Nanoparticle PEBBLE Sensors for Quantitative Nanomolar Imaging of Intracellular Free Calcium Ions

摘要

Ca²⁺ is a universal second messenger and plays a major role in intracellular signaling, metabolism and a wide range of cellular processes. To date, one of the most successful approaches for intracellular Ca²⁺ measurement involves introduction of optically sensitive Ca²⁺ indicators into living cells, combined with digital imaging microscopy. However, the use of free Ca²⁺ indicators for intracellular sensing and imaging has several limitations, such as nonratiometric measurement for the most sensitive indicators, cytotoxicity of the indicators, interference from non-specific binding caused by cellular biomacromolecules, challenging calibration and unwanted sequestration of the indicator molecules. These problems are minimized when the Ca²⁺ indicators are encapsulated inside porous and inert polyacrylamide nanoparticles. We present PEBBLE nanosensors encapsulated with rhodamine based Ca²⁺ fluorescence indicators. The here presented rhod-2 containing PEBBLEs show a stable sensing range at near-neutral pH (pH 6–9). Due to the protection of the PEBBLE matrix, the interference of protein non-specific binding to the indicator is minimal. The rhod-2 PEBBLEs give a nanomolar dynamic sensing range for both in-solution (Kd = 478 nM) and intracellular (Kd = 293 nM) measurements. These nanosensors are a useful quantitative tool for the measurement and imaging of the cytosolic nanomolar free Ca²⁺ levels.