molecule signals). DETAILED DESCRIPTION

[0026] A counting-based single-molecule detection method for high-sensitivity cardiac troponin I, comprising the following steps:

[0027] (1) Immobilizing a capture antibody capable of binding to cTnI onto magnetic beads, adding a sample containing cTnI, allowing the first site of cTnI to bind to the capture antibody, thereby capturing cTnI in the sample;

[0028] (2) adding a detection antibody capable of binding to the second site of cTnI, allowing the detection antibody to bind to the second site of cTnI, and then adding in situ signal enhancement particles capable of directly or indirectly binding to the detection antibody; or firstly combining the detection antibody with the in situ signal enhancement particles to form a complex, and then adding the complex; the in situ signal enhancement particles have a particle size of 180 nm to 350 nm, and contain a fluorescent material and a carrier, wherein the fluorescent material is coronene

[0029] (3) imaging the fluorescent signal emitted by the in situ signal enhancement particle using an optical imaging device;

[0030] (4) The number of in situ signal-enhanced particles is counted and the concentration of cTnI in the sample is further calculated.

[0031] The coronene is also called hexaphenylene, which exhibits blue fluorescence in organic solvents. For luminescent materials used in in vitro diagnosis, such fluorescent materials are rarely used in the prior art. The present invention first discovered that such luminescent materials are particularly suitable for detecting cTnI in single-molecule immunoassays characterized by counting, and their relatively large rigid structure can overcome the instability disadvantages of the aggregation-induced luminescence material used in Patent Document 2, and by using them in combination with nanoparticle carriers, excellent detection sensitivity can be obtained while meeting the specific particle size range of the present invention.

[0032] The above detection method is based on double antibody sandwich immunoassay to detect the concentration of cTnI. Magnetic beads are used to separate and clean the detection samples and reagents. The above capture antibody is fixed on the surface of the magnetic beads by physical adsorption or chemical modification, and can bind to a binding site of cTnI to separate cTnI from the sample.

[0033] The above detection antibody can bind to another binding site of the molecule to be detected, cTnI. 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 molecule 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] As used in this specification, the sensitivity of the test, also known as the limit of detection (LoD), refers to the lowest concentration of the measured variable (i.e., the number of planned measurements) that can be detected at a specific confidence level. The confidence level is usually 95%, and the probability of a false negative measurement is 5%. Sensitivity is the lowest analyte concentration that can be reliably distinguished from the blank limit (LoB) and can be detected.

[0038] The in-situ signal enhancement particle of the present invention is a material that enhances the fluorescent 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.

[0039] In the in-situ signal enhancement particles, the carrier plays an important role. For example, more fluorescent materials can be combined to make the luminescent signal stronger; it provides sites for functional modification, can combine 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-(methyl) 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.

[0040] The fluorescent material in the in situ signal enhancement particle is also necessary for achieving single molecule detection. In the present invention, the fluorescent material must be coronene, which is adsorbed or wrapped on the surface or inside of the carrier through one or more of covalent modification, chelation, spatial wrapping, 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 coronene is uniformly wrapped inside the carrier.

[0041] In the present application, the fluorescent dye is preferably a fluorescent particle formed by coronene wrapped with PEG, a fluorescent particle formed by coronene wrapped with silica, a fluorescent particle formed by coronene wrapped with polyacrylamide, a fluorescent particle formed by coronene wrapped with polystyrene, or the like.

[0042] 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 in situ signal enhancement particles 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] After in-depth research, the inventors of the present application found that the binding conditions of the first site of cTnI and the capture antibody in step (1), especially pH, also affect the sensitivity 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 follows 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, and NF-L protein. However, in actual experiments, it was found that for the cTnI detection system using coronene as a fluorescent material, although the conventional neutral conditions can also perform single-molecule detection, there is still room for improvement in terms of effect. After further in-depth research, the applicant found that when the pH of the binding of the first site of cTnI in step (1) and the capture antibody is controlled under alkaline conditions, such as 8 to 10 (especially about 9), the sensitivity and coefficient of variation can be further improved. Therefore, it is preferred that the pH at which the first site of cTnI binds to the capture antibody in step (1) be controlled within the range of 8 to 10 (particularly around 9).

