Development Of A Gold Nanoparticle-based Signal Amplification ELISA And Its Application In The Detection Of Enrofloxacin In Food Sample

Enzyme linked immunosorbent assay(ELISA)is a kind of immunological labeling technology which uses enzyme-linked antigen or enzyme-linked antibody to detect the corresponding antibody or antigen.For a long time,improving the analytical sensitivity is the research topic in the field of ELISA,which promotes the development of a series of new strategies and technologies of signal amplification.The application of new functional nanomaterials in ELISA can obtain wider detection range,lower detection limit and higher sensitivity,which has a good application prospect.In this study,an enzyme-linked immunosorbent assay(AuNPs-HRP-IgG)based on signal enhancement of gold nanoparticles was developed by using gold nanoparticles as signal carrier and horseradish peroxidase(HRP)-labeled second antibody as signal probe IC-ELISA)further improved the sensitivity of traditional ELISA,and realized the detection of enrofloxacin in large quantities,fast and highly sensitive.The main research contents and results are as follows1.Preparation and characterization of gold nanoparticles.In this study,gold nanoparticles were prepared by sodium citrate reduction method.Gold nanoparticles with different sizes of 18 nm,25 nm,33 nm and 44 nm were prepared by adding different amounts of trisodium citrate.The nanoparticles were characterized by visual inspection,UV-Vis,coalescence and transmission electron microscopy.The results show that the solution of gold nanoparticles is clear,transparent and red in appearance.There is no visible particle precipitation,and the particles are evenly dispersed.There is a single maximum absorption peak in the wavelength range of 400-700 nm,and there is a narrow half peak width.The gold nanoparticles with 25 nm,33 nm and 44 nm have different degrees of aggregation,while the gold nanoparticles with 18 nm have no aggregation.2.Preparation and characterization of AuNPs-HRP-IgG probe.Based on the principle of electrostatic adsorption between gold nanoparticles and HRP-IgG,AuNPs-HRP-IgG probe was synthesized.The results showed that the pH of gold nanoparticles and HRP-IgG was about 8.0,and the dosage of HRP-IgG was 20?g/ml.It was characterized by UV-vis,transmission electron microscopy and ELISA.The results showed that the absorbance of AuNPs combined with HRP-IgG decreased significantly,and the maximum absorption peak was red shifted from 519 nm to 525 nm.The size of the modified gold nanoparticles was uniform and the dispersion was good.It was preliminarily judged that the coupling was successful,and the binding rate of AuNPs-HRP-IgG probe was more than 70%.3.Optimization of working conditions and establishment of standard curve of AuNPs-HRP-IgG IC-ELISA.The original coating concentration,antibody dilution ratio,blocking solution,coating conditions and dilution ratio of AuNPs-HRP-IgG probe were optimized,and the optimal reaction conditions were obtained.The sensitivity(IC50)was 0.24 ng/ml,and the detection limit(IC15)was 5.0×10-4ng/ml.The cross reaction rates of norfloxacin,ofloxacin and ciprofloxacin were less than 0.1%,which indicated that the specificity of the method was good.The recoveries of the commercial ELISA kit and the ELISA kit were 102.66%-102.80%.The gold nanoparticles signal enhanced IC-ELISA established in this study has strong specificity,high sensitivity,accurate and reliable results,which can meet the detection of enrofloxacin in food.The signal amplification system strategy is mainly based on gold nanoparticles as the carrier,carrying multiple secondary antibodies to achieve signal amplification,which provides a good theoretical basis and detection platform for the development of accurate detection technology of other food hazardous substances Platform.