A preparation method of carbon dot-polystyrene fluorescent composite microspheres

Abstract

The present application belongs to the technical field of fluorescent composite microspheres, and relates to a preparation method of carbon dot-polystyrene fluorescent composite microspheres. The present application discloses a preparation method of carbon dot-polystyrene fluorescent composite microspheres, which comprises the following steps: dispersing carbon dots after freeze-drying in an alkaline solution, and uniformly stirring under heating; then adding an initiator, methyl methacrylate and styrene monomers, mixing, and performing condensation reflux reaction in an inert gas to obtain carbon dot-polystyrene fluorescent composite microspheres. The present application further discloses a rapid detection reagent, which is carbon dot-polystyrene fluorescent composite microspheres coupled with biomolecules on the surface. The present application wraps carbon dots in a polystyrene matrix through a soap-free emulsion polymerization method, simplifies the preparation process, reduces the influence of external environment on the carbon dots, improves the stability and fluorescent performance of the fluorescent composite microspheres, and can also be coupled with biomolecules, and is used for high-sensitivity biological analysis and detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent composite microspheres, and relates to a preparation method of carbon dot-polystyrene fluorescent composite microspheres. BACKGROUND

[0002] Carbon dots (CDs) are a kind of zero-dimensional carbon nanomaterials with a particle size of less than 10 nm, which were first reported in 2004. As a new type of functional nanomaterial, nanofluorescent carbon dots have unique physical and chemical properties, including small size, excellent biocompatibility, high quantum yield (QY), fluorescence adjustability, easy surface modification, and the like, and have become a promising nanomaterial for biomedical applications, and have attracted more and more attention from scientists. At present, carbon quantum dots have been widely used in the fields of biological imaging, biological sensing, drug delivery, biological catalysis and tissue engineering. However, when carbon dots are directly applied to immunoassay, many limitations will be encountered. In the process of labeling antibodies, the ultra-small size of carbon dots will cause multiple carbon dot particles to be coupled on a single antibody, thereby causing fluorescence aggregation quenching phenomenon; and the overall luminescence intensity of a single carbon dot particle is weak, which also affects the sensitivity of analysis and detection. Therefore, how to prepare a larger fluorescent microsphere by compounding carbon dots with a suitable nanocarrier material is a key to expand the application of carbon dots.

[0003] Common fluorescent carriers include silica and polystyrene. Silica has become a commonly used fluorescent carrier due to its advantages of non-toxicity, good biocompatibility, and the ability to effectively improve the stability of the material. However, the synthesis method is complex, the silica shell layer affects the active sites of the reactants, the silica shell layer is easily etched under harsh reaction conditions (high-temperature water vapor, alkaline solution), and cross-linking is easy, which makes the expandability of silica as a carrier matrix low. Polystyrene is the most common fluorescent carrier due to its advantages of large output, wide application, large specific surface area, wide particle size distribution, and easy surface modification. At present, the composite material is prepared by modifying carbon dots (fluorescent materials) on the surface of polystyrene microspheres, but carbon dot leakage is easy, and the fluorescent performance of the microspheres is easily affected by the environment. SUMMARY

[0004] The purpose of the present application is to solve the above-mentioned problems existing in the prior art, and a preparation method of carbon dot-polystyrene fluorescent composite microspheres is provided. The carbon dots after freeze-drying are added to the process of synthesizing polystyrene microspheres by a soap-free emulsion method, and the carbon dots are wrapped in the polystyrene matrix to prepare carbon dot-polystyrene fluorescent composite microspheres with good fluorescent performance.

[0005] The purpose of the present application can be achieved by the following technical solutions:

[0006] A preparation method of carbon dot-polystyrene fluorescent composite microspheres comprises:

[0007] The carbon dots after freeze-drying are dispersed in an alkaline solution, heated and stirred to be uniform; initiator, methacrylic acid and styrene monomers are added and mixed at 75-90°C, and then condensed and refluxed in inert gas to obtain carbon dot-polystyrene fluorescent composite microspheres.

[0008] As preferred, the content of carbon dots in the carbon dot-polystyrene fluorescent composite microspheres is 1-45wt%.

[0009] As preferred, the mass ratio of the carbon dots after freeze-drying to the initiator is 1:(1-20).

[0010] Further preferred, the mass ratio of the carbon dots after freeze-drying to the initiator is 1:(5-9).

[0011] As preferred, the mass-volume ratio of the carbon dots after freeze-drying to methacrylic acid and styrene monomers is (1-10)mg:(0.01-1)mL:1mL.

