Sequential detection of hypochlorous acid and sulfur dioxide derivatives by a red-emitting fluorescent probe and bioimaging applications in vitro and in vivo

Abstract

Hypochlorous acid (HOCl) and sulfur dioxide derivatives (SO₃²⁻/HSO₃⁻) play critical roles in complex signal transduction and oxidation pathways. Therefore, it is meaningful and valuable to detect both HOCl and SO₂ derivatives in biosystems by a fluorescence imaging assay. In this work, we developed a red-emitting fluorescent probe (DP) by the condensation of malononitrile and phenothiazine derivatives through a CC double bond. DP was designed with a donor–π–acceptor (D–π–A) structure, which enables absorption and emission in the long wavelength region. In the presence of HOCl, specific oxidation of the thioether of phenothiazine in DP to a sulfoxide derivative (DPO) occurs, resulting in a hypochromic shift (572 nm to 482 nm) of the absorption spectra and “OFF–ON” response of the maximum emission at 608 nm. After the activation of the CC double bond by oxidation, DPO reacts specifically with SO₃²⁻/HSO₃⁻ via a 1,4-nucleophilic addition reaction leading to a decrease in the intensity of the absorption and emission spectra, which enabled the realization of sequential detection of HOCl and SO₃²⁻/HSO₃⁻ by a single fluorescent probe. The detection limits of DP for HOCl and SO₃²⁻/HSO₃⁻ were calculated to be 81.3 nM and 70.8 nM/65.1 nm, respectively. The results of fluorescence microscopic imaging indicated that DP shows potential for the detection of intracellular HOCl and SO₃²⁻/HSO₃⁻. Using adult zebrafish and nude mice as live animal models, DP was successfully used for the fluorescence imaging of HOCl and SO₃²⁻/HSO₃⁻ in vivo.