"Mimicking the abrasion resistant sandfish epidermis" Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation. vorgelegt von Boštjan Vihar, M.Sc. Aus Maribor, Slowenien Berichter: Universitätsprofessor Prof. Dr. Peter Bräunig Universitätsprofessor Prof. Dr. Werner Baumgartner Tag der mündlichen Prüfung: 7. September 2015 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar. "L'essentiel est invisible pour les yeux." Antoine de Saint-Exup´ery Abstract The sand inhabiting skink Scincus scincus has due to its sand swimming behaviour gained some attention by the research community in recent years. The sand swimming creates ample stress on the outer layers of its skin, generating the prospect of wear. The skin itself is however very resilient towards abrasion and even facilitates smoother movement by reducing friction against the sharp sand particles. Past research has concentrated much on the characterization of structure and surface properties of the skin, however, a complete understanding of its properties is lacking up to date. In this work, a review of known data is done in addition to new measurements concerning surface properties, composition, ultrastructure and mechanics, but also other feats such as optical properties. Replication of "anti-adhesive" properties on existing technical materials was tested along with the development of new techniques for such endeavour. Chemical and structural anal- ysis shows the epidermis of the sandfish is very homogeneous and is mainly composed of compact bundles of β keratins, which are possibly interlinked by a matrix composed of α keratins. Strong glycosylation of the keratins was shown, confirming previous studies, and five glycan structures were observed in the analysis. The occurrence of glycans could also be linked with the surface properties concerning surface energy and adhesion of the epidermis towards sand. Mechanical properties of the sandfish epidermis were shown to be similar to some artificial polymers which will be interesting for the replication of the sandfish epidermis from artificial materials. The surface micro structure of the sandfish epidermis has been shown to promote diffraction of light. This might suggest possible light trapping or dispersion behaviour, which could not be conclusively confirmed. Finally, we propose a hypothesis on abrasion resistance and friction reduction, which is possibly the result of an interplay between mechanical and chemical properties of both keratins and glycans. However, additional research will be required to confirm the proposed mechanism. ii Acknowledgements I would like to express my sincere gratitude to my mentor, Prof. Dr. Werner Baumgartner, for the chance to work on this project and the guidance I needed during the process. I thank Prof. Dr. Peter Br¨aunigfor taking responsibility on and being an expert witness of my work. I also thank Prof. Dr. Lothar Elling for the valuable discussions, scientific input and for being an expert witness to this work. Special thanks goes to Agnes Weth, whose methodological expertise enabled me to do things, I otherwise could not. I thank Christian Effertz, Dennis Hirtz, Prof. Dr. Franz Georg Hanisch, Matic Tement, Umut Cakmak, Christian Reißecker, Prof. Dr. Johannes Heitz, Lisa Maria Uiberlacker, Markus Himmelsbach and Lucas Sternbauer for their indispensable cooperative work on the project, which I was not capable of doing myself. Much appreciation goes to the DFG (the German Research Foundation) and the Bionik Graduate school Bonn - Aachen - J¨ulich,which financed the project and without which this work was not possible. I owe thankfulness to all my colleagues at RWTH Aachen and JKU Linz, who often gave council, provided ideas for the research and created a pleasant working atmosphere. Special thanks also goes to my family and friends, who supported and motivated me to achieve my goals. iii Contents 1 Chapter 1 - General introduction 1 1.1 About the Sandfish . .1 1.1.1 Basic description . .1 1.1.2 Ecology . .2 1.1.3 Motivation . .4 1.2 Project goals . .5 2 Chapter 2 - What do we know about the adhesion and friction reduction, abra- sion resistance and what do these have in common? 