Study Of The Plant Polyphenols Based On Enzymes Biosensors

Being one kind of rich reserves and renewable green resources, plant polyphenols not only play an important role in agriculture, environment, food, medicine and chemical industry, but also are closely related to people'daily life, and will certainly to become of capital stocks for humanity use. Therefore, the development of simple, fast and effective methods for plant polyphenols detection has gained more and more people's attention. Combining the specifity, sensitive of enzymes and the simply, rapid of electrochemistry, enzyme-based biosensors can determine the content of certain substances in a complex system quickly and accurately, and not affected by other materials. Therefore, the development of enzyme biosensors has the very vital significance in plant polyphenols detection. However, the methods and materials used to immobilize enzymes are one of the crucial factors for improving the stability, selectivity and sensitivity of biosensor in the preparation. Three new-typed enzyme-based biosensors were prepared, and their application in detection of plant polyphenols was studied in this paper. The dissertation is divided into three main parts:(1) Using Zn(Ac)₂ and N,N-Dimethylformamide as raw materials, nanometersized ZnO was prepared at 160°C by hydrothermal reaction method. The X-ray diffraction (XRD) and Transmission electron microscopy (TEM) were used to analyse the structure of nanometersized ZnO. At the same time, the application of nanometersized ZnO using in modifying electrodes to detect the polyphenol was preliminarily studied, the results showed that the high-isoelectric point nano-ZnO promoted the direct electron transfer between the tyrosinase and the electrode to a large extent, could be effectively used for the determination of polyphenol.(2) A tyrosinase (Tyr) biosensor has been constructed by immobilizing tyrosinase on the surface of Mg-Al-CO3 hydrotalcite-like compound film (HTLc) modified glassy carbon electrode (GCE) for the determination of polyphenols. The negatively charged tyrosinase was adsorbed firmly on the surface of positively charged HTLc/GCE by the electrostatic interactions and retained its activity to a great degree. The modified electrode was characterized by cyclic voltammetry and AC impedance spectra. Polyphenols were determined by the direct reduction of biocatalytically generated quinone species. The different parameters, including pH, temperature and enzyme loading were investigated and optimized. Under the optimum conditions, Tyr/HTLc electrode gave linear response ranges of 3–300, 0.888–444 and 0.066–396μmol/L with the detection limits (S/N=3) of 0.1, 0.05 and 0.003μmol/L for catechol, caffeic acid and quercetin. In addition, the repeatability and stability of the enzyme electrode were estimated. Total polyphenol contents of real samples were also determined to study the potential applicability of the Tyr/HTLc/GCE biosensor.(3) A hemoglobin (Hb) biosensor was fabricated based on Hb immobilized onto Cu₂S nanorods/Nafion nanocomposite film for the detection of polyphenols in the presence of H₂O₂. The nanostructured inorganic–organic hybrid material formed by Cu₂S nanorods and Nafion provided a biocompatible microenvironment for Hb and increased the sensitivity for polyphenols detection. The modified electrodes were characterized by electrochemical impedance spectroscopy and linear sweep voltammetry. Parameters such as pH, H₂O₂ concentration and applied potential were optimized. Under optimum conditions, the biosensor gave linear response ranges of 7.0–110, 0.6–10 and 8–100μmol/L with the detection limits (S/N=3) of 0.5, 0.03 and 0.6μmol/L for catechol, hydroquinone and resorcin, respectively. The developed biosensor exhibited a short response time within only 8 s with good stability and reproducibility. Such new Hb biosensor showed great promise for rapid, simple analysis of polyphenols contents in real samples.