An immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes, and its preparation method and application

Abstract

The present invention discloses an immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes, and a preparation method and application thereof; the immunoassay reagent comprises an AIE magnetically encoded microsphere labeled with an antibody-1 and a water-soluble AIE dye labeled with an antibody-2. The present invention adds the AIE magnetically encoded microspheres labeled with an antibody-1 and the water-soluble AIE dye labeled with an antibody-2 to a test solution containing a corresponding antigen, and after the reaction, the AIE magnetically encoded microspheres are separated by magnetic attraction, and the encoding peak and fluorescence quantitative detection concentration are obtained by flow cytometry. The present invention utilizes the high-regionality of the AIE magnetically encoded microspheres and the signal amplification effect of the quantitative detection of the AIE molecules to realize long linear range detection in complex samples of multiple markers, and provides a new detection strategy for multiple marker immunoassay.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and in particular relates to an immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes, and a preparation method and application thereof. Background Art

[0002] The development and completion of the human genome and proteome in recent years has led to a huge demand for large-scale analysis of biomolecules, which has promoted the rapid development of liquid suspension biochips with encoded microspheres as the core. Liquid suspension biochips with rapid high-throughput flow cytometry and magnetic separation as analytical methods have gradually become a new goal for accurate diagnosis by performing rapid and high-throughput multiplex detection of markers such as proteins, nucleic acids and small molecules. Among them, the development of encoded microspheres with high encoding capacity and stability is still a huge challenge. The design of suitable encoded microspheres has the following requirements: high photostability, good anti-magnetism, large number of codes, no reabsorption and crosstalk effects, etc.

[0003] Spectral signals are widely used in the construction of liquid biochips due to their flexible encoding and fast decoding speed. At present, the commonly used spectral encoding materials are mainly organic materials, quantum dots, upconversion dots and photonic crystals. Quantum dots have become popular encoding materials due to their high brightness and narrow emission spectrum, but the encoding ability of quantum dots is limited by the reabsorption and cross-color effect between quantum dot fluorescence signals, which limits the application of quantum dot encoding microspheres. At present, commercial liquid biochip Luminex system and BD's cytometric bead array all use organic dyes for encoding, but traditional organic materials have fluorescence quenching in the aggregated state, which limits the encoding capacity and stability of traditional organic materials. The new aggregation-induced emission (AIE) material discovered by the team of Academician Tang Benzhong in 2001 is a promising encoding material for the preparation of encoding microspheres, because AIE materials can achieve in-situ enhancement of fluorescence signals during the aggregation process. Aggregation-induced emission materials have properties such as large Stokes shift, strong resistance to photobleaching, and antimagnetism, and are an excellent encoding microsphere material.

[0004] Until recently, Li Wanwan used membrane emulsification technology to prepare AIE non-magnetic encoded microspheres for the detection of multiple markers. However, the lack of magnetic materials made it impossible to achieve the goal of high-throughput testing. Nowadays, there are few public reports on the efficient preparation of AIE magnetic fluorescent encoding using AIE materials. Therefore, it is necessary to develop a technology for the efficient and direct generation of AIE magnetic encoded microspheres. Summary of the invention

[0005] In order to solve the deficiencies in the prior art, the present invention aims to provide an immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes, and a preparation method and application thereof.

[0006] The technical solution adopted by the present invention is:

[0007] An immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes comprises AIE magnetically encoded microspheres labeled with antibody-1 and water-soluble AIE dyes labeled with antibody-2.

[0008] Preferably, the mass amount of the water-soluble AIE dye labeled with antibody-2 is 0.001-0.01wt% of the mass amount of the AIE magnetically encoded microspheres labeled with antibody-1;

[0009] Preferably, the encoding capacity of the AIE magnetically encoded microspheres is ≥ 4, and the CV value of the flow fluorescence signal and the CV value of the flow FCS are both less than 10%;

[0010] Preferably, the antibody-1 and antibody-2 can specifically bind to the same antigen.

[0011] Further preferably, the antigen is an inflammatory marker, including CRP, SAA, IL-6, and PCT.

[0012] Further preferably, the antibody-1 is selected from at least one of a CRP monoclonal antibody, a PCT monoclonal antibody, an IL-6 monoclonal antibody and a SAA monoclonal antibody, and the antibody-2 is selected from the anti-antibody of antibody-1;

[0013] Further preferably, the antibody-1 and antibody-2 are mouse antibodies.

[0014] Preferably, the water-soluble AIE dye is selected from at least one of the following AIE-W1 to AIE-W4 molecules:

[0015]

[0016] Further preferably, the encoding capacity of the AIE magnetically encoded microspheres is ≥24; the AIE magnetically encoded microspheres are dual-wavelength aggregation-induced luminescence magnetically encoded microspheres, including AIE polymer microspheres and magnetic particles; the AIE polymer microspheres are obtained by embedding AIE molecules inside blank polymer microspheres, and the magnetic particles are deposited on the surface of the AIE polymer microspheres; the AIE molecules include AIE-R molecules and AIE-IR molecules;

[0017] The difference between the maximum emission wavelength of the AIE-IR molecule and the maximum emission wavelength of the AIE-R molecule is in the range of 160 nm to 290 nm, and satisfies Δλ≥185. Where I / I0 is the average value of the fluorescence intensity ratio of AIE-IR molecules and AIE-IR molecules before and after embedding, λ IR is the maximum emission wavelength of the AIE-IR molecule, λ Ris the maximum emission wavelength of the AIE-R molecule.

