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.

Keywords:

• Iron(III)
• Fe³⁺ sensor
• Rhodamine
• Cellular pH range
• Fluorescent probe
• Subcellular imaging

Disclosure statement

The IP issue related to this work is being processed by the OTCV office of University of Massachusetts, MA, USA.

Additional information

Funding

We thank the National Science Foundation (grant nos. CHE-1213838 and CHE-1229339) and NIH (grant no. 1R15GM126576-01).