A cerium single-atom nanozyme, preparation method and method for detecting organic phosphorus

Abstract

The present invention discloses a cerium single-atom nanozyme, a preparation method and a method for detecting organophosphorus, and relates to the technical field of organophosphorus residue detection. The preparation method of the cerium single-atom nanozyme comprises: mixing the raw materials dimethylimidazole, zinc nitrate and cerium source, performing a hydrothermal reaction in a reactor, collecting the precipitate, calcining and acid washing to obtain the cerium single-atom nanozyme; the cerium source is selected from cerium acetylacetonate; the temperature for the hydrothermal reaction in the reactor is 80-200°C, and the time for the hydrothermal reaction is 2-8h. The detection method provided by the present invention has a high detection selectivity for organophosphorus, and can avoid the interference of a large number of coexisting ions. In addition, the detection method provided by the present invention is simple to operate, low in cost, mild in conditions, and can meet the rapid detection requirements of organophosphorus residues of pesticides in agricultural products and the environment. The detection method provided by the present invention has a wide range of organophosphorus detection, and the cerium single-atom nanozyme provided by the present invention can be used for colorimetric detection of organophosphorus residues.

Description

Technical Field

[0001] The present invention relates to the technical field of organophosphorus residue detection, and in particular to a cerium single-atom nanozyme, a preparation method and a method for detecting organophosphorus. Background Art

[0002] Pesticides refer to chemical substances used in agriculture to prevent and control pests and diseases and regulate plant growth. There are many varieties, among which, according to chemical structure, they can be divided into several categories, such as organochlorine, organosulfur, organophosphorus, organic nitrogen, carbamate, sulfonylurea, triazine, pyrethroid, neonicotinoid, phenoxycarboxylic acid, etc. In order to avoid the loss of agricultural products due to diseases, insects and weeds, and to meet the needs of the growing population and ensure the output of agricultural products around the world, pesticides have been widely used around the world in the past 50 years. Statistics show that the use of pesticides has saved nearly 30% of the world's crops. Among them, organophosphorus pesticides have been widely used around the world due to their high efficiency and broad spectrum, accounting for about 15% of the insecticide market share; especially in my country, the use of organophosphorus pesticides is particularly common, accounting for about one-third of the global use of organophosphorus pesticides. Although the extensive use of organophosphorus pesticides has played an extremely important role in pest control and increasing crop yields, most organophosphorus pesticides are highly toxic. The extensive use of organophosphorus pesticides has also brought significant problems to the human living environment. In addition, the frequent food safety incidents caused by organophosphorus pesticide residues have also posed a huge threat to human life safety. Therefore, the development of rapid and efficient methods for detecting pesticide residues has received increasing attention.

[0003] At present, there are many methods for pesticide residue detection, and the most commonly used methods are mainly divided into two categories: one is the analysis method based on traditional instruments, mainly including gas chromatography (GC), high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (LC-MS / MS) and liquid chromatography-mass spectrometry (GC-MS / MS). These detection methods have many advantages, such as high detection sensitivity, good reproducibility, and stable test results; but at the same time, these detection methods also have some shortcomings, such as the detection instruments are generally expensive, the pretreatment and detection operation before detection are complicated, and the detection time is long, and professional training is required to complete the detection. However, these shortcomings have caused major problems in achieving low-cost and rapid detection of pesticide residues in a large number of samples. The second category is based on the principle of biological detection technology for determination, mainly including immunoassay, enzyme inhibition method, in vivo detection method, gold nanoparticles, biosensors, etc. Among them, the enzyme inhibition method has attracted much attention due to its simple operation and low cost. However, the traditional enzyme inhibition method faces the problems of low sensitivity, high false negative and false positive rates, which seriously limits its application. Applying nanozymes to colorimetric detection of organophosphorus pesticides can effectively improve sensitivity, accuracy, anti-interference ability, and environmental tolerance, and achieve high-throughput, visual, and real-time pesticide residue analysis.

[0004] In recent years, single-atom nanozymes have gradually become a research hotspot due to their advantages such as low preparation cost, high atomic utilization, and adjustable active sites. The sharply increased surface free energy, quantum size effect, unsaturated coordination environment and metal-support interaction give them excellent catalytic activity. In addition, single-atom catalysts not only have extremely low metal loading but also greatly improve the utilization of metal atoms, which can change the adsorption / desorption selectivity of active components on the catalyst for different molecules, thereby affecting the reaction kinetics. Although researchers have completed some research on single-atom nanozymes, there have been no reports on the preparation of high-loaded cerium single-atom nanozymes, and there have been no reports on their application in pesticide residues.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a cerium single-atom nanozyme, a preparation method and a method for detecting organophosphorus to meet the needs of rapid detection of organophosphorus pesticide residues in agricultural and sideline products.

[0007] The present invention is achieved in that:

[0008] In the first aspect, the present invention provides a method for preparing a cerium single-atom nanozyme, which comprises the following steps: mixing raw materials of dimethylimidazole, zinc nitrate and a cerium source, conducting a hydrothermal reaction in a reactor, collecting the precipitate, and calcining to obtain a cerium single-atom nanozyme; the cerium source is selected from one of cerium nitrate, cerium oxalate, cerium chloride, and cerium acetylacetonate; the temperature for the hydrothermal reaction in the reactor is 80, 120, 160, or 200°C, and the time for the hydrothermal reaction is 2-8h.

