| Electrochemically based enzyme biosensors are valuable analytical tools for monitoring of the enzyme as the advantages of high sensitivity, short response time, easy manipulation and high specificity, which combined the merits of electrochemical analysis method and biological technology. The enzyme biosensors have shown wide application prospect in detecting hydrogen peroxide, uric acid, glucose, cholesterol, organic phosphorus and so on. In these detected matters, the rapid and accurate determination of hydrogen peroxide is of great importance because it is not only the product of the reactions catalyzed by many highly selective oxidases but also an essential compound in food, pharmaceutical and environmental analyses. At the same time, as people living standard rise, the nutrient composition of food has been paied a great attention, cholesterol is not only a kind of important nutrients, but also an essential compound in tissue. It is an important sense to detect cholesterol in blood and food for people’s healthy. In this paper, hydrogen peroxide and choleaterol were chosen as the target analytes. An enzyme biosensor is an analytical device that responds to a suitable combination of a biological recognition system and an electrochenmical transducer. In the fabricated process of enzyme biosensors, how to choice the immobilized methods and materials of biomolecules have been the vital step in successful development of an enzyme biosensor. Therefore, this research focuses on the preparation of multi-functionalized nanomaterials, enzyme immobilization technology and the method of enzyme immobilization.The detail contents are as follows:Chapter one, in the review section, after general introduction of biosensor and enzyme biosensor, including enzyme biosensor’s principle and development course were detailed, the construction of enzyme reaction interface and the application of nanotechnology into enzyme biosensors were highlighted. Finally, the work and significance of this thesis was briefly introduced.Chapter two, a procedure for fabricating a mediator-free amperometric hydrogen peroxide biosensor has been developed based on the efficient immobilization of horseradish peroxidase (HRP) to the nano-Au(?) and L-cysteine (L-cys) modified gold electrode by electrostatic adsorption. First of all, different diameters of positively charged Au colloid nanoparticles (nano-Au(?)) were prepared and characterized by transmission electron microscopy (TEM) and microelectrophoresis. The novelty of the methodology was the use of the nano-Au(?) to effectively adsorb HRP. The presence of the nano-Au(?) provided a congenial micro environment for adsorbed HRP and reduced the electron transfer impedance, leading to a direct electrochemical behavior of the immobilized HRP. Compared with the negatively charged Au nanoparticles (nano-Au (?)), the nano-Au(?) acts as much adsorption of HRP, excellent catalytic surfaces towards the reduction of H2O2. The morphology of the enzyme biosensor was further characterized by electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM) and chronoamperometry. The performance and factors influencing the performance of the resulting biosensors were studied in detail.Chapter three, it will report a core-shell nanoparticle system coated on the carbon paste electrode (CPE) for determination of hydrogen peroxide. The amino-functionalized shell-magnetic core nanoparticles have been proven to be an effective material for Hb immobilization. The core-shell nanoparticle system was constructed by immobiling hemoglobin (Hb) on amino-functionalized shell@magnetic core composite nanoparticles (NH2-SiO2-CoFe2O4) with the bridge of gold nanoparticles (AuNPs). The electrochemical impedance spectroscopy, cyclic voltammetry and chronoamperometry were used to characterize the obtained biosensor.Chapter four, a new electrochemical biosensor with enhanced sensitivity was developed for detection of cholesterol by using platinum-palladium-chitosan-graphene hybrid nanocomposites (PtPd-CS-GS) functionalized glassy carbon electrode (GCE). An electro deposition method was applied to form PtPd nanoparticles-doped chitosan-graphene hybrid nanocomposites (PtPd-CS-GS), which were characterized by scanning electron microscopy (SEM) and electrochemical methods. The presence of the PtPd-CS-GS nanocomposites not only accelerated direct electron transfer from the redox enzyme to the electrode surface, but also enhanced the immobilized amount of cholesterol oxidase (ChOx). Under optimal conditions, the fabricated biosensor exhibited wide linear ranges of responses to cholesterol in the concentration ranges of2.2×106-5.2×10-4mol·L-1, the limit of detection was0.75μmol·L-1(S/N=3). The response time was less than7s and the Michaelis-Menten constant (KMapp) was found as0.11m mol·L-1. In addition, the biosensor also exhibited excellent reproducibility and stability. Along with these attractive features, the biosensor also displayed very high specificity to cholesterol with complete elimination of interference from UA, AA, and glucose.Chapter five, in this article, we used the TiO2-graphene-Pt-Pd hybrid nanocomposites (TGPHs) as an enhanced element of the integrated sensing platform for increasing the surface area as well as improving the electronic transmission rate. Subsequently, Au nanoparticles (AuNPS) and cholesterol oxidase (ChOx) were successively self-assembled to TGPHs with high load amount and superior biological activity. The morphology of TGPHs and stepwise fabrication processes were characterized with cyclic voltammetry (CV), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Based on the efficiently catalytic ability of TGPHs and AuNPS, the fabricated biosensor exhibited wide linear ranges of responses to cholesterol in the concentration ranges of5.0×10-8-5.9×104mol·L-1, the limit of detection was0.017×mol·L-1(S/N=3). The response time was less than7s and the Michaelis-Menten constant (KMapp) was found as0.21mmol-L"1. The fabricated biosensor was further tested using real food samples egg, meat, margarine and fish oil, showing that the biosensor has the potential to be used as a facile cholesterol detection tool in food and supplement quality control. |
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