Structure and Properties of Nanomaterials: From Inorganic ... · Structure and Properties of...

Structure and Properties of Nanomaterials: From Inorganic Boron Nitride Nanotubes to the

Calcareous Biomineralized Tubes of H. dianthus

by

Adrienne Elizabeth Tanur

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Department of Chemistry University of Toronto

© Copyright by Adrienne Elizabeth Tanur 2012

ii

Structure and Properties of Nanomaterials: From Inorganic Boron Nitride Nanotubes to the

Calcareous Biomineralized Tubes of H. dianthus


Doctor of Philosophy

Department of Chemistry

University of Toronto

2012

Abstract

Several nanomaterials systems, both inorganic and organic in nature, have been extensively

investigated by a number of characterization techniques including atomic force microscopy

(AFM), electron microscopy, Fourier transform infrared spectroscopy (FTIR), and energy

dispersive x-ray spectroscopy (EDX). The first system consists of boron nitride nanotubes

(BNNTs) synthesized via two different methods. The first method, silica-assisted catalytic

chemical vapour deposition (SA-CVD), produced boron nitride nanotubes with different

morphologies depending on the synthesis temperature. The second method, growth vapour

trapping chemical vapour deposition (GVT-CVD), produced multiwall boron nitride nanotubes

(MWBNNTs). The bending modulus of individual MWBNNTs was determined using an AFM

three-point bending technique, and was found to be diameter-dependent due to the presence of

shear effects. The second type of nanomaterial investigated is the biomineralized calcareous

iii

shell of the serpulid Hydroides dianthus. This material was found to be an inorganic-organic

composite material composed of two different morphologies of CaCO3, collagen, and

carboxylated and sulphated polysaccharides. The organic components were demonstrated to

mediate the mineralization of CaCO3 in vitro. The final system studied is the proteinaceous

cement of the barnacle Amphibalanus amphitrite. The secondary structure of the protein

components was investigated via FTIR, revealing the presence of β-sheet conformation, and

nanoscale rod-shaped structures within the cement were identified as β-sheet containing amyloid

fibrils via chemical staining. These rod-shaped structures exhibited a stiffer nature compared

with other structures in the adhesive, as measured by AFM nanoindentation.

iv

Acknowledgments

I've heard it said

That people come into our lives for a reason

Bringing something we must learn

And we are led

To those who help us most to grow

If we let them

And we help them in return

Well, I don't know if I believe that's true

But I know I'm who I am today

Because I knew you

- Glinda the Good Witch (Wicked, the musical)

I am indebted to my supervisor, Professor Gilbert Walker, for his support and guidance over the

years. Always perceptive, attentive, encouraging, and ready with a helping hand, I would not be

in the position I am now without him. His infectious enthusiasm for science and team-building

activities outside of the lab won’t be forgotten, and I am proud to be a member of his science

family.

I would also like to thank my supervisory committee members, Professors Al-Amin Dhirani and

Zhenghong Lu for their helpful input and advice. I am very appreciative of the time and

consideration they have given me over the course of my graduate studies.

To the members of Walker Labs, past and present – it is astonishing how well we all get along!

I cannot imagine what lab life would be like without you. To the postdocs, Doctors Shan Zou,

Nikhil Gunari, Weiqing Shi, Zahra Fakhraai, and Leela Reddy: thank you for the knowledge and

experience you shared with me, as both mentors and friends. To my fellow graduate students,

Doctors Shell Ip, James Li, Ruby Sullan, and Isaac Li; Melissa Paulite, Claudia Grozea,

Christina MacLaughlin, Dan Lamont, Alex Kumachev, Colin Zamecnik, Duncan Smith-

Halverson, and Alex Stewart: it has been a pleasure working with such curious, industrious, and

humorous people. In particular, I would like to thank Shell, James, Isaac, and Melissa for our

numerous insightful discussions which helped me to solve many problems and come up with

v

new ideas. To my co-authors Nikhil, Ruby, and Melissa: I have learned so much from working

with you – not only about science; but about perseverance, determination, and integrity.

Certain measurements have helped me to re-focus time and time again, and for this I am grateful

to the S4S team (you know who you are). I am also grateful to my best girls Aliza Kassam and

Courtney Smyth for always being there, sharing my highs and lows. Thanks as well to my

EngSci friends, who saw me through undergrad and beyond.

I also appreciate the support of my extended family, who sat patiently through a “what is nano?”

presentation when I tried to explain what I do, and who always made sure I was supplied with

leftovers from family parties. To my second family, the Joes: thank you for making me a part of

your family, and for your support.

Thank you Mom and Dad, for all of the sacrifices you have made for me and for your constant

encouragement and support. I appreciate that you both (quietly) expect great things from me

because you believe that I can achieve them. To my big brother Luke: you probably set me on

this path by passing on your love of reading and science fiction. Thank you for listening to my

presentations and putting up with my mess while we lived together, and for always looking out

for me. To my little sister Cheryl: you are the true chemist and I loved having you around in the

department. We must be more alike than we would care to admit, because everyone recognized

that we are sisters. Having you around gives me the confidence to rise to any occasion, because I

have to act my part as the big sister.

Finally, to my fiancé Chris Joe: Thank you for your patience with me over the past 10 years, for

understanding how much this means to me, and for your constant love.


August 7th

, 2012

vi

Table of Contents

Acknowledgments.......................................................................................................................... iv

Table of Contents ........................................................................................................................... vi

List of Symbols ............................................................................................................................. xii

List of Abbreviations ................................................................................................................... xiii

List of Tables ............................................................................................................................... xiv

List of Figures ................................................................................................................................xv

1 Introduction .................................................................................................................................1

1.1 Nanomaterials: Materials Revolution, Natural Evolution ...................................................1

1.2 Nanoscale Characterization Methods ..................................................................................2

1.3 Hexagonal Boron Nitride Nanomaterials .............................................................................2

1.4 Marine Fouling Organisms: Adhesive Nanomaterials .........................................................4

1.5 Summary of Thesis ..............................................................................................................5

1.6 References ............................................................................................................................5

2 Atomic Force Microscopy and Spectroscopy ...........................................................................10

2.1 Introduction ........................................................................................................................10

2.2 Contact Mode Imaging ......................................................................................................11

2.3 Intermittent Contact (Tapping) Mode Imaging..................................................................12

2.4 Force Spectroscopy ............................................................................................................13

2.5 References ..........................................................................................................................14

3 Synthesis of Boron Nitride Nanotubes ......................................................................................16

3.1 Permissions ........................................................................................................................16

vii

3.2 Abstract ..............................................................................................................................16

3.3 Introduction ........................................................................................................................16

3.3.1 Arc Discharge ........................................................................................................16

3.3.2 Laser Heating/Ablation ..........................................................................................17

3.3.3 Templated Synthesis ..............................................................................................19

3.3.4 Chemical Vapour Deposition .................................................................................21

3.3.5 Mechano-Thermal (Ball milling and Annealing) ..................................................23

3.3.6 Chemical Synthesis ................................................................................................24

3.3.7 Comparison of Methods .........................................................................................24

3.4 Experimental Methods .......................................................................................................25

3.4.1 Method 1: Silica-Assisted Catalytic Chemical Vapour Deposition .......................25

3.4.2 Method 2: Growth Vapour Trapping Chemical Vapour Deposition .....................26

3.5 Results ................................................................................................................................27

3.5.1 Macroscopic Description of Products ....................................................................27

3.5.2 Nanotube Morphology ...........................................................................................29

3.6 Discussion ..........................................................................................................................30

3.6.1 BNNT Growth Mechanisms in CVD Synthesis ....................................................30

3.6.2 Qualitative Comparison of Methods ......................................................................34

3.7 Conclusions ........................................................................................................................34

3.8 Contributions......................................................................................................................35

3.9 References ..........................................................................................................................35

4 Structural and Chemical Characterization of Boron Nitride Nanotubes ...................................39

4.1 Abstract ..............................................................................................................................39

4.2 Introduction ........................................................................................................................39


viii

4.3.1 Synthesis ................................................................................................................40

4.3.2 Electron Microscopy ..............................................................................................40

4.3.3 Energy Dispersive X-ray........................................................................................41

4.3.4 Fourier Transform Infrared Spectroscopy .............................................................41

4.4 Results ................................................................................................................................41

4.4.1 Scanning Transmission Electron Microscopy .......................................................41

4.4.2 EDX Characterization ............................................................................................43

4.4.3 FTIR Characterization ...........................................................................................46

4.5 Discussion ..........................................................................................................................47

4.5.1 Nanotube Morphology and Structure.....................................................................47

4.5.2 Defect Characterization .........................................................................................48

4.6 Conclusions ........................................................................................................................49

4.7 References ..........................................................................................................................49

5 Diameter-Dependent Bending Modulus of Individual Multiwall Boron Nitride Nanotubes ...50

5.1 Abstract ..............................................................................................................................50

5.2 Introduction ........................................................................................................................50


5.3.1 MWBNNT Synthesis .............................................................................................52

5.3.2 Chemical and Structural Characterization .............................................................52

5.3.3 Sample Preparation and AFM Measurements .......................................................53

5.4 Results and Discussion ......................................................................................................54

5.4.1 Characterization of MWBNNTs ............................................................................54

5.4.2 AFM Three-Point Bending ....................................................................................54

5.4.3 Elastic Properties of MWBNNTs ..........................................................................60

5.5 Conclusion .........................................................................................................................66

ix

5.6 Contribution .......................................................................................................................67

5.7 References ..........................................................................................................................67

6 Insights into the composition, morphology, and formation of the calcareous shell of the

serpulid Hydroides dianthus .....................................................................................................72

6.1 Permissions ........................................................................................................................72

6.2 Abstract ..............................................................................................................................72

6.3 Introduction ........................................................................................................................72


6.4.1 Tubeworm Collection and Preservation.................................................................74

6.4.2 X-Ray Diffraction (XRD) ......................................................................................75

6.4.3 Fourier Transform Infrared Spectroscopy (FTIR) .................................................75

6.4.4 Inductively Coupled Plasma Atomic Emission Spectrometry (ICPAES)

Analysis..................................................................................................................76

6.4.5 Electron Probe Microanalysis (EPMA) .................................................................76

6.4.6 Separation of the Organic Tube Lining and the Soluble Organic Matrix

(SOM) ....................................................................................................................76

6.4.7 Scanning Electron Microscopy (SEM) and Energy Dispersive X-Ray (EDX) .....76

6.4.8 Atomic Force Microscopy (AFM) Imaging and Nanoindentation ........................77

6.4.9 Light Microscopy and Chemical Staining .............................................................78

6.4.10 Amino Acid Analysis of the Soluble Organic Matrix (SOM) ...............................78

6.4.11 In vitro CaCO3 Mineralization Experiments ..........................................................79

6.5 Results ................................................................................................................................80

6.5.1 Bulk Composition of the Tube Shell and Adhesive Material ................................80

6.5.2 Tube Shell Ultrastructure and Spatial Composition ..............................................82

6.5.3 Adhesive Material Structure and Composition ......................................................84

6.5.4 Mechanical Properties of the Adhesive Material ...................................................86

x

6.5.5 Mechanical Properties of the Tube Shell ...............................................................88

6.5.6 Characterization of the Organic Tube Lining ........................................................88

6.5.7 Characterization of the SOM .................................................................................93

6.5.8 Characterization of the Remineralized Organic Tube Lining ................................96

6.5.9 Characterization of the SOM Mineralization Precipitates .....................................98

6.6 Discussion ........................................................................................................................101

6.6.1 Tube Layering and Mechanical Properties ..........................................................101

6.6.2 CaCO3 Polymorphs and Morphologies ................................................................104

6.6.3 Insights into the Formation and Attachment of the Adhesive Material to the

Substrate ...............................................................................................................105

6.6.4 SOM Composition ...............................................................................................106

6.6.5 IOM Composition ................................................................................................107

6.6.6 Summary of the Structure and Composition of the Tube Shell and Adhesive

Material ................................................................................................................109

6.6.7 Role of the Organic Tube Lining in Tube Formation ..........................................109

6.7 Conclusions ......................................................................................................................112

6.8 Contributions....................................................................................................................113

6.9 References ........................................................................................................................113

7 Nanoscale Structures and Properties of the Proteinaceous Cement of the Barnacle

Amphibalanus amphitrite ........................................................................................................119

7.1 Permissions ......................................................................................................................119

7.2 Abstract ............................................................................................................................119

7.3 Introduction ......................................................................................................................119

7.3.1 FTIR Characterization of Protein Secondary Structure .......................................120

7.3.2 Barnacle Cement: Proteinaceous Glue .................................................................121

7.4 Experimental Methods .....................................................................................................121

xi

7.4.1 Barnacle Rearing ..................................................................................................121

7.4.2 Fourier Transform Infrared (FTIR) Spectroscopy ...............................................122

7.4.3 Atomic Force Microscopy (AFM) Imaging and Indentation ...............................123

7.4.4 Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray (EDX) ....123

7.4.5 Chemical Staining ................................................................................................124

7.5 Results ..............................................................................................................................124

7.5.1 FTIR Spectra ........................................................................................................124

7.5.2 AFM Images, Force Curves, and Moduli Histograms .........................................125

7.5.3 SEM and EDX .....................................................................................................129

7.5.4 Amyloid-Selective Staining .................................................................................130

7.6 Discussion ........................................................................................................................130

7.6.1 Significance of β-sheet Conformation in Barnacle Cement ................................130

7.7 Conclusions ......................................................................................................................132

7.8 Contributions....................................................................................................................132

7.9 References ........................................................................................................................132

8 Summary and Outlook ............................................................................................................135

8.1 Summary of Thesis ..........................................................................................................135

8.2 Outlook ............................................................................................................................136

8.2.1 Boron Nitride Nanotubes .....................................................................................136

8.2.2 Nanomaterials in Nature ......................................................................................136

8.3 References ........................................................................................................................137

xii

List of Symbols

a suspended length to the left of applied force

b suspended length to the right of applied force

D diameter

EB bending modulus

EY Young’s modulus

F loading force

G shear modulus

I second moment of area

k spring constant

keff effective spring constant

L suspended length

R radius of curvature

δ deflection (Chapter 5)

δ indentation (Chapter 6, 7)

ν Poisson’s ratio

xiii

List of Abbreviations

AFM atomic force microscopy/microscope

BN boron nitride

BNNT boron nitride nanotube

c-BN cubic boron nitride

CNT carbon nanotube

CVD chemical vapour deposition

DCBM double clamped beam model

EDX energy dispersive x-ray spectroscopy

FTIR Fourier transform infrared spectroscopy

GVT-CVD growth vapour trapping chemical vapour deposition

h-BN hexagonal boron nitride

HR-TEM high resolution transmission electron microscopy/microscope

IR infrared

LO longitudinal optical

MSBM mixed support beam model

MWBNNT multiwall boron nitride nanotube

MWCNT multiwall carbon nanotube

SA-CVD silica-assisted chemical vapour deposition

SE standard error

SEM scanning electron microscopy/microscope

SSBM simply supported beam model

STEM scanning transmission electron microscopy/microscope

SWBNNT single wall boron nitride nanotube

SWCNT single wall carbon nanotube

TEM transmission electron microscopy/microscope

TO transverse optical

XRD x-ray diffraction

xiv

List of Tables

Table 3.1 Comparison of BNNT Synthesis Methods ................................................................... 25

Table 4.1 Infrared modes of h-BN and c-BN ............................................................................... 40

Table 4.2 FTIR Peak Positions for BN Nanomaterials. ............................................................... 48

Table 6.1 Chemical composition of seawater vs. the artificial seawater used in all CaCO3

precipitation experiments .............................................................................................................. 79

Table 6.2 Summary of IR bands for the tube shell sample FTIR spectrum. ................................ 81

Table 6.3 Summary of IR bands for the organic tube lining and the SOM FTIR spectra. .......... 94

Table 6.4 Amino acid composition for the SOM. ........................................................................ 95

Table 7.1 IR peaks and the corresponding fraction of the observed secondary structures found in

gummy barnacle cement sample ................................................................................................. 125

xv

List of Figures

Figure 1.1 Structures of h-BN and graphite. .................................................................................. 3

Figure 1.2 Structure of a carbon nanotube and a boron nitride nanotube. ..................................... 3

Figure 2.1 Schematic of atomic force microscope. ...................................................................... 11

Figure 3.1 Position of alumina boat within quartz test tube. ....................................................... 27

Figure 3.2 GVT-CVD set-up. ...................................................................................................... 27

Figure 3.3 Quartz boat after Method 1, SA-CVD (1150 oC, 1 h). ............................................... 28

Figure 3.4 Alumina boat after Method 2, GVT-CVD (1150 oC, 2 h). ......................................... 28

Figure 3.5 Electron micrographs of the products of SA-CVD.. .................................................. 29

Figure 3.6 SEM image of MWBNNTs synthesized by GVT-CVD ............................................ 30

Figure 3.7 Schematic of BNNT growth. ...................................................................................... 32

Figure 4.1 Scanning transmission electron microscopy images of BNNTs produced by SA-CVD

....................................................................................................................................................... 42

Figure 4.2 Transmission electron microscopy images of BNNTs produced by GVT-CVD. ...... 42

Figure 4.3 EDX spectrum of SA-CVD BNNTs on Si substrate prior to HF purification ........... 43

Figure 4.4 EDX spectrum of SA-CVD Bamboo BNNTs (after HF purification) on Si substrate.

....................................................................................................................................................... 44

Figure 4.5 SEM image of GVT-CVD BNNTs on lacy C TEM grid and corresponding EDX

elemental maps.............................................................................................................................. 45

Figure 4.6 EDX spectrum of GVT-CVD BNNTs on lacy C TEM grid. ..................................... 46

xvi

Figure 4.7 Fourier transform infrared spectra of BNNTs produced by SA-CVD and GVT-CVD.

....................................................................................................................................................... 47

Figure 5.1 SEM and TEM images of MWBNNTs ...................................................................... 55

Figure 5.2 FTIR spectrum of MWBNNTs. .................................................................................. 56

Figure 5.3 Beam schematics describing beam bending boundary conditions. ............................ 57

Figure 5.4 SEM and AFM images and maps of MWBNNTs on patterned Si substrate ............. 59

Figure 5.5 Representative AFM force curves. ............................................................................. 59

Figure 5.6 Tube effective stiffness (keff) vs. position along suspended tube (a/L) ....................... 60

Figure 5.7 Bending modulus vs. tube outer diameter .................................................................. 62

Figure 5.8 Determination of the Young’s modulus and shear modulus via a fit to plot of 1/EB vs

(D/L)2 ............................................................................................................................................ 65

Figure 6.1 Overview of serpulid tube structure (Hydroides dianthus) ........................................ 73

Figure 6.2 XRD and FTIR spectra for powdered sample of entire tube ...................................... 80

Figure 6.3 SEM images of tube shell transverse cross section .................................................... 84

Figure 6.4 SEM images of adhesive material, transverse cross section. ..................................... 85

Figure 6.5 SEM images of the variety of crystal morphologies observed for Mg-calcite and

aragonite. ....................................................................................................................................... 86

Figure 6.6 SEM images of the adhesive material surface, substrate side, showing the presence

and incorporation of various biofilm components ........................................................................ 87

Figure 6.7 AFM height images and elastic moduli histograms of adhesive material surface ..... 88

xvii

Figure 6.8 SEM images illustrating the different crystal morphologies and orientations present at

the adhesive material surface, adjacent to the substrate ............................................................... 89

Figure 6.9 Optical images of organic tube lining......................................................................... 89

Figure 6.10 SEM images of the adhesive material (lumen-side) and the EDTA-treated tube

lining ............................................................................................................................................. 90

Figure 6.11 EDX spectrum for the “smooth layer” (possibly an organic sheet). ........................ 91

Figure 6.12 Optical image of the insoluble organic matrix after Masson’s trichrome staining .. 91

Figure 6.13 AFM amplitude images and linescans of fibres from the insoluble organic matrix.

....................................................................................................................................................... 92

Figure 6.14 FTIR spectra of the tube shell organic matrices ....................................................... 93

Figure 6.15 SEM images of the IOM, treated overnight with 0.5 M EDTA ............................... 96

Figure 6.16 SEM images and EDX spectra of crystals formed on the EDTA-demineralized

organic tube lining sample, after 24 hours .................................................................................... 97

Figure 6.17 FTIR spectrum of the particles formed in the SOM in vitro crystallization

experiment, for the control sample ............................................................................................... 98

Figure 6.18 A) FTIR spectrum of the particles formed in the SOM in vitro crystallization

experiment, for the 48 hour SOM sample ..................................................................................... 99

Figure 6.19 SEM images and EDX spectra of the crystal products of the in vitro SOM

crystallization experiments ......................................................................................................... 100

Figure 6.20 Force profile obtained from nanoindentation on the adhesive material surface,

performed in artificial seawater .................................................................................................. 102

Figure 6.21 Summary of tube structure and composition .......................................................... 110

xviii

Figure 7.1 Typical FTIR spectrum of barnacle glue on CaF2 substrate. .................................... 120

Figure 7.2 FTIR spectra of the bulk cement from A. amphitrite. .............................................. 125

Figure 7.3 AFM topographic images of the barnacle cement. ................................................... 126

Figure 7.4 Nanoscale morphology of the bulk barnacle cement................................................ 127

Figure 7.5 Elastic modulus distribution of individual nanostructures/components observed by

AFM ............................................................................................................................................ 128

Figure 7.6 SEM images and EDX spectra of the barnacle cement resettled on aluminum foil . 129

Figure 7.7 Chemical staining images of the barnacle cement with amyloid-selective dyes ...... 130

1

1 Introduction

1.1 Nanomaterials: Materials Revolution, Natural Evolution

“Nano” has become a familiar and often used buzzword in the arenas of science and engineering.

Typically, a nanomaterial possesses features on a 1 – 100 nm length scale which may result in

properties that differ from the bulk or macroscopic form of the material. Properties of

nanomaterials are often tunable based on the size of the relevant feature. A popular example of

this is quantum dots, which are semiconducting nanoparticles small enough that quantum

confinement effects dominate the electronic properties of the particles. Whereas the band gap of

bulk semiconductors is determined by the material’s crystal structure and chemical composition,

the bandgap of quantum dots is related to their diameter. As a consequence, nanoparticles with

the same composition but different sizes can emit different wavelengths of light, with smaller

particles emitting shorter wavelengths, and larger particles emitting longer wavelengths.1

Nanomaterials such as quantum dots, fullerenes, and carbon nanotubes are relatively new

discoveries (circa mid 1980 to early 1990’s),2-4

and their unique structures and properties have

inspired a materials revolution in which researchers are designing and exploiting materials in a

fundamentally different way. On the other hand, there are many examples of remarkable

nanomaterials in the natural world that have been around for more than a billion years.

Organisms have evolved a variety of functional nanomaterials, examples which include the

superhydrophic self-cleaning surfaces of lotus leaves, exceptionally strong spider silk, and

adhesive spatulae structures on gecko toes.5-7

Discovery and study of such natural nanomaterials

has led to the design of biomimetic materials. For instance, gecko-inspired dry adhesives have

been fabricated out of polymer nanopillars and carbon nanotubes.8-10

Nanoscale systems must be studied with a different set of considerations than macroscale

systems. The properties of the surface become increasingly dominant as the surface-to-volume

ratio increases with decreasing particle size. In solids, surface atoms exist in a different

environment than bulk atoms. The symmetry of the bulk crystal is broken, and surface atoms

can have dangling bonds, functional groups, and interactions with adsorbed species. As a result,

when a significant proportion of the atoms in a particle are surface atoms, the properties of the

2

particle will deviate from bulk material properties. Some typical changes include a decrease in

melting temperature and an increase in reactivity.11

1.2 Nanoscale Characterization Methods

The development of instrumentation capable of characterizing the structure and properties of

materials at the nanoscale is responsible for the explosion of nanotechnology research over the

past few decades. Atomic force microscopy (AFM) is an important and versatile tool for the

study of surfaces at the nanoscale, in ideal cases demonstrating a lateral resolution of < 1 nm,

and a vertical resolution of < 1 Å.12

AFM force spectroscopy and nanoindentation techniques

can measure the mechanical properties of materials at the nanoscale. A particular advantage of

AFM characterization is that it is largely non-destructive, and little to no sample preparation is

required. In addition, it is capable of operating in a variety of conditions, such as in vacuum,

ambient conditions in air, and in liquids.

Electron microscopy, including scanning electron microscopy (SEM) and transmission electron

microscopy (TEM) remain standard tools for the characterization of nanomaterials, especially

when combined with integrated techniques such as energy dispersive x-ray spectroscopy, and

electron diffraction.

1.3 Hexagonal Boron Nitride Nanomaterials

The two main forms of boron nitride are cubic boron nitride (c-BN) and hexagonal boron nitride

(h-BN). These allotropes have the same basic structure as the well-known carbon allotropes

diamond (cubic form) and graphite (hexagonal form). As a result, the mechanical properties of

C and BN materials are similar – c-BN is super hard, almost as hard as diamond, and h-BN is

soft and lubricious, like graphite.13

The crystal structure of h-BN is shown in Figure 1.1 below,

along with the crystal structure of graphite as a comparison. It can be seen that the basal plane

stacking differs between h-BN and graphite, with B atoms in one layer stacking on top of N

atoms in the layer below. Stacking differences aside, both materials are anisotropic with strong

covalent bonding within an atomic plane, and weak van der Waals bonding between planes. A

major difference between h-BN and graphite is their electrical and optical properties. Whereas

3

graphite is a semimetal, h-BN is an electrical insulator and is white in colour. This is a result of

the localization of π-electrons on the nitrogen atoms, due to their higher electronegativity.

Figure 1.1 Structures of h-BN and graphite. From Souche et al.14

Boron nitride materials do not occur naturally, unlike their carbon counterparts. Hexagonal

boron nitride was first synthesized in 1842 by Balmain.15

It is a non-oxide ceramic, and

possesses a variety of useful material properties including high lubricity, good thermal

conductivity, high oxidation resistance and chemical stability, and non-wetting by molten glass

and metals. As a result, h-BN in micro-powder form is an important industrial material and is

used as a solid lubricant, as an additive in cosmetics, and in mould release applications.13, 16, 17

Figure 1.2 Structure of a carbon nanotube and a boron nitride nanotube. From Golberg et al.18

Following the discovery of carbon nanotubes (CNTs),4 boron nitride nanotubes (BNNTs) were

predicted in 1994 by Blase and Rubio, and synthesized in 1995 by Chopra et al.19-21

BNNTs can

be synthesized in various forms, such as single-wall nanotubes,22-24

double-wall nanotubes,25

multiwall nanotubes,26, 27

and bamboo morphology nanotubes.28, 29

Typically, these BNNTs have

diameters ranging from 1 – 200 nm, and lengths greater than 10 μm; recently, synthesis of

4

BNNTs with lengths greater than 1 mm has been reported.30

Like its parent material, h-BN,

BNNTs exhibit excellent oxidation resistance and thermal conductivity.31, 32

The electronic

properties of BNNTs are largely independent of chirality and diameter,19

which makes them

ready-to-use for many applications since the as-synthesized material does not have to be sorted

as is the case for carbon nanotubes. Taking advantage of their stable wide band gap, polymer

encapsulants loaded with BNNTs have been developed for the electrical insulation of

microelectronics components and optoelectronic devices.33, 34

BNNTs also show promise as

deep-UV emitters.35

Multiwall boron nitride nanotubes (MWBNNTs) have been shown to possess exceptional elastic

properties, similar to those of carbon nanotubes. The Young’s modulus of MWBNNTs has been

experimentally determined to range from several hundred GPa to over 1 TPa.36-39

With their

one-dimensional structure and high Young’s modulus, MWBNNTs are ideal reinforcement

components for plastic, glass, and ceramic composite materials.40-44

1.4 Marine Fouling Organisms: Adhesive Nanomaterials

Biofouling is a major problem for seafaring vessels, particularly those in the shipping industry.

A wide variety of marine organisms including barnacles, oysters, tubeworms, and algae will

attach to and grow on ship hulls. Accumulation of these organisms occurs while the ships are

stationary (in port) and the increased drag of the fouling layer results in significant extra fuel

consumption or slower speeds by the ships once they are underway.45

In addition, biofouling

leads to the introduction of foreign and potentially invasive species to non-native ecosystems, as

the fouling organisms are transported from port to distant port. Toxic tributyl tin-based biocidal

paints which prevent organism settlement are no longer viable anti-fouling solutions due to

environmental concerns.46

Researchers are attempting to address the biofouling problem by

developing novel non-toxic coatings that can be applied to ship hulls to repel the settlement of

fouling organisms outright, or enable the release of the organisms under the hydrodynamic forces

generated when vessels get up to cruising speed. In order to design these novel coatings, it is

necessary to have an understanding of the adhesion mechanisms that fouling organisms employ

to firmly attach themselves to surfaces.

5

Bivalves and barnacles use proteinaceous adhesives, and in some species the proteins responsible

for attachment strength have been identified.47

Oysters and serpulid tubeworms, on the other

hand, employ a cement-like layer comprised largely of calcium carbonate.48

It is becoming

apparent that the mechanical properties of these adhesive materials are often a result of

nanostructured components, by natural design. The study of these adhesive materials at the

nanoscale is therefore an active area of research, which may lead to new solutions to the

biofouling problem.

