Preparation method of fluorescent microspheres, preparation method of fluorescent microspheres and preparation method of kit

Abstract

The invention relates to the technical field of detection kits, in particular to a preparation method of fluorescent microspheres, the fluorescent microspheres and a preparation method of a kit. According to the invention, 4-vinylbenzoic acid, dibenzoylmethane and phenanthroline are used as ligands to prepare the europium complex; the prepared europium complex, styrene and lauryl methacrylate are subjected to first-stage copolymerization polymerization to obtain a polymer core with the europium complex, itaconic acid is added for continuous polymerization in the second-stage polymerization process, and the prepared fluorescent microspheres are uniform in size, high in fluorescence intensity and high in stability. The prepared detection kit has the advantages of high detection sensitivity and stable and reliable detection effect when being used for detecting the myocardial injury marker cTnI.

Description

Technical Field

[0001] The present invention relates to the technical field of detection kits, and in particular to a method for preparing fluorescent microspheres, fluorescent microspheres and a kit. Background Art

[0002] Acute myocardial infarction (AMI) is myocardial necrosis caused by acute, persistent ischemia and hypoxia in the coronary arteries. Medically, the "golden window" for emergency treatment is within six hours of onset. Beyond this window, treatment effectiveness is significantly reduced, and serious, life-threatening consequences may occur. Therefore, only by accurately diagnosing the disease as quickly as possible can early detection and treatment be achieved, minimizing disability and mortality, and improving patient prognosis and quality of life.

[0003] Time-resolved fluorescence immunoassay (TRFIA) uses lanthanide elements as tracers to label antigens or antibodies. The antigen and antibody in the reaction system form an immune complex, and the fluorescence intensity of this complex is measured using a time-resolved fluorometer to determine the analyte content. Compared to radioimmunoassay (RIA), enzyme immunoassay (EIA), chemiluminescence immunoassay (CLIA), and electrochemiluminescence immunoassay (ECLIA), TRFIA offers advantages such as high sensitivity, ease of operation, stable tracers, a wide standard curve range, immunity to interference from natural sample fluorescence, and the absence of radioactive contamination. It has become one of the most promising analytical methods in biomedical research and clinical ultra-micro biochemical testing.

[0004] Troponin I (cTnI) is a highly specific marker for diagnosing acute myocardial infarction. cTnI appears in the blood 3 to 6 hours after the onset of acute myocardial infarction and reaches a peak at 14 to 20 hours. Therefore, the development of a TRFIA kit for cTnI with a low detection limit is very important for the early diagnosis of acute myocardial infarction. The preparation of europium-labeled carboxyl polystyrene fluorescent microspheres and the preparation of a suitable kit are critical to the sensitivity of the cTnI detection TRFIA kit. The performance of the fluorescent microspheres, especially the fluorescence intensity, is one of the important factors determining the sensitivity of TRFIA detection. The strong fluorescence stability of the microspheres can avoid the loss of fluorescent substances during storage and transportation, and prevent the leakage of fluorescent substances from interfering with immune binding. Based on this, the present application aims to study the preparation method of carboxyl polystyrene fluorescent microspheres and a TRFIA kit for cTnI, so as to obtain fluorescent microspheres with high stability and fluorescence intensity, and to prepare a TRFIA kit for cTnI with high detection reliability and high sensitivity. Summary of the Invention

[0005] To address the shortcomings of the prior art, the present invention provides a method for preparing fluorescent microspheres, as well as methods for preparing fluorescent microspheres and a kit. The prepared fluorescent microspheres have uniform size, high fluorescence intensity, and strong stability. The prepared TRFIA kit for troponin I has the advantages of high sensitivity, stable, and reliable detection for the detection of cTnI, a marker of myocardial injury.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The present application provides a method for preparing fluorescent microspheres, comprising the following steps: S1. Dissolve EuCl3·6H2O in ethanol to prepare a europium precursor solution, dissolve dibenzoylmethane, o-phenanthroline, and 4-vinylbenzoic acid in ethanol to prepare a ligand solution, add the europium precursor solution to the ligand solution, adjust the pH to 6.5-7 after the addition, react at 55°C-65°C for 4-6 hours, filter, wash, and dry to obtain a europium complex; wherein the mass ratio of EuCl3·6H2O, dibenzoylmethane, o-phenanthroline, and 4-vinylbenzoic acid is 1:(1.5-2):(0.4-0.6):(0.3-0.5); S2. Add styrene, lauryl methacrylate, europium complex and anionic surfactant into water, introduce nitrogen bubbling for deoxygenation, add persulfate initiator, raise the temperature to 70-80°C, keep stirring and react for 3-5 hours to carry out the first stage polymerization; then add persulfate initiator and itaconic acid aqueous solution, continue stirring and react at the same temperature for 7-10 hours to carry out the second stage polymerization, cool to room temperature after the reaction, wash the product by centrifugation, and resuspend in deionized water to obtain; wherein, the mass ratio of styrene, lauryl methacrylate and europium complex is 1: (0.08-0.12): (0.09-0.11), and the itaconic acid in the itaconic acid aqueous solution is 4%-6% of the mass of styrene.

