CN116371395A - Cerium single-atom nano-enzyme, preparation method and method for detecting organic phosphorus - Google Patents
Cerium single-atom nano-enzyme, preparation method and method for detecting organic phosphorus Download PDFInfo
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- CN116371395A CN116371395A CN202310397411.8A CN202310397411A CN116371395A CN 116371395 A CN116371395 A CN 116371395A CN 202310397411 A CN202310397411 A CN 202310397411A CN 116371395 A CN116371395 A CN 116371395A
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- cerium
- enzyme
- organophosphorus
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- 238000000034 method Methods 0.000 title claims abstract description 40
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 title claims abstract description 23
- 229910052698 phosphorus Inorganic materials 0.000 title claims abstract description 23
- 239000011574 phosphorus Substances 0.000 title claims abstract description 23
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- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J23/00—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00
- B01J23/10—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of rare earths
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01J27/00—Catalysts comprising the elements or compounds of halogens, sulfur, selenium, tellurium, phosphorus or nitrogen; Catalysts comprising carbon compounds
- B01J27/24—Nitrogen compounds
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses cerium single-atom nano-enzyme, 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 nano-enzyme comprises the following steps: mixing raw materials of dimethyl imidazole, zinc nitrate and a cerium source, performing hydrothermal reaction in a reaction kettle, collecting precipitate, calcining and pickling to obtain cerium single-atom nano-enzyme; the cerium source is selected from cerium acetylacetonate; the temperature of the hydrothermal reaction is 80-200 ℃ and the time of the hydrothermal reaction is 2-8h. The detection method provided by the invention has higher detection selectivity for the organic phosphorus, and can avoid the interference of a large amount of coexisting ions. In addition, the detection method provided by the invention is simple to operate, low in cost and mild in condition, and can meet the requirements of rapid detection of pesticide organophosphorus residues in agricultural products and environments. The detection method provided by the invention has a wide organophosphorus detection range, and the cerium single-atom nano-enzyme provided by the invention can be used for colorimetric detection of organophosphorus residues.
Description
Technical Field
The invention relates to the technical field of organophosphorus residue detection, in particular to cerium single-atom nano-enzyme, a preparation method and a method for detecting organophosphorus.
Background
Pesticides, which are chemical substances used in agriculture to control plant diseases and insect pests and regulate plant growth, include a wide variety of species, and can be classified into organic chlorides, organic sulfides, organic phosphites, organic nitrogens, carbamates, sulfonylureas, triazines, pyrethroids, neonicotinoids, phenoxycarboxylic acids, and the like according to chemical structures. In order to avoid a great deal of loss of agricultural products due to the influence of diseases, insects and grass, and to meet the demands of ever-increasing population, the agricultural products are ensured to be produced worldwide, and pesticides are widely used worldwide in the last 50 years. Statistics indicate that the use of pesticides saves nearly 30% of crops worldwide. Among them, organophosphorus pesticides are widely used in all parts of the world due to their high efficiency and broad spectrum, and occupy about 15% of the pesticide market share; especially in China, the use of organophosphorus pesticides is very common, and the use amount of organophosphorus pesticides is about one third of the use amount of organophosphorus pesticides worldwide. Although the use of a large amount of organophosphorus pesticides plays an important role in protecting diseases and insects and improving crop yield, most organophosphorus pesticides have high toxicity, and the use of a large amount of organophosphorus pesticides brings non-negligible problems to the living environment of human beings. In addition, the food safety event caused by organophosphorus pesticide residues frequently occurs, and the safety problem of human life is also greatly threatened. Therefore, development of a method for rapidly and efficiently detecting pesticide residues has been receiving increasing attention.
At present, methods for detecting pesticide residues are various, and the most commonly used methods are mainly divided into two main types: one type is analysis based on conventional instrumentation, which mainly includes Gas Chromatography (GC), high Performance Liquid Chromatography (HPLC), gas chromatography-mass spectrometry (LC-MS/MS), and liquid chromatography-mass spectrometry (GC-MS/MS). The detection methods have many advantages, such as high detection sensitivity, good reproducibility and stable test results; however, these detection means have some disadvantages, for example, the detection instruments are generally expensive, the pretreatment and detection operation before detection are complex, the detection time is long, and the detection can be completed only through professional training. However, these deficiencies pose significant problems in achieving low cost, rapid detection of pesticide residues in a large number of samples. The second type is based on the principle of biological detection technology, and mainly comprises immunoassay, enzyme inhibition, living body detection, gold nanoparticles, biological sensing and the like. Among them, the enzyme inhibition method is of great interest because of simple operation and low cost. However, the conventional enzyme inhibition method suffers from low sensitivity and high false negative and false positive rates, which severely limits its application. The nano enzyme is applied to colorimetric detection of organophosphorus pesticides, so that the sensitivity, accuracy, anti-interference capability and environmental tolerance can be effectively improved, and high-flux, visual and instant pesticide residue analysis can be realized.
