Dandelion carbon dot nano-enzyme, preparation method thereof and application of dandelion carbon dot nano-enzyme as colorimetric probe in uric acid sensing analysis

Abstract

The invention belongs to the technical field of biomass nano-materials and sensing analysis, and provides dandelion carbon dot nano-enzyme, a preparation method thereof and application of the dandelion carbon dot nano-enzyme as a colorimetric probe in uric acid sensing analysis. The preparation method comprises the following steps: drying a whole dandelion comprising roots, stems and leaves in the sun, grinding into 50-100-mesh dandelion powder, taking the dandelion powder as a reaction precursor, taking FeCl3. 6H2O as a doping agent, obtaining a mixed solution by a one-step hydrothermal method at 180-200 DEG C, performing centrifugal dialysis purification, and performing freeze drying to obtain dandelion carbon dot nano-enzyme powder. The synthesis precursor is low in price and easy to obtain, the preparation method is simple, operation is easy and convenient, the nano-enzyme has excellent peroxidase-like activity in the presence of a catalytic substrate and hydrogen peroxide, and the concentration of uric acid can be efficiently, conveniently and quantitatively detected. The constructed method for detecting uric acid has the advantages of stable performance, wide linear range, high accuracy and sensitivity, rapidness, effectiveness, low detection cost and the like, and can rapidly, efficiently and quantitatively detect the content of uric acid in a clinical serum sample.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomass nanomaterials and sensing analysis, and particularly relates to a dandelion carbon dot nanozyme, a preparation method thereof, and an application as a colorimetric probe in uric acid sensing analysis. Background Art

[0002] Uric acid is the end product of purine metabolism and is usually excreted from the body through the kidneys. When the production of uric acid is excessive or the excretion is reduced, hyperuricemia will occur. Long-term hyperuricemia can lead to diseases such as gout, arthritis, kidney diseases, and Lesch-Nyhan syndrome. Nowadays, various analytical methods, including spectrophotometry, high performance liquid chromatography (HPLC), fluorescence probes, gas chromatography-tandem mass spectrometry (GC-MS), etc., are used to detect uric acid. However, these methods are restricted by various factors such as expensive instruments, cumbersome pretreatment, and long detection time. Therefore, it is of great significance to develop a rapid, convenient, inexpensive, and accurate method for detecting uric acid.

[0003] Nanozymes have unique enzyme-like properties and nanomaterial characteristics. Compared with bioenzymes, nanozymes are easy to store, have high stability, are easy to synthesize, have good reproducibility, are cost-effective, and have adjustable catalytic activity. These many advantages have enabled them to be widely used in fields such as medicine, chemical engineering, food, agriculture, and the environment. Carbon dot nanozymes are a branch of carbon-based nanozymes and are widely used in the fields of biocatalysis and biosensing due to their excellent water dispersibility, easy synthesis, and surface modification advantages. Using common biomass as a raw material to prepare carbon quantum dot nanozymes has the advantages of economy and environmental friendliness compared with traditional solid carbon materials and organic small molecule carbon sources. Moreover, biomass contains abundant functional groups such as carboxyl and carbonyl groups, which provide the possibility for the surface modification of carbon quantum dot nanozymes. Dandelion is a common herbaceous plant that contains various bioactive components, such as sterols and flavonoids, and these components have the effect of antioxidizing free radicals. Using dandelion to prepare carbon dot nanozymes can not only effectively utilize natural resources but also reduce the impact on the environment during the synthesis process. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a dandelion carbon dot nanozyme, a preparation method thereof, and an application as a colorimetric probe in uric acid sensing analysis; the preparation method is simple, and the raw materials are widely sourced; the prepared dandelion carbon dot-based nanozyme can be used for the sensitive detection of uric acid in clinical practice based on its peroxidase-like properties.

[0005] The present invention is realized by the following technical solutions: A kind of dandelion carbon dot nanozyme. Take the whole dandelion including roots, stems and leaves, dry it in the sun and grind it into dandelion powder with 50 - 100 meshes. Use the obtained dandelion powder as the reaction precursor, FeCl3·6H2O as the dopant, and obtain a mixed solution by one-step hydrothermal method at 180 - 200 °C. After centrifugal dialysis purification, freeze-drying is carried out to obtain the dandelion carbon dot nanozyme powder.

