Glycosylation-containing hydrophilic macromolecular polymer composite carrier, preparation method and application of glycosylation-containing hydrophilic macromolecular polymer composite carrier in HbA1c detection kit
The star-shaped hydrophilic macromolecular carrier prepared by ATRP polymerization is coupled with glycosylated peptides, which solves the problem of uncontrollable protein binding sites in traditional carriers and achieves high stability and consistency in HbA1c detection, making it suitable for accurate detection of whole blood samples.
Patent Information
- Application Number
- CN202511898358.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-16
- Publication Date
- 2026-03-13
AI Technical Summary
In existing HbA1c detection technologies, the binding sites of traditional carrier proteins are uncontrollable and easily affected by environmental factors, resulting in inconsistent and unstable detection results. Furthermore, immunoturbidimetric assays are susceptible to blood sample interference, affecting detection sensitivity.
A star-shaped hydrophilic macromolecular carrier containing double bonds was prepared by ATRP polymerization. It was then coupled with glycosylated peptides via thiol-olefin click reaction to form a stable composite carrier for use in an HbA1c detection kit.
It achieves specific binding between the carrier and the antigen, improves the reliability and consistency of the test results, is suitable for whole blood sample testing, has high stability and anti-aggregation ability, and is adaptable to complex testing environments.
Smart Images
Figure CN121652408A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of HbA1c detection technology, specifically to a glycosylated hydrophilic macromolecular polymer composite carrier and its preparation method, as well as its application in HbA1c detection kits. Background Technology
[0002] Glycated hemoglobin (HbA1c) is a core indicator for the diagnosis and blood glucose management of diabetes, and the accuracy, stability, and convenience of its detection methods have always been a research hotspot. Currently, mainstream detection technologies such as high-performance liquid chromatography (HPLC) and immunoturbidimetry each have their advantages, but also significant shortcomings: HPLC relies on expensive instruments and has a long detection cycle (30-60 minutes), making it difficult to meet the rapid detection needs of primary care facilities; immunoturbidimetry is easily affected by lipid and hemoglobin variants in the sample, and the coupling stability between antibodies and carriers in the detection reagents is poor, leading to carrier aggregation during long-term storage and a decrease in detection sensitivity.
[0003] The immunoturbidimetric competitive assay first binds excess glycated hemoglobin antibody to glycated hemoglobin antigen in a whole blood sample. A polypeptide containing glycosylation sites, bound to a carrier protein, then specifically binds to the remaining glycated hemoglobin antibody, allowing for the detection of the antigen content in the whole blood sample. The specific binding of glycated hemoglobin (HbA1c) depends on its glycosylation sites and the polypeptide composed of multiple amino acids; its unique spatial conformation allows for specific binding to glycated hemoglobin antibodies.
[0004] However, traditional carrier proteins (such as BSA, OVA, etc.) have significant limitations: on the one hand, the binding sites of carrier proteins lack controllability, resulting in significant differences between different batches of reagents; on the other hand, the performance of carrier proteins is easily affected by environmental factors such as temperature, and they are not only prone to conformational denaturation, but also have insufficient long-term storage stability, which in turn affects the consistency of subsequent immunization effects or detection performance. Summary of the Invention
[0005] The purpose of this invention is to provide a glycosylated hydrophilic macromolecular polymer composite carrier, its preparation method, and its application in an HbA1c detection kit. This invention prepares a star-shaped hydrophilic macromolecular carrier containing double bonds via ATRP polymerization. A polypeptide containing an N-terminal glycosylated and a C-terminal cysteine residue is designed and synthesized. The cysteine residue contains a thiol group, which is efficiently coupled to the carrier's double bonds via a thiol-ene click reaction. The hydrophilic macromolecular polymer composite carrier of this invention has a stable chemical structure and strong hydrophilicity. The branched chains effectively hinder intermolecular aggregation. As a highly stable composite carrier, it can be used to detect the content of glycated hemoglobin antigen in whole blood samples.
