Colorful polystyrene microspheres for detecting MxA antigen and preparation method and application thereof

Abstract

The present application relates to a kind of color polystyrene microspheres for MxA antigen detection and its preparation method and application, wherein the preparation method of color polystyrene microspheres is, first, surface aminosilica ball is prepared, then the amino group on the surface of microsphere is activated by glutaraldehyde, then covalently combined with the amino group containing color dye, then the double bond is introduced and modified using color dye, finally seed polymerization is coated with polystyrene and surface carboxyl modification. The MxA antigen test strip prepared using the color polystyrene microspheres of the present application adopts double detection line (different concentration MxA antibody) design, can realize the semi-quantitative distinction of MxA antigen concentration, can quickly judge whether MxA antigen in sample is high concentration (>100ng / mL) or low concentration (<100ng / mL), solve the defect that existing test strip cannot distinguish antigen concentration, without professional personnel and instrument, can be applied to on-site rapid detection, with wide clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of immunochromatography technology, specifically to a colored polystyrene microsphere for MxA antigen detection, its preparation method, and its application. Background Technology

[0002] MxA (Myxovirus resistance protein A) is a broad-spectrum antiviral protein induced by type I interferon and belongs to the dynamic protein superfamily. Upon viral invasion, type I interferon triggers signaling pathways, activating Mx1 / Mx2 gene expression and promoting MxA protein aggregation to form an antiviral defense barrier. MxA exhibits high viral infection specificity—it hardly increases during bacterial infections. Therefore, MxA is an ideal biomarker for differentiating between viral and bacterial respiratory infections. Thus, rapid and convenient detection of MxA protein levels is of significant value in guiding rational clinical drug use and reducing antibiotic overuse.

[0003] Currently, detection methods for MxA antigen mainly include enzyme-linked immunosorbent assay (ELISA), Western blotting, and flow cytometry. However, these methods suffer from drawbacks such as complex operation, long detection time, and the need for specialized instruments and personnel, making it difficult to meet the needs of rapid on-site testing. Immunochromatographic test strips, due to their advantages of simple operation, rapid detection, and no need for specialized equipment, are widely used in the field of rapid testing. Labeled microspheres, as the core component of the test strip, directly affect the sensitivity, specificity, and visualization of the detection.

[0004] Polystyrene microspheres are commonly used labeling carriers in immunoassays due to their uniform particle size, easy surface functionalization, good stability, and low cost. However, traditional polystyrene microspheres are mostly single-color, resulting in poor color development, susceptibility to signal interference, and complex labeling processes. Furthermore, existing MxA test strips often employ a single test line design, failing to achieve semi-quantitative differentiation of antigen concentrations and thus failing to meet the clinical need for precise detection of different concentrations of MxA antigens. When antigen concentrations vary, the color development on the test strip cannot visually indicate the concentration range, potentially leading to misdiagnosis.

[0005] Furthermore, the coupling efficiency between microspheres and antibodies, as well as the dispersibility and stability of the microspheres, directly affect the detection performance of the test strip. Current microsphere preparation processes often suffer from problems such as uneven particle size, insufficient activity of surface functional groups, and easy aggregation after antibody coupling, leading to low detection sensitivity and poor repeatability. Therefore, developing a colored polystyrene microsphere with uniform particle size, stable color development, and good functionalization, combined with a dual-detection-line design to achieve concentration-differential detection of the MxA antigen, has significant clinical application value and market potential. Summary of the Invention

[0006] To address the shortcomings of existing immunochromatographic test strips in visually distinguishing different concentration ranges of target antigen MxA, making it impossible to intuitively determine the concentration range through test strip color development in home testing; and the fact that existing microsphere preparation processes often suffer from uneven particle size, insufficient activity of surface functional groups, and easy aggregation after antibody conjugation, resulting in low detection sensitivity and poor repeatability, the present invention aims to provide a colored polystyrene microsphere for MxA antigen detection, its preparation method, and its application.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing colored polystyrene microspheres for MxA antigen detection is as follows: first, surface-amino-coated silicon spheres are prepared; then, the amino groups on the surface of the microspheres are activated by glutaraldehyde; then, they are covalently bonded with an amino-containing colored dye; then, double bond modification is introduced by the colored dye; finally, seed polymerization is used to coat polystyrene and modify the surface carboxyl groups.

