Viscoelastic Modeling of Highly Hydrated Laminin Layers at Homogeneous and Nanostructured Surfaces: Quantification of Protein Layer Properties Using QCM-D and SPR Jenny Malmstro ¨m, Hossein Agheli, Peter Kingshott, and Duncan S. Sutherland* Interdisciplinary Nanoscience Center, iNANO, UniVersity of Aarhus, Aarhus 8000, Denmark ReceiVed April 27, 2007. In Final Form: June 27, 2007 The adsorption of proteins at material surfaces is important in applications such as biomaterials, drug delivery, and diagnostics. The interaction of cells with artificial surfaces is mediated through adsorbed proteins, where the type of protein, amount, orientation, and conformation are of consequence for the cell response. Laminin, an important cell adhesive protein that is central in developmental biology, is studied by a combination of quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR) to characterize the adsorption of laminin on surfaces of different surface chemistries. The combination of these two techniques allows for the determination of the thickness and effective density of the protein layer as well as the adsorbed mass and viscoelastic properties. We also evaluate the capacity of QCM-D to be used as a quantitative technique on a nanostructured surface, where protein is adsorbed specifically in a nanopattern exploiting PLL-g-PEG as a protein-resistant background. We show that laminin forms a highly hydrated protein layer with different characteristics depending on the underlying substrate. Using a combination of QCM-D and atomic force microscopy (AFM) data from nanostructured surfaces, we model laminin and antibody binding to nanometer-scale patches. A higher amount of laminin was found to adsorb in a thicker layer of a lower effective density in nanopatches compared to equivalent homogeneous surfaces. These results suggest that modeling of QCM-D data of soft viscoelastic layers arranged in nanopatterns may be applied where an independent measure of the “dry” mass is known. Introduction The adsorption of proteins at material surfaces has been the focus of considerable research effort for several decades, and it continues to receive increasing attention 1-3 because of its importance in applications such as biomaterials, drug delivery, and diagnostics. 4 The interaction of cells with biomaterial surfaces is mediated through adsorbed proteins, where the type of protein, amount, orientation, and conformation are important to the cellular response. 5 The adsorption of cell adhesive extracellular matrix (ECM) proteins such as fibronectin and their influence on cell behavior have been widely studied. 5,6 Laminin, another major cell adhesive protein of importance in developmental biology, is often used as a coating on surfaces for the culture of neuronal cells. 7,8 Laminin is a large, flexible glycoprotein with active domains for collagen binding, cell adhesion, and heparin binding. 9 A recent active research area has been the interaction of biological systems with nanostructured materials. Both nanoscale topographic features 10,11 and nanopatterns with, for example, the nanoscale distribution of proteins 12,13 or peptides 14 have been shown to be capable of influencing cell behavior. A critical parameter in such studies is the surface density of adsorbed protein. A number of surface-sensitive techniques have been used for the quantification of protein adsorption. 4 Optical techniques such as surface plasmon resonance (SPR), 15 optical waveguide lightmode spectroscopy (OWLS), 16 ellipsometry, 16 and total internal reflection fluorescence spectroscopy (TIRF) 17 are well suited for the study of homogeneous substrates but are less likely to be useful for nanostructured substrates. In this study, we utilize a combination of the quartz crystal microbalance with dissipation (QCM-D) 18 and SPR to characterize the adsorption of laminin on substrates of different surface chemistries. The combination of these two techniques allows for the determination of not only the adsorbed mass but also the effective density of the protein layer and the viscoelastic properties of that layer. 19 We also evaluate the capacity of QCM-D to be used as a quantitative technique on a nanostructured surface, where protein is adsorbed specifically in a nanopattern exploiting PLL-g-PEG as a protein-resistant background. We show that laminin forms a highly hydrated protein layer with different characteristics depending on the underlying substrate. Using a * Corresponding author. E-mail: duncan@inano.dk. Tel: +4589425547. Fax +4589423690. (1) Andrade, J. D.; Hlady, V. AdV. Polym. Sci. 1986, 79,1-63. (2) Mrksich, M.; Whitesides, G. M. Annu. ReV. Biophys. Biomol. Struct. 1996, 25, 55-78. (3) Rechendorff, K.; Hovgaard, M. B.; Foss, M.; Zhdanov, V. P.; Besenbacher, F. Langmuir 2006, 22, 10885-10888. (4) Kasemo, B. Surf. Sci. 2002, 500, 656-677. (5) Keselowsky, B. G.; Collard, D. M.; Garcia, A. 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