Origin of the variability of the mechanical properties of silk fibres: 2 The nanomechanics of single silkworm and spider fibres

The variability in mechanical stress–strain behaviour of various silks obtained from Bombyx mori silkworm and Nephila madagascarensis spider fibres has been studied by high resolution Raman analysis using the Raman shift induced by application of a controlled strain on the νN–H mode as a probe. Silk fibres exhibiting typical 1, 2, 3 and 4 Types have been selected from their characteristic tensile stress–strain behaviour. A perfect relationship between the nanomechanic (at the scale of the chemical bond) and macroscopic (silk single fibre) behaviour is observed as in the case of other polyamide fibres (natural keratin and synthetic PA66). Copyright © 2012 John Wiley & Sons, Ltd.     

Origin of the variability of the mechanical properties of silk fibres: 3. Order and macromolecule orientation in Bombyx mori bave,hand‐stretched strings and Nephila madagascarensis spider fibres

The comparison of the low wavenumber of polarized Raman spectra (50–300 cm^–1) from Bombyx mori (fresh cocoons fibres, hand‐stretched ‘Crins de Florence’ strings from the gland content, dried gland, regenerated silk films) and Nephila madagascarensis silks reveals the high polarisation of fibre modes and the absence of polarisation for dried gland and regenerated silk films. This is consistent with X‐ray diffraction measurements. The orientation of the fibroin/spidroin chains is due to the stretching during production, as for advanced synthetic fibres. The bandwidth of the ‘ordered chains’ signature is almost the same for the different fibres. However, the degree of polarisation seems to be higher in the case of spider fibre. The huge bandwidth of low wavenumber components of regenerated films indicates high disorder. Measurements along the fibre point out conformation changes with a periodicity (~20 mm) related to the silkworm head motion during the fabrication of the cocoon. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification and synthesis of novel biomaterials based on spider structural silk fibers

The diversity in function and mechanical behavior of spider silks, and the ability to produce these silks recombinantly, have tremendous potential in creating a new class of biomimetic materials. Here we investigate the structural and mechanical properties of pyriform silks from the golden orb-weaver, Nephila clavipes. Nanoscale indentation measurements using atomic force microscopy on natural pyriform silk suggests that this biomaterial has high toughness that may be suitable for dissipating high amounts of mechanical energy. We also observed the occurrence of highly organized nanocrystals within the pyriform silk fibers that may contribute to the remarkable energy dissipation capability of these silks. It has been demonstrated that poly-(Gly–Ala) and poly-Ala stretches within the internal block repeat modules of dragline silk fibroins form nanocrystals, and these nanocrystalline structures may be responsible for the high extensibility of the dragline silks. In contrast, amino acid sequence analysis shows that PySp2 does not contain the same motifs. In the absence of poly-(Gly–Ala) and poly-Ala repeats, we hypothesized that PySp2 contains new protein motifs sufficient to polymerize into functional structures. To investigate the functional contributions of these novel motifs during pyriform fiber formation, we expressed different recombinant PySp2 fibroins with various segments spanning its block repeat units. We demonstrate that PySp2 recombinant proteins with the Pro-rich sub-block domain (PXP motifs, where X= sub-set of the amino acids A, L, or R) and/or the Ser + Gln + Ala-rich sub-block domain (QQSSVAQS motifs) are sufficient for artificial fiber formation. Moreover, we show that recombinant PySp2 proteins that contain a single block repeat unit can self-assemble into foam-like nanostructures. Collectively, our findings support the use of PySp2 recombinant proteins for a wide range of biomimetic materials with morphologies ranging from fibers to porous structures.     

