V2O5-NC nano-enzyme as well as preparation and application thereof

Abstract

The invention provides a V2O5-NC nano-enzyme as well as preparation and application thereof. The V2O5-NC nano-enzyme is dark green powder and has a stacked layered structure, the specific surface area is 16-18m < 2 > / g, mesoporous distribution is realized, the average particle size is 20-25nm, and the V2O5-NC nano-enzyme has catalase-like activity. The wide-interlayer-spacing V2O5-NC nano-enzyme based on C-O bond bridging prepared by the invention has relatively good stability, can be prepared on a large scale, is convenient to store, has good affinity to catalytic chromogenic substrates, has high stability, and has universality for the chromogenic reaction of the catalytic chromogenic substrates (TMB, ABTS, DAB and OPD). The V2O5-NC nano-enzyme disclosed by the invention is used for analyzing five organophosphorus pesticides (parathion-methyl, glyphosate, phoxim, methamidophos and chlorpyrifos), has good pesticide recognition capability and interference resistance, and also has the advantages of wide linear range, low detection limit, simplicity and convenience in operation, low detection cost and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of pesticide detection, and specifically relates to a V2O5-NC nanozyme, a preparation method thereof, and an application thereof. Background Art

[0002] In modern agricultural production, organophosphorus pesticides (OPs) are often used to control pests and regulate plant growth. However, the long-term and large-scale use of organophosphorus inevitably leaves residues in food, water bodies and soil, leading to pollution of the food chain and the environment. Long-term exposure to pesticide residues can also cause cancer and neurological diseases. Currently, the main methods for detecting organophosphorus pesticides include high performance liquid chromatography-mass spectrometry, gas chromatography, immunoassay, capillary electrophoresis, fluorescence detection, electrochemical method, etc. Although these methods have the advantages of high sensitivity, low detection limit and accurate detection results, they have limitations in rapid detection due to the complex analysis process, professional operation and high instrument price. Therefore, it is very important to develop sensitive and reliable methods for on-site detection of toxic organophosphorus pesticide residues in environmental media and food.

[0003] In recent years, nanozyme colorimetric sensing has attracted widespread attention due to its advantages such as simple, rapid detection process, low cost, visualization and instant detection. At the same time, the emergence of colorimetric sensor arrays has successfully solved the problems encountered in traditional detection methods. Sensor arrays generate complex response patterns based on non-specific recognition elements, rather than "locked key" sensors. These unique reactions can be further distinguished by LDA or other multivariate statistical methods, which provides a universal method for identifying multiple analytes in complex systems. Nanozymes are widely used in the construction of colorimetric sensor arrays due to their advantages such as easy production, good stability, low cost and high enzyme-like activity. The development of nanozymes suitable for organophosphorus pesticide detection can provide a highly sensitive, selective, simple and rapid method for on-site detection of toxic organophosphorus pesticide residues in environmental media and food. Summary of the Invention

[0004] The purpose of the present invention is to provide a wide-interlayer spacing V2O5-NC nanozyme based on CO bond bridging and its application in the detection of organophosphorus pesticides, providing a highly sensitive, selective, simple and fast method for on-site detection of toxic organophosphorus pesticide residues in environmental media and food.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a method for preparing a V2O5-NC nanozyme, the preparation method comprising the following steps:

[0007] (1) preparing an aqueous solution containing NH4VO3 and H2C2O4;

[0008] (2) adding C3H6N6 to the aqueous solution containing NH4VO3 and H2C2O4 in step (1), stirring and mixing at room temperature, and collecting the precursor by vacuum freeze drying;

[0009] (3) heating the precursor of step (2) to 350-450° C. at a heating rate of 1-5° C. / min for carbonization under an inert gas atmosphere, and further heating the precursor to 550-650° C. at a heating rate of 1-5° C. / min for calcination;

[0010] (4) cooling the calcined product of step (3) to room temperature, and then heating it to 300-350° C. at a heating rate of 0.5-1.5° C. / min in an air atmosphere;

[0011] (5) The calcined product of step (4) is washed alternately with water and alcohol and then dried to obtain the V2O5-NC nanozyme.

[0012] Further preferably, the solvent water of the aqueous solution containing NH4VO3 and H2C2O4 is ultrapure water, and the water used for washing in step (5) is ultrapure water.

