Enhanced Ratiometric Indicators for (Sub)cellular Detection of Magnesium(II) Ions by Flow Cytometry

Magnesium(II) plays essential roles in cellular physiology, yet the inner workings of its cellular distribution and transport remain challenging to study due to the scarcity of tools for Mg2+-selective detection with high spatiotemporal resolution and compatible with high-throughput analysis. We report herein MagZet2, a next-generation ratiometric fluorescent sensor featuring a quinoline dicarboxylate motif with an expanded dynamic range for Mg2+ detection by fluorescence microscopy and flow cytometry. MagZet2 displays excellent Mg2+/Ca2+ selectivity and an apparent dissociation constant, K'd = 0.4 mM, well matched to physiological free Mg2+ concentrations in mammalian cells. By conjugation to HaloTag, we further engineer MagZet2 into chemigenetic indicators that retain desirable photophysical properties of the small molecule counterpart while enabling genetically encoded control of subcellular localization. Using a hybrid sensor, MagZet2(L2)Halo, we demonstrate the first organelle-resolved Mg2+ detection by flow cytometry, a technique that does not inherently offer spatial resolution. Finally, we apply MagZet2(L2)Halo to monitor Mg2+ uptake kinetics in live Caco-2 cells, revealing transport dynamics that parallel those measured using destructive 25Mg transport assays while offering improved temporal resolution and compatibility with live-cell analysis. Together, these results establish MagZet2 and its chemigenetic derivatives as powerful tools for organelle-resolved, high-throughput interrogation of free Mg2+ in living cells.