Immobilisation strategies for the tethering of polymers and antimicrobial peptides to design multifunctional surface coatings
Andrew Boden BSc(Hons)
Submitted to the Faculty of Science, Engineering and Technology Swinburne University of Technology In partial fulfilment of the degree of Doctorate of Philosophy
2020
i
ABSTRACT
Unwanted accumulation of proteins and bacterial cells on a surface is a major concern in a number of areas including but certainly not limited to, healthcare, food preparation, and shipping industries. This type of ‘biofouling’ can significantly perturb the function of a particular device or result in material degradation. Particularly within the healthcare industry, development of non- fouling surfaces (i.e. surfaces that can resist protein and bacterial adhesion) is of great importance due to the high incidence of infection associated with the use of indwelling medical devices.
Fabrication of such surfaces have generally been achieved through lithographic or anti-adhesive techniques in order to manipulate surface topography and chemistry, however there has been little or no use of combining both of these strategies for the development of a multifunctional surface coating. As such, the research presented here examines various immobilisation strategies for the tethering of anti-adhesive polymers and antimicrobial peptides (AMPs) to different types of surfaces. This included investigating surface activation methods to generate surface-bound functional groups, and also the use of microparticles and binary colloidal crystal (BCC) layers as a platform for the covalent immobilisation of selected polymers and AMPs.
After demonstrating that puroindoline-based synthetic AMPs were active in solution against clinically relevant bacteria, subsequent experimental work provided a proof of concept that these types of AMPs can be tethered to binary colloidal crystal layers by zero-length immobilisation using EDC/NHS coupling chemistry, and still exhibit antibacterial activity with a decrease of
>70% in the viability of E.coli cells when compared to control samples (Chapter 4). Confirmation of AMP immobilisation was achieved through a range of physio-chemical characterisation techniques including zeta potential, X-ray photoelectron spectroscopy (XPS) and matrix-assisted
ii laser desorption ionisation time-of-flight mass spectrometry (MALDI-ToF MS). Interestingly,
MALDI analysis showed the importance of proper characterisation of small-molecule grafting, as traces of physically adsorbed AMP was detected for covalently immobilised samples. Considering the success of zero-length immobilisation of PuroA, subsequent experimental work was focussed on the incorporation of flexible polymer linkers to not only add an additional barrier from proteins and bacteria, but also increase AMP mobility and penetration depth. In-situ investigations using surface plasmon resonance (SPR) demonstrated that poly(ethylene glycol) (PEG) can significantly reduce the adsorption of protein compared to unmodified surfaces with graft density and protein resistant properties being able to be controlled by variations in ionic strength and polymer molecular weight (Chapter 5). It was also found that the choice of grafting method is crucial to achieve dense polymer layers, as variations in temperature, ionic strength, and pH may have positive or negative effects depending of the type of grafting method chosen for both flat and spherical surfaces (Chapter 6).
The choice of grafting method is also dependant on the availability of appropriate functional groups present, thus it was necessary to optimise various surface activation methods to generate sufficient reactive functional groups at a surface. A particular focus within this project was the generation of thiol functional groups through silanisation of inorganic surfaces with (3- mercaptopropyl)trimethoxysilane (MPTS), as thiol-based immobilisation strategies are quite versatile and show minimal interference from other functional groups (i.e. amines and carboxylic acids) (Chapter 7). Given a 2hr pre-hydrolysis period at pH 4 it was shown that relatively thick
MPTS layers can be obtained that have a high proportion of thiol functional groups providing there are minimal post-deposition treatments such as rinsing and drying. Thorough characterisation of
MPTS films was achieved using ellipsometry, water contact angle (WCA) analysis, XPS, and
Fourier transform infrared (FTIR) spectroscopy, which helped provide an in-depth description of iii
MPTS deposition methods to generate optimised thiol-containing silane layers. Such optimisation was necessary for future experiments where two immobilisation methods utilising surface-bound thiols were investigated to tether heterobifunctional PEG and AMPs to MPTS-functionalised silica colloids (Chapter 8). Both thiol-ene ‘photo-click’ and thiol-maleimide coupling methods were determined to be quite effective in PEG and AMP tethering with characterisation through zeta potential and XPS revealing that PEG and AMPs were both bound to the surface of the particles.
The AMPs; P1 and W8 were also determined to retain activity after immobilisation, with a reduction of >75% in the viability of P.aeriginosa cells compared to control samples.
This research project presents an investigation in to the various immobilisation strategies for the tethering of polymers and AMPs to design functional surface coatings for potential biomedical applications. Key findings within the project have helped contribute to the field of biomaterials demonstrating new and novel ways to generate patterned surfaces based on PEG and AMP immobilisation to colloidal crystal layers. Additionally, this research has provided valuable information regarding the success of certain grafting methods, showing that the conditions and chemistry chosen is of upmost importance to achieve successful tethering.
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ACKNOWLEDGEMENTS
The researcher wishes to extend their deepest gratitude to all people who have given their assistance and valuable time throughout the course of this research project and contributed to the completion of this Thesis.
Firstly I would like to acknowledge the Wurundjeri people who are the Traditional Custodians of the Land and pay my respects to Elders past and present. I would also like to thank the Australian
Government and Swinburne University of Technology (SUT) for funding through an Australian
Postgraduate Award (APA).
To my supervisory team; Prof. Peter Kingshott and Prof. Mrinal Bhave, I am extremely grateful and appreciative for all your guidance and valuable insights throughout the project, and also for your support in times of distress.
I would also like to acknowledge the co-operation and help provided by SUT staff members and laboratory staff for technical support and help in instrument training.
Finally, I am extremely grateful to my family for their unwavering love and patience. I could not have done this without your encouragement and support.
I thank you all, tremendously
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DECLARATION
I declare that the information within this thesis is my own work and does not contain any material that has previously been submitted elsewhere for educational purposes or publication without acknowledgement through proper referencing.
Name: Andrew Boden
Signed: Date: 10-8-20
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TABLE OF CONTENTS
ABSTRACT ...... ii ACKNOWLEDGEMENTS...... v DECLARATION ...... vi TABLE OF CONTENTS ...... vii List of Figures ...... xii List of Tables ...... xvii 1 INTRODUCTION ...... 1 1.1 Scope & aims of the PhD project ...... 1 1.2 Thesis structure ...... 4 2 LITERATURE REVIEW ...... 9 2.1 General introduction ...... 9 2.2 Interactions at the biointerface ...... 12 2.2.1 Biomaterial associated infections ...... 13 2.2.2 Biofouling and biofilm formation ...... 14 2.3 Non-fouling polymers ...... 17 2.3.1 Self-assembled monolayers ...... 17 2.3.2 Ethylene glycol based polymer brushes ...... 19 2.3.3 Other non-fouling polymers ...... 21 2.3.4 Immobilisation methods ...... 22 2.4 Antimicrobial peptides as therapeutic agents ...... 26 2.4.1 Introduction to antimicrobial peptides...... 26 2.4.2 Mode of action ...... 27 2.4.3 Immobilisation strategies ...... 28 2.5 Nano-and microstructured surfaces ...... 34 2.5.1 Bioinspired structuring ...... 34 2.5.2 Binary colloidal crystals ...... 36 2.5.2.1 Introduction to binary colloidal crystals ...... 36 2.5.2.2 Colloidal materials ...... 38 2.5.2.3 Composition of binary colloidal crystals ...... 41
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2.5.2.5 Colloidal shapes ...... 44 2.5.2.6 Mechanisms of binary colloidal crystal growth ...... 48 2.5.2.6.1 Electrostatic forces ...... 48 2.5.2.6.2 Van der Waals forces ...... 52 2.5.2.6.3 Capillary forces...... 52 2.5.2.6.4 Depletion forces ...... 53 2.5.2.6.5 Gravitational forces ...... 54 2.5.2.6.6 External forces ...... 55 2.5.2.7 Methods for binary colloidal self-assembly ...... 57 2.5.2.7.1 Sequential deposition ...... 57 2.5.2.7.2 Co-deposition ...... 63 2.5.2.8 Applications of binary colloidal crystal ...... 67 2.6 Conclusions ...... 68 2.7 References ...... 69 3 MATERIALS & METHODS ...... 101 3.1 Materials ...... 101 3.1.1 Surface materials and particles ...... 101 3.1.2 Chemicals and AMPs ...... 101 3.1.3 Bacterial strains and growth conditions...... 102 3.2 Methods ...... 103 3.2.1 Surface cleaning methods ...... 103 3.2.2 Modification of planar surfaces ...... 103 3.2.2.1 Solution-based deposition of APTES films ...... 103 3.2.2.2 Solution-based deposition of MPTS films ...... 104 3.2.2.3 Pre-hydrolysis deposition of MPTS films...... 105 3.2.2.4 In-situ grafting of PEG by reductive amination and BSA adsorption ...... 105 3.2.2.5 In-situ grafting of PEG by carbodiimide chemistry and BSA adsorption .... 107 3.2.3 Modification of colloidal particles ...... 108 3.2.3.1 Regeneration of reactive oxygen species on silica microspheres ...... 108 3.2.3.2 Silanisation of silica microspheres with 3-mercaptopropyl(trimethoxysilane) 108 3.2.3.3 Quantification of reactive thiol functional groups...... 108 3.2.3.4 Immobilisation of PuroA to PSC2 particles ...... 109
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3.2.3.5 Grafting of PEG to Si-COOH colloids via carbodiimide chemistry and BSA adsorption ...... 110 3.2.3.6 Sequential immobilisation of PEG and AMPs using thiolene ‘photoclick’ chemistry ...... 111 3.2.3.7 ‘One-Pot’ immobilisation of PEG and AMPs using thiol-maleimide chemistry 112 3.2.4 Surface fabrication through colloidal self-assembly ...... 113 3.2.4.1 Preparation of PuroA-modified BCC monolayers ...... 113 3.2.5 Bacterial cell studies ...... 115 3.2.5.1 Activity of free AMPs in solution ...... 115 3.2.5.2 Plate count assay for viable growth determination...... 116 3.2.5.3 Antibacterial activity of immobilised PuroA AMPs ...... 117 3.2.5.4 Antibacterial activity of PEG and AMP-functionalised colloids prepared using thiol-ene ‘photo-click’ and thiol-maleimide chemisty ...... 117 3.2.5.5 Scanning electron microscopy imaging of biotic surfaces ...... 118 3.2.6 Characterisation techniques ...... 119 3.2.6.1 Surface plasmon resonance (SPR) analysis ...... 119 3.2.6.2 X-ray photoelectron spectroscopy (XPS) analysis ...... 120 3.2.6.3 Attenuated total reflectance-Fourier transform infra-red (ATR-FTIR) spectroscopy ...... 121 3.2.6.4 Zeta potential measurements ...... 121 3.2.6.5 Static water contact angle (WCA) analysis...... 121 3.2.6.6 Ellipsometry analysis ...... 122 3.2.6.7 Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-ToF MS) analysis ...... 122 3.2.6.8 Scanning electron microscopy (SEM) analysis ...... 123 3.3 References ...... 124 4 Binary colloidal crystal layers as platforms for the surface patterning of Puroindoline- based AMPs and optimisation of AMP selection ...... 127 4.1 Summary ...... 127 4.2 Introduction ...... 128 4.3 Results and discussion ...... 131 4.3.1 Particle modification and characterisation ...... 131 4.3.2 Binary colloidal crystal formation and characterisation ...... 139 4.3.3 Antibacterial investigations ...... 140 ix
4.3.4 AMP selection and activity ...... 146 4.4 Conclusions ...... 147 4.5 References ...... 148 5 In-situ investigation of grafting conditions and non-specific protein adsorption to PEG layers with surface plasmon resonance ...... 158 5.1 Summary ...... 158 5.2 Introduction ...... 159 5.3 Results and Discussion...... 164 5.3.1 In-situ surface modification and protein adsorption using reductive amination 164 5.3.2 Optimisation of APTES deposition ...... 169 5.3.3 In-situ surface modification and protein adsorption using EDC/NHS chemistry 175 5.4 Conclusions ...... 178 5.5 References ...... 179 6 Investigation and characterisation of PEG immobilisation to silica microspheres and BSA adsorption ...... 185 6.1 Summary ...... 185 6.2 Introduction ...... 186 6.3 Results and discussion ...... 188 6.3.1 Determination of PEG concentration ...... 188 6.3.2 Particle modification and characterisation ...... 189 6.3.3 Protein adsorption analysis ...... 197 6.4 Conclusions ...... 199 6.5 References ...... 200 7 Analysis of MPTS deposition methods for the production of thiol-containing films ..... 205 7.1 Summary ...... 205 7.2 Introduction ...... 205 7.3 Results and discussion ...... 207 7.4 Conclusions ...... 223 7.5 References ...... 224 8 Characterisation and antibacterial assessment of ‘one-pot ‘and sequential immobilisation of PEG and AMP-functionalised colloids ...... 228 8.1 Summary ...... 228 8.2 Introduction ...... 229
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8.3 Results and Discussion...... 231 8.3.1 Particle Characterisation ...... 231 8.3.2 Assessment of antimicrobial activity ...... 238 8.4 Conclusions ...... 245 8.5 References ...... 246 9 Conclusions and Future Perspectives ...... 250 9.1 Main findings of the research ...... 250 9.2 Limitations and future perspectives ...... 254 9.3 Concluding remarks ...... 255 10 Conferences and Publications ...... 256 10.1 List of Conferences ...... 256 10.2 List of Publications ...... 256 Appendix I ...... 258 Appendix II ...... 261 Appendix III ...... 262
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List of Figures
Figure 1. Illustrative summary of the grafting methods utilised for the tethering of PEG and
AMPs to various surface types...... 4
Figure 2. Schematic illustration of biofilm formation summarised into five different stages...... 16
Figure 3. Illustrative representation of hydration and chain conformation of various polymer types ...... 18
Figure 4. Illustrative representation of a layer-by-layer (LbL) assembly containing AMPs sandwiched between polyionic polymers...... 29
Figure 5. Representation of various colloidal crystal structures ...... 37
Figure 6. Summary of different types of particle shapes used in the self-assembly of colloidal crystals ...... 44
Figure 7. Production of BCCs using various shape anisotropic particles based on convective assembly methods...... 46
Figure 8. Representative SEM images and Fourier transformations of dried monocrystals assembled from non-spherical colloids ...... 47
Figure 9. Summary of the various interactions and forces encountered during colloidal self- assembly...... 50
Figure 10. Visual representation of the electrostatically driven self-assembly of AuNPs with
TMV rods ...... 52
Figure 11. Schematic representation of crystal growth using an evaporation induced confined area assembly method ...... 61
Figure 12. Schematic representation of the production of BCCs using a template-assisted electric field induced assembly (TAEFIA) method ...... 63
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Figure 13. Schematic representation of the crystallisation methods used to fabricate BCCs based on co-deposition techniques utilising ...... 65
Figure 14. Schematic illustration demonstrating the principles of surface plasmon resonance
(SPR) to determine surface densities of adsorbed molecules...... 106
Figure 15. Illustrative representation of the two-step immobilisation used to tether PEG and
AMPs to silica particles with thiolene ‘photoclick’ chemistry...... 111
Figure 16. Reaction summary of the modification of silica colloids by thiol-maleimide chemistry used to tether PEG and AMPs...... 112
Figure 17. Overview of PuroA coupling and subsequent BCC formation using PSC2 and
PMMA011 colloids...... 115
Figure 18. Schematic illustration of the preparation of PuroA-modified BCC layers and representation of the proposed mechanisms of bacterial attachment and antimicrobial activity.
...... 128
Figure 19. Zeta potential data for unmodified and PuroA-modified PSC2 particles...... 132
Figure 20. High-resolution C1s XPS spectra of PuroA-modified colloidal particles...... 137
Figure 21. Surface MALDI-ToF MS spectra of PuroA-modified PSC2 particles over an m/z range of 1750-2250Da...... 138
Figure 22. SEM images of PSC2-PMMA011 BCC layers ...... 140
Figure 23. Representative fluorescent microscopy images after 24 h incubation showing live
(green) and membrane compromised (red) E. coli cells adhered to PuroA-modified PSC2-
PMMA011 BCCs...... 142
Figure 24. Viability of adherent E.coli cells attached to PuroA-modified PSC2PMMA011 BCC layers...... 143
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Figure 25. Illustrative representation of the proposed mechanism of bacterial attachment and antimicrobial activity to PuroA-modified BCC layers...... 144
Figure 26. Representative SEM images of adherent E.coli cells to PuroA-modified
PSC2PMMA011 BCC layers ...... 145
Figure 27. Plate images of MIC assays after 24h incubation with P1 and W8 AMPs ...... 147
Figure 28. Visual representation of the grafting of polymers under ‘good’ and ‘poor’ solvation conditions showing how high density polymer layers can be achieved to impart nonfouling properties...... 160
Figure 29. Schematic illustration of the reaction between an organosilane and surface hydroxides ...... 162
Figure 30. Schematic illustration of APTES adsorption in an aqueous solution...... 163
Figure 31. Individual thickness values as determined by SPR for PEI adsorption, BSA adsorption, and grafting of PEG (2kDa) in-situ by reductive amination at various ionic strengths.
...... 165
Figure 32. SPR sensorgrams showing the change in the total internal reflection (TIR) angle over time during a) the grafting of PEG-ald (2kDa) by reductive amination and b) subsequent
BSA adsorption...... 166
Figure 33. ATR-FTIR spectra of modified surfaces between a) 950-1250cm-1 and b) 1500-
1700cm-1...... 167
Figure 34. SPR sensorgrams showing the change in the total internal reflection (TIR) angle over time during. a) the grafting of PEG-ald (5kDa) by reductive amination under different ionic strengths, and b) subsequent BSA adsorption...... 168
Figure 35. VASE thickness measurements for APTES films prepared in toluene (2% (v/v)) at different deposition times ...... 171
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Figure 36. High-resolution N1s XPS spectra of APTES-modified Si wafers...... 174
Figure 37. High-resolution C1s spectra of APTES-modified Si wafers...... 175
Figure 38. Examples of chemical immobilisation strategies for the tethering of polymers to surface-bound functional groups ...... 187
Figure 39. Zeta potential data of unmodified and PEG-modified Si-COOH particles under various reaction conditions ...... 190
Figure 40. High-resolution C1s data for unmodified and PEG-modified Si-COOH particles ... 193
Figure 41. Representative ATR-FTIR spectra of PEG-modified Si-COOH microspheres...... 194
Figure 42. Representative SEM images of unmodified and PEG-modified Si-COOH microspheres under various grafting conditions...... 196
Figure 43. Representative SEM images of the film-like substance observed for: a) Si-PEG
(EDC/NHS), and b) Si-PEG (EDC/NHS+K2SO4) samples...... 197
Figure 44. Representative High-resolution C1s XPS spectra of BSA adsorbed to modified particles...... 199
Figure 45. MPTS film thickness values determined by VASE. Measurements were taken after
3hr deposition in toluene...... 208
Figure 46. ATR-FTIR spectra of MPTS films deposited in toluene for 3hr over a wavenumber range of a) 1070-1200cm-1, b) 840-970cm-1, and c) 2450-3500cm-1...... 209
Figure 47. Thickness values for MPTS films determined by VASE. Measurements were taken after 3hr deposition in ethanol...... 211
Figure 48. MPTS thickness values determined by VASE under pre-hydrolysis conditions at pH 4 and 8.5 with a post-deposition rinse...... 212
Figure 49. Ellipsometry thickness values of MPTS films prepared on Si wafers under various post-deposition processes...... 213
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Figure 50. Summary of static water contact angle results for MPTS films prepared on Si wafers using various post-deposition processes...... 215
Figure 51 Representative static water contact angle images of MPTS treated (2% (v/v)) Si wafers using various post-deposition treatments...... 216
Figure 52. High-resolution XPS spectra obtained from the S2p region of MPTS films prepared on Si wafers with various post-deposition treatments...... 220
Figure 53. XPS spectra of binding energies between 140-220eV of Si wafers showing overlapping spectral regions caused by plasmon structures associated with energy loss events from Si2s electrons...... 221
Figure 54. Representative ATR-FTIR spectra of MPTS films prepared with No Treatment at
MPTS concentrations of a) 0% (v/v), b) 1% (v/v), c) 2% (v/v), and d) 4% (v/v) ...... 222
Figure 55. Schematic illustration of the coupling methods used to tether PEG to thiolated surfaces by a thiol-ene ‘photo-click’ reaction or a thiol-maleimide reaction...... 230
Figure 56. Zeta potential data for PEG- and AMP-modified particles at each reaction stage.. . 232
Figure 57. High resolution C1s spectra of: a) unmodified Si particles, b) MPTS-modified Si,
Mal-PEG-NHS functionalised Si with c) W8 AMPs, and d) P1 AMPs, and Ac-PEH-NHS functionalised Si with e) W8, and f) P1 AMPs...... 235
Figure 58. Standard curve of cysteine used to determine the amount of reactive sulfhydryl functional groups ...... 236
Figure 59. Concentration and number of reactive sulfhydryl functional groups present on unmodified and modified silica colloids...... 237
Figure 60. Plate count assay indicating the number of actively growing P. aeruginosa cells incubated for 2 and 4hrs with modified and unmodified Si colloids prepared using thiolene photo-‘click’chemistry...... 240
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Figure 61. Plate count assay indicating the number of actively growing P.aeruginosa cells incubated for 2 and 4hrs with modified and unmodified Si colloids prepared using thiol- maleimide ‘click’ chemistry...... 241
Figure 62. Representative fluorescent microscopy images of P. aeruginosa cells showing live
(green) and membrane compromised (red) bacteria after being incubated for 4hrs with PEG- and
AMP-modified colloidal particles...... 243
Figure 63. Viability of P. aeruginosa cells incubated for 4hrs with the modified Si colloids.. . 244
List of Tables
Table 1. Rates of infection typically associated with indwelling medical devices and estimated cost for treatment* ...... 13
Table 2. Advantages and disadvantages of SAMs compared to polymer brushes...... 18
Table 3. Summary of polymer immobilisation strategies used to prevent non-specific protein adsorption ...... 24
Table 4. Sequence information and origin of representative AMPs ...... 27
Table 5. Summary of AMP immobilisation strategies and tested microorganisms ...... 32
Table 6. MIC ranges of selected AMPs ...... 33
Table 7. Summary of assembly methods used to fabricate BCCs and characteristics of resulting crystal structures ...... 43
Table 8. XPS atomic composition data of PuroA-modified and unmodified PSC2 particles .... 133
Table 9. Thickness and surface coverage of PuroA-modified and unmodified PSC2 particles . 135
Table 10. MICs of selected puroindoline-based synthetic AMPs ...... 146
Table 11. Calculated PEG thickness and corresponding BSA surface coverages determined by
MP-SPR ...... 169 xvii
Table 12. Atomic composition of APTES film deposited on Si wafers under different deposition conditions ...... 173
Table 13. PEG thickness and BSA surface coverage to physically adsorbed PEI and covalently bound APTES activation layers determined in-situ using MP-SPR ...... 177
Table 14. XPS atomic composition data for unmodified and PEG-modified Si-COOH particles.
...... 192
Table 15. Atomic composition data for BSA adsorbed to unmodified and PEG-modified Si-
COOH microspheres ...... 198
Table 16. XPS atomic composition data for MPTS films produced on Si wafers using various post-deposition processes...... 217
Table 17. ATR-FTIR frequencies and assignments for MPTS films prepared with No Treatment at various concentrations...... 223
Table 18. Atomic composition data for PEG- and AMP-modified Si colloids obtained using XPS
...... 234
Table 19. Binding energy positions and % area attributed to various carbon environments within the modified Si colloids ...... 236
Table 20. Total and corrected number of–SH groups on present on modified silica colloidal particles...... 238
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1 INTRODUCTION
1.1 Scope & aims of the PhD project The overall scope of this research is to investigate various immobilisation strategies for the tethering of non-fouling polymers and antimicrobial peptides to develop multifunctional surface coatings. One particular focus within this research is to determine the effects of manipulating underlying surface chemistry and grafting conditions on the immobilisation of selected polymers to facilitate subsequent AMP immobilisation. Providing optimal grafting conditions will allow for higher grafting densities which, in turn, will lead to an enhanced non-fouling ability at an interface.
While there has been several reports of the tethering of polymers and AMPs to planar and pre- existing surfaces within the current literature, the use of colloidal crystal layers as a platform for
PEG and AMP immobilisation provides an opportunity to develop versatile surface coatings to direct and control bacterial growth and attachment at an interface. Figure 1 provides a visual representation of grafting scenarios investigated within this research, which can be divided into the following research aims:
1. Surface activation to generate reactive surface-bound functional groups
As the number of tethering sites will dictate maximum grafting densities, a main focus of this research is to assess various surface activation methods to generate reactive functional groups for the subsequent tethering of non-fouling polymers and AMPs. Flat and spherical surfaces chosen within this research had pre-existing functional groups or were treated using different surface activation methods to impart the appropriate surface chemistry. Several strategies were applied to facilitate surface activation which were dependant on functional group requirements for
1 subsequent PEG grafting techniques. Moreover, both adsorptive and covalent approaches of surface activation were employed and compared for their ability to facilitate PEG grafting.
2. Polymer grafting investigations
This research stage was primarily aimed at investigating different polymer immobilisation strategies to optimise PEG grafting and non-fouling ability at an interface. Several PEG immobilisation strategies were chosen according to previous successes within current literature, which are illustratively summarised in Figure 1, and can be further separated in to the following research goals: i) To demonstrate that immobilised PEG shows non-fouling activity toward non-specific protein adsorption on planar surfaces before attempting particle functionalisation investigations. Both reductive amination and EDC/NHS chemistry was utilised for in-situ surface modifications using surface plasmon resonance, where non-fouling properties could also be assessed in real-time. ii) Investigate the effects of manipulating underlying graft layer, polymer MW and ionic strength on PEG immobilisation efficiency and resulting protein resistant properties. Optimisation of PEG grafting will allow for higher AMP loading and an increased non-fouling background, which is expected to be advantageous for bioactive coatings. iii) Demonstrate what type of reaction method can be used for the successful immobilisation of
PEG to spherical colloids.
3. AMP immobilisation and activity
The aims of this section were to first select AMPs and assess their antimicrobial activities in solution, and secondly investigate the antimicrobial activities of selected AMPs when
2 immobilised in a surface-bound state. To achieve this, separate goals were established which included the following: i) Demonstrate that puroindoline-based synthetic AMPs show antimicrobial activity in solution against clinically relevant bacterial strains using a modified broth microdilution method, and thus have potential for surface immobilisation studies. ii) Investigate the zero-length immobilisation of AMPs to demonstrate activity in an immobilised state through modification of PSC colloids with PuroA AMPs using EDC/NHS chemistry.
Confirmation of AMP immobilisation was examined using zeta potential, XPS, and MALDI-ToF-
MS, along with an assessment of surface localised antibacterial activity when AMP-modified colloids are presented in a patterned manner in the form of binary colloidal crystal layers. iii) Incorporate PEG immobilisation strategies described previously to tether AMPs to PEG- functionalised colloidal particles and assess antimicrobial activity. Based on the success of PEG immobilisation strategies and also the zero-length immobilisation of PuroA, tethering of more potent AMPs (P1 and W8) to NHS-functionalised PEG layers was utilised to develop antimicrobial coatings with superior properties compared to that of zero-length immobilisation.
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Figure 1. Illustrative summary of the grafting methods utilised for the tethering of PEG and AMPs to various surface types. Reaction summaries are separated in to three stages; 1) surface activation to produce appropriate functional groups, 2) polymer grafting to activated substrates, and 3) AMP immobilisation using selected peptides. Starting surfaces were either silicon wafers or colloidal particles. Abbreviations: MPTS: (3-mercaptopropyl)trimethoxysilane; APTES: (3-aminopropyl)triethoxysilane; PEI: polyethylenimine; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS: N-hydroxysuccinimide; AMP: antimicrobial peptide.
1.2 Thesis structure Chapter 1. Introduction
This Chapter introduces the investigations employed within this project to give the reader an overall indication of the main research aims pertained within this Thesis. A summary of the major experimental goals are also provided, in addition to highlighting gaps in current research that are attempted to be addressed by this project.
Chapter 2. Literature review
This Chapter provides an in-depth review of current literature highlighting the importance of controlling biointerfacial interactions through various surface modification techniques. A
4 particular focus of this chapter is the evaluation of current methods used to control bacterial attachment to surfaces due to the significant risks associated with bacterial colonisation. For the most part, this has entailed the immobilisation and/or release of biocidal compounds such as antibiotics and polyammonium salts, and also the use of non-fouling polymers such as poly(ethylene glycol). However, more recent studies have shown that the immobilisation of AMPs has potential for such applications due to their potency, and low probability of developing bacterial resistance. While the incorporation of AMPs in to planar surface coatings has been reported several times within the current literature, as new surface fabrication methods become available there becomes an opportunity to create novel bioactive surfaces that present various topographies and chemistries. One such method that has emerged in recent years is the use of binary colloidal crystal layers, which are surfaces created with both large and small colloidal particles that self-assemble into close-packed arrangements. Digesting this information it is apparent that there is a gap in current research that may be filled through modification of colloidal particles with non-fouling polymers and AMPs to generate multifunctional surface coatings to direct and control bacterial attachment and proliferation. Part of this Chapter is published in Advances in Colloidal and
Interface Science: Diba, F. S., Boden, A., Thissen, H., Bhave, M., Kingshott, P. & Wang, P.-Y.
Binary colloidal crystals (BCCs): Interactions, fabrication, and applications. Adv. Colloid Interface
Sci., doi:https://doi.org/10.1016/j.cis.2018.08.005 (2018).
Chapter 3. Materials and methods
This Chapter provides a complete description of the materials and experimental methods used throughout the project.
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Chapter 4. Binary colloidal crystal layers as platforms for the surface patterning of puroindoline-based AMPs and optimisation of AMP selection
Within this Chapter, an evaluation of the antimicrobial activities of several puroindoline-based
AMPs was performed against E.coli and P.aeruginosa using a modified broth microdilution method. Additionally, an investigation of the zero-length tethering of one particular AMP (PuroA) to carboxylated PS colloids was assessed to provide a proof of concept that BCC layers can be used as a platform for the immobilisation of AMPs whilst maintaining antibacterial activity. Part of this work has been published in Applied Materials and Interfaces: Boden, A., Bhave, M., Wang,
P.-Y., Jadhav, S. & Kingshott, P. Binary Colloidal Crystal Layers as Platforms for Surface
Patterning of Puroindoline-Based Antimicrobial Peptides. ACS Appl. Mater. Interfaces 10, 2264-
2274, doi:10.1021/acsami.7b10392 (2018).
Chapter 5. Effect of grafting conditions and underlying graft layer on non- specific protein adsorption to PEG layers prepared in-situ with surface plasmon resonance
To incorporate a flexible polymer linker to not only add extra mobility and penetration of immobilised AMPs, but also to impart non-fouling characteristics; the focus of this Chapter is to investigate various PEG immobilisation strategies in-situ using surface plasmon resonance.
Specifically, the use of EDC/NHS coupling chemistry and also reductive amination was assessed and compared for grafting efficiency and their ability to resist protein adsorption. In addition to this, the effect of interchanging the underlying graft layer and grafting conditions was also investigated. This was achieved using either covalently bound APTES films or physically adsorbed
PEI layers, and by manipulation of ionic strength and temperatures to promote ‘cloud-point’ conditions.
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Chapter 6. Investigation and characterisation of PEG immobilisation to silica microspheres and BSA adsorption
This Chapter presents experimental work aimed at optimising PEG grafting to carboxylated silica microspheres in preparation for future AMP immobilisation investigations. The use of EDC/NHS coupling chemistry was employed to tether PEG to ‘activated’ particles in a similar approach to
Chapter 4.1, and grafting conditions were manipulated by changing ionic strength of the polymer solution. Colloidal layers of the functionalised particles were assessed for their non-fouling ability by protein adsorption analysis using BSA as a model protein.
Chapter 7. Analysis of MPTS deposition methods for the production of thiol- containing films
Within this Chapter, an alternate surface activation method was investigated to produce sufficient pinning sites for the tethering of polymers and AMPs. Provided here is a critical evaluation of
MPTS deposition to Si wafers for the production of thiol-containing films, along with an assessment of various post-deposition treatments. Fabricated surfaces were characterised using several physio-chemical techniques to determine optimal deposition parameters that can be used in future PEG and AMP immobilisation strategies.
Chapter 8. Characterisation and antimicrobial activity of PEG and AMP- functionalised colloids prepared using thiol-ene ‘photo-click’ and thiol- maleimide chemistry
Here a broader examination of PEG and AMP immobilisation strategies was investigated utilising thiol-modified silica microspheres prepared using previously optimised conditions. A comparison between a ‘one-pot’ or a sequential immobilisation of PEG and AMPs was performed using a thiol-maleimide or thiol-ene ‘photo-click’ method, respectively. Additionally, an antimicrobial
7 assessment of the functionalised particles was also performed against P.aeruginosa to determine
AMP activity in an immobilised state.
Chapter 9. Conclusions
This Chapter provides an overall conclusion to the project highlighting major experimental findings and possible future research directions.
Chapter 10. Conferences and publications
A detailed list of publications and attended conferences can be found within this Chapter.
8
2 LITERATURE REVIEW
2.1 General introduction Fouling from proteins and microorganisms is an adverse event that impairs the function and properties of a wide range of biotechnological and biomedical devices including biosensors,1,2 implantable medical devices,3-5 drug delivery systems,6-8 and marine coatings.8,9 For example, unwanted protein adsorption can cause nonspecific responses in various types of affinity biosensors, and as adsorbed layer of proteins on medical devices can provide a suitable conditioning film for the attachment of bacteria leading to potential infection and/or device rejection. Because of this phenomena, a major aim of biointerface science is to develop material that are antifouling, i.e. materials that operate in biological media and can resist the nonspecific attachment of biomolecules and microorganisms. Within current literature there are several approaches that have been adopted to achieve this which have been based upon chemical and/or physical intervention to control the interactions that occur at the biointerface to create an environment that is unfavourable for biomolecule adsorption and bacterial colonisation. Chemical methods include the covalent or physical immobilisation of polymers or biomolecules – which can create a barrier to the surface, or can initiate specific cellular responses to prevent adhesion.
Conversely to the chemical approaches, physical intervention methods to prevent biofouling have been based on the patterning of a biointerface with topographical cues to provide an unfavourable environment for bacterial colonisation.
In the development of such an antifouling surface it is generally not practical to fabricate material with inherent antifouling properties as this may cause changes in the physical and chemical properties of the bulk and possibly perturb the original function of that material.
9
Therefore, the general approach has been to coat the surface of the desired material to impart antifouling properties while conserving the physical and chemical integrity of the bulk material.
Over the past two decades attention has been drawn to the immobilisation of hydrophilic polymers to create a physical barrier between the surface and the fouling entity. It has been shown that at sufficiently high densities a brush regime is achieved which can provide many advantages over traditional self-assembled monolayer (SAM) based coatings in the prevention of biomolecule and/or cellular adhesion. By manipulating brush characteristics such as graft density, thickness, and chemistry it is possible to tune surface properties including wettability,10,11 adsorption of biomolecules,12-15 and cellular adhesion16-19 to achieve a desired function.
Research has also investigated the immobilisation of certain biomolecules such as antimicrobial peptides (AMPs) for the development of active non-fouling surfaces. AMP-based immobilisation strategies are inspired from naturally occurring non-fouling coatings on amphibians and fish, which secrete dermal chemical defence slime incorporating various antimicrobial agents (including AMPs) to prevent colonisation by microorganisms.20 AMPs demonstrate superior antimicrobial properties compared to traditional antibiotics; showing a broad-spectrum of activity,21,22 low propensity for developing bacterial resistance,23 and high efficacy at low concentrations.22,24 These factors combine to make AMPs potential candidates in the development of antibacterial surfaces and several studies have shown that AMP immobilisation can be achieved through a number of different methods including physical or covalent immobilisation to a variety of substrates – offering a novel route for the fabrication of active antifouling surface coatings.
Aside from the manipulation of surface properties by the immobilisation of non-fouling polymers and other biomolecules, topography can also play a crucial role in manipulating the
10 adsorption and attachment profiles of bacterial cells.25-27 It has been identified that surfaces which exhibit identical surface chemistries may have vastly different colonisation rates which suggests that there is a cause and effect relationship between surface roughness and the attachment profiles of adhering bacterial cells.26 Similarly to AMPs, naturally occurring structured surfaces have evolved over millions of years to provide a beneficial function; setae on gecko feet, shark skin, and the topographical structure of insect wings have all been studied for their unique properties and antibacterial activities.26,28 These naturally occurring surfaces exert their antimicrobial activity by presenting topographical characteristics that provide unfavourable surface structures to prevent cellular attachment and/or disrupt membrane integrity leading to cell death.29 One particular focus of current research is to mimic these naturally occurring nano- and microstructured surfaces to design active antimicrobial coatings. The fabrication of such surfaces are mostly base upon modern lithographic and contact printing methods, however these processes can often be quite time consuming and involve complex equipment which may not be ideal to produce structures over large areas exceeding several cm2.30
Recently, a rapid and inexpensive approach has been discovered to fabricate nano- and microstructured surfaces by the crystallisation of particles within a confined area.31,32 This technique allows the formation of colloidal crystal layers that present complex architectures and structural hierarchies based on an evaporation induced confined area assembly (EICAA) method where extremely dilute suspension of large (L) and small (S) particles crystallise over large areas induced by capillary and convective flow forces.27 This technique is also very versatile allowing for an extraordinary range of particle sizes and chemistries to be used whilst maintaining long- range ordering.31,33
11
Because of this versatility, there is an opportunity to use binary and even multicomponent colloidal crystals as a platform for the immobilisation of nonfouliong polymers, and also a range of biomolecules such as AMPs at different ratios and densities. This is expected to allow for subtle changes in the physiochemical patterns displayed at the surface and lead to controllable cellular responses which are dependent on the particular chemical and topographical pattern presented.
2.2 Interactions at the biointerface The biointerface is considered to be the point of contact between a biomolecule, biological tissue, or living organism with another biomaterial or inorganic substrate. Physical and chemical characteristics of this interface are crucial in determining subsequent biomolecule adsorption,34 tissue integration,35 bacterial colonisation,36,37 and because of this a major focus of biointerface science has been devoted to understanding and controlling these interactions. The importance of understanding these interactions is critical in many industries as unwanted fouling from proteins and microorganisms can impair function and also compromise the integrity of a particular surface.
For example, biofouling on a ship’s hull can significantly impact the hydrodynamic performance of the vessel leading to increased fuel consumption,9,38 and can also be a contributing factor in the outbreak of food-borne diseases including Listeria monocytogenes and Salmonella.39,40 It is obvious that biofouling is of serious concern in a number of settings and environments, however there is particular risk within the healthcare industry due to the high incidence of infections associated with temporary and permanent medical devices.36 As such, the following section will describe the phenomena of biofouling and biofilm formation, and also highlight the significant impact this has on organisations and patients within the healthcare industry.
12
2.2.1 Biomaterial associated infections
In Australia there are an estimated 200,000 healthcare associated infections (HIAs) per annum with 50% of these originating from a biomaterial source.41 While in the U.S., treatment of the 1,000,000 reports of nosocomial infections associated with surgical implants are estimated to cost approximately up to 4.5 billion USD.42,43 One particular study has shown that device- associated and surgical-site infections accounted for 47.7% of all HIAs for 504 patients in 183 hospitals.44 Although the above data does not take into account infections caused by urinary catheters and urinary stents – infections associated with surgical implants are much more difficult to control and generally require prolonged antibiotic treatment and numerous surgeries. These type of HAIs can not only significantly decrease quality of life, but also crease a heavy and financial burden for patients and families. Table 1 below summarises some clinical and economical concerns associated with infections related to frequently used indwelling medical devices and surgical implants.
Table 1. Rates of infection typically associated with indwelling medical devices and estimated cost for treatment*
Type of implant Estimated no. of uses Rate of Average cost of in the US per year infection (%) treatment ($USD)
Bladder catheters >30,000,000 10-30 4,500 Mechanical heart valve 85,000 1-3 50,000 Vascular graft 450,000 1-5 40,000 Ventricular assist device 700 25-50 50,000 Joint prosthesis 600,000 1-3 30,000 Fracture-fixation devices 2,000,000 5-10 15,000
*Data presented was obtained from published studies, market reports, medical literature and device manufacturing companies.
13
With an aging population this problem is only expected to become more prominent as modern healthcare will rely more heavily on the use of implantable devices such as catheters, contact lenses, and artificial bone replacements. As these devices and implants are responsible for restoring function to diseased and damages tissues, a major focus of materials science has aimed at preventing the fouling of a biomaterials surface to ensure correct workability and reduce the risk of infection.45,46 However, to effectively manipulate such a complex phenomenon, considerable knowledge of the biological interactions that result in fouling at a biointerface is crucial for developing antifouling coatings.
Host defences within a healthy individual usually safeguards against infection by rapidly eliminating microbial contamination. The effectiveness of host defence systems is somewhat reduced in the presence of an implanted device such that the number of pathogenic organisms required to initiate infection is significantly reduced, and even ‘non-pathogenic’ species of coagulase negative staphylococci has been found to cause infection.47,48 The colonisation of such biomaterials can also drastically alter the phenotype of invading organisms; making them much more virulent and resistant to antibiotic treatments. As a result of this, there are many cases where the device needs to be removed before the infection can be treated – leading to prolonged hospital stays and higher treatment costs.49,50
2.2.2 Biofouling and biofilm formation
Ubiquitous in nature, bacterial biofilms are structured communities formed from the successful attachment and colonisation of bacterial cells to biotic and abiotic surfaces through production and encapsulation by extracellular polymeric substances (EPS).37,51 While the physiological characteristics of biofilms have been well documented in several reviews,52,53 there section herein will briefly discuss the mechanisms of biofilm formation in the context of
14 biomaterial associated infections. Biofilm formation is a complex process that comprises several stages beginning with the reversible attachment of microorganisms to a surface (Figure 2). This initial adhesion phase is dictated by several factors including; the presence of a conditioning film, hydrodynamics and properties of the surrounding medium, substratum properties, and also cell surface characteristics.37,54
As bacterial cells enter close proximity to a surface, cells will alter their phenotype in response to the surrounding microenviroment, however, it has generally been observed that bacteria will more readily attach to rougher, more hydrophobic surfaces.37,55 Once attached, sessile bacteria begin to form single- and mixed-species microcolonies where production of EPS of various compositions condition the microenvironment surrounding each bacterium. This microenvironment can show striking heterogeneity with only tens of microns separating anaerobic area from highly aerated ones, and can also show significant differences in pH, nutrient availability, temperature, osmolarity and flow conditions.56 Due to these vast differences in biofilm microenvironments, the bacteria within these biofilms will respond to their specific microenvironment, exhibiting different growth profiles and gradually forming a structurally complex mature biofilm.
15
Figure 2. Schematic illustration of biofilm formation summarised into five different stages. 1) The reversible attachment of microorganisms, 2) formation of EPS and cell-to-cell adhesion, 3) proliferation, 4) maturation, and finally 5) the detachment and reversion to planktonic growth.
