Study On The Detection Of Veterinary Drugs Based On Bio-barcode Fluorescence Immunoassay In Aquatic Products

Nutritious and healthy food is one of the important evaluation criteria for a better life.With the rapid development of our country’s economy,people’s demand for food has also changed from"eat enough"to"eat well".This transformation has led to the vigorous development of the food industry while also putting forward more requirements for food safety.In recent years,food-borne diseases have been highly valued,and the main inducement is the consumption of unsafe food,including food containing residues of agricultural and veterinary drugs,illegal addictive substances,heavy metal pollution,and microbial pollution.Therefore,food safety is the bottom line of the food industry,and detection is an important means to ensure food safety.At present,the main detection methods are instrumental methods,which use the liquid phase and gas phase as the separation system combined with detectors such as chromatography and mass spectrometry to separate and detect the target.These detection methods based on precision instruments have the characteristics of low detection limit and good repeatability,but they need complex operation,long detection time,and tedious sample pre-processing steps,which limit their application in practical detection.Immunoassay is a detection technology based on antibody-antigen specific recognition.At present,it has been used to detect hazard factors in many fields.It is easy to operate,cost-effective,sensitive,and easy to promote.Based on immunoassay,combined with the characteristics of DNA,such as amplification,hybridization,and easy modification,a variety of bio-barcode immunoprobes are constructed,which respectively realized the sensitive detection of malachite green and chloramphenicol as a single target,and also realized the simultaneous detection of four nitrofuran metabolites.The details are as follows:（1）In this study,antibody and thiol-modified DNA strands were used to prepare biological bar code immunoprobes by electrostatic adsorption and Au-S bonding,respectively.By comparing the performance of three methods（low p H method,salt aging method,and freezing method）to prepare immune probes by connecting Au NPs with DNA strands,it is determined that freezing method is superior to salt aging method and low p H method.Based on this,the process of preparing immunoprobes by freezing method was optimized.The main optimization parameters included the particle size of Au NPs,p H,the molar ratio of DNA to Au NPs,and the freezing time.The immunoprobe was prepared under the best conditions and its stability was evaluated.（2）In this study,the oligonucleotide chain（ssDNA）with amplification ability was selected as the barcode.The upstream and downstream primers were designed for amplification,and fluorescence quantitative PCR was introduced for signal amplification.The bio-barcode immunoassay combining fluorescent quantitative PCR was used for the sensitive detection of malachite green.First,suitable upstream and downstream primers were selected and the primer concentration and annealing temperature were optimized.During the preparation of immunoprobe,the amount of antibody and ss DNA was optimized.Under the optimal conditions,an indirect competitive immunoassay system was established.The standard curve was established by the number of cycles（Ct）experienced by PCR fluorescence signal reaching the set threshold and the logarithmic concentration of malachite green.The detection range of malachite green is 0.01-1000μg/L,and the LOD is 1.57×10^-3μg/L.Compared with traditional ELISA,this method has a lower detection limit,and the LOD is increased by nearly 96 times.Finally,the spiked recoveries of MG in actual samples have been confirmed by UPLC-MS/MS,and the results indicating that this method can meet the requirement of actual detection.（3）In this study,G-DNA was used as a template to synthesize green-emitting AgNCs,and a DNA strand that could complement G-DNA was selected as the barcode.The arrangement of AgNCs was adjusted by double-strand complementarity in the presence of barcode,so that the fluorescence of AgNCs changed from green to red.Ratiometric fluorescence was constructed based on this result.First,the preparation conditions of green-emitting AgNCs were optimized,the conditions include reduction time,addition ratio of G-DNA and Ag⁺,and reaction p H.Under the optimal conditions,green-emitting AgNCs were prepared and the feasibility of constructing ratiometric fluorescence was discussed.In the preparation of immunoprobe,the amount of antibody and ss DNA was optimized.Finally,a novel,sensitive and fast fluorescence immunoassay was established for the detection of chloramphenicol in aquatic products by combining bio-barcode signal amplification technology with ratiometric fluorescence.The detection range of chloramphenicol is 0.01-1000μg/L,and the LOD is 0.007μg/L.Compared with traditional ELISA,the LOD of this method is increased by nearly 38 times.The spiked recoveries of CAP in actual samples have been confirmed by UPLC-MS/MS,the results indicating that this method meet the requirement of actual detection.In this study,the ratiometric fluorescence developed by a single fluorescent group can reduce the influence of the environment on the signal and improve the detection accuracy.（4）In this study,G4 structure DNA chain that can enhance ThT fluorescence was selected as barcode.Fluorescent G4/ThT and antibodies were coupled with Au NPs to form immunoprobes,and a fluorescent immunoassay method for sensitive detection of chloramphenicol in aquatic products was established.First,the appropriate DNA strand was selected as the G4 strand,and G4/ThT was synthesized at the optimal ThT concentration.Subsequently,G4/ThT was coupled with Au NP to form a"turn off"fluorescent immunoprobe.In this process,the amount of antibody and G4/ThT was optimized.Under optimal conditions,an indirect competitive immunoassay system was established.DTT was added to dissociate and release G4/ThT,and fluorescence was restored.The quantitative analysis of chloramphenicol is realized by directly measuring the fluorescence in the well.The detection range is 0.02-10μg/L,and the LOD is 0.011μg/L.Compared with traditional ELISA,the LOD is increased by nearly 25 times.By comparing the spiking recovery of chloramphenicol in the actual sample between this method and UPLC-MS/MS,the results indicating that this method has good practical application.Compared with the previous two research methods,the fluorescent signal material was directly modified on ss DNA,which greatly simplifies the signal conversion and output process and improves the detection efficiency.（5）Based on the fluorescent immunoassay strategy constructed in the previous chapter.Four ss DNA modified with Four fluorophores were selected as barcodes,and the barcodes and four nitrofuran metabolite antibodies were conjugated to Au NPs to form multiple immunoprobes.A sensitive fluorescence immunoassay was developed for the detection of four nitrofuran metabolites（AOZ,AMOZ,AHD and SEM）in aquatic products.Four fluorescent DNA chains are selected as barcodes,and the fluorescent barcodes and four nitrofuran metabolite antibodies are coupled with Au NPs to form multiple immunoprobes.A sensitive fluorescence immunoassay was developed for the detection of four nitrofuran metabolites（AOZ,AMOZ,AHD,and SEM）in aquatic products.First,four fluorophore FAM,HEX,ROX,and Cy5 with non-overlapping excitation and emission peaks were screened.Under the optimal conditions,four immunoprobes were prepared and multiple immunoassays based on indirect competition was established.In one detection process,four fluorescence signals from multiple immunoprobes were simultaneously detected to finish simultaneous quantitative detection of four nitrofuran metabolites.The detection range of nitrofuran metabolites is 0.05-28μg/L,and the LOD is 0.01μg/L,0.02μg/L,0.02μg/L,and 0.05μg/L respectively.Compared with traditional ELISA,the lowest LOD of AOZ,AMOZ,AHD,and SEM detected by this method was increased by nearly 3 times,21 times,22times,and 25 times respectively.By comparing the spiked recovery of nitrofuran metabolites in actual samples by this method and UPLC-MS/MS,the results indicating that this method meet the requirement of actual detection.Compared with the single target detection method,this strategy reduces repeated operations and saves time.This strategy has broad application potential in food safety,drug screening,and environmental monitoring,and can effectively,quickly,and accurately detect multiple risk factors.