Preparation method and application of styrene microspheres for glycated hemoglobin detection

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a preparation method and application of a styrene microsphere for glycosylated hemoglobin detection. The styrene microsphere is prepared through a dispersion polymerization method, a seed polymerization method and a preparation step of organic high molecular particles. The microsphere prepared in the application has moderate particle size and concentrated distribution, good uniformity of the microsphere particle size, a hydroxyl covering on the surface of the microsphere to form a hydrophilic layer, and can effectively reduce the matrix effect, so as to achieve accurate analysis of glycosylated hemoglobin HbA1c. The result is accurate, stable and small in error in the detection, and the application can be applied to quantitative detection of diabetes.

Description

Technical Field

[0001] This invention belongs to the field of glycated hemoglobin detection technology, and particularly relates to a method for preparing styrene microspheres for glycated hemoglobin detection and their application. Background Technology

[0002] Glycated hemoglobin (GHb) is formed by the slow and irreversible combination of glucose with certain specific molecular sites of hemoglobin A. The amount of GHb produced is closely related to blood glucose levels. GHb is more stable than blood glucose, so GHb measurement can reflect the average blood glucose level over a period of 4 to 8 weeks prior to the blood draw, making it a good indicator of blood glucose control over a longer period.

[0003] The detection of glycated hemoglobin (HbA1c) is of great significance for the diagnosis and monitoring of diabetes. It can serve as an indicator for monitoring the severity of diabetes and as an early diagnostic indicator for mild, type 2, and latent diabetes. The normal range for HbA1c is 4% to 7% of total hemoglobin. A range higher than 7% indicates that the average blood glucose level over the past 2 to 3 months is higher than normal. For every 1% increase in HbA1c, blood glucose levels increase by 0.5 to 1.0 mmol / L. Therefore, HbA1c can be used as one of the indicators for diagnosing and screening for diabetes.

[0004] Traditional methods for detecting glycated hemoglobin mainly include high-performance liquid chromatography (HPLC), manual microcolumn manipulation, electrophoresis, ion exchange chromatography, immunoassay, and chemical methods. Each method can be effective to some extent, but each also has its own technical limitations, as detailed below:

[0005] Manual microcolumn operation is susceptible to human error, potentially leading to incomplete or excessive elution, and is also affected by ambient temperature. Furthermore, abnormally high levels of certain globulins, such as HbF, can cause simultaneous elution with glycated hemoglobin, resulting in biased results. While electrophoresis can separate and detect glycated hemoglobin and hemoglobin variants, commercially available instruments with batch sample processing capabilities are currently unavailable, limiting its clinical application. Ion exchange chromatography excels in separating hemoglobin variants and subtypes, but it too is subject to human error, potentially leading to inaccurate results. For example, abnormal increases in certain globulins can affect glycated hemoglobin detection results.

[0006] Other traditional detection methods, such as immunoassays and chemical methods, while possessing high accuracy and specificity, may still be subject to certain errors in practical operation. For example, although chemiluminescence immunoassay employs ion capture immunoassay, its results are still affected by various factors, including reagent quality and the standardization of operation.

[0007] High-performance liquid chromatography (HPLC) is one of the commonly used methods for determining glycated hemoglobin (HbA1c). Although it is renowned for its reliability and accuracy, some technical issues still exist in practical applications. For example, the presence of hemoglobin variants such as HbS and HbC can interfere with chromatographic separation, and HPLC may not be able to completely separate HbA1c from other glycated hemoglobins, leading to result deviations. Sample stability is also an issue; whole blood samples need to be immediately treated with an anti-glycation agent and refrigerated (below 4°C), otherwise red blood cell rupture or continued glycation will lead to higher results. Cell debris released from hemolyzed samples may clog the chromatographic column, while lipemic samples may affect optical absorption detection. Polystyrene latex microspheres, as the core carrier for chromatographic separation, are commonly used as solid-phase carriers. Their chemical inertness, high mechanical strength, and high specific surface area allow for effective adsorption or coupling of functional molecules, directly affecting the sensitivity and accuracy of detection.

[0008] Polystyrene latex microspheres typically range from 50 nm to 500 nm. Smaller particle sizes (e.g., 50 nm) increase specific surface area and improve separation efficiency, while larger particle sizes (e.g., 500 nm) may reduce column pressure and extend column life. Poly-L-lysine can be chemically coupled (e.g., activated by EDAC / Sulfo-NHS) to immobilize polylysine on the microsphere surface, increasing positively charged amino groups and thus enhancing adsorption to hemoglobin and reducing non-specific binding. In detection, polystyrene latex microspheres, through surface modification, reduce non-specific adsorption of sample impurities such as hemolysis and lipemia, lowering the risk of false positives. Strict control of microsphere particle size distribution and surface modification uniformity is necessary to ensure the stability of test results across different batches. Stability requirements: after coupling, microspheres must be centrifuged and sonicated to ensure uniform dispersion, and a protective agent (e.g., phosphate buffer) should be added to extend shelf life. Furthermore, in immunoassays, specific antibodies (e.g., mouse anti-human HbA1c monoclonal antibodies) can be coupled to the microsphere surface, enabling precise capture of target molecules through antigen-antibody reactions and improving detection specificity. However, the preparation of microspheres requires precise chemical coupling steps and the use of high-purity antibodies or functional molecules, resulting in high reagent costs. Furthermore, the separation efficiency of microspheres is highly dependent on parameters such as flow rate and temperature control of the HPLC system, making equipment maintenance and operation more challenging.

[0009] Chinese invention patent CN118169400A describes a method and kit for detecting glycated hemoglobin, including the following steps: S1, lysing a whole blood sample with a hemolytic agent to release hemoglobin; S2, adding an appropriate amount of the hemolyzed sample to latex reagent R1; S3, adding antibody reagent R2; S4, reading the scattering rate value under single-wavelength 655nm irradiation using a specific protein analyzer; S5, reading the glycated hemoglobin content from a calibration curve based on the scattering rate value; wherein the blank latex in latex reagent R1 is polystyrene latex microspheres with a diameter of 100-400nm. The glycated hemoglobin detection method provided by this invention uses rate scattering turbidimetry as its detection principle, has high sensitivity, and eliminates the need to separately measure the total hemoglobin and glycated hemoglobin content in the blood sample; the test time is only 68s, which is short and convenient for clinical application.