[0008] The specific method for preparing surface-aminated silicon spheres is as follows: pure water and anhydrous ethanol are added to a reaction vessel and stirred at a constant speed. Ammonia is added to the mixture and stirred. Then, silane coupling agent TEOS (tetraethoxysilane) is added and reacted at room temperature. Subsequently, silane coupling agent APTES (3-aminopropyltriethoxysilane) is added and reacted at room temperature. After the reaction is completed, the mother liquor is removed, pure water is added to the precipitate, and the precipitate is dispersed by ultrasound for later use.

[0009] Furthermore, the volume ratio of TEOS to APTES is 1:0.5-1.

[0010] The specific method for activating the amino groups on the surface of microspheres with glutaraldehyde is as follows: the prepared surface-aminated silicon spheres are centrifuged, the precipitate is dispersed in 0.1M PBS buffer (pH 7.4-7.9), glutaraldehyde is added to the dispersion, and the reaction is carried out at room temperature. After the reaction is completed, the product is dialyzed with 0.1M PBS buffer (pH 7.4-7.9) for 48 hours, and the dialysate is replaced every 8 hours to remove unreacted glutaraldehyde.

[0011] The specific method for covalently binding with amino-containing color dyes is as follows: the activated microspheres are centrifuged, the precipitate is dispersed in ethanol, oil-soluble aniline black and 1,4-diaminoanthraquinone dissolved in ethanol are added to the dispersion, glacial acetic acid is added, and the reaction is carried out at room temperature. After the reaction is completed, the precipitate is centrifuged and stored in anhydrous ethanol for later use.

[0012] Furthermore, the mass ratio of aniline black to 1,4-diaminoanthraquinone is 1:2-5.

[0013] The specific method for introducing double bond modification using colored dyes is as follows: the dyed microspheres are placed in a three-necked flask, triethylamine is added, the mixture is stirred, methacrylamide chloride is added, and the mixture is reacted at a constant temperature under ice bath conditions. After the reaction is completed, the microspheres are washed three times each with pure water and anhydrous ethanol to remove unreacted triethylamine and methacrylamide chloride. Finally, the microspheres are stored in pure water for later use.

[0014] The specific method for seed polymerization coating of polystyrene and surface carboxyl modification is as follows: the modified microspheres are added to a three-necked flask, and nitrogen gas is introduced into the flask for deoxygenation; then sodium dodecyl sulfate (SDS) is added and stirred until completely dissolved; then styrene and acrylic acid are added and stirred at high speed to ensure that the monomers are uniformly dispersed in the system; the system temperature is raised to 60°C, potassium persulfate (KPS) is added as an initiator, and then the temperature is raised to 80°C and the reaction is carried out at a constant temperature; after the reaction is completed, the product is dialyzed with pure water to remove unreacted monomers, initiators and surfactants, and colored polystyrene microspheres are obtained. Furthermore, the volume ratio of styrene to acrylic acid is 1:0.02-0.1.

[0015] The colored polystyrene microspheres prepared by this invention can be used to prepare MxA antigen test strips. The specific method is as follows: (1) Labeling of MxA antibody ① Washing: Add 50 μL of colored microspheres to a centrifuge tube containing 0.5 mL of 50 mM MES (pH 6.5) buffer, sonicate to mix, centrifuge and discard the supernatant; ② Activation: Add 0.5 mL of 50 mM MES (pH 6.5) buffer to the cleaned colored microspheres, sonicate to mix, then add 20 μL of freshly prepared EDC (10 mg / mL) and 40 μL of NHS (10 mg / mL), vortex to mix, activate at room temperature for 30 min, centrifuge and discard the supernatant. ③ Coupling: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the activated colored microspheres and sonicate to mix; add 50 μg of the MxA-labeled antibody to be coupled, vortex to mix, and couple at room temperature for 2 h. ④ Blocking: Add 300 μL of 10% BSA and 20 μL of ethanolamine to the above coupling buffer, block at room temperature for 1 h, centrifuge and discard the supernatant; ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the colored microsphere-antibody complex and store at 2-8℃ protected from light. The microsphere reconstitution solution formula is: 10 mM PBS (pH 7.4±0.05), 10 mM NaCl, 0.05% Tween-80, 1% BSA, 5% sucrose, and 0.1% ProClin 300.

