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.

Keywords: Diagnostics; EGFRvIII; FRET; Lanthanides; Nanoparticles.

Figure 1

A) Principle of the NB‐based Tb‐to‐QD FRET sandwich immunoassays (see Table 1 for explanation of abbreviations). Mixing of NB‐QD acceptor conjugates (left side from top to bottom: NB2‐H‐QD625‐CL4, NB2‐B‐QD705‐sAv, and NB2‐C‐QD705‐PEG) with Tb‐NB1 donor conjugates and sEGFR (gray arrows in the center) resulted in the formation of immunological sandwich complexes with close Tb‐QD proximity for FRET (right side). Because the sEGFR is a Fc‐chimera homodimer and NB1 and NB2 bind to different epitopes of each monomer, different donor‐acceptor distances are possible, resulting in a mixture of high, low, and no FRET. The different sEGFR conformations and NB binding sites are shown in Supporting Figure S1. B) EGFR sandwich FRET immunoassay calibration curves (rel. FRET‐ratio is the FRET‐ratio normalized to the blank sample) using Tb‐NB1 as donor conjugates and NB2‐H‐QD625‐CL4 (1.5 nM—blue), NB2‐B‐QD705‐sAv (1.5 nM—black; 3.0 nM—green), and NB2‐C‐QD705‐PEG (1.5 nM—red) as acceptor conjugates. Data points represent three (10 for the blank samples without sEGFR) independent measurements. Error bars represent standard deviations. The sEGFR concentrations (0, 0.075, 0.15, 0.30, 0.60, 0.90, 1.20, 1.50, 2.25, 3.0, 4.5, 6.0, 7.5, 9.0, 10.5, and 12.0 nM) are those in the 50 μL sample (they are 3 times lower in the total 150 μL assay volume).

Figure 2

A) Principle of Tb‐to‐QD FRET NB displacement immunoassays. Mixing of Tb‐NB1‐H donor conjugate and QD625‐CL4 acceptor results in Tb‐to‐QD FRET (left). The addition of sEGFR (gray arrow in the center) leads to a displacement of Tb‐NB1‐H from the QD surface to sEGFR and disruption of FRET (right). B) NB displacement FRET immunoassay calibration curves with an LOD (3 standard deviations below the zero concentration value – see inset) of 0.08±0.02 nM (16±4 ng mL⁻¹) sEGFR. Data points represent three (10 for the blank samples without sEGFR) independent measurements. Error bars represent standard deviations (σ). The EGFR concentrations are those in the 50 μL sample.

Figure 3

Comparison of NB displacement immunoassays for sEGFR (A) and sEGFRvIII (B) in serum (20 vol %) and buffer. Data points represent three (10 for the blank samples without sEGFR) independent measurements. Error bars represent standard deviations (σ). The EGFR concentrations are those in the 50 μL sample.