Study On The Competitive Immunosensor Technology For Small Molecular Antigens

Immunosensors are a class of integrated biological sensors with immunoreaction and sensing techniques, which can be used for monitoring the nucleophilic reaction between antigen and antibody real time, kinetic analysis. The detection time also can be shortened. With the development of biological sensitive components, many novel immunosensors such as surface plasmon resonance immunosensor, planar array immunosensor, surface acoustic wave immuosensor and quartz-crystal microbalance immunosensor have been invented in the past 20 years. They have many advantages of high efficiency, high sensitivity, easy operation and so on. Therefore, there has been increasing concern in the research field of environment, food, health and so on and there will be promising prospect in the application of immunosensors in the near future. In order to further study and explore the application of immunosensor, in this thesis, we designed a series of new competitive immunosensors, based on the high sensitivity of piezoelectric immunosensor and chemiluminescence immunoassay and the high specificity of antibody-antigen immunoreaction, as well as the the use of piezoelectric immunosensing technique and chemiluminescence immunoassay technique, which incorporates high sensitivity of piezoelectric response as well as the application about easy separation of magnetic nanoparticles and enzyme-catalyzed amplification, to used to detect small molecules in the field of environment, food and health.(1) A simple, sensitive and good reproducible competitive immunosensor was designed for the detection of small molecules based on enzyme-catalyzed amplification principle. Horseradish peroxidase labeled with hapten was first bound to the antibody by competitive pattern and was used as a biocatalyst for the oxidative precipitation of 4-chloro-1-naphthol by H2O2 to yield the insoluble product benzo-4-chlorohexadienone, amplifying the mass sensing of hapten-antibody interaction. The amount of the precipitate accumulated on the quartz crystal was controlled by the hapten concentration. The FITC could be linearly determined in the range of 0.01-1.0 ng/mL and the detection limit reached as low as 4 pg/mL.(2) Combination of enzyme-catalyzed amplification and easy separation of magnetic nanoparticles, a competitive piezoelectric immunosensor using for the detection of clenbuterol was built. The use of magnetic nanoparticles increased the fixed amount of antigen, at the same time the sensor has some advantages such as simple, no washing, easily renewable, and so on. The clenbuterol could be linearly determined in the range of 0.02-1.5 ng/mL and the detection limit reached as low as 0.01 ng/mL.(3) Another highly sensitive chemiluminescence immunosensor for the detection of clenbuterol was proposed based on an enzyme catalytic amplification procedure with magnetic particles as carrier. The magnetic nanoparticles were used for bio-sensing interfaces with fixed HRP-CL. Then, the clenbuterol could be determined via the chemiluminescence signal of HRP-catalyzed luminol/peroxide/enhancer system. The chemiluminescence intensity was directly proportional to the concentration of clenbuterol in sample solution in the range from 0.01 ng/mL to 20 ng/mL with a detection limit of 6 pg/mL.