Synthesis of nano enzyme and application of nano enzyme in detection of dichlorvos

Abstract

The invention provides a nano-enzyme synthesis method which comprises the following specific steps: sequentially heating, drying and grinding an aqueous solution containing 0-generation polyamide-amine dendritic macromolecules, potassium ferricyanide and urea, carrying out high-temperature pyrolysis twice, and grinding again to obtain the target nano-enzyme. The invention also provides a corresponding dichlorvos detection method, which comprises the following operation steps: mixing a sample solution, a buffer solution and a nano-enzyme solution for incubation, and then adding the mixed solution into a borate buffer solution containing hydrogen peroxide for reaction; and after the reaction is finished, sequentially adding a potassium iodide solution and a rhodamine 6G solution, and carrying out fluorescence detection after the reaction is finished. The nano-enzyme synthesized by the method has multiple enzymatic characteristics of oxidase-like, peroxidase-like, catalase-like and superoxide dismutase-like, has the advantages of excellent selectivity and high sensitivity in detection of dichlorvos, and is suitable for rapid detection of dichlorvos in actual samples.

Description

Technical Field

[0001] This invention relates to the preparation and application of nanoenzymes based on carbon nanodots, belonging to the field of nanomaterials technology. Background Technology

[0002] Nanozymes are a new class of next-generation artificial enzymes with enzyme-mimicking catalytic properties, playing a key role in driving innovation in the field of catalysis. Compared with natural enzymes, nanozymes have many advantages, such as simple synthesis, easy storage, high stability, high cost-effectiveness, and diverse functions. In recent years, multi-enzyme nanozymes have shown great potential in high-end applications such as biosensing, environmental monitoring, and disease diagnosis due to their multifunctional integration, synergistic catalytic effect, cascade reaction capability, and selective environmental responsiveness. However, the synthesis of multifunctional enzyme-like nanomaterials is also a common challenge in the industry (Angewandte Chemie-International Edition, 56 (2017) 14267-14271).

[0003] Dichlorvos is an organophosphorus pesticide widely distributed in the environment, posing a significant threat to water quality, ecosystems, and human health. Therefore, developing rapid, sensitive, and reliable detection methods for this pollutant is a crucial task for analytical chemists. Existing analytical methods include gas chromatography, liquid chromatography, capillary electrophoresis, atomic absorption spectrometry, ion chromatography, electrochemical methods, mass spectrometry, fluorescence spectroscopy, and colorimetry. Among these, the enzyme inhibition strategy based on acetylcholinesterase is the most commonly used detection method, offering the advantage of rapid detection without complex pretreatment. However, acetylcholinesterase is expensive and unstable, and many organophosphorus pesticides can induce non-specific enzyme inhibition, limiting the detection specificity and practical application value of this technology (Journal of Food Composition and Analysis, 143 (2025)107603). Summary of the Invention

[0004] The technical problem solved by this invention is to provide a method for synthesizing and applying nanozymes with multiple enzyme activities, enabling convenient, rapid, and selective detection of dichlorvos. The specific technical solution is as follows: A method for preparing a nanozyme includes the following steps: (1) Prepare an aqueous solution of reactants, wherein the aqueous solution of reactants contains 0-generation polyamide-amine dendritic macromolecules, potassium ferricyanide and urea; (2) The aqueous solution of the reactants is ultrasonically treated for 30 min and then heated in an oven for 3 hours; (3) After drying the product at 100 °C, transfer it to a tube furnace for a preliminary high-temperature treatment for 2 hours; (4) Grind the cooled product into fine powder, place it in a tube furnace for high-temperature treatment for 2 hours, cool it to room temperature, and grind it into fine powder again to obtain nanozyme.

[0005] Preferably, the molar ratio of 0th generation polyamide-amine dendritic macromolecules, potassium ferricyanide and urea is 1:1:(0.4~0.7).

[0006] Preferably, the concentration of the 0th generation polyamide-amine dendritic macromolecule in step (1) is 0.05~0.2 mol / L.

[0007] Preferably, the oven heating temperature in step (2) is 180~240 ℃.

[0008] Preferably, the high-temperature treatment temperature in step (3) is 350~420 °C.

[0009] Preferably, the high-temperature treatment temperature in step (4) is 600~700 °C.

[0010] Preferably, in steps (3) and (4), the heating rate of the tube furnace is 5 °C / min, and the protective atmosphere is nitrogen or argon.

[0011] A method for measuring dichlorvos includes the following steps: (1) Take 10 µL of sample and mix it with 50 µL of 0.1 mg / mL nanozyme, and incubate at 50 °C for 40 minutes; (2) Mix the resulting mixture with 790 µL borate buffer, 100 µL 50 mM H2O2 and 100 µL triple-distilled water; (3) After vortexing the obtained solution at room temperature for 30 minutes, centrifuge at 8000 rpm; take 0.5 mL of supernatant, add 90 µL of 2 M H2SO4 and 240 µL of 1 M KI, vortex for 5 minutes, add 160 µL of 100 µM Rhodamine 6G, vortex again for 5 minutes, and finally dilute to 2 mL with triple-distilled water; (4) After diluting the obtained solution by 200 times, fluorescence was measured. The excitation wavelength was 525 nm and the emission wavelength was 550 nm.

[0012] Preferably, the concentration of borate in the buffer solution in step (2) is 10~30 mmol / L.

[0013] Preferably, the centrifugation time in step (3) is 5 to 15 minutes.

