A research team led by Prof. GUO Zhen and Prof. ZHOU Lianqun from the Suzhou Institute of Biomedical Engineering and Technology (SIBET) of the Chinese Academy of Sciences has developed a nanocavity antenna that goes beyond zero-mode waveguides and revealed the interaction process of single protein molecules on the surface of exosomes.

Results of their study were published in ACS Applied Materials & Interfaces as a cover article.

The Cover from ACS Applied Materials & Interfaces, 2023, Volume 15, Number 42. (Image by ACS Applied Materials & Interfaces) 

The study of single-molecule interactions on the surface of exosomes has profound biological significance. However, the tiny size of exosomes, typically tens to hundreds of nanometers, poses a formidable challenge in detecting binding events between exosomes and nanometer-scale proteins.  

In view of this, the researchers developed a nanocavity antenna that goes beyond zero-mode waveguides, enabling more efficient excitation of single-molecule fluorescence and achieving high signal-to-noise ratios (19.5) along with commendable occupancy rates (12%-23%).  

"This innovative approach overcomes the limitations of loading large volumes of biological materials into nanometer-scale cavities," said Prof. GUO Zhen. 

The researchers then further explored the dynamic imaging of extracellular vesicle-surface protein binding events using the nanocavity antenna beyond the zero-mode waveguide. 

Using this method, they measured the binding events between a single transmembrane CD9 protein on the surface of extracellular vesicles and its monoclonal antibody, indicating that the detection range of single-molecule events has broken through the physical size limit of the zero-mode waveguide hole.  

"This method can be used to analyze the interactions between single molecules and biological materials from tens of nanometers to hundreds of nanometers, such as vesicles and apoptotic bodies," said Prof. GUO.  

The study provides a method for understanding the interactions of molecules on the surface of extracellular vesicles, which can be applied to research on the mechanism of extracellular vesicles, drug screening, tumor diagnosis and treatment. 

This work was funded by the National Key R&D Projects of China, and the National Natural Science Foundation of China, etc.   

Schematic illustration of the working principle of a nanocavity antenna beyond the zero-mode waveguide. a. Schematic diagram of the optical detection system of the nanocavity antenna; b. schematic diagram of the principle of traditional optical waveguides; c. schematic diagram of the principle of nanocavity antenna. (Image by SIBET)

Real-time recording of fluorescence signal intensity during the binding process of single protein molecules on the surface of exosomes. a, d. Fluorescence signal real-time recording results of the control group; b. fluorescence signal real-time recording results of the experimental group; c. statistical analysis of the fluorescence bleaching steps of the experimental group; e. real-time recording results of fluorescence signals obtained by microscopic imaging methods; f. statistical analysis of the fluorescence bleaching steps of the microscopic imaging method; g. image of the fluorescence changing process within a single nanocavity over time. (Image by SIBET)