[0047] 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.

[0048] 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 collection 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 collection 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.

[0049] 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).

[0050] 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.

[0051] In the present application, there are two ways to calculate the concentration of cTnI: single molecule counting mode and fluorescence intensity integration mode. Among them, with respect to 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 into the concentration information of cTnI in the sample. The so-called "directly converted into the concentration information of cTnI in the sample" refers to absolute quantification, that is, it can be converted into concentration information without standard curve correction. The so-called "indirectly converted into the concentration information of cTnI in the sample" refers to the conversion into concentration information through the number of bright spots and the standard curve (or correction parameter). As for 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 integration 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, so as to convert the approximate number of in-situ signal enhancement particles, and then convert the value into the concentration information of cTnI in the sample. Among them, the average bright spot area is obtained by counting the bright spot area of ​​a single molecule at a lower concentration 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 above-mentioned low concentration and high concentration dividing line is generally the concentration when more than one molecule to be tested is bound to the surface of a magnetic bead, and it can also be preferably the concentration when 0.5 molecules to be tested are bound to the surface of a magnetic bead on average or the concentration when 2 molecules to be tested are bound according to the standard curve fitting result.

[0052] Example

[0053] 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.

[0054] 1. Determination of particle size of in situ signal enhancement particles

[0055] 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 of Japan) to determine the particle size.

[0056] 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).

[0057] 2. Single-molecule imaging

[0058] 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.

[0059] 3. Standard curve drawing method

[0060] In the present application, the single molecule counting mode and the fluorescence intensity integration mode are used in combination, and the specific implementation method is as follows: 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, and therefore the single molecule counting mode is used to draw a standard curve for samples of the molecule to be tested of different concentrations; 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, and therefore it is more suitable to use the fluorescence intensity integration mode.

[0061] 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.

[0062] 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.

[0063] 4. Preparation of in situ signal enhancement particles

[0064] (a) Taking the preparation of in situ signal-enhancing particles with polyethylene glycol as a carrier as an example, the steps are as follows.

[0065] 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.

[0066] (b) In-situ signal enhancement particles using styrene-(meth)acrylic acid copolymer as a carrier can be prepared by the following method:

[0067] (1) a step of preparing a seed particle dispersion containing a styrene-(meth)acrylic acid copolymer,

[0068] Specifically, 400 g of pure water, 1 g of methacrylic acid, 10 g of styrene, and 0.20 g of potassium persulfate (initiator) were added to a reactor equipped with a stirrer, a reflux condenser, a temperature sensor, and a nitrogen inlet tube. After the container was purged with nitrogen, the mixture was stirred at 65° C. and 200 rpm for polymerization for 24 hours. After the polymerization was completed, the solution was filtered with filter paper, the latex particles were taken out, and then dialyzed with a dialysis membrane for 48 hours to obtain latex particles as seed particles.

[0069] (2) a step of preparing an emulsion containing the fluorescent material and an organic solvent,

[0070] Specifically, 0.4 g of a fluorescent material as a 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, and mixed to prepare an emulsion.

[0071] (3) a step of 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;

[0072] 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.

[0073] 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 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.

[0074] Example 1: Detection of cTnI in human serum (using PEG-encapsulated coronene as in situ signal enhancement particles with a particle size of 200 nm).

[0075] 1. Some experimental components

[0076] Coronene (purchased from Shanghai Baishun Biotechnology), magnetic beads (purchased from Merck), capture antibody (self-developed), detection antibody (self-developed), silane coupling agent (APTES), ammonia water, tetraethyl orthosilicate (TEOS), in situ signal enhancement particles containing PEG-encapsulated coronene (average particle size of 200 nm), serum samples to be tested, PBS buffer, Buffer C (2 mM (NH 4 ) 2 SO 4 dissolved in 10mM PBS buffer, pH=7.4), Buffer D (0.02% NaCl, 0.5% BSA dissolved in 10mM PBS, pH=7.4), Buffer E (0.009% NaCl, 0.2% BSA dissolved in 10mM PBS, pH=7.4), N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), enhanced particle preservation solution, sample diluent, Tris buffer at pH 9 and PBS washing solution.

