Study On Novel Electrochemical Immunosensors And Their Applications In Biological Analysis

Electrochemical immunosensor, combining electrochemical sensing technology and immunoassay methods, could specifically transform the immunorecognition events of antigen and antibody into electronic signals in order to achieve the quantitative determination of antigen or antibody. Due to its excellent performance such as high sensitivity, high selectivity, fast response time, simple operation, electrochemical immunosensor have become a powerful tool for the detection of biological samples in clinical diagnostics, food safety, environmental analysis and other fields.Since the1980s, nano-materials have attracted great attention. They display unique characteristics such as large surface area, surface chemical activity, good biological compatibility and so on. Therefore, they are widely used as materials for the electrode surface modification or the platform for anchoring biological molecules for designing electrochemical biosensors with ultra-high sensitivity, high selectivity. The application of nano-materials has brought a great momentum to electrochemical biosensing technology. The electrochemical determination is also coupled with photochemistry. Photoelectrochemical immunosensors, integrated the merits of photochemistry and electrochemistry, have excellent performance with high sensitivity.In this dissertation, we developed several novel immunosensors based on nanomaterials. The functionalized nano-materials played an essential role in the immobilization of the protein molecules. Thereafter, through an immunoreaction of antibody and antigen, an immunosensor was formed. Scanning electron microscopy, transmission electron microscopy, and atomic force microscopy were used to characterize the morphology of the nano-materials and the modified immunosensors. Electrochemical impedance spectroscopy, cyclic voltammetry, the current-time curve, and differential pulse voltammetry were used for characterization of electrochemical properties of the immunosensors. The designed immunosensors were successfully applied to determination of biological samples. Details are as follows: Chapter1A Photoelectrochemical Immunosensor Based on Au-Doped TiO2Nanotube Arrays for the Detection of a-Synucleina-Synuclein (a-SYN) is a very important neuronal protein that is associated with Parkinson's disease. In this paper, we utilized Au-doped TiO2nanotube arrays to design a photoelectrochemical immunosensor for the detection of a-SYN. The highly ordered TiO2nanotubes were fabricated by using an electrochemical anodization technique on pure Ti foil. After that, a photoelectrochemical deposition method was exploited to modify the resulting nanotubes with Au nanoparticles, which have been demonstrated to facilitate the improvement of photocurrent responses. Moreover, the Au-doped TiO2nanotubes formed effective antibody immobilization arrays and immobilized primary antibodies (Ab1) with high stability and bioactivity to bind target a-SYN. The enhanced sensitivity was obtained by using{Ab2-Au-GOx} bioconjugates, which featured secondary antibody (Ab2) and glucose oxidase (GOx) labels linked to Au nanoparticles for signal amplification. The GOx enzyme immobilized on the prepared immunosensor could catalyze glucose in the detection solution to produce H2O2, which acted as a sacrificial electron donor to scavenge the photogenerated holes in the valence band of TiO2nanotubes upon irradiation of the other side of the Ti foil and led to a prompt photocurrent. The photocurrents were proportional to the a-SYN concentrations, and the linear range of the developed immunosensor was from50pg/mL to100ng/mL with a detection limit of34pg/mL.Chapter2Sensitive Electrochemical Immunosensor for a-synuclein Based on Dual Signal Amplification using PAMAM Dendrimer-encapsulated Au and Enhanced Gold Nanoparticle LabelsA novel electrochemical immunosensor for sensitive detection of a-synuclein (α-SYN), a very important neuronal protein, has been developed based on dual signal amplification strategy. Herein, G4-polyamidoamine dendrimer-encapsulated Au nanoparticles (PAMAM-Au nanocomposites) were covalently bound on the poly-o-aminobenzoic acid (poly-o-ABA), which was initially electropolymerized on the electrode surface to perform abundant carboxyl groups. The formed immunosensor platform, PAMAM-Au, was proved to provide numerous amino groups to allow highly dense immobilization of antigen, and facilitate the improvement of electrochemical responses as well. Subsequently, the enhanced gold nanoparticle labels ({HRP-Ab2-GNPs}) were fabricated by immobilizing horseradish peroxidase-secondary antibody (HRP-Ab2) on the surface of gold nanoparticles (GNPs). After an immunoassay process, the{HRP-Ab2-GNPs} labels were introduced onto the electrode surface, and produced an electrocatalytic response by reduction of hydrogen peroxide (H2O2) in the presence of enzymatically oxidized thionine. On the basis of the dual signal amplification of PAMAM-Au and{HRP-Ab2-GNPs} labels, the designed immunosensor displayed an excellent analytical performance with high sensitivity and stability. This developed strategy was successfully proved as a simple, cost-effective method, and could be easily extended to other protein analysis schemes.Chapter3Electrochemical Assay of SirTl with a Novel Immunosensor Based on TiO2Nanoparticles and Hyperbranched Polymer HybridIn this paper, we report an investigation of a novel electrochemical immunosensor using TiO2-Au nanocomposite and hyperbranched polymer hybrids for SirTl assay. Thiol-functionalized hyperbranched azo-polymers (HBAP) decorated with gold colloids (GC-HBAP) via covalent bond were utilized as the biosensing platform, which has been demonstrated to anchor larger amounts of capture antibodies with high stability and bioactivity. The typical sandwich-type immunoassay procedure comprised of the immunoreaction of capture antibody Ab1#1, target SirT1, followed by the attachment of detection antibody Ab1#2, and{TiO2-Au/Ab2-HRP} bioconjugates, which featured Ab2-HRP molecules linked to TiO2-Au nanocomposites for signal amplification. The designed immunosensor displayed an excellent analytical performance with high sensitivity and stability.