Protein corona-assisted freezing enables ultrafast, programmable DNA loading on AuNPs for tunable probe performance

Abstract

Conjugating DNA onto gold nanoparticles (AuNPs) underpins numerous biomedical applications, yet mainstream routes are either slow (salt-aging, >10 h) or demand large DNA excess (freeze-thaw) to prevent aggregation, making loading control difficult. Here, we report a protein corona-assisted freezing process that completes DNA coupling in 20 min and decouples colloidal stability from DNA excess by using adsorbed protein corona as steric/electrostatic stabilizers. Ice crystallization concentrates AuNPs, proteins, DNA and salts into nanoscale pockets, accelerating reaction kinetics, while the protein corona enables precise, input-dependent tuning of DNA density on each particle. The method is compatible with multiple proteins (e.g., HRP, GOx, BSA), yielding multifunctional AuNP-DNA-protein probes. Controlled DNA densities directly modulate performance in a lateral-flow miRNA assay—shifting sensitivity and dynamic range—and in multivalent AuNP-aptamer constructs, where intermediate valency maximizes tumor-cell labeling and capture efficiency. This one-step, scalable platform streamlines probe fabrication and provides an engineering handle over ligand density, paving the way for rapid development of next-generation biosensors and targeted diagnostic agents.