Biomacromolecules 2000, 1, 622-626
622
X-ray Diffraction on Spider Silk during Controlled Extrusion under a Synchrotron Radiation X-ray Beam Christian Riekel* European Synchrotron Radiation Facility, B.P. 220, F-38043 Grenoble Cedex, France
Bo Madsen Department of Zoology, University of Aarhus, Universitetsparken B135, DK 8000, Aarhus C, Denmark
David Knight and Fritz Vollrath Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, England Received May 25, 2000; Revised Manuscript Received September 13, 2000
The structure of a single thread of Nephila edulis silk has been studied by in situ X-ray diffraction from a living spider. A systematic increase of orientational order with increasing silking speed up to 40 mm s-1 was observed. Within a few mm from the spinnerets exit, crystalline domains with a β-poly(L-alanine) structure were observed. The data also suggest an increase in crystalline fraction in the immediate vicinity of the spigot exit. Introduction Dragline spider silk combines high elasticity with high strength.1 The mechanical properties of the finished thread have been related to a crystal-reinforced network.1 The formation of the silk from its liquid crystalline precursor in the spider’s gland is, however, not well understood.2-4 For a full appreciation of the material, fiber performance must be reconciled with fiber production. We have recently shown that in situ X-ray diffraction experiments during forced silking of Nephila senigalensis spiders are feasible.5 Although the data showed a modulation of orientational order of the crystalline blocks, a systematic increase in orientational order with increasing silking speed could not be observed.5 We suggested that this modulation of orientational order at the rather slow silking speeds of e9 mm s-1 resulted from mechanical changes induced by the exit spigot, actively controlled by the spider.5 The present note reports on an extension of these experiments to silking speeds of e40 mm s-1 and a correlation of the data with mechanical properties. Experimental Section Spider Specimen and Forced Silking Techniques. Adult females of the silk spiders Nephila edulis and Nephila senegalensis were reared in-house and kept under controlled conditions. Spiders were fed by Musca domestica flies and regularly watered by spraying the webs. X-ray experiments took place within 3 days after transfer of the spiders to Grenoble. Prior to experiments, the spider was lightly anaesthetized by CO2 and fixed with soft tape facing venter
upward to a metal block. (Figure 1A) The body temperature of the spider was measured by a thermocouple at its back and was controlled by circulating a thermostatizing liquid through the metal block. A motorized spindle allowed drawing fibers in a wide range of well-defined speeds.5 (Figure 1A) Mechanical Tests. A total of 21 individuals were sampled. Single monofilaments from a major ampullate spigot located on one of the two anterior spinnerets, drawn at a specific speed at 22 ( 2 °C, were carefully transferred with a micromanipulator to a custom-built microscale materials testing machine.6 To avoid slippage, the monofilament was superglued to the crossheads of the materials testing machine. The average monofilament diameter was 3.35 ( 0.63 µm as measured uncoated under a SEM at low voltage.6 Mechanical tests were performed at a fairly constant relative humidity (around 50%). X-ray Diffraction. Dragline threads from the major ampullate glands of 10 individual N. edulis were measured at 23.5 ( 0.5 °C at the microfocus beamline (ID13) of the European Synchrotron Radiation Facility (ESRF).7 Forced silking could be maintained from a few minutes up to several hours prior to rupture of the thread. Data reported are from the individual with the longest spinning time (7 h) of a welldefined double dragline thread with good mechanical properties. The beam size at the thread was 10 µm and the wavelength 0.095 nm. Diffraction patterns were collected with a MAR CCD detector. Details of data analysis have been reported elsewhere.8 Movement of the spider made it impractical to obtain diffraction data closer than 2.4 mm from the exit spigot.
