Investigation of the molecular organisation of antibacterial insect wing surfaces
Submitted in total fulfilment of the requirements for the degree of
Doctor of Philosophy
by
Song Ha Nguyen
Department of Chemistry and Biotechnology
Faculty of Science, Engineering, and Technology
Swinburne University of Technology
May 2015
______Abstract
It has been known for some time that the surfaces of many plants and insects exhibit superhydrophobicity and self-cleaning properties. Plant leaves have been intensively studied for over a decade since the discovery of the “lotus effect”, where water droplets readily roll off the surfaces of lotus leaves. The water droplets, once landed on the surfaces of the leaves, retain their spherical shape. This arises from the superhydrophobicity of the surface. When this surface is tilted on a small angle (θtilt < 10°), the water droplets roll off the surface of the leaves, collecting all the dirt and contaminants as they go, affording the leaves the ability to ‘self-clean’. A large amount of information has been obtained regarding the surface properties of lotus leaves; however the surface properties of insects have not received as much attention in this context, despite the fact that they often present similarly effective self-cleaning properties. This work was undertaken in an effort to fill this gap in scientific knowledge.
Insect wings surfaces are covered by an epicuticle layer that is composed of hydrophobic substances, which are responsible for imparting the superhydrophobic and self-cleaning properties to some species. The surfaces of dragonfly wings are covered by an array of nanopillars, which were recently discovered to have bactericidal activity. In this work, the molecular organisation of dragonfly wings was investigated to gain an insight into how the individual nanopillars were constructed and behave when coming into contact with attaching bacterial species. Over 60 different substances were identified as comprising the insect wing surfaces, including aliphatic hydrocarbons and fatty acids. The precise chemical compositions not only directly contribute to the superhydrophobic properties of the wings, but also determine their nano-architectures. A new sub-layer of the dragonfly wing epicuticle is proposed, the meso-epicuticle, which is composed only of aliphatic hydrocarbons. This meso-epicuticle is distinct from the outer- and inner-epicuticular layers.
The bactericidal properties of dragonfly wings have been reported to arise purely due to physical means, and hence the surface morphologies and topographies of the dragonfly wings were characterised to investigate the nature of the mechanism(s) responsible for the bactericidal effect. The wing surfaces of two species of dragonfly, Hemianax papuensis and Diplacodes bipunctata, were investigated for this purpose. It
ii was suggested that mechano-bactericidal and self-cleaning properties of the wings are two distinct mechanisms that dragonflies have developed in order to cope with bacterial contamination. Wetting and adhesion are affected by both the surface topography and surface chemistry with the presence of precisely defined topographical features resulting in bactericidal properties. Moreover, a subtle change in the morphological parameters may instead enhance the ability for the surface to self-clean.
This knowledge about the relative contribution of molecular organisation and surface topology to the overall surface properties of dragonfly wings was used to inform the synthetic stage of the project. Eicosane, docosane, palmitic acid and stearic acid, all of which were found to be major components present in the epicuticular layer of dragonfly wings, were allowed to self-assemble on the surface of highly-ordered pyrolytic graphite (HOPG) to produce an ordered structure, which may replicate the antibacterial properties of dragonfly wings. Both of the alkanes assembled into ‘micro- rodlet’ type structures, while the fatty acids recrystallised into sharper ‘micro-blades’. In the case of the alkane micro-rodlets, bacterial cells coming into contact with the substrate surface containing these rodlets were found to align along the alkane crystallites. The mechanism responsible for this behaviour was determined to be a result of the air-pockets trapped between the surface structures, and hence the bacteria were limited in their colonisation to the areas of the surface that could be accessed by water. Bacterial alignment on the fatty acid interfaces was less obvious; however these surfaces were capable of killing the bacteria. The structure of the micro-blades was integral in this case; it appeared that the high radius of curvature of the sharp edge were able to rupture the bacterial cells through high stress upon contact and adhesion.
The results of this study contribute to the general knowledge available on the molecular organisation of the epicuticle of dragonfly wings, with specific contribution to the surface properties of the wings, such as their superhydrophobicity, bactericidal behaviour and their ability to self-clean. The key mechanisms responsible for each of these properties were proposed to be closely related to the interplay between the wettability of the surface and the ability of the surface to retain air between the topographical structures. This study also introduced a fast and facile method of producing artificial surfaces, being the self-assembly of lipids onto ordered substrata. The resulting surfaces were found to be successful in exerting antibacterial effects both before and after bacteria attachment. iii
Acknowledgements
First and foremost, I would like to acknowledge and send my greatest appreciation to my primary supervisor, Professor Elena Ivanova for all of her advice and support during the course of my study. Just like a mother figure, her inspiration, motivation, and encouragement were the driving forces pushing me forward to completion of this work. She does not mind to stand up for her students whenever they are treated unfairly. Despite her busy schedule, she has always taken time to talk with me whenever I stumbled across problems, and I’m really grateful for that.
Similarly, I would also like to thank Professor Russell Crawford, Professor David Mainwaring, and Dr. Peter Mahon for co-supervising the project, and for spending time helping me on various manuscripts and papers that I prepared. Being supervisors of a non-English speaking student would not be an easy job and they have always been patient with me, I really appreciate that. Special thank you to Dr. Peter Mahon, who is my supervisor and also supporter, for talking to me caringly as a friend rather than simply a teacher and I’m very thankful for that.
To Vy and Hiệp, sometimes I questioned our friendships, and felt doubtful, but when I look around I always see you guys right there, and I think that’s what matters the most. I’m thankful for this confusing friendship because when I need your supports the most, you guys never fail me, and I really appreciate that.
To Nguyên and Chris, you two are my rocks. Thanks for being here with me, for your co-operation in numerous impossible-sounding missions, and for your emotional support. When things are down, you guys have always brought me back to my ground.
I would like to send my gratitude to Dr. Jafar Hasan, Dr. Mohammad Al Kobaisi, Dr. Vi Khanh Truong, and Dr. Hooi Jun Ng for their knowledge and assistance in operating various instruments, and techniques. Also, thank you to Veselin ‘Vanya’ Boshkovikj, you are one of the funniest guys I have ever met. You guys made my PhD life more colourful and much easier.
Thank you to Dr. James Wang for his assistance in performing SEM experiments. Similarly, I would like to thank Dr. Mark Tobin and Dr. Ljiljana Puskar for helping with our work at the Australian Synchrotron, FTIR beamline; without their expertise, I would not be able to become a regular there.
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A special thanks and gratitude to chú Ngân, Soula, Savithri, Chris, Rebecca and all the laboratory technicians who have helped me throughout the course of this work. Talking to you all made me sometimes forget how tough PhD life can be.
To all my friends here at Swinburne, you guys have made my every day more fun and enjoyable. I will never forget our little office where the temperature is always above 30°, with the white board where everyone writes their thoughts. It’s a pity that around the time I write all this, you guys are not around. I would rather say my thankful words out loud individually, but I’m afraid that I’ll in tears before I complete my sentences, so I send my special gratitude here, to Yến, Matt, Kaylass, Shanthi, Phong and Qudsia.
To our lovely and handsome dog, Sammy. You are my mascot and on my wallpaper screen; looking at you make my days go by much easier, so if you can understand, I would like to say thanks to you.
Especially, thank you to my new and current family. The word by itself says a lot how much you all mean to me. To my new family, I came along and you all took me in with your kindest hearts, treated me as part of the family, giving me support and advice which always touch my heart. To my husband, Dr. Hayden Webb, I don’t know how many words are enough to express my gratitude to you for your endless emotional support, thanks for always being by my side sharing my happiness and motivating me through the toughest times despite my difficult nature. Before being my friend, my husband, you are my senior, my teacher who does not mind to be harsh to me for my improvement.
Con muốn gửi những lời cám ơn sâu sắc nhất tới ba, mẹ và bé Ngân. Con khô khan, nhưng con hy vọng mọi người hiểu được là con mang ơn mọi người như thế nào. Sự hy sinh thầm lặng của mẹ luôn là động lực vực con đứng dậy trong ư4ng tháng ngày khó khăn. Cha ít nói chuyện với con, nhưng con biết khi con thành công, cha sẽ là người nói nhiều nhất, khoe thành tích con gái cha với mọi người mà cha gặp. Nếu không có hai người, con không có mặt trên đời để giờ đây có thể ngồi đây, tự hào viết ra những dòng chữ ý nghĩa cho đời. Chị cám ơn em gái của chị, chị đi ròng rã 6-7 năm trời, mọi gánh nặng, trách nhiệm làm con đều để em gánh hết. Chị em mình tuy xa mặt, nhưng hy vọng sẽ không cách lòng.
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Declaration
I, Song Ha Nguyen, declare that this thesis is original work and contains no material that has been accepted for the award of Doctor of Philosophy, or any other degree or diploma, except where due reference is made.
I declare that to the best of my knowledge this thesis contains no material previously published or written by any other person except where due reference is made. Wherever contributions of others were involved every effort has been made to acknowledge the contributions of the respective workers or authors.
Signature ______
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List of Publications
Book Chapters
Song Ha Nguyen, Hayden K. Webb, Russell J. Crawford & Elena P. Ivanova (2014). Natural superhydrophobic surfaces. In: Elena P. Ivanova, and Russell J. Crawford, Superhydrophobic surfaces (Elsevier).
Hayden K. Webb, Song Ha Nguyen, Russell J. Crawford & Elena P. Ivanova (2014), Biological interactions with superhydrophobic surfaces. In: Elena P. Ivanova, and Russell J. Crawford, Superhydrophobic surfaces (Elsevier).
Song Ha Nguyen, Hayden K. Webb & Elena P. Ivanova (2014) Natural antibacterial surfaces. In: Elena P. Ivanova, and Russell J. Crawford, Antimicrobial surfaces (Springer).
Hayden K. Webb, Song Ha Nguyen & Elena P. Ivanova (2014) Introduction to antibacterial surfaces. In: Elena P. Ivanova, and Russell J. Crawford, Antimicrobial surfaces (Springer).
Peer-reviewed article
Song Ha Nguyen, Hayden K. Webb, Peter J. Mahon, David Mainwaring, Russell J. Crawford, and Elena P. Ivanova, “Graphite-templated self-organized alkane microcrystal interfaces regulate bacterial attachment”, Biofouling, (2015), [In press]
Song Ha Nguyen, Hayden K. Webb, Jafar Hasan, Mark J. Tobin, David E. Mainwaring, Peter J. Mahon, Richard Marchant, Russell J. Crawford, and Elena P. Ivanova, “Wing wettability of Odonata species as a function of epicuticular waxes spatial area”, Vibrational Spectroscopy, 75 (2015), p. 173-177
Song Ha Nguyen, Hayden K. Webb, Peter J. Mahon, Russell J. Crawford, Elena P. Ivanova, “Natural insect and plant micro-/nanostructured surfaces: An excellent selection of valuable templates with superhydrophobic and self-cleaning properties”, Molecules, 19 (2014), p. 13614-13630.
Song Ha T. Nguyen, Hayden K. Webb, Jafar Hasan, Mark J. Tobin, Russell J. Crawford, Elena P. Ivanova, “Dual roles of outer epicuticular lipids in determining the wettability of dragonfly wings", Colloids and Surfaces B: Biointerfaces, 106 (2013), p.126-134.
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Elena P. Ivanova, Song Ha Nguyen, Hayden K. Webb, Vi Khanh Truong, Jafar Hasan, Robert N. Lamb, Alex Duan, Mark J. Tobin, Peter J. Mahon, Russell J. Crawford, “Molecular organisation of the nanoscale surface structures of the dragonfly Hemianax papuensis wing epicuticle", PLoS One, 8 (2013), e67893.
Song Ha Nguyen, Hayden K. Webb, Jafar Hasan, Mohammed Al Kobaisi, Robert N. Lamb, Alex H.-F. Wu, Peter J. Mahon, David Mainwaring, Russell J. Crawford, Elena P. Ivanova, “Nanomorphology of dragonfly wings - finely-tuned through evolution”, PNAS [Under preparation]
Song Ha Nguyen, Hayden K. Webb, Peter J. Mahon, David Mainwaring, Russell J. Crawford, and Elena P. Ivanova, “Mechanobactericidal fatty acid microblades assembled on highly-ordered pyrolytic graphite”, Small [Under preparation]
Conference presentations with published abstracts
Song Ha Nguyen, Hayden K. Webb, Peter J. Mahon, David E. Mainwaring, Russell J. Crawford, & Elena P. Ivanova, “Antimicrobial activity of self-assembled carboxylic acid crystals on graphite”, III International Conference on Antimicrobial Research (ICAR) 2014
Song Ha Nguyen, Hayden K. Webb, Jafar Hasan, Mark J. Tobin, David E. Mainwaring, Peter J. Mahon, Richard Marchant, Russell J. Crawford, Elena P. Ivanova, “Correlation between the proportion of epicuticular waxes and wettability among various species of dragonfly wing”, 7th International Workshop on Infrared Microscopy and Spectroscopy with Accelerator Based Sources (WIRMS) 2013.
Conference posters with published abstracts
Song Ha Nguyen, Hayden K. Webb, Peter J. Mahon, David Mainwaring, Russell J. Crawford, & Elena P. Ivanova, “Regulation of bacterial attachment through graphite- templated self-organized alkane microcrystal interfaces”, Australian Society of Microbiology Meeting 2014.
Song Ha Nguyen, Hayden K. Webb, Mark J. Tobin, David E. Mainwaring, Peter J. Mahon, Russell J. Crawford, & Elena P. Ivanova, “FTIR detection of different phases of fatty acids forming 3D-assemblies”, Australia Synchrotron User Meeting 2014.
Song Ha Nguyen, Hayden K. Webb, Jafar Hasan, Robert N. Lamb, Mark J. Tobin, Peter J. Mahon, David E. Mainwaring, Russell J. Crawford, & Elena P. Ivanova, viii
“Dissimilar bactericidal activities of nanopatterned dragonfly wing surfaces”, Nano Today 2013.
Song Ha Nguyen, Hayden K. Webb, Jafar Hasan, Vi Khanh Truong, Robert N. Lamb, Xiaofei Duan, Mark J. Tobin, Peter J. Mahon, Russel J. Crawford, Elena P. Ivanova, “Hierarchical construction of the surfaces of dragonfly wings on the micro- and nano-scale”, Australian Society of Microbiology Meeting 2013.
Song Ha Nguyen, Jafar Hasan, Hayden K. Webb, Mark J. Tobin, Russell J. Crawford, Elena P. Ivanova, “Physico-Chemical characterisation of dragonfly wing nanostructures using Synchrotron Infrared Microscopy”, Australia Synchrotron User Meeting 2012
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Table of Contents
Abstract ...... ii
Acknowledgement ...... iv
Declaration ...... vi
List of Publications ...... vii
Book Chapters ...... vii
Peer-reviewed article ...... vii
Conference presentations with published abstracts ...... viii
Conference posters with published abstracts ...... viii
Table of Contents ...... x
List of Abbreviations ...... xv
List of Figures ...... xvii
List of Tables ...... xxviii
Chapter 1. Introduction ...... 1
1.1. Overview ...... 2
1.2. Aims of the study ...... 3
Chapter 2. Literature review ...... 5
2.1. Overview ...... 6
2.2. Arthropod cuticle ...... 6 2.2.1. Morphology of insect wings ...... 6 2.2.2. Aerodynamics of insect flight ...... 7
2.3. The surface of insect wings ...... 9 2.3.1. Concept of self-cleaning ...... 9 2.3.2. Wettability theory ...... 10
2.4. Surface chemistry ...... 13
2.5. Hierarchical structure induced self-cleaning ...... 22 2.5.1. Superhydrophobicity and self-cleaning surfaces in nature ...... 22 x
2.5.1.1. Plant surface structures ...... 23 2.5.1.2. Insect surface structures ...... 25 2.5.2. Superhydrophobicity with additional properties ...... 31 2.5.2.1. Superhydrophobic surfaces with high adhesive force ...... 31 2.5.2.2. Anisotropic superhydrophobic surfaces ...... 33 2.5.3. Hierarchical structure of superoleophobic surfaces ...... 35
2.6. Bacteria ...... 36 2.6.1. Bacterial physiology ...... 36 2.6.2. Biofilm formation ...... 39
2.7. New approaches for controlling biofilm formation ...... 41 2.7.1. Antibiofouling ...... 41 2.7.2. Bactericidal activity of insect wings ...... 43
Chapter 3. Materials and methods ...... 46
3.1. Overview ...... 47
3.2. Materials ...... 47
3.3. Protocols for handing of insect wings ...... 47 3.3.1. Fresh and aged insect wings ...... 47 3.3.2. Extraction of the lipids from dragonfly wings ...... 48
3.4. Surface fabrication ...... 48
3.5. Surface characterisation ...... 48 3.5.1. Atomic force microscopy ...... 48 3.5.2. Scanning electron microscopy ...... 49 3.5.2.1. Biological samples ...... 49 3.5.2.2. Synthetic surfaces ...... 49 3.5.2.3. Quantification of alignment using fast Fourier transforms (FFT) analysis ...... 50 3.5.3. Raman spectroscopy ...... 50 3.5.4. Wettability ...... 50
3.6. Chemistry characterisation techniques ...... 51 3.6.1. X-ray photoelectron spectroscopy ...... 51
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3.6.2. Synchrotron radiation Fourier Transform Infrared spectroscopy (SR- FTIR) ...... 51 3.6.2.1. FTIR in tranmission mode ...... 51 3.6.2.2. FTIR in attenuated total reflection (ATR) mode ...... 52 3.6.3. Gas chromatography – mass spectrometry ...... 52
3.7. Bacterial strains, growth, and sample preparation ...... 53
3.8. Biological activity assays ...... 54 3.8.1. Plate counting assay ...... 54 3.8.2. Quantitation of bactericidal activity using confocal images for fabricated surfaces ...... 54
3.9. Confocal laser scanning microscopy (CLSM) ...... 55
3.10. Statistical analysis ...... 55
Chapter 4. Surface characterisation and functionality of the epicuticle of insect wings ...... 56
4.1. Overview ...... 57
4.2. Surface topology and wettability of dragonfly wings ...... 57 4.2.1. Scanning electron microscopy ...... 57 4.2.2. Atomic force microscopy ...... 58 4.2.3. Surface wettability ...... 60
4.3. Correlation between surface chemistry and the wettability of a surface ...... 62 4.3.1. Variations in the surface wettability of dragonfly wings ...... 62 4.3.2. Relationship between the quantity of surface epicuticular lipids and wettability ...... 64
4.4. Dual roles of epicuticular lipids in determining the wettability of dragonfly wings ...... 69 4.4.1. Wettability of dragonfly wings as a function of aliphatic hydrocarbon content ...... 69 4.4.2. The chemical composition of untreated and chloroform extracted wings ...... 70 4.4.3. Changes in surface morphology on removal of surface aliphatic hydrocarbons ...... 74 xii
4.5. Summary ...... 79
Chapter 5. Molecular organisation of dragonfly wings ...... 81
5.1. Overview ...... 82
5.2. Physical effects on dragonfly wings resulting from extraction with chloroform ...... 82 5.2.1. Changes in surface morphologies ...... 82 5.2.2. Changes in surface topographies ...... 85
5.3. The chemical composition of the Hemianax papuensis dragonfly wing epicuticle ...... 89
5.4. Molecular organisation of H. papuensis dragonfly wing epicuticle ...... 95
5.1. Summary ...... 98
Chapter 6. Nanostructural effects on antibacterial activity and wettability of dragonfly wings ...... 100
6.1. Overview ...... 101
6.2. Bactericidal activities of dragonfly wings ...... 102
6.3. Comparative surface chemistry of the dragonfly wings ...... 105
6.4. Variation in surface structure ...... 108 6.4.1. Surface topologies ...... 108 6.4.2. Surface morphologies ...... 113
6.5. Structure-dependent wettability ...... 115
6.6. Summary ...... 122
Chapter 7. Systematic bacterial patterning using self-assembled microtextured alkanes ...... 123
7.1. Overview ...... 124
7.2. Thermodynamics of alkane recrystallisation on HOPG and silicon ...... 125
7.3. Patterns of the self-assembled crystals ...... 127 7.3.1. Crystal morphology ...... 127 7.3.2. Eicosane and docosane crystal topographies ...... 130
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7.4. Bacterial attachment to HOPG template-mediated self-organised alkane microcrystallites ...... 132 7.4.1. Local hydrophobicity for bacterial attachment and alignment ...... 135
7.5. Summary ...... 139
Chapter 8. Synthetic mechanobactericidal surfaces composed of dragonfly fatty acids ...... 141
8.1. Overview ...... 142
8.2. Crystal patterns of carboxylic acids on graphite ...... 142
8.3. Thermodynamics of crystallisation ...... 146 8.3.1. Directional crystals formed by the two fatty acids ...... 146
8.4. Biological activities ...... 150
8.5. Gold-coating of the fatty acids crystals ...... 153
8.6. Spatial wettability ...... 156
8.7. Summary ...... 160
Chapter 9. General Discussion ...... 162
9.1. Overview ...... 163
9.2. Molecular organisation of the surface of insect wings ...... 164
9.3. Assessment of the antibacterial potential of superhydrophobic insect wing surfaces ...... 166
9.4. Fabrication and evaluation of synthetic surfaces with the ability to control bacterial attachment ...... 168
Chapter 10. Conclusions and future directions ...... 171
10.1. Summary and conclusions ...... 172
10.2. Future directions ...... 173
10.3. Final remarks ...... 174
Bibliography ...... 175
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List of Abbreviations
AFM Atomic Force Microscopy
SEM Scanning Electron Microscopy
GCMS Gas Chromatography and Mass Spectrometry
XPS X-ray Photoelectron Spectroscopy
FTIR Fourier Transform Infrared Microscopy
ATR Attenuated total reflection
CLSM Confocal Laser Scanning Microscopy
CA Contact angle
WCA Water contact angle
CAH Contact angle hysteresis
CI Composite interface
SPME Solid-phase Micro-extraction
PDMS Polydimethylsiloxane
HCs Hydrocarbons
FA Fatty acid
Alcs Alcohols
Alds Aldehydes
ST Setae
SP Spatula
BR Branched
RO Radial outward
HOPG Highly ordered pyrolytic graphite
FFT Fast Fourier Transforms
UHV Ultra high vacuum
TPL Three phase line xv
MD Molecular dynamics
Au Gold
Sa Staphylococcus aureus
Pa Pseudomonas aeruginosa
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List of Figures
Figure 2.1. The role of topography and surface chemistry in determining the superhydrophobicity of a surface, according to the Cassie-Baxter model. The surface presented in A exhibits a relatively low water contact angle (WCA), as it is composed of a material that is hydrophilic, and does not possess a surface structure that can trap significant quantities of air. Changing the chemical composition of the surface (B), or the physical structure of the surface can both lead to an increased WCA, in accordance with the Cassie-Baxter equation. Surface D exhibits the highest WCA, as it combines a low surface energy material with a physical structure that can trap substantial quantities of air in the surface. Examples of plant and insect surfaces belonging to each of these surface types are presented in the inset images...... 12
Figure 2.2. Images of some superhydrophobic plant surfaces, and their corresponding high resolution SEM images: (A) lotus leaves; (B) Indian Canna leaves; (C) taro leaves; (D) perfoliate knotweed leaves. (Adapted with permission from Elsevier (Guo and Liu, 2007)) ...... 24
Figure 2.3. The surface morphologies of the Hemianax papuensis dragonfly wing. Credit photograph to © Arthur Chapman...... 26
Figure 2.4. Insect wings and their corresponding high resolution SEM images. (A-A2) Isoptera Nasutitermes sp.; (B-B2) Orthoptera Acrida cinerea cinerea; (C-C2) Hemiptera Meimuna opalifera. (Adapted with permission from ACS publication and Elsevier (Byun, Hong, Saputra et al., 2009; Watson, Cribb and Watson, 2010) and Encyclopedia of Life (2014) (eol.org, license agreements can be found at http://creativecommons.org/licenses/by-nc-sa/2.0/ and http://creativecommons.org/licenses/by-nc/2.0/), photographs taken by Kenpei and Tomomarusan)...... 30
Figure 2.5. Superhydrophobic surfaces with high water adhesion. (A) Surfaces that exhibit the ‘petal effect’ exhibit high water contact angles and high adhesion to water. (B) Rose petals, for which the effect is named, possess a similar microstructure to that of lotus leaves; however the nanostructure is comprised of cuticular folds rather than nanocrystals. (C) Gecko feet also exhibit the petal effect. The soles of their feet possess highly hierarchical surface structures that exhibit high water contact angles but the
xvii water adheres strongly to the surface. These structures also enable the geckoes to adhere to solid surfaces, enabling them to climb walls. ST: setae, SP: spatula, BR: branched. (Reproduced with permission from ACS publication and Elsevier (Gao, Wang, Yao et al., 2005; Feng, Zhang, Xi et al., 2008; Bhushan, 2012)...... 32
Figure 2.6. The anisotropic surface structures of (A) rice leaves and (B) butterfly wings. The superhydrophobic structures present on both surfaces are directionally ordered, resulting in a variable wettability that is dependent on the direction of movement. (Reproduced with permission from Elsevier (Guo and Liu, 2007; Peng, Hu and Zhang, 2011), photographs taken by Anne Ten Donkelaar and Stepanka Nemcova)...... 34
Figure 2.7. General structure of bacterial cells (Reproduced with permission for single use from ‘Molecular expression’ http://micro.magnet.fsu.edu/cells/bacteriacell.html). 37
Figure 2.8. Membrane structure of Gram-positive and Gram negative bacteria (Copyright © The McGraw-Hill Companies. Inc.) ...... 38
Figure 2.9. Schematic of the two stages biofilm formation on surfaces. Reproduced with permission from American Chemical Society (Lichter, Van Vlietpa and Rubner, 2009)...... 41
Figure 2.10. Bacterial cells were punctured by the surface of (A, A1) cicada wing Psaltoda claripennis, and (B-B3) dragonfly wing Diplacodes bipunctata. Cells were clearly punctured by nanopillars presented on the surfaces of these insect wings. (Reproduced with permissions from Encyclopedia of Life (eol.org, license agreements can be found at http://creativecommons.org/licenses/by-nc/2.0/), photographs taken by Graham Wise)...... 44
Figure 3.1. Schematic diagram of Synchrotron the FTIR in ATR mode...... 52
Figure 4.1. High resolution scanning electron micrograph of the H. papuensis and H. tau dragonfly wings. (A) The side view of the H. papuensis wing and (B) its corresponding top view; (C) and (D) are side view and top view of the H. tau wing. ... 58
Figure 4.2. Surface topography of (A) Hemianax papuensis and (B) Hemicordulia tau dragonfly wings with their corresponding line profiles. Inset scale bar = 500 nm...... 59
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Figure 4.3. A water droplet coming into contact with the H. papuensis dragonfly wing surface. The surface is sufficiently hydrophobic for the droplet to bounce after it makes initial impact...... 61
Figure 4.4. Average static water contact angle (grey) and average contact angle hysteresis (black), with example water droplet contact angles (above). The shaded area at the top of the graph represents the minimum water contact angle threshold required for self-cleaning ability. Error bars indicates standard deviation, n=15...... 63
