Preparation method and application of Cu / N-CDs

Abstract

The invention discloses a preparation method and application of Cu / N-CDs, and relates to the technical field of Cu / N-CDs preparation. The preparation method comprises the following steps: adding copper citrate, ethidene diamine and ultrapure water together, and ultrasonically stirring uniformly; transferring the obtained solution into a polytetrafluoroethylene reaction kettle, then placing the polytetrafluoroethylene reaction kettle in an electrothermal forced air drying oven, and carrying out hydrothermal reaction at 180 DEG C; naturally cooling, controlling the reaction liquid to be centrifuged, and filtering by adopting a microfiltration membrane; dialyzing the filtrate in ultrapure water for 24 hours by using a dialysis bag, and replacing new ultrapure water every 6 hours; and freeze-drying the dialyzed filtrate to obtain light brown solid powder, and marking the light brown solid powder as Cu / N-CDs. According to the preparation method, a one-step hydrothermal method is adopted, copper citrate and ethylenediamine serve as precursors, the prepared copper-nitrogen co-doped carbon quantum dots show excellent peroxidase-like catalytic activity through the synergistic effect of Cu < + > / Cu < 2 + > redox pairs, and the catalytic efficiency is remarkably improved compared with that of traditional nano-enzymes.

Description

Technical Field

[0001] The present invention belongs to the technical field of Cu / N-CDs preparation, and particularly relates to a preparation method and application of Cu / N-CDs. Background Art

[0002] The controllable synthesis and preparation of CDs are the most important and crucial steps for studying the properties of CDs and expanding their application fields. According to the different sources of raw materials, the preparation of CDs can be roughly divided into two strategies: "top-down" and "bottom-up". The "top-down" synthesis strategy often uses carbon nanotubes, carbon fibers, and graphite as carbon sources, and obtains CDs with a diameter < 10 nm through physical or chemical exfoliation methods; this method usually requires special equipment and harsh reaction conditions such as high temperature, strong acid, and strong base. In contrast, the "bottom-up" synthesis strategy uses more abundant organic small molecule compounds, including citric acid, glucose, polyethylene glycol, and urea, etc. as starting materials, and is prepared by means such as hydro / solvothermal method, microwave synthesis method, and aldol condensation polymerization method. In this strategy, ideal CDs with specific properties can be obtained by adjusting the dosage and ratio of the starting carbon-based raw materials and their combinations and optimizing the reaction conditions. Among all the starting materials, the organic acid-base combination mode with citric acid and ethylenediamine (or urea) as starting materials has been studied more. The CDs prepared by this combination have rich modifiable groups such as -COOH, -OH, -C=O, -NH2, etc. on the surface, showing more excellent fluorescence properties and a wider application field. However, such CDs still have many inherent defects such as lack of surface functional groups and single application field, and far from meeting the requirements of scientific researchers in emerging fields such as drug delivery, bioanalysis, disease treatment, and environmental monitoring. Summary of the Invention

[0003] The purpose of the present invention is to provide a preparation method and application of Cu / N-CDs. By using a one-step hydrothermal method with copper citrate and ethylenediamine as precursors, the process is simple, without harsh conditions, and avoids the high energy consumption and strong acid / alkali pollution problems of the traditional "top-down" method.

[0004] To solve the above technical problems, the present invention is realized through the following technical solutions:

[0005] The present invention provides a preparation method of Cu / N-CDs, including:

[0006] Step 1: Add copper citrate and ethylenediamine into a container, and add ultrapure water and ultrasonic for 10 min to fully dissolve and mix evenly to form a homogeneous solution;

[0007] Step 2: Transfer the above homogeneous solution into a polytetrafluoroethylene reaction kettle, and then place it in an electrothermal blast drying oven for hydrothermal reaction at 180 °C for 8 h;

[0008] Step 3: Wait for natural cooling to room temperature, take out the reaction kettle, centrifuge the reaction solution at a speed of 6000 r·min-1 for 10 min, and filter it through a 0.2 μM microporous filter membrane;

[0009] Step 4: Dialyze the filtrate in a dialysis bag with a molecular weight cut-off of 1000 Da in ultrapure water for 24 h, and replace the fresh ultrapure water every 6 h;

[0010] Step 5: Freeze-dry the dialyzed filtrate to obtain a light brown solid powder, and label it as Cu / N-CDs.

[0011] Furthermore, the dosage ratio of copper citrate to ethylenediamine in Step 1 is 1-3:1-3.

[0012] Cu / N-CDs are used for the detection of anthropogenic pollutants in the environment; specifically including:

[0013] Analyze the peroxidase-like enzyme activity of the obtained Cu / N-CDs and optimize the enzymatic reaction conditions;

[0014] Establish a quantitative determination method for H2O2 and NO2-, and use it to determine the contents of H2O2 and NO2- in water samples and food samples.

[0015] Furthermore, the quantitative analysis method for H2O2 includes:

[0016] Using a 0.1 mM acetic acid-sodium acetate buffer solution with pH 4.0 as the reaction medium, the final concentration of Cu / N-CDs is set to 40 μg·mL -1 , the final concentration of TMB is 500 μM, then add 1.0 mL of H2O2 solution with a known concentration, oscillate and react at 35 °C for 20 min, centrifuge, take the supernatant, and measure the absorbance value of the reaction solution at 652 nm;

[0017] Taking the concentration of H2O2 as the abscissa and the absorbance value at 652 nm as the ordinate, perform a linear regression on the concentration of H2O2 and the absorbance value to obtain a regression equation, and calculate the detection limit of this method according to the KSb / m equation.

[0018] Furthermore, the specific method for quantitatively determining NO2 in the Cu / N-CDs-TMB-H2O2 system - is as follows:

[0019] The final concentration of Cu / N-CDs is set to 40 μg·mL-1, the final concentrations of TMB and H2O2 are both 500 μM, react fully in a 0.1 mM acetic acid-sodium acetate buffer solution with pH 4.0 for 30 min, adjust the pH of the reaction system, and then add a series of NO2 with known concentrations- The solution reacts at a specified temperature for a certain period of time. Using the concentration of NNO2 - as the abscissa and the absorbance values at 438 nm and 652 as the ordinate, a linear regression is performed on the concentration of NO2 - and the absorbance values to obtain a regression equation, and the detection limit of this method is calculated according to the KSb / m equation.

[0020] Furthermore, the Cu / N-CDs are used for the detection of tetracycline in water; it includes colorimetric quantitative determination of TC and fluorescence quantitative determination of TC.

[0021] Furthermore, the colorimetric quantitative determination of TC includes: adding 10 μL of the Cu / N-CDs stock solution to a 0.1 mM PBS buffer solution at pH 8.0, adding different concentrations of TC to a final concentration range of 5 - 50 μM, incubating together at room temperature for 5 min, and then scanning in the wavelength range of 200 - 500 nm to determine the maximum absorption wavelength; at the maximum absorption wavelength, a linear regression is performed on the TC concentration and the absorbance value, and the detection limit of this method is calculated according to the KSb / m equation.

