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 (NP1) 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 NP1 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 (pep-NP1) was successfully targeted to nuclei and was capable of ratiometrically detecting nuclear H2O2 in living cells. These results indicated that NP1 was a highly sensitive ratiometric H2O2 dye with promising biological applications.

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

“…NP1 was reported to display one absorption band centered at 446 nm in the presence of H 2 O 2 due to its chemospecific boronate switch. 28 On the contrary, the absorption of 2, which exhibited a maximum at 413 nm, did not change after the addition of H 2 O 2 ( Figure S3b), indicating that the styryl dye itself did not react with H 2 O 2 . Consequently, the absorption band of pep3-NP1 at 442 nm could be attributed to the overlap of the absorption of the 1,8-naphthalimide (after reaction with H 2 O 2 ) and styryl fluorophores.…”

“…The peptide was synthesized on a solid-phase synthesizer by Bankpeptide biological technology Co., Ltd. NP1 was synthesized as described previously. 28 Instruments. 1 H NMR spectra were recorded with Bruker DRX 500 spectrometer at 400 MHz.…”

“…This behavior was consistent with that of NP1. 28 The fluorescence at 555 nm was ascribed to the replacement of the boronate group of pep3-NP1 by a hydroxyl group after reaction with H 2 O 2 . 28 Kinetic measurements of the H 2 O 2 -mediated boronate deprotection were performed under pseudo-firstorder conditions (1 µM pep3-NP1, 1 mM H 2 O 2 ), giving an observed rate constant of 4.87 × 10 -4 s -1 ( Figure S4).…”

“…We recently reported a versatile H 2 O 2 fluorescence reporter, NP1, based on 1,8-naphthalimide. 28 It was a highly sensitive ratiometric probe carrying an azide group, which rendered it easy to be linked to other molecules via the click reaction. To further develop fluorescence probes for monitoring the levels of H 2 O 2 near mtDNA, herein, pep3-NP1 was designed and synthesized based on NP1, by introducing a DNA-binding peptide tagged with a positively charged red-emitting styryl fluorophore (Scheme 1a).…”

Mitochondria-Targeted Triphenylphosphonium-Based Compounds: Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic Applications

“…300,301 The boronate reporter was linked to a DNA-binding peptide and a positively charged styryl fluorophore. The probe showed a colocalization with MitoTracker Green (500 nM) indicating accumulation of the probe in mitochondria in HeLa cells.…”