Highly Selective Fluorescent Probe With an Ideal pH Profile for the Rapid and Unambiguous Determination of Subcellular Labile Iron (III) Pools in Human Cells

Abstract

A convenient tool for detecting iron ions in subcellular structures is desired for better understanding its roles in biological systems. In this work, a new Fe3+ sensor, 2-(2-((1-(6-acetylpyridin-2-yl)ethylidene)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one (RhPK), which operates across the entire cellular pH range and is capable of unambiguously detecting Fe3+ ion in live human cells at subcellular resolution, is reported. The sensor exhibits high selectivity and sensitivity toward Fe3+ with a rapid fluorescence response and a 12-fold increase in intensity upon the addition of 1 equivalent Fe3+ at pH 7.3. RhPK forms a 1:1 complex with Fe3+ with an apparent binding constant 1.54 × 107 M−1 and a detection limit of 50 nM. The sensor is stable between pH 4.2 and 9.0 and operates across the whole cellular pH range. Cell imaging demonstrates the ability of the sensor to unambiguously detect basal level Fe3+ as well as its dynamic changes in real-time in live cells at subcellular resolution, with one labile Fe3+ pool identified in mitochondria in human primary fibroblast (ws1) cells for the first time and two Fe3 + pools confirmed in mitochondria and endo/lysosomes in human SH-SY5Y neuroblastoma cells, suggesting different cell types have distinctive Fe3+ storage in subcellular compartments. The RhPK probe is powerful for rapid and sensitive bioimaging of Fe3+ at subcellular level, enabling the unambiguous detection of labile Fe3+ pools at the entire cellular pH range, which is of great significance to understand the biological chemistry of Fe3+ and its roles in physiological processes and diseases.