Synchrotron FTIR Spectroscopic Characterization On Silk Proteins And Preparation Of Silk Nanofibril-based Composite Materials

Animal silks have an outstanding portfolio of mechanical properties combining high modulus, strength and extensibility. The structure of the predominant silk protein, fibroin (spidroin) has been widely studied at different levels in a range of species. In general, animal silk can be regarded as a semicrystalline biopolymer with highly organized antiparallel β-sheet nanocrystals embedded in amorphous matrix. However, the details of how these secondary structures in natural silks are affected by factors including spinning conditions and mechanical strain, and how the secondary structure helps to determine the mechanical properties of silk fibers is not completely understood. In addition, the chemical composition, fiber morphologies and mechanical properties of different silks show considerable complexity and variability, which can be traced to diversity in protein amino acid sequences, differences in spinning conditions, and also the internal and external environment of the animals. Accordingly, studies of defined microscopic regions of single natural silk fibers under different conditions would be useful to understand the relationship between protein secondary structure and mechanical properties of silk fibers.Single silk fibers have been examined by synchrotron X-ray diffraction (XRD) and Raman spectroscopy, giving some useful information. Although FTIR spectroscopy, is one of the oldest and well-established experimental techniques for the conformational analysis of polypeptides and proteins, there appear to be no reports to date of its use to characterize single natural silk fibers. The main difficultly is that the diameter of the light beam is three magnitudes larger than the 5-20 μm width of a normal single silk fiber. Thus, a single silk fiber is only exposed to a small part of the infrared beam, creating a serious signal to noise problem which results in very poor quality of the spectra rendering them almost useless. In the past twenty years, synchrotron FTIR (S- FTIR) microspectroscopy has been developed as a rapid, direct, non-destructive, and non-invasive analytical technique for micron-sized samples. This technique combines the ultra-high brightness of synchrotron infrared source (usually 100-1000 times brighter than the conventional globar source) with the powerful magnification of the microscope, allowing spectra with high signal-to-noise ratio to be obtained from micron-sized samples or sample areas.In this work (Chapter Ⅰ), we report the use of S-FTIR microspectroscopy to monitor the silk protein conformation in a range of single natural silk fibers (domestic and wild silkworm, and spider dragline silk). With the selection of suitable aperture size, we obtained high resolution S-FTIR spectra capable of semi-quantitative analysis of protein secondary structures. For the first time, we have determined from S-FTIR the β-sheet content in a range of natural single silk fibers,28±4%,23 ± 2%, and 17±4% in Bombyx mori, Antheraea pernyi, and Nephila edulis silks, respectively. The trend of P-sheet content in different silk fibers from the current study accords quite well with published data determined by XRD, Raman and 13C NMR.In order to understand the relationship between structure and properties of single silk fibers, in Chapter Ⅱ, we used S-FTIR microspectroscopy to monitor protein secondary structures (content and orientation) and their changes when subjected to different strain in single Antheraea pernyi cocoon silk fibers. The results showed that the content and orientation of p-sheet was almost unchanged for strains from 0 to 0.3. However, the orientation of a-helix and random coil improved progressively with increasing strain, with a parallel decrease in a-helix content and increase in random coil. This clearly indicates that most of the deformation upon stretching of the single fiber is due to the change of orientation in the amorphous regions coupled with a conversion of some of the α-helix to random coil. These observations provide an explanation for the supercontraction behavior of certain animal silks and are likely to facilitate understanding and optimization of post-drawing used in the conjunction with the wet-spinning of silk fibers from regenerated silk solutions. Thus our work in Chapter Ⅱ and Ⅲ demonstrates the power of S-FTIR microspectroscopy for studying biopolymers.Apart from the animal silks, silk fibroin (SF) materials also has been widely studied and is used for a variety of task-specific applications. In order to improve or adapt the properties of SF materials, SF is sometimes blended with other natural or synthetic polymers. As in other polymer blends, the phase behaviour of these