Atomic Layer Deposition on Biological Matter

Transcription

1 Atomic Layer Deposition on Biological Matter Dissertation Zur Erlangung des akademischen Grades Doktor der Ingenieurwissenschaften der Fakultät für Angewandte Wissenschaften der Albert-Ludwigs-Universität Freiburg im Breisgau vorgelegt von Seung-Mo Lee geboren in Chun-cheon, Südkorea Freiburg im Breisgau 2009

2 Dedicated to my lovely wife Ji-Sun, to my venerable father in heaven, to my mother, to my youger sister, and to my teacher Prof. Ulrich Gösele resting in peace To all those who taught me This dissertation is for you

3 If most of us are ashamed of shabby clothes and shoddy furniture, Let us be more ashamed of shabby ideas and shoddy philosophy -By Albert Einstein

4 Dekan: Prof. Dr. Hans Zappe 1. Referent: Prof. Dr. Ulrich Gösele 2. Referent: Prof. Dr. Margit Zacharias Vorsitzender der Prüfungskommission: Prof. Dr. Oliver Paul Beisitzender: Prof. Dr. Ulrich Egert Tag der Disputation: Mittwoch, Dez 16, 2009

5 Preface After invention of atomic layer deposition (ALD) by Dr. Tuomo Suntola and co-workers in 1974, the interest in ALD has strongly increased in the of 1990s and 2000s to satisfy the industrial need to scale down microelectronic devices. Thanks to increased scientific and technical interest, nowadays diverse manufacturers and institutions have performed various designs of ALD machinery together with the development of new ALD precursors. ALD can be defined as a film deposition technique which is based on the sequential use of self-terminating gas solid reactions. ALD is a subset of chemical vapor deposition (CVD) suitable for depositing inorganic layers with a thickness down to the level of a monolayer. ALD has the capability to coat complex shaped substrates with a conformal film of high quality. Due to these unique characteristics, ALD-grown materials have a wide range of applications, from catalysts to electroluminescent displays and microelectronics. ALD is recognized as one of the key technologies for the surface modification and functionalization of complex organic or inorganic nanostructures, such as nanowires, nanopores or nanotubes. Besides the excellent conformality of the ALD coating, some scientists have reported examples of inorganic nanostructures fabricated from complicated biological templates as one of the bottom up approaches for 3D nanofabrication. In this thesis, some examples will be introduced which illuminate novel applications of ALD using biological matter, such as dragline silks from Araneus spider and collagen membranes collected from chicken s eggshell matrices. It will be demonstrated that that metal oxide ALD coatings on these biological templates leads to conformally coated metal oxide films on the templates as well as to chemical/physical modifications of the inner protein structure of the silk and collagen involved. As a result, those modifications lead to an improvement of the mechanical properties. This modification process by ALD has been termed Multiple Pulsed Vapor Phase Infiltration or MPI in short. At present, the detailed mechanism associated with the chemical/physical modifications and the reasons causing those resulting property improvements are not clearly understood yet. It has, however, been deduced that this mechanical property improvement can be attributed to metal infiltration into the inner protein structure of the silk and the collagen, and this metal infiltration is related to the unique self-limiting film deposition mechanism of the ALD process which distinguishes it from other deposition techniques. On the other hand, aside from mechanical properties improvements, it is expected that, those modifications presumably could affect other physical properties, such as electrical, magnetic and optical properties. In this thesis, however, first and foremost preliminary results focusing on the improvement of mechanical properties are presented.

6 In order to give a general idea of the ALD process to the reader, in Chapter 1 the basic principle of ALD is explained using a metal oxide deposition, such as Al 2 O 3, TiO 2 and ZnO as an example. Further on, the fundamental differences between ALD and CVD are pointed out. Following the introduction of ALD, in Chapter 2, basic parameters from mechanics, required to describe and understand the mechanical deformation behaviour, are briefly described using a stress-strain curve plotted under uniaxial tensile test. The viscoelastic behaviour of materials is also introduced. In the Chapters 3, 4 and 5, ALD applications using biological matter are addressed. Firstly, in Chapter 3, an example to show the capability of ALD to conformally deposit materials (TiO 2 and ZnO) on complexshaped biological templates (macroporous collagen membranes) is presented. Moreover, some crystallographic growth features of TiO 2 and ZnO at various processing temperatures are also demonstrated. As a feasible application of those templated inorganic TiO 2 /ZnO structures, photocatalytic effects under UV illumination are presented. In Chapter 4, an example to illuminate a novel application of ALD, i.e., the chemical/physical modification of protein structure of spider dragline silk by the MPI process and the resulting mechanical property improvements are presented. In Chapter 5, similar to Chapter 4, using collagen which is a primary concern in tissue engineering, chemical/physical modification processes and subsequent improvements of the mechanical properties together with scientific validation of the modified collagen are discussed. Even though the mechanism related to the modification and subsequent improvement of the mechanical properties is not yet clear, in Chapter 4 and 5 the presumable models or mechanisms to explain mechanical deformation behaviour are proposed. Finally, in the Appendix some data figures which were not included in the main text are presented.

7 Table of Contents CHAPTER GENERAL ASPECTS OF ATOMIC LAYER DEPOSITION AND IT S APPLICATIONS INTRODUCTION PRINCIPLE AND CHARACTERISTIC FEATURES OF THE ALD PROCESS INVESTIGATED ALD MATERIALS REACTION MECHANISMS OF METAL OXIDES BY ALD COMPARISON OF ALD AND CVD MULTIPLE PULSED VAPOR PHASE INFILTRATION CHAPTER BASIC PARAMETERS IN MECHANICS STRESS AND STRAIN STRESS - STRAIN CURVE STRAIN ENERGY, BREAKING ENERGY AND TOUGHNESS STIFF MATERIALS, STRONG MATERIALS AND TOUGH MATERIALS ELASTIC DEFORMATION AND PLASTIC DEFORMATION CHAPTER METAL OXIDE DEPOSITION ON BIOTEMPLATE: MACROPOROUS PHOTOCATALYTIC TiO 2 OR ZnO MEMBRANES TEMPLATED FROM CHICKEN S EGGSHELL MATRICES BACKGROUND Historical background of photocatalysis Principle of photocatalysis and applications Crystal structure of TiO 2 and ZnO Escherichia coli (E. coli) bacteria INTRODUCTION EXPERIMENTAL Preparation of the inner shell membrane from a chicken s egg TiO 2 /ZnO atomic layer deposition on ISM Characterization Microbiology Photocatalytic experiments with ISM/TiO 2 and ISM/ZnO Tensile test of native ISM, ISM/ZnO/100 and ISM/TiO2/275 membranes RESULTS AND DISCUSSION Film quality, crystallographic features and bactericidal efficiency Mechanical flexibility and thermal stability CONCLUSION CHAPTER METAL INFILTRATION INTO SPIDER DRAGLINE SILK BACKGROUND Overview of spiders and mechanical properties of spider silk Chemical structure and macroscopic model for spider silk Models for the description of dragline silk s mechanical properties Function of metals in biological tissues EXPERIMENTAL Silk collection Multiple pulsed vapor phase infiltration process Tensile test TEM and EDX analysis Solid state nuclear magnetic resonance (NMR) spectroscopy Wide angle X-ray scattering (WAXS) RESULTS AND DISCUSSION... 67

8 4.3.1 Variation of mechanical properties under diverse conditions Scientific validation of the MPI process Model system for the metal infiltration mechanism Model system for mechanical property improvements of silk CONCLUSION CHAPTER METAL INFILTRATION INTO COLLAGEN COLLAGEN AND TISSUE ENGINEERING STRUCTURE OF COLLAGEN COLLAGEN OF A CHICKEN S EGGSHELL MEMBRANE BIOMINERALIZATION OF COLLAGEN ARCHITECTURES EXPERIMENTAL Preparation of the collagen membrane (CM) from a chicken s eggshell matrix MPI process Tensile tests Cross section sample preparation by focused ion beam (FIB) SEM, TEM and EDX Raman spectroscopy Wide angle X-ray scattering Small angle X-ray scattering RESULTS AND DISCUSSION Mechanical deformation behaviour Metal infiltration into collagen Chemical analysis via Raman shift Structural analysis via x-ray scattering Biomineralization versus metal infiltration CONCLUSION SUMMARY REFERENCES AND NOTES ACKNOWLEDGEMENT APPENDIX A1. FIGURES A2. TABLES A3. REFERENCES AND NOTES CURRICULUM VITAE...139

9 Chapter 1 1 Chapter 1 General Aspects of Atomic Layer Deposition and It s Applications With the rapid development of semiconducting devices, scientists have made an effort to develop a method to process extremely thin high-k dielectric layers with high conformality. The special self-limiting growth mechanism of Atomic Layer Deposition (ALD) facilitates the film thickness and compositional control at the atomic level, as well as the deposition on large and complicated structures. On one hand, theses unique features make ALD a promising thin film deposition method for the next generation of micro- and nanoelectronics. On the other hand, recently, ALD has been applied in other fields, such as photovolatics, sensing, proactive coatings, nanostructuring by template approach, optoelectronics, piezoelectronics, chemical surface modification of diverse materials, micro- and nanoelectromechanical systems etc. ALD is also regarded as one of the innovative tools for the development of nanotechnology. In this chapter, general features of ALD will be briefly introduced together with some examples of applications. In the last part, one of the possible new applications of ALD induced by the separation of reactants, i.e. Multiple Pulsed Vapor Phase Infiltration (MPI), which could be widely applied to biomaterials, will be introduced. 1.1 Introduction In 1974, Dr. Tuomo Suntola and co-workers introduced a new thin film deposition method which was able to improve the quality of ZnS films used in thin-film electroluminiescent flat pannel dispalys. The first display with ZnS films deposited by the new method was lit in the display board in Helsinki Airport in Since then, thanks to the new process,

10 2 ALD General more than 2 million electroluminescent displays have been produced (about m 2 (20 ha)) [1]. Nowadays we call the new deposition method Atomic Layer Deposition (ALD). Until late 1980s ALD was applied to produce compound semiconductors and efforts to make III-V compunds were lasted. However, due to the chemical instability of group III alkyl compounds and group V hydrides, only little progress was achieved with ALD, as compared to molecular beam epitaxy (MBE) or metal-organic vapor phase epitaxy (MOVPE) [2]. Meanwhile, in the middle of 1990s, with the increased interest in silicon based microelectronics devices, the large takeoff of ALD began. Miniaturizing the dimension of the devices and inceasing aspect ratios in intergrated circuits required a new thin film deposition method with high controllability of film thickness and chemical film compositions on the atomic scale. Consequently, ALD came into focus as a potential candidate to facilitate those requirements. Several comprehensive reviews [3,4] cover sucessfully deposited ALD materials and corresponding processing conditions in detail. Another review descirbes the appliation of ALD in nanoscience [5]. In the following the general outline of ALD focusing on charateristic features and principles is presented. From the review articles [3-5], some contents relevant to the main topic of this thesis are extracted and presented accordingly. In the last part, a new application of the ALD process, named Multiple Pusled Vapor Phase Infiltration (MPI) [6], is briefly introduced. 1.2 Principle and characteristic features of the ALD process ALD is a technique based on sequential surface chemistry that deposits highly conformal thin-films of materials onto substrates of varying compositions. ALD is chemically similar to Chemical Vapor Deposition (CVD), except that the ALD approach splits the CVD reaction into two half-reactions, keeping the precursor materials seperate during the reaction. Such separation leads to a self-limiting mechanism and thus a control of the film growth on the atomic/molecular level. Unlike the CVD process, the ALD process is performed in a cyclic manner. Generally, one growth cycle consists of the following four steps (in case of ALD with two reactants): Step A: Injection and exposure of the first reactant (precursor A) Step B: Purge and evacuation to remove the surplus reactant and the byproducts from the vapor phase reaction Step C: Injection and exposure of the second reactant (precursor B) Step D: Purge and evacuation to remove the surplus reactant and the byproducts from vapor phase reaction

11 Chapter 1 3 Figure 1.1 A schematic representation of the basic principle of the ALD process. This diagram shows a metal oxide, i.e. MO 2 (M can be Ti, Zr or Hf etc.) deposition by ALD. For this deposition, a metal containing precursor ML 4 (L: ligand) as the metal source and H 2 O as the oxygen source are used. Sub-step 1 (chemisorption and saturation of precursor A), Sub-step 2-3 (purge), Sub-step 4 (chemisorption and saturation of precursor B) and Sub-step 5-6 (purge) in the figure correspond to Step A, Step B, Step C and Step D, respectively (for details, see the text). In Sub-step 1, the surface is exposed to a ML 4 vapor pulse. The precursor is chosen in a way that it reacts quickly with the reactive surface sites, forming a stable saturated chemisorbed layer. Once saturation is achieved, the purging of ML 4 vapor begins. Sub-step 2 presents the situation at the beginning of the purge, while Sub-step 3 shows a completed ML 4 vapor purge. The same exposure purge sequence is repeated for H 2 O vapor in Sub-step 4 (pulse), Sub-step 5 (purge onset) and Sub-step 6 (completed purge), completing a full ALD growth cycle. In Sub-step 4, the surface bound ligands receive a proton from H 2 O and leave the surface as volatile byproduct HL, being replaced with an OH group. The recreated OH terminated surface is now available for the next ALD growth cycle. These steps are repeated with each cycle adding a sub-monolayer quantity of material to the surface until a thin MO 2 film is formed. Sub-step 3 and Sub-step 6 emphasize the purge process of each unreacted precursor and the reaction byproduct. In a case where Sub-steps 1 and 4 or 5 overlap, the self-limitation is lost and CVD growth takes place in addition to ALD.

12 4 ALD General Figure 1.1 schematically illustrates one ALD reaction cycle. Each reaction cycle adds a given amount of material to the used substrate surface. In order to grow (or deposit) a material layer (for example, metal oxide, MO 2 in the case of Figure 1.1), ALD reaction cycles are repeated as many times as required for the desired film thickness. Accordingly, with the number of ALD cycles, one can precisely control the thickness of the film. One cycle time can be also adjusted from sub seconds to few minutes, depending on (1) the objective of the process; (2) the chemical charateristics of precursors being used; (3) the structure of the substrate and the deposition temperature; (4) the reactivity of the precursor with the substrate. The cycle time (for instance, exposure time and purge time) is dependent on the reciprocal reactivities between two precursors, the spontaneity of the layer formation reaction as well as the geometric features of the used substrate. Normally, when the geometry of the used substrate is flat or rather simple, with short cycle times one can obtain high quality films. Whereas, in the case of substrates with complex geometries (e.g. porous alumina or diverse biological templates), longer cycling times are required to assure complete and uniform coverage of the template. The growth rate (deposited film thickness per cycle) is likely to be dependent on the size of the used precursor molecule, because the steric hinderance between large precursor molecules limits the number of molecules being able to adsorb on the substrate. With small molecules as precursors, monolayer growth can be achieved [7]. Apart from the precursor molecule size, chemical properties of the substrate itself (for instance, surface energy of the used substrate) or the intermediate reactions between precursors and byproducts during processing can also affect the layer growth. a b Figure 1.2 Schematic ALD process window. a, Conceptual illustration of an ALD process window with respect to the processing temperature. In order to obtain suitable chemisorption of precursors onto a substrate surface via chemical bonding, the temperature should be lower than the precursor decomposition temperature to assure stable chemisorption. On the other side, the temperature should be higher than the lower limit to prevent precursor condensation or incomplete reaction. As an example, figure b shows ALD temperature windows for diverse metal oxides depending on the precursor pairs being used (Source:

13 Chapter 1 5 In an ideal case, the injected precursor molecules chemisorb or react with surface functional groups saturatively, limiting further adsorption. The self-limiting growth mechanism brings unique following advantages to ALD [7]: 1. Film thickness depends only on the number of reaction cycles, which allows precise and simple film thickness control. 2. Unlike CVD, there is less need of reactant flux homegeniety, which gives large area deposition capability, guarantees highly conformal deposition and faciliates solid state precursors. 3. Unlike CVD, seperated injection/exposure prevents vapor phase reactions before deposition on the substrate, which allows for the use of highly reactive precursors and gives enough time for each reaction step to be finished. This also allows for films to be deposited at relatively low temperature. On the other hand, each chemical reaction cycle of ALD requires a certain activation energy to allow the used precursors to react with each other. Most of the ALD systems introduce the required activation energy by heating (usually called thermal ALD). Some systems use reactive gases like plasmas (Plasma Enhanced ALD, or PEALD) or UV irradiation. Nevertheless, the ALD process window (Figure 1.2) is often wide, which also makes the deposition process insensitive to small changes in temperature and precursor flow rate, and allows for the deposition of nanolaminates in a continous process. 1.3 Investigated ALD materials ALD can be used to deposit a large variety of materials including oxides, nitrides, sulfides and metals. The number of new ALD processes is steadily increasing. Figure 1.3 illustrates the materials grown by ALD until February 2005 in a visual form using a periodic table. The variety of deposited materials includes oxides, nitrides, sulphides, II-V and III-V compounds and elements including both metals and non-metals etc. Table 1.1 lists the frequently used precursor combinations in two reactants system ALD.

14 6 ALD General Figure 1.3 Overview of materials grown by ALD until Feb This table is reproduced and edited from the table in Puurunen s review [3]. Growth of pure elements as well as compounds with oxygen, nitrogen, sulphur, selenium, tellurium and other compounds grouped together are indicated through shadings of different types at different positions. For details, see Puurunen s article [3]. The majority of the ALD processes investigated to date rely on thermal ALD. In thermal ALD processes the reactants (precursors) have their intrinsic reactivity towards the other reactants and the overall kinetics is highly dependent on the deposition temperature. The main reasons which limit the deposition of certain materials with thermally activated ALD are the decomposition of precursors before reaching the substrate or too low a reactivity between the reactants. The deposition temperature may be further limited by the substrate, which may be a temperature-sensitive material (such as biomaterials) or device structures. Low deposition temperatures may also limit the film quality if the film forming reactions are slow or incomplete. For example, slow desorption of reaction byproducts may result in increased amounts of impurities in the films. Some processes, however, demonstrated aggressive enough half-reactions and produced high quality films even at low temperatures [8]. Others suffered from excessively long cycle times and showed high impurity contents [9]. Even though diverse types of ALD processes except the thermal ALD have been developed in order to resolve those limitations, a fundamental development of precursors, which overcome those limitations, is highly desired (information included in Table 1.2).

15 Chapter 1 7 Table 1.1 ALD materials together with corresponding precursor combinations reported in literature and list of possible applications of ALD materials. a TEMAH: Tetrakisethyl Film Precursors [3] Applications methylaminohafnium, o High-κ dielectric [10] Hf[N(C 2 H 5 )(CH 3 )] 4. AlCl 3 / H 2 O or O 3 o OLED Passivation [11] Al 2 O 3 AlBr 3 / H 2 O b o Anti-reflection and optical filters [12-14] Ti(OEt)4 : Titaniumethoxide, Al(CH) 3 / H 2 O or O 3 o Wear and corrosion inhibiting layers [15] Ti(OC 2 H 5 ) 4 HfO 2 TiO 2 HfCl 4 / H 2 O TEMAH a / H 2 O TiCl 4 / H 2 O Ti(OEt) 4 b / H2 O Ti(O i Pr) 4 c o High-κ dielectric [16-19] o High-κ dielectric [19,20] o Photocatalysis [21] o UV blocking layer [22] o Photonic crystals [23] SiO 2 SiCl 4, / H 2 O o Dielectric [18,19,24] ZrO 2 ZnO SnO 2 Ta 2 O 5 La 2 O 3 ZnS ZrCl 4 / H 2 O Zr(O t Bu) 4 d / H2 O ZrI 4 / H 2 O 2 e ZnEt 2 / H2 O f Zn(OAc) 2 / H2 O SnCl 4 / H 2 O SnI 4 / O 2 TaCl 5 / H 2 O Ta(OEt) 5 g / H2 O La(thd) 3 h / O3 La[N(SiMe 3 ) 2 ] 3 i / H2 O ZnCl 2 / H 2 S o High-κ dielectric [18,19] o Photocatalysis o Wear and corrosion inhibiting layers o Piezoelectric layers o UV blocking layer [22] o Photocatalysis [21] o Photonic crystals [24] o Optical applications (solar cells, integrated optics, optical coatings, laser) [25] o o Anti-reflection and optical filters Sensors (gas sensors, ph sensors) [26,27] o Anti-reflection and optical filters [28] o Sensors (gas sensors, ph sensors) [28] o High-κ dielectric [28] o High-κ dielectric [29,30] o Piezoelectric layers o Optoelectronic applications [31,32] WS 2 WF 6 / H 2 S o Solid Lubricant layers [33] o Biomedical coatings( biocompatible Zr 3 N 4 Zr(NMe 2 ) j 4 / NH 3 materials for in-vivo medical devices and instruments) Ta 2 N TaCl 5 / H 2 O 5 Ta(OEt) 5 / H 2 O o Photonic crystals [34] TaN Ta(N t Bu)(NEt 2 ) k / NH 3 o Diffusion barrier for Cu metallization [35] c Ti(O i Pr) 4 : Titaniumisopropoxide, Ti[OCH(CH 3 ) 2 ] 4 d Zr(O t Bu) 4 : Tetrakisethyl methylaminozirconium, Zr[N(CH 3 )(C 2 H 5 )] 4 e ZnEt2 : Diethylzinc, Zn(C 2 H 5 ) 2 f Zn(OAc)2 : Zincacetatedihydrate, Zn(O 2 CCH 3 ) 2 (H 2 O) 2 g Ta(OEt)5 :Tantalumthoxide, Ta(OC 2 H 5 ) 5 i La[N(SiMe3 ) 2 ] 3 : Lanthanumtris[bis(trimethylsil yl)amide], C 18 H 54 LaN 3 Si 6 j Zr(NMe2 ) 4 : zirconium(tetra)dimethylamid e, Zr[N(CH 3 ) 2 ] 4 AlN AlMe 3 / NH 3 AlCl 3 / NH 3 TiN TiCl 4 or Til 4 / H 2 WN WF 6 / NH 3 CuCl / H 2 Cu(thd) 2 / H 2 Cu l Cu(acac) 2 / H2 Cu(hfac) 2 x H 2 O m / Ch 3 OH MoF 6 / H 2 Mo MoCl 5 / H 2 Mo(Co) 6 / H 2 Ni(acac) 2, 2 step process Ni NiO by O 3 reduced afterwards by H 2 Ta TaCl 5 / H 2 W WF 6 / B 2 H 6 or Si 2 H 6 Ti TiCl 4 / H 2 o Piezoelectric layers o Diffusion barrier for Cu metallization [36] o Conductive gate electrodes o Biomedical coatings (biocompatible materials for in-vivo medical devices and instruments) o Diffusion barrier for Cu metallization [36] o Optical application [37] h La(thd)3,where thd is 2,2,6,6,-tetramethyl-3,5- heptanedione, (CH 3 ) 3 CCOCH 2 COC(CH 3 ) 3 k Ta(NtBu)(NEt2 ) 3 : Tris(diethylamido)(tertbutylimido)tantalum, (CH 3 ) 3 CNTa[N(C 2 H 5 ) 2 ] 3, where N t Bu is called tertbutylimido group. l Cu(acac)2 :Copper(II) acetylacetonate, (C 5 H 7 O 2 ) 2 Cu m Cu(hfac)2 H 2 O: Copper(II) hexafluoroacetylacetonate hydrate, C 10 H 2 CuF 12 O 4. H 2 O

16 8 ALD General Table 1.2 Requirements for ALD precursors. The listed information in this table is summarized from references [2,3,7]. 1 Adequate volatility Requirement 2 Reasonable thermal stability Explanation Necessary for efficient transportation of the precursor to the ALD reactor at a rough limit of 0.1 Torr and at the deposition temperature. Thermal-decomposition can destroy the selflimiting film growth. 3 Aggressiveness, Appropriate reactivity Surface reaction should ensure fast completion. Short cycle times lead to high productivity. There should be no gas phase reaction. 4 No etching of the films or substrates No competing reaction pathways. Etching prevents the film growth. 5 No dissolution into the film or substrate Dissolution would destroy the self-limiting film growth. 6 Non-reactive volatile by-products Necessary to avoid corrosion. 7 Sufficient purity, Cost effectiveness, Easy handling and synthesis, Non-toxicity, Environmental friendliness 1.4 Reaction mechanisms of metal oxides by ALD a b c Figure 1.4 Chemical structure of TIP, TMA and DEZ. a, TIP. b, TMA. c, DEZ. Up to now, the basic principle of an ALD process and investigated ALD materials were briefly introduced. In the following, focusing on metal oxides deposited by a binary ALD reaction, more detailed reaction mechanisms of ALD film formation will be described, since the present work was performed with such ALD processes (in particular with Al 2 O 3, TiO 2 and ZnO). The oxides Al 2 O 3, TiO 2 and ZnO were deposited by trimethylaluminum (TMA)/water, titaniumisopropoxide (TIP)/water and diethylzinc (DEZ)/water precursor pairs, respectively. TMA, TIP and DEZ (Figure 1.4) were used as metal source and water was used as oxygen source. It is generally well accepted that, during ALD metal oxide growth, hydroxyl groups play an important role as intermediate species remaining on the surface of the deposited film after the water exposure [38-47]. During the subsequent

17 Chapter 1 9 exposure of metal containing precursors, the hydroxyl groups react with the incoming metal compounds, thereby becoming anchored to the surface as described by the following reaction. x( OH) + ML n (g) ( O ) x ML n-x + xhl(g) (R1) And ( O ) x ML n-x further react with water molecules during the next process step ( O ) x ML n-x + (n-x)h 2 O(g) ( O ) x M( OH) n-x + (n-x)hl(g) (R2), where blue ( )/red ( ) bar denote bonds between substrate surface and L (ligand) / M (metal) and L, respectively. On the other hand, not all of the hydroxyl groups ( OH) formed in (R2) do necessarily remain as such on the surface but some of them may react with other free water. 2( OH) ( O)+ H 2 O(g) (R3) This dehydroxylation increases with temperature causing a gradual decrease of the surface hydroxyl group density [40,42,43,45,47]. The amount of metal precursor anchored to the surface, and thereby the ALD growth rate, is determined either by the steric hinderance between the ( O ) x ML n-x surface species or by the density of the hydroxyl groups. Therefore, under conditions with extensive dehydroxylation, the hydroxyl group density may become a limiting factor with respect to the film growth rate. Hydroxyl groups may form on the surface also by rehydroxylation which is a reaction essentially reverse to the (R3). M O + H 2 O(g) M( OH) 2 (R4) O M O M + H 2 O(g) HO M O M OH (R5) The resulting hydroxyl groups are bound to the same (R4) or adjacent surface metal ions. Based on the simplified reaction scheme from (R1)-(R5), it is known that Al 2 O 3, ZnO and TiO 2 film are deposited as follows: (i) Al 2 O 3 deposition from the reaction of TMA/H 2 O [9,40,42,48,49]. A binary reaction for Al 2 O 3 chemical vapor deposition, 2Al(CH 3 ) 3 +3H 2 O Al 2 O 3 + 6CH 4 is separated into two half-reactions:

18 10 ALD General a, Al-OH* + Al(CH 3 ) 3 Al-O-Al-(CH 3 ) 2 * + CH 4 ; b, Al-O-Al-(CH 3 ) 2 * + H 2 O Al-O-Al-(CH 3 )OH* + CH 4, where the asterisks designate the surface species. TMA and H 2 O reactants were employed alternatively in an ABAB..binary reaction sequence to deposit Al 2 O 3 films. (ii) ZnO deposition from the reaction of DEZ/H 2 O [50-54]. Similar to the deposition of Al 2 O 3 from TMA/H 2 O, ZnO ALD is also based on a ZnO CVD process as follows Zn(CH 2 CH 3 ) 2 + H 2 O ZnO + 2C 2 H 6 ZnO ALD is proposed to occur by speration of this binary reaction into two half-reactions: a, Zn-OH* +Zn-(CH 2 CH 3 ) 2 Zn-O-Zn-(CH 2 CH 3 )* + C 2 H 6 ; b, Zn-O-Zn-(CH 2 CH 3 )* + H 2 O Zn-O-Zn-OH* + C 2 H 6, where the asterisks designate a surface species. Alternating ABAB..reaction sequence is repeated to deposit ZnO films. (iii) TiO 2 deposition from reaction of TIP/H 2 O [55-58]. Unlike Al 2 O 3 and ZnO, the TiO 2 deposition mechanism is rather complicated. The behaviour shows strong dependency on the processing temperature and is highly affected by decomposition of TIP. It is suggested that, below a temperature of 250 C, TiO 2 grows via the two following reactions ( denotes bonds between substrate surface) [58]: 2( OH) + Ti[OCH(CH 3 ) 2 ] 4 ( O ) 2 Ti[OCH(CH 3 ) 2 ] 2 + 2(CH 3 ) 2 CHOH (R6) ( O ) 2 Ti[OCH(CH 3 ) 2 ] 2 + 2H 2 O ( O ) 2 Ti( OH) 2 + 2(CH 3 ) 2 CHOH (R7) In the reaction, half of the ligands are released during the Ti[OCH(CH 3 ) 2 ] 4 pulse anchoring on the surface hydroxyl groups (R6). The H 2 O pulse hydrolyzes the rest of the ligands and converts the surface back to being hydroxyl group terminated (R7). At higher temperatures, surface dehydroxylation becomes more intense and thus there are less OH groups at the surface after the H 2 O pulse. Therefore, the mechanism is changing. Now, only a single isopropoxide ligand is released during the titanium precursor pulse (R8), the remainder of the ligands is released during the H 2 O pulse and the surface again becomes OH terminated (R9) as follows: ( OH) + Ti(OCH(CH 3 ) 2 ) 4 O Ti[OCH(CH 3 ) 2 ] 3 + (CH 3 ) 2 CHOH(g) (R8)

19 Chapter 1 11 O Ti[OCH(CH 3 ) 2 ] 3 + 2H 2 O ( O ) 2 TiOH + 3(CH 3 ) 2 CHOH(g) (R9) 1.5 Comparison of ALD and CVD As already mentioned above, ALD is a special case of CVD. Even though ALD uses a similar chemistry to CVD, the difference between them is large. CVD involes a chemical reaction which transforms vapor phase precursor molecules into solids, depositing as thin films or powder on the surface of a substrate. As illustrated in Figure 1.5, in CVD vaporized precursors with a constant pressure (Figure 1.6) are simulataneously delivered into a reactor with a carrier gas. The precursor molecules diffuse inside the reactor to the vicinity of a substrate surface. An adsorption of the diffused molecules on surface occurs, followed by a reaction yielding solid reaction products. Since the substrate temperature is critical and can influence the type of the reaction, the reactions are activated and maintained by heat, plasma, photons, electrons, ions or a combination thereof. Vapor phase reaction products are also formed and are removed from the reaction chamber. Figure 1.5 Schematic illustration of a CVD process. For details, see the text. In contrast, in ALD the precursors are not mixed and are introduced into the reactor sequentially. Thanks to the self-limitation as a consequence of the pulsed deposition scheme, the thickness control is performed as a function of cycles, whereas, in CVD the thickness is controlled by the processing time (e.g. nm/min). A common feature of CVD and ALD is that all surfaces exposed to the precursor vapor are coated. This means that films of uniform thickness can be produced on 3D

20 12 ALD General substrates. Basically, CVD is a gas phase reaction which can cause a particle deposition. In a CVD process, the life time of precursors is not long enough for precursor molecules to be transported and diffused on the complicated 3D substrates. Therefore, CVD is rather a line-of-sight deposition method and shadowing effects lead to non-uniformity of the films along a 3D substrate. As a result, one can expect better uniformity with ALD than with CVD. By adjusting the cycle time, in particular, exposure time, the film uniformity and conformality on complicated substrates can be maximized (Figure 1.7). Including the differences mentioned above, the other differences and special features of ALD and CVD are summarized in Table 1.3. a b Figure 1.6 Schematic partial pressure profiles of ALD and CVD during a process. In the case of ALD (figure a), since the film is deposited based on exposure/purge of each precursor, the partial pressure profile has a form of a square wave function. In contrast, in classical CVD (figure b) the precursors are introduced into a reactor at the same time, the pressure profile of each precursor is kept constant. In figure a, E and P denotes exposure and purge, respectively. The pressure profile of CVD (figure b) shown here is only valid for classical CVD, but not for pulsed CVD. a b Figure 1.7 Difference of coating behaviour of ALD and CVD. In ALD (figure a), thanks to the selflimiting reaction mechanism, an extremely uniform film can be deposited. In CVD (figure b), one can expect a very uniform film but practically the film is less uniform than ALD film. Since CVD is rather a line-of-sight deposition method, shadowing effects lead to non-uniformity of the films along a 3D sample.

21 Chapter 1 13 Table 1.3 Comparison of ALD and CVD. This data is summarized after extracting information in diverse books, research articles and internet web pages [2-5,7]. Criteria ALD CVD Uniformity Control o o o Å range Controlled by counting the number of reaction cycles Ensured by the saturation mechanism o o o 10Å range, Controlled by process control and monitoring, time Requires uniform flux of reactant and uniform temperature Film Quality o o o Excellent stoichiometry Low pinhole count Stress control possible o o o Excellent stoichiometry Low pinhole count Stress control possible Conformality 100% step coverage in 60:1 aspect ratio 100% step coverage in 10:1 aspect ratio Cleanliness No particles due to separated half reaction Particles due to gas phase reactions Vacuum Requirement Medium Medium Process Window Precursor Deposition reaction Contamination < 1% dependency on 10% process parameter changes o Highly reactive precursors o Precursors must not decompose at process temperatures Surface reaction 5 ~ 30 wt % (C, O). But with PE-ALD contamination can be minimized (< 1 wt %). Strong dependency on process parameter changes o Less active precursors o Precursors can decompose at process temperature Surface reaction + Gas phase reaction < 1 wt % 1.6 Multiple pulsed vapor phase infiltration ALD has been developed for the controlled-deposition of various kinds of thin films (such as oxides, nitrides, elemental compounds etc.) with control on the atomic or molecular level. Up to now, the mainstream of ALD research has focused on expanding the variety of materials which can be deposited in a very controlled way. To this end, scientists in the field of ALD have tried to add additional systems (such as plasma) to a conventional ALD setup to activate precursors properly or they have made an effort to develop new precursors to be easily applied to a conventional thermal ALD system. On the other hand, most of the materials have been deposited mainly on solid state substrates which are normally used in the field of microelectronics devices. As the interest in nanomaterials (such as nanopores, -wires, -tubes and laminates) increased, recently some new aspects of ALD were introduced to be applied to general nanofabrication or nanotechnology. There were reports on some examples of structuring using nano-sized organic materials or biomaterials based on template approaches performed by low temperature ALD processes

22 14 ALD General [5]. A common concern of most of those ALD researchers has concentrated on the resulting ALD film and the functionality of the deposited film itself. Figure 1.8. Schematic of the difference between a conventional ALD process and the MPI process. Conventionally, by multiple pulses of ALD precursors such as TMA (green arrows) and water (pink arrows), thin films (such as alumina, illustrated by the red shell) are deposited on rigid materials (such as metals) without chemical modification of the bulk. In contrast, in the case of soft materials such as biomaterials or polymers, ALD provides a chemical modification of the bulk in addition to the thin film deposition (MPI process). As introduced above, unlike to the case of other deposition methods, an ALD film is deposited by multiple pulses of two or three precursors (binary or ternary reaction) depending on the required film. For each precursor the exposure/purge step is repeated. Since most organic materials or biomaterials are sensitive to the highly reactive metal containing precursors, one could anticipate side effects caused by ALD. Few years ago, some groups reported that, in a case of a polymer substrate, the ALD film growth is differing to the growth on other solid substrates [59]. The ALD process changed the chemical structures of the polymer [11]. More recently even more convincing results, supporting the fact that ALD can modify physical/chemical properties of soft materials (such as biomaterials), were reported [6]. It was proven that metals can infiltrate biomaterials by the alternating exposure/purge step of multiple pulses of vapor phased

23 Chapter 1 15 precursors occurring during the ALD process (Figure 1.8), named as Multiple Pulsed Vapor Phase Infiltration (MPI). In this thesis, one example of ALD on a biotemplate (Chapter 3) and two examples of MPI processes to biomaterials are presented (Chapter 4 and 5).

24 16 ALD General

25 Chapter 2 17 Chapter 2 Basic Parameters in Mechanics Mechanics is one of the well-established branches of physics. Its main focus is to investigate the behaviour of physical bodies when subjected to forces or displacements, and the subsequent effects of the bodies on their environment. The discipline has its roots in several ancient civilizations successively deveploping by learning from experience. During the early modern period, scientists such as Galileo, Kepler, and especially Newton, established the so called classical mechanics. In this chapter, several basic parameters required for understanding the mechanical behaviour of diverse physical bodies under the influence of outside force environmental conditions will briefly be introduced. Most of the contents described in this chapter are based on classical text books dealing with mechanics of materials [60-65]. 2.1 Stress and strain When a solid body with a given volume is subjected to an external force (in particular, tensile force), the material will typically elongate in the direction of the applied force. The relative elongation is called strain (often denoted by ε). Usually, this elongation leads to a contraction of the material in the direction perpendicular to the applied force, by a relative amount νε, where the coefficient ν is called Poisson ratio. For an isotropic piece of material, the relative increase of the volume during uniaxial stretching is 1-2ν which means that the Poisson ratio has an upper limit of ν 0.5, because the specimen volume is not expected to shrink under the influence of tensile forces. The load divided by the

26 18 Basic Terminologies in Mechanics surface area A is called stress (units: Pascal [Pa]). Generally, stresses and strains are not just uniaxial and need to be described by a tensor. a b c Figure 2.1 Tensile stress and shear stress. When the cubic piece of material (figure a) is subjected to tensile stress along the y-direction only, its length L is increased by Lε. The relative elongation ε is called (tensile) strain. In most cases, the dimension of the cube subjected to tensile load will contract perpendicularly to the load direction (figure b). The ratio ν of the contraction in the z-direction (or x- direction) relative to the elongation in y-direction is called the Poisson ratio. When the load is tangential to the top surface, shear deformation occurs (figure c). The shear is measured by the parameter γ, which (for small deformations) corresponds to the tilting angle of the cube edge initially parallel to the y-deformation. 2.2 Stress - strain curve A. Engineering stress-strain curve. Perhaps the most common test of a material s mechanical response is the tensile test, in which one end of a rod or wire specimen is clamped in a loading frame and the other subjected to a controlled displacement δ = (L-L 0 ) (see Figure 2.2). The engineering measures of stress and strain, denoted in this module as σ e and ε e respectively, are determined from the measured load and deflection using the original specimen cross-sectional area A 0 and length L 0 as σ e = F/A 0 and ε e = δ/l 0, respectively. In the low strain part of the curve, many materials obey Hooke s law to a reasonable approximation, so that stress is proportional to strain with the constant of proportionality being the modulus of elasticity or Young s modulus, denoted by E (= ε e /σ e ) σ e = Eε e (2.1)

27 Chapter 2 19 Figure 2.2 Schematic drawing of a tensile test and a stress-strain curve of a ductile material. By pulling on a specimen, the material s reponse to forces being applied in tension is determined. When the stress is plotted against the strain, a stress-strain curve is obtained. For the explanation, the stressstrain curve of a ductile material is exemplified. The nature of the curve varies from material to material. The stress strain behaviour of typical materials is illustrated in terms of the engineering stress (σ e ) and the engineering strain (ε e ) where the stress and strain are calculated based on the original dimensions of the specimen. The stress value calculated from instantaneous values of the specimen dimension is called true stress and the corresponding stressstrain curve is called true stress (σ t )-strain (ε t ) curve. C1: true stress (σ t )-strain (ε t ) curve; C2: engineering stress (σ e )-strain (ε e ) curve; R1: elastic deformation region (reversible); R2: strain hardening region (permanent deformation); R3: necking region (permanent deformation); P1: yielding point (σ y,ε y ), limit of the elastic region; P2: ultimate tensile strength (UTS), onset point of necking; P3: fracture point (σ f,ε f ); P4: proportional limit; E: Young s modulus, stiffness or modulus of elasticity. As strain is increased, many materials eventually deviate from this linear proportionality, the point of departure being termed the proportional limit. This nonlinearity is usually associated with stress-induced plastic flow in the specimen. Here the material is undergoing a rearrangement of its internal molecular or microscopic structure, in which atoms are moved to new equilibrium positions. This plasticity requires a mechanism for molecular mobility, which inside crystalline materials can arise from dislocation motion. Materials lacking this mobility, for instance by having internal microstructures that block dislocation motion, are usually brittle rather than ductile. The stress-strain curve for brittle materials is typically linear over the full range of strain, eventually terminating in fracture without significant plastic region. The stress needed to increase the strain beyond the proportional limit in a ductile material continues to rise beyond the proportional limit; the material requires an ever-increasing stress to continue straining, a mechanism termed strain hardening. These microstructural rearrangements associated with plastic flow are usually not reversible, even if the load is removed, so the

28 20 Basic Terminologies in Mechanics proportional limit is often the same as or at least close to the materials elastic limit. Elasticity is the property of complete and immediate recovery from an imposed displacement on release of the load, and the elastic limit is the value of stress at which the material experiences a permanent residual strain that is not lost on unloading. The residual strain induced by a given stress can be determined by drawing an unloading line from the highest point reached on the stress-strain curve back to the strain axis, drawn with a slope equal to that of the initial elastic loading line. This is done because the material unloads elastically, with no required force driving the molecular structure back to its original position. A closely related term is the yield stress, denoted σ Y in these modules; this marks the stress needed to induce plastic deformation of the specimen. Since it is often difficult to pinpoint the exact stress at which plastic deformation begins, the yield stress is often taken to be the stress needed to induce a specified amount of permanent strain, typically 0.2%. For some materials (e.g., metals and plastics), the departure from the linear elastic region cannot be easily identified. Therefore, an offset method to determine the yield strength of the material tested is allowed. These methods are discussed in ASTM E8 (metals) and D638 (plastics). An offset is specified as a percentage of strain (for metals, usually 0.2% and sometimes for plastics a value of 2% is used). The construction used to find this offset yield stress is shown in Figure 2.2 in which a line of slope E is drawn from the strain axis at ε e = 0.2%; this is the unloading line that would result in the specified permanent strain. The stress at the point of intersection with the σ e -ε e curve (P1) is the offset yield stress. The rate of strain hardening diminishes up to a point labeled ultimate tensile strength (UTS). Beyond that point, the material appears to soften, so that each increment of additional strain requires a smaller stress. The apparent change from strain hardening to strain softening is an artifact of the plotting procedure, however, as is the maximum observed in the curve at the UTS. Beyond the yield point, molecular flow causes a substantial reduction in the specimen cross-sectional area A 0, so the true stress (σ t = F/A actually borne by the material is larger than the engineering stress computed from the original cross-sectional area (σ e = F/A 0 ). The load must equal the true stress times the actual area (F = σ t A). As long as strain hardening can increase σ t enough to compensate for the reduced area A, the load and therefore the engineering stress will continue to rise as the strain increases. Eventually, the decrease in area due to flow becomes larger than the increase in true stress due to strain hardening, so the load begins to fall. This is a geometrical effect. If the true stress rather than the engineering stress were plotted, no maximum in the curve would be observed. At the UTS the differential of the load F is zero, giving an analytical relation between the true stress and the area at necking as follows:

29 Chapter 2 21 df da = d A df d A da - dσ t ( σ t ) = 0 = σ t + σ t = (2.2) A σ t The last expression states that the load and the engineering stress will reach a maximum as a function of strain when the fractional decrease in area becomes equal to the fractional increase in true stress. Figure 2.3 Necking in a tensile specimen. Under the uniaxial tensile load the aluminum specimen starts to deform and reaches necking. Finally cup and cone shaped fracture occurs (source: [65]. Even though the UTS is perhaps the materials property most commonly reported in tensile tests, it is not a direct measure of the material due to the influence of geometry as discussed above, and should be used with care. The yield stress σ Y is usually preferred to the UTS in designing ductile metals, although the UTS is a valid design criterion for brittle materials which do not exhibit these flow-induced reductions in cross-sectional area. The true stress is not quite uniform throughout the specimen, and there will always be some location-perhaps a nick or some other defect at the surface - where the local stress has a maximum. Once the maximum in the engineering curve has been reached, the localized flow at this site cannot be compensated by further strain hardening, so this area is further reduced. This increases the local stress even more, which further accelerates the flow. This localized and increasing flow soon leads to a neck in the gage length of the

30 22 Basic Terminologies in Mechanics specimen such as the one seen in Figure 2.3. Until the neck forms, the deformation is essentially uniform throughout the specimen, but after necking all subsequent deformation takes place in the neck. The neck becomes smaller and smaller with the local true stress increasing all the time, until the specimen fails. This will be the failure mode for most ductile metals. As the neck shrinks, the non-uniform geometry there alters the uniaxial stress state to a complex one, involving shear components as well as normal stresses. The specimen often fails finally with a cup and cone geometry as seen in Figure 2.3, in which the outer region fails in shear and the interior in tension. When the specimen fractures, the engineering strain at break (fracture strain, ε f ) will include the deformation in both the necked and the unnecked region. Since the true strain in the neck is larger than that in the unnecked material, the value of ε f will depend on the fraction of the gage length that has necked. Therefore, ε f is a function of the specimen geometry as well as the material, and thus is only a crude measure of material ductility. A. True stress-strain curve. As discussed above, the engineering stress-strain curve must be interpreted with care beyond the elastic limit, since the dimension of the specimen s cross section experiences a substantial change from its original value. Using the true stress σ t = F/A rather than the engineering stress σ e = F/A 0 can give a more direct measure of the material s response in the plastic flow range. A measure of strain often used in conjunction with the true stress takes the increment of strain to be the incremental increase in displacement dl divided by the current length L as follows L dl 1 L dε t = ε t = dl = In L (2.3) L L L 0 0 This is named as the true or logarithmic strain. During yield and the plastic-flow regime following yield, the material flows with negligible change in volume; increases in length are offset by decreases in cross-sectional area. Prior to necking, when the strain is still uniform along the specimen length, this volume (V) constraint can be written: A L = constant dv = 0 AL = A0 L0 = A L V 0 0 (2.4) The ratio L/L 0 is defined as extension ratio (λ). Using these relations, the relation between true and engineering measures of tensile strain can be developed as follows

31 Chapter 2 23 L L0 + dl ε t = In = In = In(1+ ε e ) = Inλ L L 0 0 (2.5) The relation between true and engineering measure of tensile stress can be also derived as follows L A0 1 + ε e = = (2.6) L A A0 A = 1 + ε e 0 F F(1 + ε e) σ t = = = σ e(1 + ε e) = σ eλ A A 0 (2.7) (2.8) These equations can be used to derive the true stress-strain curve from the engineering curve, up to the strain at which necking begins. Beyond necking, the strain is non-uniform in the gage length and computing the true stress-strain curve for greater engineering strains would not be meaningful. However, a complete true stress-strain curve could be drawn if the neck area was monitored throughout the tensile test, since for logarithmic strain we have the following relation: L L 0 A0 A L A In = Inλ = In = ε t (2.9) L A = Strain energy, breaking energy and toughness The area under the σ e ε e curve up to a given value of strain is the total mechanical energy per unit volume consumed by the material in straining it to that value. This is easily shown as follows: U 1 = V 1 A L FdL = FdL = = 0 0 L 0 L F A dl L ε 0 σ dε e e (2.10) In the absence of molecular slip and other mechanisms for energy dissipation, this mechanical energy is stored reversibly within the material as strain energy. When the stresses are low enough, so that the material remains in the elastic range, the strain energy

32 24 Basic Terminologies in Mechanics is just the triangular area in Figure 2.4a. In the elastic region, the strain energy can be described as follows: U ε ε e = σ edε e = Eε edε 1 e = Eε σ e = 2 E (2.11) a b c d Figure 2.4 Schematic description of strain energy. When the applied load is low enough for the materials to remain in an elastic region, the strain energy is equal to the triangular area shown in figure a. Even if the amount of the increased strain is the same, depending on the position of the initial strain, the corresponding strain energies vary highly as shown in figure b. Figure c shows the difference between resilience and toughness. Figure d illustrates energy loss caused by plastic deformation. As can be recognized from equation (2.11), the strain energy increases quadratically with the stress or strain. As the strain increases, the energy stored by a given increment of additional strain grows as the square of the strain. This has an important physical meaning. For example, an archery bow cannot be simply a curved piece of wood to work well. A real bow is initially straight, then bent when it is strung; this stores substantial strain energy. When it is bent further on drawing the arrow back, the energy available to throw the arrow is very much greater than if the bow was simply carved in a curved shape without actually bending it. Figure 2.4b shows schematically the amount of strain energy available for two equal increments of strain Δε, applied at different levels of existing strain. The area up to the yield point is termed the modulus of resilience, and the total area up to

33 Chapter 2 25 fracture is termed the modulus of toughness (Figure 2.4c). The term modulus is used because the units of strain energy per unit volume are Nm/m 3 or N/m 2, which are the same as stress or modulus of elasticity. The term resilience alludes to the concept that up to the point of yielding, the material is unaffected by the applied stress and upon unloading will return to its original shape. When the strain exceeds the yield point, the material is deformed irreversibly, so that some residual strain will persist even after unloading. The modulus of resilience is then the quantity of energy the material can absorb without suffering damage. Similarly, the modulus of toughness is the energy needed to completely fracture the material. Materials showing good impact resistance are generally those with a high modulus of toughness. During loading, the area under the stress-strain curve is the strain energy per unit volume absorbed by the material. Conversely, the area under the unloading curve is the energy released by the material. In the elastic range, these areas are equal and no net energy is absorbed. But if the material is loaded into the plastic range as shown in Figure 2.4d, the energy absorbed exceeds the energy released and the difference is dissipated as heat. 2.4 Stiff materials, strong materials and tough materials The elastic modulus (E, Young s modulus) describes the stiffness of a certain material. It defines the resistance against deformation when the material is subjected to a given mechanical load. Stiff materials are needed to transmit forces and to resist deformation. Stiffness is especially crucial for transmitting forces when the material is loaded in bending and it is also required for resistance against buckling when a bar is loaded in compression along its axis. But the stiffness is by no means the only critical mechanical property. The strength of a material is defined as the maximum stress it can sustain before breaking. The strength is often defined by the fracture stress, σ f. High strength is needed to allow carrying a high load. Technical materials used in construction are usually both stiff and strong. There is, however, a subtle but essential difference in how these properties are affected by defects in a homogeneous material. This may be understood by the weak link fracture model (Figure 2.5), the calculation by this model shows that if one of 100 identical elements in a chain is replaced by a link with half the stiffness and half the strength, the overall stiffness of the chain is reduced by only 1% but the overall strength is reduced by 50%. This means that the stiffness (as a bulk property) is hardly influenced by small defects while the strength depends heavily on local properties and on defects. As a consequence, the

34 26 Basic Terminologies in Mechanics strength of ceramics is almost completely controlled by the size and the amount of defects in the material, to an extent, where the strength becomes a statistical property of the ceramic (depending of the defect distribution) rather than an intrinsic one. Figure 2.5 Weak link problem in a chain. This figure is redrawn from reference [62]. Figure 2.6 Materials characteristics in terms of mechanical properties. This dependence on defects and material inhomogeneities is even worse for yet another crucial material property, the toughness. The toughness is linked to the energy needed to propagate a crack through the material and to break it. The larger the energy needed, the tougher the material is. Brittle ceramics, for example, have a very low toughness, but are typically very stiff. Indeed, a major way for a material to dissipate energy in an impact is to deform rather than to fracture. Therefore, many materials which deform easily are also tough, while very stiff materials (such as ceramics) have a higher chance to be brittle. This is a major dilemma in materials design, since both stiffness and

35 Chapter 2 27 toughness are needed for many applications. The basic difference between stiff, strong and tough materials is graphically illustrated in Figure Elastic deformation and plastic deformation Figure 2.7 Material s deformation behaviour when the load is removed after deformation. When the external load is removed after a certain deformation, an elastic material immediately recovers its initial shape. A plastic material keeps the deformed shape. Materials often have a combination of those properties: Elastoplastic materials relax partially and retain only part of the deformation. In the middle figure, when L 2 is equal to L 1, the material s behaviour is perfectly plastic. When L 1 is larger than L 2, the material s behaviour is elastoplastic. Similarly, a viscoplastic material loses gradually a part of the deformation but a fraction of it remains forever. For elastic materials, σ- ε curves measured by loading and unloading have same profile. If this line is non-linear (as often observed in polymeric materials), it is called non-linear elastic behaviour. Close to the origin (near zero stress), nearly all materials are linear elastic. For plastic materials, a residual or permanent deformation remains forever after sufficiently large deformation. For viscoelastic materials, the stress-strain curve follows a hysteresis loop (gray colored area). This area shows the amount of energy lost (as heat) in a loading and unloading cycle. The energy can be calculated by the integral σdε. Δε p denotes permanent (or residual) strain caused by plastic deformation. Δε e denotes elastic strain after plastic deformation. The general mechanical behaviour of most materials can be roughly and easily estimated by measuring the stress (σ)-strain (ε) curve. The curve provides all necessary information. Figure 2.7 shows some examples for different types of mechanical behaviour, when the material is subjected to tensile stress. Typically the stress increases first linearly, with increased strain. For larger strain, this linearity is not necessarily conserved. When the

36 28 Basic Terminologies in Mechanics external stresses producing deformation do not exceed a certain limit and the removal of external forces resumes the initial form of the materials, the mechanical behaviour of those materials is called elastic. In the special case where the σ-ε curve is linear, the material is linear elastic. In many cases, materials show a linear elastic behaviour at small deformations, but do not return to their initial shape when the external stress exceeds a critical value (often the value is called yield stress). Such a material behaviour is called plastic or permanent deformation. a b Figure 2.8 Creep and stress relaxation of viscoelastic materials. Unlike other pure elastic and plastic materials, viscoelastic materials have unique creep and stress relaxation features. Unlike elastic and plastic materials, there are also materials which after removal of external load restore the initial shape, but return along different pathway with elastic materials (leaving hysteresis) as shown in Figure 2.7. Those are called viscoelastic materials and include many biological materials, such as spider silk and collagen. Unlike other purely elastic and purely plastic materials, viscoelastic materials have an elastic component and a viscous component. They exhibit both viscous and elastic characteristics when undergoing deformation under the external load. Viscous materials, like honey or lubricant oil, resist shear strain linearly with time when a stress is applied. Elastic materials deform instantaneously when stretched and just as quickly return to their original state once the external load is removed. Viscoelastic materials have elements of both of these properties. Whereas elastic behaviour of materials is usually the result of bond stretching along crystallographic planes of an ordered solid, viscoelasticity is caused by the molecular arrangement, such as the diffusion of atoms or molecules inside of an amorphous material.

37 Chapter 2 29 The viscosity of a viscoelastic material gives the materials a strain rate dependent on time. When a load is applied and then removed, purely elastic materials do not dissipate energy (heat) as illustrated in Figure 2.7. In contrast, a viscoelastic material loses energy when a load is applied and then removed. Hysteresis is observed in the σ-ε curve. For example, when a stress is applied to a viscoelastic material such as a polymer, parts of the long polymer chain change their position. This movement or rearrangement induces specific features of viscoelastic materials such as creep and stress relaxation as illustrated in Figure 2.8.