[0013] Further preferably, in the step (3), the precursor of step (2) is heated to 380-420°C for carbonization at a heating rate of 1-3°C / min under an inert gas atmosphere, and is further heated to 580-620°C for calcination at a heating rate of 1-3°C / min, and the inert gas is preferably N2.

[0014] Further preferably, in the step (4), the calcined product of step (3) is cooled to room temperature and then heated to 310-330° C. at a heating rate of 0.8-1.2° C. / min in an air atmosphere for calcination.

[0015] Further preferably, the room temperature in step (2) and step (4) is 25±5°C.

[0016] In an embodiment of the present invention, the mass ratio of NH4VO3 and H2C2O4 in the aqueous solution containing NH4VO3 and H2C2O4 is 1:(1.5-3), for example, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, and 1:3.0.

[0017] According to some specific embodiments of the present invention, the concentration of NH4VO3 in the aqueous solution containing NH4VO3 and H2C2O4 is 0.001-0.005 g / mL, and the concentration of H2C2O4 is 0.004-0.008 g / mL.

[0018] In an embodiment of the present invention, the mass ratio of C3H6N6 in step (2) to H2C2O4 in the aqueous solution containing NH4VO3 and H2C2O4 is (8-12):1, for example, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1, and 12:1.

[0019] In an embodiment of the present invention, in step (2), the stirring speed is 400-800 r / min, such as 400 r / min, 450 r / min, 500 r / min, 550 r / min, 600 r / min, 650 r / min, 700 r / min, 750 r / min, and 800 r / min.

[0020] In an embodiment of the present invention, the stirring time in step (2) is 8 to 15 hours, for example, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, 12.5 hours, 13 hours, 13.5 hours, 14 hours, 14.5 hours, 15 hours, and 15.5 hours.

[0021] In an embodiment of the present invention, the carbonization time in step (3) is 0.8 to 1.5 h, for example, 0.8 h, 0.9 h, 1.0 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, or 1.5 h.

[0022] In an embodiment of the present invention, the calcination time in step (3) is 1.5 to 3 hours, for example, 1.5 hours, 1.6 hours, 1.7 hours, 1.8 hours, 1.9 hours, 2.0 hours, 2.1 hours, 2.2 hours, 2.3 hours, 2.4 hours, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours, and 3.0 hours.

[0023] In an embodiment of the present invention, the calcination time in step (4) is 5 to 15 hours, for example, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, 12.5 hours, 13 hours, 13.5 hours, 14 hours, 14.5 hours, and 15 hours.

[0024] In an embodiment of the present invention, the drying temperature in step (5) is 50-70°C, for example, 50°C, 55°C, 60°C, 65°C, or 70°C.

[0025] In an embodiment of the present invention, the alcohol in step (5) is ethanol.

[0026] The second aspect of the present invention provides V2O5-NC nanozyme prepared by the above preparation method.

[0027] In the embodiment of the present invention, the V2O5-NC nanozyme is a dark green powder with a stacked layer structure and a specific surface area of 16 to 18 m 2 / g, with mesoporous distribution and an average particle size of 20 to 25 nm.

[0028] In an embodiment of the present invention, the V2O5-NC nanozyme has catalase-like activity.

[0029] The third aspect of the present invention also provides the application of the above-mentioned V2O5-NC nanozyme in organophosphorus detection.

[0030] A fourth aspect of the present invention also provides a colorimetric sensor array for detecting organophosphorus pesticides, wherein the sensing elements of the colorimetric sensor array include a V2O5-NC nanozyme, an acetylcholinesterase, a choline oxidase, acetylcholine, and a chromogenic substrate. The acetylcholinesterase can hydrolyze the acetylcholine to produce choline, the choline oxidase hydrolyzes the choline to form H2O2, and the V2O5-NC nanozyme catalyzes the decomposition of the H2O2 into O2, which is further oxidized to O 2- , the chromogenic substrate is oxidized and discolored.

[0031] Specifically, the chromogenic substrate includes but is not limited to TMB, ABTS, DAB or OPD. When the chromogenic substrate is TMB, the specific wavelength is any one of 649 to 659 nm; when the chromogenic substrate is ABTS, the specific wavelength is any one of 415 to 425 nm; when the chromogenic substrate is DAB, the specific wavelength is any one of 463 to 473 nm; when the chromogenic substrate is OPD, the specific wavelength is any one of 445 to 455 nm.