Dispersal of biofilms may be caused by a number of factors such as shearing of cell aggregates due to turbulent flow, shedding of cells from actively dividing bacteria, and also active dispersal as a result of low nutrient levels or quorum sensing. While the shedding of actively growing cells from a biofilms has not been well documented, dispersal due to physical forces has been studied in more detail where the rate of erosion has been shown to increase with increasing biofilm thickness and flow velocity.57,58 Dispersal has also been shown to be species specific with
P. fluorescens dispersing and recolonising a surface after 5h, whereas V. harveyi exhibited the same phenomena after only 2h.51
Clearly there is a need to control the attachment of bacteria to biomaterial surfaces in the aim of minimising the risk of biofilm formation and infection as a result bacterial colonisation.
The sections below will outline some of the current strategies to control biointerfacial interactions
16 and also highlight new possibilities that may be used to develop an active antimicrobial coating that can be applied to a biomaterials surface.
2.3 Non-fouling polymers
2.3.1 Self-assembled monolayers It is hypothesised that if the initial nonspecific adsorption of proteins could be completely prevented that the sequential attachment of bacteria to these surfaces would not occur as there would be no conditioning film to initiate or promote bacterial growth and biofilm formation.
Arguably the most well-known chemistry used in the study of non-fouling materials and surfaces are those containing hydrophilic chemistries that create a highly hydrated, steric barrier between the fouling entity and the surface (Figure 3).59,60 Traditional approaches to apply this chemistry at the surface utilised alkanethiols terminated with oligo(ethylene glycol) (OEG) functional groups that can form self-assembled monolayers (SAMs) when chemisorbed onto gold substrates. A relatively recent publication by Emmenegger, et al. 61 corroborates previous research showing that hydroxyl-terminated OEG SAMs (Au-S-(CH2)11-(O-C2H4)2-OH) can reduce the nonspecific adsorption of single-protein solutions of fibrinogen (Fn), human serum albumin (HSA) and lysozyme (Lyz) by approximately 90% compared to uncoated Au surfaces, whereas minimal resistance was observed for SAMs prepared with 16-mercaptohexadecanoic acid due to the absence of hydrophilic oligoether moieties. Li, et al. 62, also showed that if the number of OEG moieties is increased to six ((O-C2H4)6) – hydroxyl terminated SAMs can completely resist the adsorption of Lyz, Fn, and BSA from single-protein solutions prepared at 1.5 mg/mL. This resistance is controlled by surface hydrophilicity due to terminal –OH groups, internal hydrophilicity due to the hydration of OEG chains, and also the packing density of the SAMs.
17
Figure 3. Illustrative representation of hydration layer (blue circles) and chain conformation of a) neutral polymers, b) zwitterionic polymers, and c) self-assembled monolayers, to create a physical barrier between the surface and fouling entity.
While OEG-terminated SAMs are able to reduce protein adsorption from single-protein
solutions, considerable protein adsorption from complex mixtures of proteins such as blood plasma
and serum has been observed.63 This limits the practical applications of SAMs as many diagnostic
and clinical techniques require surfaces that are completely non-fouling – as even the adsorption
of Fn at 5ng/cm2 to a surface can induce complete platelet adhesion.64,65 Furthermore, the limited
long-term stability and presence of defects in SAMs prompted the development of new strategies
to combat biofouling, and recently attention has been drawn to immobilised polymer brush layers
as they have been shown to provide many advantages when compared to SAMs (Table 2).
Table 2. Advantages and disadvantages of SAMs compared to polymer brushes
Self-assembled monolayers Polymer brushes Simple formation Long-term stability Well-defined layers Control over brush length Choice of monomer Advantage Polymerisation methods e.g. surface initiated - atom transfer radical polymerisation (SI-ATPR), ring- opening polymerisation (ROP), nitroxide-mediated polymerisation (NMP) etc. One layer Complex preparation Disadvantage Limited long-term stability Complex structures Pinholes and defects
18
2.3.2 Ethylene glycol based polymer brushes A polymer brush may be defined as a polymer chain that is tethered at one end to an underlying substrate and form at sufficiently high densities with a layer thickness that is significantly higher than the end-to-end distance when free in solution.66 Table 3 summarises several studies extending over a decade that have been conducted to assess the adsorption of proteins to polymer brush surfaces; indicating the type of polymer used, brush properties and non- fouling ability.
Applying the knowledge gained from the non-fouling properties of SAMs it is not surprising that similar hydrophilic ethylene glycol chemistries are utilised for investigating antifouling properties, and several reviews have shown that poly(ethylene glycol) (PEG) based brushes are the most popular polymer studied to prevent fouling from biomolecules and microorganisms.38,59,67-69 Early studies on PEGylated surfaces indicate that provided that the graft density of PEG chains is high enough, and that ‘cloud-point’ (CP) conditions (60°C, 0.6M K2SO4) are used, the nonspecific adsorption of Lyz, HAS, lactoferrin, and γ-globulins from a mixed- protein solution can be resisted to below detectable levels by XPS as MALDI-ToF. At non-CP conditions and at lower graft densities however, it was observed that variable amounts of irreversible protein adsorption occurred on all prepared surfaces.12 Similarly, PEG (MW 5kDa) brushes at sufficient densities prepared by reductive amination have also been shown to resist the nonspecific adsorption of β-lactoglobulins below detectable levels by XPS and time-of-flight static secondary ion mass spectroscopy (ToF-SSIMS). These surfaces were however, unable to prevent the attachment of Gram-negative (Pseudomonas sp.) and Gram-positive (Listeria monocytogenes) bacteria to these surfaces.70 Considering a detection limit of a few ng/cm2 for adsorbed proteins can be estimated for XPS, and the successful detection of proteins by MALDI-ToF relies on successful extraction of proteins from the surface,12 it is possible that low but undetectable levels 19 of protein resulted in sufficient attachment sites for bacterial colonisation, or the bacterial cells were opportunistic enough to produce their own conditioning film to facilitate colonisation.
More recent studies have investigated the kinetics of protein adsorption to PEGylated surfaces using various in-situ techniques such as quartz-crystal microbalance (QCM, optical waveguide lightmode spectroscopy (OWLS), fixed and variable angle spectroscopic ellipsometry
(FASE & VASE), and surface plasmon resonance (SPR) (See Table 3). These techniques are used in conjunction with, or even in replacement to static techniques as they provide greater sensitivity and real-time information regarding the kinetics of protein adsorption. For example, Pidhatika, et al. 71 showed that PEG (5kDa) brushes grafted to a poly(L-lysine) backbone with a dry thickness of 2.47nm was able to resist the adsorption of serum proteins down to approximately 13±4ng/cm2 as determined by OWLS. The investigated polymers brushes were observed to possess a graft density of α=0.28cm-2, where α represents the fraction of occupied sites in relation to the number of available sites per nm2. In a similar study utilising CP conditions proposed earlier,12 Emilsson, et al. 15 describes the formation of PEG (5kDa) brushes prepared by a grafting to- method that can resist nonspecific protein adsorption from diluted serum (10X dilution) down to 14ng/cm2 at a graft density of 0.54nm-2. The observed differences in serum adsorption seen between these two studies utilising PEG molecules with identical MW suggests that graft density and brush structure are critical parameters that lead to lower serum adsorption - which can be directly manipulated by changing the underlying graft layer or the grafting conditions.
Furthermore, for PEG brushes, increasing the MW of the polymer does not necessarily lead to an increased non-fouling ability. The protein repellent properties of 2, 5, 10, 20, and 30kDa PEG brushes were examined against serum proteins and results indicated that the brushes formed using
20kDa and 30kDa PEG performed far less efficiently than the 2, 5. And 10kDa brushes.15 The
20 authors suggest that the grafting kinetics of large PEG molecules becomes quite slow under CP conditions.
2.3.3 Other non-fouling polymers
In addition to PEG brushes, similar non-fouling properties are observed when considering other hydrophilic brushes such as zwitterionic and hydroxyl-functional methacrylate polymers
(See Table 3). For example, Zhao, et al. 72 investigated the antifouling properties of two types of hydroxy-functional methacrylates; poly(hydroxypropyl methacrylate) (PHPMA), and poly(2- hydroxyethyl methacrylate) (PHEMA). Both of these brushes were able to significantly reduce the adsorption of undiluted blood plasma compared to control samples. However it was found that
2 PHEMA out performed PHPMA with protein surface coverage observed to be 3.5ng/cm and
50ng/cm2 for PHEMA and PHPMA brushes, respectively. Considering the structures of the two different polymers with PHPMA having a longer aliphatic component, it was proposed that the increased non-fouling ability was attributed to the more hydrophilic nature of PHEMA brushes.
Zwitterionic-based polymers have also been investigated due to their strong hydration capacity.11,63,73 Highlighting the importance of brush thickness, poly(serine methacrylate)
(pSerMA) polymer brushes produced at an optimum thickness (37.2nm) was able to reduce protein adsorption from undiluted blood plasma down to 12.9ng/cm2.11 At greater film thicknesses however, the increased inter- and intramolecular interactions led to decreased water-polymer interactions and increased protein adsorption under these conditions. In a separate study, Li, et al.
74 investigated the non-fouling properties of poly(sulfobetaine methacrylate) zwitterionic brushes demonstrating that the adsorption from undiluted blood plasma can be reduced to below 5ng/cm2 provided that the grafting density is sufficiently high.
21
While mostly hydrophilic in nature to create a hydrated barrier between the surface and adsorbing biomolecules, it is quite apparent there are a wide range of polymer chemistries that are available to prevent the nonspecific adsorption of biomolecules at an interface. Direct comparisons between studies is often quite difficult due to differences in polymer chemistry, polymerisation method, and protein loading (See Table 3). However, current research does indicate that: hydrophilicity, thickness, and graft density are all critical factors for imparting non-fouling characteristics. Therefore, considering the established non-fouling properties and versatility of
PEG brushes, it is hypothesised that the development of an optimised PEG graft layer will be crucial for successfully preserving non-fouling activity. As such, a main focus of this research will be to investigate differences in surface properties when chemistry and topography of the underlying graft layers are manipulated.
2.3.4 Immobilisation methods
Generally, the immobilisation of polymer chains to a surface can be achieved by either a physisorption method; where the polymer is selectively adsorbed to a surface,75,76 or by chemisorption; where polymer chains are covalently tethered to the substrate.12,16 The latter of these two methods has attracted more attention recently due to the increased stability of covalently immobilised polymers, and also the wide array of immobilisation methods available. Additionally, covalently immobilised polymer chains can be prepared by a “grafting-from” or “grafting-to” technique, each having their own advantages and disadvantages.
The “grafting-from” technique uses surface-grafted initiator molecules for the in situ polymerisation of monomers from solution. Many methods have been proposed to facilitate this type of “living” polymerisation including; ring-opening polymerisation,77,78 reversible addition- fragmentation chain transfer polymerisation,79 and also atom transfer radical polymerisation,80,81
22 to name a few. “Grafting-to” however, involves the immobilisation of preformed polymers through a covalent reaction between terminal functional groups on the polymer and complementary functional groups available at the surface. This method has the distinct advantage of being a relatively simple synthesis method, and also the preformed polymers layers are more easily characterised. However, it has also been stated that it is more difficult to form dense polymer brush regimes due to the existence of previously attached chains that impedes further immobilisation, and instead a less dense “mushroom regime is formed.15,76,82 Manipulating grafting conditions can however overcome the aforementioned disadvantages to achieve a greater packing density and form strongly stretched polymer brushes. For example, Kingshott, et al. 12 has shown that increased grafting density can be achieved using cloud-point conditions to induce poor solvation of polymer chains. Similarly, Emilsson, et al. 15 was able to achieve high grafting densities using the “grafting-to” method by “salting-out” polymer chains at high salt concentrations.
23
specific protein adsorption protein specific - 13 egies used to prevent prevent to non used egies Summary of polymer immobilisation strat immobilisation polymer of Summary . 3 Table Table 24
4 1
25
2.4 Antimicrobial peptides as therapeutic agents
2.4.1 Introduction to antimicrobial peptides
The formation of biofilms upon medical devices enable sessile bacteria within these structured communities to withstand host defence systems and be drastically more resistant (up to
1000-fold) to antibiotics, biocides , and sheer forces.20 Various approaches have been employed for the fabrication of antimicrobial surfaces based on the immobilisation and/or release of biocidal substances such as polyammonium salts,83,84 metal derivatives,49,85 and antibiotics.49,86 However, considering the great resistance sessile bacteria exhibit – large concentrations of disinfectants and antibiotics are needed for the eradication of matured biofilms. This can lead to significant patient suffering, damage to the environment, and even the emergence of antibiotic resistant strains of bacteria.87 To this end, there has been a requirement to develop a broad-spectrum antibacterial coating to prevent microbial colonisation and biofilm formation on the surface of medical devices.
Forming an integral part of the innate immune system in most organisms - antimicrobial peptides (AMPs) show promising potential as a coating for medical devices as they demonstrate superior antimicrobial properties when compared to synthetic and semi-synthetic antibiotics.88
These properties include a low propensity for developing bacterial resistance,89,90 broad spectrum of activity,91 and high efficacy at low concentrations when compared to traditional antimicrobials.24 For example, Phillips, et al. 22 describes the antimicrobial activity of several puroindoline-based peptides against bacteria and fungi. Among the peptides investigated, PuroA
(FPVTRWWKWWKH-NH2) was found to be the most potent against all tested microorganisms.
Specifically for E.coli, PuroA was effective at lower concentrations (16µg/mL) when compared to the reference AMP indolicidin (20µg/mL).
26
At present, over 3,000 AMPs have been identified and isolated across six kingdoms and other synthetic sources.92 Structural information, activity, and sequence information for the AMPs is readily available from the antimicrobial peptide database
(http://aps.unmc.edu/AP/main.php).92,93 In natural settings, AMPs are generally secreted into internal bodily fluids or mucosal epithelia, and have been isolated from a number of species ranging from bacteria to mammals including humans (Table 4). AMPs are relatively short polypeptides typically between 12 to 50 amino-acids in length, and also have a net positive charge in the range of +2 to +9, which is caused by an excess of basic amino acids including lysine (K), arginine (R), and histidine (H).94,95
Table 4. Sequence information and origin of representative AMPs
Classification Sequence Species
1 10 20 30 BLP-1 G I G A S I L S A G K S A L K G L A K G L A E H F A N Frog I Magainin 1 G I G F L H S A G K F G K A F V G E I M K S Frog Chrysophsin 1 F F G W L I K G A I H A G K A I H G L I H R R R H Fish Indolicidin I L P W K W P W W P W R R Cow II Histain 5 D S H A K R H H G Y K R K F H E K H H S H R G Y Human Brevinin-1 F L P L L A G L A A N F L P K I F C K I T R K C Frog III Ranalexin F F G G L I K I V P A M I P K I F C K I T R K C Frog α-Defensin D C Y C R I P A C I A G E R R Y G T C I Y Q G R L W A F C C Human IV Human defensin A C Y C R I P A C I A G E R R Y G T C I Y Q G R L W A F C C Human Rabbit defensin V C A C R R A L C L P R E R R A G F C R I R G R I H P L C C R R Rabbit V Lactoferricin-B K C R R W Q W R M K K L G A P S I T C V Cow
2.4.2 Mode of action
While there is great structural and functional diversity among AMPs, many possess similar properties that contribute to their antimicrobial activity; they are highly cationic providing electrostatic attraction to negatively charged bacterial membranes, and are also hydrophobic
27 allowing for phospholipid displacement and destabilisation.90,96 So far there has been three major models proposed to describe AMPs mode of action; the carpet model, the barrel-stave model, and also the toroidal pore model. The carpet model proposes that the AMP covers the bacterial membrane in a “carpet-like” layer, and when a saturation point is reached abrupt lysis of the cell occurs resulting in leakage of cellular components.97 The barrel-stave and toroidal pore models are both “pore-forming’ models and result in two distinctly different structures when interacting with microbial membranes. The barrel-stave model is proposed for α-helical peptides; where they form a structure based upon lateral peptide-peptide interactions similar to that of a membrane ion channel.97 For the toroidal pore model however, specific peptide-peptide interactions are not present, and toroids form due to the local curvature of the membrane bilayer.98
There is still some conjecture regarding the interactions that ultimately lead to cell death, with hypotheses that include depolarisation of bacterial membranes, pore formation and leakage of cellular contents, activation of hydrolases that degrade the cell wall, and also intracellular mechanisms such as DNA-binding.99-101 While free AMPs can easily penetrate bacterial cells, their immobilisation to a solid substrate can reduce peptide flexibility and penetration depth. As such, current research has established general guidelines for their successful immobilisation whilst maintaining antimicrobial activity which will be discussed in the following section.
2.4.3 Immobilisation strategies
A crucial factor in the success of AMPs to be used as a coating for implant materials is their activity and stability in an immobilised state. This will be greatly influenced by the type of immobilisation method used, and the following section will detail the advantages and disadvantages of various approaches with respect to AMP activity.
28
Immobilisation of AMPs can be achieved through a number of physical and/or chemical approaches, using a variety of substrate materials. However as stated above, careful consideration in to the selected immobilisation method must be taken as this will greatly influence AMP activity.
One of the more popular physical immobilisation methods is the layer-by-layer assembly of polyionic containing AMPs on to a solid substrate.102,103 The general approach for this technique is to sequentially deposit two polyionic polymers on to a solid support where the AMPs is sandwiched between the polymers (Figure 4). This method offers great flexibility in terms of the type of polymers and AMPs that can be used, and also the number of polymer-AMP layers allowing for high and controllable AMP loading on to the surface.
Figure 4. Illustrative representation of a layer-by-layer (LbL) assembly containing AMPs sandwiched between polyionic polymers. The technique can be repeated n times to achieve an assembly of n layers of each material to allow for control over AMP loading. Alternating layers of polyanions and AMPs can also be produced by omitting polycation deposition.
While the LbL deposition of AMPs offers advantages in terms of simplicity and choice of polymer, there are however a number of disadvantages that should be considered. Diffusion of imbedded AMPs into the surrounding medium can be hindered through the LbL architectures and is influenced by a number of factors including thickness, peptide-peptide interactions, and also the 29 tortuosity of diffusion pathways.21,104,105 Long-term stability of LbL layers may also be an issue due to the short half-life and cytotoxicity associated with higher concentrations of soluble peptides.90
To overcome the disadvantages associated with the use of LbL techniques in respect to
AMP-containing surfaces, the use of covalent-based immobilisation can minimise AMP leaching from the surface and overcome short half-life and cytotoxicity problems.21,105 The basic principle for AMP covalent coupling strategies is to chemically react an available functional group present on the AMP to the desired surface – forming a stable antimicrobial coating.106,107 As modification of the amino acid sequence of AMPs may perturb function, it is more common to use functional groups inherently present on the AMP such as N-terminal amines,108-110 C-terminal carboxylic acids,111,112 and also sulfhydryl groups present on cysteine residues.113 Furthermore, surfaces that are not reactive toward AMPs may be functionalised with long spacer molecules or anti-cell adhesion molecules bearing an appropriate functional group to immobilise the AMP.
Arguably the most simple and effective method for AMP immobilisation using a covalent- based approach is via the use of AMP-reactive self-assembled monolayers (SAMs) on a suitable surface.111,114,115 The length of the SAM spacer can also be manipulated depending on the requirements for increased AMP flexibility or type of substrate used – which can be up to 12 carbon atoms in length. For example, Humblot, et al. 111 covalently immobilised Magainin I to binary SAMs prepared with 6-mercaptohexanol and 11-mercaptodecanoic acid. The antibacterial activity of the immobilised AMPs was assessed against several types of cells and showed that more than 50% of cells were propidium iodide (PI) positive due to permeabilised membranes. Similarly,
Xiao, et al. 114 investigated AMP conformation and antibacterial activity for the AMP; MSI-78 when immobilised to maleimide-terminated SAMs. It was shown that freshly prepared surfaces
30 were able to kill a substantial amount of bacterial cells after 1hr of contact. Interestingly, it was also found that after 5 days of exposure to atmospheric conditions – the AMP-SAM surface exhibited no antibacterial activity due to the decomposition of SAM surfaces.
Tethering of AMPs to using stiff or flexible spacer molecules has also been investigated to overcome the issues that arise from SAM degradation. In addition to allowing greater penetration of the AMP, in many cases these spacer molecules are polymers of anti-cell adhesion molecules, which can provide additional protection by repelling bacterial cells from the surface.
Chitosan,106,116 PEG,117-120 and other polymeric materials121 are all additions that have been used in AMP immobilisation strategies as they have generally accepted biocompatibility; being applied in research setting for quite some time. Table 5 below summarises several AMP immobilisation investigations, highlighting key aspects such as the type of AMP used, immobilisation methods, and microorganisms tested. The use of stiff spacers such as poly(methyl methacrylate) and poly(vinyl chloride) of sufficient length will also allow for penetration in to bacterial cells, however, their lateral mobility will be restricted when compared to the use of flexible spacer molecules which may inhibit interfacial AMP orientation.109 While there are reports of observing antimicrobial activity for AMPs without the incorporation of a spacer molecule,108,110,122 it is generally accepted that incorporation of a spacer molecule has more influence on antimicrobial activity than high AMP surface concentrations. 21 For example, the antimicrobial activity of AMPs; nisin and LL-37 were completely diminished when immobilised on to a solid support without the use of a spacer molecule.123,124 Additionally, Ivanov, et al. 117 reported that physical adsorption of chrysophsin-I showed a significant reduction in bactericidal activity against E. coli, whereas covalent immobilisation via a flexible (PEG)12 linker preserved 82% of the killing activity of the
AMP. Costa, et al. 106 also reports similar results where the number of viable methicillin-resistant
31
S. aureus (MRSA) cells was much higher when the AMP was physically adsorbed when compared
to covalently immobilised by a PEG spacer.
Table 5. Summary of AMP immobilisation strategies and tested microorganisms
AMP Substrate Immobilisation method Microorganism(s) Reference Magainin I Mixed self-assembled Covalently react free amines on L. ivanovii 111 monolayers (SAMs) on Au AMP to COOH using EDC/NHS (Li4pVS2), chemistry E. faecalis, S. aureus (ATCC 29213) Chrysophsin- Au crystals 1. Covalent immobilisation of E. coli (ATCC 117 I AMP via SM-(PEG)12 linker 33694)
2. Zero-length covalent immmobilisation of AMP on Au surface
3. Physical adsorption on Au surface hLF1-11 Chitosan films on Au Covalent (EDC/NHS) an physical MRSA (ATCC 106 immobilisation with and without 33591) PEG spacers
Magainin I Chitosan-terephtaldehyde Covalent immobilisation of free L. ivanovii 116 functionalised stainless amines on AMP to COOH using (Li4pVS2) steel EDC/NHS chemistry
Tet213 Poly(DMA-co-APMA) Maleimide-thiol reaction P. aeruginosa 121 brushes synthesised on a Ti (PA01) surface PuroA Carboxylated poly(styrene) Zero-length covalent E. coli (ATCC 108 microspheres immobilisation of free amines of 25922) AMP to PS-COOH using EDC/NHS
Peptide orientation can also be manipulated by using site-specific immobilisation of the
AMP. The AMPs maybe be immobilised at either their C-terminal, N-terminal, and/or N-side
chain to give rise to various peptide orientations, resulting in different antimicrobial profiles.
Gabriel, et al. 123 used site-specific immobilisation to compare the antimicrobial activities of N-
terminal and N-side chain type conjugations, and it was found that only AMPs immobilised at their
N-terminal retained antimicrobial activity. It was concluded that N-terminal immobilisation 32 allowed for parallel orientation of the AMP helices permitting bacterial membrane interactions and pore formation. In a separate study however, Bagheri, et al. 21 demonstrated that N-terminal and
N-side chain type immobilisations had similar antimicrobial activities, suggesting that blockage of
ε-lysine residues had minimal impact on the overall AMP activity. The two studies highlight how antimicrobial profiles are specific to the systems investigated, and antimicrobial activity of immobilised AMPs involves a balance of a number of different factors, where it appears that AMP orientation has less influence on activity than immobilisation through long spacer molecules.
As our understanding of the mechanisms of actions of AMPs grow, it has been possible to synthesise new and novel AMPs that exhibit superior antimicrobial activity against bacterial cells and also fungi. An example of this superior action is shown below in Table 6, where typical minimum inhibitory concentrations (MICs) of several AMPs can be compared, and shows that newly synthesised AMPs such as PuroA, P1 and W8 have MIC values ranging from 10-100 times lower than that of other AMPs commonly used in immobilisation investigations. Boden, et al. 108 has previously shown that zero-length immobilisation of PuroA to a colloidal crystal interface was active against E. coli, thus the use of more potent synthetic analogues (P1 and W8) may provide superior activity in an immobilised state.
Table 6. MIC ranges of selected AMPs
MIC range AMP Sequence Reference (µM)*
125 Melittin GIGAVLKVLTTGLPALISWIKRKRQQ-NH2 9-18 126 Magainin-II GIGKFLHSAKFGKAFVGEIMNS-NH2 40-50 127 Indolicidin ILPWKWPWWPWRR-NH2 2-16 22,108 PuroA FPVTRWWKWWKG-NH2 0.9-1.7
P1 RKRWWRWWKWWKR-NH2 0.2-0.9 Unpublished
W8 WRWWKWWK-NH2 0.85 Unpublished 23,90 *Average reported MIC values against susceptible Escherichia coli strains
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It is apparent that immobilised AMPs may well represent the next generation of antimicrobial coatings due to several advantageous properties including high potency and low cytotoxicity. Several studies summarised in the section above have elucidated to general guidelines for the effective immobilisation of AMPs, and thus will be utilised within in this research for tethering selected AMPs to non-fouling polymeric films for the development of active antimicrobial coatings.
2.5 Nano-and microstructured surfaces
2.5.1 Bioinspired structuring
It is well established that proteins and bacteria respond to changes in chemistry and topography at a biointerface.34,128 As mentioned earlier, naturally occurring surfaces (i.e. shark skin, insect wings, lotus leaves etc.) have evolved over millions of years to provide protection against fouling materials. Because of this, a main focus in materials science has been devoted to replicating and adapting these naturally occurring materials to produce artificial analogues with well-defined two- and three-dimensional (3D) structures that extend over micro-and macroscopic length scales, and can be produced with high precision at low costs and short manufacturing times.28,129,130
In many cases these natural surfaces have hierarchical surface features with superhydrophobic properties,129,131 where it has been proposed that the layer of air trapped within the dual-scale surface features minimises the contact area available to attaching bacteria.131 For example, when mimicking the surface features of the lotus (Nelumbo nucifera) leaf, Fadeeva, et al. 132 observed that no P. aeruginosa cells were able to attach to the hierarchical surface features.
However, it was seen that S. aureus cells were able to successfully colonise the same surfaces suggesting that the attachment profiles of bacteria to superhydrophobic surfaces is not as simple
34 as once thought. In a separate study Puckett, et al. 133 investigated the differences in bacterial attachment to nanorough, nanotextured, nanotubular, and unmodified Ti surfaces. Interestingly it was found that bacteria preferentially colonised nanotubular and nanotextured surfaces compared to nanorough surfaces. However the proportion of viable bacteria on tubular and textured surfaces was significantly less than that on unmodified and nanorough surfaces. These observations suggest that the size, shape and organisation of nanofeatures may affect bacterial attachment profiles in unique and controllable ways.
Traditionally, the fabrication of materials with structural hierarchies of nano/micrometer dimensions has been performed by a ‘top-down’ method where externally controlled instruments are used to direct the formation of small-scale structures from larger materials. Techniques that have been adopted for such purposes include photolithography, micro-contact printing, electron- beam lithography, and dip-pen nanolithography all which have been described previously in a number of reviews.134-137 Converse to the physical ‘top-down’ approach, the chemically inspired
‘bottom-up’ techniques begin at the molecular level – where larger entities are assembled from smaller building blocks.138,139 Similarly to the top-down approach, many fabrication methods are available using ‘bottom-up’ processes including atomic layer deposition,140,141 sol-gel,142 and molecular assembly techniques.143,144 While there are many techniques available for the fabrication of surfaces with structural hierarchies, many of these techniques involves the use of expensive equipment, cleanrooms, and have long processing times. Additionally it is also apparent that there is a requirement for a fabrication method to produce nano- and mircostrucutred surfaces that are easily controllable with no specialised equipment.
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2.5.2 Binary colloidal crystals
2.5.2.1 Introduction to binary colloidal crystals
Formation of complex structures and chemical patterns through the self-assembly of smaller entities is a phenomena that is quite ubiquitous in nature – from the self-assembly of lipid bilayers in cellular membranes to the self-assembly of collagen into macroscopic tissue networks.145 Formation of these hierarchical structures and the interesting physiochemical properties they exhibit has offered a wide-range of exciting research directions in materials and interface science. The length scales were ‘top-down’ and ‘bottom-up’ strategies converge is referred to as the colloidal domain, and its constituents are defined only by their dimensions (nm-
µm).146 Colloidal particles not only possess length scales where molecular and macroscopic systems converge, but they also exhibit interesting behaviours and characteristics due to inter- particle interactions – which govern the distinct features seen in the phenomena of colloidal self- assembly. Such self-assembly of particles into crystal structures offers a versatile approach to fabricating materials with various structures and topographies. In general, mono-component colloidal suspensions generate crystal structures of limited complexity,147 however, when colloidal crystals are formed from a multi-component suspension (i.e. binary and ternary) - a far more extensive phase behaviour is observed.148,149 This allows for greater hierarchical ordering and structural diversity to include face-centered cubic (fcc), body-centered cubic (bcc), and hexagonally close-packed (hcp) packing arrangements. This also opens the door to a vast number of potential applications in materials and interface science.
Being first observed in Brazilian opals,150 binary colloidal crystals (BCCs) consist of both large and small colloidal particles that alternately self-assemble into 2D or 3D crystal lattices
(Figure 5).151,152 With tuneable functionality and precise control over particle stiochiometries,
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BCCs provide a relatively simple approach to fabricating a large variety of novel materials with rich phase behaviours, defined structural hierarchies and chemical compositions.31,32,153,154
Materials with such structural hierarchy are ideal for the development of 3D crystals with interesting optical properties,155 and also for fabricating a range of ordered 2D and 3D structures with tunable surface chemistry, porosity, and topographies.33,149,156,157 These types of tunable structures can be ideal in a number of applications and assist in the production of novel photonic,158 biomedical,33,159-161 electrical, and magnetic devices.162
Figure 5. Representation of a) Brazilian opal, b) different stoichiometries of BCCs, and c) scanning electron microscopy images of BCCs prepared with silica and PMMA colloidal particles. Images were adapted from userblogs.ganoksin.com, , Wang, et al. 33, and Wang, et al. 161
Colloidal self-assembly is a fast but complex process that involves the balance of internal and external forces that directs crystal growth toward an equilibrium structure. Even subtle changes in the properties of a binary mixture, such as; 1) the type of material, 2) particle shape, 3) particle dimensions, and 4) dispersion medium can result in significant changes to the surface chemistry and topographies of the resulting crystal structure. Additionally, the choice of assembly method to fabricate BCCs (i.e. Langmuir-Blodgett based or evaporation based) can also affect resulting crystal structures. Such facile manipulation of the final crystal properties provides many advantages including low cost and rapid fabrication when compared to other ‘top-down’ based approaches.30,163 Furthermore, the requirement for long-range (cm2) structures is currently not
37 feasible with common lithographic techniques, however can be achieved using colloidal self- assembly.31,164
Considering the rapid fabrication times and the vast array of topographies and surface chemistries able to be presented by BCCs, the sections below discusses several aspects of BCCs in respect to the development of functional material with structural hierarchies. Additionally, fundamental concepts of colloidal properties (size, shape, chemistry etc.), developments in fabrication techniques, mechanisms of BCC growth, and applications of BCCs are also discussed below.
2.5.2.2 Colloidal materials
According to the International Union of Pure and Applied Chemistry (IUPAC) ‘Gold
Book’; a colloidal suspension is considered to be “molecules or poly-molecular particles dispersed in a medium having at least a dimension in one direction roughly between 1nm and 1µm, or that discontinuities within a system are found at distances of that order”.146 This broad definition implies that any object with size dimensions between several nanometres to several micrometres may be classified as a colloidal with the potential to subsequently be investigated for generating colloidal crystals by self-assembly.
The building blocks utilised for the assembly of BCCs can exhibit a wide-range of elemental compositions, and be fabricated through various ‘top-down’ or ‘bottom-up’ approaches.165 The ‘top-down’ approach is more appropriate for the production of larger inorganic particles with homogeneous composition; whereas the ‘bottom-up’ technique can be used to easily fabricate particles with sub-micrometre dimensions and variable composition.165-169 For the fabrication of BCCs the vast majority of research has focussed on colloids made of polymeric and silica-based materials, with quantum dots (QDs),170 and anisotropic particles167,171-173 also
38 receiving attention in recent years. The types of materials utilised when producing and investigating BCCs will ultimately contribute to the final properties and characteristics of the resulting assembly, and must therefore be carefully selected when considering downstream applications.
For the developments and discovery of more efficient colloidal crystal fabrication techniques to decrease costs and fabrication times, the most common materials for single and binary colloidal systems are polymer latex and silica-based particles. This is due to their relatively low cost, mono-dispersity, and stability,147,149,152,174 allowing researchers to reduce the time and costs associated with producing long-range ordered structures with controllable surface patterns.31,149,152 When considering silica based systems, the well understood surface chemistry allows for surface modification to impart different characteristics including hydrophobicity,175 hydrophilicity,176 porosity,177 and also fluorescence.178,179
For other applications including, but not limited to; biosensors, chromatography columns and photonic devices, alternate materials are utilised providing appropriate properties that are suitable for a particular application. Functionalised polymeric particles,32,33,156,180 along with semiconducting QDs,181 are among the different types of materials that are used for such investigations. For example, Singh, et al. 156 used carboxyl-, amino- and sulfo-functionalised polystyrene (PS) particles to immobilise proteins and antibodies with a potential for biosensing.
The authors showed that highly-ordered protein/antibody patterns could be obtained and also tuned depending on the size ratios of the large and small particles (See Table 7). Similarly, Zhao, et al.
180 used amino-functionalised silica colloidal crystal beads to successfully immobilise immunoglobulin G (IgG) probes for the detection of IgG antibodies in serum.
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The immobilisation of DNA at a particles’ surface can also lend itself to the fabrication of colloidal crystals in a controlled and programmable manner. Due to the complementary nature of
DNA, hybridisation of complementary strands can occur that drive the construction of larger scale materials.182-184 For example, Ben Zion, et al. 184 were able to create 3D microstructures of PS and
PMA colloidal particles – where the assembly was directed through an immobilised DNA origami
L-belt complex. It was demonstrated that the obtained structures exhibited a highly specific geometry that allowed fine control over particle position and chirality. Furthermore, in applications where non-compact structures are required such as photonic crystals (which operate in the visible- light range), the length of the DNA strands can control the inter-particle length upon self-assembly
– which can be ideal for the production of photonic devices.182,185
In regards to semiconducting materials of particles that are within the dimension of 1-
100nm (i.e. QDs), so called quantum confinement effects can occur, resulting in interesting optoelectric properties.170,186 Because of this phenomena, considerable research has investigated the use of lead telluride (PbTe),187,188 cadmium sulphide (CdS),189 and zinc selenide (ZnSe)190 QDs for applications in imaging and photonic bandgap (PBG) devices.170,191 To date, it appears that studies have focussed on monocomponent systems of theses QDs and also the effects of the addition of metal dopant atoms (e.g. Mn, Cu, Zn, Al), which can introduce conductivity to a poorly conductive material, and also boarded the range of luminescence properties.190 The use of binary
QD systems is relatively recent and can be used to impart optical and electrical properties to QD species within the nanocrystal. For example, CdSe and ZnS QDs imbedded in a PMMA film tended to aggregate within the polymer – initiating radiationless energy transfer due to the close- packing of the QDs.192 This phenomena could play an important role in the development of biological sensors where detection relies on radiationless energy transfer from QDs to an acceptor dye molecule. 40
Metal particles of Fe,193 Pt,194 Ti,193 Ag,195 and Au196 are also used in a variety of binary colloidal self-assembly investigations and have been discussed in more detail in a number of reviews.197,198 These structures offer many potential application in the fields of nanocatalysis,199 biomedicine,200,201 nanoelectronics,202 and biological sensors.192 Furthermore, the high valency of metal particles provides the ability to functionalise particle surfaces with a variety of ligands.200,203
2.5.2.3 Composition of binary colloidal crystals
BCCs can be produced from a mixture of large and small particles with the same composition (i.e. made from the same material), or from a mixture of particles with different compositions. For mono-material systems (where both small and large particles are made from the same material) Kitaev and Ozin 149 showed that regular arrangements were able to be produced using a binary suspension of PS particles possessing zeta potentials of -80mV and -52mV for large and small particles, respectively. Similarly, Wang, et al. 33 were able to fabricate BCC monolayers over a wide range of size ratios (small-to-large: 0.012-0.25) possessing hcp arrangements using negatively charged carboxylated0PS particles. In both cases, the negative charge, and the relatively large zeta potentials (ζ) presented by both of the colloidal particles aided in the stability of the colloidal dispersion by preventing aggregation from electrostatic attraction. This highlights one potential advantage to using mono-material systems; that as long as the size ratio of the particles is within an optimal range (dependant on the assembly technique), the surface charge on the particles will be sufficient to prevent particle aggregation. On the other hand, when materials with distinctly different chemical and physical properties, or particles with a relatively stable surface charge (-30 ≤ ζ ≤ 30 mV) are used, aggregation may occur and result in disordered structures.
Therefore, before any BCC self-assembly is performed, it is important that the particles of interest are first characterised to ensure unwanted particle aggregation is avoided.
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Conversely, particles of differing composition can be utilised for BCC fabrication. This provides the advantage of being able to selectively functionalise a particles’ surface by taking advantage of different surface chemistries that may be present.156,157 However, self-assembly becomes more complex in this case due to the diverse properties of the two particles. Wang, et al.
33 used a binary system composed of silica and polymer (e.g. PS or PMMA) for 2D monolayer self-assembly. The results suggested that besides the same surface charge particle density (i.e. gravity) dominated the self-assembly process for this particle combination. Silica particles were too heavy to interact with polymeric particles in suspension and settled on the surface within a short period of time. It was shown that capillary forces pushed all silica particles together on the surface upon solvent evaporation, and the small polymeric particles were forced to void space between silica particles to form BCC monolayers. Based on this phenomenon, BCCs will not form using a combination of large polymeric particles and small silica particles due to the large density difference between the two materials. In another investigation, not only were Singh, et al. 156 successful in the immobilisation of a number of different proteins to BCC layers including lysozyme (Lyz) and bovine serum albumin (BSA), but the immobilisation was selective to a specific particle type. This enables the effects of immobilisation density to be analysed easily by manipulation of the volume fraction of the functionalised particles. Considering the available research, it is evident that the type of material chosen for the production of BCCs is of upmost importance to achieve structures that have ordered arrangements upon self-assembly, and also provides appropriate properties for their desired applications.
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Table 7. Summary of assembly methods used to fabricate BCCs and characteristics of resulting crystal structures
Fabrication Particle(s) Surface area Size ratio Ref.
Sequential deposition Lbl growth on a vertically deposited Silica & PS 1-2mm per day 0.48-0.54 152 substrate Step-wise LbL spin-coating Silica & PS Approx. 1cm2 0.25-0.58 154 Step-wise LbL in a sandwich glass PS Several cm2 0.07-0.09 204 cell LbL deposition on a 45° incline PS-co-PMMA Approx. 2cm2 0.5 205 2 156 Confined-area evaporation-induced Silica, PS, PS-NH2, & PS-SO4 Approx. 1cm 0.10-0.90 LbL assembly Step-wise confined convective PS Several cm2 0.30-0.65 147 assembly LbL deposition at the air-water PS 10cm2 206 interface Lbl growth on a vertically deposited Silica & PS - 0.28-0.68 174 substrate Template-assisted electric-field- PS 5cm2 0.10-0.91 207 induced assembly Co-deposition
Single-step EtOH-assisted assembly PS 1cm per 4-7hr 0.110-0.227 149 at the air-EtOH-glass interface Single-step confined convective Silica & PS Several cm2 0.213-0.367 158 assembly Single-step horizontal deposition PS Approx. 2cm2 0.154-0.195 208 Single-step assembly at the air-water PS ≥2cm2 0.147-0.194 209 interface 2 157 Single-step evaporation induced Silica, PS-NH2, PS-SO4, & PS- Approx. 1cm 0.10-0.50 assembly COOH Evaporation-induced confined-area Functionalised PS and Silica Several cm2 0.10-0.55 176 assembly particles Single-step assembly at the air-water PS-COOH Limited by size of 0.19-0.4 164 interface in a Langmuir trough Langmuir trough Single-step evaporation induced Functionalised PS and Silica Several cm2 0.064-0.50 156 confined area assembly particles EtOH-assisted self-assembly at the PS > 10cm2 0.175-0.250 210 air-water interface Abbreviations: PS: polystyrene; PS-co-PMMA: polystyrene/poly(methyl methacrylate) copolymer; PS-NH2: aminated polystyrene; PS-COOH: carboxylated polystyrene; LbL: layer by layer
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2.5.2.5 Colloidal shapes
While BCCs produced from spherical particles have received the greatest attention, understanding of these shape isotropic systems will allow production of colloidal crystals using non-spherical colloids such as discs,171 rods,171 tiles173 and even quasi-crystalline211 structures
(Figure 6). Furthermore, due to the diversities seen in non-spherical colloidal arrangements, and strong polarisation effects resulting in birefringence at long wavelengths – these novel arrangements appear to be ideal for photonic applications including optical switches, filters, and low-threshold lasers.167
Figure 6. Summary of different types of particle shapes used in the self-assembly of colloidal crystals
To date, research into binary systems of shape anisotropic particles has been less common, which appears to be due to the difficulty to form well-ordered structures because of the requirements for more complex positional and orientational ordering when compared to the self- assembly of spherical particles. A Relative early study has shown that millimetre-scale
44 poly(dimethylsiloxane) (PDMS) plates with concave hydrophobic faces could self-assemble around both pentagonal and circular templates (Figure 7a-b).212 The authors demonstrated that cyclic hexameric structurres could be formed reproducibly when the diameter ratio between the
PDMS plate and the template was between 0.5 and 0.96. The assembly was driven by capillary interactions induced by the use of immiscible solvents (perfluorodecane (PFD) and water), where the template directed the spatial orientation to form the structures observed. In addition, the production of multi-component anisotropic crystals using 2D LaF3 nanodiscs and 1D CdSe/CdS nanorods through a modified liquid interface assembly method has been demonstrated for form
171 ordered arrangements (Figure 7c-f). The authors state that LS, LS2, and LS6 structures could be produced depending on the size ratios and concentrations of the anisotropic colloids, where Lx and
Sy represent the number ratio of large and small colloids, respectively, thus offering the potential to design and fabricate novel materials. It is noted that the use of two immiscible solvents
(diethylene glycol and hexane) was again an important factor to sequester and orientate the
45 colloidal particles. This drove crystal formation at the air-liquid interface upon solvent evaporation, allowing the formation of large-scale structures.
