Simultaneous electrochemical immunoassay using graphene–Au grafted recombinant apoferritin-encoded metallic labels as signal tags and dual-template magnetic molecular imprinted polymer as capture probes

• A signal amplification strategy was constructed for simultaneous detecting AFP and CEA based on graphene–Au signal tags.
• Recombinant apoferritin nanoparticles with higher binding capability towards metal ions were used as distinguishable trace labels.
• A dual-template magnetic molecularly imprinted polymers were utilized as capture probes for specific recognition with target analytes in real serum samples.

A novel electrochemical multiplexed immunoassay was designed for simultaneous determination of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) using recombinant apoferritin-encoded metallic nanoparticles (rApo-M) as labels and dual-template magnetic molecularly imprinted polymers (MMIPs) as capture probes. The labels were prepared by loading recombinant apoferritin (r-Apo) and separately immobilize primary antibodies (anti-AFP and anti-CEA) via Au nanoparticles of in site growth on graphene (G). The capture probes were synthesized by self-polymerization of dopamine (DA) on the Fe3O4 nanoparticles (Fe3O4 NPs) and using AFP and CEA as the template proteins, which were used to enrich the targets simultaneously. After a sandwich-type immunoreaction, the labels were captured to the surface of MMIPs. The subsequent electrochemical stripping analysis of the metal components from the immunocomplex provide a means for quantification of targets based on the peak currents of Cd and Pb. Experimental results showed the immunoassay enabled the simultaneous determination of AFP and CEA in a single run with wide dynamic ranges of 0.001–5 ng mL−1. And the detection limits of AFP and CEA were 0.3 and 0.35 pg mL−1 (S/N=3), respectively. These results suggested that the proposed multiplexed immunoassay would be applied for clinical screening of other biomarkers.