Copper-based nano enzyme as well as preparation method and application thereof

Abstract

The invention discloses a copper-based nano-enzyme, a preparation method thereof and application of the copper-based nano-enzyme in organophosphorus pesticide detection, and belongs to the technical field of pesticide residue detection. The copper-based nano-enzyme has a netted-like structure, a high specific surface area and many active sites, the valence state of a copper element is zero-valence Cu, Cu < + > and Cu < 2 + >, the copper-based nano-enzyme has high peroxidase-like activity and laccase-like activity, the detection limit is low, and glyphosate can be sensitively detected; and glyphosate, glufosinate-ammonium and isazofos can be well distinguished from six organophosphorus pesticides with medium concentration, and the method has high selectivity. The nano enzyme provided by the invention is prepared from ammonium citrate and copper nitrate in an N, N-dimethylformamide (DMF) solution by using a solvothermal method; the preparation process of the nano enzyme is simple.

Description

Technical Field

[0001] The present invention belongs to the technical field of pesticide residue detection, and particularly relates to a copper-based nanozyme, a preparation method thereof, and an application thereof in the detection of organophosphorus pesticides. Background Art

[0002] At present, the detection methods for organophosphorus pesticides are mainly divided into two categories. One is the instrumental analysis method, and the other is the immunoassay method. The former has the disadvantages of complex sample pretreatment, expensive instrument equipment, high detection cost, high professional operation requirements, etc. These adverse factors make it difficult for the instrumental analysis method to meet the on-site detection requirements of low cost and fast timeliness for actual samples such as agricultural products, and it is not convenient to carry out timely supervision and risk prevention and control work. The latter has the disadvantages of relatively strict catalytic conditions, easy inactivation of enzymes, and complex sample pretreatment, etc.

[0003] As a kind of nanozyme with both the unique properties of nanomaterials and catalytic functions, it has the advantages of high catalytic efficiency, stability, economy, and easy large-scale preparation, and has been widely used in the fields of medicine, chemical engineering, food, agriculture, and environment. In environmental detection, it mainly uses its peroxidase-like (POD) activity, oxidase-like (OXD) activity, catalase-like (CAT) activity, laccase-like (Lac) activity, and superoxide dismutase-like (SOD) activity to detect toxins, pesticides, heavy metal ions, etc.

[0004] The existing nanozymes for detecting organophosphorus pesticides have relatively single morphologies, mainly cubic morphologies, etc. Due to the morphological disadvantages of such materials, their catalytic selectivity is limited, the active sites are insufficiently exposed, and the electron transfer efficiency is low, making it difficult to achieve highly sensitive and selective detection of organophosphorus pesticides. For example: Song et al. disclosed a scheme for detecting pesticides, using melamine as a nitrogen source and copper nitrate as a metal ion source, and synthesizing Mel-Cu materials by a solvothermal method. The microscopic morphology of the Mel-Cu materials prepared by this method is a common rectangle, and the specific surface area of such materials is relatively low, with less contact with the substances to be detected, which will lead to low detection sensitivity. Therefore, it is necessary to find a material that can fully contact the substances to be detected to improve the detection sensitivity.

[0005] Therefore, it is of great significance to design and prepare a nanozyme with a high specific surface area, multiple active sites, and capable of sensitively and selectively detecting organophosphorus pesticides. Summary of the Invention

[0006] To overcome the deficiencies in the prior art, the first objective of the present invention is to provide a copper-based nanozyme, denoted as ACT-Cu nanozyme, which is prepared by a solvothermal method using ammonium citrate and copper nitrate as raw materials. This copper-based nanozyme has a silk-like reticular structure, a relatively high specific surface area, and a large number of active sites, in which the valence state of copper element is zero-valent Cu, Cu + and Cu 2+ existing simultaneously. This nanozyme has relatively high peroxidase-like activity (POD) and laccase-like activity (Lac), with a low detection limit and can sensitively detect glyphosate.

[0007] The second objective of the present invention is to provide a preparation method of the copper-based nanozyme.

[0008] The third objective of the present invention is to provide an application of the copper-based nanozyme.

[0009] For this reason, the first technical solution provided by the present invention is as follows:

[0010] A copper-based nanozyme, and the preparation method is as follows: Ultrasonically disperse ammonium citrate and copper nitrate into an N,N-dimethylformamide solution respectively, mix the fully dispersed solutions and stir to make them uniform; then transfer to a hydrothermal reaction kettle and carry out solvothermal reaction in a muffle furnace; after cooling to room temperature, take it out, centrifuge to collect the precipitate in sequence, wash and dry to obtain the copper-based nanozyme.

