System, method and application of multi-index digital detection sample

Abstract

The invention discloses a multi-index digital sample detection system and method and application, the system comprises a reaction pore plate, a capture biological ligand mixture, at least two detection biological ligands, a microscopic imaging system and an image processing system, the reaction pore plate is coated with the capture biological ligand mixture, the detection biological ligands are coupled with labeled particles, and the labeled particles are coupled with the detection biological ligands. The detection biological ligands are distinguished by marking particles with different spectral characteristics, analytes to be detected in a detection sample and the corresponding capture biological ligands, the detection biological ligands form a three-layer biological ligand compound on the reaction pore plate, and the microscopic imaging system is used for acquiring a fluorescence image of the reaction pore plate; and the image processing system is used for distinguishing the marked particles with different spectral characteristics in the microscopic imaging system and respectively counting the number of the marked particles. According to the multi-index digital sample detection system, the common reaction pore plate is combined with the microscopic imaging system and the image processing system to complete single molecule counting detection, and complex equipment is not needed.

Description

Technical Field

[0001] The present invention belongs to the field of bioanalysis and detection, and particularly relates to a system, method and application for detecting samples with multiple indicators numerically. Background Art

[0002] Multiple indicator detection is of great significance in clinical diagnosis. By simultaneously detecting multiple biomarkers, the accuracy of disease diagnosis can be improved, and early screening and precise diagnosis of diseases can be achieved. Digital detection is a technology that quantifies by counting the number of specifically bound or reacted molecules, such as digital PCR and digital immunoassay techniques. When the detection object is a single molecule, it is also called single molecule counting immunoassay. The classic single molecule immunoassay is digital ELISA based on the single molecule array (Simoa) system. Through the micro-well technology, single protein molecules are separated in femtoliter reaction chambers, so as to generate signals at a local high concentration for single molecule counting.

[0003] In recent years, some teams have made explorations and research on the multiple indicator digital detection system. The team of Zhen Wu from Wuhan University proposed a digital immunoassay technology based on fluorescent magnetic multifunctional nanospheres. By assembling magnetic nanoparticles and quantum dots with different emission wavelengths on the surface of copolymer nanospheres, and then binding antibodies against H9N2, H1N1 and H7N9 avian influenza viruses to green, yellow and red fluorescent magnetic nanospheres respectively, the nanospheres are dropped into polymer micro-wells, and digital analysis is carried out under a fluorescence microscope to count the number of positive micro-wells, realizing ultrasensitive digital detection of multiple viruses.

[0004] Connie Wu et al. proposed a digital ELISA platform called MOSAIC. The target protein is bound to antibody-coated magnetic microbeads, and then biotinylated detection antibodies are added. A streptavidin-DNA primer template conjugate is used for labeling, and fluorescence-labeled DNA probes are amplified and hybridized by rolling circle amplification, so as to generate strong fluorescence signals on each "positive" microbead. By using microbeads encoded with different fluorescent dyes and differentiating the microbeads with a flow cytometer, multiple target proteins can be detected simultaneously in a single sample. Stephanie J. Zhang et al. proposed a multiple digital immunoassay method based on the barcoded MOSAIC platform. The related principle is similar to the above, except that the detection antibodies are labeled with DNA barcodes - a specific primer sequence. By using different barcode and fluorescence probe combinations, the barcoded MOSAIC platform can be extended to the multiple detection of 8 or more targets.

[0005] Existing multiplex detection technologies all have certain drawbacks, such as unstable detection systems, high operation difficulty, limited sensitivity, and many other problems. Therefore, developing a method for digital detection of samples with multiple indicators that is simple, low-cost, highly sensitive, and easy to implement has important practical significance for the development of the field of simultaneous detection of multiple indicators. Summary of the Invention

[0006] To solve at least one of the problems existing in the prior art described above, especially the problems of unstable detection system and high operation difficulty, the present invention proposes a system for digital detection of samples with multiple indicators, which couples labeled particles with different spectral characteristics to detection bioligands, captures bioligands, and specifically binds the detection bioligands to the analyte to be detected, constructs a three-layer immune complex sandwich structure, uses a microscopic imaging system to obtain fluorescence or dark-field microscopic images, and combines with an image processing system to count the labeled particles with different spectral characteristics, so as to achieve the purpose of simultaneously detecting the concentrations of multiple analytes. For this purpose, the present invention also provides a method for digital detection of samples with multiple indicators. In addition, the present invention also provides the application of the system for digital detection of samples with multiple indicators in detecting interleukin-6, procalcitonin or nucleic acid molecules.

