CN111117606B - Carbon quantum dot fluorescent microsphere standard substance and preparation method thereof - Google Patents
Carbon quantum dot fluorescent microsphere standard substance and preparation method thereof Download PDFInfo
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Abstract
The invention provides a carbon quantum dot fluorescent microsphere standard substance which comprises aminated polystyrene microspheres and fluorescent substances loaded on the surfaces of the aminated polystyrene microspheres, wherein the fluorescent substances are carboxylated carbon quantum dots. The invention also provides a preparation method of the carbon quantum dot fluorescent microsphere standard substance, which comprises the following steps: (1) preparing polystyrene microspheres; (2) preparing a nitro polystyrene microsphere; (3) preparing aminated polystyrene microspheres; (4) preparing carboxylated graphene quantum dots; (5) and (3) preparing a carbon quantum dot fluorescent microsphere standard substance. The carbon quantum dot fluorescent microsphere standard substance prepared by the invention has the characteristics of difficult falling of fluorescence, short reaction time, long fluorescence activity and the like; the particle size is uniform, the monodisperse property is strict, and the stability is excellent; the method is simple, is operated at room temperature, has small environmental influence factors and low cost, and has important significance for researching fluorescent standard substances.
Description
Technical Field
The invention relates to the technical field of standard substances, in particular to a carbon quantum dot fluorescent microsphere standard substance and a preparation method thereof.
Background
The method comprises the following steps of flow cytometry linear calibration, optical path alignment, sensitivity and stability detection, laboratory quality control, detection method evaluation and the like. The Flow Cytometry (FCM) is a technique for performing rapid qualitative and quantitative analysis or sorting on single-row cells or biological particles in a rapid linear flow state one by one with multiple parameters, has the characteristics of high detection speed, multiple measurement parameters, large data acquisition amount, comprehensive analysis, high sorting purity, flexible method and the like, and has become a popular subject in recent scientific research. The technology integrates a current technology, fluorescent microspheres, laser, digital signal processing and a traditional chemical technology, and is widely applied to a plurality of fields such as metabolic process analysis, medicine invention, high-throughput analysis, disease diagnosis and the like. The realization of the technology usually requires an absolute counting fluorescent microsphere as a standard substance, and many absolute counting products of flow cytometry use microsphere with fluorescein, and the particle size range of the microsphere is required to be (1-10) mu m.
Quantum dots are a special nanomaterial, consisting of a small number of atoms. The size of three dimensions of the quantum dot is generally between 2 nm and 20nm, and the movement of electrons in the quantum dot in all directions is limited due to small particle size, so that the quantum confinement effect is particularly remarkable. The quantum dots as fluorescent materials mainly have the following characteristics: 1) the quantum dots have high fluorescence intensity and good light stability. The fluorescence intensity of the quantum dots is almost 20 times that of the organic fluorescent material rhodamine 6G, and the stability of the quantum dots is more than 100 times that of the organic fluorescent material rhodamine 6G. Therefore, the fluorescent microsphere modified by the quantum dots can be observed for a long time, and more space is provided for the use of a fluorescent microsphere standard substance; 2) quantum dots have a large stokes shift. Therefore, the fluorescent microsphere modified by the quantum dots can avoid the overlapping of an emission spectrum and an excitation spectrum, and is beneficial to improving the sensitivity of a fluorescent signal. 3) Good biocompatibility and environmental compatibility. The quantum dots can be subjected to specific connection after various chemical modifications, and have low cytotoxicity and good environmental compatibility. 4) The fluorescence lifetime is long. The fluorescence lifetime of organic fluorescent dyes is typically only a few nanoseconds (which is comparable to the time for autofluorescence of many biological samples to decay). The fluorescence lifetime of the quantum dots can last for tens of nanoseconds (20 ns-50 ns), so that most of autofluorescence is already decayed after light excitation, and the fluorescence of the quantum dots still exists, and then a fluorescence signal without background interference can be obtained. Therefore, the fluorescent microsphere modified by the quantum dots has the characteristics of high fluorescence stability, long fluorescence life, good biological and environmental compatibility and the like, and is an ideal fluorescent microsphere standard substance. Meanwhile, the quantum dot microsphere is taken as one of fluorescent microspheres, and is assembled with nano-scale or even micron-scale microspheres with functional groups on the surface, so that the quantum dot is protected, a quantum signal is amplified, and the problems of insufficient signal intensity, poor detection repeatability and the like of a single quantum dot in the detection process can be fully solved, therefore, the quantum dot microsphere has wide application in the fields of biological marking, detection, environmental tracing, immune medicine and the like. Compared with the traditional fluorescent material and other semiconductor quantum dots, the carbon quantum dot has the advantages of simple preparation process, low toxicity, good biocompatibility, environmental friendliness, easiness in surface functionalization and the like, but few reports about the standard substance of the carbon quantum dot fluorescent microsphere are provided at present.
