CN110793929A - Pesticide residue detection and distinguishing method based on multienzyme inhibition - Google Patents
Pesticide residue detection and distinguishing method based on multienzyme inhibition Download PDFInfo
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Abstract
The invention discloses a pesticide residue detection and distinguishing method based on multi-enzyme inhibition, belongs to the technical field of detection, constructs an array sensor detection system based on enzyme inhibition, and provides a new method for detecting different types of pesticide residues. A sensor array is one of multidimensional sensing technologies, and is composed of a plurality of sensing units, like a chip constructed by a plurality of functional components. The response degree of a single sensing unit to different substances is different, and different sensing units also have different responses to the same substance, so that the specific distinction of samples can be realized through characteristic maps generated by the response of different sensing units to target substances and the difference between the characteristic maps. The invention constructs the array sensor based on the enzyme inhibition system, has high detection sensitivity and strong expandability, realizes the differentiation of similar main structure pesticides and the intelligent analysis of the pesticides, has strong specificity and high accuracy, and realizes the identification and differentiation of the same-genus pesticides.
Description
Technical Field
The invention belongs to the technical field of detection, and particularly relates to a pesticide residue detection and distinguishing method based on multienzyme inhibition.
Background
China is a big country for producing and using pesticides, the types and the number of pesticide products are large, the increasing speed is high, and in addition, the unreasonable use of pesticides causes the problem of pesticide residue to be also remarkable. The detection means of pesticide residue in food is greatly changed along with the continuous improvement of chemical analysis technology and the change of social development demand. The conventional methods for detecting pesticide residues are mainly based on a precise instrument analysis method represented by a chromatography-mass spectrometry method. The method has the advantages of high sensitivity and good selectivity, but the method has the disadvantages of huge instruments and equipment, high detection cost and long time consumption, needs complex pretreatment processes such as extraction and separation of samples, needs operation of professional personnel, needs a large amount of manpower and material resources, has long detection time and expensive instrument price, needs maintenance of professional technicians, has high detection cost, is not suitable for field detection, and cannot meet the detection requirements of various agricultural products.
Therefore, with the development of modern chemical analysis techniques, pesticide residue detection is moving towards rapid detection methods. The enzyme inhibition method is the most typical and most common pesticide rapid detection method. The method becomes a hotspot of research in recent years due to the obvious advantages of portability, rapidness, simplicity, easy use, low cost and the like, but still has a plurality of defects: the sensitivity is low, and the repeatability is to be improved; due to the specificity of the 'lock and key structure', the conventional sensor cannot distinguish the types of pesticides, and even can not distinguish different pesticide brands of the same type, for example, the conventional sensor cannot distinguish whether the pesticide is an organophosphorus pesticide or a carbamate pesticide, and even can not distinguish dimethoate and chlorpyrifos which are organophosphorus pesticides.
Disclosure of Invention
In order to solve the defects of the enzyme inhibition method for detecting pesticide residues, the invention provides a method for distinguishing pesticide types and detecting pesticide residues with low cost and high sensitivity. The invention constructs an array sensor detection system based on multienzyme inhibition, and provides a new method for detecting different types of pesticide residues. A sensor array is one of multidimensional sensing technologies, and is composed of a plurality of sensing units, like a chip constructed by a plurality of functional components. The response degree of a single sensing unit to different substances is different, and different sensing units also have different responses to the same substance, so that the specific distinction of samples can be realized through characteristic maps generated by the response of different sensing units to target substances and the difference between the characteristic maps.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a pesticide residue detection and distinguishing method based on multi-enzyme inhibition comprises the following specific steps:
(1) and preparing an array sensor: preparing a sensor array with multiple sensing units by taking several different enzyme solutions as each sensing unit of the array sensor;
(2) and distinguishing and detecting different pesticides: adding a pesticide standard solution into the array sensor array in the step (1), mixing and reacting with a detection solution (an enzyme substrate, generating a colorimetric or fluorescent signal and the like) in the array sensor array, reading an ultraviolet absorption value or a fluorescent value and the like of the detection solution through an instrument, carrying out digital processing on the inhibition effect of different pesticides on different enzyme activities, introducing obtained data into SPSS software for linear discriminant analysis, and obtaining component maps of different types of pesticides by respectively taking feature vectors of first two linear discriminant functions obtained through analysis as a first factor and a second factor, and taking the first factor as a horizontal coordinate and the second factor as a vertical coordinate; then according to a composition diagram of the same kind of pesticide, the concentration of the pesticide residue is used as a horizontal coordinate, and a first factor is used as a vertical coordinate, so that a standard fitting curve of the concentration of the pesticide residue-the first factor is obtained;
(3) and distinguishing and detecting pesticides in the actual sample: adding a solution to be detected containing pesticide residues into at least one sensing unit of the array sensor for detecting different types of pesticides to be mixed with a detection solution in the array sensor for reaction, reading an ultraviolet absorption value or a fluorescence value through an instrument, carrying out digital processing on the inhibition effect of different pesticides on different enzyme activities, introducing obtained data into SPSS (SpIndustrie point-to-multipoint System) for linear discriminant analysis, obtaining component maps of different types of pesticides in a sample to be detected by taking the feature vectors of the first two linear discriminant functions as a first factor and a second factor, taking the first factor as a horizontal coordinate and the second factor as a vertical coordinate, and comparing the obtained component maps with the component map obtained in the step 2 to determine the type and the concentration of pesticide residues in an actual sample.
