CN117106852A - Detection method for rapidly detecting agricultural product pesticide residues and application thereof - Google Patents
Detection method for rapidly detecting agricultural product pesticide residues and application thereof Download PDFInfo
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- CN117106852A CN117106852A CN202311133156.2A CN202311133156A CN117106852A CN 117106852 A CN117106852 A CN 117106852A CN 202311133156 A CN202311133156 A CN 202311133156A CN 117106852 A CN117106852 A CN 117106852A
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- 238000001514 detection method Methods 0.000 title claims abstract description 73
- 239000000447 pesticide residue Substances 0.000 title claims abstract description 36
- 102000013392 Carboxylesterase Human genes 0.000 claims abstract description 39
- 108010051152 Carboxylesterase Proteins 0.000 claims abstract description 39
- 239000000758 substrate Substances 0.000 claims abstract description 26
- 230000004044 response Effects 0.000 claims abstract description 21
- 239000003987 organophosphate pesticide Substances 0.000 claims abstract description 18
- 239000000243 solution Substances 0.000 claims description 90
- 238000006243 chemical reaction Methods 0.000 claims description 36
- 150000001412 amines Chemical class 0.000 claims description 29
- 239000000047 product Substances 0.000 claims description 24
- 239000007853 buffer solution Substances 0.000 claims description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- 239000002904 solvent Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 15
- 229940122274 Carboxylesterase inhibitor Drugs 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 11
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims description 10
- AZZOSPOHDDQAFO-UHFFFAOYSA-N 5,15,15-trimethyl-3,8-dioxatetracyclo[8.8.0.02,6.011,16]octadeca-1(10),2(6),4,11(16),17-pentaene-7,9-dione Chemical compound O=C1OC(=O)C2=C(CCCC3(C)C)C3=CC=C2C2=C1C(C)=CO2 AZZOSPOHDDQAFO-UHFFFAOYSA-N 0.000 claims description 9
- 239000000126 substance Substances 0.000 claims description 9
- 238000000605 extraction Methods 0.000 claims description 8
- 239000011574 phosphorus Substances 0.000 claims description 7
- 229910052698 phosphorus Inorganic materials 0.000 claims description 7
- 238000013507 mapping Methods 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 239000003905 agrochemical Substances 0.000 claims 6
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 6
- 238000004587 chromatography analysis Methods 0.000 abstract description 4
- 150000001875 compounds Chemical class 0.000 description 29
- QCJQWJKKTGJDCM-UHFFFAOYSA-N [P].[S] Chemical compound [P].[S] QCJQWJKKTGJDCM-UHFFFAOYSA-N 0.000 description 23
- 239000008057 potassium phosphate buffer Substances 0.000 description 12
- 241000208822 Lactuca Species 0.000 description 7
- 235000003228 Lactuca sativa Nutrition 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 230000008859 change Effects 0.000 description 5
- 238000002372 labelling Methods 0.000 description 5
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 4
- 239000000575 pesticide Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000005507 spraying Methods 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 238000012271 agricultural production Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000000611 regression analysis Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 240000004201 Lactuca sativa var. crispa Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N21/62—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"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N2333/918—Carboxylic ester hydrolases (3.1.1)
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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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Chemical & Material Sciences (AREA)
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- Organic Chemistry (AREA)
- Immunology (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
The invention provides a detection method for rapidly detecting agricultural product pesticide residues and application thereof, which comprises the steps of incubating and reacting agricultural residue extracting solution, carboxylesterase solution and a substrate shown as a formula I, detecting 650-690 nm fluorescence response signals, establishing a standard curve of the concentration of organophosphorus pesticide and the fluorescence response signals of the reacted solution, and rapidly detecting the agricultural product organophosphorus pesticide residues. The detection method for rapidly detecting the pesticide residue of the agricultural product has better accuracy and sensitivity and low detection limit. The detection method for rapidly detecting the pesticide residue of the agricultural product is simple in detection, high-performance liquid chromatography and other high-performance instruments are not needed, the detection limit and sensitivity of the detection are not weaker than those of the chromatographic method, the detection cost is low, the detection time is short, and the detection method is very suitable for detecting the pesticide residue of the agricultural product up to the standard.
