Preparation method of carbon dot-polystyrene fluorescent composite microspheres

Abstract

The invention belongs to the technical field of fluorescent composite microspheres, and relates to a preparation method of carbon dot-polystyrene fluorescent composite microspheres. The invention discloses a preparation method of carbon dot-polystyrene fluorescent composite microspheres, which comprises the following steps: dispersing freeze-dried carbon dots in an alkaline solution, heating and stirring uniformly; and adding an initiator, methacrylic acid and a styrene monomer, mixing, and carrying out condensation reflux reaction in inert gas to obtain the carbon dot-polystyrene fluorescent composite microspheres. The invention also discloses a rapid detection reagent, which is the carbon dot-polystyrene fluorescent composite microspheres with biomolecules coupled on the surfaces. The carbon dots are wrapped in a polystyrene matrix through a soap-free emulsion polymerization method, 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, and meanwhile the fluorescent composite microspheres can be coupled with biomolecules and used for high-sensitivity biological analysis and detection.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent composite microspheres and relates to a method for preparing carbon dot-polystyrene fluorescent composite microspheres. Background Art

[0002] Carbon dots (CDs) are a type of zero-dimensional carbon nanomaterial with a particle size of less than 10 nm. The material was first reported in 2004. As a new type of functional nanomaterial, nano fluorescent carbon dots have unique physical and chemical properties, including small size, excellent biocompatibility, high quantum yield (QY), fluorescence tunability, and easy surface modification. They have become promising nanomaterials for biomedical applications and have attracted more and more attention from scientists. At present, carbon quantum dots have been widely used in bioimaging, biosensing, drug delivery, biocatalysis, and tissue engineering. However, when carbon dots are directly applied to immunoassays, they encounter many limitations. In the process of labeling antibodies, the ultra-small size of carbon dots will cause multiple carbon dot particles to be coupled to a single antibody at the same time, which will lead to fluorescence aggregation quenching; and the overall luminescence intensity of a single carbon dot particle is weak, which also affects the sensitivity of analytical detection. Therefore, how to prepare carbon dots into larger fluorescent microspheres by combining them with suitable nanocarrier materials is the key to expanding their application.

[0003] Common fluorescent carriers include silica, polystyrene, etc. Silica has become a commonly used fluorescent carrier due to its advantages such as non-toxicity, good biocompatibility, and ability to effectively improve material stability. However, its complex synthesis method, the silica shell affecting the active sites of the reactants, the silica shell being easily etched under harsh reaction conditions (high-temperature water vapor, alkaline solution), and easy cross-linking make it low in scalability as a carrier matrix. Polystyrene is the most common fluorescent carrier due to its advantages such as large output, wide application, large specific surface area, wide particle size distribution, and easy surface modification. At present, composite materials are mostly prepared by modifying carbon dots (fluorescent materials) on the surface of polystyrene microspheres, but it is easy to cause carbon dots to leak, and the fluorescent properties of microspheres are easily affected by the environment. Summary of the invention

[0004] The purpose of the present invention is to address the above-mentioned problems existing in the prior art and to propose a method for preparing carbon dot-polystyrene fluorescent composite microspheres. Freeze-dried carbon dots are added to the process of synthesizing polystyrene microspheres by a soap-free emulsion method, and the carbon dots are wrapped in a polystyrene matrix. The prepared carbon dot-polystyrene fluorescent composite microspheres have good fluorescent properties.

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

[0006] A method for preparing carbon dot-polystyrene fluorescent composite microspheres, comprising:

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

[0008] Preferably, the carbon dot content in the carbon dot-polystyrene fluorescent composite microspheres is 1 to 45 wt %.

[0009] Preferably, the mass ratio of the freeze-dried carbon dots to the initiator is 1:(1-20).

[0010] More preferably, the mass ratio of the freeze-dried carbon dots to the initiator is 1:(5-9).

[0011] Preferably, the mass volume ratio of the freeze-dried carbon dots, methacrylic acid, and styrene monomer is (1-10) mg: (0.01-1) mL: 1 mL.

[0012] Preferably, the heating and stirring temperature is 75 to 90° C., and the time is 1 to 60 min.

[0013] Preferably, the condensation reflux temperature is 75 to 90° C., and the time is 1 to 24 hours.

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

[0015] Preferably, the initiator is ammonium persulfate.

[0016] Preferably, the alkaline solution consists 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] Preferably, the concentration of the alkaline solution is 1-25 wt %.

[0018] Preferably, the alkaline solution is aqueous ammonia.

