The Preparation Of Five Heme Protein Modified Electrode And Direct Elecctrochemistry

Heme proteins are widely present in organism, which have many significant physiological functions, such as transfer electron, transport oxygen, the role of enzymes. Direct electrochemistry of heme proteins has aroused great interest in biological and bioelectroehemistry fields.Studies on the direct electrochemistry of heme proteins are of great importance both for studying the intrinsic heme behaviors of proteins and fabricating new kinds of biosensors without the addition of mediators.Ionic liquid (IL) is recognized as "green solvents" due to its special physical and chemical properties, such as high ionic conductivity, better chemical and thermal stability, wide electrochemical window, smaller volatility and non-toxic, etc.. IL has been widely used in the field of electroanalytical chemistry, especially electrochemical biosensors. It not only can be used as solvent and supporting electrolyte, but also the binders and modifier to prepare the modified electrode. In this paper, IL is used as modifiers and binders to fabricate five kinds of heme proteins modified electrode and the direct electrochemical behavior of heme proteins is studied in details. The thesis can be summarized as follows:1. Hemoglobin (Hb) and myoglobin (Mb) were immobilized on the surface of carbon ionic liquid electrode (CILE) by step-by-step method and drop-coating method. The direct electrochemistry of Hb and Mb was further studied. CILE was constructed based on the substitute of paraffin with 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4), 1-ethyl-3-methylimidazolium ethylsulfate ([EMIM]EtOSO3) and 1-acetic -3-methylimidazolium hexafluorophosphate (AMIMPF6) to mix with graphite powder. Hb and Mb were immobilized on the surface of CILE with different substances such as DNA, Nafion, IL, chitosan (CTS), dextran (De) to fabricate the modified electrodes such as Nafion/Hb/ DNA/CILE, CTS-IL-Mb/CILE and De/Mb/IL-DNA/CILE. The characteristics of Hb and Mb in these modified films were investigated by UV-Vis spectrum, scan electron microscopy (SEM), FT-IR spectrum and electrochemical methods. The results showed that Hb and Mb in these films retained its native structure. A pair of well-defined and quasi-reversible cyclic voltammetric peaks appeared, which indicated that the direct electrochemistry of heme proteins were realized. The electrochemical behaviors of Hb and Mb in the modified electrode were carefully investigated and the electrochemical parameters were calculated. The modified electrodes showed excellent electrocatalytic behaviors to the reduction of trichloroacetic acid (TCA), H2O2, and sodium nitrite (NaNO2) with the apparent Michaelis-Menten constants (KMapp) calculated, respectively.2. Hb and Mb were immobilized on the surface of CILE by layer-by-layer assembly (LBL) method. Direct electrochemistry and electrocatalytic activity was studied carefully by electrochemical methods. LBL method is often used for the construction of protein based electrochemical biosensors, which is mainly based on the alternative adsorption of oppositely charged species on the solid surface. Using this method, two-kind modified electrodes {IL/Hb}3/CILE and {IL/Mb}3/CILE were prepared. The characteristics of Hb and Mb in these multilayer films were investigated by UV-Vis spectrum, electrochemical impedance spectroscopy (EIS), FT-IR spectrum and electrochemical methods. The direct electrochemistry and electrocatalytic ability of the redox protein were investigated. The electrochemical parameters and the apparent Michaelis-Menten constants (KMapp) were further calculated.