Fiche publication
Date publication
septembre 2015
Journal
Sensors (Basel, Switzerland)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr COLLOT Mayeul
Tous les auteurs :
Zamaleeva AI, Despras G, Luccardini C, Collot M, de Waard M, Oheim M, Mallet JM, Feltz A
Lien Pubmed
Résumé
Semiconductor 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 Ca(2+) and H⁺ transients. Our sensors combine a commercially available CANdot(®)565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca(2+) 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 Ca(2+) 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 Ca(2+) and pH transients using live-cell fluorescence imaging.
Mots clés
FRET-based Ca2+ and H+ probes, cell-penetrating peptide, concentration microdomain, intracellular Ca2+ and H+ fluorometry, nanoparticle surface chemistry, quantum dot nanobiosensors, red-emitting indicator
Référence
Sensors (Basel). 2015 09 23;15(9):24662-80