[0006] The method for preparing the dandelion carbon dot nanozyme includes the following steps: (1) Mix 0.30 - 0.80 g of dandelion powder and 0.10 - 0.50 g of FeCl3·6H2O, add 10 - 15 mL of ultrapure water and ultrasonicate until completely dissolved. React the mixed solution at 180 - 200 °C for 4 - 12 h; (2) Naturally cool to room temperature, centrifuge at 8000 - 12000 rpm for 10 - 30 min and take the supernatant. After filtering with a 0.22 μm filter membrane, continuously dialyze with a dialysis bag for 5 - 10 h to obtain a carbon dot nanozyme solution; Freeze-drying is carried out to obtain the solid powder of the dandelion carbon dot nanozyme.

[0007] The present invention also provides the application of the dandelion carbon dot nanozyme or the dandelion carbon dot nanozyme prepared by the method in uric acid sensing analysis. The dandelion carbon dot nanozyme detects uric acid by colorimetry, and specifically includes the following steps: (1) Mix 0.02 - 0.07 mg / mL of uricase with uric acid at a concentration gradient of 0.23 - 209.30 μM, and incubate at 20 - 40 °C for 20 - 30 min; (2) Add 0.5 - 1.5 mM of o-phenylenediamine (OPD) and 15 - 35 μg / mL of dandelion carbon dot nanozyme to the mixed solution obtained in step (1), and incubate at 20 - 40 °C for 10 - 20 min; Then measure the spectrum of the mixed solution at 330 - 700 nm, and record the absorbance value at 452 nm; (3) Using Origin software, take the absorbance value measured at 452 nm in step (2) as the ordinate and the uric acid concentration as the abscissa, draw and fit the linear curve between the two. In the range of 0.23 - 93.02 μM, the linear equation is ΔA 452 = 0.0043[UA] + 0.0705, and the correlation coefficient R 2 = 0.9920, and the lowest detection limit LOD is 0.31 μM.

[0008] In step (1), the concentration of uricase is 0.05 mg / mL; incubate at 30 °C for 30 min; in step (2), OPD is 1.5 mM; the dandelion carbon dot nanozyme is 25 μg / mL; incubate at 30 °C for 20 min.

[0009] The application of the dandelion carbon dot nanozyme as a colorimetric probe in the actual detection of uric acid specifically includes the following steps: The sample to be tested is diluted with ultrapure water to a concentration of 10%-20%. The diluted serum sample is added with 0.02-0.07 mg / mL uricase, and incubated at 20-40 °C for 20-30 min. Then, 0.5-1.5 mM OPD and 15-35 μg / mL dandelion carbon dot nanozyme are added, and incubated at 20-40 °C for 10-20 min. Record the ultraviolet-visible absorption change of the mixed solution and the absorbance value at 452 nm. The uric acid concentration in the sample to be tested is calculated according to the standard curve and the measured absorbance change value.

[0010] The sample to be tested is diluted 10 times with ultrapure water; the uricase concentration is 0.05 mg / mL; incubated at 30 °C for 30 min; OPD is 1.5 mM; the dandelion carbon dot nanozyme is 25 μg / mL; incubated at 30 °C for 20 min.

[0011] Compared with the prior art, the precursor for synthesis of the present invention is cheap and easily available, the preparation method is simple, the operation is convenient, and no expensive instruments are required. The prepared nanozyme has excellent peroxidase-like activity in the presence of catalytic substrate and hydrogen peroxide. Based on this, the uric acid concentration can be detected quickly, efficiently and quantitatively. The method for detecting uric acid by the constructed nanozyme colorimetry has the advantages of stable performance, wide linear range, high accuracy and sensitivity, fast and effective, low detection cost, etc., providing a new method for uric acid detection, and can quickly, efficiently and quantitatively detect the content of uric acid in clinical serum samples. Description of the Drawings