Investigations into the self-assembly of mono-component shape anisotropic systems are much more prominent due to the more predictable interparticle interactions, and a range of shape anisotropic colloids have been investigated including rods, ellipsoids, discs and tiles. In a more recent study, the self- assembly of hexagonal and circular microtiles gave rise to hcp packing arrangements, whereas square-shaped microtiles were observed to assemble into cubic close-
Figure 7. Production of BCCs using various shape anisotropic particles based on convective assembly methods. Templated self-assembly of six PDMS plates around a) pentameric, and b) circular templates. Representative TEM images of self-assembled lattices consisting of nanodiscs and nanorods with an AB-type binary lattice self- assembled from 13.2nm LaF3 nanodiscs and CdSe/CdS nanorods c), shown with corresponding structural models d), and AB2-type binary lattice assembled from
22.3nm LaF3 nanodiscs and CdSe/CdS nanorods e), with corresponding structural model f).
packed (ccp) orientations (Figure 8i-iii).173 In this investigation, Wang, et al. 173 incorporated the use of a highly volatile sub-phase (i.e. chloroform) and co-solvent (i.e. ethanol) to control the
46 evaporation process. Conversely to previous investigations, this allowed the formation of crystals directly on to a solid support which is ideal form many materials science applications. Using this approach the authors were able to assemble microtiles of various shapes (i.e. circles, squares, and hexagons) and sizes over large areas, where the shape, aspect ratio, and particle concentration were all determined to be important factors in forming large close-packed assemblies.
Figure 8. Representative SEM images (left) and Fourier transformations of dried monocrystals (right) showing assemblies of circular, hexagonal, and square microtiles with an aspect ratio of 0.2 (i-iii), and 0.9 (iv-vi).
Furthermore, since these particles are anisotropic there is also a requirement the orientation of the colloids to facilitate particular packing arrangements and minimise crystal defects. Several approaches have been utilised to achieve this based upon ‘post-crystallisation’ techniques including high energy ion irradiation213 and uniaxial mechanical stretching.214 However, these types of post-crystallisation processes may cause deformations of possibility destruction of the lattice itself. To circumvent these challenges, the use of magnetically active building blocks have been used to allow for the alignment of the colloids along their longitudinal axis parallel to an applied magnetic field.215,216 For magnetically inactive particles however, the use of a magnetically active medium such as a ferrofluid may be used to provide each colloid with a virtual magnetic moment to impart orientation to the system.217,218
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2.5.2.6 Mechanisms of binary colloidal crystal growth
Compared to their unitary counterparts the mechanisms of BCC growth can be considered to be relatively more complex; requiring greater control over the size ratio and surface charge of the particles. However, by taking advantage of differences in chemical and physical properties between particle combinations, fabrication of binary and even multi-component colloidal crystals can be made more controllable and predictable.31 The fundamental process for producing BCCs is determined by the complex phenomena of colloidal self-assembly. This process involves the entropically driven self-assembly of colloidal particles into crystalline phase with a packing state that is directly influenced by the volume fraction and size ratio of the large and small particles.152
The forces that act upon colloidal particles during the self-assembly process involves the balance of attractive, repulsive, and external forces (Figure 9) that enable the formation of complex 2D and/or 3D structures. For example, using particle combinations of differing densities enables sequential deposition where denser particles sediment first and the lighter particles are allowed to occupy the interstitial spaces between arrangements of large particles. Additionally, differences in magnetic and electrical properties of particles in a binary system also offers more control over crystal growth; enabling the fabrication of far more complex structures that that cannot be achieved with unitary systems.207,219,220 The following section will outline the different forces that influence colloidal self-assembly and discuss how they can be used to control crystal growth.
2.5.2.6.1 Electrostatic forces
Electrostatic interactions of colloidal particles encompasses the attractive and repulsive forces that arise to protonated and deprotonated functional groups that are present on a colloids’ surface. In most circumstances, these forces are repulsive and are critical in the formation of ordered colloidal crystals; as the lack of these repulsive characteristics often results in disordered structures and/or lattice defects.221 In binary suspensions of both large and small particles, fine 48 control of the surface charge on both particles is often quite difficult due to differing degrees of hydration and ionic shielding. Oppositely charged particles will be attracted to one another and rapid aggregation will occur unless particles are stabilised by surfactants or other compounds, resulting in steric stabilisation of the colloidal suspension.164,206
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Figure 9. Summary of the various interactions and forces encountered during colloidal self-assembly. Attractive forces; a) immersion capillary forces, b) electrostatic attraction, c) flotation capillary forces, d) depletion forces, e) Van der Waals forces. Repulsive forces; f) electrostatic repulsion, g) steric repulsion. External forces h) Brownian motion, i) gravitational forces, j) magnetic fields, k) electric fields, and l) forced convection.
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Electrostatic interactions can also be manipulated and tuned by adjusting the pH or adding salts to the colloidal suspension. For example, well-ordered close-packed structures of amphoteric
PS spheres were only able to be produced in basic condition to impart sufficient repulsive character.222 Similarly, size ratios of BCCs produced by assembly at the air-water interface can be extended to 0.40 when the pH of the suspension is increased to 9.0.164 This increase in the repulsive electrostatic forces between the particles whilst minimising the attractive capillary forces allows more time for the particles to find the most energetically favourable position. For confined-area assemblies of BCCs, Wang, et al. 33 demonstrated that if the zeta potential of the particles is less than -30mV (i.e. a stable and uniform surface charge), long-range ordered crystals can be produced in H2O over size ratios of 0.005 to 0.27 without the addition of salts or manipulation of pH.
Cooperative electrostatic self-assembly of anionic tobacco mosaic virus (TMV) nanorods with cationic gold nanoparticles (AuNPs) has also been shown to produce highly ordered super lattice structures (Figure 10).223 It was found that ordered structures were likely to form under all nAuNP/nTMV ratios suggesting that the strong electrostatic interactions directs superlattice formation after nucleation. Moreover, interparticle distance between AuNPs can be controlled by altering ionic strength on the suspension (Fig 10e).
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Figure 10. Visual representation of the electrostatically driven self-assembly of AuNPs with TMV rods showing how the assembly proceeds in a zipper-like fashion a). TEM images of AuNP rows forming between two b) and four TMV rods c), and also a well-ordered AuNP-TMV supperlattice d). Upon decreasing ionic strength, AuNPS can be concentrated within the TVM rods allowing a smaller inter- particle distance to be achieved e). Scale bars: 200nm (b, c), 50nm (d). Images are adapted from Lin, et al. 223
2.5.2.6.2 Van der Waals forces
If the colloidal particles area able to approach one another closely, and overcome the repulsive electrostatic forces, the attractive van der Waals forces are able to bring particles close together and induce long-range assembly of monolayers with high quality. The magnitude and range of this force depends on both the size and shape of the colloidal particle. It has been shown however, that by matching the density and refractive index of the particles and the dispersion medium – the influence of van der Waals forces can be eliminated to tune particle-particle interaction of oppositely charged colloids.224,225
2.5.2.6.3 Capillary forces
One of the critical forces that governs the crystallisation of colloidal particles in evaporation-based assemblies in the immersion capillary force – which acts to push particles together as the height of the liquid recedes below the diameter of the colloidal particles. As solvent evaporates from the drying front of the assembly, a convective flow is created which pulls particles from the bulk to the growing assembly.226 Smaller, less dense particles will be influenced by this immersion capillary force significantly more than larger, denser particles as gravitational forces
52 affects them less. In binary systems of colloidal particles this phenomenon can be exploited using particles of significantly different densities, as the larger particles will sediment first and allow the smaller particles to freely diffuse across the preformed close-packed layer to find their lowest energy configuration.33
If particles are floating at the air-liquid interface, larger particles usually greater than 1µm in diameter will cause sufficient deformation of the liquid interface due to gravitational (FG) and
227 buoyancy (FB) forces, giving rise to flotation capillary forces and pulling the particles together.
By adding volatile solvents such ads ethanol to the suspension, evaporation rates and therefore convective forces can be tuned for faster crystallisation times,209,210 however, sometimes at the cost of BCC quality.226 For binary systems of colloidal particles, the flotation capillary force can be quite complex due to differing particle densities, surface wettability, and particle size. As such, for evaporation induced assemblies where capillary forces are dominant, it is imperative that particle-particle/particle-surface interactions are minimised to allow for particle spreading and energetically favourable configurations. This can be achieved by the use of negatively charged substrates, and colloids with negatively charged functional groups such as carboxylic acids,31,156 and sulphates33 to provide sufficient repulsive character between the particles and surface
2.5.2.6.4 Depletion forces
Attractive depletion forces also influence the aggregation of colloidal particles, particularly in the formation of BCC structures where the exclusion of small particles between the excluded volumes of large particles can facilitate BCC growth and aggregation of large particles. When the interactions between large colloids and a depletant are below a critical value (approx. 0.3kT), the adsorption of depletants on to a particle surface becomes unfavourable.228 Under such conditions, there is an exclusion volume around the large particles which overlaps as the colloids approach
53 one another. This increases the total free volume available to the depletant or other small particles
– causing their expulsion from the inter-particle region (See Fig. 9d). The resulting concentration gradient gives rise to an anisotropic osmotic pressure leading to weak, reversible particle aggregation. In binary systems consisting of both large and small particles, this depletion force plays a critical role in the self-assembly of large particles and reaches a maximum value when the
229 distance between the large particles approaches two time the radius of the small particles (≈2Rs).
Depletion forces have also been used to facilitate the self-assembly of shape anisotropic colloids of various geometries.167,171,172,230 For example, Rossi, et al. 172 demonstrated that hollow cubic particles of silica could from cubic crystal structures only in the presence of a non-adsorbing polymer acting as a depletant. These depletion forces were able to be finely tuned by using a temperature sensitive polymer; poly(N-isopropylacrylamide) (PNIPAAm) allowing nucleation to begin within minutes. Similarly, Ashton, et al. 230 showed that colloidal polymerisation of indented colloids could be induced by controlling the depletion interactions by manipulating the volume
r fraction of the depletant within the system (η s). It was shown that polymerisation occurs over quite
r r a narrow range of η s (0.1-0.15) for all indentation sizes, where at the largest value of η s almost all colloids could self-assemble into a long-range ordered network.
2.5.2.6.5 Gravitational forces
Arguably one of the most important forces that define crystal structure in a binary colloidal system are the gravitational forces that lead to sedimentation (Figure 9i). Gravitational forces exerted on a colloidal suspension will increase with increasing particle size, and also with density differences with the dispersion medium.231 For low density particles, gravitational forces has considerably less effect leading to increased sedimentation times, however, the use of a centrifuge can mitigate this effect – sometimes however at the cost of crystal quality.232,233 For binary systems
54 using a co-deposition process it is also important to consider the respective densities of the two colloids as larger particles are needed to sediment first to form a close-packed layer where the smaller particles can be deposited within the interstitial spaces.
2.5.2.6.6 External forces
External forces also allows for the directed self-assembly of particles into structural organisations beyond the energetically favourable structures seen in minimum free energy systems. These applied forces may be electric or magnetic fields which can induce particle interactions and colloidal assembly. While the exact mechanisms of these forces have been described elsewhere for unitary systems,234,235 this section will describe how these forces can be applied to binary colloidal systems.
Most colloidal particles will polarise in an electric field because their dielectric properties are mismatched from the surrounding medium. Because of this, the directed self-assembly of
BCCs under the influence of an applied electric field has offered an effective way to produce an array of crystal structures other than those seen by traditional LbL methods.162 Usually a template layer is first formed at the substrate surface to direct the assembly of the smaller particles. This may be achieved using a monolayer of large particles produced by convective assembly methods,207 or, deposition through electrophoretic methods.236 Once the template layer is established, small particles are deposited within the interstitial spaces of the large particles directed by an applied electric current. As the forces required to induce deposition only need to overcome
Brownian motion, the field strengths that are typically used are quite modest; being in the order of
1.0x104V/m. Dziomkina, et al. 236 also demonstrated that by withdrawing the electrode from the colloidal suspension during the deposition process, non-close-packed template layers could be obtained - offering a novel route to generate unconventional BCC structures.
55
The directed self-assembly of colloids under an applied magnetic field has also been investigated and offers special applications not only in the assembly of spherical particles,220,237 but also shape anisotropic colloids.216 For magnetically active colloids such as hematite, the application of an external magnetic field can direct particle orientation in space relative to the applied field. This has been used to produce symmetrical 2D and 3D crystals using asymmetric colloids not only in aqueous media,215,216 but also in polymeric matrices.162 Several new structures have been attained by the application of a magnetic field during the assembly process, for example,
Lu, et al. 216 demonstrated the formation of crystal structures from ellipsoidal and ‘peanut-shaped’ hematite colloidal particles. It was proposed that once the crystal structure is formed within the magnetic field, the medium can be removed to condense the lattice into a closely-packed 3D colloidal crystal. However, to date, the main focus of these investigations has been on mono- component rather than binary systems of colloidal particles. It is hypothesised that the same principles and mechanisms can still be applied to binary systems, considering the colloids chosen are magnetically active, or a magnetic medium such as a ferrofluid is used to impart each colloidal with a virtual magnetic moment.216,238,239
The application of wave fields triggered by ultrasound transducers aimed at nanoparticle assembly to generate 2D user specific patterns has been previously reported by Greenhall, et al.
240 . In this investigation, a theoretical calculation was used to determine the ultrasound transducer parameters needed to induce the formation of specific patterns. This theoretical computational method enables the authors to overcome optimisation issues to develop a relationship between ultrasound wave fields and the forces exerted on the nanoparticles. This kind of ultrasound directed colloidal self-assembly approach may have potential to be applied in the fabrication of material with nanoscale patterns. In a later article, the same research group demonstrated that 3D nanoparticle patterns in a fluid medium could be obtained by employing ultrasound directed 56 colloidal self-assembly methods.241 Ultrasound transducers were used to define the fluid medium’s boundary where the colloids dispersed within the liquid were driven into well-ordered patterns by the acoustic radiation forces caused by the ultrasound waves. It was hypothesised that the formation of ordered structures were formed due to the inability of particles to interact with one another due to the interference coming from larger wavelengths.
2.5.2.7 Methods for binary colloidal self-assembly
The aim of many BCC fabrication methods are to produce BCCs rapidly and inexpensively over large areas, with precise and defined surface and internal topographies. However, one of the main factors that determines the overall homogeneity and surface coverage of BCCs is the method chosen for their fabrication. Because of this, considerable research has involved employing current knowledge of colloidal self-assembly to develop alternative methods and approaches to producing reproducible BCC patterns. These techniques include LbL deposition,31,174 template-assisted assembly,162,242 electric field induced assembly,207,236 confined convective assembly,31,33 spin- coating,154 and horizontal and vertical deposition methods.174,243,244 These above strategies are based upon as sequential deposition methods; where large and small particles are sequentially deposited on the surface to build 2D and 3D hierarchical structures. Alternatively, fabrication can be carried out using a co-deposition method; where the two types of particles are assembled in- situ from a binary suspension of large and small particles. Table 7 above summarises several investigations using both sequential and co-deposition methods to fabricate BCCs, and also highlights key aspects such as particle type, size ratio and surface area obtained.
2.5.2.7.1 Sequential deposition
The fabrication of BCCs using LbL techniques is based upon a sequential deposition method. This uses an existing layer of large particles as a template layer for the subsequent
57 deposition, where the deposition and arrangement of the small particles is influenced by electrostatic, capillary, and entropic forces which have been discussed in an earlier section.
Because the underlying layer of large particles acts as a template for the deposition of small particles, this technique is often referred to as template-assisted LbL deposition. Early attempts to fabricate BCCs using LbL methods involved an evaporation induced vertical deposition method where a 2D crystal comprised of large silica particles (RL = 203nm) was used as a template onto which smaller PS or silica particles were deposited.152 During this investigation, Velikov, et al. 152 reported the growth of 2D and 3D colloidal crystals over several mm2 on glass substrates that possessed LS, LS2, and LS3 packing arrangements depending on the volume fraction and size ratios of the particles. It was found that as the volume of the the small particles decreased, less of the low-lying sites were occupied – resulting in the various pacing arrangements observed. Growth of
BCC under these conditions proceeded relatively slow (1-2mm2 per day), largely due to the slow evaporation rate of the solvent, and only a narrow range of size ratios were investigated; limiting the number of observed packing arrangements and structures.
To overcome the problem of slow solvent evaporation, Wang and Möhwald 154 reported an
LbL technique based on a step-wise spin coating method where layers of hexagonally close-packed large and small particles were formed by consecutive spin-coating onto the underlying layer.
Under these condition, BCC were able to be produced much faster than previously reported
(several minutes). While BCC structures with a broad range of size ratios were able to be achieved
(0.25-0.58), only LS2 and LS3 packing arrangements were observed and high volume fractions were used. This can limit the number of potential downstream applications, and increase costs of production. Spin coating methods have also been used in other single colloidal crystal (SCC) and
BCC investigations,208,245 and can provide some advantages compared to other crystallisation methods such as rapid fabrication times, and the ability to facilitate colloidal deposition within the 58 confined of open and continuous channels.208 However, as spin coating is a non-equilibrium process, it is difficult to achieve long-range ordering of the crystal structures and defects are usually present.246
For sequential deposition methods such as LbL, solvent evaporation is a critical parameter that must be taken into account to achieve long-range ordered crystals – as rapid evaporation can lead to crystal defects and the appearance of randomly arranged structures.152,247 By using a controlled evaporation,31,174 or the inclusion of volatile solvents,149,210 crystallisation times can be decreased while maintaining homogeneous and defect-free crystals. By confining the evaporation and assembly, Kim, et al. 147 demonstrated that well-ordered BCC layers over a broad range of size ratios (0.30-0.65) can be produced by a sequential LbL vertical deposition technique. Using this method LS2 and LS3 packing arrangements can be obtained by adjusting the size ratio in addition to the volume fraction of the small particles. The authors state that by controlling the ‘lift-up’ rate of the substrate and the concentration of the colloidal suspension, high quality 2D or 3D BCCs can be fabricated over large areas. It is noted however, that BCCs could not be produced when size ratios of the large and small particles were <0.3. Circumventing the previous limitation of the LbL method such as slow fabrication times, size-ratio restrictions, and high volume fractions, Singh, et al. 32 utilised an adapted evaporation induces confined-area assembly (EICAA) method, where the assembly process is confined to the area within a rubber O-ring (Figure 11a). The authors observed the formation of BCCs over size ratios from 0.10 to 0.90 using very low volume fractions and a range of particle sizes and types. This method is not limited to SCC and BCC structures, but also for the first time, describes the formation of multicomponent colloidal crystals (MCCs) from colloidal suspension containing two or more particle types (Figure 11b-g).
59
Clearly there are multiple techniques available for the fabrication of BCCs based upon sequential LbL deposition methods. Template-assisted, spin-coating, and confined convective assembly have all been employed from such purposes, and all provide their own distinct advantages and limitations, however only until recently has the assembly of colloidal particles with size ratios of < 0.30 been achieved using a sequential deposition technique.32 While template- assisted LbL techniques can guide the formation of complex crystal structures, without external such as an applied electric current, packing arrangements appear to be limited to LS5 and less complex structures.
60
Figure 11. Schematic representation of crystal growth using an evaporation induced confined area assembly method a), and representative SEM imaged of prepared BCCs and MCCs using: b) 50nm carboxylated-Si/200nm aminated-PS/2µm carboxylated-PS, scale bar: 1µm, c) magnified region of image (b), scale bar: 500nm, d) 50nm carboxylated-Si/200nm aminated-PS/3.1µm sulfonated-PS, scale bar: 1µm, e) 50nm carboxylated-Si/200nm aminated-PS/1µm sulfonated-PS, scale bar: 500nm, f) 25nm carboxylated- Si/722nm PS/5µm Si, scale bar 10µm, g) magnified region of (f), scale bar: 2µm.
61
Instead of relying on capillary forces to create BCCs using the typical LbL method, under the application of an electric-field the driving force for colloidal assembly is the electrophoretic mobility of the particles, which allows them to overcome potential entropic and electrostatic forces that impede crystal growth. Using a template-assisted electric field induced assembly (TAEFIA) method (Fig. 12a), Huang, et al. 207 were able to produce BCCs and even MCCs with the addition of medium sized particles (M) possessing LS8, and LM2S15 packing arrangements (Fig. 12b-e).
Similar to the confined convective assembly approach described earlier, BCCs produced using the
TAEFIA method were formed from a broad range of size ratios (0.10-0.90). However, stiochiometries are difficult to control and it was noted that intrinsic structural defects were present where small spheres did not occupy the ideal sites (“lattice points”), resulting in incomplete deposition. Similar to all other studies, regardless of the current applied, it was found that the size ratio and volume fraction were the critical parameters that influenced the stoichiometry of the observed crystal structures.248 Fabrication of BCCs and MCCs using a sequential deposition method all utilise an existing template layer onto which smaller particles are deposited. Therefore, a critical factor in crystal quality is the overall homogeneity of the template layer comprised of an existing layer of large particle deposited using the techniques described above, or alternatively may be inherently structured material, or physically modified surfaces. 244
62
Figure 12. Schematic representation of the production of BCCs using a template-assisted electric field induced assembly (TAEFIA) a), and SEM images of the resulting BCC structures comprised of PS colloids with diamters and applied voltages of; 180nm/1800nm, 2V b), 400nm/1800nm, 2.2V c), 400nm/700nm, 2.2V d), and 640nm/700nm, 2.2V e).
2.5.2.7.2 Co-deposition
In the aim of developing more rapid and controllable fabrication methods, research has gravitated toward co-deposition techniques for the production of BCCs; where particles are crystallised from a solution containing two or more particle types. Compared to sequential deposition, co-deposition also employs convective techniques of both vertical149,249 and horizontal deposition,243 though many recent reports have utilised techniques that involve confined convective based assemblies31,33 (Fig. 13a), and even assemblies at the air-water164,209,210 (Fig.
13b), or oil-water interface250,251 (Fig. 13c).
Using a convective vertical deposition technique, Kitaev and Ozin 149 were able to fabricate
BCCs with long-range ordering from particle size ratios of 0.175-0.25 with PS spheres. It was shown that the addition of ethanol provided the transport of particles to the meniscus facilitating
63 the assembly of the particles. At size ratios > 0.30 however, the smaller spheres started to disturb the packing of the large particles resulting in the formation of totally disordered layers suggesting that the formation of close-packed arrays using this technique can only be achieved with size ratios
≤0.30. Additionally, due to the sedimentation of colloids, without external forces for stabilisation, the largest colloidal particles suitable for this type of vertical deposition are 500nm and 1100nm for PS and silica colloids, respectively. Using another type of vertical co-deposition technique, 252 demonstrated the presence of cracks and defects in BCC layers could be significantly reduced by introducing a tetraethyl orthosilicate (TEOS) sol during the self-assembly process. Horizontal co- deposition directly onto a glass substrate has also been investigated and suggests there are similar size ratio and diameter limitation when compared to vertical co-deposition; with the operational window for utilising the void constriction effect to form well-ordered BCCs is within the size ratios of 0.154-0.225.243
64
Figure 13. Schematic representation of the crystallisation methods used to fabricate BCCs based on co- deposition techniques utilising a) evaporation induced confined area assembly (EICAA), b) self-assembly at the air-water interface, and c) assembly at the oil-water interface
Co-deposition at the air-water or oil-water interface requires that the exact stoichiometries of the individual particles within the suspension are known, as this will ensure proper spreading and assembly of the binary mixture at the interface. This technique involves the assembly of colloidal particles at the boundary of the air-liquid or polar-nonpolar interface and may allow for transfer of the crystal layer to a variety of substrates. 164,209,210 By assembling the crystal layer at the air-water interface, Yu, et al. 209 were able to fabricate long-range (several cm2) ordered structures using PS spheres over size ratios of 0.147-0.194 requiring no elaborate equipment. It was found that large PS colloids assembled into ordered arrays, and at the same time smaller PS
65 colloids co-self-assembled into the interstitial spaces of the large particles. A critical addition to the assembly was the introduction of ethanol, which acted as a spreading agent to facilitate to formation of ordered crystals within minutes. Dai, et al. 210 also employed the air-water interface technique based on ethanol-assisted assembly; stating that the convection and therefore the spreading of the PS particles at the air-water interface was greatly enhanced by ethanol evaporation. Assembly at the air-water interface is a fast and effective technique long range BCCs that can be transferred to a variety of substrates, however there does appear to be a size ratio limitation to this technique unless external stabilisation is introduced. Additionally, it is also limited to low density polymer colloids as denser silica colloids will feel stronger gravitational forces and undergo sedimentation.
The EICAA method described previously has also been adapted to facilitate co-deposition and BCC formation from a binary mixture on to hydrophobic/hydrophilic substrates over size ratios from 0.06 to 0.30.226 Similar to other convective based assembly methods – meniscus pinning and solvent evaporation provide the convective forces necessary to facilitate particle spreading and crystal nucleation. Within this study, solvent type, humidity, and ionic strength were all investigated and results indicated that the highest quality BCCs were produced in humid, low ionic strength (≤1mM NaCl) environments. By confining the assembly process, Wang, et al. 33 were able to extend the size ratios down to 0.005, and observe a variety of novel BCC structures.
It was also shown that BCCs produced form Si/polymer particle combinations had larger surface coverages than polymer/polymer combinations. This was due to the denser silica particles settling on the surface at an early stage due to sedimentation, allowing smaller particles to fill the voids between large particles to from close-packed BCCS. For polymer/polymer combinations, BCC formation occurs at the air-water interface, resulting in lower surface coverage as crystal islands become separated during solvent evaporation. 66
2.5.2.8 Applications of binary colloidal crystal
Due to the diversity of structures produced from a wide arrange of particle compositions, shapes, and sizes – BCCs have the potential to be utilised in many applications including but not limited to; cell culture substrates,33 photonic devices,155,191 antimicrobial surfaces,108,253 biosensors,254 and porous membranes.255,256 While more detail about these different applications can be obtained from several reviews,27,227 this section will discuss how BCCs may be applied in a biomedical setting.
As stated previously, surface topography plays a crucial role in the attachment of bacteria to biotic and abiotic surfaces, and can be employed to prevent bacterial colinsation,25,65 however it is quite difficult to impart structural hierarchies to complex geometrical features such as those on medical devices. BCCs provide an opportunity to circumvent these challenges as they can be applied to many curved surfaces, and the colloidal particles can also be used for controllable and sit-specific attachment of a range of biomolecules. For example, Boden, et al. 108 selectively immobilised the AMP: PuroA, to 2µm carboxylated-PS particles and subsequently formed BCCs with 110nm PMMA particles using an evaporation-induced assembly method. It was shown that the AMP-modified BCCs had antimicrobial activity against E. coli when compared to control surfaces. In another study the attachment profile of P. aeruginosa to plasma polymer patterns created using colloidal crystals as a mask was assessed with and without immobilised PEG.257
Results showed that while that PEG-coated patterns were able to significantly reduce the number of attached bacteria, it was also shown that the surface coverage of bacteria could be manipulated depending on the size of the colloid used to generate the plasma polymer mask. These studies provide an indication of the versatility of BCCs to be used as a surface coating for controlling bacterial attachment using specific chemical patterns. Moreover, the range of sizes and functional groups available on commercially and laboratory-synthesised particles offers countless avenues to 67 investigate bacterial responses to complex biological patterns which is not feasible with similar nano- and microscale surface patterning techniques.
2.6 Conclusions Considering the information presented in the above literature review there is a clear need to develop antifouling coatings to reduce the nonspecific adsorption of biomolecules and also minimise the risk of infection due to bacterial colonisation of medical devices. Traditional methods to achieve this have involved the incorporation of biocidal substances such as silver ions and antibiotics. This can however cause unwanted biocompatibility problems and increase the risk of cells developing antibiotic resistance, therefore research has gravitated toward more novel methods to control the interactions that occur at a biointerface. Grafting of non-fouling polymers and the immobilisation of biomolecules have all been investigated, and the use of PEG is seen as the ‘gold standard’ of non-fouling polymers due to its accepted biocompatibility and choice of terminal functional groups. Current research also suggests that immobilised AMPs represent the next generation of antimicrobial coatings; being more potent than traditional antibiotics and having a low propensity for developing bacterial resistance. Moreover, the production of synthetic AMPs that are more active compared to their natural analogues has allowed the production of such coatings to be more cost-effective as less material is used. The importance of surface topography on bacterial attachment has also been established, and the use of BCCs provides a novel yet simple way to present a wide array of chemical patterns through the immobilisation of biomolecules and the use of different sized colloids. All things considered there is an opportunity to develop a multifunctional coating based in the immobilisation of non-fouling PEG chains and AMPs to prevent bacterial attachment and also kill any bacteria that successfully attach to the surface. By performing the immobilisation to not only planar substrates but also colloidal particles, the effect of manipulating surface topography can also be investigated. 68
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3 MATERIALS & METHODS
3.1 Materials
3.1.1 Surface materials and particles
Silicon wafers (100mm in diameter, p-Si<100>, 5.0-10.0 Ω cm-1) were purchased from
M.M.R.C Pty. Ltd., (Vic, Australia), and cut into 1cm2 pieces using a diamond pen prior to use.
Au and SiO2 coated sensor slides used in SPR investigations were purchased from Bionavis
(Tampere, Finland). Glass slides and cover slips used for BCC formation and microscopy were obtained from Livingstone International Pty. Ltd. (NSW, Australia). Monodisperse silica (2.01µm;
Si2) (9.8% w/v), carboxylated silica (2.01µm; SiC2) (Dry 100%), carboxylated polystyrene (2µm;
PSC2) (4% w/v), and poly(methyl methacrylate) (0.110µm; PMMA011) (4% w/v) colloidal particles were purchased from Bangs Labs (IN, USA) or Invitrogen (NY, USA).
3.1.2 Chemicals and AMPs
Synthetic peptides PuroA (FPVTWRWWKWWKG-NH2), P1 (RKRWWRWWKWWKR-
NH2), and W8 (WRWWKWWK-NH2) (99% purity; purified in trifluoroacetic acid with high- performance liquid chromatography (HPLC)) were all obtained from Mimotopes (Vic, Australia).
Water used in all experiments was purified using a Millipore system obtained from Merck
(Darmstadt, Germnay) with a resistivity of 18MΩcm-1. Phosphate buffered saline (PBS)
(cat#P4417), 2-(N-Morpholino)ethanesulfonic acid hemisodium salt (MES) buffer (cat#M0164), sodium chloride (NaCl) ≥99.0% (cat#S9888), potassium sulphate (K2SO4) ≥99.0% (cat#PO772), sodium cyanoborohydride (NaCNBH3) 95% (cat#156159), sodium hydroxide (NaOH) pellets
≥98% (cat#S8045), polystyrene (PS) average Mw ≈ 192,000Da (cat#430102), polyethyleneimine
(PEI) average Mw ≈ 25,000Da (branched) (cat#408727), toluene; anhydrous 99.8% (cat#244511),
101 methanol; anhydrous 99.8% (cat#322415), (3-aminopropyl)triethoxysilane (APTES) 99%
(cat#440140), (3-mercaptopropyl)trimethoxysilane (MPTS) 95% (cat#175617), N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) (cat#E6383), N- hydroxysuccinimide (NHS) ≥97% (cat#130672), 2,2-dimethoxy-2-phenyacetophenone (DPAP)
99% (cat#196118), L-Cysteine hydrochloride monohydrate 98% (cat#C7880), 5,5’-Dithiobis(2- nitrobenzoic acid) (DTNB) 98% (cat#D8130), poly(ethylene glycol) (N-hydroxysuccinimide 5- pentanoate) ether N’-(3-maleimidopropionyl)aminoethane (Mal-PEG-NHS); average Mn ≈
4,000Da (cat#757853), Acrylate-PEG-NHS (Ac-PEG-NHS); average Mn ≈ 5,000Da
(cat#JKA5023), O-[2-(6-oxocaproylamino)ethyl]-O’-methylpolyethyleneglycol (M-PEG2000); average Mn ≈2,000Da (cat#54369), O-[2-(6-oxocaproylamino)ethyl]-O’- methylpolyethyleneglycol (M-PEG5000); average Mn ≈ 5,000Da (cat# 41964), α,ω-bis(2-[(3- carboxy-1-oxopropyl)amino]ethyl)polyethylene glycol (HOOC-PEG-COOH); average Mn ≈
3,000 (cat#14567), and bovine serum albumin (BSA) ≥96% (cat#A2153) were all purchased from
Sigma-Aldrich (NSW, Australia) and used without further purification or treatment. AR grade ethanol (100%) and ammonia (NH4OH) solution (30%) were obtained from Chem-Supply (SA,
Australia). Hydrochloric acid (HCl) (36%), hydrogen peroxide (H2O2) (30%) solutions, and the
LIVE/DEAD BacLightTM bacterial viability kit (cat#L7012) were purchased form ThermoFisher
(Vic, Australia) and used as received. Nitric acid (HNO3) (69%) was obtained from Merck
(Darmstadt, Germany), and ethylenediamine tetra acetic acid (EDTA) was purchased from BDH chemical (PA, USA).
3.1.3 Bacterial strains and growth conditions
Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 9027) were used for all bacterial investigations and were obtained from Swinburne University of Technology’s microbiology collection. Bacterial stock were cultivated on nutrient agar (15g/L agar, 8g/L nutrient 102 broth) at 37°C. For analysis, single colonies were inoculated into Mueller Hinton broth (MHB) or nutrient broth (NB) and grown at 37°C until the logarithmic growth phase before being diluted to the correct cell concentration based on optical density (OD) measurements at 600nm.1,2 All media were purchased from ThermoFisher (Vic, Australia).
3.2 Methods
3.2.1 Surface cleaning methods
Where appropriate, inorganic surfaces were cleaned using the Radio Corporation of
America (RCA) method as described by Cras, et al. 3 In brief, to prepare a 140mL solution, 100mL of MilliQ was transferred to a beaker followed by the addition of 20mL NH4OH (27% v/v) which was then heated to 70±5°C on a hot plate. Once the desired temperature was reached the beaker was removed from the hot plate and 20mL H2O2 (30% v/v) was added. Pre-cut Si wafers were then submerged once the solution began to bubble vigorously and allowed to react for 15min. After
15min, the wafers were then transferred to a separate container with overflowing MilliQ H2O to rinse the solution from the surface of the wafer. After several rinses the wafers were dried under a flow of N2 gas. All used RCA solutions were disposed down the drain after being heavily diluted with cold water. Where the RCA method is not applicable, surface materials were cleaned by sonicating in solutions of Decon (2% v/v) for 30min, MilliQ for 10min and finally ethanol (70% v/v) for an additional 10min. Surfaces were then given a final rinse in ethanol (70% v/v) and finally dried with compressed N2 gas.
3.2.2 Modification of planar surfaces
3.2.2.1 Solution-based deposition of APTES films
Optimisation of deposition times for the production of APTES films was performed on Si wafers cut into 1cm2 pieces using a diamond pen. Cut Si wafers were then cleaned using the RCA 103 method described previously in Section 3.2.1 followed by treatment in a UV/O3 chamber
(BioForce nanosciences, US) for 15min. Cleaned substrates were then submerged in a solution of
APTES (2% (v/v) in toluene) for 0-45min followed by sonication in toluene twice for 5min and
MilliQ to remove loosely bound APTES molecules. Surfaces were then dried under compressed
N2 gas and cured in an oven at 120°C for 20min before further analysis or functionalisation. Bulk
APTES films were prepared using a 20% (v/v) solution in toluene and deposited for 30min.
To facilitate APTES deposition on to SiO2 SPR sensors a solution of APTES (2% v/v) in toluene was transferred directly on to the surface of the sensor which had been treated in a UV/O3 chamber for 15min. Direct transfer of the APTES solution was performed to prevent APTES from adhering to the backside of the sensor which would impede SPR analysis. The reaction between
APTES and the SPR sensor was allowed to proceed for 30min with additional solvent being added every 30seconds to prevent the surface from drying out. Slides were then rinsed three times with toluene and MilliQ before being dried with compressed N2 gas and cured in an oven at 120°C for
20min.
3.2.2.2 Solution-based deposition of MPTS films
Si wafers (1cm2) were cleaned by the RCA method as described in Section 3.2.1 and treated in a UV/O3 chamber for 15min. The cleaned Si wafers were then placed in a solution of MPTS (0-
4% v/v) in anhydrous toluene or AR grade ethanol at room temperature. After an immersion time of 3hr, the samples were rinsed several times with ethanol to remove any physiosorbed MPTS and subsequently dried with compressed N2 gas. MPTS films were then cured by incubating in an oven at 100°C for 1hr and stored at room temperature until further analysis or functionalisation. A bulk
MPTS film was prepared as a control by placing 100µL of an undiluted MPTS solution on to a clean wafer and allowed to dry at room temperature.
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3.2.2.3 Pre-hydrolysis deposition of MPTS films
Deposition of MPTS under pre-hydrolysis conditions was carried out using methods adapted from Scott, et al. 4 In brief, Solutions of MPTS (0-4% v/v) were prepared in ethanol with
3% (v/v) H2O and either adjusted to pH 4 or 8.5 with concentrated HCl or NaOH, respectively.
The MPTS solutions were then left to hydrolyse for a period of 2hr, after which, cleaned Si wafers were immediately transferred to the MPTS solution and left to react for 10min. It is noted that there was no post-deposition rinse applied by Scott, et al. 4, and given that MPTS films prepared at pH 4 displayed superior properties compared to pH 8.5; the effects of various post-deposition processes were also investigated by preparing samples that were either a) rinsed and dried immediately after deposition, b) dried only after deposition, and c) not treated after deposition.
After silanisation, MPTS-treated wafers were then removed from the solution and subjected to the various post-deposition treatments described above prior to being cured by incubating in an oven at 100°C for 1hr.
3.2.2.4 In-situ grafting of PEG by reductive amination and BSA adsorption
Multi-parametric surface plasmon resonance (MP-SPR) was used to perform in-situ experiments to investigate PEG grafting and protein adsorption in real-time. SPR allows for real- time detection of (bio)molecular interactions based on a shift in the incidence angle of reflected light at a specific wavelength which is proportionate to the mass adsorbed on the sensor-slide surface (Figure 14).
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Figure 14. Schematic illustration demonstrating the principles of surface plasmon resonance (SPR) to determine surface densities of adsorbed molecules. The angle where total internal reflection occurs is dependent on the mass adsorbed to the sensor-slide and can be used to accurately determine surface mass densities
For analysis, a cleaned Au SPR sensor slide was inserted into the instrument and a baseline signal was established by allowing PBS (10mM pH 7.4) to flow through the flow-cell at a rate of
30µL/min (used for all injections) until a stable baseline was achieved. To form reacting amines on the Au sensor slide, an initial injection of polyethylenimine (PEI) (0.1mg/mL in 10mM PBS) was performed for 10 minutes followed by a post-injection rinse with 10mM PBS for 4 min. A methoxy–terminated aldehyde-PEG (M-PEG) with a MW of 2000Da or 5000Da was subsequently grafted to PEI by reductive amination using NaCNBH3 as a reducing agent. To achieve this a solution of M-PEG (1mg/mL) in 10mM PBS with NaCNBH3 (3mg/mL) and K2SO4 (0-0.6M) was injected into the flow-cell for 15 minutes followed by a post-injection rinse with 10mM PBS for
10 minutes. Protein adsorption was carried out using BSA (1mg/mL) in 10mM PBS and injected
106 into the flow-cell for 10 minutes. SPR slides were then rinsed in 10mM PBS for 10 minutes to establish a post-adsorptive baseline.
3.2.2.5 In-situ grafting of PEG by carbodiimide chemistry and BSA adsorption
MP-SPR was used to assess the grafting of PEG using carbodiimide crosslinking chemistry to covalently-bound APTES or physically-bound PEI layers. For preparation of APTES- functionalised SPR sensors; a bare SiO2 SPR sensor was placed in to the chamber of a UV/O3 cleaner and treated for 15min to form an excess of surface-bound hydroxyl groups. The SPR slides were then treated with APTES (2% (v/v) in toluene for 30min) (See Section 3.2.2.1) to produce covalently bound primary amines which were then used to facilitate PEG grafting in-situ. For physically-bound PEI layers, an initial injection of PEI (0.1mg/mL) was performed for 10min followed by a post-deposition rinse with MES buffer (10mM pH 6.5) for 4min. Upon surface activation with either APTES or PEI a baseline signal was established within the SPR by allowing
MES buffer (10mM pH 6.5) to flow through the flow-cell at 30µL/min. For polymer grafting a carboxylated homobifunctional PEG (1mg/mL) (3000Da) was activated outside the SPR in a solution containing EDC/NHS (125mM each) in MES buffer (10mM pH 6.5) and left to react for
15min. The activated PEG was then injected into the SPR onto either PEI- or APTES- functionalised sensors at 30µL/min for 30min, followed by rinsing in PBS (10mM pH 7.4) to promote amide bond formation until a stable post-injection baseline was achieved. Protein adsorption was carried out using BSA (1mg/mL) in 10mM PBS and injected into the flow-cell for
10 minutes at 30µL/min. SPR sensors were subsequently rinsed in 10mM PBS for 10 minutes to establish a post-adsorptive baseline.
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3.2.3 Modification of colloidal particles
3.2.3.1 Regeneration of reactive oxygen species on silica microspheres
Where appropriate, regeneration of hydroxyl groups on silica microspheres was performed to facilitate subsequent silanisation. In brief, Si particles (1% w/v) were washed three times in
MilliQ by centrifugation (6000rpm 3min) and then resuspended in 2M HNO3. Particles were then incubated at room temperature for 1hr with constant agitation. After the 1hr incubation period particles were washed again by centrifugation (6000rpm 3min) to remove residual acid. Particles were then resuspended in MilliQ and stored at 4°C for subsequent analysis or immediately silanised.
3.2.3.2 Silanisation of silica microspheres with 3-mercaptopropyl(trimethoxysilane)
Solutions of 3-mercaptopropyl(trimethoxysilane) (MPTS) were prepared at 4% (v/v) in ethanol with 3% (v/v) H2O and adjusted to pH 4 with concentrated HCl (1M). MPTS solutions were then left to hydrolyse for 2hr at room temperature. During the pre-hydrolysis period, particle solutions (1% w/v) were prepared according to Section 3.2.3.1 to regenerate reactive hydroxyl groups and subsequently resuspended in the above MPTS solution after the designated pre- hydrolysis period. Particle suspensions were then incubated at room temperature for 1hr with constant agitation. After being allowed to react for 1hr, Si-MPTS particles were then washed by centrifugation (6000rpm 3min) in both ethanol and MilliQ, and finally being resuspended in
MilliQ. Particles were then stored at 4°C for subsequent analysis or immediately functionalised with PEG.
3.2.3.3 Quantification of reactive thiol functional groups
The amount of reactive thiol functional groups present on modified silica colloids was determined using (5,5-dithio-bis-(2-nitrobenzoic acid)) (DTNB) which reacts with free sulfhydryl
108 groups to form a coloured product, which can be quantified using UV-Vis spectrophotometry. To allow for quantification, a standard curve of Cysteine was prepared over concentrations ranging from 0-1mM in PBS (0.1M pH 8) containing 1mM EDTA. Each standard solution was prepared to a volume of 2.950mL with 50µL of a solution of DTNB (4mg/mL in PBS pH 8; 1mM EDTA) being added to achieve a total volume of 3mL. All tubes were then incubated at room temperature with constant agitation for 15min before having their absorbance measured in a Genesys 20 UV-
Vis spectrophotometer (Thermo Scientific) at 412nm.
