Article Cite This: Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
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Real-Time Synchrotron Small-Angle X‑ray Scattering Studies of Collagen Structure during Leather Processing Yi Zhang,† Bridget Ingham,‡ Soshan Cheong,§ Nicholas Ariotti,§ Richard D. Tilley,§ Rafea Naffa,† Geoff Holmes,† David J. Clarke,‡ and Sujay Prabakar*,† †
Leather and Shoe Research Association of New Zealand, P.O. Box 8094, Palmerston North 4472, New Zealand Callaghan Innovation, P.O. Box 31310, Lower Hutt 5040, New Zealand § Electron Microscope Unit, Mark Wainwright Analytical Centre, University of New South Wales, Sydney, Australia ‡
S Supporting Information *
ABSTRACT: The collagen structure in skins is significantly influenced by the cross-linking chemistry adopted during leather processing. We have developed an in situ technique to measure real-time collagen structure changes using synchrotron-based small-angle X-ray scattering (SAXS). Three common mineral tanning systems, basic chromium sulfate (BCS), zirconium sulfate (ZIR) and an aluminosilicate-based reagent (ALS) were used to stabilize collagen in ovine skin. Studying the molecular changes by in situ SAXS revealed a range of tanning mechanisms: a complex combination of covalent cross-linking, electrostatic interactions and hydrogen bonding by BCS, hydrogen bonding interactions by ZIR, and the formation of colloidal aggregates by ALS. These results unravel the mechanisms of producing leathers with different properties, explaining why ZIR produces denser leathers while ALS produces softer leathers compared to conventional BCS leathers. ZIR and ALS are environment-friendly alternatives to BCS, and understanding their mechanisms is important for a more sustainable future for the leather industry.
1. INTRODUCTION
effective collagen stabilization systems are of increasing industrial interest.10−12 Studying the molecular level interactions of cross-linking agents with collagen can provide valuable information on the mechanisms of tanning and protein stabilization.8,13,14 Synchrotron X-ray diffraction studies by Maxwell et al. showed the effect of liming on collagen structure and the interaction of chromium salts with collagen.15,16 Using wide-angle X-ray diffraction Lin et al. observed changes in collagen structure at different concentrations of Cr3+ during tanning.17 Although Xray scattering techniques have previously been used to observe molecular level changes in collagen structure following chrome tanning, no studies looking at structural changes at the molecular level in situ during leather processing have been reported. Real-time observations of the changes in collagen structure during mineral tanning treatments could provide direct evidence of the mechanism of structure modification and stabilization by tanning processes, and understanding the mechanism could then guide the design and optimization of
Type I collagen in animal hides and skins is the primary structural element in the extracellular matrix, forming fibers with a hierarchical structure consisting of fibrils, microfibrils, and triple helical collagen molecules at the lowest level.1−3 To understand how these structures confer physical and mechanical properties to tissues such as skin, bone, and dentin, studies at the molecular level are essential.2,4−6 It is the precise polymerizing of the triple helical collagen molecules into stable, mechanically strong fibrils that provides the leather industry with its fundamental material.7 Leather tanning processes impart hydrothermal stability and enhance the organoleptic properties of the final leather product due to the structural changes of collagen in skin.7 Mineral tanning agents such as basic chromium sulfate (BCS), zirconium sulfate (ZIR), and aluminum sulfate are commonly used to cross-link with collagen molecules and stabilize the structure.8 Basic chromium sulfate is globally used and recognized as the most effective tanning agent because it imparts high hydrothermal stability and takes relatively short times to produce finished leathers.7 However, poor uptake of chromium salts by the collagen molecules in hides and skins leads to chemical waste and environmental stress.9 To alleviate the adverse effects of chromium tanning processes, greener and more © XXXX American Chemical Society
Received: Revised: Accepted: Published: A
September 17, 2017 November 13, 2017 December 15, 2017 December 15, 2017 DOI: 10.1021/acs.iecr.7b03860 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Article
Industrial & Engineering Chemistry Research environment-friendly alternatives that are equal to or better than chrome tanning or at least enhance its uptake. In our study, we first used ex situ synchrotron small-angle Xray scattering (SAXS) to study changes in the collagen structure of ovine skin after processing with one of three important mineral-based tanning methods: basic chromium sulfate (BCS), zirconium sulfate (ZIR) and an aluminosilicate/ poly(carboxylic acid) based tanning agent (ALS). We then developed a real-time synchrotron SAXS experiment to study the effect of the mineral tanning agents on collagen structure during leather processing.
