Time-resolved fluorescent microspheres, preparation method and application thereof in psp / crp / mxa quantitative detection immunochromatographic test strip

Abstract

The application relates to the technical field of immunochromatography, in particular to time-resolved fluorescent microspheres, a preparation method thereof and application of the time-resolved fluorescent microspheres in PSP / CRP / MxA quantitative detection immunochromatography test strips, wherein the preparation method of the time-resolved fluorescent microspheres comprises the following steps: 1) preparation of surface-aminated polystyrene microspheres; 2) glutaraldehyde crosslinking and 1,10-phenanthroline-5-amino combination; 3) surface grafting of rare earth fluorescent dye; and 4) polymer shell coating and carboxyl modification. Through the double fixation strategy of "surface grafting + polymer coating", the time-resolved fluorescent dye is limited in the functional region between the microsphere surface and the polymer shell layer, the diffusion leakage of the dye molecules to the external environment is blocked, and the dye falling-off problem of the traditional fluorescent microspheres is solved; the polymer shell layer can effectively isolate the external ultraviolet light, oxygen and other erosive factors, reduce the photo-oxidative degradation and non-radiative transition loss of the fluorescent molecules, and improve the light stability and fluorescent life.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunochromatography, and in particular to a time-resolved fluorescent microsphere, a preparation method thereof, and application of the time-resolved fluorescent microsphere in PSP / CRP / MxA quantitative detection immunochromatography test strips. BACKGROUND

[0002] PSP is a stress protein secreted by the pancreas, and is elevated in patients with acute and chronic pancreatitis, diabetes, inflammatory bowel disease, and digestive tract tumors, and has important value in predicting sepsis. CRP is an acute phase protein that rapidly increases in the blood when inflammation, infection, and other diseases occur, and is a commonly used clinical inflammation indicator. MxA protein is a 78kDa antiviral protein produced by cells induced by type I interferon, and its expression level can be used as a marker for viral infection.

[0003] Traditional immunochromatography test strips use colloidal gold as a marker, and have limited detection sensitivity, allowing only qualitative or semi-quantitative detection, which cannot meet the demand for accurate quantitative analysis of PSP, CRP, and MxA. In addition, existing time-resolved fluorescent immunochromatography test strips have problems such as dye leakage and poor fluorescence stability, which affect the accuracy and reliability of the detection results. Moreover, there is a lack of immunochromatography test strips on the market that can simultaneously and rapidly, accurately, and quantitatively detect the three markers PSP, CRP, and MxA, making it difficult to meet the demand for diagnosis and monitoring of multiple diseases in the clinic. SUMMARY

[0004] The first object of the present application is to provide a time-resolved fluorescent microsphere and a preparation method thereof.

[0005] The second object of the present application is to provide application of the time-resolved fluorescent microsphere in PSP / CRP / MxA quantitative detection immunochromatography test strips.

[0006] To achieve the above objects, the present application provides the following technical solutions:

[0007] A preparation method of a time-resolved fluorescent microsphere, comprising the following steps:

[0008] (1) Preparation of surface-aminated polystyrene microspheres: a reaction container is charged with pure water, and a surfactant is added under nitrogen protection. After stirring and dissolving, styrene is added, and the temperature is raised to 60℃. An initiator is added, and the temperature is further raised to 80℃ for reaction. After the reaction, the product is washed by centrifugation with anhydrous ethanol and pure water, and is redispersed in pure water to obtain an amino-modified polystyrene microsphere core;

[0009] (2) glutaraldehyde cross-linking and 1,10-phenanthroline-5-amino combination: taking the amino-modified polystyrene microspheres core, washing with PBS buffer, finally storing in buffer, ultrasonic dispersion, adding glutaraldehyde, mixing and reacting; then washing with PBS buffer, finally storing in anhydrous ethanol, adding 1,10-phenanthroline-5-amino, dropping acetic acid, washing with anhydrous ethanol and pure water after reaction, and re-dispersing in pure water;

