In-situ construction of fluorescent probes for hydrogen peroxide detection in mitochondria and lysosomes with on-demand modular assembling and double turn-on features

Due to the diverse H₂O₂ distribution in organelles, fluorescent probes were usually required to be prepared separately, which limited the convenience and practicability. Herein, we reported a flexible strategy to in-situ construct H₂O₂ fluorescent probes in different organelles. A tetrazine fused probe TP was developed with rapid click reaction capacity and sensitive H₂O₂ response. When treated with H₂O₂, the turn-on fluorescence was effectively quenched by the tetrazine part. Only after click reaction with dienophiles, the fluorescence resumed. In application, cells were firstly treated with triphenylphosphorus tagged norbornene (TPP-NB) to label mitochondria, which was followed by the introduction of probe TP to trigger click reaction. The in-situ constructed probe P1 served as a local H₂O₂ sensor. In a similar way, probe P2 was in-situ constructed in lysosomes via probe TP and morpholine tagged norbornene (MP-NB). With this on-demand modular assembling and double turn-on features, our strategy to construct fluorescent probes presented high flexibility and anti-interference performance, which was expected to inspired more applications in biological studies.