1.5 Summary of Thesis

Chapter 2 details the imaging and nanomechanical characterization capabilities of the atomic

force microscope, and the model used for analysis of indentation measurements. Chapter 3

reviews synthesis methods for boron nitride nanotubes. Two chemical vapour deposition based

methods which were utilized during the course of this thesis are described in detail. The

experimental methods are presented, together with the morphology of the synthesized nanotubes

as determined via electron microscopy. Possible growth mechanisms for the formation of boron

nitride nanotubes are discussed. In Chapter 4, the characterization of the resulting products via

FTIR, SEM, TEM, EDX is presented. In Chapter 5, the mechanical properties of individual

boron nitride nanotubes are investigated via AFM bending experiments. Euler and Timoshenko

beam models are used to calculate the bending modulus of individual tubes. A comprehensive

study of the calcareous shell of the tubeworm Hydroides dianthus in a biomineralization context

is presented in Chapter 6. Lastly, in Chapter 7, the nanoscale structures in barnacle cement are

investigated via FTIR, EDX, SEM, and AFM.

1.6 References

1. Alivisatos, A. P., Semiconductor clusters, nanocrystals, and quantum dots. Science 1996,

271, 933–937 .

2. Brus, L., Quantum crystallites and nonlinear optics. Applied Physics A: Materials Science

& Processing 1991, 53, 465–474.

3. Kroto, H. W.; Heath, J. R.; O'Brien, S. C.; Curl, R. F.; Smalley, R. E., C60:

Buckminsterfullerene. Nature 1985, 318, 162–163.

4. Iijima, S., Helical microtubules of graphitic carbon. Nature 1991, 354, 56–58.

6

5. Barthlott, W.; Neinhuis, C., Purity of the sacred lotus, or escape from contamination in

biological surfaces. Planta 1997, 202, 1–8.

6. Giesa, T.; Arslan, M.; Pugno, N. M.; Buehler, M. J., Nanoconfinement of spider silk

fibrils begets superior strength, extensibility, and toughness. Nano Lett. 2011, 11, 5038–

5046.

7. Autumn, K.; Liang, Y. A.; Hsieh, S. T.; Zesch, W.; Chan, W. P.; Kenny, T. W.; Fearing,

R.; Full, R. J., Adhesive force of a single gecko foot-hair. Nature 2000, 405, 681–685.

8. Lee, H.; Lee, B. P.; Messersmith, P. B., A reversible wet/dry adhesive inspired by

mussels and geckos. Nature 2007, 448, 338–341.

9. Yurdumakan, B.; Raravikar, N. R.; Ajayan, P. M.; Dhinojwala, A., Synthetic gecko foot-

hairs from multiwalled carbon nanotubes. Chem. Commun. 2005, 3799–3801.

10. Sethi, S.; Ge, L.; Ci, L.; Ajayan, P. M.; Dhinojwala, A., Gecko-inspired carbon nanotube-

based self-cleaning adhesives. Nano. Lett. 2008, 8, 822–825.

11. Shu, Q.; Yang, Y.; Zhai, Y.; Sun, D.; Xiang, H.; Gong, X.-g., Size-dependent melting

behavior of iron nanoparticles by replica exchange molecular dynamics. Nanoscale 2012.

12. Bhushan, B.; Marti, O., Atomic Force Microscope. In Springer Handbook of

Nanotechnology, Bhushan, B., Ed. Springer - Verlag: Berlin, 2004; pp 331–346.

13. Haubner, R.; Wilhelm, M.; Weissenbacher, R.; Lux, B., Boron Nitrides - Properties,

Synthesis and Applications. In High Performance Non-Oxide Ceramics II, Jansen, M.,

Ed. Springer Verlag: Berlin, Heidelberg, 2002; pp 1–45.

14. Souche, C.; Jouffrey, B.; Hug, G.; Nelhiebel, M., Orientation sensitive EELS-analysis of

boron nitride nanometric hollow spheres. Micron 1998, 29, 419–424.

15. Balmain, W. H., Bemerkungen uber die bildung von verbindungen des bors und siliciums

mit stickstoff und gewissen metallen. J. Prakt. Chem. 1842, 27, 422–430.

16. Greim, J.; Schwetz, K. A., Boron Carbide, Boron Nitride, and Metal Borides. In

Ullmann's Encyclopedia of Industrial Chemistry, Wiley-VCH Verlag GmbH & Co.

KGaA: 2000; pp 219–234.

17. Lipp, A.; Schwetz, K. A.; Hunold, K., Hexagonal boron nitride: Fabrication, properties

and applications. J. Eur. Ceram. Soc. 1989, 5, 3–9.

18. Golberg, D.; Bando, Y.; Tang, C. C.; Zhi, C. Y., Boron nitride nanotubes. Adv. Mater.

2007, 19, 2413–2432.

19. Blase, X.; Rubio, A.; Louie, S. G.; Cohen, M. L., Stability and band gap constancy of

boron nitride nanotubes. Europhys. Lett. 1994, 28, 335–340.

7

20. Rubio, A.; Corkill, J. L.; Cohen, M. L., Theory of graphitic boron-nitride nanotubes.

Phys. Rev. B 1994, 49, 5081–5084.

21. Chopra, N. G.; Luyken, R. J.; Cherrey, K.; Crespi, V. H.; Cohen, M. L.; Louie, S. G.;

Zettl, A., Boron-nitride nanotubes. Science 1995, 269, 966–967.

22. Lee, R. S.; Gavillet, J.; Chapelle, M. L. d. l.; Loiseau, A.; Cochon, J. L.; Pigache, D.;

Thibault, J.; Willaime, F., Catalyst-free synthesis of boron nitride single-wall nanotubes

with a preferred zig-zag configuration. Phys. Rev. B 2001, 64, 121405.

23. Smith, M. W.; Jordan, K. C.; Park, C.; Kim, J.-W.; Lillehei, P. T.; Crooks, R.; Harrison,

J. S., Very long single- and few-walled boron nitride nanotubes via the pressurized

vapor/condenser method. Nanotechnology 2009, 20, 505604.

24. Loiseau, A.; Willaime, F.; Demoncy, N.; Hug, G.; Pascard, H., Boron nitride nanotubes

with reduced numbers of layers synthesized by arc discharge. Phys. Rev. Lett. 1996, 76,

4737–4740.

25. Cumings, J.; Zettl, A., Mass-production of boron nitride double-wall nanotubes and

nanococoons. Chem. Phys. Lett. 2000, 316, 211–216.

26. Zhi, C.; Bando, Y.; Tan, C.; Golberg, D., Effective precursor for high yield synthesis of

pure BN nanotubes. Solid State Commun. 2005, 135, 67–70.

27. Lee, C. H.; Wang, J. S.; Kayatsha, V. K.; Huang, J. Y.; Yap, Y. K., Effective growth of

boron nitride nanotubes by thermal chemical vapor deposition. Nanotechnology 2008, 19,

455605–5.

28. Ma, R.; Bando, Y.; Sato, T., Bamboo‐like boron nitride nanotubes. J. Electron Microsc.

2002, 51, S259–S263.

29. Zhang, L.; Wang, J.; Gu, Y.; Zhao, G.; Qian, Q.; Li, J.; Pan, X.; Zhang, Z., Catalytic

growth of bamboo-like boron nitride nanotubes using self-propagation high temperature

synthesized porous precursor. Mater. Lett. 2012, 67, 17–20.

30. Chen, H.; Chen, Y.; Liu, Y.; Fu, L.; Huang, C.; Llewellyn, D., Over 1.0 mm-long boron

nitride nanotubes. Chem. Phys. Lett. 2008, 463, 130–133.

31. Chen, Y.; Zou, J.; Campbell, S. J.; Caer, G. L., Boron nitride nanotubes: Pronounced

resistance to oxidation. Appl. Phys. Lett. 2004, 84, 2430–2432.

32. Bando, Y.; Golberg, D.; Tang, C.; Zhang, J.; Ding, X.; Fan, S.; Liu, C., Thermal

conductivity of nanostructured boron nitride materials. J. Phys. Chem. B 2006, 110,

10354-10357.

33. Zhi, C.; Bando, Y.; Terao, T.; Tang, C.; Kuwahara, H.; Golberg, D., Towards

Thermoconductive, Electrically Insulating Polymeric Composites with Boron Nitride

Nanotubes as Fillers. Adv. Funct. Mater. 2009, 19, 1857–1862.

8

34. Ravichandran, J.; Manoj, A. G.; Liu, J.; Manna, I.; Carroll, D. L., A novel polymer

nanotube composite for photovoltaic packaging applications. Nanotechnology 2008, 19,

085712–5.

35. Li, L. H.; Chen, Y.; Lin, M. Y.; Glushenkov, A. M.; Cheng, B. M.; Yu, J., Single deep

ultraviolet light emission from boron nitride nanotube film. Appl. Phys. Lett. 2010, 97,

141104–3.

36. Suryavanshi, A. P.; Yu, M. F.; Wen, J. G.; Tang, C. C.; Bando, Y., Elastic modulus and

resonance behavior of boron nitride nanotubes. Appl. Phys. Lett. 2004, 84, 2527–2529.

37. Golberg, D.; Costa, P. M. F. J.; Lourie, O.; Mitome, M.; Bai, X.; Kurashima, K.; Zhi, C.;

Tang, C.; Bando, Y., Direct force measurements and kinking under elastic deformation of

individual multiwalled boron nitride nanotubes. Nano Lett. 2007, 7, 2146–2151.

38. Chopra, N. G.; Zettl, A., Measurement of the elastic modulus of a multi-wall boron

nitride nanotube. Solid State Commun. 1998, 105, 297–300.

39. Ghassemi, H. M.; Lee, C. H.; Yap, Y. K.; Yassar, R. S., Real-time fracture detection of

individual boron nitride nanotubes in severe cyclic deformation processes. J. Appl. Phys.

2010, 108, 024314–4.

40. Zhi, C. Y.; Bando, Y.; Wang, W. L. L.; Tang, C. C. C.; Kuwahara, H.; Golberg, D.,

mechanical and thermal properties of polymethyl methacrylate-BN nanotube composites.

J Nanomater. 2008, 642036–5.

41. Lahiri, D.; Rouzaud, F.; Richard, T.; Keshri, A. K.; Bakshi, S. R.; Kos, L.; Agarwal, A.,

Boron nitride nanotube reinforced polylactide-polycaprolactone copolymer composite:

Mechanical properties and cytocompatibility with osteoblasts and macrophages in vitro.

Acta Biomater. 2010, 6, 3524–3533.

42. Bando, Y.; Golberg, D.; Tang, C.; Terao, T.; Zhi, C. Y., Dielectric and thermal properties

of epoxy/boron nitride nanotube composites. Pure Appl. Chem. 2010, 82, 2175.

43. Choi, S. R.; Bansal, N. P.; Garg, A., Mechanical and microstructural characterization of

boron nitride nanotubes-reinforced SOFC seal glass composite. Materials Mater. Sci.

Eng., A 2007, 460–461, 509–515.

44. Qing, H.; Yoshio, B.; Xin, X.; Toshiyuki, N.; Chunyi, Z.; Chengchun, T.; Fangfang, X.;

Lian, G.; Dmitri, G., Enhancing superplasticity of engineering ceramics by introducing

BN nanotubes. Nanotechnology 2007, 18, 485706.

45. Schultz, M. P., Effects of coating roughness and biofouling on ship resistance and

powering. Biofouling 2007, 23, 331–341.

46. Champ, M. A., Economic and environmental impacts on ports and harbors from the

convention to ban harmful marine anti-fouling systems. Mar. Pollut. Bull. 2003, 46, 935–

940.

9

47. Kamino, K., Underwater adhesive of marine organisms as the vital link between

biological science and material science. Mar. Biotechnol. 2008, 10, 111–121.

48. Yamaguchi, K., Shell structure and behaviour related to cementation in oysters. Mar.

Biol. 1994, 118, 89–100.

Figure 2. sdf

Table 2. sdf

Table 3. srdfg

10

2 Atomic Force Microscopy and Spectroscopy

2.1 Introduction

The scanning tunneling microscope (STM) is credited with being the instrument that brought

about the nano-revolution because it was the first instrument capable of imaging molecules and

atoms. It was invented by Binnig and Rohrer, with the first results published in 1982.1-3

The

impact and potential of the instrument on diverse fields of science earned the inventors the Nobel

Prize just a few years later, in 1986. STM is a scanned probe technique in which a sharp metallic

probe is brought into close proximity with a conductive sample surface, and a bias is applied

between the probe and the sample. Electron tunneling occurs between the tip and the sample,

producing a small tunneling current. The topography of the sample can be traced by using the

tunneling current as a feedback parameter. As the tip raster scans across the surface (moving in

the x and y directions), the height of the tip can be raised or lowered (in the z direction) such that

the tunneling current is kept constant, thus forming a topographic image of the sample surface.

The minute translations of the tip in the x, y, and z directions are achieved with piezoelectric

transducers. Following the invention of the STM, Binnig, together with Quate and Gerber, went

on to develop another scanned probe instrument with an applicability that was not restricted to

conductive samples, the atomic force microscope (AFM).4

In AFM, a variety of forces can be probed, including van der Waals, electrostatic, interatomic,

capillary, adhesion, and mechanical contact forces. Forces (as small as 10-18

N) experienced by a

sharp probe mounted on a cantilever cause the cantilever to deflect (by as little as 10-4

Å). This

deflection is used as the feedback parameter, in contrast to the tunneling current in STM. In the

first prototype of the instrument, an STM was used to monitor the minute deflections of the

cantilever.4 The first atomic resolution image obtained was of boron nitride, an insulator.

5

Subsequently, in 1988, Meyer and Amer developed an optical method for detecting the

deflection of the cantilever. In this method, a low powered laser is reflected from the back of the

cantilever and onto a position sensitive detector.6 Commercial AFMs employ this set-up, which

is shown schematically in Figure 2.1. A photodiode with two or four segments is used as the

position sensitive detector, and voltage differences generated by the position of the laser spot on

the various diode segments are measured to monitor the cantilever deflection.

11

Figure 2.1 Schematic of atomic force microscope.7

The AFM is a very versatile instrument, capable of imaging in vacuum, ambient conditions in

air, or in liquid environments. In this chapter, the imaging modes of the AFM will be described.

The AFM force spectroscopy technique and its application to the nanomechanical

characterization of materials will also be presented.

2.2 Contact Mode Imaging

Contact mode imaging involves the raster scanning of the AFM tip while the tip is in contact

with the sample surface. The attractive and repulsive forces experienced by the tip cause the

cantilever beam to deflect, and this deflection is measured via the optical method described

above. The deflection is used as a feedback parameter in order to control the height of the tip (z

direction) such that a constant force is maintained between the tip and sample. In this manner,

the topography of the surface can be traced. The force is calculated from Hooke’s Law, F = k

∙Δx, in which F is the force, k is the spring constant of the cantilever, and Δx is the deflection of

the cantilever.

12

Low spring constant cantilevers are often used in contact mode imaging in order to increase the

sensitivity, maximizing the deflection of the cantilever. In addition to mapping surface

topography, the lateral force experienced by the cantilever can be used to map frictional forces

when a scan direction perpendicular to the cantilever axis is used.

When imaging samples in air, there is a thin water layer coating the sample surface. This layer

can influence contact mode imaging due to capillary forces. One strategy to minimize this effect

is to image the sample in water (or other liquid). However, depending on the sample of interest,

this is not always appropriate. Another problem encountered with contact mode imaging is tip

contamination and sample damage, which commonly occur when imaging soft samples such as

polymers, Langmuir Blodgett films, and lipid bilayers.

2.3 Intermittent Contact (Tapping) Mode Imaging

In order to address the issue of sample damage in contact mode imaging, an intermittent contact

mode (TappingMode, a trademark of Digital Instruments) was developed in which a high spring-

constant cantilever is driven to oscillate near its resonance frequency (~300 kHz), with an

oscillation amplitude of 20 – 100 nm.8 The tip probes the surface with each oscillation and, as a

result, the contact and lateral forces between the tip and sample is minimized. Interaction

between the tip and sample causes changes to the oscillation amplitude, phase, and frequency of

the cantilever. In tapping mode, the height of the tip is adjusted via feedback such that the

oscillation amplitude remains constant, in order to obtain a topographic image of the surface.

The phase lag between the oscillation driving the cantilever oscillation and the actual cantilever

oscillation can also be monitored to generate a phase image. Phase changes occur as a result of

energy dissipation, and are material dependent. This allows for contrast between materials with

different adhesion, elastic, and viscoelastic properties. Phase imaging is particularly useful when

there is little height variation between two distinct sample materials, as in the case of lipid

bilayers and block copolymer films, for example. In contrast to the cantilevers used in contact

mode imaging, stiffer cantilevers (~40 N/m) are used for tapping mode.9 In Chapter 5,

topographic images of boron nitride nanotubes on a patterned Si substrate are acquired using

tapping mode.

13

2.4 Force Spectroscopy

Force curves, which plot the force versus the distance between the tip and sample, can be

obtained from a given location on a sample. The AFM can be used to stretch single molecules,

such as polymer chains or proteins, between the sample and the tip. In this case, the force is

plotted against the tip-sample separation distance as the tip is withdrawn from the surface.

Phenomena such as single chain polymer elongation and hydrophobic hydration, and protein

domain unfolding can be studied with this technique.10-13

The AFM tip can also be used to apply a load to a point on the sample, in which the tip is

brought down to the sample surface and pressed a small distance into it. The force is plotted

against the tip-sample separation distance as the tip is brought down to, and into, the surface.

This nanoindentation technique can be used to determine the Young’s modulus of a sample.14, 15

Force curves are analyzed with the Sneddon-Hertz model, which describes loading vs.

indentation depth when a paraboloidal object (the AFM tip) contacts an elastic planar film of

infinite thickness (the sample). Non-adhesive contact between the two materials is assumed. In

the expression given below in Equation 1.1, F is the loading force [N], E is Young’s modulus

[Pa], R is the radius of curvature of the tip [m], δ is the indentation [m], and ν is the Poisson’s

ratio.16, 17

Equation 1.1

2/3

2 )1(3

4

v

REF

AFM nanoindentation is used in Chapters 6 and 7 to study the mechanical properties of

tubeworm and barnacle adhesive materials.15, 18

A force indentation curve is collected from a single point on the sample. In order to correlate

mechanical properties with topographic features, a topographic image may be divided into

pixels, and a force curve can be collected from each pixel. This process is known as force

mapping. In Chapter 5, force mapping is employed in order to correlate the location of the

applied force on a boron nitride nanotube suspended across a trench. This allows for the

boundary conditions of the nanotube beam to be determined, allowing for a more accurate

determination of the bending modulus using beam mechanics analysis.

14

2.5 References

1. Binnig, G.; Rohrer, H.; Gerber, C.; Weibel, E., Tunneling through a controllable vacuum

gap. Appl. Phys. Lett. 1982, 40, 178–180.

2. Binnig, G.; Rohrer, H.; Gerber, C.; Weibel, E., Surface studies by scanning tunneling

microscopy. Phys. Rev. Lett. 1982, 49, 57–61.

3. Binnig, G.; Rohrer, H., Scanning tunneling microscopy. Surf. Sci. 1983, 126, 236–244.

4. Binnig, G.; Quate, C. F.; Gerber, C., Atomic force microscope. Phys. Rev. Lett. 1986, 56,

930–933.

5. Albrecht, T. R.; Quate, C. F., Atomic resolution imaging of a nonconductor by atomic

force microscopy. J. Appl. Phys. 1987, 62, 2599–2602.

6. Meyer, G.; Amer, N. M., Novel optical approach to atomic force microscopy. Appl. Phys.

Lett. 1988, 53, 1045–1047.

7. OverlordQ Atomic force microscope block diagram.

http://en.wikipedia.org/wiki/File:Atomic_force_microscope_block_diagram.svg

(accessed August 8th, 2012).

8. Zhong, Q.; Inniss, D.; Kjoller, K.; Elings, V. B., Fractured polymer/silica fiber surface

studied by tapping mode atomic force microscopy. Surf. Sci. Lett.1993, 290, L688–L692.

9. arc a, R.; Pérez, R., Dynamic atomic force microscopy methods. Surf. Sci. Rep. 2002,

47, 197–301.

10. Bemis, J. E.; Akhremitchev, B. B.; Walker, G. C., Single polymer chain elongation by

atomic force microscopy. Langmuir 1999, 15, 2799–2805.

11. Li, I. T. S.; Walker, G. C., Effect Of temperature on the mechanical properties of

fibronectin. Biophys. J. 2009, 96, 641a.

12. Meadows, P. Y.; Bemis, J. E.; Walker, G. C., Single-molecule force spectroscopy of

isolated and aggregated fibronectin proteins on negatively charged surfaces in aqueous

liquids. Langmuir 2003, 19, 9566–9572.

13. Shi, W.; Walker, G., Mechanical desorption of single fibronectin type III module from

hydrophilic and hydrophobic surfaces. Biophys. J. 2011, 100, 480a.

14. Sun, Y.; Guo, S.; Walker, G. C.; Kavanagh, C. J.; Swain, G. W., Surface elastic modulus

of barnacle adhesive and release characteristics from silicone surfaces. Biofouling 2004,

20, 279–289.

15. Sullan, R. M. A.; Gunari, N.; Tanur, A. E.; Chan, Y.; Dickinson, G. H.; Orihuela, B.;

Rittschof, D.; Walker, G. C., Nanoscale structures and mechanics of barnacle cement.

Biofouling 2009, 25, 263–275.

16. Hertz, H., Über die berührung fester elastischer körper (On the contact of elastic solids).

J. Reine Angew. Mathematik. 1881, 92, 156–171.

17. Sneddon, I. N., The relation between load and penetration in the axisymmetric boussinesq

problem for a punch of arbitrary profile. Int. J. Eng. Sci.1965, 3, 47–57.

15

18. Tanur, A. E.; Gunari, N.; Sullan, R. M. A.; Kavanagh, C. J.; Walker, G. C., Insights into

the composition, morphology, and formation of the calcareous shell of the serpulid

Hydroides dianthus. J. Struct. Biol. 2010, 169, 145–160.

Figure 3. sadf

Equation 2 rtr

Equation 3 sdf

16

3 Synthesis of Boron Nitride Nanotubes

3.1 Permissions

The experimental material in this chapter is presented with permission from Reddy, A. L. M.;

Tanur, A. E.; Walker, G. C. Synthesis and hydrogen storage properties of different types of

boron nitride nanostructures. Int. J. Hydrogen Energy 2010, 35, 4138-4143.

3.2 Abstract

The major synthesis techniques for boron nitride nanotubes are reviewed in this chapter. Two

methods utilized over the course of this thesis are presented in detail for the synthesis of

multiwall boron nitride nanotubes (MWBNNTs), based on chemical vapour deposition (CVD).

The first method involves the mechanochemical activation of precursor powders prior to

annealing at temperatures of 1050 – 1200 oC in an NH3 atmosphere. The second method

involves an apparatus to trap the growth vapours of the precursor powders during annealing at

1200 oC in an NH3 atmosphere. Possible growth mechanisms of BNNTs are discussed.

3.3 Introduction

Despite the interest in the unique properties of boron nitride nanotubes and their similarity to

carbon nanotubes, there is much less literature published on BNNTs. A large part of this is due

to the difficulty in synthesizing high-quality BNNTs in sufficiently large quantities. As a

refractory ceramic, the commercial production of h-BN requires extremely high temperatures,

from 1000 – 5500 oC, depending on the method used.

1 Given this, it is no wonder that a major

focus of research is to achieve synthesis of BNNTs at reasonably low temperatures (i.e. < 1500

oC). The main categories of BNNT synthesis methods are summarized below.

3.3.1 Arc Discharge

Arc discharge was the first reported method for the synthesis of carbon nanotubes (CNTs).2 In

this method, two conducting electrodes are placed close together, with a gap of 1-2 mm

separating them. A DC current is applied, resulting in a potential difference between the

electrodes. At a certain threshold current, electrical arcing occurs between the two electrodes,

producing a plasma discharge. Material is vaporized from the end of the anode. The electrode

17

material is chosen based on the desired synthesis product. For carbon nanotubes, graphite

electrodes are used. After arcing, product can be found deposited as soot on the cathode, as well

as on the walls of the arc discharge chamber (different product species may be found in each

location). The quality and quantity of product synthesized is dependent on a number of

parameters, including the type and pressure (sub-atmospheric) of inert gas in the discharge

chamber, the current and voltage, the plasma temperature, the composition of the electrodes, and

the geometry of the apparatus.3

Due to its electrically insulating nature, pure h-BN cannot be used on its own as the electrodes in

arc discharge synthesis. Boron nitride nanotubes were first synthesized by Chopra et al.4 in 1995

by an arc discharge method. Currents from 50 – 140 A were applied to maintain a voltage of 30

V between the anode, an h-BN filled W rod, and the cathode, a cooled Cu electrode. The arcing

took place within a He gas atmosphere, and the temperature at the anode was in excess of the

melting point of tungsten (> 3400 oC). The method produces MWBNNTs with diameters < 10

nm, and lengths > 200 nm. Loiseau et al. reported the synthesis of few to single-wall BNNTs

using a similar arc discharge method, using HfB2 electrodes in a N2 atmosphere.5 Cumings and

Zettl produced bulk quantities of nearly monodisperse double-wall BNNTs by using electrodes

with a low metal content. Elemental B powder was mixed together with 1 at% of Ni and Co.

The powder was melted to form ingots, which were used as the electrodes. Arcing was

performed in a N2 environment.6

3.3.2 Laser Heating/Ablation

In laser heating or ablation methods, a laser is used to heat a target material to very high

temperatures (> 4000 oC). Continuous lasers heat the target and the synthesis product is

collected from the target surface. Pulsed lasers will ablate the target, and synthesis product is

transported away from the target area by a carrier gas to a collecting substrate downstream.7

In 1996, Golberg et al. used a continuous high powered CO2 laser (up to 240 W) to irradiate a

target of single crystal c-BN powder within a diamond anvil cell in a N2 environment. The area

of the target on which the laser was focused either melts as c-BN, or undergoes a phase change

to h-BN before melting, depending on the N2 pressure, which varied from 5 – 15 GPa. Short (<

30 nm) MWBNNTs with circular and polygonal cross sections 3 – 15 nm in diameter were

18

observed on both melted c-BN flakes as well as recrystallized h-BN.8 The synthesis of single to

few-wall BNNTs was achieved by Yu et al. in 1998, using an oven-laser ablation method. A

pulsed excimer laser was used to irradiate a hot pressed target composed of h-BN powder mixed

with 1 at% each of Ni and Co nanopowders. Prior to irradiation, the target was heated within a

tube furnace to 1200 oC under a flow of various carrier gases (e.g., Ar, N2, He). The type of

carrier gas was found to affect the number of walls of the BNNTs –– the use of Ar and He

resulted in predominantly SWBNNTs, and the use of N2 resulted in mainly double-wall BNNTs.

The diameters of the tubes ranged from 1.5 nm to 8 nm, and the lengths were < 100 nm.9

Following the first report by Golberg et al., continuous CO2 laser heating was used to synthesize

long ropes of BNNTs (~ 40 μm). Laude et al. used a hot pressed h-BN powder target, and heated

it with a 70 W CO2 laser for 3 min in a N2 atmosphere. BNNTs with two to four walls were

produced, with up to several tens of tubes bundled into ropes.7

Gram quantities of BNNTs were synthesized by Lee et al. using a laser ablation set-up that could

be operated continuously.10

This method employed a 1 kW CO2 laser and a N2 atmosphere. A

catalyst-free BN target was moved over the course of the synthesis to expose new areas for

continuous ablation. The yield of BNNTs was estimated to be 0.6 g/h, and consisted of mostly

SWBNNTs. HRTEM characterization revealed that the chirality of the BNNTs was

predominantly zig-zag (85%).10

A pressurized vapour/condenser (PVC) method was developed by Smith et al.11

In a pressurized

N2 environment (2 -20 atm), boron vapour is produced through the laser heating of a B-

containing target, and a condenser wire is placed within the plume of B vapour in order to

generate B droplets in its wake. The droplets encounter N2 near the shear layer of the B plume,

and BNNTs form and assemble into fibrils. The process is versatile, and was found to be

effective with a variety of targets (hot and cold pressed BN, amorphous B powder, cast B) and

condenser materials (BN, B, stainless steel, Cu, Nb, W). The BNNT fibrils produced have

diameters of ~1 mm, and are composed of individual tubes > 100 μm long and < 10 nm in

diameter (SWBNNTs, DWBNNTs, and few-wall BNNTs). In a 200 mg run, a fibril mass 15 cm

long and approximately 1.5 cm wide was produced, with an appearance similar to cotton balls.

The raw material could be processed into yarn by twisting.

19

Typically, laser heating and ablation methods are high temperature methods. However, using a

plasma enhanced pulsed laser deposition (PLD) technique, Wang et al.12

were able to synthesize

BNNTs directly on a substrate at a temperature of 600 oC. Oxidized Si substrates were coated

with a thin Fe film, and installed on a heater within the deposition chamber. The chamber was

evacuated to a high vacuum, and then backfilled with N2. After heating the substrate to 600 oC,

an RF generator generated plasma on the substrate surface, which induced a negative DC voltage

on the substrate such that positive ions were accelerated towards the substrate. After 10 min of

plasma treatment at 600 oC, the thin Fe film broke down into nano-sized particles. A pulsed

Nd:YAG laser was then used to irradiate a rotating h-BN target situated above the substrate, and

the ablated vapour was propelled towards the substrate. At substrate biases of -360 to -450 V,

BNNTs were synthesized, and observed to grow from the Fe nanoparticles on the substrate. The

BNNTs were multiwalled and had diameters of 20 nm and less. Multiple tubes from closely

spaced Fe particles were found to form vertical bundles.12

3.3.3 Templated Synthesis

BNNTs can also be produced by using other nanostructures as templates. Han et al.13

used

CNTs to synthesize bulk quantities of BNNTs via a substitution reaction (Equation 3.1) in which

the C atoms were substituted by B and N atoms.

