Real-Time Measurements of Dissolved Oxygen Inside Live Cells by Organically Modified Silicate Fluorescent Nanosensors

Abstract

Optical PEBBLE (probes encapsulated by biologically localized embedding) nanosensors have been developed for dissolved oxygen using organically modified silicate (ormosil) nanoparticles as a matrix. The ormosil nanoparticles are prepared via a sol-gel-based process, which includes the formation of core particles with phenyltrimethoxysilane as a precursor followed by the formation of a coating layer with methyltrimethoxysilane as a precursor. The average diameter of the resultant particles is 120 nm. These sensors incorporate the oxygen-sensitive platinum porphyrin dye as an indicator and an oxygen-insensitive dye as a reference for ratiometric intensity measurement. Two pairs of indicator dye and reference dye, respectively, platinum(II) octaethylporphine and 3,3'-dioctadecyloxacarbocyanine perchlorate, and platinum(II) octaethylporphine ketone and octaethylporphine, were used. The sensors have excellent sensitivity with an overall quenching response of 97%, as well as excellent linearity of the Stern-Volmer plot (r(2) = 0.999) over the whole range of dissolved oxygen concentrations (0-43 ppm). In vitro intracellular changes of dissolved oxygen due to cell respiration were monitored, with gene gun injected PEBBLEs, in rat C6 glioma cells. A significant change was observed with a fluorescence ratio increase of up to 500% after 1 h, for nine different sets of cells, which corresponds to a 90% reduction in terms of dissolved oxygen concentration. These results clearly show the validity of the delivery method for intracellular studies of PEBBLE sensors, as well as the high sensitivity, which is needed to achieve real-time measurements of intracellular dissolved oxygen concentration.

Indicators for optical oxygen sensors

“…Nanosensors have been successfully used to monitor dissolved oxygen in human plasma [34], to monitor cellular respiration [113], and to measure the real-time oxygen concentration inside tumor cells under normal and hypoxic conditions [135].…”

A Conceptual Review of Nanosensors

“…(d) Organically modified silicates (Ormosils) [58]: Ormosil PEBBLEs are prepared in two steps [58]. In the first stage, the core formation takes place by hydrolyzing phenyltrimethoxysilane within acidic environment, followed by silane condensation within alkaline environment.…”

“…Direct measurement PEBBLEs have been used for sensing H + [52], Ca 2+ [52], Mg 2+ [59], Zn 2+ [10] and glucose [60]. Sol-gel PEBBLEs are normally designed as dissolved gas sensors, and have been used for sensing dissolved molecular oxygen [57,58]. However, some analytes lack highly selective fluorescent indicators.…”

Fiber-Optic Sensor for Iodine Based on a Covalently Immobilized Aminobenzanthrone Schiff Base

“…Many optical sensors have been reported for monitoring the pH, [7][8][9] heavy metal ions 11,12 and dissolved oxygen. 13 Also, few attempts have been made to develop optical chemical sensors for iodine determination. [14][15][16] Fluorescent dyes, such as fluoranthene and carbazole derivative, have been used as carrier compounds for iodine sensing, though the number of useful fluorescent carriers remains limited.…”