Monodisperse polystyrene microspheres for glycated hemoglobin detection and preparation method thereof

Abstract

The present invention belongs to the field of protein detection and separation materials, and specifically relates to monodisperse polystyrene microspheres for glycated hemoglobin detection and a preparation method thereof. The present invention grafts hyperbranched polyglycidyl ether onto the surface of monodisperse porous polystyrene microspheres, and wraps HPG of a certain molecular weight on the surface of the microspheres by a relatively simple modification method, which can shield the hydrophobic effect on the surface of the microspheres, thereby reducing the non-specific adsorption of proteins, and also improves the biocompatibility and service life of the microspheres, and then grafts olefin chlorides, and finally sulfopropyl monomers and hydrophilic uncharged olefin monomers are surface-modified and polymerized on the HPG surface, which can form a reasonable distribution of sulfopropyl groups and uncharged hydrophilic groups. This structure separates various glycated protein components through efficient ion exchange, thereby making up for the shortcomings of poor separation effect, short service life and high cost of the current modification methods.

Description

Technical Field

[0001] The invention belongs to the field of protein detection and separation materials, and particularly relates to monodisperse polystyrene microspheres for glycosylated hemoglobin detection and a preparation method thereof. Background Art

[0002] With the improvement of living standards, changes in dietary structure, increasingly tense pace of life, and a sedentary lifestyle, the global incidence of diabetes has increased rapidly. Diabetes has become the third most serious chronic disease threatening human health after tumors and cardiovascular diseases.

[0003] The diagnosis and detection of glycated hemoglobin (GHb), measured as HbA1c, is a valuable tool for monitoring long-term glycemic control and is therefore a key point in diabetes care. HbA1c is the result of irreversible nonenzymatic glycosylation of one or both N-terminal valines of the Hbβ chain. This irreversible nonenzymatic reaction between glucose and HbA (the main Hb type in normal adults) occurs during the life cycle of red blood cells. The total amount depends on the average glucose concentration in the 2-3 months before the measurement. Several methods have been developed based on different principles, such as ion exchange chromatography, immunoagglutination, boronate affinity assay and electrophoresis. Among them, ion exchange chromatography is stable and highly reproducible and is recommended as the gold standard for glycated hemoglobin detection.

[0004] At present, many ion exchange column methods and electrophoresis methods cannot separate HbF protein from HbA1c in blood samples. HbF and HbA1c co-migrate or co-elute, which will lead to a high measured HbA1c value and affect the judgment of patient diagnosis. For example, in the invention application CN101531701A-a method for preparing a glycosylated hemoglobin separation material, the separation effect of several proteins is not ideal. The preparation of microspheres inside ion exchange columns is currently mainly divided into copolymerization and multi-step modification methods. The copolymerization method refers to the one-step polymerization of microsphere preparation monomers such as methacrylate or styrene with sulfonic acid or carboxyl monomers. Although this type of method has a simple preparation process, it is inevitably used in the separation of glycated hemoglobin and causes irreversible adsorption of some proteins in the blood, which has a great impact on the separation and detection life and results. For example, patent applications CN110314664A-A method for synthesizing a monodisperse HbA1C ion exchange chromatographic filler and its application, CN112742359A-A method for manufacturing a column filler for glycated hemoglobin analysis and CN113549183A-A method for preparing a filler for a glycated hemoglobin chromatography column, etc. The multi-step modification method generally refers to modifying a layer of hydrophilic surface on the surface of polymer microspheres to minimize the nonspecific adsorption of glycosylated proteins, thereby improving the separation and effect of detection, and also maintaining a high service life of the microspheres. This type of method includes hydrophilic modification of the microsphere surface by ozone, such as patent CN101257969B-hydrophilic polymer microparticles, and fillers for ion exchange liquid chromatography and their manufacturing methods. There are also methods for surface hydrophilic modification by grafting hydroxyl and carboxyl groups on the surface of microspheres, such as patent CN113512152B-vinyl monomer-polyvinyl crosslinker copolymer non-porous microspheres and their preparation methods and applications. However, the above hydrophilic modification methods have the disadvantages of poor effect, short service life and high cost.