In recent years, the monoatomic nano-enzyme has the advantages of low preparation cost, high atom utilization rate, adjustable active site and the like, and gradually becomes a research hot spot. The sharply increased surface free energy, quantum size effect, unsaturated coordination environment and metal-support interaction give it excellent catalytic activity. In addition, the single-atom catalyst has extremely low metal loading and greatly improves the utilization rate of metal atoms, and can change the adsorption/desorption selectivity of active components on the catalyst to different molecules, thereby influencing the reaction kinetics. Although some researches have been completed on the scientific workers of the monatomic nano-enzyme, the preparation of the high-loading cerium monatomic nano-enzyme has not been reported, and the application of the cerium monatomic nano-enzyme to pesticide residues has not been reported.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide cerium single-atom nano-enzyme, a preparation method and a method for detecting organophosphorus so as to meet the requirement of rapid detection of organophosphorus pesticide residues in agricultural and sideline products.
The invention is realized in the following way:
in a first aspect, the invention provides a method for preparing cerium single-atom nano-enzyme, which comprises the following steps: mixing raw materials of dimethyl imidazole, zinc nitrate and a cerium source, performing hydrothermal reaction in a reaction kettle, collecting precipitate, and calcining to obtain cerium single-atom nano-enzyme; the cerium source is selected from one of cerium nitrate, cerium oxalate, cerium chloride and cerium acetylacetonate; the temperature of the hydrothermal reaction in the reaction kettle is 80, 120, 160 and 200 ℃, and the time of the hydrothermal reaction is 2-8h.
The inventors provide a method for preparing cerium monoatomic nano-enzyme, which is a peroxidase mimic, and can catalyze hydrogen peroxide to generate OH, oxidize an colorimetric probe such as 3,3', 5' -Tetramethylbenzidine (TMB) through OH to form a chromogenic product, or oxidize a fluorescent probe such as o-phenylenediamine to generate a fluorescent signal. When Organic Phosphorus (OP) is added into the reaction system, the OP can effectively inhibit the activity of acetylcholinesterase, and hydrogen peroxide can be generated based on the acetylcholinesterase, the acetylcholinesterase and the cholinesterase, the OP inhibits the activity of the acetylcholinesterase, thereby reducing the amount of hydrogen peroxide, and finally the cerium single-atom nano-enzyme and a substrate system (CeN x -SAzyme+colorimetric/fluorescent probes) absorbance. By measuring the absorbance of the system with OP of different concentrations at the maximum absorption wavelength of the developer(or fluorescence intensity under the maximum emission wavelength of the fluorescent probe), drawing an organophosphorus standard working curve, and detecting the OP content of the object to be detected based on the standard working curve.
The cerium single-atom nano-enzyme prepared by the method has a specific space molecular structure and is in a regular dodecahedron structure.
In the preparation method, the dimethyl imidazole, the zinc nitrate and the cerium source can generate orange-red precipitate through hydrothermal reaction, and cerium single-atom nano-enzyme with a specific molecular structure can be generated through high-temperature calcination.
In the invention, the single-atom nano enzyme is a material with enzyme-like catalytic activity, which takes a single metal atom as an active center, and has a designable geometric structure and electronic coordination, a unique quantum size effect and maximum atom utilization efficiency. Specifically, the monoatomic nano-enzyme is a kind of nano-material containing enzymatic properties, shows the enzymatic catalysis properties of only natural enzymes on a nano scale, can catalyze biochemical reactions mediated by the natural enzymes under physiological conditions, and shows the reaction kinetics and catalysis mechanism of the natural enzymes.
In the present invention, the term "peroxisome mimic" refers to a nanomaterial having a peroxisome catalytic activity. Specifically, the oxidase analogue in the invention uses hydrogen peroxide as an electron acceptor, and generates a colored substance TMBox by oxidizing a TMB substrate for colorimetric detection.
The term "TMB" is an abbreviated name for the compound "3,3', 5' -tetramethylbenzidine", which are used interchangeably.
In the present invention, the term "CeN x SAzyme "refers to a synthetic cerium single atom nanoenzyme, both of which are used interchangeably.
In an alternative embodiment, the temperature of the hydrothermal reaction is 80 ℃ to 200 ℃.
The hydrothermal reaction time is 2-8 hours, for example 2 hours, 4 hours, 6 hours or 8 hours. Under the hydrothermal reaction conditions, cerium single-atom nano-enzyme with a target structure can be obtained.
In a preferred embodiment of the present invention, the mixed molar ratio of dimethylimidazole, zinc nitrate and cerium source is 1:1:0.1. In the proportion, the cerium single-primary nano-enzyme with higher stability and better activity is obtained, and the peroxidase enzyme activity is far greater than the oxide enzyme activity, so that the cerium single-primary nano-enzyme has excellent selectivity.
In an alternative embodiment, the dimethylimidazole and the cerium source are mixed separately in the form of a solution of methanol, ethanol, DMF or acetone.
In an alternative embodiment, the mixing volume ratio of the dimethylimidazole-containing, cerium source solution to the zinc nitrate-containing solution is 0.5-1.5:2.
In an alternative embodiment, the raw material dimethyl imidazole and cerium source are mixed, and then ultrasonic treatment and stirring are further included;
in an alternative embodiment, the sonication is for 5-30min and stirring is carried out for 0.5-3h. The dispersion uniformity of the dimethylimidazole and the cerium source is improved by ultrasonic treatment, and the stirring aim is to ensure that the raw materials are fully and uniformly mixed so as to obtain cerium single-atom nano-enzyme with higher catalytic activity and uniform site distribution.
In a preferred embodiment of the present invention, collecting the precipitate further comprises washing the precipitate with an organic solvent, and then freeze-drying the washed precipitate.
