Electrochemical Immunosensor For Determination Of Penicillin Residues In Milk

Penicillin is in beta-lactam family. It has a broad spectrum of antibacterial activity and commonly used for prophylactic or therapeutic purpose in veterinary medicine. The presence of penicillin residues in food of animal origin (milk, meat) has a serious influence on health of the public and trade of imports and exports. Electrochemical biosensors have been the most burgeoning and one of the most flourish research realms in the electrochemistry and electroanalytic chemistry. They have been widely applied in many cases such as life science, environmental science, analytic science, and material science. Because of the merits of speediness, high sensitivity, specificity and economy, electrochemical biosensors could be an alternative to commonly used assays. In this paper, Penicillinase and Penicillin antibody are immobilized by different methods on glass carbon electrodes to construct enzyme biosensors and immunobisensors. At last penicillin is detected in phosphatic buffer solution (PBS) and milk with the current as responsive signals.In detail, the thesis includes the following parts:Part one, methylene blue is electrochemical polymerized on glass carbon electrode and the polymerized membranes are studied by cyclic voltammertry at different pH values. And the changing of activation energy from various pH values on electrode modified with MB polymer film is investigated. The penicillinase electrochemical biosensor is made through penicillinase be absorbed in poly (methylene blue) membranes. Chronoamperometry and Cyclic Voltammertry methods are used to detect penicillin residues in PBS and in milk. When working potential is -0.4V, the determination limit for using Chronoamperometry is 0.05μg/mL in PBS and 0.2μg/mL in milk. When working potential is -0.6-0.6V for using cyclic voltammertry is 0.1μg/mL in PBS and 0.4μg/mL in milk. Using this method, the determination limit is lower than potentiometer electrochemical biosensors and immobilized method is simpler than glutaraldehyde-bovine serum albumin. The proposed method provides a new promising way for the penicillin residues detection.Part two, Horseradish peroxidase and antibody labeled horseradish peroxidase immobilized on the surface of nano titanium dioxide modifies glassy carbon electrode. The study indicates that HRP on nanotuble TiO₂/GC electrode can generate more effective and stable direct electron transfer reaction than nanoparticles TiO₂/GC electrode, meanwhile HRP maintains the bioactivity to H₂O₂. And the research of antibody labeled HRP provides a selected signal for antibody-antigen immunoassay.Part three, a novel immunosensor for the determination of penicillin is made by immobilization of horseradish peroxidase (HRP) labeled penicillin antibody on nanotuble TiO₂ glass carbon electrodes. The incubation of the immunosensor in 0.1 mol/L PBS (pH 7. 0) containing penicillin antigen was attributed to the formation of antigen-antibody complex, which blocked the electron transfer of HRP toward the electrode and resulted in significant decrease of peak current. Under optima conditions, the decrease of the current is proportional to penicillin concentration ranging from 0.004 to 0.1μg/mL and 0.2 to 1.0μg/mL; the determination limit is 0.004μg/mL. The penicillin immunosensor shows a good accuracy and an acceptable precision and fabrication reproducibility. The proposed method provides a new promising way for the penicillin residues detection.