Researches On The Electrochemical Detection Of BSA Damage And The Direct Electrochemical Of Heme Proteins

Protein is the basis material of life and important part of organism. It exercise or participate in almost all the life activities of human body. And it is also the main objective of free radicals. Many biological processes, such as diseases and aging, have a close relationship with free radicals immediate-damage to protein. So it is very important for investigating the mechanism of damage processes induced by unstable active specie. Room temperature ionic liquid (IL), a new green solvent, has received detail exploration in myriad of research areas owing to their unique properties including low toxicity, negligible vapor pressure, high viscosity, wide potential window, good ionic conductivity and electrochemical stability. In the field of electroanalysis, the applications of IL generally focus on as an alternative solvents or supporting electrolyte, and the electrode modified material in application of biosensors and biocatalysis. Further more, IL has greater viscosity. It is a non-proton solvent so the diffusion process of free radicals in the ionic liquid system is slow. It reduces the chances of collision with each other, thus extending their life. It provides more appropriate environment for the free radicals.The main contents and results are summarized as follows:(1) Electrochemical study of bovine serum albumin damage induced by Fenton reaction using tris (2,2’-bipyridyl) cobalt (Ⅲ) perchlorate as the electroactive indicatorThe Fenton reagents in aqueous solution mediated oxidative damage to BSA were-investigated. The generation of hydroxyl radical was validated by ultraviolet-visible (UV-vis) spectroscopy. The simple electrochemical procedure has been constructed on the glassy carbon electrode (GCE) surface by BSA direct adsorption technique. Co(bpy)33+in pH7.0tris-HCl was used as a redox indicator to monitor BSA damage induced by hydroxyl radical (·OH), which was produced from Fenton reaction. The electrochemical behaviors of the underlying electrodes were characterized by differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). UV-vis spectroscopy was used to verify the BSA damage with the universal index of the protein carbonyl group content. The optimizations of the Fe2+/H2O2ratio and incubation time on BSA damage were explored. Moreover, the protections of BSA from damage by antioxidants were investigated. The results demonstrated that BSA was damaged to the greatest extent when the concentration of Fe2+is5mM and that of H2O2is45mM for20min. The most effective protection effect of antioxidants for the concentrations of AA and catechin were about70μM and30μM.(2) An electrochemical biosensor for rapid detection of bovine serum albumin damage induced by hydroxyl radicals in the room temperature ionic liquidFenton reagents in room temperature ionic liquid-1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF6]) mediated oxidative damage to BSA were also investigated. The biosensor displayed the great degree of BSA oxidation damage at40min of the incubation time and at0.5mM FeSO4and8mM H2O2of the Fenton reagents concentrations. The optimum concentrations of AA and catechin to protect BSA from oxidation efficiently were25μM and20μM, respectively. The damage in [bmim][PF6] compared with that in an aqueous solvent media confirmed that [bmim][PF6] was a more suitable medium for the electrochemical detection of BSA damage. It indicted that the life of free radicals in ILs increased sharply due to the high viscosity of ILs. At the occasion, the damage for BSA was much serious than in the aqueous solvent.(3) Nitromethane biosensor based on heme proteins incorporated in sodium carboxymethyl cellulose/room temperature ionic liquid/multi-walled carbon nanotubes filmsA novel amperometric biosensor for nitromethane (CH3NO2) based on immobilization of heme proteins, sodium carboxymethyl cellulose (CMC), room temperature ionic liquid-1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF4]) and multi-walled carbon nanotubes (MWNTs) on the glassy carbon electrodes was constructed. The surface morphologies of the representative heme proteins hybrid films were observed by scanning electron microscopy (SEM). A pair of stable and well-defined redox peaks were observed for the heme protein incorporated film electrodes. The different parameters, including working potential, pH of supporting electrolyte and the scan rate that governed the performance of the biosensor have been studied in detail and optimized. The charge transfer coefficients (a), the apparent electron transfer rate constant (ks) and the number of electrons transferred (n) for hemoglobin (Hb), myoglobin (Mb), horseradish peroxidase (HRP) and cytochrome c (Cyt c) were calculated detailedly. Electrocatalytic parameters for the determination of CH3NO2with the four heme proteins film electrodes were evaluated by chronoamperometry. The presence of both (MWNTs and IL showed an extraordinary synergistic effect, that is, not only dramatically facilitated the electron transfer of Heme proteins, but also greatly enhanced electrocatalytic activity towards CH3NO2. Beside this, the resultant electrodes displayed low detection limit and improved sensitivity for the determination of CH3NO2.