Analytical filler for separating glycosylated hemoglobin and preparation method thereof

Abstract

The invention relates to the technical field of filler preparation, and discloses an analysis filler for separating glycosylated hemoglobin and a preparation method thereof.The analysis filler for separating glycosylated hemoglobin is obtained by synthesizing nonporous Polymer microspheres of 5 micrometers through a dispersion polymerization method, conducting hydrophilic treatment on the surfaces of the Polymer microspheres to obtain Polymer base balls and finally introducing ion exchange groups to the Polymer base balls. The hydrophilicity of the surface of the matrix is enhanced through hydrophilization treatment, non-specific adsorption is reduced, meanwhile, the swelling property of the filler in water is not affected, and the mechanical strength is not affected. The glycosylated hemoglobin analysis filler which is uniform in granularity, good in separation effect and long in service life can be obtained through the preparation method of the analysis filler.

Description

Technical Field

[0001] The invention relates to the technical field of filler preparation, and in particular to an analytical filler for separating glycated hemoglobin and a preparation method thereof. Background Art

[0002] Glycated hemoglobin (Alc) is a type of hemoglobin that reflects the average sugar concentration in the blood over the past 1 to 2 months. Therefore, it can be used as a screening test for diabetes and is widely used as a test item to assess the blood sugar control status of diabetic patients. The HPLC method is widely used as a method for determining hemoglobin Alc because of its good accuracy and the ability to be determined in a short time. In the test, the filler matrix is ​​required to have a certain hydrophobicity to reduce swelling in water, but excessive hydrophobicity will lead to poor dispersion of the filler and cause problems with nonspecific adsorption. The general method for improving the hydrophilicity of the filler is to introduce a large amount of hydrophilic monomers in the synthesis of the base ball, but this will lead to a decrease in the mechanical strength of the filler, swelling in water, and thus a decrease in accuracy.

[0003] In addition, the fillers used to measure hemoglobin A1c in the past were mostly synthesized by suspension polymerization, which had the problem of uneven particle size distribution. In order to make the particle size distribution uniform, methods such as classification operation are generally used. However, the classification operation is complicated, the product loss is large, and the current classification operation has limitations in adjusting the particle size distribution width, and cannot fully suppress the problem of peak broadening caused by sample diffusion due to uneven particle size distribution.

[0004] Therefore, the present application provides a method for preparing an analytical filler for separating glycated hemoglobin to overcome the above problems. Summary of the invention

[0005] The present application provides an analytical filler for separating glycated hemoglobin and a preparation method thereof. The analytical filler has high durability, can measure glycated hemoglobin with high precision, and can achieve good separation of hemoglobin.

[0006] In order to solve the above problems, the technical solutions adopted in this application are as follows:

[0007] In one aspect, the present application provides a method for preparing an analytical filler for separating glycated hemoglobin, comprising the following steps:

[0008] (1) mixing and emulsifying the monomer, the crosslinking agent, the initiator and the polyvinyl alcohol aqueous solution, heating and stirring, soaking and shaking with 95% ethanol to remove the porogen, and obtaining a polymer seed ball;

[0009] (2) reacting the polymer seed ball obtained in step (1) with a strong acid solution or a strong alkaline solution or a chloromethylation agent under heating conditions to obtain a polymer base ball;

[0010] (3) mixing the polymer base ball obtained in step (2), allyl glycidyl ether and Na2SO4 solution, and then adding a strong alkaline solution to react to obtain polymer-vinyl microspheres;

[0011] (4) heating the polymer-vinyl microspheres obtained in step (3) and the ligand monomer under oxygen conditions to react to obtain polymer-COOH or polymer-SO3 microspheres, i.e., 5 μm cation exchange fillers.

[0012] Furthermore, in step (1), the monomer is one or more of methyl methacrylate monomers, ethyl methacrylate, cyclohexyl methacrylate, vinyl glycidyl ether methyl, glycidyl acrylate monomers, and styrene monomers. The monomer is a hydrophobic monomer, which can make the filler not swell or almost not swell in an aqueous medium. The monomer can synthesize a hydrophobic cross-linked polymer.

[0013] Further, in step (1), the crosslinking agent is one or more of divinylbenzene, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, triethylene glycol dimethacrylate, pentaerythritol tetramethacrylate. Further, the crosslinking agent is divinylbenzene.