[0007] In the first aspect of the present invention, there is provided a system for digital detection of samples with multiple indicators, including a reaction system, a microscopic imaging system, and an image processing system.

[0008] The reaction system includes a reaction well plate, a capture bioligand mixture, and at least two detection bioligands. The analyte to be detected in the sample forms a three-layer bioligand complex with the corresponding capture bioligand and detection bioligand on the reaction well plate. The detection bioligand is conjugated with labeled particles, and the detection bioligands are distinguished by labeled particles with different spectral characteristics.

[0009] The microscopic imaging system is used to obtain microscopic images of the reaction well plate.

[0010] The image processing system is used to distinguish the labeled particles with different spectral characteristics in the microscopic image and count the number of labeled particles respectively.

[0011] Among them, the reaction well plate is a 48-well plate, a 96-well plate or a 384-well plate.

[0012] The reaction well plate is a microplate commonly used in the biological field, such as a 48-well plate, a 96-well plate, or a 384-well plate.

[0013] Among them, the capture bioligand or the detection bioligand is one or a combination of antigens, antibodies, nucleic acid aptamers, biotin, streptavidin, etc.

[0014] Among them, the labeled particles are fluorescent nanoparticles or plasmonic nanoparticles.

[0015] Preferably, the fluorescent nanoparticles are one or a combination of more than one of time-resolved fluorescent nanoparticles, organic fluorescent dye nanoparticles, fluorescent quantum dot particles, aggregation-induced emission nanoparticles, and upconversion luminescent nanoparticles.

[0016] Preferably, the plasmonic nanoparticles are one or a combination of more than one of gold, silver, palladium, and copper nanoparticles.

[0017] Among them, the particle size of the labeled particles is 10 - 500 nm.

[0018] Among them, the microscopic imaging system is a fluorescence microscopic imaging system, a dark-field microscopic imaging system, or an integrated system of the two.

[0019] In the second aspect of the present invention, there is provided a method for digitally detecting a sample with multiple indicators, which is a non-disease diagnosis method. Detection is carried out using the above system, and it includes the following steps:

[0020] S1. Coating a capture bioligand mixture in a reaction well plate to form a first-layer bioligand complex, and the mixture includes at least two capture bioligands;

[0021] S2. Adding a test sample into the reaction well plate. The test sample contains at least two analytes to be detected. The analytes to be detected specifically bind to the corresponding capture bioligands to form a second-layer bioligand complex;

[0022] S3. Adding at least two detection bioligands into the reaction well plate. The detection bioligands specifically bind to the corresponding analytes to be detected to form a third-layer bioligand complex. The detection bioligands are conjugated with labeled particles, and the detection bioligands are distinguished by conjugating labeled particles with different spectral characteristics;

[0023] S4. Using a microscopic imaging system to obtain a fluorescence image of the reaction well plate, and respectively counting the number of labeled particles with different spectral characteristics through an image processing system;

[0024] S5. Calculating the concentration of the analytes to be detected in the test sample according to the fitting relationship curve between the concentration of the analyte standard product and the number of labeled particles.

[0025] Preferably, after step S1, the following step is further included: adding a protein blocking solution into the reaction well plate to block non-specific binding sites.

[0026] In the third aspect of the present invention, there is provided an application of the system for digitally detecting a sample with multiple indicators in detecting interleukin-6 (IL-6), procalcitonin (PCT), or nucleic acid molecules.

[0027] Beneficial effects:

[0028] (1) The system for multi-index digital detection of samples in the present invention combines a common reaction well plate with a microscopic imaging system and an image processing system to complete single-molecule counting detection. It requires no complex equipment, is easy to operate, and has low costs.

[0029] (2) The method for multi-index digital detection of samples in the present invention can be used for detecting single-index in samples or for simultaneously detecting multiple indexes, providing convenience for early diagnosis and screening of diseases.