[0012] As preferred, the heating and stirring temperature is 75-90°C, and the time is 1-60min.

[0013] As preferred, the condensation and reflux temperature is 75-90°C, and the time is 1-24h.

[0014] Further preferred, the condensation and reflux temperature is 78-86°C, and the time is 10-20h.

[0015] As preferred, the initiator is ammonium persulfate.

[0016] As preferred, the alkaline solution is composed of an alkaline salt and a solvent, and the alkaline salt includes one or more of sodium bicarbonate, sodium carbonate, ammonium bicarbonate and potassium carbonate.

[0017] As preferred, the concentration of the alkaline solution is 1-25wt%.

[0018] As preferred, the alkaline solution is ammonia water.

[0019] As preferred, the carbon dots after freeze-drying are carbon dots after freeze-drying with amino groups.

[0020] As preferred, the carbon dots after freeze-drying are prepared from a carbon dot solution after freeze-drying.

[0021] Further preferred, the freeze-drying includes pre-cooling at -70 to -200°C, and then drying at -30 to -60°C for 24-72h.

[0022] Further preferred, the carbon dot solution is prepared from a carbon dot precursor and a fatty amine, an aromatic amine or an amide compound after reaction and dialysis.

[0023] More preferably, the carbon dot precursor comprises one or more of Rose Bengal (RB), glutathione (GSH), citric acid (CA).

[0024] More preferably, the fatty amine, aromatic amine, amide compound comprises one or more of ethylenediamine, butanediamine, propylenediamine, o-phenylenediamine, formamide.

[0025] More preferably, the reaction comprises one or both of a hydrothermal reaction and a microwave heating reaction; the hydrothermal reaction has a temperature of 140-220°C and a time of 1-12h; the microwave heating has a power of 400-900W and a time of 1-60min.

[0026] Preferably, the carbon dot-polystyrene fluorescent composite microspheres have an average particle size of 70-300nm.

[0027] Preferably, the carbon dots after freeze-drying have an emission wavelength of 525nm and a quantum efficiency of 85.7%.

[0028] Preferably, the method for preparing carbon dot-polystyrene fluorescent composite microspheres comprises:

[0029] (1) reacting and dialyzing a carbon dot precursor and an amine compound to obtain a carbon dot solution; freeze-drying the carbon dot solution to obtain carbon dots after freeze-drying;

[0030] (2) dispersing the carbon dots after freeze-drying in an alkaline solution, heating and stirring until uniform; adding an initiator, methacrylic acid, and styrene monomers at 75-90°C, and then condensing and refluxing in an inert gas to obtain carbon dot-polystyrene fluorescent composite microspheres.

[0031] Further preferably, the method for preparing carbon dot-polystyrene fluorescent composite microspheres comprises:

[0032] (1) reacting and dialyzing a carbon dot precursor and an amine compound to obtain a carbon dot solution; pre-cooling the carbon dot solution at -70 to -200°C, and then drying at -30 to -60°C for 24-72h to obtain carbon dots after freeze-drying;

[0033] (2) dispersing 1-10mg of the carbon dots after freeze-drying in an alkaline solution with a concentration of 1-25wt%, heating to 75-90°C, and stirring until uniform; adding 10-60mg of an initiator, 80-160μL of methacrylic acid, and 0.7-3mL of styrene monomers at 75-90°C, and then condensing and refluxing in an inert gas to obtain carbon dot-polystyrene fluorescent composite microspheres.

[0034] A carbon dot-polystyrene fluorescent composite microsphere has an average particle size of 70-300nm and a carbon dot content of 1-45wt%.

[0035] Preferably, the carbon dot-polystyrene fluorescent composite microspheres have an emission wavelength of 525 nm and a fluorescence quantum efficiency of >45%.

[0036] Further preferably, the carbon dot-polystyrene fluorescent composite microspheres have a fluorescence quantum efficiency of 59.5%.

[0037] Preferably, the carbon dot-polystyrene fluorescent composite microspheres retain 50-80% of the fluorescence quantum efficiency of the carbon dots.

[0038] A rapid detection reagent is a carbon dot-polystyrene fluorescent composite microsphere having a surface coupled to a biomolecule.

[0039] Preferably, the rapid detection reagent is prepared by activating the surface functional groups of the carbon dot-polystyrene fluorescent composite microspheres with an activating agent and then coupling the biomolecule thereto.

