Study On Near-infrared Fluorescence Detection Probe For Duck Acetylcholinesterase

Small molecule fluorescent probes have been widely used in many fields due to their advantages of high sensitivity,good selectivity,and high-throughput rapid screening.Applications refer to detection of biological enzyme activity,heavy metal ions,and small molecule compounds.Especially in recent years,near-infrared fluorescent probes have shown excellent performances in the imaging of biological tissue cells,such as low interference of fluorescent matrix,low biological toxicity,and strong tissue penetrability,and have attracted more and more attention.At present,the application of fluorescent probes in the detection of pesticide residues has also received widespread attention.Most organic phosphorus and carbamate pesticides can inhibit the activity of acetylcholinesterase(AChE)in animals.Therefore,from the perspective of fluorescent probes,we designed and synthesized fluorescent probes that can detect AChE,and uses it to quantitatively detect the AChE activity after pesticide inhibition based on the principles of enzyme inhibition,thereby indirectly detecting the content of pesticides.In the second chapter of the dissertation,a series of fluorescent probes with seminaphthorhodafluor(SNARF)as matrix were designed and synthesized.This type of probes introduce a naphthalene ring at the fluorescein end of the conventional SNARF fluorescent nucleus to extend the conjugation system of the probe and shift the emission wavelength to the near-infrared region(λem=650 nm).The ester bond is the targeting hydrolysis site of AChE,thus introducing an ester bond containing different substituent at the end of the molecular naphthalene ring.Among of these probes,DOS-1,which is the most sensitive probe for the reaction with duck plasma AChE directly,was selected.The probe is an"off-on"type fluorescent probe,and the activity of cholinesterase is characterized by detecting the change of fluorescence intensity before and after reaction with AChE by a microplate reader.In the third chapter,duck plasma AChE was used as the target enzyme.We explored the optimum reaction time and other conditions to detect the activity of AChE.Finally,the basic conditions for the reaction were determined:37 ~oC,PBS buffer p H 7.4,reaction time 15 min;probe final concentration was 10μM.When the enzyme concentration is less than 25μg/m L,there is a good linear relationship between enzyme concentration and fluorescence intensity(R~2=0.9911).The minimum detection limit is 0.32μg/m L.Two organophosphorus pesticides,methamidophos and dichlorvos;two carbamate pesticides,methombite and carbofuran,were selected in the thesis.There is a good linear relationship between pesticide concentration and inhibition rate.The limit of detection is 30μg/L,0.2μg/L,20μg/L,and 50μg/L,respectively,which is very sensitive.These pesticides were sprayed on five kinds of vegetables,respectively.Eventually,the recoveries were basically in the range of 80%-120%,which met the requirements of national standards.The experiment finally proved that this method can achieve rapid and convenient quantitative detection of common pesticides.