Molecular templates for bio-specific recognition by low-energy electron beam lithography

Protein patterning has become an important topic as advances are made in biologically integrated devices and protein chip technology. Versatile and effective patterning requires substrates that can be quantified, with active presentation of proteins and control over protein density and orientation. Herein we describe a model system and the use of low-energy electron beam lithography to pattern molecular templates for immobilization of antibodies through ligand recognition. The templates were patterned over a background of poly(ethylene glycol) (PEG) modified silicon oxide (SiO_x). These substrates were exposed to a low-voltage (2 keV) electron beam to remove PEG selectively from exposed regions. These regions were then functionalized with a dinitrophenyl (DNP) ligand and tested for specific binding of fluorescently labeled anti-DNP antibodies. The PEG modified regions in conjunction with ligand-presenting regions in the patterned arrays substantially reduces non-specific adsorption of proteins, yielding a specific/nonspecific ratio of approx 10. The surface coverage of the biologically active DNP groups on SiO_x and the amount of immobilized antibody on DNP were measured with a fluorescence-based, enzyme-linked immunosorbent assay. The specificity of the interaction between DNP ligand and fluorescently labeled anti-DNP antibodies was evaluated with fluorescence microscopy. This approach to patterning of molecular templates and assays for quantification are generally applicable to immobilization of any ligand-receptor pair on a wide range of substrates.