38 30 Basic Terminologies in Mechanics

39 Chapter 3 31 Chapter 3 Metal Oxide Deposition on Biotemplate: macroporous photocatalytic TiO 2 or ZnO membranes templated from chicken s eggshell matrices Macroporous ZnO membranes with a strong photocatalytic effect and high mechanical flexibility were prepared from inner shell membranes (ISM) of avian eggshells as templates after performing low-temperature ZnO atomic layer deposition (ALD). In order to evaluate the potential merits and general applicability of the ZnO structures, a comparative study of two membranes with coatings of either TiO 2 or ZnO, processed under similar processing conditions, was performed. The study includes crystallographic features, mechanical and thermal stability and bactericidal efficiency. Both, the ZnO and the TiO 2 coated membranes clearly exhibited bactericidal effects as well as mechanical flexibility and thermal stability even at relatively high temperatures. The ZnO membranes, even though prepared at fairly low temperatures (~100 C), exhibited polycrystalline phases and showed a good bactericidal efficiency as well as higher mechanical flexibility than the TiO 2 coated membranes. This study shows the benefits of low-temperature ZnO ALD i.e., the thermally non-destructive nature, which preserves the mechanical stability and the native morphology of the templates used, together with an added functionality, i.e. the bactericidal effect.

40 32 Photocatalytic Metal Oxide Membrane 3.1 Background Historical background of photocatalysis In 1839, the French physicist A. E. Becquerel observed that under light illumination an electric current is generated between two AgCl or AgBr coated platinum electrodes immersed in acidic solution [66]. Photoelectrochemistry is the field studying the interaction of light with electrodes. Although Becquerel s investigations were primarily motivated by photography, his article, dealing with the photoelectric effect, has been recognized as the first report in the field of photoelectrochemistry. Becquerel has also been considered as founder of semiconductor photoelectrochemistry since some of his observations are now known to be due to the semiconducting nature of silver halides he used in some experiments. However, scientists did not pay too much attention to the understanding of the phenomena observed by Becquerel until the second half of the 20 th century. In the meantime, in 1947 the first transistor was discovered. This discovery highly motivated the development of photochemistry and the relevant fundamental studies, such as the electron transfer theory by Marcus [67-73]. In the 1960 s, the photoelectrochemical investigations of further semiconducting materials such as TiO 2, ZnO, CdS, ZnS, CdSe, ZnSe, ZnTe, GaAs, GaP, and KTaO 3 were continued [74-81]. Although the photoinduced effect of semiconductors, such as Al 2 O 3 [82], MgO [8282], SiO 2 [82], CdO [82], ZnO [82], CeO 2 [82,83] and TiO 2 [82-88], in chemical reactions was reported much earlier, the practical application of semiconductor photoelectrochemistry was not well recognized. In the 1972, Fujishima and Honda reported pioneering results on electrochemical photolysis, describing that water can be split into hydrogen and oxygen using a semiconducting (rutile TiO 2 ) photoelectrode [89]. Their work promised the utilization of semiconducting materials for solar energy conversion and storage. In those days, the oil price ballooned suddenly associated with an oil embargo and oil crisis in about It is self-explaining that Fujishima and Honda s report attracted much attention not only of electrochemists but also many scientists working in other fields. Subsequently, further photovoltaic applications were introduced [90-92], such as solar cells [91]. Semiconductors have been also successfully employed in the field of photocatalysis, such as photocatalytic organic synthesis [93], photofixation of dinitrogen [94], photoreduction of carbon dioxide [95], decarboxylation of carboxylic acids [96], anti-tumor medical applications [97,98] and photooxidative reactions using oxygen from air for the removal of pollutants from ambient air [99-103].

41 Chapter Principle of photocatalysis and applications By definition, catalysis accelerates the chemical transformation of a reactant, itself remaining unaltered at the end of each catalytic cycle. The photocatalyst, with assistance of a photon, accelerates the photoreaction by interaction with the reactant in its ground or excited state and/or with a primary photoproduct, depending on the mechanism of the photoreaction. Figure 3.1 Schematic illustration of the general mechanism in photocatalytic reaction occurring on a semiconducting metal oxide photocatalyst. The detailed explanation can be found in the text. The abbreviations are as follows, CB: conduction band; VB: valence band; MOS: metal oxide semiconductor; E F,n : Fermi level of a n-type semiconductor; E F,p : Fermi level of a p-type semiconductor; A: acceptor; D: donor. Note that photocatalysis is one of the diverse semiconductor applications, which use an electron transfer phenomenon occurring at the interface between semiconducting materials and aqueous media. Fundamentally, in all applications of semiconductors, under illumination the semiconductor generates and separates charges which can subsequently undergo redox * (reduction + oxidation) reactions with substrates or induce a photocurrent. Such materials must typically have a sufficiently low bandgap to utilize solar light, but at * The terminology redox comes from the two words of reduction and oxidation. It can be explained in simple terms as follows: Oxidation: Reduction: Loss of electrons or increase in oxidation state of a molecule, atom or ion. i.e. Reductant Product + e - Gain of electrons or decrease in oxidation state of a molecule, atom or ion. i.e. Oxidant + e - Product

42 34 Photocatalytic Metal Oxide Membrane the same time the bandgap must be large enough to generate the photovoltage necessary for the activation of catalysis. For example, when in an aqueous medium the surface of a photocatalyst (such as TiO 2 or ZnO) is irradiated with light consisting of wavelengths shorter than the wavelength corresponding to the energy of its bandgap, (about 415 nm ev), the semiconductor absorbs photons with energy equal or higher than its bandgap energy. The absorbed energy is used to delocalize a valence band electron and to excite it to the conduction band of the semiconducting material. The holes formed in the valence band function as powerful oxidizing agents, thereby catalyzing some chemical reactions. Furthermore, these photoexcited charge carriers can initiate the degradation of the absorbed chemical species such as pollutants. Notwithstanding continuing research, the exact mechanism for degradation of the organic compounds adhered to the photocatalyst remains unclear except the fact that both hole and electron transfer processes are critical for the overall degradation process. Essentially, the metal oxide semiconductors immersed in liquid can be regarded as electrodes held at the open-circuit potential at which the anodic and cathodic currents are equal because two redox reactions simultaneously occur at different sites of the semiconductor an oxidation of an electron donor by a photogenerated hole and a reduction of an electron acceptor by a photogenerated electron [ ]. The key steps of a photocatalytic reaction at a semiconductor are illustrated in Figure 3.1. Upon photon absorption an electron-hole pair is generated (S1). The photogenerated charges an electron in the conduction band and a hole in the valence band can either directly recombine (primary recombination, S2), or be trapped at reactive surface sites (S3). In the case of TiO 2, trapping of holes proceeds in ns. This process is faster for electrons and requires some hundreds of picoseconds [104]. After being trapped at surface sites the charge carriers can again either recombine (secondary recombination, S4), or undergo an interfacial electron transfer process, whereby the electron reduces an electron acceptor species A to a primary reduction product A - (S5), and the hole oxidizes an electron donor species to D + (S6). The primary redox products A - (S5) and D + (S6) must then undergo a rapid conversion to the final products A re and D ox (S7 and S8) in order to avoid a reverse primary and secondary electron transfer (S9) (i.e., oxidation of A - by a reactive hole or reduction of D + by a reactive electron). Hence, in a typical photocatalytic oxidation of, for example, organic water pollutants on photocatalysts such as TiO 2 [ ] and ZnO [107], reacting holes are scavenged either directly by the pollutant or by adsorbed hydroxyl ions to produce hydroxyl radicals which then oxidize the pollutant due to their oxidizing potential. Simultaneously, the photogenerated electrons reduce molecular oxygen to a superoxide radical which can undergo further reactions to produce hydroxyl radicals via following reactions [104,105,107]:

43 Chapter 3 35 Semiconductors (such as TiO 2 or ZnO) + hv semiconductors (e CB - + h VB + ) (R1) h VB + + OH - OH (R2)* e CB - + O 2 O 2 - (R3) O H + HO 2 (R4) HO 2 + HO 2 H 2 O 2 + O 2 (R5) O HO 2 HO O 2 (R6) HO H + H 2 O 2 (R7) H 2 O 2 + O 2 - H 2 O 2 + e CB - OH + OH - + O 2 OH + OH - (R8) (R9) OH + Pollutants + O 2 - Products (CO 2, H 2 O, etc.) (R10) *The reaction (R2) is called indirect hole transfer mechanism. i.e. the photogenerated hole oxidizes an OH group on the surface of TiO 2 or ZnO to form OH. In the literature, a direct oxidation of adsorbed substances by a free hole has also been suggested to account better for some experimental observations. In this case, the holes react directly with an organic substance. Until now, researchers have used photocatalytic oxidation to break down and destroy many types of organic pollutants. It has been used to purify drinking water, destroy bacteria and viruses, remove metal ions from waste streams, and breakdown organics into simpler components of water and CO 2. Furthermore, photocatalysis has been used for removing nuisance color, taste and odor compounds and cleaning up polluted air streams. Based on these fundamental investigations in real life, photocatalysts have already been applied in cars for antibacterial purposes and to clean air. In Japan, there is a considerable number of cars with built-in photocatalysts. In hospitals, photocatalysts have been used to sterilize sickbeds and appliances. Similar use is performed in everyday life. The relevant products are gargle, toothpaste, chewing gum, contact lenses or cleaning chemicals. With increase of environmental protection consciousness and science's advance, photocatalytic materials will gain even more importance Crystal structure of TiO 2 and ZnO TiO 2. Titanium dioxide is typically found in one of its three main crystal structures: rutile (tetragonal), anatase (tetragonal) or brookite (orthorhombic). Therein, anatase and rutile are commonly used for photocatalysis, with anatase showing a higher photocatalytic activity [108]. The structure of anatase and rutile can be described in terms of chains of TiO 6 octahedra. The two crystal structures differ by the distortion of each octahedron and by the assembly pattern of the octahedral chains. Figure 3.2a and b illustrate the unit cell

44 36 Photocatalytic Metal Oxide Membrane structures of rutile and anatase crystals [109,110]. Each Ti 4+ ion is surrounded by an octahedron of six O 2- ions. The octahedron in rutile is not regular, showing a slight orthorhombic distortion. The octahedron in anatase is significantly distorted so that its symmetry is lower than orthorhombic. Ti-Ti distances in an anatase crystal (3.79 Å and 3.04 Å) are greater than the distances in rutile (3.57 Å and 2.96 Å). Whereas, the Ti-O distances in anatase (1.934 Å and Å) are shorter than in rutile (1.949 Å and Å). In the rutile structure each octahedron is in contact with 10 neighboring octahedrons (two sharing edge oxygen pairs and eight sharing corner oxygen atoms, while in the anatase structure each octahedron is in contact with eight neighbors (four sharing an edge and four sharing a corner). These differences in lattice structures cause different densities and electronic band structures between the two forms of TiO 2 as indicated in Figure 3.2a and b. a b c d Figure 3.2 Crystal structure of titanium dioxide and zinc oxide. a, Rutile phase TiO 2. b, Anatase phase TiO 2. c, Wurtzite phase ZnO. d, Zincblende phase ZnO. ZnO. Zinc oxide exists in three different crystal structures, i.e. hexagonal, wurtzite, cubic zincblende, and the rarely observed cubic rocksalt [111]. ZnO is thermodynamically stable in the wurtzite phase due to the effect that the bonding class is exactly at the borderline between covalent and ionic. The lattice constants of wurtzite mostly range from to Å for parameter a and from to Å for parameter c (see Figure 3.2c). The c/a ratio varies in a slightly wider range, from to The deviation from

45 Chapter 3 37 that of the idea wurtzite crystal is probably due to the lattice stability and electronegativity. A number of studies have been addressed to epitaxial wurtzite ZnO, while a few experimental and theoretical investigations are performed on the metastable zincblende ZnO growth and its fundamental properties. Zincblende materials have lower ionic character compared to wurtzite materials that has been related to the c/a ratio, indicating that zincblende materials are covalent. In the zincblende and wurtzite structures, each Zn (or O) has four nearest neighbors: the in-plane bonds are stronger, as indicated by higher electron density, than the out-of-plane bonds. In contrast to the zincblende/wurtzite structures, in the rocksalt structure each Zn (or O) has six nearest neighbors Escherichia coli (E. coli) bacteria Figure 3.3 Escherichia coli (abb. E. coli) bacteria. Escherichia coli (E. coli, Figure 3.3) are a large and diverse group of bacteria. The German-Austrian pediatrician and bacteriologist, Theodor Escherich, discovered E.coli from the intestines of children. E. coli was named after him in E. coli are Gramnegative bacteria, which have typically a rod-shape with a size around 2-3 μm in length Gram-negative bacteria are simply named because of their detection by the Gram s stain test in which they do not retain crystal violet dye in their cell wall. The Gram-negative bacteria cell-wall holds the pink or reddish dye once a counterstain chemical is used. On the other hand, Gram-positive bacteria retain the crystal violet dye when washed in a decolorizing solution. In terms of microbiology, from the cell wall structure Gram-positive- and negative- bacteria are classified. Gram-negativebacteria shows the following features: (1) Thin peptidoglycan layer, in the case of Gram-positive bacteria, the layer is much thicker; (2) Cytoplasmic membrane; (3) Outer membrane containing lipopolysaccharide outside the peptidoglycan layerl; (4) Porins exist in the outer membrane, which act like pores for particular molecules. Almost 90 ~95 % of Gram-negative bacteria are pathogenic, meaning that they can cause disease in a host organism. This pathogenic capability is usually associated with certain components of Gram-negative cell walls. For further detailed information, see the microbiology online lecture in University of South Carolina (

46 38 Photocatalytic Metal Oxide Membrane and 500 nm in diameter [112]. E. coli is one of the main species of bacteria found in the intestines of mammals. It is usually non-pathogenic, and can be easily isolated and grown in the laboratory. Although most strains of E. coli are harmless, some are harmful. Those can cause diarrhea, urinary tract infections, respiratory illness and pneumonia, or further illnesses. Harmless types of E. coli are even used as markers for water contamination. 3.2 Introduction During the evolution of biological creatures, numerous micro- and nanostructures with specific functionalities developed for adaptation to environmental conditions. Adoption of such structures by mimicking or templating came into focus in science in recent years [113]. Functionalization of structures by coating biological templates is one of the methods to produce more stable organic or inorganic micro/nanostructures. So far those coatings have been performed mainly by chemical vapor deposition and sol-gel strategies on various biotemplates such as, cellulose [114], wool [115], butterfly wings [116], superhydrophobic plant leaves [116] and pine wood [117]. However, these methods have some limitations in processing, such as occasional non-uniform coating of large templates or demanding film thickness control [118,119]. a b c Figure 3.4 Structure of an avian egg and scanning electron micrographs (SEM) of an inner shell membrane (ISM) of a chicken s eggshell membrane (ESM). a, Details of an avian egg and photograph of the ISM from ESM around the air cell of the egg. b, Low magnification SEM of an ISM viewing from the direction of the blue arrow shown in a. c, Magnification of several ISM fibers, showing their highly interwoven and conglutinate feature. As a promising method to overcome these processing limitations, ALD has recently attracted attention. Advantages of ALD are the conformal replication of 3D morphologies, large area uniformity, precise film thickness control on the nanometer scale and a wide

47 Chapter 3 39 range of operation temperatures [ ]. The feasibility of ALD for biological templates [ ] as well as for organic materials [127,128] has already been proven. However, the primary focus of ALD researchers was on the perfect coating of the fine structures of biotemplates with functional metal oxides such as TiO 2 [123,124] and Al 2 O 3 [ ]. Research, focusing on the optimal combination of the original functionality of the biotemplate itself and an appropriate metal oxide which can maximize the functionality of the resulting structures, has rarely been undertaken. Moreover, the mechanical stability as guarantee for easy handling and practical use has seldomly been considered. Here, an example which satisfies the above requirements is presented. Avian eggshell membranes (ESM, Figure 3.4) are processed by low temperature ALD. Those membranes were already previously used as templates for sol-gel [ ] or further deposition methods [ ]. In the present study the macroporous inner shell membrane (ISM) which is a part of an avian ESM (Figure 3.4) was used. It prevents bacterial invasions, thus protecting the embryo [ ]. TiO 2 or ZnO were deposited by ALD on this ISM, both of which show bactericidal photocatalytic effects under UV illumination (ISM/TiO 2 and ISM/ZnO) [138,139]. The bactericidal properties of those membranes were investigated and characterized quantitatively using a photocatalytic reaction which inactivated Escherichia coli (E. coli) bacteria. Both resulting membranes showed successful photocatalytic functionalization of the original ISM structure in line with good bactericidal effects. ZnO membranes, even though prepared at fairly low temperatures (~100 C), showed polycrystalline phases and exhibited stronger bactericidal effects than TiO 2 coated membranes. In addition, an improved mechanical stability of the ZnO coated membranes was observed. 3.3 Experimental Preparation of the inner shell membrane from a chicken s egg Chicken s eggs were purchased from a grocery store. They were gently broken and the ISM around their air cell portion [135] was carefully cut out and collected (Figure 3.4 a and b). The ISM was washed several times with deionized water in order to thoroughly remove the thin albumin layer and subsequently dried at room temperature for 4 hours TiO 2 /ZnO atomic layer deposition on ISM The prepared ISM was placed in an ALD chamber (Savannah 100, Cambridge Nanotech Inc) and dried at 70 C for 20 min in vacuum ( torr) with a steady Ar stream (20

48 40 Photocatalytic Metal Oxide Membrane sccm). For the TiO 2 /ZnO deposition, well established ALD processes were applied. TIP/water [55-58] and DEZ/water [50-54] were used as precursors, respectively. The TIP and DEZ were purchased from Sigma Aldrich. Each cycle was composed of a pulse, exposure and purge sequence for each precursor. For the TiO 2 deposition, for example, the TIP vapor was injected into the ALD chamber for 1.5 seconds (PULSE). Subsequently, the substrate was exposed to the TIP vapor for 30 seconds (EXPOSURE). The excess TIP was purged from the ALD chamber for 30 seconds (PURGE). In the same manner, the PULSE (1.3 second), EXPOSURE (30 seconds) and PURGE (30 seconds) sequence for H 2 O was repeated. The thickness of the deposited TiO 2 and ZnO films was adjusted to 30 nm and 55 nm, respectively, by the number of cycles. For the preparation of diverse samples with TiO 2 and ZnO, the substrate temperature was varied between 70 C and 300 C. More detailed information on the applied ALD processes and sample denotations are given in Table 3.1. Table 3.1 Detailed processing conditions of ALD process and sample denotation. Oxide Precursor Pulse (second) Exposure (second) Purge (second) Cycle Substrate temperature Sample numbering 70 C ISM/TiO2/70 Ti [OCH(CH 3 ) 2 ] C ISM/TiO2/160 TiO C ISM/TiO2/225 H 2 O C ISM/TiO2/ C ISM/TiO2/300 ZnO (C 2 H 5 ) 2 Zn H 2 O C ISM/ZnO/ C ISM/ZnO/ C ISM/ZnO/ C ISM/ZnO/ Characterization Scanning electron microscopy and transmission electron microscopy (TEM) were applied to investigate the size and morphology of the samples. The investigations were conducted using a JSM-6340F at 15 kv (SEM) and JEOL 2010 at 200kV (TEM), respectively. The crystallographic features of the metal oxide membranes were investigated by X-ray diffraction (XRD, Philips X Pert MRD) with CuK α (λ = Å) radiation. The transfer of the samples was done in air. For Ө-2Ө measurements the samples were suspended on a silicon wafer as a convenient substrate.

49 Chapter Microbiology As a test strain for all bactericidal effect studies, Escherichia coli (E. coli) strain W3110 [140] was used. A single colony was inoculated from a Luria-Bertani (LB) agar plate (Carl Roth GmbH, Germany) with 4 ml of LB broth (Carl Roth GmbH, Germany) in a 10 ml glass bottle (Figure 3.5). The bottle was incubated overnight at 30 C on an orbital shaker at 250 rpm (E.coli solution 1). After 16 h, the culture was diluted to 1:50 into fresh LB broth and incubated for 3 h at 250 rpm to obtain a logarithmic growing culture for bactericidal effect experiments (E.coli solution 2). The cell concentration of the E.coli solution 2 was determined by the spread plate method [141]. The initial logarithmic growth phase population of E.coli ranged approximately from 10 5 to 10 6 colony forming units (CFU)/ml. a b Figure 3.5 E. coli bacterium used for the experiment. a, E.coli on LB agar plate. b, 4 ml LB broth medium having a single colony of E.coli. Figure 3.6 Schematics of the reactor for Escherichia coli (E. coli) photocatalysis experiments. The whole body was made from PTFE (Polytetrafluorethylen). The sterilization was performed at high temperatures for each experiment. In order to reduce the UV absorption through the supporting part as well as to support the ISM, a PMMA (Polymethylmethacrylate) sheet (1 mm thick) was used. Through the ring shaped gasket and the mechanical clamping (spring and bolt/nut type) the leakage of E.coli solution was effectively prevented.

50 42 Photocatalytic Metal Oxide Membrane Photocatalytic experiments with ISM/TiO 2 and ISM/ZnO A reactor for the photocatalysis experiments was designed (Figure 3.6). The E.coli suspension was exposed to UV light (Osram UVC-LPS 9, peak: 365 nm, power: 2W, UV light including visible blue light) from the lower part of the reactor and was continuously stirred with 250 rpm during each experiment. A 4 ml portion of the E.coli solution was taken from the prepared stock solution and pippetted into the reactor. The photocatalytic inactivation of the E.coli cells was assessed by taking a 100 μl volume of the E.coli solution 2 from the reactor every 5 min or 15 min for 60 min and diluting the solution to 1/100 (E.coli solution 3) and to 1/1000 (E.coli solution 4) with fresh LB liquid medium. In order to count the number of viable E.coli, a 10 μl volume of the E.coli solution 4 was taken and spread onto LB agar plates as described in the previous section. The thus prepared LB agar plates were incubated overnight at 37 C. After 12 hours, the number of colonies was counted (Figure 3.7). For each experiment, three plates were used and averaged. The results were plotted as the survival ratio. a b c d Figure 3.7 Spread plate method. As described in the text, in order to count the number of viable E.coli, a 10 μl volume of the E.coli solution 4 was taken, spread onto LB agar plates, and incubated overnight at 37 C. After 12 hours, the number of colonies was counted. a shows formed E.coli colonies on a LB agar plate after 12 hours incubation of the E.coli solution on ISM/TiO2/300 without UV irradiation (ISM/TiO2/300 at t=0 min). b shows colonies on a LB agar plate after 12 hours incubation of the E.coli solution on ISM/TiO2/300 irradiated by UV for 30 min (ISM/TiO2/300 at t=30 min). c and d show colonies on LB agar plates after 12 hours incubation of the E.coli solution on ISM/ZnO/100 without irradiation and irradiated by UV for 60 min, respectively. In each picture, the red circle indicates a single colony to be counted.

51 Chapter Tensile test of native ISM, ISM/ZnO/100 and ISM/TiO2/275 membranes For the measurement of the engineering stress (σ) - strain (ε) behaviour of the prepared samples, all ISM samples were cut with a knife (BAYHA, Blades, No.24) to 2 mm 10~20 mm. Tensile tests were performed on a ZWICK 1445 tensile test machine with a 10N HBM load cell, controlled by a PC with automated testing software. The extension rate was 50% of the initial testing length (10 mm) per minute (5 mm/min). The temperature and relative humidity were C and 20-22%, respectively. SEM and optical microscopy (Leitz Aristomet) were used to measure the cross section area of the specimen. The thickness and the width of the membrane were ~100 μm and ~2000 μm, respectively. Since the thickness of the ESM was not perfectly uniform, it was averaged to yield ~100 μm from measurements at more than 20 points along the horizontal direction of each sample (2000 μm). After the tensile tests, the cross section of the fracture surface of each sample was again examined by SEM. Because of the slightly stretched length of the sample, the cross section was slightly shrunk. However, the difference was negligible. In the case of the width of each sample, a similar procedure was applied. The variation in width was also negligible. For each set of data, more than 10 samples were prepared and measured at identical conditions. Each data set showed similar stress-strain behaviour. As an average, from each measurement one typical data set was selected. All graphic works including data rescaling were performed with ORIGIN Results and discussion Film quality, crystallographic features and bactericidal efficiency Since the crystallinity of the deposited coating is temperature dependent [55,142], the composite membranes were prepared at various temperatures ranging from 70 C to 300 C. The resulting membranes, both ISM/ZnO and ISM/TiO 2, show unchanged morphological features of the original ISM. As representative SEM and TEM micrographs of the resulting ISM/ZnO and ISM/TiO 2 membranes, the images of ISM/ZnO/100 and ISM/TiO2/275 are shown in Figure 3.8 (Sample denotations can be found in Table 3.1; the reason for choosing these two samples as representative samples will be discussed in a later part). Both images show, as expected, good quality of the metal oxide deposition. The films were conformally deposited over the whole collagen membrane without any

52 44 Photocatalytic Metal Oxide Membrane distortion and shrinkage, as can be confirmed from Figure 3.8a, c and e showing fibers of ISM/ZnO/100 and Figure 3.8b, d and f showing fibers of ISM/TiO2/275. a b c d e f Figure 3.8 Electron micrographs of ISM/ZnO/100 and ISM/TiO2/275. a and b show a macroscopic view of the ISM/ZnO/100 and the ISM/TiO2/275 membrane, respectively. The morphology of the native ISM (their highly interwoven and conglutinate feature) is visible. c and d show composite membranes of collagen fibers/metal oxides at high magnification. e and f show the corresponding TEM images of the ISM/ZnO/100 and ISM/TiO2/275 membranes, respectively. The coating of the fibers with the metal oxide is visible. Apart from the deposited film quality, the bactericidal properties, as a function of the crystallographic features of the resulting macroporous membranes, are of interest. Figure 3.9a and b show X-ray diffraction (XRD) patterns of the ISM/TiO 2 and ISM/ZnO prepared at temperatures ranging from 70 C to 300 C. The TiO 2 -coated membranes (ISM/TiO 2 ) processed at 70 C and 160 C did not show any considerable diffraction peaks, whereas at higher temperatures they showed reflections from the anatase phase (ICDD card No ), anatase (101) being the strongest peak among them. The deposition

53 Chapter 3 45 temperature of 225 C shows an onset of crystallization. In contrast, the ZnO coated membranes (ISM/ZnO) showed diffraction peaks already at 70 C process temperature. The positions and the intensities of the individual peaks are in good agreement with the hexagonal wurtzite structure of ZnO according to JCPDS card No a c b d Figure 3.9 X-ray diffraction (XRD) patterns and E.coli survival rates on ISM/TiO 2 (upper row) and ISM/ZnO (lower row). a and b show XRD patterns of both membranes illustrating the crystalline structures as a function of the deposition temperature. c and d show E.coli survival rates in aqueous solution as a function of time corresponding to each membrane after UV illumination for various deposition temperatures of TiO 2 and ZnO, respectively. The photocatalytic efficiency (bactericidal effect) was evaluated from the E.coli survival ratio with respect to the illumination time with UV light for the two different types of metal oxide membranes. It is known that semiconducting metal oxides, such as ZnO and TiO 2 generate conduction electrons (e - ) and valence band holes (h + ) on the surface upon illumination with an energy higher than the band gap energy (E g,zno = 3.37 ev, E g,tio2 = 3.2 ev) in an aqueous solution [138,139]. Subsequently, holes react with the water adhering to the surface of the ZnO and TiO 2 to form highly reactive hydroxyl radicals (OH ). Oxygen acts as an electron acceptor forming super-oxide radical anions (O 2 ) which are an additional source of hydroxyl radicals upon subsequent formation of hydrogen peroxide (H 2 O 2 ) [138,139]. The generated OH, O 2 and H 2 O 2 can attack the cell walls in E.coli, which finally are damaged. After eliminating the protection of the cell