[0032] In an embodiment of the present invention, the construction method is as follows: m different organophosphorus pesticide standard solutions of different concentrations are mixed with acetylcholine and acetylcholinesterase in phosphate buffered saline with a pH value of 7.2 to 7.8, reacted in a water bath at 35 to 40°C, then choline oxidase is added to continue the reaction, and then a chromogenic substrate and the V2O5-NC nanozyme according to claim 4 or 5 and a HAc-NaAc buffer with a pH value of 3 to 3.2, 3.8 to 4.2, or 4.8 to 5.2 are added, reacted in a water bath at 20 to 30°C, and the absorbance of the reaction solution at a specific wavelength is measured by a UV-visible spectrophotometer. When the chromogenic substrate is TMB, the specific wavelength is any one of 649 to 659 nm; when the chromogenic substrate is ABTS, the specific wavelength is any one of 415 to 425 nm; when the chromogenic substrate is DAB, the specific wavelength is any one of 463 to 473 nm; when the chromogenic substrate is OPD, the specific wavelength is any one of 445 to 455 nm. Each test is set with n repetitions to obtain a data matrix of 3 pH × m organophosphorus pesticides × n repetitions, where m and n are independently 1, 2, 3, 4, 5, 6, 7 or 8.

[0033] According to some specific embodiments of the present invention, the organophosphorus pesticides include phoxim, chlorpyrifos, methamidophos, methyl parathion, and glyphosate. Organophosphorus pesticides irreversibly inhibit the activity of acetylcholinesterase, thereby inhibiting the production of H2O2, so that the chromogenic substrate cannot be oxidized and discolored. V2O5-NC nanozymes have different responses to different organophosphorus pesticides at different HAc-NaAc buffer pH values. Since different organophosphorus pesticides have different inhibitory abilities on acetylcholinesterase, this results in different degrees of catalytic inhibition of V2O5-NC nanozymes on each sensor unit. Therefore, the absorbance of each organophosphorus pesticide as a colorimetric response mode can be distinguished.

[0034] The fifth aspect of the present invention also provides a method for detecting organophosphorus pesticides, wherein a spiked or unspiked test solution is mixed with acetylcholine and acetylcholinesterase in a phosphate buffered saline solution with a pH value of 7.2 to 7.8, and the mixture is reacted in a water bath at 35 to 40°C, and then choline oxidase is added to continue the reaction, and then a HAc-NaAc buffer solution with a pH value of 3 to 5.2, a chromogenic substrate, and the V2O5-NC nanozyme according to claim 4 or 5 are added to react in a water bath at 20 to 30°C, and the reaction solution is measured at a specific wavelength by a UV-visible spectrophotometer. The absorbance value under the standard curve or the above-mentioned colorimetric sensor array is used to convert the organophosphorus pesticide content in the test solution, when the chromogenic substrate is TMB, the specific wavelength is any one of 649 to 659 nm; when the chromogenic substrate is ABTS, the specific wavelength is any one of 415 to 425 nm; when the chromogenic substrate is DAB, the specific wavelength is any one of 463 to 473 nm; when the chromogenic substrate is OPD, the specific wavelength is any one of 445 to 455 nm.

[0035] Further preferably, 80-150 μL of the test solution, 250-350 μL of acetylcholine (8-15 mM) and 80-150 μL of acetylcholinesterase (0.1-0.5 U / L) are mixed in 0.8-1.5 mL of phosphate buffered saline (pH = 7.2-7.8), reacted in a 35-40 ° C water bath for 8-15 min, 150-250 μL of choline oxidase (0.1-0.5 U / L) is added, the reaction is continued for 8-15 min, and then 1-3 mL of HAc-NaAc buffer (PH = 3 ~ 5.2), 150 ~ 250 μL chromogenic substrate (5 ~ 8 mM), 100 ~ 150 μL V2O5-NC nanoenzyme dispersion (0.08 ~ 0.5 mg / mL), react in a 20 ~ 30 ° C water bath for 15 ~ 30 minutes, and then measure the absorbance of the solution at a specific wavelength by UV-visible spectrophotometer.

[0036] According to some specific embodiments of the present invention, the chromogenic substrate is preferably TMB, and the specific wavelength is 652 nm.

[0037] Specifically, the organophosphorus pesticides include one or more of phoxim, chlorpyrifos, methamidophos, methyl parathion and glyphosate.