[0007] The preparation method of the fluorescent microspheres provided in the present application comprises the following steps: firstly, a europium precursor solution is prepared by dissolving EuCl3·6H2O in ethanol; then, 4-vinylbenzoic acid having a double bond, a phenyl group, and a carboxyl group, as well as dibenzoylmethane and o-phenanthroline are used as ligands to react in an ethanol solvent to obtain a europium complex having a terminal double bond; the results show that the 4-vinylbenzoic acid ligand can improve the fluorescence intensity and stability of the prepared fluorescent microspheres compared with acrylic acid; then, the prepared europium complex is reacted with styrene and lauryl methacrylate in the presence of a persulfate initiator and an anionic surfactant. A first-stage copolymerization is carried out to obtain a polymer core with a europium complex. After copolymerization and embedding, europium reduces leakage of fluorescent substances. Lauryl methacrylate as a comonomer can increase the hydrophobicity of the polymer core and reduce the penetration of water molecules. The long fatty chain gives the microspheres flexibility and reduces the risk of microsphere rupture. During the second-stage polymerization process, itaconic acid is added to continue the polymerization. On the one hand, the rich carboxyl functional groups of itaconic acid are utilized for cTNI antibody coupling. On the other hand, the polymer core with the europium complex is further embedded to reduce the leakage of europium fluorescent substances. The prepared fluorescent microspheres have uniform size, high fluorescence intensity and strong stability.

[0008] Furthermore, in S1, the concentration of the europium precursor solution is 0.02 g / ml to 0.04 g / ml; in the ligand solution, the concentration of dibenzoylmethane is 0.02 g / ml to 0.03 g / ml, the concentration of o-phenanthroline is 0.006 g / ml to 0.008 g / ml, and the concentration of 4-vinylbenzoic acid is 0.005 g / ml to 0.007 g / ml.

[0009] Furthermore, the washing method in S1 and S2 is to use ethanol and deionized water for washing 2 to 3 times respectively.

[0010] Furthermore, the anionic surfactant in S2 is selected from at least one of sodium dodecylbenzenesulfonate, sodium lauryl polyoxyethylene ether sulfate and potassium perfluorooctane sulfonate; The amount of anionic surfactant used in S2 is 8% to 12% of the weight of styrene.

[0011] Furthermore, the persulfate initiator is selected from at least one of potassium persulfate, sodium persulfate and ammonium persulfate; In the first stage polymerization process, the amount of persulfate initiator used is 0.1% to 0.5% of the weight of styrene; During the second stage polymerization, the amount of persulfate initiator used is 0.1% to 0.5% of the weight of styrene.

[0012] The present invention also provides carboxyl polystyrene fluorescent microspheres, which are prepared according to the above method. The fluorescent microspheres are used to prepare a TRFIA kit for detecting troponin I.

[0013] The present invention also provides a method for preparing a TRFIA kit for troponin I, comprising the following steps: (1) Sample pad preparation: pre-treat the glass fiber with a sample pad treatment solution to obtain a pre-treated glass fiber pad, spray the cTnI biotin-labeled antibody working solution onto the pre-treated glass fiber pad, and dry it to obtain a sample pad; (2) Preparation of conjugate pad: A cTnI fluorescent marker working solution is prepared using a fluorescent microsphere solution labeled with anti-cTnI antibody, and the cTnI fluorescent marker working solution is sprayed on the conjugate pad substrate and dried to obtain a conjugate pad; The fluorescent microsphere solution labeled with anti-cTnI antibody is obtained by diluting the fluorescent microspheres, activating the microspheres, quenching the activation, labeling the antibody, and purifying the fluorescent microspheres. (3) Preparation of NC coating membrane: Take a PVC plate as a support pad, with the adhesive side facing up, and stick the nitrocellulose membrane to the support pad. Prepare the test line coating liquid and the quality control line coating liquid respectively, and spray the test line coating liquid and the quality control line coating liquid on the test line and quality control line positions of the nitrocellulose membrane. After drying, the NC coating membrane is obtained; (4) Assembly of the test kit: Integrate the absorbent pad and the binding pad onto the support pad with the NC coating membrane, and encapsulate the test strip in a shell.

[0014] Furthermore, in step (1), the preparation method of the cTnI biotin-labeled antibody working solution is as follows: cTnI monoclonal antibody is diluted with 0.1M sodium bicarbonate buffer at pH 8.5 to prepare an antibody diluent with a concentration of 2 mg / ml, NHS-Biotin is diluted with DMSO to prepare a 10 mM biotin solution, the antibody diluent and the biotin solution are mixed according to a biotin to antibody molar ratio of 20:1, and the mixture is shaken in the dark at 4°C for 45 minutes, and a 10% stop solution of the biotin solution is added and allowed to stand at room temperature for 15 minutes to obtain a reaction solution; the reaction solution is transferred to an ultrafiltration tube, centrifuged at 4°C and 4000g for 15 minutes, the filtrate is discarded, and the reaction solution is resuspended and mixed with fresh labeling buffer to 90% of the initial volume, and the centrifugation-suspension is repeated 4 times to obtain a cTnI biotin-labeled antibody solution with a concentration of 1 mg / ml; the cTnI biotin-labeled antibody solution and the diluent are diluted at a ratio of 1:10 to obtain a cTnI biotin-labeled antibody working solution.

[0015] Furthermore, in step (2), the antibody labeling method is as follows: 0.08 mg of cTnI monoclonal antibody is added to the fluorescent microsphere solution after ultrasonic resuspending, dispersed, the centrifuge tube cap is tightly closed, mixed on a vortex mixer, and then placed on a full-temperature shaker at 25°C and a frequency of 280 rpm / min for 120 minutes. 1 mL of the labeled blocking solution is pipetted into a centrifuge tube, mixed on a vortex mixer, ultrasonically dispersed for 60 seconds, mixed on a vortex mixer for 5 seconds, and repeated 4 times. Then, the tube is placed on a full-temperature shaker at 25°C and a frequency of 280 rpm / min for 120 minutes.