[0009] The inventors provide a method for preparing a cerium single-atom nanozyme. The cerium single-atom nanozyme prepared by the preparation method is a peroxidase mimetic that can catalyze hydrogen peroxide to produce ·OH, and oxidize colorimetric probes such as 3,3',5,5'-tetramethylbenzidine (TMB) through ·OH to form a colorimetric product, or oxidize fluorescent probes such as o-phenylenediamine to produce a fluorescent signal. When organic phosphorus (OP) is added to the reaction system, OP can effectively inhibit the activity of acetylcholinesterase. Based on the fact that acetylcholine, acetylcholinesterase, and choline oxidase can generate hydrogen peroxide, OP inhibits the activity of acetylcholinesterase and thus reduces the amount of hydrogen peroxide produced, which ultimately causes the cerium single-atom nanozyme and substrate system (CeN x -SAzyme+colorimetric / fluorescent probe) absorbance changes. By testing the absorbance of the system with different concentrations of OP at the maximum absorption wavelength of the color developer (or the fluorescence intensity at the maximum emission wavelength of the fluorescent probe), a standard working curve of organophosphorus is drawn, and the OP content of the object to be tested can be detected based on the standard working curve.

[0010] The cerium single-atom nanozyme prepared by the above method is obtained by calcination and has a specific spatial molecular structure, presenting a regular dodecahedral structure.

[0011] In the above preparation method, dimethylimidazole, zinc nitrate and cerium source can generate orange-red precipitate through hydrothermal reaction, and cerium single-atom nanozyme with a specific molecular structure can be generated after high-temperature calcination.

[0012] In the present invention, "single-atom nanozymes" are materials with enzyme-like catalytic activity that use a single metal atom as the active center, have a designable geometric structure and electronic coordination, a unique quantum size effect, and maximum atomic utilization efficiency. Specifically, single-atom nanozymes are a type of nanomaterials that contain enzymatic properties. They exhibit enzymatic catalytic properties that only natural enzymes have at the nanoscale. They can catalyze biochemical reactions mediated by natural enzymes under physiological conditions, and exhibit the reaction kinetics and catalytic mechanisms of natural enzymes.

[0013] In the present invention, the term "peroxidase mimetic" refers to a nanomaterial having peroxidase catalytic activity. Specifically, the oxidase mimetic in the present invention uses hydrogen peroxide as an electron acceptor and generates a colored substance TMBox by oxidizing the TMB substrate for colorimetric detection.

[0014] The term "TMB" is an abbreviation of the compound "3,3',5,5'-tetramethylbenzidine", and the two are used interchangeably.

[0015] In the present invention, the term "CeN x "-SAzyme" refers to synthetic cerium single-atom nanozyme, and the two can be used interchangeably.

[0016] In an optional embodiment, the temperature of the hydrothermal reaction is 80°C-200°C.

[0017] The hydrothermal reaction time is 2-8 hours, such as 2 hours, 4 hours, 6 hours or 8 hours. Under the above hydrothermal reaction conditions, it is helpful to obtain the cerium single-atom nanozyme of the target structure.

[0018] In a preferred embodiment of the present invention, the mixing molar ratio of dimethylimidazole, zinc nitrate and cerium source is 1:1:0.01 to 1:1:0.5. Under the above ratio, it is helpful to obtain a cerium monogenous nanozyme with higher stability and better activity, and its peroxidase enzyme activity is much greater than the oxidase enzyme activity, and has excellent selectivity.

[0019] In an optional embodiment, dimethylimidazole and the cerium source are mixed in the form of solutions, and the solvent of the solution is methanol, ethanol, DMF or acetone.

[0020] In an optional embodiment, the mixing volume ratio of the solution containing dimethylimidazole and the cerium source to the solution containing zinc nitrate is 0.5-1.5:2.

[0021] In an optional embodiment, after the raw material dimethylimidazole and the cerium source are mixed, ultrasonic treatment and stirring are also included;

[0022] In an optional embodiment, ultrasonic treatment is performed for 5-30 minutes and stirring is performed for 0.5-3 hours. Ultrasonic treatment is performed to improve the dispersion uniformity of dimethylimidazole and cerium source, and the purpose of stirring is to fully mix the raw materials so as to obtain cerium single-atom nanozymes with high catalytic activity and uniform site distribution.

[0023] In a preferred embodiment of the present invention, after collecting the precipitate, the precipitate is washed with an organic solvent, and then the washed precipitate is freeze-dried.

[0024] In an alternative embodiment, the organic solvent is selected from DMF or methanol.

[0025] In a preferred embodiment of the present invention, the calcination temperature is 700-1000° C. The calcination temperature is, for example, selected from 700° C., 800° C., 900° C. or 1000° C. The above calcination conditions are helpful to obtain cerium single-atom nanozymes with target particle size and molecular structure.

[0026] In an optional embodiment, the calcination is carried out in a tubular furnace, and the calcination time is 1-3 hours.

[0027] In a second aspect, the present invention further provides a cerium single-atom nanozyme, which is prepared by the above-mentioned preparation method of the cerium single-atom nanozyme;

[0028] In an optional embodiment, the cerium single-atom nanozyme has a regular dodecahedral structure.

