Investigation Of Biosensors Based On Sol-Gel And Nanoparticles For Enzymes Immobilization

Biosensors are powerful tools for clinical medicine,biochemical research, environmental protection and food industry, owing to their small size, convenient use and fast assay. Enzymes immobilization plays an important role in design and fabrication of biosensors, which usually decides the performance of biosensors, such as sensitivity, selectivity, response time and lifetime. A good immobilization method should meet the following requirements: (1) produce immobilized enzymes that retain their biocatalytic activities; (2) produce biosensors that have a low mass-transfer resistance and a rapid response time; (3) produce immobilized enzymes that are stable and do not leach from the substrate. The purpose of this thesis is to improve the performance of biosensor by ameliorating enzymes immobilization technique. In detail, the thesis includes the following parts: Part I :Investigation of biosensors based on doped sol-gel for enzymes immobilization 1. The sol-gel encapsulation method has been widely used in enzymes immobilization since it can be prepared under mild chemical conditions, allowing the entrapped enzymes retain their activity. However, sol-gel materials may undergo cracking due to hydration stresses, and in some cases can block the accessibility of analytes to the entrapped enzymes. The use of additives can overcome such problems. In this work,during encapsulation of enzymes into sol-gel derived silica, sarcosine and D-sorbitol have been added to enhance pore sizes. So the diffusion rate for substrate molecules to entrapped enzymes was increased, and the activity of immobilized enzymes and the performance of biosensors were also improved. Based on this method, horseradish peroxidase and glucose oxidase were immobilized, and the activity of immobilized enzymes were examined by doped and undoped sol-gel. The results showed that the improvement of the catalytic capability of this two enzymes were different by using doped sol-gel method. 2. Hypoxanthine is a metabolite in the degradation of adenine nucleotide. The measurement of hypoxanthine is important in clinical diagnosis and food industry. The tradition methods are complex and costly. In addition, the detection limit of 10-7-10-6mol/L is not satisfied enough. Base on above work (PartI-1), xanthine oxidase was immobilized by the doped sol-gel method. The results suggested that the biocatalytic activity of immobilized xanthine oxidase by up to 2.54-fold relative to that of by undoped glasses. Based on the doped sol-gel technique for immobilizing enzymes, a liquid drop sensor was developed to determine hypoxanthine. In pH8.74 Tris-HCl buffer solution, the biosensor showed linear response in the range from 2.0×10-7 to 8.0×10-6mol/L, with a detection limit of 5.0×10-8mol/L. Part II:Investigation of the biosensors based on adsorption of enzymes onto silica nanoparticles Nanoparticles play important roles in enzymes immobilization due to their large specific surface area and high surface free energy. Here, we reported a method to immobilize horseradish peroxidase and glucose oxidase by combining the merits of sol-gel methods and silica nanoparticles. The results showed that silica nanoparticles could increase not only the activity but also the thermal stability, operational stability and the lifetime of the immobilized enzymes. It is to be expected that such a method will not only greatly facilitate the appearance of various types of biosensors, but also be used in other fields where immobilized enzymes are necessary. Part III: Other work Investigation of the interaction of DNA with its target molecules is very important for understanding the mechanism of drug and drug design. Here, the interaction of Lomefloxacin with calf thymus DNA was studied by fluorescence spectroscopy. The association constant of the complex formation was 4.6×103L/mol (37oC, pH=7 Tris-HCl). Metal ions influenced the interaction. For example, in the presence of magnesium ions and calcium ions, the association constant was 1.5 and 1.2 times higher, respectively. Other metal ions, such as sodium and potassium, could hardly influence the interaction. The influence of pH on the interaction was also investigated. The higher the pH value, the weaker the interaction.