[0006] Specifically, the present invention provides the following technical solution: A method for preparing a glycosylated hydrophilic macromolecular polymer composite carrier includes the following steps: (1) Preparation of star-shaped hydrophilic macromolecular polymer carriers containing double bonds by ATRP method 1,3,5-Trihydroxybenzenetri(2-bromoisobutyrate) was used as a multi-arm initiator, hydroxyethyl methacrylate and hydroxyethyl methacrylate acrylate were used as monomers, Me6TREN was used as a copper ion ligand, cuprous bromide was used as a catalyst, and isopropanol was used as a solvent. The reaction was carried out at room temperature for 1 h under nitrogen atmosphere, followed by reaction at 50-70 °C for 12-36 h to obtain a hyperbranched star polymer. The copper salt was removed by alumina column chromatography, and the polymer was precipitated with diethyl ether and filtered. (2) Design and synthesis of glycosylated peptides Based on the binding characteristics of glycated hemoglobin antigen, the amino acid sequence designed is Glc-Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Cys, as shown in SEQ ID NO:1. The C-terminal Cys provides a thiol group for coupling with the carrier double bond, and the N-terminal glucosyl Glc is used for specific binding of glycated hemoglobin antibody. Then, a linear polypeptide was synthesized using a solid-phase synthesis method, during which the thiol group of Cys was temporarily protected. After synthesis, a glucose group was attached to the Val residue via a Schiff base reaction, and finally the protecting group of the Cys thiol group was removed to obtain the glycosylated polypeptide. (3) Construction of composite carrier: The star-shaped hydrophilic macromolecular polymer carrier and the glycosylated peptide were dissolved in PBS buffer, a photoinitiator was added, and the mixture was stirred for 10 min under nitrogen protection in the dark. The reaction system was then irradiated under a 365 nm UV lamp. Through the mercapto-alkene click reaction, the mercapto group of the peptide and the double bond of the carrier formed a stable CS bond, thus achieving coupling. After the reaction was completed, the uncoupled free peptide was removed by dialysis to obtain the composite carrier solution.
[0007] In step (1), the molar ratio of hydroxyethyl methacrylate and hydroxyethyl methacrylate acrylate is 1:0.1-0.5.
[0008] In step (1), the molar ratio of cuprous bromide to the total amount of monomer is 1:20-60.
[0009] Specifically, step (2) involves using the Fmoc solid-phase synthesis method with Wang resin as a carrier to sequentially couple Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Thr-OH, Fmoc-Leu-OH, Fmoc-His-OH, and Fmoc-Val-OH. After removing the Fmoc protecting group, acetic acid and UDP-glucose are added, and the reaction is carried out at 37°C for 12 hours. Finally, all protecting groups are removed with trifluoroacetic acid eluent, and the mixture is purified by HPLC to obtain the glycosylated polypeptide Glc-Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Cys.
[0010] Furthermore, the mobile phase for HPLC purification is acetonitrile / water = 30 / 70, containing 0.1% TFA.
[0011] In step (3), the photoinitiator is water-soluble photoinitiator 819.
[0012] In step (3), the ultraviolet light irradiation distance is 10-15cm, and the irradiation time is 15-20min.
[0013] In step (3), the reaction temperature is controlled at 25-30℃.
[0014] The glycosylated hydrophilic macromolecular polymer composite carrier described in this invention is used in the preparation of an immunoturbidimetric HbA1c detection kit. Specifically, it includes: Preparation of R1 reagent: 10-50 mM pH 6.0-7.0 MES buffer containing 0.1-1.0 mg / mL HbA1c monoclonal antibody and 0.08% (v / v) Proclin 300.
[0015] Preparation of R2 reagent: 15-50 mM pH 6.0-7.0 MES buffer containing 0.01-0.1 mg / mL of the complex carrier and 0.08% (v / v) Proclin 300.
[0016] Preparation of R3 reagent: 50-100 mM acetate buffer containing 0.5% (v / v) Triton X-100, 0.1% (w / w) NaCl and 0.08% (v / v) Proclin 300, pH 4.5-6.0.
[0017] The reagent performance was determined using an immunoturbidimetric scattering instrument, and the sample type was whole blood.
[0018] Compared with the prior art, the beneficial effects of the present invention are: (1) Stable and controllable structure: The present invention prepares a novel carrier that can specifically bind to polypeptides containing glycosylation sites. It is prepared by ATRP polymerization and has a regular and stable chemical structure, avoiding the problem of uncontrollable binding sites of traditional carrier proteins. It can ensure the consistency between reagent batches and improve the reliability of detection results.
[0019] (2) Excellent hydrophilicity and anti-aggregation ability: It has strong hydrophilicity and star-shaped branched structure can effectively prevent intermolecular aggregation, reduce the risk of conformational denaturation, and adapt to the complex whole blood sample detection environment.