[0040] Preferably, the activating agent comprises one or more of EDC and NHS.

[0041] Preferably, the biomolecule comprises one or more of an antibody, an antigen, and a nucleic acid.

[0042] Compared with the prior art, the present application has the following beneficial effects:

[0043] 1. The carbon dots after freeze-drying are added to the process of synthesizing polystyrene microspheres by a soap-free emulsion method, the carbon dots are wrapped in a polystyrene matrix, and carbon dot-polystyrene fluorescent composite microspheres with uniform particle size, stable fluorescence, and high fluorescence intensity are prepared.

[0044] 2. The carbon dots are wrapped in a polystyrene matrix by a soap-free emulsion polymerization method, eliminating the influence of hydrophilic surfactants that need to be added in other polymerization methods, simplifying the preparation process, reducing the influence of the external environment on the carbon dots, improving the stability and fluorescence performance of the fluorescent composite microspheres, and also enabling coupling with biomolecules for high-sensitivity biological analysis and detection.

[0045] 3. The content of the carbon dots in the carbon dot-polystyrene fluorescent microspheres is controlled by controlling the addition amount of ammonium persulfate and the carbon dots, so that the carbon dot-polystyrene fluorescent composite microspheres have good fluorescence performance.

[0046] 4. The carbon dots after freeze-drying are first dissolved in an alkaline solution, which is conducive to uniform dispersion of the carbon dots; the alkaline substances in the alkaline solution provide a certain ionic strength for subsequent reactions, effectively adjust the pH of the emulsion, and also buffer the decrease in pH value caused by the addition of methacrylic acid.

[0047] 5. This invention controls the particle size of carbon dot-polystyrene fluorescent composite microspheres by adjusting parameters such as the reflux reaction time during the preparation process. If the average particle size of the composite microspheres is too small, i.e., the matrix thickness is small, the amount of carbon dots encapsulated in the matrix will also be reduced, and the fluorescence quantum efficiency of the carbon dot-polystyrene fluorescent composite microspheres will also decrease. If the average particle size of the composite microspheres is too large, the excessively thick polystyrene matrix will reduce the light transmittance of the carbon dot-polystyrene fluorescent composite microspheres, affecting the luminescence intensity of the internal carbon dots and reducing the luminescence efficiency. Furthermore, if the particle size of the carbon dot-polystyrene fluorescent composite microspheres is too large, it means that the amount of encapsulated carbon dots is increased, and the carbon dots are prone to aggregation and fluorescence aggregation quenching phenomenon, which will reduce the fluorescence intensity of the carbon dot-polystyrene fluorescent composite microspheres.

[0048] 6. In the preparation process, no emulsifier is added. Instead, the initiator is decomposed at high temperature, which causes the styrene monomer and methacrylic acid molecules to undergo free radical polymerization. This makes the polystyrene polymer itself surface active, eliminating the need for subsequent steps to remove the emulsifier and simplifying the operation process.

[0049] 7. The carbon dots in the carbon dot-polystyrene fluorescent composite microspheres of the present invention have amino groups, and the fluorescence quantum efficiency of the carbon dots is as high as 85.7%, which makes the fluorescence quantum efficiency of the synthesized composite microspheres also high, and can be applied to quantitative detection by fluorescence chromatography. Attached Figure Description

[0050] Figure 1 Biological transmission electron microscopy image of carbon dot-polystyrene fluorescent composite microspheres GCD@PS in Example 1 of this invention.

[0051] Figure 2 The infrared absorption spectra (a) of carbon dot GCD and carbon dot-polystyrene fluorescent composite microspheres GCD@PS in Example 1 of the present invention, and the fluorescence absorption spectrum (b) of carbon dot-polystyrene fluorescent composite microspheres GCD@PS.

[0052] Figure 3 This is a biological transmission electron microscope image of the carbon dot-polystyrene fluorescent composite microspheres GCD@PS in Example 4. Detailed Implementation

[0053] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0054] Unless otherwise specified, the materials used in this invention are commercially available products, and the methods used are conventional technical means.

[0055] Example 1

[0056] Take 30 mg of rose Bengal (RB) dissolved in deionized water, stirring ultrasonic to completely dissolved. In 0.17 mL of ethylenediamine, the solution was mixed uniformly and transferred to the reaction kettle, and reacted in a vacuum drying oven at 160℃ for 4h. After the reaction was completed, the solution was transferred to a dialysis bag, dialyzed for 48h, filtered through a filter membrane, frozen into a solid at-80℃, and then sent into a freeze dryer, and freeze-dried at-50℃ for 48h to obtain a red carbon dot powder (average particle size 5.0nm).

