Spectroscopic detection of reactive oxygen species and oxidative stress

Reactive oxygen species (ROS) include both free radicals and non-radical compounds. ESR spectroscopy, especially employing spin traps, is the unique technique allowing for detection, identification and quantification of free radicals in vitro and their imaging in vivo. However, the sensitivity of this technique is limited and essays based on the use of more sensitive fluorogenic and luminogenic probes have gained enormous popularity. Classic fluorogenic probes such as 2’,7’-dichlorodihydrofluorescein diacetate or dihydrorhodamine 123 are broadly used for detection of peroxides, but their specificity is low so often they are regarded as general indicators of oxidative stress than detectors of specific ROS. Dihydroethidine has been routinely used for detection of superoxide, but only one of its oxidation products is specific for this radical so precise identification of the product requires HPLC separation. The common problem with fluorogenic and luminogenic probes, especially when used at high concentrations, is their redox cycling; the probes not only detect but also generate ROS. Recently, a variety of new probes, i. a. boronate and BODIPY based, have been introduced, some of them allowing for a more selective detection of ROS. Probes targeted to mitochondria by a positively charged (usually phosphonium) moiety allow for detection of ROS produced by the main endogenous cellular source of ROS. A promising novel approach consists in the use of ROS-reactive fluorescent proteins. HyPer is a unique genetically engineered yellow fluorescent protein (YFP) for ratiometric measurement of hydrogen peroxide; cpYFP protein has been proposed as a superoxide sensor and pn-Green Fluorescent Protein (GFP) as a sensor of peroxynitrite. Other proteins have been constructed for measurement of other indices of oxidative stress like changes in the redox state of main cellular redox systems. RexYFP is sensitive to changes in the NAD+/NADH ratio and rxYFP measures changes in the oxidation state of glutathione. Fluorescent proteins can be targeted to various cell compartments allowing for studies of oxidative stress at the subcellular level. However, they have also drawbacks related, i. a., to the effects of other factors on their fluorescence and possible effects of their presence on cellular redox homeostasis. L13.2