Disclosure of Invention
In view of the above technical problems, an object of the present invention is to provide a carbon quantum dot fluorescent microsphere standard substance and a method for preparing the same, in which polystyrene microspheres and carbon quantum dots are bonded by chemical bonds, thereby improving the stability of the carbon quantum dot fluorescent microsphere standard substance.
The technical scheme adopted by the invention is as follows:
a carbon quantum dot fluorescent microsphere standard substance is composed of aminated polystyrene microspheres and fluorescent substances loaded on the surfaces of the aminated polystyrene microspheres, wherein the fluorescent substances are carboxylated carbon quantum dots.
The carbon quantum dot fluorescent microsphere standard substance provided by the invention is characterized in that the particle size of the aminated polystyrene microsphere is 2.2-5.2 mu m.
The carbon quantum dot fluorescent microsphere standard substance is characterized in that the carboxylated carbon quantum dots are carboxylated graphene quantum dots.
The carbon quantum dot fluorescent microsphere standard substance provided by the invention is characterized in that the wavelength range of the luminescence of the carboxylated graphene quantum dot is 300-800 nm.
The preparation method of the carbon quantum dot fluorescent microsphere standard substance comprises the following steps:
(1) preparing polystyrene microspheres: preparing polystyrene microspheres by using styrene as a monomer, azodiisobutyronitrile as an initiator, polyvinylpyrrolidone as a dispersing agent and a mixed solution of ethanol and deionized water as a dispersing medium through a dispersion polymerization method;
(2) preparation of the nitro polystyrene microsphere: carrying out nitration reaction on the polystyrene microspheres in the step (1) in concentrated sulfuric acid and concentrated nitric acid solution to prepare nitrated polystyrene microspheres;
(3) preparation of aminated polystyrene microspheres: with Na2S2O4Reducing the nitrated polystyrene microspheres in the step (2) to obtain aminated polystyrene microspheres as a reducing agent;
(4) preparing carboxylated graphene quantum dots: preparing graphene oxide by a Hummers method, and then modifying the graphene oxide by chloroacetic acid to obtain a carboxylated graphene quantum dot;
(5) preparing a carbon quantum dot fluorescent microsphere standard substance: mixing the dispersion liquid of the carboxylated graphene quantum dots in the step (4) with the aminated polystyrene microspheres in the step (3) for reaction; the product is obtained through the chemical condensation reaction of amino and carboxyl.
The preparation method of the carbon quantum dot fluorescent microsphere standard substance comprises the following steps of (1) mixing ethanol, deionized water, a dispersing agent, an initiator and a monomer in a ratio: (90-100) mL: (5-10) mL: (1.0-2.5) g: (0.2-0.8) g: (15-25) mL; in the step (2), the volume ratio of the concentrated sulfuric acid to the concentrated nitric acid is (0.01-100): 1.
The preparation method of the carbon quantum dot fluorescent microsphere standard substance comprises the following steps of (1) preparing a carboxylated graphene quantum dot and an aminated polystyrene microsphere in a mass ratio of 1: 10; the volume ratio of the concentrated sulfuric acid to the concentrated nitric acid in the step (2) is 3: 2.
The preparation method of the carbon quantum dot fluorescent microsphere standard substance comprises the step (5), wherein the solvent in the dispersion liquid is 400mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 100mM N-N-hydroxysuccinimide (NHS) 2- (N-morpholine) ethanesulfonic acid buffer (MES buffer, pH 5.2).
The preparation method of the carbon quantum dot fluorescent microsphere standard substance comprises the following steps:
(1) preparing polystyrene microspheres: adding ethanol and deionized water into a three-neck flask with a condenser tube, adding polyvinylpyrrolidone, stirring, and introducing nitrogen to completely dissolve the polyvinylpyrrolidone; then adding styrene dissolved with azodiisobutyronitrile, and electrically stirring and reacting for 8-16 h in a water bath kettle at 65-75 ℃ to obtain polystyrene emulsion; washing off polyvinylpyrrolidone to obtain polystyrene microspheres;
(2) preparation of the nitro polystyrene microsphere: adding the polystyrene microspheres in the step (1) into a flask, adding deionized water, stirring at a high speed in a water bath at 45-52 ℃, adding a mixed acid of sulfuric acid and nitric acid in a ratio of 3:2, and reacting for 1-4 hours; adding deionized water after the reaction is finished, centrifugally filtering, washing with deionized water until the pH value is neutral, vacuum drying to obtain light yellow nitro polystyrene microspheres, and storing in a refrigerator;
(3) preparation of aminated polystyrene microspheres: putting the nitrosation polystyrene microspheres in the step (2) into a flask, adding 50-150 mL of 2mol/L NaOH solution, and then adding 2-6 g of reducing agent Na2S2O4Stirring at high speed in a water bath at 70-75 ℃ for reacting for 4 h; centrifuging and collecting the solution after reaction, washing with deionized water for 3-5 times, washing with 95% ethanol for 2-4 times, vacuum drying to obtain aminated polystyrene microspheres, and storing in a refrigerator;
(4) preparing carboxylated graphene quantum dots: preparing graphene oxide by a Hummers method, and then modifying the graphene oxide by chloroacetic acid to obtain a carboxylated graphene quantum dot;
(5) preparing a carbon quantum dot fluorescent microsphere standard substance: and (3) adding 100-200 mg of the aminated polystyrene microsphere in the step (3) into the dispersion liquid of the carboxylated graphene quantum dot in the step (4), stirring at room temperature for reaction, carrying out chemical condensation reaction through amino and carboxyl, centrifuging after the reaction is completed, and washing with deionized water to obtain the carbon quantum dot fluorescent microsphere standard substance.
