Application Of Cholinesterase Fluorescent Probe In Combined Toxicity Detection Of Pesticides

The mixed use of multiple pesticides has been widely used in agricultural production due to its advantages of high efficiency,economy and convenience,which also increases the coexistence of multiple pesticide residues and pollutants in fresh food.At present,the research on the harm of a single pesticide to human body has been in-depth,but the combined toxicity effect of pesticides remains unclear,which may induce synergistic toxicity and cause potential harm to human health.Therefore,exploring the combined toxicity of multiple mixed pesticides and building a pesticide mixed pollution screening technology are of great significance to ensure public health and safety.Fluorescence detection technology is widely used in the field of biological enzyme activity detection due to its advantages of high sensitivity,strong selectivity,convenience and quickness.Cholinesterase is an important hydrolase in human body,and it is also the target of organophosphorus and carbamate pesticides.In this study,a fluorescent probe for detecting cholinesterase activity was synthesized,and an efficient and convenient screening method for combined toxicity of mixed pesticides was established based on the principle of enzyme inhibition,which provided scientific basis for the evaluation of multi-pesticide combined effect and risk assessment.In second chapter,a fluorescent probe FPO for detection acetylcholinesterase（ACh E）was synthesized with 3-O-Methylfluorescein as fluorophore.The product after the probe decomposed by the enzyme was excited by a 455 nm light source,and a strong emission fluorescence was detected at 510-560 nm.Its kinetic parameters were V_max=1.27mmol/min/mg,K_m=11.49μM,indicating that ACh E had good catalytic activity towards the probe FPO.A pesticide combined toxicity detection method was established based on the principle of enzyme inhibition,and the combined inhibitory effects of unit,binary and ternary mixed pesticides of methamidophos,dimethoate,omethoate,chlorpyrifos,chlorpyrifos-methyl,carbofuran and m-tolyl methylcarbamate on ACh E activity were determined,it was found that dimethoate or m-tolyl methylcarbamate could produce synergistic toxic effect when mixed with other pesticides.In third chapter,a fluorescent probe FPN with specific selectivity for detection of butylcholinesterase（BCh E）was synthesized by using 3-O-Methylfluorescein as fluorophore,and introducing N,N-dimethyl group through ester bond as recognition site.Being metabolized by BCh E,the probe had a maximum emission wavelength at 516 nm under the excitation of440 nm excitation light,and the fluorescence intensity was enhanced by 11 times.In the range of p H=7～9,the fluorescence intensity was not affected by p H change,and had good anti-interference ability.Enzymatic reaction exhibited Hill kinetics,with kinetic parameters:V_max=0.17μmol/min/mg,K_m=1.58μM,indicating that the probe FPN had a good affinity for BCh E.Probe FPN meets the demand of detecting BCh E activity in complex systems,and is expected to be applied to the study of the combined toxicity of pesticides.