[0011] Further, in the reaction system of the above preparation method of a copper-based nanozyme, the molar ratio of copper nitrate to ammonium citrate is 3 - 7:1, preferably 5:1.

[0012] Further, in the above preparation method of a copper-based nanozyme, the ultrasonic time is 15 min, the mixing and stirring time is 10 min, and both ultrasonic and mixing and stirring are carried out at room temperature.

[0013] Further, in the above preparation method of a copper-based nanozyme, the reaction temperature is 100 - 180 °C, preferably 100 °C.

[0014] Further, in the above preparation method of a copper-based nanozyme, the solvothermal reaction time is 9 - 15 h, preferably 12 h.

[0015] The second technical solution of the present invention is the copper-based nanozyme prepared by the first technical solution. The said copper-based nanozyme has a silk-like reticular structure, is composed of ammonium citrate and copper, and the diameter size is 10 - 200 nm.

[0016] The third technical solution of the present invention is to provide the application of the copper-based nanozyme for detecting organophosphorus pesticides.

[0017] The specific method for using the copper-based nanozyme to detect organophosphorus pesticides is as follows: The copper-based nanozyme is dispersed in a buffer solution and a solution of organophosphorus pesticides. After incubating at room temperature for 15 minutes, it is divided into two centrifuge tubes. 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide are added to the first centrifuge tube, and the reaction is carried out at room temperature for 30 minutes, denoted as Channel 1; 4-aminoantipyrine (4-AAP) and 2,4-dichlorophenol (2,4-DCP) are added to the second centrifuge tube, and the reaction is carried out at 60 °C for 60 minutes, denoted as Channel 2.

[0018] Furthermore, in the above application method of a copper-based nanozyme, the pH value of the buffer solution is 5-7, preferably 6.

[0019] Furthermore, in the above application method of a copper-based nanozyme, the concentration of the nanozyme is 5-15 μg / mL, preferably 10 μg / mL.

[0020] Furthermore, in the above application method of a copper-based nanozyme, the concentration of TMB is 20 mM, the concentration of hydrogen peroxide is 50 mM, the concentration of 4-AAP is 1 mg / mL, and the concentration of 2,4-DCP is 1 mg / mL.

[0021] Compared with the prior art, the technical solution provided by the present invention has the following technical advantages:

[0022] 1. The copper-based nanozyme provided by the present invention is prepared by a one-step reaction using ammonium citrate as the nitrogen source and copper nitrate as the copper source, and is used for sensitive detection of organophosphorus pesticides. Ammonium citrate used in the technical solution provided by this application has both the dual functions of nitrogen source supply and organic ligand compared with urea, ammonia water, etc. It can form a stable coordination structure with copper ions and expose more peroxidase-like active sites. In addition, as a nitrogen source, ammonium citrate can capture the phosphate group of organophosphorus molecules through nitrogen defect sites, which can provide the selectivity of detection. Under the electron microscope, the nanozyme has a filamentous network-like structure. Compared with the traditional cubic-shaped nanozyme, the filamentous network-like structure has a higher specific surface area and more active sites.

[0023] 2. The copper-based nanozyme provided by the present invention has both peroxidase-like activity and laccase-like activity at the same time, and organophosphorus pesticides can inhibit both activities of the nanozyme simultaneously. Compared with other nanozymes for detecting organophosphorus pesticides, where only one type of enzyme activity is inhibited for detection, detection with dual signals of two types of enzyme activities will be more accurate. In addition, the detection limit of the copper-based nanozyme provided by the present invention for detecting organophosphorus pesticides is relatively low, and it can sensitively detect glyphosate.

[0024] 3. The copper-based nanozyme provided by the present invention has the advantages of low toxicity, simple preparation process, low cost, and high stability, can achieve sensitive detection of organophosphorus pesticides, has good application prospects, and provides a reference for the development of new nanozymes for detecting pesticide residues. Description of the Drawings

[0025] Figure 1 SEM images (a) and elemental overlay distribution (b), EDS elemental distribution maps of Cu (c), O (d), N (e), and C (f) of the ACT-Cu nanozyme prepared in Example 1;

[0026] Figure 2 TEM image of the ACT-Cu nanozyme prepared in Example 1;

[0027] Figure 3 SEM images of the ACT-Cu nanozyme prepared in Examples 1, 4, and 5;

[0028] Among them: SEM image (a) of the ACT-Cu nanozyme prepared in Example 1, SEM image (b) of the ACT-Cu nanozyme prepared in Example 4, and SEM image (c) of the ACT-Cu nanozyme prepared in Example 5;

