A multi-mode aggregation-induced fluorescent immunochromatographic test paper and preparation method thereof

Abstract

The present invention discloses an aggregation-induced fluorescent particle, a composite, a multi-mode immunochromatographic test paper and a preparation method thereof. The aggregation-induced fluorescent particle has a core-shell structure under a transmission electron microscope, a particle size of 50-500nm, and is mainly composed of an aggregation-induced fluorescent material and a polymer having both color and fluorescence characteristics. The present invention adopts a mini-emulsion solvent evaporation method to prepare aggregation-induced fluorescent particles, the equipment and process are simple, the prepared composite particles have uniform particle size, controllable conditions, good repeatability, and the concentration of aggregation-induced fluorescent materials in the particles is easy to control. The most important thing is that the obtained particles have both color and fluorescence characteristics. The immunochromatographic test strip prepared with the particles as signal carriers can not only be qualitatively detected by the naked eye, but also quantitatively detected by fluorescence.

Description

Technical Field

[0001] The present invention belongs to the technical field of rapid immunoassay, and specifically relates to an aggregation-induced fluorescent particle, a complex, a multi-mode chromatography test paper and a preparation method thereof. Background Art

[0002] Immunochromatographic test strips are currently the main technical means for rapid on-site detection and analysis due to their advantages such as simplicity, speed and low cost. They are widely used in technical fields such as clinical diseases, food safety, and environmental monitoring. The basic principle is to use nitrocellulose membrane as the chromatographic reaction membrane. The analyte to be tested in the sample undergoes a specific immune reaction with the antigen or antibody fixed in the detection area of ​​the membrane. The signal strength of the immune marker in the detection area is analyzed to detect the analyte to be tested. Traditional immunochromatography uses colloidal gold as a marker, and the test results can be read with the naked eye without the need for complex equipment and medical professionals. Although this detection method can easily obtain qualitative or semi-quantitative results, the immunochromatographic detection based on colorimetry is difficult to reflect subtle changes in the target concentration in color depth, so accurate quantification and high sensitivity cannot be achieved.

[0003] The quantitative performance of immunochromatographic tests can be improved by using fluorescent materials including organic fluorophores or quantum dots as markers to obtain higher signals, better contrast, and lower background interference. However, fluorescent materials have low light absorption capacity, so fluorescent test strips generally lack visual detection capabilities. In addition, the reliance on additional excitation light sources and sensing devices for signal readout also limits the use scenarios of fluorescent immunochromatographic tests. The shortcomings of colorimetry and fluorescence methods have prompted the emergence of a new universal detection method with independent strong visible / fluorescent dual signals, which is suitable for a range of application scenarios. In 2020, Chongwen Wang et al. reported a polyethyleneimine (PEI)-mediated assembly method (Analytical Chemistry, 2020, 92(23): 15542-15549.), with SiO2 as the core, a shell layer formed by colloidal gold to produce a strong colorimetric signal, and a shell layer formed by carboxylated quantum dots to provide fluorescent signals and protein coupling surface sites, to prepare nanoparticles with good stability, high luminescence, and good biocompatibility. The immunochromatographic test strips made with such nanoparticles as markers can achieve colorimetric and fluorescence detection. The disadvantage is that the preparation process is relatively complicated, and there is aggregation fluorescence quenching phenomenon of quantum dots.

[0004] Traditional fluorescent materials have the phenomenon of aggregation fluorescence quenching (ACQ): they show strong fluorescence in dilute solutions, but their luminescence performance tends to weaken or even disappear completely in high-concentration solutions or when they are made into solids. Aggregation-induced emission (AIE) is a phenomenon accidentally discovered by the team of Academician Tang Benzhong in 2001: the luminescence of materials is enhanced in the aggregated state, which provides a new idea for solving the fluorescence quenching problem of traditional ACQ materials; the advantages of AIE materials for biomolecule labeling are mainly reflected in: low background, high signal-to-noise ratio, good sensitivity, etc. In 2019, Weihua Lai et al. first reported (Biosensors and Bioelectronics, 2019, 135: 173–180), using AIE fluorescent microspheres for fluorescent immunochromatography detection. Compared with traditional fluorescent microspheres, AIE has a larger Stokes shift, higher fluorescence intensity and fluorescence quantum yield. In 2021, Li Fuyou's team from Fudan University reported (ACS nano, 2021, 15(5): 8996-9004.) that fluorescent microspheres were prepared using AIE materials in the near-infrared II region for immunochromatographic detection. This method can reduce the background fluorescence interference of the sample. In recent years, it has been reported that aggregation-induced fluorescent materials also have colors visible to the naked eye and are used in the development of organic optoelectronic materials. However, the existing methods of preparing fluorescent microspheres with AIE materials mainly utilize the fluorescence properties of AIE materials to prepare fluorescent immunochromatographic test strips, which limits their application in on-site naked eye detection. Summary of the invention

