Detection kit and application, marker detection system, marker detection method

Abstract

The application provides a detection kit and application, a marker detection system and a marker detection method. The detection kit comprises carrier particles, detection function objects, peroxidase conjugates and a substrate solution. The carrier particles have magnetism, and the surface of the carrier particles is coated with capture function objects. The capture function objects can specifically bind to target objects. Each carrier particle is provided with a fluorescent point, and fluorescent points of the same color correspond to the same capture function object. The substrate solution comprises a dendritic polymer. The dendritic polymer is labeled with a plurality of fluoresceins, and the dendritic polymer is covalently coupled with a plurality of tyramide molecules. The detection kit can be used for detection of low-concentration markers. The detection kit has a low detection lower limit, is simple to operate and has a low detection cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay, in particular, to a detection kit and application, a marker detection system and a marker detection method. BACKGROUND

[0002] In the field of clinical diagnosis, the accurate detection of low-abundance proteins and disease-related biomarkers in biological fluids (such as blood) is a core technical challenge to achieve early diagnosis of major diseases such as cardiovascular and cerebrovascular diseases, neurodegenerative diseases, and tumors.

[0003] Traditional detection systems such as magnetic particle chemiluminescence immunoassay (CLIA) have the advantages of convenient operation and significant cost-effectiveness, but their detection sensitivity is usually limited to the pg / mL level, which is difficult to meet the analysis requirements of trace biomarkers. In recent years, digital bioanalysis technology based on single molecule detection principles (such as digital enzyme-linked immunosorbent assay, Digital ELISA) has improved the detection sensitivity to the fg / mL level (more than 3 orders of magnitude higher than traditional ELISA) through antibody-antigen specific recognition and fluorescence signal amplification mechanisms. However, existing single molecule immunoassay systems still face significant technical bottlenecks: they rely on microcavity array chips to achieve spatial isolation of single molecule reaction units, resulting in a significant increase in chip consumable costs, a significant increase in the complexity of the optical system of the instrument, and difficulty in achieving high-throughput detection without physical barriers. SUMMARY

[0004] The present application provides a detection kit and application, a marker detection system and a marker detection method. The detection kit of the present application can be used for the detection of low-concentration markers, which not only has a low detection lower limit, but also is simple to operate and has a low detection cost.

[0005] In a first aspect, the present application provides a detection kit, which comprises:

[0006] Carrier particles, the carrier particles have magnetism, and the surface of the carrier particles is coated with a capture functional substance, the capture functional substance can specifically bind to a target substance, and each carrier particle is provided with a fluorescent dot, and the same color fluorescent dot corresponds to the same capture functional substance;

[0007] Detection functional substance, the antibody can specifically bind to the target substance;

[0008] Peroxidase conjugate, the peroxidase conjugate is a peroxidase conjugated with streptavidin;

[0009] Substrate solution, the substrate solution comprises a dendrimer, the dendrimer is labeled with a plurality of fluoresceins, and the dendrimer is covalently conjugated with a plurality of tyramide molecules.

[0010] In the technical solution, when the detection kit detects the sample to be detected, the carrier particles are first mixed with the sample to be detected, so that the capture function in the sample to be detected specifically binds to the expected marker (i.e., the target) in the sample to be detected. After binding, the detection function is added, and the detection function specifically binds to the target. Then, the peroxidase conjugate is added, and the detection function binds to the part of streptavidin in the peroxidase conjugate. Then, the substrate solution is added. Since the dendrimer is covalently coupled with multiple fluorescein molecules, under the action of peroxidase in the peroxidase conjugate, the tyramide-labeled dendrimer is deposited on the surface of the carrier particles to realize signal accumulation. In this way, the detection function for low-concentration markers can be realized.

[0011] In a possible implementation, the number of carrier particles corresponding to each capture function is 5000-50000.

[0012] In a possible implementation, the equivalent particle diameter of the carrier particles is 0.5-8 μm.

[0013] In a possible implementation, the peroxidase includes at least one of dextran-conjugated horseradish peroxidase or natural peroxidase.

[0014] In a possible implementation, the substrate solution includes substrate solution A and substrate solution B. The substrate solution A includes tyramide-labeled dendrimer, and the substrate solution B includes hydrogen peroxide with a volume fraction of 0.001%-0.005%.

[0015] In a possible implementation, the dendrimer includes any one of inert branched DNA polymer, polyamidoamine, polyethyleneimine, or polypropyleneimine.

[0016] In a possible implementation, the fluorescein includes at least one of Alexa Fluor 647, iFluor 647, or Cy5.

[0017] In a second aspect, the present application provides a marker detection system, which includes the detection reagent, a data acquisition module, and an algorithm processing module. The data acquisition module is used to acquire data, and the algorithm processing module is used to analyze the data and output content.

[0018] In a third aspect, the application provides a detection method of a marker, which uses the marker single-molecule detection system of the second aspect, and comprises the following steps: first incubating the carrier particles with a sample to be detected, then adding a detection function substance for reaction and performing magnetic separation and cleaning, subsequently adding a peroxidase conjugate and performing magnetic separation and enhancement cleaning, then adding a substrate solution, subsequently using a data acquisition module to detect a signal, and then using an algorithm processing module to analyze data and output content.

[0019] In a fourth aspect, the application provides an application of the detection kit in detecting a marker of heart and brain organ injury.

[0020] The application adopts single-molecule immunodetection, and compared with a conventional protein detection method, the digital technology improves the sensitivity by 1000 times, and realizes super-sensitive multiple detection of low-abundance proteins. The beneficial effects of the application include but are not limited to:

[0021] 1) The (single-molecule) detection method of the application is based on a single-molecule algorithm of Poisson distribution principle. The cascade signal amplification technology of nano-enzyme and tyramide deposition is adopted, and the in-situ nano-enzyme catalysis enrichment on the surface of microspheres is utilized. The reactivity of free radicals is higher, and the tyramide is catalyzed to form a dendritic fluorescein deposition, thereby improving the fluorescence intensity. The markers can be uniformly combined on the surface of the target microspheres, the aggregation of the magnetic coding microspheres is reduced, the non-specificity is obviously reduced, the sensitivity of the detection system is improved, and the sensitivity reaches the femto range (10 fg / ml level).

