Nano-visualization of oriented-immobilized IgGs on immunosensors by high-speed atomic force microscopy

Oriented immobilization of sensing molecules on solid phases is an important issue in biosensing. In case of immunosensors, it is essential to scrutinize not only the direction and shape of immunoglobulin G (IgG) in solution but also the real-time movement of IgGs, which cannot be achieved by conventional techniques. Recently, we developed bio-nanocapsules (BNCs) displaying a tandem form of the IgG Fc-binding Z domain derived from Staphylococcus aureus protein A (ZZ-BNC) to enhance the sensitivity and antigen-binding capacity of IgG via oriented-immobilization. Here, we used high-speed atomic force microscopy (HS-AFM) to reveal the fine surface structure of ZZ-BNC and observe the movement of mouse IgG3 molecules tethered onto ZZ-BNC in solution. ZZ-BNC was shown to act as a scaffold for oriented immobilization of IgG, enabling its Fv regions to undergo rotational Brownian motion. Thus, HS-AFM could decipher real-time movement of sensing molecules on biosensors at the single molecule level.