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

• Published in: Science advances 2016-06, Vol.2 (6), p.e1600265
• Main Authors: Afsari, Hamid Samareh, Cardoso Dos Santos, Marcelina, Lindén, Stina, Chen, Ting, Qiu, Xue, van Bergen En Henegouwen, Paul M P, Jennings, Travis L, Susumu, Kimihiro, Medintz, Igor L, Hildebrandt, Niko, Miller, Lawrence W
• Format: Article
• Language: English
• Subjects: Biosensors; Cell Line; Cell-Penetrating Peptides; Chemical Sciences; Endocytosis; Extracellular Space; Fluorescence Resonance Energy Transfer - methods; Humans; Intracellular Space; Life Sciences; Microinjections; Molecular Imaging - methods; Molecular Imaging - standards; Nanostructures - chemistry; Optical Imaging - methods; Optical Imaging - standards; Physics; Quantum Dots; SciAdv r-articles; Semiconductors; Sensitivity and Specificity; Single-Domain Antibodies; Terbium
• ISSN: 2375-2548; 2375-2548
• DOI: 10.1126/sciadv.1600265

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.