[0018] More preferably, the blank polymer microspheres are selected from at least one of polystyrene microspheres, polymethyl methacrylate microspheres and polyglycidyl acrylate microspheres;

[0019] More preferably, the blank polymer microspheres are selected from polystyrene microspheres or polyglycidyl acrylate microspheres.

[0020] More preferably, the magnetic particles are Fe3O4;

[0021] More preferably, the AIE-R molecule is selected from at least one of the following AIE-R1 to AIE-R16 molecules:

[0022]

[0023]

[0024]

[0025] Alkyl chain in AIE-R 1 molecule -C8H 17 Each is independently a branched chain alkyl or a straight chain alkyl.

[0026] Alkyl chain in AIE-R 11 molecule -C6H 13 Each is independently a branched chain alkyl or a straight chain alkyl.

[0027] The AIE-IR molecule is selected from at least one of the following AIE-IR1 to AIE-IR10 molecules:

[0028]

[0029]

[0030] More preferably, the AIE molecules are embedded in the blank polymer microspheres by an active swelling method, wherein the amount of the AIE molecules used in the active swelling method is 0.01-10wt% of the amount of the blank polymer microspheres, wherein the mass amount of the AIE-R molecules accounts for 0.01% to 1000% of the mass amount of the AIE-IR molecules;

[0031] The active swelling method is to allow the AIE molecules to swell with the blank polymer microspheres in a swelling agent so that the AIE molecules are embedded in the blank polymer microspheres.

[0032] In the active swelling method, the swelling agent is selected from at least one of dichloromethane, tetrahydrofuran, chloroform, anisole, benzyl alcohol and toluene, and the particle size of the blank polymer microspheres ranges from 0.03 μm to 50 μm;

[0033] The particle size of the blank polymer microspheres is preferably in the range of 0.5 μm to 20 μm, more preferably in the range of 0.5 μm to 10 μm.

[0034] More preferably, the magnetic particles are deposited on the surface of the AIE polymer microspheres by an in-situ precipitation method of iron salts, wherein the amount of iron salts used in the in-situ precipitation method is 0.1-25 wt % of the amount of blank polymer microspheres used.

[0035] The in-situ precipitation method is to add an alkaline solution to react with the iron salt chelated on the surface of the AIE polymer microspheres in situ to generate magnetic particles that are deposited on the surface of the AIE polymer microspheres;

[0036] In the in-situ precipitation method, the surface of the AIE polymer microspheres is modified by sulfonation, the alkaline solution is selected from at least one of sodium hydroxide solution, ammonia water, potassium hydroxide solution and sodium bicarbonate solution; the iron salt is selected from at least one of trivalent iron salt and divalent iron salt; the trivalent iron salt is selected from at least one of ferric chloride hexahydrate and anhydrous ferric chloride; the divalent iron salt is selected from at least one of ferrous chloride tetrahydrate, ferrous chloride dihydrate, ferrous sulfate and ferrous sulfate heptahydrate.

[0037] Considering the uniformity of magnetic particles deposited on the surface of the AIE polymer microspheres, the iron salt is a compound of a trivalent iron salt and a divalent iron salt, wherein the mass amount of the trivalent iron salt is 10% to 60% of the mass amount of the divalent iron salt.

[0038] More preferably, the preparation method of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres comprises the following steps:

[0039] S1. Preparation of AIE polymer microspheres by active swelling method

[0040] (1) dispersing blank polymer microspheres in water to obtain a microsphere dispersion;

[0041] (2) dissolving the AIE molecules in a swelling agent to obtain an AIE swelling agent solution;

[0042] (3) adding the AIE swelling agent solution described in step (2) to the microsphere dispersion described in step (1), and after ultrasonic treatment, closing the reactor to carry out swelling reaction. After the reaction is completed, volatilizing and removing the swelling agent to obtain AIE polymer microspheres;

[0043] S2. Preparation of dual-wavelength aggregation-induced luminescence magnetically encoded microspheres by in situ precipitation

[0044] The AIE polymer microspheres of step S1 are dissolved in a sulfuric acid solution, the temperature is increased for reaction, and the supernatant is removed by centrifugation to obtain AIE polymer microspheres with sulfonation-modified surfaces. The AIE polymer microspheres are mixed with iron salt and water for reaction, and the supernatant is removed by centrifugation to obtain iron salt-chelated AIE polymer microspheres. An alkaline solution is added, and the temperature is increased. The iron salt reacts in situ to generate magnetic particles that are deposited on the surface of the AIE polymer microspheres. The supernatant is removed by centrifugation to prepare dual-wavelength aggregation-induced luminescence magnetically encoded microspheres.

