0 Preface
Pesticides, as an important agricultural means of production, play a very important role in the prevention and control of diseases, insects, grasses and rodents in the production of agricultural products. They have created tremendous economic benefits for humans, but at the same time they have also brought environmental and human health risks. A certain threat. Pesticide pesticides used in agricultural production can be divided into four major categories: The first is organochlorine pesticides, which have been discontinued due to defects such as teratogenicity, carcinogenesis, and mutagenesis. Production and use; the second category is organophosphorus pesticides; the third category is carbamate insecticides; the fourth is pyrethroid insecticides. Among all pesticides, organophosphorus pesticides are mainly killed. Insecticides are widely used in the production of various crops, especially fruits and vegetables, because of their large variety, many control objects, and high insecticidal efficiency. Some of the organophosphorus pesticides are highly toxic and are used in large quantities. It will cause environmental pollution, crop injury and human and animal poisoning. According to statistics of the World Health Organization, the number of pesticide poisoning in the world is about 3 million people each year, of which organic phosphorus pesticide poisoning accounts for 70%. China's experts have numerous pesticide species. Among the ten strongly polluting pesticides screened out from the list, seven of them belong to organophosphates. It can be seen that the focus of pesticide residue detection should be on organophosphorus pesticides. Currently, the rapid detection technology for pesticide residues in China is the most widely used. Is the enzyme inhibition method, its high specificity, sensitivity and accuracy and low cost has attracted widespread attention. The author mainly for the rapid detection of organophosphorus pesticides for two enzymes acetylcholinesterase and plant esterase A review of applied research in residues.
1 Organophosphorus pesticides
1. 1 Characteristics of Organophosphorus Pesticides
As early as the 1930s, Germany's Schrader synthesized a series of organophosphate compounds and found that some of them had insecticidal and acaricidal activity. At present, there are more than 100 species of organic phosphorus insecticides in the world. There are more than 50 species. There are more than 30 organic phosphorus species in China's pesticide market, accounting for 38% of China's pesticide species, while the output accounts for 75%. Organophosphorus pesticides are the most widely used and most used category of pesticides in China. Insecticides, organic pesticides generally remain in the soil, or drinking water, and then enter the plants, animals, and human body through the biological chain. So how to determine the amount of pesticide residues in the soil, we can determine by the soil tester. It can measure the content of nitrogen and phosphorus and infer whether it contains pesticide components. There are many kinds of pesticides, and their performance varies greatly. In summary, they have the following characteristics:
(1) It is easy to degrade in natural environment, animals and plants. When it is properly used, there is little residual problem, it will not pollute the environment and there is no cumulative toxicity in higher animals. (2) High insecticidal efficiency, broad spectrum, and various modes of action (such as contact, Stomach poisoning and fumigation) (3) Large differences in toxicity. Phoxim, malathion and trichlorfon are low in toxicity, while parathion and phorate are highly toxic species. In general, organophosphorus kills. Insecticides have high toxicity; (4) Easy to detoxify. Special detoxification agents for acute poisoning caused by organophosphate insecticides, such as pralidoxime and atropine; (5) Insecticides with organochlorine pesticides and pyrethroids. Compared to pesticides, insect pest resistance to organophosphate insecticides has been slow.
1.2 Toxicity of Organophosphorus Pesticides
As typical enzyme poisons, organophosphorus pesticides can be ingested through the digestive tract and can also be absorbed through the skin, mucous membranes, and respiratory tract. The acute toxicity of organic phosphorus is mainly attributed to the activity of acetylcholinesterase on the postsynaptic membrane of cholinergic nerves. Inhibition prevents the enzyme from catalyzing the hydrolysis of acetylcholine, resulting in excessive accumulation of acetylcholine in the tissue that can overexcite the nerves. This metabolic disorder of the acetylcholine transporter can cause delayed neurotoxicity, causing movement disorders, coma, and respiratory centers. Paralysis, delirium, and even death. Experiments have shown that paraoxonase and parathionylation of human brain cholinesterase is generally "aged" after about 4 days, the enzyme activity is not easy to recover.
2 enzyme inhibition method for the detection of organophosphorus pesticides
At present, the commonly used methods for the detection of organophosphorous pesticide residues are: chromatography, spectrometry and enzyme inhibition. Chromatographic detection is accurate, high sensitivity, but the cost is high, the measurement time is long, and requires professionals with a certain level of skill to operate , Only suitable for analysis in the laboratory; Spectrometry interference factors, sensitivity is not high, prone to false negative results, generally only as a method of rough selection; enzyme inhibition method is simple, fast, suitable for on-site detection and a large number of sample screening The detection and frequent on-site rapid detection in China's small-scale, decentralized distribution system are widely used as a common method for detecting pesticides. How to select the enzyme source is the key to achieve rapid and accurate detection.
