title: Bioactivity of immobilized EGF on self-assembled monolayers: optimization of the immobilization process.
abstract: Last trends in Biomaterials focus the mimic of cellular environments capable to control cellular responses. Epidermal growth factor (EGF) is a pleiotropic cytokine known to regulate cell proliferation, differentiation, and death. This study aims to optimize the immobilization of EGF on 11-mercapto-1-undecyl-tetra(ethylene)glycol (EG4)-self-assembled monolayers (SAMs) and to establish a new model surface to study EGF-mediated signaling. Gold substrates were modified with a monolayer of EG4 and N,N'-carbonyldiimidazole (CDI) was used to activate hydroxyl terminated groups of EG4-SAMs. EGF was then immobilized on activated EG4-SAMs at pH 7.4, 4 degrees C, and 100 rpm. Different immobilization reaction times were tested as well as different CDI concentrations to optimize the reaction conditions and obtain a range of immobilized EGF concentrations on the surfaces. Surface characterization of EGF-SAMs was performed using radiolabeling, water contact angle measurements, X-ray photoelectron spectroscopy, and ELISA. Phosphorylation of EGFR on BT-20 breast cancer cell line by EGF-SAMs was tested by immunostaining. EGF was successfully immobilized on EG4-SAMs, at 4 degrees C and pH 7.4 in a range of concentrations from 3.6 +/- 0.8 to 17.6 +/- 1.5 ng/cm(2). The concentration of EGF increases with immobilization time and with the CDI concentration reaching the maximum for surfaces activated with 30 mg/mL of CDI after 48 h. The bioactivity of EGF-SAMs was confirmed by immunostaining of phospho-EGFR of BT-20 cells. This study described EGF immobilization on EG4-SAMs at different concentrations, which could be important surface models to study specific protein interactions at the molecular level evolving EGF-family of proteins.
authors.affiliation: INEB, Biomaterials Division, NEWTherapies Group, R. Campo Alegre, 823, Porto 4150-180, Portugal. raquelg@ineb.up.pt
authors.forename: Raquel
authors.initials: R
authors.lastname: Gonçalves
authors.forename: Maria Cristina Lopes
authors.initials: MC
authors.lastname: Martins
authors.forename: Maria José
authors.initials: MJ
authors.lastname: Oliveira
authors.forename: Graça
authors.initials: G
authors.lastname: Almeida-Porada
authors.forename: Mário Adolfo
authors.initials: MA
authors.lastname: Barbosa
mesh_terms: D000327:Adsorption; D001672:Biocompatible Materials / Q000737:chemistry* / Q000378:metabolism; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D006046:Gold / Q000737:chemistry; D006801:Humans; D007093:Imidazoles / Q000737:chemistry; D010766:Phosphorylation; D056951:Photoelectron Spectroscopy; D011092:Polyethylene Glycols / Q000737:chemistry; D011869:Radioligand Assay; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013499:Surface Properties
chemical_list: D001672:Biocompatible Materials; D007093:Imidazoles; D013438:Sulfhydryl Compounds; D011092:Polyethylene Glycols; D004815:Epidermal Growth Factor; C006900:N,N-carbonyldiimidazole; D006046:Gold; D066246:ErbB Receptors