[0012] Figure 1 It is the transmission electron microscope image of the dandelion carbon dot nanozyme prepared in Example 1; Figure 2 It is the particle size distribution diagram of the dandelion carbon dot nanozyme prepared in Example 1; Figure 3 It is the X-ray photoelectron spectroscopy of the dandelion carbon dot nanozyme prepared in Example 1; Figure 4 It is the infrared spectrum of the dandelion carbon-based nanozyme prepared in Example 1; Figure 5 It is the steady-state kinetic analysis of the dandelion carbon-based nanozyme on o-phenylenediamine (OPD) in Example 2; Figure 6 It is the steady-state kinetic analysis of the dandelion carbon-based nanozyme on H2O2 in Example 2; Figure 7 It is the Lineweaver-Burk curve of the dandelion carbon-based nanozyme on OPD in Example 2; Figure 8 The Lineweaver - Burk curve of dandelion carbon - based nanozyme for H2O2 in Example 2; Figure 9 The selective detection of different substances in the environment by the dandelion carbon - based nanozyme - OPD - H2O2 sensing system in Example 3. The results show that it has selectivity only for uric acid; Figure 10 The change in selectivity for uric acid when there are interfering substances in the environment by the dandelion carbon - based nanozyme - OPD - H2O2 sensing system in Example 4. The results show that the sensing system has excellent anti - interference performance; Figure 11 The graph of the change in absorbance value of the dandelion carbon - based nanozyme - OPD - H2O2 in solutions with different uric acid contents in Example 5; Figure 12 The linear graph of uric acid concentration and the absorbance value of the whole system in Example 5. Detailed implementation manners

[0013] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The materials cited herein and their citations will be incorporated by reference.

[0015] Equivalent technologies of the specific embodiments described that can be understood by those skilled in the art through routine experiments will be included in this application.

[0016] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The instrument and equipment used in the following embodiments are all conventional laboratory instrument and equipment unless otherwise specified; the experimental materials used in the following embodiments are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0017] Example 1: Preparation and characterization of dandelion carbon dot nanozyme. The specific method is as follows: (1)Take the whole dandelion including roots, stems and leaves, dry it and grind it into dandelion powder with 50 - 100 mesh. Put 0.50 g of dandelion powder and 0.30 g of FeCl3·6H2O into a beaker, add an appropriate amount of ultrapure water and ultrasonicate until completely dissolved. Transfer the above mixed solution into a polytetrafluoroethylene reaction kettle and place it in an oven, and react at 200 ºC for 6 h.

[0018] (2)Let the reaction kettle cool to room temperature naturally. After centrifuging at 8000 rpm for 15 min, take the supernatant, filter it with a 0.22 μm filter membrane, and then continuously dialyze it with a dialysis bag for 6 h to obtain a carbon dot nanozyme solution. The solid powder of dandelion carbon dot nanozyme is prepared by freeze-drying method.

[0019] The property characterization of the obtained solid powder of dandelion carbon dot nanozyme is shown in Figure 1 、 Figure 2 and Figure 3 。

[0020] Figure 1 Figure Figure 2 is the transmission electron microscope image of the dandelion carbon dot nanozyme prepared in Example 1.

[0021] Figure 3 Figure

[0022] Figure 4 is the X-ray photoelectron spectroscopy image of the dandelion carbon dot nanozyme prepared in Example 1, indicating that the dandelion carbon dot nanozyme is mainly composed of five elements: Cl (2p), C (1s), N (1s), O (1s) and Fe (2p).

[0023] Example 2: Determination of the peroxidase-like activity of dandelion carbon dot nanozyme. The specific method is as follows: (1)Taking H2O2 as the substrate, kinetic analysis is carried out on the dandelion carbon dot nanozyme. Add different concentrations of OPD (concentrations are 0.025, 0.05, 0.10, 0.20, 0.30 mM respectively) to the mixed solution containing 25 μg / mL dandelion carbon dot nanozyme and 0.25 mM H2O2. After incubating for 15 min, measure the ultraviolet absorption value of different solution systems at 452 nm, and calculate the reaction rate corresponding to different concentrations of OPD through the formula. Using Origin software, with the OPD concentration in the mixed solution as the abscissa and the calculated reaction rate as the ordinate, draw the relationship diagram between the two, and the results are shown in Figure 5 。

[0024] (2) Using OPD as the substrate, kinetic analysis was performed on the dandelion carbon dot nanozyme. Different concentrations of H2O2 (concentrations were 0.20, 0.30, 0.40, 0.50, 0.80 mM respectively) were added to the mixed solution containing 25 μg·mL -1 of dandelion carbon dot nanozyme and 1.5 mM OPD. The ultraviolet absorption values of different solution systems were measured at 452 nm, and the reaction rates corresponding to different concentrations of H2O2 were calculated through the formula. Using Origin software, with the concentration of H2O2 in the mixed solution as the abscissa and the reaction rate as the ordinate, the relationship graph between the two was plotted, and the results are shown in Figure 6 .

