Study On Lateral Flow Immunoassay For Detection Of Furazolidone Based On Nano-signal Probe

Furazolidone(FZD),a broad-spectrum antibiotic of nitrofuran,is widely used as a feed additive for prevention and treatment of gastrointestinal infections caused by Escherichia coli and Salmonella in pigs,poultry and aquatic products.However,FZD has a very short half-life in animals,the furazolidone metabolite 3-amino-2-oxazolidinone(AOZ)can bind to proteins in the body and exist for a long time,thereby causing carcinogenic,teratogenic,mutagenic and other toxic effects.Therefore,the real-time detection of FZD is very important in food samples.Lateral flow immunoassay has been widely used as an on-site real-time detection technology,owing to the advantages of simple operation,rapid detection,portability and low price.However,traditional Au NP-based immunoassay has low sensitivity and stability,which further limits in trace analysis and complex systems.Therefore,in order to detect the residues of AOZ in different food systems,a derivative of AOZ(CPAOZ)as the target,we used three different signal probes(small particle size Co₃O₄NPs,Au-SiO₂ Janus NPs and MnO₂-Au NPs)to construct novel immunoassay,which realize highly sensitive detection of AOZ in food samples.The research content and result of this paper are as follows:1.Construction and performance of LFIA based on Co₃O₄ NPs signal probe:firstly,Co₃O₄ NP with small particle size was prepared by a hydrothermal method.Co₃O₄ NPs were coupled with anti-CPAOZ m Ab by electrostatic adsorption to obtained Co₃O₄-m Ab probe.Owing to the small particle size and bright color,Co₃O₄ NPs can effectively reduce steric hindrance and promote the antigen-antibody reaction.Only a small amount of probes can produce clearly color on the test line;the reduced amount of antibody can also trigger more intense competition between the free analysis and the immobilized antigen for the antibody,thereby improving the sensitivity of detection.Under the optimal conditions,the visual detection limit of the CPAOZ standard solution based on the Co₃O₄ NPs-LFIA is 1.0 ng m L^-1,which is 3-fold more sensitive than the traditional Au NPs-LFIA.The Co₃O₄NPs-LFIA has strong specificity and no cross-reactivity with furazolidone analogs.Finally,the method is successfully applied in the detection of furazolidone residues in honey,chicken,pork and milk powder samples,with the detection limits of 3 ng m L^-1,3 ng m L^-1,3ng m L^-1 and 1 ng m L^-1,respectively.This work opens up a new way for the signal amplification and performance improvement of LFIA.2.Construction and performance of LFIA based on the asymmetric Au-SiO₂ Janus signal probe:firstly,we used 4-mercaptophenylacetic acid(4-MPAA)and polyacrylic acid(PAA)to modify Au NP by ligand competition method.Under the catalysis of ammonia,Au NP was partially encapsulated by hydrolyze of TEOS to synthesis Au-SiO₂ Janus NP.Au-SiO₂ Janus NP was coupled with anti-CPAOZ m Ab to prepare Au-SiO₂ Janus-m Ab signal probe.Owing to asymmetric structure and function of Au-SiO₂ Janus NP,only the Au NP side is coupled with the antibody,and the introduction of silica can increase the stability of the probe.In addition,Au-SiO₂ Janus NP can effectively reduce the uncomplete competition reaction in traditional gold lateral flow immunoassay.Meanwhile,a small amount of antibody can generate a clearly signal,making the competition reaction between antigen and small molecule more intense,which further improve the analysis sensitivity.The constructed immunoassay method have a minimum visual detection limit of 0.8 ng m L^-1 for CPAOZ standard solution,which is 3.75 times lower than that of traditional Au NP-LFIA.Five other structural analogs were used as interferences,the results show that the Au-SiO₂Janus NP-LFIA has a good specificity for CPAOZ.Finally,the test strip successfully detecte CPAOZ in honey,chicken,pork and beef food samples with the visual detection limits of 1ng m L^-1 respectively.This work provides an effective method for the imperfect competition reaction in the competitive immuoassay at low concentrations of residues.3.Construction and performance of colorimetric/photothermal dual-signal LFIA based on MuO₂-Au signal probe:First,MuO₂ nanoflowers were used as templates,and sodium citrate was used to reduce chloroauric acid to form Au NPs on MuO₂ nanoflowers.MuO₂-Au NPs and anti-CPAOZ m Ab were coupled to prepare MuO₂-Au m Ab signal probes.MnO₂-Au has a higher photothermal effect due to a large amount of Au NPs,which contribute to overcome the complexity of antibody coupling.In addition,the dual-signal immunoassay can not only visually detect vio color changes,but also record thermal signal through thermal infrared imagers.Compared with single-signal immunoassay,the colorimetric/photothermal method has more diverse detection methods due to the changes of two signals,which can meet the requirements for accurate and visual detection.The minimum visual detection limit of the constructed immunoassay method for AOZ standard solution is 1 ng m L^-1,and the quantitative detection limit is 0.43 ng m L^-1.In addition,other CPAOZ analogs had no obvious inhibition on the color development of the test strip.The dual-signal immunoassay biosensor is successfully applied to the detection of AOZ contamination in milk powder and shrimp samples.The recovery rate is 80.6%?106%,and the relative standard deviation is less than 0.67%.This work provides a dual-signal detection method to improve the accuracy and diversity of analysis,and effective avoid the existence of false positives or false negatives.