54 46 Photocatalytic Metal Oxide Membrane wall, oxidation of the underlying cytoplasmic membrane and the intracellular contents takes place and eventually leads to death of the E.coli [143]. As illustrated in Figure 3.9c and d, in the dark without ZnO and TiO 2, the survival ratio was constant or slightly increased due to the natural replication of the E.coli. Under UV illumination both membranes clearly showed the capability to inactivate E.coli in aqueous solution. In agreement with previous literature, it was observed that the bactericidal effect of ZnO and TiO 2 has a stronger dependence on the crystallinity than on the film thickness [144,145]. Specifically, the bactericidal efficiency of the ISM/TiO 2 with anatase / rutile phases and the ISM/ZnO with the hexagonal phase revealed proportional relationship to the ALD deposition temperature and the relative intensity of the (100) crystal direction, respectively. The membrane itself has a strong impact on the photocatalytic behaviour. From the respective E.coli survival ratio, it can be seen that, in terms of bactericidal efficiency under UV illumination, the ISM without metal oxide coating is more effective than the suspension only, i.e. without a membrane. The metal oxide coating enhances the efficiency considerably. Comparing the bactericidal efficiency of the coated membranes with TiO 2 particles (Degussa P25, average particle size: 30 nm) [146] or home-made TiO 2 films [147] reported in the literature, the ISM/TiO2/300 is much more efficient when considering the irradiation intensity and the area exposed to UV light. Similar to TiO 2, the ISM/ZnO/100 also shows a higher efficiency, as compared to ZnO powder [148] with a much lager surface area. Presumably this is caused by the macroporous structure of the ISM. Probably, E.coli bacteria can easily adhere to the macroporous ISM, leading to an increased concentration of bacteria close to the UV source. Thus more bacteria are destroyed in the same time period. It is noteworthy that ISM/TiO 2 reveals a higher bactericidal efficiency than ISM/ZnO. This result is consistent with a previous publication comparing the photocatalytic efficiency of TiO 2 and ZnO [149,150], however, opposite results have been also reported [151,152]. The relative photocatalytic behaviour of TiO 2 and ZnO still seems to be ambiguous. The graphs in Figure 3.9c and d show that, as expected, ISM/TiO2/300 has the strongest bactericidal effects. However, with decreasing processing temperature, the efficiency of the TiO 2 -coated membrane also decreases. Already at 275 C (ISM/TiO2/275), the efficiency is comparable to that of the ZnO-coated one processed at much lower temperatures (ISM/ZnO/100). Therefore, for the strongest effects, one has to coat the membrane at a high temperature, thereby somewhat damaging the morphology of the original ISM. Since most biological materials have a tendency to decompose or deform at high temperatures (pyrolysis temperature of ISM 240 C) [153], a deposition at such temperatures is not suitable in most cases. A good compromise can be found if the sensitive membranes are coated with ZnO at 100 C, showing reasonably efficient

55 Chapter 3 47 photocatalytic behaviour. Thus, in terms of bactericidal efficiency as well as preservation of the original morphology, the ISM/ZnO is more beneficial than ISM/TiO Mechanical flexibility and thermal stability Even though the ISM is stable against the reaction byproducts of the ALD process (e.g. isopropanol) [136,137], as stated above, upon heating (around 240 C) it undergoes pyrolysis [153]. For coating of the ISM with ZnO, the pyrolysis is not a critical issue, the highest photocatalytic efficiency and preservation of original structures of ISM can be assured by virtue of low processing temperatures (< 240 C). In contrast, in the case of TiO 2, due to the required higher processing temperature (> 240 C), even though the photocatalytic efficiency can be assured, the mechanical stability was reduced by the pyrolytic damage to the original structures of the ISM. Figure 3.10 Tensile test data of the native ISM, ISM/ZnO/100 and ISM/TiO2/275 membrane and a photograph of the ISM/ZnO/100. As shown in the σ-ε curve, after low temperature (100 C) ALD of ZnO, the mechanical properties of ISM/ZnO/100 were enhanced and showed high flexibility as can also be seen from the inset photograph. In contrast, in the case of ISM/TiO2/275, it was observed that the membranes become stiffer after high deposition temperature (275 C). The process presumably caused a thermal damage of the collagen structure. Table 3.2 Maximum stress (MPa) and strain (%) value of Native ISM, ISM/TiO2/275 and ISM/ZnO/100. (Average ± Standard deviation) Maximum Stress[MPa] (σ max ) Maximum Strain[%] (ε max ) Native ISM 6.21 ± ± 0.52 ISM/TiO2/ ± ± 0.43 ISM/ZnO/ ± ± 0.83 The results from our experiments are shown in Figure 3.10 and Table 3.2. The resulting ISM/TiO2/275 membranes deposited at 275 C were more brittle and stiffer than

56 48 Photocatalytic Metal Oxide Membrane the native ISM (decreased maximum strain ε max and increased stiffness (initial Young s modulus), E ini (ISM/TiO2/275)). In contrast, the ISM/ZnO/100 membrane showed an even higher flexibility and mechanical stability against external load than the native ISM (increased maximum stress σ max and strain ε max together with stiffness E ini (ISM/ZnO/100)). Considering that most of the metal oxides are generally brittle even at nanometer thicknesses [154], the ISM membranes after ALD are expected to show lower flexibility and stability to external load, if the contribution of the metal oxide layer itself and thermal effects are considered. However, this is not always the case. It is known that the collagen based ESM contains functional groups, such as amines, amides and carboxylates [136,137], which may interact with the ALD precursors (TIP or DEZ/water) during the ALD process. Highly reactive ALD precursor pairs, can chemically interact with the ISM fiber surface as well as the bulk of the protein structures (results will be discussed in Chapter 5). Hence the flexibility of those composite membranes can presumably be ascribed to anchored metal containing precursors such as, Zn or Ti, similar to the mechanical properties enhancing effects of insect s cuticles by small amounts of impregnated metals [155]. Detailed investigations of the mechanical properties of ALD infiltrated collagen structures will be reported in Chapter Conclusion In conclusion, eggshell membranes were used as templates for coatings with TiO 2 and ZnO, respectively, via ALD. The resulting structures satisfy the optimal combination of original functionality of the biotemplate and appropriate metal oxides which can improve the functionality of the resulting structures. The membranes show good mechanical flexibility for practical use. Depending on the deposition temperature of the metal oxides, the resulting films were either amorphous or polycrystalline. Upon UV illumination, the ISM/TiO 2 and ISM/ZnO membranes clearly exhibited bactericidal effects. Above all, it was found that polycrystalline ISM/ZnO membranes can be prepared at a fairly low temperature (100 C) and nevertheless show a bactericidal efficiency which is competitive to that of ISM/TiO 2 membranes prepared at a much higher temperature (275 C). Furthermore the ZnO-coated membranes are mechanically more stable than the TiO 2 - coated ones. It is concluded that for coatings of diverse temperature sensitive templates (such as biological materials or polymers), low-temperature ALD of ZnO is more suitable than the deposition of TiO 2, i.e. thermally less destructive and photocatalytically competitive.

57 Chapter 4 49 Chapter 4 Metal Infiltration into Spider Dragline Silk In nature, tiny amounts of inorganic impurities, such as metals, are incorporated in the protein structures of some biomaterials and lead to unusual mechanical properties of those materials [155]. A desire to produce these biomimicking new materials has stimulated materials scientists, and diverse approaches to produce those superior materials have been attempted. In contrast, research to improve the mechanical properties of biomaterials themselves by direct metal incorporation into inner protein structures has rarely been tried, presumably because of the difficulty to develop a method to infiltrate metals into biomaterials. Here it is demonstrated that metals can be intentionally infiltrated into inner protein structures of biomaterials through multiple pulsed vapor-phase infiltration (MPI) performed with equipment conventionally used for atomic layer deposition (ALD). Zinc, titanium, or aluminum was infiltrated into spider dragline silks and a greatly improved toughness of the resulting silks was observed. The presence of the infiltrated metals such as Al or Ti was verified measuring the treated silk by energy-dispersive x-ray (EDX) and nuclear magnetic resonance spectroscopy. This result of enhanced toughness of spider silk could potentially serve as a model for a more general approach to enhance the strength and toughness of further biomaterials. 4.1 Background Overview of spiders and mechanical properties of spider silk It is commonly believed that the first spiders appeared during the Devonian period, about 400 million years ago [156,157]. Taxonomists say that at present (as of 2008),

58 50 Metal Infiltration into Spider Dragline Silk approximately 4000 different spider species, which are grouped in 109 families [158], are found all over the world on every continent except for Antarctica. They have conquered all ecological environments, perhaps with the exception of the air and open sea. The order of spiders (Araneae) is usually classified into three suborders: the Mesothelae, the Mygalomorphae and the Araneomorphae [158]. More than 90 %, thus most of the spiders that people encounter in daily life, are Araneomorphae. Among those, the Araneidae family is known to be the most impressive orb-weaver spider which builds spiral wheel shaped webs very often found in gardens. The spider either sits right in the center of the web or hides in a retreat outside of it. Insects flying into the web become trapped by sticky threads long enough for the spider to rush out from the hub to bite or wrap its victim. It is known that some other spiders build an orb web that is very similar to the webs of the araneids, however, differing in one critical aspect: the catching threads are not coated with glue droplets but are instead decorated with an extremely fine mesh of silk known as hackle band [159]. Figure 4.1 Different types of silks of Nephila golden spider (reproduced from reference [160]). The illustration shows silk glands of each silk and corresponding stress-strain curves, where the highlighted line in each graph is that of the graph label. D and W in the flagelliform graph are dry and wet, respectively.

59 Chapter 4 51 a b c d e Figure 4.2 Mechanical properties of spider dragline silks from major ampullate and sticky silks from flagelliform [161]. a, b, c and d show typical values of the initial Young s modulus (E ini ), maximum tensile stress (σ max ), maximum tensile strain (ε max ) and toughness of a dragline silk together with a comparison with other natural and manmade materials, respectively. e, depicts basic terminologies from mechanics in a typical stress-strain curve of a dragline silk under an uniaxial tensile test. Silks are defined as protein biopolymers that are spun into fibers by Lepidoptera larvae such as silkworms, spiders, scorpions, mites and flies [162]. Among those, spider silk has been noted for its extraordinary properties. During the last century, a number of scientific reports were published on spider silk. But it is only in the past few years that a serious understanding of the reason for the extraordinary mechanical properties that silk possesses has emerged. In particular, the dragline silk of the orb weaving tribe of araneid spiders, such as the garden spider Araneus diadematus or the golden silk spider Nephila clavipes, is known to outperform almost any man-made material in its combination of tensile strength and extensibility (Figure 4.1 and Figure 4.2) (so called, extreme toughness, i.e. σdε, where σ and ε are stress and strain, respectively) [161,163]. It is noteworthy that dragline and sticky silk (fragelliform silk) absorb more energy prior to fracture than nearly any commonly used material. Due to those unique mechanical properties, the dragline silk

60 52 Metal Infiltration into Spider Dragline Silk has been regarded as the benchmark of spider silk and has played a vital role in understanding spider silks. On the other hand, it is known that the dragline silk is produced by the major ampullate gland of a spider and provides a safety-rope that the spider can rely on if it falls down. The term dragline relates to the fact that the spider constantly drags its safety-rope behind. Typical orb weaving spiders (Araneus or Nephila) construct their web from different silks, each of which is produced in a separate gland and has a wide range of unique properties and applications as shown in Figure 4.1 [160,164] Chemical structure and macroscopic model for spider silk Figure 4.3. Macroscopic to molecular structure of spider dragline silk. a, Picture of Araneus diadematus sitting on its web. b, Thickness comparison between a human hair and a spider dragline silk (~5μm). c and d, 2D and 3D schematic drawings of the inner silk structure at the nanometer

61 Chapter 4 53 scale. As can be seen in the figure, the silk is composed of long polypeptide chains. The spider silk is generally regarded as a biopolymer containing two major parts, i.e. highly-/less-ordered β-sheet crystalline parts consisting of hydrophobic polyalanine sequences, and highly-/less-oriented amorphous parts composed of amino acid residues, linked with hydrogen bonds. AMNC: Amorphous Molecular Network Chain; HOBSC: Highly-Ordered β-sheet Crystal; LOBSC: Less- Ordered β-sheet Crystal; HOAMNC: Highly-Oriented AMNC; LOAMNC: Less-Oriented AMNC. Figure c is redrawn from reference [165]. e, The lattice constant of a β-sheet crystal (unit cell structure * ) is known to be 15.7Å (chain direction) x 9.44Å (hydrogen bond direction) x 6.95Å (Van der Waals bond direction) [166]. f, Molecular structure of a parallel / anti-parallel arrangement of β - sheet strands. The strands are connected by hydrogen-bonds (dotted lines). R=CH 3. g, Schematic of an dipeptide consisting of glycine and alanine. The peptide bond is formed between the molecules carbonyl group ( COOH) and the amine group ( NH 2 ) by dehydration (COOH + H 2 N CONH + H 2 O). Table 4.1 Amino acid composition of silk from Araneus diadematus. The amino acids are described with a three-letter code. The table is written based on the reference [167]. Amino acid Major Ampullate Minor Ampullate Flagelliform Aciniform Cylindriform Asp Thr Ser Glu Pro trace Gly Ala Val Ile Leu Tyr Phe Lys His trace trace trace Arg In 1907, Fischer reported on the main component of spider silk being proteins [168]. It is known that except for the sticky materials on capturing silks (flagelliform silks), further compounds including sugars, mineral, and lipids, are not covalently bound to the silk protein [169]. In spite of different species of spiders, different types of silk, having different protein sequences, a general trend in the structure of spider silk is a sequence of alternating Gly and Ala (or Ala only), which self-assembles into a β-sheet structure (Figure 4.3). In particular, the dragline silk from a major ampullate gland is composed predominantly of Gly and Ala, as shown in Table 4.1 [167]. These two simplest amino acids are connected by peptide bonds. This peptide unit forms a β-sheet, which stack to form crystals, whereas the other segments form amorphous domains. The ratio of crystalline fraction to amorphous fraction is known to vary between 3:7 and 2:8 (in the * As far as the unit cell size is concerned, the value is not consistent. For example, Grubb et al.[170] reported different unit cell size of 10.6 Å 9.44 Å 6.95 Å.

62 54 Metal Infiltration into Spider Dragline Silk case of dragline silk of Nephila clavipes [170]. The β-sheet crystals have a pseudo orthorhombic unit cell conformation [171] with a size of 15.7Å x 9.44Å x 6.95Å (see Figure 4.3e for details) [166] Models for the description of dragline silk s mechanical properties Over the past decades, the dragline silk has been recognized as a semicrystalline biopolymer composed of amorphous flexible protein chains reinforced by strong and stiff crystallites, as depicted in Figure 4.3. The crystallites are believed to be made of hydrophobic polyalanine sequences arranged into β-sheets [163]. On the other hand, the amorphous chain is known to be composed of kinetically free oligopeptides rich in Gly. The question, how a silk with a comparatively simple molecular structure reveals such remarkable mechanical properties, seriously occupied the scientists. In order to validate the interplay between the hard crystalline segments and the elastic semi amorphous regions, many scientists proposed diverse models to describe such extraordinary mechanical properties of spider silks [172,173]. Nevertheless, none of them has been able to properly describe the mechanical behaviour of spider silk. Attempts to describe deformation behaviour of the dragline silk under mechanical load from the point of view of unfolding of proteins were perfomed [174,175]. Among the proposed models, predictions for the thermo-mechanical properties of silk fiber from mean field theory were made. This approach is called Group Interaction Modeling and is used for polymers in terms of chemical composition and the degree of order in the polymer structure. It has been developed to formulate structure-property relationships in polymer processing rheology [172]. This model system assumes that spider silk is composed of hydrogenbonded protein chains arranged in an ordered and a disordered manner. The density of hydrogen bond defines ordered and disordered regions inside the spider silk. This model allows structure-property relations to be predicted in terms of chemical composition and simple morphological structure. Gersappe [173] tried to analyze the molecular toughening mechanism based on the model assuming the silk being a polymer reinforced with nanoscopic filler particles. In the model, the mobility of the nanoscale filler particles was found to be critical for increasing the toughness of the polymer. He claimed that nanoscale crystallites observed in spider silk presumably play the same role as the nanoscale filler particles. On the other hand, the hierarchical chain model for the capture silk [176,177], which shows a similar behaviour to wetted dragline silk (except its extensibility), was very recently proposed, based on the assumption that spider capture silk is composed of a number of different structural motifs which are organized in hierarchical levels.

63 Chapter 4 55 a b Figure 4.4 Molecular model of a spider dragline silk proposed by Termonia [178,179]. a, The left hand figure shows an actual molecular structure of a spider dragline silk and the right hand figure shows a simplified molecular structure. b, Calculated stress-strain curve, based on the model from a molecular dynamics simulation. This result shows the effect of the crystallite size on the deformation behaviour. Figure a and b are reproduced and redrawn figures from reference [179]. In addition, in 1994, Termonia [178,179] perceived results from X-ray measurements [180] and represented the dragline silk with a large number of small crystallites separated by amorphous regions made of rubber-like chains. He recognized that, for a defined volume fraction of crystallites in the silk, the conformation of amorphous parts of silk is similar to the entangled conformation of synthetic polyethylene. In addition to being entangled, the amorphous chains are linked together by hydrogen bonds. Based on these considerations, he proposed a molecular model of a spider dragline silk, which is in the meantime well accepted. He simplified the molecular structure of silk to a twodimensional network of flexible amorphous chains reinforced by stiff crystallites, as shown in Figure 4.4a. Termonia theoretically presented the reinforcing effect of the crystallites in spider dragline silk in terms of crystallite size variation (Figure 4.4b).

64 56 Metal Infiltration into Spider Dragline Silk a b c d Figure 4.5 Zn bearing Nereis seaworm jaws. a, Picture of a Nereis seaworm and its jaw in different magnifications. b, Zn distribution in a Nereis seaworm jaw. (From to left to right) The first picture shows the whole jaw under the light microscope. The second figure shows an X-ray absorption image of a jaw. The tip part is darker than the rest of the jaw. The third image shows the Zn K α fluorescence with highly concentrated Zn at the tip of the jaw (for details, see the reference [181]). c, Scanning electron microscopy (SEM) image of a complete jaw. The jaw was trimmed to the plane indicated by the red disc. The upper right figure shows energy dispersive X-ray spectroscopy (EDX) maps of a native, a Zn removed and a Zn re-infiltrated jaw, respectively. Bright parts indicate high Zn concentrations. The lower right figure shows hardness (H) and stiffness (E) profiles as a function of the lateral position along the lines 1 and 2. A comparison of native (red line) and Zn removed (blue line) indicates that both H and E are substantially decreased in regions where Zn has been removed. A significant recovery of initial H and E is observed when Zn is re-infiltrated (green line). d, Proposed model for Zn-meditated cross-linking of histidine-rich proteins in jaws (for details, refer to reference [182]). b and c, d are reproduced and edited figures from references [181,182].

65 Chapter Function of metals in biological tissues Many biological tissues of diverse living organisms, such as those in mandibles, stingers, claws and ovipositors of invertebrates are known for their mechanical stiffness and toughness. Whether used for support (e.g. bone and cuticle), protection and defense (e.g. shell, cuticle and jaws/stings of arthropods, some worms) or nutrition (e.g. mandible of grasshopper, claws of arthropods), the evolution of hardened or stiffened material is driven by the need to maximize mechanical properties for particular natural circumstances. These stiffening/hardening/toughening mechanisms are known to be accomplished by metal incorporation, such as, Zn, Mn, Cu and Ca, into protein matrix of tissues. Even though the organic components are mineralized by only a few percent of the mineral components, the fracture toughness exceeds that of single crystals of the pure mineral by two or three orders of magnitude [181]. For example, Nereis seaworm (commonly known as a clam worm) has one pair of jaws with a size of around 3 mm (Figure 4.5 a and b). All Zn inside the jaw is concentrated in the tip region of the jaw (where the mechanical impact and the abrasion are expected to be highest), with no or very low levels of Zn at the base of the jaw [181]. In 2006 Broomell et al. [182] directly demonstrated that zinc plays a critical role in mechanical properties of these histidine-rich jaws performing nanoindentation measurements on Zn chelated/re-infiltrated jaws (Figure 4.5c). Finally, they concluded that hardening/stiffening mechanisms appear to be meditated by reversible Zn binding to bundles of histidine-rich protein fibers. Many other results showed that the metals inside of biological tissues have a positive effect on the mechanical property improvements. The efforts to elucidate correlations between the presence of some metals, such as Zn, Mn, Ca, or Cu, accumulated in diverse biological tissues. Investigations of their enhanced mechanical properties have been extensively performed. Still, however, only little is known about the chemical/physical mechanisms responsible for the astonishing mechanical property improvements.

66 58 Metal Infiltration into Spider Dragline Silk a b c d e f h g Figure 4.6 Overview of the experimental procedure. a, Photograph of the Araneus spider. b and c, Dragline silk collection using a paper clip as a spool. d, Al 2 O 3, TiO 2 or ZnO ALD processes. e, Tensile test using a cardboard jig for supporting silks during the test (pictured by Canon EOS 350D with Canon EF 100mm f/2.8 USM macro lens). Usually it is very difficult to take a photograph of a dragline silk with 5 μm thickness even though a very fine macro lens was used. So multiple (fourfold) draglines were used for imaging purposes. f, NMR measurement. g, WAXS measurement. h, EDX measurement.

67 Chapter 4 59 Table 4.2 Detailed processing conditions of prepared samples and their denotation. All vapor infiltration experiments were performed at 70 C substrate temperature Resulting Vapor pulse Pulse Exposure Purge Cycle Sample # materials Pairs (second) (second) (second) [1] SS/N [2] SS/H [3] SS/TIP [4] SS/W 5 SS/Al2O3/ TMA [5] SS/TP/100 [6] SS/WP/100 8 SS/Al2O3/300 9 Al 2 O [7] PF/Al2O3/300 [8] SS/Al2O3/300/H 10 H 2 O SS/Al2O3/ [9] 11 SS/Al2O3/500/H SS/Al2O3/700 SS/TiO2/ TIP SS/TiO2/ TiO SS/TiO2/500/H H 2 O SS/TiO2/700 [10] SS/TiO2/700/C ZnO DEZ SS/ZnO/100 SS/ZnO/ H 2 O SS/ZnO/700 [1] SS/N: Native dragline silk of Araneus spider. [2] SS/H: Annealed SS/N in ALD chamber at 70 C and 0.01torr for 15 hours. [3] SS/TIP and [4] SS/W : Native silks which are dipped into TIP or water at room conditions (T=15, P=P atm ) for 10 hours, followed by drying at same conditions, respectively. [5] SS/TP/100 and [6] SS/WP/100: TMA or water pulse exposed silk without water or TMA pulse exposure for 100 cycles at same processing conditions as SS/Al2O3/100, respectively. [7] PF/Al2O3/300 (for NMR): Para film on which Al 2 O 3 layer is deposited at same processing conditions as SS/Al2O3/300. [8] SS/Al2O3/300/H and [9] SS/Al2O3/500/H: Annealed SS/Al2O3/300 and SS/Al2O3/500 at same conditions as SS/H for 15 hours, respectively. [10] SS/TiO2/700/C: SS/TiO2/700 which are produced without the self-limiting mechanism (CVD). For ease in preparation and handling of these samples, when we dipped the silk into TIP or water and subsequently dried the silk, we used a paper clip on which silk fibers are wound as a sample carrier for dipping and drying. During this process, unintentionally the sample, in particular SS/W, is subjected to an axial restraint during drying at room temperature and in ambient atmosphere.

68 60 Metal Infiltration into Spider Dragline Silk 4.2 Experimental Silk collection The complete experimental procedure is schematically described in Figure 4.6. Firstly, in order to collect dragline silk fibers of an Araneus spider (Figure 4.6a), the spider was caught in the garden of the Max-Planck-Institut für Mikrostrukturphysik (Halle, Germany) and housed in a cm 3 transparent glass box. The silk was obtained within two days after capture, during which time the spider was fed on a diet of houseflies. Before starting silking, the spider was sedated in the refrigerator for around 1h at T = 4 C since otherwise the spider was too nervous to reel the silk. Then the spider was gently fixed on a piece of thick and soft paper using a small piece of white colored 3M scotch tape. Under an optical microscope (Leitz Aristomet, Type , LEITZ WETZLAR GERMANY) the spinnerets on its abdomen were carefully stimulated with a soft stick. After emerging two dragline silk threads from the spigots of the ampullate glands, the scotch tape was carefully removed and the spider was allowed to roam and spin freely over a black colored paper in a closed paper box ( cm 3 ). The dragline silks left behind as a safety line were carefully wound to minimize initial stretching of the silk, using a paper clip as a spool at ambient room conditions (23 ± 3 C and 35 ± 4 % relative humidity). The walking speed of the spider was around 1 cm/s. After silk reeling, under the optical microscope (Leitz Aristomet) and a magnifying glass ( 10) the wound silk was reexamined for separate rewinding of a single dragline silk thread. Finally the prepared silk was stored in a desiccator. Since the mechanical behaviour of a silk is highly sensitive to the silk reeling conditions [183] and due to lacking access to specialized silking equipment [184], each step was performed very carefully to minimize uncertainty Multiple pulsed vapor phase infiltration process The prepared silk was placed in an ALD reactor (Savannah 100, Cambridge Nanotech Inc.) and dried at 70 C for 5 min in vacuum ( torr) with a steady Ar gas stream (20 sccm). For the infiltration, TMA/water, TIP/water, and DEZ/water (H 2 O) pairs were used as sources for aluminum/oxygen, titanium/oxygen and zinc/oxygen, respectively. TMA, and DEZ were purchased from Strem Chemicals and TIP from Sigma Aldrich. Each cycle was composed of a pulse, exposure and purge sequence for each precursor. More specific information on the ALD processes is given in Table 4.2.

69 Chapter 4 61 Figure 4.7 Schematic description of the pressure profile for the MPI of SS/Al2O3/100, SS/TP/100 and SS/WP/100. Pressure profile of SS/Al2O3/100 during ALD. Normally, in an ALD process, the exposure/purge steps of two precursors (e.g. TMA and Water) are repeated continuously. In order to prepare SS/TP/100 and SS/WP/100, the TMA/water pulse valve is closed with water/tma valve opened under conditions identical to the preparation of SS/Al2O3/100, respectively. Therefore, during the preparation of SS/TP/100, there are no water pulse exposure/purge signals, likewise, during the preparation of SS/WP/100, there is no TMA pulse exposure/purge signals. E: Exposure and P: Purge of each precursor. A. SS/TMA/100 and SS/H2O/100. The same procedure and same source of infiltration (TMA and water) like in the sample SS/Al2O3/100 was repeated. As illustrated in Figure 4.7, during the ALD procedure the valve of the water source bottle/the valve of aluminum source bottle was closed while the valve of the aluminum source bottle/the valve of the respective other precursor was opened, respectively. B. SS/H, SS/Al2O3/300/H and SS/Al2O3/500/H. In the ALD chamber, the samples SS/N, SS/Al2O3/300 and SS/Al2O3/500 were annealed for 15 hours at 70 C and 0.01torr. C. SS/TiO2/700/C. For the preparation of this sample, under identical processing conditions like SS/TiO2/700, controlling the stop valve in the ALD tool (Figure 4.8), overlapping pulses of TIP and water are applied.

