AIPE phosphorescent microspheres and their preparation method and application

Abstract

The present invention relates to the field of fluorescent immunochromatography technology, and in particular to an AIPE phosphorescent microsphere and its preparation method and application. The preparation method comprises first synthesizing an AIPE phosphorescent metal platinum complex monomer containing a double bond, wherein the AIPE phosphorescent metal platinum complex monomer uses 2-phenylpyridine as a primary ligand and a Schiff base as an auxiliary ligand, wherein the auxiliary ligand is double-bond modified, and then copolymerizing the complex monomer with styrene and a functional monomer by emulsion polymerization; wherein the functional monomer is methacrylic acid and / or acrylic acid. The AIPE phosphorescent microsphere prepared by the method of the present invention has the advantages of long luminescence lifetime, large Stokes shift, good photostability and no background interference, and can be used in the field of immunochromatography for quantitative detection of a target object.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent immunochromatography, and specifically relates to AIPE phosphorescent microspheres and a preparation method and application thereof. Background Art

[0002] Fluorescence immunochromatography is a novel membrane-based detection technology based on antigen-antibody specific immune reactions. It uses a strip of fibrous chromatography material immobilized with a test line (coated antibody or antigen) and a control line (antibody) as the stationary phase. The test fluid serves as the mobile phase, and fluorescently labeled antibodies or antigens are immobilized on a connection pad. Capillary action allows the analyte to migrate along the strip. For large antigens with multiple antigenic determinants (proteins, viruses, pathogenic bacteria, etc.), a "sandwich" double-antibody immunochromatography approach is typically employed. This involves the analyte first binding to a fluorescently labeled antibody in the mobile phase. Upon reaching the test line, it then binds to the coated antibody, forming a double-antibody sandwich.

[0003] Currently, the most widely reported fluorescent microspheres are typically prepared by first preparing surface-functionalized polystyrene microspheres, and then embedding traditional fluorescent dyes into the microspheres through a swelling method. However, microspheres prepared by this method often have the problem of easy leakage of dyes, and the use of swelling agents causes serious environmental pollution and is harmful to human health. In addition, these dyes have a short luminescence lifetime and a high background value. They do not emit light at high concentrations or in a solid state, resulting in luminescence quenching, which is often referred to as the fluorescence quenching effect. Time-resolved fluorescent microspheres are made by doping polystyrene microspheres with rare earth complexes. Rare earth complex fluorescent dyes have advantages such as high Stokes shift and long fluorescence lifetime. However, too much or too little of these dyes can reduce the fluorescence intensity of the microspheres.

[0004] CN113321758A discloses carboxyl-modified aggregation-induced luminescent polymer microspheres and their preparation and application. The method involves mixing an aqueous solution containing an emulsifier with an oil-phase solution containing an AIE molecule, a stabilizer, and a cross-linker dissolved in a carboxyl-functional monomer and a hydrophobic monomer, followed by stirring and pre-emulsification to obtain a coarse emulsion. The container containing the coarse emulsion is placed in an ice-water bath and ultrasonically treated to obtain a monomer miniemulsion. Free radical polymerization is initiated by adding an initiator (either an oil-soluble initiator is added to the oil-phase solution or a water-soluble initiator is added to the monomer miniemulsion). The reaction is then conducted by nitrogen gas flow to expel oxygen, resulting in an AIE fluorescent microsphere emulsion with surface carboxyl groups modified. However, these microspheres have a short luminescence lifetime, poor photostability, and high background interference.

[0005] CN101787276A discloses surface-functionalized phosphorescent microspheres, which are prepared by using a polymethyl methacrylate polymer or copolymer to form an encapsulation matrix and embedding phosphorescent molecules within the microspheres via a swelling method. However, the phosphorescent microspheres prepared by this method, due to the use of polymethyl methacrylate as the encapsulating matrix, have a relatively high density, which is not conducive to the monodisperse state of the microspheres in an aqueous system and is prone to sedimentation and agglomeration. Furthermore, the commonly used swelling method has the problem of dye leakage, and the use of swelling agents is harmful to the human body and the environment.