Description
Technical Field
The invention relates to the field of environment detection, in particular to a detection method for rapidly detecting pesticide residues in agricultural products and application thereof.
Background
Organophosphorus pesticides are commonly used in agricultural production to improve agricultural work efficiency. However, improper use of pesticides can cause serious exceeding of pesticide residues, thereby causing environmental pollution and endangering life and health of human beings. Therefore, the efficient and accurate detection of organophosphorus pesticides is of great significance to human agricultural production activities. The high performance liquid chromatography and the gas chromatography are widely applied to the detection, the qualitative and the quantitative of organophosphorus pesticides, and can detect the accurate quantitative of pesticide residues in agricultural products and target substances.
However, the high performance liquid chromatography and the gas chromatography have high precision of the instrument, high price of the instrument, high cost of sample measurement consumable, long detection time, high economic cost and high time cost when being applied to the up-to-standard pesticide residue detection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a detection method for rapidly detecting pesticide residues of agricultural products and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a detection method for rapidly detecting pesticide residues in agricultural products, the method comprising the steps of:
(1) Extracting organophosphorus pesticide residues in quantitative agricultural product samples by using a quantitative extraction solvent, and collecting supernatant, wherein the extraction solvent is a mixed solution of a buffer solution with the pH of 6.8-7.5 and ethanol, and the volume ratio of the buffer solution to the ethanol is 1: (0.5-1);
(2) Uniformly mixing 10-25 mU L of supernatant obtained in the quantifying step (1) with 5-25 mU of carboxylesterase solution, and then incubating for 15-25 minutes at the temperature of 35-38 ℃;
(3) Adding a substrate into the mixed solution after the incubation in the step (2), and fixing the volume to 150-300 mu L by using a buffer solution with the pH value of 6.8-7.5, wherein the concentration of the substrate after the volume fixing is 2.5-8.0 mu M; continuing to incubate at 35-38 ℃ for 20-35 minutes, and then adding a sufficient and quantitative carboxylesterase inhibitor to terminate the reaction; the chemical structure of the substrate is shown as a formula I;
(4) Measuring a fluorescence response signal A1 of the solution after the reaction is stopped in the step (3) at 650-690 nm;
(5) And (3) establishing a mapping standard curve of the concentration of the organophosphorus and a fluorescence response signal of the solution at 650-690 nm after the termination reaction, and calculating the concentration of organophosphorus pesticide residues in the sample to be detected.
According to the detection method for rapidly detecting agricultural product pesticide residues, the carboxylesterase is utilized to convert the substrate with the chemical structure shown in the formula I, the reaction process is shown in the formula II, the carboxylesterase can hydrolyze and decarboxylate the substrate shown in the formula I to obtain the compound shown in the formula III, the compound shown in the formula III has a strong fluorescence signal at 650-690 nm, and the highest peak value is 665nm, so that the conversion yield of the compound shown in the formula III reacts with the conversion activity of the carboxylesterase under the fixed conditions of fixed time. Through researches, the organophosphorus pesticide has the function of inhibiting carboxylesterase activity. However, if the reaction can be applied to the detection of an organophosphorus pesticide, further studies have to be made on whether the inhibition of carboxylesterase activity by the concentration of the organophosphorus pesticide can be quantified, and whether the quantitative correlation with the yield of the compound represented by formula (III) has a substantial influence on the establishment of a detection method. The conversion yield of the compound shown in the formula (III) obtained by the conversion after the carboxylesterase is inhibited is reduced by researching that the concentration of the organophosphorus pesticide has positive correlation with the inhibition degree of the carboxylesterase, and the conversion yield of the carboxylesterase to the compound shown in the formula (III) is researched to have linear correlation, and the concentration of the organophosphorus pesticide has linear correlation with the conversion amount of the carboxylesterase to the compound shown in the formula (III) under the condition of a certain concentration of the carboxylesterase and a certain concentration of the substrate in a specific organophosphorus concentration range by regression analysis. And because the concentration of the compound shown in the formula (III) has strong sensitivity to the fluorescence intensity, the change of the concentration of the organophosphorus pesticide can be more sensitively reflected by the change of the concentration of the compound shown in the formula (III), and the detection method for rapidly detecting the pesticide residue of the agricultural product has better accuracy and sensitivity and has low detection limit. The detection method for rapidly detecting the pesticide residue of the agricultural product is simple in detection, high-performance liquid chromatography and other high-cost instruments are not needed, the detection limit and sensitivity of the detection are not weaker than those of the chromatographic method, the detection cost is low, the detection time is short, and the detection method is very suitable for detecting the pesticide residue of the agricultural product up to the standard.