Equation 3.1 B2O3 + 3C (nanotubes) + N2 2 BN (nanotubes) + 3CO

A crucible containing a layer of MWCNTs (~10 nm diameter) over B2O3 powder was heated to

1500 oC under a flow of N2. The resulting BNNTs had similar dimensions to the starting CNTs,

but displayed a higher crystallinity.

Another type of one-dimensional nanostructure that has been used to template BNNT growth

consists of SiC nanowires. Zhong et al.14

demonstrated that the surface of SiC nanowires can be

coated by a liquid B-N containing polymer, produced from the decomposition of ammonia

borane at 1450 oC in an N2 environment. The decomposition products of ammonia borane

include H2, BH2NH2 (monomeric aminoborane), (BHNH)3 (borazine), and B2H6 (diborane). As

the products coating the SiC nanowires polymerizes into BN, H2 is evolved and trapped beneath

the film. At 1450 oC, H2 etches SiC, and by the end of the reactions the SiC nanowire is

20

completely etched away, leaving a hollow BN tube behind. These BNNTs had slightly larger

diameters compared with the SiC nanowires (150 nm vs. 100 nm), and possessed a unique

beaded structure in which sections of straight tubular walls are joined together by spheric shells.

The authors hypothesize that surface tension causes the liquid film to bead on the nanowire

surface, resulting in the final beaded morphology.14

Nanoporous materials such as mesoporous silica and anodic aluminum oxide (AAO) have been

used to template BNNT growth by deposition on the inner walls of the pores. The BNNTs can be

subsequently freed from the template using template-specific etchants. One particular advantage

that this membrane-assisted template process has over other techniques is the ability to form

monodisperse vertically aligned nanotubes, with control over the tube diameter, spacing, and

length. This is desirable for many applications, because BNNTs have anisotropic properties such

as thermoconductivity and Young’s modulus, both of which are greater in the direction of the

tube axis.15, 16

Therefore, to exploit these properties to their fullest, alignment of the nanotubes

in devices and composite materials is necessary. Li et al.17

used a chemical vapour deposition

(CVD) technique by reacting BCl3 and NH3 gases which formed BN via the reaction in Equation

3.2:

Equation 3.2 BCl3 (g) + NH3 (g) BN (s) + HCl (g)

The silica template was dissolved by treatment with 5% HF. The pore size of the mesoporous

silica used was ~7 nm, and TEM analysis of the freed BNNTs revealed that they had similar

diameters.

Bechelany et al.18

synthesized vertically aligned supported MWBNNT arrays via the infiltration

of AAO with liquid polymeric borazine. After infiltration, the impregnanted AAO samples were

heated to 200 oC to create thermoset polymer films coating the walls of the AAO channels. The

samples were then annealed in N2 at 1200 oC, and the polymer films underwent solid state

thermolysis to produce BN coatings on the channel walls. A thin film of BN covered the

surface of the template, such that the nanotube array was supported by it once freed from the

template by 48% HF. To improve the crystallinity of the BNNTs, samples after HF treatment

were annealed in N2 at temperatures between 1450 – 1800 oC. The AAO membrane used had a

200 nm pore size and pore lengths of ~60 μm, and the MWBNNTs were found to have similar

21

dimensions. Other AAO membranes with different pore diameters and channel length could be

used to control the dimensions of the BNNTs.

3.3.4 Chemical Vapour Deposition

MWBNNTs can be produced with chemical vapour deposition (CVD) based techniques. These

techniques involve the introduction of gaseous precursors into a reaction chamber containing a

heated substrate. Gas-phase reactions occur, forming intermediate species; these species can

form solids in the gas phase which are deposited onto the substrate, where they can become

crystal nucleation centers. Alternatively, gaseous intermediate species can adsorb directly onto

the substrate and heterogeneous reactions can occur at the gas-solid interface, forming the solid

species which become nucleation centers.19

The majority of CVD methods developed for BNNT synthesis involve the use of a metal-based

catalyst, and relatively mild reaction temperatures < 1300 oC. Compared with other techniques

such as arc discharge or laser ablation, the BNNTs produced by CVD are predominantly

multiwalled and larger in diameter (~10 – 100 nm). The first report of CVD synthesis of

BNNTs, by Lourie et al.,20

used borazine gas (B3N3H6) and Co, Ni, NiB, and Ni2B catalyst

particles. The catalyst particles were suspended in ethanol and deposited onto oxidized Si

substrates. The reaction was carried out at 1000 – 1100 oC under N2 flow. It was found that the

NiB and Ni2B catalysts produced the highest yield and quality of MWBNNTs, compared with

Co and Ni. The MWBNNTs grew directly from these catalyst particles, were 10 – 50 nm in

diameter, and possessed lengths up to 5 μm.

In other works, the most commonly used gas precursors for the source of N are N2 and NH3. For

the source of B, amorphous B powders, B thin films, and alloy particles such as Fe-B can be

used.21-24

Tang et al.25

combined B and MgO powders and heated the mixture to 1300 oC in an

induction furnace. At this temperature, the following reaction takes place:

Equation 3.3 2B (s) + 2MgO (s) B2O2 (g) + 2Mg (g)

The B2O2 vapour was then transported via an Ar carrier gas flow into a cooler (1100 oC) reaction

chamber, where NH3 gas was introduced. BNNTs were produced via Equation 3.4.

22

Equation 3.4 B2O2 (g) + 2NH3 (g) BN (s) + 2H2O (g) + H2 (g)

Following the work of Tang et al., a high-yield process for MWBNNTs was developed by Zhi et

al.,26

using a mix of FeO, MgO, and B powders at the precursor material. When heated in an

induction furnace under NH3 flow at temperatures between 1100 – 1700 oC, the MgO precursor

was found to effectively produce B2O2 vapour, while the FeO precursor was reduced to Fe and

served as a catalyst for the growth of BNNTs. These two processes resulted in a high product

yield (~200 mg/h), and the yield was found to increase with increasing temperature. This

method is known as boron oxide chemical vapour deposition (BOCVD).

Transition metals such as Fe, Co, and Ni are typical catalysts, and can be used in nanoparticle

form, or as thin films coated on substrates (which de-wet and form nanoparticles of a

characteristic size, depending on the film thickness, when heated).24, 27, 28

There are several CVD methods which are notable due to their unique precursors, products,

and/or equipment. Kim et al.29

used a floating nickelocene catalyst (solid nickelocene was

heated at 80 oC to form a vapour) and borazine gas. These two gases were reacted in the

presence of N2 and NH3 at 1200 oC, and at low borazine pressures, highly crystalline doublewall

BNNTs were formed (~2 nm diameter). The yield of BNNTs was estimated to be ~20 mg/h,

with 70% being comprised of doublewall BNNTs, with SWBNNTs and few-wall BNNTs

making up the other fraction. Ma et al.30

showed that a metal-based catalyst is not necessary for

the synthesis of BNNTs. They used a melamine diborate precursor (B4N3O2H) powder, which

was heated to 1700 oC in an induction furnace under N2 flow. MWBNNT product was found

downstream, in a zone of the furnace where the temperature was ~1200 oC. A common feature

of the MWBNNTs was the presence of bulbous tips on the nanotube ends. HRTEM revealed

that these tips were BN cages filled with amorphous material. In another study without metal

catalysts, Gan et al.22

grew BNNT films on oxidized Si substrates. The precursors were B films

deposited on the substrates, and NH3 gas. Synthesis was carried out at 1175 oC. It was observed

that growth was preferential on SiO2 as opposed to plain Si, and that SiO2 played a catalytic role

in the growth of BNNTs.22

Films up to 1 mm thick were synthesized, comprised of tubes with

diameters < 100 nm.

23

Assisted CVD methods have been developed in order to achieve low temperature synthesis of

BNNTs (< 1000 oC). A microwave plasma-enhanced CVD method was used by Guo et al.

27, 28

to synthesize MWBNNTs on Ni and Co coated oxidized Si substrates at a temperature of 800 oC.

The gas precursors used were diborane (B2H6, diluted in H2 to 5 vol%), NH3, and H2. Under

optimum process conditions, MWBNNTs several microns long with diameters of 5 – 20 nm

were produced on the substrates. Su et al.31

synthesized MWBNNTs at 900 oC using a home-

built plasma-assisted CVD system. In addition to the plasma, finely dispersed Fe catalyst

particles were prepared on high surface area SiO2/Al2O3 supports, in order to promote nanotube

growth at the lower temperature. B2H6, NH3, O2, and Ar were used as process gases.

3.3.5 Mechano-Thermal (Ball milling and Annealing)

A synthesis method with promise for industrial scale-up is the mechano-thermal method, in

which precursor powders are subjected to ball-milling for extended periods in order to break

them down into nano-sized particles with highly activated surfaces prior to thermal annealing.

High-energy ball milling involves a stainless steel cell into which the powder is loaded along

with several hard balls (usually made out of stainless steel or tungsten carbide). The cell is then

mechanically shaken, such that the powder gets mixed and broken down by a variety of

processes including grinding, fracturing, and plastic deformation. Due to these processes the

surfaces of the particles become very reactive with many dangling bonds, and the particles have

a high density of structural defects such as dislocations, grain boundaries, etc. The disordered

surfaces and defects allow for non-equilibrium chemical reactions to proceed at low

temperatures.

Chen et al. introduced the technique for BNNTs in 1999,32

as an alternative to arc discharge and

laser heating. The milling cell was loaded with B powder and NH3 gas and milled for 150 h.

The gas pressure within the cell was monitored, and was observed to decrease during the first 40

h of milling, and then increase until it stabilized at 100 h. The pressure changes were a signature

of a nitriding reaction between the B and NH3, in which NH3 was first adsorbed onto the B

particles, followed by the reaction shown in Equation 3.5:

Equation 3.5 B + NH3 BN + 3/2 H2

24

The milled sample is then annealed at 1000 oC and higher under N2 flow for 6 h, allowing the

nitriding reaction to proceed to completion. XRD analysis of the products confirms the presence

of h-BN, and also indicates that a Fe-B phase is present. Small amounts (5 wt%) of Fe were

introduced from the stainless steel cell and milling balls, and served as catalysts for the BNNT

synthesis. MWBNNTs with diameters ranging from 20 – 100 nm were produced with this

method, and higher temperatures were found to increase the tube diameters.

Other variations on the mechano-thermal method include the milling and annealing of h-BN

powder in NH3 and N2,33, 34

of B powder in N2,35

and of B2O3 powder in N2 and NH3.36

Milling

times were typically 100 h and longer, and annealing was carried out at temperatures of 1000 –

1300 oC for periods of 6 h and longer. A mixture of BNNT types was produced, consisting of

bamboo and cylindrical MWBNNTs. A yield of 600 mg BNNTs per 1 g milled h-BN powder

was demonstrated by Li et al.36

As further evidence for the catalyst hypothesis, these studies

reported the incorporation of impurities into the milled powders; namely Fe, Cr, and Ni (or W in

the case of WC milling balls and cell), and traces of Si and Al.

3.3.6 Chemical Synthesis

Another method which is promising for industrial scale-up is the self-propagation high-

temperature synthesis (SHS) and annealing process, developed by Wang et al.37

The key

element of this method is the synthesis of a porous precursor via SHS. B2O3, Mg, and CaB6

powders are mixed in a blender, then heated to 750 oC in an Ar atmosphere. These precursors

undergo the reaction described in Equation 3.6 to form the porous precursor, B18Ca2(MgO)9.

Equation 3.6 3B2O3 + 9Mg + 2 CaB6 B18Ca2(MgO)9

The precursor is then annealed in a horizontal tube furnace at 1150 oC under a NH3 flow for 6 h.

Gram quantities of MWBNNTs (20 – 300 nm in diameter) were obtained after synthesis, with a

product yield above 80 wt%. The process was further developed to yield kilogram quantities of

BNNTs with various morphologies (MWBNNTs, bamboo BNNTs), using a B31Fe17(MgO)27

porous precursor synthesized by SHS.38

3.3.7 Comparison of Methods

A summary of the synthesis methods discussed above is presented in Table 3.1.

25

Table 3.1 Comparison of BNNT Synthesis Methods

Synthesis Method Temperature

(oC)

Morphology Tube Diameter

(nm) Yield

Arc Discharge > 3400 SW, DW,

few-wall BNNT < 10

Low4, 5

Moderate6

Laser

Heating/Ablation > 4000

SW, DW,

few-wall BNNT < 10

Low8, 9

Moderate7

High10, 11

CVD/Chemical

Synthesis 800 – 1700

MWBNNT,

bamboo BNNT38

10 – 200

Moderate

High26, 37, 38

Mechano-Thermal 1000 – 1300 MWBNNT,

bamboo BNNT 50 – 200

Moderate

High36

3.4 Experimental Methods

3.4.1 Method 1: Silica-Assisted Catalytic Chemical Vapour Deposition

The first method employed in this thesis and presented here is based on the work of Reddy et al.

(2010).39

Silica-assisted chemical vapour deposition (SA-CVD) is a bulk synthesis method

combining the use of a Fe2O3-SiO2/Al2O3 supported catalyst, ball milling, and CVD.

3.4.1.1 Preparation of the Precursor Powder

In a typical preparation, 3.5 g of Fe(NO3)3∙9H2O, 5 g of fumed Al2O3, and 80 mL of methanol

are combined. The mixture is covered and stirred overnight, following which the solvent is

evaporated, leaving behind a powder. Following this, 0.65 g of the Fe2O3-Al2O3 catalyst, 1 g of

amorphous B powder, and 1 g of fumed SiO2 are combined with 80 mL of methanol. Again, the

mixture is covered and stirred overnight. After solvent evaporation, the precursor powder is

ready for ball milling treatment. The powder is loaded into a stainless steel cell with several 1

cm stainless steel balls, and milled for 20 h.

26

3.4.1.2 CVD Process

The ball milled precursor, if used some time after the milling process, is ground with a mortar

and pestle, then sprinkled evenly into a quartz boat. The boat is then placed in the centre of a

tube furnace, and heated to 1000 – 1150 oC under 80 sccm Ar gas flow. Upon reaching the set

temperature, 50 sccm of NH3 is flowed into the process tube, and the Ar flow is stopped. The

reaction is allowed to proceed under these conditions for 2 h, then the NH3 flow is stopped and

the Ar flow restarted, to protect the products from oxidation while the system cools down to

room temperature.

3.4.1.3 Purification

After synthesis, the quartz boat is filled with a grey powder. In order to remove the SiO2 and

Al2O3 components, the product is washed with 1 M HF, and filtered several times. HCl can be

used to dissolve Fe impurities, while HNO3 can be used to dissolve any B.40

3.4.2 Method 2: Growth Vapour Trapping Chemical Vapour Deposition

The second method employed in this thesis follows the method of Lee et al (2008).24

Growth

vapour trapping chemical vapour deposition (GVT-CVD) is a simple thermal CVD process using

a conventional tube furnace set-up, with the critical components being a vacuum pump and a

quartz test tube which is used to trap the growth vapours evolved during the reactions close to

the nucleation sites.

3.4.2.1 Preparation of the Precursor Powder

Amorphous B, MgO, and Fe2O3 nanopowders (Sigma Aldrich, MO) are combined in a mortar

and pestle in a 4:1:1 molar ratio.

3.4.2.2 CVD Process

In a typical run, 100-150 mg of precursor powder is measured into a 3 mL alumina boat. Si

substrates can be placed over top of the boat to partially cover it. The boat is then placed at the

closed end of a quartz test tube, as shown in Figure 3.1 below.

27

Figure 3.1 Position of alumina boat within quartz test tube.

The quartz test tube is then placed within the quartz process tube of the tube furnace

(ThermoScientific), such that the closed end of the test tube and alumina boat are located within

the middle of the heating element region. The process tube is then sealed, and evacuated to ~25

kPa via a mechanical pump. The vacuum pump is kept running for the duration of the process.

A flow of NH3 gas is started at 200 sccm and kept flowing for the duration of the temperature

ramp-up (~10 oC/min) and dwell time at 1200

oC (1-2 h). At the end of the dwell time, the NH3

flow is shut off, and the system is allowed to cool to room temperature under vacuum. A

schematic of the set-up is shown in Figure 3.2.

Figure 3.2 GVT-CVD set-up.

After the synthesis, a white powder can be found in the alumina boat, covering the remains of the

precursor powder (the reaction does not go to completion in 1-2 h).

3.4.2.3 Purification

Product collected from Si substrates can be used without purification. For the product in the

boat, HCl can be used to dissolve Fe impurities, while HNO3 can be used to dissolve any B.40

3.5 Results

3.5.1 Macroscopic Description of Products

After a typical SA-CVD run, the quartz boat and its contents are as shown in Figure 3.3. A white

powder covers the transparent colourless quartz boat, and a grey powder covers the remnants of

the precursor powders, which were rust-coloured prior to the synthesis and black afterward.

BNNT products were found within the grey powder, and not the white powder.

28

Figure 3.3 Quartz boat after Method 1, silica-assisted CVD (1150 oC, 1 h).

After a typical GVT-CVD run, the alumina boat and its contents are as shown in Figure 3.4. A

white powder covers the remnants of the precursor powders, which were rust-coloured prior to

the synthesis and black afterward. The walls of the white alumina boat remained largely clean of

material. BNNTs were found to be present in the white powder (see Chapter 4 for

characterization details).

Figure 3.4 Alumina boat after Method 2, GVT-CVD (1150 oC, 2 h).

29

3.5.2 Nanotube Morphology

The BN products synthesized via SA-CVD are shown in Figure 3.5 below. Different synthesis

temperatures resulted in different BN nanostructure morphologies.

Figure 3.5 Electron micrographs of the products of SA-CVD. a) TEM image of BN flower structure, synthesized at 1000

oC. b) TEM image of short bamboo BNNTs, synthesized at 1050

oC. c) TEM image of long bamboo BNNTs,

synthesized at 1100 oC. d) SEM image of long bamboo BNNTs. e) TEM image of straight MWBNNT, synthesized at

1150 oC. f) SEM image of MWBNNTs.

30

The BN products synthesized via GVT-CVD are shown in Figure 3.6 below.

Figure 3.6 a) SEM image of MWBNNTs synthesized by GVT-CVD. b) TEM image of MWBNNTs.

3.6 Discussion

3.6.1 BNNT Growth Mechanisms in CVD Synthesis

3.6.1.1 Redox Reactions – Generation of Growth Vapours

At temperatures above 600 oC, a number of redox reactions occur amongst the solid precursor

materials and the NH3 process gas. In SA-CVD and GVT-CVD, B reacts with Fe2O3 to form

B2O2 vapour and metallic Fe, as shown in Equation 3.7.21

B2O2 vapour is also produced from a

reaction of B and SiO2, in the SA-CVD method (Equation 3.8).22

In GVT-CVD, MgO reacts

with B to produce B2O2 vapour, as shown in Equation 3.9.25

Equation 3.7 6B (s) + 2Fe2O3 (s) 3B2O2 (g) + 4Fe (s)

Equation 3.8 2B (s) + SiO2 (s) B2O2 (g) + Si (s)

Equation 3.9 2B (s) + 2MgO (s) B2O2 (g) + 2Mg (g)

BNNTs can be formed from the reaction between B2O2 and NH3 gases, as described by Equation

3.10:

Equation 3.10 B2O2 (g) + 2NH3 (g) 2BN (s) + 2H2O (g) + H2 (g)

31

However, the dominant growth mechanism of BNNTs in the CVD synthesis methods explored in

this work involves the Fe nanoparticles which are formed after reduction of the Fe2O3 precursor

powder. As in the case of CNTs, in which transition metals including Fe have been

demonstrated to catalyze nanotube growth in CVD methods,41

the Fe nanoparticles catalyze

BNNT growth by serving as a vessel for the concentration of B and N and controlling the

precipitation of BN via the nanoparticle morphology.

3.6.1.2 Formation of Low Melting Point Eutectic Nanoparticles

The catalytic ability of the Fe nanoparticles relies in part on the particles being in a liquid or

semi-molten state, in which the particles are quasi-spherical. The melting point of Fe is 1538

oC,

42 which is a higher temperature than the ones employed in Methods 1 and 2 (~1200

oC). Fe

nanoparticles can exhibit melting point depression, with smaller particles melting at a lower

temperature than the bulk material. However, for nanoparticles 25 nm and larger, which is the

estimated size of the Fe nanoparticles in Methods 1 and 2, the melting point would only be

depressed by ~150 oC.

43 Therefore, pure Fe nanoparticles would remain solid at the synthesis

temperature of ~1200 oC. When B and N diffuse into the Fe nanoparticles, alloys can form,

namely Fe2B and Fe4N. Fe4N does not possess catalytic properties. It can also be reduced back

to Fe by B at high temperatures (> 700 oC). On the other hand, Fe2B is a low melting point

eutectic, with a melting point of ~1200 oC.

42 It is more thermodynamically stable than Fe4N, and

is therefore the dominant alloy formed under the reaction conditions. Fe2B alloy particles can

form via solid state diffusion between adjacent B particles and Fe particles, as well as via

decomposition of B2O2 on the surface of Fe particles, and the subsequent diffusion of B into the

Fe particles, as depicted in Figure 3.7a.40

In the SA-CVD method, Si can also alloy with Fe to

produce low melting point eutectics (FeSix), with melting points ~1200 oC.

44,45

3.6.1.3 Vapour-Liquid-Solid Hypothesis

Many studies of CVD methods propose that BNNTs are formed via a vapour-liquid-solid (VLS)

process. The Fe2B nanoparticles constitute the liquid phase, while the gaseous B and N sources

(B2O2, NH3) make up the vapour phase. The process gases or growth vapours (N2 or NH3; B2O2)

decompose onto the metal nanoparticle surfaces, allowing for the diffusion of N and B atoms

into the nanoparticles. When the concentration of B and N within the nanoparticle reaches

32

supersaturation, B and N precipitate out on the surface of the metal nanoparticles in a layer-by-

layer fashion to form a cap of BN (the solid phase), as shown in Figure 3.7b.23

It is also a

possibility that the B atoms which diffused into the Fe2B particle eventually segregate to form a

shell on the outside of the nanoparticle, and then react directly with N from the decomposed NH3

vapour to form the BN cap.31, 40

As BN growth continues, the liquid nanoparticle undergoes a shape change from spherical to

elongated, due to capillary forces from the BN cap/growing BNNT (Figure 3.7c). This shape

change allows for the growth of cylindrical BN walls, forming a BNNT.46

Figure 3.7 Schematic of BNNT growth, showing (a) the Fe-B alloy catalyst particle and its uptake of B and N from gaseous sources (and also B via solid state diffusion), (b) the precipitation of a BN cap, (c) BNNT growth from an

elongated Fe-B alloy particle, and (d) contraction of Fe-B alloy particle and formation of a new BN cap. The growth process repeats to form the bamboo BNNT structure.

Bamboo BNNTs are typically formed at lower temperatures than straight-walled cylindrical

MWBNNTs. The VLS hypothesis can explain the formation of the bamboo structure by a

stuttered MWBNNT growth process in which the shape of the metal nanoparticle is dictated by

the balance of surface tension and capillary forces. In this process, a BN cap is formed and

growth of a short segment of cylindrical walls occurs with the metal nanoparticle elongating due

to capillary forces. The elongated shape of the nanoparticle is not stable below a certain

temperature, and surface tension causes the metal nanoparticle to contract, reforming a sphere.

The process then repeats, with the formation of a new BN cap and the growth of another short

segment of cylindrical tube wall. This process is illustrated in Figure 3.7d above.

33

The colour change of the precursor powders from rust-coloured to black indicates that the iron

oxide component changes from Fe2O3 to Fe to Fe3O4 over the course of the reaction.

3.6.1.4 Achieving BNNT Growth at Low Temperatures

In Method 1, the design and synthesis of the supported catalyst material and subsequent ball

milling result in the successful growth of BNNTs at relatively low temperatures (1100 – 1150

oC). The choice of catalyst support has been found to influence nanotube growth in studies of

CNT synthesis. SiO2 and Al2O3 are two support materials that appear to promote growth. The

high surface area of the fumed Al2O3 enables a high dispersion of Fe2O3 particles with minimal

aggregation. Furthermore, there is a strong interaction between the two oxides. The Al2O3

stabilizes the Fe2O3 particles to high temperatures by inhibiting their reducibility.47

Therefore,

the formation of the catalytic Fe nanoparticles occurs at temperatures closer to the optimal

synthesis temperature, which gives the particles less time to sinter and aggregate together. This

ensures that the particle sizes of the catalyst remain small and amenable to nanotube growth.48

SiO2 has a similar interaction with Fe2O3, although not as strong as Al2O3. The introduction of

SiO2 into the Fe2O3/Al2O3 support promotes efficient BNNT growth by creating larger pore

volumes and increasing the surface area of the supported catalyst exposed to growth vapours

during synthesis.31

The relatively short (20 h) ball milling treatment of the precursor powders

breaks down large aggregates and thoroughly mixes together all components, resulting in a fine

powder with a uniform distribution of precursors and high surface area. The redox reaction

between SiO2 and B also increases the concentration of B2O2 vapour, which leads to a higher

nucleation rate.

Three strategies are employed to increase the nucleation rate for BNNTs at 1200 oC in the GVT-

CVD method. The first strategy is the efficient generation of B2O2 vapour in the early stages of

the synthesis via the redox reaction between MgO and B, following the boron oxide chemical

vapour deposition (BOCVD) method developed by Zhi et al.26

The second strategy is to trap the

growth vapours evolved from the redox reactions between the precursors, such that there is a

high concentration of B source in the vicinity of the Fe catalyst particles within the alumina boat.

The trapping is accomplished by means of a quartz test tube, with the alumina boat located

within the test tube at the closed end. The closed end faces the flow of incoming NH3 gas, and

therefore protects the growth vapours from being transported downstream away from the boat.

34

The open end is 60 cm away from the closed end, and allows the NH3 gas to diffuse into the test

tube and reach the alumina boat. Lastly, the third strategy for promoting nucleation is to carry

out the synthesis under vacuum conditions. The mild vacuum (~0.025 MPa) created by the

mechanical pump increases the vapour pressure of the growth species, which in turn results in a

higher probability of nuclei formation. The probability of nuclei formation is described in

Equation 3.11, in which A is a constant, σ is the surface energy of the catalyst particles, k is the

Boltzmann constant, T is the temperature in Kelvin, and α = p/po (p = vapour pressure of the

growth species, po = equilibrium vapour pressure of the condensed phase).

Equation 3.11

For constant T and σ, the probability of nuclei formation can be increased by increasing the

vapour pressure of the growth species, and therefore α.24

3.6.2 Qualitative Comparison of Methods

Methods 1 and 2 have different strengths and weaknesses as synthesis methods for BNNTs. SA-

CVD possesses a higher growth rate/yield than the GVT-CVD method, and is more amenable to

industrial scale-up. The control over BN nanostructure morphology via synthesis temperature is

useful for the study of each type of nanostructure, and also gives insight into the growth

mechanisms involved. On a laboratory scale, the preparation of the precursor powder is a time

intensive process, and the many components make purification of the final product more

difficult. The GVT-CVD method has the advantage of very quick and simple precursor powder

preparation, and BNNTs collected from substrates covering the reaction boat can be used for

most applications without further purification. However, the growth rate and yield of GVT-CVD

are lower than those of SA-CVD, and as a result make GVT-CVD not as suitable to industrial

scale-up.

3.7 Conclusions

The main methods for synthesizing BNNTs were summarized. Two methods utilized over the

course of this thesis were presented in detail, namely a silica-assisted chemical vapour deposition

technique, and a growth vapour trapping chemical vapour deposition technique. The resulting

35

products are described qualitatively (to be presented in further detail in Chapter 4), and possible

growth mechanisms are discussed.

3.8 Contributions

The author set up the synthesis equipment and process for the methods of Reddy et al. and Lee et

al. at the University of Toronto. BNNTs were synthesized according to the protocols,

characterized (see next chapter), and used in various experiments.

3.9 References



Ed. Springer Verlag: Berlin, Heidelberg, 2002; pp 1–45.

2. Iijima, S., Helical microtubules of graphitic carbon. Nature 1991, 354, 56–58.

3. Prasek, J.; Drbohlavova, J.; Chomoucka, J.; Hubalek, J.; Jasek, O.; Adam, V.; Kizek, R.,

Methods for carbon nanotubes synthesis - review. J. Mater. Chem. 2011, 21, 15872–

15884.


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5. Loiseau, A.; Willaime, F.; Demoncy, N.; Hug, G.; Pascard, H., Boron nitride nanotubes

with reduced numbers of layers synthesized by arc discharge. Phys. Rev. Lett. 1996, 76,

4737–4740.

6. Cumings, J.; Zettl, A., Mass-production of boron nitride double-wall nanotubes and

nanococoons. Chem. Phys. Lett. 2000, 316, 211–216.

7. Laude, T.; Matsui, Y.; Marraud, A.; Jouffrey, B., Long ropes of boron nitride nanotubes

grown by a continuous laser heating. Appl. Phys. Lett. 2000, 76, 3239–3241.

8. Golberg, D.; Bando, Y.; Eremets, M.; Takemura, K.; Kurashima, K.; Yusa, H.,

Nanotubes in boron nitride laser heated at high pressure. Appl. Phys. Lett. 1996, 69,

2045–2047.

9. Yu, D. P.; Sun, X. S.; Lee, C. S.; Bello, I.; Lee, S. T.; Gu, H. D.; Leung, K. M.; Zhou, G.

W.; Dong, Z. F.; Zhang, Z., Synthesis of boron nitride nanotubes by means of excimer

laser ablation at high temperature. Appl. Phys. Lett. 1998, 72, 1966–1968.

