Methods for the detection of ROS Species in cells

Reactive oxygen species (ROS) exert a dual role in cellular biology, functioning both as signaling molecules at basal levels and cytotoxic agents under oxidative stress conditions. This thesis explores the evolution of different ROS detection methods, examining strengths and limitations associated with each approach. The initial section of this thesis aims to delineate the nature and functions of ROS, including the central role of antioxidant systems in preventing the onset of oxidative stress and in maintaining physiological redox balance in cells. Particularly, it highlights the necessity and challenges involved in identifying effective methods for ROS detection. The second chapter provides an overview of traditional methods for detecting ROS species, which mainly involve the use of fluorescent, chemiluminescent, and spectrophotometric probes. The third chapter of this thesis focuses on an in-depth analysis of Electron Paramagnetic Resonance (EPR) spectroscopy, examining principles of biospectroscopy underlying this technique and the application of spin traps and spin probes. Moreover, two recent case studies concerning in vivo EPR application are presented to support its promising potential as a clinical tool. Finally, innovative methods for the detection of ROS are illustrated, which rely on the usage of genetically engineered recombinant proteins as probes, as well as the application of innovative immunochemical techniques. The thesis concludes with a reflection on potential research lines that should be substantiated to overcome the existing shortcomings of these methodologies.