Unraveling the Impact of Subsurface and Surface Properties of a Material on Biological Adhesion—A Multi-Scale Approach

Unraveling the impact of subsurface and surface properties of a material on biological adhesion—a multi-scale approach

Dissertation zur Erlangung des Grades des Doktors der Naturwissenschaften der Naturwissenschaftlich-Technischen Fakultät II - Physik und Mechatronik - der Universität des Saarlandes

von

Peter Moritz Loskill

Saarbrücken 2012 Tag des Kolloquiums: 29.10.2012

Dekan: Univ.-Prof. Dr. rer. nat. Ch. Wagner

Mitglieder des Prüfungsausschusses:

Vorsitzender: Univ.-Prof. Dr. rer. nat. L. Santen

Gutachter: Univ.-Prof. Dr. rer. nat. K. Jacobs Univ.-Prof. Dr. med. M. Herrmann Univ.-Prof. Dr. rer. nat. Ch. Ziegler, TU Kaiserslautern

Akademischer Beisitzer: Dr. J.-B. Fleury Kurzzusammenfassung

Das Verständnis der Adhäsion biologischer Objekte an anorganischen Materialien ist ein wichtiges Forschungsziel in der Physik und den Lebenswissenschaften. Um biologische Adhäsion zu beschreiben, berücksichtigen viele Studien lediglich die Eigenschaften der Oberfläche; die Materialzusammensetzung unterhalb der Oberfläche wird häufig überse- hen. Langreichweitige Van der Waals (VdW)-Kräfte werden somit vernachlässigt. Die vorliegende Arbeit zeigt, dass Unterschiede im Grenzflächenpotential einen Einfluss auf biologische Objekte (Proteine, Bakterien, Geckos) haben. Mithilfe von Siliziumwafern mit unterschiedlich dicken Oxidschichten wird der VdW-Anteil des Grenzflächenpotentials un- abhängig von den Oberflächeneigenschaften variiert. Durch Funktionalisierung der Wafer mit einer Silan-Monolage wird auch die Oberflächenchemie gesondert verändert. Auf diesen Modelloberflächen wurden Adhäsions- und Adsorptionsexperimente durchgeführt. Dabei wurde die Proteinadsorption mittels in situ Röntgenreflektometrie, die Bakterien- adhäsion mittels AFM-Kraftspektroskopie mit Bakteriensonden und die Geckoadhäsion mittels einer mechanischen Testplattform charakterisiert. Zudem wurde in der vorliegen- den Arbeit ermittelt, inwiefern Veränderungen der Oberfläche, wie die Fluorierung von künstlichen Zähnen oder Umordnungen in der bakteriellen Zellwand, die Bakterienadhä- sion beeinflussen und inwiefern eine verringerte Quervernetzung der bakteriellen Zellwand deren Elastizität verändert.

III IV Abstract

Understanding the adhesion of biological objects to inorganic surfaces is an important research objective in physics and the life sciences. To characterize biological adhesion, most studies describe a substrate solely by its surface properties; the composition of the material beneath the surface is frequently overlooked. That way, long-range van der Waals (vdW) interactions are disregarded. This work reveals that biological objects of all scales—nanoscopic proteins, microscopic bacteria, and macroscopic geckos—are influenced by nanoscale differences in the interface potential. By using tailored silicon wafers with a variable silicon oxide layer thickness, the vdW part of the interface potential is tuned independently from the surface properties. By modifying the wafers with silane monolay- ers, the surface chemistry can be varied separately as well. On these model substrates, adsorption and adhesion experiments were performed. Protein adsorption was investigated by in situ X-ray reflectometry, bacterial adhesion was explored via AFM force spectroscopy with bacterial probes, and gecko adhesion was characterized using a mechanical testing platform. Moreover, this work investigates whether or not bacterial adhesion is influenced by changes in surface properties such as the fluoridation of artificial teeth or contact- induced rearrangements in the bacterial cell wall and whether or not a reduction of the peptidoglycan crosslinking affects the elasticity of the bacterial cell wall.