[0014] The nanozymes synthesized using the method of this invention not only possess multiple enzyme properties, including oxidase-like, peroxidase-like, catalase-like, and superoxide dismutase-like characteristics, but also exhibit selective response to dichlorvos (DDVP) based on their catalase-like activity. Other organophosphorus pesticides do not interact with the synthesized nanozymes. The nanozymes provided by this invention possess multiple enzyme activities, enabling highly selective and sensitive detection of dichlorvos in complex samples. Attached Figure Description

[0015] Figure 1 Transmission electron microscopy (TEM) image of PAMAM-Fe carbon dots (A); PAMAM-Fe carbon dot size distribution (B); Fourier transform infrared (FT-IR) spectrum of PAMAM-Fe carbon dots (C); X-ray photoelectron spectroscopy (XPS) full spectrum of PAMAM-Fe carbon dots (D); and high-resolution XPS spectra of C 1s (E), O 1s (F), Fe 2p (G) and N 2p (H).

[0016] Figure 2 High-resolution transmission electron microscopy image of PAMAM-Fe carbon dots

[0017] Figure 3 (A) UV-Vis absorption spectra of the oxidase-like colorimetric system after 15 minutes of reaction: (a) PAMAM-FeCDs + TMB, (b) TMB, (c) PAMAM-FeCDs; (B) Absorbance spectra of the colorimetric system after 15 minutes of reaction under different gas environments (oxygen, air, nitrogen); (C) UV-Vis absorption spectra of the peroxidase-like colorimetric system after 15 minutes of reaction: (a) PAMAM-FeCDs + TMB + H2O2, (b) PAMAM-FeCDs + TMB, (c) TMB + H2O2, (d) PAMAM-FeCDs + H2O2; (D) UV-Vis absorption spectra of the catalase-like activity assessment system after 10 minutes of reaction: (a) PAMAM-FeCDs + H2O2 + NaVO3, (b) H2O2 + NaVO3.

[0018] Figure 4 (A) ·OH verification experiment (a) without IPA, (b) with IPA; (B) ¹O2 verification analysis (a) without HD, (b) with HD; (C) h (a) without EDTA, (b) with EDTA. + Verification experiment; (D) Fluorescence spectra of PAMAM-Fe CDs, DHE, and PAMAM-Fe CDs reacting with DHE for 30 minutes.

[0019] Figure 5 (A) Visible absorption spectra of mixed solutions of NBT, riboflavin, methionine and EDTA under different conditions: (a) Blank control without light, (b) Illuminated but without nanomaterials, (c) Illuminated and with PAMAM-Fe CDs present; (B) Scavenging efficiency of different concentrations of PAMAM-Fe CDs for superoxide anion radicals: (a) 0 mg / mL, (b) 0.5 mg / mL, (c) 1.0 mg / mL, (d) 1.5 mg / mL, (e) 2.0 mg / mL, (f) 3.0 mg / mL, (g) 4.0 mg / mL; (C) Relative superoxide dismutase (SOD)-like activity of PAMAM-Fe CDs.

[0020] Figure 6 Enzyme kinetics of PAMAM-FeCDs nanozymes with catalase-like activity

[0021] Figure 7 (A) Representative fluorescence spectra of the sensing system at different dichlorvos concentrations; (B) Linear correlation between fluorescence intensity variation (ΔF) and the logarithm of dichlorvos concentration. Error bars represent the standard deviation of three independent experiments.

[0022] Figure 8 The selective detection platform of this invention for dichlorvos Detailed Implementation

[0023] The inventors accidentally discovered during their research that mixing 0th generation polyamide-amine dendrimer (GO-PAMAM), potassium ferricyanide, and urea in a specific ratio, followed by appropriate treatment, yielded nanomaterials exhibiting properties similar to oxidases, peroxidases, catalases, and superoxide dismutases. No reports have been found of GO-PAMAM-based nanozymes possessing four or more enzymatic properties. More interestingly, the catalase activity of this nanozyme selectively responds to dichlorvos, while other organophosphorus pesticides do not interact with the synthesized nanozyme. Based on this, the present invention provides a method for the synthesis and application of multi-active nanozymes, achieving highly selective and sensitive detection of dichlorvos in complex samples. The specific scheme is as follows: A method for preparing a nanozyme includes the following steps: (1) Prepare an aqueous solution of reactants, wherein the aqueous solution of reactants contains 0-generation polyamide-amine dendritic macromolecules, potassium ferricyanide and urea; (2) The aqueous solution of the reactants is ultrasonically treated for 30 min and then heated in an oven for 3 hours; (3) After drying the product at 100 °C, transfer it to a tube furnace for a preliminary high-temperature treatment for 2 hours; (4) Grind the cooled product into fine powder, place it in a tube furnace for high-temperature treatment for 2 hours, cool it to room temperature, and grind it into fine powder again to obtain nanozyme.

[0024] The molar ratio of the mixture of the 0th generation polyamide-amine dendritic macromolecule, potassium ferricyanide, and urea is 1:1:(0.4~0.7); the concentration of the 0th generation polyamide-amine dendritic macromolecule is 0.05~0.2 mol / L; the oven heating temperature is 180~240 °C; the initial high-temperature treatment temperature is 350~420 °C; the subsequent high-temperature treatment temperature is 600~700 °C; the heating rate of the tube furnace is 5 °C / min; and the protective atmosphere is nitrogen or argon.

