ASSESSMENT OF THE BACTERICIDAL EFFECT OF BIOMIMICKED NANOPILLARS OF CICADA WINGS ON TITANIUM IMPLANTS
Hesam Shahali M.Phil., BSc
Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy
School of Mechanical, Medical and Process Engineering Science and Engineering Faculty Queensland University of Technology 2020
Keywords
Bactericidal surface, nanopillars, natural bactericidal surface, surface characteristics, antibacterial nanostructure, cytocompatibility, biocompatibility, titanium, electron beam lithography, helium ion microscopy, scanning electron microscopy, atomic force microscopy.
Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants i
Abstract
Bacteria colonization and biofilm formation are the major causes of infection in implants. The annual cost of implant-associated infections in the US is 150-200 million USD and 7-11 million Euro in the UK. It is therefore vital to eliminate the bacterial attachment and biofilm formation from the surface of implants. Currently, chemical- based detergents and traditional antibacterial/antibiotic coatings are used to produce antibacterial surfaces. These products are, however, not always effective since biofilm can still form on the implant surfaces. Moreover, there is an additional problem of long-term efficiency. Nanopillars on cicada wings have recently drawn the interest of scholars due to their bactericidal, self-cleaning and superhydrophobic characteristics. This research aimed to (i) systematically characterise and assess the bactericidal and cytocompatible characteristics of the insect wings of three cicada species, (ii) mimic the wings’ nanopillars architecture on titanium substrates using electron beam lithography and (iii) perform a simulation analysis to find the most optimum fabrication method. The nano topography of three Australian cicadas (Psaltoda claripennis (PC), Aleeta curvicosta (AC) and Palapsalta eyrei (PE)) were characterised using scanning electron microscopy (SEM), helium ion microscopy (HIM), atomic force measurement (AFM) and transmission electron microscopy (TEM). Chemical characteristics of nanopillar surface were investigated using X-ray photoelectron spectroscopy (XPS) and infrared spectroscopy (FTIR). Pseudomonas aeruginosa (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria were selected for the antibacterial study and human osteoblast cell lines were used to study the biocompatibility of the insect wing. Bactericidal efficiency and biocompatibility were evaluated through the plate count method and AlamarBlueTM assay, respectively. The nanopillars on cicada wings were mimicked on titanium substrates using Electron Beam Lithography (EBL) and the process variables were optimised to achieve the closest nanopillar architecture to the cicada wings. The wings of all the tested cicada species possessed unique nanopillars architecture on the vein and membrane. The geometry of nanopillars (e.g. diameter, height, centre to centre distance, density, and aspect ratio) differed among the species as well as among the membrane and veins. The aspect ratio and density of nanopillars were considerably higher on membranes than on veins. Microscopy analysis of
ii Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants
bacteria attachment showed that both bacteria were killed on nanopillars. Nanopillars of cicada wings significantly reduced P. aeruginosa colonies after 18 hrs compared to the control surface. A considerable reduction in S. aureus was found after 2 and 4 hrs compared to the control surface. Species PE and AC produced the highest bactericidal effect after 18 hrs with values of 7.3 × 105 and 1.43 × 106 (CFU/mL) against P. aeruginosa and S. aureus, respectively. All three cicada species produced cytocompatibility in response to human osteoblasts. The human osteoblast cell morphology remained undamaged, demonstrating the biocompatibility of the insect wing surfaces. In the second stage, EBL was employed to mimic the cicada nanopillars on titanium substrates through a systematic design and modelling approach. Monte Carlo simulation was used to optimize the beam energy and pattern design (dot and circle) prior to the experimental study. EBL process variables including write field, pitch, and more importantly, exposure factor (EF) were optimized to fabricate titanium nanopillars close to those of the PE species as the most effective antibacterial surface. Three groups of titanium nanopillar arrays were fabricated: (i) Based on the simulation of a circle-pattern diameter of 70 nm and centre to centre distance of 160 nm, the fabricated nanopillar array had a base diameter of 94.4 nm, top diameter 12.6 nm, centre to centre distance 165.8 nm, height of 115.6 nm and aspect ratio 2.16. (ii) Based on the simulation of a circle pattern diameter of 120 nm and centre to centre distance of 200 nm, the fabricated nanopillar array had a base diameter of 148.6 nm, spike diameter of 21.05 nm, centre to centre distance of 200.3 nm, height of 221.6 nm and aspect ratio 2.32. (iii) Based on the simulation of a circle pattern diameter of 200 nm and centre to centre distance of 320 nm, the fabricated nanopillar array had a base diameter of 214 nm, spike diameter of 48.9 nm, centre to centre distance of 324.9 nm, height of 288 nm and an aspect ratio of 2.19. While the three fabricated groups of titanium nanopillar surfaces damaged the membrane of P. aeruginosa and S. aureus, the first group produced the best bacteria killing performance in the same manner as PE cicada wings. The titanium nanopillars were biocompatible with human osteoblasts, enhancing cell anchorage and proliferation. To conclude, the assessment of bactericidal activity and the biocompatibility of natural cicada nanopillars and the versatile design of biomimicked titanium nanopillars will enable biomedical engineers to identify an ideal solution for design and manufacture of biomedical devices, such as orthopaedic implants.
Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants iii
List of Publications
Journal articles
• Hesam Shahali, Jafar Hasan, Asha Mathews, Hongxia Wang, Cheng Yan, Tuquabo Tesfamichael, Prasad KDV Yarlagadda, Multi-biofunctional properties of three species of cicada wings and biomimetic fabrication of nanopatterned titanium pillars, 2019, Journal of Materials Chemistry B, 7(8), 1300-1310 (DOI: 10.1039/C8TB03295E).
• Hesam Shahali, Jafar Hasan, Hongxia Wang, Tuquabo Tesfamichael, Cheng Yan, Prasad KDV Yarlagadda, Evaluation of particle beam lithography for fabrication of metallic nano-structures, 2019, Procedia Manufacturing, 30, 261-267 (DOI: 10.1016/j.promfg.2019.02.038).
• Hesam Shahali, Alka Jaggessar, Prasad KDV Yarlagadda, Recent Advances in Manufacturing and Surface Modification of Titanium Orthopaedic Applications, 2017, Procedia Engineering, 174, 1067-1076 (DOI: 10.1016/j.proeng.2017.01.259).
• Alka Jaggessar, Hesam Shahali, Asha Mathew, Prasad KDV Yarlagadda, Bio-mimicking nano and micro-structured surface fabrication for antibacterial properties in medical implants, 2017, Journal of Nanobiotechnology, 15(1), 64 (DOI: 10.1186/s12951-017-0306-1). • Shahali, Hesam, Hasan, Jafar, Cheng, Han-Hao, Ramakrishna, Seeram, Yarlagadda, Prasad KDV, A systematic approach towards biomimicry of nanopatterned cicada wings on titanium using Electron Beam Lithography (under review in Nanotechnology)
Conference paper • Hesam Shahali, Alka Jaggessar, Prasad KDV Yarlagadda, Recent Advances in Manufacturing and Surface Modification of Titanium Orthopaedic Applications, 2016 Global Congress on Manufacturing and Management, China.
iv Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants
• Hesam Shahali, Jafar Hasan, Hongxia Wang, Tuquabo Tesfamichael, Cheng Yan, Prasad KDV Yarlagadda, Evaluation of particle beam lithography for fabrication of metallic nanostructures, 14th Global Congress on Manufacturing and Management, Brisbane, Australia (GCMM-2018).
Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants v
Table of Content
Keywords ...... i Abstract ...... ii List of Publications ...... iv Table of Content ...... vi List of Figures ...... x List of Tables ...... xxi List of Abbreviations ...... xxiii Statement of Original Authorship ...... xxv Acknowledgements ...... xxvi Chapter 1: Introduction ...... 1 1.1 Background ...... 1 1.2 Research Problem ...... 4 1.3 Aims and Objectives ...... 5 1.4 Significance and Outcomes ...... 5 1.5 Thesis Outline ...... 6 Chapter 2: Literature Review ...... 8 2.1 Overview ...... 8 2.2 Infection and Biofilm Formation on Orthopaedic Implants ...... 8 2.2.1 Bacterial infection ...... 8 2.2.2 Biofilm formation ...... 9 2.3 Biocompatibility and Osseointegration of Titanium Implants ...... 11 2.4 Traditional Antibacterial Coating and Surface Modification ...... 12 2.4.1 Antibiotic releasing coating ...... 12 2.4.2 Inorganic antibacterial coating ...... 13 2.4.3 Electron beam evaporation, electrochemical etching and anodization ...... 13 2.4.4 Drawbacks of traditional antibacterial method and surface modification ...... 14 2.5 Natural Antibacterial and Biomimicked Surfaces ...... 14 2.5.1 Taro and lotus leaves ...... 15 2.5.2 Sharkskin ...... 18 2.5.3 Gecko skin ...... 21 2.5.4 Dragonfly ...... 23 2.5.5 Antimicrobial Natural Peptides (AMPs) ...... 26 2.5.6 Cicada wing ...... 27 2.6 The Interface of Nanopillars and Bacteria ...... 33 2.7 Nanofabrication of Biomimicked Nanopillars ...... 37 2.7.1 Soft lithography ...... 37 2.7.2 Vacuum casting ...... 43 2.7.3 Femtosecond laser and laser interface lithography ...... 44 vi Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants
2.7.4 Reactive Ion Etching (RIE) ...... 45 2.7.5 Hydrothermal Method ...... 47 2.7.6 Photolithography (PL) ...... 48 2.7.7 Glancing Angle Sputter Deposition (GLAD) ...... 49 2.7.8 Particle Beam Lithography (PBL) ...... 50 2.8 Summary ...... 57 2.8.1 Literature review finding ...... 57 2.8.2 Knowledge gap ...... 58 Chapter 3: Research Methodology ...... 60 3.1 Overview ...... 60 3.2 Research Methodology Design ...... 61 3.3 Material ...... 61 3.3.1 Cicada wing ...... 61 3.3.2 Titanium sample ...... 63 3.3.3 Bacteria ...... 64 3.3.4 Osteoblast human cells ...... 65 3.4 Surface Chemical Characterization ...... 66 3.4.1 Fourier Transform Infrared Spectroscopy (FTIR) ...... 66 3.4.2 X-ray photoelectron spectroscopy (XPS) ...... 67 3.5 Nano Topography Analysis ...... 68 3.5.1 Nano topography analysis of cicada wings using Helium Ion Microscopy (HIM) ...... 68 3.5.2 Transmission Electron Microscopy (TEM): cross-sectional analysis and sample preparation ...... 69 3.5.3 Atomic Force Microscopy (AFM) for topography analysis and cell conformity ...... 70 3.6 Bacteria preparation and Bacterial viability testing ...... 71 3.7 Osteoblast cell culture and AlamarBlueTM assay ...... 72 3.8 Statistical Analysis ...... 73 3.9 Electron Beam lithography (EBL) ...... 73 3.9.1 PMMA selection ...... 74 3.9.2 PMMA thickness measurement ...... 76 3.9.3 Titanium coating surface roughness measurement ...... 77 3.9.4 TESCAN EBL system and process parameters ...... 77 3.9.5 Monte Carlo simulation ...... 80 3.9.6 Electron scattering in EBL ...... 80 3.10 Titanium deposition ...... 81 3.10.1 Thermal evaporation ...... 81 3.10.2 DC and RF sputtering ...... 81 3.10.3 Electron beam evaporation ...... 83 3.10.4 Step coverage in metal deposition ...... 84 Chapter 4: Surface Characteristics, Cell Interaction, Bactericidal Properties and Biocompatibility of Nanopillars of Three Cicada Species...... 87 4.1 Overview ...... 87 4.2 Cicada Wing Nano Topography ...... 87 4.3 Chemical Characteristics of Wing Nanopillars ...... 92 4.3.1 FTIR analysis ...... 92
Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants vii
4.3.2 XPS analysis ...... 93 4.4 Mechanical Characteristics of Cicada Wing Nanopillars using AFM ...... 94 4.5 Analysis of Bacteria Interaction with Nanopillars ...... 96 4.6 Analysis of Bactericidal Efficiency using the Plate Counting Method ...... 104 4.6.1 Analysis of bactericidal efficiency of P. aeruginosa ...... 104 4.6.2 Analysis of bactericidal efficiency of S. aureus ...... 105 4.7 Cytocompatibility of the Cicada Wing Nanopillars ...... 107 4.8 Main Findings and Remarks ...... 109 Chapter 5: Optimization of Electron Beam Lithography and Process Parameters for the Fabrication of Biomimicked Nanopillars ...... 112 5.1 Introduction ...... 112 5.2 Optimization Flowchart of Electron Beam Lithography (EBL) ...... 113 5.3 Monte Carlo Simulation ...... 114 5.3.1 Monte Carlo simulation on 300 nm resist (dot and circle design) ...... 115 5.3.2 Monte Carlo simulation on 400 nm resist (dot and circle design) ...... 117 5.3.3 Monte Carlo simulation on 500 nm resist (dot and circle design) ...... 118 5.3.4 Monte Carlo simulation on 670 nm resist (dot and circle design) ...... 120 5.3.5 Monte Carlo simulation on 700 nm resist (dot and circle design) ...... 122 5.4 Optimization of Electron Beam Lithography on PMMA Resist ...... 124 5.4.1 EBL on one layer of PMMA 950 A4 ...... 125 5.4.2 EBL on multi-layer (two and three layers) of PMMA 950 A4 ...... 128 5.5 Experimental Evaluation of EBL on Multilayer PMMA Resist ...... 131 5.5.1 One layer PMMA 495 A4 and one layer PMMA 950 A4 ...... 131 5.5.2 Two layers PMMA 495 A4 and one layer PMMA 950 A4 (Write field 50 µm × 50 µm) ...... 133 5.5.3 Two layers PMMA 495 A4 and one layer PMMA 950 A4 (Write field 30 µm × 30 µm) ...... 135 5.5.4 Two layers PMMA 495 A4 and one layer PMMA 950 A2 (Write field 30 µm × 30 µm and 150 nm titanium coating thickness) ...... 138 5.5.5 Three layers PMMA 495 A4 and one layer PMMA 950 A2 (Write field 25 µm × 25 µm) with 250 nm coating ...... 141 5.6 Summary ...... 147 Chapter 6: Evaluation of Bacteria Interaction and Biocompatibility of Titanium Fabricated Nanopillars ...... 150 6.1 Overview ...... 150 6.2 Nanotopography Analysis of Fabricated Nanopillars using SEM ...... 150 6.3 Chemical Characteristics of Fabricated Nanopillars using XPS ...... 153 6.4 Mechanical Characteristics of Fabricated Nanopillars using AFM ...... 153 6.5 Analysis of Bacteria Interaction with Fabricated Nanopillars using SEM ...... 154 6.6 Cytocompatibility of Osteoblast Cells on Fabricated Nanopillars using AlamarBlueTM Assay ...... 158 6.7 Analysis of Osteoblast Cell Interactions with Fabricated Nanopillars using SEM .... 159 6.8 Main Findings and Remarks ...... 160 Chapter 7: Analysis and Discussion ...... 162
viii Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants
7.1 Overview ...... 162 7.2 Cicada Wing Surface Characterization ...... 163 7.3 Analysis of Bactericidal Efficiency of Nanopillars Against P. aeruginosa and S. Aureus ...... 170 7.4 Analysis of Bacterium Interaction with Natural Cicada Wing Nanopillars...... 172 7.5 Effect of the Geometry of Nanopillar on Bacteria Interaction ...... 172 7.6 Biocompatibility of Cicada Nanopillar ...... 175 7.7 Selecting the Ideal Method to Mimic and Fabricate Titanium Nanopillars ...... 176 7.8 Systematic Approach to Optimize the EBL Process Variables to Fabricate Titanium Nanopillars ...... 179 7.9 Comparison of Bacteria Interaction on Cicada Wing Nanopillars and Titanium Nanopillars ...... 182 7.10 Biocompatibility of Titanium Nanopillars ...... 184 Chapter 8: Conclusion and Future Work ...... 185 8.1 Introduction ...... 185 8.2 Conclusion Statement ...... 185 8.3 Outlook for Future Research ...... 187 Bibliography ...... 189 Appendices ...... 201
Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants ix
List of Figures
Figure 2.1: Schematic of the five stages of biofilm formation (adapted from D.Davis) (Bixler et al., 2014)...... 10 Figure 2.2: Biofilm SEM cross-sectional image representing the EPS morphology (Bixler and Bhushan, 2012)...... 10 Figure 2.3: Formation of biofilm by Staphylococcus aureus on (a) Surface of a catheter (Chung and Toh, 2014), (b) Patient ventilation tube and (c) Patient Pacemaker (Bixler and Bhushan, 2012)...... 11 Figure 2.4: SEM image of taro leaves. (a) liquid substitution and (b) air-dry and sputter coating (Ma et al., 2011)...... 16 Figure 2.5: Relationship between nanostructure density of taro leaves and bacteria adhesion (Ma et al., 2011)...... 16 Figure 2.6: SEM image of lotus leaf: (a) and (c) untreated micro-size bulge shaped pattern plus nanocrystal with different magnification, (b) and (d) treated micro-size bulge shaped pattern (Cheng et al., 2006)...... 17 Figure 2.7: Comparison of whale (left) and shark (right) skin (Bixler and Bhushan, 2013)...... 18 Figure 2.8: SEM image of a single scale of six shark species: (a) smooth hammerhead, (b) banded hound shark, (c) Jaws- (d) shortfin mako (Mako shark), (e) sand shark, and (f) mud shark (Spiny Dogfish) (Pu et al., 2016)...... 19 Figure 2.9: Negative biomimicked sharklet micro-pattern (4 µm height, 2 µm width and spacing) (Carman et al., 2006)...... 20 Figure 2.10: Zoospore settlement on: (A) smooth surface, (B) Chanel micro pattern (5 µm width and height and spacing), (C) Sharklet (Scale bar 25 µm) (Carman et al., 2006)...... 20 Figure 2.11: SEM images of the gecko skin including a micro/nanostructure consisting of nano-hairs with a submicron spacing and a spike radius < 20 nm (A, B and C), (D and F) Interaction between bacteria (Porphyromonas gingivalis) and the nanopattern (Watson et al., 2015)...... 22 Figure 2.12: (A) The active number of active Streptococcus mutan bacteria (Gram-positive) on the gecko skin, replicated surface and Acrylic substrate, (B) The active number of Porphyromonas gingivalis (Gram- negative) bacteria gecko skin, replicated surface and Acrylic substrate (three and seven days exposure periods) (Li et al., 2016)...... 22 Figure 2.13: (A) SEM image of the interaction between Porphyromonas gingivalis bacteria and replicated nanostructure, (B) SEM images of Streptococcus mutan bacteria positioned between replicated nanostructures (Li et al., 2016)...... 23
x Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants
Figure 2.14: Four areas for nanopillars analysis on the dragonfly wing (Sympetrum vulgatum) (Rajendran et al., 2012)...... 23 Figure 2.15: (a) Nanopillars of Pantala flavescens and its contact angle, (b) biomimicked nanopillars via nano-imprint and its contact angle (Cho et al., 2013)...... 24 Figure 2.16: Bactericidal activity on hydrothermal etched titanium after 19 hrs for S. aureus (left) and P. aeruginosa (right). (Scale bar in SEM images is 200 nm and in confocal laser scanning microscopy (CLSM) is 10 µm) (Bhadra et al., 2015)...... 25 Figure 2.17: SEM image of the upper surface of (a) Fabricated nanopillars on black silicon and (b) dragonfly forewings (Scale bars: 200 nm) (Hasan et al., 2013)...... 26 Figure 2.18: Bactericidal efficiency of black silicon and dragonfly wings (Hasan et al., 2013)...... 26 Figure 2.19: SEM images of four cicada species and their wings. A: C. maculata, B: M. conica, C: M. microdon, D: T. jinpingensis). The SEM o topography surfaces in Aj, Bj, Cj, Dj were tilted 30 from those in A, B, C, D, respectively. (Scale bars: 1μm)...... 28 Figure 2.20: Penetration of nanopillars into P.aeruginosa (a) without coating, (b) with the gold coating (scale bar: 200 nm) (Ivanova et al., 2012)...... 29 Figure 2.21: SEM Top-view images of fixed Pseudomonas fluorescens cells on (a) M. intermedia, (c) C. aguila, and (e) A. spectabile cicada wings. (Scale Bars: 2 μm); AFM images from interaction (b) M. intermedia, (d) C. aguila, and (f) A. spectabile wings on P. fluorescens bacteria (Area is 3.2× 3.2 μm) (Kelleher et al., 2015)...... 30 Figure 2.22: Bacteria/surface interaction in (a) Gram-negative bacteria, (b) Gram-positive bacteria. Scale bars in SEM micrographs is 1µm and in CLSM is 5 µm (Hasan et al., 2013d)...... 31 Figure 2.23: The interaction of Gram-positive bacteria and nanopillars after microwave irradiation (Scale bars in SEM micrographs is 1 µm and in CLSM is 5 µm), dead bacteria are shown in red colour (right) in CLSM viability analysis (Pogodin et al., 2013)...... 32 Figure 2.24: P. aeruginosa interaction with (a) nanopillars on the control silicon wafer, (b) surface A diamond nanopillars on a silicon wafer (height:1.6µm, width = 350-750 nm), and (c) surface B diamond nanopillars on a silicon wafer with low height (100 nm), large height (3–5 µm) and width <100 nm to 1.2 µm nanocones (Fisher et al., 2016)...... 33 Figure 2.25: Interaction between bacteria and nanopillars of three cicada species wings with different pitch and diameter (Kelleher et al., 2015)...... 34 Figure 2.26: AFM micrographs and height profiles of bacteria (E. coli) on (a) cap diameter: 215 nm, centre to centre distance: 595 nm, base diameter: 380 nm, height: 300 nm, (b) cap diameter: 190 nm, centre to centre distance: 320 nm, base diameter: 130 nm, height: 300 nm (c) cap diameter: 70 nm, centre to centre distance: 170 nm, base diameter: 100
Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants xi
nm, height: 210 nm, and (d) flat film: Scale bar : 2 µm (Dickson et al., 2015)...... 35 Figure 2.27: Proposed mechanism of killing bacteria in the hair-like nanopillars of gecko skin. (A) Interaction between nanopillars and P.gingivalis, i: the adhesion area is not enough to penetrate ii: the adhesion contact area is enough for penetration (Li et al., 2016)...... 36 Figure 2.28: (a) Penetration and killing P. gingivalis bacteria by gecko replica nanopillars, (b) trapping the S. mutans bacteria and cellular damage, (c) the impairment mechanism through (i) compression and (ii) stretching (Li et al., 2016)...... 36 Figure 2.29: (a) Schematic of the NIL method, (b) fabricated silicon mold with electron beam lithography and nano-imprinted pattern with 10 nm hole (Guo, 2007, Rodríguez-Hernández and Cortajarena, 2015)...... 38 Figure 2.30: SEM image of (a) sample 1, (b) sample 2, and (c) sample 3 – tilt angle is 30o and the scale bar is 1µm (Dickson et al., 2015)...... 39 Figure 2.31: Schematic of UV-NIL (Cho et al., 2013)...... 39 Figure 2.32: SEM image of (a) top view of the dragonfly wing, (b) fabricated nanopillars of the dragonfly wing on glass using UV-NIL (Cho et al., 2013)...... 40 Figure 2.33: Micro-molding process (adapted from Kim, 2014)...... 40 Figure 2.34: Biomimicked nanopattern of sharkskin using (a) micro-molding, (b) NIL (Kim, 2014)...... 41 Figure 2.35: The SEM images of (a) sharkskin surface, (b) replicated sharkskin by miro-moulding (Liu and Li, 2012)...... 41 Figure 2.36: SEM images of replica sharkskin after flame treatment. It includes 300-500 nm submicron pattern and 20-50 nm silica particle (Liu and Li, 2012)...... 41 Figure 2.37: Top SEM images of (a) gecko skin and (b) replica gecko skin (Li et al., 2016)...... 42 Figure 2.38: Synthetic bio-replication of sharkskin process (Zhang et al., 2011). .... 42 Figure 2.39: Schematic of vacuum casting for sharkskin replication (Zhao et al., 2012)...... 43 Figure 2.40: Structure fabricated by femtosecond laser on AISI 304L (the same scale applies to all images) (Kietzig et al., 2009)...... 44 Figure 2.41: (a) P. aeruginosa cell attachment on fabricated titanium, (b) S. aureus cell attachment on fabricated titanium substrate (Fadeeva et al., 2011)...... 45 Figure 2.42: SEM images of (a) nanopattern fabricated with RIE in -200 V, (b) nanopattern fabricated with RIE in -150 V (Fisher et al., 2016)...... 46 Figure 2.43: Biomimicked nanopillars of dragonfly wings on silicon wafer (Hasan et al., 2015)...... 46 Figure 2.44: Schematic of photolithography...... 48
xii Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants
Figure 2.45: A: pillar (1.5-5 µm height, 5 µm width, 5-20 µm spacing), B: pit (5 µm height, 5 µm width, 5-20 µm spacing), C: channel (5 µm height, 5- 20 µm width, 5 µm spacing), D: ridges (1.5-5 µm height, 5 µm width, 5-20 µm spacing), and E: Sharklet or bioinspired surface of shark wing (4 µm height, 2 µm width spacing) (Carman et al., 2006)...... 49 Figure 2.46: Interaction volume difference among charged particle beams used for imaging (Hlawacek et al., 2014)...... 50 Figure 2.47: SEM images showing FIB milled nanopillars on GaN (Wu et al., 2008)...... 52 Figure 2.48: (a) Diffraction comparison between e-, He+ and Ne+ (GmbH, 2016)...... 53 Figure 2.49: (a) System of Orion NanoFab column and GFIS, (b) Source tip is formed by three atoms (trimer) emitting helium ions (Chen, 2010, Hlawacek et al., 2014, Hines and Wolf, 2016)...... 54 Figure 2.50: The principle of Electron Beam Lithography...... 56 Figure 2.51: SEM image of gold nanopillars fabricated by EBL, (a) cross section, (b) top view (Jindai et al., 2019)...... 57 Figure 3.1: Overall schematic of the research methodology...... 62 Figure 3.2: The cell membrane structure of (a) P. aeruginosa and (b) S. aureus and (Goldman and Green, 2008)...... 65 Figure 3.3: Spin coating speed versus film thickness for (a) PMMA 495 A4, (b) PMMA 950 A2 and PMMA 950 A4. (PMMA 495 A2/A6 and PMMA 950 A7 also can be applied to make different thicknesses) (MicroChem, 2011)...... 74 Figure 3.4: The fabrication process for biomimicked nanopillars using EBL...... 76 Figure 3.5: Schematic of the MIRA3 EBL system (FEG-SEM, 2013)...... 78 Figure 3.6: Electron scattering phenomena in EBL (Cheng, 2018)...... 80 Figure 3.7: Schematic of thermal deposition...... 82 Figure 3.8: DC sputtering operation principle in PVD 75 Kurt J. Lesker magnetron sputtering...... 83 Figure 3.9: E-beam evaporation system...... 83 Figure 3.10: Simple schematic of step coverage in the physical deposition method, (a) thermal evaporation, (b) E-beam evaporation, and (c) sputtering...... 84 Figure 4.1 Psaltoda claripennis (PC), Aleeta curvicosta (AC), and Palapsalta eyrei (PE) (scale bars = 1 cm) (left). HIM images of nanopillars of wing membranes from the top view and tilt angle 300 (middle). HIM top images of veins nanopillars (scale bars = 200 nm) (right) (Shahali et al., 2019)...... 88 Figure 4.2: Density of nanopillars (n/µm2) in veins and membranes for the three cicada species wings (Shahali et al., 2019)...... 89
Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants xiii
Figure 4.3: Aspect ratio of nanopillars in veins and membranes for the three cicada species wings (Shahali et al., 2019)...... 89 Figure 4.4: AFM image (left) and line profiles (right) of nanopillars of PC wings (scanning area: 2 µm × 2 µm) (Shahali et al., 2019)...... 90 Figure 4.5: AFM image (left) and line profiles (right) of nanopillars of AC wings (scanning area: 2 µm × 2 µm). (Shahali et al., 2019)...... 90 Figure 4.6: AFM image (left) and line profiles (right) of nanopillars of PE wings (scanning area: 2 µm × 2 µm) (Shahali et al., 2019)...... 90 Figure 4.7: TEM cross-section image of PC wing membranes (Shahali et al., 2019)...... 91 Figure 4.8: TEM cross-section image of AC wing membranes (Shahali et al., 2019)...... 91 Figure 4.9: TEM cross-section image of PE wing membranes (Shahali et al., 2019)...... 92 Figure 4.10: Chemical characteristics of cicada wings using FTIR (Shahali et al., 2019)...... 93 Figure 4.11: XPS analysis of the wing surfaces of the three cicada species (Shahali et al., 2019)...... 94 Figure 4.12: Force mapping on 2 μm × 2 μm of AC wing: (a) adhesion force map, (b) reduced modulus force map: (c) and (d) calculated reduced modulus, (d) Force cure on one point (Applied force is 5 Nn)...... 95 Figure 4.13: Force mapping on 2 μm × 2 μm of PC wing: (a) adhesion force map, (b) reduced modulus force map...... 95 Figure 4.14: Force mapping on 2 μm × 2 μm of PE wing: (a) adhesion force map, (b) reduced modulus force map...... 96 Figure 4.15: (A, B) SEM images of P. aeruginosa attachment on vein and membrane of PC after 18 hrs, (C, D) SEM images of S. aureus attachment on vein and membrane of PC after 18 hrs (left column: membrane, right column: vein, Scale bars = 1 µm) (Shahali et al., 2019)...... 97 Figure 4.16: (E, F) SEM images of P. aeruginosa attachment on vein and membrane of AC after 18 hrs, (G, H) SEM images of S. aureus attachment on vein and membrane of AC after 18 hrs (left column: membrane, right column: vein, Scale bars = 1 µm) (Shahali et al., 2019)...... 98 Figure 4.17: (I, J) SEM images of P. aeruginosa attachment on vein and membrane of PE after 18 hrs, (K, L) SEM images of S. aureus attachment on vein and membrane of PE after 18 hrs (left column: membrane, right column: vein, Scale bars = 1 µm) (Shahali et al., 2019)...... 99 Figure 4.18: Interaction of P. aeruginosa and the control surface (glass, unpatterned) after 18 hrs (Shahali et al., 2019)...... 99
xiv Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants
Figure 4.19: Interaction of S. aureus and control surface (glass, unpatterned) after 18 hrs (Shahali et al., 2019)...... 100 Figure 4.20: AFM image of the interaction between P. aeruginosa and nanopillars of PE after 18 hrs, (a) 2D AFM profile of punctured P. aeruginosa on nanopillars, (b) 3D AFM image of punctured P. aeruginosa on nanopillars, (c) Line profile for areas A, B, C...... 101 Figure 4.21: AFM analysis of P. aeruginosa on nanopillars and control surface after 18 hrs: (a) AFM analysis of penetrated bacteria and unpenetrated P. aeruginosa on wing nanopillars, (b) 3D AFM image of penetrated bacteria and unpenetrated P. aeruginosa on nanopillars, (c) AFM analysis of penetrated bacteria and unpenetrated P. aeruginosa on the control surface (glass), (d) AFM profile of line A, (e) AFM profile of line B, (f) AFM profile of line C...... 102 Figure 4.22: AFM analysis of S. aureus on nanopillars and control surface after 18 hrs: (a) AFM analysis of undamaged S. aureus on nanopillars, (b) AFM line profile (Line A), (c) punctured colony of S. aureus on nanopillars, (d) AFM line profile (Line B), (e) AFM analysis of S. aureus on the control surface (glass), (f) AFM line profile (Line C)...... 103 Figure 4.23: Colony-forming unit (bactericidal activity) of P. aeruginosa on control media (CM), control surface (glass), PC, AC and PE. (Statistically significant differences (p < 0.05) compared to CM, glass, PC and AC are indicated by the symbols +, *, φ and Ɵ, respectively)...... 104 Figure 4.24: Calculated colony-forming unit of P. aeruginosa on well plates for control media (CM), control surface (glass) and PE cicada wing for time intervals 0, 2, 4, and 18 hrs...... 105 Figure 4.25: Colony-forming unit (bactericidal activity) of S. aureus on control media (CM), control surface (glass), PC, AC and PE (Statistically significant differences (p < 0.05) compared to CM, glass, PC and AC are indicated by the symbols +, *, φ and Ɵ, respectively)...... 106 Figure 4.26: Calculated colony-forming unit of S. aureus on well plates for control media (CM), control surface (glass) and AC cicada wing for time intervals 0, 2, 4, 18 hrs...... 106 Figure 4.27: SEM image of osteoblast and PC wing membranes after 24 hrs (Scale bars, left = 10 µm, right = 2 µm) (Shahali et al., 2019)...... 108 Figure 4.28: SEM image of osteoblast and AC wing membranes after 24 hrs (Scale bars, left = 10 µm, right = 2 µm) (Shahali et al., 2019)...... 108 Figure 4.29: SEM image of osteoblast and AC wing membranes after 24 hrs (Scale bars, left = 10 µm, right = 2 µm) (Shahali et al., 2019)...... 108 Figure 4.30: AlamarBlueTM assay results of osteoblasts on control media (CM), glass, PC, AC and PE after 4 and 24 hrs (Statistically significant differences (p < 0.05) compared to CM and glass are shown by the symbols + and *, respectively) (Shahali et al., 2019)...... 109 Figure 5.1: Optimization flowchart of EBL (Yes: lift-off is desirable, and the result is repeatable and reliable, No: lift-off is not desirable and the
Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants xv
result is not repeatable and reliable, LMW: Low molecular weight and HMW: High molecular weight)...... 114 Figure 5.2: Monte Carlo simulation of electron PMMA (300 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and dot pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter 2.8 nm)...... 116 Figure 5.3: Monte Carlo simulation of electron PMMA (300 nm)/ Ti/ SiC scattering interaction during E-beam exposure at different accelerating voltages and circle pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter 2.8nm)...... 116 Figure 5.4: Monte Carlo simulation of electron PMMA (400 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and dot pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter: 2.8 nm)...... 117 Figure 5.5: Monte Carlo simulation of electron PMMA (400 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and circle pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter 2.8 nm)...... 118 Figure 5.6: Monte Carlo simulation of electron PMMA (500 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and dot pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter: 2.8 nm)...... 119 Figure 5.7: Monte Carlo simulation of electron PMMA (500 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and circle pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter 2.8 nm)...... 120 Figure 5.8: Monte Carlo simulation of electron PMMA (670 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and dot pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter 2.8 nm)...... 121 Figure 5.9: Monte Carlo simulation of electron PMMA (670nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and circle pattern simulated in CASINO v2.5.1.0. The blue lines indicate the forward scattered electron path while the red lines indicate the path of the backscattered electron (beam diameter 2.8 nm)...... 122 Figure 5.10: Monte Carlo simulation of electron PMMA (700 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and dot pattern simulated in CASINO v2.5.1.0. Blue lines
xvi Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants
indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter 2.8 nm)...... 123 Figure 5.11: Monte Carlo simulation of electron PMMA (700 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and circle pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron...... 124 Figure 5.12: One layer of PMMA 950 A4 on a silicon wafer coated by 30 nm titanium...... 125 Figure 5.13: Circle design pattern, centre to centre distance 160 nm, 4×5 Field, Field size 50 µm × 50 µm, the distance between field 50 µm, EF 0.5- 2.4...... 126 Figure 5.14: Dot design pattern, centre to centre distance 160 nm, 4×5 Field, Field size 50 µm × 50 µm, the distance between field 50 µm, EF 1-10.5...... 126 Figure 5.15: SEM images of the pattern after coating and lift off, circle pattern EF 0.5-2.4, (a) pattern and coating remain on the surface, (b) partial lift- off...... 127 Figure 5.16: SEM images of dot pattern after coating and lift off EF 0.5-2.4...... 127 Figure 5.17: Defects in primary EBL experiment: (a) Blistering and crack, (b) field overlapping ...... 128 Figure 5.18: Two and three-layers of PMMA 950 A4 on a silicon wafer coated by 30 nm titanium...... 129 Figure 5.19: Pattern design on two and three layers PMMA 950 A4: (a) Circle design pattern, centre to centre distance 160 nm, 4×5 Field, Field size 50 µm × 50 µm, distance between field 53 µm, EF 0.5-2.4, (b) Dot design pattern, centre to centre distance 160 nm, 4 × 5 Field, Field size 50 µm × 50 µm, distance between field 53 µm, EF 1-10.5...... 129 Figure 5.20: SEM images of the pattern after coating and lift off, (a) two layers of PMMA 950, (b) three layers of PMMA 950 A4...... 130 Figure 5.21: SEM images of the pattern after coating and lift off, (a) two layers of PMMA 950 with EF1.2, (b) three layers of PMMA 950 A4 with EF 1.5...... 130 Figure 5.22: One layer of PMMA 495 A4 and one layer PMMA 950 A4 on a silicon wafer coated by 30 nm titanium...... 132 Figure 5.23: Top view SEM image of the sample with one layer of PMMA 495 A4 and one layer of PMMA 950 A4 with an overall thickness of 494 nm after lift-off...... 133 Figure 5.24: Two layers of PMMA 495 A4 and one layer of PMMA 950 A4 on a silicon wafer coated by 30 nm titanium...... 133 Figure 5.25: Top SEM images of circle pattern after coating and lift off. (a): repeated pattern, (b) pattern number 1, (c) pattern number 3...... 134
Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants xvii
Figure 5.26: SEM image of the top view and tilt angle (45o) for EF of 1.3 with a diameter of 75 nm, a centre to centre distance of 160 nm, and a height of 100 nm...... 135 Figure 5.27: Circle design pattern, diameter 70 nm, centre to centre distance 160 nm, 6 × 10 Field, Field size 30 µm × 30 µm, the gap between field 5 µm at EF 1.3...... 136 Figure 5.28: SEM images of the pattern after coating and lift-off (a): 6 × 10 fields with EF of 1.3 after lift-off, (b) high magnification of nanopillars on one field (30 µm × 30 µm) with EF of 1.3...... 137 Figure 5.29: SEM image of nanopillars fabricated on two layers of PMMA 495 A4 and one layer PMMA 950 A2, EF of 1.3, lift-off after 200 nm Ti coating at 45-degree tilt...... 137 Figure 5.30: Two layers of PMMA 495 A4 and one layer of PMMA 950 A2 on Ti and SiC substrate...... 138 Figure 5.31: The circle pattern design with 70 nm circle, centre to centre distance 160 nm and EF0.1-2...... 139 Figure 5.32: SEM images of the pattern after coating and lift off. (a) repeated pattern, (b) write field with 1.6 EF, (c) write field with EF of 1.2...... 139 Figure 5.33: SEM image of the top view and tilt angle (45o) for EF of 1.3, diameter of 75 nm centre to centre distance of 160 nm and height of 100 nm...... 140 Figure 5.34: SEM image of top view for the fabricated pattern with EF of 1.3 with 20 × 20 fields...... 140 Figure 5.35: Three layers of PMMA 495 A4 and one layer of PMMA 950 A2 on Ti and SiC substrate...... 141 Figure 5.36: Design patterns for circle 70 nm, 120 nm and 320 nm according to Table 5.8 (dimensions in nanometres)...... 143 Figure 5.37: 70 nm circle pattern design with 110 nm and 160 nm centre to centre distance and EF of 1-2.1...... 143 Figure 5.38: 120 nm circle pattern design with 160 nm and 200 nm centre to centre distance and EF of 1-2.1...... 144 Figure 5.39: 200 nm circle pattern design with 320 nm and 260 nm centre to centre distance and EF of 1-2.1...... 144 Figure 5.40: (a) Top SEM image of fabricated pattern based on circle design diameter of 70 nm and centre to centre distance of 110 nm at the top and circle design diameter of 70 nm and centre to centre distance of 160 nm at the bottom with EF 1-2.1, (b) Top SEM image of fabricated nanopillars based on circle design diameter of 70 nm and centre to centre distance of 160 nm with EF of 2, (c) SEM image of fabricated nanopillars based on circle design diameter of 70 nm and centre to centre distance of 160 nm with EF of 2 with tilt angle of 45...... 145 Figure 5.41: (a) Top SEM image of fabricated pattern based on circle design diameter of 120 nm and centre to centre distance of 160 nm at the top and circle design diameter of 120 nm and centre to centre distance of
xviii Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants
200 nm at the bottom with EF 1-2.1, (b) Top SEM image of fabricated nanopillars based on circle design diameter of 120 nm and centre to centre distance of 200 nm with EF of 1.3, (c) SEM image of fabricated nanopillars based on circle design diameter of 120 nm and centre to centre distance of 200 nm with EF of 1.3 with tilt angle of 45...... 146 Figure 5.42: (a) Top SEM image of fabricated pattern based on circle design diameter of 200 nm and centre to centre distance of 260 nm at the top and circle design diameter of 200 nm and centre to centre distance of 320 nm at the bottom with EF 1-2.1, (b) Top SEM image of fabricated nanopillars based on circle design diameter of 200 nm and centre to centre distance of 320 nm with EF of 1, (c) SEM image of fabricated nanopillars based on circle design diameter of 200 nm and centre to centre distance of 320 nm with EF of 1 with tilt angle of 45...... 146 Figure 6.1: SEM image of the patterned titanium nanopillars (base diameter 94.4 ± 6 nm, top diameter 12.6 ± 2 nm, centre to centre distance 165.8 ± 5.6 nm, aspect ratio 2.16 ± 0.2): (a) Top SEM image, (b) 45 degree tilt SEM image...... 151 Figure 6.2: SEM image of the patterned titanium nanopillars (base diameter 148.6 ± 4.7 nm, top diameter 21.05 ± 3.6 nm, centre to centre distance 205.9 ± 4.7 nm, aspect ratio 2.35 ± 0.1): (a) Top SEM image, (b) 45 degree tilt SEM image...... 152 Figure 6.3: SEM image of the patterned titanium nanopillar (base diameter 214 ± 10 nm, top diameter 48.9 ± 7 nm, centre to centre distance 324.9 ± 6.9 nm, aspect ratio 2.19 ± 0.1): (a) Top SEM image, (b) 45 degree tilt SEM image...... 152 Figure 6.4: XPS spectrum of the titanium nanopillar surface...... 153 Figure 6.5: SEM images of titanium fabricated surface: (a) 0.5 mm × 0.5 mm composed of 20 × 20 fields of size 25 μm × 25 μm, (b) fabricated nanopillars from Ø70 nm, centre to centre distance 160 nm design in 25 μm × 25 μm, (c) fabricated nanopillars from Ø120 nm, centre to centre distance of 200 nm design in 25 μm × 25 μm, (d) fabricated nanopillars from Ø200 nm, centre to centre distance 320 nm design in 25 μm × 25 μm...... 154 Figure 6.6: Interaction of P.aeruginosa with fabricated nanopillars based on design Ø70 nm, centre to centre distance 160 nm and final geometry with base diameter 94.4 ± 6 nm, spike diameter 12.6 ± 2 nm, centre to centre distance 165.8 ± 5.6 nm and aspect ratio 2.16...... 155 Figure 6.7: Interaction of P.aeruginosa with fabricated nanopillar based on design Ø120 nm, centre to centre 200 nm and final geometry with base diameter 148.6 ± 4.7 nm, spike diameter 21.05 ± 3.6 nm, centre to centre distance 205.9 ± 4.7 nm and aspect ratio 2.35 ± 0.1...... 155 Figure 6.8: Interaction of P.aeruginosa with fabricated nanopillars based on design Ø200 nm, centre to centre distance 320 nm and final geometry with base diameter 214 ± 10 nm, spike diameter 48.9 ± 7 nm, centre to centre distance 324.9 ± 6.9 nm and aspect ratio 2.19...... 156
Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants xix
Figure 6.9: Interaction of S.aureus with fabricated nanopillars based on design Ø70 nm, centre to centre distance 160 nm and final geometry with base diameter 94.4 ± 6 nm, spike diameter 12.6 ± 2 nm, centre to centre distance 165.8 ± 5.6 nm and aspect ratio 2.16...... 156 Figure 6.10: Interaction of (a) P.aroginosa and (b) S.aures with the titanium control surface ...... 157 Figure 6.11: SEM image of P.aeruginosa interaction with fabricated nanopillars in tilt angle 45 degree based on design Ø70 nm, centre to centre 160 nm and final geometry with base diameter 94.4 ± 6 nm, spike diameter 12.6 ± 2 nm, centre to centre distance 165.8 ± 5.6 nm and aspect ratio 2.16. ... 157 Figure 6.12: SEM image of S.aureus interaction with fabricated nanopillars in tilt angle 45 degree based on design Ø70 nm, centre to centre distance of 160 nm and final geometry with base diameter 94.4 ± 6 nm, spike diameter 12.6 ± 2 nm, centre to centre distance 165.8 ± 5.6 nm and aspect ratio 2.16...... 158 Figure 6.13: Cellular metabolic activity of the osteoblast cells on fabricated titanium nanopillars and titanium control surface after time intervals of 1 and 3 days...... 158 Figure 6.14: SEM of osteoblast attachment on titanium nanopillars and titanium control surfaces: (a) SEM image of attached and well-spread osteoblast cells on titanium nanopillars in low magnification, (b,c) SEM image of attached and well-spread osteoblast cells on titanium nanopillars in high magnification, (d) SEM image of attached and well-spread osteoblast cells on the titanium control surface...... 160 Figure 7.1: Comparison of the nanopillar geometry on the membranes and the veins of Psaltoda claripennis (PC), Aleeta curvicosta (AC), and Palapsalta eyrei (PE)...... 165 Figure 7.2: Bacteria interaction with nanopillars on veins and membranes: (a) left, schematic of P. aeruginosa interaction with AC membrane and right, associated SEM image, (b) left, schematic of P. aeruginosa interaction with AC vein and right, associated SEM image, (c) left, schematic of S. aureus interaction with AC membrane and right, associated SEM image, (d) left, schematic of S. aureus interaction with AC vein and right, associated SEM image...... 173 Figure 7.3: Effect of the geometry of cicada nanopillars (on veins and membranes) on P. aeruginosa...... 174 Figure 7.4: Effect of the geometry of cicada nanopillars (on veins and membranes) on S. aureus...... 175 Figure 7.5: Effect of write field on pitch and spacing (note: spacing is a ratio and unitless)...... 179 Figure 7.6: Schematic of possible scenarios during electron beam lithography...... 181 Figure 7.7: Process optimization for nanopillar fabrication using electron beam lithography...... 182 Figure 7.8: Comparison of cicada nanopillars and titanium nanopillars and their bacteria interaction...... 183 xx Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants
List of Tables
Table 2.1: Nanopillars analysis including average height and diameter of four areas on the dragonfly wing (Rajendran et al., 2012)...... 23 Table 2.2: Surface characterization of the wings of 15 cicada species (Height values were calculated in 30-degree tilt angle). (Sun et al., 2009). (C.A. = contact angle)...... 28 Table 2.3: Geometry of cicada wing nanopillars and their contact angle (C.A) (Kelleher et al., 2015)...... 30 Table 2.4: Bactericidal activity in cicada wing, black silicon and dragonfly wing (Hasan et al., 2013). C.A. is contact angle...... 32 Table 2.5: Geometry of nanopillars fabricated by NIL (Dickson et al., 2015)...... 38 Table 2.6: Main imaging parameters and their definitions in HIM (Helium Ion Microscopy) (Hines and Wolf, 2016)...... 54 Table 2.7: Main patterning parameters and their definitions in He+-FIB milling (Hines and Wolf, 2016)...... 55 Table 2.8: Resolution, throughput, advantage and limitation of PBL methods (Shahali et al., 2019)...... 57 Table 3.1: Specification of the three cicada species used in the study...... 63 Table 3.2: Bacteria specification (Harrington, 2011, Harris and Richards, 2006, NCEZID, 2011)...... 65 Table 3.3: Common components, elements and wavenumbers of an insect wing (Aliofkhazraei, 2015, Mistry, 2009, Tobin et al., 2013)...... 67 Table 3.4: Main element of the wings and their binding energy...... 68 Table 3.5: Flood gun parameters used in Helium Ion Microscopy (HIM)...... 69 Table 3.6: Optimum parameter for imaging titanium nanopillars and nanopillars with cell interaction...... 69 Table 3.7: Standard operation procedure for biological samples in HIM, SEM and AFM...... 72 Table 3.8: TESCAN MIRA3 EBL system specifications (EBL, 2014)...... 78 Table 3.9: Fixed-Parameter and definition in TESCAN-EBL (EBL, 2014)...... 79 Table 3.10: Variable parameters in TESCAN-EBL (EBL, 2014)...... 79 Table 3.11: E-beam evaporation parameters for titanium deposition...... 84 Table 4.1: Nanopillar geometry of veins and membranes for three cicada species wings (Shahali et al., 2019)...... 88 Table 4.2: Surface roughness of the three cicada species wings and control surface analysed by AFM analysis (Shahali et al., 2019)...... 91
Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants xxi
Table 4.3: Atomic content and binding energy extracted by XPS spectra for the three cicada species wings (b.e: binding energy, a.c: atomic number) (Shahali et al., 2019)...... 94 Table 4.4: The average reduced Young's modulus for AC, PC, PE and glass...... 96 Table 5.1: Fixed parameter in one layer PMMA 950 A4...... 125 Table 5.2: EBL Fixed parameter in one layer PMMA 495 A4 and one layer of PMMA 950 A4 with an overall thickness of 494 nm...... 132 Table 5.3: EBL Fixed parameters in two layers of PMMA 495 A4 and one layer of 950 A4...... 134 Table 5.4: The fixed parameters in two-layer PMMA 495 A4 and one layer 950 A4...... 136 Table 5.5: Fixed parameters in two layers PMMA 495 A4 and one layer 950 A2...... 138 Table 5.6: Fabrication geometry of two layers PMMA 495 A4, one layer PMMA 950 A2 and two layers PMMA 495 A4 and one layer PMMA 950 A2. .... 140 Table 5.7: EBL Fixed parameters in three layers PMMA 495 A4 and one layer 950 A2...... 142 Table 5.8: EBL design pattern parameter...... 142 Table 5.9: Geometry of patterns and EBL result...... 147 Table 6.1: Optimum geometry of fabricated titanium nanopillars...... 151 Table 6.2: Atomic content and binding energy extracted by XPS for fabricated titanium nanopillars (b.e: binding energy, a.c: atomic number)...... 153 Table 7.1 summarized and compared the geometry of different species of cicada and dragonfly...... 167 Table 7.2: Comparing FTIR data and assigning vibration absorbance bands associated with compounds (vs: Symmetric and vas: antisymmetric C-H stretch absorbance)...... 169 Table 7.3: Comparison of bactericidal efficiency of natural nanopillar of dragonfly and cicada wings...... 171 Table 7.4: Comparison of nanopillars fabrication methods...... 178
xxii Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants
List of Abbreviations
AFM Atomic force microscope CFU Colony-forming unit CLSM Confocal laser scanning microscopy EPS Extracellular polymeric substances HIM Helium ion microscope IM Inner membrane OM Outer membrane PG Peptidoglycan PI Propidium iodide SE Secondary electron SEM Scanning electron microscope TEM Transmission electron microscope EBL Electron beam lithography PMMA Poly (methyl methacrylate) NMP N-Methyl-2-Pyrrolidone XPS X-ray photoelectron spectroscopy FTIR Fourier-transform infrared spectroscopy PC Psaltoda claripennis AC Aleeta curvicosta PE Palapsalta eyrei FIB Focused ion beam Ga Gallium He Helium Ne Neon S. aureus Staphylococcus aureus P. aeruginosa Pseudomonas aeruginosa RIE Reactive Ion Etching NIL Nanoimprint lithography UV-NIL UV Nanoimprint Lithography PL Photolithography
Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants xxiii
PBL Particle Beam lithography IPA Isopropyl alcohol MIBK Methyl isobutyl ketone NMP N-Methyl-2-pyrrolidone EBID Electron Beam Induced Deposition (EBID) DI Deionized water
xxiv Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants Statement of Original Authorship
The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other higher education institution. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made.
