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

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 ch...

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Main Authors:	Zamaleeva, Alsu I., Despras, Guillaume, Luccardini, Camilla, Collot, Mayeul, de Waard, Michel, Oheim, Martin, Mallet, Jean-Maurice, Feltz, Anne
Format:	Online
Language:	English
Published:	MDPI 2015
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4610457/

id	pubmed-4610457
recordtype	oai_dc
spelling	pubmed-46104572015-10-26 FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains Zamaleeva, Alsu I. Despras, Guillaume Luccardini, Camilla Collot, Mayeul de Waard, Michel Oheim, Martin Mallet, Jean-Maurice Feltz, Anne Article 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 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. MDPI 2015-09-23 /pmc/articles/PMC4610457/ /pubmed/26404317 http://dx.doi.org/10.3390/s150924662 Text en © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Zamaleeva, Alsu I. Despras, Guillaume Luccardini, Camilla Collot, Mayeul de Waard, Michel Oheim, Martin Mallet, Jean-Maurice Feltz, Anne
spellingShingle	Zamaleeva, Alsu I. Despras, Guillaume Luccardini, Camilla Collot, Mayeul de Waard, Michel Oheim, Martin Mallet, Jean-Maurice Feltz, Anne FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains
author_facet	Zamaleeva, Alsu I. Despras, Guillaume Luccardini, Camilla Collot, Mayeul de Waard, Michel Oheim, Martin Mallet, Jean-Maurice Feltz, Anne
author_sort	Zamaleeva, Alsu I.
title	FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains
title_short	FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains
title_full	FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains
title_fullStr	FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains
title_full_unstemmed	FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains
title_sort	fret-based nanobiosensors for imaging intracellular ca2+ and h+ microdomains
description	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 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.
publisher	MDPI
publishDate	2015
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4610457/
_version_	1613489808774004736

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

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