Figure 4.5. High resolution scanning micrographs of (A) H. papuensis, (B) H. tau, (C) D. melanopsis dragonfly wings and their corresponding 3D image...... 64
Figure 4.6. Infrared absorbance spectra of the wings of three species of dragonfly. Most bands in the spectra were very similar for all three species, however the increased intensity of the CH2 stretching bands in the spectrum of the H. papuensis dragonfly provides evidence that there is a greater amount of aliphatic hydrocarbons present on the wing surface...... 65
Figure 4.7. Infrared absorbance spectra of two species of damselfly wings, Xanthagrion erythroneurum and Ischnura heterosticta, and their corresponding WCA. The intensity of the CH2 stretching bands provides an indication of the amount of aliphatic hydrocarbons present on the wing surface...... 68
Figure 4.8. Loss of the superhydrophobic and self-cleaning properties of dragonfly wings after extraction with chloroform. The average water contact angles are presented in grey and average contact angle hysteresis presented in black. The shaded area at the top of the graph represents the minimum water contact angle threshold required for classification as superhydrophobic surfaces. Error bars indicates standard deviations, n=20...... 70
Figure 4.9. Infrared spectra of the H. tau dragonfly wings before and after chloroform extraction. The decrease in aliphatic hydrocarbons present on the surface of the dragonfly wings occurs with increasing chloroform extraction time. The intensity of the
CH2 stretching bands is a good approximation of the concentration of aliphatic hydrocarbons, i.e. lipids (waxes) present on the wing surface. The spectra were acquired at the Australian Synchrotron...... 71
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Figure 4.10. Relative proportion of all identified chemical classes present in the epicuticular layer of the H. tau dragonfly wing at three extraction times. The epicuticle was dominated by n-alkanes...... 72
Figure 4.11. Cross-section (A, C, E, G) and surface (B, D, F, H) morphology of Hemicordulia tau dragonfly wing surfaces before and after chloroform extraction over 10, 20 and 30 s. Decreased water contact angles were observed with increased extraction time (inset). Scale bars = 400 nm ...... 75
Figure 4.12. Surface topography of Hemicordulia tau dragonfly wings after various periods of chloroform extraction, inferred from typical two-dimensional AFM scans (5 µm × 5 µm areas) and corresponding cross-sectional profiles. The inset images are AFM scans of 1 m × 1 m areas. Scale bar = 1 m...... 77
Figure 5.1. Cross-sectional images of H. papuensis dragonfly wing membranes prior to, and post-extraction with chloroform, and corresponding images of the WCA. Extraction for 10 s had only a minor effect on the surface morphology, however the WCA decreased by more than 20°. After 1 h extraction, there was no trace of the surface nanostructures, indicating the complete removal of the epicuticular lipids. The WCA on the 1 h extracted surface was 93°, nearly 70° lower than the original, untreated wing. Scale bars = 400 nm...... 83
Figure 5.2. Top view micrographs and 3D images of Hemianax papuensis dragonfly wings prior to, and after 10 s and 1 h of chloroform extraction. The general structure of the dragonfly wings surface largely remained intact after 10 s of chloroform extraction, however, the structure disappeared completely after a 1 h extraction period...... 85
Figure 5.3. Surface topography of Hemianax papuensis dragonfly wings after various periods of extraction with chloroform. Typical two-dimensional AFM scans (1.0 µm × 1.0 µm areas) and cross-sectional profiles of the untreated (A), 10 s (B) and 1 h (C) chloroform-extracted wing surfaces are presented. The surface features became broader and more spatially distributed after 10 s of extraction, however after 1 h of extraction the nanostructures were no longer visible. Scale bars = 100 nm...... 87
Figure 5.4. Representative infrared spectra of untreated and chloroform-extracted wing membranes of the Hemianax papuensis dragonfly. The intensity of the CH stretching bands (shaded), and carbonyl peak (indicated by arrows) decreased successively with extended extraction time. Spectra were acquired in transmission mode...... 90 xx
Figure 5.5. Relative proportions of the major compound classes (top) and chain length (bottom) of dragonfly wing epicuticle components, extracted in 10 s and 1 h...... 94
Figure 5.6. Atomic proportions of carbon, oxygen and nitrogen in the wings of Hemianax papuensis...... 96
Figure 5.7. Proposed model of the epicuticle of the Hemianax papuensis wing membranes. Three layers are contained within the epicuticle: the outer epicuticle, the mesoepicuticle and the inner epicuticle...... 98
Figure 6.1. Bactericidal effects of H. papuensis (top) and D. bipunctata (bottom) dragonfly wings against various bacterial strains. The dead cells in CLSM images were stained with propidium iodide, indicated in red, while live cells were stained with SYTO 9, indicated in green (Scale bar = 5µm). Yellow fluorescence indicated the binding of both dyes, which indicates that sufficient propidium iodide had passed through the membrane, indicating cell rupture and death...... 103
Figure 6.2. Representative scanning electron micrographs of various bacterial cells on the surface of H. papuensis (left column) and D. bipunctata (right column) dragonfly wings, demonstrating the attachment behaviour of P. aeruginosa (A, B), S. aureus (C, D), vegetative B. subtilis (E, F) cells and its spores (G, H). All cell types were deformed when adhered to D. bipunctata wings, however only P. aeruginosa cells were similarly affected on the H. papuensis wings. Scale bars = 1 µm, inset scale bars = 200 nm. ... 104
Figure 6.3. Bactericidal efficiency of D. bipunctata and H. papuensis dragonfly wing surfaces against four different bacterial cell types. The values presented are the number of cells killed per cm2 of wing surface per minute of incubation, over the first three hours...... 105
Figure 6.4. Representative infrared spectra of the wing membranes of H. papuensis (red) and D. bipunctata (blue) dragonflies. The spectra of two dragonfly wings showed their highly similarities in fundamental chemical compositions...... 106
Figure 6.5. High resolution XPS spectra of H. papuensis and D. bipunctata dragonfly wings. High-resolution scans were performed in approximately 20 eV intervals across the O 1s and C 1s peaks. The C 1s peaks of both species are dominated by C–C and C– H bonds, whereas there is an approximately 3:4 ratio between C–O and C=O components within the O 1s peaks...... 107
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Figure 6.6. Photographs of (A) H. papuensis and (B) D. bipunctata dragonflies and corresponding scanning electron micrographs of their wing surfaces (C, D). According to SEM analysis, their structures appeared to be highly similar, however the H. papuensis wings appeared to have more connecting structures between the pillars and the peaks of D. bipunctata wings appeared slightly sharper and thinner. Insets are 3D representations of sections of the micrographs. (A, and B are reproduced with permission from the Encyclopedia of Life (2014))...... 109
Figure 6.7. Fast Fourier transform analysis of (A) H. papuensis and (B) D. bipunctata wing surface images. The grayscale plots of cross-sections of the 2D FFT at 0° show a longer range directional periodicity up to 2.5 µm for D. bipunctata and 4 µm for H. papuensis wings. The area % and population distribution of the wings surface features demonstrated that the feature size of H. papuensis wings is about 70 nm and 100 nm for D. bipunctata wings...... 110
Figure 6.8. Cross-sections of the wings of H. papuensis and D. bipunctata dragonflies. The nanopillar arrays on the surface of D. bipunctata wings appeared relatively consistent in height, whilst those on the surface of H. papuensis wings appeared to vary more in their height...... 111
Figure 6.9. Typical two-dimensional AFM scans (2.5 µm × 2.5 µm areas) and the cross-sectional profiles of (A) H. papuensis and (B) D. bipunctata wings. The surface of the H. papuensis wing appeared highly irregular, with an increased degree of hierarchy of the nanopillars, whilst the line profile of D. bipunctata wings was comprised of regular peaks with a similar height and relatively consistent spatial distribution...... 112
Figure 6.10. Demonstration of bearing analysis. The bearing ratio of a surface is defined as the proportion of the projected area that is occupied by the surface at a given depth from the highest point on the surface (demonstrated in A-C, shaded areas). (D) a demonstration of how three statistical parameters were extracted directly from the plot, i.e. peak height (Rpk), core roughness (Rk) and valley depth (Rvk)...... 114
Figure 6.11. Fourier-transform analysis of H. papuensis dragonfly wing structures at various magnification levels. The inflection points (indicated) demonstrate the dominant feature sizes on the wings of H. papuensis wing at each magnification. The feature size indicated for the 15000× magnification corresponds to the negative space and provides an indication of the spacing of the nanopillars, whilst the size indicated at 35000× and xxii
70000× magnification levels correspond to the nanopillars themselves. The presence of two different nanopillar heights is indicative of the presence of a hierarchical surface structure...... 116
Figure 6.12. Fourier-transform analysis of D. bipunctata dragonfly wing structures at various magnification levels. The inflection points (indicated) demonstrate the dominant feature size on the wings of D. bipunctata at each magnification. The feature size indicated for 15000× magnification corresponds to the negative space and provides an indication of the spacing of the nanopillars, whilst the size indicated at 35000× and 70000× magnification levels correspond to the nanopillars themselves. The nanopillars appeared to be relatively similar at both the larger magnifications...... 117
Figure 6.13. Cassie-Baxter wettability of H. papuensis and D. bipunctata dragonfly wings based on their bearing profiles. If the surface is assumed to adhere to the Cassie- Baxter model of wettability, the theoretical wettability of the surface can also be plotted as a function of the depth of impingement of a water droplet between the surface features of the wing...... 119
Figure 7.1. The process of making ordered microcrystalline interfaces ...... 124
Figure 7.2. Topographical analysis of the surfaces of silicon wafer and HOPG. Scans of 5µm x 5µm areas of (a) HOPG, and (b) silicon with corresponding line profiles. Both substrata have highly smooth surfaces...... 125
Figure 7.3. Scanning electron micrographs of (A, C) eicosane and (B, D) docosane self- assembled on the surface of (A, B) HOPG and (C, D) silicon wafer. Rodlet directionality was observed for both lipids on the surface of the HOPG, suggesting an epitaxial relationship; whilst on the surface of silicon wafer, the lipids appeared as irregular clumps...... 127
Figure 7.4. HOPG template-mediated recrystallisation patterns of eicosane and docosane. Scanning electron micrographs of (a) eicosane and (b) docosane and their corresponding crystallite orientations demonstrate the tendency of the crystals to align in certain directions, often forming triangular shapes. This pattern is attributable to the alignment of alkane molecules with the molecular order of the HOPG substratum. ... 128
Figure 7.5. Schematic representation of alkane alignment when they are adsorbed on graphite in both zigzag and armchair orientations. Two alkane molecules in the same
xxiii orientation will align an angle of 60°, while molecules in the opposite orientations will be aligned at 30° or 90°...... 129
Figure 7.6. 2D-FFT analysis of micrographs of (A) eicosane and (B) docosane on HOPG, Preferential orientation of the crystals can be observed, with predominant directionalities 60° apart, suggesting that most of the initially adsorbed molecules adopted either the zigzag or armchair configurations...... 130
Figure 7.7. Topography of eicosane (A) and docosane (B) alkane microcrystals self- organised on the graphite templates and their corresponding cross-sectional profiles. The directionality of the alkane microcrystals are indicated with arrows...... 131
Figure 7.8. Bacterial attachment to the self-organised alkane microcrystallites. Bacterial cells attached only along the interfaces formed by the microcrystallites. The white ovals indicate the alignment of bacterial cells in straight lines, following the same patterns that were evident in the SEM and AFM images of the alkane microcrystals (see Figure 7.4 and 7.6). SYTO®9 fluorescent dye was used to stain the live bacteria, whilst non-viable cells were labelled red using propidium iodide...... 133
Figure 7.9. Attachment of S. aureus and P. aeruginosa cells on the HOPG surface. Bacterial cells attached randomly on the surface without any apparent pattern...... 134
Figure 7.10. Alkane microcrystallite-directed bacterial cell attachment. (A) Raman microspectroscopic mapping indicated air retention on the surface of eicosane microcrystallites when immersed in water. Lighter areas indicate a high intensity of - OH bands (attributable to higher quantities of water) and darker areas indicate regions lacking water (i.e. air pockets) (B) Schematic representation of spherical bacterial cells attaching to the microcrystallite-water-air interface. The force of attraction to alkanes is characterised by van der Waals forces, resulting in an effective radial adhesion force
Fadh. The contact angle θ and the resultant capillary force Fcap established the depth of penetration at the water-air interface. The net force between Fadh and Fcap as represented by the penetration angle ψ illustrates the interplay between bacterial hydrophilicity and lipid adhesion. The rod-shaped bacteria at this TPL can add additional complexity due to further deformations induced in the fluid-fluid interface...... 136
Figure 7.11. (A) Density of attached bacterial cells as inferred from confocal analysis and (B) Surface wettability of eicosane, docosane, S. aureus and P. aeruginosa. The S. xxiv aureus cells appear to more readily attach onto the eicosane, whereas the P. aeruginosa cells more readily attach onto the docosane...... 138
Figure 8.1. The molecular structures of palmitic (left) and stearic (right) acids, and the SEM morphologies of their corresponding microcrystallites. Palmitic acid self- assembled to form thin microblades, whilst stearic acid appeared to form interjoined pillars. The differences in the sharpness of the features formed by the two fatty acids are apparent in the 3D images...... 143
Figure 8.2. The topography of palmitic acid (A) and stearic acid (B) self-organised microcrystals on HOPG templates, and their corresponding cross-sectional profiles. From the AFM images it can be seen that palmitic acid crystallised into relatively parallel features whilst the stearic acid self-assembled into structures pointing in various directions. The line profile of palmitic acid demonstrated the sharp edges of each crystallite, whilst stearic acid features consisted of flattened high peaks...... 144
Figure 8.3. Morphological analysis of the crystallites of (A) palmitic acid, and (B) stearic acid. These data were obtained from particle size analysis using Gwyddion data processing software (Nečas and Klapetek, 2012). Any particles below 200 nm in width were excluded to minimise the effects of noise...... 146
Figure 8.4. FFT analysis of scanning electron micrographs of palmitic acid and stearic acid microcrystallites. Some directionality was observed in palmitic acid (white ovals) but the stearic acid microcrystallites appeared to orient somewhat randomly...... 147
Figure 8.5. Adsorption alignment of straight chain carboxylic acid molecules self- assembled into monolayers on graphite. The alignments shown are all in zigzag configuration. The adjacent chains within one domain will have their –COOH group pointing either in the same direction or opposite directions. Reproduced with permission from AIP Publishing LLC (Yang, Berber, Liu et al., 2008)...... 148
Figure 8.6. Chemical composition of FA interfaces. (A) FTIR spectra of palmitic acid and stearic acid obtained in mapping mode, scan area 50 µm × 50 µm; and the 2D spectral maps of the C-H stretching bands of (B) palmitic acid, (C) stearic acid, and the C=O stretching band of stearic acids...... 149
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Figure 8.7. Peak shift of the C-H stretching band of stearic acid. (A) Peak shift spectra, (B) the 2D map of C-H stretching band, (C) the SEM image of stearic acid, and (D) the 2D map indicating the relative position of the peak maximum...... 150
Figure 8.8. Bactericidal activities of palmitic and stearic fatty acids crystallite interfaces. Confocal Laser Scanning Microscopy (CLSM) images show the viability of bacterial cells on each of the fatty acids. Dead cells are indicated in red, viable cells in green. There was relative large number of red signals in all cases (particularly S. aureus on palmitic acid). Scale bars = 5µm...... 151
Figure 8.9. Bactericidal efficiency of the two surfaces against P. aeruginosa and S. aureus cells. In most cases, the surfaces succeeded in killing greater than 60% of the bacteria, except in the case of S. aureus on the surface of stearic acid, which killed 49%...... 152
Figure 8.10. Bactericidal activities of the gold-coated surfaces. Both surfaces showed strong bactericidal activities against both P. aeruginosa and S. aureus. Dead cells are indicated in red, viable cells in green. Scale bar = 5µm...... 154
Figure 8.11. Killing efficiency of palmitic and stearic acid interfaces against Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Sa). In most cases, the bactericidal efficiency increased noticeably on the gold-coated surface compared to the native surfaces...... 155
Figure 8.12. Attachment rates of Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Pa) on palmitic and stearic acid interfaces. The number of attached cells increased substantially as observed under confocal microscopy. This increase in number of cells is most likely due to enhancement of fluorescent signals on metal surface (gold-coated)...... 155
Figure 8.13. Water contact angles on native fatty acids and after coating with a thin layer of gold...... 157
Figure 8.14. Raman spectra of palmitic acid and stearic acid crystallites immersed under water for air imaging...... 159
Figure 8.15. Image scans of fatty acid (FA) crystallites and water using Raman microspectroscopy. The regions, which are darker in both images, indicated the presence of air pockets...... 159
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Figure 8.16. Schematic representation of forces applied to a S. aureus cell ‘riding’ the ridge of a palmitic acid microblade. The wetting force of the three phase lines (Ftpl) on either side of the microblade pull the cell in opposing directions, however the component of each force normal to the microcrystal interface (Fnor) are cumulative. The lateral force components (Flat) are 180° opposed to each other, and may aid in tearing the cell...... 160
Figure 9.1. Proposed model of the epicuticle of dragonfly wing membranes. Three layers are contained within the epicuticle: the outer-epicuticle, the meso-epicuticle, and the inner-epicuticle...... 166
Figure 9.2. Photographs of H. papuensis (left) and D. bipunctata (right) dragonflies in their natural habitats, and their corresponding geographic distributions. The yellow spots in the world map indicate their general global distributions whilst colour coding of the Australian map indicates their common and preferable habitats, based on surveys of the number of dragonflies present at the sampled locations. Reproduced with permission from the Encyclopedia of Life (2014) and Atlas of Living Australia (National Research Infrastructure for Australia (NCRIS), 2014)...... 168
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List of Tables
Table 2.1. Different types of solvents used for extraction of the lipid components from the insect body and insect wings surfaces...... 15
Table 2.2. Micro- and nano-scale wax crystal morphologies on the epidermal cells of insect wing surfaces and their WCA ...... 28
Table 4.1. Assignment of chemical components to the major absorption bands present in the IR spectra of the wing membranes ...... 66
Table 4.2. Wax components of the Hemicordulia tau wing epicuticle isolated and identified by GCMS. Components that could not be unambiguously identified are grouped together...... 73
Table 4.3. Roughness analysis of the untreated and chloroform extracted H. tau wing surfaces...... 78
Table 5.1. Roughness analysis of the untreated and chloroform extracted Hemianax papuensis wing surfaces ...... 88
Table 5.2. Individual lipid components of the Hemianax papuensis wing epicuticle isolated by extraction with chloroform, identified by GCMS. Components that could not be unambiguously identified are grouped together...... 91
Table 6.1. Roughness analysis data for the two dragonfly wing surfaces, H. papuensis and D. bipunctata dragonflies...... 113
Table 6.2. Bearing statistics of the H. papuensis and D. bipunctata dragonfly wing surfaces...... 114
Table 7.1. AFM roughness analysis of the graphite and silicon wafer surfaces ...... 126
Table 7.2. AFM roughness analysis of the eicosane and docosane crystals on HOPG surfaces ...... 131
Table 8.1. AFM roughness analysis of the eicosane and docosane crystals on HOPG surfaces ...... 145
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Chapter 1. Introduction
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1.1. Overview
It is well-documented that biofilm formation on medical implants by pathogenic bacteria can have dramatic consequences, leading to the failure of the device, often resulting in the necessity to surgically remove the implant. Subsequent complications that can then arise include systemic infection, loss of organ or limb function, amputation or death (Ong, Razatos, Georgiou et al., 1999; Donlan, 2001; Diaz, Cortizo, Schilardi et al., 2007). The problems are exacerbated by the enormous number of resistant strains that have emerged as the result of the heavy chemical input in current sterilisation methods. For these reasons, there is a need for innovative approaches and alternatives for controlling bacterial colonisation.
Natural occurring surfaces have always provided great inspiration for innovative technologies that can be used in a wide range of potential applications (Watson, Cribb and Watson, 2010; Watson, Cribb and Watson, 2011). These surfaces have evolved and have been optimised for millions of years in order to adapt to ever-changing environments (Barthlott and Neinhuis, 1997; Barthlott, Neinhuis, Cutler et al., 1998; Cheng, Rodak, Wong et al., 2006; Byun, Hong, Saputra et al., 2009; Koch, Bhushan, Ensikat et al., 2009; Boeve, Voigt and Gorb, 2011; Hu, Watson, Cribb et al., 2011). Nature is therefore an excellent resource for the development of innovative technologies that can be applied widely in the related fields of chemistry, medicine and material science. During the past few decades, the ‘lotus effect’ has arisen as the archetype for surfaces that possess highly important properties, such as superhydrophobicity, and the ability to self-clean (Yoshimitsu, Nakajima, Watanabe et al., 2002; Sun, Feng, Gao et al., 2005; Dani, 2006; Watson, Cribb and Watson, 2010).
The term ‘self-cleaning’ originates from the combination of superhydrophobicity and a low water droplet sliding angle (Sun, Feng, Gao et al., 2005). Furthermore, superhydrophobicity arises as a result of the combination of the surface chemistry and the surface roughness, with small changes in either property having the potential to significantly influence the extent of surface wettability (Barthlott and Neinhuis, 1997; Bhushan and Jung, 2008; Bhushan, Jung and Koch, 2009b; Ensikat, Ditsche-Kuru, Neinhuis et al., 2011; Watson, Cribb and Watson, 2011). A great deal of effort has been made in obtaining a better understanding of these phenomena in order to adapt them to dealing with current issues surrounding the formation of biofilms (Bico, Marzolin and
2
Quéré, 1999; Herminghaus, 2000; Öner and McCarthy, 2000; Nakajima, Hashimoto and Watanabe, 2001; Lafuma and Quéré, 2003; Patankar, 2004; Ma and Hill, 2006; Watson, Cribb and Watson, 2010). The concepts of self-cleaning and superhydrophobicity, whilst being closely related, are distinct phenomena that are not usually successfully distinguished in the literature. This shortcoming has led to confusion between the separate concepts of superhydrophobicity and self-cleaning. Fürstner and Barthlott were the first to assert that superhydrophobicity and self-cleaning were two different phenomena (Fürstner, Barthlott, Neinhuis et al., 2005). A self-cleaning surface must possess superhydrophobicity but a self-cleaning surface may not necessarily exhibit superhydrophobicity. Therefore, when designing self-cleaning materials, superhydrophobicity is an important consideration.
A number of insects and plants contain surfaces that exhibit both superhydrophobicity and self-cleaning properties. While a number of studies have been conducted on the surface properties of plant leaves to obtain an insight into the mechanisms responsible for these properties, this is not the case for the surfaces of insects. For example, while the surface chemistry of insects has been investigated for many years from an agricultural perspective, these studies did not have a focus on surface wettability. Many of these insect surfaces are also bactericidal, the mechanisms of which are purely physical. This recently obtained knowledge warrants further study into the ways by which these surfaces kill bacteria and may provide an insight into the mechanisms by which the formation of biofilms on surfaces may be controlled. It may then be viable to mimic these environmentally-friendly processes that do not require the use of chemical synthesis or fabrication techniques.
1.2. Aims of the study
The ultimate aim of this work is to successfully construct an artificial surface that is based on a naturally occurring insect wing that can kill bacterial cells via a process of mechanical stresses. As mentioned above, controlling biofilm formation and bacterial infections would be of great benefit to society. In order to achieve this aim, three intermediate objectives are identified as key stages toward making such a surface capable of acting to prevent bacterial proliferation and infection.
The first objective is to physically and chemically characterise the surfaces of superhydrophobic various insect wings. This involves a number of complementary
3 methods including atomic force microscopy (AFM), scanning electron microscopy (SEM), gas chromatography and mass spectrometry (GCMS), x-ray photoelectron spectroscopy (XPS) and Fourier transform infrared microscopy (FTIR) to investigate the composition, architecture and morphology of the wings of several species of Odonata in order to obtain a general overview of the interspecies consistency. This information provides clearer insight into how the surfaces of dragonfly wings are structured.
The second objective is to assess the bactericidal potential of different dragonfly wings. Several different strains of pathogenic bacteria will be used for this assessment. Primarily, three techniques will be used, including SEM, confocal laser scanning microscopy (CLSM) and plate counting to quantify the efficiency of each wing in controlling the extent of bacterial attachment. This is an important consideration as it provides an insight into the mechanisms by which the bacteria are able to be killed when coming into contact with the surface. This will be of great benefit when designing antibacterial surfaces.