Signature: QUT Verified Signature
Date: 11/09/2020
Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants xxv Acknowledgements
I would like to acknowledge the Science and Engineering Faculty of Queensland University of Technology (QUT) for support through the QUT Higher Degree Research Tuition Fee Sponsorship and also the Australian Government Research Training Program Scholarship for its financial support.
My deepest gratitude goes to my supervisory team for their outstanding supervision, motivation, encouragement throughout and their assistance whenever required. My greatest appreciation goes to Prof. Prasad KDV Yarlagadda for guiding and helping me complete this project and thesis. I would like to thank my co- supervisors Prof. Hongxia Wang, Dr Tuquabo Tesfamichael and Prof. Cheng Yan for their guidance during the project.
I am also grateful to Dr Jafar Hassan and Dr Asha Mathews for their valuable support throughout my study and their excellent advice, making this research more effective and productive.
My deep appreciation goes to Dr Chris East, Dr Peter Hines and Dr Annalena Wolff and all staff members at the Central Analytical Research Facility (CARF) for their technical help and support to enhance my research quality.
I thank Dr Elliot Cheng for his support and provision of a training course for EBL in the Centre for Microscopy and Microanalysis (CMM). I also acknowledge the Australian National Fabrication Facility (QLD Node) facilities and the scientific and technical assistance of Dr Elena Taran. I also thank Dr Clare Morrison for editing and proofreading my thesis.
Finally, my sincere appreciation goes to my wife, mother, father and sister who supported and encouraged me throughout my whole PhD journey.
xxvi Assessment of the bactericidal effect of biomimicked nanopillars of cicada wings on titanium implants Chapter 1: Introduction
This chapter presents an introduction to the research and provides the background (Section 1.1) and research problem of the study (Section 1.2) followed by the aims and objectives (Section 1.3). The significance of the research is explained in Section 1.4. Summary of the chapter and the thesis outlines are presented in Section 1.5.
1.1 BACKGROUND
The surfaces of orthopaedic implants host human cells as well as pathogenic microorganisms like bacteria. Bacterial colonies attach to each other as well as substrate surfaces, then secrete Extracellular Polymeric Substances (EPS) and cause biofilm formation and implant infection (Costerton et al., 1999, Bandara et al., 2017a, Flemming et al., 2007). Once the biofilm is formed, the immune system and antibiotic therapy are no longer effective in eliminating the infection. The infection leads to inflammation, implant failure and revision surgery, which impose significant cost and intense patient pain (Dickson et al., 2015a, Ercan et al., 2011). Each treatment of infected arthroplasty costs more than $50,000 (Berbari et al., 1998). In the USA, the annual cost of infected revision jumped from $320 million to $566 million during 2001 to 2009 and it is expected to increase to $1.62 billion by 2020 (M. Kurtz, 2012). In the United Kingdom, infectious implants cost 7-11 million Euros per year (Harris and Richards, 2006). Due to the intense pain and expensive cost for patients, it is important to reduce or eliminate the biofilm formation from implants because it helps the host tissue cells preserve the implant surface from bacterial colonization. Bacterial infections associated with orthopaedic and dental implants are the priority of current research (Arciola et al., 2018, Franz et al., 2011, Gittens et al., 2014, Jaggessar et al., 2017, Shahali et al., 2017). The economic significance of biofilm formation is not only limited to the biomedical and health industry. Oil and gas industries spend approximately one-third of their corrosion-associated costs on preventing microbial corrosion while Marine spends 3 billion USD per year on antibiofouling projects (de Carvalho, 2018, Shahali et al., 2019).
Chapter 1: Introduction 1
In order to reduce and eliminate bacterial adherence and biofilm formation on surfaces, many attempts have been made to produce antibacterial surfaces (Bandara et al., 2017a, Hasan, 2013, Shahali et al., 2019). Traditional methods like antibiotic coating and chemical coating (e.g. Ag, Au and Cu), E-beam evaporation, sputtering, plasma spray, electrochemical, and anodization have been applied to produce antibacterial surfaces in titanium implants. These methods do not possess long term effects and they are not effective once the resistant biofilm layer is formed (Hasan and Chatterjee, 2015, Hasan et al., 2013). The main problems related to the coating method is bio-resorption and delamination on implants (Ferraris and Spriano, 2016). Bioengineers and scientists have looked to create potential intrinsic surfaces which have long-lasting antibacterial effects (Shahali et al., 2019b, Bandara et al., 2017a, Hasan et al., 2015, Watson et al., 2017). Flora and fauna surfaces are at the centre of scientist’s attention for producing durable antibacterial surfaces. Surfaces like lotus (Cheng et al., 2006) and taro leaf (Ma et al., 2011) surfaces and sharkskin(Kim, 2014)produce antibiofouling effects because of their micro/nano superhydrophobic structure. Nanopillars on dragonfly (Ivanova et al., 2013)and cicada wings and gecko skin deactivate and disturb bacteria cells in contact with nanopillars through the mechanical interaction mechanism. Cicada wings possess uniform and well-organized nanopillars compared to dragonfly wings which aid the fabrication of nanopillars (Ivanova et al., 2012). This structure also helps in studying the precise correlation between the geometry of nanopillars and bacteria killing. The natural and biomimicked surface of cicada wings has superhydrophobic, self-cleaning and bactericidal capabilities because it is covered by an array of nanopillars with a top diameter of 60 nm, base diameter of 100 nm, centre to centre distance of 170 nm and height of 200 nm (Ivanova et al., 2012b, Pogodin et al., 2013). It is claimed that the mechanism of killing bacteria is based on mechanical rupture or penetration which occurs between nanopillars and bacterial membranes and it takes minutes to kill the bacteria (Ivanova et al., 2012). The surface of the implant and biomedical devices should possess both bactericidal effect and cytocompatibility before implant into the human body (Hasan et al., 2017a, Jaggessar et al., 2017). Therefore, biomimicked nanopillars also necessitate having ideal human cell proliferation, cytocompatibility plus bactericidal effect.
2 Chapter 1: Introduction
The throughput, resolution (fabrication of features less than 100 nm) and material are key parameters when selecting the best fabrication method and design strategy for mimicking the cicada wing nanopillar on titanium. High throughput fabrication methods, like hydrothermal (Jaggessar et al., 2020) and reactive ion etching (Ghosh et al., 2019), can only produce random nanopillars and even when controlling the process parameters, fabrication accuracy is a big challenge. Early research on the fabrication of biomimicked nanopillars shows that replication methods like vacuum casting and nanoimprint lithography produce high throughput, but they are limited to soft material like polymers and resins and the resolution remains a challenge. Among the fabrication methods, Particle Beam Lithography (PBL) including Electron Beam Lithography (EBL) and Focused Ion Beam lithography (FIB) can fabricate high- resolution titanium nanopillars but their throughputs are less than hydrothermal and reactive ion etching. This research has focused on the surface characteristics of three cicada species’ wings including both vein and membrane, bactericidal effect against Staphylococcus aureus and Pseudomonas aeruginosa and cytocompatibility with human osteoblasts. In order to mimic the cicada nanopillars with optimum bactericidal effect, in-depth analysis of nano topography and surface characteristics of the cicada wing was crucial. Helium ion microscopy (HIM), Transmission electron microscopy (TEM), Scanning electron microscopy (SEM) and Atomic Force Microscopy (AFM) were used to analyse the geometry of cicada nanopillars (e.g. shape, spike diameter, base diameter, height, aspect ratio, density). AFM was employed to identify the mechanical properties of nanopillars. X-ray photoelectron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FTIR) were also performed to ensure that no chemical reactions were involved in the bactericidal effects. Bactericidal efficiency of cicada wings against S. aureus and P. aeruginosa was evaluated by plate counting methods followed by the analysis of bacterium interactions using AFM and SEM analysis. Cytocompatibility of cicada wings to osteoblast human cells was carried out by AlamarBlueTM assay. After analysis of the wing nanopillars and their bactericidal effect, the optimum nanopillar geometry was selected for the design strategy of fabrication. EBL was applied to mimic titanium nanopillars through the systematic approach and Monte Carlo simulations were used to simulate the beam interaction and optimize the design geometry and beam energy, saving time and money prior to the experimental stage. A
Chapter 1: Introduction 3
systematic experimental approach optimized the process variables including exposure factor )EF), write field and pitch. Finally, the bacteria interactions and cytocompatibility of the biomimicked nanopillars were analysed by AlamarBlueTM assay and SEM, respectively. This research is significant for its in-depth analysis of three cicada species wings and establishing a systematic method to mimic the cicada wing nanopillars on titanium to reduce orthopaedic implant infection, implant failure, revision surgery and enhance biocompatibility. Recent research is limited to random metallic nanopillars with limited resolution and soft material like polymer and resin. This research provides the versatile design of titanium nanopillars for long-lasting effect in controlling bacteria attachment, preventing biofilm formation and cytocompatibility with human cells. The research outcomes will reduce patient pain, the effects of antibiotic therapy, hospital stay time and the cost of invasive and revision surgeries. It also will provide optimum design solutions for manufacturing antibacterial and biocompatible nanopillar surfaces used in biomedical devices such as orthopaedic implants.
1.2 RESEARCH PROBLEM
Infection is one of the main reasons for implant failure, resulting in pain and stress for the patient, prolonging antibiotic therapy, increasing time and cost of hospitalization and revision surgery (Shahali et al., 2019). Infection in orthopaedic implants is caused by bacteria attachment that forms a biofilm in the interaction of the implant surface with tissue, leading to inflammatory problems for joints and bones. Implantation of titanium in the human body is always associated with the risk of infection, so the antibacterial effect is vital for implant surfaces in interactions with bacteria and biofilm. However, while surface modification methods based on coating and antibiotic therapy can produce antibacterial surfaces, they may have side effects, particularly during long periods, such as bio-resorption and delamination for coating and antibiotic-resistant bacterial strains for antibiotic therapy. Natural antibacterial surfaces, like cicada wings, have motivated scientists to produce long-lasting bactericidal surfaces because the nanopillars of the cicada wing can penetrate the bacterial membrane, killing the bacteria through mechanical rather than chemical processes. Existing research is mainly focused on natural insect wings and is limited to materials like silicon and polymer, all of which have resolution limits. These limits
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and problems highlight the requirement for a new systematic, in-depth assessment of the natural surface of cicada wings, followed by biomimicking of the nanopillar arrays on titanium with a high-resolution method. The titanium biomimicked nanopillars can be used in future biomedical devices and titanium implants.
1.3 AIMS AND OBJECTIVES
The main aim of this research is to evaluate the bactericidal effect and cytocompatibility of biomimicked titanium nanopillars for biomedical implants. This aim would be achieved by undertaking the following two research objectives: 1. Investigation of the surface nanotopography, chemical characteristics, mechanical properties, bacteria interaction, bactericidal efficiency (against P. aeruginosa and S. aureus) and cytocompatibility (in response to human osteoblast cells) of three cicada species wings. 2. Mimic the cicada wing nanopillars to maximize the bactericidal effect and cytocompatibility on titanium by systematically optimizing the fabrication process to achieve the closest geometry to cicada nanopillars. Optimization is based on surface characteristics, mechanical properties, bacteria interaction and cytocompatibility of the mimicked titanium nanopillars.
1.4 SIGNIFICANCE AND OUTCOMES
This research will significantly enhance the in-depth knowledge of the effect of nanopillar topography on cell interactions as well as bactericidal and biocompatibility of the cicada wing. The results will enable the design and fabrication of titanium biomimicked nanopillars with the same bactericidal and cytocompatibility levels as natural cicada nanopillars. This research also develops the systematic study of fabrication methods allowing optimisation of the process variables and versatile design of nanopillars for various applications, especially orthopaedic applications. The output of this research will help to fabricate a long-lasting bactericidal surface for medical devices, like orthopaedic implants, helping reduce patient pain, the side effects of antibiotic therapy, hospital time and the cost of invasive and revision surgery.
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1.5 THESIS OUTLINE
Chapter 1 starts with an introduction to the research background and literature review followed by the research problem, aims and objectives. The significance and outcomes of the research and its application in the field of biomedical devices are described. Chapter 2 provides a comprehensive literature review based on recently published data. This chapter starts with the introduction of infection and biofilm formation on orthopaedic implants and biocompatibility of titanium implants. Then, the chapter introduces traditional antibacterial coating and surface modification including antibiotic releasing coating, inorganic antibacterial coating and their associated disadvantages and drawbacks. Subsequently, this chapter presents the latest research on natural antibacterial surfaces (taro and lotus leaves, shark and gecko skin and dragonfly and cicada wings). This is followed by a review of nanofabrication methods to mimic natural nanostructures to identify the ideal method for fabrication of cicada nanopillars. Finally, the chapter outlines the research gaps found in the literature. Chapter 3 presents the research methodology design to achieve the research aims and objectives. The chapter then discusses the materials and methods used including surface characteristics methods, bactericidal efficiency and bacteria interaction analysis methods, cytocompatibility analysis of cicada wings and biomimicked titanium nanopillars and finally, a detailed description of nanofabrication methods to mimic the cicada nanopillars on titanium. Chapter 4 demonstrates the results of the nano topography analysis, chemical characteristics, mechanical properties, bactericidal efficiency (against P. aeruginosa and S. aureus) and bacteria interactions of three cicada species wings. This chapter also discusses the results of the cytocompatibility of wings in response to human osteoblast cells and compares them with control surfaces. Finally, this chapter introduces the optimum geometry of natural nanopillar arrays with high bactericidal effects and biocompatibility. Chapter 5 provides the systematic method for mimicking the cicada nanopillars (based on Chapter 4) on titanium substrate using Electron Beam Lithography (EBL). This chapter includes the optimization flowchart of EBL, Monte Carlo simulation to identify the beam energy and design profile, and a detailed description of the
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experimental approach to optimize the process variables of EBL and optimum titanium fabricated nanopillars. Chapter 6 evaluates bacteria interaction and biocompatibility of the biomimicked titanium nanopillars. Chapter 7 presents the detailed results and discussions based on Chapters 4, 5 and 6 and how they relate to recently published research. Chapter 8 provides the conclusions and makes recommendations for future work.
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Chapter 2: Literature Review
2.1 OVERVIEW
The chapter begins with an introduction to bacterial infection, biofilm formation, related costs and biocompatibility of titanium orthopaedic implants. This includes the importance of antibacterial surfaces on implants and criticism of the traditional methods for antibacterial coating and surface modification. This chapter also evaluates and analyses natural antibacterial surfaces (e.g. taro leaves, lotus leaves, gecko skin, sharkskin, dragonfly wings and cicada wings) and bioinspired surfaces as well as the interface of nanostructures and bacteria. This chapter assesses the capability of current fabrication techniques for producing natural antibacterial structures on different materials. Finally, the chapter highlights the knowledge gaps in the literature in relation to appropriate methodology.
2.2 INFECTION AND BIOFILM FORMATION ON ORTHOPAEDIC IMPLANTS
2.2.1 Bacterial infection Infection is one of the principal issues in orthopaedics implants (e.g. hip and knee), resulting in inflammation, implant failure and revision surgery (Ercan et al., 2011). Implantation of titanium in the human body is connected with the risk of infection, especially for bone fracture fixation and revision surgery (Sinha, 2002, Gustilo et al., 1990). Inflammatory problems in orthopaedic replacement comprise 50% of chronic disease in people more than 50 years old in developed countries (Ribeiro et al., 2012). The annual rate of revision hip replacement is 37,000 people in the US with hospital expenses for each person of $31,000 (Katz et al., 2007). Between 1993-2004, the amount of revision and primary total hip replacements grew 50% in the US (Katz et al., 2007). Infection rates after hip replacements can increase from 0.5- 1% to 5% over 10 years because the infection rate grows over time (Unosson, 2015). Primary hip surgery takes only three hrs while revision hip surgery prolongs the surgery time to five hrs, leading to loss of 2 L of blood instead of 1.5 L, increasing hospitalization time from one week to one month, and costs of $100,000 instead of $20,000 (Bozic and Ries, 2005). The infection rate for a closed fracture is 1.5 %, while
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it is 3 to 40% for open fractures which is attributed to bacterial contamination during the surgery (Harris and Richards, 2006).
2.2.2 Biofilm formation Microorganisms like bacteria are old, uninvited guests on our planet from a long time ago and over time have established various behaviours for settlement on surfaces (Hasan et al., 2013). Bacterial attachment is the primary cause of implant infection and the process of bacteria attachment is complicated and affected by environment, bacterial characteristics, material and surface properties and tissue cells. Surface characteristics like chemical composition, surface roughness, surface structure, hydrophobicity and the presence of a particular protein on the surface can influence the attachment process (Ribeiro et al., 2012). The bacteria infection in implants can be attributed to the operation room environment, surgical tools, medical devices and bacteria attachment on patient skin and body infections (Chevalier and Gremillard, 2009). Biofilm formation initiates after microorganisms like bacteria are placed on implant surfaces. Microorganisms attach to each other and the surface with a sticky substance named Extracellular Polymeric Substance (EPS). The biofilm keeps growing and forming three-dimensional structures by further attachment. Biofilm can adhere to a regular hard substrate like glass (Zobell, 1943), and can also attach to critical hard substrates used in implants, marine applications and food industries (Bixler et al., 2014). The surfaces of implants can host human cells as well as pathogenic microorganisms like bacteria. When the bacterial colonies are attached to each other and the substrate, the body’s immune system cannot respond effectively. As illustrated in Figure 2.1, formation of biofilm involves five stages: Stage 1: Floating bacteria attach to the substrate. Stage 2: Accumulated bacteria cells form microcolonies and EPS. This irreversible adherence is generated as EPS is secreted. EPS is an insoluble and sticky substance which is secreted by bacteria cells, surrounding and covering many attached cells in the matrix. The EPS matrix has a high amount of nutrients, which is an ideal place for growing cells, and can protect cells from the external environment (Veerachamy et al., 2014).
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Figure 2.2 illustrates the cross-section of the biofilm and highlights the EPS morphology. Stage 3: Mature biofilm is formed from a multilayer of cells. Stage 4: Biofilm accumulates to form 3D growth. At this stage, the immune system and antibiotic therapy are not effective. Stage 5: The biofilm reaches maximum mass and floating bacteria will scatter and become ready to settle on other surfaces (Unosson, 2015).
Figure 2.1: Schematic of the five stages of biofilm formation (adapted from D.Davis) (Bixler et al., 2014).
Figure 2.2: Biofilm SEM cross-sectional image representing the EPS morphology (Bixler and Bhushan, 2012).
Staphylococcus aureus and Pseudomonas aeruginosa are active biofilm formers and are resistant to antibiotic therapy on titanium orthopaedic implants (Veerachamy et al., 2014). Staphylococcus encompasses up to 66% of all pathogens in infections of the orthopaedic implant (Ribeiro et al., 2012). Figure 2.3 (a, b and c) shows biofilm formation on different medical devices. In conclusion, it is important to reduce and eliminate biofilm formation because it helps the host tissue cell to keep the implant surface free from bacteria colonization.
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(a) (b) (c) Figure 2.3: Formation of biofilm by Staphylococcus aureus on (a) Surface of a catheter (Chung and Toh, 2014), (b) Patient ventilation tube and (c) Patient Pacemaker (Bixler and Bhushan, 2012).
2.3 BIOCOMPATIBILITY AND OSSEOINTEGRATION OF TITANIUM IMPLANTS
Biocompatibility is a general technical term in biomaterial and biomedical devices. It refers to the ability of biomaterial to provide effective harmonic behaviour or biological performance in contact with human cell tissues and body fluids (Black, 2005). Considering the current focus on biomaterials for tissue engineering, complex cell, drug and gene delivery, Williams (2008) decided to change the definition of biocompatibility to the capability of biomaterial to implement its desirable function with respect to medical therapy (e.g. gene and drug delivery, etc.), without undesirable local and systematic influence from therapy but producing the most effective response from living cells and tissues (Williams, 2008). Implants should be non-toxic and not cause infection. The success of implants is essentially evaluated by the response of the human body to the implant. Two main parameters that have a great impact on biocompatibility are “host response” which is caused by materials and “material degradation” in the human body. In orthopaedic implants, osseointegration shows the ability of the implant surface to interact with the surrounding bone tissue. Chemical composition, surface morphology and characteristics are the most vital factors in the development of ideal osseointegration (Black, 2005, Geetha et al., 2009). Orthopaedic titanium implants must have ideal surface and structural compatibility with host tissue (Ribeiro et al., 2012). Titanium and its alloys are mostly employed for non-bearing surface parts rather than metals because of their remarkable corrosion resistance as well as their similar stiffness to the bone. They are manufactured with a porous structure, causing less stress shielding of bone compared with other materials (Ribeiro et al., 2012). This research assessed the antibacterial characteristics of biomimicked titanium surfaces and cytocompatibility of the antibacterial surface in interactions with human
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osteoblast cells as a main cellular element of bone. These assessments enabled achievement of the ideal titanium implant surface with long-lasting bactericidal effects and good biocompatibility.
2.4 TRADITIONAL ANTIBACTERIAL COATING AND SURFACE MODIFICATION
To reduce the rate of implant infections and failure and increase biocompatibility, coating methods and surface modifications can be applied to implant surfaces. An antibacterial surface on titanium implants can be achieved by antibiotic releasing coating and antibacterial surface modification (Odekerken et al., 2013). Antibacterial surface modification can be created in three ways: 1. Coating the surface with inorganic antibacterial agents without applying a layer (e.g. ion implantation, alloy).
2. Developing a TiO2 layer followed by loading the surface with inorganic antibacterial agents (e.g. ion beam assistant deposition, plasma electrolytic oxidation, anodic spark deposition).
3. Depositing TiO2 or hydroxyapatite and loading surfaces with an inorganic antibacterial agent (e.g. plasma spray, sputtering) (Ferraris and Spriano, 2016). In the next section, relevant antibacterial methods are discussed and critiqued in detail.
2.4.1 Antibiotic releasing coating In this method, different antibiotics, including gentamicin, vancomycin, rifampicin and tobramycin, are applied to orthopaedic implants. Mixing hydroxyapatite and tobramycin generates a good antibacterial effect as well as osseointegration (Moojen et al., 2009). One of the methods for preventing infection in implants is combining antibiotics in PMMA (Poly (methyl methacrylate)) bone cement. In antibiotic-loaded PMMA filler, the antibiotic is moderately released to transfer a more local focus and this method can be applied in both primary and revision surgery (Belt et al., 2001). The disadvantages of these methods are the rising proportion of antibiotic-resistant bacterial strains over time, slow leaching times, lack of delivery concentration and poor durability. Moreover, antibacterial therapy loses its
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effectiveness when biofilm forms on the surface (Kelleher et al., 2015b, Odekerken et al., 2013).
2.4.2 Inorganic antibacterial coating The most popular inorganic antibacterial agents on titanium implant surfaces are Ag, Cu and Zn (Ferraris and Spriano, 2016). Ionic states of Ag, Cu and Zn ( Ag+, Cu2+ and Zn2+) easily react with amines and bacteria DNA to avert bacteria reproduction as well as inactivation (Odekerken et al., 2013). Silver is a potential antibacterial metal which is used in medical applications and is applied to the titanium substrate through ion implantation, electrochemical methods, synthesis of nanoparticles, sputtering, plasma spray and alloys (Ferraris and Spriano, 2016, Odekerken et al., 2013). Li enhanced the antibacterial properties of the porous structure of titanium with silver loaded gelatin microspheres. First, the porous titanium is manufactured by powder metallurgy. Then, microanodization is applied and finally, gelatin microspheres are synthesized in a water-in-oil emulsion. The silver-loaded specimens possessed great antibacterial capability against both E. coli and S. aureus bacteria (Li et al., 2016) Generally, inorganic coatings like silver, do not produce antibacterial effectiveness in vivo because preclinical investigation and external fixation implants cannot evaluate the efficiency of silver coating (Odekerken et al., 2013).
2.4.3 Electron beam evaporation, electrochemical etching and anodization E-beam (electron beam) evaporation is used as a coating process in which a layer of the antibacterial surface is deposited on the substrate. Puckett et al. compared the antibacterial effect of rough titanium nanostructures produced by E-beam evaporation and two different anodization processes (first anodization process: 1 min in 0.5% HF at 20 V, second anodization process: 10 min in 1.5% HF at 20 V) against Gram- negative and positive bacteria. The E-beam evaporated nanostructures reduced the attachment of both bacteria compared to other surfaces. Rough titanium and E-beam evaporated nanostructures have anatase and rutile phases of crystalline TiO2 while the anodized surface is amorphous TiO2. Amorphous TiO2 enhanced bacteria adherence compared to anatase TiO2 (Puckett et al., 2010).
S-H. Uhm et al. fabricated antibacterial nanoparticles on TiO2 nanotubes. Nanotube diameter was controlled by the voltage of the anodization process from 70-
Chapter 2: Literature Review 13
100 nm diameter. They found that silver nanoparticles on the larger diameter of the nanotube are not cytotoxic and produce a better antibacterial feature appropriate for bio-implants (Uhm et al., 2013). Electrochemical etching and anodization can fabricate different types of nanostructured surfaces. Liang et al. produced antibacterial hierarchical cell-sized micro-hole arrays mixed with a nanopattern structure using electrochemical etching. Two-stage acid etching was applied to increase the roughness and finally, an anodization process was applied in the electrolyte to generate a nanostructure porous titanium layer at the top of the microstructure (Liang et al., 2015).
2.4.4 Drawbacks of traditional antibacterial method and surface modification Considering the aforementioned methods and their capabilities for creating an antibacterial surface on Ti implants, the following disadvantages are: 1. Accumulating nanoparticles in tissue can generate toxic build-up which cannot be easily eliminated by white blood cells and cleared in a physiological way. 2. Chemical coating or immobilizing has short time activity on the surface (Li et al., 2016). 3. Thin layer antibacterial coating should not be worn down in interactions with human tissue, resulting in low durability. Moreover, the main problem related to the coating method is bio-resorption and delamination on implants (Dong et al., 2011, Ferraris and Spriano, 2016). 4. The inorganic coating does not show antibacterial effectiveness in vivo because preclinical investigations and external fixation implants cannot evaluate the efficiency of coating (Odekerken et al., 2013). While traditional antibacterial methods and surface modification can produce an antibacterial effect, their drawbacks necessitate focusing on natural antibacterial and bioinspired surfaces as reliable and long-lasting antibacterial surfaces.
2.5 NATURAL ANTIBACTERIAL AND BIOMIMICKED SURFACES
Natural surfaces demonstrate an ever-increasing source of motivation for scholars to mimic antibacterial surfaces because it is believed that antibacterial biomimicked surfaces have a long-lasting effect (Ivanova et al., 2012). Natural surfaces can reduce the attachment and proliferation rate of algal spores, particles and
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bacteria and can be classified into antibiofouling surfaces and bactericidal surfaces. In antibiofouling surfaces (e.g. lotus leaves, taro leaves and sharkskin) bacteria cells are repelled from attachment mainly because of the chemical composition and micro/nano superhydrophobic structure. Bactericidal surfaces, like dragonfly wings, cicada wings and gecko skin deactivate, disturb and kill bacteria cells in contact with nanostructures like nanopillars through mechanical mechanisms (Hasan et al., 2013). This section evaluates the natural antibacterial surfaces (biofouling and bactericidal) as well as biomimicked surfaces.
2.5.1 Taro and lotus leaves
Some plant leaves possess antibiofouling, and self-cleaning abilities because of their micro and nanopatterns (Ma et al., 2011). The hydrophobic surface of plant leaves, like taro and lotus, is composed of a hydrophobic layer with a high density of nanoscale wax crystals that are covered with convex microstructures, causing a high contact angle (C.A>150o) on the surface. Therefore, bacteria can easily adhere to the nearest water droplet on the surface and be cleared away from the surface when the drop detaches from the surface (Barthlott and Neinhuis, 1997, Ma et al., 2011). The hydrophobic surface has a contact angle higher than 120o while the superhydrophobic surface has a contact angle higher than 150o. In underwater conditions, the air is trapped among nanopatterns so this mechanism needs air which is trapped among the nanopattern (Cassie and Baxter, 1944). Figure 2.4 shows the topography of the taro leaf surface. This figure shows the polygon shape of cells constituting a bulge of 15 to 30 μm diameter associated with a papilla at the middle with 10-15μm diameter. The cell surface is capped with nanoscale wax crystals. The crystal structure is an important player in the self-cleaning procedure of taro and lotus leaves. This nanocrystal distribution is not homogenous, meaning that maximum density is on the papilla and minimum density exists at the edge of every cell or between cells.
Chapter 2: Literature Review 15
Figure 2.4: SEM image of taro leaves. (a) liquid substitution and (b) air-dry and sputter coating (Ma et al., 2011).
The nanopatterns on taro leaf show resistance to particle and bacteria attachment in submerged conditions and can be divided into high density and low density. The high density nanopatterns can reduce bacteria adherence under water in comparison with low-density nanopatterns. Figure 2.5 shows the influence of nanostructure density on the level of adhesion. Trapped air among nanopatterns has an active role in preventing bacteria and particle attachment. When the nanopattern of taro leaf is entirely wet, it has a repelling effect for both bacteria and particles (Ma et al., 2011). The higher density nanocrystal produces more resistance to P. aeruginosa, meaning that lower bacteria adhesion in wet conditions could be observed in nanopatterns with higher density (Ma et al., 2011).
Figure 2.5: Relationship between nanostructure density of taro leaves and bacteria adhesion (Ma et al., 2011).
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Although micro/nanostructures of taro leaves have an antibiofouling effect by preventing attachment of bacteria on the surface, it does not have a bactericidal effect to kill the bacteria in the surrounding environment. The antibiofouling and self-cleaning effect of the lotus leaf (e.g. Nelumbo nucifera) is called the "Lotus effect” and has been the subject of intensive research. Self-cleaning paint and cloths and bio-repellent coating are applications of the lotus leaf in human life (Ma and Hill, 2006, Patankar, 2004). The superhydrophobicity of the lotus leaf is associated with the micro/nanostructure of the surface. When a bead of water is placed on the lotus leaf with a high contact angle, it immediately rolls off the surface, gathering up the contamination and germs. The lotus leaf structure is composed of a micro-size bulge shaped pattern which sticks out of the leaf surface and nanocrystal structure that covers the entire surface. Figure 2.6 shows the two classes of lotus leaf surface, including the micro-size bulge shaped pattern (Figure 2.6 (a)) and the nanocrystal structure (Figure 2.6 (c)) (Cheng et al., 2006). Cheng produced the self-cleaning characteristics of the micro/nanostructure were examined by separately considering untreated (Figure 2.6 (a,c)) and annealed lotus leaf (Figure 2.6 (b,d). Annealing (1 hr in 150oC) eliminates all nanocrystals whereas micro-bulges (5-10 µm height) remain on the surface. Untreated surface (micro/nanostructure) is superhydrophobic with a water contact angle of 142.4o ± 8.6o compared to treated lotus leaf with 126.3o and smooth wax surface with 74o (Cheng et al., 2006).
Figure 2.6: SEM image of lotus leaf: (a) and (c) untreated micro-size bulge shaped pattern plus nanocrystal with different magnification, (b) and (d) treated micro-size bulge shaped pattern (Cheng et al., 2006).
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The results show that the micro-size bulge-shaped pattern is more effective in increasing water C.A with 70%, however, the share of the nanocrystal increase in hydrophobicity is only 13%. On the annealed dried surface, the droplet becomes sticky, which shows that the nanocrystal is vital for the self-cleaning effect as the sticky droplet cannot be avoided by nanocrystals in high condensation of water on the surface (Cheng et al., 2006). In summary, even though the nanostructure of the lotus leaf is superhydrophobic with antibiofouling effect, it can only repel particles and bacteria rather than kill them.
2.5.2 Sharkskin Sharkskin possesses antibiofouling and drag reduction due to its 3D riblet microstructure. This microstructure provides swimming speed of 90 km/h (Pu et al., 2016) and enhances the antibiofouling effect to decrease bacterial and microorganism colonization, especially those that are larger than the gap between riblets (Bixler and Bhushan, 2013, Kim, 2014). These exceptional surface characteristics of sharkskin distinguish it from other species like whales that are covered by tiny barnacles. (Bixler and Bhushan, 2013). Figure 2.7 shows the self-cleaning effect of sharkskin compared to whale skin. The size, shape and number of riblets differ among species (Bixler and Bhushan, 2013). Figure 2.8 illustrates the riblet structure among different shark species. For example, Spiny Dogfish (mud shark) skin consists of riblets with a width of 100-300 µm, radius of 15 µm, height of 200-500 nm and 100-300 µm distance between riblets (Jung and Bhushan, 2009).
Figure 2.7: Comparison of whale (left) and shark (right) skin (Bixler and Bhushan, 2013).
The sharkskin covered by micro riblets can reduce the friction in turbulent water flow through drag reduction (Zhao et al., 2012) and can save energy and increase speed
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effectively, which makes sharkskin useful for engineering applications. The drag reduction effect of sharkskin can be used for ships and submarines to reduce fuel consumption rates and increase speed (Pu et al., 2016). It is discovered that the engineered surface of shark riblets can decrease aeroplane drag up to 8% producing a 1.5% fuel-saving (Ball, 1999).
Figure 2.8: SEM image of a single scale of six shark species: (a) smooth hammerhead, (b) banded hound shark, (c) Jaws- (d) shortfin mako (Mako shark), (e) sand shark, and (f) mud shark (Spiny Dogfish) (Pu et al., 2016).
Pu et al showed that hydrophobicity of the biomimicked surface of sharkskin on Polydimethylsiloxane (PDMS) improved 18% compared to a flat surface (Pu et al., 2016). Li found that biomimicked superhydrophobic surfaces of sharkskin and lotus leaf based on micro-moulding and flame treatment could exhibit 1600 contact angles which were considerably higher than previous studies (Liu and Li, 2012). In marine applications, the attachment of cell-like algae can reduce the drag resistance of submarines and ships by up to 15%. Kesel et al. found that micro-patterns (40 µm-2 mm) on silicone surface based on the biomimicked surface of sharkskin achieved the best antibiofouling effect, reducing cell attachment by 67% (Kesel and Liedert, 2007). Carmen et al. evaluated the relationship between wettability and cell adherence to bioinspired sharkskin microstructure. This research compared the wettability of PDMS fabricated negative (pit, sharklet and channels) and positive
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(pillars and ridges) micro-patterns by the photolithography method. As illustrated in Figure 2.9, a bioinspired shark wing (negative sharklet) produce the highest wettability of all micro-patterns by raising the contact angle to 135o (Carman et al., 2006).
Figure 2.9: Negative biomimicked sharklet micro-pattern (4 µm height, 2 µm width and spacing) (Carman et al., 2006).
Attachment of zoospore cells decreases by 85% on negative biomimicked sharklet (Carman et al., 2006). As illustrated in Figure 2.10, in the sharklet pattern the 2 µm feature size is less than the size of zoospore cells while in the channel pattern, the feature size is 5 µm and a smaller cell can be trapped in the channels (Carman et al., 2006). The algae attachment and antibiofouling effect of the biomimicked surface of PDMS are reduced gradually over time, but it is preferable to the flat-PDMS (Pu et al., 2016). Although 3D riblet microstructure of sharkskin has a self-cleaning and antibiofouling effect and could save energy and time, it can only decrease the attachment of large cell-like algae and spores by repelling them rather than killing them.
Figure 2.10: Zoospore settlement on: (A) smooth surface, (B) Chanel micro pattern (5 µm width and height and spacing), (C) Sharklet (Scale bar 25 µm) (Carman et al., 2006).
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2.5.3 Gecko skin Geckos possess antiwetting characteristics and bactericidal effects due to the nanopattern on their skin, particularly their feet. As illustrated in Figure 2.11, the hair- like structure on gecko skin has 4 µm length, a top radius of 10-20 nm and submicron spacing (Figure 2.11 (A, B and C)). This nanopattern produces a bactericidal effect against Gram-negative bacteria Porphyromonas gingivalis (Figure 2.11 (D and F)) (Watson et al., 2015). Li et al. found that the nanopillars on gecko skin and its replica (made of acrylic) using the moulding method can kill bacteria after one week of repeated bacterial incubation. The C.A in gecko skin, replica and control surface are 150o, 108o and 83o, respectively. The length of the hair-like nanopillars varies from 2-4 µm in different areas of gecko skin. The results showed that the gecko skin and the replicated surface had a bactericidal effect against S. mutan and P. gingivalis. Figure 2.12 shows the number of active bacteria (S. mutan and P. gingivalis) on natural gecko skin, the gecko replica and a control surface (Li et al., 2016). S. mutan (Gram-positive) and P. gingivalis (Gram-negative) bacteria react differently to replica surface. The nanopillars fully penetrate the cell membrane of P. gingivalis whereas lack of penetration is observed against S. mutan. Figure 2.13 illustrates the difference in reaction of the replica surface to the two bacteria. The resistance of the Gram-positive bacteria (S. mutan) to the replica surface is attributed to the higher stiffness and thickness of Gram-positive bacteria structure as well as the size of the bacteria. In summary, the gecko skin and its replica were successful in killing 88% of Gram- negative and 66% of Gram-positive bacteria (Li et al., 2016). Although nanopillars of gecko skin (with 4 µm length and submicron spacing) have antiwetting, self-cleaning and bactericidal effects, this structure is not efficient enough to kill Gram-positive and small bacteria because Gram-positive bacteria membranes possess higher stiffness and thickness and small bacteria cannot spread over and be penetrated by the hair-like nanopillars due to its submicron spacing.