[0005] Hyperbranched polyglycidyl ether (HPG) is a highly hydrophilic polymer material with a highly branched structure. On the one hand, in terms of physical and chemical properties, the characteristics of HPG are very similar to those of PEG (polyethylene glycol), with excellent hydrophilicity and good biocompatibility, applicable to a high pH range, stable chemical properties, and safe and non-toxic compared to PEG. On the other hand, since HPG has a large number of hydroxyl unit structures, it can be modified by simple reactions, such as surface modification by acyl chloride or ring-opening polymerization grafting side chains, and surface free radical polymerization or click chemistry modification to give HPG more properties. Based on the above characteristics, hyperbranched polyglycidyl ether (HPG) is an ideal modification material for improving the hydrophilicity of the microsphere surface. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides monodisperse polystyrene microspheres for detecting glycated hemoglobin and a method for preparing the same, in order to solve the problem that in the existing method for separating glycated hemoglobin from the microspheres inside the ion exchange column, the copolymerization method causes irreversible adsorption of some proteins in the blood during hemoglobin separation, which has a great impact on the separation and detection life and results; the traditional multi-step modification method has the technical problems of poor effect, short service life and high cost.

[0007] The present invention provides a method for preparing monodisperse polystyrene microspheres for detecting glycosylated hemoglobin, and the specific technical scheme is as follows:

[0008] The invention discloses a method for preparing monodisperse polystyrene microspheres for detecting glycated hemoglobin. The method comprises the following steps: grafting hyperbranched glycidyl ether onto the surface of monodisperse porous polystyrene microspheres, and then performing surface modification with sulfopropyl monomers and hydrophilic uncharged olefin monomers to obtain monodisperse polystyrene microspheres for detecting glycated hemoglobin.

[0009] Further, the following steps are included:

[0010] S1, 3 μm polystyrene seed microspheres are dispersed in 0.3 wt% sodium dodecylbenzene sulfonate solution, nitrogen is introduced to deoxygenate, styrene, crosslinking agent and porogen solution are added, and after swelling at room temperature for 24 hours, an equal volume of 10 wt% hydroxymethyl cellulose is added and mixed evenly, and the mixture is heated to 70° C. and reacted for 24 hours, and then washed with ethanol and deionized water to obtain monodisperse porous polystyrene microspheres;

[0011] S2, ultrasonically mixing the monodisperse porous polystyrene microspheres in step S1 with dichloromethane, adding a chloroethyl ether reagent and stirring evenly, adding a catalyst, and reacting at a temperature of 5-10° C. for 12-24 hours to obtain chlorinated polystyrene microspheres;

[0012] S3, dispersing 2 ml of a mixed solution of glycidol and 1,2-propylene glycol in 50 ml of dichloromethane, and adding a trace amount of boron trifluoride etherate as a catalyst to initiate a polymerization reaction, and performing the polymerization reaction at room temperature for 24 hours. After removing the dichloromethane solvent by rotary evaporation, a light yellow transparent viscous substance is obtained, and then the light yellow transparent viscous substance is dissolved in a mixed solvent comprising 10 ml of water, 5 ml of polyethylene glycol and 30 ml of isopropanol, respectively, to obtain a hyperbranched polyglycidol solution;

[0013] S4, dispersing the hyperbranched polyglycidol solution in step S3 in a 3M potassium hydroxide solution, adding the chlorinated polystyrene microspheres in step S2, stirring and mixing at room temperature, heating to 50° C. for one hour, cooling to room temperature and continuing to react for 12-24 hours, washing and drying with pure water and methanol, and obtaining microspheres with surface grafted HPG;

[0014] S5, dispersing the microspheres with surface grafted HPG in step S4 into tetrahydrofuran solvent, adding olefin acyl chloride reagent and acid binding agent at 2-5° C., reacting for 12-24 hours, washing with methanol and drying, to obtain microspheres with rich surface olefin groups;

[0015] S6, dispersing the microspheres with olefin groups on the surface in step S5 into an organic solvent, adding sulfopropyl monomers, hydrophilic uncharged monomers and initiators, stirring and introducing nitrogen to react at 70°C for 12-24 hours, and then washing and drying with pure water and methanol to obtain polystyrene microspheres with hydrophilic sulfonic acid groups modified on the surface, i.e., monodisperse polystyrene microspheres for glycated hemoglobin detection.

[0016] In certain embodiments, in step S1, the ratio of styrene to the cross-linking agent is 2:1 to 1:1, and the ratio of styrene to the porogen is 1:1 to 1:2; the cross-linking agent is any one of divinylbenzene, 3-diisopropenylbenzene, and 2-butenylbenzene; the porogen is any one of n-octanol, isooctyl alcohol, dodecanol, diethyl phthalate, dibutyl phthalate, dioctyl phthalate, and didecyl phthalate.

[0017] In certain embodiments, in step S2, the mixing ratio of monodisperse porous polystyrene microspheres and dichloromethane is 1:5-1:10, the amount of chloroethyl ether reagent added is 5wt% to 10wt% of the total weight, the amount of the catalyst added is 0.5wt% to 2wt% of the total weight, the chloromethyl ether reagent is any one of chloromethyl ethyl ether, chloroethyl ethyl ether, and chlorobutyl ethyl ether, and the catalyst is any one of anhydrous tin tetrachloride, zinc chloride, acetic acid, and phosphoric acid.

