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.

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.…”

Advancements in semiconductor quantum dots: expanding frontiers in optoelectronics, analytical sensing, biomedicine, and catalysis

“…264 Cancer biomarkers and antigen sensing. Cancer biomarkers like heparin, heparinase, metalloproteinase-14 (MMP-14), and miRNA have been selectively sensed by CQDs through methods like aggregation-induced emission (AIEE), 265 Fo ¨rster resonance energy transfer (FRET), [266][267][268][269][270][271][272][273][274] immunosensor like electrochemiluminescence (ECL), 268 and photoelectrochemical (PEC). 275 Dan Yan et al designed a novel dual-detection probe based on a ''turn-on-off'' fluorescence method for sensitive and selective detection of heparin and heparinase I using polyethyleneimine (PEI)-modified Ag 2 S (PEI-Ag 2 S CQDs).…”

“…268 FRET strategies can be effectively utilized with CQDs for sensing applications by functionalizing them with specific molecules such as dyes 267,274 or nanobodies (NBs). 266 For instance, in a study by Zhicheng Jin et al, CQDs were conjugated with rhodamine derivative dyes for sensing MMP-14. The fluorescence intensity of the CQDs exhibited a gradual increase, while concurrently, the intensity of the dye gradually decreased in response to the concentration of MMP-14.…”

“…They achieved a remarkable LOD of 80 AE 20 pM (16 AE 4 ng mL À1 ) for quantification of the epidermal growth factor receptor (EGFR). 266 Yanbin Zhang et al synthesized InP-based core/shell CQDs for the detection of alpha-fetoprotein (AFP, serum markers of hepatocellular carcinoma [HCC]) antigen by coupling the alpha-fetoprotein by using a CQD-based fluorescence-linked immunosorbent assay (FLISA), which enabled a low detection limit of 0.58 ng mL À1 to be achieved. The fluorescence intensity of CQDs increases as the concentration of AFP increases.…”

Structural Insights into the Stability and Recognition Mechanism of the Antiquinalphos Nanobody for the Detection of Quinalphos in Foods

“…As with other immunoassays, Nb-based immunoassays are most easily generated for the detection of large molecules. , However, numerous assays for low-molecular-weight compounds have now been reported, including insecticides, , toxins, ,, herbicides, , and byproducts in food processing . Despite increasing research, there is limited structural information revealing the detailed mechanisms of hapten recognition with Nbs.…”