A dual-color synergistic colored microsphere, its preparation method and application

Abstract

This invention relates to the field of immunochromatography, specifically to a dual-color synergistic colorimetric microsphere, its preparation method, and its applications. The method involves first preparing a red silica core using the Stöber method, and then coating it with a blue polystyrene shell. The dual-color synergistic colorimetric microspheres prepared by this invention can be used for high-sensitivity single- or multiple-detection of infection biomarkers. Due to the synergistic effect of the blue and red colors, the colorimetric microspheres prepared by this invention exhibit an order-of-magnitude increase in sensitivity compared to colorimetric microspheres embedded with a single dye, thereby improving the accuracy and sensitivity of high-sensitivity detection items.

Description

Technical Field

[0001] This invention relates to the field of immunochromatography technology, specifically to a dual-color synergistic color microsphere, its preparation method, and its application. Background Technology

[0002] Immunochromatography is a simple, rapid, and highly specific diagnostic method that has been widely applied in clinical diagnosis, food safety, drug testing, and environmental pollution control. It primarily relies on immunochromatographic test strips as a carrier, using solid-phase markers to qualitatively, semi-qualitatively, or quantitatively detect analytes in samples. While traditional colloidal gold immunochromatographic test strips offer advantages such as ease of use and low cost, they suffer from low sensitivity and poor accuracy, making them unsuitable for quantitative detection.

[0003] To improve detection sensitivity, researchers have developed various novel signal probes, such as fluorescent latex microspheres, quantum dot fluorescent microspheres, and time-resolved microspheres. These fluorescent probes overcome the limitations of traditional colloidal gold test strips by enhancing fluorescence signals and improving optical stability. However, these fluorescent probes still have some problems, such as requiring an additional ultraviolet excitation source and only being able to achieve a single signal output.

[0004] In recent years, colored microsphere technology, through innovations such as internal embedding staining processes, precise particle size control, and high-density surface modification, has become a core engine for the transformation of immunochromatography from qualitative to quantitative analysis. This technology combines dyes with polymer microspheres to form composite materials with high sensitivity and specificity, and is widely used in fields such as bioagglutination assays and lateral chromatography. However, existing methods for preparing colored microspheres still have some problems, such as low dye loading, difficulty in removing surfactants, and difficulty in controlling the core-shell structure. Especially for some highly sensitive detection items, such as the preparation of blue and purple microspheres, existing technologies are insufficient, affecting the accuracy and sensitivity of the detection results. Therefore, developing a method for preparing colored microspheres with high sensitivity, uniform particle size, high dye loading, and low leakage is of great significance for promoting the application and development of immunochromatographic technology.

[0005] Several patent documents have been published regarding solutions to the sensitivity and color depth issues of colored microspheres in immunochromatographic detection. For example, CN116550311A discloses a method for synthesizing colored microspheres using a hydrothermal synthesis method and its application. This method prepares monodisperse polystyrene latex microspheres through emulsion polymerization, mixes oil-soluble dyes with blank polystyrene latex microspheres, and then synthesizes colored latex microspheres in one step using a hydrothermal synthesis method under high temperature and pressure. This method is simple to operate, does not use emulsifiers, and produces colored microspheres with uniform particle size, good dispersibility, high dye loading, and low leakage. However, the colored microspheres prepared by this method suffer from the problem of shallow coloring of single-color microspheres, especially for single-color microspheres such as red, blue, and purple, which are difficult to meet the requirements of high-sensitivity detection.

[0006] Human myxovirus resistance protein A (MxA) is a cytoplasmic protein induced by interferon type I / III in human cells. This protein possesses GTPase activity, has a molecular weight of approximately 75 kDa, and can be induced by small amounts of virus. It exhibits broad-spectrum antiviral activity and is a highly sensitive and specific biomarker for viral infections. The determination of MxA protein is of great significance for avoiding antibiotic overuse and improving the diagnosis of clinical infectious diseases. Combined detection of MxA protein with other inflammatory markers such as C-reactive protein (CRP), procalcitonin (PCT), pancreatin (PSP), interleukin-6 (IL-6), and heparin-binding protein (HBP) can significantly improve the clinical sensitivity and specificity for differentiating between viral and bacterial infections.