[0045] Preferably, the preparation method of the dual-wavelength aggregation-induced luminescence magnetically encoded microspheres comprises the following steps:

[0046] S1. Preparation of AIE polymer microspheres by active swelling method

[0047] (1) dispersing blank polymer microspheres in water to obtain a microsphere dispersion;

[0048] (2) dissolving the AIE molecules in a swelling agent to obtain an AIE swelling agent solution;

[0049] (3) adding the AIE swelling agent solution described in step (2) to the microsphere dispersion described in step (1), and after ultrasonic treatment at an ultrasonic power of 50 W to 400 W for 1 min to 30 min, a closed reactor is used for swelling reaction for 0.5 h to 4 h. After the reaction is completed, the swelling agent is volatilized to obtain AIE polymer microspheres;

[0050] S2. Preparation of dual-wavelength aggregation-induced luminescence magnetically encoded microspheres by in situ precipitation

[0051] The AIE polymer microspheres of step S1 are dissolved in a sulfuric acid solution, the temperature is increased to 30°C to 100°C and the reaction is carried out for 4h to 12h, and the supernatant is removed by centrifugation to obtain AIE polymer microspheres with sulfonation modified on the surface. The microspheres are mixed with iron salt and water, and the mixture is immersed in the solution for reaction for 0.5h to 10h, and the supernatant is removed by centrifugation to obtain iron salt chelated AIE polymer microspheres. An alkaline solution is added, and the temperature is increased to 60°C to 120°C and the reaction is carried out for 0.5h to 4h. The iron salt reacts in situ to generate magnetic particles which are deposited on the surface of the AIE polymer microspheres. The supernatant is removed by centrifugation to prepare aggregation-induced luminescence magnetically encoded microspheres.

[0052] Preferably, in step S1, the ultrasonic power of step (3) is 300W, the ultrasonic treatment time is 5 minutes, and the time for volatilizing and removing the swelling agent is 2 hours to 10 hours. More preferably, the time for volatilizing and removing the swelling agent is 4 hours.

[0053] Preferably, in step S2, the soaking reaction time is 2 h to 4 h.

[0054] Preferably, in step S1, the mass amount of blank polymer microspheres in the microsphere dispersion is 0.1% to 20% of the mass amount of water;

[0055] Further preferably, the mass amount of blank polymer microspheres in the microsphere dispersion is 0.5% to 10% of the mass amount of water.

[0056] Preferably, in step S1, the mass amount of the AIE molecules in the AIE swelling agent solution is 0.1% to 50% of the mass amount of the swelling agent;

[0057] Preferably, in step S2, the mass concentration of the sulfuric acid solution is 70% to 99%; the mass amount of the AIE polymer microspheres is 1% to 30% of the mass amount of the sulfuric acid solution;

[0058] Further preferably, the mass concentration of the sulfuric acid solution is 98.3%; and the mass amount of the AIE polymer microspheres is 5% to 20% of the mass amount of the sulfuric acid solution.

[0059] In step S2, the mass dosage of the iron salt is 0.0005% to 25% of the mass dosage of water; the mass dosage of the alkaline solution is 0.1% to 50% of the mass dosage of water; the mass concentration of the alkaline solution is 1% to 10%;

[0060] The water in step S1 and step S2 is ultrapure water; the centrifugal speed is 3000 rpm to 6000 rpm, the centrifugal time is 5 min to 60 min, and the number of centrifugation is more than three times.

[0061] The preparation method of the above-mentioned immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes comprises the following steps:

[0062] (1) Carboxyl modification, carboxyl activation, and antibody-1 coupling of the AIE magnetically encoded microspheres to obtain antibody-1 labeled AIE magnetically encoded microspheres;

[0063] (2) activating the carboxyl group of the water-soluble AIE dye and coupling it with antibody-2 to obtain a water-soluble AIE dye labeled with antibody-2;

[0064] The immunoassay reagent based on the AIE magnetically encoded microspheres and the AIE dye is obtained.

[0065] Preferably,

[0066] In step (1), the carboxyl modification comprises: mixing and reacting the AIE magnetic encoded microspheres, a base, TEOS and a silane coupling agent, magnetically separating, and adding a monomer containing a double bond and a carboxyl group to react to obtain the carboxyl modified AIE magnetic encoded microspheres;

[0067] In step (1), the activation of carboxyl groups comprises: dispersing carboxyl-modified AIE magnetic encoded microspheres in an acidic buffer, adding a carbodiimide condensation reagent, and stirring to activate the carboxyl groups, thereby obtaining carboxyl-activated AIE magnetic encoded microspheres;

[0068] In step (1), the antibody-1 coupling comprises: dispersing the AIE magnetically encoded microspheres with activated carboxyl groups in an alkaline buffer, adding the antibody-1, incubating with stirring, separating by magnetic absorption, adding a blocking solution and a preservation solution, and obtaining the AIE magnetically encoded microspheres labeled with the antibody-1;

[0069] In step (2), the activation of the carboxyl group comprises: dispersing the carboxyl-modified water-soluble AIE dye in an acidic buffer, adding a carbodiimide condensation reagent, and stirring the reaction to activate the carboxyl group to obtain a water-soluble AIE dye with activated carboxyl group;

[0070] In step (2), the antibody-2 coupling includes: dispersing the water-soluble AIE dye with activated carboxyl group in an alkaline buffer, adding antibody-2, incubating with stirring, separating by magnetic absorption, adding blocking solution and preservation solution, and obtaining the water-soluble AIE dye labeled with antibody-2.

[0071] More preferably,

[0072] In the carboxyl modification of step (1), the silane coupling agent is selected from at least one of the following: 3-vinyltrimethoxysilane, methacryloxypropyltrimethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane;

[0073] In the carboxyl modification of step (1), the monomer containing a double bond and a carboxyl group is selected from at least one of the following: acrylic acid, methacrylic acid, and maleic anhydride;

[0074] In the carboxyl modification of step (1), the base is aqueous ammonia with a concentration of 20% to 28%;

[0075] In the carboxyl modification of step (1), the mass volume ratio of AIE magnetic encoded microspheres, base, TEOS, silane coupling agent and monomer containing double bonds and carboxyl groups is 0.1g:2-3mL:1-1.5mL:0.45-0.6mL:0.3-0.5g; the temperature of the mixed reaction is 25°C-55°C, and the time is 4h-12h; the temperature of adding the monomer containing double bonds and carboxyl groups is 60°C-80°C, and the time is 6h-24h.