70 62 Metal Infiltration into Spider Dragline Silk a b c d Figure 4.8 Comparison of SS/TiO2/700 and SS/TiO2/700/C. a and c, SEM micrographs of SS/TiO2/700 and SS/TiO2/700/C. b and d, Pressure profiles with respect to time during the deposition process of the corresponding samples, respectively. Firstly, b shows an ALD-type of pressure profile, i.e. well-separated and self-limited precursor exposure / purge of each precursor. With a proper control of the stop valve, as shown in d, a mixture of TIP and water is present in the ALD chamber. As a consequence, a TiO 2 film is deposited by continuous and not self-limiting process, thereby causing much higher TiO 2 layer thickness and coarse film quality as can be seen in c. E: Exposure and P: Purge of each precursor Tensile test For the measurement of the engineering stress (σ) - strain (ε) behaviour of the prepared samples, all samples were mounted in a thick paper jig having 4 mm punched holes (Figure 4.6e). The paper jigs were used to facilitate alignment and clamping of the specimens during the tensile test. With the support of the jigs, the silk was able to be held straight as it was clamped, furthermore the jigs allowed to cut easily through the cutting line so that silk specimen and paper jigs were not loaded together during the actual test. Pattex Blitz Kleber (Henkel, Germany) was used as glue to fix the silk to the edge of the jigs. The tensile test was performed on a ZWICK 1445 tensile test machine with 10N HBM load cell, controlled by a PC having automated testing software. Firstly, the clamping part 1 of the jig was fixed to the load cell by a screw type clamping system and subsequently the jig was cut through the cutting line with scissors. After controlling the vertical alignment of the silk specimen attached to the jig, the clamping part 2 was also fixed in the same way as the clamping part 1. The extension rate was 50 % of the initial

71 Chapter 4 63 length per minute (2 mm/min). The temperature and relative humidity were C and %, respectively, and the fiber was extended until fracture occurred. Force (mn)- strain (%) data for each specimen were exported from the software of the machine and subsequently the data were rescaled into the engineering stress (σ)-strain (ε). For the measurement of one sample, initially more than 40 silk samples fixed on a paper jig were prepared. Under an optical microscope (Leitz Aristomet) and a magnifying glass ( 10) the sample conditions such as tight fixation by glue and surface damage of silk during fixation were controlled. At this stage, all inferior samples were excluded. Subsequently the tensile tests were performed. During the test, the samples for which the location, where the fracture occurred, was not in a center of the jig, were also excluded from data processing. Finally, up to 10 identical samples were prepared and measured under the same conditions. Most of the data showed similar stress-strain behaviour and at each measurement one typical data set was selected. All graphic works including data rescaling, were performed with ORIGIN 7.5. A JSM-6340F scanning electron microscope was used to measure the silk diameters before and after an actual tensile test or to characterize the surfaces of the ruptured silk specimen after the tensile test. For the measurement of the diameter of the native silk and Al 2 O 3, TiO 2 or ZnO treated silk, after gold sputtering on top, at least five diameters were measured on different positions and averaged. The average diameter was 5.0 ± 0.2 μm TEM and EDX analysis To prepare specimens of SS/TiO2/500 for TEM and EDX (energy dispersive x-ray), the used spool (paper clip) wound by SS/TiO2/500 was fixed with 3 % glutaraldehyde (Sigma, Taufkirchen, Germany) in 0.1 M sodium cacodylate buffer (SCB; ph 7.2) for two hours at room temperature, rinsed with SCB, dehydrated in a graded ethanol series, infiltrated with epoxy resin according to Spurr [185] and polymerized at 70 C for 24 hours. Ultrathin sections (90 nm) were made with an Ultracut R ultramicrotome (Leica, Wetzlar, Germany) and transferred to copper grids coated with a carbon film. TEM observations were performed with a JEOL JEM-1010 and Philips CM20FEG TEM/STEM microscope, operating at a voltage of 100 kv and 200 kv, respectively. The CN20FEG electron microscope is equipped with an EDX-detector enabling the detection of light elements (IDFix-system, SAMx-Germany). Point analyses as well as EDX-line scans on the silk disks were performed with this equipment.

72 64 Metal Infiltration into Spider Dragline Silk Solid state nuclear magnetic resonance (NMR) spectroscopy From the samples SS/Al2O3/300 and PF/Al2O3/300 (Table 4.2) 27 Al-NMR spectra were recorded at MHz resonance frequency on a BRUKER AVANCE 750 spectrometer equipped with a 17.6T magnet. A BRUKER magic-angle spinning (MAS) probe with 2.5 mm rotors was employed, spinning at a MAS rate of 20 khz. The spectra were acquired with a Hahn-echo sequence, using an echo delay of one rotor period (50 μs) and a 16-step phase cycle. The relaxation delay was 5s, and 16,000 to 32,000 transients were accumulated. All spectra were referenced against an aqueous solution of AlCl 3 at 0 ppm Wide angle X-ray scattering (WAXS) a b Figure 4.9 WAXS sample preparation and WAXS profile of native silk (SS/N). a, The photograph of a specimen. Approximately 200 silk filaments are glued on a slide glass of 1mm thickness with a double-faced soft tape. (See text for details). b, Diffraction profile of a native silk, SS/N. Approximately 200 spider dragline silk filaments were carefully glued on glass slides (1mm thick) with a double-faced soft tape. By means of a conventional laboratory wide angle X-ray diffractometer (Philips X Pert MRD with 50kV, 30mA) with Ni-filtered CuKα (λ = Å) radiation, diffraction profiles were measured in 2Ө scans across the fiber diameter. The beam was perpendicular to the fiber axis (Figure 4.9a). Firstly, X-Ray diffraction (XRD) profiles of SS/N and the background (slide glass without silk) were measured over an angular range of 5 < 2Ө < 85 with a step size of 0.1 and a fixed incident angle (Ө i ) of 2. The applied exposure times were varied between 60 s/step and 90 s/step depending on the intensity of the diffraction signal. Figure 4.9b shows the XRD profile of SS/N after subtraction of the background XRD profile. Three main peaks appeared at the angular position of 2Ө A = 16.97, 2Ө B = and 2Ө C = In the case of the other samples, XRD profiles were measured over an angular range of 5 < 2Ө < 35 with the same measurement parameters like SS/N, followed by subtraction of the background XRD profile. Using a least square algorithm, all XRD profiles were interpolated with a linear combination of six Gaussian functions as follows [186].

73 [(2θ ) (2θ ) ] n I(2θ ) = I + 0 exp 2, n= 1 2wn Chapter 4 65 where Ө 0 and ω n are Bragg angle and FWHM (full width at half maximum), respectively. a b c d e f Figure 4.10 WAXS profiles of diverse samples with Gaussian interpolation functions. Using a least square algorithm, each profile was fitted with a linear combination of six Gaussian functions (See details in the text). Figure 4.10 shows the resulting Gaussian functions of each XRD profile. From the FWHM value, i.e. ω n, the average crystallite size was estimated by the Scherrer equation [187,188], L = 0.9λ / (ω n cosө 0 ). For each XRD profile, at least four profile measurements were carried out and among them one profile which had the minimum relative error to the raw data was selected. In order to obtain Gaussian interpolation functions corresponding to each XRD profile, a Compaq Visual Fortran compiler (version 7.0) and Maple (version 10.0) was used and graphical tasks were performed with ORIGIN 7.5. The two main

74 66 Metal Infiltration into Spider Dragline Silk peaks, P A and P B could be indexed as (210) and (020) according to a model system of the silks s crystal structure provided by Marsh et al. [171] or Warwicker [166]. a b c d e f g Figure 4.11 Tensile test curves of diverse silk composites. a-c, Stress(σ) - strain (ε) curves of multiple pulsed vapor phase infiltrated [(a) TMA / H 2 O, (b) TIP / H 2 O and (c) DEZ/H 2 O] dragline silks of the Araneus spider (SS/Al2O3, SS/TiO2, SS/ZnO) with a varying number of cycles ranging from 100 to 700 cycles. d, (σ - ε) curves of silk composites treated with individual pulses (SS/TP/100: TMA without water, SS/WP/100: water without TMA, see Figure 4.7) together with a curve of SS/Al2O3/100 for comparison. e, (σ - ε) curves of silks which are dipped separately into TIP (SS/TIP) or H 2 O (SS/W) for 10 hours, followed by drying at room conditions, and SS/TiO2/300 as a comparative (σ - ε) curve (the operation time of SS/TiO2/300 in the ALD chamber was around 10 hours). f, (σ - ε) curves of SS/TiO2/700/C and SS/TiO2/700 which are prepared by

75 Chapter 4 67 continuous precursor pulses with overlapping precursor vapors and discrete precursor pulses, respectively. The detailed information of SS/TiO2/700/C is described in Figure 4.8. g, (σ - ε) curves of annealed silk composites (SS/H, SS/Al2O3/300/H and SS/Al2O3/500/H) together with curves of SS/N, SS/Al2O3/300 and SS/Al2O3/500 for comparison. In all graphs, the green line indicates a (σ ε) curve of the native spider silk without any treatment (Details are described in Table 4.3). Table 4.3 Values of maximum stress (σ max ), maximum strain (ε max ) and toughness ( σdε) of the diverse samples. By linear approximation in the range of 0< ε < 0.2 %, the initial modulus was calculated. Since the silk material is combined with the outer metal oxide layer, it is difficult to define the exact strain range to calculate the initial modulus. Therefore, a slight difference to literature values might appear. (Average ± Standard Deviation) Sample # Maximum strain (%) Maximum stress (GPa) Initial Modulus (GPa) Toughness [J/m^3] *10^6 1 SS/N ± ± ± ± SS/H ± ± ± ± SS/TIP ± ± ± ± SS/W ± ± ± ± SS/Al2O3/ ± ± ± ± SS/TP/ ± ± ± ± SS/WP/ ± ± ± ± SS/Al2O3/ ± ± ± ± SS/Al2O3/300/H ± ± ± ± SS/Al2O3/ ± ± ± ± SS/Al2O3/500/H ± ± ± ± SS/Al2O3/ ± ± ± ± SS/TiO2/ ± ± ± ± SS/TiO2/ ± ± ± ± SS/TiO2/ ± ± ± ± SS/TiO2/700/C 21.49± ± ± ± SS/ZnO/ ± ± ± ± SS/ZnO/ ± ± ± ± SS/ZnO/ ± ± ± ± Results and discussion Variation of mechanical properties under diverse conditions Since an ALD process leads to thin deposited layers on the silk fibers, the question naturally arises whether the increased tensile strength is a result of these deposited layers. In the following it will be demonstrated that this is not the case. In addition, it will also be

76 68 Metal Infiltration into Spider Dragline Silk shown that (i) simple annealing or (ii) pulses of just one of the two required precursors/reactants of the ALD process or (iii) dipping the silk into the ALD precursors at ambient conditions (T = 15 and P = P atm ) or (iv) continuous mixture-pulses of two ALD precursors without a self-limiting process (similar to CVD process) do not lead to the observed large improvements of the mechanical properties. These questions were examined in experiments with three kinds of vapor-pulse pairs (i.e. TMA/H 2 O, TIP/H 2 O and DEZ/H 2 O) infiltrated dragline silks of the Araneus spider varying the numbers of pulse cycles (sample information and denotation can be found in Table 4.2). In the following, the experimental results illustrated in Figure 4.11 will be discussed shortly. A. Multiple pulses. As a first experiment, SS/Al2O3 (spider dragline silk + Al 2 O 3 by a TMA/H 2 O pulse pair) composite samples were prepared. First of all, the question whether the extensibility of the silk composites is changed, was examined. As shown in Figure 4.11a, when compared with the native dragline silk (SS/N), the maximum tensile stress before rupture as well as the extensibility were increased. As a result, the mechanical toughness was remarkably enhanced (see Table 4.3). Taking into account that thin Al 2 O 3 films generally fail by brittle fracture (fracture strain, ε f O (10-3 )) [189], it seems that, due to the outer Al 2 O 3 coating of the silk, the initial stiffness (E ini ) in the range of 0 < ε < 0.02 was increased to E ini (SS/Al2O3) 50.1~68.2 GPa (when E ini (SS/N) 9.7 GPa). However, in the further region, the increase of the maximum tensile stress and strain is independent of the contribution of the Al 2 O 3 coating. In the case of other vapor-pulse pairs, i.e. TIP/H 2 O (Figure 4.11b) and DEZ/H 2 O (Figure 4.11c), similar results were observed. From the experimental results of those three kinds of vapor pulse pairs, it was concluded that both strength and extensibility of the native spider dragline silk can be increased by MPI and the contribution of the outer metal-oxide layer coat on the silk fiber to the improvement of the mechanical properties is of minor importance. B. Individual pulses. Observing the mechanical data, the question whether separated individual pulses, e.g. water pulse exposure without TMA pulse exposure and vice versa, would also affect the silk properties, arises. In order to find out, samples subjected to individual precursor vapors (SS/TP/100 and SS/WP/100, See Table 4.2 and Figure 4.7) under the same processing conditions as the MPI samples (SS/Al2O3/100) were prepared. As shown in Figure 4.11d, the overall stiffness and the initial modulus of both SS/TP/100 and SS/WP/100 are somewhat decreased and, in contrast, the extensibility increased slightly. However, the toughness is much smaller than in the case of SS/Al2O3/100. It appears that pulses of only one reactant (e.g. TMA or H 2 O) are not effective for the enhancement of the mechanical properties of the silk and only the combination of the

77 Chapter 4 69 precursor pulses has a serious effect on the mechanical properties. Additionally, exposure times indicate to play a significant role. For this process a chamber-type ALD-setup with adjustable exposure times (exposure of the silk to the precursor vapor) was used. Exposure times of up to 30 seconds were used (see also Table 4.2). Control experiments with a flow-type ALD-reactor, where pulsing time and exposure time are identical and usually below 1 second, did not yield considerable enhancement of the mechanical properties, clearly indicating that a more complex process than simple coating of structures as it would be expected in a regular ALD process takes place. C. Dipping. In order to find out whether simple dipping of the silks into the highly reactive ALD precursor would enhance the mechanical properties of the silk, an additional experiment was performed. Various samples were prepared, which were separately dipped into the individual precursor solutions (SS/TIP: dipped into TIP and SS/W: dipped into water, See Table 4.2) under room conditions (T = 15 and P = P atm ) for 10 hours, followed by drying under the same conditions. As shown in Figure 4.11e, the initial modulus and maximum stress was increased and the maximum strain was slightly decreased as compared to SS/N. However, similar to the samples produced with individual pulses under ALD conditions, the overall mechanical properties of those composites (SS/TIP and SS/W) were still not comparable to those of SS/TiO2/300. D. Overlapping pulses. As already mentioned in Chapter 1, ALD is a self-limiting process which relies on a sequential surface chemistry. It is similar to the CVD technique, except that the ALD reaction separates the CVD reaction into two half-reactions, keeping the precursor materials separate during the reaction. Since CVD is also a thin film deposition technique and is based on continuous multiple pulsed vapor phase exposure without self-limitation, it is of interest to verify whether the mechanical improvement is particular for an MPI process or whether CVD processes lead to similar results, too. Two samples, i.e. SS/TiO2/700/C (continuous exposure) and SS/TiO2/700 (discrete pulsing by MPI) (The detailed information for the sample preparation is described in Figure 4.8) were prepared to compare the effects of CVD and MPI. As shown in Figure 4.11f, the initial modulus and the maximum stress of SS/TiO2/700/C increased. In contrast, the maximum strain decreased as compared to SS/N. However, the curve shape of SS/TiO2/700/C looks similar to the one of SS/TiO2/700 except for the reduced strain. Presumably the increase In this experiment, we have tried both a chamber-type ALD-setup with adjustable exposure times (Savannah 100, Cambridge Nanotech Inc) and a flow-type ALD-setup (SUNALE R75, Picosun, Finland) with negligible exposure times. Considerable enhancement of the mechanical properties of the silk was observed only when the Savannah 100 was used.

78 70 Metal Infiltration into Spider Dragline Silk of the maximum strain could be attributed to the discretely exposed pulses. Similar phenomena were also observed in SS/ZnO composites shown in Figure 4.11c. In principle, since the ALD process is a self-limiting process, the growth rate is relatively low as compared to CVD and the deposited film has better uniformity. In this experiment, the Al 2 O 3, TiO 2 and ZnO growth showed self-limited behaviour and their growth rates were around 1Å/cycle (Al 2 O 3 ), 0.5Å/cycle (TiO 2 ) and 2Å/cycle (ZnO), respectively. Taking into account that the growth rate of ZnO was comparatively high and the films showed relatively poor uniformity as compared to Al 2 O 3 and TiO 2, the ZnO ALD process has shown a lower self-limiting behaviour and also revealed combined features of an ALD and CVD process. As can be recognized from Figure 4.11c, all SS/ZnO composites show an increased maximum stress but the increase of the maximum strain is small as compared to SS/N or even has negative values. E. Annealing. It has been reported that heat treatment of the native silk induces structural changes in the less-oriented amorphous regions, thereby causing severe variations of the mechanical properties [ ], even though the native silk shows high thermal stability against denaturation [193]. In a further experiment, in order to investigate the effects of annealing on the mechanical properties of the resulting silk composites produced by MPI, tensile tests with silk composites, i.e. SS/H, SS/Al2O3/300/H and SS/Al2O3/500/H, which were fabricated by annealing the prepared SS/N, SS/Al2O3/300 and SS/Al3O3/500 at P = torr and T = 70 C for 15 hours were performed. The three resulting σ-ε curves are shown in Figure 4.11g together with comparative curves of the as-prepared silks. In the case of the MPI samples (SS/Al2O3/300/H and SS/Al3O3/500/H), both maximum tensile strength and maximum strain decreased, as compared to non-annealed samples (Δσ max = 25%, Δε max = 60% and Δσ max = 31%, Δε max = 44%). In contrast, SS/H showed an increased maximum stress (Δσ max = 27%) and a decreased maximum strain (Δε max = 60%) (Table 4.3). As an additional concern, the question arises whether after the MPI process the silks are contracted due to the common water precursor or other volatile precursors such as TMA, TIP and DEZ. In this experiment, only around 3 % of silk contraction was observed. Accordingly the diameter change of the silk after MPI and thus the influence of supercontraction were almost negligible. From the above mechanical property data measured at various conditions, it can be concluded that only multiple pulsed vapor phase exposure can significantly enhance the mechanical properties of the silk and the process should be performed in the pressure/temperature range applied for regular low temperature ALD processing. Further

79 Chapter 4 71 on, the process should be carried out in the chamber-type ALD-equipment allowing for long enough precursor exposure times (~30 seconds) Scientific validation of the MPI process a b c d Figure 4.12 Wide Angle X-Ray Scattering (WAXS) profile. a and b show the crystal structure evolution of silk after annealing (SS/H), MPI (SS/Al2O3/300) and additional post annealing of MPI samples (SS/Al2O3/300/H). The FWHM (w n, Full With at Half Maximum) of the native sample (SS/N) slightly decreased after annealing (SS/H) (See Figure 4.13 and Table 4.4) and the FWHM of the MPI sample (SS/Al2O3/300) clearly decreased after annealing (SS/Al2O3/300/H), as shown in b. But the FWHM of SS/Al2O3/300 from MPI (Al 2 O 3 deposition + simultaneous annealing during deposition) increased as compared to the sample simply annealed without metal oxide deposition (SS/H) as well as to the native sample (SS/N). Similar behaviour was observed in the TiO 2 deposited silk (c and d). b and d are drawn from the superposition of Gaussian profiles which are illustrated in Figure A. Validation via WAXS. The tensile strength is determined by both crystal size and orientation, and the extensibility is dependent on the molecular configuration of amorphous regions of the silk [178,179,194]. In order to detect changes in the crystal structure of the samples after annealing only and regular MPI, WAXS measurements were performed. As shown in Figure 4.12, some noteworthy differences of the crystal structure

80 72 Metal Infiltration into Spider Dragline Silk between the two types of samples can be observed. Firstly, the overall peak intensity of SS/Al2O3/300 and SS/TiO2/500 was reduced presumably due to X-ray absorption of the amorphous Al 2 O 3 and TiO 2 layer coating the silks. Further it can be observed that on peak A and B, the peak intensities of both SS/N and SS/Al2O3 decrease and the peaks get sharper at the same time after annealing, i.e. FWHM (w n, Full Width at Half Maximum) values decrease. As a result, the average size of the crystallite (which can be estimated by the Scherrer equation) increases (Table 4.4 and Figure 4.13) [ ]. In contrast, comparing SS/Al2O3 with SS/N, it can be clearly recognized that FWHM values increase after MPI, i.e. the average sizes of crystallites decrease. In general, the peak broadening of X-Ray Diffraction (XRD) patterns is determined by two factors; a) crystallite size [195], or b) lattice distortion inside the crystallites [196,197]. The broadening observed in this work, however, could mainly be attributed to the crystallite size since the spider silk is known for having high thermal stability [193]. The peak intensity ratio, i.e. I [I B,max / I A,max ], also revealed variations, as shown in Table 4.4 and Figure 4.13b. Initially, the intensity ratio of SS/N, i.e. I SS/N = [I B,max / I A,max ] SS/N, was but by MPI both I SS/Al2O3/300 and I SS/TiO2/500 increased to and 0.462, respectively. In contrast, after annealing, a differing behaviour in the intensity ratio was found; I SS/H and I SS/TiO2/500/H decreased as compared to I SS/N and I SS/TiO2/500, respectively, but I SS/Al2O3/300/H increased as compared to I SS/Al2O3/300. Furthermore, the peak positions in both cases were invariant upon annealing. Consequently, it may be assumed that the variation of I is caused by the change of the total number of the β-crystallites or by a slight change of β-crystallite s amino acid composition [193] after annealing or MPI. The regular ALD process can be regarded as a combined process of metal oxide deposition and simultaneous annealing during the process. As can be recognized from the σ-ε curve of SS/H in Figure 4.11g, simple annealing of the native silk does not contribute to an enhancement of those properties (decreased ε max ). The contribution of the outer metal oxide layer coating the silk to the increase of ε max can also be neglected since in general metal oxides, such as Al 2 O 3, TiO 2 and ZnO are very brittle. Therefore, the probability to induce the enhancement (increased σ max and increased ε max ) of the mechanical properties of the silks under a regular ALD process (i.e. the combined process of metal oxide deposition and simultaneous annealing during deposition) is low. As an evidence to support our assumption, the σ-ε curve of all MPI samples in Figure 4.11 revealed opposite stress-strain behaviour to the samples that can be assumed to be fabricated by a regular ALD process. Moreover, from XRD measurements, it was clearly observed that the size of the silk crystallites decreased after MPI and increased after annealing. Hence it can be concluded that there is a distinct difference between the regular ALD process and the MPI process, namely an additional effect beyond metal oxide deposition and annealing. In the

81 Chapter 4 73 following, it will be shown that such an additional effect can be attributed to the infiltration of ALD precursors into the molecular protein structure of the spider silk. Table 4.4 X-ray diffraction data of diverse samples. 2Ө: diffraction angles, I: intensity of each peak and FWHM: Full With at Half Maximum of each peak. (Average ± Standard Deviation) SS/N SS/H SS/Al2O3/300 SS/Al2O3/300/H SS/TiO2/500 SS/TiO2/500/H 2Ө A 2Ө B I B,max /I A,max (FWHM) A (FWHM) B ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.01 a b Figure 4.13 Evolution of the average size of silk crystallites and relative peak intensity ratio. a, The average size of silk crystallites estimated by means of the Scherrer equation [187,188], which relates the XRD peak broadening to the crystal size, i.e. L = 0.9λ / (ω n cosө 0 ) where ω n is the FWHM value (See Table 4.4) of the peak at the Bragg angle (Ө 0 ) and X-ray wave length, λ = Å (Cu K α ). b, Relative peak intensity ratio between peak A and B, i.e. I B,max /I A, max. B. Validation via NMR. In order to show the evidence for the presence of metal inside the silk, two approaches (Al NMR and TEM-EDX) were performed. Figure 4.14a and b show 27 Al MAS (Magic Angle Spinning) NMR spectra obtained from PF/Al2O3/300 and a control sample (alumina deposited on parafilm under identical processing conditions as SS/Al2O3/300 [see Table 4.2]). Figure 4.14c and d show the corresponding spectra of the MPI treated silk composite SS/Al2O3/300. Here, Al 2 O 3 treated samples were chosen, since the sensitivity of the NMR to 27 Al is much higher compared to 47 Ti or 49 Ti. A comparison with data from literature shows that the line shapes do not originate from a single type of alumina, as they do not show the spectra typically observed for α-al 2 O 3

82 74 Metal Infiltration into Spider Dragline Silk [198,199], γ-alumina [200,201] or θ-alumina [199]. Instead, the central-transition spectra in that figure appear to be a superposition of different contributions. For a more detailed analysis, the spectra were deconvoluted and fitted, using the DMFIT program [202]. The program assumes a Gaussian distribution of the chemical shift [202,203], so that line fitting delivers a value for the isotropic chemical shift, δ iso, plus the width of the distribution, dδ. For the quadrupolar coupling, a Czjzek distribution [201,204] is used in a simplified form [202,203], which results in an average quadrupolar coupling parameter, <C q >. The results of the fitting are shown in Table 4.5. Firstly, the spectrum of PF/Al2O3/300 shows contributions from three different species, which look similar to the spectrum of aluminosilicate zeolites [200] and porous alumina fabricated by anodic oxidation of aluminum [205]. From their positions in the spectrum, these three species can be assigned to four- [AlO 4 ], five- [AlO 5 ], and six-fold [AlO 6 ] oxygen-coordinated aluminum, respectively [198,199, ]. On the other hand, the spectral shape of SS/Al2O3/300 (Figure 4.14c) shows one additional component with an isotropic chemical shift of δ iso = - 4.9ppm, which indicates that in SS/Al2O3/300 the aluminum nuclei of species 4 experience a different coordination environment to PF/Al2O3/300 (species 1 to 3). Although the value of δ iso is comparatively low, it is still within the range reported in the literature for six-fold coordinated aluminum, [AlO 6 ] [208]. Significantly, a negative value of δ iso has also been observed for 27 Al in the presence of organic ligands [207], which may indicate the vicinity of organic material for species 4. Moreover, by integration of the relative line intensities of the contribution of all signals (Table 4.5), it can be seen that only 9% of the observed aluminum is located in such surroundings. All these data are consistent with the scenario of aluminum being infiltrated into the spider silk protein and/or interacting with the surface of the protein. In order to elucidate the inter-molecular bonding states or interaction states between aluminum and silk protein molecules, Raman measurements were performed, yet unfortunately no conclusive results have been obtained. C. Validation via EDX. Figure 4.15a shows a TEM image of SS/TiO2/500. As shown in this figure, along the TiO 2 shell, a region of ~100nm in depth shows in a high image contrast. Considering the relative weight ratio of carbon, oxygen, and Ti, a large amount of Ti was infiltrated in this shell region, which can also be confirmed from the result of the EDX line scan (Figure 4.15b). In the central part of the silk (folded region), EDX point analysis (Figure 4.15c) showed weak but clear Ti signals (1.42 ~ 2.83 % by relative weight ratio). Because the resolution limit of the system amounts to about 0.5 ~ 1%, the spectrum under those limits has not been quantified. Qualitatively, the small amount of Ti shown as the Ti-K peak is well above the background.