Scheme 1. Setup for the in Situ Tanning Experiments
2. EXPERIMENTAL METHODS 2.1. Ex Situ Sample Preparation. Ovine skins were fleshed and acidified during the pickling process (stage 1, pH = 2.5−3.0) to protonate the amino acid side chain groups of collagen molecules in preparation for tanning.7 The pickled samples were then treated with Zoldine ZE (Oxazolidine E, stage 2, pH = 7.5−8.0), a five-membered heterocyclic compound derived from the reaction between an aminohydroxy compound and aldehyde to pretan the skins.18,19 Such pretanning agents are commonly used to improve the penetration of main tanning agents through the collagen matrix by decreasing the availability of amino acid side chain groups on the collagen molecule. The main tanning (stage 3) was carried out with either basic chromium sulfate (BCS, pH = 3.8−4.2), zirconium sulfate (ZIR, pH = 3.8−4.2), or aluminosilicate/poly(carboxylic acid) based tanning system (ALS, pH = 4.2−4.5) followed by retanning (stage 4, pH = 4.0−4.5) and fatliquoring (stage 5, pH = 4.0−4.5) to improve the organoleptic properties of the final leather. Fixation of the latter steps were carried out by pH adjustments, followed by washing, to produce the wet leather (stage 6, pH = 3.5−4.0). Finally, the leather was dried (stage 7) in air or vacuum. For the ex situ analysis on the SAXS, samples of size 2 cm × 2 cm × 3 mm (L × W × H) for the isotropic measurements and 2 cm × 2 mm × 3 mm strips for the cross-section were collected at each processing stage. Detailed processing steps can be found in the Supporting Information. 2.2. In Situ Sample Preparation. Square shaped sections (2 cm × 2 cm × 3 mm) of pretanned ovine skins (stage 2) were used as the starting materials. These were attached to a purpose-designed plastic sample holder, suspended in a tanning-bath solution in a temperature-controlled beaker with continuous and vigorous stirring at 80 rpm (Scheme 1 and SI video). The beaker was located directly underneath the X-ray beam measurement position and the sample holder was attached to a linear stage to allow periodic raising of the sample out of the solution to record SAXS measurements before returning it to the solution. Each measurement took 10 s to complete. The main tanning step of either BCS, ZIR, or ALS was carried out and after the experiment, samples were cut into 2 cm × 2 mm × 3 mm strips for cross-section measurements. 2.3. Small-Angle X-ray Scattering (SAXS). SAXS experiments were performed at the SAXS/WAXS beamline at the Australian Synchrotron. An X-ray energy of 12 keV and camera length of 3345.83 cm was used. Data were collected using a Pilatus 1 M detector with 1 s exposures. Each sample was sandwiched between two pieces of Kapton tape to prevent skin dehydration, and mounted on an aluminum plate in a transmission geometry. For isotropic and cross-section measurements, the incident X-ray beam was normal to the grain surfaces and the plane of section, respectively. Scans were
recorded in “gapless” mode by translating the detector. Ten such scans were performed for each sample, averaged, and radially integrated using the software package Scatterbrain v.2.82. Fitting of the 1D integrated data was performed using in-house software to obtain the fibril diameter, D-period spacing, and relative intensities of the collagen diffraction peaks. Additional details of the sample preparation and data analysis/fitting can be found in the Supporting Information. 2.4. Electron Microscopy and Elemental Analysis. Scanning transmission electron microscopy coupled with energy dispersive X-ray spectroscopy (STEM-EDS) analysis was carried out at UNSW Australia on a JEOL JEM-F200 fieldemission gun transmission electron microscope operated at 200 kV. The microscope is equipped with a high-angle annular dark field (HAADF) detector for STEM-HAADF imaging and a windowless JEOL silicon drift detector (SDD) for STEMEDS elemental mapping analysis. EDS data was analyzed using the NSS Noran System 7 Software (Thermo Scientific). TEM specimens were prepared from a tanned ALS sample (stage 3). Strips of the sample were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at room temperature for 1 h, and then washed in 0.1 M sodium cacodylate in 1% osmium tetroxide and dehydrated in increasing percentages of ethanol. The fixed and stained samples were serially infiltrated with Procure 812 resin (ProSciTech, Kirwan, Queensland, Australia) and polymerized to hardness at 60 °C for 48 h; 100 nm thick sections were cut on a Leica EMUC6 ultramicrotome and placed onto carbon-coated copper grids for imaging and analysis.