[0076] In the activated carboxyl groups in steps (1) and (2), the acidic buffer is glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer, disodium hydrogen phosphate-citric acid buffer or 2-(N-morpholine)ethanesulfonic acid buffer; the pH value of the acidic buffer is 6.0-6.5;

[0077] In the activated carboxyl group of steps (1) and (2), the carbodiimide condensing agent is selected from at least one of N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0078] In the steps (1) and (2) of activating the carboxyl group, the activation time of the carboxyl group is 5 min to 3 h;

[0079] In the activated carboxyl group of step (1), the mass amount of the acidic buffer is 10 to 100 times the mass amount of the AIE magnetically encoded microspheres;

[0080] In the activated carboxyl group of step (1), the mass amount of the carbodiimide condensation reagent is 1% to 90% of the mass amount of the AIE magnetically encoded microspheres;

[0081] In the antibody-1 coupling of step (1) and the antibody-2 coupling of step (2), the alkaline buffer is tris-hydrochloric acid buffer, boric acid buffer or phosphate buffered saline; the pH value of the alkaline buffer is 7.0 to 9.0;

[0082] In the antibody-1 coupling of step (1), the mass amount of the alkaline buffer is 5 to 90 times the mass amount of the AIE magnetically encoded microspheres.

[0083] In the antibody-1 coupling of step (1), the mass amount of the antibody-1 is 0.005 to 0.02% of the mass amount of the AIE magnetically encoded microspheres.

[0084] In the antibody-1 coupling of step (1), the stirring incubation temperature is 25°C to 35°C, and the time is 5 minutes to 3 hours.

[0085] The application of the above-mentioned immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes in antigen immunoassay comprises the following steps:

[0086] The AIE magnetic encoding microspheres labeled with antibody-1 and the water-soluble AIE dyes labeled with antibody-2 were added to the test solution containing the corresponding antigens, and after the reaction, they were separated by magnetic absorption, and the encoding peak and fluorescence quantitative detection concentration were obtained by flow cytometry.

[0087] Preferably, the mass amount of the corresponding antigen is 0.01-1wt% of the mass amount of the water-soluble AIE dye labeled with antibody-2.

[0088] The present invention can use AIE magnetic encoded microspheres labeled with antibody-1 of multiple different antigens and water-soluble AIE dyes labeled with antibody-2 of corresponding antigens to detect the solution to be tested respectively, so as to realize the detection of multiple antigens in the solution to be tested; that is, the AIE magnetic encoded microspheres labeled with antibody-1 of one antigen and the water-soluble AIE dyes labeled with antibody-2 of the corresponding antigen are used to detect the solution to be tested each time, and the process is repeated multiple times.

[0089] The present invention can use AIE magnetic encoded microspheres with different coding strengths respectively labeled with antibodies-1 of multiple different antigens and water-soluble AIE dyes labeled with antibodies-2 corresponding to the antigens to perform one detection on the solution to be tested, thereby realizing the detection of multiple antigens in the solution to be tested; that is, the AIE magnetic encoded microspheres labeled with antibodies-1 include AIE magnetic encoded microspheres with different coding strengths respectively labeled with antibodies-1 of multiple different antigens, and the water-soluble AIE dyes labeled with antibodies-2 include water-soluble AIE dyes respectively labeled with antibodies-2 corresponding to multiple different antigens; the solution to be tested is detected once.

[0090] The above-mentioned immunoassay reagents based on AIE magnetically encoded microspheres and AIE dyes are used in the field of in vitro non-disease diagnostic testing, such as food testing and public safety testing.

[0091] The present invention marks different types of antibodies-1 on the surface of AIE magnetically encoded microspheres, and marks corresponding antibodies-2 with water-soluble AIE dyes as quantitative detection markers for analysis. Through sandwich immune reaction, the aggregation-induced luminescence property of AIE materials is utilized to realize multi-item quantitative immunodetection of four inflammatory markers (CRP, SAA, IL-6, PCT), and the detection limit concentration ranges from pg·mL -1 ~mg·mL -1 Measure within the range.

[0092] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0093] (1) Compared with the fluorescent materials of traditional magnetic encoded microspheres, the AIE materials of the aggregation-induced luminescence magnetic encoded microspheres of the present invention improve the discrimination of the magnetic encoded microspheres and solve the fluorescence reabsorption effect of traditional fluorescent materials inside the microspheres. At the same time, the AIE materials have high photostability, so that there is no obvious fluorescence attenuation after repeated irradiation.

[0094] (2) Compared with the traditional ACQ molecules, the water-soluble AIE molecules of the present invention have the aggregation-induced emission property. The signal amplification effect of AIE is utilized during the detection process, and long linear range detection can be achieved in high-concentration samples even in complex samples with multiple markers.

[0095] (3) The AIE material of the present invention is used as a coding element. Due to the large Stoss shift, there is no reabsorption effect. At the same time, the dipole moment interaction between the two AIE molecules is greater than the FRET process, and the two different AIE molecules have a fluorescence enhancement effect inside the microsphere. At the same time, the AIE material has a typical aggregation-induced emission property. The two different AIE molecules are embedded in the microsphere and can emit strong fluorescence in the aggregate.

