Thermo-Hygro-Mechanical Behavior of Spider Dragline Silk: Glassy and Rubbery States

GUSTAVO R. PLAZA, GUSTAVO V. GUINEA, JOSE PEREZ-RIGUEIRO, MANUEL ELICES Departamento de Ciencia de Materiales, Universidad Politecnica de Madrid, ETSI de Caminos, Canales y Puertos. Ciudad Universitaria. E-28040 Madrid, Spain

Received 6 September 2005; revised 21 November 2005; accepted 3 January 2006 DOI: 10.1002/polb.20751 Published online in Wiley InterScience (www.interscience.wiley.com).

Spider silk is considered as the basis of a new family of high performance bers that would reproduce the excellent mechanical properties of the silk, in particular its extreme toughness. However, it has been observed that the mechanical properties of spider silk are severely inuenced by humid environments that give rise to signicant decreases in its length and elastic modulus. The change from stiff to compliant tensile properties is associated with the glass transition from a glassy to a rubbery state; here, we have found that it depends on both temperature and relative humidity. The glass transition was identied at different temperatures and relative humidities by monitoring the variation of the elastic modulus and observing the emergence of supercontracC tion. V 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 994999, 2006 Keywords: glass transition; mechanical properties; proteins; spider silk; thermal properties
ABSTRACT:

INTRODUCTION
Spider dragline silk has exceptional mechanical properties in terms of tensile strength and breaking energy,13 and it is considered as the basis of a new family of polymeric bers.47 However, both the microstructure and the mechanisms underlying these outstanding properties remain obscure. Spider silk is described as a semicrystalline polymer8,9 composed of stacked b-pleated sheets,10 known as b-microcrystallites, with less ordered regions referred to as amorphous regions. The different secondary structures that have been assigned to the amorphous regions, such as 310 helix,11 b-turn,12 or random coil,11,13 and their role in the mechanical behavior of the bers are still unexplained.

Correspondence to: M. Elices (E-mail: melices@mater. upm.es)

Journal of Polymer Science: Part B: Polymer Physics, Vol. 44, 994999 (2006)
C V 2006 Wiley Periodicals, Inc.

Progress has been made in the identication of the interactions that determine the properties of spider silk, based on the analysis of the large shrinkage experienced by unrestrained spider silk bers in wet environments, known as supercontraction.14 Wet bers are extremely compliant, with an elastomeric tensile behavior,15 and the effect of water was justied as the result of breaking a network of proteinprotein hydrogen bonds.16,17 From these observations, spider silk in dry environments can be modeled as a double network of elastomeric chains frozen by hydrogen bonds bridging chains. Both temperature and humidity have a strong inuence on the mechanical behavior of spider silk bers. Most previous works have studied the mechanical properties at room temperature (20 258C), with the bers in dry conditions or immersed in water, and recently the inuence of temperature has been studied with no control of humidity.18,19 To the authors knowledge, this article is the rst systematic report of the combined effect of temperature and humidity.

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Figure 1. Technique used to examine the effect of temperature and humidity: (a) initial set up, with a loose ber, (b) the ber is clamped at its maximum length (L0) under zero force, (c) stabilization stage, (d) conditioning stage, and (e) nal length of the ber after conditioning (LC).

A transition from a stiff to a compliant tensile behavior is characteristic of polymeric materials in their glass transition.20 This transition has been identied in spider silk by comparing the elastic modulus of dry bers, Edry % 10 GPa,6,15,21 with that of wet spider bers, E % 0.01 GPa.15 The overall compliance of the stressstrain curve is much greater in the wet bers. In most polymers, the glass transition sets in at a given temperature, known as the glass transition temperature, that depends on the microstructural details of the polymer. In spider silk, however, the glass transition should be inuenced not only by temperature but also by the environmental relative humidity (RH), as observed in other hygroscopic polymers.22 This is the rst study of the tensile mechanical properties of spider silk bers in different conditions of temperature and RH, considered together. The results show an evolution of the shape of the stressstrain curve, of the amount of supercontraction, and of the mechanical parameters, and from these a glass transition is identied and characterized in terms of temperature and RH.