The third objective is to design, fabricate and evaluate synthetic surfaces that are constructed according to the knowledge generated from the previous stages. The intermediate aim is then to prepare surfaces that contained ordered structures that can control bacterial attachment, using fast and facile methods such as self-assembly. A number of the surface characteristics that were identified in the initial stages of this research will be used to produce self-assembled surfaces that are able to limit the extent of bacterial adhesion. The knowledge generated regarding the mechanisms by which the bactericidal action takes place represent an important guide for designing surfaces that can control the formation of biofilms.
In the following chapters, the current state of knowledge regarding the fields of superhydrophobicity, self-cleaning, insect wings and anti-bacterial surfaces will be discussed. Following this discussion, chapters discussing the surface characteristics and properties of dragonfly wings will be presented, along with an analysis of the mechanisms whereby these surfaces are able to control bacterial attachment. Finally, the possible impact of the knowledge generated here and the future perspectives of this work will be addressed.
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Chapter 2. Literature review
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2.1. Overview
Surfaces with superhydrophobic and self-cleaning properties have gained much attention during the past few decades because of their high potential as templates in the design of biomimetic artificial surfaces and in material sciences where products and devices that can exhibit similar properties (Yoshimitsu, Nakajima, Watanabe et al., 2002; Sun, Feng, Gao et al., 2005; Dani, 2006; Watson, Cribb and Watson, 2010). Such surfaces have applications that range from daily life applications, such as self-cleaning glass, anti-fouling coatings and oil/water separation, to high-technology devices, such as anti-wetting and lab-on-a-chip devices. Insect wings are one of many examples of natural surfaces that possess superhydrophobic and self-cleaning properties, and as such they have attracted great attention from the scientific community interested in this wide range of potential applications.
Surfaces found in nature have been optimised through the process of evolution for millions of years. Insects have evolved the ability to fly for at least 400 million years (Grimaldi and Engel, 2005) while they represent approximately half of all the living macro-organisms on earth. Insect wings are composed of lightweight materials, which can withstand extremely heavy loadings with minimal resource requirements (Wan, Cong, Wang et al., 2008). In order to adapt to an environment that provides little protection against wetting by rain and other aqueous sources that they may encounter, insects evolved wings into geometric, non-smooth structures (Wan, Cong, Wang et al., 2008; Watson, Cribb and Watson, 2011). These structures are comprised of a range of different waxes, which impart anti-wetting and self-cleaning properties as the result of their superhydrophobic properties (Buckner, Nelson and Mardaus, 1994; Gibbs, 1998; Buckner, Hagen and Nelson, 1999; Nelson, Freeman and Buckner, 2000; Arsene, Schulz and Van Loon, 2002; Nelson and Charlet, 2003; Boeve, Voigt and Gorb, 2011). It is now generally accepted that the impenetrability of an insect to water is due to the presence of a thin, superficial layer of wax in the cuticle as originally reported by Ramsay in 1935 (Ramsay, 1935).
2.2. Arthropod cuticle
2.2.1. Morphology of insect wings
Insects were the first organism that developed the ability to fly and took to the skies at least 90 million years prior to the earliest winged vertebrates (Grimaldi and Engel, 6
2005). Currently, flying insects are organised in the superorder Pterygota. Insect cuticles consist of two main components: a crystalline polymer, chitin, embedded in fibrillar form in a matrix (Kreuz, Arnold and Kesel, 2001). This arrangement builds up three distinct layers of insect wing cuticle: the epicuticle, the endocuticle and the exocuticle (Xiao, Bai, Wang et al., 2007; Wan, Cong, Wang et al., 2008). The cuticle is secreted by a single layer of epidermal cells, the hypodermis (Lockey, 1988), and transported to the cuticle surface by a system of pore canals (Gorb, 1997). The epicuticle is the thinnest and outermost layer at the dorsal and ventral surfaces, being less than 1 µm in thickness. According to Lockey, the epicuticle may consist of two thinner layers: outer epicuticle and inner epicuticle (Lockey, 1988). The outer epicuticle (cuticulin layer) is about 10 to 18 nm thick while the inner layer is where the waxy components are formed. The components of these waxes vary depending on the species and their living environment. Generally, cuticle waxes are composed of varying proportions of fatty alcohols, fatty acids, fatty aldehydes, esters, steroids, long chain hydrocarbons and sometimes aromatic compounds (Lapointe, Hunter and Alessandro, 2004). The endocuticle and the exocuticle together can be considered as one layer, known as the procuticle (Wootton, 1992; Wagner, Neinhuis and Barthlott, 1996; Nelson, Freeman, Buckner et al., 2003; Vukusic, Wootton and Sambles, 2004; Jarrold, Moore, Potter et al., 2007; Gołebiowski, Maliński, Boguś et al., 2008; Gorb, Tynkkynen and Kotiaho, 2009; Gołebiowski, Bogus, Paszkiewicz et al., 2011). The endocuticle (middle layer of the cuticle) is where proteins are sclerotized whereas the exocuticle, the inner most layer, consists of chitin and occupies about 25 % - 40 % of insect cuticle by dry-weight (Wan, Cong, Wang et al., 2008).
2.2.2. Aerodynamics of insect flight
Insects are one of the most wonderful organisms on Earth. They are abundant yet very elusive, they are fascinating to watch but difficult to obtain. Their flying mechanisms are combinations of various motions that are assisted by the sophisticated architecture of their wings. Their wings are framed by a system of veins which aid in stabilising the wing as a whole (Kreuz, Arnold and Kesel, 2001). Its basic framework consists of chitin, in the form of a long chain crystalline polymer providing support for the membrane and bearing the forces applied to the wings during flight (Kesel, Philippi and Nachtigall, 1998; Rajabi, Moghadami and Darvizeh, 2011). The junctions between the vein and the wing membrane are comprised of the protein resilin, which enhances 7 the flexibility of the wing (Andersen and Weis-Fogh, 1964; Neville, Parry and Woodhead-Galloway, 1976; Gorb, 1999). The vein system of the wings not only provides support for the membrane but also bears a majority of the bending and twisting forces applied to the wings. There are three different types of veins existing in the wings, including ambient, longitudinal and cross veins. The ambient veins function as the stabilising framework and surround the wing, while longitudinal veins act as girders which divide the wings into small cells (Rajabi, Moghadami and Darvizeh, 2011). This venation system, together with their light-weight building materials supports routine flights as well as longer colonisation flights (Song, Xiao, Bai et al., 2007).
The main features of the flapping motions seen in most insects include back and forth strokes along the horizontal plane, an incline stroke plane or up and down strokes against wind (Wang, 2005). In order to support their body weight, insect wings typically produce 2-3 times more lift than conventional aerodynamics (Ellington, 1999). To minimise the materials needed to form the wing but still be able to protect themselves from wetting and pollutants, insect wing surface membranes are optimised to display a disordered, rough microstructure, composed of numerous nanometre-scale columns (Song, Xiao, Bai et al., 2007; Wan, Cong, Wang et al., 2008). Researchers have been long fascinated by the excellent flying abilities of insect wings despite their limitations in size and weight. Among all species of insects, dragonflies and damselflies belonging to the Order Odonata are unsurpassed in the insect world for flying abilities. Just as the wings of the airplane are the determining factor in their aerodynamics, dragonfly wings determine their flying ability (Zhao, Yin and Zhong, 2011).
Odonata represents the Order that contains one the oldest forms of insect flight in which their two pairs of wings can beat independently. For this reason, the wings provide the insect the availability of various motions which mark their wonderful signature in nature. Even though damselflies and dragonflies are very similar in size, their wings are constructed completely differently. This enables dragonflies to become predators rather than prey, which is also the case for some species of damselflies (Wakeling and Ellington, 1997). Dragonfly wings were designed in a way that allows their stroke planes to be nearly normal to the direction of the resultant force, which makes their velocities and accelerations greater than those of damselflies (Wakeling and Ellington, 1997).
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Studies of the aerodynamic behaviour of insect wings have provided valuable ideas for micro-air vehicles. These flying abilities also provide new instrumental advances at the nano- and micro-scale enables characterisation of more valuables properties of insect wings. For example, the intricate hierarchical nanoarchitecture of insect wings can be used to increase the water contact angle of incoming droplets and/or reduce adhesion on the surface during rainy conditions that enhance their resistance against the wetness of the semi-aquatic environments in which some insects reside (e.g. cranefly and water-striders) (Cheng, 1973; Suter, Rosenberg, Loeb et al., 1997; Hu, Chan and Bush, 2003; Watson, Myhra, Cribb et al., 2008; Rothstein, 2010; Su, Ji, Zhang et al., 2010; Watson, Cribb and Watson, 2010; Hu, Watson, Cribb et al., 2011; Watson, Cribb and Watson, 2011). These structures can often contribute to colouration exhibited by some insects, such as the iridescence in the hindwings of the damselfly Neurobasis chinensis chinensis, the blue iridescence in the wings of the giant tropical wasp Megascolia procer javanensis, and the transparent and translucent wings of butterflies (Vukusic, Wootton and Sambles, 2004; Hooper, Vukusic and Wootton, 2006; Sarrazin, Vigneron, Welch et al., 2008; Perez Goodwyn, Maezono, Hosoda et al., 2009; Shevtsova, Hansson, Janzen et al., 2011). This colourisation phenomenon is caused by the unique photonic properties of insect wings arising as a result of the combination of ‘chemical’ and ‘physical’ colours (Huang, Wang and Wang, 2006).
2.3. The surface of insect wings
2.3.1. Concept of self-cleaning
During the past decade, lotus leaves have become the benchmark for superhydrophobic and self-cleaning surfaces (Ensikat, Ditsche-Kuru, Neinhuis et al., 2011). The ‘lotus effect’ is the phenomenon in which water droplets form pearl-like shapes and move freely across the surface of the leaf, collecting dirt and contaminants as they roll off the surface (Barthlott and Neinhuis, 1997; Riedel, Eichner and Jetter, 2003; Koch, Neinhuis, Ensikat et al., 2004; Sun, Feng, Gao et al., 2005; Cheng, Rodak, Wong et al., 2006; Koch and Ensikat, 2008; Koch, Bhushan, Ensikat et al., 2009; Rothstein, 2010; Buschhaus and Jetter, 2011; Ensikat, Ditsche-Kuru, Neinhuis et al., 2011). According to concept of the ‘lotus effect’, self-cleaning is explained as the result of the presence of superhydrophobicity and low sliding contact angle (Feng, Li, Li et al., 2002; Sun, Feng, Gao et al., 2005). For example, Nelumbo nucifera (lotus) and
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Colocasia esculenta (taro) leaves exhibit water contact angles (WCA) of θ ≈ 160º. These leaves have been the subject of various applications in the fabrication of bio- mimetic artificial surfaces.
The majority of species of the Odonata Order inhabit environments which are both wet and dirty, therefore needed to evolve strategies for coping with potential fouling particles, in the form of superhydrophobic and self-cleaning surfaces (Wagner, Neinhuis and Barthlott, 1996; Simonsen, 2001; Koch, Dommisse and Barthlott, 2006; Byun, Hong, Saputra et al., 2009; Sun, Watson, Zheng et al., 2009; Wan, Cong, Jin et al., 2009; Watson, Watson, Hu et al., 2010; Hu, Watson, Cribb et al., 2011; Watson, Cribb and Watson, 2011). Recently, cranefly and water-striders have been receiving increased attention, as they have the ability to walk freely on water without penetrating the surface due to the presence of fine structures of their cuticle (Hu, Watson, Cribb et al., 2011). Other biological surfaces, such as duck feathers and butterfly wings, contain corrugated surfaces that form air pockets that prevent water from touching the surface (Nosonovsky and Bhushan, 2005; Nosonovsky and Bhushan, 2007b).
2.3.2. Wettability theory
When a surface exhibits a high water contact angle (WCA > 150°) and low contact angle hysteresis (CAH < 10°), the surfaces are regarded as being not only superhydrophobic but also self-cleaning (Shirtcliffe, McHale, Atherton et al., 2010; Su, Ji, Zhang et al., 2010; Bhushan, 2012). CAH is the difference between the advancing and receding contact angles (Nosonovsky and Bhushan, 2008; Ferrari and Ravera, 2010). Superhydrophobicity occurs when the water droplets form an almost spherical shape on the surface and therefore will readily move when subjected to low tilting angles, resulting in self-cleaning. Any dirt or contaminants on the surface are then collected by the droplets and are removed as the droplet rolls off the surface. This characteristic is usually associated with low-drag (Bhushan, 2012) and low adhesion properties (Mei, Luo, Guo et al., 2011). Depending on the structure and chemical composition of the surface, antibiofouling (Webb, Truong, Hasan et al., 2012), anisotropic wetting (Nishimoto and Bhushan, 2013), anti-reflection (Chattopadhyay, Huang, Jen et al., 2010) and anti-icing (Mei, Luo, Guo et al., 2011) properties may also be present.
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Whilst Wenzel’s theory explains that surface hydrophobicity is a function of surface roughness as well as chemical composition (Wenzel, 1936), it is the theory developed by Cassie and Baxter (Cassie and Baxter, 1944) that is most often applicable for superhydrophobic surfaces. The Cassie-Baxter model also considers the topography and chemistry of a surface in determining the hydrophobicity. According to this model, surface hydrophobicity is a function of surface chemical heterogeneity according to the following equation: cos θ = f1cos θ1 + f2cos θ2 Equation 2.1. where θ is the observed contact angle of the heterogeneous surface, f1 and f2 are the area fractions of surface components 1 and 2, with θ1 and θ2 being their respective contact angles. In the case of superhydrophobic surfaces, one of the components is typically air, which exhibits a water contact angle of 180°. This allows the simplification of equation (1) to: cos θ = f1(cos θ1 + 1) – 1 Equation 2.2.
Superhydrophobicity arises from the combination of hierarchical surface structures (i.e. surfaces with multiples scales of roughness) that enable the entrapment of air on low surface energy materials. The sliding angle, the other parameter that is important in determining hydrophobicity, is defined as the critical angle at which the water droplets start to slide along a tilted surface (Jung and Bhushan, 2006; Bhushan, Jung and Koch, 2009b; Yan, Gao and Barthlott, 2011). A superhydrophobic surface would effectively form a composite interface (CI) with air residing between the asperities on the surface. The CI can be destabilised and irreversibly transformed into a homogeneous interface, for example, by the application of sonication (Nosonovsky and Bhushan, 2007a). There are three factors which can destroy a CI; (i) a capillary wave formed at the liquid-air interface, (ii) nanodroplet accumulation in the valleys on the surface and (iii) hydrophilic surface regions arising from the chemical heterogeneity of the surface. Hierarchical roughness or ‘Cassie-Baxter structure’ needs to exist in order for the CI to be stabilised (Neinhuis and Barthlott, 1997; Nosonovsky and Bhushan, 2007a).
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Figure 2.1. The role of topography and surface chemistry in determining the superhydrophobicity of a surface, according to the Cassie-Baxter model. The surface presented in A exhibits a relatively low water contact angle (WCA), as it is composed of a material that is hydrophilic, and does not possess a surface structure that can trap significant quantities of air. Changing the chemical composition of the surface (B), or the physical structure of the surface can both lead to an increased WCA, in accordance with the Cassie-Baxter equation. Surface D exhibits the highest WCA, as it combines a low surface energy material with a physical structure that can trap substantial quantities of air in the surface. Examples of plant and insect surfaces belonging to each of these surface types are presented in the inset images.
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According to the Cassie-Baxter expression, any variation in the chemical composition or the topographical structure of a surface will affect the values of θ1 and f1 respectively, which will result in a change in the observed composite water contact angle (Figure 2.1). The surface illustrated in (B) possesses a different chemical composition to that of (A) and exhibits a higher water contact angle θ1. The surface shown in (C) is composed of the same material as (A), however the altered surface topography enables it to trap a greater quantity of air, decreasing f1, and causing the observed angle to approach that of air, i.e. 180°. Surface (D) is composed of a low energy material in addition to possessing the Cassie-Baxter structure, which results in a surface that exhibits an even greater water contact angle.
2.4. Surface chemistry
Three quarters of all insect species on Earth are Arthropoda (Grimaldi and Engel, 2005) and their existence has always directly or indirectly affected farming and food production. Therefore, studies on insect chemistry have progressed for a long period of time. Recent advances in the knowledge of insect cuticular wax chemistry have revealed that the waxes not only provide a significant barrier to protect insects from desiccation and the adverse effects of microorganisms but also function as communication cues. This chemical knowledge is relevant for a number of applications in medicine and biomedicine. Unlike plants, insects can only produce cuticular waxes once in their life time and they can neither be renewed nor added at a later date (Kreuz, Arnold and Kesel, 2001; Wan, Cong, Jin et al., 2009). The border between the outer epicuticle and the inner epicuticle is thin, so that the chemical information of individual layers is difficult to distinguish. However, with many advances in technologies, alternative methods are being developed to study epicuticle waxes.
Traditionally, insect cuticles have been subjected to extraction using various organic solvents in order to remove the waxes for analysis. There have been many studies conducted on the chemical composition of insect cuticle based on solvent extraction and these studies are summarised in Table 2.1. Depending on polarity of the targeted waxes, the solvent can be varied from mild, i.e., acetone, ethanol, hexane and heptane, to very strong solvents such as benzene and chloroform (Wan, Cong, Wang et al., 2008). Therefore, the use of different solvents will result in the extraction of different components of the waxes. For example, chloroform was described as the most
13 suitable solvent for extraction of aliphatic hydrocarbons and wax esters on the epicuticular layer of the wing surfaces. A newer technique has been developed recently for surface wax extraction, known as solid-phase micro-extraction (SPME). This method involves a single SPME fibre coated with polydimethylsiloxane (PDMS), which is rubbed directly on the surface for approximately 5 minutes to remove the waxes of the insect cuticles (Zhang, Yang and Pawliszyn, 1994; Moneti, Dani, Pieraccini et al., 1997; Liebig, Peeters, Oldham et al., 2000; Dani, 2006). The fibre is then inserted directly into injection port of the GCMS for chemical analysis. Another technique that has been suggested to be one of the best methods for surface wax extraction is silica- rubbing, since it allows the greatest recovery of a majority of the epicuticular waxes (Choe, Ramírez and Tsutsui, 2012). The targeted surface, which is placed with silica gel and subsequently vortex for 30 s, is removed from the gel and the gel is then extracted with hexane (Choe, Ramírez and Tsutsui, 2012).
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Table 2.1. Different types of solvents used for extraction of the lipid components from the insect body and insect wings surfaces.
Comments Dielectric Extraction Solvents Insect body Insect wings References constant time Chemistry Physical General
Damselflies Pyrrhosoma 1,2- (Gorb, Kesel nymphuls and Coenagrion No specific chemistry was Destroyed up to Propylene 1h 70% area dissolved and Berger, puella; Dragonfly Aeshna identified 30% surface pillae oxide 2000) cyanea
(Geiselhardt, Leaf beetle HCs* – n- and branched To remove pillae No morphology change Geiselhardt n-Pentanea 1.84 Gastrophysa 15 m alkanes, alkenes (C16 - Odd number of C in was reported and Peschke, viridula C37) backbone skeleton 2009)
Goldenrod gall Branched alkane (C28- To extract HCs* on (Nelson and fly Eurosta 1 m C32). Long chain Alds† epicuticular waxes Lee Jr, 2004) solidaginis (C30, C32)
Saturated homologous n- Identified Booklouse (C21-C34), mono- (C28-C42) epicuticular lipids: (Howard and Liposcelis 30 s and dimethyl-alkanes (C31- HCs* and FA# Lord, 2003) bostrychophila C43) amides (stearoyl No morphology change Hexanea 1.89 # amides) FA amides (C16-C22) was reported
Homologous alkanes (C21- C45) Ectoparasitoid Wax ester: FAs# - short To extract (Howard and Habrobracon 3 m chain, unbranched, even epicuticular HCs* Baker, 2003) hebetor numbered carbons (C8- and wax esters ‡ C20), with secondary Alcs moieties (C22-C25)
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Comments Dielectric Extraction Solvents Insect body Insect wings References constant time Chemistry Physical General
To dissolve (Wan, Cong, Dragonfly Pantala No specific chemistry was Dissolved 30-40% of 2 h insoluble Wang et al., flavescens fabricius identified surface area compounds 2008) Heptane 1.92 Damselflies Pyrrhosoma (Gorb, Kesel nymphuls and Coenagrion No specific chemistry was Dissolved 20% of area, Affected 70% wax 1 h and Berger, puell; Dragonfly Aeshna identified 10% areas looked intact covering 2000) cyanea
Toxic organic solvent dissolve ester Dragonfly Pantala No specific chemistry was Dissolved 50-60% of Benzene 2.28 2 h flavescens fabricius identified surface structure Very non-polar Be able to remove HCs*
Damselflies Pyrrhosoma (Gorb, Kesel nymphuls and Coenagrion 1 h and Berger, puella; Dragonfly Aeshna Very strong solvent. 2000) cyanea No specific chemistry was Chloroform 4.81 100 % of area dissolved Treated for 2 h was identified too long. (Wan, Cong, Dragonfly Pantala 2 h Wang et al., flavescens fabricius 2008)
Periplaneta To extract brunnea * (Saïd, P. americana HCs* ranged epicuticular HCs Dichloro- No morphology change Costagliola, d 9.08 P. fuliginosa 2 m C -C while other three * methane 24 43 was reported C19-C25 HCs Leoncini et al., species ranged C -C . P. australasiae, 21 41 profile is species 2005) P. american specific
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Comments Dielectric Extraction Solvents Insect body Insect wings References constant time Chemistry Physical General
Damselflies Pyrrhosoma (Gorb, Kesel 1,2-Dichloro nymphuls and Coenagrion No specific chemistry was 5% of area dissolved, Can destroy up to 10.42 1 h and Berger, ethane puella; Dragonfly Aeshna identified 40% area looked intact 50% surface pillae 2000) cyanea
Damselflies Pyrrhosoma (Gorb, Kesel nymphuls and Coenagrion Pillae appeared 1 h and Berger, puella; Dragonfly Aeshna amorphous Least destroying 2000) cyanea effects. No specific chemistry was Acetone 20.7 (25) Relative polar, not identified Subtle changes on very effective for (Wan, Cong, Dragonfly Pantala surface non-polar 2 h Wang et al., flavescens fabricius compounds Pillae appeared non- 2008) uniform
Damselflies Pyrrhosoma (Gorb, Kesel nymphuls and Coenagrion 40% of area dissolved, Relative polar, not 1 h and Berger, puella; Dragonfly Aeshna 10% looked intact very effective to non-polar 2000) cyanea No specific chemistry was Ethanol 24.6 compounds. identified Subtle changes Unmatched results (Wan, Cong, Dragonfly Pantala 2 h obtained with Wang et al., flavescens fabricius Pillae appeared different species amorphous 2008)
Damselflies Pyrrhosoma 30% of area looked More polar solvent (Gorb, Kesel nymphuls and Coenagrion No specific chemistry was intact Methanol 32.6 (25) 1 h Unmatched results and Berger, puella; Dragonfly Aeshna identified 70% was substantially obtained with 2000) cyanea destroyed different species
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(Wan, Cong, Dragonfly Pantala 15-20% of the area 2 h Wang et al., flavescens fabricius dissolved 2008)
Comments Dielectric Extraction Solvents Insect body Insect wings References constant time Chemistry Physical General
Saturated and unsaturated Silverleaf # whitefly FA with even numbered Hexanec/CH To remove Bemisia 1-2/ carbons( C14-C20) No morphology change (Buckner and Cl :MeOH epicuticular lipids 3 argentifolii was reported Hagen, 2003) (2:1) 30 m Phosphatidylethanolamine and surface pillae Belows and (PE), phosphatidylcholine Perring (PC)
Saturated wax ester (C44- (Nelson, Giant whitefly C60) Freeman and Aleurodicus † ‡ Buckner, dugesii Long chain Alds and Alcs (C30) 2000)
1.5/0.5 m Alds† and Alcs‡ with even Hexane used for * numbered carbons (C26- HCs and wax esters Giant whitefly C32) on epicuticular layer (Nelson, Aleyrodes Guershon and Hexanec/ Wax esters: even numbered No morphology change Chloroform singularis Gerling, 1998) Chloroformb carbons FAs# (C16-C24), was reported removed and Alcs‡ (C26, C28) intracuticular waxes, i.e., long chain Alds† and Even carbon numbered wax ‡ esters (C38-C64) Alcs Silverleaf (Buckner, whitefly Odd numbered carbons n- 1/1 m Hagen and Bemisia alkanes (C25-C35) Nelson, 1999) argentifolii Even carbon numbered Alds† and Alcs‡ (C28-C34)
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Comments Dielectric Extraction Solvents Insect body Insect wings References constant time Chemistry Physical General
Adult whitefly Even numbered carbons † ‡ (Buckner, Bemisia tabaci Alds and Alcs (C30 - Nelson and and 1/1-2 m C34) Mardaus, Trialeurodes Even numbered carbons 1994) vaporariorum wax esters (C38-C54) Hexane used for * Free FAs# (C14-C28) HCs and wax esters on epicuticular layer (Nelson, n-, and branched alkanes Dustywing Freeman, Hexanec/ 1.5/ 0.5 m (C23-C33) No morphology change Chloroform Semidalis flinti Buckner et al., Chloroformb was reported removed Even carbon numbered wax intracuticular 2003) esters (C34-C44) waxes, i.e., long chain Alds† and Methyl-branched alkanes Alcs‡ (C25-C53) Potato beetles (Yocum, Leptinotarsa 2 m/ 45 s Triacylglycerol (C50, C52 Buckner and decemlineata and C54) with even carbon- Fatland, 2011) numbered acid isomers (C16, C18)
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Comments Dielectric Extraction Solvents Insect body Insect wings References constant time Chemistry Physical General
Dried-bean Even carbons numbered (Gołebiowski, beetle FAs#, Alds†, Alcs‡ (C16- Malinski, 10 s/ 5 m Acanthoscelides C18); triacylglycerols and Nawrot et al., obtectus sterol Petroleum ether 2008) used to minimise dissolution of Calliphora intracuticular lipid vicina, (free FAs#, (Gołebiowski, Petroleum Dendrolimus Saturated and unsaturated e glycerides) which Maliński, ether / pini and 10 s/ 5 m C5-C20 in alkyl chain of No morphology change removed by Boguś et al., Dichloro- Galleria FAs#. was reported f dichloromethane 2008) methane mellonella larvae High contents of FA# were explained # FAs C15-C34 found in as the result of anti- (Gołebiowski, Dendrolimus petroleum ether fraction fungal infections. Bogus, 10 s/ 5 m pini FAs# C8-C34 found in Paszkiewicz et dichloromethane fraction al., 2010) a: 0.5mL-1mL, b: 4-8mL, c: 5-10mL, d: 50mL, e: 150mL, f: 200mL *HCs: Hydrocarbons, #FAs: Fatty acids, †Alds: Aldehydes, ‡Alcs: Alcohols The polarity of a solvent reflects the balance between the polar and non-polar components of a single molecule, and solvents ranging in polarity can be used to extract the corresponding wax components from the wing surfaces. High polarity solvents will dissolve polar compounds and low polarity solvents dissolve low polarity compounds. There are different parameters for classifying the polarity of a solvent, i.e., dipole moment, dielectric constant and miscibility with water. The greater the dielectric constant, the greater the solvent polarity (water = high, gasoline = low).
