A novel antibody-aptamer sandwich-type electrochemical biosensor for femtomolar detection of VEGF165 in patients with lymphoma cancer

Abstract

Vascular endothelial growth factor (VEGF165) is a homodimeric protein that plays a key role in tumor formation and progression. Therefore, early determination of blood levels of VEGF165 is vital in the diagnosis and successful treatment of different types of cancers. In the present study, a novel sandwich-type biosensor was designed for sensitive and selective determination of VEGF165 in human blood serum samples. Carbon dots (CDs) were used as inexpensive and conductive nanomaterials for immobilization of the antibody (Ab) on the electrode surface. The prepared carbon dots were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), FT-IR, UV–Visible spectrophotometry, and spectrofluorimetry. Highly fluorescent particles were formed due to the quantum restrictions brought about by their ultrasmall size (average size ~10 nm). The high surface-to-volume ratio of carbon dots, as well as the presence of many oxygen-containing functional groups, was appropriate for attachment of large amounts of Ab moieties. In the presence of VEGF165, a highly selective VEGF165-Ab complex was formed on the electrode surface, which was then bound to the added selective aptamer to form Aptamer-VEGF₁₆₅-Ab. As the electrochemical probe, methylene blue (MB) was used, which has a high binding tendency to guanine bases of the aptamer (MB-Aptamer-VEGF165-Ab) on the electrode surface, which resulted in well-known MB⁺/MB redox peaks. A linear relationship was obtained between peak currents and log [VEGF165] in the wide concentration range of 0.01 pM to 10 nM (0.34–34 × 10⁶ pg/mL) by differential pulse voltammetry (pH 7.4, 0.1 M PBS). The limit of detection (LOD) was calculated as 3.3 fM (0.112 pg/mL) based on S/N = 3. The proposed biosensor was successfully applied to the determination of VEGF165 in blood serum samples of patients with lymphoma cancer.