[0022] Furthermore, for the fluorescence quantitative determination of TC: fluorescence measurement is carried out using a 0.1 mM PBS buffer solution at pH 8.0 as the medium. Add 10 μL of the Cu / N-CDs solution and TC control solutions with different concentration gradients into a quartz fluorescence cuvette containing the PBS buffer solution, adjust the final volume of the reaction system to 3.0 mL, after incubating together for 5 min, excite at the optimal excitation wavelength of 358 nm, and obtain a fluorescence spectrum in the emission wavelength range of 375 - 600 nm; at the maximum emission wavelength, a linear regression is performed on the TC concentration and the fluorescence intensity, and the detection limit of this method is calculated according to the KSb / m equation.

[0023] The present invention has the following beneficial effects:

[0024] The present invention adopts a one-step hydrothermal method with copper citrate and ethylenediamine as precursors. The process is simple and does not require harsh conditions. And the copper-nitrogen co-doped carbon quantum dots (Cu / N-CDs) of the present invention exhibit excellent peroxidase-like (POD) catalytic activity through the synergistic effect of the Cu + / Cu 2+ redox pair, and the catalytic efficiency is significantly improved compared with traditional nanozymes; at the same time, the rich oxygen-containing / nitrogen functional groups and amorphous graphite structure on the surface of the quantum dots ensure the chemical stability of the material in a complex environment.

[0025] Of course, it is not necessary for any product implementing the present invention to simultaneously achieve all the above-mentioned advantages. Description of the Drawings

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0027] Figure 1 Effect of different factors on the fluorescence intensity of Cu / N-CDs;

[0028] Figure 2 XRD and FT-IR spectra of Cu / N-CDs;

[0029] Figure 3 TEM, HR-TEM and particle size distribution of Cu / N-CDs;

[0030] Figure 4 XPS survey spectrum and high-resolution spectra of C 1s, N 1s, O 1s, Cu 2p of Cu / N-CDs;

[0031] Figure 5 UV-Vis absorption spectrum, excitation / emission spectrum and fluorescence spectrum of Cu / N-CDs;

[0032] Figure 6 UV-Vis absorption spectra of different systems;

[0033] Figure 7 Effect of quantum dot concentration, pH, reaction time and reaction temperature on the catalase-like activity of Cu / N-CDs;

[0034] Figure 8 Catalase-like enzyme catalytic reaction mechanism of Cu / N-CDs: (a) Effect of radical scavengers on the enzyme activity, (b) ESR determination of the generation of ·OH;

[0035] Figure 9 Cu / N-CDs-TMB-H2O2 system: (a) Visible light scanning spectra at different H2O2 concentrations, (b) Linear relationship between H2O2 concentration and absorbance value;

[0036] Figure 10 For NO2 - During the determination: (a) Effect of reaction pH, (b) Reaction time and (c) Reaction temperature;

[0037] Figure 11 For different concentrations of NO2 --TMBox system: (a) Color change, (b) UV-Vis spectrum, (c) Absorbance at 438 nm and (d) Absorbance at 652 nm vs. NO2 - Concentration linear regression curves;

[0038] Figure 12 Schematic diagram of dual-mode determination of tetracycline by Cu / N-CDs;

[0039] Figure 13 Full wavelength scanning spectra of different systems: (a) TC, TC + Cu / N-CDs, (b) Different concentrations of TC, (c) Different concentrations of TC + Cu / N-CDs, (d) Linear regression curve of absorbance at 402 nm vs. TC concentration;

[0040] Figure 14 Reaction systems of different concentrations of TC-Cu / N-CDs: (a) Fluorescence spectrum, (b) Change in fluorescence intensity at 436 nm;

[0041] Figure 15 For the TC and Cu / N-CDs systems: (a) UV absorption spectrum and (b) Time-resolved fluorescence spectrum;

[0042] Figure 16 Stability of Cu / N-CDs: (a) pH, (b) Storage time, (c) Sodium chloride concentration, and (d) Effect of storage temperature on fluorescence intensity. Detailed implementation mode

[0043] Next, the technical solutions will be described in conjunction with the accompanying drawings in the embodiments of the present invention.

[0044] Using copper citrate and ethylenediamine as starting materials, Cu / N-CDs were synthesized by a classical hydrothermal reaction. The specific preparation method is as follows: Accurately weigh 0.36 g of copper citrate (MW 360.22, 1 mM) and 0.06 g of ethylenediamine (MW 60.10, 1 mM) into a 100 mL clean beaker, add 20 mL of ultrapure water, and ultrasonicate for 10 min to fully dissolve and mix evenly. Transfer the above homogeneous solution to a 100 mL polytetrafluoroethylene reaction kettle, and then place it in an electrothermal blast drying oven for hydrothermal reaction at 180 °C for 8 h. Wait for it to cool naturally to room temperature, take out the reaction kettle, centrifuge the reaction solution at a speed of 6000 r·min-1 for 10 min, and filter it through a 0.2 μM microporous filter membrane to remove insoluble precipitates and macromolecular polymers. Subsequently, the filtrate was dialyzed in ultrapure water using a dialysis bag with a molecular weight cut-off of 1000 Da for 24 h, and fresh ultrapure water was replaced every 6 h to remove the residual copper citrate and ethylenediamine monomers in the filtrate. Finally, the dialyzed filtrate was freeze-dried to obtain a light brown solid powder, which was labeled as Cu / N-CDs.

[0045] The basic principle of synthesizing CDs by hydrothermal reaction is the polymerization reaction of organic monomers under high temperature and high pressure. Therefore, factors such as the type of organic monomers, hydrothermal reaction temperature, and reaction time will affect the formation process, polymerization degree, particle size, and surface functional groups of CDs, and further affect the fluorescence properties of CDs. The effects of hydrothermal reaction temperature, reaction time, and the molar ratio of copper citrate to ethylenediamine on the preparation of Cu / N-CDs were investigated.

[0046] XRD, FT-IR, TEM, XPS and other means were used to systematically test and characterize the crystal plane structure, surface functional groups, appearance morphology, and the surrounding chemical environment of surface elements of Cu / N-CDs. The specific measurement conditions of XRD were: current 20 mA, voltage 40 kV, scanning rate 5°·min -1 , and the scanning range was 10 - 90°. The specific measurement conditions of FT-IR were: KBr tablet pressing, scanning interval 1 cm -1 , and the scanning range was 4000 - 400 cm -1 . The specific test conditions of XPS were: using Al-Kα radiation source, and all data were corrected with C1s (284.6 eV) as the standard. UV-vis and FL were used to measure the optical properties of Cu / N-CDs. The scanning step of UV-vis was 2 nm, and the scanning range was 200 - 400 nm. The scanning step of FL was 1 nm, and the scanning range was 350 - 650 nm.