10.1021/bm000047c CCC: $19.00 © 2000 American Chemical Society Published on Web 10/28/2000
X-ray Diffraction on Spider Silk
Biomacromolecules, Vol. 1, No. 4, 2000 623
Figure 1. (A) Experimental setup for in situ X-ray diffraction during forced silking. The spider is fixed by soft tape and Mylar bandages to a metal support. The path of the thread from the spinnerets to the motorized reel is schematically indicated. Distance indications (to the spigot exit) correspond to points were X-ray diffraction data were recorded. (B) SEM image of draw-down of N. edulis spider silk at a drawing speed of 20 mm s-1. (C) Diffraction pattern obtained at 23.5 ( 0.5 °C. Miller’s indices indicated for selected reflections.12
Figure 1A shows the support structure holding the spider to allow sampling by the X-ray beam at discrete points along the spinline by the X-ray beam. In a separate experiment, a SEM image of a monofilament (single fiber) extracted at a drawing speed of 20 mm s-1, a speed comparable to natural spinning rates,9 was obtained. Figure 1B shows that the fiber becomes more regular and thinner with increasing distance from the exit spigot. Results and Discussion Crystallinity along Spinline. The diffraction pattern obtained at a distance of 4.9 mm from the exit spigot, after the first guiding needle, at a drawing speed of 7 mm s-1 (Figure 1C) corresponds at this point to the pattern obtained from freely foraging spiders8 and has been modeled as consisting of small, anisotropic crystalline domains with the β-poly(L-alanine) structure and an oriented amorphous fraction.8,10-13 At 2.4 mm from the spigot exit, as close as we could get, we observed that β-poly(L-alanine) crystallites (Figure 2) were already present which implies that this crystalline phase has formed inside or at or just after the exit spigot. The observation that the oriented halo is particularly important at 2.4 mm distance from the exit spigot (Figure 2), suggested that the maximum crystallinity had not been reached at 2.4 mm and that draw-down crystallization continues a few millimeters from the exit spigot along the spinline. Orientational Order. The azimuthal width of equatorial reflections is related to the orientational order of the
anisotropic crystalline domains along the fiber axis.8,13 Diffraction data recorded at distances g2.4 mm from the exit spigot (Figure 2) qualitatively show no change in azimuthal width, which suggests that the additional drawdown at these distances is rather small. A more quantitative analysis requires diffraction data to be collected from closer to the spigot exit as the SEM data (Figure 1B) show a reduction in cross sectional area of the fiber by about a factor of 3 within the first 1.4 mm from the exit spigot. We note that the principal orientation mechanism of silk as it progresses through the spider’s production pathway is assumed to be due to the internal draw-down taper within the spinning duct, commencing approximately 4 mm before the exit spigot.4,14 The diffraction patterns show, however, a systematic reduction of azimuthal width of the equatorial reflections with increasing drawing speed (Figure 3A) in contrast to previous experiments on a N. senigalensis spider.5 This corroborates the suggestion that the spider modulates orientation in the fiber at low drawing speeds by a mechanical action of the spinnerets.5 Orientational order can be expressed quantitatively in terms an average angle 〈cos2φ〉 of the crystalline domains with respect to the spinline. 〈cos2φ〉 in spider silk can be derived by X-ray diffraction from the azimuthal width of the equatorial (020)/(210) reflections8,13 by Herman’s orientation function (fc):15 fc ) (3〈cos2φ〉 - 1)/2
(1)
Herman’s orientation function was determined as a function
624
Biomacromolecules, Vol. 1, No. 4, 2000
Riekel et al.
Figure 2. Variation of diffraction pattern as a function of distance from the exit of the spigot (drawing speed: 7 mm s-1). The azimuthal profile on the right side is derived from an annulus centered on the (210) reflection, which also covers the oriented halo, and partially the (021) reflections. (Figure 1C) The rotation of the fiber axis (F) is due to the change in orientation of the thread between the spinnerets and the spindle (Figure 1A).
