Surface charge density measurement of a single protein molecule with a controlled orientation by AFM.

The spatial distribution of functional groups causes a charge distribution that often has a close relationship with its biofunctions. To understand them of the protein molecules, measurements of the charge distribution under physiological conditions are desired. Atomic force microscopy (AFM) has been utilized to measure the surface charge density by measuring the electric double layer (EDL) force caused by the overlap of the EDLs on the surfaces of the AFM tip and the biomolecule. Here, we demonstrated the surface charge density measurement of a single streptavidin (SA) protein molecule by the three-dimensional force mapping method based on frequency modulation AFM (FM-AFM). The SA has a strong affinity to biotin because of the electrostatic interactions between the molecules. Therefore, the surface charge density measurements of the biotin-binding sites and other surface areas of the molecule have been anticipated. However, the surface charge density of the surfaces other than the biotin-binding side has never been measured. We demonstrate the surface charge density measurement of the top surface of the single SA molecule, which is perpendicular to the biotin-binding sides, with a controlled orientation using DNA origami as a template by FM-AFM in an electrolyte solution. The surface charge density of the top surface of the SA molecule was estimated by fitting the experimental force curves to the Derjaguin-Landau-Verwey-Overbeck theory. We found that the surface charge density of the top surface of the SA molecule is comparable to those reported earlier for the biotin-binding sides of the molecule. We expect that, by using the DNA origami technology, one can control the orientation of a biomolecule attached to the substrate and measure the surface charge density of the specific surface areas of the biomolecule to obtain information that will help us to understand the relationship between their structures and functions.