Superoxide dismutase-based third-generation biosensor for superoxide anion

A third-generation biosensor for superoxide anion (O₂^-) was developed by immobilizing superoxide dismutase (SOD) on a self-assembled monolayer of cysteine on gold electrode; i.e., a SOD/cysteine-modified gold electrode (SOD/Cys/Au) was fabricated. A rapid and direct electron transfer of SOD was realized at the gold electrode by using the cysteine molecule as an electron-transfer promoter. The promoted direct electron transfer of SOD and biomolecular recognition by the exploitation of specific and significant enzyme-substrate reactivity of SOD toward O₂^- combined with the low operating potential enabled a sensitive measurement of O₂^-. At SOD/Cys/Au, O₂^- could be specifically oxidized and reduced to O₂ and hydrogen peroxide, respectively, through the inherent catalytic reaction of SOD. This allowed us to measure O₂^- by polarizing the electrode both anodically and cathodically. We could successfully measure O₂^- by suitably polarizing the electrode, typically at 300 and -200 mV versus Ag/ AgC1 without the virtual interference from physiological levels of H₂O₂, ascorbic acid, uric acid, and metabolites of neurotransmitters. The response mechanism of SOD/Cys/Au to O₂^- and its sensor characteristics are also presented and discussed.