title: Visible light excitable Zn2+ fluorescent sensor derived from an intramolecular charge transfer fluorophore and its in vitro and in vivo application.
abstract: The UV- and sensor-induced interferences to living systems pose a barrier for in vivo Zn(2+) 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 Zn(2+) sensor, NBD-TPEA. This sensor demonstrates a visible ICT absorption band, a large Stokes shift, and biocompatibility. It emits weakly (Phi = 0.003) without pH dependence at pH 7.1-10.1, and the lambda(ex) and lambda(em) are 469 (epsilon(469) = 2.1 x 10(4) M(-1) cm(-1)) and 550 nm, respectively. The NBD-TPEA displays distinct selective Zn(2+)-amplified fluorescence (Phi = 0.046, epsilon(469) = 1.4 x 10(4) M(-1) cm(-1)) with emission shift from 550 to 534 nm, which can be ascribed to the synergic Zn(2+) coordination by the outer bis(pyridin-2-ylmethyl)amine (BPA) and 4-amine. The Zn(2+) 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 Zn(2+)-induced photoinduced electron transfer blockage as well as for the enhanced Zn(2+) binding ability of 4-amine. Successful intracellular Zn(2+) 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 Zn(2+) 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 Zn(2+) storage for the zebrafish development. Moreover, high Zn(2+) 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 Zn(2+) fluorescent sensor based on ICT fluorophore other than xanthenone analogues. Current data show that NBD-TPEA staining can be a reliable approach for the intact in vivo Zn(2+) imaging of zebrafish larva as well as for the clarification of subcellular distribution of Zn(2+) in vitro.
authors.affiliation: State Key Laboratory of Coordination Chemistry, Coordination Chemistry Institute, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China.
authors.forename: Fang
authors.initials: F
authors.lastname: Qian
authors.forename: Changli
authors.initials: C
authors.lastname: Zhang
authors.forename: Yumin
authors.initials: Y
authors.lastname: Zhang
authors.forename: Weijiang
authors.initials: W
authors.lastname: He
authors.forename: Xiang
authors.initials: X
authors.lastname: Gao
authors.forename: Ping
authors.initials: P
authors.lastname: Hu
authors.forename: Zijian
authors.initials: Z
authors.lastname: Guo
mesh_terms: D000818:Animals; D002413:Cations, Divalent / Q000737:chemistry; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D007814:Larva; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D051381:Rats; D013050:Spectrometry, Fluorescence; D015027:Zebrafish; D015032:Zinc / Q000737:chemistry*
chemical_list: D002413:Cations, Divalent; D005456:Fluorescent Dyes; D015032:Zinc