Studies On The Establishment Of The Detection Of Veterinary Drug Residues By ELISA And The Application Of Nano Functional Probes

Objective The phenomenon of overdosed Veterinary drug is serious in china,and animal tissues of veterinary drug residues on human health caused great threat,C hloramphenicol(C AP),diethylstilbestrol(DES)and C lenbuterol(CL)were widely used in veterinary animal.Therefore,we make the preparation antigen of veterinary drugs and the establishment of traditional enzyme-linked immunosorbent assay(ELISA)detection technology.The formation of complexes between Quantum dots(QDs)and Gold nanoparticles(AU)which will cause the fluorescence of QDs quenched,established a Highly selectivity detection of target.In the presence of the target molecule,the combination between of them is decrease,and hinders the transfer process,recover the fluorescence resonance energy transfer(FRET),we can calculation of the content of the target by measuring the change of fluorescence intensity.Methods Preparation of antigen,by active ester method,using the method of UV scan and gel electrophoresis to proved the successful coupling,Then optimized conditions of ELISA,and calculated the sensitivity and specificity.Two kinds of nano functional probes were prepared by the coupling of fluorescent nanoparticles and metal nanoparticles with biological antibodies.To optimize the reaction conditions,we studied the fluorescence intensity changes when the concentration of the target was added,and then applied it to the detection of insulin..Results The synthesis of coated antigen which used the method of active ester has been proved to be successful by UV scanning and gel electrophoresis,and BSA-DES and BSA-CL have been proved to be successful by UV scanning.The conditions of ELISA method were optimized to choose the optimum conditions.The optimal coating liquid was salt buffer of p H=9 and the concentration was 0.01 M,the best sealing liquid was casein,the closed volume was 120μL and incubated time was 90 min.The optimal concentration of Tetramethylbenzidine(TMB)and urea hydrogen peroxide was 1g/m L and 0.3g/m L.When the concentration of CAP was 0-10000ng/m L,R2 = 0.991,IC50=11.65ng/m L,LOD=0.415ng/m L,when DES and CL was 0-1000ng/m L,R2=0.991,IC50=0.7405ng/m L,LOD=0.348ng/m L and R2=0.9965,IC50=0.9629ng/m L,LOD=0.357ng/m L,and the sensitivity of method was high..The preparation method of fluorescent nanoparticles was determined by the change of fluorescence intensity,the activation process was only added with activator 1-ethyl-3-dimethylaminopropyl carbodiimide(EDC),the concentration was 5 mg/m L,the dilution formula of BSA was 0.5%,Polyethylene glycol(PEG)was 0.3%.Fluorescent probe of Antibodies,human albumin,insulin and DES were prepared,and the four conjugates had biological activity and fluorescence intensity.The changes of fluorescence intensity of probe of Antibodies and probe of insulin were different when adding different concentrations of competitive products.Conclusions In this study,three kinds of typical veterinary drug residues were successfully detected by ELISA,and showed high sensitivity,specificity and stability.In addition,two kinds of nano functional probes were successfully prepared by active ester method.The probes have high selectivity and sensitivity,and are especially suitable for the analysis of trace co mponents in complex matrices and molecular recognition elements.The results showed that the two kinds of nano functional probes were applied to the detection of insulin,and the results were good.