Particle suspensions (1% w/v) were washed twice by centrifuging at 3000rpm for 3min and resuspended in PBS (0.1M pH 8; EDTA 1mM). 100µL of the particle suspension was then added to 850µL of PBS (0.1M pH 8; EDTA 1mM) followed by the addition of 50µL of DTNB to achieve a final particle concentration of 0.1% (w/v). Samples were then incubated at room temperature for 15min with constant agitation. After the incubation period all samples were centrifuged at 3000rpm for 3min, where the absorbance of the supernatant was measured at 412nm.
3.2.3.4 Immobilisation of PuroA to PSC2 particles
EDC/NHS zero-length coupling chemistry was used for the covalent coupling of carboxyl groups of PS particles to terminal amines found on PuroA AMPs. The activation of COOH groups with EDC is most efficient at pH 4.5-7.2; however, because of the instability of the o-acylisourea active ester intermediate, NHS was also used as this will form an active ester with o-acylisourea.
This active ester is much more stable (half-life 4-5hr) than EDC-activated COOH groups and is therefore expected to increase the coupling efficiency with the COOH groups on PS particles. To perform the immobilisation, 1mL of diluted PSC2 particles (0.4% (w/v)) were centrifuged at
1.5x103 rpm for 15min and the supernatant was discarded and replaced with 1mL of MilliQ (pH
5.4) containing EDC/NHS (125mM each). Samples were allowed to react whilst shaking for 15min
109 at 4°C to activate COOH groups. The particle suspensions were again centrifuged at 1.5x103 rpm for 15min, and the supernatant was again discarded. For PuroA coupling, the activated PSC2 particles were resuspended in 1mL of diluted PBS (1mM, pH 7.4), containing 250µg/mL PuroA and allowed to react for 20h at 4°C whilst shaking. The particles were then washed three times by centrifugation at 1.5x103 rpm and replacing the supernatant with 1mL of fresh MilliQ after each wash. Samples were then suspended in 1mL of MilliQ and stored at 4°C prior to subsequent analysis.
3.2.3.5 Grafting of PEG to Si-COOH colloids via carbodiimide chemistry and BSA adsorption
The grafting of PEG to carboxylated Si (SiC) colloidal particles was investigated using
EDC/NHS coupling chemistry with their non-fouling properties being subsequently assessed by their ability to resist BSA adsorption. For PEG immobilisation, stock SiC particles (2.01µm) where first suspended in MilliQ at 1% (w/v) and then washed by centrifugation (6000rpm 3min) to remove any impurities and surfactants and finally resuspended in 1mL of MilliQ. Particle suspensions were again centrifuged (6000rpm 3min) and resuspended in MilliQ (pH 5.4) containing EDC/NHS at 125mM each. Samples were then left to react at 4°C for 15 min with constant agitation to activate –COOH groups on Si particles. After activation, particles were centrifuged (6000rpm 3min) and resuspended in PBS (10mM pH 7.4) containing HOOC-PEG5000-
NH2 at 1mg/mL and then left to react overnight at 4°C with constant agitation. To remove any unbound PEG and residual EDC.NHS particle suspensions were washed three times in MilliQ by centrifugation (6000rpm 3min) and finally resuspended in 1mL of the same. Samples were then stored at 4°C for subsequent analysis.
Protein adsorption to the particles prepared above was assessed using BSA (1mg/mL).
Firstly, particle suspensions were diluted to 0.1% (w/v) using MilliQ and 100µL of this diluted
110 suspension was drop-cast on to a PS-coated glass slide which had been treated in a UV/O3 chamber.
Samples were then left to dry overnight and then heat-treated for stabilisation on a hot-plate at
130°C for 30seconds. Stabilised colloidal layers were then placed in to the wells of a sterile 6-well flat-bottomed plate and submerged in a solution of BSA (1mg/mL) for 2hrs at room temperature.
After 2hr the surfaces were then washed three times in MilliQ to remove any loosely bound BSA and stored at 4°C for subsequent analysis.
3.2.3.6 Sequential immobilisation of PEG and AMPs using thiolene ‘photoclick’ chemistry
Thiolene ‘photoclick’ chemistry was used to sequentially immobilise PEG and AMPs to
MPTS-modified silica colloids using a method adapted from Bini, et al. 5 and Russo, et al. 6. A heterobifunctional PEG (Ac-PEG5000-NHS) was utilised to facilitate reactions between thiols on
MPTS-modified colloids and acrylate groups on PEG, and also the immobilisation of primary amines of AMPs to NHS-terminated PEG chains (Figure 15).
Figure 15. Illustrative representation of the two-step immobilisation used to tether PEG and AMPs to silica particles with thiolene ‘photoclick’ chemistry.
For a typical reaction, MPTS-modified colloids (1% w/v) were prepared according to
Section 3.2.3.2 and resuspended in a solution of MeOH:H2O (1:2) containing Ac-PEG5000-NHS
(1mg/mL) and 2,2-dimethoxy-2-phenylacetophenone (DPAP) (0.5mg/mL) which was used as a radical initiator. Samples were then irradiated by blue light (λ=365nm) using a Translux® EC
111 dental wand (Kulzer, DEU) info here for approximately 10min followed by being washed twice in
PBS (10mM pH7.4) by centrifugation (6000rpm 3min). For AMP immobilisation, washed samples were resuspended in PBS (10mM pH 7.4) containing either P1 or W8 AMPs at 250µg/mL and allowed to react for 4hr at room temperature with constant agitation. After the incubation period samples were washed three times in PBS (10mM pH 7.4) by centrifugation (6000rpm 3min) and finally resuspended in 1mL of MilliQ. Samples were then stored at 4°C for subsequent analysis.
3.2.3.7 ‘One-Pot’ immobilisation of PEG and AMPs using thiol-maleimide chemistry
Thiol-maleimide coupling was used as a ‘one-pot’ synthesis method for the immobilisation of PEG and AMPs to MPTS-modified silica colloids using a method adapted from Northrop, et al.
7 Here, a maleimide PEG succinimidyl carboxymethyl ester (Mal-PEG4000-NHS) was used to facilitate immobilisation of PEG through thiol-maleimide conjugation, and also AMP immobilisation through reaction with primary amines and activated NHS esters (Figure 16).
Figure 16. Reaction summary of the modification of silica colloids by thiol-maleimide chemistry used to tether PEG and AMPs.
In brief, MPTS-modified silica colloids (1% w/v) were prepared according to Section
3.2.3.2 and the resulting solution was resuspended in PBS (10mM pH7.4) containing Mal-PEG4000-
NHS (1mg/mL) and either P1 or W8 AMPs at a concentration of 250µg/mL. The contents of the
112 microcentrifuge tubes were then allowed to react overnight at room temperature with constant agitation. Particle suspensions were then washed three times in PBS (10mM pH 7.4) by centrifugation (6000rpm 3min) and finally resuspended in 1mL of MilliQ before being stored at
4°C.
3.2.4 Surface fabrication through colloidal self-assembly
3.2.4.1 Preparation of PuroA-modified BCC monolayers
2 Glass slides were used as the BCC substrate and cut to approximately 1cm . Prior to use, the slides were cleaned by sonicating in solutions of Decon (2% (v/v)) (30min), MilliQ (10min), and ethanol (70% (v/v)) and were then given a final rinse in ethanol (70% (v/v)) and dried under compressed N2 gas. Cleaned slides were then coated in polystyrene by applying 30µL of a polystyrene solution (5% (w/v) in toluene) to the centre of the glass substrate followed by spin coating at 800rpm for 100s. All slides were the treated in a UV-ozone cleaner (Bioforce
Nanosicence, Iowa, USA) for 15min to increase surface hydrophilicity to a level where colloidal crystal growth is possible.8
BCC layers were prepared from PuroA-modified and unmodified particle solutions (0.4%
(w/v)) by the evaporation-induced confined area assemble (EICAA) method.8,9 Approximate volumes of the large particles (VP in µL) confined in a rubber O-ring of diameter DR (in cm) needed to create a single monolayer can be calculated according to Equation 1 as reported previously.8
Equation 1. Calculation of the volume of colloidal solution required to generate a single colloidal monolayer.
2 10 × � × � × �� × �� � = � 12 × �
113
3 Where ρ is the particle density (g/cm ), DP is the diameter (µm), and w is the % solid weight content of the particle suspension.
Large and small particle combinations chosen for this investigation were 2µm PSC (PSC2) and 0.110µm PMMA (PMMA011) particles, respectively. These combination were chosen as
PuroA could be selectively tethered to SPC2 particles and the BCC topographical features are similar to or smaller than the dimensions of a typical bacterial cell (≈3x1µm for E.coli).10
Typically, to produce a PSC2-PMMA BCC monolayer covering the area of an O-ring of
1cm in diameter (0.785cm2), 20.9µL of PSC2 (0.4% (w/v)) and 29.6µL of PMMA011 (0.4%
(w/v/)) particles were mixed and diluted in MilliQ to a final volume of 100µL. After being thoroughly vortexed to ensure particles were well-suspended, the resulting binary colloidal suspension was then pipetted inside the rubber O-ring in an EICAA apparatus and left at room temperature until complete solvent evaporation (≈12hr). The surfaces were then removed from the
EICAA apparatus and the resultant BCC layers were heat-treated from stabilisation by placing the prepared surface on a hot plate at 130°C for 60s. Figure 17 below summarised the steps taken to immobilise PuroA to PSC2-PMMA011 BCC layers using EDC/NHS as the coupling agent and
EICAA as the method for BCC formation.
114
Figure 17. Overview of PuroA coupling and subsequent BCC formation. PSC2 and PMMA011 represent 2µm carboxylated polystyrene and 0.110µm poly(methyl methacrylate) particles, respectively.
3.2.5 Bacterial cell studies
3.2.5.1 Activity of free AMPs in solution
The minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial agent that can inhibit the visile growth of a microorganisms after an overnight
11 culture. The MIC of three AMPs; PuroA (FPVTWRWWKWWKG-NH2), P1
(RWRWWRWWKWWKR-NH2), and W8 (WRWWKWWK-NH2) in solution against
Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 9027) using a modified broth microdilution method,12 with AMP concentrations ranging from 250µg/mL to 0.5µg/mL. In brief, 50µL of the stock peptide solution (1mg/mL in 1mM PBS pH 7.4) was added to the first empty well of a sterile 96-well microtitre plate, and 25µL of this was transferred to the next row already containing 25µL of 1mM PBS. This process was repeated until a two-fold serial dilution of the stock peptide solution was achieved with concentrations ranging from 1000µg/mL to
115
2µg/mL. Bacterial suspensions were grown to late log-phase of growth in MHB and cell densities were adjusted to 1x108CFU/mL before being diluted to approximately 6.66x105CFU/mL. 75µL of the bacterial suspension was then transferred to the appropriate wells achieving a final cell density of 5.0x105CFU/mL and AMP concentrations of 0.5-250µg/mL. Control wells were prepared with no bacterial cells (25µL of 1mM PBS and 75µL of MHB), and also cells treated with no AMPs
(25µL of 1mM PBS and 75µL of cell suspension). Plates were then incubated at 37°C for 24h.
The MIC values were determined by measuring the absorbance of samples at 600nm after the 24h incubation period using a POLARstar Omega microplate reader (BMG LABTECH, DEU). All samples were prepared and analysed in triplicate (n=3).
3.2.5.2 Plate count assay for viable growth determination
A viable cell count assay was performed to assess the antimicrobial activity of modified particles in solution against P. aeruginosa cells. Cells were first obtained and grown in nutrient broth (NB) to a cell concentration of 2x105CFU/mL according to Section 3.1.3. For a typical analysis 100µL of a particle suspension (1% w/v) was centrifuged at 6000rpm for 3min and the supernatant replaced with 100µL of NB before being thoroughly resuspended. 100µL of the bacterial suspension (2x105CFU/mL) prepared above was added to the particle solution and mixed thoroughly to obtain a final cell density of 1x105CFU/mL. Samples were then incubated in a shaking incubator at 37°C for 2 or 4hr. After the designated incubation period a ten-fold serial dilution of the suspension was prepared from 10-1 to 10-4 using NB and 100µL the resulting diluted suspensions were plated on separate nutrient agar plates and incubated overnight at 37°C. Control samples were prepared using unmodified particle samples and also cell only suspensions. To quantify cell numbers, plates where single colony growth could be distinctly identified were counted and used to calculate undiluted cell concentrations. All samples were prepared in duplicate
(n=2) and three replicates were individually plated at each concentration. 116
3.2.5.3 Antibacterial activity of immobilised PuroA AMPs
The viability of E. coli (ATCC 25922) attached to PuroA-modified PSC2PMMA011 BCC layers was assessed using the BacLightTM Bacterial Viability Kit. For a typical analysis, BCC layers prepared according to Section 3.2.4 were placed into wells of a sterile 12-well flat-bottomed plate and submerged in suspensions of E. coli (1x108 CFU/mL) in MHB. The plates were then incubated at 37°C for 24hr. After incubation, samples were washed three times in PBS (10mM pH
7.4) to remove any non-adherent cells and treated with 200µL of a Syto9/PI solution (0.3% v/v in
1mM PBS pH 7.4) and left in the dark for 15min at room temperature. Samples were again washed three times in PBS to remove any excess staining solution and subsequently imaged using a Nikon
Eclipse 50i fluorescence microscope with excitation wavelengths of 485nm for Syto9 (green) and
561nm for PI (red). The emission wavelengths for the excited fluorophores were detected through spectra emission filters of 505 and 630/622nm for green and ref fluorescence, respectively. All samples were prepared in triplicate (n=3) and three images were taken per sample. Control surfaces were prepared using i) unmodified BCC layers, ii) PuroA physically adsorbed to BCC layers, and iii) flat PS-coated glass slides.
3.2.5.4 Antibacterial activity of PEG and AMP-functionalised colloids prepared using thiol-
ene ‘photo-click’ and thiol-maleimide chemisty
Viability of P. aeruginosa (ATCC 9027) cells incubated with P1- and W8-modifeid Si microspheres were assessed using the BacLightTM Bacterial Viability Kit. Modified particles were prepared using both the thiol-ene ‘photo-click’ (See Section 3.2.3.6) and thiol-maleimide (See
Section 3.2.3.7) reactions with the resulting Si-PEG-AMP microspheres being suspended in 1mL of MilliQ. 100µL of the particle suspensions were washed twice in MilliQ by centrifugation
(6000rpm 3min) before being resuspended in 500µL of nutrient broth. To this solution, 500µL of a suspension of P. aeruginosa cells (2x105 CFU/mL) was added resulting in a full cell density of 117
1x105 CFU/mL. All samples were then incubated in a shaker incubator at 37°C for 4hr. After incubation, all samples were washed three times with a NaCl solution (0.85% w/v) by centrifugation (6000rpm 8min) and resuspended in 1mL of 0.85% NaCl. 10µL of this solution was taken and use for a plate count assay according to section 3.2.5.2. To the remaining solutions, 3µL of dye mixture was added containing equal parts Syto9/PI and incubated in the dark at room temperature for 15min. 5µL of the sample was placed on a microscope slide and covered with a coverslip and imaged using a FLUOVIEW FV1000 Confocal microscope (Olympus, JP) with excitation wavelengths of 488nm for Syto9 (green) and 561nm for PI (red). Both red and green fluorescence emission was detected through 500/550nm filters. All samples were prepared in duplicate (n=2) and six images were taken per sample. Control samples were prepared using i) cells only, ii) Bare Si microspheres, and PEG-modified Si microspheres prepared using iii) thiol- ene ‘photo-click’ and iv) thiol-maleimide chemistry.
3.2.5.5 Scanning electron microscopy imaging of biotic surfaces
For imaging of biotic surfaces, bacterial cells were seeded onto surfaces at 1x108 CFU/mL and incubated at 37°C for 24hr. After the incubation period cell suspensions and substrates were removed and washed three times in PBS (10mM pH 7.4), followed by fixing in 3.0% (v/v) glutaraldehyde for 3h. Surfaces were then dehydrated under an increasing ethanol gradient (60,
70, 80, 90, and 100%) being incubated for 10min at each concentration. Dehydrated surfaces were then coated in Au (≈10nm) by evaporation using a K975X Turbo Evaporator and then imaged using field emission SEM with a ZEISS SUPRA 40 VP FE-SEM (Carl Zeiss, DEU) operating at
3keV.
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3.2.6 Characterisation techniques
3.2.6.1 Surface plasmon resonance (SPR) analysis
All SPR measurements were performed using an MP-SPR-NAVI 210A VASA instrument
(BioNavis, Finland) operating at wavelengths of 670nm and 785nm. The instrument is fitted with a four-channel flow-cell with independent parallel flow channels, with measurements being obtained in angular scan mode within the liquid internal reflection range (50.01-77.91°). Layer analysis was performed using LayerSolver software (V1.2.) and thickness values were determined based on the wavelength shift between pre-injection and post-injection baselines.
For calculating exact surface concentration (surface mass densities), the de Freiter equation was utilised:13
(� − � )� Γ = � � � � �� ⁄��
Where np is the sample layer refractive index (RI), nm is the medium RI, dp is the sample layer thickness (nm) and dn/dc is the (mass) concentration dependency. Deriving this further to include instrument signal:
�� − �� = ∆� = ∆� × �
∆� × � × � Γ = � � �� ⁄��
Where the constant k is the instrument (sensor, wavelength) dependant coefficient for sensitivity, and θ is the angular response in the measurement. For thin layers (<100nm), k*dp can be approximated to be constant and can be obtained from theoretical modelling of SPR peaks, or by empirical experiments. For the MP-SPR-NAVI 210A, the k*dp values in aqueous-based buffers
119 are approximately 1.0x10-7 nm/degree and 1.9x10-7 nm/degree at wavelengths of 670nm and
785nm, respectively.
For proteins with dn/dc values of 0.182cm3/g such as BSA, the surface coverage can be calculated as follows:
ng Protein surface coverage ( ) =change in TIR at 670nm ×550, or cm2
2 Γp670=∆θ×550ng/cm
3.2.6.2 X-ray photoelectron spectroscopy (XPS) analysis
XPS was used to investigate the elemental composition and chemical sates of atoms in the outer most 10nm of the prepared samples. Data was obtained using a Kratos Axis Nova spectrometer (Kratos Analytical, UK) equipped with a monochromated aluminium X-ray source
14,15 (Alkα; hυ = 1486.6eV) operating at a power of 15mA and 15kV (225W). Survey and high- resolution spectra were acquired at detector pass energies of 160eV and 20eV, respectively. Each sample was prepared in triplicate (n=3) and three spots were analysed per sample over an elliptical area of approximately 0.3 x 0.7mm2. Corresponding XPS data was analysed using Casa XPS software (V2.3.16) (Casa Software Ltd. Teignmouth, UK). Concentrations of each atomic species were calculated using the relative peak intensities and sensitivity factors provided by the instrument manufacturer. For effective charge compensation, operating conditions employed were; filament current: 1.8A, charge balance: 3.3V and a bias voltage of 1.3V. For analysis of high-resolution spectra, a linear type background was chosen for quantification and a Gaussian broadened Lorentzian line shape (GL(30)) was used for curve-fitting. All spectra were calibrated to the C-C/C-H component at 285.0eV and peaks widths were restricted to a full width half maximum (FWHM) between 1.1-1.9eV. A residual STD of ≤1.0 was used to indicate calculated data is in concordance with experimental data. 120
3.2.6.3 Attenuated total reflectance-Fourier transform infra-red (ATR-FTIR) spectroscopy
Investigations by ATR-FTIR spectroscopy were performed to assess the chemical composition of modified surfaces and microspheres where appropriate. For analysis of modified microspheres, 100µL of the colloidal suspension was transferred on to a glass substrate inside the area of an O-ring 1cm in diameter and allowed to dry prior to analysis. All spectra were collected using a Nicolet iS5 FT-IR spectrometer (ThermoFisher, US) operating in ATR mode with up to
64 scans being collected at 4cm-1 resolution. Background spectra were collected using unmodified surfaces and microspheres with automatic atmospheric suppression employed to minimise interference from H2O and CO2. All samples were prepared in triplicate with a minimum of two spots being analysed per sample.
3.2.6.4 Zeta potential measurements
Zeta potential analysis was used to assess the surface charge of PEG- and AMP-modified colloidal particles and all measurements were taken using a 90 Plus particle analyser (Brookhaven,
US). In brief, for a typical analysis, 100µL of the particle suspension was diluted to 1% (v/v) using
MilliQ (pH 5.4), and the resulting particle solutions (10mL) were used for equilibrating the electrochemical (EC) cell and also for analysis. The reported zeta potential of each colloidal suspension is the average of five data points of two independent samples, and control samples were prepared using unmodified particles.
3.2.6.5 Static water contact angle (WCA) analysis
Static WCA analysis was used to assess the surface hydrophobicity of MPTS films using an FTA1000 C Class contact angle and surface tension instrument (First Ten Angstroms, US)
16 using the manual drop method. In brief, 1µL of deionised H2O was carefully dropped onto the surface of the samples using a micropipette. An image was immediately captured using a CCD
121 camera (25Hz) and the contact angle was calculated automatically using FTA instrument software.
All measurements were performed under normal atmospheric conditions. All samples were prepared in duplicate (n=2) and three spots were analysed per sample.
3.2.6.6 Ellipsometry analysis
Ellipsometry was used to determine the thickness values of silane and PEG-modified surfaces. All data was obtained using an M-2000 variable angle spectroscopic ellipsometer
(VASE) (J.A. Woolam, US), and measurements of Psi (Ψ) and Delta (Δ) were used to model thicknesses of the prepared samples. Samples were analysed at 60-75° in increments of 5° with a
5 second acquisition time. A cleaned unmodified Si wafer was used to determine the thickness of the underlying SiO2 layer and fixed for each subsequent measurement. Thickness values of sample layers were calculated using supplied Cauchy model parameters, and optical constants of n=1.465 and k=0 were used for both PEG/silane and underlying SiO2 layers. All samples were prepared in triplicate (n=3) and at least three spots were analysed per sample. To calibrate and ensure workability of the ellipsometer before sample analysis, a commercial Si calibration wafer having an oxide layer thickness of 22nm was used.
3.2.6.7 Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-
ToF MS) analysis
MALDI-ToF MS analysis was used to confirm the success of the covalent immobilisation of PuroA to PSC2 colloidal particles. The so-called surface-MALDI technique can be used to directly analyse peptides and proteins immobilised at a surface.17,18 It is known that the soft ionisation process of MALDI-ToF analysis does not have enough energy to break covalent bonds; thus, successful reaction between PuroA and the PSC2 colloids should result in no signals being detected in the resultant spectra.19
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PuroA-modified PSC2 particles were prepared as described in Section 3.2.3.3, and
MALDI-ToF analysis was performed as outlined previously.20 For analysis, each sample was spotted on a MALDI target plate in duplicate (n=2) by adding 1µL of sample and 1µL of matrix onto the target plate and mixing by pipetting up and down several times. The matrix used was
10mg/mL DHB diluted in a 1:1 water/acetonitrile solution with 0.1% TFA. The sample-matrix mixture (2µL) was set to dry on the sample plate, and spectra were acquired in a Shimadzu Axima
Performance MALDI-ToF MS (Shimadzu Corp., JPN) using a 337nm nitrogen laser operating in reflectron mode over a mass range between 100 and 4000m/z. Control samples were prepared using PuroA (250µg/mL in 1mM PBS), unmodified PSC2 particles, and physically adsorbed
AMPs (i.e. no EDC/NHS).
Generated spectra were analysed using Shimadzu Launchpad software (V2.9.2), and the
MALDI-ToF MS was externally calibrated using standards over a mass range of 190-3658Da
(C10H8NO3 (190.05Da), angiotensin II (1046.45Da), angiotensin I (1296.69Da), Glu-1-fibrinogen
(1570.98Da), N-acetyl renin (1800.94Da), ACTH 1-17 (2093.08Da), ACTH 18-39 (2465.20Da), and ACTH 7-38 (3657.93Da)).
3.2.6.8 Scanning electron microscopy (SEM) analysis
Topographical surface structures of prepared samples were observed using field emission
SEM with a ZEISS SUPRA 40 VP FE-SEM (Carl Zeiss, DEU) operating at 3keV. Prior to SEM imaging, all surfaces were coated with Au (≈10nm) by evaporation using a K975X Turbo
Evaporator.
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3.3 References 1 Shao, J., Xiang, J., Axner, O. & Ying, C. Wavelength-modulated tunable diode-laser absorption spectrometry for real-time monitoring of microbial growth. Appl. Opt. 55, 2339-2345, doi:10.1364/AO.55.002339 (2016).
2 Biesta-Peters, E. G., Reij, M. W., Joosten, H., Gorris, L. G. M. & Zwietering, M. H. Comparison of Two Optical-Density-Based Methods and a Plate Count Method for Estimation of Growth Parameters of <em>Bacillus cereus</em>. Appl. Environ. Microbiol. 76, 1399, doi:10.1128/AEM.02336-09 (2010).
3 Cras, J. J., Rowe-Taitt, C. A., Nivens, D. A. & Ligler, F. S. Comparison of chemical cleaning methods of glass in preparation for silanization. Biosensors Bioelectron. 14, 683-688, doi:http://dx.doi.org/10.1016/S0956-5663(99)00043-3 (1999).
4 Scott, A. F., Gray-Munro, J. E. & Shepherd, J. L. Influence of coating bath chemistry on the deposition of 3-mercaptopropyl trimethoxysilane films deposited on magnesium alloy. J. Colloid Interface Sci. 343, 474-483, doi:10.1016/j.jcis.2009.11.062 (2010).
5 Bini, D., Russo, L., Battocchio, C., Natalello, A., Polzonetti, G., Doglia, S. M., Nicotra, F. & Cipolla, L. Dendron Synthesis and Carbohydrate Immobilization on a Biomaterial Surface by a Double-Click Reaction. Org. Lett. 16, 1298-1301, doi:10.1021/ol403476z (2014).
6 Russo, L., Battocchio, C., Secchi, V., Magnano, E., Nappini, S., Taraballi, F., Gabrielli, L., Comelli, F., Papagni, A., Costa, B., Polzonetti, G., Nicotra, F., Natalello, A., Doglia, S. M. & Cipolla, L. Thiol-ene mediated neoglycosylation of collagen patches: a preliminary study. Langmuir 30, 1336-1342, doi:10.1021/la404310p (2014).
7 Northrop, B. H., Frayne, S. H. & Choudhary, U. Thiol–maleimide “click” chemistry: evaluating the influence of solvent, initiator, and thiol on the reaction mechanism, kinetics, and selectivity. Polymer Chemistry 6, 3415-3430, doi:10.1039/C5PY00168D (2015).
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8 Wang, P. Y., Pingle, H., Koegler, P., Thissen, H. & Kingshott, P. Self-assembled binary colloidal crystal monolayers as cell culture substrates. J. Mater. Chem. B 3, 2545-2552, doi:10.1039/c4tb02006e (2015).
9 Singh, G., Pillai, S., Arpanaei, A. & Kingshott, P. Multicomponent colloidal crystals that are tunable over large areas. Soft Matter 7, 3290-3294, doi:10.1039/c0sm01360a (2011).
10 Reshes, G., Vanounou, S., Fishov, I. & Feingold, M. Cell shape dynamics in Escherichia coli. Biophys. J. 94, 251-264, doi:10.1529/biophysj.107.104398 (2008).
11 Andrews, J. M. Determination of minimum inhibitory concentrations. J. Antimicrob. Chemother. 48, 5-16 (2001).
12 Wiegand, I., Hilpert, K. & Hancock, R. E. W. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 3, 163-175, doi:10.1038/nprot.2007.521 (2008).
13 Willem M. Albers, I. V. L. Surface Plasmon Resonance on Nanoscale Organic Films. Vol. 1st (Springer, 2010).
14 Seah, M. P. The quantitative analysis of surfaces by XPS: A review. Surf. Interface Anal. 2, 222-239, doi:10.1002/sia.740020607 (1980).
15 Boden, A., Bhave, M., Wang, P.-Y., Jadhav, S. & Kingshott, P. Binary Colloidal Crystal Layers as Platforms for Surface Patterning of Puroindoline-Based Antimicrobial Peptides. ACS Appl. Mater. Interfaces 10, 2264-2274, doi:10.1021/acsami.7b10392 (2018).
16 Yuan, Y. & Lee, T. R. in Surface Science Techniques (eds Gianangelo Bracco & Bodil Holst) 3-34 (Springer Berlin Heidelberg, 2013).
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17 Kingshott, P., St. John, H. A. W. & Griesser, H. J. Direct detection of proteins adsorbed on synthetic materials by matrix- assisted laser desorption ionization-mass spectrometry. Anal. Biochem. 273, 156-162, doi:10.1006/abio.1999.4201 (1999).
18 Boyd, A. R., Burke, G. A., Duffy, H., Holmberg, M., O'Kane, C., Meenan, B. J. & Kingshott, P. Sputter deposited bioceramic coatings: Surface characterisation and initial protein adsorption studies using surface-MALDI-MS. J. Mater. Sci. Mater. Med. 22, 74- 84, doi:10.1007/s10856-010-4180-8 (2011).
19 McLean, K. M., McArthur, S. L., Chatelier, R. C., Kingshott, P. & Griesser, H. J. Hybrid biomaterials: Surface-MALDI mass spectrometry analysis of covalent binding versus physisorption of proteins. Colloids Surf. B. Biointerfaces 17, 23-35, doi:10.1016/S0927- 7765(99)00055-7 (2000).
20 Jadhav, S., Sevior, D., Bhave, M. & Palombo, E. A. Detection of Listeria monocytogenes from selective enrichment broth using MALDI–TOF Mass Spectrometry. J. Proteomics 97, 100-106, doi:http://dx.doi.org/10.1016/j.jprot.2013.09.014 (2014).
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4 Binary colloidal crystal layers as platforms for the surface patterning of Puroindoline- based AMPs and optimisation of AMP selection
4.1 Summary The combination of topographical surface features and immobilised bioactive compounds has great potential for the development of multifunctional surface coatings in a range of application areas. Surface immobilisation of AMPs in particular presents itself as the ‘new generation’ of antimicrobial surfaces due to their low cytotoxicity and low propensity for developing bacterial resistance. As such, the chapter herein investigates the antibacterial activities of several puroindoline-based synthetic AMPs (PuroA, P1 and W8) against E. coli and P. aeruginosa.
Additionally, the zero-length immobilisation of PuroA to colloidal crystal layers was performed to assess immobilised AMP activity and also to elucidate the mechanisms of action through visualisation of cell structures using SEM. In addition to showing a significant reduction in the amount of attached viable cells, it was found that affected bacterial cells were always associated with the larger PuroA-functionalised colloidal particles within the BCC layers. This suggests that antimicrobial activity can be attributed to surface-bound AMPs rather than unfavourable surface topography provided by the BCC layer. The experimental work presented here was prudent for the proposed findings to be published in Applied Materials & Interfaces1: Boden, A., Bhave, M.,
Wang, P.-Y., Jadhav, S. & Kingshott, P. Binary Colloidal Crystal Layers as Platforms for Surface
Patterning of Puroindoline-Based Antimicrobial Peptides. ACS Appl. Mater. Interfaces 10, 2264-
127
2274, doi:10.1021/acsami.7b10392 (2018). The outcomes of these investigations are schematically shown below in Figure 18.
Figure 18. Schematic illustration of the preparation of PuroA-modified BCC layers and representation of the proposed mechanisms of bacterial attachment and antimicrobial activity. Reprinted with permission from Boden, et al. 1 Copyright (2018) American chemical society. 4.2 Introduction The prevention of bacterial attachment and growth by modification of both surface chemistry and topography has the potential to play an important role in a number of different application areas including implantable medical devices,2-5 water purification systems,6,7 and food processing equipment.8 There is however particular risks within the biomedical industry due to chronic infections associated with the use of indwelling medical devices such as catheters, stents and artificial bone replacements. Such infections caused by the attachments of bacteria to these devices can subsequently lead to increased medical costs, patient suffering, prolonged hospitalisation, and even patient death.9-11 Whilst the use of proper aseptic techniques and
128 antibiotics has reduced the incidence of such infections, the emergence of antibiotic-resistant bacteria and/or the formation of tenacious biofilms has prompted the need to develop a ‘new- generation’ of antimicrobial coatings.
Recent studies have indicated that two main approaches are used to control the attachment and growth of bacterial cells at an interface. These involve either; i) prevention of bacterial attachment (i.e. fouling) through unfavourable surface topographies and chemistry, and ii) incorporation of biocidal or biostatic agents to prevent and/or inhibit the growth of any attached bacteria.12,13 Several types of antifouling surfaces have been proposed which include the grafting of hydrophilic polymer brushes,14-16 and also the fabrication of nano/micro-structured topographies inspired from naturally occurring surfaces.17-19 The use of antifouling polymer brushes provides further functionalisation possibilities – as a range of antibiotic molecules can potentially be immobilised to terminal functional groups via numerous coupling strategies.20
However, long polymerisation and processing times,7,21 and the presence of brush defects currently limits the use of such coating in a practical setting.
Bacterial attachment to nano/microstructured surfaces prepared by a range of lithographic techniques have also been investigated, with results strongly suggesting that surface topography plays an important role in the attachment of bacterial cells.22,23 Previous research has shown that certain nanometer sized topographies on titanium surfaces are capable of reducing the adhesion of
Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa when compared to flat control surfaces.24 Additionally, it has also been shown that nanometer sized topographical features are responsible for the bactericidal properties of cicada (Psaltoda calripennis) wings.19 More recently, a novel approach has been developed for producing ordered micro- and nano-topographies over large areas using binary mixtures of colloidal particles that
129 self-assemble into layers with defined stoichiometries.25,26 These binary colloidal crystal (BCC) layers enable the fabrication of surfaces with specific topographies and chemistries, which is highly desirable in many applications including photonics,27,28 biomedicine,29 and have even been applied in the fabrication of surface-enhanced Raman scattering (SERS) substrates.30,31 Recently, these BCC layers have been used as a platform for the for the production of highly-ordered mixed protein patterns, which also suggests that a range of other (bio)molecules may be immobilised to these layers in a selective and patterned manner.32
Traditionally, the incorporation of bactericidal and bacteriostatic agents into biomedical coatings has been limited to antibiotics,33,34 quaternary ammonium salts35 and metal ions including silver,36 copper and zinc.37 However, these approaches pose some problems such as cytotoxic side- effects and the development of antibiotic resistant strains of bacteria. Antimicrobial peptides
(AMPs) may be able to overcome such obstacles as these highly cationic peptides possess a range of unique properties including a broad-spectrum of activity, high efficacy at low concentrations, and an inherently low propensity for developing bacterial resistance.20,38 AMPs are part of the innate immune system of many organisms and target the negatively charged membranes of bacterial cells through electrostatic interactions from positively charged amino acids.39 Due to their susceptibility to proteolytic degradation and peptide self-aggregation at high concentrations,40 in a biomedical setting, covalent immobilisation and their synthetic derivatives is the preferred method to overcome such problems whilst maintaining antimicrobial activity. For initial immobilisation investigations the AMP; PuroA (FPVTWRWWKWWKG-NH2) - which is a highly cationic peptide derived from the tryptophan-rich-domain (TRD) of wheat puroindoline proteins, was chosen due to its broad-spectrum of antimicrobial activity,41,42 and also its stability over a wide pH range (pH 2-12) and high temperature (130°C).43 However, as our knowledge of AMPs expand it has been possible to produce synthetic analogues that possess specific antimicrobial 130 activity and enhanced performance to their predecessors.44-46 Two such AMPs; P1 and W8, which are based upon the amino acid sequence of PuroA, show promising potential for AMP immobilisation to solid supports as they have shown to be active against a number of bacteria and fungi,41 and may be immobilised by their N-terminus via a number of different chemical reactions.
Similar to PuroA, P1 (RKRWWRWWKWWKR-NH2) and W8 (WRWWKWWK-NH2) have an overall net positive charge and high Trp (W) content to attract and destabilise bacterial membranes, however it is important to assess the AMPs activity in solution against clinically relevant organisms prior to their immobilisation.
The experimental results outlined below utilise BCC layers as a platform with inherent nano/microstructures for the covalent immobilisation of AMPs and present a surface coating method based on AMP-functionalised nano/micro-rough surfaces, along with an examination of their antibacterial activities. Additionally, the minimum inhibitory concentration (MIC) of two synthetic AMPs; P1 and W8, which are based upon the amino acid sequence of PuroA, were assessed against E. coli (ATCC 25922) and P. aeruginosa (ATCC 10145) using a modified broth microdilution method.47
4.3 Results and discussion 4.3.1 Particle modification and characterisation
Immobilisation of PSC2 colloidal particles with PuroA was assessed using zeta potential measurements, XPS, and surface MALDI-ToF-MS. The zeta potential data at pH 5.4 (Figure 19) shows that there is a large change in surface charge of the colloidal particles suggesting that PuroA was successfully immobilised the particle surface. Zeta potentials of the unmodified PSC2 particles (-25±9mV) were similar to that of PSC2 particles treated with EDC/NHS but without
PuroA (-28 ±9mV), suggesting that activation of COOH groups and subsequent hydrolysis of NHS
131 active esters does not significantly affect surface charge, and any change observed is likely to be due to the AMPs themselves. Results for unmodified PSC2 particles are seen to be somewhat different to previously published data (≈-45mV),29 which was likely caused by the lower pH used here, which may have increased the proportion of protonated COOH groups, or the particles may have a lower than expected COOH surface density. Zeta potentials of samples processed without the addition of coupling reagents (i.e. no EDC/NHS) suggested a considerable amount of PuroA was physically adsorbed to the particle surface – which was not surprising considering the highly cationic nature of PuroA and negative zeta potential of unmodified PSC2 particles. There was however a larger change in surface charge seen for covalently immobilised PuroA samples
(+48±8mV) when compared to physically immobilised samples (+28±5mV). This could have been caused by a different orientation of the AMPs; as physically immobilised peptides will orientate themselves to a position that is entropically favourable and minimises the electrostatic repulsive forces.48
Figure 19. Zeta potential data for unmodified and PuroA-modified PSC2 particles. Sample type is indicated on the y-axis and zeta potential in mV is indicated on the x-axis. Values presented are mean (mV) ± standard deviation (n=5). Reprinted with permission from Boden, et al. 1 Copyright (2018) American Chemical Society.
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XPS analysis was used to determine the elemental composition and chemical states of atoms within the outer 10nm of the particles surfaces. The atomic composition results (Table 8) indicate that there is a moderate increase in At% N for both covalent (4.1±0.5%) and physically immobilised (2.7±0.2%) samples when compared to controls, which is expected after successful protein immobilisation.32 Low levels of sodium and chloride (≈ 0.1%) was also detected and is not surprising considering NaCl is a major component of the PBS buffer used. Data shown for the analysis of bulk films of PuroA (Table 8) showed the presence of fluorine which was likely due to some residual trifluoroacetic acid that was present after PuroA purification by HPLC by the supplier. The presence of fluorine is also confirmed by high-resolution C1s spectra (Fig. 20a) with
49 peaks being assigned to CF3 at a binding energy of 292.6eV. For calculations of the thickness and surface coverage of the PuroA films on PSC2 colloidal monolayers, the contribution of fluorine in these samples was omitted (w/o F) as to obtain more accurate representations of the
%N in the bulk PuroA film.
Table 8. XPS atomic composition data of PuroA-modified and unmodified PSC2 particles
Atomic % Sample ID O C N F Si PuroA 14.2 ± 2.7 65.2 ± 4.6 12.4 ± 3.5 8.2 ± 3.5 - PuroA (w/o F) 15.6 ± 3.6 71.0 ± 2.3 13.4 ± 1.4 - - PSC2 9.5 ± 2.9 87.6 ± 3.8 1.6 ± 0.9 - 1.5 ± 1.2 PSC2-PuroA 37.8 ± 1.1 46.8 ± 2.6 4.1 ± 0.5 - 11.3 ± 2.0 PSC2-PuroA Phys 26.0 ± 1.1 63.9 ± 2.2 2.7 ± 0.2 - 8.0 ± 1.2
Table 8 also shows the presence of Si for all PuroA-modified PSC2 particles. This was however, attributed to the glass (SiO2) substrate rather than contamination as the increase in Si was also accompanied by a substantial increase in %O. Elemental composition of unmodified
PSC2 colloids also had small traces of nitrogen, which is most likely attributed to residual surfactant that is removed by rinsing before PuroA immobilisation, or chemical groups used to 133 generate COOH on the PS surface. Compared to theoretical O/C ratios for carboxylated PS (≈0.2), the results gave a lower ratio (≈0.1) suggesting that the number of COOH groups may be quite low, which is also supported by zeta potential results that were of lower magnitude than expected for unmodified PSC2 particles. Moreover, for covalently immobilised samples there was a considerable increase in %N after PuroA incubation (4.1%) when compared to unmodified PSC2 particles (1.6%). This reaffirms zeta potential data that PuroA immobilisation was successful, albeit below the expected value for PuroA (≈13%) as determined by the %N obtained from bulk films of PuroA.
Using atomic composition data obtained from XPS survey spectra (Table 8) the thickness of PuroA layers (z) (in a dry state) and surface coverage can be calculated using the overlayer equation (See Equation 2).50 This is under the assumption that the particle surface has random roughness with a range of emission angles51
� Equation 2: � = −����� × cos � × ln (1 − ) �∞ where I and I are the %N determined from the AMP-coated samples and the reference AMP, respectively. Θ is the angle between the sample and analyser, which is estimated to be 57.3° for
51 randomly rough particle surfaces, and λIMFP is the inelastic mean free path of N1s photoelectrons emitted from the AMP (2.5nm).32 Subsequent surface coverages (Θ) of the AMPs can be calculated from z values assuming an AMP density of 1.4g/cm3.50
Table 9 shows the calculated thickness and surface coverage values for unmodified and
PuroA–modified PSC2 particles. For more accurate estimation on the amount of immobilised
PuroA, the calculated PuroA surface coverage from unmodified PSC2 particles was subtracted from the other samples before calculating the immobilisation density of PuroA peptides
(molecules/cm2). These concessions indicate that the densities of PuroA immobilised on PSC2 134 colloidal particles were 1.93x1013 and 7.14x1012 molecules/cm2 for the covalently and physically immobilised PuroA, respectively. Using product specifications for PSC2 colloids (Lot no.
1071790) (www.thermofisher.com), the total number of COOH groups/cm2 can be calculated
(1.11x1014) and compared to observed PuroA densities for physically and covalently immobilised samples. It appears that the efficiency of the immobilisation was considerably low; being approximately 17% and 6% for covalently and physically immobilised samples, respectively.
However, assuming that less than half of the occupied COOH groups will be available for analysis by XPS when presented as a colloidal multilayer, in reality, the immobilisation efficiency is expected to be higher than what was observed.
