Design And Applications Of Visualized Fluorescence Assays For The Detection Of Organophosphate Pesticides Based On Gold Nanoclusters-anchored MnO₂ Nanosheets

Nowadays,the global demand for food continues to increase,resulting in the large use of pesticides.Among them,organophosphorus pesticide is one of the most widely used pesticides because of its high efficacy,variety and wide use.However,organophosphorus pesticides have the disadvantages of long residual period and high toxicity to humans and animals in the use process.Organophosphorus pesticides residues in water and agricultural products can enter the human body through the food chain,inhibit the activity of cholinesterase,and then cause serious harm to the nervous system.Therefore,effective monitoring of organophosphorus pesticides in food is very important.The traditional detection methods of organophosphorus pesticides mainly include gas chromatography（GC）,high performance liquid chromatography（HPLC）,gas chromatography-mass spectrometry（GC-MS）,liquid chromatography-mass spectrometry（LC-MS）,etc.These methods have the advantages of high accuracy,high precision and low detection limit.However,it is difficult to achieve large-scale detection in a short time due to the limitations of high requirements for pretreatment,high requirements for operators and long time consuming.Fluorescence analysis method is simple,convenient,rapid and sensitive,which can solve the shortcomings of traditional detection methods.However,there are some problems in the construction of fluorescent probes,such as carboxyl fluorescein,rhodamine derivatives and fluorescent nanomaterials（Carbon Dots）.In order to avoid the above problems,two kinds of multi signal output fluorescence detection methods（fluorometric-colorimetric assay and ratiometric fluorometric assay）were constructed by using AuNCs-MnO₂ NSs composite materials with simple synthesis and excellent luminescent properties,combined with the specific inhibitory effect of organophosphorus pesticides（methyl-paraoxon,chlorpyrifos）on acetylcholinesterase（AChE）.Through the mutual verification of multiple signals,the sensitive and efficient visual detection of organophosphorus pesticides can be realized,which provides new analytical methods and technical means for the detection of pesticide residues in food.The main research contents include:（1）Based on the efficient quenching effect of MnO₂ nanosheets（MnO₂ NSs）on gold nanoclusters（AuNCs）and the inhibition effect of methyl-paraoxon（MP）on AChE,the visualization and highly sensitive detection of MP were realized by fluorescence colorimetric analysis.In the absence of MP in the system,thiocholine（TCh）was synthesized from Acetylthiocholine（ATch）under the catalysis of AChE,TCh can reduce MnO₂ NSs to Mn²⁺,which makes it lose the ability of fluorescence quenching.The fluorescence of AuNCs was recovered,the fluorescence intensity of the system increased,and the solution was colorless.In the presence of MP,MP could inhibit the activity of AChE,the formation of TCh and the decomposition of MnO₂NSs.The fluorescence of AuNCs was quenched again,the fluorescence intensity of the system decreased,and the solution appeared light yellow.According to the change of fluorescence intensity and color,the visible and sensitive detection of MP was realized.Under the optimal conditions,the linear range of MP was 0.005-200 ng m L^-1,and the detection limit was 0.0031 ng m L^-1.The method has been successfully applied to the determination of MP in real samples（tap water,Songhua River water and orange juice）.Through the color change of the system,the naked eye of MP was qualitatively tested.At the same time,the hydrogel platform was constructed to provide a new method and reliable basis for the development of portable reagent box.（2）Based on the fluorescence quenching of MnO₂ NSs and the oxidation of o-phenylenediamine（OPD）catalyzed by oxidase like activity,A fluorescence ratio probe was constructed with AuNCs and 3-diaminophenazine(the oxidized product of OPD,OPD_OX),combined with the inhibition between AChE and chlorpyrifos（CP）,a ratio fluorescence method for the detection of CP was developed.In the absence of CP,AChE catalyzes ATch to produce TCh,which reduces MnO₂ NSs to Mn²⁺.The fluorescence quenching ability of MnO₂ NSs was greatly reduced and the activity of oxidase like enzymes was greatly reduced.In the system,the fluorescence of AuNCs is increased at 645 nm,and the fluorescence of OPD_OX at 550 nm is lowered.In the presence of CP,the activity of AChE was inhibited and the formation of TCh was prevented.The quenching ability and oxidase like activity of MnO₂ NSs were restored.The fluorescence of AuNCs was quenched again.The amount of OPD catalytically oxidized to OPD_OX increased.The fluorescence decreased at 645 nm and increased at550 nm.The fluorescence ratio(F₅₅₀/F₆₄₅)of the system has a good linear relationship with the logarithm of CP concentration in the range of 0.005-0.25μg m L^-1,and the minimum detection limit for CP is 0.27 ng m L^-1.The naked eye of CP was qualitatively tested with the help of a handheld UV lamp.The method has been successfully applied to the determination of CP in real samples（tap water and Songhua River water）.This method can effectively reduce the influence of environmental changes,matrix light scattering,instrument error and other interference on fluorescence detection by using ratio fluorescence probe,and the detection results are more accurate and reliable.