AFM Imaging of Immobilized Fibronectin: Does the Surface Conjugation Scheme Affect the Protein Orientation/Conformation?

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• 作者：Karine Valliè ; res ; Pascale Chevallier ; Christian Sarra-Bournet ; St&eacute ; phane Turgeon ; Ga&eacute ; tan Laroche
• 刊名：Langmuir
• 出版年：2007
• 出版时间：September 11, 2007
• 年：2007
• 卷：23
• 期：19
• 页码：9745 - 9751
• 全文大小：393K
• 年卷期：v.23,no.19(September 11, 2007)
• ISSN：1520-5827

文摘

Covalent grafting of biomolecules could potentially improve the biocompatibility of materials. However, thesemolecules have to be grafted in an active conformation to play their biological roles. The present work aims at verifyingif the surface conjugation scheme of fibronectin (FN) affects the protein orientation/conformation and activity. FNwas grafted onto plasma-treated fused silica using two different crosslinkers, glutaric anhydride (GA) or sulfosuccinimidyl4-(p-maleimidophenyl)butyrate (SMPB). Fused silica was chosen as a model surface material because it presents aroughness well below the dimensions of FN, therefore allowing AFM analyses with appropriate depth resolution. Celladhesion assays were performed to evaluate the bioactivity of grafted FN. Cell adhesion was found to be higher onGA-FN than on SMPB-FN. Since FN-radiolabeling assays allowed us to rule out a surface concentration effect(approximately 80 ng/cm² of FN on both crosslinkers), it was hypothesized that FN adopted a more active conformationwhen grafted via GA. In this context, the FN conformation on both crosslinkers was investigated through AFM andcontact angle analyses. Before FN grafting, GA- and SMPB-modified surfaces had a similar water contact angle,topography, and roughness. However, water contact angles of GA-FN and SMPB-FN surfaces clearly show differencesin surface hydrophilicity, therefore indicating a dependence of protein organization toward the conjugation strategy.Furthermore, AFM results demonstrated that surface topography and roughness of both FN-conjugated surfaces weresignificantly different. Distribution analysis of FN height and diameter confirmed this observation as the proteindimensions were significantly larger on GA than SMPB. This study confirmed that the covalent immobilizationscheme of biomolecules influences their conformation and, hence, their activity. Consequently, selecting the appropriateconjugation strategy is of paramount importance in retaining molecule bioactivity.