V VI Contents

1 Introduction 1

2 Overview and Connectivity 3

3 Context and State of the Art 5 3.1 Surface Forces ...... 5 3.1.1 Van der Waals Interactions ...... 6 3.1.2 Electrostatic Interactions ...... 7 3.1.3 Hydrogen Bonds ...... 10 3.1.4 Hydration and Hydrophobic Forces ...... 10 3.1.5 Steric Repulsion ...... 11 3.1.6 A Thermodynamical Approach: Work of Adhesion, Interfacial Energy, and Surface Energy ...... 12 3.1.7 Covalent and Chemical bonds ...... 13 3.1.8 Capillary Forces ...... 13 3.2 Proteins ...... 14 3.2.1 Protein Adsorption ...... 15 3.3 Bacteria ...... 16 3.3.1 Cell Wall of Gram-positive Bacteria ...... 18 3.3.2 Bacterial Adhesion ...... 18 3.3.3 Staphylococci ...... 22 3.3.4 Streptococci ...... 25 3.4 Geckos ...... 26 3.4.1 Gecko Adhesion ...... 26 3.5 Friction and Contact Mechanics ...... 28

4 Materials and Methods 35 4.1 Substrates ...... 35 4.1.1 Silicon wafers ...... 35 4.1.2 ‘Everyday Substrates’ ...... 38 4.2 Atomic Force Microscopy ...... 39 4.2.1 Force Spectroscopy ...... 41 4.2.2 Bacterial Probes for AFM Force Spectroscopy ...... 44 4.2.3 Implementation of Force Spectroscopy Experiments ...... 47 4.2.4 PeakForce QNM® ...... 48

VII 4.3 Parallel Plate Flow Chambers ...... 49

5 Inﬂuence of Substrate Properties on Surface Processes 53 5.1 The “Subsurface Energy” ...... 53 5.1.1 Impact on Protein Adsorption ...... 53 5.1.2 Impact on Bacterial Adhesion ...... 54 5.1.3 Impact on Gecko Adhesion ...... 54 5.2 Inﬂuence of the Fluoridation of Hydroxyapatite on Bacterial Adhesion . 55 5.3 Bacterial Adhesion to ‘Everyday Surfaces’ ...... 55

6 Surface Properties of Bacteria 59 6.1 Dynamic Adhesion of Diﬀerent Staphylococci Species ...... 59 6.2 Dependence of the Cell Wall Elasticity on the Degree of Peptidoglycan Crosslinking ...... 64

7 Summary and Outlook 67

Bibliography 69

Publications 99 Addendum I - Is adhesion superficial?! Silicon wafers as a model system to study van der Waals interactions ...... 101 Addendum II - Subsurface Influence on the Structure of Protein Adsorbates as Revealed by in Situ X-ray Reflectivity ...... 109 Addendum III - The Influence of the Subsurface Composition of a Material on the Adhesion of Staphylococci ...... 121 Addendum IV - Adhesion of gecko setae reflects nanoscale differences in subsurface energy ...... 129 Addendum V - Fluoridation of hydroxyapatite leads to a reduced adhesion of oral bacteria ...... 141 Addendum VI - Reduction of the peptidoglycan crosslinking causes a de- crease in stiffness of the Staphylococcus aureus cell envelope ...... 149 Abbreviations

AFM atomic force microscope BSA bovine serum albumin CW cell wall DLVO Derjaguin-Landau-Verwey-Overbeek DMT Derjaguin-Muller-Toporov JKR Johnson-Kendall-Roberts MD Maugis-Dugdale MRSA methicillin resistant Staphylococcus aureus OTS octadecyltrichlorosilane PBP4 penicillin-binding protein 4 PBS phosphate buﬀered saline PDA poly(dopamine) PG peptidoglycan PLL poly-L-lysine QCM quartz crystal microbalance QCM-D quartz crystal microbalance with dissipation QNM quantitative nanomechanical property mapping SAM self assembled monolayer SD surface delay SERAMs secretable expanded repertoire adhesive molecules TM tapping mode™ vdW van der Waals

IX X 1 Introduction

The adhesion of biological objects to artificial materials, also referred to as bioadhesion, is a process that is of major importance in many different areas: Primarily in medical research, the investigation of bioadhesion is crucial for the performance of im- plants and catheters or the development of biosensors and biological glues. Yet, many other branches of industry are also affected: In oil or gas pipelines, in the shipping industry, and in the food production, for instance, the adhesion of biofilms (bacteria, proteins) or mussels is a major concern [Kum1998, Zhu2003, Ner2006, Sch2011]. Con- sequently, it is the objective of various studies to prevent or control the adhesion of biological objects such as proteins, bacteria and eukariotic cells.

Moreover, the capability of organisms, such as mussels, insects, frogs, or geckos, to adhere extremely strong to all kinds of surfaces inspired engineers to mimic the respective responsible structure [Fed2001, Gei2003, Lee2007b, Bar2011]. The hunt for gecko-like adhesives, for instance, enjoys great popularity nowadays, especially because gecko adhesion is self-cleaning and completely reversible [Han2005,Lee2008,Boe2010].