10. Lee, R. S.; Gavillet, J.; Chapelle, M. L. D. L.; Loiseau, A.; Cochon, J. L.; Pigache, D.;

Thibault, J.; Willaime, F., Catalyst-free synthesis of boron nitride single-wall nanotubes

with a preferred zig-zag configuration. Phys. Rev. B 2001, 64, 121405.

11. Smith, M. W.; Jordan, K. C.; Park, C.; Kim, J.-W.; Lillehei, P. T.; Crooks, R.; Harrison,

J. S., Very long single- and few-walled boron nitride nanotubes via the pressurized

vapor/condenser method. Nanotechnology 2009, 20, 505604.

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12. Wang, J.; Kayastha, V. K.; Yap, Y. K.; Fan, Z.; Lu, J. G.; Pan, Z.; Ivanov, I. N.;

Puretzky, A. A.; Geohegan, D. B., Low temperature growth of boron nitride nanotubes on

substrates. Nano Lett. 2005, 5, 2528–2532.

13. Han, W.; Bando, Y.; Kurashima, K.; Sato, T., Synthesis of boron nitride nanotubes from

carbon nanotubes by a substitution reaction. Appl. Phys. Lett. 1998, 73, 3085–3087.

14. Zhong, B.; Song, L.; Huang, X. X.; Wen, G. W.; Xia, L., Synthesis of boron nitride

nanotubes with SiC nanowire as template. Mater. Res. Bull. 2011, 46, 1521–1523.


conductivity of nanostructured boron nitride materials. J. Phys. Chem. B 2006, 110,

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nitride nanotube. Solid State Commun. 1998, 105, 297–300.

17. Li, N.; Li, X.; Geng, W.; Zhao, L.; Zhu, G.; Wang, R.; Qiu, S., Template synthesis of

boron nitride nanotubes in mesoporous silica SBA-15. Mater. Lett. 2005, 59, 925–928.

18. Bechelany, M.; Bernard, S.; Brioude, A.; Cornu, D.; Stadelmann, P.; Charcosset, C.;

Fiaty, K.; Miele, P., Synthesis of boron nitride nanotubes by a template-assisted polymer

thermolysis process. J. Phys. Chem. C 2007, 111, 13378–13384.

19. Choy, K. L., Chemical vapour deposition of coatings. Prog. Mater Sci. 2003, 48, 57–170.

20. Lourie, O. R.; Jones, C. R.; Bartlett, B. M.; Gibbons, P. C.; Ruoff, R. S.; Buhro, W. E.,

CVD growth of boron nitride nanotubes. Chem. Mater. 2000, 12, 1808–1810.

21. Li, J.; Lin, H.; Chen, Y.; Su, Q.; Huang, Q., The effect of iron oxide on the formation of

boron nitride nanotubes. Chem. Eng. J. 2011, 174, 687–692.

22. Gan, Z. W.; Ding, X. X.; Huang, Z. X.; Huang, X. T.; Cheng, C.; Tang, C.; Qi, S. R.,

Growth of boron nitride nanotube film in situ. Appl. Phys. A. 2005, 81, 527–529.

23. Fu, J. J.; Lu, Y. N.; Xu, H.; Huo, K. F.; Wang, X. Z.; Li, L.; Hu, Z.; Chen, Y., The

synthesis of boron nitride nanotubes by an extended vapour–liquid–solid method.

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25. Tang, C.; Bando, Y.; Sato, T.; Kurashima, K., A novel precursor for synthesis of pure

boron nitride nanotubes. Chem. Commun. 2002, 1290–1291.

26. Zhi, C.; Bando, Y.; Tan, C.; Golberg, D., Effective precursor for high yield synthesis of

pure BN nanotubes. Solid State Commun 2005, 135, 67–70.

27. Guo, L.; Singh, R. N., Selective growth of boron nitride nanotubes by plasma-enhanced

chemical vapor deposition at low substrate temperature. Nanotechnology 2008, 19,

065601.

28. Guo, L.; Singh, R. N., Catalytic growth of boron nitride nanotubes using gas precursors.

Physica E 2009, 41, 448–453.

37

29. Kim, M. J.; Chatterjee, S.; Kim, S. M.; Stach, E. A.; Bradley, M. G.; Pender, M. J.;

Sneddon, L. G.; Maruyama, B., Double-walled boron nitride nanotubes grown by floating

catalyst chemical vapor deposition. Nano Lett. 2008, 8, 3298–3302.

30. Ma, R.; Bando, Y.; Sato, T., CVD synthesis of boron nitride nanotubes without metal

catalysts. Chem. Phys. Lett. 2001, 337, 61–64.

31. Su, C.-Y.; Chu, W.-Y.; Juang, Z.-Y.; Chen, K.-F.; Cheng, B.-M.; Chen, F.-R.; Leou, K.-

C.; Tsai, C.-H., Large-scale synthesis of boron nitride nanotubes with iron-supported

catalysts. The Journal of Physical Chemistry C 2009, 113, 14732–14738.

32. Chen, Y.; Fitz Gerald, J.; Williams, J. S.; Bulcock, S., Synthesis of boron nitride

nanotubes at low temperatures using reactive ball milling. Chem. Phys. Lett. 1999, 299,

260–264.

33. Chen, Y.; Chadderton, L. T.; Gerald, J. F.; Williams, J. S., A solid-state process for

formation of boron nitride nanotubes. Appl. Phys. Lett. 1999, 74, 2960–2962.

34. Singhal, S.; Srivastava, A.; Pant, R.; Halder, S.; Singh, B.; Gupta, A., Synthesis of boron

nitride nanotubes employing mechanothermal process and its characterization. J. Mater.

Sci. 2008, 43, 5243–5250.

35. Kim, J.; Lee, S.; Uhm, Y. R.; Jun, J.; Rhee, C. K.; Kim, G. M., Synthesis and growth of

boron nitride nanotubes by a ball milling–annealing process. Acta Mater. 2011, 59, 2807–

2813.

36. Li, Y.; Zhou, J. e.; Zhao, K.; Tung, S.; Schneider, E., Synthesis of boron nitride

nanotubes from boron oxide by ball milling and annealing process. Mater. Lett. 2009, 63,

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by self-propagation high-temperature synthesis and annealing method. J. Nanomater.

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synthesis of boron nitride nanotubes by self-propagation high-temperature synthesis and

annealing process. J. Solid State Chem. 2011, 184, 2478–2484.

39. Reddy, A. L. M.; Tanur, A. E.; Walker, G. C., Synthesis and hydrogen storage properties

of different types of boron nitride nanostructures. Int. J. Hydrogen Energy 2010, 35,

4138–4143.

40. Koi, N.; Oku, T.; Inoue, M.; Suganuma, K., Structures and purification of boron nitride

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43, 2955–2961.

41. Öncel, Ç.; Yürüm, Y., Carbon nanotube dynthesis via the vatalytic CVD method: A

review on the effect of reaction parameters. Fullerenes Nanotubes Carbon Nanostruct.

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42. Okamoto, H., B-Fe (Boron-Iron). J. Phase Equilib. Diffus. 2004, 25, 297–298.

38

43. Shu, Q.; Yang, Y.; Zhai, Y.; Sun, D.; Xiang, H.; Gong, X.-G., Size-dependent melting

behavior of iron nanoparticles by replica exchange molecular dynamics. Nanoscale 2012

(accepted manuscript).

44. Tang, C. C.; Ding, X. X.; Huang, X. T.; Gan, Z. W.; Qi, S. R.; Liu, W.; Fan, S. S.,

Effective growth of boron nitride nanotubes. Chem. Phys. Lett. 2002, 356, 254–258.

45. Schlesinger, M. E., Thermodynamics of solid transition-metal silicides. Chem. Rev. 1990,

90, 607–628.

46. Loh, K. P.; Lin, M.; Yeadon, M.; Boothroyd, C.; Hu, Z., Growth of boron nitride

nanotubes and iron nanowires from the liquid flow of FeB nanoparticles. Chem. Phys.

Lett. 2004, 387, 40–46.

47. Berry, F. J.; Lin, L.; Liang, D.; Wang, C.; Tang, R.; Zhang, S., An investigation of metal-

support interactions in some iron, ruthenium, and iron-ruthenium catalysts by in situ iron-

57 Mossbauer spectroscopy. Appl.Catal. 1986, 27, 195–205.

48. Esmaieli, M.; Khodadadi, A.; Mortazavi, Y., Catalyst support and pretreatment effects on

carbon nanotubes synthesis by chemical vapor deposition of methane on iron over SiO2,

Al2O3 or MgO. Int. J. Chem. Reactor Eng. 2009, 7, A36–17.

Equation 4 f

Figure 4. t

Table 4.

39

4 Structural and Chemical Characterization of Boron Nitride Nanotubes

4.1 Abstract

In this chapter, the structural and chemical characterization of boron nitride nanotubes via

electron microscopy, FTIR, and EDX is presented.

4.2 Introduction

Electron microscopy and its related techniques are invaluable characterization tools for the study

of nanomaterials. In general, scanning electron microscopy (SEM) has the capability to image at

magnifications ranging from x 10 – 100 000, while high resolution transmission electron

microscopy (HR-TEM) can achieve magnifications up to x 10 000 000. TEM can measure the

spacing between planes of atoms (lattice imaging), yielding crystallographic information.1, 2

In

addition to imaging, electron microscopy is often performed together with integrated techniques

such as energy dispersive x-ray (EDX) analysis. EDX provides chemical analysis through the

detection of characteristic x-ray lines emitted by the sample due to interaction with the electron

beam. Elements with Z ≥ 5 can be detected qualitatively, with quantitative analysis possible for

Z ≥ 11. The combination of SEM-EDX allows for chemical analysis on the microscale, and

correlation between micro-nano structures and elemental composition.3

For bulk characterization of chemical composition, Fourier transform infrared (FTIR)

spectroscopy is a commonly used technique.4 FTIR can differentiate between hexagonal and

cubic boron nitride phases. The sp2 bonding in h-BN gives rise to two characteristic lattice

vibrational modes which are infrared-active: E1u, corresponding to in-plane stretching between B

and N atoms, and A2u, corresponding to out-of-plane stretching between B and N atoms. In c-

BN, the sp3 bonding results in one infrared-active mode, F2. The frequencies of the modes for h-

BN and c-BN are summarized in Table 4.1 below.5

40

Table 4.1 Infrared modes of h-BN and c-BN

Material Infrared Mode Wavenumber (cm-1

)

h-BN E1u (TO) 1367

E1u (LO) 1510

A2u (TO) 767

A2u (LO) 783

c-BN F2 1045


4.3.1 Synthesis

Boron nitride nanotubes were synthesized by two methods, as described in Chapter 3. Briefly, a

silica-assisted chemical vapour deposition technique (SA-CVD) was used to produce two types

of BNNTs. Bamboo morphology tubes were grown at a temperature of 1100 oC, while multiwall

BNNTs (MWBNNTs) were grown at 1150 oC. Following synthesis, the products were purified

by HNO3 and HF treatment. MWBNNTs were also synthesized using a growth-vapour-trapping

chemical vapour deposition technique (GVT-CVD).

4.3.2 Electron Microscopy

BNNT samples were dispersed in DI H2O or ethanol via sonication for ~30 min. Drops of the

BNNT suspension were placed on Si substrates or lacy C-coated TEM grids and allowed to dry.

SEM images were acquired with accelerating voltages of 10 – 15 kV, and currents of 20 – 30 μA

(S-5200, Hitachi, Japan). STEM images were obtained at 200 kV and ~40 μA emission current

(HD-2000, Hitachi, Japan).

41

4.3.3 Energy Dispersive X-ray

EDX spectra and element maps were obtained with accelerating voltages of 10-15 kV (Inca

System, Oxford Instruments, United Kingdom).

4.3.4 Fourier Transform Infrared Spectroscopy

SA-CVD BNNT samples were dispersed in KBr powder and pellets were made. Transmission

FTIR was performed on the pellets (Spectrum BX, Perkin Elmer, MA). GVT-CVD BNNTs and

h-BN nanoparticles (< 100 nm diameter, MKNano, Canada) were dispersed in ethanol via

sonication, and deposited onto a Ge attenuated total internal reflection (ATR) crystal. The

solvent was allowed to dry, and then spectra were obtained in the ATR configuration (Spectrum

BX, Perkin Elmer, MA).

4.4 Results

4.4.1 Scanning Transmission Electron Microscopy

STEM images obtained in Z-contrast mode are shown in Figure 4.1, showing two nanotubes

produced by Method 1. Figure 4.1a shows a ~60 nm diameter straight-wall BNNT several

microns long. A magnified image of the root of the nanoparticle, attached to a round catalyst

particle, is shown in Figure 4.1b. The tip of the nanotube in Figure 4.1a in shown in greater

detail in Figure 4.1c. Two bamboo segments can be seen to be attached to the straight-wall

portion of the nanotube. Figure 4.1d shows a ~1 μm long fragment of a bamboo BNNT with a

diameter of ~100 nm.

A low resolution TEM image of BNNTs produced by GVT-CVD is shown in Figure 4.2a. The

nanotubes are hollow, with diameters ranging from 30 – 60 nm. Periodic dark spots are visible

on the nanotube walls. A high resolution TEM image of one such dark spot is shown in Figure

4.2b. This particular tube has ~23 walls. The corresponding fast Fourier transform is given in

Figure 4.2c. The two main diffraction spots correspond to [0002] reflections in h-BN, and

indicate an inter-wall spacing of 0.34 nm.6

42

Figure 4.1 Scanning transmission electron microscopy images of BNNTs produced by SA-CVD. (a) Nanotube with catalyst particle attached at root. (b) Higher magnification image of catalyst particle in (a). (c) Higher magnification

image of nanotube tip in (a). (d) Broken bamboo morphology nanotube.

Figure 4.2 Transmission electron microscopy images of BNNTs produced by GVT-CVD. (a) Low-resolution TEM image showing hollow nanotubes with periodic dark spots within their walls. (b) High resolution TEM image of a dark

spot from (a). (c) Fast Fourier transform of nanotube wall from (b).

43

4.4.2 EDX Characterization

The EDX spectrum for SA-CVD MWBNNTs prior to HF purification is given in Figure 4.3a.

The dominant peaks are O, Al, and Si, with Si having the highest number of counts due to the Si

substrate upon which the MWBNNTs are deposited. These EDX signals indicate the presence of

Al2O3 and SiO2, precursor materials which can be removed by HF treatment. Figure 4.3b shows

an expanded view of the spectrum below 1 keV, in which B, N, and O signals are present. The

sample region from which the EDX spectrum was collected is indicated by the pink box in

Figure 4.3c.

Figure 4.3 (a) EDX spectrum of SA-CVD BNNTs on Si substrate prior to HF purification. (b) Low energy region of spectrum from (a). (b) SEM image indicating the area over which the EDX spectrum was collected (pink box).

44

A bamboo BNNT sample produced via SA-CVD was analyzed with EDX following HF

purification. The spectrum shown in Figure 4.4a shows B, N, O, and Si signals (again, the Si

signal is largely due to the substrate). No Al is apparent, and the O signal is lower than the N

signal, indicating that the majority of the oxide precursors have been eliminated via HF

treatment. An expanded view of the B, N, and O peaks is given in Figure 4.4b, and the sample

area from which the measurement was taken is indicated by the pink box in Figure 4.4c.

Figure 4.4 EDX spectrum of SA-CVD Bamboo BNNTs (after HF purification) on Si substrate.

In order to obtain spatially correlated elemental composition, EDX element maps were acquired.

Results for unpurified MWBNNTs produced via GVT-CVD, are shown in Figure 4.5 below,

45

with an SEM image of the region of interest presented at the top of the figure, followed by

individual EDX element maps. These MWBNNTs were deposited on a lacy C coated TEM grid.

The nanotube features are clearly visible in the B and N maps. Some detector overlap between

the B and C x-ray lines appears to cause some nanotube features to be visible in the C map. The

O, Mg, and Fe maps show that the material in the upper left corner of the SEM image is

comprised of residual precursors, namely MgO and Fe (likely in Fe3O4 form, see previous

chapter for details the of synthetic redox reactions)

Figure 4.5 SEM image of GVT-CVD BNNTs on lacy C TEM grid and corresponding EDX elemental maps.

46

The corresponding EDX spectrum acquired over the area shown above in Figure 4.5 is given in

Figure 4.6a below. The dominant peaks in the spectrum are of C, O, Mg, Fe, and Cu. The high

C signal is due to the lacy C coating on the TEM grid, and the Cu signal is from the TEM grid

itself. An expanded view from 0 to 2.5 keV is shown in Figure 4.6b. No B peak can be seen due

to overlap with the C peak. The N signal is apparent, and small Al and Si peaks are also present.

Al and Si may have been introduced into the BNNT sample from the Al2O3 boat and the Si

substrate which partially covered the boat during the synthesis. The small peak at ~2.1 keV could

not be assigned to any characteristic x-ray lines.

Figure 4.6 EDX spectrum of GVT-CVD BNNTs on lacy C TEM grid.

4.4.3 FTIR Characterization

The FTIR spectra for SA-CVD bamboo BNNTs, SA-CVD MWBNNTs, GVT-CVD

MWBNNTs, and h-BN nanoparticles are given in Figure 4.7. All materials exhibit two major

bands at ~800 cm-1

and ~1375 cm-1

which is characteristic of h-BN (A2u, E1u TO). The GVT-

CVD MWBNNT sample exhibits another peak at ~1500 cm-1

, which corresponds to the E1u LO

mode. The other samples do not exhibit two distinct TO and LO modes, but a shoulder which

47

likely corresponds to the LO mode is apparent in the h-BN nanoparticle spectrum. The broad

asymmetric lineshapes of the E1u TO peaks in the SA-CVD sample spectra indicate that some

contribution from the LO mode is present.

Figure 4.7 Fourier transform infrared spectra of BNNTs produced by SA-CVD and GVT-CVD. The spectrum for <100 nm h-BN nanoparticles is shown for comparison.

4.5 Discussion

4.5.1 Nanotube Morphology and Structure

The observation of the SA-CVD MWBNNT with a catalyst particle attached at its root, and

bamboo segments at its tip (Figure 4.1a-c) supports the growth mechanism hypothesis described

in Chapter (see Figure 3.7). In the initial stage of growth, the catalyst particle could not maintain

48

a stable morphology for the growth of a straight-walled tube, and as a result, bamboo segments

were formed. The catalyst particle was eventually able to maintain an elongated shape, perhaps

due to an increase in temperature, such that the remainder of the nanotube formed a MWBNNT

morphology. At the end of the growth process, as the temperature decreased, the catalyst particle

formed a sphere once again. Note that the diameter of the catalyst particle is larger than the

diameter of the nanotube. When elongated, the particle’s diameter decreases (in the direction

perpendicular to the nanotube axis) which templates the diameter of the nanotube.

4.5.2 Defect Characterization

The peak position of the E1u (TO) mode is sensitive to defects within the h-BN lattice. As lattice

defects increase, the peak will shift to higher wavenumber. The peak positions of the BNNTs

synthesized by SA-CVD and GVT-CVD are summarized in Table 4.2 together with the peak

positions for the h-BN nanoparticles. For comparison, the peak positions for a thin film of single

crystalline h-BN are also listed.7

Table 4.2 FTIR Peak Positions for BN Nanomaterials.

BN Material/Infrared Mode E1u, TO (cm-1

) E1u, LO (cm-1

) A2u (cm-1

)

Bamboo BNNTs (SA-CVD) 1384 - 797

MWBNNTs (SA-CVD) 1381 - 798

MWBNNTs (GVT-CVD) 1367 1513 805/819

h-BN nanoparticles 1377 ~1480 shoulder 783

Single crystal h-BN thin film

(from Shi et al.7)

1370 - 823

Based on the E1u, TO peak position, the bamboo BNNTs have the most defective structure,

followed by the SA-CVD MWBNNTs and the h-BN nanoparticles. The GVT-CVD MWBNNTs

appear to be highly crystalline, with a peak position of 1367 cm-1

. The narrow band width and

the distinctly separate LO peak at 1510 cm-1

are also strong indications of the high crystallinity

of the GVT-CVD MWBNNT sample.

49

4.6 Conclusions

The morphology of the BNNTs produced by SA-CVD and GVT-CVD were investigated with

SEM. EDX is a useful tool for detecting elements of interest within the material, and correlating

the elemental composition with nanostructures. The crystal structure of GVT-CVD BNNTs was

investigated with HR-TEM, and confirmed the tubular h-BN nature of the nanotubes. FTIR was

employed in order to provide bulk characterization of the chemical composition of the BNNTs.

4.7 References

1. Verhoeven, J. D., Scanning Electron Microscopy. In ASM Handbook, Volume 10 -

Materials Characterization, Whan, R. E., Ed. ASM International: 1986; pp 490–515.

2. Romig, A. D. J., Analytical Transmission Electron Microscopy. In ASM Handbook,

Volume 10 - Materials Characterization, Whan, R. E., Ed. ASM International: 1986; pp

429–489.

3. Heinrich, K. F. J.; Newbury, D. E., Electron Probe X-Ray Microanalysis. In ASM

Handbook, Volume 10 - Materials Characterization, Whan, R. E., Ed. ASM

International: 1986; pp 516–535.

4. Marcott, C., Infrared Spectroscopy. In ASM Handbook, Volume 10 - Materials

Characterization, Whan, R. E., Ed. ASM International: 1986; pp 109–125.

5. Geick, R.; Perry, C. H.; Rupprech.G, Normal modes in hexagonal boron nitride. Phys.

Rev. 1966, 146, 543–547.

6. Terauchi, M.; Tanaka, M.; Matsuda, H.; Takeda, M.; Kimura, K., Helical nanotubes of

hexagonal boron nitride. J. Electron Microsc.1997, 46, 75–78.

7. Shi, Y.; Hamsen, C.; Jia, X.; Kim, K. K.; Reina, A.; Hofmann, M.; Hsu, A. L.; Zhang, K.;

Li, H.; Juang, Z.-Y.; Dresselhaus, M. S.; Li, L.-J.; Kong, J., Synthesis of few-layer

hexagonal boron nitride thin film by chemical vapor deposition. Nano. Lett. 2010, 10,

4134–4139.

Figure 5. sds

50

5 Diameter-Dependent Bending Modulus of Individual Multiwall Boron Nitride Nanotubes

5.1 Abstract

The mechanical properties of individual multiwall boron nitride nanotubes (MWBNNTs)

synthesized by a growth-vapor-trapping chemical vapor deposition method are investigated by a

three-point bending technique via atomic force microscopy. Multiple locations on suspended

tubes are probed in order to determine the boundary conditions of the supported tube ends. The

bending moduli (EB) calculated for 20 tubes with diameters ranging from 18 to 55 nm confirm

the exceptional mechanical properties of MWBNNTs, with an average EB of 760 ±30 GPa. For

the first time, the bending moduli of MWBNNTs are observed to increase with decreasing

diameter, ranging from 100 ± 20 GPa to as high as 1800 ± 300 GPa. This diameter dependence is

evaluated by Timoshenko beam theory. The Young’s modulus and shear modulus were

determined to be 1800 ± 300 GPa and 7 ± 1 GPa, respectively for a trimmed data set of 16 tubes.

The low shear modulus of MWBNNTs is the reason for the detected diameter-dependent

bending modulus and is likely due to the presence of inter-wall shearing between the crystalline

and faceted helical nanotube structures of MWBNNTs.

5.2 Introduction

Boron nitride nanotubes (BNNTs), first predicted in 19941,2

and synthesized in 1995,3 have

attracted increasing attention in recent years due to their unusual properties. Although

structurally similar to carbon nanotubes (CNTs), BNNTs have significantly different optical and

electronic properties. BNNTs are much more insulating than CNTs, with a band gap of 5 – 6 eV

which is largely independent of tube chirality or diameter.2 Theoretical studies have indicated

that the axial Young’s modulus of single wall BNNTs (SWBNNTs) is of the same order as that

of carbon nanotubes (~1 TPa).4,5

BNNTs’ mechanical properties, together with their high

aspect ratio, high thermal conductivity,6 optical transparency, electrically insulating character,

and high resistance to oxidation (up to 1100oC)

7 make them ideal fillers for technologically

relevant composite materials such as seals and encapsulants,8-11

and biomaterials.12

In addition,

BNNTs show promise for a diverse range of other applications, including hydrogen storage,13,14

targeted drug delivery,15

and optoelectronic devices such as lasers and light emitting diodes.16,17

51

For BNNTs to be successfully employed in the aforementioned applications, a better

understanding of their mechanical properties is required. This is particularly important for

applications which rely on the mechanical properties of individual tubes, such as resonators and

sensors,18

and microtubule mimics.19

In contrast to CNTs, only a handful of experimental

studies have been conducted on BNNTs to determine their Young’s modulus. Chopra and Zettl20

used the resonance technique of Treacy et al.21

to determine that an arc-discharge multiwall

BNNT (MWBNNT) (3.5 nm outer diameter) had a modulus of ~1.22 TPa. Electric-field-

induced resonance experiments by Suryavanshi et al.22

yielded moduli of 505 – 1031 GPa for a

set of 18 tubes, with outer diameters ranging from 34 – 94 nm. Golberg et al.23

determined

moduli of 0.5 – 0.6 TPa (40 nm and 100 nm outer diameter tubes) via in-situ bending

experiments using an atomic force microscope (AFM) set-up within a transmission electron

microscope (TEM). Using a similar set-up, Ghassemi et al.24

measured 5 MWBNNTs with outer

diameters of 38 – 51 nm, and found that the average modulus was ~0.5 TPa. Depending on the

choice of shell thickness, the Young’s modulus of a 1.9 nm diameter SWBNNT was found to

range from 0.87 to 1.11 TPa.25

The wide range of moduli observed indicates a need for further

study in order to elucidate the influence of factors such as synthesis technique, nanotube

structure, and morphology on the Young’s modulus.

Three-point bending tests conducted with AFM have been used to characterize the modulus of a

variety of high aspect ratio structures, including CNTs,26-28

nanowires,29,30

and electrospun

polymers.31

Typically, the nanotubes or wires are deposited onto a stiff substrate with a

topographical pattern, such as polished porous Al2O3 membranes or Si gratings patterned with

trenches. The tubes occasionally lie over pores or trenches, and the midpoint of the suspended

portion is subjected to a downward force applied by the AFM tip. Force-displacement curves are

obtained, and the bending modulus can be calculated directly from the slope of the force curve

together with the geometrical parameters of the tube’s diameter and suspended length. In most

studies, the supported beam ends are assumed to have clamped boundary conditions due to the

adhesion between the nanomaterial and the substrate. However, this assumption can be

unfounded and can be a source of systematic error in the determination of the bending modulus.

Other beam end boundary conditions include simply supported and mixed support in which one

end is clamped, and the other end is simply supported. Depending on the support conditions, the

solution of the Euler-Bernoulli beam equation takes on different forms, yielding different

52

expressions for the bending modulus. The appropriate boundary conditions for an individual

tube can be determined if multiple locations along the length of the suspended tube are probed.

This allows for a more accurate determination of the modulus value, as demonstrated by

Shanmugham et al.,32

Chen et al.,33

Kluge et al.,34

and Gangadean et al.35

In this study, we use AFM to measure the bending modulus Eb of MWBNNTs synthesized by a

growth-vapor-trapping chemical vapor deposition (GVT-CVD) technique.36

A force mapping

technique is used in order to collect force curves from various locations along the length of the

suspended tube. We show that for our sample the majority of tubes possess simply supported

ends instead of clamped ends. Based on these boundary conditions, we calculate the bending

moduli for tubes of various diameters, and we present a discussion about the diameter

dependence that is observed.


5.3.1 MWBNNT Synthesis

MWBNNTs were synthesized via the growth vapor trapping chemical vapor deposition

technique previously described by Lee and coworkers.36

The MWBNNTs were collected on Si

substrates and sonicated in ethanol to form a MWBNNT suspension.

5.3.2 Chemical and Structural Characterization

MWBNNTs were synthesized via the growth vapor trapping chemical vapor deposition

technique previously described by Lee and coworkers.36

The MWBNNTs were collected on Si

substrates and sonicated in ethanol to form a MWBNNT suspension.

The MWBNNTs were characterized with scanning electron microscopy (SEM), low and high

resolution transmission electron microscopy (TEM, HR-TEM), and Fourier transform infrared

spectroscopy (FTIR). The morphology of the as-synthesized MWBNNTs was characterized with

SEM (S-4700, Hitachi, Japan). For the TEM measurements, the MWBNNT suspension was

dropped onto a holey carbon TEM grid and allowed to dry. Bright-field low resolution TEM

images were acquired at 30 kV, 17.5 μA emission current (S-5200, Hitachi, Japan). Bright-field

HR-TEM images were acquired at 200kV, 39 μA emission current (HD-2000, Hitachi, Japan).

FTIR spectra were taken using an attenuated total internal reflection (ATR) set-up. The

53

MWBNNT suspension was dropped onto a ZnSe ATR crystal and allowed to dry. For

comparison purposes, hexagonal boron nitride (h-BN) nanoparticles (MK-hBN-N70, MK Impex

Canada, Mississauga, Ontario) were also characterized by FTIR. Spectra were recorded on an

FTIR spectrometer (Spectrum BX, Perkin-Elmer, Waltham, MA) at a resolution of 1 cm-1

.