[0022] 2) The kit of the application uses magnetic coding microspheres as a solid carrier, and compared with single microspheres, the detection of 6 indexes in a single tube can be realized, the detection flux can be significantly improved, and the detection efficiency is improved.

[0023] 3) The optical equipment used in the detection method is a conventional detector, which realizes non-compartment detection, the optical module is simple, the structure of the instrument is relatively simple, the cost is low, and the instrument can be maintained by itself. The problems of complex instrument structure and high cost in the prior art are solved. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiments of the application. It should be understood that the following drawings only show some embodiments of the application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0025] Figure 1 It is a detection flowchart of the first embodiment of the application.

[0026] Figure 2A schematic diagram of a dendrimer having multiple tyramide molecules covalently coupled in an embodiment of the present application.

[0027] Figure 3 A three-item flow cytometric cluster diagram of myocardial infarction in the first embodiment of the present application. DETAILED DESCRIPTION

[0028] Before describing the technical solutions of the present application, some basic concepts in the related field need to be introduced, as follows:

[0029] The branched DNA (bDNA) technology is a nucleic acid-based signal amplification technology, and its core advantage lies in the unique cascade amplification effect, which can realize high-sensitivity detection of extremely low-concentration nucleic acid molecules without PCR (Polymerase Chain Reaction) amplification. The bDNA of the present application refers to the DNA double-stranded structure molecule used in the bDNA technology, which is an artificially synthesized special oligodeoxyribonucleic acid. The design core is to introduce branch points (such as nucleoside derivatives with protected hydroxyl groups) to form a nonlinear multi-chain topological structure, which is not a double helix structure; for example, the bDNA can be a multi-chain structure of tree or bifurcation. In the bDNA, each extension chain is connected by a phosphodiester bond, and the backbone composition is the same as that of natural DNA, and the nucleotides at the branch points are connected to the 5' or 3' ends of different extension chains by multiple phosphodiester bonds.

[0030] Streptavidin (SA) is a protein with similar biological properties to avidin, which is the secretion of streptomyces avidinii bacteria. It has high affinity and can tightly bind to biotin (Biotin), so it is widely used in biological detection. By labeling biotin on the target molecule, the high affinity of streptavidin can be used for capture, such as labeling streptavidin with fluorescein, which can achieve efficient detection of target molecules.

[0031] In the present application, "M" represents mol / L, "mM" represents mmol / L, and "μM" represents μmol / L.

[0032] In the present application, "EDC" represents 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide, i.e. 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and "NHS" represents N-Hydroxy succinimide, i.e. N-hydroxy succinimide. EDC and NHS can be used to activate carboxyl groups to react with amino groups to produce amides.

[0033] In the present application, "PBST" stands for Phosphate Buffered Saline with Tween-20, "MES" stands for 2-Morpholinoethanesulfonic Acid, and "PBS" stands for Phosphate Buffered Solution.

[0034] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturer are adopted. If the manufacturers of the reagents or instruments are not specified, they are all the conventional products that can be purchased in the market. If not otherwise specified, the pH in the present application is the value measured at 25°C.

[0035] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturer are adopted. If the manufacturers of the reagents or instruments are not specified, they are all the conventional products that can be purchased in the market.

[0036] In the field of clinical diagnosis, the detection of low-abundance proteins and disease-related biomarkers is often an important basis for diagnosing major diseases such as cardiovascular and cerebrovascular diseases, neurodegenerative diseases, and tumors. However, traditional immunoassays such as CLIA are difficult to detect low-concentration proteins. Currently, there are single-molecule immunoassay techniques for detecting low-concentration proteins, such as: (1) fluorescent nanoprobe labeling system: by covalently coupling specific antibodies to the surface of fluorescent nanoparticles, the signal probe is associated with the target protein at the molecular level through the formation of antigen-antibody complexes, and finally the quantitative detection is realized through fluorescent microsphere counting. Its signal amplification mechanism can be divided into two categories: 1. Rigid fluorescent microspheres (particle size about 180 nm) as signal carriers, but there is a signal resolution problem due to the optical diffraction limit of small particle size particles, while large particle size microspheres (> 200 nm) are prone to spatial steric hindrance effects when combined with capture magnetic beads (such as 3 μm), resulting in increased dispersion of binding efficiency; 2. Flexible fluorescent polymer nanoparticles (particle size > 100 nm) enhance fluorescence intensity, but large size polymer particles are prone to non-specific cross-linking aggregation between magnetic beads, resulting in loss of effective signal and decrease in detection sensitivity. (2) In situ nano-enzyme catalytic amplification system: by labeling signal initiators on the detection antibody, enzyme cascade catalytic reaction is used to realize signal amplification, and single-molecule quantification is completed by combining Poisson distribution statistical model. Main technical branches include: 1. Microcavity chip enzyme catalytic fluorescence product detection, which faces the industrialization obstacles of high cost of consumables and instruments; 2. Signal amplification technology (Tyramide Signal Amplification, TSA), which realizes signal accumulation by horseradish peroxidase (HRP) catalyzing tyramide-fluorescent substrate deposition on the surface of microspheres, but there is a problem of non-specific deposition leading to background signal enhancement; 3. Amplification-assisted signal amplification (such as rolling circle amplification technology, Rolling Circle Amplification, RCA), which is limited by cost and slow enzyme reaction kinetics (reaction time > 0.5 hours). The above methods generally have technical defects of insufficient clinical transformation adaptability. (3) Multiple immunoassay technology based on spectrally encoded microspheres (such as Luminex xMAP system) in the field of multiple detection, through multi-dimensional coding strategy, it can realize the synchronous detection of dozens of biomarkers, significantly improving the detection throughput and reducing the unit detection cost. However, the existing detection system is still limited by the key performance defects: the detection sensitivity of low-abundance targets is difficult to break through the fg / mL level threshold. As can be seen, the existing low-concentration detection technology is complex, the detection cost is high, and the lower limit of the detection concentration is still relatively high.

