Article pubs.acs.org/Langmuir
AFM-based Force Spectroscopy on Polystyrene Brushes: Effect of Brush Thickness on Protein Adsorption Carsten Hentschel,† Hendrik Wagner,‡ Jens Smiatek,§ Andreas Heuer,*,§ Harald Fuchs,† Xi Zhang,∥ Armido Studer,*,‡ and Lifeng Chi*,† †
Physikalisches Institut, Westfälische Wilhelms-Universität, Wilhelm-Klemm-Straße 10, 48149 Münster, Germany and Center for Nanotechnology (CeNTech), Heisenbergstraße 11, 48149 Münster, Germany ‡ Organisch-Chemisches Institut and §Institut für Physikalische Chemie, Westfälische Wilhelms-Universität, Corrensstraße 40, 48149 Münster, Germany ∥ Key Laboratory of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084, People’s Republic of China S Supporting Information *
ABSTRACT: Herein we present a study on nonspecific binding of proteins at highly dense packed hydrophobic polystyrene brushes. In this context, an atomic force microscopy tip was functionalized with concanavalin A to perform single-molecule force spectroscopy measurements on polystyrene brushes with thicknesses of 10 and 60 nm, respectively. Polystyrene brushes with thickness of 10 nm show an almost two times stronger protein adsorption than brushes with a thickness of 60 nm: 72 pN for the thinner and 38 pN for the thicker layer, which is in qualitative agreement with protein adsorption studies conducted macroscopically by fluorescence microscopy.
1. INTRODUCTION The development of protein repellent interfaces has emerged to a heavily investigated research field, and its applications range from bionanotechnology to medical engineering and the design of advanced biomaterials. Protein repellent and antifouling surfaces are used for diverse purposes, such as the construction of protein biochips, drug discovery, diagnostics, sensors, and microarrays.1−5 Furthermore, protein antifouling properties enable a target-orientated surface patterning of specific medical devices and implants.6−8 General approaches for the design of antifouling materials are accomplished by applying proteinrepellent self-assembled monolayers (SAMs) or by covering surfaces with polymers, which bear specific functional groups. In particular polymers, which are covalently bound to surfaces, so-called polymer brushes are a robust class of coatings for tuning surface properties, such as protein repellency.9 Polymer brushes can generally be prepared by the “grafting to” or by the “grafting from” approach. The “grafting to” strategy is based on the covalent linkage of prefabricated polymers to a solid substrate. This approach is straightforward, but it is difficult to gain thick and dense coatings due to steric repulsion between grafted and approaching polymers during the adhesion event.10 In contrast, “grafting from” processes can lead to dense and thick polymer brushes with high grafting densities because polymerization is initiated by a surface-bound initiator, and the growth of the polymer leads to chain stretching rather than a coil-type conformation. By applying controlled radical polymerization techniques, such as nitroxide-mediated polymerization (NMP), it is also possible to adjust the coat thickness of the resulting polymer thin film.9,11 In particular, polymer brushes with hydrophilic, zwitterionic, or fluorinated hydrophobic side chains lead to low-fouling © XXXX American Chemical Society
interfaces and are therefore an interesting class of biomaterials.12−14 Hydrophilic polymer brushes show protein-repellent properties, and it is assumed that an interfacial water layer acts as a barrier. Therefore, adhesion of proteins at hydrophilic surfaces is not preferred due to low interfacial energy.15,16 Proteins can be generally regarded as bioamphiphiles so that adsorption at hydrophobic surfaces is favored via hydrophobic interactions.17 However, superhydrophobic polymer brushes bearing fluorinated side chains show protein-repellent behavior.18,19 Interfacial features for protein repellency have not yet been fully clarified and can be regarded as a complex interplay of multiple factors, such as surface structure, hydrophilicity, exposure time, and coat thickness.20−25 Furthermore, sizeexclusion effects may also play an important role to reduce protein adhesion.26,27 The density of the grafted coating has a strong influence on the protein adsorption behavior, which leads to the conclusion that polymer brushes have a great potential for the design of new antifouling materials. In particular, poly(ethylene glycol) (PEG)-covered surfaces have shown strong protein-repellent properties, but the stability toward oxidation is a limiting factor for their long-time applications.28 Herein we present a qualitative and quantitative examination of the protein adsorption behavior on polystyrene (PS) brushes by using fluorescence microscopy and atomic force microscopy (AFM). We chose PS brushes as model system for the investigation of the adhesion of proteins at purely hydrophobic Received: May 31, 2012 Revised: December 24, 2012
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dx.doi.org/10.1021/la302212h | Langmuir XXXX, XXX, XXX−XXX
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Article
Scheme 1. Molecular Structure of Surface-Active NMP Initiators 1 and 2a
a Surface-initiated nitroxide mediated polymerization for the preparation of polystyrene brushes with different coat thicknesses and table for different polystyrene brushes prepared by NMP.
polymer brushes. PS brushes are, in general, easily accessible and are considered to be highly robust coatings.
2. RESULTS AND DISCUSSION To investigate the coat-thickness-dependent protein adsorption behavior on PS brushes, we first synthesized silanes 1 and 2 in multistep sequences and subsequently prepared NMP-initiatorbased monolayers A on oxidized silicon wafers as previously described.29 These monolayers A were successfully used as initiators for the preparation of polymer brushes B in the presence of sacrificial initiator 3. Surface-initiated NMP allowed for the control of the brush thickness (Scheme 1) by simply varying the concentration of external initiator 3.29 The coat thickness was determined by AFM, and the grafting density was evaluated by the analysis of the concomitant unbound polymer by size exclusion chromatography. This soluble polymer was used as a reference to roughly estimate the chain length of the polymers bound to the surface.29−31 The PS brushes were then treated with fluorescence-tagged Concanavalin A (Con A), streptavidin, and bovine serum albumin (BSA). To this end, the polymer brush was exposed to a solution containing the protein by placing the polymer coated wafer into the protein solution for 24 h at room temperature to guarantee a sufficient exposure time. The surface of the wafer was then carefully rinsed with water and protein adsorption behavior was qualitatively studied by fluorescence microscopy. We found counterintuitively coat-thickness-dependent protein adsorption: with a brush thickness larger than 45 nm, the surfaces were protein-repellent, whereas the expected protein adsorption behavior was observed for brushes with thicknesses