5.3.3 Sample Preparation and AFM Measurements

For the AFM sample preparation, the MWBNNT suspension was dropped onto clean Si

substrates patterned with trenches 400 nm wide and 200 nm deep (LightSmyth Technologies,

Eugene OR) and was allowed to dry. AFM height images of the tubes on the patterned substrate

were acquired in air under ambient conditions using AC (intermittent contact) mode (MFP-3D,

Asylum Research, Santa Barbara CA). Si probes (NCH, Nano World, Neuchâtel Switzerland)

with tip radii of ~20 nm were used. The optical lever sensitivity of the cantilevers was calibrated

by acquiring force curves in contact mode on a clean Si substrate. The spring constant of each

cantilever used was determined by the thermal method and found to range from 33 to 46 N/m.37

A discussion of the applicability of the thermal method for high spring constant cantilevers is

presented in the Supporting Information. AFM force maps (typically 2 μm x 0.5 μm with 32 x

16 points) were obtained of MWBNNTs spanning trenches. A force curve (applied force F

versus tip-sample separation) was collected at each point on the map. The force curves

corresponding to the points along the suspended portion of the tube (as determined from the

height map and the force curves themselves) were analyzed to extract the effective tube stiffness,

keff, by a linear fit to the slope of the force curve. A more detailed description of the force

mapping method is given in the Supporting Information.

The suspended length L for a given tube was determined from the AFM height map as well as

from higher-resolution AFM height images acquired in tapping mode. The lateral dimension of

the pixels making up the force map was used to estimate the errors associated with the values of

position (a, b) and suspended length. The tube diameter was determined by the height of the

tube on the substrate from the tapping mode height images.

Equation 5

54

5.4 Results and Discussion

5.4.1 Characterization of MWBNNTs

Electron microscopy images of the MWBNNTs produced by the GVT-CVD method are shown

in Figure 5.1. The scanning electron microscope (SEM) image in Figure 1a shows straight fibers

with diameters ranging from ~15 to 60 nm. Figure 5.1b depicts a low resolution bright field

TEM image of the as-synthesized MWBNNTs, and confirms the hollow tubular nature of the

fibers. A high resolution TEM image of a dark region in a tube wall is shown in Figure 5.1c.

The layers appear crystalline with an interlayer spacing of ~0.34 nm, as determined from the

(002) diffraction spots in the fast Fourier transform for this region (not shown). This spacing is

consistent with the crystal structure of hexagonal boron nitride and BNNTs.24,36

The FTIR spectrum of the MWBNNTs is shown in Figure 5.2. For comparison, the spectrum for

commercially available hexagonal BN (h-BN) nanoparticles is also given. The MWBNNT

spectrum exhibits peaks at ~1368 cm-1

and ~1510 cm-1

, which correspond to the in-plane

transverse optical (TO) and longitudinal optical (LO) E1u modes of h-BN. The TO E1u mode is a

stretching mode along the tube axis, while the LO E1u mode is a stretching mode along the tube

circumference. A weak feature at around 800 cm-1

is shown enlarged in the inset of Figure 5.2.

A shoulder is visible at ~819 cm-1

, and a peak at ~806 cm-1

. These spectral features correspond

to the out-of-plane TO and LO A2u modes of h-BN. 24,38

5.4.2 AFM Three-Point Bending

In three-point bending experiments, slender wires can be modeled as an elastic string (pure

stretching), a stiff beam (pure bending), or a combination of the two. Heidelberg et al.39

presented a generalized approximation for these behaviors, in which a force F is applied to the

mid-point of the suspended wire and the wire ends are assumed to be clamped:

Equation 5.1 ) 24I

A+(1

L

I192E=F 2

centercenter3

Bcenter

In the above expression, δ is the deflection of the wire, EB is the bending modulus, A is the cross

sectional area of the wire, and I is the second moment of area. At small displacements, the wire

undergoes pure bending which is described by the first linear term. At large displacements,

55

Figure 5.1 (a) SEM image of MWBNNTs. Inset: Higher magnification SEM image showing straight, slender fibres 15 – 60 nm in diameter. (b) Low resolution TEM image of MWBNNTs. (c) High resolution TEM image of a MWBNNT

wall near a typical dark spot shown in (b).

56

Figure 5.2 FTIR spectrum (blue line) of MWBNNTs. FTIR spectrum of hBN nanopowders (red line) is also shown for comparison.

axial tensile stresses are induced as the wire stretches which are described by the cubic second

term (F δ3). In this study, only pure bending is considered because the experiments are

conducted within the small deflection regime, in which the maximum deflection does not exceed

the radius of the wire.

To model the MWBNNTs in this study as stiff beams undergoing pure bending, Euler-Bernoulli

beam theory was employed. It should be noted that this theory assumes a homogeneous isotropic

material, which is not the case for multiwall nanotubes. Nevertheless, simulations indicate that

this approximation offers an adequate description of nanotube bending mechanics prior to

buckling.40

As a result, this approach has been widely used in AFM bending experiments on

nanowires and nanotubes.26-29,31

Unique solutions to the beam equation depend on the boundary

conditions of the beam ends, which can be considered to be clamped (no deflection or slope at

beam end), or simply supported (no deflection or bending moment at beam end). Beam

schematics are presented in Figure 5.3, which summarize the three models considered in this

work: simply supported beam model (a, SSBM), double clamped beam model (b, DCBM), and

mixed support beam model (c, MSBM).41

57

Figure 5.3 Beam schematics describing beam bending boundary conditions.

The corresponding equations are as follows:

Equation 5.2

22

3

ba

ILEF B

(SSBM)

Equation 5.3

33

33

ba

IELF B

(DCBM)

Equation 5.4

aLba

IELF B

3

1232

3

(MSBM)

In these equations, L is the suspended length of the beam, and a and b are the suspended lengths

on both sides of the applied force F, where a + b = L. I, the second moment of area, is taken to

be I = πD4/64 which is defined for a solid cylindrical wire with a circular cross section, where D

is the diameter. Hence, in this approximation of the multiwall nanotube beam, only the outer

diameter is taken into account and not the inner diameter. In order to determine the appropriate

boundary conditions for each tube, the AFM tip is used to apply a force at different positions

along the suspended tube, not just at the midpoint. Therefore, AFM force curves (plots of F

versus the tip-sample separation (= beam deflection, δ)) are collected at multiple locations along

the tube. The linear slope of a force curve directly yields the effective tube stiffness, keff = F/ δ.

The boundary conditions for the tube are determined by plotting keff versus the position along the

58

tube (a/L) and performing fits to the various beam models (Equations 2-4). The bending

modulus EB is then determined using the appropriate beam model.

Figure 5.4 shows SEM (a) and AFM images (b-d) of suspended MWBNNTs on patterned Si

trenches (400 nm wide and 200 nm deep). An AFM height image of a typical MWBNNT

spanning a trench is shown in Figure 5.4b. The height image is subsequently divided up into

pixels (typically 32 x 16 or 64 x 32) by the AFM software, and force curves are collected at each

point (pixel) during the force mapping procedure. The corresponding AFM height map image

illustrating the spatial location (x,y) of each of the force measurements is shown in Figure 5.4c.

The height in each pixel is determined from the Z range distance at which the tip first engages

the sample during the extend portion of the force curve. Figure 5.4d shows the AFM height

image of the MWBNNT after the force map was performed, and its similarity to Figure 5.4b

indicates that the tube did not shift or deform as a result of the force measurements. Typical

force curves collected from different locations on a MWBNNT are shown in Figure 5.5. The red

dotted line corresponds to a force curve obtained from a location where the tube is supported by

the Si substrate (red ‘x’ in Figure 5.4c, illustrative purpose only), while the blue solid line

corresponds to a force curve obtained from a position where the tube is suspended over a trench

(blue ‘x’ in Figure 5.4c, illustrative purpose only). The slope of the blue solid line in Figure 5.5

is equivalent to keff.

Plots of the effective tube stiffness (keff) versus position along the suspended tube (a/L) fitted

with Equations (1) to (3) corresponding to SSBM, DCBM, and MSBM are given in Figure 5.6.

The values of keff located near the ends (a/L < 0.2, a/L > 0.8) of the suspended tubes were not

included in the fits, because of the large error associated with fitting force curves with large

slopes (theoretically, at the ends, keff approaches infinity). Figure 5.6a shows that SSBM fits the

data better than DCBM signifying that the MWBNNT is an example of a simply supported tube.

On the other hand, data in Figure 5.6b fits the MSBM model well and is therefore an example of

a tube which is fixed on one end (its left side) and simply supported on the opposite end (its right

side). Despite the fact that all of the tubes examined were on the same sample, various support

conditions were observed which demonstrates the importance of determining the boundary

conditions for each individual tube.

59

Figure 5.4 (a) SEM image of MWBNNTs on patterned Si substrate. (b) AFM height image before force mapping was performed. (c) Height map image corresponding to a force map acquired at a deflection trigger of 1 nm. (d) AFM

height image after force map was acquired.

Figure 5.5 (Red dotted line) Force curve obtained from a point on a tube supported by substrate. (Blue solid line) Force curve obtained from a point on a tube suspended over a trench.

60

Figure 5.6 Tube effective stiffness (keff) vs. position along suspended tube (a/L). (a) A simply supported tube. (b) A

mixed support tube, with left side fixed and right side simply supported.

5.4.3 Elastic Properties of MWBNNTs

5.4.3.1 Bending Modulus

The bending moduli EB determined for 20 tubes with diameters ranging from 18 – 55 nm are

shown in Figure 5.7. EB ranged from 100 ± 20 to 1800 ± 300 GPa, with an average of 760 ± 30

GPa. The error in EB was determined via error propagation, using an error of 10% for the tube

61

outer diameter D, half the lateral pixel width in the force map for the tube length L and lengths

on each side of the loading position (a, b), and an error of 20% for effective spring constant of

the tube keff.

It is worth noting that there is the possibility that the calculated bending moduli may be

underestimated due to inaccurate assumptions about the cross sectional geometry. The

nanotubes were approximated to be solid wires, with a solid circular cross section. This model

was chosen because it was not possible for us to determine the inner diameter of the nanotubes

we probed with AFM, given that we deposited the tubes on a substrate that is not amenable to

TEM analysis. A more accurate model would be a hollow cylinder, with an inner diameter Di

and an outer diameter Do, which results in a second moment of area expression of I = π/64(Do4 –

Di4). Modeling the tube as a solid wire as opposed to a hollow cylinder underestimates the

modulus; however, in the most extreme case (i.e. very large diameter tube with only a few walls)

the underestimation is on the order of 30%. Based on the range of Di/Do ratios observed in

TEM data for 24 tubes in the same MWBNNT production batch as the tubes used in the bending

experiments (presented in the Supporting Information), the underestimation is closer to 10% for

our particular sample.

From the plot of EB versus tube outer diameter shown in Figure 5.7, it is evident that there is a

decreasing trend for the bending modulus with increasing tube diameter. For both CNTs and

BNNTs, the Young’s modulus should theoretically approach an upper limit defined by the in-

plane elastic constant of graphite and h-BN, respectively.4,42

For h-BN single crystals, this

constant was measured to be c11 = 811 GPa,43

while for single crystal graphite, c11 = 1109

GPa.4,42

These measurements correlate well with various theoretical calculations.44-46

This limit

is expected to apply to MWBNNTs as well, because the modulus depends mainly on intra-wall

bonds. Simulations suggest that the Young’s modulus of a MWCNT is slightly higher than that

of a SWCNT, for the same outer diameter, due to the effect of inter-wall van der Waals forces in

MWCNTs.47

The average EB of the MWBNNTs studied in the present investigation matches the

c11 elastic constant of h-BN quite closely. However, the origin of the wide range of bending

moduli and the diameter dependence requires further analysis.

62

Figure 5.7 Bending modulus vs. tube outer diameter. The beam model used for calculating EB is denoted by black

(SSBM), blue (MSBM), and red (DCBM).

5.4.3.2 Investigation of Diameter Dependence: Shear Effects

In experimental studies of multiwall nanotubes a wide range of modulus values has been

measured. For MWCNTs, Treacy et al.21

were first to show that CNTs have Young’s moduli in

the TPa range, using a thermal excitation method. They found that arc-discharge MWCNTs with

outer diameters ranging from 5.4 nm to 24.8 nm had Young’s moduli of 0.4 to 4.15 TPa. A

number of other studies have also produced Young’s moduli in the TPa range, for arc-discharge

MWCNTs.26,27,48,49

Within these studies, despite the focus on the ~1 TPa measurements as

validation of the superior mechanical properties of CNTs, there are many instances of tubes with

lower moduli, on the order of tens to hundreds of GPa. In the work of Salvetat et al.27

catalytic

CVD MWCNTs were also studied and found to have an average modulus of 27 GPa, which is

dramatically lower than the average modulus of 810 GPa measured for arc-discharge MWCNTs.

63

Additional studies also observe lower moduli for catalytic CVD and pyrolytic MWCNTs, in

certain cases as low as tens of GPa.28,50-52

Typically, catalytic CVD and pyrolysis synthesis

methods produce tubes with defective structures compared with the highly crystalline tubes

synthesized by arc-discharge. While point defects do not affect the modulus by more than a few

percent,53

extended defects can cause the modulus to drop by as much as two orders of

magnitude.27,51

In some studies, within sample sets of nanotubes produced under the same conditions, the

modulus is observed to drop with increasing tube diameter. This diameter dependence can be

attributed to three possibilities; namely, the probing of an elastic rippling mode in bending

experiments,49

the presence of defects,28,52

or shear effects. Due to the linearity of the force

curves obtained in the present study, it is unlikely that rippling modes are the cause of the low

moduli measurements observed for larger tubes. Although it was not rare to acquire force curves

which exhibited kinks, potentially due to tube buckling or tip slipping events, fits were only

made to the initial linear portion of the force curves (for deflections less than 10 nm) after

contact. In terms of defects, the low resolution TEM image (Figure 5.1b) shows long, straight

nanotubes with uniform diameters. The high resolution TEM image (Figure 5.1c) shows that the

dark spots present in the tube walls in Figure 1b are crystalline. The MWBNNTs do not appear

to exhibit the type of pronounced structural defects that were found to affect the modulus of

catalytic or pyrolytic MWCNTs, as discussed above. In beam bending experiments, shear must

always be considered for short, stocky beams – those which have a length-to-diameter ratio L/D

< 10. The length-to-diameter ratio L/D was measured to be greater than 10 for all tubes in this

study, which indicates that if shear effects are present, they are not a result of the experimental

geometry. Rather, they can be an indication of a material’s anisotropy.28,54

If shear effects are present, then the bending modulus is not equivalent to the Young’s modulus.

In order to determine whether the Young’s modulus of the MWBNNTs is diameter dependent,

the contribution of shear deflection to the total deflection in the bending experiment must be

quantified. This approach follows Salvetat and coworkers’ bending and shear analysis of single-

wall CNT ropes.55

64

The bending modulus is related to the Young’s modulus EY and the shear modulus G using the

following relationship, determined by Timoshenko beam theory:28,55-57

Equation 5.5

2

211

L

D

G

f

EE

s

YB

In this expression, fs is a shape factor which has a value of 10/9 for a cylindrical beam, and γ is a

shear term coefficient with values of 3, 1.715, and 0.75 for DCBM, MSBM, and SSBM

respectively. The Timoshenko beam theory converges to the Euler-Bernoulli beam theory when

the beam is rigid in shear (G ∞). In this case, the bending modulus is equal to the Young

modulus and is not diameter dependent (which is not the case here). EY and G in our case can be

estimated by plotting 1/EB against (D2/L

2), as shown in Figure 5.8. A linear fit weighted by the

error in 1/EB was obtained for a trimmed data set of 16 tubes. The shear coefficient was taken as

γ = 1.152, determined by the number of tubes exhibiting each type of boundary condition (16

tubes total = 12 simply supported tubes + 2 mixed support tubes + 2 doubly clamped tubes). EY

and G were determined to be 1800 ± 300 GPa and 7 ± 1 GPa, respectively. The expected shear

modulus for a MWBNNT should be on the order of several hundred GPa, based on the

calculations for MWCNTs47,58

which find that GMWCNT ~ 500 GPa. This value is on the order of

the intralayer shear modulus. However, the value of G that we determined for MWBNNTs is

much lower than this, and is close to the value of the c44 elastic constant of h-BN, c44 = 7.7 ± 5,

measured by Bosak et al.43

This elastic constant is equivalent to the interlayer shear modulus of

h-BN, and describes the shear between basal planes. In the case of the MWBNNT structure this

corresponds to shearing between tube walls, which can only occur if there are discontinuities due

to the presence of extended defects within the tube walls.

Although no extensive defects are apparent from the TEM characterization of the MWBNNTs,

as discussed above, the dark spots within the tube walls in the low resolution TEM image (Figure

5.1b) and their somewhat regular pattern within a given tube warrant additional consideration. A

detailed electron diffraction study by Celik-Aktas et al.59

determined that the dark spots can be

attributed to a helical nanotube structure in which the tube is comprised of two or more helices

(each comprised of multiple walls) which wrap to form the entire nanotube. In this structure, the

dark spots correspond to a strongly diffracting helix, which is locally highly crystalline. The

highly crystalline regions are joined together by line defects which result in a faceted helix.

65

Figure 5.8 Determination of the Young’s modulus and shear modulus via a fit to plot of 1/EB vs (D/L)2. The beam

model used for calculating 1/EB is denoted by black (SSBM), blue (MSBM), and red (DCBM).

The lighter regions of the tube wall form the other helix, which possesses the conventional

nested coaxial cylindrical structure expected for multiwall nanotubes. Based on this multi-helix

nanotube structure, it is conceivable that the line defects within the faceted helix as well as the

interface between faceted and cylindrical helices make inter-wall shearing a possibility.

Therefore, as our analysis of the bending data suggests, shear cannot be ignored in the

calculation of the elastic modulus, and shear effects arise from nanotube anisotropy (G << EY)

and the presence of defects within the nanotubes, and not from the experimental geometry.

Our finding that the shear modulus of MWBNNTs is orders of magnitude smaller than the

Young’s modulus indicates that the existing theoretical models are not sufficient in predicting

the mechanical properties of such extremely anisotropic structures, particularly when structural

66

defects are present.47

Experiments performed on MWCNTs support this assertion. Guhados et

al.60

determined that EY = 350 ± 110 GPa and G = 1.4 ± 0.3 GPa for 13 MWCNTs grown by a

CVD method, while Wei et al.61

found that EY ranged from 300 – 900 GPa while G ranged from

30 to 800 MPa, for a sample of 8 tubes. Both studies attribute the low shear modulus to defects

in the structure of the nanotubes. There are several possible benefits of having a low shear

modulus: (1) Taking advantage of its high melting temperature, the shear modulus of

MWBNNTs cast within metals or ceramics would enable damping of vibrations. This could

result in quieter, more durable materials.62-64

(2) Local distortions allowed due to the low shear

modulus could enable MWBNNTs to adapt to local structure variations while maintaining

rigidity on long length scales (longitudinal distortions), imparting toughness to otherwise brittle

composite materials.48

(3) The mutual compensation of shear modulus and Young’s modulus,

where by tubes of different diameters have similar bending stiffness could allow for lower purity

BNNT materials in BNNT coated interfaces for release applications.65,66

(4) With a shear

modulus on the order of the value for h-BN, MWBNNTs can be used as a high-temperature solid

lubricant additive in industrially relevant composites.67

The nanotube structures would have the

added advantages of enabling more efficient heat transport on longitudinal length scales,6 and

increasing the wear resistance of the composite due to reinforcement of the matrix.68

5.5 Conclusion

The bending modulus of individual multiwall boron nitride nanotubes (MWBNNTs) was

measured via AFM bending experiments. Boundary conditions for the beam bending model

were determined by using a force mapping technique. MWBNNTs were found to have excellent

mechanical properties, with an average bending modulus of 760 ± 30 GPa, which is consistent

with the theoretically predicted value for BNNTs. Shear effects were found to be non-negligible,

and the Young’s modulus and shear modulus were determined to be 1800 ± 300 GPa and 7 ± 1

GPa, respectively. The experimental geometry and the dimensions of the nanotubes were not

major contributors to the shear effects; rather, it is likely that inter-wall shearing occurred

between crystalline and faceted cylindrical helices in these MWBNNTs.

67

5.6 Contribution

The author performed the majority of the work described above. A.E.T. carried out the

characterization and AFM measurements and undertook the data analysis to determine the elastic

properties of the MWBNNTs. A.E.T. was the first author on a paper based on the above

submitted to a refereed journal for publication. The MWBNNTs were provided by coauthors J.

Wang and Y.K. Yap, and the preliminary AFM experiments upon which the above work was

based were performed by coauthors A.L.M. Reddy and D. N. Lamont.

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Equation 6 fg

Table 5. sdf

Table 6. sd

72

6 Insights into the composition, morphology, and formation of the calcareous shell of the serpulid Hydroides dianthus

6.1 Permissions

The material in this chapter is presented with permission from Tanur, A. E.; Gunari, N.; Sullan,

R. M. A.; Kavanagh, C. J.; Walker, G. C. J. Struct. Biol. 2010 169, 145-160. Copyright 2009

Elsevier Inc.

6.2 Abstract

To date, the calcareous tubes of serpulid marine worms have not been studied extensively in a

biomineralization context. The structure and composition of the tube shell and adhesive cement

of the marine tubeworm Hydroides dianthus were studied using a variety of characterization

techniques, including powder XRD, FTIR, SEM, EDX, and AFM. The tube and cement were

determined to be inorganic-organic composite materials, consisting of inorganic aragonite

(CaCO3) and Mg-calcite ((Ca0.8,Mg0.2)CO3) crystals, and both soluble and insoluble organic

matrices (SOM and IOM). SEM imaging revealed a variety of crystal morphologies. AFM

nanoindentation of the inorganic components yielded Young’s moduli of ~20 Pa in the wet

state, and ~50 GPa in the dry state. Amino acid analysis of the SOM indicated substantial

amounts of acidic and non-polar neutral amino acids. Part of the insoluble organic tube lining

was identified as being composed of collagen-containing fibres aligned in a criss-crossed

structure. The SOM and organic tube lining were found to contain carboxylated and sulphated

polysaccharides. In an artificial seawater solution, the SOM and the organic tube lining

mediated CaCO3 mineralization in vitro.

6.3 Introduction

Recently, much interest has been paid to the structure and formation of inorganic-organic

composite materials by various organisms. Biomineralization processes in nature produce

inorganic crystalline materials with exceptionally fine control over nucleation and growth

processes, and overall material organization. All of this is achieved under ambient conditions

73

largely through the use of organic macromolecules, which are also incorporated into the

crystalline structures to form the inorganic-organic composite material.1-3

Calcium carbonate, in both calcitic and meta-stable aragonitic forms, is one of the most common

and abundant biominerals and is widely used by many marine species, such as in certain

echinoderms, brachiopods, scleractinian corals, and molluscs as a structural material.4

Numerous studies have been conducted on the material structure and properties of nacre, sea

urchin spines, oyster shell folia, and coral, and models for the biomineralization processes in

each of these organisms have been proposed and developed.5-11

Unlike the aforementioned

marine invertebrates, the calcareous tube-building marine worms in the serpulid family

(Annelida, Polychaeta) have not been studied extensively with regards to CaCO3

biomineralization. Serpulids are sessile animals that spend their adult lives within tubes they

have built, which are permanently attached to a substrate (Figure 6.1). The thin material which

is in contact with the substrate is referred to in this paper as the adhesive material, and the thicker

cylindrical portion is referred to as the tube shell. The tubes are composed of calcite, aragonite,

or a combination of these two CaCO3 polymorphs, although typically one polymorph is

dominant. Organic sheets have also been occasionally observed within the tube shell walls and

lining the inside of the tubes.12, 13

Figure 6.1 Overview of serpulid tube structure (Hydroides dianthus). A) Cartoon of tubeworm, transverse cross section. Thickness of adhesive material is exaggerated circa 20X, relative to the shell wall. B) Bottom view of

tubeworm, with white coloured tube walls, and light brown (grey in Figure) adhesive materials. The wider end of the tube is the head section (most recent growth), and the thinner end is the tail section (oldest part of the tube).

Ultrastructural studies of tube shell cross-sections from a number of recent and fossil serpulid

species by Vinn et al.12

indicate that roughly 75% of serpulid tubes consist of a single layer, with

74

the remaining 25% being composed of two to four layers. Layers are classified by the overall

orientation and organization of the inorganic crystals making up the tube. The current model for

serpulid tube formation involves the secretion of calcareous granules and organic components

from the calcium-secreting glands in the anterior portion of the worm, located on the

peristomium near the base of the collar. The glandular openings are within a region known as

the ventral shield, whose epithelial mucocytes secrete additional organic components. New

material is secreted and added to the anterior end of the existing tube, and moulded by the

worm’s collar.14-18

Although the calcium-secreting glands play a pivotal role in the tube

formation process, it is unclear how certain serpulids achieve layered structures within the tube,

some with very intricate organization, through this mechanism alone. It has been hypothesized

that extracellular mediation may be involved, such as in the case of molluscs, but to date the

macromolecules and their roles in the biomineralization process have not been studied in

detail.12, 19

In this paper, we identify several organic components of the calcareous tube of the serpulid

Hydroides dianthus, and provide evidence for their participation in an extracellular

biomineralization mechanism. In addition, we present a description of both the tube shell and

the adhesive material in terms of their ultrastructure, crystal polymorph and morphology, and

mechanical properties.


6.4.1 Tubeworm Collection and Preservation

Tubeworms were collected near the Sebastian inlet in the Indian River lagoon system in Florida,

USA (27 oN, 80

oW). Rectangular 10.16 x 20.32 cm (4 x 8 in) glass panels were coated with a

polydimethylsiloxane-based anti-fouling material (Sylgard 184, Dow Corning, Midland, MI) in

thicknesses ranging from 110 – 380 μm. The panels were fastened to PVC frames and

suspended from a floating raft at a depth of 1 m. The panels were submerged until they were

encrusted with a variety of marine biofoulers, including H. dianthus.

Upon removal from the lagoon, the encrusted panels were kept moist and immediately shipped

overnight to the University of Toronto on September 9th

, 2008. The panels were placed into a 45

L aquarium filled with 35 ppt artificial seawater (Instant Ocean®

Sea Salt, Marineland,

75

Cincinnati, OH) and kept at room temperature. The tubeworms were fed daily with rotifers (Bio-

Pure, Hikari, Hayward, CA). For all experiments, tubeworms were pushed off of the substrate in

shear, and the worms removed. For experiments in which the samples were required to be in a

dry state, tube shells were rinsed with deionized water and then dried in an oven set at 80 oC for

1 hour, under 30 mmHg vacuum.

6.4.2 X-Ray Diffraction (XRD)

Dry tube shells were crushed into a fine powder using a mortar and pestle. Two types of samples

were prepared, one consisting of both the shell and adhesive material from a single tube, and the

other consisting of only adhesive material. For the latter sample type, the adhesive material was

collected from 3 different worms on the same panel, and ground up separately from the rest of

the tube material. Samples were subjected to powder XRD analysis, using an automated

diffractometer (AXS D5000, Siemens/Bruker, Madison, WI) system and Cu-Kα radiation (50

kV, 35 mA). A solid-state Si/Li Kevex detector was used for the removal of Kβ lines.

Diffraction patterns were collected on a θ/2θ Bragg-Brentano reflection geometry, with fixed

slits. A step scan mode was used for data acquisition, with a step size of 0.02o 2θ, and a counting

time of 2.5 s per step. Qualitative identification of the crystalline components was performed

using the Search/MatchTM

routine, part of the data processing software EvaTM

v.8.0

(Siemens/Bruker, Madison, WI). Rietveld refinement was carried out with profile fitting

software (TopasTM

, Siemens/Bruker, Madison, WI).

6.4.3 Fourier Transform Infrared Spectroscopy (FTIR)

Powdered tube shell samples (from the same samples that were used for XRD analysis) were

dispersed in KBr pellets. Spectra were obtained with a Fourier transform infrared spectrometer

(FTIR) (Spectrum BX, Perkin-Elmer, Waltham, MA), using a resolution of 2 cm-1

. Spectra of

the organic tube lining were taken using an attenuated total reflection set-up. The soluble

organic matrix (SOM) was deposited onto a CaF2 window and allowed to dry under ambient

conditions, and its spectrum was taken in transmission mode. For the in vitro mineralization

experiments, the Au substrates were analyzed via FTIR using a reflectance set-up.

76

6.4.4 Inductively Coupled Plasma Atomic Emission Spectrometry (ICPAES) Analysis

25 mg of powdered tube shell was dissolved in 5 mL of concentrated HCl and diluted with

deionized water to 100 mL in a volumetric flask. Solutions were assayed by Inductively

Coupled Plasma Atomic Emission Spectrometry (ICP AES), on a spectrometer (Optima

3000DV, Perkin-Elmer, Waltham, MA).

6.4.5 Electron Probe Microanalysis (EPMA)

Polished cross-sectioned samples were carbon coated and subjected to electron probe

microanalysis (EPMA), using an electron microprobe instrument (SX50, Cameca, France). Ca

and Mg molar ratios were calculated and averaged from measurements at six different locations

within the tube shell cross-section.

6.4.6 Separation of the Organic Tube Lining and the Soluble Organic Matrix (SOM)

After removal of the worm from the tube, the inner organic lining of the tube was removed with

tweezers. The lining constitutes part of the insoluble organic matrix (IOM) of the tube shell

along with other organic sheets within the tube which were not extracted. After separation from

the tube shell, the organic tube lining was subjected to treatment with 0.5 M EDTA overnight.

For the extraction of the SOM, 1 mL of 0.5 M EDTA (adjusted to a pH of 8) was added to 110

mg of powdered tube shell in an eppendorf tube. The sample was vortexed for 1 min every 30

min for 6 hrs, and subsequently refrigerated at 4 oC overnight. Afterwards, the eppendorf tube

was centrifuged for 10 min at 13.5 g. The supernatant was removed and diluted with deionized

water to form a 0.05 M EDTA solution, which was then used for the AA analysis and the in vitro

mineralization experiments.