[0037] Based on this, this application provides a single-molecule detection kit, comprising carrier particles, a detection functional group, a peroxidase conjugate, and a substrate solution. The carrier particles are magnetic, and their surfaces are coated with a capture functional group that specifically binds to the target. Each carrier particle has a fluorescent dot, with fluorescent dots of the same color corresponding to the same capture functional group. The detection functional group specifically binds to the target. The peroxidase conjugate is streptavidin coupled with peroxidase. The substrate solution comprises a dendritic polymer labeled with multiple fluorophores, and the dendritic polymer is covalently coupled with multiple tyramine molecules.

[0038] like Figure 1 As shown, when the single-molecule detection kit of this application detects the sample, the carrier particles and the sample are first mixed. This allows the capture functional component in the sample to specifically bind to the expected marker (i.e., target) in the sample. After binding, the detection functional component is added, which binds specifically to the target. Next, a peroxidase conjugate is added, which binds to a portion of the streptavidin in the peroxidase. Then, the substrate solution is added. Because the dendritic polymer is covalently coupled with multiple fluorophores, under the action of the polyperoxidase in streptavidin, the dendritic polymer deposits on the surface of the carrier particles, achieving signal accumulation. This enables the detection of low concentrations of the marker. In the figure, "coated antibody carrier particles" represent the carrier particles of this application, and "antibody" is the capture functional component in the carrier particles; "tyramine-fluorophore polymer" represents the dendritic polymer of this application, where "tyramine" consists of multiple tyramine molecules covalently coupled to the dendritic polymer, and "fluorophore" consists of multiple fluorophores labeled on the dendritic polymer.

[0039] In specific embodiments of this application, the type of capturing functional substance can be one or more, and can be adjusted according to actual detection needs; this application does not impose any particular limitation. However, it should be noted that the surface of each carrier particle can only be coated with the same capturing functional substance, and the color of the fluorescent spot corresponding to the capturing functional substance must be consistent to accurately determine the observation results. For better observation of the detection results, as an example, in the embodiments of this application, the number of carrier particles corresponding to each capturing functional substance is generally between 5000 and 50000. Furthermore, in the embodiments of this application, the equivalent particle size of the carrier particles is typically in the range of 0.5 to 8 μm, which can both suppress non-specific cross-linking and aggregation between magnetic beads and improve detection sensitivity; for single-molecule detection methods, the equivalent particle size of the carrier particles is typically 3 μm.

[0040] The detection function material in the present application can specifically bind to the target material, and therefore the detection function material in the present application generally comprises a plurality of specific binding units covalently coupled to the periphery of the core structure, wherein the specific binding unit is preferably an antibody or streptavidin.

[0041] In the peroxidase conjugate in the present application, the streptavidin part is used to recognize and bind to the biotinylated antibody, and the peroxidase part can catalyze the accumulation of the substrate solution on the surface of the carrier particle to enhance the detection sensitivity; the peroxidase can be at least one of horseradish peroxidase (HRP), natural peroxidase, etc. In addition, in some embodiments of the present application, in order to better achieve the accumulation effect, the peroxidase can be a polyperoxidase, that is, a complex formed by coupling a plurality of peroxidases to a polymer matrix as a carrier, wherein the polymer matrix includes but is not limited to polysaccharides, dendritic polymers, and polyamino acid polymers; preferably, dextran is used as the carrier, and the molecular structure thereof is a long-chain cross-linked polymer.

[0042] The substrate solution in the present application can play a role in signal cascade amplification. Specifically, the dendritic polymer in the substrate solution includes any one of branched DNA polymer (bDNA), polyamidoamine (PAMAM), polyethylenimine (PEI), and poly(propylene imine) (PPI), and is preferably a bDNA polymer, and more preferably a carboxylated bDNA. When the bDNA is used, the substrate solution can combine the high loading capacity of bDNA, the enzyme deposition characteristics of tyramide, and the high sensitivity detection advantage of fluorescein, and is suitable for high sensitivity detection of single molecules.

[0043] In addition, the substrate solution of the present application generally comprises substrate solution A and substrate solution B, wherein the substrate solution A contains a dendritic polymer, and in a preferred technical solution, the dendritic polymer forms a three-dimensional core structure, and the surface thereof is covalently connected with an array of tyramide molecules. The fluorescein is selected from the group consisting of Alexa Fluor 647, iFluor 647, and Cy5 series, and is preferably Alexa Fluor 647. In addition, in some other embodiments, the dendritic polymer can also be labeled with biotin, but at this time the dendritic polymer needs to be used together with streptavidin labeled with fluorescein. The substrate solution B is generally hydrogen peroxide with a concentration of 0.001% to 0.005% (volume fraction level).

[0044] In addition, the detection kit of the present application also comprises a calibrator and a quality control product, the calibrator comprises low, medium and high concentration protein solutions, and the quality control product comprises zero protein solution, low protein solution, and medium concentration protein solution.

