Fluorescent nanosensors for intracellular measurements: synthesis, characterization, calibration, and measurement

Abstract

Measurement of intracellular acidification is important for understanding fundamental biological pathways as well as developing effective therapeutic strategies. Fluorescent pH nanosensors are an enabling technology for real-time monitoring of intracellular acidification. The physicochemical characteristics of nanosensors can be engineered to target specific cellular compartments and respond to external stimuli. Therefore, nanosensors represent a versatile approach for probing biological pathways inside cells. The fundamental components of nanosensors comprise a pH-sensitive fluorophore (signal transducer) and a pH-insensitive reference fluorophore (internal standard) immobilized in an inert non-toxic matrix. The inert matrix prevents interference of cellular components with the sensing elements as well as minimizing potentially harmful effects of some fluorophores on cell function. Fluorescent nanosensors are synthesized using standard laboratory equipment and are detectable by non-invasive widely accessible imaging techniques. The outcomes of studies employing this technology are dependent on reliable methodology for performing measurements. In particular, special consideration must be given to conditions for sensor calibration, uptake conditions and parameters for image analysis. We describe procedures for: (1) synthesis and characterization of polyacrylamide and silica based nanosensors, (2) nanosensor calibration and (3) performing measurements using fluorescence microscopy.

New generation of bioreactors that advance extracellular matrix modelling and tissue engineering

“…Fluorescent nanosensors are sub-micron sized optical sensors specifically designed for non-invasive analyte monitoring in real time (Desai et al 2014 ). They generally based on porous matrices composed of crosslinked polyacrylamide which encapsulate a sensing component that is responsive to analytes (Buck et al 2004 ).…”

Augmenting Automated Analytics Using Fluorescent Nanosensors

“…could be extracted and then treated with nanosensors to determine intra or extracellular biochemical parameters [88]. For bioreactors, intracellular biochemical parameter characterization would consist of a very large time lag as particles would have to be initially delivered and validated to ensure delivery of particles has not induced biochemical changes [89], and therefore may not be valid for automated analytics.…”

Intracellular processing of silica-coated superparamagnetic iron nanoparticles in human mesenchymal stem cells

“…Polyacrylamide-based fluorescent nanosensors are an example of a smart measurement system that allow for complex sensory data to be acquired with minimal sample interference 21. They are spherical particles, ∼50 nm in diameter, which allow for many particles to be delivered to intracellular spaces and provide a high signal-to-noise ratio 22. Due to their size and inert matrix, combined with their ratiometric measurement properties, they can collect valuable subcellular real-time metrics for parameters, such as pH and oxygen (Fig.…”