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Homogeneous And Duplexed Immunoassay Of EGFR Receptors Based On Terbium Complex To Quantum Dot FRET | 18711
Journal of Analytical & Bioanalytical Techniques
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Förster resonance energy transfer (FRET) has attracted much research interest in bioanalytical application, because its transfer
distances (1-20 nm) are in the range of biomolecular interactions. Combining terbium complexes (Tbs) and semiconductor
quantum dots (QDs) for FRET biosensors has many advantages. Tbs provide multiple narrow photoluminescence (PL) emission
lines in a broad wavelength range and exceptionally long PL excited-state lifetimes (ms), which enable time-gated detection
void of autofluorescence background. QDs add very strong absorption and narrow and symmetric PL bands, whose colors can
be tuned by the composition and the size of the QDs. The combination in Tb-to-QD FRET offers homogeneous (no washing
and separation steps) and multiplexed biosensors. Here we exploit the nanosurface features of the QD by applying small single
domain antibodies (V
H nanobodies) as biological recognition molecules. Nanobodies do not only offer high surface coating
density but also a possibly reduced FRET distance compared to large IgG antibodies. We present a systematic investigation
of random vs. oriented nanobody-QD conjugation for FRET-based immunoassays. The homogeneous assays provide sub-
nanomolar (few ng/mL) detection limits of the two epidermal growth factor receptors EGFR and Her2 in 50 μL buffer or serum
samples. These very low EGFR and Her2 concentrations measured on a KRYPTOR diagnostic plate reader system under ?real-
life? sample conditions demonstrate the direct applicability of our nanobody-based Tb-to-QD FRET immunoassays for fast
and sensitive biomarker detection in both point-of-care and high throughput
Xue Qiu received her MS degree from Fudan University (P. R. China) in 2012 and now she is a PhD student under the supervision of Prof. Hildebrandt at Universit?
Paris-Sud (France). Her research interests include development of assays and sensors for biomarkers (proteins and DNA/RNAs) based on time-resolved Forster
resonance energy transfer.