A preparation method of carbon dot nanoscale enzyme based on response surface method optimization, carbon dot nanoscale enzyme and application thereof

Abstract

The application discloses a preparation method of carbon dot nanoscale enzyme based on response surface method optimization, the carbon dot nanoscale enzyme and application thereof, and belongs to the technical field of carbon dot nanoscale enzyme preparation.The preparation method comprises the following steps: stirring and dissolving glucose, ferrous sulfate and concentrated sulfuric acid in water, then performing hydrothermal reaction, and obtaining the carbon dot nanoscale enzyme through purification after the reaction is completed; measuring the fluorescence intensity of the carbon dot nanoscale enzyme; selecting the molar ratio of glucose to ferrous sulfate, the addition amount of concentrated sulfuric acid and the reaction time as influencing factors, taking the fluorescence intensity of the carbon dot nanoscale enzyme as the observation value, performing response surface method optimization, and obtaining a regression equation; and finally obtaining optimal reaction conditions through software optimization.The carbon dot nanoscale enzyme synthesized by the method has high fluorescence quantum yield and excellent peroxidase-like activity, and can be applied to tetracycline detection as a colorimetric probe, and has high detection sensitivity and accuracy.

Description

Technical Field

[0001] This invention relates to the field of carbon dot nanozyme preparation technology, and in particular to a method for preparing carbon dot nanozymes optimized by response surface methodology, carbon dot nanozymes and their applications. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Pharmaceuticals and personal care products, as a new type of pollutant, pose a serious threat to the health of microorganisms and plants and animals if they persist in the environment for extended periods. Therefore, the detection and analysis of new pollutants in the environment is of significant research value. Traditional pollutant detection methods require expensive instruments and complex operations. In recent years, with the rapid development of nanotechnology, carbon dot nanozymes, as a novel nanomaterial, have gained widespread attention in fields such as bioimaging, sensors, and environmental monitoring due to their unique optical properties, good biocompatibility, and ease of preparation.

[0004] Researchers have utilized various methods, including hydrothermal, microwave-assisted, and electrochemical methods, to prepare carbon dot nanozymes with superior properties. However, most of these methods rely on trial-and-error approaches driven by human experience, leading to resource waste, environmental impact, and low synthesis efficiency, which is detrimental to sustainable development. Currently, the controllable and efficient preparation of carbon dot nanozymes remains a significant challenge. Summary of the Invention

[0005] In view of this, the present invention provides a method for preparing carbon dot nanozymes based on response surface methodology, carbon dot nanozymes and their applications. The present invention can rapidly and efficiently optimize the preparation conditions of carbon dot nanozymes, and can obtain high-performance carbon dot nanozymes, which can be used as probes to analyze and detect new pollutants in the environment.

[0006] In a first aspect, the present invention provides a method for preparing carbon dot nanozymes optimized based on response surface methodology, comprising the following steps:

[0007] Glucose, ferrous sulfate, and concentrated sulfuric acid were dissolved in water by stirring, and then a hydrothermal reaction was carried out. After the reaction was completed, carbon dot nanozymes were obtained by purification. The fluorescence intensity of the carbon dot nanozymes was measured.

[0008] The molar ratio of glucose to ferrous sulfate, the amount of concentrated sulfuric acid added, and the reaction time were selected as influencing factors. The fluorescence intensity of carbon nanozymes was used as the evaluation value, and the response surface methodology was used for optimization to obtain the regression equation.

[0009] Finally, the maximum fluorescence intensity of the carbon dot nanozyme and the corresponding molar ratio of glucose to ferrous sulfate, the amount of concentrated sulfuric acid added, and the reaction time were obtained through software optimization.

[0010] Preferably, the regression equation is as follows:

[0011] Y = 2.08 × 10 5 +10.63A-34622.5B+1.22×10 5 C+812.25AB+32007.5AC+12704.75B

[0012] C+44001.6A 2 -9534.15B 2 +1.11×10 5 C 2 .

[0013] Preferably, the molar ratio of glucose to ferrous sulfate is 1 to 3, the amount of concentrated sulfuric acid added is 0.2 to 0.6 vol% of the amount of water added, and the reaction time is 8 to 12 hours.

