Research ArticleMATERIALS SCIENCE

Biomimetic composites with enhanced toughening using silk-inspired triblock proteins and aligned nanocellulose reinforcements

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Science Advances  13 Sep 2019:
Vol. 5, no. 9, eaaw2541
DOI: 10.1126/sciadv.aaw2541
  • Fig. 1 Protein design, phase separation, and composite fiber formation.

    (A) Schematic representation of the triblock spidroin protein architecture consisting of terminal CBMs, separated by spidroin sequences (eADF3 and ADF3). Reference architectures are also presented. (B) Phase contrast light microscopy and phase atomic force microscopy (AFM) images of the liquid-liquid phase separated coacervates. (C) Scanning electron microscopy (SEM) and height atomic force microscopy images of a CNF network. (D) Infiltration of CNF network by coacervates at various time points. (E) Light microscopy image of deformation of coacervates during shear stress. (F) Two extruded fibers (CBM-ADF3-CBM:CNF, 1:2 w:w) in a coagulation bath. Fibers made with CNF alone or any of the CBM-containing proteins could be collected in lengths of several meters (fig. S2). (G) If the monoblock eADF3 (lacking CBM) was used, the resulting fiber was fragmented and could not be collected as a continuous fiber.

  • Fig. 2 Properties of composite fibers.

    (A) Stress-strain curves of CNF fibers with the triblock spidroin proteins and reference compositions. Mean values ± SD (n = 8) are shown. (B) Representative stress-strain curves of fibers spun at different protein-to-CNF ratios. RH, relative humidity. (C) Representative stress-strain curves of composite fiber spun with different protein variants. (D) Spider chart representing the performance space for the composite fibers spun from different protein variants. (E) Toughness values extracted from the work of fracture for all variants shown in (C). (F) Representative stress-strain curves of the oriented (fiber) and nonoriented (film) composites and pure CNF. The graphs show representative curves. Further data with SDs are shown in figs. S3 to S5.

  • Fig. 3 Effects of ethanol on protein conformation and CBM binding to cellulose.

    (A) CD spectra of triblock protein CBM-eADF3-CBM in the coacervated state (top curve in blue) and after coagulation with ethanol (bottom curve in orange). The spectra show a switch that is characteristic of a change from random coil or α helix toward high β sheet content. (B) Comparison of 13C NMR spectra (aliphatic part) of CBM-eADF3-CBM as coacervate (top curve in blue) and after ethanol coagulation (bottom curve in orange). The spectra shown are a projection along the 13C dimension of a 2D 13C-1H heteronuclear single-quantum coherence spectrum of the coacervate and a 1H-13C cross-polarization–magic angle spinning spectrum of the coagulate. Comparison of signals shows that Ala residues are mostly in random coil conformation before coagulation and in β sheets after coagulation. *An artifact resulting from the methyl resonance of ethanol. **Solution NMR spectra were referenced to 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) signal at 0.00 parts per million (ppm). ***Solid-state NMR spectra were referenced to adamantane CH2 signal at 38.48 ppm. ****See (23). *****See (24). (C) Attenuated total reflection (ATR)–FTIR spectra of CBM-eADF3-CBM coacervate showing the amide I band peak at 1638 cm−1 (top curve in blue) and, after coagulation and drying, showing a shift of the amide I peak to 1622 cm−1 (bottom curve in orange). (D) Cartoon illustrating the shift of conformation of CBM-eADF3-CBM from the coacervate (top in blue) and coagulation and drying (bottom in orange). (E) SDS–polyacrylamide gel electrophoresis gel showing that the CBM binds with high efficiency to CNF and is not removed from CNF by water or ethanol. S1 is the supernatant after CBM adsorption to CNF. P1 is the CNF pellet with the adsorbed CBM. S2a and S2b are the supernatants from two wash steps with water, and P2 is the CNF pellet after the water wash. S3a and S3b are the supernatants from two wash steps with ethanol, and P3 is the CNF pellet after ethanol washing. MW shows molecular weight standards.

  • Fig. 4 SEM fractography of the fibers.

    (A) Fracture cross section of the fibers after tensile measurement testing. Samples with the triblock proteins CBM-eADF3-CBM and CBM-ADF3-CBM show surfaces with less tear-out than other samples. (B) High-magnification images of fracture surfaces of the CNF control, CBM-CBM and CNF, and CBM-eADF3-CBM and CNF composite fibers. At this magnification, the fibril tips of the CBM-eADF3-CBM show distinguishable rounded tips and tight bundles with blunt ends.

  • Fig. 5 Hypothesis for the mechanism of fracture surface morphology.

    The reduced fiber pullout seen in the fracture surfaces with triblock protein could be explained by cohesiveness that the silk material provides and allows mechanisms for hindering crack propagation. The left column represents the cellulose fiber without protein, and the column to the right represents a fiber with the triblock protein as a matrix. The regions with different colors highlight regions with strongly associated fibrils having regions of more loosely associated fibrils forming in between. The loosely associated regions are more easily separated and show up as voids in the fracture surfaces in the samples without triblock protein.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaaw2541/DC1

    Fig. S1. Coacervation of proteins, coacervate infiltration of cellulose, and viscosity of mixtures.

    Fig. S2. Double-injection CNF-protein fiber spinning and apparatus for making CNF-protein films.

    Fig. S3. Mean values and SDs of mechanical properties for CNF-only and composite CBM-eADF3-CBM-CNF and CBM-ADF3-CBM-CNF fibers at a protein-to-CNF ratio of 1:2.

    Fig. S4. Mean values and SDs of mechanical properties for CNF-only and composite CBM-eADF3-CBM-CNF and CBM-ADF3-CBM-CNF fibers at different mixing ratios.

    Fig. S5. Mean values and SDs of mechanical properties for CNF-only and composite fibers made from mono-, di-, and triblock protein variants.

    Fig. S6. Effect of orientation on the mechanical properties of the oriented CNF-only and composite CBM-eADF3-CBM-CNF fibers and nonoriented corresponding films.

    Fig S7. Conformational studies by NMR.

    Fig S8. Mechanical properties of composite fibers at different RHs.

    Fig. S9. SEM fractography for all fibers after tensile measurement test.

    Movie S1. Infiltration of CNFs by CBM-eADF3-CBM coacervates.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Coacervation of proteins, coacervate infiltration of cellulose, and viscosity of mixtures.
    • Fig. S2. Double-injection CNF-protein fiber spinning and apparatus for making CNF-protein films.
    • Fig. S3. Mean values and SDs of mechanical properties for CNF-only and composite CBM-eADF3-CBM-CNF and CBM-ADF3-CBM-CNF fibers at a protein-to-CNF ratio of 1:2.
    • Fig. S4. Mean values and SDs of mechanical properties for CNF-only and composite CBM-eADF3-CBM-CNF and CBM-ADF3-CBM-CNF fibers at different mixing ratios.
    • Fig. S5. Mean values and SDs of mechanical properties for CNF-only and composite fibers made from mono-, di-, and triblock protein variants.
    • Fig. S6. Effect of orientation on the mechanical properties of the oriented CNF-only and composite CBM-eADF3-CBM-CNF fibers and nonoriented corresponding films.
    • Fig S7. Conformational studies by NMR.
    • Fig S8. Mechanical properties of composite fibers at different RHs.
    • Fig. S9. SEM fractography for all fibers after tensile measurement test.

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Infiltration of CNFs by CBM-eADF3-CBM coacervates.

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