Carboxyl polystyrene microspheres, preparation method and application thereof in quantitative detection MxA kit

Abstract

The present application relates to the technical field of in vitro diagnostic kit, especially to a carboxyl polystyrene microsphere and a preparation method thereof and application of the carboxyl polystyrene microsphere in a quantitative detection MxA kit, the preparation method of the carboxyl polystyrene microsphere is as follows: first, preparing polystyrene microspheres rich in aldehyde groups on the surface through a soap-free emulsion polymerization method; then, through Schiff base reaction, the surface of the microspheres is modified with 2-aminoethyl methacrylate hydrochloride, and meanwhile, the ester group and imino group can improve the hydrophilicity of the microspheres; finally, through a thiol-double bond addition reaction, the surface of the microspheres is generated with carboxyl-rich polystyrene microspheres through mercaptobutane dioic acid. The carboxyl polystyrene microsphere prepared by the present application can improve the binding efficiency of the latex and MxA antibody, thereby improving the detection sensitivity, and the purpose of high-sensitivity quantitative detection of MxA protein can be achieved, and the detection time is greatly shortened.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of in vitro diagnostic kits, in particular to a carboxyl polystyrene microsphere, a preparation method thereof and application of the carboxyl polystyrene microsphere in a MxA quantitative detection kit. BACKGROUND

[0002] Polystyrene microspheres have been widely concerned and applied in the fields of medical engineering, chemical industry, environmental protection, electronic information and the like. The polystyrene microspheres have important positions in the field of biological medicine due to the advantages of easy functionalization of the surface, strong reaction capacity and strong reagent fixing performance. Various functionalized microspheres, magnetic microspheres and fluorescent polystyrene microspheres have attracted attention of many researchers and clinical experts, and the prepared microspheres have shown good application effects in the directions of clinical testing, cell tracking and drug release.

[0003] MxA protein is an interferon-induced antiviral protein, and the serum level of the MxA protein can reflect the antiviral immune state of the body, and has important value in the diagnosis of infectious diseases. The latex immunoturbidimetry for detecting MxA currently used in the clinic has the problem of insufficient sensitivity, especially poor detection accuracy for low-concentration samples.

[0004] At present, the carboxyl polystyrene microspheres on the market have low binding efficiency with MxA antibodies, resulting in poor sensitivity of the latex immunoturbidimetry for detecting MxA biochemical reagents. SUMMARY

[0005] The present application aims to provide a carboxyl polystyrene microsphere, a preparation method thereof and application of the carboxyl polystyrene microsphere in a MxA quantitative detection kit.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0007] A preparation method of a carboxyl polystyrene microsphere comprises the following steps: first, preparing polystyrene microspheres rich in aldehyde groups on the surface by a soap-free emulsion polymerization method; then, modifying the surface of the microspheres by a Schiff base reaction with 2-aminoethyl methacrylate hydrochloride; and finally, generating polystyrene microspheres rich in carboxyl groups on the surface by a thiol-double bond addition reaction with mercaptobutane dioic acid.

[0008] Specific steps are as follows: a three-necked flask is added with pure water, and oxygen is removed by nitrogen for 30 min; an ionic strength regulator is added, and then styrene and an aldehyde functional monomer are dissolved; the temperature is raised to 60 DEG C; an initiator is added; and the reaction is carried out at 75-85 DEG C for 12 h. After the reaction, the product is washed with pure water three times, washed with anhydrous ethanol three times, and finally stored in anhydrous ethanol.

[0009] The microspheres stored in anhydrous ethanol are transferred to a reaction bottle, 2-aminoethyl methacrylate hydrochloride and glacial acetic acid are added, after 30 min of reaction at room temperature, mercaptobutane acid and azobis diisopropyl cyanide are added, and the reaction is continued at room temperature for 24 h, after the reaction is completed, the anhydrous ethanol is washed for three times, the pure water is washed for three times, and finally the microspheres are stored in pure water, and the solid content is adjusted to 10%.

[0010] The ionic strength regulator is at least one of NaCl, Na2CO3 and NaHCO3.

[0011] The aldehyde functional monomer is methacrolein and / or propylene aldehyde, and the volume ratio of the aldehyde functional monomer to the styrene monomer is 1:5-10.

[0012] The initiator is a water-soluble initiator, and is any one of potassium persulfate, sodium persulfate and ammonium persulfate.

[0013] The molar ratio of the aldehyde functional monomer to 2-aminoethyl methacrylate hydrochloride is 1:1.0-1.5.

[0014] The molar ratio of 2-aminoethyl methacrylate hydrochloride to mercaptobutane acid is 1:1.0-1.5.

