Revealing the Binding Events of Single Proteins on Exosomes Using Nanocavity Antennas beyond Zero-Mode Waveguides.

Exosomes (EXOs) play a crucial role in biological action mechanisms. Understanding the biological process of single-molecule interactions on the surface of the EXO membrane is essential for elucidating the precise function of the EXO receptor. However, due to dimensional incompatibility, monitoring the binding events between EXOs of tens to hundreds of nanometers and biomolecules of nanometers using existing nanostructure antennas is difficult. Unlike the typical zero-mode waveguides (ZMWs), this work presents a nanocavity antenna (λNAs) formed by nanocavities with diameters close to the visible light wavelength dimensions. Effective excitation volumes suitable for observing single-molecule fluorescence were generated in nanocavities of larger diameters than typical ZMWs; the optimal signal-to-noise ratio obtained was 19.5 when the diameter was 300 nm and the incident angle was ∼50°. EXOs with a size of 50-150 nm were loaded into λNAs with an optimized diameter of 300-500 nm, resulting in appreciable occupancy rates that overcame the nanocavity size limitation for large-volume biomaterial loading. Additionally, this method identified the binding events between the single transmembrane CD9 proteins on the EXO surface and their monoclonal antibody anti-CD9, demonstrating that λNAs expanded the application range beyond subwavelength ZMWs. Furthermore, the λNAs provide a platform for obtaining in-depth knowledge of the interactions of single molecules with biomaterials ranging in size from tens to hundreds of nanometers.