Details

Autor(en) / Beteiligte

• Wegner, K. David
• Lindén, Stina
• Jin, Zongwen
• Jennings, Travis L.
• Khoulati, Rachid el
• van Bergen en Henegouwen, Paul M. P.
• Hildebrandt, Niko

Titel

Nanobodies and Nanocrystals: Highly Sensitive Quantum Dot-Based Homogeneous FRET Immunoassay for Serum-Based EGFR Detection

Ist Teil von

• Small (Weinheim an der Bergstrasse, Germany), 2014-02, Vol.10 (4), p.734-740

Ort / Verlag

Germany: Blackwell Publishing Ltd

Erscheinungsjahr

2014

Link zum Volltext

Quelle

Wiley Online Library Journals Frontfile Complete

Beschreibungen/Notizen

• 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. Nano‐squared: The combination of single domain antibodies (nanobodies) and semiconductor quantum dots leads to highly sensitive and homogeneous Förster resonance energy transfer (FRET) immunoassays for epidermal growth factor receptor (EGFR).