A novel electrochemical immunosensor that amplifies Poly(o-phenylenediamine) signal by pH-driven cascade reaction...

The present protocol develops an electrochemical immunosensor with poly(o-phenylene diamine) attached gold nanoparticles (PPD@Au NPs) as the immune platform, polydopamine-loaded cobalt ions (Co²⁺-PDA) as the immune probe, and K₂S₂O₈ as the signal amplifying substance with pH-driven cascade reaction. The application of conventional immunosensors often leads to easy leakage of the current signal and increases the impedance due to assembly. However, this new immunosensor offers the following advantages: (1) The signal substance PPD is modified on the electrode surface, effectively reducing the signal loss and leakage of the immunosensor; (2) The pH response reduces the impedance of the immunosensor while destroying the Co²⁺-PDA secondary antibody label; (3) The pH response releases a small amount of Co²⁺, leading to SO₄^-· generation by K₂S₂O₈ through a cascade reaction, further amplifying the PPD response current signal; (4) The pH response generates excess Co²⁺ and the by-product PDA fragments can consume the SO₄^-· generated by K₂S₂O₈, so that the final response signal decreases with the increasing antigen concentration. The experimental results showed that the immunosensor exhibited good selectivity, long-term stability, and reproducibility for AFP detection in the range of 1 pg/mL-100 ng/mL, with a detection limit of 0.214 pg/mL. Interestingly, it is expected to be used for detecting AFP in actual blood samples.