[0030] (3) The method for multi-index digital detection of samples in the present invention has high sensitivity. In PCT detection, compared with a multifunctional microplate reader, the sensitivity is increased by 28 times; in a mixed sample of IL-6 and PCT, compared with a multifunctional microplate reader, the detection sensitivity of IL-6 is increased by 32 times; in a mixed sample of IL-6, PCT, and 2,4-dinitrophenyl (DNP), the detection sensitivity of IL-6 is increased by 15 times, and the detection sensitivity of PCT is increased by 7 times.

[0031] (4) The method for multi-index digital detection of samples in the present invention has a sensitivity increased by about 98 times when detecting nucleic acid molecules.

[0032] The concept, specific structure, and technical effects of the present invention will be further described below in conjunction with the drawings to fully understand the purpose, features, and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0034] Figure 1 Showing a transmission electron microscope micrograph of aggregation-induced emission (AIE) nanospheres.

[0035] Figure 2 Showing a spectrogram of AIE nanospheres.

[0036] Figure 3 Showing the fluorescence microscope images and the obtained standard curves in Example 1, where A is the fluorescence microscope image, B is the standard curve obtained by using a multifunctional microplate reader, C is the standard curve obtained by using the method of the present application, and D is the comparison of the standard curves obtained by the two methods.

[0037] Figure 4 Showing a transmission electron microscope micrograph of quantum dot nanospheres (red) in Example 2.

[0038] Figure 5 Shows the spectral diagram of the quantum dot nanospheres (red) in Example 2.

[0039] Figure 6 Shows the fluorescence microscope images and the obtained standard curves in Example 2. Among them, A is the fluorescence microscope image, B is the standard curve obtained by using a multi-functional microplate reader, C is the standard curve obtained by using the method of the present application, and D is the comparison of the standard curves obtained by the two methods.

[0040] Figure 7 Shows the fluorescence microscope images and the obtained standard curves in Example 3.

[0041] Figure 8 Shows the transmission electron microscope micrograph of the quantum dot nanospheres (green) in Example 4.

[0042] Figure 9 Shows the spectral diagram of the quantum dot nanospheres (green) in Example 4.

[0043] Figure 10 Shows the fluorescence microscope images and the obtained standard curves in Example 4. Among them, A is the comparison of the standard curves of the IL-6 reagent and the PCT reagent obtained when detected by a multi-functional microplate reader; B is the comparison of the standard curves of the IL-6 reagent and the PCT reagent obtained when detected by the method of the present application; C is the comparison of the standard curves obtained by using a multi-functional microplate reader and the method of the present application when testing the PCT reagent; D is the comparison of the standard curves obtained by using a multi-functional microplate reader and the method of the present application when testing the IL-6 reagent.

[0044] Figure 11 Shows the fluorescence microscope images and the obtained standard curves in Example 5. Detailed implementation manners

[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0046] The terms "first", "second", etc. are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first", "second" may explicitly or implicitly include at least one such feature. In the description of this application, if the term "plurality" appears, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise specifically defined.

[0047] As used herein, "comprising" and "including" cover cases where only the recited elements are present and cases where there are other unrecited elements in addition to the recited elements.

[0048] All percentages in the present invention are mass percentages, unless otherwise specified.

[0049] Multiplex detection is of great significance in clinical diagnosis. By simultaneously detecting multiple biomarkers, analyzing the contents of multiple biomarkers, and based on the content analysis results, it is determined whether subsequent tests are necessary and the specific items of the tests, which has a certain guiding role for the early diagnosis and screening of diseases.

[0050] Clinically, when the concentration of the analyte to be measured in the test sample is low, with traditional detection methods, due to strong signal interference, it is very likely that accurate detection results cannot be obtained. Single-molecule immunoassay, which uses single molecules as the detection objects, well solves the above technical problems. However, single-molecule immunoassay generally has problems of great operation difficulty and high cost.

[0051] This application provides a system for multiplex digital detection of samples, which can achieve single-molecule detection through conventional equipment, has simple operation, relatively low cost, and high sensitivity, and well solves the above technical problems.

[0052] A system for multiplex digital detection of samples includes a reaction system, a microscopic imaging system, and an image processing system.

[0053] The reaction system includes a reaction well plate, a capture bioligand mixture, and at least two detection bioligands. The reaction well plate is a 48-well plate, a 96-well plate, or a 384-well plate. The reaction well plate is used to coat the capture bioligand mixture, and the analyte to be measured in the test sample forms a three-layer bioligand complex with the corresponding capture bioligand and detection bioligand on the reaction well plate.