[0019] Preferably, the freeze-dried carbon dots are freeze-dried carbon dots with amino groups.

[0020] Preferably, the freeze-dried carbon dots are obtained by freeze-drying a carbon dot solution.

[0021] More preferably, the freeze-drying comprises precooling at -70 to -200°C and then drying at -30 to -60°C for 24 to 72 hours.

[0022] More preferably, the carbon dot solution is prepared by reacting a carbon dot precursor with aliphatic amines, aromatic amines, or amide compounds and dialyzing them.

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

[0024] More preferably, the aliphatic amines, aromatic amines and amide compounds include one or more of ethylenediamine, butanediamine, propylenediamine, o-phenylenediamine and formamide.

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

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

[0027] Preferably, the emission wavelength of the freeze-dried carbon dots is 525 nm, and the quantum efficiency is 85.7%.

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

[0029] (1) A carbon dot precursor and an amine compound are reacted and dialyzed to obtain a carbon dot solution; the carbon dot solution is freeze-dried to obtain freeze-dried carbon dots;

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

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

[0032] (1) reacting a carbon dot precursor with an amine compound and dialyzing to obtain a carbon dot solution; precooling the carbon dot solution at -70 to -200° C., and drying it at -30 to -60° C. for 24 to 72 hours to obtain freeze-dried carbon dots;

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

[0034] A carbon dot-polystyrene fluorescent composite microsphere, the average particle size of which is 70-300nm and the carbon dot content is 1-45wt%.

[0035] Preferably, the emission wavelength of the carbon dot-polystyrene fluorescent composite microspheres is 525 nm, and the fluorescent quantum efficiency is greater than 45%.

[0036] More preferably, the fluorescence quantum efficiency of the carbon dot-polystyrene fluorescent composite microspheres reaches 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 with biological molecules coupled to the surface.

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

[0040] Preferably, the activator includes one or more of EDC and NHS.

[0041] Preferably, the biomolecules include one or more of antibodies, antigens, and nucleic acids.

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

[0043] 1. The present invention adds freeze-dried carbon dots into the process of synthesizing polystyrene microspheres by soap-free emulsion method, wraps the carbon dots in a polystyrene matrix, and prepares carbon dot-polystyrene fluorescent composite microspheres with uniform particle size, stable fluorescence and high fluorescence intensity.

[0044] 2. The present invention encapsulates carbon dots in a polystyrene matrix through 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, and improving the stability and fluorescence performance of the fluorescent composite microspheres. At the same time, it can also be coupled with biological molecules for high-sensitivity biological analysis and detection.

[0045] 3. The present invention controls the amount of ammonium persulfate and carbon dots added to adjust the content of carbon dots in the carbon dot-polystyrene fluorescent microspheres, so that the carbon dot-polystyrene fluorescent composite microspheres have better fluorescence properties.

[0046] 4. The present invention dissolves the freeze-dried carbon dots in an alkaline solution first, which is beneficial to the uniform dispersion of the carbon dots; the alkaline substance in the alkaline solution provides a certain ionic strength for the subsequent reaction, effectively adjusts the pH of the emulsion, and can also buffer the decrease in pH value caused by the addition of methacrylic acid.

[0047] 5. The present invention controls the particle size of the carbon dot-polystyrene fluorescent composite microspheres by adjusting parameters such as the time of the condensation reflux reaction during the preparation process; if the average particle size of the composite microspheres is too small, that is, the matrix thickness is small, the amount of carbon dots coated in the matrix will also be reduced, and the fluorescence quantum efficiency of the carbon dot-polystyrene fluorescent composite microspheres will also be reduced; 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, affect the luminous intensity of the internal carbon dots, and reduce the luminous efficiency; and if the particle size of the carbon dot-polystyrene fluorescent composite microspheres is too large, it means that the amount of coated carbon dots increases, the carbon dots are prone to aggregation and fluorescence aggregation quenching occurs, which in turn reduces the fluorescence intensity of the carbon dot-polystyrene fluorescent composite microspheres.