6.4.7 Scanning Electron Microscopy (SEM) and Energy Dispersive X-Ray (EDX)

For cross-sectioned samples, tube shells were embedded in a low-viscosity embedding media

(Spurr, Canemco, Quebec, Canada), sectioned, and polished. Samples were etched for 1-2 min

in 1% acetic acid. For measurements on the tube adhesive material, tube shells were fixed using

77

a 0.1M glutaraldehyde-paraformaldehyde phosphate buffer (pH 7.2) solution, serially dehydrated

using 50%, 70%, 90%, and 100% ethanol solutions, then critical point dried with CO2 using a

critical point dryer (Autosamdri 810, Tousimis Research Corporation, Rockville, MD). All

samples were mounted directly onto aluminum stubs using carbon tape and/or carbon paint, and

carbon coated. Measurements were obtained with a field emission scanning electron

microscope (SEM) (S-5200, Hitachi, Japan) equipped with an energy dispersive x-ray (EDX)

system (Inca, Oxford Instruments, United Kingdom). EDX measurements were performed at 10

kV, with a probe current of 20 μA.

6.4.8 Atomic Force Microscopy (AFM) Imaging and Nanoindentation

Tapping mode AFM images and nanoindentation measurements were obtained in 35 ppt artificial

seawater employing atomic force microscopy (AFM) (MFP 3D, Asylum Research, Santa

Barbara, CA). AFM images were obtained using V-shaped silicon nitride cantilevers (MLCT-

AUNM, Veeco Inc., Santa Barbara, CA) with nominal spring constants of ~ 0.5 N/m.

The mechanical properties of the tubeworm shell and the adhesive region were measured by

nanoindentation experiments using rectangular shaped silicon tips (NCH_W, Nano World,

Switzerland). Prior to use, individual cantilever spring constants were determined using the

thermal noise spectrum method.20

The spring constant was determined to be 39 N/m. The

resulting force-indentation curves were analyzed with custom-programmed analysis software

(Igor Pro, Wavemetrics, Portland, OR), as described previously.21, 22

Indentation measurements on the adhesive section were carried out by first obtaining several 20

x 20 μm images in a ~8000 μm2 region in artificial seawater using a contact-mode tip (0.01 N/m)

and then replacing the tip with a rectangular silicon cantilever (39 N/m) for the mechanical tests.

In addition to the measurements performed on the adhesive material, unetched polished cross-

sectioned samples were also examined via nanoindentation, in order to minimize the error in the

determination of the modulus due to topographic variations. Indentation measurements were

also performed on the cross-sectioned samples in the dry state as well as the wet state. The

polished samples were immersed in artificial sea water for 8 days prior to the measurements

performed in the wet state.

78

Tapping mode AFM imaging in air was performed on samples of the organic tube lining. For

these samples, the IOM was sonicated in DI water for 2 min (Bransonic 2510, Branson,

Danbury, CT) in order to break it up into fragments. The fragments were then deposited onto

freshly cleaved mica substrates, and allowed to dry before imaging.

6.4.9 Light Microscopy and Chemical Staining

A light microscope (CME, Leica Microsystems Inc., Bannockburn, IL) was used for optical

imaging of the organic tube lining. Thin sections of the lining were glued to a piranha cleaned

glass slide using epoxy glue. The glue was allowed to cure for 2 hours at room temperature in a

laminar flow hood. The glass slide was then washed with ethanol and then rinsed in distilled

water. The tube lining was then subjected to Masson’s trichrome staining (Trichrome stain AB

solution, Sigma-Aldrich, St. Louis, MO), per the Sigma-Aldrich protocol.

6.4.10 Amino Acid Analysis of the Soluble Organic Matrix (SOM)

Amino acid analysis of the EDTA extract was performed on an UltraPerformance Liquid

Chromatograph (UPLC) (Acquity, Waters Corporation, Milford, MA). Dried protein sample

was hydrolyzed by a vapour phase or liquid phase reaction, using 6 M HCl with 1% phenol at

110 oC for 24 hrs. Glycoprotein sample was hydrolyzed using 6 M HCl with 1% phenol at 110

oC for 28 hrs (to quantify amino sugars and obtain accurate serine measurements). A work

station (PICO-TAG, Waters Corporation, Milford, MA) was used for the drying and hydrolysis

procedures. After hydrolysis, the excess HCl was removed from the hydrolysis tube under

vacuum, and the sample was treated with a re-drying solution consisting of

methanol:water:triethylamine (2:2:1) and dried under vacuum. The sample was then derivatized

for 20 min at room temperature using a derivatizing solution of

methanol:water:triethylamine:phenylisothiocyanate (PITC, 7:1:1:1). The solution was removed

under vacuum, and the sample was treated once again with the re-drying solution and dried to

remove any traces of PITC.

The derivatized sample was dissolved in a given amount of sample diluents (pH 7.4) and an

aliquot was injected into the UPLC BEH C18 column (1.7 μm, 2.1 mm x 100 mm) running on a

gradient for UPLC. The column temperature was 48 oC. The PITC-amino acids were detected at

254 nm. Data acquisition and collection was obtained using a TUV detector. The whole system

79

was controlled using chromatography software (Empower 2.0, Waters Corporation, Milford,

MA).

6.4.11 In vitro CaCO3 Mineralization Experiments

For the organic tube lining remineralization experiment decalcified lining material was rinsed in

distilled water and then placed in an uncovered crystallization dish containing 4 mL of 35 ppt

artificial seawater solution. For the SOM mineralization experiment, 50 μL of the SOM extract

was deposited onto a flame-annealed Au substrate, and allowed to adsorb for 10 min.

Afterwards, the substrate was placed into an uncovered crystallization dish to which 4 mL of 35

ppt artificial sea water solution was added. Flame-annealed Au substrates (no SOM deposition)

were placed into dishes with 4 mL of 35 ppt artificial seawater solution as well, as controls.

Crystallization was performed using the ammonium carbonate diffusion method.23

The dishes

were placed in a chamber containing powdered (NH4)2CO3 and sealed using parafilm.

Precipitates were collected after various time intervals. For the organic tube lining experiment,

the IOM was removed after 24 hr, and for the SOM experiments, the Au substrates were

removed after 90 min and after 48 hr. Samples were rinsed in distilled water and allowed to dry

at ambient conditions.

For reference, the chemical compositions of sea water and artificial seawater are given in Table

6.1.

Table 6.1 Chemical composition of seawater vs. the artificial seawater used in all CaCO3 precipitation experiments. (Atkinson, M.J., Bingman, C., 1997. Elemental composition of commercial seasalts. J. Aquaricult. Aquat. Sci. 8, 39-

43.)

Salinity Na Mg Ca K Sr Cl- SO4- BO3

HCO3-,

CO32-

Molar Mass

(g/mol)

Seawater 35 470 53 10.3 10.2 0.09 550 28 0.42 1.90 22.9898

Instant

Ocean 29.65 462 52 9 9.4 0.19 521 23 0.44 1.90 24.305

80

6.5 Results

6.5.1 Bulk Composition of the Tube Shell and Adhesive Material

The XRD spectra for the tube shell and the tube adhesive both showed the same peaks,

indicating that the crystal structure and distribution is similar for the bulk tube shell material and

for the adhesive material. The XRD spectrum for the bulk tube shell is shown in Figure 6.2A.

The peaks correspond to a combination of two polymorphs of CaCO3; namely, aragonite and

magnesium calcite (Mg-calcite). After Rietveld refinement of the spectrum, the relative

proportions of the two polymorphs were found to be 40 ± 1 % aragonite, and 60 ± 1 % Mg-

calcite. The unit cell parameters of the Mg-calcite component were found to be a = 4.921 ±

0.002 Å and c = 16.714 ± 0.007 Å (rhombohedral unit cell, space group R c), with a

corresponding unit cell volume of 350.5 ± 0.2 Å. Based on the site occupancies of Ca and Mg in

the 6b position, the empirical formula of the Mg-calcite was determined to be (Ca0.8Mg0.2)CO3.

Figure 6.2 A) XRD spectrum for powdered sample of entire tube. Aragonite and Mg-calcite peaks are labeled A and C respectively. B) FTIR spectrum for powdered sample of entire tube. Inset: Features of ν4 bands.

81

The FTIR spectra for the tube shell and the tube adhesive exhibited the same features, and thus

only the tube shell spectrum is shown in Figure 6.2B. As shown in Table 6.2, the peak positions

of the carbonate ion modes confirm the presence of both aragonite and calcite. Comparison of

the observed calcite peaks with those of an abiogenic calcite sample indicates that the biogenic

sample contains Mg-calcite. Shifts in the peak positions and line width broadening of the ν2 and

ν4 modes, as compared to those of pure calcite, are characteristic of the presence of magnesium

in the calcite structure. In particular, the ν4 mode is ideal for estimation of the Mg content in Mg-

calcite because it is both a function of composition and independent of formation conditions.24, 25

The ν2 and ν4 modes in pure calcite are 876 cm-1

and 712 cm-1

, respectively, and these modes are

shifted to 873 cm-1

, and 713cm-1

, with a shoulder at 723cm-1

(see inset of Figure 6.2B). Given

that the ν4 mode of aragonite is at 713 cm-1

, the shoulder at 723 cm-1

is attributed to Mg-calcite.

Using the relationship developed by Bottcher et al.,25

ν4 [cm-1

] = 39.40XMg + 712.20, our FTIR

results indicate a Mg content of ~27 mol%. However, the relationship is only valid for XMg <

0.23; hence, qualitatively the FTIR data confirms the XRD identification of a high Mg-calcite

present in the tubeworm shell.

Table 6.2 Summary of IR bands for the tube shell sample FTIR spectrum and comparison with geological calcite and aragonite samples, in units of wavenumbers (cm

-1). Abbreviations: vvs = very very strong, vs = very strong, s =

strong, m = medium, vw = very weak, sh = shoulder, br = broad.

Carbonate ion mode H. dianthus Geological Calcite Geological Aragonite

ν1 + ν4 ~1796 (vw,br) 1800 (m) 1788 (m)

ν3 1482 (vvs), 1429 (sh) 1422 (vvs) 1477 (vvs)

ν1 1082 (m) - 1083 (s)

ν2

873 (vs) 876 (vs) -

858 (s) - 855 (vs)

844 (sh) 848 (sh) 843 (sh)

ν4

723 (sh,br), 713 (m) 712 (s) 713 (s)

700 (m), ~693 (sh,br) 696 (sh) 700 (m)

82

The presence of Mg was also confirmed by electron probe microanalysis and ICP analysis,

which yielded estimates of 16 ± 2 mol% and ~11 mol%, respectively. The ICP result is low in

comparison to the other results because the aragonite phase could not be separated from the

calcite phase for the analysis.

6.5.2 Tube Shell Ultrastructure and Spatial Composition

An overview of a transverse tube cross-section is shown in Figure 6.3A. The adhesive material

is only a few tens of microns thick; in contrast, the tube shell is on the order of a hundred to

several hundred microns thick. Concentric layers are evident within the tube shell, and the

layers terminate roughly perpendicular to the adhesive material at the base of the shell. Figure

6.3B shows the cross-section of one of the tube shell walls in greater detail. The direction of the

exterior (ext) and interior (lumen) of the tube shell is indicated by the arrow label. As previously

described by Vinn et al.,26

the shell of H. dianthus consists of three distinct layers, each with a

specific crystal organization. In the following description, we use the terminology established by

Vinn et al.12

The outermost layer, in the top left of Figure 6.3B, possesses a spherulitic prismatic

structure (SPHP). Below this is a layer with an irregularly oriented prismatic structure (IOP).

The last layer, terminating in the inner wall of the tube shell (shown as the bottom two-thirds of

Figure 6.3B), has a lamello-fibrillar (LF) structure, in which the orientation of crystals changes

with successive growth increments.

Figure 6.3C-F show the structural details of the three layers and the transitions between layers.

The local chemical composition of or within the layers is indicated, as determined through EDX.

First, in Figure 6.3C, the SPHP layer was found to be composed of aragonite (bottom left in

image), while the IOP layer was a mixture of aragonite (adjacent to the SPHP layer) and Mg-

calcite (top left in image). Next, Figure 6.3D shows the interface between the IOP layer (bottom

left of image) and the LF layer (top right of image). Despite the difference in crystal

morphology and organization, both regions were found to consist of Mg-calcite. Inside the LF

layer, Figure 6.3E shows that both aragonite and Mg-calcite can be present in this particular

structure. Lastly, Figure 6.3F shows the LF layer terminating into the inner wall of the shell

(upper right), on which there is an organic layer (the tube lining). In the lower left of the image,

among the Mg-calcite crystals, another organic layer is present. Both organic layers appear

smooth and unaffected by the acetic acid etching process.

84

Figure 6.3 SEM images of tube shell transverse cross section, showing a layered structure with each layer consisting of a different crystal morphology and organization, and/or polymorph. The head of the arrows indicate the direction of the tube interior (lumen). In Figs. C-F, the local chemical composition is indicated by A – aragonite, MC – Mg-calcite,

O = organic. A) Large scale view of cross section, with fragments of adhesive material also visible. The arrow indicates the region over which Figs. C-F were taken. B) Tube shell wall, showing the three distinct layers (SPHP =

spherulitic prismatic structure, IOP = irregularly oriented prismatic structure, LF = lamello-fibrillar structure). C) Details of outer tube wall, showing the SPHP and IOP layers. D) Details of the middle region of the tube wall,

showing the transition from the IOP layer to the LF layer. E) Details of the LF layer, showing the presence of both aragonite and Mg-calcite within the layer. F) Details of the inner tube wall, showing several organic membranes. G) Representative EDX spectrum for Mg-calcite crystals. H) Representative EDX spectrum for aragonite crystals. I)

Representative EDX spectrum for organic material.

Representative EDX spectra for Mg-calcite, aragonite, and organic material are shown in Figure

6.3G-I. Any Cd signal present in the spectra is the result of contamination within the SEM

chamber. Both CaCO3 polymorphs exhibit strong Ca peaks as well as C and O peaks, but can be

distinguished by the presence of a Mg peak in the case of Mg-calcite, and a Sr peak (with no Mg

peak visible) in the case of aragonite. Sr is able to substitute into the aragonite lattice because

the Ca-O distances are larger than they are in the calcite lattice.27

The organic membranes are

distinguished by high C, O, and S signals.

6.5.3 Adhesive Material Structure and Composition

The adhesive material has an apparently different structural organization than the tube shell.

Although its thickness is comparable to that of the SPHP layer (on the order of 10-20 μm), it

appears to be made up of multiple thinner layers, each several microns thick, as shown in Figure

6.4. The adhesive layers appear crystalline, like the tube wall layers, but a greater amount of

organic material (sheet and fibre features) is associated with the crystals, as seen in Figure 6.4B-

D. The transition between the bulk tube wall crystals and the adhesive material is shown in

Figure 6.4A, with an organic sheet separating the two regions. The adhesive material at the

outermost edge of the tube wall has a layer consistent with the outer SPHP structure (Figure

6.4B). Cross sections of the adhesive fragments ‘a’ and ‘b’ (labelled in Figure 6.3A) are shown

in Figure 6.4C and 3D respectively.

A wide variety of different crystal morphologies was observed within the adhesive material, with

several distinct forms observed for both Mg-calcite and aragonite polymorphs. Figure 6.5A-C

show SEM images of Mg-calcite crystals and Figure 6.5D-F are images of aragonite crystals, as

determined by EDX. Besides the rhombohedral crystal form shown in Figure 6.5C, Mg-calcite

can also take the form of cauliflower-like aggregates made up of triangular layers (Figure 6.5A),

and dumbbells, shown in various stages of growth in Figure 6.5B.

85

Figure 6.4 SEM images of adhesive material, transverse cross section. Layers are evident, some with apparently different structures than those observed in the bulk tube. Sample subjected to a 2 min etching with 1% acetic acid.

A) Base of tube wall, showing transition between adhesive region and the crystals of the bulk wall. B) Cross section of adhesive material extending out from the outer tube wall. C) Cross section of adhesive fragment ‘a’ (labeled in

Fig. 6.3A). D) Cross section of adhesive fragment ‘b’ (from Fig. 6.3A).

In fact, the latter two forms were observed much more often than the rhombohedral form. Most

aragonite crystals observed had an acicular habit, whether in a randomly oriented arrangement as

in Figure 6.5D, or in bundles with a common orientation, as in Figure 6.5E. However, globular

aggregates of aragonite were also observed, as shown in Figure 6.5F. Crystals observed on the

substrate side of the adhesive material were often found to be associated with thin fibre and sheet

structures (possibly organic), as shown in Figure 6.5C and Figure 6.5E-F. Bacteria and diatoms

were also observed on or embedded within the adhesive material, as shown in Figure 6.6.

86

Figure 6.5 SEM images of the variety of crystal morphologies observed for A-C) Mg-calcite and D-F) aragonite.

6.5.4 Mechanical Properties of the Adhesive Material

The mechanical properties of the tubeworm adhesive material were measured using AFM

indentation measurements. The Young’s modulus was determined by considering load-

indentation dependence for a paraboloidal tip shape given by Equation 6.1:

Equation 6.1 2/3

2 )1(3

4

v

REF Y

Here, F is the loading force in nN, EY is Young’s modulus in Pa, R is the radius of curvature of

the tip in nm, is the indentation in nm, and is Poisson’s ratio. In order to estimate the

Young’s modulus, a value of 0.2 was used for Poisson’s ratio, derived from averaging the bulk

Poisson’s ratios for calcite and aragonite (0.32 and 0.16, respectively).28, 29

AFM topography images collected in seawater of defects in the adhesive material surface which

was in contact with the substrate are shown in Figure 6.7A and Figure 6.7B. The defects

illustrate the different crystal morphologies and orientations present in the material. SEM

images of the adhesive material surface, showing similar features to those observed in the AFM

images, are shown in Figure 6.8.

87

Figure 6.6 SEM images of the adhesive material surface, substrate side, showing the presence and incorporation of various biofilm components. A-B) Bacteria among crystals and on surface. C-D) Diatoms embedded in adhesive

material. E-F) Fibrous networks on the surface and within the adhesive material, some showing signs of mineralization.

88

Figure 6.7 A) 5 x 5 μm and B) 3 x 3 μm AFM height images of adhesive material surface (substrate side), taken in solution (sea water). C) 20 x 20 μm AFM height image of adhesive material area, representative of area over which indentation measurements in solution were taken (adhesive material). D) “Wet state, adhesive”: Histogram of elastic moduli derived from indentation measurements in solution (adhesive material, substrate side). E) “Wet state, shell”: Histogram of elastic moduli derived from indentation measurements in solution (transverse tube shell cross-section,

polished). F) “Dry state, shell”: Histogram of elastic moduli derived from indentation measurements in air (transverse tube shell cross-section, polished).

Figure 6.7D shows a histogram of elastic moduli obtained via nanoindentation measurements

made over the area shown in the representative AFM topographic image Figure 6.7C. A

modulus of 3 ± 1 GPa was obtained from these measurements.

6.5.5 Mechanical Properties of the Tube Shell

Nanoindentation on polished cross-sectioned samples of the tube shell in artificial seawater

solution and in air was performed, yielding Young’s modulus values of 22 ± 3 Pa and 51 ± 8

GPa respectively, as shown in the histograms in Figure 6.7E-F.

6.5.6 Characterization of the Organic Tube Lining

The insoluble organic tube lining was characterized via light microscopy, SEM, chemical

staining, FTIR, and AFM. Figure 6.9A shows a partially broken tube, revealing the lining which

also has a tubular form. Figure 6.9B-C show the insoluble organic tube after removal from the

calcareous tube shell, at 10X magnification (Figure 6.9B) and 40X magnification (Figure 6.9C).

89

Figure 6.8 SEM images illustrating the different crystal morphologies and orientations present at the adhesive material surface, adjacent to the substrate. All images were obtained away from the tube walls. Note the smooth

regions where the crystal growth was terminated by the substrate. A) Columnar-type crystals, roughly perpendicular to the substrate. B) Spherulitic needles, terminating at the substrate in various angles. C) Aggregates with a layered

triangular habit. D) Bundles of acicular crystals, amongst smaller crystals with a triangular habit.

Figure 6.9 Optical images of organic tube lining. A) Partially broken tube, showing exposed organic matrix, 10X magnification. B) Tube lining after removal from the tube, 10X magnification. C) IOM, 40X magnification.

90

SEM observations of the lumen-side of the adhesive material fragments reveal that criss-crossed

layers of fibres are located on the inner wall of the tube, and that the fibres are covered by a

smooth thin layer, possibly an organic sheet, as shown in Figure 6.10A. The smooth layer is

shown in detail in the inset of Figure 6.10A, and EDX measurements indicate the presence of C,

O, Ca, Mg, Na, P, and S (Figure 6.11). A region in which a layer of fibres is in intimate contact

with calcareous crystals is shown in Figure 6.10B. The structure of the fibres is more clearly

seen in Figure 6.10C. Each ~3 μm fibre is made up of many smaller fibres, some in what

appears to be a twisted conformation. EDX measurements of the fibres yielded results similar

those obtained for the smooth layer, with varying levels of S. Cross-sectional details of the

organic layer lining the inside of the tube are shown in Figure 6.10D. Layers of fibre and sheet

structures are apparent.

Figure 6.10 SEM images of the adhesive material, lumen-side (A-C) and of the EDTA-treated tube lining (D). Fibre structures similar to those observed via optical microscopy are evident. A) Fibres covered by a smooth sheet-like structure. Inset: Details of sheet surface. B) Fibres associated with crystalline aggregates. C) Details of fibres,

showing them to be composed of smaller fibres. D) Cross-section of the EDTA-treated organic tube lining, showing layers of fibres and sheets.

91

Figure 6.11 EDX spectrum for the “smooth layer” (possibly an organic sheet) shown in the inset of Fig. 6.10A, showing the presence of C, O, Ca, Mg, Na, P, and S. The Cd signal is due to contamination within the SEM

chamber.

Masson’s trichrome staining was performed in order to identify the organic tube lining. Some

areas were observed to be stained blue, which indicates a positive result for the presence of

collagen, as shown in Figure 6.12. Sonicated fragments of the lining were deposited onto mica

substrates and examined via AFM, in order to characterize the diameter and structure of the

smaller fibres. Two types of fibres were distinguished via AFM, as shown in Figure 6.13. The

first type consisted of fibres with a diameter ~ 150 nm, which exhibited banding with a 67 nm

spacing, which is similar to vertebrate collagen type I.30

The second type was unstriated, with a

diameter of ~100 nm.

Figure 6.12 Optical image of the insoluble organic matrix after Masson’s trichrome staining, 40X magnification. The blue colour indicates a positive result for the presence of collagen.

92

Figure 6.13 1 x 1 μm AFM amplitude images and linescans of fibres from the insoluble organic matrix. A) Striated fibre. B) Smooth fibre.

The FTIR spectrum of the tube lining is given in Figure 6.14, and the observed band frequencies

are summarized in Table 6.3. The absence of calcite bands at 1416 cm-1

, 870 cm-1

, and 719 cm-1

(ν3, ν2, and ν4 carbonate modes) indicate that the lining is fully de-mineralized. The dominant

features of the spectrum appear to be signatures of carbohydrates. The broad peak at 3284 cm-1

is due to bonded –OH, while the components in the range of 2850 – 2950 cm-1

are attributed to

CH stretching. In the 1000 – 1100 cm-1

region, the band at 1096 cm-1

arises from C-O and C-C

stretching in pyranose rings, while the peaks at 1030 and 1007 cm-1

are assigned to C-O-C and

C-C stretches, somewhat similar to modes observed in α and β-glucan.31, 32

Peaks at 797 and 777

cm-1

could be due to α and β-galactose skeletal bending.33

The strong bands at 1594 and 1400

cm-1

are attributed to –COO- (carboxylic acid salt), while the bands at 1323 and 1231 cm

-1

correspond to sulphate ester vibrations.34

93

Figure 6.14 FTIR spectra of the tube shell organic matrices. A) Organic tube lining spectrum. B) SOM spectrum.

6.5.7 Characterization of the SOM

The FTIR spectrum of the SOM is given in Figure 6.14B, and a summary of the bands is

presented in Table 6.3 (below). The broad bands at 3377 and 3260 cm-1

are due to bonded –OH,

and the bands around 2850 – 2950 cm-1

are assigned as above in the organic tube lining FTIR

spectrum. Carbohydrate signatures arising from C-C, C-O-C, and C-O stretches are present in

the region between 980 – 1050 cm-1

. The lower frequency components at 925 and 851 cm-1

are

assigned to ring vibration and α-anomeric CH deformation, respectively. The band at 851 cm-1

may also be assigned to a C-O-S bending mode of sulphate esters, and additional sulphate ester

modes are observed at 1332, 1258, 1181, and 1114 cm-1

. Carboxylic acid salt (-COO-) bands are

observed at 1601 and 1407 cm-1

, and the bands at 1739 and 963 cm-1

are assigned to COOH

modes.

94

Table 6.3 Summary of IR bands for the organic tube lining and the SOM FTIR spectra.

IR Band (cm-1

) Assignment IR Band (cm-1

) Assignment

Organic

Tube

Lining SOM

Organic

Tube

Lining SOM

- 3377 Bonded –OH, -NH, -

NH2 1231 1258

Sulphate ester (S-O

stretch)

3284 3260 Bonded –OH, -NH, -

NH2 - 1181

Phenol, sulphonic

acid

2962 2955

-CH2, -CH3, bonded

OH in COOH

- 1114 SO42: S=O stretch

2921 2881 1096 - C-O, C-C stretch

2849 2838 - 1050 C-C bond in

alcohols, C-O stretch

- 2731 -CHO, bonded –OH

in COOH 1030 1029

C-C bond in

alcohols, C-O-C and

C-O stretch

- 2651 Bonded –OH in

COOH 1007 1003 C-C stretch

2350 - CO2 (background) - 984 C-O stretch, -

CH=CH2

- 1739 -CHO, -COOH - 963 -COOH

1594 1601 -COO

- asymm.

stretch 915 925 ring vibration

- 1489 Aromatic - 851

Sulphate ester (C-O-

S bending), α-

anomeric CH

deformation, C1-OH

1400 1407 -COO- - 832 Aromatic

1323 1332 SO2 asymm. stretch 797 - α-galactose skeletal

bending

- 1282 H-C-C, C-C 777 - β-galactose skeletal

bending

- 710 Aromatic

95

In addition to the FTIR measurements, the SOM extract was subjected to amino acid analysis.

The relative proportions of each type of residue are shown in 0. The majority of the amino acids

present (~61 %) are nonpolar and neutral, including significant amounts of glycine and proline.

Aspartic and glutamic acid, both polar and negatively charged, make up ~19 % of the

composition.

Table 6.4 Amino acid composition for the SOM.

Amino Acid Composition (mol%)

Asp 8.42

Glu 10.20

Ser 6.36

Gly 14.47

Arg 2.76

Thr 6.54

Ala 7.75

Pro 21.42

Tyr 3.06

Val 4.89

Met 0.87

Ile 3.69

Leu 5.34

Phe 2.21

Lys 1.94

96

6.5.8 Characterization of the Remineralized Organic Tube Lining

Prior to the remineralization experiment, the demineralised organic tube lining was subjected to

SEM and EDX analysis. The fibre and sheet features were found to be smooth, and C, O, Na,

and S were detected via EDX, as shown in Figure 6.15.

Figure 6.15 SEM images of the IOM, treated overnight with 0.5 M EDTA. A-B) Fibre structures, showing the smooth fibre surface. C) EDX spectrum of IOM (associated image not shown).

The absence of Ca indicates that the lining was fully decalcified following the overnight EDTA

treatment, which is consistent with the FTIR spectrum of the lining. SEM examination of the

97

remineralized tube lining revealed that the sheet-like structures were covered in crystals

exhibiting dumbbell morphology and a cauliflower-like texture, all in the same size range of ~5

μm in length and diameter. The cauliflower-like texture of the crystals is shown in detail in the

bottom left of Figure 6.16A, and a crystal in an earlier stage of growth before the formation of

the dumbbell shape is shown in the upper right of Figure 6.16A. Although the density of

coverage varied slightly from site to site, the morphology and size of the crystals was generally

constant along the surface of the lining. The EDX spectrum of the dumbbell-shaped crystals is

shown below the SEM image in Figure 6.16A. Strong C, O, and Ca signals indicate that they are

composed of CaCO3, but both Mg and Sr signals are present. The higher Sr signal indicates the

composition is predominantly aragonite. Crystals with a different morphology were observed

near fibre structures, as shown in Figure 6.16B. Ellipsoidal leaf-like features are evident. The

EDX spectrum of these crystals is shown in the bottom half of Figure 6.16B and the presence of

a strong Mg signal in addition to the C, O, and Ca signals suggest that the crystals are composed

of Mg-calcite. Details of the fibre structures are shown in Figure 6.16C, showing granular

features along the fibres. In addition to the strong S signal, there are small Ca and Mg signals,

which suggest that the fibres are slightly mineralized.

Figure 6.16 SEM images and EDX spectra of crystals formed on the EDTA-demineralized organic tube lining sample, after 24 hours. A) Crystals with cauliflower-like texture and dumb-bell morphology, with a high Sr content. B)

Crystal with ellipsoidal leaf-like features, with a high Mg content. C) Organic tube lining fibres, beginning to show signs of mineralization (Ca, Mg signals).

