Atomic Layer Deposition on Biological Materials BarrierPhD Thesis Seung Mo Lee Final 20091224

7/25/2019 Atomic Layer Deposition on Biological Materials BarrierPhD Thesis Seung Mo Lee Final 20091224

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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



D

edicated 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



“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



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



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 ofapplications, 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 collagenmembranes 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.



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 Al2O3, TiO2

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 (TiO2 and ZnO) on complex-

shaped biological templates (macroporous collagen membranes) is presented. Moreover,

some crystallographic growth features of TiO2 and ZnO at various processing

temperatures are also demonstrated. As a feasible application of those templated inorganic

TiO2/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.



Table of Contents

CHAPTER 1 .................................................................. ................................................................. ... 1

GENERAL ASPECTS OF ATOMIC LAYER DEPOSITION AND IT’S APPLICATIONS .......... 1 1.1 I NTRODUCTION........................................................ ........................................................ 1 1.2 PRINCIPLE AND CHARACTERISTIC FEATURES OF THE ALD PROCESS ................................ 2 1.3 I NVESTIGATED ALD MATERIALS ............................................................. ........................ 5 1.4 R EACTION MECHANISMS OF METAL OXIDES BY ALD ...................................................... 8 1.5 COMPARISON OF ALD AND CVD .......................................................... ........................ 11 1.6 MULTIPLE PULSED VAPOR PHASE INFILTRATION........................................................... . 13

CHAPTER 2 .................................................................. ................................................................ .. 17

BASIC PARAMETERS IN MECHANICS................................. .................................................... 17

2.1 STRESS AND STRAIN ........................................................... ........................................... 17 2.2 STRESS - STRAIN CURVE ............................................................... ................................. 18 2.3 STRAIN ENERGY, BREAKING ENERGY AND TOUGHNESS ................................................. 23 2.4 STIFF MATERIALS, STRONG MATERIALS AND TOUGH MATERIALS .................................. 25 2.5 ELASTIC DEFORMATION AND PLASTIC DEFORMATION ................................................... 27

CHAPTER 3 .................................................................. ................................................................ .. 31

METAL OXIDE DEPOSITION ON BIOTEMPLATE: MACROPOROUS PHOTOCATALYTICTiO2 OR ZnO MEMBRANES TEMPLATED FROM CHICKEN’S EGGSHELL MATRICES... 31

3.1 BACKGROUND ......................................................... ...................................................... 32 3.1.1 Historical background of photocatalysis ............................................................. .... 32 3.1.2 Principle of photocatalysis and applications.......... ................................................. 33 3.1.3 Crystal structure of TiO2 and ZnO........................... ................................................ 35 3.1.4 Escherichia coli ( E. coli) bacteria ............................................................ ............... 37

3.2 I NTRODUCTION........................................................ ...................................................... 38 3.3 EXPERIMENTAL ....................................................... ...................................................... 39 3.3.1 Preparation of the inner shell membrane from a chicken’s egg .............................. 39 3.3.2 TiO2 /ZnO atomic layer deposition on ISM........................................................ ....... 39 3.3.3 Characterization ............................................................... ....................................... 40 3.3.4 Microbiology..................................... ..................................................................... .. 41 3.3.5 Photocatalytic experiments with ISM/TiO2 and ISM/ZnO ....................................... 42 3.3.6 Tensile test of native ISM, ISM/ZnO/100 and ISM/TiO2/275 membranes............... 43

3.4 R ESULTS AND DISCUSSION............................................................ ................................. 43 3.4.1 Film quality, crystallographic features and bactericidal efficiency ........................ 43 3.4.2 Mechanical flexibility and thermal stability........................................................ ..... 47

3.5 CONCLUSION ........................................................... ...................................................... 48

CHAPTER 4 .................................................................. ................................................................ .. 49 METAL INFILTRATION INTO SPIDER DRAGLINE SILK....................................................... 49

4.1 BACKGROUND ......................................................... ...................................................... 49 4.1.1 Overview of spiders and mechanical properties of spider silk................................. 49 4.1.2 Chemical structure and macroscopic model for spider silk..................................... 52 4.1.3 Models for the description of dragline silk’s mechanical properties....................... 54 4.1.4 Function of metals in biological tissues................................................................ ... 57

4.2 EXPERIMENTAL ....................................................... ...................................................... 60 4.2.1 Silk collection......................... ..................................................................... ............. 60 4.2.2 Multiple pulsed vapor phase infiltration process...................................... ............... 60 4.2.3 Tensile test ................................................................... ............................................ 62 4.2.4 TEM and EDX analysis............................. ............................................................... 63

4.2.5 Solid state nuclear magnetic resonance (NMR) spectroscopy ................................ 64 4.2.6 Wide angle X-ray scattering (WAXS) .......................................................... ........... 64 4.3 R ESULTS AND DISCUSSION............................................................ ................................. 67



4.3.1 Variation of mechanical properties under diverse conditions ................................. 67 4.3.2 Scientific validation of the MPI process.............................................................. ..... 71 4.3.3 Model system for the metal infiltration mechanism.................................................. 76 4.3.4 Model system for mechanical property improvements of silk .................................. 78

4.4 CONCLUSION ........................................................... ...................................................... 80

CHAPTER 5 .................................................................. ............................................................... ... 81

METAL INFILTRATION INTO COLLAGEN ................................................................ .............. 81

5.1 COLLAGEN AND TISSUE ENGINEERING ............................................................... ............ 81 5.2 STRUCTURE OF COLLAGEN ........................................................... ................................. 83 5.3 COLLAGEN OF A CHICKEN’S EGGSHELL MEMBRANE ...................................................... 85 5.4 BIOMINERALIZATION OF COLLAGEN ARCHITECTURES.................................................... 86 5.5 EXPERIMENTAL........................................................ ...................................................... 88

5.5.1 Preparation of the collagen membrane (CM) from a chicken’s eggshell matrix ..... 88 5.5.2 MPI process ............................................................. ................................................ 88 5.5.3 Tensile tests .............................................................. ................................................ 90 5.5.4 Cross section sample preparation by focused ion beam (FIB)................................. 91 5.5.5 SEM, TEM and EDX .......................................................... ...................................... 92 5.5.6 Raman spectroscopy.............................................................................................. ... 92 5.5.7 Wide angle X-ray scattering................................... .................................................. 92 5.5.8 Small angle X-ray scattering........................ ............................................................ 93

5.6 R ESULTS AND DISCUSSION ............................................................ ................................. 93 5.6.1 Mechanical deformation behaviour ................................................................. ........ 93 5.6.2 Metal infiltration into collagen ................................................................. ............... 95 5.6.3 Chemical analysis via Raman shift ............................................................. ............. 97 5.6.4 Structural analysis via x-ray scattering .............................................................. ..... 98 5.6.5 Biomineralization versus metal infiltration.......................... .................................. 101