Table 9. Thickness and surface coverage of PuroA-modified and unmodified PSC2 particles
z Θ Corrected Θ Density Sample ID (nm) (mg/m2) (mg/m2) (molecules/cm2) PSC2 0.26 0.36 - - PSC2-PuroA 0.72 1.01 0.65 2.11E+13* PSC2-PuroA Phys 0.45 0.63 0.27 8.88E+12* *Densities of PuroA were calculated using a MW of 1862.0Da obtained from supplier’s specifications (Mimotopes, Clayton,Vic) High-resolution C1s spectra generated from PuroA-modified PSC2 colloidal layers (Figure
20) showed certain peaks for all samples including those corresponding to C=C (284.9eV), C-C/C-
H (285.5eV), and also a weak shake-up satellite peak attributed to π-π* transitions (291.6eV) in the benzene ring of PSC2 colloidal particles. As the majority of PSC2 colloids are composed of aromatic benzene rings, peak intensities for C=C at approximately 284.9eV are observed to be greater than those for C-C/C-H photoelectrons due to the high proportion of aromatic carbons.
These peaks are also present in the PuroA only sample which is attributed to aromatic Trp and Phe residues.52 High-resolution C1s spectra also show that there is an increase in the components that can be assigned to N-C=O photoelectrons at approximately 288.5eV, for both physically (Fig. 20d) and covalently (Fig. 20c) immobilises samples, which again suggests that PuroA immobilisation 135 was successful to the PSC2 colloidal particles. Interestingly, the high-resolution C1s spectra obtained for unmodified PSC2 colloids (Fig. 20b) shows no peak that can be assigned to O=C-O binding energies at approximately 289.5eV, which would be expected to be present for carboxyl- containing particles.53 Considering the lower than expected zeta potentials and the considerably low contribution of oxygen obtained for bare PSC2 colloids, it is likely that the surface concentration of COOH groups is actually quite low resulting in the lower than expected PuroA surface coverage. Furthermore, films of PuroA (Fig.20a) show that the contribution of C=C photoelectrons at approximately 284.8eV has much less prominence than that of C-C/C-H, which was unexpected considering that 46 of the 97 carbons (47%) from PuroA possess aromaticity. This may be explained by the presence of trifluoroacetic acid that is used during the peptide purification process which is also confirmed using XPS survey spectra indicating the presence of fluorine within these samples.
136
Figure 20. High-resolution C1s XPS spectra of: a) PuroA, b) unmodified PSC2 particles, c) PuroA covalently immobilised to PSC2 particles, and d) PuroA physically adsorbed to PSC2 particles. Reprinted with permission from Boden, et al. 1 Copyright (2018) American Chemical Society.
Surface-MALDI-ToF-MS was utilised to assess if the PuroA peptides were successfully and covalently immobilised to the surface of the PSC2 colloidal particles - as no peptide signal should be detected in subsequent MALDI spectra due to the soft ionisation process that does not have enough energy to break the amide bond formed between PuroA and the PSC2 colloid.54,55
Corresponding MALDI spectra are shown below in Figure 21, with the presence of PuroA being confirmed by the peak observed at an m/z of 1860.8 – which corresponds to the protonated molecular ion [M+H]+ (Fig. 21b), where M corresponds to PuroA
+ + (FPVTWRWWKWWKWWKG-NH2). Sodium [M-H+Na] and potassium [M-H+K] adducts can also be seen at m/z values of 1883.4 and 1899.1, respectively, which was not surprising as Na and
K are components in PBS which was used during the coupling procedure. Peaks observed at m/z values of 1893.0 and 1908.0 that were seen in physically adsorbed samples are yet to be
137 definitively assigned but it is speculated that these could have been caused by PuroA complexes
+ + + 56 formed with the loss of multiple H2O molecules and the addition of H ,K , or Na ions.
0.3 a) 0.2 0.1 0.0
500 1860.8(sn749745) 1883.4 b) 1899.1 250
0 1860.9(sn353) c) Intensity (%) Intensity 1.0
0.5
0.0 1860.8(sn2540) 1883.0 1893.0 50 1899.0 d)
25 1908.0
0 1800 1900 2000 2100 2200 m/z
Figure 21. Surface MALDI-ToF MS spectra of PuroA-modified PSC2 particles over an m/z range of 1750- 2250Da. Spectra shown are of a) unmodified PSC2 particels, b) free PuroA, c) PuroA covalently immobilised to PSC2 particles, and d) PuroA physically adsorbed to PSC2 particles. Major peaks are labelled with their detected molecular weighs (Da) and signal to noise ratio (sn) is noted for the molecular ion [M+H]+. Reprinted with permission from Boden, et al. 1 Copyright (2018) American Chemical Society.
For the mass range of 1750-2250Da, the MALDI spectra shows clear peaks at m/z = 1860.9 and 1860.8 which confirms the presence of PuroA in both physically adsorbed (Fig. 21d) and covalently coupled samples (Fig. 21c). The considerably low signal-to-noise ratio (S/N) for
[M+H]+ in covalently immobilised sample (S/N = 353), when compared to physically immobilised samples (S/N = 2540) indicates that a small proportion of PuroA molecules are physically adsorbed to the PSC2 particle surface. Thus, MALDI-ToF-MS analysis provides a way to quantitatively confirm that the covalent binding of PuroA to PSC2 particles using EDC/NHS coupling chemistry
138 was somewhat successful, however that were still some traces of physically adsorbed PuroA in the covalently immobilised samples. Although this finding, to the best of our knowledge, has yet to be reported for the covalent immobilisation of AMPs, comparable results have been observed with
DNA-based immobilisation55 and small proteins.57 This highlights the importance of thorough characterisation of immobilised AMPs and antimicrobial agents as potential leaching of physically adsorbed molecules from functionalised surfaces could by cytotoxic in certain applications.38 In addition to this, given the potential for leaching of physically adsorbed AMPs in covalently immobilised samples, the mode of action may be incorrectly interpreted if not all molecules are irreversibly attached to surfaces.
4.3.2 Binary colloidal crystal formation and characterisation
Surface topographies and structures of binary colloidal crystal (BCC) layers prepared with unmodified 2µm PSC particles and 0.110µm PMMA particles (PSC2-PMMA011) were observed using SEM (Fig. 22a). The BCC layers were observed to form a hexagonally close-packed arrangement comprised of large PSC2 particles with the smaller PMMA particles assembling within the interstitial spaces between the larger particles. This structure was similar to previous observation using particle combinations with similar size ratios (γ = 0.055).29 SEM micrographs of BCC layers also indicate that the prepared particle combinations can form long-range ordered structures which facilitates patterned immobilisation of PuroA AMPs and potentially a wide-range of other biomolecules. A similar long-range ordering was observed for PuroA-modified BCC layers (Fig. 22b), however, it was evident that defect areas and disordered arrangements were more prominent within the modified-BCC layers (red box). This suggests that the large reversal in surface charge on the modified particles may have perturbed BCC formation, since is it is well established that the repulsive electrostatic interactions are important for the production of well- ordered BCCs.29 Furthermore, the areas between the larger particles, which are occupied by small 139
PMMA particles appear to increase after functionalisation with PuroA. This was likely due to the smaller PMMA particles adsorbing to PuroA-modified colloids through electrostatic interactions, or a relative increase in the proportion of smaller PMMA particles compared to larger PSC2 particles as some larger PSC2 colloids may have been lost in the multiple washing steps during functionalisation.
Figure 22. SEM images of PSC2-PMMA011 BCC layers showing a) unmodified BCCs, and b) PuroA- modified BCC layers. Size ratio (γ) of the BCC layer is 0.055. Reprinted with permission from Boden, et al. 1 Copyright (2018) American Chemical Society.
4.3.3 Antibacterial investigations
To assess the activity of unbound PuroA, the minimum inhibitory concentration (MIC) was first determined against E.coli. Using the modified broth micro-dilution method described previously, PuroA was found to be active at a MIC of 32µg/mL (data not shown), which is somewhat higher than the previously reported MIC against E.coli of 16µg/mL.41 The antibacterial activity of PuroA, and also a vast majority of other AMPs, is attributed to their hydrophobic and cationic properties, allowing them to be attracted to negatively charged bacterial cell walls and insert their hydrophobic motifs into the lipophilic portion of the membrane.38 As this process is highly dependent on AMP concentration and also the starting inoculum of bacterial cells, this can
140 make MIC values difficult to compare between studies. In this respect, the control of AMP indolicidin also showed a higher MIC value against E.coli (64µg/mL) when compared to that of previous studies, where the observed differences in activities seen here may be caused by small variations in starting inoculums rather than differences in AMP activity.
The antimicrobial properties of PuroA-modified BCCs after 24h incubation with E.coli was assessed using propidium iodide (PI) uptake as an indicator of compromised membranes (Fig.
23). Similar to MIC investigations, a 24h incubation period was chosen for this experiment as an end-point to determine if the coating can remain active over sustained periods of time, which is required for in vivo applications. The results show a substantial increase in the number of PI- positive cells for all PuroA-modified BCCs (Fig. 23c,d) compared to that of unmodified BCCs
(Fig. 23b) and PS-coated glass slides (Fig. 23a). The apparent number of adherent E.coli cells per field of view was similar for all samples except the PS coated glass, which exhibited a slightly lower number of adhered cells. This observation was somewhat expected considering E.coli cells may withstand washing procedures when attached to micro- and nanoscale structures found within the BCCs. Moreover, the highly cationic nature of PuroA would provide a more favourable electrostatic environment for attraction to the negatively charged bacterial membranes.39 The visual observations were also substantiated using ImageJ software, which shows a clear decrease in the viability of E.coli cells for all PuroA-modified BCCs when compared to that of control samples (Figure 24). More than 70 and 50% of adherent E.coli cells were PI-positive for covalently and physically immobilised samples, respectively. These values were significantly higher than that of PS-coated glass and unmodified BCCs, which exhibited approximately 10 and 15% PI-positive cells, respectively. Furthermore, considering the presence of small traces of physically adsorbed
PuroA in covalently immobilised samples - which was confirmed by MALDI – it is difficult to attribute the activity solely due to covalently immobilised PuroA. However, it is clear that PuroA 141 presents surface-localised activity toward E.coli cells when covalently and/or physically immobilised to PSC2-PMMA011 BCC layers.
Although the significant decrease seen in the viability of E.coli cells for PuroA physically or covalently immobilised to PSC2-PMMA BCCs is promising for the production of effective antimicrobial coatings, direct comparisons with other literature reports are not always appropriate because of different experimental conditions,58 starting inoculum,40,48,58 and strain-or species- specific differences in bacterial responses to various surface topographies and chemistries.18
Therefore, it is necessary to investigate responses of these surfaces to a number of bacterial species/strains for a better understanding of their spectrum of antimicrobial potential.
Figure 23. Representative fluorescent microscopy images after 24 h incubation showing live (green) and membrane compromised (red) E. coli cells adhered to: a) PS-coated glass slides, b) unmodified PSC2PMMA011 BCCs, c) PuroA covalently immobilised to PSC2-PMMA011 BCCs, and d) PuroA physically adsorbed to PSC2-PMMA011 BCCs. Images were taken at 1000X magnification under immersion oil, and six images were taken per sample. Reprinted with permission from Boden, et al. 1 Copyright (2018) American chemical society. 142
Figure 24. Viability of adherent E.coli cells attached to PuroA-modified PSC2PMMA011 BCC layers. Values presented are percentage Live/Dead ± standard deviation (preliminary y-axis) and the total number of bacteria per field ± standard deviation (secondary y-axis). Sample description is indicated on the x-axis, and statistical analysis was performed using a paired-samples Students T. Test. Values of p < 0.05 were considered significant and indicated using *. Reprinted with permission from Boden, et al. 1 Copyright (2018) American Chemical Society.
Figure 24 also shows the average number of bacteria observed per field of view in the microscopy images after the 24h incubation period and results suggest an increased attachment to the modified surfaces compared to that of the relatively planar PS controls. It is widely known that bacteria will alter their phenotype and attachment profiles depending on the surface topography presented,59,60 and it suggested that the inherent micro- and nano-topographies presented here not only facilitate AMP penetration but also provides attachments sites for bacterial cells which is shown schematically in Figure 24.
143
Figure 25. Illustrative representation of the proposed mechanism of bacterial attachment and antimicrobial activity. Planktonic cells may adhere to AMP-coated particles causing membrane destabilisation and death, or alternatively may attach within the interstitial spaces of the AMP-modified PSC2 colloids. Reprinted with permission from Boden, et al. 1 Copyright (2018) American Chemical Society.
SEM images in Figure 26 show representative micrographs of adherent E. coli cells to the different substrate types. Upon incubation with PuroA-modified BCCs, differences in cell structure were seen when compared to adherent cells on unmodified BCC layers – with cells appearing to show the leakage of cellular components after membrane disruption (green ellipse). Interestingly, deflated cells were only observed on PuroA-modified samples only and the majority of the time they were associated with a nearby large PSC2 particle, which suggests that the antibacterial activity observed was likely due to the immobilised AMP. Additionally, the images shown in
Figure 26 confirm our hypothesis that nano-and micro-topographies can influence bacterial attachment as it was observed that many of the adherent cells were found within the low-lying sites of the BCC layers as opposed to the protruding PSC2 particles. This result is quite interesting, as it suggests that site-specific attachment of bacterial cells can be achieved using patterned BCC layers, which has several potential applications in a number of clinical and diagnostic materials.61-
63
144
Figure 26. Representative SEM images of adherent E. coli cells to a) PC coated glass slides, b) unmodified PSC2PMMA011 BCC layers, c-d) PuroA-modified PSC2PMMA011 BCC layers. Scale bar: 1µm. Reprinted with permission from Boden, et al. 1 Copyright (2018) American Chemical Society.
While surface-immobilised AMPs have only been reported a modest number of times within current literature, there is a general consensus that AMP immobilisation using flexible spacer molecules will enhance activity when compared to zero-length immobilisation.20,40,48 For example, zero-length immobilisation of the AMP; chrysophsis-1 (CHY1) resulted in approximately 34% killing of E. coli (ATCC 33694), whereas the addition of a flexible poly(ethylene glycol) (PEG) linker resulted in approximately 80% killing of adherent cells.48 The relative amounts of immobilised AMPs in each case were determined by quartz crystal microbalance with dissipation monitoring and were calculated to be 1.01x1015 and 4.56x1014 molecules per cm2 for zero-length and PEG-linker immobilisations, respectively. This indicates that a higher AMP loading onto a planar surface is not sufficient to counteract the decrease in lateral mobility and penetration depth.
Another research group also showed that the AMP; IG-25 grafted at a density of 1.6x1013 molecules per cm2 via a flexible PEG linker could significantly reduce the viability of P. aeruginosa (GFP-PA01) cells by more than 80% when compared to control samples. It is noted however that the relative amount of AMP covalently immobilised to the surface within this study is compared only to the total amount of AMP used in the reaction of these reports. Therefore, without any subsequent surface analysis it is difficult to determine the respective amounts of physically and covalently immobilised AMP and also makes it difficult to ascertain the source of the antibacterial activity. 145
Considering the absence of a flexible linker molecule used in this work, yet the relatively high activity seen for the PuroA-modified surfaces, the surface activity noted here is likely to be due to the inherent nano- and micro-topographies of BCC layers,25,29 potential AMP leaching, in addition to the high Trp content and potency of PuroA.41 The combination of these properties may have allowed efficient entrance and subsequent destabilisation of bacterial membranes, and it is hypothesised that the necessity for spacer molecules may be reduced by tethering AMPs to surfaces with inherent nano- and micro-structures – thus allowing for greater penetration depth compared to zero-length immobilisation to planar surfaces.
4.3.4 AMP selection and activity
Shown below in Table 10 are the determined MIC values for the assessed AMPs against
E.coli and P. aeruginosa. For E. coli, results show that both P1 and W8 have lower MIC values
(16µg/mL) when compared to PuroA (32µg/mL). Similarly, for P. aeruginosa, MIC values were observed to be at a lower value for P1 (8µg/mL) and W8 (16µg/mL) when compared to PuroA
(32µg/mL).
Table 10. MICs of selected puroindoline-based synthetic AMPs MIC (µg/mL) Peptide ID Sequence E. coli P. aeruginosa
PuroA FPVTWRWWKWWKG-NH2 32 32
P1 RKRWWRWWKWWKR-NH2 16 8
W8 WRWWKWWK-NH2 16 16
Figure 27 below also shows plate images taken for the P1 and W8 MIC assays after the
24h incubation period. It can be clearly be seen that bacterial growth occurs at low AMP concentrations due to the presence of turbidity in the wells. It is also apparent that turbidity was present in wells that contained 250µg/mL of AMPs, however this not due to bacterial growth as
146 an aliquot of these wells were taken and plated on NA plates – and no growth was observed. It is likely that at high AMP concentrations the peptide/peptide and cell/peptide interactions are greatly increased leading to agglomeration of AMP-cell complexes.
Figure 27. Plate images of MIC assays after 24h incubation with P1 and W8 AMPs. The above image shows MIC results obtained against E. coli (left) and P. aeruginosa (right) with AMP concentrations (in µg/mL).
Using the molecular weights of AMPs calculated using ExPASy
(https://web.expasy.org/compute_pi/) the molarity MIC can be determined, which indicates that
P1 and W8 are active against E.coli at concentrations of 0.2µM and 1.7µM, respectively. When comparing these MIC values to AMPs previously used in immobilisation studies, the puroindoline- based AMPs clearly outperform the more commonly used AMPs such as indolicidin, magainin I, magainin II, and melamine, whose MIC values against susceptible E. coli strains are all within the range of 2-50µM.40,64,65 Also, considering Boden, et al. 1 has demonstrated retained activity of
PuroA when immobilised to BCC layers, it is expected to be advantageous to investigate to immobilisation of more potent AMPs: P1 and W8 for increased antimicrobial performance.
4.4 Conclusions In conclusion, PuroA was successfully immobilised to 2µm PSC particles using EDC/NHS coupling chemistry, and when presented as BCC layers using smaller 110nm PMMA particles this 147 led to a significant decrease (>70%) in the viability of surface-adherent E. coli on the modified surfaces. Thus, the presentation of PuroA using PSC2-PMMA011 BCC layers shows promise for the potential development of antibacterial coatings, and also provide a suitable platform for the site-specific tethering of other biomolecules to surfaces with a range of well-defined surface chemistries and topographies. In addition, these investigations also highlighted that declaring the antibacterial activity solely based on the action of covalently immobilised AMPs can be misleading, it was shown here that small amounts of physically bound PuroA was detected using
MALDI-ToF-MS, which may have contributed to the surface-localised antibacterial activity observed. While PuroA demonstrated activity against E.coli in an immobilised state, it was shown that other synthetic AMPs demonstrated superior antibacterial properties in solution when compared to PuroA. Considering this, future investigations will employ the more potent AMPs;
P1 and W8, and also address the use of spacer molecules to reduce steric hindrance and allow for greater penetration into bacterial membranes. Additionally, optimisation of the underlying graft layer and spacer immobilisation will need to be investigated with the aim of achieving high AMP loading for exceedingly active antimicrobial surface coatings.
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29 Wang, P. Y., Pingle, H., Koegler, P., Thissen, H. & Kingshott, P. Self-assembled binary colloidal crystal monolayers as cell culture substrates. J. Mater. Chem. B 3, 2545-2552, doi:10.1039/c4tb02006e (2015).
30 Zhang, X., Xiao, X., Dai, Z., Wu, W., Zhang, X., Fu, L. & Jiang, C. Ultrasensitive SERS performance in 3D "sunflower-like" nanoarrays decorated with Ag nanoparticles. Nanoscale 9, 3114-3120, doi:10.1039/c6nr09592e (2017).
31 Dai, Z. G., Xiao, X. H., Wu, W., Zhang, Y. P., Liao, L., Guo, S. S., Ying, J. J., Shan, C. X., Sun, M. T. & Jiang, C. Z. Plasmon-driven reaction controlled by the number of graphene layers and localized surface plasmon distribution during optical excitation. Light Sci. Appl. 4, doi:10.1038/lsa.2015.115 (2015).
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33 Kumar, A. S., Sornambikai, S., Deepika, L. & Zen, J. M. Highly selective immobilization of amoxicillin antibiotic on carbon nanotube modified electrodes and its antibacterial activity. J. Mater. Chem. 20, 10152-10158, doi:10.1039/c0jm02262d (2010).
34 Yoo, H. S., Kim, T. G. & Park, T. G. Surface-functionalized electrospun nanofibers for tissue engineering and drug delivery. Adv. Drug Del. Rev. 61, 1033-1042, doi:10.1016/j.addr.2009.07.007 (2009).
35 Zhang, X., Ma, J., Tang, C. Y., Wang, Z., Ng, H. Y. & Wu, Z. Antibiofouling Polyvinylidene Fluoride Membrane Modified by Quaternary Ammonium Compound: Direct Contact-Killing versus Induced Indirect Contact-Killing. Environ. Sci. Technol. 50, 5086-5093, doi:10.1021/acs.est.6b00902 (2016).
36 Sotiriou, G. A. & Pratsinis, S. E. Antibacterial activity of nanosilver ions and particles. Environ. Sci. Technol. 44, 5649-5654, doi:10.1021/es101072s (2010).
37 Kim, T. N., Feng, Q. L., Kim, J. O., Wu, J., Wang, H., Chen, G. C. & Cui, F. Z. Antimicrobial effects of metal ions (Ag+, Cu2+, Zn2+) in hydroxyapatite. J. Mater. Sci. Mater. Med. 9, 129-134, doi:10.1023/A:1008811501734 (1998).
38 Costa, F., Carvalho, I. F., Montelaro, R. C., Gomes, P. & Martins, M. C. L. Covalent immobilization of antimicrobial peptides (AMPs) onto biomaterial surfaces. Acta Biomater. 7, 1431-1440, doi:10.1016/j.actbio.2010.11.005 (2011).
39 Reddy, K. V. R., Yedery, R. D. & Aranha, C. Antimicrobial peptides: Premises and promises. Int. J. Antimicrob. Agents 24, 536-547, doi:10.1016/j.ijantimicag.2004.09.005 (2004).
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40 Costa, F., Maia, S., Gomes, J., Gomes, P. & Martins, M. C. L. Characterization of hLF1- 11 immobilization onto chitosan ultrathin films, and its effects on antimicrobial activity. Acta Biomater. 10, 3513-3521, doi:10.1016/j.actbio.2014.02.028 (2014).
41 Phillips, R. L., Palombo, E. A., Panozzo, J. F. & Bhave, M. Puroindolines, Pin alleles, hordoindolines and grain softness proteins are sources of bactericidal and fungicidal peptides. J. Cereal Sci. 53, 112-117, doi:10.1016/j.jcs.2010.10.005 (2011).
42 Alfred, R., Shagaghi, N., Palombo, E. & Bhave, M. 1395-1405 (2013).
43 Alfred, R. L., Palombo, E. A., Panozzo, J. F., Bariana, H. & Bhave, M. Stability of puroindoline peptides and effects on wheat rust. World J. Microbiol. Biotechnol. 29, 1409-1419, doi:10.1007/s11274-013-1304-6 (2013).
44 López-Pérez, P. M., Grimsey, E., Bourne, L., Mikut, R. & Hilpert, K. Screening and Optimizing Antimicrobial Peptides by Using SPOT-Synthesis. Frontiers in Chemistry 5, doi:10.3389/fchem.2017.00025 (2017).
45 Sinha, R. & Shukla, P. Antimicrobial Peptides: Recent Insights on Biotechnological Interventions and Future Perspectives. Protein Pept Lett 26, 79-87, doi:10.2174/0929866525666181026160852 (2019).
46 Fox, J. L. Antimicrobial peptides stage a comeback. Nat. Biotechnol. 31, 379-382, doi:10.1038/nbt.2572 (2013).
47 Wiegand, I., Hilpert, K. & Hancock, R. E. W. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 3, 163-175, doi:10.1038/nprot.2007.521 (2008).
48 Ivanov, I. E., Morrison, A. E., Cobb, J. E., Fahey, C. A. & Camesano, T. A. Creating antibacterial surfaces with the peptide chrysophsin-1. ACS Appl. Mater. Interfaces 4, 5891-5897, doi:10.1021/am301530a (2012).
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50 Paynter, R. W., Ratner, B. D., Horbett, T. A. & Thomas, H. R. XPS studies on the organization of adsorbed protein films on fluoropolymers. J. Colloid Interface Sci. 101, 233-245, doi:10.1016/0021-9797(84)90023-7 (1984).
51 Frydman, A., Castner, D. G., Schmal, M. & Campbell, C. T. A Method for Accurate Quantitative XPS Analysis of Multimetallic or Multiphase Catalysts on Support Particles. J. Catal. 157, 133-144, doi:10.1006/jcat.1995.1274 (1995).
52 Dettin, M., Herath, T., Gambaretto, R., Iucci, G., Battocchio, C., Bagno, A., Ghezzo, F., Di Bello, C., Polzonetti, G. & Di Silvio, L. Assessment of novel chemical strategies for covalent attachment of adhesive peptides to rough titanium surfaces: XPS analysis and biological evaluation. J. Biomed. Mater. Res. A 91, 463-479, doi:10.1002/jbm.a.32222 (2009).
53 Maeda, S., Corradi, R. & Armes, S. P. Synthesis and characterization of carboxylic acid- functionalized polypyrrole-silica microparticles. Macromolecules 28, 2905-2911 (1995).
54 McLean, K. M., McArthur, S. L., Chatelier, R. C., Kingshott, P. & Griesser, H. J. Hybrid biomaterials: Surface-MALDI mass spectrometry analysis of covalent binding versus physisorption of proteins. Colloids Surf. B. Biointerfaces 17, 23-35, doi:10.1016/S0927- 7765(99)00055-7 (2000).
55 O'Donnell, M. J., Tang, K., Köster, H., Smith, C. L. & Cantor, C. R. High-Density, Covalent Attachment of DNA to Silicon Wafers for Analysis by MALDI-TOF Mass Spectrometry. Anal. Chem. 69, 2438-2443 (1997).
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56 Keller, B. O. & Li, L. Discerning matrix-cluster peaks in matrix-assisted laser desorption/ionization time-of-flight mass spectra of dilute peptide mixtures. J. Am. Soc. Mass Spectrom. 11, 88-93, doi:10.1016/S1044-0305(99)00126-9 (2000).
57 Kingshott, P., St. John, H. A. W. & Griesser, H. J. Direct detection of proteins adsorbed on synthetic materials by matrix- assisted laser desorption ionization-mass spectrometry. Anal. Biochem. 273, 156-162, doi:10.1006/abio.1999.4201 (1999).
58 Héquet, A., Humblot, V., Berjeaud, J. M. & Pradier, C. M. Optimized grafting of antimicrobial peptides on stainless steel surface and biofilm resistance tests. Colloids Surf. B. Biointerfaces 84, 301-309, doi:10.1016/j.colsurfb.2011.01.012 (2011).
59 Pingle, H., Wang, P. Y., Thissen, H., McArthur, S. & Kingshott, P. Colloidal crystal based plasma polymer patterning to control pseudomonas aeruginosa attachment to surfaces. Biointerphases 10, 1-11, doi:10.1116/1.4936071 (2015).
60 Crawford, R. J., Webb, H. K., Truong, V. K., Hasan, J. & Ivanova, E. P. Surface topographical factors influencing bacterial attachment. Adv. Colloid Interface Sci. 179– 182, 142-149, doi:http://dx.doi.org/10.1016/j.cis.2012.06.015 (2012).
61 Stavis, C., Clare, T. L., Butler, J. E., Radadia, A. D., Carr, R., Zeng, H., King, W. P., Carlisle, J. A., Aksimentiev, A., Bashir, R. & Hamers, R. J. Surface functionalization of thin-film diamond for highly stable and selective biological interfaces. Proc. Natl. Acad. Sci. U. S. A. 108, 983-988, doi:10.1073/pnas.1006660107 (2011).
62 Lobaina, T., Zhurbenko, R., Alfonso, I., Rodríguez, C., Gala-García, A., Gontijo, S. L., Cortés, M. E., Gomes, A. & Sinisterra, R. D. Efficacy of coral-hydroxyapatite and biphasic calcium phosphate for early bacterial detection. Biointerphases 9, 029018, doi:10.1116/1.4880616 (2014).
63 Anselme, K., Davidson, P., Popa, A. M., Giazzon, M., Liley, M. & Ploux, L. The interaction of cells and bacteria with surfaces structured at the nanometre scale. Acta Biomater. 6, 3824-3846, doi:10.1016/j.actbio.2010.04.001 (2010). 156
64 Dathe, M., Nikolenko, H., Meyer, J., Beyermann, M. & Bienert, M. Optimization of the antimicrobial activity of magainin peptides by modification of charge. FEBS Lett. 501, 146-150, doi:https://doi.org/10.1016/S0014-5793(01)02648-5 (2001).
65 Alves, D. & Olívia Pereira, M. Mini-review: Antimicrobial peptides and enzymes as promising candidates to functionalize biomaterial surfaces. Biofouling 30, 483-499, doi:10.1080/08927014.2014.889120 (2014).
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5 In-situ investigation of grafting conditions and non-specific protein adsorption to PEG layers with surface plasmon resonance
5.1 Summary The incorporation of a flexible polymer linker can add greater mobility and penetration of immobilised AMPs into bacterial cell membranes, which in turn will also increase their efficacy as an active antimicrobial coating. Due to the extensive polymer immobilisation methods available, this chapter will investigate and characterise the grafting of PEG by reductive amination and carbodiimide crosslinking in-situ using SPR to monitor molecular interactions in real-time.
Bovine serum albumin (BSA) is used as a model protein to assess the polymer layers’ non-fouling ability. As functional group density of the underlying graft layer will subsequently dictate polymer grafting densities, interchanging the underlying graft layer from physically adsorbed PEI layers to covalently bound APTES layers was also assessed in order to optimise grafting conditions. It was shown that PEG grafting by reductive amination to physically adsorbed PEI layers was far more effective and exhibited superior non-fouling properties compared to other conditions.
Additionally, the use of high salt concentration (0.6M K2SO4) and elevated temperature (40°C) was found to be a crucial factor to achieve dense PEG layers by reductive amination, however this was determined to hinder grafting using carbodiimide crosslinking chemistry in these circumstances. Results presented in this Chapter have been presented at the Australasian Society for Biomaterials and Tissue Engineering (ASBTE) conference 2017, Canberra, Australia; Boden,
A., Bhave, M., Wang, P.-Y., & Kingshott, P. In-situ investigation of PEG grafting using surface plasmon resonance (SPR) to optimise coatings to prevent non-specific protein adsorption. ASBTE,
Canberra (2017). 158
5.2 Introduction One of the aims of this research is the addition of flexible polymer linkers (so-called non- fouling polymers) to provide an additional barrier against protein and bacterial interaction with surfaces, and also to allow for greater penetration of AMPs into bacterial membranes when they are tethered to the end of the polymer chains. Current research has shown that there are many types of polymers with a variety of chemical structures that are available for such purposes. The general design criteria for non-fouling polymers are that they should be hydrophilic and electrically neutral, and also have hydrogen bond acceptors but little or no hydrogen bond donors.1-3 As described previously, poly(ethylene glycol) (PEG) is one such polymer that has been used frequently in surface modification investigations and displays several desirable characteristics including hydrophilicity and no cytoxicity.4,5 However, it is also important that the polymer chains are grafted at sufficient densities to impart non-fouling properties at the surface, which in turn will also lead to higher AMP loading. Using reductive amination, Kingshott, et al. 6 has shown that the use of ‘cloud-point’ (CP) or poor solvation conditions can significantly reduce the hydrodynamic radius of PEG chains to facilitate high density grafting at an interface (Figure 28). It was shown that PEG layers prepared at ‘non-CP’ conditions exhibited some degree of protein adsorption, whereas for those grafted at ‘CP’ conditions (60°C, 0.6M K2SO4), there was no protein adsorption detected as confirmed by XPS. This highlights that the solvation of PEG chains is a critical parameter that can be manipulated to control grafting density, which can be ideal in attempting to immobilise multiple types of (bio)molecules at an interface.7,8
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Figure 28. Visual representation of the grafting of polymers under ‘good’ and ‘poor’ solvation conditions showing how high density polymer layers can be achieved to impart nonfouling properties.
While it is crucial that polymers be immobilised at sufficient densities, it is also just as important that sufficient numbers of pinning sites exist, and that they possess the appropriate chemical functional groups that are already present on the substrate.6,9 For the most part, this is achieved by either; physically adsorbing an oppositely charged molecule to the surface, or covalent immobilisation to surface functional groups. While physically adsorbing molecules to facilitate polymer grafting can be a versatile approach with a large variety of molecules and surfaces being available,9,10 long-term stability may be with problem marine coatings, and food preparation surfaces where prolonged stability is required. On the other hand, covalent immobilisation of molecules to produce reactive functional groups is more robust, however without prior surface treatments is somewhat limited to metal and semi-conducting metal hydroxides9,11,12
Arguably the most common treatment for inorganic surfaces is the grafting of organosilanes in organic solvents to surface hydroxides, with many types of organosilanes being 160 available for such purposes depending on functional group requirements needed for a particular application.13,14 However, the reaction is quite a complex process involving the hydrolysis of methoxy or ethoxy groups on silane molecules followed by the condensation with surface hydroxides (Figure 29). Additionally, there are some factors such as humidity and history of laboratory glassware that can result in film properties that deviate under seemingly identical experimental conditions.15 For the most part, methods that have been previously described for organosilane deposition have focussed on solution-based (SD),16-18 and vapour-based deposition
(VD) methods.19-21 The SD technique involves deposition in organic solvents to prevent rapid hydrolysis and polymerisation of silanes in solution. Under many circumstances, hydrolysis occurs at the substrate interface where surface-bound H2O initiates the reaction followed by condensation with surface hydroxides. On the other hand, VD methods involve increasing the partial pressure of the organosilane within a closed system by either; increasing the temperature of the system or decreasing the pressure using a vacuum pump. In general, silane films produced using VD methods tend to exhibit higher order and greater quality, with less probability of forming multilayers.20,22
However, this method also require additional equipment such as pumps, furnaces, lasers, and cooling systems to dissipate large amounts of thermal energy. Regardless of the deposition technique applied, a curing process is always applied to the silane film post-deposition to remove excess solvent, which is usually performed in an oven at temperatures ranging from 100°C-120°C for 5-60min.12,17,23
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Figure 29. Schematic illustration of the reaction between an organosilane and surface hydroxides. The reaction proceeds by: a) hydrolysis of methoxy of organosilanes, followed by b) condensation with surface hydroxides with methanol being formed as a by-product.
For silanisation using APTES, the majority of research has investigated the effects of concentration, solvent type, reaction time and reaction temperature, suggesting that solvent type and deposition time are the critical parameters that influence film structure and reactivity.13,15,24
For example, Kim, et al. 13 has shown for deposition in toluene, ethoxy groups are not completely hydrolysed until near the surface and subsequently condense with surface silanols and adjacent
APTES leading to lateral film growth. However, deposition in an aqueous solution (PBS) readily hydrolyses ethoxy groups in solution, and partially protonated amino groups are likely to adsorb to surface hydroxides to promote vertical film growth (Figure 30). Additionally, Howarter and
Youngblood 24 showed that at limited deposition times (≤1hr), smooth films of similar thickness could be obtained regardless of the concentration used. At longer deposition times and higher concentrations however, films were of lower quality, being much rougher and containing APTES agglomerates.
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Figure 30. Schematic illustration of APTES adsorption in an aqueous solution. The initial adsorption is driven by the electrostatic interactions between surface silanols and protonated amino groups with minimal condensation to form siloxane bonds. a) Subsequent deposition then proceeds by hydrogen bonding and/or electrostatic interactions to establish APTES multilayers b).
The incorporation of a flexible PEG linker to impart non-fouling properties and an extra degree of AMP mobility is important for the development of highly active antimicrobial surface coatings.25,26 As such, the Chapter herein investigates the effect of chain length and solvation conditions on the immobilisation of PEG to physically adsorbed poly(ethylenimine) (PEI) layers using reductive amination. However, as the integrity and quality of the underlying graft layer will subsequently dictate the maximum immobilisation densities of PEG and AMPs, and it is also necessary to investigate the effects of manipulating the underlying graft layer to increase the efficiency of downstream grafting processes. Therefore, this Chapter will also describe the optimisation and characterisation of APTES films using SD techniques to prepare covalently bound amino-terminated films which will then be compared to physically adsorbed PEI layers for their ability to facilitate PEG immobilisation. All the prepared coatings were assessed to determine
PEG thickness and their protein resistant properties in-situ using SPR as it is a powerful tool to
163 assess molecular interactions and binding kinetics at an interface,27,28 and can also provide valuable quantitative information regarding polymer layer thickness and surface coverage.
5.3 Results and Discussion
5.3.1 In-situ surface modification and protein adsorption using reductive amination
The principle behind CP grafting of polymer chains is the use of elevated temperatures and high ionic strength to overcome the repulsive forces preventing PEG molecules to come within close proximity to one another. Under such conditions polymers have limited solubility, significantly reducing their hydrodynamic radius to minimise interactions with H2O. This phenomena is dependent on the MW of the polymer however and it is expected that as conditions approach the ‘cloud-point’, thicker PEG layers and decreased BSA adsorption will be observed.
Figure 31 shows thickness values for the individual layers using Au SPR chips as determined by
SPR based upon the difference in pre- and post-injection baselines. When considering PEI layers, it is apparent that thickness values were consistently similar being 1.6±0.4nm on average, thus providing a uniform layer for subsequent PEG grafting. For PEG grafting to PEI layers by reductive amination, there is a gradual increase in thickness as the ionic strength is increased.
Subsequently, there is a corresponding decrease in BSA adsorption to surfaces that contained more
PEG when compared to PEI surfaces with only NaCNBH3 in the solution and no K2SO4. PEG thickness values for grafting in 0.3M and 0.6M K2SO4 were determined to be 1.27±0.40nm and
2.37±0.40nm, and the corresponding BSA thicknesses for adsorption to those PEG surfaces was
0.88±0.23nm and 0.16±0.04nm, respectively. This result was expected and in accordance with previously published work indicating that as conditions approach the ‘cloud-point’; PEG chains are de-shielded from one another allowing for chains to come within closer proximity to one another, and also the surface. This will result in higher grafting densities providing there are 164 sufficient pinning sites,6 where the grafted PEG layer acts as a steric barrier to prevent protein adsorption.
Figure 31. Individual thickness values as determined by SPR for PEI adsorption, BSA adsorption, and grafting of PEG (2kDa) in-situ by reductive amination at various ionic strengths.
SPR measurements were also used to monitor the grafting in real-time, with sensorgrams for PEG immobilisation and BSA adsorption being shown in Figure 32. Interestingly, when only
NaCNBH3 (No PEG) is injected into the system after PEI adsorption, a ΔTIR of similar magnitude to 0M K2SO4 was observed indicating that the strong reducing agent may have an influence on the
PEI structure. Initial association kinetics with the SPR sensor are similar for all conditions with strong interactions occurring at the surface within the first minute after injection. For grafting at
0.6M K2SO4 however, the binding kinetics deviate from the other conditions at a critical ΔTIR
(≈2.9 Θ). At this point, the grafting continues, yet becomes substantially slower. As PEG chains have a greater degree of freedom and mobility as ionic strength is increases, it is hypothesised that
165 under these conditions PEG chains have more opportunity to rearrange themselves to adopt a conformation that is thermodynamically more favourable.
Figure 32. SPR sensorgrams showing the change in the total internal reflection (TIR) angle over time during a) the grafting of PEG-ald (2kDa) by reductive amination and b) subsequent BSA adsorption.
ATR-FTIR was also used to provide chemical information regarding the modified surfaces.
To assess the presence of PEG, spectra were taken between a wavenumber range of 950-1250cm-
1 (Fig. 33a). The PEG reference spectrum shows the presence of a strong peak occurring at 1100cm-
1 corresponding to the C-O-C stretching vibrations (blue line). IR spectra of SPR slides functionalised with only PEI (magenta line) show the presence of two major peaks occurring at
980cm-1 and 1080cm-1 which are consistent with PEI reference spectra (red line) for C-N stretching vibrations. For PEG-modified samples, the diminishment in the IR peak intensities occurring at
980cm-1 and 1080cm-1, and a shift toward 1100cm-1 may suggest that PEG grafting was successful under these conditions. Interestingly, there appears to be no distinct differences between spectra taken of 0.3M K2SO4 and 0.6M K2SO4 samples considering the large differences in thickness seen from SPR analysis. This could be due to the sensitivity of the IR instrument or conformation of
PEG chains under the prepared conditions as it is hypothesised that while PEG chains may have lower densities when prepared at 0.3M K2S04, they may be collapsed at the interface due to a lack
166 of steric hindrance and inter-chain interactions leading to similar interfacial densities to conditions of 0.6M K2SO4, or on the other hand that SPR is a ‘wet’ thickness measurements as opposed to a
‘dry’ state for IR measurements.
One of the most important regions in the IR spectra for analysing proteins falls between
1500 and 1700cm-1 corresponding to the amide I (1600-1700cm-1) and amide II (1510-1580cm-1) bands primarily governed by the stretching vibrations of C=O and C-N, and in-plane N-H bending, respectively.29 Fig. 33b shows ATR-FTIR spectra taken between the wavenumber range of 1500 to 1700cm-1 with amide I and II modes being identified at approximately 1660cm-1 and 1550cm-1 when observing control BSA samples (black line). Similar peaks can be observed in spectra of modified samples, particularly for “NaCNBH3 Only” and 0M K2SO4 samples suggesting a higher
BSA surface coverage to these samples, which is also supported by SPR data. Moreover, the intensity of these peaks is significantly reduced for PEG-modified surface grafted at 0.3M K2SO4 and 0.6M K2SO4 indicating reduced BSA adsorption and superior non-fouling properties compared to other conditions.
Figure 33. ATR-FTIR spectra of modified surfaces between a) 950-1250cm-1 and b) 1500-1700cm-1. The axis for absorbance shown in a) is split to easily observe peaks of relatively low absorbance.
167
Due to the SPR instrument only being able to perform experiments up to 40°C, it is most likely that the CP conditions could not be achieved within the SPR. This is likely to have resulted in lower graft densities and thicknesses leading to increased protein adsorption when compared to similar studies.6,30,31 To increase the non-fouling ability at the interface, the MW of PEG was increased to 5kDa and corresponding sensorgrams for PEG grafting and BSA adsorption are shown in Figure 34. PEG grafting sensorgrams show a similar trend when compared to the grafting of
PEG 2kDa; with strong surface interactions occurring within the first minute. Similarly, at 0.6M
K2SO4, interactions with the SPR sensor show that higher grafting densities were able to be achieved when compared to 0M and 0.3M K2SO4 (Fig.34a) which also corresponded to lower BSA adsorption as indicated by a smaller ΔTIR under these conditions (Fig. 34b).
Figure 34. SPR sensorgrams showing the change in the total internal reflection (TIR) angle over time during. a) the grafting of PEG-ald (5kDa) by reductive amination under different ionic strengths, and b) subsequent BSA adsorption.
The de Freiter equation32 was utilised to determine surface coverage of adsorbed protein for each experimental condition and indicates that while all samples showed some level of protein adsorption, there was significantly less BSA on PEGylated surfaces compared to controls (Table
11). These results show that using PEG 2kDa, BSA surface coverage can be reduced down to
168
2 15±12ng/cm when grafting is performed at 0.6M K2SO4 providing further evidence that the non-
fouling ability of PEG is partially determined by graft density and therefore grafting conditions.