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Insect cuticular waxes are composed of an intricate mixture of lipids, including wax esters, triglycerides, hydrocarbons and other derivatives of aliphatic hydrocarbons (Lockey, 1959; Soliday, Blomquist and Jackson, 1974; Lockey, 1980; Lockey, 1985; Buckner, Mardaus and Nelson, 1996; Nelson, Fatland, Buckner et al., 1999; Lapointe, Hunter and Alessandro, 2004; Buschhaus and Jetter, 2011; Gołebiowski, Bogus, Paszkiewicz et al., 2011; Yocum, Buckner and Fatland, 2011). Advances in analytical chemistry techniques (GC-MS, FTIR, etc.) have enabled studies on the chemistry of surfaces to become more accessible. Many previous studies have reported that more than 40% of the total waxes extracted from whiteflies are wax esters, except for the exuviae of Aleyrodes singularis, Bemisia tabaci, Aleurodicus dugesii and Trialeurodes vaporariorum, which have long chain aldehydes and long chain alcohols as the main components (Buckner, Nelson and Mardaus, 1994; Nelson, Buckner and Fatland, 1994; Nelson, Walker, Buckner et al., 1997; Nelson, Guershon and Gerling, 1998; Buckner, Hagen and Nelson, 1999; Nelson, Fatland, Buckner et al., 1999; Nelson, Freeman and Buckner, 2000; Buckner and Hagen, 2003). In the case of beetles and grasshoppers, long chain hydrocarbons are the key cuticle constituents (Soliday, Blomquist and Jackson, 1974; Gołebiowski, Malinski, Nawrot et al., 2008; Geiselhardt, Geiselhardt and Peschke, 2009). The cuticles of some dustywings and booklouse species are mainly composed of fatty acids (Howard and Lord, 2003; Nelson, Freeman, Buckner et al., 2003). Recently, terpenes were detected on the surface of Acanthoscelides obtectus (Gołebiowski, Malinski, Nawrot et al., 2008). The length of the carbon chain in these terpenes varies from less than 10 to up to 30-35 carbon atoms. In the case of esters, some species can have up to 54 carbon atoms in their wax components. Insects have special mechanisms to adapt to tough environmental conditions (Van Dooremalen and Ellers, 2010). For example, the goldenrod gall fly Eurosta soliginis modifies the hydrocarbon backbone of their wax components by methylation to lower the melting and transition temperature during winter, and the potato beetle Leptinotarsa decemlineata stores a large amount of triacylglycerol for energy (Nelson and Lee Jr, 2004; Yocum, Buckner and Fatland, 2011).
Numerous investigations have been conducted on the chemistry of insect surfaces for various purposes. Much of this research has reported the functionality of cuticle chemistries with a view to providing an insight into the social communication of colonised insects, including colony membership, hierarchical dominance, fertility status 21 and task group membership (Moneti, Dani, Pieraccini et al., 1997; Liebig, Peeters, Oldham et al., 2000; Endler, Liebig, Schmitt et al., 2004; Ferveur, 2005; Howard and Blomquist, 2005; Dani, 2006; Martin and Drijfhout, 2009; Choe, Ramírez and Tsutsui, 2012). Recently, fatty acids, terpenoids, and chitins were found to perform significant roles as anti-microbial agents (Kramer and Muthukrishnan, 1997; Tapia, Vallejo, Gouiric et al., 1997; Powell and Raffa, 1999; Keeling and Bohlmann, 2006; Jarrold, Moore, Potter et al., 2007; Savluchinske-Feio, Nunes, Pereira et al., 2007; Carballeira, 2008; Gołebiowski, Maliński, Boguś et al., 2008; Bogus, Czygier, Golbiowski et al., 2010; Gołebiowski, Bogus, Paszkiewicz et al., 2010; Gołebiowski, Bogus, Paszkiewicz et al., 2011; Van Dyck, Caulier, Todesco et al., 2011). Some reports have distinguished insect species within the same order based on the differences in their hydrocarbon compositions (Buckner, Nelson, Hakk et al., 1984; Bernier, Carlson and Geden, 1998; Howard and Baker, 2003; Lapointe, Hunter and Alessandro, 2004; Saïd, Costagliola, Leoncini et al., 2005). Despite the long history of studying insect surfaces, there are very few works that have expressed the chemistry of the surface in terms of the context of surface wettability.
2.5. Hierarchical structure induced self-cleaning
2.5.1. Superhydrophobicity and self-cleaning surfaces in nature
In 1936, Fogg published the observation that very high water contact angles (WCA) would form on the upper surface of the leaves of Triticum (wheat) plants, representing the first reported natural surface that possessed WCAs above 150°. This property is now commonly known as ‘superhydrophobicity’ (Fogg, 1944). Since then, nature has been the source of many valuable templates used in the design of synthetic hydrophobic materials. Shark skin, bird feathers, gecko feet, plant leaves and insects are some examples which have been shown to exhibit highly hydrophobic properties. A synthetic replica of shark skin, which formed a WCA of 146°, has been used as a model for the reduction of drag in fluid flow environments (Bhushan, 2011; Bhushan, 2012). Gecko feet (WCA of 128° at the setae) are known to exhibit reversible adhesion with surfaces (Bhushan and Sayer, 2008; Liu, Du, Wu et al., 2012; Stark, Badge, Wucinich et al., 2013). Bird and duck feathers (WCA from 114° to 126°) possess corrugated surfaces that create air pockets that prevent water from touching the surface. These have been used as a model for water repellency treatments (Cassie and Baxter, 1944;
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Bormashenko, Bormashenko, Stein et al., 2007; Liu, Chen and Xin, 2008). Each of these surfaces exhibited a WCA lower than 150° and as such are not considered superhydrophobic. From the published literature, it appears that plant leaves and the surfaces of some insects are the only natural surfaces that exhibit both superhydrophobic and self-cleaning properties.
2.5.1.1. Plant surface structures
As in the case with insect surfaces, plant surfaces are also covered by a cuticular surface layer. Depending on the plant species, this layer is secreted by the epidermal cells either at certain stages of their development throughout the lifetime of the plant, or on a single occasion only. The cuticular layer consists of two compartments: a layer of epicuticlar wax crystals and a ‘cutin’ layer, which can be considered to be analogous to the epicuticle and intracuticle present on the surfaces of insects. The composition of the plant epicuticular waxes is very similar to that found with insects, whilst ‘cutin’ is comprised of a polymer of predominantly ω- and mid-chain hydroxyl and epoxy C16 and C18 fatty acids together with glycerol (Samuels, Kunst and Jetter, 2008). Some plants that demonstrate superhydrophobic surfaces are presented in Figure 2.2. The upper surface of the lotus leaf is covered by a layer of microscale papillae which is, in turn, covered by an array of nanoscale asperities (Figure 2.2A) that are composed of epicuticular hydrophobic wax tubules (Nosonovsky and Bhushan, 2007b). The WCA and CAH are 164° and 3°, respectively (Koch, Bhushan, Jung et al., 2009).
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Figure 2.2. Images of some superhydrophobic plant surfaces, and their corresponding high resolution SEM images: (A) lotus leaves; (B) Indian Canna leaves; (C) taro leaves; (D) perfoliate knotweed leaves. (Adapted with permission from Elsevier (Guo and Liu, 2007))
Other plant leaf surfaces that exhibit superhydrophobic and self-cleaning properties include the Indian Canna (Figure 2.2B), Taro (Figure 2.2C) and perfoliate knotweed (Figure 2.2D). Indian Canna leaves are covered by many wax platelets, distributed randomly on a series of rod-like structures. This increases the proportion of air that can be trapped within the surface, producing water contact angles in excess of the 150° contact angle condition for superhydrophobicity (i.e. 165°). The surface of taro leaves (Figure 2.2C) is composed of a layer of elliptic protrusions, which are uniformly arranged on micro-scale cave-like structures, resulting in a WCA of 159° and CAH of 24
3°. Similar surface hydrophobicity characteristics were observed for perfoliate knotweed leaves (Guo and Liu, 2007).
The examples shown in Figure 2.2 are just a few of the plants that have been identified as possessing superhydrophobic surfaces. Neinhuis and Barthlott reported the static WCAs of 200 water repellent plant species (Neinhuis and Barthlott, 1997), most of which were classified as having superhydrophobic properties. The common feature shared by these surfaces is their very dense layer of three-dimensional cuticular wax crystals arranged either randomly or uniformly on their corresponding micro-scale surface features (papillae).
The wax compositions found on plant surfaces, together with their orientation, are species specific with their characteristic composition and orientation being used in taxonomic classification (Barthlott, Neinhuis, Cutler et al., 1998; Barthlott, Theisen, Borsch et al., 2003; Koch, Bhushan and Barthlott, 2008). The morphology of the waxes can range from being irregularly placed to being arranged in highly organised structures on the surface, prompting the question: what factors are responsible for determining the three dimensional structure of the surface wax crystals? Despite the lack of information available on these factors, it has been postulated that the cutin network may play a role in controlling the orientation of the wax crystals (Jeffree, 2006).
2.5.1.2. Insect surface structures
The surfaces of insect wings have also attracted a great deal of attention from researchers due to the sophisticated structures that exist on these surfaces. Such surfaces serve to afford the wings with unique properties, such as anti-fogging and anti-wetting behaviour. Insect surfaces have also been the source of a great deal of valuable information regarding the mechanisms responsible for superhydrophobicity and self- cleaning behaviour. In the past few years, insects such as the butterfly, cicada, water strider, dragonfly and damselfly have been recognised as insects that possess these two unique properties. Their outermost layer, the cuticle, is the barrier that directly interacts with environment. This layer has been optimised through millions of years of evolution to protect the insects from ever-changing environments. The cuticle is secreted by a single layer of epidermal cells, forming a lipophilic structure (Lockey, 1980; Lockey, 1985; Nelson and Blomquist, 1995; Buschhaus, Herz and Jetter, 2007; Jetter and Kunst, 2008; Samuels, Kunst and Jetter, 2008; Buckner, 2010; Moussian, 2010). This structure
25 consists of two major components, which can be distinguished by their solubility in organic solvents (Buschhaus and Jetter, 2011). The outermost layer of the cuticle, the epicuticle, is composed of a mixture of aliphatic hydrocarbons and their derivatives; these compounds contain one or more oxygen functional groups including esters, ketones, alcohols, aldehydes and fatty acids (Koch and Ensikat, 2008; Samuels, Kunst and Jetter, 2008). The intracuticular layer, located beneath the epicuticle is a mixture of chitin (poly-N-acetylglucosamine) and protein (Lockey, 1980; Lockey, 1985; Lockey, 1988). The mixture of organic components is self-organised in the epicuticular layer of the cuticle, producing either a smooth two-dimensional (2D) wax film or a three- dimensional (3D) wax crystal structure. These structures have afforded the organisms the ability to adapt to their environmental living conditions.
The surface of dragonfly wings is covered by an array of nanopillars that form a fractal structure (Figure 2.3). This structure enables the surface to retain pockets of air within its surface when it comes into contact with water, producing a high WCA. Moreover, low CAH and water droplet sliding angles less than 10°, indicating the ability to self-clean, can be observed in many different Odonata species (Ivanova, Hasan, Webb et al., 2012; Ivanova, Hasan, Webb et al., 2013; Pogodin, Hasan, Baulin et al., 2013a). These self-cleaning properties can be further accelerated by the wings being moved in turbulent conditions during flight (Nishimoto and Bhushan, 2013).
Figure 2.3. The surface morphologies of the Hemianax papuensis dragonfly wing. Credit photograph to © Arthur Chapman.
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To date, there is not a universally adopted system for the description of the surface morphologies of insect wings. Byun et al. used the terms ‘layered cuticle’, ‘setae’, ‘denticles’ and ‘fractal’ to describe the morphological features present on the surfaces of the insect wings in their study. A majority of the insect surfaces characterised to date could be readily classified according to this description. The term ‘layered cuticle’ generally refers to a surface that contains scale-type structures that overlap, such as those typically found on butterfly wings. Surfaces with ‘setae’ contain high aspect ratio needles or hairs. ‘Denticle’ structures refer to tooth-like projections and these can vary in their morphology ranging from small hemispheres to taller pillars. ‘Fractal’ structures, as described by Byun et al., are not true fractals but composed of an irregular array of fine nanoscale protrusions (Byun, Hong, Saputra et al., 2009). Amongst these structural types, the presence of layered cuticles, denticles and fractal structures appear to result in the production of the most superhydrophobic surfaces, whilst the presence of setae alone on a surface does not induce superhydrophobic properties (Table 2.2). Fractal and layered cuticle typically display surface hierarchy and therefore exhibit WCA >150°, whilst setae and denticles do not.
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Table 2.2. Micro- and nano-scale wax crystal morphologies on the epidermal cells of insect wing surfaces and their WCAa)
Structural Order Species WCA (°) morphology Coleoptera Allomyrina dichotoma Setae 54 Coleoptera Chrysolina virgata Setae 71 Isoptera Schedorhinotermes sp. Setae 71 Coleoptera Amphizoa sinica Setae 109 Hymenoptera Vespa simillima xanthoptera Setae 121 Hymenoptera Vespa dybowskii Setae 126 Hemiptera Meimuna microdon Denticle 140 Orthoptera Atractomorpha lata Denticle 148 Orthoptera Acrida cinerea cinerea Denticle 151 Odonata Hemicordulia tau Fractal 157 Odonata Hemianax papuensis Fractal 161 Lepidoptera Artogeia canidia Layered cuticle 162 Lepidoptera Papilio xuthus Layered cuticle 168 a)This table was modified and updated from (Byun, Hong, Saputra et al., 2009).
Terminology regarding structural morphology was also adopted from Byun et al.
The surface topologies of the wings of several insects are presented in Figure 2.4. The wings of the Nasutitermes sp., which belongs to the order Isoptera, exhibits superhydrophobic properties (WCA = 180°, Figure 2.4A) (Watson, Cribb and Watson, 2010). The surface of their wings is covered by micro-scale setae together with a star- shaped nanoscale structure (micraster), which produces a combined hierarchical structure that exhibits superhydrophobic properties. Similarly, the surface of the grasshopper wing, Acrida cinerea cinerea (WCA = 151°), is covered by micro-denticles that are enclosed by nanoscale wax-crystals (Figure 2.4B). High resolution SEM images indicate the presence of a binary structure (containing micro-and nano-features), which results in the wings possessing superhydrophobic properties. Cicada Meimuna opalifera (Walker) wings also exhibit superhydrophobicity (WCA 165°) despite not having a
28 hierarchical surface structure (Figure 2.4C) (Byun, Hong, Saputra et al., 2009). The surface is covered with a layer of dense nanopillars that result in a high degree of air entrapment on the surface. It is noteworthy that not all cicada wings surfaces exhibit the same properties; Hemiptera Meimuna microdon cicada wings are also covered by layer of denticle structures, however exhibits a WCA of only 140° (Table 2.2). In this case, the density, size, shape and composition of the denticles clearly contribute significantly to the extent of surface hydrophobicity. It is clear that the presence of surface topographical hierarchy is an important factor in imparting superhydrophobicity to a surface but other factors also play an important role. According to the Cassie-Baxter theory, the two important components to determine the hydrophobicity of a surface are the surface chemistry of the materials comprising the surface and the ability of the surface to entrap air, this ability being a function of the roughness and topology of the surface. The presence of a hierarchical surface clearly assists in the entrapment of air; however this is also a function of the density, size and shape of the hierarchical roughness features, as seen for the cicada wing surface shown in Figure 2.4C.
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Figure 2.4. Insect wings and their corresponding high resolution SEM images. (A-A2) Isoptera Nasutitermes sp.; (B-B2) Orthoptera Acrida cinerea cinerea; (C-C2) Hemiptera Meimuna opalifera. (Adapted with permission from ACS publication and Elsevier (Byun, Hong, Saputra et al., 2009; Watson, Cribb and Watson, 2010) and Encyclopedia of Life (2014) (eol.org, license agreements can be found at http://creativecommons.org/licenses/by-nc-sa/2.0/ and http://creativecommons.org/licenses/by-nc/2.0/), photographs taken by Kenpei and Tomomarusan).
The superhydrophobicity of an insect wing surface, together with its ability to self- clean, are very important factors that contribute to an insect’s ability to survive. The nanoarray structures present on the surfaces of some insect wings such as those of the cicada and dragonfly afford the insect antireflective properties, which can assist in protecting them from attack from predators (Watson, Myhra, Cribb et al., 2008). The superhydrophobic and self-cleaning properties can assist to keep their surfaces clean and free from contaminants that may adversely impact their antireflective properties. Moreover, dragonfly wings were recently reported to possess bactericidal properties
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(Ivanova, Hasan, Webb et al., 2012; Ivanova, Hasan, Webb et al., 2013). The mechanism by which this occurs is unclear, however it was postulated that this bactericidal behaviour was related to the wettability of the surface (Pogodin, Hasan, Baulin et al., 2013a).
2.5.2. Superhydrophobicity with additional properties
2.5.2.1. Superhydrophobic surfaces with high adhesive force
The hierarchical surface structure of the petals of some flowers imparts superhydrophobic properties to their surface, resulting in high CAH (greater than 10°) values. These surfaces possess sufficient micro- and nano-structure to induce superhydrophobic properties; however these surfaces also display a high adhesive force towards water. Water droplets in contact with these petal surfaces remain spherical in shape, however these water droplets do not roll off the surface, even when the petal is turned upside down (Figure 2.5A). This is referred to as the ‘petal effect’ (Feng, Zhang, Xi et al., 2008; Schulte, Droste, Koch et al., 2011). The micro- and nanostructure of the petals of the rose, Chinese Kafir lily and sunflower, have a smaller pitch value compared to that of the lotus leaf (i.e. pitch value is the distance between the micropapillae, which is approximately 20 µm compared to 40-50 µm of the lotus leaf). As a result, water droplets coming in contact with the surface can penetrate between the microstructure and partially penetrate between the nanostructures, inducing the ‘Cassie impregnating wetting state’. The wettability of this type of surface is less than that of a surface exhibiting the Wenzel regime but greater than that of a surface exhibiting the Cassie-Baxter regime of wetting (Bhushan and Her, 2010). This also implies that the CAH of such surfaces will increase with increasing wetting area, which is governed by the hierarchical structure of the surface.
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Figure 2.5. Superhydrophobic surfaces with high water adhesion. (A) Surfaces that exhibit the ‘petal effect’ exhibit high water contact angles and high adhesion to water. (B) Rose petals, for which the effect is named, possess a similar microstructure to that of lotus leaves; however the nanostructure is comprised of cuticular folds rather than nanocrystals. (C) Gecko feet also exhibit the petal effect. The soles of their feet possess highly hierarchical surface structures that exhibit high water contact angles but the water adheres strongly to the surface. These structures also enable the geckoes to adhere to solid surfaces, enabling them to climb walls. ST: setae, SP: spatula, BR: branched. (Reproduced with permission from ACS publication and Elsevier (Gao, Wang, Yao et al., 2005; Feng, Zhang, Xi et al., 2008; Bhushan, 2012).
Bhushan and Her identified the surface structural properties that distinguish the ‘petal effect’ from the ‘lotus effect’ (Bhushan and Her, 2010). It was demonstrated that if a microstructure possesses a large pitch value, i.e. small peak to base height and low density nanostructure, water droplets coming in contact with the surface can impregnate the microstructure and partially wet the nanostructure. This will result in high CAH and the development of high adhesive forces that have minimal effect on the magnitude of
32 the high static CA (Figure 2.5B-B2). On the other hand, a microstructure possessing a small pitch and high density of nanostructure will create a surface that exhibits an increased propensity to form air pockets, leading to low adhesion of water droplets in contact with the surface. Like lotus leaves, rose petals, shown in Figure 2.5B-B2, are covered with micro papillae; however instead of having nanocrystals on the surface, the papillae on rose petals have cuticular wrinkles or folds that increase the degree of surface roughness. This structure again enables the surface to trap air-pockets, inducing superhydrophobicity; however this structure is not self-cleaning since they have large CAH (Figure 2.5 A, A1).
The foot attachment pads of several animals are capable of repeatedly attaching and detaching from a variety of surfaces. This is useful for locomotion on vertical walls or on inverted surfaces (Hu, Chan and Bush, 2003; Liu, Du, Wu et al., 2012). This dynamic attachment mechanism is referred to as reversible or ‘smart’ adhesion (Bhushan, 2012). There are two kinds of foot pads, either relatively smooth or with a hair-like coating. The former can be found in some amphibians, for example tree frogs, cockroaches and grasshoppers (Federle, Barnes, Baumgartner et al., 2006). The latter can be found in many insects, for example, beetles, spiders and lizards (Bhushan, 2012). Gecko feet have a hair-like surface and are considered an animal equivalent to the plant surfaces that exhibit the petal effect (Figure 2.5C-C3). These surfaces exhibit a static water contact angle larger than 150° whilst their adhesive force to water is reported to be approximately 66 μN (Liu, Du, Wu et al., 2012). The explanation for these properties originates from the morphology of the surface of the skin on their toes (Gao, Wang, Yao et al., 2005) (Figure 2.5C), which are covered by hundreds of thousands of setae (ST). Each ST contains hundreds of spatulae and their hierarchical structure ranges over 5 orders of magnitude, from macroscale (mm) to nanoscale (nm). This hierarchical structure allows geckos to adjust the shear adhesion of their feet when in contact with a range of surfaces under different environmental conditions (Stark, Badge, Wucinich et al., 2013).
2.5.2.2. Anisotropic superhydrophobic surfaces
Surfaces that possess directionally dependent or anisotropic superhydrophobicity have been the subject of a number of studies due to their potential application in microfluidic devices, their ability to form evaporation-driven surface patterns and their
33 potential to form coatings that are readily cleaned (Higgins and Jones, 2000; Liu, Zhai and Jiang, 2006; Liu, Yao and Jiang, 2010). Anisotropic superhydrophobicity has been observed on the surfaces of a number of plants and insects, including rice leaves and butterfly wings (Figure 2.6). These surfaces were found to exhibit properties associated with anisotropic hydrophobicity, that is low water drag, superhydrophobicity and the ability to self-clean (Bixler and Bhushan, 2012; Bixler and Bhushan, 2013), properties that arise from the unique hierarchical structures present on the surface. (Bixler and Bhushan, 2013).
Figure 2.6. The anisotropic surface structures of (A) rice leaves and (B) butterfly wings. The superhydrophobic structures present on both surfaces are directionally ordered, resulting in a variable wettability that is dependent on the direction of movement. (Reproduced with permission from Elsevier (Guo and Liu, 2007; Peng, Hu and Zhang, 2011), photographs taken by Anne Ten Donkelaar and Stepanka Nemcova).
Rice leaves possess a hierarchical structure similar to that of the lotus leaf, however the arrangement of the micro- and nanostructure on the rice leaves is anisotropic (Figure 2.6A-A2), whereas the arrangement is homogeneous on the upper surface of lotus leaves. The surfaces of rice leaves are patterned with sinusoidal grooves covered by 34 micropapillae that are arranged in a quasi-one-dimensional order parallel to the edge of the leaf (Figure 2.6 A, A1). These micropapillae also contain wax nanobumps on their surface (Figure 2.6 A2) (Feng, Li, Li et al., 2002). As a result of this hierarchical structure, water droplets making contact with the surface roll off the leaf along the parallel grooves rather than in a direction perpendicular to these grooves, enhancing the ability of the leaf to self-clean. The tilting angles associated with these two directions of movement have been reported to be 4° and 12°, respectively (Nishimoto and Bhushan, 2013).
Butterfly wing surfaces have been shown to demonstrate anisotropic superhydrophobicity and are known for their multifunctional properties such as structural colour, chemical sensing capacity and ability to fluoresce (Gu, Uetsuka, Takahashi et al., 2003; Vukusic and Sambles, 2003; Vukusic and Hooper, 2005). The directional adhesion of water droplets on the surface of the wings, both along and against the radial outward (RO) direction away from the body centre can be observed (Figure 2.6B-B2). When the wing is tilted along the RO direction, the water droplets initiated the process of ‘roll off’ at an angle of 9°, whereas when tilted perpendicular to the RO direction, the water droplets were tightly pinned to the wing surface. This unique property of butterfly wings arises from the alignment of the anisotropic shingle- like microscales, together with the radially aligned nanogrooves.
