Optical PEBBLE nanosensors and fiber optic sensors for real-time intracellular imaging and analysis of magnesium and oxygen.

Abstract

A highly selective magnesium fluorescent optical nanosensor, made possible by PEBBLE (Probe Encapsulated By Biologically Localized Embedding) technology is presented. Ratiometric sensors were prepared, via a microemulsion polymerization process, by co-immobilizing a highly selective magnesium dye with a reference dye inside a polymer matrix. The resultant spherical sensors are &sim;40 nm in diameter. Several dyes were investigated with coumarin 343 (C343) providing the best selectivity towards Mg<super>2+</super> vs. ions such as Ca<super> 2+</super>, Na<super>+</super>, and K<super>+</super>. The dynamic range of the sensors was 1 to 30 mM (linear from 1 to 10 mM) with a response time of less than 4 s. The fully reversible sensors exhibit minimal leaching and photobleaching. <italic> In vitro</italic> intracellular changes in Mg<super>2+</super> concentration were monitored in C6 Glioma cells. Preliminary experiments demonstrated the capability of these sensors thus they were used to investigate the PhoP/Q transmembrane protein system on the internal membrane of <italic>salmonella </italic>. The sensors were used to monitor the magnesium levels inside <italic> salmonella</italic> containing vacuoles. Previously believed hypotheses on changes in magnesium concentration are challenged with the newly obtained results. The selectivity for Mg<super>2+</super> along with the biocompatibility of the matrix of these sensors provides a new and reliable tool for intracellular magnesium measurements. A second sensor platform was developed for the detection of intracellular dissolved oxygen, using a fiber optic probe. The design and fabrication of an oxygen fiber optic sensor based on the fluorescence quenching properties of the oxygen sensitive platinum (II) octaethylporphine ketone (PtOEPK) is presented. Octaethyl porphyrin (OEP) or bodipy maleimide 577/618, was also entrapped, as a reference dye, in a polyvinyl chloride matrix including the plasticizing agent bis 2-ethylhexyl sebacate (DOS). The multi mode fibers were pulled down to submicron dimensions and a dip coating procedure was used to apply the sensing membrane to the tip. This sensor was fully characterized and exhibits excellent reversibility, minimal photobleaching and leaching, and fast response times. This oxygen sensor is one of the most sensitive optical oxygen sensors to date, with exceptional linearity in the Stern-Volmer plot calibrations. This paper presents the full characterization of this sensor ultimately intended for intracellular measurements.