[0038] The sixth aspect of the present invention also provides an organophosphorus pesticide detection kit, characterized in that the kit comprises the V2O5-NC nanozyme according to claim 4 or 5, acetylcholinesterase, choline oxidase, acetylcholine and TMB.

[0039] Preferably, in the kit, the V2O5-NC nanozyme is stored in the form of a dispersion with a concentration of 0.08 to 0.5 mg / mL (the solvent is water), the chromogenic substrate is stored in the form of a solution with a concentration of 5 to 8 mM (the solvent is DMSO), the concentration of acetylcholinesterase is 0.1 to 0.5 U / L, the concentration of choline oxidase is 0.1 to 0.5 U / L, and acetylcholine is stored in the form of a solution with a concentration of 5 to 15 mM (the solvent is water).

[0040] Preferably, the kit further comprises an organophosphorus pesticide standard solution or an organophosphorus pesticide standard stock solution.

[0041] Preferably, the organophosphorus pesticide is selected from one or more of phoxim, chlorpyrifos, methamidophos, methyl parathion and glyphosate.

[0042] Further preferably, the organophosphorus pesticide standard stock solution uses methanol as a solvent and water as a dilution solvent to prepare an organophosphorus pesticide standard solution with a concentration ranging from 1 to 100 μM.

[0043] Compared with the prior art, the present invention has the following advantages:

[0044] The wide-interlayer-spacing V2O5-NC nanozyme based on CO bond bridging prepared by the present invention has good stability, can be prepared on a large scale, and is easy to store.

[0045] The V2O5-NC nanozyme prepared by the present invention is an oxidase-like and catalase-like enzyme with high enzyme-like catalytic activity, good affinity for catalytic chromogenic substrates, high stability, and universal applicability to catalytic chromogenic substrates (TMB, ABTS, DAB, OPD).

[0046] The V2O5-NC nanozyme of the present invention was used to analyze five organophosphorus pesticides (methyl parathion, glyphosate, phoxim, methamidophos, and chlorpyrifos), demonstrating advantages such as a wide linear range, low detection limit, ease of use, and low cost. A colorimetric sensor array constructed using a cascade reaction based on the V2O5-NC nanozyme for identifying these five organophosphorus pesticides exhibited excellent pesticide recognition and interference resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the preparation process of V2O5-NC nanozyme;

[0048] Figure 2 The morphological and structural characteristics of V2O5-NC nanozymes: (a) SEM image; (b) HRTEM image; (c) XRD image; (d) adsorption-desorption curve and pore size distribution; (e) Fourier transform infrared spectrum; (f) Raman spectrum;

[0049] Figure 3To explore the enzymatic activity of V2O5-NC nanozyme: (a), (b) Enzyme activity diagram of V2O5-NC nanozyme; (c) Effect of different atmospheric conditions (O2, Air, N2) on the enzymatic activity of V2O5-NC nanozyme; (d) Effect of V2O5-NC nanozyme concentration on the enzymatic activity of V2O5-NC nanozyme; (e) Effect of incubation temperature on the enzymatic activity of V2O5-NC nanozyme; (f) Effect of buffer pH on the enzymatic activity of V2O5-NC nanozyme;

[0050] Figure 4 Steady-state kinetics of V2O5-NC nanozymes: (a) Michaelis-Menten curves at different TMB concentrations; (b) Lineweaver-Burk plot of TMB concentration; (c) Michaelis-Menten curves at different H2O2 concentrations; (d) Lineweaver-Burk plot of H2O2 concentration;

[0051] Figure 5 Detection of organophosphorus pesticides by V2O5-NC nanozymes: (a) Schematic diagram of the detection of organophosphorus pesticides by V2O5-NC nanozymes; (b) Colorimetric response diagram of five organophosphorus pesticides at 10 μM; (c) Heat map of five organophosphorus pesticides at 10 μM; (d) PCA map of five organophosphorus pesticides at 10 μM; (e) Colorimetric response map of five organophosphorus pesticides at 100 μM; (f) Heat map of five organophosphorus pesticides at 10 μM and 100 μM; (g) PCA map of five organophosphorus pesticides at 10 μM and 100 μM;

[0052] Figure 6 Figure 4: Standard curves for the detection of five organophosphorus pesticides by UV-visible spectrophotometry: (a) linear relationship between absorbance and methyl parathion concentration; (b) linear relationship between absorbance and glyphosate concentration; (c) linear relationship between absorbance and phoxim concentration; (d) linear relationship between absorbance and methamidophos concentration; (e) linear relationship between absorbance and chlorpyrifos concentration.