[0016] Furthermore, in step (3), The test line coating solution was prepared by using a pipette to take the NC membrane coating buffer and add it to a clean centrifuge tube, then adding the streptavidin solution and mixing to obtain a test line coating solution with a streptavidin concentration of 1 mg / ml. The quality control line coating solution was prepared by using a pipette to take the NC membrane coating buffer and add it to a clean centrifuge tube, then adding rabbit anti-mouse IgG and mixing to obtain a quality control line coating solution with an antibody concentration of 1 mg / ml.

[0017] The detection principle of the TRFIA kit for troponin I provided in the present application is that the sample to be tested is combined with the immobilized biotin-labeled cTnI antibody in the sample pad to form a complex, and further combined with the fluorescent-labeled antibody in the fluorescent microspheres labeled with anti-cTnI antibodies on the conjugate pad to form a biotinylated antibody-antigen-fluorescently labeled antibody sandwich structure, which is finally captured by streptavidin immobilized on the nitrocellulose membrane detection line. The more cTnI in the sample, the more complex accumulates on the detection line, and the intensity of the fluorescent signal is proportional to the concentration of the captured cTnI. After analysis by a fluorescent immunoassay analyzer, the concentration of cTnI in the sample can be determined.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. The present application provides a method for preparing fluorescent microspheres, which utilizes 4-vinylbenzoic acid, dibenzoylmethane, and o-phenanthroline as ligands to react in ethanol to obtain a europium complex; the prepared europium complex is copolymerized with styrene and lauryl methacrylate in the presence of a persulfate initiator and a nonionic surfactant to obtain a polymer core having the europium complex, wherein europium is copolymerized and embedded to reduce leakage of the fluorescent substance; in the second stage polymerization, itaconic acid is added to continue polymerization to obtain fluorescent microspheres. The prepared fluorescent microspheres have uniform size, high fluorescence intensity, and strong stability.

[0019] 2. This application provides a method for preparing a TRFIA kit for troponin I. Biotinylated anti-cTnI antibodies are immobilized in the sample pad, fluorescent microspheres labeled with anti-cTnI antibodies are immobilized in the conjugate pad, and streptavidin is immobilized on the nitrocellulose membrane detection line. This method shifts the sample binding site forward, helps improve the capture efficiency of low-concentration cTnI, and amplifies the fluorescent signal. Furthermore, the antigen is first bound to the biotinylated antibody in the sample pad, which reduces interference of the sample matrix with subsequent reactions. Using streptavidin instead of traditional macromolecular capture antibodies to immobilize on the detection line reduces steric hindrance of complex binding and improves the efficiency of the complex antibody. The detection limit for the myocardial injury marker cTnI is as low as 10.67 pg / ml, demonstrating high sensitivity and stable and reliable detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a flow chart of the preparation process of the TRFIA kit for troponin I in Example 2; Figure 2 This is a schematic structural diagram of the TRFIA test strip for troponin I in Example 2; Figure 3 This is the linear curve of the TRFIA kit for troponin I in Example 3. DETAILED DESCRIPTION

[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. The embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] Unless otherwise specified, the methods are conventional methods, and the raw materials can be obtained from public commercial channels unless otherwise specified.

[0023] Example 1: Preparation of fluorescent microspheres A europium precursor solution was prepared by dissolving 1.464 g of EuCl₃·6H₂O in 50 ml of ethanol. A ligand solution was prepared by dissolving 2.692 g of dibenzoylmethane, 0.72 g of o-phenanthroline, and 0.592 g of 4-vinylbenzoic acid in 100 mL of ethanol. The europium precursor solution was slowly added dropwise to the ligand solution. The pH was adjusted to 6.5 with 0.5 mol / L sodium hydroxide ethanol solution. The mixture was reacted at 60°C for 5 h, filtered, washed twice with ethanol and deionized water, respectively, and dried under vacuum at 45°C to obtain a europium complex. 10 g of styrene, 1 g of lauryl methacrylate, 1 g of europium complex, and 1 g of sodium dodecyl sulfate were added to 100 ml of deionized water, and nitrogen was bubbled in for 30 min for deoxygenation. 1 g of a 4% aqueous solution of ammonium persulfate was added, and the temperature was raised to 75°C. After stirring at 300 rpm for 4 h, 1 g of a 4% aqueous solution of ammonium persulfate and 5 g of a 10% aqueous solution of itaconic acid were added and the stirring reaction was continued for 8 h. After the reaction was completed, the temperature was cooled to room temperature. The product was centrifuged at 10,000 rpm for 20 min. The obtained solid was washed twice with ethanol and deionized water, respectively, and resuspended with deionized water to form a 50 mg / ml fluorescent microsphere dispersion, which was stored at 4°C for later use.

[0024] Example 2: Preparation of fluorescent microspheres A europium precursor solution was prepared by dissolving 1.464 g of EuCl₃·6H₂O in 50 ml of ethanol. A ligand solution was prepared by dissolving 2.692 g of dibenzoylmethane, 0.72 g of o-phenanthroline, and 0.592 g of 4-vinylbenzoic acid in 100 mL of ethanol. The europium precursor solution was slowly added dropwise to the ligand solution. The pH was adjusted to 7 with 0.5 mol / L sodium hydroxide ethanol solution. The mixture was reacted at 55°C for 6 h, filtered, washed twice with ethanol and deionized water, respectively, and dried in vacuo at 45°C to obtain a europium complex. 10 g of styrene, 0.8 g of lauryl methacrylate, 0.9 g of europium complex, and 0.8 g of sodium dodecyl sulfate were added to 100 ml of deionized water, and nitrogen was bubbled in for 30 min for deoxygenation. 1 g of a 4% ammonium persulfate aqueous solution was added, and the temperature was raised to 70°C. After stirring at 300 rpm for 5 h, 1 g of a 4% ammonium persulfate aqueous solution and 4 g of a 10% itaconic acid aqueous solution were added and the stirring reaction was continued for 7 h. After the reaction was completed, the temperature was cooled to room temperature. The product was centrifuged at 10,000 rpm for 20 min. The obtained solid was washed twice with ethanol and deionized water, respectively, and resuspended with deionized water to form a 50 mg / ml fluorescent microsphere dispersion and stored at 4°C for later use.

