Ultrasensitive Label-Free Electrochemical Immunosensor for Carbohydrate Antigen-125 (CA125) Employing Cu(II)-Polyethyleneimine (PEI)/Silica Nanoparticle@Overoxidized Polypyrrole (OPPy)/Multi-Walled Carbon Nanotubes (MWNTs) Hybrid Nanomaterial

Novel electroactive nanotags of Cu(II) functionalized polyethylenimine (PEI)/SiO₂ nanoparticles (abbreviated as Cu²⁺/PEI/SiO₂ NPs) were conveniently synthesized using a strong coordination reaction between Cu(II) and the multi-amino groups of PEI. These electroactive nanotags were easily coated on the overoxidized polypyrrole (OPPy) modified Nafion/multi-walled carbon nanotubes (abbreviated as Nafion/MWNTs) electrode surface through Cu-N bonds and strong electrostatic interactions. The Cu²⁺/PEI/SiO₂ NPs@OPPy/MWNTs hybrid nanomaterial was formed without the addition of extra membrane-forming substances. The experimental results demonstrate that the OPPy/MWNTs composite membrane coupled with electrocatalytic Cu(Tris)₄²⁺ complexes in tris(hydroxymethyl)aminomethane (Tris)-HCl buffer medium amplified the electrochemical signals of nanotags based on the mutual synergistic effects. As an application, a label-free electrochemical immunosensor for epithelial ovarian cancer biomarker-carbohydrate antigen-125 (CA125) was constructed by covalently immobilizing CA125 antibody onto the hybrid nanomaterial modified electrode. With the increasing concentrations of CA125 antigen, different decreases in the electrochemical signals of the nanotags were observed due to the formation of antigen-antibody immune complexes. The electrochemical immunosensor showed a linear relationship from 0.001 to 500 U·mL⁻¹ for CA125 with a detection limit of 3.4 × 10⁻⁴ U·mL⁻¹. In addition, the proposed immunosensor was applied to the determination of CA125 in human serum with satisfactory results.