Study On The Impedimetric Immuno-Electrochemical Detection Method For Tetracyclines

Tetracyclines (TCs) are a group of broad-spectrum antibiotics produced by Streptomyces and commonly used for medical purposes as well as animal growth promotion agent. At present more and more serious abusing of TCs resulted serious question of residue tetracycline exceeded the standard which posed a serious threat to human health. Therefore, there is an urgent need to develop a fast and accurate method to detection of them. This thesis through the preparation of specific antibodies to tetracyclines, a new method of immunosensor which based on electrochemical impedance was developed.In my study two different methods was used to derive the tetracycline, and synthesized a completely immune antigen and coating antigen, respectively. Meanwhile, the carrier protein BSA was undergone carboxylation, which reduced self-polymerization of BSA and improved the purity of the conjugation. After identification by HPLC-MASS, gel chromatography, infrared spectra, UV map, the synthesis of complete immune antigen and coating antigen were successful, and the coupling ratio of artificial antigen(TC-aBSA) synthesized with carboxylic BSA was higher than with BSA without carboxylation(TC-BSA), 11:1 and 7:1, respectively, the coupling ratio of coating antigen was 5:1.Two synthesized artifical antigen TC-aBSA and TC-BSA were used to immune two New Zealand rabbits, respectively. Three of them produced antiserum for TC and after purification by the methody of SAS, the titers of the three antiserum was 144000, 256000 and 300000, respectively. Indirect competitive enzyme-linked immunosorbent assay method was developed based on the highest titer serum which found that OD450 value is linear with the concentration of TC between 40～120 ng/kg(R2=0.9937), IC50 value was 97μg/mL, cross reactivities with two similar chemical structure compounds OTC, CTC were 100% and 85%.Using the prepared antibody as sensitive material, through self-assembling of L-Cys and nano-Au particles, antibody was immobilized onto the working gold electrode. The optimum processing conditions were as following: The self-assembling concentration of L-Cys was 0.5%, pH value was 4.5 and time was 2 h; The nano-Au particles diameter used for self-assembling was 20 nm, pH value was 6.0; The concentration of antibody was 100μg/mL, pH value was 7.0, time was 1 h; Detetion time was 15 min; Initial voltage was 0.2 V. The immunosensor has a good linear relationship with the concentration of TC between 5～85μg/kg(R2=0.9908), LOD was 1μg/kg, recovering rate from chicken was 79.37%; The working electrodes was stable within one month.