[0025] (3) The reaction rate of the dandelion carbon dot nanozyme was calculated using the Lineweaver Burk double reciprocal plot: 1 / V = K m / V max [S] + 1 / V max , where K m , [S], V and V max represent the Michaelis constant, substrate concentration, initial reaction rate and maximum reaction rate respectively. Figure 5 and Figure 6 The corresponding double reciprocal plots are Figure 7 and Figure 8 .

[0026] From Figure 5 and Figure 6 , it can be seen that the catalytic reactions of the dandelion carbon dot nanozyme with the two substrates both conform to the standard Michaelis model. The intercept and slope of the linear fit of the Burk double reciprocal plot ( Figure 7 and Figure 8 ) were used to obtain the K m and V max corresponding to OPD and H2O2. The K m of OPD and H2O2 were 0.022 μM and 1.94 μM respectively, and the V max of OPD and H2O2 was 2.74×10 -8 M·s -1 and 13.33×10 -8 M·s -1 .

[0027] Example 3: To investigate the selectivity of the dandelion carbon dot nanozyme for uric acid, the specific method was as follows: (1) Dissolve dandelion carbon dot nanozyme in ultrapure water to prepare a dandelion carbon dot nanozyme solution with a concentration of 5 mg / mL. Prepare aqueous solutions with a concentration of 0.01 M of different substances (such as alanine / Ala, valine / Val, lysine / Lys, arginine / Arg, leucine / Leu, homocysteine / Hcy, isoleucine / Ile, glutamine / Gln, glutathione / GSH, tryptophan / Try, glycine / Gly, proline / Pro, cysteine / Cys, aspartic acid / Asp, serine / Ser, methionine / Met, Na + 、K + 、Mg 2+ 、Zn 2+ 、Ca 2+ 、Cl - 、ascorbic acid / AA, uric acid and urea) respectively for later use.

[0028] (2) Place different substances (0.16 mM) from step (1) and uricase (0.05 mg / mL) at 30 °C for 30 min, then add OPD (1.5 mM) and dandelion carbon dot nanozyme (0.025 mg / mL) into the above mixed solution and place it at 30 °C for 20 min. Then use a UV-visible spectrometer to measure the spectrum of the mixed solution in the range of 330 - 700 nm and record the absorbance at 452 nm.

[0029] (3) Using Origin software, with the absorbance values measured at 452 nm in step (2) as the ordinate, compare the color development effects of different interfering substances on the system. The results are shown in Figure 9 .

[0030] Figure 9 shows the changes in the sensing system after the addition of different substances. The results show that in the uric acid environment of Example 4, the absorption value increases significantly and the color changes from colorless to yellow. Other substances have no effect on the absorbance of the sensing system, indicating that dandelion carbon dot nanozyme has excellent selectivity for uric acid.

[0031] Example 4: Investigate the anti-interference ability of dandelion carbon dot nanozyme for uric acid. The specific method is as follows: (1) Dissolve dandelion carbon dot nanozyme in ultrapure water to prepare a dandelion carbon dot nanozyme solution with a concentration of 5 mg / mL. Prepare aqueous solutions with a concentration of 0.01 M of interfering substances that may exist in the actual environment (such as alanine / Ala, valine / Val, lysine / Lys, arginine / Arg, leucine / Leu, homocysteine / Hcy, isoleucine / Ile, glutamine / Gln, glutathione / GSH, tryptophan / Try, glycine / Gly, proline / Pro, cysteine / Cys, aspartic acid / Asp, serine / Ser, methionine / Met, ascorbic acid / AA, Na + 、K + 、Mg 2+ 、Zn 2+ 、Ca 2+ 、Cl - and urea) respectively for later use.