[0096] (4) The present invention provides a simple method, which utilizes the high specificity of AIE encoded microspheres and the signal amplification effect of AIE molecule quantitative detection to obtain a multi-item immunoassay technology based on AIE magnetic encoded microspheres and AIE signal amplification, thereby providing a new detection strategy for multiple marker immunoassays. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 This is a scanning electron microscope image of the dual-wavelength AIE magnetic encoding microspheres prepared in Example 1.

[0098] Figure 2 Emission spectra of AIE-R1 and AIE-IR1.

[0099] Figure 3 Schematic diagram of flow encoding of dual-wavelength AIE magnetic encoding microspheres prepared in Example 1. DETAILED DESCRIPTION

[0100] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0101] The blank polymer microspheres in the examples and comparative examples are polystyrene polymer microspheres with a diameter of 5 μm, from AIE Research Institute, with the product number NWKPB-500. Antibody-1, Antibody-2 and Antigen are from Maichuan (Guangzhou) Biotechnology Co., Ltd., with the product number AB10 series.

[0102] Example 1

[0103] 1. Preparation of AIE magnetically encoded microspheres

[0104] Weigh 1 g of blank polymer microspheres and disperse them in 100 g of water to obtain a microsphere dispersion;

[0105] 5 mg of AIE-R1 molecules (alkyl chain -C8H 17The AIE polymer microspheres were prepared by mixing the two solutions (the total mass of AIE molecules accounted for 1 wt% of the mass of the blank polymer microspheres) and ultrasonically treating them at a power of 100 W for 1 min. After the closed swelling for 2 h, the solvent was evaporated for 4 h, and the supernatant was removed by centrifugation to obtain the AIE polymer microspheres. The obtained AIE polymer microspheres were added to 50 mL of concentrated sulfuric acid (98.3 wt%), the temperature was raised to 80 ° C, and the reaction was carried out for 8 h. The microspheres were centrifuged at 5000 rpm and resuspended in water. The reaction was repeated three times to obtain the AIE polymer microspheres with sulfonated surface.

[0106] Ferrous sulfate heptahydrate and ferric chloride hexahydrate were mixed with 100g water in a mass ratio of 2:1 (the total mass of iron salt accounted for 5wt% of the mass of blank polymer microspheres), and the AIE polymer microspheres with sulfonation modified on the surface obtained above were added. After soaking and reacting for 1h, the microspheres were centrifuged at 5000rpm and resuspended in water. The process was repeated three times to obtain iron salt chelated AIE polymer microspheres; 50mL of 1mol / L sodium hydroxide solution was added, the temperature was raised to 80℃, and the reaction was carried out for 2h. The iron salt reacted in situ to generate magnetic particles that were deposited on the surface of the AIE polymer microspheres; the microspheres were centrifuged at 5000rpm and resuspended in water. The process was repeated three times to obtain dual-wavelength aggregation-induced luminescence magnetically encoded microspheres. 3 mL of ammonia water (28%), 1.5 mL of TEOS and 0.6 mL of 3-vinyltrimethoxysilane were added to the above-mentioned aggregation-induced emission magnetically encoded microspheres (0.1 g). After reacting for 6 hours and magnetic separation, 0.5 g of acrylic acid monomer was added, the temperature was raised to 80°C, and after reacting for 8 hours and magnetic separation, carboxyl-modified AIE magnetically encoded microspheres were obtained.

[0107] The dosage of AIE-R1 molecules was changed to 0.05 mg, 0.1 mg, and 0.5 mg respectively to obtain carboxyl-modified AIE magnetic encoded microspheres with different encoding intensities (other parameters remained unchanged).

[0108] 2. Antibody-1 labeled AIE magnetically encoded microspheres

[0109] The prepared carboxyl modified AIE magnetic encoded microspheres (AIE-R1 molecule dosage is 5 mg) were added to 10 g MES buffer (pH = 6.5), and after ultrasonic dispersion, 0.0095 g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, and the carboxyl groups were activated in a mixer at a speed of 300 rpm for 5 min. The supernatant was removed by magnetic suction to obtain the activated AIE magnetic encoded microspheres, 3 g boric acid buffer (pH = 8) and 10 μg mouse antibody-1 of the inflammatory marker SAA were added, and the mixture was placed in a mixer for incubation for 30 min, the supernatant was removed by magnetic suction, 0.5 mL of blocking solution BSA (0.5%) and Tris (0.1%) microsphere preservation solution were added, and ultrasonic dispersion was performed to obtain the AIE magnetic encoded microsphere solution (0.2 g / mL) labeled with SAA antibody-1.

[0110] The preparation method of the AIE magnetically encoded microsphere solution labeled with antibody-1 corresponding to the inflammatory marker CRP is the same as above, except that the mouse antibody-1 of the inflammatory marker SAA is replaced with the mouse antibody-1 of the inflammatory marker CRP, and the microspheres are replaced with AIE magnetically encoded microspheres with different encoding strengths (the amount of AIE-R1 molecules used is 0.05 mg);

[0111] The preparation method of the AIE magnetically encoded microsphere solution labeled with antibody-1 corresponding to the inflammatory marker IL-6 is the same as above, except that the mouse antibody-1 of the inflammatory marker SAA is replaced with the mouse antibody-1 of the inflammatory marker IL-6, and the microspheres are replaced with AIE magnetically encoded microspheres with different encoding strengths (the amount of AIE-R1 molecules used is 0.1 mg);

[0112] The preparation method of the AIE magnetically encoded microsphere solution labeled with antibody-1 corresponding to the inflammatory marker PCT is the same as above, the only difference is that the mouse antibody-1 of the inflammatory marker SAA is replaced by the mouse antibody-1 of the inflammatory marker PCT, and the microspheres are replaced with AIE magnetically encoded microspheres with different coding strengths (the amount of AIE-R1 molecules used is 0.5 mg).

