Calixarene-engineered gold nanoparticles for reliable thiol chemistry and simplified nucleic acid grafting

Thiol chemistry is the most widely used strategy for functionalizing gold nanoparticles (AuNPs). However, the limited stability of Au-S anchoring often prevents a robust and reproducible surface modification, a problem exacerbated by the poor tolerance of citrate-stabilized AuNPs, which are commonly used as a starting material, to harsh experimental conditions. Herein, calix[4]arene-coated AuNPs with covalently grafted Au-C bonds are investigated as a robust starting platform for thiol-mediated functionalization. Using a fluorescent PEG-thiol probe, the stability of the thiol anchoring was evaluated under thermal, competitive, and chemical stresses. The Au-S bond exhibited enhanced resistance to ligand desorption on calixarene-coated nanoparticles compared with conventional citrate-stabilized AuNPs as the starting material. Leveraging the high colloidal stability imparted by the calixarene interlayer, thiolated DNA and RNA were successfully grafted under abrupt single-step high-salt conditions (900 mM NaCl), thereby bypassing conventional salt-aging procedures. In contrast, citrate-stabilized AuNPs exhibited pronounced aggregation under equivalent functionalization conditions. On calixarene-coated AuNPs, both nucleic acids reach similarly high surface densities (∼0.44-0.46 strands per nm²) without inducing nanoparticle aggregation. Interestingly, these densities are similar to those obtained from citrate-stabilized AuNPs, suggesting that nucleic acids assemble in a packing-limited regime dictated by interstrand electrostatic repulsion and steric crowding rather than by the absolute number of available gold binding sites. Moreover, DNA-functionalized calixarene-coated AuNPs retain full hybridization capability in lateral flow and solution-phase assays, while citrate-stabilized AuNPs functionalized under the same single-step high-salt conditions show reduced functionality due to aggregation. These results demonstrate that the calixarene interlayer provides robust colloidal stability independently of nucleic acid coverage, enabling rapid, reproducible, and high-density nucleic acid functionalization under conditions that typically destabilize citrate-stabilized systems. Overall, calixarene-coated AuNPs emerge as a robust and generalizable alternative to citrate-stabilized nanoparticles as starting nanomaterials for thiol-based functionalization.