Single-dye-doped fluorescent nanoprobe enables self-referenced ratiometric imaging of hypochlorous acid in lysosomes

The design of novel ratiometric nanoprobes with two well-resolved fluorescence bands for accurate imaging of lysosomal hypochlorous acid (HClO) is significant to understand its biological and pathological behaviors. Herein, we develop a single-dye-doped self-referenced ratiometric nanoprobe (SSRN) for imaging of HClO in lysosomes under a single-wavelength excitation (lambda(ex)= 405 nm). The referenced unit (benzothiazole moiety) and HClO-responsive unit (rhodamine B) are integrated into the single dye (HB-Py) to achieve the self-referenced ratiometric effect. The well-dispersed SSRN are prepared by a simple coprecipitation of the morpholine moiety modified amphiphilic polymers (CO-720-MA) and HB-Py. Of note, the two well-resolved fluorescence bands (similar to 130 nm) of SSRN could provide accurate imaging of analyte in live cells. Additionally, other distinct merits with SSRN are also simultaneously realized, including high water solubility, high sensitivity (detection limit: 52 nM), excellent selectivity, good biocompatibility and excellent photostability. Significantly, benefiting from the morpholine moiety, this nanoprobe SSRN can specially locate lysosomes and enable ratiometric imaging of exogenous/endogenous HClO in lysosomes.