Impedance spectroscopy and conductometric biosensing for probing catalase reaction with cyanide as ligand and inhibitor.

In this work, a new biosensor was prepared through immobilization of bovine liver catalase in a photoreticulated poly (vinyl alcohol) membrane at the surface of a conductometric transducer. This biosensor was used to study the kinetics of catalase-H(2)0(2) reaction and its inhibition by cyanide. Immobilized catalase exhibited a Michaelis-Menten behaviour at low H(2)0(2) concentrations (<100mM) with apparent constant K(M)(app)=84±3mM and maximal initial velocity V(M)(app)=13.4μS min(-1). Inhibition by cyanide was found to be non-competitive and inhibition binding constant K(i) was 13.9±0.3μM. The decrease of the biosensor response by increasing cyanide concentration was linear up to 50μM, with a cyanide detection limit of 6μM. In parallel, electrochemical characteristics of the catalase/PVA biomembrane and its interaction with cyanide were studied by cyclic voltammetry and impedance spectroscopy. Addition of the biomembrane onto the gold electrodes induced a significant increase of the interfacial polarization resistance R(P). On the contrary, cyanide binding resulted in a decrease of Rp proportional to KCN concentration in the 4 to 50μM range. Inhibition coefficient I(50) calculated by this powerful label-free and substrate-free technique (24.3μM) was in good agreement with that determined from the substrate-dependent conductometric biosensor (24.9μM).