[0010] The main technical problems of this patent are: (1) Sensitivity to interference factors. Rate scattering turbidimetry is easily affected by non-target components in the sample (such as hyperlipidemic samples, residual cell debris from incomplete hemolysis), which may lead to abnormal scattering signals and affect detection accuracy. Single wavelength (655 nm) detection has limited ability to eliminate background noise. If other light-absorbing substances (such as bilirubin or drug metabolites) are present in the sample, they may interfere with signal specificity. (2) Dependence and universality of calibration curve. Calibration curve depends on standard. If there are batch differences in standard or improper storage conditions, systematic bias may occur. Differences in hemoglobin characteristics among different populations (such as anemic patients, hemoglobin variant carriers) may not be covered by the calibration curve, leading to result bias. (3) Potential risk of antibody specificity. If antibody reagent R2 has cross-reactivity with epitope recognition of glycated hemoglobin (such as binding with hemoglobin F or certain variants), false positive or false negative results may occur. (4) Limitations of reagent stability and reaction kinetics. The reaction time (68 seconds) between the latex reagent and the antibody may be temperature-sensitive and requires strict temperature control; otherwise, changes in the reaction rate will affect the reproducibility of the results. The long-term storage stability of the latex microspheres and antibody is not clearly defined, and aggregation or degradation may lead to a decrease in sensitivity.

[0011] The main technical problems of latex microspheres are: (1) Insufficient uniformity of particle size distribution. The diameter range of latex microspheres (100-400 nm) is large, and the difference in particle size distribution between different batches may cause fluctuations in scattering signals, affecting the repeatability and precision of detection. (2) Surface modification and functionalization defects. If polystyrene latex microspheres are not hydrophilized or modified with functional groups, the coupling efficiency of antibody (R2) may be low, affecting the sensitivity and specificity of reagents. The exposed hydrophobic surface may non-specifically adsorb other proteins (such as albumin or immunoglobulins) in the sample, increasing background noise. (3) Latex stability risk. Polystyrene microspheres are prone to aggregation in high-salt or complex biological matrices, resulting in a shortened shelf life of reagents or abnormal signals during detection. Uneven surface charge distribution of microspheres may cause sedimentation problems during long-term storage, requiring the addition of additional stabilizers (such as surfactants), which may introduce new interfering factors. (4) Biocompatibility limitations. The bioinertness of polystyrene materials may make it difficult to fix antibodies in a targeted manner, requiring chemical coupling (such as carboxylation / aminoation modification). If the process is not mature, it may reduce antibody activity.

[0012] Chinese invention patent CN 201710135164.9 describes the preparation of cyclodextrin-styrene-divinylbenzene microspheres and their application in chiral separation. Seed microspheres of different particle sizes were synthesized using dispersion polymerization. A multi-step swelling polymerization method was employed, using styrene as the monomer and divinylbenzene as the crosslinking agent. Modified cyclodextrin was added during the swelling process to introduce double bonds into the microspheres, thus obtaining cyclodextrin-styrene-divinylbenzene microspheres. Optimal preparation conditions were determined by investigating the dispersant, stabilizer, and swelling temperature, ultimately yielding β-CD-PS-DVB and γ-CD-PS-DVB microspheres of different particle sizes. The microspheres contain cyclodextrin, exhibit good dispersibility, uniform particle size, and complete sphericity and pore structure. These microspheres can be used as chiral stationary phases in high-performance liquid chromatography. This method for preparing chiral chromatographic columns is inexpensive and simple.

[0013] The main technical problems with this patent are: (1) Insufficient cyclodextrin modification efficiency. Cyclodextrin needs to be chemically modified to introduce double bonds to participate in polymerization. If the reaction efficiency is low or there are many side reactions, the cyclodextrin may not be effectively bonded to the surface of the microspheres, affecting the chiral recognition ability. Residual unreacted modifiers may contaminate the microspheres, requiring additional purification steps and increasing costs. (2) Complex swelling process control. The multi-step swelling method requires precise control of temperature, dispersant / stabilizer ratio and swelling time. The operation has a low fault tolerance rate and is prone to microsphere particle size inhomogeneity or structural defects (such as pore blockage or collapse). (3) Risk of residual dispersant and stabilizer. If the dispersant (such as polyvinyl alcohol) or stabilizer is not completely removed, it may remain on the surface of the microspheres, reducing the column efficiency or causing baseline noise.

[0014] The main technical problems of the microspheres themselves are: (1) Uneven distribution of cyclodextrin and limited loading. The distribution of cyclodextrin on the surface or inside the microspheres may be uneven, resulting in local overloading or underloading of chiral recognition sites and fluctuations in separation selectivity. Excessive cyclodextrin loading may clog the pores and reduce mass transfer efficiency; too low loading will weaken the separation ability. (2) Insufficient mechanical strength and chemical stability. The microspheres need to withstand the long-term high-pressure environment of high-performance liquid chromatography (HPLC). If the degree of cross-linking is insufficient (such as a low proportion of divinylbenzene), it may cause the microspheres to break or swell and deform, shortening the column life. Cyclodextrin may detach under extreme pH or high temperature conditions, affecting the stability of the stationary phase. (3) Balance between pore structure and mass transfer efficiency. If the pore size and distribution are not optimized for the target analyte, it may lead to high mass transfer resistance (small pore size) or insufficient retention time (large pore size), reducing separation efficiency. (4) Batch-to-batch repeatability challenges. Multi-step preparation processes can easily introduce variables (such as fluctuations in swelling temperature and differences in stirring speed), leading to differences in the performance of microspheres from different batches and affecting the reproducibility of the chromatographic column.

[0015] The specification of Chinese Invention Patent CN 112851866 B describes a method for preparing functionalized surface-coated polystyrene microspheres by grafting, comprising the following steps: (1) obtaining polystyrene microspheres by polymerization of styrene and divinylbenzene, the surface of which has several dangling double bonds; (2) coating or immersing the surface of the polystyrene microspheres with a mixture containing glycidyl methacrylate and an initiator, the dangling double bonds and glycidyl methacrylate polymerizing on the surface, resulting in several epoxy groups on the surface, or treating the surface of the polystyrene microspheres with peroxide, so that some or all of the dangling double bonds are formed into epoxy groups; (3) coating or immersing the surface of the polystyrene microspheres with several epoxy groups obtained in step (2) with a curing agent for curing, thereby forming a network polymer coating layer on the surface, the outer surface of the network polymer coating layer having several amino groups; (4) converting the amino groups obtained in step (3) into active groups through a functional group conversion process.

[0016] The functionalized polystyrene microspheres described in this patent can theoretically be used as HPLC column packing materials because their surface is modified with functional groups such as epoxy and amino groups, providing selective adsorption capacity. Furthermore, the polystyrene matrix itself has potential advantages such as resistance to organic solvents and chemical stability. However, the following key issues exist in practical applications: (1) Insufficient particle size uniformity (the patent does not specify a method for controlling monodispersity, which may lead to reduced column efficiency and peak broadening); (2) Insufficient mechanical strength (polystyrene microspheres are easily deformed under high pressure, requiring additional cross-linking treatment to enhance rigidity); (3) Limited chemical stability (epoxy and amino groups are easily hydrolyzed under strong acid / alkali conditions, and the applicable pH range may be narrower than that of silica gel packing materials); (4) Poor surface modification repeatability (fluctuations in reaction conditions between batches may lead to inconsistent functional group densities, affecting chromatographic reproducibility); (5) Missing validation data (key performance parameters such as column efficiency and resolution are not provided, making it impossible to evaluate the actual separation effect). Therefore, further optimization of particle size control, enhancement of mechanical strength, expansion of pH tolerance, and validation of separation performance are needed to meet the stringent requirements of HPLC packing materials. Summary of the Invention

[0017] To address the above technical problems, this invention provides a method for preparing styrene microspheres for glycated hemoglobin detection and their application. The prepared microspheres have a moderate and concentrated particle size distribution, good particle size uniformity, and good mechanical strength. The surface of the microspheres is covered with hydroxyl groups to form a hydrophilic layer, which can effectively reduce the matrix effect, thereby achieving accurate analysis of glycated hemoglobin HbA1c. The detection results are accurate, stable, and have small errors, making it suitable for quantitative detection of diabetes.