[0007] In summary, existing methods for preparing colored microspheres often result in shallow staining of single-color microspheres (such as red, blue, and purple), which is insufficient for high-sensitivity detection requirements and affects the accuracy and sensitivity of test results. Acute respiratory infections are common in respiratory outpatient clinics, and early differentiation between bacterial and viral infections is crucial for appropriate treatment. Combining the detection of MxA protein with CRP, PCT, PSP, IL-6, and HBP can improve diagnostic accuracy, avoid antibiotic overuse, optimize treatment strategies, reduce the risk of drug resistance, and ensure patient safety. Summary of the Invention

[0008] The purpose of this invention is to provide a dual-color synergistic color microsphere, its preparation method, and its application.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A method for preparing dual-color synergistic colored microspheres includes the following steps:

[0011] (1) Preparation of the core of red silica microspheres: Add pure water and anhydrous ethanol to the reaction vessel, add red oil-soluble dye, dissolve by sonication, fix the reaction vessel on an electric stirrer and stir, add ammonia water, add silane coupling agent TEOS after a certain time, react at room temperature overnight, after the reaction is completed, centrifuge to remove the mother liquor, add pure water and disperse by sonication for later use.

[0012] (2) Coating with blue polymer shell: Add pure water to the reaction vessel, purge with nitrogen to remove oxygen, add anionic surfactant and stir to dissolve, add the red silica microsphere core prepared in step (1); weigh out the blue oil-soluble dye and dissolve it in a mixed solution of purified styrene and functional monomers, slowly add it to the reaction vessel, stir and emulsify at high speed, heat to 60℃, add potassium persulfate, and react at 75-85℃ for 4-12h.

[0013] In step (1), the volume ratio of pure water to anhydrous ethanol is 1:4-9, the red oil-soluble dye includes solvent red or disperse red, and the mass ratio of the red oil-soluble dye to the silane coupling agent TEOS is 1:2-3.

[0014] In step (2), the blue oil-soluble dye includes Disperse Blue and Solvent Blue, and the mass ratio of the blue oil-soluble dye to all monomers is 1:10-50; the anionic surfactant is sodium dodecyl sulfate; the functional monomer is methacrylic acid or acrylic acid, preferably methacrylic acid. The volume ratio of styrene to the functional monomer is 1:0.04-0.2.

[0015] The dual-color synergistic color microspheres prepared by the above-described method of the present invention can be used in high-sensitivity single-detection or multi-detection of infection biomarkers.

[0016] The infection markers include viral infection markers and bacterial infection markers. The viral infection marker is MxA protein, and the bacterial infection markers include CRP, PCT, PSP, IL-6, and HBP.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] This invention first prepares a red silica core using the Stöber method, and then coats it with a blue polystyrene shell. Due to the synergistic effect of the blue and red colors, the colored microspheres prepared by this method have an order of magnitude higher sensitivity than colored microspheres with a single embedded dye, thus improving the detection accuracy and sensitivity of high-sensitivity detection items. Attached Figure Description

[0019] Figure 1 Color microspheres prepared for Examples 1-3 and Comparative Examples 1-2. 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 dual-color synergistic colored microspheres by emulsion polymerization

[0022] (1) Preparation of the core of red silica microspheres: 10 mL of pure water and 90 mL of anhydrous ethanol were added to a three-necked flask, along with 2 g of Solvent Red 26. After sonication to dissolve the solvent, the three-necked flask was fixed on an electric stirrer and stirred. 5 mL of ammonia was added, and after 10 min, 5 mL of silane coupling agent TEOS was added. The mixture was reacted overnight at room temperature. After the reaction was completed, the mother liquor was removed by centrifugation, and the mixture was dispersed by sonication with pure water for later use.

