Bioelectrochemistry Of Redox Protein Based On Micro/nanostructure Materials

Direct electrochemistry of redox proteins has aroused great interest in biological and bioelectrochemical fields. Protein film voltammetry (PFV) is a newly effective method in studying the direct electrochemistry of redox proteins, which can offer essential physicochemical data concerning the kinetics and energetics of protein redox reactions. Moreover, direct electron transfer between immobilized protein and electrode can establish a foundation for fabricating new kinds of mediator-free biosensors. In this work, microbial exocellular polysaccharide-gellan gum (GG), poly (ethylene glycol) (PEG) grafted multi-walled carbon nanotubes (PEG-g-MWCNTs), ordered mesoporous crystalline C-TiO₂ (MCT) composites, and the novel worm-like titanium oxide (wTiO₂) were used to immobilize the protein and the obtained results are as follows:1. A new composite film of microbial exocellular polysaccharide-gellan gum (GG) and room temperature ionic liquid (IL) 1-butyl-3-methyl-imidazolium hexafluorophosphate (BMIMPF6) was firstly used as an immobilization matrix to entrap proteins and its bioelectrochemical properties were studied. Hemoglobin (Hb) was chosen as a model protein to investigate the composite system. UV-vis spectroscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the composite film. The obtained results demonstrated that the Hb molecule in the film kept its native structure and showed its good electrochemical behavior. A pair of well-defined, quasi-reversible cyclic voltammetric peaks appeared in pH7.0 phosphate buffer solutions (PBS, 0.1M), with the formal potential (E°') of -0.368V (vs. SCE), which was the characteristic of Hb Fe (III)/Fe (II) redox couples. The Hb- IL-GG modified electrode also showed an excellent electrocatalytic behavior to the reduction of hydrogen peroxide (H₂O₂).2. The novel poly (ethylene glycol) (PEG) grafted multi-walled carbon nanotubes (PEG-g-MWCNTs) were synthesized by the covalent functionalization of MWCNTs with hydroxyl-terminated PEG chains, exhibited excellent hydrophilicity and biocompatibility. The PEG-g-MWCNTs were characterized by Fourier transform infrared spectra, transmission electron microscopy, and thermogravimetric analysis, which verified that PEG chains were grafted onto the surface of the MWCNTs. The PEG-g-MWCNTs were then used as substrates for the immobilization of hemoglobin (Hb) and their bioelectrochemical behaviors were studied. Electrochemical impedance spectroscopy was used to confirm the adsorption of Hb onto the surface of PEG-g-MWCNTs. The cyclic voltammetry results of Hb/PEG-g-MWCNT-modified electrode showed a pair of well-defined and quasi reversible redox peaks centered at approximate -0.34 V (vs. saturated calomel electrode) , which was the characteristic peaks of Hb Fe(III)/Fe(II), in pH 7.0 phosphate buffer solution. Hb immobilized onto the surface of PEG-g-MWCNTs demonstrated good bioelectrocatalytic activities for the reduction of nitrite.3. A direct-triblock-copolymer templating method is demonstrated to synthesize ordered mesoporous crystalline C-TiO₂ (MCT) composites using phenolic resins and acid-base pairs [acidic TiCl₄ and basic counterpart Ti (OC₄H₇)₄] as carbon and titanium sources, respectively. The composites possess highly crystalline anatase pore walls that are"glued"by amorphous carbon, ordered mesostructure, high surface areas (200 m²/g), and large pore volumes (0.15 cm³/g). The titania content is as high as 87 wt%. The MCT composites were then used as substrates for the immobilization of hemoglobin (Hb) and their bioelectrochemical behaviors were studied. The cyclic voltammetry results of MCT modified GC electrode showed a pair of well-defined and quasi reversible redox peaks centered at approximate -0.336 V (vs.SCE), which was the characteristic peaks of Hb Fe(III)/Fe(II), in pH 7.0 phosphate buffer solution. Hb immobilized onto the surface of MCT demonstrated good bioelectrocatalytic activities for the reduction of H₂O₂.4. A novel composite film of worm-like titanium oxide (wTiO₂) combined with ionic liquid was firstly used for horseradish peroxidase (HRP)) immobilization and its bioelectrochemical properties were studied. UV-Vis absorption and electrochemical impedance spectroscopy (EIS) were used to characterize the composite film. The results showed the wTiO₂ and IL composite film provided a friendly microenvironment to retain the bioactivity of the immobilized HRP. The resulting electrode (HRP/wTiO₂/IL) gave a pair of well-defined, quasi reversible redox peaks centered at approximate -0.349V (vs. SCE) in pH 7.0 phosphate buffer solution. It reflects the characteristic of HRP heme Fe (III)/Fe (II) redox couple with fast heterogeneous electron transfer rate. The immobilized HRP also displayed excellent electrocatalytical responses to the reduction of hydrogen peroxide.