[0014] Furthermore, the molar ratio of glucose to ferrous sulfate is 3, the amount of concentrated sulfuric acid added is 0.52 vol% of the amount of water added, and the reaction time is 12 h.

[0015] Preferably, the temperature of the hydrothermal reaction is 150–250°C.

[0016] Preferably, the ratio of ferrous sulfate to water is (0.2-2) g: 50 mL.

[0017] Preferably, the purification step specifically involves: centrifuging the product after the hydrothermal reaction, then dialyzing the supernatant, and drying the dialysate to obtain carbon dot nanozymes.

[0018] Preferably, the step of determining the fluorescence intensity of the carbon dot nanozyme specifically involves: dissolving the carbon dot nanozyme in a solvent to obtain a carbon dot nanozyme solution, fixing the excitation wavelength at 370–450 nm, performing fluorescence spectroscopy detection on the carbon dot nanozyme solution, and recording the intensity of the maximum fluorescence emission peak.

[0019] Secondly, the present invention provides carbon dot nanozymes prepared by the above preparation method.

[0020] Thirdly, the present invention provides the application of the above-mentioned carbon dot nanozyme in tetracycline detection.

[0021] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0022] (1) This invention uses glucose as a carbon source, ferrous sulfate as a metal precursor, and concentrated sulfuric acid as an additive to prepare S,Fe doped carbon dot nanozymes by hydrothermal synthesis. At the same time, the relationship between reaction conditions and fluorescence intensity of carbon dot nanozymes is established by response surface methodology, and the optimal reaction conditions are obtained. It has the advantages of controllability and high efficiency, high synthesis efficiency, and avoids resource waste.

[0023] (2) The carbon dot nanozyme optimized and synthesized in this invention has high fluorescence quantum yield, excellent peroxidase-like activity and good catalytic activity. It can be used as a colorimetric probe for the detection of tetracycline with high detection sensitivity and accuracy. Attached Figure Description

[0024] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0025] Figure 1 This is a correlation analysis graph between the predicted and experimental values ​​of Embodiment 1 of the present invention;

[0026] Figure 2 This is a response surface plot showing the effect of the interaction between the molar ratio of glucose to ferrous sulfate and the concentration of concentrated sulfuric acid on fluorescence intensity in Example 1 of the present invention.

[0027] Figure 3 This is a response surface plot showing the effect of the interaction between the molar ratio of glucose to ferrous sulfate and the reaction time on the fluorescence intensity in Example 1 of this invention.

[0028] Figure 4 This is a response surface plot showing the effect of the interaction between concentrated sulfuric acid content and reaction time on fluorescence intensity in Example 1 of the present invention.

[0029] Figure 5 This is a transmission electron microscope image of the S,Fe doped carbon nanoparticle nanozyme prepared under the optimal conditions in Example 1 of the present invention.

[0030] Figure 6 These are the UV-Vis absorption, fluorescence excitation, and fluorescence emission curves of the S,Fe doped carbon nanoparticle nanozyme prepared under the optimal conditions in Example 1 of this invention.

[0031] Figure 7 This is a graph showing the enzyme activity test results of the S,Fe doped carbon nanoparticle nanozyme prepared under the optimal conditions in Example 1 of this invention.

[0032] Figure 8This invention fits steady-state kinetic curves with o-PD and H2O2 as substrates using the Michaelis-Menten equation, where a is the steady-state kinetic curve with o-PD as substrate and b is the steady-state kinetic curve with H2O2 as substrate;

[0033] Figure 9 This is a graph showing the relationship between the change in absorbance of the present invention and the concentration of tetracycline. Detailed Implementation

[0034] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0035] This invention provides a method for preparing carbon dot nanozymes optimized based on response surface methodology, comprising the following steps:

[0036] Glucose, ferrous sulfate, and concentrated sulfuric acid were dissolved in water by stirring, and then a hydrothermal reaction was carried out. After the hydrothermal reaction was completed, carbon dot nanozymes were obtained by purification. The fluorescence intensity of the carbon dot nanozymes was measured.

[0037] The molar ratio of glucose to ferrous sulfate, the amount of concentrated sulfuric acid added, and the reaction time were selected as influencing factors. The fluorescence intensity of carbon nanozymes was used as the evaluation value, and the response surface methodology was used for optimization to obtain the regression equation.

