Electrochemical analysis of gold nanoparticles multifunctionalised with Cytochrome c and a zinc Porphyrin

Cytochrome c (Cyt c), known for its functional redox capabilities, plays a pivotal role in biological processes such as the electron transport chain and apoptosis. However, understanding how different conjugation strategies impact its structural and redox characteristics is limited. To fill this gap, we investigated the effects of conjugating Cyt c and a zinc(II) porphyrin (Zn Porph) to gold nanoparticles (AuNPs). We used circular dichroism (CD) spectroscopy to detect structural conformational changes in Cyt c upon conjugation and time-of-flight secondary ion mass spectrometry (ToF-SIMS) to identify protein orientation. Cyt c was predicted to have different orientations depending on the size of AuNPs and methods used to conjugate the protein, it was hypothesised that the orientation of Cyt c may influence the redox properties of the protein. The electrochemical properties of Cyt c were assessed using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). We used DPV-based to determine the heterogeneous rate constant (k0). The results show a lower k0for conjugated Cyt c than free Cyt c, likely due to structural changes in the protein. The spatial orientation of Cyt c had minimal influence on k0, while ligand density and AuNP size had an effect. The k0 value of Zn Porph did not decrease on conjugation. Despite these changes, Cyt c and Zn Porph maintained their electrochemical capabilities after conjugation.