Studies On Third-generation Biosensors Based On Direct Electrochemistry Of The Natural Biomolecule With Iron Porphyrin

Biomacromolecule including proteins and enzymes are the primary groups of life. Direct electrochemistry of proteins and enzymes has aroused great interest in biological and bioelectrochemical field. Studies on direct electrochemistry of redox proteinslenzymes can be used to gain their thermodynamics and the kinetic properly, understand the mechanistic of electron exchange among proteins in biological systems and physiological action. Moreover, direct electron transfer between immobilized enzyme and underlying electrode can establish a foundation for fabricating new kinds of mediator-free biosensor, biofuel cell and bioreactors. In a sense, the essence of studying life process is to study the electron transfer process. Therefore, uses electrochemistry method to study the electron transfer process of proteins have the special superiority. Unfortunately, it is difficult for an enzyme to carry out a direct electrochemical reaction due to several factors. For example, enzymes would be adsorbed on the electrode surface, resulting in the denaturation and loss of their electrochemical, it is usual that the active sites are deeply buried in the molecules, and so on. So, in this dissertation, the third-generation electrochemical biosensors based on direct electron transfer between the enzyme and the underlying electrode were fabricated and developed, which may be widely used in chemical, biological, clinical, food, and environmental fields. We explored the application of nanomaterials and proper method in the fabrication of the third-generation biosensors, and the performance of these novel materials and the resulting biosensors was evaluated. These researches may open up new opportunities for designing novel biosensors and enhancing the performance of existing bioanalytical devices. The main points are summarized as follows:1. A poly (L-tyrosine)/cysteine/gold colloid/Hemoglobin modified electrode has been prepared. The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. The modified process was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The linear range for the determination of H₂O₂ is 3.5×10^-7 - 2.0×10^-3 mol/L with a detection limit of 1.0×10^-7 mol/L. The sensor exhibited high sensitivity, selectivity and stability. The method is applied to the determination of H₂O₂ with satisfactory results.2. We had prepared a hemoglobin (Hb)/gold colloid (nano-Au)/l-cysteine (l-cys)/nano-Au/nanoparticles Pt (nano-Pt)-chitosan (CHIT) composite film-modified platinum disk electrode to construct a biosensor for determination of H₂O₂. The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. The modified process was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The morphologies of different composite film were investigated with scanning electron microscopy (SEM) and the element of composite film was investigated with X-ray photoelectron spectroscopy (XPS). Analytical parameters such as pH and temperature were also studied. The linear range for the determination of H₂O₂ is 1.4×10^-7 to 6.6×10³ mol/L with a detection limit of 4.5×10^-8 mol/L (S/N = 3). The sensor achieved 95% of the steady-state current within 10 s. The sensor exhibited high sensitivity (17.62μA/(mmol/ L)), selectivity and stability. The method is applied to the determination of H₂O₂ with satisfactory results.3. A new hydrogen peroxide (H₂O₂) biosensor with high sensitivity was fabricated by immobilizing HRP on Pt disk substrate modified the multiple membrane matrixes consisting of CoFe₂O₄ nanoparticles and polyvinyl butyl (PVB) via a sol-gel method. The electrochemical properties of the modified Pt electrodes were characterized by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and chronoamperometry (CA). Results showed that the presence of CoFe₂O₄ nanoparticles could enhance the peak currents of redox species. Moreover, the H₂O₂ biosensor exhibited an excellent sensitivity and fast response time for H₂O₂ with a wide linear response range from 3.5×10^-6 mol/L to 2.0×10^-2mol/L.4. A HRP/ porosity gold colloid (nano-Au)/l-cysteine (1-cys) modified gold electrode has been prepared to construct a biosensor for determination of H₂O₂. Analytical parameters such as pH and temperature were also studied. The linear range for the determination of H₂O₂ is 3.5×10^-7 to 1.28×10^-3 mol/L with a detection limit of 1.0×10^-7 mol/L (S/N = 3). The method is applied to the determination of H₂O₂ with satisfactory results.