Differential pulse voltammetric immunosensor for direct and label-free detection of VEGF using variable domain of heavy-chain antibody displaying phage

Abstract

Herein the pioneering label-free electrochemistry-based immunosensor with exceptional sensitivity for the rapid and efficient detection of vascular endothelial growth factor (VEGF) was precisely designed based on the phage display technique. The specific variable domain of a heavy-chain antibody (VHH)-displaying phage against VEGF8–109 was successfully immobilized on Au nanoparticles deposited gold electrode modified by 3-mercapto propionic acid and 11-mercaptoundecanoic acid and then, carbodiimide coupling reaction using N-Hydroxysuccinamide and N-(3-dimethyl aminopropyl)-N′-ethyl carbodiimide hydrochloride. Electrochemical characterizations of the proposed immunosensor were performed by PM-FT-IRRAS, scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy techniques. Electrochemical measurements were performed using differential pulse voltammetry in phosphate buffer containing [Fe(CN)6]3–/4– ions as a probe, where the decline in the peak current value was related to a concentration of the antigen cast on the surface of the immunosensor. Under the optimum conditions (pH 7.4, interval time 20 s, and step potential 0.1 V), a linear range of response for the detection of VEGF8–109 by immunosensor was 0.03–10,000 pg/mL (in log concentration), with a limit of detection of 11 fg/mL. The specificity of the immunosensor was also investigated against some typical blood growth factors. The novel immunosensor was applied to evaluate VEGF levels in human serum specimens clinically. The recommended immunosensor was uncomplicated, reasonably priced, and showed adequate accuracy and specificity, which could be applied in the early diagnosis of tumors.