Bioactivity of immobilized EGF on self-assembled monolayers: Optimization of the immobilization process

✍ Scribed by Raquel Gonçalves; Maria Cristina Lopes Martins; Maria José Oliveira; Graça Almeida-Porada; Mário Adolfo Barbosa

• Book ID: 102296880
• Publisher: John Wiley and Sons
• Year: 2010
• Tongue: English
• Weight: 370 KB
• Volume: 9999A
• Category: Article
• ISSN: 1549-3296
• DOI: 10.1002/jbm.a.32723

✦ Synopsis

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°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°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. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 2010