SF-based polymer blends is thought to be very important towards their related properties. Conventional methods used to study phase behaviour in SF based polymer blends include scanning electron microscopy (SEM), atomic force microscopy (AFM), differential scanning calorimetry (DSC), and dynamic thermomechanical analysis (DTMA). Although these analytical methods have provided a great deal of useful information about the phase behaviour of SF-based blends, they cannot directly provide data on chemical structure information within these blends. FTIR imaging is a good candidate for providing such information as it offers the possibility of combining spectral and spatial information, thereby enabling a spatial chemical visualization of the sample. Thus, in Chapter IV and Chapter V, we use this technique to study SF-based polymer blends in the work.In Chapter IV, we used FTIR imaging to study the phase behavior of three silk protein-based polymer blends, silk fibroin/chitosan (SF/CS) blend, silk fibroin/sodium alginate (SF/SA) blend, and silk fibroin/polyvinyl alcohol (SF/PVA) blend. FTIR images of the films prepared from these polymer blends indicated that SF/CS blend was compatible, SF/SA blend was partially compatible, and SF/PVA blend was incompatible. The results accord with the conclusions from the conventional analysis methods like SEM, DSC, and DTMA reported in the literature. Moreover, we show that FTIR images of the blends can provide additional useful information on the composition of the individual components, and the conformation of SF at defined locations with a spatial resolution of 4 μm.In Chapter V, we integrated FTIR imaging and scanning transmission X-ray microscopy (STXM) imaging techniques to monitor the phase separation of silk fibroin/polyethylene oxide (SF/PEO) blend. We demonstrate that FTIR and STXM imaging provide complimentary chemical sensitivities, resolution ranges and sample thickness requirements that can enable greater understanding of polymer blend films. From the FTIR images, we find SF shows random coil and/or helical conformation in the SF-rich domains, and β-sheet conformation in the PEO-rich matrix. In the meantime, the SF content in SF-rich domains is 74±4%, and 38±6% in PEO-rich matrix from the STXM images. These findings support and give further evidence to the conclusions of the previous studies on SF/PEO blends in the literature. Our results in Chapter IV and V strongly suggest that FTIR and STXM imaging techniques are two promising approaches for the study of phase behavior and molecular conformation in SF-based blend materials. On the other hand, as with many others techniques, both FTIR and STXM imaging have their own limitation. Normally, the basis of the imaging technique is the difference in the absorption of the characteristic peaks of the components, so it may encounter difficulties when study SF/protein blends. However, this drawback can be solved by use far-infrared spectroscopy, because we showed in Chapter VI that different silk proteins, such as B. mori SF and A. pernyi SF, have different specific absorption in far-infrared region.At the last part of work (Chapter VII and VIII), we prepared two kinds of silk nanofibrils composites:(1) silk nannofibrils/graphene hybrids. Under carefully selected experimental conditions, we showed that graphene nanosheets are able to direct one-dimensional self-assembly of silk fibroin, forming an unprecedented type of nano-hybrids. These graphene/silk hybrids combine physical properties of both constituents and form functional composites with well-ordered hierarchical structures. Due to the facile fabrication process and their tunable nanostructures, the resultant hybrids show promise in applications as diverse as tissue engineering, drug delivery, nanoelectronics, nanomedicine, biosensors and functional composites. (2) SF nanofibrils/amyloid fibrils composites. Amyloid fibrils and SF nanofibrils are proteinaceous aggregates occurring either naturally or as artificially reconstituted fibrous systems, in which the constituent P-strands are aligned either orthogonal or parallel to the fibril main axis, respectively, conferring complementary physical properties. In Chapter VIII, we showed how the combination of these two classes of protein fibrils with orthogonally oriented β-strands results in composite materials with controllable physical properties at the molecular, mesoscopic and continuum length scales. Furthermore, by either adding magnetic nanoparticles or by removing enzymatically one of the two constituents, we showed how the amyloid-silk hybrids can be used as sensors, magnetic actuators or separation membranes of tuneable cut-off, which could serve in applications as diverse as molecular filters, microfluidics devices, magnetic valves or artificial skin.