[0018] To address the above technical problems, this invention provides a method for preparing styrene microspheres for glycated hemoglobin detection, comprising the following steps:

[0019] Step 1: Preparation of seed microspheres using dispersion polymerization:

[0020] (1) In an argon atmosphere, PVP (polyvinylpyrrolidone) was dissolved in anhydrous ethanol to form mixture I, and AIBN (azobisisobutyronitrile) was dissolved in styrene to form mixture II. Mixture II was added to mixture I and mixed evenly. The mixture was carried out in a water bath with mechanical stirring at 180-220 rpm. The system was initially transparent. Polymerization began when the temperature was raised. Pre-initiation was carried out at 48-52℃ for 28-32 min, and the temperature was raised to 67-73℃ for 10-13 h. The mass ratio of PVP: anhydrous ethanol: AIBN: styrene was 3-6.5: 100-103: 0.25-0.35: 30-34, and the PVP was PVP-30T.

[0021] (2) After the polymerization reaction is completed, the polymer microspheres are washed three times with ethanol / water, and the unreacted styrene and PVP raw materials are removed by centrifugation. Then the polymer microspheres are dispersed in ethanol / water solution and microspheres with uniform particle size are obtained by sedimentation separation.

[0022] (3) Wash the bulbs with ethanol, centrifuge and let them settle, then dry them at 58-62℃ for 6-10 hours to obtain dried bulbs;

[0023] In this invention, the dispersion polymerization of styrene uses AIBN as an initiator, PVP as a stabilizer / dispersant, anhydrous ethanol as a dispersion medium, and argon as a protective gas, and is carried out in a water bath under mechanical stirring.

[0024] Step 2: Preparation of GMA-DVB microspheres using seed polymerization:

[0025] A. Take seed microspheres and add them to a 0.25% (w / w) SDS (sodium dodecyl sulfate) solution. Sonicate for 0.8-1.2 h, maintain water bath heating at 32-38℃ and mechanical stirring at 120-150 rpm, and set aside. Check the results of the microscope and particle size analyzer to confirm that there is no aggregation. Add DBP (dibutyl phthalate) to the reaction solution, sonicate for 8-12 min, and swell at 33-36℃ for 10-13 h under mechanical stirring at 120-150 rpm to obtain polystyrene microspheres in a swollen state. The ratio of seed microspheres: SDS solution: DBP is 0.8-1.2: 98-102: 2.3-2.6 by mass.

[0026] B. After ultrasonically mixing the swelling monomers GMA (glycidyl methacrylate), DVB (divinylbenzene), and BPO (benzoyl peroxide) for 5 min, add the mixture to the reaction solution and ultrasonically mix for 8-12 min. Then, under mechanical stirring at 120-150 rpm, continue swelling at a constant temperature of 34-36℃ for 10-13 h. Finally, add a 5% (w / w) PVA (polyvinyl alcohol) solution and polymerize at 68-72℃ for 10-13 h. The ratio of GMA:DVB:BPO:PVA by mass is 15-20.5:3.2-3.5:0.3-0.5:18-22.

[0027] C. The product is centrifuged, washed repeatedly with deionized water and ethanol 2-3 times, and the microspheres after sedimentation are dried at 98-102℃ for 10-13h to obtain dried GMA-DVB microspheres.

[0028] SDS is sodium dodecyl sulfate, used as an emulsifier / surfactant; DBP is dibutyl phthalate, used as a swelling agent; DVB is divinylbenzene, used as a crosslinking agent; and BPO is benzoyl peroxide, used as an initiator. BPO, as an oil-soluble free radical initiator, decomposes under heating conditions to generate free radicals, initiating the polymerization reaction of the monomers.

[0029] Step 3: Preparation of styrene microspheres:

[0030] Add 2-propanol (isopropanol) and 1,3-propanesulfonyl lactone to GMA-DVB microspheres and heat to 48-52℃; then add 8 mol / L sodium hydroxide aqueous solution, stir for 5-7 h, centrifuge and discard the supernatant, wash with 0.5 mol / L hydrochloric acid, water, 0.5 mol / L sodium hydroxide aqueous solution and water in sequence to obtain styrene microspheres; wherein, by mass, the ratio of GMA-DVB microspheres:isopropanol:1,3-propanesulfonyl lactone:sodium hydroxide aqueous solution is 2.8-3.2:23-25:2.8-3.2:1-1.3.

[0031] 2-Propanol, i.e., isopropanol, is used as the reaction solvent; 1,3-propanesulfonic acid lactone is a sulfonating agent used to modify the surface of the microspheres with sulfonic acid groups. In this invention, the styrene microspheres are non-porous organic polymer particles with cation exchange groups.

[0032] In the optimized solution, the raw materials underwent pretreatment:

[0033] Purification of the styrene: Take about 200 mL of styrene in a 1000 mL separatory funnel, wash it three times with 1 M sodium hydroxide aqueous solution, each time using about 400 mL, to remove the polymerization inhibitor, then wash it with deionized water until neutral, dry it with anhydrous calcium chloride, then perform vacuum distillation, and store it in a refrigerator for later use.

[0034] Purification of the initiator AIBN: 10 g of AIBN was dissolved in 100 mL of chloroform by recrystallization to form a saturated solution. The solution was filtered using a Buchner funnel. Methanol was added to the resulting solution, and white AIBN crystals precipitated. The solution was filtered to obtain white AIBN crystals, which were then dried in a vacuum drying oven and then placed in a beaker covered with tin foil and stored in a desiccator for later use.

[0035] In the further optimized scheme, the purification method of the initiator BPO is the same as the purification method of AIBN.

[0036] The methacrylic acid is distilled under reduced pressure and then kept for later use; other reagents are used directly.

[0037] In step one, the mass ratio of PVP: anhydrous ethanol: AIBN: styrene is 3.738: 101.25: 0.325: 32.5.

[0038] In step one, the process is carried out in a water bath with mechanical stirring at 200 rpm.

[0039] Furthermore, by mass, the ratio of seed microspheres:SDS solution:DBP is 1:100:2.5.

[0040] The mass measurement meter has the following ratios: GMA:DVB:BPO:PVA = 16.4:3.4:0.4:20.

