Single-Protein Identification by Simultaneous Size and Charge Imaging Using Evanescent Scattering Microscopy

Separation and identification of different proteins is one of the most fundamental tasks in biochemistry that is typically achieved by electrophoresis and Western blot techniques. Yet, it is challenging to perform such an analysis with a small sample size. Using a principle analogous to these conventional approaches, we present a label-free, single-molecule technique to identify different proteins based on the difference in their size, charge, and antibody binding. We tether single protein molecules to a sensor surface with a flexible polymer and drive them into oscillation by applying an alternating electric field. By tracking the nanometer-scale oscillation of each protein molecule via high-resolution scattering microscopy, the size and charge of each protein molecule can be determined simultaneously. Changes induced by varying the buffer pH and antibody binding are also investigated, which allows us to further expand the separation ability and identify two different proteins in a mixture. We anticipate our technique will contribute to single protein analysis and biosensing.