Protein Patterns Fabricated by Affinity-Based Surface Ligand Selection from Protein Solution Mixtures on a Polymer Hydrogel Substrate

摘要

We review a recent surface patterning, modification and protein-surface affinity selection strategy that yields high-fidelity protein patterns by protein-ligand selection from solutions at surfaces - so-called affinity-based surface "protein sorting". The approach exploits pre-patterned high affinity ligands immobilized on polymer surface chemistry known to effectively inhibit non-specific protein adsorption and cell adhesion, while providing a reliable capacity for specific, dense, uniform immobilization of desired molecules to pre-designed patterns of reactive chemistry. Soluble proteins select ligands at these surfaces from solution by affinity-matched surface engagement, producing two distinct types of protein monolayer organization on surfaces: spatial (e.g., two different proteins selecting their respective ligands in spatial patterns on surfaces) and orientational (e.g., antibody binding to ligands specific to their Fab versus Fc domains). These ligand patterns and surface-protein interactions are analyzed, and spatially and orientationally verified using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Photolithographic patterning of reactive ester groups on a non-fouling PEG-coated surface facilitate ligand coupling with high fidelity or patterns of peptides, proteins, and mammalian cells. Furthermore, two different surface-patterned affinity ligands, facilitate binding of two different proteins (e.g., streptavidin and HaloTag?), co-patterned self-selectively from their mixed solution on the non-fouling surface. As a unique label-free chemically selective surface imaging technique, TOF-SIMS analysis can distinguish differences in amino acid composition between bound streptavidin and HaloTag? proteins, and also between Fab and Fc domains on surface-immobilized antibodies. Since antibody orientation and spatial patterning remains important to antibody-based surface capture assays, TOF-SIMS imaging is useful to correlate immobilized biomolecule bioactivity. Patterned RGD peptides can also be imaged, and maintain high-fidelity cell patterns in long-term serum-containing cultures.