Direct electrochemistry of cytochrome c immobilized on gold electrode surface via Zr(IV) ion glue and its activity for ascorbic acid

• Zr(IV) ion helps immobilization of Cyt c on Au, preserving its activity over 32 days.
• Γ (1.15 × 10− 11 mol cm− 2) and ket (8.62 s− 1) of Cyt c are found electrochemically.
• The method is simple, effective and free of immobilizing activators and reagents.
• Immobilized Cyt c shows a linear response to ascorbic acid: 10–1300 μM AA, DL 5 μM.

Direct electrochemistry of cytochrome c (Cyt c) is achieved via Zr(IV) ion as an immobilization matrix to interface Cyt c on gold surface via thiol self-assembled monolayers. Steps of surface modification and electrocatalytic activity of the immobilized Cyt c are followed by voltammetry, impedance spectroscopy, chronoampetrometry, and attenuated total reflectance Fourier transform infrared (ATR–FTIR) spectroscopy. The results indicate that the native structure of Cyt c is conserved during the immobilization process. The immobilization method is rather simple, effective and free of immobilizing activators and reagents. Direct electron transfer rate constant and surface coverage of the immobilized Cyt c are found as 8.62(± 1.98) s− 1 and 1.15(± 0.38) × 10− 11 mol cm− 2, respectively. Bioactivity studies of the immobilized Cyt c toward oxidation of the ascorbic acid (AA) substrate show a linear response, from 10.0 μM to 1.30 mM AA, with a detection limit of 5.0(± 1.8) μM AA and mean relative standard deviations varied from 13.7% to 3.7% for n = 4 at each point. A value of 1.6(± 0.8) mM AA is found for the Michaelis–Menten constant of Au-MPA-Zr(IV)-Cyt c toward AA for the first time. The tightly immobilized Cyt c maintains its bioactivity for more than 32 days storage at 4 °C.