We present a strategy for the irreversible and oriented immobilization of native antibodies (Abs) onto magnetic nanoparticles (MNPs) by integrating Ni2+-NTA chelation with diazirine (Dia)-mediated photo-crosslinking. MNPs were co-functionalized with nitrilotriacetic acid (NTA) and photoreactive Dia-2 to create a mixed monolayer NTA/Dia-2@MNPs that selectively binds the His-rich Fc domain of unmodified Abs. Short UV exposure activates Dia-2, generating reactive carbenes that covalently anchor proximal residues and permanently lock the Ab in an oriented configuration. This dual-mode immobilization preserves Fab accessibility, enhances binding performance, and prevents Ab dissociation during stringent washing. We validated the platform using two cancer therapy Abs (trastuzumab and cetuximab) and one cancer biomarker (anti-serum amyloid A, anti-SAA) in cancer cells and human serum. Anti-SAA MNPs fabricated by the NTA-Ni2+ method showed a 1.5-fold increase in antigen binding in the serum sample compared to the boronate affinity-based method and a significant (22-fold) improvement over random immobilization. Cetuximab-functionalized oriented MNPs by the current immobilization strategy achieved a 4.7-6-fold enhancement in EGFR pulldown efficiency from human embryonic kidney (HEK293T) and non-small cell lung cancer (NSCLC) models, compared to randomly immobilized controls. Notably, the oriented MNPs enabled co-purification of markedly high interactome coverage of >1000 proteins and differential abundance of downstream proteins. Importantly, this platform requires no prior Ab modification and is compatible with full-length native Abs and stable in complex biological samples (cell or serum). By combining chelation-guided orientation with photoinduced covalent fixation, this strategy addresses key challenges in Ab surface engineering and offers a robust, versatile solution for applications in immunoprecipitation, proteomics, and biomarker discovery.
Huo et al. (Wed,) studied this question.