Magnetic Beads-based Electrochemical Immunosensor

Special attention has been paid in magnetic beads-based electrochemical immunosensor in the recent years. However, one major obstacle for magnetic beads-based electrochemical immunoassay is how to confine the magnetic beads on the detection surface for the direct electrochemical sensing. The use of magnetic electrodes for magnetic beads manipulation is an effective solution. Focusing on the preparation of magnetic electrodes, and the construction of the novel magnetic beads-based electrochemical immunoassay with nanoparticles amplification for animal diseases diagnosis, this thesis carried out some researches as follows.(1) The fabrication of magnetic electrodesThe simple and low cost methods were developed to fabricate the magnetic gold electrode and magnetic glass carbon electrode. The properties of the magnetic electrodes were carefully examined. The results showed that magnetic electrode could effectively confine the magnetic beads on their detection surfaces for the direct sensing. It is telling that the developed magnetic electrodes can be applied in the magnetic beads-based electrochemical immunoassay.(2) Magnetic beads-based electrochemical immunoassay for the detection of Japanese encephalitis virus using multiwalled carbon nanotubes s as dopantA novel magnetic beads-based electrochemical immunoassay strategy using multiwalled carbon nanotubes as dopant has been developed for the detection of Japanese encephalitis virus. Gold-coated magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and horseradish peroxidase was chosen as an enzymatic tracer. After immunological reaction, multiwalled carbon nanotubes were introduced to improve electric conductivity and the sensitivity of the assay. With the help of the magnetic gold electrode, magnetic beads mixed with multiwalled carbon nanotubes were confined on the detection surface for the direct sensing. The Japanese encephalitis virus was selected as the model. Under the optimal conditions, the detection limit of Japanese encephalitis virus was 2.0×103 PFU/mL, which was 2 orders of magnitude lower than that of immunochromatographic strip and similar to that obtained from RT-PCR. Finally, the proposed method was applied in the clinical diagnosis of Japanese encephalitis virus, and had a good diagnostic agreement (95%) with the results from RT-PCR. This is telling that the present method has a great promise for the clinical diagnosis of Japanese encephalitis virus.(3) Magnetic beads-based electrochemical immunoassay for the detection of pseudorabies virus antibody using gold nanoparticles as tagsA novel magnetic beads-based electrochemical immunoassay has been developed for the detection of pseudorabies virus antibody using gold nanoparticles as electroactive labels. COOH-coated magnetic beads were employed as the platforms. After immunological reaction, the magnetic beads were captured and focused on the detection surface with the help of magnetic glass carbon electrode. The gold nanoparticles were first oxidized electrochemically to produce AuC14-, and then DPV was employed for the determination of AuC14-. Pseudorabies virus antibody was chosen as the model. Under the optimal conditions, the detection limit of pseudorabies virus antibody was 1:1000 (standard positive serum), which was lower than that obtained from ELISA. Finally, this developed immunoassay method was successfully applied in the detection of pseudorabies virus antibody in swine serum, and had a good diagnostic accordance in comparison with ELISA.(4) Magnetic beads-based electrochemical immunoassay with silver amplification for the detection of antibody against porcine circovirus type 2A novel magnetic beads-based electrochemical immunoassay with silver amplification has been developed for the detection of antibody against porcine circovirus type 2. ITC-coated magnetic beads were employed as the platforms, and gold nanoparticles were selected as labels. After immunological reaction, the silver deposition solution was introduced to deposit silver on the gold nanoparticle tags. Magnetic beads were then confined on the detection surface with help of magnetic glass carbon electrode. The deposited silver was measured by anodic stripping analysis. Antibody against porcine circovirus type 2 was employed as model analytes. Under the optimal conditions, the detection limit of antibody against porcine circovirus type 2 was 1:4096, which was lower than that obtained from ELISA. Finally, this developed immunoassay method was successfully applied in the detection of antibody against porcine circovirus type 2 in swine serum, and had a good diagnostic accordance in comparison with ELISA.