Electrochemical Sensing Of Hydrogen Peroxide And Electrochemical Investigation Of Hemeproteins

The investigations of the properties for the unfolding proteins induced by chemical denaturant, interface induced and other factors by electrochemistry, electrochemical biosensing and spectroscopic methods can help to obtain the dynamic information between the protein structure and biological activity and have more in-depth understanding the relationship of protein structure and performance. In this dissertation, nano materials of PtNPs and Ag@C-MnO₂ were synthesized. Two kinds of hydrogen peroxide biosensors based on these nano materials were constructed and new analysis methods of determination of H₂O₂ were established. The conformational changes of Cyt-c, Hb and Mb were studied by electrochemistry, electrochemical biosensing and spectroscopic methods. Electrochemical biosensors based on uMb/clay and uHb/Co-GE were investigated for H₂O₂ determination with much high sensitivity and low detection limit. The studies enrich the research contents of electrochemistry, which can provide references for elucidating the mechanism of protein electron transfer, interface adsorption and medical application. There are four chapters in the dissertation, and the main contributions of the author are presented as follow:1. The PtNPs was synthesized by controlled gas-liquid interface reaction and the nonenzymatic electrochemical sensor of PtNPs/GCE was constructed. The material properties of sensing interface, the relationship between electrochemical and electrocatalytic properties and the sensor response to H₂O₂ were investigated. A new method for the determination of H₂O₂ was established. The results of TEM and XRD on the surface properties of the materials showed that the PtNPs dispersion was high, the size of the particle was uniform, and the particle size was about 5 nm. Electrochemical studies showed that the conductivity of PtNPs/GCE was better than GCE. PtNPs had obvious electrocatalytic reduction on H₂O₂. The linear range for the determination of H₂O₂ was 0.5～375 μmol·L^-1, and the detection limit 0.05 μmol·L^-1 （S/N=3）.Colloidal C kernel covering of Ag nanoparticles was synthesized through hydrothermal method, and the electrochemical sensor based on Ag@C-MnO₂ by using MnO₂ nanotube and Ag@C was constructed. The material properties and components of sensing interface, the relationship between the sensor and electrochemical response to H₂O₂ were investigated. A new method for the determination of H₂O₂ was established. The surface properties research of the materials by SEM and EDS showed that the Ag@C was spherical with uniform particle size with a diameter of about 250 nm. the diameter of MnO₂ nanotubes was about 60～110 nm. Electrochemical studies showed that Ag@C-MnO₂ composites could effectively improve the electrode electron transfer efficiency, and the sensor had good electrocatalytic activity toward H₂O₂. The linear range for the determination of H₂O₂ was 5.0×10^-7～5.7×10^-3 mol·L^-1, the detection limit 0.17 μmol·L^-1 （S/N=3）, and the response time was less than 3 s.2. The conformational changes of Cyt-c induced by guanidine hydrochloride was investigated by CV using bare electrode. Electrochemical studies showed that the denatured ability of guanidine hydrochloride was very strong, which could strongly induce conformational change of Cyt-c with heme fallen from Cyt-c. The comparison of electrochemistry, UV-Vis and MFS showed that the unfolding rate curves using the electrochemical and spectroscopic methods had a good consistency. Therefore, the method was simple and effective to study the conformational change of Cyt-c.The conformation change of bovine Hb during the unfolding process induced by urea and acid was investigated by MPA modified Au electrode. An electrochemical method for the study of Hb conformational change was established. The change of unfolding percentage showed that the Hb unfolding induced by urea was a two-step, three-state transition process, while the unfolding induced by acid was a two-state transition process, which confirmed that the denatured ability of acid was stronger than that of urea. In addition, the free energy change of Hb in water solution was 9.157 kJ·mol^-1, and the difference between the oxidation state and reduction state of unfolding Hb was -2.8 kJ·mol^-1. The present work holds great promise for the design of novel sensitive biosensors.The conformational changes of Mb induced by urea and acid were investigated using electrochemical and spectroscopic methods and the controlled unfolding Mb was obtained, which was used to construct the electrochemical biosensor of uMb/clay/GCE. The relationships between the unfolding Mb on sensing interface and the type of denaturant, the concentration of denaturant and the response performance were investigated. A new method for the determination of H₂O₂ was established. The linear range for the determination of H₂O₂ was 8.0×10^-7～1.8×10^-4 mol·L^-1, the detection limit 0.3 μmo·L^-1 （S/N=3）, and the sensitivity 151.5 μA·(mmol·L^-1)^-1. The response time was less than 3 s. Comparison with electrochemical biosensor based on natural state Mb, the electroactive centers in Mb on the sensor exposed greatly with the synergistic effect of the two kinds of denaturants. The efficiency of electron transfer was improved and sensitivity for the detection of H₂O₂ was nearly increased 10-fold.Co-GE nanocomposites were synthesized by electrodeposition method. The Hb folding intermediate was obtained induced by urea and acid and the electrochemical biosensor based on uHb/Co-GE was constructed. The relationships between the unfolding Hb on sensing interface and the type of denaturant, the concentration of denaturant and the response performance were investigated. A new method for the determination of H₂O₂ was established. The research results showed that the sensor had good electrocatalytic activity toward H₂O₂. The linear range for the determination of H₂O₂ was 2.5×10^-7～1.9×10^-4 mol·L^-1, the detection limit 0.08 μmol·L^-1 （S/N=3）, and the sensitivity 116.3 μA·(mmol·L^-1)-1. The sensitivity for the detection of H₂O₂ was increased 8-fold compared to the electrochemical biosensor based on natural Hb, and the detection limit was decreased 4-fold compared to the electrochemical biosensor based on uMb/clay.