[0025] Example 3: Preparation of fluorescent microspheres A europium precursor solution was prepared by dissolving 1.464 g of EuCl₃·6H₂O in 50 ml of ethanol. A ligand solution was prepared by dissolving 2.692 g of dibenzoylmethane, 0.72 g of o-phenanthroline, and 0.592 g of 4-vinylbenzoic acid in 100 mL of ethanol. The europium precursor solution was slowly added dropwise to the ligand solution. The pH was adjusted to 6.5 with 0.5 mol / L sodium hydroxide ethanol solution. The mixture was reacted at 60°C for 4 h, filtered, washed twice with ethanol and deionized water, respectively, and dried in vacuo at 45°C to obtain a europium complex. 10 g of styrene, 1.2 g of lauryl methacrylate, 1.1 g of europium complex, and 1.2 g of sodium dodecyl sulfate were added to 100 ml of deionized water, and nitrogen was bubbled in for 30 min for deoxygenation. 1 g of a 4% aqueous solution of ammonium persulfate was added, and the temperature was raised to 80°C. After stirring at 300 rpm for 3 h, 1 g of a 4% aqueous solution of ammonium persulfate and 6 g of a 10% aqueous solution of itaconic acid were added and the stirring reaction was continued for 10 h. After the reaction was completed, the temperature was cooled to room temperature. The product was centrifuged at 10,000 rpm for 20 min. The obtained solid was washed twice with ethanol and deionized water, respectively, and resuspended with deionized water to form a 50 mg / ml fluorescent microsphere dispersion, which was stored at 4°C for later use.

[0026] Comparative Example 1: Preparation of fluorescent microspheres A europium precursor solution was prepared by dissolving 1.464 g of EuCl₃·6H₂O in 50 ml of ethanol. A ligand solution was prepared by dissolving 2.692 g of dibenzoylmethane, 0.72 g of o-phenanthroline, and 0.592 g of acrylic acid in 100 mL of ethanol. The europium precursor solution was slowly added dropwise to the ligand solution. The pH was adjusted to 6.5 with 0.5 mol / L sodium hydroxide ethanol solution. The mixture was reacted at 60°C for 5 h, filtered, washed twice with ethanol and deionized water, respectively, and dried in vacuo at 45°C to obtain a europium complex. 10 g of styrene, 1.5 g of acrylic acid, 1 g of europium complex, and 1 g of sodium dodecyl sulfate were added to 100 ml of deionized water, and nitrogen was bubbled in for 30 min for deoxygenation. 2 g of a 4% aqueous solution of ammonium persulfate was added, and the temperature was raised to 75°C. The mixture was stirred at 300 rpm for 12 h. After the reaction was completed, the temperature was cooled to room temperature. The product was centrifuged at 10,000 rpm for 20 min. The obtained solid was washed twice with ethanol and deionized water, respectively, and resuspended in deionized water to form a 50 mg / ml fluorescent microsphere dispersion, which was stored at 4°C for later use.

[0027] Comparative Example 2: Preparation of fluorescent microspheres A europium precursor solution was prepared by dissolving 1.464 g of EuCl₃·6H₂O in 50 ml of ethanol. A ligand solution was prepared by dissolving 2.692 g of dibenzoylmethane, 0.72 g of o-phenanthroline, and 0.592 g of acrylic acid in 100 mL of ethanol. The europium precursor solution was slowly added dropwise to the ligand solution. The pH was adjusted to 6.5 with 0.5 mol / L sodium hydroxide ethanol solution. The mixture was reacted at 60°C for 5 h, filtered, washed twice with ethanol and deionized water, respectively, and dried in vacuo at 45°C to obtain a europium complex. 10 g of styrene, 1 g of lauryl methacrylate, 1 g of europium complex, and 1 g of sodium dodecyl sulfate were added to 100 ml of deionized water, and nitrogen was bubbled in for 30 min for deoxygenation. 1 g of a 4% aqueous solution of ammonium persulfate was added, the temperature was raised to 75°C, and the reaction was stirred at 300 rpm for 4 h. Then, 1 g of a 2% aqueous solution of ammonium persulfate and 5 g of a 10% aqueous solution of itaconic acid were added and the reaction was continued with stirring for 8 h. After the reaction was completed, the temperature was cooled to room temperature. The product was centrifuged at 10,000 rpm for 20 min. The obtained solid was washed twice with ethanol and deionized water, respectively, and resuspended with deionized water to form a 50 mg / ml fluorescent microsphere dispersion, which was stored at 4°C for later use.

[0028] Comparative Example 3: Preparation of fluorescent microspheres A europium precursor solution was prepared by dissolving 1.464 g of EuCl₃·6H₂O in 50 ml of ethanol. A ligand solution was prepared by dissolving 2.692 g of dibenzoylmethane, 0.72 g of o-phenanthroline, and 0.592 g of 4-vinylbenzoic acid in 100 mL of ethanol. The europium precursor solution was slowly added dropwise to the ligand solution. The pH was adjusted to 6.5 with 0.5 mol / L sodium hydroxide ethanol solution. The mixture was reacted at 60°C for 5 h, filtered, washed twice with ethanol and deionized water, respectively, and dried under vacuum at 45°C to obtain a europium complex. 10 g of styrene, 1.5 g of acrylic acid, 1 g of europium complex, and 1 g of sodium dodecyl sulfate were added to 100 ml of deionized water, and nitrogen was bubbled in for 30 min for deoxygenation. 2 g of a 4% aqueous solution of ammonium persulfate was added, and the temperature was raised to 75°C. The mixture was stirred at 300 rpm for 12 h. After the reaction was completed, the temperature was cooled to room temperature. The product was centrifuged at 10,000 rpm for 20 min. The obtained solid was washed twice with ethanol and deionized water, respectively, and resuspended in deionized water to form a 50 mg / ml fluorescent microsphere dispersion, which was stored at 4°C for later use.

