Glutathione peroxidase-based amperometric biosensor for the detection of S-nitrosothiols

摘要

A new biosensor is described for the detection of S-nitrosothiols (RSNOs) based on their decomposition by immobilized glutathione peroxidase (GPx), an enzyme containing selenocysteine residue that catalytically produces nitric oxide (NO) from RSNOs. The enzyme is entrapped at the distal tip of a planar amperometric NO sensor. The new biosensor shows good sensitivity, linearity, reversibility, and response times towards various RSNO species in PBS buffer, pH 7.4. In most cases, the response time is less than 5 min, and the response is linear up to 6 mu M of the tested RSNO species. The lowest detection limit is obtained for S-nitrosocysteine (CysNO), at approx. 0.2 mu M. The biosensor's sensitivity is not affected by the addition of EDTA as a chelating agent; an advantage over other potential catalytic enzymes that contain copper ion centers, such as CuZn-superoxide dismutase and xanthine oxidase. However, lifetime of the new sensor is limited, with sensitivity decrease of 50% after two days of use. Nonetheless, the new amperometric GPx based RSNO sensor could prove useful for detecting relative RSNO levels in biological samples, including whole blood.