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

A novel biosensor for superoxide radical (O(2)(*-)) detection based on Pseudomonas aeruginosa azurin immobilized on gold electrode was designed. The rate constant of azurin reduction by O(2)(*-) was found to be 10(5)M(-1)s(-1) in solution and five times lower, i.e., 0.2 x 10(5)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 6s(-1). The sensitivity of this biosensor to O(2)(*-) was 6.8 x 10(2)Am(-2)M(-1). The response to the interference substances, such as uric acid, H(2)O(2), and dimethylsulfoxide was negligible below 10 microM. The electrode was applied in three O(2)(*-) generating systems: (i) xanthine oxidase (XOD), (ii) potassium superoxide (KO(2)), and (iii) stimulated neutrophil granulocytes. The latter was compared with luminol-amplified chemiluminescence. The biosensor responded to O(2)(*-) in all three environments, and the signals were antagonized by superoxide dismutase.