| Quality and safety of agricultural products is a matter of public health and the development of agricultural industry,pesticides meet the needs of agricultural production and quality and promote the development of modern agriculture at the same time,due to the irrational use of pesticides and their own slow degradation and other problems caused by pesticide residues exceed the standard and other safety issues,will cause great damage to the environment,biological and human health.Therefore,the establishment of a simple,efficient,sensitive and accurate method for the detection of organophosphorus pesticide residues is of great importance to meet the demand for pesticide residue detection and environmental monitoring as well as national health in China.In this study,with bio-barcode immunoassay and catalytic hairpin self-assembly signal amplification technology,the quantitative detection of organophosphorus pesticides by indirect competition reaction and catalytic hybridization reaction was developed.The main contents and results of the study are as follows:A uniform particle size and well-dispersed gold nanoparticle and its probes labeled with triazophos antibodies and bio-barcodes were prepared,hairpin structures H1 and H2 and bio-barcode target DNA strands were synthesized,and the feasibility of this experiment was verified.The concentration of antibody protein modified on the gold nanoparticle surface,the bio-barcode immune competitive reaction conditions and the parameters of the catalytic hairpin reaction system were optimized for the rapid quantitative detection of triazophos.The linear range of the method was 0.01-50 ng/m L,and the detection limit(IC10)was 0.0048 ng/m L.Spiked recovery experiments and linearity studies were performed using this method and LC-MS/MS method on five actual samples including water,apple,cabbage,cucumber and rice.The spiked recoveries of the method in the actual samples ranged from81.0 to 104.1%with CV of 5.0-17.4%,and the results of the two detection methods were correlated.It was verified that the immune competitive method has good accuracy and sensitivity and can be applied to the detection in water samples and agricultural products.Based on the single-residue bio-barcode detection of triazophos,a hairpin self-assembly based multi-residue bio-barcode immunoassay was developed for triazophos,parathion and chlorpyrifos.Two sets of colloidal gold probes and hairpin structures were prepared for parathion and chlorpyrifos,and the addition of antibodies to the gold nanoparticle probes was optimized.The specificity of the antigen-antibody and hairpin structures of the three pesticides was tested to demonstrate the feasibility of the experiment in immune competitive and catalytic hairpin reactions for multi-residue detection.The working dilution concentrations of the antigen-antibody in the immune competitive system were also optimized.Under the optimized experimental conditions,the standard curves of the pesticide multi-residue immunoassay method were established with the linear ranges of 0.01-50 ng/m L and the detection limits(IC10)of 0.012 ng/m L,0.0057 ng/m L and 0.0074 ng/m L,respectively.Four actual samples,such as apple,cabbage,cucumber and rice,were selected for spiked recovery experiments,and instrumental confirmation was performed by LC-MS/MS.The spiked recoveries of the four samples measured by this method were between 82.8-110.6%,with CV of 5.5-18.5%.The results of the two detection methods were correlated,which proved the applicability and accuracy of the method for the actual samples.At the same time,it provides new ideas and directions in the analysis of multi-residue trace detection of other small molecules such as pesticides. |
