A two-photon ratiometric fluorescent probe for the synergistic detection of the mitochondrial SO ₂ /HClO crosstalk in cells and in vivo

Known as a new member of the reactive sulphur species (RSS), endogenous sulfur dioxide (SO₂) plays potentially reductive roles such as antioxidation, anti-aging, anti-inflammation, and cytoprotection; however, SO₂ is also a metabolite of antioxidation. Hypochlorous acid (HClO) has powerful bio-effects on the innate immune system, and its uncontrolled production leads to adverse damage in cells. To illuminate the potential crosstalk between SO₂ and HClO in redox homeostasis, we designed a two-photon ratiometric fluorescent probe for dual-response to mitochondrial SO₂/HClO crosstalk in cells and in vivo. Our probe can effectively achieve ratio-type fluorescence dual-response to SO₂ and HClO with desirable properties such as large two-photon absorption cross sections, rapid response times (SO₂ < 50 s, HClO < 20 s), high sensitivities (limit of detection 8.0 nM for SO₂ and 15.2 nM for HClO), and favorable selectivity. The probe has been successfully applied to quantitatively detect endogenous SO₂ and HClO in HeLa and Raw 264.7 cells. We have verified that there exists a crosstalk between SO₂ and HClO during the process of oxidative stress in mitochondria. We have also detected that SO₂ can be endogenously produced through oxidation of intracellular sulfur-containing amino acids by HClO in zebrafish in real-time.