[0032] (2) Respectively mix different substances (0.16 mM) in step (1) with uric acid and uricase (0.05 mg / mL) and place them at 30 °C for 30 min. Then put OPD (1.5 mM) and dandelion carbon dot nanozyme (0.025 mg / mL) into the above mixed solution and place it at 30 °C for 20 min. Then use a UV-visible spectrometer to measure the spectrum of the mixed solution at 330 - 700 nm and record the absorbance at 452 nm.

[0033] (3) Using Origin software, with the absorbance values measured at 452 nm in step (2) as the ordinate, compare the influence of different interfering substances on the color development effect of the system. The results are shown in Figure 11 .

[0034] Figure 10 is the selectivity of the sensing system for uric acid after introducing different interfering substances. The results show that all interfering substances have no effect on the absorbance, indicating that the dandelion carbon dot nanozyme has excellent anti-interference performance and can achieve stable detection of uric acid in complex environments.

[0035] Example 5: Construct a uric acid sensor based on the peroxidase-like properties of dandelion carbon dot nanozyme. The specific method is as follows: (1) After mixing uricase (0.05 mg / mL) with different concentrations of uric acid, incubate at 30 °C for 30 min, and the uric acid concentration increases in a gradient within the range of 0.23 - 209.30 µM.

[0036] (2) Continuing to add OPD (1.5 mM) and dandelion carbon-based nanozyme (0.025 mg / mL) to the solution in step (1), incubating for 20 min. Then, the spectrum of the mixed solution in the range of 330 - 700 nm was measured with an ultraviolet-visible spectrometer, and the result is shown in Figure 9 , and the absorbance value at 452 nm was recorded.

[0037] (3) Using Origin software, with the absorbance value at 452 nm measured in step two as the ordinate and the uric acid concentration as the abscissa, the linear relationship between the two was plotted and fitted, and the result is shown in Figure 12 .

[0038] Figure 11 It shows that the absorbance (A 452 ) at 452 nm increases with the increase of uric acid concentration. Figure 12 is the linear relationship diagram between the absorbance of the mixed solution at 452 nm and the uric acid concentration. In the range of 0.23 - 93.02 μM, the linear equation is ΔA 452 = 0.0043[UA] + 0.0705, and the correlation coefficient R 2 = 0.9920, and the lowest detection limit (LOD) is 0.31 μM.

[0039] Example 6: Detection of uric acid content in clinical samples using peroxidase-like colorimetric method: (1) Collecting clinical serum samples from healthy volunteers and those with elevated uric acid levels, storing them at -20 °C, diluting them 10 times with ultrapure water and keeping them for later use. The serum sample numbers are 1 # , 2 # , 3 # , 4 # , 5 # , 6 # , 7 # .

[0040] (2) After mixing uricase (0.05 mg·mL -1 ) with different diluted serum samples, incubating at 30 °C for 30 min. Continuing to add OPD (1.5 mM) and dandelion carbon-based nanozyme (0.025 mg / mL) to this mixed solution, incubating for 20 min. Using ultraviolet-visible spectroscopy to measure the absorption value A0 of the mixed solution at 452 nm, substituting A0 into the linear equation (ΔA 452 = 0.0043[UA] + 0.0705), calculating the detected concentration of uric acid in the serum sample. All experiments were repeated three times, and the results are shown in Table 1.

[0041] Table 1: Detection of uric acid content in clinical samples using colorimetric method The uric acid concentrations in seven clinical serum samples measured by the national standard method were 584, 465, 525, 533, 585, and 449 μM, respectively. Table 1 lists the uric acid concentrations in the serum samples calculated in Example 6. The results are basically consistent with those of the national standard method, and the relative standard deviations are all less than 8.5%, showing good reproducibility.

[0042] Example 7: Preparation of dandelion carbon dot nanozyme, which specifically includes the following steps: 0.30 g of dandelion powder and 0.10 g of FeCl3·6H2O are placed in a beaker and reacted at 180 °C for 12 h. After centrifuging at 10000 rpm for 30 min, the supernatant is taken, and the remaining method is the same as that described in Example 1. The obtained dandelion carbon dot nanozyme used as a colorimetric probe has no significant difference in the detection effect in actual uric acid detection compared with the dandelion carbon dot nanozyme described in Example 1.