[0029] The fluorescence intensity of the fluorescent microspheres prepared in Examples 1 to 3 and Comparative Examples 1 to 3 was tested using a fluorescence spectrometer. The fluorescence intensity measurement was set at 364 nm as the laser light wavelength, and the emission light wavelength of 613 nm was detected. The fluorescent microspheres prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were placed at 35° C. for 10 days and then the fluorescence intensity was measured again. The particle size and PDI value of the fluorescent microspheres prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were measured using a Malvern Zeta particle size analyzer. The surface carboxyl content of the fluorescent microspheres prepared in Examples 1 to 3 and Comparative Examples 1 to 3 was determined by the following method: the fluorescent microspheres were taken and titrated with 0.01 mol / L sodium hydroxide. The conductivity first decreased and then increased. The volume of the sodium hydroxide solution when the conductivity turned was recorded. The molar amount of sodium hydroxide at this time was the molar mass of carboxyl groups on the surface of the fluorescent microspheres. The calculation formula for the surface carboxyl content of the fluorescent microspheres was as follows: Carboxyl content (mmol / mg) = …… ... Where: C NaOH ——Molar concentration of sodium hydroxide solution used for titration (mmol / mL); V NaOH ——The volume of sodium hydroxide solution used for titration (mL); m 微球 ——Mass of fluorescent microspheres (mg); Table 1: Fluorescent microsphere test results

[0030] As shown in Table 1, the fluorescent microspheres prepared in Examples 1 to 3 have a particle size of about 200 nm, a uniform particle size distribution, high initial fluorescence intensity and stability, and a high carboxyl content on the surface of the fluorescent microspheres.

[0031] Comparing Example 1 with Comparative Example 1, in Comparative Example 1, acrylic acid is used to construct the double bond in the europium complex, and acrylic acid is used as a comonomer in the styrene polymerization process. The prepared fluorescent microspheres have a smaller particle size, but the initial fluorescence intensity, the fluorescence intensity after 10 days, and the surface carboxyl content are all reduced; Comparing Example 1 with Comparative Example 2, in Comparative Example 2, an equal amount of acrylic acid is used to replace 4-vinylbenzoic acid to prepare the europium complex. The prepared fluorescent microspheres have a smaller particle size, and the surface carboxyl content of the fluorescent microspheres is not much different, while the initial fluorescence intensity and the 10d fluorescence intensity are both reduced; Comparing Example 1 with Comparative Example 3, acrylic acid is used as a comonomer in the polystyrene polymerization process of Comparative Example 3. The prepared fluorescent microspheres have a smaller particle size, the surface carboxyl content of the fluorescent microspheres is reduced, and the initial fluorescence intensity and the 10d fluorescence intensity are both reduced.

[0032] Example 2: Preparation of TRFIA Kit for Troponin I Please combine Figure 1 , Figure 1 The present invention provides a method for preparing a TRFIA kit for troponin I, and the specific process is as follows: 1. Preparation of sample pad: Prepare a sample pad treatment solution (0.01M PBS pH 7.4 + 1% BSA + 0.1% Tween-20 + 3% sucrose), slowly drip the sample pad treatment solution onto the glass fiber to completely wet it. After standing at room temperature for 30 minutes, place it in a low humidity drying room at a relative humidity of ≤30% and a temperature of 35°C and dry it for 24 hours to obtain the pretreated glass fiber mat, cut it, and set aside. Prepare cTnI biotin-labeled antibody working solution and spray the cTnI biotin-labeled antibody working solution onto the pretreated glass fiber mat. The amount of cTnI biotin-labeled antibody working solution used is 0.1 μL / cm 2 Place the sprayed sample pad on a clean screen net, transfer it to a blast drying oven, dry it at 37±1℃, humidity ≤30% for 24 hours, take out the dried sample pad, seal it and store it at relative humidity ≤30% and 4℃.

[0033] The preparation method of cTnI biotin-labeled antibody working solution is as follows: cTnI monoclonal antibody (purchased from Asquier (Guangzhou) Biotechnology Co., Ltd.) is diluted to a concentration of 2 mg / ml using 0.1 M sodium bicarbonate buffer at pH 8.5, NHS-Biotin is diluted with DMSO to prepare a 10 mM biotin solution, the antibody diluent and biotin solution are mixed at a biotin to antibody molar ratio of 20:1, and the mixture is shaken in the dark at 4°C for 45 minutes. A stop solution (1 M Tris-HCl pH 7.5) containing 10% of the volume of the biotin solution is added, and the mixture is allowed to stand at room temperature for 15 minutes to obtain a reaction solution; the reaction solution is transferred to an ultrafiltration tube (MWKD of 20 kDa), centrifuged at 4°C and 4000g for 15 minutes, the filtrate is discarded, and fresh labeling buffer (pH 7.4) is used. The cTnI biotin-labeled antibody solution was diluted with diluent (0.01 M PBS + 1% BSA + 0.05% Tween-20) at a ratio of 1:10 to obtain the cTnI biotin-labeled antibody working solution.

