Study And Application Of Biosensors With High Sensitivity

With the human genome Project and proteomics research progress, the rapid development of life science bring forward to a large number of new issues for analytical chemistry. The analysis of biological macromolecules such as the peptides, proteins, nucleic acids has become one of the most important frontier areas in the modern development of biochemical analysis. Quantitative determination of protein and rapid identification of single-base mutations are of particular importance for the pathogeny and early therapy of corresponding diseases.In this thesis, we proposed a new specific, sensitive fluorescence immunosensing method based on aptamer-plasimid complex amplification for the microanalysis of protein and a novel electrochemical method for DNA point mutation detection based on allele-specific extension which are described in following sections:(1)We developed a new simple, sensitive method which utilized the specific recognition between antibody and antigen as well as aptamer-plasmid complex and the intercalation of fluorescence dye SYBR Green ? in the groove of duplex plasmid DNA in detection of PDGF-BB. The aptamer-plasmid DNA complex can be prepared with ease via hybridization of the plasmid vector PUC19 with the extension of an aptamer to PDGF-BB. The immunoassay was performed in the microtiter wells in which rabbit anti PDGF-BB antibody is immobilized. The PDGF-BB analyte is captured by the primary antibody and then sandwiched by the aptamer-plasmid DNA complex. The introduction of fluorescence dye SYBR Green ? allows for the detection of the sandwiched immunocomplex of antibody/anigen/aptamer-plasmid complex. The proposed method offers a sensitive detection for PDGF-BB in serum.(2)A novel electrochemical method based on allele-specific extension in detection of point mutation has been proposed. Briefly, allele-specific primer perfectly matching with template can be extended to result in an increase of current, whereas allele-specific primer mismatching with the template at 3'terminal bases cannot be extended to result in a unchanged of the current. The present approach has been demonstrated with the identification of a single-base mutation in -28 site (A to G) forβ-thalassemia gene and the wild type and mutant type were successfully scored.(3)we developed a novel voltammetric immunosensing method based on colloidal gold enhancement. This method demonstrates the implementation of a sandwich immunoassay of a model analyte, Human immunoglobulin G (HIgG), using the colloidal gold-labeled goat anti-human immunoglobulin G (GAH IgG) as the detection probe. The electroless catalytic deposition of gold ions on the gold nanoparticals enables the growth of the gold nanoparticals at the electrode surface. The developed method is expected to hold great promise in immunosensing due to the ease of implementation, high sensitivity and specificity.