Alkyne Nitro Tag Enables Stable and Efficient Protein Functionalization of Gold Nanoparticles

Surface ligands play a critical role in the preparation of stable, covalently bound nanoparticle-protein conjugates. However, large surface ligands often introduce steric and antifouling effects that reduce protein conjugation yields, whereas small ligands tend to induce nanoparticle aggregation during activation. Here, we report Alkyne Nitro Tag (ANT), a compact heterobifunctional ligand that represents a design principle for nanoparticle surface chemistry. ANT presents a preinstalled reactive nitrophenyl ester that undergoes nucleophilic acyl substitution with protein residues under mild aqueous conditions, while its nitro substituent imparts colloidal stability to AuNPs through electrostatic repulsion. In ANT, the terminal alkyne is essential for strong attachment to the AuNP surface, as thiol groups are incompatible with the reactive ester. Robust conjugation of ANT-capped AuNPs (Au@ANT) with streptavidin (SA), horseradish peroxidase (HRP), and immunoglobulin G (IgG) was demonstrated by lateral flow assays, Western blotting, enzymatic sensing, and immunoprecipitation. Compared to the conventional physical adsorption and EDC/NHS chemistry, Au@ANT conjugates consistently exhibited higher yields, improved stability under physiological and denaturing conditions, and greater retention of protein activity. These results establish ANT as a generalizable and high-performance strategy for generating stable and active AuNP-protein conjugates, offering significant advantages for nanomedicine and advanced materials science.