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.

Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?

“…3-phenoxybenzoic acid 4 , aflatoxin 10 , ochratoxin 8 , and tetrabromobisphenol A 9,11 . However, for analysis of large analytes, a number of sandwich immunoassays using nanobodies showed moderate performance for procalcitonin 12 , epidermal growth factor receptor 13 , Bacillus thuringiensis Cry1Ac and Cry1Fa toxins 14,15 , influenza H3N2 and H5N1 16,17 , and hepatitis B surface antigen 18 , with the LOD in the ng/mL range or even much higher. Thus, it is often important to increase the sensitivity of nanobody based immunoassays.…”

Kinetic and Sensitive Analysis of Tyrosinase Activity Using Electron Transfer Complexes: In Vitro and Intracellular Study

“…Quantum dots (QDs) are favorable platforms as energy donors that can be functionalized with multiple bio‐recognition groups as acceptors to construct Förster resonance energy transfer (FRET) system in response to target analyte . Besides FRET, electron‐transfer (ET) has also been involved in fluorescence (FL) modulation.…”

Improved Immunoassay Sensitivity in Serum as a Result of Polymer-Entrapped Quantum Dots: ‘Papaya Particles’

“…However the most routinely used methods use carbodiimide chemistry or the high affinity interaction between biotin and streptavidin, whose limitations have been recently highlighted 22,23 . Examples of QD nanoparticle-based immunoassay working directly in complex matrices are rare 20,21,24,25 .…”