5.7 CONCLUSION ........................................................... .................................................... 105

SUMMARY........... ..................................................................... ................................................... 107

REFERENCES AND NOTES ................................................................... .................................... 109

ACKNOWLEDGEMENT ..................................................................... ........................................ 129 APPENDIX................ ................................................................ .................................................... 131

A1. FIGURES........................................................ ............................................................... 131 A2. TABLES......................................................... ............................................................... 134 A3. R EFERENCES AND NOTES .............................................................. ............................... 135

CURRICULUM VITAE ............................................................. ................................................... 139



Chapter 1 1

Chapter 1

General Aspects of Atomic Layer Depositionand 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 1982. Since then, thanks to the new process,



2 ALD General

more than 2 million electroluminescent displays have been produced (about 200.000 m2

(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 by-

products 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 by-

products from vapor phase reaction



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. MO2 (M can be Ti, Zr or Hf etc.) deposition by ALD. For this deposition, a

metal containing precursor ML4 (L: ligand) as the metal source and H2O 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 ML4 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 isachieved, the purging of ML4 vapor begins. Sub-step 2 presents the situation at the beginning of the

purge, while Sub-step 3 shows a completed ML4 vapor purge. The same exposure–purge sequence

is repeated for H2O 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 H2O 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 MO2 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.



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, MO2 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: http://www.beneq.com).





6 ALD General

Figure 1.3 Overview of materials grown by ALD until Feb 2005. 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 cycletimes 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).



Chapter 1 7

Table 1.1 ALD materials together with corresponding precursor combinations reported in

literature and list of possible applications of ALD materials.

Film Precursors [3] Applications

Al2O3

AlCl3 / H2O or O3

AlBr 3 / H2O Al(CH)3 / H2O or O3

o High-κ dielectric [10]

o OLED Passivation [11]

o Anti-reflection and optical filters [12-14] o Wear and corrosion inhibiting layers [15]

HfO2 HfCl4 / H2O

TEMAHa / H2O

o High-κ dielectric [16-19]

TiO2 TiCl4 / H2O

Ti(OEt)4b / H2O

Ti(OiPr)4c

o High-κ dielectric [19,20]

o Photocatalysis [21]

o UV blocking layer [22]

o Photonic crystals [23]

SiO2 SiCl4, / H2O o Dielectric [18,19,24]

ZrO2 ZrCl4 / H2O

Zr(OtBu)4d / H2O

ZrI4 / H2O2


o Photocatalysis

o Wear and corrosion inhibiting layers

ZnO ZnEt2e / H2O

Zn(OAc)2f / H2O

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]

SnO2 SnCl4 / H2OSnI4 / O2

o Anti-reflection and optical filters

o Sensors (gas sensors, pH sensors)

[26,27]

Ta2O5 TaCl5 / H2O

Ta(OEt)5g / H2O

o Anti-reflection and optical filters [28]

o Sensors (gas sensors, pH sensors) [28]

o High-κ dielectric [28]

La2O3 La(thd)3

h / O3

La[N(SiMe3)2]3i / H2O


ZnS ZnCl2 / H2So Piezoelectric layers

o Optoelectronic applications [31,32]

WS2 WF6 / H2S o Solid Lubricant layers [33]

Zr 3N4 Zr(NMe2)4 j / NH3

o Biomedical coatings( biocompatible

materials for in-vivo medical devices and

instruments)

Ta2N5 TaCl5 / H2OTa(OEt)5 / H2O

o Photonic crystals [34]

TaN Ta(NtBu)(NEt2)k / NH3 o Diffusion barrier for Cu metallization [35]

AlN AlMe3 / NH3

AlCl3 / NH3 o Piezoelectric layers

TiN TiCl4 or Til4 / H2

o Diffusion barrier for Cu metallization [36]

o Conductive gate electrodes

o Biomedical coatings (biocompatible

materials for in-vivo medical devices and

instruments)

WN WF6 / NH3 o Diffusion barrier for Cu metallization [36]

o Optical application [37]

Cu

CuCl / H2 Cu(thd)2 / H2

Cu(acac)2l / H2

Cu(hfac)2 x H2Om

/ Ch3OH

MoMoF6 / H2 MoCl5 / H2

Mo(Co)6 / H2

NiNi(acac)2, 2 step processNiO by O3 reducedafterwards by H2

Ta TaCl5 / H2

W WF6 / B2H6 or Si2H6

Ti TiCl4 / H2

a TEMAH: Tetrakisethyl

methylaminohafnium,

Hf[N(C2H5)(CH3)]4.

b Ti(OEt)4: Titaniumethoxide,

Ti(OC2H5)4

c Ti(OiPr)4:

Titaniumisopropoxide,

Ti[OCH(CH3)2]4

d Zr(OtBu)4: Tetrakisethyl

methylaminozirconium,

Zr[N(CH3)(C2H5)]4

e ZnEt2: Diethylzinc,

Zn(C2H5)2

f Zn(OAc)2:

Zincacetatedihydrate,

Zn(O2CCH3)2(H2O)2

g Ta(OEt)5:Tantalumthoxide,

Ta(OC2H5)5

h La(thd)3,where thd is

2,2,6,6,-tetramethyl-3,5-

heptanedione,

(CH3)3CCOCH2COC(CH3)3

i La[N(SiMe3)2]3:

Lanthanumtris[bis(trimethylsil

yl)amide], C18H54LaN3Si6

j Zr(NMe2)4:

zirconium(tetra)dimethylamid

e, Zr[N(CH3)2]4

k Ta(NtBu)(NEt2)3 :

Tris(diethylamido)(tert-

butylimido)tantalum,

(CH3)3CNTa[N(C2H5)2]3,

where NtBu is called tert-

butylimido group.

l Cu(acac)2:Copper(II)

acetylacetonate, (C5H7O2)2Cu

m

Cu(hfac)2•H2O: Copper(II)

hexafluoroacetylacetonate

hydrate, C10H2CuF12O4.•H2O



8 ALD General

Table 1.2 Requirements for ALD precursors. The listed information in this table is summarized

from references [2,3,7].

Requirement Explanation

1 Adequate volatilityNecessary for efficient transportation of theprecursor to the ALD reactor at a rough limit of 0.1Torr and at the deposition temperature.

2 Reasonable thermal stabilityThermal-decomposition can destroy the self-limiting film growth.

3Aggressiveness,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 substratesNo competing reaction pathways.Etching prevents the film growth.