[0054] For the convenience of subsequent distinction, the detection bioligands are conjugated with labeled particles, and different detection bioligands are conjugated with labeled particles having different spectral characteristics, so that the types of analytes to be measured can be distinguished according to the colors of the labeled particles in the microscopic image, and the concentration of the analyte to be measured can be calculated.

[0055] The labeled particles are fluorescent nanoparticles or plasmonic nanoparticles. The fluorescent nanoparticles are one or a combination of more than one of time-resolved fluorescent nanoparticles, organic fluorescent dye nanoparticles, fluorescent quantum dot particles, aggregation-induced emission nanoparticles, and upconversion luminescent nanoparticles. The plasmonic nanoparticles are one or a combination of more than one of gold, silver, palladium, and copper nanoparticles. The particle size of the labeled particles is 10 - 500 nm.

[0056] The capture bioligand or detection bioligand is one or a combination of more than one of antigen, antibody, nucleic acid aptamer, biotin, and streptavidin.

[0057] A microscopic imaging system for acquiring microscopic images of reaction well plates. The microscopic imaging system is a fluorescence microscopic imaging system, a dark field microscopic imaging system, or an integrated system of the two. A common fluorescence microscopic imaging system, such as an inverted fluorescence microscope.

[0058] An image processing system for distinguishing labeled particles with different spectral characteristics in the microscopic imaging system and separately counting the number of labeled particles. A common image processing system such as Photoshop. Manually import the images acquired by the microscopic imaging system into Photoshop, use Photoshop to distinguish particles of different colors, and count them separately.

[0059] This application also provides a method for detecting samples with multiple indicators numerically, a non-disease detection method, which uses the above system for detection, including the following steps:

[0060] S1. Coating a capture bioligand mixture in a reaction well plate to form a first-layer bioligand complex, and the mixture includes at least two capture bioligands;

[0061] S2. Adding a test sample into the reaction well plate, the test sample contains at least two analytes to be measured, and the analytes to be measured specifically bind to the corresponding capture bioligands to form a second-layer bioligand complex;

[0062] S3. Adding at least two detection bioligands into the reaction well plate, the detection bioligands specifically bind to the corresponding analytes to be measured to form a third-layer bioligand complex, the detection bioligands are conjugated with labeled particles, and the detection bioligands are distinguished by conjugating labeled particles with different spectral characteristics;

[0063] S4. Using the microscopic imaging system to acquire microscopic images of the reaction well plate, and separately counting the number of labeled particles with different spectral characteristics through the image processing system;

[0064] S5. Calculating the concentration of the analytes to be measured in the test sample according to the fitting relationship curve between the concentration of the analyte standard product and the number of labeled particles.

[0065] Preferably, after step S1, the following steps are further included: adding a protein blocking solution into the reaction well plate to block non-specific binding sites.

[0066] The above method for detecting samples with multiple index numbers may be used in disease diagnosis methods. This application excludes disease diagnosis methods and only obtains the contents of multiple indicators through the above method. By analyzing the contents of each indicator, it provides guidance for subsequent detections or examinations, such as whether further examinations are needed, rather than directly obtaining a disease diagnosis result.

[0067] Example 1: Aggregation-induced emission (AIE) nanospheres conjugates for the detection of procalcitonin (PCT)

[0068] 1. Experimental raw materials or instruments

[0069] AIE nanospheres, PCT capture antibody, PCT detection antibody, PCT recombinant protein; The analytical instruments include a multifunctional microplate reader (excitation wavelength 365 nm and emission wavelength 570 nm are used in this test process), and an inverted fluorescence microscope (10× objective lens is used in this test process).

[0070] 2. Preparation of AIE fluorescent nanospheres

[0071] A mixed solution of AIE dye (the AIE dye here is 1,1,2,2-tetrakis(4-(diphenylamino)phenyl)ethylene, and other tetraphenylethylene derivatives can also be selected) (8 mg / mL) and polystyrene maleic anhydride copolymer (number average molecular weight ~28000) (8 mg / mL) prepared with chloroform is used as the oil phase. Take 1 mL of the oil phase and mix it with 2 mL of dodecyltrimethylammonium bromide aqueous phase (15 mg / mL), and shake well. Volatilize the chloroform solvent at room temperature or under reduced pressure to obtain AIE fluorescent nanospheres.