[0048] 6. The present invention does not add an emulsifier during the preparation process. Instead, the initiator is decomposed at high temperature to allow free radical polymerization of styrene monomers and methacrylic acid molecules, so that the polystyrene polymer itself has surface activity, eliminating the subsequent step of removing 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%, so that the fluorescence quantum efficiency of the synthesized composite microspheres is also relatively high, and can be applied to fluorescence chromatography quantitative detection. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0052] Figure 3 This is the biological transmission electron microscopy image of the carbon dot-polystyrene fluorescent composite microspheres GCD@PS in Example 4. DETAILED DESCRIPTION

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

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

[0055] Example 1

[0056] Take 30 mg of Bengal Rose Red (RB) and dissolve it in deionized water, stir and ultrasonic until it is completely dissolved. Add 0.17 mL of ethylenediamine, mix the solution evenly and transfer it to a reactor, and react in a vacuum drying oven at 160°C for 4 hours. After the reaction is completed, transfer the solution to a dialysis bag, dialyze for 48 hours, filter through a filter membrane, freeze it into a solid at -80°C, and then send it to a freeze dryer, freeze-dry it at -50°C for 48 hours to obtain red carbon point powder (average particle size of 5.0 nm).

[0057] Take 5 mg of red carbon dot powder, dissolve it in 15 mL of deionized water, add 0.03 g of sodium bicarbonate, and stir at 80 ° C for 10 min to make it evenly stirred; add 35 mg of initiator ammonium persulfate and 100 μL of methacrylic acid, stir for 20 min; add 1 mL of styrene monomer, and pass nitrogen for 30 min to remove oxygen in the three-necked flask; condense and reflux at 80 ° C for 14 h, wash the product with deionized water to obtain carbon dot-polystyrene fluorescent composite microspheres (GCD@PS).

[0058] Figure 1 This is a biological transmission electron microscopy image of the carbon dot-polystyrene fluorescent composite microspheres GCD@PS in this embodiment, with an average particle size of 117nm;

[0059] Figure 2 The infrared absorption spectra of carbon dots GCD and carbon dots-polystyrene fluorescent composite microspheres GCD@PS (a), and the fluorescence absorption spectrum of carbon dots-polystyrene fluorescent composite microspheres GCD@PS (b). 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-polystyrene fluorescent composite microspheres GCD@PS in this embodiment is 59.5%.

[0061] Example 2

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

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

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

[0065] Example 3

[0066] Weigh 0.21g of citric acid (CA) and 0.216g of o-phenylenediamine (o-PDA) and dissolve them in 10mL of water. Stir at 60°C for 10min to completely dissolve them. Transfer the solution to a high-pressure reactor and react in a vacuum drying oven at 160°C for 4h. After the reaction, centrifuge the crude product at 10000rpm for 10min to remove the crude product. Filter the reaction solution and dialyze it with deionized water for 12h. Freeze the reaction solution into a solid at -80°C and send it to a freeze dryer. Freeze-dry at -50°C for 48h to obtain a light yellow carbon dot powder (average particle size of 3.0nm).

[0067] Take 5 mg of light yellow carbon dot powder, dissolve it in 15 mL of deionized water, add 0.03 g of sodium bicarbonate, and stir at 80 ° C for 10 min to make it evenly stirred; add 35 mg of initiator ammonium persulfate and 100 μL of methacrylic acid, stir for 20 min; add 1 mL of styrene monomer, and pass nitrogen for 30 min to remove oxygen in the three-necked flask; condense and reflux at 80 ° C for 14 h, wash the product with deionized water to obtain carbon dot-polystyrene fluorescent composite microspheres (BCD@PS).

[0068] The average particle size of the carbon dot-polystyrene fluorescent composite microspheres GCD@PS in this embodiment is 116 nm, and the fluorescence quantum efficiency is 30.1%.

[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, and 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 stirred for 20 min; 1 mL of styrene monomer is added, and nitrogen is passed through for 30 min to remove oxygen in the three-necked flask; the reaction 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).

[0071] Figure 3 This is a biological transmission electron microscopy image of the carbon dot-polystyrene fluorescent composite microspheres GCD@PS in this embodiment, with an average particle size of 188 nm.

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

[0073] Example 5

[0074] Compared with Example 1, the difference is that 5 mg of red carbon dot powder is taken, dissolved in 15 mL of deionized water, 37.52 μL of ammonia water (25%) is added, and 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 stirred for 20 min; 1 mL of styrene monomer is added, and nitrogen is passed through for 30 min to remove oxygen in the three-necked flask; the reaction 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 in this embodiment is 122 nm, and the fluorescent 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 taken, dissolved in 15 mL of deionized water, 0.03 g of sodium bicarbonate is added, and stirred at 80°C for 10 min to make it uniform; 20 mg of initiator ammonium persulfate and 100 μL of methacrylic acid are added, and stirred for 20 min; 1 mL of styrene monomer is added, and nitrogen is passed through for 30 min to remove oxygen in the three-necked flask; the reaction 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 in this embodiment is 235 nm, and the fluorescent 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 taken, dissolved in 15 mL of deionized water, 0.03 g of sodium bicarbonate is added, and stirred at 80°C for 10 min to make it uniform; 100 mg of initiator ammonium persulfate and 100 μL of methacrylic acid are added, and stirred for 20 min; 1 mL of styrene monomer is added, and nitrogen is passed through for 30 min to remove oxygen in the three-necked flask; the reaction is condensed and refluxed at 80°C for 14 h, and the product is washed once with deionized water to obtain carbon dot-polystyrene fluorescent composite microspheres (GCD@PS).

