Studies On Fluorescence Quenching/Electrochemical Immunoassay Based On Enzyme And Nanoparticles Label

It is evident that the development of simple and sensitive strategy is one of the important researches in the field of immunoassay. Enzyme and nanoparticles, because of their special properties, have such potential clinical application. This research focuses on the details as follow:1. A novel, simple and sensitive immunoassay was reported using fluorescence quenching caused by gold nanoparticles coated with antibody. The method is based on a non-competitive heterogeneous immunoassay of human IgG conducted by the typical procedure of sandwich immunocomplex formation. Goat anti-human IgG was first adsorbed on polystyrene microwells, and human IgG analyte was captured by the primary antibody and then sandwiched by antibody labeled with gold nanoparticles. The sandwich-type immunocomplex was subsequently dissociated by the mixed solution of sodium hydroxide and trisodium citrate. The solution obtained, which contains gold nanoparticles coated with antibody, was used to quench fluorescence. The fluorescence intensity of fluorescein at 517 nm was inversely proportional to the logarithm of the concentration of human IgG in the dynamic range of 10~5000 ng/ml with a detection limit of 4.7 ng/ml. The electrochemical experiments and the UV-vis measurements were applied to demonstrate whether the immunogold was dissociated completely and whether the gold nanoparticles aggregated after being dissociated, respectively. The proposed system can be extended to detect target molecules such as other kinds of antigens and DNA strands, and has broad potential applications in disease diagnosis.2. A sensitive immunosensor using colloidal gold as electrochemical label is described. In this method, the capture protein was first immobilized on a carbon paste electrode surface through passive adsorption to bind quantitatively with corresponding antigen and colloidal gold labeled antibody to perform a sandwich assay. To detect the amount of the colloidal gold captured on the electrode surface, the colloid was first oxidized electrochemically to produce AuCl4? ions which were adsorbed strongly on the electrode surface. Adsorptive voltammetry was then employed for the determination of the adsorbed AuCl4? ions. A linear relationship between reduction wave peak current and the antigen concentration (human IgG) from 10 to 500 ng/ml is obtained with a detection limit of 4.0 ng/ml. 3. An electrochemical amplification immunoassay is reported using biocatalytic metal deposition coupled with anodic stripping voltammetric detection. In this method, the captured antibody was first immobilized onto a gold electrode via a self-assembled layer. After a sandwich immunoreaction, alkaline phosphatase-labeled antibody was bound to the gold electrode. The alkaline phosphatase on the electrode catalyzes the hydrolysis of ascorbic acid 2-phosphate to produce ascorbic acid. The latter, in turn, reduced silver ions on the electrode surface, leading to the deposition of silver onto the protein-modified electrode surfaces. The deposited metal was electrochemically stripped into solution and then measured by anodic stripping voltammmetry. Compared with the direct voltammetric detection of ascorbic acid, anodic stripping voltammetric detection of metal ions is more sensitive. For the amount of deposited silver relates to the amount of enzyme-generated ascorbic acid, which was controlled by the amount of enzyme bound on the electrode surface, the stripping current signal reflects the amount of target protein, achieving a linearly relationship in the range from 5 to 1000 ng/ml in a logarithmic plot with a detection limit of 2.2 ng/ml. The utilization of the high biocatalytic activity of enzyme and the sensitive anodic stripping voltammetry to detect metal ions dramatically enhanced the sensitivity in immunoassay.4. Asuccessively signal-amplified electrochemical immunoassay has been reported on the basis of the biocatalytic deposition of silver nanoparticles with their subsequent amplifacation by nanoparticle-promoted catalytic precipitation of silver from the silver-enhancer solution. The immunoassay was carried out based on a heterogeneous sandwich procedure using polystyrene microwells to immobilize antibodies. After all the processes comprising the formation of immunocomplexes, biocatalytic deposition of silver nanoparticles and following silver enhancement were completed, the silver on polystyrene microwells was dissolved and quantified by anodic stripping voltammetry (ASV). The effect of relevant experimental conditions, including the concentration of ascorbic acid 2-phosphate (AA-p) substrate and Ag(I) ions, the biocatalytic deposition time, and of crucial importance, the silver enhancement time, were investigated and optimized. The anodic stripping peak current was proportional to the concentration of human IgG in a dynamic range of 0.1~10 ng/ml with a detection limit of 0.03 ng/ml. Scanning electron microscope (SEM) was applied to characterize the silver nanoparticles before and after silver enhancement on the surface of polystyrene microplates. By coupling the highly catalytic effect of enzyme and nanoparticles to successively amplify the analytical signal, the sensitivity of immunoassay was enhanced so dramatically that this approach would be a promising strategy to achieve a lower detection limit for bioassays.