Anti-CD44 antibodies grafted immunoaffinity Fe3O4@MnO2 nanozymes with highly oxidase-like catalytic activity for specific detection of triple-negative breast cancer MDA-MB-231 cells

Abstract

Cell-enzyme-linked immunosorbent assay (CELISA) is extensively applied for cancer diagnosis and screening because of its simple operation, high sensitivity, and intuitive color change. However, the unstable horseradish peroxidase (HRP), hydrogen peroxide (H₂O₂) and non-specificity have led to a high false negative rate, which limits its application. In this study, we have developed an innovative immunoaffinity nanozyme aided CELISA based on anti-CD44 monoclonal antibodies (mAbs) bioconjugated manganese dioxide-modified magnetite nanoparticles (Fe₃O₄@MnO₂ NPs) for the specific detection of triple-negative breast cancer MDA-MB-231 cells. The ^CD44FM nanozymes were fabricated to replace unstable HRP and H₂O₂ to counteract possible negative effects in conventional CELISA. Results suggested that ^CD44FM nanozymes displayed remarkable oxidase-like activities over an extensive pH and temperature range. The bioconjugation of CD44 mAbs enabled ^CD44FM nanozymes to enter MDA-MB-231 cells selectively via over-expressed CD44 antigens on the membrane surface of these cells, and then catalyzed oxidation of the chromogenic substrate TMB, further achieving specific detection of these cells. Additionally, this study exhibited high sensitivity and low detection limit for MDA-MB-231 cells with a quantitation range of just 186 cells. To sum up, this report developed a simple, specific and sensitive assay platform based on ^CD44FM nanozymes, which could provide a promising strategy for targeted diagnosis and screening of breast cancer.

Keywords: Breast cancer detection; CELISA; Immunoaffinity (CD44)FM nanozymes; Oxidase-like activity.