[0077] 2.1 Covalent coupling of magnetic beads and capture antibodies

[0078] (1) Take 166.6 μL of 20 mg / mL magnetic beads purchased from Merck, wash them five times with 6 mM PBS buffer, and remove the buffer.

[0079] (2) Take 80 μg of capture antibody, dilute it with 100 μL of 10 mM PBS buffer, add it to the magnetic beads in (1), mix well, add 90 μL of Buffer C, rotate and mix at 37°C, and react for 50 minutes.

[0080] (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 50 min.

[0081] (4) Wash five times with 10 mM PBS buffer and store in 100 μL Buffer E for later use.

[0082] 2.3 Covalent coupling of detection antibodies to in situ signal enhancement particles

[0083] (1) Take 10 μL of the in situ signal enhancement particles stored in the enhancement particle storage solution, add 60 μL of PBS buffer and sonicate for 3 min.

[0084] (2) Take 0.008 g of EDC and dissolve it in 60 μL of PBS buffer. Take 0.015 g of NHS and dissolve it in 80 μL of PBS buffer.

[0085] (3) The in situ signal enhancement particles were centrifuged at 12000 rpm, the supernatant was removed, and 60 μL of PBS buffer was added for resuspending. After ultrasonication for 1 min, 2.8 μL of EDC solution was added. After ultrasonication for 1 min, 10 μL of NHS solution was added. After mixing, the mixture was rotated at 37°C for 10 min. The mixture was centrifuged at 12000 rpm for 10 min, the supernatant was removed, and the nanoparticles were resuspended in 100 μL of PBS buffer.

[0086] (4) Add 30 μg of detection antibody (self-developed) and rotate to mix at 37°C for 2 h.

[0087] (5) Add 30 μL of Buffer D, incubate the reaction at 37°C for 60 min, centrifuge at 12,000 rpm for 15 min, and resuspend in 100 μL of Buffer E.

[0088] 3. Experimental methods

[0089] Preparation of standard curve

[0090] (1) Fetal bovine serum was used to dilute the concentration of cTnI antigen to 0, 0.78, 1.56, 3.13, 6.25, 12.50, 25.00, 50.00, and 100 fg / mL, respectively.

[0091] (2) Dilute the magnetic beads labeled with the 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.

[0092] (3) Add 10 μL of in situ signal enhancement particles bound to the detection antibody, incubate at 37° C. for 45 min, wash 4 times with washing buffer to wash away the residual in situ signal enhancement particles, and remove the supernatant.

[0093] (4) Add 6 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.

[0094] (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.

[0095] 4. Experimental results

[0096] Standard curve results

[0097] like Figure 1 As shown, in this embodiment, the detection range of cTnI is 0.5fg / mL to 100fg / mL, and within this range, the number of single molecule signals and the sample concentration show a good linear relationship (close to 1), and its detection limit can reach 0.5fg / mL, and the detection sensitivity is very high, which is better than the best sensitivity of Patent Document 2. Moreover, the coefficient of variation CV value is only 3%.

[0098] Example 2

[0099] 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 cTnI was 1 fg / mL to 100 fg / mL. In this range, the number of single molecule signals showed a good linear relationship with the sample concentration, and the detection limit was 1 fg / mL. The detection sensitivity was also very excellent. The coefficient of variation CV value was 8%.

[0100] Example 3

[0101] 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 cTnI was 0.8 fg / mL to 100 fg / mL. In this range, the number of single molecule signals showed a good linear relationship with the sample concentration, and the detection limit was 0.8 fg / mL, indicating excellent detection sensitivity. The coefficient of variation CV value was 4%.

[0102] Example 4

[0103] 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 cTnI was 0.9 fg / mL to 100 fg / mL. In this range, the number of single molecule signals showed a good linear relationship with the sample concentration, and the detection limit was 0.9 fg / mL, indicating excellent detection sensitivity. The coefficient of variation CV value was 5%.

[0104] Example 5

[0105] 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 cTnI was 1fg / mL to 100fg / mL. In this range, the single molecule signal number (i.e., CPN) and the sample concentration showed a good linear relationship, and the detection limit was 1fg / mL. The detection sensitivity was acceptable. The coefficient of variation CV value was 7%, which met the requirement of less than 10%.