[0023] (2) Coating with a blue polymer shell: Add 200 mL of pure water to a three-necked flask, purge with nitrogen to remove oxygen, add 50 mg of SDS and stir to dissolve, then add the silica red microsphere core from step (1); weigh 0.3 g of Sudan Blue B and dissolve it in 10 mL of purified styrene and 2 mL of methacrylic acid mixed solution, slowly add it to the reaction flask, and emulsify by high-speed stirring. Raise the temperature to 60℃, add 120 mg of potassium persulfate, and react at 80℃ for 8 h.

[0024] Products such as Figure 1 As shown.

[0025] Example 2: Preparation of dual-color synergistic colored microspheres by emulsion polymerization

[0026] (1) Preparation of the core of red silica microspheres: 10 mL of pure water and 90 mL of anhydrous ethanol were added to a three-necked flask, along with 2 g of Solvent Red 26. After sonication to dissolve the solvent, the three-necked flask was fixed on an electric stirrer and stirred. 5 mL of ammonia was added, and after 10 min, 5 mL of silane coupling agent TEOS was added. The mixture was reacted overnight at room temperature. After the reaction was completed, the mother liquor was removed by centrifugation, and the mixture was dispersed by sonication with pure water for later use.

[0027] (2) Coating with a blue polymer shell: Add 200 mL of pure water to a three-necked flask, purge with nitrogen to remove oxygen, add 50 mg of SDS and stir to dissolve, then add the silica red microsphere core from step (1); weigh 0.5 g of Sudan Blue B and dissolve it in 10 mL of purified styrene and 2 mL of methacrylic acid mixed solution, slowly add it to the reaction flask, and emulsify by high-speed stirring. Raise the temperature to 60 °C, add 120 mg of potassium persulfate, and react at 80 °C for 8 h.

[0028] Products such as Figure 1 As shown.

[0029] Example 3: Preparation of dual-color synergistic colored microspheres by emulsion polymerization

[0030] (1) Preparation of the core of red silica microspheres: 10 mL of pure water and 90 mL of anhydrous ethanol were added to a three-necked flask, along with 2 g of Solvent Red 26. After sonication to dissolve the solvent, the three-necked flask was fixed on an electric stirrer and stirred. 5 mL of ammonia was added, and after 10 min, 5 mL of silane coupling agent TEOS was added. The mixture was reacted overnight at room temperature. After the reaction was completed, the mother liquor was removed by centrifugation, and the mixture was dispersed by sonication with pure water for later use.

[0031] (2) Coating with a blue polymer shell: Add 200 mL of pure water to a three-necked flask, purge with nitrogen to remove oxygen, add 50 mg of SDS and stir to dissolve, then add the silica red microsphere core from step (1); weigh 1 g of Sudan Blue B and dissolve it in 10 mL of purified styrene and 2 mL of methacrylic acid mixed solution, slowly add it to the reaction flask, and emulsify by high-speed stirring. Raise the temperature to 60℃, add 120 mg of potassium persulfate, and react at 80℃ for 8 h.

[0032] Products such as Figure 1 As shown.

[0033] To highlight the beneficial effects of the present invention, the following comparative experiments are provided.

[0034] Comparative Example 1: Preparation of Red Microspheres

[0035] (1) Add 10 mL of pure water and 90 mL of anhydrous ethanol to a three-necked flask, add 2 g of Solvent Red 26, and dissolve by sonication. Then 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 overnight. After the reaction is complete, centrifuge to remove the mother liquor, add pure water and sonicate for dispersion.

[0036] (2) Add 200 mL of pure water to a three-necked flask, purge with nitrogen to remove oxygen, add 50 mg of SDS and stir to dissolve, then add the silica red microsphere core from step (1); dissolve 0.5 g of Solvent Red 26 in 10 mL of purified styrene and 2 mL of methacrylic acid mixture, slowly add to the reaction flask, and emulsify by high-speed stirring. Heat to 60℃, add 120 mg of potassium persulfate, and react at 80℃ for 8 h. The product is as follows Figure 1 As shown.

