Immobilization of hemoglobin on zirconium dioxide nanoparticles for preparation of a novel hydrogen peroxide biosensor

Direct electrochemistry and thermal stability of hemoglobin (Hb) immobilized on a nanometer-sized zirconium dioxide (ZrO₂) modified pyrolytic graphite (PG) electrode were studied. The immobilized Hb displayed a couple of stable and well-defined redox peaks with an electron transfer rate constant of (7.90±0.93)s^-1 and a formal potential of -0.361V (-0.12V versus NHE) in 0.1M pH 7.0 PBS. Both nanometer-sized ZrO₂ and dimethyl sulfoxide (DMSO) could accelerate the electron transfer between Hb and the electrode. Spectroscopy analysis of the Hb/ZrO₂/DMSO film showed that the immobilized Hb could retain its natural structure. This modified electrode showed a high thermal stability up to 74°C and an electrocatalytic activity to the reduction of hydrogen peroxide (H ₂O₂) without the aid of an electron mediator. The electrocatalytic response showed a linear dependence on the H₂O ₂ concentration ranging from 1.5 to 30.2μM with a detection limit of 0.14μM at 3σ. The apparent Michaelis-Menten constant K _M^app for H₂O₂ sensor was estimated to be (0.31±0.02) mM, showing a high affinity.