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Figure 2.11: SEM images of the gecko skin including a micro/nanostructure consisting of nano-hairs with a submicron spacing and a spike radius < 20 nm (A, B and C), (D and F) Interaction between bacteria (Porphyromonas gingivalis) and the nanopattern (Watson et al., 2015).
Figure 2.12: (A) The active number of active Streptococcus mutan bacteria (Gram-positive) on the gecko skin, replicated surface and Acrylic substrate, (B) The active number of Porphyromonas gingivalis (Gram-negative) bacteria gecko skin, replicated surface and Acrylic substrate (three and seven days exposure periods) (Li et al., 2016).
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Figure 2.13: (A) SEM image of the interaction between Porphyromonas gingivalis bacteria and replicated nanostructure, (B) SEM images of Streptococcus mutan bacteria positioned between replicated nanostructures (Li et al., 2016).
2.5.4 Dragonfly Dragonfly wing has a superhydrophobic, self-cleaning and bactericidal effect because of its nanopillars which are composed of chitin and lipid (Hasan et al., 2013). Rajendran et al. characterised the nanopillars of dragonfly (Sympetrum vulgatum) accurately in four areas using the AFM-tapping mode (Figure 2.14 and Table 2.1) (Rajendran et al., 2012).
Figure 2.14: Four areas for nanopillars analysis on the dragonfly wing (Sympetrum vulgatum) (Rajendran et al., 2012).
Table 2.1: Nanopillars analysis including average height and diameter of four areas on the dragonfly wing (Rajendran et al., 2012). Region of wing Height (nm) Diameter (nm) Surface roughness (nm) D1 188.3 195.08 22.47 D2 133.1 135.85 24.25 D3 79.63 83.25 18.58 D4 170.2 145.2 23.21
Bandara conducted an in-depth analysis of dragonfly nanopillars (Orthetrum villosovittatum) using Helium Ion Microscopy (HIM). Results showed that the wings
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are covered by short nanopillars with a diameter of 37 nm and a height of 189 nm and tall nanopillars with a diameter of 57 nm and height of 311 nm. The antibacterial efficiency of the wing against E. coli demonstrated a remarkable reduction from 4.99 × 105 to 1.65 × 105 cells min-1cm-2 within 4 hrs. (Bandara et al., 2017). Cho et al. fabricated a 100 nm diameter dragonfly nanopillars (Pantala flavescens) on glass, silicon, polymer via nanomoulding and nanoimprint lithography. The hydrophobic fabricated surface on the polymer has a contact angle of 135o which is close to a real dragonfly, at 145o (Figure 2.15) (Cho et al., 2013).
Figure 2.15: (a) Nanopillars of Pantala flavescens and its contact angle, (b) biomimicked nanopillars via nano-imprint and its contact angle (Cho et al., 2013).
The nanopillars on the dragonfly wing possess antibacterial effects against different bacteria. Bhadra evaluated the interaction of bacteria cells (Pseudomonas aeruginosa and Staphylococcus aureus) and fibroblasts with biomimicked dragonfly nanopillars on titanium fabricated by hydrothermal etching. Figure 2.16 shows that the biomimicked surface of titanium eliminated about 50% of P. aeruginosa and 20% of S. aureus. The hydrothermal etched titanium surface also showed enhanced cell attachment and proliferation of primary human fibroblasts over 10 days of growth. (Bhadra et al., 2015).
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Figure 2.16: Bactericidal activity on hydrothermal etched titanium after 19 hrs for S. aureus (left) and P. aeruginosa (right). (Scale bar in SEM images is 200 nm and in confocal laser scanning microscopy (CLSM) is 10 µm) (Bhadra et al., 2015).
An engineered surface fabricated by deep reactive ion etching (DRIE) (height: 4 µm, 220 nm diameter and random distance) can kill approximately 85% of both S. aureus and E. coli cells after 3 hrs, which is 6 times more than the control surface. This surface also has a fatal effect on mammalian osteoblast cells with 88% of osteoblast cells killed on the surface. The mechanism of killing both bacteria is due to excessive stretching on the nanopillars, leading to cell rupture (Hasan et al., 2015). Ivanova et al. produced the biomimicked dragonfly surface (Diplacodes bipunctata) on black silicon via reactive ion etching. Figure 2.17 shows the nanopillars of biomimicked black silicon and dragonfly wing. Both dragonfly wing and biomimicked black silicon have the same bactericidal effect against S.aureus with about 45 × 104 cell cm-2 min-1 killing efficiency over 3 hrs, while fabricated black silicon shows higher bactericidal effect against P. aeruginosa with about 42 × 104 cell cm-2 min-1 killing efficiency over the 3 hrs (Ivanova et al., 2013). Figure 2.18 shows the efficiency of natural dragonfly wing and black silicon against four types of bacteria.
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Figure 2.17: SEM image of the upper surface of (a) Fabricated nanopillars on black silicon and (b) dragonfly forewings (Scale bars: 200 nm) (Hasan et al., 2013).
Figure 2.18: Bactericidal efficiency of black silicon and dragonfly wings (Hasan et al., 2013).
In conclusion, random nanopillars of dragonfly wing and its biomimicked surface possess self-cleaning, superhydrophobicity and bactericidal effects. Moreover, Gram-negative and Gram-positive bacteria are both susceptible to dragonfly wing nanopillars.
2.5.5 Antimicrobial Natural Peptides (AMPs) AMPs encompass light molecules of protein with 5 to 100 various amino acids (Bahar and Ren, 2013) which show a wide range of antimicrobial characteristics against antibiotic-resistant bacteria, viruses, and fungi (Izadpanah and Gallo, 2005, Nguyen et al., 2011). Natural sources of AMPs can be found in the tissue of some animals that are in contact with airborne pathogens like bacteria. For instance, frog skin which includes more than three hundred different AMPs.
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One of the main sources of antimicrobial AMPs is insects. Samia cynthia and Ceropia mouth were the first known species with AMP (cecropin). AMPs in insects are tiny and mostly catonic and they have an antimicrobial effect in interaction with bacteria, fungi and parasites (Yi et al., 2014). (Fantner et al., 2010) demonstrated that CM15 AMPs have an antibacterial effect against E. coli cells with both mechanisms of time-variable incubation and rapid execution. (Onaizi and Leong, 2011, Fantner et al., 2010). Onaizi et al. claimed that low concentrations of AMPs as coating agents can be more beneficial for decreasing pathogenic bacteria, especially for implants, compared to antibiotic therapy (Onaizi and Leong, 2011). After AMPs concentration reaches the threshold, the bacteria membrane is disrupted through different mechanisms and models involving disordered and toroidal pore, electroporation, membrane thinning or thickening which cause metabolic disruption and cytoplasm attacking (Nguyen et al., 2011). While AMPs can disrupt bacteria through various mechanisms, the main problem of AMPs are durability and long-lasting effect.
2.5.6 Cicada wing The cicada wing has captured scientists’ attention due to its unique wing nanopillars. Cicadas can survive in different situations from subterranean to tree-tops, in high temperature and humidity. Survival in this challenging situation can be attributed to their self-cleaning and superhydrophobic wings. The cicada wing mainly consists of wax, chitin and protein which is covered with nanopillars (Ivanova et al., 2012b, Tobin et al., 2013). The hydrophobicity of the cicada wing surface is affected by its nanopillars and wax layer composition. Sun et al. characterized the superhydrophobicity of 15 cicada species in which the different areas on the cicada wing produced high and low hydrophobicity ranging from 76.80 to 146.00. Figure 2.19 illustrates the surface topography of four major cicada species and Table 2.2 shows the surface geometry and contact angle for the 15 cicada species. Environmental Scanning Electron Microscope (ESEM) analysis showed the various geometries of the nanopillars among the 15 cicada species with diameter 82-148 nm, spacing 44-117 nm, and height 159-146 nm. The nanopillars without wax are hydrophobic only while the nanopillars with the wax layer are superhydrophobic. Dense and tall nanopillars show higher hydrophobic
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characteristics. The arranged and uniformed nanopillars also cause a greater contact angle compared to disordered nanopillars (Sun et al., 2009).
Figure 2.19: SEM images of four cicada species and their wings. A: C. maculata, B: M. conica, C: M.
o microdon, D: T. jinpingensis). The SEM topography surfaces in Aj, Bj, Cj, Dj were tilted 30 from those in A, B, C, D, respectively. (Scale bars: 1μm).
Table 2.2: Surface characterization of the wings of 15 cicada species (Height values were calculated in 30-degree tilt angle). (Sun et al., 2009). (C.A. = contact angle).
Name of Cicada Origin Diameter Spacing (between two Height C.A (nm) pillars) (nm) (nm) (degree) Chremistica maculata China 97±5 92±11 309±24 76.8±13.9 Pomponia scitula China 84±5 84±11 282±18 91.9±5.9 Mogannia hebes China 85±7 95±11 164±8 78.4±5 Leptopsalta bifuscata China 90±5 117±13 200±52 81.3±8.3 Mogannia conica China 95±7 115±15 159±13 93.9±8.3 Meimuna durga China 89±5 89±9 257±24 134.8±5.7 Aola bindusara China 84±4 91±13 234±18 135.5±5.2 Meimuna microdon China 82±3 89±7 208±8 139.8±4.5 Meimuna mongolica China 128±4 47±5 417±26 123.3±12.7 Platylomia radha China 137±5 44±3 288±12 136.5±5.2 Dundubia vaginata China 132±6 56±7 363±22 141.3±3.3 Dundubia nagarasingna China 128±6 47±5 316±18 141.6±4.5 Meimuna opalifer China 148±6 48±5 418±38 143.8±6 Terpnosia vacua Japan 141±5 44±4 446±28 144.2±6.8 Terpnosia jinpingensis China 141±5 46±4 391±24 146±2.6
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Ivanova characterized the wettability of Psaltoda claripennis with a contact angle of 158.8o which is higher than the cicadas investigated by Sun et al. Ivanova et al. also noted that a 10 nm gold coating on the cicada wing changes the contact angle from 158.8° to 105.5° (Ivanova et al., 2012). In addition to superhydrophobicity of the cicada wing, which is attributed to the wax layer and nanopillars, it is claimed that the nanopillars of the cicada wing have bactericidal effects. Ivanova discovered that the nanopillars of the cicada wing (Psaltoda claripennis) possess a bactericidal effect against Pseudomonas aeruginosa rather than an antibiofouling influence. They found that the mechanism of killing bacteria is mechanical rather than chemical. The cell membrane tends to spread over the nanopillars until cell rupture occurs. The whole killing process takes only 3 minutes. It is also determined that 10 nm gold-coated cicada wing does not affect the bactericidal effect, meaning that surface chemistry does not have a role in killing bacteria (Ivanova et al., 2012). Figure 2.20 shows the same bactericidal effect between coated and uncoated cicada wing against P. aeruginosa.
Figure 2.20: Penetration of nanopillars into P.aeruginosa (a) without coating, (b) with the gold coating (scale bar: 200 nm) (Ivanova et al., 2012).
Kelleher studied the relationship between Gram-negative Pseudomonas fluorescens bacteria cells and wing nanopillars of three cicada species, Cryptotympana aguila, Ayuthia spectabile, and Megapomponia intermedia. The geometry of nanopillars for the three cicadas is illustrated in Table 2.3 (Kelleher et al., 2015).
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Table 2.3: Geometry of cicada wing nanopillars and their contact angle (C.A) (Kelleher et al., 2015).
Cicada species wing Height Pitch Diameter Spacing C.A (nm) (nm) (nm) (nm) (degree) Cryptotympana aguila 182 187±13 159 ± 47 28 95.65 Ayuthia spectabile 182 251±31 207 ± 62 44 113.2 Megapomponia 241 165±8 156 ± 29 9 135.5 intermedia
The results show that the bactericidal effect of M. intermedia and C. aguila is greater than the A. spectabile sample. Pseudomonas fluorescens cells interact with the nanopillars, drawing the pillars and at times puncturing and collapsing around the nanopillars. Dense nanopillars (close to each other) like M. intermedia caused more interactions with bacteria cells. SEM images and AFM analysis of the three cicada species nanopillars are shown in Figure 2.21 (Kelleher et al., 2015).
Figure 2.21: SEM Top-view images of fixed Pseudomonas fluorescens cells on (a) M. intermedia, (c) C. aguila, and (e) A. spectabile cicada wings. (Scale Bars: 2 μm); AFM images from interaction (b) M. intermedia, (d) C. aguila, and (f) A. spectabile wings on P. fluorescens bacteria (Area is 3.2× 3.2 μm) (Kelleher et al., 2015).
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Hasan evaluated the bactericidal effect of the nanopillars of cicada wings against different cell shapes (rod and coccoid) and cell structures (Gram-negative and positive bacteria). Regardless of the shape of bacteria (rod shape or coccus), the nanopillars of cicada wing have a bactericidal effect on Gram-negative bacteria. Gram-positive cells, such as S. aureus, B. subtilis and P. maritimus, show resistance to the nanopillars because the thickness of the peptidoglycan layer of Gram-positive bacteria is higher than in Gram-negative bacteria and this layer is more rigid, so the Gram-positive bacteria cell structure has greater resistance (Hasan et al., 2013d). Figure 2.22 shows the interaction of bacteria cells (Gram-negative and positive bacteria) with cicada wing nanopillars.
(a) (b) Figure 2.22: Bacteria/surface interaction in (a) Gram-negative bacteria, (b) Gram-positive bacteria. Scale bars in SEM micrographs is 1µm and in CLSM is 5 µm (Hasan et al., 2013d).
Generally, the peptidoglycan layer in Gram-negative bacteria has a thickness of 2-3 nm whereas it is 20-80 nm for Gram-positive bacteria (Kelleher et al., 2015). As illustrated in Figure 2.23, Pogodin et al. employed microwave irritation to reduce the turgor pressure and rigidity of Gram-positive bacteria, enhancing the sensitivity to be killed (Pogodin et al., 2013). Table 2.4 illustrates the bactericidal performance of cicada wing nanopillars against Gram-negative and positive bacteria compared to the dragonfly and biomimicked dragonfly wing on black silicon.
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Figure 2.23: The interaction of Gram-positive bacteria and nanopillars after microwave irradiation (Scale bars in SEM micrographs is 1 µm and in CLSM is 5 µm), dead bacteria are shown in red colour (right) in CLSM viability analysis (Pogodin et al., 2013).
Table 2.4: Bactericidal activity in cicada wing, black silicon and dragonfly wing (Hasan et al., 2013). C.A. is contact angle.
Nanopillars type C.A Nanopillars height (nm) Bactericidal activity Cicada wing 159o 200 Gram-negative Dragonfly wing 153o 240 Gram-negative, Gram-positive, Spores Black silicon 80o 500 Gram-negative, Gram-positive, Spores
Dickson et al. found that biomimicked nanopillars on PMMA (Poly (methyl methacrylate)) using nano-imprint lithography have a bactericidal effect against Gram- negative E. coli bacteria. The results show that dense and small nanopillars are more bactericidal because bacterial cells interact with more nanopillars and more stress is generated in the contact area (Dickson et al., 2015). Fisher et al evaluated the bactericidal effect of biomimicked diamond nanocones using a hybrid method of Microwave Plasma Chemical Vapour Deposition (MPCVD) and Reactive Ion Etching (RIE). Figure 2.24 shows that sharp nanopillars with random length size, disordered structure and lower density (surface B) are efficient for killing P. aeruginosa. (Fisher et al., 2016).
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Figure 2.24: P. aeruginosa interaction with (a) nanopillars on the control silicon wafer, (b) surface A diamond nanopillars on a silicon wafer (height:1.6µm, width = 350-750 nm), and (c) surface B diamond nanopillars on a silicon wafer with low height (100 nm), large height (3–5 µm) and width <100 nm to 1.2 µm nanocones (Fisher et al., 2016).
In summary, the nanopillars on the dragonfly wing have random dimension and distribution while cicada wing nanopillars have a well-organized structure with an ordered distribution. Both cicada wing and dragonfly wing nanopillars have a bactericidal effect, however, the nanopillars of cicada wings are more effective for killing Gram-negative bacteria and are more superhydrophobic than those of dragonfly wings.
2.6 THE INTERFACE OF NANOPILLARS AND BACTERIA
The mechanism of killing bacteria on the nanopillars of a natural surface like cicada wings, dragonfly wings and gecko skin is complex because it is highly dependent on the geometry of the nanopillars, bacteria behaviour and structure (Gram- negative or positive), the mechanical properties of bacteria, and the nanopillars and adhesion force. (Ivanova et al., 2012) found that the mechanism of killing bacteria is based on the rupture of bacteria membranes stretched between nanopillars. Cell rupture occurs when the bacteria membrane sinks onto a 200 nm height nanopillar (Ivanova et al., 2012). When the bacteria cell touches the nanopillars on the cicada wing, the membrane of the cell is drawn between negative areas of nanopillars, resulting in rupture of the cell membrane (Pogodin et al., 2013). Pogodin proposed biophysical model for the interface between the nanopattern and bacterial cell indicates that the mechanical properties of bacteria are an important parameter in the bactericidal characteristics of nanopillars Reducing the rigidity of bacteria with MI (microwave
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irritation) results in reduced turgor pressure, causing the bacteria to be killed more easily (Pogodin et al., 2013). The level of bacteria attachment highly depends on the geometry of the surface nanopillars (Hasan et al., 2013). As shown in Figure 2.25, the geometry of nanopillars has a decisive role in the bactericidal effect. Kelleher compared the bactericidal effect of three cicada species with different geometry. They found that the number of nanopillars that interact with a bacteria cell is dependent on the pitch and diameter of the nanopillars (Kelleher et al., 2015).
Figure 2.25: Interaction between bacteria and nanopillars of three cicada species wings with different pitch and diameter (Kelleher et al., 2015).
Dickson et al. presents two reasons for killing the bacteria (E. coli) through AFM analysis. The movement of bacteria on the flat surface is by twitching through the pili but a nanopillar can prevent the twitching movement of bacteria. The average height of bacteria on nanopillars is reduced about 1/3 compared to the flat surface. The bacteria are fitted on nanopattern orientation, which causes minimum contact, leading to less penetration and killing of bacteria. Figure 2.26(a) shows the well-settled bacteria on a surface with distant nanopillars and high diameter (cap diameter: 215 nm, centre to centre distance: 595 nm, base diameter: 380 nm, height: 300 nm). On the smaller and closed nanopillars (Figure 2.26(b) and (c)), bacteria cannot be conformed preferentially and nanopillars can transfer more stress to bacteria cells. Bacterial cells become longer on the smaller and closed nanopillars. Variable cell length on smaller and closed nanopillars indicate that cells cannot divide easily and grow. These factors,
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such as DNA damage, oxidate damage, and nutrient shortage, occur because of stress (Dickson et al., 2015).
Figure 2.26: AFM micrographs and height profiles of bacteria (E. coli) on (a) cap diameter: 215 nm, centre to centre distance: 595 nm, base diameter: 380 nm, height: 300 nm, (b) cap diameter: 190 nm, centre to centre distance: 320 nm, base diameter: 130 nm, height: 300 nm (c) cap diameter: 70 nm, centre to centre distance: 170 nm, base diameter: 100 nm, height: 210 nm, and (d) flat film: Scale bar : 2 µm (Dickson et al., 2015).
Li et al. proposed a thermodynamic model based on evaluating the overall free energy variation of bacterial cells attached to the wing nanopillars. The bactericidal effect can be improved by increasing the density, radius and height of the nanopillars. The key factor of bacteria damage is an increase in adhesion contact area with nanopillars. More than one bactericidal mechanism was identified on gecko skin which is dependent on the type of bacteria and nanopillar topology. The effective parameters for bactericidal efficiency and penetration size of bacteria are the density of nanopillars and cell structure (Gram-negative and Gram-positive). The density of nanopillars and bacteria size can indicate the amount of contact (Li et al., 2016). When a larger bacteria like P. gingivalis (> 500 nm) is placed on a replica of gecko skin, two situations are possible (Figures 2.27 and 2.28): (i) the adhesion contact area and adhesion energy are not enough to penetrate and the bending of nanopillars occur at the top, (ii) the adhesion contact area and adhesion energy are enough to penetrate the bacteria cell. When a small bacterium like S. mutans resides on nanopillars, bacteria membrane damage and death can happen through compression, stretching or cellular damage.
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the interaction of Gram-negative and Gram-positive bacteria with cicada wing nanopillars: 1. The adhesion contact area and adhesion energy are not enough to penetrate and the bending of nanopillars occurs at the top. 2. Gram-positive bacteria with stiffer and thicker layers are not too vulnerable to being killed and can stay at the top of the nanopillars without any damage. 3. The adhesion contact area and adhesion energy are enough to penetrate the bacteria cell.
2.7 NANOFABRICATION OF BIOMIMICKED NANOPILLARS
The rapid development in miniaturization has drawn scientist's attention to focus on nanofabrication (less than 100 nm) over the past two decades in a wide range of applications from biomedical devices (Shahali et al., 2019), drug delivery (Fu et al., 2018), biosensing (Karimian and Ugo, 2019), solar cells (Borgström et al., 2018), semiconductors (Cheng et al., 2006), optics (Liu et al., 2019), flexible electronic devices (Kang et al., 2018) to quantum computing (Hendrickx et al., 2018). Nanofabrication can be classified into the bottom-up and top-down approaches. Bottom-up approaches commence with nano-particles and atoms and produce upward to fabricate nanopillars like self-assembly (Rodríguez-Hernández and Cortajarena, 2015) while top-down approaches begin with large materials and fabricates nanoscale. Top-down nano-fabrication approaches can be divided into generative and replicative approaches. Generative techniques are UV photolithography (Wiley et al., 2010), particle beam lithography (Cheng et al., 2006), laser lithography (Sugioka and Cheng, 2014), ice lithography (Zhao et al., 2019), reactive ion etching (Hasan et al., 2017) and hydrothermal while the replicative technique is nanoimprint lithography (Dickson et al., 2015). In this section, relevant methods for fabrication of biomimicked nanopillars are discussed and compared to find an appropriate method to fabricate the nanopillars of the cicada wing.
2.7.1 Soft lithography Soft lithography is an advanced technique used to replicate a micro and nanostructure onto a polymer substrate. Embossing (nanoimprint lithography) and micro-moulding are the common techniques used for fabrication of natural surfaces.
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This method is not expensive because it does not need an expensive clean processing environment (Rodríguez-Hernández and Cortajarena, 2015).
❖ Nanoimprint Lithography (NIL)
NIL (or hot embossing) is a simple high throughput and economical fabrication method which can provide nanopatterns by transferring the pattern from mold to deposited polymer under pressure and temperature (higher than glass transition temperature Tg). Figure 2.29 shows the process of nanoimprint lithography (Guo, 2007, Rodríguez-Hernández and Cortajarena, 2015). Dickson produced the biomimicked nanopillars of a cicada wing on PMMA substrate using NIL. Spin coating is applied for deposition of PMMA film and an annealing process is carried out on a hot plate at 100 0C before imprinting. The nanopillars are fabricated in three geometries, as shown in Table 2.5. The nanopattern of sample 1 is created by silicon nano-hole mould, sample 2 is produced by commercially available nickel stamp and sample 3 is replicated natural cicada wing. Figure 2.30 shows the SEM image of the three biomimicked nanopatterns (Dickson et al., 2015).
(a) (b) Figure 2.29: (a) Schematic of the NIL method, (b) fabricated silicon mold with electron beam lithography and nano-imprinted pattern with 10 nm hole (Guo, 2007, Rodríguez-Hernández and Cortajarena, 2015).
Table 2.5: Geometry of nanopillars fabricated by NIL (Dickson et al., 2015).
Sample Cap-diameter (nm) Centre to centre distance (nm) Base diameter (nm) Height (nm) 1 215 595 380 300 2 190 320 130 300 3 70 170 100 210
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Figure 2.30: SEM image of (a) sample 1, (b) sample 2, and (c) sample 3 – tilt angle is 30o and the scale bar is 1µm (Dickson et al., 2015).
Rosenzweig et al. used the same NIL approach to fabricate PMMA nanopillars and evaluate the bactericidal effect against filamentous fungi. The results show that A. fumigatus and F. oxysporum were depleted on the biomimicked nanopillars in the period of 6 to 24 hr. The fabricated nanopillars also can decrease the upstream mobility and accumulation of P. aeruginosa compared to the flat surface (Rosenzweig et al., 2019a, Rosenzweig et al., 2019).
❖ UV Nanoimprint Lithography (UV-NIL)
High temperature (> Tg) and pressure are applied in the NIL method while in UV-NIL, UV radiation is employed to crosslink polymers to avoid deformation. However, as UV-NIL prevents deformation by using UV, this method is limited to cross-linkable polymers (Lan and Ding, 2010, Glinsner et al., 2010). Cho et al. biomimicked the nanopillars of dragonfly (Pantala flavescens) wing on glass, Si by using UV-NIL. Figure 2.31 illustrates the process of fabrication using UV-NIL. Figure 2.32 illustrates natural dragonfly wing nanopillars as well as fabricated nanopillars on glass using UV-NIL(Cho et al., 2013).
Figure 2.31: Schematic of UV-NIL (Cho et al., 2013).
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Figure 2.32: SEM image of (a) top view of the dragonfly wing, (b) fabricated nanopillars of the dragonfly wing on glass using UV-NIL (Cho et al., 2013).
❖ Micro molding Micro molding is a high throughput technique to replicate the nanostructure on polymer substrates. Kim et al. replicated the microstructure of sharkskin on epoxy resin substrate. First, the pre-treatment of real sharkskin sample is applied to keep the rigidity of scale and riblets. The skin template is fixed to eliminate shrinkage and deformation. Fixed and pre-treated sharkskin is used to produce the mold made of PDMS. Finally, a replicated sharkskin made of epoxy resin is extracted from the PDMS mold (Figure 2.33). (Kim, 2014).
Figure 2.33: Micro-molding process (adapted from Kim, 2014).
Kim also compared micro molding and the NIL method (with pressure 1.2 MPa and T = 1060C). The dimensional accuracy of micro molding method is 7% and micro molding can replicate sharkskin far better than hot embossing, However, NIL has a better performance in accurately fabricating the outer edge of scales and fine structures (Kim, 2014). Figure 2.34 illustrates the performance of micro molding and NIL in biomimicking the nanopattern of sharkskin.
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Figure 2.34: Biomimicked nanopattern of sharkskin using (a) micro-molding, (b) NIL (Kim, 2014).
Liu employed a hybrid method of micro molding and flame treatment to fabricate a biomimicked surface with both lotus leaf and shark wing effects. First, the biomimicked surface of a shark riblet is produced by micro molding. Then flame treatment is applied to generate the nano/micro pattern of the lotus leaf. The duration of the flame treatment is important to achieve the desired structure (Liu and Li, 2012). Figure 2.35 shows the replicated surface using micro molding and Figure 2.36 shows the replicated surface after flame treatment.
Figure 2.35: The SEM images of (a) sharkskin surface, (b) replicated sharkskin by miro-moulding (Liu and Li, 2012).
Figure 2.36: SEM images of replica sharkskin after flame treatment. It includes 300-500 nm submicron pattern and 20-50 nm silica particle (Liu and Li, 2012).
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Li et al. used micro molding to replicate gecko skin by producing a negative mold made of Poly-vinyl siloxane (PVS) and filling epoxy resin into the mold to produce replica gecko skin. As illustrated in Figure 2.37, the replicated nanopillars and their nano-hair density, bottom thickness, spacing (500 nm) and surface roughness are close to natural gecko skin. The replicated hair-like nanopattern is a little smaller than the real one and the top radius was two times greater than natural skin.
(a) (b) Figure 2.37: Top SEM images of (a) gecko skin and (b) replica gecko skin (Li et al., 2016).
Zhang employed a synthetic method comprised of micro molding and linkage co-polymerization in which nano chains were interlinked with micro-grooves of replicated shark riblet. Figure 2.38 shows the synthetic replication method includes three stages: 1. Sharkskin treatment (cleaning, fixing, drying). 2. Preparing female silicon die by soft lithography in which vacuum degassing has been applied to remove bubbles from the silicone mold. 3. Preparing the pre-polymer from polyacrylamide (PAM) and resin followed by adding a curing agent and plasticizer (Zhang et al., 2011).
Figure 2.38: Synthetic bio-replication of sharkskin process (Zhang et al., 2011).
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The replication error in ridge height and grove widths are -2.6% and -0.4%, respectively (negative values represent shrinkage). The error in synthetic replication was noticeably improved over previous studies on micro molding and micro embossing. The micro-embossing process applies high pressure which causes bending and shrinkage in riblet width and ridge height and prevents high-resolution fabrication. The synthetic replication method uses silicon which is easily fabricated without any pressure or high temperature (Zhang et al., 2011). While the soft lithography methods are used as high throughput replication methods, they do not have an ideal resolution for nanopillars, and they are limited to polymers and soft material with low melting temperature.
2.7.2 Vacuum casting One of the common methods for replication of natural surfaces made with polymer and silicon is vacuum casting. Zhao replicated sharkskin with silicon using vacuum casting as shown in Figure 2.39. Fresh sharkskin is preheated, fixed and baked at 60oC for 3 hrs. Multi glass fibre is added to the resin to eliminate cracks in the resin mold and increase the quality of fabrication. After the sharkskin and resin are put into a container, 0.02 MPa vacuum pressure is applied, followed by demolding the sharkskin from the resin. Then, melted silicon is poured into a replicated mold at 100oC and 0.92 MPa vacuum pressure. The error of replication with vacuum casting in width of groove and height of riblet is 5.19% and 7.04%, respectively, because of shrinkage of the mold during the process (Zhao et al., 2012).
Figure 2.39: Schematic of vacuum casting for sharkskin replication (Zhao et al., 2012).
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Although the vacuum enhances the dimensional accuracy of the fabricated surface in vacuum casting compared to soft lithography methods, this method can only replicate polymers and soft materials.
2.7.3 Femtosecond laser and laser interface lithography A femtosecond laser can fabricate superhydrophobic structures on different metals like stainless steel, high speedy steel and mold steel. Kietzig used the Ti- sapphire laser with a wavelength of 800 nm, 1 Khz repetition rate, pulse width of 150 fs and 30 µm spot size. Three different radiant exposures including 5.16, 2.83 and 0.78 J cm-2 were applied to create the structure. Higher radiant exposure creates a rougher pattern and the maximum contact angle is reported as 147oC on stainless steel (304L) with 0.78 J cm-2 (Figure 2.40). (Kietzig et al., 2009).
Figure 2.40: Structure fabricated by femtosecond laser on AISI 304L (the same scale applies to all images) (Kietzig et al., 2009).
Fadeeva et al. biomimicked the micro/nanopattern of lotus leaf on titanium substrate via femtosecond laser fabrication. Ti-sapphire laser, pulse width of 50 fs, 800 nm wavelength, 1 kHz repetition rate and radiant exposure of 100 J cm-2 were applied to fabricate the structure. The biomimicked surface is composed of a micro bulge with 10-20 µm size and nanostructure of less than 200 nm and this surface produces a 144o water contact angle. As illustrated in Figure 2.41, accumulation of S. aureus is noticeable on the fabricated surface while P. aeruginosa does not tend to attach to the surface. Two requirements should be provided for bacteria attachment, first, sufficient cell contact with the pattern and the capability of EPS to attach the cell to the interface area. Small bacteria like S. aureus possesses these two specifications while P. aeruginosa is incapable because it is a large cell and EPS formation will not be sufficient for cell attachment (Fadeeva et al., 2011).
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Figure 2.41: (a) P. aeruginosa cell attachment on fabricated titanium, (b) S. aureus cell attachment on fabricated titanium substrate (Fadeeva et al., 2011).
Laser interference lithography can be used to fabricate nanopillars and nano- columnar patterns on the surface (200 nm to 1 µm length). Du et al. fabricated photoresist (PR) as a mold on the substrate via laser interference lithography. The metal film is directly coated on the PR nanopattern by E-beam evaporation. A PR layer is etched by immersion in acetone. The height of nanopillars using laser interference lithography is 350 nm. (Du et al., 2011). While laser lithography can fabricate metals like stainless steel, fabricating features less than 200 nm, especially with a sharp edge, is a big challenge.
2.7.4 Reactive Ion Etching (RIE) RIE is a micro/nano etching technique in which plasma sputters the material on the substrate. Plasma is generated under vacuum conditions via the electromagnetic field producing energized ions which hit the surface, causing material removal (Franssila, 2010, Tan et al., 2019). Fisher fabricated the nanopillars of cicada wings on a diamond using a hybrid method of microwave plasma chemical vapour deposition (MPCVD) and RIE. First, the polycrystalline layer is coated on a silicon wafer using MPCVD followed by applying Ar and H2 gas as a reactive gas to produce plasma. The etching process is carried out under the 1 × 10-3 torr vacuum, 1400 w power. Two groups of nanopatterns are fabricated with -150 and -200 V. Surface (a) included nanopatterns with the average height of 1.6 µm and 350-750 nm width. Surface (b) is divided into two groups
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involving a small nanopattern with 100 nm height and a large nanopattern with a height of 3 to 5 µm. The width of nanostructures on Surface B is about 100 nm to 1.2 µm (Figure 2.42) (Fisher et al., 2016).
Figure 2.42: SEM images of (a) nanopattern fabricated with RIE in -200 V, (b) nanopattern fabricated with RIE in -150 V (Fisher et al., 2016).
Hasan et al. produced biomimicked nanopillars of dragonfly wings on black silicon using reactive ion etching. SF6 and O2 gas are used to produce plasma etching on black silicon. Figure 2.7 illustrates the fabricated nanopillars on black silicon via RIE and the natural nanostructure of dragonfly wings (Hasan et al., 2013). Hasan engineered the biomimicked nanostructure of dragonfly wing nanopillars (height: 4 µm, 220 nm diameter and random distance according to Figure 2.43) on silicon wafer by deep reactive ion etching (DRIE). A Mixture of SF6 and O2 with the power of 2000-2150 W was used for fabrication (Hasan et al., 2015).
Figure 2.43: Biomimicked nanopillars of dragonfly wings on silicon wafer (Hasan et al., 2015).
To achieve better versatility in RIE, nano-sphere lithography is used as a hybrid method of coating, etching, and cleaning to reach different sharpness and aspect ratios
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on the substrate like quartz used in antibacterial optical devices. Results show that quartz fabricated nanopillars with a height of 300 nm and a diameter of 10 nm have a better bactericidal performance against P. aeruginosa and E. coli (Han et al., 2018). In summary, RIE is an advantageous method to fabricate nanopillars with good resolution and high throughput, but because it requires mask coating and mask fabricating on the surface, it is expensive. Moreover, controlling the geometry and mask fabrication on brittle material, like titanium, is a challenging issue.
2.7.5 Hydrothermal Method
The hydrothermal method was developed to generate TiO2 nano/microstructures and nanowire arrays for solar cell application. TMAOH (tetramethylammonium hydroxide) was used in a hydrothermal solution as a complex agent and the autoclave reaction temperature varied from 160 to 220oC. At 200oC and 8 hr reaction time, uniform orientated TiO2 nanopillars array can be fabricated. The nanopillar dimensions after 12 hr reaction time are width 250 nm width and length 700 nm (Dong et al., 2010). Wei et al. studied the formation of nano-brushite and nanotubes by hydrothermal and anodic oxidation (SBAOTH). This structure can enhance both superhydrophobicity and bioactivity. The SBAOTH process includes sandblasting, acid etching, anodic oxidation, heat treatment and finally hydrothermal treatment. -1 Anodic oxidation is performed in 10 g L NH4F at 20 V for 1 hr and hydrothermal o treatement is carried out in saturated Ca (OH)2 solution for 2 hr at 200 C. The length of nano-brushite is 250-300 nm (Wei et al., 2015). Bahadra investigated the effect of antibacterial nano-textured titanium surfaces by using a hydrothermal etching approach on bacteria and human cells. The results show that half of P. aeruginosa and about a quarter of S. aureus are killed in contact with the surface. Additionally, the nano-textured surface generated by the hydrothermal method improve the osseointegration, cell adherence and proliferation of fibroblast human cells (Bhadra et al., 2015). In summary, the hydrothermal method can fabricate nanowires rather than nanopillars on titanium substrate with high throughput but controlling nanowire structures to achieve organized structures with the same direction and size is challenging.
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2.7.6 Photolithography (PL) The principle of photolithography is illustrated in Figure 2.44. The process starts with cleaning the surface and spin coating photoresist on the substrate. Two classes of photoresist, positive and negative photoresist, are used in the PL process. In positive photoresist, the portion of photoresist exposed to the light source becomes soluble by changing the chemical structure, while in negative photoresist the parts of photoresist exposed to the light become insoluble via polymerising. The baking process is used to strengthen the photoresist and enhance adhesion of the photoresist to the substrate. The substrate is glass which is coated by chromium as a mask. Then, the mask is coated by the photoresist layer, followed by light exposure. The soluble part of photoresist is removed by a solution called "developer" and etching the area which is not covered by the photoresist (Rodríguez-Hernández and Cortajarena, 2015). While PL is widely applied in the semiconductor industry, two dimensional and cell-encapsulating scaffolds are a limited area in biomedical applications. Negative PL requires a photo cross-linkable polymer but biocompatible cross-linkable polymers are not extensive (Bae et al., 2014).
Figure 2.44: Schematic of photolithography.
Although the PL technique can create a micro/nanopattern in various complex devices in a minimum amount of time, it has drawbacks such as high process cost and inability to achieve smaller sizes because of light diffraction. Because diffraction of light and resolution is affected by light wavelength and mask type, resolution varies
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from 45 nm to microscale (Bae et al., 2014). To achieve small sizes, the wavelength can be reduced from UV (450 nm) to deep UV (193 nm) and X-ray (0.5 nm). (Carman et al., 2006) fabricated biomimicked sharkskin surface by negative and positive PL. As illustrated in Figure 2.45, negative PL is used for pit, sharklet and channels, and positive PL is employed for pillars and ridges (Carman et al., 2006).
Figure 2.45: A: pillar (1.5-5 µm height, 5 µm width, 5-20 µm spacing), B: pit (5 µm height, 5 µm width, 5-20 µm spacing), C: channel (5 µm height, 5-20 µm width, 5 µm spacing), D: ridges (1.5-5 µm height, 5 µm width, 5-20 µm spacing), and E: Sharklet or bioinspired surface of shark wing (4 µm height, 2 µm width spacing) (Carman et al., 2006).
2.7.7 Glancing Angle Sputter Deposition (GLAD) A recent advance in sputtering deposition for producing antibacterial surfaces in the implant and food industry is glancing angle sputter deposition, which can fabricate nano-columnar or nanopillar shapes. Sengstock et al. fabricated a bioinspired antibacterial nano-columnar surface based on cicada wings using GLAD. The top cathode is located at 27o for the deposition of thin film and the 88o GLAD cathode generated nanopattern. The revolution speed of the substrate is adjusted to 25 rev /min. The height of the fabricated columnar structure is about 478 ± 6 nm, the width is 33 ± 7 nm. The results show that E. coli bacteria is dramatically reduced after 3 hr on the nano-columnar surface (Sengstock et al., 2014). Motemani developed a GLAD method to fabricate nano- columnar titanium and titanium dioxide in a study investigating the adherence of human stem cells on a nano-columnar surface. A large surface area and higher inter- column spacing are more biocompatible and this is an acceptable choice for bio- functionalization (Motemani et al., 2014).
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In summary, the main drawback of the GLAD method is that the deposited nanostructure is not straight (oblique structure) and controlling the size of nanostructure in a thin layer is almost impossible.
2.7.8 Particle Beam Lithography (PBL) Particle beam lithography applies electron or ions (Ga+, He+, Ne+) to fabricate nanopillars and can be classified into Electron Beam Lithography (EBL) and Focused Ion Beam Milling (FIB). Particles (electrons and ions) with high energy (> 2 keV) and nanoscale wavelength can decrease the diffraction which increases resolution compared to photolithography. Being maskless can reduce the cost of the process, which is another benefit of PBL (Rodríguez-Hernández and Cortajarena, 2015).
❖ Focused ion beam milling (FIB)
FIB is a popular technique for imaging, sample preparation and direct writing on different materials (e.g. polymers, metals) with high resolution (10-100 nm). The FIB system applies the ions (e.g. Ga+, Ne+ or He+) to fabricate the surface (Shahali et al., 2019). The interaction area of Ga+, Ne+ and He+ is presented in Figure 2.46. He+ has the smaller interaction area due to the lower secondary electron (SE) compared to electron and gallium, resulting in the highest imaging resolution and capability of sub- nanometre fabrication in 30 Kev. Ga+ possesses more momentum which can have a high volume of scattering on different materials but the resolution is lower in than electron and helium (Hlawacek et al., 2014).
Figure 2.46: Interaction volume difference among charged particle beams used for imaging (Hlawacek et al., 2014).
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FIB is a maskless high-resolution fabrication method that can be used as direct milling on metal, but its throughput is less than EBL. FIB is not only used in the semiconductor industry but is also applied in biomaterial and biological matter for imaging and sample preparation for TEM (Grandfield and Engqvist, 2011). FIB milling is beneficial due to being maskless, its ability to direct writing on metal and lower proximity. However, the lower resolution compared to EBL caused by the high momentum and beam diameter of Ga ion, generating surface damage and contamination due to ion implantation is a key issue (Maas et al., 2010, Shahali et al., 2019).
❖ Gallium Ion Milling Ga+-FIB Milling has been widely applied for more than two decades in semiconductor device applications. In this method, low energized Ga is used for imaging while the higher energized Ga ion can be used for direct writing of milling as well as preparing biological TEM samples coated by platinum (Suutala, 2013, Volkert and Minor, 2007). Yadav et al. fabricated nanopillars in the shape of nanotube arrays on titanium (Yadav et al., 2015) by the Ga+-FIB method assisted by anodization in an aqueous electrolyte. The implanted Ga on the surface is eliminated by the anodization process. Important factors in Ga+-FIB are dwell time (t: 0.3 to 2.3 s), FIB spacing(s), voltage (30 kV Ga+), beam current (30, 300 pA), Ga+ dose (1 × 108 to 1.7 × 109 Ga/point) (Yadav et al., 2015). Wu et al. applied FIB to fabricate nanopillars with a diameter of 95 nm and length of 150-160 nm on InGaN/GaN used in semiconductor devices with fabrication parameters of voltage 30 keV and 300 pA beam current. Potassium hydroxide with 20% (weight percentage) dissolved in ethylene glycol was applied to eliminate the debris and outer damage to nanopillars. Figure 2.47 shows the nanopillars fabricated on GaN.(Wu et al., 2008).
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Figure 2.47: SEM images showing FIB milled nanopillars on GaN (Wu et al., 2008).
Oyama et al. fabricated PLLA polymer nano-holes with a diameter of 80-490 nm using Ga+ FIB. Milling current is optimized to reduce the amount of debris and thermal deformation. In this work, fabricated biocompatible and biodegradable PLLA produced good cell adherence because of carbonizing during the atom sputtering. (Oyama et al., 2013).
❖ He and Ne ion milling (HIM)
HIM employs nano spot size via gas field ion source (GFIS) to capture high- resolution images of sub-nanometre features like biological samples without surface damage (Hlawacek et al., 2014, Maas et al., 2010). Diffraction of the electron is higher than He+ and Ne+ because electrons are lighter. Therefore, He+ and Ne+ have the capability of focusing on a very small point compared to SEM (GmbH, 2016). As illustrated in Figure 2.48, while He+ and Ne+ ion beams hit the sample with their higher mass, the particles do not spread near the surface, meaning that a smaller area of surface interaction and much higher resolution is achievable. Figure 2.50 (a) illustrates the Orion NanoFab HIM column and its instrument, including beam source, accelerator, aperture, stigmator, scanning coil, lenses, detector and flood gun.
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Figure 2.48: (a) Diffraction comparison between e-, He+ and Ne+ (GmbH, 2016).
