Direct electrochemistry and electrocatalysis of hemoglobin immobilized in a magnetic nanoparticles-chitosan film

Magnetic nanoparticles (Fe₃O₄) were synthesized by a chemical coprecipitation method. X-ray diffraction (XRD) and transmission electron microscope (TEM) were used to confirm the crystallite structure and the particle's radius. The Fe₃O₄ nanoparticles and chitosan (CS) were mixed to form a matrix in which haemoglobin (Hb) can be immobilized for the fabrication of H₂O₂ biosensor. The Fe₃O₄-CS-Hb film exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks due to the redox of Hb-heme Fe (III)/Fe (II) in a pH 7.0 phosphate buffer. The formal potential of Hb-heme Fe(III)/Fe(II) couple varied linearly with the increase of pH in the range of 4.0-10.0 with a slope of 46.5 mV pH⁻¹, indicating that electron transfer was accompanied with single proton transportation in the electrochemical reaction. The surface coverage of Hb immobilized on Fe₃O₄-CS film glassy carbon electrode was about 1.13 × 10⁻¹⁰ mol cm⁻². The heterogeneous electron transfer rate constant (k_s) was 1.04 s⁻¹, indicating great facilitation of the electron transfer between Hb and magnetic nanoparticles-chitosan modified electrode. The modified electrode showed excellent electrocatalytic activity toward oxygen and hydrogen peroxide reduction. The apparent Michaelis-Menten constant for H₂O₂ was estimated to be 38.1 μmol L⁻¹.