[0020] (3) High coupling efficiency: The double bond contained in the carrier can be efficiently coupled with the thiol group of the C-terminal cysteine of the polypeptide through the thiol-ene click reaction, which can accurately bind to the target polypeptide containing the glycosylation site and achieve specific binding of the antigen.
[0021] (4) Strong storage and environmental tolerance: It has better chemical stability than traditional carrier proteins, is less affected by environmental factors such as temperature, and has good long-term storage stability, which can ensure the continuous stability of subsequent immune response efficiency and detection performance.
[0022] (5) Adaptable to clinical testing scenarios: As a highly stable composite carrier, it can adapt to the complex system of whole blood samples, efficiently realize the accurate detection of glycated hemoglobin antigen content, and has clear clinical application value. Attached Figure Description
[0023] Figure 1 It has a star-shaped hydrophilic macromolecular polymer carrier structure.
[0024] Figure 2 This is a calibrator test diagram for the reagent kit.
[0025] Figure 3 Correlation test plot for 40 whole blood samples. Detailed Implementation
[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, 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.
[0027] Example 1: Preparation of star-shaped hydrophilic macromolecular polymer carriers containing double bonds by ATRP method 2 mL of the monomer hydroxyethyl methacrylate, 1 mL of hydroxyethyl methacrylate acrylate, 35 g of the initiator 1,3,5-trihydroxybenzenetris(2-bromoisobutyrate), and 16 g of the copper ion ligand Me6TREN (tris[2-(dimethylamino)ethyl]amine) were sequentially added to a reaction flask, along with 200 mL of isopropanol as a solvent. The reaction flask was placed in a liquid nitrogen environment and subjected to a freezing-vacuuming-nitrogen purging cycle, repeated three times to completely remove oxygen from the system. Subsequently, 10 g of the catalyst CuBr was quickly added, and the system was placed in liquid nitrogen again, repeating the freezing-vacuuming-nitrogen purging cycle three times to ensure a stable oxygen-free environment.
[0028] After the reaction system was restored to room temperature, the reaction was carried out at room temperature for 1 hour; then the reaction temperature was raised to 60℃ and the reaction was continued for 24 hours to obtain the target hyperbranched star polymer. After the reaction, the crude polymer product was purified by alumina column chromatography to remove copper salt impurities from the system; the purified polymer solution was slowly added dropwise to excess diethyl ether for precipitation, and after filtration and vacuum drying, the purified hyperbranched star polymer product T0 was obtained, with the structure shown below. Figure 1 As shown.
[0029] Example 2 Synthesis of Glycosylated Peptides Glycosylated peptide synthesis: The Fmoc solid-phase synthesis method was employed, using Wang resin (0.2 g) as a carrier. Fmoc-Cys(Trt)-OH (351.4 mg), Fmoc-Lys(Boc)-OH (221.1 mg), Fmoc-Glu(OtBu)-OH (246.9 mg), Fmoc-Glu(OtBu)-OH (246.9 mg), Fmoc-Pro-OH (202.4 mg), Fmoc-Thr-OH (212.0 mg), Fmoc-Leu-OH (210.9 mg), Fmoc-His-OH (230.1 mg), and Fmoc-Val-OH (202.4 mg) were sequentially coupled, followed by the removal of Fmoc... After the protecting group was removed, a few drops of acetic acid and 169.9 mg of UDP-glucose were added, and the mixture was reacted at 37 °C for 12 h. Finally, all protecting groups were removed with 2 mL of trifluoroacetic acid (TFA) elution buffer, and the mixture was purified by HPLC (mobile phase: acetonitrile / water = 30 / 70, containing 0.1% TFA) to obtain the glycosylated peptide (Glc-Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Cys, the sequence of which is shown in SEQ ID NO:1) at a final concentration of 5%.
[0030] Example 3 Construction of composite carrier T1 50 mg of the star-shaped carrier T0 prepared in Example 1 was dissolved in 10 mL of 10 mM pH 7.4 PBS buffer, 50 mg of the glycosylated peptide prepared in Example 2 was added, and 10 mg of water-soluble photoinitiator 819 was added as a photoinitiator. The mixture was stirred for 10 min under nitrogen protection in the dark. The reaction system was then irradiated with 365 nm ultraviolet light for 18 min at a distance of 10 cm. The reaction temperature was controlled at 25-30 °C. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 10000 Da and dialyzed with 10 mM pH 7.4 PBS buffer for 48 h to obtain a composite carrier solution with a concentration of 1.0 mg / mL.