[0016] (2) Preparation of nitrocellulose membrane The prepared MxA-coated antibody solution was applied to a nitrocellulose membrane at a coating volume of 1 μL / cm using a streak sprayer with two different concentrations (1 mg / mL and 0.4 mg / mL) and a goat anti-mouse IgG antibody solution (1 mg / mL). These were used as the detection line and the control line, respectively. The membrane was dried overnight at 45°C.

[0017] (3) Preparation of the binding pad The MxA antibody complex labeled with colored microspheres from step (1) above was sprayed onto a glass cellulose membrane at a coating amount of 5 μL / cm and dried overnight at 45°C to prepare a conjugation pad.

[0018] (4) Reagent card assembly ① Place the absorbent pad, conjugate pad, and sample pad sequentially onto the PVC substrate coated with the nitrocellulose membrane. Attach the absorbent pad to one end of the control line, which rests on the NC membrane. Attach the conjugate pad to one end of the test line, which rests on the NC membrane. Place the sample pad on top of the conjugate pad.

[0019] ② Cut the large plate assembled in ① into reagent strips with a cutting machine with a width of 3mm. Put the reagent strips into the corresponding card holders. Place the filled reagent cards and a desiccant into an aluminum foil bag, seal it, and store it for later use.

[0020] Compared with the prior art, the beneficial effects of the present invention are: The colored polystyrene microspheres prepared by this invention are dyed with a combination of aniline black and 1,4-diaminoanthraquinone by introducing glutaraldehyde, resulting in uniform and stable color development. Furthermore, double bonds are introduced by reacting the amino group of the dye 1,4-diaminoanthraquinone with methacryloyl chloride, which serve as seeds for seed polymerization to re-ply polystyrene on the surface and carboxylate it. The process is simple, the steps are controllable, the reaction conditions are mild (mostly room temperature or mild heating), no complex equipment is required, and it is easy to carry out large-scale industrial production.

[0021] The MxA antigen test strip prepared using the colored polystyrene microspheres of this invention features a dual-detection-line design (different concentrations of MxA antibody), enabling semi-quantitative differentiation of MxA antigen concentration. It can quickly determine whether the MxA antigen in the sample is at a high concentration (>100 ng / mL) or a low concentration (<100 ng / mL), overcoming the shortcomings of existing test strips that cannot distinguish antigen concentration. It requires no professional personnel or instruments and is suitable for rapid on-site testing, showing broad clinical application prospects. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the interpretation of immunochromatographic test strips at different concentrations of MxA antigen.

[0023] Figure 2The results of the stability test of the chromatography test strip prepared in Example 1 after 14 days of accelerated aging at 37°C are shown. Detailed Implementation

[0024] 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.

[0025] Example 1 (1) Add 10 mL of pure water and 90 mL of anhydrous ethanol to a three-necked flask, fix the three-necked flask on an electric stirrer and stir, add 5 mL of ammonia water, and after 10 min, add 5 mL of silane coupling agent TEOS. React at room temperature for 8 h, then add 5 mL of silane coupling agent APTES and continue to react for 8 h. After the reaction is completed, centrifuge to remove the mother liquor, add pure water and sonicate for dispersion for later use.

