CN112964706A - Porous Co3O4Rapid colorimetric detection method of peroxidase applied to glyphosate - Google Patents
Porous Co3O4Rapid colorimetric detection method of peroxidase applied to glyphosate Download PDFInfo
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Abstract
The invention discloses a method for applying porous Co3O4 peroxidase to rapid colorimetric detection of glyphosate, which comprises the following steps: (1) mixing the glyphosate-containing sample with porous Co with the concentration of 1ug/mL3O4Mixing the solutions, and incubating at 30 deg.C for 10 min; (2) subsequently, a NaAc buffer solution having a pH of 6.0 and a concentration of 5mM and H having a concentration of 8mM were added to the mixed solution2O2And TMB substrate solution with the concentration of 1.5mM is subjected to color reaction for 10min, and the content of glyphosate is judged by measuring the change of the absorbance value of the mixed solution at 650 nm. The glyphosate detection reagent has the minimum detection limit of 2.37ug/L for glyphosate, has the advantages of good detection specificity, discernable naked eyes, simple and convenient operation and the like, can realize the rapid detection of glyphosate pesticide residue without depending on a large instrument, and can be widely applied to the detection of organophosphorus pesticides in agricultural products.
Description
Technical Field
The invention belongs to the field of detection of organophosphorus pesticide residues in agricultural products, and particularly relates to a detection method for determining the characteristic absorption peak change of a detection system at 650nm by using a colorimetric method in an aqueous phase buffer solution system and judging glyphosate pesticide by naked eyes.
Background
Glyphosate is a broad-spectrum organophosphorus herbicide that causes plant death by inhibiting the activity of 5-enolpyruvylshikimate-3-phosphatase in important metabolic pathways in plants, and has been widely used in agricultural vegetation control for the past few decades due to its high herbicidal activity. Although the evaluation agency lists this as a low-risk compound, studies have shown that glyphosate can affect cell regulation, cause chronic kidney disease, and even cause cancer. At present, the abuse of glyphosate causes the pollution of soil, water and agricultural products. The harmful trace residue can easily enter human bodies through agricultural products, and potential threat to human health is caused. Therefore, the Chinese Ministry of agriculture and the United states Environmental Protection Agency (EPA) release relevant standards that set the maximum limit standard for glyphosate residue in agricultural products to be 500 μ g/L.
The traditional pesticide detection methods comprise a gas chromatography method, a liquid chromatography method and a chromatography-mass spectrometry combined method, and although the methods have the advantages of high sensitivity and high reliability, detection instruments are expensive, long in detection time, and required to be operated by professionals, so that the requirements of field detection cannot be met. The field rapid detection method comprises an immunoassay method and an enzyme inhibition method, wherein the immunoassay method comprises an enzyme immunoassay method, a fluorescence immunoassay method, a radioimmunoassay and a flow injection immunoassay method, but the application of the immunoassay method has certain limitation because the development process of the antigen and the antibody needs to invest more funds and longer time. The enzyme inhibition method is a pesticide rapid detection method developed based on pesticide targeting enzyme, acetylcholinesterase inhibition reaction and hydrolysis reaction, but the methods utilize acetylcholinesterase, plant lipase and the like, and the biological proteins have the defects of high preparation cost, limitation of working conditions of natural proteins and the like, so long-term application cannot be realized.
Porous Co3O4(cobaltosic oxide) is a new artificial enzyme with activity simulating peroxidase and capable of catalyzing a substrate to produce a color change. In recent years, based on porous Co3O4The established analysis method of peroxidase catalytic activity has been successfully applied in the fields of environment and food detection. However,heretofore, porous Co3O4Peroxidase (porous Co)3O4Nanoenzymes) has not been used in glyphosate assays. We have found that glyphosate is able to inhibit porous Co3O4The catalytic activity of the peroxidase leads to a significant change in the color of the sensing solution. Accordingly, a porous Co-based material was established3O4A glyphosate detection method of peroxidase activity.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a macroscopic colorimetric detection method for glyphosate, has the advantages of macroscopic identification, high sensitivity, strong specificity, convenience in operation, rapidness in detection and the like, and can be widely applied to rapid detection of glyphosate residues in agricultural products.
The purpose of the invention is realized by the following technical scheme:
porous Co3O4The method for applying peroxidase to glyphosate rapid colorimetric detection comprises the following steps:
(1) mixing the glyphosate-containing sample with porous Co with the concentration of 1ug/mL3O4Mixing the solutions, and incubating at 30 deg.C for 10 min;
(2) subsequently, a NaAc buffer solution having a pH of 6.0 and a concentration of 5mM and H having a concentration of 8mM were added to the mixed solution2O2And TMB (3,3 ', 5, 5' -tetramethylbenzidine) substrate solution with the concentration of 1.5mM are subjected to color reaction for 10min, and the content of glyphosate is judged by measuring the change of the absorbance value of the mixed solution at 650 nm.