Carboxylesterase can hydrolytically decarboxylate a substrate of formula (I) to give a compound of formula (III)
Preferably, in the step (5), the standard curve is established by preparing aqueous amine sulfur phosphorus solutions with different concentrations of 0-100 μg/L, replacing 5-25 μl of quantitative supernatant in the step (2) with 5-25 μl of aqueous amine sulfur phosphorus solutions with different concentrations of 0-100 μg/L, and repeating the step (2), the step (3) and the step (4) to obtain the mapping relation between the aqueous amine sulfur phosphorus solutions with different concentrations and different fluorescence response signals.
In the detection method for rapidly detecting agricultural product pesticide residues, the fluorescence intensity of the aqueous amine sulfur phosphorus solution and the fluorescence intensity of the solution after the reaction in the step (3) have good linear correlation, the linear correlation degree of the aqueous amine sulfur phosphorus solution concentration is 0-50 mug/L and is 0.9974, and the minimum detection limit is 0.1025 mug (aqueous amine sulfur phosphorus equivalent)/L, which is far lower than the maximum residual quantity of the national standard of 0.5mg/kg. And the method is calculated by taking the water amine sulfur phosphorus as an equivalent, so that the content of the water amine sulfur phosphorus of all the organophosphorus pesticide residues can be detected as the equivalent.
Preferably, in the step (4), a fluorescence response signal of the solution after the termination reaction in the step (3) is measured at 660 to 670 nm.
The maximum response of the fluorescence intensity of the compound shown in the formula (III) is 665nm, and the concentration change is more sensitive and the sensitivity is higher in the range of 660-670 nm.
Preferably, in the step (3), the carboxylesterase inhibitor is bis-p-nitrophenyl phosphate, O-dimethyl-O- (2, 2-dichloroethylene) phosphate, tanshinone IIA anhydride or phenylmethylsulfonyl fluoride.
The detection method for rapidly detecting agricultural product pesticide residues uses bis-p-nitrophenyl phosphate, O-dimethyl-O- (2, 2-dichloroethylene) phosphate, tanshinone IIA anhydride or phenylmethylsulfonyl fluoride as a carboxylesterase inhibitor, and after the reaction is carried out for a fixed time in the step (3), the carboxylesterase is completely inhibited to be deactivated. The problems of poor linear relation and inaccurate detection result of a detection method caused by uncertain reaction nodes due to continuous reaction of strong carboxylesterase in the solution to be detected in fluorescence detection are avoided.
Preferably, the solvent of the carboxylesterase solution is a buffer solution with pH of 6.8-7.5, and the buffer solution is a phosphate buffer solution.
Preferably, the solvent of the carboxylesterase solution is a buffer solution having a pH of 7.0 to 7.5.
More preferably, the solvent of the carboxylesterase solution is a buffer solution having a pH of 7.4.
Preferably, in the step (2) and the step (3), the incubation temperature is 36-37 ℃.
