In Situ Reactive Oxygen Species‐Generating Semiconductor Nanopore for Real Time Peptide Oxidation Analysis

Abstract Photo‐induced damage impairs protein activity and leads to functional deterioration in organisms and tissues. In situ studies of peptide motifs oxidation are pivotal for elucidating molecular mechanisms of photodamage and identifying therapeutic targets. Although nanopores for analyzing peptides and amino acids are well established, no platform generates reactive oxygen species (ROS) in situ and delivers real‐time readouts of oxidative events. Here, a novel in situ oxidation detection platform combines a photocatalytically active TiO 2 nanopipette with a mesoporous MIL‐125(Ti) nanosheet, integrating a photocatalytic nanoreactor and solid‐state nanopore sensing. The MIL‐125/TiO 2 heterojunction produces abundant high‐valent ROS under light irradiation, while the MIL‐125(Ti) nanosheet at the nanopipette tip serves as a solid‐state nanopore, whose ionic current signatures reveal surface charge and conformational dynamics of peptides before and after oxidation. Using the Ω‐loop D motif of cytochrome c as a model, transient current measurements and molecular dynamics simulations reveal that ROS oxidation markedly increases peptide surface charge and induces a folded‐to‐extended transition. Methionine and tyrosine residues within Ω‐loop D are identified as key antioxidants, while exogenous antioxidants preserve their conformation in real time. This work establishes a novel in situ molecular‐level method for studying oxidative modification, photocatalyst evaluation, and antioxidant drug screening.