[0010] (3) surface grafting of rare earth fluorescent dye: dispersing the product of step (2) in ethanol, adding rare earth metal chloride and auxiliary ligand, dissolving, then adding acid binding agent, washing with anhydrous ethanol and pure water after reaction, and re-dispersing in pure water;

[0011] (4) coating of polymer shell and carboxyl modification: dispersing the microspheres of surface grafted rare earth fluorescent dye in anhydrous ethanol, ultrasonic dispersion, deoxidizing by nitrogen, adding polyacrylic acid, and stirring and dissolving; taking styrene, divinylbenzene and acrylic acid in an EP tube, adding initiator, ultrasonic dissolving, then adding into a reaction container, washing with ethanol and pure water after reaction, and finally storing in pure water.

[0012] In step (1), the surfactant is an anionic surfactant, the initiator is water-soluble persulfate, and the mass ratio of styrene to 2-aminoethyl methacrylate hydrochloride is 1:0.05-0.5.

[0013] In step (1), the surfactant is sodium dodecyl sulfate, and the initiator is potassium persulfate.

[0014] In step (2), the PBS buffer is 50mM pH7.4 PBS buffer.

[0015] In step (2), the molar ratio of 1,10-phenanthroline-5-amino to glutaraldehyde is 1:0.6-1.

[0016] In step (3), the rare earth metal chloride is EuCl3·6H2O, the auxiliary ligand is dibenzoylmethane, and the acid binding agent is triethylamine.

[0017] In step (3), the molar ratio of EuCl3·6H2O, dibenzoylmethane and triethylamine is 1:3-3.6:1-1.2.

[0018] In step (4), the mass ratio of styrene, divinylbenzene and acrylic acid is 1:0.01-0.05:0.02-0.2.

[0019] The time-resolved fluorescent microspheres prepared by the above preparation method can be used to prepare PSP / CRP / MxA quantitative detection immunochromatographic test strips, and the specific preparation method is as follows:

[0020] (1) Labeling of PSP antibody

[0021] ① Washing: Take 50 μL time-resolved fluorescent microspheres into a centrifuge tube containing 0.5 mL 50 mM MES (pH 6.5) buffer, mix by ultrasonic, and centrifuge to discard the supernatant;

[0022] ② Activation: Add 0.5 mL 50 mM MES (pH 6.5) buffer to the washed microspheres, mix by ultrasonic, then add 20 μL of freshly prepared EDC (50 mg / mL) and 40 μL of NHS (50 mg / mL), mix by vortex, activate at room temperature for 30 min, and centrifuge to discard the supernatant;

[0023] ③ Coupling: Add 0.5 mL 50 mM MES (pH 6.5) buffer to the activated microspheres, mix by ultrasonic; add 50 μg of PSP-labeled antibody to be coupled, mix by vortex, and couple at room temperature for 2 h;

[0024] 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 to discard the supernatant;

[0025] ⑤ Resuspension: Add 500 μL of microsphere resuspension solution to the microsphere-antibody complex, and store at 2-8°C in the dark. The microsphere resuspension solution is formulated as follows: 10 mM PBS (pH 7.4±0.05), 10 mM NaCl, 0.05% Tween-80, 1% BSA, 5% sucrose, and 0.1% ProClin 300.

[0026] (2) Labeling of CRP antibody

[0027] ① Washing: Take 50 μL time-resolved fluorescent microspheres into a centrifuge tube containing 0.5 mL 50 mM MES (pH 6.5) buffer, mix by ultrasonic, and centrifuge to discard the supernatant;

[0028] ② Activation: Add 0.5 mL 50 mM MES (pH 6.5) buffer to the washed microspheres, mix by ultrasonic, then add 20 μL of freshly prepared EDC (50 mg / mL) and 40 μL of NHS (50 mg / mL), mix by vortex, activate at room temperature for 30 min, and centrifuge to discard the supernatant;

[0029] ③ Coupling: Add 0.5 mL 50 mM MES (pH 6.5) buffer to the activated microspheres, mix by ultrasonic; add 50 μg of CRP-labeled antibody to be coupled, mix by vortex, and couple at room temperature for 2 h;

[0030] 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;

[0031] ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the 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.

