Zn2+ ions posess important roles in living organisms, they are involved in cell division, neurotransmission, immunity, etc. They also play roles in diseases as Alzheimer's or Parkinson's disease and in viral infections such as SARS-CoV-2. Detection of metal ions in biological systems ususally relies on fluorescent microscopy aided by fluorescent chemosensors, which illuminate the analytes. The number of fluorescent probes for Zn2+ used in Two-Photon (2P) microscopy is limited and many of the known probes are compromised by issues such as weak signal to noise ratio, selectivity or solubility. This work presents the synthesis of a library of fluorescent Zn2+ sensors for 2P microscopy based on the fluorescent chromophore of the green fluorescent protein (GFP). A detailed spectroscopical characterization is provided by spectrophotometric and fluorimetric studies. Fluorescence-enchancements up to 75-fold are reported by excitation at λex = 450 nm, while emission at λem = 510 nm was detected selectively in presence of Zn2+ at the physiologically relevant pH range. The Zn2+ complexes display high stability (Kd = 0.15-15 nM) and water-solubility of the probes was excellent (&gt; 1 g/L), strong fluorescence under two-photon excitation was detected as well. Finally, the practical applicability of the probes has been demonstrated by live cell imaging of Zn2+ in fluorescent microscopy. In conclusion, this work presents the preparation and characterization of a novel library of selective, two-photon active fluorescent chemosensors for Zn2+.

Novel fluorescent Zn2+ chemosensors based on locked GFP-chromophore for two-photon microscopy

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