When the MW of PEG is increased to 5kDa however, greater PEG thicknesses and lower BSA
surface coverage can be achieved when compared to PEG 2kDa. Under the condition of PEG
2 grafting at 0.6M K2SO4 BSA surface coverage was further reduced to 2±2ng/cm , which is quite
significant as it is known that fibrinogen (Fg) adsorption as low as 7ng/cm2 is capable of inducing
platelet adhesion and thrombosis on medical implants.33
Table 11. Calculated PEG thickness and corresponding BSA surface coverages determined by MP-SPR
PEG - 2kDa PEG - 5kDa PEG thickness BSA surface coverage PEG thickness BSA surface coverage Sample (nm) (ng/cm2) (nm) (ng/cm2)
NaCNBH3 Only 0.71±0.3 239±7 - -
0M K2SO4 0.94±0.2 132±14 1.13±0.4 104±10
0.3M K2SO4 1.27±0.4 83±9 1.83±0.4 45±13
0.6M K2SO4 2.37±0.4 15±12 3.56±0.3 2±2
5.3.2 Optimisation of APTES deposition
Preliminary investigations in the development of a stable antimicrobial coating based upon the
immobilisation of non-fouling polymers and AMPs requires that sufficient grafting sites are first
established to maximise polymer and AMP loading.6 Due to the flexibility and popularity of
EDC/NHS chemistry for the chemical immobilisation of primary amines to carboxylic acids, this
method was chosen to immobilise carboxylated poly(ethylene glycol) to APTES-functionalised
surfaces. For the production of optimised APTES layers with high retention of amine functional
groups, within these experiments, the effect of deposition time on the quality of APTES films was
assessed using:
169
1. Ellipsometry to determine films thickness.
2. XPS to determine elemental composition and chemical states of atoms in the outer most
10nm of APTES films.
Deposition was performed on silicon wafers with toluene being used as a solvent, and the optimal deposition parameters were subsequently used to produce amino-functionalised Si wafers and SiO2-SPR sensor slides for further ex-situ and in-situ modifications.
Thicknesses of the prepared APTES films (2% (v/v)) after various deposition times were measured using variable angle spectroscopic ellipsometry (VASE) and the results show that there are small differences in the calculated thickness depending on the model parameters used (Fig.
35a). Though the experimental deviations observed for the different models are extremely similar, treating APTES as a separate layer and modelling its thickness using a Cauchy substrate with a refractive index of n=1.465 exhibited a relatively larger thickness compared to APTES layers that were treated as an extension of the SiO2 layer. Considering that similar results were obtained using the different parameters described, fitting for only SiO2 is determined to be a good approximation of true film thickness and this has been described previously.13,24 Even when comparisons are made between four experimental results, deviations in film thickness under seemingly identical conditions were observed (Fig. 35b). These results were somewhat expected because (as stated previously) the reactivity of APTES with surface hydroxides is quite difficult to control; being influenced by small changes in temperature, humidity, and the history of glassware.15 There was however an increase in film thickness from ≈0.5nm to 1.5nm as the deposition time was extended from 15min to 45min. This result indicates that as deposition time proceeds, the formation of
APTES multilayers occurs due to further condensation with surface-bound APTES and/or the adsorption of solution-phase APTES molecules and oligomers.
170
Figure 35. VASE thickness measurements for APTES films prepared in toluene (2% (v/v)) at different deposition times. (a) Differences in film thickness can be obtained depending on the model and used to analyse VASE data, and also under seemingly identical experimental conditions (b).
The average thickness values for APTES films were calculated to be 0.75±0.19nm,
1.01±0.21nm, and 1.38±0.40nm for deposition times of 15, 30, and 45min, respectively.
Monolayer coverage of APTES is expected to be 0.79nm based upon molecular projection models,34 and therefore is seen to be achieved between 15 and 30 minute deposition times. While the APTES concentration used here for deposition was 2% (v/v), similar thickness values have been observed at APTES concentrations of 0.4% (v/v) in toluene for 15min,15 and also at 1% (v/v) in toluene for 15min.13 Similarities in thickness and deposition times were achieved, yet the observed differences in concentration values between this and reported studies once again indicates that consistency in film properties are difficult to obtain.
Atomic composition and chemical states of the prepared films were assessed using XPS, and the atomic composition data is shown in Table 12. As expected, as the deposition time increased so did the carbon and nitrogen content within the films suggesting successful deposition.
Additionally, due to the low sampling depth of XPS (<10nm) it also suggests that many of the reactive amino-propyl chains are in the outer-most layers of the film. Atomic composition of a
171 bulk APTES (20% (v/v)) film shows that under much higher APTES concentrations, a higher proportion of ethoxy chains were not hydrolysed due to the higher C% (50.2±0.2%), when compared to the theoretical C% for unhydrolysed APTES (64%), and fully-condensed APTES
(27%). At lower APTES concentrations however, it appears that many ethoxy groups are hydrolysed as the atomic composition data more closely represents theoretical data for completely condensed APTES (See Table 12). It was also observed that increased C% was seen when compared to theoretical values, which was not surprising due to the presence of adventitious carbon contamination (≈10%), which can also be seen on cleaned Si wafers.
The XPS Overlayer Equation (See Equation 2) was also used to estimate thickness values of APTES films (z) based upon the reduction in substrate (Si) signal after silane deposition. This is under the assumption that the electron take-off angle is 0° for flat Si wafers, and the inelastic mean free path length of Si2p photoelectrons is 3.9nm.35 Similar to VASE measurements it is seen that as the deposition time and concentration of APTES increases so does the thickness of the silane films. For modest APTES concentrations (2% w/v), the thickness measurements determined using XPS atomic composition data shows a somewhat larger thickness compared to VASE results, with thicknesses of 1.7nm, 2.3nm, and 2.7nm observed for deposition times of 15, 30, and
45min, respectively. It is known that ellipsometry can systematically underestimate film thickness due to the use of bulk optical constants, which in fact may be different to those of very thin films.36,37 Therefore, it is suggested that XPS thickness measurements may provide a more accurate representation of APTES thickness. Comparisons to bulk APTES films however, could not be made as VASE was unable to obtain sensible data for these films possibly due to internal reflection within the film, which can scatter the light before entering the detector.
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Table 12. Atomic composition of APTES film deposited on Si wafers under different deposition conditions Atomic composition (%) Time (min) O C N Si z (nm) 0 32.4 ± 0.1 9.6 ± 0.9 0 ± 0 58.0 ± 0.7 - 15 29.4 ± 0.7 29.8 ± 2.3 3.3 ± 0.5 37.6 ± 2.0 1.7 30 26.6 ± 0.1 34.9 ± 0.3 6.4 ± 0.2 32.1 ± 0.4 2.3 45 24.7 ± 0.2 38.2 ± 0.4 7.8 ± 0.3 29.3 ± 0.4 2.7 30* 22.7 ± 0.2 50.2 ± 0.2 13.8 ± 0.2 13.3 ± 0.1 5.7 Fully-condensed 27.3 27.3 9.0 36.4 NA APTES *Bulk APTES films prepared at 20% (v/v) in toluene and deposited for 30 min
Further information regarding APTES films was obtained using high-resolution XPS analysis, and spectra of N1s binding energies are shown in Figure 36, which provides useful information regarding the chemical states of the prepared films. All of the obtained spectra show that there are two chemical states that can be identified. These occur at approximately 400.0eV for
+ primary (-NH2) amines, and also at 401.5eV which can be attributed protonated (-NH3 ) and hydrogen-bonded amines. For 15min deposition times it appears that while the predominant form of N was the primary amine (59.4%), a large proportion was present as a protonated or hydrogen- bonded amine (40.6%) (Fig. 36a). At longer deposition times however, the proportion of primary amines present within the films substantially increases to 80.2% and 72.9% for 30min and 45min depositions, respectively (Fig. 36b-c). A possible reason for this observation could be that the initial interactions involve amines that hydrogen bond to the silicon oxide surface and/or utilise
- + protons from the surface to form SiO --- H --- NH2 species, which has been proposed previously.15,38
For bulk films of APTES the predominant from of N was the protonated or hydrogen- bonded amine (75.0%), with a small proportion being the primary amine (25%) (Fig. 36d). This result was not surprising as it has been shown previously that higher concentrations of surface
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39 amines can form bicarbonate salts with atmospheric CO2. This phenomenon can be minimised by performing the deposition in an N2 saturated atmosphere whilst maintaining sufficiently thick films.13
Figure 36. High-resolution N1s XPS spectra of APTES-modified Si wafers. Spectra shown above indicate the chemical states of films prepared using APTES (2% (v/v)) and deposited for a) 15min, b) 30min, c) 45min, and d) APTES (20% (v/v)) deposited for 30min.
High-resolution XPS C1s spectra were also acquired to provide more information regarding the chemical environments of the APTES films (Figure 37). At 2% (v/v), an increase in the C-N component (≈286eV) can be seen as the deposition time increases, which was expected as longer deposition times should result in a higher proportion of N deposited at the surface. There is also a small contribution from photoelectrons at approx. 288.2eV seen for all samples, which is likely due to adventitious carbon contamination or the formation of bicarbonate salts from atmospheric CO2. Bulk films of APTES (Fig. 37d) show the presence of another carbon environment that was not seen for other sample occurring at approx. 283.8eV, which can be 174 attributed to Si-C photoelectrons. Under the high concentrations used here, polymerisation of
APTES monomers at the surface is not easily controlled, resulting in the formation of aggregates and multilayers where APTES adopts a conformation where silanol groups are orientated away from the surface.13
Figure 37. High-resolution C1s spectra of APTES-modified Si wafers deposited for a) 15min. b) 30min, c) 45min, and d) bulk APTES films prepared at 20% (v/v) and deposited for 30min.
5.3.3 In-situ surface modification and protein adsorption using EDC/NHS chemistry
EDC/NHS coupling chemistry was employed for the in-situ modification of APTES- modified SPR sensor slides using a homo-bifunctional carboxylated-PEG (Mn = 3000). The modified surfaces were then assessed for their ability to resist protein adsorption (BSA 1mg/mL) in-situ via SPR. In addition, covalently immobilised APTES layers was compared to physically adsorbed PEI to determine which pre-modification approach may be best to use as a platform for subsequent surface modification with polymer chains. Table 13 shows the calculated PEG 175 thickness and BSA surface coverages to the modified surfaces after the designated injection period.
When considering PEG thickness values for the different activation layers it can be seen that there are similar thicknesses are achieved for samples with and without EDC/NHS; where PEG thicknesses on PEI layers were observed to be 0.89±0.4nm and 0.86±0.3nm with and without
EDC/NHS activation, respectively. A similar trend is also seen for APTES activation layers where
PEG thicknesses were 1.26±0.4nm and 1.32±0.4nm with and without EDC/NHS activation, respectively. The similarities in thicknesses seen between activated and non-activated samples indicates that prior activation of –COOH groups with EDC/NHS does not lead to an increase in the amount of PEG grafted to the aminated surfaces. It is suggested that under these experimental conditions the ‘activated’ NHS ester is hydrolysed much more readily within the SPR fluidic system possibly due to, pressure fluctuations, buffer diffusion rates, and/or differences between local microenvironments of the running buffer and the surface which gives rise to unwanted pH gradients and decreased binding capacity.40 The presence of PEG seen with “No activation” at
0.86±0.3nm and 1.32±0.4nm for PEI and APTES activation layers, respectively also suggests some physically adsorbed PEG to these surfaces that are of similar thickness to “Activated” samples Even the incorporation of 0.6M K2SO4 to provide osmotic balance and ‘poor’ solvation of PEG chains did not lead to an increased graft density, which was observed to be a crucial parameter to achieve dense PEG layers by reductive amination (See Section 5.3.1). With the addition of 0.6M K2SO4 it was seen that PEG thickness values were lower than other conditions being 0.79±0.3nm on physically adsorbed PEI layers and 0.96±0.3 on covalently bound APTES layers. It is expected that the solubility of K2SO4 at the instrument temperature of 22°C is quite low, which perturbs the interactions of PEG and the functionalised SPR sensor slide. However, increasing the temperature within the SPR instrument will also drastically increase the rate of hydrolysis of any PEG-NHS esters and lower grafting density even further.
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Table 13. PEG thickness and BSA surface coverage to physically adsorbed PEI and covalently bound APTES activation layers determined in-situ using MP-SPR
PEI APTES PEG PEG BSA coverage BSA coverage thickness thickness (ng/cm2) (ng/cm2) (nm) (nm) Bare SPR slide - 232±47 - No PEG - 271±28 - 114±20 No activation 0.86±0.3 119±23 1.32±0.4 45±13 Activation 0.89±0.4 116±32 1.26±0.4 53±22 Activation + 0.6M 0.79±0.3 142±18 0.96±0.3 152±38 K2SO4
When considering the amount of BSA adsorbed to the SPR sensor slides under each of the conditions it is not surprising that the bare SPR slides exhibited high BSA adsorption
(232±47ng/cm2) as the net charge of BSA at pH 7.4 would be negative and is known to reversibly bind to negatively charged substances such as Au.41 A similar phenomena is seen for BSA adsorption to PEI layers where surface coverage was seen to be 271±28ng/cm2, rationalised down to the favourable electrostatic interactions between negatively charged BSA and positively charged amines within the PEI layer. Compared to the bare Au SPR slide there is actually more BSA adsorbed to the PEI surfaces, which is most likely due to the branched polymeric structure and increased void volume that resulted in more sites for BSA adsorption. For APTES only there is a significant reduction in BSA surface coverage (114±20ng/cm2) when compared to the other controls which is surprising as terminal amines will also be positively charged and promote protein adsorption. Due to the relatively non-polar nature of alkyl chains on APTES films however, protein-surface interactions are expected to be less favourable when compared to the branched polymeric nature of PEI which may trap BSA and resulted in the BSA adsorption patterns observed.
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Similarly to reductive amination PEG grafting studies, there is a general trend where an increased PEG thickness correlates to an increase in non-fouling ability which, is an expected property of PEGylated surfaces.42 Interestingly, using EDC/NHS appeared to have little effect on the thickness of PEG layers and also BSA adsorption; with negligible differences in surface coverage seen. PEG grafted to APTES layers did however show lower BSA adsorption when compared to physically adsorbed PEI layers once again rationalised down to the lower PEG thicknesses observed, and also the branched polymeric nature of PEI. Clearly, it appears that covalent immobilisation of underlying graft layers is an important step for improving PEG grafting and stability and lower protein adsorption, however results also show that the choice of grafting method is even more crucial when performing in-situ surface modifications; as flow-rate, buffer composition, pH and temperature will all influence the rate of grafting and hydrolysis of any
‘activated esters’ used to attach PEG molecules to the underlying fictionalised substrate.
5.4 Conclusions This Chapter investigated PEG grafting utilising reductive amination in conjunction with
‘poor solvation’ conditions for the in-situ grafting of PEG-ald to physically deposited PEI layers and assessed the ability of these surfaces to resist protein adsorption. While it was declared that
CP conditions could not be achieved using SPR due to temperature restrictions encountered limiting grafting to 40°C, it was shown that under certain conditions the adsorption of BSA could be reduced significantly compared to control samples. It was found that the critical parameters to achieve non-fouling properties was the use of high ionic strength in conjunction with elevated temperatures to allow for higher density grafting. It is also noted that the CP for the PEGs used will be different due to the differences in MW, however as PEG 5kDa shows superior protein resistant properties compared to PEG 2kDa these results demonstrate that the increased MW can negate the temperature limitations to a certain extent. 178
EDC/NHS covalent coupling chemistry was also assessed to mediate the grafting of PEG to either physically adsorbed PEI or covalently bound APTES graft layers. This method was shown to be far inferior to reductive amination investigations for both PEI and APTES, with significantly lower PEG thicknesses and increased BSA adsorption seen on these surfaces. Moreover, it was shown that PEG grafting to covalently bound graft layers of minimal thickness is advantageous when compared to PEG grafting to physically adsorbed polymeric structures. Due to the lower than expected thickness values calculated for PEG layers using EDC/NHS chemistry, and the inherent complications when performing this type of tethering in-situ it is prudent to investigate other PEG grafting and surface activation methods to further optimise these surfaces to achieve dense PEG layers and high AMP loading. This is reported in subsequent chapters.
5.5 References 1 Chen, S., Li, L., Zhao, C. & Zheng, J. Surface hydration: Principles and applications toward low-fouling/nonfouling biomaterials. Polymer 51, 5283-5293, doi:http://dx.doi.org/10.1016/j.polymer.2010.08.022 (2010).
2 Zhang, H. & Chiao, M. Anti-fouling Coatings of Poly(dimethylsiloxane) Devices for Biological and Biomedical Applications. J Med Biol Eng 35, 143-155, doi:10.1007/s40846-015-0029-4 (2015).
3 Chapman, R. G., Ostuni, E., Liang, M. N., Meluleni, G., Kim, E., Yan, L., Pier, G., Warren, H. S. & Whitesides, G. M. Polymeric Thin Films That Resist the Adsorption of Proteins and the Adhesion of Bacteria. Langmuir 17, 1225-1233, doi:10.1021/la001222d (2001).
4 Vermonden, T., Censi, R. & Hennink, W. E. Hydrogels for Protein Delivery. Chem. Rev. 112, 2853-2888, doi:10.1021/cr200157d (2012).
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5 Yoshikawa, C., Qiu, J., Huang, C. F., Shimizu, Y., Suzuki, J. & van den Bosch, E. Non- biofouling property of well-defined concentrated polymer brushes. Colloids Surf. B. Biointerfaces 127, 213-220, doi:10.1016/j.colsurfb.2015.01.026 (2015).
6 Kingshott, P., Thissen, H. & Griesser, H. J. Effects of cloud-point grafting, chain length, and density of PEG layers on competitive adsorption of ocular proteins. Biomaterials 23, 2043-2056, doi:10.1016/S0142-9612(01)00334-9 (2002).
7 Hetemi, D. & Pinson, J. Surface functionalisation of polymers. Chem. Soc. Rev. 46, 5701- 5713, doi:10.1039/C7CS00150A (2017).
8 Brady, D. & Jordaan, J. Advances in enzyme immobilisation. Biotechnol. Lett. 31, 1639, doi:10.1007/s10529-009-0076-4 (2009).
9 Kingshott, P., Wei, J., Bagge-Ravn, D., Gadegaard, N. & Gram, L. Covalent attachment of poly(ethylene glycol) to surfaces, critical for reducing bacterial adhesion. Langmuir 19, 6912-6921, doi:10.1021/la034032m (2003).
10 Li, Y., Wei, S., Wu, J., Jasensky, J., Xi, C., Li, H., Xu, Y., Wang, Q., Marsh, E. N. G., Brooks, C. L. & Chen, Z. Effects of peptide immobilization sites on the structure and activity of surface tethered antimicrobial peptides. Journal of Physical Chemistry C 119, 7146-7155, doi:10.1021/jp5125487 (2015).
11 Thompson, W. R., Cai, M., Ho, M. & Pemberton, J. E. Hydrolysis and Condensation of Self-Assembled Monolayers of (3-Mercaptopropyl)trimethoxysilane on Ag and Au Surfaces. Langmuir 13, 2291-2302, doi:10.1021/la960795g (1997).
12 Scott, A. & Gray-Munro, J. E. The surface chemistry of 3- mercaptopropyltrimethoxysilane films deposited on magnesium alloy AZ91. Thin Solid Films 517, 6809-6816, doi:http://dx.doi.org/10.1016/j.tsf.2009.05.044 (2009).
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13 Kim, J., Seidler, P., Wan, L. S. & Fill, C. Formation, structure, and reactivity of amino- terminated organic films on silicon substrates. J. Colloid Interface Sci. 329, 114-119, doi:http://dx.doi.org/10.1016/j.jcis.2008.09.031 (2009).
14 Hu, M., Noda, S., Okubo, T., Yamaguchi, Y. & Komiyama, H. Structure and morphology of self-assembled 3-mercaptopropyltrimethoxysilane layers on silicon oxide. Appl. Surf. Sci. 181, 307-316, doi:http://dx.doi.org/10.1016/S0169-4332(01)00399-3 (2001).
15 Vandenberg, E. T., Bertilsson, L., Liedberg, B., Uvdal, K., Erlandsson, R., Elwing, H. & Lundström, I. Structure of 3-aminopropyl triethoxy silane on silicon oxide. J. Colloid Interface Sci. 147, 103-118, doi:http://dx.doi.org/10.1016/0021-9797(91)90139-Y (1991).
16 Singh, J. & Whitten, J. E. Adsorption of 3-Mercaptopropyltrimethoxysilane on Silicon Oxide Surfaces and Adsorbate Interaction with Thermally Deposited Gold. The Journal of Physical Chemistry C 112, 19088-19096, doi:10.1021/jp807536z (2008).
17 Scott, A. F., Gray-Munro, J. E. & Shepherd, J. L. Influence of coating bath chemistry on the deposition of 3-mercaptopropyl trimethoxysilane films deposited on magnesium alloy. J. Colloid Interface Sci. 343, 474-483, doi:10.1016/j.jcis.2009.11.062 (2010).
18 Crudden, C. M., Sateesh, M. & Lewis, R. Mercaptopropyl-Modified Mesoporous Silica: A Remarkable Support for the Preparation of a Reusable, Heterogeneous Palladium Catalyst for Coupling Reactions. J. Am. Chem. Soc. 127, 10045-10050, doi:10.1021/ja0430954 (2005).
19 R Glass, N., Tjeung, R., Chan, P., Yeo, L. & Friend, J. Organosilane deposition for microfluidic applications. Vol. 5 (2011).
20 Semaltianos, N. G., Pastol, J. L. & Doppelt, P. Copper chemical vapour deposition on organosilane-treated SiO2 surfaces. Appl. Surf. Sci. 222, 102-109, doi:https://doi.org/10.1016/j.apsusc.2003.08.003 (2004).
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21 Mahapatro, A. K., Scott, A., Manning, A. & Janes, D. B. Gold surface with sub-nm roughness realized by evaporation on a molecular adhesion monolayer. Appl. Phys. Lett. 88, 151917, doi:10.1063/1.2183820 (2006).
22 Szili, E. J., Kumar, S., Smart, R. S. C. & Voelcker, N. H. Generation of a stable surface concentration of amino groups on silica coated onto titanium substrates by the plasma enhanced chemical vapour deposition method. Appl. Surf. Sci. 255, 6846-6850, doi:10.1016/j.apsusc.2009.02.092 (2009).
23 Terracciano, M., Rea, I., Politi, J. & De Stefano, L. Optical characterization of aminosilane-modified silicon dioxide surface for biosensing. Journal of the European Optical Society 8, doi:10.2971/jeos.2013.13075 (2013).
24 Howarter, J. A. & Youngblood, J. P. Optimization of silica silanization by 3- aminopropyltriethoxysilane. Langmuir 22, 11142-11147, doi:10.1021/la061240g (2006).
25 Onaizi, S. A. & Leong, S. S. J. Tethering antimicrobial peptides: Current status and potential challenges. Biotechnol. Adv. 29, 67-74, doi:10.1016/j.biotechadv.2010.08.012 (2011).
26 Costa, F., Maia, S., Gomes, J., Gomes, P. & Martins, M. C. L. Characterization of hLF1- 11 immobilization onto chitosan ultrathin films, and its effects on antimicrobial activity. Acta Biomater. 10, 3513-3521, doi:10.1016/j.actbio.2014.02.028 (2014).
27 Green, R. J., Davies, J., Davies, M. C., Roberts, C. J. & Tendler, S. J. Surface plasmon resonance for real time in situ analysis of protein adsorption to polymer surfaces. Biomaterials 18, 405-413, doi:10.1016/s0142-9612(96)00141-x (1997).
28 Willem M. Albers, I. V. L. Surface Plasmon Resonance on Nanoscale Organic Films. Vol. 1st (Springer, 2010).
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29 Ganim, Z., Hoi, S. C., Smith, A. W., Deflores, L. P., Jones, K. C. & Tokmakoff, A. Amide I two-dimensional infrared spectroscopy of proteins. Acc. Chem. Res. 41, 432- 441, doi:10.1021/ar700188n (2008).
30 Hucknall, A., Rangarajan, S. & Chilkoti, A. In pursuit of zero: Polymer brushes that resist the adsorption of proteins. Adv. Mater. 21, 2441-2446, doi:10.1002/adma.200900383 (2009).
31 Emilsson, G., Schoch, R. L., Feuz, L., Hook, F., Lim, R. Y. H. & Dahlin, A. B. Strongly Stretched Protein Resistant Poly(ethylene glycol) Brushes Prepared by Grafting-To. ACS Appl. Mater. Interfaces 7, 7505-7515, doi:10.1021/acsami.5b01590 (2015).
32 Albers, W. & Lundin, I. Surface Plasmon Resonance on Nanoscale Organic Films. 1 edn, (2010).
33 Jaffer, I. H., Fredenburgh, J. C., Hirsh, J. & Weitz, J. I. Medical device-induced thrombosis: what causes it and how can we prevent it? J. Thromb. Haemost. 13, S72-S81, doi:10.1111/jth.12961 (2015).
34 Myungsook Kim, F. B., Jiwoong Park, Tae-Ho Yoon, Yun Hee Jang. (eds Gw Department of Materials Science and Engineering, Gwangju 500-712 angju Institute of Science and Technology, & Korea).
35 Al-Ani, A., Pingle, H., N, P. R., Wang, P. Y. & Kingshott, P. Tuning the Density of Poly(ethylene glycol) Chains to Control Mammalian Cell and Bacterial Attachment. Polymers 9, doi:10.3390/polym9080343 (2017).
36 Hilfiker, J. N., Stadermann, M., Sun, J., Tiwald, T., Hale, J. S., Miller, P. E. & Aracne- Ruddle, C. Determining thickness and refractive index from free-standing ultra-thin polymer films with spectroscopic ellipsometry. Appl. Surf. Sci. 421, 508-512, doi:10.1016/j.apsusc.2016.08.131 (2017).
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37 McCrackin, F. L., Passaglia, E., Stromberg, R. R. & Steinberg, H. L. Measurement of the Thickness and Refractive Index of Very Thin Films and the Optical Properties of Surfaces by Ellipsometry. J Res Natl Bur Stand A Phys Chem 67A, 363-377, doi:10.6028/jres.067A.040 (1963).
38 White, L. D. & Tripp, C. P. Reaction of (3-Aminopropyl)dimethylethoxysilane with Amine Catalysts on Silica Surfaces. J. Colloid Interface Sci. 232, 400-407, doi:10.1006/jcis.2000.7224 (2000).
39 Peña-Alonso, R., Rubio, F., Rubio, J. & L. Oteo, J. Study of the hydrolysis and condensation of ??- Aminopropyltriethoxysilane by FT-IR spectroscopy. Vol. 42 (2007).
40 Schuck, P. & Zhao, H. The role of mass transport limitation and surface heterogeneity in the biophysical characterization of macromolecular binding processes by SPR biosensing. Methods in molecular biology (Clifton, N.J.) 627, 15-54, doi:10.1007/978-1-60761-670- 2_2 (2010).
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42 Li, L., Chen, S., Zheng, J., Ratner, B. D. & Jiang, S. Protein adsorption on oligo(ethylene glycol)-terminated alkanethiolate self-assembled monolayers: The molecular basis for nonfouling behavior. J. Phys. Chem. B 109, 2934-2941, doi:10.1021/jp0473321 (2005).
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6 Investigation and characterisation of PEG immobilisation to silica microspheres and BSA adsorption 6.1 Summary Topographical cues and surface patterns exhibited by colloidal crystal layers is a versatile way of modulating and controlling interactions that occur at an interface, and as shown in Chapter
4, these fabricated surfaces can also be used as a platform for AMP immobilisation to generate active antimicrobial coatings. To translate the previous PEG immobilisation strategies of planar surfaces to spherical colloids, the aim of this Chapter is to investigate the tethering of PEG to silica colloids and assess their ability to resist protein adsorption. Due to its versatility and mild conditions, carbodiimide coupling was used here to immobilise a heterobifunctional polymer
(HOOC-PEG-NH2) to carboxylated silica colloids, with the incorporation of high salt concentrations (0.6M K2SO4) also being used in an attempt to reduce the hydrodynamic radius of
PEG chains to control grafting density. It was shown that, similar to planar surface investigations, that the tethering of PEG using EDC/NHS chemistry was determined to be somewhat inefficient resulting in extremely low polymer thicknesses and minimal reduction in protein adsorption compared to control samples. These results suggest that the efficiency of EDC/NHS chemistry for conjugation of carboxyl groups to primary amines is minimised when; using polymers that have functional groups that can enter competing interactions with one another, when using relatively large and dense Si colloids, and also when using high ionic strength, which results in lower grafting densities.
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6.2 Introduction The formation of defined surface topographical features and chemistries by the application of functionalised colloidal crystals lends itself to many application areas including biosensing,1,2 cell culture substrates,3,4 and antimicrobial coatings.5 In regards to antimicrobial surface coatings, many of the investigations into the use of AMPs have been to impart antimicrobial properties to a planar surface using zero-length immobilisation.6,7 However, only until quite recently has there become a general consensus that the incorporation of a flexible polymer linker can allow for enhanced penetration and mobility.8,9 The choice of grafting method to facilitate polymer immobilisation to a surface is dependent on both; the terminal and side chain functional groups present on polymer molecules, and also the surface-bound functional groups that are inherent to the surface or produced by ‘activation’ through chemical modification. While not a complete or exhaustive list of potential coupling methods is presented here, Figure 38 below summarises some of the chemical immobilisation methods used for tethering polymer chains to a surface, with common approaches utilising surface-bound amine, or thiol functional groups.
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Figure 38. Examples of chemical immobilisation strategies for the tethering of polymers to surface-bound functional groups: a) the use of primary amines for immobilisation through activated NHS-esters or aldehydes, and b) the use of thiols for covalent immobilisation to maleimide or acrylate functional groups.
Similar techniques and immobilisation methods that are described in Figure 38 can also be applied to colloidal particles to impart desired properties and functionality, which is a main focus of this research. While results from previous chapters have suggested that the grafting of PEG to planar surfaces using EDC/NHS chemistry was not as effective as reductive amination for in-situ modifications, this method has however been shown to be quite effective in many circumstances for the immobilisation of several classes of biomolecules and uses relatively mild conditions.5,10,11
As such, within these experiments, the immobilisation of a hetero-bifunctional PEG (HOOC-
PEG5000-NH2) to 2µm carboxylated silica particles was assessed using EDC/NHS coupling chemistry. To overcome the drawbacks of this method seen previously during in-situ investigations, low temperatures and activation of surface bound carboxyl groups was chosen to minimise hydrolysis and prevent auto-immobilisation of PEG chains, respectively. PEG grafting was assessed by zeta potential, XPS, ATR-FTIR, and SEM, and the incorporation of K2SO4 was
187 also assessed with the aim of minimising the hydrodynamic radius of PEG chains to facilitate high- density grafting. The ability of PEG-modified colloids to resist protein adsorption was subsequently investigated by XPS using BSA as a model protein to determine any non-fouling properties.
6.3 Results and discussion
6.3.1 Determination of PEG concentration
To determine the amount of PEG required for surface coverage of the colloidal particles it is first necessary to determine their respective surface area, and also the cross-sectional area of
PEG molecules. The calculated surface area of 2µm particles is 1.25x109Å and the cross-sectional area of a helical structured PEG has been previously determined to be 22Å2.12 Thus, the approximate number of PEG molecules required to cover the surface area of one Si particle is calculated to be 5.7x107 molecules per particle. Assuming the solid weight content of the particle in each reaction is 1% (w/v) (0.01 g/mL), and 1mL reaction volumes were used – we can approximate the volume composition of Si to be 0.005 cm3 assuming the density of silica colloids is 2 g/cm3.13,14 Using the total volume of Si in a 1 mL reaction mixture (0.005 cm3) and knowing the volume of one particle (4.2x10-12 cm3 per particle) the total number of particles in the reaction is calculated to be:
0.005 8 ⁄4.2 × 10−12 = 1.2 × 10 Si particles in a 1mL reaction mixture
Therefore, the total number of PEG molecules required in a 1 mL reaction can be approximated to be:
(5.7 × 107) × (1.2 × 108) = 6.8 × 1016 PEG molecules
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This can then be converted to moles by dividing Avogadro’s number (NA) (6.022x1023) to yield 0.113 µmol, which would have a concentration of 0.113 mM in a 1mL reaction. Thus, considering the above calculations PEG immobilisation experiments will be performed with PEG at an excess concentration of 0.25 mM.
6.3.2 Particle modification and characterisation
Results presented below in Figure 39 show zeta potential results for unmodified and PEG- modified Si-COOH particles in MilliQ (pH 7). As expected, the unmodified Si-COOH colloids possessed a negative surface charge (≈-40mV) due to carboxyl groups present on the particles surface and is within the expected range for this particle type; with previous research suggesting
15 that 0.1µm SiO2-COOH colloids presented a zeta potential of -42.2mV. When compared to the modified particles there are only small differences in zeta potentials between the different grafting conditions with particles being slightly less negative than the unmodified control. While samples prepared with no EDC/NHS and EDC/NHS+K2SO4 were seen to be most similar to the unmodified particles with zeta potentials of approximately -38mV and -39mV respectively, there was a larger change in surface charge seen for particles modified with only EDC/NHS. Under these conditions zeta potentials were observed to be approximately -34mV and somewhat confirms the presence of
PEG at the surface as there is expected to be a reduction in magnitude of surface charge as the neutral PEG chains will mask the carboxyl groups on the particle surface.16,17
189
Figure 39. Zeta potential data of unmodified and PEG-modified Si-COOH particles under various reaction conditions. X-axis indicates zeta potential in mV and Y-axis indicates sample type. Data reported are the average of two independent samples with five measurements being taken per sample.
The similarities in surface charge seen between unmodified particles and samples with no
EDC/NHS and with EDC/NHS+ K2SO4 provide little evidence that any grafting occurred under these conditions, which is not that surprising as EDC/NHS is a necessary reagent for activating
10 carboxylic acids to covalently bond to primary amines, and the addition of K2SO4 would also supress molecular interactions due to the shielding of surface charges.18,19 Additionally, without elevated temperatures to promote CP conditions, the solubility of K2SO4 will be quite modest – hindering PEG-surface interactions further.
To characterise the modified particles further, XPS was used to determine elemental composition and chemical states of the outer-most 10 nm with atomic composition data being shown below in Table 14. These results are consistent with zeta potential data presented in Figure
190
39, which suggests that no PEG grafting took place for no EDC/NHS and EDC/NHS+K2SO4 samples, however there was evidence of PEG grafting seen for EDC/NHS samples with a substantial increase in %C (33.5±2.0%) which was accompanied by a decrease in %O (45.1±2.2%) when compared to unmodified samples. There was also a decrease in %Si (18.1±1.0%) seen for
EDC/NHS samples compared to all other conditions (≈23%) which was expected if some of the surface Si-COOH groups had been consumed during PEG grafting. This was not however substantiated by high-resolution C1s spectral data (Fig. 40a-d) which clearly shows that there is minimal difference in the carbon environments between all samples.
Interestingly, there were small traces of nitrogen seen for all samples in the range of 2-3% which was unexpected as the only reasonable source of nitrogen would be from terminal NH2 on
PEG chains. Considering this phenomena was seen for unmodified as well as PEG-modified samples, it suggests that this may be due residual functional groups that are used to form carboxyl groups at the particles surface. Additionally, there were no traces of K+ in survey XPS spectra which indicates that zeta potential results for EDC/NHS+K2SO4 samples were not affected by ionic shielding from K+ ions in solution and can be considered accurate. Assuming PEG grafting was successful for EDC/NHS samples, the XPS overlayer equation can be used to estimate the thickness (z) of PEG layers based upon the attenuation of Si signal from the particle surface. This is under the assumption that the electron take-off angle is 57.3° for randomly rough particle surfaces,5,20 and the inelastic mean free path length of Si2p photoelectrons is 3.9nm.21 From these calculations the thickness of PEG layers can be approximated to 0.6nm which supports previous evidence that there is minimal, if any PEG bound to the surface. These are dry-state thickness values, however and are expected to be significantly lower than the thickness of hydrated PEG layers.
191
Table 14. XPS atomic composition data for unmodified and PEG-modified Si-COOH particles.
Atomic composition (%) Sample ID O C N Na Si z (nm) Bare Si-COOH 51.1±2.4 22.8±3.4 2.3±0.3 0.2±0.2 23.6±1.0 - PEG (no EDC/NHS) 51.9±1.6 21.4±2.4 2.5±0.2 0.3±0.3 23.8±1.1 - PEG (EDC/NHS) 45.1±2.2 33.5±2.0 2.9±0.5 0.4±0.3 18.1±1.0 0.6 0.1 PEG (EDC/NHS+K2SO4) 49.57±1.9 26.1±2.8 2.1±0.4 0.2±0.1 22.1±1.1
Figure 40 shows high-resolution XPS C1s spectra of unmodified and PEG-modified Si-
COOH colloids indicating the different chemical states of each of the samples. Obtained spectra
indicates that there is no discernible difference between the different chemical states of the
unmodified and PEG-modified particles. All samples were seen to possess C-C/C-H (285eV), C-
O/C-N (286.2eV), and also O-C=O/N-C=O (288.5eV) environments according to C1s spectra
suggesting a relatively aliphatic environment, which is typical for adventitious carbon
contamination.22,23 There was however small differences seen in the relative contributions of these
chemical environments, which shows that the C-O/C-N carbon environments slightly increases for
Si-PEG (no EDC/NHS) and Si-PEG (EDC/NHS) compared to other samples. Considering that the
same increase was not seen for Si-PEG (EDC/NHS+K2SO4) samples it is likely that the PEG
thickness estimated in Table 14 is due to a relative increase in adventitious carbon contamination
compared to the other experimental conditions.
192
Figure 40. High-resolution C1s data for unmodified and PEG-modified Si-COOH particles. Spectra shown above are of a) unmodified Si-COOH colloids, b) Si-PEG (no EDC/NHS), c) Si-PEG (EDC/NHS), and d) Si-PEG (EDC/NHS+K2SO4).
Further characterisation of the PEG-modified Si colloids was performed using ATR-FTIR analysis, with a HOOC-PEG-NH2 control being used to identify any traces of PEG on the modified particles. Obtained spectra are shown in Figure 41. The peaks seen for the PEG control are typical for this type of polymer with peaks being seen at ca.1100cm-1, 1453cm-1, and 2950cm-1, which can be attributed to C-O stretching, C-H bending, and –CH stretching vibrational modes, respectively.24,25 For PEG-modified surfaces FTIR spectra only show the presence of one major peak occurring at ca. 1100cm-1, which is in the expected region for C-O stretching. Considering the absence of peaks attributed to –CH stretching and C-H bending it is expected that these peaks are likely to come from the Si colloids as the Si-O-Si stretching modes also occur at ca. 1100cm-
1. This is also supported by additional peaks seen at ca. 795cm-1 which is due to Si-O bending.26
The presence of these peaks were somewhat unexpected as unmodified Si was used to zero the
FTIR instrument and should therefore not be present if the particle chemistry was homogeneous.
193
The likely explanation for this phenomena is that the washing and treatment procedures potentially hydrolysed COOH groups yielding relatively more Si-O-Si compared to bare Si-COOH colloids.
Considering these results, it is apparent that there is minimal or no PEG tethered to the surface of the Si colloidal particles, which is in agreement to previous results.
Figure 41. Representative ATR-FTIR spectra of PEG-modified Si-COOH microspheres. Unmodified Si- COOH microspheres were used as blank for the detection of bound PEG, and a PEG only control was also used.
Surface morphology and topography of the PEG-modified particles was assessed using
SEM to observe any morphological changes upon crystallisation with obtained micrographs being shown in Figure 42. Such characterisation is necessary to determine whether the functionalised surface coating exhibit long-range ordering and forms close-packed arrangements. According to
194 zeta potential results all colloidal suspension should form ordered layers due to the sufficient repulsive character to prevent particle aggregation and promote the formation of ordered structures.3 As can be seen at moderate magnifications (Figure 42 middle column), many regions of the colloidal layers showed areas of long-range ordering and closely-packed arrangements as expected. There were also small areas of defects present, which were more apparent in PEG- modified samples which is quite interesting considering the similar surface charge seen for all samples. One possible explanation for this observation if that during the functionalisation procedures small amounts of colloidal suspension were lost, which will result in a lower wt% compared to the unmodified Si-COOH particles leading to a potential increase in the number of pin-holes and defects for these samples.
The act of tethering polymers such as PEG to the surface of colloidal particles is known to sterically stabilise a colloidal suspension and prevent aggregation.27,28 Considering this phenomena, it would be expected that the addition of PEG would result in large areas of ordered arrangements - similar to those seen for unmodified Si-COOH colloids. The apparent absence of this ordering within these samples could suggest either: i) little or minimal PEG is tethered to these surfaces, and/or ii) that small proportions of PEG are tethered (physically and/or covalently), but in such a small quantity that some aggregation did occur, which would account for the areas of aggregated particles seen for Si-PEG (EDC/NHS) and Si-PEG (EDC/NHS+K2SO4) at moderate and high magnifications. However, considering that there are negligible differences in surface charge seen the likely reasoning for this is that some colloidal suspension is lost during functionalisation and washing procedures, and that negligible amount of PEG are tethered.
The presence of a film-like material was observed in high-magnification SEM images of
Si-PEG (EDC/NHS) and Si-PEG (EDC/NHS+K2SO4) samples, which was not apparently present
195 in other samples and appears to bind groups of particles together (Figure 43). It is still yet to be elucidated what the cause and identity of this substance is, however considering that this was only observable in samples that contained EDC/NHS it could be possible that cross-linking of particles occurred, where the terminal carboxyl groups of PEG were activated by residual EDC/NHS and conjugated to either: a) primary amines on PEG chains, or b) residual amines on the particles surface that were used to form carboxyl groups. This is supported by XPS survey spectra, which showed the presence of nitrogen (2-3%) for unmodified Si-COOH particles.
Figure 42. Representative SEM images of unmodified and PEG-modified Si-COOH microspheres under various grafting conditions. Scale of the images shown are at 100µm (left), 10µm (middle), and 2µm (right).
196
Figure 43. Representative SEM images of the film-like substance observed for: a) Si-PEG (EDC/NHS), and b) Si-PEG (EDC/NHS+K2SO4) samples. Scale bar: 2µm. 6.3.3 Protein adsorption analysis
Protein adsorption to colloidal crystal layers prepared with unmodified and PEG-modified particles was assessed using BSA as a model protein. A solution of 1mg/mL BSA was used and adsorption was allowed to proceed for 2hr before being analysed with XPS. Atomic composition data for BSA adsorption to the various surfaces are shown in Table 15, where the At%N can be indirectly used to infer protein adsorption. Considering the presence of nitrogen at approximately
2% prior to adsorption (See Table 14), the results in Table 15 indicate that surfaces had varying levels of adsorption, with %N being in the range of 5.2-6.8%. Si-PEG (EDC/NHS) samples did show significantly lower levels of adsorption compared to the other surfaces, which suggests that there were small amounts of PEG tethered to the particle surface creating a physical barrier between the adsorbing protein and the surface. Using theoretical elemental composition of BSA
(C2932N780O898S39) (H excluded) it can be calculated that the %N for an infinitely thick film of
BSA would be 16.8%, which is much higher than what was observed for the prepared surfaces. As the prepared surfaces are not planar and have inherent nano- and micro-topographies it is expected that some protein was adsorbed within the grooves and interstitial spaces of the Si colloids and not be visible by XPS analysis. This in itself is quite interesting as it is known that an adsorbed layer
197 of protein can provide a suitable conditioning film for subsequent bacterial attachment,29,30 Thus colloidal crystal layers may be used to direct and control protein adsorption using particles of various dimensions, which in turn may reduce or direct bacterial colonisation to specific areas within these surfaces.
