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.

本协议开发了一种电化学免疫传感器，以聚（邻苯二胺）附着的金纳米粒子（PPD@Au NPs）为免疫平台，以聚多巴胺负载的钴离子（Co ²⁺ -PDA）为免疫探针，K ₂ S ₂ O ₈作为具有pH驱动的级联反应的信号放大物质。常规免疫传感器的应用往往会导致电流信号容易泄漏，并且由于组装而增加了阻抗。然而，这种新型免疫传感器具有以下优点：（1）在电极表面修饰了信号物质PPD，有效减少了免疫传感器的信号丢失和泄漏；(2) pH响应降低了免疫传感器的阻抗同时破坏了Co ²⁺-PDA二抗标签；(3) pH响应释放少量Co ²⁺，导致K ₂ S ₂ O ₈通过级联反应生成SO ₄ ^{- · ，进一步放大PPD响应电流信号；}(4) pH响应产生过量的Co ²⁺副产物PDA碎片可以消耗K ₂ S ₂ O ₈产生的SO ₄ ^{− ·}，因此最终的反应信号随着抗原浓度的增加而降低。实验结果表明，免疫传感器在1 pg/mL-100 ng/mL范围内对AFP检测表现出良好的选择性、长期稳定性和重现性，检测限为0.214 pg/mL。有趣的是，它有望用于检测实际血液样本中的 AFP。