The preparation method of the carbon quantum dot fluorescent microsphere standard substance comprises the following steps:
(1) preparing polystyrene microspheres: adding 95mL of ethanol and 5mL of deionized water into a three-neck flask with a condenser tube, adding 1.5-2.0g of polyvinylpyrrolidone, stirring, and introducing nitrogen to completely dissolve the polyvinylpyrrolidone; then adding 15-25mL of styrene dissolved with azobisisobutyronitrile, and electrically stirring and reacting in a water bath kettle at 70 ℃ for 8-16 h to obtain polystyrene emulsion; settling the prepared polystyrene emulsion for one day, removing supernatant, continuously adding absolute ethyl alcohol, performing ultrasonic dispersion, repeating the step for 2-4 times, and washing off attached polyvinylpyrrolidone; obtaining polystyrene microspheres; the dosage ratio of the azodiisobutyronitrile to the styrene is 0.02g to 1 mL;
(2) preparation of the nitro polystyrene microsphere: adding 1.0-1.2 g of the polystyrene microspheres in the step (1) into a flask, adding 50mL of deionized water, stirring at a high speed in a water bath at 50 ℃, adding 20mL of concentrated nitric acid and 30mL of concentrated sulfuric acid, and reacting for 3 hours; adding deionized water after the reaction is finished, centrifugally filtering, washing with deionized water until the pH value is neutral, vacuum drying to obtain light yellow nitro polystyrene microspheres, and storing in a refrigerator at 4 ℃;
(3) preparation of aminated polystyrene microspheres: putting 1g of the nitrated polystyrene microspheres in the step (2) into a flask, adding 100mL of 2mol/L NaOH solution, and then adding 5g of reducing agent Na2S2O4Stirring at high speed in a water bath at 70-75 ℃ for reacting for 4 h; then centrifugally collecting the reacted solution, washing with deionized water for 3-5 times, washing with 95% ethanol for 3 times, vacuum drying to obtain aminated polystyrene microspheres, and storing in a refrigerator at 4 ℃;
(4) preparing carboxylated graphene quantum dots: preparing graphene oxide by a Hummers method, and then modifying the graphene oxide by chloroacetic acid to obtain a carboxylated graphene quantum dot;
(5) preparing a carbon quantum dot fluorescent microsphere standard substance: and (4) adding 200mg of aminated polystyrene microspheres obtained in the step (3) into 200mL of carboxylated graphene quantum dot dispersion liquid obtained in the step (4), stirring at room temperature for reaction for 4-24 h, carrying out chemical condensation reaction through amino and carboxyl, centrifuging after complete reaction, and washing with deionized water to obtain the carbon quantum dot fluorescent microsphere standard substance, wherein the concentration of carboxylated graphene quantum dots in the dispersion liquid is 0.1 mg/mL.
The invention has the following beneficial effects:
(1) the carbon quantum dot fluorescent microsphere standard substance prepared by the invention has the characteristics of difficult falling of fluorescence, short reaction time, stable fluorescence and the like.
(2) The carbon quantum dot fluorescent microsphere standard substance prepared by the invention has uniform particle size, standard deviation of particle size distribution controlled within 2 percent, strict monodispersity and excellent stability.
(3) The method is simple, is operated at room temperature, has small environmental influence factors and low cost, and has important significance for researching fluorescent standard substances.
(4) The carbon quantum dots and the polystyrene standard particles are connected through chemical bonds, so that the stability of the carbon quantum dot fluorescent microsphere standard substance is improved.
The preparation method of the carbon quantum dot fluorescent microsphere standard substance comprises the following steps of (1) mixing ethanol, deionized water, a dispersing agent, an initiator and a monomer in a ratio: (90-100) mL: (5-10) mL: (1.0-2.5) g: (0.2-0.8) g: (15-25) mL; the dosage is different, the particle size of the microspheres is different, and the dispersibility is different; in the step (2), the volume ratio of the concentrated sulfuric acid to the concentrated nitric acid is (0.01-100): 1, the optimal volume ratio is 3:2, and the volume ratio of the concentrated sulfuric acid to the concentrated nitric acid is too low, so that the product is not completely nitrated and the microspheres are damaged if the product is too large.
