Study On Electrochemiluminescence Enzyme Biosensor Based On Novel Carbon Nanocomposite

Electrochemiluminescence (ECL) is a process whereby species electrochemically generated on the electrode surface through an applied potential and then undergo high-energy electron transfer (redox or enzymatic) reactions to emit light from the excited states.ECL is a new analytical detection method combined with the electrochemical technique and luminescent technique, and its advantages are high sensitivity, good selectivity, ease of control and simple equipment. The enzyme biosensor is a common analytical device which is based on the immobilized enzymes as molecular recognition elements, modified electrodes as the signal conversion device. Therefore, the enzyme biosensor has the function of molecular recognition and selective catalytic, and as also the electrode response of the detection substrate is fast, the experimental process is easy to operate. As a novel combination product of ECL technique and highly specific and efficient catalytic of enzyme, the ECL enzyme biosensor becomes a available and powerful mean to detect substance with high sensitivity and selectivity.With the rapid development of nanotechnology, various nanomaterials have been widely applied to the biosensing system. Nanomaterials exhibit various advantageous properties, such as large specific surface area, unique electrochemical properties, good biocompatibility, which would provide a promising approach to develop efficient biosensor to detect biologicaly important compounds in clinical applications.Thereinto, carbon nanotubes and graphene both have special physical and chemical properties, good electrical conductivity and electrocatalytic activity, promote the electron transfer between the immobilized enzyme and the electrode.At the same time, the application of carbon-based nanocomposites in the construction of enzyme biosensor can provide a good microenvironment for the immobilized enzyme, which can maintain the biological activity of enzyme. Based on the above considerations, this paper developed a series of sensitive ECL enzyme biosensor based on the carbon-based nanocomposites, and the performance of these ECL enzyme biosensors improved to a large extent compared with the previous reported enzyme biosensors.The main works are included as follows:Part 1 Hemin functionalized multi-walled carbon nanotubes as a matrix for sensitive electrogenerated chemiluminescence cholesterol biosensorBased on Hemin-MWCNTs nanocomposite and Hemin-catalysed luminol-H2O2 reaction, a sensitive ECL cholesterol biosensor was proposed in this paper. Firstly, hemin-MWCNTs was prepared via π-π stacking and modified on the surface of GCE. Subsequently, cholesterol oxidase (ChOx) was adsorbed on the modified electrode to achieve a cholesterol biosensor. Hemin-MWCNTs nanocomposite provided the electrode with a large surface area to load ChOx, and endowed the nanostructured interface on the electrode surface to enhance the performance of biosensor. The biosensor responded to cholesterol in the linear range from 0.3 μmol·L-1 to 1.2 mmol·L-1 with a detection limit of 0.1 μmol·L-1. In addition, the proposed biosensor has good accuracy, high sensitivity and good stability. Therefore, this work provides a novel ECL signal amplication strategy in the application of clinical diagnostics and bioanalytical testing, successfully expanded the application of luminol-H2O2 ECL system in the biosensing system.Part 2 Synthesis of multiwall carbon nanotubes-graphene oxide-thionine-Au nanocomposites for electrochemiluminescence detection of cholesterolIn this work, we developed a facile approach to synthesize multiwall carbon nanotubes-graphene oxide-thionine-Au (MWCNTs-GO-Thi-Au) nanocomposites with the synergistic effect of GO and Thi, resulting in reducing AuCl4- to Au nanoparticles (AuNPs). Meanwhile, the AuNPs reduced by GO and Thi promoted the ECL of luminol-H2O2 system and offered active sites for immobilization of numerous enzymes. Additionally, it was also found that the synergetic interactions of Thi with MWCNTs and GO were employed for enhancing ECL of luminol-H2O2, which was applied to develop an ECL biosensor for the first time. Using cholesterol oxidase as model enzyme, the proposed biosensor employed by MWCNTs-GO-Thi-Au nanocomposites showed a high sensitivity for cholesterol in a concentration range of 0.15～828 μmol·L-1 with a detection limit of 50 nmol·L-1.Besides, the proposed biosensor exhibited high sensitivity, good selectivity and excellent stability. Taking into account the integrated advantages of MWCNTs-GO-Thi-Au nanocomposites and ECL detection, we confidently expect that this biosensing approach would have potential applications in clinical diagnosis.Part 3 A novel electrochemiluminescence choline biosensor based on biofunctional AMs-ChO biocompositeIn this work, a novel ECL biosensing system for the sensitive and selective detection of choline was creatively designed based on the smart integration of multifunctional nanocomposites and a certain enzymatic reaction. Firstly, graphene-Au-chitosan (GR-Au-CS) nanocomposite, which possesses the property of intensification effect on luminol ECL, was electrochemically deposited on the bare GCE. Then, a biofunctional Fe3O4-TiO3-ChO biocomposite was unprecedentedly synthesized with combining the mimic peroxidase activity of Fe3O4 and the intensification effect of TiO2 on the luminol ECL. Thus, on the basis of smart integration of the above nanocomposites and the detection of H2O2 generated by ChO enzymatic reactions under the optimal conditions, the obtained ECL biosensor for choline showed wide linear range from 3 nmol·L-1 to 1.12 mmol·L-1 with detection limit of 1 nmol·L-1. The excellent performance of the biosensor shows that this method opens up a new avenue in the practical application.