Further, the enzyme solution in the step (1) is prepared by mixing acetylcholinesterase, thioacetylcholine iodide and 5,5 '-dithiobis (2-nitrobenzoic acid) solutions according to a volume ratio of 30-40:2-4:1-2, mixing butyrylcholinesterase, thiobutyrylcholine iodide and 5,5' -dithiobis (2-nitrobenzoic acid) solutions according to a volume ratio of 6-7:2-4:1-2 or mixing alkaline phosphatase and disodium 4-nitrophenylphosphate solutions according to a volume ratio of 5-7: 1-2;
furthermore, the concentration of the acetylcholine esterase solution is 10U/L-100U/L, the concentration of the thiocholine iodide solution is 0.1mM-1mM, the concentration of the butyrylcholine esterase solution is 0.2U/L-2U/L, the concentration of the thiocyanechol iodide solution is 0.1mM-1mM, the concentration of the 5,5' -dithiobis (2-nitrobenzoic acid) solution is 0.05mM-0.5mM, the concentration of the alkaline phosphatase solution is 5U/L-100U/L, and the concentration of the disodium 4-nitrophenyl phosphate solution is 0.005mM-0.5 mM.
Further, the iodized thioacetylcholine reacts with 5,5' -dithiobis (2-nitrobenzoic acid) under the action of acetylcholinesterase, the reaction temperature is 36-38 ℃, the reaction time is 30-40 min, and a characteristic absorption peak exists at a position of 410-415 nm; the iodized thio-butyrylcholine reacts with 5,5' -dithiobis (2-nitrobenzoic acid) under the action of butyrylcholinesterase, the reaction temperature is 36-38 ℃, the reaction time is 30-40 min, and a characteristic absorption peak exists at a position of 410-415 nm; the alkaline phosphatase and 4-nitrophenyl phosphate disodium salt have color reaction at 36-38 deg.c for 30-40 min and characteristic absorption peak at 400-405 nm.
Further, the pesticide standard solution in the step (2) is one or more of dimethoate, chlorpyrifos, phosmet, carbaryl, isoprocarb, pirimicarb, hexachloro cyclohexane, dichlorodiphenyl trichloroethane and the like. Wherein, dimethoate, malathion, isocarbophos, chlorpyrifos and phosmet belong to organophosphorus pesticide, carbaryl, isoprocarb and pirimiphos belong to carbamate pesticide, and hexachloro cyclohexane hexachloride and dichlorodiphenyl trichloroethane belong to organochlorine pesticide.
In embodiments, the sensing unit comprises a 96-well plate, and the 96-well plate is a fully transparent microplate, but is not limited thereto.
The principle of the invention is as follows: different types of pesticides, (organophosphorus pesticides, carbamate pesticides, etc.) have inhibitory effects on different types of enzymes (acetylcholinesterase, butyrylcholinesterase, alkaline phosphatase, etc.), and the inhibitory effects are different, and based on the above properties, an array sensor based on inhibition by a plurality of enzymes is constructed.