Drawings
FIG. 1, a is the UV-VIS absorption spectrum of the aminated polystyrene microsphere described in example 1; b is an ultraviolet-visible absorption spectrogram of the carbon quantum dot fluorescent microsphere standard substance;
FIG. 2 is a fluorescence spectrum of a carbon quantum dot fluorescent microsphere standard substance in example 1;
FIG. 3 is a Scanning Electron Microscope (SEM) image of the aminated polystyrene microsphere described in example 1;
FIG. 4 is a fluorescence microscope image of a carbon quantum dot fluorescent microsphere standard substance described in example 1;
FIG. 5 is a Scanning Electron Microscope (SEM) image of the aminated polystyrene microsphere described in example 2;
FIG. 6 is a Scanning Electron Microscope (SEM) image of aminated polystyrene microspheres described in example 3;
FIG. 7 is a graph of particle size distribution for aminated polystyrene microspheres described in example 3;
FIG. 8 is a graph of fluorescence stability of the carbon quantum dot fluorescent microsphere standard substance described in example 3.
The invention will be further illustrated with reference to specific embodiments and the accompanying drawings.
Detailed Description
The microstructure of the product was analyzed in the following examples using a Zeiss field emission scanning electron microscope SIGMA 300 and a fluorescence microscope HGP-001 (old glossing Instrument factory); analyzing the particle size distribution of the product by using a Malvern laser particle size analyzer; the optical properties of the product were analyzed using an ultraviolet-visible spectrophotometer PE Lambda 950 and Hitachi fluorescence spectrometer F-7000F-7000.
Example 1
The carbon quantum dot fluorescent microsphere standard substance is composed of aminated polystyrene microspheres and fluorescent substances loaded on the surfaces of the aminated polystyrene microspheres, wherein the fluorescent substances are carboxylated carbon quantum dots, the carboxylated carbon quantum dots are carboxylated graphene quantum dots, and the particle size of the aminated polystyrene microspheres is 5.2 mu m.
The preparation method of the carbon quantum dot fluorescent microsphere standard substance comprises the following steps:
(1) preparing polystyrene microspheres: adding 95mL of ethanol and 5mL of deionized water into a 250mL three-neck flask with a condenser tube, adding 1.5g of polyvinylpyrrolidone, stirring, and introducing nitrogen to completely dissolve the polyvinylpyrrolidone; then adding 15mL of styrene dissolved with azobisisobutyronitrile, wherein the dosage ratio of the azobisisobutyronitrile to the styrene is 0.02g:1mL, and electrically stirring and reacting for 8h in a 70 ℃ water bath kettle to obtain polystyrene emulsion; settling the prepared polystyrene emulsion for one day, removing supernatant, continuously adding absolute ethyl alcohol, performing ultrasonic dispersion, repeating the step for 3 times, and washing off attached polyvinylpyrrolidone; obtaining polystyrene microspheres;
(2) preparation of the nitro polystyrene microsphere: adding 1.2g of the polystyrene microspheres in the step (1) into a 500mL flask, adding 50mL of deionized water, stirring at a high speed in a water bath at 50 ℃, adding 20mL of concentrated nitric acid and 30mL of concentrated sulfuric acid, and reacting for 3 h; adding deionized water after the reaction is finished, centrifugally filtering, washing with deionized water until the pH value is neutral, vacuum drying to obtain light yellow nitro polystyrene microspheres, and storing in a refrigerator at 4 ℃;
(3) preparation of aminated polystyrene microspheres: putting 1g of the nitrated polystyrene microspheres in the step (2) into a flask, adding 100mL of 2mol/L NaOH solution, and then adding 5g of reducing agent Na2S2O4Stirring in a water bath at 70 ℃ at high speed, and reacting for 4 h; then centrifugally collecting the reacted solution, washing with deionized water for 3-5 times, washing with 95% ethanol for 3 times, vacuum-drying at 40 ℃ to obtain aminated polystyrene microspheres, and storing in a refrigerator at 4 ℃;
(4) preparing carboxylated graphene quantum dots: graphene oxide is prepared by a reported Hummers method (the specific preparation method is shown in the literature Hummers, W.; Offeman, R.J.Am.chem.Soc.,1958,80,1339-1339), and then the graphene oxide is modified by chloroacetic acid to obtain carboxylated graphene quantum dots (the specific preparation method is shown in the literature Sun X M, Li Z.Nano Res,2009,1: 203-212); the carboxylated graphene quantum dots can be uniformly dispersed in an aqueous solution;
(5) preparing a carbon quantum dot fluorescent microsphere standard substance: adding 200mg of the aminated polystyrene microsphere in the step (3) into 200mL of the dispersion liquid of the carboxylated graphene quantum dot in the step (4), stirring at room temperature for reaction for 24h, carrying out chemical condensation reaction through amino and carboxyl, centrifuging after the reaction is completed, and washing with deionized water for 3 times to obtain the carbon quantum dot fluorescent microsphere standard substance, wherein the concentration of the carboxylated graphene quantum dot in the dispersion liquid is 0.1mg/mL, and the solvent in the dispersion liquid is 400mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 100mM N-N-hydroxysuccinimide (NHS) 2- (N-morpholine) ethanesulfonic acid buffer (MES buffer, pH 5.2).
