A preparation method of fluorescent microspheres, fluorescent microspheres and a preparation method of a kit

Abstract

The present application relates to the technical field of detection kit, in particular to a preparation method of fluorescent microspheres, fluorescent microspheres and a preparation method of a kit. The present application utilizes 4-vinylbenzoic acid, benzoyl methane and o-phenanthroline as ligands to prepare europium complexes; the prepared europium complexes are subjected to first-stage copolymerization with styrene and lauryl methacrylate to obtain a polymer inner core with europium complexes; in the second-stage polymerization process, itaconic acid is added for continuous polymerization; the prepared fluorescent microspheres are uniform in size, high in fluorescence intensity and strong in stability. The prepared detection kit has the advantages of high detection sensitivity and stable and reliable detection effect in the detection of myocardial injury marker cTnI.

Description

Technical Field

[0001] This invention relates to the field of detection kit technology, specifically to a method for preparing fluorescent microspheres, and a method for preparing fluorescent microspheres and a kit. Background Technology

[0002] Acute myocardial infarction (AMI) is myocardial necrosis caused by acute and persistent ischemia and hypoxia of the coronary arteries. Medically, the first 6 hours after the onset of an AMI are considered the "golden time" for treatment. Beyond this period, the effectiveness of treatment is greatly reduced, and serious consequences, even life-threatening ones, may occur. Therefore, only by correctly diagnosing the disease as quickly as possible can early detection and treatment be achieved, minimizing the risk of disability and death, and improving the patient's prognosis and quality of life.

[0003] Time-resolved fluoroluminescence immunoassay (TRFIA) uses lanthanide elements as tracers to label antigens or antibodies. The antigen-antibody complexes in the reaction system form immune complexes, and the fluorescence intensity of these complexes is measured using a time-resolved fluorescence spectrometer to determine the analyte concentration. Compared to radioimmunoassay (RIA), enzyme immunoassay (EIA), chemiluminescent immunoassay (CLIA), and electrochemiluminescent immunoassay (ECLIA), TRFIA offers advantages such as high sensitivity, ease of operation, stable tracers, a wide standard curve range, immunity to natural fluorescence interference, and no radioactive contamination. It has become one of the most promising analytical methods for ultra-micro biochemical testing in biomedical research and clinical practice.

[0004] Troponin I (cTnI) is a highly specific biomarker for diagnosing acute myocardial infarction. cTnI appears in the blood 3-6 hours after an acute myocardial infarction, peaking at 14-20 hours. Therefore, developing a TRFIA kit with a low detection limit for cTnI is crucial for the early diagnosis of acute myocardial infarction. The preparation of europium-labeled carboxylated polystyrene fluorescent microspheres and suitable kit preparation methods are key 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. Strong fluorescence stability of the microspheres can avoid loss of fluorescent material during storage and transportation and prevent leakage of fluorescent material from interfering with immune binding. Based on this, this application aims to study the preparation method of carboxylated polystyrene fluorescent microspheres and a TRFIA kit for cTnI, to obtain fluorescent microspheres with high stability and fluorescence intensity, and to use them to prepare a TRFIA kit for cTnI with high detection reliability and high sensitivity. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing fluorescent microspheres, as well as a method for preparing fluorescent microspheres and a reagent kit. The prepared fluorescent microspheres exhibit uniform size, high fluorescence intensity, and strong stability. The prepared troponin I TRFIA kit demonstrates high detection sensitivity and stable, reliable detection results for the detection of the myocardial injury marker cTnI.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This application provides a method for preparing fluorescent microspheres, comprising the following steps:

[0008] S1. Prepare a europium precursor solution by dissolving EuCl3·6H2O in ethanol. Prepare a ligand solution by dissolving dibenzoylmethane, o-phenanthroline, and 4-vinylbenzoic acid in ethanol. Add the europium precursor solution to the ligand solution. After addition, adjust the pH to 6.5-7. React at 55℃-65℃ for 4-6 hours. Filter, wash, and dry to obtain the europium complex. 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).

[0009] S2. Styrene, lauryl methacrylate, europium complex, and anionic surfactant are added to water, and nitrogen gas is bubbled through to remove oxygen. Persulfate initiator is added, and the temperature is raised to 70℃~80℃. The reaction is carried out at this temperature with stirring for 3h~5h for the first stage of polymerization. Then, persulfate initiator and itaconic acid aqueous solution are added, and the reaction is continued at this temperature with stirring for 7h~10h for the second stage of polymerization. After the reaction is completed, the temperature is lowered to room temperature, and the product is centrifuged, washed, and resuspended in deionized water to obtain the product. 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.

[0010] The method for preparing the fluorescent microspheres provided in this application first involves dissolving a europium precursor solution in ethanol using EuCl3·6H2O. Then, 4-vinylbenzoic acid, which has double bonds, phenyl, and carboxyl functional groups, along with dibenzoylmethane and o-phenanthroline as ligands, are reacted in an ethanol solvent to obtain a europium complex with terminal double bonds. Results show that, compared to acrylic acid, the 4-vinylbenzoic acid ligand can improve the fluorescence intensity and stability of the prepared fluorescent microspheres. Finally, the prepared europium complex is reacted with styrene and lauryl methacrylate under the action of a persulfate initiator and anionic surfactant. The first-stage copolymerization yields a polymer core with europium complexes. Europium encapsulation reduces leakage of fluorescent substances. Lauryl methacrylate, as a comonomer, enhances the hydrophobicity of the polymer core, reducing water molecule penetration. Long fatty chains impart flexibility to the microspheres, reducing the risk of microsphere rupture. In the second-stage polymerization, itaconic acid is added for further polymerization. On the one hand, itaconic acid's abundant carboxyl functional groups provide a basis for cTNI antibody conjugation. On the other hand, it further encapsulates the europium complex-containing polymer core, reducing europium fluorescent substance leakage. The prepared fluorescent microspheres exhibit uniform size, high fluorescence intensity, and strong stability.

