Construction And Application Of Several Hemoglobin Modified Electrodes

Hemoglboin(Hb) is an important respiratory protein in red cells, which can be used as the carrier of oxygen. Studies on the direct electrochemical behaviors of Hb can be used for the understanding of the mechanisms of electron transfer reactions and oxidation reduction in life processes. Construction of sensitive and selective sensing interface is key for the study of electrochemical behaviors of Hb. In this thesis, electrochemical biosensors of Hb were fabricated based on three types of sensing interfaces, and its electrochemical behaviors were studied in details. The biosensors were used for amperometric determination of H2O2. These studies may provide new thoughts for the construction of high sensitive and selective electrochemical biosensing interfaces and extending the application of electroanalytical techniques in bioanalysis. The paper can be summarized as follows:1. Direct electrochemistry and electrocatalytic behaviors of Hb, which entrapped on ionic liquid carbon paste electrode modified with nano-nickel oxide and chitosan-nano-CaCO3, were carefully studied. The results indicated that Hb remained its bioactivity on the modified electrode, showing a pair of well-defined, quasi-reversible cyclic voltammetric peaks with the apparent standard potential (Eo) at about-0.214 V (vs. SCE) in pH 7.0 PBS. The number of electron transferred (n) was calculated as 0.93, the coefficient of electron transferred (α) was 0.573, the average apparent heterogeneous electron transfer rate constant (Ks) obtained by fitting CVdate for Hb was 1.74 s-1 and the Michaelies-Menten constant Km value was estimated to be 1.245 mmol/L. The biosensor had good long-term stability, high sensitive and selective, and showed excellent electrocatalytic responses to the reduction of H2O2.2. A hemoglobin modified electrode was constructed by using room temperature ionic liquid [bmim]PF6 as modifier. Hb was immobilized on the CILE with an agarose-DMF composite film. Ultraviolet visible spectroscopy, fourier transform infrared spectroscopy and cyclic voltammetry were used to characterize the hemoglobin on the modified electode. Hb on the modified electrode kept it bioactivity and showed good direct electrochemical behavior. A fast direct electron transfer with the formal potential (Eo) of-0.15 V(vs.SCE) and a pair of well-defied and nearly reversible cyclic voltammetric peaks for HbFe(III)/Fe(II) redox couple in phosphate buffered saline solution(pH= 7.0) was obtained. The fabricated agarose/CILE exhibited excellent electrocatalytic ability to the reduction of H2O2. The calibration range was 2.0×10-6 mol/L to 1.2×10-3mol/L, and the detection limit was 2.0×10-7 mol/L (S/N= 3). The apparent Michaelis-Menten constant (KMapp) was calculated to be 1.495 mmol/L. The biosensor can be renewed by simple polishing step and showed good reproducibility.3. The direct electrochemistry of Hb was achieved by immobilizing Hb-chitosan (chit) on a gold colloid (AuNPs)/3-aminopropyl triethylene silane (APS)/prussian blue (PB) composite film modified glassy carbon electrode (GCE). UV-Vis absorption spectra, FT-IR spectra and cyclic voltammetry analysis indicated that the immobilized Hb could retain its native structure in the chitosan/gold colloid/3-aminopropyl triethylene silane/prussian blue composite film. The composite film showed significant promotion to the direct electron-transfer of Hb. It exhibited a pair of well defined and quasi-reversible peaks with a formal potential of 0.205 V (vs. SCE). The formal potential of Hb heme Fe(Ⅲ)/Fe(Ⅱ) couple shifted linearly between pH 4.0-9.0 with a slope of 44.5 mV/pH, suggesting that one proton was coupled to each electron transfer in the electrochemical reaction. The Hb-chitosan/gold colloid/3-aminopropyl triethylene silane/prussian blue composite film exhibited good electrocatalytic activity for the reduction of H2O2. The catalytic currents were linear to the concentrations of H2O2 in the ranges of 2.0×10-6-4.8×10-4 mol/L, and the detection limit was 1.0×10-7 mol/L (S/N= 3). The apparent Michaelis-Menten constant(KMapp) of the sensor was calculated to be 1.992 mmol/L. The biosensor exhibited high sensibility, good reproducibility, and long-term stability.