[0038] Finally, the maximum fluorescence intensity of the carbon dot nanozyme and the corresponding molar ratio of glucose to ferrous sulfate, the amount of concentrated sulfuric acid added, and the reaction time were obtained through software optimization.

[0039] This invention utilizes glucose as the carbon source, ferrous sulfate as the metal precursor, and concentrated sulfuric acid as an additive to prepare S,Fe-doped carbon nanozymes via hydrothermal synthesis. Simultaneously, response surface methodology (RSM) was employed to establish the relationship between reaction conditions and the fluorescence intensity of the carbon nanozymes, optimizing the reaction conditions. RSM is a statistical analytical method that comprehensively handles the relationship between experimental variables and response values, including experimental optimization design, analysis, and multidimensional modeling. Its principle is based on Box-Benhnken central composite design, arranging experimental schemes and considering random errors. It expresses the complex and unknown implicit functional relationship between variables and response values ​​within a small region using a multiple quadratic regression equation, employing a second-order polynomial relationship. The results present a multidimensional image that is intuitive and simple. Therefore, this invention can quickly and efficiently determine the optimal preparation conditions for carbon nanozymes, shortening the research cycle and avoiding resource waste.

[0040] In this invention, the regression equation is as follows:

[0041] Y = 2.08 × 10 5 +10.63A-34622.5B+1.22×10 5 C+812.25AB+32007.5AC+12704.75B

[0042] C+44001.6A 2 -9534.15B 2 +1.11×10 5 C 2 Where Y is the fluorescence intensity of the carbon nanozyme, A is the molar ratio of glucose to ferrous sulfate, B is the amount of concentrated sulfuric acid added, and C is the reaction time.

[0043] In this invention, the molar ratio of glucose to ferrous sulfate is 1–3, the amount of concentrated sulfuric acid added is 0.2–0.6 vol% of the amount of water added, and the reaction time is 8–12 h. Through optimization, the optimal molar ratio of glucose to ferrous sulfate is 3, the optimal amount of concentrated sulfuric acid added is 0.52 vol% of the amount of water added, and the optimal reaction time is 12 h.

[0044] In this invention, the temperature of the hydrothermal reaction is 150–250°C, more preferably 180–220°C, and most preferably 200°C. This invention does not impose special limitations on the specific steps of the hydrothermal reaction process; commonly used hydrothermal reaction processes in the art can be employed.

[0045] In this invention, the ratio of ferrous sulfate to water is (0.2-2) g: 50 mL, more preferably (0.2-0.8) g: 50 mL.

[0046] In this invention, the purification step specifically involves: centrifuging the product after the hydrothermal reaction, then dialyzing the supernatant, and drying the dialysate to obtain the carbon dot nanozyme. This invention does not impose any special limitations on the specific centrifugation, dialysis, and drying processes; commonly used centrifugation, dialysis, and drying steps in the art can be employed.

[0047] In this invention, the step of determining the fluorescence intensity of the carbon dot nanozyme specifically involves: dissolving the carbon dot nanozyme in a solvent to obtain a carbon dot nanozyme solution; fixing the excitation wavelength at 370–450 nm; performing fluorescence spectroscopy on the carbon dot nanozyme solution; and recording the intensity of the maximum fluorescence emission peak. This invention does not impose any special limitations on the specific type of solvent, as long as it can dissolve the carbon dot nanozyme.

[0048] This invention also provides carbon dot nanozymes prepared by the above-described method. The carbon dot nanozymes obtained by optimizing reaction conditions using response surface methodology exhibit high fluorescence quantum yield, excellent peroxidase-like activity, and good catalytic activity.

[0049] This invention also provides the application of the aforementioned carbon dot nanozyme in tetracycline detection. The carbon dot nanozyme prepared by this invention exhibits excellent detection sensitivity and accuracy in tetracycline detection. This invention does not impose any special limitations on the specific application process; commonly used application methods in the field can be employed.