[0106] Comparative Example 1

[0107] The same operation as in Example 1 was carried out except that the aggregation-induced emission material 4-C3O-TPE disclosed in Patent Document 2 was used instead of coronene as the fluorescent material. As a result, the detection range of cTnI was 2 fg / mL to 100 fg / mL. Within this range, the number of single-molecule signals (i.e., CPN) and the sample concentration showed a good linear relationship, and the detection limit was 2 fg / mL. However, the coefficient of variation CV value was 15%, which could not meet industrial requirements.

[0108] Comparative Example 2

[0109] The same operation as in Example 1 was carried out except that fluorescein isothiocyanate, a luminescent material disclosed in Patent Document 1, was used instead of coronene as the fluorescent material. As a result, the detection range of cTnI was 3 fg / mL to 100 fg / mL. Within this range, the number of single-molecule signals (i.e., CPN) and the sample concentration showed a good linear relationship, and the detection limit was 3 fg / mL. However, the coefficient of variation CV value was 12%, which could not meet industrial requirements.

[0110] Comparative Example 3

[0111] The same operation as in Example 1 was carried out except that the luminescent material quantum dots disclosed in Patent Document 1 were used instead of coronene as the fluorescent material. As a result, the detection range of cTnI was 10 fg / mL to 100 fg / mL. Within this range, the number of single-molecule signals (i.e., CPN) and the sample concentration showed a good linear relationship, and the detection limit was 10 fg / mL. However, the coefficient of variation CV value was 18%, which could not meet industrial requirements.

Claims

1. A counting-based single-molecule detection method for high-sensitivity cardiac troponin I, comprising the following steps: (1) Immobilize the capture antibody that can bind to cTnI onto the magnetic beads, add the sample containing cTnI, and allow the first site of cTnI to bind to the capture antibody, thereby capturing cTnI in the sample; (2) adding a detection antibody capable of binding to the second site of cTnI, allowing the detection antibody to bind to the second site of cTnI, and then adding in situ signal enhancing particles capable of directly or indirectly binding to the detection antibody; or firstly combining the detection antibody with the in situ signal enhancing particles to form a complex, and then adding the complex; the in situ signal enhancing particles have a particle size of 180 nm to 350 nm and contain a fluorescent material and a carrier, wherein the fluorescent material is coronene, (3) imaging the fluorescent signal emitted by the in situ signal enhancing particle using an optical imaging device; (4) Count the number of in situ signal-enhanced particles and further calculate the concentration of cTnI in the sample.

2. The single molecule detection method according to claim 1, wherein: The particle size of the in situ signal enhancement particles is 200-300 nm.

3. The single molecule detection method according to claim 1 or 2, wherein: The carrier is polyethylene glycol, styrene-(meth)acrylic acid copolymer, silicon dioxide, polystyrene, or polyacrylamide.

4. The single molecule detection method according to any one of claims 1 to 3, wherein: The binding of the first site of cTnI to the capture antibody in step (1) is carried out in the pH range of 8 to 10.

5. Use of the single molecule detection method according to any one of claims 1 to 4 in detecting the concentration of high-sensitivity cardiac troponin I.

6. A single-molecule counting-based kit for detecting high-sensitivity cardiac troponin I, comprising in situ signal-enhanced particles and magnetic beads, wherein the in situ signal-enhanced particles comprise a fluorescent material and a carrier, and have an average particle size of 180 to 350 nm, and the fluorescent material is coronene.

7. The kit according to claim 6, wherein The carrier is polyethylene glycol, styrene-(meth)acrylic acid copolymer, silicon dioxide, polystyrene, or polyacrylamide.

8. The kit according to claim 6 or 7, further comprising a capture antibody that binds to the first site of cTnI and a detection antibody that binds to the second site of cTnI, respectively.

9. The kit according to claim 6 or 7, further comprising a buffer system, wherein the pH of the buffer system is 8-10, and the binding of the first site of cTnI from the sample to the capture antibody can be performed within the pH range of 8-10.

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