A Nanobody‐on‐Quantum Dot Displacement Assay for Rapid and Sensitive Quantification of the Epidermal Growth Factor Receptor (EGFR)

Abstract

Biosensing approaches that combine small, engineered antibodies (nanobodies) with nanoparticles are often complicated. Here, we show that nanobodies with different C‐terminal tags can be efficiently attached to a range of the most widely used biocompatible semiconductor quantum dots (QDs). Direct implementation into simplified assay formats was demonstrated by designing a rapid and wash‐free mix‐and‐measure immunoassay for the epidermal growth factor receptor (EGFR). Terbium complex (Tb)‐labeled hexahistidine‐tagged nanobodies were specifically displaced from QD surfaces via EGFR‐nanobody binding, leading to an EGFR concentration‐dependent decrease of the Tb‐to‐QD Förster resonance energy transfer (FRET) signal. The detection limit of 80±20 pM (16±4 ng mL−1) was 3‐fold lower than the clinical cut‐off concentration for soluble EGFR and up to 10‐fold lower compared to conventional sandwich FRET assays that required a pair of different nanobodies.

Plasmonic quenching and enhancement: metal–quantum dot nanohybrids for fluorescence biosensing

“…5). 74 A small genetically engineered antibody with a size of approximately 15 kDa (nanobody) and specific for the epidermal growth factor receptor EGFR was labeled with a terbium complex (Tb). A hexahistidine (His 6 ) tag on the opposite end of the EGFR binding site of the nanobody allowed for efficient polyhistidine-metal affinity mediated self-assembly to a 625 nm emitting core–shell QD, which served as a FRET acceptor for the Tb FRET donor.…”

Targeted Single Particle Tracking with Upconverting Nanoparticles

“…The recently developed lipid-based coating of UCNPs could also be of interest, as it allows for better protection of the emitting centers against quenching by water molecules and provides better stability of UCNPs in cell buffers and media . Further improvements could also be anticipated by replacing the bulky antibodies (150–180 kDa, ∼15 nm) with much smaller and flexible aptamers (6–30 kDa, ∼2 nm), nanobodies (∼15 kDa, ∼3 nm), or other artificial small protein binders which were successfully conjugated to nanoparticules . Also the large streptavidin–biotin link (60 kDa) could be replaced by an irreversible conjugation using genetically encoded protein tags like the SpyTag/SpyCatcher system (12 kDa) or even a smaller link via click chemistry. , On the instrumental side, other excitation strategies could further maximize the upconversion efficiency, resulting in the collection of more photons and thus allowing for better localization accuracy and/or higher frame rates …”

Pursuing excitonic energy transfer with programmable DNA-based optical breadboards

“…Indeed, many of the examples described in the preceding section epitomize such applications where, for example, dye-labeled DNA structures either on their own or as interfaced with NPs such as QDs are providing for a variety of targeted sensors for monitoring force on the nanoscale, 432 pH, 429 voltage changes, 435 multiplexed enzyme activity, 431,445,448 along with providing for multiplexed diagnostics, and biomarker detection. 419,436,460 The always growing library of available fluorescent materials, ranging from new dye families to noble metal clusters and NPs manifesting unique quantum confined or other complex photophysical properties such as upconversion will also contribute to new FRET-based applications and probes that utilize DNA scaffolds. 4,5,117,461,462 Interestingly, DNA scaffolding often proves itself critically useful in helping to confirm that new fluorescent materials actually engage in FRET by providing a defined nanoscale ruler to do the requisite distance-dependent ET experiments.…”