83 Chapter 4 75 a b c d Figure 4.14 Nuclear Magnetic Resonance (NMR) spectra of silk composites, SS/Al2O3/ Al NMR central-transition spectra, recorded with a magic-angle spinning (MAS) rate of 20 khz. Deconvolution and fitting of the spectra was performed using the DMFIT program [202], assuming a statistical distribution of both chemical shift and quadrupolar interaction for each species present. a and b, Experimental spectrum of parafilm, PF/Al2O3/300 and its line shape fitted with 3 contributing species. c and d, Experimental spectrum of SS/Al2O3/300 and its line shape fitted with 4 contributing species. Table 4.5 NMR parameters. These parameters are obtained from deconvoluting and fitting the 27 Al NMR spectra shown in Figure 4.14 with the DMFIT program. Listed are the isotropic chemical shifts, δ iso, the width of the distribution, dδ, and the average quadrupolar coupling parameter, <C q >, plus the relative integrated intensities of the spectral lines. Samples Signal δ iso / ppm dδ / ppm <C q > Integrated Line Intensity % PF/Al2O3/ % % % SS/Al2O3/ % % %

84 76 Metal Infiltration into Spider Dragline Silk a b c Figure 4.15 EDX measurements of a silk composite, SS/TiO2/500. a, TEM image of microtomed SS/TiO2/500. The prepared samples originally should show a disk shape, but the silk folded and the TiO 2 layer broke and dispersed in some regions. It is assumed that such distortions arose during the microtoming process because of the small thickness (~90 nm) of the silk disk. The TiO 2 layer and the silk part (SS/TiO2/500) showed similar image contrast. Ti infiltration into the silk was observed along the whole TiO 2 shell [inset (I)]. b, Element concentration from EDX scanned across the boundary region along the TiO 2 layer (A to B in the pink box; R1, R2, and R3 designate the region of the carbon grid, TiO 2 layer, and silk part of SS/TiO2/500, respectively). c, EDX spectrum measured on the folded silk part located at the center of the silk, Ti X-ray emission peak K α at 4.5 kev and K β at 4.9 kev Model system for the metal infiltration mechanism The exact infiltration mechanism and the intermolecular bonding states between proteins and metals have not yet been determined. However, considering the severe attack of water occurring at the hydrogen bonds that interconnect the proteins [209], a global weakening of the hydrogen bonds in proteins with increasing water vapor temperature [210] can be assumed. In addition, considering the strong reactivity with functional sites [11] and deep penetrating capability of the metal-containing ALD precursors (such as TMA, TIP and DEZ) into soft materials such as polymers [59] and the metal ions preferential binding features to proteins [211,212], this could lead to stable metal-protein compounds by chelating ions [213,214] such as Al 3+ [215], Ti 4+ [216] or Zn 2+ [217]. During the long exposure times to water vapor (5 to 40 s) **, the inner hydrogen bonds of the silk protein ** In the ALD process, ZnO deposition has shown relatively poor self-limitation. With longer exposure times, the process shows parasitic chemical vapor deposition (high film thickness with relatively poor uniformity). Therefore, in the case of ZnO, an exposure time of only 5 s was applied.

85 Chapter 4 77 are likely to weaken or break in some regions upon water vapor attack at 70 C. Subsequently after long-term exposure to the metal precursor vapor, Al 3+, Ti 4+ or Zn 2+ is likely to infiltrate the protein and bind to the broken bonding sites, thereby resulting in the formation of metal-coordinated or even covalent bonds. As a minor additional effect, methane (TMA/H 2 O) or isopropanol (TIP/H 2 O) as reaction byproducts may have additional weakening effects on the remaining hydrogen bonds. Consequently, the recoverable hydrogen bonds may be changed to permanent covalently bound or coordinated Al-, Ti- or Zn-protein composites. Therefore, unlike native dragline silks, which are highly sensitive to environmental conditions such as humidity and temperature (mainly caused by the hydrogen bond breaking and recovery feature) [161], the metalinfiltrated silks, which presumably may have covalent or coordinated bonds, are hardly affected by these conditions. formation of metal-coordinated or even covalent bonds with Al. Figure 4.16 Schematic of a proposed metal infiltration mechanism into the silk protein. It is likely that, due to the applied vacuum and elevated temperatures, the silk looses a significant amount of incorporated water, thus opening channels for the infiltration of the gas phase precursors such as TMA and water. Subsequently, the proteins strongly interact with the highly reactive TMA species during repeated longterm exposure (reaction of TMA with unsaturated bonds was already shown in [23]), at elevated temperatures, which, together with the water pulses, weakens the hydrogen bonds. Consequently, Al 3+ is likely to be inserted at those sites, thereby resulting in the It has been observed that in the case of the metal-infiltrated silks, the contraction ratio produced by humidity up to 100% or temperature up to 70 C is in an almost negligible range (<3%).

86 78 Metal Infiltration into Spider Dragline Silk Model system for mechanical property improvements of silk Figure 4.17 Schematic description of proposed molecular changes in the silk produced by MPI. The detailed explanation can be found in the text. By the above described WAXS measurements of SS/Al2O3/300 and SS/TiO2/500, a structural change of the inner-protein matrix, caused by metal incorporation, was observed. More specifically, the size of silk protein β-sheet crystallites decreased after the MPI treatment. The strength of the silk is strongly dependent on the sizes of the polyalanine crystals, which are either small and perfect or large and imperfect [218], as proposed by Termonia [178,179] and experimentally validated by Du et al. [194]. The extensibility of the silk is determined by the orientation and the amount of amorphous chains, as proposed by Termonia [178,179] and elucidated by Lefèvre et al. [219]. From these WAXS results (a decrease in the size of the protein crystallites), it could be easily conjectured that parts of the protein crystals (probably large imperfect crystallites) are likely to be converted into protein chains by hydrogen bond breaking and metal substitution caused by MPI, thus resulting in additional amorphous regions. From the theoretical model by Termonia and supportive experimental results by Du et al. [194] and Lefèvre et al. [219], it is likely that additional amorphous regions and size-reduced protein crystallites, induced by metal

87 Chapter 4 79 infiltration, function as factors to enhance σ max and ε max of MPI-treated silks (Figure 4.17 and Figure 4.18). a b c Figure 4.18 The relation between the size of silk crystallites/the orientation and amount of amorphous parts and corresponding mechanical properties of silk. a, Experimental results by Du et al. [194]. The faster speed of silk drawing makes the silk stronger and stiffer. Based on this idea, they verified the variation of silk crystallite sizes after varying the silk reeling speed and measured the corresponding mechanical behaviour. Since the silk drawing speed does not affect the amount of amorphous parts of silk [194], their finding proves that smaller crystallite sizes in silk protein increases the strength of the silk. b,theoretical model by Y. Termonia [178,179]. From computational results he proposed that silk with smaller crystallites and an increased amorphous fraction can increase both strength and extensibility. c, The stress (σ)-strain (ε) curves of native silk, SS/N and MPI treated silk, SS/Al2O3/300. As compared to the σ-ε curve C2 in Termonia s model (in b), the σ-ε curve of SS/Al2O3/300 (curve C4 in c) corresponds well except for the increase of the yielding stress, Δσ y, and initial Young s modulus, E i of SS/Al2O3/300. On the other hand, it was observed that the individual TMA or H 2 O pulses produced no major enhancement of the mechanical properties of the silk composites (Figure 4.11d). It appears that even though the individually pulsed vapor molecules contribute to the severe hydrogen bond breaking in the silk, because of the strong recovery behaviour of the hydrogen bonds, some of them are reestablished. The resulting silks show an analogous curve profile to those of slightly contracted silk fibers, caused by local hydrogen bond breaking [164]. It was also observed that direct dipping of the silk into the pure liquid TIP

88 80 Metal Infiltration into Spider Dragline Silk precursor at ambient conditions shows a slight enhancement in mechanical properties (increased σ max of SS/TIP in Figure 4.11e), similar to the hardness enhancement of the polychaete worm s jaws caused by Zn incorporation through incubation in ZnCl 2 solution at 25 C [182]. Unlike TIP, which has low polarity [220], water causes a serious shrinkage of the silks by destruction of hydrogen bonds in silk proteins (mainly amorphous regions) [221]. It is speculated that subsequent drying of the contracted silk fibers under axially restrained conditions, due to fixing the silk fibers on a paper clip (see Table 4.2 for the details of the sample preparation), leads to better alignment of the amorphous protein chains [222], resulting in decreased strain and slightly increased stress [223] (see result for SS/W in Figure 4.11e). 4.4 Conclusion By Multiple Pulsed Vapor Phase Infiltration (MPI) the native spider silk can be chemically or physically modified and the resulting silk composites show a surprising level of enhancement of the mechanical properties. Although many scientific questions, such as the diffusion mechanisms of ALD precursors into the silk protein structure, variation of internal bonding states induced by infiltrated precursors and its corresponding chemical/physical property changes (like protein mechanics), remain open, it is believed that the MPI process has strong potential to be widely used. Applied to other biomaterials, such as B.mori silk fibers and artificial spider silks as well as collagen aimed at tissue remodeling (in Chapter 5), might allow for an improvement of their mechanical properties.

89 Chapter 5 81 Chapter 5 Metal Infiltration into Collagen Creation of collagen-based tissue equivalents, which are functionally similar to natural tissues, is a longstanding challenge in the field of tissue engineering. Biochemical approaches lead to artificial collagen fibril production based on self-assembly of synthetic collagen gels. However, since such collagen fibrils do not yet show satisfactory mechanical properties to replace native collagen tissues, controlling the chemical composition appears to be the only way to overcome such restrictions. In this work we demonstrate a method to improve the mechanical properties of collagen tissues by modifying the molecular structure of collagen which can be performed by metal infiltration. The metal (Ti, Al and Zn)-infiltrated collagen tissues show simultaneous enhancement of both strength and ductility (more than 3-fold increase of breaking energy), as compared to native collagen. The approach to infiltrate metals into collagen could be a means to improve the mechanical properties of collagen-based tissue equivalents. The fracture behavior of metal infiltrated collagen tissue could serve as an example of a designed improvement of protein biomechanics. 5.1 Collagen and tissue engineering The term collagen is derived from the Greek word for glue and was initially described as the constituent of connective tissue, which yield gelatin on boiling. However, now it has been proven that in some tissues, collagen is present in either heavily cross-linked or covalently bound in some other stable structures, which prevents an extraction by just heating. About one quarter of all proteins in most mammals are classified as collagen. Collagen is a fibrious protein. Fibers (primarily type I collagen), which are composed of bundles of collagen, are a major component of the extracellular matrix [224,225] (ECM).

90 82 Metal Infiltration into Collagen The structures support most of the tissues in animals and act as benign native scaffolding for arranging cells within connective tissues. For most of the common connective tissues in the body such as bone, teeth, tendon, cornea, cartilage, blood vessels and skin, the collagen fibers and their networks, which have a highly organized 3D architecture and surround cells, function as the ECM. a b Figure 5.1 Metal (e.g. Ti) infiltration by MPI process. a, Two alternating pulses of reactant vapors, i.e. TIP and water, are introduced into a vacuum chamber and purged from the chamber in multiple repeating cycles. b, From the gas phase, Ti can infiltrate soft materials such as collagen tissues and subsequently the physical/chemical characteristics of those materials can be modified. In a, (E) and (P) denote exposure and purge, respectively. The main objective of tissue engineering is to develop artificial tissues which are functionally equivalent to damaged or lost tissues in diverse vertebrates. Because the function of those tissues is highly dependent on the structure of the ECM, it is necessary to recreate the required ECM with an appropriate micro-architecture. Since the collagen is the primary component of the ECM, research on collagen has inspired tissue engineering with a considerable attention being paid to the artificial creation of collagen-based tissue equivalents. Such collagen based materials promise biologically mimicked tissues with large physical and chemical similarity to the native ones. Properly designed, they may replace damaged or lost tissues [ ]. Up to now diverse strategies for the biochemical synthesis of such artificial tissues with various forms of gels, fibers and membranes have been reported [228,229]. Mechanical property tests of those artificial tissues under a variety of conditions have proven that at least the mechanical response of the artificial tissue shows similar behaviour to the native one. Although similar in behaviour, the collagen-based tissue equivalents do not yet exhibit mechanical properties good enough to replace native tissues, significantly hindering the progress of tissue engineering [226,227]. Here a method for improvement of the mechanical properties of collagen tissue by metal infiltration performed with Multiple Pulsed Vapor Phase Infiltration (MPI) is introduced (Figure 5.1) [6]. By MPI, Ti, Al and Zn were infiltrated

91 Chapter 5 83 into natural collagen membranes (primarily composed of type I and V collagen) collected from shell membranes of chicken s eggs [ ]. Upon infiltration the deformation behaviour under uniaxial tension showed a simultaneous increase of strength and ductility. ε The toughness (breaking energy, i.e. F where σ, ε and ε F are stress, strain and 0 σdε fracture strain, respectively) increased over 3 times as compared to untreated collagen membranes. Rather than macroscopic fracture, the fracture behaviour appeared to be governed by molecular fracture resulting from the stretching of the protein backbone and uncoiling of collagen triple helices. 5.2 Structure of Collagen Figure 5.2 Molecular structure of glycine (Gly), proline (Pro) and Hydroxyproline (Hyp). The 1950s was the golden period for structural biology. The correct structure for the α- helix and the β-sheet in proteins were proposed in 1951 by Linus Pauling s group and immediately confirmed to be correct by X-ray diffraction analysis. Several groups were actively working on solving the structure of the fibrous protein collagen, as well as of deoxyribonucleic acid (DNA). At that time, the structure of collagen was realized to be unique among other fibrous or globular proteins. Many models, which fit the high angle diffraction pattern, were proposed. After the theory of the helix diffraction was elucidated, in 1953 Cohen and Bear [233] recognized that kangaroo tail tendon collagen had a helical conformation with a 7 2 symmetry. However, its three-stranded nature was not realized until the sequence data and steric constraints were taken into account. The critical assumptions for the correct model were that glycine (Gly) (Figure 5.2), which constitutes about 33% of all residues in collagen, would be present as every third residue in the sequence and that high amounts of proline (Pro) and hydroxyproline (Hyp) could be accommodated while maintaining planar peptide bonds and not violating the standard atomic distances and angles set out by Pauling and Corey [234]. The close packing of the three chains near the axis explained the requirement for Gly as every third residue along each chain, generating the observed (Gly-X-Z) n pattern, where X and Z are frequently Pro

92 84 Metal Infiltration into Collagen or Hyp, respectively. Indeed, the most commonly found triplet in collagen chains is Gly- Pro-Hyp. It was subsequently proposed by Ramachandran et al. [235] and Rich et al. [236] that this three-chain structure has to be supercoiled to fit the observed fiber diffraction data. Improved fiber diffraction patterns and analysis led to a refined model for the triple-helix [237], but it was not possible to crystallize collagen or large collagen fragments. With the advance in solid phase synthesis, collagen-like peptides proved to be a valuable resource for biophysical studies and finally for crystal structure determination. In 1981, crystals of (Gly-Pro-Pro) 10 were used to obtain a high resolution molecular structure for an average tripeptide unit [238], although they turned out not to be single crystals. More recently, a single crystal of a peptide containing Gly-Pro-Hyp triplet repeats, as well as a single Gly Ala substitution, was solved to high resolution, providing the first detailed parameters of a triple-helix [239]. Figure 5.3 Supercoiled structure of collagen and its radial projection. A collagen molecule is a right-handed triple helix, composed of three left-handed polypeptide chains in the form of (Gly-X-Z) n, which are themselves helical, coiled around each other. For the demonstration of the detailed molecular structure of collagen, a radial projection of a single polypeptide chain which has 10 protein motifs in three turns with a translation of 2.86 ~ 3.00 Å is drawn based on the description by Rich and Crick [236]. Dashed arrows (blue, black and red) in the right figure represent single polypeptide chains (chain 1, 2 and 3) and dotted lines represent the backbone of the collagen triple helix.

93 Chapter 5 85 Based on the above results, it is recognized that the collagen superfamily contains at least 19 proteins that are formally defined as collagen and additional 10 proteins that have collagen-like domains. Notwithstanding the remarkable progress in structural biology, the collagen defies any simple definition and still the collagen is characterized on the basis of above-mentioned constitutive and structural features. So, two sets of main characteristics can differentiate collagen from other proteins. First is the amino acid composition which is distinctive in its very high contents of glycine residues (~ 33%) and the proline (Pro) and hydroxyproline (Hyp) residues (Figure 5.2). Second is the composition of three polypeptide chains (as already mentioned above), each of which contains regions with a repeating amino acid motif [Gly-X-Z] [ ]. Each polypeptide chain (α-chain) has left-handed helical conformation with three identical α-chains constituting a right-handed coiled-coil triple-helical structure (Figure 5.3). All Gly residues are buried inside the core of the protein and residues X and Z are exposed to the surface. 5.3 Collagen of a chicken s eggshell membrane Table 5.1 Amino acid composition of inner shell membrane of a chicken s egg. The data in this table is extracted from the report of Nakano et al. [232]. Amino acid Mol % Amino acid Mol % Asn/Asx (Asparagine/Aspartic acid) 8.4 ± 0.4 Leu (Leucine) 5.6 ± 0.5 Thr (Threonine) 6.9 ± 0.0 Tyr (Tyrosine) 2.2 ± 0.1 Ser (Serine) 9.2 ± 0.2 Phe (Phenylalanine) 1.6 ± 0.1 Gln/Glx (Glutamine/Glutamic acid) 11.1 ± 0.4 His (Histidine) 4.1 ± 0.4 Gly (Glycine) 11.1 ± 0.2 Lys (Lysine) 3.6 ± 0.2 Ala (Alanine) 4.6 ± 0.2 Arg (Arginine) 5.7 ± 0.3 Val (Valine) 7.2 ± 0.2 Pro (Proline) 11.6 ± 0.7 Met (Methionine) 2.3 ± 1.0 Hyp (Hydroxyproline) 1.5 ± 0.3 Ile (Isoleucine) 3.3 ± 0.4 As already described in Chapter 3, the chicken eggshell comprises a calcified shell and inner/ outer shell membranes (Figure 3.4). The two shell membranes surround the egg of most avian species in a way that a thick outer membrane is attached to the shell and a thin inner membrane is exposed to the egg. Each of those membranes is composed of a network of fibers. The membranes retain albumen and prevent penetration of bacteria. The shell membranes are also essential for the formation of the eggshell. The constituents of the eggshell and the shell membranes are proteins with small amounts of carbohydrates and lipids [240]. From several studies, it has been reported that the chicken eggshell membrane contains type I, V and X collagens [ ]. The detailed amino acid composition of the inner shell membrane is summarized in Table 5.1.

94 86 Metal Infiltration into Collagen 5.4 Biomineralization of collagen architectures As already described, collagen is one of the most common proteins found in nature and an abundant constituent of many of the tissues in the body of humans and other vertebrates. It provides a number of important functions, among them the capability to become mineralized in tissues such as bone, cartilage, tendon, dentin and cementum. Mineralized collagen offers (1) the framework for mechanical support subjected by the skeleton; (2) a reservoir for cations, anions and small molecules; and (3) a means for strain energy storage [241,242]. Mineralized collagen has also the potential to be utilized as a biomimetic material to augment or repair calcified tissues that may be impaired, diseased, or damaged. A number of scientists made an effort to fabricate such a functional structure and contemporarily an effort to verify the mechanism by which collagen becomes mineralized has continuously been attempted. However, up to now the precise molecular events leading to collagen mineral interactions are not completely understood. The structure of the major type I collagen in vertebrates and its assembly into higher order arrays appear to dictate the location and organization of the mineral associated with it. Type I collagen is a triple helix consisting of three left-handed polyproline peptide chains interwined in a right-handed fashion. Two of the three chains have an identical amino acid sequence. These two α1(i) chains are distinct from the slightly different amino acid composition of the α2(i) chain. The triple helix of type I collagen is approximately 300 nm long and 1.5 nm wide and consists of about 1000 amino acids per chain. In vivo, type I collagen self-assembles in two-dimensions by forming molecules staggered by approximately 22% of their length with respect to their nearest neighbor. This so-called quarter-staggered model [243] (by Hodge and Petruska), has two defined regions, a hole or gap (~47 nm in length) and an overlap (~20 nm in length) zone, that comprised the periodic staggered distance (Figure 5.4) [244,245]. Recently collagen was found to be packed in three dimensions through strict and contiguous alignment of its composite hole and overlap zones. This exact registration of the hole and overlap sites provides channels or gaps in their assemblies where the detectable crystals of apatite nucleate [246]. On subsequent independent growth and development of crystals within many hole zone channels and overlap sites, the mineral is elaborated as thin, irregularly shaped platelets [247]. These small particles develop preferentially longitudinal along their crystallographic c-axes. In addition, they become oriented with their c-axes generally parallel to each other as well as to the long axis of the collagen fibrils accommodating them [247,248]. Growth and development of the width of the platelets occur along the contiguous collagen hole zone channels [247]. A further result of the development of platelets is that they are coplanar and form a series of parallel crystal sheets [247,249].

95 Chapter 5 87 The mechanism by which type I collagen assembles in two- and three-dimensions in vivo is not entirely clear. But it has been believed that these features of collagen mineral interaction presumably depend in part on at least two factors: (1) the cross-linking feature of the molecules [250] as well as specific stereochemical properties of the particular amino acid residues; (2) the nature of their bonding comprising the collagen hole and overlap zones and the hole zone channels. Figure 5.4 Structure of the collagen assembly and the mineralization process. This model was proposed by Landis et al. [246]. The schematic shows the cross-linked pattern of individual molecules (S2) into two-dimensional quarter-staggered arrays of holes or gaps (~40nm) and overlap zones (~27nm) suggested by Hodge and Petruska [243]. These collagen arrays pack and assemble in three-dimensions to create hole zone channels (marked by yellow arrows in S3) or gaps as a consequence of the exact registration of hole and overlap sites among molecules (S3). The plateletlike crystals of mineral (apatite) nucleate principally within the hole zone channels and become oriented in the process (S4). The development of crystals is characterized by preferential growth in their crystallographic c-axial length along to the collagen long axis and growth in width along the hole zone channels (S5). The mineral platelets are coplanar as they grow, a result presumably dictated both by the specific stereochemistry, including the nature of the flexible regions, comprising the hole channels and by the cross-linking character of the collagen assembly. Research has been performed to verify the effect of mineralization on changes in mechanical properties of collagen fibers. Among those collagens, the type I collagen fiber has been of primary concern. It has been known that for natural tissues, such as insect s cuticles, generally mineral compounds make materials stiff (or hard). This stiffening, induced by mineralization, brings the undesirable mechanical property of brittleness (reduced ductility) [ ]. Similar to mineralized insect s cuticles, type I collagen fibers show similar mechanical behaviour. Figure 5.5 shows the mechanical deformation behaviour of mineralized turkey tendon (composed primarily of type I collagen) under uniaxial tensile load. As the mineral contents increase, the stiffness (initial Young s

96 88 Metal Infiltration into Collagen modulus) increases, but the fracture strain value decreases. Namely, with rising mineral content, the materials become stiff, but one must pay for reduced ductility. Figure 5.5 Stress-strain curves of mineralizing turkey tendons with different mineral weight fractions. This graph is reproduced from the report by Silver et al. [254]. The values shown in parentheses are the infused mineral weight fraction into the tendon. Further details of this curve and explanation are given in the reference. 5.5 Experimental Preparation of the collagen membrane (CM) from a chicken s eggshell matrix An overview of the experimental steps is given in Figure 5.6. Some of the steps were already described in Chapter 3. Chicken s eggs were purchased from a grocery store. They were gently broken and the collagen around their air cell portion [135] was carefully collected and cut out (Figure 5.6a). The collagen membrane was washed with deionized water several times in order to thoroughly remove the thin albumin layer and subsequently dried at room temperature for 4 hours MPI process Sample CM/Al 2 O 3, CM/TiO 2 and CM/ZnO. The prepared collagen membrane was placed in an ALD reactor (Savannah 100, Cambridge Nanotech Inc.) and dried at 70 C for 5 min in vacuum ( torr) with a steady Ar gas stream (20 sccm). For the infiltration, TMA/water, TIP/water, and DEZ/water pairs were used as sources for aluminum/oxygen, titanium/oxygen and zinc/oxygen, respectively. The TMA, TIP, DEZ were purchased from Sigma Aldrich. Each cycle was composed of a pulse, exposure and purge sequence for each precursor. More specific information on the ALD processes is given in Table 5.2

97 Chapter 5 89 a b c d e f g Figure 5.6 Overview of the experimental procedure. a, Photograph of a native collagen membrane from a chicken s eggshell. b, Al, Ti and Zn infiltration by the MPI processes. c, Tensile test using a cardboard jig for supporting collagen membranes during the test. d, Energy dispersive X-ray (EDX) analysis. e, Raman measurement. f, Wide angle X-ray scattering (WAXS) measurement. g, Small angle X-ray scattering (SAXS) measurement.

98 90 Metal Infiltration into Collagen Table 5.2 Detailed processing conditions of prepared samples and their denotation. All vapor infiltration experiments were performed at 70 C substrate temperature. Resulting materials Vapor pulse Pairs Pulse (sec) Exposure (sec) Purge (sec) Cycle Samples 1 2 [1] CM/N [2] SG/N 3 TMA CM/Al2O3 Al 2 O 3 4 H 2 O [3] Si/Al2O3 5 TIP CM/TiO2 TiO H 2 O [4] Si/TiO2 7 DEZ CM/ZnO ZnO 8 H 2 O [5] Si/ZnO [1] CM/N: Native collagen membrane (CM) of a chicken s eggshell. [2] SG/N: Bare slide glass (SG). [3] Si/Al2O3, [4] Si/TiO2, and [5] Si/ZnO: Silicon wafer (110) on which Al 2 O 3, TiO 2 and ZnO are deposited together with CM/Al2O3, CM/TiO2, and CM/ZnO under the same processing conditions, respectively Tensile tests The experimental conditions for tensile test were partly described already in Chapter 3. A more detailed description is given here. For the measurement of the engineering stress (σ) - strain (ε) behaviour of the prepared samples, all eggshell membrane samples (2 mm 1cm) were cut with a knife (BAYHA, Blades, No.24). All samples were mounted in a thick paper jig having 20 mm punched holes (Figure 5.6c). The paper jigs were used to facilitate alignment and clamping of the specimens during the tensile test. With the support of the jigs, the sample was held straight as it was clamped, furthermore the jigs allowed to cut easily through the cutting line so that specimen and paper jigs were not loaded together during the actual test. Pattex Blitz Kleber (Henkel, Germany) was used as glue to fix the sample to the edge of the jig. The tensile test was performed on a ZWICK 1445 tensile test machine with 500g HBM load cell with 0.1mN resolution and 0.5% uncertainty, controlled by a PC having automated testing software. The upper part of the jig was fixed to the load cell by a screw type clamping system and subsequently the jig was cut through the middle line with scissors. After controlling the vertical alignment of the specimen attached to the jig, the lower part of the jig was also fixed in the same way as the upper part. The extension rate was 50 % of the initial length per minute (10 mm/min). The temperature and relative humidity were C and %, respectively. The

99 Chapter 5 91 sample was extended until fracture occurred. Force (mn)-strain (%) data for each specimen were exported from the software of the machine and subsequently the data were rescaled into the engineering stress (σ)-strain (ε). A JSM-6340F scanning electron microscope and optical microscope (Leitz Aristomet) were used to measure and confirm the cross section area of the collagen membrane specimen [thickness (~100 μm) and width (~2000 μm) of the membrane]. Since the thickness of the membrane was not perfectly uniform, along the horizontal direction of each sample (2000 μm), typically at more than 20 points the thicknesses were measured (with 1 μm range of minimum resolution limit) and averaged (~100 μm). After the tensile tests, the cross section of the fracture surface of each sample was again investigated by SEM to measure the thickness. Because of the slightly stretched length of the sample, the cross section was slightly shrunk. However, the area difference was almost negligible. In the case of the width of each sample, a similar procedure was applied. Before the tensile test, using an optical microscope and an SEM, the widths of the sample at more than 30 points were carefully measured (with 10 μm range of minimum resolution limit) and averaged. Like the measurement of the thickness, after the tensile test, the fractured cross sections were again observed to measure the width by an optical microscope and a SEM. The variation of the width was found to be negligible. For the measurement of each sample, more than 10 individual samples were prepared in the same way and measured at identical conditions. Most of the data showed similar stress-strain behaviour. For each measurement one typical data set was selected. All graphic works, including data rescaling, were performed with ORIGIN Cross section sample preparation by focused ion beam (FIB) Collagen membrane samples were selected and pieces of about 5 x 5 mm were cut out. Those pieces were attached to a sample holder with carbon paste. To avoid electrostatic charges by the electron beam of the SEM, a platinum layer (~10 nm thick) was deposited on each sample by PECS (precision etch and coating system). Areas for cross sectioning were selected and a bar of platinum with an area of 20 x 2 µm 2 was deposited in two steps: initially about 50 nm by electron beam deposition and afterwards about 2 µm by ion beam deposition. Slices were cut out with an ion beam under a high beam current and the samples were transported to a special TEM grid with a mounted OmniProbe manipulator which was placed on a holder at the sample stage. Finally the samples were fixed by platinum deposition.