[0034] 2. Preparation of conjugate pad: Prepare cTnI fluorescent marker working solution and spray the cTnI fluorescent marker working solution on the cut conjugate pad substrate. The amount of cTnI fluorescent marker working solution used is 0.2 μL / cm 2 Place the sprayed binding pad on a clean screen net and transfer it to a blast drying oven. Dry it at 37±1℃ and humidity ≤30% for 24 hours. Take out the dried sample pad, seal it and store it at relative humidity ≤30% and 4℃.

[0035] The method for preparing the cTnI fluorescent marker working solution comprises the following steps: (1) Diluting the fluorescent microspheres: Take a 2 ml centrifuge tube, use a pipette to draw 0.9 ml of labeling buffer A (50 mM MES pH 6.0), draw 0.1 ml of the fluorescent microsphere dispersion prepared in Example 1 into the centrifuge tube, mix in a vortex mixer, and ultrasonically disperse for 60 seconds each time, mix on the vortex mixer for 5 seconds, repeat 4 times, and set aside; (2) Microsphere activation: Use labeling buffer A (50 mM MES pH 6.0) to prepare EDC solution with a concentration of 10 mg / ml and NHS solution with a concentration of 10 mg / ml, respectively. Use a pipette to draw 0.016 mL of EDC solution and 0.044 mL of NHS solution and quickly add them into the centrifuge tube containing the diluted fluorescent microspheres. Close the lid tightly, mix on a vortex mixer, and then place on a full-temperature shaker (25°C, frequency 280 rpm / min) for reaction for 30 minutes.

[0036] (3) Activation and quenching: After activation, tightly cover the centrifuge tube cap with the fluorescent microspheres and place them in a centrifuge. Turn on the centrifuge, adjust the centrifugal speed to 20,000 g, the temperature to 8 ° C, and the centrifugal time to 15 min, and start centrifugation. After the centrifugation is completed, gently remove the centrifuge tube, slowly aspirate the supernatant with a pipette and discard it. Keep the precipitate, use a pipette to aspirate 1 mL of labeling buffer B (0.02M PB buffer, pH 7.4) and add it to the centrifuge tube. Ultrasonicate and disperse: 60 seconds each time, mix on a vortex mixer for 5 seconds, and repeat 4 times. Mix on a vortex mixer. After the ultrasonication is completed, use a purple flashlight to illuminate and carefully observe the fluorescent microsphere solution. If bright spots are still found in the solution, repeat the ultrasonication 2 to 3 times.

[0037] (4) Antibody labeling: Take 0.08 mg of cTnI monoclonal antibody and quickly add it to the fluorescent microsphere solution after ultrasonic resuspension. Use a pipette to disperse it, cover the centrifuge tube tightly, and mix it on a vortex mixer. Then place it on a full-temperature shaker (25°C, frequency 280 rpm / min) for 120 minutes. Use a pipette to draw 1 mL of labeled blocking solution (0.05M Tris-HCL pH8.0 + 2% BSA) into the centrifuge tube, mix it on a vortex mixer, ultrasonically disperse it for 60 seconds, mix it on a vortex mixer for 5 seconds, and repeat 4 times. After the ultrasonication is completed, use a purple flashlight to irradiate it. If bright spots are still found in the solution, repeat the ultrasonication 2 to 3 times. Then place it on a full-temperature shaker (25°C, frequency 280 rpm / min) for 120 minutes.

[0038] (5) Purification: After the blocking reaction is completed, tightly cover the centrifuge tube cap, place it on the centrifuge, turn on the centrifuge, adjust the centrifugal speed to 20,000g, the temperature to 8°C, the centrifugal time to 20 minutes, and start centrifugation; after the centrifugation is completed, gently remove the centrifuge tube, use a pipette to slowly aspirate the supernatant and discard it; retain the precipitate, use a pipette to aspirate 1 mL of fluorescent marker dilution solution (0.05MTris-HCL pH8.0+2%BSA+15% trehalose) and add it to the centrifuge tube, ultrasonically disperse for 60 seconds, mix on a vortex mixer for 5 seconds, and repeat 4 times. After the ultrasonication is completed, use a purple flashlight to irradiate. If bright spots are still found in the solution, repeat the ultrasonication 2 to 3 times.

[0039] (6) Prepare the cTnI fluorescent marker working solution. Ultrasonicate the prepared fluorescent microsphere solution labeled with anti-cTnI antibody for 60 seconds, then mix it on a vortex mixer for 5 seconds, and repeat 4 times. Take a clean centrifuge tube, use a pipette to measure the above-treated fluorescent microsphere solution labeled with anti-cTnI antibody into the centrifuge tube, then add the fluorescent marker diluent (0.05MTris-HCL pH8.0+2%BSA+15%trehalose), seal it, mix it on a vortex mixer, ultrasonicate for 60 seconds, mix it on a vortex mixer for 5 seconds, and repeat 4 times.

[0040] 3. Preparation of NC coating membrane: Take a PVC plate as a support pad, with the adhesive side facing up, and stick the nitrocellulose membrane onto the support pad; Prepare the test line coating solution and the quality control line coating solution separately. Spray the test line coating solution and the quality control line coating solution on the nitrocellulose membrane test line and the quality control line at a temperature of 18℃~28℃ and a humidity of 45%~65%. The usage of the test line coating solution and the quality control line coating solution is 0.05μL / cm 2 Place the sprayed coating film on the film drying rack and transfer it to a blast drying oven. Dry it for 48 hours at 50±1℃ and humidity ≤30%. Take out the dried coating film, seal it and store it at relative humidity ≤30% and 4℃.