[0045] The carrier particles, detection functionals, peroxidase conjugates, and dendrimers of the present application can be obtained commercially or prepared in-house. By way of example, the present application provides methods for preparing them, as follows:

[0046] (I) Preparation of carrier particles

[0047] The magnetic particles containing carboxyl groups and 0.05 mol / L coating buffer (pH 5.5) are added to a microtube and vortexed using a vortex mixer. After magnetic separation, the supernatant is removed. Then 0.05 mol / L coating buffer is added and mixed. An appropriate amount of EDC and NHS is added, and the mixture is shaken at 1000 rpm for 30 minutes at room temperature. After magnetic separation, the supernatant is removed, and the washing buffer is added. The washing process is repeated three times. Then the corresponding capture functional (usually an antibody) is added and mixed. The mixture is mixed for 2 hours at room temperature in the dark. The washing process is repeated three times. Then a blocking agent is added, and the mixture is mixed for 16 hours or more at 2-8°C in the dark. After magnetic separation, the supernatant is removed, and the washing buffer is added. The washing process is repeated three times. Finally, the suspension is stored in a preservation solution at 2-8°C.

[0048] In the above steps, the parameters can be adjusted as needed, for example: the blocking solution contains polyaromatic amino acids, usually polytyrosine, phenylalanine or tryptophan; it can also contain 1wt% bovine serum albumin and 5wt% polyaromatic amino acids. The rest of the adjustments are not described here, as long as they meet the purpose of the present application.

[0049] (II) Preparation of dendrimers

[0050] Taking carboxylated bDNA as an example, preferably, a fluorescein-modified nucleotide is directly involved in the synthesis of bDNA, and the fluorescein is chemically modified to become part of the DNA backbone or base in advance. The preparation of the substrate solution includes three steps of material pretreatment, coupling, purification and verification, as follows:

[0051] S100, material pretreatment.

[0052] S110, pretreatment of carboxylated bDNA: select bDNA with terminal carboxyl groups (-COOH) (molecular weight 20-100 kDa), dissolve in 0.1 M MES buffer at pH 5.5, and the concentration is 1-5 mg / mL.

[0053] S120, aminolysis of fluorescein: Alexa Fluor 647 NHS Ester (1 mg) was dissolved in anhydrous DMSO (100 μL), and 10-fold molar ratio of NH2-PEG-NH2 (2 kDa) was added, and the mixture was reacted at room temperature in the dark for 2 h to generate the long-arm Alexa Fluor 647-PEG-NH2 intermediate.

[0054] S200, coupling.

[0055] S210, carboxyl activation: EDC (10 mM) and NHS (20 mM) were added to the bDNA solution, and the mixture was shaken at room temperature for 30 min to activate the carboxyl group into a reactive NHS ester.

[0056] S220, tyramide coupling: tyramide hydrochloride (5 mg) was dissolved in 0.1 M phosphate buffer at pH 8.0, and added to the activated DNA dendrimer (mass ratio 1:10), and the mixture was reacted at room temperature in the dark for 30 min to form a DNA-tyramide conjugate.

[0057] S230, fluorescein labeling: long-arm Alexa Fluor 647-PEG-NH2 (molar ratio 1:5) was added to the above reaction system, and the mixture was reacted at 4°C in the dark for 12 h to complete the fluorescein labeling and blocking through amino-carboxyl condensation.

[0058] In addition, the fluorescein can also be iFluor 647 or Cy5 series, but the effect of Alexa Fluor 647 is better.

[0059] S300, purification and detection.

[0060] S310, purification: the reaction solution was subjected to ultrafiltration centrifugation (30 kDa molecular weight cutoff) to remove unreacted free tyramide and fluorescein, and then further purified by HPLC (C18 reverse phase column, acetonitrile / water gradient elution) to obtain the target product.

[0061] S320, characterization: UV-Vis spectroscopy (UV-Vis) was used to detect the characteristic absorption peak of AF647 (647 nm), dynamic light scattering (DLS) was used to determine the particle size, and fluorescence spectroscopy was used to verify the signal intensity (excitation / emission: 638 / 670 nm).

[0062] In the single-molecule detection kit of the present application, enzyme-mediated deposition and multivalent loading are combined, and the detection limit reaches the fg / mL level; the rigid structure of the dendrimer limits non-specific adsorption, and the background signal is reduced by 60%; various markers such as biotin can be adapted, and the kit can be expanded to various detection modes.

[0063] The detection kit of the present application can be used for detecting markers, for example, can be used for detecting peripheral blood central organ injury markers.

[0064] The detection kit of the present application can be used in a marker detection system, specifically, the marker detection system comprises the above-mentioned detection kit, and a flow cytometer, wherein the flow cytometer comprises a data acquisition module and an algorithm processing module, the data acquisition module is used for acquiring data, and the algorithm processing module is used for analyzing data and outputting content.

[0065] Specifically, in the marker detection system of the present application, the analyzer is used to collect the original signal of the above-mentioned fluorescent complex, capture and decode the cluster, and the appropriate data processing algorithm is selected according to the medical decision level value during product design. Specifically as follows:

[0066] I. For target markers with clinical expected concentration of fg level

[0067] When detecting target markers of fg level, the detection kit is usually set to a single molecule detection form, and the specific detection algorithm is as follows:

[0068] (1) For low-abundance proteins, use digital algorithm: when the average fluorescence intensity of the carrier particles is lower than the preset threshold (such as 5000), enable single molecule counting mode. The single molecule detection data processing is the proportion or number of "1" carrier particles, which means that after detecting the fluorescence value of a single target molecule, all detection carrier particles in the detection system are marked by a binary system according to the signal marking mode of "0" for non-target marker carrier particles and "1" for target marker carrier particles. More preferably, the concentration is calculated by calculating the proportion of "1" in the total of "0" and "1" carrier particles. By setting an intensity threshold to identify individual fluorescence peaks, the number of peaks in the carrier particles is counted, and the concentration of the analyte as low as fg level is calculated by Poisson distribution. The preset threshold value will fluctuate up and down for different instruments and reagents.

[0069] (2) For medium-abundance proteins, use analog algorithm: when the average fluorescence intensity of the carrier particles exceeds the threshold, switch to the median mode of the fluorescence intensity of the carrier particles. Calculate the fluorescence intensity on each carrier particle, and realize pg / mL level quantification through standard curve.