In HIM, three atoms come from the source tip, called trimer as shown in Figure 2.49 (b). The cryogenic cooling system is used to mitigate the gas to form a stable source tip (Hines and Wolf, 2016). A flood gun in HIM generates negative electron charge on the non-conductive sample surface to compensate the accumulated positive charge from the He+ or Ne+ beam. The floodgun facilitates the imaging of non- conductive samples like biological samples (Joens et al., 2013, Hines and Wolf, 2016). Table 2.6 illustrates the significant parameters used to achieve high-resolution imaging.
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Figure 2.49: (a) System of Orion NanoFab column and GFIS, (b) Source tip is formed by three atoms (trimer) emitting helium ions (Chen, 2010, Hlawacek et al., 2014, Hines and Wolf, 2016).
Table 2.6: Main imaging parameters and their definitions in HIM (Helium Ion Microscopy) (Hines and Wolf, 2016).
Parameter Definition and description Focus The focus is a point where all beam ions reach a small spot. It is controlled by objective lens 2, as illustrated in Figure 2.50. Astigmatism Lenses bring about the distortion of the beam in the elliptical shape. The perfect shape of a beam is circular and a stigmator is being used to ensure the circular shape of the beam. Scan size Scan size is defined as a pixel resolution and it refers to scan point per line. The standard resolution is 1024 × 1024 Dwell time/ Scan Dwell time is the remaining time of beam in a single scan point before scanning the speed next point. Averaging and This option is used to enhance the image quality and can be combined with shorter filter dwell in beam sensitive samples. Contrast and Brightness and contrast are vital for a high-quality image that demonstrates Brightness informative small features. Working distance It describes the distance between sample and pole piece. The ideal amount of working distance is 8 mm (eccentric height). Flood gun A Flood gun is applied for non-conductive samples like insect wings. Important parameters parameters of the Flood gun include Flood gun mode, X/Y deflection, Flood energy, flood time and flood voltage.
He+ milling possesses a low rate of sputtering and high resolution (<5 nm) due to its lower mass compared to Ga+ (Scholder et al., 2013). Process variables, like current, voltage, aperture size, spot size, dwell time, and dose factor, are important for
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high performance and resolution in patterning. Table 2.7 shows the important parameters and their definitions in He+-FIB milling. Beam alignment parameters including focusing and adjusting astigmatism are the key to stable and accurate patterning (Hines and Wolf, 2016). Among all parameters, the dose is an essential parameter that demonstrates the resolution, size of milling (depth and width) that could be achieved by Equation (2.1).
Ion stAI][][ N ch ce][arg Dose = = Equation (2.1) marea2 ][ marea2 ][
In Equation 2.1, IIon is current of the ion beam, t is the dwell time, the N is the number of ions, the elemental charge is 1.602 × E-19 C (coulomb), the area is milling area. In most applications, nC/µm2 (nano coulomb per micro square meter) is obtained from Equation (2.2). nC I pA T(sec))( Dose ][= Equation (2.2) m2 mA2 )(1000
Table 2.7: Main patterning parameters and their definitions in He+-FIB milling (Hines and Wolf, 2016).
Parameter Definition and description Beam alignment Focus and stigmator should be adjusted to reach the best patterning condition Beam overlap Bema overlap indicates how much beam overlaps between different scan points. Ion beam regularly need more than 50% overlap to ensure smooth edge cutting Acceleration voltage It demonstrated how much Ion passes through the sample and specifies the resolution. (Ion beam energy) Aperture and spot size Aperture shows the ion beam current and beam diameter. Larger aperture provides more current for milling and beam is larger. Small aperture provides less current but a smaller beam diameter which means that smaller structure can be produced. Dwell time Dwell time is the amount of time that ion beam stays in a single point. Longer dwell time mills more material Number of passes This parameter is linked to the dwell time. It shows the amount of repeat that ion beam is scanned over the patterning area. In small dwell time (1µs) several thousand passes are necessary to reach pattern depth but sometimes single pass with a very long-time dwell time can be used Dose Determine the number of ions which go into a specific area of the surface.
❖ Electron Beam lithography (EBL) EBL is an efficient technique for fabricating different nanostructure shapes, like nanoholes, nanowires, nanodots and nanopillars, with high resolution, throughput and lower proximity effect (Maas et al., 2010). The electron can be used for imaging in SEM (scanning electron microscopy) or fabricate the resist in the EBL system. In EBL,
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the electron can only fabricate polymer resist like PEG or PMMA due to its lower energy. EBL can be applied in a negative and positive tone via crosslinking and degradation of E-beam resist as shown in Figure 2.50. Development is performed to dissolve and remove the undesirable area. Nanoscale features (1 mm - 10 nm) on a quite large area can be fabricated by EBL. Exposure area is affected by secondary electrons and resolution depends on the scattering area and resist molecule size (Rodríguez-Hernández and Cortajarena, 2015).
Figure 2.50: The principle of Electron Beam Lithography.
Jindai et al. biomimicked cicada nanopillars on gold substrate with a diameter of 300 nm, height of 400 nm and pitch of 500 nm using EBL (Figure 2.51). Target diameter was 100 nm greater than the set value for the diameter. The results show that bacteria adherence depends on nanostructure and bacteria properties (Jindai et al., 2019). The electron can also be used for depositing materials like platinum on the substrate through electron beam induced deposition (EBID). Ganjian et al. (2019) fabricated nanopillars made of platinum, carbon and oxygen with 190 nm height, 80 nm diameter and 170 nm centre to centre distance which had 97% and 36% killing efficiency against E. coli and S. aureus, respectively. While EBID can achieve high- resolution nanopillars in the systematic study, the throughput is very low compared to EBL (Modaresifar et al., 2019, Ganjian et al., 2019).
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Figure 2.51: SEM image of gold nanopillars fabricated by EBL, (a) cross section, (b) top view (Jindai et al., 2019).
Among PBL methods, EBL has higher resolution and throughput to fabricate polymers while FIB (e.g. Ga+/ He+/ Ne+-FIB) can directly write on small scale areas of metal. Table 2.8 shows the resolution and throughput limitations of PBL methods (Shahali et al., 2019).
Table 2.8: Resolution, throughput, advantage and limitation of PBL methods (Shahali et al., 2019). PBL Resolution Throughput Advantage Limitation method EBL *** *** High throughput Need resist (polymer electron resist) Low aspect ratio for close nanopillars Ga+-FIB ** ** Metal Direct writing Surface damage and contamination He+-FIB **** * Metal Direct writing Low throughput Ne+-FIB *** ** Metal Direct writing Low stability of Ne+ Low throughput EBID *** ** Metal Direct writing Low throughput
EBL has a high resolution and acceptable throughput for the fabrication of metallic nanopillars. Fabrication of dense nanopillars with the lowest diameter, good proximity effect and high aspect ratio are challenging subjects which require a systematic approach to optimize the process variables.
2.8 SUMMARY
2.8.1 Literature review finding The major findings of the literature review are summarised below. • Infection is the leading reason for revision surgery and failure of the titanium implant. Therefore, identification of long-lasting antibacterial surfaces is essential.
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• Antibiotic therapy has various drawbacks and side effects. 1. When biofilm forms on titanium implants, the immune system and antibiotic therapy are no longer effective. 2. The growing amount of antibiotic-resistant bacterial strains over time. 3. Lack of control of concentration upon delivery. Traditional methods and surface modifications are not effective due to their low durability. • Recent research demonstrates that natural surfaces like cicada wings have bactericidal effects due to their nanopillars. Nanopillars on cicada wings penetrate the bacterial membrane, causing a mechanical rupture. Several factors including the mechanical properties of bacteria, bacteria structure, mechanical characteristics of nanopillars, the geometry of nanopillars (e.g. length, centre to centre and diameter and spike radius) are important for the evaluation of bactericidal activity of cicada wing nanopillars and biomimicked nanopillars. • Scholars have recently concentrated on biomimicking nanopillars using different materials. The most common methods, such as soft lithography (e.g. NIL and UV-NIL), micro-moulding, photolithography, and vacuum casting are limited to fabricating the nanopillars of cicada wings on polymer, resin and silicon. The hydrothermal method can fabricate nanowires on metal, but it is hard to control the size by changing the process variable. RIE also can fabricate nanopillars on metal but the mask fabrication on metal is very expensive. While electron beam lithography has higher throughput to fabricate polymers, focused beam ion lithography can fabricate precise and controlled nanopillars on a small area of titanium.
2.8.2 Knowledge gap The gaps identified in the comprehensive literature review include: 1. Most research studies have been limited to nanopillars of natural wings, like cicada and dragonfly wings, while limited studies have focused on fabricating high resolution titanium nanopillars (accurate size of nanopillars less than 100 nm). 2. Existing research on biomimicking antibacterial nanopillars is limited to polymer and black silicon and is rarely applied to metals like titanium.
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3. Current biomimicking of natural nanopillars on metal is restricted to hydrothermal etching and laser fabrication, EBL as a high-resolution and efficient fabrication method is rarely applied for biomimicking precise nanopillars on titanium. 4. The EBL fabrication process is expensive and requires a high level of skill; hence, it needs a systematic approach to optimize the process. 5. Few research studies have used TEM and HIM microscopy to understand nanopillar topography with precise resolution.
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Chapter 3: Research Methodology
3.1 OVERVIEW
This chapter explains the research methodology, research design, materials, and analysis to achieve the aims and objective of the thesis (stated in Section 3.2). The microscopic study of bacteria interactions on nanopillars of the cicada wing and biomimicked nanopillars is essential to evaluate the bactericidal effect of the wing, leading to identification of the effective geometry of nanopillars with the highest bactericidal effect. Cicada wings have well-oriented nanopillars which are an ideal structure for bactericidal study and biomimicked nanopillars and there is limited research investigating the effect of the geometry of cicada wing nanopillars on bactericidal effect. Current research studies have mainly focused on bactericidal mechanisms and effects of natural wing nanostructure while others were limited to biomimicked nanostructures made of polymer and silicon. Mimicking high resolution titanium nanopillars is challenging due to the lack of accuracy of most nano-fabrication techniques. Recent studies have shown that the chemistry of nanopillars does not have an impact on the bactericidal effect even with the coating of gold. In this research, the chemical properties of wing nanopillars were characterized to confirm that there is no chemical reaction involved in the bactericidal effect of cicada wings. Then, bacteria and osteoblast cells were cultured on the surface to evaluate the bactericidal effect of nanopillars. Using SEM imaging, bacteria and cell interactions and plate counting methods, the bactericidal efficiency and effect of nanopillar geometry on bactericidal effect were determined, to identify the best strategy to fabricate the biomimicked nanopillars. In this chapter, Section 3.2 explains the research design and research methodology. Section 3.3 discusses the material preparation (e.g. wing preparation, bacteria and cell culture, titanium substrate preparation). Sections 3.4, 3.5, 3.6, 3.7 and 3.8 describe surface chemical characterization, nano topography analysis, bacteria efficiency test, osteoblast cell culture and AlmarBlueTM assay and statistical analysis
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methods, respectively. Finally, Sections 3.9 and 3.10 present the process variables and electron beam lithography used to fabricate titanium nanopillars.
3.2 RESEARCH METHODOLOGY DESIGN
This research studies the bactericidal effect and biocompatibility of nanopillars of three cicada species wings to (i) find the optimum geometry of natural wing nanopillars with high bactericidal effect and biocompatibility, and (ii) mimic and fabricate the most effective nanopillars on titanium. The process variables of electron beam lithography are optimized to achieve the best bactericidal effect and biocompatibility. A schematic of the research methodology, research design, materials, analysis and aims and objectives are given in Figure 3.1. As shown in Figure 3.1, surface characteristics, chemical properties, bactericidal efficiency, cell conformity and interaction, and osteoblast cell proliferation of nanopillars of three cicada species are studied in objective one. Objective two aims to mimic the nanopillars of the cicada wings obtained from Objective 1 on titanium substrate using Electron Beam Lithography (EBL). The process variables of EBL are optimized to achieve a titanium nanopillar geometry (diameter, height, aspect ratio, tope diameter and centre to centre distance) as close as possible to wing nanopillars. The osteoblast cell and bacteria conformity on titanium nanopillars are assessed by qualitative and quantitative analysis.
3.3 MATERIAL
3.3.1 Cicada wing The first objective is to study the surface characteristics, bactericidal interactions, and bactericidal efficiency of three cicada species (Table 3.1). Psaltoda claripennis (PC), Aleeta curvicosta (AC) and Palapsalta eyrie (PE) were supplied by Australian Insect Farm (www.insectfarm.com.au/). Cicada wings were selected for this study because they have uniform and well-organized nanopillars which facilitates the fabrication of nanopillars compared to random dragonfly wing nanopillars and it also helps to study the effect of geometry in killing bacteria.
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Figure 3.1: Overall schematic of the research methodology.
Table 3.1 summarises information about the three cicada species. Forty-five cicadas (15 for each species) were caught by the supplier on the same date and at the same locality (30-38 km west of Mount Garnet, QLD 4872). All samples were stored in the household deep freezer for two days until insects were packed and dried to A1 quality. The wings were detached carefully from the cicada body using tweezers without damaging the wing. For qualitative analyses like HIM and SEM, plate counting and AlamarBlueTM assay analysis, wing samples were cut to 8 mm diameters using a Biopsy punch (8 mm) and rinsed in DI water (deionised water) (specific resistance 18.2 MΩ in 25oC from the Milli-Q system from Millipore) for 15 min and dried overnight under a fume hood. For AFM analysis, the wing sample was cut using scissors and cured in PDMS and solidified at the room temperature under a fume hood.
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Table 3.1: Specification of the three cicada species used in the study.
Name of Species Size (mm) Image Psaltoda claripennis (PC) 35-40
Aleeta curvicosta (AC) 30-35
Palapsalta eyrei (PE) 12-15
3.3.2 Titanium sample The second objective is to evaluate the biomimicked nanopillars of cicada wings on pure titanium. In this research, high pure titanium was used as it is the most widely used material in orthopaedic implants. For the titanium control surface and substrate preparation for EBL, 10 mm × 10 mm silicon wafers were coated with thickness of 30 nm pure titanium (99.99%) using E-beam evaporation. In E-Beam evaporation the surface roughness of the Si substrate and deposition rate are two important parameters that lead to a smooth surface. The film thickness of titanium also has an effect on the surface quality of the film. Surface roughness reduces with decreasing deposition rate and deposition thickness (Bordo and RUBAHN, 2012, Cai et al., 2005). In this research, the lowest deposition rate (0.1 A/s) was applied by the E-beam on a smooth Si substrate to achieve the desired surface properties of the titanium film.
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3.3.3 Bacteria In this research, the bactericidal effect of cicada wings and biomimicked nanopillars of cicada wings on titanium were evaluated against two main sources of infection: Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853). S. aureus, a Gram-positive bacteria with a cocci shape, and P. aeruginosa, a Gram-negative bacteria with a rod shape, are substantial sources of orthopaedic implant infection (Belt et al., 2001). Specifications of both bacteria in this study are given in Table 3.2. The structure of S. aureus differs from P. aeruginosa (Figure 3.2). The cell wall of bacteria is responsible for protecting the cell from the external environment and physically resisting changes like osmotic pressure up to 25 atm (Kim et al., 2015). The cell wall of Gram-positive bacteria has a thickness between 20-40 nm which is thicker than Gram-negative bacteria. The cell envelope in Gram-negative bacteria (P. aeruginosa) is composed of the outer membrane (OM), peptidoglycan cell wall (CW), periplasm (PP) and inner membrane (IM). In Gram-positive bacteria (S. aureus), there is no outer membrane, instead, Gram-positive bacteria are covered by a thick layer of PG (Peptidoglycan) that helps the cell to resist turgor pressure on the cell membrane. Turgor pressure (TP) is the force applied by stored water against a cell wall (Silhavy et al., 2010). External physical changes like nanopillars can increase the TP resulting in membrane damage (Pogodin et al., 2013). S. aureus has a 16 nm inner wall zone, 5.4 nm plasma membrane thickness and PG layer of 19 nm (variable 20- 40 nm thickness) which has 10 nm high-density thickness. The Gram-negative bacteria has 5-7 nm cell walls and the PG layer of P.aeruginosa is 2.4 nm thick (Goldman and Green, 2008). Pili are hairlike and slender proteinaceous appendages on the surface of the Gram-negative bacteria which play an important role in surface adhesion during infection, biofilm formation, and twitching movements of bacteria (Van Schaik et al., 2005).
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Table 3.2: Bacteria specification (Harrington, 2011, Harris and Richards, 2006, NCEZID, 2011).
Name of Type and shape size Image bacteria Staphylococcus Gram-positive, Cocci shape 0.5-1 µm diameter aureus and grape-like
Pseudomonas Gram-negative, Rod-shape 0.5 to 0.8 µm by 1.5 aeruginosa to 3.0 µm
(a) (b) Figure 3.2: The cell membrane structure of (a) P. aeruginosa and (b) S. aureus and (Goldman and Green, 2008).
3.3.4 Osteoblast human cells Osteoblast cells are the major cellular component of bone as well as a functional part of the bones. In this research, the cell metabolic activity of osteoblasts was studied on natural cicada wing nanopillars as well as titanium biomimicked nanopillars. Human osteoblast cells were selected from a female donor (ethical approval: QUT HREC 1400001024). Bone with the lowest defect was selected and rinsed in PBS. Then it was mixed with Penicillin/Streptomycin (Pen/Strep), continuing this process until the cloudy state of PBS was removed after shaking. Afterwards, the bone flakes were cultured in a tissue culture flask with culture media including MEM-alpha, 10% FBS and Pen/Strep. After a few days, osteoblast cells were grown and were ready for cellular metabolic activity analysis (Shahali et al., 2019). An 8 mm diameter sample of cicada wing was
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cut by Biopsy punch (8 mm) and rinsed in DI water (deionised water). Then, the osteoblast media were cultured on surfaces including cicada wing samples, fabricated titanium nanopillars, and a control surface in 48 well plates.
3.4 SURFACE CHEMICAL CHARACTERIZATION
XPS and FTIR spectroscopy were used to characterise the chemical composition of the three cicada species wings.
3.4.1 Fourier Transform Infrared Spectroscopy (FTIR) Infrared spectroscopy is a widely used analytical method to analyse organic components, polymers, bacteria, and insect wings including wax and chitin. FTIR is a physical/chemical method based on the measurement of molecular vibration stimulated by infrared radiation at a particular wavelength range (Davis and Mauer, 2010, Tobin et al., 2013). Infrared (IR) microscopy can measure the chemical composition of the material based on the infrared absorbance spectrum from a specified area on the surface. When the FTIR passes through the sample, a wavelength is absorbed by chemical bonds in the sample to produce molecular vibration including bending, contracting and stretching. Regardless of the different structures in a molecule, all existing groups in one molecule absorb the infrared radiation in the sample wavenumber range. Then, peaks are extracted as a result of the absorption of bond vibrational energy in the IR region as a fingerprint (Davis and Mauer, 2010). An IR spectrum is obtained from calculating the intensity of IR before and after it goes through the sample. The spectrum is conventionally plotted with Y-axis as transmittance or absorbance versus X-axis as wavenumber units (Davis and Mauer, 2010). The main composition of the insect wing is protein and chitin and they have a wax layer on the top, which is responsible for the high water contact angle (Gorb et al., 2000, Kreuz et al., 2001, LOCKEY, 1960). It is believed that the distribution of wax on the wing has an essential impact on self-cleaning and wettability of wing. Chitin is a main component of the exoskeleton, or external skeleton, of many arthropods such as insects, spiders, and crustaceans. This element is firm, durable, and can protect and support soft tissue. Wax is an organic component which creates the
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hydrophobic effect in the environment. It is composed of alkanes and lipid. The bactericidal effect of nanopillars attributed to surface topography and superhydrophobicity is affected by both surface chemistry and surface topography. Nanostructures and their chemistry can define the energy emitted from surface materials (Zhang et al., 2008). A Nicolet iS50 FT-IR Spectrometer (Thermo Fisher Scientific, Madison WI, USA) equipped with an infrared microscope and in-built diamond single-bounce sampling accessories was used to identify the chemical composition of three cicada species wings. FTIR spectra were collected for 128 scans at a resolution of 8 cm-1 through the 4000-1000 cm-1 region. Nicolet OMNIC V 9 software was used for analysing and extracting data (Shahali et al., 2019). FTIR spectra (wavenumber versus absorbance) were used to identify the chemical composition. Table 3.3 shows the common components, elements and range of wavenumbers (cm-1) existing on an insect wing. Both Amide I and II can be associated with chitin and protein. The existence of the C-H stretching band is associated with wax, which is the main component of cicada wings according to previous studies (Table 3.3) (Movasaghi et al., 2008a, Singh et al., 1993, Tobin et al., 2013).
Table 3.3: Common components, elements and wavenumbers of an insect wing (Aliofkhazraei, 2015, Mistry, 2009, Tobin et al., 2013).
Component Description and range of wavenumber Amide I Mainly C=O stretching coupled to N-H bending, 1610-1695 cm-1 Amide II C-N stretch coupled to N-H bending, 1480-1575 cm-1
-1 CH2, CH3 Symmetric (vs) and anti-symmetric (vas), 2840-3000 cm (CH2-vas 2931-2913)
3.4.2 X-ray photoelectron spectroscopy (XPS) XPS is a high-resolution technique to characterize the quantitative atomic chemical composition of conducting and non-conducting samples. In this technique, monochromatic X-rays in vacuum condition irradiate the surface, and then the intensity and energy of emitted photoelectrons are analyzed to interpret the concentration of the element. These photoelectrons are emitted from 10 nm depth of substrate (Hollander and Jolly, 1970, Wagner and Muilenberg, 1979, Watts, 1994). XPS was used to identify chemical properties of three cicada species wings using
Kratos Axis Supra with a monochromatic AlKα source (1486.7 eV). CasaXPS software
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was used for data analysis for survey and resolution mode. While the main elements of the wing are carbon, oxygen and nitrogen, there are differences in atomic weight and the presence of other elements like silicon. Table 3.4 illustrates the main elements of the wing and their binding energy in XPS (Shahali et al., 2019).
Table 3.4: Main element of the wings and their binding energy.
Element Peak (binding Energy) ev O1s 531 N1s 398 C1s 285 P2s 188 O2s 23 Si2p 103.3
3.5 NANO TOPOGRAPHY ANALYSIS
3.5.1 Nano topography analysis of cicada wings using Helium Ion Microscopy (HIM) Conductive thin film, like gold and platinum, are regularly are used on biological samples to reduce sample charging and damage. Visualizing the nano-topographical features of cicada wings is a big challenge as the coating can produce artefacts on the surface. HIM is a suitable technique for characterising the nanopattern of insect wings without traditional metal coating. The Flood gun compensates for the charge which is generated by the insulation characteristics of the wing sample. Surface topography and geometry of cicada wing surface nanopillars and bacteria interaction with nanopillars were characterized using HIM. High-resolution HIM images from membrane and vein of forewings were obtained using Zeiss Orion Helium Ion Microscope (Zeiss Orion Nanofab, Zeiss, Peabody MA, USA), at 25 kV with a 0.3 PA blanker current. Wing samples were cut into three pieces and fixed on carbon tape to calculate the geometry of nanopillars including base and cap diameter, centre to centre distance, height, nanopillar density, aspect ratio (height/mean diameter) in the membrane and vein. Fifty fields (area of 1 µm × 1 µm) were studied to identify the density of nanopillars (number of pillars/µm). One hundred nanopillars were evaluated to measure the geometry of the nanopillars (e.g. cap diameter, base diameter, centre by centre) for each species in the top view as well as height in 45- degree tilt angle via image J software. The decisive parameters for the Flood gun and
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its definitions are illustrated in Table 3.5. A good starting parameter is Beam current 0.5 pA and Short flood time 1000 ms.
Table 3.5: Flood gun parameters used in Helium Ion Microscopy (HIM).
Parameters Description X, Y deflections Deflects Flood gun electron beam in up/down and lateral direction used to position flood beam onto the desired area of the sample (set the best contrast) - iterate between x and y deflection until best possible contrast is achieved. Flood energy The acceleration voltage of the Flood gun electrons. There is a maximum value, lower and higher energies are less efficient (check contrast/brightness efficiency), recommended value 677 V. Flood time Determines how long the sample is flooded (1000 ms). Focus voltage Focusses the Flood gun electron beam onto the sample surface. Source voltage Heating voltage of filament, usually at 1.53 (recommended). Grid blank time Time between ETD off and Flood gun on, recommended value is 300 ms.
Nano topography analysis of the cicada wings and sample preparation using Scanning Electron Microscopy (SEM) was also employed to image and characterise the wing nanopillars of the wings, fabricated titanium nanopillars and cell interaction because it was easy to set-up and more available than HIM. SEM images of wing nanopillars, fabricated nanopillars and cell interactions were taken using a TESCAN FEG-SEM instrument. Wing samples were fixed on carbon tape on aluminium stud. Samples with the interaction of nanopillars and cells were coated with 8 nm gold using a gold sputter Leica EM SCD005 instrument before imaging. Table 3.6 shows the SEM parameters for imaging the fabricated nanopillars and biological samples including cells.
Table 3.6: Optimum parameter for imaging titanium nanopillars and nanopillars with cell interaction.
Imaging condition kV Beam Spot size Mode Working intensity (nm) distance (mm) Imaging titanium 10-15 7-8 2.8 Resolution (In 4-5 nanopillars beam SEM) Imaging wing and 3-5 4-6 5 Resolution (In 4-5 titanium nanopillars beam SEM) with cell
3.5.2 Transmission Electron Microscopy (TEM): cross-sectional analysis and sample preparation TEM is a great technique for cross-sectional analysis of the biological sample. In TEM microscopy, the electron beam is passed through an extremely thin sample. Interaction of electron beam transmitted through sample forms a high-resolution image
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compared to optical microscopy (Saxena et al., 2010). Electrons are transmitted from a tungsten filament followed by accelerated voltage from 80-100 kV. Magnetic lenses are applied to concentrate the beam to produce an image of the sample (JEOL, 2011). TEM was employed to measure the nanopillar height in wing membranes, validating the tilt image captured by SEM or HIM. In this technique, cross-sectional analysis of 100 nm thick embedded cicada wings in resin was conducted using a JEOL JEM1400. TEM cross-sectional analysis of cicada wings, wing samples with 3-5 mm width and length 10 mm were embedded in LX112 resin, heated at 70oC overnight in the oven before sectioning and imaging. Sectioning was carried out using a microtome (Leica EM UC6 and UC7 equipment) for 100 nm thickness and the geometry of the nanopillars was analysed by Image J. For delicate structures and biological samples, 80 kV was used to minimize damage to the sample and its structure (Shahali et al., 2019).
3.5.3 Atomic Force Microscopy (AFM) for topography analysis and cell conformity AFM is an ultra-high-resolution technique to characterise surface morphology and is composed of a cantilever and a probe with a sharp tip (nano-size tip radius). While the probe approaches the surface, the force between the sharp tip of the probe and surface causes deflections in the cantilever. Tapping and contact mode are mostly used in AFM analysis (Haugstad, 2012, Li, 1997). Tapping mode is a recent technology in AFM, which facilitates high-resolution surface morphology of delicate samples like insect wing and bacteria interaction. Tapping mode is performed in atmosphere air and while the tip of the probe does not have any contact with the surface, the piezo movement makes oscillations in the cantilever with high amplitude (> 20 nm). When the oscillated probe starts to tap the surface lightly, the tip begins to oscillate and move up with high frequency, so vibration amplitude is employed to indicate and evaluate the surface structure (Haugstad, 2012). Generally, the tapping mode system avoids surface adhesion and damage from the probe. AFM analysis was carried out to precisely measure the nanopillar height and analyse bacteria conformity, interaction and mechanical properties of the wing. Wing samples (10 ×10 mm) and the control surface (glass) were analysed using AFM on a MFP-3DTM Asylum instrument. Tapping mode (non-contact) and contact mode were used for imaging (height of nanopillars, bacteria interaction and conformity) and
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mechanical properties of nanopillars, respectively. A HA-NC-B ETALON polysilicon cantilever with silicon tip from TipsNano with length 124 µm and a tip radius of 10 nm was used for contact and non-contact AFM analysis. Tip resonance of 140 kHz and force constant of 3.5 N/m were used for mechanical properties and imaging using Asylum software (Shahali et al., 2019). The Young’s modulus was obtained by analysing the retract curve using the Derjaguin-Muller-Toporov (DMT) model in Asylum software (Equation (3.1)) (Bandara et al., 2017).
4 * 3 =− − ddREFF)( Equation (3.1) adh 3 0
− FFadh is the force that measured the tip in relation to adhesion force, R is the
* tip end radius. − dd0 is the deformation of the sample and E is reduced modulus.
Having poisson's ratio, Young modulus (Es) can be obtained. Because the poisson's ratio is unknown, it is assumed that its value is zero.
3.6 BACTERIA PREPARATION AND BACTERIAL VIABILITY TESTING
Gram-positive bacteria, S. aureus (ATCC 25923) and Gram-negative bacteria, P. aeruginosa (ATCC 27853) were selected as two main sources of orthopaedic infection. Standard plate counting method was used to assess the bactericidal efficiency of PC, AC and PE, control media (CM) and control surface (glass) over 0 hr, 2 hr, 4 hr and 18 hr. This method was previously employed for evaluating the bactericidal effect of dragonfly wing, biomimicked nanopillars on black silicon using RIE (Ivanova et al., 2013) and biomimicked nanopillars of cicada wing on PMMA using NIL (Dickson et al., 2015). All wing samples were rinsed in ethanol and UV sterilized before the plate counting test. Both bacteria were suspended in 5 mL sterile nutrient broth at 37oC overnight to reach OD600 of 0.3. Then OD600 was decreased and fixed at 0.1 by dilution in PBS. The re-suspended cell media with a dilution ratio of 1:10 was incubated at 37°C in triplicate with the samples. Bacteria media in the tube without samples were selected as control media (CM) and glass was used as the control surface. Each triplicate cicada sample and glass sample were put in separate 24 well plates. Each experiment had 500 µL culture volume where a 100 µL of suspension was diluted (1:1000) and spread on a fresh nutrient agar plate. The number of bacteria colonies
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was counted using Image-J software and colony-forming units (CFU/mL) were calculated based on culture volume and dilution factor as shown in Equation (3.2) (Postgate, 1969). PBS is used as test media to limit the exponential growth of bacteria as in nutrient broth. Moreover, it is a widely accepted protocol for bacteria testing in insect wings (Ivanova et al., 2013a, Liao and Shollenberger, 2003).
number of colonies×dilution factor number of colonies×1000 CFU/mL = = volume of cultured on plate 0.1 Equation (3.2)
For bacteria interactions and cell conformity in AFM, SEM and HIM analyses, the bacteria media were discarded, and the sample washed three times with PBS and fixed using 3% glutaraldehyde. Table 3.7 illustrates the post fixing procedure used before SEM, HIM and AFM analyses. After post fixing, all samples were dried in the fume hood overnight. Then, the samples were gold coated (10 nm) for cell interaction and cell conformity analyses (Shahali et al., 2019).
Table 3.7: Standard operation procedure for biological samples in HIM, SEM and AFM.
Post fixing Procedure Time 1. Buffer Rinse (0.1 M cacodylate buffer) 10 min×3 2. Osmium Tetroxide post fixation (1% Osmium Tetroxide in cacodylate buffer) 1 hr 3. Rinse (UHQ water) 10 min×2 5. Dehydration Ethanol 50% 10 min×2 Ethanol 70% 10 min×2 Ethanol 90% 10 min×2 Ethanol 100% 15 min×2 6. HMDS (Hexamethyldisilazane) 30 min×2
3.7 OSTEOBLAST CELL CULTURE AND ALAMARBLUETM ASSAY
The AlamarBlueTM cell proliferation assay is widely used as an important index of cell metabolic rate for quantitative measurement of proliferation and cytotoxicity of the cell in which dye reduction is proportional to the cell metabolic activity. The aim behind testing osteoblasts is to see whether nanopillars have an adverse effect on osteoblast proliferation under normal conditions. The metabolic activity of human osteoblasts was assessed on the control surface (glass), control media (cells only) and three cicada species wings, control titanium (smooth) and nanopillar titanium surfaces. Samples were rinsed with ethanol and UV sterilized prior to the test.
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The cells were transferred to each sample in 48 well plates with a cell density of 10000, followed by incubation with 200µL cell culture including MEM-alpha, 10% FBS and 1×Pen/Strep for the specific time interval. Pen/Strep is added to culture media to avoid bacteria contamination of cells. This protocol is extensively applied in previous studies and the Pen/Strep does not have a negative influence on osteoblast cells (Kizhner et al., 2011, Jaggessar et al., 2018, Elsafadi et al., 2016). However, the effect of nanopillars in inhibiting bacteria contamination in osteoblast cell culture was not studied in this experiment. Eighteen cicada samples (6 samples for each cicada species) and 3 glass samples as control surfaces were used and put in separate wells for the analysis of the metabolic activity of the cicada wing. The 6 titanium nanopillar samples plus 6 titanium control samples were selected for the analysis of the metabolic activity of the titanium. The AlamarBlueTM assay was carried out in accordance with the manufacturer’s instruction. For all time intervals, the cell media were taken out and discarded from each well and samples were washed three times with sterile PBS then incubated for 2 hr with 10% AlamarBlueTM solution. Absorbance in wavelength of 550 and 595 nm was measured using a BIO-RAD Benchmark PlusTM instrument and dye percentage decrease was determined to assess the metabolic activity (Shahali et al., 2019).
3.8 STATISTICAL ANALYSIS
Statistical analyses were performed using 2-Way ANOVAs and t-tests with Tukey’s multiple comparison tests in Microsoft Excel to compare two means from two independent samples (Shahali et al., 2019). Significant results were illustrated in each figure.
3.9 ELECTRON BEAM LITHOGRAPHY (EBL)
In EBL, the reflected electron beam from the sample can be used to fabricate the E beam resist which is previously applied on the substrate. Due to the low momentum of electrons, EBL can fabricate polymer resist such as PMMA (Poly (methyl methacrylate)), PEG Poly (ethylene glycol) and PAA Poly (acrylic acid). As shown in Figure 2.50, the electron can produce negative tone by crosslinking and positive tones by degradation depending on the type of mask. Developer solution is applied to remove
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Laurell Technologies Corporation was used to apply PMMA resist on the substrate for EBL sample preparation (Shahali et al., 2019). 10 mm × 10 mm silicon wafers were sonicated and cleaned in Acetone, Isopropyl
Alcohol (IPA) and deionized water for 5 min, followed by drying with N2 gas for 1 min. Cleaned silicon wafers were heated on the hot plate at 100oC for 5 min to remove impurities and moisture from the surface prior to spin coating. Samples were kept in the vacuum chamber for E-beam evaporation a night before deposition. Samples were coated by 30 nm pure titanium using E-beam evaporation (PVD 75 K.J. Lesker) with the rate of 0.1 A/s based on the pressure of 4.8 ± 10-7 Torr and voltage of 10 kV. Samples were heated again on a hot plate at 100oC for 10 min prior to spin coating to remove organic material and impurities and enhance resist bonding. The substrate was spin-coated with PMMA to achieve the desired thickness in 45s (Figure 3.3). To apply the multilayer PMMA, each layer was soft-baked at 170oC for 10 min except the last layer to remove the solvent from the resist layer and enhance lift-off. Afterwards, samples were exposed by E-beam in a TESCAN-MIRA3 EBL system to create the pattern on PMMA. Exposed samples were developed in MIBK: IPA (1:3). After development, the sample was coated again with 200-250 nm titanium under a deposition rate of 0.1 A/s. The lift-off was performed to remove the PMMA from the substrate after coating by using acetone or NMP (1-methyl-2-pyrrolidinone). NMP had a better lift-off performance at 70 oC for 4 hrs compared to acetone. The fabrication process for biomimicked nanopillars using EBL is illustrated in Figure 3.4.
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Figure 3.4: The fabrication process for biomimicked nanopillars using EBL.
3.9.2 PMMA thickness measurement Ellipsometry is a noncontact, and non-invasive optical technique, which can measure the film thickness from sub-nanometres to a few microns. In this method, the interface between the reflection of light from the surface and light passing through the film determines the film thickness. The interference includes both amplitude and phase information. Film thickness is extracted by measuring Psi (Ψ) and Delta (∆) through
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different algorithms and equations to generate the model, which defines the interaction of light with the substrate (Tompkins and Hilfiker, 2016). In this research, ellipsometry was used to measure the thin titanium and PMMA film on the silicon wafer. The Cauchy model was employed to measure the thickness of PMMA on titanium and the B-Spline model was used for measuring titanium film on the silicon wafer. Titanium film, single layer and multilayers of PMMA 495 A4, PMMA 950 A4 and PMMA 950 A2 were measured using a M-2000 Ellipsometer (J. A. Woollam Co., Inc.) (Shahali et al., 2019).
3.9.3 Titanium coating surface roughness measurement Titanium deposition thickness and surface roughness were measured by Stylus profilometer (Bruker Dektak XT). Stylus profilometers apply physical contact between tip and surface to extract the topographical information of surfaces like surface roughness and deposition thickness (Poon and Bhushan, 1995). The surface roughness of titanium coated samples was measured with the surface roughness of
Ra = 4.5 ± 2.5 nm.
3.9.4 TESCAN EBL system and process parameters EBL has been developed as a flexible and reliable technique for nano-fabrication applications. In general, the EBL system is composed of SEM, pattern generator and beam blanker. In this research, TESCAN-MIRA3 field emission electron gun SEM was used for both imaging and EBL. MIRA3 equipped with electrostatic beam blanker and pattern generator was employed to control the electron beam intensity (FEG-SEM, 2013, Tesarova, 2015, Khajehpour, 2014). In MIRA3, the pattern generator software (DrawBeam) controls the electron beam and creates a feature design from centimetre to nanometre-scale on the electron beam resist. DrawBeam in MIRA3 is equipped with minimal pixel dwell time (20 ns) and proximity effect correction. It enables drawing the design in the software and then importing the graphic in AutoCAD formats (GDSII and DXF) (EBL, 2014) (Figure 3.5). The specifications of TESCAN MIRA3 are given in Table 3.8.
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Figure 3.5: Schematic of the MIRA3 EBL system (FEG-SEM, 2013).
Table 3.8: TESCAN MIRA3 EBL system specifications (EBL, 2014).
Filament type Schottky field emission electron Acceleration Voltage 200 eV-30 KeV Tip size 20 nm Frequency 50 MHz Emission current 300 µm Maximum pixel dwell time 20 ns Write field size 1 × 1 µm – 1 × 1 mm Spot size 2.8 nm at 30 Kev Beam blanker Electrostatic beam blanker (10 MHz) Working distance 2 mm - 15 mm Sample size 2 inch × 2 inches Pattern generator Resolution 16-bit scanning ramp DACs (65,536 x 65,536 virtual write field) Step size or Exp. Pitch Write field and it should be less than 4*DAC
Fixed parameters, variable parameters and their description in the EBL- MIRA3-TESCAN are summarized below in Tables 3.9 and 3.10, respectively. The exposure factor which multiplies the dwell time is considered as dose test in TESCAN system.
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Table 3.9: Fixed-Parameter and definition in TESCAN-EBL (EBL, 2014).
Parameters Definition Scan Number of electron beam scans per object. In this project, it was fixed at 1. Scanning Different types of scanning path in DrawBeam (flyback mode, Zigzag) path Settle factor Period needed for the beam to return from the end of the scanning line to the beginning of the next one to minimize dynamic distortions. It was set to 1 for all experiments. Orientation Required for the circular feature and indicates the orientation of the beam (clockwise or counter clockwise). It was set to clockwise in all experiments. Scan angle Indicates the scan angle of the beam and was set to 0 for all experiments. Beam Beam current can be read using the Faraday Cup located on the stage and calculated current based on the mathematical model in the software. EBL Mode Four modes are available in MIRA3 for imaging and EBL including Resolution, depth mode, a field mode and wide-field mode. In this research, the resolution mode was selected to achieve the highest resolution. Spot size Beam spot diameter in resolution mode after alignment of the EBL system. Dose Amount of delivered energy per exposure area. According to the material library, it is (µC/cm2) fixed at 350 µC/cm2 for PMMA. Accuracy Indicates the resolution of the pixel map and there str three options coarse and fine. It was fixed at fine. Proximity Principally the result of back-scattered electrons, which are reflected from the substrate correction back into the resist layer above. Proximity effect correction is a great tool for precise exposure of small feature (less than 50µm) Angular An important parameter of the gun which depends mainly on the extractor voltage. The intensity value of this parameter is important for electron emitter lifetime. The angular intensity is also used for the precise calculation of the beam current on the sample for EBL.
Table 3.10: Variable parameters in TESCAN-EBL (EBL, 2014).
Parameters Definition Write field size The area of nano feature design. Low file sizes increase resolution and decrease (µm) dwell time Exp. factor Number that multiplies the dwell time (period which the electron beam remains at one exposure point during the scanning) Dwell time Time needed for a beam to stay in one spot. Spacing Relative value indicating step exposure regarding spot size. DAC resolution Resolution of pattern generator that changes with pitch and write field size. (pitch:4 × DOC) Exp. Pitch Distance between two exposed pixels calculated by Spacing × Spot size
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3.9.5 Monte Carlo simulation Monte Carlo simulation was performed in CASINO v2.5.1.0 to optimize the process parameters, such as beam energy, before the fabrication experiment. The simulation was carried out based on 5000 x number of the electrons, with an average beam diameter of 2.8 nm as the average spot size in the MIRA3 system. Both the forward and backward election scattering trajectories on different thicknesses of PMMA on 30 nm titanium and SiC substrate were carried out during the simulation.
3.9.6 Electron scattering in EBL Electron scattering in EBL can be categorized as forward and backward scattering events. Forward scattering (shown in black arrow, Figure 3.6) occurs almost all the time under the small angles and it is mainly inelastic, generating secondary electrons (SEI) with a few eV kinetic energy. Backward scattering (shown in red, Figure 3.6) occurs occasionally under the large angles and is mainly elastic, generating high kinetic energy. Electrons with a typical few eV kinetic energy generated from secondary electrons (SEI and SEII) are the main reason for the resist exposure (Cheng, 2018). Figure 3.6 shows the electron scattering to the resist. The proximity effect in EBL originates from backscattered electrons, which possess higher energy and expose the area of resist out of the designed pattern. It can be affected by the geometry of the pattern, beam acceleration voltage, resist material and beam spot size. This phenomenon occurs mostly in highly dense features present in a small area (Cheng, 2008).
Figure 3.6: Electron scattering phenomena in EBL (Cheng, 2018).
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Surface charging effect is another common problem in EBL in which the extra charges are trapped close to the substrate surface, causing pattern distortion on the resist. Coating the substrate with the conductive layer before applying the E-beam resist can minimize the charging effect (Cheng, 2008). In this research, 30 nm titanium is deposited on a silicon wafer to decrease the charging effect. In summary, in this research, the features were small (diameter less than 200 nm) and the highest voltage was applied (30 kV), therefore the proximity effect did not cause beam diffraction and overlap of the feature during the fabrication process. The substrate was also coated with titanium to minimize the surface charging.
3.10 TITANIUM DEPOSITION
In the fabrication of metallic biomimicked nanopillars, the material can be deposited by using thermal, electron beam evaporation and magnetron sputtering techniques.
3.10.1 Thermal evaporation Thermal evaporation is a popular metal deposition technique in which the chamber should be vacuumed before the deposition process and the target material in the crucible is heated using two possible systems, resistive or inductive. When the target is heated to a specific temperature, it is vaporized and deposited on the substrate. The deposition thickness is normally evaluated by a quartz crystal oscillator which is positioned near the substrate (Figure 3.7). The thermal evaporation process normally processes high temperatures in the chamber which is not suitable for metal deposition on PMMA resist.
3.10.2 DC and RF sputtering Sputtering is a common evaporation technique in the solar industry due to its capability to deposit different materials and better step coverage. Sputtering coating equipment is equipped with direct current (DC) and radio frequency (RF) sputtering magnetron sources (Figure 3.8). In DC sputtering, Argon gas is applied to produce plasma which is constrained near the source target using a magnetic field. The surface of the target is bombarded by energetic plasma ions and target atoms are detached from the target surface and
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bounced off in the chamber and finally, the bounced off atoms are deposited in the substrate just above the source target. Substrate rotation and heating (for metal oxide) are applied for uniform surface with high step coverage. Kurt J. Lesker recommended E-beam evaporation (as an excellent condition) and DC sputtering for deposition of titanium on the substrate. In this research, E-beam evaporation and DC sputtering could be used to producing thin film deposition on PMMA by electron beam lithography.