[0031] Example 4 Construction of composite carrier T2 50 mg of the star-shaped carrier T0 prepared in Example 1 was dissolved in 10 mL of 10 mM pH 7.4 PBS buffer, 75 mg of the glycosylated peptide prepared in Example 2 was added, and 10 mg of water-soluble photoinitiator 819 was added as a photoinitiator. The mixture was stirred for 10 min under nitrogen protection in the dark. The reaction system was then irradiated with 365 nm ultraviolet light for 18 min at a distance of 10 cm. The reaction temperature was controlled at 25-30 °C. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 10000 Da and dialyzed with 10 mM pH 7.4 PBS buffer for 48 h to obtain a composite carrier solution with a concentration of 1.0 mg / mL.
[0032] Example 5 Construction of composite carrier T3 50 mg of the star-shaped carrier T0 prepared in Example 1 was dissolved in 10 mL of 10 mM pH 7.4 PBS buffer, 100 mg of the glycosylated peptide prepared in Example 2 was added, and 10 mg of water-soluble photoinitiator 819 was added as a photoinitiator. The mixture was stirred for 10 min under nitrogen protection in the dark. The reaction system was then irradiated with 365 nm ultraviolet light for 18 min at a distance of 10 cm. The reaction temperature was controlled at 25-30 °C. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 10000 Da and dialyzed with 10 mM pH 7.4 PBS buffer for 48 h to obtain a composite carrier solution with a concentration of 1.0 mg / mL.
[0033] Example 6: Preparation of HbA1c Reagent Kit K1 R1 reagent preparation: 15mM pH 6.5 MES buffer containing 0.5mg / mL HbA1c monoclonal antibody (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD-mAb-HbA1c-01) and 0.08% (v / v) Proclin 300.
[0034] Preparation of R2 reagent: 15mM pH 6.5 MES buffer containing 0.05mg / mL of the composite carrier T1 prepared in Example 3 and 0.08% (v / v) Proclin 300.
[0035] Preparation of R3 reagent: 60mM acetate buffer containing 0.5% (v / v) Triton X-100, 0.1% (w / w) NaCl and 0.08% (v / v) Proclin 300, pH 4.5.
[0036] Example 7 Preparation of HbA1c Reagent Kit K2 R1 reagent preparation: 15mM pH 6.5 MES buffer containing 0.5mg / mL HbA1c monoclonal antibody (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD-mAb-HbA1c-01) and 0.08% (v / v) Proclin 300.
[0037] Preparation of R2 reagent: 15mM pH 6.5 MES buffer containing 0.05mg / mL of the composite carrier T2 prepared in Example 4 and 0.08% (v / v) Proclin 300.
[0038] Preparation of R3 reagent: 60 mM acetate buffer containing 0.5% (v / v) Triton X-100, 0.1% (w / w) NaCl and 0.08% (v / v) Proclin 300, pH 5.0. Example 8: Preparation of HbA1c Reagent Kit K3 R1 reagent preparation: 15mM pH 6.5 MES buffer containing 0.5mg / mL HbA1c monoclonal antibody (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD-mAb-HbA1c-01) and 0.08% (v / v) Proclin 300.
[0039] Preparation of R2 reagent: 15mM pH 6.5 MES buffer containing 0.05mg / mL of the composite carrier T3 prepared in Example 5 and 0.08% (v / v) Proclin 300.
[0040] Preparation of R3 reagent: 60mM acetate buffer containing 0.5% (v / v) Triton X-100, 0.1% (w / w) NaCl and 0.08% (v / v) Proclin 300, pH 5.0.
[0041] Comparative Example 1 The polypeptide Glc-Val-His-Leu-Thr-Pro-Glu-Glu-Lys was synthesized using the method described in Example 2.
[0042] Comparative Example 2: Construction of Glc-Val-His-Leu-Thr-Pro-Glu-Glu-Lys (Biotin) Weigh 5 mg of Glc-Val-His-Leu-Thr-Pro-Glu-Glu-Lys (Comparative Example 1) into a 10 mL brown centrifuge tube, dissolve it in 10 mM pH 7.4 PBS buffer, and bring the volume to 5 mL to prepare a peptide stock solution of approximately 1 mmol / L. Simultaneously, weigh the corresponding amount of Biotin-NHS ester according to a peptide to Biotin-NHS molar ratio of 1:1.5, and prepare a 2 mmol / L stock solution with a small amount of chromatographically pure DMSO. Take 4 mL of the peptide stock solution into a 15 mL brown centrifuge tube, slowly add 3 mL of Biotin-NHS stock solution, and gently vortex to mix. In a dark environment at room temperature (25 °C), react at a low speed of 60 r / min for 4 h to prepare Glc-Val-His-Leu-Thr-Pro-Glu-Glu-Lys (Biotin).
