A highly sensitive immunosensor for calmodulin assay based on enhanced biocatalyzed precipitation adopting a dual-layered enzyme strategy

• A highly sensitive immunosensor adopting a dual-layered enzyme strategy was proposed.
• This immunosensor exhibited satisfactory analytical performances for CaM detection.
• It was successfully applied to the CaM analysis in two cancer cells (HepG2 and MCF-7) with high sensitivity, which has shown great potency for cancer study.

Calmodulin (CaM) is a ubiquitous protein in eukaryotic cells, and it plays an important role in cancer progression. In this paper, a highly sensitive immunosensor adopting a dual-layered enzyme strategy was proposed for electrochemical detection of CaM. This immunosensor was constructed by introducing honeycomb-like mesoporous carbon (HMPC) as a sensor platform to sequentially immobilize antibody (Ab1), CaM and a multi-functionalized label. The label (HRP-PAupc-Ab1) was synthesized by covalently binding Ab1 and horseradish peroxidase (HRP) to poly(acrylic acid)-functionalized Au popcorn (PAupc) nanoparticles. A novel dual-layered enzyme strategy was employed by incubating HRP-secondary antibody (HRP-Ab2) onto the label surface and the enhanced biocatalyzed precipitation was therefore induced. This immunosensor exhibited satisfactory analytical performances for CaM detection with a linear response ranging from 5.0 pg mL−1 to 100 ng mL−1 and a detection limit of 1.5 pg mL−1. The immunosensor has also been successfully applied to the CaM analysis in two cancer cells (HepG2 and MCF-7) with high sensitivity, which has shown great potency for cancer study.