Development and characterization of a high-throughput system for assessing cell-surface receptor-ligand engagement

A nonfouling interfacial interpenetrating polymer network (IPN) of poly(acrylamide-co-ethylene glycol/acrylic acid) [p(AAm-co-EG/AAc)] was grafted to polystyrene for use as a novel platform for the development of high-throughput assays for screening of specific bimolecular interactions (i.e., receptor-ligand engagement). For the development of the IPN, a water-soluble hydrogen-abstracting photoinitiator was investigated: (4-benzoylbenzyl)trimethylammonium chloride. IPN-modified polystyrene surfaces were characterized using XPS, contact angle goniometry, and protein adsorption analysis. These IPN surfaces minimized fibrinogen adsorption compared to tissue culture polystyrene (>96% reduction), prevented mammalian cell adhesion, and served as nonfouling surfaces to graft biological ligands. For bimolecular interaction studies, a model peptide ligand from bone sialoprotein (Ac-CGGNGEPRGDTYRAY-NH(2)) was grafted to p(AAm-co-EG/AAc) via a 3400 M(w) linear pEG spacer. Ligand density measurements, cell culture, and a centrifugal adhesion assay were used to study cell adhesion to peptide-modified IPNs (i.e., receptor-ligand engagement). Ligand density (Gamma) was controllable from approximately 1 to 20 pmol/cm(2) by modulating the peptide input concentration (0.02-20 microM). Cell adhesion was directly dependent on the ligand density. This technology creates a powerful high-throughput system to simultaneously probe a myriad of cell-surface receptor-ligand interactions.