[0041] The ratio of GMA-DVB spheres: isopropanol: 1,3-propanesulfonyl lactone: sodium hydroxide aqueous solution is 3:24:3:1.2.

[0042] The present invention discloses a kit for the detection of glycated hemoglobin, which is applicable to the preparation method of styrene microspheres for the detection of glycated hemoglobin described in the present invention. The kit is characterized in that it comprises microspheres prepared as described in the present invention and antibody reagents.

[0043] This invention provides a method for detecting glycated hemoglobin, applicable to the microsphere preparation method for detecting glycated hemoglobin described in this invention. The mobile phase A consists of 50 mmol / L MES, 1.0 mmol / L EDTA.3K, and 120 mmol / L KCI, adjusted to pH 5.4 using NaOH; mobile phase B consists of 50 mmol / L MOPS, 1.0 mmol / L EDTA.3K, and 120 mmol / L KCI, adjusted to pH 7.9 using NaOH; and mobile phase C consists of 50 mmol / L MES, 1.0 mmol / L EDTA.3K, and 0.10% Tween 20, adjusted to pH 7.0 using NaOH.

[0044] The application of styrene microspheres for glycated hemoglobin detection in this invention is the application of the styrene microspheres in the preparation of chromatographic column packing.

[0045] The column packing method in the HPLC detection includes the following steps:

[0046] (a) Preparation of materials: Clean the homogenizing tank, prepare one chromatographic column and two sieve plates;

[0047] Homogenization preparation: Mix 20ml of water with 2.5g of microspheres using ultrasonication;

[0048] Turn on the compressor and cooler to preheat, and fill the column packer with water up to half (white bucket next to the column packer).

[0049] (ii) Install a sieve plate on one side of the chromatographic column and mark the liquid inlet direction. The liquid inlet direction is the same as that in high performance liquid chromatography. Connect the other side to the homogenizer and ensure a tight connection to prevent leakage or pressure loss.

[0050] The homogenizing tank is filled with a uniform homogenate and then filled with filtered water.

[0051] Connect the homogenizing tank to the column packing machine, ensuring a tight connection to prevent leakage or pressure loss.

[0052] (iii) Open the drive speed regulating valve, close the high pressure shut-off valve, and adjust the drive pressure regulating valve until the high pressure gauge displays 4-5 MPa. Open the high pressure shut-off valve and wait for two and a half minutes. During the waiting period, observe whether the water output speed is uniform. If the water output speed is slow or no water is output and the high pressure gauge rises abnormally, it is determined that the column is blocked and the column needs to be reinstalled.

[0053] (iv) After the column is packed, close the drive pressure regulating valve and wait for the high pressure gauge to reach 0 MPa. First, remove the column from the homogenizer, and then remove the homogenizer from the column packer. During the whole process, place a clean beaker under the homogenizer to catch any remaining microspheres that fall. After the column is removed, observe whether the packing effect is good. If it is determined to be full, remove the excess microspheres with a blade and observe whether the cut is smooth. If it is smooth, replenish the sieve plate in time. The column is used for high performance liquid chromatography detection.

[0054] The preparation of styrene microspheres in the chromatographic column packing material of this invention employs a three-step method to improve the overall size uniformity of the microspheres. Selecting appropriate crosslinking agents and controlling their proportions ensures the morphology and rigidity of the microspheres. Choosing a mild sulfonating agent effectively enhances the safety of the reaction. Adjusting the buffer formulation, with suitable pH and salt concentration, effectively improves the analytical results for glycated hemoglobin.

[0055] This invention enables the quantitative detection of glycated hemoglobin, achieving accurate analysis of glycated hemoglobin HbA1c, and can be used for diabetes detection.

[0056] In this invention, the concentration of sulfonic acid groups on the surface of styrene microspheres was determined by titration with 1 M NaOH solution, and the concentration was found to be 2.56 mmol / g. The reasonable density of sulfonic acid groups on the surface allows for the formation of a hydrophilic layer with hydroxyl groups, effectively mitigating the matrix effect and achieving accurate analysis of glycated hemoglobin (HbA1c).

[0057] In the detection method of this invention, according to the standards of the American Diabetes Association, an HbA1c level of less than 5.7% is considered normal; between 5.7% and 6.4% indicates prediabetes; and greater than 6.5% indicates diabetes. Attached Figure Description

[0058] Figure 1 This is a microscopic image of seed microspheres prepared by dispersion polymerization in this invention.

[0059] Figure 2 This is a microscopic image of GMA-DVB swollen microspheres prepared by the seed polymerization method in this invention.

[0060] Figure 3 For the present invention Figure 2 Microscopic data of styrene microspheres after processing.

[0061] Figure 4 The results are based on HPLC analysis of commercially available microsphere packing material used in this invention.

[0062] Figure 5 The results are HPLC analysis of the microsphere packing material prepared in this invention.

[0063] Figure 6 This is a graph showing the particle size distribution of polystyrene seed microspheres in this invention.

[0064] Figure 7 This is a data graph from the GMA-DVB swelling microsphere particle size analyzer used in this invention.

[0065] Figure 8 This is a graph showing the particle size distribution of the final product microspheres in this invention.

[0066] Figure 9 The data for seed microsphere size analyzer when PVP-K30 is 1.869 g in this invention are as follows.

[0067] Figure 10 The data for seed microsphere size analyzer when PVP-K30 is 7.476 g is presented in this invention.

[0068] Figure 11 The data obtained from the seed microsphere particle size analyzer were obtained when the stirring speed was 250 rpm in this invention.

[0069] Figure 12 The data obtained from the seed microsphere particle size analyzer were obtained when the stirring speed was 300 rpm in this invention.

[0070] Figure 13 These are the particle size microscopy data of the microspheres after swelling with DBP for 12 h in this invention.

[0071] Figure 14 These are the particle size microscopy data of the microspheres after swelling in cyclohexane for 12 hours in this invention.

[0072] Figure 15 This is an HPLC chromatogram of glycated hemoglobin in the final product microspheres prepared using St as the swelling monomer in this invention.

[0073] Figure 16 This is a magnified view of the HPLC analysis spectrum of glycated hemoglobin in the final product microspheres prepared using St as the swelling monomer in this invention.

[0074] Figure 17 These are the microscopic data of the swollen microspheres obtained when the amount of GMA added in this invention is 12.3 g.

[0075] Figure 18 The data obtained from the particle size analyzer for the swollen microspheres when the GMA feed amount was 12.3 g in this invention.

[0076] Figure 19 These are the microscopic data of the swollen microspheres obtained when the amount of GMA added in this invention is 20.5 g.

[0077] Figure 20 These are the microscopic data of the swollen microspheres obtained when the DVB feed amount was 2.8 g in this invention.

[0078] Figure 21 The data are microscopic data of the swollen microspheres obtained when the DVB feed amount was 2.2 g in this invention.

[0079] Figure 22 The data obtained from the particle size analyzer for the swollen microspheres when the GMA feed amount was 20.5 g in this invention.