[0041] The test line coating solution (streptavidin concentration is 1 mg / ml) is prepared by using a pipette to add NC membrane coating buffer (0.01M PBS pH 7.4) to a clean centrifuge tube, then adding streptavidin solution, mixing, and storing at 4°C for later use. The quality control line coating solution (antibody concentration 1 mg / ml) was prepared by pipetting NC membrane coating buffer (0.01M PBS pH 7.4) into a clean centrifuge tube, then adding rabbit anti-mouse IgG, mixing, and storing at 4°C for later use. 4. Kit Assembly In an environment with relative humidity ≤ 30%, tear off the adhesive surface of the support pad, and sequentially stick and laminate the absorbent pad, conjugate pad, and sample pad onto the support pad with the NC coating film. The absorbent pad partially overlaps one end of the NC coating film, and the conjugate pad partially overlaps the other end of the NC coating film. Ensure that each sticking part is flat and even. The schematic diagram of the prepared test strip is shown below. Figure 2 As shown, T is the detection line and C is the quality control line. The prepared test strips are sealed and stored at a relative humidity of ≤30% and 4°C.

[0042] The test strips are packaged in a shell, an ID card is made, and the final assembly is completed.

[0043] Example 3: Linear Relationship Samples with concentrations of 20, 125, 625, 3125, 12500, and 52500 (unit: pg / mL) were prepared and tested using the kit of Example 2. The sample of each concentration was repeated 3 times, and the fluorescence signal was read in a fluorescence immunoassay. The mean of the fluorescence signal results was calculated, and a fitting curve was drawn with the dilution concentration as the ordinate and the mean of the fluorescence signal as the abscissa to obtain a standard curve. Figure 3 , R 2 =0.9998.

[0044] The results showed that the standard curve had good linearity in the range of 20 pg / mL to 52500 pg / mL. Example 4: Detection Limit The kit prepared in Example 2 was used to detect 5 samples with low values ​​of high-sensitivity cardiac troponin I (hs-cTnI) at concentrations close to the limit of detection (LOD), and the detection limit of the samples was determined to be 10.67 pg / ml.

[0045] Example 5: Repeatability test Prepare samples at three concentration levels (unit: pg / mL): low, medium, and high. Use the kit of Example 2 for detection. Repeat 10 times for each concentration of sample. Read the concentration in the fluorescence immunoassay analyzer. Calculate the average value of the measured value using formulas (2), (3), and (4) respectively ( ), standard deviation (SD), and coefficient of variation (CV). The coefficient of variation (CV) was calculated according to the formula (CV = SD / M × 100%). The results are shown in Table 2.

[0046] ……… ... SD= ……… ... CV= ……… ... Where: --average value; ——various test data; n——number of data; SD—standard deviation; CV - coefficient of variation.

[0047] Table 2: Repeatability test results (unit: pg / ml)

[0048] As shown in Table 2, the CV values ​​obtained for samples of different concentrations were all below 10%, indicating that the cTnI detection kit had good reproducibility.

[0049] Example 6: Accuracy The company's reference product was used as the sample for testing. Each sample was measured three times. The average of the three measurement results was calculated and recorded as (X). The relative deviation (B) was calculated according to formula (5). The results are shown in Table 3 below: B=(XT) / T×100%…………………………………………(5) Where: B——relative deviation; X——the average value of the measurement results; T——calibrated concentration.

[0050] Table 3: Accuracy test results (unit: pg / ml)

[0051] As shown in Table 3, when using different concentrations of the company's reference products as sample tests, the relative deviation did not exceed ±5%, and the cTnI detection kit had good detection accuracy.

[0052] Comparative Example 4: Preparation of TRFIA Kit for Troponin I A troponin I TRFIA kit was prepared according to the method of Example 2, except that the cTnI fluorescent marker working solution in Example 2 used the fluorescent microsphere dispersion prepared in Comparative Example 1 as a raw material.

[0053] According to the method of Example 4 above, the detection limit of the TRFIA kit for troponin I prepared in Comparative Example 4 was 30.51 pg / ml, which was lower than the sensitivity of the TRFIA kit for troponin I prepared in Example 2.

[0054] According to the method of Example 5 above, the repeatability of the TRFIA kit for troponin I prepared in Comparative Example 4 was measured. The results are shown in Table 4 below: Table 4: Repeatability test results (unit: pg / ml)

[0055] As shown in Table 4, the CV values ​​obtained for samples with lower and higher concentrations were greater than 10%, indicating that the repeatability of the cTnI detection kit was inferior to that of Example 2.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit them. Although the present application has been described in detail with reference to preferred embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present application can still be modified or some technical features can be replaced by equivalents, which should all be included in the scope of the technical solutions requested for protection in this application.

Claims

1. A method for preparing fluorescent microspheres, characterized in that: The following steps are involved: S1. Dissolve EuCl3·6H2O in ethanol to prepare a europium precursor solution, dissolve dibenzoylmethane, o-phenanthroline, and 4-vinylbenzoic acid in ethanol to prepare a ligand solution, add the europium precursor solution to the ligand solution, adjust the pH to 6.5-7 after the addition, react at 55°C-65°C for 4-6 hours, filter, wash, and dry to obtain a europium complex; wherein the mass ratio of EuCl3·6H2O, dibenzoylmethane, o-phenanthroline, and 4-vinylbenzoic acid is 1:(1.5-2):(0.4-0.6):(0.3-0.5); S2. Add styrene, lauryl methacrylate, europium complex and anionic surfactant into water, introduce nitrogen bubbling for deoxygenation, add persulfate initiator, raise the temperature to 70-80°C, keep stirring and react for 3-5 hours to carry out the first stage polymerization; then add persulfate initiator and itaconic acid aqueous solution, continue stirring and react at the same temperature for 7-10 hours to carry out the second stage polymerization, cool to room temperature after the reaction, wash the product by centrifugation, and resuspend in deionized water to obtain; wherein, the mass ratio of styrene, lauryl methacrylate and europium complex is 1: (0.08-0.12): (0.09-0.11), and the itaconic acid in the itaconic acid aqueous solution is 4%-6% of the mass of styrene.

