Direct electron-transfer and electrochemical catalysis of hemoglobin immobilized on mesoporous Al2O3

The direct electrochemistry of hemoglobin (Hb) has been achieved by immobilizing Hb on mesoporous Al₂O₃ (meso-Al₂O₃) film modified glassy carbon (GC) electrode. Meso-Al₂O₃ shows significant promotion to the direct electron-transfer of Hb, thus it exhibits a pair of well defined and quasi-reversible peaks with a formal potential of −0.345 V (vs. SCE). The electron-transfer rate constant (k_s) is estimated to be 3.17 s⁻¹. The immobilized Hb retains its biological activity well and shows high catalytic activity to the reduction of hydrogen peroxide (H₂O₂) and nitrite (NO₂⁻). Under the optimized experimental conditions, the catalytic currents are linear to the concentrations of H₂O₂ and NO₂⁻ in the ranges of 0.195-20.5 μM and 0.2-10 mM, respectively. The corresponding detection limits are 1.95 × 10⁻⁸ M and 3 × 10⁻⁵ M (S/N = 3). The resulting protein electrode has high thermal stability and good reproducibility due to the protection effect of meso-Al₂O₃. Ultraviolet visible (UV-vis) absorption spectra and reflection-absorption infrared (RAIR) spectra display that Hb keeps almost natural structure in the meso-Al₂O₃ film. The N₂ adsorption-desorption experiments show that the pore size of meso-Al₂O₃ is about 14.4 nm, suiting for the encapsulation of Hb (average size: 5.5 nm) well. Therefore, meso-Al₂O₃ is an alternative matrix for protein immobilization and biosensor preparation.