[0080] Figure 23 The data obtained from the swollen microsphere particle size analyzer are when the DVB feed amount is 2.8 g in this invention.

[0081] Figure 24 The data obtained from the swollen microsphere particle size analyzer are when the DVB feed amount is 2.2 g in this invention. Detailed Implementation

[0082] The present invention will be further described below with reference to specific embodiments:

[0083] In the following examples, the morphology of the microspheres was observed using a microscope, and the particle size and uniformity of the microspheres were characterized using a Malvern laser particle size analyzer MS3000 with distilled water as the reagent.

[0084] Example 1:

[0085] Step 1: Pretreatment of raw materials:

[0086] (1) Purification of styrene: Take about 200 mL of styrene into a 1000 mL separatory funnel, wash it three times with 1 M sodium hydroxide aqueous solution, each time using about 400 mL, to remove the polymerization inhibitor, then wash it with deionized water until neutral, dry it with anhydrous calcium chloride, then distill it under reduced pressure, and store it in a refrigerator for later use.

[0087] (2) Purification of initiator azobisisobutyronitrile (AIBN): 10 g of AIBN was dissolved in 100 mL of chloroform by recrystallization to form a saturated solution. The solution was filtered by Buchner funnel. Methanol was added to the solution, and white AIBN crystals precipitated. The solution was filtered to obtain white AIBN crystals, which were dried in a vacuum drying oven and then placed in a beaker covered with tin foil and stored in a desiccator for later use.

[0088] (3) The purification method for the initiator benzoyl peroxide (BPO) is the same as that for azobisisobutyronitrile (AIBN). Methacrylic acid is distilled under reduced pressure and then set aside; other reagents are used directly.

[0089] Step 2: Preparation of seed microspheres by dispersion polymerization:

[0090] The dispersion polymerization of styrene was carried out in a 250 mL three-necked round-bottom flask with AIBN as the initiator, PVP as the stabilizer, anhydrous ethanol as the dispersion medium, and argon as the protective gas in a water bath with mechanical stirring at 200 rpm. First, argon was introduced to remove oxygen from the reaction system. Then, 3.738 g of PVP-30T was dissolved in 101.25 g of anhydrous ethanol and added to the round-bottom flask. Next, 0.325 g of AIBN was dissolved in 32.5 g of styrene and added to the system. After thorough mixing, the system was initially transparent. Polymerization was initiated by heating, with pre-initiation at 50 °C for 30 min, followed by stabilization at 70 °C for 12 hours. The proportions of each raw material are shown in Table 1.

[0091] After the polymerization reaction, the microspheres were washed three times with ethanol / water, and unreacted styrene and PVP were removed by centrifugation. The polymer microspheres were then dispersed in an ethanol / water solution, and uniformly sized microspheres were obtained by sedimentation. Alternatively, the microspheres were washed with ethanol, centrifuged, and then dried at 60 °C for 6-10 h to obtain dried seed microspheres. Figure 1 As shown.

[0092] Table 1 Formulation for Dispersion Polymerization

[0093] ;

[0094] The method described in this embodiment can produce seed microspheres with good monodispersity, a Span value of 0.671, and a particle size of approximately 2.3 μm. Microscopic data are as follows: Figure 1 As shown (4x scale display), the particle size analyzer data is as follows: Figure 6 As shown.

[0095] Step 3: Preparation of GMA-DVB microspheres by seed polymerization:

[0096] (1) Take 1 g of seed microspheres and add them to 100 g of SDS solution with a mass fraction of 0.25%. Mix ultrasonically for 1 h, maintain heating in a water bath at 35°C and mechanical stirring at 140 rpm, and set aside for use. Check the results of the microscope and particle size analyzer to confirm that there is no aggregation. Add 2.5 g of DBP to the reaction solution, sonicate for 10 min, and swell at 35°C for 12 h under mechanical stirring at 140 rpm.

[0097] (2) Mix 16.4 g of GMA, 3.4 g of DVB and 0.4 g of BPO by sonication for 5 min, add the mixture to the reaction solution, sonicate for 10 min, continue to swell at 35°C for 12 h under mechanical stirring at 140 rpm, add 20 mL of 5% PVA solution, and polymerize at 70°C for 12 h.

[0098] (3) The product was centrifuged, washed three times with deionized water and ethanol, and the microspheres were separated by sedimentation. The microspheres were then dried at 100℃ for 12 hours to obtain dried GMA-DVB swollen microspheres, as shown below. Figure 2 and Figure 7 As shown.

[0099] Step Four

[0100] 3g of GMA-DVB spheres were mixed with 24g of 2-propanol (isopropanol) and 3g of 1,3-propanesulfonyl lactone, and heated to 50℃. 1.2g of 8mol / L sodium hydroxide aqueous solution was added, and the mixture was stirred for 6 hours. The supernatant was discarded by centrifugation, and the mixture was washed sequentially with 0.5mol / L hydrochloric acid, water, 0.5mol / L sodium hydroxide aqueous solution, and water again to obtain non-porous organic polymer particles with cation exchange groups, such as... Figure 3 and Figure 8 As shown, this step yields swollen microspheres with a particle size of 3.53 μm and good uniformity (Span value of 1.057).

[0101] In the above embodiments Figures 6-8 The graphs show particle size distribution data for three types of microspheres: seed microspheres, GMA-DVB microspheres, and the final product styrene. These data reflect the particle size and uniformity of the microspheres. The smaller the Span value in the data, the higher the uniformity.

[0102] Example 2:

[0103] A method for preparing styrene microspheres for glycated hemoglobin detection, with other steps as described in Example 1, wherein step one is carried out in a water bath with mechanical stirring at 180 rpm; pre-initiation is performed at 48°C for 28 min, followed by heating to 67°C and reacting for 10 h; wherein, by mass, the ratio of PVP:anhydrous ethanol:AIBN:styrene is 3:100:0.25:30. After washing the microspheres with ethanol, centrifuging and settling, drying at 58°C for 10 h yields dried seed microspheres.

[0104] In step A of step 2, the mixture was ultrasonically mixed for 0.8 h, heated in a water bath at 38 °C, and mechanically stirred at 120 rpm, and set aside. DBP (dibutyl phthalate) was added to the reaction solution, ultrasonicated for 8 min, and then swollen at 33 °C for 10-13 h under mechanical stirring at 150 rpm to obtain polystyrene microspheres in a swollen state. The ratio of seed microspheres: SDS solution: DBP was 0.8:98:2.3 by mass.

[0105] B. After ultrasonically mixing the swelling monomers GMA (glycidyl methacrylate), DVB (divinylbenzene), and BPO (benzoyl peroxide) for 5 min, the mixture was added to the reaction solution and ultrasonicated for 8 min. The mixture was then kept at 34℃ for 13 h under mechanical stirring at 150 rpm. Finally, a 5% PVA (polyvinyl alcohol) solution was added, and polymerization was carried out at 68℃ for 13 h. The mass ratio of GMA:DVB:BPO:PVA was 15:3.2:0.3:18.