In an alternative embodiment, the organic solvent is selected from DMF or methanol.
In a preferred embodiment of the present invention, the calcination temperature is 700-1000 ℃. The calcination temperature is selected, for example, from 700 ℃, 800 ℃, 900 ℃ or 1000 ℃. The cerium single-atom nano-enzyme with the target particle size and molecular structure can be obtained under the calcination condition.
In an alternative embodiment, the calcination is carried out in a tube furnace for a period of time ranging from 1 to 3 hours.
In a second aspect, the invention also provides a cerium single-atom nano-enzyme, which is prepared by the preparation method of the cerium single-atom nano-enzyme;
in an alternative embodiment, the cerium single atom nanoenzyme has a regular dodecahedron structure.
In a third aspect, the present invention also provides a method for detecting organic phosphorus based on cerium single-atom nano-enzyme, the method comprising the steps of:
mixing and incubating organophosphorus with different concentrations with acetylcholinesterase, adding choline oxidase and acetylcholine, incubating, adding cerium single-atom nano-enzyme and substrate for incubation, and measuring absorbance/fluorescence signals; drawing a standard working curve of phosphorus concentration and IR value; the substrate is a chromogenic substrate or a fluorogenic substrate;
the IR value is calculated by the following formula:
in which A 0 For blank absorbance/fluorescence values without addition of acetylcholine and organophosphorus, A 1 The maximum absorbance value/fluorescence value of the sample without adding organic phosphorus but with adding acetylcholine is that of the sample after incubation with the organic phosphorus and acetylcholine added simultaneously;
and mixing and incubating a sample to be tested with acetylcholinesterase, adding choline oxidase and acetylcholinesterase, incubating, adding the cerium single-atom nano-enzyme and a substrate (chromogenic substrate or fluorogenic substrate) for incubating, measuring the absorbance of the sample, calculating an IR value of the obtained sample, and obtaining the organophosphorus content of the sample according to a standard working curve.
The IR value is the Inhibition Rate (IR) of AChE activity. The linear relationship between IR and OP concentration is used to detect the residual amount of OP in crops or vegetables. And drawing an organophosphorus concentration-absorbance standard working curve.
The detection method provided by the invention has higher detection selectivity and strong anti-interference capability for the organophosphorus, can still keep higher accuracy for the detection of the organophosphorus under the environment of pyrethroid, anabasine, other pesticides such as chlorfenapyr, herbicides such as bispyribac-sodium, various metal ions, sulfate ions and chloride ions, and can avoid the interference of a large amount of coexisting ions. In addition, the detection method provided by the invention has the advantages of simple operation, low cost, mild condition and no pollutionOther reagents are needed to be added, so that the requirement of rapid detection of pesticide organophosphorus residues in agricultural products and the environment can be met. The detection method provided by the invention has wide organophosphorus detection range, and the detection range is 1ng-1000ng mL -1 Or 0.5ng-1000ng mL -1 Sensitivity is not more than 0.8ng mL -1 The method is far lower than the MRLs (organic phosphorus chemical reaction) values specified in national food safety standards in China, is superior to the existing literature reporting method, can completely meet the minimum detection requirements of pesticide residue limit standards of the agricultural products in China on organophosphorus pesticides such as dichlorvos, chlorpyrifos, malathion and the like, and has important significance in the quality safety supervision of agricultural products.
In a preferred embodiment of the use according to the invention, the chromogenic substrate is selected from TMB, N-diethyl-p-phenylenediamine, o-tolidine, N-ethyl N- (3-sulfopropyl) -3-methylaniline sodium salt and 4-aminoantipyrine (which need to work together), sodium 3, 5-dichloro-2-hydroxybenzenesulfonate and 4-aminoantipyrine, or 2,2' -azo-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt.
The fluorogenic substrate is selected from the group consisting of o-phenylenediamine (OPD), o-phenylenediamine, amplixRed, terephthalic acid, 10-acetyl 3, 7-hydroxy-fiver.
In a preferred embodiment of the invention, the conditions for mixed incubation of the organophosphorus and acetylcholinesterase are: incubating at 35-37deg.C for 5-40min; the mixing mole ratio of the organic phosphorus and the acetylcholinesterase is 0.015ng-90ng:2.5mU-50mU; the mixing volume ratio of the acetylcholinesterase, the choline oxidase and the acetylcholinesterase is 2.5mU-50mU, 25mU-50mU and 60mmol-900mmol.
In an alternative embodiment, the incubation is performed for 10-60min after adding choline oxidase, acetylcholine;
in an alternative embodiment, the cerium single atom nanoenzyme and the substrate are mixed in a ratio of: 5.4 μg-21.6 μg:108mmol-324mmol;
in an alternative embodiment, the cerium single atom nanoenzyme and the substrate are added and incubated for 10-40min in a buffer selected from an acetate buffer, a PBS buffer, a Tris-maleic buffer or a citrate buffer.
In a preferred embodiment of the use of the invention, when the chromogenic substrate is selected from TMB, A 0 、A 1 And A are absorbance values at 652 nm. 652nm is the maximum ultraviolet absorption wavelength of TMB, and can be used for detecting the residual amount of OP in fruits, vegetables, grains or the environment.