[0014] Further, in step (1), the initiator is one or more of azobisisobutyronitrile, dimethyl azobisisobutyrate, benzoyl peroxide, and diacyl peroxide. Further, the initiator is azobisisobutyronitrile.

[0015] Furthermore, in step (1), the stabilizer is selected from one or more of polyvinyl alcohol, polyoxyethylene, polyethylene glycol, polyvinyl pyrrolidone, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl propyl cellulose, and sodium polyaspartate. Furthermore, the stabilizer is polyvinyl alcohol.

[0016] Furthermore, in step (1), the heating and stirring temperature is 70-90°C and the time is 10-13 hours. Furthermore, the time is 12 hours.

[0017] Furthermore, in step (1), the shaking time is 6 hours.

[0018] Furthermore, in step (1), the polymer ball is a polymer ball with a particle size of 5 μm and no pores on the surface.

[0019] Further, in step (2), the strong acid solution is selected from one or more of sulfuric acid, hydrochloric acid, and nitric acid. Further, in step (2), the strong acid solution is sulfuric acid.

[0020] Furthermore, in step (2), the strong alkaline solution is selected from one or more of NaOH, KOH or LiOH. Furthermore, in step (2), the strong alkaline solution is NaOH.

[0021] Furthermore, in step (2), the chloromethylation agent is selected from one or more of chloromethyl methyl ether and chloromethyl ethyl ether.

[0022] Furthermore, in step (2), the heating temperature is 25-70°C and the time is 20-24h.

[0023] Furthermore, in step (3), the strong alkaline solution is NaOH.

[0024] Furthermore, in step (3), the stirring conditions are: rotation speed is 160 rpm, and time is 2 h.

[0025] Furthermore, in step (3), the reaction conditions are: rotation speed of 160 rpm, temperature of 25-30° C., and time of 14-16 h.

[0026] Further, in step (4), the ligand monomer is selected from one or more of mercaptosuccinic acid, mercaptoglutaric acid, acrylic acid, butenoic acid, butenedioic acid, glutaric acid, NaHSO3, and Na2SO3. Further, the ligand monomer is selected from one or more of mercaptosuccinic acid and NaHSO3.

[0027] Furthermore, in step (4), the heating temperature is 40-70° C. and the heating time is 10-12 h.

[0028] The mechanism of action of this application is as follows:

[0029] 1. Synthesis of 5μm non-porous Polymer-based spheres by dispersion polymerization of hydrophobic monomers:

[0030] In order to make the filler not swell or hardly swell in aqueous media, a hydrophobic monomer is selected to synthesize a hydrophobic cross-linked polymer as the base ball. The specific method is as follows:

[0031] Using hydrophobic monomers such as methyl methacrylate (MMA), glycidyl methacrylate monomers (GMA), styrene monomers (St), etc., through dispersion polymerization, PMMA microspheres, PGMA microspheres, and PSDVB microspheres with uniform particle size of 5 μm are synthesized, which are collectively referred to as polymer seed balls;

[0032] PMMA microspheres are hydrolyzed through ester bonds under alkaline conditions to generate PMMA-COOH microspheres, or PGMA microspheres are hydrolyzed through epoxy groups under acidic conditions to generate PGMA-OH microspheres, or PSDVB is synthesized by chloromethylation to replace H on the benzene ring to synthesize PSDVB-Cl microspheres. The above three types of microspheres are collectively referred to as Polymer-based microspheres, that is, hydrophobic cross-linked polymer-based microspheres are obtained.

[0033] 2. Treat the Polymer base ball with hydrophilicity:

[0034] The surface of the hydrophobic cross-linked polymer base ball is hydrophilized. After the base ball is functionalized, a hydrophilic block is connected for hydrophilization. For example, the hydrophobic cross-linked polymer base ball is treated with carboxylation, hydroxylation, chloromethylation, etc., and allyl glycidyl ether compounds with dual reactive functional groups of allyl and epoxy are selected to perform hydrophilization on the surface of the base ball through epoxy ring-opening reaction and functional group reaction.

[0035] By using allyl glycidyl ether, the functional groups of the polymer base ball and the epoxy group undergo a ring-opening reaction to obtain PMMA-vinyl or PGMA-vinyl or PSDVB-vinyl microspheres containing hydrophilic groups on the surface, which are collectively referred to as polymer-vinyl microspheres.