[0032] (3) Labeling of MxA antibodies

[0033] ① Washing: Take 50 μL of time-resolved fluorescent microspheres 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 to discard the supernatant.

[0034] ② Activation: Add 0.5 mL of 50 mM MES (pH 6.5) buffer to the cleaned microspheres, sonicate to mix, then add 20 μL of freshly prepared EDC (50 mg / mL) and 40 μL of NHS (50 mg / mL), vortex to mix, activate at room temperature for 30 min, centrifuge and discard the supernatant.

[0035] ③ Coupling: Add 0.5 mL of 50 mM MES (pH 6.5) buffer to the activated 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.

[0036] 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;

[0037] ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the 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.

[0038] (4) Preparation of nitrocellulose membrane

[0039] The prepared PSP coated antibody (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) solution is drawn on the nitrocellulose membrane with a membrane drawing and gold spraying integrated machine at a coating amount of 1 μL / cm to serve as a detection line and a quality control line of the first test strip respectively, the prepared CRP coated antibody (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) solution is drawn on the nitrocellulose membrane with a membrane drawing and gold spraying integrated machine at a coating amount of 1 μL / cm to serve as a detection line and a quality control line of the second test strip respectively, the prepared MxA coated antibody (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) solution is drawn on the nitrocellulose membrane with a membrane drawing and gold spraying integrated machine at a coating amount of 1 μL / cm to serve as a detection line and a quality control line of the third test strip respectively, and the prepared products are dried at 45°C overnight.

[0040] (5) Preparation of a conjugate pad

[0041] The microsphere-labeled PSP antibody complex, CRP antibody complex and MxA antibody complex in the above step are sprayed on the glass cellulose membrane at a coating amount of 5 μL / cm respectively, and dried at 45°C overnight to prepare a conjugate pad.

[0042] (6) Assembly of a reagent strip

[0043] ① The above coated nitrocellulose membrane is pasted on a PVC bottom plate in turn with a water absorption pad, a conjugate pad and a sample pad. The quality control line is pasted with a water absorption pad at one end, the water absorption pad is placed on the NC membrane, the detection line is pasted with a conjugate pad at one end, the conjugate pad is placed on the NC membrane at one end, and the sample pad is placed on the conjugate pad.

[0044] ② The large plate assembled in step ① is cut into a reagent strip with a width of 3 mm by a cutting machine, the reagent strip is loaded into a card shell, and the loaded reagent card and a desiccant are placed in an aluminum foil bag for sealing, and stored for standby use.

[0045] The detection method of the PSP / CRP / MxA quantitative detection immunochromatographic test strip prepared in the application is specifically as follows:

[0046] ① The aluminum foil bag is opened, and the detection card is taken out from the aluminum foil bag.

[0047] ② 5 μL of the sample is taken and added to the sample diluent, and after being fully mixed, 70 μL (4 drops of a dropper) is vertically added to the sample addition hole of the detection card, and the reaction time is 8 min.

[0048] ③ The detection result is read.

[0049] Compared with the prior art, the application has the beneficial effects that:

[0050] The application prepares high-performance time-resolved fluorescent microspheres through "step-by-step functional modification + core-shell structure design", and the technical advantages include:

[0051] (1) Polystyrene microspheres as a substrate, by surface amination modification to build a reactive platform, so that the microsphere surface is uniformly loaded with active amino sites, laying the foundation for subsequent covalent connection.

[0052] (2) By crosslinking agent glutaraldehyde to realize the directional grafting of the main ligand: using the dialdehyde group characteristics of glutaraldehyde, one end forms a Schiff base covalent bond with the amino group on the surface of the microsphere, and the other end connects 1,10-phenanthroline-5-amino ligand, to construct a stable connection structure of "microsphere-glutaraldehyde-ligand", providing a target binding site for the immobilization of fluorescent dyes.

