Directional immobilization of antibody onto magnetic nanoparticles by Fc-binding protein-assisted photo-conjugation for high sensitivity detection of antigen

• Published in: Analytica chimica acta 2021-11, Vol.1184, p.339054-339054, Article 339054
• Main Authors: Tang, Jin-Bao, Yang, Hong-Ming, Gao, Xiao-Yi, Zeng, Xian-Zhong, Wang, Feng-Shan
• Format: Article
• Language: English
• Subjects: Antibody; Directional immobilization; Immunosorbent assay; Magnetic nanoparticles; Photo-conjugation
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2021.339054

Immobilized antibodies with site-specific, oriented, and covalent pattern are of great significance to improve the sensitivity of solid-phase immunoassay. Here, we developed a novel antibody conjugation strategy that can immobilize antibodies in a directional and covalent manner. In this study, an IgG-Fc binding protein (Z domain) carrying a site-specific photo-crosslinker, p-benzoyl-L-phenylalanine, and a single C-terminal cysteine (Cys) handle was genetically engineered. Upon UV irradiation, the chimeric protein enables the Cys handle to couple with the native antibody in Fc-specific and covalent conjugation pattern, resulting in a novel thiolated antibody. Thus, an approach for the covalent, directional immobilization of antibodies to maleimide-modified magnetic nanoparticles (MNPs) was developed on the basis of the crosslinking between sulfhydryl and maleimide groups. The antibody-conjugated MNPs were applied in MNP-based enzyme-linked immunosorbent assay (ELISA) for the detection of carcinoembryonic antigen. The MNP-based ELISA presented a quantification linear range of 0.1–100 ng mL−1 and detection limit of 0.02 ng mL−1, which was approximately 100 times more sensitive than the traditional microplate ELISA (2.0 ng mL−1). Thus, the proposed antibody immobilization approach can be used in surface functionalization for the sensitive detection of various biomarkers. [Display omitted] •A photoactivable ZBpa-Cys recombinant was genetically engineered by aaRS/tRNA technique.•A thiol modified antibody with Fc-specific and covalent conjugation was fabricated by employing ZBpa-Cys.•An approach for directional immobilization of antibody onto MNPs was proposed.•A MNPs-based ELISA for enhanced antigen detection was developed.•The sensitivity was about 100 times more than the traditional microplate ELISA.