[0072] 3. Material characterization of AIE fluorescent nanospheres

[0073] Characterize the obtained AIE fluorescent nanospheres by transmission electron microscopy, and the results are as Figure 1 shown. As can be seen from Figure 1 , the morphology and size of the AIE fluorescent nanospheres are uniform, the average particle size is about 179 nm, and the dispersion state is good. The fluorescence spectrum measurement data is as Figure 2 shown, and there is a fluorescence emission peak at 570 nm.

[0074] 4. Conjugation of AIE fluorescent nanospheres with detection antibodies

[0075] 1) Dilute 100 μL of the as-prepared AIE nanospheres with carboxyl groups on the surface (emission wavelength: 570 nm, shown as yellow in fluorescence microscopy images) to 300 μL with phosphate buffer at pH 6.0. Add 0.3 mg of the activator 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), place it on a rotary mixer, and react at room temperature for 0.5 h.

[0076] 2) After activation of the carboxyl nanospheres, centrifuge at 12,000 rpm for 10 min. After removing the supernatant, disperse the activated AIE nanospheres in 300 μL of phosphate buffer, add approximately 100 μg of the PCT detection antibody, place it on a rotary mixer, and react at room temperature for 1 h.

[0077] 3) After the coupling reaction of the PCT detection antibody and the quantum dot nanospheres, centrifuge at 12,000 rpm for 10 min. After removing the supernatant, disperse the antibody-AIE nanospheres in 300 μL of phosphate buffer, add 10 mg of BSA (bovine serum albumin), place it on a rotary mixer, and perform a blocking reaction at room temperature for 2 h to obtain the PCT detection antibody conjugated to the AIE nanospheres.

[0078] 5. Quantitative detection of single PCT molecules

[0079] Balance all reagents to room temperature before use.

[0080] (1) Use the antibody diluent (Sangon Biotech (Shanghai) Co., Ltd., model D608501) to dilute the concentrations of the PCT antigen calibrator (PCT recombinant protein) (concentration: 1 mg / mL) to 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, 0.195, 0.0975, 0.0488, and 0 ng / mL respectively. Set 3 replicates for each gradient concentration.

[0081] (2) Dilute the PCT capture antibody (Xiamen Tongrenxin Biotechnology Co., Ltd., GR23 capture antibody) (5.4 mg / mL) to 10 μg / mL with the antibody coating buffer (Shanghai Yuanye Bio-Technology Co., Ltd.), add it to a 96-well plate (100 μL per well), and incubate overnight at 4 °C.

[0082] (3) Wash the 96-well plate three times with PBST (PBS buffer containing 0.1% Tween), discard the residual liquid, and pat dry the 96-well plate. Add 200 μL of the blocking solution (2% bovine serum albumin) to each well for blocking for 30 min, then wash three times with PBST and pat dry.

[0083] (4) Add 100 μL of the calibrated products with various concentrations obtained in step (1) and serially diluted into each well of the 96-well plate, and incubate at 37 °C for 2 h. Wash three times with PBST and pat dry.

[0084] (5) Add 100 μL of the PCT detection antibody conjugated with AIE nanospheres (the PCT detection antibody conjugated with AIE nanospheres is simply referred to as the PCT detection antibody conjugate, the concentration of the PCT detection antibody conjugate is 10 μg / mL, the original concentration is 2.5 mg / mL, diluted with the antibody diluent, the PCT detection antibody is purchased from Xiamen Tongrenxin Biotechnology Co., Ltd., GR24-labeled antibody) into each well, and incubate at room temperature for 2 h. Wash three times with PBST and pat dry.

[0085] (6) Use a Leica inverted fluorescence microscope, under ultraviolet excitation, use a 10X objective lens, take a microscopic photo of the bottom of the microplate (the above-mentioned 96-well plate), and obtain a fluorescence image as Figure 3 (A) shown, and then perform single molecule counting statistics and analysis. To highlight the advantages of digital single molecule detection in this detection method, at the same time use a multifunctional microplate reader to immediately measure the fluorescence intensity of each well, the excitation wavelength is 365 nm, and the emission wavelength is 620 nm.