[0029] In a third aspect, the present invention also provides a method for detecting organic phosphorus based on cerium single-atom nanozyme, the method comprising the following steps:

[0030] Mix different concentrations of organophosphorus with acetylcholinesterase for incubation, then add choline oxidase and acetylcholine for incubation, add the above-mentioned cerium single-atom nanozyme and substrate for incubation, and measure the absorbance / fluorescence signal; draw a standard working curve of phosphorus concentration and IR value; the substrate is a color substrate or a fluorescent substrate;

[0031] The IR value is calculated by the following formula: , where A0 is the blank absorbance value / fluorescence value without adding acetylcholine and organophosphate, A1 is the maximum absorbance value / fluorescence value without adding organophosphate but adding acetylcholine, and A is the absorbance value / fluorescence value after incubation of the sample with both organophosphate and acetylcholine added;

[0032] The sample to be tested is mixed with acetylcholinesterase for incubation, and then choline oxidase and acetylcholine are added for incubation, and the above-mentioned cerium single-atom nanozyme and substrate (color substrate or fluorescent substrate) are added for incubation, the absorbance of the sample is measured and the IR value of the sample is calculated, and the organophosphorus content of the sample is obtained according to the standard working curve.

[0033] The IR value is the inhibition rate (IR) of AChE activity. The linear relationship between IR and OP concentration is used to detect the OP residue in crops or vegetables. Draw the standard working curve of organophosphorus concentration-absorbance.

[0034] The detection method provided by the present invention has high detection selectivity for organophosphorus and strong anti-interference ability. In the environment of other types of insecticides such as pyrethroids, neonicotinoids, butyronitrile, herbicides such as bispyribac-sodium, various metal ions, sulfate ions, and chloride ions, the detection of organophosphorus can still maintain high accuracy and avoid interference from a large number of coexisting ions. In addition, the detection method provided by the present invention is simple to operate, low in cost, and mild in conditions. It does not require the addition of other reagents to meet the needs of rapid detection of organophosphorus residues in agricultural products and pesticides in the environment. The detection method provided by the present invention has a wide range of organophosphorus detection, ranging from 1 ng to 1000ng mL -1 or 0.5 ng-1000 ng mL -1 , sensitivity is no more than 0.8 ng mL -1 , which is far lower than the organophosphorus MRLs value stipulated in my country's national food safety standards and is better than the existing methods reported in the literature. It can fully meet the minimum detection requirements of my country's pesticide residue limit standards for organophosphorus pesticides such as dichlorvos, chlorpyrifos, and malathion, and is of great significance in the quality and safety supervision of agricultural products.

[0035] In a preferred embodiment of the present invention, the chromogenic substrate is selected from TMB, N,N-diethyl-p-phenylenediamine, o-phenylenediamine, o-tolidine, N-ethyl N-(3-sulfopropyl)-3-methylaniline sodium salt and 4-aminoantipyrine (need to work together), 3,5-dichloro-2-hydroxybenzenesulfonate sodium, 3,5-dichloro-2-hydroxybenzenesulfonate sodium and 4-aminoantipyrine, or 2,2′-azo-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt.

[0036] The fluorescent substrate is selected from o-phenylenediamine (OPD), o-phenylenediamine, Amplex Red, terephthalic acid, and 10-acetyl 3,7-hydroxyphenoxazine.

[0037] In a preferred embodiment of the present invention, the conditions for mixed incubation of organophosphorus and acetylcholinesterase are: incubation at 35-37°C for 5-40 min; the mixed molar ratio of organophosphorus and acetylcholinesterase is 0.015ng-90ng:2.5mU-50mU; the mixed volume ratio of acetylcholinesterase, choline oxidase and acetylcholine is 2.5mU-50 mU:25mU-50mU:60umol-900umol.

[0038] In an optional embodiment, choline oxidase and acetylcholine are added and incubated for 10-60 min;

[0039] In an optional embodiment, the mixing ratio of the cerium single-atom nanozyme and the substrate is: 5.4 μg-21.6 μg: 108 nmol-324 nmol;

[0040] In an optional embodiment, the cerium single-atom nanozyme and the substrate are added and incubated in a buffer for 10-40 minutes, and the buffer is selected from acetate buffer, PBS buffer solution, Tris-maleic acid buffer or citric acid buffer.

[0041] In a preferred embodiment of the present invention, when the chromogenic substrate is selected from TMB, A0, A1 and A are all absorbance values ​​at 652 nm. 652 nm is the maximum ultraviolet absorption wavelength of TMB, which can be used to detect the residual amount of OP in fruits, vegetables, food or the environment.

[0042] In an optional embodiment, the detectable organophosphorus concentration of the sample to be tested by the method is in the range of 0.5-1000 ng / mL -1 Or 1-1000ng / mL -1 , sensitivity does not exceed 0.8 ng mL -1 , which is far lower than the MRLs of organophosphorus pesticides in food stipulated by my country, and can meet the quality and safety supervision needs of agricultural products. In the fourth aspect, the present invention also provides an application of a cerium single-atom nanozyme in the fluorescence and / or colorimetric detection of organophosphorus residues. The single-atom enzyme colorimetric detection of organophosphorus pesticide residues has mild conditions, strong anti-interference ability, good specificity, high sensitivity, and no need to add other reagents, low detection cost, and simple operation; compared with the existing national standard method, it has higher sensitivity, shorter detection time, higher detection throughput, simpler operation, and is easy to promote. It can play an important role in the daily supervision and protection of agricultural products, and has good economic and social benefits.

