Visible light excitable zn²⁺ fluorescent sensor derived from an intramolecular charge transfer fluorophore and its in vitro and in vivo application

The UV- and sensor-induced interferences to living systems pose a barrier for in vivo Zn²⁺ imaging. In this work, an intramolecular charge transfer (ICT) fluorophore of smaller aromatic plane, 4-amino-7-nitro-2,1,3- benzoxadiazole, was adopted to construct visible light excited fluorescent Zn²⁺ sensor, NBD-TPEA. This sensor demonstrates a visible ICT absorption band, a large Stokes shift, and biocompatibility. It emits weakly (φ=0.003) without pH dependence at pH 7.1-10.1, and the λ _eX and λ_em are 469 (£₄₆₉ =2.1 × 10⁴ M^-1 cm^-1) and 550 nm, respectively. The NBD-TPEA displays distinct selective Zn²⁺-amplified fluorescence (φ=0.046, £469 =1.4 × 10⁴ M^-1 cm ^-1) with emission shift from 550 to 534 nm, which can be ascribed to the synergic Zn²⁺ coordination by the outer bis(pyridin-2-ylmethyl) amine (BPA) and 4-amine. The Zn²⁺ binding ratio of NBD-TPEA is 1:1. By comparison with its analogues NBD-BPA and NBD-PMA, which have no Zn ²⁺ affinity, the outer BPA in NBD-TPEA should be responsible for the Zn²⁺-induced photoinduced electron transfer blockage as well as for the enhanced Zn²⁺ binding ability of 4-amine. Successful intracellular Zn²⁺ imaging on living cells with NBD-TPEA staining exhibited a preferential accumulation at lysosome and Golgi with dual excitability at either 458 or 488 nm. The intact in vivo Zn²⁺ fluorescence imaging on zebrafish embryo or larva stained with NBD-TPEA revealed two zygomorphic luminescent areas around its ventricle which could be related to the Zn²⁺ storage for the zebrafish development. Moreover, high Zn²⁺ concentration in the developing neuromasters of zebrafish can be visualized by confocal fluorescence imaging. This study demonstrates a novel strategy to construct visible light excited Zn²⁺ fluorescent sensor based on ICT fluorophore other than xanthenone analogues. Current data show that NBD-TPEA staining can be a reliable approach forthe intact in vivo Zn ²⁺ imaging of zebrafish larva as well as for the clarification of subcellular distribution of Zn²⁺ in vitro.