[0086] (7) According to the detection results, count and take the average value, and draw a standard curve as Figure 3 (B, C, D), and calculate the LOD value.

[0087] The above standard curve is a four-parameter fitting (4PL) curve. Use the four-parameter fitting (4PL) curve to analyze the relationship between the fluorescence intensity or the number of fluorescent particles and the concentration, and calculate the LOD value (LOD, which is the signal of the blank well plus three times the standard deviation of the blank group).

[0088] 6. Detection Results

[0089] As Figure 3 shown, the LOD value, that is, the limit of blank, is used to determine the detection limit of the method of this application and the detection method of the multifunctional microplate reader through the value of the limit of blank. When detected by the multifunctional microplate reader, the calculated limit of blank is 691.7 pg / mL; while using the method of this application, the obtained limit of blank is 24.9 pg / mL. It can be seen that the sensitivity of the method of this application can be increased by about 28 times.

[0090] Example 2: Detection of Multiple Indicators (IL-6 and PCT)

[0091] The inventor used quantum dot nanospheres conjugated with an IL-6 (Interleukin-6) detection antibody (Haitai Biotechnology (Shanghai) Co., Ltd., IL6-L395), and AIE nanospheres conjugated with a PCT detection antibody to detect IL-6 and PCT respectively in the same well plate, verifying single molecule counting detection of multiple indicators. The preparation methods of the quantum dot nanospheres conjugated with the IL-6 (Interleukin-6) detection antibody and the AIE nanospheres conjugated with the PCT detection antibody are the same as those in Example 1.

[0092] The quantum dot nanospheres (QBC620x) were provided by Shanghai Sai Fanke Separation Technology Co., Ltd. and characterized by transmission electron microscopy. The results are as Figure 4 shown. It can be seen from Figure 4 that the morphology and size of the quantum dot nanospheres are uniform, the particle size distribution is about 190 nm, and the dispersion state is good. The fluorescence spectrum of the quantum dot nanospheres is as Figure 5 shown. Observed under an inverted fluorescence microscope, the quantum dot nanospheres (QBC620x) appear red.

[0093] 1. Detecting mixed samples of gradient concentration IL-6 and constant concentration PCT

[0094] (1) A series of gradient concentration IL-6 antigen standards were set, which were 4000, 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49, 0.25, and 0 pg / mL respectively. The specific steps are similar to those in Example 1 and will not be elaborated here in detail.

[0095] (2) Replace the PCT capture antibody in Example 1 with a mixture of a PCT capture antibody and an IL-6 capture antibody (Haitai Biotechnology (Shanghai) Co., Ltd., IL6-L152), and replace the AIE nanospheres conjugated with the PCT detection antibody with a mixture of the AIE nanospheres conjugated with the PCT detection antibody (PCT detection antibody conjugate) and the quantum dot conjugated IL-6 detection antibody (IL-6 detection antibody conjugate). The specific concentrations used are shown in Table 1. The specific detection method is the same as that in Example 1.

[0096] The experimental results are as Figure 6 shown. When detected by a multifunctional microplate reader, the calculated limit of blank is 118.77 pg / mL; while using the method of the present application, the obtained limit of blank is 3.65 pg / mL. It can be seen that the sensitivity of the method of the present application can be increased by about 32 times.

[0097] Table 1

[0098] IL-6 PCT Capture antibody 1.25 μg / mL 2.5 μg / mL Sample Gradient concentration 2.5 ng / mL Detection antibody conjugate 10 μg / mL 10 μg / mL

[0099] 2. Detect the mixed samples of gradient concentration PCT and constant concentration IL-6

[0100] Set a series of PCT standards with gradient concentrations, namely 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.20, 0.10, 0.05, 0.02, 0.01, and 0 ng / mL. The specific steps are similar to those in Example 1 and will not be elaborated here in detail.

[0101] Replace the PCT capture antibody in Example 1 with a mixture of PCT capture antibody and IL-6 capture antibody, and replace the PCT detection antibody conjugated with AIE nanospheres with a mixture of PCT detection antibody conjugated with AIE nanospheres and IL-6 detection antibody conjugated with quantum dots. The specific concentrations used are shown in Table 2. The specific detection method is the same as that in Example 1.