Affinity of protein fibres towards sulfation

Wool, Bombyx mori and Antheraea pernyi (Tussah) silk fibres were treated with chlorosulfonic acid in pyridine and investigated by FT‐IR and FT‐Raman spectroscopies as well as mechanical measurements. The reactivity towards sulfation was found to decrease along the series: wool > Bombyx mori silk fibroin > Tussah silk fibroin, in agreement with weight gain which decreased along the same series. Accordingly, Tussah silk maintained its intrinsic tensile properties essentially unchanged upon the treatment, while for Bombyx mori silk fibroin, the tensile performance decreased sharply especially at longer reaction times. Sulfated wool was characterized by an increased fibre extensibility. New IR and Raman bands attributable to various vibrations of sulfated groups were detected in sulfated wool and to a lower extent in Bombyx mori silk fibroin fibres; all the fibres underwent conformational rearrangements upon sulfation, independent of the sulfation yield. Wool fibres treated with chlorosulfonic acid in pyridine bound considerable amounts of sulfate mainly through the hydroxyl groups of serine, threonine and tyrosine. Also, tryptophan and basic amino acids were found to participate to the reaction. B. mori silk fibroin fibres appeared to bind a minor amount of sulfate groups mainly trough the hydroxyl groups of Ser. Weight gain, spectroscopic and mechanical data are discussed in relation to the difference in fibre morphology, structure and crystallinity, as well as to the amount and accessibility of potentially reactive amino acids. Copyright © 2012 John Wiley & Sons, Ltd.     

Rheological characterization of hydrogels formed by recombinantly produced spider silk

Many fibrous proteins such as spider silks exhibit impressive mechanical properties and are highly biocompatible leading to many potential biomaterial applications. For applications such as tissue engineering, polymer hydrogels have been proposed as an effective means of producing porous but stable scaffolds. Here, nanofiber-based hydrogels were produced from engineered and recombinantly produced spider silk proteins. The silk nanofibers are stable semi-flexible polymers which assemble into hydrogel networks. We studied the hydrogel rheology and determined the concentration dependence of the elastic modulus. AFM images indicate that the nanofibers might assemble into branch-like structures, which would also be consistent with the measured rheological behavior. Since the developed spider silk hydrogels are stable over weeks and show a high elastic modulus at low volume fractions, they are well suited for a broad variety of applications.     

Raman and surface enhanced Raman scattering of a black dyed silk

The Raman and surface enhanced Raman scattering (SERS) spectra of a black dyed silk sample (BDS) were registered. The spectral analysis was performed on the basis of Raman and SERS spectral data of isolated samples of Bombyx mori silk fibroin, its motif peptide component (GAGAGS) and the synthetic reactive black 5 dye (RB5). The macro FT‐Raman spectrum of the silk sample is consistent with a silk II‐Cp crystalline fraction of Bombyx mori silk fibroin; the SERS spectrum is highly consistent with conformational modifications of the fibroin due to the interactions with the Ag nanoparticles. The GAGAGS peptide sequence dominates the Raman spectrum of the silk. The SERS spectrum of the peptide suggests a random coil conformation imposed by the surface interaction; the serine residue in the new conformation is exposed to the surface. Quantum chemical calculations for a model of the GAGAGS–Ag surface predict a nearly extended conformation at the Ag surface. The Raman spectrum of the dye was analysed, and a complete band assignment was proposed; it was not possible to propose a preferential orientation or organization of the molecule on the metal surface. Quantum chemical calculations for a model of the dye interacting with a silver surface predict a rather coplanar orientation of the RB5 on the Ag metal surface. The Raman spectrum of the BDS sample is dominated by signals from the dye; the general spectral behaviour indicates that the dye mainly interacts with the silk through the sulphone (–SO₂–) and sulphonate (–SO₂–O–) groups. Besides the presence of dye signals, mainly ascribed to the sulphone and sulphonate bands, the SERS spectrum of the BDS sample also displays bands belonging to the amino acids alanine, glycine, serine and particularly tyrosine. Copyright © 2013 John Wiley & Sons, Ltd.     

Origin of the variability of the mechanical properties of silk fibers: 4. Order/crystallinity along silkworm and spider fibers

The poor crystallinity of proteic fibers has fuelled an ongoing debate over their exact organization. We present a full‐range Raman comparison of Nephila madagascariensis spider and Bombyx mori silkworm silks that sheds some new light on that matter. On the one hand, a large variability is observed along the fibers in the −200 to 200 cm⁻¹ spectral window, which is sensitive to the long‐range order signature of polyamide chains. This questions the validity of previous literature data considering silk fiber as a homogeneous material. On the other hand, the ‘amide I’ band is almost independent of the targeted point, which sets a limit to this widely used structure probe. In‐line mapping of the fibers showed that the extension of the ordered zones ranges between 1 and 3 µm. The correlation between the macromechanical behavior (the stress–strain curves) and the nanomechanics (Raman low wavenumber signatures) under controlled tensile strain demonstrates a Prevorsek's type microstructure: the macromolecular chains belong to both ordered and amorphous ‘regions’. Copyright © 2014 John Wiley & Sons, Ltd.     

Origin of the variability of the mechanical properties of silk fibres: 1 ‐ The relationship between disorder,hydration and stress/strain behaviour

The causes of the variability in mechanical behaviour of various silks obtained from silkworms (Bombyx mori, Antheraea/Tussah) and spiders (Nephila madagascarensis) have been studied by tensile uniaxial tests, differential scanning calorimetry and Raman microspectrometry. Analyses of tensile stress–strain curves recorded for different silkworm and spider baves and single fibres, undergoing different histories (age, degumming, thermal/chemical treatments), allow the behaviour to be classified into five groups and correlated with the fibroin/spidroin structural status: (1) Type I exhibits linear elastic behaviour and then a quasi plateau, (2) Type II has a two‐step linear elastic behaviour with kinks then a quasi plateau, (3) Type III exhibits a smooth transition from a linear behaviour to a plateau, (4) Type IV has a behaviour rather similar to that of Type I but above ~8–12% hardening occurs, and (5) Type V results in the breakage of fibres during the first elastic stage. Unambiguously, Type IV is more frequent for degummed, very dried fibres and Type III for water‐saturated fibres. The most striking Raman signature differences are observed in the Raman intensity of the amorphous/‘ordered’ νN–H and H₂O band components and correlated to the water content and disorder degree according to the calorimetry study. Types I and II are frequently observed for fresh (largely amorphous) spun fibres/baves. Type V is characteristic of degraded fibres. Type IV represents the most ordered state. The similar stress–strain types for dried silkworm and spider fibres show that the general difference in amino acid contents is not the most pertinent parameter for the mechanical behaviour. Copyright © 2011 John Wiley & Sons, Ltd.     

Formation of silk fibroin nanoparticles in water-miscible organic solvent and their characterization

When Silk fibre derived from Bombyx mori, a native biopolymer, was dissolved in highly concentrated neutral salts such as CaCl₂, the regenerated liquid silk, a gradually degraded peptide mixture of silk fibroin, could be obtained. The silk fibroin nanoparticles were prepared rapidly from the liquid silk by using water-miscible protonic and polar aprotonic organic solvents. The nanoparticles are insoluble but well dispersed and stable in aqueous solution and are globular particles with a range of 35–125 nm in diameter by means of TEM, SEM, AFM and laser sizer. Over one half of the ɛ-amino groups exist around the protein nanoparticles by using a trinitrobenzenesulfonic acid (TNBS) method. Raman spectra shows the tyrosine residues on the surface of the globules are more exposed than those on native silk fibers. The crystalline polymorph and conformation transition of the silk nanoparticles from random-coil and α-helix form (Silk I) into anti-parallel β-sheet form (Silk II) are investigated in detail by using infrared, fluorescence and Raman spectroscopy, DSC, ¹³C CP-MAS NMR and electron diffraction. X-ray diffraction of the silk nanoparticles shows that the nanoparticles crystallinity is about four fifths of the native fiber. Our results indicate that the degraded peptide chains of the regenerated silk is gathered homogeneously or heterogeneously to form a looser globular structure in aqueous solution. When introduced into excessive organic solvent, the looser globules of the liquid silk are rapidly dispersed and simultaneously dehydrated internally and externally, resulting in the further chain–chain contact, arrangement of those hydrophobic domains inside the globules and final formation of crystalline silk nanoparticles with β-sheet configuration. The morphology and size of the nanoparticles are relative to the kinds, properties and even molecular structures of organic solvents, and more significantly to the looser globular substructure of the degraded silk fibroin in aqueous solution. It is possible that the silk protein nanoparticles are potentially useful in biomaterials such as cosmetics, anti-UV skincare products, industrial materials and surface improving materials, especially in enzyme/drug delivery system as vehicle.     

Resilient silk captures prey in black widow cobwebs

The gumfoot thread of a black widow (Latrodectus hesperus) spider’s cob web is a spring-loaded trap that yanks walking insects into the web. Since spider silks are known as energy dissipating materials, we investigated this trap to find out where the energy is stored. Using previously measured material properties, we modeled the gumfoot thread as a damped harmonic oscillator and compared it to high speed video analysis of prey capture. These measurements show that the gumfoot thread is plastically deformed during prey capture and cannot be the site of energy storage. We then measured the material properties of scaffolding silk that makes up the upper portion of the cob web. Scaffolding silk is highly resilient (90%) at strains less than 3%. This energy storage is sufficient to drive the oscillations seen in prey capture and is consistent with the measured kinematics. This study is the first demonstration of energy-storage as a primary biological function for spider silk. PACS 87.15.La; 81.40.Jj; 81.05.Lg     

Statistical approaches for investigating silk properties

Amino acid repeats or motifs have engendered interest because of their significance for protein physical characteristics as well as folding properties. Spider dragline silk proteins are unique because they are composed of long repetitive sections and relatively short non-repetitive sections that are known to interact to generate the very peculiar mechanical and solubility properties of silk. Computational analysis compared with in vitro measurements suggest that the silks achieve their unique pattern of extreme solubility inside the spider glands/complete insolubility outside by correlating their repetitive hydrophobic regions through a type of stochastic resonance, generated by the addition of the non-repetitive sequences to a basically periodic hydrophobicity pattern. PACS 87.14.Ee; 87.15.Cc; 87.15.He; 02.50.Ey; 05.40.Ca     

Stability toward alkaline hydrolysis of B. mori silk fibroin grafted with methacrylamide

Bombyx mori silk fibroin fibers were grafted with methacrylamide (MAA) and characterized by Raman and infrared (IR) vibrational spectroscopy before and after hydrolysis in NaOH 5% to elucidate the possible interactions between the two components and the stability of the fibers toward alkaline hydrolysis. Upon grafting, the fibers underwent conformational rearrangements toward a more unordered state and lost orientation at weight gains higher than 60%. Vibrational spectroscopy disclosed the occurrence of intermolecular interactions (mainly hydrogen bonds) between B. mori silk fibroin and polyMAA in the grafted fibers, and the formation of covalent bonds has been explored. These strong interactions made the grafted fibers as a whole more stable toward alkaline hydrolysis because they prevented the solubilization of the polymer upon hydrolysis and made slower the transformation of its CONH₂ groups into COOH and COO⁻ groups. Upon hydrolysis, silk fibroin underwent an enrichment in the β‐sheet crystalline domains, because of the preferential removal of the unordered domains, which were more prone to the OH⁻ attack. IR and Raman spectroscopy proved valid techniques to investigate the degradation mechanism and kinetics of grafted silk fibroin fibers and so for designing high‐performing silk‐based materials. The A₇₃₁/A₁₀₀₄ Raman intensity ratio was proposed to spectroscopically evaluate the composition of the grafted samples; its value was found to linearly increase with weight gain (R² = 0.998), envisaging the possibility of using Raman spectroscopy as a routine analytical technique for qualitative and quantitative characterization of grafted industrial samples. Copyright © 2016 John Wiley & Sons, Ltd.     

Surface modification and properties of Bombyx mori silk fibroin films by antimicrobial peptide

The Bombyx mori silk fibroin films (SFFs) were modified by a Cecropin B (CB) antimicrobial peptide, (NH₂)-NGIVKAGPAIAVLGEAAL-CONH₂, using the carbodiimide chemistry method. In order to avoid the dissolution of films during the modification procedure, the SFFs were first treated with 60% (v/v) ethanol aqueous solution, resulting a structural transition from unstable silk I to silk II. The investigation of modification conditions showed that the surface-modified SFFs had the satisfied antimicrobial activity and durability when they were activated by EDC·HCl/NHS solution followed by a treatment in CB peptide/PBS buffer (pH 6.5 or 8) solution at ambient temperature for 2 h. Moreover, the surface-modified SFFs showed the smaller contact angle due to the hydrophilic antimicrobial peptides coupled on the film surface, which is essential for the cell adhesion and proliferation. AFM results indicated that the surface roughness of SFFs was considerably increased after the modification by the peptides. The elemental composition analysis results also suggested that the peptides were tightly coupled to the surface of SFFs. This approach may provide a new option to engineer the surface-modified implanted materials preventing the biomaterial-centered infection (BCI).     

Comparative Raman spectroscopic analysis of orientation in fibers and regenerated films of Bombyx mori silk fibroin

This study is focused on the Raman spectroscopic analysis of degummed silk fibroin (SF) fibers and regenerated Bombyx mori silk fibroin films: a correlation was found between some spectral features related to the methylene deformation modes and the molecular orientation of the samples. Polarized Raman spectra on SF fibers were used to obtain the orientation distribution function of carbonyl groups along the protein backbone. The variation of the intensity ratio of 1400/1450 cm⁻¹ for the peaks attributed to the wagging and bending deformation modes of CH₂ groups with respect to the angular orientation of the fiber was measured and quantitatively correlated with the orientation distribution function of the carbonyl groups. Unpolarized Raman spectra were measured for regenerated silk fibroin films and lyophilized solutions. The variation of the intensity ratio of 1415/1455 cm⁻¹, which is related to the deformation modes of CH₂ groups in SF regenerated materials, was qualitatively related to the microstructural orientation of the samples observed by scanning electron microscopy (SEM), and to the presence of Silk I phase as suggested by the analysis of samples obtained in different casting conditions and also by the measurements on mechanically deformed films. The results obtained showed the utility of the spectroscopic intensity ratio of 1400/1450 cm⁻¹ for the rapid assessment of molecular orientation in silk fibers, which could be useful for quality and process control of regenerated silk‐based textiles. Moreover, the qualitative dependence of the intensity ratio of 1415/1455 cm⁻¹ was found to be sensitive to both the microstructural orientation and Silk I content of regenerated silk fibroin films, suggesting a possible correlation of this Raman marker of the Silk I phase with the degree of molecular order brought about by this polymorph. Copyright © 2006 John Wiley & Sons, Ltd.     

Precise laser ablation processing of black widow spider silk

Contact-free precise laser processing of black widow spider (Latrodectus hesperus) dragline silk by laser ablation at =157 nm is achieved. At this wavelength, the small optical penetration depth, below 100 nm, allows efficient and gentle material removal above the ablation threshold of _th=29 mJ/cm². The ablation rate in nm/pulse is measured against laser fluence and simultaneously calibrated with ultrapure polymethylmethacrylate (PMMA). Ripple formation in fiber ablation can be overcome by a suitable combination of ablation removal and laser polishing steps using exposure sequences at different irradiation angles , such as vertical at 0° followed by oblique irradiation at 70°. In this way ripples are destroyed until the fiber is automatically smoothed over the entire laser-exposed length. This allows precise laser ablation processing of spider silk fibers and other highly absorbing materials without affecting the bulk mechanical, and other material, properties. PACS 61.41.+e; 61.46.+w; 61.80.Ba; 61.82.Pv; 62.25.+g     

Processing and modification of films made from recombinant spider silk proteins

Protein films represent an interesting class of materials with various possibilities for applications. We investigated films made of two different synthetic spider silk proteins derived from the garden spider’s (Araneus diadematus) two dragline silk proteins ADF-3 and ADF-4. Protein films cast from hexafluoroisopropanol solutions displayed a predominantly α-helical secondary structure. Processing such films with potassium phosphate or methanol resulted in a transition to a β-sheet rich structure. While as-cast films could be dissolved in water, processed β-sheet rich films were water insoluble. The chemical stability of processed films depended on the amino acid sequence of the respective protein employed. As a proof of principle, fluorescent probes or enzymes were covalently attached to the film surface. The presented approach provides a basis for designing tailor-made protein films using silk proteins as scaffold, in which the film properties can be controlled by genetic engineering of the underlying silks. PACS 68.55.Jk; 81.15.Lm; 87.14.Ee     

Influence of grafting with acrylate compounds on the conformational rearrangements of silk fibroin upon electrospinning and treatment with aqueous methanol

Silk fabrics from Bombyx mori silkworm were grafted with 2‐hydroxyethyl methacrylate (HEMA) as well as a binary system of HEMA and 4‐hydroxybutyl acrylate (HBA) and then analysed by Raman and infrared (IR) spectroscopy to elucidate the interactions between the components and their possible conformational changes. The samples were then dissolved in trifluoroacetic acid and electrospun; the influence of the grafted polymers on the silk fibroin rearrangements upon these treatments was investigated by vibrational spectroscopy. Upon grafting, the fabrics underwent conformational rearrangements towards a more unordered state, although they kept their prevailing β‐sheet conformation; also the polymeric component underwent hydrogen bonding and backbone rearrangements upon interaction with silk fibroin and the occurrence of strong covalent bonds cannot be excluded. By immersing the as‐electrospun grafted and pure fibroin nanofibres (prevalently unordered) in aqueous methanol, they partially recovered the β‐sheet content observed in the corresponding starting fabrics; the percentage of recovery decreased along the series: pure silk > HEMA‐grafted silk > HEMA and HBA‐grafted silk. This trend suggests that the presence of the polyHEMA grafted component hinders the silk fibroin recrystallization into β‐sheet upon aqueous methanol treatment; moreover, the addition of the more sterically hindered HBA monomer in the grafting system further prevented this process. Copyright © 2016 John Wiley & Sons, Ltd.     

Effect of gamma irradiation on synthesis and characterization of bio-nanocomposite SF/Ag nanoparticles

This study describes the synthesis of silver nanoparticles (AgNPs) using aqueous silk fibroin (SF) solution obtained from Bombyx mori silk under gamma radiation environment. The obtained AgNPs were characterized using UV–visible (UV–Vis) spectroscopy, X-ray diffraction (XRD) measurements, dynamic light scattering experiment (DLS) and transmission electron microscope (TEM) images. The UV–Vis absorption spectra of the samples confirmed the formation of AgNPs by showing surface plasmon resonance (SPR) band in the range of (= 428–435?nm. The XRD study revealed metal silver with the face-centered cubic (FCC) crystal structure. DLS measurements showed the dose-dependent average size of the AgNPs. TEM images showed formed AgNPs are nearly spherical in shape with smooth edges. From this study, it was found that the increasing radiation dose increases the rate of reduction and decreases the particle size. The size of the AgNPs can be tuned by controlling the radiation dose.     

Spider silk as a model biomaterial

Spider silk combines strength and extensibility, and a wide range of mechanical properties can be achieved with only minute (if any) changes in chemical structure. It appears that the full range of thermo-mechanical properties of such silk fibres can be predicted by examining the energy imparted during stretching with the theoretical framework provided by Mean Field Theory for Polymers. This approach attempts to integrate strain and tensile stress with a range of relevant energetic and mechanical parameters such as the loss tangent and potential energy of atomic inter-chain bonding as well as the tensile and bulk elastic moduli. The model reveals that the underlying design principle of silks seems to share an inherent and surprising simplicity at the macromolecular level. We conclude that our modelling approach allows in-depth analysis of natural silks as well as a comparison with synthetic fibres.     

Structural changes of thin films from recombinant spider silk proteins upon post-treatment

Engineering of spider silk proteins offers the possibility to control their molecular sequence and thus their material properties. Spin coating was used to prepare films of engineered spider silk protein derived from the garden spider’s (Araneus diadematus) dragline silk protein ADF-4. A conformational transition from α-helix to β-sheet-rich structures upon methanol treatment of the films was detected by external reflection IR spectroscopy. We present direct evidence for this structural transformation using grazing-incidence X-ray diffraction (GIXRD) and small-angle scattering (GISAXS). The protein film structure after the methanol treatment consists mainly of β-sheet polyalanine crystals dispersed in an amorphous protein matrix. The GIXRD intensity profiles show Bragg peaks from β-sheet polyalanine crystallites having an average size of 7.5 nm. The non-uniform and large crystal size distributions within the film were explained based on the protein composition. The effect of the chemical nature of the interface on the protein film structure was investigated as well. PACS 87.15.-v; 68.55.-a; 61.10.Eq