A Highly Sensitive Ratiometric Fluorescent Probe for the Detection of Cytoplasmic and Nuclear Hydrogen Peroxide

Abstract

As a marker for oxidative stress and a second messenger in signal transduction, hydrogen peroxide (H₂O₂) plays an important role in living systems. It is thus critical to monitor the changes in H₂O₂ in cells and tissues. Here, we developed a highly sensitive and versatile ratiometric H₂O₂ fluorescent probe (<b>NP1</b>) based on 1,8-naphthalimide and boric acid ester. In response to H₂O₂, the ratio of its fluorescent intensities at 555 and 403 nm changed 1020-fold within 200 min. The detecting limit of <b>NP1</b> toward H₂O₂ is estimated as 0.17 μM. It was capable of imaging endogenous H₂O₂ generated in live RAW 264.7 macrophages as a cellular inflammation response, and especially, it was able to detect H₂O₂ produced as a signaling molecule in A431 human epidermoid carcinoma cells through stimulation by epidermal growth factor. This probe contains an azide group and thus has the potential to be linked to various molecules via the click reaction. After binding to a Nuclear Localization Signal peptide, the peptide-based combination probe (<b>pep-NP1</b>) was successfully targeted to nuclei and was capable of ratiometrically detecting nuclear H₂O₂ in living cells. These results indicated that <b>NP1</b> was a highly sensitive ratiometric H₂O₂ dye with promising biological applications