[0070] II. For target markers with clinical expected concentration of pg, ng level

[0071] When detecting target markers of pg, ng level, the detection kit is usually set to a liquid chip detection form, and the data processing preferentially selects analog algorithm to realize high-throughput accurate quantification of multiple targets through median statistics and curve fitting, specifically as follows:

[0072] The double-laser excitation signal of each carrier particle is collected by the optical detection module: classification fluorescence (classification channel, such as red laser), based on the fluorescent code (such as different proportions of two fluorescent dyes) embedded in the carrier particle, to identify the corresponding detection target (i.e. project ID) of the carrier particle. The reporter fluorescence (detection channel, such as green laser) reflects the fluorescence signal intensity (MFI, Median Fluorescence Intensity) of the target molecule binding amount.

[0073] Data preprocessing and background correction, removing invalid carrier particles, removing abnormal carrier particles with signal-to-noise ratio below threshold or deviating from the classification fluorescence cluster center. Background subtraction: calculate the median of the reporter fluorescence (Median MFI) of the corresponding project carrier particles in the negative control wells (such as blank or homotypic control), and subtract this value from the sample MFI to eliminate non-specific signal interference.

[0074] Median fluorescence intensity (Median MFI) calculation: for all valid carrier particles (usually ≥100 / well) of the same detection project (i.e. the same carrier particle classification ID), calculate the median of the reporter fluorescence intensity values (Median MFI). Select the median instead of the mean to avoid the influence of extreme outliers (such as high-fluorescence carrier particles that are not completely eluted) on data distribution, and improve the robustness of the results.

[0075] Standard data modeling: using the Median MFI values of the series of concentration standards, establish the product factory master curve, and through the four-parameter fitting of the fluorescence intensity-concentration relationship curve. Substitute the Median MFI of the target project in the sample to be tested into the calibration curve, and calculate the corresponding target concentration in the sample to be tested by interpolation or numerical optimization algorithm. For values outside the linear range of the standard curve, it needs to be marked as "higher than the upper limit of quantification (ULOQ)", "lower than the lower limit of quantification (LLOQ)", to avoid non-quantitative speculation.

[0076] When using the marker detection system of the present application to detect markers, it usually includes the following steps:

[0077] First incubate the carrier particles with the sample to be tested, then add the detection function substance reaction and perform magnetic separation and washing, then add the substrate solution, and then perform signal detection and data analysis.

[0078] The technical solutions of the present application will be specifically described below in conjunction with examples.

[0079] First embodiment

[0080] <Test kit>

[0081] The cardiac and cerebral organ injury markers detected in this embodiment are cardiac infarction three markers or cerebral injury marker S100. The single molecule detection kit comprises carrier particles, detection function objects, peroxidase conjugates, substrate solution (including substrate solution A and substrate solution B), calibrators and quality control products, as shown in Table 1:

[0082] Table 1 Main components of the detection kit

[0083]

[0084] In Table 1, different types of capture function objects are labeled on the surface of different fluorescent carrier particles. In actual application, flexible combinations can be made according to detection needs, for example, when only part of the cardiovascular markers need to be detected, a single or combined scheme of multiple cardiovascular marker probes can be selectively used. The equivalent particle diameter of the carrier particles corresponding to the cTnI and CK-MB indicators is 3 μm, and the working concentration of each indicator carrier particle is 10,000 particles / test; the diameter of the carrier particles corresponding to the Myo indicator is 6 μm, and the working concentration thereof is 1,500 particles / test.

[0085] The specific preparation method of the carrier particles is referred to the above content. The blocking treatment is as follows: the cTnI and CK-MB carrier particles use a blocking solution containing polyaromatic amino acids (including polytyrosine, phenylalanine or tryptophan), and the more preferred formula is a mixed solution of 1% bovine serum albumin, 50% CE510 and 5% polytyrosine. The Myo carrier particles use a blocking solution containing 1% human serum albumin, 50% CE510 and 5% glycine. The storage solution is prepared as follows: after blocking, the carrier particles are suspended in a phosphate buffer (pH 7.4) containing 40 μg / mL blocking agent HBR I, 1% BSA, 0.05% Tween-20 and 0.1% ProClin 300 preservative, and finally stored in a 4°C environment.

[0086] In the detection function object, the concentration of each labeled antibody indicator is 3.0 μg / mL.

[0087] The working concentration of streptavidin conjugated with peroxidase is 5.0 μg / mL.

[0088] The preparation method of the dendrimer in the substrate solution A can be seen from the above content, and the preparation method is as follows: 10% glycerol, 0.05% Tween-20 and distilled water are mixed, and the final concentration is 20 μmol / L (calculated by tyramine fluorescein).

[0089] The volume of the substrate solution B is not less than 200 ul.

[0090] Calibrator: cTnI, CK-MB and Myo calibrator, main component is recombinant protein, calibrator composition of two containing low, medium high concentration of calibrator, for the establishment of calibration curve.

[0091] Quality control: cTnI, CK-MB and Myo quality control, the main component is protein, quality control through three containing zero, low, medium concentration of protein to achieve internal quality control.

[0092] <Signal detection process>

[0093] 1. Detection operation process

[0094] The use of the above magnetic attraction magnetic rod transfer rapid detection method can improve the magnetic ball retention rate; at the same time, the single serving of full automatic liquid chip analyzer instrument iMatrix Fusion is used, and the automatic detection is realized. The specific detection steps are as follows:

[0095] (1) add the sample to be tested (or calibrator and quality control) to the reaction tank R1 containing carrier particle mixture, stir and mix uniformly, and then react at 37℃. Directly transfer to the reaction tank R2 containing detection function material by magnetic rod, stir and mix uniformly, and then continue to react at 37℃.