2.1 Sources of enzymes
Cholinesterase is widely present in animal tissues. In 1953, Stedman et al. first isolated acetylcholinesterase from horse serum. Since then, different sources of acetylcholinesterase have been purified and studied. Currently, animal-derived acetylcholinesterates have been isolated and purified. Enzymes are mainly concentrated in animal livers and blood, such as pig liver, chicken blood, etc. There are also the use of earthworms and marine fish, crustacean nerve tissue, and brain tissue to isolate acetylcholinesterase. In recent years, many scholars have devoted their efforts to extract acetylcholine from insects. Esterases such as houseflies, wheat bifurcations, yellow-orange flies, tobacco horn moths, Culex pipiens pallidae, yellow-powdered aphids, Spodoptera frugiperda, Cardamom mydas, potato leafworms, harassment horn fly, and cotton bollworm So far, enzyme inhibition assays for pesticide residues detection have shown that acetylcholinesterase extracted from sensitive house flies has the best effect, and its sensitivity is better than that of acetylcholinesterase from other sources. Further research shows that after housefly emergence, 2～ At 3 days, the acetylcholinesterase sensitivity of the head was the highest. Plant-derived esterase was one of the three enzyme markers for pesticide residue detection due to its wide range of sources. The price is low and has received more attention in recent years. However, almost all of the studies are directly using wheat flour as an enzyme source, screening the best wheat variety for pesticide residue detection or using flour as an enzyme marker to make an enzyme tablet. A comparative study was conducted on the temperature, time, and the choice of chromogenic reagents for detecting color reaction of pesticide residues in enzyme tablets. The sensitivity and minimum detection limit of a large number of organophosphorus pesticides using purified and purified esterase preparations have not been seen yet. Research reports.
2. 2 Comparison of two enzyme inhibition methods
The inhibition of organophosphorus pesticides by enzyme inhibition method is based on the inhibition of organophosphorus pesticides on the enzyme-decomposing substrates. The enzymes are cholinesterase from animals and plant esterases extracted from plants. The esterase method is based on the toxicity of organophosphorus pesticides inhibiting acetylcholinesterase catalyzed hydrolysis of the substrate to acetic acid and choline, the latter reacting with 5,52-dithio-2-succinate 222 nitrobenzoic acid to yield the yellow product 52 Thio 222 Nitrate The benzoic acid, which has a maximum absorption at 410 nm, was measured for its production per unit time. The activity of acetylcholinesterase was measured. Phytoesterase can catalyze the hydrolysis of naphthyl acetate to naphthol and acetic acid, naphthol. The azo compound reacts with the chromogenic agent Blue B to form a purplish red azo compound and a color reaction occurs.
Organophosphorous pesticides can inhibit the activity of plant esterases and change the reaction rate. The yield of purplish red azo compounds is different, and the absorbance values ​​are also different. That is, the degree of inhibition of the enzyme activity is different. Many scholars have compared animals through extensive experiments. Esterase and plant esterase reaction conditions, total esterase activity and sensitivity, and its detection conditions were optimized, such as in 2004 Huang Zhiyong, etc. compared the rapid determination of organophosphorus pesticide residues in cholinesterase inhibition method and plant As a result of esterase inhibition, two rapid assay methods for enzyme inhibition were found to be applicable to the determination of organophosphorus pesticide residues in vegetables. The spiked recoveries were all above 85%, but the determination of plant esterase was less than 3%. Precision The degree of activity of the plant esterase and animal esterase at different p H values, the degree of inhibition, and other properties of the experimental study also showed that the flies enzyme, maggot enzyme and plant in animal esterases. Compared with wheat esterases in esterases, their viability and their inhibition by pesticides are similar. Fly esterases have a maximum inhibition of 71% when p H is 6.5, and plant esterases are 6. 0 times The degree of system reached 73%. The minimum detection limit of phytoesterase inhibition method for monocrotophos, dimethoate and phoxim was 0.03～0. 40mg/kg, and the animal esterase inhibition method was applied to the corresponding pesticide. The minimum detection limit is 0.04～0. 50 mg/kg. It can be seen that both plant esterase and animal esterase can be used for rapid determination of organophosphorus pesticide residues in vegetables. Compared with animal esterases, plant esterase enzymes The indicators of liveness, inhibition, etc. are not inferior, and they have the advantages of convenient source and low cost.