[0018] In some embodiments, in step S3, the volume ratio of glycidol to 1,2-propylene glycol is 1:1 to 1:5, and the volume of boron trifluoride ether added accounts for 0.01 to 0.05 of the total volume. HPG solutions with different molecular weights can be prepared by adjusting the ratio of glycidol, 1,2-propylene glycol and catalyst. When the ratio of glycidol (2,3-epoxy-1-propanol) to 1,2-propylene glycol is 1:1 and the added volume of the catalyst boron trifluoride etherate accounts for 0.01 of the volume ratio in the whole reaction, the prepared HPG has a molecular weight of 500; when the ratio of glycidol (2,3-epoxy-1-propanol) to 1,2-propylene glycol is 2:1 and the added volume of the catalyst boron trifluoride etherate accounts for 0.01 of the volume ratio in the whole reaction, the prepared HPG has a molecular weight of 1500; when the ratio of glycidol (2,3-epoxy-1-propanol) to 1,2-propylene glycol is 5:1 and the added volume of the catalyst boron trifluoride etherate accounts for 0.05 of the volume ratio in the whole reaction, the prepared HPG has a molecular weight of 4500. By optimizing and selecting HPG with a suitable molecular weight to achieve the optimal hydrophilic modification result, the nonspecific adsorption of protein to the surface of the microsphere can be avoided.

[0019] In certain embodiments, in step S4, the molecular weight of the hyperbranched glycidyl ether is 500, 1500 or 4500, and the weight ratio of the chlorinated polystyrene microspheres to the hyperbranched glycidyl ether is 1:10-1:50.

[0020] In certain embodiments, in step S5, the ratio of the microspheres grafted with HPG on the surface to the tetrahydrofuran is 1:5-1:10, the ratio of the acyl chloride reagent to the microspheres grafted with HPG on the surface is 1:1-1:5, the ratio of the acid binding agent to the microspheres grafted with HPG on the surface is 1:10-1:50, the acyl chloride reagent is any one of acryloyl chloride, methacryloyl chloride, crotonoyl chloride and 4-pentenoyl chloride; the acid binding agent is any one of triethylamine, tripropylamine, diisopropylethylamine and pyridine.

[0021] In certain embodiments, in step S6, the mass ratio of the vinyl-rich microspheres, the sulfopropyl monomer, the hydrophilic uncharged monomer and the initiator is 10:5:5:1, the sulfopropyl monomer is any one of sodium allyl sulfonate, sodium methacrylic sulfonate, sodium ethyl allyl sulfonate, 2-acrylamide-2-methylpropane sulfonic acid and 2-acrylamide-2-ethylpropane sulfonic acid; the hydrophilic uncharged monomer is any one of acrylamide, crotonamide, methacrylamide, N-ethylacrylamide and N-methyl-2-acrylamide, acryl alcohol, and isobutylene alcohol; the initiator is one of benzoyl peroxide, azobisisobutyronitrile, azobisisovaleronitrile and azobisisoheptylonitrile, and the organic solvent is one of N, N-dimethylformamide, N, N-dimethylacetamide, tetrahydrofuran and dimethyl sulfoxide.

[0022] The present invention also provides another technical solution, namely, monodisperse polystyrene microspheres for detecting glycosylated hemoglobin, which are prepared by the above method.

[0023] The present invention has the following beneficial effects: the preparation method of monodisperse polystyrene microspheres for glycated hemoglobin detection provided by the present invention adopts polystyrene microspheres with high uniformity as the substrate, and has the following advantages: In addition, hyperbranched polyglycidyl ether (HPG) is grafted onto the surface of polystyrene microspheres, which can avoid the non-specific adsorption of proteins on the surface of microspheres. For the hydrophilic modification of the surface of polystyrene microspheres, HPG is an ideal material for modification. HPG with a certain molecular weight is wrapped on the surface of microspheres by a relatively simple multi-step modification method, which can shield the hydrophobic effect of the surface of microspheres to the maximum extent, thereby reducing the non-specific adsorption of proteins, and improving the biocompatibility and service life of microspheres is very important. Then, olefin acyl chloride is grafted, and finally sulfopropyl monomers and hydrophilic uncharged monomers are polymerized on the surface of HPG, which can form a reasonable distribution of sulfopropyl and uncharged hydrophilic groups. This structure can separate various glycated protein components through efficient ion exchange, thereby making up for the shortcomings of poor separation effect, short service life and high cost in the current modification method. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic flow chart of a method for preparing monodisperse polystyrene microspheres for detecting glycosylated hemoglobin of the present invention;

[0025] Figure 2 It is a schematic diagram of the electron microscope structure of monodisperse polystyrene microspheres for detecting glycosylated hemoglobin of the present invention;

[0026] Figure 3 This is a diagram showing the hydrophilicity test results of monodisperse polystyrene microspheres for glycated hemoglobin detection of the present invention;

[0027] Figure 4This is a detection diagram of monodisperse polystyrene microspheres packed in a column for glycated hemoglobin detection according to the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the following is a detailed description of the present invention in conjunction with the following specific embodiments. Figure 1-4 , the present invention is further described in detail.

