AVS 49th International Symposium
    Biomaterials Wednesday Sessions
       Session BI-WeA

Paper BI-WeA10
Development of a Fluorescent Based Assay for Quantifying Ligand Surface Density on IPN-Modified PS for High Throughput Applications

Wednesday, November 6, 2002, 5:00 pm, Room C-201

Session: Polyelectrolyte Surfaces/Cell-Surface Interactions
Presenter: G.M. Harbers, Northwestern University
Authors: G.M. Harbers, Northwestern University
T.A. Barber, University of California, Berkeley and University of California, San Francisco
K.E. Healy, University of California, Berkeley
S.L. Golledge, University of Washington
D.G. Castner, University of Washington
Correspondent: Click to Email
Biomimetic surface engineering exploits the power of specific ligand-receptor engagement to control cell-biomaterial interactions independent of bulk material characteristics. Accurate characterization of ligand surface density (@GAMMA@) is crucial for interpreting cellular response to these engineered surfaces. Currently, low throughput techniques including ellipsometry, SPR, and radiolabeling are employed to make these measurements. Lack of high throughput alternatives provided the motivation for the development of a fluorescence microplate reader based assay to measure @GAMMA@ on a modular biomimetic surface developed to rapidly screen the adhesive potential of bioactive peptides. Poly(acrylamide-co-ethylene glycol/acrylic acid) interpenetrating polymer networks [p(AAm-co-EG-/AAc) IPNs] were grafted on to 96-well polystyrene (PS) plates. Fluorescently labeled peptides were subsequently coupled to the IPN using different input concentrations (0.01-100 µM) to modulate @GAMMA@. Surface characterization (contact angle goniometry and XPS) and cell-surface interactions were consistent with the results on previously developed IPN modified metal oxide surfaces. Reproducible control of @GAMMA@ was observed over four orders of magnitude (~ 0.1-100 pmol/cm@super 2@). Furthermore, competitive binding experiments using labeled and unlabeled peptides facilitated the determination of the equilibrium dissociation constants (K@sub d@) of the various peptides. Although this technique may not be as sensitive as the others mentioned above, it allows for the characterization and rapid development of well defined biomimetic surfaces for high throughput applications.