[0043] Comparative Example 3: Preparation of HbA1c Reagent Kit C1 R1 reagent preparation: 15mM pH 6.5 MES buffer containing 0.5mg / mL HbA1c monoclonal antibody (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD-mAb-HbA1c-01) and 0.08% (v / v) Proclin 300.
[0044] Preparation of reagent R2: 15mM pH 6.5 MES buffer containing 0.05mg / mL of Glc-Val-His-Leu-Thr-Pro-Glu-Glu-Lys (Biotin) prepared in Comparative Example 2 and 0.08% (v / v) Proclin 300.
[0045] Preparation of R3 reagent: 60mM acetate buffer containing 0.5% (v / v) Triton X-100, 0.1% (w / w) NaCl and 0.08% (v / v) Proclin 300, pH 4.5.
[0046] Example 9: Reagent Performance Determination 1) Detection instrument: Immunoturbidimetric scattering analyzer; 2) Instrument manufacturer: Anhui Yipunokang Biotechnology Co., Ltd.; 3) Instrument model: Dream T-200PLUS.
[0047] 4) Sample parameters: R1, R2, R3 and sample size are 160μL, 55μL, 250μL and 20μL respectively.
[0048] Table 1 presents the precision test results. Ten repeated tests of the three sample kits (K1, K2, K3) and one control kit (C1) show that the sample kits exhibit superior performance in both low-concentration (L) and high-concentration (H) samples. Regarding repeatability, the coefficients of variation (CV) of K1, K2, and K3 all meet the requirement of ≤10% for quantitative detection of in vitro diagnostic reagents. The CV for low concentration is 4.2%-7.0%, and the CV for high concentration is only 1.8%-2.4%, significantly better than control C1 (10% for low concentration, 9% for high concentration). Furthermore, K2 and K3 show more outstanding repeatability at low concentrations (CV < 5%). In terms of signal intensity, K3... The highest average signal was observed at high concentrations (11.25). The overall signal levels of the example kits (4.10-4.90 for low concentrations and 10.62-11.25 for high concentrations) were superior to those of the control C1 (3.95 for low concentrations and 10.14 for high concentrations). In terms of data stability, the standard deviation (SD) of the self-developed kits was smaller (0.19-0.29 for low concentrations and 0.19-0.27 for high concentrations), while the SD of C1 was significantly larger (0.39 for low concentrations and 0.91 for high concentrations), indicating that the detection consistency and reliability of the example kits were superior to those of the control products.
[0049] Table 1. Precision testing of samples using the HbA1c detection kit. The fitted curve from the HbA1c calibrator test shows ( Figure 2 As shown), the coefficients of determination (R²) of the calibration curves for the three reagent kits (K1, K2, K3) are... 2 The values of K1, K2, and K3 were all at relatively high levels (K1: 0.9971, K2: 0.9977, K3: 0.9982), indicating that the detection concentrations of all three exhibited good linear correlation with the calibrator concentrations, meeting the basic requirement for linearity of the calibration curve in clinical testing (typically R0). 2 ≥0.995). Where R of K3 2 The value closest to 1 indicates the best linear fit, meaning a more accurate correspondence between the "detection signal and calibration concentration" and smaller systematic bias; the R-values of K1 and K2 are... 2 The values were slightly lower but still within the reliable range, and the linearity was also stable. Overall, the calibration performance of all three kits met the specifications for IVD testing, with K3 showing superior calibration linearity, which helps improve the accuracy and traceability of test results.
[0050] In the correlation test of 40 whole blood samples ( Figure 3 The coefficient of determination (R²) between the detection concentration and the actual concentration of the sample for the three HbA1c detection kits (K1, K2, K3) 2 All three (K1: 0.983, K2: 0.989, K3: 0.987) showed a high correlation with the actual concentration of HbA1c in the samples, indicating good consistency and a stable ability to reflect the actual HbA1c level in clinical samples. Specifically, the R-value of K2 was above 0.98. 2 The highest value (0.989) indicates the best fit between the sample data points and the fitted curve, resulting in more prominent correlation and reliability of the detection results; the R-values of K1 and K3 are... 2 Although the value is slightly low, it still meets the requirements for sample correlation in clinical testing (usually R0). 2 (≥0.98). Overall, all three kits showed good correlation performance in actual whole blood sample testing and can be effectively applied to clinical HbA1c testing scenarios. Among them, K2 has a greater advantage in sample matching and detection consistency.