[0029] Figure 4 Near-infrared spectra of the ACT-Cu nanozyme prepared in Examples 1, 4, and 5;

[0030] Figure 5 XRD pattern of the ACT-Cu nanozyme prepared in Example 1;

[0031] Figure 6 Survey spectrum (a), C 1s (b), N 1s (c), Cu 2p (d) of the XPS spectrum of the ACT-Cu nanozyme prepared in Example 1;

[0032] Figure 7 Cu Auger spectrum of the ACT-Cu nanozyme prepared in Example 1;

[0033] Figure 8 Two kinds of enzyme-like activity inhibition comparison diagrams of glyphosate on the ACT-Cu nanozyme prepared in Application Example 1, Application Example 2, and Application Example 3;

[0034] Among them: (a) is the inhibition rate of Channel 1, and (b) is the inhibition rate of Channel 2;

[0035] Figure 9 Absorbance at 652 nm and 510 nm in ultraviolet of the ACT-Cu nanozyme prepared in Example 1 when detecting 2.5, 2, 1, 0.5, 0.25, 0.05 μg / mL glyphosate;

[0036] Figure 10 The inhibition rates of glyphosate at 2.5, 2, 1, 0.5, 0.25, and 0.05 μg / mL on the two kinds of enzyme-like activities of the ACT-Cu nanozyme prepared in Example 1;

[0037] Figure 11 The standard working curve of glyphosate prepared for detecting organophosphorus pesticides in Channel 1 of the ACT-Cu nanozyme prepared in Example 1;

[0038] Figure 12 The standard working curve of glyphosate prepared for detecting organophosphorus pesticides in Channel 2 of the ACT-Cu nanozyme prepared in Example 1;

[0039] Figure 13 The linear discriminant analysis diagram of the responses of a sensor array based on the ACT-Cu nanozyme prepared in Example 1 to six different kinds of organophosphorus pesticides at 1 μg / mL;

[0040] Figure 14 The linear discriminant analysis diagram of the responses of a sensor array based on the ACT-Cu nanozyme prepared in Example 1 to three different kinds of organophosphorus pesticides at 10 μg / mL. Detailed implementation manners

[0041] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0042] Example 1

[0043] This example provides a copper-based nanozyme, and the synthesis method sequentially includes the following steps:

[0044] (1) Add 10 mL of N,N-dimethylformamide, 5 mL of deionized water, and 0.1215 g of ammonium citrate to beaker 1 and ultrasonicate for 15 min. Add 10 mL of N,N-dimethylformamide and 0.604 g of copper nitrate to beaker 2 and ultrasonicate for 15 min. Then mix the two solutions and stir for 10 min to obtain a mixed solution;

[0045] (2) Then transfer the mixed solution prepared in step (1) to a hydrothermal reaction kettle and carry out a solvothermal reaction at 100 °C in a muffle furnace for 12 h; after cooling to room temperature, take it out, centrifuge to collect the precipitate in sequence, wash it 3 times with deionized water, and place it in an oven at 60 °C to dry for 12 h to obtain the copper-based nanozyme, denoted as ACT-Cu nanozyme.

[0046] Example 2

[0047] This example provides a copper-based nanozyme, and the synthesis method sequentially includes the following steps:

[0048] (1) Add 10 mL of N,N-dimethylformamide, 5 mL of deionized water, and 0.1215 g of ammonium citrate to beaker 1 and ultrasonicate for 15 min. Add 10 mL of N,N-dimethylformamide and 0.8456 g of copper nitrate to beaker 2 and ultrasonicate for 15 min. Then mix the two solutions and stir for 10 min to obtain a mixed solution;

[0049] (2) Then transfer the mixed solution prepared in step (1) to a hydrothermal reaction kettle and carry out a solvothermal reaction at 100 °C in a muffle furnace for 12 h; after cooling to room temperature, take it out, centrifuge to collect the precipitate in sequence, wash it 3 times with deionized water, and place it in an oven at 60 °C to dry for 12 h to obtain the ACT-Cu nanozyme.