[0029] Example 1

[0030] The method for preparing monodisperse polystyrene microspheres for glycated hemoglobin detection provided in this embodiment comprises the following steps:

[0031] Step 1, preparation of monodisperse porous polystyrene microspheres: first, 3 micron polystyrene seed microspheres are dispersed in 0.3wt% sodium dodecylbenzene sulfonate solution, nitrogen is introduced for 30 minutes for deoxygenation, and a certain proportion of styrene, crosslinking agent and porogen solution are added (the ratio of styrene to crosslinking agent is 2:1, and the ratio of styrene to porogen is 1:1). After swelling at room temperature for 24 hours, 10wt% of hydroxymethyl cellulose is added and mixed evenly, and the mixture is heated to 70°C for reaction for 24 hours. After the reaction is completed, the monodisperse porous crosslinked polystyrene microspheres are washed with ethanol and deionized water to obtain.

[0032] Step 2, add monodisperse porous polystyrene microspheres into a reactor, add dichloromethane for ultrasonic mixing (the mixing ratio of monodisperse porous polystyrene microspheres to dichloromethane is 1:5), then add 5wt% of chloromethyl ether reagent and mechanically stir until the mixture is uniformly mixed, add 0.5wt% of catalyst, react at 5°C for 24 hours to obtain chlorinated polystyrene microspheres.

[0033] Step 3, dissolve 12 ml of a mixture of glycidol and 1,2-propylene glycol (the volume ratio of glycidol to 1,2-propylene glycol is 1:1) in 38 ml of dichloromethane, and add 0.5 ml of boron trifluoride etherate as a catalyst to initiate a polymerization reaction. The polymerization reaction is carried out at room temperature for 24 hours, and then the dichloromethane solvent is removed by rotary evaporation to obtain a light yellow transparent viscous substance, which is then dissolved in a mixed solvent comprising 10 ml of water, 5 ml of polyethylene glycol and 30 ml of isopropanol. By adjusting the ratio of each reaction component, three HPGs with different molecular weights of 600, 1500 and 4500 were prepared.

[0034] Step 4, dispersing the HPG solution in a 3M potassium hydroxide solution, adding the chlorinated polystyrene microspheres prepared in step 2, stirring and mixing at room temperature, first heating to 50°C to react for one hour, then cooling to room temperature to react for 12 hours, and then washing and drying with pure water and methanol to obtain microspheres with surface grafted HPG;

[0035] Step 5, dispersing the microspheres rich in HPG on the surface into tetrahydrofuran solvent, the ratio of the microspheres grafted with HPG on the surface to tetrahydrofuran is 1:5, adding acyl chloride reagents and acid binding agents at 5°C, the ratio of the acyl chloride reagents to the microspheres grafted with HPG on the surface is 1:1, and the ratio of the acid binding agent to the microspheres grafted with HPG on the surface is 1:10, reacting for 12 hours, washing with methanol and drying, to obtain microspheres rich in vinyl on the surface;

[0036] Step 6, dispersing the vinyl-rich microspheres in an organic solvent, adding sulfopropyl and acrylamide monomers and initiators, the mass ratio of the vinyl-rich microspheres, sulfopropyl monomers, hydrophilic uncharged monomers and initiators is 10:5:5:1, mechanically mixing and stirring, introducing nitrogen to react for 12 hours, and finally washing and drying with pure water and methanol to obtain polystyrene microspheres with surface modified hydrophilic sulfonic acid groups.

[0037] In this embodiment, in step 1, the cross-linking agent is 3-diisopropenylbenzene; and the porogen is diethyl phthalate.

[0038] In step 2, the chloromethyl ether reagent is chloromethyl methyl ether; and the catalyst is zinc chloride.

[0039] In step 3, when the ratio of glycidol (2,3-epoxy-1-propanol) to 1,2-propylene glycol is 1:1 and the added volume of the catalyst boron trifluoride ether is 0.01 in the entire reaction volume ratio, HPG with a molecular weight of 500 is prepared.

[0040] In step 4, the molecular weight of HPG is selected to be 500, and the weight ratio of polystyrene microspheres to HPG addition is 1:10.