Further, the specific steps comprise the following:
(1) preparing a detection system with known glyphosate concentration: taking 11 1.5mL calibrated centrifuge tubes, respectively adding 10uL glyphosate standard solution and 40uL Co with the concentration of 1ug/mL3O4Mixing the solution, incubating at 30 deg.C for 10min, adding 415uLpH 5.0 mM NaAc buffer solution, and adding 35 μ L8 mM H2O2And 1.5mM TMB so that the total volume of the entire sensing solution reaches 500 mu L of the extract, fully and uniformly mixing the extract, incubating the extract for 10min at the temperature of 30 ℃, and determining the incubated solution to be tested;
(2) taking another 1 detection solution prepared according to the step (1), adding 10 mu L of ultrapure water to replace the glyphosate standard solution, and treating to obtain a blank control system solution;
(3) respectively placing 200 mu L of the standard solution prepared in the step (1) and the blank reference solution prepared in the step (2) in a 96-hole enzyme label plate, and scanning and measuring an absorption peak at 650nm by using an enzyme label reader to obtain an absorption spectrum of the standard solution;
(4) plotting glyphosate with different concentrations and the measured delta A, and drawing a standard curve; the delta A is the light absorption value of the sample to be detected and the light absorption value of the blank sample;
(5) preparing a sample detection system: taking 10 mu L of actual tea, vegetable and fruit sample liquid, fully and uniformly mixing the detection solution prepared in the step (1), and measuring the absorption peak value of the detection solution in the step (3);
(6) and (4) checking a standard curve according to the absorbance value of the sample in the NaAc buffer solution system to obtain the content of the glyphosate in different samples.
The content of glyphosate in the whole detection system in the step (1) is maintained at 5-800 ug/L.
The basic principle of the invention is as follows: the addition of glyphosate can prevent porous Co3O4Catalysis H2O2The substrate TMB can not be normally oxidized due to the decomposition of OH, so that the color of the sensing solution is changed, and the color change degree is in direct proportion to the concentration of the glyphosate, thereby being used for colorimetric detection of the glyphosate.
Compared with the prior art, the detection method provided by the invention does not need to rely on large-scale instruments and equipment, has the advantages of high detection sensitivity, good selectivity, simple and quick operation and identification by naked eyes, and can be widely applied to the quick detection of glyphosate pesticide residue in agricultural products and the like.
Drawings
FIG. 1. based on suppressing porous Co3O4Schematic diagram of colorimetric detection of glyphosate by peroxidase activity;
FIG. 2. color change of sensing solution (gradual change from blue to light blue or colorless) when glyphosate concentration changes;
FIG. 3. Standard Curve (reflecting linearity) of sensing solution with changes in glyphosate concentration;
FIG. 4. Effect of other organophosphorus pesticides on detecting glyphosate (reflecting specificity).
Detailed Description
The following detailed description of specific embodiments, features and effects according to the present invention will be described with reference to the accompanying drawings and preferred embodiments.
Example 1:
a method for applying porous Co3O4 peroxidase to rapid colorimetric detection of glyphosate comprises the following steps:
(1) preparing a detection system with known glyphosate concentration: taking 11 1.5mL calibrated centrifuge tubes, respectively adding 10uL glyphosate standard solution (the glyphosate content in the whole detection system is maintained at 5-800ug/L) and 40uL Co with the concentration of 1ug/mL3O4Mixing the solution, incubating at 30 deg.C for 10min, adding 415uLpH 5.0 mM NaAc buffer solution, and adding 35 μ L8 mM H2O2And mixing the solution with a substrate of 1.5mM TMB to ensure that the total volume of the whole sensing solution reaches 500 mu L of the mixed solution, fully and uniformly mixing, incubating for 10min at the temperature of 30 ℃, and determining the incubated solution to be tested.
(2) And (3) adding 10 mu L of ultrapure water into another 1 detection solution prepared according to the step (1) to replace the glyphosate standard solution, and treating to obtain a blank control system solution.
(3) And (3) respectively putting 200 mu L of the standard solution prepared in the step (1) and the blank control solution prepared in the step (2) into a 96-hole enzyme label plate, and scanning and measuring an absorption peak at 650nm by using an enzyme label instrument to obtain an absorption spectrum of the standard solution.
(4) And (3) plotting glyphosate with different concentrations and the measured delta A (the absorbance value of the sample to be measured-the absorbance value of the blank sample), and drawing a standard curve to obtain a linear range of 5-80ug/L, wherein the corresponding regression equation is that y is 0.13655+0.00793x, and x is the glyphosate concentration (ug/L).
(5) Preparing a sample detection system: and (3) taking 10 mu L of actual tea leaf sample, fully and uniformly mixing the detection solution prepared in the step (1), and then measuring the absorption peak value of the detection solution according to the step (3).