[0081] The average particle size of the carbon dot-polystyrene fluorescent composite microspheres in this embodiment is 86 nm, and the fluorescent quantum efficiency is 58.2%.

[0082] In this embodiment, when the initiator is slightly excessive, the gradient distribution becomes wider, the particle size of the microspheres decreases, the shell thickness decreases, the transmittance will increase relatively, and when the amount of carbon dots is constant, the fluorescence quantum efficiency will increase relatively; however, due to the decrease in matrix thickness, the amount of carbon dots encapsulated in the matrix will also decrease, so the quantum efficiency will decrease; therefore, the overall fluorescence quantum efficiency of the carbon dot-polystyrene fluorescent composite microspheres does not change much.

[0083] Example 8

[0084] Compared with Example 1, the difference is that the reaction is carried out under condensation reflux at 80° C. for 24 hours.

[0085] The average particle size of the carbon dot-polystyrene fluorescent composite microspheres in this embodiment is 241 nm, and the fluorescent quantum efficiency is 46.7%.

[0086] Comparative Example 1

[0087] Nitrogen was 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 were added to the three-necked flask, and the mixture was stirred at 80° C. for 10 min. 35 mg of initiator ammonium persulfate was added and mixed, and the mixture was condensed and refluxed at 80° C. for 10 h. 5 mL of a mixed solution of 5 mg of red carbon dot powder and 35 mg of initiator ammonium persulfate in Example 1 was added, and the mixture was condensed and refluxed at 80° C. for 4 h. The product was washed once with deionized water to obtain carbon dot-polystyrene fluorescent composite microspheres (GCD@PS).

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

[0089] The carbon dot-polystyrene fluorescent composite microspheres of the present invention are activated by EDC and NHS and coupled with a labeled antibody of cardiac troponin (CTnI). The sandwich method is used in lateral flow immunochromatography to quantitatively detect CTnI, which has a good effect.

[0090] In summary, the present invention wraps carbon dots layer by layer in a polystyrene matrix through a soap-free emulsion polymerization method, which simplifies the preparation process, reduces the influence of the external environment on the carbon dots, and improves the stability and fluorescence performance of the fluorescent composite microspheres; at the same time, the surface of the carbon dot-polystyrene fluorescent composite microspheres can be coupled with biological molecules to obtain rapid detection reagents for high-sensitivity biological analysis and detection.

[0091] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A method for preparing carbon dot-polystyrene fluorescent composite microspheres, characterized in that: The preparation method comprises: dispersing freeze-dried carbon dots in an alkaline solution, heating and stirring evenly; adding an initiator, methacrylic acid and styrene monomer at 75-90°C, mixing, and condensing and refluxing in an inert gas to obtain carbon dot-polystyrene fluorescent composite microspheres.

2. The method for preparing carbon dot-polystyrene fluorescent composite microspheres according to claim 1, characterized in that: The mass ratio of the freeze-dried carbon dots to the initiator is 1:(1-20).

3. The method for preparing carbon dot-polystyrene fluorescent composite microspheres according to claim 1, characterized in that: The mass volume ratio of the freeze-dried carbon dots, methacrylic acid and styrene monomer is (1-10) mg: (0.01-1) mL: 1 mL.

4. The method for preparing carbon dot-polystyrene fluorescent composite microspheres according to claim 1, characterized in that: The condensation reflux temperature is 75 to 90° C., and the time is 1 to 24 hours.

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

6. The method for preparing carbon dot-polystyrene fluorescent composite microspheres according to claim 1, characterized in that: 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.

7. The carbon dot-polystyrene fluorescent composite microsphere as claimed in claim 1, characterized in that: 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. A carbon dot-polystyrene fluorescent composite microsphere as claimed in claim 7, characterized in that: The emission wavelength of the carbon dot-polystyrene fluorescent composite microspheres is 525 nm, and the fluorescence quantum efficiency is greater than 45%.

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

10. The rapid detection reagent according to claim 9, characterized in that It is a carbon dot-polystyrene fluorescent composite microsphere with biological molecules coupled on the surface.

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