In an alternative embodiment, the detectable concentration of organophosphorus in the sample to be tested is in the range of 0.5-1000ng/mL -1 Or 1-1000ng/mL -1 Sensitivity is not more than 0.8ng mL -1 Is far lower than MRLs of organophosphorus pesticides in foods specified in China, and can meet the quality safety supervision requirement of agricultural products. In a fourth aspect, the invention also provides application of the cerium single-atom nano-enzyme in fluorescence and/or colorimetric detection of organophosphorus residues. The monoatomic enzyme is adopted for colorimetric detection of organophosphorus pesticide residues, the conditions are mild, the anti-interference capability is strong, the specificity is good, the sensitivity is high, no other reagent is needed, the detection cost is low, and the operation is simple; compared with the existing national standard method, the method has the advantages of higher sensitivity, shorter detection time, higher detection flux, simpler and more convenient operation, easy popularization, important function in daily supervision and guarantee of agricultural products, and good economic and social benefits.
In a fifth aspect, the present invention also provides a nanoenzyme colorimetric/fluorescence sensor comprising an array sensor comprising a colorimetric array comprising a cerium monoatomic nanoenzyme.
In a sixth aspect, the invention also provides application of the cerium single-atom nano-enzyme in preparing a nano-enzyme colorimetric/fluorescent sensor.
The invention has the following beneficial effects:
the cerium single-atom nano-enzyme with a specific molecular structure is prepared by mixing dimethyl imidazole, a zinc source and a cerium source, performing hydrothermal reaction to obtain a precipitate and calcining the precipitate. The cerium monoatomic enzyme is a peroxidase mimic capable of catalyzing hydrogen peroxide production of OH by which a color-developing agent such as 3,3', 5' -Tetramethylbenzidine (TMB) and a fluorogenic substrate are oxidized, developed or emit a strong fluorescent signal. When added into the reaction systemIn the case of Organic Phosphorus (OP), OP can effectively inhibit the activity of acetylcholinesterase, and based on the fact that acetylcholinesterase, acetylcholine and choline oxidase can generate hydrogen peroxide, OP inhibits the activity of acetylcholinesterase, thereby reducing the amount of hydrogen peroxide generated, and finally causes cerium single-atom nano-enzyme and a color-developing agent system (CeN) x -SAzyme+chromogenic/fluorescent system) absorbance. And (3) testing the absorbance/fluorescence value of a system added with the OP with different concentrations under the maximum absorption wavelength of the color reagent, drawing an organophosphorus concentration-absorbance/organophosphorus concentration-fluorescence standard working curve, and detecting the OP content of the object to be detected based on the standard working curve.
The detection method provided by the invention has higher detection specificity and selectivity for organophosphorus and hardly responds to non-organophosphorus pesticides; the anti-interference capability is strong, the detection of the organic phosphorus can still keep higher accuracy under the environment of various metal ions, sulfate ions and chloride ions, and the interference of a large number of coexisting ions and other pesticides can be effectively avoided. In addition, the detection method provided by the invention is simple to operate, low in cost and mild in condition, and can meet the requirements of rapid detection of organophosphorus pesticide residues in agricultural products and environments without adding other reagents. The detection method provided by the invention has wide organophosphorus detection range, and the detection range is 0.5ng-1000ng mL -1 Or 1ng-1000ng mL -1 Sensitivity is not more than 0.8ng mL -1 The method can completely meet the minimum detection limit of the national food safety limit standard of China on organophosphorus pesticides such as dichlorvos.
In addition, the cerium single-atom nano-enzyme provided by the invention can be used for detecting organophosphorus residues by ratio fluorescence and/or colorimetry, and can also be used for preparing nano-enzyme colorimetric sensors.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a high resolution SEM image of cerium monoatomic enzyme prepared in example 1;
FIG. 2 is a full spectrum of ultraviolet absorption of cerium monoatoms and a control group prepared in example 1;
FIG. 3 is an XRD pattern of cerium monoatomic enzyme prepared in example 1;
FIG. 4 is an infrared spectrum of cerium monoatomic enzyme prepared in example 1;
FIG. 5 is a graph showing the statistical results of the anti-interference performance test and the selective test of the cerium monoatomic enzyme prepared in example 1;
FIG. 6 is a standard graph of organophosphorus pesticide detection in rice using cerium monoatomic enzyme prepared in example 1.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The embodiment provides a preparation method of cerium single-atom nano-enzyme and a colorimetric method for detecting organophosphorus pesticide residues based on the cerium single-atom nano-enzyme, which comprises the following steps:
1. preparation of cerium single-atom nano enzyme:
3.942g of dimethyl imidazole, 0.2619g of cerium acetylacetonate and 3.542g of zinc nitrate are respectively weighed and dissolved in 45mL of methanol solution and 90mL of methanol solution for mixing, ultrasonic treatment is carried out for 15min, mechanical stirring is carried out for 1h, and then the mixture is placed in a high-pressure reaction kettle for reaction for 4h at 120 ℃. Calcining the freeze-dried product in a tube furnace at 900 ℃ and pickling with 1mol/L nitric acid to obtain cerium single-atom nano-enzyme (CeN) X -SAzyme)。
Taking 10.8 mu L cerium single atom nano-enzyme (1 mg mL) -1 Dissolved in deionized water) was dispersed in acetic acid + acetate buffer (10 mM, ph=4.06) and 30 μl TMB (4.8 mM, deionized water dissolved) was dissolved with H 2 O 2 Adding the solution into the mixed solution, and incubating for 10min to obtain CeN x -SAzyme+TMB+H 2 O 2 。
Another 10.8. Mu.L cerium single atom nano-enzyme (1 mg mL) was prepared -1 Dissolved in deionized water) was dispersed in acetic acid+acetate buffer (10 mM, pH=4.06), 30. Mu. LTMB (4.8 mM, dissolved in deionized water) was added to acetic acid+acetate buffer containing cerium-containing monoatomic nanoenzyme, and incubated for 10min to obtain CeN x -SAzyme+TMB。
Determination of CeN by UV-visible absorption Spectrophotometer x -SAzyme、CeN x -SAzyme+TMB+H 2 O 2 、CeN x The total ultraviolet absorbance spectrum of SAzyme+TMB, recorded absorbance, and the total ultraviolet absorbance spectrum is shown in FIG. 2. FIG. 2 shows that cerium single-atom nano-enzyme and TMB and H 2 O 2 Has a characteristic absorption peak at 652 nm. By inhibiting H 2 O 2 The absorption peak of the mixture of cerium single-atom nano-enzyme and TMB is obviously reduced. Based on the above, a corresponding colorimetric method for detecting organophosphorus pesticide residues is developed.