Figure 3.7: Schematic of thermal deposition.
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Figure 3.8: DC sputtering operation principle in PVD 75 Kurt J. Lesker magnetron sputtering. 3.10.3 Electron beam evaporation In E-beam evaporation, electron beam flux bent is generated by a strong magnetic field applied to heat the charge in the crucible. In most E-beam evaporation systems, the tungsten filament is responsible for the emission of the electron beam with high energy (Jonker, 1990). Electron beam flux can generate the focused heating spot on the crucible, where the only charge is heated, and crucible is cooled to eliminate contamination (Figure 3.9).
Figure 3.9: E-beam evaporation system.
While E-beam evaporation can be applied to deposit a wide range of materials, it can cause electron beam radiation damage and blistering or peeling in PMMA (Broers et al., 1996, Melville, 2006). Kurt J. Lesker suggested using E-beam evaporation to generate excellent thin films of titanium. In this research, an E-beam evaporation system with 10 kV (Kurt J. Lesker PVD75) was mainly employed for deposition of titanium used to coat the nanoholes, fabricated by EBL (Table 3.11).
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Table 3.11: E-beam evaporation parameters for titanium deposition.
Parameters Value Z factor 0.628 Density 4.5 (gm/cc) Vacuum pressure 2.4-3.6 × 10-7 Ramp 1 power 10% Ramp 2 power 12% Final Thickness 0.3 KÅ for sample preparation (silicon wafer coating) and 2 KÅ for coating PMMA after EBL. Deposition Rate 0.2 Å/s for sample preparation - 0.4 Å/s for final deposition P, I, D 40, 1 ,0.01
3.10.4 Step coverage in metal deposition Step coverage shows the conformity and uniformity of deposited material at the top of the rough surface or fabricated area (top, bottom and wall). Step coverage is a key player in selecting the coating method for titanium on PMMA electron beam exposed resist because lower step coverage can produce better lift-off and repeatable results. In thermal and E-beam evaporation methods (Figure 3.10 (a), (b)) discontinuous coating is produced only at the edge and bottom of the hole while the wall is not affected with deposition, confirming that poor step coverage had better lift-off performance (Cheng, 2008, Campbell, 1996). The thermal coating has a limitation of coating on PMMA due to the high temperature in the chamber during titanium deposition.
Figure 3.10: Simple schematic of step coverage in the physical deposition method, (a) thermal evaporation, (b) E-beam evaporation, and (c) sputtering.
E-beam evaporation can cause radiation damage and blistering or peeling damage for polymers like PMMA (Broers et al., 1996, Melville, 2006). New
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technology in Kurt J. Lesker PVD75 reduces the damaging effect by using high vacuumed chamber and the large distance between the target and substrate resulting in minimal blistering and peeling. In sputtering (Fig. 3.10 (c)), the continuous coating occurs at the edge, wall and bottom of the hole, causing high step coverage which is not ideal for lift-off. On the other hand, sputtering is a more aggressive deposition method compared to the E-beam evaporation, which can produce the early blockage at the top of the nano-holes on the PMMA, meaning that evaporated material accumulates at the top of the nano-hole gate causing a blockage. In conclusion, the E-beam evaporation system with 10 kV (Kurt J. Lesker PVD75) was used to generate the lower step coverage compared to sputtering.
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Chapter 4: Surface Characteristics, Cell Interaction, Bactericidal Properties and Biocompatibility of Nanopillars of Three Cicada Species
4.1 OVERVIEW
This chapter presents the experimental results of objective 1 (stated in section 3.2). In this chapter, the geometry and topography of the cicada wing’s nanopillars were analysed by HIM, TEM and AFM and the chemical properties of the wing surfaces were characterised by FTIR and XPS. The mechanical properties of cicada nanopillars were then measured by AFM. Cell morphology and bacterial interaction (P. aeruginosa and S. aureus) with nanopillars were evaluated by SEM and AFM. Finally, the bactericidal efficiency and cytocompatibility were determined by plate counting method and the AlamarBlueTM assay test, respectively.
4.2 CICADA WING NANO TOPOGRAPHY
To identify the correlation between nanopillars and bactericidal characteristics, an in-depth analysis of nanotopography is vital. In this research SEM, HIM, TEM and AFM were employed to analyse of the density and geometry (e.g. diameter, height, centre to centre distance and aspect ratio) of nanopillars of three cicada species wings. Analysis of both hindwings and forewings show that veins and membranes of both sides of wings have consistent and uniform nanopillar arrays without damage and defect (Figure 4.1). The geometry of nanopillars differs among the species (Table 4.1). The dimensions of nanopillars including cap and base diameter, height and centre to centre distance on the vein are two times higher than in the membrane in all three species. The heights of the wing membrane nanopillars of PC, AC and PE are 202.6 nm, 213 nm and 211.2 nm, respectively (Table 4.1).
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Figure 4.1 Psaltoda claripennis (PC), Aleeta curvicosta (AC), and Palapsalta eyrei (PE) (scale bars = 1 cm) (left). HIM images of nanopillars of wing membranes from the top view and tilt angle 300 (middle). HIM top images of veins nanopillars (scale bars = 200 nm) (right) (Shahali et al., 2019).
Table 4.1: Nanopillar geometry of veins and membranes for three cicada species wings (Shahali et al., 2019).
Cap Base Height Centre to Aspect Density diameter diameter (nm) centre distance ratio (n/µm2) (nm) (nm) (nm) PC Membrane 72.4±5.6 136.9±11.4 202.6±10.9 176.4±12.7 1.9±0.2 32.2±2.4 Vein 149.4±13.9 300±34.3 345.6±39.2 393.8±50.3 1.5±0.2 5.2±0.7 AC Membrane 65.4±4.6 125.7±7.8 213±17.4 171.3±12.5 2.2±0.2 40.2±1.3 Vein 153.9±25.2 322.6±30.6 244.4±20.9 387.3±46.7 1.2±0.1 7.6±1.8 PE Membrane 60.3±3.7 123.2±7.5 211.2±19.1 155.9±13.4 2.3±0.3 40±1.6 Vein 110.7±9.5 218.7±25 281.6±22.5 335.8±38.9 1.7±0.2 11.3±1.1
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Density (number of nanopillars/μm2) and aspect ratio of nanopillars for veins and membranes in all three species wings are illustrated in Figures 4.2 and 4.3. In general, the density of nanopillars is higher in the membrane compared to the vein. The density of nanopillars on the membrane of AC is 40.2 ± 1.3 n/µm2 and PE is 40 ± 1.6 n/µm2, both of which are higher than PC with 32.2 ± 2.4 n/µm2. The aspect ratios of wing nanopillars are roughly 2 among the three wings membranes and the aspect ratio of PE (2.3 ± 0.3) is slightly higher than PC and PE. The aspect ratio is a key player in the bactericidal effect of nanopillars (Modaresifar et al., 2019). While the topography of nanopillars on veins and membranes are the same, the density and aspect ratio of nanopillars on the membrane are considerably higher than on veins in all species. The vein had a course pattern which provides flexibility and support during flight (Appel et al., 2015, Dirks and Taylor, 2012).
Figure 4.2: Density of nanopillars (n/µm2) in veins and membranes for the three cicada species wings (Shahali et al., 2019).
Figure 4.3: Aspect ratio of nanopillars in veins and membranes for the three cicada species wings (Shahali et al., 2019).
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AFM analysis was employed to confirm the geometry of the nanopillars of the three cicada wings. Nano topography of PC, AC and PE are illustrated in Figures 4.4, 4.5 and 4.6, respectively. The results show that PE has a finer nanopillar compared to PC and AC.
Figure 4.4: AFM image (left) and line profiles (right) of nanopillars of PC wings (scanning area: 2 µm × 2 µm) (Shahali et al., 2019).
Figure 4.5: AFM image (left) and line profiles (right) of nanopillars of AC wings (scanning area: 2 µm × 2 µm). (Shahali et al., 2019).
Figure 4.6: AFM image (left) and line profiles (right) of nanopillars of PE wings (scanning area: 2 µm × 2 µm) (Shahali et al., 2019).
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AFM analysis also shows that the three cicada wings have a similar surface roughness value with little difference among the species (Table 4.2).
Table 4.2: Surface roughness of the three cicada species wings and control surface analysed by AFM analysis (Shahali et al., 2019).
Sample Ra (nm) Rq (nm) Rmax(nm) Glass 0.008 ± 0.001 0.07 ± 0.08 1.08 ± 0.5 PC 27.7 ± 3.6 34.6 ± 4.4 119.7 ± 3.2 AC 39.5 ± 12.2 48.7 ± 12.8 159.6 ± 10.7 PE 39.2 ± 13.1 47.9 ± 17.5 168.6 ± 6.1
TEM was also applied for in-depth analysis of nanopillar topography. Pear- shapes of nanopillars and the difference between top and base diameter on the membrane are observed in the TEM analysis. Figures 4.7, 4.8 and 4.9 show a cross- sectional view of nanopillars on the membrane of PC, AC and PE, respectively, confirming a higher aspect ratio of AC and PE compared to PC.
Figure 4.7: TEM cross-section image of PC wing membranes (Shahali et al., 2019).
Figure 4.8: TEM cross-section image of AC wing membranes (Shahali et al., 2019).
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Figure 4.9: TEM cross-section image of PE wing membranes (Shahali et al., 2019).
4.3 CHEMICAL CHARACTERISTICS OF WING NANOPILLARS
In this research, in order to confirm that no chemical substance associated with the bactericidal effect of natural cicada wings, the chemical characteristic of the cicada wing is crucial. In this research, FTIR and XPS were employed to characterize the chemical composition of the nanopillars.
4.3.1 FTIR analysis Chemical characteristic analyses of the nanopillars show that all wings have a similar chemical composition with minor deviation. As shown in Figure 4.8, the peak between 1600-1700 cm-1 is amide I and it is due to C=O bond stretching weakly coupled with N-H bending and C-N stretching. Amide II ranges from 1500- 1600 cm-1, including C-N stretching strongly coupled with N-H bending. Amide III ranges from 1200-1350 cm-1 which contains N-H in-plane bending coupled with C-N stretching and C-H and N-H deformation vibration (Movasaghi et al., 2008a, Singh et al., 1993, Tobin et al., 2013). The peak between -1 2970-2950 cm is attribute to C-H (asym/sym/stretch) and it is methyl (-CH3). The peaks in the range of 2935-2915 cm-1 and 2865-2845 cm-1 are assigned to C-H
(asym./sym/stretch) and are methylene >CH2 (Coates, 2006, Lin-Vien et al., 1991, Tobin et al., 2013). The peak between 1470-1430 cm-1 and 1380-1370 cm-1 is attributed to C-H (asym./sym) and methyl (-CH3). Peak 1072 is the Phosphate I band for two different C-O vibrations of deoxyribose in DNA in a disordering structure (Theophilou et al., 2015). In summary, the FTIR analysis shows that the wings of the
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three cicada species possess amide (I, II and III), carbonyl, methyl, methylene, chitin (OH stretch), waxes and carboxylic acids (Figure 4.10).
Figure 4.10: Chemical characteristics of cicada wings using FTIR (Shahali et al., 2019).
4.3.2 XPS analysis The XPS analysis, identified carbon, nitrogen and oxygen as the major components of the three cicada species wings (Figure 4.11). The atomic content of carbon, oxygen and nitrogen differed slightly among the three species (Table 4.3). Carbon (C1s), with the highest peak of ~285 eV, is a major component of the wing while the second and third components are oxygen (~530 eV for O1s and 23 eV for O2s) and nitrogen (N1s) with ~397 eV. The amount of carbon is approximately the same in the three cicada wings. AC has the highest amount of O1s with the value of 12.7 while it is 9.5 and 9.1 for PC and AC, respectively. The atomic content of N1s is highest in PC with the value of 1.7 while it is 1.27 and 0.5 for AC and PE, respectively (Table 4.3). XPS analysis also confirms the existence of amide, protein and carboxylic acids on cicada wings. In summary, the chemical characteristics of nanopillars agreed with the published results and these elements do not have a bactericidal effect (Ivanova et al., 2013, Mainwaring et al., 2016, Nguyen et al., 2013, Ivanova et al., 2012).
Chapter 4: Surface Characteristics, Cell Interaction, Bactericidal Properties and Biocompatibility of Nanopillars of Three Cicada Species 93
Figure 4.11: XPS analysis of the wing surfaces of the three cicada species (Shahali et al., 2019).
Table 4.3: Atomic content and binding energy extracted by XPS spectra for the three cicada species wings (b.e: binding energy, a.c: atomic number) (Shahali et al., 2019).
Element Parameters PC AC PE C1s b.e 285 285 285 a.c 86.5 84 88.7 O1s b.e 529 530 529 a.c 9.5 12.7 9.1 N1s b.e 397 397 397 a.c 1.7 1.27 0.5
4.4 MECHANICAL CHARACTERISTICS OF CICADA WING NANOPILLARS USING AFM
While the direct influence of the mechanical properties of nanopillars is still unknown, the mechanical stability of nanopillars can affect the killing mechanism. AFM contact mode was employed to analyse the mechanical properties of the nanopillars for AC, PC, PE and glass as a control surface. HA-NC-B ETALON polysilicon cantilever with silicon tip form TipsNano with the length of 124 µm and a tip radius of 10 nm was used for contact and non-contact AFM analysis. The Young’s modulus was obtained by analysing the retract curve using the Derjaguin-Muller- Toporov (DMT) model in Asylum software. Applied force for calibration on glass was
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50 nN and on the surface of the cicada wings was 3 nN. Force mapping was employed to analyse the adhesion force and extract the average reduced modulus in 2 μm × 2 μm samples of the three cicada species wings and control surface (Figures 4.12, 4.13 and 4.14). The average reduced Young's modulus for all substrates is shown in Table 4.4. PC, AC and PE have approximately the same Young's modulus with the value of 45.34±6.7 MPa, 48.16±7 MPa and 35.69±5.2 MPa respectively which is consonant with recent findings on dragonfly wings (Bandara et al., 2017).
Figure 4.12: Force mapping on 2 μm × 2 μm of AC wing: (a) adhesion force map, (b) reduced modulus force map: (c) and (d) calculated reduced modulus, (d) Force cure on one point (Applied force is 5 Nn).
Figure 4.13: Force mapping on 2 μm × 2 μm of PC wing: (a) adhesion force map, (b) reduced modulus force map.
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Figure 4.14: Force mapping on 2 μm × 2 μm of PE wing: (a) adhesion force map, (b) reduced modulus force map.
Table 4.4: The average reduced Young's modulus for AC, PC, PE and glass.
Substrate Reduced Modulus (MPa) PC 45.34±6.7 AC 48.16±7 PE 35.69±5.2 Glass 218 ± 26
4.5 ANALYSIS OF BACTERIA INTERACTION WITH NANOPILLARS
The arrays of nanopillars of the three cicada species wings produce different levels of damage on the membrane of both P. aeruginosa and S. aureus. As seen in Figures 4.15, 4.16 and 4.17, the nanopillars of wing membranes sink into the bacterial membrane, causing damage to the membranes of both bacteria species within 18 hrs. Spherical and rod shape bacteria are trapped among the vein nanopillars, remaining undamaged due to lower density and aspect ratios.
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Figure 4.15: (A, B) SEM images of P. aeruginosa attachment on vein and membrane of PC after 18 hrs, (C, D) SEM images of S. aureus attachment on vein and membrane of PC after 18 hrs (left column: membrane, right column: vein, Scale bars = 1 µm) (Shahali et al., 2019).
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Figure 4.16: (E, F) SEM images of P. aeruginosa attachment on vein and membrane of AC after 18 hrs, (G, H) SEM images of S. aureus attachment on vein and membrane of AC after 18 hrs (left column: membrane, right column: vein, Scale bars = 1 µm) (Shahali et al., 2019).
Interactions between P. aeruginosa and S. aureus and the control surface (glass) are shown in Figures 4.18 and 4.19 after 18 hrs. The bacteria cells tend to have strong interactions with nanopillars, sinking into nanopillars, while on the control surface (glass), bacteria can find no feature to attach to, remaining undamaged on the surface. These results are consistent with recent research on cicada wings (Kelleher et al., 2015, Hasan et al., 2012, Ivanova et al., 2012).
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Figure 4.17: (I, J) SEM images of P. aeruginosa attachment on vein and membrane of PE after 18 hrs, (K, L) SEM images of S. aureus attachment on vein and membrane of PE after 18 hrs (left column: membrane, right column: vein, Scale bars = 1 µm) (Shahali et al., 2019).
Figure 4.18: Interaction of P. aeruginosa and the control surface (glass, unpatterned) after 18 hrs (Shahali et al., 2019).
Chapter 4: Surface Characteristics, Cell Interaction, Bactericidal Properties and Biocompatibility of Nanopillars of Three Cicada Species 99
Figure 4.19: Interaction of S. aureus and control surface (glass, unpatterned) after 18 hrs (Shahali et al., 2019).
Nanopillars of wing membranes damage the bacterial cells more than vein nanopillars due to the higher density and aspect ratio of membrane nanopillars. Even with the lower density and aspect ratio of nanopillars, the cell membrane is ruptured on the vein. However, due to limitations of biological techniques and the small area of vein, a quantitative comparison between vein and membrane for the bactericidal effect of nanopillars cannot be conducted. AFM tapping mode was also applied for the analysis of the interaction between nanopillars and bacteria in more detail. P. aeruginosa strongly interacts with nanopillars and was punctured (Figure 4.20). Bright zones (A and B) are not fully punctured while the dark zones (B) are fully punctured. The partially depleted area has a height of 150 nm (C) while the fully depleted area has a height of 50 nm (B), P. aeruginosa needs to sink 100 nm to be fully punctured.
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Figure 4.20: AFM image of the interaction between P. aeruginosa and nanopillars of PE after 18 hrs, (a) 2D AFM profile of punctured P. aeruginosa on nanopillars, (b) 3D AFM image of punctured P. aeruginosa on nanopillars, (c) Line profile for areas A, B, C.
The morphology of P. aeruginosa is affected by the surface texture (Figure 4.21). When the bacteria attached on nanopillars (A) the height of bacteria is 150 nm while the height is 59 nm for punctured bacteria (B) and 314 nm for bacteria on glass (C). When the bacteria attach to nanopillars, the height decreased by 52% due to the initial attachment and when it sinks into the nanopillar, the height is reduced by 80% due to cytoplasm leakage compared to the bacteria on the glass.
Chapter 4: Surface Characteristics, Cell Interaction, Bactericidal Properties and Biocompatibility of Nanopillars of Three Cicada Species 101
Figure 4.21: AFM analysis of P. aeruginosa on nanopillars and control surface after 18 hrs: (a) AFM analysis of penetrated bacteria and unpenetrated P. aeruginosa on wing nanopillars, (b) 3D AFM image of penetrated bacteria and unpenetrated P. aeruginosa on nanopillars, (c) AFM analysis of penetrated bacteria and unpenetrated P. aeruginosa on the control surface (glass), (d) AFM profile of line A, (e) AFM profile of line B, (f) AFM profile of line C.
S. aureus bacteria cells also interact with nanopillars and its morphology is affected by the nanopillars (Figure 4.22). Undamaged S. aureus cells attach to nanopillars (Figure 4.22 (a) and (b)) have a height of 453 nm while punctured S. aureus have a height of 176 nm on nanopillars due to cell wall damage and cytoplasm leakage. Undamaged S. aureus on glass have a height of 753 nm.
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Figure 4.22: AFM analysis of S. aureus on nanopillars and control surface after 18 hrs: (a) AFM analysis of undamaged S. aureus on nanopillars, (b) AFM line profile (Line A), (c) punctured colony of S. aureus on nanopillars, (d) AFM line profile (Line B), (e) AFM analysis of S. aureus on the control surface (glass), (f) AFM line profile (Line C).
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4.6 ANALYSIS OF BACTERICIDAL EFFICIENCY USING THE PLATE COUNTING METHOD
Bactericidal efficiency analyses of PC, AC and PE, control media and control surfaces against P. aeruginosa and S. aureus were carried out using the standard plate count method over time intervals of 2, 4, and 18 hrs.
4.6.1 Analysis of bactericidal efficiency of P. aeruginosa Cicada wings have considerably higher bactericidal efficiency (p < 0.0001) against Gram-negative bacteria P. aeruginosa after 18 hrs in comparison with 2 hr and 4 hr. While there is no noticeable difference among the three species wings in killing P. aeruginosa cells, PE shows considerable bactericidal efficiency (p < 0.0001) after 4 hrs and 18 hrs compared to the other species (Figure 4.23, Figure 4.24 and Appendix 1). As shown in Figure 4.23, the bacteria did not grow significantly on the control media and control surface within 4 hrs because PBS was used as a dilution media in this study thereby, bacteria can be preserved for a long time without growing and multiplying (Liao and Shollenberger, 2003).
Figure 4.23: Colony-forming unit (bactericidal activity) of P. aeruginosa on control media (CM), control surface (glass), PC, AC and PE. (Statistically significant differences (p < 0.05) compared to CM, glass, PC and AC are indicated by the symbols +, *, φ and Ɵ, respectively).
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Figure 4.24: Calculated colony-forming unit of P. aeruginosa on well plates for control media (CM), control surface (glass) and PE cicada wing for time intervals 0, 2, 4, and 18 hrs.
4.6.2 Analysis of bactericidal efficiency of S. aureus The growth pattern of S. aureus (coccoid) differs from P. aeruginosa (rod- shaped) cells. While there is no noticeable difference among the wings for killing S. aureus cells, AC shows considerable bactericidal efficiency (p < 0.0001) after 18 hrs. The number of S. aureus colonies on PC, AC and PE decrease to 2.32 × 106, 2.12 × 106 and 1.85 × 106 (CFU/mL), respectively, compared to glass and control media with 2.52 × 106 (CFU/mL). AC nanopillar surfaces produce the highest bactericidal efficiency with 1.43 × 106 after 18 hrs compared to PC with 1.53 × 106 and PE with 1.48 × 106 (Figures 4.25 and 4.26 and Appendix 1).
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Figure 4.25: Colony-forming unit (bactericidal activity) of S. aureus on control media (CM), control surface (glass), PC, AC and PE (Statistically significant differences (p < 0.05) compared to CM, glass, PC and AC are indicated by the symbols +, *, φ and Ɵ, respectively).
Figure 4.26: Calculated colony-forming unit of S. aureus on well plates for control media (CM), control surface (glass) and AC cicada wing for time intervals 0, 2, 4, 18 hrs.
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In summary, nanopillars of PE wings produced the highest bactericidal efficiency against P. aeruginosa in all intervals and S. aureus after 2 and 4 hrs. These results are likely due to the higher nanopillar aspect ratio and density in comparison with PC and AC. The results of bacterial interaction analysis using SEM also confirm that the reduction in colony counting is due to the existence of nanopillars on the surfaces and concurs with earlier research studies. While the bacterial membrane damage and bactericidal effect of nanopillars are significant in qualitative analyses, the valid number of dead bacteria is not indicated in the plate counting method because the colony-forming unit is calculated per millilitre of nutrient broth. On the other hand, selected 1 mL media samples contain only suspended bacteria not attached bacteria and divide near the nanopillar surface, causing a small reduction in colonies forming compared to the whole volume of the bacteria suspension. The bactericidal effect of nanopillars on any substrate cannot be technically compared to bactericidal characteristics of antibiotics or any antibacterial solution. The killing mechanism of chemical-based antibacterial solutions (e.g. antibiotics and detergents) occurs in media, causing 1, 2 and 3 log decreases of colony-forming but it is not mechanical surface-based. It is important to standardize the level of the bactericidal effect of physical-mechanical surfaces (nanostructures and nanopillars) and chemical media (antibiotics). For instance, a 90% (2 log) decrease of colony units in contact with an antibiotic solution can be equivalent to a 50% reduction in contact with nanopillar surfaces.
4.7 CYTOCOMPATIBILITY OF THE CICADA WING NANOPILLARS
Orthopaedic titanium implants and other biomedical devices should possess not only antibacterial properties but also biocompatibility for human cells like osteoblasts. In this research, cytocompatibility (cellular metabolic activity) of osteoblast cells on the wings of three cicada species was performed by AlamarBlueTM assay. To evaluate the cytotoxicity of cicada wings, osteoblast cells were incubated on the wings, followed by measuring cellular metabolic activity in 4 and 24 hr intervals. As shown in Figures 4.27, 4.28 and 4.29, the osteoblast cells spread and grew on the nanopillar arrays of cicada wings within 24 hrs and their actin filaments proliferated uniformly on the nanopillars without any defects on the cell structure.
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Figure 4.27: SEM image of osteoblast and PC wing membranes after 24 hrs (Scale bars, left = 10 µm, right = 2 µm) (Shahali et al., 2019).
Figure 4.28: SEM image of osteoblast and AC wing membranes after 24 hrs (Scale bars, left = 10 µm, right = 2 µm) (Shahali et al., 2019).
Figure 4.29: SEM image of osteoblast and AC wing membranes after 24 hrs (Scale bars, left = 10 µm, right = 2 µm) (Shahali et al., 2019).
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From the AlamarBlueTM assay analysis, it was observed that nanopillars of cicada wings do not influence osteoblast metabolic activity (Figure 4.30), presenting cytocompatibility which concurs with previous studies on planthopper wings (Watson et al., 2017). The three cicada species wings possessed higher cellular metabolic activity compared to the control surface.
Figure 4.30: AlamarBlueTM assay results of osteoblasts on control media (CM), glass, PC, AC and PE after 4 and 24 hrs (Statistically significant differences (p < 0.05) compared to CM and glass are shown by the symbols + and *, respectively) (Shahali et al., 2019).
4.8 MAIN FINDINGS AND REMARKS
In this chapter, the nano topography of the three cicada wings and their chemical composition were characterised using HIM, SEM, TEM, AFM, FTIR and XPS. The interactions of the two types of bacteria (S. aureus and P. aeruginosa) with the nanopillars of cicada wings were investigated by SEM and AFM. Bactericidal efficiency of cicada wings against the two types of bacteria was studied using the plate counting method. Finally, the interaction of human osteoblasts with nanopillars and cytocompatibility of the cicada wings were analysed by SEM and AlamarBlueTM assay. All analyses in this chapter were carried out to achieve the first objective mentioned in Chapter 3. The following remarks can be concluded from this chapter: • Nanopillar arrays cover veins and membranes of both hindwings and forewings of the three cicada species. • Density and aspect ratio of membrane nanopillars are higher than for veins.
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• The aspect ratio of membrane nanopillars is approximately 2 in the three species while the aspect ratio of PE is slightly higher than the other two species with a cap diameter of 60.3 nm, base diameter of 123.2 nm, height of 211.2 nm, centre to centre distance of 155.9 nm, aspect ratio of 2.3 and density of 40 n/µm2. • Veins with lower density and aspect ratio have a courser texture than membranes which provide strength and flexibility for cicada wings. • The surface roughness of the three species wings is similar, ranging from 27.7 to 39.5 nm. • HIM is a high-resolution technique used to characterise biological samples like cicada wing nanopillars without the need for coating and generating artefacts. • From TEM analysis, it is observed that nanopillar architecture is straight and has a pear-like profile with the round shape at the top. Analysis also confirmed that the heights and aspect ratios of PE and AC nanopillars are higher than PC. • From FTIR analysis, it was found that wings are composed of amide (I&II), carbonyl, methyl, methylene, chitin, waxes and carboxylic acids. • From XPS analysis, it was found that cicada wings are composed of three main elements including carbon (C1s), nitrogen (N1s) and oxygen (O1s), confirming the existence of chitin, protein, amide and carboxylic acid. • This is the first report of the mechanical properties of cicada wing nanopillars. PC, AC and PE have similar Young's modulus ranges with values of 45.34 ± 6.7 MPa, 48.16 ± 7 MPa and 35.69 ± 5.2 MPa, respectively. • Once bacteria attach to the nanopillars, deformation (e.g. overspreading and wrinkling) can be observed on nanopillars compared to the control surface where bacteria remained undamaged without any deformation. • Both bacteria species adhere strongly to nanopillars with nanopillars gradually sinking into membranes causing membrane damage while bacteria can find no feature to attach to on control surfaces (glass), remaining undeformed and undamaged. • The wing membrane has more effective geometry with higher density and aspect ratio to puncture and kill the bacteria.
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• Both bacteria species are trapped among the vein nanopillars, however, despite some deformation, they remain undamaged because veins possess lower nanopillar density and aspect ratios. • From AFM analysis, it was found that P. aeruginosa sinks at least 100 nm to be fully punctured. Once the P. aeruginosa is attached to nanopillars, it has a 52% change in height and needs 80% penetration to be fully punctured. • When S. aureus attaches to nanopillars its height decreases by 66% and when sunk to 76% of its height, it is fully depleted. • While all three species wings demonstrate similar bactericidal efficiency against P. aeruginosa, PE shows considerable bactericidal efficiency against P. aeruginosa (p < 0.0001) after 4 and 18 hrs compared to other cicada wings. Colony-forming units of P. aeruginosa on PE decrease 1.98 × 106 to 7.264 × 105 (63%) after 18 hrs. • The three species wings have similar levels of bactericidal effect against S. aureus cells. AC represents the maximum colony-forming reduction of 43% (2.25 × 106 to 1.43 × 106) against S. aureus in 18 hrs. • The osteoblast cells grow and expand on the nanopillar arrays of cicada wings and actin filaments proliferate uniformly on the nanopillars without any defects on the cell structure. • The three wings produce higher cellular metabolic activity compared to the control surface (glass). PE produces better cellular metabolic activity that is 2.7 times higher after 4 hrs and 2.5 times higher after 24 hrs than glass.
In summary, the nanopillars of the cicada wings have an ideal bactericidal effect against P. aeruginosa and S. aureus and are biocompatible with human osteoblasts. Therefore, the outcomes reported in this chapter can create the roadmap to implement nanopillar arrays for titanium implants. The next chapter focuses on the fabrication of titanium nanopillars that mimic the arrays of nanopillars of cicada wings for orthopaedic applications.
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Chapter 5: Optimization of Electron Beam Lithography and Process Parameters for the Fabrication of Biomimicked Nanopillars
5.1 INTRODUCTION
This chapter demonstrates the evaluation of the Electron Beam Lithography (EBL) process parameters to fabricate biomimicked cicada wing nanopillars based on the optimum surfaces with the best bactericidal effect and biocompatibility shown in Chapter 4 (nanopillar geometry: top diameter: 70 nm, base diameter: 120 nm, height: 200 nm). The main goal of this chapter is to design the titanium nanopillars with the closest geometry to the cicada wing nano topography with the highest response. In this chapter, the EBL process parameters are optimized to achieve the optimum design of nanopillars, which can contribute towards the highest bactericidal effect and biocompatibility. Here, Monte Carlo simulation was carried out to find the best resist thickness and voltage before the experimental study. The capability of EBL to fabricate the nanopillars was evaluated after metal deposition based on the performance of lift-off. The process variables including dose, exposure factor, dwell time and pitch exposure were optimized to achieve the precise nanopillar architecture closest to the cicada wing nanopillars architecture. A TESCAN-MIRA3 FE-SEM equipped with EBL was used for nanopillar fabrication. This chapter is divided into three sections, Section 5.1 presents the optimization flowchart of EBL, Section 5.2 describes the Monte Carlo simulation and Section 5.3 presents the optimization of EBL on PMMA resist. The results of this research are based on the hypothesis that nanopillars have mechanical disruptive interactions with the membranes of bacteria. The nanopillars rupture the bacterial membrane through irreversible physical damage that lyses the bacterial cell.
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5.2 OPTIMIZATION FLOWCHART OF ELECTRON BEAM LITHOGRAPHY (EBL)
The experimental strategy was performed to optimize the EBL process variables and reduce the experimental cost and time. Figure 5.1 shows the optimization flowchart of EBL. In the first step, Monte Carlo simulation is used to optimize kV and pattern design (circle and dot) on different thickness of PMMA. The results of the first step are used in the subsequent experimental sections to reduce expensive and time- consuming experiments. The EBL is evaluated through lift-off performance for repeatable and reliable results. In each step, if the lift-off is not desirable and the result is not repeatable (No), the strategy is changed accordingly based on applying different thickness and types of PMMA (High Molecular Weight (HMW) and Low Molecular Weight (LMW)) as well as optimizing EBL parameters (exposure factor (EF), write field size, pitch). If the lift-off is desirable and the result is repeatable, the optimization ends with introducing the optimum process variable. As shown in Figure 5.1, the experimental section starts with a single layer and multilayer of PMMA 950 A4 (HMW). If the lift-off process was not desirable, the process continues with multi-layers of PMMA 950 A4 or A2 (HMW) as high- resolution resists used as a top layer and PMMA 495 A4 (low molecular weight) used as a bottom layer to create better lift-off.
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Figure 5.1: Optimization flowchart of EBL (Yes: lift-off is desirable, and the result is repeatable and reliable, No: lift-off is not desirable and the result is not repeatable and reliable, LMW: Low molecular weight and HMW: High molecular weight).
5.3 MONTE CARLO SIMULATION
To optimize the process parameter (beam energy) to mimic the cicada wing nanoarchitecture in the MIRA3 EBL system, Monte Carlo simulation was performed in CASINO v2.5.1.0. Electron exposure using different beam energy (kV), with the varying circle and dot pattern dimensions on different thickness were simulated in CASINO v2.5.1.0. This software was employed for Monte Carlo simulation of electron exposure in any substrate and resist. Both the forward and backward election scattering trajectories on different thicknesses of PMMA on 30 nm titanium and SiC substrate were simulated based on 5000 number of the electrons, with an average beam diameter of 2.8 nm as the average spot size in MIRA3 system.
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5.3.1 Monte Carlo simulation on 300 nm resist (dot and circle design) The first simulation was performed on 300 nm of PMMA resist using dot and circle design. Figures 5.2 and 5.3 show the simulated results of electron interaction based on dot pattern (beam diameter 2.8 nm) and circle pattern (circle diameter of 70 nm), respectively, on 300 nm PMMA/Ti/SiC substrate with beam energy ranging from 5 kV to 30 kV (blue lines indicate the forward scattered electron path while the red lines indicate the path of the backscattered electron). To expose the 300 nm resist for a dot pattern, beam energy with a minimum of 15 kV is required. For beam energy higher than 20 kV, a higher attainable resolution can be achieved due to a narrower- angled, forward scattered electron. Dot pattern generates the small circle at the top of the resist and wider diffraction at the bottom of the resist, causing early gate blockage at the top during metal deposition. It also creates the beam overlap between adjacent features. However, the simulation shows that 30 kV create better electron beam exposure with better wall angle compared to 15-25 kV (Figure 5.2). The circle pattern simulation was performed with the same voltages. The circle patterns represent the better resolution, lower wall angle at 30 kV compared to the dot pattern, providing lower overlap between two features next to each other (Figure 5.3).
(a) 5 kV, Dot pattern. (b) 10 kV, Dot pattern.
(c) 15 kV, Dot pattern (d) 20 kV, Dot pattern
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(e) 25 kV, Dot pattern (f) 30 kV, Dot pattern Figure 5.2: Monte Carlo simulation of electron PMMA (300 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and dot pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter 2.8 nm).
(a) 5 kV, circle pattern (ϕ70 nm). (b) 10 kV, circle pattern (ϕ70 nm)
(c) 15 kV, circle pattern (ϕ 70 nm) (d) 20 kV, circle pattern (ϕ 70 nm)
(e) 25 kV, circle pattern (ϕ70nm) (f) 30 kV, circle pattern (ϕ70 nm). Figure 5.3: Monte Carlo simulation of electron PMMA (300 nm)/ Ti/ SiC scattering interaction during E-beam exposure at different accelerating voltages and circle pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter 2.8nm).
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5.3.2 Monte Carlo simulation on 400 nm resist (dot and circle design) The simulation was also performed on the 400 nm thickness of PMMA. To expose the 400 nm resist for a dot pattern, beam energy with a minimum of 15 kV is required. For beam energy higher than 20 kV, a higher attainable resolution can be achieved due to a narrower-angled, forward scattered electron. Dot pattern generates the small circle at the top of the resist and wider diffraction at the bottom of the resist, causing early gate blockage at the top during metal deposition. It also creates the beam overlap between adjacent features. The simulation shows that 30 kV creates better electron beam exposure with better wall angle compared to 15-25 kV (Figure 5.4).
(a) 5 kV, Dot pattern (b) 10 kV, Dot pattern
(c) 15 kV, Dot pattern (d) 20 kV, Dot pattern.
(e) 25 kV, Dot pattern (f) 25 kV, Dot pattern Figure 5.4: Monte Carlo simulation of electron PMMA (400 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and dot pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter: 2.8 nm).
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The circle pattern simulation was performed with the same voltages. The circle patterns represent the better resolution, lower wall angle at 30 kV compared to the dot pattern, providing lower overlap between two features next to each other (Figure 5.5).
(a) 5 kV, circle pattern (ϕ 70 nm) (b) 10 kV, circle pattern (ϕ 70 nm)
(c) 15 kV, circle pattern (ϕ 70 nm) (d) 20 kV, circle pattern (ϕ 70 nm)
(e) 25 kV, circle pattern (ϕ 70 nm) (f) 30 kV, circle pattern (ϕ 70 nm) Figure 5.5: Monte Carlo simulation of electron PMMA (400 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and circle pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter 2.8 nm).
5.3.3 Monte Carlo simulation on 500 nm resist (dot and circle design) The simulation was also performed on 500 nm thickness of PMMA. To expose the 500 nm resist for a dot pattern, beam energy with a minimum of 15 kV is required. For beam energy higher than 20 kV, a higher attainable resolution can be achieved due to a narrower-angled, forward scattered electron. Dot pattern generates the small circle
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at the top of the resist and wider diffraction at the bottom of the resist, causing early gate blockage at the top during metal deposition. It also creates the beam overlap between adjacent features. However, the simulation shows that 30 kV creates better electron beam exposure with better wall angle compared to 15-25 kV (Figure 5.6). The circle pattern simulation was performed with the same voltages. The circle patterns represent the better resolution, lower wall angle at 30 kV compared to the dot pattern, providing lower overlap between two features next to each other (Figure 5.7).
(a) 5 kV, Dot pattern (b) 10 kV, Dot pattern.
(c) 15 kV, Dot pattern (d) 20 kV, Dot pattern
(e) 25 kV, Dot pattern. (f) 30 kV, Dot pattern. Figure 5.6: Monte Carlo simulation of electron PMMA (500 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and dot pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter: 2.8 nm).
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(a) 5 kV, circle pattern (ϕ 70 nm). (b) 10 kV, circle pattern (ϕ 70 nm).
(c) 15 kV, circle pattern (ϕ 70 nm) (d) 20 kV, circle pattern (ϕ 70 nm)
(e) 25 kV, Circle pattern (ϕ 70 nm) (f) 25 kV, Circle pattern(ϕ 70 nm) Figure 5.7: Monte Carlo simulation of electron PMMA (500 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and circle pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter 2.8 nm).
5.3.4 Monte Carlo simulation on 670 nm resist (dot and circle design) Similarly, the simulation was performed on 670 nm thickness of PMMA with dot and circle designs. When the beam energy was 20 kV and higher, the simulation of the dot design represents better resolution with the narrower forward scattered electron at the bottom of the resist (Figure 5.8). The dot pattern generates the small circle at the top of the resist and wider circle at the bottom of resist. This causes an early blockage at the top during metal deposition and creates the beam overlap between adjacent features. From the simulation, it was found that 30 kV creates a better electron beam exposure with better wall compared to 15-25 kV (Figure 5.8). For the circle
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pattern, 5 kV and 10 kV beam energies possess low energy of forward scattering as well as high diffraction of backscattered electron whereas 15 kV and 20 kV have low diffraction of backscattered electrons, followed by low diffraction of forward scattered electrons (Figure 5.9). Comparatively, the beam energy at 30 kV shows better results for circle patterns on 670 nm PMMA compared to the dot pattern. The circle patterns represent lower diffraction wall angle at 30 kV compared to the dot pattern, providing better resolution and lower overlap between two features next to each other.
(a) 5 kV, Dot pattern (b) 10 kV, Dot pattern
(c) 15 kV, Dot pattern (d) 20 kV, Dot pattern
(e) 25 kV, Dot pattern (f) 30 kV, Dot pattern Figure 5.8: Monte Carlo simulation of electron PMMA (670 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and dot pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter 2.8 nm).
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(a) 5 kV, circle pattern (ϕ 70 nm) (b) 10 kV, circle pattern (ϕ 70 nm)
(c) 15 kV, circle pattern (ϕ 70 nm) (d) 20 kV, circle pattern (ϕ 70 nm)
(e) 25 kV, circle pattern (ϕ 70 nm) (f) 30 kV, circle pattern (ϕ 70 nm) Figure 5.9: Monte Carlo simulation of electron PMMA (670nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and circle pattern simulated in CASINO v2.5.1.0. The blue lines indicate the forward scattered electron path while the red lines indicate the path of the backscattered electron (beam diameter 2.8 nm).
5.3.5 Monte Carlo simulation on 700 nm resist (dot and circle design) The simulation was also performed on the 700 nm thickness of PMMA with dot and circle design. When the beam energy was 20 kV and higher, the simulation of the dot design represents better resolution with the narrower forward scattered electron at the bottom of the resist (Figure 5.10). The dot pattern generates the small circle at the top of the resist and wider circle at the bottom of resist. This causes an early blockage at the top during metal deposition and it also creates the beam overlap between adjacent features. From the simulation, it was found that 30 kV creates a better electron beam exposure with better wall compared to 15-25 kV (Figure 5.10). For the circle
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pattern, 5 kV and 10 kV beam energies possess low energy of forward scattering as well as high diffraction of backscattered electron whereas 15 kV and 20 kV have low diffraction of backscattered electrons, followed by low diffraction of forward scattered electron (Figure 5.11). In summary, the beam energy at 30 kV produced a better result for the circle pattern on 700 nm PMMA compared to the dot pattern. The circle patterns represent lower diffraction wall angle at 30 kV compared to dot pattern, providing better resolution and lower overlap between two features next to each other. Monte Carlo simulation could save 20 days of the primary experiment which translates to $9,600 including labour and equipment costs.
(a) 5 kV, Dot pattern (b) 10 kV, Dot pattern
(c) 15 kV, Dot pattern (d) 20 kV, Dot pattern
(e) 25 kV, Dot pattern (f) 30 kV, Dot pattern Figure 5.10: Monte Carlo simulation of electron PMMA (700 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and dot pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron (Beam diameter 2.8 nm).
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(a) 5 kV, Circle pattern, (ϕ 70 nm) (b) 10 kV, Circle pattern, (ϕ 70 nm).
(c) 15 kV, Circle pattern (ϕ 70 nm) (d) 20 kV, Circle pattern (ϕ 70 nm)
(e) 25 kV, Circle pattern (ϕ 70 nm) (f) 30 kV, Circle pattern (ϕ 70 nm)
Figure 5.11: Monte Carlo simulation of electron PMMA (700 nm)/Ti/SiC scattering interaction during E-beam exposure at different accelerating voltages and circle pattern simulated in CASINO v2.5.1.0. Blue lines indicate the forward scattered electron path while red lines indicate the path of the backscattered electron.
5.4 OPTIMIZATION OF ELECTRON BEAM LITHOGRAPHY ON PMMA RESIST
In this section, EBL was performed on single and multi-layers of PMMA resist to find the closest geometry to cicada wing nanopillars in a repeatable and reliable approach. The experimental methodology is based on the systematic approach shown in Figure 5.1.
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5.4.1 EBL on one layer of PMMA 950 A4 Initially, one layer of PMMA 950 A4 was spin-coated on titanium substrate (30 nm) which is coated using E-beam evaporation on a silicon wafer as shown in Figure 5.12. The final thickness of the PMMA was 287 nm (Mean Squared Error (MSE = 3.8)). The EBL fixed parameters are illustrated in Table 5.1. Circle and dot pattern designs are employed with Exposure Factor (EF) 0.5-2.4 and 0.1-4, respectively, as shown in Figures 5.13 and 5.14. The field size 50 µm × 50 µm and spot size of 2.8 nm were selected to have the highest resolution. The circle pattern was designed at a diameter of 70 nm circle, centre to centre distance of 160 nm and EF of 0.5-2.4 (Figure 5.13). The dot pattern was designed with 160 nm between each dot and EF1-10.5 (Figure 5.14).
