A biosensor based on cytochrome c immobilization on a poly-3-methylthiophene/multi-walled carbon nanotubes hybrid-modified electrode. Application to the electrochemical determination of nitrite

A cytochrome c (Cyt c) electrochemical biosensor constructed by immobilization of the biomolecule on a hybrid material consisting of the conducting polymer poly-(3-methylthiophene) (P3MT) and multi-walled carbon nanotubes (MWCNT) is reported. Cyt c was immobilized onto l-cysteine (l-Cys) self-assembled monolayer-modified P3MT/MWCNT/GCE at neutral pH, where positively charged Cyt c strongly interacts with l-Cys negatively charged sites. The electron transfer rate constant, ks, was 0.49 ± 0.01 s−1, and the surface coverage of Cyt c, Γ, (1.6 ± 0.8) × 10−11 mol cm−2. Under the optimized conditions, the mediatorless biosensor exhibits good electrocatalytic activity towards H2O2 reduction. The amperometric measurements of H2O2 at an applied potential of 0.0 V in 0.05 M Tris–HCl buffer of pH 8.0 exhibited good repeatability (RSD = 3.1%, n = 10) and an acceptable reproducibility for the biosensor preparation (RSD = 8.1%, n = 5). The lifetime of the biosensor was evaluated. A linear calibration graph for H2O2 (r = 0.998) was obtained between 0.7 and 400 μM, with a detection limit of 0.23 μM. The analytical characteristics for H2O2 determination were found to be better than those reported for other nanostructured cytochrome c biosensors. The apparent Michaelis–Menten constant for the reaction between Cyt c and H2O2 was 451 μM. The developed Cyt c/l-Cys/P3MT/MWCNT/GCE biosensor was applied for the biocatalytic determination of nitrite through the oxidation of NO 2 - to NO 3 - by electrogenerated [Fe4+-Cyt c]·species. Amperometry in stirred PBS solutions at a potential of +0.9 V allowed nitrite to be determined in a linear range between 10 and 100 μM, with a detection limit of 0.5 μM.