title: Ligand density characterization of peptide-modified biomaterials.
abstract: A simple fluorescence based characterization method was developed to assess ligand density on peptide-modified biomaterials. The method exploits the exquisite sensitivity of proteolysis for the purpose of liberating a fluorescently labeled probe fragment from an immobilized peptide. The released fragment can then be detected in solution using high-throughput fluorometry. In silico screening tools identified the enzyme chymotrypsin as a promising candidate for releasing a detectable probe fragment from the fluorescently labeled peptide, Ac-CGGNGEPRGDTYRAYK(FITC)GG-NH(2). After chymotrypsin digestion of the peptide in solution was first characterized using mass spectrometry and HPLC, a basic enzyme mediated release protocol was developed and implemented to generate peptide-binding isotherms on various peptide-modified biomaterials. The new method is sensitive, has good signal-to-noise ratio (S/N), and is easily standardized. Furthermore, the technique can be applied independent of material chemistry and geometry, making it a suitable alternative to radiolabeling for a wide range of biomaterial applications.
authors.affiliation: Department of Bioengineering, University of California at Berkeley, 94720, USA.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Barber
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Harbers
authors.forename: Susan
authors.initials: S
authors.lastname: Park
authors.forename: Michele
authors.initials: M
authors.lastname: Gilbert
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Healy
mesh_terms: D001665:Binding Sites; D020099:Coated Materials, Biocompatible / Q000032:analysis* / Q000737:chemistry*; D005456:Fluorescent Dyes; D008024:Ligands; D008422:Materials Testing / Q000379:methods*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D020099:Coated Materials, Biocompatible; D005456:Fluorescent Dyes; D008024:Ligands; D010455:Peptides