[0057] Take 5mg of red carbon dot powder, dissolved in 15mL deionized water, add 0.03g of sodium bicarbonate, stir at 80℃ for 10min to make it stir uniformly; add 35mg of initiator ammonium persulfate, and 100μL of methacrylic acid, stir for 20min; add 1mL of styrene monomer, and blow nitrogen for 30min to remove oxygen in the flask; 80℃ condensation reflux reaction for 14h, the product was washed with deionized water to obtain carbon dot-poly styrene fluorescent composite microspheres (GCD@PS).

[0058] Figure 1 The average particle size of the carbon dot-poly styrene fluorescent composite microspheres GCD@PS in this embodiment is 117nm;

[0059] Figure 2 The infrared absorption spectrum (a) of carbon dots GCD and carbon dot-poly styrene fluorescent composite microspheres GCD@PS, and the fluorescence absorption spectrum (b) of carbon dot-poly styrene fluorescent composite microspheres GCD@PS, it can be seen that the optimal excitation wavelength of the composite microspheres is 480nm, and the emission wavelength is 525nm.

[0060] The fluorescence quantum efficiency of the carbon dot-poly styrene fluorescent composite microspheres GCD@PS in this embodiment is 59.5%.

[0061] Example 2

[0062] Take 0.5g of glutathione (GSH) dissolved in 20mL of formamide, stirring ultrasonic to completely dissolved, in a microwave oven at 700W, microwave reaction for 3min. The reaction solution was centrifuged (10000rpm, 5min) to remove the large particle precipitate, and the reaction solution was dialyzed in deionized water for 5 days. The reaction solution was frozen into a solid at-80℃, and then sent into a freeze dryer, and freeze-dried at-50℃ for 48h to obtain a dark green carbon dot powder (average particle size 3.8nm).

[0063] Take 5 mg of greenish carbon dot powder, dissolve in 15 mL of deionized water, add 0.03 g of sodium bicarbonate, stir at 80°C for 10 min to make it stir evenly; add 35 mg of initiator ammonium persulfate, and 100 μL of methacrylic acid, stir for 20 min; add 1 mL of styrene monomer, purged with nitrogen for 30 min to remove oxygen in the flask; 80°C condensation reflux reaction for 14h, the product is washed with deionized water, to obtain carbon dot-poly styrene fluorescent composite microspheres (RCD@PS).

[0064] The average particle size of the carbon dot-poly styrene fluorescent composite microspheres GCD@PS of this example is 123 nm, and the fluorescence quantum efficiency is 10.8%.

[0065] Example 3

[0066] Take 5 mg of greenish carbon dot powder, dissolve in 15 mL of deionized water, add 0.03 g of sodium bicarbonate, stir at 80°C for 10 min to make it stir evenly; add 35 mg of initiator ammonium persulfate, and 100 μL of methacrylic acid, stir for 20 min; add 1 mL of styrene monomer, purged with nitrogen for 30 min to remove oxygen in the flask; 80°C condensation reflux reaction for 14h, the product is washed with deionized water, to obtain carbon dot-poly styrene fluorescent composite microspheres (RCD@PS).

[0067] Take 5 mg of greenish carbon dot powder, dissolve in 15 mL of deionized water, add 0.03 g of sodium bicarbonate, stir at 80°C for 10 min to make it stir evenly; add 35 mg of initiator ammonium persulfate, and 100 μL of methacrylic acid, stir for 20 min; add 1 mL of styrene monomer, purged with nitrogen for 30 min to remove oxygen in the flask; 80°C condensation reflux reaction for 14h, the product is washed with deionized water, to obtain carbon dot-poly styrene fluorescent composite microspheres (RCD@PS).

[0068] The average particle size of the carbon dot-poly styrene fluorescent composite microspheres GCD@PS of this example is 123 nm, and the fluorescence quantum efficiency is 10.8%.

[0069] Example 4

[0070] Compared with Example 1, the difference is that 50 mg of red carbon dot powder is taken, dissolved in 15 mL of deionized water, 0.03 g of sodium bicarbonate is added, stirred at 80°C for 10 min to make it stir evenly; add 35 mg of initiator ammonium persulfate, and 100 μL of methacrylic acid, stir for 20 min; add 1 mL of styrene monomer, purged with nitrogen for 30 min to remove oxygen in the flask; 80°C condensation reflux reaction for 14h, the product is washed with deionized water, to obtain carbon dot-poly styrene fluorescent composite microspheres (GCD@PS).