[0011] 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.

[0012] Furthermore, the washing method in S1 and S2 is to wash with ethanol and deionized water 2 to 3 times respectively.

[0013] Furthermore, the anionic surfactant in S2 is selected from at least one of sodium dodecylbenzenesulfonate, sodium lauryl polyoxyethylene ether sulfate, and potassium perfluorooctyl sulfonate;

[0014] The amount of anionic surfactant used in S2 is 8% to 12% of the weight of styrene.

[0015] Furthermore, the persulfate initiator is selected from at least one of potassium persulfate, sodium persulfate, and ammonium persulfate;

[0016] In the first stage of polymerization, the amount of persulfate initiator used is 0.1% to 0.5% of the weight of styrene;

[0017] In the second stage of polymerization, the amount of persulfate initiator used is 0.1% to 0.5% of the weight of styrene.

[0018] The present invention also provides a carboxylated polystyrene fluorescent microsphere, which is prepared according to the above method, and the fluorescent microsphere is used to prepare a TRFIA kit for detecting troponin I.

[0019] This invention also provides a method for preparing a TRFIA kit for troponin I, comprising the following steps:

[0020] (1) Sample pad preparation: Glass fiber is pretreated with sample pad treatment solution to obtain pretreated glass fiber pad. cTnI biotin-labeled antibody working solution is sprayed onto the pretreated glass fiber pad and dried to obtain sample pad.

[0021] (2) Preparation of conjugate pad: A working solution of cTnI fluorescent labeling was prepared by using a fluorescent microsphere solution labeled with anti-cTnI antibody. The working solution of cTnI fluorescent labeling was sprayed onto the conjugate pad substrate and dried to obtain the conjugate pad.

[0022] The fluorescent microsphere solution labeled with anti-cTnI antibody was obtained by diluting, activating, quenching, labeling, and purifying the fluorescent microspheres described above.

[0023] (3) Preparation of NC coating membrane: Take a PVC board as a support pad with the adhesive side facing up, and paste the nitrocellulose membrane onto the support pad. Prepare the detection line coating solution and the quality control line coating solution respectively. Spray the detection line coating solution and the quality control line coating solution onto the detection line and quality control line positions of the nitrocellulose membrane. After drying, the NC coating membrane is obtained.

[0024] (4) Kit assembly: Integrate the absorbent pad and conjugate pad onto the support pad with NC coating membrane, and encapsulate the test strip with a shell.

[0025] Further, in step (1), the preparation method of cTnI biotin-labeled antibody working solution is as follows: take cTnI monoclonal antibody and dilute it with 0.1M sodium bicarbonate buffer at pH 8.5 to prepare an antibody dilution solution with a concentration of 2mg / ml. Dilute NHS-Biotin with DMSO to prepare a 10mM biotin solution. Mix the antibody dilution solution and the biotin solution at a biotin to antibody molar ratio of 20:1. Shake at 4℃ in the dark for 45min. Add 10% biotin solution as a stop solution and let stand at room temperature for 15min to obtain the reaction solution. Transfer the reaction solution to an ultrafiltration tube and centrifuge at 4℃ and 4000g for 15min. Discard the filtrate and resuspend it with fresh labeling buffer to mix it to 90% of the initial volume. Repeat the centrifugation-resuspension 4 times to obtain a cTnI biotin-labeled antibody solution with a concentration of 1mg / ml. Dilute the cTnI biotin-labeled antibody solution with the diluent at a ratio of 1:10 to obtain the cTnI biotin-labeled antibody working solution.

[0026] Further, in step (2), the antibody labeling method is as follows: take 0.08 mg of cTnI monoclonal antibody, add it to the fluorescent microsphere solution after ultrasonic resuspension, disperse it, tighten the centrifuge tube cap, mix it on a vortex mixer, and then place it on a full-temperature shaker at 25°C and 280 rpm / min for 120 minutes. Use a pipette to draw 1 mL of the labeling blocking solution into the centrifuge tube, mix it on a vortex mixer, sonicate it for 60 seconds, mix it on a vortex mixer for 5 seconds, repeat this process 4 times, and then place it on a full-temperature shaker at 25°C and 280 rpm / min for 120 minutes.

[0027] Furthermore, in step (3),

[0028] The preparation method of the detection line coating solution is as follows: use a pipette to add NC membrane coating buffer into a clean centrifuge tube, then add streptavidin solution, mix well, and obtain a detection line coating solution with a streptavidin concentration of 1 mg / ml.

[0029] The preparation method of the control line coating solution is as follows: use a pipette to add NC membrane coating buffer into a clean centrifuge tube, then add rabbit anti-mouse IgG, mix well, and obtain a control line coating solution with an antibody concentration of 1 mg / ml.