Compared with the prior art, the invention has the following advantages:
the enzyme inhibition method for detecting organophosphorus pesticide and carbamate pesticide is a conventional method in national standard, but an array sensor constructed based on the multi-enzyme inhibition method is not reported. Due to the specific inhibition of organophosphorus pesticide and carbamate pesticide on acetylcholinesterase, the array sensor has good anti-interference performance when detecting pesticide in the presence of other components such as protein, vitamins, pigments and the like. The invention constructs the array sensor based on the enzyme inhibition system, has high detection sensitivity and strong expandability, realizes the differentiation of similar main structure pesticides and the intelligent analysis of the pesticides, has strong specificity and high accuracy, and realizes the identification and differentiation of the same-genus pesticides.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings used in the prior art will be briefly described below.
FIG. 1 is a linear discriminant analysis spectrum of the response of the array sensor to pesticide residues with the same concentration and different types according to the invention, and the experiments are performed in parallel for 5 times;
FIG. 2 is a graph of a first factor of a linear discriminant analysis of the present invention, which is a feature vector of a first linear discriminant function, as fitted to a variation in the concentration of a pesticide residue.
Detailed Description
In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
A pesticide residue detection and distinguishing method based on multi-enzyme inhibition comprises the following specific steps:
(1) and preparing an array sensor: mixing acetylcholinesterase, thioacetylcholine iodide and 5,5' -dithiobis (2-nitrobenzoic acid) solution according to the volume ratio of 30-40:2-4: 1-2; mixing butyrylcholine esterase, iodized thio-butyrylcholine and 5,5' -dithiobis (2-nitrobenzoic acid) solution according to the volume ratio of 6-7:2-4: 1-2; mixing alkaline phosphatase and disodium 4-nitrophenyl phosphate solution in the volume ratio of 5-7 to 1-2; adding the three mixed solutions into each sensing unit of the array sensor respectively to prepare a sensor array with three sensing units; the concentration of the acetylcholinesterase solution is 10-100U/L, the concentration of the iodothioacetylcholine solution is 0.1-1 mM, the concentration of the butyrylcholinesterase solution is 0.2-2U/L, the concentration of the iodothiobutyrylcholine solution is 0.1-1 mM, the concentration of the 5,5' -dithiobis (2-nitrobenzoic acid) solution is 0.05-0.5 mM, the concentration of the alkaline phosphatase solution is 5-100U/L, and the concentration of the disodium 4-nitrophenyl phosphate solution is 0.005-0.5 mM.
(2) And distinguishing and detecting different pesticides: adding a pesticide standard solution into at least one sensing unit in the array sensor array in the step (1), mixing and reacting with the detection solution, reading an ultraviolet absorption value through an enzyme-labeling instrument, and performing linear discriminant analysis to obtain component diagrams of different types of pesticides, so as to obtain a pesticide residue concentration-first factor standard fitting curve; the linear discriminant analysis is that different types of pesticide standard solutions use a data set obtained by an array sensor as a given training set, and the training set is projected on a straight line through a dimension reduction thought of the linear discriminant analysis, so that the projection points of the same type of pesticide data sets are as close as possible, and the projection points of the different types of pesticide data sets are as far away as possible. And (4) deriving a linear discriminant function, arranging the eigenvalues from large to small, and taking the eigenvectors corresponding to the two former eigenvalues, namely the linear discriminant function. And reducing the data of the training set to a two-dimensional space by using the two linear discriminant functions, namely the obtained component map. The first factor is a feature vector of a first linear discriminant function.
(3) And distinguishing and detecting pesticides in the actual sample: adding the solution to be detected containing pesticide residues into at least one sensing unit of the array sensor for detecting different types of pesticides and the detection solution in the array sensor for mixing reaction, reading the ultraviolet absorption value by an enzyme-labeling instrument and carrying out linear discriminant analysis to obtain the composition diagram of different types of pesticides in the sample to be detected. The data sets obtained by the array sensors of different types of pesticide standard solutions are used as given training sets, and the training sets are projected onto a straight line through the dimension reduction thought of linear discriminant analysis, so that the projection points of the same type of pesticide data sets are as close as possible, and the projection points of different types of pesticide data sets are as far away as possible. The data set of the actual sample measured by the array sensor is the sample set. And when the new sample is classified by linear discriminant analysis, projecting the sample set onto the same straight line as the training set, and determining the class of the new sample according to the position of the projection point. And (3) comparing the obtained composition diagram with the composition diagram obtained in the step (2), thereby identifying the types and the concentrations of the pesticide residues in the actual samples.