The invention also provides a kit for rapidly detecting agricultural product pesticide residues, which comprises a substrate solution with a chemical structure as shown in formula I, a carboxylesterase solution, a carboxylesterase inhibitor solution, a water amine thiophosphorus solution and a buffer solution with a pH value of 7.0-7.5;
the chemical structure of the substrate is shown as a formula I;
the kit for rapidly detecting agricultural product pesticide residues is prepared by matching a substrate solution, a carboxylesterase inhibitor solution, a water amine sulfur phosphorus solution and a buffer solution with the pH of 7.0-7.5, can be used for sample preparation immediately after sampling, can be detected by using a portable fluorometer, or can be used for sample preparation in the process of transferring after sampling, and can be used for detecting immediately after reaching a detection station, so that the detection time is shortened as a whole, and the detection result can be obtained earlier.
Preferably, the solvent of the substrate solution is a buffer solution with a pH of 7.0-7.5, the solvent of the carboxylesterase inhibitor solution is a buffer solution with a pH of 7.0-7.5, the buffer solution is a phosphate buffer solution, and the molar concentration of the phosphate is 10-200 mM.
Preferably, the carboxylesterase inhibitor is bis-p-nitrophenyl phosphate, O-dimethyl-O- (2, 2-dichloroethylene) phosphate, tanshinone IIA anhydride or phenylmethylsulfonyl fluoride.
Preferably, the concentration of the aqueous amine sulfur phosphorus solution is 0-100 mug/L.
Preferably, the concentration of the carboxylesterase solution is 50-250U/L.
Preferably, the substrate solution of formula I is at a concentration of 10 to 20. Mu.M and the carboxylesterase inhibitor solution is at a concentration of 0.1 to 1. Mu.M.
Preferably, the kit comprises instructions describing the detection method for rapidly detecting pesticide residues in agricultural products according to any one of the above.
The invention has the beneficial effects that: the invention provides a detection method for rapidly detecting agricultural pesticide residues, which utilizes a reaction of carboxylesterase to convert a substrate with a chemical structure shown as a formula I, wherein the reaction process is shown as a formula (II), the carboxylesterase can hydrolyze and decarboxylate the substrate shown as the formula (I) to obtain a compound shown as a formula (III), the compound shown as the formula (III) has a strong fluorescent signal at 650-690 nm and a peak value at 665nm, and the conversion yield of the compound shown as the formula (III) reacts with the conversion activity of the carboxylesterase under fixed conditions for fixed time. Through further research, the organophosphorus pesticide has the function of inhibiting carboxylesterase activity, the concentration of the organophosphorus pesticide has positive correlation on the inhibition degree of carboxylesterase, the conversion yield of the compound shown in the formula (III) obtained by conversion after the carboxylesterase is inhibited is reduced, the concentration of the organophosphorus pesticide has linear correlation with the conversion yield of the carboxylesterase on the compound shown in the formula (III), and through regression analysis, the concentration of the organophosphorus pesticide has linear correlation with the conversion amount of the carboxylesterase on the compound shown in the formula (III) under the conditions of a certain concentration of the carboxylesterase and a certain concentration of a substrate in a specific organophosphorus concentration range. The concentration of the compound shown in the formula (III) has strong sensitivity to the fluorescence intensity, so that the change of the concentration of the organophosphorus pesticide can be more sensitively reflected by the change of the concentration of the compound shown in the formula (III), and the detection method for rapidly detecting the pesticide residue of the agricultural product has better accuracy and sensitivity and has low detection limit. The detection method for rapidly detecting the pesticide residue of the agricultural product is simple in detection, high-performance liquid chromatography and other high-performance instruments are not needed, the detection limit and sensitivity of the detection are not weaker than those of the chromatographic method, the detection cost is low, the detection time is short, and the detection method is very suitable for detecting the pesticide residue of the agricultural product up to the standard.
Drawings
FIG. 1 is a graph showing fluorescence response signals of a solution of a compound of formula (III) in a detection method for rapidly detecting agricultural pesticide residues according to an embodiment of the present invention.