[0005] The purpose of the present invention is to solve the deficiencies of the above-mentioned prior art and provide a multi-mode aggregation-induced fluorescent particle with good stability and high sensitivity, an aggregation-induced fluorescent particle-antibody complex, and an immunochromatographic test paper coated with the aggregation-induced fluorescent particle-antibody complex and a preparation method thereof. The preparation method of the present invention is simple, highly sensitive, reproducible, and environmentally friendly.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] An aggregation-induced fluorescent particle, which has a core-shell structure under a transmission electron microscope, a particle size of 50 to 500 nm, and is mainly composed of an aggregation-induced fluorescent material and a polymer having both color and fluorescence properties. Preferably, the aggregation-induced fluorescent particle also contains a carboxyl functional group on its surface.

[0008] Furthermore, the aggregation-induced fluorescent material having both color and fluorescence properties is selected from one or more of tetraphenylethylene AIE derivatives, silole AIE derivatives, 1,4-stilbene AIE derivatives, five-membered heterocyclic AIE derivatives and furan AIE derivatives.

[0009] Furthermore, the polymer is selected from at least one of polystyrene-acrylic acid copolymer, polystyrene-maleic anhydride copolymer and polymethyl methacrylate-methacrylic acid copolymer.

[0010] Furthermore, the molecular weight of the polymer is 1500 to 40000. Preferably, the molecular weight of the polymer is 1900 to 37000.

[0011] Furthermore, the surface of the aggregation-induced fluorescent particles has carboxyl groups, which are derived from polymers. The present invention also provides an aggregation-induced fluorescent particle-antibody complex, which contains the aggregation-induced fluorescent particles and antibodies as described above.

[0012] The present invention also provides an immunochromatographic test paper, the coupling pad of which is coated with the aggregation-induced fluorescent particle-antibody complex as described above.

[0013] The present invention also provides a method for preparing the above-mentioned aggregation-induced fluorescent particles, comprising the following specific steps: the oil phase is an aggregation-induced fluorescent material and a polymer dissolved in chloroform, wherein the concentration of the aggregation-induced fluorescent material is 1-20 mg / mL, and the concentration of the polymer is 5-100 mg / mL; the aqueous phase is an aqueous surfactant solution with a mass concentration of 0.1-1%; the oil phase / aqueous phase mixing volume ratio is 1:3-6 (i.e., oil phase:aqueous phase=1:3-6), and after magnetic stirring for 1-10 minutes, ultrasonic probe emulsification is performed for 1-10 minutes to obtain a microemulsion; then the chloroform solvent is removed to solidify the microemulsion droplets, thereby obtaining aggregation-induced fluorescent particles.

[0014] Furthermore, after the microemulsion droplets are solidified, the aggregation-induced fluorescent particles are centrifuged, the precipitate is collected, washed with a phosphate buffer, and then dispersed in a phosphate buffer for later use.

[0015] Furthermore, the ratio of the aggregation-induced fluorescent material to the polymer in the oil phase is 1:1-8.

[0016] Furthermore, the concentration of the aggregation-inducing fluorescent material in the oil phase is 2.5-5 mg / mL; and the concentration of the polymer is 20 mg / mL.

[0017] Furthermore, the surfactant is selected from at least one of sodium dodecyl sulfate, Tween 20, Tween 80 and Triton X-100.

[0018] On the other hand, the present invention also provides a method for preparing the aggregation-induced fluorescent particle-antibody complex: the aggregation-induced fluorescent particles as described above are dispersed in a phosphate buffer solution with a pH of 4 to 6, an activator is added, an activation reaction is carried out, the supernatant is removed by centrifugation, the activated particles are dispersed in a phosphate buffer solution, antibodies are added, the reaction is incubated at room temperature, the supernatant is removed by centrifugation to remove free antibodies, and the precipitated particle-antibody conjugate is dispersed in a blocking solution for incubation to obtain an aggregation-induced fluorescent particle-antibody complex.