[0041] In step 5, the acyl chloride reagent is methacryloyl chloride; and the acid binding agent is tripropylamine.

[0042] In step 6, the sulfopropyl monomer is sodium methacrylic sulfonate; the amide monomer is crotonamide; the initiator is benzoyl peroxide; and the organic solvent is N,N-dimethylacetamide.

[0043] Example 2

[0044] The method for preparing monodisperse polystyrene microspheres for glycated hemoglobin detection provided in this embodiment comprises the following steps:

[0045] Step 1, preparation of monodisperse porous polystyrene microspheres: first, 3-micron polystyrene seed microspheres are dispersed in a 0.3wt% sodium dodecylbenzene sulfonate solution, and after nitrogen is introduced for 30 minutes for deoxygenation, a certain proportion of styrene, crosslinking agent and porogen solution are added (the ratio of styrene to crosslinking agent is 1.5:1, and the ratio of styrene to porogen is 1:1.5), and after swelling at room temperature for 24 hours, the solution is heated to 70°C for reaction for 24 hours. After the reaction is completed, the solution is washed with ethanol and deionized water to obtain monodisperse porous crosslinked polystyrene microspheres.

[0046] Step 2, adding monodisperse porous polystyrene microspheres into a reactor, and adding dichloromethane for ultrasonic mixing (the mixing ratio of monodisperse porous polystyrene microspheres to dichloromethane is 1:7), then adding 8wt% of chloromethyl ether reagent and mechanically stirring until the mixture is uniformly mixed, adding 1wt% of catalyst, and reacting at 5°C for 24 hours to obtain chlorinated polystyrene microspheres;

[0047] Step 3, preparation of HPG (hyperbranched polyglycidol) with different molecular weights: 12 ml of a mixture of glycidol and 1,2-propylene glycol (the volume ratio of glycidol to 1,2-propylene glycol is 1:2) is dissolved in 38 ml of dichloromethane, and 0.5 ml of boron trifluoride etherate is added as a catalyst to initiate the polymerization reaction. The polymerization reaction is carried out at room temperature for 24 hours, and then the dichloromethane solvent is removed by rotary evaporation to obtain a light yellow transparent viscous substance, and then the viscous polymer is dissolved in a mixed solvent containing 10 ml of water, 5 ml of polyethylene glycol and 30 ml of isopropanol. By adjusting the ratio of each reaction component, three HPGs with different molecular weights are prepared, namely 600, 1500 and 4500.

[0048] Step 4, dispersing the HPG solution in a 3M potassium hydroxide solution, adding the chlorinated polystyrene microspheres prepared in step 2, stirring and mixing at room temperature, first heating to 50°C to react for one hour, then cooling to room temperature to react for 12-24 hours, and then washing and drying with pure water and methanol to obtain microspheres with surface grafted HPG;

[0049] Step 5, dispersing the microspheres rich in HPG on the surface into tetrahydrofuran solvent, the ratio of the microspheres grafted with HPG on the surface to tetrahydrofuran is 1:8, adding acyl chloride reagents and acid binding agents at 5°C, the ratio of the acyl chloride reagents to the microspheres grafted with HPG on the surface is 1:2, the ratio of the acid binding agent to the microspheres grafted with HPG on the surface is 1:20, after reacting for 12 hours, washing and drying with methanol to obtain microspheres rich in vinyl on the surface;

[0050] Step 6, dispersing the vinyl-rich microspheres in an organic solvent, adding a certain amount of sulfopropyl and acrylamide monomers and initiators, the mass ratio of the vinyl-rich microspheres, sulfopropyl monomers, hydrophilic uncharged monomers and initiators is 10:5:5:1, and after mechanical mixing and stirring, nitrogen is introduced to react for 12 hours, and finally, the polystyrene microspheres with surface modified hydrophilic sulfonic acid groups are obtained after washing and drying with pure water and methanol.

[0051] In this embodiment, in step 1, the cross-linking agent is divinylbenzene; and the porogen is dibutyl phthalate.

[0052] In step 2, the chloromethyl ether reagent is chloromethyl ethyl ether; and the catalyst is anhydrous tin tetrachloride.

[0053] In step 3, when the ratio of glycidol (2,3-epoxy-1-propanol) to 1,2-propylene glycol is 2:1 and the added volume of the catalyst boron trifluoride ether is 0.01 in the entire reaction volume ratio, HPG with a molecular weight of 1500 is prepared.

[0054] In step 4, the molecular weight of HPG is selected to be 1500, and the weight ratio of polystyrene microspheres to HPG addition is 1:20.

[0055] In step 5, the acyl chloride reagent is acryloyl chloride; the acid binding agent is triethylamine; the reaction temperature is preferably 2° C., and the reaction time is preferably 16 hours.