[0030] The TRFIA kit for troponin I provided in this application works by combining the sample to be tested with an immobilized biotinylated cTnI antibody in the sample pad to form a complex. This complex then further binds to a fluorescently labeled antibody in fluorescent microspheres labeled with anti-cTnI antibodies on the binding pad, forming a biotinylated antibody-antigen-fluorescently labeled antibody sandwich structure. Finally, the complex is 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 fluorescence signal intensity is directly proportional to the concentration of captured cTnI. Analysis using a fluorescence immunoassay analyzer reveals the concentration of cTnI in the sample.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] 1. The method for preparing fluorescent microspheres provided in this application involves reacting 4-vinylbenzoic acid, dibenzoylmethane, and o-phenanthroline as ligands in ethanol to obtain europium complexes; then, the prepared europium complexes are copolymerized with styrene and lauryl methacrylate in the presence of a persulfate initiator and a nonionic surfactant to obtain a polymer core containing europium complexes, wherein europium is encapsulated in the copolymerization process to reduce leakage of fluorescent substances; in the second stage of 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.

[0033] 2. This application provides a method for preparing a TRFIA kit for troponin I. Biotinylated anti-cTnI antibody is immobilized in the sample pad, fluorescent microspheres labeled with anti-cTnI antibody are immobilized in the binding pad, and streptavidin is immobilized on the nitrocellulose membrane detection line. This method shifts the binding site of the sample forward, improving the capture efficiency for low concentrations of cTnI and amplifying the fluorescence signal. Furthermore, the fact that the antigen binds to the biotinylated antibody first in the sample pad reduces interference from the sample matrix on subsequent reactions. Using streptavidin instead of traditional macromolecular capture antibodies immobilized on the detection line reduces steric hindrance in 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, exhibiting advantages such as high detection sensitivity and stable and reliable detection results. Attached Figure Description

[0034] Figure 1 This is a flowchart illustrating the preparation process of the TRFIA kit for troponin I in Example 2.

[0035] Figure 2 This is a schematic diagram of the structure of the TRFIA test strip for troponin I in Example 2;

[0036] Figure 3 The linear curve for the TRFIA kit of troponin I in Example 3 is shown. Detailed Implementation

[0037] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Unless otherwise specified, all methods described herein are conventional methods, and all raw materials described herein are available from publicly available commercial sources.

[0039] Example 1: Preparation of fluorescent microspheres

[0040] Europium precursor solution was prepared by dissolving 1.464 g EuCl3·6H2O in 50 mL ethanol. Ligand solution was prepared by dissolving 2.692 g dibenzoylmethane, 0.72 g o-phenanthroline, and 0.592 g 4-vinylbenzoic acid in 100 mL ethanol. Europium precursor solution was slowly added dropwise to ligand solution. pH was adjusted to 6.5 with 0.5 mol / L sodium hydroxide ethanol solution. The reaction was carried out at 60 °C for 5 h. After filtration, the product was washed twice with ethanol and deionized water, and dried under vacuum at 45 °C to obtain europium complex.

[0041] 10g styrene, 1g lauryl methacrylate, 1g europium complex, and 1g sodium dodecyl sulfate were added to 100ml deionized water. Nitrogen gas was bubbled through the mixture for 30min to remove oxygen. 1g of 4% ammonium persulfate aqueous solution was added, and the mixture was heated to 75℃ and stirred at 300rpm for 4h. Then, 1g of 4% ammonium persulfate aqueous solution and 5g of 10% itaconic acid aqueous solution were added, and the mixture was stirred for another 8h. After the reaction was completed, the mixture was cooled to room temperature, and the product was centrifuged at 10000rpm for 20min. The obtained solid was washed twice with ethanol and deionized water, respectively, and resuspended in deionized water to form a 50mg / ml fluorescent microsphere dispersion, which was then stored at 4℃ for later use.

[0042] Example 2: Preparation of fluorescent microspheres

[0043] Europium precursor solution was prepared by dissolving 1.464 g EuCl3·6H2O in 50 mL ethanol. Ligand solution was prepared by dissolving 2.692 g dibenzoylmethane, 0.72 g o-phenanthroline, and 0.592 g 4-vinylbenzoic acid in 100 mL ethanol. Europium precursor solution was slowly added dropwise to ligand solution. pH was adjusted to 7 with 0.5 mol / L sodium hydroxide ethanol solution. The reaction was carried out at 55 °C for 6 h. After filtration, the mixture was washed twice with ethanol and deionized water, respectively, and dried under vacuum at 45 °C to obtain europium complex.

[0044] 10g styrene, 0.8g lauryl methacrylate, 0.9g europium complex, and 0.8g sodium dodecyl sulfate were added to 100ml deionized water. Nitrogen gas was bubbled through the mixture for 30min to remove oxygen. 1g of 4% ammonium persulfate aqueous solution was added, and the mixture was heated to 70℃ and stirred at 300rpm for 5h. Then, 1g of 4% ammonium persulfate aqueous solution and 4g of 10% itaconic acid aqueous solution were added, and the mixture was stirred for another 7h. After the reaction was completed, the mixture was cooled to room temperature, and the product was centrifuged at 10000rpm for 20min. The obtained solid was washed twice with ethanol and deionized water, respectively, and resuspended in deionized water to form a 50mg / ml fluorescent microsphere dispersion, which was then stored at 4℃ for later use.