98

6.5.9 Characterization of the SOM Mineralization Precipitates

The precipitates collected after 90 min and 48 hr in the crystallization chamber were analyzed

via FTIR, SEM, and EDX. The FTIR spectrum for the 90 min control and SOM samples

identified the precipitates as being composed of amorphous calcium carbonate (ACC). The

control sample appeared to contain more precipitate than the SOM sample, from its higher

absorbance. The FTIR spectrum for the control sample is given in Figure 6.17. Broad peaks at

862 and 1070 cm-1

were observed, as well as the splitting of the ν3 carbonate ion mode into two

bands at 1410 and 1470 cm-1

. The band at 1654 cm-1

is assigned to water, indicating that the

ACC is partially hydrated. These features are all consistent with the spectrum for ACC.35, 36

For

the 48 hr precipitates, the FTIR spectrum for the SOM sample (Figure 6.18) indicated the

presence of aragonite. The control sample could not be identified via FTIR due to low surface

coverage of the Au substrate.

2000 1800 1600 1400 1200 1000 800 6000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Absorb

ance / A

Wavenumber / cm-1

Figure 6.17 FTIR spectrum of the particles formed in the SOM in vitro crystallization experiment, for the control sample. The broad peaks at 862 and 1070 cm

-1 as well as the splitting of the ν3 carbonate ion mode into two bands

at 1410 and 1470 cm-1

correspond to amorphous calcium carbonate (ACC).

99

Figure 6.18 A) FTIR spectrum of the particles formed in the SOM in vitro crystallization experiment, for the 48 hour SOM sample. The peaks correspond to aragonite. B) Details of ν2 and ν4 carbonate ion modes.

The morphology of the ACC particles formed on the control sample differed from that of the

SOM sample, as shown in Figure 6.19AI-II and Figure 6.19BI-II. The control ACC particles

were spherical (0.5 – 1 μm in diameter) and smooth, and there was a high coverage density of the

particles on the substrate. Ca, C, and O signals in the EDX spectrum (Figure 6.19AIII) confirm

B

A

100

the CaCO3 composition of the particles. A high Mg signal and a low Sr signal were also

observed. In contrast, the SOM ACC particles were irregularly shaped and formed micron-sized

aggregates with sub-particles sizes of ~100 nm. C, O, Ca, and Mg were detected via EDX, as

shown in Figure 6.19BIII. The surface coverage of the substrate was sparser for the SOM ACC

particles than for the control ACC particles.

Figure 6.19 SEM images and EDX spectra of the crystal products of the in vitro SOM crystallization experiments. AI-

III) Control sample after 90 min. Spherical ACC particles with a high Mg content. BI-III) SOM sample after 90 min. Irregularly shaped ACC particles. CI-III) Control sample after 48 hr. Flower-like crystals with dumb-bell morphology,

and rhombic “petals”. Higher Mg signal than Sr. DI-III) Crystals with a bundle-of-needles morphology. Higher Sr signal than Mg.

101

The morphology of the 48 hr precipitates also differed between the control and SOM samples, as

shown in Figure 6.19CI-II and Figure 6.19DI-II. The control crystals were larger, about 10-15

μm in diameter, and possessed a flower-like morphology, with the “petals” exhibiting the

rhombic symmetry typical of calcite. From the EDX spectrum given in Figure 6.19CIII, C, O,

and Ca signals indicate a CaCO3 composition, and Mg and Sr were also detected, with the Mg

signal being higher than the Sr one. The surface coverage for the control particles was quite low,

which explains the difficulty with the FTIR spectrum. The SEM and EDX data suggest a Mg-

calcite composition. In comparison, the surface coverage for the SOM particles was quite high,

and the crystals had a needle-aggregate morphology. The particle size was consistently ~5 μm

on the long axis of the crystals. The EDX spectrum given in Figure 6.19CIII shows relatively

high Mg and Sr signals, in addition to the C, O, and Ca signals. The Sr signal is slightly higher

than that for Mg. Despite the presence of both Mg and Sr, the FTIR spectrum only exhibited

peaks for aragonite, and not calcite.

6.6 Discussion

6.6.1 Tube Layering and Mechanical Properties

The relatively low modulus of 3 ± 1 GPa obtained via nanoindentation on the adhesive material

surface (substrate side) could have two different origins. First, it could indicate the presence of a

thin organic film adsorbed onto the adhesive material, originating from the biofilm coating the

substrate upon which the worm settled, or from organic secretions from the worm. Second, the

low Young’s modulus could be due to hydration of the adhesive material, in particular, by the

outermost few nanometers. The modulus may be observed in the force profile shown in Figure

6.20 that shows two regions. The first region (i) of indentation from 0 nm to 7 nm exhibits a

good fit to the Sneddon model, giving a modulus of 6.3 ± 0.3 GPa (fit shown as the grey solid

line). The second region (ii) of indentation from 7 nm to 8 nm shows a sharp increase in force.

Using 6 nm as an estimate for the contact point, this second region was fit (shown as the red

solid line), yielding a Young’s modulus of ~36 Pa. This value is consistent with literature

values for biogenic calcite and aragonite in the wet state,37, 38

whereas the modulus obtained from

region (i) cannot be attributed to calcite or aragonite because it is too low. Therefore, the

Young’s modulus of 3 ± 1 Pa (which was obtained from analyses of region (i) of the force

profiles) arises from the presence of a thin organic film on the adhesive material surface.

102

Figure 6.20 Force profile obtained from nanoindentation on the adhesive material surface, performed in artificial seawater. The solid grey line denotes the fit for region (i) of indentation from 0 nm to 7 nm, yielding a Young’s

modulus of 6.3 +/- 0.3 GPa. The solid red line denotes the fit for region (ii) between 7 nm and 8 nm, calculated using a contact point set at 6 nm. The Young’s modulus for this region is ~36 GPa.

The Young’s modulus range of 40 – 65 GPa (FWHM) obtained via nanoindentation

measurements on the polished tube cross-section (in air) is consistent with literature values for

inorganic calcite and aragonite, for directions other than the c-axis of the crystals.39, 40

The range

is also consistent with biogenic calcite and aragonite measurements, such as those made on

gastropod nacre (aragonite) and calcitic fibre structures in brachiopods.38, 41

Both calcite and

aragonite are anisotropic materials, and therefore the measured Young’s modulus will depend on

the orientation of the crystals. Although the Young’s modulus of aragonite is typically higher

than that of calcite, due to the various unknown crystal orientations within the shell, regions of

calcite and aragonite could not be distinguished via the nanoindentation measurements. In order

to estimate the Young’s modulus, the bulk Poisson’s ratios for each polymorph were averaged.

Nanoindentation performed on the same sample in artificial seawater solution yielded lower

Young’s modulus values, with a range of 18 – 24 GPa (FWHM). This is consistent with studies

of nacre, in which the Young’s modulus is lower in the wet state compared with the dry state.38,

42 It is the organic phase of the nacre composite that becomes softer upon hydration; for the

tubeworm composite, the same effect is likely. For nacre, the wet Young’s modulus value is

typically ~10 Pa lower than the dry value. The tube shell exhibits a larger decrease in Young’s

modulus due to hydration compared with nacre (~20 GPa), which is likely due to its different

structure and composition.

103

Due to crystal anisotropy, the crystal organization in each ultrastructural layer plays a role in the

overall mechanical properties of the tube. This has been observed in studies of two calcitic

brachiopods, one with a semi-nacre structure, and the other with a calcitic fibre structure. The

semi-nacre structure was found to have a higher Young’s modulus as well as a greater hardness,

which correlates with the observation that the dominant orientation of crystals in the semi-nacre

structure consisted of the c-axis of the crystals being perpendicular to the surface.41

The spatial distribution of aragonite and Mg-calcite is moderately correlated with the three

ultrastructural layers, as shown in Figure 6.3C-F. The outer SPHP layer is composed of

aragonite, and the innermost layer in the LF region is composed of Mg-calcite. The middle

LF/IOP region of the tube wall is predominantly composed of Mg-calcite, although aragonite

was also occasionally detected. This polymorph distribution could offer some benefits for the

mechanical stability of the tube. Aragonite is denser, harder and less brittle than calcite,43

properties which make it beneficial for the outermost layer of the tube. The Mg content of the

calcite, at 20 mol%, is high in comparison to other biogenic forms of Mg-calcite. The elastic

modulus and hardness of calcite is known to increase with increasing Mg content.44-46

The

incorporation of such a large amount of Mg could have evolved as a means of developing a

stronger tube.

In general, laminar structures have been found to exhibit superior fracture resistance and

toughness. Propagating cracks will tend to arrest or deflect at the interface between layers due to

the differences in the structure or composition of each layer.47, 48

Serpulids that create multi-

layered tubes, such as H. dianthus, would therefore likely have an advantage over serpulids with

single-layered tubes, since the laminar organization of the shell increases its fracture resistance to

loads normal to the tube surface. Robust tubes would provide greater protection from predators,

such as sea urchins, clam worms, and fish.49, 50

It is interesting to consider the fact that the teeth

of such predators are also biomineralized structures, which have evolved to be exceptionally hard

and tough materials; in particular, Mg-calcite sea urchin teeth have hardnesses in the range of 3.5

– 4 GPa,51-53

which is on par with the hardness of aragonite. From an evolutionary perspective,

serpulid phylogeny is divided into two major clades, and multi-layered serpulid tubes with

complex oriented structures are found to occur in only one of the clades. It is thought that the

104

clade which is comprised of organisms with simpler structures and single-layered tubes is the

plesiomorphic condition for Serpulidae.12

6.6.2 CaCO3 Polymorphs and Morphologies

The presence of both aragonite and Mg-calcite, in nearly equal proportions, is unusual because

most biomineralized marine materials consist of one dominant polymorph, such as in the cases of

sea urchin spines (Mg-calcite), mollusc shells (aragonite), and coral (aragonite).10, 54, 55

Although

serpulid tubes are commonly bimineralic,12, 56, 57

in most species, either calcite or aragonite

comprises 75% or more of the tube material. To our knowledge, only one other serpulid species,

Pyrgopolon ctenactis, has a tube which consists of equal proportions of calcite and aragonite

(50.9 and 49.1 %, respectively).12

In his study of two serpulid worms, H. brachyacantha and E.

dianthus (= H. dianthus), Neff observed that tubes from each species contained large amounts of

both high Mg-calcite and aragonite, but did not specify their relative proportions.58

Similarly,

Vinn et al.12

observed that H. dianthus was bimineralic, but their XRD data did not include

results for H. dianthus.

The variety of crystal morphologies observed in the adhesive material for each polymorph is also

significant because it implies that there are a number of different macromolecular interactions or

biomineralization pathways. Most of the morphologies, with perhaps the exception of the

rhombohedral crystals shown in Figure 6.5C-D, are markedly different from the abiogenic

crystal forms for calcite and aragonite. For abiogenic crystals, under equilibrium conditions the

final morphology is closely related to the symmetry of crystal structure of the material, due to the

different surface energies of various crystal planes. High surface energy planes grow quickly

and are thermodynamically driven to minimize their surface area, while low surface energy

planes grow more slowly and therefore form the faces of the crystal with the largest surface

areas.3, 59

In biogenic systems, macromolecules such as aspartic acid-rich proteins and sulphated

glycosaminoglycans (GAGs, formerly known as mucopolysaccharides) can interact with certain

crystal planes, altering growth rates and therefore the final crystal morphology.3, 60

Ions such as

Mg2+

and Sr2+

can also influence the polymorph and morphology of the crystals by the same

thermodynamic principles.27

105

In the adhesive material, the acicular aragonite forms observed in H. dianthus are consistent with

the typical habit of aragonite formed by cyanobacteria and corals.61, 62

The dumbbell Mg-calcite

forms are similar to calcite precipitates obtained through in vitro mineralization by marine

bacteria and to Mg-calcite precipitates formed in the presence of the SOM extracted from the

giant seastar P. giganteus.63, 64

The crystal morphologies of the precipitates formed in the in vitro mineralization experiments in

the present study were different than those observed in the tube shell and adhesive material.

However, the precipitates produced in the control experiments (without the presence of organic

components from the tubeworm) did exhibit morphologies or features consistent with non-

biogenic forms of ACC and Mg-calcite, while those formed in the SOM and IOM experiments

possessed biogenic characteristics. Furthermore, the morphology and polymorph of the

precipitates formed in the SOM in vitro mineralization experiment after 48 hrs were different

between the control sample and the SOM sample. Lastly, the different relative abundances of

precipitate in the control versus the SOM sample for each time interval indicate that there are

different rates of formation for each calcium carbonate polymorph, due to the absence or

presence of the SOM. Therefore, the in vitro mineralization experiments show that both the

SOM and IOM components mediate the calcification process.

6.6.3 Insights into the Formation and Attachment of the Adhesive Material to the Substrate

In considering the biomineralization mechanisms contributing to the formation of the adhesive

material, one cannot rule out the possible role of the biofilm present on the substrate. Biofilms

tend to form on hard substrates exposed to aqueous solution, such as seawater, and are composed

of organic molecules and microorganisms such as bacteria, and diatoms. It is known that

biofilms influence marine invertebrate larvae settlement, and in the case of H. elegans, a serpulid

species closely related to H. dianthus, the larvae settle preferentially on substrates covered in a

biofilm.65-67

Some components of the biofilm, such as the extracellular polysaccharides

produced by bacteria, are capable of inducing CaCO3 mineralization.68, 69

It is possible that the

adhesive material forms in an environment containing macromolecules produced by the

inhabitants of the biofilm as well as the tubeworm, which could explain the variety of crystal

morphologies observed within the adhesive material.

106

The calcareous tube of H. elegans exhibited increased adhesion strength for individuals settled

on biofilms versus clean substrates.70

Nanoindentation measurements in this study on H.

dianthus revealed a thin soft layer on the surface of the adhesive material, suggesting the

presence of a biofilm or organic secretion. In addition, SEM images show bacteria and diatoms

incorporated within the adhesive material (Figure 6.6), and fibre and sheet structures, likely

organic, are observed to be associated with crystals (Figure 6.5C, Figure 6.5E-F). These

observations of the intimate contact and merging of the adhesive material with the underlying

biofilm could explain the increased adhesion strength of tubes attached to biofilm-coated

substrates.

6.6.4 SOM Composition

No amide bands are immediately visible in the FTIR spectrum, although it is possible that they

are obscured due to band overlap with carboxylic acid salt and sulphate ester resonances. Due to

these dominant bands and the carbohydrate signatures, carboxylated and sulphated

polysaccharides constitute the majority of the SOM, and proteins form a minority component.

The most abundant amino acids in the SOM of H. dianthus were found to be Asp (8.4 mol%),

Glu (10.2 mol%), Gly (14.5 mol%), and Pro (21.4 mol%). This is largely consistent with the

SOM amino acid composition averaged from data for three serpulid species,71

in which Asp,

Glu, and Gly are major components, although the Asp content is higher (~20 mol%) and the Pro

content is much lower (~5 mol%) than observed for H. dianthus. Pro plays a role in the

stabilization of collagen conformation,72

and therefore the high Pro content could be due to

collagen from the tube lining, some of which could have been solubilised.

The presence of Asp, Glu, and Gly is also consistent with studies of various calcareous

biomineralizing organisms such as molluscs and coral. Analyses of these organisms typically

reveal that acidic amino acids (aspartic acid and glutamic acid) and neutral amino acids (such as

serine and glycine) are major components of the SOM, with the acidic amino acids making up 30

– 50% of the total.60, 73-75

In those studies, Asp in particular was prominent, making up 20% or

more of the total amino acid composition. Sequences of aspartic acid separated by neutral amino

acids, as well as poly-Asp molecules have been shown to be able to bind with Ca2+

; in addition,

poly-Asp takes on a beta-sheet structure upon binding with Ca2+

. This structure exposes the

107

negatively charged amino acids in such a way that it can interact with specific crystal faces,

matching the distances between Ca2+

ions in calcite or aragonite lattices.76

In H. dianthus, the

acidic amino acid content is low in comparison with the aforementioned biomineralizing

organisms, including other serpulids. However, the Asp and Glu content in H. dianthus is

similar to that observed in the SOM of P. giganteus ossicles (Echinodermata), as reported by

Gayathri et al.64

In their work, the seastar’s SOM was found to accelerate the conversion of

amorphous calcium carbonate into crystalline Mg-calcite, despite the relatively low acidic amino

acid content (~18 mol%). Therefore, although the general observation among CaCO3

biomineralizing species is that high amounts of negatively charged amino acids correlates with

control over crystal growth and morphology, there are other factors to consider. As mentioned

above, macromolecules such as acidic or sulphated polysaccharides can also play a role in Ca2+

binding and mineralization, and indeed the SOM contains significant amounts of carboxylated

and sulphated polysaccharides as determined via FTIR.

6.6.5 IOM Composition

The relatively high S signals observed in the EDX spectra for the organic tube lining and other

organic membranes within the tube of H. dianthus could indicate the presence of sulphated

GAGs, which are negatively charged linear polysaccharide chains covalently bound to a core

protein, with the resulting complex known as a proteoglycan.77

It is unlikely that such a high

signal would arise from proteins alone (disulphide bonds), unless there were a substantial amount

of cysteine residues; furthermore, previous studies have noted the presence of high amounts of

sulphated polysaccharides associated with annelid collagen.15, 78-80

The polysaccharide and

sulphate ester signatures in the FTIR spectrum of the organic tube lining (Figure 6.14) appear to

provide further evidence for the presence of sulphated GAGs. However, no protein signatures

are immediately apparent in the FTIR spectrum, despite the fact that collagen was also detected

within the lining. It is possible that the amide bands are obscured by the absorptions

corresponding to carboxylated polysaccharides, particularly by the carboxylic acid salt bands at

1594 and 1400 cm-1

. Upon close inspection of the 1594 cm-1

band, which is not symmetric, there

appear to be higher and lower frequency components contributing to the shape of the broad band,

with the higher frequency component possibly corresponding to the amide I band. Regardless,

the intensity of the polysaccharide bands and the lack of obvious amide bands suggest that

108

proteins are a relatively minor component of the lining, and that the polysaccharides are mainly

composed of sugars which lack amino groups and hence are not GAGs. Overall, the spectrum

bears a strong resemblance to that of cationic salts (Na+, Ca

2+) of alginic acid, an acidic

polysaccharide found in seaweed,31, 81

with additional peaks indicating the presence of sulphated

polysaccharides, such as sulphated fucans.82

Cations can cross-link individual polysaccharide chains, resulting in the formation of gels.

Acidic polysaccharides and alginates are known to self-assemble into a variety of structures,

including lamellar, sponge-like, and sheet structures,83

in part through this cation cross-linking

process. The organic sheet structures observed via SEM could therefore be composed of

polysaccharides. It is also possible that the fibre structures are partially composed of

polysaccharides, in addition to the collagen fibrils.

Based on the above, in conjunction with the SEM data, the organic tube lining is comprised of

layers of collagen-containing fibres and organic sheets, which are composed of sulphated and

carboxylated polysaccharides. This is consistent with a description of a “coating membrane”

given by Muzii,84

for Eupomatus (=Hydroides) dianthus, which tentatively identifies the

membrane as being composed of protein fibres and dispersed sulphomucins. In the present

study, however, although the presence of sulphated polysaccharides is confirmed, the presence of

GAGs is not. The organic membranes within the tube shell may be similar, based on the S EDX

signal, although the presence of collagen or fibre features has not been verified in these

membranes.

Both polysaccharides and collagen have been found to influence biomineralization. Sulphated

and carboxylated polysaccharides have the capability to bind cations, thereby playing roles in the

nucleation process.23, 76, 85

Studies by Cuif et al.86

have identified varying distributions of

sulphated proteoglycans spatially correlated with varying CaCO3 polymorph distribution.

Collagen gels as well as solubilised collagen have been used for in vitro mineralization

experiments, and found to affect the polymorph and morphology of the resulting CaCO3

crystals.87-89

In the case of the collagen gels, the gel was found to have two effects on the crystal

morphology. Firstly, the gel creates constricted volumes within its fibre network, which

provides supersaturated microenvironments for nucleation and growth, as well as physical

109

boundaries for constraining crystal size or orientation. Secondly, deformation of the gel can also

change the alignment of the collagen fibres, which could then interact with the crystal faces

themselves to alter the morphology. Solubilised collagen influences calcite morphology by

adsorbing to different crystal planes, resulting in a variety of crystal habits depending on the

collagen concentration.

6.6.6 Summary of the Structure and Composition of the Tube Shell and Adhesive Material

The preceding observations are summarized in Figure 6.21, which shows cartoons of the

transverse cross-sections of both the tube shell and the adhesive material. In Figure 6.21A, the

three distinct ultrastructural layers, SPHP, IOP, and LF, are shown to be composed

predominantly of aragonite, Mg-calcite, and Mg-calcite, respectively. Organic sheets are shown

within the mineralized layers of the shell, and the organic tube lining is shown on the inner wall

of the tube shell. In Figure 6.21B, although no distinct ultrastructural layers could be easily

distinguished, the observed spatial distribution of aragonite and Mg-calcite is shown, with

aragonite closer to the substrate side and Mg-calcite closer to the organic tube lining and the

lumen side. The abundance of organic sheets is also depicted within the mineral layer. Both the

organic sheets and the organic tube lining contain sulphated polysaccharide components.

6.6.7 Role of the Organic Tube Lining in Tube Formation

The structure and composition of the organic tube lining of H. dianthus is reminiscent of

descriptions of polychaete cuticles, with the exception of the observation of banding in

individual collagen fibrils. In many polychaete worms, including serpulids, the cuticle consists

of unstriated collagen fibrils in an amorphous or fine filamentous matrix, with the fibrils

frequently present in a criss-cross layered structure, located in the basal layer of the cuticle. The

collagenous cuticle is produced by secretory cells in the epidermis, and is adherent to the

epidermis in most polychaetes. Striated collagen has been observed in polychaetes, but only for

non-cuticular interstitial forms, located under the epidermal cells in the extracellular

matrix.80,90,91

Because the organic tube lining was found to be present and intimately associated

with the mineralized tube wall after worm removal, it is not cuticular material. However, the

110

similarities in composition and structure suggest that the organic tube lining is also produced by

secretory cells in the epidermis.

Figure 6.21 Summary of tube structure and composition. A) Cartoon of tube shell, transverse cross-section. LF = lamello-fibrillar structure, IOP = irregularly oriented prismatic structure, SPHP = spherulitic prismatic structure. B)

Cartoon of adhesive material, transverse cross-section.

Most observations of the tubes of various serpulid species have found the inner tube walls to be

smooth, with no mention of criss-crossed fibre structures.12, 13

However, organic sheets lining

the inner tube walls were observed in the serpulids M. cavatica and S. giganteus, with a mesh-

like structure noted for the latter species.12

Furthermore, the inner tube walls of one polychaete

species, Chaetopterus variopedatus, were found to consist of aligned protein fibres with a criss-

crossed layered structure, in an amorphous matrix. The worm itself was found to be lacking a

cuticle entirely, and it was hypothesized that C. variopedatus sloughs off its cuticle and uses it as

a template for tube construction.92

It has not been verified whether the fibre layers are cuticular,

although they appeared to be formed by the epidermis. Although the tube of C. variopedatus is

111

not calcareous, being composed of proteins and GAGs, C. variopedatus demonstrates an

example of a tube formation strategy utilizing a framework of aligned protein fibres.

H. dianthus may employ a similar strategy, using its organic tube lining as a scaffold for the

biomineralization of CaCO3. A further similarity between H. dianthus and C. variopedatus is the

observation that the protein fibrils in the tube lining exhibit striation, which is unusual since the

vast majority of annelid cuticular collagen has been found to be smooth. A banding of 64.1 nm

was observed for C. variopedatus via SEM, which is close to the 67 nm banding we observed in

H. dianthus via AFM. These values are consistent with the values for vertebrate collagen type I,

which has a spacing of 64 – 67 nm.30

The association of the tube lining’s collagen-containing fibres with CaCO3 crystals at the tube

wall interface (Figure 6.10), and the detection of elements consistent with Mg-calcite in the

fibres and on the smooth sheet at the lumen interface further support the idea that H. dianthus

employs the organic tube lining as a scaffold for biomineralization. A previous study showed

that the decalcified IOM of H. dianthus was capable of becoming recalcified in an inorganic

solution mimicking the ion concentrations found in molluscan extrapallial fluid.93

The IOM was

described as a tube-shaped structure, and presumably consisted of both the tube lining and the

organic sheets within the tube walls. Whether mineralization is induced by collagen

components, the polysaccharide components or a combination of the two remains to be

determined conclusively; however, the results of the in vitro recalcification of the organic tube

lining in artificial seawater (Figure 6.19) confirm that the lining as a whole can induce CaCO3

mineralization. The majority of the Sr-rich crystals observed on the surface of the IOM smooth

sheet structures had the same morphology and were similar in size and chemical composition.

This indicates that the nucleation and growth conditions for these CaCO3 crystals were very

similar, which suggests that the macromolecules associated with or comprising the sheet

structures play a role in the biomineralization process. The IOM fibre structures also appear to

influence the form of the CaCO3 precipitates, based on observations of Mg-rich crystals near the

fibres with a different morphology than the Sr-rich crystals. The fibres themselves exhibited

granular features which were not observed on EDTA-treated IOM fibres, and the detection of

Mg and Ca signals implies that the fibres were becoming mineralized, possibly via a Mg2+

selective mechanism.

112

As a last point to consider, from the serpulid studies of Hedley and Vovelle,15-17

it was observed

that the mucus-secreting cells of the ventral and lateral epithelium contained a high amount of

calcium, with only the cells of the calcium-secreting glands containing more. As mentioned

earlier, it is possible that the organic tube lining is also produced by epidermal secretory cells.

Therefore, the worm could create a high local concentration of Ca2+

ions adjacent to its body

through epidermal mucus secretions, which could then bind to the highly negatively charged

sulphated and carboxylated polysaccharides in the organic tube lining, initializing the

biomineralization process on the organic tube lining.

6.7 Conclusions

The calcareous tube shell and adhesive material of the serpulid H. dianthus were studied from a

biomineralization perspective. The tube shell consists of three layers: an outer layer composed

of aragonite crystals with a spherulitic prismatic (SPHP) structure, a middle irregularly oriented

prismatic (IOP) Mg-calcite layer, and an inner lamello-fibrillar (LF) Mg-calcite layer. The

Young’s moduli of the inorganic components, as measured via AFM nanoindentation on

polished cross-sectioned samples, ranged from ~22 GPa in the wet state, to ~51 GPa in the dry

state. Both soluble and insoluble organic matrices were extracted from the tube shells.

H. dianthus appears to utilize its organic tube lining, which is composed of striated and smooth

collagen and carboxylated and sulphated polysaccharides, as an extracellular scaffold for the

biomineralization of Mg-calcite. This is consistent with a speculation by Neff,58

who

hypothesized that the calcium-secreting glands of H. dianthus formed the aragonitic outer wall

(SPHP layer) of the tube, while the ventral epithelium played a role in the formation of the Mg-

calcitic inner layers (IOP and LF layers). In vitro mineralization experiments in artificial

seawater confirmed the ability of the organic tube lining and the SOM to mediate calcification,

with some degree of control over the final morphology and polymorph of the resulting crystals.

Further characterization of the soluble and insoluble organic matrices is required in order to

better understand their roles in the biomineralization process. Purification of the matrices in

order to separate the various proteins and polysaccharides would allow for the determination of

the specific roles of each component in the biomineralization process.

113

6.8 Contributions

The author performed the SEM, EDX, and FTIR measurements and analysis and wrote the

manuscript. A.E.T. also undertook an extensive literature survey in order to present and interpret

all of the experimental results in the context of calcium carbonate biomineralization.

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Figure 7. sfdtg

Table 7. swrdtg

119

7 Nanoscale Structures and Properties of the Proteinaceous Cement of the Barnacle Amphibalanus amphitrite

7.1 Permissions

The material in this chapter is presented with permission from Sullan, R. M. A.; Gunari, N.;

Tanur, A. E.; Chan, Y.; Dickinson, G. H.; Orihuela, B.; Rittschof, D.; Walker, G.C. Nanoscale

structures and mechanics of barnacle cement. Biofouling 2009, 25, 263-275.

7.2 Abstract

In this work, we investigate the proteins comprising barnacle cement, which is an adhesive

nanomaterial for firmly attaching the organism to a substrate. The presence of β-sheet

components within the cement was established via FTIR spectroscopy. Rod shaped structures

were identified by AFM and SEM imaging, and their organic nature was determined with EDX.

Chemical staining revealed that the rods contained beta-sheet structure components. AFM

indentation on the rods indicated a stiffer nature of these structures compared with other

structures within the cement.

7.3 Introduction

Proteins are nature’s designer nanomaterial, formed by directed self-assembly of amino acids

into a polypeptide chain (via transcription and translation from the blueprints encoded within

genes). An immense variety of polypeptides is possible, due to the combinatorial possibilities

afforded by the 20 types of amino acids. Adding to this richness of diversity, polypeptides fold

into specific structures, dictated by the interactions between their constituent amino acids and

with their surrounding solvent. Furthermore, individual proteins can associate to form larger

structures. This so-called hierarchical design is an important concept and goal in

nanotechnology, in which nanoscale components come together to form functional materials. It

is a protein’s structure which dictates its function, and as a result, an active area of research

concerns the determination of protein structure in order to better understand how it functions in

its particular physiological roles.

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The sequence of amino acids within a polypeptide is known as its primary structure. When a

protein folds, local segments form repeating structures, stabilized by hydrogen bonds. These

structures are known as secondary structures, of which the most common are α-helices, random

coils, and β-sheets. The overall three-dimensional structure of a folded protein, consisting of a

number of secondary structures, is called the tertiary structure. Proteins with a globular tertiary

structure are generally water soluble and many function as enzymes. Proteins with a fibrous

tertiary structure, such as collagen and keratin, tend to function as structural materials.1

7.3.1 FTIR Characterization of Protein Secondary Structure

Characterization of proteins by FTIR can yield information about the secondary structure by

probing the vibrational modes of functional groups and atomic configurations. A resulting

spectrum contains several well-known features denoted as amide bands, as shown in Figure 7.1.