3. RESULTS AND DISCUSSION 3.1. Isotropic Measurements on ex Situ Samples. The ex Situ SAXS results presented in Figure 1A show the changes in scattering intensity after the different processing steps. No changes were observed between the pickled and pretanned skins, but after tanning with BCS, ZIR, or ALS there was a significant increase in the intensity of the diffraction peaks. This can be attributed to the enhanced electron density contrast caused by the metal ions from the respective tanning agents; a similar observation was previously reported for BCStanned bovine hide.15 The most significant changes in intensities were observed for the 5th−11th order peaks, along with a decrease in the intensity of the third order peak. B
DOI: 10.1021/acs.iecr.7b03860 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
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following a long-range ordered packing model.23 On the basis of the model, the axial staggering periodicity of the gap/overlap region of collagen molecules (D-period) is directly related to the positions of the diffraction peaks. Changes in D-period associated with the different processing stages are shown in Figure 1B. Three features were observed: (1) a decrease in the D-period between the pickling and pretanning stages (from 65.5 to 64.5 nm), (2) a steady D-period from the main tanning through to the fatliquoring stages, and (3) a substantial decrease in D-period between wet and dry leather. The decrease in D-period observed following pretanning can be attributed to the reaction between the aldehyde groups of Zoldine ZE with the amino groups of the lysine side chains on collagen, forming a Schiff’s base adduct which are then covalently cross-linked with other amino acid residues.19 This could disrupt the electrostatic environment of the hydrogen bonds between amino acid side chains and water molecules, thus affecting the gap regions of the axial staggering of the collagen molecules, resulting in a decrease in the D-period.10 During the main tanning step (stage 3) only slight changes in D-period were observed for all three tanning agents. However, previous studies on BCS treated bovine samples showed an apparent increase in D-period.15 This discrepancy can be explained by the pretanning using Zoldine ZE in our study, which was not applied in the studies mentioned above. This resulted in an exhaustion of the terminal amino acid side chain groups and therefore only limited active sites remained for BCS to affect the axial staggering periodicity. The ZIR samples had slightly lower D-periods compared to the BCS and ALS samples. While BCS forms cross-links with the collagen molecules on the carboxylic acid side chain groups, ZIR is characterized by a combination of cationic, neutral, and anionic species (Scheme 2) that interact with amino side chain groups by electrostatic interactions and hydrogen bonding.7 Although the mechanism of ZIR is unclear, we speculate that the binding of Zr4+ species with residual terminal amino side chains leads to small changes in the gap/overlap regions of the axially staggered collagen molecules, resulting in the D-period being slightly smaller. While no significant changes were observed for the retanning and fatliquoring steps (i.e., between stages 3−6), a large decrease in the axial periodicities were observed in each case when the leathers were dried (stage 6 → 7). Leather is normally dried at ∼45 °C to remove unbound water in preparation for the final product. In the absence of glycosaminoglycans and natural cross-links, the removal of unbound water from the collagen fibrils in tanned hides and skin is known to cause a structure collapse of the collagen matrix, resulting in a concomitant decrease in D-period.24−28 However, the decrease was much larger for the ZIR tanned leather (64.5 to 61.6 nm) than the BCS (64.7 to 63.3 nm) and
Figure 1. (A) Ex situ SAXS data showing differences in scattering intensity between pickled (stage 1), pretanned (stage 2), and main tanned (stage 3) samples. Selected peaks corresponding to q = 2πn/D where n is the peak order and D is the D-period are labeled. These data were analyzed to obtain (B) changes in D-period and (C) changes in fibril diameter after each processing stage (1, pickled; 2, pretanned; 3, after main tanning; 4, retanned; 5, fat-liquored; 6, wet leather; 7, dry leather).
