Elucidating the Mechanism of Interactions Between Aminoglycosides and AuNPs: Why the Classical Colorimetric Assay May Falsely Report Aptamer Affinity.

Gold nanoparticles (AuNPs) are widely used in aptasensors because of their high extinction coefficient and aggregation-dependent color differences. However, recent studies have indicated that nonspecific interactions between target molecules and AuNPs may dominate the detection signal rather than aptamer-target specific binding. This study systematically investigated the interactions between 13 aminoglycoside antibiotics and AuNPs. We found that all aminoglycoside antibiotics interacted strongly with AuNPs, considerably reducing their salt stability. Furthermore, methoxy polyethylene glycol thiol reversed AuNP aggregation induced by aminoglycoside antibiotics, indicating that it occurs at the secondary minimum. Using density functional theory, we analyzed the molecular structures and charge distribution characteristics of the aminoglycoside antibiotics, elucidating that they replace citrate ions on AuNP surfaces via a ligand exchange mechanism, thereby inducing aggregation. Additionally, both aptamer targets and complementary DNA struggled to desorb the aptamer (KAN6-1) from the AuNP surfaces. Our study demonstrates that the label-free colorimetric assay based on aggregation of unmodified citrate-AuNPs is neither suitable for characterizing the binding affinity of aminoglycoside aptamers nor viable for constructing corresponding colorimetric sensors to detect this class of antibiotics. Thus, researchers should incorporate mechanistic verification and rigorous controls when employing this system to ensure reliable results.