100 92 Metal Infiltration into Collagen SEM, TEM and EDX The morphology of as-prepared collagen membrane samples was examined by scanning electron microscopy (JEOL JSM-6340F). The characterization of cross-sections of membrane samples (CM/N, CM/Al2O3, CM/ZnO and CM/TiO2) prepared by FIB was performed with a JEOL JEM-1010 (100 kv). EDX examinations (line scans and point analyses) were carried out with a Philips CM20FEG and FEI TITAN microscope in scanning mode Raman spectroscopy Micro-Raman scattering spectroscopy was employed in order to decipher the structural and chemical modifications of collagen membranes induced by metal infiltration. Raman measurements were carried out at room temperature in backscattering geometry with a LabRam HR800 UV spectrometer with following laser lines: 633 nm He-Ne laser, 514 and 488 nm Ar ion laser, and 325 nm He-Cd laser. To avoid local heating effects, the laser power density was kept below 100 μw/μm 2. Data obtained from different objectives, corresponding to different laser spot diameters on the sample at a fixed laser power, confirmed the absence of the heating effects. The backscattered Raman light is diffracted by a 1800 g/mm grating and detected by a charged coupled device camera. The spectral distance between adjacent channels was ~0.5 cm -1. The spectra were recorded in a wavenumber interval of cm -1 under the extended range configuration. The spectra measured with 488 and 514 nm lasers show a strong fluorescence background, which limits the extraction of meaningful data. In this work, only data recorded with the 633 nm laser are presented Wide angle X-ray scattering Collagen membrane samples were carefully attached to a slide glass (76 mm length x 26 mm width x 1 mm thickness) with a soft tape (Figure 5.6f). By means of a conventional laboratory wide angle X-ray diffractometer (Philips X Pert MRD with 50kV, 30mA) with Ni-filtered CuK α (λ = Å) radiation, diffraction profiles were measured in 2Ө scans. Firstly, X-Ray diffraction (XRD) profiles of all samples were measured in an angular range of 10 < 2Ө < 45 with a step size of 0.1. The applied exposure time was 90 s/step. In the case of CM/N and CM/TiO2, additionally measurements with a step size of 0.1 and an exposure time of 120 s/step in an angular range of 12 < 2Ө < 20 and 26 < 2Ө < 42 were performed. For each sample, to confirm the reproducibility of the spectra under the same conditions, the measurement was repeated at least 4 times. After measuring

101 Chapter 5 93 diffraction profiles of each sample, the background diffraction profile was subtracted from each profile. Graphical tasks were performed with ORIGIN Small angle X-ray scattering All Small-angle X-ray scattering experiments were performed using a RIGAKU rotating anode. The X-ray beam was monochromated with an Osmic X-ray optics (λ=1.54 Å). The size of the beam on the sample was approximately μm. The samples were placed in a chamber which was evacuated to a pressure of approximately 10 1 mbar (right figure in (Figure 5.6g). A Siemens area detector with channels was used to count the scattered intensity. Typical measuring times were 90 min. All measurements were performed at room temperature. 5.6 Results and discussion Mechanical deformation behaviour As already mentioned, mineralization of the collagen occurs only within limited zones (60~70 nm distance) mainly by electrostatic interactions between charged amino acids and minerals on the fibrils surface [255,256]. Mineralization does not occur inside the bulk of the collagen [255]. Accordingly, the mineral distribution in bones and tendons is macroscopically not uniform [255,257], which makes mineralized collagen structures strong but also leads to brittleness (loss of ductility) [ ]. However, under uniaxial tensile tests, the metal (Ti, Al or Zn) infiltrated collagen membranes showed rather different deformation behaviour from those mineralized collagen architectures. The metal infiltrated collagen membranes showed both an increased strength and an increased ductility as compared to native collagen membranes (CM/N) [6] (Figure 5.7, sample denotation and mechanical data can be found in Table 5.2 and Table 5.3). While, under uniaxial tension, the fracture stress and strain values of CM/N were around 6.2 MPa and 6.1 %, respectively, the values for metal infiltrated collagen membranes rose to ranges 8.8 ~ 11.8 MPa and 8.1 % ~ 10.2 %. Since ALD processes lead to very thin films, one may question whether the increased fracture stress is caused by the deposited outer metal oxide layer. Taking into account that a metal oxide film fails by brittle fracture, the contribution of the outer metal oxide layer appears to be minor. Among those collagen membranes, Ti infiltrated membranes (CM/TiO2) showed the best improvement in terms of both σ F (fracture stress) and ε F and therefore will be discussed in more detail. It is notable that the Ti infiltrated collagen membranes in Chapter 3 showed decreased mechanical properties. This seems to be caused by the thermal decomposition or damage of collagen during

102 94 Metal Infiltration into Collagen processing at 275. Data for collagen membranes infiltrated with Al and Zn can be found in the Appendix. Figure 5.7 Deformation behaviour of metal infiltrated (Al, Zn and Ti) collagen membranes under the uniaxial tensile test. Sample denotations and fracture stress/strain, yield stress/strain and toughness values can be found in Table 5.2. and Table 5.3. The arrows on each stress-strain curve mark saw tooth patterns (See details in the text). Inset: SEM picture of a Ti infiltrated (CM/TiO2) membrane. Table 5.3 Values of fracture stress(σ F ) / strain(ε F ), yield stress(σ Y ) / strain(ε Y ), stiffness(e s ), dissipative strain (ε F -ε Y ), brittleness measure [BM, (ε F -ε Y ) / ε Y ] and toughness ( σdε) of the diverse samples.. For the dissipative strain, a definition given by Buehler [262] was used. The ratio between (ε F ε Y ) and ε Y was defined as a brittleness measure (BM). The smaller BM is, the more brittle-like is the material. (Average ± Standard Deviation) Samples stress [MPa] Fracture strain [%] *stress [GPa] *Yield *strain [%] *E s (MPa) (ε F ε Y ) (ε F ε Y ) /ε Y Toughness σdε F [MJ/m^3] 1 CM/N 6.2 ± ± ± CM/Al2O3 8.8 ± ± ± CM/TiO ± ± ± CM/ZnO 10.5 ± ± ± 5.6 * Since a yielding point is rather subjective, for the extraction of the yielding point of each curve in Figure 5.7, only one stress-strain curve for each sample was used. Therefore, ε F, ε Y and E s do not have standard deviation values and those values might be different to those from literature. For the calculation of yielding points of each curve, based on least square algorithm, interpolated third order polynomials ( σ ( ε ) = 3 anεn,[0 ε ε ] n = 0 F )of 2 d σ ( ε ) each curve in Figure 5.7 were obtained and the inflection points ε i,( = 0 ) were calculated. On these 2 dε

103 Chapter 5 95 inflection points, from the slopes (α) of each curve defined by dσ ( ε ) and the σ-intercept values β, line α = dε equations, f ( ε ) = αε + β, corresponding to each curve were calculated. From the absolute convergence value defined by ξ ε ) = σ ( ε ) f ( ε ) in the range of ( n n n that point was defined as a yielding point σ, ε ). ( Y Y i ε F ε = εi ε < ε, when the value ξ ( ε n+ 1) becomes larger than 2, ξ ( ε ) n Looking at the stress-strain curves, a noteworthy feature of the infiltrated collagen membranes are saw-tooth peaks, (marked by arrows in Figure 5.7) which are also frequently observed in similar measurements of other biomaterials such as titin [258], spectrin [259], abalone shell [260] and tenascin [261]. The number of observable sawtooth peaks increases after the metal infiltration. Presumably, the increase of the number of those peaks, induced by the stress fluctuation, is a matter of the rupture of a larger number of metal-mediated interprotein bonds or the slip pulse of interfibrillar cross-links inside the collagen membrane (in a later part, this will be discussed further), originating from the metal infiltration. Ti infiltration into collagen can also be derived from scanning transmission electron microscopy (STEM) energy dispersive X-ray (EDX) analysis as shown in Figure Metal infiltration into collagen By scanning transmission electron microscopy-energy dispersive X-ray (STEM-EDX) point analysis of a cross-sectioned (by focused ion beam) native collagen membrane (CM/N), sulphur and potassium aside from carbon, oxygen and hydrogen could be identified (Figure 5.8a and Figure 5.8b). In the case of CM/TiO2, as shown in Figure 5.8c, a gradient in the mass concentration of Ti between the TiO 2 shell layer and the bulk collagen was observed. Along the TiO 2 shell layer, in some regions, it was difficult to discriminate a sharp boundary between the TiO 2 shell and the collagen. From an EDX line scan from point A to B in Figure 5.8c it could be derived that in proximity to the TiO 2 shell layer the collagen contains a substantial amount of Ti. In the central region, a weaker signal (1.5 ~ 3.4% by atomic weight ratio), but still above the resolution limit of the EDX (0.5 ~ 1.0 %), was observed. Similar results were also observed after infiltration with Al or Zn (Figure A1 and Figure A2). The exact infiltration mechanism during MPI and the potential binding sites for Ti are not yet clear. However, several effects might be considered to be responsible for the significant modification of the bonding structure of the collagen. Those effects include: The severe attack of water at elevated temperatures occurring at the hydrogen bonds [263] which interconnect the three polypeptide chains in the triple helix of the collagen, the resulting global weakening of hydrogen bonds with

104 96 Metal Infiltration into Collagen increasing water temperature [264], the strong reactivity of titanium isopropoxide, amine and hydroxyl groups and the metal ions affinity to bind to the collagens [265], thus leading to Ti-collagen complexes [ ]. The bonding structure of the collagen seems to be significantly modified after Ti infiltration. New bonds are formed by mediation of Ti, which can also be derived from Raman shifts of the Ti infiltrated collagen (Figure 5.9, Figure A3 and Table A1). a b c d Figure 5.8 Scanning Transmission electron microscopy (STEM) images and corresponding energy dispersive X-ray (EDX) analysis. a, STEM image of a cross sectioned native collagen fiber of CM/N prepared by focused ion beam (FIB) using platinum (Pt) as an electrode. b, EDX point analysis spectrum measured at position A in figure a. c, STEM image of a cross sectioned Ti infiltrated collagen fiber (CM/TiO2). d, Element concentration profile from EDX analysis scanned along the line from A to B in figure c. The dotted lines are actual data and the corresponding solid line is an interpolated curve using those data.

105 Chapter 5 97 Figure 5.9 Raman shifts of a native collagen membrane and a Ti infiltrated collagen membrane in the region of 1800 to 800 and 700 to 200 cm -1. Raman shifts and corresponding tentative assignments of further samples can be found in Figure A3 and Table A1. Key to Abbreviation: ν(stretching), δ(bending) and τ(twisting) Chemical analysis via Raman shift Raman spectra of the collagen membranes (Figure 5.9) show signals from amide I (1668 cm -1 ) and amide III (1271cm -1 ) [270], (C-C) stretching vibration modes of the ring of Hyp and Pro at 875 and 855 cm -1 [270,271], and the vibration mode at 1582 cm -1 which can be assigned to Pro and Hyp [270,271]. The data support the presence of a helical conformation in the collagen of the chicken s eggshell membrane [269,272]. Interestingly, some changes are detected for Ti-infiltrated collagen membranes. In particular, a decrease of the amide I (C=O stretching) and amide III (N-H bending) band intensities for the Ti infiltrated collagen at 1668 cm -1 and 1271cm -1, respectively, can be observed (Further changes and the corresponding assignments can be found in Table A1). It is known that, while its coiled-coil triple helix structure is dominated by hydrophobic interactions, the responsible interactions for stabilizing collagen are hydrogen bonds. In triple helices, each individual α-chain is stabilized by Pro and Hyp, and the trimerization of α-chains is favored by close packing and intermolecular hydrogen bonding. There is only one hydrogen bond per Gly-X-Z triplet, namely between the amine group of Gly and the carbonyl group of the residue in position X. The remaining two backbone carbonyl groups

106 98 Metal Infiltration into Collagen in each triplet and any backbone amine group of X and Z are not involved in hydrogen bonding [273]. Vapor phase TIP and water might affect the hydrogen bonds connecting Gly and X in a way as the (presumably N H O=C) bonds are weakened and hydrogen is substituted with Ti. The newly formed bonds, mediated by Ti, are stronger than hydrogen bonds (Figure 5.10). This assumption is also supported by the Raman spectra, particularly from the shifts at 578, 338 cm -1 which can tentatively be assigned to vibration modes of a Ti-N bonding (right figure in Figure 5.9) [274]. In addition, the WAXS (wide angle X-ray scattering) pattern of CM/TiO2 shows crystallographic changes presumably caused by Timediated bonding between nitrogen and carbonyl groups (Figure 5.11). a Figure 5.10 Two dimensional representation of the conformation of collagen α-chain. a, The repeating sequence shown is Gly-Pro-Hyp. Based on the results from Rich et al. [236] and Fraser et al. [237], z-coordinates are listed. b and c are schematically sketched based on the collagen description by Bella et al. [239] and the Raman data (Figure A3 and Table A1). b c Structural analysis via x-ray scattering During the past 50 years, research focused on highly ordered collagen structures, like rat tendons [236]. X-ray diffraction (XRD) was the method of choice to resolve the molecular

107 Chapter 5 99 structure of collagen. The structure itself is relatively well understood both on the macroscopic as well as on the atomic level [ ,273,275]. Unlike the rat tendon, the present collagen membrane is poorly ordered. Nevertheless, protein crystal structures were observed from reflections by WAXS (peak A of CM/N in Figure 5.11, Table 5.4). The value of spacing (d) was around 3.0 Å, which is well corresponding to the translation length per one amino acid in a single α-chain of the collagen (see the schematic drawing in Figure 5.3) [235,237,239]. Peaks B and C can potentially be associated to the diffraction between the separate chains. It can be observed that the crystal structure of the collagen membrane is notably changed after Ti infiltration: Two new peaks, D (d=5.30å) and E (d=6.31å), were observed with an intensity of D being stronger than of peak E. It means that the change is quite pronounced. WAXS on reference samples showed that the peaks D and E reveal only from metal infiltrated collagen and not from TiO 2 or the slide glass used as sample holder. Considering the spacing of 5.30 Å and helix parameters of the collagen models by Rich and Crick [236] or Fraser et al. [237], this distance is very close to the vertical distance of hydrogen bonds [(N H) Gly (O=C) Pro ] connecting single collagen α- chains (see the detailed parameters for the helix in references [236,237,239] and the schematic drawing of the atomic arrangements in Figure 5.3 and Figure 5.10). In agreement with the Raman shifts, after Ti infiltration, the intra-helical hydrogen bonds of collagen are most likely broken or transformed into new Ti mediated bonds (N-Ti O=C). According to the strong peak intensity, the newly formed bonds should have a very high regularity. Figure 5.11 Wide angle x-ray scattering (WAXS) patterns of diverse samples. The spectra are vertically shifted for clarity. The CM/N shows three diffraction peaks (A, B and C). In the case of metal infiltrated CM/Al2O3, CM/TiO2 and CM/ZnO, new diffraction peaks (D and E) are observed. Some already existing peaks (A, B and C) disappear (details can be found in Table 5.4). Both Al 2 O 3 and TiO 2 deposited at 70 C are amorphous. ZnO shows wurtzite phases at the same temperature [21]. To verify that the peaks D and E do not arise from metal oxides or a slide glass used as a substrate, reference samples were prepared and measured. As shown in the figure, Al 2 O 3, TiO 2 and the slide glass reveal amorphous phases, and ZnO shows only the wurtzite phase.

108 100 Metal Infiltration into Collagen a b c Figure 5.12 Small angle x-ray scattering (SAXS) patterns of the diverse samples. a, SAXS pattern of four different samples. Native collagen membranes (CM/N) do not show any obvious Bragg diffraction patterns related to the collagen s internal structure on the nanometer scale. It is assumed that the scattering signal is attributed to interfacial scattering from the fiber surfaces. Within the profiles of Ti (CM/TiO2) and Al infiltrated collagen (CM/Al2O3), scattering was detected. Since ALD usually produces very uniform metal oxide layers regardless of geometric complexity of the used substrates, it cannot be assured that the scattering signals arise from the changed collagen structure after metal infiltration. In order to clarify the source of detected diffraction, the X-ray form factor, F(q) was calculated with isotropically oriented lamellae using the 2 K sin( qd / 2) equation, F q) = [276], where d and K are the thickness of the lamellae 2 ( q q and the fitting parameter, respectively. b and c show an asymptotic Porod limit I ~ q -4 and a calculated form factor plot together with the diffraction pattern of CM/TiO2 and CM/Al2O3, respectively. The actual thicknesses of deposited Al 2 O 3 and TiO 2 films on the collagen membrane were around nm and nm, respectively. These values were relatively close to the d values from the form factor (d CM/Al2O3 = 44 nm, d CM/TiO2 = 46 nm). Therefore, the detected signals of CM/TiO2 and CM/Al2O3 may arise from the metal oxide film. In the case of Zn infiltrated collagen membranes, since the thickness of the deposited ZnO layer was around 100 nm, it is apparently out of the detection range of the machine.

109 Chapter Table 5.4 Peak locations and distances between planes in crystals of native and metal infiltrated collagen membranes. 2Ө: diffraction angles of each peak, d: spacing. Peak Location (2θ) [ ] Spacing d (=1.54/2sinθ) [Å] CM/N CM/Al2O3 CM/TiO2 CM/ZnO A Yes Yes Yes No B Yes No Yes No C Yes No Yes No D No No Yes Yes E No No Yes No Investigation of selachian egg capsules, which are similar in structure and functionality to the present membranes, showed fine XRD patterns from a highly ordered longitudinal and transverse arrangement of nanometer scaled collagen fibrils [277]. In contrast, the present samples did not show such diffraction patterns from small angle x-ray scattering (SAXS). Therefore, it is likely that the crystallographic changes of the Ti infiltrated collagen membrane, although distinguished on a sub-nanometer scale, on a macroscopic scale are minor or below the detection limit (Figure 5.12) Biomineralization versus metal infiltration As already mentioned above, mineralization in the collagen architectures such as bone [241,244,247,252,253,255,257] and tendon [242,246, ], periodically occurs only within limited zones (hole and overlap, Figure 5.4) with nm of distance. No mineralization inside the bulk of the collagen was observed so far. The distribution of minerals in those architectures is macroscopically non-uniform. Accordingly, from continuum based standard composite theory, one can derive the following: Once a tensile stress is applied to those structures, mineral parts tolerate micro-cracks within the range of the mineral s endurable fracture strain limit, thus increasing the overall fracture stress of the structure. If the exerted stress exceeds the limit of the mineral s fracture stress, a macroscopic failure of the structure will result. With minerals having a very low facture strain value as compared to non-mineralized collagen, one has to pay with reduced ductility even for a remarkably increased stiffness. Accordingly, the stiffness and the fracture strain as well as the overall deformation behaviour of collagen tissues are highly dependent on the mineral deposited on the collagen matrix [252,253,255]. Based on the simple rule of mixture one can predict the mechanical properties of those mineralized collagen tissues.

110 102 Metal Infiltration into Collagen a b c Figure 5.13 Experimental and computational results which have investigated the effects of the cross-linking density on mechanical properties of the collagen tissue. a and b shows experimental results by Charulatha et al. [278] and Rajini et al. [279] to demonstrate the effect of cross-linking on the mechanical properties of a collagen tissue. a, Stress/strain varies in direct/inverse proportion to cross-link density inside the collagen fibrils, respectively. b shows the opposite case. c, Computational results from Buehler [262]: Larger cross-linking density leads to larger yield strain. Above a critical cross-linking density (β>25) the fracture behaviour changes to molecular fracture resembling from unfolding tropocollagen molecules under rupture of hydrogen bonds (See details in the reference.). a b Figure 5.14 Variation of yield behaviour of metal infiltrated collagen membranes and comparison to the computational results by Buehler [262]. a, The metal infiltrated collagens have larger yield stress / strain values than the native collagen membrane. These experimental results correspond well to the computational results for increased cross-linking, shown in b. It is noteworthy that the values, (ε F ε Y ) as well as (ε F ε Y )/ε Y increased after metal-infiltration (Table 5.3), which means that the metal infiltrated collagen membranes show less brittle-like behaviour than the native collagen membrane.

111 Chapter In contrast to the mineralized collagen, the present results indicate that by metal infiltration the inter-helix bonding state is changed and subsequently the mechanical properties of collagen in terms of both σ F and ε F are enhanced. Unlike bones and tendons, after infiltration the metal is distributed inside the collagen with a concentration gradient as a function of the penetration depth. Hence, one can assume that the deformation and fracture behaviour of metal infiltrated collagens would be different to those of mineralized bones and tendons. A simple explanation cannot be derived from classical composite theory. Experimentally, it was proven that the mechanical properties of collagen tissues are primarily dependent on the formation of cross-links between the collagen molecules preventing slippage under load. Charulatha et al. [278] showed that the fracture stress/strain varies in direct/inverse proportion to the cross-link density inside collagen fibrils, respectively. However, Rajini et al. [279] demonstrated the opposite behaviour (Figure 5.13). In the metal infiltrated collagen, the fracture stress increased in agreement with the results of Chaulatha, and the fracture strain increased in agreement with the results of Rajini, respectively. The mechanical behaviour seems to be dependent on the cross-linking agent or the collagen tissues used. From computational results, Buehler [262] reported that larger cross-link density leads to larger yield stress/strain (increased elastic region). If a critical cross-linking density is exceeded, the fracture behaviour shifts to molecular fracture resulting from unfolding of collagen molecules caused by rupture of hydrogen bonds. Similar to those results, the metal infiltrated collagen membranes have larger yield stress/strain values, i.e. an increased elastic region as compared to a native collagen membrane. In contrast to Buehler s theoretical results, however, the metal infiltrated membrane shows less brittle-like deformation behaviour (Figure 5.14 and Table 5.3). Although there is a certain discrepancy between the deformation behaviour of metal infiltrated membranes and the results discussed above, it appears reasonable that crosslinking is one key factor for the mechanical properties of collagen tissues. An exact mechanism for the simultaneous enhancement of both fracture stress and strain by metal infiltration is not clear yet. It, however, seems that cross-linking of collagen fibrils and structural changes of collagen molecules upon infiltration with metals are crucial parameters for the enhancement. Even though Raman and WAXS results confirmed changes in the molecular structure, ways to clearly show that the metal infiltration increased the cross-linking density have not been found yet. However, considering the increased number of saw-tooth peaks (Figure 5.7) and the increased yield stress/strain value (Figure 5.14 and Table 5.3) of metal infiltrated collagen, from Buehler s results it can be indirectly inferred that the cross-linking density increased during metal infiltration.

112 104 Metal Infiltration into Collagen Chemically one would expect an increase as Hyp with its functional hydroxyl group (-OH) should strongly react with the chemicals used (trimethylaluminum, diethylzinc and TIP). Furthermore, functional side groups of amino acids X and Z play a key role for permitting the twisting of the collagen helix. Those groups protrude from the chain and are believed to be exposed to the surrounding and available for intermolecular/interchain interaction, such as cross-links between tropocollagen fibers [224,225]. The collagen of the chicken s eggshell membrane contains Gly, Pro and Hyp with 11.1, 11.6 and 1.5 % of the total amino acid composition, providing large amounts of functional groups. Additionally there are further amino acids with functional side groups available for cross-linking [232]. The interfibrillar interactions caused by increased cross-linking density and the intrafibrillar interactions caused by metal-mediated interprotein bonds determines the mechanical deformation behaviour of collagen. The deformation behaviour of metal infiltrated collagen could possibly be governed by the molecular fracture related to the stretching of the protein backbone and uncoiling of collagen triple helices, which results in increased fracture stress as well as fracture strain, as already suggested by Buehler (Figure 5.15) [ ]. Figure 5.15 Deformation map of collagen fibrils. The diagram is reproduced from the article by Buehler et al. [280]. In the reference, it is described that the molecular fracture can occur in the case of strong molecular interactions, such as increased cross-link density. For a detailed explanation, see the reference. A more comprehensive review is given in reference [282].

113 Chapter Conclusion Metal (Ti, Al and Zn) infiltration into collagen via the multiple pulsed vapor phase infiltration process, induced crystallographic changes of collagen likely related to interchain hydrogen bonds and presumably induced an increase of the interfibrillar crosslinking density. This metal infiltration is different to the mineralization, which was observed in diverse collagen-based tissues, as the infiltration resulted in a simultaneous enhancement of strength and ductility of the collagen tissues. The deformation and fracture behaviour of metal infiltrated collagen tissues under uniaxial tension were found to be possibly close to molecular fracture after stretching the protein backbone and uncoiling the triple helix of the collagen. The process to infiltrate metals into collagen could be a means to improve the mechanical properties of collagen-based tissue equivalents good enough to replace native tissues, although the biocompatibility of such structures still has to be investigated in detail.