Table 15. Atomic composition data for BSA adsorbed to unmodified and PEG-modified Si-COOH microspheres
Atomic composition (%) Sample ID O C N Si Na Bare Si-COOH 22.0±4.0 67.7±7.3 6.5±0.4 3.3±3.3 0.5±0.2 PEG (no EDC/NHS) 24.3±4.7 61.6±8.1 6.2±0.4 4.3±3.6 0.4±0.2 PEG (EDC/NHS) 30.1±6.9 54.1±11.5 5.2±0.2 9.6±4.9 0.3±0.1 PEG (EDC/NHS + K2SO4) 24.7±4.9 64.3±7.1 6.8±0.5 3.9±3.7 0.3±0.2
High-resolution XPS C1s spectra were also obtained of BSA adsorbed to the modified surfaces and indicated the presence of three major carbon environments, which are attributed to
C-C/C-H (285.0eV), C-O/C-N (286.4eV), and also O=C-N/O-C=O (288.5-289.2eV) photoelectrons (Figure 44). The contributions of each of these environments were extremely similar between all samples with the exception of Si-PEG (EDC/NHS) samples (Fig.44c), which has a lower percent area for O=C-N/O=C-O environments (11.9%) compared to all other surfaces
(14.1-16.5%). These results support previous data, which suggests that samples with EDC/NHS have small amounts of tethered PEG and capable of reducing protein adsorption to a small degree when compared to other experimental conditions, although not to an extent where these surfaces would be useful for non-fouling applications.
198
Figure 44. Representative High-resolution C1s XPS spectra of BSA adsorbed to modified particles. Spectra above are of BSA adsorbed to a) bare Si-COOH, b) Si-PEG (no EDC/NHS), c) Si-PEG (EDC/NHS), and d) Si-PEG (EDC/NHS + K2SO4). 6.4 Conclusions
This Chapter investigated the tethering of a heterobifunctional PEG (HOOC-PEG5000-NH2) to carboxylated silica colloidal particles and assessed the protein resistant properties when presented as a surface in the form of a colloidal crystal layer. The obtained experimental results suggest that little or no PEG was bound to the surface for Si-PEG (no EDC/NHS) and Si-PEG
(EDC/NHS+K2SO4) samples; as no significant change in the surface charge was seen after immobilisation, and XPS data showed little or no increase in the C-O component (286.4eV) which would definitively confirm the presence of PEG at the surface. The only condition that was likely to be somewhat effective was EDC/NHS only samples, which showed changes in atomic composition consistent with small amount of tethered PEG that was determined to be 0.6nm thick.
199
This was also supported by protein adsorption analysis suggesting that Si-PEG (EDC/NHS) samples had lower protein adsorption compared to all other conditions.
While it was determined that EDC/NHS only samples had small amounts of tethered PEG and showed some protein resistant properties, the extent of grafting and non-fouling ability was quite inefficient. In the case of no EDC/NHS and EDC/NHS+K2SO4 samples this is rationalised down to the fact that PEG was not adsorbed strongly to the Si-COOH colloids and washed away in the multiple centrifugation steps, and that the use of K2SO4 at such moderate temperatures hindered PEG grafting rather than providing ‘poor’ solvation conditions. Moreover, the tethering of a α-amine ω-carboxy hetero-bifunctional PEG through EDC/NHS grafting is determined to be inherently inefficient due to competitive interactions and the likelihood of particle cross-linking as seen in SEM images.
6.5 References 1 Vaisocherova, H., Brynda, E. & Homola, J. Functionalizable low-fouling coatings for label-free biosensing in complex biological media: advances and applications. Anal. Bioanal. Chem. 407, 3927-3953, doi:10.1007/s00216-015-8606-5 (2015).
2 Singh, G., Pillai, S., Arpanaei, A. & Kingshott, P. Highly ordered mixed protein patterns over large areas from self-assembly of binary colloids. Adv. Mater. 23, 1519-1523, doi:10.1002/adma.201004657 (2011).
3 Wang, P. Y., Pingle, H., Koegler, P., Thissen, H. & Kingshott, P. Self-assembled binary colloidal crystal monolayers as cell culture substrates. J. Mater. Chem. B 3, 2545-2552, doi:10.1039/c4tb02006e (2015).
4 Diba, F. S., Reynolds, N., Thissen, H., Wang, P.-Y. & Kingshott, P. Tunable Chemical and Topographic Patterns Based on Binary Colloidal Crystals (BCCs) to Modulate MG63 Cell Growth. Adv. Funct. Mater. 29, 1904262, doi:10.1002/adfm.201904262 (2019).
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5 Boden, A., Bhave, M., Wang, P.-Y., Jadhav, S. & Kingshott, P. Binary Colloidal Crystal Layers as Platforms for Surface Patterning of Puroindoline-Based Antimicrobial Peptides. ACS Appl. Mater. Interfaces 10, 2264-2274, doi:10.1021/acsami.7b10392 (2018).
6 Lim, K., Chua, R. R. Y., Saravanan, R., Basu, A., Mishra, B., Tambyah, P. A., Ho, B. & Leong, S. S. J. Immobilization studies of an engineered arginine-tryptophan-rich peptide on a silicone surface with antimicrobial and antibiofilm activity. ACS Appl. Mater. Interfaces 5, 6412-6422, doi:10.1021/am401629p (2013).
7 Onaizi, S. A. & Leong, S. S. J. Tethering antimicrobial peptides: Current status and potential challenges. Biotechnol. Adv. 29, 67-74, doi:10.1016/j.biotechadv.2010.08.012 (2011).
8 Ivanov, I. E., Morrison, A. E., Cobb, J. E., Fahey, C. A. & Camesano, T. A. Creating antibacterial surfaces with the peptide chrysophsin-1. ACS Appl. Mater. Interfaces 4, 5891-5897, doi:10.1021/am301530a (2012).
9 Xiao, M., Jasensky, J., Gerszberg, J., Chen, J., Tian, J., Lin, T., Lu, T., Lahann, J. & Chen, Z. Chemically Immobilized Antimicrobial Peptide on Polymer and Self- Assembled Monolayer Substrates. Langmuir 34, 12889-12896, doi:10.1021/acs.langmuir.8b02377 (2018).
10 Bartczak, D. & Kanaras, A. G. Preparation of peptide-functionalized gold nanoparticles using one pot EDC/Sulfo-NHS coupling. Langmuir 27, 10119-10123, doi:10.1021/la2022177 (2011).
11 Grabarek, Z. & Gergely, J. Zero-length crosslinking procedure with the use of active esters. Anal. Biochem. 185, 131-135, doi:http://dx.doi.org/10.1016/0003-2697(90)90267- D (1990).
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12 Arcot, L., Ogaki, R., Zhang, S., Meyer, R. L. & Kingshott, P. Optimizing the surface density of polyethylene glycol chains by grafting from binary solvent mixtures. Appl. Surf. Sci. 341, 134-141, doi:http://dx.doi.org/10.1016/j.apsusc.2015.02.156 (2015).
13 Kimoto, S., Dick, W. D., Hunt, B., Szymanski, W. W., McMurry, P. H., Roberts, D. L. & Pui, D. Y. H. Characterization of nanosized silica size standards. Aerosol Sci. Technol. 51, 936-945, doi:10.1080/02786826.2017.1335388 (2017).
14 Zou, H., Wu, S. & Shen, J. Polymer/Silica Nanocomposites: Preparation, characterization, propertles, and applications. Chem. Rev. 108, 3893-3957, doi:10.1021/cr068035q (2008).
15 An, Y., Chen, M., Xue, Q. & Liu, W. Preparation and self-assembly of carboxylic acid- functionalized silica. J. Colloid Interface Sci. 311, 507-513, doi:https://doi.org/10.1016/j.jcis.2007.02.084 (2007).
16 Gref, R., Lück, M., Quellec, P., Marchand, M., Dellacherie, E., Harnisch, S., Blunk, T. & Müller, R. H. ‘Stealth’ corona-core nanoparticles surface modified by polyethylene glycol (PEG): influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption. Colloids Surf. B. Biointerfaces 18, 301-313, doi:https://doi.org/10.1016/S0927-7765(99)00156-3 (2000).
17 Wan, F., You, J., Sun, Y., Zhang, X.-G., Cui, F.-D., Du, Y.-Z., Yuan, H. & Hu, F.-Q. Studies on PEG-modified SLNs loading vinorelbine bitartrate (I): Preparation and evaluation in vitro. Int. J. Pharm. 359, 104-110, doi:https://doi.org/10.1016/j.ijpharm.2008.03.030 (2008).
18 Lindman, S., Xue, W.-F., Szczepankiewicz, O., Bauer, M. C., Nilsson, H. & Linse, S. Salting the charged surface: pH and salt dependence of protein G B1 stability. Biophys. J. 90, 2911-2921, doi:10.1529/biophysj.105.071050 (2006).
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19 Gittings, M. R. & Saville, D. A. The determination of hydrodynamic size and zeta potential from electrophoretic mobility and light scattering measurements. Colloids Surf. Physicochem. Eng. Aspects 141, 111-117, doi:https://doi.org/10.1016/S0927- 7757(98)00207-6 (1998).
20 Frydman, A., Castner, D. G., Schmal, M. & Campbell, C. T. A Method for Accurate Quantitative XPS Analysis of Multimetallic or Multiphase Catalysts on Support Particles. J. Catal. 157, 133-144, doi:10.1006/jcat.1995.1274 (1995).
21 Al-Ani, A., Pingle, H., N, P. R., Wang, P. Y. & Kingshott, P. Tuning the Density of Poly(ethylene glycol) Chains to Control Mammalian Cell and Bacterial Attachment. Polymers 9, doi:10.3390/polym9080343 (2017).
22 Kim, J., Seidler, P., Wan, L. S. & Fill, C. Formation, structure, and reactivity of amino- terminated organic films on silicon substrates. J. Colloid Interface Sci. 329, 114-119, doi:http://dx.doi.org/10.1016/j.jcis.2008.09.031 (2009).
23 Yamamoto, H. S. & Yamamoto, H. S. Enhanced Growth of Thermal Oxide Due to Impurity Absorption from Adjoining Contaminated Silicon Wafers. Jpn. J. Appl. Phys. 31, 1756-1757, doi:10.1143/JJAP.31.1756 (1992).
24 Shameli, K., Ahmad, M. B., Jazayeri, S. D., Sedaghat, S., Shabanzadeh, P., Jahangirian, H., Mahdavi, M. & Abdollahi, Y. Synthesis and characterization of polyethylene glycol mediated silver nanoparticles by the green method. Int. J. Mol. Sci. 13, 6639-6650, doi:10.3390/ijms13066639 (2012).
25 Alcantar, N. A., Aydil, E. S. & Israelachvili, J. N. Polyethylene glycol–coated biocompatible surfaces. J. Biomed. Mater. Res. 51, 343-351, doi:10.1002/1097- 4636(20000905)51:3<343::AID-JBM7>3.0.CO;2-D (2000).
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27 Vukovic, L., Khatib, F. A., Drake, S. P., Madriaga, A., Brandenburg, K. S., Kral, P. & Onyuksel, H. Structure and dynamics of highly PEG-ylated sterically stabilized micelles in aqueous media. J. Am. Chem. Soc. 133, 13481-13488, doi:10.1021/ja204043b (2011).
28 Szleifer, I. Protein adsorption on tethered polymer layers: effect of polymer chain architecture and composition. Physica A: Statistical Mechanics and its Applications 244, 370-388, doi:https://doi.org/10.1016/S0378-4371(97)00293-8 (1997).
29 Shirtliff, M. E., Mader, J. T. & Camper, A. K. Molecular interactions in biofilms. Chem. Biol. 9, 859-871, doi:10.1016/S1074-5521(02)00198-9 (2002).
30 Donlan, R. M. Biofilms: Microbial life on surfaces. Emerging Infect. Dis. 8, 881-890 (2002).
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7 Analysis of MPTS deposition methods for the production of thiol-containing films 7.1 Summary This Chapter studies the deposition of MPTS in organic solvents on to Si substrates and characterises film properties using ellipsometry, XPS, static water contact angle, and ATR-FTIR.
Condensation of MPTS onto Si surfaces was most efficient in more anhydrous solvents such as toluene, with monolayer thickness values (≈0.6nm) being observed at MPTS concentrations of
0.08% (v/v) when compared to 1% (v/v) when deposition was carried out in ethanol. The shortcomings of ethanol deposition can be overcome by applying a pre-hydrolysis period at low pH to achieve similar film thicknesses and properties compared to toluene at shorter deposition times - which is important when functionalisation needs to be carried out in plastic consumables.
In addition to assessing the effects of different MPTS concentrations and solution chemistries, it was also necessary to investigate solution chemistry and post-deposition treatments as these methods vary widely within current literature resulting in vastly different film properties.1,2 With minimal post-deposition treatments it was shown that controllable film thicknesses can be achieved with a high-retention of thiol functional groups. Results presented are currently being adapted for publication. Additionally, the procedures developed here are important for many applications such as metal bonding, the functionalisation of planar substrates and nanoparticles, where the organosilane thickness, degree of order, reactivity, and surface concentration of thiols influence film functionality.
7.2 Introduction As indicated within the thesis introduction, two silanes; 3-aminopropyl(triethoxysilane)
(APTES) and 3-mercaptoproply(trimethoxysilane) (MPTS) were used to adorn (or “activate”)
205 inorganic surfaces with amine and thiol functional groups, respectively. This was performed to facilitate subsequent polymer and AMP immobilisation as it is necessary that the appropriate chemical functional groups are present and at sufficient densities. Ideally, a single silanisation method would be in place to facilitate APTES and MPTS immobilisation, however this is generally not possible due to the differences in reactivity of terminal functional groups and their likelihood to enter competing reactions.3 For example, amino groups on APTES molecules can interact with silanols present on the surface and/or on hydrolysed APTES through electrostatic interactions or hydrogen bonding.4,5
Deposition of MPTS has been proven to be a promising technique in a number of application areas including corrosion prevention,6 protein immobilisation,7 molecular electronics,8 and (bio)sensing devices.9 In addition, MPTS shows versatility when compared to other silanes as the methoxy silane component can bond to many metal oxide surfaces10,11 while the terminal thiol group can bond to gold,12,13 and silver,12 and also be involved in a number of other reactions. As with the deposition of many silanes, MPTS deposition is highly sensitive to pH, silane concentration, and also moisture content. Thus changes in any of the aforementioned properties will also greatly affect the rates of hydrolysis and condensation. Specifically for MPTS deposition, the relatively non-polar nature and higher pKa of the thiol functional groups results in reaction kinetics that are somewhat different to that of APTES. For example, using a solution-based deposition method, Scott, et al. 6 has shown that initial pre-hydrolysis of MPTS at pH 4 for 2h provides a balance between hydrolysis of methoxy groups and condensation with surface hydroxides allowing rapid bonding to surfaces on many metallic and semiconducting materials.2,6
Under these conditions, there is a high retention of thiol functional groups present at the surface available for subsequent bonding, and deposition times can be as low as 5 min. Furthermore, the
206 use of post-deposition process processes such as rinsing and drying seem to be inconsistent within current literature which may significantly alter film properties.1,2
Considering the above information and the versatility of thiol-based immobilisations, this section will assess the deposition of MPTS in organic solvents aimed at the production of thiol- containing films which will then be subsequently used as a platform for polymer and AMP immobilisation. Additionally, the effect of different post-deposition treatments, and pre-hydrolysis conditions for MPTS deposition is also assessed in order to optimise surface coverage and film quality. Chemical and physical properties of the resulting films were evaluated using a combination of surface analytical techniques including ellipsometry, water contact angle, XPS, and ATR-FTIR.
7.3 Results and discussion Preliminary investigations in to MPTS films used solution-based deposition in toluene at varying concentrations directly on cleaned Si wafers. Thickness and surface chemistry of the resulting MPTS films was subsequently determined using ellipsometry and ATR-FTIR, respectively. Thickness values determined by VASE shown in Figure 45 below indicate that film thickness increases with increasing MPTS concentration as expected, where thickness values ranged from 0.55-1.75nm over concentrations of 0.04-4% (v/v). Based upon molecular projection models monolayer coverage (0.6nm) is seen to be achieved at MPTS concentrations between 0.08 and 0.2 (%(v/v)) and at a deposition time of 3hr. Interestingly, it appears that the growth of MPTS films proceeds much slower than APTES at the same concentration (2% (v/v)) (See Section 5.3.2), with similar thickness values (≈1nm) being observed after 30min compared to 3hr for MPTS. This could be caused by the strong electrostatic interactions that occur between amine groups and surface silanols, resulting in greater lateral film growth and films that may be more resistant to
207 washing procedures. Additionally, there also appears to be significant deviation in the thickness of MPTS films at each concentration investigated, which once again highlights how film thicknesses can be quite varied under seemingly identical experimental conditions due to humidity and history of glassware.
2.0
1.5
1.0 Thickness (nm) 0.5
0.0 0.040.080.2124 MPTS concentration (% (v/v))
Figure 45. MPTS film thickness values determined by VASE. Measurements were taken after 3hr deposition in toluene. Data reported are the average of three independent samples, with three spots being analysed per sample.
In assessing the quality of MPTS films deposited in toluene there are certain spectral regions of interest when interpreting ATR-FTIR data which correspond to Si-O-CH3 and Si-O-Si vibrational modes (Figure 46). However, it is noted that due to the similarities in chemical environments between silane films and the underlying SiO2 substrate, in some circumstances these regions can be quite difficult to interpret. Theoretically, as hydrolysis continues there will be a
-1 -1 reduction in methoxy (Si-O-CH3) bands (1089cm ) and the appearance of Si-O-Si (1116cm ) bands upon polymerisation.11 Fig. 46a shows there are no observable peaks at 1089cm-1 under any
208 reaction condition, however bands attributed to Si-O-Si can be seen for all samples – suggesting successful condensation with surface silanols.
When comparing Figure 46a and 46b it can be seen that relative peak intensities for Si-
OH/Si-O bands at approximately 900cm-1 are lower than those seen for Si-O-Si indicating that a larger proportion of MPTS had condensed with surface silanols or other hydrolysed MPTS molecules. Interestingly, there was no peak observed at 2550cm-1 corresponding to S-H stretching modes which would definitively confirm the presence of MPTS at the surface (Fig. 456). It is noted that S-H stretching modes are quite weak in ATR-FTIR setups which may have contributed to this peak not being seen in the data obtained. It could also be possible that the depth resolution of ATR-
FTIR is not high enough to accurately determine film chemistry that are ≤2nm thick.
Figure 46. ATR-FTIR spectra of MPTS films deposited in toluene for 3hr over a wavenumber range of a) -1 -1 -1 1070-1200cm , b) 840-970cm , and c) 2450-3500cm .
209
The above results suggest that when deposited in toluene the thickness of MPTS films increases with increasing MPTS concentration, and monolayer coverage is seen to be achieved at
MPTS concentrations between 0.08% and 0.2% (v/v) with successful condensation with surface hydroxides. However, as this project will also investigate polymer and AMP colloidal particles it is a requirement that plastic lab consumables be used during functionalisation and centrifugations procedures. Therefore to avoid damage to plastic lab consumables and to facilitate colloidal functionalisation, deposition of MPTS in ethanol also was investigated.
Shown below in Figure 47 are ellipsometry thickness measurements for MPTS films prepared in ethanol which indicates that ethanol is less effective in silanisation processes with consistently lower thickness values when compared to deposition in toluene. Minimum and maximum Thickness values for deposition in ethanol were observed to be 0.32±0.05nm and
0.83±0.15nm at concentrations of 0.04% and 4% (v/v), respectively. It is suggested that ethanol being a more polar solvent than toluene readily hydrolyses methoxy groups leading to increased polymerisation in solution over the 3hr deposition period. These solution-phase oligomers and polymers would be only loosely bound to the surface and most likely washed away during the rinsing procedures leading to the relatively lower thickness values observed.
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1.0
0.8
0.6
0.4 Thickness (nm)
0.2
0.0 0.040.080.2124 Concentration (% v/v)
Figure 47. Thickness values for MPTS films determine by VASE. Measurements were taken after 3hr deposition in ethanol and data reported are the average of three independent samples, with three spots being analysed per sample.
It is also interesting to note that the curing conditions (100°C for 1hr) will result in a thermal oxide layer forming on bare Si <100> of an approximate thickness of approximately 0.5-
1.5nm,14-17 and has also been reproduced experimentally at 1.5±0.4nm (data not presented on graph). This may suggest that at low MPTS concentrations (0.04-0.2% (v/v)), deposition of MPTS in ethanol did not occur, and instead thickness measurements were a result of thermal oxide growth. Considering these unfavourable results and the need to facilitate silanisation of colloidal particles deposition in ethanol was pursued by applying a 2hr pre-hydrolysis period at high MPTS concentrations and adjusted pH using a method adapted from Scott, et al. 6. Additionally, as this study does not mention the use of any post-deposition treatments (e.g, rinsing and drying), the effects of different treatments were also assessed in regards to the quality of MPTS films.
Ellipsometry was again used to determine the thickness of MPTS films produced in ethanol at pH 4 or 8.5 with a 2hr pre-hydrolysis period and with and without a post-deposition rinsing procedure with thickness measurements for films produced with a post-deposition rinse are shown
211 below in Figure 48. Clearly it can be seen that thicker films are obtained at pH 4 when compared to pH 8.5 which was expected as previous studies have shown that at pH 4; not only is the rate of hydrolysis much faster than at pH 8.5, there is also an equilibrium between available surface silanols and minimal interference with solution-phase oligomers.6 Thickness values observed at pH 4 were seen to between 0.7nm and 1.5nm over a concentration range of 1-4% (v/v), which indicates that by applying a pre-hydrolysis period and low pH, thickness values that are similar to deposition in toluene can be achieved at shorter reaction times (See Figure 45). At pH 8.5 however, there appeared to be a negligible difference in film thickness regardless of the concentration used, suggesting the higher pH used slowed down the hydrolysis rate of methoxy groups considerably, leading to minimal or no deposition under these conditions.
Figure 48. MPTS thickness values determined by VASE under pre-hydrolysis conditions at pH 4 and 8.5 with a post-deposition rinse. Data reported is the average of two independent samples (n=2) and three measurements were taken per sample.
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In order to achieve thick MPTS films with a high retention of thiol functional groups, the effects of various post-deposition processes were investigated which involved either a) Rinse and
Dry after deposition, b) Dry Only after deposition, and c) No Treatment. Ellipsometry thickness measurements for the prepared films at high MPTS concentrations and using various post- deposition treatments are shown in Figure 49, which shows that film thicknesses increase with increasing MPTS concentration. For films that were immediately rinsed and dried after deposition, this phenomena was not as significant; where film thickness values were observed to be 1.1, 1.1 and 1.7nm at concentration of 1, 2, and 4 % (v/v), respectively. This result provides little information if deposition was successful under these conditions because, as stated previously, it is known that oxidation of Si <100> will continue to occur under ambient conditions, and will also be accelerated during the curing process, which will result in an oxide layer of similar thickness to that observed here.14-17
Figure 49. Ellipsometry thickness values of MPTS films prepared on Si wafers under various post- deposition processes. All samples were prepared in triplicate (n=3) and three individual spots were analysed per sample. 213
For MPTS films prepared using other post-deposition treatments a clear and large increase in film thickness can be seen as the solution MPTS concentration increases, and films prepared with no post-deposition treatment were generally higher than other conditions at the same MPTS concentrations. This result was expected as the physical force created through drying and rinsing procedures would have removed loosely bound MPTS molecules and oligomers leading to decreased film thickness. While thickness measurements obtained using minimal post-deposition methods suggest multilayer coverage of MPTS – it is critical that there are sufficient concentrations of reactive thiol (-SH) functional groups present at the surface for subsequent tethering reactions such as those with polymer, protein, or peptides. As such, further characterisation of MPTS films by water contact angle (WCA) measurements, XPS, and ATR-
FTIR was performed to assess surface hydrophobicity, elemental composition, and surface chemistry, respectively.
Figure 50 shows the static WCA measurements for MPTS films prepared under the various experimental conditions described. The contact angle for UV/O3 treated Si wafers was observed to be 5.4° ± 0.4°, which was expected for treated silicon surfaces due to the removal of organic
18 contaminants and conversion of O-Si-C bonds to the more hydrophilic SiOx (1.6 ≤ x ≤ 2.0). For samples that were prepared without the addition of MPTS (0% (v/v)), results show that similar contact angles regardless of the post-deposition treatment used which are most likely due to residual ethanol from either the deposition or rinsing process. As the deposition solutions were prepared at pH 4, it is also likely that surface oxides were reduced to hydroxides leading to a more hydrophobic chemical environment.
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Figure 50. Summary of static water contact angle results for MPTS films prepared on Si wafers using various post-deposition processes. Samples were prepared in triplicate (n=3) and three separate spots were analysed each sample.
When considering results for Dry Only and No (post) Treatment, a significant increase in contact angle is observed for all MPTS concentrations compared to the UV/O3 control. For Dry
Only samples, WCA measurements were 79.2 ± 6.4°, 80.5 ± 3.4°, and 85.8 ± 2.5°, for MPTS concentrations of 1, 2, and 4% (v/v), respectively. Similarly, for No Treatment samples, contact angles were seen to be 72.1 ± 1.8°, 80.1 ± 3.4°, and 86.2 ± 5.3° at MPTS concentrations of 1, 2, and 4% (v/v), respectively. While these values are somewhat higher than those previously reported in the literature for monolayer coverage of MPTS films, ellipsomety results indicate multilayer coverage of MPTS where there are a high proportion of mercaptopropyl side-chains at the interface resulting in the increased contact angles seen here.
215
For Rinse and Dry samples there was minimal difference seen in contact angles under all
MPTS concentrations, where results were 62.4 ± 2.8°, 55.8 ± 2.5° and 58.8 ± 3.3° for MPTS concentrations of 1, 2, and 4% (v/v), respectively. Comparing these values to the mean contact angle for negative control samples (0% (v/v) MPTS) of 55.8 ± 7.3°, the result suggests there is no considerable change in surface chemistry between unmodified and modified surfaces under these conditions. This result may be deceiving as it has been previously reported that monolayers of
MPTS films have contact angles of 58 ± 3°19, 54 ± 2°20, and 53 ± 2°21, which are comparative to values obtained here for UV/O3 control samples, providing little evidence that silanisation can be declared successful using extensive washing procedures. Representative contact angle images for untreated and MPTS treated (2 % (v/v)) silicon wafers under the various post-deposition processes are shown in Figure 51 which clearly shows the significant increase in static contact angle for samples with minimal post-deposition treatments (i.e. Dry Only and No Treatment) when compared to more extensive post-deposition treatments and unmodified samples.
Figure 51 Representative static water contact angle images of MPTS treated (2% (v/v)) Si wafers using various post-deposition treatments.
To characterise MPTS coatings further, XPS was used to investigate the elemental composition and chemical states of the outer 10nm of modified surfaces and atomic composition data are shown below in Table 16. Results show that the elemental composition for untreated Si
(0% MPTS) consisted of oxygen (34.8 ± 0.7%), carbon (16.3 ± 1.3%), and silicon (48.7 ± 0.8%)
216
with small traces on nitrogen (0.1 ± 0.1%). Such atomic composition data is expected for Si
surfaces with adventitious contamination which may have occurred during washing or curing
procedures, and is consistent with previously published data.3,17 For MPTS treated samples
utilising Rinse and Dry post-deposition methods there was negligible difference in elemental
composition data for all MPTS concentrations and were similar to untreated (0% MPTS) samples.
However the presence of sulfur was also detected at levels 12.2-12.8%. When considering atomic
composition data for Dry Only and No Treatment samples there are clear differences when
compared to unmodified samples; with a substantial decrease in both %Si and %O, which was
likely due to the immobilisation of hydrolysed MPTS to surface hydroxides or already existing
MPTS layers.
Table 16. XPS atomic composition data for MPTS films produced on Si wafers using various post- deposition processes.
Atomic composition (%) O C S Si N Bare Si 34.8 ± 0.7 16.3 ± 1.3 0 ± 0 48.7 ± 0.8 0.1 ± 0.1 1%( v/v) 30.7 ± 0.5 11.8 ± 0.7 12.2 ± 0.5 45.3 ± 0.7 0 ± 0 Rinse and 2%( v/v) 29.4 ± 0.3 13.2 ± 1.7 12.7 ± 0.3 44.7 ± 1.5 0 ± 0 Dry 4%( v/v) 28.3 ±0.6 12.8 ± 0.9 12.8 ± 0.1 46.2 ± 0.5 0 ± 0 1%( v/v) 29.3 ± 0.5 25.4 ± 4.1 11.6 ± 0.5 33.7 ± 3.1 0 ± 0 Dry Only 2%( v/v) 30.2 ± 0.4 16.8 ± 1.6 12.8 ± 0.3 40.2 ± 1.1 0 ± 0 4%( v/v) 30.2 ± 0.5 17.8 ±1.4 12.5 ± 0.1 39.5 ± 1.2 0 ± 0 1%( v/v) 24.5 ± 0.3 50.5 ± 0.2 11.2 ± 0.4 13.8 ± 0.1 0 ± 0 No 2%( v/v) 24.4 ± 0.1 50.1 ± 0.5 11.8 ± 0.1 13.6 ± 0.1 0 ± 0 Treatment 4%( v/v) 26.4 ± 1.8 41.3 ± 8.8 11.0 ±1.0 21.3 ± 7.6 0 ± 0 MPTS 27 55 9 9 0 monomer* Condensed 25 37.5 12.5 25 0 MPTS*
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*Denotes theoretical atomic composition data for MPTS monomers and also MPTS polymer fully condensed with surface silanols ([C3O2SSi2]n).
Theoretical atomic composition data for MPTS monomers and polymerised MPTS films were used as a guide to assess the quality of MPTS films (Table 16) and shows that the most notable changes are the decrease of carbon content from 55% to 37.5% due to the hydrolysis of methoxy groups and subsequent proportional in silicon upon polymerisation from 9% to 25%.
When comparing these values to data obtained from No Treatment samples it can be seen that elemental composition falls between the two calculated theoretical values indicating there are possibly areas of partially polymerised MPTS. These results are also supported by ellipsometry data which suggests there are MPTS multilayers - which are more likely to form through deposition of partially polymerised oligomers to already existing MPTS layers.2,22 Considering the similarities in atomic composition for all MPTS concentrations of samples with No Treatment it is expected that film properties will also be similar in the outermost 10nm for all concentrations suggesting that functionalisation with MPTS at 1% (v/v) or higher and minimal post-deposition treatments may be suitable for generating thiol-containing surfaces.
In assessing the quality of MPTS films it is observed that C/Si ratios for Dry Only samples
(≈0.6) are lower than that of both MPTS monomers and (≈6.0) MPTS polymers (≈1.5), which would only occur if substrate signals are present. This observation is quite interesting as ellipsometry data indicates that MPTS thicknesses are greater than that of the sampling depth of
XPS (≈10nm) and substrate signals should not be present. Results also show areas of inhomogeneity at high MPTS concentrations for No Treatment samples as indicated by the large deviation in atomic composition seen for carbon and silicon. The above observations may be explained by the presence of film defects and pinholes caused by the physical removal of unbound
218
MPTS oligomers and polymers by N2 drying processes – which are undetected by ellipsomety due to its larger sampling area.23
Interestingly, all samples, regardless of the type of post–deposition treatment applied, showed somewhat similar amounts of %S within the ranges of 11-13%. This is interesting considering the vastly different ellipsometry thicknesses and water contact angles seen for the different post-deposition treatments. To investigate this further and confirm the presence of thiol- functional groups at the surface, high-resolution S2p spectra of MPTS-modified and unmodified
Si were obtained and results are presented in Figure 52. For unmodified samples (Fig. 52a) a peak is clearly visible occurring at approximately 166.9eV and corresponds to the plasmon lines due to energy loss events of Si2s photoelectrons.24,25 Unfortunately these plasmon features overlap with the spectral region of S2p photoelectrons (See Figure 53), which can make data interpretation somewhat more complex. However, it is still possible to visualise these S2p peaks after curve- fitting with associated binding energies for the S2p1/2 peak. For –C-SH groups, these are expected to occur at 164.1eV with a doublet arising due to the spin-orbit splitting; S2p1/2/S2p3/2 (Δ =
1.2eV).26
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Figure 52. High-resolution XPS spectra obtained from the S2p region of a) bare Si wafers, b) MPTS films prepared by Rinse and Dry methods at 2% (v/v), MPTS films prepared by Dry Only at c) 1% (v/v), d) 2% (v/v), e) 4% (v/v), and films prepared with No Treatment at f) 1% (v/v), g) 2% (v/v), and h) 4% (v/v). *Data for Rinse and Dry samples at 1% (v/v) and 4% (v/v) are not shown as spectra obtained showed no presence of photoelectrons attributed to S2p orbitals.
For MPTS films prepared by Dry Only, high-resolution S2p spectra show that substrate signals are clearly present under all MPTS concentrations (Fig.52c-e); with Si plasmon loss features being observed at 167.9eV. Interestingly these features do not become less prominent as the MPTS concentration increases, once again suggesting the presence of pinholes and defects under these conditions, which expose the underlying Si substrate. MPTS films prepared with No
Treatment however show negligible (1% (v/v)) (Fig.52f) or no (2-4% (v/v)) (Fig.52g-h) substrate signals from Si2s photoelectrons and the only peaks observed were attributed to thiol (R-SH) functional groups at approximately 164.0eV. This indicates that there is a high proportion of thiol functional groups present at the surface which can be used for subsequent grafting investigations.
220
Figure 53. XPS spectra of binding energies between 140-220eV of Si wafers showing overlapping spectral regions caused by plasmon structures associated with energy loss events from Si2s electrons.
Since XPS analysis showed that No (post) Treatment MPTS films were of high-quality with a high retention of thiol functional groups, ATR-FTIR was also employed to characterise the chemical properties of these surfaces further, and representative spectra are shown below in Figure
54. When characterising organosilane films using IR spectroscopy there are certain spectral regions of interest, however, interpretation and data collection for thin films can be difficult due to the large penetration depths in ATR mode. This can be observed for MPTS films prepared at lower concentrations (1% (v/v)) (Fig. 54b), where representative spectra are similar to that of the negative control (Fig. 54a).
221
Figure 54. Representative ATR-FTIR spectra of MPTS films prepared with No Treatment at MPTS concentrations of a) 0% (v/v), b) 1% (v/v), c) 2% (v/v), and d) 4% (v/v). Samples were analysed at 4cm-1 resolution and 64 scans were performed each measurement.
For substantially thicker MPTS films such as those prepared at 2% (v/v) (Fig. 54c) and 4%
(v/v) (Fig. 54d), ATR-FTIR results show characteristic peaks that are typical for organosilane films
-1 -1 including Si-O-Si stretching modes at 797 and 1100cm , C-S stretching at 689cm , and S-H stretching at ca. 2550cm-1 which are summarised below in Table 17. Interestingly, while it was predicted that all films would contain areas of partially hydrolysed MPTS being indicated by Si-
O-C asymmetric stretching at ca. 820cm-1, these were not observed at 4% (v/v). However, as the
IR signals from Si-O-Si (ca. 794cm-1) can overlap with the stretching modes of Si-O-C it is assumed that there are still areas of partially hydrolysed MPTS moieties within the film as indicated by the XPS data. When comparing peak assignments and intensities for MPTS films prepared at 2% (v/v) and 4% (v/v) it can be seen that there are very few differences in chemical 222 assignments at these concentrations suggesting similar surface chemistry for films prepared under these conditions. Additionally, previous research has suggested that solutions of MPTS at 0.02-
0.04% (v/v)2, and 4% (v/v)27 can produce thiol-containing films which are suitable for subsequent reactions. Also, considering the evidence shown to support successful deposition for films prepared at high MPTS concentrations with No Treatment, it is expected that these films will provide an adequate support for the grafting of additional functional layers such as non-fouling polymers and bioactive peptides.
Table 17. ATR-FTIR frequencies and assignments for MPTS films prepared with No Treatment at various concentrations.
Wavenumber (cm-1) 0% (v/v) 1% (v/v) 2% (v/v) 4% (v/v) Assignment Reference27,28 - 689 689 v(C-S) 659 - 795 796 796 v(Si-O-Si) 794
- 821sh 816sh - va(Si-O-C) 820 - 1003 1002sh 1002sh v(C-C) 1006 - 1033 1035 1030 CH2 rock 1032 - - 1102 1100 v(Si-O-Si) 1100
- - 1116sh 1118sh CH2 wag 1116 - 2552 2553 v(S-H) 2543
- - 2845 2847 vs(CH3) 2842
- - 2885 2886 vs(CH2) 2889
- - 2928 2928 va(CH2) 2928
- - 3348 3345 v(H2O) 3350
Nomenclature: v = stretching, vs = symmetric stretching, va = assymetric stretching, sh = shoulder
7.4 Conclusions Results presented within this Chapter clearly indicate that there are certain chemical and physical changes in properties of MPTS films depending on the type of deposition method and post-deposition treatment employed. It is apparent that utilising a pre-hydrolysis period in conjunction with low pH (pH 4) for MPTS formation, a balance between hydrolysis of methoxy groups and subsequent condensation with surface silanols is achieved. Under these conditions 223 substantially thicker films can be obtained with thicknessed increasing as MPTS concentration increases. Without the pre-hydrolysis period and low pH it appears that the film thickness becomes extremely low and difficult to characterise due to chemical similarities with the underlying Si substrate. A similar phenomena is seen when investigating films prepared by the Rinse and Dry post-deposition method where film characteristics are seen to be similar to that of the control Si.
The combination of a pre-hydrolysis period at low pH and minimal post-deposition treatments allows for thick MPTS films to be obtained which can be controlled with MPTS concentration. Additionally, a high retention of thiol functional groups can be achieved with this method which can then be used as a platform for the immobilisation of polymers and AMPS to not only planar surface, but also spherical colloidal particles. From the results presented here we can also propose a method for silanisation in relatively polar solvents such as ethanol, which can be beneficial when the use of plastic consumables or the need for using solvents of less toxicity is required.
7.5 References 1 Hu, M., Noda, S., Okubo, T., Yamaguchi, Y. & Komiyama, H. Structure and morphology of self-assembled 3-mercaptopropyltrimethoxysilane layers on silicon oxide. Appl. Surf. Sci. 181, 307-316, doi:http://dx.doi.org/10.1016/S0169-4332(01)00399-3 (2001).
2 Singh, J. & Whitten, J. E. Adsorption of 3-Mercaptopropyltrimethoxysilane on Silicon Oxide Surfaces and Adsorbate Interaction with Thermally Deposited Gold. The Journal of Physical Chemistry C 112, 19088-19096, doi:10.1021/jp807536z (2008).
3 Kim, J., Seidler, P., Wan, L. S. & Fill, C. Formation, structure, and reactivity of amino- terminated organic films on silicon substrates. J. Colloid Interface Sci. 329, 114-119, doi:http://dx.doi.org/10.1016/j.jcis.2008.09.031 (2009).
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4 White, L. D. & Tripp, C. P. Reaction of (3-Aminopropyl)dimethylethoxysilane with Amine Catalysts on Silica Surfaces. J. Colloid Interface Sci. 232, 400-407, doi:10.1006/jcis.2000.7224 (2000).
5 Kamisetty, N. K., Pack, S. P., Nonogawa, M., Devarayapalli, K. C., Kodaki, T. & Makino, K. Development of an efficient amine-functionalized glass platform by additional silanization treatment with alkylsilane. Anal. Bioanal. Chem. 386, 1649-1655, doi:10.1007/s00216-006-0741-6 (2006).
6 Scott, A. F., Gray-Munro, J. E. & Shepherd, J. L. Influence of coating bath chemistry on the deposition of 3-mercaptopropyl trimethoxysilane films deposited on magnesium alloy. J. Colloid Interface Sci. 343, 474-483, doi:10.1016/j.jcis.2009.11.062 (2010).
7 Perret, E., Leung, A., Morel, A., Feracci, H. & Nassoy, P. Versatile Decoration of Glass Surfaces To Probe Individual Protein−Protein Interactions and Cellular Adhesion. Langmuir 18, 846-854, doi:10.1021/la015601y (2002).
8 Selzer, Y., Salomon, A. & Cahen, D. The Importance of Chemical Bonding to the Contact for Tunneling through Alkyl Chains. The Journal of Physical Chemistry B 106, 10432-10439, doi:10.1021/jp026324m (2002).
9 Snow, A. W., Ancona, M. G., Kruppa, W., Jernigan, G. G., Foos, E. E. & Park, D. Self- assembly of gold nanoclusters on micro- and nanoelectronic substrates. J. Mater. Chem. 12, 1222-1230, doi:10.1039/B108859A (2002).
10 Lee, J. P., Kim, H. K., Park, C. R., Park, G., Kwak, H. T., Koo, S. M. & Sung, M. M. Photocatalytic Decomposition of Alkylsiloxane Self-Assembled Monolayers on Titanium Oxide Surfaces. The Journal of Physical Chemistry B 107, 8997-9002, doi:10.1021/jp030077k (2003).
11 Scott, A. & Gray-Munro, J. E. The surface chemistry of 3- mercaptopropyltrimethoxysilane films deposited on magnesium alloy AZ91. Thin Solid Films 517, 6809-6816, doi:http://dx.doi.org/10.1016/j.tsf.2009.05.044 (2009). 225
12 Lee, K.-H., Huang, K.-M., Tseng, W.-L., Chiu, T.-C., Lin, Y.-W. & Chang, H.-T. Manipulation of the Growth of Gold and Silver Nanomaterials on Glass by Seeding Approach. Langmuir 23, 1435-1442, doi:10.1021/la061880j (2007).
13 Piwoński, I., Grobelny, J., Cichomski, M., Celichowski, G. & Rogowski, J. Investigation of 3-mercaptopropyltrimethoxysilane self-assembled monolayers on Au(111) surface. Appl. Surf. Sci. 242, 147-153, doi:https://doi.org/10.1016/j.apsusc.2004.08.009 (2005).
14 Lin, C.-H. in Encyclopedia of Microfluidics and Nanofluidics (ed Dongqing Li) 1584- 1584 (Springer US, 2008).
15 Massoud, H. Z. The onset of the thermal oxidation of silicon from room temperature to 1000°C. Microelectron. Eng. 28, 109-116, doi:https://doi.org/10.1016/0167- 9317(95)00026-5 (1995).
16 Sarti, G. C., Santarelli, F. & Camera Roda, G. Kinetics of thermal growth of thin silicon oxide films. Chem. Eng. Sci. 41, 2699-2705, doi:10.1016/0009-2509(86)80059-8 (1986).
