Nanobodies and nanocrystals: highly sensitive quantum dot-based homogenous FRET immunoassay for serum-based EGFR detection

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 fl uorescence-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 fl uorescence plate
reader and provides sub-nanomolar (few ng/mL) detection limits of soluble epidermal
growth factor receptor (EGFR) in 50 μ L buffer or serum samples. Apart from the fi rst
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.