[0071] Figure 3 The average particle size of the carbon dot-polystyrene fluorescent composite microspheres GCD@PS of this example is 188 nm.

[0072] The fluorescence quantum efficiency of the carbon dot-polystyrene fluorescent composite microspheres GCD@PS of this example is 45.8%.

[0073] Example 5

[0074] Compared with Example 1, the difference is that 5 mg of red carbon dot powder is dissolved in 15 mL of deionized water, 37.52 μL of ammonia water (25%) is added, and the mixture is stirred at 80°C for 10 min to make it uniform; 35 mg of initiator ammonium persulfate and 100 μL of methacrylic acid are added, and the mixture is stirred for 20 min; 1 mL of styrene monomer is added, and nitrogen gas is introduced for 30 min to remove oxygen in the three-necked flask; the mixture is condensed and refluxed at 80°C for 14 h, and the product is washed with deionized water to obtain carbon dot-polystyrene fluorescent composite microspheres (GCD@PS).

[0075] The average particle size of the carbon dot-polystyrene fluorescent composite microspheres of this example is 122 nm, and the fluorescence quantum efficiency is 45.6%.

[0076] Example 6

[0077] Compared with Example 1, the difference is that 5 mg of red carbon dot powder is dissolved in 15 mL of deionized water, 37.52 μL of ammonia water (25%) is added, and the mixture is stirred at 80°C for 10 min to make it uniform; 35 mg of initiator ammonium persulfate and 100 μL of methacrylic acid are added, and the mixture is stirred for 20 min; 1 mL of styrene monomer is added, and nitrogen gas is introduced for 30 min to remove oxygen in the three-necked flask; the mixture is condensed and refluxed at 80°C for 14 h, and the product is washed with deionized water to obtain carbon dot-polystyrene fluorescent composite microspheres (GCD@PS).

[0078] The average particle size of the carbon dot-polystyrene fluorescent composite microspheres of this example is 235 nm, and the fluorescence quantum efficiency is 48.7%.

[0079] Example 7

[0080] Compared with Example 1, the difference is that 5 mg of red carbon dot powder is dissolved in 15 mL of deionized water, 37.52 μL of ammonia water (25%) is added, and the mixture is stirred at 80°C for 10 min to make it uniform; 35 mg of initiator ammonium persulfate and 100 μL of methacrylic acid are added, and the mixture is stirred for 20 min; 1 mL of styrene monomer is added, and nitrogen gas is introduced for 30 min to remove oxygen in the three-necked flask; the mixture is condensed and refluxed at 80°C for 14 h, and the product is washed with deionized water to obtain carbon dot-polystyrene fluorescent composite microspheres (GCD@PS).

[0081] The carbon dot-polystyrene fluorescent composite microspheres of the embodiment have an average particle size of 86 nm and a fluorescence quantum efficiency of 58.2%.

[0082] In the embodiment, when the initiator is slightly excessive, the gradient distribution is widened, the microsphere particle size is reduced, the shell thickness is reduced, the light transmittance is relatively increased, the fluorescence quantum efficiency is relatively increased when the carbon dot amount is constant, the matrix thickness is reduced, and the amount of carbon dots coated into the matrix is also reduced, so the quantum efficiency is reduced, and the overall fluorescence quantum efficiency of the carbon dot-polystyrene fluorescent composite microspheres changes little.

[0083] Example 8

[0084] Compared with Example 1, the difference lies in that the condensation reflux reaction is performed at 80℃ for 24h.

[0085] The carbon dot-polystyrene fluorescent composite microspheres of the embodiment have an average particle size of 241 nm and a fluorescence quantum efficiency of 46.7%.

[0086] Comparative Example 1

[0087] Nitrogen is introduced for 30 min to remove oxygen in the three-necked flask, 100 μL of methacrylic acid, 1 mL of styrene monomer, 0.03 g of sodium bicarbonate, and 15 mL of deionized water are added into the three-necked flask, stirring is performed at 80℃ for 10 min, 35 mg of initiator ammonium persulfate is added, after mixing, the condensation reflux reaction is performed at 80℃ for 10h, 5 mL of a mixed solution of 5 mg of red carbon dot powder and 35 mg of initiator ammonium persulfate in Example 1 is further added, the condensation reflux reaction is performed at 80℃ for 4h, and the product is washed once with deionized water to obtain carbon dot-polystyrene fluorescent composite microspheres (GCD@PS).