[0006] In summary, the existing technologies have many shortcomings, such as: (1) the dye is easily leaked in the swelling encapsulation technology, and the swelling agent is harmful to the environment and human body; (2) the concentration quenching effect is easy to occur, resulting in low fluorescence intensity and poor linearity; (3) the lifetime is short and the background interference is strong; (4) the microspheres are dense, easy to settle and agglomerate, and the monodispersity is poor. Therefore, how to prepare phosphorescent microspheres with long luminescence lifetime, large Stokes shift, good photostability and no background interference has become one of the technical problems that need to be solved urgently in this field. Summary of the Invention

[0007] The purpose of the present invention is to provide AIPE phosphorescent microspheres and a preparation method and application thereof.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A method for preparing AIPE phosphorescent microspheres comprises the following steps: firstly synthesizing an AIPE phosphorescent metal platinum complex monomer containing a double bond, wherein the AIPE phosphorescent metal platinum complex monomer uses 2-phenylpyridine as a main ligand and a Schiff base as an auxiliary ligand, wherein the auxiliary ligand is double-bond modified, and then copolymerizing the complex monomer with styrene and a functional monomer by emulsion polymerization; wherein the functional monomer is methacrylic acid and / or acrylic acid.

[0010] Furthermore, the preparation method of the AIPE phosphorescent metal platinum complex monomer is to first condense 2,4-dihydroxybenzaldehyde with amine, then perform Friedel-Crafts alkylation on the para-hydroxyl group and halogen to modify the double bond, and then react with platinum dichloride bridge to prepare it.

[0011] Further, the following steps are included:

[0012] (1) Prepare the aqueous phase: Add pure water to the reaction vessel, pass nitrogen to remove oxygen in the system, then add the surfactant and stir to dissolve;

[0013] (2) Preparation of oil phase: Add AIPE phosphorescent metal platinum complex monomer to styrene and dissolve it by ultrasonication;

[0014] (3) Emulsification: slowly drop the oil phase into the water phase and stir at high speed for 30 minutes to emulsify;

[0015] (4) Initiation reaction: Add water-soluble initiator at 60℃, react at 70-85℃ for 0.5h, then add functional monomer and continue the reaction for 4-20h.

[0016] The functional monomer is acrylic acid, and the mass fraction of the functional monomer is 4%-20%, preferably 8%-16%, of styrene.

[0017] The mass fraction of the AIPE phosphorescent metal platinum complex monomer is 0.2%-10% of styrene, preferably 1%-2%.

[0018] Wherein, the surfactant is an anionic surfactant, preferably sodium lauryl sulfate; and its usage is 0.01%-0.25% of the mass of pure water.

[0019] Wherein, the initiator is at least one of sodium persulfate, potassium persulfate and ammonium persulfate, preferably potassium persulfate; the amount of the initiator used is 0.2%-2% of the total mass of the monomers.

[0020] The AIPE phosphorescent microspheres prepared by the above preparation method of the present invention have a particle size of 100-400 nm.

[0021] The AIPE phosphorescent microspheres of the present invention can be used for fluorescent immunochromatography, wherein the specific process of labeling the antibody (HbA1c mb) is as follows:

[0022] (1) Take 50 μL of AIPE phosphorescent microspheres and add them to a centrifuge tube containing 1 mL of coupling buffer (50 mmol MES, pH 6.0) and mix them by ultrasonication.

[0023] (2) Add 5 μL each of freshly prepared EDC (100 mg / mL) and NHS (100 mg / mL) and vortex to mix. Activate at room temperature in the dark for 30 min.

[0024] (3) Centrifuge at 15,000 rpm for 20 min, remove the supernatant, add coupling buffer, and mix by ultrasonication.

[0025] (4) Add 50 μg of antibody, vortex to mix, and couple at room temperature in the dark for 2 h.

[0026] (5) Add 500 μL of microsphere blocking solution (20 mM boric acid buffer pH 8.0, 1% BSA, 0.2% ethanolamine), vortex mix, and block at room temperature in the dark for 1 h.

