Fluorescent nano-PEBBLE sensors designed for intracellular glucose imaging

Abstract

Polyacrylamide-based, ratiometric, spherical, optical nanosensors, or polyacrylamide PEBBLEs (Probes Encapsulated By Biologically Localized Embedding), have been fabricated, aimed at real-time glucose imaging in intact biological systems, i.e. living cells. These nanosensors are prepared using a microemulsion polymerization process, and their average size is about 45 nm in diameter. The sensors incorporate glucose oxidase (GOx), an oxygen sensitive fluorescent indicator (Ru[dpp(SO3Na)2]3)Cl2, and an oxygen insensitive fluorescent dye, Oregon Green 488-dextran or Texas Red-dextran, as a reference for the purpose of ratiometric intensity measurements. The enzymatic oxidation of glucose to gluconic acid results in the local depletion of oxygen, which is measured by the oxygen sensitive ruthenium dye. The small size and inert matrix of these sensors allows them to be inserted into living cells with minimal physical and chemical perturbations to their biological functions. The PEBBLE matrix protects the enzyme and 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 larger variety of dyes can be used in optimal fashion. Furthermore, the PEBBLE matrix enables the synergistic approach in which there is a steady state of local oxygen consumption, and this cannot be achieved by separately introducing free enzyme and dyes into a cell. The work presented here describes the production and characterization of glucose sensitive PEBBLEs, and their potential for intracellular glucose measurements. The sensor response is determined in terms of the linear range, ratiometric operation, response time, sensor stability, reversibility and immunity to interferences.

Enhancing cell and gene therapy manufacture through the application of advanced fluorescent optical sensors (Review)

“…Cellular delivery can be challenging as molecules may permeate the cell membrane unassisted. 43 Furthermore, there could be interference from cellular components that generate measurement artifacts 44 and subsequently nonratiometric measurements. 45 Finally, isolated molecules of fluorescent sensors spread throughout a biological system may bleach to a level below detectable background limits more rapidly than packed clusters of sensors in one location (Fig.…”

A fluorescence lifetime-based fibre-optic glucose sensor using glucose/galactose-binding protein

“…Several receptors for glucose have been investigated for fluorescence-based glucose sensing 4 including boronic acid derivatives, 4 concanavalin A 4,17,19,21 and other lectins, 8 glucose oxidase 4,13,16 and bacterial glucose/galactose-binding protein (GBP).…”

“…Moreover, measurement of fluorescence decay lifetime, as opposed to intensity, has special potential for in vivo sensing 6,7 because it is relatively independent of light scattering in the tissues, signal amplitude fluctuations and fluorophore concentration-thus coating of an implanted sensor, which may reduce fluorescence intensity, will leave the lifetime unaltered. Fluorescence glucose sensors may be envisaged as totally implanted, 8 formulated as microcapsules for impregnation in the skin as a 'smart tattoo', 9-12 as nanocapsules (pebbles or polymersomes) for intracellular sensing, 13,14 as an intravenous catheter, 15 and as a fibre-optic probe. [16][17][18][19] Fibre-optic glucose sensors have the attraction of ease of implantation into and removal from the subcutaneous tissue, containment of the sensing element, and communication between the tissues and instrumentation outside the body via a waveguide which is minimally invasive, electrical isolated and generally biocompatible.…”

A Conceptual Review of Nanosensors

“…These allow the analyte to permeate the matrix and interact with the indicator dye directly and selectively, thereby causing stimulation or quenching of fluorescence. 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].…”