Efficient mediatorless superoxide sensors using cytochrome c-modified electrodes: surface nano-organization for selectivity and controlled peroxidase activity

Amperometric superoxide anion sensor electrodes were prepared by immobilizing cytochrome c (Cyt c) on COOH-terminated Au–alkanethiolate monolayers. Monolayers and mixed-monolayers of 3-mercaptopropionic acid (MPA) with the coadsorbate, 3-mercaptopropanol (MP), were constructed on the surface of Au electrodes. The electrochemical characteristics and the superoxide anion sensor activities of cytochrome c immobilized on the monolayers and mixed-monolayers of MPA were found to depend largely on the structure of the underlying alkanethiolate monolayer. While cytochrome c on a MPA monolayer, Au/MPA/Cyt c, showed a reversible redox wave at 0.08 V (vs. Ag ∣ AgCl ∣ NaCl (sat.)), cytochrome c on the mixed-monolayer of MPA and MP, Au/MPA+MP/Cyt c, showed a wave at −0.01 V. Cytochrome c immobilized on the Au–alkanethiolate layer was supposed to acquire different conformations at each of the Au/MPA/Cyt c and Au/MPA+MP/Cyt c electrodes, leading to a difference in the thermodynamic potential of cytochrome c. The apparent heterogeneous electron-transfer rate constant, khet, is determined by fast-scan cyclic voltammetry to be (2.8±0.4)×103 s−1 for the Au/MPA+MP/Cyt c electrode. Both Au/MPA/Cyt c and Au/MPA+MP/Cyt c electrodes show anodic currents to O2− anion generated by enzymatic reaction with a response time of ∼15 s, and the magnitude of the current response is higher at Au/MPA+MP/Cyt c relative to that at Au/MPA/Cyt c. While H2O2 did not give any current response at the Au/MPA electrode at the applied potential of 0.15 V, cytochrome c immobilized on the Au–alkanethiolate layers was found to reduce H2O2; the cathodic current recorded for the reduction of H2O2 is very much higher at Au/MPA/Cyt c than that at Au/MPA+MP/Cyt c at all the applied potentials. The O2− anion sensor activity of the two electrodes was compared with that of Au/HS(CH2)10COOH/Cyt c. Among the cytochrome c-modified electrodes tested, Au/MPA+MP/Cyt c showed a high anodic current for O2− anion and low interferences, 17 and 7%, due to the electrochemical interferents, H2O2 and uric acid, respectively.