[0113] 3. Antibody-2 labeled water-soluble AIE dye

[0114] 0.5 mg of carboxyl-modified water-soluble AIE-W1 dye was activated, coupled with SAA antibody-2, and blocked according to the method of the above step (ii) to obtain a SAA antibody-2 labeled water-soluble AIE dye solution (0.1 μg / mL) (wherein SAA antibody-2 is a mouse antibody-2 of the inflammatory marker SAA).

[0115] The preparation method of the water-soluble AIE dye solution labeled with antibody-2 corresponding to the inflammatory markers CRP, IL-6 and PCT is the same as above, the only difference is that the mouse antibody-2 of the inflammatory marker SAA is replaced by the mouse antibody-2 of the inflammatory marker CRP, IL-6 or PCT.

[0116] 4. Determination of multiple marker immunoassays

[0117] 10 μL of each AIE magnetic encoded microsphere solution labeled with four different types of antibody-1 and 10 μL of each water-soluble AIE dye solution labeled with the corresponding antibody-2 were added to the same flow tube, and then 5 μL of the sample to be tested (a mixed solution of four inflammatory markers SAA, CRP, IL-6, and PCT) was added. After magnetic separation, the coding peak was obtained through the coding channel of the flow cytometer, and the types of inflammatory markers in the test solution were distinguished by the coding channel and intensity; the fluorescence intensity of the water-soluble dye was detected to achieve quantitative determination of each inflammatory marker in the test sample.

[0118] The morphology of AIE magnetically encoded microspheres was observed using a field emission SEM electron microscope. The microspheres showed a regular spherical structure with a statistical particle size of about 5 μm (e.g. Figure 1 The fluorescence spectrum of the AIE magnetically encoded microspheres was tested using a fluorescence spectrometer. The emission wavelength of AIE-R1 was 620 nm and the emission wavelength of AIE-IR1 was 780 nm (e.g. Figure 2 ), the difference in center wavelength is 160nm. The emission wavelength of the water-soluble dye labeled with antibody-2 is 550nm. The detection concentration of multiple markers was tested using an analytical flow cytometer. The minimum detection concentrations were: SAA-0.2mg / mL, CRP-0.4mg / mL, IL-6-0.1ng / mL, PCT-1pg / mL, and the multiple analysis detection effect was good.

[0119] Table 1 Effects of AIE-encoded microspheres with different types of encoding dyes and detection dyes on multiple marker immunoassays

[0120]

[0121]

[0122] Comparative Example 1

[0123] 1. Preparation of APC magnetically encoded microspheres

[0124] The AIE-R1 molecule and the AIE-IR1 molecule in step (I) of Example 1 were substituted for the APC molecule (allophycocyanin) and the APC-cy7 molecule. The rest was the same as step (I) of Example 1.

[0125] 2. Antibody-1 labeled APC magnetically encoded microspheres

[0126] The method for preparing the APC magnetically encoded microsphere solution labeled with antibody-1 corresponding to the inflammatory markers SAA, CRP, IL-6 and PCT is the same as step (ii) of Example 1, except that the carboxyl-modified magnetically encoded microspheres are different (this comparative example is the carboxyl-modified APC magnetically encoded microspheres prepared as described above).

[0127] 3. Antibody-2 labeled water-soluble PE (phycoerythrin) dye

[0128] The preparation method of the water-soluble PE dye solution labeled with antibody-2 corresponding to the inflammatory markers SAA, CRP, IL-6 and PCT is the same as step (iii) of Example 1, except that the dye is different (the comparative example is a carboxyl-modified water-soluble PE dye).

[0129] 4. Determination of multiple marker immunoassays

[0130] The determination operation of the multiple marker immunoassay is the same as step (iv) of Example 1.

[0131] The morphology of AIE magnetically encoded microspheres was observed using a field emission SEM electron microscope. The microspheres showed a regular spherical structure with a statistical particle size of about 5 μm. The fluorescence spectrum of AIE magnetically encoded microspheres was tested using a fluorescence spectrometer. The APC emission wavelength was 648 nm, the APC-cy7 emission wavelength was 780 nm, and the difference in center wavelength was 132 nm. The emission wavelength of the water-soluble PE dye labeled with antibody-2 was 550 nm. The detection concentration of multiple markers was tested using an analytical flow cytometer. Because non-AIE dyes were used, the encoding peaks could not be distinguished, the encoding failed, the quantitative detection limit was zero, and the multiple analysis detection effect failed.

[0132] Comparative Example 2

[0133] 1. Preparation of AIE magnetically encoded microspheres

[0134] Same as step (a) of Example 1.

