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

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 5.0pgmL^-1 to 100ngmL^-1 and a detection limit of 1.5pgmL^-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.