Carboxyl polystyrene microsphere, preparation method and application of carboxyl polystyrene microsphere in kit for quantitatively detecting MxA

Abstract

The invention relates to the technical field of in-vitro diagnostic kits, in particular to carboxyl polystyrene microspheres, a preparation method and application of the carboxyl polystyrene microspheres in a kit for quantitatively detecting MxA. The preparation method of the carboxyl polystyrene microspheres comprises the steps that firstly, polystyrene microspheres with the surface rich in aldehyde groups are prepared through a soap-free emulsion polymerization method; then carrying out a Schiff alkali reaction with 2-aminoethyl methacrylate hydrochloride to carry out double bond modification on the surface of the microsphere, and meanwhile, improving the hydrophilicity of the microsphere by an ester group and an imino group; and finally, carrying out sulfydryl-double bond addition reaction on the polystyrene microspheres and mercaptosuccinic acid to generate the polystyrene microspheres rich in carboxyl on the surfaces. The carboxyl polystyrene microspheres prepared by the invention can improve the binding efficiency of latex and an MxA antibody, so that the detection sensitivity is improved, the purpose of high-sensitivity quantitative detection of MxA protein can be achieved, and the detection time is greatly shortened.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagent kit technology, specifically to a carboxylated polystyrene microsphere and its preparation method, as well as its application in a quantitative detection kit for MxA. Background Technology

[0002] Polystyrene microspheres have received widespread attention and application in fields such as pharmaceutical engineering, chemical engineering, environmental protection, and electronic information. Their advantages, including easy surface functionalization, strong reactivity, and excellent reagent immobilization properties, determine their important position in the biomedical field. Various functionalized microspheres, magnetic microspheres, and fluorescent polystyrene microspheres have attracted considerable attention from researchers and clinical experts, and the prepared microspheres have shown promising applications in clinical testing, cell tracking, and drug sustained release.

[0003] MxA protein, as an interferon-induced antiviral protein, has serum levels that reflect the body's antiviral immune status and is of significant value in the diagnosis of infectious diseases. However, the commonly used clinical latex immunoturbidimetric assay for MxA detection suffers from insufficient sensitivity, particularly poor accuracy in low-concentration samples.

[0004] Currently, the binding efficiency of commercially available carboxylated polystyrene microspheres with MxA antibodies is low, resulting in poor sensitivity of latex immunoturbidimetric assay for MxA biochemical reagents. Summary of the Invention

[0005] The purpose of this invention is to provide a carboxylated polystyrene microsphere, a preparation method thereof, and its application in a quantitative detection kit for MxA.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing carboxylated polystyrene microspheres: First, polystyrene microspheres with aldehyde-rich surfaces are prepared by soap-free emulsion polymerization; then, the microsphere surface is modified with double bonds by reacting with 2-aminoethyl methacrylate hydrochloride through a Schiff base reaction; finally, polystyrene microspheres with carboxyl-rich surfaces are generated by reacting with mercaptosuccinic acid through a mercapto-double bond addition reaction.

[0007] The specific steps are as follows: Add pure water to a three-necked flask, purge with nitrogen to remove oxygen for 30 minutes, add an ionic strength regulator, dissolve the flask, add styrene and aldehyde functional monomers, heat to 60℃, add an initiator, and react at 75-85℃ for 12 hours. After the reaction is complete, wash three times with pure water and three times with anhydrous ethanol, and finally store in anhydrous ethanol. The microspheres stored in anhydrous ethanol were transferred to a reaction flask, and 2-aminoethyl methacrylate hydrochloride and glacial acetic acid were added. After reacting at room temperature for 30 min, mercaptosuccinic acid and azobisisoheptanenitrile were added, and the reaction was continued at room temperature for 24 h. After the reaction was completed, the microspheres were washed three times with anhydrous ethanol and three times with pure water. Finally, the microspheres were stored in pure water and the solid content was adjusted to 10%.

[0008] The ionic strength modifier is at least one of NaCl, Na2CO3, and NaHCO3.

[0009] The aldehyde functional monomer is methacrolein and / or acrolein, wherein the volume ratio of the aldehyde functional monomer to the styrene monomer is 1:5-10.

[0010] The initiator is a water-soluble initiator, and is any one of potassium persulfate, sodium persulfate, and ammonium persulfate.

[0011] The molar ratio of the aldehyde functional monomer to 2-aminoethyl methacrylate hydrochloride is 1:1.0-1.5.

[0012] The molar ratio of 2-aminoethyl methacrylate hydrochloride to mercaptosuccinic acid is 1:1.0-1.5.

[0013] The amount of azobisisoheptanenitrile added is 1%-5% of 2-aminoethyl methacrylate hydrochloride.

[0014] The carboxylated polystyrene microspheres prepared by the above-described preparation method of the present invention have a particle size range of 350-450 nm.