EXPERIMENTAL
Major ampullate gland silk bers (MAS) were collected by forced silking23,24 at a reeling speed of 1.5 cm/s, 20 8C, and 35% relative humidity (RH). The silking procedure was implemented to retrieve only one of the two monolaments (brin) that form the ber. The samples were stored at 20 8C and 35% RH until testing. All samples were silked from the same Argiope trifasciata speci-

men. The spider was captured near the Mediterranean coast and fed on a diet of crickets. The initial cross-sectional area of the samples, Ao, was measured from scanning electron microscopy micrographs. The cross section of one out of every ve samples was measured and used to rescale force into stress for adjacent samples, as adjacent MAS bers show similar tensile properties.14 The tested samples were glued on aluminum foil frames, with a nominal gauge length of 20 mm. Samples were xed in the grips of the testing machine (Instron 3309-622/8501), which was appended to an environmental chamber Dycometal CCK-25/300 to control temperature and RH. Forces were measured with a 100 mN load cell HBM Q11 with 0.1 mN resolution. Temperature and moisture control was achieved within 60.5 8C and 61.5% RH. Further details are given elsewhere.17,25 Reproducibility was checked by testing at 20 8C and 35% RH one out of every ve samples as a control. We used only bers with no signicant deviation from the tensile properties of the control samples. The effect of temperature and humidity was ascertained by the procedure sketched in Figure 1: before testing, the ber was fully extended at zero load and xed at 20 8C and 35% RH. This length was labeled as L0. The chamber was allowed to stabilize in the desired environmental conditions for at least 1 h (stabilization period) and then the ends of the ber were approached so that the ber could contract freely for an additional hour (conditioning period). The length of the fully extended ber at zero load after the conditioning period, LC, was taken as the reference length to compute the strain. Tensile tests were carried out at 1 mm/min.

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As the bers have a micrometric diameter (%4 lm) and a large surface/volume ratio, 1 h is considered a suitable period of time for the stabilization and conditioning stages. It had been observed, in fact, that the effects of the water absorption develop in just a few seconds.17 In addition, the selected tensile test rate of 1 mm/min is considered low enough to allow for equilibrium with the wet environment. Tensile tests were performed in no less than 360 s (up to 2100 s), whereas in ref. 26 it was shown that for a nylon ber of similar diameter 95% of equilibrium water content is reached after 12 s.

RESULTS AND DISCUSSION

Figure 2 illustrates the analogous effect of temperature and humidity on the tensile behavior of spider silk. Strain is dened as (L LC)/LC, where L is the instantaneous length during testing and LC is the length of the ber after conditioning, as explained earlier. Stress was computed by dividing the force by the cross-sectional area of the ber after conditioning, AC, which was computed from the initial cross-sectional area of the ber before conditioning, Ao, under the hypothesis of constant volume. This hypothesis has been validated by measuring the cross-sectional areas of samples subjected to supercontraction and different degrees of stretching.26 The inuence of RH on the tensile behavior of spider silk at constant temperature is shown in Figure 2(a) (all tests were performed at 55 8C), and Figure 2(b) shows the effect of temperature, at constant RH (all tests performed at 50% RH). At constant temperature, the shape of the stressstrain curve changes from a curve characteristic of glassy polymers, at the lowest values of RH, to a curve similar to that of an elastomer at the highest RH. Simultaneously, the overall stiffness and the elastic modulus decrease when the RH increases. At constant RH, the change in the shape of the stressstrain curve parallels the previous situation; the shape of the curve changes from glassy at 20 8C to rubbery at 90 8C [Fig. 2(b)]. The overall stiffness and the elastic modulus decrease with the temperature increase. Under conditions of high humidity or temperature, a reduction of the initial length of the ber during the conditioning stage was observed. In bers submerged in water, the reduction of the