5No dissolution into the film orsubstrate

Dissolution would destroy the self-limiting filmgrowth.

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 2O3,

TiO2 and ZnO).

The oxides Al2O3, TiO2 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



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) + MLn(g) → ( ─ O ─ )xMLn-x + xHL(g) (R1)

And ( ─ O ─ )xMLn-x further react with water molecules during the next process step

( ─ O ─ )xMLn-x + (n-x)H2O(g) → ( ─ O ─ )xM( ─ 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)+ H2O(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 ─ )xMLn-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 + H2O(g) → ─ M( ─ OH)2 (R4)

O ─ M ─ O ─ M + H2O(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 Al2O3, ZnO and

TiO2 film are deposited as follows:

(i) Al2O3 deposition from the reaction of TMA/H2O [9,40,42,48,49]. A binary reaction

for Al2O3 chemical vapor deposition, 2Al(CH3)3 +3H2O → Al2O3 + 6CH4 is separated intotwo half-reactions:



10 ALD General

a, Al-OH* + Al(CH3)3 → Al-O-Al-(CH3)2* + CH4 ;

b, Al-O-Al-(CH3)2* + H2O → Al-O-Al-(CH3)OH* + CH4,

where the asterisks designate the surface species. TMA and H2O reactants were employed

alternatively in an ABAB…..binary reaction sequence to deposit Al2O3 films.

(ii) ZnO deposition from the reaction of DEZ/H2O [50-54]. Similar to the deposition of

Al2O3 from TMA/H2O, ZnO ALD is also based on a ZnO CVD process as follows

Zn(CH2CH3)2 + H2O → ZnO + 2C2H6

ZnO ALD is proposed to occur by speration of this binary reaction into two half-reactions:

a, Zn-OH* +Zn-(CH2CH3)2 → Zn-O-Zn-(CH2CH3)* + C2H6 ;

b, Zn-O-Zn-(CH2CH3)* + H2O → Zn-O-Zn-OH* + C2H6,

where the asterisks designate a surface species. Alternating ABAB…..reaction sequence is

repeated to deposit ZnO films.

(iii) TiO2 deposition from reaction of TIP/H2O [55-58]. Unlike Al2O3 and ZnO, the

TiO2 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, TiO2 grows via the two following

reactions (“ ─ ” denotes bonds between substrate surface) [58]:

2( ─ OH) + Ti[OCH(CH3)2]4 → ( ─ O ─ )2Ti[OCH(CH3)2]2 + 2(CH3)2CHOH (R6)

( ─ O ─ )2Ti[OCH(CH3)2]2 + 2H2O → ( ─ O ─ )2Ti( ─ OH)2 + 2(CH3)2CHOH (R7)

In the reaction, half of the ligands are released during the Ti[OCH(CH3)2]4 pulse

anchoring on the surface hydroxyl groups (R6). The H2O 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 H2O 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 H2O pulse and the surface again becomes

─ OH terminated (R9) as follows:

( ─ OH) + Ti(OCH(CH3)2)4 → ─ O ─ Ti[OCH(CH3)2]3 + (CH3)2CHOH(g) (R8)



Chapter 1 11

─ O ─ Ti[OCH(CH3)2]3 + 2H2O → ( ─ O ─ )2TiOH + 3(CH3)2CHOH(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



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 self-

limiting 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.



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 Å range

o

Controlled by counting thenumber of reaction cycles

o Ensured by the saturation

mechanism

o 10Å range,

o

Controlled by process control andmonitoring, time

o Requires uniform flux of reactant

and uniform temperature

Film Quality

o Excellent stoichiometry

o Low pinhole count

o Stress control possible

o Excellent stoichiometry

o Low pinhole count

o Stress control possible

Conformality100% step coverage in 60:1 aspect

ratio

100% step coverage in 10:1 aspect

ratio

CleanlinessNo particles due to separated half

reaction

Particles due to gas phase reactions

Vacuum Requirement Medium Medium

Process Window< 1% dependency on 10% process

parameter changes

Strong dependency on process

parameter changes

Precursor

o Highly reactive precursors

o Precursors must not

decompose at process

temperatures

o Less active precursors

o Precursors can decompose at

process temperature

Deposition reaction Surface reactionSurface reaction +

Gas phase reaction

Contamination

5 ~ 30 wt % (C, O).

But with PE-ALD contamination canbe minimized (< 1 wt %).

< 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 thefield 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



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



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).



16 ALD General



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 1-2 ν 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



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-L0)

(see Figure 2.2). The engineering measures of stress and strain, denoted in this module as

σe and εe respectively, are determined from the measured load and deflection using theoriginal specimen cross-sectional area A0 and length L0 as σe = F/A0 and εe = δ/L0,

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 (= εe/σe)

ee E ε σ = (2.1)



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 stress-

strain 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 (σe) and the engineering strain (εe) 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 stress-

strain curve is called true stress (σt)-strain (εt) curve. C1: true stress (σt)-strain (εt) curve; C2:

engineering stress (σe)-strain (εe) curve; R1: elastic deformation region (reversible); R2: strain

hardening region (permanent deformation); R3: necking region (permanent deformation); P1:

yielding point (σy,εy), limit of the elastic region; P2: ultimate tensile strength (UTS), onset point ofnecking; P3: fracture point (σf ,εf ); 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 rearrangementsassociated with plastic flow are usually not reversible, even if the load is removed, so the




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 σY 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 εe = 0.2%; this is the unloading line that would result in the specified

permanent strain. The stress at the point of intersection with the σe-εe 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 A0, so the true stress (σt

= F/A actually borne by the material is larger than the engineering stress computed fromthe original cross-sectional area (σe = F/A0). The load must equal the true stress times the

actual area (F = σtA). As long as strain hardening can increase σt 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:



Chapter 2 21

t

t t t t

d

A

dAdA Ad dF Ad dF

σ

σ σ σ σ =⇒+==⇒= - 0 )( (2.2)

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:

http://web.mst.edu/~ide120/lessons/tension/fractures/index.html) [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 asdiscussed above, and should be used with care. The yield stress σY 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 theflow. This localized and increasing flow soon leads to a neck in the gage length of the




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, εf ) 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 εf will depend on the fraction of the gage length

that has necked. Therefore, εf 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 σt = F/A rather than the engineering stress σe = F/A0 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

0

In1

0

L

LdL

L

L

dLd

L

L

t t ==⇒= ∫ε ε (2.3)

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 stilluniform along the specimen length, this volume (V) constraint can be written:

L

L

A

A L A ALdV V 0

0

00 0 constant =⇒=⇒=⇒= (2.4)