[0026] (2) Centrifuge the above microspheres and disperse them in 50 mL of 0.1 M pH 7.4 PBS buffer. Add 100 μL of glutaraldehyde and react at room temperature for 4 h. Dialyze the product with 0.1 M pH 7.4 PBS buffer for 48 h, changing the dialysis buffer every 8 h.

[0027] (3) Centrifuge the product from step (2), disperse it in 100 mL of ethanol, add 0.1 g of aniline black (oil-soluble) and 0.3 g of 1,4-diaminoanthraquinone dissolved in ethanol, and then add 0.5 mL of glacial acetic acid. React at room temperature for 2 h. After centrifugation, store in 100 mL of anhydrous ethanol.

[0028] (4) Place the microspheres from step (3) into a three-necked flask, add 0.1 mL of triethylamine, add methacrylamide chloride after 10 min, and react under ice bath conditions for 2 h. After the reaction is complete, wash three times with pure water and three times with anhydrous ethanol, and finally store in pure water.

[0029] (5) Add the microspheres from step (4) to a three-necked flask, purge with nitrogen for 20 min to remove oxygen, add 100 mg sodium dodecyl sulfate, add 5 mL styrene and 0.2 mL acrylic acid after 10 min, stir at high speed for 30 min, add 30 mg KPS at 60℃, and react at 80℃ for 8 h. Dialyze with pure water for 3 days.

[0030] Example 2

[0031] (1) Add 10 mL of pure water and 90 mL of anhydrous ethanol to a three-necked flask, fix the three-necked flask on an electric stirrer and stir, add 5 mL of ammonia water, and after 10 min, add 5 mL of silane coupling agent TEOS. React at room temperature for 8 h, then add 5 mL of silane coupling agent APTES and continue to react for 8 h. After the reaction is completed, centrifuge to remove the mother liquor, add pure water and sonicate for dispersion for later use.

[0032] (2) Centrifuge the above microspheres and disperse them in 50 mL of 0.1 M pH 7.4 PBS buffer. Add 100 μL of glutaraldehyde and react at room temperature for 4 h. Dialyze the product with 0.1 M pH 7.4 PBS buffer for 48 h, changing the dialysis buffer every 8 h.

[0033] (3) Centrifuge the product from step (2), disperse it in 100 mL of ethanol, add 0.1 g of aniline black (oil-soluble) and 0.4 g of 1,4-diaminoanthraquinone dissolved in ethanol, and then add 0.5 mL of glacial acetic acid. React at room temperature for 2 h. After centrifugation, store in 100 mL of anhydrous ethanol.

[0034] (4) Place the microspheres from step (3) into a three-necked flask, add 0.1 mL of triethylamine, add methacrylamide chloride after 10 min, and react under ice bath conditions for 2 h. After the reaction is complete, wash three times with pure water and three times with anhydrous ethanol, and finally store in pure water.

[0035] (5) Add the microspheres from step (4) to a three-necked flask, purge with nitrogen for 20 min to remove oxygen, add 100 mg sodium dodecyl sulfate, add 5 mL styrene and 0.2 mL acrylic acid after 10 min, stir at high speed for 30 min, add 30 mg KPS at 60℃, and react at 80℃ for 8 h. Dialyze with pure water for 3 days.

[0036] Example 3

[0037] (1) Add 10 mL of pure water and 90 mL of anhydrous ethanol to a three-necked flask, fix the three-necked flask on an electric stirrer and stir, add 5 mL of ammonia water, and after 10 min, add 5 mL of silane coupling agent TEOS. React at room temperature for 8 h, then add 5 mL of silane coupling agent APTES and continue to react for 8 h. After the reaction is completed, centrifuge to remove the mother liquor, add pure water and sonicate for dispersion for later use.

[0038] (2) Centrifuge the above microspheres and disperse them in 50 mL of 0.1 M pH 7.4 PBS buffer. Add 100 μL of glutaraldehyde and react at room temperature for 4 h. Dialyze the product with 0.1 M pH 7.4 PBS buffer for 48 h, changing the dialysis buffer every 8 h.