[0045] Example 3: Preparation of fluorescent microspheres

[0046] Europium precursor solution was prepared by dissolving 1.464 g EuCl3·6H2O in 50 mL ethanol. Ligand solution was prepared by dissolving 2.692 g dibenzoylmethane, 0.72 g o-phenanthroline, and 0.592 g 4-vinylbenzoic acid in 100 mL ethanol. Europium precursor solution was slowly added dropwise to ligand solution. pH was adjusted to 6.5 with 0.5 mol / L sodium hydroxide ethanol solution. The reaction was carried out at 60 °C for 4 h. After filtration, the product was washed twice with ethanol and deionized water, respectively, and dried under vacuum at 45 °C to obtain europium complex.

[0047] 10g styrene, 1.2g lauryl methacrylate, 1.1g europium complex, and 1.2g sodium dodecyl sulfate were added to 100ml deionized water. Nitrogen gas was bubbled through the mixture for 30min to remove oxygen. 1g of 4% ammonium persulfate aqueous solution was added, and the mixture was heated to 80℃ and stirred at 300rpm for 3h. Then, 1g of 4% ammonium persulfate aqueous solution and 6g of 10% itaconic acid aqueous solution were added, and the mixture was stirred for another 10h. After the reaction was completed, the mixture was cooled to room temperature, and the product was centrifuged at 10000rpm for 20min. The obtained solid was washed twice with ethanol and deionized water, respectively, and resuspended in deionized water to form a 50mg / ml fluorescent microsphere dispersion, which was then stored at 4℃ for later use.

[0048] Comparative Example 1: Preparation of Fluorescent Microspheres

[0049] Europium precursor solution was prepared by dissolving 1.464 g EuCl3·6H2O in 50 mL ethanol. Ligand solution was prepared by dissolving 2.692 g dibenzoylmethane, 0.72 g o-phenanthroline and 0.592 g acrylic acid in 100 mL ethanol. Europium precursor solution was slowly added dropwise to ligand solution. pH was adjusted to 6.5 with 0.5 mol / L sodium hydroxide ethanol solution. The reaction was carried out at 60 °C for 5 h. After filtration, the product was washed twice with ethanol and deionized water, and dried under vacuum at 45 °C to obtain europium complex.

[0050] 10g styrene, 1.5g acrylic acid, 1g europium complex, and 1g sodium dodecyl sulfate were added to 100ml deionized water. Nitrogen gas was bubbled through the mixture for 30min to remove oxygen. 2g of 4% ammonium persulfate aqueous solution was added, and the mixture was heated to 75℃ and stirred at 300rpm for 12h. After the reaction was completed, the mixture was cooled to room temperature, and the product was centrifuged at 10000rpm for 20min. The obtained solid was washed twice with ethanol and deionized water, respectively, and resuspended in deionized water to form a 50mg / ml fluorescent microsphere dispersion, which was then stored at 4℃ for later use.

[0051] Comparative Example 2: Preparation of Fluorescent Microspheres

[0052] Europium precursor solution was prepared by dissolving 1.464 g EuCl3·6H2O in 50 mL ethanol. Ligand solution was prepared by dissolving 2.692 g dibenzoylmethane, 0.72 g o-phenanthroline, and 0.592 g acrylic acid in 100 mL ethanol. Europium precursor solution was slowly added dropwise to ligand solution. pH was adjusted to 6.5 with 0.5 mol / L sodium hydroxide ethanol solution. The reaction was carried out at 60 °C for 5 h. After filtration, the mixture was washed twice with ethanol and deionized water, respectively, and dried under vacuum at 45 °C to obtain europium complex.

[0053] 10g styrene, 1g lauryl methacrylate, 1g europium complex, and 1g sodium dodecyl sulfate were added to 100ml deionized water. Nitrogen gas was bubbled through the mixture for 30min to remove oxygen. Then, 1g of 4% ammonium persulfate aqueous solution was added. The mixture was heated to 75℃ and stirred at 300rpm for 4h. After that, 1g of 2% ammonium persulfate aqueous solution and 5g of 10% itaconic acid aqueous solution were added and the mixture was stirred for another 8h. After the reaction was completed, the mixture was cooled to room temperature. The product was centrifuged at 10000rpm for 20min. The obtained solid was washed twice with ethanol and deionized water, respectively, and resuspended in deionized water to form a 50mg / ml fluorescent microsphere dispersion, which was then stored at 4℃ for later use.

[0054] Comparative Example 3: Preparation of Fluorescent Microspheres

[0055] Europium precursor solution was prepared by dissolving 1.464 g EuCl3·6H2O in 50 mL ethanol. Ligand solution was prepared by dissolving 2.692 g dibenzoylmethane, 0.72 g o-phenanthroline, and 0.592 g 4-vinylbenzoic acid in 100 mL ethanol. Europium precursor solution was slowly added dropwise to ligand solution. pH was adjusted to 6.5 with 0.5 mol / L sodium hydroxide ethanol solution. The reaction was carried out at 60 °C for 5 h. After filtration, the product was washed twice with ethanol and deionized water, and dried under vacuum at 45 °C to obtain europium complex.