[0027] (6) Centrifuge at 15000 rpm for 15 min, discard the supernatant, add 500 μL microsphere preservation solution, disperse evenly by ultrasonication, and store at 2-8°C in the dark until use.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The preparation method of AIPE phosphorescent microspheres of the present invention first synthesizes an AIPE phosphorescent metal platinum complex containing a double bond. This type of platinum-containing complex uses 2-phenylpyridine as a main ligand and a Schiff base as an auxiliary ligand. The auxiliary ligand is double-bond modified, and a luminescent monomer is copolymerized with styrene and a functional monomer through a simple emulsion polymerization method.

[0030] AIPE phosphorescent metal platinum complex has a planar quadrilateral structure, which can effectively inhibit the "aggregation-induced quenching" (ACQ) phenomenon and maintain a high luminescence intensity in the aggregated state, thereby improving its photostability. In addition, with phenylpyridine as the main ligand and Schiff base as the auxiliary ligand, it is possible to achieve effective energy transfer from the platinum complex to the phosphorescent material, thereby improving the luminescence effect of the phosphorescent material. This energy transfer mechanism not only enhances the intensity and stability of phosphorescence, but also improves the photostability of the material. Moreover, the platinum complex itself has a high photostability, which can effectively inhibit the photodegradation and photodamage of the phosphorescent material during the excitation process, thereby extending the service life of the phosphorescent material. The antioxidant and anti-photocorrosion properties of the platinum complex protect the structural integrity of the phosphorescent material and further improve its long-term stability.

[0031] In summary, the AIPE phosphorescent microspheres prepared by the method of the present invention have the advantages of long luminescence lifetime, large Stokes shift, good photostability and no background interference. Moreover, the microspheres have uniform particle size and good monodispersity, and can be used in the field of immunochromatography for quantitative detection of the target object. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the preparation mechanism diagram of AIPE phosphorescent microspheres.

[0033] Figure 2 Schematic diagram of the structure of AIPE phosphorescent microspheres.

[0034] Figure 3 This is the UV-visible absorption and fluorescence spectral properties of AIPE phosphorescent metal platinum complex.

[0035] Figure 4 This is a correlation test of HbA1c antigen calibrator after AIPE phosphorescent microspheres are combined with HbA1c monoclonal antibody. DETAILED DESCRIPTION

[0036] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0037] Example 1 Preparation of AIPE phosphorescent platinum complex

[0038]

[0039] (1) Preparation of compound 1: 5 mmol of 2,4-dihydroxybenzaldehyde and 5.5 mmol of p-trifluoromethylaniline were dissolved in 20 mL of ethanol under the catalysis of 200 μL of acetic acid and reacted at room temperature for 3 h to obtain the product;

[0040] (2) Preparation of compound 2: 5 mmol of compound 1 and 5.5 mmol of 2-chloroethyl methacrylate were dissolved in 50 mL of DMF under the action of 5 mmol of K2CO3 and reacted at 90°C for 12 h under anaerobic conditions.

[0041] (3) Preparation of compound 3: 2.2 mmol of 2-phenylpyridine and 1 mmol of K2PtCl4 were dissolved in 50 mL of ethylene glycol ether / H2O (v / v, 3:1) in the absence of oxygen and reacted at 80°C for 24 h.

[0042] (4) Preparation of AIPE phosphorescent metal platinum complex: 2.2 mmol of compound 2 and 1 mmol of compound 3 were dissolved in 20 mL of ethylene glycol ethyl ether under the action of Na2CO3, in the absence of oxygen, and reacted at 100°C for 1.5 h to obtain the product.