[0047] Cu / N-CDs were dissolved and diluted with PBS buffer solution with pH 3 - 9 and sodium chloride solutions with different molar concentrations to analyze their fluorescence stability in different environments. The specific steps were: add 20 μL CDs solution into a centrifuge tube containing 3.0 mL of different pH buffer solutions or different molar concentration sodium chloride solutions, shake well for 10 min, and then perform fluorescence spectrum measurement. The prepared Cu / N-CDs with a certain concentration were stored in a 4℃ refrigerator, sampled and measured at regular intervals to investigate their storage stability. The prepared Cu / N-CDs with a certain concentration were placed in a shaker and shaken at 10, 20, 30, 40, 50, and 60℃ for 60 min respectively, sampled and measured to investigate the effect of temperature on their fluorescence stability.

[0048] The reaction temperature, reaction time, and the molar ratio of copper citrate to ethylenediamine in the hydrothermal synthesis process of Cu / N-CDs were changed to investigate the effects of the above conditions on the fluorescence intensity of the synthesized Cu / N-CDs. The experimental results are shown in Figure 1 shown.

[0049] The structure of copper citrate is rich in –COOH, –OH, and –C=O, while the structure of ethylenediamine is rich in –NH2. Under high temperature and high pressure, they are more likely to undergo a polymerization reaction to form Cu / N-CDs.

[0050] Fix the molar ratio of copper citrate to ethylenediamine at 1:1 and the hydrothermal reaction time at 8 h. The results of the influence of the reaction temperature on the fluorescence intensity of Cu / N-CDs are shown in Figure 1 a. When the hydrothermal temperature is 100 °C, the fluorescence intensity of the product is very low, only 16.2% of that at 200 °C, indicating that too low a hydrothermal reaction temperature cannot fully polymerize copper citrate and ethylenediamine to form a quantum dot-like structure. Continuing to increase the hydrothermal reaction temperature, the fluorescence intensity of the obtained product increases significantly. When the temperature reaches 180 °C, the fluorescence intensity of the obtained Cu / N-CDs is 96.4% of that at 200 °C, indicating that copper citrate and ethylenediamine have completely undergone a polymerization reaction at this reaction temperature, forming a large amount of Cu / N-CDs; increasing the reaction temperature to 200 °C, although the fluorescence intensity of the product is the strongest, the increase is not significant. Considering the tolerance of the polytetrafluoroethylene reaction kettle and the electrothermal blast drying oven, 180 °C is selected as the optimal temperature for the hydrothermal reaction.

[0051] Fix the molar ratio of copper citrate to ethylenediamine at 1:1 and the hydrothermal reaction temperature at 180 °C. The results of the influence of different hydrothermal reaction times on the fluorescence intensity of Cu / N-CDs are shown in Figure 1 b. When the reaction time is 2 h, the fluorescence intensity of the reaction solution is 34.6% of the maximum value, indicating that at 2 h, partial polymerization has occurred between ethylenediamine and copper citrate, forming a certain amount of Cu / N-CDs. However, the number of Cu / N-CDs formed is small or the degree of polymerization is insufficient, so the fluorescence intensity is low. Subsequently, as the hydrothermal reaction time is extended, the fluorescence intensity of the obtained product increases. When the reaction time reaches 8 h, the fluorescence intensity of the product reaches the maximum value, meaning that the maximum Cu / N-CDs yield is obtained at this time. Continuing to extend the hydrothermal reaction time, the fluorescence intensity of the sample shows an obvious downward trend. The fluorescence intensity at 12 h drops to 81.3% of that at 8 h. The reason may be that the formed Cu / N-CDs undergo a re-polymerization reaction, the particle size becomes larger, and the unique fluorescence effect of the quantum dots is lost; or the reaction time is too long, and the luminescent groups and defects on the surface of Cu / N-CDs are damaged to a certain extent.

[0052] In the molecular structure of copper citrate, –COOH and –C=O are prone to condensation reactions with –NH2 of ethylenediamine during the hydrothermal reaction to form amide groups, and they are coupled with each other, ultimately leading to the generation of quantum dots. Therefore, only when the two are in an appropriate molar ratio can they rapidly undergo a polymerization reaction to obtain Cu / N-CDs with uniform size and good photoluminescence properties. The results of the influence of the molar ratio between copper citrate and ethylenediamine on the fluorescence intensity of Cu / N-CDs are shown in Figure 1 c. When the proportion of copper citrate is relatively high and the ratio between the two is 3:1, the synthesized sample has strong fluorescence emission, indicating that copper citrate as the carbon source in the reaction system plays a key role in mediating the synthesis of Cu / N-CDs. When the ratio between the two is 2:1, the fluorescence intensity of the obtained sample is the strongest, indicating that under this condition, the two are most likely to polymerize to form Cu / N-CDs. When the proportion of ethylenediamine in the reaction system is further increased, the fluorescence intensity of the obtained sample decreases. When the ratio between the two is 1:3, the fluorescence intensity of the sample is only 56.8% of that under the optimal conditions. Therefore, the molar ratio of 2:1 between copper citrate and ethylenediamine is selected for the synthesis of Cu / N-CDs.

[0053] XRD, FT-IR, TEM, XPS and other testing methods are used to systematically characterize and analyze the crystal plane structure, morphological characteristics, surface functional groups, surface element composition and its surrounding chemical environment of the prepared Cu / N-CDs.

[0054] First, XRD and FT-IR are used to measure and analyze the crystal structure and surface functional groups of the prepared Cu / N-CDs.

[0055] Figure 2 a is the XRD pattern of Cu / N-CDs. It can be seen from the pattern that a relatively obvious diffraction peak appears at 2θ = 26.2°, which is highly consistent with the crystal plane of amorphous graphite carbon, indicating that the doping of Cu and N elements does not destroy the amorphous graphite-like structure of the quantum dots. At the same time, it is found that there are no characteristic diffraction peaks of CuO, Cu2O or elemental Cu in the XRD pattern of Cu / N-CDs. It is preliminarily judged that Cu elements may exist in the form of highly dispersed single atoms or ions in the quantum dots. At the same time, it is found that the intensity of the crystal plane diffraction peak of Cu / N-CDs is significantly lower than that of graphene and carbon nitride reported in the literature, indicating that the crystallinity of the prepared quantum dots is low and exists in an amorphous form, meaning that Cu / N-CDs are more likely to exist in the form of smaller particle sizes. The amorphous form of Cu / N-CDs ensures the high dispersion and full exposure of Cu centers, laying a foundation for its subsequent peroxidase-like enzyme activity and application in the detection of exogenous pollutants.