17 Yamamoto, H. S. & Yamamoto, H. S. Enhanced Growth of Thermal Oxide Due to Impurity Absorption from Adjoining Contaminated Silicon Wafers. Jpn. J. Appl. Phys. 31, 1756-1757, doi:10.1143/JJAP.31.1756 (1992).
18 Egitto, F. D., Matienzo, L. J., Spalik, J. & Fuerniss, S. J. in Materials Research Society Symposium - Proceedings. 245-250.
19 Bhatia, S. K., Teixeira, J. L., Anderson, M., Shriver-Lake, L. C., Calvert, J. M., Georger, J. H., Hickman, J. J., Dulcey, C. S., Schoen, P. E. & Ligler, F. S. Fabrication of surfaces resistant to protein adsorption and application to two-dimensional protein patterning. Anal. Biochem. 208, 197-205 (1993).
20 Hong, H. G., Jiang, M., Sligar, S. G. & Bohn, P. W. Cysteine-specific surface tethering of genetically engineered cytochromes for fabrication of metalloprotein nanostructures. Langmuir 10, 153-158, doi:10.1021/la00013a023 (1994). 226
21 Cras, J. J., Rowe-Taitt, C. A., Nivens, D. A. & Ligler, F. S. Comparison of chemical cleaning methods of glass in preparation for silanization. Biosensors Bioelectron. 14, 683-688, doi:http://dx.doi.org/10.1016/S0956-5663(99)00043-3 (1999).
22 Crudden, C. M., Sateesh, M. & Lewis, R. Mercaptopropyl-Modified Mesoporous Silica: A Remarkable Support for the Preparation of a Reusable, Heterogeneous Palladium Catalyst for Coupling Reactions. J. Am. Chem. Soc. 127, 10045-10050, doi:10.1021/ja0430954 (2005).
23 in Handbook of Vibrational Spectroscopy.
24 Tougaard, S. Energy loss in XPS: Fundamental processes and applications for quantification, non-destructive depth profiling and 3D imaging. J. Electron. Spectrosc. Relat. Phenom. 178-179, 128-153, doi:https://doi.org/10.1016/j.elspec.2009.08.005 (2010).
25 Kusunoki, I. & Igari, Y. XPS study of a SiC film produced on Si(100) by reaction with a C2H2 beam. Appl. Surf. Sci. 59, 95-104, doi:https://doi.org/10.1016/0169- 4332(92)90293-7 (1992).
26 Dodero, G., De Michieli, L., Cavalleri, O., Rolandi, R., Oliveri, L., Daccà, A. & Parodi, R. l-Cysteine chemisorption on gold: an XPS and STM study. Colloids Surf. Physicochem. Eng. Aspects 175, 121-128, doi:https://doi.org/10.1016/S0927- 7757(00)00521-5 (2000).
27 Thompson, W. R., Cai, M., Ho, M. & Pemberton, J. E. Hydrolysis and Condensation of Self-Assembled Monolayers of (3-Mercaptopropyl)trimethoxysilane on Ag and Au Surfaces. Langmuir 13, 2291-2302, doi:10.1021/la960795g (1997).
28 Kurth, D. G. & Bein, T. Surface reactions on thin layers of silane coupling agents. Langmuir 9, 2965-2973, doi:10.1021/la00035a039 (1993).
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8 Characterisation and antibacterial assessment of ‘one-pot ‘and sequential immobilisation of PEG and AMPs to silica colloids
8.1 Summary Considering the need for versatile surface coatings that can display multiple bioactive signals and chemistries, the use of more novel surface modification methods is starting to emerge.
Thiol-mediated conjugation of biomolecules has been shown to be quite advantageous for such purposes due to the reactivity and selectivity towards thiol functional groups. As such, this Chapter investigates the immobilisation of PEG and AMPs to silica colloidal particles based on thiol- mediated conjugation techniques, along with an assessment of the antimicrobial potential of the functionalised particles against P. aeruginosa. Silica colloids were first modified with MPTS to generate surface reactive thiol functional groups, which were then used to tether PEG by either a thiol-ene ‘photo-click’ reaction or a thiol-maleimide type conjugation using terminal acrylate or maleimide functional groups, respectively. Selected AMPs were subsequently immobilised by their N-terminus to NHS-terminated PEG chains. It was demonstrated that both types of immobilisation techniques were effective at tethering PEG and AMP, in which a significant reduction in the number of viable bacterial cells compared to unmodified samples was observed after incubation with the colloidal suspension. The findings presented in this Chapter provide a promising outlook for the fabrication of multifunctional surfaces based upon the tethering of PEG and AMPs to colloidal particles through thiol-mediated conjugation, which has potential for use as implant coatings or as antibacterial formulations that can be incorporated into wound dressings to prevent or control bacterial infections.
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8.2 Introduction The use of thiol-mediated surface reactions offers a versatile approach for the tethering of many types of (bio)molecules including polymers,1,2 proteins,3 and DNA.4,5 Due to its selectivity and reactivity, the use of such coupling methods may be advantageous for the tethering of PEG and AMPs to develop multifunctional surface coatings based on AMP-functionalised colloidal crystal layers or drug delivery formulations. While the immobilisation of PEG and AMPs have been discussed in general terms within previous chapters, here the specific use of thiol-mediated coupling is discussed in more detail.
Previous research has clearly shown that Au-thiol self-assembled monolayers (SAMs) can form defined and ordered non-fouling layers with minimal defects,6,7 however as the need for more complex surfaces that can display multiple signals and various topographies are required, different approaches are needed to immobilise specific types of (bio)molecules. For example, Bini, et al. 8 utilised a thiol-ene ‘photo-click’ reaction to facilitate the immobilisation of a multi-antennary glycidic structure on to thiolated collagen. In a separate study, Russo, et al. 9 also demonstrated that neoglycosylated collagen patches prepared by thiol-ene ‘photo-click’ chemistry was shown to have a positive effect on murine cartilage regeneration compared to control surfaces. In both of these studies the effective tethering of carbohydrate moieties by a thiol-mediated ‘photo-click’ reaction was demonstrated, allowing efficient conjugation in a chemo-selective manner, which can be quite useful for regenerative medicine and also ‘cell-responsive’ materials.9,10 The Michael addition reaction between thiol and maleimide functional groups has also been shown to be quite a reproducible method for the immobilisation of (bio)molecules, with maleimide groups used for thiol conjugation having relatively good stability and reactivity compared to other functional groups.1
229
With little or no precedence in current literature, the Chapter herein uses thiol-mediated conjugation reactions for the tethering of PEG and AMPs to silica particles, while assessing immobilisation efficiency and antimicrobial activity. Specifically, the knowledge of MPTS deposition methods gained from the previous Chapter was applied to functionalise spherical microparticles for the subsequent immobilisation of ethylene glycol-based polymers using thiol- mediated reactions, namely; thiol-ene ‘photo-click’ and thiol-maleimide ‘click’ chemistry (Figure
55). To facilitate the immobilisation of selected AMPs to PEG layers an NHS-terminated PEG was used to chemically react with N-terminus amines of AMPs with little interference from surface- bound thiols. The antimicrobial activity of the AMP-PEG functionalised colloids was also assessed over various time periods using P. aeruginosa as a model bacterial strain.
Figure 55. Schematic illustration of the coupling methods used to tether PEG (represented by R) to thiolated surfaces by a thiol-ene ‘photo-click’ reaction (red arrow) or a thiol-maleimide reaction (blue arrow).
230
8.3 Results and Discussion
8.3.1 Particle Characterisation
Zeta potential analysis was used to assess the surface charge of unmodified and modified particles with results being shown below in Figure 56. For bare silica particle zeta potential results were -74.8±5.9mV which is somewhat more negative than expected for this particle type with previous research suggesting a zeta potential of approximately -40mV and -50mV at pH 7 for
20nm and 100nm silica particles, respectively.11,12 These differences may be explained by the respective sizes of the colloidal particles between these experiments as the hydrodynamic size of
2.01µm Si colloids would be significantly larger- increasing their relative surface charge in relation to smaller particles. Additionally, atomic composition data presented later show negligible amounts of Na+, K+, and Mg2+ - minimising the shielding effect of the ion cloud surrounding the colloids.13,14 After immobilisation of MPTS the surface charge on Si particles becomes less negative suggesting that surface hydroxides were used in the formation of siloxane bonds with
MPTS molecules. XPS data presented in Table 18 however shows no significant changes in atomic composition between MPTS-coated and unmodified Si colloids. Considering the chemical similarities between adventitious carbon contamination and silanes – it is expected that without an extremely thick coverage of MPTS there would be negligible differences in atomic composition.
Moreover, as shown previously; confirming the presence of MPTS through the detection of sulphur for such coating can be quite problematic due to the overlapping spectral regions of Si 2s and S 2p photoelectrons.
231
Figure 56. Zeta potential data for PEG- and AMP-modified particles at each reaction stage. Sample type is indicated on the x-axis and zeta potential (mV) is indicated on the y-axis. Values presented are mean (mV) ± standard deviation (n=3).
For PEGylated particles a far more substantial change in surface charge was observed for both maleimide-terminated (-38.8 ± 2.7mV) and acrylate-terminated (-32.2 ± 2.3mV) which is similar to previously published data stating that PEG hydrogel-coated magnetic iron oxide nanoparticles possessed a surface charge of approximately -30mV,15 and suggests that PEG immobilisation was successful under both reaction types and grafting conditions. Furthermore, the presence of PEG can be detected from analysis of high-resolution C1s data obtained by XPS
(Figure 57) which shows a high-contribution of photoelectrons originating from C-O (C2) bonds occurring at approximately 286 eV which is typical for PEG coatings. 232
With regards to AMP immobilisation, a clear reversal in surface charge can be seen for all
AMP functionalised particles, which ranged from 12.1 ± 3mV to 29.6 ± 3.9mV suggesting that
AMP immobilisation was successful. For thiol-maleimide grafting this reversal was of lower magnitude when compared to thiol-ene grafting conditions, however, regardless of the grafting conditions the surface charge of P1-functionalised particles (19.7 ± 3.1mV and 29.6 ± 3.9mV) was consistently more positive than W8-functionalised particles (12.1 ± 3.1mV and 22.1 ± 1.1mV).
This result was expected due to the more cationic nature of P1; which contains seven positively charged amino acid residues compared to three in W8. The surface charge observed here for AMP- functionalised particles is slightly lower than what has been previously published for zero-length
16 immobilisation of PuroA (FPVTWRWWKWWKG-NH2) (+49 ±9mV), which was likely caused by the presence of PEG itself, and also small differences in pH and ionic strength which can greatly affect zeta potential measurements.13,14
AMP immobilisation was also confirmed using XPS atomic composition data which shows the presence of nitrogen for all AMP-modified samples arising from the amide backbone of AMPs.
Atomic composition data in Table 18 indicate that nitrogen content was 2.8 ± 0.9% and 3.4 ± 0.8% for W8-modified particles, and 3.1 ± 1.0% and 3.9 ± 0.7% for P1-modified samples using thiol- maleimide and thiol-ene ‘click’ chemistry, respectively. These results are somewhat lower than theoretical atomic composition data represented in Table 18 indicating that At% N for infinitely thick AMP coatings is 18% and 21% for P1 and W8 AMPs, respectively. The significant increase in At% N for AMP-modified samples after PEG and AMP immobilisation is also accompanied by an increase in At% C and decrease in At% O which would be expected after successful immobilisation. However, as XPS data also indicates the presence of Si for all samples, this suggests that combined PEG and AMP thickness is smaller than the sampling depth of XPS leading to substrate signals being seen from the underlying Si particle layers. This result is comparable to 233
previously published data which shows that antimicrobial activity of AMP-modified polystyrene
microspheres is retained at nitrogen compositions ranging from 2.7 ± 0.2% to 4.1 ± 0.5%, which
is promising for current results as the incorporation of a flexible PEG space should allow for
greater access and penetration into bacterial membranes.17-19.
Table 18. Atomic composition data for PEG- and AMP-modified Si colloids obtained using XPS
Atomic composition (%) O C N Si Na Mg Si 57.3±6.1 16.9±8.7 0.7±0.7 23.7±5.1 0.1±0.1 1.1±1.1 Si-MPTS 56.1±5.5 16.4±4.9 0.7±0.7 25.1±2.9 0.4±0.1 0.8±0.8 Si-PEG-Mal-W8 43±3.1 36.5±4.3 2.8±0.9 15.9±2.5 1.0±0.3 0.9±0.9 Si-PEG-Mal-P1 38±2.8 40±4.4 3.1±1.0 17±1.1 0.8±0.4 0.6±0.6 Si-PEG-Ac-W8 37.5±4.1 41.8±3.6 3.4±0.8 15.1±3.2 1.0±0.5 0.9±0.4 Si-PEG-Ac-P1 37.2±4.3 42.3±2.9 3.9±0.7 14.4±2.8 1.3±0.7 0.7±0.3 W8 theoretical* 9 73 18 0 0 0 P1 theoretical* 9 69 21 0 0 0
Furthermore, high-resolution C1s XPS data was also used to characterise the chemical
composition of AMP-modified colloids and results are shown below in Figure 57. For PEG- and
AMP-functionalised colloids (Fig 57c-f) there is observed to be a significant increase in the
proportion of C1s photoelectron attributed to C-O/C-N environments occurring at approximately
286.4eV which was expected after successful PEG immobilisation due to the high proportion of
C-O bonds present in PEG molecules.20,21 It can also be seen that that there is an increase in the
peak occurring at approximately 288.2eV, which is attributed to carbonyl and amide C1s
photoelectrons which again supports successful AMP immobilisation. Comparing C2 (C-O/C/N)
and C3 (C=O/N-C=O) carbon environments between PEG-AMP-functionalised samples in Table
19 it is observed that PEGAc-P1 has relatively lower C2:C3 ratios (≈1.1) compared to all other
samples. This may be due to the respective lengths of the of the AMPs used as P1 is five amino
234 acids longer than W8, but may also suggest higher AMP loading under these conditions which is supported by atomic composition data showing higher At% N and lower C:O ratios. Interestingly the same trend was not observed for PEGAc-W8 functionalisation which exhibited the highest
C2:C3 ratio (≈1.9) suggesting lower AMP to PEG ratios. This was also seen for PEGMal-W8 which indicates that the AMPs degree of hydrophobicity may play a role in reaction kinetics and efficiency due to differences in solvent/substrate interactions.22,23
Figure 57. High resolution C1s spectra of: a) unmodified Si particles, b) MPTS-modified Si, Mal-PEG- NHS functionalised Si with c) W8 AMPs, and d) P1 AMPs, and Ac-PEG-NHS functionalised Si with e) W8, and f) P1 AMPs.
235
Table 19. Binding energy positions and % area attributed to various carbon environments within the modified Si colloids
C1 C2 C3 C4 C5 C6 2- (C-C) (C-O/C-N) (C=O/N-C=O) (O-C=O) (CO3 /Charging) (CF2/CF3) Position Area Position Area Position Area Position Area Position Area Position Area
(eV) (%) (eV) (%) (eV) (%) (eV) (%) (eV) (%) (eV) (%)
Si-OH 285 42.7 286.5 27.8 288.4 17.8 289.8 11.7 - - - -
Si-MPTS 285 39 286.5 28.5 288 17.6 289.4 14.9 - - - -
Si-PEGMal-W8 284.9 19.5 286.3 34.5 287.9 22.7 289.3 16.6 290.6 4.5 292.8 2.3
Si-PEGMal-P1 284.8 17.0 286.3 34.9 288 25.7 289.4 17.9 290.8 4.6 - -
Si-PEGAc-W8 284.9 20 286.4 38.1 288.2 19.8 289.4 13.3 290.6 5.8 292.9 2.9
Si-PEGAc-P1 284.9 14.2 286.3 24.8 288.1 22.3 289.5 24.1 291 12.3 292.8 2.4
Ellman’s reagent was used to determine the amount of reactive sulfhydryl groups on
modified particles with a standard curve of cysteine being utilised to allow for quantification
(Figure 58). This standard curve shows that as expected there is a linear relationship between
absorbance and the amount of free (or reactive) sulfhydryl groups, and also that the trendline
represents the data quite well with an r2 value of 0.9886. Therefore, this can be accurately used to
determine the amount of sulfhydryl groups present on the surface of modified particles.
Figure 58. Standard curve of cysteine used to determine the amount of reactive sulfhydryl functional groups
236
Figure 59 shows calculated sulfhydryl concentrations for unmodified and modified silica colloids, and also the number of sulfhydryl groups present per particle. These results clearly indicate that there is a significantly high amount of –SH groups present on MPTS-modified particles (1.04x1010 per particle) when compared to all other samples, which have a much lower
9 numbers of –SH groups (2.03-2.17x10 per particle). This result was somewhat expected as there should be a much higher quantity of –SH groups on MPTS-modified silica colloids before functionalisation with PEG and AMPs. What is interesting about these results is the relatively similar amounts of –SH groups present on bare silica colloids when compared to PEG and AMP- modified particles. This suggests that a vast majority of reactive –SH groups were used for PEG and AMP immobilisation and that the absorbance values seen were from a small amount of particles that were present in the suspension, and provides promising evidence that modification of silica colloids with MPTS and also PEG and AMPs was successful.
Figure 59. Concentration and number of reactive sulfhydryl functional groups present on unmodified and modified silica colloids. Data reported here is the average of two independent samples (n=2) with three aliquots being measured per sample. 237
Considering the inherent complications with analysing Si- and thiol-containing films on
silicon substrates that arise from chemical similarities between the surface and adhering silane,
and also the overlapping spectral regions of S 2p and Si 2S photoelectrons in XPS analysis – the
information presented here clearly demonstrates a high degree of sulfhydryl functionality, which
are then subsequently used to facilitate PEG and AMP tethering. For a more accurate estimation
of the number of thiol functional groups present of the particle surface, the number of –SH groups
calculated for bare Si-OH particles was subtracted from other samples as this was attributed to
light scattering from small amounts of particles within the suspension. Corrected values are shown
below in Table 20.
Table 20. Total and corrected number of–SH groups on present on modified silica colloidal particles.
Si-OH Si-MPTS Si-PEGAc-P1 Si-PEGAc-W8 Si-PEGMal-P1 Si-PEGMal-W8 Thiol concentration 0.103±0.01 0.516±0.07 0.108±0.01 0.11±0.01 0.106±0.01 0.109±0.01 (mM)
Total number of -SH 6.22E+16 3.10E+17 6.48E+16 5.99E+16 6.33E+16 6.07E+16 Number of -SH per 2.08E+09 1.04E+10 2.17E+09 2.01E+09 2.12E+09 2.03E+09 particle Corrected* - 8.32E+09 8.84E+07 -7.57E+07 3.79E+07 -5.05E+07
8.3.2 Assessment of antimicrobial activity
The antimicrobial activity of modified particles was assessed against P. aeruginosa using a
viable cell count method to identify the number of actively dividing cells within the samples after
various incubations times using P1 and W8 AMPs. Data in Figure 60 show the cell count results
for P. aeruginosa incubated for two and four hours with Si colloidal particles modified using the
thiolene ‘photo-click’ chemistry. For MPTS- and PEG-modified microspheres there is no
significant difference in the number of viable cells compared to samples without microspheres
present at both time points. This result indicates that bare Si and PEG coatings do not adversely
238 affect cell viability and any decrease seen in cell viability for AMP-modified samples have been caused by the action of the AMPs themselves. After the two hour incubation period the number of actively dividing cells was observed to be 2.9x105 ± 0.1CFU/mL for sample without the addition of microspheres which drastically increased at four hours with viable cell count numbers observed to be 1.7x106 ± 0.2CFU/mL. This was likely caused by the induction of lag-phase growth where bacterial cells adapt themselves to growth conditions at the start of seeding. This same trend can be seen for bare Si and PEG-modified colloids where the number of viable cells is seen to drastically increase after two hours as bacterial cells adjust to their environment.24,25
For W8- and P1-modified samples a clear reduction in the number of viable cells is observed after the two hour period, where viable cell numbers were observed to be 4.4x104 ± 0.6CFU/mL and 4.5x104 ± 0.4CFU/mL, which is below the starting inoculum of 1.0x105 CFU/mL indicating a significant amount of bacterial cells are lysed by the action of AMPs. This activity is seen to be maintained through the four hour period, where viable cell numbers were observed to be 8.2x104
±0.1CFU/mL and 7.9x104 ± 0.1CFU/mL for W8- and P1-modified colloids, respectively. This result indicates that the antimicrobial coating is stable over extended periods without losing activity. It is also hypothesised that the action of AMPs may inhibit growth of planktonic cells; as the leakage of intracellular components may promote quorum sensing in planktonic cells resulting in the inhibition of growth due to the hostile environment created by the AMP-functionalised surfaces. Comparing results between AMPs to determine which may be more potent in a surface immobilised state suggests that both AMPs work well in significantly decreasing the viability of bacterial cells over extended periods as there is no substantial difference in viable cell count numbers between the two different AMPs.
239
Figure 60. Plate count assay indicating the number of actively growing P. aeruginosa cells incubated for 2 and 4hrs with modified and unmodified Si colloids prepared using thiolene photo-‘click’chemistry.
For thiol-maleimide functionalised particles an similar trend was observed for viable cell count numbers showing that after an initial lag-phase, bacterial growth increases significantly for cell only and unmodified particle samples (Figure 61). For AMP-functionalised colloids however, there were small differences seen using this immobilisation strategy which suggests that the bacterial cells were able to recover better from the initial lag-phase growth period when compares to the thiolene ‘photo-click’ reaction method with viable cell count numbers determined to be
3.9x105 ± 0.9CFU/mL and 3.3x105 ± 0.9CFU/mL for W8- and P1-modified colloids, respectively.
Cell count numbers after the two hour incubation period were also somewhat higher compared to the thiolene reaction method being 7.4x104 ± 0.9CFU/mL and 3.3x105 ± 0.9CFU/mL for W8- and
P1-functionalised colloids, respectively (See Figure 61). Considering zeta potential results in
Figure 56 indicating a more positive surface charge, and also XPS data showing slightly higher 240
%N for AMP-modified samples prepared using thiolene-mediated immobilisation strategies, it is declared that higher AMP loading was achieved through the two-step thiolene methods rather than the ‘one-pot’ method used in thiol-maleimide immobilisation. It is suggested that the competing interactions of reactants will be more pronounced in the ‘one-pot’ synthesis method leading to the relatively lower AMP loading and antimicrobial activity observed here.
Figure 61. Plate count assay indicating the number of actively growing P. aeruginosa cells incubated for 2 and 4hrs with modified and unmodified Si colloids prepared using thiol-maleimide ‘click’ chemistry.
The viability of P. aeruginosa cells incubated with the modified colloids for 4hr was assessed using propidium iodide (PI) uptake as an indicator of compromised bacterial membranes using the live/dead assay (Figure 62). The 4hr incubation used here provides a direct relationship to plate count results shown in Figures 60-61, and gives an indication as to the proportion of viable
241 cells in each sample. Fluorescent microscopy images provided in Figure 62 show a clear decrease in the number of total bacteria per field for AMP-modified samples (Fig. 62e-h) when compared to that of all other experimental conditions, suggesting that there is a reduction in the number of actively dividing cells within these samples. When making a comparison between AMP-modified samples, there were slightly higher cell numbers per field for thiol-maleimide immobilisation strategies (Fig. 62g-h) compared to thiol-ene photo-click chemistry (Fig. 62e-f), suggesting higher
AMP-loading due to higher amounts of grafted PEG. This is also supported by XPS data (Table
18) showing a higher %N for thio-ene based immobilisations with 3.9±0.7% and 3.4±0.8%, compared to thiol-maleimide strategies with 3.1±1.0% and 2.8±0.9% for PI and W8 AMPs, respectively. Additionally, apparent cell numbers for unmodified (Fig. 62b) and PEG-modified
(Fig. 62c-d) colloids were somewhat similar to cell only samples, indicating that PEGylated Si and
Si colloids do not adversely affect bacterial growth conditions, and any antibacterial activity is caused by immobilised AMPs, which is also supported by plate count results.
242
Figure 62. Representative fluorescent microscopy images of P. aeruginosa cells showing live (green) and membrane compromised (red) bacteria after being incubated for 4hrs with: a) nutrient broth, b) 2µm Si colloids, c) Si-PEGAc, d) Si-PEGMal, Si-PEGAc modified with e) P1and, f) W8 AMPs, and Si-PEGMal modified with g) P1 and h) W8 AMPs. Images were taken with a confocal microscope using a 40X objective and six images were taken per sample. Scale bar: 10µm
Fluorescent microscopy images were also supported through ImageJ analysis which was used to determine the proportion of live and dead cells using PI uptake to indicate compromised membranes (Figure 63). Compared to all other conditions a clear decrease in the viability of P. aeruginosa cells is seen for AMP-modified samples with approximately 78% and 72% PI uptake seen for thiol-ene based tethering of P1 and W8 AMPs, respectively. PI uptake for thiol-maleimide immobilisations was slightly lower, being approximately 60% and 55% for P1 and W8 AMPs, respectively, which was somewhat expected as previous results have suggested a slightly lower immobilisation efficiency using the ‘one-pot’ thiol-maleimide reaction. Regardless of the lower PI uptake, both immobilisation strategies show a significant decrease in the viability of P. aeruginosa cells when incubated for periods up to 4hrs. Unmodified and PEG-modified Si colloids all exhibited similar viability results compared to the cell only control with an approximate PI uptake of 25%. It is noted that total cell numbers for AMP-modified samples was relatively low resulting
243 in a smaller data size among these samples. However, considering the agreement with plate count data it is determined that viability of P. aeruginosa cells seen here provides an accurate representation of the antimicrobial activity of the AMP-modified colloids.
90 Live 18 Dead 80 16 CFU/mL
70 14 CFU/mL) 5 60 12 50 10 40 8 30 6 20 4 PercantageLive/Dead 10 2 0 0 Colony forming units(x10 Colonyforming
Figure 63. Viability of P. aeruginosa cells incubated for 4hrs with the modified Si colloids. Values presented here are percentage Live/Dead ± standard deviation (preliminary y-axis) and the number of colony forming units (CFU/mL) (secondary y-axis).
As stated in previous chapters, direct comparisons of viability results to other AMP-based immobilisation reports can be somewhat difficult due to differences in incubation times26,27 and starting inoculums.27 However, previous immobilisation strategies using similar puroindoline- based AMPs reports that PI uptake for E.coli cells can be up to 70% after 24hr incubation periods.16
Considering that many applications require an initial antimicrobial activity to prevent initial colonisation; for example during implantation of medical devices, the activity observed here is expected to be sufficient for such purposes.
244
8.4 Conclusions The experimental results presented in the Chapter explored two different grafting methods for the immobilisation of PEG-based polymers and AMPs which were: thiol-maleimide and thiolene-photo ‘click’ chemistry, which both offer a versatile approach for the immobilisation of various types of biomolecules. Results show that a clear reversal in surface charge can be seen for both W8- and P1-modified particles due to the highly cationic properties of selected AMPs. XPS data also confirmed the immobilisation of AMPs as survey spectra for elemental composition shows the presence of nitrogen for all AMP-modified samples at % compositions ranging between
2.8-3.9%.
The antimicrobial activity of PEG-AMP-modified particles were then assessed using a viable cell count method after two and four hour incubation periods. For unmodified and PEG- modified samples no significant difference in the number of viable P. aeruginsa cells were observed when compared to cell only samples which shows that MPTS- and PEG-modified colloids do not adversely affect bacterial growth. With regards to AMP-modified particles there was a significant decrease in the number of viable cells after both the two and four hour incubation periods showing that AMPs are still active in a surface-bound state. Results also show that after four hour incubation an increased antimicrobial activity was observed for thiolene-based modifications with a 1.6- and 1.7-log reduction in viable organisms for W8 and P1 AMPs, respectively. These values were slightly higher than thiol-maleimide samples having a 1.3- and
1.6-log reduction in viable bacteria for W8 and P1 AMPs, respectively. This indicates that a two- step immobilisation process may be advantageous for higher AMP loading to minimise competitive interactions that hinder the immobilisation process. However, based on the clear antimicrobial activity seen for both immobilisation methods it can be determined that both
245 thiolene- and thiol-maleimide-based coupling strategies are effective methods for the immobilisation of PEG and AMPs whilst maintaining antimicrobial activity.
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19 Li, Y., Wei, S., Wu, J., Jasensky, J., Xi, C., Li, H., Xu, Y., Wang, Q., Marsh, E. N. G., Brooks, C. L. & Chen, Z. Effects of peptide immobilization sites on the structure and activity of surface tethered antimicrobial peptides. Journal of Physical Chemistry C 119, 7146-7155, doi:10.1021/jp5125487 (2015).
20 Pingle, H., Wang, P. Y., Thissen, H., McArthur, S. & Kingshott, P. Colloidal crystal based plasma polymer patterning to control pseudomonas aeruginosa attachment to surfaces. Biointerphases 10, 1-11, doi:10.1116/1.4936071 (2015).
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23 Otto, S. & Engberts, J. B. F. N. Hydrophobic interactions and chemical reactivity. Org. Biomol. Chem. 1, 2809-2820, doi:10.1039/B305672D (2003).
24 Lu, Y., Yue, Z., Wang, W. & Cao, Z. Strategies on designing multifunctional surfaces to prevent biofilm formation. Frontiers of Chemical Science and Engineering 9, 324-335, doi:10.1007/s11705-015-1529-z (2015).
25 Subbiahdoss, G., Kuijer, R., Grijpma, D. W., van der Mei, H. C. & Busscher, H. J. Microbial biofilm growth vs. tissue integration: "The race for the surface" experimentally studied. Acta Biomater. 5, 1399-1404, doi:10.1016/j.actbio.2008.12.011 (2009).
26 Xiao, M., Jasensky, J., Gerszberg, J., Chen, J., Tian, J., Lin, T., Lu, T., Lahann, J. & Chen, Z. Chemically Immobilized Antimicrobial Peptide on Polymer and Self- Assembled Monolayer Substrates. Langmuir 34, 12889-12896, doi:10.1021/acs.langmuir.8b02377 (2018).
27 Li, Y., Santos, C. M., Kumar, A., Zhao, M., Lopez, A. I., Qin, G., McDermott, A. M. & Cai, C. "Click" Immobilization on alkylated silicon substrates: Model for the study of surface bound antimicrobial peptides. Chem. Eur. J. 17, 2656-2665, doi:10.1002/chem.201001533 (2011).
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9 Conclusions and Future Perspectives
9.1 Main findings of the research In conclusion, the research presented within this thesis provides an in-depth investigation of the various immobilisation strategies used for the tethering of polymers and AMPs to develop multifunctional surface coatings that can resist and control the adhesion of proteins and bacterial cells. The fabrication of such coatings and functionalised surfaces provides great promise in many biomaterial applications, with the results provided here adding further insight to our already existing knowledge within the area. Several key findings have been made throughout the research that demonstrates the viability of immobilised AMPs as an active antimicrobial coating, but also analysis of the efficiency of several grafting methods for polymer and AMP tethering. It is hypothesised that this research can be used and developed further for in vivo applications such as coatings on medical implants or drug delivery vehicles.
Preliminary work identified several puroindoline-based AMPs that were shown to be active against both E. coli and P. aeruginosa. The three AMPs; PuroA (FPVTWRWWKWWKG-NH2),
P1 (RKRWWRWWKWW-NH2), and W8 (WRWWKWWK-NH2) were observed to have MIC values that are up to 250X lower than commonly used AMPs such as Magainin-II. The parent
AMP, PuroA was then able to be immobilised to the surface of carboxylated PS colloids using
EDC/NHS coupling chemistry at sufficient densities to exhibit an antibacterial effect when presented through surface-stabilised BCC layers. Here it was shown that although PuroA densities were relatively low (1.93x1013 molecules/cm2), a significant decrease in the viability of adherent
E. coli cells (>70%) was observed when compared to that of unmodified BCC layers. This proof of concept that BCC surface patterns together with PuroA immobilisation can be used for
250 development of antimicrobial surface coatings provided the basis for future investigations within this project, as it was hypothesised that the incorporation of a flexible PEG linker and the use of slightly more potent AMPs (P1 and W8) would result in enhanced non-fouling properties and increased antimicrobial activity.
PEG grafting investigations were performed to both flat and spherical surfaces using a variety of immobilisation and surface activation methods to determine which may be most efficient. Initial experiments utilising SPR provided a novel way to investigate several polymer grafting methods in real-time showing how grafting kinetics and protein adsorption to PEG layers can be manipulated depending on the type of reaction method and the conditions applied during immobilisation. Within these initial investigations reductive amination was shown to be far superior when compared to carbodiimide coupling chemistry for the grafting of PEG to physically adsorbed PEI layers, with optimised conditions (40°C, 0.6M K2SO4) showing that PEG thicknesses of approximately 4 nm are able to significantly reduce BSA adsorption down to
2±2ng/cm2 when compared to 116±32ng/cm2 for EDC/NHS reactions.
The addition of K2SO4 and elevated temperatures to promote ‘cloud-point’ conditions was shown to be a critical factor to achieve the non-fouling ability observed for reductive amination reactions – being rationalised down to the ‘poor solvation’ of PEG chains to reduce their hydrodynamic radius and facilitate high-density grafting. The addition of K2SO4 was however, found to greatly hinder grafting using carbodiimide chemistry, where protein adsorption to the
2 prepared surfaces reached densities of 142±16 ng/cm . These vast differences seen between the two grafting methods for in situ surface modifications highlights the necessity to employ immobilisation methods that are compatible with surface-bound functional groups, but also has
251 minimal interferences from functional groups present on PEG chains that are not meant to participate in the reaction.
These investigations also provided valuable information regarding what type of surface activation method results in more efficient PEG grafting and therefore increased non-fouling properties. Here it was found that the grafting to covalently-bound APTES layers resulted in PEG thickness values that were considerably higher than grafting to physically adsorbed PEI layers regardless of the experimental conditions applied. Moreover, this was also accompanied by a decrease in protein adsorption to these surfaces, as the branched polymeric structure of PEI will provide more sites for adsorption when compared to the covalently-bound APTES films. These results highlight that the use of grafted layers comprised of relatively small covalently-bound molecules such as silanes is more advantageous than the use of polyelectrolyte multilayers, providing that silane deposition is first optimised.
Considering the previous success of EDC/NHS coupling chemistry for the zero-length immobilisation of PuroA to carboxylated PS microspheres, additional PEG immobilisation investigations used a similar method to immobilise HOOC-PEG-NH2 through activation of carboxylated silica microspheres using EDC/NHS. Similar to in situ experiments, it was found that this particular approach to PEG tethering was somewhat inefficient, with little or no evidence to support the presence of bound PEG through zeta potential, XPS, and FTIR analysis. Once again, this emphasises the importance in the choice of grafting method and also terminal functional groups to minimise interference from competitive interactions.
An optimised silane coating was shown to be more beneficial for the tethering of PEG compared to that of branched polymeric structures that may trap biomolecules leading to a decreased non-fouling ability. This knowledge, and the inherent complications with previous 252
EDC/NHS grafting experiments prompted investigations to use a thiol-terminated silane (MPTS) as a graft layer for the subsequent immobilisation of PEG and AMPs via thiol-maleimide or thiol- ene ‘photo-click’ reactions. Several key findings were made in the optimisation of MPTS layers, which has provided great insights into the complicated process of silane deposition. Major results show that a 2hr pre-hydrolysis period at low pH (pH 4) provided a balance between hydrolysis of methoxy groups and condensation with surface silanols leading to increased film thicknesses with a high retention of thiol functional groups as seen in high-resolution S 2p XPS spectra.
Additionally, it was seen that rinsing and drying procedures (which have somewhat inconsistent reports within current literature) were observed to greatly affect the quality and thickness of MPTS films, with an increased presence of defects and pinholes as more post-deposition treatments were applied to the MPTS films.
Coupling of PEG and AMPs to thiolated silica microspheres using thiol-ene ‘photo-click’ and thiol-maleimide was found to be a very effective method for the production of antibacterial surface coatings. Both methods were observed to result in a significant decrease in the number of viable P. aeruginosa cells after incubation with the PEG-AMP modified colloids. A 1.3-1.7-log reduction in viable organisms after 4hr was seen showing that the AMPs are still active in a surface-bound state. With no precedent in current literature, such modification of colloidal particles by PEG and AMPs offers a novel route to develop surface coatings that can present bioactive patterns and topographical cues through the selective immobilisation to colloidal crystal layers.
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9.2 Limitations and future perspectives
While the experimental design and methods used within this research were carefully considered prior to implementation, it is also important to be aware of the limitations of the research, and speculate on future research that may be warranted. One particular limitation that was encountered was the availability of resources needed for conducting experiments or characterisation of samples. Significant delays in obtaining data was experienced in regards to
SEM and XPS characterisation due to instrument breakdown. Furthermore, the continuation of
PEG and AMP immobilisation through reductive amination was not explored due to difficulties at the time in obtaining PEG with compatible functional groups for AMP immobilisation. This is worth investigating in future research as it was shown that PEG grafting by reductive amination was extremely effective at creating non-fouling layers.
Grafting via EDC/NHS chemistry was shown to be effective for the zero-length immobilisation of PuroA to PSC colloids, however, while all PEG grafting investigations using
EDC/NHS chemistry were observed to be informative, they also exhibited undesirable outcomes.
This limited the future work within this area due to the need to progress with other experimental designs that were subsequently proven to be more effective for the tethering of PEG and AMPs; i.e. thiol-based immobilisation strategies.
Future research will need to address several aspects that were not able to be investigated through the course of this project, which will help to provide a more encompassing picture of the use of immobilised PEG and AMPs to develop bioactive surface coatings. One aspect that this research does not address is the cytocompatibility toward AMPs and AMP-functionalised surfaces.
This is of critical importance if this research is to be translated for in vivo applications as it is imperative that the AMPs do not adversely affect the growth of mammalian cells. As such, future 254 studies should investigate the attachment and growth of fibroblast and fibroblast-like cells to AMP- modified surfaces. Additionally, the use of BCC layers was shown to be a novel way to pattern
AMPs and present topographical cues which is advantageous for manipulating and controlling biointerfacial interactions. It is hypothesised that the use of AMP-PEG-modified BCCs of varying size ratios will further elucidate how topography can affect bacterial attachment, and this will also enable the fine-tuning of AMP densities by altering distances between AMP-functionalised colloids.
9.3 Concluding remarks
To conclude, the research presented here provides an in-depth analysis of the various polymer and AMP immobilisation strategies that can be applied to develop novel surface coatings, with several key findings being made that has contributed to the fields of surface fabrication and biomaterial science. With no precedence within current literature, it was shown that BCC layers were a suitable platform for AMP immobilisation, while still exhibiting antibacterial activity against E. coli. Investigating various surface activation methods provided valuable results and information regarding MPTS deposition which has expanded our knowledge for the generation of high quality silane layers. Finally, while various successes and failures with regards to PEG immobilisation were observed throughout his research, the effective grafting of PEG and AMPs to colloidal particles through thiol-based immobilisation strategies is reported here for the first time. These immobilisation strategies demonstrated antibacterial activity against P. aeruginosa at each time interval investigated, and hold great potential for the future development of antibacterial and non-fouling coatings on implants or drug delivery devices based on AMP-PEG-functionalised colloidal crystal layers.
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10 Conferences and Publications
10.1 List of Conferences
1. Poster presentation BioFest 2016, Melbourne – Completed
Boden, A., Bhave, M., Wang, P.-Y. & Kingshott, P. New antibacterial coatings based on the immobilisation of PuroA antimicrobial peptides to colloidal crystal layers.
2. Poster presentation ASBTE 2017, Canberra - Completed
Boden, A., Bhave, M., Wang, P.-Y. & Kingshott, P. In-situ investigation of PEG grafting using surface plasmon resonance (SPR) to optimise coatings to prevent non-specific protein adsorption
3. Poster presentation MISE 2020, Melbourne – Feb 2020
Boden, A., Bhave, M. & Kingshott, P. Thiol-mediated tethering of antimicrobial peptides
as potential antibacterial surface coatings
10.2 List of Publications 1. Boden, A., Bhave, M., Wang, P.-Y., Jadhav, S. & Kingshott, P. Binary Colloidal Crystal Layers as Platforms for Surface Patterning of Puroindoline-Based Antimicrobial Peptides. ACS Appl. Mater. Interfaces 10, 2264-2274, doi:10.1021/acsami.7b10392 (2018) (See Appendix I).
2. Diba, F. S., Boden, A., Thissen, H., Bhave, M., Kingshott, P. & Wang, P.-Y. Binary colloidal crystals (BCCs): Interactions, fabrication, and applications. Adv. Colloid Interface Sci., doi:https://doi.org/10.1016/j.cis.2018.08.005 (2018) (See Appendix II).
3. Al-Ani, A., Boden, A., Al Kobaisi, M., Pingle, H., Wang, P.-Y. & Kingshott, P. The influence of PEG-thiol derivatives on controlling cellular and bacterial interactions with
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gold surfaces. Appl. Surf. Sci. 462, 980-990, doi:https://doi.org/10.1016/j.apsusc.2018.08.136 (2018).
4. Boden, A., Bhave, M. & Kingshott, P. Investigation of organosilane deposition methods for the production of thiol-containing films. Under review.
5. Boden, A., Bhave, M. & Kingshott, P In-situ investigation of grafting conditions and non- specific protein adsorption to PEG layers with surface plasmon resonance. Under review.
6. Boden, A., Dart, A., Russo, L., Bhave, M. & Kingshott, P. antimicrobial activity of PEG
and AMP-functionalised colloids prepared using thiol-ene ‘photo-click’ and thiol-
maleimide. Under review.
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Appendix I
Copyright Statements
The following pages contain Copyright Statements for published content that has been reproduced within this Thesis and presented in a sequential order.
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Appendix II
Authorship indication and citation for original publication
This section contains Authorship Indication and citation for the journal article entitled “Binary
Colloidal Crystal Layers as Platforms for the Surface Patterning of Puroindoline-Based
Antimicrobial Peptides” by Andrew Boden, Mrinal Bhave, Peng-Yuan Wang, Snehal Jadhav and
Peter Kingshott, and published in Applied Material and Interfaces 2018, 10(3), 2264-2274.
Citation: Boden, A.; Bhave, M.; Wang, P.-Y.; Jadhav, S.; Kingshott, P., Binary Colloidal
Crystal Layers as Platforms for Surface Patterning of Puroindoline-Based Antimicrobial
Peptides. ACS Appl. Mater. Interfaces 2018, 10 (3), 2264-2274.
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Appendix III
Authorship indication and citation for original publication
This section contains Authorship Indication and citation for the review article entitled “Binary
Colloidal Crystals (BCCs): Interactions, Fabrication, and Applications” by Andrew Boden,
Farhana Diba, Helmut Thissen, Mrinal Bhave, Peter Kingshott, and Peng-Yuan Wang, and published in Advances in Colloid and Interface Science 2018, 261, 102-127.
Citation: Diba, F. S.; Boden, A.; Thissen, H.; Bhave, M.; Kingshott, P.; Wang, P.-Y., Binary colloidal crystals (BCCs): Interactions, fabrication, and applications. Adv. Colloid Interface Sci.
2018.
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