Molecular Wiring of Chlorophyll a and Cytochrome c on Carbon Black for Amplified Photocurrent Generation and ROS Profiling in Cancer Cells

Abstract

Natural processes like photosynthesis involve complex electron transfer pathways, including both direct electron transfer (DET) and molecular wiring, which are essential for biological energy conversion and storage. Replicating such systems in artificial platforms remains a major scientific challenge. In this study, we successfully fabricated a bioelectrode comprising chlorophyll, specifically plant-derived chlorophyll a (Chla), molecularly wired with cytochrome c (Cytc) on a carbon black (CB)-modified electrode surface. This hybrid electrode, designated as CB@Cytc-Chla, was prepared using a simple solution-phase approach. The system demonstrated efficient DET between the protein ensemble and the electrode surface. Cyclic voltammetry in nitrogen-purged pH 7 phosphate buffer revealed a well-defined and reversible redox couple at E° = -0.2 V vs Ag/AgCl, with a surface coverage value of 2.96 nmol cm⁻². Control experiments using electrodes modified with individual proteins (Cytc or Chla alone) showed no such redox behavior, highlighting the importance of molecular wiring in the composite assembly. To probe specific interactions between Chla and Cytc, in situ electrochemical quartz crystal microbalance (EQCM) analysis was performed, confirming strong binding affinity. Furthermore, in situ scanning electrochemical microscopy (SECM) in feedback mode revealed distinct electroactive sites on the bioelectrode surface. Under simulated solar illumination, the CB@Cytc-Chla bioelectrode produced a significantly enhanced photocurrent compared to the control electrodes with individual components, indicating effective photo-induced charge transfer. The selective electrochemical reduction of hydrogen peroxide (H₂O₂) was explored as a model reaction at neutral pH, where the hybrid bioelectrode exhibited the highest current response relative to the protein-only modified electrodes. As a demonstration of practical utility, a selective batch-injection analysis of H₂O₂ was carried out using a three-in-one disposable screen-printed electrode modified with the CB@Cytc-Chla composite. This system, integrated with a prototype wireless device, enabled sensitive onedrop detection of reactive oxygen species (ROS) released from chemically stressed cancer cells.