[0096] (2) after the reaction is completed, the magnetic rod is adsorbed, and the system is transferred to R3 cleaning buffer, stirred and mixed, then the magnetic rod is adsorbed, and then transferred to another hole R4 or R5; repeat the cleaning step, a total of three times.

[0097] (3) transfer the magnetic rod to the reaction tank R6 containing peroxidase conjugate, stir and mix uniformly, and then react at 37℃; repeat the cleaning step, a total of three times; the cleaning step can be seen in step (2).

[0098] (4) mix substrate liquid B and substrate liquid A in the reaction tank R7, then transfer the system in step (3) to the reaction tank R7 by magnetic rod, stir and mix uniformly, and then react at 37℃; repeat the cleaning step, a total of three times; the cleaning step can be seen in step (2).

[0099] (5) transfer the system in step (4) to the reaction tank R13 detection liquid by magnetic rod, stir and mix uniformly, and then place the detection liquid in the liquid chip analyzer for reading, and measure the fluorescence value of each carrier particle.

[0100] The sample is usually pretreated, and the pretreatment is preferably performed in a whole blood pretreatment device to achieve rapid result output. Specifically, the whole blood pretreatment device comprises a sample suction needle module, a sample adding needle module, a vacuum extraction module, a photoelectric detection module, a separation cup and a rotating disc mechanism; the separation cup comprises a cup body, a filter membrane assembly arranged in the cup body and a reagent arranged at the bottom of the cup body; the reagent comprises freeze-dried immunomagnetic beads and an antibody reagent; and the rotating disc mechanism comprises a fixed base, a rotating shaft arranged at the center of the fixed base and a separation cup tray located above the fixed base and performing rotary motion with the rotating shaft.

[0101] <Flow cytometry detection instrument and detection method>

[0102] The solid-state markers enriched on the surface of the carrier are fluidly introduced into a flow cytometry detection instrument for detection, and the detection does not include droplet encapsulation treatment. The flow cytometry instrument realizes high-efficiency detection of three-channel fluorescence signals at a single molecule level by optimizing the wavelength segmentation strategy of the dichroic mirror chain structure and the fluorescence collection detection area.

[0103] The flow cytometry instrument comprises an optical system for flow fluorescence collection, and the specific structure is as follows:

[0104] (1) Spot shaping area: two laser light sources (488 nm and 638 nm) are adopted, and preliminary spot shaping is performed through independent primary shaping lens groups. After being combined by a reflecting mirror and a beam combiner, a compound spot with consistent size and uniform energy distribution is formed by a secondary achromatic shaping lens (curvature radius 24-28 mm / 200-210 mm), so as to ensure that the fluorescence signal forms of different wavelengths excited when exciting the fluorescence carrier particles are consistent.

[0105] (2) Fluorescence collection detection area extension design:

[0106] Dichroic mirror chain reconstruction: a fourth dichroic mirror is added on the basis of the dichroic mirror chain to form a four-stage light splitting structure:

[0107] 1. Dichroic mirror (reflecting wavelengths below 505 nm and transmitting wavelengths above 505 nm): reflecting side scatter light (<505 nm) to a PD detector, and transmitting fluorescence signals to a subsequent dichroic mirror chain.

[0108] 2. Dichroic mirror (reflecting 506-555 nm and transmitting > 555 nm): reflecting the first fluorescence channel (FITC marker, emission peak 530 nm) to PMT1, and transmitting long-wavelength fluorescence to the third dichroic mirror.

[0109] 3. Dichroic mirror (reflecting 556-655 nm and transmitting > 655 nm): reflecting the second channel (PE marker, emission peak 575 nm) to PMT2, and transmitting to the fourth dichroic mirror.

[0110] 4. Dichroic mirror (reflect 656-780 nm, transmit >680 nm): reflect the third channel (APC label, emission peak 670 nm) to PMT3, transmit the fourth channel to PMT4.

[0111] Filter configuration: narrow-band filter (bandwidth ±15 nm) is set before each PMT, for example, PMT1 is configured with 535 / 40 nm, PMT2 is configured with 585 / 40 nm, PMT3 is configured with 670 / 30 nm, and PMT4 is configured with 785 / 20 nm, to eliminate spectral cross interference.

[0112] (3) Scattered light collection optimization: the conical diaphragm of the forward scattered light collection module is designed to have a 25° collection angle, matching the detection requirements of the carrier particle size; the lateral scattered light collection lens (aspheric curvature radius 6.1 mm, diameter 9 mm) cooperates with the PD detector to represent the surface complexity of the carrier particle.

[0113] Detection method:

[0114] 1. Sample processing: the carrier particles to be tested are labeled with three kinds of fluorescent dyes (FITC, PE, APC, etc.), and a single-column carrier particle flow is formed by focusing through sheath flow technology.

[0115] 2. Signal excitation and collection: double-wavelength laser synchronously excites the carrier particles to generate forward / lateral scattered light and four-color fluorescence:

[0116] The forward scattered light is filtered by the conical diaphragm and the filter (<640 nm) and then the particle size information is obtained by the PD detector.

[0117] The lateral scattered light is reflected by the dichroic mirror and then the surface structure signal is obtained by another PD detector.

[0118] The four-color fluorescence is sequentially split by the dichroic mirror chain, and then synchronously collected by the corresponding PMT detector, and the quantitative analysis of the four-channel fluorescence intensity is realized through the signal processing unit.

[0119] After each sample detection is completed, the liquid channel needs to be cleaned multiple times with sheath liquid to improve the detection accuracy.

[0120] In the flow cytometer of the embodiment, through the cascade light splitting of the dichroic mirror chain and the narrow-band filtering technology, the fluorescence detection channel is expanded to more than three while the original modular structure is maintained, the crosstalk rate of each channel is less than 1%, the detection sensitivity is less than 100 MESF, and single-molecule level detection is realized. At the same time, the achromatic lens of the multiplexing spot shaping area ensures the spatial consistency of the fluorescence signals under different excitation wavelengths, and improves the accuracy of multi-color detection. Figure 3 The flow cytometry scatter diagram of the three items of myocardial infarction of the embodiment.

