An ICT-FRET fluorescent probe for ratiometric recognition of hydrogen peroxide utilizing coumarin-naphthalimide derivative

Being an important reactive oxygen species, hydrogen peroxide (H₂O₂) is involved in regulating biological processes. Thus, it is vital to develop highly sensitive and selective detection methods for monitoring H₂O₂ levels. In this work, a new fluorescent probe for the ratiometric recognition of H₂O₂ was fabricated by integrating intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET) mechanisms. Coumarin was chosen as energy donor, while naphthalimide was selected as energy acceptor, with pentafluorobenzenesulfonate serving as H₂O₂ recognition moiety. Before the introduction of H₂O₂, the pentafluorobenzenesulfonate group effectively quenched the electron transfer of probe, disabling both the ICT effect and FRET process. Therefore, when H₂O₂ was absent, the probe demonstrated intense blue fluorescence originating from the coumarin moiety. After the introduction of H₂O₂, the pentafluorobenzenesulfonate group underwent a chemical transformation to generate a hydroxyl group, thereby activating the ICT mechanism and initiating the FRET effect. Therefore, upon introducing H₂O₂ characteristic bright yellow fluorescence was seen from the probe. Moreover, quantitative detection of H₂O₂ was obtained in the 0.01–1 μM range, as evidenced by the linear correlation of the I_{550 nm}/I_{476 nm} with H₂O₂ concentration, yielding a 7.7 nM detection limit. The probe exhibited excellent selectivity, outstanding sensitivity and rapid response time. And the designed probe demonstrated successful application in ratiometric imaging of both intracellularly generated and externally introduced H₂O₂ in A549 cell lines. Organophosphorus pesticides (OPs) suppress acetylcholinesterase (AChE) activity, hindering the ability of AChE and choline oxidase (ChOx) to generate H₂O₂ from acetylthiocholine chloride (ATChCl). Therefore, the developed probe had been also utilized for the ratiometric detection of organophosphorus pesticides.