[0015] The amount of azobis diisopropyl cyanide is 1%-5% of 2-aminoethyl methacrylate hydrochloride.

[0016] The carboxyl polystyrene microspheres prepared by the preparation method have a particle size range of 350-450 nm.

[0017] The carboxyl polystyrene microspheres can be used for preparing a latex immunoturbidimetry quantitative MxA detection kit, and the specific preparation method is as follows:

[0018] (a) Preparation of reagent R1

[0019] In the MES buffer solution with a concentration of 20 mM and a pH of 6.0, three reagents of Tween 20, PEG20000 and Proclin 300 are sequentially added, and the final concentrations of the reagents in the system are controlled to be: a volume fraction of Tween 20 of 0.1%, a mass concentration of PEG20000 of 1%, and a volume fraction of Proclin 300 of 0.08%, and after the sample is added, the system is uniformly mixed to ensure uniformity.

[0020] (b) Preparation of reagent R2

[0021] ①Microsphere dilution: the carboxyl polystyrene microspheres prepared in the application are diluted to a concentration of 2 mg / mL with 50 mM pH 6.0 MES buffer solution;

[0022] ② Activation: EDC was weighed and prepared into a 10 mg / mL mother liquor with 50 mM pH 6.0 MES buffer solution; EDC mother liquor was taken and added into the above solution to make the final concentration 0.5 mg / mL, and incubated at 37℃ for 0.5 hours, then centrifuged at 13000 r / min for 25 min to remove the supernatant, and resuspended with PB buffer solution with pH 7.2;

[0023] ③ Coupling: the MxA antibody was diluted to 1 mg / mL with PB buffer solution with pH 7.2, and added into the above activated microspheres according to the volume ratio of 1:5, and incubated at 37℃ for 2 hours;

[0024] ④ Blocking: 1% bovine serum albumin was added, and incubated at 37℃ for 1 hour; the above blocking reagent was centrifuged to remove the supernatant, and resuspended with 10 mM Tris buffer solution with pH 7.5 containing 4-8% trehalose, and mixed uniformly, and then dispersed by ultrasonic, and the MxA antibody-microsphere complex was obtained, that is, R2 solution.

[0025] Compared with the prior art, the beneficial effects of the present application are:

[0026] (1) Carboxyl density optimization: the surface of the carboxyl polystyrene microspheres of the present application is rich in carboxyl groups, and the surface carboxyl density is further improved through specific process regulation, which provides sufficient reaction sites for subsequent functional modification and active substance combination.

[0027] (2) Single dispersion improvement: the introduction of multiple hydrophilic groups such as ester groups, imino groups and mercapto groups in the hydrophilic segment on the surface of the microspheres can effectively improve the dispersion stability between particles and significantly improve the monodispersity of the microsphere system, reducing the agglomeration phenomenon.

[0028] (3) Detection performance enhancement: the carboxyl polystyrene microspheres have high specific binding capacity of the surface carboxyl groups with MxA antibodies, which greatly improves the antibody immobilization efficiency, can improve the binding efficiency of the latex and MxA antibodies, thereby improving the detection sensitivity, and can realize the purpose of high-sensitivity quantitative detection of MxA protein, greatly shortening the detection time and providing support for accurate detection of low-concentration target substances. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The preparation flow chart of the carboxyl polystyrene microspheres of the present application is shown in the figure. DETAILED DESCRIPTION

[0030] With reference to the accompanying drawings on which the embodiments of the application are illustrated, the technical solutions in the embodiments of the application will be described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments of the application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the protection scope of the application.

[0031] Example 1 Preparation of carboxyl polystyrene microspheres PS-1

[0032] In combination Figure 1 As shown in the figure, 200 mL of pure water was added to a three-necked flask, and oxygen was removed by nitrogen for 30 min. After 50 mg of an ion strength regulator NaCl was dissolved, 20 mL of styrene and 2 mL of methyl acrylate were added. The temperature was raised to 60°C, 100 mg of potassium persulfate was added, and the reaction was carried out at 80°C for 12 h. After the reaction, the microspheres were washed with pure water three times and anhydrous ethanol three times, and finally stored in 100 mL of anhydrous ethanol.

[0033] The microspheres stored in anhydrous ethanol were transferred to a reaction bottle, 4.4 g of 2-aminoethyl methacrylate hydrochloride and 1 mL of glacial acetic acid were added, and the reaction was carried out at room temperature for 30 min. Then 4.4 g of mercaptobutane acid and 0.1 g of azobis isopropyl cyanide were added, and the reaction was continued at room temperature for 24 h. After the reaction, the microspheres were washed with anhydrous ethanol three times and pure water three times, and finally stored in pure water, with the solid content adjusted to 10%.