[0036] 3. Introducing ion exchange groups into Polymer-vinyl microspheres:

[0037] The ion exchange groups, such as sulfonic acid groups, carboxyl groups, etc., are introduced into the hydrophilized polymer base balls through chemical reactions between functional groups, such as the reaction between double bonds and thiol groups, or the oxidation reaction of double bonds, etc. Specifically:

[0038] Polymer-vinyl microspheres and ligand monomers containing thiol groups, such as mercaptosuccinic acid or mercaptoglutaric acid, are reacted to form polymer-COOH microspheres through the reaction of double bonds on the microsphere surfaces with thiol groups;

[0039] Alternatively, the double bonds on the surface of the microspheres can be oxidized by NaHSO3 to generate Polymer-SO3 microspheres.

[0040] On the other hand, the present application also provides an analytical filler for separating glycated hemoglobin, and the analytical filler is prepared using the above-mentioned preparation method.

[0041] Beneficial effects of this application:

[0042] The present application provides an analytical filler for separating glycated hemoglobin, which uses hydrophobic monomers to synthesize hydrophobic cross-linked polymers, i.e., 5 μm non-porous microspheres as base balls by a dispersion polymerization method, and at the same time, the hydrophilicity of the matrix surface is enhanced by hydrophilization treatment, which reduces nonspecific adsorption, does not affect the swelling of the filler in water, and does not affect the mechanical strength. The polymer after hydrophilization treatment can improve salt resistance and reduce nonspecific adsorption. Nonspecific adsorption is actually an adsorption caused by a non-covalent bond force, which may be caused by the hydrophobic effect between the test compound in the solution and the solid surface. In the application of separation fillers, this nonspecific adsorption may cause nonspecific binding between the target molecule and the filler, thereby affecting the efficiency and purity of the separation, compared to specific adsorption, which is a unique combination between the filler and the target molecule, based on specific interactions between molecules. The analytical filler prepared by the preparation method of the present application has uniform particle size, good separation effect and long service life. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the SEM image of the PMMA microspheres prepared in Example 1-2.

[0044] Figure 2 This is the SEM image of the PGMA microspheres prepared in Example 3-4.

[0045] Figure 3 This is the SEM image of the PSDVB microspheres prepared in Example 5-6.

[0046] Figure 4 HPLC linear elution profile of the filler prepared in Example 1 injected into a GlyHb-HNmicro 4.6*20mm metal prepacked column. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0048] Before introducing the embodiments of the present application, the relevant terms involved in the embodiments of the present application are explained as follows:

[0049] Hemoglobin: It is the product of the combination of hemoglobin in red blood cells and glucose in serum. This combination is formed by non-enzymatic glycosylation reaction, in which the most important carbohydrate is glucose. Hemoglobin glycosylation is a slow and relatively irreversible process, usually lasting more than three months. The production of glycosylated hemoglobin is related to blood sugar concentration and the contact time between blood sugar and hemoglobin, but has nothing to do with factors such as the time of blood drawing, whether the patient is fasting or whether insulin is used.

[0050] Strong cation exchange fillers: Strong cation exchange fillers are a class of highly hydrophilic materials, usually based on matrices such as poly(glycidyl methacrylate) microspheres, and their performance is enhanced by surface modification. This type of filler is mainly used for protein analysis, rapid and efficient separation, analysis and purification of biomacromolecules.

[0051] SEM test: Scanning Electron Microscope test is a microscopic analysis technology that uses a high-energy electron beam to scan the sample surface and capture signals to generate high-resolution images. SEM emits a high-energy electron beam to the sample surface, and these electrons interact with the sample to generate secondary electrons, backscattered electrons and other signals. These electronic signals are captured by the detector and converted into electrical signals, which then form an image to show the microscopic structure of the sample surface.

[0052] O2 bubbling: It is a technology that injects oxygen into liquid in the form of bubbles to increase the solubility and utilization of oxygen.

[0053] Chromatographic column: A chromatographic column is a key component used in high performance liquid chromatography (HPLC) and other chromatographic techniques. Its main function is to separate different components in a mixture.