[0043] In a fifth aspect, the present invention also provides a nanozyme colorimetric / fluorescence sensor, which includes an array sensor, the sensor includes a colorimetric array, and the colorimetric array includes a cerium single-atom nanozyme.

[0044] In a sixth aspect, the present invention also provides the use of cerium single-atom nanozymes in the preparation of nanozyme colorimetric / fluorescence sensors.

[0045] The present invention has the following beneficial effects:

[0046] The present invention obtains a precipitate by a mixed hydrothermal reaction of raw materials dimethylimidazole, a zinc source and a cerium source, and obtains a cerium single-atom nanozyme with a specific molecular structure by calcining the precipitate. The cerium single-atom enzyme is a peroxidase mimetic, which can catalyze hydrogen peroxide to produce ·OH, and oxidize color developers and fluorescent substrates such as 3,3',5,5'-tetramethylbenzidine (TMB) by ·OH to develop color or emit a strong fluorescent signal. When organic phosphorus (OP) is added to the reaction system, OP can effectively inhibit the activity of acetylcholinesterase. Based on the fact that acetylcholinesterase, acetylcholine and choline oxidase can generate hydrogen peroxide, OP inhibits the activity of acetylcholinesterase and thus reduces the amount of hydrogen peroxide produced, which ultimately causes the cerium single-atom nanozyme and color developer system (CeN x -SAzyme+color / fluorescence system) absorbance changes. By testing the absorbance / fluorescence value of the system with different concentrations of OP added at the maximum absorption wavelength of the color developer, a standard working curve of organophosphorus concentration-absorbance / organophosphorus concentration-fluorescence is drawn. Based on the standard working curve, the OP content of the object to be tested can be detected.

[0047] The detection method provided by the present invention has high detection specificity and selectivity for organophosphorus, and has almost no response to non-organophosphorus pesticides; it has strong anti-interference ability, and can still maintain high accuracy in the detection of organophosphorus in the environment of multiple metal ions, sulfate ions, and chloride ions, and can effectively avoid interference from a large number of coexisting ions and other pesticides. In addition, the detection method provided by the present invention is simple to operate, low in cost, and mild in conditions. It does not require the addition of other reagents, and can meet the needs of rapid detection of organophosphorus pesticide residues in agricultural products and the environment. The detection method provided by the present invention has a wide range of organophosphorus detection, with a detection range of 0.5 ng-1000 ng mL -1 or 1 ng-1000 ng mL -1 , sensitivity does not exceed 0.8 ngmL -1 , which can fully meet the minimum detection limit of organophosphorus pesticides such as DDT in my country's national food safety limit standards.

[0048] In addition, the cerium single-atom nanozyme provided by the present invention can be used for ratiometric fluorescence and / or colorimetric detection of organophosphorus residues, and can also be used to prepare nanozyme colorimetric sensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0050] Figure 1 This is a high-resolution SEM image of the cerium single-atom enzyme prepared in Example 1;

[0051] Figure 2 The full ultraviolet absorption spectra of the cerium single atom prepared in Example 1 and the control group;

[0052] Figure 3 The XRD pattern of the cerium single-atom enzyme prepared in Example 1;

[0053] Figure 4 This is the infrared spectrum of the cerium single-atom enzyme prepared in Example 1;

[0054] Figure 5 The statistical result diagram of the anti-interference performance test of the detection system and the statistical result diagram of the selectivity test of the cerium single-atom enzyme prepared in Example 1;

[0055] Figure 6 This is a standard curve diagram for detecting organophosphorus pesticides in rice using the cerium single-atom enzyme prepared in Example 1. DETAILED DESCRIPTION

[0056] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0057] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc.); Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0058] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0059] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0060] Example 1

[0061] This example provides the preparation of cerium single-atom nanozymes and a method for detecting organophosphorus pesticide residues based on cerium single-atom nanozyme colorimetry:

[0062] 1. Preparation of cerium single-atom nanozymes:

[0063] 3.942 g of dimethylimidazole, 0.2619 g of cerium acetylacetonate and 3.542 g of zinc nitrate hexahydrate were accurately weighed and dissolved in 45 mL and 90 mL of methanol solution respectively, mixed, ultrasonicated for 15 min, mechanically stirred for 1 h, and then placed in a high-pressure reactor at 120 ° C for 4 h. The freeze-dried product was calcined at 900 ° C in a tube furnace and washed with 1 mol / L nitric acid to obtain cerium single-atom nanozyme (CeN X -SAzyme).

[0064] Take 10.8 μL of the prepared cerium single-atom nanozyme (1 mg mL -1 , dissolved in deionized water) was dispersed in acetic acid + acetate buffer solution (10 mM, pH = 4.06), 30 μL TMB (4.8 mM, dissolved in deionized water) and H2O2 solution were added to the mixed solution and incubated for 10 min to obtain CeN x -SAzyme+TMB+ H2O2.