[0053] Figure 7 for Figure 6 Enlarged view of the linear relationship graph between absorbance value and phoxim concentration. DETAILED DESCRIPTION

[0054] The present invention will be further described below with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific applications. The implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other. The implementation conditions used in the examples can be further adjusted according to specific requirements. The implementation conditions not specified are generally the conditions used in routine experiments.

[0055] In the following examples, all chemicals were of analytical or chromatographic grade. Ultrapure water (>18.2 MΩ) was produced using a Milli-Q water purifier (Bedford, MA, USA). Scanning electron microscopy (SEM, Quanta 250; FEI, USA); transmission electron microscopy (Talos F200X G2; FEI, USA); X-ray diffractometer (D8-Advance, Bruker, Germany); X-ray photoelectron spectroscopy (K-Alpha+; Thermo Scientific, USA); and Raman spectroscopy (DXR microscope, Thermo Scientific, USA) were performed. Enzyme kinetic data and UV-visible spectra were obtained using a UV-8000 spectrophotometer (Shanghai, China).

[0056] In the present invention, room temperature refers to 25±5°C.

[0057] Example 1

[0058] Preparation of V2O5-NC nanozymes:

[0059] according to Figure 1 The preparation process shown in the figure is to dissolve 0.23g NH4VO3 and 0.5g H2C2O4 in 80mL ultrapure water and sonicate to form a yellow homogeneous solution. 5.0g C3H6N6 is then added, and the mixture is stirred at 600 rpm for 10 hours at room temperature using a magnetic stirrer. The mixture is then freeze-dried in a vacuum oven for 24 hours. The precursor is then collected and placed in a tube furnace. Under a nitrogen atmosphere, the temperature is increased to 400°C at a heating rate of 2°C / min for carbonization for 1 hour. The temperature is then increased to 600°C at a heating rate of 2°C / min for 2 hours. After the tube furnace cools to room temperature, the nitrogen flow is stopped and the mixture is heated to 320°C in an air atmosphere at a heating rate of 1°C / min for 8 hours. The resulting product is washed several times with ultrapure water and ethanol alternately and dried at 60°C. The resulting dark green powder is V2O5-NC nanosheets.

[0060] Morphological and structural characteristics of V2O5-NC nanozymes:

[0061] like Figure 2As shown, the V2O5-NC nanozyme is a stacked layered structure. From the high-resolution cross-sectional image, it can be seen that two types of layers are stacked alternately. (V2O5), the other is (NC), showing the total spacing is XRD spectrum shows that the orthorhombic crystal structure ( JCPDS card number: 41-1426). For the original V2O5, the diffraction peak in the low-angle region (2θ = 15.3°) corresponds to the (200) plane of the layered V2O5. The results show that after the carbonization process, the interlayer spacing of V2O5-NC increases from 15.3° to 7.3°, corresponding to a d-spacing increase from Increase to The reason is that the NC layer enters the middle layer, which is conducive to the diffusion of H2O2 molecules between the layers. The specific surface area of V2O5-NC is 17.23m 2 / g, showing a typical type II adsorption isotherm. The pore size curve shows that V2O5-NC is mesoporous with an average particle size of 23.20nm. Fourier transform infrared spectroscopy (FTIR) further analyzed the microstructure and functional groups of V2O5-NC. -1 stretching vibration at 945cm -1 and 1015cm -1 The VOV bond is at 587 cm -1 and 818cm -1 The stretching vibration at 1315cm -1 、1546cm -1 and 1654cm -1 The peak at 1369 cm is caused by the stretching vibration of C=N and CN, which further confirms the formation of nitrogen-doped carbon (NC). -1 and 1612cm -1 The positions correspond to COC and C=O, which confirms that V2O5 and NC are connected by CO bonds. Raman spectroscopy shows that V2O5-NC has a -1 The same characteristic peak as that of V2O5 appears, which further confirms the existence of V2O5 in the material. -1 (D belt) and 1610cm -1 (G band), showing two characteristic peaks, I D :I G The value is 1.41.