[0039] (3) Centrifuge the product from step (2), disperse it in 100 mL of ethanol, add 0.1 g of aniline black (oil-soluble) and 0.5 g of 1,4-diaminoanthraquinone dissolved in ethanol, and then add 0.5 mL of glacial acetic acid. React at room temperature for 2 h. After centrifugation, store in 100 mL of anhydrous ethanol.

[0040] (4) Place the microspheres from step (3) into a three-necked flask, add 0.1 mL of triethylamine, add methacrylamide chloride after 10 min, and react under ice bath conditions for 2 h. After the reaction is complete, wash three times with pure water and three times with anhydrous ethanol, and finally store in pure water.

[0041] (5) Add the microspheres from step (4) to a three-necked flask, purge with nitrogen for 20 min to remove oxygen, add 100 mg sodium dodecyl sulfate, add 5 mL styrene and 0.2 mL acrylic acid after 10 min, stir at high speed for 30 min, add 30 mg KPS at 60℃, and react at 80℃ for 8 h. Dialyze with pure water for 3 days.

[0042] To highlight the beneficial effects of the present invention, the following comparative experiments are provided.

[0043] Comparative Example 1 (1) Add 10 mL of pure water and 90 mL of anhydrous ethanol to a three-necked flask, fix the three-necked flask on an electric stirrer and stir, add 5 mL of ammonia water, and after 10 min, add 5 mL of silane coupling agent TEOS. React at room temperature for 8 h, then add 5 mL of silane coupling agent APTES and continue to react for 8 h. After the reaction is completed, centrifuge to remove the mother liquor, add pure water and sonicate for dispersion for later use.

[0044] (2) Centrifuge the above microspheres and disperse them in 50 mL of 0.1 M pH 7.4 PBS buffer. Add 100 μL of glutaraldehyde and react at room temperature for 4 h. Dialyze the product with 0.1 M pH 7.4 PBS buffer for 48 h, changing the dialysis buffer every 8 h.

[0045] (3) Centrifuge the product from step (2), disperse it in 100 mL of ethanol, add 0.1 g of aniline black (oil-soluble) dissolved in ethanol, and then add 0.5 mL of glacial acetic acid. React at room temperature for 2 h. After centrifugation, store it in 100 mL of anhydrous ethanol.

[0046] (4) Place the microspheres from step (3) into a three-necked flask, add 0.1 mL of triethylamine, add methacrylamide chloride after 10 min, and react under ice bath conditions for 2 h. After the reaction is complete, wash three times with pure water and three times with anhydrous ethanol, and finally store in pure water.

[0047] (5) Add the microspheres from step (4) to a three-necked flask, purge with nitrogen for 20 min to remove oxygen, add 100 mg sodium dodecyl sulfate, add 5 mL styrene and 0.2 mL acrylic acid after 10 min, stir at high speed for 30 min, add 30 mg KPS at 60℃, and react at 80℃ for 8 h. Dialyze with pure water for 3 days.

[0048] Comparative Example 2 (1) Add 10 mL of pure water and 90 mL of anhydrous ethanol to a three-necked flask, fix the three-necked flask on an electric stirrer and stir, add 5 mL of ammonia water, and after 10 min, add 5 mL of silane coupling agent TEOS. React at room temperature for 8 h, then add 5 mL of silane coupling agent APTES and continue to react for 8 h. After the reaction is completed, centrifuge to remove the mother liquor, add pure water and sonicate for dispersion for later use.

[0049] (2) Centrifuge the above microspheres and disperse them in 50 mL of 0.1 M pH 7.4 PBS buffer. Add 100 μL of glutaraldehyde and react at room temperature for 4 h. Dialyze the product with 0.1 M pH 7.4 PBS buffer for 48 h, changing the dialysis buffer every 8 h.

[0050] (3) Centrifuge the product from step (2), disperse it in 100 mL of ethanol, add 0.3 g of 1,4-diaminoanthraquinone dissolved in ethanol, and then add 0.5 mL of glacial acetic acid. React at room temperature for 2 h. After centrifugation, store it in 100 mL of anhydrous ethanol.