2. 3 animal cholinesterase method
Since the discovery in 1951 that organophosphorus pesticides also inhibited cholinesterase in vitro, the vast majority of studies have been based on the principle of animal cholinesterase. At present, there are mainly methods for the detection of organophosphorus by cholinesterase method. Enzyme tablet method, enzyme inhibition spectrophotometry and cholinesterase biosensor method.
2. 3. 1 enzyme plate method
The enzymatic tablet method applies the cholinesterase and chromogenic substrate solution of sensitive organisms to a filter paper or similar carrier material after being solidified, and the matrix is ​​rapidly decomposed under the action of cholinesterase to generate choline (blue ) and acetic acid. Organophosphorus and carbamate pesticides have an inhibitory effect on cholinesterase, which changes the process of catalysis, hydrolysis and discoloration. If cholinesterase is inhibited, the matrix cannot be hydrolyzed and does not change color. Contains organic phosphorus and carbamate pesticides (positive). Currently used rapid measurement box, rapid card method are all belong to the enzyme tablet method. In recent years, domestic common curing with cholinesterase and nonylphenol acetate The rapid test card method for the on-site supervision and detection of some pesticide residues in Chinese vegetables established by the test paper of ester reagents has a detection limit of 0.3-3. 5 mg/kg and a detection time of 15 min. , Fast speed, low test cost. Some scholars also use the acetylcholinesterase in horse serum and substrate acetylcholine action, according to the pH value before and after the reaction and the color change of the indicator, the established one can be used for qualitative and Semi-quantitative analysis Rapid detection of enzyme tablets is effective in the practical detection of pesticide residues in cucumbers. In foreign countries, Hwa2Young No and Young Ah Kim used test paper coated with acetylcholinesterase to detect the content of pesticide residues in samples. The experiment showed that Hybond N was used. The enzyme carrier and phenylacetate were the most suitable substrates. The detection limits of organophosphorus and carbamate pesticides were 10 -6 to 102 μg/mL and 10 -6 to 100 μg/mL, respectively. It has also been found that the test paper method is more sensitive to oxidized paraoxon compared with paraoxon.
2.3.2 enzyme inhibition spectrophotometry
Organophosphorus pesticides have inhibitory effects on acetylcholinesterase activity. The higher the concentration, the higher the inhibition rate. The absorbance was measured by spectrophotometer with time, and compared with the blank control, the inhibition rate of the enzyme was determined to determine whether the sample was The existence of organophosphorus or carbamate pesticides. This method requires simple equipment, high sensitivity, simple pretreatment, and short detection time. It can detect pesticides containing vegetables in time. It has been reported that chicken brain is used as the enzyme source extraction. Acetylcholinesterase, using enzyme inhibition spectrophotometric method to detect the residues of organophosphorus pesticides (parathion, phoxim, omethoate) in Chinese cabbage, can make the recoveries of the three pesticides reach 97% and 101% respectively. 99%. IngridWalt et al. extracted cutinases from Fisi sp. using enzyme inhibition spectrophotometry and 96-well plate method to simultaneously detect the content of organophosphorus and carbamate insecticides in multiple samples. This method is for chlorpyrifos and The detection limits of oxyphosphorus were 2.6 mg/L and 0.04 mg/L, respectively.
2.3.3 Cholinesterase Biosensor
The cholinesterase biosensor method is a research hotspot in the field of pesticide residue rapid measurement technology. It has great advantages in terms of research method diversity, sensitivity, accuracy, and response time.
(1) Electrochemical type: The electrochemical biosensor is based on the reaction of cholinesterase catalyzed hydrolysis of the substrate to form an electroactive substance. Usually electrochemical oxidization is used to directly determine the thiocholine generated or to use choline oxidation. Enzymatically oxidized choline, further electrochemically measure the O2 consumed in this catalytic oxidation process or the generated H2O2. Andreescu et al. use cholinesterase extracted from Drosophila as the enzyme source, based on phenylacetate ester. Electrodes, Ag/Al reference electrodes, developed a screen-printed electrochemical sensor with detection limits for paraoxon and chlorpyrifos of 5. 2 × 10 - 3 mg/L and 0. 56 × 10 - 3 mg, respectively. / L.
