Mechanical properties of spun fibers

During wet-spinning the spinning dope is extruded into a coagulation bath containing a mixture of isopropanol and water in which the spidroin precipitates and a fiber is formed.

After wet-spinning from both BSD and CSD, the spun fibers were post-stretched in order to improve their mechanical properties. In nature, spiders use their hind legs to post-stretch the fiber directly when it leaves the spinneret. The size and orientation of the β-sheet crystals in the fiber change with the reeling speed and, thus, directly influence the mechanical properties of the fiber. Du et al.^[154] showed that a high reeling speed leads to a high β-sheet crystal nucleus density in natural silk, resulting in fibers containing smaller crystals but with an increased crystal proportion. In this work, the recombinant fibers were stretched up to 600 % of their initial length after spinning in order to align the spidroins.

Tensile testing of all recombinant fibers showed that the post-stretching significantly improved the mechanical properties of the fibers (see table 1 in publication I). While recombinant fibers spun from CSD, resulted in mostly inhomogeneous and brittle fibers (Figure 9), especially when spun from dopes containing spidroins without the carboxy-terminal domain NR3, wet-spinning of BSD led to very homogeneous and long fibers.

Tensile testing revealed that a) the molecular set-up of the spidroins and b) the type of dope used for spinning had a great impact on the mechanical properties of the recombinant fibers.

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Figure 9: A,B) Real stress–real strain curves of recombinant and natural spider silk fibers. A) As-spun (inset) and 600%

post-stretched N1L(AQ)12 NR3-fibers, spun from “classical” (CSD) as well as “biomimetic” (BSD) spinning dopes (both 10% (w/v)) and B) 600% post-stretched (AQ)12NR3- and N1L(AQ)12NR3-fibers from CSD as well as BSD in comparison to natural A. diadematus dragline silk fibers. C) Average toughness of natural dragline silk fibers (blue), fibers spun from CSD (red) and BSD (green).^[15] By courtesy of the publisher John Wiley and sons.

Post-stretching of (AQ)12-fibers spun from CSD was only possible up to 400 % of their initial length, and the fibers appeared very brittle. In comparison, fibers spun from CSD of N1L(AQ)12 and (AQ)12NR3, showed a higher extensibility and strength reaching a higher overall toughness than (AQ)12 fibers. For fibers wet-spun from CSD, the highest toughness (111 ± 33 MJ/m³) was achieved with N1L(AQ)12NR3 fibers that were post-stretched to 600 % of their initial length. While post-stretched (AQ)12NR3 and N1L(AQ)12NR3 fibers had the same strength, N1L(AQ)12NR3 fibers showed an increase in extensibility and a lower stiffness than the (AQ)12NR3 fibers. Determining the mechanical properties of post-stretched (AQ)₁₂NR3 and N1L(AQ)₁₂NR3 fibers spun from BSD revealed a significant increase in extensibility and toughness compared to the corresponding fibers spun from CSD. The toughness was equal to ((AQ)₁₂NR3, 171.6 ± 52.7 MJ/m³) or even slightly exceeding (N1L(AQ)₁₂NR3, 189.0 ± 33.4 MJ/m³) that of natural spider silk fibers (167.0 ± 65.3 MJ/m³) (Figure 9).

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Since the mechanical properties of polymer fibers are strongly influenced by the molecular weight of the polymer,^[236,237] the mechanical properties of fibers spun from (AQ)24 -variants were also determined, possessing almost twice the molecular weight of the corresponding (AQ)12-variants. As anticipated, (AQ)24-fibers spun from CSD showed improved mechanical properties compared to (AQ)12-fibers. Surprisingly, during the presence of one or both terminal domains N1L/NR3, the mechanical properties were no longer solely influenced by the molecular weight of the underlying core domain, but by their assembly process. N1L(AQ)24, (AQ)24NR3 and N1L(AQ)24NR3 fibers spun from CSD all revealed inferior mechanical properties in comparison to (AQ)24 fibers (see table 1 in publication I). This indicates that spider silk proteins cannot solely be treated like polymers, but that their “protein” features have a considerable influence on fiber mechanics. Since the amino-terminal domain only dimerizes upon a pH drop in the spinning duct, it is still in its monomeric form in the spinning dope, preventing a correct alignment of the repetitive parts of the spidroins. This effect is even amplified during the absence of the carboxy-terminal domain, yielding brittle fibers (see table 2 in publication I). Corresponding to the discoveries with (AQ)₁₂-variants, the mechanical properties of (AQ)₂₄NR3 and N1L(AQ)₂₄NR3 fibers spun from BSD showed a significant increase in strength and toughness when compared to those spun from CSD. Overall, fibers spun from both CSD and BSD of (AQ)24-variants comprising either one or both non-repetitive terminal domains showed inferior mechanical properties in comparison to that spun from the corresponding (AQ)12-variants. This indicates an entanglement of the large spidroin molecules once the assembly-controlling terminal domains are present, which cannot be straightened out when using the limited and non-biomimetic wet-spinning process.

The highest mean toughness (189.0 ± 33.4 MJ/m³) was obtained with post-stretched N1L(AQ)12NR3-fibers spun from BSD. Even though the toughness of the recombinant fibers lies in the same range as that of natural spider silk fibers, it has to be noted that strength, extensibility and stiffness of the recombinant fibers differ significantly. The recombinant fibers are not as strong as the natural fibers, but are far more extensible. This may be due to two major discrepancies between the used set-up for this work and the natural role-model, namely 1) the application of only one spidroin and 2) the applied wet-spinning process. As mentioned in chapter 1.2.2, natural spider dragline fibers contain at least two spidroins, which likely are both crucial in order to obtain outstanding mechanical properties. Accordingly, the highly complex natural spinning process (chapter 1.4.2) cannot possibly be mimicked by a simple wet-spinning process. The development of a biomimetic spinning technology, as well as the application of more than one recombinant

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spidroin will likely achieve the production of recombinant fibers with mechanical properties superior to that of the natural role-model, opening up new possibilities for fibrous materials.

3.4. Molecular structure of artificial spider silk fibers compared to natural ones