[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.
Claims
1. A method for preparing a glycosylated hydrophilic macromolecular polymer composite carrier, characterized in that, Includes the following steps: (1) Preparation of star-shaped hydrophilic macromolecular polymer carriers containing double bonds by ATRP method 1,3,5-Trihydroxybenzenetri(2-bromoisobutyrate) was used as a multi-arm initiator, hydroxyethyl methacrylate and hydroxyethyl methacrylate acrylate were used as monomers, Me6TREN was used as a copper ion ligand, cuprous bromide was used as a catalyst, and isopropanol was used as a solvent. The reaction was carried out at room temperature for 1 h under nitrogen atmosphere, followed by reaction at 50-70 °C for 12-36 h to obtain a hyperbranched star polymer. The copper salt was removed by alumina column chromatography, and the polymer was precipitated with diethyl ether and filtered. (2) Design and synthesis of glycosylated peptides Based on the binding characteristics of glycated hemoglobin antigen, the amino acid sequence was designed as Glc-Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Cys, where the C-terminal Cys provides a thiol group for coupling with the carrier double bond, and the N-terminal glucosyl Glc is used for specific binding of glycated hemoglobin antibody. Then, a linear polypeptide was synthesized using a solid-phase synthesis method, during which the thiol group of Cys was temporarily protected. After synthesis, a glucose group was attached to the Val residue via a Schiff base reaction, and finally the protecting group of the Cys thiol group was removed to obtain the glycosylated polypeptide. (3) Construction of composite carrier: The star-shaped hydrophilic macromolecular polymer carrier and the glycosylated peptide were dissolved in PBS buffer, a photoinitiator was added, and the mixture was stirred for 10 min under nitrogen protection in the dark. The reaction system was then irradiated under a 365 nm UV lamp. Through the mercapto-alkene click reaction, the mercapto group of the peptide and the double bond of the carrier formed a stable CS bond, thus achieving coupling. After the reaction was completed, the uncoupled free peptide was removed by dialysis to obtain the composite carrier solution.
2. The method for preparing the glycosylated hydrophilic macromolecular polymer composite carrier according to claim 1, characterized in that: In step (1), the molar ratio of hydroxyethyl methacrylate and hydroxyethyl methacrylate acrylate is 1:0.1-0.
5.
3. The method for preparing the glycosylated hydrophilic macromolecular polymer composite carrier according to claim 1, characterized in that: In step (1), the molar ratio of cuprous bromide to the total amount of monomer is 1:20-60.
4. The method for preparing the glycosylated hydrophilic macromolecular polymer composite carrier according to claim 1, characterized in that: Specifically, step (2) involves using the Fmoc solid-phase synthesis method with Wang resin as a carrier to sequentially couple Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Thr-OH, Fmoc-Leu-OH, Fmoc-His-OH, and Fmoc-Val-OH. After removing the Fmoc protecting group, acetic acid and UDP-glucose are added, and the reaction is carried out at 37°C for 12 hours. Finally, all protecting groups are removed with trifluoroacetic acid eluent, and the mixture is purified by HPLC to obtain the glycosylated polypeptide Glc-Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Cys.
5. The method for preparing the glycosylated hydrophilic macromolecular polymer composite carrier according to claim 4, characterized in that: The mobile phase for HPLC purification was acetonitrile / water = 30 / 70, containing 0.1% TFA.
6. The method for preparing the glycosylated hydrophilic macromolecular polymer composite carrier according to claim 1, characterized in that: In step (3), the photoinitiator is water-soluble photoinitiator 819.
7. The method for preparing the glycosylated hydrophilic macromolecular polymer composite carrier according to claim 1, characterized in that: In step (3), the ultraviolet light irradiation distance is 10-15cm, and the irradiation time is 15-20min.
8. The method for preparing the glycosylated hydrophilic macromolecular polymer composite carrier according to claim 1, characterized in that: In step (3), the reaction temperature is controlled at 25-30℃.
9. A glycosylated hydrophilic macromolecular polymer composite carrier prepared by the preparation method according to any one of claims 1-8.
10. The application of the glycosylated hydrophilic macromolecular polymer composite carrier of claim 9 in the preparation of an immunoturbidimetric HbA1c detection kit.