[0037] Comparative Example 2: Preparation of Blue Microspheres

[0038] (1) Add 10 mL of pure water and 90 mL of anhydrous ethanol to a three-necked flask, add 2 g of Sudan Blue B, dissolve by sonication, 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 overnight. After the reaction is complete, centrifuge to remove the mother liquor, add pure water and sonicate for dispersion, then set aside for later use.

[0039] (2) Add 200 mL of pure water to a three-necked flask, purge with nitrogen to remove oxygen, add 50 mg of SDS and stir to dissolve, then add the silica microsphere core from step (1); dissolve 0.5 g of Sudan Blue B in 10 mL of purified styrene and 2 mL of methacrylic acid mixture, slowly add to the reaction flask, and emulsify by high-speed stirring. Heat to 60°C, add 120 mg of potassium persulfate, and react at 80°C for 8 hours. The product is as follows: Figure 1 As shown.

[0040] Table 1 Microsphere Particle Size and Dispersion Index

[0041]

[0042] Example 4

[0043] The colored microspheres of Examples 1-3 and Comparative Examples 1-2 were used to prepare human myxovirus resistance protein A (MxA) detection cards. The specific steps are as follows:

[0044] (1) Labeling of MxA antibody

[0045] ① Washing: Take 50 μL of colored microspheres from Examples 1-3 and Comparative Examples 1-2 respectively and add them to centrifuge tubes containing 0.5 mL of 50 mM MMES (pH 6.1) buffer. Mix well by sonication and centrifugation to discard the supernatant.

[0046] ② Activation: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the cleaned colored microspheres of Examples 1-3 and Comparative Examples 1-2, 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;

[0047] ③ Coupling: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the activated colored microspheres of Examples 1-3 and Comparative Examples 1-2, 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;

[0048] 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;

[0049] ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the colored microsphere-antibody complexes of Examples 1-3 and Comparative Examples 1-2, 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.

[0050] (2) Preparation of nitrocellulose membrane

[0051] The prepared MxA-coated antibody (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) solutions were applied to nitrocellulose membranes at a coating volume of 1 μL / cm using a streak sprayer, serving as the detection line and control line, respectively. The membranes were then dried overnight at 45°C.

[0052] (3) Preparation of the binding pad

[0053] The MxA antibody complexes labeled with colored microspheres from Examples 1-3 and Comparative Examples 1-2 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.

[0054] (4) Reagent card assembly

[0055] 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.

[0056] 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.

[0057] (5) Reagent card testing

[0058] ① Tear open the aluminum foil bag seal and take out the test card from the aluminum foil bag.

[0059] ② Add 5 μL of sample to the sample diluent, mix thoroughly, and then add 70 μL (4 drops) vertically into the sample well of the test card. The reaction time is 8 min.

[0060] ③ Read the test results.

[0061] Application Example 1

[0062] MxA Sensitivity Detection

[0063] Different concentrations of MxA calibrators were used for standard curve testing. The reaction time was 8 min. 70 μL of diluent was added to the sample or calibrator, and 80 μL of the diluent was used to add the sample to the test cards prepared in Examples 1-3 and Comparative Examples 1-2. The experimental results are shown in Table 2.

[0064] Table 2. Performance comparison of microspheres in Examples 1, 2, and 3 with microspheres in Comparative Examples 1 and 2

[0065]

[0066] Note: The signal values ​​are obtained by the card reader. The higher the value, the more obvious the color display.

[0067] Based on the performance comparison results of the microspheres in Examples 1-3 and Comparative Examples 1-2, it can be seen that the microspheres in Examples 1-3 are significantly better than the red microspheres in Comparative Example 1 and the blue microspheres in Comparative Example 2 in terms of MxA detection performance. Considering both detection performance and visual perception, the purple-red microspheres in Example 2 should be given priority in MxA and PCT joint detection, MxA and PSP joint detection, MxA and IL-6 joint detection, and MxA and HBP joint detection.

