Article pubs.acs.org/Langmuir
Subsurface Influence on the Structure of Protein Adsorbates as Revealed by in Situ X-ray Reflectivity Hendrik Haḧ l,† Florian Evers,‡,§ Samuel Grandthyll,† Michael Paulus,‡ Christian Sternemann,‡ Peter Loskill,† Matthias Lessel,† Anne K. Hüsecken,‡ Thorsten Brenner,‡ Metin Tolan,‡ and Karin Jacobs*,† †
Department of Experimental Physics, Saarland University, 66041 Saarbrücken, Germany Fakultät Physik/DELTA, TU Dortmund, 44221 Dortmund, Germany
‡
S Supporting Information *
ABSTRACT: The adsorption process of proteins to surfaces is governed by the mutual interactions among proteins, the solution, and the substrate. Interactions arising from the substrate are usually attributed to the uppermost atomic layer. This actual surface defines the surface chemistry and hence steric and electrostatic interactions. For a comprehensive understanding, however, the interactions arising from the bulk material also have to be considered. Our protein adsorption experiments with globular proteins (α-amylase, bovine serum albumin, and lysozyme) clearly reveal the influence of the subsurface material via van der Waals forces. Here, a set of functionalized silicon wafers enables a distinction between the effects of surface chemistry and the subsurface composition of the substrate. Whereas the surface chemistry controls whether the individual proteins are denatured, the strength of the van der Waals forces affects the final layer density and hence the adsorbed amount of proteins. The results imply that van der Waals forces mainly influence surface processes, which govern the structure formation of the protein adsorbates, such as surface diffusion and spreading.
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INTRODUCTION The unspecific adsorption of proteins on solid/liquid interfaces is a well-known and often studied phenomenon.1−4 Its importance in and influence on clinical, biological, and technical applications is enormous. For instance, biofilm formation on surfaces adherent to biological solutions (saliva, blood, etc.) begins with and depends on the primary adsorption by the proteins. Therefore, to control the biofilm development, it is desirable to gain control of the protein adsorption process. Recent studies have shown that the substrate strongly influences the enzymatic activity of adsorbed proteins,5 their orientation on the surface,6,7 and the kinetics of the adsorption process.5,8,9 The latter studies have shown that slight changes in the substrate’s subsurface composition (i.e., the material composition below the uppermost surface layer) influence the kinetics of adsorption. It is the aim of our study to yield structural information about the adsorption process and the influence of the subsurface composition of the substrate and connect the results to kinematical studies.8−10 Parameters that influence protein adsorption are numerous: the pH value, ionic strength, temperature, protein concentration, and the concentration of cosolvents and additives are the most prominent properties of the solution.2−4,11−13 The influence of the substrate on adsorption is in most studies described by and restricted to the sign and value of the surface © 2012 American Chemical Society
charge, the roughness of the surface, and the surface free energy (i.e., the chemical composition of the surface).4,14−16 The above-named substrate properties belong to the actual surface of the offered substrate. Nevertheless, the influence of the bulk substrate should not be neglected because van der Waals forces range over tens of nanometers, depending mainly on the geometry of the interacting objects.17,18 It could already be shown that van der Waals forces govern the stability of thin films.19,20 Furthermore, they influence the adhesion strength even in wet surroundings.21,22 Thus, their influence on protein adsorbates is expected. In recent studies, it was shown that the adsorption kinetics of large, deformable proteins such as α-amylase and bovine serum albumin (BSA) are affected by different van der Waals forces.8,9 By using silicon wafers with different silicon oxide thicknesses, the van der Waals forces can be varied while keeping the surface free energy constant. On thick silicon oxide wafers, regardless of whether they were hydrophilic or hydrophobic, the proteins showed Langmuir-like kinetics, whereas on thin silicon oxide “stepped” kinetics was observed with a distinct change in the adsorption rate at low coverage. Such behavior was not recorded for the lysozyme,8 which is generally regarded as a stiff Received: February 28, 2012 Revised: April 25, 2012 Published: April 25, 2012 7747
dx.doi.org/10.1021/la300850g | Langmuir 2012, 28, 7747−7756
Langmuir
Article
mainly of stabilizing salts such as sulfates. This results in final protein solutions with a consequently decreased protein content and marginally increased ionic strength of the solution.) BSA and lysozyme were stored at 2 °C and α-amylase was stored at −20 °C until used.
protein (Gibbs energy for unfolding: 60 kJ/mol).2 On the basis of this fact, it is expected that the final state of the protein films also diversifies (e.g., in protein orientation or conformation) with different subsurface compositions. There are only a few techniques, however, that allow us to resolve in situ the structure of films with thicknesses of only a few nanometers. In this study, we applied X-ray reflectometry at high X-ray energies to analyze the film structure with angström resolution. In former studies,23−25 this technique has proven to be able to resolve the film structure of similar protein films.
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Table 2. Characteristics of the Proteins Studied α-amylase31 molar mass/kg/ mol molar volume/ cm3/mol electron density/ e/Å3 isoelectric point (IEP) diameters/Å3
EXPERIMENTAL SECTION
Materials and Setup. As substrates for protein adsorption, two types of silicon wafers were used: wafers with a natural silicon oxide layer of 1.7(3) nm (Wacker Siltronic AG, Burghausen, Germany) and wafers with a thermally grown amorphous silicon oxide layer with a thickness of 150(3) nm (Silchem, Freiberg, Germany), as characterized by ellipsometry. Prior to use, the wafers were cleaned for 30 min in a fresh 1:1 H 2 SO4 (conc)/H 2 O 2 (30%) solution and subsequently rinsed in hot deionized water to remove hydrocarbon residues from the polishing process. After this procedure, the wafers were hydrophilic with a water contact angle of
