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In-depth understanding of the intricate interactions between biomolecules and nanoparticles is hampered by a lack of analytical methods providing quantitative information about binding kinetics. Herein, we demonstrate how label-free evanescent light-scattering microscopy can be used to temporally resolve specific protein binding to individual surface-bound (∼100 nm) lipid vesicles. A theoretical model is proposed that translates protein-induced changes in light-scattering intensity into bound mass. Since the analysis is centered on individual lipid vesicles, the signal from nonspecific protein binding to the surrounding surface is completely avoided, offering a key advantage over conventional surface-based techniques. Further, by averaging the intensities from less than 2000 lipid vesicles, the sensitivity is shown to increase by orders of magnitude. Taken together, these features provide a new avenue in studies of protein-nanoparticle interaction, in general, and specifically in the context of nanoparticles in medical diagnostics and drug delivery.

Time-Resolved and Label-Free Evanescent Light-Scattering Microscopy for Mass Quantification of Protein Binding to Single Lipid Vesicles