Figure 2. Stressstrain curves of spider silk (MAS from A. trifasciata): (a) at constant temperature of 55 8C, (b) at constant RH of 50%. The bers were conditioned as explained in the text. Engineering strain is dened as (L LC)/LC, where L is the instantaneous length and LC is the length after conditioning. Engineering stress is calculated by dividing load by the cross-sectional area after conditioning, AC. The cross section after conditioning is calculated from the initial cross section, Ao, assuming that volume remains constant throughout the supercontraction process (AoLo ACLC).26

length in unrestrained silk bers reaches up to 60% of the initial length, a fact known as supercontraction.14,27 Exposition of bers to increasing levels of RH, at constant temperature, leads also to a signicant reduction of the ber length, reaching maximum supercontraction only at RH 100%. The inuence of environmental conditions on the progress of supercontraction can be characterized by the ratio of supercontraction, dened as (LC L0)/L0. All these experimental results show that during the evolution of the mechanical properties of spider silk in different environmental conditions two parameters appear signicant: the elastic

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Figure 3. Evolution of elastic modulus and percentage of supercontraction as a function of temperature and RH. (a) Evolution of the elastic modulus, E, obtained from the tensile tests as described in the text. (b) Evolution of the ratio of supercontraction after conditioning.

modulus, dened as the initial slope of the stressstrain curves, and the ratio of supercontraction. The evolution of these two parameters with RH, at different temperatures, is drawn in Figure 3(a,b). At constant temperature, the elastic modulus shows a slight decrease with increasing humidity until the RH threshold is reached, where a sharp decrease is observed. At higher humidities a slight decrease is observed again. All three curves are similar and can be superimposed by horizontal shift. In all the cases, the elastic modulus decreases from $10 GPa at the lowest values of RH to $0.03 GPa at the highest RH values. At constant RH, the initial elastic modulus decreases with temperature, as shown in Figure 3(a). The evolution of the ratio of supercontraction with increasing humidity at a given temperature

parallels qualitatively that of the elastic modulus, although with some signicant differences. Supercontraction initiates at a certain value of RH that decreases with increasing temperature. From this point on, supercontraction increases gradually with increasing RH until the maximum supercontraction is reached at 100% RH. A remarkable feature is found at high RH: the supercontraction ratio coincides at all three temperatures (lower line), indicating that at high RH the role of temperature on supercontraction is negligible. The maximum supercontraction was achieved only at RH 100%, when immersed in water. Stressstrain curves show a transition driven by environmental conditions; this transition is characterized by a change in the overall shape (from glassy to rubbery) and in the global compliance of the curves, particularly with a strong decrease of the initial elastic modulus as shown in Figure 3(a). It was also found that the transition in the tensile properties is coupled to the emergence of supercontraction. Figure 3(a,b) shows that the conditions under which supercontraction appears coincide with the sharp decrease of the elastic modulus. The curves on Figure 3(a,b) suggest the occurrence of a glass transition in spider silk. This transition would correspond to the change from glassy to rubbery behavior shown in Figure 2. The glass transition of an amorphous material is related to a change in the mobility of the molecules, and the temperature at which the transition occurs is identied as the glass transition temperature (Tg). It can be modied by the presence of a plasticizer, which reduces the interchain interactions. The aforementioned results indicate that water acts as a plasticizer for spider silk, leading to signicant disruption of the hydrogen bonds between molecular chains in the amorphous regions.8,15,16 Reducing the number of hydrogen bonds increases the mobility of the chains, and so Tg of the plasticized bers would depend on the water content, which depends on the RH at a given temperature. Therefore, it is not surprising that the glass transition depends both on temperature and RH. The glass transition diagram, deduced from the data shown in Figure 3, is shown in Figure 4. The mean values of temperature and RH at which both the drop of the elastic modulus and the supercontraction threshold occurred have been used to dene the glassrubber transition. As expected, the RH required to complete the

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Figure 4. Transition from the glassy to the rubbery state as a function of temperature and RH. The mean conditions of the transition of the initial elastic modulus (full circles), and for the supercontraction drop (open squares) at 20, 55, and 90 8C are dened from the results presented in Figure 3.