FIG. 2 is a schematic diagram showing carboxylesterase conversion of fluorescent compounds in a detection method for rapidly detecting pesticide residues in agricultural products according to an embodiment of the present invention.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.
Example 1
As a detection method for rapidly detecting pesticide residues of agricultural products, the embodiment of the invention comprises the following steps:
(1) 1.00g of sample is weighed into a 10mL glass centrifuge tube, 2mL of extraction solvent is added, the extraction solvent is a mixed solution of potassium phosphate buffer solution with pH of 7.4 and ethanol, and the volume ratio of the potassium phosphate buffer solution to the ethanol is 1:0.5; shaking for about 30s, and centrifuging;
(2) Taking 20 mu L of the supernatant in the step (1) and adding the supernatant and 100 mu L of carboxylesterase solution with the concentration of 100U/L into a 96-well plate, uniformly mixing, and then incubating at 37 ℃ for 15 minutes;
(3) Adding 50 μl of 10 μM compound as substrate, adding 30 μ LpH 7.4 potassium phosphate buffer solution to 200 μl, incubating at 37deg.C for 25 min, and adding 10 μL 0.2 μM tanshinone IIA anhydride solution to terminate the reaction;
(4) Measuring a fluorescence response signal A1 of the solution after the termination reaction in the step (3) at 665 nm;
(5) The method comprises the steps of (1) establishing a mapping standard curve of the concentration of the organic phosphorus and fluorescence response signals of a solution after termination reaction at 665nm, wherein the standard curve is established by preparing aqueous amine sulfur phosphorus solutions with different concentrations of 0-100 mu g/L, respectively replacing 20 mu L of quantitative supernatant fluid in the step (2) with 20 mu L of aqueous amine sulfur phosphorus solutions with different concentrations of 0-100 mu g/L, repeating the step (2), the step (3) and the step (4) to obtain the mapping relation between the aqueous amine sulfur phosphorus solutions with different concentrations and the different fluorescence response signals, and calculating the concentration of the organic phosphorus pesticide residues in a sample to be detected.
Experimental method
1. Establishment of a Standard Curve
1. And (5) solution preparation.
The aqueous amine sulfur phosphorus solution with the concentration of 0, 0.5 mug/L, 1 mug/L, 2 mug/L, 5 mug/L, 10 mug/L, 25 mug/L and 50 mug/L is prepared for standby.
A20 mM potassium phosphate buffer solution at pH 7.4 was prepared for use.
A carboxylesterase solution of 100U/L was prepared with 20mM potassium phosphate buffer solution having a pH of 7.4.
A10. Mu.M solution of a compound of formula I was prepared using 20mM potassium phosphate buffer at pH 7.4.
A solution of tanshinone IIA anhydride at 0.2. Mu.M was prepared with 20mM potassium phosphate buffer at pH 7.4 as the inhibitor.
A solution of a compound of the formula (III) was prepared in a concentration of 5. Mu.M in a potassium phosphate buffer solution of 20mM pH 7.4.
2. Fluorescence response of a solution of the compound represented by formula (III).
The fluorescence response signal of a solution of a compound of formula (III) having a chemical structure of 5. Mu.M was scanned in the range of 600 to 750nm, and as a result, the compound of formula (III) showed a strong fluorescence signal at 650 to 690nm and a peak at 665nm as shown in FIG. 1.
3. Standard curve establishing method
(1) Adding 20 mu L of aqueous amine sulfur phosphorus solution with concentration of 0, 0.5 mu g/L, 1 mu g/L, 2 mu g/L, 5mu g/L, 10 mu g/L, 25mu g/L and 50 mu g/L and 100 mu L of carboxylesterase solution with concentration of 100U/L into a 96-well plate, uniformly mixing, and then incubating at 37 ℃ for 15 minutes;
(2) Adding 50 μl of 10 μM compound as substrate, adding 30 μ LpH 7.4 potassium phosphate buffer solution to 200 μl, incubating at 37deg.C for 25 min, and adding 10 μL 0.2 μM tanshinone IIA anhydride solution to terminate the reaction;
(3) The fluorescence response intensity F of the solution after termination of the reaction at 665nm was measured by a Semer-fly Multiskan Skyhigh full-wavelength microplate reader, respectively.
