Time-resolved fluorescent microspheres, preparation method and application of time-resolved fluorescent microspheres in PSP / CRP / MxA quantitative detection immunochromatographic test strip

Abstract

The invention relates to the technical field of immunochromatography, in particular to time-resolved fluorescent microspheres, a preparation method and application of the time-resolved fluorescent microspheres in PSP / CRP / MxA quantitative detection immunochromatography test strip, and the preparation method of the time-resolved fluorescent microspheres comprises the following steps: 1) preparing surface aminated polystyrene microspheres; (2) carrying out glutaraldehyde crosslinking and 1, 10-phenanthroline-5-amino combination; 3) grafting a rare earth fluorescent dye on the surface; and 4) coating a polymer shell layer and modifying carboxyl. According to the invention, through a dual fixation strategy of surface grafting and polymer coating, the time-resolved fluorescent dye is confined to a functional region between the surface of the microsphere and a polymer shell layer, diffusion and leakage of dye molecules to the external environment are blocked, and the problem of dye shedding of traditional fluorescent microspheres is solved; the polymer shell layer can effectively isolate external ultraviolet light, oxygen and other corrosive factors, reduce photooxidative degradation and non-radiative transition loss of fluorescent molecules, and improve the light stability and fluorescent lifetime of the fluorescent molecules.

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 tumor patients, and has important value in predicting sepsis. CRP is an acute phase protein that rapidly increases in blood when inflammation, infection, and other diseases occur, and is a commonly used inflammation indicator in clinical practice. MxA protein is a 78kDa antiviral protein produced by I-type interferon-induced cells, 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 clinical practice. 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: A preparation method of a time-resolved fluorescent microsphere, comprising the following steps: (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. The surfactant is added again, mixed, and then 2-aminoethyl methacrylate hydrochloride is added. After reaction, the product is cooled and terminated, and the product is washed by centrifugation with anhydrous ethanol and pure water, and then dispersed in pure water to obtain an amino-modified polystyrene microsphere core; (2) glutaraldehyde cross-linking and 1,10-phenanthroline-5-amino combination: the amino-modified polystyrene microspheres core was taken, washed with PBS buffer, finally stored in the buffer, ultrasonic dispersion, mixed with glutaraldehyde, reaction; then washed with PBS buffer, finally stored in anhydrous ethanol, added with 1,10-phenanthroline-5-amino, dropped with acetic acid, washed with anhydrous ethanol and pure water after reaction, and then re-dispersed in pure water; (3) surface grafting of rare earth fluorescent dye: the product of step (2) was dispersed in ethanol, added with rare earth metal chloride and auxiliary ligand, dissolved, added with acid binding agent, washed with anhydrous ethanol and pure water after reaction, and then re-dispersed in pure water; (4) polymer shell coating and carboxyl modification: the microspheres of surface grafted rare earth fluorescent dye were dispersed in anhydrous ethanol, ultrasonic dispersion, deoxidized by nitrogen, and added with polyacrylic acid, and stirred and dissolved; styrene, divinylbenzene and acrylic acid were taken in an EP tube, added with an initiator, ultrasonic dissolved, and then added into a reaction container, washed with ethanol and pure water after reaction, and finally stored in pure water.

[0007] 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.

[0008] In step (1), the surfactant is sodium dodecyl sulfate, and the initiator is potassium persulfate.

[0009] In step (2), the PBS buffer is 50mM pH7.4 PBS buffer.

[0010] In step (2), the molar ratio of 1,10-phenanthroline-5-amino to glutaraldehyde is 1:0.6-1.

[0011] In step (3), the rare earth metal chloride is EuCl3·6H2O, the auxiliary ligand is dibenzoylmethane, and the acid binding agent is triethylamine.

[0012] In step (3), the molar ratio of EuCl3·6H2O, dibenzoylmethane and triethylamine is 1:3-3.6:1-1.2.

[0013] In step (4), the mass ratio of styrene, divinylbenzene and acrylic acid is 1:0.01-0.05:0.02-0.2.