[0050] Example 3

[0051] This example provides a copper-based nanozyme, and the synthesis method sequentially includes the following steps:

[0052] (1) Add 10 mL of N,N-dimethylformamide, 5 mL of deionized water, and 0.1215 g of ammonium citrate to beaker 1 and ultrasonicate for 15 min. Add 10 mL of N,N-dimethylformamide and 0.3624 g of copper nitrate to beaker 2 and ultrasonicate for 15 min. Then mix the two solutions and stir for 10 min to obtain a mixed solution;

[0053] (2) Then transfer the mixed solution prepared in step (1) to a hydrothermal reaction kettle and carry out a solvothermal reaction at 100 °C in a muffle furnace for 12 h; after cooling to room temperature, take it out, centrifuge to collect the precipitate in sequence, wash it 3 times with deionized water, and place it in an oven at 60 °C to dry for 12 h to obtain the ACT-Cu nanozyme.

[0054] Example 4

[0055] This example provides a copper-based nanozyme, and the synthesis method sequentially includes the following steps:

[0056] (1) Add 10 mL of N,N-dimethylformamide, 5 mL of deionized water, and 0.1215 g of ammonium citrate to beaker 1 and ultrasonicate for 15 min. Add 10 mL of N,N-dimethylformamide and 0.604 g of copper nitrate to beaker 2 and ultrasonicate for 15 min. Then mix the two solutions and stir for 10 min to obtain a mixed solution;

[0057] (2) Then transfer the mixed solution prepared in step (1) to a hydrothermal reaction kettle and carry out a solvothermal reaction at 150 °C in a muffle furnace for 12 h; after cooling to room temperature, take it out, centrifuge to collect the precipitate in sequence, wash it 3 times with deionized water, and place it in an oven at 60 °C to dry for 12 h, thus obtaining the ACT-Cu nanozyme.

[0058] Example 5

[0059] This example provides a copper-based nanozyme, and the synthesis method sequentially includes the following steps:

[0060] (1) Add 10 mL of N,N-dimethylformamide, 5 mL of deionized water, and 0.1215 g of ammonium citrate to beaker 1 and ultrasonicate for 15 min. Add 10 mL of N,N-dimethylformamide and 0.604 g of copper nitrate to beaker 2 and ultrasonicate for 15 min. Then mix the two solutions and stir for 10 min to obtain a mixed solution;

[0061] (2) Then transfer the mixed solution prepared in step (1) to a hydrothermal reaction kettle and carry out a solvothermal reaction at 180 °C in a muffle furnace for 12 h; after cooling to room temperature, take it out, centrifuge to collect the precipitate in sequence, wash it 3 times with deionized water, and place it in an oven at 60 °C to dry for 12 h, thus obtaining the ACT-Cu nanozyme.

[0062] Example 6

[0063] This example provides a copper-based nanozyme, and the synthesis method sequentially includes the following steps:

[0064] (1) Add 10 mL of N,N-dimethylformamide, 5 mL of deionized water, and 0.1215 g of ammonium citrate to beaker 1 and ultrasonicate for 15 min. Add 10 mL of N,N-dimethylformamide and 0.604 g of copper nitrate to beaker 2 and ultrasonicate for 15 min. Then mix the two solutions and stir for 10 min to obtain a mixed solution;

[0065] (2) Then transfer the mixed solution prepared in step (1) to a hydrothermal reaction kettle and carry out a solvothermal reaction at 100 °C in a muffle furnace for 9 h; after cooling to room temperature, take it out, centrifuge to collect the precipitate in sequence, wash it 3 times with deionized water, and place it in an oven at 60 °C to dry for 12 h, thus obtaining the copper-based nanozyme, denoted as ACT-Cu nanozyme.

[0066] Example 7

[0067] This example provides a copper-based nanozyme, and the synthesis method sequentially includes the following steps:

[0068] (1) Add 10 mL of N,N-dimethylformamide, 5 mL of deionized water, and 0.1215 g of ammonium citrate to beaker 1 and ultrasonicate for 15 min. Add 10 mL of N,N-dimethylformamide and 0.604 g of copper nitrate to beaker 2 and ultrasonicate for 15 min. Then mix the two solutions and stir for 10 min to obtain a mixed solution;

[0069] (2) Then transfer the mixed solution prepared in step (1) to a hydrothermal reaction kettle and carry out a solvothermal reaction at 100 °C in a muffle furnace for 15 h; after cooling to room temperature, take it out, centrifuge to collect the precipitate in sequence, wash it 3 times with deionized water, and place it in an oven at 60 °C to dry for 12 h to obtain a copper-based nanozyme, denoted as ACT-Cu nanozyme.

[0070] The SEM image and EDS element distribution of the ACT-Cu nanozyme prepared in Example 1 are referred to Figure 1 , where the SEM image (a) and the elemental overlay distribution (b), Cu (c), O (d), N (e), and C (f). It can be seen from (c), (d), (e), and (f) that the Cu, O, N, and C elements are evenly distributed in the material.