[0056] Figure 2b is the FT-IR spectrum of Cu / N-CDs. The broad absorption peak centered at 3415 cm -1 should be attributed to the stretching vibrations of O–H and N–H; the two weak absorption peaks at 2942 cm -1 and 2831 cm -1 can be attributed to the symmetric and asymmetric stretching vibrations of C–H in –CH2; the strongest absorption peak at 1635 cm -1 is strong evidence for the presence of C=O on the surface of Cu / N-CDs. The two absorption peaks centered at 1407 cm -1 and 1235 cm -1 are generated by C-N and C-O-C stretching vibrations, indicating that coupling occurred between copper citrate and ethylenediamine in the form of amide groups during the hydrothermal synthesis process. Finally, the two absorption peaks at 809 cm -1 and 621 cm -1 come from the stretching vibrations of Cu–N and Cu–C, indicating that Cu forms a catalytic active center in coordination with surrounding C and N groups in the form of covalent bonds.

[0057] In summary, ethylenediamine has been successfully functionalized on the surface of Cu / N-CDs. The synthesized Cu / N-CDs surface has a large number of hydrophilic groups containing O and N such as –COOH, –C=O, –NH2, and –OH, while Cu is anchored on the surface of the quantum dots in the form of covalent bonds.

[0058] The morphology of the prepared Cu / N-CDs was carefully analyzed by TEM and HR-TEM, and the scanning results are shown in Figure 3 .

[0059] From Figure 3 a and Figure 3 c, it can be seen that Cu / N-CDs are uniformly distributed in a spherical structure with a diameter of about 5-10 nm, and the average particle size is 6.05±0.24 nm, which is consistent with the XRD prediction results: Cu / N-CDs exist in the form of smaller particle sizes. Figure 3 b is the HR-TEM scanning pattern of Cu / N-CDs. The lattice spacing of the quantum dots is 0.21 nm, which is consistent with the diffraction plane of graphite carbon (sp2), further proving the amorphous graphite-like structure of Cu / N-CDs.

[0060] Figure 4 a is the XPS full scan spectrum of Cu / N-CDs. It can be clearly found from the scanning pattern that there are four elements, C, N, O, and Cu, on the surface of Cu / N-CDs, indicating the successful doping of Cu and N elements. Figure 4The high-resolution XPS absorption peaks of C1s were fitted to obtain four absorption peaks centered at 284.1, 285.0, 285.6, and 287.1 eV, which were attributed to the C atoms in C–C / C=C, C–O / C–N, C=O / C=N, and C–O–C chemical bonds, respectively. Figure 4 c is the result of fitting the high-resolution XPS absorption peaks of N1s. The absorption peaks at 398.7 eV and 399.7 eV correspond to the N atoms in C=N–C and C–N chemical bonds, respectively. The above results further confirm that amide bonds are formed between copper citrate and ethylenediamine during the hydrothermal reaction. Figure 4 Three absorption peaks can be fitted from the high-resolution XPS spectrum of O1s in d, which are C=O at 530.0 eV, C–O–C at 531.3 eV, and C–O–H at 532.4 eV, respectively. Figure 4 The high-resolution XPS spectrum of Cu2p in e can be fitted into four different absorption peaks: 931.8 eV and 951.4 eV are from Cu+, and 933.6 eV and 953.8 eV are from Cu 2+ . Both Cu + and Cu 2 + can undergo the following reactions (Equations 2-1 and 2-2). The cycle between Cu+ and Cu 2+ can accelerate the electron transfer and cycle during the catalytic reaction, indicating that Cu / N-CDs have potential high POD-like enzyme activity.

[0061] Cu + + H2O2 → Cu2 + + ·OH Equation 2-1

[0062] Cu2 + + O2 → Cu + + ·O2 – Equation 2-2.

[0063] The ultraviolet absorption spectrum of Cu / N-CDs was obtained by the full-wavelength scanning method. The scanning range was 200 - 400 nm, with an interval of 2 nm. The scanning results are shown in Figure 5a As can be seen from the experimental results, Cu / N-CDs do not show relatively independent absorption peaks, but there are two relatively obvious absorption shoulders at 240 and 268 nm. The absorption peaks in this range theoretically originate from the π→π* transition of C=C double bonds in the quantum dots. The main reason why Cu / N-CDs do not show relatively independent absorption peaks in this range may be the complex chemical environment caused by the diverse functional groups on its surface: the π→π* transition process becomes extremely complex due to C=C, C=O, C=N, C–O–H, and the metal complexation of Cu. Cu / N-CDs still have a certain absorption intensity above 300 nm, and this part of the absorption mainly comes from the defects caused by doped N atoms and the n→π* transition triggered by C–OH near C=C. Figure 5 b Schematic diagram of the maximum excitation wavelength and maximum emission wavelength scanning of Cu / N-CDs. As can be seen from the scanning results, the maximum excitation wavelength of Cu / N-CDs is 358 nm, and the maximum emission wavelength is 436 nm. The inset is a photo of Cu / N-CDs excited at a wavelength of 365 nm using a triple-purpose UV analyzer. It can be seen from the photo that Cu / N-CDs exhibit bright blue fluorescence.

[0064] Change the excitation wavelength and analyze the fluorescence characteristics of Cu / N-CDs at different excitation wavelengths. It is found from the scanning results Figure 5 c that Cu / N-CDs have excitation-light-dependent fluorescence characteristics. As the excitation wavelength increases, the emission wavelength significantly redshifts. When the excitation wavelength is 440 nm, its emission wavelength redshifts to 500 nm, which greatly expands the application range of this type of quantum dot.

[0065] It is known that the best substrate for measuring the POD-like enzyme activity of transition metal ion-doped CDs is TMB. In this reaction system, TMB is oxidized to TMB-ox, showing a bright blue color. This compound can undergo a diazo coupling reaction with NO2 - to obtain an orange-yellow azo compound. NO2 - can be quantitatively determined according to the change in color or absorbance.

[0066] First, analyze the POD-like enzyme activity of the obtained Cu / N-CDs, optimize factors such as pH, temperature, reaction time, and substrate concentration in the enzymatic reaction process, establish a quantitative determination method for H2O2 and NO2 - and use it for the content determination of H2O2 and NO2 - in water samples and food samples.

[0067] The analysis of the POD-like enzyme activity performance of nanozymes mostly uses colorless TMB as the reaction substrate. TMB can accept ·O2 generated during the enzymatic reaction process -Or ·OH forms the oxidation product TMB-ox with a maximum absorption wavelength at 652 nm; TMB-ox has the advantages of high molar extinction coefficient, stable properties, and less interference at the maximum absorption wavelength position, so it is widely used.

[0068] The POD-like enzymatic reaction process of nanozymes is affected by multiple factors, mainly including the pH of the enzymatic reaction system, reaction temperature, reaction time, substrate, and nanozyme concentration. In this chapter, single-factor experiments are used to analyze the effects of the above factors on the POD-like enzyme activity of Cu / N-CDs.