[0014] 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: (1) labeling of PSP antibody ①Washing: 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; ②Activation: 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; ③Coupling: Add 0.5 mL 50 mM MES (pH 6.5) buffer to the activated microspheres, mix by ultrasonic, then add 50 μg of the PSP-labeled antibody to be coupled, mix by vortex, and couple at room temperature for 2 h; ④Blocking: Add 300 μL of 10% BSA and 20 μL of ethanolamine to the above coupling buffer, block at room temperature for 1 h, and centrifuge to discard the supernatant; ⑤Resuspension: 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 has the following formulation: 10 mM PBS (pH 7.4±0.05), 10 mM NaCl, 0.05% Tween-80, 1% BSA, 5% sucrose, and 0.1% ProClin 300.

[0015] (2) Labeling of CRP antibody ①Washing: 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; ②Activation: 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; ③Coupling: Add 0.5 mL 50 mM MES (pH 6.5) buffer to the activated microspheres, mix by ultrasonic, then add 50 μg of the CRP-labeled antibody to be coupled, mix by vortex, and couple at room temperature for 2 h; ④Blocking: Add 300 μL of 10% BSA and 20 μL of ethanolamine to the above coupling buffer, block at room temperature for 1 h, and centrifuge to discard the supernatant; ⑤Resuspension: 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 has the following formulation: 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] (3) Labeling of MxA antibody 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; 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 prepared EDC (50 mg / mL) and 40 μL of prepared NHS (50 mg / mL), mix by vortex, activate at room temperature for 30 min, centrifuge to discard the supernatant; ③ Coupling: Add 0.5 mL 50 mM MES (pH 6.5) buffer to the activated microspheres, mix by ultrasonic; add 50 μg of MxA labeled antibody to be coupled, mix by vortex, 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 to discard the supernatant; ⑤ Resuspension: Add 500 μL of microsphere resuspension solution to the microsphere-antibody complex, store at 2-8°C in the dark. The microsphere resuspension solution is 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.

[0017] (4) Preparation of nitrocellulose membrane Prepare the PSP coated antibody (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) solution at a coating amount of 1 μL / cm, and use a membrane drawing and gold spraying integrated machine to draw them onto a nitrocellulose membrane as the detection line and the quality control line of the first test strip, respectively. Prepare the CRP coated antibody (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) solution at a coating amount of 1 μL / cm, and use a membrane drawing and gold spraying integrated machine to draw them onto a nitrocellulose membrane as the detection line and the quality control line of the second test strip, respectively. Prepare the MxA coated antibody (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) solution at a coating amount of 1 μL / cm, and use a membrane drawing and gold spraying integrated machine to draw them onto a nitrocellulose membrane as the detection line and the quality control line of the third test strip, respectively. Dry at 45°C overnight.

[0018] (5) Preparation of binding pad Spray the microsphere labeled PSP antibody complex, CRP antibody complex, and MxA antibody complex prepared in the above steps onto a glass fiber membrane at a coating amount of 5 μL / cm, and dry at 45°C overnight to prepare a binding pad.

[0019] (6) Assembly of reagent strip 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.

[0020] ② Cut the large plate assembled in step ① into reagent strips with a 3mm width using a chopper. 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.

[0021] The detection method for the PSP / CRP / MxA quantitative detection immunochromatographic test strip prepared in this invention is as follows: ① Tear open the aluminum foil bag seal and take out the test card from the aluminum foil bag.

[0022] 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.

[0023] ③ Read the test results.

[0024] Compared with the prior art, the beneficial effects of the present invention are: This invention prepares high-performance time-resolved fluorescent microspheres through "stepwise functionalization modification + core-shell structure design," and its technical advantages include: (1) Using polystyrene microspheres as a substrate, a reactive platform is constructed by surface amination modification, so that the surface of the microspheres is uniformly loaded with active amino sites, laying the foundation for subsequent covalent connection.

[0025] (2) Directional grafting of main ligands by crosslinking agent glutaraldehyde: Utilizing the dual aldehyde properties of glutaraldehyde, one end forms a Schiff base covalent bond with the amino group on the surface of the microsphere, and the other end is connected to the 1,10-phenanthroline-5-amino ligand to construct a stable connection structure of "microsphere-glutaraldehyde-ligand", providing a targeted binding site for the fixation of fluorescent dyes.

