Study Of Immunochromatographic Strip For Rapid Detection Furazolidone In Milk

Furazolidone,a kind of nitrofuran antibacterial antibiotics,has been used to prevent and treat gastrointestinal,bacterial and protozoal infections in breeding industry,particularly in pigs and poultry.However,the drug can increase animal vigour and may act as a growth promoter.Some research has also revealed that its residue has potential mutagenic,carcinogenic,and teratogenic effects on human health and could induce cancer.Such evidences have been taken as the basis to forbid the use of the nitrofuran antibiotic in food animal production in the European Union,USA,and China.However,driven by profit,nitrofuran drugs are still often used illegally,resulting in food safety incidents.Furazolidone can be rapidly metabolized in vivo and its parental drug residues are hardly detected.However,its metabolite of 3-amino-2-oxazolidinone?AOZ?is stable,can maintain for a considerable period of time,and is commonly used to detect the presence of furazolidone.In this work,to monitor the residues of furazolidone in milk,the CPAOZ,derivative of AOZ,was used as the detection target.Nanogold-based and magnetic nanoparticles-based signal amplified lateral flow test strip were established for rapid detection of furazolidone in milk.The following results were obtained in this study:1.The ascites of anti-CPAOZ antibody was prepared by induction method in mice and then purified and identified.Results showed that when the OD_450nm50nm value is 1.0,titer of the monoclonal antibody?MAb?was 64000.Sensitivity(expressed by IC₅₀)of the MAb was 2 ng m L^-1.The MAb provided a core material for the development of immunochromatographic strips.2.A nanogold-based immunochromatographic assay for the rapid detection of CPAOZ was developed.The gold nanoparticles?GNPs?were prepared according to the classical method by reducing HAuCl₄·3H₂O through trisodium citrate.After optimization,the optimum coating solution for the antigen on test line,the best blocking solutions for conjugation pad and sample pad were determined.Under the optimal conditions,the detection limit for CPAOZ standard was 1 ng mL^-1?0.44 ng mL^-11 for AOZ?.Besides,the test strip had a satisfied specificity with no cross reaction to SEM,AMOZ,AHD,chloramphenicol and penicillin.For the residual monitoring of furazolidone in food sample,the detection limit for CPAOZ in milk was 2 ng mL^-1(0.88 ng mL^-1for AOZ).3.A magnetic nanoparticle-based signal amplified immunochromatographic assay was developed for the rapid detection of CPAOZ.Magnetic nanoparticles?MNPs?with carboxyl groups were synthesized using a one-step hydrothermal synthesis method.The MAb was immobilized onto the MNPs by the classical EDC/NHS method,The amplified signal benefited from high affinity between two probes of MNPs labeled murine MAb?MNPs-MAb?and MNPs labeled goat anti-mouse antibody?MNPs-GAMA?and was achieved by the generation of dual-probe network complex in which more MNPs contained.Assay conditions for the strip test were optimized,under the optimal conditions,the detection limit for CPAOZ standard could be read out as 0.1 ng mL^-1(0.044 ng mL^-1for AOZ).The sensitivity of the signal amplified strip assay was 5-fold higher than that of the traditional MNP-based lateral flow strip test and 10-fold higher than that of the traditional nanogold-based lateral flow strip test.The actual detection limit for CPAOZ was 0.25 ng mL^-1(0.11 ng mL^-1for AOZ)in raw whole milk,the detection limits were all 0.1 ng mL^-1?0.044 ng mL^-11 for AOZ?in pasteurized skimmed milk and skimmed milk powder.