Construction Of Fluorescence Immunoassay Based On AuNCs-MnO₂ NFs And Application In The Highly Sensitive Detection Of Fenitrothion

Fenitrothion（FNT）is a kind of organophosphorus pesticides that protects crops by controlling the damaging behavior of insects.It is widely used because of its low cost and good performance,but improper use can lead to contamination of water,soil and crops,which in turn increases the possibility of entering humans through the food chain and endangers human health.Fenitrothion can cause neuromuscular system toxicity by inhibiting acetylcholinesterase activity in the human body and even affect male reproductive health.Therefore,based on human health and public health safety considerations,the development of a rapid detection method for fenitrothion is of great practical importance.At present,the methods applied to detect fenitrothion mainly include high performance liquid chromatography,gas chromatography-mass spectrometry,enzyme-linked immunosorbent assay,electrochemical sensing,fluorescence sensing,etc.Although all of the above methods have their own advantages,they also have their own defects in realizing the rapid detection of crops,food and other complex matrices.Such as relying on large instruments and high-level operators,high cost,complicated pre-treatment,and poor anti-interference ability.To remedy these drawbacks,we simply synthesized the gold nanoclusters anchored manganese dioxide nanoflakes（Au NCs-Mn O₂ NFs）composite with good fluorescence performance in our paper,combining with alkaline phosphatase-labeled nanobody to construct a sensitive and anti-interference fluorescence immunoassay.The assay was successfully applied to the detection of fenitrothion in different samples by competitive immune recognition of nanobody against FNT linked-alkaline phosphatase（Nb-FNT-ALP）.Besides,the residues and degradation of fenitrothion in Chinese cabbage were also investigated by using this method,which provides new ideas to achieve rapid detection of organophosphorus pesticides in complex matrices.The main studies in this paper are as follows:1.Establishment of a fluorescence signal response platform based on gold nanoclusters-manganese dioxide nanoflakes（Au NCs-Mn O₂ NFs）.The mechanism of gold nanoclusters（Au NCs）anchoring manganese dioxide nanoflakes（Mn O₂ NFs）leads to Au NCs fluorescence burst.ALP can catalyze the hydrolysis of L-ascorbic acid-2-phosphate（AAP）to form Ascorbic acid（AA）,and the strong reducibility of AA leads to the formation of Mn O₂ NFs.The strong reducing property of AA leads to the decomposition of Mn O₂ NFs,thus restoring the burst fluorescence of Au NCs.Under the optimal conditions,the linear range of the fluorescence signal response platform constructed based on Au NCs-Mn O₂ NFs was 0.001-1 U/L（R²=0.9942）for the detection of ALP,indicating that the fluorescence signal response platform was successfully constructed.Meanwhile,the detection platform has strong anti-interference ability against common interfering substances in biological samples,and can be used as a stable fluorescence signal response platform.2.Construction of a fluorescence immunoassay for the highly sensitive detection of fenitrothion.A fluorescent immunoassay was established for the detection of fenitrothion using the above constructed fluorescent signal response platform in combination with the Nb-FNT-ALP immune recognition system.In the immunoassay system,the antigen was encapsulated on a 96-well plate.The Nb-FNT-ALP was successfully bound to the encapsulated antigen by the competitive immune reaction of fenitrothion and the encapsulated antigen against Nb-FNT-ALP.Then ALP was used to trigger the fluorescence signal response,and the quantitative detection of the target was achieved by the change of fluorescence signal.Under the optimal experimental conditions,the detection limit（IC10）of fenitrothion was 5.78 pg/m L,which was about 56 times more sensitive than the conventional colorimetric immunoassay under the same experimental conditions.The method was successfully applied to the detection of fenitrothion in seven actual samples of tap water,river water,apple,cabbage,lettuce,rice and tomato with the recoveries of 90.49-107.21%and the relative standard deviations（RSDs）≤7.45%.Meanwhile,the method achieved the residue and degradation monitoring of fenitrothion in leaves and roots of cabbage.The method exhibited high sensitivity,strong anti-interference ability and good reproducibility in the application,which provided a new solution for the construction of the detection method of organophosphorus pesticides in complex matrices.