[0062] Example 2

[0063] Investigation of the enzyme activity of V2O5-NC nanozymes:

[0064] Experimental procedure: 120 μL of a 0.1 mg / mL dispersion of V2O5-NC nanozyme (solvent: water), 200 μL of 6 mM TMB (solvent: DMSO), and 80 μL of 5 mM H2O2 (solvent: water) were added to 2 mL of HAc-NaAc buffer (0.03 M, pH 4.0). The mixture was incubated at 25°C for 20 minutes, and the absorbance at 652 nm was measured using a UV-visible spectrophotometer.

[0065] like Figure 3 As shown in Figure 2, V2O5-NC nanozymes exhibit excellent catalase-like activity, which is due to the fact that the wider interlayer spacing of V2O5-NC greatly promotes the diffusion of H2O2 molecules between the layers, fully utilizes the storage sites in the interlayers to promote electron transfer, thereby improving the catalytic efficiency of the nanozymes. m The values were 0.37mM (TMB) and 0.35mM (H2O2), V max is 7.64×10 -8 M / s(TMB) and 5.56×10 -8 M / s(H2O2).

[0066] Example 3

[0067] Construction of a colorimetric sensor array for organophosphorus pesticide (OPs) detection based on V2O5-NC:

[0068] Based on the cascade reaction, a colorimetric sensing array was constructed for identifying five organophosphorus pesticides. Figure 5 As shown in the figure, acetylcholinesterase (AChE) can first hydrolyze acetylcholine (ACh) to produce choline. Then choline oxidase (ChOx) hydrolyzes choline to form H2O2. Then V2O5-NC nanozyme catalyzes the decomposition of H2O2 into oxygen, and its OXD-like activity further oxidizes O2 into O 2-, oxidizing TMB to blue oxTMB. However, Ops irreversibly inhibit AChE activity, thereby inhibiting the production of H2O2, preventing TMB from being oxidized to blue oxTMB. V2O5-NC nanozymes respond differently to different OPs at different HAc-NaAc buffer pH values. Due to the different inhibitory abilities of different OPs on acetylcholinesterase, this results in different degrees of catalytic inhibition of each sensor unit by V2O5-NC nanozymes. Therefore, the absorbance [(A-A0) / A0] of each organophosphorus pesticide as a colorimetric response mode can be distinguished, where A is the absorbance of the blank control group (no organophosphorus pesticide added) and A0 is the absorbance value of the experimental group (organophosphorus pesticide added). At the same time, a heat map of the responses of the five OPs was generated, called a "fingerprint". These results demonstrate the feasibility of the colorimetric sensor array for identifying OPs in the 100 μM range. Therefore, three different types of [(A-A0) / A0] (pH = 3.2, pH = 4 and pH = 5) were used to test phoxim (PHO), chlorpyrifos (CPF), methamidophos (MAP), methyl parathion (MPH) and glyphosate (GLY) five times each, forming a matrix of 5 OPs × 3 channels × 5 repetitions. LDA was used to separate the matrix into two typical factors, forming a two-dimensional PCA graph. The five organophosphorus pesticides were well clustered into five groups and separated from each other. Excellent pesticide recognition ability was shown over a wide concentration range. At the same time, UV-visible spectroscopy was used to study the detection performance of the V2O5-NC nanozyme colorimetric sensor system for the five OPs. Standard curves for the detection of the five organophosphorus pesticides by UV-visible spectrophotometry were established, and the detection limits and linear ranges were calculated. Figure 6 A standard curve at pH = 4 is shown.

[0069] (1) Use water as solvent and prepare a concentration of 0.1 mg mL -1 V2O5-NC nanozyme dispersion; DMSO was used as solvent to prepare 6mM TMB solution;

[0070] (2) Using water as solvent, prepare 0.3 U / L acetylcholinesterase (AChE), 0.4 U / L choline oxidase (ChOx) and 10 mM acetylcholine (ACh);

[0071] (3) Using methanol as the solvent, prepare 0.2 mM stock solutions of five organophosphorus pesticides. Then, using ultrapure water as the solvent, dilute the stock solutions to prepare working solutions of the five organophosphorus pesticides with concentrations ranging from 1 to 100 μM.

