Microplate Format Protein Nanopatterning for High-Throughput Screening of Cellular Microenvironments

Abstract

An advanced protein nanopatterned cell culture platform is engineered to emulate the extracellular matrixs complexity, enabling precise nanoscale biomolecule copatterning to mimic environments analogous to native tissue for cellular assays. Nanopatterns fabricated through sparse colloidal lithography, with 100 nm to 800 nm features in separate wells, are seamlessly integrated into standard microplate formats (96-well/384-well). Robust patterns are built from fully PEGylated, passivated thin glass coverslips optimized for minimal nonspecific interactions. Biotin-avidin binding and click chemistry to ensure the accurate and robust localization of bioligands. The transparent, metal-free substrates are free of topographical interference, rendering them ideal for diverse fluorescence microscopy techniques encompassing single-molecule TIRFM and extensive high-throughput imaging. The structural stability of these nanopatterns persists beyond a year in storage and long-term in cell culture conditions, endorsing their application for prolonged experimental studies and potential for widespread academic and industrial use. The platform has been demonstrated for nanopatterning an array of biomolecules, from small molecules to proteins, DNA, and extracellular matrix components, instrumental for studying cell signaling. Experiments with C2C12 cells demonstrated the exceptional specificity of the nanopatterned microplates, with nonspecific adhesion remaining below 2% and the platforms ability to elicit size-dependent cellular reactions when interfaced with nanopatterned fibronectin.