2.5.3. Hierarchical structure of superoleophobic surfaces
Not all surfaces that possess hierarchical structure are superhydrophobic. Several aquatic species exhibit superoleophobicity rather than superhydrophobicity, exhibiting an oil CA greater than 150° when submerged in water. In principle, superoleophobicity is very similar to superhydrophobicity, in that the oil CA on a surface is determined by the interfacial energies between the oil and the surface, the oil and the surrounding medium (i.e. water), and the surface and the medium. These organisms possess hierarchical surface structures that are self-cleaning, are antifouling and promote low- drag when moving through water (Bixler and Bhushan, 2013). The skin of a shark is one such example; it is covered by minute scales known as dermal denticles, which are shaped like small ribs (or ‘riblets’). They are positioned such that they align with the direction of fluid flow as the shark swims through the water. It is believed that sharks have the ability to remain clean due to their flexible, low drag riblet microstructure
35 together with a mucous layer on the skin (Bushnell and Moore, 1991; Bechert, Bruse, Hage et al., 1997; Dean and Bhushan, 2010). The design of the riblets, together with the mucous layer, enables the shark to move through water with reduced drag. This surface structure also assists in the protection of the sharks from abrasion, which in turn minimises the opportunities for microscopic organisms to adhere (Bhushan, 2012).
The scales of other fish species are further examples of self-cleaning surfaces in aquatic environments. Their living conditions can be subjected to pollution arising from oil leaks or garbage originating from branched rivers etc. (Hay, 1996). The body of the fish body is, however, well protected from the attachment of microorganisms and hence remains clean. The scales of fish display a similarity to the shark skin in that they are covered by a hierarchical structure, consisting of sector-like scales (diameter of 4-5 mm) covered by papillae (100-300 µm in length and 30-40 µm in width), and exhibit a particularly high oil contact angle in water (163°).
The surfaces of snail shells also possess the ability to remain clean, despite their dwelling environments and their appearance on rainy days. The surface of snail shells is comprised of a rough structure consisting of line grooves (pitch of 0.5 mm), smaller grooves crossing the line groove (pitch of 0.1 mm) and micro-grooves between the line grooves (pitch of 10 µm). As a result snail shells have a rough but regular hierarchical structure. There is one major difference, however, between these structures and those found on superhydrophobic surfaces that entrap air within their hierarchical structure; the snail shells trap water droplets within their rough surface. This macroscopic surface roughness, in fact, facilitates the entrapment of water, ensuring that the shells remain in a wet condition. This is a key factor that contributes to their ability to self-clean, in that their usually wetted surface is rarely able to be contaminated (Nishimoto and Bhushan, 2013). With this approaches, not only contaminants, but also biological organisms, i.e. bacterial cells, can also be cleaned by very similar mechanisms.
2.6. Bacteria
2.6.1. Bacterial physiology
Bacterial cells, which are classified as prokaryotes, were first observed in 1676 under a microscope by Anton van Leeuwenhoek. Bacterial cells assume four main morphologies, including coccus (spherical), bacillus (rod-shape), spirillum (spiral) and filamentous (elongated). Their general structure includes a cell wall surrounding genetic 36 materials, i.e. plasmids and chromosomes. Some bacteria have flagella which help them move in aquatic environments (Figure 2.7).
Figure 2.7. General structure of bacterial cells (Reproduced with permission for single use from ‘Molecular expression’ http://micro.magnet.fsu.edu/cells/bacteriacell.html).
Based on cell wall structure, bacteria are generally divided into Gram-positive and Gram-negative (Figure 2.8). The cell wall of Gram-positive bacteria consists of many layers of peptidoglycan, which have the ability to retain the violet coloured stain ‘crystal violet’ (Madigan, Martinko and Parker, 2009). Peptidoglycan is embedded in teichoic acid and accounts for about half of the dried mass of the cell wall (Shai, 2002; Epand, Rotem, Mor et al., 2008; Madigan, Martinko and Parker, 2009). The peptidoglycan is located outside the cytoplasmic membrane and provides a strong barrier from attachment of antibodies or antibacterial agents. The rigidity of the bacterial cell is due to multiple layers of peptidoglycan, which is a polymer of β (1-4)- linked N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc). It consists of four alternating L- and D-amino acid with all the lactyl groups substituted by stem peptides (Royet and Dziarski, 2007). Most Gram-positive bacteria have L- lysine as the third amino acid in their stem peptides composition (Royet and Dziarski, 2007). Teichoic acids are polymers which are linked by phosphodiester groups of
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adjacent glycerol residues at position one and three or adjacent ribitol residues at position one and four. These make up a poly-glycerolphosphate or poly- ribitolphosphate backbone (Knox and Wicken, 1973). Most Gram-positive bacteria have their teichoic acid covalently bound to membrane glycolipids; these contain a lipoteichoic acid complex providing immunogenic ability which is derived from the teichoic acid component. However, the main role of teichoic acids is to attract cations, such as magnesium (Mg2+) and sodium (Na+), and to strengthen the rigidity of the cell membrane (Knox and Wicken, 1973).
Figure 2.8. Membrane structure of Gram-positive and Gram negative bacteria (Copyright © The McGraw-Hill Companies. Inc.)
Different from Gram-positive bacteria, the Gram-negative bacterial cell wall has a much thinner peptidoglycan layer. They contain additional outer membrane layers composed of phospholipids and lipopolysaccharides, which interact with the external environment. The negative charges that cover the cell wall are derived from lipopolysaccharides, which are often species specific for different bacterial strains. The toxicity of many Gram-negative bacterial cells is usually associated with the lipopolysaccharide layer (Beveridge, 1999). Even though their cell wall is thin, they are sufficiently strong to withstand approximately 3 atm of turgor pressure (Gumbart, Wiener and Tajkhorshid, 2007), and can be elastic enough to expand several times their
38 normal surface area (Koch and Woeste, 1992; Doyle and Marquis, 1994; Yao, Jericho, Pink et al., 1999). The differences in membrane structures of Gram-positive and Gram- negative bacteria cells cause them to behave differently when in contact with a surface. Once bacteria successfully invade a host surfaces they form aggregations called biofilms, which can be detrimental from both an economic and public health perspective. Therefore, it is important to understand the underlying interactions in order to find suitable approaches that control their formation and proliferation.
2.6.2. Biofilm formation
Biofilms are defined as the attachment and development of a microorganism community embedded in extracellular matrix on a substrate surface (O'Toole, Kaplan and Kolter, 2000). The organisms undergo a transition state between being free swimming in their native environment (planktonic cells) to being cells that form part of a surface-attached community. The essential factors for formation of biofilms are microbes and a substratum (Garrett, Bhakoo and Zhang, 2008). There are numerous advantages to bacteria from taking part in the formation of a biofilm. These advantages include: resistance against antibiotics (Schmidt, Winter and Gallert, 2012), resistance to disinfectants (Ryu and Beuchat, 2005; Simões, Simões and Vieira, 2009) and sheltering from dynamic environments (Liu and Tay, 2002; Di Iaconi, Ramadori, Lopez et al., 2005). The intercellular communication within the biofilm community enhances the regulation of gene expression, which enables the bacterial cells to temporally adapt their phenotypic variation to suit the conditions of the surrounding environment, such as the deficiency of nutrients (Dalton and March, 1998; Kjelleberg and Molin, 2002; Daniels, Vanderleyden and Michiels, 2004).
Biofilm formation can involve a single microbial species or multiple microbial species adhering onto a range of surfaces. On most environmental surfaces, mixtures of various species dominate biofilms. It is usually single species that are responsible for the infection of medical devices and implants (Behlau and Gilmore, 2008; Holmes, Doré, Saraswatula et al., 2008; Seo, Lee, Rayamahji et al., 2008; Wu, Sendamangalam, Xue et al., 2012; Bulgarelli, Schlaeppi, Spaepen et al., 2013). According to a public health report published in 2002 (Klevens, Edwards, Richards Jr et al., 2007), approximately 64% of hospital attending cases were due to the presence of a viable bacterial infection of medical devices and implants, which was associated with 100,000
39 mortalities annually in the US alone. Effort toward the studies of the nature of biofilms started over 3 decades ago with the discovery that in natural living conditions, microorganisms were predominantly found attached to surfaces (Geesey, Richardson, Yeomans et al., 1977).
The first recorded observation of a biofilm was published in 1933 by Henrici (Henrici, 1933). The impact of biofilm formation had, however, been recognised prior to this date as a result of ships being fouled in marine environments (Angst, 1923). It has been estimated that the costs associated with the fouling of ship hulls in the US Navy alone is approximately $180M-$260M per year, which represents only 0.5% of the total number of ships world-wide (Schultz, Bendick, Holm et al., 2011).
The basic development of biofilms is described by a two stage kinetic binding model (Figure 2.9). The first stage involves the initial reversible interaction between bacterial cells and the material surface, followed by the second stage when specific and non-specific interactions at the molecular level take place (Bos, Van Der Mei and Busscher, 1999; Lichter, Van Vlietpa and Rubner, 2009). The interactions that occur in stage two involve proteins that are expressed on bacterial surface and molecules on the material surfaces. The second stage occurs slowly and irreversibly once a mature biofilm is formed. Additional to these two main steps of biofilm maturation, O’Toole et al. proposed that the starvation response pathway can also be considered as part of the biofilm development (O'Toole, Kaplan and Kolter, 2000). This happens when the source of nutrients becomes depleted, and single microbial cells detach from the surface and return to their planktonic mode which may to infect new areas.
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Figure 2.9. Schematic of the two stages biofilm formation on surfaces. Reproduced with permission from American Chemical Society (Lichter, Van Vlietpa and Rubner, 2009).
For these reasons, controlling bacterial attachment on material surfaces has been a long-standing challenge for science. Several approaches have been developed to limit colonisation of microbes on surfaces; however most of these approaches have focussed on the use of chemical-based methods, which has led to the new and rising problem of bacterial resistance. Preventing bacterial adhesion by modifying the topography of a surface, e.g. imparting a superhydrophobic and self-cleaning surface structure, has been identified as being a more attractive solution for controlling biofilm formation.
2.7. New approaches for controlling biofilm formation
2.7.1. Antibiofouling
As noted, biofouling has remained a complex and problematic issue for some time due to its significant impact on the economy and public health. As a result, the design of antibacterial materials has focussed on advancing strategies designed to limit the extent of bacterial colonisation onto a surface (Zhang, Wang and Levänen, 2013). Traditionally, antibacterial surfaces were designed so that they leached biocides
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(commonly known as cytotoxins) from host cells, which caused the death of bacterial cells on site and in areas surrounding the surface. For example, peptides and chitosan have been used as chemical-based agents for controlling the bacterial colonisation of surfaces (Qi, Xu, Jiang et al., 2004; Gazit, 2007). Antibacterial metal nanoparticles such as Ag (Rai, Yadav and Gade, 2009), Cu (Hsiao, Chen, Shieh et al., 2006), and Mo (Yasuyuki, Kunihiro, Kurissery et al., 2010) have also been used for controlling bacterial attachment, as they are toxic to the bacteria in small concentrations. Their effects on human health and the environment, however, are of concern. Stronger chemical-based methods for disinfection of surfaces are being required in order to control bacterial colonisation, which has resulted in bacterial species developing resistance to such reagents.
More recently, new approaches that use photocatalytic metal oxides such as TiO2 (Gelover, Gómez, Reyes et al., 2006) and ZnO (Franklin, Rogers, Apte et al., 2007; Jones, Ray, Ranjit et al., 2008) have been employed. These materials produce highly reactive chemical species such as hydroxyl radicals, hydrogen peroxide and superoxide, which are lethal to Escherichia coli and some other types of bacterial cells (Maness, Smolinski, Blake et al., 1999; Ibáñez, Litter and Pizarro, 2003). These metal oxides are mainly activated by UVA light, which have limited their potential application (Fu, Vary and Lin, 2005; In, Orlov, Berg et al., 2007).
Superhydrophobic surfaces based on natural surfaces such as plant leaves and insect cuticles are current attractive approaches under study for controlling the extent of bacterial colonisation. Traditionally, materials that could induce bacterial cell death were considered antibacterial materials (Zhang, Wang and Levänen, 2013). Recently, however, many superhydrophobic surfaces have been categorised as being antibacterial materials due to their potential application in controlling bacterial attachment. These natural surfaces have become responsive to external living conditions and are therefore constantly in contact with pollutants as well as harsh living environments. Observing these unique surfaces allows us to understand their antimicrobial action and therefore design surfaces that exhibit resistance to bacterial colonisation.
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2.7.2. Bactericidal activity of insect wings
Ivanova et al. recently found that the robust hexagonal arrays of 'nanopillars' present on the surface of the Psaltoda claripennis cicada wings exhibit bactericidal behaviour (Ivanova, Hasan, Webb et al., 2012). This array of nanopillars penetrated Pseudomonas aeruginosa cells in contact with the surface, killing them with great efficiency (Figure 2.10A, A1). The surface of the cicada wings retained its lethality against these Gram-negative pathogenic bacteria even after the surface was coated with a 10 nm-thick layer of gold, which indicated that the bactericidal properties of the surface were due to physical rather than chemical action. It was also reported that the wings consistently killed other Gram-negative bacteria, i.e., Branhamella cartarrhlis, E. coli, and Pseudomonas fluorescens, however Gram-positive cells (Bacillus subtilis, Planococcus maritimus, and Staphylococcus aureus) were resistant to the action of these nanopillars (Hasan, Webb, Truong et al., 2012a). These cicada wings were the first example of a surface that possessed bactericidal properties that relied on only a physical interaction with the bacteria.
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Figure 2.10. Bacterial cells were punctured by the surface of (A, A1) cicada wing Psaltoda claripennis, and (B-B3) dragonfly wing Diplacodes bipunctata. Cells were clearly punctured by nanopillars presented on the surfaces of these insect wings. (Reproduced with permissions from Encyclopedia of Life (eol.org, license agreements can be found at http://creativecommons.org/licenses/by-nc/2.0/), photographs taken by Graham Wise).
To explain this phenomenon, biophysical models were constructed to describe the interaction between bacterial cells and the nanopatterned pillars present on the cicada wing surfaces (Pogodin, Hasan, Baulin et al., 2013a). Modelling calculations revealed that the nanopillars did not actually pierce the cell wall but rather the cell wall was stretched to the point of breakage in the regions between the nanopillars as the cells adsorbed onto the wing surface. It was also found that the more rigid the cell membrane, the more difficult it was for the cell wall to rupture, which was consistent with the persistence obtained with Gram-positive bacteria that have a more rigid peptidoglycan layer. This theory was supported experimentally by decreasing the rigidity of Gram- positive cells through the application of microwave radiation. B. subtilis, S. aureus and Planococcus maritimus bacteria were tested as representive bacterial species. After exposure to microwave radiation, all three bacterial species showed a higher level of
44 susceptibility to the action of the cicada wing surfaces (Pogodin, Hasan, Baulin et al., 2013a).
In contrast to cicada wings, dragonfly wings rupture a large range of bacterial species, including Gram-negative (Pseudomonas aeruginosa), Gram-positive (Staphylococcus aureus and Bacillus subtilis) and even endospores. The dragonfly wing surfaces, like the cicada wing surfaces, were covered by a layer of nanopillar-like structures that showed activity against all bacterial cells coming into contact with the surface, as demonstrated in Figure 2.10, B1-3. A synthetic material (black silicon), which mimicked the surface structure of the dragonfly wings also showed high effectiveness at killing the same bacterial cells (Ivanova, Hasan, Webb et al., 2013). This project will focus on understanding the mechanisms by which the superhydrophobic and self-cleaning insect surfaces can exhibit mechano-bactericidal properties with a view to designing artificial surfaces that have the ability to control bacterial attachment for potential use in biomedical device and other industrial applications.
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Chapter 3. Materials and methods
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3.1. Overview
This thesis focusses experimentally on the chemical and structural characterisations as well as the biological activity and physical surface properties of dragonfly (mainly Hemianax papuensis and Hemicordulia tau) wings. Additionally, experiments that attempt to model how these surface structures are generated are used to explore the mechanisms involved in their biological activities.
3.2. Materials
Chloroform (>99.8%, ethanol stabilised), n-eicosane (CH3(CH2)18CH3, anhydrous,
≥99%) and docosane (CH3(CH2)20CH3, 99%) were purchased from Sigma Aldrich (Castle Hill, NSW, Australia). Highly ordered pyrolytic graphite (HOPG - 0.8 grade, Atomgraph-Crystal, RF) was obtained from AtomGraphCrystal (ATC) (Moscow, Russian Federation). A p-type boron-doped 100 mm diameter commercial Si wafer with specific resistivity of 10-20 Ω cm-1, (100) oriented surface and 525 ± 25 µm thickness was obtained from Atecom Ltd (Taipei, Taiwan). Single-sided Kapton® polyimide tape, purchased from Proscitech (Thuringowa Central, QLD Australia), was used to cleave the HOPG surfaces, as it has been found to be suitable for experiments conducted over a wide range of temperatures.
3.3. Protocols for handing of insect wings
3.3.1. Fresh and aged insect wings
The Hemicordulia tau and Hemianax papuensis dragonfly wing samples, which commonly dwell in suburban regions of Melbourne, Australia, were provided by the Melbourne Museum. The Diplacodes bipunctata dragonfly and Ischnura heterosticta damselfly wing samples were obtained from the suburban regions of South Australia. These fresh samples were collected in early to mid-2011 and preserved in sterile Petri dishes at room temperature (ca. 22 °C). Aged Diplacodes melanopsis dragonfly and aged Xanthagrion erythroneurum damselfly wing samples that had been preserved at the Melbourne Museum in the 1970s were also used in this study. When required, the wings were aseptically removed from the body and stored at room temperature in sterile plastic containers.
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3.3.2. Extraction of the lipids from dragonfly wings
An approximately 0.5 cm2 section of the dragonfly wings was used for stepwise extraction with chloroform. The epicuticular layer of the H. tau wing was extracted with chloroform for 10 s, 20 s, and 30 s periods, respectively, to investigate the impact of surface topography and surface chemistry on the wettability of the wing surface. The surface of H. papuensis wings was extracted for 10 s and 1 hr in chloroform to allow the chemical components that comprise the nanoscale structures of the wing to be identified. Ten second extractions were used in order to limit the extraction depth to just below the outer epicuticular surface, whereas the extractions performed over one hour ensured that the entire lipid fraction of the epicuticle was removed from the wing surface. The lipid-chloroform mixture was then filtered through glass wool to remove any contaminating particles that may have been present. The chloroform extracted wing samples were retained for further surface characterisation. A 20 µg volume of n- tetracosane was added to each of the chloroform fractions as an internal standard before performing GC-MS analysis.
3.4. Surface fabrication
Freshly cleaved highly ordered pyrolytic graphite (HOPG - 0.8 grade) surfaces were obtained by stripping their outermost layers with an adhesive tape. The lipids, eicosane (C20H42), docosane (C22H46), hexadecanoic acid (palmitic acid - C16H32O2), and octadecanoic acid (stearic acid - C18H36O2) were then individually dissolved in chloroform at a concentration of 3 mg mL-1. Approximately 10 µL of the alkane solution was then applied on the surfaces of the silicon wafer and freshly-cleaved HOPG, the lipids were allowed to self-assemble at ambient temperature (ca. 22°C).
3.5. Surface characterisation
3.5.1. Atomic force microscopy
Atomic force microscope (Innova, Veeco, USA) in tapping mode was employed to examine the surface topographic profiles. Phosphorus-doped silicon probes (MPP- 31120-10, Veeco) with a spring constant of 0.9 N m-1, radius of curvature (tip) of 8 nm and a resonance frequency of approximately 20 kHz were utilised for surface imaging. Scanning was carried out at a right angle to the axis of the cantilever at 1 Hz. The topographical data of the investigated surfaces were processed with first order horizontal and vertical levelling. In order to analyse the surface topography, different 48 surface roughness parameters were employed: the average roughness (Ra), root-mean- square (rms) roughness (Rq) and maximum peak height (Rmax) were performed using Gwyddion data processing software (Nečas and Klapetek, 2012). Two additional parameters, skewness (Rsk) and kurtosis (Rku), which are useful for describing surface morphology, have also been utilised. Skewness measures the symmetry of the height probability density function and kurtosis measures the ‘peakedness’ of the profile (Gadelmawla, Koura, Maksoud et al., 2002; Lamolle, Monjo, Lyngstadaas et al., 2009; Truong, Rundell, Lapovok et al., 2009; Ivanova, Truong, Wang et al., 2010).
Bearing statistics, including peak height (Rpk), valley depth (Rvk), core roughness
(Rk) and Rm were also analysed, as described elsewhere (Crawford, Webb, Truong et al., 2012). These were able to distinguish the differences between surfaces with similar average roughness. All images were produced using an Innova atomic force microscope (Veeco) and the raw data was then extracted to the Gwyddion software for processing (Nečas and Klapetek, 2012).
3.5.2. Scanning electron microscopy
3.5.2.1. Biological samples
The surface morphologies of the both lipid-extracted and untreated wings were observed using a field emission scanning electron microscope (FeSEM - SUPRA 40VP, Carl Zeiss GmbH, Jena, Germany) at fixed voltage of 3 kV. The wing sample was attached to a metallic substratum using conductive double-sided adhesive tape. Samples were sputter coated with gold using a JEOL NeoCoater (model MP-19020NCTR) prior to imaging, using a method described elsewhere (Ivanova, Hasan, Webb et al., 2012). To observe the structure of cross-sections of the wing membranes, the wing was snap- frozen in liquid nitrogen and then broken perpendicular to the main veins of the wing using tweezers. Broken wings were attached to metallic discs at one end, to enable the exposed end to face away from the substratum, enabling optimum imaging. Samples were then sputter coated with gold in an identical manner as above.
3.5.2.2. Synthetic surfaces
Samples for analysis were first coated with a thin layer of gold film (approximately 3 -5 nm thickness) using a Dynavac CS300. The high resolution scanning electron microscopy images were obtained using field-emission SEM (FESEM, SUPRA 40VP) at 3kV under 15,000× and 35,000× magnification. A two-dimensional fast Fourier- 49 transform was applied to the micrographs to identify the dominant patterns of alkane self-assembly.
3.5.2.3. Quantification of alignment using fast Fourier transforms (FFT) analysis
The quantitative topographical analysis of the SEM images was carried out using Gwyddion software (Open source, www.gwyddion.net).(Nečas and Klapetek, 2012) A two-dimensional fast Fourier-transform was applied to the micrographs to identify the dominant patterns of alkane self-assembly. Rectangular regions on top view SEM images with resolution of 744 × 502 pixels were processed for 2D FFT. The flat-top window function was chosen to obtain an insight into the spatial arrangements and direction of the self-assembled alkanes onto the surface of HOPG.
3.5.3. Raman spectroscopy
The self-assembled alkanes present on the surface of the graphite were immersed in 5 mL of MilliQ water for 1 hour. The air retained on the surfaces after this time was observed using an Alpha 300R Raman micro-spectrometer (WiTEC) with a 532.1 nm wavelength laser (hν = 2.33 eV). A water-immersion 63× objective lens (numerical aperture = 0.9, Zeiss) was used. A grid of 50 spectra × 50 spectra was acquired over a scanning area of 25 µm × 25 µm. The integration time for each spectrum was 0.5 seconds. Independent scanning was repeated twice on each of the two different samples (containing self-assembled eicosane and docosane). The signal was collected at an angle of 90° relative to the sample plane. The intensity of the spectral peak at 3100 – 3600 cm-1, which corresponds to the O-H bonds of water molecules, was used for qualitatively assessing the concentration of air bubbles (i.e. lack of water) present on the surface (Truong, Webb, Fadeeva et al., 2012; Webb, Boshkovikj, Fluke et al., 2013).
3.5.4. Wettability
The contact angles of Milli-Q water on both biological and synthetic surfaces was measured using the sessile drop method (Crawford, Koopal and Ralston, 1987; Van Oss, Good and Chaudhury, 1988; Guy, Crawford and Mainwaring, 1996; Öner and McCarthy, 2000). The contact angle measurements were carried out in air using an FTA1000 (First Ten Ångstroms, Inc., Portsmouth, VA, USA) instrument. An average of at least ten measurements was obtained for each sample, in duplicate. The evaluation of contact angles was performed by recording 50 images in 2 seconds with a Pelcomodel 50
PCHM 575–4 camera using FTA Windows Mode 4 software. As for the insect wings, to eliminate effect of the vein structure on the surface, the water droplet was placed on regions sufficiently large to accommodate the droplet footprint. The measurement was taken at ambient conditions of 21ºC and relative humidity of 60-70%.
3.6. Chemistry characterisation techniques
3.6.1. X-ray photoelectron spectroscopy
A VG ESCALAB 220i-XL X-ray Photoelectron Spectrometer equipped with a hemispherical analyser was used for XPS data acquisition. Samples underwent alternating data acquisition and ion beam etching cycles. Monochromatic Al Kα X-rays (1486.6 eV) at 220 W (22 mA and 10 kV) were used as incident radiation for data acquisition. Survey scans were carried out at pass energies of 100 eV over a binding energy range of 1200 eV. Base pressure in the analysis chamber was below 7.0 × 10-9 mbar and during sample depth profile analysis rose to 1.5 × 10-7 mbar. Ion beam etching was performed using a 4 keV Argon beam over a 2 mm × 2 mm area for intervals of 120 seconds. Data acquisition and etching was performed for 5 cycles, with a total etching time of 3000 seconds. XPS data were fitted using mixed Gaussian Lorentzian peak shape and linear background subtraction with CasaXPS.