[0056] In step 6, the sulfopropyl monomer is sodium ethyl allyl sulfonate; the amide monomer is acrylamide; the initiator is azobisisobutyronitrile, and the organic solvent is N,N-dimethylformamide.

[0057] Example 3

[0058] The method for preparing monodisperse polystyrene microspheres for glycated hemoglobin detection provided in this embodiment comprises the following steps:

[0059] Step 1: Preparation of monodisperse porous polystyrene microspheres: First, 3-micron polystyrene seed microspheres are dispersed in 0.3wt% sodium dodecylbenzene sulfonate solution, and nitrogen is introduced for 30 minutes for deoxygenation. Then, a certain proportion of styrene, crosslinker and porogen solution are added (the ratio of styrene to crosslinker is 1:1, and the ratio of styrene to porogen is 1:2). After swelling at room temperature for 24 hours, the solution is heated to 70°C for reaction for 24 hours. After the reaction, the solution is washed with ethanol and deionized water to obtain monodisperse porous crosslinked polystyrene microspheres.

[0060] Step 2, adding monodisperse porous polystyrene microspheres into a reactor, and adding dichloromethane for ultrasonic mixing (the mixing ratio of monodisperse porous polystyrene microspheres to dichloromethane is 1:10), then adding 10wt% of chloromethyl ether reagent and mechanically stirring until the mixture is uniformly mixed, adding 0.5wt% of a catalyst, and reacting at 5°C for 24 hours to obtain chlorinated polystyrene microspheres;

[0061] Step 3: Preparation of HPG (hyperbranched polyglycidol) with different molecular weights: Dissolve a mixture of glycidol and 1,2-propylene glycol (the volume ratio of glycidol to 1,2-propylene glycol is 1:5) in 38 ml of dichloromethane, and add 0.5 ml of boron trifluoride etherate as a catalyst to initiate the polymerization reaction. The polymerization reaction is carried out at room temperature for 24 hours, and then the dichloromethane solvent is removed by rotary evaporation to obtain a light yellow transparent viscous substance, and then the viscous polymer is dissolved in a mixed solvent containing 10 ml of water, 5 ml of polyethylene glycol and 30 ml of isopropanol. By adjusting the ratio of each reaction component, three HPGs with different molecular weights of 600, 1500 and 4500 were prepared.

[0062] Step 4, dispersing the HPG solution in a 3M potassium hydroxide solution, adding the chlorinated polystyrene microspheres prepared in step 2, stirring and mixing at room temperature, first heating to 50°C to react for one hour, then cooling to room temperature to react for 24 hours, and then washing and drying with pure water and methanol to obtain microspheres with surface grafted HPG;

[0063] Step 5, dispersing the microspheres rich in HPG on the surface into tetrahydrofuran solvent, the ratio of the microspheres grafted with HPG on the surface to tetrahydrofuran is 1:5-1:10, adding acyl chloride reagents and acid binding agents at 5°C, the ratio of the acyl chloride reagents to the microspheres grafted with HPG on the surface is 1:5, and the ratio of the acid binding agent to the microspheres grafted with HPG on the surface is 1:50, reacting for 12 hours, washing with methanol and drying, to obtain microspheres rich in vinyl on the surface;

[0064] Step 6, dispersing the vinyl-rich microspheres in an organic solvent, adding a certain amount of sulfopropyl and acrylamide monomers and initiators, the mass ratio of the vinyl-rich microspheres, sulfopropyl monomers, hydrophilic uncharged monomers and initiators is 10:5:5:1, and after mechanical mixing and stirring, nitrogen is introduced to react for 12 hours, and finally, the polystyrene microspheres with surface modified hydrophilic sulfonic acid groups are obtained after washing and drying with pure water and methanol.

[0065] In this embodiment, in step 1, the cross-linking agent is 2-butenylbenzene; and the porogen is dioctyl phthalate.

[0066] In step 2, the chloromethyl ether reagent is chloromethyl butyl ether; and the catalyst is acetic acid.

[0067] In step 3, when the ratio of glycidol (2,3-epoxy-1-propanol) to 1,2-propylene glycol is 5:1 and the added volume of the catalyst boron trifluoride ether is 0.05 in the entire reaction volume ratio, HPG with a molecular weight of 4500 is prepared.

[0068] In step 4, the molecular weight of HPG is 4500; the weight ratio of polystyrene microspheres to HPG is 1:50; and the reaction time is 12-24 hours, preferably 16 hours.

[0069] In step 5, the acyl chloride reagent is crotonoyl chloride; and the acid binding agent is diisopropylethylamine.