[0043] Example 2 Preparation of AIPE Phosphorescent Microspheres 1 (200 nm)

[0044] Using the emulsion polymerization method, 100 mL of pure water was added to a three-necked flask, nitrogen was introduced for 20 minutes to remove oxygen from the system, 75 mg of the surfactant sodium dodecyl sulfate was added, and the mixture was stirred to dissolve. 100 mg of the AIPE phosphorescent metal platinum complex prepared in Example 1 was weighed into an EP tube, 10 mL of styrene was added and ultrasonically dissolved, and the mixture was added to the reaction flask and stirred at high speed for 30 minutes. 80 mg of the initiator KPS was added at 60°C, and after reacting at 80°C for 30 minutes, 1 mL of the functional monomer acrylic acid was added and the reaction was continued for 16 hours. The preparation mechanism diagram and structural schematic diagram of AIPE phosphorescent microspheres are shown in the figure. Figure 1-2 shown.

[0045] Example 3 Preparation of AIPE Phosphorescent Microspheres 2 (200 nm)

[0046] Using an emulsion polymerization method, 100 mL of pure water was added to a three-necked flask and nitrogen was introduced for 20 minutes to remove oxygen from the system. 70 mg of the surfactant sodium dodecyl sulfate was added and stirred to dissolve. 150 mg of the AIPE phosphorescent metal platinum complex prepared in Example 1 was weighed into an EP tube and dissolved in 10 mL of styrene under ultrasonication. The mixture was then added to the reaction flask and stirred at high speed for 30 minutes. At 60°C, 80 mg of the initiator KPS was added. After reacting at 80°C for 30 minutes, 1 mL of the functional monomer acrylic acid was added and the reaction continued for 16 hours.

[0047] Example 4 Preparation of AIPE Phosphorescent Microspheres 3 (300 nm)

[0048] Using an emulsion polymerization method, 100 mL of pure water was added to a three-necked flask and nitrogen was introduced for 20 minutes to remove oxygen from the system. 40 mg of the surfactant sodium dodecyl sulfate was added and stirred to dissolve. 100 mg of the AIPE phosphorescent metal platinum complex prepared in Example 1 was weighed into an EP tube and dissolved in 10 mL of styrene under ultrasonication. The mixture was then added to the reaction flask and stirred at high speed for 30 minutes. At 60°C, 80 mg of the initiator KPS was added. After reacting at 80°C for 30 minutes, 1 mL of the functional monomer acrylic acid was added and the reaction continued for 16 hours.

[0049] Example 5 Labeled Antibody (Taking HbA1c Monoclonal Antibody as an Example, Nanjing Liding Medical Technology Co., Ltd.)

[0050] (1) Take 50 μL of AIPE phosphorescent microspheres 1, 2, and 3 in Example 2-4 and add them to a centrifuge tube containing 1 mL of coupling buffer (50 mmol MES, pH 6.0) and mix them by ultrasonication;

[0051] (2) Add 5 μL each of freshly prepared EDC (100 mg / mL) and NHS (100 mg / mL) and vortex to mix. Activate at room temperature in the dark for 30 min.

[0052] (3) Centrifuge at 15,000 rpm for 20 min, remove the supernatant, add coupling buffer, and mix by ultrasonication;

[0053] (4) Add 50 μg of antibody, vortex to mix, and couple at room temperature in the dark for 2 h;

[0054] (5) Add 500 μL of microsphere blocking solution (20 mM boric acid buffer pH 8.0, 1% BSA, 0.2% ethanolamine), vortex mix, and block at room temperature in the dark for 1 h;

[0055] (6) Centrifuge at 15000 rpm for 15 min, discard the supernatant, add 500 μL microsphere preservation solution, disperse evenly by ultrasonication, and store at 2-8°C in the dark until use.

[0056] Example 6 Test

[0057] (1) Determination of the photophysical properties of AIPE phosphorescent platinum complexes

[0058] a. UV-visible absorption spectroscopy: A tetrahydrofuran solution of the polymer was prepared in a cuvette with a concentration of approximately 1×10 - 5 mol / L. The absorption and emission spectra were measured using a Shimadzu UV-1750 ultraviolet-visible spectrometer and a Hitachi F-4600 fluorescence spectrometer.

