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

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.