A Real-Time Ratiometric Method for the Determination of Molecular Oxygen Inside Living Cells Using Sol−Gel-Based Spherical Optical Nanosensors with Applications to Rat C6 Glioma

TLDR

The authors develop sol‑gel based, ratiometric optical nanosensors (PEBBLEs) to enable reliable, real‑time measurements of subcellular molecular oxygen. The sensors were fabricated by a modified Stöber process with PEG stabilization, yielding 50–300 nm spherical PEBBLEs that embed an oxygen‑sensitive Ru(II) complex and an oxygen‑insensitive Oregon Green 488‑dextran for ratiometric readout. The PEBBLE sensors exhibit excellent reversibility, wide dynamic range, and resistance to leaching and photobleaching, and their small, inert matrix permits minimally invasive insertion into living cells, enabling reliable real‑time intracellular oxygen measurements in rat C6 glioma cells while protecting dyes from protein interference and reducing dye toxicity.

Abstract

The first sol−gel-based, ratiometric, optical nanosensors, or sol−gel probes encapsulated by biologically localized embedding (PEBBLEs), are made and demonstrated here to enable reliable, real-time measurements of subcellular molecular oxygen. Sensors were made using a modified Stöber method, with poly(ethylene glycol) as a steric stabilizer. The radii of these spherical PEBBLE sensors range from about 50 to 300 nm. These sensors incorporate an oxygen-sensitive fluorescent indicator, Ru(II)−tris(4,7-diphenyl-1,10-phenanthroline) chloride ([Ru(dpp)3]2+), and an oxygen-insensitive fluorescent dye, Oregon Green 488-dextran, as a reference for the purpose of ratiometric intensity measurements. The PEBBLE sensors have excellent reversibility, dynamic range, and stability to leaching and photobleaching. The small size and inert matrix of these sensors allow them to be inserted into living cells with minimal physical and chemical perturbations to their biological functions. Applications of sol−gel PEBBLEs inserted in rat C6 glioma cells for real-time intracellular oxygen analysis are demonstrated. Compared to using free dyes for intracellular measurements, the PEBBLE matrix protects the fluorescent dyes from interference by proteins in cells, enabling reliable in vivo chemical analysis. Conversely, the matrix also significantly reduces the toxicity of the indicator and reference dyes to the cells, so that a wide variety of dyes can be used in optimal fashion.