FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains

Alsu Zamaleeva (1) , Guillaume Despras (2) , Camilla Luccardini (1) , Mayeul Collot (2) , Michel de Waard (3) , Martin Oheim (4) , Jean-Maurice Mallet (2) , Anne Feltz (1)

1 IBENS - Institut de biologie de l'ENS Paris
2 UPMC - Université Pierre et Marie Curie - Paris 6
3 Canaux calciques, Fonctions et Pathologies
4 LPC - UMR 8118 - Laboratoire de physiologie cérébrale

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 OPEN ACCESS Sensors 2015, 15 24663 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: quantum dot nanobiosensors; nanoparticle surface chemistry; FRET-based Ca 2+ and H + probes; red-emitting indicator; intracellular Ca 2+ and H + fluorometry; cell-penetrating peptide; concentration microdomain

Domaines: Chimie

Alsu Zamaleeva, Guillaume Despras, Camilla Luccardini, Mayeul Collot, Michel de Waard, et al.. FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains. Sensors, 2015, 15 (9), pp.24662-24680. ⟨10.3390/s150924662⟩. ⟨hal-01303366⟩