Use of Modified Graphite for Single-Molecule Atomic Force Microscopy of Biomacromolecules

Abstract

Atomic force microscope (AFM) provides 3D topography images of single biomacromolecules, such as nucleic acids, proteins and polysaccharides. The resolution and quality of images depend strongly on the properties of a substrate namely its smoothness, charge, and hydrophobicity. Mica and graphite are the two substrates commonly used for AFM; however, both of them have disadvantages. Mica often produces imaging artifacts due to a high surface charge and adsorption of the water vapor from air. Graphite is highly hydrophobic and can induce conformational changes in adsorbed protein molecules. Here we describe the use of a graphite modifier (GM) that makes graphite a universal substrate for visualization of proteins, DNAs and polysaccharides. The modifier has a hydrocarbon part that drives adsorption to graphite and an oligo-glycine part that attenuates hydrophobicity. The procedure of graphite modification is simple and fast (within 10 minutes) and reduces the hydrophobicity of graphite as confirmed by a decrease of the water contact angle from 60.2±0.2° on bare freshly cleaved graphite to 45.5±0.5° on GM-coated graphite. The surface of graphite covered with a thin layer of the modifier has unchanged roughness, such that the root mean square roughness (Rq) is equal to 0.09±0.02 nm before and after GM application. GM-coated graphite allows for reproducible, high-resolution imaging of single molecules that was shown by AFM of proteins (fibrinogen and factor XIII), DNA, and glycosaminoglycans (heparin). The use of GM-graphite provides imaging with an unprecedented resolution that allows for visualization of flexible and/or unstructured regions of proteins and other molecules that are not seen with other AFM substrates.