FIGS. 1a and b are UV-VIS absorption spectra of the aminated polystyrene microsphere and the carbon quantum dot fluorescent microsphere standard substance in this example, respectively; as can be seen from FIG. 1, the ultraviolet-visible absorption spectrum of the carbon quantum dot fluorescent microsphere standard substance has a characteristic absorption peak around 330nm, while the aminated polystyrene microsphere has substantially no ultraviolet absorption. FIG. 2 is a fluorescence spectrum of a standard substance of the fluorescent microsphere with carbon quantum dots, wherein when the excitation wavelength is 365nm, the emission wavelength is 513 nm. Fig. 3 is a Scanning Electron Microscope (SEM) image of the aminated polystyrene microsphere in this example, and it can be seen from fig. 3 that the aminated polystyrene microsphere has uniform particle size, strict monodispersion, and standard deviation of particle size distribution within 2%. Fig. 4 is a fluorescence microscopic image of the carbon quantum dot fluorescent microsphere standard substance of the present embodiment magnified 800 times, and it can be seen from fig. 4 that the particle size of the carbon quantum dot fluorescent microsphere standard substance is uniform. In addition, the carbon quantum dot fluorescent microsphere standard substance emits green fluorescence.
Example 2
The carbon quantum dot fluorescent microsphere standard substance is composed of aminated polystyrene microspheres and fluorescent substances loaded on the surfaces of the aminated polystyrene microspheres, wherein the fluorescent substances are carboxylated carbon quantum dots, the carboxylated carbon quantum dots are carboxylated graphene quantum dots, and the particle size of the aminated polystyrene microspheres is 4.0 mu m.
The preparation method of the carbon quantum dot fluorescent microsphere standard substance comprises the following steps:
(1) preparing polystyrene microspheres: adding 95mL of ethanol and 5mL of deionized water into a 250mL three-neck flask provided with a condenser tube, adding 2.0g of polyvinylpyrrolidone, stirring, and introducing nitrogen to completely dissolve the polyvinylpyrrolidone; then 20mL of styrene dissolved with azobisisobutyronitrile is added, the dosage ratio of the azobisisobutyronitrile to the styrene is 0.02g:1mL, and the mixture is electrically stirred and reacts for 12 hours in a water bath kettle at the temperature of 70 ℃ to obtain polystyrene emulsion; settling the prepared polystyrene emulsion for one day, removing supernatant, continuously adding absolute ethyl alcohol, performing ultrasonic dispersion, repeating the step for 3 times, and washing off attached polyvinylpyrrolidone; obtaining polystyrene microspheres;
(2) preparation of the nitro polystyrene microsphere: adding 1.2g of the polystyrene microspheres in the step (1) into a 500mL flask, adding 50mL of deionized water, stirring at a high speed in a water bath at 50 ℃, adding 20mL of concentrated nitric acid and 30mL of concentrated sulfuric acid, and reacting for 3 h; after the reaction is finished, adding deionized water, carrying out centrifugal filtration, washing with the deionized water until the pH value is neutral, carrying out vacuum drying to obtain light yellow nitro polystyrene microspheres, and storing in a refrigerator at 4 ℃;
(3) preparation of aminated polystyrene microspheres: putting 1g of the nitrated polystyrene microspheres in the step (2) into a flask, adding 100mL of 2mol/L NaOH solution, and then adding 5g of reducing agent Na2S2O4Stirring in a water bath at 70 ℃ at high speed, and reacting for 4 h; then centrifugally collecting the reacted solution, washing with deionized water for 3-5 times, washing with 95% ethanol for 3 times, vacuum-drying at 40 ℃ to obtain aminated polystyrene microspheres, and storing in a refrigerator at 4 ℃;
(4) preparing carboxylated graphene quantum dots: the same as example 1;
(5) preparing a carbon quantum dot fluorescent microsphere standard substance: adding 200mg of the aminated polystyrene microsphere in the step (3) into 200mL of the carboxylated graphene quantum dot dispersion liquid in the step (4), stirring at room temperature for reaction for 24h, carrying out chemical condensation reaction through amino and carboxyl, centrifuging after the reaction is completed, and washing with deionized water for 3 times to obtain the carbon quantum dot fluorescent microsphere standard substance, wherein the concentration of the carboxylated graphene quantum dot in the dispersion liquid is 0.1mg/mL, and the solvent in the dispersion liquid is 400mM EDC and 100mM NHS 2- (N-morpholine) ethanesulfonic acid buffer (MES buffer, pH 5.2).