[0135] 2. Antibody-1 labeled AIE magnetically encoded microspheres

[0136] The preparation method of the AIE magnetically encoded microsphere solution labeled with antibody-1 corresponding to the inflammatory markers SAA, CRP, IL-6 and PCT is the same as step (ii) of Example 1, the only difference being that the carboxyl-modified AIE magnetically encoded microspheres are different (this comparative example is the carboxyl-modified AIE magnetically encoded microspheres prepared as described above).

[0137] 3. Antibody-2 labeled with water-soluble PE dye

[0138] The preparation method of the water-soluble PE dye solution labeled with antibody-2 corresponding to the inflammatory markers SAA, CRP, IL-6 and PCT is the same as step (iii) of Example 1, except that the dye is different (the comparative example is a carboxyl-modified water-soluble PE dye).

[0139] 4. Determination of multiple marker immunoassays

[0140] The determination operation of the multiple marker immunoassay is the same as step (iv) of Example 1.

[0141] The morphology of the AIE magnetically encoded microspheres was observed using a field emission SEM electron microscope. The microspheres showed a regular spherical structure with a statistical particle size of about 5 μm. The fluorescence spectrum of the AIE magnetically encoded microspheres was tested using a fluorescence spectrometer. The emission wavelength of AIE-R1 was 620 nm, and the emission wavelength of AIE-IR1 was 780 nm (e.g. Figure 2 ), the difference in center wavelength is 160nm. The emission wavelength of the water-soluble PE dye labeled with antibody-2 is 550nm. The four-fold encoding peaks were well distinguished using analytical flow cytometry, but because the PE dye could not amplify the signal, the minimum detection limits of PCT and IL-6 were as high as 50ng / mL and 500pg / mL, and quantitative detection failed.

[0142] Comparative Example 3

[0143] 1. Preparation of APC magnetically encoded microspheres

[0144] The AIE-R1 molecule and the AIE-IR1 molecule in step (I) of Example 1 were substituted for the APC molecule (allophycocyanin) and the APC-cy7 molecule. The rest was the same as step (I) of Example 1.

[0145] 2. Antibody-1 labeled APC magnetically encoded microspheres

[0146] The method for preparing the APC magnetic encoded microsphere solution labeled with antibody-1 corresponding to the inflammatory markers SAA, CRP, IL-6 and PCT is the same as step (ii) of Example 1, except that the carboxyl-modified magnetic encoded microspheres are different (this comparative example is the carboxyl-modified APC encoded microspheres prepared as described above).

[0147] 3. Antibody-2 labeled water-soluble AIE dye

[0148] The preparation method of the water-soluble AIE dye solution labeled with antibody-2 corresponding to the inflammatory markers SAA, CRP, IL-6 and PCT is the same as step (iii) of Example 1 (the water-soluble AIE dye is the water-soluble AIE-W1 dye).

[0149] 4. Determination of multiple marker immunoassays

[0150] The determination operation of the multiple marker immunoassay is the same as step (iv) of Example 1.

[0151] The morphology of AIE magnetically encoded microspheres was observed using a field emission SEM electron microscope. The microspheres showed a regular spherical structure with a statistical particle size of about 5 μm. The fluorescence spectrum of APC magnetically encoded microspheres was tested using a fluorescence spectrometer. The APC emission wavelength was 648 nm, the APC-cy7 emission wavelength was 780 nm, the difference in center wavelength was 132 nm, and the emission wavelength of the water-soluble dye labeled with antibody-2 was 550 nm. Since APC dye was used as the code, the quadruple coding peaks could not be distinguished using an analytical flow cytometer, and the coding and concentration quantitative detection failed.

[0152] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. An immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes, characterized in that: The method comprises an AIE magnetic encoding microsphere labeled with antibody-1 and a water-soluble AIE dye labeled with antibody-2; The AIE magnetically encoded microspheres are dual-wavelength aggregation-induced luminescence magnetically encoded microspheres, including AIE polymer microspheres and magnetic particles; the AIE polymer microspheres are obtained by embedding AIE molecules inside blank polymer microspheres, and the magnetic particles are deposited on the surface of the AIE polymer microspheres; the AIE molecules include AIE-R molecules and AIE-IR molecules; The difference between the maximum emission wavelength of the AIE-IR molecule and the maximum emission wavelength of the AIE-R molecule is in the range of 160 nm to 290 nm, and satisfies Δλ≥185, wherein Δλ=0.1*I / I0+ , where I / I0 is the average value of the ratio of the fluorescence intensity of AIE-R molecules and AIE-IR molecules before and after embedding, is the maximum emission wavelength of the AIE-IR molecule, is the maximum emission wavelength of the AIE-R molecule; The antibody-1 and antibody-2 specifically bind to the same antigen.

2. The immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes according to claim 1, characterized in that: The mass amount of the water-soluble AIE dye labeled with the antibody-2 is 0.001-0.01 wt % of the mass amount of the AIE magnetic encoding microspheres labeled with the antibody-1; The encoding capacity of the AIE magnetically encoded microspheres is ≥ 4, and the CV value of the flow fluorescence signal and the CV value of the flow FCS are both less than 10%.

3. The immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes according to claim 2, characterized in that: The antibody-1 is selected from at least one of a CRP monoclonal antibody, a PCT monoclonal antibody, an IL-6 monoclonal antibody and a SAA monoclonal antibody, and the antibody-2 is selected from the anti-antibody of the antibody-1; The antibody-1 and antibody-2 are mouse antibodies.