[0050] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0051] Example 1

[0052] 1. Preparation process of S,Fe-doped carbon dot nanozymes (S,Fe-doped CDs nano-enzymes):

[0053] Glucose, 0.5 g ferrous sulfate (0.0018 mol), and concentrated sulfuric acid were added to a beaker containing 50 mL of distilled water. After stirring until completely dissolved, the mixture was transferred to a PTFE-lined stainless steel reactor and reacted at 200 °C for a set time in a forced-air drying oven. After the reaction was complete, the mixture was allowed to cool naturally to room temperature, centrifuged at high speed to collect the supernatant, dialyzed for 24 hours, and then freeze-dried to obtain carbon dot nanozymes under different conditions.

[0054] Fluorescence intensity detection of carbon dot nanozymes: 0.005 g of the carbon dot nanozymes prepared above was dissolved in 5 mL of ethanol and sonicated for 1 min to obtain a homogeneous solution; at 25 °C, the excitation wavelength was fixed at 370-450 nm, and the fluorescence spectrum of the solution was detected, and the intensity of the maximum fluorescence emission peak was recorded.

[0055] 2. Optimization process of preparation method of S,Fe doped carbon dot nanozymes:

[0056] Traditional methods for obtaining high-performance carbon dot nanozymes require screening of precursors and precise control of preparation conditions. This embodiment utilizes the Box-Behnken Design (BBD) model to optimize the preparation conditions of carbon dot nanozymes. Design-Expert.V8.0.6 software was used, with the molar ratio of glucose to ferrous sulfate (factor A, n) as the optimal parameters. G :n Fe The three factors, concentrated sulfuric acid content (factor B) and reaction time (factor C, Time), are input variables. They are entered in the Box-Behnken Design interface, and the response output value is fluorescence intensity. The required experimental procedure design can be obtained through simulation. The experimental procedure design is shown in Table 1.

[0057] Table 1 Experimental design based on the response surface methodology (Box-Behnken)

[0058]

[0059] Based on the model design, the experiment was conducted 17 times. The fluorescence intensity of S,Fe-doped carbon nanoparticle nanozymes prepared under different conditions is shown in Table 2. The determination coefficient R0 2 and the adjusted R 2 The values ​​are 0.9195 and 0.8161 respectively, predicting R... 2 The value of -0.2466 indicates a good correlation between the predicted and actual fluorescence intensities. The F and P values ​​for the model significance test were 8.89 and 0.0044 (P < 0.005), respectively, indicating that the model is highly significant and can be used for analysis. The goodness of fit for the missing terms of the F and P values ​​were 3.65 and 0.23 (p > 0.05), respectively, indicating that the model differs little from the experiment and can be analyzed using regression equations.

[0060] Table 2. Fluorescence intensity changes based on Box-Behnken experimental design and fluorophore intensity changes.

[0061]

[0062] By fitting the model, the binomial equilibrium equation between the fluorescence intensity Y of the prepared carbon dot nanozyme and the experimental factors can be obtained, as shown in the following equation:

[0063] Y = 2.08 × 10 5 +10.63A-34622.5B+1.22×10 5 C+812.25AB+32007.5AC+12704.75B

[0064] C+44001.6A 2 -9534.15B 2 +1.11×10 5 C 2 ;

[0065] Where A represents the molar ratio of glucose to ferrous sulfate, B represents the concentration of concentrated sulfuric acid, and C represents the reaction time. Figure 1 As can be seen, the experimental results have a strong correlation with the predicted values, such as... Figure 1 As shown.

[0066] The interaction effects among three factors—the molar ratio of glucose to ferrous sulfate, the concentration of concentrated sulfuric acid, and the reaction time—were further analyzed. Figure 2 The effect of the interaction between the molar ratio of glucose to ferrous sulfate and the concentration of concentrated sulfuric acid on fluorescence intensity was shown. Figure 2 As can be seen, H2SO4 has a greater effect on fluorescence intensity than the molar ratio of glucose to ferrous sulfate. With increasing H2SO4 content, the fluorescence intensity first increases and then decreases. Figure 3 The effect of the interaction between the molar ratio of glucose to ferrous sulfate and reaction time on fluorescence intensity was shown. Figure 3 As can be seen, the fluorescence intensity increases with increasing reaction time and the molar ratio of glucose to ferrous sulfate, and the effect of reaction time is more significant. Figure 4 The effect of the interaction between concentrated sulfuric acid content and reaction time on fluorescence intensity was shown. Figure 4 As can be seen, with the increase of sulfuric acid content, the fluorescence intensity of the prepared nanozyme first increases and then decreases; with the increase of reaction time, the fluorescence intensity of the nanozyme gradually increases, and the effect of reaction time is more obvious.