FIG. 5 is a Scanning Electron Microscope (SEM) image of the aminated polystyrene microsphere of this example, and it can be seen from FIG. 5 that the aminated polystyrene microsphere is strictly monodisperse, has a particle size of 4.0 μm, a standard deviation of particle size distribution within 2%, and has uniform particle size.
Example 3
The carbon quantum dot fluorescent microsphere standard substance is composed of aminated polystyrene microspheres and fluorescent substances loaded on the surfaces of the aminated polystyrene microspheres, wherein the fluorescent substances are carboxylated carbon quantum dots, the carboxylated carbon quantum dots are carboxylated graphene quantum dots, and the particle size of the aminated polystyrene microspheres is 2.2 mu m.
The preparation method of the carbon quantum dot fluorescent microsphere standard substance comprises the following steps:
(1) preparing polystyrene microspheres: adding 95mL of ethanol and 5mL of deionized water into a 250mL three-neck flask provided with a condenser tube, adding 1.8g of polyvinylpyrrolidone, stirring, and introducing nitrogen to completely dissolve the polyvinylpyrrolidone; then adding 23mL of styrene dissolved with azobisisobutyronitrile, wherein the dosage ratio of the azobisisobutyronitrile to the styrene is 0.02g:1mL, and electrically stirring and reacting for 16h in a 70 ℃ water bath kettle to obtain polystyrene emulsion; settling the prepared polystyrene emulsion for one day, removing supernatant, continuously adding absolute ethyl alcohol with the volume 2 times that of the polystyrene emulsion, performing ultrasonic dispersion, repeating the step for 3 times, and washing off attached polyvinylpyrrolidone; obtaining polystyrene microspheres;
(2) preparation of the nitro polystyrene microsphere: adding 1.0g of the polystyrene microspheres in the step (1) into a 500mL flask, adding 50mL of deionized water, stirring at a high speed in a water bath at 50 ℃, adding 20mL of concentrated nitric acid and 30mL of concentrated sulfuric acid, and reacting for 3 h; adding deionized water after the reaction is finished, centrifugally filtering, washing with deionized water until the pH value is neutral, vacuum drying to obtain light yellow nitro polystyrene microspheres, and storing in a refrigerator at 4 ℃;
(3) preparation of aminated polystyrene microspheres: putting 1g of the nitrated polystyrene microspheres in the step (2) into a flask, adding 100mL of 2mol/L NaOH solution, and then adding 5g of reducing agent Na2S2O4Stirring in a water bath at 70 ℃ at high speed, and reacting for 4 h; then centrifugally collecting the reacted solution, washing with deionized water for 3-5 times, washing with 95% ethanol for 3 times, vacuum-drying at 40 ℃ to obtain aminated polystyrene microspheres, and storing in a refrigerator at 4 ℃;
(4) preparing carboxylated graphene quantum dots: the same as example 1;
(5) preparing a carbon quantum dot fluorescent microsphere standard substance: adding 200mg of the aminated polystyrene microsphere in the step (3) into 200mL of the carboxylated graphene quantum dot dispersion liquid in the step (4), stirring at room temperature for reaction for 24h, carrying out chemical condensation reaction through amino and carboxyl, centrifuging after the reaction is completed, and washing with deionized water for 3 times to obtain the carbon quantum dot fluorescent microsphere standard substance, wherein the concentration of the carboxylated graphene quantum dot in the dispersion liquid is 0.1mg/mL, and the solvent in the dispersion liquid is 400mM EDC and 100mM NHS 2- (N-morpholine) ethanesulfonic acid buffer (MES buffer, pH 5.2).