[0068] Example 6

[0069] The preparation method of the purple-red microspheres used in Example 2 for the MxA and PCT detection card is as follows:

[0070] (1) Labeling of MxA and PCT antibodies

[0071] ① Washing: Take 50 μL of the purple-red microspheres from Example 2 and add them to a centrifuge tube containing 0.5 mL of 50 mM MES (pH 6.1) buffer. Mix well by sonication and centrifugation to discard the supernatant.

[0072] ② Activation: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the cleaned purple-red microspheres from 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.

[0073] ③ Coupling: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the activated purple-red microspheres of Example 2 and mix by sonication; add 50 μg of MxA-labeled antibody and 30 μg of PCT-labeled antibody to be coupled, vortex to mix, and couple at room temperature for 2 h.

[0074] 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;

[0075] ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the purple-red microsphere-antibody complex from Example 2 and store at 2-8°C protected from light. The microsphere reconstitution solution formulation 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.

[0076] (2) Preparation of nitrocellulose membrane

[0077] The prepared MxA-coated antibody, PCT-coated antibody (1 mg / mL), and goat anti-mouse IgG antibody (1 mg / mL) solutions were applied to nitrocellulose membranes at a coating volume of 1 μL / cm using a streak-spraying gold sprayer, serving as the detection line and control line, respectively. The membranes were then dried overnight at 45°C.

[0078] (3) Preparation of the binding pad

[0079] The MxA antibody and PCT antibody complex labeled with purple-red 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.

[0080] (4) Reagent card assembly

[0081] 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.

[0082] 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.

[0083] (5) Reagent card testing

[0084] ① Tear open the aluminum foil bag seal and take out the test card from the aluminum foil bag.

[0085] ② Add 5 μL of sample to the sample diluent, mix thoroughly, and then add 70 μL (4 drops) vertically into the sample well of the test card. The reaction time is 8 min.

[0086] ③ Read the test results.

[0087] In this embodiment, the product was compared with similar products already approved for marketing in 212 clinical whole blood samples. The positive concordance rate of MxA was 96.15%, the negative concordance rate was 93.28%, and the total concordance rate was 94.34%; the positive concordance rate of PCT was 95.52%, the negative concordance rate was 91.72%, and the total concordance rate was 92.92%. The test results showed that the concordance rate was good.

[0088] Example 7

[0089] The method for preparing the MxA and PSP detection card using the purple-red microspheres from Example 2 is as follows:

[0090] (1) Labeling of MxA and PSP antibodies

[0091] ① Washing: Take 50 μL of the purple-red microspheres from Example 2 and add them to a centrifuge tube containing 0.5 mL of 50 mM MES (pH 6.1) buffer. Mix well by sonication and centrifugation to discard the supernatant.

[0092] ② Activation: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the cleaned purple-red microspheres from 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.

[0093] ③ Coupling: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the activated purple-red microspheres of Example 2 and mix by sonication; add 50 μg of MxA-labeled antibody and 50 μg of PSP-labeled antibody to be coupled, vortex to mix, and couple at room temperature for 2 h.

[0094] 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;

[0095] ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the purple-red microsphere-antibody complex from Example 2 and store at 2-8°C protected from light. The microsphere reconstitution solution formulation 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.

[0096] (2) Preparation of nitrocellulose membrane

[0097] The prepared MxA-coated antibody (1 mg / mL), PSP-coated antibody (2 mg / mL), and goat anti-mouse IgG antibody (1 mg / mL) solutions were applied to nitrocellulose membranes at a coating volume of 1 μL / cm using a streak coater, serving as the detection line and control line, respectively. The membranes were then dried overnight at 45°C.

[0098] (3) Preparation of the binding pad

[0099] The MxA antibody and PSP antibody complex labeled with purple-red microspheres from 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.

[0100] (4) Reagent card assembly

[0101] 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.

[0102] 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.

[0103] (5) Reagent card testing

[0104] ① Tear open the aluminum foil bag seal and take out the test card from the aluminum foil bag.