[0070] In step 6, the sulfopropyl monomer is 2-acrylamide-2-methylpropanesulfonic acid; the amide monomer is N-methyl-2-acrylamide; the initiator is azobisisovaleronitrile; and the organic solvent is dimethyl sulfoxide.

[0071] The present invention obtains highly uniform monodisperse polymer microspheres for glycated hemoglobin detection, such as Figure 2 As shown, the prepared microspheres are monodisperse and have uniform particle size.

[0072] Hydrophilicity test: The synthesized microspheres were assembled into a 4.6 mm*50 mm chromatographic column, installed on a high performance liquid chromatography instrument, and the lysozyme protein was injected under pH=7 phosphate buffer conditions. At a flow rate of 1 ml / min, the penetration curve was recorded. Figure 3 As shown, the peak height and peak area of ​​the three injections are approximately the same, indicating that the surface of the microspheres has good hydrophilicity and the hydrophilic layer modified with HPG on the surface almost eliminates the hydrophobic interaction between the microspheres and proteins.

[0073] Detection effect test: 1. Take 1g of the prepared monodisperse polymer microspheres for glycated hemoglobin detection and load them into the column. Use 0.1M sodium chloride solution to make a 10wt% homogenate. After ultrasonication for 5 minutes, pour the homogenate into a 10ml homogenate tank. Use pure water as the displacement liquid. Fill the microspheres into a 4.6mm×30mm PEEK chromatographic column tube at a pressure of 2000psi.

[0074] 2. Install the assembled chromatographic column on the liquid chromatograph, use 200mM citric acid + 200mM sodium chloride as the mobile phase to balance the chromatographic column, inject 20 microliters of the diluted glycated blood sample, and perform isocratic elution. The detection spectrum is as follows: Figure 4 As shown: In about 1 minute, various components such as HbF, L-A1c, HbA1c and HbA0 in glycated blood samples can be efficiently and quickly detected.

[0075] In summary, the preparation method of monodisperse polystyrene microspheres for glycated hemoglobin detection provided by the present invention adopts highly uniform monodisperse polystyrene microspheres as a substrate, has the advantages of being able to tolerate a wide range of pH values, low chromatographic column back pressure, high separation efficiency, long service life, and being washable and recyclable; in addition, hyperbranched polyglycidyl ether (HPG) is grafted onto the surface of polystyrene microspheres, which can avoid nonspecific adsorption of proteins on the surface of microspheres. HPG is an ideal material for hydrophilic modification of the surface of monodisperse polystyrene microspheres. HPG with a certain molecular weight is wrapped on the surface of the microspheres through a relatively simple multi-step modification method, which can shield the hydrophobic effect on the surface of the microspheres to the maximum extent, thereby reducing the non-specific adsorption of proteins, which is crucial to improving the biocompatibility and service life of the microspheres. Then, olefin acyl chlorides are grafted and sulfopropyl monomers are polymerized on the HPG surface to form a reasonable distribution of sulfopropyl and uncharged hydrophilic groups. This structure can separate various glycated protein components through efficient ion exchange, thereby making up for the shortcomings of current modification methods, such as poor separation effect, short service life and high cost.

[0076] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. The present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for preparing monodisperse polystyrene microspheres for detecting glycosylated hemoglobin, characterized in that: The steps include: S1, 3 μm polystyrene seed microspheres are dispersed in 0.3 wt% sodium dodecylbenzene sulfonate solution, nitrogen is introduced to deoxygenate, styrene, crosslinking agent and porogen solution are added, and after swelling at room temperature for 24 hours, an equal volume of 10 wt% hydroxymethyl cellulose is added and mixed evenly, and the mixture is heated to 70° C. and reacted for 24 hours, and then washed with ethanol and deionized water to obtain monodisperse porous polystyrene microspheres; S2, ultrasonically mixing the monodisperse porous polystyrene microspheres in step S1 with dichloromethane, adding a chloroethyl ether reagent and stirring evenly, adding a catalyst, and reacting at a temperature of 5-10° C. for 12-24 hours to obtain chlorinated polystyrene microspheres; S3, dispersing a mixed solution of glycidol and 1,2-propylene glycol in dichloromethane, adding boron trifluoride etherate, and polymerizing at room temperature for 24 hours, removing the dichloromethane solvent by rotary evaporation to obtain a light yellow transparent viscous substance, and then dissolving the light yellow transparent viscous substance in a mixed solvent comprising 10 ml of water, 5 ml of polyethylene glycol and 30 ml of isopropanol, respectively, to obtain a hyperbranched polyglycidol solution; S4, dispersing the hyperbranched polyglycidol solution in step S3 in a 3M potassium hydroxide solution, adding the chlorinated polystyrene microspheres in step S2, stirring and mixing at room temperature, heating to 50° C. for one hour, cooling to room temperature and continuing to react for 12-24 hours, washing and drying with pure water and methanol, and obtaining microspheres with surface grafted HPG; S5, dispersing the microspheres with surface grafted HPG in step S4 into tetrahydrofuran solvent, adding olefin acyl chloride reagent and acid binding agent at 2-5° C., reacting for 12-24 hours, washing with methanol and drying, to obtain microspheres with rich surface olefin groups; S6, dispersing the microspheres with olefin groups on the surface in step S5 into an organic solvent, adding sulfopropyl monomers, hydrophilic uncharged monomers and initiators, stirring and introducing nitrogen to react at 70°C for 12-24 hours, and then washing and drying with pure water and methanol to obtain polystyrene microspheres with hydrophilic sulfonic acid groups modified on the surface, i.e., monodisperse polystyrene microspheres for glycated hemoglobin detection.