FIG. 6 is a Scanning Electron Microscope (SEM) image of the aminated polystyrene microsphere of this example. As can be seen from FIG. 6, the particle size of the aminated polystyrene microsphere is 2.2 μm, the dispersibility is good, the standard deviation of the particle size distribution is within 2%, and the particle size is uniform; fig. 7 is a distribution diagram of the particle size of the aminated polystyrene microsphere in this example, and it can be seen from fig. 7 that the particle size of the aminated polystyrene microsphere is normally distributed, has a narrow distribution, and has a good monodispersity. Fig. 8 is a fluorescence stability chart of the carbon quantum dot fluorescent microsphere standard substance in this embodiment, and as can be seen from fig. 8, the fluorescence intensity of the carbon quantum dot fluorescent microsphere standard substance is kept unchanged within 60 days, and the fluorescence stability is good.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (8)
1. A carbon quantum dot fluorescent microsphere standard substance is characterized in that: the material consists of aminated polystyrene microspheres and fluorescent substances loaded on the surfaces of the aminated polystyrene microspheres, wherein the fluorescent substances are carboxylated carbon quantum dots; the carboxylated carbon quantum dots are carboxylated graphene quantum dots; the particle size of the aminated polystyrene microsphere is 2.2-5.2 mu m; the carbon quantum dot fluorescent microsphere standard substance is prepared by the following preparation method, and the preparation method comprises the following steps:
(1) preparing polystyrene microspheres: preparing polystyrene microspheres by using styrene as a monomer, azodiisobutyronitrile as an initiator, polyvinylpyrrolidone as a dispersing agent and a mixed solution of ethanol and deionized water as a dispersing medium through a dispersion polymerization method;
(2) preparation of the nitro polystyrene microsphere: carrying out nitration reaction on the polystyrene microspheres in the step (1) in concentrated sulfuric acid and concentrated nitric acid solution to prepare nitrated polystyrene microspheres;
(3) preparation of aminated polystyrene microspheres: with Na2S2O4Reducing the nitrated polystyrene microspheres in the step (2) to obtain aminated polystyrene microspheres as a reducing agent;
(4) preparing carboxylated graphene quantum dots: preparing graphene oxide by a Hummers method, and then modifying the graphene oxide by chloroacetic acid to obtain a carboxylated graphene quantum dot;
(5) preparing a carbon quantum dot fluorescent microsphere standard substance: mixing the dispersion liquid of the carboxylated graphene quantum dots in the step (4) with the aminated polystyrene microspheres in the step (3) for reaction; the product is obtained through the chemical condensation reaction of amino and carboxyl.
2. The carbon quantum dot fluorescent microsphere standard substance of claim 1, wherein: the wavelength range of the light emitted by the carboxylated graphene quantum dots is 300-800 nm.
3. The method for preparing the carbon quantum dot fluorescent microsphere standard substance as claimed in claim 1 or 2, which is characterized in that: the method comprises the following steps:
(1) preparing polystyrene microspheres: preparing polystyrene microspheres by using styrene as a monomer, azodiisobutyronitrile as an initiator, polyvinylpyrrolidone as a dispersing agent and a mixed solution of ethanol and deionized water as a dispersing medium through a dispersion polymerization method;
(2) preparation of the nitro polystyrene microsphere: carrying out nitration reaction on the polystyrene microspheres in the step (1) in concentrated sulfuric acid and concentrated nitric acid solution to prepare nitrated polystyrene microspheres;
(3) preparation of aminated polystyrene microspheres: with Na2S2O4Reducing the nitrated polystyrene microspheres in the step (2) to obtain aminated polystyrene microspheres as a reducing agent;
(4) preparing carboxylated graphene quantum dots: preparing graphene oxide by a Hummers method, and then modifying the graphene oxide by chloroacetic acid to obtain a carboxylated graphene quantum dot;
(5) preparing a carbon quantum dot fluorescent microsphere standard substance: mixing the dispersion liquid of the carboxylated graphene quantum dots in the step (4) and the aminated polystyrene microspheres in the step (3) for reaction; the product is obtained through the chemical condensation reaction of amino and carboxyl.
4. The preparation method of the carbon quantum dot fluorescent microsphere standard substance according to claim 3, which is characterized in that: in the step (1), the dosage ratio of the ethanol, the deionized water, the dispersant, the initiator and the monomer is as follows: (90-100) mL: (5-10) mL: (1.0-2.5) g: (0.2-0.8) g: (15-25) mL; in the step (2), the volume ratio of the concentrated sulfuric acid to the concentrated nitric acid is (0.01-100): 1.
5. The preparation method of the carbon quantum dot fluorescent microsphere standard substance according to claim 3, which is characterized in that: in the step (5), the mass ratio of the carboxylated graphene quantum dots to the aminated polystyrene microspheres is 1: 10; the volume ratio of the concentrated sulfuric acid to the concentrated nitric acid in the step (2) is 3: 2.
6. The preparation method of the carbon quantum dot fluorescent microsphere standard substance according to claim 3, which is characterized in that: the solvent in the dispersion in step (5) was 400mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 100mM N-N-hydroxysuccinimide in 2- (N-morpholine) ethanesulfonic acid buffer.