For both purposes, the control and the mimicry of bioadhesion, a comprehensive understanding of the interface between the artificial materials and biological objects is necessary. To gain this understanding, researchers from various fields tackle this topic, situated at the border of physics, chemistry, and biology. Unfortunately, however, bioadhesion is usually an interplay of multiple different interactions. Hence, the only way to gain a comprehensive picture of the involved players is to unravel the various responsible parameters.

The main objective of this thesis is to study single parameters or interactions in terms of their impact on the adhesion of biological objects. Thereto, selected parameters of the surface and the subsurface of substrates are independently examined. To cover a wide range of length scales, diﬀerent types of biological objects featuring a broad variety of dimensions were studied: nanoscopic proteins, microscopic bacteria, and macroscopic geckos. Proteins and bacteria account for the principal components of most bioﬁlms. Geckos are, as mentioned above, one of the most prominent examples of nature that engineers try to mimic.

The major focus of this thesis lies on van der Waals (vdW) interactions, especially on those arising from a material that is hidden by the topmost surface layer. VdW forces

1 1 Introduction are the sole type of intermolecular forces that are present in every system and arise— unlike most other interactions—not only from the surface, but from the complete material of which the substrate is constituted. Particularly in the presence of thin surface coatings, the overall vdW interactions can depend strongly on the underlying material, a fact that is frequently overlooked. The main objective of the here presented work is to determine whether a variation in the subsurface characteristics of a substrate affects the adhesion of biological objects and whether an effect is limited to a specific scale.

A further parameter focussed on in this thesis is the impact of the fluoridation of artificial tooth surfaces on bacterial adhesion. It is well known that the application of fluoride compounds has a cariostatic effect. The origin of this effect, however, is usually solely attributed to a decreased demineralization of the teeth. Hence, another objective of the here presented work is to determine whether the fluoridation affects the strength of the force by which oral bacteria adhere to the tooth surfaces. Such an effect would account for an additional source of the cariostatic character of fluoridation.

Besides the modiﬁcation of substrate properties, the characteristics of the interacting biological objects can be altered. In the case of bacteria, this can be done on diﬀerent levels of separation of single characteristics: It is either possible to study

• alive and dead examples of the same type of bacteria, • different species of one specific bacterial genus, • different strains of one species, or • genetically modified bacteria together with the respective wild-type strain.

A combination of the ﬁrst two options is utilized in this thesis to compare the time dependence of alive and dead bacteria of the two Staphylococcal species S. aureus and S. carnosus. The objective, thereby, is to gain insight in the adhesion process and to examine possible diﬀerences for pathogenic and non-pathogenic Staphylococci.

The other two options are also applied in the here presented work, with the aim to investigate the impact of a reduction in the crosslinking of the cell wall of S. aureus on the stiffness of the bacterial cell envelope. To determine if the stiffness or a change in it is correlated with the resistance against antimicrobials, two S. aureus strains featuring different degrees of resistance and the respective mutants are investigated.

2 2 Overview and Connectivity

The work at hand engulfs six publications, three of them already published in and three submitted to peer reviewed journals. Although emerged from multiple projects, the publications are closely connected: In all of them, one parameter of the (sub)surface of materials is separately studied with respect to their impact on biological adhesion. In the publications in Addenda II-IV, the van der Waals forces that arise from interactions with the subsurface material are studied. These studies are preluded by a review of the state of the art in the field of vdW interactions in general and in multilayer systems in particular in Addendum I. The study presented in Addendum V focusses on the effect of a fluoridation of tooth surfaces and the publication in Addendum VI concentrates on the surface properties of bacteria, namely the elasticity of the cell envelope. The biological objects studied cover a wide range of length scales; the publication in Addendum II focusses on nanocopic proteins, the ones in Addenda III, V, VI on microscopic bacteria, and the one in Addendum IV on macroscopic geckos.

Overall, the thesis is organized in ﬁve chapters:

• In the ‘Context and State of the Art’ chapter, the studied objects and applied concepts are introduced and a general literature review is given. A detailed and project-tailored overview of the theoretical concepts and the state of the art is then given in the respective publications. • The ‘Materials and Methods’ chapter elucidates the primarily utilized methods and materials that are obligatory for the understanding of the presented unpublished results. Project speciﬁc applied methods and used materials are accounted for in the respective publications. • In the two result chapters ‘Inﬂuence of Substrate Properties on Surface Pro- cesses’ and ‘Surface Properties of Bacteria’, a brief review of each attached publication is given and further unpublished results are presented and discussed. • The thesis is concluded by the ‘Summary and Outlook’ chapter, comprising a summary of the presented results and an outlook on possible future and already ongoing follow-up projects.

