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 λ_ex = 450 nm, while emission at λ_em = 510 nm was detected selectively in presence of Zn²⁺ at the physiologically relevant pH range. The Zn²⁺ complexes display high stability (K_d = 0.15-15 nM) and water-solubility of the probes was excellent (> 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⁺.

A) Concept of the locked GFP-chromophore fluorescence B) Mechanism of action of the presented probes

Synthetic route and structure of the novel fluorescent Zn2+ sensors

Tamás Visnovitz

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