The ratio L/L0 is defined as extension ratio (λ ). Using these relations, the relation between

true and engineering measures of tensile strain can be developed as follows



Chapter 2 23

λ ε ε In)In(1InIn0

0

0

=+=+

== et L

dL L

L

L (2.5)

The relation between true and engineering measure of tensile stress can be also derived as

follows

A

A

L

L e

0

0

1 ==+ ε (2.6)

e

A A

ε +=

10 (2.7)

λ σ ε σ ε

σ eeee

t

A

F

A

F =+=

+== )1(

)1(

0

(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:

t A

A

L

L

A

A

L

L ε λ ===⇒= 0

0

0

0

InInIn (2.9)

2.3 Strain energy, breaking energy and toughness

The area under the σe−εe 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 shownas follows:

∫∫∫∫ ====ε

ε σ 00 00000

1

1 ee

L L

d L

dL

A

F FdL

L A FdL

V U (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




is just the triangular area in Figure 2.4a. In the elastic region, the strain energy can be

described as follows:

E E d E d U e

eeeee

22

002

1

2

1

σ

ε ε ε ε σ

ε ε

==== ∫∫ (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



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/m3 or N/m2, 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. Itdefines 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, σf . High strength is needed to allow carrying a highload. 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




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



Chapter 2 27

toughness are needed for many applications. The basic difference between stiff, strong and

tough materials is graphically illustrated in Figure 2.6.

2.5 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 immediatelyrecovers 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 L2 is equal to L1, the material’s behaviour is perfectly plastic.

When L1 is larger than L2, 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. Δεp denotes permanent (or residual) strain caused by plastic deformation. Δεe 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




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 andplastic 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.



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.






Chapter 3 31

Chapter 3

Metal Oxide Deposition on Biotemplate:

macroporous photocatalytic TiO2 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 TiO2 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 TiO2 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 TiO2 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.



32 Photocatalytic Metal Oxide Membrane

3.1 Background

3.1.1 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 TiO2,

ZnO, CdS, ZnS, CdSe, ZnSe, ZnTe, GaAs, GaP, and KTaO3 were continued [74-81].

Although the photoinduced effect of semiconductors, such as Al2O3 [82], MgO [8282],

SiO2 [82], CdO [82], ZnO [82], CeO2 [82,83] and TiO2 [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 TiO2) 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 1973. 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].



Chapter 3 33

3.1.2 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; EF,n: Fermi level of a n-type semiconductor; EF,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, underillumination 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 simpleterms as follows:

Oxidation: Loss of electrons or increase in oxidation state of a molecule, atom or ion. i.e. Reductant → Product + e-

Reduction: Gain of electrons or decrease in oxidation state of a molecule, atom or ion. i.e. Oxidant + e- → Product




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 TiO2 or ZnO) is irradiated with light consisting of wavelengths

shorter than the wavelength corresponding to the energy of its bandgap, (about 415 nm -

3.0 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 [104-107]. 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 TiO2, trapping of holes proceeds in 10-100 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 arapid conversion to the final products Are and Dox (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 TiO2 [104-106] 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 hydroxylradicals via following reactions [104,105,107]:



Chapter 3 35

Semiconductors (such as TiO2 or ZnO) + hv → semiconductors (eCB- + hVB

+) (R1)

hVB+ + OH- → •OH (R2)*

eCB- + O2 → •O2

- (R3)

•O2- + H+ → HO2• (R4)

HO2• + HO2• → H2O2 + O2

(R5)

•O2- + HO2• → HO2

- + O2

(R6)

HO2- + H+ → H2O2

(R7)

H2O2 + •O2- → •OH + OH- + O2

(R8)

H2O2 + eCB- → •OH + OH-

(R9)

•OH + Pollutants + •O2- → Products (CO2, H2O, etc.) (R10)

*The reaction (R2) is called “indirect hole transfer mechanism”. i.e. the photogenerated hole oxidizes an OH

group on the surface of TiO2 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 CO2. Furthermore, photocatalysis has been used forremoving 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.

3.1.3 Crystal structure of TiO2 and ZnO

TiO2. 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

TiO6 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




structures of rutile and anatase crystals [109,110]. Each Ti4+ ion is surrounded by an

octahedron of six O2- 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 1.980 Å) are shorter than in rutile (1.949 Å and 1.980 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 TiO2 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 TiO2. b, Anatase

phase TiO2. 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 3.2475 to

3.2501 Å for parameter a and from 5.2042 to 5.2075 Å for parameter c (see Figure 3.2c).

The c/a ratio varies in a slightly wider range, from 1.593 to 1.6035. The deviation from



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.

3.1.4 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 1919. E. coli are Gram-

negative 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

negativebacteria 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 (http://media.med.sc.edu/microbiology2007).




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 thosecoatings 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].

b

a

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



Chapter 3 39

range of operation temperatures [120-122]. The feasibility of ALD for biological

templates [123-126] 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 TiO2 [123,124] and Al2O3

[124-128]. 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 [129-131]

or further deposition methods [132-134]. 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 [135-137]. TiO2 or ZnO were deposited by

ALD on this ISM, both of which show bactericidal photocatalytic effects under UV

illumination (ISM/TiO2 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 TiO2 coated membranes. In addition, an improved mechanical stability of the

ZnO coated membranes was observed.

3.3 Experimental

3.3.1 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.

3.3.2 TiO2/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 (1×10-2 torr) with a steady Ar stream (20




sccm). For the TiO2/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 TiO2 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

H2O was repeated. The thickness of the deposited TiO2 and ZnO films was adjusted to 30

nm and 55 nm, respectively, by the number of cycles. For the preparation of diverse

samples with TiO2 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 PrecursorPulse

(second)

Exposure

(second)

Purge

(second)Cycle

Substrate

temperature

Sample

numbering

70°C ISM/TiO2/70

160°C ISM/TiO2/160Ti [OCH(CH3)2]4 1.5 30 30

225°C ISM/TiO2/225

275°C ISM/TiO2/275

TiO2

H2O 1.3 30 30

500

300°C ISM/TiO2/300

70°C ISM/ZnO/70(C2H5)2Zn 0.1 5 15

100°C ISM/ZnO/100

125°C ISM/ZnO/125ZnO

H2O 1.5 15 150

500

200°C ISM/ZnO/200

3.3.3 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 = 1.5421 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.



Chapter 3 41

3.3.4 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 105 to 106 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.




3.3.5 Photocatalytic experiments with ISM/TiO2 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 ofcolonies 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.



Chapter 3 43

3.3.6 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 preparedsamples, 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 26-28 °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 toyield ~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® 7.5.

