3.5 C ONCLUSION
4.3.2 Scientific validation of the MPI process
a b
c d
Figure 4.12 Wide Angle X-Ray Scattering (WAXS) profile. a and b show the crystal structure evolution of silk after annealing (SS/H), MPI (SS/Al2O3/300) and additional post annealing of MPI samples (SS/Al2O3/300/H). The FWHM (wn, Full With at Half Maximum) of the native sample (SS/N) slightly decreased after annealing (SS/H) (See Figure 4.13 and Table 4.4) and the FWHM of the MPI sample (SS/Al2O3/300) clearly decreased after annealing (SS/Al2O3/300/H), as shown in b.
But the FWHM of SS/Al2O3/300 from MPI (Al2O3 deposition + simultaneous annealing during deposition) increased as compared to the sample simply annealed without metal oxide deposition (SS/H) as well as to the native sample (SS/N). Similar behaviour was observed in the TiO2 deposited silk (c and d). b and d are drawn from the superposition of Gaussian profiles which are illustrated in Figure 4.10.
A. Validation via WAXS. The tensile strength is determined by both crystal size and orientation, and the extensibility is dependent on the molecular configuration of amorphous regions of the silk [178,179,194]. In order to detect changes in the crystal structure of the samples after annealing only and regular MPI, WAXS measurements were performed. As shown in Figure 4.12, some noteworthy differences of the crystal structure
72 Metal Infiltration into Spider Dragline Silk
between the two types of samples can be observed. Firstly, the overall peak intensity of SS/Al2O3/300 and SS/TiO2/500 was reduced presumably due to X-ray absorption of the amorphous Al2O3 and TiO2 layer coating the silks. Further it can be observed that on peak A and B, the peak intensities of both SS/N and SS/Al2O3 decrease and the peaks get sharper at the same time after annealing, i.e. FWHM (wn, Full Width at Half Maximum) values decrease. As a result, the average size of the crystallite (which can be estimated by the Scherrer equation) increases (Table 4.4 and Figure 4.13) [186-188]. In contrast, comparing SS/Al2O3 with SS/N, it can be clearly recognized that FWHM values increase after MPI, i.e. the average sizes of crystallites decrease. In general, the peak broadening of X-Ray Diffraction (XRD) patterns is determined by two factors; a) crystallite size [195], or b) lattice distortion inside the crystallites [196,197]. The broadening observed in this work, however, could mainly be attributed to the crystallite size since the spider silk is known for having high thermal stability [193]. The peak intensity ratio, i.e. ∂I ≡ [IB,max / IA,max], also revealed variations, as shown in Table 4.4 and Figure 4.13b. Initially, the intensity ratio of SS/N, i.e. ∂ISS/N = [IB,max / IA,max]SS/N, was 0.370 but by MPI both
∂ISS/Al2O3/300 and ∂ISS/TiO2/500 increased to 0.385 and 0.462, respectively. In contrast, after annealing, a differing behaviour in the intensity ratio was found; ∂ISS/H and ∂ISS/TiO2/500/H
decreased as compared to ∂ISS/N and ∂ISS/TiO2/500, respectively, but ∂I SS/Al2O3/300/H increased as compared to ∂ISS/Al2O3/300. Furthermore, the peak positions in both cases were invariant upon annealing. Consequently, it may be assumed that the variation of ∂I is caused by the change of the total number of the β-crystallites or by a slight change of β-crystallite’s amino acid composition [193] after annealing or MPI.
The regular ALD process can be regarded as a combined process of metal oxide deposition and simultaneous annealing during the process. As can be recognized from the σ-ε curve of SS/H in Figure 4.11g, simple annealing of the native silk does not contribute to an enhancement of those properties (decreased εmax). The contribution of the outer metal oxide layer coating the silk to the increase of εmax can also be neglected since in general metal oxides, such as Al2O3, TiO2 and ZnO are very brittle. Therefore, the probability to induce the enhancement (increased σmax and increased εmax) of the mechanical properties of the silks under a regular ALD process (i.e. the combined process of metal oxide deposition and simultaneous annealing during deposition) is low. As an evidence to support our assumption, the σ-ε curve of all MPI samples in Figure 4.11 revealed opposite stress-strain behaviour to the samples that can be assumed to be fabricated by a regular ALD process. Moreover, from XRD measurements, it was clearly observed that the size of the silk crystallites decreased after MPI and increased after annealing. Hence it can be concluded that there is a distinct difference between the regular ALD process and the MPI process, namely an additional effect beyond metal oxide deposition and annealing. In the
following, it will be shown that such an additional effect can be attributed to the infiltration of ALD precursors into the molecular protein structure of the spider silk.
Table 4.4 X-ray diffraction data of diverse samples. 2Ө: diffraction angles, I: intensity of each peak and FWHM: Full With at Half Maximum of each peak.
(Average ± Standard Deviation) SS/N SS/H SS/Al2O3/300 SS/Al2O3/300/H SS/TiO2/500 SS/TiO2/500/H 2ӨA 14.09°
Figure 4.13 Evolution of the average size of silk crystallites and relative peak intensity ratio. a, The average size of silk crystallites estimated by means of the Scherrer equation [187,188], which relates the XRD peak broadening to the crystal size, i.e. L = 0.9λ / (ωn cosӨ0) where ωn is the FWHM value (See Table 4.4) of the peak at the Bragg angle (Ө0) and X-ray wave length, λ = 1.5418 Å (Cu Kα). b, Relative peak intensity ratio between peak A and B, i.e. IB,max /IA, max.