(6) According to the absorbance value of the sample measured in a NaAc buffer solution system, looking up a standard curve to obtain the content of glyphosate in the tea, wherein the detection result is shown in the following table:
and (3) verifying the detection effect of the actual sample: the method is used for measuring 3 different tea samples, 1mg/kg of glyphosate is added into the samples, the obtained recovery rate is 91.8% -93.2%, and the reliability of the method is proved.
Example 2:
the procedure of example 1 was repeated using tomatoes, cucumbers and cabbage as actual samples, as shown in the following table:
and (3) verifying the detection effect of the actual sample: the method of the invention is used for determining the tomato, the cucumber and the cabbage as actual samples, and the obtained recovery rate is between 86.4 and 95.2 percent.
The above recovery rate calculation method is as follows:
in conclusion, the invention uses porous Co in NaAc buffer solution3O4And H2O23,3 ', 5, 5' -Tetramethylbenzidine (TMB) and target four-target interaction catalysis colorimetric specificity detection glyphosate, wherein the used reagent mainly comprises random porous Co3O4、NaAc、H2O2And 3,3 ', 5, 5' -Tetramethylbenzidine (TMB). When the target is contacted with porous Co3O4After a certain incubation time, it can be covered in porous Co3O4Surface, inhibition of porous Co3O4Catalysis H2O2Oxidizing to form OH, thereby inhibiting the substrate TMB from being oxidized normally, and enabling the color of the sensing solution to be changedThe original blue color is changed into light blue or colorless, the characteristic absorption peak of the product is 650nm, and when the concentration of the glyphosate is in the range of 5-80ug/L, the variation amplitude of the light absorption value at 650nm is in direct proportion to the concentration of the glyphosate. The glyphosate detection reagent has the minimum detection limit of 2.37ug/L for glyphosate, has the advantages of good detection specificity, discernable naked eyes, simple and convenient operation and the like, can realize the rapid detection of glyphosate pesticide residue without depending on a large instrument, and can be widely applied to the detection of organophosphorus pesticides in agricultural products.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are within the scope of the present invention without departing from the technical spirit of the present invention.
Claims (3)
1. Porous Co3O4The method for applying peroxidase to rapid colorimetric detection of glyphosate is characterized by comprising the following steps: the method comprises the following steps:
(1) mixing the glyphosate-containing sample with porous Co with the concentration of 1ug/mL3O4Mixing the solutions, and incubating at 30 deg.C for 10 min;
(2) subsequently, a NaAc buffer solution having a pH of 6.0 and a concentration of 5mM and H having a concentration of 8mM were added to the mixed solution2O2And TMB substrate solution with the concentration of 1.5mM is subjected to color reaction for 10min, and the content of glyphosate is judged by measuring the change of the absorbance value of the mixed solution at 650 nm.
2. Porous Co according to claim 13O4The method for applying peroxidase to rapid colorimetric detection of glyphosate is characterized by comprising the following steps: the steps specifically include the following:
(1) preparing a detection system with known glyphosate concentration: taking 11 1.5mL calibrated centrifuge tubes, respectively adding 10uL glyphosate standard solution and 40uL Co with the concentration of 1ug/mL3O4Mixing the solution, incubating at 30 deg.C for 10min, and adding 415uLpH6.0 NaAc buffer solution at 5mM concentration, and 35. mu.L of 8mM H2O2Mixing the solution with a substrate of 1.5mM TMB to ensure that the total volume of the whole sensing solution reaches 500 mu L, fully and uniformly mixing, incubating for 10min at the temperature of 30 ℃, and determining the incubated solution to be tested;
(2) taking another 1 detection solution prepared according to the step (1), adding 10 mu L of ultrapure water to replace the glyphosate standard solution, and treating to obtain a blank control system solution;
(3) respectively placing 200 mu L of the standard solution prepared in the step (1) and the blank reference solution prepared in the step (2) in a 96-hole enzyme label plate, and scanning and measuring an absorption peak at 650nm by using an enzyme label reader to obtain an absorption spectrum of the standard solution;
(4) plotting glyphosate with different concentrations and the measured delta A, and drawing a standard curve; the delta A is the light absorption value of the sample to be detected and the light absorption value of the blank sample;
(5) preparing a sample detection system: taking 10 mu L of actual tea, vegetable and fruit sample liquid, fully and uniformly mixing the detection solution prepared in the step (1), and measuring the absorption peak value of the detection solution in the step (3);
(6) and (4) checking a standard curve according to the absorbance value of the sample in the NaAc buffer solution system to obtain the content of the glyphosate in different samples.
3. Porous Co according to claim 23O4The method for applying peroxidase to rapid colorimetric detection of glyphosate is characterized by comprising the following steps: the content of glyphosate in the whole detection system in the step (1) is maintained at 5-800 ug/L.
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| CN114272862A (en) * | 2021-12-23 | 2022-04-05 | 江苏省特种设备安全监督检验研究院 | Ruthenium-based metal ion liquid polymer microsphere artificial enzyme and preparation method and application thereof |
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