[0053] (3) Synthesis and grafting of time-resolved fluorescent dyes on the surface of microspheres: using 1,10-phenanthroline-5-amino as the ligand skeleton, through coordination reaction, the rare earth complex time-resolved fluorescent dye is fixed on the surface of the microsphere, ensuring that the dye molecules are evenly distributed and firmly combined.

[0054] (4) In order to solve the key problems of dye leakage and insufficient light stability, further adopt the process of core-shell coating and surface modification: a dense polymer shell is coated on the outside of the dye-loaded microspheres, forming a core-shell structure of "inner core functional layer-outer shell protective layer"; at the same time, by copolymerization, carboxyl active groups are introduced into the polymer shell, giving the microspheres good bioconjugation ability.

[0055] The preparation method has two core technical advantages: first, through the double fixation strategy of "surface grafting + polymer coating", the time-resolved fluorescent dye is confined in the functional area between the surface of the microsphere and the polymer shell, completely blocking the diffusion and leakage of the dye molecules to the external environment, solving the problem of dye shedding of traditional fluorescent microspheres; second, the dense polymer shell can effectively isolate the external ultraviolet light, oxygen and other erosive factors, reducing the photo-oxidative degradation and non-radiative transition loss of fluorescent molecules, significantly improving the light stability and fluorescence lifetime.

[0056] Based on the method of the present application, the time-resolved fluorescent microspheres are prepared, and the carboxyl on the surface of the shell and the amino of the antibody form an amide bond, successfully realizing the efficient labeling of PSA, CRP and MxA antibodies. The labeled fluorescent microspheres are applied to the immune detection system, and the time-resolved fluorescent signal can be quantitatively read, realizing the high specificity and high sensitivity synchronous quantitative detection of the three biomarkers of PSP, CRP and MxA, providing a probe tool with excellent performance for multi-index joint diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 It is a schematic diagram of the preparation process of time-resolved fluorescent microspheres.

[0058] Figure 2The image shows a SEM image of the time-resolved fluorescent microspheres prepared in Example 4.

[0059] Figure 3 The image shows a SEM image of the time-resolved fluorescent microspheres prepared in Example 5.

[0060] Figure 4 A schematic diagram of an immunochromatographic test strip for PSP / CRP / MxA quantitative detection. Detailed Implementation

[0061] 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.

[0062] Example 1

[0063] like Figure 1 As shown, 100 mL of pure water was added to a three-necked flask, and nitrogen gas was purged for 20 min to remove oxygen. Under nitrogen protection, 100 mg of sodium dodecyl sulfate (SDS) was added, stirred until dissolved, and then 5 mL of styrene was added. The temperature was raised to 80 °C. At 60 °C, 100 mg of potassium persulfate (KPS) (dissolved in 5 mL of pure water) was added, and the reaction was continued at 80 °C for 2 h. 50 mg of SDS was added and mixed well, followed by 2 g of 2-aminoethyl methacrylate hydrochloride. The reaction was continued for 4 h, and then cooled to terminate the reaction. The product was washed three times each with anhydrous ethanol and pure water by centrifugation, redispersed in pure water, and the solid content was adjusted to 10% to obtain the core of amino-modified polystyrene microspheres.

[0064] Example 2

[0065] like Figure 1 As shown, 1g of the amino-modified polystyrene microsphere core from Example 1 was taken, washed twice with 50mM pH7.4 PBS buffer, and finally stored in 20mL buffer. It was then ultrasonically dispersed, 1mL of glutaraldehyde was added and mixed, and the mixture was reacted at room temperature for 1h. Then, it was washed three times with 50mM pH7.4 PBS buffer, and finally stored in 50mL of anhydrous ethanol. 2.5g of 1,10-phenanthroline-5-amino was added, and then a few drops of acetic acid were added as a catalyst. The mixture was reacted at room temperature for 30min, washed three times each with anhydrous ethanol and pure water, and then redispersed in 10mL of pure water.

[0066] Example 3

[0067] like Figure 1As shown, the microspheres grafted with the host ligand from Example 2 were dispersed in 90 mL of ethanol, and 3.75 g of EuCl3∙6H2O and 7.5 g of dibenzoylmethane were added. After dissolution, 1.67 mL of triethylamine was added, and the reaction was carried out at 65 °C for 4 h. After washing three times each with anhydrous ethanol and pure water, the microspheres were redispersed in 10 mL of pure water.