3.4 Results and discussion

3.4.1 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/TiO2, show unchanged morphological

features of the original ISM. As representative SEM and TEM micrographs of the

resulting ISM/ZnO and ISM/TiO2 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




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/TiO2 and ISM/ZnO

prepared at temperatures ranging from 70°C to 300°C. The TiO2-coated membranes

(ISM/TiO2) 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 (ICDDcard No. 21-1272), anatase (101) being the strongest peak among them. The deposition



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. 36-1451.

a c

b d

Figure 3.9 X-ray diffraction (XRD) patterns and E.coli survival rates on ISM/TiO2 (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 TiO2 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 asZnO and TiO2 generate conduction electrons (e-) and valence band holes (h+) on the

surface upon illumination with an energy higher than the band gap energy (Eg,ZnO = 3.37

eV, Eg,TiO2 = 3.2 eV) in an aqueous solution [138,139]. Subsequently, holes react with the

water adhering to the surface of the ZnO and TiO2 to form highly reactive hydroxyl

radicals (OH•). Oxygen acts as an electron acceptor forming super-oxide radical anions

(O2•−) which are an additional source of hydroxyl radicals upon subsequent formation of

hydrogen peroxide (H2O2) [138,139]. The generated OH•, O2•− and H2O2 can attack the

cell walls in E.coli, which finally are damaged. After eliminating the protection of the cell




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 TiO2, 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

TiO2 has a stronger dependence on the crystallinity than on the film thickness [144,145].

Specifically, the bactericidal efficiency of the ISM/TiO2 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 TiO2 particles (Degussa P25, average particle size: 30 nm) [146] or home-made TiO2

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 TiO2, 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/TiO2 reveals a higher

bactericidal efficiency than ISM/ZnO. This result is consistent with a previous publication

comparing the photocatalytic efficiency of TiO2 and ZnO [149,150], however, opposite

results have been also reported [151,152]. The relative photocatalytic behaviour of TiO2

and ZnO still seems to be ambiguous.

The graphs in Figure 3.9c and d show that, as expected, ISM/TiO2/300 has thestrongest bactericidal effects. However, with decreasing processing temperature, the

efficiency of the TiO2-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 thesensitive membranes are coated with ZnO at 100 °C, showing reasonably efficient



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/TiO2.

3.4.2 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

TiO2, 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] (σmax) Maximum Strain[%] (εmax)

Native ISM 6.21 ± 0.62 6.18 ± 0.52

ISM/TiO2/275 6.02 ± 0.25 3.45 ± 0.43

ISM/ZnO/100 9.09 ± 0.71 9.02 ± 0.83

The results from our experiments are shown in Figure 3.10 and Table 3.2. Theresulting ISM/TiO2/275 membranes deposited at 275°C were more brittle and stiffer than




the native ISM (decreased maximum strain εmax and increased stiffness (initial Young’s

modulus), Eini(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 σmax and strain εmax together with stiffness Eini(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 5.

3.5 Conclusion

In conclusion, eggshell membranes were used as templates for coatings with TiO2 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/TiO2 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/TiO2 membranes prepared at a much higher temperature (275 °C).

Furthermore the ZnO-coated membranes are mechanically more stable than the TiO2-

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 TiO2, i.e. thermally less destructive and photocatalytically

competitive.



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

4.1.1 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),



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.



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 (Eini), maximum tensile

stress (σmax), maximum tensile strain (εmax)

and toughness of a dragline silk together with

a comparison with other natural and man-made 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




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].

4.1.2 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



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=CH3. 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 (−NH2) by dehydration (COOH + H2N →

CONH + H2O).

Table 4.1 Amino acid composition of silk from Araneus diadematus. The amino acids aredescribed with a three-letter code. The table is written based on the reference [167].

Amino acidMajor

Ampullate

Minor

AmpullateFlagelliform Aciniform Cylindriform

Asp 1.04 1.91 2.68 8.04 6.26

Thr 0.91 1.35 2.48 8.66 3.44

Ser 7.41 5.08 3.08 15.03 27.61

Glu 11.49 1.59 2.89 7.22 8.22

Pro 15.77 trace 20.54 2.99 0.59

Gly 37.24 42.77 44.16 13.93 8.63

Ala 17.60 36.75 8.29 11.30 24.44

Val 1.15 1.73 6.68 7.37 5.97

Ile 0.63 0.67 1.01 4.27 1.69Leu 1.27 0.96 1.40 10.10 5.73

Tyr 3.92 4.71 2.56 1.99 0.95

Phe 0.45 0.41 1.08 2.79 3.22

Lys 0.54 0.39 1.35 1.90 1.76

His trace trace 0.68 0.31 trace

Arg 0.57 1.69 1.13 4.09 1.49

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, havingdifferent 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.




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].

4.1.3 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 suchremarkable 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 weremade. 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 hydrogen-

bonded 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.



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 two-

dimensional 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).




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 Kα 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].



Chapter 4 57

4.1.4 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 knownas 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.





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

materials

Vapor pulse

Pairs

Pulse

(second)

Exposure

(second)

Purge

(second)Cycle Sample† #

1 [1] SS/N

2 [2]SS/H

3 [3]SS/TIP

4 [4]SS/W‡

5 SS/Al2O3/100

6 [5] SS/TP/100

7

100

[6] SS/WP/100

8

TMA 0.3 30 30

SS/Al2O3/300

[7] PF/Al2O3/300

9 300[8] SS/Al2O3/300/H

10 SS/Al2O3/500

11500

[9] SS/Al2O3/500/H

12

Al2O3

H2O 1.5 40 40

700 SS/Al2O3/700

13 300 SS/TiO2/300

14 SS/TiO2/500

15

TIP 1.5 30 30500

SS/TiO2/500/H

16 700 SS/TiO2/700

17

TiO2

H2O 1.3 30 30

700[10]SS/TiO2/700/C

18 100 SS/ZnO/100

19DEZ 0.1 5 10

300 SS/ZnO/300

20

ZnO

H2O 1.5 15 150 700 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=Patm) 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 Al2O3 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.




4.2 Experimental

4.2.1 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 25×15×10 cm3 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 307-148.001, 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 (70×70×30 cm3). 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.

4.2.2 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 (1×10-2 torr) with a steady Ar gas stream (20sccm). For the infiltration, TMA/water, TIP/water, and DEZ/water (H2O) 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.



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 conditionsidentical 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.




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 TiO2 film is deposited by continuous and not self-limiting

process, thereby causing much higher TiO2 layer thickness and coarse film quality as can be seen inc. E: Exposure and P: Purge of each precursor.

4.2.3 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



Chapter 4 63

length per minute (2 mm/min). The temperature and relative humidity were 22-24 °C and

32-36 %, 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 Al2O3, TiO2 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.

