Ratiometric Optical PEBBLE Nanosensors for Real-Time Magnesium Ion Concentrations Inside Viable Cells

This paper presents the development and characterization of a highly selective magnesium fluorescent optical nanosensor, made possible by PEBBLE (probe encapsulated by biologically localized embedding) technology. A ratiometric sensor has been developed by co-immobilizing a dye that is sensitive to and highly selective for magnesium, with a reference dye in a matrix. The sensors are prepared via a microemulsion polymerization process, which entraps the sensing components inside a polymer matrix. The resultant spherical sensors are ~40 nm in diameter. The Coumarin 343 (C343) dye, which by itself does not enter the cell, when immobilized in a PEBBLE is used as the magnesium-selective agent that provides the high and necessary selectivity over other intracellular ions, such as Ca^2+, Na+, and K+. The dynamic range of these sensors was 1-30 mM, with a linear range from 1 to 10 mM, with a response time of <4 s. In contrast to free dye, these nano-optodes are not perturbed by proteins. They are fully reversible and exhibit minimal leaching and photobleaching over extended periods of time. In vitro intracellular changes in Mg^2+ concentration were monitored in C6 glioma cells, which remained viable after PEBBLE delivery via gene gun injection. The selectivity for Mg^2+ along with the biocompatibility of the matrix provides a new and reliable tool for intracellular magnesium measurements.