[0034] Example 2 Preparation of carboxyl polystyrene microspheres PS-2

[0035] In combination Figure 1 As shown in the figure, 200 mL of pure water was added to a three-necked flask, and oxygen was removed by nitrogen for 30 min. After 50 mg of an ion strength regulator NaCl was dissolved, 20 mL of styrene and 2 mL of methyl acrylate were added. The temperature was raised to 60°C, 100 mg of potassium persulfate was added, and the reaction was carried out at 80°C for 12 h. After the reaction, the microspheres were washed with pure water three times and anhydrous ethanol three times, and finally stored in 100 mL of anhydrous ethanol.

[0036] The microspheres stored in anhydrous ethanol were transferred to a reaction bottle, 4.4 g of 2-aminoethyl methacrylate hydrochloride and 1 mL of glacial acetic acid were added, and the reaction was carried out at room temperature for 30 min. Then 4.4 g of mercaptobutane acid and 0.1 g of azobis isopropyl cyanide were added, and the reaction was continued at room temperature for 24 h. After the reaction, the microspheres were washed with anhydrous ethanol three times and pure water three times, and finally stored in pure water, with the solid content adjusted to 10%.

[0037] Example 3 Preparation of carboxyl polystyrene microspheres PS-3

[0038] In combination Figure 1As shown, 200 mL of pure water was added to a three-necked flask, deoxygenated by nitrogen for 30 min, 50 mg of ion strength regulator NaCl was added, after dissolution, 20 mL of styrene and 4 mL of methyl acrylate were added, the temperature was raised to 60°C, 100 mg of potassium persulfate was added, and the reaction was carried out at 80°C for 12 h. After the reaction was completed, the solution was washed with pure water three times and anhydrous ethanol three times, and finally stored in 100 mL of anhydrous ethanol.

[0039] The microspheres stored in anhydrous ethanol were transferred to a reaction flask, 8.8 g of 2-aminoethyl methacrylate hydrochloride and 1 mL of glacial acetic acid were added, and the reaction was carried out at room temperature for 30 min. Then 8.8 g of mercaptobutane sulfonic acid and 0.1 g of azobis isopropyl cyanide were added, and the reaction was continued at room temperature for 24 h. After the reaction was completed, the solution was washed with anhydrous ethanol three times and pure water three times, and finally stored in pure water, and the solid content was adjusted to 10%.

[0040] Preparation of MxA detection kit (latex immunoturbidimetry) of Example 4

[0041] (1) Preparation of reagent R1

[0042] In a MES buffer (2-morpholinoethanesulfonic acid buffer) with a concentration of 20 mM and a pH of 6.0, three reagents, Tween 20, PEG20000 and Proclin 300, were added in sequence. 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 system was thoroughly mixed to ensure uniformity.

[0043] (2) Preparation of reagent R2

[0044] 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;

[0045] ② Activation: EDC was weighed and prepared into a 10 mg / mL stock solution with 50 mM pH 6.0 MES buffer; EDC (ethyl (3-(dimethylamino) propyl) carbodiimide hydrochloride) stock solution was taken and added to the above solution to a final concentration of 0.5 mg / mL, and incubated at 37°C for 0.5 h. After completion, centrifugation was performed at 13000 r / min for 25 min to remove the supernatant, and resuspended with PB buffer (phosphate buffer) with a pH of 7.2;

[0046] ③Coupling: two strains of MxA antibodies (Nanjing Liding Medical Technology Co., Ltd., Catalog No.: mAb03, mAb04) were diluted to 1 mg / mL with PB buffer at pH 7.2, added to the above activated microspheres at a volume ratio of 1:5, and incubated at 37°C for 2 hours;

[0047] ④Blocking: add bovine serum albumin at a final concentration of 1%, incubate at 37°C for 1 hour; centrifuge the above blocking reagent to remove the supernatant, resuspend and mix well with 10 mM Tris buffer at pH 7.5 containing 4-8% trehalose, and then disperse with ultrasonic waves. The MxA antibody-microsphere complex, i.e. R2 solution, is obtained.

[0048] Example 5 Test

[0049] (1) Particle size test: OTSUKA ELECTRONICS was used to test the particle size and dispersion index. Three samples were diluted with deionized water and measured three times to test the average particle size and dispersion index (PDI).

[0050] (2) Carboxyl content test: the surface carboxyl density was quantitatively analyzed by conductivity detection acid-base titration method (DDS-S07A, Raydist).