[0026] (3) Synthesis and grafting of time-resolved fluorescent dyes on the surface of microspheres: using the surface-bound 1,10-phenanthroline-5-amino as the ligand skeleton, the rare earth complex time-resolved fluorescent dyes are fixed on the surface of microspheres through coordination reaction to ensure that the dye molecules are uniformly distributed and firmly bound.

[0027] (4) To solve the key problems of dye leakage and insufficient photostability, a core-shell coating and surface modification process is further adopted: a dense polymer shell is coated on the outside of the dye-loaded microspheres to form a core-shell structure of "core functional layer - outer protective layer"; at the same time, carboxyl active groups are introduced into the polymer shell through copolymerization to give the microspheres good bio-coupling ability.

[0028] The preparation method has two core technical advantages: first, by means of the double fixation strategy of “surface grafting + polymer coating”, the time-resolved fluorescent dye is limited in the functional region between the surface of the microsphere and the polymer shell layer, the diffusion leakage of the dye molecules to the external environment is completely blocked, and the problem of dye falling off of the traditional fluorescent microsphere is solved; second, the dense 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 significantly improve the light stability and fluorescence lifetime.

[0029] The time-resolved fluorescent microsphere prepared based on the method of the application can form an amide bond between the carboxyl on the surface of the shell layer and the amino group of the antibody, and successfully realize efficient labeling of PSA, CRP and MxA antibodies. When the labeled fluorescent microsphere is applied to an immune detection system, the high specificity and high sensitivity of the PSP, CRP and MxA three biomarkers can be realized by quantitative reading of the time-resolved fluorescent signal, and an excellent probe tool for multi-index joint diagnosis is provided. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 FIG. 1 is a schematic diagram of the preparation process of the time-resolved fluorescent microsphere.

[0031] Figure 2 FIG. 3 is a SEM image of the time-resolved fluorescent microsphere prepared in Example 4.

[0032] Figure 3 FIG. 4 is a SEM image of the time-resolved fluorescent microsphere prepared in Example 5.

[0033] Figure 4 FIG. 5 is a schematic diagram of the PSP / CRP / MxA quantitative detection immunochromatographic test strip. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0035] Example 1 As Figure 1As 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.

[0036] Example 2 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.

[0037] Example 3 like Figure 1 As 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.

[0038] Example 4 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.

[0039] Example 5 like Figure 1The microspheres 2 mL in Example 3 were dispersed in 20 mL of anhydrous ethanol, ultrasonically dispersed, and deoxygenated by nitrogen for 20 min. 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 taken into an EP tube, 30 mg of initiator AIBN was added, ultrasonically dissolved, and then added into the flask. The reaction was carried out at 70°C for 24 h. The product was washed with ethanol for 3 times and pure water for 3 times, and finally stored in pure water with a solid content of 1%. The SEM image of the prepared time-resolved fluorescent microspheres is shown in FIG. 6. Figure 3

[0040] Example 6 The time-resolved fluorescent microspheres of Examples 4-5 were used for the preparation of PSP, CRP, and MxA quantitative detection immunochromatographic test strips. The specific steps are as follows: (1) Labeling of PSP antibody ① Washing: 50 μL of the time-resolved fluorescent microspheres of Example 5 were added into a centrifuge tube containing 0.5 mL of 50 mM MES (pH 6.5) buffer, ultrasonically mixed, and centrifuged to discard the supernatant; ② Activation: 0.5 mL of 50 mM MES (pH 6.5) buffer was added into the washed microspheres, ultrasonically mixed, and then 20 μL of freshly prepared EDC (50 mg / mL) and 40 μL of NHS (50 mg / mL) were added. After vortex mixing, the mixture was activated at room temperature for 30 min, and then centrifuged to discard the supernatant; ③ Coupling: 0.5 mL of 50 mM MES (pH 6.5) buffer was added into the activated microspheres, ultrasonically mixed, and then 50 μg of the PSP-labeled antibody to be coupled was added. After vortex mixing, the mixture was coupled at room temperature for 2 h; ④ Blocking: 300 μL of 10% BSA and 20 μL of ethanolamine were added into the above coupling buffer, and the mixture was blocked at room temperature for 1 h. After centrifugation, the supernatant was discarded; ⑤ Resuspension: 500 μL of microsphere resuspension solution was added into the microsphere-antibody complex, and the mixture was 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.

