Mitochondria-Directed Fluorescent Probe for the Detection of Hydrogen Peroxide near Mitochondrial DNA

It is important to detect hydrogen peroxide (H₂O₂) near mitochondrial DNA (mtDNA) because mtDNA is more prone to oxidative attack than nuclear DNA (nDNA). In this study, a mitochondria-targeted fluorescence probe, <b>pep3-NP1</b>, has been designed and synthesized. The probe contains a DNA-binding peptide, a H₂O₂ fluorescence reporter, and a positively charged red emissive styryl dye to facilitate accumulation in mitochondria. Due to groove binding of the peptide with DNA, the styryl dye of <b>pep3-NP1</b> intercalated into the bases of DNA, leading to an increase in red fluorescence intensity (centered at 646 nm) and quantum yield. In this case, <b>pep3-NP1</b> was a turn-on probe for labeling DNA. Subcellular locations of <b>pep3-NP1</b> and MitoTracker suggested that <b>pep3-NP1</b> mostly accumulated in the mitochondria of live cells. Namely, as an intracellular DNA marker, <b>pep3-NP1</b> bound to mtDNA. In the presence of H₂O₂, <b>pep3-NP1</b> emitted green fluorescence (centered at 555 nm). Thus, the ratio of green with red fluorescence of <b>pep3-NP1</b> was suitable to reflect the change of the H₂O₂ level near mtDNA in living cells. The detecting limit for H₂O₂ was estimated at 2.9 and 5.0 μM in vitro and in cultured cells, respectively. The development of <b>pep3-NP1</b> could help in studies to protect mtDNA from oxidative stress

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

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