Garlic peroxidase immobilized into chitosan matrix suitable for biosensors applications

Peroxidase X1 (POX1) isoenzyme was purified from garlic bulb (Allium sativum L.). Native-PAGE profile showed two isoforms partially purified (designated POX1A and POX1B). A POX1B-based electrode showed great potential for monitoring hydrogen peroxide in biological samples. Chitosan was used as a matrix for enzyme immobilization. The enzyme activities were studied by photometry. Immobilization was accomplished by either inclusion in a thin film or adsorption to cross-linked microspheres. Two linkage agents were used: glutaraldehyde and glyoxal. The best immobilization and activity yields (82%, 95%) were obtained when POX1B was incorporated within a chitosan/glyoxal film. The effect of temperature on the immobilized enzyme was tested. Results showed that full activity was retained after 40 min incubation at 40 °C. Anchored POX1B inside chitosan was used for biosensor design. Cyclic voltammetry and impedance spectroscopy were employed to analyze electrochemical properties of the modified gold electrode and to monitor hydrogen peroxide. The biosensor was very sensitive and attained a detection limit of 100 nM.