The relationship between the fluorescence response intensity F at 665nm and the concentration of the aqueous amine sulfur phosphorus solution through linear regression analysis is as follows:
F＝-160398c+3709164，R 2 =0.9974. The minimum detection limit was calculated to be 0.1025 μg (aqueous amine sulfur phosphorus equivalent)/L according to the 3-fold blank response method.
2. Detection of labeled pesticide residue sample
1. Taking 20mL of 5mu g/L aqueous amine sulfur phosphorus solution, using a small sprayer, namely, an empty alcohol sprayer with the volume of 50mL, airing, filling 20mL of 5mu g/L aqueous amine sulfur phosphorus solution, uniformly spraying 20mL of 5mu g/L aqueous amine sulfur phosphorus solution on the surface of 100g of lettuce three times, spraying every 30 minutes, spraying 6.5-6.8 mL each time, standing for 2 hours after spraying 20mL in total, and measuring.
20mL of 50 mu g/L aqueous amine sulfur phosphorus solution is taken as 100g of lettuce for marking. The method is the same as above.
2. Sample detection
(1) Cutting lettuce into small pieces with the length of about 3mm, weighing 1g of the sample into a 10mL glass centrifuge tube, adding 2mL of extraction solvent, recording the accurate mass of the sample, wherein the extraction solvent is a mixed solution of potassium phosphate buffer solution with the pH of 7.4 and ethanol, and the volume ratio of the potassium phosphate buffer solution to the ethanol is 1:0.5; shaking for about 30s, and centrifuging;
(2) Taking 20 mu L of the supernatant in the step (1) and 100 mu L of carboxylesterase solution with the concentration of 100U/L, adding the mixture into a 96-well plate, uniformly mixing the mixture, and then incubating the mixture at 37 ℃ for 15 minutes;
(3) Adding 50 μl of 10 μM compound as substrate, adding 30 μ LpH 7.4 potassium phosphate buffer solution to 200 μl, incubating at 37deg.C for 25 min, and adding 10 μL 0.2 μM tanshinone IIA anhydride solution to terminate the reaction;
(4) The fluorescence response intensity F of the solution after termination of the reaction at 665nm was measured by a Semer-fly Multiskan Skyhigh full-wavelength microplate reader, respectively.
8 times of parallel are carried out on the marked sample with each concentration, and the concentration of the aqueous amine sulfur and phosphorus in the marked supernatant fluid is calculated.
The concentration of the aqueous amine thiophosphorus in the unlabeled lettuce was determined as described above.
Calculating the concentration of the water amine sulfur and phosphorus in the marked lettuce, and calculating and detecting the marked recovery rate (%). The experimental results are shown in table 1.
