Development of an Iminocoumarin-Based Zinc Sensor Suitable for Ratiometric Fluorescence Imaging of Neuronal Zinc

Abstract

Ratiometric imaging is a technique to reduce artifacts by minimizing the influence of extraneous factors on the fluorescence of a sensor and is particularly useful for cellular imaging studies. Here we characterized the iminocoumarin fluorophore as a new scaffold for sensors for ratiometric imaging. The iminocoumarin 4 showed a high quantum yield in aqueous media on excitation in the visible wavelength region, while its coumarin analogue showed little fluorescence. We therefore developed a novel fluorescence probe, ZnIC, for ratiometric imaging of Zn2+, using iminocoumarin as a fluorophore and (ethylamino)dipicolylamine as a Zn2+ chelator. ZnIC exhibited almost the same fluorescence properties as 4, and the emission spectrum of this probe was red-shifted on addition of Zn2+ under physiological conditions. ZnIC is selective for Zn2+ over other biologically important metal ions, such as Ca2+ and Mg2+, and has high affinity for Zn2+. To confirm the suitability of ZnIC for biological applications, we employed it for the ratiometric detection of changes in intracellular Zn2+ in cultured cells and in rat hippocampal slices. The results indicate that iminocoumarin is a useful fluorophore for fluorescence microscopic imaging and that ZnIC should be useful for studies on the biological functions of Zn2+.

Sensing Inside the Living Cells

“…13.32 ). The DPA-bound Zn 2+ interacts with an iminocoumarin moiety, thereby infl uencing the internal charge transfer process connected to the fl uorescence emission (Komatsu et al 2007 ).…”

Recent Progress in Abasic Site-binding Small Molecules for Detecting Single-base Mutations in DNA

“…[43][44][45][46] Significant efforts have been devoted to develop this class of probes for a wide range of analytes, including metal ions, 43,47 inorganic and organic anions, 48,49 enzyme activities, 50 and nucleic acids. [51][52][53][54][55][56][57][58] A typical way to produce the ratiometric signal is by using double fluorescent dyes, in which techniques based on fluorescence resonance energy transfer (FRET) are widely used.…”

Design of Cation‐Selective Synthetic Fluorescent Indicators

“…Compared to 22, the imino-coumarin 23 exhibits a reduced acceptor strength, yielding a less polarized 1 CT state and reduced absorption and emission shifts. Nevertheless, the indicator proved suitable for monitoring dynamic Zn 2+ fluxes in live cells and hippocampal tissue [42]. As implied by the large Stokes shift for 24, the 1 CT state of this indicator is highly polarized [44].…”