Methods for detection and measurement of hydrogen peroxide inside and outside of cells

Hydrogen peroxide (H2O2) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of its production. Characterization of the cellular functions of H2O2 requires measurement of its concentration selectively in the presence of other oxygen metabolites and with spatial and temporal fidelity in live cells. For the measurement of H2O2 in biological fluids, several sensitive methods based on horseradish peroxidase and artificial substrates (such as Amplex Red and 3,5,3’5’-tetramethylbenzidine) or on ferrous oxidation in the presence of xylenol orange (FOX) have been developed. For measurement of intracellular H2O2, methods based on dihydro compounds such as 2’,7’-dichlorodihydrofluorescein that fluoresce on oxidation are used widely because of their sensitivity and simplicity. However, such probes react with a variety of cellular oxidants including nitric oxide, peroxynitrite, and hypochloride in addition to H2O2. Deprotection reaction-based probes (PG1 and PC1) that fluoresce on H2O2-specific removal of a boronate group rather than on nonspecific oxidation have recently been developed for selective measurement of H2O2 in cells. Furthermore, a new class of organelle-targetable fluorescent probes has been devised by joining PG1 to a substrate of SNAP-tag. Given that SNAP-tag can be genetically targeted to various subcellular organelles, localized accumulation of H2O2 can be monitored with the use of SNAP-tag bioconjugation chemistry. However, given that both dihydro- and deprotection-based probes react irreversibly with H2O2, they cannot be used to monitor transient changes in H2O2 concentration. This drawback has been overcome with the development of redox-sensitive green fluorescent protein (roGFP) probes, which are prepared by the introduction of two redox-sensitive cysteine residues into green fluorescent protein; the oxidation of these residues to form a disulfide results in a conformational change of the protein and altered fluorogenic properties. Such genetically encoded probes react reversibly with H2O2 and can be targeted to various compartments of the cell, but they are not selective for H2O2 because disulfide formation in roGFP is promoted by various cellular oxidants. A new type of H2O2-selective, genetically encoded, and reversible fluorescent probe, named HyPer, was recently prepared by insertion of a circularly permuted yellow fluorescent protein (cpYFP) into the bacterial peroxide sensor protein OxyR.