As shown in Table 1, the linear correlation degree of the aqueous amine sulfur phosphorus solution concentration of 0-50 mug/L is 0.9974, the minimum detection limit is 0.1025 mug (aqueous amine sulfur phosphorus equivalent)/L, and the maximum residual quantity is far lower than the national standard by 0.5mg/kg. The concentration of the fenpyrad in the unlabeled lettuce was determined according to the method described above and none of the results were detected. Through the detection of a labeling sample, the labeling recovery rate under the condition of low concentration labeling (1 mug/kg of lettuce) is 82.0% -88.4%, the standard deviation is 2.66%, and the labeling recovery rate under the condition of high concentration labeling (10 mug/kg of lettuce) is 76.8% -87.5%, and the standard deviation is 4.16%, so that the detection method meets the requirements of the detection method. Although the standard recovery rate is lower than 100%, the uniformity of solid standard and the loss factor of standard are lower than that of solution standard, the standard deviation between parallel samples is smaller than 75% -125%, the accuracy and sensitivity of the detection method are better, the detection limit is lower, the detection method is simple, high-performance liquid chromatography and other high-performance instruments are not needed, the detection limit and sensitivity are not weaker than those of the chromatographic method, the detection cost is low, the detection time is short, and the method is very suitable for standard detection of pesticide residues of agricultural products.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A detection method for rapidly detecting pesticide residues in agricultural products, which is characterized by comprising the following steps:
(1) Extracting organophosphorus pesticide residues in quantitative agricultural product samples by using a quantitative extraction solvent, and collecting supernatant, wherein the extraction solvent is a mixed solution of a buffer solution with the pH of 6.8-7.5 and ethanol, and the volume ratio of the buffer solution to the ethanol is 1: (0.5-1);
(2) Uniformly mixing 10-25 mU L of supernatant obtained in the quantifying step (1) with 5-25 mU of carboxylesterase solution, and then incubating for 15-25 minutes at the temperature of 35-38 ℃;
(3) Adding a substrate into the mixed solution after the incubation in the step (2), and fixing the volume to 150-300 mu L by using a buffer solution with the pH value of 6.8-7.5, wherein the concentration of the substrate after the volume fixing is 2.5-8.0 mu M; continuing to incubate at 35-38 ℃ for 20-35 minutes, and then adding a sufficient and quantitative carboxylesterase inhibitor to terminate the reaction; the chemical structure of the substrate is shown as a formula I;
(4) Measuring a fluorescence response signal A1 of the solution after the reaction is stopped in the step (3) at 650-690 nm;
(5) And (3) establishing a mapping standard curve of the concentration of the organophosphorus and a fluorescence response signal of the solution at 650-690 nm after the termination reaction, and calculating the concentration of organophosphorus pesticide residues in the sample to be detected.
2. The method for rapidly detecting agricultural pesticide residues according to claim 1, wherein in the step (5), a standard curve is established by preparing aqueous amine-sulfur-phosphorus solutions with different concentrations of 0-100 μg/L, and 5-25 μl of the aqueous amine-sulfur-phosphorus solutions with different concentrations of 0-100 μg/L are replaced with 5-25 μl of quantitative supernatant in the step (2), and the mapping relation between the aqueous amine-sulfur-phosphorus solutions with different concentrations and different fluorescence response signals is obtained in the steps (2), (3) and (4).
3. The method for rapidly detecting agricultural chemical residue according to claim 1, wherein in the step (4), a fluorescence response signal of the solution after the termination of the reaction in the step (3) is measured at 660 to 670 nm.
4. The method for rapidly detecting agricultural chemical residues according to claim 1, wherein in the step (3), the carboxylesterase inhibitor is bis-p-nitrophenylphosphate, O-dimethyl-O- (2, 2-dichlorovinyl) phosphate, tanshinone IIA anhydride or phenylmethylsulfonyl fluoride.
5. The method for rapidly detecting agricultural chemical residues according to claim 1, wherein the solvent of the carboxylesterase solution is a buffer solution with a pH of 6.8-7.5, and the buffer solution is a phosphate buffer solution.
6. The method for rapidly detecting agricultural chemical residue according to claim 5, wherein the carboxylesterase solution is a buffer solution having a pH of 7.0 to 7.5, and the incubation temperature in step (2) and step (3) is 36 to 37 ℃.