[0121] [Concentration calculation]

[0122] cTnI, CK-MB use single molecule detection mode, signal processing according to the intelligent switching digital algorithm and analog algorithm, the specific content of this paper. Myo using liquid chip method detection form, data processing directly use analog algorithm. Using the calibration curve obtained by calibration, iMatrix Fusion analysis software will "1" carrier particle ratio or fluorescence intensity into the curve to calculate the corresponding target concentration in the sample to be tested.

[0123] <Performance evaluation>

[0124] 1. Linear range

[0125] From the detection results of this example, the detection range of cTnI is 0.08 pg / mL~100 pg / mL, the detection range of CK-MB is 0.02 ng / mL~100 ng / mL, and the detection range of Myo is 21 ng / mL~3000 ng / mL. The linear coefficient r is greater than 0.9900 in this interval.

[0126] 2. Precision evaluation

[0127] The precision reference was detected 10 times in parallel, and the variation coefficient (CV) of the determination results was calculated to be ≤10%.

[0128] 3. Specificity evaluation

[0129] The sample has hemoglobin <400 mg / L, triglyceride or cholesterol <1500 mg / dL, bilirubin <5.0 mg / dL, and rheumatoid factor <1000 IU / mL, which has no effect on the detection results.

[0130] The application also provides a use of the above-mentioned cardiovascular single molecule quantitative detection reagent. The reagent is used for quantitative detection of at least one of the markers of myocardial infarction three items (cTnI, CK-MB and Myo) or S100 in blood.

[0131] The detection method and reagent of the embodiment are based on the single molecule detection method enhanced by in-situ nanometer enzyme catalysis, and the magnetic rod transfer is used to improve the number of molecules of the sample to be tested, and the flow principle is combined to detect the signal, so that the super-sensitive detection is realized. It is suitable for POCT detection, and can realize rapid result in 20 minutes when combined with whole blood pretreatment, and is suitable for early diagnosis of myocardial infarction patients in chest pain center.

[0132] Second embodiment

[0133] <Detection kit>

[0134] The markers for heart and brain organ injury detected in this embodiment are markers for neurodegenerative diseases, specifically, Aβ proteins Aβ40 and Aβ42, phosphorylated tau proteins p-Tau217 and p-Tau181, nerve injury marker neurofilament light chain protein (NfL), and glial activation marker glial fibrillary acidic protein (GFAP). The single-molecule detection kit includes carrier particles, detection functions, peroxidase conjugates, substrate solution (including substrate solution A, substrate solution B, and other substrates), calibrators, and quality control samples, as shown in Table 2:

[0135] Table 2 Main components of the detection kit

[0136]

[0137] In Table 2, different types of capture functions are labeled on the surfaces of different fluorescent carrier particles. In actual applications, they can be flexibly combined according to detection needs, for example, when only some cardiovascular markers need to be detected, a single or combined scheme of multiple cardiovascular marker probes can be selectively used. The diameter of each carrier particle is 3 μm, and the working particle number of each index carrier particle is 6000. In addition, the carrier particle mixture also contains a phosphate buffer solution of bovine serum albumin, Tween-20, and ProClin300.

[0138] The specific preparation method of the carrier particles is described above.

[0139] In the detection function, the concentration of the biotinylated detection antibody for each index is 2.0 μg / mL.

[0140] The working concentration of streptavidin conjugated with peroxidase for each index is 5.0 μg / mL.

[0141] The preparation method of the dendrimer in substrate solution A is described above. The preparation method is as follows: 10% glycerol, 0.05% Tween-20, and distilled water are mixed to a final concentration of 5 μmol / L (calculated based on tyramide biotin).

[0142] The volume of substrate solution B is not less than 240 ul.

[0143] In other substrates, the preparation method of the fluorescently labeled streptavidin is as follows: the following components with a certain weight percentage are mixed: 0.5 ~ 3% bovine serum albumin, 1 ~ 10% trehalose, 0.05 ~ 0.2% Tween-20, 0.06 ~ 0.3% Proclin300, and then a certain proportion of 0.1 M phosphate buffer (pH = 7 ~ 8) is added to prepare 2.0 μg / mL for each index.

[0144] Calibrators are Aβ40, Aβ42, p-tau217, p-tau181, NfL and GFAP calibrators, the main component is protein, and the calibrator is composed of two low, medium and high concentration calibrators.

[0145] Quality control is Aβ40, Aβ42, p-tau217, p-tau181, NfL and GFAP quality control, the main component is protein, and the quality control is achieved by three proteins containing zero, low, medium concentration for internal quality control.

[0146] <Signal detection process>

[0147] 1. Detection operation process

[0148] The following magnetic attraction magnetic ball separation method is used. The specific detection steps are as follows:

[0149] (1) Add the sample to be tested (or calibrators and quality control), reaction buffer and carrier particle mixture into the reaction tube, stir and mix uniformly, and then react at 37℃. Then add detection function to the reaction tube, stir and mix uniformly, and then continue to react at 37℃.

[0150] (2) After the reaction is completed, magnetic separation is performed, the supernatant is discarded, and washing buffer is added to the reaction tube, stirred and mixed, and then magnetic separation is performed, and the supernatant is discarded again; Repeat the washing step for a total of three times.

[0151] (3) Add peroxidase conjugate to the reaction tube, stir and mix, and then react at 37℃; wash five times according to the operation of step (2).

[0152] (4) Add substrate solution A and substrate solution B to the reaction tube, stir and mix, and then react at 37℃; wash three times according to the operation of step (2).

[0153] (5) Then add other substrates to the reaction tube, stir and mix, and then react at 37℃; wash three times according to the operation of step (2).

