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

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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 influ- enced 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 bioadhe- sion, 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 ad- here 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
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