[0068] Example 4

[0069] like Figure 1 As shown, 2 mL of the microspheres from Example 3 were dispersed in 20 mL of anhydrous ethanol, ultrasonically dispersed, and nitrogen gas was purged for 20 min to remove oxygen from the system. Then, 0.2 g of polyacrylic acid was added and stirred to dissolve. 2 mL of styrene, 0.05 mL of divinylbenzene, and 0.1 mL of acrylic acid were placed in an EP tube, and 30 mg of azobisisobutyronitrile (AIBN) initiator was added. After ultrasonic dissolution, the mixture was added to a flask and reacted at 70 °C for 24 h. The mixture was washed three times with ethanol and three times with pure water, and finally stored in pure water with the solid content adjusted to 1%. The SEM image of the prepared time-resolved fluorescent microspheres is shown below. Figure 2 As shown.

[0070] Example 5

[0071] like Figure 1 As shown, 2 mL of the microspheres from Example 3 were dispersed in 20 mL of anhydrous ethanol, ultrasonically dispersed, and nitrogen gas was purged for 20 min to remove oxygen from the system. Then, 0.2 g of polyacrylic acid was added and stirred to dissolve. 2 mL of styrene, 0.05 mL of divinylbenzene, and 0.2 mL of acrylic acid were placed in an EP tube, and 30 mg of initiator AIBN was added. After ultrasonic dissolution, the mixture was added to a flask and reacted at 70 °C for 24 h. The mixture was washed three times with ethanol and three times with pure water, and finally stored in pure water with the solid content adjusted to 1%. The SEM image of the prepared time-resolved fluorescent microspheres is shown below. Figure 3 As shown.

[0072] Example 6

[0073] The time-resolved fluorescent microspheres from Examples 4-5 above were used in the preparation of immunochromatographic test strips for quantitative detection of PSP, CRP, and MxA. The specific steps are as follows:

[0074] (1) Labeling of PSP antibodies

[0075] ① Washing: Take 50 μL of time-resolved fluorescent microspheres from Example 5 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.

[0076] ② Activation: 0.5 mL of 50 mM MES (pH 6.5) buffer was added to the washed microspheres, and after ultrasonic mixing, 20 μL of freshly prepared EDC (50 mg / mL) and 40 μL of NHS (50 mg / mL) were added, and mixed by vortex, activated at room temperature for 30 min, centrifuged and discarded the supernatant;

[0077] ③ Coupling: 0.5 mL of 50 mM MES (pH 6.5) buffer was added to the activated microspheres, and after ultrasonic mixing, 50 μg of the PSP-labeled antibody to be coupled was added, and mixed by vortex, coupled at room temperature for 2 h;

[0078] ④ Blocking: 300 μL of 10% BSA and 20 μL of ethanolamine were added to the above coupling buffer, and blocked at room temperature for 1 h, centrifuged and discarded the supernatant;

[0079] ⑤ Resuspension: 500 μL of microsphere resuspension solution was added to the microsphere-antibody complex, and stored at 2-8°C in the dark. The microsphere resuspension solution was prepared as follows: 10 mM PBS (pH 7.4±0.05), 10 mM NaCl, 0.05% Tween-80, 1% BSA, 5% sucrose, and 0.1% ProClin 300.