Example 1 identification and differentiation of various pesticide residues
The sensing unit 1: 0.05mg/mL acetylcholinesterase solution, 0.1mM thioacetylcholine iodide solution, 0.05mM5,5' -dithiobis (2-nitrobenzoic acid) solution; the sensing unit 2: 0.5. mu.g/mL butyrylcholinesterase solution, 0.1mM thiobutyrylcholine iodide solution, 0.05mM5,5' -dithiobis (2-nitrobenzoic acid) solution; the sensing unit 3: 5mU/mL alkaline phosphatase solution, 0.075mM disodium 4-nitrophenylphosphate solution;
respectively adding 0.125mg/L dimethoate standard solution, 0.125mg/L malathion standard solution, 0.125mg/L isocarbophos standard solution, 0.125mg/L chlorpyrifos standard solution, 0.125mg/L phosmet standard solution, 0.125mg/L carbaryl standard solution, 0.125mg/L isoprocarb standard solution and 0.125mg/L pirimicarb standard solution into each sensing unit of the array sensor, mixing the sensing units with the detection liquid for reaction, putting a certain volume into a 96-well plate, respectively generating different color changes after the sensing units of the array sensor for detecting the pesticide react with different pesticides, reading the ultraviolet absorption value of each mixed reaction system by an enzyme reader, carrying out experiments for 5 times in parallel, carrying out digital treatment on the inhibition of different pesticides on different enzyme activities, introducing the obtained data into SPSS software for linear discrimination analysis, the feature vectors of the first two linear discriminant functions obtained by analysis are a first factor and a second factor, the first factor is used as a horizontal coordinate, the second factor is used as a vertical coordinate, the composition diagram of different pesticides is obtained as shown in fig. 1, different types of pesticides can be correctly identified and distinguished, and further, different brands of pesticides of the same species can be well identified and distinguished.
Example 2 establishment of Standard fitting Curve for pesticides
The sensing unit 1: 0.05mg/mL acetylcholinesterase solution, 0.1mM thioacetylcholine iodide solution, 0.05mM5,5' -dithiobis (2-nitrobenzoic acid) solution; the sensing unit 2: 0.5. mu.g/mL butyrylcholinesterase solution, 0.1mM thiobutyrylcholine iodide solution, 0.05mM5,5' -dithiobis (2-nitrobenzoic acid) solution; the sensing unit 3: 5mU/mL alkaline phosphatase solution, 0.075mM disodium 4-nitrophenylphosphate solution;
respectively adding 0mg/L,0.125mg/L,0.25mg/L,0.375mg/L,0.5mg/L,0.75mg/L and carbaryl pesticide standard solution into each sensing unit of the array sensor, mixing and reacting with the detection liquid in the sensing units, putting a certain volume into a 96-well plate, respectively generating different color changes after the sensing units of the array sensor for detecting pesticide react with different pesticides, reading the ultraviolet absorption value of each mixed reaction system through a microplate reader, performing digital treatment on the inhibition effect of different pesticides on different enzyme activities in an experiment for 5 times in parallel, introducing the obtained data into SPSS software for linear discriminant analysis, fitting a curve by taking the pesticide concentration as a horizontal coordinate and the first factor of the linear discriminant analysis as a vertical coordinate, wherein the first factor is a characteristic vector of a first linear discriminant function, as shown in FIG. 2, LOD of the pesticide was detected to be < 80. mu.g/L.