3 4 3 Context and State of the Art

3.1 Surface Forces

In principle, solely the four fundamental forces, namely

• strong and weak interactions, acting between elementary particles, and • gravitational and electromagnetic interactions, acting between atoms and molecules, are sufficient to describe all processes in nature. Thereby, the first two interactions may be classified as ‘sub-atomar’ forces and the latter two as ‘super-atomar’. The role of the two ‘super-atomar’ interactions, gravitation and electromagnetism, depends on the scale of the described system. On a macroscopic scale (stellar systems, tides, and falling bodies), gravitation is the major factor. On a microscopic scale, electromagnetism plays a key role and is therefore often referred to as the ‘true’ intermolecular force.

Although all forces between objects on a microscopic scale are essentially of electromagnetic origin, it is useful to further classify these forces into distinct categories of intermolecular forces. Individually, all of these forces are theoretically described, yet an overall solution for the theoretical description of all electromagnetic interactions in arbitrary systems (from single atoms to multi atom bodies) has not been achieved so far. Due to this classiﬁcation, an interaction (e. g. adhesion or cohesion) between two objects is the interplay of many diﬀerent intermolecular forces.

In the following sections, some of the major ‘players’ in the adhesion process are reviewed. A more comprehensive description of intermolecular forces is given, in general by the textbooks of Israelachvili [Isr1992] and Lyklema [Lyk1991], focused on biology by Leckband & Israelachvili [Lec2001], and on nanotechnology by French et al. [Fre2010].

5 3 Context and State of the Art

Figure 3.1: Van der Waals potentials for diﬀerent geometries. Adapted from [Isr1992].

3.1.1 Van der Waals Interactions

Van der Waals (vdW) forces describe the interactions between permanent and induced dipoles. Usually, three types of interactions contribute to the vdW interactions [Isr1992]:

Keesom interactions: Dipol-dipol interactions of freely rotating molecules that carry permanent dipoles. Debye interactions: Forces between a freely rotating permanent dipole and a non- polar molecule, wherein a dipole moment is induced by the permanent dipole. London interactions, also referred to as dispersion interactions: Electrodynamic forces between instantaneously induced dipoles. They are of quantum mechanical origin [Lon1937].

The common characteristic of all three types of vdW interactions is the scaling with x−6. Due to this, vdW forces in general are often considered to be of short-range. Yet, this is only true for molecular systems, since the exponent 6 persists only on −

6 3.1 Surface Forces

Table 3.1: Hamakerconstants A123 = A12−32 describing various systems that are of relevance in this thesis. a Material 1 Material 2 Material 3 Hamaker constant [kBT ] Source b b c SiO2 vacuum SiO2 17.4 [Tan2005] b c water SiO2 1.9 [Tan2005] Si vacuum Si 51.7 [Fre1995]c c α Al2O3 40.4 [Fre1995] − Proteind water Proteind 3.1 [Rot1996]c Quartz 1.65 [Rot1996]e

a −20 at room temperature 10 J = 2.4 kBT b amorphous c Full spectral method d BSA e Hough and White method [Hou1980] this scale. On a macroscopic scale, however, vdW forces are of long-range and the exponent of the scaling law increases up to 1 depending on the geometry as depicted − in Figure 3.1 . Between two semi-inﬁnite half slabs, for instance, the interactions scale with x−2 [Isr1992]. The strength of the vdW interactions is determined by the Hamaker constant of the system, which depends on the polarizabilties of the involved materials (cf. Table 3.1).

Van der Waals forces are the only other interactions—besides gravitation—that exist in every system. They can never be completely shielded and are always attractive1. Moreover, vdW forces act not only with the surface but with all atoms in the bulk. This is especially of interest for multilayer systems, since in these, layers below the surface can interact with a probe object via vdW forces.

A more comprehensive review of the theory and history of vdW interactions in general and in multilayer systems is given in the publication in Addendum I.

3.1.2 Electrostatic Interactions

Two charged atoms, ions, or surfaces interact by ‘classical’ electrostatic forces. The Coulomb force can be both attractive or repulsive. It is commonly considered as both

1A repulsion due to vdW forces is indeed also possible. Yet, this repulsive interaction is a result of a stronger attractive interaction with the medium than between the interacting objects.

7 3 Context and State of the Art

Figure 3.2: Schematic illustration of the electrokinetic double layer overlaid with a typical potential: Adjacent to the surface, a layer of bound counterions forms the Helmholtz or Stern layer, followed by the shear layer and the diﬀuse layer. The potential is usually described by the ζ-potential. Adapted from [Hof2012]. the strongest and the longest ranging intermolecular force, since, in simple systems, its potential scales with x−1.

In liquids, however, electrolytes strongly aﬀect the electrostatic interactions. Due to the ionization or dissociation of surface groups (e. g. carboxylic or amino groups) and the adsorption of ions, virtually all nat