Figure 7.1 Typical FTIR spectrum of barnacle glue on CaF2 substrate.

The band used most often for the determination of secondary structure is the amide I Band,

which spans the 1600 – 1700 cm-1

spectral region. This band arises primarily from C=O

stretching modes, with minor contributions from out-of-phase CN stretching, CCN deformation,

and NH in-plane bending. Because of these modes, the amide I Band is sensitive to protein

backbone structure, and is not affected by side chains. The amide I Band is centered at around

1650 cm-1, and for proteins with β-structures, band splitting occurs due to a transition dipole

coupling (TDC) mechanism. TDC is essentially a resonance interaction between oscillating

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dipoles of neighbouring groups, in this case amide groups, and the coupling depends on their

relative orientation and distance.

In terms of interpreting FTIR spectra of proteins, two techniques are commonly used: the second

derivative method,2 and the deconvolution method.

3 Taking the second derivative of a spectrum

enhances subtle changes in the line shapes of bands, thereby revealing distinct secondary

structure band positions within the broad amide I Band. The band center positions obtained from

the second derivative method can be further used as input parameters for the deconvolution

method. This method assumes a Lorentzian line shape for the original unresolved components,

and Gaussian band shapes for deconvolved components.

7.3.2 Barnacle Cement: Proteinaceous Glue

Sessile marine fouling organisms such as mussles and barnacles employ proteinaceous glues in

order to anchor themselves to substrates. In some species, the proteins responsible for the

attachment strength have been identified.4 In this study, the link between adhesive

composition/morphology and mechanical toughness is explored.


7.4.1 Barnacle Rearing

Adult A. amphitrite (= B. amphitrite) on silicone (Silastic® T2 (Dow Corning) or Veridian®

(International Paint)) panels were prepared at Duke Marine Laboratory (Rittschof et al. 1984;

Holm et al. 2005), and shipped overnight to the University of Toronto. The panels were placed in

a 45 L fish tank filled with 35 ppt artificial seawater (Marineland, Instant Ocean Mix, Ohio) at

room temperature under a constant 12 h light/dark cycle. Aliquots (1.5–2 ml) of nauplius larvae

of the brine shrimp Artemia sp. were fed to the barnacles daily. Before performing experiments,

the barnacles were removed from the silicone panels in shear using a mechanical force gauge

(Imada, Northbrook, Illinois), following the procedure in ASTM D5618–94,5 to ensure future

reference capabilities.

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7.4.2 Fourier Transform Infrared (FTIR) Spectroscopy

Barnacles with opaque glue were detached from Veridian panels and reattached onto CaF2

windows for two days and kept in 35 ppt seawater at room temperature. Reattached barnacles

were forced off the CaF2 windows before IR spectroscopy was performed on the remaining

gummy cement. Fresh glue secreted by the adhesive ducts of barnacles was also collected and

smeared on CaF2 windows.

IR spectra at 2 cm-1

resolution on the cement were collected using an FTIR spectrometer

equipped with a liquid nitrogen cooled MCT detector and dry air purger (Nicolet, Nexus 470,

Minnesota). The region from 1600 to 1700 cm-1

(corresponding to the amide I band) was

examined in detail. A clean CaF2 window with no cement sample was scanned to provide a

background spectrum.

The resulting spectra were processed via Fourier self-deconvolution, followed by peak-fitting, as

per the well-established method of Byler and Susi.3 First, the second derivative of the original

FTIR spectrum was taken in order to identify the peak positions of the multiple secondary

structure components within the Amide I band. Next, Fourier self-deconvolution was performed

on the original FTIR spectrum in order to sharpen the spectral features (secondary structure

components) present in the Amide I band. This process was performed with the FTIR analysis

software (GRAMS, Salem, NH), which uses the Griffiths-Pariente method. This method utilizes

two filters, an exponential filter of the form e2πγx

, and a low pass smoothing filter. The

exponential filter performs the deconvolution; but in doing so, it also increases the noise in the

spectrum. Therefore, the smoothing filter is used to compensate for the increased noise. The

software accepts two input parameters: “γ” (= full width at half height of the widest resolvable

peak), and “Smoothing %”. Values of γ = 6.5 cm-1

(following the parameters chosen by Byler

and Susi3), and Smoothing % = 75 were used. The frequencies of the resulting resolved bands

were checked against those identified in the second derivative spectrum. The deconvolved

spectrum was then subjected to peak fitting in order to determine the integrated areas of the

resolved bands. The Levenberg–Marquadt method was used, assuming a Lorentzian line shape

for the deconvolved spectrum and Gaussian peak shapes for the fit. The integrated areas were

then used to estimate the percentage of each major type of secondary structure (eg. α-helix,

random coil, β-sheet, turns) found in the barnacle protein. Uncertainties when using this

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approach are typically 10%.6 Bands were assigned to their respective secondary structures based

on the summary of amide I band IR spectral features in H2O by Barth.7

7.4.3 Atomic Force Microscopy (AFM) Imaging and Indentation

Barnacles with opaque glue were detached from Veridian panels and reattached onto glass

coverslips for two days and kept in 35 ppt seawater at room temperature. Reattached barnacles

were forced off the glass coverslips in shear, and AFM experiments were carried out on the

remaining cement in artificial seawater.

AFM topographic images of the cement were obtained using tapping (non-contact) mode

(Asylum Research, MFP 3D, Santa Barbara, CA). A Si3N4 probe (Veeco, DNP, California) was

used, with a tip radius of ~25 nm. The spring constant was determined to be 0.58 N/m by the

thermal noise method.8 Indentation measurements were obtained in contact mode on various

morphologies identified in the topographic images. Force versus indentation curves were

obtained from the deflection of the AFM cantilever as a function of the piezo vertical

displacement plots. The data were processed using Igor Pro software (Wavemetrics, Portland,

OR) and fit with the Sneddon-Hertz model (Equation 7.1) in order to extract the Young’s

modulus.

Equation 7.1 2/3

2 )1(3

4

v

REF Y

In the expression above, F is the loading force [N], EY is Young’s modulus [Pa], R is the radius

of curvature of the tip [m], δ is the indentation [m], and ν is the Poisson’s ratio, which is taken

here as 0.5.9, 10

7.4.4 Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray (EDX)

Barnacles with opaque glue were removed from the silicone panels and reattached onto

aluminum foil for longer than one week during which time they were fed with nauplius larvae of

the brine shrimp Artemia sp. daily. The barnacles were detached and the remaining cement on

the foil was coated with carbon for imaging in the SEM (S-570, Hitachi, Japan). EDX spectra

(Oxford Instruments, Inca Systems, United Kingdom), were then obtained on the fibrillar

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features observed under the SEM. In all SEM-EDX measurements, a 5 keV beam energy was

used, resulting in an interaction volume of ~1 μm3.

7.4.5 Chemical Staining

Thioflavin T (ThT) and Congo red were used as purchased (Sigma-Aldrich, Madison). ThT

binding was carried out at room temperature as described by Mostaert et al.11

Barnacles with

opaque glue were detached from Veridian panels and reattached onto CaF2 windows for a

maximum of two days and kept in artificial seawater at room temperature. The cement remaining

on a CaF2 window after the barnacle was removed was stained with 10 mM ThT for 5 min. It was

then rinsed with excess 18 MΩ deionized water (Millipore, Milli-Q), and air-dried. Images were

taken with a laser confocal microscope (Leica, TCS SP2, Germany). For Congo red staining, the

barnacles were reattached to glass coverslips for two days and then removed; glass was used

instead of CaF2 because of the birefringence of CaF2. The cement remaining on the coverslip was

stained with 0.5% Congo red in 50% ethanol in water for 5 min. It was then rinsed with excess

18 MΩ water and blow dried with N2 gas prior to examination using the same microscope as

before, now equipped with crossed polarizers.

7.5 Results

7.5.1 FTIR Spectra

The transmission FTIR spectrum of barnacle cement on a CaF2 substrate gave signatures of β-

sheet and a large fraction of random coil conformations. Figure 7.2 shows the deconvoluted

FTIR spectrum for cement of A. amphitrite, fit with four bands (SE: 0.0003, correlation value R2

= 0.999). To check the validity of this fit, multiple fits were performed on the same deconvoluted

FTIR spectrum, from two to 11 bands. The SE did not undergo significant improvement for fits

with more than four bands. Assignment of the four bands to their corresponding secondary

structure resulted in the identification of a large random coil component at 1655 cm-1

(79%), and

smaller low and high frequency β-sheet components, at 1623, 1637 and 1692 cm-1

. The

distribution of secondary structures obtained from the FTIR spectra by the peak fitting algorithm

is summarized in Table 7.1. Note that this distribution applies to the barnacle cement as a whole,

which is made up of a number of different proteins in unknown proportions.

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Figure 7.2 FTIR spectra of the bulk cement from A. amphitrite. Top spectrum, the original data before processing, offset for clarity; Bottom spectrum, the deconvoluted data, with a four-band peak fit showing peaks corresponding to random coil structure (1655 cm

-1) and low (1623, 1637 cm

-1) and high (1692 cm

-1) frequency β-sheet components.

Table 7.1 IR peaks and the corresponding fraction of the observed secondary structures found in gummy barnacle cement sample

Secondary structure band positions in H2O (cm-1

) Relative peak area fraction (%)

Low-frequency β-sheet components (1623, 1637) 17

Random coil (1655) 79

High-frequency β-sheet component (1692) 4

7.5.2 AFM Images, Force Curves, and Moduli Histograms

After reattachment to glass substrates, barnacles typically to produced ~1 mm wide glue deposits

below the periphery of their baseplate, consistent with the location of the glue ducts. After

pushing these barnacles off the substratum, the residual glue on the substratum was imaged with

AFM. Typical topography images are shown in Figure 7.3. Mesh structures such as those

reported earlier by Weigemann and Watermann were observed.12

AFM images of the barnacle

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cement in both 35 ppt artificial seawater (Figure 7.3A) and in air (Figure 7.3B) exhibit this mesh

morphology. The mesh structure in Figure 7.3 is the general picture of the adhesive when

scanned in a larger area.

Figure 7.3 AFM topographic image of the barnacle cement in (A) 35 ppt sea water and (B) air. Gray-scale provides

height reference (right hand of each image). Images are 15 x 15 m2.

Upon zooming in, the mesh in Figure 7.3A was observed to be composed of a mixture of

different structures such as those shown in Figure 7.4A–C. Figure 7.4A shows clusters of

globular structures with diameters ranging from 60 to 100 nm. Pearl-like arrangements of the

globular aggregates are clearly seen. Smaller globular structures (10–30 nm) and a small, rod-

like structure of 11 nm in diameter and ~300 nm length were also observed (Figure 7.4B).

Figure 7.4C shows a larger, more regular rod-shaped structure. An unstructured aggregate

(Figure 7.4D) is also seen in the matrix (Figure 7.4E). Thus, the bulk cement is composed of the

mesh comprised of the structures in Figure 7.4A–C and of the matrix (Figure 7.4E) with some

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unstructured aggregates (Figure 7.4D). There is no specific pattern or localization of those

structures. The relative fraction of those occurring is variable from one barnacle sample to

another, even from within the same species.

Figure 7.4 The bulk barnacle cement is composed of both the mesh structure and the matrix. A: Globular aggregates. B: Smaller rod-like and smaller globular features. (C) A larger, more regular rod-shaped structure

comprising the mess. (D) An unstructured aggregate in the matrix. (E) Bulk glue. The black dots in figures (C) to (E) indicate the point of indentation.

AFM nanoindentation was performed on the rod-shaped structure (Figure 7.5A), the unstructured

aggregate (Figure 7.5B), and the matrix (Figure 7.5C), to determine and compare their elastic

moduli. The black dot in each figure indicates the point of indentation. Individual fits for the

larger, more regular rod-shaped structure, the unstructured aggregate, and the matrix and the

corresponding histogram of elastic modulus are shown below the images of each structure in

Figure 7.5A–C.

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Figure 7.5 In situ determination of the elastic modulus of the barnacle cement. Elastic modulus distribution of individual nanostructures/components observed by AFM. (A) Fit to the force-indentation plot with the corresponding histogram of the elastic modulus for larger, more regular rod-shaped structure. (B) The unstructured aggregate. (C)

The matrix. Insets in (B) and (C) are linescans (taken across the feature, as shown by the line drawn across it) of the larger, more regular rod-shaped structure and unstructured aggregate, respectively. For both rod-shaped and

unstructured aggregate structures, the height of the feature is ~250 nm.

The corresponding force-indentation plots were fit by Sneddon mechanics.9 The solid line

represents the fit to indentation data by the paraboloidal tip model (Equation 7.1). The larger,

more regular rod-shaped structure (Figure 7.5A) was found to have a Young’s modulus in the

range of 20–90 MPa; the unstructured aggregate, 0.20–2 MPa; and the matrix, 1–10 MPa. Insets

in Figure 7.5A and B are the linescans (taken across the feature as shown by the line drawn

across it) of the larger, more regular rod-shaped structure and unstructured aggregate,

respectively. For both rod-shaped and unstructured aggregate structures, the height of the

indented feature is ~250 nm and the rod had a diameter of ~600 nm. Indentations were <20% of

the sample thickness so finite thickness effects that can affect the predicted modulus are

minimal.13

129

7.5.3 SEM and EDX

To determine the chemical composition of the rod-shaped features observed in the AFM, SEM

images coupled to EDX were obtained. The rod-shaped features observed under SEM (Figure

7.6A) showed high counts of carbon, nitrogen and oxygen in the EDX line scan spectrum (Figure

7.6B) suggestive of the organic nature of the components. Figure 7.6C and D shows an EDX

area mapping, indicating the spatial location of the elements in a given region. Consistent with

the EDX line scan in Figure 7.6A and B, the rod-shaped features in Figure 7.6C showed signals

of C, N and O (Figure 7.6D) coming from the rod-shaped structures, again indicative of the

organic nature of the components. The high counts of Al for both the EDX line scan and area

mapping are attributed to the background aluminum foil where the cement was attached. The Mg

background was similar to Al. The F elemental map is shown as a control. The elemental X-ray

lines are indicated for each line scan and map (a = α).

Figure 7.6 SEM images and EDX spectra of the barnacle cement resettled on aluminum foil. (A) SEM image showing the rod-shaped structures and the corresponding (B) EDX line scan spectra showing high counts of elemental

carbon, nitrogen and oxygen coming from the rod-shaped structures. (C) SEM image of the rod-shaped structures and the corresponding (D) elemental maps of the elements. Fair signals of C, N and O coming from the rod-shaped structures are obtained. The elemental X-ray lines from which the EDX signals were derived are indicated for each

line scan and map (a = α)

130

7.5.4 Amyloid-Selective Staining

To test for the presence of amyloids within the cement, chemical staining with amyloid-selective

dyes was performed. ThT staining resulted in fluorescence of the rod-shaped structures (Figure

7.7B), and Congo-red staining resulted in apple-green birefringence under cross-polarized light

(Figure 7.7D). For comparison, an image of the barnacle cement in the absence of ThT is shown

in Figure 7.7A, and a bright field image of the cement stained with Congo red is shown in Figure

7.7C.

Figure 7.7 Chemical staining images of the barnacle cement with amyloid-selective dyes. (A) Confocal images of the barnacle cement on a CaF2 window without ThT. (B) Stained with ThT. (C) Bright field image of the barnacle cement stained with Congo red. (D) Polarized light image of Congo red sample. Fluorescence and apple-green birefringence

of the rod-shaped structures are observed when the barnacle cement is stained with ThT and Congo red, respectively.

7.6 Discussion

7.6.1 Significance of β-sheet Conformation in Barnacle Cement

AFM indentation on some of the morphologies seen in Figure 7.4A–E gave a wide range of

elastic modulus values as shown in Figure 7.5A–C, indicating that the elasticity of the cement is

heterogeneous. From the force indentation plot in Figure 7.5A, the depth of indentation changes

only slightly as the force is increased for the rod-shaped structure. In the case of the unstructured

aggregate and of the matrix, the depth of indentation increases as the force is increased. This is

131

reflective of the more compliant nature of these morphologies. These results indicate the stiffer

nature of the larger, more regular rod-shaped structure than both the unstructured aggregate and

the matrix. This is also evident from the magnitude of their elastic moduli. The rod-shaped

structure has an elastic modulus in the range of 20 – 90 MPa, which is similar in range to what

has been observed for the amyloid fibrils in insulin (5–50 MPa).14

Materials with higher elastic modulus typically require more stress to fracture.15

The higher

elastic modulus value for the rod-shaped structure suggests that it has a stiffer nature with higher

cohesive forces. This causes the load applied to it at any point to be distributed across its length,

making a composite more resistant to fracture. A simple way to understand how the rod-shaped

structures could contribute to the cement strength is to consider that they distribute load across a

relatively wide region of the bulk cement. The results do not allow determination of how

important this effect is for barnacle cement toughness, but their presence needs to be considered

in a detailed model for fracture. Kamino et al.16

previously suggested that the 100 kDa protein

present in the barnacle cement of M. rosa may be similar to the proteins involved in the

formation of amyloid fibrils. This was based on the abundance of β-sheet structures in the 100

kDa barnacle protein, its alternating hydrophobic and hydrophilic profile, and its very insoluble

nature. It was reported that the pattern of alternating polar and non-polar residues in a cross-β

structure is essential in the formation of the insoluble amyloid fibrils.17

Recently, amyloid-like sequences have been found in the primary structure of the protein in the

bulk cement of the M. rosa.4 Moreover, the 100 kDa cement protein of M. rosa is particularly

rich in isoleucine (Ile), valine (Val) and threonine (Thr) residues, which are the three amino acid

residues reported to have the highest propensity to form the β-sheet structure.18

Chemical staining of the barnacle cement with amyloid-selective dyes was done to check for the

possible presence of amyloids in the cement proteins of A. amphitrite. The apple-green

birefringence under polarized light when stained with Congo red (Figure 7.7C), and fluorescence

of ThT (Figure 7.7B) indicate the presence of an amyloid fibril structure in the barnacle cement.

Both ThT and Congo red dyes are known to be amyloid-selective.19-22

The fluorescence observed

in the presence of ThT and the apple-green birefringence with Congo red are related to the cross-

132

β core structure, both of which are indications of the binding of the dyes to an amyloid fibril

structure in the barnacle cement.

A variety of rod-shaped structures (or fibre-like features) were observed under the optical

microscope, but only a small portion gave an apple-green birefringence and fluorescence when

stained with Congo red and ThT, respectively. This indicates that the amyloid fibrils in the

barnacle cement only comprise a small fraction of the bulk cement. Such a small fraction is still

significant because, in most fiber reinforced composite materials, fibers need to be present in

only a minute fraction to lead to a considerable increase in the toughness of the material.23, 24

Non-amyloid fibers could also play a similar mechanical role.

7.7 Conclusions

Different nanoscale structures were observed in the bulk cement of the barnacle A. amphitrite

through AFM, viz. a mesh which is composed of rod-shaped, threadlike and globular structures

and a matrix with unstructured aggregates. Indentation data suggested a stiffer nature of the rod-

shaped structure over the other components of the bulk cement. The EDX spectrum of the rod-

shaped structures was suggestive of its organic nature. The FTIR spectra supported the presence

of a β-sheet conformation, whereas the results of chemical staining with both ThT and Congo red

confirmed the presence of a small fraction of amyloid fibrils in the bulk cement.

7.8 Contributions

The author performed the FTIR, SEM, and EDX measurements and analysis. The material in

this chapter, based on Sullan, R. M. A.; Gunari, N.; Tanur, A. E.; Chan, Y.; Dickinson, G. H.;

Orihuela, B.; Rittschof, D.; Walker, G.C. Nanoscale structures and mechanics of barnacle

cement. Biofouling 2009, 25, 263-275, has been presented with an emphasis on A. E. T.’s

contributions.

7.9 References

1. Matthews, H. R.; Freedland, R. A.; Miesfeld, R. L., Proteins. In Biochemistry - A Short

Course, John Wiley & Sons: New York, 1997; pp 25–43.

2. Susi, H.; Michael Byler, D., Protein structure by Fourier transform infrared spectroscopy:

Second derivative spectra. Biochem. Bioph. Res. Co. 1983, 115, 391–397.

133

3. Byler, D. M.; Susi, H., Examination of the secondary structure of proteins by

deconvolved FTIR spectra. Biopolymers 1986, 25, 469–487.

4. Kamino, K., Underwater adhesive of marine organisms as the vital link between

biological science and material science. Mar. Biotechnol. 2008, 10, 111–121.

5. International, A., Standard test method for measurement of barnacle adhesion strength in

shear. 1997, 2005; Vol. Designation D5618–94.

6. Dousseau, F.; Pezolet, M., Determination of the secondary structure content of proteins in

aqueous solutions from their amide I and amide II infrared bands. Comparison between

classical and partial least-squares methods. Biochemistry 1990, 29, 8771–8779.

7. Barth, A., Infrared spectroscopy of proteins. Biochimica et Biophysica Acta (BBA) -

Bioenergetics 2007, 1767, 1073–1101.

8. Hutter, J. L.; Bechhoefer, J., Calibration of atomic-force microscope tips. Rev. Sci.

Instrum. 1993, 64, 1868–1873.

9. Sneddon, I. N., The relation between load and penetration in the axisymmetric boussinesq

problem for a punch of arbitrary profile. Int. J. Eng. Sci. 1965, 3, 47–57.



20, 279–289.

11. Anika, S. M.; Suzanne, P. J., Beneficial characteristics of mechanically functional

amyloid fibrils evolutionarily preserved in natural adhesives. Nanotechnology 2007, 18,

044010.

12. Wiegemann, M.; Watermann, B., Peculiarities of barnacle adhesive cured on non-stick

surfaces. J. Adhes. Sci. Technol. 2003, 17, 1957–1977.

13. Akhremitchev, B. B.; Walker, G. C., Finite sample thickness effects on elasticity

determination using atomic force microscopy. Langmuir 1999, 15, 5630–5634.

14. Guo, S.; Akhremitchev, B. B., Packing density and structural heterogeneity of insulin

amyloid fibrils measured by AFM nanoindentation. Biomacromolecules 2006, 7, 1630–

1636.

15. Griffith, A. A., The phenomena of rupture and flow in solids. Philos. Trans. R. Soc.

London, Ser. A 1921, 221, 163–198.

16. Kamino, K.; Inoue, K.; Maruyama, T.; Takamatsu, N.; Harayama, S.; Shizuri, Y.,

Barnacle cement proteins. J. Biol. Chem. 2000, 275, 27360–27365.

17. West, M. W.; Wang, W.; Patterson, J.; Mancias, J. D.; Beasley, J. R.; Hecht, M. H., De

novo amyloid proteins from designed combinatorial libraries. Proc. Natl. Acad. Sci.

1999, 96, 11211–11216.

18. Xiong, H.; Buckwalter, B. L.; Shieh, H. M.; Hecht, M. H., Periodicity of polar and

nonpolar amino acids is the major determinant of secondary structure in self-assembling

oligomeric peptides. Proc. Natl. Acad. Sci. U.S.A.1995, 92, 6349–6353.

19. Missmahl, H. P.; Hartwig, M., Polarisationsoptische untersuchungen an der

amyloidsubstanz. Virchows Arch. Path. Anat. 1953, 324, 489–508.

134

20. Janigan, D. T., Experimental amyloidosis. Structural relationships of amyloid and

reticulin in tissue sections and isolated preparations. Am. J. Pathol. 1966, 49, 657–678.

21. Stopa, B.; Piekarska, B.; Konieczny, L.; Rybarska, J.; Spolnik, P.; Zemanek, G.;

Roterman, I.; Krol, M., The structure and protein binding of amyloid-specific dye

reagents. Acta Biochim. Polonica 2003, 50, 1213–1227.

22. Khurana, R.; Coleman, C.; Ionescu-Zanetti, C.; Carter, S. A.; Krishna, V.; Grover, R. K.;

Roy, R.; Singh, S., Mechanism of thioflavin T binding to amyloid fibrils. J. Struct. Biol.

2005, 151, 229–238.

23. Melanitis, N.; Galiotis, C.; Tetlow, P. L.; Davies, C. K. L., Interfacial shear stress

distribution in model composites part 2: Fragmentation studies on carbon fibre/epoxy

systems. J. Compos. Mater.1992, 26, 574–610.

24. Savastano Jr, H.; Warden, P. G.; Coutts, R. S. P., Potential of alternative fibre cements as

building materials for developing areas. Cem. Concr. Compos. 2003, 25, 585–592.

135

8 Summary and Outlook

8.1 Summary of Thesis

The study of nanomaterials requires a multi-faceted approach in order to determine the

morphology, chemical composition, and mechanical properties of the nanoscale features. Each

technique provides complementary information, helping to build a fuller understanding of the

links between material structure, properties, and function. For the study of boron nitride

nanotubes (Chapters 3-5), scanning and transmission electron microscopy (SEM and TEM) were

essential techniques for the characterization of nanotube morphology and structure. Energy

dispersive x-ray (EDX) spectroscopy and mapping, combined with SEM, identified the presence

of elements of interest that were expected to be present in the synthesized nanotubes. B and N

signals were observed from the nanotubes, and O, Fe, Mg, and Al were also detected within the

synthesized product prior to purification as remnants of the precursor and catalyst powders.

SEM and EDX were also indispensable tools in the identification of aragonite and Mg-calcite

phases within the calcareous tube of H. dianthus (Chapter 6). It was possible to choose specific

locations to probe with EDX based on the SEM images, such that the different CaCO3

polymorphs could be identified and correlated with layers within the tube based on the Sr signal

for aragonite, and the Mg signal for Mg-calcite. In Chapter 7, EDX was used to determine that

the nanoscale rods observed within the barnacle cement were organic and proteinaceous in

nature, through the observation of C, N, and O signals.

Fourier transform infrared spectroscopy (FTIR) was useful for characterizing the bulk chemical

composition of boron nitride nanotubes (BNNTs) in Chapters 4 and 5. Shifts in the peak

position of the E1u TO mode for h-BN were qualitative indications of the crystallinity of the

nanotubes. It was also able to provide signatures of nanoscale structures, namely the β-sheet

conformation within proteins in barnacle cement (Chapter 7). In addition, in Chapter 6, FTIR

determined that the soluble and insoluble organic matrices within the calcareous shell of H.

dianthus contained substantial amounts of carboxylated and sulphated polysaccharides,

molecules which have been demonstrated to mediate CaCO3 mineralization through the

interaction between their functional groups and ions within seawater.

136

Atomic force microscopy (AFM) was used to determine the bending modulus of individual

BNNTs in Chapter 5. The force mapping technique was employed in order to determine the

boundary conditions of each nanotube, and revealed that the assumption made in many studies

that such systems possessed only clamped ends was false. As a result, the bending modulus was

determined using the appropriate beam models for each type of boundary condition. AFM

nanoindentation was used to measure the Young’s modulus of biological materials in Chapters 6

and 7, in the investigation of tubeworm and barnacle adhesives both in air, and in an artificial sea

water solution. The versatility of the AFM is showcased through the above studies.

8.2 Outlook

8.2.1 Boron Nitride Nanotubes

The potential of BNNTs as an advanced functional material remains to be fully realized. Most

synthesis methods require further development in order to produce industrial scale yields. The

unique set of properties that BNNTs possess makes it a worthwhile endeavour, however. Several

exciting possible applications for BNNTs include their incorporation as an emitting layer in

organic light emitting diode architectures, outputting UV light; thermally conductive electrically

insulating composite polymer encapsulants for electronics; additives to ceramics and metals to

enhance damping characteristics; and targeted boron neutron capture therapy agents for the

treatment of cancerous tumors.1-4

8.2.2 Nanomaterials in Nature

The study of the materials design principles employed by organisms such as tubeworms and

barnacle continues to inspire the design and synthesis of novel biomimetic materials. In

particular, biomineralization offers lessons in the growth of nano and microcrystals in mild

aqueous conditions, with exceptional control over chemical composition and morphology. The

organic molecules which mediate biomineralization, including polysaccharides and proteins,

give materials scientists new ideas and new tools with which to create new materials.5-7

137

8.3 References



141104–3.

2. Zhi, C.; Bando, Y.; Terao, T.; Tang, C.; Kuwahara, H.; Golberg, D., Towards

thermoconductive, electrically insulating polymeric composites with boron nitride

nanotubes as fillers. Adv. Funct. Mater. 2009, 19, 1857–1862.

3. Sueyoshi, H.; Rochman, N. T.; Kawano, S., Damping capacity and mechanical property

of hexagonal boron nitride-dispersed composite steel. J. Alloy Compd. 2003, 355, 120–

125.

4. Ciofani, G.; Raffa, V.; Yu, J.; Chen, Y.; Obata, Y.; Takeoka, S.; Menciassi, A.;

Cuschieri, A., Boron nitride nanotubes: A novel vector for targeted magnetic drug

delivery. Curr. Nanosci. 2009, 5, 33–38.

5. Barnard, A. S.; Russo, S. P., Modeling the environmental stability of FeS2 nanorods,

using lessons from biomineralization. Nanotechnology 2009, 20, 115702.

6. Oaki, Y.; Adachi, R.; Imai, H., Self-organization of hollow-cone carbonate crystals

through molecular control with an acid organic polymer. Polym. J. 2012, 44, 612–619.

7. Nudelman, F.; Sommerdijk, N. A. J. M., Biomineralization as an inspiration for materials

chemistry. Angew. Chem. Int. Ed. 2012, 51, 6582–6596.

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