4.2.4 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.




4.2.5 Solid state nuclear magnetic resonance (NMR) spectroscopy

From the samples SS/Al2O3/300 and PF/Al2O3/300 (Table 4.2) 27Al-NMR spectra were

recorded at 195.31MHz 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 AlCl3 at 0 ppm.

4.2.6 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

CuK α (λ = 1.5421 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 (Өi) 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 2ӨA = 16.97°, 2ӨB = 14.09° and 2ӨC = 18.65°. 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].



Chapter 4 65

[ ]∑= ⎪⎭

⎪⎬⎫

⎪⎩

⎪⎨⎧

⎟⎟ ⎠

⎞⎜⎜⎝

⎛ −−+=

6

12

2

00

2

)2()2(exp)2(

n n

n

w I I

θ θ θ ,

where Ө0 and ωn 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. ωn, the average crystallite size was estimated by the Scherrer equation

[187,188], L = 0.9λ / (ωn cosӨ0). 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 (version10.0) was used and graphical tasks were performed with ORIGIN® 7.5. The two main




peaks, PA and PB 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

gFigure 4.11 Tensile test curves of diverse silk

composites. a-c, Stress(σ) - strain (ε) curves of

multiple pulsed vapor phase infiltrated [(a) TMA /

H2O, (b) TIP / H2O and (c) DEZ/H2O] 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 H2O (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



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 (σmax), maximum strain (εmax) 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 25.72 ± 4.50 0.90 ± 0.12 9.7 ± 1.4 141.8 ± 20.1

2 SS/H 10.42 ± 1.70 1.15 ± 0.15 59.1 ± 3.1 73.5 ± 13.1

3 SS/TIP 27.48 ± 3.17 1.57 ± 0.44 15.0 ± 2.8 203.0 ± 18.6

4 SS/W 21.94 ± 1.95 1.24 ± 0.06 20.2 ± 3.2 180.4 ± 13.6

5 SS/Al2O3/100 26.64 ± 8.36 3.33 ± 0.16 50.1 ± 3.7 498.9 ± 186.2

6 SS/TP/100 27.78 ± 4.91 0.49 ± 0.14 1.7 ± 0.8 81.8 ± 29.0

7 SS/WP/100 22.08 ± 7.38 0.34 ± 0.08 3.5 ± 0.5 49.3 ± 19.9

8 SS/Al2O3/300 65.07 ± 1.42 3.97 ± 0.06 53.8 ± 4.5 1362.9 ± 85.1

9 SS/Al2O3/300/H 22.25 ± 2.54 3.20 ± 0.37 55.2 ± 2.1 459.2 ± 53.1

10 SS/Al2O3/500 55.75 ± 1.69 3.53 ± 0.21 63.4 ± 6.4 1095.1 ± 62.6

11 SS/Al2O3/500/H 28.12 ± 2.41 2.51 ± 0.12 65.4 ± 4.2 462.3 ± 54.7

12 SS/Al2O3/700 44.62 ± 3.91 4.58 ± 0.00 68.2 ± 5.7 1007.4 ± 143.6

13 SS/TiO2/300 46.98 ± 3.92 3.80 ± 0.60 53.1 ± 4.4 900.9 ± 150.2

14 SS/TiO2/500 71.28 ± 1.20 4.24 ± 0.36 58.0 ± 4.8 1493.4 ± 22.0

15 SS/TiO2/700 58.09 ± 5.26 4.20 ± 0.44 66.3 ± 5.5 1294.6 ± 154.3

16 SS/TiO2/700/C 21.49± 9.86 1.05 ± 0.38 62.2 ± 3.5 175.9 ± 4.62

17 SS/ZnO/100 33.56 ± 5.99 3.71 ± 0.48 63.4 ± 6.5 710.8 ± 171.5

18 SS/ZnO/300 25.21 ± 0.15 3.64 ± 0.02 70.6 ± 5.3 534.7 ± 26.5

19 SS/ZnO/700 23.89 ± 0.12 3.18 ± 0.28 87.7 ± 3.4 437.7 ± 26.6

4.3 Results and discussion

4.3.1 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





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 = Patm) 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 byMPI) (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 negligibleexposure times. Considerable enhancement of the mechanical properties of the silk was observed only when the

Savannah 100 was used.




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

Al2O3, TiO2 and ZnO growth showed self-limited behaviour and their growth rates were

around 1Å/cycle (Al2O3), 0.5Å/cycle (TiO2) 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 Al2O3 and TiO2, 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 [190-192], 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 =

1×10-2 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 (Δσmax =

−25%, Δεmax = −60% and Δσmax = −31%, Δεmax = −44%). In contrast, SS/H showed an

increased maximum stress (Δσmax = 27%) and a decreased maximum strain (Δεmax =

−60%) (Table 4.3).

As an additional concern, the question arises whether after the MPI process the silksare 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



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).

4.3.2 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 (wn, 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 (Al2O3 deposition + simultaneous annealing duringdeposition) 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 TiO2 deposited

silk (c and d). b and d are drawn from the superposition of Gaussian profiles which are illustrated in

Figure 4.10.

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




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 Al2O3 and TiO2 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 (wn, 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) [186-188]. 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 ≡ [IB,max /

IA,max], also revealed variations, as shown in Table 4.4 and Figure 4.13b. Initially, the

intensity ratio of SS/N, i.e. ∂ISS/N = [IB,max / IA,max]SS/N, was 0.370 but by MPI both

∂ISS/Al2O3/300 and ∂ISS/TiO2/500 increased to 0.385 and 0.462, respectively. In contrast, after

annealing, a differing behaviour in the intensity ratio was found; ∂ISS/H and ∂ISS/TiO2/500/H

decreased as compared to ∂ISS/N and ∂ISS/TiO2/500, respectively, but ∂I SS/Al2O3/300/H increased

as compared to ∂ISS/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 εmax). The contribution of the outer metal

oxide layer coating the silk to the increase of εmax can also be neglected since in general

metal oxides, such as Al2O3, TiO2 and ZnO are very brittle. Therefore, the probability toinduce the enhancement (increased σmax and increased εmax) 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






[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, δiso, 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,

<Cq>. 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- [AlO4], five- [AlO5], and six-fold [AlO6] oxygen-coordinated

aluminum, respectively [198,199,205-208]. On the other hand, the spectral shape of

SS/Al2O3/300 (Figure 4.14c) shows one additional component with an isotropic chemical

shift of δiso = - 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 δiso is comparatively low, it is still within the range reported in

the literature for six-fold coordinated aluminum, [AlO6] [208]. Significantly, a negative

value of δiso has also been observed for 27Al 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 inthis figure, along the TiO2 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.