[0054] Example 1

[0055] This embodiment provides a method for preparing an analytical filler for separating glycated hemoglobin, comprising the following steps:

[0056] (1) 10 g of methyl methacrylate (MMA), 3 g of divinylbenzene (DVB), 0.2 g of azobisisobutyronitrile (AIBN) and 5% polyvinyl alcohol aqueous solution were mixed and emulsified at 1000 rpm for 5 min. The average droplet size was 6 μm. The temperature was raised to 70°C and stirred for 12 h. The unreacted monomers and oligomers were washed repeatedly with ethanol and water for 3 times to remove the unreacted monomers and oligomers. PMMA microspheres with a particle size of 5 μm and no pores on the surface were obtained. The microspheres were dried at 80°C for future use. The SEM test image is shown in the figure below. Figure 1 As shown. Through SEM observation, the surface morphology and particle size of the microspheres can be intuitively seen. Figure 1 It can be seen that the PMMA microspheres are evenly distributed and have an average particle size of 5 μm.

[0057] (2) Take 10 g of PMMA microsphere powder, add 20 g of 0.8 M NaOH aqueous solution, heat to 70 ° C, react at 180 rpm for 24 h, add 40 g of 0.8 M HCl, wash repeatedly with ethanol and water three times to obtain PMMA-COOH microspheres, i.e., polymer-based microspheres, wash repeatedly with ethanol and water three times, and dry at 80 ° C for storage.

[0058] (3) Take 10 g of Polymer base ball, 5 g of allyl glycidyl ether, 0.2 g of Na2SO4, and 100 g of 50% dimethyl sulfoxide aqueous solution, mix the solutions, stir at 160 rpm for 2 h, add NaOH solution, react at 30°C for 16 h to obtain Polymer-vinyl microspheres, wash them repeatedly with ethanol and water for 3 times, and dry them at 80°C for storage.

[0059] (4) Take 5 g of Polymer-vinyl microspheres, 2.5 g of mercaptosuccinic acid, 100 g of water, and 0.1 g of azobisisobutyronitrile, react at 180 rpm and 70°C for 12 h to obtain Polymer-COOH microspheres, wash them repeatedly with ethanol and water for 3 times, and dry them in a forced air drying oven at 90°C to obtain a weak cation exchange filler with an average particle size of 5 μm.

[0060] Example 2

[0061] This embodiment provides a method for preparing an analytical filler for separating glycated hemoglobin, comprising the following steps:

[0062] (1) 10 g of methyl methacrylate (MMA), 3 g of divinylbenzene (DVB), 0.2 g of azobisisobutyronitrile (AIBN) and 5% polyvinyl alcohol aqueous solution were mixed, emulsified at 1000 rpm for 5 min, the average droplet size was 6 μm, the temperature was raised to 70°C, and the reaction was stirred for 12 h. Ethanol and water were repeatedly washed 3 times to remove unreacted monomers and oligomers, and PMMA microspheres with a particle size of 5 μm and no pores on the surface were obtained. They were dried at 80°C and stored for future use. The SEM test image is shown in the figure below. Figure 1 As shown. Through SEM observation, the surface morphology and particle size of the microspheres can be intuitively seen. Figure 1 It can be seen that the PMMA microspheres are evenly distributed and have an average particle size of 5 μm.

[0063] (2) Take 10 g of PMMA microsphere powder, add 20 g of 0.8 M NaOH aqueous solution, heat to 70 ° C, react at 180 rpm for 24 h, add 40 g of 0.8 M HCl, wash repeatedly with ethanol and water three times to obtain PMMA-COOH microspheres, i.e., polymer-based microspheres, wash repeatedly with ethanol and water three times, and dry at 80 ° C for storage.

[0064] (3) Take 10 g of polymer base ball, 5 g of allyl glycidyl ether, 0.2 g of Na2SO4, and 100 g of 50% dimethyl sulfoxide aqueous solution, mix the solutions, stir at 160 rpm for 2 h, add NaOH solution, react at 30°C for 16 h to obtain polymer-vinyl microspheres, wash them repeatedly with ethanol and water for 3 times, and dry them at 80°C for storage.

[0065] (4) Take 5 g of Polymer-vinyl microspheres, 3.2 g of NaHSO3, and 100 g of water, and react with O2 bubbling at 2 ng / s, 180 rpm, and 40°C for 12 h to obtain Polymer-SO3 microspheres. Wash them repeatedly with ethanol and water for 3 times, and dry them in a forced air drying oven at 90°C to obtain a strong cation exchange filler with an average particle size of 5 μm.