[0071] The TEM image of the nanozyme prepared in Example 1 is referred to Figure 2 , and the SEM image is referred to Figure 3 in (a). It can be seen from Figure 2 and Figure 3 in (a) that the ACT-Cu nanozyme has a silk-net-like structure.

[0072] The SEM images of the nanozymes prepared in Examples 4 and 5 are referred to Figure 3 in (b) and (c). It can be seen from Figure 3 in (b) and (c) that compared with (a), the silk-net-like structure in (b) and (c) is not as fluffy as that in (a) and has a tendency to gradually agglomerate with increasing temperature. Therefore, the material prepared in Example 1 is selected as the subsequent material.

[0073] It can be seen from Figure 4 that the ACT-Cu-100 nanozyme detects the characteristic hydroxyl peak of ammonium citrate at 3500 cm -1 . At the same time, the ACT-Cu-100 nanozyme detects the characteristic COO- antisymmetric stretching peak of ammonium citrate at 1560 cm -1 . It can be seen from Figure 5 that the XRD pattern of this nanozyme is highly similar to that of Cu 2+1 O in the standard card PDF#05-0667. Cu 2+1 O has rich defects and vacancies, which can provide multivalent Cu. Therefore, this nanozyme should also have these advantages, thus providing higher density active sites for the reaction.Figure 6 The XPS spectrum of ACT-Cu. (a) is the full spectrum of the XPS of the material, from which it can be seen that the material contains elements such as C, N, O, and Cu. (b) is the fine spectrum of C1s, from which it can be seen that the material contains O-C=O, which corroborates the data in the infrared spectrum. Spectrum (c) is the fine spectrum of N1s, from which it can be seen that the material contains -NH2, -N-O, and Cu-N bonds. (d) is the fine spectrum of Cu 2p, and the spectrum contains Cu 2+ The unique satellite peak and Cu + The characteristic peak, so it can be concluded that there is Cu in the Cu 2p of this nanozyme + and Cu 2+ , which is consistent with the results in XRD. Since the peaks of zero-valent Cu and Cu + in the XPS spectrum are very close, it is necessary to use the Auger spectrum of Cu and the standard spectrum to fit by the non-linear least squares method to judge whether there is zero-valent Cu in monovalent copper Figure 7 is the Cu Auger spectrum of the ACT-Cu nanozyme. After fitting by the non-linear least squares method, it can be seen from the figure that monovalent copper accounts for the majority, but zero-valent copper also makes a certain contribution, thus proving the existence of zero-valent copper in the material

[0074] Application Example 1

[0075] This application provides an application of a copper-based nanozyme in detecting organophosphorus pesticides. The specific steps are as follows

[0076] Disperse the ACT-Cu nanozyme prepared in Example 1 in deionized water to prepare a nanozyme solution with a concentration of 10 μg / mL. Take two centrifuge tubes, add 200 μL of the nanozyme solution, 790 μL of MES buffer (pH 6), and 10 μL of 100 μg / mL glyphosate solution to each. Another blank group without adding glyphosate solution is set. Both the blank group and the experimental group are incubated at room temperature for 15 min. Add 200 μL of TMB solution (20 mM) and 200 μL of hydrogen peroxide (50 mM) to centrifuge tube 1 and react at room temperature for 30 min, denoted as Channel 1. Add 200 μL of 4-AAP (1 mg / mL) and 200 μL of 2,4-DCP (1 mg / mL) to centrifuge tube 2. Another blank group without adding organophosphorus pesticide is set. Both the blank group and the experimental group are reacted in an oven at 60 °C for 60 min, denoted as Channel 2. After the reaction, measure the absorbance of the solution in centrifuge tube 1 at a wavelength of 652 nm and the absorbance of the solution in centrifuge tube 2 at a wavelength of 510 nm by an ultraviolet spectrophotometer. Test in parallel three times and calculate the inhibition rate. The results are shown in Table 1 and Figure 8 .

[0077] The inhibition rate calculation formula is as follows

[0078]

[0079] Among them, A0 is the absorbance of the blank group (without adding organophosphorus pesticides) at the corresponding wavelength.