Chapter 4 75

a b

c d

Figure 4.14 Nuclear Magnetic Resonance (NMR) spectra of silk composites, SS/Al2O3/500.27 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,

δiso, the width of the distribution, dδ, and the average quadrupolar coupling parameter, <Cq>, plus

the relative integrated intensities of the spectral lines.

Samples Signal δiso / ppm dδ / ppm <Cq>Integrated

Line Intensity

1 69.0 9.2 9.55 36.9%

2 43.6 15.2 8.06 46.3%PF/Al2O3/300

3 16.0 10.4 6.40 16.8%

1 71.8 13.2 9.54 29.4%

2 45.2 11.8 9.04 37.9%

3 9.6 16.7 5.24 23.9%SS/Al2O3/300

4 -4.9 9.3 4.81 8.8%




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 TiO2 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 TiO2 layer and the silk part (SS/TiO2/500)

showed similar image contrast. Ti infiltration into the silk was observed along the whole

TiO2 shell [inset (I)]. b, Element concentration from EDX scanned across the boundary

region along the TiO2 layer (A to B in the pink box; R1, R2, and R3 designate the region

of the carbon grid, TiO2 layer, and silk part of SS/TiO2/500, respectively). c, EDXspectrum measured on the folded silk part located at the center of the silk, Ti X-ray

emission peak Kα at 4.5 keV and Kβ at 4.9 keV.

4.3.3 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 beassumed. 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 Al3+[215], Ti4+[216] or Zn2+[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.



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, Al3+, Ti4+ or Zn2+ 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/H2O) or isopropanol (TIP/H2O) 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 metal-

infiltrated silks, which presumably may have covalent or coordinated bonds, are hardly

affected by these conditions††.

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 long-

term exposure (reaction of

TMA with unsaturated bonds

was already shown in [23]), atelevated temperatures, which,

together with the water pulses,

weakens the hydrogen bonds.

Consequently, Al3+ is likely to

be inserted at those sites,

thereby resulting in the

formation of metal-coordinated or even covalent bonds with Al.

†† 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%).




4.3.4 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



Chapter 4 79

infiltration, function as factors to enhance σmax and εmax of MPI-treated silks (Figure 4.17

and Figure 4.18).

b

a

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 theyielding stress, Δσy, and initial Young’s modulus, Ei of SS/Al2O3/300.

On the other hand, it was observed that the individual TMA or H2O 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




precursor at ambient conditions shows a slight enhancement in mechanical properties

(increased σmax 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 ZnCl2 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 ConclusionBy “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.





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 [225-227]. 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



Chapter 5 83

into natural collagen membranes (primarily composed of type I and V collagen) collected

from shell membranes of chicken’s eggs [230-232]. Upon infiltration the deformation

behaviour under uniaxial tension showed a simultaneous increase of strength and ductility.

The toughness (breaking energy, i.e. ∫

F

d

ε

ε σ 0

where σ, ε and εF are stress, strain and

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 72 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 alongeach chain, generating the observed (Gly-X-Z)n pattern, where X and Z are frequently Pro




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.



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] [235-239]. 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 [230-232]. The detailed amino acid

composition of the inner shell membrane is summarized in Table 5.1.





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 assemblein 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 platelet-

like 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) [251-253]. 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) underuniaxial tensile load. As the mineral contents increase, the stiffness (initial Young’s




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

5.5.1 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 carefullycollected 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.

5.5.2 MPI process

Sample CM/Al2O3, CM/TiO2 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 (1×10-2 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






Table 5.2 Detailed processing conditions of prepared samples and their denotation. All vapor

infiltration experiments were performed at 70°C substrate temperature.

Resultingmaterials

Vapor pulsePairs

Pulse(sec)

Exposure(sec)

Purge(sec)

Cycle Samples

1

[1]

CM/N

2 [2] SG/N

3 TMA 0.3 30 30 CM/Al2O3

4Al2O3

H2O 1.5 40 40 [3] Si/Al2O3

5 TIP 1.5 30 30 CM/TiO2

6TiO2

H2O 1.3 30 30 [4] Si/TiO2

7 DEZ 0.1 5 10 CM/ZnO

8 ZnO H2O 1.5 15 150

500

[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 Al2O3, TiO2 and ZnO are

deposited together with CM/Al2O3, CM/TiO2, and CM/ZnO under the same processing conditions,

respectively.

5.5.3 Tensile tests

The experimental conditions for tensile test were partly described already in Chapter 3. Amore 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 26-28 °C and 20-22 %, respectively. The



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® 7.5.

5.5.4 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 µm2 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.




5.5.5 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 80-300 microscope

in scanning mode.

5.5.6 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/μm2. 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 50 – 2000 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.

5.5.7 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 conventionallaboratory wide angle X-ray diffractometer (Philips X’Pert MRD with 50kV, 30mA) with

Ni-filtered CuK α (λ = 1.5421 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







Chapter 5 95

inflection points, from the slopes (α) of each curve defined by

id

d

ε ε ε

ε σ α

=

=)( and the σ-intercept values β, line

equations, β αε ε +=)( f , corresponding to each curve were calculated. From the absolute convergence value

defined by )()()( nnn f ε ε σ ε ξ −= in the range of F i ε ε ε ≤< , when the value

)(

)( 1

n

n

ε ξ

ε ξ + becomes larger than 2,

that point was defined as a yielding point ),( Y Y ε σ .

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 saw-

tooth 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 5.8.

5.6.2 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 TiO2 shell layer and the bulk

collagen was observed. Along the TiO2 shell layer, in some regions, it was difficult to

discriminate a sharp boundary between the TiO2 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 TiO2

shell layer the collagen contains a substantial amount of Ti. In the central region, a weakersignal (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




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 [265-269]. 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.



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).

5.6.3 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




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 Ti-

mediated 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

5.6.4 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



Chapter 5 99

structure of collagen. The structure itself is relatively well understood both on the

macroscopic as well as on the atomic level [233-239,273,275]. 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 TiO2 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 Al2O3 and TiO2 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, Al2O3, TiO2 and the slide glass reveal amorphous phases,

and ZnO shows only the wurtzite phase.




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, )(q F was calculated with isotropically oriented lamellae using the

equation,

2

2

)2/sin()( ⎟⎟

⎠

⎞⎜⎜

⎝

⎛ =

q

qd

q

K q F [276], where d and K are the thickness of the lamellae

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 Al2O3 and TiO2 films on the collagen

membrane were around 45-55 nm and 30-40 nm, respectively. These values were relatively

close to the d values from the form factor (dCM/Al2O3 = 44 nm, dCM/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.



