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Two-Photon Fluorescent Chemosensors Based on the GFP-Chromophore for the Detection of Zn2+ in Biological Samples – From Design to Application

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A library of twelve novel fluorescent GFP chromophore based chemosensors (GFZnPs) was prepared for the detection of Zn2+ in biological samples with two-photon microscopy. The presented compounds are the hybrids of the Zn2+ chelator 8-aminoquinoline motif and the GFP chromophore, exploiting and enhancing the properties of the natural chromophore. In the presented probes, the quenching rotation around the benzylidene carbon of the GFP chromophore is conditionally blocked upon Zn2+ binding, resulting in a fluorescent response to the ion. GFZnPs have an efficient and simple synthesis making them one of the most easily accessible two-photon (2P) Zn2+ sensors. We present the comprehensive spectroscopical characterization of the probe family highlighting their bright fluorescent emission (ε × Φ > 200) at λem∼520 nm upon excitation by λex∼450 nm light. The sensors feature high selectivity for Zn2+ with an affinity in the nanomolar range, excellent water solubility, and usability at a wide pH-range (pH > 6). The relationship between the structure of compounds and their properties was studied and rationalized by theoretical considerations. The 2P-action cross section of the probes reaches δ × Φcomp > 5 GM at λex, 2P = 900 nm, with an increase up to 200-fold, which makes them one of the best two-photon probes for Zn2+. The applicability of selected probes was demonstrated by epifluorescent and 2P microscopy on cultured cells and brain slices. This work expands the toolbox of efficient two-photon zinc sensors with a valuable new family.€¦