Claims (5)
1. A pesticide residue detection and distinguishing method based on multi-enzyme inhibition is characterized by comprising the following specific steps:
(1) and preparing an array sensor: preparing a sensor array with multiple sensing units by taking several different enzyme solutions as each sensing unit of the array sensor;
(2) and distinguishing and detecting different pesticides: adding a pesticide standard solution into at least one sensing unit in the array sensor array in the step (1), mixing and reacting with detection liquid (enzyme substrate, generating colorimetric or fluorescent signals and the like) in the array sensor array, reading ultraviolet absorption or fluorescence values and the like of the detection liquid through an instrument, carrying out digital processing on inhibition effects of different pesticides on different enzyme activities, introducing obtained data into SPSS software for linear discriminant analysis, and obtaining component maps of different pesticides by respectively taking feature vectors of first two linear discriminant functions obtained through analysis as a first factor and a second factor, wherein the first factor is used as a horizontal coordinate and the second factor is used as a vertical coordinate; then according to a composition diagram of the same kind of pesticide, the concentration of the pesticide residue is used as a horizontal coordinate, and a first factor is used as a vertical coordinate, so that a standard fitting curve of the concentration of the pesticide residue-the first factor is obtained;
(3) and distinguishing and detecting pesticides in the actual sample: adding a solution to be detected containing pesticide residues into at least one sensing unit of the array sensor for detecting different types of pesticides to be mixed with a detection solution in the array sensor for reaction, reading an ultraviolet absorption value or a fluorescence value through an instrument, carrying out digital processing on the inhibition effect of different pesticides on different enzyme activities, introducing obtained data into SPSS (SpIndustrie point-to-multipoint System) for linear discriminant analysis, obtaining component maps of different types of pesticides in a sample to be detected by taking the feature vectors of the first two linear discriminant functions as a first factor and a second factor, taking the first factor as a horizontal coordinate and the second factor as a vertical coordinate, and comparing the obtained component maps with the component map obtained in the step 2 to determine the type and the concentration of pesticide residues in an actual sample.
2. The method for detecting and distinguishing pesticide residues based on multi-enzyme inhibition as claimed in claim 1, wherein the enzyme solution in step (1) is acetylcholinesterase, thioacetylcholine iodide, 5 '-dithiobis (2-nitrobenzoic acid) solution mixed according to a volume ratio of 30-40:2-4:1-2, butyrylcholinesterase, thiobutyrylcholine iodide, 5' -dithiobis (2-nitrobenzoic acid) solution mixed according to a volume ratio of 6-7:2-4:1-2 or alkaline phosphatase, disodium phosphate-4-nitrophenyl ester solution mixed according to a volume ratio of 5-7: 1-2.
3. The method for detecting and distinguishing pesticide residues based on multi-enzyme inhibition according to claim 2, wherein the concentration of the acetylcholine esterase solution is 10U/L to 100U/L, the concentration of the thioacetyl iodide solution is 0.1mM to 1mM, the concentration of the butyrylcholinesterase solution is 0.2U/L to 2U/L, the concentration of the thiobutyrylcholine iodide solution is 0.1mM to 1mM, the concentration of the 5,5' -dithiobis (2-nitrobenzoic acid) solution is 0.05mM to 0.5mM, the concentration of the alkaline phosphatase solution is 5U/L to 100U/L, and the concentration of the disodium 4-nitrophenylphosphate solution is 0.005mM to 0.5 mM.
4. The method for detecting and distinguishing pesticide residues based on multi-enzyme inhibition as claimed in claim 2, wherein the thioacetyl choline iodide is reacted with 5,5' -dithiobis (2-nitrobenzoic acid) under the action of acetylcholinesterase, the reaction temperature is 36-38 ℃, the reaction time is 30-40 min, and a characteristic absorption peak is found at 410-415 nm; the iodized thio-butyrylcholine reacts with 5,5' -dithiobis (2-nitrobenzoic acid) under the action of butyrylcholinesterase, the reaction temperature is 36-38 ℃, the reaction time is 30-40 min, and a characteristic absorption peak exists at a position of 410-415 nm; the alkaline phosphatase and 4-nitrophenyl phosphate disodium salt have color reaction at 36-38 deg.c for 30-40 min and characteristic absorption peak at 400-405 nm.
5. The method for detecting and distinguishing pesticide residues based on multi-enzyme inhibition as claimed in claim 1, wherein the pesticide standard solution in the step (2) is one or more of dimethoate, chlorpyrifos, phosmet, carbaryl, isoprocarb, pirimicarb, hexachloro-hexa, dichlorodiphenyl trichloroethane and the like. Wherein, dimethoate, malathion, isocarbophos, chlorpyrifos and phosmet belong to organophosphorus pesticide, carbaryl, isoprocarb and pirimiphos belong to carbamate pesticide, and hexachloro cyclohexane hexachloride and dichlorodiphenyl trichloroethane belong to organochlorine pesticide.
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