[0080] A t is the absorbance of the experimental group (added with organophosphorus pesticides) at the corresponding wavelength;

[0081] Application Example 2

[0082] This application provides an application of a copper-based nanozyme in the detection of organophosphorus pesticides. The specific steps are as follows:

[0083] Disperse the ACT-Cu nanozyme prepared in Example 4 in deionized water to prepare a nanozyme solution with a concentration of 10 μg / mL. Take two centrifuge tubes, add 200 μL of the nanozyme solution, 790 μL of MES buffer (pH 6), and 10 μL of 100 μg / mL glyphosate solution to both. Additionally, set a blank group without adding the glyphosate solution. Both the blank group and the experimental group are incubated at room temperature for 15 min; add 200 μL of TMB solution (20 mM) and 200 μL of hydrogen peroxide (50 mM) to centrifuge tube 1 and react at room temperature for 30 min, denoted as Channel 1; add 200 μL of 4-AAP (1 mg / mL) and 200 μL of 2,4-DCP (1 mg / mL) to centrifuge tube 2. Additionally, set a blank group without adding organophosphorus pesticides. Both the blank group and the experimental group react in an oven at 60 °C for 60 min, denoted as Channel 2. After the reaction, measure the absorbance of the solution in centrifuge tube 1 at a wavelength of 652 nm and the absorbance of the solution in centrifuge tube 2 at a wavelength of 510 nm using a UV spectrophotometer. Conduct parallel tests three times and calculate the inhibition rate according to the inhibition rate calculation formula in the application example. The results are shown in Table 1 and Figure 8 .

[0084] Application Example 3

[0085] This application provides an application of a copper-based nanozyme in the detection of organophosphorus pesticides. The specific steps are as follows:

[0086] Disperse the ACT-Cu nanozyme prepared in Example 5 in deionized water to prepare a nanozyme solution with a concentration of 10 μg / mL. Take two centrifuge tubes, add 200 μL of the nanozyme solution, 790 μL of MES buffer (pH 6), and 10 μL of 100 μg / mL glyphosate solution to both tubes. Additionally, set up a blank group without adding the glyphosate solution. Incubate both the blank group and the experimental groups at room temperature for 15 min. Add 200 μL of TMB solution (20 mM) and 200 μL of hydrogen peroxide (50 mM) to centrifuge tube 1 and react at room temperature for 30 min, denoted as Channel 1. Add 200 μL of 4-AAP (1 mg / mL) and 200 μL of 2,4-DCP (1 mg / mL) to centrifuge tube 2. Also set up a blank group without adding the organophosphorus pesticide. Incubate both the blank group and the experimental groups in an oven at 60 °C for 60 min, denoted as Channel 2. After the reaction, measure the absorbance of the solution in centrifuge tube 1 at a wavelength of 652 nm using an ultraviolet spectrophotometer, and measure the absorbance of the solution in centrifuge tube 2 at a wavelength of 510 nm. Conduct parallel tests three times and calculate the inhibition rate according to the inhibition rate calculation formula in the application example. The results are shown in Table 1 and Figure 8 。

[0087] Table 1

[0088]

[0089] From Table 1 and Figure 8 it can be seen that the inhibition rates of Application Example 1 and Comparative Application Example 2 are similar. The value of Channel 1 in Application Example 3 is lower, while the value of Channel 2 is higher. Considering comprehensively, the nanozyme prepared in Example 1 is selected for subsequent experiments.

[0090] Application Example 4

[0091] This application provides an application of a copper-based nanozyme in the detection of organophosphorus pesticides. The specific steps are as follows:

[0092] The ACT-Cu nanozyme prepared in Example 1 was dispersed in deionized water to prepare a nanozyme solution with a concentration of 10 μg / mL. Two centrifuge tubes were taken, and 200 μL of the nanozyme solution, 790 μL of MES buffer (pH 6), and 10 μL of glyphosate solutions with different concentrations (250, 200, 100, 50, 25, and 5 μg / mL) were added thereto, so that the concentration of the glyphosate solution in the system was 2.5, 2, 1, 0.5, 0.25, and 0.05 μg / mL. In addition, a blank group without adding the glyphosate solution was set. The blank group and the experimental groups were both incubated at room temperature for 15 min; 200 μL of TMB solution (20 mM) and 200 μL of hydrogen peroxide (50 mM) were added to centrifuge tube 1, and the reaction was carried out at room temperature for 30 min, denoted as Channel 1; 200 μL of 4-AAP (1 mg / mL) and 200 μL of 2,4-DCP (1 mg / mL) were added to centrifuge tube 2. In addition, a blank group without adding the organophosphorus pesticide was set. The blank group and the experimental groups were both reacted in an oven at 60 °C for 60 min, denoted as Channel 2. After the reaction, the absorbance of the solution in centrifuge tube 1 at a wavelength of 652 nm was measured by an ultraviolet spectrophotometer, and the absorbance of the solution in centrifuge tube 2 at a wavelength of 510 nm was measured. The measurement was carried out in parallel nine times, and the inhibition rate was calculated according to the inhibition rate calculation formula in the application example. The standard curve graphs of glyphosate in Channel 1 and Channel 2 were respectively made, and the absorbance results are shown in Table 2, Figure 9 , and the inhibition rate results are shown in Figure 10 , and the standard curve results are shown in Figure 11 and Figure 12 .

