Construction Of Electrochemiluminescence Immunosensor Based On The Strategy Of Enhancing Reacted Efficiency Of Co-reactants

Electrochemiluminescence（ECL） immunoassay technology, combined the high sensitivity and controllability of ECL analysis technique with high specificity of immune recognition, can significantly improve the detection sensitivity and selectivity of biological protein molecules, which shows great potential in immunobiology and clinical laboratory science. The ECL intensity could be significantly enhaced when co-reactants are introduced into ECL system. However, some co-reactants are instable and difficult to be labeled, increasing the operation difficulty and measurement error. Furthermore, the traditional ECL types are mainly based on intermolecular interactions between luminescent agents and co-reactants, which have some drawbacks, such as long distance for electron transfer, high energy loss and poor stability. Accordingly, in this thesis, in asisitance of kinds of nano-materials, multiple ECL immunosensors are contructed for sensitive detection of a variety of disease-related proteins by using enzyme-catalyzed reaction and synthesizing novel self-enhanced ECL reagent to improve the reacted efficiency of co-reactants. The studies of this thesis are mainly divided into the following sections: 1. The research on electrochemiluminescence immunosensor based on bi-enzyme synergetic catalysis to in situ generate coreactantThe drawbacks of some co-reactants in unstablity and difficulty in labeling limit the application of ECL system. In this work, it has been resolved by using enzyme-catalyzed reaction to in situ generate co-reactants around the electrode surface. Studies have shown that glucose oxidase and horseradish peroxidase can catalyze the hydrolysis of glucose to produce O₂. Based on this, the strategy of in situ generating O₂ by bi-enzyme synergetic catalysis is proposed, which has been successfully applied in S₂O₈^2--O₂ ECL system to achieve the enhancement of stability and signal amplification. A simple label-free ECL immunosensor is fabricated for sensitive detection of alpha-fetoprotein（AFP） based on this bi-enzyme catalyzed stratege combined with the promotion of L-cysteine and gold nanoparticles. A novel signal amplification method is presented for S₂O₈^2--O₂ ECL system, broadening its application. 2. The research on electrochemiluminescence immunosensor based on multi-fullerenes encapsulated palladium nanocage and mimicking bi-enzyme synergetic catalysis to in situ generate coreactantUsing nanomaterials as mimicking enzyme could overcome shortcomings of native enzyme, for instance easy deactivation and harsh reaction conditions, which broadens the application scope of enzyme-catalyzed reaction. In this study, palladium nanocages（PdNCs） with porous structure are modified with L-cysteine（L-Cys） functionalized fullerenes(C₆₀) to obtain C₆₀-L-Cys-PdNCs nanocomposite. Due to its good electro-catalytic activity and large specific surface area, C₆₀-L-Cys-PdNCs can be well applied to construct biosensor. Thus, in this work, a sandwich-type ECL immunosensor was constructed to detect streptococcus suis（SS2） by using C₆₀-L-Cys-PdNCs to immobilize glucose oxidase and secondary antibody protein. C₆₀-L-Cys, as a new co-reactant for S₂O₈^2--O₂ ECL system, could greatly enhance the ECL intensity. In the presence of glucose, glucose oxidase catalyzes glucose to generate hydrogen peroxide（H2O₂）, and PdNCs as horseradish peroxidase mimetic enzyme further catalyze H2O₂ decomposition to produce O₂, thereby significantly enhancing of ECL signal of S₂O₈^2--O₂ system. The linear range of the immunosensor is 0.1 pg mL^-1 100 ng mL^-1, and the detection limit is 33.3 fg mL^-1. 3. The research on self-enhanced electrochemiluminescence immunosensor based on nanowires obtained by a green approachThe conventional ECL reactions are mainly based on intermolecular interactions between luminescent agents and co-reactants, which are usually accompanied with high energy loss and poor stability. In view of this, a new self-enhanced ECL derivative（PAMAM-Ru） is prepared by crosslinking reaction between carboxylated bipyridine ruthenium（Ru） and polyamidoamine（PAMAM） dendrimer. The ECL derivative has high luminous efficiency by intramolecular interactions between luminescent agents and co-reactants, which shorten the distance of electron transfer, reduce energy losses and greatly improve luminous stability. Using palladium nanowires, obtained by green way with high large specific surface area, to immobilize the ECL derivative and secondary antibody, an ECL immunosensor is successfully fabricated for detection of carcinoembryonic antigen（CEA）. The synthesis of the novel self-enhanced ECL derivative with high luminous efficiency and stability makes ECL technology to better apply in clinical assays. 4. The research on self-enhanced electrochemiluminescence immunosensor based on polymer chains grafted on palladium nanocagesIn the synthesis of self-enhanced ECL derivatives, the polymerization reaction has been included to increase the content of luminophore, further significantly improving the luminous efficiency. Palladium nanocages（PdNCs） with porous structure are used to load a large number of polyethyleneimine（PEI）, and a polymeric chain containing Ru（II） complex in situ grow on PEI by atomic radical polymerization reaction to form a ECL self-enhanced composite（PdNCs-PEI-PSRu）. The self-enhanced composite has high luminous efficiency through the intramolecular interactions between luminescent agents（Ru（II） complex） and co-reactant（PEI）, which shorten the distance of electron transfer, reduce energy losses and greatly improve luminous stability. Furthermore, the amount of Ru（II） complex is geatly increased by the atom radical polymerization, further enhancing the luminous efficiency. A sandwiched ECL immunosensor for detection of carbohydrate antigen 15-3（CA 15-3） is constructed by using the obtained composite labeled with secondary antibody as signal probe, and gold nanoparticles functionalized graphene as substrate. The linear range of the immunosensor is 0.01 U mL^-1 120 U mL^-1, and the detection limit is 0.003 U mL^-1. 5 The research on immunosensor using self-enhanced tris（bipyridine） ruthenium（II） derivative nanorods as electrochemiluminescence materialHere, we firstly use ruthenium（II） complexe with self-enhanced ECL property as precursor to directly synthesize nanorod with high luminous efficiency through a solvent evaporation induced self-nucleation process. The synthesis of the nanomaterial with luminous efficiency is simple, effective, and has the following advantages: firstly, the self-enhanced precursor has high luminous efficiency through the intramolecular interactions between luminophore and co-reactive group. Secondly, after the formation of rod-like structure, the amount of self-enhanced ruthenium（II） complexes is significantly increased, leading to further improvement of luminous efficiency. In addition, since the nanorods are positive charged and contain amino groups, these are easy for post-modification. To enhance the conductivity, platinum nanoparticles with negatively charged are adsorbed on the surface of the nanorods to label secondary antibody in this study. Through sandwiched immunoreactions, a signal on ECL immunosensor is constructed for determination of N-acetyl-β-D-glucosaminidase（NAG）, a marker of diabetic nephropathy. The synthesis of the new ECL nanomaterials combined efficient immobilization and high luminous efficiency provides a new idea and platform for the establishment of ultrasensitive ECL clinical testing system.