4. The immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes according to claim 2, characterized in that: The encoding capacity of the AIE magnetically encoded microspheres is ≥24; The water-soluble AIE dye is selected from at least one of the following AIE-W1 to AIE-W4 molecules:

5. The immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes according to claim 4, characterized in that: The blank polymer microspheres are selected from at least one of polystyrene microspheres, polymethyl methacrylate microspheres and polyglycidyl acrylate microspheres; The magnetic particles are Fe3O4; The AIE-R molecule is selected from one of the following AIE-R1 to AIE-R16 molecules: The AIE-IR molecule is selected from one of the following AIE-IR1 to AIE-IR10 molecules: The AIE molecules are embedded in the blank polymer microspheres by an active swelling method, wherein the amount of the AIE molecules used in the active swelling method is 0.01-10 wt% of the amount of the blank polymer microspheres, wherein the mass amount of the AIE-R molecules accounts for 0.01% to 1000% of the mass amount of the AIE-IR molecules; The magnetic particles are deposited on the surface of the AIE polymer microspheres by an in-situ precipitation method of iron salts, wherein the amount of iron salts used in the in-situ precipitation method is 0.1-25 wt % of the amount of blank polymer microspheres used.

6. The immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes according to claim 5, characterized in that: The active swelling method is to allow the AIE molecules to swell with the blank polymer microspheres in a swelling agent so that the AIE molecules are embedded in the blank polymer microspheres. In the active swelling method, the swelling agent is selected from at least one of dichloromethane, tetrahydrofuran, chloroform, anisole, benzyl alcohol and toluene, and the particle size of the blank polymer microspheres ranges from 0.03 µm to 50 µm; The in-situ precipitation method is to add an alkaline solution to react with the iron salt chelated on the surface of the AIE polymer microspheres in situ to generate magnetic particles that are deposited on the surface of the AIE polymer microspheres; In the in-situ precipitation method, the surface of the AIE polymer microspheres is modified by sulfonation, the alkaline solution is selected from at least one of sodium hydroxide solution, ammonia water, potassium hydroxide solution and sodium bicarbonate solution; the iron salt is selected from at least one of trivalent iron salt and divalent iron salt; the trivalent iron salt is selected from at least one of ferric chloride hexahydrate and anhydrous ferric chloride; the divalent iron salt is selected from at least one of ferrous chloride tetrahydrate, ferrous chloride dihydrate, ferrous sulfate and ferrous sulfate heptahydrate.

7. The method for preparing an immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Carboxyl modification, carboxyl activation, and antibody-1 coupling of the AIE magnetically encoded microspheres are performed to obtain antibody-1 labeled AIE magnetically encoded microspheres; (2) activating the carboxyl group of the water-soluble AIE dye and coupling it with antibody-2 to obtain a water-soluble AIE dye labeled with antibody-2; The immunoassay reagent based on the AIE magnetically encoded microspheres and the AIE dye is obtained.

8. The preparation method according to claim 7, characterized in that: In step (1), the carboxyl modification comprises: mixing and reacting the AIE magnetically encoded microspheres, a base, TEOS and a silane coupling agent, magnetically separating, and adding a monomer containing a double bond and a carboxyl group to react, thereby obtaining the carboxyl-modified AIE magnetically encoded microspheres; In step (1), the activation of carboxyl groups comprises: dispersing carboxyl-modified AIE magnetically encoded microspheres in an acidic buffer, adding a carbodiimide condensation reagent, and stirring the reaction to activate the carboxyl groups, thereby obtaining AIE magnetically encoded microspheres with activated carboxyl groups; In step (1), the antibody-1 coupling comprises: dispersing the AIE magnetically encoded microspheres with activated carboxyl groups in an alkaline buffer, adding the antibody-1, incubating with stirring, separating by magnetic absorption, adding a blocking solution and a preservation solution, and obtaining the AIE magnetically encoded microspheres labeled with the antibody-1; In step (2), the activation of the carboxyl group comprises: dispersing the carboxyl-modified water-soluble AIE dye in an acidic buffer, adding a carbodiimide condensation reagent, and stirring the reaction to activate the carboxyl group, thereby obtaining a water-soluble AIE dye with activated carboxyl group; In step (2), the antibody-2 coupling includes: dispersing the water-soluble AIE dye with activated carboxyl group in an alkaline buffer, adding antibody-2, incubating with stirring, separating by magnetic absorption, adding blocking solution and preservation solution, and obtaining the water-soluble AIE dye labeled with antibody-2.

9. Use of the immunoassay reagent based on AIE magnetically encoded microspheres and AIE dyes according to any one of claims 1 to 6 in antigen immunoassay, characterized in that: The following steps are involved: The AIE magnetic encoding microspheres labeled with antibody-1 and the water-soluble AIE dyes labeled with antibody-2 were added to the test solution containing the corresponding antigens, and after the reaction, they were separated by magnetic absorption, and the encoding peak and fluorescence quantitative detection concentration were obtained by flow cytometry.

10. The use according to claim 9, characterized in that: The mass amount of the corresponding antigen is 0.01-1 wt % of the mass amount of the water-soluble AIE dye labeled with antibody-2; The antibody-1 labeled AIE magnetic encoded microspheres include AIE magnetic encoded microspheres with different encoding intensities respectively labeled with antibodies-1 corresponding to multiple different antigens, and the antibody-2 labeled water-soluble AIE dyes include water-soluble AIE dyes respectively labeled with antibodies-2 corresponding to multiple different antigens.

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