[0093] Table 2

[0094]

[0095] The standard curve of glyphosate in Channel 1 is as shown in Figure 10 . The linear range is 0.05 - 2.5 μg / mL, y = 0.30116x + 0.02999 (R 2 = 0.99086). The detection limit (LOD) calculated according to the 3σ / k principle is 0.044 μg / mL. The standard curve of glyphosate in Channel 2 is as shown in Figure 11 . The linear range is 0.05 - 2.0 μg / mL, y = 0.4277x + 0.03753 (R 2 = 0.99737). The detection limit (LOD) calculated according to the 3σ / k principle is 0.063 μg / mL.

[0096] Application Example 5

[0097] This application provides an application of copper-based nanozyme in the detection of organophosphorus pesticides. The specific steps are as follows:

[0098] The organophosphorus pesticides detected in this application are six organophosphorus pesticides, namely glyphosate, chlorpyrifos, phoxim, glufosinate, profenofos, and fenitrothion, aiming to test whether this detection array can distinguish the six organophosphorus pesticides at low concentrations.

[0099] Disperse the ACT-Cu nanozyme prepared in Example 1 in deionized water to prepare a nanozyme solution with a concentration of 10 μg / mL. Take two centrifuge tubes, and add 200 μL of the nanozyme solution and 790 μL of MES buffer (pH 6) to each centrifuge tube. Add 10 μL of glyphosate (100 μg / mL) to make the concentration of glyphosate in the system 1 μg / mL. Additionally, set up a blank group without adding organophosphorus pesticides. Both the blank group and the experimental group are incubated at room temperature for 15 min. Add 200 μL of TMB solution (20 mM) and 200 μL of hydrogen peroxide (50 mM) to centrifuge tube 1 and react at room temperature for 30 min, denoted as Channel 1. Add 200 μL of 4-AAP (1 mg / mL) and 200 μL of 2,4-DCP (1 mg / mL) to centrifuge tube 2. Additionally, set up a blank group without adding organophosphorus pesticides. Both the blank group and the experimental group are reacted in an oven at 60 °C for 60 min, denoted as Channel 2. After the reaction, measure the absorbance of the solution in centrifuge tube 1 at a wavelength of 652 nm and the absorbance of the solution in centrifuge tube 2 at a wavelength of 510 nm using a UV spectrophotometer. The detection of the other five organophosphorus pesticides is also carried out according to the above protocol, and each organophosphorus pesticide is measured in parallel nine times. The obtained absorbance results are shown in Table 3.

[0100] Table 3

[0101]

[0102] Process the obtained absorbance data using linear discriminant analysis (LDA). The eigenvectors of the first and second linear discriminant functions obtained are used as the first factor and the second factor respectively. With the first factor as the abscissa and the second factor as the ordinate, a linear discriminant analysis graph of the responses of different types of pesticide residues with the same concentration to the array sensor is obtained.

[0103] The obtained linear discriminant analysis graph is shown in Figure 12 , and it can be seen that when this ACT-Cu nanozyme detects the six organophosphorus pesticides at a concentration of 1 μg / mL, it can distinguish glyphosate from the other five organophosphorus pesticides.

[0104] Application Example 6

[0105] This application provides an application of copper-based nanozyme in the detection of organophosphorus pesticides. The specific steps are as follows:

[0106] The organophosphorus pesticides detected in this application are three organophosphorus pesticides, glyphosate, glufosinate, and isazofos, aiming to test whether this detection array can distinguish the three organophosphorus pesticides.