[0041] Labeling of CRP antibody ① Washing: 50 μL of the time-resolved fluorescent microspheres of Example 4 were added into a centrifuge tube containing 0.5 mL of 50 mM MES (pH 6.5) buffer, ultrasonically mixed, and centrifuged to discard the supernatant; ​② Activation: 0.5 mL of 50 mM MES (pH 6.5) buffer was added to the washed microspheres, and then 20 μL of freshly prepared EDC (50 mg / mL) and 40 μL of NHS (50 mg / mL) were added after ultrasonic mixing. The mixture was vortexed and activated at room temperature for 30 min, and then centrifuged to discard the supernatant; ③ Coupling: 0.5 mL of 50 mM MES (pH 6.5) buffer was added to the activated microspheres, and then 50 μg of the CRP-labeled antibody to be coupled was added after ultrasonic mixing. The mixture was vortexed and coupled at room temperature for 2 h; ④ Blocking: 300 μL of 10% BSA and 20 μL of ethanolamine were added to the above coupling buffer, and the mixture was blocked at room temperature for 1 h. The mixture was then centrifuged to discard the supernatant; ⑤ Resuspension: 500 μL of microsphere resuspension solution was added to the microsphere-antibody complex, and the mixture was 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.

[0042] Labeling of MxA antibody ① Washing: 50 μL of the time-resolved fluorescent microspheres of Example 5 were added to a centrifuge tube containing 0.5 mL of 50 mM MES (pH 6.5) buffer, and then ultrasonically mixed and centrifuged to discard the supernatant; ② Activation: 0.5 mL of 50 mM MES (pH 6.5) buffer was added to the washed microspheres, and then 20 μL of freshly prepared EDC (50 mg / mL) and 40 μL of NHS (50 mg / mL) were added after ultrasonic mixing. The mixture was vortexed and activated at room temperature for 30 min, and then centrifuged to discard the supernatant; ③ Coupling: 0.5 mL of 50 mM MES (pH 6.5) buffer was added to the activated microspheres, and then 50 μg of the CRP-labeled antibody to be coupled was added after ultrasonic mixing. The mixture was vortexed and coupled at room temperature for 2 h; ④ Blocking: 300 μL of 10% BSA and 20 μL of ethanolamine were added to the above coupling buffer, and the mixture was blocked at room temperature for 1 h. The mixture was then centrifuged to discard the supernatant; ⑤ Resuspension: 500 μL of microsphere resuspension solution was added to the microsphere-antibody complex, and the mixture was 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.

[0043] Preparation of nitrocellulose membrane The prepared PSP coated antibody (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) solution was 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 the detection line and the 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 was 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 the detection line and the 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 was 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 the detection line and the quality control line of the third test strip, respectively. The membrane was dried at 45°C overnight.

[0044] (5) Preparation of the conjugate pad The microsphere-labeled PSP antibody complex, CRP antibody complex and MxA antibody complex prepared in the above step were sprayed on the glass fiber membrane at a coating amount of 5 μL / cm, respectively, and dried at 45°C overnight to prepare the conjugate pad.

[0045] (6) Assembly of the reagent card ①The water absorption pad, the conjugate pad and the sample pad were sequentially attached to the PVC bottom plate on which the nitrocellulose membrane was attached. The quality control line was attached to one end of the water absorption pad, and the water absorption pad was placed on the NC membrane. The conjugate pad was attached to one end of the detection line, and the conjugate pad was placed on the NC membrane. The sample pad was placed on the conjugate pad.

[0046] ②The large plate assembled in step ① was cut into a reagent strip with a width of 3 mm by a cutting machine, as shown in FIG. 2. Figure 4 The reagent card was placed into the card shell, and the assembled reagent card and a desiccant were placed into an aluminum foil bag for sealing. The sealed aluminum foil bag was stored for later use.

[0047] (7) Detection of the reagent card ①The aluminum foil bag was opened, and the detection card was taken out of the aluminum foil bag.

[0048] ②5 μL of the sample was taken and added to the sample diluent. After being mixed thoroughly, 70 μL (4 drops of the dropper) was added vertically to the sample addition hole of the detection card, and the reaction time was 8 min.

[0049] ③The detection result was read.