6 2.1 Introduction . .6 2.1.1 From interaction to friction . .6 2.1.2 The ”sandfish effect” . .7 2.1.3 What we know so far . .8 2.1.4 Determining the surface energy . 11 2.2 Methods . 12 2.2.1 Epidermis preparation . 12 2.2.2 Contact angle measurement . 12 2.2.3 Surface energy calculation . 13 2.3 Results and discussion . 14 3 Chapter 3 - Surface and ultrastructure of the epidermis 16 3.1 Introduction . 16 3.2 Methods . 18 3.2.1 Polychromatic staining and light microscopy . 18 3.2.2 Transmission electron microscopy . 21 3.3 Results and discussion . 24 4 Chapter 4 - Optical properties 28 4.1 Introduction . 28 4.2 Methods . 29 4.2.1 Creating keratin thin films . 29 4.2.2 Refractive index . 30 iv 4.2.3 Scale diffraction . 30 4.2.4 Light transmission . 30 4.2.5 Heat absorption . 31 4.2.6 Simulations of light trapping behaviour . 31 4.3 Results . 32 4.3.1 Refraction and diffraction . 32 4.3.2 IR transmission . 33 4.3.3 Heat absorption . 34 4.3.4 Simulations . 36 5 Chapter 5 - Mechanical properties 37 5.1 Introduction . 37 5.2 Methods . 39 5.2.1 Tensile testing . 39 5.2.2 Nanoindentation . 40 5.3 Results and discussion . 41 5.3.1 Tensile testing . 41 5.3.2 Nanoindentation . 44 6 Chapter 6 - Biochemical composition and properties 47 6.1 Introduction . 47 6.2 Methods . 51 6.2.1 Skin lysis and transfer to a watery medium . 51 6.2.2 SDS-PAGE . 52 6.2.3 Western blotting . 55 6.2.4 Lectin blotting . 57 6.2.5 Capillary electrophoresis . 58 6.2.6 Mass spectrometry . 59 6.2.7 Raman spectroscopy . 60 6.3 Results and discussion . 61 6.3.1 SDS-PAGE . 61 6.3.2 Western blotting . 62 6.3.3 Lectin blotting . 63 6.3.4 Capillary electrophoresis . 64 6.3.5 Mass spectroscopy . 66 6.3.6 Raman spectroscopy . 67 7 Chapter 7 - Understanding and replication of the adhesion/friction reduction 76 7.1 Introduction . 76 7.2 Methods . 77 7.2.1 Glycan extraction from alternative sources . 77 v 7.2.2 Additional glycan purification . 79 7.2.3 Sample evaluation . 79 7.2.4 Surface modification of glass . 80 7.2.5 Surface modification of PMMA . 81 7.3 Results and discussion . 86 7.3.1 Glass . 88 7.3.2 PMMA . 89 8 Chapter 8 - Conclusion and outlook 97 9 References 102 A Materials list 110 A.1 Materials . 110 A.1.1 Chemicals . 110 A.1.2 Laboratory equipment . 113 A.1.3 Miscellaneous . 114 A.1.4 Software . 114 A.1.5 Animal care . 115 vi List of Figures 1.1 Scincus scincus...................................2 1.2 Geographic distribution of S. scincus........................3 2.1 DLVO theory. .7 2.2 Friction angles comparison of sand with sandfish skin and other materials. .9 2.3 AFM measurement comparison of sandfish and corn snake scales. 10 2.4 Sand blasting comparison of sandfish skin and technical materials. 11 2.5 Young's equation and contact angle. 12 2.6 Surface energy plot. 15 3.1 A comparison of sand swimming reptiles and their skin. 16 3.2 A comparison of ventral and dorsal scales of four species under SEM. 17 3.3 The epidermal structure of lizards. 18 3.4 Polychromatic staining of the sandfish epidermis. 25 3.5 TEM image of a sandfish scale in cross section . 26 3.6 TEM image of a sandfish scale in cross section, labelled with WGA-Strepatividin gold ........................................ 27 4.1 A sample measurement of light diffraction on a sandfish scale. 32 4.2 Diffraction of monochromatic light on sandfish and Berber skinks scales. 33 4.3 Light transmission of dorsal and ventral sandfish scales . 34 4.4 Atmospheric transmission of EM radiation in the spectrum from UV to IR . 35 4.5 Greenhouse effect of the sandfish scales. 35 4.6 Rayleigh points on the simulated sandfish scale. ..