A Novel Fluorescent Immune Bio-barcode Assay For Detection Of Atrazine

Atrazine（ATZ）has been widely used around the world since its discovery in the mid-twentieth century.The population has increased the demand for food,so the widespread use of herbicides in agriculture is inevitable,but in the process of increasing food production,the use of herbicides has also increased environmental pollution.Atrazine in the soil can eventually contaminate crops and water through natural circulation systems.High concentrations of atrazine can affect the neuroendocrine and reproductive development of aquatic animals and humans.Statistics from several epidemiological studies of drinking water indicate that atrazine poses a carcinogenic risk to humans.In consideration of the potential hazard of atrazine to humans,there is a need to develop a rapid and efficient detection method.For atrazine and other pesticide residue detection,in the laboratory commonly used instruments such as liquid chromatography（LC）etc.,these traditional large instruments have been relatively mature,and China’s food safety testing standards are given in the use of liquid chromatography detection method for atrazine.Although large instruments have many advantages in the detection of atrazine,they demand high sample performance in the detection process,usually require complicated pre-treatment procedures,and samples are tested in a longer time compared to rapid detection methods.These instruments are often costly and require personnel that are trained to perform the assay,which limited the application of rapid analytical methods in actual practice.In recent years,immunoassays have been gradually introduced into the field of biosensor detection,and their main core key consists of antigen-antibody combination,which helped to improve the selectivity of the detection method for complex substrate samples.On the other hand,traditional immunoassay methods have the benefits of high specificity,but their detection sensitivity and detection methods still are not sufficient for the rapid detection of atrazine,and the combination of nanomaterial and signal amplification means is crucial in the development of new immunoassay methods.In this paper,an immunoassay fluorescence detection method based on immunomagnetic beads and a functionalized probe-based competitive immune bio-barcode fluorescence detection method were established as targets for atrazine.（1）Based on immunomagnetic bead enzyme-linked fluorescence assay for atrazine was used.A magnetic nanoprobe modified with Bovine Serum Albumin-atrazine complete antigen（BSA-ATZ complete antigen）was prepared with a small molecule of atrazine competitively bound to an anti-atrazine monoclonal antibody（anti-ATZ m Ab）.The magnetic separation mixture isolates the MMPs/anti-ATZ m Ab complex,and then the labeled horseradish peroxidase on the enzyme-labeled secondary antibody catalyzes the oxidation of Amplex red to produce an intensely fluorescent substance using hydrogen peroxide as the substrate.The detection range of the method was between 2 ng·m L^-1 and 100 ng·m L^-1,and the spiked recoveries were between 96.43%and 102.40%and 94.28-107.42%for the Haihe water samples and tap water samples,respectively.（2）A functionalized gold nanoprobe competitive immune bio-barcode method for atrazine detection was used.A dual functional probe（DFP）modified with both antibodies and DNA barcodes was prepared to recognize the target and act as a signal amplifier.In this method,a cyclodextrin-modified gold nanomaterial was utilized to covalently modify an antibody and a DNA strand modified with a pyrene fluorescent group at the 5’end on the gold nanomaterial in a one-step process using its excellent host-guest interaction.The constructed magnetic nanoprobes can then be competitively bound to functionalized gold nanoprobes prepared by small molecules.After magnetic separation of the MMP/DFP complex,the G-quadruplexes modified in the DFP are folded in the presence of hemin and K⁺to form a stable G-quadruplex/heme chloride DNAzyme,which catalyzes the hydrogen peroxide-mediated oxidation of the fluorescent red dye to produce the strongly fluorescent substance resorufin.The detection range of the method is between 0.01 ng·m L^-1 and 100 ng·m L^-1.The minimum detection limit was 0.0047 ng·m L^-1.In this study,two fluorescent immunoassay methods were established for the detection of ATZ based on bio-barcode assay technology and immunomagnetic separation technology.Compared with simple antibody recognition,the preparation of formed functionalized nanoprobes is beneficial to improve the sensitivity of detection and signal amplification.The above methods,provide a new approach and platform for rapid detection and field monitoring of ATZ.