[0069] The freeze-dried Cu / N-CDs were ultrasonically suspended in different pH acetate-sodium acetate buffer solutions to prepare a stock solution of 1 mg·mL -1 for standby. In a 25 mL reaction system, first add a certain amount of Cu / N-CDs stock solution, then add 1 mL of TMB solution dissolved in DMSO (except as otherwise specified, the final concentration of TMB is 500 μM), add 22 mL of the corresponding buffer solution, and finally add 1 mL of H2O2 solution. Place the above system in a thermostatic oscillator and oscillate and react at a certain temperature at a frequency of 100 rpm·min -1 Take a 3.0 mL sample at a specified time, centrifuge, and immediately measure the absorbance value of the system at 652 nm. The higher the absorbance value, the stronger the POD-like enzyme activity.

[0070] After obtaining the optimal reaction conditions for the POD-like enzyme activity of Cu / N-CDs, a radical quenching experiment was used to analyze the types of ROS generated in the Cu / N-CDs-TMB-H2O2 system. Add 10 mM isopropanol, tyrosine, and SOD as ·OH, 1 O2 and ·O2 - quenching agents respectively, analyze their effects on the POD-like enzyme activity of Cu / N-CDs, and determine the types of ROS that mediate the oxidation of TMB in the reaction system.

[0071] The Cu / N-CDs-H2O2 system can oxidize TMB to TMB-ox, and TMB-ox can undergo a diazotization reaction with NO2 - to generate a diazo compound with a maximum absorption wavelength of 438 nm; the increase in the absorbance value of the reaction system at 438 nm and the decrease in the absorbance value at 652 nm have a good correlation with the concentration of NO2 - in the system, which can be used for the quantitative determination of NO2 - in samples.

[0072] The specific method for quantitatively determining NO2 - in the Cu / N-CDs-TMB-H2O2 system is as follows: The final concentration of Cu / N-CDs is set to 40 μg·mL -1, with the final concentrations of TMB and H2O2 both being 500 μM, react fully for 30 min in a 0.1 mM acetic acid - sodium acetate buffer solution at pH 4.0, adjust the pH of the reaction system, and then add a series of NO2 solutions with known concentrations. React for a certain period of time at the specified temperature. Using the concentration of NO2 as the abscissa and the absorbance values at 438 nm and 652 as the ordinate, perform a linear regression on the concentration of NO2 and the absorbance values to obtain a regression equation, and calculate the detection limit of this method according to the KSb / m equation. - solutions, react for a certain period of time at the specified temperature. With the concentration of NO2 - as the abscissa and the absorbance values at 438 nm and 652 as the ordinate, perform a linear regression on the concentration of NO2 - and the absorbance values to obtain a regression equation, and calculate the detection limit of this method according to the KSb / m equation.

[0073] The POD - like enzyme activity of Cu / N - CDs was determined by the full - wavelength scanning method. From the experimental results Figure 6 it can be seen that there is no obvious absorption in the wavelength range of 450 - 800 nm for the TMB and TMB + H2O2 experimental groups, indicating that O2 in the air or H2O2 alone is not sufficient to oxidize TMB to bright - blue TMB - ox. When Cu / N - CDs are added, an obvious absorption peak appears at the center wavelength of 652 nm in the reaction system, which indicates that Cu / N - CDs have significant POD - like enzyme catalytic activity and can catalyze the cleavage of H2O2 to produce ROS, and then ROS oxidizes TMB to bright - blue TMB - ox.

[0074] After determining the POD - like enzyme activity of Cu / N - CDs, the enzymatic reaction conditions, including the dosage of quantum dots, the pH of the reaction system, the reaction temperature, and the reaction time, were optimized.

[0075] Fix the pH of the reaction system at 4.0, with the concentrations of TMB and H2O2 being 500 μM. The experimental results of the effect of the dosage of quantum dots on the POD - like enzyme activity of Cu / N - CDs are shown in Figure 7 a. As the concentration of Cu / N - CDs increases, the absorbance value at 652 nm in the reaction system also increases, indicating that it has concentration - dependent POD - like enzyme activity. When the concentration is 40 μg·mL -1 , the absorbance value reaches 0.92 ± 0.07, showing strong enzymatic reaction performance. When the concentration of quantum dots is further increased to 50 μg·mL -1 , the absorbance value of the reaction system increases to 1.05 ± 0.08, and the increasing amplitude is not significant, indicating that Cu / N - CDs are basically saturated at this concentration. Therefore, in the subsequent factor investigation experiments, fix the concentration of Cu / N - CDs at 40 μg·mL -1 .

[0076] Figure 7b shows the experimental results of the effect of the pH of the reaction system on the activity of Cu / N-CDs mimicking POD. Cu / N-CDs exhibit excellent POD-like activity under weakly acidic conditions. When the pH of the reaction system > 4.5, the POD-like activity of the quantum dots decreases sharply, which is consistent with the catalytic properties of POD-like nanozymes reported in most literature. In-depth analysis of the POD-like enzymatic reaction mechanism shows that when pH ≤ 3.0, H2O2 is easily protonated to H3O2 + , H3O2 + and Cu in Cu / N-CDs + / Cu 2+ active centers carry the same charge, and the nucleophilic reaction between them will be greatly reduced. When pH > 4.5, Cu+ in Cu / N-CDs is easily oxidized and disproportionated to Cu 2+ , and Cu + proportion reduction leads to the blockage of the Cu+ / Cu 2+ cycle, which will greatly reduce the POD-like activity of Cu / N-CDs. Therefore, the pH of the reaction system was fixed at 4.0 in the subsequent factor investigation experiments.

[0077] With other conditions in the system unchanged, the effect of reaction time on the POD-like activity of Cu / N-CDs was investigated, and the experimental results are shown in Figure 7 c. It can be seen from the experimental results that Cu / N-CDs can rapidly catalyze the cleavage of H2O2 in the reaction system to produce ROS, which in turn oxidizes TMB to blue TMB-ox. At 20 min, the absorbance value of the system at 652 nm reaches 0.96 ± 0.08. When the reaction time is further extended, the absorbance value of the system does not increase significantly, indicating that the enzymatic reaction endpoint is basically reached at 20 min, and H2O2 in the system is almost consumed. Therefore, 20 min was determined as the optimal reaction time.

[0078] The experimental results of the effect of different reaction temperatures on the POD-like activity of Cu / N-CDs are shown in Figure 7 d. In the range of 15 - 35 °C of the reaction system, as the temperature increases, the POD-like activity of Cu / N-CDs also increases. When the reaction temperature rises to 45 °C, the POD-like activity of Cu / N-CDs shows an obvious downward trend, and the absorbance value of the reaction system decreases from 1.09 ± 0.04 at 35 °C to 0.87 ± 0.06. When the temperature is further increased to 55 °C, the POD-like activity of Cu / N-CDs is only 52.5% of that at 35 °C. The main reason for the significant decrease in enzyme activity at this time is that H2O2 decomposes at too high a temperature.

[0079] Comprehensively analyzing the results of the single-factor experiments, it was determined for H2O2 and NO2 -The most suitable reaction conditions for the POD-like enzyme activity of Cu / N-CDs in quantitative determination are: Cu / N-CDs concentration of 40 μg·mL -1 , in acetate buffer, pH 4.0, at 35°C for 20 min.

