Hydrogen Peroxide (H₂O₂) Detection Probes

Hydrogen peroxide (H₂O₂) is a type of reactive oxygen species formed by the two-electron reduction of molecular oxygen. In biological systems, H₂O₂ is primarily generated through the dismutation of superoxide (O₂^⁻･) catalyzed by superoxide dismutase (SOD). When hydrogen peroxide reacts with iron or other transition metals in vivo, hydroxyl radicals (^･OH) are generated, causing damage to DNA, proteins, lipids, and other biomolecules. Therefore, cells must eliminate H₂O₂, a role primarily fulfilled by catalase, which catalyzes the dismutation of hydrogen peroxide into water and oxygen.

Hydrogen peroxide is more stable than other reactive oxygen species such as superoxide, and a variety of measurement methods have been established that utilize enzymes such as catalase, peroxidase, and alcohol dehydrogenase¹⁾. Among these approaches, fluorescence probe–based methods are widely used in the life sciences because they enable real-time monitoring of trace amounts of hydrogen peroxide present in cells. Common fluorescent probes for hydrogen peroxide include dichlorodihydrofluorescein (DCFH) and its diacetylated derivative, DCFH-DA. DCFH is oxidized by H₂O₂ in the presence of peroxidase or heme-containing proteins to produce the fluorescent compound dichlorofluorescein (DCF)¹⁾. However, these probes exhibit limited specificity for H₂O₂, and have been reported to be activated by hydroperoxides in the presence of peroxidase, nitric oxide, or peroxynitrite²⁾. Consequently, a strong need exists for the development of fluorescent probes with improved specificity for hydrogen peroxide.

BES-H₂O₂-Ac and BES-H₂O₂ are fluorescent probes for the detection of hydrogen peroxide (H₂O₂) in living cells²⁾. Compared with the conventional fluorescent probe dichlorodihydrofluorescein (DCFH), they exhibit greater specificity for hydrogen peroxide, resulting in reduced background fluorescence. These probes are suitable for live-cell imaging by fluorescence microscopy as well as for analysis by flow cytometry.

Features

• Detection of hydrogen peroxide in living cells
• Higher specificity for hydrogen peroxide than conventional fluorescent probes (DCFH)
• Water-soluble, allowing direct use in aqueous media
• Ex/Em=485±20 nm/515±20 nm

Principle

Data

Detection of Hydrogen Peroxide in Jurkat T cells with BES-H₂O₂-Ac

Jurkat T cells were incubated in medium containing 50 µM BES-H₂O₂-Ac at 37°C for 1 hour to allow cellular uptake of the reagent. Subsequently, hydrogen peroxide production was induced by the addition of 5 mM butyrate, followed by an additional 1-hour incubation.

	Without H2O2 production	With H2O2 production
Fluorescence image
Phase-contrast image

Data by courtesy of Dr.Maeda, School of Pharmacy, Hyogo University of Health Sciences

1. 「活性酸素実験プロトコール」ed. by Suma, H., Shujunsha, Tokyo, p.18(1994). (Japanese)
2. Maeda, H. et al.: Angew. Chem. Int. Ed., 43, 239(2004).
   Fluorescent probes for hydrogen peroxide based on a non-oxidative mechanism