2. Colorimetric detection method for organophosphorus pesticide residues based on cerium single-atom nano-enzyme
30. Mu.L of various concentrations of organophosphorus (dichlorvos) 1, 5, 30, 100, 300, 500, 1000ng/mL were incubated with 5. Mu.L of acetylcholinesterase solution at 37℃for 20min. Then 5. Mu.L of choline oxidase solution was added and incubated with 15. Mu.L of acetylcholine for 40min. 1mg/mL was prepared with deionized water -1 Cerium monoatomic enzyme solution, 10.8 mu L of nano enzyme solution, 120 mu L of acetic acid and sodium acetate buffer solution and 30 mu L of TMB are respectively taken and incubated in the incubated solution for 30min again, and absorbance change at 652nm is measured by using an enzyme-labeled instrument.
For the detection of OP, inhibition of AChE activity (IR) was used. A control group without ACh and OP was added to obtain a blank absorbance value (A0). The other group was not supplemented with OP, and as a control group, the maximum absorbance value (A1) was obtained under the experimental parameters. A is the absorbance value after incubation of the sample with the simultaneous addition of organophosphorus and acetylcholine. The equation for calculating the IR value is then as follows:
the IR value and the concentration of OP are in a linear relation, and the concentration of OP in the sample to be detected can be detected based on the linear relation. And drawing an organophosphorus concentration-absorbance standard working curve.
Example 2
The embodiment provides a preparation method of cerium single-atom nano-enzyme and a colorimetric method for detecting organophosphorus pesticide residues based on the cerium single-atom nano-enzyme, which comprises the following steps:
1. the preparation method of the cerium single-atom nano-enzyme comprises the following steps:
3.942g of dimethyl imidazole, 0.131g of cerium acetylacetonate and 3.542g of zinc nitrate are respectively dissolved in 45mL and 90mL of methanol solution for mixing, ultrasonic treatment is carried out for 10min, mechanical stirring is carried out for 1h, and the mixture is placed in a high-pressure reaction kettle for reaction for 4h at 120 ℃. And (3) placing the freeze-dried product into a tube furnace for calcination at 700 ℃, and pickling with 1mol/L nitric acid to obtain the cerium single-atom nano-enzyme.
2. Colorimetric detection method for organophosphorus pesticide residues based on cerium single-atom nano-enzyme
mu.L of organophosphorus (chlorpyrifos) 1, 5, 30, 100, 300, 500, 1000ng/mL were incubated with 5. Mu.L of acetylcholinesterase solution at 37℃for 5min. Then 5. Mu.L of choline oxidase solution was added and incubated with 15. Mu.L of acetylcholine for 45min. 1mg/mL was prepared with deionized water -1 Cerium monoatomic enzyme solution, 10.8 mu L of nano enzyme solution, 120 mu L of acetic acid and sodium acetate buffer solution and 30 mu L of TMB are respectively taken and incubated in the incubated solution for 30min again, and absorbance change at 652nm is measured by using an enzyme-labeled instrument. And drawing an organophosphorus concentration-absorbance standard working curve.
Example 3
The embodiment provides a preparation method of cerium single-atom nano-enzyme and a colorimetric method for detecting organophosphorus pesticide residues based on the cerium single-atom nano-enzyme, which comprises the following steps:
1. the cerium monoatomic nanoenzyme was prepared with the difference compared to example 1 in the hydrothermal temperature:
3.942g of dimethyl imidazole, 0.2619g of cerium acetylacetonate and 3.542g of cerium nitrate are respectively dissolved in 45mL and 90mL of methanol solution, mixed, ultrasonically stirred for 0.5h, mechanically stirred for 1h, and then placed in a high-pressure reaction kettle for reaction at 160 ℃ for 4h. And (3) placing the freeze-dried product into a tube furnace for calcination at 900 ℃, and pickling with 1mol/L nitric acid to obtain the cerium single-atom nano-enzyme.
