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Sensors (Basel). 2015 Sep 23;15(9):24662-80. doi: 10.3390/s150924662.

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

Author information

1
Ecole Normale Supérieure, Institut de Biologie de l'ENS (IBENS), Inserm U1024, CNRS UMR 8197, Paris F-75005, France. alsu.zamaleeva@curie.fr.
2
Department de Chimie, École Normale Supérieure-PSL Research University, CNRS UMR 7203 LBM, 24, rue Lhomond, and Sorbonne University, UPMC Univ Paris 06 LBM, 4 place Jussieu, Paris F-75005, France. gdespras@oc.uni-kiel.de.
3
Ecole Normale Supérieure, Institut de Biologie de l'ENS (IBENS), Inserm U1024, CNRS UMR 8197, Paris F-75005, France. Camilla.luccardini@univ-lyon1.fr.
4
Department de Chimie, École Normale Supérieure-PSL Research University, CNRS UMR 7203 LBM, 24, rue Lhomond, and Sorbonne University, UPMC Univ Paris 06 LBM, 4 place Jussieu, Paris F-75005, France. mayeul.collot@unistra.fr.
5
Inserm U836, Grenoble Neuroscience Institute, Research Group 3, LabEx Ion Channel Science and Therapeutics, Joseph Fourier University, BP170, Grenoble Cedex 09 38042, France. Michel.dewaard@ujf-grenoble.fr.
6
Brain Physiology Laboratory, CNRS UMR 8118, Faculté des Sciences Fondamentales et Biomédicales, Fédération de Neurosciences FR3636, Paris Descartes University, PRES Sorbonne Paris Cité, Paris F-75006, France. Martin.oheim@parisdescartes.fr.
7
Department de Chimie, École Normale Supérieure-PSL Research University, CNRS UMR 7203 LBM, 24, rue Lhomond, and Sorbonne University, UPMC Univ Paris 06 LBM, 4 place Jussieu, Paris F-75005, France. jean-maurice.mallet@ens.fr.
8
Ecole Normale Supérieure, Institut de Biologie de l'ENS (IBENS), Inserm U1024, CNRS UMR 8197, Paris F-75005, France. anne.feltz@ens.fr.

Abstract

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.

KEYWORDS:

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

PMID:
26404317
PMCID:
PMC4610457
DOI:
10.3390/s150924662
[Indexed for MEDLINE]
Free PMC Article

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