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

## TL;DR

A new ligand called Alkyne Nitro Tag improves the stable and efficient attachment of proteins to gold nanoparticles.

## Contribution

ANT is a compact, heterobifunctional ligand that enables stable and efficient protein functionalization of gold nanoparticles.

## Key findings

- Au@ANT conjugates showed higher yields and improved stability compared to conventional methods.
- Protein activity was better retained in Au@ANT conjugates under various conditions.
- ANT demonstrated effectiveness with multiple proteins like streptavidin and horseradish peroxidase.

## Abstract

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.