2. The method for preparing fluorescent microspheres according to claim 1, wherein: In S1, the concentration of the europium precursor solution is 0.02 g / ml to 0.04 g / ml; in the ligand solution, the concentration of dibenzoylmethane is 0.02 g / ml to 0.03 g / ml, the concentration of o-phenanthroline is 0.006 g / ml to 0.008 g / ml, and the concentration of 4-vinylbenzoic acid is 0.005 g / ml to 0.007 g / ml.

3. The method for preparing fluorescent microspheres according to claim 1, wherein: The washing method in S1 and S2 is to use ethanol and deionized water for 2 to 3 times respectively.

4. The method for preparing fluorescent microspheres according to claim 1, wherein: The anionic surfactant in S2 is at least one selected from sodium dodecylbenzenesulfonate, sodium lauryl polyoxyethylene ether sulfate and potassium perfluorooctane sulfonate; The amount of anionic surfactant used in S2 is 8% to 12% of the weight of styrene.

5. The method for preparing fluorescent microspheres according to claim 1, wherein: The persulfate initiator is selected from at least one of potassium persulfate, sodium persulfate and ammonium persulfate; In the first stage polymerization process, the amount of persulfate initiator used is 0.1% to 0.5% of the weight of styrene; During the second stage polymerization, the amount of persulfate initiator used is 0.1% to 0.5% of the weight of styrene.

6. A fluorescent microsphere, characterized in that: The fluorescent microspheres are prepared according to the method according to any one of claims 1 to 5, and are used to prepare a TRFIA kit for detecting troponin I.

7. A method for preparing a TRFIA kit for troponin I, characterized in that: The following steps are involved: (1) Sample pad preparation: pre-treat the glass fiber with a sample pad treatment solution to obtain a pre-treated glass fiber pad, spray the cTnI biotin-labeled antibody working solution onto the pre-treated glass fiber pad, and dry it to obtain a sample pad; (2) Preparation of conjugate pad: A cTnI fluorescent marker working solution is prepared using a fluorescent microsphere solution labeled with anti-cTnI antibody, and the cTnI fluorescent marker working solution is sprayed on the conjugate pad substrate and dried to obtain a conjugate pad; Wherein, the fluorescent microsphere solution labeled with anti-cTnI antibody is obtained by diluting the fluorescent microspheres according to claim 6, activating the microspheres, quenching the activation, labeling with antibodies, and purifying; (3) Preparation of NC coating membrane: Take a PVC plate as a support pad, with the adhesive side facing up, and stick the nitrocellulose membrane to the support pad. Prepare the test line coating liquid and the quality control line coating liquid respectively, and spray the test line coating liquid and the quality control line coating liquid on the test line and quality control line positions of the nitrocellulose membrane. After drying, the NC coating membrane is obtained; (4) Assembly of the test kit: Integrate the absorbent pad and the binding pad onto the support pad with the NC coating membrane, and encapsulate the test strip in a shell.

8. The method for preparing a TRFIA kit for troponin I according to claim 7, wherein: In step (1), the preparation method of the cTnI biotin-labeled antibody working solution is as follows: cTnI monoclonal antibody was diluted with 0.1 M sodium bicarbonate buffer (pH 8.5) to prepare an antibody diluent at a concentration of 2 mg / ml. NHS-Biotin was diluted with DMSO to prepare a 10 mM biotin solution. The antibody diluent and biotin solution were mixed at a biotin:antibody molar ratio of 20:1, and the mixture was shaken in the dark at 4°C for 45 min. A 10% biotin stop buffer was added and the reaction mixture was allowed to stand at room temperature for 15 min. The reaction solution was transferred to an ultrafiltration tube and centrifuged at 4000 g at 4°C for 15 min. The filtrate was discarded and resuspended in fresh labeling buffer to 90% of the initial volume. The centrifugation and resuspension were repeated four times to obtain a cTnI biotin-labeled antibody solution at a concentration of 1 mg / ml. The cTnI biotin-labeled antibody solution was diluted with the diluent at a ratio of 1:10 to obtain a cTnI biotin-labeled antibody working solution.

9. The method for preparing a TRFIA kit for troponin I according to claim 7, wherein: In step (2), the antibody labeling method is: Take 0.08 mg of cTnI monoclonal antibody and add it to the fluorescent microsphere solution after ultrasonic resuspending, disperse it, cover the centrifuge tube tightly, mix it on a vortex mixer, and then place it on a full-temperature shaker at 25°C and a frequency of 280 rpm / min for 120 minutes. Use a pipette to draw 1 mL of labeled blocking solution into a centrifuge tube, mix it on a vortex mixer, ultrasonically disperse it for 60 seconds, mix it on a vortex mixer for 5 seconds, repeat 4 times, and then place it on a full-temperature shaker at 25°C and a frequency of 280 rpm / min for 120 minutes.

10. The method for preparing a TRFIA kit for troponin I according to claim 7, wherein: In step (3), The test line coating solution was prepared by using a pipette to take the NC membrane coating buffer and add it to a clean centrifuge tube, then adding the streptavidin solution and mixing to obtain a test line coating solution with a streptavidin concentration of 1 mg / ml. The quality control line coating solution was prepared by using a pipette to take the NC membrane coating buffer and add it to a clean centrifuge tube, then adding rabbit anti-mouse IgG and mixing to obtain a quality control line coating solution with an antibody concentration of 1 mg / ml.

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