[0050] Table 1 Detection result of the calibration sample of the PSP / CRP / MxA quantitative detection immunochromatographic test strip in Example 6

[0051] As shown in Table 1, the PSP / CRP / MxA quantitative detection immunochromatographic test strip prepared in Example 6 showed excellent performance in the calibration sample detection, and the three correlation coefficients R2 >0.99, and the blank calibrator reaction is very low, and there is no obvious non-specific interference, which proves that the test strip has good linearity and strong specificity, the concentration gradient of the three indexes covers the common detection interval in clinic, and the PSP, CRP and MxA can be accurately quantified, which provides reliable data support for the diagnosis of respiratory system diseases and the identification of infection types, and is suitable for clinical detection scenes.

[0052] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are merely divergences of the principles and spirit of the application and that numerous modifications, changes, substitutions, and alterations can be made thereto without departing from the spirit and scope of the application as defined by the appended claims and their equivalents.

Claims

1. A method for preparing time-resolved fluorescent microspheres, characterized in that, It comprises the following steps: (1) Preparation of surface aminated polystyrene microspheres: pure water is added into a reaction container, and a surfactant is added under nitrogen protection. After stirring and dissolving, styrene is added. When the temperature is raised to 60℃, an initiator is added, and the temperature is continuously raised to 80℃ for reaction. After adding a surfactant, 2-aminoethyl methacrylate hydrochloride is added, and the reaction is terminated after cooling. The product is washed by centrifugation with anhydrous ethanol and pure water, and is dispersed in pure water again to obtain an amino-modified polystyrene microsphere core; (2) Glutaraldehyde crosslinking and 1,10-phenanthroline-5-amino combination: the amino-modified polystyrene microsphere core is washed with PBS buffer and finally stored in the buffer, and is ultrasonically dispersed. Glutaraldehyde is added and mixed, and then is reacted. Then, the product is washed with PBS buffer and finally stored in anhydrous ethanol. 1,10-phenanthroline-5-amino is added, acetic acid is added dropwise, and then is reacted. After washing with anhydrous ethanol and pure water, the product is dispersed in pure water again; (3) Surface grafting of rare earth fluorescent dye: the product of step (2) is dispersed in ethanol, and rare earth metal chloride and auxiliary ligand are added. After dissolving, an acid binding agent is added, and then is reacted. After washing with anhydrous ethanol and pure water, the product is dispersed in pure water again; (4) Coating of a polymer shell layer and carboxyl modification: the microspheres grafted with rare earth fluorescent dye are dispersed in anhydrous ethanol, and are ultrasonically dispersed. Nitrogen is used to remove oxygen, and polyacrylic acid is added and stirred and dissolved. Styrene, divinylbenzene and acrylic acid are taken into an EP tube, an initiator is added, and then is ultrasonically dissolved and added into a reaction container. After reaction, the product is washed with ethanol and pure water, and is finally stored in pure water.

2. The method of claim 1, wherein: In step (1), the surfactant is an anionic surfactant, and the initiator is a water-soluble persulfate. The mass ratio of styrene to 2-aminoethyl methacrylate hydrochloride is 1:0.05-0.

5.

3. The method of claim 2, wherein: In step (1), the surfactant is sodium dodecyl sulfate, and the initiator is potassium persulfate.

4. The method of claim 1, wherein: In step (2), the PBS buffer is a 50mM PBS buffer with pH 7.

4.

5. The method of claim 1, wherein: In step (2), the molar ratio of 1,10-phenanthroline-5-amino to glutaraldehyde is 1:0.6-1.

6. The method of claim 1, wherein: In step (3), the rare earth metal chloride is EuCl3·6H2O, the auxiliary ligand is dibenzoylmethane, and the acid binding agent is triethylamine.

7. The method of claim 6, wherein: In step (3), the molar ratio of EuCl3·6H2O, dibenzoylmethane and triethylamine is 1:3-3.6:1-1.

2.

8. The method of claim 1, wherein: In step (4), the mass ratio of styrene, divinylbenzene and acrylic acid is 1:0.01-0.05:0.02-0.

2.

9. The time-resolved fluorescent microspheres prepared by the preparation method of any one of claims 1-8.

10. Application of the time-resolved fluorescent microspheres of claim 9 in PSP / CRP / MxA quantitative detection immunochromatographic test strips.

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