(2) Photochemical type: The basis for the construction of photochemical biosensors is the reaction of cholinesterase catalyzed hydrolysis of substrates to form photoactive substances. During the enzymatic hydrolysis of the substrate acetylcholine, the resulting acetic acid causes the decrease of the solution pH, which can cause p The color change of the H indicator bromcresol purple. According to this principle, a "sandwich" type biosensor with a three-layer bioactive membrane has been constructed. Cholinesterase is immobilized on the outer layer of hydrophilically modified poly. On the vinylidene fluoride film, the middle layer is a sol-gel layer mixed with bromocresol violet, and the lower layer is a glass sheet. Similarly, the principle of changing the color of the chlorophenol red caused by the p H indicator according to the decrease of the solution p H is also based on the principle of AChE. Chlorophenol red was covalently immobilized on a controlled porous glass sphere, and a flow injection analysis method was established. Pardo2Yissar et al. designed a photocurrent sensing system combining AChE with a chemically modified CdS nanoparticle. In the presence of AChE inhibitors, the catalytic activity of AChE is inhibited, the amount of hydrolyzed mercaptocholine is reduced, and the photocurrent is reduced. The photocurrent sensing system that combines this enzyme with CdS nanoparticle has It can be developed into a general class of biosensor mode.
(3) Piezoelectric: The basic idea of ​​designing a piezoelectric biosensor is to immobilize cholinesterase (ChE) or its inhibitor on the surface of quartz crystal, when the ChE to be tested and its inhibitor and the fixed identifier When the specific binding occurs due to the interaction, it will cause a change in the mass loading of the crystal surface and be detected by the frequency change of the sensor. Abad et al. used the direct adsorption and covalent bonding to fix the AChE on the quartz crystal surface to make a piezoelectric biosensor. The use of organophosphorus to inhibit the reaction of immobilized AChE with indole acetic acid to form insoluble indigo dye, and to determine the change in the oscillation frequency of the piezoelectric crystal. Makower et al. established a competitive affinity analysis method using paraoxon as a recognition element through chelation. Cooperation is fixed on the surface of the gold electrode of the piezoelectric sensor. The surface modified by paraoxon can be combined with BChE. When the insecticide is present in the sample, the amount of BChE bound to the surface of the electrode is reduced. ChE can be cocaine type The combination of compounds, the enzyme catalyzes the rate of hydrolysis constant is very small. Based on this, Knosche et al. fixed the benzoylecgonine, a derivative of cocaine, at pressure. The surface of the transistor and the kinetic characteristics of its binding to AChE were studied. The results showed that benzoyl Agonine can be used as an affinity ligand for biosensing analysis. Nunes et al. made a cholinesterase (ChE). Current-type biosensor for the determination of carbamate pesticides aldicarb, carbaryl, chlorbenzulam and propoxur in cereals, with a linear range of 5 × 10 -5μg/g, detection limit 1 × 10 - 4 to 3. 5μg / g. Holger Schulze et al. applied the AchE2 sensor directly to plant samples with N2 bromosuccinimide as oxidant and iso-octane as extractant. The detection time was At 2 h, the repeat rate was 84%, and the detection limit of paraoxon was 2 μg/kg, reaching the EU food safety limit for pesticide residues.
2.4 Plant Esterase Inhibition
In the method of rapid detection of pesticide residues by enzymatic methods, acetylcholinesterase obtained from the head of sensitive housefly is usually used, but animal esterases often exhibit short retention periods and need to be cryopreserved at low temperatures. The plant esterase is rich in enzymes, extraction and preservation are more Conveniently, only the supernatant obtained by centrifugation with a certain proportion of water or buffer can be used as the crude enzyme solution, and can be stored at -4°C. Furthermore, compared with the animal esterase, the measurement of the plant esterase inhibition method Accuracy and precision are not inferior, can achieve the purpose of rapid detection of pesticide residues.
In 1987, the Ministry of Agriculture of the People's Republic of China developed an enzyme sheet and a color-developing matrix sheet, which allows the rapid determination of organophosphorus pesticides in vegetables and fruits without the use of instruments. The detection limit is 0.5. ～10 mg/ kg. Then, some people used homemade plant enzyme tablets and high-sensitivity chemical detection solution (rapid measurement spirit) to make the sensitivity of detection of organophosphorus pesticide residues reach 0.4~10 mg/L and 0.18~10. Mg/kg, detection time is 5 ~ 10 min, detection cost is 0.2 ~ 0.3 yuan. So far, the plant esterase method for the detection of organophosphorus pesticide residues has begun to attract the attention of researchers, and various aspects of research also Then expand.
