title: Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging.
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.
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607-7061, USA.
authors.forename: Hamid Samareh
authors.identifier: 0000-0002-5839-4765
authors.initials: HS
authors.lastname: Afsari
authors.affiliation: NanoBioPhotonics ( www.nanofret.com ), Institut d'Electronique Fondamentale, Université Paris-Saclay, Université Paris-Sud, CNRS, 91405 Orsay Cedex, France.
authors.forename: Marcelina
authors.initials: M
authors.lastname: Cardoso Dos Santos
authors.affiliation: NanoBioPhotonics ( www.nanofret.com ), Institut d'Electronique Fondamentale, Université Paris-Saclay, Université Paris-Sud, CNRS, 91405 Orsay Cedex, France.
authors.forename: Stina
authors.initials: S
authors.lastname: Lindén
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607-7061, USA.
authors.forename: Ting
authors.identifier: 0000-0002-3124-4337
authors.initials: T
authors.lastname: Chen
authors.affiliation: NanoBioPhotonics ( www.nanofret.com ), Institut d'Electronique Fondamentale, Université Paris-Saclay, Université Paris-Sud, CNRS, 91405 Orsay Cedex, France.
authors.forename: Xue
authors.identifier: 0000-0003-0635-6801
authors.initials: X
authors.lastname: Qiu
authors.affiliation: Cell Biology, Department of Biology, Science Faculty, Utrecht University, 3584 CH Utrecht, Netherlands.
authors.forename: Paul M P
authors.initials: PM
authors.lastname: van Bergen En Henegouwen
authors.affiliation: Affymetrix Inc., 10255 Science Center Drive, San Diego, CA 92121, USA.
authors.forename: Travis L
authors.identifier: 0000-0002-6845-2041
authors.initials: TL
authors.lastname: Jennings
authors.affiliation: Optical Sciences Division, Code 5611, U.S. Naval Research Laboratory, Washington, DC 20375, USA.; Sotera Defense Solutions, Columbia, MD 21046, USA.
authors.forename: Kimihiro
authors.initials: K
authors.lastname: Susumu
authors.affiliation: Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, USA.
authors.forename: Igor L
authors.initials: IL
authors.lastname: Medintz
authors.affiliation: NanoBioPhotonics ( www.nanofret.com ), Institut d'Electronique Fondamentale, Université Paris-Saclay, Université Paris-Sud, CNRS, 91405 Orsay Cedex, France.
authors.forename: Niko
authors.initials: N
authors.lastname: Hildebrandt
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607-7061, USA.
authors.forename: Lawrence W
authors.identifier: 0000-0003-4501-9357
authors.initials: LW
authors.lastname: Miller
mesh_terms: D002460:Cell Line; D057846:Cell-Penetrating Peptides; D004705:Endocytosis; D005110:Extracellular Space; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D042541:Intracellular Space; D008845:Microinjections; D057054:Molecular Imaging / Q000379:methods / Q000592:standards; D049329:Nanostructures / Q000737:chemistry*; D061848:Optical Imaging / Q000379:methods* / Q000592:standards; D045663:Quantum Dots; D012666:Semiconductors; D012680:Sensitivity and Specificity; D061905:Single-Domain Antibodies; D013725:Terbium
chemical_list: D057846:Cell-Penetrating Peptides; D061905:Single-Domain Antibodies; D013725:Terbium
keywords: FRET relays; cell penetrating peptides; cytosol; endocytosis; immunostaining; microinjection; nanobodies; quantum dots; terbium