[0154] (6) Add an equal volume of detection solution to the reaction tube, stir and mix, and place the reaction tube in a liquid chip analyzer for single molecule reading;

[0155] <Planar imaging detector detection and detection method>

[0156] The solid-state label enriched on the surface of the carrier is introduced into the planar imager in a fluid manner for detection. The detection does not include micro-reaction cavities with a volume of nanoliter level, picoliter level or femtoliter level.

[0157] Based on the multispectral fluorescence imaging technology, the carrier particles are excited by two LED light sources (such as 488 nm and 638 nm) on the flat and smooth surface of the solid support in turn, and the fluorescence signals of the carrier particles in the static state are captured by a high-resolution CMOS camera or a CCD camera. The encoding channel (such as 530 nm) identifies the identity of the carrier particles, and the detection channel (such as 670 nm) quantifies the binding amount of the target. The image segmentation algorithm is combined to extract the fluorescence intensity of each channel, and the image is analyzed by clustering according to the encoding channel to decode the identity of the carrier particles, and then the pixel intensity extracted under the report channel of each cluster carrier particle is output respectively. The pixel intensity of each carrier particle is obtained, and finally statistical calculation is performed to realize multiple detection and complete digital detection of the number of single molecules in the target.

[0158] The optical imaging device of the planar imaging detector includes a light source and an optical signal acquisition unit, and the detection system does not include a total internal reflection microscope, a near-field microscope and an Airy spot focusing detection device. Low-cost single molecule detection is achieved.

[0159] The signal processing is intelligently switched between digital algorithm and analog algorithm according to the fluorescence intensity, and the specific method is described above. The calibration curve is obtained by using the calibrators, and the analysis software of iMatrix Fusion brings the “1” carrier particle ratio or the fluorescence intensity into the curve to calculate the corresponding target concentration in the sample to be detected.

[0160] <Performance evaluation>

[0161] 1. Linear range

[0162] The negative sample is detected in parallel for 20 times, and the average value plus 2 times the standard deviation is taken as the determination result. The lowest detection limit of the neurodegenerative disease marker in this embodiment is Aβ40 of 0.91 pg / mL, Aβ42 of 0.76 pg / mL, p-tau217 of 0.53 pg / mL, p-tau181 of 0.46 pg / mL, NfL of 1.51 pg / mL and GFAP of 12.39 pg / mL. The lowest detection limit of IL-6 of the cytokine in this embodiment is 9.73 fg / mL.

[0163] 2. Precision evaluation

[0164] The precision reference is detected in parallel for 10 times, and the coefficient of variation (CV) ≤ 10% is calculated.

[0165] 3. Specificity evaluation

[0166] The sample has hemoglobin <400 mg / L, triglyceride or cholesterol <1500 mg / dL, bilirubin <5.0 mg / dL, rheumatoid factor <1000 IU / mL, and does not affect the test results.

[0167] The embodiment also provides an application of the above-mentioned Alzheimer's disease quantitative detection reagent. The reagent is used for quantitative detection of at least one of the markers of Aβ40, Aβ42, p-tau217, p-tau181, NfL and GFAP in peripheral blood. The detection method and reagent are based on a single-molecule detection method of enzyme catalytic deposition fluorescence and cascade signal amplification, adopt a conventional mechanical arm sample adding module, and combine imaging principle detection signal to realize single-molecule immunodetection method fg-level hypersensitive detection. The detection system has very low cost and is suitable for medical institutions to carry out popular early screening of Alzheimer's disease.

[0168] Third embodiment

[0169] Compared with the first embodiment, the embodiment is based on a single-molecule quantitative detection system of cascade signal amplification, and can detect target molecules including proteins and nucleic acids. The nucleic acid detection reagent includes a capture functional substance, a mixed solution of carrier particles coated with different capture functional substances, which can specifically bind to different first sequences to be detected; a detection functional substance, a mixed solution of different detection probes combined with different second sequences, the detection probes being directly or indirectly combined with a label; through in-situ nano-enzyme catalytic enrichment, the binding on the surface of the carrier particles reacts with the substrate solution to generate photons; finally, an optical detection device is used to collect and report.

[0170] The above is only an embodiment of the present application and does not limit the protection scope of the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A test kit comprising, It comprises: carrier particles, the carrier particles having magnetism, and the surface of the carrier particles being coated with capture functionals capable of specifically binding to target materials, each of the carrier particles being provided with fluorescent dots, and the fluorescent dots of the same color corresponding to the same capture functionals; detection functionals capable of specifically binding to the target materials; peroxidase conjugates, the peroxidase conjugates being peroxidase conjugated with streptavidin, the peroxidase being dextran-conjugated horseradish peroxidase; substrate liquids, the substrate liquids comprising substrate liquid A and substrate liquid B, the substrate liquid A comprising dendrimers, the dendrimers being labeled with a plurality of fluoresceins, and the dendrimers being covalently conjugated with a plurality of tyramine molecules; wherein the dendrimers are branched chain DNA polymers, and the fluoresceins are Alexa Fluor 647; the substrate liquid B comprising 0.001% to 0.005% by volume of hydrogen peroxide.

2. The test kit according to claim 1, characterized in that, The number of the carrier particles corresponding to each of the capture functionals is 5000 to 50000.

3. The test kit according to claim 1 or 2, characterized in that, The equivalent particle diameter of the carrier particles is 0.5 to 8 μm.

4. A marker detection system characterized by, It comprises a data acquisition module, an algorithm processing module and the detection kit of any one of claims 1 to 3, the detection kit being used for detecting markers, the data acquisition module being used for acquiring detection data of the detection kit, and the algorithm processing module being used for analyzing the detection data and outputting contents.

5. Use of the detection kit of any one of claims 1 to 3 in the preparation of a detection kit for detecting markers of heart and brain organ injury.

Images