Study On Rapid Detection Method Of Pesticide Residues Based On Plant Esterase And Fluorescence Analysis

Organophosphorus and carbamate pesticides are widely used in the production of agricultural products,which are important grasps for controlling pests and diseases and ensuring the safety of agricultural products.However,some pesticides have been banned or restricted due to their high toxicity.Over the years,the agricultural products quality and safety incidents caused by the illegal and excessive use of organophosphorus and carbamate pesticides by producers has highest proportion.Rapid detection is an important scientific and technological support to find problems in the first place and achieve effective supervision.Enzyme inhibition method has become the most official rapid detection method for organophosphorus and carbamate pesticide in the past two decades because of its low cost,convenience and fast speed.However,the restriction of key enzymes employed in this method and complex substrates can easily lead to the occurrence of a high proportion of false positive and false negative test results,which cannot be used for emergency supervision and daily support in practical work.Therefore,it is urgent to innovate the traditional enzyme inhibition method,and it is also a current major national demand.Organophosphorus and carbamate pesticides are the important guarantees for increasing the output of agricultural products because of their various types and wide range of control.At the same time,the unreasonable use of pesticides by farmers has brought huge safety risks to the quality and safety of agricultural products.Enzyme inhibition method is a rapid detection method that is currently the most widely used and is listed as the official detection standard for organophosphorus and urethane pesticides in many countries and regions.Due to the reason of enzyme used in detection,false positive and false negative results are likely to occur.Therefore,it is urgent to improve the traditional enzyme inhibition method.Based on the above background,this paper takes high-activity plant esterase as the research target,and screens out red kidney bean esterase from plant seeds.After separation and purification,red kidney bean esterase is prepared and its enzymatic properties are studied.Based on this,the traditional visible spectrophotometry method was replaced by fluorescence analysis method,and a rapid detection method for organophosphorus and carbamate pesticide residues based on red kidney bean esterase and fluorescence analysis was established.The main research contents and results are as follows:1.By comparing the crude enzyme activity,specific enzyme activity,and pesticide sensitivity of various plant-derived plant esterases,the optimal plant source was red kidney beans.Further separation and purification were performed by ion exchange chromatography.SDS-PAGE electrophoresis showed that the target esterase was purified well and its molecular weight was 100 kD.2.The enzymatic properties of red kidney bean esterase were characterized,and the optimum pH of red kidney bean esterase was found to be 7.5,and the enzyme activity remained stable between pH 6.0?8.0;the optimal temperature was 35?and the enzyme activity remained stable between 25??35?.The tolerance of red kidney bean esterase to different organic solvents was quite different.The esterase has poor tolerance to acetonitrile,but similar tolerance to ethanol and methanol,and low-concentration methanol shows promotional effect on enzyme activity.The effect of different metal ions on red kidney bean esterase is also different.Cu²⁺has a significant inhibitory effect on the esterase,and this inhibitory effect can be relieved to a certain extent by chelating agents EDTA.3.By studying the hydrolysis of red kidney bean esterase on several common fluorescent substrates MUA,MUB,FDA,and indole acetate,the substrates'own stability and kinetic parameters,indole acetate was finally selected as the best substrate.A rapid test method for the detection of pesticide residues using red kidney bean esterase as the enzyme source and indole acetate as the substrate was established,and the reaction system was optimized.Under the best detection conditions,the selectivity of various organophosphorus pesticides such as dichlorvos,trichlorfon and profenofos,was tested.The results showed that the detection method established in this study had good selectivity to 8 kinds of organophosphorus and carbamate pesticides,such as dichlorvos and trichlorfon,whose IC₅₀ were lower than the relevant national standards.Moreover,the practical application of the method was studied based on Chinese cabbage.The results showed that the method has a certain ability to resist matrix interference and can meet the needs of practical detection.4.Based on the good sensitivity of plant esterase,acetylcholinesterase and microbial esterase to organophosphorus and carbamate pesticides,a ternary OR logic gate was constructed.Taking whether the inhibition rate of pesticides to different enzymes is greater than 50%as the input signal,different output signals are obtained to realize pesticides detection.The detection range of pesticide residues in samples is increased from 6?8 to 16,and the reliability of the results is greatly improved.Meanwhile,a binary INHIBIT functional enzyme logic gate was constructed using acetylcholinesterase and peroxo mimetic enzyme PtNPs.Signal amplification was achieved through the secondary catalysis of the peroxo mimic enzyme PtNPs,which improved the sensitivity of the detection method.The detection limits of dichlorvos and carbofuran were as low as 2.3?g/L and 1.4?g/L.