[0102] The detection results are as Figure 7 shown. When detected by a multifunctional microplate reader, the calculated limit of blank is 49.29 pg / mL; while using the method of the present application, the calculated limit of blank is 13.22 pg / mL. It can be seen that the sensitivity can be increased by about 3.7 times by using the method of the present application.

[0103] Table 2

[0104] PCT IL-6 Capture antibody 2.5 μg / mL 1.25 μg / mL Sample Gradient concentration 100 pg / mL Detection antibody conjugate 10 μg / mL 10 μg / mL

[0105] Example 3: Detection of multiple indicators (IL-6, PCT, and DNP)

[0106] 1. Experimental raw materials or instruments

[0107] 2,4-dinitrophenyl-conjugated bovine serum albumin (DNP-BSA) (Xiamen Tongrenxin Biotechnology Co., Ltd., ZKC11), quantum dot nanospheres (Shanghai Sai Fanke Separation Technology Co., Ltd., QBC520), DNP detection antibody (Xiamen Tongrenxin Biotechnology Co., Ltd., ZKC21); the rest are the same as in Example 2.

[0108] 2. Material characterization of quantum dot nanospheres (QBC520x)

[0109] The transmission electron microscope test image of the quantum dot nanospheres applied in this example is as Figure 8 shown. It can be seen from Figure 8 that the morphology and size of the quantum dot nanospheres are uniform, the average particle size is about 78 nm, and the dispersion state is good. The fluorescence spectrum measurement data is as Figure 9 shown. Observed under a fluorescence microscope, the quantum dot nanospheres appear green.

[0110] The preparation methods of the IL-6 detection antibody conjugated with quantum dot nanospheres, the PCT detection antibody conjugated with AIE nanospheres, and the DNP detection antibody conjugated with quantum dot nanospheres are the same as those in Example 1.

[0111] 3. Multiplex detection of IL-6, PCT, and quality control DNP

[0112] A series of calibration standards with gradient concentrations of IL-6 and PCT are set up. The concentrations of IL-6 are 1000, 250, 62.5, 31.25, 7.81, 3.61, 1.95, 0.98, and 0.24 pg / mL respectively. The concentrations of PCT calibration standards are 100, 25, 12.5, 6.25, 3.13, 0.78, 0.39, 0.098, and 0.024 ng / mL respectively. They are mixed in the order of decreasing IL-6 concentration and increasing PCT calibration standard concentration to obtain 9 groups of mixed samples of IL-6, PCT, and DNP.

[0113] Replace the PCT capture antibody in Example 1 with a mixture of PCT capture antibody, IL-6 capture antibody, and DNP-BSA. Replace the PCT detection antibody conjugated with AIE nanospheres with a mixture of PCT detection antibody conjugated with AIE nanospheres (PCT detection antibody conjugate), IL-6 detection antibody conjugated with quantum dot nanospheres (QBC620x) (IL-6 detection antibody conjugate), and DNP detection antibody conjugated with quantum dot nanospheres (QBC520x) (DNP detection antibody conjugate). The specific concentrations used are shown in Table 3, and the specific detection method is the same as that in Example 1.

[0114] The experimental results are as Figure 10 shown. When detected using a multifunctional microplate reader, the calculated limit of blank for the IL-6 reagent is 24.73 pg / mL; while using the method of the present application, the calculated limit of blank for the IL-6 reagent is 1.69 pg / mL. It can be seen that the sensitivity can be increased by about 15 times using the method of the present application. When detected using a multifunctional microplate reader, the calculated limit of blank for the PCT reagent is 384.03 ng / ml; while using the method of the present application, the calculated limit of blank for the PCT reagent is 56.69 pg / mL. It can be seen that the sensitivity can be increased by about 7 times using the method of the present application.

[0115] Table 3

[0116]

[0117] Example 4: Detection of nucleic acid molecules using quantum dot nanosphere conjugates

[0118] 1. Experimental raw materials or instruments

[0119] Quantum dot nanospheres (Shanghai Sai Fanke Separation Technology Co., Ltd., QBC620x), FAM detection antibody (Chongqing Tansheng Technology Co., Ltd., FAB-C001-2A1), streptavidin (Sangon Biotech (Shanghai) Co., Ltd., A610492), single-stranded DNA (with biotin and fluorescein tags at both ends) (purchased from Shanghai Tulugang Biotechnology Co., Ltd., model number CAT#31201); the rest is the same as in Example 1.