[0080] Nanozymes with POD-like enzyme activity are enzymes that catalyze substrate oxidation using H2O2 as an electron acceptor. In order to determine whether the blue TMB-ox oxidized TMB in the Cu / N-CDs reaction system is produced by catalytic cleavage of H2O2 to produce ·OH, a free radical quenching experiment was first used to verify it. Figure 8 aAfter adding different free radical quenchers, the absorbance change of the reaction system shows that when ·O2 - and 1 The absorbance of the reaction system did not decrease significantly when O2 quenchers SOD and Try were used, indicating that O2 - and 1 O2 is not the main ROS produced by the reaction system. When IPA was added, the absorbance value of the reaction system decreased significantly, indicating that ·OH was the main ROS produced in the reaction system, which mediated the oxidation of TMB. Subsequently, ESR technology was used to further confirm the generation of ·OH in the Cu / N-CDs-H2O2 reaction system. Figure 8 b It can be seen that H2O2 alone did not lead to the generation of any signal. When Cu / N-CDs was added, a strong DMPO-·OH paramagnetic resonance signal with a ratio of 1:2:2:1 was immediately generated, and the generation of ·OH in the Cu / N-CDs-H2O2 system was fully confirmed.

[0081] Figure 9 a is the scanning curve of the reaction system in the visible light region under the optimal reaction conditions with the change of H2O2 concentration. From the experimental results, it can be seen that the system has a maximum absorption at 652nm, and the absorbance value at this position increases with the increase of H2O2 concentration. In the concentration range of 2.5-50μM, the H2O2 concentration in the reaction system shows a good linear correlation with the absorbance value. Figure 9 b, indicating that within the concentration range, Cu / N-CDs can react with H2O2 quantitatively, and then obtain a quantitative TMB-ox blue product. Figure 9 c, the linear equation of H2O2 in the concentration range of 2.5-50μM is: A=0.0125C+0.0248, r=0.9990. The blank reference is scanned continuously 20 times, the standard deviation N of the blank reference is calculated, and the detection limit of H2O2 by this method is 0.11μM according to S / N=3.

[0082] The established Cu / N-CDs-based POD-mimicking colorimetric method can visually detect H2O2. This method is simple, rapid, has a low detection limit, high sensitivity, and shows good application potential.

[0083] The POD enzyme-catalyzed cascade reaction with TMB as the substrate can be used for the quantitative detection of NO2 - To establish a quantitative analysis method for NO2 - Firstly, a single-factor analysis and optimization of the reaction conditions affecting the detection process were carried out. Taking the change in the absorbance value of the reaction system at 438 nm as the evaluation index, the effect of the pH of the reaction system on the determination of NO2 - content was investigated. As shown in Figure 10 a, different pH values have a significant impact on the diazotization reaction, and the reaction activity is the strongest at pH 2.5; when the pH value is further increased, the absorbance of the reaction system at 438 nm decreases significantly, indicating that the diazonium salt produced decreases significantly. This is related to the mechanism of the diazotization reaction: the diazotization reaction is a series of proton transfer processes. The diazotizing reagent is protonated nitrous acid and nitrosyl cation, which requires a strong acidic environment; at the same time, the diazonium salt formed by aryl primary amine and NO2 - will decompose into N2 in a higher pH environment. Therefore, during the determination of NO2 - the pH of the reaction system needs to be adjusted to 2.5 with hydrochloric acid.

[0084] The experimental results of the effect of reaction time on the determination of NO2 - are shown in Figure 10 b. As the reaction time prolongs, the absorbance of the reaction system at 438 nm also increases. However, after 30 min, the absorbance value of the reaction system basically remains unchanged, indicating that the diazotization at 30 min has completely reacted. It is appropriate to select 30 min for the determination of NO2-.

[0085] The experimental results of the effect of reaction temperature on the determination of NO2 - are shown in Figure 10 c. At a relatively low temperature of 10 - 15 °C, the diazotization reaction is relatively complete. When the reaction temperature is further increased, the absorbance of the reaction system decreases significantly. The main reason for the decrease is that the diazonium salt formed at a higher temperature will decompose rapidly. In addition, NO2 - is also prone to decomposition at a higher temperature. Therefore, the temperature for the quantitative determination of NO2 - is selected as 15 °C.

[0086] Under the above optimized conditions, after the Cu / N-CDs-TMB-H2O2 system reacts at 35 °C for 20 min, the pH of the system is adjusted to 2.5 with hydrochloric acid, and different concentrations of NO2 - are added and shaken at 15 °C for 30 min. The color change of the reaction system is shown inFigure 11 a. Without the addition of NO2 - , the reaction system still remained a bright blue color; when NO2 - was added, as the concentration of NO2 - increased, the bright blue color of the reaction system became weaker, gradually turning green and then into golden yellow. The change in color above indicated that NO2 - had undergone a diazotization reaction with TMB-ox in the system, gradually forming a golden yellow diazonium salt.

[0087] The above reaction system was scanned at full wavelength, and the scanning results are shown in Figure 11 b. From the scanning results, it can be seen that as the concentration of NO2 - in the reaction system increased, the absorbance value of the solution at 652 nm decreased proportionally, while the absorbance value at 438 nm increased proportionally, indicating that the concentration of TMB-ox in the reaction system gradually decreased, and the concentration of the diazonium salt formed by the reaction of TMB-ox and NO2 - gradually increased.

[0088] The absorbance values of the reaction system at 652 nm and 438 nm were linearly regressed and fitted with the final concentration of NO2 - respectively, and the linear regression equations were obtained, as shown in Figure 11 c and 11d. From the fitting results, it can be seen that in the concentration range of 5 - 300 μM, the increase in the absorbance of the reaction system at 438 nm showed a good positive linear relationship with the concentration of NO2 - , A = 0.0056C + 0.0017, r = 0.9990;

[0089] Similarly, the decrease in the absorbance of the sample solution at 652 nm showed a good positive linear relationship with the concentration of NO2 - , A = -0.0027C + 0.9745, r = 0.9997. The blank reference was continuously scanned 20 times, the standard deviation N of the blank reference was calculated, and the detection limit of this method for NO2 - was calculated according to S / N = 3: the LOD measured at 438 nm was 2.3 μM, and the LOD measured at 652 nm was 3.6 μM. The above experimental results indicated that the NO2 - in the sample could be quantitatively determined based on the changes in the absorbance values of the reaction system at 438 nm and 652 nm.

[0090] In the following text, TC is the abbreviation of tetracycline, which is a broad-spectrum antibiotic with a benzanthracene ring structure.