[0105] ② Add 5 μL of sample to the sample diluent, mix thoroughly, and then add 70 μL (4 drops) vertically into the sample well of the test card. The reaction time is 8 min.

[0106] ③ Read the test results.

[0107] In this embodiment, the product was compared with similar products already approved for marketing in 202 clinical whole blood samples. The positive concordance rate of MxA was 96.92%, the negative concordance rate was 92.7%, and the total concordance rate was 94.06%; the positive concordance rate of PSP was 93.52%, the negative concordance rate was 91.42%, and the total concordance rate was 92.92%. The test results show that the concordance rate is good.

[0108] Example 8

[0109] The preparation method of the purple-red microspheres used in Example 2 for the MxA and IL-6 detection card is as follows:

[0110] (1) Labeling of MxA and IL-6 antibodies

[0111] ① Washing: Take 50 μL of the purple-red microspheres from Example 2 and add them to a centrifuge tube containing 0.5 mL of 50 mM MES (pH 6.1) buffer. Mix well by sonication and centrifugation to discard the supernatant.

[0112] ② Activation: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the cleaned purple-red microspheres from 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.

[0113] ③ Coupling: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the activated purple-red microspheres of Example 2 and mix by sonication; add 50 μg of MxA-labeled antibody and 50 μg of IL-6-labeled antibody to be coupled, vortex to mix, and couple at room temperature for 2 h.

[0114] 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;

[0115] ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the purple-red microsphere-antibody complex from Example 2 and store at 2-8°C protected from light. The microsphere reconstitution solution formulation 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.

[0116] (2) Preparation of nitrocellulose membrane

[0117] The prepared MxA-coated antibody (1 mg / mL), IL-6-coated antibody (1.5 mg / mL), and goat anti-mouse IgG antibody (1 mg / mL) solutions were applied to nitrocellulose membranes at a coating volume of 1 μL / cm using a streak sprayer, serving as the detection line and control line, respectively. The membranes were then dried overnight at 45°C.

[0118] (3) Preparation of the binding pad

[0119] The MxA antibody and IL-6 antibody complex labeled with purple-red 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.

[0120] (4) Reagent card assembly

[0121] 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.

[0122] 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.

[0123] (5) Reagent card testing

[0124] ① Tear open the aluminum foil bag seal and take out the test card from the aluminum foil bag.

[0125] ② Add 5 μL of sample to the sample diluent, mix thoroughly, and then add 70 μL (4 drops) vertically into the sample well of the test card. The reaction time is 8 min.

[0126] ③ Read the test results.

[0127] In this embodiment, the product was compared with similar products already approved for marketing in 225 clinical whole blood samples. The positive concordance rate for MxA was 94.2%, the negative concordance rate was 92.31%, and the total concordance rate was 92.89%; the positive concordance rate for IL-6 was 94.37%, the negative concordance rate was 92.21%, and the total concordance rate was 92.89%. The test results showed that the concordance rate was good.

[0128] Example 9

[0129] The method for preparing the MxA and HBP detection card using the purple-red microspheres from Example 2 is as follows:

[0130] (1) Labeling of MxA and HBP antibodies

[0131] ① Washing: Take 50 μL of the purple-red microspheres from Example 2 and add them to a centrifuge tube containing 0.5 mL of 50 mM MES (pH 6.1) buffer. Mix well by sonication and centrifugation to discard the supernatant.

[0132] ② Activation: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the cleaned purple-red microspheres from 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.

[0133] ③ Coupling: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the activated purple-red microspheres of Example 2 and mix by sonication; add 50 μg of MxA-labeled antibody and 30 μg of HBP-labeled antibody to be coupled, vortex to mix, and couple at room temperature for 2 h.

[0134] 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;

[0135] ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the purple-red microsphere-antibody complex from Example 2 and store at 2-8°C protected from light. The microsphere reconstitution solution formulation 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.

