Article pubs.acs.org/JPCB
Protein Adsorption at Nanopatterned Surfaces Studied by Quartz Crystal Microbalance with Dissipation and Surface Plasmon Resonance Stine H. Kristensen,† Gitte A. Pedersen,‡ Lene N. Nejsum,†,‡ and Duncan S. Sutherland*,† †
Interdisciplinary Nanoscience Center (iNANO) and ‡Department of Molecular Biology and Genetics, Aarhus University, Denmark S Supporting Information *
ABSTRACT: This paper presents the use of the quartz crystal microbalance with dissipation (QCM-D) combined with surface plasmon resonance (SPR) to probe protein adsorption at nanopatterned surfaces. Three different types of adsorbing materials, representing rigid discrete nanoparticles, dense protein films, and soft low density films have been studied on systematic varied circular nanostructures in the 100−1000 nm size range. Analysis and quantification of the QCM-D response from larger nanostructures could be understood and quantified in the same way as for homogeneous surfaces, while that for nanostructures of 100 and 200 nm diameter was significantly underestimated. Our findings suggest a size limitation of those techniques in analysis of adsorption at nanofeatures.
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INTRODUCTION The interaction and adsorption of proteins with interfaces is of importance in a range of research fields such as biosensors, food industry, and drug delivery.1−3 For example, a medical implant is surrounded by bodily fluids which rapidly generate a complex layer of different proteins in varied conformations at the interface.1 The interface material properties influence protein adsorption,4 and the combination of conformation, orientation, and quantity of adsorbed proteins define the cellular response of adhering cells.5 Both nanoscale topographies and nanoscale protein patterns have been shown to influence the behavior of adherent cells,6−9 and thus, focus on interactions between biological systems and nanostructured surfaces has greatly increased over the last decades. An important experimental parameter in studies of the cellular response to nanoscale protein patterned interfaces is the surface density of adsorbed proteins. A number of surface sensitive techniques have been used to quantify protein adsorption, for example, quartz crystal microbalance with dissipation (QCM-D),10,11 surface plasmon resonance (SPR),12 and ellipsometry (ELM).13 These techniques are well suited for studying protein adsorption at homogeneous surfaces; however, optically based approaches (e.g., SPR and ELM) may not be ideally suited for studying protein adsorption at nanostructured surfaces. The size scale of the structural features which is significant compared to the wavelength of the photons can interfere with the measurement itself through, for example, scattering, which may prevent quantitative interpretation. Thus, a common approach in cell studies has been to measure protein adsorption at a homogeneous surface of similar chemistry and use those measurements to estimate protein adsorption at the nanostructured interfaces. © 2013 American Chemical Society
Protein adsorption may differ significantly between nanostructures and homogeneous substrates14 as it has been shown by a number of studies that nanoscale curvature affects protein adsorption and kinetics. Thus, protein adsorption must be quantified at the actual nanostructured materials.15,16 QCM-D can measure real time protein and lipid adsorption onto nanostructured materials14,17,18 and has been used extensively to study soft, hydrated interfaces.19 Appropriate selection of analytical models to interpret QCM data is critical, and recent advances have allowed a more structured approach for specific experimental situations.20 Functionalized gold nanoparticles have recently been shown to enhance the sensitivity of ions at a QCM-D sensor.21 Here, we directly compare different types of adsorbing objects of relevance to interfaces in bioscience between substrates and adhering cells. Using QCM-D and SPR, we systematically analyzed adsorption on varied nanostructures of sizes from 100 to 1000 nm. The adsorbing materials represent rigid, discrete nanoparticles (16 nm Au-NP), “dense” protein films (vitronectin), and soft “low density” protein films (E-cadherin:Fc). The results revealed that measurements of protein binding within pits of larger diameter (>300 nm diameter for 14 nm depth) were unaffected by the structure, whereas the measurements of adsorption into smaller pit diameters ( 300 nm both for small dense filmlike layers and larger low density discrete protein films. This indicates that for specific systems caution should be taken for smaller pits sizes.
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ASSOCIATED CONTENT
* Supporting Information S
Tables of used proteins, their concentration and incubation, Fluorescence micrographs of VN patterns, schematic drawing of protein patterns, and QCM-D spectra including ΔΓ/-Δf,-Δf plots. This material is available free of charge via the Internet at http://pubs.acs.org. 10382
dx.doi.org/10.1021/jp4038528 | J. Phys. Chem. B 2013, 117, 10376−10383
The Journal of Physical Chemistry B
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Article
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AUTHOR INFORMATION
Corresponding Author
*Mailing address: 14 Gustav Wieds Vej, Aarhus C 8000 Denmark. Telephone: +45 23 38 57 89. E-mail: duncan@inano. au.dk. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Jacques Chevallier and Folmer Lyckegaard for performing the sputtering and evaporation and Rasmus Vendelboe for help with fabrication. This work was funded through the Danish Research Council (645-05-0016 and 27408-0464) to Duncan S. Sutherland and a Lundbeck Foundation Junior Group Leader Fellowship to Lene N. Nejsum.
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