glass transition is lower at higher temperatures, since water molecules are more likely to disrupt hydrogen bonds weakened by higher temperatures. This tendency is conrmed by the previous data on the glass transition in spider silk at temperatures above 100 8C performed under unstatedbut supposedly negligiblewater vapor concentration and estimated at Tg % 160 8C (Nephila clavipes19) and 198 8C (Nephila edulis18). These gures compare well with a rough extrapolation from data in Figure 4, which yields a Tg between 150 and 200 8C for 0% RH. It is worth noting that performing a direct measurement of Tg at 0% RH is far from trivial, since the environmental chambers cannot control humidity at temperatures close to 100 8C (actually the chamber used in this study cannot control humidity beyond 95 8C). The observed glass transition occurs in a range of RH that spans from the initiation of transition to its completion. This range of RH must be a consequence of the heterogeneity of the local chemical environments in which the proteinprotein hydrogen bonds are embedded. A sudden glass transition would be expected if all proteinprotein hydrogen bonds were in a similar chemical environment, since all hydrogen bonds would be simultaneously disrupted when their chemical potential equals the chemical potential of free water molecules. One example of the heterogeneity in the conformation of protein chains appears when the distance of the chain to the b-microcrystallites is considered: the alignment of the chain

increases with decreasing distance from the microscrystallites.28 However, it is likely that local chemical environments of proteinprotein hydrogen bonds include some or all of the conformations previously assigned to the amorphous regions of spider silk.11,12 The effect of water, as a plasticizer that induces a decrease in the elastic modulus, has been studied in other natural polymers such as wool29 and silkworm silk30; in all cases, the effect of water has been assigned to the disruption of hydrogen bonds between protein chains. However, a complete glass transition at room temperature appears as a distinct feature of spider silk. Wet silkworm silk bers show a four-fold reduction in their elastic modulus and no signicant reduction in their initial length. Moreover, the stressstrain curves do not match with the corresponding curves of an elastomer. The tensile behavior of wet spider silk bers can be justied from the properties of the protein chains of the amorphous regions in the rubbery state,15 while the mechanical behavior of wet silkworm silk has been justied considering van der Waals interactions in the amorphous matrix.30 Amorphous regions of silkworm silk bers do not reach the rubbery state by immersion in water. This difference from spider silk must be attributed essentially to two factors: the amino acid sequence in the amorphous regions, which determines interactions between chains, and the degree of crystallinity, which is 3050% in spider silk8,31 and nearly 60% in silkworm silk.32 A lower degree of crystallinity favors the appearance of the glass transition in milder conditions.20

SUMMARY AND CONCLUSIONS

Tensile tests of spider silk bers have been performed systematically under controlled temperature and RH. On increasing the temperature or the RH, the behavior of silk bers changes from stiff, typical of glassy polymers, to compliant, similar to that of elastomeric bers. Compliance can be characterized by means of the initial elastic modulus. At each temperature, this parameter shows a sudden decrease within a small range of values of RH, a behavior indicative of a glassrubber transition. Supercontraction also shows a sudden increase within a narrow range of values of RH in tests performed at constant temperature. Curiously,

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the average values of temperature and RH with this transition are very similar to the values of the glassrubber transition. All these results agree with a model of silk bers as a semicrystalline material made of amorphous exible chains reinforced by crystallites, and they provide a plausible explanation of the glassrubber transition. The chains are strongly hydrogen-bonded and the presence of water and/or high temperature inuences the hydrogen bonding; water is known to have a plasticizing effect by preventing the formation of hydrogen bonds between chains, and temperature works in a similar way.
A. trifasciata specimens were kindly provided by Jesus Minano. We thank Oscar Campos and Ivan Blanco (Naturaleza Misteriosa, Parque Zoologico de Madrid, Spain) for rearing the spiders, and Jose Miguel Martnez for help with testing samples and drawing gures. This work was funded by Ministerio de Ciencia y Tec nologa (Spain) through projects MAT 2003-04906, and by Comunidad de Madrid through project GR/MAT/ 0038/2004.