[0136] (2) Preparation of nitrocellulose membrane

[0137] The prepared MxA-coated antibody (1 mg / mL), HBP-coated antibody (1 mg / mL), and goat anti-mouse IgG antibody (1 mg / mL) solutions were applied to nitrocellulose membranes at a coating volume of 1 μL / cm using a streak coater, serving as the detection line and control line, respectively. The membranes were then dried overnight at 45°C.

[0138] (3) Preparation of the binding pad

[0139] The MxA antibody and HBP antibody complex labeled with purple-red 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.

[0140] (4) Reagent card assembly

[0141] 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.

[0142] 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.

[0143] (5) Reagent card testing

[0144] ① Tear open the aluminum foil bag seal and take out the test card from the aluminum foil bag.

[0145] ② Add 5 μL of sample to the sample diluent, mix thoroughly, and then add 70 μL (4 drops) vertically into the sample well of the test card. The reaction time is 8 min.

[0146] ③ Read the test results.

[0147] In this embodiment, the product was compared with similar products already approved for marketing in 214 clinical whole blood samples. The positive concordance rate for MxA was 95.45%, the negative concordance rate was 92.57%, and the total concordance rate was 93.46%; the positive concordance rate for HBP was 93.94%, the negative concordance rate was 91.22%, and the total concordance rate was 92.06%. The test results show that the concordance rate is good.

[0148] 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 dual-color synergistic colored microspheres, characterized in that, Includes the following steps: (1) Preparation of the core of red silica microspheres: Add pure water and anhydrous ethanol to the reaction vessel, add red oil-soluble dye, dissolve by sonication, fix the reaction vessel on an electric stirrer and stir, add ammonia water, add silane coupling agent TEOS after a certain time, react at room temperature overnight, after the reaction is completed, centrifuge to remove the mother liquor, add pure water and disperse by sonication for later use. (2) Coating with blue polymer shell: Add pure water to the reaction vessel, purge with nitrogen to remove oxygen, add anionic surfactant and stir to dissolve, add the red silica microsphere core prepared in step (1); weigh out the blue oil-soluble dye and dissolve it in a mixed solution of purified styrene and functional monomers, slowly add it to the reaction vessel, stir and emulsify at high speed, heat to 60℃, add potassium persulfate, and react at 75-85℃ for 4-12h.

2. The method for preparing dual-color synergistic colored microspheres according to claim 1, characterized in that: In step (1), the volume ratio of pure water to anhydrous ethanol is 1:4-9.

3. The method for preparing dual-color synergistic colored microspheres according to claim 1, characterized in that: In step (1), the red oil-soluble dye includes Solvent Red or Disperse Red.

4. The method for preparing dual-color synergistic colored microspheres according to claim 1, characterized in that: In step (1), the mass ratio of the red oil-soluble dye to the silane coupling agent TEOS is 1:2-3.

5. The method for preparing dual-color synergistic colored microspheres according to claim 1, characterized in that: In step (2), the blue oil-soluble dyes include Disperse Blue and Solvent Blue.

6. The method for preparing dual-color synergistic colored microspheres according to claim 1, characterized in that: In step (2), the mass ratio of the blue oil-soluble dye to all monomers is 1:10-50; the anionic surfactant is sodium dodecyl sulfate; the functional monomer is methacrylic acid or acrylic acid, wherein the volume ratio of styrene to the functional monomer is 1:0.04-0.

2.

7. The method for preparing dual-color synergistic colored microspheres according to claim 6, characterized in that: The functional monomer is methacrylic acid.

8. The dual-color synergistic color microspheres prepared by the preparation method according to any one of claims 1-7.

9. The application of the dual-color synergistic color microspheres as described in claim 8 in the preparation of detection cards for highly sensitive single- or multiple-detection infection markers.

10. The application of the dual-color synergistic color microspheres according to claim 9 in the preparation of detection cards for highly sensitive single- or multiple-detection infection biomarkers, characterized in that: The infection markers are viral infection markers and bacterial infection markers. The viral infection marker is MxA protein, and the bacterial infection markers include CRP, PCT, PSP, IL-6, and HBP.

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