The Research Of An Electrochemical Immunosensor Based On New Methylene Blue As Mediator For Determination Of Penicillin In Milk

The penicillin is a kind of important β-lactam antibiotics. Penicillin and other β-lactam antibiotics constitute the antimicrobial drugs are most frequently used in veterinary medicine in the treatment of infections of lactating cows and other disease of dairy catties, so that the presence of penicillin residues have severely influenced the health of human being and restricted the development of the economy of our country. Therefore, the development of sensitive, relatively simple and inexpensive penicillin sensor for the diagnosis of penicillin in milk is necessary for health protection and for further medical treatment. The electrochemical immunosensor uses specificity of the reaction of antigen and antibody to combine traditional method for immunoassay with the technology of modern sensor, which both has the high specificity of the immunochemical system and has the high sensitivity of electrochemical analysis and has been broadly applied in many fields such as health and medical community, environment. In this thesis, two type amperometric immunosensor were prepared by covalently bonding and electropoly merization and based on new methylene blue as mediator. The sensor electrochemical behavior, checking principle, conditional optimization, influencing factors and detection in milk have been investigated.The detailed materials are summarized as follows:1. The electrochemical behavior of new methylene blue (NMB) in solution has been studied, and selected new methylene blue as mediator because of its excellent redox reversibility. The reaction belongs to the one-proton and two-electron reaction in the neuter or weak basicity solution, and the electrochemical response provided good character within broader pH range. And that acidity range which is to keep biomolecule activity and the enzyme catalytic activity having provided the proper condition.2. The penicillin sensor was developed with the adsorption of nano-Au through the poly (new methylene blue) and nano-Au was polymerized on the surface of electrode. The modified surface was studied by means of Fourier Transform Infrared Reflection Spectroscopy (FTIR) , Ultraviolet visible( UV-vis) spectroscopy, Scanning ElectronMicroscope (SEM) and electrochemistry method. The linearity range of the sensor to penicillin in phosphate buffer solution was 0.5～6 ng/mL, and the detection limit was 0.067 ng/mL.3. A novel amperometric hydrogen peroxide sensor was proposed by co-immobilizing new methylene blue (NMB) and Horseradish peroxidase (HRP) on a glassy carbon electrode through covalent Immobilization. The response of sensor on H₂O₂ exhibited some advantages including quick response, wide linear range and high sensitivity. The electrochemistry and dynamics character of the sensor in 0.1 mol/L phosphate buffer solution (pH=7.0) were investigated and the electron transfer coefficient and apparent reaction rate constant were determined to be 0.861 and 1.27 s^-1. And the linear dependence of current on H₂O₂ is in the range of 2.5～100mol/L. At the same time, the detection limit was 2.07mol/L from the 10 times detection value of the blank solution.4. New methylene blue (NMB) and Horseradish peroxidase (HRP)-labeled penicillin antibody on a glassy carbon electrode through covalent Immobilization and then a penicillin immunosensor was made. The sensor was used to detect penicillin in milk system and the result showed that the linear range and the limit of detection of the assay for penicillin was determined to 0.25～3 ng/mL and 0.298 ng/mL. In addition, we also investigated the antibody's purification and label, antibody titer, the confirmation and selectivity of the sensor, the optimization of condition, reproducibility and stability of the sensor and the factors of effects on the sensor.