[0025] A method for measuring dichlorvos includes the following steps: (1) Take 10 µL of sample and mix it with 50 µL of 0.1 mg / mL nanozyme, and incubate at 50 °C for 40 minutes; (2) Mix the resulting mixture with 790 µL borate buffer, 100 µL 50 mM H2O2 and 100 µL triple-distilled water; (3) After vortexing the obtained solution at room temperature for 30 minutes, centrifuge at 8000 rpm; take 0.5 mL of supernatant, add 90 µL of 2 M H2SO4 and 240 µL of 1 M KI, vortex for 5 minutes, add 160 µL of 100 µM Rhodamine 6G, vortex again for 5 minutes, and finally dilute to 2 mL with triple-distilled water; (4) After diluting the obtained solution by 200 times, fluorescence was measured. The excitation wavelength was 525 nm and the emission wavelength was 550 nm.

[0026] The concentration of borate in the buffer solution is 10-30 mmol / L; the centrifugation time is 5-15 min.

[0027] To further understand the present invention, the synthesis and measurement methods provided by the present invention will be described in detail below with reference to the embodiments. The scope of protection of the present invention is not limited by the following embodiments.

[0028] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0029] Example 1: Synthesis and Surface Physicochemical Characterization of Nanozymes 1.1 Synthesis of Nanozymes Two mL solutions containing 0.1 mol / L G0-PePAMAM, 0.1 mol / L potassium ferricyanide, and 0.05 mol / L urea were sonicated for 30 minutes. The mixture was then transferred to a 10 mL PTFE-lined stainless steel reactor (Xi'an Gauge Instruments, Shaanxi, China) and heated in an oven at 220 °C for 3 hours. After cooling to room temperature, the solution was dried overnight at 100 °C. The dried product was transferred to a quartz boat and placed in a tube furnace. Under a nitrogen atmosphere, the temperature was increased to 400 °C at a rate of 5 °C / min and held for 2 hours. The solid product was then removed, ground into a fine powder, and placed back into the quartz boat. The furnace was then heated to 650 °C at a rate of 5 °C / min and held for 2 hours under a nitrogen atmosphere. After cooling to room temperature, the product was ground into a fine powder to obtain the final PAMAM-FeCDs nanozyme.

[0030] 1.2 Characterization of PAMAM-FeCDs nanozymes 1.2.1 Transmission Electron Microscopy Characterization The morphology, size, and structural characteristics of PAMAM-Fe nanozymes were analyzed using transmission electron microscopy (TEM). TEM images ( Figure 1 A) shows that the carbon dots are uniformly sized, well-dispersed quasi-spherical particles. Statistical analysis of the particle diameter indicates that their size distribution is narrow, ranging from 1.4 to 2.8 nm, with an average diameter of 2.1 nm. Figure 1 B). High-resolution transmission electron microscopy (HRTEM) images ( Figure 2 Further investigation confirmed that the particle has a clear lattice structure with a lattice spacing of 0.21 nm, corresponding to the (100) crystal plane of graphite carbon, indicating that the carbon dot has a graphite-like structure.

[0031] 1.2.2 Infrared Spectroscopic Characterization The composition and surface functional groups of PAMAM-Fe carbon dots were identified and characterized using Fourier transform infrared spectroscopy (FTIR). Figure 1 As shown in C, 3430 cm -1 1606 cm -1 and 1008 cm -1 The characteristic peaks at 1375 cm⁻¹ correspond to the stretching vibrations of OH, C=O, and COC bonds, respectively, indicating the presence of oxygen-containing functional groups on the surface of the carbon dot nanozyme. Furthermore, the characteristic peaks at 1375 cm⁻¹... -1 and 1176 cm -1 The peaks at 2983 cm⁻¹ correspond to the stretching vibrations of CN and CNC bonds, respectively, indicating the presence of nitrogen-containing functional groups. -1 and 2835 cm -1The vibration peak at approximately 2100 cm⁻¹ is attributed to the stretching vibration of the CH bond, while the peak at approximately 2100 cm⁻¹ is attributed to the stretching vibration of the CH bond. -1 The peak at that location is a characteristic signal of the C≡C bond. Additionally, at approximately 800 cm⁻¹... -1 The vibration peak at that point corresponds to the stretching vibration of the Fe-OH bond.

[0032] 1.2.3 X-ray photoelectron spectroscopy characterization The elemental composition and chemical state of PAMAM-Fe carbon dot nanozymes were analyzed using X-ray photoelectron spectroscopy (XPS). Full spectrum ( Figure 1 D) shows characteristic peaks corresponding to C 1s, K 2p, N 1s, O 1s, and Fe 2p, indicating that the carbon dot nanozyme contains C, K, N, O, and Fe elements, with Fe content at 3.44%, indicating that iron has been successfully doped into the carbon dots. High-resolution C1s spectrum ( Figure 1 E) can be decomposed into three characteristic peaks, corresponding to the functional groups CC / C≡C (284.25 eV), CN / CO (285.12 eV), and C=O (287.69 eV), respectively. Furthermore, the O 1s spectrum ( Figure 1 F) shows two characteristic peaks at 530.88 eV and 531.94 eV, corresponding to C=O and C-OH / COC, respectively. (Fe 2p spectrum) Figure 1 G) indicates the presence of Fe. 2+ and Fe 3+ Two valence states, existing in six forms: Fe 2+ 2p 3 / 2 (708.14 eV), Fe 3+ 2p 3 / 2 (711.06 eV), Fe 2+ 2p 1 / 2 (720.76 eV), Fe 3+ 2p 1 / 2 (724.43 eV), Fe 3+ 2p 3 / 2 (716.28 eV) and Fe 3+ 2p 1 / 2 (732.09 eV). Additionally, the N 1s spectrum ( Figure 1 H) shows three characteristic peaks at 397.2 eV, 398.13 eV and 400.73 eV, corresponding to pyridine nitrogen, pyrrole nitrogen and graphitic nitrogen, respectively.

