Format

Send to

Choose Destination
Sci Adv. 2016 Jun 10;2(6):e1600265. doi: 10.1126/sciadv.1600265. eCollection 2016 Jun.

Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging.

Author information

1
Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607-7061, USA.
2
NanoBioPhotonics ( www.nanofret.com ), Institut d'Electronique Fondamentale, Université Paris-Saclay, Université Paris-Sud, CNRS, 91405 Orsay Cedex, France.
3
Cell Biology, Department of Biology, Science Faculty, Utrecht University, 3584 CH Utrecht, Netherlands.
4
Affymetrix Inc., 10255 Science Center Drive, San Diego, CA 92121, USA.
5
Optical Sciences Division, Code 5611, U.S. Naval Research Laboratory, Washington, DC 20375, USA.; Sotera Defense Solutions, Columbia, MD 21046, USA.
6
Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, USA.

Abstract

Time-gated Förster resonance energy transfer (FRET) using the unique material combination of long-lifetime terbium complexes (Tb) and semiconductor quantum dots (QDs) provides many advantages for highly sensitive and multiplexed biosensing. Although time-gated detection can efficiently suppress sample autofluorescence and background fluorescence from directly excited FRET acceptors, Tb-to-QD FRET has rarely been exploited for biomolecular imaging. We demonstrate Tb-to-QD time-gated FRET nanoassemblies that can be applied for intra- and extracellular imaging. Immunostaining of different epitopes of the epidermal growth factor receptor (EGFR) with Tb- and QD-conjugated antibodies and nanobodies allowed for efficient Tb-to-QD FRET on A431 cell membranes. The broad usability of Tb-to-QD FRET was further demonstrated by intracellular Tb-to-QD FRET and Tb-to-QD-to-dye FRET using microinjection as well as cell-penetrating peptide-mediated endocytosis with HeLa cells. Effective brightness enhancement by FRET from several Tb to the same QD, the use of low nanomolar concentrations, and the quick and sensitive detection void of FRET acceptor background fluorescence are important advantages for advanced intra- and extracellular imaging of biomolecular interactions.

KEYWORDS:

FRET relays; cell penetrating peptides; cytosol; endocytosis; immunostaining; microinjection; nanobodies; quantum dots; terbium

PMID:
27386579
PMCID:
PMC4928903
DOI:
10.1126/sciadv.1600265
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for PubMed Central
Loading ...
Support Center