Reactive oxygen species (ROS) are tightly involved in normal cell functions as well as in development of a wide variety of pathologies. Commonly used for ROS detection, dichlorofluorescein (DCF) derivatives have several serious disadvantages: they are not specific, i.e., they are sensitive to multiple types of ROS; they cannot be targeted to specific intracellular compartments; and, most important, they can produce ROS upon light exposure , which results in artifactual ROS generation and signal amplification.
HyPer is the first fully genetically encoded fluorescent sensor capable of detecting of intracellular hydrogen peroxide (H₂O₂), one of the main ROS generated by cells [Belousov et al., 2006]. Developed on the basis of yellow fluorescent protein inserted into the regulatory domain of E. coli protein OxyR (OxyR-RD) [Choi et al., 2001], HyPer demonstrates submicromolar affinity to hydrogen peroxide and, at the same time, it is insensitive to other oxidants tested, such as superoxide, oxidized glutathione, nitric oxide, and peroxinitrite. HyPer does not cause artifactual ROS generation and can be used for detection of fast changes of H₂O₂ concentration in different cell compartments.