[0065] In addition, 10.8 μL of the prepared cerium single-atom nanozyme (1 mg mL -1 , dissolved in deionized water) was dispersed in an acetic acid + acetate buffer solution (10 mM, pH = 4.06), and 30 μL of TMB (4.8 mM, dissolved in deionized water) was added to the acetic acid + acetate buffer solution containing the cerium single-atom nanozyme and incubated for 10 min to obtain CeN x -SAzyme+TMB.

[0066] The CeN x -SAzyme, CeN x -SAzyme+TMB+ H2O2、CeN x -UV absorption spectrum of SAzyme+TMB, record the absorbance, UV absorption spectrum as Figure 2 shown. Figure 2 It can be seen that the mixed solution of cerium single-atom nanozyme, TMB and H2O2 has a characteristic absorption peak at 652nm. The absorption peak of the mixture of cerium single-atom nanozyme and TMB can be significantly reduced by inhibiting the production of H2O2. Based on this, a corresponding colorimetric method for detecting organophosphorus pesticide residues was developed.

[0067] 2. Colorimetric detection of organophosphorus pesticide residues based on cerium single-atom nanozymes

[0068] 30 μL of different concentrations of organophosphate (DDV) 1, 5, 30, 100, 300, 500, 1000 ng / mL were incubated with 5 μL of acetylcholinesterase solution at 37°C for 20 min. Then 5 μL of choline oxidase solution and 15 μL of acetylcholine were added and incubated for 40 min. 1 mg / mL was prepared with deionized water -1 Ce single-atom enzyme solution, take 10.8 μL of nanozyme solution, 120 μL of acetic acid and sodium acetate buffer solution and 30 μL of TMB into the above incubated solution and incubate again for 30 min, and use a microplate reader to measure the absorbance change at 652 nm.

[0069] For the detection of OP, the inhibition rate (IR) of AChE activity was used. A control group without the addition of ACh and OP was used to obtain the blank absorbance value (A0). Another group without the addition of OP was used as the control group to obtain the maximum absorbance value (A1) under the experimental parameters. A is the absorbance value after incubation of the sample with the addition of organophosphate and acetylcholine at the same time. Then the equation for calculating the IR value is as follows: .

[0070] There is a linear relationship between the IR value and the concentration of OP, and the OP concentration in the sample to be tested can be detected based on this linear relationship. Draw a standard working curve of organophosphorus concentration-absorbance.

[0071] Example 2

[0072] This example provides the preparation of cerium single-atom nanozymes and a method for detecting organophosphorus pesticide residues based on cerium single-atom nanozyme colorimetry:

[0073] 1. The preparation method of cerium single-atom nanozyme is as follows:

[0074] 3.942g of dimethylimidazole, 0.131g of cerium acetylacetonate and 3.542g of zinc nitrate hexahydrate were dissolved in 45mL and 90mL of methanol solution respectively, mixed, ultrasonicated for 10min, mechanically stirred for 1h, and placed in a high-pressure reactor at 120℃ for 4h. The freeze-dried product was calcined at 700℃ in a tube furnace and acid-washed with 1mol / L nitric acid to obtain the cerium single-atom nanozyme.

[0075] 2. Colorimetric detection of organophosphorus pesticide residues based on cerium single-atom nanozymes

[0076] 30 μL of different concentrations of organophosphate (chlorpyrifos) 1, 5, 30, 100, 300, 500, 1000 ng / mL were incubated with 5 μL of acetylcholinesterase solution at 37°C for 5 min. Then 5 μL of choline oxidase solution and 15 μL of acetylcholine were added and incubated for 45 min. 1 mg / mL was prepared with deionized water. -1 Ce single atom enzyme solution, take 10.8 μL nanozyme solution and 120 μL acetic acid and sodium acetate buffer solution and 30 μL TMB into the above incubated solution and incubate again for 30 min. Use a microplate reader to measure the absorbance change at 652 nm. Draw the standard working curve of organophosphorus concentration-absorbance.

[0077] Example 3

[0078] This example provides the preparation of cerium single-atom nanozyme and a method for detecting organophosphorus pesticide residues based on cerium single-atom nanozyme colorimetry:

[0079] 1. Preparation of cerium single-atom nanozyme, the difference from Example 1 is that the hydrothermal temperature is different:

[0080] 3.942g of dimethylimidazole, 0.2619g of cerium acetylacetonate and 3.542g of zinc nitrate hexahydrate were dissolved in 45mL and 90mL of methanol solution respectively, mixed, ultrasonicated for 0.5h, mechanically stirred for 1h, and then placed in a high-pressure reactor at 160℃ for 4h. The freeze-dried product was calcined at 900℃ in a tube furnace and acid-washed with 1mol / L nitric acid to obtain the cerium single-atom nanozyme.

[0081] 2. Colorimetric detection method for organophosphorus pesticide residues based on cerium single-atom nanozymes, the difference from Example 1 is that the incubation time is different

[0082] 30 μL of different concentrations of organophosphorus (parathion) were incubated with 5 μL of acetylcholinesterase solution at 37°C for 15 min. Then 5 μL of choline oxidase solution and 15 μL of acetylcholine were added and incubated for 40 min. 1 mg / mL was prepared with deionized water. -1 Ce single atom nanozyme solution, take 10.8 μL nanozyme solution and 120 μL acetic acid and sodium acetate buffer solution and 30 μL TMB into the above incubated solution and incubate again for 30 min. Use a microplate reader to measure the absorbance change. Draw the standard working curve of organophosphorus concentration-absorbance.