[0033] Example 2: Multi-enzyme activity characterization of nanozymes 2.1 Oxidase

[0034] 2.1.1 Synthesis of Nanozymes Two mL solutions containing 0.05 mol / L G0-PAMAM, 0.05 mol / L potassium ferricyanide, and 0.02 mol / L urea were ultrasonically treated for 30 minutes. The mixture was then transferred to a 10 mL PTFE-lined stainless steel reactor and heated at 180 °C for 3 hours in an oven. After cooling to room temperature, the solution was dried overnight at 100 °C. The dried product was transferred to a quartz boat and placed in a tube furnace. Under a nitrogen atmosphere, the temperature was increased to 350 °C at a rate of 5 °C / min and held for 2 hours. The solid product was then removed, ground into a fine powder, and placed back into the quartz boat. The furnace was then heated to 600 °C at a rate of 5 °C / min and held for 2 hours under a nitrogen atmosphere. After cooling to room temperature, the product was ground into a fine powder to obtain the final PAMAM-FeCDs nanozyme.

[0035] 2.1.2 Enzyme activity detection To investigate the oxidase-like activity of PAMAM-Fe carbon dots, a 1 mL reaction mixture was prepared, consisting of 50 μL PAMAM-Fe carbon dot suspension (0.25 mg / mL), 50 μL TMB (5 mM), 50 μL NaH2PO4 / H3PO4 buffer (pH 3.0), and 850 μL triple-distilled water. After incubation at room temperature for 15 minutes, the mixture was analyzed using UV-Vis spectroscopy, and the absorbance at 652 nm was recorded. Furthermore, PAMAM-Fe carbon dots (0.25 mg / mL) were incubated with DHE (10 µM) for 30 minutes to detect superoxide anion radicals (O2). •- To elucidate the potential mechanism of oxidase activity in the PAMAM-Fe carbon dot colorimetric sensor, isopropanol (IPA, 20 mM), histidine (HD, 20 mM), and ethylenediaminetetraacetic acid (EDTA, 10 mM) were used as hydroxyl radicals (·OH) and singlet oxygen (·OH), respectively. 1 O2) and photogenerated holes (h + ) cleaning agent.

[0036] The oxidase-like activity of PAMAM-Fe carbon dots was evaluated by directly catalyzing the oxidation of TMB in the absence of hydrogen peroxide. Compared with the control group containing only TMB, the catalytic system composed of carbon dots and TMB showed a significantly higher absorbance at 652 nm. Figure 3 A). This enhanced absorbance is attributed to the oxidized TMB product (ox-TMB), indicating that PAMAM-FeCDs possess intrinsic oxidase-like activity.

[0037] To determine whether the oxidase-like activity of PAMAM-FeCDs depends on oxygen, a series of gas-controlled experiments were conducted under a fixed reaction time of 15 minutes. Figure 3 B illustrates the catalytic oxidation of TMB in solutions saturated with different gases (air, oxygen, and nitrogen). Under these conditions, the oxygen-saturated system exhibits the highest absorbance at 652 nm. This indicates that oxygen plays a crucial role in modulating the oxidase-like activity of PAMAM-FeCDs nanozymes, and that the efficient transfer of electrons from PAMAM-FeCDs nanozymes to dissolved oxygen leads to the generation of highly catalytically active reactive oxygen species (ROS).

[0038] To further elucidate the oxidase-like catalytic mechanism of PAMAM-FeCDs nanozymes, it is essential to identify the reactive oxygen species involved in the process. Initially, we focused on detecting potentially generated reactive oxygen species during catalysis, particularly hydroxyl radicals (·OH) and singlet oxygen, by using isopropanol (IPA), histidine (HD), and ethylenediaminetetraacetic acid (EDTA) as scavengers. 1 O2) and photogenerated holes (h + ).like Figure 4 As shown in Figures A, B, and C, the absorbance curves of the oxidase catalytic system did not change after the addition of IPA, HD, or EDTA. This indicates that no ·OH groups were generated in the CDs + TMB catalytic system. 1 O2 and h + A key characteristic of oxidases is their ability to catalyze the conversion of dissolved oxygen (O2) into superoxide anion radicals (O2⁻). •- We use diethylhydrogen ether (DHE) to detect O2. •- Because DHE undergoes two-electron oxidation to generate the fluorescent substance ethidium, ethidium produces significant fluorescence emission at 610 nm under 520 nm excitation. For example... Figure 4 As shown in Figure D, the PAMAM-FeCD solution without DHE showed no fluorescence after incubation, while the DHE-only solution also exhibited only weak fluorescence. In contrast, the mixture of CDs and DHE resulted in a significant increase in fluorescence intensity. These results indicate that in the CDs + TMB system, dissolved oxygen accepts electrons transferred from PAMAM-FeCDs and is subsequently converted into O2. •- This mechanism supports the high catalytic activity of CDs, thereby promoting the direct oxidation of TMB to the chromogenic product ox-TMB. 2.2 Peroxidase

[0039] 2.2.1 Synthesis of Nanozymes Except for the initial solution being a 2 mL mixed solution containing 0.05 mol / L, 0.05 mol / L, and 0.028 mol / L GO-PePAMAM, potassium ferricyanide, and urea, respectively, the rest is the same as in 2.1.1.