[0015] The carboxylated polystyrene microspheres of this invention can be used to prepare a latex immunoturbidimetric assay kit for the quantitative detection of MxA. The specific preparation method is as follows: (a) Preparation of reagent R1 Tween 20, PEG20000, and Proclin 300 were added sequentially to a 20 mM, pH 6.0 MES buffer solution. The final concentrations of each reagent in the system were controlled as follows: Tween 20 volume fraction 0.1%, PEG20000 mass concentration 1%, and Proclin 300 volume fraction 0.08%. After adding the samples, the mixture was thoroughly mixed to ensure the system was homogeneous.

[0016] (b) Preparation of reagent R2 ① Microsphere dilution: Dilute the carboxylated polystyrene microspheres prepared in this invention to a concentration of 2 mg / mL with 50 mM pH 6.0 MES buffer; ② Activation: Weigh EDC and prepare a stock solution of 10 mg / mL with 50 mM pH 6.0 MES buffer; add the EDC stock solution to the above solution to make the final concentration 0.5 mg / mL, incubate at 37℃ with shaking for 0.5 hours, centrifuge at 13000 r / min for 25 min to remove the supernatant, and resuspend in PB buffer at pH 7.2; ③ Coupling: Dilute the MxA antibody to 1 mg / mL with PB buffer at pH 7.2, add it to the activated microspheres at a volume ratio of 1:5, and incubate at 37°C with shaking for 2 hours; ④ Blocking: Add 1% bovine serum albumin to a final concentration, incubate at 37°C with shaking for 1 hour; centrifuge the above blocking reagent to remove the supernatant, resuspend and mix with 10 mM Tris buffer (pH 7.5) containing 4-8% trehalose, and then disperse by sonication to obtain the MxA antibody-microsphere complex, i.e., R2 solution.

[0017] Compared with the prior art, the beneficial effects of the present invention are: (1) Optimization of carboxyl density: The surface of the carboxyl polystyrene microspheres of the present invention is rich in carboxyl groups. The surface carboxyl density is further improved by specific process control, providing sufficient reaction sites for subsequent functionalization modification and binding with active substances.

[0018] (2) Improved monodispersity: The introduction of multiple hydrophilic groups such as ester, imino, and thiol groups into the hydrophilic segments on the surface of microspheres can effectively improve the dispersion stability between particles, significantly improve the monodispersity of the microsphere system, and reduce agglomeration. (3) Enhanced detection performance: The carboxylated polystyrene microspheres have a high efficiency and specific binding ability between the carboxyl groups on their surface and MxA antibodies, which greatly improves the antibody immobilization efficiency and can improve the binding efficiency between latex and MxA antibodies, thereby improving the detection sensitivity. This enables the high-sensitivity quantitative detection of MxA protein, greatly shortens the detection time, and provides support for the accurate detection of low-concentration target substances. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the preparation process of the carboxylated polystyrene microspheres of the present invention. Detailed Implementation

[0020] 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.

[0021] Example 1 Preparation of carboxylated polystyrene microspheres PS-1 Combination Figure 1 As shown, 200 mL of pure water was added to a three-necked flask, and nitrogen gas was purged for 30 min to remove oxygen. 50 mg of NaCl, an ionic strength modifier, was added and dissolved. Then, 20 mL of styrene and 2 mL of methacrolein were added. The mixture was heated to 60 °C, and 100 mg of potassium persulfate was added. The reaction was carried out at 80 °C for 12 h. After the reaction was complete, the mixture was washed three times with pure water and three times with anhydrous ethanol, and finally stored in 100 mL of anhydrous ethanol.

[0022] The microspheres preserved in anhydrous ethanol were transferred to a reaction flask, and 4.5 g of 2-aminoethyl methacrylate hydrochloride and 1 mL of glacial acetic acid were added. After reacting at room temperature for 30 min, 4.4 g of mercaptosuccinic acid and 0.1 g of azobisisoheptanenitrile were added, and the reaction was continued at room temperature for 24 h. After the reaction was completed, the microspheres were washed three times with anhydrous ethanol and three times with pure water. Finally, the microspheres were stored in pure water, and the solid content was adjusted to 10%.

[0023] Example 2 Preparation of carboxylated polystyrene microspheres PS-2 Combination Figure 1 As shown, 200 mL of pure water was added to a three-necked flask, and nitrogen gas was purged for 30 min to remove oxygen. 50 mg of NaCl, an ionic strength modifier, was added and dissolved. 20 mL of styrene and 3 mL of methacrolein were then added. The mixture was heated to 60 °C, and 100 mg of potassium persulfate was added. The reaction was carried out at 80 °C for 12 h. After the reaction was complete, the flask was washed three times with pure water and three times with anhydrous ethanol, and finally stored in 100 mL of anhydrous ethanol.