[0066] Example 3

[0067] This embodiment provides a method for preparing an analytical filler for separating glycated hemoglobin, comprising the following steps:

[0068] (1) 10 g of glycidyl methacrylate (GMA), 3 g of divinylbenzene (DVB), 0.15 g of azobisisobutyronitrile (AIBN) and 5% polyvinyl alcohol aqueous solution were mixed and emulsified at 1200 rpm for 5 min, with an average droplet size of 6 μm. The mixture was heated to 70°C and stirred for 12 h. The mixture was washed with ethanol and water three times to remove unreacted monomers and oligomers, and PGMA microspheres with a particle size of 5 μm and no pores on the surface were obtained. The microspheres were dried at 80°C and stored for future use. The SEM test image is shown in the figure below. Figure 2 As shown. Through SEM observation, the surface morphology and particle size of the microspheres can be intuitively seen. Figure 2 It can be seen that the PGMA microspheres are evenly distributed and have an average particle size of 5 μm.

[0069] (2) Take 10 g of PGMA ball powder and add 20 g of 0.8 M H2SO4 aqueous solution, heat to 70°C, react at 180 rpm for 24 h, wash repeatedly with ethanol and water 3 times to obtain PGMA-OH microspheres, i.e., polymer-based microspheres, wash repeatedly with ethanol and water 3 times, and dry at 80°C for storage.

[0070] (3) Take 10 g of polymer base ball, 5 g of allyl glycidyl ether, 0.2 g of Na2SO4, and 100 g of 50% dimethyl sulfoxide aqueous solution, mix the solutions, stir at 160 rpm for 2 h, add NaOH solution, react at 30°C for 16 h to obtain polymer-vinyl microspheres, wash them repeatedly with ethanol and water for 3 times, and dry them at 80°C for storage.

[0071] (4) Take 5 g of Polymer-vinyl microspheres, 2.5 g of mercaptosuccinic acid, 100 g of water, and 0.1 g of AIBA, react at 180 rpm and 70°C for 12 h to obtain Polymer-COOH microspheres, wash them repeatedly with ethanol and water for 3 times, and dry them in a forced air drying oven at 90°C to obtain a weak cation exchange filler with an average particle size of 5 μm.

[0072] Example 4

[0073] This embodiment provides a method for preparing an analytical filler for separating glycated hemoglobin, comprising the following steps:

[0074] (1) 10 g of glycidyl methacrylate (GMA), 3 g of divinylbenzene (DVB), 0.15 g of azobisisobutyronitrile (AIBN) and 5% polyvinyl alcohol aqueous solution were mixed and emulsified at 1200 rpm for 5 min, with an average droplet size of 6 μm. The mixture was heated to 70°C and stirred for 12 h. The mixture was washed with ethanol and water three times to remove unreacted monomers and oligomers, and PGMA microspheres with a particle size of 5 μm and no pores on the surface were obtained. The microspheres were dried at 80°C and stored for future use. The SEM test image is shown in the figure below. Figure 2 As shown. Through SEM observation, the surface morphology and particle size of the microspheres can be intuitively seen. Figure 2 It can be seen that the PGMA microspheres are evenly distributed and have an average particle size of 5 μm.

[0075] (2) Take 10 g of PGMA microsphere powder and add 20 g of 0.8 M H2SO4 aqueous solution, heat to 70°C, react at 180 rpm for 24 h, wash repeatedly with ethanol and water 3 times to obtain PGMA-OH microspheres, i.e., polymer-based microspheres, wash repeatedly with ethanol and water 3 times, and dry at 80°C for storage.

[0076] (3) Take 10 g of polymer base ball, 5 g of allyl glycidyl ether, 0.2 g of Na2SO4, and 100 g of 50% dimethyl sulfoxide aqueous solution, mix the solutions, stir at 160 rpm for 2 h, add NaOH solution, react at 30°C for 16 h to obtain polymer-vinyl microspheres, wash them repeatedly with ethanol and water for 3 times, and dry them at 80°C for storage.

[0077] (4) Take 5 g of Polymer-vinyl microspheres, 3.2 g of NaHSO3, and 100 g of water, and react with O2 bubbling at 2 ng / s, 180 rpm, and 40°C for 12 h to obtain Polymer-SO3 microspheres. Wash them repeatedly with ethanol and water for 3 times, and dry them in a forced air drying oven at 90°C to obtain a strong cation exchange filler with an average particle size of 5 μm.