[0120] 2. Material Characterization of Quantum Dot Nanospheres

[0121] The same as in Example 2.

[0122] The applicant used quantum dot nanospheres conjugated with fluorescein (FAM) detection antibody to detect single-stranded DNA. Specifically, streptavidin was coated on the 96-well plate in Example 1, quantum dot nanospheres conjugated with fluorescein detection antibody (fluorescein detection antibody conjugate), and single-stranded DNA was used as a sample for detection.

[0123] The use concentration of streptavidin here is 2 μg / mL, and the concentration of fluorescein detection antibody conjugate is 10 μg / mL (the preparation method is the same as in Example 1).

[0124] Experiments were carried out similarly to Example 1, and the experimental results are as Figure 11 shown. When detected by a multifunctional microplate reader, the calculated blank limit of the reagent is 1.08 pM (picomole); while when using the method of this application, the calculated blank limit of the reagent is 0.011 pM. It can be seen that by using the method of this application, the sensitivity can be increased by about 98 times.

[0125] The above-described embodiments only represent several implementation manners of the present invention, and the description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A system for detecting samples of multi-index numbers, characterized in that It includes a reaction system, a microscopic imaging system and an image processing system. The reaction system includes a reaction well plate, a capture bioligand mixture and at least two detection bioligands. The analyte to be measured in the test sample forms a three-layer bioligand complex with the corresponding capture bioligand and detection bioligand on the reaction well plate. The detection bioligand is conjugated with labeled particles, and the detection bioligands are distinguished by labeled particles with different spectral characteristics. The microscopic imaging system is used to obtain the microscopic image of the reaction well plate. The image processing system is used to distinguish the labeled particles with different spectral characteristics in the microscopic image and count the number of labeled particles respectively.

2. The system for detecting a sample with multiple index numbers according to claim 1, characterized in that The reaction well plate is a 48-well plate, a 96-well plate or a 384-well plate.

3. The system for detecting samples with multiple index numbers according to claim 1, characterized in that The capture bioligand or the detection bioligand is one or a combination of more of antigen, antibody, nucleic acid aptamer, biotin, streptavidin.

4. The system for detecting samples with multiple index numbers according to claim 1, characterized in that, The labeled particles are fluorescent nanoparticles or plasmonic nanoparticles.

5. The system for detecting a sample with multiple index numbers according to claim 4, wherein The fluorescent nanoparticles are one or a combination of more of time-resolved fluorescent nanoparticles, organic fluorescent dye nanoparticles, fluorescent quantum dot particles, aggregation-induced emission nanoparticles, upconversion luminescent nanoparticles.

6. The system for detecting samples with multiple index numbers according to claim 4, wherein, The plasmonic nanoparticles are one or a combination of more of gold, silver, palladium, copper nanoparticles.

7. The system for detecting samples with multiple index numbers according to claim 1, characterized in that, The particle size of the labeled particles is 10 - 500 nm.

8. A method for detecting samples of multiple-index numbers, which is not a method for diagnosing diseases, using the system according to any one of claims 1-7, characterized in that, It includes the following steps: S1. Coating the capture bioligand mixture in the reaction well plate to form the first-layer bioligand complex, and the mixture includes at least two capture bioligands. S2. Adding the test sample into the reaction well plate. The test sample contains at least two analytes to be measured, and the analytes to be measured specifically bind to the corresponding capture bioligands to form the second-layer bioligand complex. S3. Adding at least two detection bioligands into the reaction well plate. The detection bioligands specifically bind to the corresponding analytes to be measured to form the third-layer bioligand complex. The detection bioligand is conjugated with labeled particles, and the detection bioligands are distinguished by conjugated labeled particles with different spectral characteristics. S4. Using the microscopic imaging system to obtain the microscopic image of the reaction well plate, and counting the number of labeled particles with different spectral characteristics through the image processing system. S5. Calculating the concentration of the analyte to be measured in the test sample according to the fitting relationship curve between the concentration of the analyte standard product and the number of labeled particles.

9. The method for detecting a sample with multiple index numbers according to claim 8, wherein After step S1, the following step is also included: adding a protein blocking solution into the reaction well plate to block non-specific binding sites.

10. Application of the system for multiplex digital detection of samples in detecting interleukin-6, procalcitonin or nucleic acid molecules.

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