Electrochemical Immunosensor Based On Amplification Of Enzyme-catalyzed Precipitation And Novel Nanomaterials

The enhancement of immunoassay signal is an important aspect of immunoassay study, which is vital to improvement of the assay sensitivity. Meanwhile, the key point for the fabrication of biosensors with excellent property is the efficient immobilization of bio-species on the surface of transducers. So it is undoubtable that the explorer of new immunization immobilization material with good stability and excellent property is one of the main purposes of researchers. Based on the considerations above and comprehensive literature, this research focuses on new methods for fabrication of sensing interface of biosensor and the enhancement of the signal of immunoassay, especially on the enhancement of the signal of electrochemical immunoassay. The details are summarized as follows:(1) A highly sensitive electrochemical immunosensor based on combination of chitosan (CHIT) and coral-shaped AuNPs (C-AuNPs) to form an immobilization matrix has been developed using human IgG as a model analyte (in Chapter 2). The inorganic-organic hybrid film with abundant adsorbing sites and large surface area can reserve the biocompatibility of the biomaterials which greatly increase loading amounts of assembling, thus, significantly improves the performance of biosensing. The morphology is studied by scanning electron microscopy (SEM). Under the optimized experimental conditions, the immunosensor exhibits excellent performance (e.g., a detection limit of 5 pg/mL, a linear dynamic range of 3 orders of magnitude, high specificity). This possibly makes it an attractive platform for the direct immunoassay of human IgG or other biomolecules.(2) A sensitive electrochemical immunosensor for the determination of human IgG was proposed in this paper (in Chapter 3). The electrochemical enzyme immunoassay is based on amplification of enzyme-catalyzed precipitation of HRP-H2O2-OPD (o-pheneylenediamine) system, in which the product DAP (2, 3-Diaminophenazine) of HRP-catalyzed H2O2 oxidizing OPD is measured by electrochemical methods.The electropolymerization of thionine in neutral phosphate buffer solutions has been carried out by a two-step method. A polythionine film containing large amounts of amino groups was formed, which can combine with gold nanoparticles through covalent interactions. Because gold nanoparticles have a large surface area, a large number of antibody molecules can be adsorbed to gold particles surface, and horseradish peroxidase labeled human IgG is captured to the electrode surface by sandwich-type immunoreaction. Then HRP biocatalyzed OPD in the presence of H2O2, resulting in an insoluble product on the electrode surface, which changes the electrochemical properties of the electrode surface and can be measured by electrochemical methods. The immunosensor exhibits excellent performance, The proposed immunosensor in optimal conditions has a linear range of 100 pg/mL to 50 ng/mL for hIgG detection with a detection limit of 10 pg/mL.(3) In this paper, an amplified immunosensor with highly sensitivity has been proposed based on precipitation of an insoluble product on functionalized electrode (in Chapter 4). First, the glassy carbon electrode surface is modified by a polythionine film, providing a rich amino interface for the construction of sensor. Then 3D Au clusters with good stability, large surface area, good biocompatibility and performance were synthesized by electrodeposited on polythionine modified glassy carbon electrode. The electrodeposited Au nanoclusters build directly immobilization interface for protein molecules, and can be combined with the antibody molecules through amino-Au bond with high loading amounts and high immune activity. The electrochemical immunosensor uses human IgG as a model analyte, and has a detection limit as low as 5 pg/mL.