[0051] (4) Place the microspheres from step (3) into a three-necked flask, add 0.1 mL of triethylamine, add methacrylamide chloride after 10 min, and react under ice bath conditions for 2 h. After the reaction is complete, wash three times with pure water and three times with anhydrous ethanol, and finally store in pure water.

[0052] (5) Add the microspheres from step (4) to a three-necked flask, purge with nitrogen for 20 min to remove oxygen, add 100 mg sodium dodecyl sulfate, add 5 mL styrene and 0.2 mL acrylic acid after 10 min, stir at high speed for 30 min, add 30 mg KPS at 60℃, and react at 80℃ for 8 h. Dialyze with pure water for 3 days.

[0053] Table 1 Microsphere Particle Size and Dispersion Index Table 1 shows the microsphere size and dispersion index. The microspheres prepared in Examples 1-3 and Comparative Examples 1-2 have a particle size of about 300 nm and a dispersion index of less than 0.05, indicating that the particle size is uniform and the dispersion is good.

[0054] Example 4

[0055] The method for preparing a human myxovirus resistance protein A (MxA) detection card using the colored microspheres from the above examples and comparative examples includes the following steps: (1) Labeling of MxA antibody ① Washing: Take 50 μL of colored microspheres from Examples 1, 2, 3, Comparative Example 1, and Comparative Example 2 and add them to a centrifuge tube containing 0.5 mL of 50 mM MES (pH 6.5) buffer. Mix well by sonication and centrifugation, then discard the supernatant. ② Activation: Add 0.5 mL of 50 mM MES (pH 6.5) buffer to the cleaned colored microspheres of Examples 1, 2, 3, Comparative Example 1, and Comparative Example 2, sonicate to mix, then add 20 μL of freshly prepared EDC (10 mg / mL) and 40 μL of NHS (10 mg / mL), vortex to mix, activate at room temperature for 30 min, centrifuge and discard the supernatant; ③ Coupling: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the activated colored microspheres of Examples 1, 2, 3, Comparative Example 1, and Comparative Example 2, and sonicate to mix; add 50 μg of the MxA-labeled antibody (Merk, MABF938) to be coupled, vortex to mix, and couple at room temperature for 2 h; ④ Blocking: Add 300 μL of 10% BSA and 20 μL of ethanolamine to the above coupling buffer, block at room temperature for 1 h, centrifuge and discard the supernatant; ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the colored microsphere-antibody complexes of Examples 1, 2, 3, Comparative Example 1, and Comparative Example 2, and store at 2-8°C protected from light. The microsphere reconstitution solution formula is: 10 mM PBS (pH 7.4±0.05), 10 mM NaCl, 0.05% Tween-80, 1% BSA, 5% sucrose, and 0.1% ProClin 300.

[0056] (2) Preparation of nitrocellulose membrane The prepared MxA-coated antibody solution was applied to a nitrocellulose membrane at a coating volume of 1 μL / cm using a streak sprayer with two different concentrations (1 mg / mL and 0.4 mg / mL) and a goat anti-mouse IgG antibody solution (1 mg / mL). These were used as the detection line and the control line, respectively. The membrane was dried overnight at 45°C.

[0057] (3) Preparation of the binding pad The MxA antibody complexes labeled with colored microspheres from Examples 1, 2, 3, Comparative Example 1, and Comparative Example 2 in step (1) above were sprayed onto a glass cellulose membrane at a coating amount of 5 μL / cm and dried overnight at 45°C to prepare a conjugation pad.

[0058] (4) Reagent card assembly ① Place the absorbent pad, conjugate pad, and sample pad sequentially onto the PVC substrate coated with the nitrocellulose membrane. Attach the absorbent pad to one end of the control line, which rests on the NC membrane. Attach the conjugate pad to one end of the test line, which rests on the NC membrane. Place the sample pad on top of the conjugate pad.