[0051] (3) Sample test: the PA120 full-automatic specific protein analyzer of Jinrui was used to test the whole blood sample.

[0052] The particle size, dispersion index and carboxyl density of the carboxyl polystyrene microspheres prepared in Examples 1-3 and the control microspheres (P0425, purchased from JSR Corporation) in Table 1 are as follows: the particle sizes are 418 nm, 426 nm, 432 nm and 428 nm, respectively, and the dispersion index PDI is <0.05, indicating good monodispersity. The carboxyl densities of the microspheres of Examples 1-3 are 0.355 mmol / g, 0.402 mmol / g and 0.437 mmol / g, respectively, all higher than the carboxyl density of the control microspheres.

[0053] In the process of preparing carboxyl polystyrene microspheres by emulsion polymerization, there is a key technical bottleneck: as the particle size of the microspheres increases, it is difficult for the carboxyl groups to be effectively grafted onto the surface of the microspheres. This phenomenon directly leads to the common problem of low carboxyl content in large-particle-size carboxyl polystyrene nanomicrospheres on the market. In the examples, polystyrene microspheres with high aldehyde group content on the surface were first synthesized, and then through a two-step process of Schiff base reaction and thiol-double bond addition reaction, efficient grafting of carboxyl groups on the surface of the microspheres was realized. This strategy has the advantage of a clear stoichiometric relationship: one aldehyde group can be connected to one double bond, and one double bond can further introduce two carboxyl groups through addition reaction, and finally polystyrene microspheres with high carboxyl content on the surface are successfully prepared.

[0054] Table 1 Particle size and dispersion index of microspheres

[0055]

[0056] Table 2 shows the detection reactivity of the MxA detection reagent prepared in Example 4 on the whole blood calibration sample. From the experimental results, it can be seen that the reactivity of the MxA detection reagent assembled by the microspheres prepared in Examples 1-3 is significantly higher than that of the reagent prepared by the control microspheres (model P0425, purchased from JSR Corporation), which indicates that the MxA detection reagent corresponding to the microspheres of the examples has better sensitivity. It is speculated that the core reason for this difference is that the microspheres of the examples have higher carboxyl content on the surface, and carboxyl as a key binding site can effectively improve the binding efficiency with MxA antibody, thereby enhancing the reactivity of the reagent and the detection sensitivity. In terms of linearity, the linearity R2 of Examples 1-3 is 0.9997, 0.9998, and 0.9997, respectively, which is better than that of the control example 0.9967.

[0057] Table 2 Detection reactivity of the MxA detection reagent prepared in Example 4 on the whole blood calibration sample

[0058]

[0059] Although embodiments of the present application 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 therein without departing from the principles and spirit of the application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A process for the preparation of carboxypolystyrene microspheres, characterized in that: First, the surface of the polystyrene microspheres rich in aldehyde groups is prepared by a soap-free emulsion polymerization method; then the surface of the microspheres is modified by a Schiff base reaction with 2-aminoethyl methacrylate hydrochloride; finally, the surface of the microspheres rich in carboxyl groups is generated by a thiol-double bond addition reaction with mercaptosuccinic acid.

2. The method for preparing carboxylated polystyrene microspheres according to claim 1, characterized in that: The reaction vessel is added with pure water, oxygen is removed by nitrogen, an ionic strength regulator is added, after dissolution, styrene and aldehyde functional monomers are added, the temperature is raised to 60 DEG C, an initiator is added, and the reaction is carried out at 75-85 DEG C for 12 hours; after the reaction is completed, the microspheres are washed with pure water three times and anhydrous ethanol three times, and finally stored in anhydrous ethanol; The microspheres stored in anhydrous ethanol are transferred to the reaction vessel, 2-aminoethyl methacrylate hydrochloride and glacial acetic acid are added, the reaction is carried out at room temperature, then mercaptosuccinic acid and azobisisoheptane nitrile are added, the reaction is continued at room temperature, after the reaction is completed, the microspheres are washed with anhydrous ethanol three times and pure water three times, and finally stored in pure water, and the solid content is adjusted to 10%.

3. The method for preparing carboxylated polystyrene microspheres according to claim 2, characterized in that: The ionic strength regulator 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 propylene aldehyde, 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, which 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 addition amount of azobisisoheptane nitrile is 1%-5% of 2-aminoethyl methacrylate hydrochloride.

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

10. The carboxyl polystyrene microspheres according to claim 9 are used in the preparation of a latex immunoturbidimetry quantitative detection MxA kit.

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