Preparation and biochemical characterisation of nanoconjugates of functionalized carbon nanotubes and cytochrome c

Objective(s): The present work deals with the preparation of nanobioconjugates based on the immobilization of cytochrome c (cyt c) on functionalized multi-wall carbon nanotubes (f-MWCNTs). The effect of the nanosupport and the immobilization procedure on the biochemical and structural characteristics of the immobilized protein was investigated. Methods: The MWCNTs were functionalized to provide alkyl chains with different length and terminal functional groups on their surface. The immobilization of cyt c was achieved through physical adsorption and covalent binding. Cyt-c-based nanoconjugates were characterized in terms of peroxidase activity and stability of protein, while UV-visible spectroscopy was used to investigate the structural characteristics of the immobilized protein. Results: The loading of cyt c on f-MWCNTs was effectively achieved, with immobilization yields reaching up to 77%. The peroxidase activity of cyt c was higher in the case of non covalent immobilization compared to that of covalent procedure. Immobilized cyt c exhibited higher thermal stability than the native protein after 24 h incubation at 40oC, while it preserved up to 100% of its initial activity after incubation in the presence of a denaturing agent such as H2O2. No significant changes in the heme microenvironment of cyt c were observed in the presence of f-MWCNTs. Conclusions: This study has demonstrated that f-MWCNTs are effective supports for the immobilization of cyt c, providing a universally applicable platform for the development of bionanoconjugates with potential use in a wide variety of fields in nanobiocatalysis, biosensing and nanomedicine.