The ALS samples were observed to have lower peak intensities overall compared to the BCS and ZIR samples. This can be explained on the basis that while the chromium and zirconium ions in BCS and ZIR tanning have higher electron density, the Al/Si from the aluminosilicates in ALS have comparatively lower electron density.7,10,20−22 The overall diffraction peak intensity is also related to the extent of covalent cross-linking and collagen long-range order; the aggregates formed in the ALS tanning process do not enhance the diffraction, but contribute to background scatter. Collagen molecules within the fibrils are staggered relative to each other
Scheme 2. Tetrameric Zirconium Sulfate Complex with Water Exhibiting Anionic, Neutral, and Cationic Character
C
DOI: 10.1021/acs.iecr.7b03860 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
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3.2. Isotropic Measurements during in Situ Samples. To understand the characteristic reaction mechanism of each tanning agent with collagen, changes that occur to the collagen structure during leather tanning processes were measured in a series of in situ experiments on the beamline. Figure 3 shows the change in D-period with time during the main tanning process along with the changes in relative
ALS (64.7 to 63.7 nm) tanned leathers. Chromium sulfate in BCS plays multiple roles during tanning, forming both covalent cross-links with collagen molecules in addition to electrostatic interactions in the collagen matrix.7 In ALS tanning, the poly(carboxylic acid) component interacts covalently with collagen similar to BCS, leading to a similar decrease in Dperiod by drying. On the other hand, in ZIR tanning, hydrogen bonds are predominantly formed7 within the collagen structure, therefore removing unbound water during drying will result in a greater collapse of the collagen structure that can be associated with a larger reduction in D-period. Figure 1C shows the change in collagen fibril diameters at each stage of processing. The BCS and ZIR samples showed similar fibril diameter changes; however, the ALS samples exhibited a higher percentage increase. Additionally, the ALS tanned leathers had a plump feel after drying, which can be explained by the larger fibril diameter. The larger fibril diameters of the ALS samples are believed to be due to the aluminosilicate/poly(carboxylic acid) tanning agent forming a coating on the outside of the fibrils. STEMEDS was performed to probe the presence of the tanning agent in the ALS samples. The STEM-HAADF image in Figure 2A
Figure 2. (A) STEM-HAADF image of the ALS sample after main tanning (stage 3). (B) Overlays of STEMEDS maps of silicon (Si), aluminum (Al), and oxygen (O) showing the uniform distribution of these elements across the collagen fibrils and the presence of an aluminosilicate particle. (C−E) Individual maps of Si, Al, and O, respectively.
Figure 3. In situ SAXS analysis showing relative intensity changes (open circles) and change in D-period (black lines) obtained from the collagen diffraction peaks during the main tanning step (stage 2−3) for the (A) BCS, (B) ZIR, and (C) ALS tanning methods.
shows the characteristic D-banding structure of the collagen molecules and their fibrillar arrangement. STEM-EDS maps (Figures 2B−E) show a uniform distribution of silicon, aluminum, and oxygen over the fibrils, as well as the presence of an aluminosilicate colloidal particle (∼100 nm) in the interfibrillar region. Observation from the overlays of the elemental maps onto the electron image indicates that these elements have likely formed a layer of coating on the fibrils (Figure S1 in the Supporting Information), which is consistent with the measured increase in fibril diameter in the dry leather samples. Colloidal particles were not indicated in the SAXS pattern because of the wide distribution of particle sizes, confirmed by STEM-EDS imaging and analyses (Figure S2 in the Supporting Information).