3.6.2. Synchrotron radiation Fourier Transform Infrared spectroscopy (SR- FTIR)
3.6.2.1. FTIR in tranmission mode
The distribution of organic functional groups present across the wing membrane before and after chloroform extraction was analysed using SR-FTIR, on the Infrared microscopy beamline at the Australian Synchrotron. The samples were scanned in transmission mode over several areas of approximately 50 µm × 50 µm using a Bruker Hyperion 2000 FTIR microscope (Bruker Optic GmbH, Ettlingen, Germany), equipped with a narrow-band, high-sensitivity mercury cadmium telluride detector. A long-pass filter selected the detection range from 4950 cm-1 to 750 cm-1 and the microscope was fitted with a sample chamber purged with dry air to maintain low constant humidity (approximately 20%). OPUS version 6.5 software was employed for operating the microscope and spectrometer as well as for analysing the data.
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3.6.2.2. FTIR in attenuated total reflection (ATR) mode
The infrared beam passes through the ATR crystal and reflects back, producing an evanescent wave which penetrates no more than a few micrometres into the upper surface of the sample (in this case ~1 µm). This method provides information on the chemical functionality of the surface in contact with the crystal (Figure 3.1).
Figure 3.1. Schematic diagram of Synchrotron the FTIR in ATR mode.
The ATR measurements were made with a hemispherical ATR element (germanium and zinc selenide (ZnSe)) with the flat side of the hemisphere pressed into contact with the sample (Beattie, Beaussart, Mierczynska-Vasilev et al., 2012). Similar with FTIR in transmission mode, the data was analysed using OPUS version 6.5 software.
3.6.3. Gas chromatography – mass spectrometry
Prior to GC-MS analysis, any hydroxyl-containing components of the wing extracts (e.g. alcohols and carboxylic acids) were converted to trimethylsilyl (TMSi) derivatives by means of reaction with N,N-bis-trimethylsilyl-triflouro-acetamide (BSTFA,
C8H21NOSi2) in the presence of pyridine for 45 minutes at 70°C according to previously 52 optimised laboratory protocols (Gołebiowski, Bogus, Paszkiewicz et al., 2010; Van Dooremalen and Ellers, 2010; Gołebiowski, Bogus, Paszkiewicz et al., 2011; Murphy and Axelsen, 2011; Yocum, Buckner and Fatland, 2011; Küçükbay, Kuyumcu, Bilenler et al., 2012). The composition of the sample extract was determined by injecting 1 µL of the solution onto a capillary GC column (Rxi-5SIL MS column fused silica; column 30.0 m, 0.25 mm i.d., df = 0.25 µm, Restek Corporation, Bellefonte PA, U.S.A.), using He as the carrier gas at constant flow of 1.4 mL min-1 and a mass spectrometric detector (GCMS-QP2010, Shimadzu Scientific Instruments, Columbia, U.S.A). The chromatograph was programmed as follows: on-column injection at 50°C, oven at 50°C for 5 minutes, then increasing to 280°C at the rate 3°C min-1, and then held for 30 minutes at 280°C. Mass spectra were recorded from m/z 14 to 800. The percentage composition reported for each component is based on the peak area from the Total Ion Current (TIC) chromatogram without standardisation. A standard mixture of n-alkanes
(C8-C40) in chloroform was used as an external standard to verify the retention times (Van Den Dool and Kratz, 1963). The retention indices (RI) for each component were calculated based on:
RTxn RT 0 RI100 n0 Equation 3.1. RTnn10 RT where n0 is the number of carbons for an n-alkane standard and n1 corresponds to the next n-alkane in the series. RTx, RTn0 and RTn1 are the retention times for the component being measured relative to the two closest n-alkane standards. The RI was compared with retention index databases (Sadtler Research Laboratories, 1984; Pacakova and Feltl, 1992) to identity the components not contained in the Wiley 7th Edition mass spectral database.
3.7. Bacterial strains, growth, and sample preparation
Pseudomonas aeruginosa ATCC 23246, Staphylococcus aureus CIP65.8T, Bacillus subtilis NCIMB 3610T and B. subtilis spores were used in this study. The bacterial strains were obtained from NCIMB (Aberdeen, UK), The Collection of Institut Pasteur (Paris, France), and American Type Culture Collection (Manassas, VA, USA). Prior to individual experiments, bacterial cultures were refreshed from stock onto nutrient agar (BD, Franklin Lakes, NJ, USA). To prepare spore, B. subtilis culture was left in the incubator at 37 °C for a week prior to experimentation to depress the oxygen levels,
53 hence stimulating the formation of spores. For individual experiments, bacterial cell suspensions of each strain were prepared in 5 mL nutrient broth (BD, Franklin Lakes, NJ, USA) from fresh culture grown overnight in an incubator at 37 °C. Bacterial cells were collected at the logarithmic stage of growth and the suspensions were adjusted to
OD600 = 0.3 for vegetative cells or OD600 = 1.0 for spores. The mounted insect wings were then immersed in approximately 5 mL bacterial suspension and incubated for 18 hours at room temperature (ca. 22 °C). The control samples consisted of the glass cover slip immersed in the same bacterial suspension.
3.8. Biological activity assays
3.8.1. Plate counting assay
Viability assays were performed by standard plate counts (Postgate, 1969) . P. aeruginosa, S. aureus and B. subtilis cells and spores were suspended in 5 mL of phosphate buffered saline (PBS) and adjusted to OD600 = 0.1. Re-suspended cells were diluted 1:10 and then incubated in 3.5 cm diameter wells, in triplicate, with each well containing a 1 cm2 area substrate sample of a D. bipunctata and H. papuensis wing, black silicon, smooth silicon wafer or a glass cover slip. The cell suspensions were then sampled (100 µL) at discrete time intervals (3 hours and 18 hours), serially diluted 1:10 and each dilution spread on three nutrient agar plates. Resulting colonies were then counted and the number of colony forming units per mL was calculated. The number of colony forming units was assumed to be equivalent to the number of live cells in suspension (Postgate, 1969). The maximum bactericidal efficiency was measured as the number of inactivated cells per square centimetre of sample per minute of incubation time, relative to the control surfaces.
3.8.2. Quantitation of bactericidal activity using confocal images for fabricated surfaces
Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus CIP 65.8T were the bacterial strains used in this study. Three independent experiments were performed for each bacterium on each surface. In each, bacterial cell suspensions were prepared in 5 mL nutrient broth (BD, Franklin Lakes, NJ, USA) from fresh culture, grown overnight at 37°C. Bacterial cells were collected at the logarithmic stage of growth, and the suspensions were adjusted to OD600 = 0.1. The mounted self-assembled surfaces were then immersed in 5 mL of bacterial suspension and incubated for 2 hours at room 54 temperature (ca. 22°C). Control samples consisting of untreated, freshly cleaved HOPG were also immersed in the same bacterial suspensions.
3.9. Confocal laser scanning microscopy (CLSM)
Confocal laser scanning microscopy was used to visualise the proportions of live cells and dead cells using a LIVE/DEAD® BacLight™ Bacterial Viability Kit, L7012. SYTO® 9 permeated both intact and damaged membranes of the cells, binding to nucleic acids and fluorescing green when excited by a 485 nm wavelength laser. Propidium iodide only entered the cells that had sustained significant membrane damage (and therefore considered to be non-viable) and bound with higher affinity to the intracellular nucleic acids than the SYTO® 9. Bacterial suspensions were stained according to the manufacturer’s protocol and imaged using a Fluoview FV10i inverted microscope (Olympus, Tokyo, Japan) (Ivanova, Hasan, Webb et al., 2013).
3.10. Statistical analysis
The results obtained were expressed in terms of their mean values and the corresponding standard deviations following commonly used protocols. Statistical data processing was performed using paired Student’s two-tailed t-tests to evaluate the consistency of results (asterisked if p < 0.05) in figures and tables. To obtain the biological activities of bacterial cells onto fabricated surfaces, imaging processing software (the Gwyddion), was used to estimate the number of cells attached at each microscopic view. At least 17 individual scan areas were used for statistical analysis.
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Chapter 4. Surface characterisation and functionality of the epicuticle of insect wings
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4.1. Overview
A number of natural surfaces possess superhydrophobic and self-cleaning properties that would be extremely beneficial in industrial applications. The surface of dragonfly wings is one of such examples. Here, the surface characteristics of the wings of various species of dragonflies, including more recently collected samples in 2011 and aged samples collected in the 1970s, were investigated in order to gain an insight into their surface structures, with a view to their potential applications of these surfaces. Surface characterisation methods, including water contact angle (WCA), scanning electron microscopy (SEM) and atomic force microscopy (AFM) which detail the wettability and morphology of these dragonfly wings. The chemical functionality of the surface of the wings was determined using the Fourier Transform Infrared beam line at the Australian Synchrotron and gas chromatography mass spectrometry (GCMS). It was determined that the surfaces of the wings are largely comprised of aliphatic hydrocarbons, which contribute to not only surface hydrophobicity of the surface, but also to the formation of the wing surface nano-structure.
4.2. Surface topology and wettability of dragonfly wings
4.2.1. Scanning electron microscopy
High-resolution scanning electron micrographs of a cross-section of the H. papuensis and H. tau dragonfly wings shown the presence of three distinct layers; these are the epicuticular layer on both the dorsal and ventral surface, and the intracuticular layer (procuticle) (Figure 4.1A). The epicuticular layers were estimated to be 180 - 400 nm in thickness, whilst the intracuticle was found to be approximately 1.5 µm thick, giving a total thickness of 2.0 – 2.5 µm for the wing membrane. A top view of the wing shown that the surface of consisted of multiple nanoscale pillars that have round tops with a diameter of approximately 80 ± 20 nm, and spaced approximately 180 ± 30 nm apart. These observations were consistent with those reported previously by Watson et al. (Watson, Watson, Hu et al., 2010) for the Rhyothemis phyyllischloe dragonfly species.
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Figure 4.1. High resolution scanning electron micrograph of the H. papuensis and H. tau dragonfly wings. (A) The side view of the H. papuensis wing and (B) its corresponding top view; (C) and (D) are side view and top view of the H. tau wing.
4.2.2. Atomic force microscopy
AFM was employed to measure the surface topography as shown in Figure 4.2. This method provided a comparison of the surface nano-structures of the different species of insect wings.
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Figure 4.2. Surface topography of (A) Hemianax papuensis and (B) Hemicordulia tau dragonfly wings with their corresponding line profiles. Inset scale bar = 500 nm.
Typical two-dimensional images and cross-sectional profiles of 2.5 µm x 2.5 µm AFM scan areas of each of the wing surfaces are presented in Figure 4.2. Each cross- sectional profile was generated by selecting one line from the corresponding two- dimensional AFM scan and plotting peak height as the function of translation along the line. The mean planes of corresponding scans were defined as 0 in cross-sectional profiles. The nanopillars appeared to be more regular on the surface of H. papuensis compared to the of H. tau dragonfly wings. The line profile of H. papuensis showed that there are more splits within the same peaks compared to the line profile of the H. tau wing. The maximum height of the H. papuensis wing was approximately 130 - 250 nm whilst that of the H. tau wing was 150 - 300 nm in height. AFM is able to provide accurate dimensions of the nanopillars on the surfaces of the wings, where the data also highlights the consistency between dragonfly species, in which the general structures of the wing surface appear to be very similar.
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4.2.3. Surface wettability
Dragonfly wings have been gaining significant research attention recently due to their superhydrophobic and self-cleaning properties. Whereas Ivanova et al. reported for the first time a mechanical-bactericidal surface that arises from the sophisticated structure of insect wings (Ivanova, Hasan, Webb et al., 2013). This new finding prompted further research here to understand the underlying mechanism(s) responsible for this mechanical-bactericidal phenomenon. As a starting point, the wettability of these surfaces was investigated. To eliminate the effect of the vein structure on the surface, a water droplet was placed onto the regions of the surface that were sufficiently large that the droplet footprint could be accommodated. Contact angles (CA) were measured using the sessile drop method. The image of the droplet was captured immediately after it came to rest on the surface and the contact angle (CA) determined. When a drop is allowed to fall onto the H. papuensis dragonfly wing surface, it bounces as a result of being repelled by the self-cleaning nature of the wing surface (Figure 4.3).
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Figure 4.3. A water droplet coming into contact with the H. papuensis dragonfly wing surface. The surface is sufficiently hydrophobic for the droplet to bounce after it makes initial impact.
Hemianax papuensis and Hemicordulia tau dragonflies are common inhabitants of Australia. The wings of both dragonflies exhibited WCA greater than 150° (162°, and 151° respectively), which highlight their superhydrophobic properties. Water droplets are able to roll easily across the surface when it is tilted on an angle of less than 10° (6.3° in the case of H. papuensis and 2.3° for H. tau), which also supports their self- cleaning properties. It well established that superhydrophobic surfaces tend to adhere to the Cassie-Baxter regime, where a droplet sits on top of the textured surface with air
61 being trapped between the texture features. In order to maintain a stable superhydrophobic state, hierarchical structure is important (Bhushan, 2007; Berne, Weeks and Zhou, 2009). Therefore, further investigation into the surface characteristics of these dragonflies’ wings is suggested.
4.3. Correlation between surface chemistry and the wettability of a surface
4.3.1. Variations in the surface wettability of dragonfly wings
Three different dragonfly wings, two of which were collected in 2011 (Hemianax papuensis and Hemicordulia tau), and one collected in the 1970s (Diplacodes melanopsis) were analysed to compare the surface wettability between relatively fresh and aged samples. The WCA was found to be 162°, 151°, and 138° for H. papuensis, H. tau and D. melanopsis dragonfly wings, respectively. Whilst the two wings collected in 2011 both exhibited superhydrophobic properties (i.e. having a contact angle above 150°), the wing collected in the 1970s fell just short of this threshold as illustrated in Figure 4.4.
The fact that the aged dragonfly wing appeared to be less hydrophobic than the more recently collected wings in term of their wettability prompted two possible questions. The first was whether ageing of the wing might have affected the chemistry of the wing surface; the second was whether the differences that were detected were simply a function of the natural diversity between the species. In order to obtain an insight into these questions, various analytical methods, including Synchrotron FTIR, SEM and AFM were employed to compare and contrast their surface properties. FTIR analysis provides information regarding the chemistry across the wing membrane, which may reflect on any changes that may have taken place on the wing surface over time. In addition, SEM and AFM would provide an insight into the differences in surface architecture that could be correlated with data obtained using FTIR spectroscopy.
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Figure 4.4. Average static water contact angle (grey) and average contact angle hysteresis (black), with example water droplet contact angles (above). The shaded area at the top of the graph represents the minimum water contact angle threshold required for self-cleaning ability. Error bars indicates standard deviation, n=15.
The SEM images of the three dragonfly wings show that all of the surfaces are covered by a layer of nanopillars (Figure 4.5). There are, however, some minor differences in the individual structures. The pillars on the surface of the D. melanopsis wings appeared to form larger clusters, whilst the more recently collected wings appeared to possess more distinct pillars with round tips (Figure 4.5). Based on these micrographs, there is no visible evidence to suggest that long-term storage had any impact on the surface structure of the wings.
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Figure 4.5. High resolution scanning micrographs of (A) H. papuensis, (B) H. tau, (C) D. melanopsis dragonfly wings and their corresponding 3D image.
4.3.2. Relationship between the quantity of surface epicuticular lipids and wettability
Synchrotron FTIR in the transmission mode was used to determine the chemical composition of the wing membrane. Spectra representing three different species of insect wings including recently collected (H. papuensis, and H. tau) and aged (D. melanopsis) wings were found to contained three major bands at 3480-3230 cm-1, 3000- 2800 cm-1 and 1750-1480 cm-1 These bands corresponded to hydroxyl, alkyl hydrocarbons and amide groups (Figure 4.6, Table 4.1).
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Figure 4.6. Infrared absorbance spectra of the wings of three species of dragonfly. Most bands in the spectra were very similar for all three species, however the increased intensity of the CH2 stretching bands in the spectrum of the H. papuensis dragonfly provides evidence that there is a greater amount of aliphatic hydrocarbons present on the wing surface.
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Table 4.1. Assignment of chemical components to the major absorption bands present in the IR spectra of the wing membranes
Band position (cm-1) Functional groups Characteristic Out-of-phase combination of the N-H in plane bend and the C-N stretching 1480-1580 Amide II vibration Carbonyl stretching band due to ketone, carboxylic acid (Barth, 2007) C=O stretch vibration causes CCN Amide I 1570-1700 deformation and the N-H in-plane bend Carbonyl groups (Jackson and Mantsch, 1995) Carbonyl stretching band (Lasch, Boese, 1700-1750 Ester Pacifico et al., 2002) CH , CH symmetric and asymmetric 2800-3000 Alkanes or alkyl groups 2 3 stretch Unsaturated and aromatic C-H stretch (C is sp2) (Coury and Dillner, 3000-3100 C-H 2008)
O–H and N-H stretching, which can be due O-H stretch from free to carbohydrates, carboxylic acids, amines 3200-3500 alcohol or phenol or alcohols/phenols (Caruso, Furlong, Ariga et al., 1998)
The spectra of all three wings were dominated by Amide I and Amide II absorption bands due to C=O bond stretching coupled to N-H bending (1610 - 1695 cm-1) and C-N stretching coupled to N-H bending (1480 - 1575 cm-1), respectively (Caruso, Furlong, Ariga et al., 1998; Brugnerotto, Lizardi, Goycoolea et al., 2001; Ganim, Hoi, Smith et al., 2008; Sajomsang and Gonil, 2010). The presence of amide groups can be attributed to chitin and proteins, as they represent the major structural components that make up the bulk of insect cuticle (Neville, Parry and Woodhead-Galloway, 1976; Hackman and Goldberg, 1987; Gorb, 1997; Kramer and Muthukrishnan, 1997; Kreuz, Arnold and Kesel, 2001). The broad, overlapping O-H and N-H stretching bands within 3500 - 3200 cm-1 are likely also to result from absorption by the chitin and protein components of the wings (Coury and Dillner, 2008). A small ester carbonyl stretching band was present at approximately 1735 cm-1 in all of the recorded spectra. This may be due to the
66 presences of carbonyl compounds, such as carboxylic acids and esters, on the wing membrane.
The C-H stretching region (2840 - 3000 cm-1) contains three bands representing the symmetric (s) and anti-symmetric (as) stretching vibrations of the CH2 and CH3 functional groups (Figure 4.6). The presence of C-H stretching bands with a prevalence of methylene bands is indicative of long-chain aliphatic hydrocarbons (Merk, Blume and Riederer, 1998; Zeier and Schreiber, 1999). Standardisation of the spectra using the Amide I peak was used to compare the proportion of aliphatic hydrocarbons relative to the thickness of the wing. The results revealed that their intensity reasonably aligned with their WCA. Both H. papuensis and H. tau exhibited high WCA, which also appeared to contain the higher proportion of aliphatic hydrocarbons. The opposite was the case for D. melanopsis, which exhibited the lowest WCA and hence, appeared with lower aliphatic hydrocarbon content. The data presented here suggested, not unexpectedly, that there was a correlation between the WCA and amount of aliphatic hydrocarbons; however this information does not provide an insight into whether the differences in both wettability and surface chemistry have arisen as a result of long periods of storage.
To investigate whether preservation had an impact on the amount of aliphatic hydrocarbon present on the wing surfaces, the wings of two species of damselfly, belonging to the Odonata order, were analysed. The species included samples collected in 2011 (Ischnura heterosticta) and aged samples collected in the 1970s (Xanthagrion erythroneurum). The infrared spectra and corresponding WCA analyses are presented in Figure 4.7.
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Figure 4.7. Infrared absorbance spectra of two species of damselfly wings, Xanthagrion erythroneurum and Ischnura heterosticta, and their corresponding WCA.
The intensity of the CH2 stretching bands provides an indication of the amount of aliphatic hydrocarbons present on the wing surface.
The two damselfly wings including the aged sample X. erythroneurum, both exhibited a WCA greater than 150°, which indicated that long time storage did not affect the wettability of the surface. Also, the intensity of the C-H stretching regions in the IR spectra supports the suggestion that the quantity of aliphatic hydrocarbons is correlated with the extent of their wettability. These results also suggest that differences in the wettability between Odonata wings arose as a result of variations within species, which was also related to the amount hydrocarbons on the surface.
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4.4. Dual roles of epicuticular lipids in determining the wettability of dragonfly wings
4.4.1. Wettability of dragonfly wings as a function of aliphatic hydrocarbon content
In previous experiments it was shown that there was a correlation between the surface wettability and the quantity of aliphatic hydrocarbons presented on the outermost layer of dragonfly wing. It was well-known that surface composition plays an important role in controlling surface wettability according to the Cassie-Baxter theory of wetting. Therefore, it is important to identify the precise composition present on the surface of the dragonfly wings. In order to do so, the epicuticular layer of dragonfly wings was extracted using chloroform, and then the mixtures were analysed using GCMS to identify their chemical components. The wettability of the dragonfly before and after the extraction process was measured to determine the extent to which the presence of aliphatic carbons affected the surface wettability.
Removal of the hydrophobic materials in the form of lipids (waxes) from the H. papuensis and H. tau dragonfly wing surfaces was predicted to lead to an increase in wettability. An area of approximately 0.5 mm × 0.5 mm of each wing was extracted with chloroform for 10, 20, and 30 s in order to investigate how the extraction process would affect the properties of the wings. The WCAs measured on the chloroform extracted wings decreased as a function of increasing chloroform extraction time. This decrease was accompanied by an increase in the contact angle hysteresis (CAH) (Figure 4.8). Within 10 s of extraction, the WCA of the wing fell below the threshold for superhydrophobic materials (shaded area, Figure 4.8), with further decreases in WCA and CAH occurring for the samples that were subjected to longer extraction times.
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Figure 4.8. Loss of the superhydrophobic and self-cleaning properties of dragonfly wings after extraction with chloroform. The average water contact angles are presented in grey and average contact angle hysteresis presented in black. The shaded area at the top of the graph represents the minimum water contact angle threshold required for classification as superhydrophobic surfaces. Error bars indicates standard deviations, n=20.
The chloroform extracted wings were found to remain relatively hydrophobic, which suggests that the newly exposed outer layer was composed of a material with low surface energy.
4.4.2. The chemical composition of untreated and chloroform extracted wings
Synchrotron FTIR spectroscopy was used in transmission mode to determine the chemical composition of the wing membranes, before and after extraction of the wing components using chloroform. The spectra in Figure 4.9 represent the C-H stretching region of the untreated and chloroform-extracted wings of H. tau dragonfly wings (Figure 4.9). The C-H stretching IR absorption bands appeared with relatively high intensity in the spectra of the untreated wings; however their intensity considerably decreased as the extraction time was increased. This observation suggests that the chloroform extraction removed the hydrophobic wax-like components present on the epicuticular layer of the wing membrane.
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Figure 4.9. Infrared spectra of the H. tau dragonfly wings before and after chloroform extraction. The decrease in aliphatic hydrocarbons present on the surface of the dragonfly wings occurs with increasing chloroform extraction time. The intensity of the
CH2 stretching bands is a good approximation of the concentration of aliphatic hydrocarbons, i.e. lipids (waxes) present on the wing surface. The spectra were acquired at the Australian Synchrotron.
The identities of the lipid components that were extracted from the epicuticular layer of Hemicordulia tau dragonfly wings were determined using GC-MS (Table 4.2). Data analysis confirmed the presence of aliphatic hydrocarbons and fatty acids; the major components of all fractions were n-alkanes (Figure 4.10). They represented approximately 71 % of the total compounds present in the chloroform-extracted fraction (10 s extraction time) and the proportion of n-alkanes in the total extraction remained constant for both the 20 and 30 s extraction times (71 % and 69 %, respectively). The extracted alkanes ranged in length from C10 to C34, and were dominated by alkanes with an even number of carbon atoms, with smaller quantities of C19, C23, C25, and C29 (Table
4.2). Among the n-alkanes, n-hexacosane (C26) alone accounted for 26.8 % of the total detected compounds in the 10 s extract, and this increased slightly to approximately 29 % in the 20 s extract, and dropped to 18 % in the 30 s extract. Methylalkanes were also prevalent amongst the aliphatic hydrocarbons extracted from the epicuticular layer of the wing membrane. They comprised up to 19 % of the total compounds present in the
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10 s extract, however, their proportion decreased as the time of extraction increased. The only oxygen-containing compound detected in the 10 s extract was palmitic acid (hexadecanoic acid), which made up 7 % of the extracted epicuticular lipids. The proportion of palmitic acid in the wing extracts increased to 18 % and 14 % after 20 se and 30 s of extraction, respectively.
Figure 4.10. Relative proportion of all identified chemical classes present in the epicuticular layer of the H. tau dragonfly wing at three extraction times. The epicuticle was dominated by n-alkanes.
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Table 4.2. Wax components of the Hemicordulia tau wing epicuticle isolated and identified by GCMS. Components that could not be unambiguously identified are grouped together.