[0024] The microspheres preserved in anhydrous ethanol were transferred to a reaction flask, and 6.1 g of 2-aminoethyl methacrylate hydrochloride and 1 mL of glacial acetic acid were added. After reacting at room temperature for 30 min, 6.6 g of mercaptosuccinic acid and 0.1 g of azobisisobutyronitrile were added, and the reaction was continued at room temperature for 24 h. After the reaction was completed, the microspheres were washed three times with anhydrous ethanol and three times with pure water. Finally, the microspheres were stored in pure water, and the solid content was adjusted to 10%.

[0025] Example 3 Preparation of carboxylated polystyrene microspheres PS-3 Combination Figure 1 As shown, 200 mL of pure water was added to a three-necked flask, and nitrogen gas was purged for 30 min to remove oxygen. 50 mg of NaCl, an ionic strength modifier, was added and dissolved. 20 mL of styrene and 4 mL of methacrolein were then added. The mixture was heated to 60 °C, and 100 mg of potassium persulfate was added. The reaction was carried out at 80 °C for 12 h. After the reaction was complete, the flask was washed three times with pure water and three times with anhydrous ethanol, and finally stored in 100 mL of anhydrous ethanol.

[0026] The microspheres preserved in anhydrous ethanol were transferred to a reaction flask, and 8.8 g of 2-aminoethyl methacrylate hydrochloride and 1 mL of glacial acetic acid were added. After reacting at room temperature for 30 min, 8.8 g of mercaptosuccinic acid and 0.1 g of azobisisoheptanenitrile were added, and the reaction was continued at room temperature for 24 h. After the reaction was completed, the microspheres were washed three times with anhydrous ethanol and three times with pure water. Finally, the microspheres were stored in pure water, and the solid content was adjusted to 10%.

[0027] Example 4: Preparation of MxA Detection Kit (Latex Immunoturbidimetric Assay) (1) Preparation of reagent R1 Add Tween 20, PEG20000, and Proclin 300 sequentially to a 20 mM, pH 6.0 MES buffer (2-morpholine ethanesulfonic acid buffer). Control the final concentrations of each reagent in the system as follows: Tween 20 volume fraction 0.1%, PEG20000 mass concentration 1%, and Proclin 300 volume fraction 0.08%. Mix thoroughly after adding the samples to ensure system homogeneity.

[0028] (2) Preparation of reagent R2 ① Microsphere dilution: The microspheres prepared in Examples 1-3 and the control microspheres (model P0425, purchased from JSR Corporation) were diluted with 50 mM pH 6.0 MES buffer to a concentration of 2 mg / mL. ② Activation: Weigh EDC and prepare a stock solution of 10 mg / mL with 50 mM pH 6.0 MES buffer; add the EDC (ethyl (3-(dimethylamino)propyl)carbodiimide hydrochloride) stock solution to the above solution to make a final concentration of 0.5 mg / mL, incubate at 37℃ with shaking for 0.5 hours, centrifuge at 13000 r / min for 25 min to remove the supernatant, and resuspend in pH 7.2 PB buffer (phosphate buffer); ③Conjugation: Dilute the two MxA antibodies (Nanjing Liding Medical Technology Co., Ltd., catalog number: mAb03, mAb04) to 1 mg / mL with PB buffer at pH 7.2, and add them to the activated microspheres at a volume ratio of 1:5. Incubate at 37°C with shaking for 2 hours. ④ Blocking: Add 1% bovine serum albumin to a final concentration, incubate at 37°C with shaking for 1 hour; centrifuge the above blocking reagent to remove the supernatant, and resuspend in 10 mM Tris buffer (pH 7.5) containing 4-8% trehalose, mix well, and then disperse by sonication to obtain the MxA antibody-microsphere complex, i.e., R2 solution.

[0029] Example 5 Test (1) Particle size test: Particle size and dispersion index were tested using an Otsuka Electronics particle size analyzer. Three samples were diluted with deionized water and measured three times to determine the average particle size and dispersion index (PDI).

[0030] (2) Carboxyl content test: The surface carboxyl density was quantitatively analyzed by acid-base titration method with conductivity detection (DDS-S07A, Leici).

[0031] (3) Sample testing: Whole blood samples were tested using the Jinrui PA120 fully automated specific protein analyzer.

[0032] Table 1 shows the particle size, dispersion index, and carboxyl density of the carboxylated polystyrene microspheres prepared in Examples 1-3 and the control microspheres (model P0425, purchased from JSR Corporation). The particle sizes were 418 nm, 426 nm, 432 nm, and 428 nm, respectively, and the dispersion index PDI < 0.05, indicating good monodispersity. The carboxyl densities of the microspheres in Examples 1-3 were 0.355 mmol / g, 0.402 mmol / g, and 0.437 mmol / g, respectively, all higher than the carboxyl density of the control microspheres.