7. The kit for rapidly detecting agricultural product pesticide residues is characterized by comprising a substrate solution with a chemical structure as shown in a formula I, a carboxylesterase solution, a carboxylesterase inhibitor solution, a water amine thiophosphorus solution and a buffer solution with a pH value of 7.0-7.5;
the chemical structure of the substrate is shown as a formula I;
8. the kit for rapid detection of agricultural pesticide residues according to claim 7, wherein the solvent of the substrate solution is a buffer solution with a pH of 7.0-7.5, the solvent of the carboxylesterase inhibitor solution is a buffer solution with a pH of 7.0-7.5, the buffer solution is a phosphate buffer solution, the molar concentration of phosphate is 10-200 mM, and the carboxylesterase inhibitor is bis-p-nitrophenyl phosphate, O-dimethyl-O- (2, 2-dichloroethylene) phosphate, tanshinone IIA anhydride or phenylmethylsulfonyl fluoride.
9. The kit for rapid detection of agricultural pesticide residues according to claim 7 or 8, wherein the concentration of the aqueous amine thiophosphorus solution is 0 to 100 μg/L, the concentration of the carboxylesterase solution is 50 to 250U/L, the concentration of the substrate solution of formula i is 10 to 20 μΜ, and the concentration of the carboxylesterase inhibitor solution is 0.1 to 1 μΜ.
10. A kit for rapid detection of agricultural chemical residue according to claim 7 or 8, wherein the kit comprises instructions describing the method for rapid detection of agricultural chemical residue according to any one of claims 1 to 6.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050054691A1 (en) * | 2003-08-29 | 2005-03-10 | St. Jude Children's Research Hospital | Carboxylesterase inhibitors |
| CN101776601A (en) * | 2010-01-25 | 2010-07-14 | 泸州老窖集团有限责任公司 | Method for fast detecting pesticide residue |
| CN102788776A (en) * | 2012-08-09 | 2012-11-21 | 中国科学院长春应用化学研究所 | Detection method for activity of carboxylesterase and activity of carboxylesterase inhibitor |
| CN103251571A (en) * | 2004-03-30 | 2013-08-21 | 瑞立普萨公司 | Potassium ion binding polymers for pharmaceutical compositions |
| CN106124433A (en) * | 2016-06-22 | 2016-11-16 | 上海理工大学 | A kind of method that quick detection is pyrethroid pesticide remained |
| CN111705110A (en) * | 2020-04-28 | 2020-09-25 | 北京智云达科技股份有限公司 | Rapid detection kit for organophosphorus pesticide residues, preparation method and detection method thereof |
| CN114235792A (en) * | 2021-11-30 | 2022-03-25 | 广东省科学院测试分析研究所(中国广州分析测试中心) | Rapid detection method for pyrethroid pesticide residue |
-
2023
- 2023-09-04 CN CN202311133156.2A patent/CN117106852B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050054691A1 (en) * | 2003-08-29 | 2005-03-10 | St. Jude Children's Research Hospital | Carboxylesterase inhibitors |
| CN103251571A (en) * | 2004-03-30 | 2013-08-21 | 瑞立普萨公司 | Potassium ion binding polymers for pharmaceutical compositions |
| CN101776601A (en) * | 2010-01-25 | 2010-07-14 | 泸州老窖集团有限责任公司 | Method for fast detecting pesticide residue |
| CN102788776A (en) * | 2012-08-09 | 2012-11-21 | 中国科学院长春应用化学研究所 | Detection method for activity of carboxylesterase and activity of carboxylesterase inhibitor |
| CN106124433A (en) * | 2016-06-22 | 2016-11-16 | 上海理工大学 | A kind of method that quick detection is pyrethroid pesticide remained |
| CN111705110A (en) * | 2020-04-28 | 2020-09-25 | 北京智云达科技股份有限公司 | Rapid detection kit for organophosphorus pesticide residues, preparation method and detection method thereof |
| CN114235792A (en) * | 2021-11-30 | 2022-03-25 | 广东省科学院测试分析研究所(中国广州分析测试中心) | Rapid detection method for pyrethroid pesticide residue |
Non-Patent Citations (1)
| Title |
|---|
| VALENTINA DE LUCA等: "Development of a Qualitative Test to Detect the Presence of Organophosphate Pesticides on Fruits and Vegetables", 《LIFE》, vol. 13, no. 2, pages 490 * |
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