FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains
AbstractSemiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca2+ and H+ transients. Our sensors combine a commercially available CANdot®565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca2+ or H+ probes. These ‘Rubies’ are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid) for H+ or Ca2+ sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery) at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca2+ and pH transients using live-cell fluorescence imaging. View Full-Text
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Zamaleeva, A.I.; Despras, G.; Luccardini, C.; Collot, M.; de Waard, M.; Oheim, M.; Mallet, J.-M.; Feltz, A. FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains. Sensors 2015, 15, 24662-24680.
Zamaleeva AI, Despras G, Luccardini C, Collot M, de Waard M, Oheim M, Mallet J-M, Feltz A. FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains. Sensors. 2015; 15(9):24662-24680.Chicago/Turabian Style
Zamaleeva, Alsu I.; Despras, Guillaume; Luccardini, Camilla; Collot, Mayeul; de Waard, Michel; Oheim, Martin; Mallet, Jean-Maurice; Feltz, Anne. 2015. "FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains." Sensors 15, no. 9: 24662-24680.