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

Hypochlorous acid (HOCl) and sulfur dioxide derivatives (SO(3)(2−)/HSO(3)(−)) play critical roles in complex signal transduction and oxidation pathways. Therefore, it is meaningful and valuable to detect both HOCl and SO(2) 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 C[double bond, length as m-dash]C 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 (DP[double bond, length as m-dash]O) 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 C[double bond, length as m-dash]C double bond by oxidation, DP[double bond, length as m-dash]O reacts specifically with SO(3)(2−)/HSO(3)(−)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 SO(3)(2−)/HSO(3)(−) by a single fluorescent probe. The detection limits of DP for HOCl and SO(3)(2−)/HSO(3)(−) 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 SO(3)(2−)/HSO(3)(−). Using adult zebrafish and nude mice as live animal models, DP was successfully used for the fluorescence imaging of HOCl and SO(3)(2−)/HSO(3)(−)in vivo.