[0083] Example 4

[0084] This example provides the preparation of cerium single-atom nanozymes and a method for detecting organophosphorus pesticide residues based on cerium single-atom nanozyme colorimetry:

[0085] 1. The preparation of cerium single-atom nanozyme is different from that in Example 1 in that the calcination temperature is different.

[0086] 3.942g of dimethylimidazole, 0.2619g of cerium acetylacetonate and 0.566g of zinc nitrate hexahydrate were dissolved in 45mL and 100mL of methanol solution respectively, mixed, ultrasonicated for 1 min, mechanically stirred for 1h, and then placed in a high-pressure reactor at 120℃ for 4h. The freeze-dried product was calcined at 800℃ in a tube furnace to obtain cerium single-atom nanozyme.

[0087] 2. The colorimetric detection method for organophosphorus pesticide residues based on cerium single-atom nanozymes is different from that in Example 1 in that the incubation time is different.

[0088] 30 μL of different concentrations of organophosphate (fenthion) were incubated with 5 μL of acetylcholinesterase solution at 37°C for 15 min. Then 5 μL of choline oxidase solution and 15 μL of acetylcholine were added and incubated for 60 min. 1 mg / mL was prepared with deionized water. -1 Ce single atom nanozyme solution, take 10.8 μL nanozyme solution and 120 μL acetic acid and sodium acetate buffer solution and 30 μL TMB into the above incubated solution and incubate again for 30 min. Use a microplate reader to measure the absorbance change. Draw the standard working curve of organophosphorus concentration-absorbance.

[0089] Example 5

[0090] This example provides the preparation of cerium single-atom nanozymes and a method for detecting organophosphorus pesticide residues based on cerium single-atom nanozyme colorimetry:

[0091] The preparation of the cerium single-atom nanozyme is different from that in Example 1 in that the calcination temperature is different.

[0092] 3.942g of dimethylimidazole and 0.2619g of cerium acetylacetonate were dissolved in 45mL and 90mL of methanol solution respectively, mixed, ultrasonicated for 1 min, mechanically stirred for 1h, and then placed in a high-pressure reactor at 120℃ for 4h. The freeze-dried product was calcined at 1000℃ in a tube furnace and washed with 1mol / L nitric acid to obtain cerium single-atom nanozyme.

[0093] 2. The colorimetric detection method for organophosphorus pesticide residues based on cerium single-atom nanozyme is different from that in Example 1 in that the incubation time is different.

[0094] 30 μL of different concentrations of organophosphate (malathion) were incubated with 5 μL of acetylcholinesterase solution at 37°C for 15 min. Then 5 μL of choline oxidase solution and 15 μL of acetylcholine were added and incubated for 60 min. 1 mg / mL was prepared with deionized water. -1Ce single atom nanozyme solution: 10.8 μL of nanozyme solution, 120 μL of acetic acid and sodium acetate buffer solution, and 30 μL of TMB were added to the above incubated solution and incubated again for 30 min. The absorbance change was measured using an enzyme reader.

[0095] Draw the standard working curve of organophosphorus concentration-absorbance.

[0096] Example 6

[0097] This example provides the preparation of cerium single-atom nanozymes and a method for detecting organophosphorus pesticide residues based on cerium single-atom nanozymes fluorescence method:

[0098] 1. Preparation of cerium single-atom nanozyme, compared with Example 1, the difference lies in the difference in hydrothermal temperature and raw material ratio:

[0099] 3.942g of dimethylimidazole, 0.1964g of cerium acetylacetonate and 3.542g of zinc nitrate were dissolved in 45mL and 90mL of methanol solution respectively, mixed, ultrasonicated for 0.5h, mechanically stirred for 1h, and then placed in a high-pressure reactor at 160℃ for 4h. The freeze-dried product was calcined at 900℃ in a tube furnace and washed with 1mol / L nitric acid to obtain the cerium single-atom nanozyme.

[0100] 2. Colorimetric detection method for organophosphorus pesticide residues based on cerium single-atom nanozymes, the difference from Example 1 is that the incubation time is different

[0101] Incubate 30 μL of organophosphate (DDV) at different concentrations with 5 μL of acetylcholinesterase solution at 37°C for 15 min. Then add 5 μL of choline oxidase solution and 15 μL of acetylcholine and incubate for 40 min. Prepare 1 mg / mL with deionized water. -1 Ce single atom nanozyme solution, take 10.8 μL of nanozyme solution, 130 μL of acetic acid and sodium acetate buffer solution and 20 μL of terephthalic acid into the above incubated solution and incubate again for 30 min, and use fluorescence spectrometer to measure the change of fluorescence value. Draw the organophosphorus concentration-fluorescence standard working curve.

[0102] Comparative Example 1

[0103] The preparation of cerium single-atom nanozyme is different from that in Example 1 only in that the hydrothermal temperature is different.