[0107] Disperse the ACT-Cu nanozyme prepared in Example 1 in deionized water to prepare a nanozyme solution with a concentration of 10 μg / mL. Take two centrifuge tubes, add 200 μL of the nanozyme solution and 790 μL of MES buffer (pH 6) to both. Add 10 μL of glyphosate (1000 μg / mL) to make the concentration of the organophosphorus pesticide in the system 10 μg / mL. Another blank group without adding organophosphorus pesticide is set. Both the blank group and the experimental group are incubated at room temperature for 15 min; add 200 μL of TMB solution (20 mM) and 200 μL of hydrogen peroxide (50 mM) to centrifuge tube 1 and react at room temperature for 30 min, denoted as Channel 1; add 200 μL of 4-AAP (1 mg / mL) and 200 μL of 2,4-DCP (1 mg / mL) to centrifuge tube 2. Another blank group without adding organophosphorus pesticide is set. Both the blank group and the experimental group are reacted in an oven at 60 °C for 60 min, denoted as Channel 2. After the reaction, measure the absorbance of the solution in centrifuge tube 1 at a wavelength of 652 nm and the absorbance of the solution in centrifuge tube 2 at a wavelength of 510 nm using a UV spectrophotometer. The detection of the other two organophosphorus pesticides is also carried out according to the above scheme. Each organophosphorus pesticide is measured in parallel twelve times, and the obtained absorbance results are shown in Table 4.

[0108] Table 4

[0109]

[0110] Process the obtained absorbance data using linear discriminant analysis (LDA). The eigenvectors of the first and second linear discriminant functions obtained are used as the first factor and the second factor respectively. With the first factor as the abscissa and the second factor as the ordinate, a linear discriminant analysis graph of the responses of pesticide residues of the same concentration but different types to the array sensor is obtained.

[0111] The obtained linear discriminant analysis graph is shown in Figure 13 , and it can be seen that when this ACT-Cu nanozyme detects six organophosphorus pesticides at a concentration of 10 μg / mL, it can distinguish the three organophosphorus pesticides, glyphosate, glufosinate, and isazofos.

[0112] The experimental results prove that the present invention for the first time prepares a copper-based nanozyme ACT-Cu by a one-step solvothermal reaction of ammonium citrate and copper nitrate. This nanozyme has a filamentous reticular structure, a relatively high specific surface area, and many active sites. The copper in this nanozyme exists in the form of zero-valent Cu, Cu + and Cu2+ Existing simultaneously, it has high peroxidase-like activity and laccase-like activity, can selectively detect glyphosate with a low detection limit, can sensitively detect glyphosate at low concentrations, and at slightly higher concentrations, can distinguish among three pesticides, namely glyphosate, glufosinate, and isazofos. The nanozyme provided by the present invention has the advantages of simple preparation process, low cost, special morphology, and high enzyme-like activity, can realize sensitive detection of organophosphorus pesticide residues, and has good practical application prospects.

[0113] As described above, the above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. For those skilled in the art of this technology, changes or substitutions that can be easily thought of are also regarded as the protection scope of the present invention.

Claims

1. A preparation method of a copper-based nanozyme, characterized in that, The steps are as follows: (1) Ultrasonic dispersion of ammonium citrate and copper nitrate in N,N-dimethylformamide solution respectively, and mixing and stirring the fully dispersed solutions to make them uniform; (2) mixing the above solutions and transferring them to a hydrothermal reactor to carry out a solvothermal reaction in a muffle furnace; cooling them to room temperature and then taking them out, centrifuging and collecting the precipitate, washing and drying them in sequence to obtain the copper-based nanozyme; The molar ratio of the copper nitrate to the ammonium citrate is 3-7:

1.

2. The preparation method of a copper-based nanozyme according to claim 1, wherein In step (1), the time for ultrasonically dispersing ammonium citrate and copper nitrate into the N,N-dimethylformamide solution is 15 minutes, and the time for mixing the two solutions is 10 minutes, and the temperature is room temperature.

3. The preparation method of a copper-based nanozyme according to claim 1, wherein, In step (2), the solvent thermal reaction time is 9 to 15 hours, and the temperature is 100 to 180 °C.

4. The preparation method of a copper-based nanozyme according to claim 1, wherein, In step (2), the drying is carried out in an oven at 60°C for 12 h.

5. A copper-based nanozyme, characterized in that, The copper-based nanozyme obtained by the synthesis method according to any one of claims 1 to 4.

6. The copper-based nanozyme according to claim 5, wherein, The described copper-based nanozyme has a silk-like network structure, is composed of ammonium citrate and copper, and has a diameter size of 10-200 nm, where the valence states of copper elements are zero-valent Cu, Cu + and Cu 2+ existing simultaneously.

7. The copper-based nanozyme described in claim 5 is used to detect organophosphorus pesticides.

8. The method for detecting organophosphorus pesticides by copper-based nanozymes according to claim 5, wherein the copper-based nanozymes are added to the test solution, mixed evenly and incubated at room temperature; then, the corresponding color developer is added to react, and the organophosphorus pesticides are quantitatively detected by detecting the absorbance value of the supernatant within 400-800 nm.

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