A label-free amperometric immunosensor based on horseradish peroxidase functionalized carbon nanotubes and bilayer gold nanoparticles

In this work, a novel label-free amperometric immunosensor has been constructed for detecting α-1-fetoprotein (AFP) based on nanocomposite of horseradish peroxidase (HRP) labeled carbon nanotubes (CNTs). First, the gold nanoparticles (AuNPs) were electrodeposited on the surface of the glass carbon electrode by electrochemical reduction of gold chloride tetrahydrate (HAuCl₄) to immobilize horseradish peroxidase labeled carbon nanotubes (HRP-CNTs). Then HRP-CNTs bioconjugate was immobilized on the surface of the electrodeposited AuNPs layer by the combination of forces (coordination and electrostatic force). Subsequently, it was immersed into gold colloidal nanoparticles (GNPs) solution, which was used to immobilize antibody biomolecules (anti-AFP). Enhanced sensitivity was obtained by using bioconjugates featuring HRP labeled (HRP-CNTs), which had lager specific surface area and good electronic catalysis (current response signal) compared to carbon nanotubes. Under optimized conditions, the linear ranges were from 0.2 to 200 ng mL⁻¹ with a detection limit of 0.067 ng mL⁻¹ (at an S/N of 3). The proposed immunosenor showed good precision, acceptable stability and reproducibility and could be used for the detection AFP in normal human serum, which provided a potential alternative tool for the detection of protein in clinical diagnosis.