Figure 5.12: One layer of PMMA 950 A4 on a silicon wafer coated by 30 nm titanium.
Table 5.1: Fixed parameter in one layer PMMA 950 A4.
Parameter Value Working distance 7.578 mm Beam intensity 10 Spot size: 2.8 nm Angular intensity 0.233 (mA/srad) Current 0.266 nA Dose 350 µC/cm2 Dwell time 0.16 µs Ex pitch 3.4 nm Spacing 1.202 DAC resolution 0.84 EBL accuracy Fine Write field 4 × 5 Field, Size: 50 µm × 50 µm
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Figure 5.13: Circle design pattern, centre to centre distance 160 nm, 4×5 Field, Field size 50 µm × 50 µm, the distance between field 50 µm, EF 0.5-2.4.
Figure 5.14: Dot design pattern, centre to centre distance 160 nm, 4×5 Field, Field size 50 µm × 50 µm, the distance between field 50 µm, EF 1-10.5.
After E-beam exposure on PMMA 950 A4, 200 nm titanium was coated on the exposed area. Acetone and NMP (1-methyl-2-pyrrolidone) were used as a lift-off medium and it was found that NMP had a better stripper performance compared to acetone in the high thickness of PMMA. Here, the lift-off is not desirable (Figure 5.15). Low ratio of resist thickness and coating thickness (300 nm/200 nm) causes a high step coverage that prevents a good lift-off which causes the resist and coating to remain partially or fully on the surface.
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(a) (b) Figure 5.15: SEM images of the pattern after coating and lift off, circle pattern EF 0.5-2.4, (a) pattern and coating remain on the surface, (b) partial lift-off.
No pattern and no resist remained on the surface after lift-off of the dot pattern due to overlapping of two patterns next to each other, as simulated by the Monte Carlo method (Figure 5.16).
Figure 5.16: SEM images of dot pattern after coating and lift off EF 0.5-2.4.
In summary, the one layer of PMMA 950 A4 (thickness 287 nm) and a coating of 200 nm of titanium cause high step coverage, resulting in an undesirable lift-off. In the next experiments, two and three layers of PMMA 950 were coated to achieve lower step coverage and better lift-off. In primary experiments, blistering and crack were found on PMMA after E-beam exposure and coating as shown in Figure 5.17 (a) as well as overlapping of the fields shown in Figure 5.17 (b). Blistering occurs from E-beam exposure during the E-beam deposition and from moisture trapped on the surface before coating. In the PVD 75
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Kurt J. Lesker E-beam evaporation system, the distance between the target and sample holder is large and it is almost impossible to cause E-beam blistering, however, the samples are grounded to the holder and metal gripper is used to fix the sample to reduce the chance of charging and E-beam blistering. To reduce bubbling, samples are placed under the vacuum in the chamber overnight to enhance the quality of vacuum and eliminate any moisture and contamination. Overlapping of write field is because the EBL stage is mechanical (based in screw and spring), so that the stage can have microscale movement. The best method to eliminate the overlapping is to consider the 2-5 µm between fields.
(a) (b) Figure 5.17: Defects in primary EBL experiment: (a) Blistering and crack, (b) field overlapping
5.4.2 EBL on multi-layer (two and three layers) of PMMA 950 A4 Two and three layers of PMMA950 A4 were spin-coated on titanium substrate (30 nm) as shown in Figure 5.18. The final thickness of E-beam resist for two layers and three layers were 585 nm (MSE 5.47) and 863 nm (MSE 7.3), respectively. The EBL fixed parameters are the same as in Table 5.1. Circle and dot pattern designs were employed with EF 0.5-2.4 and 0.1- 4, respectively, as shown in Figure 5.19 (a, b). The field size of 50 µm × 50 µm, with the distance between fields at 53 µm and a spot size of 2.8 nm were selected for the highest resolution. The patterns are repeated at least 4 times on the same substrate to analyse the repeatability and reliability of the process parameter.
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Figure 5.18: Two and three-layers of PMMA 950 A4 on a silicon wafer coated by 30 nm titanium.
(a) (b) Figure 5.19: Pattern design on two and three layers PMMA 950 A4: (a) Circle design pattern, centre to centre distance 160 nm, 4×5 Field, Field size 50 µm × 50 µm, distance between field 53 µm, EF 0.5-2.4, (b) Dot design pattern, centre to centre distance 160 nm, 4 × 5 Field, Field size 50 µm × 50 µm, distance between field 53 µm, EF 1-10.5.
Two and three layers of PMMA 950 A4 do not have desirable lift-off (Figure 5.20). As illustrated in Figure 5.20, exposure factor of 0.5-0.8 is not enough, resulting in no residual (PMMA and coating) on the substrate. For all other exposure factors (EF 0.9-2.4), the lift-off is not desirable with the coating and the resist remaining on the substrate.
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(a) (b) Figure 5.20: SEM images of the pattern after coating and lift off, (a) two layers of PMMA 950, (b) three layers of PMMA 950 A4.
However, the lift-off is not entirely desirable, in some small areas a partial lift- off was found with no repeatable manner (Figure 5.21). As shown in Figure 5.21, EF 1.2 in two layers of PMMA 950 A4 and EF 1.5 in three layers of PMMA 950 A4 have partial lift-off in which nanopillars remained on the surface.
(a) (b) Figure 5.21: SEM images of the pattern after coating and lift off, (a) two layers of PMMA 950 with EF1.2, (b) three layers of PMMA 950 A4 with EF 1.5.
Similar to one layer of PMMA 950 A4, the lift-off result of the dot pattern on two layers and three layers of PMMA 950 A4 is not desirable and no resist remained
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on the surface due to overlapping of two patterns next to each other, which was also verified by Monte Carlo simulation. In summary, PMMA 950 A4 is high-resolution resist for EBL but because it is a high molecular resist, the lift-off is not desirable and repeatable for the high thickness of deposition (>100 nm). EBL of circle patterns on one layer of PMMA 950 A4 (thickness ̴ 300 nm) can generate the straight wall (according to Monte Carlo simulation). Additionally, 200 nm titanium deposition on 300 nm PMMA generates a low ratio of deposition thickness to resist thickness (200/300 nm), causing high step coverage resulting in an unsuccessful lift off. EBL of the circle pattern in two and three layers of PMMA 950 A4 (thickness ̴ 600 nm and ̴ 900 nm, respectively) does not produce a repeatable result. Step coverage of two layers and three layers of PMMA 950 A4 is lower than a single layer, causing partial lift-off in a few areas of two layers of PMMA 950 A4 (EF 1.2), and three layers of PMMA 950 A4 and EF 1.5. There is no consistent repeatability observed by applying two and three layers of PMMA 950 A4. To have a better lift-off and repeatability, low molecular resist (PMMA 495 A4) is used as the base layer and high molecular resist (PMMA 950 A4 and A2) is used as the top thin layer. PMMA 495 A4 would provide an ideal lift-off while PMMA 950 A4 and A2 would provide ideal resolution.
5.5 EXPERIMENTAL EVALUATION OF EBL ON MULTILAYER PMMA RESIST
In Section 5.5, the results of EBL in single and multi-layer of PMMA 950 A4 did not have desirable lift-off and repeatability. In this section, the experiments were carried out based on multi-layers of PMMA 495 A4 as low molecular weight resist to have better lift-off and PMMA 950 A2 and A4 as high molecular resist to achieve a high-resolution results and minimize the top layer to decrease the step coverage.
5.5.1 One layer PMMA 495 A4 and one layer PMMA 950 A4 First, one layer of PMMA 495 A4 and one layer of PMMA 950 A4 were spin- coated on a titanium substrate as shown in Figure 5.22. The final thickness was 494 nm (MSE = 4.5) and the EBL fixed parameters are illustrated in Table 5.2. Circle and
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dot pattern designs were fabricated on the resists with EF 0.5-2.4 and 0.1-4 respectively, as in Section 5.3.1.
Figure 5.22: One layer of PMMA 495 A4 and one layer PMMA 950 A4 on a silicon wafer coated by 30 nm titanium.
Table 5.2: EBL Fixed parameter in one layer PMMA 495 A4 and one layer of PMMA 950 A4 with an overall thickness of 494 nm. Parameter Value Working distance 7.574 mm Beam intensity 10 Spot size: 2.83 nm Angular intensity 0.25 (mA/srad) Current 0.266 nA Dose 350 µC/cm2 Dwell time 0.16 µs Ex pitch 3.4 nm Spacing 1.202 DAC resolution 0.84 EBL accuracy Fine Write field 50 µm × 50 µm
Figure 5.23 illustrates the SEM image of the sample (one layer of PMMA 495 A4 and one layer of PMMA 950 A4 with an overall thickness of 494 nm) after 200 nm coating of titanium followed by lift-off in NMP. It is observed that the EF of 0.5-1.1 does not generate enough exposure to affect the whole thickness (494 nm) of the resist. On the other hand, EF of 1.2-2.4 causes excessive exposure and a high amount of step coverage after 200 nm titanium coating on 494.45 nm thick PMMA. From EBL of the dot pattern, it was found that no pattern and no resist remained on the surface due to overlapping of patterns next to each other, which was also verified by Monte Carlo simulation. In summary, EBL with one layer of PMMA 495 A4 as the base layer, one top layer of PMMA 950 A4, and 200 nm coating of titanium do not produce a desirable lift off because the overall thickness of PMMA was low, causing high step coverage.
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Figure 5.23: Top view SEM image of the sample with one layer of PMMA 495 A4 and one layer of PMMA 950 A4 with an overall thickness of 494 nm after lift-off.
5.5.2 Two layers PMMA 495 A4 and one layer PMMA 950 A4 (Write field 50 µm × 50 µm) Two layers of PMMA 495 A4 and one layer of PMMA 950 A4 were spin-coated on titanium substrate (Figure 5.24). The final thickness was 651 nm (MSE = 4.5). The EBL fixed parameters are illustrated in Table 5.3. Circle and dot pattern designs were employed with EF of 0.5-2.4 and 0.1-4, respectively, as in Section 5.5.1.
Figure 5.24: Two layers of PMMA 495 A4 and one layer of PMMA 950 A4 on a silicon wafer coated by 30 nm titanium.
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Table 5.3: EBL Fixed parameters in two layers of PMMA 495 A4 and one layer of 950 A4.
Parameter Value Working distance 7.578 mm Beam intensity 10 Spot size: 2.8 nm Angular intensity 0.233 (mA/srad) Current 0.266 nA Dose 350 µC/cm2 Dwell time 0.16 µs Ex pitch 3.4 nm Spacing 1.202 DAC resolution 0.84 EBL accuracy Fine Write field 50 µm × 50 µm
Figure 5.25 shows the results after 200 nm coating and lift off. The EBL of the circle pattern is repeated 5 times (Figure 5.25 (a)) on the multilayered resist with the same pattern (Figure 5.25(a)).
(a) (b) (c) Figure 5.25: Top SEM images of circle pattern after coating and lift off. (a): repeated pattern, (b) pattern number 1, (c) pattern number 3.
EF of 0.5, 0.6, 0.7, 0.8, 0.9 and 1 do not have enough energy to expose the resist and no nanopillars remained on the surface while EF of 1.5-2.4 has undesirable lift-off due to the overexposure. Optimum and repeatable results were achieved in EF of 1.2 and 1.3 after coating 200 nm and lift-off (Figure 5.26).
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(a) (b) Figure 5.26: SEM image of the top view and tilt angle (45o) for EF of 1.3 with a diameter of 75 nm, a centre to centre distance of 160 nm, and a height of 100 nm.
No nanopillars and no resist remained on the surface from the dot pattern experiments due to overlapping of two patterns next to each other which was also verified by Monte Carlo simulations in which dot pattern designs cause high beam diffraction at the bottom of resist followed by overlapping and unsuccessful lift-off. As shown in Figure 5.26 (b), some fabricated nanopillars have defects in the edge. In the next experiments, the results for EF of 1.3 are repeated in a different area of resist, with smaller field size to decrease pitch, thereby improving resolution.
5.5.3 Two layers PMMA 495 A4 and one layer PMMA 950 A4 (Write field 30 µm × 30 µm) In this experiment, the results of Section 5.5.2 were repeated on the same resist (Figure 2.24) with the write field of 30 µm × 30 µm. The EBL fixed parameters are illustrated in Table 5.4. Figure 5.27 shows the pattern design of the circle with 70 nm diameter, centre to centre distance of 160 nm. The circle pattern is repeated for EF of 1.3 (Figure 5.27).
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Table 5.4: The fixed parameters in two-layer PMMA 495 A4 and one layer 950 A4.
Parameter Value Working distance 7.578 mm Beam intensity 10 Spot size: 2.8 nm Angular intensity 0.265 (mA/srad) Current 0.252 Dose 350 µC/cm2 Dwell time 0.06 µs Ex pitch 2.04 nm Spacing 0.702 DAC resolution 0.51 EBL accuracy Fine Write field 30 µm × 30 µm
Figure 5.27: Circle design pattern, diameter 70 nm, centre to centre distance 160 nm, 6 × 10 Field, Field size 30 µm × 30 µm, the gap between field 5 µm at EF 1.3.
EF 1.3 with the field size of 30 µm × 30 µm and pitch of 2.04 nm has better lift- off performance and resolution compared to the results of Section 5.5.2 with 50 µm × 50 µm with pitch of 3.4 nm. The lift-off results at EF 1.3 and 1.7 are illustrated in Figure 5.28.
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Figure 5.28: SEM images of the pattern after coating and lift-off (a): 6 × 10 fields with EF of 1.3 after lift-off, (b) high magnification of nanopillars on one field (30 µm × 30 µm) with EF of 1.3.
Then the nanopillars with a base diameter of 100 nm, height of 100 ± 5 nm and spike radius of less than 20 nm were fabricated on titanium substrate (Figure 5.29).
Figure 5.29: SEM image of nanopillars fabricated on two layers of PMMA 495 A4 and one layer PMMA 950 A2, EF of 1.3, lift-off after 200 nm Ti coating at 45-degree tilt.
Multilayer PMMA, including two base layers of PMMA 495 A4 and top layer PMMA 950 A4, had a desirable and repeatable result at EF of 1.3 and field size of 30 µm × 30 µm. The maximum achievable aspect ratio was 1.92 ± 0.23 because when the deposition thickness reached 100 nm, the top gate of the fabricated hole on PMMA 950 A4 was blocked and it did not allow nanopillars to exceed more than 100 nm in height.
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In the next experiment, to increase the aspect ratio and the height of nanopillar, top layer of PMMA 950 A2 was added to produce the thinner top layer as well as overall thickness.
5.5.4 Two layers PMMA 495 A4 and one layer PMMA 950 A2 (Write field 30 µm × 30 µm and 150 nm titanium coating thickness) In this experiment, high-resolution molecular resist of PMMA 950 A2 was applied as a top layer to produce a top thin layer. The lower thickness of the top layer produces low step coverage after the titanium coating which is beneficial for lift-off and reducing the overall thickness. Two layers of PMMA 495 A4 and one layer of PMMA 950 A2 were spin-coated on titanium substrate according to Figure 5.30. The final thickness was 455 nm (MSE = 6.244). The EBL fixed parameters are illustrated in Table 5.5. Circle pattern (70 nm diameter circle) were employed with EF0.1-2. EBL process parameters were as shown in Figure 5.31.
Figure 5.30: Two layers of PMMA 495 A4 and one layer of PMMA 950 A2 on Ti and SiC substrate.
Table 5.5: Fixed parameters in two layers PMMA 495 A4 and one layer 950 A2.
Parameter Value Working distance 7.577 mm Beam intensity 10 Spot size: 2.82 nm Angular intensity 0.246 (mA/srad) Current 0.262 nA Dose 350 µC/cm2 Dwell time 0.06 µs Ex pitch 2.04 nm Spacing 0.724 DAC resolution 0.51 EBL accuracy Fine Write field 30 µm × 30 µm Coating thickness 150 nm
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Figure 5.31: The circle pattern design with 70 nm circle, centre to centre distance 160 nm and EF0.1- 2.
Figure 5.32 shows the results after 150 nm coating and lift off. The circle pattern repeated 8 times on resist with the same pattern and the reliable and repeatable results were achieved after lift-off (Figure 5.32). EF of 0.1, 0.2, 0.3 and 0.4 cannot produce enough exposure on PMMA resist. Exposure factors higher than 1.6 produce overexposure, resulting in resist peeling and defective nanopillars at the edge of the field (Figure 5.32 (a) and (b)) EF1.2 and 1.3 produced uniform nanopillars without defects (Figure 5.32 (c)).
(a) (b) (c) Figure 5.32: SEM images of the pattern after coating and lift off. (a) repeated pattern, (b) write field with 1.6 EF, (c) write field with EF of 1.2.
EF of 1.3 produced better and more consistent shapes of nanopillars as shown in Figure 5.33. The nanopillars have a sharp edge and the aspect ratio is slightly better than Section 5.4.3 with an aspect ratio of 2.31 ± 0.2. Figure 5.34 shows the pattern with EF of 1.3 fabricated in the larger area.
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Figure 5.33: SEM image of the top view and tilt angle (45o) for EF of 1.3, diameter of 75 nm centre to centre distance of 160 nm and height of 100 nm.
Figure 5.34: SEM image of top view for the fabricated pattern with EF of 1.3 with 20 × 20 fields.
Table 5.6 shows the fabrication geometry of two layers PMMA 495 A4 plus one layer PMMA 950 A4 and two layers PMMA 495 A4 plus one layer PMMA 950 A2 in Sections 5.6.3 and 5.6.4, respectively.
Table 5.6: Fabrication geometry of two layers PMMA 495 A4, one layer PMMA 950 A2 and two layers PMMA 495 A4 and one layer PMMA 950 A2.
Geometry Two layers PMMA 495 A4, one Two layers PMMA 495 A4 and layer PMMA 950 A4 one layer PMMA 950 A2 Base diameter 88.31±7.5 nm 97.35±5.2 nm Top diameter 16.13±2.9 nm 14.43±2.8 nm Height 101.35±9.2 nm 127.48±6.6 nm Centre by centre distance 166.5±4.64 nm 165.94±5.5 nm Aspect ratio 1.92±0.23 2±0.2
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Using PMMA 950 A2 as a top layer increases the aspect ratio and base diameter because PMMA 950 A2 produced thin layers (61 nm) compared to the thick layer (250 nm) produced by PMMA 950 A4, postponing the material gate blockage on the top layer. This experiment was repeated for the titanium coating of 200 nm and lift-off was not desirable as resist and coating remained on the surface because the overall thickness of resist (455 nm) was low, resulting in high step coverage with a coating thickness of 200 nm. In next experiment, another base layer of PMMA 495 A4 was added to make three layers of PMMA 495 A4 at the bottom and a single layer of PMMA 950 A2 was added at the top to change the geometry of the circle and reach the optimum process parameter for each geometry.
5.5.5 Three layers PMMA 495 A4 and one layer PMMA 950 A2 (Write field 25 µm × 25 µm) with 250 nm coating Three layers of low molecular weight PMMA 495 A4 were used for the bottom layer to have desirable lift-off and one layer of PMMA 950 A2 was applied as high resolution and high molecular resist to reduce the step coverage generated by coating and having desirable lift-off. The schematic of applied resist and thickness is shown in Figure 5.35. The final thickness was 667 nm (MSE = 6.24). The EBL fixed parameter is illustrated in Table 5.7.
Figure 5.35: Three layers of PMMA 495 A4 and one layer of PMMA 950 A2 on Ti and SiC substrate.
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Table 5.7: EBL Fixed parameters in three layers PMMA 495 A4 and one layer 950 A2.
Parameter Value Working distance 7.578 mm Beam intensity 10 Spot size 2.8 nm Angular intensity 0.247 (mA/srad) Current 0.264 nA Dose 350 µC/cm2 Dwell time 0.04 µs Ex pitch 1.7 nm Spacing 0.602 DAC resolution 0.42 EBL accuracy Fine Write field 25 µm × 25 µm EF 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 Coating thickness 250 nm
From the primary experiment, the process of fabrication of nanopillars had limited aspect ratios because the EBL fabricated gate was blocked during the coating process. Three pattern diameters (e.g. 70 nm, 120 nm and 320 nm) and two centre to centre distances for each diameter were designed to evaluate the lift-off performance to achieve higher aspect ratios (Table 5.8 and Figure 5.36). Circle patterns with diameter 70 nm (centre to centre distance of 110 nm, 160 nm), 120 nm (centre to centre distance of 160 nm, 200 nm) and 200 nm (centre to centre distance of 260 nm, 320 nm) were employed with EF of 1-2.1. EBL design pattern parameters are given in Table 5.8 and Figure 5.36. Pattern designs of diameters of 70 nm, 120 nm and 200 nm in Drawbeam are illustrated in Figures 5.37, 5.38 and 5.39, respectively.
Table 5.8: EBL design pattern parameter.
Circle diameter design (nm) Centre to centre distance (nm) 70 110 70 160 120 160 120 200 200 260 200 260
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Figure 5.36: Design patterns for circle 70 nm, 120 nm and 320 nm according to Table 5.8 (dimensions in nanometres).
Figure 5.37: 70 nm circle pattern design with 110 nm and 160 nm centre to centre distance and EF of 1-2.1.
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Figure 5.38: 120 nm circle pattern design with 160 nm and 200 nm centre to centre distance and EF of 1-2.1.
Figure 5.39: 200 nm circle pattern design with 320 nm and 260 nm centre to centre distance and EF of 1-2.1.
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As shown in Figure 5.40, for the circle pattern with a diameter of 70 nm and centre to centre distance of 110 nm, the lift-off is not desirable (Figure 5. 40 (a) right) because the exposed areas on the resist are too close to each other, causing overlap of two patterns next to each other followed by unsuccessful lift-off. No overlap is observed between nanopattern on the resist, followed by fabricated titanium nanopillars on the surface. The best result was achieved in EF of 2 as shown in Figure 5.40 (b and c).
(a) (b) (c) Figure 5.40: (a) Top SEM image of fabricated pattern based on circle design diameter of 70 nm and centre to centre distance of 110 nm at the top and circle design diameter of 70 nm and centre to centre distance of 160 nm at the bottom with EF 1-2.1, (b) Top SEM image of fabricated nanopillars based on circle design diameter of 70 nm and centre to centre distance of 160 nm with EF of 2, (c) SEM image of fabricated nanopillars based on circle design diameter of 70 nm and centre to centre distance of 160 nm with EF of 2 with tilt angle of 45.
As shown in Figure 5.41, for the circle pattern 120 nm diameter and centre to centre distance of 160 nm, the lift-off is not desirable because the exposed areas on the resist are too close to each other, causing overlap of two patterns next to each other. No overlap is observed between nanopattern on the resist, followed by fabricated titanium nanopillars on the surface. The best result is achieved in EF 1.3 for circle pattern 120 nm diameter and centre to centre distance of 200 nm as shown in Figure 5.41 (b and c).
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(a) (b) (c) Figure 5.41: (a) Top SEM image of fabricated pattern based on circle design diameter of 120 nm and centre to centre distance of 160 nm at the top and circle design diameter of 120 nm and centre to centre distance of 200 nm at the bottom with EF 1-2.1, (b) Top SEM image of fabricated nanopillars based on circle design diameter of 120 nm and centre to centre distance of 200 nm with EF of 1.3, (c) SEM image of fabricated nanopillars based on circle design diameter of 120 nm and centre to centre distance of 200 nm with EF of 1.3 with tilt angle of 45.
As shown in Figure 5.42, for circle pattern 200 nm diameter and 260 nm centre to centre distance, the lift-off is not desirable because the exposed areas on the resist are too close to each other, causing overlap of two patterns next to each other. No overlap is observed between nanopattern on the resist, followed by fabricated titanium nanopillars on the surface. The optimum result is achieved in EF of 1 for circle pattern 200 nm diameter and 320 nm centre to centre distance as shown in Fig 5.42 (b).
(a) (b) (c) Figure 5.42: (a) Top SEM image of fabricated pattern based on circle design diameter of 200 nm and centre to centre distance of 260 nm at the top and circle design diameter of 200 nm and centre to centre distance of 320 nm at the bottom with EF 1-2.1, (b) Top SEM image of fabricated nanopillars based on circle design diameter of 200 nm and centre to centre distance of 320 nm with EF of 1, (c) SEM image of fabricated nanopillars based on circle design diameter of 200 nm and centre to centre distance of 320 nm with EF of 1 with tilt angle of 45.
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The geometries of pattern design and fabricated nanopillars are summarized in Table 5.9. For circle pattern diameter of 70 nm and centre to centre distance of 160 nm, the optimum result is achieved at EF of 2, followed by fabricated nanopillars with base diameter 94.4 ± 6 nm, top diameter of 12.6 ± 2 nm, centre to centre distance of 165.8 ± 5.6 nm and aspect ratio of 2.16 ± 0.2 nm. For circle pattern diameter of 120 nm and centre to centre distance of 200 nm, the optimum result is achieved at EF of 1.3, followed by base diameter 148.6 ± 4.7 nm, top diameter of 21.05 ± 3.6 nm, centre to centre distance of 205.9 ± 4.7 nm and aspect ratio of 2.35 ± 0.1 nm. For circle pattern diameter of 200 nm and centre to centre distance of 320 nm, the optimum result is achieved at EF of 1, followed by base diameter 214 ± 9 nm, top diameter of 324.9 ± 6.9 nm, centre to centre distance of 202.2 ± 6.6 nm and aspect ratio of 1.53 ± 0.1 nm (Table 5.9).
Table 5.9: Geometry of patterns and EBL result.
EBL Fabricated nanopillars Exposure Comments pattern Base Top centre to Aspect Density factor geometry diameter diameter centre ratio (n/μm2) (nm) (nm) distance (nm) Ø70 nm, ˗ ˗ ˗ ˗ ˗ No dis110 nm nanopillars Ø70 nm, 94.4±6 12.6±2 165.8±5.6 2.16±0.2 43±1 EF2 dis160 nm Ø120 nm, ˗ ˗ ˗ ˗ ˗ No dis160 nm nanopillars Ø120 nm, 148.6±4. 21.05±3.6 205.9±4.7 2.35±0.1 30±1 EF1.3 dis200 nm 7 Ø200 nm, ˗ ˗ ˗ ˗ ˗ No dis260 nm nanopillars Ø200 nm, 214±10 48.9±7 324.9±6.9 2.19±0.1 10±1 EF1 dis320 nm
5.6 SUMMARY
In this research, EBL was used to fabricate the titanium biomimicked nanopillars and then EBL process variables were evaluated and optimized to achieve desirable lift- off and repeatability. Following conclusions can be drawn from the present chapter:
• E-beam exposure was simulated by Monte Carlo simulation to reduce experiment cost and fabrication time. Monte Carlo simulation optimizes the beam energy (Kv) and pattern design. As a result of the simulation, 30 Kv and circle pattern design were identified as the best practice to fabricate the thick PMMA resist. Circle
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pattern generated less diffraction on resist while 30 Kv can fully expose the nanopattern on PMMA with thickness higher than 300 nm.
• EBL of single and multilayer PMMA 950 A4 do not produce the desirable lift-off and repeatable results because PMMA 950 A4 is high molecular and high- resolution resist, causing high step coverage for the fabrication of nano circle pattern (70 nm) on thick resist (>300 nm), resulting in undesirable lift-off. High molecular weight resist is hard to lift-off in higher thickness (>300 nm).
• In the primary experiment, prior to lift-off, crack and resist blistering were observed due to the moisture and quenching on PMMA resist after coating. For this purpose, the samples were kept in dry conditions overnight under vacuum before coating. After coating, the samples are kept for half an hour in the chamber to reach room temperature, avoiding crack due to quenching. E-beam blistering is almost impossible during the coating of fabricated PMMA because, in the coating system, the centre to centre distance between the sample holder and E-beam source is too great. However, to eliminate the chance of resist blistering, samples were grounded to the sample holder using metal grippers. Overlapping of the write field was observed during the experiment, causing undesirable lift-off because the EBL stage is mechanical (based in screw and spring) and there is microscale movement on stage. The best practice to eliminate the overlapping is to consider the 2-5 µm between fields.
• As a result of unsuccessful lift-off and unrepeatable results from EBL of single and multi-layers of PMMA 950 A4, the strategy was changed to apply low molecular PMMA 495 A4 as a base layer and PMMA 950 A4 or A2 as the top layer. PMMA 495 A4 is low molecular weight resist, which enhances lift-off while PMMA 950 A4 and A2 are the high-resolution E-beam resist which can generate precise wall geometry. PMMA 950 A2 generates the thinner top layer (65 nm) compared to PMMA 950 A4 (200 nm) which can cause lower step coverage, followed by a higher aspect ratio. The best result was three layers of PMMA 495 A4 as the bottom layer and one layer of PMMA 950 A2 as the top layer.
• The top layer of PMMA 950 A2 with the thickness of 60-65 nm represented lower step coverage and high aspect ratio of nanopillars compared to PMMA 950 A4 as
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the top layer with a thickness of 250 nm. Applying PMMA 950 A2 can increase aspect ratio from 1.92 to 2.31.
• In this research, to have the highest resolution, the main parameters were fixed including beam energy of 30 Kv, the spot size of 2.8 nm, doses of 350 µC/cm2, beam intensity of 10, and working distance of 7.6 mm.
• The most important EBL parameters are EF, write field size, pitch and dwell time during the experiment. When the field size decreased from 50 µm × 50 µm to 25 µm × 25 µm, followed by decreasing dwell from 0.16 µm to 0.04, and decreasing pitch from 3.4 nm to 1.7 nm, resolution improved. The optimum fixed EBL parameters include beam energy of 30 Kv, 25 µm × 25 µm write field, pitch of 1.7 nm, dose of 350 µC/cm2, beam intensity of 10, working distance of 7.6 mm, and spot size 2.8. Exposure factor (EF) was optimized based on the geometry of each nanopattern. For circle pattern diameter of 70 nm and centre to centre distance of 160 nm, the optimum result was achieved at EF of 2, followed by fabricated nanopillars with base diameter 94.4 ± 6 nm, top diameter of 12.6 ± 2 nm, centre to centre distance of 165.8 ± 5.6 nm and aspect ratio of 2.16 ± 0.2 nm. For circle pattern diameter of 120 nm and centre to centre distance of 200 nm, the optimum result was achieved at EF of 1.3, followed by base diameter 148.6 ± 4.7 nm, top diameter of 21.05 ± 3.6 nm, centre to centre distance of 205.9 ± 4.7 nm and aspect ratio of 2.35 ± 0.1 nm. For circle pattern diameter of 200 nm and centre to centre distance of 320 nm, the optimum result was achieved at EF of 1, followed by base diameter 214 ± 9 nm, top diameter of 324.9 ± 6.9 nm, centre to centre distance of 202.2 ± 6.6 nm and aspect ratio of 1.53 ± 0.1 nm.
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Chapter 6: Evaluation of Bacteria Interaction and Biocompatibility of Titanium Fabricated Nanopillars
6.1 OVERVIEW
This chapter describes the assessment of bacteria interaction and biocompatibility of titanium fabricated nanopillars. To identify the bactericidal function of cicada wing nanopillars and extend this function to medical device applications, it is vital to mimic the array of nanopillars using other materials like titanium. When titanium nanopillars have the same bactericidal effect and biocompatibility as cicada wings, the first step in nanopillar application in medical devices is achieved. Therefore, bacteria interaction and biocompatibility evaluation of titanium nanopillars can help scientists and bioengineers implement the array of nanopillars on orthopaedic implants and biomedical devices. In the previous chapter, nanopillars of cicada wings were biomimicked on titanium and EBL process parameters were optimized to achieve the optimum design of nanopillars. In this chapter, nanotopography, chemical composition and mechanical properties of the fabricated titanium nanopillars are analysed using SEM, XPS and AFM, respectively. Interactions of P. aeruginosa, S. aureus and human osteoblast cells are investigated by SEM and cellular metabolic analysis of osteoblast cells on fabricated nanopillars is evaluated using AlamarBlueTM assay. The results of this research are based on the hypothesis that titanium fabricated nanopillars have the same killing mechanisms as cicada wings based on mechanical disruptive interactions with bacteria membranes.
6.2 NANOTOPOGRAPHY ANALYSIS OF FABRICATED NANOPILLARS USING SEM
As a result of the systematic optimization of nanopillar arrays using electron beam lithography (EBL) in Chapter 5, the optimum geometry of fabricated titanium nanopillars based on PE cicada wing is summarized in Table 6.1 and shown in Figures
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6.1, 6.2 and 6.3. Three groups of titanium nanopillars were fabricated with a base diameter of 94.4 ± 6 nm, 148.6 ± 4.7 nm and 214 ± 10 nm, top (spike) diameter of 12.6 ± 2 nm, 21.05 ± 3.6 nm and 48.9 ± 7 nm, centre to centre distance of 165.8 ± 5.6 nm, 205.9 ± 4.7 nm and 324.9 ± 6.9 nm, aspect ratio of 2.16 ± 0.2, 2.35 ± 0.1 and 2.19 ± 0.1. Of the three groups of titanium nanopillars, the closest geometry of titanium nanopillars to the nanopillars of PE wing has base diameter 94.4 ± 6 nm, top diameter of 12.6 ± 2 nm, centre to centre distance of 165.8 ± 5.6 nm and aspect ratio of 2.16 ± 0.2 (Table 6.1, Figures 6.1, 6.2 and 6.3).
Table 6.1: Optimum geometry of fabricated titanium nanopillars.
Design Base Top diameter Centre to Height Aspect Density dimension diameter (nm) centre (nm) ratio (n μm-2) (nm) (nm) distance (nm) Ø70 nm, 94.4±6 12.6±2 165.8±5.6 115.6±6 2.16±0.2 43±1 Dis 160 nm Ø120 nm, 148.6±4.7 21.05±3.6 200.3±4.7 221.6±7 2.35±0.1 30±1 dis 200 nm Ø200 nm, 214±10 48.9±7 324.9±6.9 288±7 2.19±0.1 10±1 dis 320 nm
(a) (b) Figure 6.1: SEM image of the patterned titanium nanopillars (base diameter 94.4 ± 6 nm, top diameter 12.6 ± 2 nm, centre to centre distance 165.8 ± 5.6 nm, aspect ratio 2.16 ± 0.2): (a) Top SEM image, (b) 45 degree tilt SEM image.
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(a) (b) Figure 6.2: SEM image of the patterned titanium nanopillars (base diameter 148.6 ± 4.7 nm, top diameter 21.05 ± 3.6 nm, centre to centre distance 205.9 ± 4.7 nm, aspect ratio 2.35 ± 0.1): (a) Top SEM image, (b) 45 degree tilt SEM image.
(a) (b) Figure 6.3: SEM image of the patterned titanium nanopillar (base diameter 214 ± 10 nm, top diameter 48.9 ± 7 nm, centre to centre distance 324.9 ± 6.9 nm, aspect ratio 2.19 ± 0.1): (a) Top SEM image, (b) 45 degree tilt SEM image.
The aspect ratio of the fabricated titanium nanopillars ranged from 2.16 to 2.35 which was in the range of cicada wing nanopillars.
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6.3 CHEMICAL CHARACTERISTICS OF FABRICATED NANOPILLARS USING XPS
The XPS spectrum shows that oxygen (O1s) with the highest peak of ~533 eV and titanium (Ti2p) with the peak of 461 eV are the main components of titanium fabricated nanopillars which confirms the existence of TiO2 on the surface (Figure 6.4). Carbon (C1s) possesses the lowest peak with 287 eV which is attributed to contamination on the surface. The atomic content of O1s, Ti2p and C1s were 58.35%, 31.83% and 9.82%, respectively (Table 6.2). The XPS results of fabricated titanium nanopillars were consistent with previously published studies (Aronsson et al., 1996, Diebold and Madey, 1996).
Table 6.2: Atomic content and binding energy extracted by XPS for fabricated titanium nanopillars (b.e: binding energy, a.c: atomic number).
Element Binding energy Atomic % O1s 533 58.35 Ti2p 461 31.83 C1s 287 9.82
Figure 6.4: XPS spectrum of the titanium nanopillar surface.
6.4 MECHANICAL CHARACTERISTICS OF FABRICATED NANOPILLARS USING AFM
AFM contact mode was used to analyse the mechanical properties of titanium. A HA-NC-B ETALON polysilicon cantilever with silicon tip from TipsNano with
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length 124 µm and a tip radius of 10 nm was used for contact and non-contact AFM analysis. The Young’s modulus was obtained by analysing the retract curve using the Derjaguin-Muller-Toporov (DMT) model in Asylum software. Applied force for calibration on titanium was 50 nN. Force mapping was employed to analyse the adhesion force and extract the average reduced modulus in 2 μm × 2 μm samples from three groups of titanium nanopillar surfaces. The average reduced Young's modulus for the titanium nanopillar was 250 ± 12 MPa.
6.5 ANALYSIS OF BACTERIA INTERACTION WITH FABRICATED NANOPILLARS USING SEM
To analyse the bacteria interaction the three design groups were fabricated in 0.5 × 0.5 mm composed of 20 fields of size of 25 μm × 25 μm as shown in Figure 6.5.
Figure 6.5: SEM images of titanium fabricated surface: (a) 0.5 mm × 0.5 mm composed of 20 × 20 fields of size 25 μm × 25 μm, (b) fabricated nanopillars from Ø70 nm, centre to centre distance 160 nm design in 25 μm × 25 μm, (c) fabricated nanopillars from Ø120 nm, centre to centre distance of 200 nm design in 25 μm × 25 μm, (d) fabricated nanopillars from Ø200 nm, centre to centre distance 320 nm design in 25 μm × 25 μm.
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Titanium nanopillars (three groups of nanopillars in Table 6.1) interacted with P. aeruginosa, damaging the bacteria membrane in the same way as the three cicada species wings (Figures 6.6, 6.7 and 6.8).
Figure 6.6: Interaction of P.aeruginosa with fabricated nanopillars based on design Ø70 nm, centre to centre distance 160 nm and final geometry with base diameter 94.4 ± 6 nm, spike diameter 12.6 ± 2 nm, centre to centre distance 165.8 ± 5.6 nm and aspect ratio 2.16.
Figure 6.7: Interaction of P.aeruginosa with fabricated nanopillar based on design Ø120 nm, centre to centre 200 nm and final geometry with base diameter 148.6 ± 4.7 nm, spike diameter 21.05 ± 3.6 nm, centre to centre distance 205.9 ± 4.7 nm and aspect ratio 2.35 ± 0.1.
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Figure 6.8: Interaction of P.aeruginosa with fabricated nanopillars based on design Ø200 nm, centre to centre distance 320 nm and final geometry with base diameter 214 ± 10 nm, spike diameter 48.9 ± 7 nm, centre to centre distance 324.9 ± 6.9 nm and aspect ratio 2.19.
Titanium mimicked nanopillars also interacted with S. aureus, damaging the bacteria membrane in the same way as the three cicada species wings (Figure 6.9).
Figure 6.9: Interaction of S.aureus with fabricated nanopillars based on design Ø70 nm, centre to centre distance 160 nm and final geometry with base diameter 94.4 ± 6 nm, spike diameter 12.6 ± 2 nm, centre to centre distance 165.8 ± 5.6 nm and aspect ratio 2.16.
As is shown in Figure 6.10, the titanium control surface with the smooth surface did not possess any features to trap and penetrate the bacteria membranes.
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(a) (b) Figure 6.10: Interaction of (a) P.aroginosa and (b) S.aures with the titanium control surface
The first group of fabricated nanopillars with circle design array (Ø70 nm, centre to centre distance of 160nm) and the final fabricated nanopillars with a base diameter of 94.4 ± 6 nm, spike diameter of 12.6 ± 2 nm, centre to centre distance of 165.8 ± 5.6 nm and aspect ratio of 2.16 have optimum killing performance against P. aeruginosa (Figure 6.11) and S. aureus (Figure 6.12).
Figure 6.11: SEM image of P.aeruginosa interaction with fabricated nanopillars in tilt angle 45 degree based on design Ø70 nm, centre to centre 160 nm and final geometry with base diameter 94.4 ± 6 nm, spike diameter 12.6 ± 2 nm, centre to centre distance 165.8 ± 5.6 nm and aspect ratio 2.16.
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Figure 6.12: SEM image of S.aureus interaction with fabricated nanopillars in tilt angle 45 degree based on design Ø70 nm, centre to centre distance of 160 nm and final geometry with base diameter 94.4 ± 6 nm, spike diameter 12.6 ± 2 nm, centre to centre distance 165.8 ± 5.6 nm and aspect ratio 2.16.
6.6 CYTOCOMPATIBILITY OF OSTEOBLAST CELLS ON FABRICATED NANOPILLARS USING ALAMARBLUETM ASSAY
Cytocompatibility and cellular metabolic activity of fabricated titanium nanopillars were analysed via AlamarBlueTM assay through the attachment of osteoblasts on the fabricated surface with a base diameter of 94.4 ± 6 nm, spike diameter of 12.6 ± 2 nm, centre to centre distance of 165.8 ± 5.6 nm as well as a control surface over time intervals of 1 and 3 days. The great cytocompatibility and cellular activity of osteoblast cells are observed on titanium nanopillar surfaces after 1 day and 3 days (Figure 6.13 and Appendix C).
Figure 6.13: Cellular metabolic activity of the osteoblast cells on fabricated titanium nanopillars and titanium control surface after time intervals of 1 and 3 days.
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The fabricated titanium nanopillars and titanium control surface possessed similar levels of cellular activity after 1 and 3 days, meaning that titanium nanopillar surfaces have the same cytocompatibility as the titanium control surface. Titanium materials and substrates have been employed widely in biomedical devices and surgical implants and have great cytocompatibility with human cells like osteoblasts (Hasan et al., 2017, Geetha et al., 2009). In summary, the titanium nanopillars do not demonstrate any cytotoxic properties to human osteoblasts which is consistent with recently published studies and proves the excellent capability of titanium nanopillars for orthopaedic implants.
6.7 ANALYSIS OF OSTEOBLAST CELL INTERACTIONS WITH FABRICATED NANOPILLARS USING SEM
Osteoblast human cell interactions with nanopillar surfaces (base diameter 94.4 ± 6 nm, spike diameter 12.6 ± 2 nm, centre to centre distance 165.8 ± 5.6 nm and height 115.6 ± 6 nm) were analysed by SEM to identify the quality of cell proliferation on titanium nanopillars. As seen in Figure 6.14, the osteoblast cell spreads on the fabricated titanium nanopillar surface and its actin filaments attach uniformly on the titanium nanopillars without any damage to the cell structure. No anchorage was observed for the titanium control surface (Figure 6.8 (d)) compared to the nanopillar surface (Figure 6.8 (a-c)), confirming that as with the cicada nanopillars, the titanium nanopillars provide more anchorage for osteoblast cells.
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Figure 6.14: SEM of osteoblast attachment on titanium nanopillars and titanium control surfaces: (a) SEM image of attached and well-spread osteoblast cells on titanium nanopillars in low magnification, (b,c) SEM image of attached and well-spread osteoblast cells on titanium nanopillars in high magnification, (d) SEM image of attached and well-spread osteoblast cells on the titanium control surface.
6.8 MAIN FINDINGS AND REMARKS
In this chapter, the nano topography of three groups of titanium fabricated nanopillars was characterised using SEM and XPS. The interactions of the two types of bacteria with titanium nanopillars were investigated by SEM. Finally, the interaction of human osteoblasts with titanium nanopillars and cytocompatibility of the titanium nanopillars were analysed by SEM and AlamarBlueTM assay. All analyses in this chapter were carried out to achieve the second objective mentioned in Chapter 3. The following remarks can be concluded from this chapter:
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• Based on the results of Chapter 5, three groups of nanopillars were fabricated and their geometries precisely characterised. The first group was fabricated based on design dimensions of the circle (Ø70 nm, centre to centre distance 160 nm). The final fabricated nanopillar had a base diameter of 94.4 ± 6 nm, spike diameter of 12.6 ± 2 nm, centre to centre distance of 115.6 ± 6 nm and height of 115.6 ± 6 nm. The second group was fabricated based on design dimension of a circle (Ø120 nm, centre to centre distance 200 nm) with final nanopillars base diameter of 148.6 ± 4.7 nm, spike diameter of 21.05 ± 3.6 nm, centre to centre distance of 200.3 ± 4.7 nm and height of 221.6 ± 7 nm. The third group was fabricated based on design dimension of a circle (Ø200 nm, centre to centre distance 320 nm) with final nanopillars base diameter of 214 ± 10 nm, spike diameter of 48.9 ± 7 nm, centre to centre distance of 324.9 ± 6.9 nm and height of 288 ± 7 nm. • In this research, for the first time, the mechanical properties of titanium nanopillars are reported with reduced Young's modulus of 250 ±12 MPa which produces the rigid nanopillars It is comparable with Young's modulus of dragonfly ranging from 23-38 MPa (Bandara et al., 2017). Due to the nanopillars surface architecture, the mechanical properties such as Young's modulus of the fabricated surface maybe different from the Young's modulus of the bulk material. • While both P. aeruginosa and S. aureus interacted with fabricated titanium nanopillars in the same way as they do with cicada nanopillars, fabricated nanopillars with circle design array (Ø70 nm, centre to centre distance 160 nm) with final fabricated geometry with base diameter of 94.4 ± 6 nm, spike diameter of 12.6 ± 2 nm, centre to centre distance of 165.8 ± 5.6 nm and aspect ratio of 2.16 produced the best killing efficiency against P. aeruginosa and S. aureus compared to the two other groups of nanopillars. • The AlamarBlueTM analysis demonstrated that osteoblast cells have great cellular activity on titanium nanopillar surfaces after 1 and 3 days the same level as titanium control surfaces which are widely used in biomedical devices. Moreover, osteoblast cells spread and proliferate on titanium nanopillars without damage to cells.