According to the sensitivity of organophosphorus pesticides, many researchers directly extract enzymes from flour. However, different wheat enzymes have different sensitivities to pesticides. Even for the same variety of wheat, the origin is different and the method of cultivation is different. Different, different growth period, its sensitivity to pesticides will also have a big difference. Therefore, further research is needed to determine the plant esterase enzyme sources that can be quantitatively analyzed and have interchangeability.
In terms of the coloring solution, the detection of the pesticide residue by the enzyme inhibition method often requires the coloration of the chromogenic matrix liquid composed of the solid blue B salt and naphthyl acetate. However, the coloring solution is very unstable after 40 min at room temperature. The color rendering ability is greatly reduced, and it cannot be used for measurement. It needs to be used nowadays. This brings a lot of inconvenience to the inspection work. For this reason, Dong agent greatly prolongs the effective use period of the color developer and makes it at room temperature. After 3 months of placement, the performance was unchanged, and the detection volume fraction of methamidophos, dichlorvos, parathion and phoxim was 10 - 4 - 10 - 7 . Some scholars switched to other color reagents such as 2 , 62 dichloroacetophenol, first with plant esterases to hydrolyze orange 2,62 dichloroacetophenone, and then spectrophotometrically determine the absorbance of decomposed indole phenol (blue) to calculate the available organic phosphorus content. The determination limits for several commonly used organophosphorus and carbamate pesticides were in the range of 0.001 5 to 0.33 mg/L. Further experiments were conducted to compare two different color reagents (2,62 dichlorine). The color-developing principle and performance of ethyl phthalate ethyl ester and solid blue B salt confirmed the use of 2, 62 dichloroindophenolate ethyl ester. The dual function of the developer and the developer, and the ease of use of the chromogenic substrate solution, the stability, and the convenience of on-site rapid detection, use of 2,62 dichloroindophenol-ethyl ester color reagent than solid blue B-salt To color more superiority.
Further research also found that the relationship between esterase activity of wheat and storage temperature: only 59.6 % of enzyme activity was stored at 20 °C for 5 d, and 57.6 % was stored at 4 °C for 5 d, and the enzyme activity declined rapidly. The enzyme was stored at -196 °C for 8 d with liquid nitrogen, and the enzyme activity was still 97. 5%. It was evident that the enzyme requires lower temperature for preservation. In recent years, researchers mainly used enzyme immobilization technology to solve the problem of difficult storage of enzyme liquid. If the ion exchange resin is used as an immobilized carrier to immobilize the plant esterase, not only the stability of the enzyme can be greatly improved, but also the sensitivity to inhibition of the pesticide is significantly improved compared with the free esterase. The styrene microporous reaction plate was used as an enzyme carrier to immobilize the self-made plant esterase on the surface of the microporous inner wall of the carrier to make a rapid detection plate for pesticides. The sensitivity of detection of organic phosphorus and carbamate pesticides was up to 0.01. ~ 0. 1 mg/kg. It has been recently reported that PVA/SiO2 organic/inorganic hybrid sols are prepared by in situ sol-gel method, and the color developing agent and plant esterase are dispersed in sol to form a kind of hydrophilic 2 Pro-acetone adjustable, good storage film. Fixed on the hybrid membrane Enzyme stability significantly than mentioned in a solution state at room temperature and refrigerated. Thus, the enzyme immobilization methods play an important role in detecting pesticide residues.
3 Conclusion
As people pay attention to pesticide residues and food safety problems in vegetables, only by rapidly updating and improving the rapid detection technology of pesticide residues can meet human health and food trade requirements. The enzyme inhibition method is relatively effective in detecting organophosphorus pesticide residues. In the traditional spectrum method, the chromatographic method has advantages in simplicity, rapidity, and detection cost. The accuracy and sensitivity of the cholinesterase-based enzyme sheet method, spectrophotometry, and biosensor method are still good. One of the research hotspots in recent years --- the biosensor method, China still stays in the research and development stage, and it should focus on solving its problems of stability, accuracy, etc. and put the research results into practical use as soon as possible. Compared with animal enzymatic methods, the plant esterase method The cost of extraction, preservation, and detection is superior, and the sensitivity and accuracy are not inferior, so it is popularized rapidly in our country. However, different plant esterases have different inhibition strengths for organophosphorus pesticides, and more effective separation should be explored. The purification method and its structure were studied to clarify the role of the target site, in order to increase the detection sensitivity of plant esterase to a greater extent. Efforts to develop cheap, simple and accurate speed measurement equipment, and strive to excessive residues of vegetables, fruits blocked before eating.