[0091] In this paper, the fluorescence of the prepared Cu / N-CDs can also be specifically quenched by TC. At the same time, it was found that after co-incubation of Cu / N-CDs with TC, the maximum absorption wavelength of TC shifted to a longer wavelength, showing a visible yellow color to the naked eye. A colorimetric method can be used for the quantitative determination of TC, such as Figure 12 . Based on this, a fluorescence / colorimetric dual-mode method for the determination of TC using Cu / N-CDs was established in this chapter. The test conditions were optimized, and the TC residues in different water bodies were quantitatively determined.

[0092] Colorimetric quantitative analysis method for TC: Add 10 μL of the Cu / N-CDs stock solution to a 0.1 mM PBS buffer solution at pH 8.0. Add different concentrations of TC to a final concentration range of 5 - 50 μM, and incubate together at room temperature for 5 min. Subsequently, scan in the wavelength range of 200 - 500 nm to determine the maximum absorption wavelength. At the maximum absorption wavelength, perform a linear regression of the TC concentration against the absorbance value, and calculate the detection limit of this method according to the KSb / m equation. At the same time, investigate the precision, reproducibility, and stability of this method.

[0093] Analysis of detection selectivity: Fix the concentration of TC in the determination system at 30 μM, and other potential interfering substances at 300 μM (10 times) (Li + , Na + , K + , Ca 2+ , Mg 2+ and other cations, Cl - , NO3 - , SO4 2- , CO3 2- , PO4 3- and other anions, as well as antibiotics such as AMX, CN, CL, EM, and STR) to analyze the change in the absorbance value of the system and determine the anti-interference ability of this method.

[0094] The fluorescence quantitative determination method for TC is carried out according to the following steps: Use a 0.1 mM PBS buffer solution at pH 8.0 as the medium for fluorescence measurement. Add 10 μL of the Cu / N-CDs solution and different concentration gradients of the TC control solution to a quartz fluorescence cuvette containing the PBS buffer solution. Adjust the final volume of the reaction system to 3.0 mL. After co-incubation for 5 min, excite at the optimal excitation wavelength of 358 nm, and obtain the fluorescence spectrum in the emission wavelength range of 375 - 600 nm. At the maximum emission wavelength, perform a linear regression of the TC concentration against the fluorescence intensity, and calculate the detection limit of this method according to the KSb / m equation. At the same time, investigate the precision, reproducibility, and stability of this method.

[0095] Cu / N-CDs may undergo a complexation reaction with TC, resulting in changes in the UV-visible absorption spectrum of TC. Consequently, a colorimetric method for the determination of TC can be established.

[0096] Figure 13 a shows the UV-visible scanning spectra of 30 μM TC and TC + Cu / N-CDs. From the scanning spectra, it can be seen that TC has a maximum absorption at 356 nm. However, when TC and Cu / N-CDs are mixed, the maximum absorption wavelength of the mixture redshifts to 402 nm, and the absorption intensity increases significantly. Moreover, a new absorption peak appears at 304 nm, indicating that TC and Cu / N-CDs form a stable complex. The absorption peak of the TC + Cu / N-CDs solution at the maximum absorption wavelength of 402 nm is not interfered by TC. Therefore, TC can be quantitatively determined based on the relationship between the absorbance value and concentration of this absorption peak.

[0097] Figure 13 b and 13c are the full-wavelength scanning spectra of different concentrations of TC and TC + Cu / N-CDs, respectively. From the scanning results, it can be seen that the absorbance values of TC and TC + Cu / N-CDs at their respective maximum absorption wavelengths increase with the increase in concentration. The inset is a photo of the above solutions. TC remains almost colorless in the concentration range of 5 - 50 μM, but the color of the TC + Cu / N-CDs system changes from colorless to light yellow that is clearly visible to the naked eye, and the color gradually deepens with the increase in the concentration of TC. This can also visually confirm that TC and Cu / N-CDs form a complex.

[0098] As Figure 13 shown in d, a linear regression was performed on the absorbance values and concentrations of the above complex at different concentrations, and the regression equation was obtained as A = 0.0281C + 0.1007, r = 0.9990, indicating that in the concentration range of 5 - 50 μM, the absorbance value of TC shows a good linear correlation with the concentration. The blank control was continuously scanned 20 times, and the LOD value of this colorimetric method was calculated to be 0.71 μM according to the KSb / m equation.

[0099] Cu / N-CDs have excellent blue fluorescence properties. The compound to be detected can cause a decrease in the fluorescence intensity of Cu / N-CDs through different fluorescence quenching methods. In a certain concentration range, the decrease value of the fluorescence intensity of Cu / N-CDs shows a good correlation with the concentration of the compound to be detected. Based on this, the compound to be detected can be quantitatively determined. As Figure 14 shown in a, with the increase in the addition amount of TC in the reaction system, the fluorescence intensity of Cu / N-CDs at 436 nm gradually decreases, indicating that TC has a relatively obvious quenching effect on the fluorescence emission of Cu / N-CDs. A ratio fluorescence detection method can be constructed with the change in fluorescence intensity at 436 nm as the response signal for the quantitative detection of TC.Figure 14 b is the change in the fluorescence intensity of Cu / N-CDs at 436 nm under the action of different TCs. It can be seen from the experimental results that in the concentration range of 0 - 80 μM, the decrease in the fluorescence intensity of Cu / N-CDs at 436 nm has a good linear relationship with the concentration of TC. The regression equation is F = -3.525C + 879.13, and r = 0.9995. The blank control was continuously scanned 20 times, and the LOD value for the determination of TC by this fluorescence ratio method was calculated to be 0.31 μM according to the KSb / m equation.

[0100] UV-vis absorption spectroscopy and time-resolved fluorescence spectroscopy were used to analyze the principle of the fluorescence ratio method for determining the content of TC with Cu / N-CDs. First, the UV-vis absorption spectra of TC and Cu / N-CDs were measured. As Figure 15 shown in a, TC has a strong absorption in the range of 200 - 400 nm, almost covering all the excitation spectra of Cu / N-CDs. This enables the excitation energy of Cu / N-CDs to be effectively absorbed by TC, thereby leading to fluorescence quenching. Therefore, IFE may be one of the main reasons for the quenching of Cu / N-CDs fluorescence by TC. To further elaborate the quenching mechanism, the time-resolved fluorescence spectra of Cu / N-CDs and TC + Cu / N-CDs were measured 15b. By fitting, the fluorescence lifetime of Cu / N-CDs is 6.37 ns, and the fluorescence lifetime of the TC + Cu / N-CDs system is 6.32 ns, with almost no change, indicating that there is no energy transfer between the two. This further confirms that the fluorescence quenching of Cu / N-CDs caused by TC is triggered by the IFE effect.

[0101] The fluorescence intensity of Cu / N-CDs was measured at the most suitable excitation wavelength, and with a quinine sulfate solution as the reference, its fluorescence quantum efficiency was calculated according to Equation 2 - 3.

[0102]

[0103] Among them, is the fluorescence quantum yield, F is the fluorescence integral area, A is the absorbance value at the corresponding excitation wavelength, θ is the refractive index of the solvent, R represents quinine sulfate, and X represents Cu / N-CDs.