[0019] Furthermore, the activator is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC); the working concentration of EDC is 0.1-1 mg / mL.

[0020] Specifically, the aggregation-induced fluorescent particles are dispersed in a phosphate buffer with a pH of 4 to 6, and an activator 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride is added at a concentration of 0.1 to 1 mg / mL. The activation reaction is carried out for 20 to 40 minutes (preferably 30 minutes). After centrifugation to remove the supernatant, the activated particles are dispersed in a phosphate buffer, antibodies are added, and the reaction is incubated at room temperature for 1 hour. The free antibodies are then removed by centrifugation, and the precipitated particle-antibody conjugate is dispersed in a blocking solution and incubated at room temperature for more than 2 hours (including 2 hours) to obtain an aggregation-induced fluorescent particle-antibody complex.

[0021] Furthermore, after blocking, the obtained aggregation-inducing fluorescent particle-antibody complex was added with bovine serum albumin (BSA) with a mass concentration of 5% and stored for later use.

[0022] Furthermore, the blocking solution is a casein solution. Preferably, the blocking solution is a casein solution with a concentration of 5 mg / mL.

[0023] The present invention also provides an immunochromatographic test paper: comprising a sample pad, a coupling pad, a nitrocellulose membrane and absorbent paper which are sequentially overlapped on a bottom plate; wherein the coupling pad is coated with the aggregation-induced fluorescent particle-antibody complex as described above.

[0024] Compared with the existing technology, the present invention has the following advantages:

[0025] 1. Compared with the existing immunochromatographic detection method using colloidal gold as a marker, the aggregation-induced fluorescence chromatography detection has the characteristics of higher sensitivity, smaller batch differences, and more stable markers.

[0026] 2. Compared with the existing immunochromatographic detection method using fluorescent materials including organic fluorophores or quantum dots as markers, it has the characteristics of strong light absorption ability and can be observed by naked eyes; high fluorescence intensity and not easy to quench; high signal-to-noise ratio and good sensitivity.

[0027] 3. The present invention adopts a miniemulsion-solvent evaporation method to prepare aggregation-induced fluorescent particles. Compared with the particles prepared by the electrostatic self-assembly method, the equipment and process are simple, the prepared composite particles have uniform particle size, controllable conditions, good repeatability, and easy to control material concentration in the particles.

[0028] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 These are transmission electron micrographs of aggregation-induced fluorescent particles at different magnifications. The scale of the large icon is 500 nanometers, and the scale of the small icon in the lower right corner is 50 nanometers.

[0030] Figure 2 These are the results of immunochromatographic detection of C-reactive protein using aggregation-induced fluorescent particles that have both color and fluorescence properties as markers. a is the result under ultraviolet light excitation, and b is the result under natural light.

[0031] Figure 3 These are the results of immunochromatographic detection of interleukin 6 using time-resolved fluorescent microspheres as markers. a is the result under natural light, and b is the result under ultraviolet light excitation. DETAILED DESCRIPTION

[0032] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further explained below with reference to specific diagrams. However, the invention is not limited to the following implementation cases.

[0033] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with this technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantive technical significance. Any structural modification, change in proportion or adjustment of size, without affecting the effects and purposes that can be achieved by the present invention, should still fall within the scope of the technical contents disclosed by the present invention.

[0034] Example 1: Preparation of aggregation-induced fluorescent particles and method for labeling C-reactive protein antibodies:

[0035] A. Preparation of oil phase components: Disperse tetraphenylethylene AIE derivatives and polystyrene-maleic anhydride copolymer (molecular weight 1900, purchased from Sigma-Aldrich) in chloroform solution, the concentration of tetraphenylethylene AIE derivatives is 10 mg / mL, and the concentration of polymer is 10 mg / mL;

[0036] B. Prepare aqueous phase components: prepare sodium dodecyl sulfate and Triton X-100 into an aqueous solution, wherein the mass concentration of sodium dodecyl sulfate is 0.1% and the mass concentration of Triton X-100 is 0.5%;

[0037] C. Add the oil phase to the aqueous phase solution, stir magnetically until uniform, and then use an ultrasonic crusher to ultrasonicate the mixture for 2 minutes to obtain a uniform and stable microemulsion;

[0038] D. Place the microemulsion in an open container at room temperature and allow the solvent to evaporate gradually under magnetic stirring for 16 hours. Wash by centrifugation and disperse the precipitate by ultrasonication to obtain material-polymer composite particles. The electron microscope photo is shown in FIG. Figure 1 , emulsion photos such as Figure 2 ;