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Chapter 7: Analysis and Discussion
7.1 OVERVIEW
This chapter explains the results presented in Chapters 4-6 and how they pertain to the recent findings indicated in the literature. The main results and discussions obtained from Chapters 4-6 are divided into three main categories: (a) finding the optimum natural bactericidal and cytocompatible nanopillars of the three cicada species, (b) simulation and optimization of EBL process variables to fabricate titanium nanopillars as close as possible to natural nanopillars, and (c) evaluation of bacterial interaction and cytocompatibility with titanium nanopillars. Section 7.1 explains and compares surface characterization and nanopillar geometry of the wings of the three cicada species to previous studies. Section 7.2 analyses the bactericidal efficiency of three cicada species wings against both P. aeruginosa and S. aureus bacteria strains and compares these results to previous studies. Sections 7.3 and 7.4 discuss the bacteria interactions with cicada nanopillars and the effect of geometry and its correlation with bactericidal efficiency. Section 7.5 explains the biocompatibility of the natural cicada wing nanopillars and compares it with published data. The outcomes of Sections 7.2, 7.3 and 7.4 helped with fabrication of the most accurate geometry to mimic natural cicada nanopillars. Section 7.6 explains the selection of the ideal method with high resolution and good throughput to fabricate titanium nanopillars mimicked from cicada wings. Section 7.7 explains the systematic approach used to optimize the EBL process for fabricating the titanium nanopillars. Section 7.8 compares the bacteria interaction on cicada wing nanopillars with titanium nanopillars and finally, Section 7.9 discusses the biocompatibility of titanium nanopillars.
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7.2 CICADA WING SURFACE CHARACTERIZATION
The surface characterization of the wings of the three cicada species, Psaltoda claripennis (PC), Aleeta curvicosta (AC), and Palapsalta eyrei (PE), was conducted and analysed as reported in Chapter 4. The veins and membranes of hindwings and forewings possessed consistent and uniform nanopillar arrays without damage, which is consistent with previous literature (Kelleher et al., 2015). Nanopillar geometry varies among insects (cicada and dragonfly) as well between species in the same taxonomic groups (Table 7.1). To the best of our knowledge, this is the most comprehensive nano topography analysis of cicada wings, which identifies all geometric parameters such as cap diameter (spike diameter), base diameter, height, centre to centre distance, as well as density and aspect ratio on both the membrane and vein. Most research on cicada wings has focused on PC (Ivanova et al., 2012), while AC and PE have rarely been investigated in recent studies. The PC membrane had a spike diameter of 72.4 ± 5.6 nm, base diameter of 136.9 ± 11.4 nm, height of 202.6 ± 10.9 nm, centre to centre distance of 176.4 ± 12.7 nm, aspect ratio of 1.9 ± 0.2 nm and density of 32.2 ± 2.4 n/µm2 (Table 4.1), which is consistent previous results (Hasan, 2013, Ivanova et al., 2012). The most noticeable difference between the nanopillar structure of dragonfly and cicada wings are observed in diameter and height. Bandara has categorized dragonfly wing nanopillars into two groups, the first group are short with a diameter of 37 ± 6 nm and height of 189 ± 67 nm and the second group are taller, with a diameter of 57 ± 8 nm and height of 311 ± 52 nm (Bandara et al., 2017, Mudiyanselage and Bandara, 2017). Cicada Wing nanopillar geometry is constant in height and centre to centre distance but had cap and base diameters, giving a pear-like shape to the nanopillars (Shahali et al., 2019). The nanopillars on the dragonfly wing are not completely perpendicular to the surface, whereas cicada wing nanopillars have an upright direction (Bandara et al., 2017a, Shahali et al., 2019). Hasan found that damselfly wings are thinner (0.57 μm) than dragonfly wings (2 μm) (Hasan, 2013). The diameter of dragonfly wing nanopillars (37-57 nm) on the membrane is generally smaller than cicada wing nanopillars (60-200 nm), among all species. HIM was employed for the first time to precisely measure dragonfly wing nanopillars without artefacts and metal coating (Bandara et al., 2017a, Mudiyanselage and Bandara, 2017). This research was the first to employ HIM for in-depth analysis
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of cicada nanopillar nano topography. AFM analysis and TEM were also employed to confirm the HIM results. The nano topography analysis demonstrated that the density and aspect ratio of membrane nanopillars was higher than vein nanopillars, for all three species (Figure 7.1). PE has a nanopillar density of 40 and 11.3 n/μm2 on the membrane and vein, respectively, AC has nanopillar density of 40 and 7.6 n/μm2, on the membrane and vein, respectively, while PC has a membrane nanopillar density of 32 n/μm2 and a vein density of 5.2 N/μm2. These are higher than the densities reported for cicada wings in previously published literature (Figure 4.2 and Table 7.1) (Sun et al., 2012). PE also had the highest aspect ratio on membranes and veins with 2.3 and 1.7, respectively, compared to PC and AC (Figure 4.3) which were not analysed in previous studies (Table 7.1).
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Figure 7.1: Comparison of the nanopillar geometry on the membranes and the veins of Psaltoda claripennis (PC), Aleeta curvicosta (AC), and Palapsalta eyrei (PE).
AFM analysis was employed to measure surface roughness (Table 4.2 and Table
7.1). PC, AC and PE have a surface roughness (Ra) of 27.7, 34.6 and 119.7 nm, respectively. (Ivanova et al., 2012) reported a surface roughness of 25 nm for PC which was close to the surface roughness reported in this research (27.7 nm). In the latest published results on dragonfly wings, the surface roughness (Ra) was reported to be 32 nm (Bandara et al., 2017), which close to that of AC and PE in this research with values of 39.5 and 39.2 nm, respectively (Shahali et al., 2019). To prove that the bactericidal mechanism of natural nanopillars is not related to chemistry, chemical characterization of the three cicada species wings was conducted using FTIR and XPS (Figures 4.10 and 4.11). FTIR spectrum information from the
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three wings presented particular absorbance peaks which relate to specific chemical compounds. FTIR peaks show the molecular data related to various functional groups (Figure 4.10 and Table 7.2). From the FTIR spectra, similar absorbance peaks were found for the three cicada species wings, indicating similar chemistry for all wings (Figure 4.10). In all cicada wings, amide I, II and III peaks were identified at 1621, 1528 and 1226 cm-1, respectively, which are associated with protein and chitin. Methyl and methylene were found at 2945, 2920 and 2853 cm-1, which is attributed to wax and hydrocarbons. An -1 absorbance peak of 3290 cm represents O-H or N-H stretch, typically associated with phenols, alcohols or amines (Table 7.2). In summary, all wings possessed amide (I, II and III), carbonyl, methyl and methylene, and O-H stretch which is associated with protein, chitin, wax (hydrocarbon) and carboxylic acids. These results are consistent with previous studies (Ganim et al., 2008, Movasaghi et al., 2008b, Mistry, 2009, Tobin et al., 2013, Hasan, 2013, Sajomsang et al., 2010).
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Table 7.1 summarized and compared the geometry of different species of cicada and dragonfly.
Insect Species Cap diam. Base diam. Height Centre to centre Aspect Density Surface Roughness Reference 2 (nm) (nm) (nm) distance (nm) ratio (n/µm ) (Ra, Rq, Rmax) nm Cicada P. claripennis (PC) 60 100 200 170 - - 25, 31.8, 257 (Ivanova et al., 2012) Cicada P. claripennis (PC) 70 100 230 170 (Hasan, 2013) Cicada C. maculata 97 309 92 - - (Sun et al., 2009) Cicada P. scitula 84 282 84 - - (Sun et al., 2009) Cicada M. hebes 85 164 95 - - (Sun et al., 2009) Cicada L. bifuscata 90 200 117 - - (Sun et al., 2009) Cicada M. conica 95 159 115 - - (Sun et al., 2009) Cicada M. durga 89 89 257 - - (Sun et al., 2009) Cicada A. bindusara 84 234 91 - - (Sun et al., 2009) Cicada M. microdon 82 208 89 - - (Sun et al., 2009) Cicada M. mongolica 128 417 47 - - (Sun et al., 2009) Cicada P. radha 137 288 44 - - (Sun et al., 2009) Cicada D. vaginata 132 363 56 - - (Sun et al., 2009) Cicada D. nagarasingna 128 316 47 - - (Sun et al., 2009) Cicada M. opalifer 148 418 48 - - (Sun et al., 2009) Cicada T. vacua 141 446 44 - - (Sun et al., 2009) Cicada T. jinpingensis 141 391 46 - - (Sun et al., 2009) Cicada M. intermedia 156±29 241 165±8 1.55 - (Kelleher et al., 2015) Cicada C. aguila 159±47 182 187±13 1.15 - (Kelleher et al., 2015) Cicada A. spectable 207±62 182 251±31 0.88 - (Kelleher et al., 2015) Cicada L. bifuscata 90 200 117 30 (Sun et al., 2012) Cicada A. bindusara 84 234 91 42 (Sun et al., 2012) Cicada M. opalifer 148 418 48 33 (Sun et al., 2012) Cicada C. atrata 85 462 90 42 (Sun et al., 2012) Cicada N. pruinosus 197.2 405 217.16 - - - (Oh et al., 2017)
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Cicada N. tibicen 210.8 345 224.4 - - - Oh et al., 2017) Cicada Me. dorsatus 195.8 250 226.7 - - - Oh et al., 2017) Cicada Ma. septendecim 210.2 50 227.1 - - - Oh et al., 2017) Cicada P. claripennis 72.4±5 136.9±11 202.6±11 176.4±13 1.9±0.2 32.2±2 27.7,34.6, 119.7 (Shahali et al., 2019) (PC)-Membrane P. claripennis 149.4±14 300±34 345.6±39 393.8±50 1.5±0.2 5.2±0.7 (Shahali et al., 2019) (PC)-vein Cicada A. curvicosta (AC)- 65.4±4.6 125.7±7.8 213±17.4 171.3±12.5 2.2±0.2 40.2±1.3 39.5, 48.7, 159.6 (Shahali et al., 2019) Membrane A. curvicosta (AC)- 153.9±25.2 322.6±30.6 244.4±20.9 387.3±46.7 1.2±0.1 7.6±1.8 (Shahali et al., 2019) Vein Cicada P. eyrei (PE)- 60.3±3.7 123.2±7.5 211.2±19.1 155.9±13.4 2.3±0.3 40±1.6 39.2, 47.9, 168.6 (Shahali et al., 2019) Membrane P. eyrei (PE)- Vein 110.7±9.5 218.7±25 281.6±22.5 335.8±38.9 1.7±0.2 11.3±1.1 (Shahali et al., 2019) Dragonfly - 40 240 - - - - (Hasan, 2013) & Damselfly Dragonfly - 37±6 189±67 - - - 32 (Bandara et al., 2017) 57±8 311±52
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Table 7.2: Comparing FTIR data and assigning vibration absorbance bands associated with
compounds (vs: Symmetric and vas: antisymmetric C-H stretch absorbance).
Absorbance Peak obtained Corresponding bond Chemical Reference Peak Range in this research compound (cm-1) 1600-1700 1621 C=O stretch Protein and chitin (Hasan, 2013, weakly coupled Sajomsang et al., with N-H 2010) bending and C-N stretch (Amide I) 1500-1600 1528 C-N stretch strongly Protein and chitin (Ganim et al., 1490-1590 coupled with N-H 2008, Tobin et bending (amide al., 2013, Hasan, II) 2013, Sajomsang et al., 2010) 1200-1350 1226 N-H in-plane Protein and chitin (Lin-Vien et al., bending coupled 1991) with C-N stretching & C-H and N-H deformation vibration (Amide III)
2840-3000 2945, 2920, vs C-H and vas C-H Wax as long- (Hasan, 2013,
2853 stretch (CH2 and chain Lin-Vien et al.,
CH3)- Methyl and hydrocarbon 1991) Methylene 3200-3500 3290 O-H stretch or N-H Phenols, alcohols (Hasan, 2013, stretch or amine Lin-Vien et al., 1991)
Carbon (C1s with the highest peak of ~285 eV), nitrogen (N1s with ~397 eV) and oxygen (~530 eV for O1s and 23 eV for O2s) were the major components of all three wings (Figure 4.11). These results identify the existence of protein, chitin and wax on the three cicada wings, confirming that the chemical composition of nanopillars is not associated with bactericidal properties (Ivanova et al., 2013, Mainwaring et al., 2016, Nguyen et al., 2013, Ivanova et al., 2012). To evaluate the mechanical stability of cicada wing nanopillars, AFM contact mode was employed to measure the mechanical properties of nanopillars. The reduced Young's modulus of PC, AC and PE were 45.34 ± 6.7, 48.16 ± 7 and 35.69 ± 5.2 MPa, respectively, while published work on dragonfly wings shows that short and tall nanopillars have Young’s moduli of 38 ± 3.8 MPa and 23 ± 5.7 MPa, respectively (Bandara et al., 2017).
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7.3 ANALYSIS OF BACTERICIDAL EFFICIENCY OF NANOPILLARS AGAINST P. AERUGINOSA AND S. AUREUS
Bactericidal efficiency of PC, AC and PE wings, control media and control surface against P. aeruginosa and S. aureus was determined using the standard plate count method reported in Figures 4.23 and 4.25 at 2, 4 and 18 hr time intervals. All three wings had a remarkable bactericidal effect (p < 0.0001) against P. aeruginosa after 4 and 18 hrs. However, there is no considerable difference in bactericidal efficiency between the cicada species. PE produced the highest bactericidal efficiency, starting from 1.98 × 106 and decreasing to 1.15 × 106 after 4 hrs and then to 7.3 × 105 CFU/mL after 18 hrs (Figure 4.23 and Appendix A). The three wings had similar levels of bactericidal efficiency against Gram- positive S. aureus cells with a coccoid shape, however, AC and PE showed higher bactericidal efficiency (p < 0.0001) after 18 hrs compared to PC. Colony-forming units 6 6 of S. aureus on AC decreased from 2.54 × 10 CFU/mL to 1.43 × 10 CFU/mL after 6 6 18 hrs while those on PC reduced from 2.52 × 10 CFU/mL to 1.53 × 10 CFU/mL (Figure 4.25 and Appendix 1). However, published works claim that Gram-positive bacteria including B. subtilis, P. maritimus, and S. aureus are resistant to cicada wing nanopillars (Hasan et al., 2013d) and only dragonfly wings have bactericidal properties against Gram-positive bacteria (Table 7.3) (Ivanova et al., 2013). The reason for inconsistency of result can be attributed to the dilution factor in plate counting as well as lack of in-depth analysis. Bandara et al. observed that the bactericidal efficiency against E. coli decreased from 4.99 × 105 cells min-1 cm-1 after 1 hr to 1.65 × 105 after 4 hrs (Bandara et al., 2017). Ivanova found that bactericidal efficiency of cicada wings over 3 hrs was 2 × 105 against P. aeruginosa, while the other bacterial strains such as S. aureus and B. subtilis were resistant to cicada wing nanopillars. It is also claimed that bactericidal efficiency of dragonfly wings over 3 hrs was 3 × 105 cells min-1 cm-2 against P. aeruginosa, 4.6 × 105 cells min-1 cm-2 against S. aureus and 1.4 × 105 cells min-1 cm-2 against B. subtilis (Ivanova et al., 2013)(Table 7.3).
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Table 7.3: Comparison of bactericidal efficiency of natural nanopillar of dragonfly and cicada wings.
Type of insect Cell tested Bactericidal efficiency Finding Reference Cicada P. aeruginosa ATCC 9027 Not tested Needs 200 nm penetration and 220 s for (Ivanova et al., membrane damage. 2012) Attachment and killing process starts from 20 min. Cicada P. aeruginosa- ATCC 9027 2 × 105 cells min-1 cm-2 in 3hrs S. aureus and B. subtilis are resistant to (Ivanova et al., cicada nanopillars. 2013) Cicada B. subtilis- (NCIMB) 3610T 6.14 × 106 cfu cm-2 in 30 min for P. aeruginosa Killed Gram-negative bacteria, e.g. E. coli (Hasan et al., 2013d) P. maritimus- (KMM) 3738 and P. aeruginosa, S. aureus- (CIP) 65.8T B. subtilis, P. maritimus, and S. aureus were E. coli- K12 resistant. P. aeruginosa- ATCC9027 Cicada P. fluorescens PCL1701 The bacterial dead: live ratio: The distance of nanopillar and diameter (Kelleher et al., - 0.222 for M. intermedia affect the bactericidal effect. 2015) - 0.123 for C. aguila - 0.067 for A. spectable Cicada P. aeruginosa - ATCC 27853 PC: 1.98 × 106 to 0.91 × 106 CFU/mL in 18 hrs - (Shahali et al., 2019) AC: 1.98 × 106 to 0.82 × 106 CFU/mL in 18 hrs PE: 1.98 × 106 to 0.72 × 106 CFU/mL in 18 hrs Cicada S. aureus - ATCC 25923 PC: 2.52 × 106 to 1.53 ×106 CFU/mL in 18 hrs - (Shahali et al., 2019) AC: 2.52 × 106 to 1.43 × 106 CFU/mL in 18 hrs PE: 2.52 × 106 to 1.48 × 106 CFU/mL in 18 hrs Dragonfly P. aeruginosa - ATCC 9027 3×105 cells min-1 cm-2 - (Ivanova et al., S. aureus (CIP) 65.8T 4.6×105 cells min-1 cm-2 2013) B. subtilis (NCIMB 3610) 1.4×105 cells min-1 cm-2 (in 3 hrs) Dragonfly E. coli 4.99×105 cells min-1 cm-1 in 1 hr. - (Bandara et al., 1.65×105 cells min-1 cm-1 in 4 hrs 2017)
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7.4 ANALYSIS OF BACTERIUM INTERACTION WITH NATURAL CICADA WING NANOPILLARS
SEM and AFM analysis showed that both bacteria strain membranes (P. aeruginosa and S. aureus) strongly attached and interacted with the nanopillars of the three cicada species wings (Figures 4.15, 4.16, 4.17, 4.20, 4.21 and 4.22). Increased turgor pressure applied by nanopillars, stretched and damaged the bacterial cell membrane (Pogodin et al., 2013). Nanopillars penetrated the bacteria membrane, causing depletion and damage (Figure 4.15, 4.16 and 4.17). Qualitative results were consistent with published literature (Ivanova et al., 2012b, Hasan et al., 2013d). However, (Hasan et al., 2013d) found that Gram-positive bacteria were resistant to cicada wing nanopillars. From qualitative analysis using SEM, it was found that S. aureus cells were also vulnerable to cicada wing nanopillars. Both bacteria species remained undamaged at the top or in the middle of the vein nanopillars (mainly on PC and AC) due to the lower density and aspect ratio of nanopillars (Figure 7.2).
7.5 EFFECT OF THE GEOMETRY OF NANOPILLAR ON BACTERIA INTERACTION
Nanopillar geometry (e.g. centre to centre distance, height, diameter) was a key parameter in the bacteria killing mechanism of nanopillars. Kelleher found that the number of nanopillars interacted with bacteria during the attachment process is an important factor in bactericidal activity. Denser nanopillars and smaller sized pillars produce better killing effects on Gram-negative bacteria, P. fluorescens (Kelleher et al., 2015), with similar results reported by Dickson on PMMA nanopillars (Dickson et al., 2015). In this research, the effect of the geometry of the cicada wing nanopillars (e.g. contact number, spike diameter, aspect ratio and nanopillar density) on bacteria interaction was analysed. Based on the fixed size of P. aeruginosa at 2 × 0.5 μm (obtained from the average measurement of P. aeruginosa on glass), the killing capability of the cicada wings was compared (Figure 7.3). PE had the highest contact number of 11, lowest spike diameter of 60 nm, the highest aspect ratio of 2.3 with density of 40 n/μm2 compared to AC and PC which created more contact points to increase turgor pressure, causing damage to the bacteria membrane. The plate count method results (Figure 4.23) confirmed that PE produced the highest bactericidal effect
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after 18 hrs. The nanopillars of the vein in the three species had higher contact numbers (contact number of 5 and 6) and spike diameter, and lower density and aspect ratio compared to the membrane, resulting in less turgor pressure. P. aeruginosa remained undamaged on the nanopillars.
Figure 7.2: Bacteria interaction with nanopillars on veins and membranes: (a) left, schematic of P. aeruginosa interaction with AC membrane and right, associated SEM image, (b) left, schematic of P. aeruginosa interaction with AC vein and right, associated SEM image, (c) left, schematic of S. aureus interaction with AC membrane and right, associated SEM image, (d) left, schematic of S. aureus interaction with AC vein and right, associated SEM image.
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As S. aureus, with a diameter of 678 nm (obtained from average measurement of S. aureus on glass), had two contact numbers in contact with nanopillars on veins and membranes of the three species, it had more chance of being killed by nanopillars with smaller spike diameters like AC with 65 nm and PE with 60 nm (Figure 7.4). In summary, sharp nanopillars with high density and aspect ratio produced higher bactericidal effects on both P. aeruginosa and S. aureus strains. The size, shape and the structure of bacteria also influenced the bactericidal effect (Dickson et al., 2015a, Hasan et al., 2013d). S. aureus is a cocci Gram-positive bacteria with a size of 0.5-1.5 µm (Harris et al., 2002), resulting in two contact points with cicada nanopillars (Figure 7.3), increasing the chance of being killed by sharp nanopillars. P. aeruginosa is a rod-shaped Gram-negative bacteria with a length of 1- µm and width of 0.5-1.0 µm (Costerton et al., 1994), generating 9 to 11 contact points with cicada nanopillars, resulting in an increased chance of death compared to S. aureus.
Figure 7.3: Effect of the geometry of cicada nanopillars (on veins and membranes) on P. aeruginosa.
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Figure 7.4: Effect of the geometry of cicada nanopillars (on veins and membranes) on S. aureus.
The cell structure (e.g. cell wall, peptidoglycan layer PG) of S. aureus and P. aeruginosa play an important role in the bactericidal effect (Pogodin et al., 2013a, Ivanova et al., 2012). Plate counting results (Figure 4.23 and Figure 4.25) and SEM analysis showed that P. aeruginosa is more susceptible to being killed by nanopillars compared to S.aureus, because the thickness of the peptidoglycan (PG) layer of P. aeruginosa cells is 2.4 nm, while S. aureus has a PG layer of 19 nm (variable 20-40 nm, Figure 3.2) (Goldman and Green, 2008), requiring less turgor pressure for Gram- negative bacteria to be killed on nanopillars. Gram-negative bacteria like P. aeruginosa also have pili which increase the chances of adhesion and biofilm formation (Van Schaik et al., 2005).
7.6 BIOCOMPATIBILITY OF CICADA NANOPILLAR
In addition to analysis of bactericidal efficiency and bacteria interaction, biocompatibility or cytocompatibility (cellular metabolic activity) of osteoblast cells was analysed using AlamarBlueTM assay. The cicada wing nanopillars have no negative effect on osteoblast cells, and possessed higher cellular metabolic activity
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compared to the control surface. To the best of our knowledge, this was the first time that cytocompatibility of human osteoblast cells was studied on cicada wings. (Watson et al., 2017) analysed the biocompatibility of stem cells and epithelial cells on Planthopper wings. Planthopper wings have a microstructure with a height of 6.5 μm, width of 3.8 μm, spacing of 14.4 μm, density of 57 (n/10000 μm2), and nano protuberances 520 nm long, and 47 nm wide. The wing membrane of planthoppers produced biocompatibility for growth, division and proliferation of both stem cells and epithelial cells (Watson et al., 2017). The results of cytocompatibility and SEM human osteoblast cells on the cicada wings (Figures 4.27, 4.28, 4.29 and 4.30) agreed with this published literature on planthopper wings (Watson et al., 2017).
7.7 SELECTING THE IDEAL METHOD TO MIMIC AND FABRICATE TITANIUM NANOPILLARS
The first objective of this research aimed to study natural cicada nanopillars, leading to the second objective: to select the best biomimicking method for fabricating titanium nanopillars. For this purpose, selecting the ideal method to fabricate the high resolution of nanopillars (diameter size less than 100 nm) is crucial and necessitated more in-depth study on all currently used methods. Earlier research mimicking cicada wing nanopillars employed soft lithography methods like nanoimprint lithography (NIL) (Dickson et al., 2015), UV Nanoimprint Lithography (Cho et al., 2013) and micro-moulding (Li et al., 2016) to replicate nanopillars on soft material such as PMMA, Poly-vinyl siloxane (PVS) and epoxy resin. These methods have good throughput, low resolution, and are limited to polymer and soft materials with low melting temperatures (Figure 7.5). Reactive Ion Etching (RIE) was recently used to fabricate nanopillars on silicon wafers with 500 nm to 4 µm height, 220 nm diameter and random spacing (Ivanova et al., 2013, Hasan et al., 2015), and titanium with 1 µm height and 80 nm diameter (Ghosh et al., 2019, Hasan et al., 2017). The hydrothermal method has been recently used for fabrication of titanium nanostructures (nanofiber/nanowire). Nanowires and nanostructures fabricated by the hydrothermal method had heights of 250-945 nm and diameters of 20-52 nm with random spacing (Jaggessar et al., 2018, Jaggessar et al., 2020) (Table 7.4). However, RIE and hydrothermal methods are high throughput
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methods for fabrication of titanium nanopillars and nanowires and the resolution is limited as there is no control on centre to centre distance and nanopillar diameter. Among all methods for titanium nanopillar fabrication, EBL can mimic titanium nanopillars of cicada wings with high-resolution (<100 nm) and high versatility to design various geometries. This is the first time that EBL has been employed to fabricate titanium biomimicked nanopillars. EBL fabricated titanium nanopillars with a uniform shape like cicada wings, and different geometry design (diameter, height and centre to centre distance) that are difficult to generate with RIE and the hydrothermal method (Shahali et al., 2019).
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Table 7.4: Comparison of nanopillars fabrication methods. Fabrication Nanopillar geometry (diameter, height, centre to centre Material Throughput Resolution Comments Reference method distance) NIL - 215, 300, 595 nm PMMA **** ** Only soft material with low (Dickson et al., 2015) - 190, 300, 320 nm melting temperature. - 70, 210, 170 nm
UV-NIL 100 nm diameter PET film **** ** Only soft material with low (Cho et al., 2013), melting temperature. Micro-moulding Base dia:540 nm, Cap dia: 200 nm, height: 1.2 μm, Poly-vinyl **** * Only on soft material with low (Li et al., 2016) centre to centre distance: 1 μm siloxane (PVS) melting temperature. RIE - 100 nm height and width 100 nm Diamond ***** ** No control on geometry like (Fisher et al., 2016). - 3 to 5µm height, the width of 1.2µm height. RIE 4 µm height, 220 nm diameter, random distance Silicon wafer ***** ** Random distance. (Hasan et al., 2015). RIE 500 nm height Silicon wafer ***** ** No control on geometry. (Ivanova et al., 2013). RIE Height: 1 μm, diameter: 80 nm Titanium ***** ** Randomly oriented. No control (Ghosh et al., 2019) on geometry. Hydrothermal Height: 250-945 nm, diameter: 20-52 nm Titanium ***** ** Randomly oriented. No control (Jaggessar et al., 2018) on geometry. EBL Diameter 300 nm, height 400 nm and pitch 500 nm Gold *** *** Design geometry is far beyond (Jindai et al., 2019) fabricated geometry. EBID Height 190 nm, dia: 80 nm, centre to centre distance: Platinum and ** *** Low throughput. (Ganjian et al., 2019) 170 nm carbon EBL Base dia: 93.6 nm, spike dia:13.3, height: 117 nm, Titanium *** **** Control on resolution - ability to (Shahali et al., 2019) centre to centre dist: 166 nm. design. Need a clean room - Base dia:148 nm, spike dia: 22.2 nm, height: 222 nm, high cost of fabrication. centre to centre dist:206 nm. Base dia: 214 nm, spike dia: 49 nm, height: 282 nm, centre to centre dist: 325 nm
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7.8 SYSTEMATIC APPROACH TO OPTIMIZE THE EBL PROCESS VARIABLES TO FABRICATE TITANIUM NANOPILLARS
EBL is an expensive fabrication method which needs a cleanroom and high operation skill. In this research, the systematic study was performed to reduce costs and time (Figure 5.1). To the best of our knowledge, this is the first time EBL has been employed with a systematic approach to create titanium nanopillars biomimicking cicada wings. Monte Carlo simulation was performed to find the optimum kV and pattern design (dot and circle) on different resist thicknesses. From the simulation, it was found that the circle design and 30 kV were ideal to fabricate on large thickness PMMA (>500 nm). The simulation results potentially saved 20 days of primary experimentation and $9,600 (labour and equipment costs). Process variables for EBL include thickness of the resit, dose (µC/cm2), spot size, beam density, the working distance, dwell time, field size, DAC resolution, and pitch, which affect fabrication performance. Beam intensity, working distance, spot size and current were fixed (Table 5.1) while the resist thickness, dose, spacing, pitch, field size were optimized during experimentation. For all resist thicknesses, dose test (e.g. Exposure Factor (EF)) was applied and write field (which affects resolution parameters including pitch, spacing, DAC resolution and dwell time) were optimized until satisfactory results were achieved. For spot size 2.8 nm, the lower field size (25 μm × 25 μm) decreased the pitch from 7.74 nm to 1.7 nm and spacing from 2.4 nm to 0.602, resulting in higher resolution (Figure 7.5).
Figure 7.5: Effect of write field on pitch and spacing (note: spacing is a ratio and unitless).
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Neither using one layer or multi-layers of PMM 950 A4 met satisfactory results, as PMM 950 A4 has a high molecular weight, resulting in desirable lift-off, agreeing with previous results on the fabrication of metallic nano-transistors (Cheng, 2008). Using a thick layer of low molecular weight PMMA 450 A4 as a base layer and a thin layer of high molecular weight PMMA 950 A2 as a top layer, provided high resolution, desirable lift-off and repeatable results. From experimental results, it was found that three layers of PMMA 495 A4 (602 nm) and one layer of PMMA 950 A2 (65 nm) with write field 25 µm × 25 µm produced the optimum result to fabricate three groups of nanopillar array designs. (1). Circle design patterns with diameters of 70 nm and distances of 160 nm fabricated nanopillars with a base diameter of 94.4 nm, spike diameter of 12.6 nm, and distance of 166 nm based on exposure factor EF 2. (2). Circle design patterns with a diameter of 120 nm and distance of 200 nm fabricated nanopillars with a base diameter of 149 nm, spike diameter 21 nm, and distance of 206 nm based on exposure factor EF 1.3. (3). Circle design patterns with a diameter of 200 nm and distance of 320 nm fabricated nanopillars with a base diameter of 214 nm, spike diameter 49, and distance of 325 nm based on exposure factor EF 1 (Table 5.9). The outcome of the experiment also helped to fabricate three sets of nanopillar array designs on the same thickness of PMMA but with different EF, saving 3 hrs of sample preparation for each sample.
From EBL simulation and experimental results, the following scenarios may occur during electron exposure, development, coating and lift-off (Figure 7.6):
Scenario 1: E-beam exposure cannot fully penetrate the resist due to lack of exposure (low level of EF) or lack of energy of the electron (lack of kV), causing the removal of the resist and coating during lift-off. Simulation showed that 30 kV possessed suitable energy to make a pattern on the thick resist.
Scenario 2: Dot patterns on thick resists cause higher diffraction of electrons, resulting in overlapping (partial or full of two features close together) followed by unsatisfactory fabrication. Simulation confirmed that the dot pattern generates the high diffraction of the beam, causing overlapping on the resist.
Scenario 3: Overexposure (high-level of EF and kV) causes full overlapping and no nanopillars fabricated on the surface after lift-off.
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Scenario 4: High step coverage occurred due to using low resist thickness and high coating thickness and using a single layer of high molecular weight resist, resulting in full coverage of the resist by a coating, which does not allow the lift-off.
Scenario 5: The optimum condition with low step coverage in which the thickness and EF are optimized to achieve desirable lift-off and repeatable results.
Figure 7.6: Schematic of possible scenarios during electron beam lithography.
The process optimization of electron beam lithography of bioinspired titanium is illustrated in Figure 7.7.
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Figure 7.7: Process optimization for nanopillar fabrication using electron beam lithography.
7.9 COMPARISON OF BACTERIA INTERACTION ON CICADA WING NANOPILLARS AND TITANIUM NANOPILLARS
The optimum geometry of fabricated titanium nanopillars in three groups is shown in Table 6.1 and Figure 7.7. SEM analysis shows that P. aeruginosa and S. aureus cells interact with titanium nanopillars in the same way as cicada nanopillars. The smallest titanium nanopillar had a spike diameter of 12.6 nm, base diameter of 94.4 nm, height of 115.6 nm, centre to centre distance of 165.8 nm, aspect ratio of 2.15 and density of 43 n/µm2 (Figure 7.8 (b)). This was closer to PE nanopillars with a spike diameter of 60.3 nm, base diameter of 123.2 nm, height of 211.2 nm, centre to centre distance of 155.9 nm, aspect ratio of 2.3 and density of 40 n/µm2 (Figure 7.8 (e)), producing enhanced killing performance compared to two other groups of titanium nanopillars (Figure 7.8 (d,f)). The contact point between PE and P. aeruginosa and the smallest titanium nanopillars with P. aeruginosa were at similar levels (10 and 9), the
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spike diameter of the titanium nanopillar was 12.6 nm which was 4.8 times smaller than PE, generating more turgor pressure for membrane damage. The contact point of all nanopillar arrays is two interactions with S. aureus, however, nanopillar arrays with a sharp spike and high density showed a better killing performance (Figure 7.8 (b)). Sharper nanopillars with high density (lower centre by centre distance) produced better killing performance, which is supported by previous research by Kelleher (Kelleher et al., 2015). We also found that nanopillars with heights of 115.6 nm, spike diameter of 12.6 nm and density of 43 n/µm2 had a killing mechanism. It was not required for the nanopillar to be 200 nm tall, as initially proposed by Ivanova et al (Ivanova et al., 2012).
Figure 7.8: Comparison of cicada nanopillars and titanium nanopillars and their bacteria interaction.
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7.10 BIOCOMPATIBILITY OF TITANIUM NANOPILLARS
Cytocompatibility and proliferation tests (in vitro test) are necessary before implantation of titanium biomedical devices into the human body. Cytocompatibility of fabricated titanium nanopillars was assessed by AlamarBlueTM assay through the cellular metabolic activity of human osteoblast cells. Titanium control surfaces (without nanopillars) and titanium nanopillar surfaces had the same level of cellular activity after 1 and 3 days (Figure 6.7). Moreover, osteoblast cells could spread and have increased cell proliferation with titanium nanopillars compared to the flat titanium surface (Figure 6.8). Pure titanium and its alloys are widely used in biomedical devices and orthopaedic implants which produce ideal biocompatibility with human cells like osteoblasts (Shahali et al., 2017, Geetha et al., 2009). Titanium fabricated nanopillars also have good biocompatibility and cytocompatibility. Nanopillars with 1 μm height and 80 nm diameter fabricated by RIE produced the greatest cell attachment and cell proliferation with Human Mesenchymal Stem Cells (hMSCs) (Hasan et al., 2017), and hydrothermal nanopillars with 298 nm length and 52 nm diameter increased metabolic activity, compared to flat titanium from 4 to 24 hrs (Jaggessar et al., 2018, Jaggessar et al., 2020). In this research, titanium nanopillars fabricated by EBL have ideal cytocompatibility (Shahali et al., 2019), supported by published data for nanopillars fabricated by hydrothermal and REI (Jaggessar et al., 2018, Ghosh et al., 2019, Hasan et al., 2017). Therefore, the findings of this research show that fabricated titanium nanopillars by EBL can be suitable for next generation orthopaedic implants, which have both bactericidal and cytocompatibility properties.
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Chapter 8: Conclusion and Future Work
8.1 INTRODUCTION
This chapter presents the general findings and conclusions of the research project and suggests future work directions based on the research findings. The bactericidal effect and cytocompatibility of both natural cicada and biomimicked nanopillars were studied and evaluated to find an optimum surface with high antibacterial effect and biocompatibility. The research findings show that natural nanopillars and biomimicked titanium nanopillars can conceivably be employed as an antibacterial surface with ideal biocompatibility in biomedical devices like orthopaedic implants. The risk of infections and biofilm formation can be reduced and stopped at the fist attachment of microorganisms based on mechanical interaction (depletion or puncturing) without the side effects of traditional methods like antibiotic therapy and coating. Antibacterial nanopillars can enhance health in biomedical devices and would have economic benefits in food, pipeline and marine industries by preventing the first attachment and subsequent biofilm formation. The inherent antibacterial effect of nanopillars originated from the mechanical and physical interaction between bacteria and nanopillars, increasing the turgor pressure and damaging the membrane of bacteria. This chapter concludes with recommendations for the optimum design of surfaces with the highest antibacterial effect.
8.2 CONCLUSION STATEMENT
The research commenced with the in-depth analysis of surface characteristics and evaluation of the bactericidal effect of three cicada wing species against P. aeruginosa and S. aureus. Advance microscopy (HIM, TEM and SEM) were extensively employed to analyse the geometry of nanopillars as well as bacteria and human osteoblast cell interactions. The cicada wings are covered with uniform arrays of pear-like nanopillars on both membranes and veins. However, the vein possesses lower nanopillar density and aspect ratios in all three cicadas compared to the membrane, Palapsalta eyrei (PE) and Aleeta curvicosta (AC) membranes have higher density and aspect ratios than Psaltoda claripennis (PC).
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Among the three cicada species, PE and AC produced the highest bactericidal effect against P. aeruginosa and S. aureus, respectively. The results of the bactericidal effects of cicada nanopillars against S. aureus contradicts previous research in which S. aureus were found to be resistant to cicada wing nanopillars. In this study, the cicada nanopillars had better killing performance against P. aeruginosa than S. aureus. Analysis of bacteria interaction with nanopillars showed that membrane of cicada nanopillars have more interactions bacteria due to its higher density and aspect ratio. XPS and FTIR analysis confirmed that the chemical composition of cicada wings included wax, chitin and protein which do not have chemical substances for killing bacteria. Nanopillar arrays of all cicada species possessed ideal cytocompatibility compared to the control surface in interactions with human osteoblasts. Biomimicking cicada nanopillars on titanium substrate was conducted by Electron Beam Lithography (EBL) as a high-resolution technique. For this purpose, a systematic study was conducted to optimize the EBL process and save time and cost. Monte Carlo simulation could save time and labour costs associated with the primary experiment (20 days experiment and cost of $9,600). As a result of the simulations and experiments, three sets of titanium nanopillar arrays were fabricated with base diameter ranging from 94.4-214 nm, spike diameter from 12.6-48.9 nm, centre to centre distance from 165.8-324.9 nm and height from 115-288 nm and the process variables, including exposure factor (EF), write field and pitch were optimized to achieve repeatable results. Biomimicked titanium nanopillars with a base diameter of 94.4 nm, spike diameter of 12.6 nm, height of 115.6 nm, density of 43 n/μm2 fabricated with process variables of EF2, write field of 25 µm × 25 µm produced the best killing performance against the two species of bacteria. The geometry of the nanopillar plays an important role in the killing mechanism. Nanopillars with high density and aspect ratio and sharp tip effectively penetrated the membrane and killed the bacteria in the same manner as cicada nanopillars. Titanium fabricated nanopillars not only had a bactericidal effect but also possessed good cytocompatibility with human osteoblast cells in the same manner as the cicada wing nanopillars. Moreover, the SEM analysis confirmed that the titanium nanopillars enhanced cell anchorage and cell proliferation compared to a flat titanium surface. This research provided a systematic strategy to design desired nanopillar arrays and help biomedical device engineers develop a versatile antibacterial surface.
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8.3 OUTLOOK FOR FUTURE RESEARCH
In this research, SEM, HIM and AFM were used to study the cell interface and cell morphology interacting with natural and fabricated nanopillars. To have an in- depth analysis of bacteria interaction and remove the metal coating in traditional TEM and focused ion beam milling which damaged the sample, this research can be extended to use Cryo-TEM tomography to analyse cross-sections at cryogenic temperatures without coating and intense staining. While Cryo-TEM is an expensive technique for the study of cell interactions with nanopillars, it can answer many questions behind the bactericidal mechanism. Contact angle analysis and wettability tests should also be conducted to identify the correlation between the geometry of nanopillars and hydrophobicity. The Tescan SEM system was equipped with a 30 kV EBL system based on a mechanical stage which caused microscale movement during fabrication. Inaccurate stage and chamber contamination are significant challenges in this research. While plasma cleaning of the chamber and selecting a small write field (25 × 25 μm) with a gap of 3 μm can solve most of the issues, it is recommended to fabricate larger samples (> 1 × 1 cm2) using advanced ultra-high-performance systems like the Raith EBL system equipped with a laser interferometer based stage and 100 kV beam energy to halve fabrication duration, resulting in high accuracy and reliability. High fabrication speed at 100 kV can fabricate a larger sample with the more reliable and repeatable results, providing more in-depth quantitative analysis of bactericidal effects and biocompatibility. In this research, due to the available capability of the EBL system, titanium nanopillars were fabricated by EBL in the millimetre scale which was small for quantitative analysis. For each 1 mm × 1 mm sample, 40 × 40 fields (each field 25 μm × 25 μm) should be fabricated. Each field of 25 μm × 25 μm is covered by 29,348 circles. The number of circle patterns on 1 mm × 1 mm is 41,520,000 which takes approximately 6 hrs to fabricate on PMMA resist while other particle beam lithography (PBL) methods, like EBID, can fabricate 42 × 42 μm field sizes in 6-7 hrs. While the EBL method is an ideal high-resolution method, the throughput is low, and it is also expensive with high skill requirements. It is suggested to direct the application of this research to small-size biomedical devices to save time and increase cost-effectiveness. Since nanopillars with a sharp tip are used as highly sensitive detection for biosensors,
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future work could extend the systematic EBL fabrication study and utilize the different geometry of nanopillar arrays to biosensor applications as well as the fabrication of microfluidics and quantum integrated circuits. The mechanical stability of titanium nanopillars, adhesion between the nanopillar and titanium substrate and the correlation between the mechanical properties and bactericidal effect can be a novel edge for future directions. It is recommended that more in vitro studies, such as cell viability, and in vivo testing, be conducted before testing it in the human body. Moreover, the different responses of blood cells and stem cells can be investigated to generalize the application in various biomedical industries. Co-culturing of osteoblasts and bacteria can be considered as a future direction to evaluate the osteogenic and anti-infective characteristics of titanium nanopillars. It also can assess competition of bacteria and osteoblasts in the attachment to the titanium nanopillars. In conclusion, this research has studied the bactericidal effect and biocompatibility of natural cicada wings and developed a versatile method to design and fabricate titanium nanopillar arrays with bactericidal effects and ideal biocompatibility. The fabricated titanium nanopillars are an ideal tool to reduce bacteria attachment, biofilm formation and infection on the surface of titanium orthopaedic implants without applying chemicals and antibiotic therapy.
