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Published on: July 25, 2025
A Ratiometric Two-Photon Fluorescent Cysteine Probe with Well-Resolved Dual Emissions Based on Intramolecular Charge
Sheng Yang1, Chongchong Guo1, Yuan Li2
1School of Chemistry and Biological Engineering, Hunan Provincial Key Laboratory of Materials Protection for Electric Power and Transportation , Changsha University of Science and Technology , Changsha , 410114 , P. R. China.
Abstract:
The development of an efficient ratiometric two-photon fluorescence imaging probe is crucial for in situ monitoring of biothiol cysteine (Cys) in biosystems, but the current reported intramolecular charge transfer (ICT)-based one suffers from serious overlap between the shifted emission bands. To address this issue, we herein for the first time constructed an ICT-mediated two-photon excited fluorescence resonance energy transfer (TP-FRET) system consisting of a two-photon fluorogen benzo[ h]chromene and a Cys-responsive benzoxadiazole-analogue dye. Different from a previous mechanism that utilized single two-photon fluorogen to acquire a ratiometric signal, ICT was used to switch on the TP-FRET process of the energy transfer dyad by eliciting an absorption shift of benzoxadiazole with Cys to modulate the spectral overlap level between benzo[ h]chromene emission and benzoxadiazole absorption, resulting in two well-separated emission signal changes with large emission wavelength shift (120 nm), fixed two-photon excitation maximum (750 nm), and significant variation in fluorescence ratio (over 36-fold). Therefore, it can be successfully employed to ratiometrically visualize Cys in HeLa cells and liver tissues. Importantly, this new ICT-mediated TP-FRET integration mechanism would be convenient for designing ratiometric two-photon fluorescent probes with two well-resolved emission spectra suitable for high resolution two-photon fluorescence bioimaging.
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