[0072] (4) 100 μL of 5 organophosphorus pesticide standard solutions of different concentrations, 300 μL ACh (10 mM) and 100 μL AChE (0.3 U / L) were mixed in 1 mL phosphate buffered saline (0.01 M, pH = 7.4). After reacting in a 37 °C water bath for 10 min, 200 μL ChOx (0.4 U / mL) was added and the reaction continued for 10 min. Then, 2 mL HAc-NaAc buffer (0.03 M, pH = 3.2 / 4.0 / 5.0), 200 μL TMB (6 mM) and 120 μL V2O5-NC nanozyme (0.1 mg / mL) were added. After reacting in a 25 °C water bath for 20 min, the absorbance of the solution at a wavelength of 652 nm (denoted as A) was measured by UV-visible spectrophotometer. 652 ). Repeat 5 samples to obtain a data matrix (3pH×5OPs×5 repeats).

[0073] Example 4

[0074] Detection of 5 organophosphorus pesticides in actual samples:

[0075] Four actual lake water samples were extracted from different areas of Suzhou, filtered with a 0.22 μm filter membrane, and the filtrate was collected as the test liquid. Lettuce, spinach, greens, broccoli, apples, pears, oranges and tomatoes were purchased from a market in Suzhou. After soaking in tap water for 10 minutes, the soaked water was filtered with a 0.22 μm filter membrane, and the filtrate was collected as the test liquid. A certain volume of the test liquid was added with 4.0 μM of different organophosphorus pesticide standards. The V2O5-NC nanozyme colorimetric sensor system in Example 4 was used to detect the concentrations of 5 organophosphorus pesticides in the test liquid using ultraviolet-visible spectroscopy. The organophosphorus pesticide standard solution in step (4) of Example 4 was replaced with a spiked test liquid, and the pH of the HAc-NaAc buffer used was 4. The measured A 652 Bring in Figure 6 and Figure 7 The corresponding standard curve was used to convert the detection concentration of the spiked test solution, and the spike recovery was calculated. Each sample was tested three times, and the relative standard deviation of the three test results was calculated. The results were compared with those of the traditional high performance liquid chromatography method. The results are shown in Table 1.

[0076] Table 1

[0077]

[0078]

[0079]

[0080] Table 1 shows that the UV-visible spectroscopy results for five organophosphorus pesticides in real samples using the V2O5-NC nanozyme colorimetric sensor system described in Example 4 were essentially consistent with those obtained using traditional HPLC, demonstrating accurate results. Compared to traditional HPLC, UV-visible spectroscopy offers the advantages of rapidity, efficiency, low cost, and the ability to perform on-site detection.

[0081] Based on the above examples, it can be seen that the wide interlayer spacing V2O5-NC nanozyme based on CO bond bridging prepared in Example 1 has good stability, can be prepared on a large scale, and is easy to store. The wider interlayer spacing of V2O5-NC greatly promotes the diffusion of H2O2 molecules between the layers, fully utilizes the storage sites in the interlayer to promote electron transfer, and thus the V2O5-NC nanozyme exhibits excellent catalase-like activity. The colorimetric sensor array for organophosphorus pesticide detection constructed with basic V2O5-NC nanozymes has good recognition ability and anti-interference properties for five organophosphorus pesticides (methyl parathion, glyphosate, phoxim, methyl parathion, and chlorpyrifos), and all have the advantages of wide linear range, low detection limit, simple operation, and low detection cost.

[0082] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing V2O5-NC nanozyme, characterized in that: The preparation method comprises the following steps: (1) preparing an aqueous solution containing NH4VO3 and H2C2O4; (2) adding C3H6N6 to the aqueous solution containing NH4VO3 and H2C2O4 in step (1), stirring and mixing at room temperature, and collecting the precursor by vacuum freeze drying; (3) heating the precursor of step (2) to 350-450° C. at a heating rate of 1-5° C. / min for carbonization under an inert gas atmosphere, and further heating the precursor to 550-650° C. at a heating rate of 1-5° C. / min for calcination; (4) cooling the calcined product of step (3) to room temperature, and then heating it to 300-350° C. at a heating rate of 0.5-1.5° C. / min in an air atmosphere; (5) The calcined product of step (4) is washed alternately with water and alcohol and then dried to obtain the V2O5-NC nanozyme.

2. The preparation method according to claim 1, characterized in that The mass ratio of NH4VO3 to H2C2O4 in the aqueous solution containing NH4VO3 and H2C2O4 is 1:(1.5-3); and / or, the concentration of NH4VO3 in the aqueous solution containing NH4VO3 and H2C2O4 is 0.001 to 0.005 g / mL, and the concentration of H2C2O4 is 0.004 to 0.008 g / mL; And / or, the mass ratio of C3H6N6 in the step (2) to H2C2O4 in the aqueous solution containing NH4VO3 and H2C2O4 is (8-12):

1.

