A two-photon ratiometric fluorescence probe for cupric ions in live cells and tissues

Development of sensitive and selective probes for cupric ions (Cu(2+)) at cell and tissue level is a challenging work for progress in understanding the biological effects of Cu(2+). Here, we report a ratiometric two-photon probe for Cu(2+) based on the organic-inorganic hybrids of graphene quantum dots (GQDs) and Nile Blue dye. Meanwhile, Cu-free derivative of copper-zinc superoxide dismutase (SOD) - E2Zn2SOD is designed as the unique receptor for Cu(2+) and conjugated on the surface of GQDs. This probe shows a blue-to-yellow color change in repose to Cu(2+), good selectivity, low cytotoxicity, long-term photostability, and insensitivity to pH over the biologically relevant pH range. The developed probe allows the direct visualization of Cu(2+) levels in live cells as well as in deep-tissues at 90-180 μm depth through the use of two-photon microscopy. Furthermore, the effect of ascorbic acid is also evaluated on intracellular Cu(2+) binding to E2Zn2SOD by this probe.