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Bibliography
APPEL, E., HEEPE, L., Lin, Chung-Ping. & GORB, Stanislav N .2015. Ultrastructure of dragonfly wing veins: composite structure of fibrous material supplemented by resilin. Journal of Anatomy, 227, 561-582. ARCIOLA, C. R., CAMPOCCIA, D. & MONTANARO, L. 2018. Implant infections: adhesion, biofilm formation and immune evasion. Nature Reviews Microbiology, 16, 397-409. ARONSSON, B.-O., KROZER, A., LAUSMAA, J. & KASEMO, B. J. S. S. S. 1996. Commercially Pure Titanium and Ti6Al 4V: XPS Comparison Between Different Commercial Ti Dental Implants and Foils Prepared by Various Oxidation Procedures. 4, 42-89.Surface Science Spectra. BAE, H., CHU, H., EDALAT, F., CHA, J. M., SANT, S., KASHYAP, A., AHARI, A. F., KWON, C. H., NICHOL, J. W. & MANOUCHERI, S. 2014. Development of functional biomaterials with micro‐and nanoscale technologies for tissue engineering and drug delivery applications. Journal of Tissue Engineering and Regenerative Medicine, 8, 1-14. BAHAR, A. A. & REN, D. 2013. Antimicrobial peptides. Pharmaceuticals, 6, 1543- 1575. BALL, P. 1999. Engineering shark skin and other solutions. Nature, 400, 507-509. BANDARA, C. D., SINGH, S., AFARA, I. O., TESFAMICHAEL, T., WOLFF, A., OSTRIKOV, K. & OLOYEDE, A. 2017a. Bactericidal Effects of Natural Nanotopography of Dragonfly Wing on Escherichia Coli. ACS Applied Materials & Interfaces. BARTHLOTT, W. & NEINHUIS, C. 1997. Purity of the sacred lotus, or escape from contamination in biological surfaces. Planta, 202, 1-8. BELT, H. V. D., NEUT, D., SCHENK, W., HORN, J. R. V., MEI, H. C. V. D. & BUSSCHER, H. J. 2001. Infection of orthopedic implants and the use of antibiotic-loaded bone cements: a review. Acta Orthopaedica Scandinavica, 72, 557-571. BERBARI, E. F., HANSSEN, A. D., DUFFY, M. C., STECKELBERG, J. M., ILSTRUP, D. M., HARMSEN, W. S. & OSMON, D. R. 1998. Risk factors for prosthetic joint infection: case-control study. Clinical Infectious Diseases, 27, 1247-1254. BHADRA, C. M., KHANH TRUONG, V., PHAM, V. T. H., AL KOBAISI, M., SENIUTINAS, G., WANG, J. Y., JUODKAZIS, S., CRAWFORD, R. J. & IVANOVA, E. P. 2015. Antibacterial titanium nano-patterned arrays inspired by dragonfly wings. Scientific Reports, 5, 16817. BIXLER, G. D. & BHUSHAN, B. 2012. Biofouling: lessons from nature. Philosophical Transactions of the Royal Society of London A: Mathematical, Physical and Engineering Sciences, 370, 2381-2417. BIXLER, G. D. & BHUSHAN, B. 2013. Fluid Drag Reduction with Shark‐Skin Riblet Inspired Microstructured Surfaces. Advanced Functional Materials, 23, 4507- 4528. BIXLER, G. D., THEISS, A., BHUSHAN, B. & LEE, S. C. 2014. Anti-fouling properties of microstructured surfaces bio-inspired by rice leaves and butterfly wings. Journal of Colloid and Interface Science, 419, 114-133.
Bibliography 189
BLACK, J. 2005. Biological performance of materials: fundamentals of biocompatibility, CRC Press. BORDO, K. & RUBAHN, H.-G. 2012. Effect of deposition rate on structure and surface morphology of thin evaporated Al films on dielectrics and semiconductors. Materials Science, 18, 313-317. BORGSTRÖM, M. T., MAGNUSSON, M. H., DIMROTH, F., SIEFER, G., HÖHN, O., RIEL, H., SCHMID, H., WIRTHS, S., BJÖRK, M. & ÅBERG, I. 2018. Towards nanowire tandem junction solar cells on silicon. IEEE Journal of Photovoltaics, 8, 733-740. BOZIC, K. J. & RIES, M. D. 2005. The impact of infection after total hip arthroplasty on hospital and surgeon resource utilization. The Journal of Bone & Joint Surgery, 87, 1746-1751. BROERS, A. N., HOOLE, A. C. F. & RYAN, J. M. 1996. Electron beam lithography—Resolution limits. Microelectronic Engineering, 32, 131-142. CAI, K., MÜLLER, M., BOSSERT, J., RECHTENBACH, A. & JANDT, K. D. 2005. Surface structure and composition of flat titanium thin films as a function of film thickness and evaporation rate. Applied Surface Science, 250, 252-267. CAMPBELL, S. A. 1996. The science and engineering of microelectronic fabrication, Oxford University Press, USA. CARMAN, M. L., ESTES, T. G., FEINBERG, A. W., SCHUMACHER, J. F., WILKERSON, W., WILSON, L. H., CALLOW, M. E., CALLOW, J. A. & BRENNAN, A. B. 2006. Engineered antifouling microtopographies - correlating wettability with cell attachment. Biofouling, 22, 11-21. CASSIE, A. & BAXTER, S. 1944. Wettability of porous surfaces. Transactions of the Faraday Society, 40, 546-551. CHEN, P. 2010. Three-dimensional Nanostructures Fabricated by Ion-Beam-Induced Deposition, TU Delft, Delft University of Technology. CHENG, E. 2018. DIRECT WRITE LITHOGRAPHY (DWL) E-BEAM, ION- BEAM, TWO PHOTON NANOLITHOGRAPHY. ANFF-Qld Node. CHENG, H. H., ANDREW, C. N. & ALKAISI, M. M. 2006. The fabrication and characterisation of metallic nanotransistors. Microelectronic Engineering, 83, 1749- 1752. CHENG, Y. T., RODAK, D. E., WONG, C. A. & HAYDEN, C. A. 2006b. Effects of micro- and nano-structures on the self-cleaning behaviour of lotus leaves. Nanotechnology, 17, 1359-1362. CHEVALIER, J. & GREMILLARD, L. 2009. Ceramics for medical applications: A picture for the next 20 years. Journal of the European Ceramic Society, 29, 1245-1255. CHO, J. Y., KIM, G., KIM, S. & LEE, H. 2013. Replication of surface nano-structure of the wing of dragonfly (Pantala Flavescens) using nano-molding and UV nanoimprint lithography. Electronic Materials Letters, 9, 523-526. CHUNG, P. Y. & TOH, Y. S. 2014. Anti-biofilm agents: recent breakthrough against multi-drug resistant Staphylococcus Aureus. Pathogens and Disease, 70, 231- 239. COATES, J. 2006. Interpretation of infrared spectra, a practical approach. Encyclopedia of Analytical Chemistry. DOI: https://doi.org/10.1002/9780470027318.a5606. COSTERTON, J. W., STEWART, P. S. & GREENBERG, E. P. 1999. Bacterial biofilms: a common cause of persistent infections. Science, 284, 1318-1322.
190 Bibliography
COSTERTON, J. W. J. P. A., INFECTION & TREATMENT 1994. Pseudomonas Aeruginosa; the microbe and pathogen. 1-20. DAVIS, R. & MAUER, L. 2010. Fourier transform infrared (FT-IR) spectroscopy: a rapid tool for detection and analysis of foodborne pathogenic bacteria. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, 2, 1582-1594. DE CARVALHO, Carla CCR. 2018. Marine biofilms: a successful microbial strategy with economic implications. Frontiers in Marine Science, 5, 126. DICKSON, M. N., LIANG, E. I., RODRIGUEZ, L. A., VOLLEREAUX, N. & YEE, A. F. 2015a. Nanopatterned polymer surfaces with bactericidal properties. Biointerphases, 10, 021010. DIEBOLD, U. & MADEY, TE. 1996. TiO2 by XPS. 4, 227-231. Surface Science Spectra, 4, 227. DIRKS, J.-H. & TAYLOR, D. 2012. Veins improve fracture toughness of insect wings. PLOS ONE. 7, 8, e43411. DONG, X., TAO, J., LI, Y. Y. & ZHU, H. 2010. Oriented single crystalline TiO2 nano- pillar arrays directly grown on titanium substrate in tetramethylammonium hydroxide solution. Applied Surface Science, 256, 2532-2538. DONG, Y., LI, X., TIAN, L., BELL, T., SAMMONS, R. L. & DONG, H. 2011. Towards long-lasting antibacterial stainless steel surfaces by combining double glow plasma silvering with active screen plasma nitriding. Acta Biomaterialia, 7, 447-457. DU, K., WATHUTHANTHRI, I., MAO, W. D., XU, W. & CHOI, C. H. 2011. Large- area pattern transfer of metallic nanostructures on glass substrates via interference lithography. Nanotechnology, 22, 28. EBL, T. D. F. 2014. DrawBeam for Electron Beam Lithography Instructions for use. In: TESCAN ORSAY HOLDING, A. S., BRNO, CZECH REPUBLIC (ed.). ELSAFADI, M., MANIKANDAN, M., DAWUD, R., ALAJEZ, N., HAMAM, R., ALFAYEZ, M., KASSEM, M., ALDAHMASH, A., MAHMOOD, A. J. C. D. & DISEASE 2016. Transgelin is a TGF β-inducible gene that regulates osteoblastic and adipogenic differentiation of human skeletal stem cells through actin cytoskeleston organization. Cell Death and Disease, 7, e2321- e2321. EMAMI, S. H., SALOVEY, R. & HOGEN‐ESCH, T. E. 2002. Peroxide‐mediated crosslinking of poly (ethylene oxide). Journal of Polymer Science Part A: Polymer Chemistry, 40, 3021-3026. ERCAN, B., KUMMER, K. M., TARQUINIO, K. M. & WEBSTER, T. J. 2011. Decreased Staphylococcus aureus biofilm growth on anodized nanotubular titanium and the effect of electrical stimulation. Acta Biomaterialia, 7, 3003- 3012. FADEEVA, E., TRUONG, V. K., STIESCH, M., CHICHKOV, B. N., CRAWFORD, R. J., WANG, J. & IVANOVA, E. P. 2011. Bacterial Retention on Superhydrophobic Titanium Surfaces Fabricated by Femtosecond Laser Ablation. Langmuir, 27, 3012-3019. FANTNER, G. E., BARBERO, R. J., GRAY, D. S. & BELCHER, A. M. 2010. Kinetics of antimicrobial peptide activity measured on individual bacterial cells using high-speed atomic force microscopy. Nature Nanotechnology, 5, 280-285. FEG-SEM, T.-M. 2013. MIRA3 FEG-SEM Instruction of Use. In: TESCAN, A. S., BRNO, CZECH REPUBLIC (ed.). Australia: AXT Pty. Ltd.
Bibliography 191
FERRARIS, S. & SPRIANO, S. 2016. Antibacterial titanium surfaces for medical implants. Materials Science & Engineering C-Materials for Biological Applications, 61, 965-978. FISHER, L. E., YANG, Y., YUEN, M.-F., ZHANG, W., NOBBS, A. H. & SU, B. 2016. Bactericidal activity of biomimetic diamond nanocone surfaces. Biointerphases, 11, 011014. FLEMMING, H.-C., NEU, T. R. & WOZNIAK, D. 2007. The EPS matrix: the “house of biofilm cells”. Journal of Bacteriology, 189, 7945-7947. FRANSSILA, S. 2010. Deep Reactive Ion Etching. Introduction to Microfabrication, Second Edition, 255-270. FRANZ, S., RAMMELT, S., SCHARNWEBER, D. & SIMON, J. C. 2011. Immune responses to implants – A review of the implications for the design of immunomodulatory biomaterials. Biomaterials, 32, 6692-6709. FU, X., CAI, J., ZHANG, X., LI, W.-D., GE, H. & HU, Y. 2018. Top-down fabrication of shape-controlled, monodisperse nanoparticles for biomedical applications. Advanced Drug Delivery Reviews, 132, 169-187. GANIM, Z., CHUNG, H. S., SMITH, A. W., DEFLORES, L. P., JONES, K. C. & TOKMAKOFF, A. 2008. Amide I two-dimensional infrared spectroscopy of proteins. Account of Chemical Research, 41, 432-441. GANJIAN, M., MODARESIFAR, K., LIGEON, M. R., KUNKELS, L. B., TÜMER, N., ANGELONI, L., HAGEN, C. W., OTTEN, L. G., HAGEDOORN, P. L. & APACHITEI, I. 2019. Nature helps: Toward bioinspired bactericidal nanopatterns. Advance Material Interface, 6, 1900640. GEETHA, M., SINGH, A. K., ASOKAMANI, R. & GOGIA, A. K. 2009. Ti based biomaterials, the ultimate choice for orthopaedic implants–a review. Progress in Materials Science, 54, 397-425. GHOSH, L. D., HASAN, J., JAIN, A., SUNDARESAN, N. R. & CHATTERJEE, K. J. N. 2019. A nanopillar array on black titanium prepared by reactive ion etching augments cardiomyogenic commitment of stem cells. Nanoscale, 11, 20766-20776. GITTENS, R. A., SCHEIDELER, L., RUPP, F., HYZY, S. L., GEIS-GERSTORFER, J., SCHWARTZ, Z. & BOYAN, B. D. 2014. A review on the wettability of dental implant surfaces II: biological and clinical aspects. Acta Biomaterialia, 10, 2907-2918. GLINSNER, T., KREINDL, G. & KAST, M. 2010. Nanoimprint Lithography. Optik & Photonik, 5, 42-45. GMBH, C. Z. M. 2016. ORION NanoFab (ORION NanoFab Three Ion Beams for Total Flexibility in Sub10-nm Fabrication). In: GMBH, C. Z. M. (ed.). ZEISS. GOLDMAN, E. & GREEN, L. H. 2008. Practical handbook of microbiology, CRC Press. GORB, S. N., KESEL, A. & BERGER, J. 2000. Microsculpture of the wing surface in Odonata: evidence for cuticular wax covering. Arthropod Structure & Development, 29, 129-135. GRANDFIELD, K. & ENGQVIST, H. 2011. Focused ion beam in the study of biomaterials and biological matter. Advances in Materials Science and Engineering, 2012, 6. GUO, L. J. 2007. Nanoimprint lithography: Methods and material requirements. Advanced Materials, 19, 495-513. GUSTILO, R. B., MERKOW, R. L. & TEMPLEMAN, D. 1990. The management of open fractures, The Journal of Bone & Joint Surgery, 72, 299-304.
192 Bibliography
HAN, S., JI, S., ABDULLAH, A., KIM, D., LIM, H. & LEE, D. J. A. S. S. 2018. Superhydrophilic nanopillar-structured quartz surfaces for the prevention of biofilm formation in optical devices. Applied Surface Science, 429, 244-252. HARRINGTON, J. D. 2011. Spacebound Bacteria Inspire Earthbound Remedies [Online].Available:https://www.nasa.gov/topics/shuttle station/features/pseu domonas.html [Accessed]. HARRIS, L. G., FOSTER, S. & RICHARDS, R. 2002. An introduction to Staphylococcus aureus, and techniques for identifying and quantifying S. Aureus adhesins in relation to adhesion to biomaterials: review. 4, 39-60. L.G. Harris European Cells and Materials. HARRIS, L. G. & RICHARDS, R. G. 2006. Staphylococci and implant surfaces: a review. Injury, 37, S3-S14. HASAN, J. 2013. Investigation of artificial and natural antibacterial surfaces. Doctor of Philosophy, Swinburne University of Technology, Available: https://researchbank.swinburne.edu.au/file/5823efa9-a4a9-48ca-a451- 1c069d238e64/1/Jafar%20Hasan%20Thesis.pdf. HASAN, J. & CHATTERJEE, K. 2015. Recent advances in engineering topography mediated antibacterial surfaces. Nanoscale, 7, 15568-15575. HASAN, J., CRAWFORD, R. J. & IVANOVA, E. P. 2013. Antibacterial surfaces: The quest for a new generation of biomaterials. Trends in Biotechnology, 31, 295-304. HASAN, J., JAIN, S. & CHATTERJEE, K. 2017. Nanoscale Topography on Black Titanium Imparts Multi-biofunctional Properties for Orthopedic Applications. Scientific Reports, 7, 41118.. HASAN, J., RAJ, S., YADAV, L. & CHATTERJEE, K. 2015. Engineering a nanostructured "super surface" with superhydrophobic and superkilling properties. RSC Advances, 5, 44953-44959. HASAN, J., WEBB, H. K., TRUONG, V. K., POGODIN, S., BAULIN, V. A., WATSON, G. S., WATSON, J. A., CRAWFORD, R. J. & IVANOVA, E. P. 2012. Selective bactericidal activity of nanopatterned superhydrophobic cicada Psaltoda Claripennis wing surfaces. Applied Microbiology and Biotechnology, 97, 9257-9262. HAUGSTAD, G. 2012. Atomic force microscopy: understanding basic modes and advanced applications, John Wiley & Sons. HENDRICKX, N., FRANKE, D., SAMMAK, A., KOUWENHOVEN, M., SABBAGH, D., YEOH, L., LI, R., TAGLIAFERRI, M., VIRGILIO, M. & CAPELLINI, G. 2018. Gate-controlled quantum dots and superconductivity in planar germanium. Nature Communications, 9, 2835. HINES, P. & WOLF, A. 2016. HIM ORION NanoFab working instruction. In: (CARF)-QUT. HLAWACEK, G., VELIGURA, V., VAN GASTEL, R. & POELSEMA, B. 2014. Helium ion microscopy. Journal of Vacuum Science & Technology B, 32, 020801. HOLLANDER, J. M. & JOLLY, W. L. 1970. X-ray photoelectron spectroscopy. Accounts of Chemical Research, 3, 193-200. IVANOVA, E. P., HASAN, J., WEBB, H. K., GERVINSKAS, G., JUODKAZIS, S., TRUONG, V. K., WU, A. H. F., LAMB, R. N., BAULIN, V. A., WATSON, G. S., WATSON, J. A., MAINWARING, D. E. & CRAWFORD, R. J. 2013a. Bactericidal activity of black silicon. Nature Communications, 4.
Bibliography 193
IVANOVA, E. P., HASAN, J., WEBB, H. K., TRUONG, V. K., WATSON, G. S., WATSON, J. A., BAULIN, V. A., POGODIN, S., WANG, J. Y. & TOBIN, M. J. 2012. Natural bactericidal surfaces: mechanical rupture of Pseudomonas Aeruginosa cells by cicada wings. Small, 8, 2489-2494. IVANOVA, E. P., NGUYEN, S. H., WEBB, H. K., HASAN, J., TRUONG, V. K., LAMB, R. N., DUAN, X., TOBIN, M. J., MAHON, P. J. & CRAWFORD, R. J. 2013b. Molecular Organization of the Nanoscale Surface Structures of the Dragonfly Hemianax Papuensis Wing Epicuticle. PLOS ONE, 8, e67893. IZADPANAH, A. & GALLO, R. L. 2005. Antimicrobial peptides. Journal of the American Academy of Dermatology, 52, 381-390. JAGGESSAR, A., MATHEW, A., WANG, H., TESFAMICHAEL, T., YAN, C. & YARLAGADDA, P. 2018. Mechanical, bactericidal and osteogenic behaviours of hydrothermally synthesised TiO2 nanowire arrays. The Journal of the Mechanical Behaviour of Biomedical Materials, 80, 311-319. JAGGESSAR, A., SHAHALI, H., MATHEW, A. & YARLAGADDA, P. K. 2017. Bio-mimicking nano and micro-structured surface fabrication for antibacterial properties in medical implants. Journal of Nanobiotechnology, 15, 64. JAGGESSAR, A., YARLAGADDA, P. K. 2020. Modelling the growth of hydrothermally synthesised bactericidal nanostructures, as a function of processing conditions. Materials Science and Engineering: C, 108, 110434. JEOL 2011. Preparation of Biological Specimens for Electron Microscopy. JEOL. JINDAI, K., NAKADE, K., SAGAWA, T., KOJIMA, H., SHIMIZU, T., SHINGUBARA, S. & ITO, T. J. M. T. P. 2019. Investigation of nanostructure- based bactericidal effect derived from a cicada wing by using QCM-D. Materials Today, 7, 492-496. JOENS, M. S., HUYNH, C., KASUBOSKI, J. M., FERRANTI, D., SIGAL, Y. J., ZEITVOGEL, F., OBST, M., BURKHARDT, C. J., CURRAN, K. P. & CHALASANI, S. H. 2013. Helium Ion Microscopy (HIM) for the imaging of biological samples at sub-nanometer resolution. Scientific reports, 3. JONKER, B. 1990. A compact flange‐mounted electron beam source. Journal of Vacuum Science & Technology A: Vacuum, Surfaces, and Films, 8, 3883- 3886. JUNG, Y. C. & BHUSHAN, B. 2009. Biomimetic structures for fluid drag reduction in laminar and turbulent flows. Journal of Physics: Condensed Matter, 22, 035104. KANG, K., CHO, Y. & YU, K. 2018. Novel nano-materials and nano-fabrication techniques for flexible electronic systems. Micromachines, 9, 263. KARIMIAN, N. & UGO, P. 2019. Recent advances in sensing and biosensing with arrays of nanoelectrodes. Current Opinion in Electrochemistry, 16, 106. KATZ, J. N., WRIGHT, J., WRIGHT, E. A. & LOSINA, E. 2007. Failures of total hip replacement: a population-based perspective. Orthop J Harvard Med Sch, 9, 101-106. KELLEHER, S. M., HABIMANA, O., LAWLER, J., O’ REILLY, B., DANIELS, S., CASEY, E. & COWLEY, A. 2015. Cicada Wing Surface Topography: An Investigation into the Bactericidal Properties of Nanostructural Features. ACS Applied Materials & Interfaces, 8, 14966-14974. KESEL, A. & LIEDERT, R. 2007. Learning from nature: Non-toxic biofouling control by shark skin effect. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 146, S130. KHAJEHPOUR, K. 2014. RE: Electron Beam Lithography, TESCAN Co.
194 Bibliography
KIETZIG, A.-M., HATZIKIRIAKOS, S. G. & ENGLEZOS, P. 2009. Patterned superhydrophobic metallic surfaces. Langmuir, 25, 4821-4827. KIM, S. J., CHANG, J. & SINGH, M. J. B. E. B. A.-B. 2015. Peptidoglycan architecture of Gram-positive bacteria by solid-state NMR. Biochimica et Biophysica Acta (BBA)- Biomembranes, 1848, 350-362. KIM, T. W. 2014. Assessment of hydro/oleophobicity for shark skin replica with riblets. Journal of Nanoscience and Nanotechnology, 14, 7562-7568. KIZHNER, T., BEN-DAVID, D., ROM, E., YAYON, A., LIVNE, E, D. 2011. Effects of FGF2 and FGF9 on osteogenic differentiation of bone marrow-derived progenitors. In Vitro Cellular & Developmental Biology - Animal 47, 294-301. KREUZ, P., ARNOLD, W. & KESEL, A. 2001. Acoustic microscopic analysis of the biological structure of insect wing membranes with emphasis on their waxy surface. Annals of Biomedical Engineering, 29, 1054-1058. KRSKO, P., SUKHISHVILI, S., MANSFIELD, M., CLANCY, R. & LIBERA, M. 2003. Electron-beam surface-patterned poly (ethylene glycol) microhydrogels. Langmuir, 19, 5618-5625. LAN, H. & DING, Y. 2010. Nanoimprint lithography. Lithography. InTech. LI, H.-Q. 1997. The Common AFM Modes. LI, M. T., WANG, Y., GAO, L. L., SUN, Y. H., WANG, J. X., QU, S. X., DUAN, K., WENG, J. & FENG, B. 2016. Porous titanium scaffold surfaces modified with silver loaded gelatin microspheres and their antibacterial behavior. Surface & Coatings Technology, 286, 140-147. LI, X. 2016. Bactericidal mechanism of nanopatterned surfaces. Physical Chemistry Chemical Physics, 18, 1311-1316. LI, X., CHEUNG, G., WATSON, G., WATSON, J., LIN, S., SCHWARZKOPF, L. & GREEN, D. 2016b. The nanotipped hairs of gecko skin and biotemplated replicas impair and/or kill pathogenic bacteria with high efficiency. Nanoscale, 8, 18860-18869. LIANG, J. H., SONG, R., HUANG, Q. L., YANG, Y., LIN, L. X., ZHANG, Y. M., JIANG, P. L., DUAN, H. P., DONG, X. & LIN, C. J. 2015. Electrochemical construction of a bio-inspired micro/nano-textured structure with cell-sized microhole arrays on biomedical titanium to enhance bioactivity. Electrochimica Acta, 174, 1149-1159. LIAO, C. H. & SHOLLENBERGER, L. J. L. I. A. M. 2003. Survivability and long‐ term preservation of bacteria in water and in phosphate‐buffered saline. Letters in applied microbiology37, 45-50. LIN-VIEN, D., COLTHUP, N. B., FATELEY, W. G. & GRASSELLI, J. G. 1991. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules, Elsevier. LINKLATER, D. P., JUODKAZIS, S., RUBANOV, S., IVANOVA, E. P. J. A. A. M. & INTERFACES 2017. Comment on “Bactericidal effects of natural nanotopography of dragonfly wing on Escherichia coli”. ACS Applied Materials & Interfaces 9, 29387-29393. LIU, X. Q., YU, L., YANG, S. N., CHEN, Q. D., WANG, L., JUODKAZIS, S. & SUN, H. B. 2019. Optical Nanofabrication of Concave Microlens Arrays. Laser & Photonics Reviews, 13, 1800272. LIU, Y. & LI, G. 2012. A new method for producing “Lotus Effect” on a biomimetic shark skin. Journal of Colloid and Interface Science, 388, 235-242. LOCKEY, K. H. 1960. The thickness of some insect epicuticular wax layers. Journal of Experimental Biology, 37, 316-329.
Bibliography 195
MA, J. W., SUN, Y. K., GLEICHAUF, K., LOU, J. & LI, Q. L. 2011. Nanostructure on Taro Leaves Resists Fouling by Colloids and Bacteria under Submerged Conditions. Langmuir, 27, 10035-10040. MA, M. & HILL, R. M. 2006. Superhydrophobic surfaces. Current Opinion in Colloid & Interface Science, 11, 193-202. MAAS, D., VAN VELDHOVEN, E., CHEN, P., SIDORKIN, V., SALEMINK, H., VAN DER DRIFT, E. & ALKEMADE, P. Nanofabrication with a helium ion microscope. SPIE Advanced Lithography, 2010. International Society for Optics and Photonics, 763814-763814-10. MAINWARING, D. E., NGUYEN, S. H., WEBB, H., JAKUBOV, T., TOBIN, M., LAMB, R. N., WU, A. H. F., MARCHANT, R., CRAWFORD, R. J. & IVANOVA, E. P. 2016. The nature of inherent bactericidal activity: insights from the nanotopology of three species of dragonfly. Nanoscale, 8, 6527-6534. MELVILLE, D. O. 2006. Planar Lensing Lithography: Enhancing the Optical Near Field. MICROCHEM 2011. PMMA Data Sheet In: MICROCHEM (ed.). MicroChem Corp. Copyright 2001. MISTRY, B. 2009. A Handbook of spectroscopic data Chemistry (UV, IR, PMR, CNMR and Mass Spectroscopy), Science College, Va/sad (Gujarat). Oxford Book Company. M. KURTZ, S., LAU MS, E., WATSON, H., SCHMIER MA, K.J., PARVIZI MD, J. 2012. Economic Burden of Periprosthetic Joint Infection in the United States, The Journal of Arthroplasty, 27, Issue 8, 61-65. MODARESIFAR, K., AZIZIAN, S., GANJIAN, M., FRATILA-APACHITEI, L. E. & ZADPOOR, A. A. J. A. B. 2019. Bactericidal effects of nanopatterns: A systematic review. Acta Biomaterialia, 83, 29-36. MOOJEN, D. J. F., VOGELY, H. C., FLEER, A., NIKKELS, P. G., HIGHAM, P. A., VERBOUT, A. J., CASTELEIN, R. M. & DHERT, W. J. 2009. Prophylaxis of infection and effects on osseointegration using a tobramycin‐periapatite coating on titanium implants—An experimental study in the rabbit. Journal of Orthopaedic Research, 27, 710-716. MOTEMANI, Y., GREULICH, C., KHARE, C., LOPIAN, M., BUENCONSEJO, P. J. S., SCHILDHAUER, T. A., LUDWIG, A. & KÖLLER, M. 2014. Adherence of human mesenchymal stem cells on Ti and TiO2 nano-columnar surfaces fabricated by glancing angle sputter deposition. Applied Surface Science, 292, 626-631. MOVASAGHI, Z., REHMAN, S. & UR REHMAN, D. I. 2008a. Fourier transform infrared (FTIR) spectroscopy of biological tissues. Applied Spectroscopy Reviews, 43, 134-179. MUDIYANSELAGE, I. & BANDARA, C. C. D. 2017. Characterisation of the bactericidal efficacy of natural nano-topography using dragonfly wing as a model. Queensland University of Technology. NCEZID. 2011. Staphylococcus aureus in Healthcare Settings [Online]. Centers for Disease Control and Prevention. Available: https://www.cdc.gov/hai/organisms/staph.html [Accessed]. NGUYEN, L. T., HANEY, E. F. & VOGEL, H. J. 2011. The expanding scope of antimicrobial peptide structures and their modes of action. Trends in Biotechnology, 29, 464-472. NGUYEN, S. H. T., WEBB, H. K., HASAN, J., TOBIN, M. J., CRAWFORD, R. J. & IVANOVA, E. P. 2013. Dual role of outer epicuticular lipids in determining
196 Bibliography
the wettability of dragonfly wings. Colloids and Surfaces B: Biointerfaces, 106, 126-134. ODEKERKEN, J. C., WELTING, T. J., ARTS, J. J., WALENKAMP, G. & EMANS, P. J. 2013. Modern orthopaedic implant coatings—their pro’s, con’s and evaluation methods. Modern Surface Engineering Treatments. New York: InTech, 45-73. OH, J., DANA, C. E., HONG, S., ROMÁN, J. K., JO, K. D., HONG, J. W., NGUYEN, J., CROPEK, D. M., ALLEYNE, M., MILJKOVIC, N. J. A. A. M. & INTERFACES 2017. Exploring the role of habitat on the wettability of cicada wings. ACS Applied Materials & Interfaces. 9, 27173-27184. ONAIZI, S. A. & LEONG, S. S. J. 2011. Tethering antimicrobial peptides: Current status and potential challenges. Biotechnology Advances, 29, 67-74. OYAMA, T. G., HINATA, T., NAGASAWA, N., OSHIMA, A., WASHIO, M., TAGAWA, S. & TAGUCHI, M. 2013. Micro/nanofabrication of poly (L-lactic acid) using focused ion beam direct etching. Applied Physics Letters, 103, 163105. PATANKAR, N. A. 2004. Mimicking the lotus effect: influence of double roughness structures and slender pillars. Langmuir, 20, 8209-8213. POGODIN, S., HASAN, J., BAULIN, V. A., WEBB, H. K., TRUONG, V. K., NGUYEN, T. H. P., BOSHKOVIKJ, V., FLUKE, C. J., WATSON, G. S., WATSON, J. A., CRAWFORD, R. J. & IVANOVA, E. P. 2013a. Biophysical Model of Bacterial Cell Interactions with Nanopatterned Cicada Wing Surfaces. Biophysical Journal, 104, 835-840. POON, C. Y. & BHUSHAN, B. 1995. Comparison of surface roughness measurements by stylus profiler, AFM and non-contact optical profiler. Wear, 190, 76-88. POSTGATE, J. 1969. Chapter XVIII Viable counts and Viability. Methods in Microbiology, 1, 611-628. PU, X., LI, G. & HUANG, H. 2016. Preparation, anti-biofouling and drag-reduction properties of a biomimetic shark skin surface. Biology Open, bio. 016899. PU, X., LI, G. & LIU, Y. 2016. Progress and perspective of studies on biomimetic shark skin drag reduction. ChemBioEng Reviews, 3, 26-40. PUCKETT, S. D., TAYLOR, E., RAIMONDO, T. & WEBSTER, T. J. 2010. The relationship between the nanostructure of titanium surfaces and bacterial attachment. Biomaterials, 31, 706-713. RAJENDRAN, S., KARUPPANAN, K. K. & PEZHINKATTIL, R. 2012. Analysis on surface nanostructures present in hindwing of dragon fly (Sympetrum vulgatum) using atomic force microscopy. Micron, 43, 1299-1303. RIBEIRO, M., MONTEIRO, F. J. & FERRAZ, M. P. 2012. Infection of orthopedic implants with emphasis on bacterial adhesion process and techniques used in studying bacterial-material interactions. Biomatter, 2, 176-194. RODRÍGUEZ-HERNÁNDEZ, J. & CORTAJARENA, A. L. 2015. Design of Polymeric Platforms for Selective Biorecognition, Springer. ROSENZWEIG, R., PERINBAM, K., LY, V. K., AHRAR, S., SIRYAPORN, A., YEE, A. Nanopillared Surfaces Disrupt Pseudomonas aeruginosa Mechanoresponsive Upstream Motility. ACS Applied Materials & Interfaces. 11, 10532-10539. SAJOMSANG, W., GONIL, P. 2010. Preparation and characterization of α-chitin from cicada sloughs. Materials Science and Engineering: C. 30, 357-363.
Bibliography 197
SAXENA, A., TRIPATHI, R. & SINGH, R. 2010. Biological synthesis of silver nanoparticles by using onion (Allium cepa) extract and their antibacterial activity. Dig J Nanomater Bios, 5, 427-432. SCHOLDER, O., JEFIMOVS, K., SHORUBALKO, I., HAFNER, C., SENNHAUSER, U. & BONA, G.-L. 2013. Helium focused ion beam fabricated plasmonic antennas with sub-5 nm gaps. Nanotechnology, 24, 395301. SENGSTOCK, C., LOPIAN, M., MOTEMANI, Y., BORGMANN, A., KHARE, C., BUENCONSEJO, P. J. S., SCHILDHAUER, T. A., LUDWIG, A. & KOLLER, M. 2014. Structure-related antibacterial activity of a titanium nanostructured surface fabricated by glancing angle sputter deposition. Nanotechnology, 25. SHAHALI, H., HASAN, J., MATHEWS, A., WANG, H., YAN, C., TESFAMICHAEL, T. & YARLAGADDA, P. K. 2019. Multi-biofunctional properties of three species of cicada wings and biomimetic fabrication of nanopatterned titanium pillars. Journal of Materials Chemistry B, 7, 1300- 1310. SHAHALI, H., HASAN, J., WANG, H., TESFAMICHAEL, T., YAN, C. & YARLAGADDA, P. K. 2019c. Evaluation of particle beam lithography for fabrication of metallic nano-structures. Procedia Manufacturing, 30, 261-267. SHAHALI, H., JAGGESSAR, A. & YARLAGADDA, P. K. 2017. Recent advances in manufacturing and surface modification of Titanium orthopaedic applications. Procedia Engineering, 174, 1067-1076. SILHAVY, T. J., KAHNE, D. & WALKER, S. J. C. S. H. P. I. B. 2010. The bacterial cell envelope. Cold Spring Harbor Perspective in Biology. 2, a000414. SINGH, B. R., DEOLIVEIRA, D. B., FU, F.-N. & FULLER, M. P. Fourier transform infrared analysis of amide III bands of proteins for the secondary structure estimation. Biomolecular Spectroscopy III, 1993. International Society for Optics and Photonics, 47-56. SINHA, R. K. 2002. Hip replacement: current trends and controversies, CRC Press. SUGIOKA, K. & CHENG, Y. 2014. Femtosecond laser three-dimensional micro-and nanofabrication. Applied Physics Reviews, 1, 041303. SUN, M., LIANG, A., WATSON, G. S., WATSON, J. A., ZHENG, Y., JU, J. & JIANG, L. 2012. Influence of cuticle nanostructuring on the wetting behaviour/states on cicada wings. PLoS One, 7, e35056. SUN, M., WATSON, G. S., ZHENG, Y., WATSON, J. A. & LIANG, A. 2009. Wetting properties on nanostructured surfaces of cicada wings. Journal of Experimental Biology, 212, 3148-3155. SUUTALA, A. 2013. Focused ion beam technique in nanofabrication. The Meeting of National Graduate School of Nanoscience. https:// www. jyu. fi/ science/ muut_ yksikot/ nsc/ en/ studies/ ngs/ course/ meeting09/ suutala_ esitys. Accessed, 19. TAN, Q., LU, F., XUE, C., ZHANG, W., LIN, L., XIONG, J. J. S. & PHYSICAL, A. A. 2019. Nano-fabrication methods and novel applications of black silicon. Sensors and Actuators A: Physical, 295, 560-573. TESAROVA, H. 2015. RE: Electron Beam Litographym (EBL) Description-Current Status-Futire Prospects. THEOPHILOU, G., LIMA, K. M., BRIGGS, M., MARTIN-HIRSCH, P. L., STRINGFELLOW, H. F. & MARTIN, F. L. 2015. A biospectroscopic analysis
198 Bibliography
of human prostate tissue obtained from different time periods points to a trans- generational alteration in spectral phenotype. Scientific Reports, 5, 13465. TOBIN, M. J., PUSKAR, L., HASAN, J., WEBB, H. K., HIRSCHMUGL, C. J., NASSE, M. J., GERVINSKAS, G., JUODKAZIS, S., WATSON, G. S. & WATSON, J. A. 2013. High-spatial-resolution mapping of superhydrophobic cicada wing surface chemistry using infrared microspectroscopy and infrared imaging at two synchrotron beamlines. Journal of Synchrotron Radiation, 20, 482-489. TOMPKINS, H. G. & HILFIKER, J. 2016. Spectroscopic Ellipsometry. Practical Application to Thin Film Characterization. UHM, S. H., SONG, D. H., KWON, J. S., LEE, S. B., HAN, J. G., KIM, K. M. & KIM, K. N. 2013. E-beam fabrication of antibacterial silver nanoparticles on diameter-controlled TiO2 nanotubes for bio-implants. Surface & Coatings Technology, 228, S360-S366. UNOSSON, E. 2015. Antibacterial Strategies for Titanium Biomaterials, ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2015. VAN SCHAIK, E. J., GILTNER, C. L., AUDETTE, G. F., KEIZER, D. W., BAUTISTA, D. L., SLUPSKY, C. M., SYKES, B. D. & IRVIN, R. 2005. DNA binding: a novel function of Pseudomonas Aeruginosa type IV pili. Journal of Bacteriology.187, 1455-1464. VEERACHAMY, S., YARLAGADDA, T., MANIVASAGAM, G. & YARLAGADDA, P. K. 2014. Bacterial adherence and biofilm formation on medical implants: a review. Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine, 228, 1083-1099. VOLKERT, C. A. & MINOR, A. M. 2007. Focused ion beam microscopy and micromachining. MRS Bulletin, 32, 389-399. WAGNER, C. D. & MUILENBERG, G. 1979. Handbook of X-ray Photoelectron Spectroscopy, Perkin-Elmer. WATSON, G. S., GREEN, D. W., CRIBB, B. W., BROWN, C. L., MERITT, C. R., TOBIN, M. J., VONGSVIVUT, J., SUN, M., LIANG, A.-P. & WATSON, J. A. 2017. Insect Analogue to the Lotus Leaf: A Planthopper Wing Membrane Incorporating a Low-Adhesion, Nonwetting, Superhydrophobic, Bactericidal, and Biocompatible Surface. ACS Applied Materials & Interfaces, 9, 24381- 24392. WATSON, G. S., GREEN, D. W., SCHWARZKOPF, L., LI, X., CRIBB, B. W., MYHRA, S. & WATSON, J. A. 2015. A gecko skin micro/nano structure–A low adhesion, superhydrophobic, anti-wetting, self-cleaning, biocompatible, antibacterial surface. Acta Biomaterialia, 21, 109-122. WATTS, J. F. 1994. X-ray photoelectron spectroscopy. Vacuum, 45, 653-671. WEI, D. Q., FENG, W., DU, Q., ZHOU, R., LI, B. Q., WANG, Y. M., ZHOU, Y. & JIA, D. C. 2015. Titania nanotube/nano-brushite composited bioactive coating with micro/nanotopography on titanium formed by anodic oxidation and hydrothermal treatment, Ceramics International, 41, 13115-13125. WILEY, B. J., QIN, D. & XIA, Y. 2010. Nanofabrication at high throughput and low cost. ACS Nano, 4, 3554-3559. WILLIAMS, D. F. 2008. On the mechanisms of biocompatibility. Biomaterials, 29, 2941-2953. WU, S. E., HUANG, Y. W., HSUEH, T. H. & LIU, C. P. 2008. Fabrication of nanopillars comprised of InGaN/GaN multiple quantum wells by focused ion beam milling. Japanese Journal of Applied Physics, 47, 4906-4908.
Bibliography 199
YADAV, P. K., LEMOINE, P., DALE, G., HAMILTON, J. W., DUNLOP, P. S., BYRNE, J. A., MAILLEY, P. & BOXALL, C. 2015. Hierarchical titania nanostructures prepared with focused ion beam-assisted anodisation of titanium in an aqueous electrolyte. Applied Physics A, 119, 107-113. YI, H.-Y., CHOWDHURY, M., HUANG, Y.-D. & YU, X.-Q. 2014. Insect Antimicrobial Peptides and Their Applications. Applied Microbiology and Biotechnology, 98, 5807-5822. ZHANG, D., LI, Y., HAN, X., LI, X. & CHEN, H. 2011. High-precision bio- replication of synthetic drag reduction shark skin. Chinese Science Bulletin, 56, 938-944. ZHANG, J., SHENG, X. & JIANG, L. 2008. The dewetting properties of lotus leaves. Langmuir, 25, 1371-1376. ZHAO, D.-Y., HUANG, Z.-P., WANG, M.-J., WANG, T. & JIN, Y. 2012. Vacuum casting replication of micro-riblets on shark skin for drag-reducing applications. Journal of Materials Processing Technology, 212, 198-202. ZHAO, D., HAN, A. & QIU, M. 2019. Ice lithography for 3D nanofabrication. Science Bulletin. ZOBELL, C. E. 1943. The effect of solid surfaces upon bacterial activity. Journal of Bacteriology, 46, 39.
200 Bibliography
Appendices
Appendix A
Colony-forming unit (CFU/mL) result for P. aeruginosa and S. aureus
Colony-forming unit (CFU/mL) of P. aeruginosa on CM (control media), Glass (control surface), PC, AC and PE (CFU presented in the order of 106 and Error is in the order of 105).
CM (× CM err Gl PC AC PE Interval 106) (× 105) GL (err) PC (err) AC (err) PE (err) 0 1.98 3.35 1.97 3.35 1.98 3.35 1.98 3.35 1.98 3.35 2 1.99 3.19 1.97 1.52 1.98 1.36 1.98 1.47 1.98 1.21 4 2.40 1.86 1.98 1.34 1.23 8.37 1.18 9.71 1.15 1.00 18 2.00 1.92 1.99 1.67 0.91 1.08 0.82 0.84 0.73 1.02
Colony-forming unit (CFU/mL) of S. aureus on CM (control media), Glass (control surface), PC, AC and PE (CFU presented in the order of 106 and Error is in the order of 105).
CM (× CM err Gl PC AC PE Interval 106) (× 105) GL (err) PC (err) AC (err) PE (err) 0 2.25 1.17 2.52 1.17 2.52 1.17 2.52 1.17 2.52 1.17 2 2.54 1.21 2.49 1.05 2.31 9.27 2.22 7.74 2.19 6.57 4 2.54 1.46 2.41 1 2.32 8.24 2.12 6.94 1.85 6.57 18 2.19 0.87 2.09 7.4 1.53 0.5 1.43 0.28 1.48 3.85
Appendices 201
Appendix B
Cellular metabolic activity of three cicada species wing in 4 and 24 hrs.
Cellular metabolic activity of three cicada species wing in 4 and 24 hrs.
Interval CM CM err Gl GL err PC PC err AC AC Err PE PE err 4 hrs 80.57 13.55 37.62 10.74 93.42 13.75 98.73 10.25 101.28 14.27 24 hrs 65.54 10.48 33.76 13.64 78.48 11.18 81.16 13.74 85.71 15.81
202 Appendices
Appendix C
Cellular metabolic activity of titanium nanopillars and titanium control surface
Titanium nanopillar Titanium Titanium nanopillar fabricated by EBL Ti control- control Error Interval fabricated by EBL Error Control surface 1 Day 93.35 7.81 85.90 3.540849 3 Days 107.42 19.3 108.16 9.087116
Appendices 203