2. Colorimetric detection method for organophosphorus pesticide residues based on cerium monoatomic nano-enzyme, which is different from example 1 in incubation time
30. Mu.L of various concentrations of organophosphorus (parathion) were incubated with 5. Mu.L of acetylcholinesterase solution at 37℃for 15min. Then 5. Mu.L of choline oxidase solution was added and incubated with 15. Mu.L of acetylcholine for 40min. 1mg/mL was prepared with deionized water -1 And (3) taking 10.8 mu L of nano enzyme solution, 120 mu L of acetic acid and sodium acetate buffer solution and 30 mu L of TMB from the cerium single-atom nano enzyme solution, incubating the solution for 30min again, and measuring the absorbance change by using an enzyme-labeled instrument. And drawing an organophosphorus concentration-absorbance standard working curve.
Example 4
The embodiment provides a preparation method of cerium single-atom nano-enzyme and a colorimetric method for detecting organophosphorus pesticide residues based on the cerium single-atom nano-enzyme, which comprises the following steps:
1. the cerium monoatomic nanoenzyme was prepared with the difference in calcination temperature compared to example 1.
3.942g of dimethyl imidazole, 0.2619g of cerium acetylacetonate and 0.566g of zinc nitrate are respectively dissolved in 45mL and 100mL of methanol solution for mixing, ultrasonic treatment is carried out for 1min, mechanical stirring is carried out for 1h, and the mixture is placed in a high-pressure reaction kettle for reaction for 4h at 120 ℃. And (3) placing the freeze-dried product into a tube furnace for calcination at 800 ℃ to obtain the cerium single-atom nano-enzyme.
2. Colorimetric detection methods based on cerium monoatomic nano-enzyme organophosphorus pesticide residues are different from example 1 in incubation time.
30. Mu.L of various concentrations of organic phosphorus (phosphorus sesquioxide) were incubated with 5. Mu.L of acetylcholinesterase solution at 37℃for 15min. Then 5. Mu.L of choline oxidase solution was added and incubated with 15. Mu.L of acetylcholine for 60min. 1mg/mL was prepared with deionized water -1 And (3) taking 10.8 mu L of nano enzyme solution, 120 mu L of acetic acid and sodium acetate buffer solution and 30 mu L of TMB from the cerium single-atom nano enzyme solution, incubating the solution for 30min again, and measuring the absorbance change by using an enzyme-labeled instrument. PaintingAnd (5) preparing an organophosphorus concentration-absorbance standard working curve.
Example 5
The embodiment provides a preparation method of cerium single-atom nano-enzyme and a colorimetric method for detecting organophosphorus pesticide residues based on the cerium single-atom nano-enzyme, which comprises the following steps:
the cerium monoatomic nanoenzyme enzyme was prepared with the difference in calcination temperature compared to example 1.
3.942g of dimethyl imidazole and 0.2619g of cerium acetylacetonate are respectively dissolved in 45mL and 90mL of methanol solution for mixing, ultrasonic treatment is carried out for 1min, mechanical stirring is carried out for 1h, and the mixture is placed in a high-pressure reaction kettle for reaction for 4h at 120 ℃. And (3) placing the freeze-dried product in a tube furnace for calcining at 1000 ℃, and pickling with 1mol/L nitric acid to obtain the cerium single-atom nano-enzyme.
2. Colorimetric detection methods based on cerium monoatomic nano-enzyme organophosphorus pesticide residues are different from example 1 in incubation time.
30. Mu.L of various concentrations of organophosphorus (malathion) were incubated with 5. Mu.L of acetylcholinesterase solution at 37℃for 15min. Then 5. Mu.L of choline oxidase solution was added and incubated with 15. Mu.L of acetylcholine for 60min. 1mg/mL was prepared with deionized water -1 And (3) taking 10.8 mu L of nano enzyme solution, 120 mu L of acetic acid and sodium acetate buffer solution and 30 mu L of TMB from the cerium single-atom nano enzyme solution, incubating the solution for 30min again, and measuring the absorbance change by using an enzyme-labeled instrument.
And drawing an organophosphorus concentration-absorbance standard working curve.
Example 6
The embodiment provides a preparation method of cerium single-atom nano-enzyme and a method for detecting organophosphorus pesticide residues based on a cerium single-atom nano-enzyme fluorescence method:
1. the preparation of cerium single-atom nano-enzyme is different from that of example 1 in the differences of hydrothermal temperature and raw material ratio:
3.942g of dimethyl imidazole, 0.1964g of cerium acetylacetonate and 3.542g of cerium nitrate are respectively dissolved in 45mL and 90mL of methanol solution, mixed, ultrasonically stirred for 0.5h, mechanically stirred for 1h, and then placed in a high-pressure reaction kettle for reaction at 160 ℃ for 4h. And (3) placing the freeze-dried product into a tube furnace for calcination at 900 ℃, and pickling with 1mol/L nitric acid to obtain the cerium single-atom nano-enzyme.
2. Colorimetric detection method for organophosphorus pesticide residues based on cerium monoatomic nano-enzyme, which is different from example 1 in incubation time
30. Mu.L of various concentrations of organophosphorus (dichlorvos) were incubated with 5. Mu.L of acetylcholinesterase solution at 37℃for 15min. Then 5. Mu.L of choline oxidase solution was added and incubated with 15. Mu.L of acetylcholine for 40min. 1mg/mL was prepared with deionized water -1 And (3) taking 10.8 mu L of nano enzyme solution, 130 mu L of acetic acid and sodium acetate buffer solution and 20 mu L of terephthalic acid into the incubated solution, incubating for 30min again, and measuring the change of fluorescence value by using a fluorescence spectrometer. And (5) drawing an organic phosphorus concentration-fluorescence standard working curve.
