title: Nanoparticle PEBBLE sensors for quantitative nanomolar imaging of intracellular free calcium ions.
abstract: Ca(2+) 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 Ca(2+) measurement involves the introduction of optically sensitive Ca(2+) indicators into living cells, combined with digital imaging microscopy. However, the use of free Ca(2+) indicators for intracellular sensing and imaging has several limitations, such as nonratiometric measurement for the most-sensitive indicators, cytotoxicity of the indicators, interference from nonspecific binding caused by cellular biomacromolecules, challenging calibration, and unwanted sequestration of the indicator molecules. These problems are minimized when the Ca(2+) indicators are encapsulated inside porous and inert polyacrylamide nanoparticles. We present PEBBLE nanosensors encapsulated with rhodamine-based Ca(2+) fluorescence indicators. The rhod-2-containing PEBBLEs presented here show a stable sensing range at near-neutral pH (pH 6-9). Because of the protection of the PEBBLE matrix, the interference of protein-nonspecific binding to the indicator is minimal. The rhod-2 PEBBLEs give a nanomolar dynamic sensing range for both in-solution (K(d) = 478 nM) and intracellular (K(d) = 293 nM) measurements. These nanosensors are useful quantitative tools for the measurement and imaging of the cytosolic nanomolar free Ca(2+) levels.
authors: Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, United States.
authors: Di
authors: D
authors: Si
authors: Tamir
authors: T
authors: Epstein
authors: Yong-Eun Koo
authors: YE
authors: Lee
authors: Raoul
authors: R
authors: Kopelman
mesh_terms: D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008297:Male; D053758:Nanoparticles; D036103:Nanotechnology; D011471:Prostatic Neoplasms / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D002118:Calcium