[0106] C. The product is centrifuged, washed repeatedly with deionized water and ethanol 2-3 times, and the microspheres after sedimentation are dried at 98℃ for 13h to obtain dried GMA-DVB microspheres.

[0107] Step 3: The ratio of GMA-DVB balls to isopropanol to 1,3-propanesulfonyl lactone to sodium hydroxide aqueous solution is 2.8:23:2.8:1, based on mass.

[0108] Example 3:

[0109] A method for preparing styrene microspheres for glycated hemoglobin detection, with other steps as described in Example 1, wherein step one is carried out in a water bath with mechanical stirring at 220 rpm; pre-initiation is performed at 52℃ for 28 min, followed by heating to 73℃ and reacting for 10 h; wherein, by mass, the ratio of PVP:anhydrous ethanol:AIBN:styrene is 6.5:103:0.35:34. After washing the microspheres with ethanol, centrifuging and settling, drying at 62℃ for 6 h yields dried seed microspheres.

[0110] In step A of step 2, the mixture was ultrasonically mixed for 1.2 hours, heated in a water bath at 32°C, and mechanically stirred at 150 rpm, and set aside. DBP (dibutyl phthalate) was added to the reaction solution, ultrasonicated for 12 minutes, and then swollen at 36°C for 10 hours under mechanical stirring at 120 rpm to obtain polystyrene microspheres in a swollen state. The ratio of seed microspheres: SDS solution: DBP was 1.2:102:2.6 by mass.

[0111] B. After ultrasonically mixing the swelling monomers GMA (glycidyl methacrylate), DVB (divinylbenzene), and BPO (benzoyl peroxide) for 5 min, add the mixture to the reaction solution, ultrasonicate for 12 min, and continue swelling at 36℃ for 10 h under mechanical stirring at 120 rpm. Then, add a 5% PVA (polyvinyl alcohol) solution and polymerize at 68-72℃ for 10-13 h. The mass ratio of GMA:DVB:BPO:PVA is 20.5:3.5:0.5:22.

[0112] C. The product is centrifuged, washed repeatedly with deionized water and ethanol 2-3 times, and the microspheres after sedimentation are dried at 102℃ for 10h to obtain dried GMA-DVB microspheres.

[0113] Step 3: The mass ratio of GMA-DVB balls: isopropanol: 1,3-propanesulfonyl lactone: sodium hydroxide aqueous solution is 3.2:25:3.2:1.3.

[0114] Example 4

[0115] The column packing process using the styrene microspheres prepared in this invention was determined after fully considering factors such as homogenate concentration, column packing pressure, column packing time, and the actual pressure of the HPLC column.

[0116] (a) Preparation of materials: Clean the homogenizing tank, prepare one chromatographic column and two sieve plates;

[0117] Homogenization preparation: Mix 20ml of water with 2.5g of microspheres using ultrasonication;

[0118] Turn on the compressor and cooler to preheat, and fill the column packer with water up to half (white bucket next to the column packer).

[0119] (ii) Install a sieve plate on one side of the chromatographic column and mark the liquid inlet direction. The liquid inlet direction is the same as that in high performance liquid chromatography. Connect the other side to the homogenizer and ensure a tight connection to prevent leakage or pressure loss.

[0120] The homogenizing tank is filled with a uniform homogenate and then filled with filtered water.

[0121] Connect the homogenizing tank to the column packing machine, ensuring a tight connection to prevent leakage or pressure loss.

[0122] (iii) Open the drive speed regulating valve, close the high pressure shut-off valve, and adjust the drive pressure regulating valve until the high pressure gauge displays 4-5 MPa. Open the high pressure shut-off valve and wait for two and a half minutes. During the waiting period, observe whether the water output speed is uniform. If the water output speed is slow or no water is output and the high pressure gauge rises abnormally, it is determined that the column is blocked and the column needs to be reinstalled.

[0123] (iv) After the column is packed, close the drive pressure regulating valve and wait for the high pressure gauge to reach 0 MPa. First, remove the column from the homogenizer, and then remove the homogenizer from the column packer. During the whole process, place a clean beaker under the homogenizer to catch any remaining microspheres that fall. After the column is removed, observe whether the packing effect is good. If it is determined to be full, remove the excess microspheres with a blade and observe whether the cut is smooth. If it is smooth, replenish the sieve plate in time. The column is used for high performance liquid chromatography detection.

[0124] The unique column packing process in this invention ensures a flow rate of 1.5 mL / min in subsequent HPLC analysis.

[0125] The buffer solution used was a conventional formulation, in which Bis-tris was the buffer salt, EDTA-2Na was the metal ion chelating agent, KCl provided the salt ionic strength, Tween20 was the surfactant, and the pH was adjusted with hydrochloric acid.

[0126] Sample diluent: distilled water

[0127] Buffer A: 20 mmol / L Bis-tris, 1 mmol / L EDTA-2Na, 120 mmol / L KCl, pH 5.4.

[0128] Buffer B: 20 ​​mmol / L Bis-tris, 1 mmol / L EDTA-2Na, 120 mmol / L KCI, 0.10% Tween 20, pH 6.4.

[0129] Example 5:

[0130] HPLC Analysis: In this step of the HPLC analysis, commercially available glycated hemoglobin packing material was purchased and tested using the same analytical method. Through result comparison, the packing material prepared in this invention has advantages in terms of accuracy and analytical efficiency in analyzing glycated hemoglobin. Details are as follows:

[0131] (1) Select the appropriate chromatographic column according to the requirements of the test item, prepare the required mobile phase, and filter all mobile phases.

[0132] (2) Degas the filtered mobile phase by ultrasonication for 30 min.

[0133] (3) Connect the selected column to the system and place the mobile phase in the mobile phase tray of the corresponding instrument.

[0134] (4) Check whether the flow path is normal, and focus on checking for leaks.

[0135] (5) Turn on the machine and check if the circuit is working properly. First, turn on the computer, then turn on the power switches of the instrument, pump, autosampler, detector and column oven respectively. Finally, double-click the desktop icon, enter the username and password, and click OK.

[0136] (6) Check the baseline and perform a single analysis after the baseline has stabilized.

[0137] (7) Click "Run Once". A dialog box will appear. Select the edited method, enter the file name, and enter the sample vial, sample rack, and sample volume. Click "OK" to start the sample analysis. Click the "Stop" button on the work interface to stop the analysis.

[0138] (8) Specimen diluent: 20 mmol / L CHES, 1.0 mmol / L EDTA.3K and 0.10% Triton X-100 were prepared with NaOH to pH 9.6.

[0139] Mobile phase A: 50 mmol / L MES, 1.0 mmol / L EDTA.3K, and 120 mmol / L KCl were adjusted to pH 5.4 using NaOH.

[0140] Mobile phase B: 50 mmol / L MOPS, 1.0 mmol / L EDTA.3K, and 120 mmol / L KCl were adjusted to pH 7.9 using NaOH.