diffraction peak intensity. The relative intensity was calculated based on the intensities of the third order peak and the fifth order peak (see the Supporting Information), relative to the two endmembers of the ex situ series for each tanning agent: pretanning (stage 2, identified as 0) and main tanning (stage 3, identified as 1). Thus, a relative intensity of 0 corresponds to the pattern observed after the pretanning step, and a relative intensity of 1 corresponds to that observed after the main tanning step. Fractional values denote the extent of the change from one to the other. Similar trends to those displayed in Figure 3 were also observed by comparing the third order peak to other strong diffraction peaks (except the sixth order peak, which is highly sensitive to hydration levels).29 D
DOI: 10.1021/acs.iecr.7b03860 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
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Industrial & Engineering Chemistry Research For the BCS treated samples, gradual increases in relative intensity as well as D-period were observed following the addition of chromium sulfate. Unlike the relative intensity, which increased steadily throughout the experiment, the Dperiod increased during the first 20 min, then reached a plateau, and then increased again as the temperature was raised from 25 to 40 °C. (During conventional chrome tanning, the reaction initially proceeds at room temperature to facilitate uniform penetration of chromium sulfate into the collagen matrix. After 30 min, the temperature is gradually increased to 40 °C to promote the cross-linking mechanism.30) The Dperiod eventually stabilized, with an overall 0.4 nm increase from the value for the pretanned stage. The D-period in the in situ experiments is mainly affected by the molecular level crosslinking reactions between chromium and collagen, while the diffraction peak intensities indicate that both long-range order and electron density of the sample increases with the introduction of Cr3+ species into the collagen matrix. The fact that they behave differently is strong evidence that two separate processes are occurring. We hypothesize that during the initial stages of uptake, chromium reacts with the amino acid side chains to form covalent cross-links as indicated by changes in D-period and diffraction peak intensity. Once the amino acid side chains are saturated, any additional chromium binds to the collagen molecules by secondary interactions, including electrostatic interactions and hydrogen bonding indicated by the changes in the diffraction peak intensity only. These insights can only be obtained through in situ experiments. In the case of the ZIR-treated samples, no significant change in D-period was observed throughout the in situ experiment, confirming the ex situ observations and the predominant hydrogen-bond interactions with the collagen molecule on multiple active side chains. Interestingly, the scattering intensity of the ZIR samples increased sharply during the first 10 min indicating that the Zr4+ species are readily taken up by the collagen matrix. After 10 min the scattering intensity plateaued, which could be attributed to a dense structure being formed on the surface of the skin due to the inherent astringency of tetrameric zirconium complexes, which suppressed further uptake of zirconium into the collagen matrix.7 For the ALS treated samples a gradual increase in D-period was observed, indicating gradual binding to the collagen molecules by the aluminosilicate/poly(carboxylic acid) in the ALS tanning agent. However, only a slight increase in the relative intensity was observed, implying fewer cross-links being formed, highlighting the low uptake, and suggesting the importance of mechanical action in the uptake of the ALS tanning agent. 3.3. Cross-Section Scans on ex Situ and in Situ Samples. To further investigate the characteristic function of penetration of BCS, ZIR, and ALS tanning agents, measurements were performed on cross sections of the samples after the in situ experiments, together with ex situ samples after the main tanning step (stage 3). The relative diffraction peak intensities through the cross sections are shown in Figure 4, normalized from 0 to 1 corresponding to the isotropic pretanning (stage 2) and main tanning (stage 3) samples for each tanning agent, as for the analysis of the in situ experiments. While the ex situ samples showed high relative intensities throughout the cross-section, the in situ treated samples had lower relative intensities, indicating that the penetration of Cr/
Figure 4. Normalized relative intensities through the cross sections of ex situ (closed triangles) and in situ (open circles) samples after the main tanning step (stage 3) treated with (A) BCS, (B) ZIR, and (C) ALS. 0% depth corresponds to the grain surface, while 100% corresponds to the flesh.
Zr/Al−Si species in the in situ samples was poorer. This again highlights the requirement for mechanical action during leather processing as a significant driver of uptake and penetration of the tanning agents in skin. Relative intensities for the BCS treated samples (Figure 4A) demonstrate that the ex situ samples were completely converted through the entire depth of the sample from the grain surface (0−25%) through to the center (25−75%) and into the flesh region (75−100%). In the in situ samples, while the flesh region had comparatively higher relative intensities than the grain surface, the gradual decrease in relative intensity from both regions through to the center region suggests that penetration was driven by the concentration gradient of the BCS tanning agent. Note that the larger uncertainties in the grain-center region (0−50%) are due to the diffraction peaks from that part of the samples having much lower overall absolute intensity; the in situ experiment results give an average through the entire sample thickness and so will be weighted toward values of the regions with higher intensity. For the ZIR treated samples (Figure 4B), the ex situ samples also showed an even distribution to the ex situ BCS samples. The in situ ZIR, however, shows a steep intensity change from the grain and flesh regions of the skin samples to near-zero E
DOI: 10.1021/acs.iecr.7b03860 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
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from a structural perspective into the tanning mechanisms associated with each of the three tanning agents that produce leather with different properties. An effective and environmentfriendly pathway can be optimized for collagen stabilization to achieve a more sustainable future for the leather industry. Our in situ SAXS method enables the structural analysis of similar collagen-based systems.
