title: Nanobodies and nanocrystals: highly sensitive quantum dot-based homogeneous FRET immunoassay for serum-based EGFR detection.
abstract: Semiconductor quantum dot nanocrystals (QDs) for optical biosensing applications often contain thick polyethylene glycol (PEG)-based coatings in order to retain the advantageous QD properties in biological media such as blood, serum or plasma. On the other hand, the application of QDs in Förster resonance energy transfer (FRET) immunoassays, one of the most sensitive and most common fluorescence-based techniques for non-competitive homogeneous biomarker diagnostics, is limited by such thick coatings due to the increased donor-acceptor distance. In particular, the combination with large IgG antibodies usually leads to distances well beyond the common FRET range of approximately 1 to 10 nm. Herein, time-gated detection of Tb-to-QD FRET for background suppression and an increased FRET range is combined with single domain antibodies (or nanobodies) for a reduced distance in order to realize highly sensitive QD-based FRET immunoassays. The "(nano)(2) " immunoassay (combination of nanocrystals and nanobodies) is performed on a commercial clinical fluorescence plate reader and provides sub-nanomolar (few ng/mL) detection limits of soluble epidermal growth factor receptor (EGFR) in 50 μL buffer or serum samples. Apart from the first demonstration of using nanobodies for FRET-based immunoassays, the extremely low and clinically relevant detection limits of EGFR demonstrate the direct applicability of the (nano)(2-) assay to fast and sensitive biomarker detection in clinical diagnostics.
authors.affiliation: NanoBioPhotonics, Institut d'Electronique Fondamentale, Université Paris-Sud, 91405, Orsay Cedex, France.
authors.forename: K David
authors.initials: KD
authors.lastname: Wegner
authors.forename: Stina
authors.initials: S
authors.lastname: Lindén
authors.forename: Zongwen
authors.initials: Z
authors.lastname: Jin
authors.forename: Travis L
authors.initials: TL
authors.lastname: Jennings
authors.forename: Rachid
authors.initials: R
authors.lastname: el Khoulati
authors.forename: Paul M P
authors.initials: PM
authors.lastname: van Bergen en Henegouwen
authors.forename: Niko
authors.initials: N
authors.lastname: Hildebrandt
mesh_terms: D002138:Calibration; D066246:ErbB Receptors / Q000097:blood*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D053758:Nanoparticles / Q000737:chemistry*; D045663:Quantum Dots / Q000737:chemistry*; D061905:Single-Domain Antibodies / Q000737:chemistry*; D013057:Spectrum Analysis
chemical_list: D061905:Single-Domain Antibodies; D066246:ErbB Receptors
keywords: FRET; VHH; immunoassays; quantum dots; terbium