[0141] (9) Injection method: Glycated hemoglobin standard (commonly used 2.5% glycated hemoglobin filler material purchased from the market):

[0142] Injection volume: 5 μL

[0143] Flow rate: 1.5 ml / min

[0144] Method: 0.01~1.00 min, b concentration 0%

[0145] 1.01~3.00 min b concentration 0%

[0146] The results are as follows Figure 4 And as shown in Table 2 below:

[0147] Table 2. Detailed HPLC analysis data of commonly used microsphere packing materials purchased from the market.

[0148] ;

[0149] (10) Glycated hemoglobin standard (the microsphere filler content prepared in this invention is 10.0%):

[0150] Injection volume: 10 μL

[0151] Flow rate: 1.5 ml / min

[0152] Method: 0.01~1.00 min, B concentration 0%

[0153] 1.01~3.00 min B concentration 0%

[0154] The results are as follows Figure 5 And as shown in Table 3 below:

[0155] Table 3. Detailed HPLC analysis data of the microsphere packing material prepared in this invention.

[0156] ;

[0157] (11) Post-injection procedures: After all samples have been processed, flush the column with water and wait for baseline equilibration.

[0158] Shutdown: First turn off the computer, then turn off the liquid chromatography system.

[0159] In the detection method of this invention, according to the standards of the American Diabetes Association, an HbA1c level of less than 5.7% is considered normal; between 5.7% and 6.4% indicates prediabetes; and greater than 6.5% indicates diabetes.

[0160] The HPLC analysis method in this invention is unaffected by variant hemoglobin and its derivatives, making it particularly suitable for monitoring diabetic patients. It can provide important information about the patient's average blood glucose levels over the past two to three months, which is of great significance for the management and treatment of diabetes.

[0161] Experiment 1

[0162] Repeatability tests were performed on the glycated hemoglobin standard (commonly available microsphere filler material) in Example 5, as shown in Table 4 below:

[0163] Table 4. Repeatability Test of Glycated Hemoglobin Standards (Commonly Used Microsphere Fillers Available Commercially)

[0164] ;

[0165] Based on the HPLC data results of the 10 sets in the table above, the RSD value is 0.385%, indicating that the reagent kit and HPLC analysis method of this invention have good repeatability, accuracy and high stability for detecting glycated hemoglobin standards.

[0166] Experiment 2: Adjustment of PVP Addition Amount

[0167] Using the scheme in Example 1, only the equivalent of PVP-K30 was adjusted. Experiments were conducted with addition amounts of 1.869 g (0.5 eq in Table 1) and 7.476 g (2 eq in Table 1), respectively. Figure 9 As shown, the uniformity of the seed microspheres decreased. On the one hand, the Span value increased to 0.940; on the other hand, a small peak appeared before the main peak in the particle size analyzer data, indicating the formation of impurity spheres with a particle size of approximately 0.5 μm. This is highly detrimental to maintaining and controlling the size and uniformity of the microspheres in subsequent synthesis steps. However, when the PVP-K30 was adjusted to 7.476 g, as... Figure 10 As shown, the microspheres still maintain good uniformity, but the particle size of the microspheres has decreased slightly. Considering the actual economic cost, it is still preferable to use a feed amount of 3.738 g of PVP-K30.

[0168] Experiment 3: Adjustment of stirring speed

[0169] Rotation speed is a crucial parameter affecting the particle size and uniformity of microspheres. Using the seed microsphere preparation method described in Example 1, only the stirring speed was adjusted. In addition to the 200 rpm specified in the original method, the stirring speed was also adjusted to 250 rpm and 300 rpm for experiments. Figure 11 and Figure 12 As shown, when the stirring speed is increased to 250 rpm, two sets of impurity spheres of about 0.15 μm and about 0.6 μm are generated in the reaction; when the stirring speed is further increased to 300 rpm, the proportion of the two sets of impurity peaks is further increased, and the uniformity of the seed microspheres becomes extremely poor. Therefore, a stirring speed of 200 rpm is still preferred.

[0170] Experiment 4: Types of Swelling Agents

[0171] The main innovation of the seed polymerization method for preparing swollen microspheres lies in the selection of appropriate types of swelling agents, swelling monomers, the amount of swelling monomers added, and the amount of crosslinking agents added. These factors have a significant impact on the morphology and particle size uniformity of the microspheres prepared by seed polymerization. Specific experimental data and comparisons are as follows:

[0172] Other aspects are as described in Example 1. Besides dibutyl phthalate (DBP) used in the microsphere preparation method, other swelling agents, such as cyclohexane, have also been used in swelling experiments. Figure 13 and Figure 14 The particle size changes observed under a microscope after 12 h of swelling with DBP and cyclohexane were respectively shown. It can be seen that the swelling effect of DBP is very obvious, with the seed microspheres swelling from 2.3 μm to about 3.5 μm; while cyclohexane has almost no obvious swelling effect, with the microsphere particle size only changing from 2.3 μm to about 2.5 μm, which is a poor effect. Therefore, DBP is still the preferred swelling agent for the seed swelling reaction.

[0173] Five monomer types in the experiment

[0174] Other aspects are as described in Example 1. Besides glycidyl methacrylate, the monomer used in the scheme, polystyrene (St) was also attempted as a monomer. However, in subsequent analytical experiments, the HPLC results obtained were as follows... Figure 15 and Figure 16 As shown, using the same analytical method, no peak was observed 15 minutes prior, making it impossible to analyze glycated hemoglobin. This is presumably due to the non-specific interaction between the hydrophobicity of styrene and the protein. Therefore, GMA is still the preferred monomer.

[0175] Experiment 6: GMA monomer addition amount

[0176] Other details are as described in Example 1. In addition to using the 16.4 g GMA addition amount described above, lower (12.3 g) and higher (20.5 g) amounts of the swelling monomer were also tested to investigate the effect of GMA monomer addition on microsphere preparation. When the GMA addition amount was 12.3 g, the data for the obtained GMA-DVB microspheres are as follows: Figure 17 and Figure 18 As shown, especially from the microscopic data, the bowl-shaped morphology of the microspheres is very clearly visible, indicating that insufficient GMA monomer addition cannot fully absorb into the microspheres to achieve a filling effect. When the GMA monomer addition amount is 20.5 g, the data results for the obtained GMA-DVB microspheres are as follows... Figure 19 and Figure 22 As shown, the difference between spherical microspheres and those with 16.4 g of GMA is not significant, but the uniformity of the microspheres is reduced. Considering the economic cost of the reagents used, the optimal amount of GMA was finally determined to be 16.4 g.

[0177] Experiment 7: DVB crosslinking agent addition amount

[0178] Other conditions were the same as in Example 1, except that the DVB addition amount was 3.4 g as described above. All conditions remained unchanged, except that the DVB addition amount was adjusted to 2.8 g and 2.2 g for testing. When the DVB addition amount decreased, the uniformity of the microsphere size was observed to decrease in the particle size analyzer data. Furthermore, theoretically, insufficient crosslinking agent addition may lead to a decrease in the rigidity and mechanical strength of the microspheres, making them unable to withstand the high pressure conditions of subsequent HPLC analysis. Therefore, the DVB addition amount of 3.4 g was still preferred. Specific experimental results are as follows... Figure 20 , Figure 21 , Figure 23 and Figure 24 As shown.