values at the center, that is, that only the outer surfaces had been modified by ZIR. This observation illustrates the rapid reaction of the ZIR tanning agent with collagen and the astringency of the tetrameric Zr4+ complex that discourages uniform penetration across the full cross-section of the skin sample. Our finding supports the conventional zirconiumbased tanning methods, in which the amount of water applied at first is minimal to help the zirconium species penetrate through the substrate, followed by additional water to initiate the polymerization process through hydrolysis, thereby increasing the tanning agents’ molecular weight and astringency.7 The ex situ ALS treated samples (Figure 4C) exhibited a rather different relative intensity distribution through the skin cross-section: from the grain surface to the center, the relative intensity increased above the isotropic value and then decreased from the center to the flesh region. This indicates that the effect of ALS is inhomogeneous throughout different regions of the skin. During the reaction of the ALS tanning agent with collagen, the aluminosilicate components penetrate through the skin, binding to the collagen molecules as well as forming colloidal aggregates within the collagen matrix.20 In regions of high density of collagen molecules, such as the graincenter region (0−50%), we observed high relative intensities. This indicates that in this region the aluminosilicates are more likely to bind with the long-range ordered collagen molecules, rather than form colloidal aggregates. Therefore, the uneven distribution of diffraction peak relative intensities can be explained by the colloidal aggregation mechanism being facilitated in the more open fiber structure of the skin in the flesh region (75−100%). In the in situ ALS sample, although we observed partially converted collagen on the surfaces, the tanning agent did not penetrate through to the center of the skin. This highlights the importance of mechanical action for the even distribution and reaction of tanning agents with the collagen molecules in skin. To summarize, we have studied the characteristic molecular level changes in the collagen structure of skins, induced by common industrial mineral tanning agents. Isotropic ex situ SAXS analysis demonstrated the significant effect of cross-links and the presence of water on the D-period and the diffraction peak intensity changes due to the changing electron density contrasts caused by mineral tanning. The observations from in situ SAXS revealed a range of different tanning mechanisms. BCS exhibited a complex combination of covalent crosslinking, electrostatic interactions, and hydrogen bonding. During in situ SAXS experiments, BCS showed a gradual uptake and relatively uniform penetration, with possible noncovalent interactions between the excess chromium species and collagen molecules. ZIR however, fixed rapidly to the surfaces discouraging further penetration in the in situ experiments and saw a dramatic decrease in the D-period on drying in the ex situ measurements, both of which highlighted the astringency of zirconium species as tanning agents. These results suggest that ZIR forms mainly hydrogen bonds by its tetrameric complexes within the collagen matrix. ALS, in addition to inducing covalent cross-linking by its poly(carboxylic acid) component, caused an increase in the collagen fibril diameters by the formation of a coating. The ALS-treated sample also displayed a nonuniform relative intensity distribution through the cross-section because of the propensity of ALS to form colloidal aggregates in the open structured regions of the skins. This method provides insights
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.7b03860. Detailed experimental procedures, materials, SAXS data analysis, additional STEM images PDF) Movie of SAXS measurements (MP4)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Yi Zhang: 0000-0002-4196-3036 Richard D. Tilley: 0000-0003-2097-063X Geoff Holmes: 0000-0002-9864-1150 Sujay Prabakar: 0000-0003-4371-9085 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS S.P, Y.Z, R.N, and G.H would like to thank the Ministry of Business, Innovation and Employment (MBIE) for providing funding through Grant LSRX-1301. Portions of this work were conducted on the SAXS beamline at the Australian Synchrotron, Victoria, Australia. Part of this research used the facilities at the Electron Microscope Unit at UNSW.
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REFERENCES
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