Direct Electrochemistry Of Hemoglobin On Ionic Liquid Modified Electrode

Hemoglobin (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, which can also be proposed as a sensitive method for Hb determination. Room temperature ionic liquids (RTILs) have excellent physical and chemical properties, which can also be applied in the field of analytical chemistry. RTILs can be used either as supporting electrolyte or modifier for electrode. Since electroanalysis has inherent advantages such as lower detection limit and. wider dynamic range, it can be used to investigate the electrochemical behaviors of biomolecules. In this thesis the direct electrochemical investigation of Hb was connected with electroanalytical method. The paper can be summarized as follows:1. Two room temperature ionic liquids (RTILs) modified carbon paste electrode (CPE) were constructed based on the substitute of paraffin with 1-butyl-3-methyl imidazolium hexafluorophate ([BMIM]PF₆) or N-butylpyridinium hexafluoro phosphate (BPPF₆) to mix with graphite power. The optimal conditions for preparation were selected and the surface morphologies of the CILE were scanned by a scanning electron microscopy. The electrochemical behaviors of the CILE was investigated by using K₃[Fe(CN)₆] as probe and compared with that of traditional CPE. The result showed that the electrochemical response was greatly improved with the high conductivity of RTILs and the modified CILE showed good mechanic property and stability.2. The direct electrochemistry of Hb is carefully studied on the CILE. Hb was immobilized on the surface of CILE with clay and polyvinyl alcohol (PVA) film or Nafion/Nano-CaCO₃ film by step-by-step method. The characters of Hb in these modified films were investigated by electrochemical, spectra and scan electron microscopic methods. The results showed that Hb in these films retained the essential features of its native structure. A pair of well-defined and quasi-reversible cyclic voltammetric peaks were appeared. The electrochemical behaviours of Hb in the modified electrode were carefully investigated. The formal potential (E^o') was almost independent of the scan rate and the electrochemical parameters were also calculated. Hb has an iron-porphyrin heme as the active center, so it has the inherent reactivity as a peroxidase, which can catalyze the reduction of hydrogen peroxide. The fabricated Hb modified electrode exhibited excellent electrocatalytic behaviors to the reduction of H₂O₂, trichloroacetic acid (TCA) and NaNO₂.3. A new hemoglobin (Hb) modified electrode was constructed by using room temperature ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF₆) as binder. By mixing Hb with [BMIM]PF₆ and graphite powder together, a carbon paste electrode containing Hb and IL (Hb-IL-CPE) was prepared and further characterized by scanning electron microscopy (SEM) and electrochemical method. The Hb in the carbon paste kept it bioactivity and showed good direct electrochemical behaviors. A pair of well-defined, quasi-reversible redox peaks was appeared at pH 7.0 phosphate buffer solution (PBS). The electrochemical parameters such as the electron transfer number (n), the electron transfer coefficient (α) and the heterogeneous electron transfer kinetic constant (ks) of the electrode reaction were calculated as 1.2, 0.465 and 0.434 s^-1, respectively. The fabricated Hb-IL-CPE exhibited excellent electrocatalytic behaviors to the reduction of H₂O₂. The calibration range was between 8.0×10^-6 mol/L to 2.8×10^-4 mol/L with the linear regression as Iss (μA)=0.12 C (μmol/L)+0.73 (γ=0.997, n=18) and the detection limit as 1.0×10^-6 mol/L (3σ). The apparent Michaelis -Menten constant (K_M^app) of this sensor was calculated to be 1.103 mmol/L. The surface of biosensor can be renewed by simple polishing step and showed good reproducibility.