Studies On Enzyme-inhibited And Copper-mediated Fluorescence Analysis For The Detection Of Organophosphorus Pesticides

Organophosphorus pesticide is a highly toxic neurotoxin,which can damage human function at low concentrations.It is of great importance to develop simple and sensitive detection methods for organophosphorus pesticides to safeguard human health and safety.Organophosphorus pesticides can specifically inhibit the catalytic activity of acetylcholinesterase and hinder the hydrolysis of acetythiocholine.To achieve the signal conversion,the copper-mediated analysis methods are relay on the combination of Cu²⁺and special functional group such as the thiol group,and the strong oxidation and catalysis of Cu²⁺.Advantages of fast detection speed,simple operation and high sensitivity,the copper-mediated analysis has been one of the hotspots in the analysis field in recent years.Fluorescence analysis is high sensitivity,simplicity and rapidity,which has greatly used in analytical chemistry,environmental science and other fields.In this paper,three copper-mediated fluorescence analysis methods are established for the quantitative detection of organophosphorus pesticides based on the inhibition of the activity of acetylcholinesterase.The main contents of these works can be concluded as follows:Part one:A highly sensitive fluorescence assay was developed for the detection of organophosphorus pesticides based on the inhibition of acetylcholinesterase activity and copper-catalyzed click chemical reaction.Two hybridized DNA probes can be ligated by copper ions catalyzed click chemical reaction,inducing a fluorescence quenching during the strand displacement reaction.Acetylcholinesterase can catalyze the hydrolysis of acetythiocholine to form thiocholine which contains a thiol group.Thiocholine will react with copper ions,blocking the click reaction and a high fluorescence signal is observed.But in the presence of organophosphorus pesticides,the activity of acetylcholinesterase is inhibited,releasing of a high concentration of copper ions that catalyze the click chemistry reaction,and resulting in decreased fluorescence signals.Take the advantage of copper-mediated signal amplification effect,the sensitivity was improved.The detection limit of organophosphorus pesticides is 1.4 ng/mL for omethoate.This assay has also been applied to detect organophosphorus pesticides in river water samples with satisfactory results,which demonstrates that the method has great prospects for practical application in environmental protection and food safety field.Part two:A novel fluorescence assay for rapid detection of organophosphorus pesticides was developed based on the inhibition of acetylcholinesterase activity and DNA template copper nanoclusters.In the absence of target pesticides,acetylcholinesterase catalyzes the hydrolysis reaction of acetythiocholine to generate thiocholine,which has a higher affinity to copper ions.Copper nanoclusters are failed to form in the lack of copper ions,so the fluorescence signal is weak.However,in the presence of organophosphorus pesticides,the activity of the acetylcholinesterase is inhibited.High concentration of Cu²⁺is reduced to Cu⁺,then copper nanoclusters are formed by DNA template.Therefore,the fluorescence signal is greatly enhanced.The organophosphorus pesticides can be quantitatively detected by the change of fluorescence intensity.The detection limit is 0.75?g/mL for omethoate.In addition,the recovery test for organophosphorus pesticides in river water is also studied.The results show that the proposed method has potential for application in the complex environmental samples.Part three:A simple and sensitive fluorescence assay was developed for detecting organophosphorus pesticides based on the inhibition of acetylcholinesterase activity and the oxidation reaction of Cu²⁺and o-phenylenediamine.In the absence of organophosphorus pesticides,thiocholine which is the hydrolysate of acetythiocholine combines with free Cu²⁺.At this time,the fluorescence signal is weak because the o-phenylenediamine is failed to be oxidized.However,in the presence of organophosphorus pesticides,thiocholine is not produced.Free Cu²⁺will oxidize o-phenylenediamine to generate 2,3-diaminophenazine with orange fluorescence.Therefore,the fluorescence signal increased markedly.According to the change of fluorescence intensity,this method can be used for the sensitive determination of organophosphorus pesticides with high specificity.The detection limit is 8 ng/mL for dichlorvos.In addition,it has potential application prospect for the detection of organophosphorus pesticide residues in real samples.