[0043] Example 8: Preparation of dandelion carbon dot nanozyme, which specifically includes the following steps: 0.80 g of dandelion powder and 0.50 g of FeCl3·6H2O are placed in a beaker and reacted at 190 °C for 4 h. After centrifuging at 12000 rpm for 10 min, the supernatant is taken, and the remaining method is the same as that described in Example 1. The obtained dandelion carbon dot nanozyme used as a colorimetric probe has no significant difference in the detection effect in actual uric acid detection compared with the dandelion carbon dot nanozyme described in Example 1.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dandelion carbon dot nanozyme, characterized in that: Take the whole dandelion including roots, stems and leaves, dry it in the sun and grind it into dandelion powder with 50-100 meshes. Use the obtained dandelion powder as the reaction precursor, FeCl3·6H2O as the dopant, and obtain a mixed solution by hydrothermal method at 180-200 °C. After centrifugal dialysis purification, freeze-drying is the dandelion carbon dot nanozyme powder.

2. The method for preparing the dandelion carbon dot nanozyme according to claim 1, characterized in that: The steps are as follows: (1) Mix 0.30-0.80 g of dandelion powder and 0.10-0.50 g of FeCl3·6H2O, add 10-15 mL of ultrapure water and ultrasonicate until completely dissolved. The mixed solution reacts at 180-200 °C for 4-12 h; (2) Naturally cool to room temperature, centrifuge at 8000-12000 rpm for 10-30 min, take the supernatant, filter it through a 0.22 μm filter membrane, and continuously dialyze it with a dialysis bag for 5-10 h to obtain a carbon dot nanozyme solution; freeze-drying gives the dandelion carbon dot nanozyme solid powder.

3. Use of the dandelion carbon dot nanozyme according to claim 1 or the dandelion carbon dot nanozyme prepared by the method according to claim 2 in uric acid sensing analysis, characterized in that: The dandelion carbon dot nanozyme uses a colorimetric method to detect uric acid, and specifically includes the following steps: (1) Mix 0.02-0.07 mg / mL of uricase with uric acid at a concentration gradient of 0.23-209.30 μM, and incubate at 20-40 °C for 20-30 min; (2) Add 0.5-1.5 mM of o-phenylenediamine OPD and 15-35 μg / mL of dandelion carbon dot nanozyme to the mixed solution obtained in step (1), and incubate at 20-40 °C for 10-20 min; then measure the spectrum of the mixed solution at 330-700 nm and record the absorbance value at 452 nm; (3) Using Origin software, with the absorbance value at 452 nm measured in step (2) as the ordinate and the uric acid concentration as the abscissa, plot and fit the linear curve between the two. In the range of 0.23 - 93.02 μM, the linear equation is ΔA 452 = 0.0043[UA] + 0.0705, and the correlation coefficient R 2 = 0.9920, and the lowest detection limit LOD is 0.31 μM.

4. The application according to claim 3, characterized in that: In step (1), the concentration of uricase is 0.05 mg / mL; incubate at 30 °C for 30 min; in step (2), OPD is 1.5 mM; the dandelion carbon dot nanozyme is 25 μg / mL; incubate at 30 °C for 20 min.

5. The application according to claim 3, wherein: The application of the dandelion carbon dot nanozyme as a colorimetric probe in the actual detection of uric acid specifically includes the following steps: The test sample is diluted with ultrapure water to a concentration of 10%-20%, the diluted serum sample is added with 0.02-0.07 mg / mL of uricase, and mixed and incubated at 20-40 °C for 20-30 min. Then add 0.5-1.5 mM of OPD and 15-35 μg / mL of dandelion carbon dot nanozyme, and mix and incubate at 20-40 °C for 10-20 min. Record the ultraviolet-visible absorption change of the mixed solution and the corresponding absorbance value at 452 nm, and calculate the uric acid concentration in the test sample according to the standard curve and the measured absorbance change value.

6. The application according to claim 5, characterized in that: The test sample is diluted 10 times with ultrapure water; the concentration of uricase is 0.05 mg / mL; incubate at 30 °C for 30 min; OPD is 1.5 mM; the dandelion carbon dot nanozyme is 25 μg / mL; incubate at 30 °C for 20 min.

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