2. The method for preparing monodisperse polystyrene microspheres for detecting glycosylated hemoglobin according to claim 1, characterized in that: In step S1, the ratio of styrene to the cross-linking agent is 2:1 to 1:1, and the ratio of styrene to the porogen is 1:1 to 1:2; the cross-linking agent is any one of divinylbenzene, 3-diisopropenylbenzene, and 2-butenylbenzene; the porogen is any one of n-octanol, isooctyl alcohol, dodecanol, diethyl phthalate, dibutyl phthalate, dioctyl phthalate, and didecyl phthalate.

3. The method for preparing monodisperse polystyrene microspheres for detecting glycosylated hemoglobin according to claim 1, characterized in that: In step S2, the mixing ratio of monodisperse porous polystyrene microspheres and dichloromethane is 1:5-1:10, the amount of chloroethyl ether reagent added is 5wt%-10wt% of the total weight, the amount of the catalyst added is 0.5wt%-2wt% of the total weight, the chloromethyl ether reagent is any one of chloromethyl ethyl ether, chloroethyl ethyl ether, and chlorobutyl ethyl ether, and the catalyst is any one of anhydrous tin tetrachloride, zinc chloride, acetic acid, and phosphoric acid.

4. The method for preparing monodisperse polystyrene microspheres for detecting glycosylated hemoglobin according to claim 1, characterized in that: In step S3, the volume ratio of glycidol to 1,2-propylene glycol is 1:1 to 1:5, and the ratio of the added volume of boron trifluoride ether to the total volume is 0.01 to 0.

05.

5. The method for preparing monodisperse polystyrene microspheres for detecting glycosylated hemoglobin according to claim 1, characterized in that: In step S4, the molecular weight of the hyperbranched glycidyl ether is 500, 1500 or 4500, and the weight ratio of the chlorinated polystyrene microspheres to the hyperbranched glycidyl ether is 1:10-1:

50.

6. The method for preparing monodisperse polystyrene microspheres for detecting glycosylated hemoglobin according to claim 1, characterized in that: In step S5, the ratio of the microspheres grafted with HPG on the surface to the tetrahydrofuran is 1:5-1:10, the ratio of the acyl chloride reagent to the microspheres grafted with HPG on the surface is 1:1-1:5, the ratio of the acid binding agent to the microspheres grafted with HPG on the surface is 1:10-1:50, the acyl chloride reagent is any one of acryloyl chloride, methacryloyl chloride, crotonoyl chloride and 4-pentenoyl chloride; the acid binding agent is any one of triethylamine, tripropylamine, diisopropylethylamine and pyridine.

7. The method for preparing monodisperse polystyrene microspheres for detecting glycosylated hemoglobin according to claim 1, characterized in that: In step S6, the mass ratio of the vinyl-rich microspheres on the surface, the sulfopropyl monomer, the hydrophilic uncharged monomer and the initiator is 10:5:5:1, the sulfopropyl monomer is any one of sodium allyl sulfonate, sodium methacrylic sulfonate, sodium ethyl allyl sulfonate, 2-acrylamide-2-methylpropanesulfonic acid and 2-acrylamide-2-ethylpropanesulfonic acid; the hydrophilic uncharged monomer is any one of acrylamide, crotonamide, methacrylamide, N-ethylacrylamide and N-methyl-2-acrylamide, acryl alcohol, and isobutylene alcohol; the initiator is one of benzoyl peroxide, azobisisobutyronitrile, azobisisovaleronitrile and azobisisoheptylonitrile, and the organic solvent is one of N, N dimethylformamide, N, N dimethylacetamide, tetrahydrofuran and dimethyl sulfoxide.

8. Monodisperse polystyrene microspheres for glycated hemoglobin detection, characterized in that: Microspheres prepared by the method according to any one of claims 1 to 7.

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