[0078] Example 5

[0079] This embodiment provides a method for preparing an analytical filler for separating glycated hemoglobin, comprising the following steps:

[0080] (1) 10 g of styrene (St), 3 g of divinylbenzene (DVB), 0.1 g of benzoyl peroxide (BPO) and 4% polyvinyl alcohol aqueous solution were mixed and emulsified at 900 rpm for 5 min, with an average droplet size of 6 μm. The mixture was heated to 90°C and stirred for 12 h. The unreacted monomers and oligomers were washed repeatedly with ethanol and water for 3 times to obtain PSDVB microspheres with an average particle size of 5 μm and no pores on the surface. The microspheres were dried at 80°C for future use. The SEM test image is shown in the figure below. Figure 3 As shown. Through SEM observation, the surface morphology and particle size of the microspheres can be intuitively seen. Figure 3 It can be seen that the PSDVB microspheres are evenly distributed with an average particle size of 5 μm.

[0081] (2) Take 10g of PSDVB microsphere powder, add 2.8g of paraformaldehyde, 90mL of dichloroethane, and 20g of concentrated hydrochloric acid, heat to 25°C and react at 180rpm for 24h, wash with ethanol and water three times to obtain PSDVB-Cl microspheres, i.e., polymer-based microspheres, wash with ethanol and water three times, and dry at 80°C for later use. (Among them, dichloroethane is used as a solvent, and paraformaldehyde and concentrated hydrochloric acid are used to chloromethylate the PSDVB microspheres, and the benzene ring is electrophilically substituted by paraformaldehyde, and formaldehyde reacts with hydrogen chloride to generate chloromethyl aromatic compounds with aromatic compounds)

[0082] (3) Take 10 g of polymer base ball, 2.2 g of ethylene glycol, 5 g of allyl glycidyl ether, 0.2 g of Na2SO4, and 100 g of 50% dimethyl sulfoxide aqueous solution, stir the solution at 160 rpm for 2 h, then add NaOH solution, react at 30°C for 16 h to obtain polymer-vinyl microspheres, wash them repeatedly with ethanol and water for 3 times, and dry them at 80°C for storage.

[0083] (4) Take 5 g of Polymer-vinyl microspheres, 2.5 g of mercaptosuccinic acid, 100 g of water, and 0.1 g of AIBA, react at 180 rpm and 70°C for 12 h to obtain Polymer-COOH microspheres, wash them repeatedly with ethanol and water for 3 times, and dry them in a forced air drying oven at 90°C to obtain a weak cation exchange filler with an average particle size of 5 μm.

[0084] Example 6

[0085] This embodiment provides a method for preparing an analytical filler for separating glycated hemoglobin, comprising the following steps:

[0086] (1) 10 g of styrene (St), 3 g of divinylbenzene (DVB), 0.1 g of benzoyl peroxide (BPO) and 4% polyvinyl alcohol aqueous solution were mixed and emulsified at 900 rpm for 5 min. The average droplet size was 6 μm. The temperature was raised to 90°C and stirred for 12 h. Ethanol and water were repeatedly washed three times to remove unreacted monomers and oligomers. PSDVB microspheres with an average particle size of 5 μm and no pores on the surface were obtained. They were dried at 80°C and stored for future use. The SEM test image is shown in the figure below. Figure 3 As shown. Through SEM observation, the surface morphology and particle size of the microspheres can be intuitively seen. Figure 3 It can be seen that the PSDVB microspheres are evenly distributed with an average particle size of 5 μm.

[0087] (2) Take 10g of PSDVB microsphere powder, add 2.8g of paraformaldehyde, 90mL of dichloroethane, and 20g of concentrated hydrochloric acid, heat to 25°C and react at 180rpm for 24h, wash repeatedly with ethanol and water 3 times to obtain PSDVB-C1 microspheres, i.e., polymer-based microspheres, wash repeatedly with ethanol and water 3 times, and dry at 80°C for later use. (Among them, dichloroethane is used as a solvent, and paraformaldehyde and concentrated hydrochloric acid are used to chloromethylate the PSDVB microspheres, and the benzene ring is electrophilically substituted by paraformaldehyde, and formaldehyde reacts with hydrogen chloride to generate chloromethyl aromatic compounds with aromatic compounds)

[0088] (3) Take 10 g of polymer base ball, 2.2 g of ethylene glycol, 5 g of allyl glycidyl ether, 0.2 g of Na2SO4, and 100 g of 50% dimethyl sulfoxide aqueous solution, stir the solution at 160 rpm for 2 h, then add NaOH solution, react at 30°C for 16 h to obtain polymer-vinyl microspheres, wash them repeatedly with ethanol and water for 3 times, and dry them at 80°C for storage.

