Development of single molecule binding kinetics immunosensor based on surface plasmon resonance microscopy

Surface plasmon resonance (SPR) technology plays a crucial role in kinetic analysis of molecular interactions due to the advantages of real-time, label-free, and high sensitivity. However, most SPR sensors detects the protein interactions with average value of multiple molecules. In this study, we developed a single molecule binding kinetics immunosensor based on surface plasmon resonance microscopy (SPRM). A BSA based biosensor was prepared for immobilizing single molecules and the interaction between human immunoglobulin G (IgG) and its antibodies was investigated showing good concentration response and selectivity. And the molecular height influence on imaging has been observed. Finally, the single, micro, and macroscopic molecule binding kinetics parameters (ka, kd and KD) were calculated and compared revealing higher affinity in single molecule region than the expanded region. It demonstrates the importance of single molecule kinetics, which can be applied to the study of protein heterogeneity analysis.