Electrochemical characterization and application of azurin-modified gold electrodes for detection of superoxide

A novel biosensor for superoxide radical (O2−) detection based on Pseudomonas aeruginosa azurin immobilized on gold electrode was designed. The rate constant of azurin reduction by O2− was found to be 105 M−1 s−1 in solution and five times lower, i.e., 0.2 × 105 M−1 s−1, for azurin coupled to gold by 3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP). The electron transfer rate between the protein and the electrode ranged from 2 to 6 s−1. The sensitivity of this biosensor to O2− was 6.8 × 102 A m−2 M−1. The response to the interference substances, such as uric acid, H2O2, and dimethylsulfoxide was negligible below 10 μM. The electrode was applied in three O2− generating systems: (i) xanthine oxidase (XOD), (ii) potassium superoxide (KO2), and (iii) stimulated neutrophil granulocytes. The latter was compared with luminol-amplified chemiluminescence. The biosensor responded to O2− in all three environments, and the signals were antagonized by superoxide dismutase.