WGM Single-Molecule Validation of pH-Dependent Molecular Energetics at Gold Nanointerfaces: Influence of Buffer...

Single-molecule sensors are increasingly applied to small multifunctional molecules whose nanoparticle interactions are governed by multiple functional groups and ionic states. At this scale, ligand and buffer molecules become chemically comparable, raising a fundamental question: how does a small multifunctional ligand compete with buffer molecules for nanoparticle binding sites when both carry similar reactive groups? We address this question using whispering gallery mode (WGM) optoplasmonic sensors, which resolve individual glyphosate (GLP) binding events at gold nanorods in real time and without labels. We show that glyphosate interactions shift from permanent cooperative binding at alkaline pH, where amine and oxyanion groups outcompete carbonate for gold surface sites, to transient physisorption mediated by surface-adsorbed buffer molecules at lower pH. For the first time, a label-free single-molecule technique consolidates these observed kinetics with complexation energies, binding geometries, and coordination motifs predicted by density functional theory (DFT) and confirmed by surface- enhanced Raman spectroscopy (SERS), establishing a general framework for probing competitive ligand binding at nanointerfaces.