Chapter 5 101

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.

PeakLocation

(2θ) [°]

Spacing

d (=1.54/2sinθ) [Å]CM/N CM/Al2O3 CM/TiO2 CM/ZnO

A 29.4 3.02 Yes Yes Yes No

B 36.2 2.47 Yes No Yes No

C 39.5 2.27 Yes No Yes No

D 16.7 5.30 No No Yes Yes

E 14.0 6.31 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).

5.6.5 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 [242,246,254-256], periodically occurs only

within limited zones (hole and overlap, Figure 5.4) with 60-70 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.




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, (εF – εY) as well as (εF – εY)/εY 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.






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)

[230-282].

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].








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 TiO2 or ZnO, processed under similar processing conditions, was performed. The

study included a comparison of crystallographic features of TiO2 and ZnO deposited byALD, mechanical/ thermal stability and bactericidal efficiency of the resulting inorganic

structures. Both, the ZnO and the TiO2 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 TiO2 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 hadalready been reported that in nature, tiny amounts of inorganic impurities, such as metals,





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130



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 Al2O3 layer show clear

contrast difference. The thickness of the deposited Al2O3 layer was about 45 nm. The border

between Al2O3 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.



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.



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.



134

A2. Tables

Table A1 Observed Raman shifts and corresponding tentative assignments.

Region Raman shift(cm-1)

Tentativeassignments 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]

1451 δ(CH2) scissoring in lipids andδ(CH3),(CH2) in protein

[4]-[5], [15]–[19]

1324 τ(CH3, CH2)[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)[4]-[5], [8],[10],[13],[15], [19], [22]-[24],[25]

970 ν(C-C) [15], [19]

875 ν(C-C) of Hydroxyproline ring [4], [10], [19]

855

Proline, Hydroxyproline, Tyrosineν(C-C) in Proline ringSide chain vibrations of Proline,Hydroxyproline

[8],[4]–[5],[19][8]

1800to

800(cm-1)

831

Proline, Hydroxyproline, TyrosineC-H out of plan bending of benzoid ringTyrosine (Fermi resonance of ringfundamental and overtone)

[8],[26][13]

578 TiN [27]-[31]

570 ?

488 ?

338 TiN [27]-[32]

324 ZnO [33]-[35]

700to

200(cm-1)

235 AlN [36]-[37]

Key to Abbreviation: ν(stretching), δ(bending) and τ(twisting)



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, 2107-2116 (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,

1313-1320 (2007).4. Frushour, B. G. & Koenig, J. L. Raman scattering of collagen, gelatin, and elastin.

Biopolymers 14, 379-391 (1975).

5. Buschman, H. P. Raman microspectroscopy of human coronary atherosclerosis:

Biochemical assessment of cellular and extracellular morphologic structures in situ.

Cardiovasc. Pathol. 10, 69-82 (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, 5509-5518 (1989).7. Chi, Z., Chen, X. G., Holtz, J. S. W. & Asher, S. A. UV resonance Raman-

selective amide vibrational enhancement: quantitative methodology for

determining protein secondary structure. Biochemistry 37, 2854-2864 (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, 75–

79 (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.) 3335–3362 (John

Wiley and Sons, 2002).

10. Huang, Z. et al. Near-infrared Raman spectroscopy for optical diagnosis of lung

cancer. Int. J. Cancer 107, 1047–1052 (2003).

11. Stone, N. et al. Raman spectroscopy for identification of epithelial cancers.

Faraday Discuss. 126, 141-157 (2004).

12. Stone, N. et al. Near-infrared Raman spectroscopy for the classification of

epithelial pre-cancers and cancers. J. Raman Spectrosc. 33, 564-573 (2002).







138



Curriculum Vitae 139

Curriculum Vitae

Seung-Mo, Lee

Personal Information

Place of Birth :

Chunchon, Korea

Gender : Male, Married

Nationality: Korea

Address: Max‐Planck‐Institut fuer Mikrostrukturphysik

Experimental Dept. II

Weinberg 2, D‐06120 Halle(Saale), Germany

E‐mail: 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)



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 Scholarship and Prize Honour Student ( full 4 years scholarship) from Inha University

o Deutscher Akademischer Austausch Dienst (DAAD) Scholarship

Additional Capabilities

Language: English, German

Computer

skill:

I‐

DEAS,

Auto

CAD,

Fortran

and

C

language

Sport: Tennis, Badminton, Soccer

Extracurricular activity: Calligraphy, Hiking, Acoustic Guitar, Photography

Field of Interests

o Atomic Layer Deposition process

o Bio‐inspired organic/organic hybrid materials

o

LIGA process, electroforming, Injection molding, Stereolithography, Microfluidics, optics,

Polymer processing, Micro optics, Mirosensor and actuator

o Patterning of nano‐ and micro size structure and its applications, Laser Interference

Lithography

o 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, 553–559 (2006)

“Fabrication of hydrophobic films replicating plant leaves in nature”

[02] S.‐M. Lee, T. H. Kwon

Nanotechnology 17, 3189–3196 (2006)

“Mass‐producible replication of highly‐hydrophobic surfaces from plant leaves”

‐Highlighted

in

nanowerk.com





J. Micromech. Microeng. 17, 687–692 (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, 114‐118 (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,

3085

‐3091

(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, 3487‐3494 (2008)

“Hierarchical three‐dimensional ZnO and their shape‐preserving transformation into hollow

ZnAl2O4 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, 700208 (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,

103108

(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, 2796–2810 (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



142

Phys. Chem. Chem. Phys. 11, 3608‐3614 (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, 488‐492 (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: p369‐372)

“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: p247‐250)

“Replication of highly‐hydrophobic surface with micro/nano combined structures from

nature”




[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



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) 이 , 이현섭, 김동성, 권태헌

The 7th Korean MEMS Conference, April 7‐9, 2005, Jeju Island (Korea) (Proceeding: p 57‐

60)




“식물잎을 이용한 소수성 필름 제작”

[02] (Poster) 이 , 권태헌

The 7th Korean MEMS Conference, April 6‐8, 2006, Jeju Island (Korea) (Proceeding: p 605‐

608)

“식물 잎의 표면 구조 모사를 이용한 소수성 필름의 대량 생산 방법”

Patents


Application number: 11/225,150

Publication number: US 2007/0013106 A1

Filing date:

13

Sep

2005

U.S. Classification 264338000

“Method of preparing hydrophobic polymer substrate and hydrophobic polymer”



146



Documents

Atomic Layer Deposition on Biological Materials BarrierPhD Thesis Seung Mo Lee Final 20091224