Characterization of a mediated amperometric biosensor for hypoxanthine using deflavo xanthine oxidase

Deflavo xanthine oxidase was prepared by removing the flavine adenine dinucleotide (FAD) group from the native enzyme using 2 M CaCl₂. The deflavo enzyme was effectively stabilized by 5 mM EDTA in Tris buffer solution, pH 8, and retained about 80% of its activity after several days when stored in this medium at 4°C. The deflavo enzyme was then used for the development of a mediated amperometric electrode for hypoxanthine/xanthine using 1,1′‐dimethylferricinium as mediator, and its performance characteristics were compared to a native enzyme electrode. The resulting deflavo xanthine oxidase electrode was not noticeably sensitive to oxygen in the presence of hypoxanthin while its capability to interact with ferricinium remained intact. The electrode sensor with immobilized deflavo enzyme showed high operational and storage stabilities with a detection limit of about 0.1 μM. No significant sensitivity loss was observed on the enzyme electrode after 150 repeated analyses during a 7.5 hour operation. The deflavo enzyme electrode retained 80% of its sensitivity when stored at 4°C after a month.