[0104] 3.942g of dimethylimidazole, 0.2619g of cerium acetylacetonate and 3.542g of zinc nitrate hexahydrate were dissolved in 45mL and 90mL of methanol solution respectively, mixed, ultrasonicated for 0.5h, mechanically stirred for 1h, and then placed in a high-pressure reactor at 50℃ for 4h. The freeze-dried product was calcined at 800℃ in a tube furnace. Due to the low hydrothermal temperature and the low autogenous pressure in the reactor, single-atom nanozymes with uniform morphology and excellent catalytic performance could not be generated after calcination and acid washing.

[0105] Comparative Example 2

[0106] 1. Preparation of cerium single-atom nanozyme, which is different from Example 1 in that no cerium source is added.

[0107] 3.942g of dimethylimidazole and 3.542g of zinc nitrate hexahydrate were dissolved in 45mL and 90mL of methanol solution respectively, mixed, ultrasonicated for 0.5h, mechanically stirred for 1h, and then placed in a high-pressure reactor at 120℃ for 4h. The freeze-dried product was calcined at 800℃ in a tube furnace. Due to the high calcination temperature, the organic metal framework without cerium acetylacetonate was damaged, and the original morphology could not be maintained, and there was almost no enzyme-like catalytic activity.

[0108] Experimental Example 1

[0109] In this experiment, the cerium single-atom enzyme prepared in Example 1 was observed by scanning electron microscopy. Figure 1 As shown, it has a regular dodecahedral microstructure and a particle size of 300-500nm.

[0110] The XRD pattern of the cerium single-atom enzyme prepared in Example 1 is shown in Figure 3 As shown, Figure 3 There are only two diffraction peaks at 26.5° and 42°, which can be attributed to carbon (002) and (101), respectively, indicating that there is no crystalline cerium. Figure 4 As shown, there are no characteristic peaks of ZIF-8 and Ce-O, proving the successful preparation of cerium single-atom nanozyme.

[0111] Experimental Example 2

[0112] The cerium single-atom enzyme prepared in Example 1 was tested for anti-interference performance and selectivity of the detection system.

[0113] The specific test process is as follows:

[0114] OP detection selectivity was tested by adding different ions (Mg) at 10 μg / mL in the buffer system. 2+ , Ca 2+ Glu, Ka + 、Zn 2+ 、SO4 2-, Cl - ) was used to investigate the absorbance of the CeNx-SAzyme+TMB system.

[0115] Results reference Figure 5 As shown, the results show that the detection method provided by the present invention has almost no response to pesticides such as herbicides and non-organophosphorus insecticides, proving that it has high detection selectivity and specificity for organophosphorus and strong anti-interference ability. In the environment of multiple metal ions and sulfate ions and chloride ions, the detection of organophosphorus can still maintain a high accuracy and can avoid the interference of a large number of coexisting ions.

[0116] Experimental Example 3

[0117] The method for detecting organophosphorus residues provided in Example 1 of the present invention was used to detect pesticide residues in rice. The standard curve of organophosphorus concentration-IR value was drawn with reference to Figure 6 shown.

[0118] Experimental Example 4

[0119] The cerium single-atom nanozyme and the method for detecting organophosphorus provided by the present invention can realize the rapid detection of the organophosphorus pesticide dichlorvos in rice in a short time. When the dichlorvos addition level in the rice is 0.04 mg / kg, the standard curve of Experimental Example 3 is used to detect the organophosphorus residue, and the results show that the addition recovery rate and relative standard deviation have satisfactory results (see Table 1).

[0120] As shown in Table 2, the quantitative limit of the rapid detection method provided by the present invention is much lower than the maximum residual limit value of dichlorvos in rice specified in the national standard (0.2 mg / kg, GB2763-2021), indicating that the method provided by the present invention has low detection cost, high sensitivity and high detection throughput.

[0121] The method involved in the present invention is applied to the detection in actual samples, and the recovery rate is in the range of 89.2%-94.3%, which has high reliability and practicality; through correlation analysis with the detection results of the GC-MS method, the results show that the correlation is good, which proves that the method provided by the present invention can realize the accurate detection of organophosphorus pesticide residues.

[0122] Table 1 Statistical table of the results of the addition recovery experiment using the cerium single-atom enzyme prepared in Example 1 for pesticide residue analysis.

[0123]

[0124] Table 2 is a sensitivity comparison table of the detection method provided in Example 1, the current national standard method, and the method reported in the literature.

[0125]

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a cerium single-atom nanozyme, characterized in that: The method comprises the following steps: mixing raw materials of dimethylimidazole, zinc nitrate and a cerium source, carrying out a hydrothermal reaction in a reactor, collecting a precipitate, calcining and washing with 0.5-1.5 mol / L nitric acid to obtain a cerium single-atom nanozyme; the cerium source is selected from cerium nitrate, cerium oxalate, cerium chloride and cerium acetylacetonate; the temperature for the hydrothermal reaction in the reactor is 80°C-200°C, and the time for the hydrothermal reaction is 2-8h; the molar ratios of the dimethylimidazole, zinc nitrate and cerium source are 48:11.9:0.6, 48:11.9:0.3, 48:1.9:0.6 and 48:11.9:0.

449.

2. The method for preparing the cerium single-atom nanozyme according to claim 1, characterized in that: The dimethylimidazole, zinc nitrate and cerium source are mixed in the form of solutions, and the solvent of the solution is methanol, ethanol, DMF or acetone.