[0056] 10g styrene, 1.5g acrylic acid, 1g europium complex, and 1g sodium dodecyl sulfate were added to 100ml deionized water. Nitrogen gas was bubbled through the mixture for 30min to remove oxygen. 2g of 4% ammonium persulfate aqueous solution was added, and the mixture was heated to 75℃ and stirred at 300rpm for 12h. After the reaction was completed, the mixture was cooled to room temperature, and the product was centrifuged at 10000rpm for 20min. The obtained solid was washed twice with ethanol and deionized water, respectively, and resuspended in deionized water to form a 50mg / ml fluorescent microsphere dispersion, which was then stored at 4℃ for later use.

[0057] Fluorescence intensity was measured using a fluorescence spectrometer for the fluorescent microspheres prepared in Examples 1-3 and Comparative Examples 1-3. The fluorescence intensity was measured with a laser wavelength of 364 nm and an emission wavelength of 613 nm. The fluorescent microspheres were placed at 35°C for 10 days, and the fluorescence intensity was measured again. The particle size and PDI value of the fluorescent microspheres prepared in Examples 1-3 and Comparative Examples 1-3 were determined using a Malvern Zeta particle size analyzer. The carboxyl content on the surface of the fluorescent microspheres prepared in Examples 1-3 and Comparative Examples 1-3 was determined using the following method: Fluorescent microspheres were titrated with 0.01 mol / L sodium hydroxide. The conductivity initially decreased and then increased. The volume of the sodium hydroxide solution at the point of inflection was recorded. The molar mass of sodium hydroxide at this point is the molar mass of the carboxyl groups on the surface of the fluorescent microspheres. The formula for calculating the carboxyl content on the surface of the fluorescent microspheres is as follows:

[0058] Carboxyl content (mmol / mg) = …………………(1)

[0059] In the formula: C NaOH —The molar concentration (mmol / mL) of the sodium hydroxide solution used in the titration;

[0060] V NaOH —The volume (mL) of sodium hydroxide solution used in the titration;

[0061] m 微球 —Mass of fluorescent microspheres (mg);

[0062] Table 1: Test results of fluorescent microspheres

[0063]

[0064] As shown in Table 1, the fluorescent microspheres prepared in Examples 1 to 3 have a particle size of about 200 nm, uniform particle size distribution, high initial fluorescence intensity and stability, and high carboxyl content on the surface of the fluorescent microspheres.

[0065] Comparing Example 1 and Comparative Example 1, Comparative Example 1 used acrylic acid to construct the double bonds in the europium complex and used acrylic acid as a comonomer in the styrene polymerization process. The prepared fluorescent microspheres had smaller particle sizes, but the initial fluorescence intensity, fluorescence intensity after 10 days, and surface carboxyl content were all reduced. Comparing Example 1 and Comparative Example 2, Comparative Example 2 used an equal amount of acrylic acid to replace 4-vinylbenzoic acid to prepare the europium complex. The prepared fluorescent microspheres had smaller particle sizes, and the surface carboxyl content of the fluorescent microspheres was not significantly different, but the initial fluorescence intensity and the fluorescence intensity after 10 days were both reduced. Comparing Example 1 and Comparative Example 3, Comparative Example 3 used acrylic acid as a comonomer in the polystyrene polymerization process. The prepared fluorescent microspheres had smaller particle sizes, lower surface carboxyl content, and lower initial fluorescence intensity and the fluorescence intensity after 10 days.

[0066] Example 2: TRFIA kit for preparing troponin I

[0067] Please combine Figure 1 , Figure 1 The preparation method of the TRFIA kit for troponin I provided in this embodiment is as follows:

[0068] 1. Preparation of the sample pad:

[0069] Prepare the sample pad treatment solution (0.01M PBS pH 7.4 + 1% BSA + 0.1% Tween-20 + 3% sucrose). Slowly drop the sample pad treatment solution onto the glass fiber to completely wet the glass fiber. After placing it at room temperature for 30 minutes, place it in a low humidity drying room at a relative humidity ≤30% and a temperature of 35℃ for 24 hours to dry. The pretreated glass fiber pad is then cut and set aside.

[0070] Prepare the cTnI biotin-labeled antibody working solution and spray it onto the pretreated glass fiber mat. The volume of cTnI biotin-labeled antibody working solution used is 0.1 μL / cm². 2 Place the sprayed sample pad on a clean screen and transfer it to a forced-air drying oven. Dry it for 24 hours at 37±1℃ and ≤30% humidity. Remove the dried sample pad, seal it, and store it at ≤30% relative humidity and 4℃.

[0071] The preparation method of the cTnI biotin-labeled antibody working solution is as follows: cTnI monoclonal antibody (purchased from Asquith (Guangzhou) Biotechnology Co., Ltd.) was diluted to a concentration of 2 mg / ml with 0.1M sodium bicarbonate buffer (pH 8.5). NHS-Biotin was diluted with DMSO to prepare a 10 mM biotin solution. The antibody dilution and biotin solution were mixed at a biotin to antibody molar ratio of 20:1. The mixture was shaken at 4°C in the dark for 45 min. 10% (by volume) of stop solution (1M Tris-HCl, pH 7.5) was added, and the mixture was allowed to stand at room temperature for 15 min to obtain the reaction solution. The reaction solution was transferred to an ultrafiltration tube (MWKD 20 kDa), centrifuged at 4°C and 4000g for 15 minutes, and the filtrate was discarded. Fresh labeling buffer (pH 7.4) was then used. Resuspend the cTnI biotin-labeled antibody solution in PBS and mix well to 90% of the initial volume. Repeat the centrifugation-resuspending process 4 times to obtain a cTnI biotin-labeled antibody solution with a concentration of 1 mg / ml. Dilute the cTnI biotin-labeled antibody solution with diluent (0.01M PBS + 1% BSA + 0.05% Tween-20) at a ratio of 1:10 to obtain the cTnI biotin-labeled antibody working solution.

[0072] 2. Preparation of the binding pad:

[0073] Prepare a cTnI fluorescent labeling working solution and spray it onto the pre-cut bonding pad substrate. The amount of cTnI fluorescent labeling working solution used is 0.2 μL / cm². 2 Place the sprayed bonding pad on a clean screen and transfer it to a forced-air drying oven. Dry it for 24 hours at 37±1℃ and ≤30% humidity. Remove the dried sample pad, seal it, and store it at ≤30% relative humidity and 4℃.

[0074] The preparation method of the cTnI fluorescently labeled working solution includes the following steps:

[0075] (1) Diluting fluorescent microspheres: Take a 2ml centrifuge tube, use a pipette to draw 0.9ml of labeling buffer A (50mM MES pH 6.0), draw 0.1ml of the fluorescent microsphere dispersion prepared in Example 1 into the centrifuge tube, mix in a vortex mixer, sonicate for 60 seconds each time, mix in a vortex mixer for 5 seconds, repeat 4 times, and set aside for use;

[0076] (2) Microsphere activation: EDC solution with a concentration of 10 mg / ml and NHS solution with a concentration of 10 mg / ml were prepared using labeling buffer A (50 mM MES pH 6.0). 0.016 mL of EDC solution and 0.044 mL of NHS solution were quickly added to centrifuge tubes containing diluted fluorescent microspheres using a pipette. The caps were tightened, and the mixture was vortexed and then placed on a full-temperature shaker (25℃, 280 rpm / min) for 30 minutes.

[0077] (3) Activation and quenching: After activation, tighten the cap of the centrifuge tube and place it on the centrifuge. Turn on the centrifuge, adjust the centrifugation speed to 20000g, the temperature to 8℃, and the centrifugation time to 15min, and start centrifugation. After centrifugation, 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, sonicate to disperse: 60 seconds each time, mix for 5 seconds on a vortex mixer, and repeat 4 times. After sonication, use a UV flashlight to carefully observe the fluorescent microsphere solution. If bright spots are still found in the solution, repeat sonication 2 to 3 more times.

[0078] (4) Antibody labeling: Take 0.08 mg of cTnI monoclonal antibody and quickly add it to the fluorescent microsphere solution after sonication and resuspension. Disperse the microspheres using a pipette, tighten the centrifuge tube cap, and mix thoroughly on a vortex mixer. Then, place the tube on a full-temperature shaker (25℃, 280 rpm / min) and react for 120 minutes. Use a pipette to draw 1 mL of the labeling blocking solution (0.05 M Tris-HCl pH 8.0 + 2% BSA) into the centrifuge tube, mix thoroughly on a vortex mixer, sonicate for 60 seconds, mix again on a vortex mixer for 5 seconds, and repeat 4 times. After sonication, irradiate the solution with a UV flashlight. If bright spots are still found in the solution, repeat sonication 2 to 3 times. Then, place the tube on a full-temperature shaker (25℃, 280 rpm / min) and react for 120 minutes.

[0079] (5) Purification: After the blocking reaction is completed, tighten the centrifuge tube cap, place it on the centrifuge, turn on the centrifuge, adjust the centrifugation speed to 20000g, the temperature to 8℃, and the centrifugation time to 20min, and start centrifugation; after centrifugation, gently remove the centrifuge tube, slowly aspirate the supernatant with a pipette and discard it; keep the precipitate, and use a pipette to add 1 mL of fluorescent label dilution solution (0.05M Tris-HCl pH8.0 + 2% BSA + 15% trehalose) to the centrifuge tube, sonicate for 60 seconds, mix on a vortex mixer for 5 seconds, and repeat 4 times. After sonication, irradiate with a UV flashlight. If bright spots are still found to be gathered in the solution, sonication can be repeated 2 to 3 times.

[0080] (6) Prepare the working solution of cTnI fluorescent label. First, sonicate the prepared fluorescent microsphere solution labeled with anti-cTnI antibody for 60 seconds, then mix it in a vortex mixer for 5 seconds. Repeat 4 times. Take a clean centrifuge tube, use a pipette to measure the prepared fluorescent microsphere solution labeled with anti-cTnI antibody into the centrifuge tube, then add the fluorescent label diluent (0.05M Tris-HCl pH8.0 + 2% BSA + 15% trehalose), seal, mix in a vortex mixer, sonicate for 60 seconds, mix in a vortex mixer for 5 seconds, and repeat 4 times.

[0081] 3. Preparation of NC-coated membrane:

[0082] Use a PVC board as a support pad, with the adhesive side facing up, and attach the nitrocellulose film to the support pad;

[0083] Prepare separate coating solutions for the test line and the control line. At a temperature of 18℃–28℃ and a humidity of 45%–65%, spray the coating solutions onto the test line and control line positions of the nitrocellulose membrane. The volume of each coating solution used is 0.05 μL / cm². 2 Place the sprayed coating film on the drying rack and transfer it to a forced-air drying oven. Dry it for 48 hours at 50±1℃ and ≤30% humidity. Remove the dried coating film, seal it, and store it at ≤30% relative humidity and 4℃.

[0084] The preparation method of the detection line coating solution (streptavidin concentration of 1 mg / ml) is as follows: use a pipette to add NC membrane coating buffer (0.01M PBS pH7.4) to a clean centrifuge tube, then add streptavidin solution, mix well, and store at 4℃ for later use.

[0085] The preparation method of the quality control line coating solution (antibody concentration of 1 mg / ml) is as follows: use a pipette to add NC membrane coating buffer (0.01M PBS pH7.4) to a clean centrifuge tube, then add rabbit anti-mouse IgG, mix well, and store at 4℃ for later use;

[0086] 4. Reagent kit assembly

[0087] In an environment with relative humidity ≤30%, peel off the adhesive side of the support pad, and then sequentially attach and laminate the absorbent pad, bonding pad, and sample pad onto the support pad with the NC coating membrane already applied. The absorbent pad should partially overlap one end of the NC coating membrane, and the bonding pad should partially overlap the other end, ensuring that all bonding areas are flat and uniform. A 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℃.

[0088] The test strips are encapsulated in a shell, an ID card is made, and then assembled.

[0089] Example 3: Linear Relationship

[0090] Samples were prepared at concentrations of 20, 125, 625, 3125, 12500, and 52500 (pg / mL). The kit described in Example 2 was used for detection. Each concentration was tested three times. Fluorescence signals were read using a fluorescence immunoassay analyzer, and the mean value of the fluorescence signals was calculated. A fitted curve was plotted with the dilution concentration on the ordinate and the mean fluorescence signal on the abscissa to obtain the standard curve. (See [link to standard curve]). Figure 3 R 2 =0.9998.

[0091] The results showed that the standard curve exhibited good linearity in the range of 20 pg / mL to 52500 pg / mL.

[0092] Example 4: Detection Limit

[0093] The kit prepared in Example 2 was used to detect five samples with low concentrations of high-sensitivity troponin I (hs-cTnI) at near-limit of detection (LOD), and the LOD of the samples was determined to be 10.67 pg / ml.

[0094] Example 5: Repeatability Test

[0095] Samples at three concentration levels (low, medium, and high, unit: pg / mL) were prepared and tested using the kit described in Example 2. Each concentration was repeated 10 times, and the concentrations were read in a fluorescence immunoassay analyzer. The average value of the measured values ​​was calculated using formulas (2), (3), and (4). The standard deviation (SD) and coefficient of variation (CV) were calculated using the formula CV = SD / M × 100%. The results are shown in Table 2.

[0096] ……………………………………………(2)

[0097] SD= ………………………………………(3)

[0098] CV= ……………………………………………(4)

[0099] In the formula:

[0100] --average value;

[0101] —Various test data;

[0102] n — the number of data items;

[0103] SD – Standard Deviation;

[0104] CV – Coefficient of variation.

[0105] Table 2: Repeatability test results (unit: pg / ml)

[0106]

[0107] As shown in Table 2, the CV values ​​obtained from samples of different concentrations were all below 10%, indicating that the cTnI detection kit has good repeatability.

[0108] Example 6: Accuracy

[0109] Using the enterprise reference sample as the sample for testing, each sample was measured three times, and the average of the three measurements was calculated and denoted as (X). The relative deviation (B) was calculated according to formula (5), and the results are shown in Table 3 below:

[0110] B=(XT) / T×100%…………………………………………(5)

[0111] In the formula:

[0112] B – Relative deviation;

[0113] X — the average value of the measurement results;

[0114] T – Calibration concentration.

[0115] Table 3: Accuracy Test Results (Unit: pg / ml)

[0116]

[0117] As shown in Table 3, when using enterprise reference materials of different concentrations as samples for testing, the relative deviation did not exceed ±5%, indicating that the cTnI detection kit has good detection accuracy.

[0118] Comparative Example 4: TRFIA kit for preparing troponin I

[0119] The TRFIA kit for troponin I was prepared according to the method in Example 2, except that the working solution for cTnI fluorescent labeling in Example 2 used the fluorescent microsphere dispersion prepared in Comparative Example 1 as the raw material.

[0120] Following the method described in Example 4 above, the detection limit of the TRFIA kit for determining troponin I prepared in Comparative Example 4 was 30.51 pg / ml, and the sensitivity was lower than that of the TRFIA kit for troponin I prepared in Example 2.

[0121] Following the method described in Example 5 above, the reproducibility of the troponin I TRFIA kit prepared in Comparative Example 4 was determined, and the results are shown in Table 4 below:

[0122] Table 4: Repeatability test results (unit: pg / ml)

[0123]

[0124] As shown in Table 4, the CV values ​​obtained for samples with lower and higher concentrations were >10%, indicating that the repeatability of this cTnI detection kit was not as good as that of Example 2.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and not to limit them; although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of this application or equivalent substitutions can be made to some technical features, all of which should be covered within the scope of the technical solutions claimed in this application.

