Synthesis of a highly Mg2+-selective fluorescent probe and its application to quantifying and imaging total intracellular magnesium

Abstract

Magnesium plays a crucial role in many physiological functions and pathological states. Therefore, the evolution of specific and sensitive tools capable of detecting and quantifying this element in cells is a very desirable goal in biological and biomedical research. We developed a Mg-selective fluorescent dye that can be used to selectively detect and quantify the total magnesium pool in a number of cells that is two orders of magnitude smaller than that required by flame atomic absorption spectroscopy (F-AAS), the reference analytical method for the assessment of cellular total metal content. This protocol reports itemized steps for the synthesis of the fluorescent dye based on diaza-18-crown-6-hydroxyquinoline (DCHQ5). We also describe its application in the quantification of total intracellular magnesium in mammalian cells and the detection of this ion in vivo by confocal microscopy. The use of in vivo confocal microscopy enables the quantification of magnesium in different cellular compartments. As an example of the sensitivity of DCHQ5, we studied the involvement of Mg in multidrug resistance in human colon adenocarcinoma cells sensitive (LoVo-S) and resistant (LoVo-R) to doxorubicin, and found that the concentration was higher in LoVo-R cells. The time frame for DCHQ5 synthesis is 1-2 d, whereas the use of this dye for total intracellular magnesium quantification takes 2.5 h and for ion bioimaging it takes 1-2 h.

Analysis of Intracellular Magnesium and Mineral Depositions during Osteogenic Commitment of 3D Cultured Saos2 Cells

“…Recently, we reported that high extra-cellular Mg levels potentiate osteoclastic differentiation, while decrease osteoblastogenesis. A plausible hypothesis is that Mg might reprogram cholecalciferol (vitamin D3) activity on bone remodelling, causing an unbalanced activation of osteoclasts and osteoblasts [19]. On the other hand, we also demonstrated that magnesium deprivation accelerates the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells [31].…”

“…Osteogenic phenotype of SaOS2 cells was monitored via RT-qPCR analysis of credited osteogenic markers (RUNX2, COL1A1, BGLAP, SPP1) and histological analysis of extracellular calcium depositions. Magnesium intracellular concentration was assessed by a fluorimetric method [19], while the presence and the distribution of the cation in mineral depositions was investigated by X-ray fluorescence microscopy [20,21]. Finally, a morphological characterization of samples was obtained by X-ray computed micro-tomography (µCT) techniques allowing to observe, by a non-destructive method, the distribution of extracellular mineral depositions, and their shape and size within the sample volume.…”

Colorimetric detection of magnesium (II) ions using tryptophan functionalized gold nanoparticles

“…To our knowledge, no other small-molecular receptors were reported for Mg 2+ in urine and serum samples. Selective detection of Mg 2+ is appropriate to identify the significance of the Mg 2+ in many physiological functions and pathological conditions 27 . Thus, results demonstrated reliability of the tryptophan capped AuNPs for detecting Mg 2+ contents in real samples.…”

The Remarkable Effect of Halogen Substitution on the Membrane Transport of Fluorescent Molecules in Living Cells

“…[1] Particularly,s mall-molecule-based fluorescent probes have found diverse applications in the areas of cell biology,d rug discovery,chemical biology, and clinical diagnosis. [2] Although al arge number of smallmolecular fluorescent probes have been developed for various cellular applications, [3][4][5] the mechanisms by which these molecules enter the cells are not well understood in many cases.F luorescence probes based on 4-amino-1,8naphthalimide have attracted significant attention as these compounds can be synthesized in high yields from readily available starting materials and their solubility and fluorescence properties can be altered by substitution at the amino and imide moieties ( Figure 1). [6] Thet wo-photon fluorescent probe for the detection of formaldehyde, [7] the lysosometargetable probe for monitoring the endogenous nitric oxide (NO), [8] the solvatochromic fluorophore for the development of ap rotein-based pH sensor, [9] the ratiometric fluorescent probe for monitoring the mitochondrial NO level, [10] the pantetheine probe for the visualization of the chain-flipping mechanism in fatty-acid biosynthesis [11] represent af ew selected examples of fluorescent probes that have been employed successfully for cellular applications.Inthis paper, we report for the first time that as imple substitution of hydrogen atoms with halogens remarkably enhances the cellular uptake of naphthalimide-based fluorescent molecules in mammalian cells.T his study also suggests that halogen bonding may facilitate the active transport of halogenated compounds,p articularly iodinated compounds,a cross the plasma membrane.…”