[0059] ② Cut the large plate assembled in ① into reagent strips with a cutting machine with a width of 3mm. Put the reagent strips into the corresponding card holders. Place the filled reagent cards and a desiccant into an aluminum foil bag, seal it, and store it for later use.

[0060] (5) Reagent card testing ① Tear open the aluminum foil bag seal and take out the test card from the aluminum foil bag.

[0061] ② Add 5 μL of sample to the sample diluent, mix thoroughly, and then add 80 μL (4 drops) vertically into the sample well of the test card. The reaction time is 8 min.

[0062] ③ Read the test results.

[0063] Schematic diagram of interpreting immunochromatographic test strips at different concentrations of MxA antigen, as shown below. Figure 1 As shown.

[0064] Example 5 MxA Sensitivity Detection Different concentrations of MxA calibrators were used for standard curve testing. The reaction time was 8 min. 5 μL of sample or calibrator was added to 80 μL of diluent, and then 80 μL of the diluted sample was added to the test card. The experimental results are shown in Table 2.

[0065] Table 2. Performance comparison of microspheres in Examples 1, 2, and 3 with microspheres in Comparative Examples 1 and 2 Note: "+" indicates that the color is developed, and "-" indicates that the color is not developed.

[0066] Table 2 shows the comparison of the chromatographic performance of the microspheres in each example and the comparative example under different concentrations of calibrators, where "+" indicates a positive signal and "-" indicates no signal. The results show that the microspheres prepared in Examples 1-3 of this invention showed no signal (negative) on both the T1 and T2 detection lines at 0 ng / mL calibrator, with no false positive interference; at a low concentration of 50 ng / mL calibrator, the T1 line showed a clear positive signal and the T2 line showed no signal, achieving effective identification of low-concentration samples; at concentrations of 100 ng / mL and above, both the T1 and T2 lines showed positive signals, achieving a gradient response to medium- and high-concentration samples. In contrast, Comparative Example 1 showed no signal at a low concentration of 50 ng / mL, indicating insufficient detection sensitivity; Comparative Example 2 showed only a single linear signal at low concentrations, failing to achieve gradient interpretation. Based on the particle size and dispersion index data in Table 1, it can be seen that the microspheres prepared by this invention have excellent particle size uniformity, as well as higher detection sensitivity and gradient recognition ability, which can effectively meet the needs of immunochromatographic test strips for accurate detection of low-concentration samples.

[0067] Figure 2 The results of the stability test of the chromatography test strip prepared in Example 1 after 14 days of accelerated aging at 37°C show that the deviation between the high and low values ​​in the 14-day accelerated stability test is within ±10%, which proves that it has good stability.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing colored polystyrene microspheres for MxA antigen detection, characterized in that: First, surface-aminated silicon spheres are prepared. Then, the amino groups on the surface of the microspheres are activated by glutaraldehyde. Next, they are covalently bonded with amino-containing color dyes. Then, double bond modification is introduced by color dyes. Finally, seed polymerization is used to coat polystyrene and modify the surface carboxyl groups. The specific method for glutaraldehyde to activate the amino groups on the surface of microspheres is as follows: the prepared surface-amino-coated silicon spheres are centrifuged, the precipitate is dispersed in 0.1M PBS buffer (pH 7.4-7.9), glutaraldehyde is added to the dispersion, and the reaction is carried out at room temperature. After the reaction is completed, the product is dialyzed with 0.1M PBS buffer (pH 7.4-7.9) for 48 hours, and the dialysate is replaced every 8 hours to remove unreacted glutaraldehyde. The specific method for covalent bonding with amino-containing color dyes is as follows: centrifuge the activated microspheres, disperse the precipitate in ethanol, add oil-soluble aniline black and 1,4-diaminoanthraquinone dissolved in ethanol to the dispersion, add glacial acetic acid, and react at room temperature; after the reaction is completed, centrifuge the precipitate and store it in anhydrous ethanol for later use. The specific method for introducing double bond modification using colored dyes is as follows: place the dyed microspheres into a three-necked flask, add triethylamine, stir, add methacrylamide chloride, and react at a constant temperature under ice bath conditions; after the reaction is completed, wash the microspheres three times each with pure water and anhydrous ethanol to remove unreacted triethylamine and methacrylamide chloride, and finally store the microspheres in pure water for later use. The specific method for seed polymerization coating of polystyrene and surface carboxyl modification is as follows: Modified microspheres are added to a three-necked flask, and nitrogen gas is introduced into the flask for deoxygenation. Sodium dodecyl sulfate is then added and stirred until completely dissolved. Styrene and acrylic acid are then added, and the mixture is stirred at high speed to ensure uniform dispersion of the monomers in the system. The system temperature is raised to 60°C, potassium persulfate is added as an initiator, and the temperature is then raised to 80°C for constant-temperature reaction. After the reaction is complete, the product is dialyzed with pure water to remove unreacted monomers, initiators, and surfactants, yielding colored polystyrene microspheres. The volume ratio of styrene to acrylic acid is 1:0.02-0.