[0067] Based on the results analysis, the influence of factors on fluorescence intensity was in the following order: Factor C (reaction time) > Factor B (H2SO4 content) > Factor A (molar ratio of glucose to ferrous sulfate), consistent with the model prediction (F values ​​were 41.72, 3.37, and 3.175 × 10⁻⁶, respectively). -7 Based on model optimization, the optimal preparation conditions were determined as follows: A: molar ratio of glucose to ferrous sulfate = 3; B: H2SO4 content = 0.26 mL (i.e., volume fraction relative to water solvent = 0.52%); C: reaction time = 12 h. Under these optimal conditions, S,Fe doped carbon nanodot nanozymes were prepared and used in subsequent detection, analysis and application processes.

[0068] Test case

[0069] 1. Transmission electron microscopy (TEM) analysis:

[0070] The structure and particle size of the prepared S,Fe-doped carbon nanoparticle nanozymes were analyzed by transmission electron microscopy (TEM), such as... Figure 5 As shown in the figure, the prepared S,Fe-doped carbon nanoparticles are uniformly distributed, with a lattice size of approximately 0.21 nm, consistent with the (100) lattice distance of graphite carbon. Measured using Nano Measurer 1.2 software, the particle size is approximately 3-6 nm.

[0071] 2. Optical property analysis:

[0072] Figure 6 The UV-Vis absorption, fluorescence excitation, and fluorescence emission curves of S,Fe-doped carbon nanozymes prepared under optimal conditions are shown in the figure. As can be seen from the figure, the S,Fe-doped carbon nanozymes exhibit a broad absorption band in the 220–400 nm range. The optimal excitation wavelength is 366 nm, and the optimal emission wavelength is 440 nm. The calculated fluorescence quantum yield of the S,Fe-doped carbon nanozymes is 50.14%.

[0073] 3. Enzyme activity evaluation:

[0074] The enzyme activity of the prepared S,Fe-doped carbon dot nanozyme (o-PD) was evaluated using o-phenylenediamine (o-PD) as a substrate. Figure 7 As shown, S,Fe-doped carbon nanodot nanozymes can promote the oxidation of o-PD to brown oxidized o-PD in the presence of H2O2, and a significant absorption peak appears at 450 nm. However, in different systems such as S,Fe-doped carbon nanodot nanozymes, o-PD, H2O2, o-PD+H2O2, S,Fe-doped carbon nanodot nanozymes+o-PD, and S,Fe-doped carbon nanodot nanozymes+H2O2, no absorption peak appears at 450 nm, further demonstrating that S,Fe-doped carbon nanodot nanozymes possess excellent peroxidase-like activity.

[0075] To further evaluate the activity of S,Fe-doped carbon nanoparticle nanozymes, steady-state kinetic analysis was employed under optimal reaction conditions using o-PD and H₂O₂ as substrates. Michaelis-Menten curves related to o-PD and H₂O₂ were obtained based on the Michaelis-Menten equation. Figure 8 As shown in a and b, the reaction rate of the system increases linearly with increasing H2O2 and o-PD concentrations, and gradually reaches equilibrium after the concentrations exceed a certain range. This indicates that the enzyme-catalyzed reaction of S,Fe-doped carbon nanoparticle nanozymes conforms to the Michaelis-Menten model. According to the fitting calculations, using o-PD as the substrate, K... m and V m 2.85 mM and 36 × 10, respectively -8 M·s -1 When H2O2 is used as the substrate, K m and V m The values ​​were 0.27 mM and 5.61 × 10⁻⁸ M·s, respectively. -1 By comparing with K in existing technologies m and V m In comparison, the S,Fe-doped carbon nanoparticle nanozymes prepared in this application exhibit good catalytic activity.