Comparative example 1
The cerium monoatomic nanoenzyme was prepared only by comparison with example 1: the hydrothermal temperatures are different.
3.942g of dimethyl imidazole, 0.2619g of cerium acetylacetonate and 3.542g of zinc nitrate are respectively dissolved in 45mL and 90mL of methanol solution, mixed, ultrasonically stirred for 0.5h, mechanically stirred for 1h, and then placed in a high-pressure reaction kettle for reaction for 4h at 50 ℃. And placing the freeze-dried product in a tube furnace for calcination at 800 ℃, wherein the hydrothermal temperature is too low, and the autogenous pressure in the reaction kettle is too low, so that the monoatomic nano enzyme with uniform morphology and excellent catalytic performance cannot be generated after calcination and acid washing.
Comparative example 2
1. The cerium single-atom nano-enzyme was prepared, differing from example 1 in that no cerium source was added.
3.942g of dimethyl imidazole and 3.542g of zinc nitrate are respectively dissolved in 45mL and 90mL of methanol solution for mixing, ultrasonic treatment is carried out for 0.5h, mechanical stirring is carried out for 1h, and then the mixture is placed in a high-pressure reaction kettle for reaction for 4h at 120 ℃. The freeze-dried product is placed in a tube furnace for calcination at 800 ℃, and the organic metal framework without cerium acetylacetonate is damaged due to the too high calcination temperature, so that the original appearance can not be maintained, and the enzyme-like catalytic activity is almost absent.
Experimental example 1
In this experimental example, the cerium monoatomic enzyme prepared in example 1 was observed by a scanning electron microscope, and the cerium monoatomic enzyme has a microstructure of a regular dodecahedron and a particle size of 300 to 500nm, as shown in FIG. 1.
The XRD patterns of the cerium monoatomic enzyme prepared in example 1 are shown with reference to FIG. 3, and only two diffraction peaks at 26.5℃and 42℃in FIG. 3, which can be assigned to carbons (002) and (101), respectively, indicate the absence of crystalline cerium. The infrared spectrogram of the cerium monoatomic enzyme is shown in the figure 4 without characteristic peaks of ZIF-8 and Ce-O, and the successful preparation of the cerium monoatomic nano enzyme is proved.
Experimental example 2
The cerium monoatomic enzyme prepared in example 1 was subjected to the anti-interference performance test and the selectivity test of the detection system.
The specific test procedure is as follows:
OP detection Selectivity by detecting the addition of different ions 10. Mu.g/mL (Mg in a buffer System 2+ 、Ca 2+ 、Glu、Ka + 、Zn 2+ 、SO 4 2- 、Cl - ) The absorbance of the CeNx-SAzyme+TMB system of (C).
The results are shown by referring to FIG. 5, and the results show that the detection method provided by the invention has almost no response to pesticides such as herbicides, non-organophosphorus pesticides and the like, proves that the detection method has higher detection selectivity and specificity to organophosphorus, has strong anti-interference capability, can still keep higher accuracy in the detection of organophosphorus under the environment of various metal ions, sulfate ions and chloride ions, and can avoid the interference of a large amount of coexisting ions.
Experimental example 3
The organophosphorus residue detection method provided by the embodiment 1 of the invention is adopted to detect pesticide residues in rice. The standard curve of the concentration of organic phosphorus-IR value is shown with reference to FIG. 6.
Experimental example 4
The cerium single-atom nano-enzyme and the method for detecting the organic phosphorus can realize rapid detection of the organic phosphorus pesticide dichlorvos in the rice in a short time, and when the adding level of the dichlorvos in the rice is 0.04mg/kg, the organic phosphorus residual quantity is detected by adopting the standard curve of the experimental example 3, and the result shows that the adding recovery rate and the relative standard deviation are satisfactory (shown in the table 1).
As shown in Table 2, the quantitative limit of the rapid detection method provided by the invention is far lower than the maximum residual limit value (0.2 mg/kg, GB 2763-2021) of dichlorvos in rice specified in national standards, and the method provided by the invention is low in detection cost, high in sensitivity and high in detection flux.
The method is applied to detection in an actual sample, the recovery rate is 89.2% -94.3%, and the method has high reliability and practicability; by carrying out correlation analysis with a detection result of a GC-MS method, the result shows that the correlation is good, and the method provided by the invention can be used for accurately detecting the organophosphorus pesticide residues.
Table 1 statistical table of the results of addition recovery experiments for pesticide residue analysis using cerium monoatomic enzyme prepared in example 1.
Table 2 shows the sensitivity of the detection method provided in example 1 compared with the current national standard method and the reported method in the literature.
| Sequence number | Method | Sensitivity of | |
| 1 | Colorimetric method | 2.4μg/L | doi.org/10.1007/s00604-019-3485-7 |
| 2 | Gas chromatography | 4μg/kg | GB/T 5009.145-2003 |
| 3 | Paper sheet process | 0.3mg/kg | GB/T 5009.199-2003 |
| 4 | Spectrophotometry | 0.1mg/kg | GB/T 5009.199-2003 |
| 5 | Colorimetric method | 0.8μg/L | Example 1 |
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The preparation method of the cerium single-atom nano-enzyme is characterized by comprising the following steps of: mixing raw materials of dimethyl imidazole, zinc nitrate and a cerium source, performing hydrothermal reaction in a reaction kettle, collecting precipitate, calcining and pickling with 0.5-1.5 mol/L nitric acid to obtain cerium single-atom nano-enzyme; the cerium source is selected from cerium nitrate, cerium oxalate, cerium chloride and cerium acetylacetonate; the temperature of the hydrothermal reaction in the reaction kettle is 80, 120, 160 and 200 ℃, and the time of the hydrothermal reaction is 2-8h.