Proportion (%) Formula Nomenclature 10 s 20 s 30 s
C10H22 n-decane 1.2
C11H24 4-methyldecane 0.1
C14H30 n-tetradecane 4.7 5.1 2.9
C16H34 3-methylpentadecane 0.3 1.0 0.6
C16H34 n-hexadecane 7.0 6.4 5.2
C18H38 3-methylheptadecane 0.9 2.5 1.9
C18H38 n-octadecane 7.7 7.8 7.7 3,15- C H 0.1 19 40 dimethylheptadecane
C19H40 n-nonadecane 2.3
C20H42 7-methylnonadecane 0.1
C20H42 8-methylnonadecane 0.1
C20H42 3-methylnonadecane 1.2 1.6 2.2
C20H42 n-eicosane 6.5 6.0 9.5
C16H32O2 Hexadecanoic acid 7.1 17.9 14.4
C21H44 n-heneicosane 2.8
C22H46 4-methylheneicosane 0.4
C22H46 3-methylheneicosane 0.7 1.6 1.9
C23H48 7,11-dimethylheneicosane 0.7 1.6 1.9
C22H46 n-docosane 12.2 11.2 13.5
C23H48 n-tricosane 0.1 1.8
C24H50 11-methyltricosane 0.1
C24H51 7-methyltricosane 0.1
C24H50 5-methyltricosane 0.1 0.1
C24H50 3-methyltricosane 12.0 2.2 5.9
C25H52 n-pentacosane 1.6 2.0
C27H56 7,11-dimethylpentacosane 2.2 0.3 1.9
C26H54 n-hexacosane 26.8 28.9 18.4
C28H58 2,6-dimethylhexacosane 0.1 0.3 0.1
C26H54 3-methylpentacosane 4.3 0.3 0.2
C28H58 n-octacosane 3.4 3.3 1.7
C29H60 n-nonacosane 0.1
C30H62 n-triacontane 2.1 0.3 0.9
C34H70 n-tetratriacontane 0.3 0.7 0.1 %Composition based on measured TIC peak areas without standardisation.
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Aliphatic hydrocarbons represented approximately 93 % of the 10 s, 82 % of the 20 s, and 86 % of the 30 s extract. The change in proportions of aliphatic hydrocarbons allowed an insight into determining the location of certain compounds from different layers of the dragonfly wing epicuticle. For example, in the 10 s extraction, most compounds in the outermost layer of the wing surface were extracted and their proportions did not increase as the extraction time increased. This highlighted the importance that aliphatic hydrocarbons have in determining the wettability of dragonfly wings. Removal of a large portion of the n-alkanes from the surface in the 10 s extracted surface appeared to be the principal cause of the loss of the wing’s self-cleaning ability. Longer extraction times led to an increasing loss of lipid components, and this resulted in further decreases in the measured WCA. Even-numbered n-alkanes were present in high proportion in 10 s extract and this remained stable or slightly decreased in the longer extraction time samples. The dominance of even-numbered aliphatic hydrocarbons can be explained by the addition of acetyl groups to a fatty acid precursor, whereby chain extension occurs in multiples of C2, with the final length of the molecule being dependent on the fatty acid precursor (Howard and Blomquist, 2005; Samuels, Kunst and Jetter, 2008).
4.4.3. Changes in surface morphology on removal of surface aliphatic hydrocarbons
The surface morphology of the wing samples that were subjected to the 10 s chloroform extraction changed as a result of the extraction process, after which time slight changes to the pillar structures was observed. In these samples, the three distinct layers were still clearly visible, however the round tops on the pillar structures present on the untreated wings were no longer present (Figure 4.11C, D). The extraction process caused the pillars to sharpen, and become more sparse, suggesting that the outermost portions of the pillars were dissolved by chloroform extraction. As the extraction time was increased, the progressive loss of surface structure became more obvious. After 20 s (Figure 4.11E, F), the epicuticular layers of the wing appeared thinner in their cross-sectional view, and circular patterns became visible in the surface images. After 30 s of chloroform extraction, minimal structure remained, and this was scattered across the surface, whilst the internal structure remained unaffected (Figure 4.11G, H).
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Figure 4.11. Cross-section (A, C, E, G) and surface (B, D, F, H) morphology of Hemicordulia tau dragonfly wing surfaces before and after chloroform extraction over 10, 20 and 30 s. Decreased water contact angles were observed with increased extraction time (inset). Scale bars = 400 nm
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The changes in the surface morphologies observed in the electron micrographs were also reflected in the AFM images. Typical two-dimensional images and cross- sectional profiles of 5 µm × 5 µm scan areas of each of the wing surfaces and insets corresponding to 1 µm × 1 µm scan areas are presented in Figure 4.12. Each cross- sectional profile was generated by selecting one line from the corresponding two- dimensional AFM scan and plotting height as a function of translation along the line. The surface features in the profiles appeared to decrease in height and sharpness as the time of extraction increased. Untreated wing surface profiles contained features ranging from 50 - 150 nm in width. After 10 s of chloroform extraction, the features appeared rounder and shorter (50 - 100 nm). Notably, while scanning the surface of the chloroform-extracted wings, the AFM tip experienced decreased lateral deflection, i.e. the tip adhered to the wing surface to a lesser extent after lipid extraction, suggesting that less adhesive lipid material was present on the surface. As the time of extraction was increased to 20 and 30 s, the surface features decreased further to about 80 nm in height, appeared broader and were spaced further apart (Figure 4.12).
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Figure 4.12. Surface topography of Hemicordulia tau dragonfly wings after various periods of chloroform extraction, inferred from typical two-dimensional AFM scans (5 µm × 5 µm areas) and corresponding cross-sectional profiles. The inset images are AFM scans of 1 m × 1 m areas. Scale bar = 1 m. 77
A statistical roughness analysis was performed on the data measured on both the untreated and chloroform extracted wings. Six statistical parameters, Rmax, Ra, Rq, Rsk,
Rku and Rdr, were calculated for each surface (Table 4.3) (Crawford, Webb, Truong et al., 2012). Rmax is the maximum roughness, is the vertical distance between the highest and lowest points on a topographical profile or surface. Ra and Rq are the average roughness; Ra, is the average deviation of the height values from the mean line/plane, whilst Rq is the root-mean-square deviation from the mean line/plane, i.e. the standard deviation from the mean. Skewness (Rsk) and kurtosis (Rku) describe the shape of the height distribution. Positive skewness indicates the surface having high peaks with shallower and broader valleys, whilst the surfaces with negative skewness tend to have small peaks with deep, narrow valleys. Kurtosis is a measure of the ‘sharpness’ of the height distribution, or the sharpness of the peaks in a profile or surface. Rdr is a measure of the surface area relative to the projected area of the scan(Crawford, Webb, Truong et al., 2012).
Table 4.3. Roughness analysis of the untreated and chloroform extracted H. tau wing surfacesa.
Roughness Untreated Chloroform extraction time (s) b statistics sample 10 s 20 s 30 s
Rmax (nm) 814 ± 72 409 ± 40 285 ± 14 332 ± 14
Ra (nm) 47 ± 3 38 ± 11 25 ± 3 40 ± 8
Rq (nm) 61 ± 3 50 ± 11 32 ± 3 49 ± 10
Rdr (%) 278 ± 51 157 ± 54 121 ± 3 143 ± 10 aResults presented are averages of three measurements taken from 25 µm2 scanning areas and errors are the standard deviations. bA technical description of the roughness parameters used can be found in Crawford et al. (Crawford, Webb, Truong et al., 2012).
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It can be seen that Rdr decreased as the extraction time increased, and compared to the untreated sample. A similar trend was observed for Rmax, Ra, and Rq. The average roughness (Ra) of the untreated wing surface was found to be 47.0 nm, which gradually decreased to as low as 25.2 nm after 20 s of chloroform extraction. The roughness of the wings subjected to a 10 s chloroform extraction also appeared to decrease, however the changes were not significant. In contrast with the 10 s and the 20 s extracted samples, the roughness parameters of the 30 s extracted wing increased. Extended extraction time may have led to the removal of a greater amount of material from between the pillars than on their upper regions. This would enable the AFM tip to reach deeper into the sample, hence causing the height of the pillars to appear to increase in size. The loss of surface nanostructure with extended extraction time highlights the fact that the components of the wing that are rapidly soluble in chloroform, i.e. the epicuticular lipids, play a major role in forming the wing nanoarchitecture.
4.5. Summary
The dragonfly wing membranes consist of three distinct layers, including two epicuticles on the dorsal and ventral surfaces and an intracuticle located in between the epicuticles. Through Synchrotron FTIR spectroscopy, it was found that the epicuticular layers consisted mainly of aliphatic hydrocarbons, with protein and chitin making up the bulk of intracuticle. Combining the data obtained using two techniques, FTIR and WCA measurement, revealed a correlation between the quantity of lipids and wettability of the wing surface. Step-wise extractions of the wings using chloroform resulted in decreased surface hydrophobicity, with the WCA of the treated samples falling below the threshold that defines the condition of superhydrophobicity within 10 s of extraction. The loss of surface structure on the wing surface due to extraction with chloroform leads to two important conclusions. Firstly, it is not only the loss of hydrophobic material from the epicuticle that caused the wing to lose its superhydrophobic and self- cleaning properties; loss of structure was almost certainly also a contributing factor. Secondly, the removal of the wing nanostructures via chloroform extraction enables the conclusion to be drawn that the nanostructures present on the wing surfaces are composed of the materials that were identified in the chloroform extracts. It can be concluded that the nanoscale pillars present on the surface of Hemicordulia tau dragonfly wing membranes are composed primarily of aliphatic hydrocarbons, especially n-alkanes with even-numbered chain lengths ranging between C18 and C26, 79 and a relatively small proportion of palmitic acid. These components are vital in determining the superhydrophobic and self-cleaning properties of Hemicordulia tau wing membranes, as they not only present a hydrophobic lipid layer to the external environment, but are the primary building materials that form the nanostructures on the wing surface.
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Chapter 5. Molecular organisation of dragonfly wings ______
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5.1. Overview
As noted the chemical composition of dragonfly wings is a critical factor in determining their surface properties and the molecular organisation of the wing surfaces is an important parameter when it comes to constructing dragonfly wing-inspired materials. A combination of chromatographic and spectroscopic techniques was employed to analyse the wings. Lipids were extracted from the wing surfaces and analysed using gas chromatography-mass spectrometry (GCMS), whilst Synchrotron- sourced Fourier-transform infrared microspectroscopy (FTIR) and x-ray photoelectron spectroscopy (XPS) depth profiling were performed directly on the intact wings. The identities of over 60 chemical components that constitute the nanoscale structures on the surface of the wings of the dragonfly, Hemianax papuensis, were defined. The wing samples were immersed in chloroform for either 10 s or 1 h. Immersion of the wing in chloroform for just 10 s enabled the outermost components of the wing to be preferentially extracted, whilst the 1 h extractions enabled the analysis of the chemical composition of the entire epicuticular layer. To confirm that the intended regions of the epicuticle were successfully extracted, the pre- and post-extracted surfaces were analysed for changes in their surface topologies using SEM and AFM.
5.2. Physical effects on dragonfly wings resulting from extraction with chloroform
5.2.1. Changes in surface morphologies
Scanning electron micrographs confirmed that a chloroform extraction period of 10 s was sufficient to extract the outer epicuticle of the wing. It can be seen from cross- sections of the untreated and the 10 s extracted sample (Figure 5.1) that the wing surface structures remained largely unchanged after the 10 s extraction period. The cross- sectional views of the wing were obtained in order to visualise the wing surface as well as the internal structure of the dragonfly wing membrane. The extraction process did not appear to cause visible damage to the intracuticle, even after a 1 h extraction period. The nanostructure of the epicuticular layer, however, was completely eradicated after 1 h of extraction.
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Figure 5.1. Cross-sectional images of H. papuensis dragonfly wing membranes prior to, and post-extraction with chloroform, and corresponding images of the WCA. Extraction for 10 s had only a minor effect on the surface morphology, however the WCA decreased by more than 20°. After 1 h extraction, there was no trace of the surface nanostructures, indicating the complete removal of the epicuticular lipids. The WCA on the 1 h extracted surface was 93°, nearly 70° lower than the original, untreated wing. Scale bars = 400 nm.
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Surface view electron micrographs of the untreated and chloroform-extracted wings were also obtained, and are presented with their corresponding 3D images (Figure 5.2). The 3D images were generated from the micrographs using Gwyddion data processing software (Nečas and Klapetek, 2012). After a 10 s chloroform extraction period, the nanostructures on the wing surfaces were still visible, however the spatial distribution between individual pillars appeared to have increased. This observation was due to removal of material from the surface of these nanostructures during the extraction process, causing them to decrease in size. The wing surface appeared to be completely devoid of nanostructures after a 1 h chloroform extraction period. The results obtained here demonstrate that the 10 s extraction period was successful at removing just the outermost layer of lipids, whereas a 1 h extraction period enabled the entire epicuticle layer to be removed.
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Figure 5.2. Top view micrographs and 3D images of Hemianax papuensis dragonfly wings prior to, and after 10 s and 1 h of chloroform extraction. The general structure of the dragonfly wings surface largely remained intact after 10 s of chloroform extraction, however, the structure disappeared completely after a 1 h extraction period.
5.2.2. Changes in surface topographies
The changes in the surface topographies of the Hemianax papuensis wing surface that were observed in the electron micrographs were also reflected in the AFM analysis. Typical two-dimensional images and cross-sectional profiles of 1.0 µm × 1.0 µm AFM scan areas of each of the wing surfaces are presented in Figure 5.3. Each cross-sectional profile was generated by selecting one line from the corresponding two-dimensional
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AFM scan and plotting height as a function of translation. The surface features in the profiles appeared to decrease in sharpness as the period of chloroform extraction increased. Untreated wing surface profiles contained features ranging from 50 - 100 nm in width. After 10 s of extraction with chloroform, the features appeared to have increased in their spatial distribution and the peaks appeared broader and higher compared to that of the untreated wing. These AFM data were in agreement with the SEM images. Extraction with chloroform may preferentially remove more material from the regions between the pillars rather than from the areas nearer to the apices of the pillars. This would enable the AFM tip to reach deeper into the surface of the samples that had been subjected to chloroform extraction for a 10 s period compared to that of the untreated samples, and therefore the height of the pillars appeared to increase in size. As the time of extraction was increased to 1 h, the surface nanopillars completely disappeared. The maximum height of the features on the 1 h extracted surface ranged from 5 - 10 nm. This most likely reflected the morphology of the intracuticular surface on which the epicuticular lipids had been assembled. It has been reported in the literature that the lipids found on the surface of wings are transported to the surface of insect cuticles through a series of pore channels, where they form the epicuticular nanopillar structures (Lockey, 1980; Lockey, 1985).
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Figure 5.3. Surface topography of Hemianax papuensis dragonfly wings after various periods of extraction with chloroform. Typical two-dimensional AFM scans (1.0 µm × 1.0 µm areas) and cross-sectional profiles of the untreated (A), 10 s (B) and 1 h (C) chloroform-extracted wing surfaces are presented. The surface features became broader and more spatially distributed after 10 s of extraction, however after 1 h of extraction the nanostructures were no longer visible. Scale bars = 100 nm.
The roughness of the wings was quantitatively assessed by calculating a number of roughness parameters for each of the samples (Table 5.1). These parameters were Rmax,
Ra, Rq, Rsk, Rku, and Rdr, as detailed in Section 4.4.3. Relative to the untreated control
87 wing surface, the Rdr increased slightly after a 10 s extraction period and then sharply decreased when the extraction time was increased to 1 h. A similar trend was observed for Rmax, Ra and Rq. The average roughness (Ra) of the untreated wing surface was found to be 49.2 nm, which then increased to 67.5 nm after 10 s of extraction. The wing surface after a 1 h extraction period was found to be very smooth, with both Ra and Rq decreasing significantly (2.4 nm and 3.1 nm respectively) compared to both the untreated and samples that had been chloroform extracted for 10 s. These observations appear to correlate well with the selected line profiles presented in Figure 5.3.
Table 5.1. Roughness analysis of the untreated and chloroform extracted Hemianax papuensis wing surfacesa
Roughness Chloroform Chloroform Untreated statistics extraction for 10 s extraction for 1 h
Rmax (nm) 396 ± 30 491 ± 40 21 ± 5
Ra (nm) 49.2 ± 7.4 67.5 ± 5.8 2.4 ± 0.6
Rq (nm) 61.6 ± 7.6 83.2 ± 6.2 3.1 ± 0.8
Rsk 0.3 ± 0.1 0.4 ± 0.1 0.2 ± 0.1
Rku 2.6 ± 0.1 2.7 ± 0.2 2.7 ± 0.2
Rdr (%) 277 ± 24 306 ± 45 103 ± 2 aResults presented are averages of five measurements taken from 1 µm2 scanning areas and errors are the standard deviations.
The relative percentages of surface area compare to projected area (Rdr) were approximately 277 – 306 % in the case of the untreated and 10 s extracted wing (Table 5.1). This directly indicates the rough nanoscale nature of the surfaces of the wings. After the wings were extracted with chloroform for 1 h, removal of the lipids led to elimination of the nanostructure, and therefore the measured surface areas were found to be much closer to the projected area (i.e. Rdr = 103 %). This demonstrates that the underlying intracuticle of H. papuensis dragonfly wings is relatively smooth before the epicuticular lipids are transported to the surface to form the epicuticular structures.
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5.3. The chemical composition of the Hemianax papuensis dragonfly wing epicuticle
Synchrotron-radiation FTIR microspectroscopy was initially used to confirm that subjecting the dragonfly wings to chloroform extraction led to a decrease in the lipid content on the surface of the wings but had minimal effect on the non-lipid components. Synchrotron radiation allows for the generation of small infrared beam spots that enable the analysis of small areas of the wing. Multiple scans can be conducted in a short period of time within a certain area to validate the consistency of the chemical composition as well as the reliability of quantitative changes of the chemistry over different chloroform extraction times. The characteristic FTIR spectra of the dragonfly wings before and after extraction with chloroform contained three major groups of bands (Figure 5.4) that were very similar to those obtained for the Hemicordulia tau dragonfly wings presented in Section 4.4.2. Of particular interest are the two peaks at -1 -1 2830 cm and 2930 cm , which correspond to symmetrical and antisymmetrical CH2 stretching vibrations. These two bands are of relatively high intensity in the spectra recorded for the untreated wings and are indicative of long chain aliphatic hydrocarbons with repeating CH2 units, i.e. lipid components. After 10 s of extraction with chloroform, the intensity of both peaks decreased visibly, indicating that the lipids were successfully extracted by the chloroform. After 1 h of extraction, the peaks decreased in intensity further but were still present. This is to be expected, as proteins, chitin and other organic molecules that constitute part of the wing membrane contribute to the extent of absorption in this region of the spectrum (Andersen and Weis-Fogh, 1964; Hepburn and Chandler, 1976; Neville, Parry and Woodhead-Galloway, 1976; Hackman and Goldberg, 1987; Gorb, 1997; Nelson and Charlet, 2003; Bogus, Czygier, Golbiowski et al., 2010; Sajomsang and Gonil, 2010). The small ester carbonyl peak, which appeared at approximately 1700 cm-1, also disappeared from the sample that was subjected to 1 h of chloroform extraction, whilst it was still visible in the spectrum for the wing that had been subjected to the shorter 10 s of chloroform extraction (Figure 5.4, indicated by arrows).
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Figure 5.4. Representative infrared spectra of untreated and chloroform-extracted wing membranes of the Hemianax papuensis dragonfly. The intensity of the CH stretching bands (shaded), and carbonyl peak (indicated by arrows) decreased successively with extended extraction time. Spectra were acquired in transmission mode.
While surface analytical techniques such as FTIR microspectroscopy are very useful for generating maps and locating specific chemical functionalities, they usually return a signal or spectrum that represents a sum of all of the chemical components at that interface. This makes detailed molecular identification difficult or impossible, particularly with complex samples. To overcome this limitation, a chromatographic analysis of the lipid extracts was conducted using GCMS. Analysis of the GCMS data revealed that the major lipid components of the wings were comprised mostly of aliphatic hydrocarbons, with a significant contribution from fatty acids (Table 5.2).
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Table 5.2. Individual lipid components of the Hemianax papuensis wing epicuticle isolated by extraction with chloroform, identified by GCMS. Components that could not be unambiguously identified are grouped together.
Proportion (%) Formula RI* Chemical name 10 s 1 h
CHCl3 extraction CHCl3 extraction
C7H12O 965 2-Heptenal 0.1 0.2
C12H26 1200 n-dodecane 0.2
C14H30 1371 3-methyltridecane 0.1
C14H30 1400 n-tetradecane 5.4 2.3
C15H32 1540 3-methyltetradecane 0.1
C16H34 1571 3-methylpentadecane 1.5 0.7
C16H32 1593 1-hexadecene 0.1
C16H34 1600 n-hexadecane 10.6 5.7
C18H38 1728 8-ethylhexadecane 0.1
C18H38 1728 7-ethylhexadecane 0.1
C18H38 1772 3-methylheptadecane 4.2 1.5
C18H36 1793 1-octadecene 0.1
C18H38 1800 n-octadecane 10.3 6.7
C18H38 1799 6,12-dimethylheptadecane 10.3 6.7
C18H38 1888 7,12-dimethylhexadecane 0.1
C20H42 1888 7,12-dimethyloctadecane 0.1
C20H42 1915 6,7-dimethyloctadecane 0.1
C20H42 1921 5,6-dimethyloctadecane 0.1
C20H42 1928 6,10,14-trimethyloctadecane 0.2
C20H42 1944 7-methylnonadecane 0.1
C20H42 1944 8-methylnonadecane 0.1
C20H42 1966 2-methylnonadecane 0.2
C20H42 1973 3-methylnonadecane 2.1 1.7
C20H42 2000 n-eicosane 8.5 6.3
C16H32O1 2046 Hexadecanoic acid 24.9 32.7
C22H46 2116 7-ethyleicosane 0.2
C22H46 2133 11-methylheneicosane 0.1
C22H46 2142 5-methylheneicosane 0.1
C22H46 2142 7-methylheneicosane 0.1
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C22H46 2142 9-methylheneicosane 0.1 (E)-3,7-dimethyl-2,6- C10H16O2 2162 0.1 octadienoic acid
C22H46 2170 3-methylheneicosane 1.7 1.9
C23H48 2170 7,11-dimethylheneicosane 1.7 1.9
C22H46 2200 n-docosane 6.9 6.7
C18H36O2 2244 Octadecanoic acid 13.0
C24H50 2282 9-propylheneicosane 0.2
C24H50 2315 9-ethyldocosane 0.3
C24H50 2373 3-methyltricosane 1.6 2.0
C25H52 2415 12-ethyltricosane 0.1
C25H52 2431 11-methyltetracosane 0.2 0.2
C26H54 2480 12-propyltricosane 0.1
C26H54 2507 11-ethyltetracosane 0.1
C27H56 2525 11,15-dimethylpentacosane 0.1
C26H54 2532 9-methylpentacosane 0.1
C26H54 2532 10-methylpentacosane 0.1
C26H54 2532 11-methylpentacosane 0.1
C26H54 2532 13-methylpentacosane 0.1
C26H54 2544 7-methylpentacosane 0.1
C26H54 2544 5-methylpentacosane 0.1
C27H56 2544 9,13-dimethylpentacosane 0.1
C26H54 2575 3-methylpentacosane 0.7 1.6
C26H54 2600 n-hexacosane 9.9 7.1
C28H58 2704 2,6-dimethylhexacosane 0.1
C28H58 2800 n-octacosane 2.9 4.9
C29H60 2900 n-nonacosane 0.1
C29H60 2973 3-methyloctacosane 0.4 1.0
C30H62 3000 n-triacontane 2.4 3.1
C32H66 3097 2,10-dimethyltriacontane 0.5 0.1
C32H66 3097 2,12-dimethyltriacontane 0.5 0.1
C31H64 3100 n-hentriacontane 0.1 0.1
C33H68 3172 7,23-dimethylhentriacontane 0.1 0.1
C32H66 3172 3-methylhentriacontane 0.1 0.1
C33H68 3172 7,25-dimethylhentriacontane 0.1 0.1
C32H66 3200 n-dotriacontane 0.3 2.1
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C35H72 3369 7,19-dimethyltritriacontane 0.1
C35H72 3369 7,21-dimethyltritriacontane 0.1
C34H70 3400 n-tetratriacontane 0.1 0.1
C32H52O2 3578 Retinyl dodecanoate 0.1
C36H74 3600 n-hexatriacontane 0.1 % Composition based on measured TIC peak areas without standardisation. *RI = Retention index
Palmitic acid (Hexadecanoic acid) was found to be the major component of both the 10 s and 1 h extracts (approximately 24 % and 33%, respectively). A higher proportion of palmitic acid was detected in the 1 h extracts, suggesting that 10 s of extraction was not sufficient to remove this compound completely from the surface. This indicated that palmitic acid was localised not only to the outer epicuticular layer but was also present deeper within the wing. Stearic acid (Octadecanoic acid) was only present in the 1 h extract and had relatively large contribution to the total components of the epicuticular lipids in that sample (13%). Apart from these carboxylic acids components, straight chain alkanes as a group were the most abundant components, among which hexadecane, octadecane, eicosane and docosane were present in the greatest quantities. These six components (palmitic acid, stearic acid, hexadecane, octadecane, eicosane and docosane) were selected for study of their molecular organisation and assembly on synthetic substrata, which is the focus of Chapters 7 and 8.
Straight-chain alkanes made up approximately 50 % of the compounds detected in the 10 s chloroform extracts, with an additional 25 % being monomethylated (11 %) and dimethylated alkanes (14%). The remaining 23 % of the extract could be attributed to oxygenated compounds; more specifically this group was almost entirely comprised of palmitic acid. Palmitic acid, is a fatty acid widely distributed among plants, animals and insects (Smith, 1994; Milligan, Parenti and Magee, 1995; Dowhan, 1997; Buckner and Hagen, 2003; Carballeira, 2008; Samuels, Kunst and Jetter, 2008; Küçükbay, Kuyumcu, Bilenler et al., 2012; Mariod and Abdelwahab, 2012). After 1 h of chloroform extraction, the proportion of oxygenated compounds increased to 38 % of the identified compounds, largely due to the quantity of stearic acid. Since stearic acid was totally
93 undetected in the 10 s extracts, therefore it suggests that stearic acid is primarily situated deeper within the wing membrane, beneath the epicuticular surface.
Figure 5.5. Relative proportions of the major compound classes (top) and chain length (bottom) of dragonfly wing epicuticle components, extracted in 10 s and 1 h.
