A highly sensitive and fast-response fluorescence nanoprobe for in vivo imaging of hypochlorous acid

Fluorescent probes for in vivo hypochlorous acid (HClO) imaging often face challenges of low selectivity and high cytotoxicity, largely due to poor analyte recognition and water-insoluble aromatic skeletons. To address this, we synthesized fluorescein hydrazide by introducing a spiro-lactam unit into fluorescein, which offers high emission intensity and molar absorption. The five-membered heterocycle in fluorescein hydrazide is selectively disrupted by HClO, enhancing the conjugated system and electron delocalization of the fluorophore, resulting in highly sensitive fluorescence detection of HClO. For in vivo imaging, fluorescein hydrazide was covalently grafted onto the surface of silica nanoparticles via nucleophilic substitution reaction, avoiding complex modifications. This fluorescent nanoprobe (Si-FL) leverages the high-density fluorophores and hydroxyl groups on the silica surface to enrich low-concentrations of HClO through weak supramolecular interactions, thereby accelerating the reaction between HClO and the recognition sites. Compared to other molecular probes, Si-FL demonstrates a superior response speed (within 20 s) and a lower detection limit (72 nM), alongside excellent biocompatibility and water solubility. The nanoprobe Si-FL was successfully applied for HClO detection in living cells, zebrafish, and plants, significantly improving the stability of fluorescence imaging.