[0039] E. Activation of the carboxyl groups on the particle surface: the particles were dispersed in a phosphate buffer solution at pH 4.0 with a particle concentration of 20 mg / mL, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) was added at a concentration of 1.0 mmol / L and incubated at room temperature for 30 min;

[0040] F. After the incubation, the supernatant was removed by centrifugation, and the precipitate was dispersed by adding phosphate buffer, and then C-reactive protein antibody (purchased from Hangzhou Yibaixin Biotechnology Co., Ltd.) was added to the phosphate buffer, and the concentration of C-reactive protein antibody was 30 μg / ml. Then, the aggregation-inducing fluorescent particle solution was added to the C-reactive protein antibody solution, and incubated at room temperature for 30 minutes;

[0041] G. After incubation, remove excess antibodies by centrifugation, add casein for ultrasonic dispersion at a concentration of 5 mg / mL, and block (incubate) at room temperature for more than 2 hours;

[0042] H. After blocking, BSA with a mass concentration of 5% was added to obtain aggregation-induced fluorescent particles coupled with C-reactive protein antibodies.

[0043] Example 2: Preparation of aggregation-induced fluorescent particles and labeling of goat anti-rabbit immunoglobulin (IgG) antibodies:

[0044] A. Preparation of oil phase components: Disperse tetraphenylethylene AIE derivatives and polystyrene-acrylic acid copolymer (molecular weight 37000, purchased from Sigma-Aldrich) in chloroform solution, the concentration of tetraphenylethylene AIE derivatives is 10 mg / mL, and the concentration of polymer is 10 mg / mL;

[0045] B. preparing an aqueous phase component, preparing sodium dodecyl sulfate into an aqueous solution, wherein the mass concentration of sodium dodecyl sulfate is 0.5%;

[0046] C. Add the oil phase to the aqueous phase solution, stir magnetically until uniform, and then use an ultrasonic crusher to ultrasonicate the mixture for 5 minutes to obtain a uniform and stable microemulsion;

[0047] D. Place the microemulsion in an open container at room temperature and allow the solvent to evaporate gradually under magnetic stirring for 24 hours. Wash by centrifugation and disperse the precipitate by ultrasonic water to obtain material-polymer composite particles. The electron microscope photo is shown in FIG. Figure 1 , emulsion photos such as Figure 2 ;

[0048] E. Activation of the carboxyl groups on the particle surface: the particles were dispersed in phosphate buffer at pH 4.0 with a particle concentration of 20 mg / mL, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) was added at a concentration of 1.0 mmol / L and incubated at room temperature for 30 min;

[0049] F. After the incubation, the supernatant was removed by centrifugation, and the precipitate was dispersed by adding phosphate buffer, and then goat anti-rabbit IgG polyclonal antibody (purchased from Hangzhou Longji Biotechnology Co., Ltd.) was added to the phosphate buffer, and the concentration of goat anti-rabbit IgG antibody was 50ug / ml. Then, the aggregation-induced fluorescent particle solution was added to the goat anti-rabbit antibody solution, and incubated at room temperature for 30 minutes;

[0050] G. After incubation, remove excess antibodies by centrifugation, add casein for ultrasonic dispersion at a concentration of 5 mg / mL, and block at room temperature for more than 24 hours;

[0051] H. After blocking, BSA with a mass concentration of 5% was added for storage, thereby obtaining aggregation-inducing particles coupled with goat anti-rabbit IgG antibodies.

[0052] Example 3: C-reactive protein sample detection

[0053] The aggregation-inducing fluorescent particles labeled with C-reactive protein antibodies in Example 1 (i.e., aggregation-inducing fluorescent particles-C-reactive protein antibody complex) and the aggregation-inducing particles labeled with goat anti-rabbit IgG antibodies in Example 2 (i.e., aggregation-inducing fluorescent particles-goat anti-rabbit IgG antibody complex) were used to prepare a highly sensitive C-reactive protein aggregation-inducing fluorescent particle multimodal immunochromatographic test paper. The test paper preparation steps are as follows:

[0054] The aggregation-inducing fluorescent particle-C-reactive protein antibody complex and the aggregation-inducing fluorescent particle-goat anti-rabbit IgG antibody complex were coated on the coupling pad for later use; the concentrations of the other strain of C-reactive protein antibody and the anti-rabbit antibody were adjusted to 1.0 mg / mL respectively with phosphate buffered saline (PBS);

