# Chelation-Controlled Oriented and Irreversible Immobilization of Native Antibodies on Photoreactive Magnetic Nanoparticles

## TL;DR

A new method for attaching antibodies to magnetic nanoparticles improves their performance in capturing proteins from complex biological samples.

## Contribution

A novel dual-mode immobilization strategy combining Ni2+-NTA chelation and photo-crosslinking for oriented antibody immobilization.

## Key findings

- Oriented immobilization enhances antibody binding performance and prevents dissociation during washing.
- The method achieves 4.7–6-fold higher EGFR pulldown efficiency compared to random immobilization.
- The platform enables co-purification of over 1000 proteins with high interactome coverage.

## Abstract

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.