[0179] The above embodiments / experimental examples are merely illustrative and not intended to limit the implementation methods. Those skilled in the art will recognize that various variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementation methods. However, obvious variations or modifications derived therefrom remain within the scope of this invention.

Claims

1. A method for preparing styrene microspheres for glycated hemoglobin detection, characterized in that: Includes the following steps: Step 1: Preparation of seed microspheres using dispersion polymerization: (1) In an argon atmosphere, PVP is dissolved in anhydrous ethanol to form mixture I, and AIBN is dissolved in styrene to form mixture II. Mixture II is added to mixture I and mixed evenly. The mixture is carried out in a water bath with mechanical stirring at 200 rpm. The reaction is pre-initiated at 48-52℃ for 28-32 min, then heated to 67-73℃ and reacted for 10-13 h. The mass ratio of PVP:anhydrous ethanol:AIBN:styrene is 3-6.5:100-103:0.25-0.35:30-34. (2) After the polymerization reaction is completed, the unreacted styrene and PVP raw materials are removed, and the polymer microspheres are dispersed in ethanol / water solution. Microspheres with uniform particle size are obtained by sedimentation separation. (3) Wash the pellets with ethanol, centrifuge and let them settle, then dry them at 58-62℃ for 6-10 hours to obtain dried seed microspheres; Step 2: Preparation of GMA-DVB microspheres using seed polymerization: A. Take seed microspheres, add SDS solution, sonicate for 0.8-1.2 h, maintain water bath heating at 32-38℃ and mechanical stirring at 120-150 rpm; then add DBP to the reaction solution, sonicate for 8-12 min, and swell at 33-36℃ for 10-13 h under mechanical stirring at 120-150 rpm to obtain polystyrene microspheres in a swollen state; wherein, by mass, seed microspheres: SDS solution: DBP = 0.8-1.2: 98-102: 2.3-2.6; B. After ultrasonically mixing GMA, DVB, and BPO for 5 min, add the mixture to the reaction solution and ultrasonicate for 8-12 min. Then, under mechanical stirring at 120-150 rpm, continue swelling at a constant temperature of 34-36℃ for 10-13 h. Finally, add a 5% PVA solution and polymerize at 68-72℃ for 10-13 h. The mass ratio of GMA:DVB:BPO:PVA is 15-20.5:3.2-3.5:0.3-0.5:18-22. C. The product is centrifuged, washed repeatedly with deionized water and ethanol 2-3 times, and the microspheres after sedimentation are dried at 98-102℃ for 10-13h to obtain dried GMA-DVB microspheres. Step 3: Preparation of styrene microspheres: Add 2-propanol and 1,3-propanesulfonyl lactone to GMA-DVB microspheres and heat to 48-52℃; then add 8 mol / L sodium hydroxide aqueous solution, stir for 5-7 h, centrifuge and discard the supernatant, wash with 0.5 mol / L hydrochloric acid, water, 0.5 mol / L sodium hydroxide aqueous solution and water in sequence to obtain styrene microspheres; wherein, by mass, the ratio of GMA-DVB microspheres:isopropanol:1,3-propanesulfonyl lactone:sodium hydroxide aqueous solution is 2.8-3.2:23-25:2.8-3.2:1-1.

3.

2. The method for preparing styrene microspheres for glycated hemoglobin detection according to claim 1, characterized in that: The styrene and azobisisobutyronitrile undergo the following pretreatment: Purification of the styrene: Take 200 mL of styrene into a 1000 mL separatory funnel, wash it three times with 1 M sodium hydroxide aqueous solution, each time using 400 mL, to remove the polymerization inhibitor, then wash it with deionized water until neutral, dry it with anhydrous calcium chloride, then perform vacuum distillation, and store it in a refrigerator for later use. The purification of the azobisisobutyronitrile (AIBN) was carried out by recrystallization. 10 g of AIBN was dissolved in 100 mL of chloroform to form a saturated solution. The solution was filtered using a Buchner funnel. Methanol was added to the resulting solution, and white AIBN crystals precipitated out. The solution was filtered to obtain white AIBN crystals, which were then dried in a vacuum drying oven and then placed in a beaker covered with tin foil and stored in a desiccator for later use.

3. The method for preparing styrene microspheres for glycated hemoglobin detection according to claim 1, characterized in that: In step one, the mass ratio of PVP: anhydrous ethanol: AIBN: styrene is 3.738: 101.25: 0.325: 32.

5.

4. The method for preparing styrene microspheres for glycated hemoglobin detection according to claim 1, characterized in that: By mass, the ratio of seed microspheres:SDS solution:DBP is 1:100:2.5; and the ratio of GMA:DVB:BPO:PVA is 16.4:3.4:0.4:

20.

5. A method for preparing styrene microspheres for glycated hemoglobin detection according to any one of claims 1-3, characterized in that: The ratio of GMA-DVB microspheres: isopropanol: 1,3-propanesulfonyl lactone: sodium hydroxide aqueous solution is 3:24:3:1.

2.

6. A kit for detecting glycated hemoglobin, characterized in that: The kit includes antibody reagents and styrene microspheres prepared by any one of the methods described in claims 1-5.

7. The application of styrene microspheres obtained by any one of claims 1 to 5, characterized in that: The application of the styrene microspheres in the preparation of chromatographic column packing.

8. The application of the styrene microspheres as described in claim 7, characterized in that: The column packing method includes the following steps: (a) Preparation of materials: Clean the homogenizing tank, prepare one chromatographic column and two sieve plates; Homogenization preparation: Mix 20ml of water with 2.5g of microspheres using ultrasonication; Turn on the compressor and cooler to preheat, and add half of the backup water to the column loading machine; (ii) Install a sieve plate on one side of the chromatographic column and mark the liquid inlet direction, i.e., the liquid inlet direction is the same as the liquid inlet direction in high performance liquid chromatography, and connect the other side to the homogenizing tank; fill the homogenizing tank with a uniform homogenate and fill it with filtered water; connect the homogenizing tank to the column packer; (iii) Open the drive speed regulating valve, close the high pressure shut-off valve, and adjust the drive pressure regulating valve until the high pressure gauge displays 4-5 MPa. Then open the high pressure shut-off valve and wait for two and a half minutes. (iv) After the column is packed, close the drive pressure regulating valve and wait for the high pressure gauge to reach 0 MPa. First, remove the column from the homogenizer, and then remove the homogenizer from the column packer. After the column is removed, observe whether the packing effect is good. If it is determined to be full, remove the excess microspheres and observe whether the cut is flat. If it is flat, replenish the sieve plate in time. The column is used for high performance liquid chromatography detection.

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