Claims

1. A method for preparing fluorescent microspheres, characterized by, The method comprises the following steps: S1, dissolving EuCl3·6H2O in ethanol to prepare an europium precursor solution, dissolving dibenzoylmethane, o-phenanthroline and 4-vinylbenzoic acid in ethanol to prepare a ligand solution, adding the europium precursor solution into the ligand solution, adjusting the pH to 6.5-7, and reacting at 55-65°C for 4-6h, and then filtering, washing and drying to obtain an 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, adding styrene, lauryl methacrylate, the europium complex and an anionic surfactant into water, bubbling nitrogen to remove oxygen, adding a persulfate initiator, heating to 70-80°C, and reacting for 3-5h under stirring to perform first-stage polymerization, then adding a persulfate initiator and an itaconic acid aqueous solution to continue the reaction for 7-10h under stirring to perform second-stage polymerization, and then cooling to room temperature, and washing the product by centrifugation, and then resuspending in deionized water to obtain the product; wherein the mass ratio of styrene, lauryl methacrylate and the europium complex is 1:(0.08-0.12):(0.09-0.11), and the content of 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, characterized in that, In S1, the concentration of the europium precursor solution is 0.02-0.04g / ml; in the ligand solution, the concentration of dibenzoylmethane is 0.02-0.03g / ml, the concentration of o-phenanthroline is 0.006-0.008g / ml, and the concentration of 4-vinylbenzoic acid is 0.005-0.007g / ml.

3. The method for preparing fluorescent microspheres according to claim 1, characterized in that, In S1 and S2, the washing method is to use ethanol and deionized water for 2-3 times of washing respectively.

4. The method for preparing fluorescent microspheres according to claim 1, characterized in that, In S2, the anionic surfactant is at least one selected from sodium dodecylbenzenesulfonate, sodium lauryl alcohol polyoxyethylene ether sulfate and potassium perfluorooctylsulfonate; In S2, the use amount of the anionic surfactant is 8-12% of the weight of styrene.

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

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

7. A method for preparing a TRFIA kit for troponin I, characterized by, The method comprises the following steps: (1) sample pad preparation: pretreating glass fibers with a sample pad treatment solution to obtain pretreated glass fiber pads, spraying a cTnI biotin-labeled antibody working solution onto the pretreated glass fiber pads, and drying to obtain sample pads; (2) binding pad preparation: preparing a cTnI fluorescent marker working solution by using a fluorescent microsphere solution labeled with an anti-cTnI antibody, spraying the cTnI fluorescent marker working solution on a binding pad substrate, and drying to obtain a binding pad. The fluorescent microsphere solution marked with the anti-cTnI antibody is obtained by dilution, microsphere activation, activation quenching, antibody marking, and purification using the fluorescent microsphere of claim 6. (3) NC coating film preparation: take a PVC plate as a support pad, with the adhesive surface facing up, paste the nitrocellulose membrane onto the support pad, respectively prepare the detection line coating liquid and the quality control line coating liquid, spray the detection line coating liquid and the quality control line coating liquid onto the positions of the detection line and the quality control line of the nitrocellulose membrane, and dry to obtain the NC coating film; (4) Reagent kit assembly: integrate the water absorption pad and the combination pad onto the support pad with the NC coating film, and package the test strip with the shell.

8. The method for preparing a TRFIA kit for troponin I according to claim 7, characterized in that, In step (1), the preparation method of the cTnI biotin-labeled antibody working solution is as follows: Take the cTnI monoclonal antibody, dilute it with 0.1M sodium bicarbonate buffer solution at pH 8.5 to prepare an antibody diluent with a concentration of 2mg / ml, dilute the NHS-Biotin with DMSO to prepare a 10mM biotin solution, mix the antibody diluent and the biotin solution according to a biotin-to-antibody molar ratio of 20:1, avoid light at 4℃, shake for 45 minutes, add a 10% termination liquid to the biotin solution, and stand at room temperature for 15 minutes to obtain a reaction solution; transfer the reaction solution to an ultrafiltration tube, centrifuge at 4℃ and 4000g for 15 minutes, discard the filtrate, resuspend and mix the solution with fresh labeling buffer to 90% of the initial volume, repeat the centrifugation-mixing for 4 times, and obtain a cTnI biotin-labeled antibody solution with a concentration of 1mg / ml; dilute the cTnI biotin-labeled antibody solution with the diluent according to a ratio of 1:10 to obtain the cTnI biotin-labeled antibody working solution.

9. The method for preparing a TRFIA kit for troponin I according to claim 7, characterized in that, In step (2), the antibody labeling method is as follows: Take 0.08mg of the cTnI monoclonal antibody, add it to the ultrasonically resuspended fluorescent microsphere solution, disperse, tightly cover the centrifuge tube cap, mix on the vortex mixer, then place it on the constant temperature shaker at 25℃, at a frequency of 280rpm / min, and react for 120 minutes, use a pipette to take 1mL of the labeling blocking solution into the centrifuge tube, mix on the vortex mixer, ultrasonically disperse for 60 seconds, mix on the vortex mixer for 5 seconds, repeat for 4 times, then place it on the constant temperature shaker at 25℃, at a frequency of 280rpm / min, and react for 120 minutes.

10. The method for preparing a TRFIA kit for troponin I according to claim 7, characterized in that, In step (3), The preparation method of the detection line coating liquid is as follows: take the NC membrane coating buffer into a clean centrifuge tube with a pipette, then add the streptavidin solution, mix, and obtain the detection line coating liquid with a streptavidin concentration of 1mg / ml; The preparation method of the quality control line coating liquid is as follows: take the NC membrane coating buffer into a clean centrifuge tube with a pipette, then add the rabbit anti-mouse IgG, mix, and obtain the quality control line coating liquid with an antibody concentration of 1mg / ml.

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