114 106 Metal Infiltration into Collagen

115 Summary 107 Summary The intended message of this thesis is to show a new and promising application of ALD. As can be recognized from the tile of this thesis, ALD on Biological Matter, the results presented here are mainly related to side effects of the ALD process presumably caused by the deposition mechanism of ALD (in particular, the separation of precursors) rather than conformally and uniformly deposited materials with a thickness control on a monolayer level. As an alternative to bottom-up approaches for nanofabrication, so far few scientists have reported on inorganic nanostructures fabricated by ALD on diverse organic templates. However, they have been mainly interested in conformally deposited films and their quality. The side effects which could occur during an ALD process have been rarely recognized. Furthermore, side effects are usually considered to bring rather negative consequences than positive ones. However, in this research it has been shown that those side effects can induce very positive chemical/physical modifications of the inner protein structure of biotemplates and that the modification drastically enhances the mechanical properties of the templates. This process is different to a thin film deposition and was thus named as Multiple Pulsed Vapor Pulsed Infiltration (MPI). Using two different biotemplates (spider dragline silks and collagen membranes), the modifications by MPI and the corresponding results were presented. The first example in this thesis to show the capability of ALD to conformally coat materials on complex-featured biological templates was presented in Chapter 3. Using low temperature ZnO ALD, inorganic macroporous ZnO membranes with a photocatalytic effect and mechanical flexibility were fabricated from inner shell membranes of chicken s eggshell matrices as templates. In order to evaluate the potential merits and general applicability of the ZnO structures, a comparative study of two membranes with coatings of either TiO 2 or ZnO, processed under similar processing conditions, was performed. The study included a comparison of crystallographic features of TiO 2 and ZnO deposited by ALD, mechanical/ thermal stability and bactericidal efficiency of the resulting inorganic structures. Both, the ZnO and the TiO 2 coated membranes clearly exhibited bactericidal effects as well as mechanical/thermal stability even if deposited at relatively high temperatures. The ZnO membranes, even though prepared at fairly low temperatures (~100 C), exhibited polycrystalline phases and showed a good bactericidal efficiency as well as better mechanical properties than the TiO 2 coated membranes. From this study, the benefits of low-temperature ZnO ALD on biological templates were demonstrated. The focus of Chapter 4 was to illuminate a new application of ALD, i.e. MPI. It had already been reported that in nature, tiny amounts of inorganic impurities, such as metals,

116 108 are incorporated in protein structures of some biomaterials and can lead to improved mechanical properties of those structures. In Chapter 4, it was demonstrated that metals can be intentionally infiltrated into protein structures through the MPI approach performed with equipment conventionally used for ALD. By MPI metals such as Al, Ti or Zn were infiltrated into the dragline silk of an Araneus spider. It was observed that infiltrated metals change the crystallographic feature of the inner protein structure in the dragline silk and that this crystallographic change leads to a greatly increased toughness (increased strength and ductility) of the resulting silks. In Chapter 5, for collagen with a rather different protein structure than in spider silk, a similar approach to Chapter 4 was performed. Unlike spider silk being composed of polyalanine on a molecular level, collagens are composed of three polypeptide chains, each of which contains regions with a repeating amino acid motif [Gly (Glycine)-X-Z], where X and Z stands for any amino acid. Frequently X and Z are proline (Pro) and hydroxyproline (Hyp), respectively. Each polypeptide chain (α-chain) has left-handed helical conformation with three identical α-chains constituting a right-handed coiled-coil triple-helical structure. It had been reported that the mechanical properties of connective tissues constituted by collagen are highly dependent on the proteins hierarchical structures from the molecular to the macroscopic level. It was observed that similar to the metal infiltrated spider silk, metal infiltrated collagen structure also shows simultaneous increase of strength and ductility. In the case of metal infiltrated silk, it was assumed that the increased ductility is attributed to the increased amorphous fraction inside the silk protein structures by metal infiltration. In the case of metal infiltrated collagen, it appeared that the factor to increase the ductility is possibly related to molecular fracture accompanying stretching the protein backbone and uncoiling the collagen triple helices. With the development of nanoscience, in the field of thin film technology, diverse coating techniques have been developed and many efforts to increase the film quality and controllability have been attempted. Among those techniques, ALD very recently came into the spotlight. Even though the general ALD-based research has grown remarkably, still the research trend has mainly been focused towards expanding the diversity of ALDdeposited materials or increasing the film yield and quality. In this thesis few examples were presented for new applications which can be achieved by ALD. It cannot yet be judged whether the studies presented here will be recognized as a promising cornerstone for future applications of ALD and similar approaches would be adapted.

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135 References and notes In the related literature, various reported shifts of the vibration modes of titanium nitride, in particular in references [27-32] in Appendix, are not perfectly corresponding or even show large differences. In addition, Raman shifts at 578, and 338 cm -1 shows poor relevance to the shifts of amorphous TiO 2 in the literature Ricard-Blum, S., Ruggiero, F. & van der Rest, M. The collagen superfamily. Top. Curr. Chem. 247, (2005) Guinier, A. X-Ray Diffraction: In Crystals, Imperfect Crystals, and Amorphous Bodies (Dover Publications, 1994) Knight, D. P & Hunt, S. Fibril structure of collagen in egg capsule of dogfish. Nature 249, (1974) Charulatha, V & Rajaram, A. Influence of different crosslinking treatments on the physical properties of collagen membranes. Biomaterials 24, (2003) Rajini, K. H., Usha, R., Arumugam, V. & Sanjeevi, R. Fracture behaviour of cross-linked collagen fibers. J. Mater. Sci. 36, (2001) Buehler, M. J. Nature designs tough collagen: explaining the nanostructure of collagen fibrils. Proc. Natl. Acad. Sci. USA 103, (2006) Buehler, M. & Ackbarow, T. Fracture mechanics of protein materials. Mater. Today 10, (2007) Buehler, M. J., Keten, S. & Ackbarow, T. Theoretical and computational hierarchical nanomechanics of protein materials: deformation and fracture. Prog. Mater. Sci. 53, (2008).

136 128

137 Acknowledgement 129 Acknowledgement I have too many people to whom I want to express my deep gratitude. It can hardly imagine how this thesis it might have been completed without the confidence, encouragement and support of Professor Ulrich Gösele in heaven. And to my supervisor, Mato, I would like to express special gratitude. Thanks to his selection, I got a chance to work in Halle. Even if he is my supervisor during last two and half year, firstly he was my best research colleague for serious scientific discussion and sometimes he was my close friend. I am very grateful to various mentors who laid the foundation on which this thesis is based: Hee Han, Woo Lee, Eckhard Pippel, Gerd Hause, Gyeong-Man Kim, Christian Dresbach, Norbert Schammelt, Oussama Moutanabbir, Marcel Mittag, Liangbing Zhang, Yong Qin, Adriana Szeghalmi, Pratyush Das Kanungo, Gregor Grass, Stephan Senz, Ilja Gunkel and Thomas Thurn-Albrecht. I learned much from many others, and I apologize to them for not explicitly recognizing my debt to them. I am also very grateful to my friends in Korea, and to many others who helped in one way or the other but are not mentioned here by name.

138 130

139 Appendix 131 Appendix A1. Figures Figure A1 Cross section sample preparation of CM/Al2O3 by FIB and EDX analysis. a, Pt sputtered CM/Al2O3. b and c, SEM and TEM image of a cross sectioned sample of CM/Al2O3, respectively. d, Magnified TEM of the marked (blue) square in c. Pt and Al 2 O 3 layer show clear contrast difference. The thickness of the deposited Al 2 O 3 layer was about 45 nm. The border between Al 2 O 3 and the collagen fiber is not sharp. e, Magnified STEM images of the marked (yellow) square in c. f, The presence of infiltrated Al ions is further confirmed by an EDX line scan along the line from A to B in e. The dotted lines are actual data and the corresponding solid line is interpolated curve using those data.

140 132 Figure A2 Cross section sample preparation of CM/ZnO by FIB and EDX analysis. a, Pt sputtered CM/ZnO. b and c, SEM and TEM image of a cross section sample of CM/ZnO, respectively. d, Magnified TEM image of the marked (blue) square in c. ZnO layer and collagen fiber show clear contrast difference. The thickness of the deposited ZnO layer was about 80 nm. The border between the ZnO layer and the collagen fiber is not sharp. e and f, The presence of infiltrated Zn ions is further confirmed by an EDX line scan (f) along the line from A to B in e. The dotted lines are actual data and the corresponding solid line is interpolated curve using those data.

141 Appendix 133 Figure A3 Raman shifts of diverse samples in the region of 1800 to 800 and 700 to 200 cm -1. Tentative assignments of peaks and shoulders can be found in Table A1.

142 134 A2. Tables Table A1 Observed Raman shifts and corresponding tentative assignments. Region Raman shift (cm-1) Tentative assignments of vibration mode Reference 1762 ν(c=o) in Ester [1]-[3] 1668 ν(c=o) in Amide І [1], [4], [5] 1642 ν(c=o) in Amide І [4] 1610 Tyrosine, Phenylalanine, Tryptophan [4], [6]-[7] 1582 Proline, Hydroxyproline [4], [8] 1549 Amide ІI (β-turns) or Tryptophan [9]-[12] 1463 δ(ch3, CH2) [4], [13]-[15] 1800 to 800 (cm-1) τ(ch3, CH2) δ(ch2) scissoring in lipids and δ(ch3),(ch2) in protein [4]-[5], [15] [19] [4],[10], [20]- [25721] 1271 δ(n-h) and ν(c-n) in Amide III [4], [22]-[23] 1169 Tyrosine [8], [10]-[12], [15] 1152 ν(c-c), ν (C-N) [10] 1125 ν(c-c), ν (C-N) [8]-[9] 1004 Aromatic ring breathing (Phenylalanine) 970 ν(c-c) [15], [19] [4]-[5], [8],[10],[13], [15], [19], [22]-[24], [25] 875 ν(c-c) of Hydroxyproline ring [4], [10], [19] Proline, Hydroxyproline, Tyrosine ν(c-c) in Proline ring Side chain vibrations of Proline, Hydroxyproline Proline, Hydroxyproline, Tyrosine C-H out of plan bending of benzoid ring Tyrosine (Fermi resonance of ring fundamental and overtone) [8], [4] [5],[19] [8] [8], [26] [13] 578 TiN [27]-[31] 700 to 200 (cm-1) 570? 488? 338 TiN [27]-[32] 324 ZnO [33]-[35] 235 AlN [36]-[37] Key to Abbreviation: ν(stretching), δ(bending) and τ(twisting)

143 Appendix 135 A3. References and notes 1. Engleson, S. B. & Nørgaard, L. Comparative vibrational spectroscopy for determination of quality parameters in amidated pectins as evaluated by chemometrics. Carbohydr. Polym. 30, 9-24 (1996). 2. Pellow-Jarman, M. V., Hendra, P. J. & Hetem, M. J. J. Poly(butylene terephthalate) polycarbonate transecsterification: monitoring its progess with Fourier transform Raman spectroscopy. Spectroc. Acta Pt. A-Molec. Biomolec. Spectr. 51, (1995). 3. Panicker, C. Y. et al. Raman, IR and SERS spectra of methyl(2-methyl-4,6- dinitrophenylsulfanyl)ethanoate. Spectroc. Acta Pt. A-Molec. Biomolec. Spectr. 67, (2007). 4. Frushour, B. G. & Koenig, J. L. Raman scattering of collagen, gelatin, and elastin. Biopolymers 14, (1975). 5. Buschman, H. P. Raman microspectroscopy of human coronary atherosclerosis: Biochemical assessment of cellular and extracellular morphologic structures in situ. Cardiovasc. Pathol. 10, (2000). 6. Fodor, S. P. A., Copeland, R. A., Grygon, C. A. & Spiro, T. G. Deep-ultraviolet Raman excitation profiles and vibronic scattering mechanisms of phenylalanine, tyrosine, and tryptophan. J. Am. Chem. Soc. 111, (1989). 7. Chi, Z., Chen, X. G., Holtz, J. S. W. & Asher, S. A. UV resonance Ramanselective amide vibrational enhancement: quantitative methodology for determining protein secondary structure. Biochemistry 37, (1998). 8. Cheng, W. -T., Liu, M. -T., Liu, H. -N. & Lin, S. -Y. Micro-Raman spectroscopy used to identify and grade human skin pilomatrixoma. Microsc. Res. Tech. 68, (2005). 9. Dukor, R. K. Vibrational spectroscopy in the detection of cancer in Handbook of Vibrational Spectroscopy Vol. 5 (eds Griffiths, J.M.C.A.P.R.) (John Wiley and Sons, 2002). 10. Huang, Z. et al. Near-infrared Raman spectroscopy for optical diagnosis of lung cancer. Int. J. Cancer 107, (2003). 11. Stone, N. et al. Raman spectroscopy for identification of epithelial cancers. Faraday Discuss. 126, (2004). 12. Stone, N. et al. Near-infrared Raman spectroscopy for the classification of epithelial pre-cancers and cancers. J. Raman Spectrosc. 33, (2002).

144 Shetty, G. et al. Raman spectroscopy: evaluation of biochemical changes in carcinogenesis of oesophagus. Br. J. Cancer 94, (2006). 14. Shim, M. G., Song, L. M., Marcon, N. E. & Wilson, B. C. In-vivo near infrared Raman spectroscopy: demonstration of feasibility during clinical gastrointestinal endoscopy. Photochem. Photobiol. 72, (2000). 15. Lakshimi, R. J. et al. Tissue Raman spectroscopy for the study of radiation damage: brain irradiation of mice. Radiat. Res. 157, (2002). 16. Shafer-Peltier, K. E. et al. Raman microspectroscopic model of human breast tissue: Implications for breast cancer diagnosis in vivo. J. Raman Spectrosc. 33, (2002). 17. Kaminaka, S. et al. Near infrared multichannel Raman spectroscopy toward realtime in vivo cancer diagnosis. J. Raman Spectrosc. 33, (2002). 18. Malini, R. et al. Discrimination of normal, inflammatory, premalignant, and malignant oral tissue: a Raman spectroscopy study. Biopolymers 81, (2006). 19. Frank, C. J., McCreecy, R. L. & Redd, D. C. B. Raman spectroscopy of normal and diseased human breast tissues. Anal. Chem. 67, (1995). 20. Huang, Z. et al. Effect of formalin fixation on the near-infrared Raman spectroscopy of normal and cancerous human bronchial tissues. Int. J. Oncol. 23, (2003). 21. Lau, D. P. et al. Raman spectroscopy for optical diagnosis in the larynx: preliminary findings. Lasers Surg. Med. 37, (2005). 22. Baraga, J. J., Feld, M. S. & Rava, R. P. In situ optical histochemistry of human artery using near-infrared Fourier-transform Raman-spectroscopy. Proc. Natl. Acad. Sci. USA 89, (1992). 23. Kupper, L., Heise, H. M., Bechara, F. G. & Stucker, M. Micro-domain analysis of skin samples of moor-mummified corpses by evanescent wave infrared spectroscopy using silver halide fibers. J. Mol. Struct. 565, (2001). 24. Kateinen, E. et al. Qualification of the amphetamine content in seized street samples by Raman spectroscopy. J. Forensic Sci. 52, (2007). 25. Notingher, I., Green, C. & Dyer, C. Discrimination between ricin and sulphur mustard toxicity in vitro using Raman spectroscopy. J. R. Soc. Interface 1, (2004). 26. Laska, J. & Widlarz, J. Spectroscopic and structural characterization of loe molecular weight fractions of polyaniline. Polymer 46, (2005).

145 Appendix Kazmanli, K. Daryal, B. & Urgen, M. Characterization of nano-composite TiN-Sb coating produced with hybrid physical vapor deposition system. Thin Solid Films 515, (2007). 28. Constable, C. P., Yarwood, J. & Münz, W. -D. Raman microscopic studies of PVD hard coatings. Surf. Coat. Technol , (1999). 29. Spengler, W. & Kaiser, R. First and second order Raman scattering in transition metal compounds. Solid State Commun. 18, (1976). 30. Spengler, W. & Kaiser, R. Resonant Raman scattering in a superconducting transition metal compound-tin. Solid State Commun. 17, (1975). 31. Spengler, W., Kaiser, R., Christensen, A. N. & Muller-Vogt, G. Raman scattering superconductivity and phonon density of states of stoichiometric and nonstoichiometric TiN. Phys. Rev. B 17, (1978). 32. Cheng, Y. H. et al. Substrate bias dependence of Raman spectra for TiN films deposited by filtered cathodic vacuum arc. J. Appl. Phys. 92, (2002). 33. Dong, G. et al. Hierarchical metal oxides assembled by nanocrystallites via a simple bio-inspired route. J. Am. Ceram. Soc. 90, (2007). 34. Hasuike, N. et al. Raman scattering studies on ZnO doped with Ga and N(codoping), and magnetic impurites. J. Phys.: Condens. Matter 16, S5807-S5810 (2004). 35. Budesmann, C. et al. Raman scattering in ZnO thin films doped with Fe, Sb, Al, Ga, and Li. Appl. Phys. Lett. 83, (2003). 36. Perlin, P., Polian, A. & Suski, T. Raman-scattering studies of aluminum nitride at high pressure. Phys. Rev. B 47, (1993). 37. Bergman, L, Alexson, D., Murphy, P. L. & Nemanich, R. J. Raman analysis of phonon lifetimes in AlN and GaN of wurtzite structure. Phys. Rev. B 59, (1999).

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147 Curriculum Vitae 139 Curriculum Vitae Seung-Mo, Lee Personal Information Place of Birth : Gender : Nationality: Address: E mail: Chunchon, Korea Male, Married Korea Max Planck Institut fuer Mikrostrukturphysik Experimental Dept. II Weinberg 2, D Halle(Saale), Germany smlee@mpi halle.mpg.de Educational Background Mar / 2003 ~ Feb / 2005 M.S. in Mechanical Engineering, Pohang University of Science and Technology (POSTECH), South Korea Thesis: Fabrication of Hydrophobic Films Using a Plant Leaf in Nature (Academic Advisor: Prof. Tai Hun Kwon) Mar / 1995 ~ Feb / 2003 B.S. in Mechanical Engineering, Inha University, South Korea Thesis: A design of Vibration Absorber (Academic Advisor: Prof. Usik Lee) Research Experiences Feb/ 2007 ~ Present Max Planck Institute of Microstructure Physics, Halle, Germany (under the supervision of Prof. Ulrich Gösele and Dr. Mato Knez) Jun / 2006 ~ Feb/ 2007 Lehr und Forschungsgebiet Konstruktion und Entwicklung von Mikrosystemen (KEmikro), Rheinisch Westfälische Technische Hochschule (RWTH) Aachen, Germany (under the supervision of Prof. Werner Karl Schomburg)

148 140 Mar / 2005 ~ May / 2006 Advanced Materials Processing Lab, Pohang University of Science and Technology (POSTECH), South Korea (under the supervision of Prof. Tai Hun Kwon) Honors and Awards o o Scholarship and Prize Honour Student (full 4 years scholarship) from Inha University Deutscher Akademischer Austausch Dienst (DAAD) Scholarship Additional Capabilities Language: Computer skill: Sport: Extracurricular activity: English, German I DEAS, Auto CAD, Fortran and C language Tennis, Badminton, Soccer Calligraphy, Hiking, Acoustic Guitar, Photography Field of Interests o o o o o Atomic Layer Deposition process Bio inspired organic/organic hybrid materials LIGA process, electroforming, Injection molding, Stereolithography, Microfluidics, optics, Polymer processing, Micro optics, Mirosensor and actuator Patterning of nano and micro size structure and its applications, Laser Interference Lithography UV nanoimprint lithography, Hot embossing Publications International Journal [01] S. M. Lee, H. S. Lee, D. S. Kim, T. H. Kwon Surf. Coat. Technol. 201, (2006) Fabrication of hydrophobic films replicating plant leaves in nature [02] S. M. Lee, T. H. Kwon Nanotechnology 17, (2006) Mass producible replication of highly hydrophobic surfaces from plant leaves Highlighted in nanowerk.com

149 Curriculum Vitae 141 [03] S. M. Lee, T. H. Kwon J. Micromech. Microeng. 17, (2007) Effects of intrinsic hydrophobicity on wettibility of polymer replicas of superhydrophobic lotus leaf [04] Y. Qin, S. M. Lee, A. Pan, U. Gösele, M. Knez Nano Lett. 8, (2008) Rayleigh instability induced metal nanoparticle chains encapsulated in nanotubes produced by atomic layer deposition Highlighted in nanowerk.com [05] G. M. Kim, S. M. Lee, G. H. Michler, H. Roggendorf, U. Gösele, M. Knez Chem. Mater. 20, (2008) Nanostructured pure anatase titania tubes replicated from electrospun polymer fiber templates by atomic layer deposition [06] Y. Yang, D. S. Kim, R. Scholz, M. Knez, S. M. Lee, U. Gösele, M. Zacharias Chem. Mater. 20, (2008) Hierarchical three dimensional ZnO and their shape preserving transformation into hollow ZnAl 2 O 4 nanostructures [07] A. Bielawny, P. T. Miclea, R. B. Wehrspohn, S. M. Lee, M. Knez, C. Rockstuhl, M. Lisca, F. L. L ederer, R. Carius Proceedings of SPIE, 7002, (2008) Three dimensional photonic crystals as intermediate filter for thin film tandem solar cells [08] D. S. Kim, S. M. Lee, R. Scholz, M. Knez, U. Gösele, J. Fallert, H. Kalt, M. Zacharias Appl. Phys. Lett. 93, (2008) Synthesis and optical properties of ZnO and carbon nanotube based coaxial heterostructures [09] A. Bielawny, J. Üpping, P. T. Miclea, R. B. Wehrspohn, C. Rockstuhl, F. Lederer, M. Peters, L. Steidl, R. Zentel, S. M. Lee, M. Knez, A. Lambertz, R. Carius phys. stat. sol.(a) 205, (2008) 3D photonic crystal intermediate reflector for micromorph thin film tandem solar cell [10] S. M. Lee, G. Grass, G. M. Kim, C. Dresbach, L. Zhang, U. Gösele, M. Knez

150 142 Phys. Chem. Chem. Phys. 11, (2009) Low temperature ZnO atomic layer deposition on biotemplates: flexible photocatalytic Zn O structures from eggshell membranes Invited article [11] S. M. Lee, E. Pippel, U. Gösele, C. Dresbach, Y. Qin, C. V. Chandran, T. Bräuniger, G. Hause, M. Knez Science 324, (2009) Greatly increased toughness of infiltrated spider silk Reported and highlighted in many newspapers, magazines, internet websites and journals of many countries [12] S. M. Lee, E. Pippel, O. Moutanabbir, I. Gunkel, U. Gösele, T. Thurn Albrecht, M. Knez submitted for publication Increase of strength and ductility of collagen after metal infiltration [13] Y. Qin, Y. Kim, L. B. Zhang, S. M. Lee, R. B. Yang, A. L. Pan, K. Mathwig, M. Alexe, U. Gösele, M. Knez submitted for publication Helical oxide nanotubes templated from carbon nanocoils by atomic layer deposition International Conference (Talk and Posters) [01] (Poster) S. M. Lee, H. S. Lee, T. H. Kwon NSTI Nanotech 2005, May 8 12, 2005, Anaheim(USA) (Proceeding: p ) Fabrication of hydrophobic films using a plant leaf from nature [02] (Invited Talk) S. M. Lee, T. H. Kwon Nanoengineering symposium 2005, October 27 29, 2005, Deajeon (Korea) (Proceeding: p 29 32) Replication of micro/nano combined structure from nature [03] (Talk) S. M. Lee, T. H. Kwon NSTI Nanotech 2006, May 7 11, 2006, Boston(USA) (Proceeding: p ) Replication of highly hydrophobic surface with micro/nano combined structures from nature

151 Curriculum Vitae 143 [04] (Poster) S. M. Lee, T. H. Kwon NSTI Nanotech 2007, May 20 24, 2007, Santa Clara (USA) Intrinsic effects of materials on superhydrophobicity of polymer replicas from nature [05] (Invited Talk) M. Knez, L. Zhang, S. M. Lee, A. J. Patil, S. Mann, K. Nielsch, U. Gösele Annuals AVS ALD meeting, June 24 27, 2008, San Diego (USA) (Bio)organic inorganic hybrid nanostructures by ALD [06] (Talk) S. M. Lee, U. Gösele, C. Dresbach, Y. Qin, M. Knez Annuals AVS ALD meeting, June 29 July 2, 2008, Bruges (Belgium) Improved mechanical properties of spider silk by multiple pulsed vapor phase infiltration [07] (Invited Talk) M. Knez, S. M. Lee, Y. Qin, R. Scholz, E. Pippel, G. Hause, J. Woltersdorf, U. Gösele Transatlantic Frontiers of Chemistry, July 31 August 3, 2008, Cheshire (UK) Novel nanostructures by atomic layer deposition [08] (Talk) Y. Qin, L. Liu, S. M. Lee, U. Gösele, M. Knez Annuals AVS ALD meeting, June 29 July 2, 2008, Bruges (Belgium) Rayleigh instability inducted metal nanoparticle chains encapsulated in nanotubes produced by ALD [09] (Talk) S. M. Lee, U. Gösele, C. V. Chandran, T. Braeuniger, C. Dresbach, G. Hause, Y. Qin, M. Knez 3rd Interdisciplinary Max Planck PhDnet Workshop, August 29 31, 2008, Munich (Germany) Drastic toughness increase of doped spider silk [10] (Talk) Y. Yang, D. S. Kim, R. Scholz, M. Knez, S. M. Lee, U. Gösele, M. Zacharias European Materials Research Society (E MRS) Fall Meeting, September 15 19, 2008, Warsaw (Poland) Hierarchical 3D ZnO and their shape preserving transformation into zinc spinel nanostructures [11] (Talk) A. Bielawny, J. Üpping, P. T. Miclea, R. B. Wehrspohn, S. M. Lee, M. Knez, M. Peters, A. Lalnbertz, R. Carius 2008 Materials Research Society (MRS) Fall Meeting, December 1 5, 2008, Boston (USA) 3D photonic spectrally selective and diffractive intermediate filter for micromorph tandem

152 144 cell [12] (Invited Seminar) M. Knez, Y. Qin, S. M. Lee, A. V. Szeghalmi, U. Gösele Universität Bielefeld, February 05, 2009, Bielefeld (Germany) Coating and infiltration of materials by atomic layer deposition [13] (Invited Seminar) S. M. Lee BMBF Junior Research Groups NanoFutur Workshop, February 6, 2009, TU Braunschweig, Braunschweig (Germany) Application of atomic Layer Deposition (ALD) to biological matter [14] (Invited Talk) Y. Yang, D. S. Kim, E. Pippel, M. Knez, R. Scholz, S. M. Lee, Y. Qin, L. F. Liu, W. Lee, U. Gösele Materials Research Society (MRS) Spring Meeting, April 13 17, 2009, San Francisco (USA) The Kirkendall effect revisited in the nanoworld [15] (Invited Seminar) S. M. Lee Gwangju Institute of Science and Technology (GIST), October 28, 2009, Gwangju (Korea) Atomic Layer Deposition on Biological Matter [16] (Talk) S. M. Lee, E. Pippel, U. Gösele, C. Dresbach, Y. Qin, C. V. Chandran, T. Bräuniger, G. Hause, M. Knez 2009 Materials Research Society (MRS) Fall Meeting, November 29 December 4, 2009, Boston (USA) Greatly increased toughness of infiltrated spider silk [17] (Poster) S. M. Lee, G. Grass, G. M. Kim, C. Dresbach, L. Zhang, U. Gösele, M. Knez 2009 Materials Research Society (MRS) Fall Meeting, November 29 December 4, 2009, Boston (USA) Low temperature ZnO atomic layer deposition on biotemplates: flexible photocatalytic ZnO structures from eggshell membranes Domestic Conference (in Korea) Talks and Posters [01] (Talk) 이승모, 이현섭, 김동성, 권태헌 The 7th Korean MEMS Conference, April 7 9, 2005, Jeju Island (Korea) (Proceeding: p 57 60)

153 Curriculum Vitae 145 식물잎을 이용한 소수성 필름 제작 [02] (Poster) 이승모, 권태헌 The 7th Korean MEMS Conference, April 6 8, 2006, Jeju Island (Korea) (Proceeding: p ) 식물 잎의 표면 구조 모사를 이용한 소수성 필름의 대량 생산 방법 Patents [01] S. M. Lee, T. H. Kwon Application number: 11/225,150 Publication number: US 2007/ A1 Filing date: 13 Sep 2005 U.S. Classification Method of preparing hydrophobic polymer substrate and hydrophobic polymer

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155 147 Eidesstattliche Erklärung Ich erkläre, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe der Quelle gekennzeichnet. Insbesondere habe ich hierfür nicht die entgeltliche Hilfe von Vermittlungs- oder Beratungsdiensten Promotionsberaterinnen oder Promotionsberater oder anderer Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen. Die Arbeit wurde bisher weder im In- noch im Ausland in gleicher oder ähnlicher form einer anderen Prüfungsbehörde vorgelegt. Ich erkläre hiermit, dass ich mich noch nie an einer in- oder ausländischen wissenschaftlichen Hochschule um die Promotion beworben habe oder gleichzeitig bewerbe. Seung-Mo Lee Halle (Saale), September 21, 2009