Enhancing Antigen Recognition of Living Cells by Site‐Specific VHH Antibody Conjugation

Biosensors and microdevices using living cells have gained significant attention in recent years, including methods for cell immobilization on antibody-coated surfaces. Random antibody fixation may lead to suboptimal exposure of recognition sites, reducing interaction efficiency. In contrast, aligned antibody immobilization enhances antigen recognition and binding capacity during cell immobilization. Site incorporation of azido-phenylalanine (AzF) into antibodies facilitates site-specific conjugation, enabling aligned antibody immobilization. In this study, we expressed and purified anti-mCherry variable domains of heavy-chain-only antibodies (VHHs), incorporating AzF at the C-terminus using an unnatural amino acid system to ensure its placement away from the antigen-binding site for mCherry. Anti-mCherry VHH-AzF was conjugated to dibenzylcyclooctyne (DBCO)-coated glass via click chemistry, followed by incubation with HeLa cells expressing mCherry on their surfaces (mCherry-coated cells). This resulted in increased immobilization of mCherry-coated cells on the VHH-coated glass, whereas normal HeLa cells did not adhere.