[0089] (4) Take 5 g of Polymer-vinyl microspheres, 3.2 g of NaHSO3, and 100 g of water, and bubble O2 at 2 ng / s, 180 rpm, and react at 40°C for 12 h to obtain Polymer-SO3 microspheres, which are then washed with water and alcohol and dried in a 90°C forced air drying oven to obtain a strong cation exchange filler with an average particle size of 5 μm.

[0090] Performance Test:

[0091] 1. Load test

[0092] The filler prepared in Example 1 was loaded into the column (4.6*20 mmmL), and the loading capacity test was carried out at a flow rate of 0.5 mL / min.

[0093] The solution preparation method is as follows:

[0094] Balance solution: 10 mM phosphate buffer solution (pH 6.3);

[0095] Loading solution: 1 mg / mL lysozyme solution;

[0096] Washing solution: 10 mM phosphate buffer solution (pH 6.3);

[0097] Eluent: 1 M NaCl 10 mM phosphate buffer solution (pH 7.3).

[0098] The specific experimental methods are as follows:

[0099] (1) Column installation and equilibration: Install the GlyHb-HNmicro 4.6×20 mm metal prepacked column correctly into the HPLC system. Use 10 mM phosphate buffer solution (pH 6.3) at an appropriate flow rate of 0.5-1.0 mL / min to flush the column until the baseline is stable to remove possible impurities in the column and reach equilibrium.

[0100] (2) Sample preparation: Accurately weigh a certain amount of lysozyme standard, dissolve it in 10 mM phosphate buffer solution (pH 6.3) and dilute it to an appropriate concentration (e.g., 0.1-1.0 mg / mL). Use a microinjector to inject the sample into the injector of the HPLC system.

[0101] (3) Chromatographic analysis: Start the HPLC system, set the appropriate flow rate, column temperature and detection wavelength (usually 280 nm). After injecting the sample, observe and record the chromatogram. Note the elution time, peak shape and purity of lysozyme.

[0102] During the entire experiment, all operations should be performed under sterile conditions to avoid contamination.

[0103] The loading capacity is calculated by the following formula:

[0104] Loading capacity = OD 洗脱液 / OD 上样液 ×V 洗脱液 (mg / mL),

[0105] In this Example 1, the loading capacity reaches 11.2 mg / mL, and the filler has excellent adsorption performance.

[0106] 2. Pressure resistance test

[0107] The specific experimental methods are as follows:

[0108] (1) The filler prepared in Example 1 was loaded into the column (4.6*20 mmmL), and a series of different flow rate values ​​were set, usually starting from the minimum flow rate recommended for the column and gradually increasing to the maximum flow rate or approaching the system pressure limit. The composition and pH value of the mobile phase were set and adjusted according to the specific requirements of the GlyHb-HNmicro column. Parameters such as column temperature and detection wavelength were set.

[0109] (2) Run the column at each set flow rate in turn and record the system pressure at each flow rate. The pressure data can be recorded automatically by the HPLC system software or manually. Observe and record the elution curve of the column to check for any abnormalities (such as peak distortion, tailing, etc.).

[0110] Plot the relationship between flow rate and pressure and analyze the pressure change trend with flow rate. Evaluate the pressure stability of the column at different flow rates.

[0111] The pressure flow rate test results of the GlyHb-HNmicro 4.6*20mm metal prepacked column are shown in Table 1. The results show that the filler prepared in Example 1 has excellent pressure resistance.

[0112] Table 1 Pressure and flow rate data table

[0113] Flow rate (mL / min) <![CDATA[1]]> <![CDATA[2]]> <![CDATA[3]]> <![CDATA[4]]> <![CDATA[5]]> <![CDATA[6]]> <![CDATA[7]]> Pressure(Mpa) 1.96 3.63 5.25 6.75 8.16 9.43 10.80

[0114] 3. Linear elution test

[0115] Test fluid: whole blood

[0116] Solution A: pH 5.0 phosphate buffer solution;

[0117] Solution B: pH 7.2 phosphate buffer solution;

[0118] Solution C: EDTA hemolytic agent.