[0076] Application examples

[0077] To further verify the usability of S,Fe-doped carbon dot nanozymes, this application example constructs an S,Fe-doped carbon dot nanozyme colorimetric probe for the detection of tetracycline. The specific method is as follows:

[0078] The prepared carbon dot nanozyme was mixed with a tetracycline hydrochloride sample and allowed to react fully. During the reaction, tetracycline molecules bound to the functional groups on the surface of the carbon dot nanozyme, leading to a change in the catalytic activity of the carbon dot nanozyme. The absorbance of the oxidized substrate (ox-oPD) after the reaction was measured at 450 nm using a UV-Vis spectrometer. A standard curve was established based on the relationship between the change in absorbance and the concentration of tetracycline (TC), as shown in the figure. Figure 9 As shown in the figure, the absorbance gradually increases with increasing tetracycline concentration. Fitting analysis of absorbance and tetracycline concentration shows high reliability within the range of 6-100 μg / mL, with R0... 2 =0.9902.

[0079] Further investigation into the practical applicability of the established probe was conducted by analyzing the tetracycline content in actual aquaculture wastewater samples. The experimental contents measured are shown in Table 3. It can be seen that the recovery rate is between 103.22% and 110.31%, and the RSD is <6%, indicating that the constructed colorimetric probe has high sensitivity and accuracy in detecting actual samples.

[0080] Table 3 Performance evaluation of probe construction using aquaculture wastewater samples

[0081]

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing carbon dot nanozymes based on response surface methodology optimization, characterized in that, Includes the following steps: Glucose, ferrous sulfate, and concentrated sulfuric acid were dissolved in water by stirring, and then a hydrothermal reaction was carried out. After the reaction was completed, carbon dot nanozymes were obtained by purification. The fluorescence intensity of the carbon dot nanozymes was measured. The molar ratio of glucose to ferrous sulfate, the amount of concentrated sulfuric acid added, and the reaction time were selected as influencing factors. The fluorescence intensity of carbon nanozymes was used as the evaluation value, and the response surface methodology was used for optimization to obtain the regression equation. Finally, the maximum fluorescence intensity of the carbon dot nanozyme and the corresponding molar ratio of glucose to ferrous sulfate, the amount of concentrated sulfuric acid added, and the reaction time were obtained through software optimization. The regression equation is as follows: Y = 2.08 × 10 5 +10.63A-34622.5B+1.22×10 5 C+812.25AB+32007.5AC+12704.75BC+44001.6A 2 -9534.15B 2 +1.11×10 5 C 2 Where Y is the fluorescence intensity of the carbon nanozyme, A is the molar ratio of glucose to ferrous sulfate, B is the amount of concentrated sulfuric acid added, and C is the reaction time. The molar ratio of glucose to ferrous sulfate is 1 to 3, the amount of concentrated sulfuric acid added is 0.2 to 0.6 vol% of the amount of water added, and the reaction time is 8 to 12 hours. The ratio of ferrous sulfate to water is (0.2~0.8) g : 50 mL; S,Fe co-doped carbon dot nanozymes were prepared by hydrothermal synthesis.

2. The preparation method according to claim 1, characterized in that, The molar ratio of glucose to ferrous sulfate is 3, the amount of concentrated sulfuric acid added is 0.52 vol% of the amount of water added, and the reaction time is 12 h.

3. The preparation method according to claim 1, characterized in that, The temperature of the hydrothermal reaction is 150~250℃.

4. The preparation method according to claim 1, characterized in that, The purification steps are as follows: after the hydrothermal reaction is completed, the product is centrifuged, the supernatant is dialyzed, and the dialysate is dried to obtain carbon dot nanozyme.

5. The preparation method according to claim 1, characterized in that, The specific steps for determining the fluorescence intensity of carbon dot nanozymes are as follows: dissolving the carbon dot nanozymes in a solvent to obtain a carbon dot nanozyme solution, fixing the excitation wavelength at 370~450nm, performing fluorescence spectroscopy detection on the carbon dot nanozyme solution, and recording the intensity of the maximum fluorescence emission peak.

6. The carbon dot nanozyme prepared by the preparation method according to any one of claims 1 to 5.

7. The application of the carbon dot nanozyme as described in claim 6 in tetracycline detection.

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