B. Validation via NMR. In order to show the evidence for the presence of metal inside the silk, two approaches (Al NMR and TEM-EDX) were performed. Figure 4.14a and b show 27Al MAS (Magic Angle Spinning) NMR spectra obtained from PF/Al2O3/300 and a control sample (alumina deposited on parafilm under identical processing conditions as SS/Al2O3/300 [see Table 4.2]). Figure 4.14c and d show the corresponding spectra of the MPI treated silk composite SS/Al2O3/300. Here, Al2O3 treated samples were chosen, since the sensitivity of the NMR to 27Al is much higher compared to 47Ti or 49Ti. A comparison with data from literature shows that the line shapes do not originate from a single type of alumina, as they do not show the spectra typically observed for α-Al2O3
74 Metal Infiltration into Spider Dragline Silk
[198,199], γ-alumina [200,201] or θ-alumina [199]. Instead, the central-transition spectra in that figure appear to be a superposition of different contributions. For a more detailed analysis, the spectra were deconvoluted and fitted, using the DMFIT program [202]. The program assumes a Gaussian distribution of the chemical shift [202,203], so that line fitting delivers a value for the isotropic chemical shift, δiso, plus the width of the distribution, dδ. For the quadrupolar coupling, a Czjzek distribution [201,204] is used in a simplified form [202,203], which results in an average quadrupolar coupling parameter,
<Cq>. The results of the fitting are shown in Table 4.5. Firstly, the spectrum of PF/Al2O3/300 shows contributions from three different species, which look similar to the spectrum of aluminosilicate zeolites [200] and porous alumina fabricated by anodic oxidation of aluminum [205]. From their positions in the spectrum, these three species can be assigned to four- [AlO4], five- [AlO5], and six-fold [AlO6] oxygen-coordinated aluminum, respectively [198,199,205-208]. On the other hand, the spectral shape of SS/Al2O3/300 (Figure 4.14c) shows one additional component with an isotropic chemical shift of δiso = - 4.9ppm, which indicates that in SS/Al2O3/300 the aluminum nuclei of species 4 experience a different coordination environment to PF/Al2O3/300 (species 1 to 3). Although the value of δiso is comparatively low, it is still within the range reported in the literature for six-fold coordinated aluminum, [AlO6] [208]. Significantly, a negative value of δiso has also been observed for 27Al in the presence of organic ligands [207], which may indicate the vicinity of organic material for species 4. Moreover, by integration of the relative line intensities of the contribution of all signals (Table 4.5), it can be seen that only 9% of the observed aluminum is located in such surroundings. All these data are consistent with the scenario of aluminum being infiltrated into the spider silk protein and/or interacting with the surface of the protein. In order to elucidate the inter-molecular bonding states or interaction states between aluminum and silk protein molecules, Raman measurements were performed, yet unfortunately no conclusive results have been obtained.
C. Validation via EDX. Figure 4.15a shows a TEM image of SS/TiO2/500. As shown in this figure, along the TiO2 shell, a region of ~100nm in depth shows in a high image contrast. Considering the relative weight ratio of carbon, oxygen, and Ti, a large amount of Ti was infiltrated in this shell region, which can also be confirmed from the result of the EDX line scan (Figure 4.15b). In the central part of the silk (folded region), EDX point analysis (Figure 4.15c) showed weak but clear Ti signals (1.42 ~ 2.83 % by relative weight ratio). Because the resolution limit of the system amounts to about 0.5 ~ 1%, the spectrum under those limits has not been quantified. Qualitatively, the small amount of Ti shown as the Ti-K peak is well above the background.
a b
c d
Figure 4.14 Nuclear Magnetic Resonance (NMR) spectra of silk composites, SS/Al2O3/500.
27Al NMR central-transition spectra, recorded with a magic-angle spinning (MAS) rate of 20 kHz.
Deconvolution and fitting of the spectra was performed using the DMFIT program [202], assuming a statistical distribution of both chemical shift and quadrupolar interaction for each species present. a and b, Experimental spectrum of parafilm, PF/Al2O3/300 and its line shape fitted with 3 contributing species. c and d, Experimental spectrum of SS/Al2O3/300 and its line shape fitted with 4 contributing species.
Table 4.5 NMR parameters. These parameters are obtained from deconvoluting and fitting the 27Al NMR spectra shown in Figure 4.14 with the DMFIT program. Listed are the isotropic chemical shifts, δiso, the width of the distribution, dδ, and the average quadrupolar coupling parameter, <Cq>, plus the relative integrated intensities of the spectral lines.
Samples Signal δiso / ppm dδ / ppm <Cq> Integrated Line Intensity
1 69.0 9.2 9.55 36.9%
2 43.6 15.2 8.06 46.3%
PF/Al2O3/300
3 16.0 10.4 6.40 16.8%
1 71.8 13.2 9.54 29.4%
2 45.2 11.8 9.04 37.9%
3 9.6 16.7 5.24 23.9%
SS/Al2O3/300
4 -4.9 9.3 4.81 8.8%
76 Metal Infiltration into Spider Dragline Silk
a b
c Figure 4.15 EDX measurements of
a silk composite, SS/TiO2/500. a, TEM image of microtomed SS/TiO2/500. The prepared samples originally should show a disk shape, but the silk folded and the TiO2 layer broke and dispersed in some regions. It is assumed that such distortions arose during the microtoming process because of the small thickness (~90 nm) of the silk disk. The TiO2 layer and the silk part (SS/TiO2/500) showed similar image contrast. Ti infiltration into the silk was observed along the whole TiO2 shell [inset (I)]. b, Element concentration from EDX scanned across the boundary region along the TiO2 layer (A to B in the pink box; R1, R2, and R3 designate the region of the carbon grid, TiO2 layer, and silk part of SS/TiO2/500, respectively). c, EDX spectrum measured on the folded silk part located at the center of the silk, Ti X-ray emission peak Kα at 4.5 keV and Kβ at 4.9 keV.