[0119] The specific experimental methods are as follows:

[0120] The filler prepared in Example 1 was injected into the injector of the HPLC system, and the system was started to begin analysis.

[0121] During the analysis, the HPLC system will pump the mobile phase into the GlyHb-HNmicro 4.6*20mm metal pre-packed column according to the set parameters. The components in the sample will be separated in the column and detected by the detector in turn. The software of the PLC system will automatically record the elution time (or elution volume) and corresponding signal intensity (such as peak height or peak area) of each component.

[0122] Export these data and plot them into a linear elution graph. Figure 4As shown, the abscissa generally represents the elution time and the ordinate represents the signal intensity. The results show that the filler has excellent separation performance for alb1, F, LA1C and SA1C proteins in whole blood.

[0123] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an analytical filler for separating glycated hemoglobin, characterized in that: The following steps are involved: (1) mixing and emulsifying the monomer, the cross-linking agent, the initiator and the stabilizer, heating and stirring, soaking and shaking with ethanol, and obtaining a polymer seed ball; (2) reacting the polymer seed ball obtained in step (1) with a strong acid solution or a strong alkaline solution or a chloromethylation agent under heating conditions to obtain a polymer base ball; (3) mixing the polymer base ball obtained in step (2), allyl glycidyl ether and Na2SO4 solution, and then adding a strong alkaline solution to react to obtain polymer-viny1 microspheres; (4) reacting the polymer-vinyl microspheres obtained in step (3) with ligand monomers under heating conditions to obtain polymer-COOH or polymer-SO3 microspheres, i.e., analytical fillers for separating glycated hemoglobin; In step (1), the monomer is one or more of methyl methacrylate monomers, ethyl methacrylate, cyclohexyl methacrylate, vinyl glycidyl ether methyl, glycidyl acrylate monomers, and styrene monomers; In step (2), the strong acid solution is one or more of sulfuric acid, hydrochloric acid, and nitric acid, the strong alkaline solution is one or more of NaOH, KOH, and LiOH, and the chloromethylation agent is one or more of chloromethyl methyl ether or chloromethyl ethyl ether; In step (4), the ligand monomer is one or more of mercaptosuccinic acid, mercaptoglutaric acid, acrylic acid, butenoic acid, butenedioic acid, glutaric acid, NaHSO3, and Na2SO3.

2. The method for preparing an analytical filler for separating glycated hemoglobin according to claim 1, characterized in that: In the step (1), the crosslinking agent is one or more of divinylbenzene, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, triethylene glycol dimethacrylate, and pentaerythritol tetramethacrylate.

3. The method for preparing an analytical filler for separating glycated hemoglobin according to claim 1, characterized in that: In the step (1), the initiator is one or more of azobisisobutyronitrile, dimethyl azobisisobutyrate, benzoyl peroxide, and diacyl peroxide.

4. The method for preparing an analytical filler for separating glycated hemoglobin according to claim 1, characterized in that: In the step (1), the stabilizer is selected from one or more of polyvinyl alcohol, polyoxyethylene, polyethylene glycol, polyvinyl pyrrolidone, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl propyl cellulose, and sodium polyaspartate.

5. The method for preparing an analytical filler for separating glycated hemoglobin according to claim 1, characterized in that: In the step (1), the temperature of heating and stirring is 70-90° C. and the time is 10-13 h.

6. The method for preparing an analytical filler for separating glycated hemoglobin according to claim 1, characterized in that: In the step (2), the heating temperature is 25-70° C. and the heating time is 20-24 h.

7. The method for preparing an analytical filler for separating glycated hemoglobin according to claim 1, characterized in that: In the step (3), the reaction temperature is 25-30°C and the reaction time is 14-16h.

8. The method for preparing an analytical filler for separating glycated hemoglobin according to claim 1, characterized in that: In the step (4), the heating temperature is 40-70° C. and the heating time is 10-12 h.

9. An analytical filler for separating glycated hemoglobin, characterized in that: The analytical filler is prepared by the preparation method described in any one of claims 1 to 8.

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