of drawing speed at a distance of 4.89 mm from the exit spigot (Figure 3B). As expected from the diffraction patterns (Figure 3A), fc increases with increasing drawing speed. At the upper end of the drawing speeds, spider silk reaches an fc value observed for the high performance polymer Kevlar.16 An effective draw ratio λ has been included in Figure 3B which was calculated from17 〈cos2φ〉 = 1 - 0.5πλ-3/2
(2)
Equation 2 is based on an affine deformation model for anisotropic particles in an elastic medium.18,19 This model has been shown to hold for lyotropic main chain liquid crystal polymers.17 The application of the affine deformation model to forced silking is reasonable as it can also be applied to changes in orientational order of the anisotropic crystalline domains in N. senigalensis silk, induced by tensile strain.13
The stress of about e0.5 GPa, induced by forced silking experiments, corresponds roughly to tensile strain values of e0.1.6,13 Effect of Drawing Rate on Tensile Properties. The tensile strength as measured (Figure 4) shows an increase with drawing speeds up to about 100 mm s-1. Statistical models for the fiber tensile strength assume that failure is due to rapid growth of defects, which are initially randomly distributed throughout the fiber.21 It is reasonable to assume that the increase in orientation of the crystalline domains with spinning speed (Figure 3) reflects a general increase of chain orientation, which reduces the number of defects, and therefore reduces failure probability. Conclusions In situ X-ray diffraction during silking gives a first glimpse into two important aspects of this extraordinary polymer
X-ray Diffraction on Spider Silk
Biomacromolecules, Vol. 1, No. 4, 2000 625
Figure 3. (A) Variation of diffraction patterns of (210) and (020) reflections as a function of drawing speed. (see box in Figure 1C). (B) Variation of degree of orientation, fc, and effective draw ratio (λ) as a function of the drawing speed. The dashed curve is a guideline to the eye.
References and Notes
Figure 4. Variation of tensile strength as a function of drawing speed. The dashed line is a guideline to the eye.
synthesis and fiber-spinning factory. First, post-extrusion draw down enhances crystallinity in a monofilament that is already largely solidified as it leaves the spinneret. Second, the speed of drawing constrains molecular order and thus can affect the mechanical properties of the final thread. Acknowledgment. We wish to thank D. Rolles for his help with data analysis.
(1) Gosline, J. M.; DeMont, M. E.; Denny, M. W. EndeaVour 1986, 10 (1), 37-43. (2) Vollrath, F.; Wen Hu, X.; Knight, D. P Proc. Roy Soc. 1998, 263, 817-820. (3) Vollrath, F.; Knight, D. P. Internat. J. Biol. Macromol. 1999, 24 (2-3), 243-249. (4) Knight, D. P.; Vollrath, F. Proc. R. Soc. London B 1999, 266, 519523. (5) Riekel, C.; Mu¨ller, M.; Vollrath, F. Macromolecules 1999, 32, 44644466. (6) Madsen, B.; Vollrath, F. Naturwissenschaften 2000, 87, 148-153. (7) Riekel, C. Rep. Prog. Phys. 2000, 63, 233-262. (8) Riekel, C.; Bra¨nden, C.; Craig, C.; Ferrero, C.; Heidelbach, F.; Mu¨ller, M. Int. J. Mol. Biol. 1999, 24, 187-195. (9) Shao, Z.; Young, R. J.; Vollrath, F. Int. J. Biol. Macromol. 1999, 24, 295-300. (10) Simmons, A. H.; Michal, C. A.; Jelinski, L. W. Science 1996, 271, 84-87. (11) Arnott, S.; Dover: S. D.; Elliott, A. J. Mol. Biol. 1967, 30, 201208. (12) Warwicker, J. O. J. Mol. Biol. 1960, 2, 350-362. (13) Grubb, D. T.; Jelinski, L. W. Macromolecules 1997, 30, 2860-2867. (14) Knight, D.; Knight, M. M.; Vollrath, F. Int. J. Biol. Macromol. 2000, 27, 205-210. (15) Stein, R. S.; Wilkes, G. L. In Physico-Chemical Approaches to the Measurement of Anisotropy; Stein, R. S., Wilkes, G. L., Ed.; Applied Science Published Ltd.: London, 1975; pp 57-145. (16) Wu, T. M.; Blackwell, J. Macromolecules 1996, 29, 5621-5627.
626
Biomacromolecules, Vol. 1, No. 4, 2000
(17) Northolt, M. G.; Sikkema, D. J. In Lyotropic Main Chain Liquid Crystal Polymers; Northolt, M. G., Sikkema, D. J., Ed.; SpringerVerlag: Berlin, 1991; Vol. 98, pp 115-177. (18) Kuhn, W.; Gruen, F. Kolloid Z. 1942, 101, 248-271. (19) Ward, I. M. Proc. Phys. Soc. 1962, 80, 1176-1188.
Riekel et al. (20) Termonia, Y. Macromolecules 1994, 27, 7378-7381. (21) Weibull, W. J. Appl. Mech. 1951, 18, 293-297.
BM000047C
