This study devised a label-free electrochemical immunosensor for the quantitative detection of alpha-fetoprotein (AFP). 3-Polythiophene acetic acid (3-PTAA) nanoparticles were anchored onto a few-layer graphene (FLG) nanosheet, and the resulting nanocomposite was utilized as the immunosensor platform. The AFP antibody (anti-AFP) was immobilized on 3-PTAA@FLG via a covalent interaction between the amine group of anti-AFP and the carboxylic group of 3-PTAA via ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling. FLG is largely responsible for providing electrochemical signals, whereas 3-PTAA nanoparticles are well-known for their ability to be compatible with biological molecules in neutral aqueous solutions. Moreover, the carboxyl group present in 3-PTAA effectively binds anti-AFP through EDC/NHS conjugation. Owing to good dispersibility and higher surface area of 3-PTAA, it is very convenient for casting the polymer directly on the electrode substrate followed by immobilization of anti-AFP. Thus, it is feasible to regulate the activity of AFP proteins and control the spatial distribution of the immobilized anti-AFP proteins. The electrochemical sensing performance was assessed via cyclic voltammetry and electrochemical impedance spectroscopy. For an increase in the bioconjugate concentration, the results demonstrated a surge in charge-transfer resistance and a consequent decline in the current response. This approach effectively detected AFP at an extended dynamic range of 0.0001-250 ng/mL with a detection limit of 0.047 pg/mL. Furthermore, the sensing capacity of the immunosensor for AFP detection has been demonstrated to be steady in real human serum cultures. Our approach exhibits good electrochemical performance in terms of reproducibility, selectivity, and stability, which would surely impart budding applications in the clinical diagnosis of several other tumor markers.
Keywords: 3-PTAA; CV; EIS; FLG; immunosensing; nanocomposite; nanosheet.