[0033] A key technical bottleneck exists in the preparation of carboxylated polystyrene microspheres using emulsion polymerization: as the microsphere particle size increases, it becomes difficult to effectively graft carboxyl groups onto the microsphere surface. This directly leads to the common problem of low carboxyl content in commercially available large-particle-size carboxylated polystyrene nanospheres. In this example, polystyrene microspheres with high aldehyde content on the surface are first synthesized. Subsequently, a two-step process involving a Schiff base reaction and a thiol-double bond addition reaction is used to achieve efficient grafting of carboxyl groups onto the microsphere surface. This strategy has the advantage of a clear stoichiometric reaction relationship: an aldehyde group can be linked to a double bond through the reaction, and a double bond can further introduce two carboxyl groups through an addition reaction, ultimately successfully preparing polystyrene microspheres with a surface rich in carboxyl groups.

[0034] Table 1 Microsphere Particle Size and Dispersion Index

[0035] Table 2 shows the reactivity of the MxA detection reagent prepared in Example 4 to whole blood calibrated samples. The results indicate that the MxA detection reagents assembled using the microspheres prepared in Examples 1-3 all exhibited significantly higher reactivity than those prepared using the control microspheres (model P0425, purchased from JSR Corporation). This suggests that the MxA detection reagents using the microspheres from the examples have superior sensitivity. The core reason for this difference is presumably that the microspheres from the examples have a higher carboxyl group content on their surface. Carboxyl groups, as key binding sites, can effectively enhance the binding efficiency with MxA antibodies, thereby increasing the reagent's reactivity and detection sensitivity. In terms of linearity, the R² values ​​for Examples 1-3 were 0.9997, 0.9998, and 0.9997, respectively, all superior to the control example's 0.9967.

[0036] Table 2. Detection reactivity of the MxA detection reagent prepared in Example 4 to whole blood calibrated samples.

[0037] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing carboxylated polystyrene microspheres, characterized in that: First, polystyrene microspheres with aldehyde-rich surfaces were prepared by soap-free emulsion polymerization. Then, the surface of the microspheres was modified with double bonds by reacting with 2-aminoethyl methacrylate hydrochloride through a Schiff base reaction. Finally, polystyrene microspheres with carboxyl-rich surfaces were generated by reacting with mercaptosuccinic acid through a mercapto-double bond addition reaction.

2. The method for preparing carboxylated polystyrene microspheres according to claim 1, characterized in that: Add pure water to the reaction vessel, purge with nitrogen to remove oxygen, add ionic strength regulator, dissolve and add styrene and aldehyde functional monomer, heat to 60℃, add initiator, react at 75-85℃ for 12h, after the reaction is complete, wash three times with pure water and three times with anhydrous ethanol, and finally store in anhydrous ethanol. The microspheres stored in anhydrous ethanol were transferred to a reaction vessel, and 2-aminoethyl methacrylate hydrochloride and glacial acetic acid were added. After reacting at room temperature, mercaptosuccinic acid and azobisisoheptanenitrile were added, and the reaction was continued at room temperature. After the reaction was completed, the microspheres were washed three times with anhydrous ethanol and three times with pure water. Finally, the microspheres were stored in pure water, and the solid content was adjusted to 10%.

3. The method for preparing carboxylated polystyrene microspheres according to claim 2, characterized in that: The ionic strength modifier is at least one of NaCl, Na2CO3, and NaHCO3.

4. The method for preparing carboxylated polystyrene microspheres according to claim 3, characterized in that: The aldehyde functional monomer is methacrolein and / or acrolein, wherein the volume ratio of the aldehyde functional monomer to the styrene monomer is 1:5-10.

5. The method for preparing carboxylated polystyrene microspheres according to claim 4, characterized in that: The initiator is a water-soluble initiator, and is any one of potassium persulfate, sodium persulfate, and ammonium persulfate.

6. The method for preparing carboxylated polystyrene microspheres according to claim 5, characterized in that: The molar ratio of the aldehyde functional monomer to 2-aminoethyl methacrylate hydrochloride is 1:1.0-1.

5.

7. The method for preparing carboxylated polystyrene microspheres according to claim 6, characterized in that: The molar ratio of 2-aminoethyl methacrylate hydrochloride to mercaptosuccinic acid is 1:1.0-1.

5.

8. The method for preparing carboxylated polystyrene microspheres according to claim 7, characterized in that: The amount of azobisisobutyronitrile added is 1%-5% of 2-aminoethyl methacrylate hydrochloride.

9. The carboxylated polystyrene microspheres prepared by the method according to any one of claims 1-8, characterized in that: The particle size range is 350-450 nm.

10. The application of the carboxylated polystyrene microspheres according to claim 9 in the preparation of a latex immunoturbidimetric assay kit for quantitative detection of MxA.

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