[0080] Labeling of CRP antibody

[0081] ① Washing: 50 μL of the time-resolved fluorescent microspheres of Example 4 were taken and added to a centrifuge tube containing 0.5 mL of 50 mM MES (pH 6.5) buffer, and mixed by ultrasonic, and centrifuged and discarded the supernatant;

[0082] ② Activation: 0.5 mL of 50 mM MES (pH 6.5) buffer was added to the washed microspheres, and after ultrasonic mixing, 20 μL of freshly prepared EDC (50 mg / mL) and 40 μL of NHS (50 mg / mL) were added, and mixed by vortex, activated at room temperature for 30 min, centrifuged and discarded the supernatant;

[0083] ③ Coupling: 0.5 mL of 50 mM MES (pH 6.5) buffer was added to the activated microspheres, and after ultrasonic mixing, 50 μg of the PSP-labeled antibody to be coupled was added, and mixed by vortex, coupled at room temperature for 2 h;

[0084] ④ Blocking: 300 μL of 10% BSA and 20 μL of ethanolamine were added to the above coupling buffer, and blocked at room temperature for 1 h, centrifuged and discarded the supernatant;

[0085] ⑤Resuspension: 500 μL of microsphere resuspension solution was added to the microsphere-antibody complex, which was stored at 2-8°C in the dark. The microsphere resuspension solution was formulated as follows: 10 mM PBS (pH 7.4 ± 0.05), 10 mM NaCl, 0.05% Tween-80, 1% BSA, 5% sucrose, 0.1% ProClin 300.

[0086] Labeling of MxA antibody

[0087] ①Washing: 50 μL of time-resolved fluorescent microspheres of Example 5 were taken and added to a centrifuge tube containing 0.5 mL of 50 mM MES (pH 6.5) buffer, and mixed by ultrasonication and centrifuged to discard the supernatant;

[0088] ②Activation: 0.5 mL of 50 mM MES (pH 6.5) buffer was added to the washed microspheres, mixed by ultrasonication, and then 20 μL of freshly prepared EDC (50 mg / mL) and 40 μL of NHS (50 mg / mL) were added, mixed by vortexing, and activated at room temperature for 30 min, and then centrifuged to discard the supernatant;

[0089] ③Coupling: 0.5 mL of 50 mM MES (pH 6.5) buffer was added to the activated microspheres, mixed by ultrasonication, and then 50 μg of the MxA labeled antibody to be coupled was added, mixed by vortexing, and coupled at room temperature for 2 h;

[0090] ④Blocking: 300 μL of 10% BSA and 20 μL of ethanolamine were added to the above coupling buffer, blocked at room temperature for 1 h, and then centrifuged to discard the supernatant;

[0091] ⑤Resuspension: 500 μL of microsphere resuspension solution was added to the microsphere-antibody complex, which was stored at 2-8°C in the dark. The microsphere resuspension solution was formulated as follows: 10 mM PBS (pH 7.4 ± 0.05), 10 mM NaCl, 0.05% Tween-80, 1% BSA, 5% sucrose, 0.1% ProClin 300.

[0092] Preparation of nitrocellulose membrane

[0093] The prepared PSP-coated antibody (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) solutions were applied to a nitrocellulose membrane at a coating volume of 1 μL / cm using a streak coater, serving as the test line and control line for the first test strip, respectively. The prepared CRP-coated antibody (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) solutions were applied to a nitrocellulose membrane at a coating volume of 1 μL / cm using a streak coater, serving as the test line and control line for the second test strip, respectively. The prepared MxA-coated antibody (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) solutions were applied to a nitrocellulose membrane at a coating volume of 1 μL / cm using a streak coater, serving as the test line and control line for the third test strip, respectively. The strips were dried overnight at 45°C.

[0094] (5) Preparation of the conjugate pad

[0095] The microsphere-labeled PSP antibody complex, CRP antibody complex, and MxA antibody complex from the above steps 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.

[0096] (6) Reagent card assembly

[0097] 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.

[0098] ② Cut the large plate assembled in step ① into reagent strips with a width of 3mm using a chopper, such as... Figure 4 As shown. Insert the reagent strip into the corresponding card holder, then place the prepared reagent card and a desiccant into an aluminum foil bag, seal it, and store it for later use.

[0099] (7) Reagent card testing

[0100] ① Tear open the aluminum foil bag seal and take out the test card from the aluminum foil bag.

[0101] 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.

[0102] ③ Read the test results.

[0103] Table 1. Calibration test results of the PSP / CRP / MxA quantitative detection immunochromatographic test strip in Example 6.

