Development Of Electrochemical Immunosensors For Schistosoma-Japonicum Diagnosis And Protein Determination

Schistosomiasis Japonicum (Sj), a wide-disseminated and serious parasitic disease, still threatens human health at some lake reqious in Hunan, Hubei, Jangxi and Anhui provinces. The development of sensitive, fast, robust, selective assay methods for the diagnosis of Schistosoma Japonicum is necessary for health protection and for further medical treatment. The electrochemical immunosensing devices combine the high specificity of the immunochemical system with the advantages of electrochemical transducer. They offer some advantages such as robustness, sensitivity,possibility of miniaturization, relatively simple and inexpensive instrumentation. In this thesis, the electrochemical immunosensors based on different immobilizatiom methods and different transducers have been developed for Schistosoma-Japonicum diagnosis and protein determination. The detailed materials are summarized as follows:A renewable amperometric immunosensor based on graphite-parafin-.Sc/n.sfo.y0ma Japonium antibody (SjAb) biocomposite electrode has been prepared for the detection of Schistosoma Japonium antigen (SjAg). The measuring range of SjAg was 0.5~30 ug/ml under the optimum condition. Based on the adsorptive immobilization of anti-complement III antibody onto an electrochemically pretreated carbon-paraffin electrode, an amperometric immunosensor was developed for the detection of complement HI (C3). The system was optimized to realize a reliable determination of C3 in the range of 0.06 jag/ml to 10 ug/ml. An amperometric immunosensor based on the adsorptive immobilization of SjAg onto the Nafion-modified electrode surface has been prepared for the detection of SjAb. The measuring range of SjAb was 0.02~4.8 ^g/ml under the optimum condition. An regenerable amperometric immunosensor based on the covalently immobilization of anti-Tf antibody to protein A embedded in Nafion film was preparedfor the detection of transferrin (Tf). The system was optimized to realize a reliable determination of Tf in the range of 0.12 |ig ml"1 to 14.4 ug ml"1. A seperation-free heterogenerous ELISA is described for determining IgM based on the Nafion film used to immobilize antibody andaccumulated o-AP as the electron media. In this system, surface bound conjugate can be spatially resolved from unbound conjugate in measurement solution, because only the Nafion film surface bound coujugate can encounter the o-AP in nafion film, catalyzing electrochemical reducetion of H2O2 and producing in amperometric response. Thus, no washing step for separation of free and bound enzyme labels are needed and the assay procedures are simplified. A capacitive immunosensor based on Schistosoma Japonicum antibody (SjAb) non-covalently immobilized on the gold electrode surface via protein A modified layer has been prepared for the detection of Schistosoma Japonicum antigen (SjAg). This method allows measurement of SjAg directly without the use of labeled compounds in the range of 0.4 to 18 ng/mL. An amplified mass enzyme linked immunosensor with quartz crystal microbalance as transducer was developed to determine IgM. The quartz crystal microbalance is highly sensitive to the surface mass. The amount of HRP-IgM was measured by enzyme-catalyze TMB oxidized to form precipitate products. This system has the advantages of low cost and simplicity.