A novel bidirectional (anodic and cathodic) stripping voltammetric immunoassay (SVI) was designed for the simultaneous determination of multiple model cancer biomarkers (AFP, CEA and CA19-9) in a single run, based on the integration of Envision complex loaded metal nanoparticles as signal tags and immunomagnetic beads as capture probes. The Envision complex, which is a long branching polymer consisting of numerous secondary antibodies and horseradish peroxidase (HRP), was employed for signal amplification by labeling corresponding detection antibodies and further loading metal nanoparticles (CdS, PbS and gold) to prepare distinguishable metal signal tags. Herein, the generation of immunosensing probes involved the co-immobilization of three types of primary anti-AFP, anti-CEA, and anti-CA19-9 antibodies onto a single magnetic Dynabead. After a two-binding step sandwich-type immunoassay, the Envision/CdS, Envision/PbS and Envision/Au signal tags were introduced onto the surface of the Dynabeads. The subsequent bidirectional voltammetric analysis of stripping metal components from immunocomplexes for quantification of tumor biomarkers was performed in a microcell with minimum capacity of 50 μL. Experimental results showed the immunoassay enabled the simultaneous determination of multiple biomarkers over a broad range of concentrations (AFP, 1 pg mL−1–50 ng mL−1; CEA, 1 pg mL−1–50 ng mL−1; CA19-9, 5 pg mL−1–100 ng mL−1) with detection limits reaching 0.02 pg mL−1 for AFP, 0.05 pg mL−1 for CEA, and 0.3 pg mL−1 for CA19-9. The results indicated that the proposed bidirectional multiplexed immunoassay can increase the number of analytes by SVI and has high sensitivity, excellent stability, and great promise for applications in clinical cancer diagnosis.