3. The preparation method according to claim 1, characterized in that In the step (2), the stirring speed is 400 to 800 r / min; And / or, the stirring time in step (2) is 8 to 15 hours; and / or, the carbonization time in step (3) is 0.8 to 1.5 h; and / or, the calcination time in step (3) is 1.5 to 3 hours; and / or, the calcination time in step (4) is 5 to 15 hours; and / or, the drying temperature in step (5) is 50-70° C.; And / or, the alcohol in step (5) is ethanol.

4. The V2O5-NC nanozyme prepared by the preparation method according to any one of claims 1 to 3.

5. The V2O5-NC nanozyme according to claim 4, characterized in that The V2O5-NC nanozyme is a dark green powder with a stacked layer structure and a specific surface area of 16 to 18 m 2 / g, mesoporous distribution, average particle size of 20-25nm; And / or, the V2O5-NC nanozyme has catalase-like activity.

6. Use of the V2O5-NC nanozyme as claimed in claim 4 or 5 in organophosphorus detection.

7. A colorimetric sensor array for detecting organophosphorus pesticides, characterized in that: The elements of the colorimetric sensor array include V2O5-NC nanozyme, acetylcholinesterase, choline oxidase, acetylcholine and a chromogenic substrate, and the chromogenic substrate is TMB, ABTS, DAB or OPD.

8. The colorimetric sensor array according to claim 7, wherein: Its construction method is: m different organophosphorus pesticide standard solutions of different concentrations are mixed with acetylcholine and acetylcholinesterase in phosphate buffered saline with a pH value of 7.2 to 7.8, and reacted in a water bath at 35 to 40° C., and then choline oxidase is added to continue the reaction, and then a chromogenic substrate and the V2O5-NC nanozyme according to claim 4 or 5 and a HAc-NaAc buffer with a pH value of 3 to 3.2, 3.8 to 4.2, or 4.8 to 5.2 are added, and the reaction is carried out in a water bath at 20 to 30° C. The absorbance of the reaction solution at a specific wavelength is measured by a UV-visible spectrophotometer. When the chromogenic substrate is When TMB is used, the specific wavelength is any wavelength between 649 and 659 nm; when the chromogenic substrate is ABTS, the specific wavelength is any wavelength between 415 and 425 nm; when the chromogenic substrate is DAB, the specific wavelength is any wavelength between 463 and 473 nm; when the chromogenic substrate is OPD, the specific wavelength is any wavelength between 445 and 455 nm. Each test is set with n repetitions to obtain a data matrix of 3 pH × m organophosphorus pesticides × n repetitions, where m and n are independently 1, 2, 3, 4, 5, 6, 7 or 8.

9. A method for detecting organophosphorus pesticides, characterized in that: The spiked or unspecified test solution is mixed with acetylcholine and acetylcholinesterase in phosphate buffered saline with a pH value of 7.2 to 7.8, and reacted in a water bath at 35 to 40° C., and then choline oxidase is added to continue the reaction, and then HAc-NaAc buffer with a pH value of 3 to 5.2, a chromogenic substrate and the V2O5-NC nanozyme according to claim 4 or 5 are added, and reacted in a water bath at 20 to 30° C., and the absorbance of the reaction solution at a specific wavelength is measured by a UV-visible spectrophotometer, and the absorbance is inserted into the standard curve or the colorimetric sensor array according to claim 7. The content of the organophosphorus pesticide in the test solution is converted in the column, the chromogenic substrate is TMB, ABTS, DAB or OPD, when the chromogenic substrate is TMB, the specific wavelength is any wavelength of 649 to 659 nm; when the chromogenic substrate is ABTS, the specific wavelength is any wavelength of 415 to 425 nm; when the chromogenic substrate is DAB, the specific wavelength is any wavelength of 463 to 473 nm; when the chromogenic substrate is OPD, the specific wavelength is any wavelength of 445 to 455 nm.

10. The colorimetric sensor array according to claim 7 or the organophosphorus pesticide detection method according to claim 9, characterized in that: The organophosphorus pesticides include one or more of phoxim, chlorpyrifos, methamidophos, methyl parathion and glyphosate.

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