[0040] 2.2.2 Enzyme activity detection To evaluate the peroxidase-like activity of PAMAM-Fe carbon dots, a 1 mL reaction mixture was prepared, consisting of: 50 μL PAMAM-Fe carbon dot suspension (0.25 mg / mL), 100 μL hydrogen peroxide (H2O2, 10 mM), 50 μL TMB (5 mM), 50 μL NaH2PO4 / H3PO4 buffer (pH 3.0), and 750 μL triple-distilled water. After incubating the mixture at room temperature for 15 minutes, the absorbance of the CDs + H2O2 + TMB system was measured at 652 nm.

[0041] The peroxidase-like activity of PAMAM-Fe carbon dots was assessed by incubating suspensions containing different combinations (CDs + TMB + H2O2, CDs + TMB, TMB + H2O2, CDs + H2O2) for 15 minutes and then monitoring the changes in absorbance. Figure 3 As shown in Figure C, the absorbance of the suspensions of TMB + H2O2 and CDs + H2O2 was extremely low (negligible). In contrast, the absorbance of the suspensions of CDs + TMB + H2O2 (absorbance A = 0.460 at 652 nm) and CDs + TMB (absorbance A = 0.208 at 652 nm) was significantly increased. These results indicate that PAMAM-FeCDs possess significant peroxidase-like activity. 2.3 Catalase

[0042] 2.3.1 Synthesis of Nanozymes Two mL solutions containing 0.2 mol / L G0-PePAMAM, 0.2 mol / L potassium ferricyanide, and 0.10 mol / L urea were ultrasonically treated for 30 minutes. The mixture was then transferred to a 10 mL PTFE-lined stainless steel reactor and heated at 240 °C for 3 hours. After cooling to room temperature, the solution was dried overnight at 100 °C. The dried product was transferred to a quartz boat and placed in a tube furnace. Under an argon atmosphere, the temperature was increased to 420 °C at a rate of 5 °C / min and held for 2 hours. The solid product was then removed, ground into a fine powder, and placed back into the quartz boat. The furnace was then heated to 700 °C at a rate of 5 °C / min and held for 2 hours under an argon atmosphere. After cooling to room temperature, the product was ground into a fine powder to obtain the final PAMAM-FeCDs nanozyme.

[0043] 2.3.2 Enzyme activity test The catalase-like (CAT) activity of PAMAM-Fe carbon dots was evaluated according to the method described in the literature. The simplified procedure was as follows: A 1 mL reaction mixture was prepared consisting of 50 μL of PAMAM-Fe carbon dot nanozyme suspension (0.25 mg / mL), 100 μL of hydrogen peroxide (H₂O₂, 100 mM), and 850 μL of borax buffer (pH 9.8). The mixture was vortexed thoroughly at room temperature for 10 minutes and then centrifuged at 8000 rpm for 5 minutes. Subsequently, 0.5 mL of the supernatant was carefully collected and mixed with 160 μL of 4 M hydrochloric acid (HCl) and 320 μL of 1 mg / mL sodium vanadate (NaVO₃). After standing for 8 minutes, the resulting solution was analyzed by UV-Vis spectroscopy.

[0044] The catalase-like activity of PAMAM-Fe carbon dot nanozymes was assessed by monitoring the absorbance change of an orange-red vanadium peroxide complex, a product of the reaction of hydrogen peroxide (H₂O₂) with sodium metavanadate (NaVO₃). Under acidic conditions, the absorbance of the H₂O₂ + NaVO₃ system (curve b) was measured. Figure 3 Compared to system D, CDs + H2O2 + NaVO3 (curve a, Figure 3 The decrease in absorbance at 452 nm corresponding to the vanadium peroxide complex in D) indicates that the PAMAM-FeCDs nanozyme has catalase-like activity. 2.4 Superoxide dismutase

[0045] 2.4.1 Synthesis of Nanozymes Except that the initial solution was a 2 mL mixed solution containing 0.2 mol / L of G0-PePAMAM, 0.2 mol / L of potassium ferricyanide, and 0.14 mol / L of urea, the rest was the same as in 2.3.1.

[0046] 2.4.2 Enzyme activity test To investigate the superoxide dismutase (SOD)-like activity of PAMAM-Fe carbon dot nanozymes, a 1 mL reaction mixture was prepared, consisting of: 100 μL of PAMAM-Fe carbon dot suspensions at different concentrations (0.25, 0.5, 1, 1.5, 2, 3, and 4 mg / mL), 100 μL of nitroblue tetrazolium (NBT, 0.75 mM), 100 μL of riboflavin (0.2 mM), 100 μL of methionine (1.3 mM), 100 μL of ethylenediaminetetraacetic acid (EDTA, 1 mM), and 500 μL of Tris-HCl buffer (pH 8.0). The mixture was then incubated for 10 minutes under a YG-175W UV lamp (Guangdong Yugu, China) at a wavelength of 365 nm and an intensity of 12.80 mW / cm². Finally, the absorbance of the reaction system at 560 nm was measured.

[0047] The SOD activity of PAMAM-Fe carbon dot nanozymes was evaluated using a standard photoreduction assay. This assay consisted of riboflavin (VB2), nitroblue tetrazolium (NBT), methionine, and ethylenediaminetetraacetic acid (EDTA). Under aerobic light irradiation, VB2 promoted the activity of superoxide anion radicals (O2). •- The generation of O2 •- This further reduces NBT to the blue-violet iodonitrobetrazolium formazan (INT-formazan). The solution exhibits a characteristic absorption peak at 560 nm. Figure 5 Curve A (c) shows the absorption peak, while the same absorption peak was not observed under dark conditions (curve a). Importantly, the absorbance decreased significantly after the addition of PAMAM-FeCDs (curve b), indicating that PAMAM-FeCDs possess SOD-like activity and can compete with NBT for O2. •- This inhibits the reduction of NBT. Furthermore, Figure 5 B shows that the superoxide anion scavenging capacity of carbon dots is positively correlated with their concentration. Specifically, when the concentration of PAMAM-FeCDs exceeds 4 mg / mL, approximately 81% of O2 is scavenged. •- Cleared ( Figure 5 C).

[0048] Example 3: Evaluation of catalase activity Using the PAMAM-Fe CDs nanozyme from Example 1, the catalase activity of the PAMAM-Fe carbon dot nanozyme was evaluated by systematically changing the H2O2 substrate concentration. Subsequently, a Lineweaver-Burk double reciprocal plot was constructed. Figure 6 The Michaelis constant (Km) of the PAMAM-FeCDs nanozyme was determined to be 59.3 mM, and the maximum initial rate (Vmax) was 1.73 × 10⁻⁶. -5 M·s -1Table 1 shows that the performance of our synthesized nanozymes is superior to the values ​​reported in the literature.

[0049] Table 1. Comparison of kinetic parameters of PAMAM-Fe carbon dot nanozymes with other reported catalase-like materials.

[0050] References cited in the table: [1] M. Gharib, A. Kornowski, H. Noei, WJ Parak, I. Chakraborty,Protein-Protected Porous Bimetallic AgPt Nanoparticles with pH-SwitchablePeroxidase / Catalase-Mimicking Activity, ACS Mater. Lett., 1 (2019) 310-319. [2] JS Mu, L. Zhang, M. Zhao, Y. Wang, Catalase Mimic Property ofCo3O4 Nanomaterials with Different Morphology and Its Application as a CalciumSensor, ACS Appl. Mater. Interfaces, 6 (2014) 7090-7098. [3] QQ Qiao, JY Wang, K. Long, LW Li, JH Chen, YH Guo, ZQXu, Y. Kuang, TJ Ji, C. Li, A cascaded enzyme system based on the catalase-like activity of Ti3C2T x MXene nanosheets for the efficient combination cancertherapy, Nano Today, 54 (2024) 16, 102059. [4] T.T. Zhang, N.N. Lu, C. Wang, R.S. Zhang, W.J. Chen, Z.Q. Zhang,D.M. Xing, Biomimetic cascade nanoreactor with triple-enzyme mimeticactivities for colorimetric detection of acid phosphatase, Chem. Eng. J., 437(2022) 8, 135267. [5] K.L. Fan, J.Q. Xi, L. Fan, P.X. Wang, C.H. Zhu, Y. Tang, X.D. Xu,M.M. Liang, B. Jiang, X.Y. Yan, L.Z. Gao, In vivo guiding nitrogen-dopedcarbon nanozyme for tumor catalytic therapy, Nat. Commun., 9 (2018) 11, 1440. [6] C.C. Ge, G. Fang, X.M. Shen, Y. Chong, W.G. Wamer, X.F. Gao, Z.F.Chai, C.Y. Chen, J.J. Yin, Facet Energy versus Enzyme-like Activities: TheUnexpected Protection of Palladium Nanocrystals against Oxidative Damage, ACSNano, 10 (2016) 10436-10445. [7] M.Z. Jiao, Z.J. Li, X.L. Li, Z.J. Zhang, Q.P. Yuan, F.Vriesekoop, H. Liang, J.W. Liu, Solving the H2O2 by-product problem using acatalase-mimicking nanozyme cascade to enhance glycolic acid oxidase, Chem.Eng. J., 388 (2020) 9, 124249. Example 4: Study on the detection performance of dichlorvos based on nanozymes Take 10 µL of dichlorvos standard solution and mix it with 50 µL of the PAMAM-FeCDs nanozyme solution synthesized in Example 1 (concentration 0.1 mg / mL). Incubate at 50°C for 40 minutes. Then add 790 µL of 10 mM borate buffer (pH 9.8), 100 µL of 50 mM H2O2, and 100 µL of triple-distilled water to the mixture. Vortex at room temperature for 30 minutes, then centrifuge at 8000 rpm for 5 minutes. Take 0.5 mL of the supernatant and add 90 µL of 2 M H2SO4 and 240 µL of 1 MKI sequentially. Vortex for 5 minutes. Then add 160 µL of 100 µM rhodamine 6G, vortex for 5 minutes, and bring the volume to 2 mL with triple-distilled water. Dilute the resulting solution 200 times and perform fluorescence detection, setting the excitation wavelength to 525 nm and the emission wavelength to 550 nm. The blank control group used triple-distilled water instead of the test solution, and quantitative analysis was performed by calculating the change in fluorescence intensity (ΔF) between the test solution and the blank group.

