Hemoglobin (Hb) was successfully immobilized on a glassy carbon electrode modified with nickel oxide (NiO) nanoparticles. The electrodeposition of metallic nickel was carried out using cyclic voltammetry (20 scans between 1 and 1.0 V at a scan rate of 50 mV s1) in pH 4 acetate buffer solution containing 1 mM nickel nitrate. Then, the electrode was immersed in fresh phosphate solution containing 5 mgml1 hemoglobin and the potential was repetitively cycled (30 scans) from 1 to 0.5 V at a scan rate of 100 mV s1 for electrodissolution and passivation of nickel oxide layer and immobilization of hemoglobin. The presence of pair of well defined and nearly reversible CV peaks at about 0.07 V vs. reference electrode (pH 7) indicates the character of Hb heme Fe(III)/Fe(II) redox couple. The formal potentials of Hb in nickel oxide film was linearly varied in the range 2–11 with a slope of 58 mV/pH, indicating that the electron transfer is accompanied by single proton transportation. The surface coverage of Hb immobilized on nickel oxide film glassy carbon electrode was about 1.73 · 1011 mol cm2. The transfer coefficient (a) was calculated to be 0.45 and the heterogeneous electron transfer rate constant (ks) was 5.2 ± 0.5 s1, indicating great facilitation of the electron transfer between Hb and nickel oxide nanoparticles deposited on the electrode surfaces. The modified electrode shows excellent electrocatalytic activity toward hydrogen peroxide reduction. The Michaelis– Menten constant KM of 1.37 mM indicates that the Hb immobilized on to nickel oxide film retained its native activity. According to the direct electron transfer property and enhanced activity of Hb in nickel oxide film, it can be used as a new type of regretless biosensor without using any electron transfer mediator or specific reagent.