The aliphatic hydrocarbon constituents of both the 10 s and the 1 h extracts contained several main compounds in relatively high abundance. In addition, both extracts contained significant proportions of n-alkanes with even numbers of carbon atoms ranging from C14 to C30. Biosynthesis of n-alkanes occurs by the addition of acetyl groups to a fatty acyl precursor conjugated to co-enzyme A (Howard and Blomquist, 2005; Samuels, Kunst and Jetter, 2008). The chain-lengths of the resulting hydrocarbons are therefore in multiples of 2 and are dependent on the length of the fatty-acyl chain of the precursor. The composition of the minor constituents varied
94 significantly between the two extraction times, with considerably more compounds being detected in small quantities after 1 h of extraction. Most of these minor constituents were identified as various methyl-substituted alkanes, however three alkenes were detected in the 1 h extract; 1-hexadecene, 1-octadecene, and 1-eicosene. In addition, two esters, one aldehyde and one terpenoid were detected in very small proportions.
5.4. Molecular organisation of H. papuensis dragonfly wing epicuticle
X-ray photoelectron spectroscopy (XPS) is capable of nanometre resolution in the z- dimension (Wagner, 1978; Wagner, Gale and Raymond, 1979; 1983; Escobar Galindo, Gago, Duday et al., 2010). Depth-profiling can be performed using XPS by alternating data acquisition and ion-beam etching. The resulting spectra were found to be dominated by carbon (Figure 5.5). This was to be expected, as it was already well evidenced by FTIR and GCMS analyses that organic compounds were the major components of the wing membrane. Further analysis of the nitrogen and oxygen contents were of greater interest. After the first etching cycle, the oxygen content on the wing surface decreased to almost zero. After 6 etching cycles, the detected oxygen levels began to increase and quickly stabilised at around 2 %. The contribution of nitrogen components was insignificant until after the tenth etching cycle, after which it increased to around 6 %. This is an indication that after 10 etching cycles the protein and chitin structural components in the bulk of the wing were close enough in proximity to the surface to be detected in the spectra. Based on this observation, a tentative estimate of the etching rate can be made; dragonfly wings are typically ~3 μm thick (Kreuz, Arnold and Kesel, 2001; Song, Xiao, Bai et al., 2007; Wan, Cong, Wang et al., 2008; Chen, Wang, Ren et al., 2011) and from the images presented in Figure 4.2 the epicuticular layers of the wings of the H. papuensis dragonfly can be estimated to account for one tenth of the cross-section of the wing. Thus, in 10 etching cycles, i.e. 20 minutes, the wings were etched to a depth of approximately 300 nm, which corresponds to an etching rate of 15 nm min-1.
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Figure 5.6. Atomic proportions of carbon, oxygen and nitrogen in the wings of Hemianax papuensis.
When one considers the XPS depth profiling data in conjunction with the GC-MS data, a tentative model of the molecular organisation of the wing membrane can be proposed. According to the XPS analysis, the oxygen content dropped to zero after just one etching cycle (Figure 5.5). From the GC-MS analysis, the only significant oxygen
96 containing compound in the 10 s lipid extracts was palmitic acid, which suggested the presence of a thin layer at the outer surface of the wing containing palmitic acid (Figure 5.6, Table 5.2). Lipid extracts after 1 h of chloroform extraction contained a significant proportion of stearic acid, along with an increased proportion of palmitic acid. XPS data indicated that the oxygen content only began to increase again after 6 etching cycles, suggesting the presence of a deeper layer that contained both palmitic acid and stearic acid. Aliphatic hydrocarbons were detected in large quantity in all extracts and in the regions between these two fatty acid-containing layers, carbon was the only element detected in XPS spectra.
Exposure of biological samples to ultra-high vacuum (UHV) can cause dehydration, which may conceivably lead to the collapse of the intracuticle. This work focuses on the epicuticular layer of the wing, which is much less hydrated as it does not contain polar components such as sugar or protein (Lapointe, Hunter and Alessandro, 2004). Therefore, UHV should not interfere with the reliability of data obtained here. Based on this information and the etching rate given above, a model of the epicuticle of the H. papuensis dragonfly wing is proposed that includes three distinct layers, one of which was never previously reported for insect epicuticles (Figure 5.7). It is proposed that the new layer be referred to as the mesoepicuticle, that it is positioned between the outer and inner epicuticles, and contains almost exclusively saturated aliphatic hydrocarbons.
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Figure 5.7. Proposed model of the epicuticle of the Hemianax papuensis wing membranes. Three layers are contained within the epicuticle: the outer epicuticle, the mesoepicuticle and the inner epicuticle.
5.1.Summary
While the outer epicuticle is generally accepted in literature to be composed of lipids, the precise identities of the compounds that form the surface nanostructures of the H. papuensis dragonfly wings have not previously been identified. Here, using a combination of surface analytical techniques, including SEM and AFM, and the complementary chemical analytical techniques of Synchrotron-sourced Fourier- transform infrared microspectroscopy (FTIR), gas chromatography-mass spectrometry (GCMS) and x-ray photoelectron spectroscopy (XPS), the epicuticular lipids of dragonfly wings were identified. Removal of the epicuticular lipids led to the disappearance of the surface nanostructures, revealing that the nanostructures themselves were composed of lipids and that the native surface structure of intracuticle was relatively smooth. XPS depth profiling, in conjunction with GCMS analysis of the
98 chloroform extracts, enabled the detection of three distinct layers within the epicuticle: the outer epicuticle, containing mainly palmitic acid; the mesoepicuticle, composed of aliphatic hydrocarbons; and the inner epicuticle, which contained palmitic acid and stearic acid. The identification of a third sub-layer of the epicuticle of insect wings, i.e. the mesoepicuticle, is now reported from this work.
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Chapter 6. Nanostructural effects on antibacterial activity and wettability of dragonfly wings ______
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6.1. Overview
In Chapters 4 and 5, the surface chemistry and the surface topography of different species of dragonfly wings were investigated and characterised in the context of their wettability. This chapter will address how this wettability relates to the antibacterial properties of the wings. The bactericidal activities of Hemianax papuensis and Diplacodes bipunctata dragonfly wings were evaluated to determine the consistency of bacterial activity between different dragonfly species. It was found that H. papuensis dragonfly wings were only moderately effective at inactivating some types of bacterial cells, whilst D. bipunctata dragonfly wings were highly lethal to all tested cell types. This chapter presents the results of work that was undertaken to determine whether the surface chemistry and/or surface topography of the dragonfly wings contribute to this variation in bactericidal activity. Complementary techniques were employed to investigate the mechanisms responsible for these differences; specifically, the surface morphologies of the wings were characterised using SEM and AFM, whilst the surface chemistry was assessed using Synchrotron-based FTIR and XPS.
The two dragonfly wing samples were found to be comprised of very similar chemical components, primarily aliphatic hydrocarbons. These compounds are hydrophobic in nature, partially contributing to the superhydrophobic properties of the wings. The surface roughness of the substrata was found to be the factor that played the greatest role in determining the differences in functionality between the wings of the two species. Despite the visual similarities of the wings, subtle differences were found in (i) their surface morphologies and (ii) their surface topographies. H. papuensis dragonfly wings possessed a higher surface area, were generally rougher and possessed a more hierarchical surface structure than the wings of the D. bipunctata wings, hence enabling the surface features to trap more air pockets within their structure, contributing to the surface hydrophobicity. For this reason, the surface of H. papuensis dragonfly wings were less accessible to bacterial attachment compare to D. bipunctata. The amount of trapped air determined how deep water droplets placed in contact with the surface could penetrate the surface in between the pillars, as evidenced in their differences in water contact angles. This may also be the case for bacterial cells; the depth to which they could impinge upon the surface would likely determine their susceptibility to the bactericidal action of the wings.
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6.2. Bactericidal activities of dragonfly wings
The wings of the H. papuensis dragonfly were found to have moderate bactericidal activity. They were somewhat effective at killing P. aeruginosa, S. aureus cells and spores of B. subtilis, however the surface was found to be ineffective against vegetative B. subtilis cells. This can be observed in the confocal laser scanning microscopy (CLSM) images presented in Figure 6.1. On the other hand, D. bipunctata wings were found to be very effective against all tested cell types (P. aeruginosa, S. aureus, B. subtilis cells and spores). In general, the green fluorescence signal, which indicated the presence of live bacterial cells, was greater on the H. papuensis wings, while the cells on D. bipunctata wings almost exclusively exhibited red fluorescence, indicating the presence of dead cells. As for the largely resilient B. subtilis spores, a lower killing efficiency was observed on the wings of both dragonfly species, however, a greater number of dead cells were found to be present on the surface of D. bipunctata wings compared to that of H. papuensis wings. Yellow-fluorescing cells indicated the binding of both dyes, which is an indication that those cells are in fact dead, as the red propidium iodide is only able to enter cells with significant membrane damage.
The adhesion and morphology of bacterial cells after they had adsorbed onto the surfaces of the two dragonfly wings were observed using scanning electron microscopy (Figure 6.2). In both cases, P. aeruginosa cells appeared to sink deeply between the nano-features of the two dragonfly wings. The structure of these cells appeared severely deformed on both surfaces, which would explain the red fluorescence in CLSM images; as such strong deformation would very likely be accompanied by cell rupture. S. aureus, B. subtilis and spores also appeared deformed on the wings of D. bipunctata, however they were relatively unaffected by the action of the surface of the H. papuensis wings.
Bacterial viability assays in the form of serial dilutions and colony counts on agar plates which was employed to identify the bactericidal efficiency of the two dragonfly wings, based on the 3 hours assay. The data revealed that the bactericidal efficiency of the H. papuensis wings against S. aureus and B. subtilis spores after 3 hours was 5.3 × 104 and 4.5 × 104 cells killed per min-1 per cm-2 of wing surface, respectively (Figure 6.3). The next highest activity of this wing was against P. aeruginosa cells, however the efficacy against all types of cells was much less than that of D. bipunctata cells, which
102 in all cases was capable of killing at least twice as many cells in the same period of time.
Figure 6.1. Bactericidal effects of H. papuensis (top) and D. bipunctata (bottom) dragonfly wings against various bacterial strains. The dead cells in CLSM images were stained with propidium iodide, indicated in red, while live cells were stained with SYTO 9, indicated in green (Scale bar = 5µm). Yellow fluorescence indicated the binding of both dyes, which indicates that sufficient propidium iodide had passed through the membrane, indicating cell rupture and death.
The two dragonflies being studied are native to Australia. The surface morphologies of H. papuensis and D. bipunctata wings appeared to be highly similar when viewed using SEM, however the difference in their ability to kill bacterial cells is both striking and clear. Based on the confocal and SEM images, the surfaces of D. bipunctata wings were much more effective at killing bacterial cells than those of H. papuensis wings. In order to determine how such seemingly similar wing morphologies result in vastly different functionalities, various techniques were employed to further discriminate between the nanostructures present on each of these wing types.
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Figure 6.2. Representative scanning electron micrographs of various bacterial cells on the surface of H. papuensis (left column) and D. bipunctata (right column) dragonfly wings, demonstrating the attachment behaviour of P. aeruginosa (A, B), S. aureus (C, D), vegetative B. subtilis (E, F) cells and its spores (G, H). All cell types were deformed when adhered to D. bipunctata wings, however only P. aeruginosa cells were similarly affected on the H. papuensis wings. Scale bars = 1 µm, inset scale bars = 200 nm. 104
Figure 6.3. Bactericidal efficiency of D. bipunctata and H. papuensis dragonfly wing surfaces against four different bacterial cell types. The values presented are the number of cells killed per cm2 of wing surface per minute of incubation, over the first three hours.
6.3. Comparative surface chemistry of the dragonfly wings
Despite their differences in efficiency of killing bacterial cells, the elemental chemical compositions across the wing membranes of H. papuensis and D. bipunctata wings were highly similar. Typical infrared spectra containing the characteristic peaks of the two dragonflies across the wing membrane were dominated by the three major bands, which represent aliphatic hydrocarbons, carbonyl compounds and amides (Figure 6.4).
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Figure 6.4. Representative infrared spectra of the wing membranes of H. papuensis (red) and D. bipunctata (blue) dragonflies. The spectra of two dragonfly wings showed their highly similarities in fundamental chemical compositions.
XPS is a surface sensitive method for quantifying elemental compositions, therefore it is an important technique for this study. The XPS data showed that the major elements present on the surface of the wing were carbon and oxygen. This is consistent with what was observed from FTIR data. The carbon peaks were dominant over the oxygen peaks, which indicated that there is much larger proportion of carbon at the outer surface of the wing. The high resolution scans of the C 1s peak showed the presence of, saturated carbon (i.e. C–C and C–O), and O-C꞊O chemical configurations, whereas two oxygen configurations were detected (O–C and O꞊C), consistent with the data presented in Chapter 5. This indicated that the major components present on the outer layer of the wings were aliphatic alkanes and fatty acids (Section 5.3 and 5.4).
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Figure 6.5. High resolution XPS spectra of H. papuensis and D. bipunctata dragonfly wings. High-resolution scans were performed in approximately 20 eV intervals across the O 1s and C 1s peaks. The C 1s peaks of both species are dominated by C–C and C– H bonds, whereas there is an approximately 3:4 ratio between C–O and C=O components within the O 1s peaks.
The chemical analyses based on data obtained from Synchrotron-radiation sourced FTIR and XPS demonstrated the existence of high similarities between the two dragonfly species. No substantial differences in the chemical composition of the wings of H. papuensis and D. bipunctata could be detected, which further supports the concept that the bacterial killing action is mechanobactericidal in nature. It follows that the difference in functionality of the wings is based primarily on morphology and structure, therefore further investigation of their surface topographies were conducted in order to determine the cause of the variation in bactericidal activity. 107
6.4. Variation in surface structure
6.4.1. Surface topologies
The SEM images of the two dragonfly wings provide an indication of their surface morphology. The surfaces of the wings of both species appeared similar, however differences are evident visually (Figure 6.6). Here it can be seen in the high resolution SEM images that the nanopillars on the surface of H. papuensis wings appeared thicker and more densely packed than those of D. bipunctata wings. The tips of the pillars on the H. papuensis wings also appeared comparatively round and thick whereas the pillars appeared sharper and thinner in case of the D. bipunctata wings.
A 2D Fourier Transform (FT) analysis was applied to electron micrographs of both wings at various magnifications to provide a quantifiable measurement of their respective topographies. Fourier transformation is a tool in image analysis used to quantify the size and spacing between surface features (Burger and Burge, 2007). Figure 6.7 shows the FT power spectrum of SEM images of the wings of H. papuensis and D. bipunctata dragonflies.
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Figure 6.6. Photographs of (A) H. papuensis and (B) D. bipunctata dragonflies and corresponding scanning electron micrographs of their wing surfaces (C, D). According to SEM analysis, their structures appeared to be highly similar, however the H. papuensis wings appeared to have more connecting structures between the pillars and the peaks of D. bipunctata wings appeared slightly sharper and thinner. Insets are 3D representations of sections of the micrographs. (A, and B are reproduced with permission from the Encyclopedia of Life (2014)).
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Figure 6.7. Fast Fourier transform analysis of (A) H. papuensis and (B) D. bipunctata wing surface images. The grayscale plots of cross-sections of the 2D FFT at 0° show a longer range directional periodicity up to 2.5 µm for D. bipunctata and 4 µm for H. papuensis wings. The area % and population distribution of the wings surface features demonstrated that the feature size of H. papuensis wings is about 70 nm and 100 nm for D. bipunctata wings.
The data obtained from FFT of the two wings provided quantitative estimate for the dimension of the pillars and their spatial distribution based on the vertical view of their scanning electron micrographs. This showed a maximum pillar diameter of 70 nm for the H. papuensis wing compared to about 100 nm for D. bipunctata wing; the latter exhibiting a greater degree of tailing to the larger diameters of the distribution, which was evidence of a higher level of aggregation of these features at the tips. Since the analysis is based on a vertical image from SEM, it only reflects patterning in the x- and y-plane. By imaging cross sections of the wing membranes of the two species, the dimensions of the pillars in the z-dimension can be directly measured (Figure 6.8).
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Figure 6.8. Cross-sections of the wings of H. papuensis and D. bipunctata dragonflies. The nanopillar arrays on the surface of D. bipunctata wings appeared relatively consistent in height, whilst those on the surface of H. papuensis wings appeared to vary more in their height.
The nanopillars on the H. papuensis wings appeared to have multiple layers with respect to their height, whilst those on the D. bipunctata wings appeared more regular. The height of pillars present on the H. papuensis wings ranged from 155 nm to 290 nm, whereas the pillars on the D. bipunctata wings fluctuated in height around 265 nm. This data correlated relatively well with the results obtained from the AFM analysis. Typical two-dimensional AFM images and cross-sectional profiles of 2.5 µm × 2.5 µm scan areas of the two dragonfly wings are presented in Figure 6.9. As can be seen in the two- dimensional AFM images, the pillars appeared irregular, with more outliers (bright and dark areas based on colour scales at the right of each AFM images) for H. papuensis compared to D. bipunctata wings. The zero in the line profiles was defined as the mean planes of corresponding scans. The surface features in the profiles appeared taller and
111 more irregular for the H. papuensis wings. The maximum features were 300 nm for H. papuensis and 250 nm for D. bipunctata wings.
Figure 6.9. Typical two-dimensional AFM scans (2.5 µm × 2.5 µm areas) and the cross-sectional profiles of (A) H. papuensis and (B) D. bipunctata wings. The surface of the H. papuensis wing appeared highly irregular, with an increased degree of hierarchy of the nanopillars, whilst the line profile of D. bipunctata wings was comprised of regular peaks with a similar height and relatively consistent spatial distribution.
Again, the six standard roughness parameters, i.e., Rmax, Ra, Rq, Rsk, Rku and Rdr, were calculated for each wing surface and these are summarised in Table 6.1. The maximum roughness (Rmax) of H. papuensis wings appeared larger in comparison to those of the D. bipunctata wings (481 nm compared to 371 nm). There were minor variations in Ra and Rq between the two species; e.g. the average roughness (Ra) of the H. papuensis wing was 50 nm, whereas this was 43.38 nm for the D. bipunctata wing.
Skewness (Rsk) and kurtosis (Rku), which have been used as an indication of the spatial variation in height, show that high similarity exists between the two species. Minor differences were observed with D. bipunctata wings possessing a slightly higher degree
112 of skewness. The Rdr measured 245% for H. papuensis and 204% for D. bipunctata wings, which demonstrated that the real surface area of the former species was significantly higher. These differences in surface roughness can be considered to be statistically significant in comparison to the surface roughness variations that can occur on wing surfaces within the same species, as indicated by the error values. Previously reported data also suggests that variations in the surface morphologies of insect wings within a single species is negligible (Watson, Cribb and Watson, 2011).
Table 6.1. Roughness analysis data for the two dragonfly wing surfacesa, H. papuensis and D. bipunctata dragonflies.
Roughness parameterb H. papuensis D. bipunctata
Rmax (nm) 482 ± 7 371 ± 8
Ra (nm) 50.4 ± 0.7 43.4 ± 0.8
Rq (nm) 64 ± 1 55 ± 1
Rsk 0.4 ± 0.1 0.4 ± 0.1
Rku 3.2 ± 0.1 3.2 ± 0.1
Rdr (%) 245 ± 6 204 ± 2 aResults presented are averages of five measurements taken from 2.5 µm2 scanning areas and errors are the standard deviations. bA technical description of the roughness parameters used can be found in Section 4.4.3 and in Crawford et al. (Crawford, Webb, Truong et al., 2012).
6.4.2. Surface morphologies
Other parameters often used to evaluate surfaces in materials engineering are the bearing statistics (Gadelmawla, Koura, Maksoud et al., 2002; Butt, Cappella and Kappl, 2005). Bearing area is determined as the area occupied by the surface in a single plane at a given depth measured from the highest point of the surface, and is usually expressed as the ratio of bearing area to the projected area of the plane, i.e. the bearing ratio, tp. The bearing ratios of the two dragonfly species were plotted as a function of depth (Figure 6.10). From these two graphs, a series of bearing statistics was extracted, i.e. peak height Rpk, core roughness Rk and valley depth Rvk, each of which is given in Table 6.2.
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Figure 6.10. Demonstration of bearing analysis. The bearing ratio of a surface is defined as the proportion of the projected area that is occupied by the surface at a given depth from the highest point on the surface (demonstrated in A-C, shaded areas). (D) a demonstration of how three statistical parameters were extracted directly from the plot, i.e. peak height (Rpk), core roughness (Rk) and valley depth (Rvk).
Table 6.2. Bearing statistics of the H. papuensis and D. bipunctata dragonfly wing surfaces.
Bearing parameter H. papuensis (nm) D. bipunctata (nm)
Rpk 28.6 28.3
Rk 135.6 98.2
Rvk 50.8 65.2
The parameters Rk, Rpk and Rvk are defined as core roughness, peak height, and valley depth, respectively (Crawford, Webb, Truong et al., 2012). In order to identify these parameters, the height was plotted as a function of the bearing area. The tangent with the maximum slope within the middle 40% of the bearing area was extended in both directions until it intersected tp = 0 and tp = 100%. The difference in height of those
114 two intercepts was identified as the core roughness. The value on the bearing curve at the depth of both tangential intercepts was located and tangents to these points were added to intersect tp = 0 at the base of the curve and tp = 100 at the upper end. The difference between these two additional intercepts and the intercepts of the original tangent gave the peak height Rpk (at the base of the curve) and valley depth Rvk (at the top of the curve). Detailed descriptions of these parameters can be found in the literature (Thomas, 1998; Gadelmawla, Koura, Maksoud et al., 2002; Crawford, Webb, Truong et al., 2012). All of the bearing parameters were larger for the H. papuensis compared to D. bipunctata wings, particularly with respect to the core roughness and valley depth. The bearing statistics suggested that the surface of the H. papuensis wings was more irregular and rougher than that of the D. bipunctata wings. Also, the core roughness Rk is lowest on the surface of D. bipunctata wing (98.2 compared to 135.6 of H. papuensis wing) suggesting that the sides of the D. bipunctata wing pillars are much steeper than those of the surface of H. papuensis wings, which was in agreement with the roughness analysis and electron micrographs previously discussed.
6.5. Structure-dependent wettability
An additional 2D Fourier Transform analysis was applied to SEM images of both wing samples at various magnifications to provide a further quantifiable measurement of their respective topographies. The FT power spectrum of SEM images at various magnifications of H. papuensis and D. bipunctata wings is given in Figure 6.11 and Figure 6.12, respectively. At the two lower magnifications (15000× and 35000×), the inflection points in the data indicated that the surface of H. papuensis wings had larger peak features compared to those of the D. bipunctata wings. Specifically at 15000× magnification, inflection points were observed at approximately 110 nm and 200 nm for D. bipunctata, and H. papuensis wings, respectively, which represents the average distance between the asperities on the surface. At higher magnification (35000×) a second inflection point was observed for each species, at approximately 42 nm and 70 nm for D. bipunctata and H. papuensis wings, respectively, which corresponds to the typical diameter of the asperities on each of the respective wings. At the highest magnification of 70000×, the most common length scale remains 42 nm for the D. bipunctata wings but a third feature was observed on the wings of the H. papuensis samples at 34 nm, which is indicative of the presence of a hierarchical structure (Figure 6.12). 115
Figure 6.11. Fourier-transform analysis of H. papuensis dragonfly wing structures at various magnification levels. The inflection points (indicated) demonstrate the dominant feature sizes on the wings of H. papuensis wing at each magnification. The feature size indicated for the 15000× magnification corresponds to the negative space and provides an indication of the spacing of the nanopillars, whilst the size indicated at 35000× and 70000× magnification levels correspond to the nanopillars themselves. The presence of two different nanopillar heights is indicative of the presence of a hierarchical surface structure.
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Figure 6.12. Fourier-transform analysis of D. bipunctata dragonfly wing structures at various magnification levels. The inflection points (indicated) demonstrate the dominant feature size on the wings of D. bipunctata at each magnification. The feature size indicated for 15000× magnification corresponds to the negative space and provides an indication of the spacing of the nanopillars, whilst the size indicated at 35000× and 70000× magnification levels correspond to the nanopillars themselves. The nanopillars appeared to be relatively similar at both the larger magnifications.
The D. bipunctata wing possessed only a single scale of roughness, which consists of pillars that are approximately 42 nm in diameter and spaced 110 nm apart. Conversely, dual scales of roughness were detected on the H. papuensis wings, which consist of pillars approximately 70 nm in diameter and spaced approximately 200 nm apart, they are interwoven with bridges between the major asperities that are approximately 34 nm in width. This hierarchical structure of the H. papuensis wing indicated by this Fourier transform analysis matches well with the description of Cassie- Baxter structure (Cassie and Baxter, 1944; Nishimoto and Bhushan, 2013; Papadopoulos, Mammen, Deng et al., 2013; Yang, Jin, Liu et al., 2013; Zhang, Wang and Levänen, 2013) and hence gives a stable superhydrophobic and self-cleaning surface. Thus the H. papuensis wing exhibited a higher static water contact angle (161°) compared to that of the surface of the D. bipunctata wings (151°).
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Insect wings, including cicada, damselflies, and dragonflies are well-known for their superhydrophobic nature. It was also well established in the literature that for superhydrophobic surfaces, water droplets are most likely to be in the Cassie-Baxter wetting state. Briefly, the Cassie-Baxter theory explains the wetting behaviours of water droplets on a composite or highly rough solid surface. According to this theory, changes in hydrophobicity of a surface can be induced by the combined effects of surface topography and surface chemistry (Cassie and Baxter, 1944; Shirtcliffe, McHale, Atherton et al., 2010; Su, Ji, Zhang et al., 2010; Bhushan, 2012). The wettability of a surface containing a large proportion of entrapped air is described by the Cassie-Baxter equation: