On the detection of carbon fibre storage contamination and its effect on the fibre-matrix interface.

Contamination caused by inappropriate carbon fibre (CF) storage may have an impact on their end use in reinforced composite materials. Due to the chemical complexity of CFs it is not easy to detect potential contaminants, especially at the early stage during manufacturing and handling. In this paper, X-ray Photoelectron Spectroscopy (XPS), Fourier Transform Infrared (FTIR) spectroscopy and Surface Energy Analysis (IGC-SEA) were used to assess the surfaces of CFs stored in polyolefin zip-lock bags for possible contamination. Only after over 2 months in-bag storage, was XPS capable of detecting a minor increase in nitrogen on the CF surface while FTIR revealed the presence of fatty acid amides and fatty acids, both associated with the storage media. However neither of these techniques were sensitive enough to show significant evolution of the amount of contamination as a function of storage time. In contrast, IGC-SEA distinguished surface energy differences between CFs before and after storage. These differences were found to change as a function of storage time, which were attributed to increases in contamination amounts. Single fibre fragmentation tests indicated that the surface contamination had potential to disrupt the fibre-matrix interface. These findings provide a new method for assessing the surface contamination of CFs with potential application to other materials.

Graphene based room temperature flexible nanocomposites from permanently cross-linked networks.

Graphene based room temperature flexible nanocomposites were prepared using epoxy thermosets for the first time. Flexible behavior was induced into the epoxy thermosets by introducing charge transfer complexes between functional groups within cross linked epoxy and room temperature ionic liquid ions. The graphene nanoplatelets were found to be highly dispersed in the epoxy matrix due to ionic liquid cation-π interactions. It was observed that incorporation of small amounts of graphene into the epoxy matrix significantly enhanced the mechanical properties of the epoxy. In particular, a 0.6 wt% addition increased the tensile strength and Young's modulus by 125% and 21% respectively. The electrical resistance of nanocomposites was found to be increased with graphene loading indicating the level of self-organization between the ILs and the graphene sheets in the matrix of the composite. The graphene nanocomposites were flexible and behave like ductile thermoplastics at room temperature. This study demonstrates the use of ionic liquid as a compatible agent to induce flexibility in inherently brittle thermoset materials and improve the dispersion of graphene to create high performance nanocomposite materials.

Confirmation of Bioinformatics Predictions of the Structural Domains in Honeybee Silk.

Honeybee larvae produce a silk made up of proteins in predominantly a coiled coil molecular structure. These proteins can be produced in recombinant systems, making them desirable templates for the design of advanced materials. However, the atomic level structure of these proteins is proving difficult to determine: firstly, because coiled coils are difficult to crystalize; and secondly, fibrous proteins crystalize as fibres rather than as discrete protein units. In this study, we synthesised peptides from the central structural domain, as well as the N- and C-terminal domains, of the honeybee silk. We used circular dichroism spectroscopy, infrared spectroscopy, and molecular dynamics to investigate the folding behaviour of the central domain peptides. We found that they folded as predicted by bioinformatics analysis, giving the protein engineer confidence in bioinformatics predictions to guide the design of new functionality into these protein templates. These results, along with the infrared structural analysis of the N- and C-terminal domain peptides and the comparison of peptide film properties with those of the full-length AmelF3 protein, provided significant insight into the structural elements required for honeybee silk protein to form into stable materials.

Design of silk proteins with increased heme binding capacity and fabrication of silk-heme materials.

In our previous studies, heme was bound into honeybee silk to generate materials that could function as nitric oxide sensors or as recoverable heterogeneous biocatalysts. In this study, we sought to increase the heme-binding capacity of the silk protein by firstly redesigning the heme binding site to contain histidine as the coordinating residue and secondly, by adding multiple histidine residues within the core of the coiled coil core region of the modified silk protein. We used detergent and a protein denaturant to confirm the importance of the helical structure of the silk for heme coordination. Aqueous methanol treatment, which was used to stabilize the materials, transformed the low-spin, six-coordinate heme to a five-coordinate high-spin complex, thus providing a vacant site for ligand binding. The optimal aqueous methanol treatment time that simultaneously maintains the helical protein structure and stabilizes the silk material without substantial leaching of heme from the system was determined.

Recombinant Structural Proteins and Their Use in Future Materials.

Recombinant proteins are polymers that offer the materials engineer absolute control over chain length and composition: key attributes required for design of advanced polymeric materials. Through this control, these polymers can be encoded to contain information that enables them to respond as the environment changes. However, despite their promise, protein-based materials are under-represented in materials science. In this chapter we investigate why this is and describe recent efforts to address this. We discuss constraints limiting rational design of structural proteins for advanced materials; advantages and disadvantages of different recombinant expression platforms; and, methods to fabricate proteins into solid-state materials. Finally, we describe the silk proteins used in our laboratory as templates for information-containing polymers.

Structural Analysis of Hand Drawn Bumblebee Bombus terrestris Silk.

Bombus terrestris, commonly known as the buff-tailed bumblebee, is native to Europe, parts of Africa and Asia. It is commercially bred for use as a pollinator of greenhouse crops. Larvae pupate within a silken cocoon that they construct from proteins produced in modified salivary glands. The amino acid composition and protein structure of hand drawn B. terrestris, silk fibres was investigated through the use of micro-Raman spectroscopy. Spectra were obtained from single fibres drawn from the larvae salivary gland at a rate of 0.14 cm/s. Raman spectroscopy enabled the identification of poly(alanine), poly(alanine-glycine), phenylalanine, tryptophan, and methionine, which is consistent with the results of amino acid analysis. The dominant protein conformation was found to be coiled coil (73%) while the ß-sheet content of 10% is, as expected, lower than those reported for hornets and ants. Polarized Raman spectra revealed that the coiled coils were highly aligned along the fibre axis while the ß-sheet and random coil components had their peptide carbonyl groups roughly perpendicular to the fibre axis. The protein orientation distribution is compared to those of other natural and recombinant silks. A structural model for the B. terrestris silk fibre is proposed based on these results.

The purple coloration of four late 19th century silk dresses: A spectroscopic investigation.

Prior to the 19th century the use of purple dyes for textile coloration was expensive and usually limited to royalty. The discovery of several synthetic purple dyes during the 19th century made the production of purple textiles more affordable and thus more readily available. The identification of the source of the purple coloration is of historical interest. Small yarn samples from four late 19th century silk dresses were analyzed using a combination of thin layer chromatography and surface enhanced Raman spectroscopy, Fourier transform infrared spectroscopy and energy dispersive x-ray spectroscopy. This combination of techniques enabled the analysis of the complex extraction products. While three of the dresses were found to be dyed using methyl violet, the fourth dress was found to be constructed from a warp yarn dyed with methyl violet in the presence of a tannic acid mordant, and a weft yarn dyed with mauve and a tin mordant.

Click chemistry modification of natural keratin fibers for sustained shrink-resist performance.

This paper introduces a novel chemical treatment for achieving sustained shrink-resist performance on natural keratin fibers. The new treatment involves the controlled reduction of keratin in the cuticle region of the fiber, and the application of a water soluble diacrylate, namely glycerol 1,3-diglycerolate diacrylate (GDA), on the reduced keratin substrate. The acrylate groups of the GDA react with cysteine residues in the reduced keratin through thiol-ene click reactions at room temperature, leading to GDA grafting and the formation of GDA crosslinks in the keratin structure. The modified substrates were characterized by infrared spectroscopy and scanning electron microscopy, and assessed for its shrink-resistance and wet burst strength. This chemical modification has shown to alter the fiber surface morphology and hydrophilicity, resulting in substantially improved shrink-resistance with good fiber strength retention. Possible shrink-resistance mechanisms were also discussed.

De Novo Engineering of Solid-State Metalloproteins Using Recombinant Coiled-Coil Silk.

To achieve the sophisticated chemistry required for life, nature uses metal containing proteins (metalloproteins). However, despite intensive research efforts, very few of these metalloproteins have been exploited for biotechnological applications. One major limiting factor is the poor stability of these proteins when they are removed from their cellular environment. To produce stable metalloproteins, we have developed an engineering strategy that uses structural proteins which can be fabricated into a number of different solid-state materials. Here we demonstrate that a recombinant silk protein (AmelF3 - Apis mellifera Fibroin 3) binds heme and other metal macrocycles in a manner reminiscent of naturally occurring metalloproteins, whereby an amino acid coordinates directly to the metal center. Our strategy affords design at four different levels: the metal center, the organic macrocycle, the protein scaffold, and the material format structure. The solid-state metalloproteins produced remained functional when stored at room temperature for over one year.

The effects of physical and chemical treatments on Na2S produced feather keratin films.

The industrial utilisation of feather keratin as a biopolymer has proven difficult due to the lack of a viable extraction technique and the poor mechanical properties of the regenerated products. Here, pure keratin films were produced from chicken feathers using sodium sulphide as sole extraction reagent in a scheme that allows films to be formed without residual chemicals. In a comparison to other films, those produced using Na2S extraction were found to be superior to other regenerated protein films and were similar to un-oriented commercial polymers. However, there was considerable variation in tensile properties between twenty repetitions of extracting and casting films which was attributed to variations in chain entanglement caused by the drying conditions. Chemical and physical treatments including crosslinking, dehydration and addition of nano-particles were investigated as means to enhance these properties. Significant increases were achieved by soaking films in isopropyl alcohol or weak acid (13 to 50% increases) or by formaldehyde or glutaraldehyde crosslinking (24 to 40% increases). The wide range of values across the pure keratin films indicates that the best route to further strength improvement may be from optimising self-assembly via controlling drying conditions, rather than from chemical treatment.

Stabilization of viruses by encapsulation in silk proteins.

Viruses are important for a range of modern day applications. However, their utility is limited by their susceptibility to temperature degradation. In this study, we report a simple system to compare the ability of different dried protein films to stabilize viruses against exposure to elevated temperatures. Films from each of three different silks, silkworm, honeybee silk and hornet silk, stabilized entrapped viruses at 37 °C better than films of albumin from bovine serum (BSA) and all four proteins provided substantially more stabilization than no protein controls. A comparison of the molecular structure of the silks and BSA films showed no correlation between the ability of the proteins to stabilize the virus and the secondary structure of the protein in the films. The mechanism of stabilization is discussed and a hypothesis is suggested to explain the superior performance of the silk proteins.

Micromolar biosensing of nitric oxide using myoglobin immobilized in a synthetic silk film.

In this work we investigate the use of coiled-coil silk proteins, produced in recombinant Escherichia coli, as a new material for immobilizing biosensors. Myoglobin was embedded in transparent honeybee silk protein films. Immobilized myoglobin maintained a high affinity for nitric oxide (KD NO=52 µM) and good sensitivity with a limit of detection of 5 µM. The immobilized myoglobin-silk protein film was stable and could be stored as a dry film at room temperature for at least 60 days. The effect of immobilization on the structure of myoglobin was fully investigated using UV/visible, Fourier Transform Infrared and Raman spectroscopy, which indicated a weakening in the strength of the iron-histidine bond. This study demonstrates that recombinant coiled-coil silk proteins provide a safe and environmentally friendly alternative to sol-gels for stabilizing heme proteins for use as optical biosensors.

Novel immobilization of a quaternary ammonium moiety on keratin fibers for medical applications.

This paper introduces a new approach for immobilizing a quaternary ammonium moiety on a keratinous substrate for enhanced medical applications. The method involves the generation of thiols by controlled reduction of cystine disulfide bonds in the keratin, followed by reaction with [2-(acryloyloxy)ethyl]trimethylammonium chloride through thiol-ene click chemistry. The modified substrate was characterized with Raman and infrared spectroscopy, and assessed for its antibacterial efficacy and other performance changes. The results have demonstrated that the quaternary ammonium moiety has been effectively attached onto the keratin structure, and the resultant keratin substrate exhibits a multifunctional effect including antibacterial and antistatic properties, improved liquid moisture management property, improved dyeability and a non-leaching characteristic of the treated substrate.

Infrared and Raman spectroscopic studies of tris-[3-(trimethoxysilyl)propyl] isocyanurate, its sol-gel process, and coating on aluminum and copper.

Tris-[3-(trimethoxysilyl)propyl] isocyanurate (TTPI) has been used as a precursor to prepare a sol using ethanol as the solvent under acidic conditions. The sol-gel was applied for the surface treatment of aluminum and copper. Infrared and Raman spectra have been recorded for pure TTPI and the TTPI sol, xerogel and TTPI sol-gel coated metals. From the vibrational spectra, TTPI is likely to have the C1 point group. Vibrational assignments are suggested based on group frequencies, the expected reactions in the sol-gel process and the vibrational studies of some related molecules. From the experimental infrared spectra of xerogels annealed at different temperatures and from the thermal-gravimetric analysis, it is found that the TTPI xerogel decomposes at around 450°C with silica being the major decomposition product. A cyclic voltammetric study of the metal electrodes coated with different concentrations of TTPI ranging from 5% to 42% (v/v) has shown that the films with high concentrations of sol would provide better corrosion protection for aluminum and copper.

Raman spectroscopy in the analysis of food and pharmaceutical nanomaterials.

Raman scattering is an inelastic phenomenon. Although its cross section is very small, recent advances in electronics, lasers, optics, and nanotechnology have made Raman spectroscopy suitable in many areas of application. The present article reviews the applications of Raman spectroscopy in food and drug analysis and inspection, including those associated with nanomaterials. Brief overviews of basic Raman scattering theory, instrumentation, and statistical data analysis are also given. With the advent of Raman enhancement mechanisms and the progress being made in metal nanomaterials and nanoscale metal surfaces fabrications, surface enhanced Raman scattering spectroscopy has become an extra sensitive method, which is applicable not only for analysis of foods and drugs, but also for intracellular and intercellular imaging. A Raman spectrometer coupled with a fiber optics probe has great potential in applications such as monitoring and quality control in industrial food processing, food safety in agricultural plant production, and convenient inspection of pharmaceutical products, even through different types of packing. A challenge for the routine application of surface enhanced Raman scattering for quantitative analysis is reproducibility. Success in this area can be approached with each or a combination of the following methods: (1) fabrication of nanostructurally regular and uniform substrates; (2) application of statistic data analysis; and (3) isotopic dilution.

Convergently-evolved structural anomalies in the coiled coil domains of insect silk proteins.

The use of coiled coil proteins as the basis of silk materials is an engineering solution that has evolved convergently in at least five insect lineages-the stinging hymenopterans (ants, bees, hornets), argid sawflies, fleas, lacewings, and praying mantises-and persisted throughout large radiations of these insect families. These coiled coil silk proteins share a characteristic distinct from other coiled coil proteins, in that they are fabricated into solid materials after accumulating as highly concentrated solutions within dedicated glands. Here, we relate the amino acid sequences of these proteins to the secondary and tertiary structural information available from biophysical methods such as X-ray scattering, nuclear magnetic resonance and Raman spectroscopy. We investigate conserved and convergently evolved features within these proteins and compare these to the features of classic coiled coil proteins including tropomyosin and leucine zippers. Our analysis finds that the coiled coil domains of insect silk proteins have several common structural anomalies including a high prevalence of alanine residues in core positions. These atypical features of the coiled coil fibrous proteins - which likely produce deviations from canonical coiled-coil structure - likely exist due to selection pressures related to the process of silk fabrication and the final function of the proteins.

Thermal annealing effects on multi-walled carbon nanotube yarns probed by Raman spectroscopy.

The realized mechanical properties of CNT macrostructures such as webs and yarns remain significantly lower than those of the individual CNTs. Structural changes induced by thermal annealing under inert atmosphere were assessed using Raman spectroscopy. Annealing above 1000 °C resulted in a marked decrease in the D/G ratio which can be attributed to an increase in the crystallite size or the distance between defects. The band component parameters obtained by spectral deconvolution reveal that the D band peak maximum shifts to slightly higher energy with increased annealing temperature. In contrast, the energy of the G band did not change. The full widths at half height (FWHH) of the D and G bands are seen to decrease with increasing annealing temperature. The tensile properties of the yarns have been investigated and it was found that the yarn tenacity did not improve with these structural changes. The effect of impurities in the annealing system such as oxygen, adsorbed water or organic surface contamination was also investigated.

A comparison of convergently evolved insect silks that share ß-sheet molecular structure.

Raspy crickets produce silk webs that are used to build shelters. These webs have been found to consist of both fiber and film components. Raman spectra obtained from both components were found to be very similar for a given species. The protein structure of the fibers and films produced by both species was predominately ß-sheet with lesser amounts of ß-turns, unordered and α-helical protein also detected. The orientation of the ß-sheet backbone in the fiber was determined to be parallel to the fiber axis. Compared to cocoon and dragline silk the orientation distribution exhibits a significant randomly orientated protein component. Amino acid analysis confirmed the presence of glycine, serine, and alanine in both species, which are known to form antiparallel ß-sheet structures. Both species, although at significantly different concentrations, where found to contain proline. This amino acid is uncommon in insect silks, and likely involved in increasing fiber elasticity.

Modifying surface resistivity and liquid moisture management property of keratin fibers through thiol-ene click reactions.

This paper reports on a new method for improving the antistatic and liquid moisture management properties of keratinous materials. The method involves the generation of thiols by controlled reduction of cystine disulfide bonds in keratin with tris(2-carboxyethyl) phosphine hydrochloride and subsequent grafting of hydrophilic groups onto the reduced keratin by reaction with an acrylate sulfonate or acrylamide sulfonate through thiol-ene click chemistry. The modified substrates were characterized with Raman spectroscopy and scanning electron microscopy and evaluated for their performance changes in liquid moisture management, surface resistivity, and wet burst strength. The results have revealed that the thiol-acrylate reaction is more efficient than the thiol-acrylamide reaction, and the keratinous substrate modified with an acrylate sulfonate salt exhibits significantly improved antistatic and liquid moisture management properties.

Continuous production of flexible fibers from transgenically produced honeybee silk proteins.

Flexible and solvent stable fibers are produced after concentrated recombinant honeybee protein solutions are extruded into a methanol bath, dried, drawn in aqueous methanol, then covalently cross-linked using dry heat. Proteins in solution are predominantly coiled coil. Significant levels of non-orientated ß-sheets form during drying or after coagulation in aqueous methanol. Drawing generally aligns the coiled coil component parallel with the fibre axis and ß-sheet component perpendicular to the fiber axis. The fibres are readily handled, stable in the strong protein denaturants, urea and guanidinium, and suitable for a range of applications such as weaving and knitting.