[0059] b. Fluorescence Emission Spectra: Fluorescence emission spectra of the materials (solutions and thin films) were measured on a Hitachi F-4600 spectrometer. The test samples were either solutions or solids, and the excitation wavelength was the maximum wavelength of the UV-visible absorption spectrum. Thin films were prepared using a KW-4A spin coater developed by the Institute of Microelectronics, Chinese Academy of Sciences.

[0060] (2) Particle size test

[0061] The Otsuka nanoSAQLA particle size analyzer was used for the test. Each sample was tested three times and the average value was taken.

[0062] (3) Calibrator testing

[0063] The test was performed using a Lanbo AFS-1000 dry-type fluorescent immunoassay analyzer with an excitation wavelength of 260 nm and an emission wavelength of 590 nm to test the luminescence intensity of four calibrators.

[0064] Figure 3 Graphs showing the absorption and emission spectra of the AIPE phosphorescent metal platinum complex monomer prepared in Example 1 show that the monomer has a maximum absorption at 260 nm and an emission wavelength of approximately 590 nm.

[0065] Table 1 Particle size and dispersion index of AIPE phosphorescent microspheres

[0066]

[0067] Table 2 HbA1c antigen calibrator testing

[0068]

[0069] Table 1 shows the average particle size and dispersion index (PDI) of AIPE phosphorescent microspheres 1, 2, and 3 prepared in Examples 2-4. The particle sizes were 208 nm, 210 nm, and 308 nm, respectively, with a dispersion index (PDI) < 0.05, indicating uniform particle size and good monodispersity. Table 2 shows the data for the detection of four serially diluted HbA1c antigen calibrators using AIPE phosphorescent microspheres bound to an HbA1c monoclonal antibody, demonstrating detection of all HbA1c antigens. Figure 4 It is the correlation data. It can be seen from the figure that it has a good correlation. 2 >0.98.

[0070] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing AIPE phosphorescent microspheres, characterized by: First, synthesize the double bond-containing AIPE phosphorescent metal platinum complex monomer, the structural formula of the AIPE phosphorescent metal platinum complex monomer is: ； The AIPE phosphorescent metal platinum complex monomer uses 2-phenylpyridine as the main ligand and a Schiff base as the auxiliary ligand. The auxiliary ligand is double-bond modified, and the complex monomer is copolymerized with styrene and a functional monomer by emulsion polymerization. The functional monomer is methacrylic acid and / or acrylic acid, and the mass fraction of the AIPE phosphorescent metal platinum complex monomer is 0.2%-10% of styrene.

2. The method for preparing AIPE phosphorescent microspheres according to claim 1, characterized in that: The following steps are involved: (1) Prepare the aqueous phase: Add pure water to the reaction vessel, pass nitrogen to remove oxygen from the system, then add a surfactant and stir to dissolve; (2) Preparation of oil phase: Add AIPE phosphorescent metal platinum complex monomer to styrene and dissolve it by ultrasonication; (3) Emulsification: slowly drop the oil phase into the water phase and stir at high speed for 30 minutes to emulsify; (4) Initiation reaction: Add water-soluble initiator at 60℃, react at 70-85℃ for 0.5h, then add functional monomer and continue the reaction for 4-20h.

3. The method for preparing AIPE phosphorescent microspheres according to claim 2, characterized in that: The functional monomer is acrylic acid.

4. The method for preparing AIPE phosphorescent microspheres according to claim 2, wherein: The mass fraction of the functional monomer is 4%-20% of styrene.

5. The method for preparing AIPE phosphorescent microspheres according to claim 4, characterized in that: The surfactant is an anionic surfactant, and its usage is 0.01%-0.25% of the mass of pure water.

6. The method for preparing AIPE phosphorescent microspheres according to claim 5, characterized in that: The initiator is at least one of sodium persulfate, potassium persulfate and ammonium persulfate, and the amount of the initiator is 0.2%-2% of the total weight of the monomers.

7. AIPE phosphorescent microspheres prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The particle size is 200-300 nm.

8. Use of the AIPE phosphorescent microspheres according to claim 7 in the preparation of fluorescent immunochromatographic reagents.

Images