Study On Novel Enzyme Biosensors Based On Nano Materials And Their Applications In Biological Analysis

With the development of life science, the isolation and analysis of complex systems has become an important research field of analytical chemistry. In order to obtain comprehensive and accurate data for complex samples, more effective analytical methods which are sensitive and selective are urgently needed. Biosensor is a kind of technology that incorporates a living organism or product derived from living systems (e.g., enzyme or antibody) and a transducer to provide an indication signal of the presence of the wanted substance. Biosensor has played a significant role in clinical diagnosis, environmental analysis, food safety inspection, drug analysis and other fields,due to its miniature size, high sensitivity, good selectivity and fast response time.In recent decades, nanomaterials were widely used in various areas with their unique properties such as large surface area, catalytic property, biological compatibility. Higher sensitivity and better stability of the biosensor can be received with the utilization of nanotechnology. What's more, with the development of photoelectrochemistry, fabrication of photoelectrochemical biosensor is a focusing analytical study, which can enhance the sensitivity of analytical method.In this paper, incorporating nanotechonology, photoelectrotechnology with biosenser, we constructed several novel biosensors with good selectivity, high sensitivity and long term stability for the determination of complex biological samples. The primary research work is as follows:Chapter 1. PrefaceA critical review with regard to biosensors and the development of biosensors is given, which introduced the concepts, fundamental principles and applications of the biosensors, and the characteristics of electrochemical enzyme biosensor and immunosensor were further exposited. Then, the principle and application of photoelectro-chemistry and nanotechnology was highlighted. We also introduced the development of several common aged diseases. Finally, the purpose and significance of the dissertation was briefly introduced.Chapter 2. A dual enzyme biosensor for simultaneous determination of glucose and cholesterol in serum and peritoneal macrophages of diabetic mice: evaluation of the diabetes-accelerated atherosclerosis riskIn this chapter, we reported a novel dual enzyme biosensor for simultaneous determination of glucose and cholesterol in serum and peritoneal macrophages (PMs) of diabetic mice to evaluate the diabetes-accelerated atherosclerosis risk. The biosensor was firstly modified with a poly-thionine (PTH) film as electron transfer mediator (ETM), then the gold nanoparticles (GNPs) were covered on the surface of PTH to act as tiny conduction centers for facilitating the electron transfer between enzymes and electrode. The developed dual biosensor had good electrocatalytic activity toward the oxidations of glucose and cholestero. The results of the diabetic mice demonstrated that the cholesterol level was not changed obviously with the increase of glucose level in serum, while the cholesterol level was enhanced together with the increase of the glucose level in PMs. As we know, the large accumulation of cholesterol in macrophage could lead to macrophage foam cell formation, the hallmark of early atherosclerosis. This study provided useful experimental evidences for the diabetes-accelerated atherosclerosis development.Chapter 3. A novel semiconductor photoelectrochemical immunosensor based on Au-doped TiO2 nanotubes for the sensitive detection of a-synucleina-Synuclein is a very important neuronal protein which is associated with Parkinson's disease. In this chapter, we utilized Au-doped TiO2 nanotubes to design a novel photoelectrochemical immunosensor for sensitive detection ofα- synuclein. The highly ordered TiO2 nanotube arrays were fabricated by electrochemical anodization technique on a 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 the photoecurrent responses. Moreover, the Au-doped TiO2 nanotubes constructed an effective antibody immobilization array and made the primary antibodies (Ab1) immobilized with high stability and bioactivity to bind the targetα- synuclein (a-SYN). The enhanced sensitivity was obtained by using{Ab2-Au-GOx} bioconjugates, which was featured with secondary antibody (Ab2) and glucose oxidase (GOx) labels linked to Au nanoparticles for signal amplification. The GOx enzyme immobilized on the prepared immunosensor can catalyze glucose in detection solution to produce H2O2, which acts as a sacrificial electron donor to scavenge the photogenerated holes generated in the valence band of TiO2 nanotubes upon irradiation of the other side of Ti foil and leads to a remarkable photocurrent. The increased photocurrents, highly reproducible for on/off cycles of illumination, were proportional to the a-synuclein concentrations. This proposed method showed high sensitivity, stability, reproducibility, and could become a promising technique for protein detection.Chapter 4. Study of acetylcholinesterase inhibition with photoelectrochemical biosensor based on multiple N/F-Ag-TiO2 nanotube structureAcetylcholinestrase (AChE) plays a critical role in cholinergic neurotransmission and the abnormalitiy in its concentration is related to Parkinson's disease. In this chapter, N/F doped TiO2 multiple nanotubes were fabricated by electrochemical anodic oxidation and supported with Ag nanoparticles by microwave assisted heating polyol process. Then the biomolecule AChE was crosslinked on the TiO2 nanotubes. The obtained N/F-Ag-TiO2-AChE hybrid system provides a new, valid and rapid photoelectrochemical approach to the determination of AChE inhibition induced by endogenous neurotoxin, (R)-Sal (R)-NMSal. Due to the separation of excitation and detection signal, the photoelectrochemical method has been proved to be quite sensitive. Furthermore, the TiO2 multiple nanotubes can be also coupled with other enzymes to design photoelectrochemical sensors for the inhibition of enzymes.