[0051] As the concentration of dichlorvos increases, the fluorescence intensity at 550 nm gradually decreases. Figure 7 A). In 1×10 -4 Within the concentration range of μg / mL to 10 μg / mL, the change in fluorescence intensity (ΔF) showed a good linear relationship with the logarithm (lgC) of the dichlorvos concentration, and the correlation coefficient (R) was [missing value]. 2 The value is 0.9979. Figure 7 B). The limit of detection (LOD) of the sensing platform was calculated using the formula LOD = 3σ / S, where σ is the standard deviation of the fluorescence intensity of the blank solution (n=11), and S is the slope of the calibration curve. The result showed that the LOD was 1.69 × 10⁻⁶. -5 μg / mL. It is noteworthy that the detection limit reported in this study is comparable to, or even better than, data from recent literature (see Table 2). Table 2 Comparison of different methods for detecting dichlorvos

[0052] References cited in the table: [8] G.Q. Deng, H.Y. Chen, Q. Shi, L.X. Ren, K. Liang, W.J. Long, W.Lan, X.L. Han, Y.B. She, H.Y. Fu, Colorimetric assay based on peroxidase-likeactivity of dodecyl trimethylammonium bromide-tetramethyl zinc (4-pyridinyl)porphyrin for detection of organophosphorus pesticides, Microchim. Acta, 189(2022) 11, 375. [9] G.T. Yi, Z.W. Tao, W.F. Fan, H. Zhou, Q.F. Zhuang, Y. Wang,Copper Ion-Induced Self-Assembled Aerogels of Carbon Dots as Peroxidase-Mimicking Nanozymes for Colorimetric Biosensing of OrganophosphorusPesticide, ACS Sustain. Chem. Eng., 12 (2024) 1378-1387.

[10] S.G. Liu, H.Y. Wang, Q. Zhao, W.L. Gao, X.B. Shi, Z.H. Liu, Aportable colorimetric sensing platform for rapid and sensitive quantificationof dichlorvos pesticide based on Fe-Mn bimetallic oxide nanozyme-participatedhighly efficient chromogenic catalysis, Anal. Chim. Acta, 1292 (2024) 10,342243.

[11] H.F. Cui, T.T. Zhang, Q.Y. Lv, X.J. Song, X.J. Zhai, G.G. Wang,An acetylcholinesterase biosensor based on doping Au nanorod@SiO2nanoparticles into TiO2-chitosan hydrogel for detection of organophosphatepesticides, Biosens. Bioelectron., 141 (2019) 8, 111452.

[12] Y. Wan, H.T. Wang, L. Zhang, Y.X. Chen, S. Li, J. Zhou, Q.Zhang, L.X. Xia, Highly stable acetylcholinesterase electrochemical biosensorbased on polymerized ionic liquids microgel for pesticides detection,Microchim. Acta, 189 (2022) 12, 300.

[13] G.Z. Zhao, B.H. Zhou, X.W. Wang, J. Shen, B. Zhao, Detection oforganophosphorus pesticides by nanogold / mercaptomethamidophos multi-residueelectrochemical biosensor, Food Chem., 354 (2021) 7, 129511.

[14] S. Huang, J.D. Yao, B. Li, G. Ning, Q. Xiao, Integrating target-responsive CD-CdTe QD-based ratiometric fluorescence hydrogel with smartphonefor visual and on-site determination of dichlorvos, Microchim. Acta, 188(2021) 10, 318.

[15] H. Mei, XL Zhu, ZQ Li, JH Jiang, HL Wang, XD Wang,PP Zhou, Manganese dioxide nanosheet-modulated ratiometric fluoroprobebased on carbon quantum dots from okra for selective and sensitive dichlorvosdetection in foods, Food Chem., 434 (2024) 9, 137507.

[16] BY Su, WW Wang, XY Yang, DY Chen, XM Chen, Construction of copper nanoclusters / silicon quantum dots-based ratiometric fluorescent platform for the detection of dichlorvos, J. Food Compos. Anal., 143 (2025) 9, 107603.

[17] YY Yang, JJ Xu, RR Zhou, ZY Qin, CH Liao, SY Shi, YX Chen, Y. Guo, SH Zhang, Coordinated carbon dots-Fe featuring synergistically enhanced peroxidase-like activity at neutral pH for specific and background-free detection of dichlorvos, Carbon, 219 (2024) 12, 118831. Example 5, Method Selectivity Interference resistance is a key attribute for ensuring the reliable performance of the analytical platform. Using the PAMAM-FeCDs nanozyme from Example 1, the selectivity for dichlorvos was evaluated using a series of related substances, including inorganic ions (Cl...). - NO3 - SO4 2- Na + K + Ca2+ Ba 2+ Mn 2+ Mg 2+ Zn 2+ Fe 3+ And Cr(VI)), amino acids (methionine, glycine, and sarcosine), and other organophosphorus pesticides (glyphosate, dibromophos, and acephate). The concentrations of inorganic ions and amino acids were all set at 10 mM, and the concentration of pesticide interfering substances was set at 10 µg / mL, which is 2.2 × 10⁻⁶ times the concentration of dichlorvos (0.1 µg / mL, 0.45 µM). 4 The results showed that the analytical platform responded significantly more strongly to dichlorvos than to the potential interfering substances tested (see [reference needed]). Figure 8 ).

[0053] Example 6: Detection of dichlorvos in actual samples 6.1 Sample preparation

[0054] Samples of tap water (from our laboratory), river water (from Shichahai Lake in Beijing, China), and rainwater (from the campus of Beijing Normal University) were all filtered through a 0.45 μm filter membrane (Beijing Jiude Technology Co., Ltd.) to remove large particulate matter. Subsequently, dichlorvos standard solution (final concentrations of 0.0005, 0.1, and 0.15 μg / mL, respectively) was added to the samples before analysis for spiking.

