Sub-10 nm Resolution Patterning of Pockets for Enzyme Immobilization with Independent Density and Quasi-3D Topography Control

The ability to precisely control the localization of enzymes on a surface is critical for several applications including biosensing, bionanoreactors, and single molecule studies. Despite recent advances, fabrication of enzyme patterns with resolution at the single enzyme level is limited by the lack of lithography methods that combine high resolution, compatibility with soft, polymeric structures, ease of fabrication, and high throughput. Here, a method to generate enzyme nanopatterns (using thermolysin as a model system) on a polymer surface is demonstrated using thermochemical scanning probe lithography (tc-SPL). Electrostatic immobilization of negatively charged sulfonated enzymes occurs selectively at positively charged amine nanopatterns produced by thermal deprotection of amines along the side-chain of a methacrylate-based copolymer film via tc-SPL. This process occurs simultaneously with local thermal quasi-3D topographical patterning of the same polymer, offering lateral sub-10 nm resolution, and vertical 1 nm resolution, as well as high throughput (5.2 × 10⁴ μm²/h). The obtained single-enzyme resolution patterns are characterized by atomic force microscopy (AFM) and fluorescence microscopy. The enzyme density, the surface passivation, and the quasi-3D arbitrary geometry of these patterned pockets are directly controlled during the tc-SPL process in a single step without the need of markers or masks. Other unique features of this patterning approach include the combined single-enzyme resolution over mm² areas and the possibility of fabricating enzymes nanogradients.