[0104]

[0105] From Table 1, the PSP / CRP / MxA quantitative detection immunochromatography test strip prepared in Example 6 shows excellent performance in calibration detection, three correlation coefficients R 2 > 0.99, and the blank calibration sample has very low reaction degree and no obvious non-specific interference, proving that the test strip has excellent linearity and strong specificity, the three index concentration gradients cover the common detection interval in clinic, can accurately quantify PSP, CRP and MxA, provides reliable data support for the diagnosis of respiratory diseases and the identification of infection types, and is suitable for clinical detection scenes.

[0106] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing time-resolved fluorescent microspheres, characterized in that, Includes the following steps: (1) Preparation of surface-aminated polystyrene microspheres: Pure water was added to the reaction vessel, and a surfactant was added under nitrogen protection. After stirring and dissolving, styrene was added. When the temperature was raised to 60°C, an initiator was added, and the temperature was raised to 80°C for further reaction. The surfactant was added and mixed well, and then 2-aminoethyl methacrylate hydrochloride was added. The reaction was terminated by cooling. The product was washed by centrifugation with anhydrous ethanol and pure water, and redispersed in pure water to obtain the core of amino-modified polystyrene microspheres. (2) Glutaraldehyde crosslinking and 1,10-phenanthroline-5-amino binding: Take the core of amino-modified polystyrene microspheres, wash with PBS buffer, and finally store in buffer, sonicate and disperse, add glutaraldehyde and mix well, and react; then wash with PBS buffer, and finally store in anhydrous ethanol, add 1,10-phenanthroline-5-amino, add acetic acid dropwise, react and wash with anhydrous ethanol and pure water, and redisperse in pure water; (3) Surface grafting of rare earth fluorescent dyes: The product of step (2) is dispersed in ethanol, rare earth metal chloride and auxiliary ligand are added, and after dissolution, an acid-binding agent is added. After reaction, the product is washed with anhydrous ethanol and pure water and redispersed in pure water. The rare earth metal chloride is EuCl3∙6H2O, the auxiliary ligand is dibenzoylmethane, and the acid-binding agent is triethylamine. (4) Coating the polymer shell and carboxyl modification: Disperse the microspheres grafted with rare earth fluorescent dyes in anhydrous ethanol, sonicate, remove oxygen by purging with nitrogen, add polyacrylic acid, and stir to dissolve; take styrene, divinylbenzene and acrylic acid in EP tube, add initiator, sonicate to dissolve and add to reaction vessel, wash with ethanol and pure water after reaction, and finally store in pure water.

2. The method for preparing time-resolved fluorescent microspheres according to claim 1, characterized in that: In step (1), the surfactant is an anionic surfactant, the initiator is a water-soluble persulfate, and the mass ratio of styrene to 2-aminoethyl methacrylate hydrochloride is 1:0.05-0.

5.

3. The method for preparing time-resolved fluorescent microspheres according to claim 2, characterized in that: In step (1), the surfactant is sodium dodecyl sulfate and the initiator is potassium persulfate.

4. The method for preparing time-resolved fluorescent microspheres according to claim 1, characterized in that: In step (2), the PBS buffer is a 50mM pH7.4 PBS buffer.

5. The method for preparing time-resolved fluorescent microspheres according to claim 1, characterized in that: In step (2), the molar ratio of 1,10-phenanthroline-5-amino to glutaraldehyde is 1:0.6-1.

6. The method for preparing time-resolved fluorescent microspheres according to claim 1, characterized in that: In step (3), the molar ratio of EuCl3∙6H2O, dibenzoylmethane and triethylamine is 1:3-3.6:1-1.

2.

7. The method for preparing time-resolved fluorescent microspheres according to claim 1, characterized in that: In step (4), the mass ratio of styrene, divinylbenzene, and acrylic acid is 1:0.01-0.05:0.02-0.

2.

8. Time-resolved fluorescent microspheres prepared by the method according to any one of claims 1-7.

9. The application of the time-resolved fluorescent microspheres as described in claim 8 in a quantitative detection immunochromatographic test strip for PSP / CRP / MxA.

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