[0055] Cabbage, rapeseed, and spinach were purchased from a local supermarket. Each sample was chopped and then mixed with dichlorvos standard solution to achieve final concentrations of 0.0005, 0.1, and 0.15 μg / g, respectively. After incubation at 4 °C for 1 hour, 1 g of spiked sample was mixed with 15 mL of acetonitrile and homogenized for 30 minutes. Subsequently, 5 mL of 1 M sodium chloride solution was added to the mixture, and the mixture was vigorously shaken and allowed to stand to promote phase separation. The supernatant was collected and centrifuged at 12,000 rpm for 30 minutes. The resulting supernatant was evaporated to dryness under vacuum, and the residue was reconstituted with 1 mL of borate buffer solution (10 mM, pH 9.8) before analysis. 6.2 Sample Measurement

[0056] Take 10 µL of sample solution and mix it with 50 µL of the PAMAM-FeCDs nanozyme solution synthesized in Example 1 (0.1 mg / mL). Incubate at 50°C for 40 minutes. Then add 790 µL of 30 mM borate buffer (pH 9.8), 100 µL of 50 mM H2O2, and 100 µL of triple-distilled water to the mixture. Vortex at room temperature for 30 minutes, then centrifuge at 8000 rpm for 15 minutes. Take 0.5 mL of the supernatant and add 90 µL of 2 M H2SO4 and 240 µL of 1 M KI sequentially. Vortex for 5 minutes. Then add 160 µL of 100 µM Rhodamine 6G, vortex for 5 minutes, and bring the volume to 2 mL with triple-distilled water. Dilute the resulting solution 200 times and perform fluorescence detection, setting the excitation wavelength to 525 nm and the emission wavelength to 550 nm. The blank control group used triple-distilled water instead of the test solution, and quantitative analysis was performed by calculating the change in fluorescence intensity (ΔF) between the test solution and the blank group. 6.3 Measurement Results and Evaluation

[0057] The detection capability of the constructed sensing platform for trace amounts of dichlorvos in actual samples was studied. Dichlorvos was found in river water and spinach samples (see Tables 1 and 2), but the detected concentrations were all below the limit of quantitation of this method and far below the maximum residue limits (MRLs) specified in Chinese national standards GB 5749-2022 and GB 2763-2021. According to these standards, the maximum residue limits for dichlorvos in cabbage, rapeseed, and spinach are 0.1 mg / kg, 0.2 mg / kg, and 0.5 mg / kg, respectively.

[0058] The accuracy of the method was further evaluated using the standard addition method. Three concentration levels of the target contaminant (including one below the maximum residue limit) were added to each sample. The recoveries of dichlorvos ranged from 91.9% to 106%, with relative standard deviations (RSDs) ranging from 0.716% to 4.03% (Table 3). The results indicate that this method is suitable for the detection of dichlorvos in real-world samples.

[0059] Table 3. Detection of dichlorvos in actual samples (n=3)

[0060] The above embodiments are merely illustrative of the method and core ideas of this invention. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims of this invention.

Claims

1. A method for synthesizing a nano-enzyme, comprising the following steps: (1) preparing a water solution of reactants, wherein the water solution of reactants contains 0-generation polyamidoamine dendrimer, potassium ferricyanide and urea; (2) after ultrasonic treatment of the water solution of reactants for 30 min, placing the water solution of reactants in an oven for heating for 3 hours; (3) after drying the product at 100 ℃, transferring the product to a tube furnace for preliminary high-temperature treatment for 2 hours; (4) after grinding the cooled product into fine powder, placing the fine powder in the tube furnace for later high-temperature treatment for 2 hours, and cooling to room temperature, grinding the fine powder into fine powder again, a nano-enzyme is obtained.

2. The production method according to claim 1, characterized by, The molar ratio of 0-generation polyamidoamine dendrimer, potassium ferricyanide and urea is 1:1:(0.4-0.7).

3. The preparation method according to claim 1, characterized in that, The concentration of 0-generation polyamidoamine dendrimer in step (1) is 0.05-0.2 mol / L.

4. The method of claim 1, wherein, The heating temperature of the oven in step (2) is 180-240 ℃.

5. The preparation method according to claim 1, characterized in that, The temperature of the preliminary high-temperature treatment in step (3) is 350-420 ℃.

6. The method of claim 1, wherein, The temperature of the later high-temperature treatment in step (4) is 600-700 ℃.

7. The preparation method according to claim 1, characterized in that, The heating rate of the tube furnace in steps (3) and (4) is 5 ℃ / min, and the protective atmosphere is nitrogen or argon. 8.A method for measuring dichlorvos, comprising the following steps: mixing 10 µL of a sample with 50 µL of 0.1 mg / mL nano-enzyme, and incubating at 50 ℃ for 40 min; mixing the obtained mixture with 790 µL of a borate buffer, 100 µL of 50 mM H2O2 and 100 µL of triple distilled water; after vortexing the obtained solution at room temperature for 30 min, centrifuging at 8000 rpm; taking 0.5 mL of supernatant, adding 90 µL of 2 M H2SO4 and 240 µL of 1 M KI, vortexing for 5 min, adding 160 µL of 100 µM rhodamine 6G, vortexing again for 5 min, and finally diluting to 2 mL with triple distilled water; after diluting the obtained solution by 200 times, performing fluorescence determination, wherein the excitation wavelength is 525 nm and the emission wavelength is 550 nm.

9. The detection method according to claim 8, characterized in that, The concentration of borate in the buffer in step (2) is 10-30 mmol / L.

10. The detection method of claim 8, wherein, The centrifugation time in step (3) is 5-15 min.

Images