A two-photon “turn-on” fluorescent probe based on carbon nanodots for imaging and selective biosensing of hydrogen sulfide in live cells and tissues

Determination of hydrogen sulfide (H₂S) in live cells and tissues is still a challenge for evaluating the key roles that H₂S plays in physiological and pathological processes. In this work, a “turn-on” two-photon fluorescent (TPF) sensor for H₂S is developed, in which carbon nanodot (C-Dot) was employed as a two-photon fluorophore due to its large two-photon absorption cross-section (σ), and AE-TPEA–Cu²⁺ complex [AE-TPEA = N-(2-aminoethyl)-N, N, N′-tris(pyridin-2-ylmethyl)ethane-1,2-diamine] was first designed as a specific receptor for H₂S. The fluorescence of C-Dot conjugated with AE-TPEA (C-Dot-TPEA) was quenched upon the addition of Cu²⁺. Then, the fluorescence was restored after the addition of H₂S, because Cu²⁺ could be released from TPEA binding site when H₂S interacted with the Cu²⁺ ion. The designed C-Dot-TPEA–Cu²⁺ fluorescent sensor exhibited high specificity for H₂S over biothiols, sulfur-containing compounds, reactive oxygen species (ROS), and other biological interferences. Meanwhile, a broad linear range from 5 πM to 100 πM was obtained and the detection limit was achieved to 0.7 πM. In addition, the C-Dot-based TPF probe exhibited bright two-photon fluorescence, favourable photostability against light illumination and pH change, and low cytotoxicity. Accordingly, the nanohybridized TPF sensor with high selectivity and sensitivity, as well as the fascinating properties of C-Dot themselves, successfully provided a new way for TPF imaging and biosensing of H₂S in live cells and tissues. We believe this is the first report of TPF imaging and biosensing of H₂S in live cells and tissues using a specially engineered C-Dot-based nanosystem.