3. The method for preparing the cerium single-atom nanozyme according to claim 2, characterized in that: The mixing volume ratio of the mixed solution containing the dimethylimidazole and the cerium source to the solution containing the zinc nitrate is 0.5-1.5:2; after the raw materials dimethylimidazole and the cerium source are mixed, ultrasonic treatment and stirring are also included.

4. The method for preparing the cerium single-atom nanozyme according to claim 3, characterized in that: Ultrasonic treatment was performed for 5-30 min and stirring was performed for 0.5-3 h.

5. The method for preparing the cerium single-atom nanozyme according to claim 2, characterized in that: The temperature for the hydrothermal reaction in the reactor is 80°C, 120°C, 160°C or 200°C.

6. The method for preparing the cerium single-atom nanozyme according to claim 5, characterized in that: After collecting the precipitate, the method further comprises washing the precipitate with an organic solvent, and then freeze-drying the washed precipitate.

7. The method for preparing the cerium single-atom nanozyme according to claim 6, characterized in that: The organic solvent is selected from DMF or methanol; The calcination temperature is 700-1000°C; The calcination is carried out in a tubular furnace, and the calcination time is 1-3 hours.

8. A cerium single-atom nanozyme, characterized in that: It is prepared by the preparation method of the cerium single-atom nanozyme described in any one of claims 1 to 7.

9. The cerium single-atom nanozyme according to claim 8, characterized in that: The cerium single-atom nanozyme has a 100-500 nm regular dodecahedral structure.

10. A method for detecting organic phosphorus based on cerium single-atom nanozymes, characterized in that: The method comprises the following steps: Mix different concentrations of organophosphorus with acetylcholinesterase for incubation, then add choline oxidase and acetylcholine for incubation, add the cerium single-atom nanozyme and substrate described in any one of claims 8-9 for incubation, and measure the absorbance / fluorescence signal; draw a standard working curve of organophosphorus concentration and IR value; the substrate is a chromogenic substrate or a fluorescent substrate; The IR value is calculated by the following formula: , where A0 is the blank absorbance or fluorescence value without adding acetylcholine and organophosphate, A1 is the maximum absorbance or fluorescence value without adding organophosphate but adding acetylcholine, and A is the absorbance or fluorescence value after incubation of the sample with both organophosphate and acetylcholine added; The sample to be tested is mixed with acetylcholinesterase for incubation, and then choline oxidase and acetylcholine are added for incubation, and the cerium single-atom nanozyme and substrate described in any one of claims 8-9 are added for incubation, the absorbance of the sample is measured and the IR value of the sample is calculated, and the organophosphorus content of the sample is obtained according to the standard working curve.

11. The method for detecting organic phosphorus based on cerium single-atom nanozyme according to claim 10, characterized in that: The color developing substrate is selected from 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine, N,N-diethyl-p-phenylenediamine, o-tolidine, N-ethyl N-(3-sulfopropyl)-3-methylaniline sodium salt and 4-aminoantipyrine, 3,5-dichloro-2-hydroxybenzenesulfonate sodium and 4-aminoantipyrine, or 2,2'-azo-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt.

12. The method for detecting organic phosphorus based on cerium single atom nanozyme according to claim 10, characterized in that: The fluorescent substrate is selected from o-phenylenediamine (OPD), Amplex Red, terephthalic acid, o-phenylenediamine, and 10-acetyl 3,7-hydroxyphenoxazine.

13. The method for detecting organic phosphorus based on cerium single-atom nanozyme according to claim 10, characterized in that: The conditions for mixed incubation of the organophosphorus and acetylcholinesterase are: incubation at 35-37° C. for 5-40 minutes; the mixing ratio of the organophosphorus and acetylcholinesterase is 0.015ng-90ng:2.5mU-50mU; the mixing ratio of acetylcholinesterase, choline oxidase and acetylcholine is 2.5mU-50 mU:25mU-50mU:60umol-900umol.

14. The method for detecting organic phosphorus based on cerium single-atom nanozyme according to claim 13, characterized in that: After adding choline oxidase and acetylcholine, incubate for 10-60 min; The mixing ratio of the cerium single-atom nanozyme and the substrate is: 5.4 μg-21.6 μg: 108 nmol-324 nmol.

15. The method for detecting organic phosphorus based on cerium single atom nanozyme according to claim 13, characterized in that: The cerium single-atom nanozyme and the substrate are added and incubated in a buffer solution for 10-40 minutes. The buffer solution is selected from acetate buffer, Tris-maleic acid buffer, PBS buffer solution or sodium citrate buffer.

16. The method for detecting organic phosphorus based on cerium single atom nanozyme according to claim 10, characterized in that: When the chromogenic substrate is selected from TMB, A0, A1 and A are all absorbance values ​​at 652 nm; The detectable organic phosphorus concentration range of the sample to be tested by the method is 0.5-1000ng / mL -1 .

17. Use of the cerium single-atom nanozyme as described in any one of claims 8 to 9 in fluorescence and / or colorimetric detection of organophosphorus residues.

18. A nanozyme colorimetric sensor, characterized in that: It comprises an array sensor, wherein the sensor comprises a colorimetric array, and the colorimetric array comprises the cerium single-atom nanozyme according to any one of claims 8-9.

19. Use of the cerium single-atom nanozyme according to any one of claims 8 to 9 in preparing a nanozyme colorimetric sensor.

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