[0055] Another strain of C-reactive protein antibody was sprayed on the lower part of the nitrocellulose membrane as the test line (T line), and rabbit IgG was sprayed on the upper part as the quality control line (C line). The membrane was scratched at a speed of 1 μL / cm, and then dried at 37°C for use;

[0056] Finally, the sample pad, coupling pad, nitrocellulose membrane and absorbent paper were overlapped on the bottom plate, and cut into appropriate widths with a knife to obtain the C-reactive protein aggregation-induced fluorescent particle multimodal immunochromatographic test paper.

[0057] The detection principle and process are as follows:

[0058] Detection principle: When the sandwich method detects positive samples, the marker-coupled antibody and the antibody marked by the T line are two antibodies targeting different sites of the same antigen. The antigen to be tested first binds to the marker coupled with the corresponding antibody, so that when the antigen-antibody complex passes through the T line, it binds to another antibody on the T line to form an antibody-antigen-antibody sandwich structure, so the T line of the positive sample is colored. On the contrary, in the negative sample, there is no antigen to be tested in the negative sample that binds to the antibody on the marker, so the marker cannot bind to the antibody on the T line when passing through the T line, resulting in no color of the T line.

[0059] Detection process: Take 5μL serum / plasma sample (or 8.5μL whole blood sample), add the sample to phosphate buffer, and mix thoroughly for 30s to 1min. Use a pipette to take 75μL from the above solution and add it to the sample pad of the immunochromatographic test paper, wait for the sample to react for 2 to 8 minutes, and observe the test paper results with naked eyes ( Figure 2 a) Or use a laser fluorescence reader to scan the test paper fluorescence intensity to calculate the quantitative results (fluorescence excitation: Figure 2 b)

[0060] The upper line of the test strip is the quality control line (C line), and the lower line is the test line (T line). Since C-reactive protein is a large molecular antigen, it is detected by sandwich method, so the color of T line becomes darker and darker as the detection concentration increases. The rabbit IgG fixed on the independent C line undergoes immune binding reaction with the aggregation-induced fluorescent particles labeled with goat anti-rabbit IgG antibody, so the color of C line remains unchanged.

[0061] Comparative Example 1: Method for labeling interleukin-6 antibody with time-resolved fluorescent microspheres

[0062] A. Disperse the purchased time-resolved fluorescent microspheres in phosphate buffer at pH 4.0 at a particle concentration of 20 mg / mL, then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) at a concentration of 1.0 mmol / L and incubate at room temperature for 30 minutes;

[0063] B. After the incubation, the supernatant was removed by centrifugation, and the precipitate was dispersed by adding phosphate buffer, and then the interleukin-6 antibody was added to the phosphate buffer at an antibody concentration of 50ug / ml. Then, the time-resolved fluorescent microspheres were added to the interleukin-6 antibody solution and incubated at room temperature for 30 minutes;

[0064] C. After incubation, remove excess antibodies by centrifugation, add casein for ultrasonic dispersion at a concentration of 5 mg / mL, and block at room temperature for more than 24 hours;

[0065] D. After blocking, BSA (purchased from Sinopharm Chemical Reagent Co., Ltd.) with a mass concentration of 5% was added to the preservation solution to obtain time-resolved fluorescent microspheres coupled with interleukin-6 antibodies.

[0066] Comparative Example 2: Detection of Interleukin-6 Samples

[0067] The time-resolved fluorescent microspheres labeled with interleukin-6 antibody obtained in Comparative Example 1 (i.e., time-resolved fluorescent microsphere-interleukin-6 antibody complex) were used to prepare a highly sensitive interleukin-6 immunochromatographic test paper. The test paper preparation steps are as follows:

[0068] The time-resolved microsphere-IL-6 antibody complex is coated on the coupling pad for later use;

[0069] The concentration of another interleukin-6 antibody was adjusted to 1.0 mg / mL with PBS solution;

[0070] Spray another antibody against interleukin-6 on the lower part of the nitrocellulose membrane as the detection line, and draw the membrane at a speed of 0.5 μL / cm, then dry at 37°C for later use;

[0071] Finally, the sample pad, coupling pad, nitrocellulose membrane and absorbent paper are overlapped on the bottom plate, and the interleukin-6 immunochromatographic test paper is obtained after being cut into appropriate widths with a knife.