[0104] It was determined that the fluorescence quantum efficiency of Cu / N-CDs is 15.6%, showing good fluorescence emission ability. CDs with great application value should have broad environmental adaptability and good stability; Figure 16a shows the change in the fluorescence intensity of Cu / N-CDs under different pH conditions. It can be seen from the experimental results that except when the pH is 3.0 and the fluorescence intensity decreases slightly, within the relatively wide pH range of 4.0 - 9.0, the fluorescence intensity of Cu / N-CDs remains almost unchanged, indicating that it is not sensitive to pH. The Cu / N-CDs solution was stored in a refrigerator at 4 °C, taken out at regular intervals, and the change in its fluorescence intensity with storage time was measured. From the experimental results Figure 16 as shown in b, the storage time has no significant effect on the fluorescence intensity of Cu / N-CDs, and no precipitation appears in the Cu / N-CDs solution, indicating that it has strong hydrophilicity, can be fully dissolved and highly stably dispersed in aqueous solution. The presence of electrolytes will affect the suspension performance of CDs in the liquid phase, cause flocculation or aggregation of CDs, and thus affect the fluorescence excitation and emission of CDs. Figure 16 c shows the effect of ionic strength on the fluorescence intensity of Cu / N-CDs. When the concentration of NaCl in the system gradually increases from 0 to 1.0 mol·L -1 , the fluorescence intensity of Cu / N-CDs hardly changes at all, and the solution remains clear and transparent, indicating that Cu / N-CDs has good salt tolerance and still maintains good stability and fluorescence characteristics in an environment with a high ionic strength. Figure 16 d shows the effect of different temperatures on the fluorescence intensity of Cu / N-CDs. It can be seen from the experimental results that within the temperature range of 10 - 40 °C, Cu / N-CDs maintains good fluorescence stability. When the temperature is higher than 40 °C, its fluorescence intensity shows an obvious downward trend. The main reason is that as the temperature increases, the irregular Brownian motion of CDs is enhanced, and the collision between CDs leads to energy loss.

[0105] In summary, Cu / N-CDs prepared from copper citrate and ethylenediamine as raw materials have good stability, can tolerate high ionic concentrations, can be stored for a long time, can be used within a relatively wide pH range, and still maintain good stability when applied in an environment below 40 °C.

Claims

1. A preparation method of Cu / N-CDs, characterized in that, Including: Step 1: Add copper citrate and ethylenediamine into a container, and add ultrapure water and ultrasonicate for 10 min to fully dissolve and mix them evenly to form a homogeneous solution; Step 2: Transfer the above homogeneous solution into a polytetrafluoroethylene reaction kettle, then place it in an electrothermal blast drying oven and carry out hydrothermal reaction at 180 °C for 8 h; Step 3. Wait for natural cooling to room temperature, take out the reaction kettle, and centrifuge the reaction solution at a speed of 6000 r·min -1 for 10 min and filter it through a 0.2 μM microporous filter membrane; Step 4: Dialyze the filtrate with a dialysis bag with a molecular weight cut-off of 1000 Da in ultrapure water for 24 h, and replace the fresh ultrapure water every 6 h; Step 5: Freeze-dry the dialyzed filtrate to obtain a light brown solid powder, which is labeled as Cu / N-CDs.

2. The preparation method of a kind of Cu / N-CDs according to claim 1, characterized in that, In the said Step 1, the dosage ratio of copper citrate to ethylenediamine is 1-3:1-3.

3. Application of Cu / N-CDs prepared by the preparation method of Cu / N-CDs according to claim 1 or 2, characterized in that, The said Cu / N-CDs are used for the detection of anthropogenic pollutants in the environment.

4. The application according to claim 3, wherein Specifically including: Analyze the peroxidase-like enzyme activity of the obtained Cu / N-CDs and optimize the enzymatic reaction conditions; Establish a quantitative determination method for H2O2 and NO 2- and apply it to the determination of the contents of H2O2 and NO 2- in water samples and food samples.

5. The application according to claim 4, wherein The quantitative analysis method of the said H2O2 includes: Using 0.1 mM acetic acid - sodium acetate buffer solution with pH 4.0 as the reaction medium, the final concentration of Cu / N-CDs was set at 40 μg·mL -1 , the final concentration of TMB was 500 μM, then 1.0 mL of H2O2 solution with a known concentration was added, and the reaction was oscillated at 35 °C for 20 min, centrifuged, the supernatant was taken, and the absorbance value of the reaction solution was measured at 652 nm; Taking the concentration of H2O2 as the abscissa and the absorbance value at 652 nm as the ordinate, conduct a linear regression on the concentration of H2O2 and the absorbance value to obtain a regression equation, and calculate the detection limit of this method according to the KSb / m equation.

6. The application according to claim 4, characterized in that, Quantitative determination of NO by Cu / N-CDs-TMB-H2O2 system 2- The specific method is as follows: The final concentration of Cu / N-CDs was set at 40 μg·mL -1 , and the final concentrations of TMB and H2O2 were both 500 μM. They were fully reacted in a 0.1 mM acetic acid-sodium acetate buffer solution at pH 4.0 for 30 min. Then, the pH of the reaction system was adjusted, and a series of NO 2- solutions were added. The reaction was carried out at a specified temperature for a certain time. With the concentration of NO 2- as the abscissa and the absorbance values at 438 nm and 652 as the ordinate, a linear regression was performed on the concentration of NO 2- and the absorbance values to obtain the regression equation, and the detection limit of this method was calculated according to the KSb / m equation.

7. The application according to claim 3, wherein The said Cu / N-CDs are also used for the detection of tetracycline in water; including colorimetric quantitative determination of TC and fluorescence quantitative determination of TC.

8. The application according to claim 7, wherein The colorimetric quantitative determination of TC includes: Add 10 μL of the Cu / N-CDs stock solution into a 0.1 mM PBS buffer solution with pH 8.0, add different concentrations of TC to a final concentration range of 5-50 μM, incubate together at room temperature for 5 min, then scan in the wavelength range of 200-500 nm to determine the maximum absorption wavelength; at the maximum absorption wavelength, conduct a linear regression on the TC concentration and the absorbance value, and calculate the detection limit of this method according to the KSb / m equation.

9. The application according to claim 7, wherein The fluorescence quantitative determination of TC: Conduct fluorescence measurement with a 0.1 mM PBS buffer solution with pH 8.0 as the medium. Add 10 μL of the Cu / N-CDs solution and different concentration gradients of the TC control solution into a quartz fluorescence cuvette filled with the PBS buffer solution, adjust the final volume of the reaction system to 3.0 mL, after incubating together for 5 min, excite at the optimal excitation wavelength of 358 nm, and obtain a fluorescence spectrum in the emission wavelength range of 375-600 nm; at the maximum emission wavelength, conduct a linear regression on the TC concentration and the fluorescence intensity, and calculate the detection limit of this method according to the KSb / m equation.

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