[0088] The carbon dot-polystyrene fluorescent composite microspheres of the comparative example have an average particle size of 107 nm and a fluorescence quantum efficiency of 52.2%.

[0089] The carbon dot-polystyrene fluorescent composite microspheres of the application are activated after using the method of EDC and NHS, and the labeled antibody of cardiac troponin (CTnI) is coupled, a sandwich method is used in lateral flow immunochromatography to quantitatively detect CTnI, and good effects are obtained.

[0090] In summary, the carbon dots are wrapped in the polystyrene matrix layer by layer by the method of the application, the preparation process is simplified, the influence of the external environment on the carbon dots is reduced, the stability and fluorescence performance of the fluorescent composite microspheres are improved, the carbon dot-polystyrene fluorescent composite microspheres can be coupled with biomolecules on the surface, a rapid detection reagent is obtained, and the rapid detection reagent is used for high-sensitivity biological analysis and detection.

[0091] The specific embodiments described herein are merely illustrative of the spirit of the application. Various modifications or changes in the specific embodiments described herein can occur to those skilled in the art to which the application pertains without departing from the spirit of the application, and it is understood that such modifications or changes are to be considered as within the scope of the application as defined by the appended claims.

Claims

1. A method for preparing carbon dot-polystyrene fluorescent composite microspheres, characterized in that, The preparation method comprises the following steps: dispersing the freeze-dried carbon dots in an alkaline solution, uniformly heating and stirring, adding an initiator, methyl methacrylate and styrene monomers at 75-90 DEG C, mixing, condensing and refluxing in an inert gas, and obtaining carbon dot-polystyrene fluorescent composite microspheres. The mass-volume ratio of the freeze-dried carbon dots, methyl methacrylate and styrene monomers is (1-10) mg:(0.01-1) mL:1 mL; and the mass ratio of the freeze-dried carbon dots to the initiator is 1:(1-20). The alkaline solution is composed of an alkaline salt and a solvent, and the alkaline salt comprises one or more of sodium bicarbonate, sodium carbonate and potassium carbonate. The freeze-dried carbon dots are prepared from a carbon dot solution after freeze-drying; the carbon dot solution is prepared from one or more of rose Bengal (RB), glutathione (GSH) and citric acid (CA) after reaction and dialysis; and the fatty amine, aromatic amine and amide compound comprises one or more of ethylenediamine, butanediamine, propylenediamine, o-phenylenediamine and formamide.

2. The method for preparing carbon dot-polystyrene fluorescent composite microspheres according to claim 1, characterized in that, The carbon dot content in the carbon dot-polystyrene fluorescent composite microspheres is 1-45 wt%.

3. The method for preparing carbon dot-polystyrene fluorescent composite microspheres according to claim 1, characterized in that, The condensing and refluxing temperature is 75-90 DEG C, and the time is 1-24 h.

4. The method for preparing carbon dot-polystyrene fluorescent composite microspheres according to claim 1, characterized in that, The initiator is ammonium persulfate.

5. The method for preparing carbon dot-polystyrene fluorescent composite microspheres according to claim 1, characterized in that, The concentration of the alkaline solution is 1-25 wt%.

6. The method for preparing carbon dot-polystyrene fluorescent composite microspheres according to claim 1, characterized in that, The freeze-dried carbon dots are freeze-dried carbon dots with amino groups.

7. The carbon dot-polystyrene fluorescent composite microspheres of claim 1, wherein, The average particle size of the carbon dot-polystyrene fluorescent composite microspheres is 70-300 nm, and the carbon dot content is 1-45 wt%. 8.The carbon dot-polystyrene fluorescent composite microsphere of claim 7, wherein, The emission wavelength of the carbon dot-polystyrene fluorescent composite microspheres is 525 nm, and the fluorescence quantum efficiency is >45%.

9. A rapid detection reagent, characterized by, The rapid detection reagent is prepared by coupling a biological molecule to the carbon dot-polystyrene fluorescent composite microspheres prepared by the preparation method of any one of claims 1-6 and / or the carbon dot-polystyrene fluorescent composite microspheres of any one of claims 7-8 after activating the surface functional groups of the carbon dot-polystyrene fluorescent composite microspheres with an activator.

10. The rapid detection reagent according to claim 9, characterized in that, The carbon dot-polystyrene fluorescent composite microspheres are coupled with a biological molecule on the surface.

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