Engineered synthetic nanobody-based biosensors for electrochemical detection of epidermal growth factor receptor

Two engineered synthetic nanobody-based nanobiocomposite platforms were developed for label-free electrochemical detection of the epithelial growth factor receptor (EGFR) biomarker. Screen-printed carbon electrodes (SPCE) were decorated either with NiO nanoparticles (NPs) or poly(thiophene acetic acid) (PTAA) to link the anti-EGFR nanobody (Nb) and form nanobiocomposites for detecting the EGFR biomarker by electrochemical impedance spectroscopy (EIS). The nanoarchitectures were prepared by in situ electrosynthesis of NiO NPs or PTAA layers at SPCEs. A modified version of the 9G8 Nb (Nb9G8m), specific for the EGFR (anti-EGFR), was designed and produced as the nanobiosensor bioreceptor. This Nb was engineered to provide a hexahistidine tag (6xHis-tag) and a lysine (Lys) dual functionality to form a (6xHis-tag)/Ni²⁺ or Lys/PTAA interface. The biosensing interfaces were characterized by field-emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, cyclic voltammetry, and EIS. The nanobody/nanobiocomposite-based biosensors detected EGFR proteins in a linear range from 0.25 to 50 μg mL⁻¹ and 0.5 to 50 μg mL⁻¹, with limits of detection of 0.46 μg mL⁻¹ and 1.14 μg mL⁻¹, for NiO- and PTAA-based platforms, respectively. The biosensing platforms offer high simplicity, specificity, and selectivity to detect EGFR, but Nbs can be readily engineered to detect other (glycol)proteins. Finally, as a proof of concept, the EGFR was detected in several tumor cell lines, differentiating biomarker expression among them.