In this chemical contrast nanopattern design for selective immobilization of streptavidin, effects from potential insertion of the second polymer brush into the nanopatterned first polymer brush monolayer during the backfill step needs to be mitigated. It is found to be necessary that the biotinylated polypeptoid brush monolayer is lithographically patterned first, and the nonbiotinylated polypeptoid brush for background passivation is backfilled as the second polymer brush; otherwise, the interdigitated biotinylated polypeptoid brush will bind a sufficient amount of streptavidin, making the background non-passivating against streptavidin (Supporting Information, Figure ). This adjusted patterning strategy indicates that the covalently attached biotin functionality on polypeptoid brushes is preserved through the lithographic patterning workflow, enabling specific biotin-streptavidin binding in the final surface chemical contrast nanopatterns. We believe this bioinspired, sequence-defined polypeptoid brush platform will be of interest as surface modification materials beyond the current scope of this study aimed at semiconductor/bio interfaces. The thermal stability, 32 enzymatic resistance, and control over solubility make polypeptoids advantageous over natural biomacromolecules like polypeptides and nucleotides for more tolerant processing conditions, while sequence-definition renders polypeptoids the same level of programmability and precision in molecular design as biomacromolecules, and potentially more flexibility in incorporation of chemical functionalities. We expect this polypeptoid brush platform could find immediate applicability in biophysical research and nanobiotechnology applications that utilize nanopatterned surfaces and structures such as for cell adhesion and signaling, semi-bio hybrid sensing systems, as well as peptide/protein sequencing. EXPERIMENTAL SECTION