REFERENCES AND NOTES

1. Kaplan, D. L.; Lombardi, S. J.; Muller, W. S.; Fossey, S. A. In Biomaterials; Byron, D., Ed.; Stockton Press: New York, 1991; pp 153. 2. Lewis, R. V. Acc Chem Res 1992, 25, 392398. 3. Structural Biological Materials; Elices, M., Ed.; Pergamon Materials Science: Amsterdam, 2000; pp 293334. 4. Colgin, M. A.; Lewis, R. V. Chem Ind 1995, 1009 1012. 5. Lazaris, A.; Arcidiacono, S.; Huang, Y.; Zhou, J. F.; Duguay, F.; Chretien, N.; Welsh, E. A.; Soares, J. W.; Karatzas, C. N. Science 2002, 295, 472476. 6. Vollrath, F.; Knight, D. Nature 2001, 410, 541548. 7. Elices, M.; Perez-Rigueiro, J.; Plaza, G. R.; Guinea, G.V. JOM 2005, 57, 6066. 8. Simmons, A. H.; Michal, C. A.; Jelinsky, L. W. Science 1996, 271, 8487. 9. Thiel, B. L.; Viney, C.; Mater Res Bull 1995, 20, 5256. 10. Marsh, R. B.; Corey, L.; Pauling, L. Biochem Biophys Acta 1955, 16, 134.

11. Van Beek, J. D.; Kummerlen, J.; Vollrath, F.; Meier, B. H. Int J Biol Macromol 1999, 24, 173178. 12. Jelinski, J. W.; Blye, A.; Liivak, O.; Michal, C.; LaVerde, G.; Seidel, A.; Shah, N.; Yand, Z. Int J Biol Macromol 1999, 24, 197201. 13. Hinman, M. B.; Jones, J. A.; Lewis, R. V. TIBTECH 2000, 18, 374379. 14. Work, R. W. Textile Res J 1977, 47, 650662. 15. Gosline, J. M.; Denny, M. W.; DeMont, M. E. Nature 1984, 309, 551552. 16. Termonia, Y. Macromolecules 1994, 27, 73787381. 17. Guinea, G. V.; Elices, M.; Perez-Rigueiro, J.; Plaza, G. Polymer 2003, 44, 57855788. 18. Yang, Y.; Chen, X.; Shao, Z.; Zhou, P.; Porter, D.; Knight, D. P.; Vollrath, F. Adv Mater (Weinheim, Germany) 2005, 17, 8488. 19. Guess, K. B.; Viney, C. Thermochim Acta 1998, 315, 6166. 20. Riande, E.; Daz-Calleja, R.; Prolongo, M. G.; Masegosa, R. M.; Salom, C. Polymer Viscoelasticity, Stress and Strain in Practice. Marcel Dekker: New York, 2000. 21. Perez-Rigueiro, J.; Elices, M.; Llorca, J.; Viney, C. J Appl Polym Sci 2001, 82, 22452251. 22. Nielsen, L. E. Mechanical Properties of Polymers. Van Nostrand Reinhold: New York, 1962. 23. Work, R. W.; Emerson, P. D. J Arachnol 1982, 10, 110. 24. Guinea, G. V.; Elices, M.; Real, J. I.; Gutierrez, S.; Perez-Rigueiro, J. J Exp Zool A 2005, 303, 3744. 25. Guinea, G. V.; Elices, M.; Perez-Rigueiro, J.; Plaza, G. R. J Exp Biol 2005, 208, 2530. 26. Plaza, G. R. Thermo-hygro-mechanical behaviour of spider silk Fibers; Ph.D. thesis (in Spanish). Universidad Politecnica de Madrid, 2004. 27. Perez-Rigueiro, J.; Elices, M.; Guinea, G. V. Polymer 2003, 44, 7333736. 28. Struick, L. C. E. Physical Ageing in Amorphous Polymers and Other Materials; Elsevier: Amsterdam, 1978. 29. Feughelman, M. Mechanical Properties and Structure of a-Keratin Fibers. University of New South Wales Press: Sydney, 1997. 30. Perez-Rigueiro, J.; Viney, C.; Llorca, J.; Elices, M. Polymer 2000, 41, 84338439. 31. Izuka, E. J Appl Polym Sci 1985, 41, 173185. 32. Gillespie, D. B.; Viney, C.; Yager, P. In Silk Polymers, Materials Science and Technology; Kaplan, D.; Adams, W. W.; Farmer, B., Eds.; ACS Symposium Series: Washington D.C., 1994; 544, pp 155167.