2. The method for preparing cerium single-atom nano-enzyme according to claim 1, wherein the mixed molar ratio of dimethyl imidazole, zinc nitrate and cerium source is 1:1:0.01-1:1: 0.5;
preferably, the dimethylimidazole, zinc nitrate and cerium source are mixed in the form of solutions respectively, and the solvents of the solutions are methanol, ethanol, DMF or acetone;
preferably, 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; preferably, after the raw material dimethyl imidazole and the cerium source are mixed, ultrasonic treatment and stirring are further included;
preferably, the ultrasonic treatment is carried out for 5-30min, and stirring is carried out for 0.5-3h.
3. The method for preparing cerium single-atom nano-enzyme according to claim 2, wherein collecting the precipitate further comprises washing the precipitate with an organic solvent, and then freeze-drying the washed precipitate;
preferably, the organic solvent is selected from DMF or methanol;
preferably, the temperature of the calcination is 700-1000 ℃;
preferably, the calcination is carried out in a tube furnace for a period of 1-3 hours.
4. A cerium single-atom nano-enzyme, characterized in that it is produced by the method for producing a cerium single-atom nano-enzyme according to any one of claims 1 to 3;
preferably, the cerium single-atom nano-enzyme has a structure of 100-500nm regular dodecahedron.
5. The method for detecting the organic phosphorus based on cerium single-atom nano-enzyme is characterized by comprising the following steps of:
mixing and incubating organophosphorus with acetylcholinesterase at different concentrations, adding choline oxidase and acetylcholine, incubating, adding cerium single-atom nano-enzyme and substrate according to claim 4, incubating, and measuring absorbance/fluorescence signals; drawing a standard working curve of the concentration of the organic phosphorus and the IR value; the substrate is a chromogenic substrate or a fluorogenic substrate;
the IR value is calculated by the following formula:
in which A 0 For blank absorbance or fluorescence values without addition of acetylcholine and organophosphorus, A 1 The maximum absorbance value or fluorescence value of the sample without adding the organic phosphorus but with adding the acetylcholine is that of the sample after incubation with the organic phosphorus and the acetylcholine simultaneously;
mixing and incubating a sample to be tested with acetylcholinesterase, adding choline oxidase and acetylcholinesterase, incubating, adding cerium single-atom nano-enzyme and a substrate according to claim 4, incubating, measuring absorbance of the sample, calculating an IR value of the obtained sample, and obtaining the organophosphorus content of the sample according to a standard working curve.
6. The method for detecting organophosphorus based on cerium monoatomic nanoenzyme according to claim 5, wherein the chromogenic substrate is selected from 3,3', 5' -Tetramethylbenzidine (TMB), o-phenylenediamine, N-diethyl-p-phenylenediamine, o-tolidine, N-ethyl N- (3-sulfopropyl) -3-methylaniline sodium salt and 4-aminoantipyrine, sodium 3, 5-dichloro-2-hydroxybenzenesulfonate and 4-aminoantipyrine, or 2,2' -azo-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt;
preferably, the fluorogenic substrate is selected from the group consisting of o-phenylenediamine (OPD), duplex Red, terephthalic acid, o-phenylenediamine, 10-acetyl 3, 7-hydroxy-phenostring;
preferably, the conditions under which the organophosphorus and acetylcholinesterase are mixed and incubated are: incubating at 35-37deg.C for 5-40min; the mixing ratio of the organic phosphorus to the acetylcholinesterase is 0.015ng-90ng:2.5mU-50mU; the mixing ratio of the acetylcholinesterase, the choline oxidase and the acetylcholinesterase is 2.5mU-50mU, 25mU-50mU, 60mmol-900mmol;
preferably, adding choline oxidase and acetylcholine, and incubating for 10-60min;
preferably, the mixed molar ratio of the cerium single-atom nano-enzyme to the substrate is as follows: 5.4 μg-21.6 μg:108mmol-324mmol;
preferably, the cerium monoatomic nanoenzyme and the substrate are added and incubated for 10-40min in a buffer selected from acetic acid buffer, tris-maleic acid buffer, PBS buffer or sodium citrate buffer.
7. The method for detecting organic phosphorus based on cerium single atom nanoenzyme according to claim 6, wherein when said chromogenic substrate is selected from TMB, said A 0 、A 1 And A is the absorbance at 652 nm;
preferably, the detectable organophosphorus concentration of the sample to be detected by the method is in the range of 0.5-1000ng/mL -1 。
8. Use of the cerium single-atom nano-enzyme according to claim 4 for fluorescence and/or colorimetric detection of organophosphorus residues.
9. A nanoenzyme colorimetric sensor comprising an array sensor comprising a colorimetric array comprising the cerium monoatomic nanoenzyme of claim 4.
10. The use of cerium single-atom nano-enzyme according to claim 4 for preparing a nano-enzyme colorimetric sensor.
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