Studies Of Ultrasensitive Electrochemical Immunosensers Based On Enzymetically Catalytic Amplification

Immunoassay plays an important role in various areas including clinical medicine, food industry and environmental supervision. Traditional immunoassay methods are frequently criticized toward for their long assay time, low detection sensitivity, complicated procedures and expensive instrumentation. The development of new strategies with high sensitivity, low cost, and rapid outcome is of considerable significance for practical problem solving in biomedical studies and clinical diagnosis.Immunosensors is a transducer that could specifically transform the immuno- recognition events into electronic and optic signals. Due to its portability, low cost and high sensitivity, immunosensors have become an attractive subject in bioanalytical chemistry. Among them, electrochemical enzyme-linked immunosensors are of particular significance because of the possibility of hyphenation with simple instrumentation as electrochemical systems and combination with high efficiency of enzyme in signal amplification. The basic scheme in electrochemical enzyme-linked immunosensors includes the conjugation of a certain enzyme with antibody or antigen, the formation of antibody-antigen immuno-compelx and the enzyme-mediated reaction that generates an electrochemically measurable product, furnishing a redox signal correlated to the analyte. This technique is recognized for very high sensitivity, ease in miniaturization and implementation, therefore, has provided a promising tool for biomedical researches and clinical diagnosis. Based on this consideration, the present thesis is directed toward to the development of novel electrochemical immunosensing techniques. The main studies include the following aspects:First, a novel sensitive electrochemical immunoassay method was proposed based on gold nanoparticle mediated biocatalytic deposition of platinum followed by stripping voltammetric determination. The feasibility of the approach was investigated using a"sandwich"immunoassay format with human immunoglobulin G (h IgG) as the analyte. H IgG was firstly captured by primary goat anti-h IgG polyclonal antibody (h IgG Ab) immobilized on polystyrene microwells. Gold nanopartcile-labeled alkaline phosphatase (ALP)-h IgG Ab was then bound to the microwells through sandwiched h IgG. The surface-bound alkaline phosphate catalyzed the generation of ascorbic acid, which, in turn, reduced platinum ions into its metal form in the presence of gold nanoparticles. The deposited metal was released in aqua regia (three parts HCl, one part HNO3), and reduced on glassy-carbon electrode, which generated a significant cathodic current due to the platinum-catalyzed hydrogen evolution. The cathodic current was observed to show linear correlation to logarithmic h IgG concentration over the range from 100ng/ml to 2μg/ml and from 100pg/mL to 100ng/mL respectively, the detection limit was as low as 22pg/ml. The high performance of the method is attributed to the sensitive determination of platinum and the catalytic precipitation-based amplification mediated by gold nanopartcile-labeled ALP-h IgG Ab.Second, a new, sensitive electrochemical immunoassay has been established based on the precipitation of copper onto platinum nanoparticles on the surface of glass-carbon electrode to inhibit the electrocatalytic activity of platinum, which catalytically reduces the protons to hydrogen in acidic medium. A negative shift of hydrogen evolution potential can be obtained after determination using linear sweep voltammetry (LSV) in 0.1 M HCl solution. The method was evaluated for a sandwich heterogeneous immunoassay of immunoglobulin G (IgG) as a model target. The influence of relevant experimental variables, including the concentration of H2PtCl6, Cu2+ and ascorbic aid 2-phosphatase, and the reaction time of enzymes catalytic copper deposition on the shift of potential was examined and optimized. The analytical signal was observed to be dependent linearly upon the IgG concemtration over the range of 0.1ng/mL to 10μg/mL in a logarithmic plot. This method can be extended to the application of environmental and food monitoring as well as clinical diagnosis because of the advantages such as the simple operation, inexpensive apparatus, and the high sensitivity.Third, platelet-derived growth factor (PDGF) has been correlated with many diseases. We attempted to establish a new method for the determination of PDGF in human serum using a label-free electrochemical immunosensor. The rabbit anti-human PDGF-B polyclonal antibody was used as capture antibody and the analyte was captured onto electrode, the PDGF aptamer, amplified by polymerase and then formed a ds-DNA with template molecula, bind to electrode surface by forming the protein/DNA complex with PDGF-BB. Electroactive mark MB intercalating into the ds-DNA, thus the Oxidation peak current of square wave voltammetry was obtained and used as quantitative analysis of PDGF-BB. There has a good linear response within the range of PDGF-BB concentration from 50pg.mL-1 to 500ng.mL-1 and the resulting equation was y =44.67x-31.81 with correlation coefficient R2 = 0.9904. The detection limit was reached at 18 pg ml?1. The method stated in this article exhibited a variety of good performance characteristics including high sensitivity, fast response, Moreover, the short time of assay and low the cost, it may be benefit for applying this method in clinic measurement.