[0072] The detection principle is the same as that of Example 3.

[0073] Test process: Take 100μL of interleukin-6 quality control product and add it to the sample pad of the test strip, wait for the sample to react for 2 to 8 minutes, and observe the test strip results with naked eyes ( Figure 3 a) Or use a laser fluorescence reader to scan the test paper fluorescence intensity and calculate the quantitative results (fluorescence excitation results: Figure 3 b).

[0074] The line below the test strip in the results is the test line (T line). Because it is only used as a comparison for Example 3, no quality control line (C line) is set. Since interleukin 6 is a large molecular antigen, the sandwich method is used for detection, so the color of the T line becomes darker and darker as the detection concentration increases. Unlike Example 3, where the test results can be observed in both modes, the test results of Comparative Example 2 can only be seen under excitation of the excitation light, and the test results cannot be observed by the naked eye.

[0075] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. An aggregation-induced fluorescent particle, characterized in that: The particle size is 50 to 500 nm, and comprises an aggregation-induced fluorescent material and a polymer having both color and fluorescence characteristics; the aggregation-induced fluorescent material having both color and fluorescence characteristics is a tetraphenylethylene AIE derivative; the polymer is a polystyrene-acrylic acid copolymer and / or a polystyrene-maleic anhydride copolymer; the molecular weight of the polymer is 1500 to 40000; the aggregation-induced fluorescent particles are prepared by the following method: The oil phase is the aggregation-induced fluorescent material and the polymer dissolved in chloroform, wherein the concentration of the aggregation-induced fluorescent material is 1-20 mg / mL, and the concentration of the polymer is 5-100 mg / mL; the ratio of the aggregation-induced fluorescent material to the polymer in the oil phase is 1:1-8; the water phase is a surfactant aqueous solution with a mass concentration of 0.1-1%; the oil phase / water phase mixed volume ratio is 1:3-6, and after magnetic stirring for 1-10 minutes, ultrasonic probe emulsification is performed for 1-10 minutes to obtain a microemulsion; then the chloroform solvent is removed to solidify the microemulsion droplets, so as to obtain the aggregation-induced fluorescent particles; the surface of the aggregation-induced fluorescent particles contains carboxyl functional groups, and the carboxyl groups come from the polymer.

2. An aggregation-induced fluorescent particle-antibody complex, characterized in that: The method comprises the aggregation-induced fluorescent particles according to claim 1.

3. An immunochromatographic test paper, characterized in that: The coupling pad of the immunochromatographic test paper is coated with the aggregation-induced fluorescent particle-antibody complex described in claim 2.

4. A method for preparing the aggregation-induced fluorescent particles according to claim 1, characterized in that: The method comprises the following specific steps: the oil phase is the aggregation-induced fluorescent material and the polymer dissolved in chloroform, wherein the concentration of the aggregation-induced fluorescent material is 1-20 mg / mL, and the concentration of the polymer is 5-100 mg / mL; the ratio of the aggregation-induced fluorescent material to the polymer in the oil phase is 1:1-8; the water phase is a surfactant aqueous solution with a mass concentration of 0.1-1%; the oil phase / water phase mixed volume ratio is 1:3-6, and a microemulsion is obtained by magnetic stirring for 1-10 minutes and then emulsifying with an ultrasonic probe for 1-10 minutes; and then the chloroform solvent is removed to solidify the microemulsion droplets, so as to obtain the aggregation-induced fluorescent particles.

5. The method for preparing aggregation-induced fluorescent particles according to claim 4, characterized in that: The concentration of the aggregation-induced fluorescent material in the oil phase is 2.5-5 mg / mL; the concentration of the polymer is 20 mg / mL.

6. A method for preparing the aggregation-inducing fluorescent particle-antibody complex according to claim 2, characterized in that: The following steps are involved: The aggregation-induced fluorescent particles are dispersed in a phosphate buffer with a pH of 4 to 6, an activator is added, an activation reaction is carried out, and after centrifugation to remove the supernatant, the activated particles are dispersed in a phosphate buffer, antibodies are added, and the reaction is incubated at room temperature. The free antibodies are then removed by centrifugation, and the precipitated particles-antibody conjugates are dispersed in a blocking solution, and incubated at room temperature to obtain aggregation-induced fluorescent particles-antibody complexes.

7. The method according to claim 6, characterized in that The activator is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, and the blocking solution is casein solution.

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