7. The preparation method of the carbon quantum dot fluorescent microsphere standard substance according to claim 3, which is characterized in that: the method comprises the following steps:
(1) preparing polystyrene microspheres: adding ethanol and deionized water into a three-neck flask with a condenser tube, adding polyvinylpyrrolidone, stirring, and introducing nitrogen to completely dissolve the polyvinylpyrrolidone; then adding styrene dissolved with azodiisobutyronitrile, and electrically stirring and reacting for 8-16 h in a water bath kettle at 65-75 ℃ to obtain polystyrene emulsion; washing off polyvinylpyrrolidone to obtain polystyrene microspheres;
(2) preparation of the nitro polystyrene microsphere: adding the polystyrene microspheres in the step (1) into a flask, adding deionized water, stirring at a high speed in a water bath at 45-52 ℃, adding a mixed acid of sulfuric acid and nitric acid in a ratio of 3:2, and reacting for 1-4 hours; adding deionized water after the reaction is finished, centrifugally filtering, washing with deionized water until the pH value is neutral, vacuum drying to obtain light yellow nitro polystyrene microspheres, and storing in a refrigerator;
(3) preparation of aminated polystyrene microspheres: putting the nitrosation polystyrene microspheres in the step (2) into a flask, adding 50-150 mL of 2mol/L NaOH solution, and then adding 2-6 g of reducing agent Na2S2O4Stirring at high speed in a water bath at 70-75 ℃ for reacting for 4 h; centrifuging and collecting the solution after reaction, washing with deionized water for 3-5 times, washing with 95% ethanol for 2-4 times, vacuum drying to obtain aminated polystyrene microspheres, and storing in a refrigerator;
(4) preparing carboxylated graphene quantum dots: preparing graphene oxide by a Hummers method, and then modifying the graphene oxide by chloroacetic acid to obtain a carboxylated graphene quantum dot;
(5) preparing a carbon quantum dot fluorescent microsphere standard substance: and (3) adding 100-200 mg of the aminated polystyrene microsphere in the step (3) into the dispersion liquid of the carboxylated graphene quantum dot in the step (4), stirring at room temperature for reaction, carrying out chemical condensation reaction through amino and carboxyl, centrifuging after the reaction is completed, and washing with deionized water to obtain the carbon quantum dot fluorescent microsphere standard substance.
8. The method for preparing the carbon quantum dot fluorescent microsphere standard substance according to any one of claims 3 to 7, wherein the method comprises the following steps: the method comprises the following steps:
(1) preparing polystyrene microspheres: adding 95mL of ethanol and 5mL of deionized water into a three-neck flask with a condenser tube, adding 1.5-2.0g of polyvinylpyrrolidone, stirring, and introducing nitrogen to completely dissolve the polyvinylpyrrolidone; then adding 15-25mL of styrene dissolved with azobisisobutyronitrile, and electrically stirring and reacting in a water bath kettle at 70 ℃ for 8-16 h to obtain polystyrene emulsion; settling the prepared polystyrene emulsion for one day, removing supernatant, continuously adding absolute ethyl alcohol, performing ultrasonic dispersion, repeating the step for 2-4 times, and washing off attached polyvinylpyrrolidone; obtaining polystyrene microspheres; the dosage ratio of the azodiisobutyronitrile to the styrene is 0.02g to 1 mL;
(2) preparation of the nitro polystyrene microsphere: adding 1.0-1.2 g of the polystyrene microspheres in the step (1) into a flask, adding 50mL of deionized water, stirring at a high speed in a water bath at 50 ℃, adding 20mL of concentrated nitric acid and 30mL of concentrated sulfuric acid, and reacting for 3 hours; adding deionized water after the reaction is finished, centrifugally filtering, washing with deionized water until the pH value is neutral, vacuum drying to obtain light yellow nitro polystyrene microspheres, and storing in a refrigerator at 4 ℃;
(3) preparation of aminated polystyrene microspheres: putting 1g of the nitrated polystyrene microspheres in the step (2) into a flask, adding 100mL of 2mol/L NaOH solution, and then adding 5g of reducing agent Na2S2O4Stirring at high speed in a water bath at 70-75 ℃ for reacting for 4 h; then centrifugally collecting the reacted solution, washing with deionized water for 3-5 times, washing with 95% ethanol for 3 times, vacuum drying to obtain aminated polystyrene microspheres, and storing in a refrigerator at 4 ℃;
(4) preparing carboxylated graphene quantum dots: preparing graphene oxide by a Hummers method, and then modifying the graphene oxide by chloroacetic acid to obtain a carboxylated graphene quantum dot;
(5) preparing a carbon quantum dot fluorescent microsphere standard substance: adding 200mg of the aminated polystyrene microspheres in the step (3) into 200mL of the carboxylated graphene quantum dot dispersion liquid in the step (4), stirring at room temperature for reaction for 4-24 h, carrying out chemical condensation reaction through amino and carboxyl, centrifuging after complete reaction, and washing with deionized water to obtain the carbon quantum dot fluorescent microsphere standard substance, wherein the concentration of the carboxylated graphene quantum dots in the dispersion liquid is 0.1 mg/mL.
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