1.

2. The method for preparing colored polystyrene microspheres according to claim 1, characterized in that: The specific method for preparing surface-aminated silicon spheres is as follows: pure water and anhydrous ethanol are added to a reaction vessel and stirred at a constant speed. Ammonia is added to the mixture and stirred. Then, silane coupling agent TEOS is added and reacted at room temperature. Subsequently, silane coupling agent APTES is added and the reaction is continued at room temperature. After the reaction is completed, the mother liquor is removed, pure water is added to the precipitate, and the precipitate is dispersed by ultrasound for later use.

3. The method for preparing colored polystyrene microspheres according to claim 2, characterized in that: The volume ratio of TEOS to APTES is 1:0.5-1.

4. The method for preparing colored polystyrene microspheres according to claim 1, characterized in that: The mass ratio of aniline black to 1,4-diaminoanthraquinone is 1:2-5.

5. Colored polystyrene microspheres prepared by the method according to any one of claims 1-4.

6. The application of the colored polystyrene microspheres according to claim 5 in the preparation of MxA antigen test strips, characterized in that: The specific method is as follows: (1) Labeling of MxA antibody ① Washing: Add 50 μL of colored microspheres to a centrifuge tube containing 0.5 mL of 50 mM MES buffer, sonicate to mix, centrifuge and discard the supernatant; ② Activation: Add 0.5 mL of 50 mM MES buffer to the cleaned colored microspheres, sonicate to mix, then add 20 μL of freshly prepared EDC and 40 μL of NHS, vortex to mix, activate at room temperature for 30 min, centrifuge and discard the supernatant. ③ Coupling: Add 0.5 mL of 50 mM MES buffer to the activated colored microspheres and sonicate to mix. Add 50 μg of the MxA-labeled antibody to be coupled, vortex to mix, and couple at room temperature for 2 h. ④ Blocking: Add 300 μL of 10% BSA and 20 μL of ethanolamine to the above coupling buffer, block at room temperature for 1 h, centrifuge and discard the supernatant; ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the colored microsphere-antibody complex and store at 2-8℃ protected from light; the microsphere reconstitution solution formula is: 10 mM PBS, 10 mM NaCl, 0.05% Tween-80, 1% BSA, 5% sucrose, 0.1% ProClin 300; (2) Preparation of nitrocellulose membrane The prepared MxA-coated antibody solution, at two different concentrations and a goat anti-mouse IgG antibody solution, was applied to a nitrocellulose membrane using a streak coater at a coating volume of 1 μL / cm. These served as the detection line and control line, respectively, and the membrane was dried overnight at 45°C. The two different concentrations were 1 mg / mL and 0.4 mg / mL, respectively. (3) Preparation of the binding pad The MxA antibody complex labeled with colored microspheres in step (1) was sprayed onto a glass cellulose membrane at a coating amount of 5 μL / cm and dried overnight at 45°C to prepare a conjugation pad. (4) Assemble the reagent cards.

Images