ANIMAL SILKS: FRACTURE MECHANISM AND FORMATION MECHANISM Gong Li (B. Sc, Sichuan University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSICS NATIONAL UNIVERSITY OF SINGAPORE (2013) Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously.” Gong Li August 02 2013 i Acknowledgements I would like to express my deepest gratitude to my supervisor, Prof. Liu Xiangyang, and co-supervisor, Prof. Wang Haifeng, for their constant guidance, support and help during the last four years. This thesis would not have been possible without their persistent help. Prof. Liu provided me a wide research area from fundamental biophysics to application, and his creative thinking inspired me a lot in my research career. He also taught me the skill of presenting scientific data and converting experimental reports into publishable manuscripts. His encouragement and inspiration will be benefit for me throughout my whole life. Prof. Wang helped me in exact experimental skills. His rigorous attitude towards research deeply affected me, and his wide knowledge always helped me when I met puzzles. I would also like to thank my seniors, colleagues and friends, Mr. Teo Hoon Hwee, Ms. Sin Yin, Du Ning, Li Yang, Pan Haihua, Wang Hui, Yuan Bing, Hu Wen, Wu Xiang, Zhao Xiaodan, Xu Gangqin, Yang Zhen, Diao Yingying, Deng Qinqiu, Ye Dan, Lin Naobo, Zhang Desuo, Tian Liyong, William, Wengong, Tuan, Viet, Paul, Luo Yuan, and Li Huanan, for their help in by research life. Meanwhile, I am indebted to my parents. My father is a spare time musician who let me know how to feel the beauty of the world like an artist, and my mother stimulated my initial interest in science by showing me magic about ii physics and chemistry and triggered my enthusiasm to explore the nature. I would like to give special thanks to my beloved wife Chen Yanlin for her love and support during these four years. Last but not least, I would like to express my acknowledgement to National University of Singapore for offering the scholarship to support my study. iii List of Publications 1. Paul Kumar Upputuri, Jian Lin, Li Gong, Xiang-Yang Liu, Haifeng Wang, and Zhiwei Huang Circularly polarized coherent anti-Stokes Raman scattering microscopy. OPTICS LETTERS, Vol. 38, No. 8, April 15, 2013 2. Gangqin Xu, Li Gong, Zhen Yang, and X.Y. Liu Helically Twisted Nano Fibrils of Silk Fibers: from Nano Organization to Performance (Accepted by Soft Matter, G. Xu and L. Gong have equal contributions to this work) 3. Li Gong, Zhen Yang, and X. Y. Liu Electrostatic confined One Dimensional Growth of Nano-Fibrils in Fibroin Networks (To be submitted) 4. Gangqin Xu, Li Gong, and X.Y. Liu Intra molecular -sheet Enhanced Elastic Recovery in Silkworm and Spider Silks (To be submitted, G. Xu and L. Gong have equal contributions to this work) 5. Zhen Yang, Li Gong, Xiang Yang Liu Nucleation Controlled Fiber Spinning (To be submitted) iv Table of Contents Declaration .i Acknowledgements ii List of Publications iv Table of Contents . v Summary viii List of Tables xi List of Figures .xii List of Symbols . xvii Chapter 1: Introduction .1 1.1 General description of silkworm silk and spider silk . 1.2 Hierarchical structure of spider and silkworm silk .3 1.3 The mechanical properties of silk and previous modeling studies .6 1.4 The application of silk and the one dimensional growth of fibroin nano-fibrils . 11 1.5 The shear induced spinning process of natural silk fiber formation 13 1.5.1 Observations of silk spinning process . 13 1.5.2 In-vitro observation of the fibroin nano-fibril formation in aqueous solution under shear by small angle X-ray scattering (SAXS) . 15 1.6 Motivations and Objectives 17 1.7 Outline of the Thesis 18 Chapter 2: Breaking Mechanism of Silk Fibers by Hierarchical Modeling 20 2.1 Introduction .20 v 2.2 The Network–Non-slipping Fibril Bundle (N–NFB) model of the breakage of silk fibers 22 2.2.1 The splitting of β-crystallites . 22 2.2.2 The fracture of nano-fibrils 25 2.2.3 The eventual fracture of silk fibers 28 2.2.4 Brief summary of the N-NFB modeling 30 2.3 The results of N-NFB modeling . 31 2.4 The role of different structural parameters . 33 2.5 The synergy of the hierarchical structures via crack-stopping mechanism 39 2.6 Summary .44 Chapter 3: Electrostatic Confined One Dimensional Growth of Nano-Fibrils 46 3.1. Introduction . 46 3.2. Ionization of the fibroin molecule in aqueous solution . 49 3.3. Modelling 52 3.4. Results and discussion .56 3.5. Kinetic characterization of fibroin formation 60 3.6. Summary 64 Chapter 4: The Mechanism of Shear Induced Fibroin Nano-fibril Formation in Aqueous Solution 65 4.1 Introduction: . 65 4.2 Phenomenological study of shear induced fibril formation . 66 4.3 Shear induced fluctuation enhancement (SIFE) in triggering the phase transition . 68 4.3.1 Theory of SIFE in polymer solution 69 4.3.2 The estimation of the SIFE in fibroin solution 70 4.3.3 The simulation of the SIFE and potential experimental verification method 75 4.4 SAXS analysis of fibroin solution under shear by synchrotron radiation 78 4.4.1 Experimental schedule .78 4.4.2 Results and discussion . 80 vi 4.5 Summary .82 Chapter 5: Conclusions and Outlook . 84 5.1 Conclusions .84 5.2 Outlook . 87 Reference .89 vii Summary Spider and silkworm silks attract more and more attention from multiple disciplines, including physics, biology, chemistry, material science and engineering due to the exceptional mechanical properties, biocompatibility and environment-friendly nature. However, at the biophysical point of view, the structure-property relation, the self-assemble of fibroin molecules and the formation mechanism of silk fiber are still unclear. In this thesis, a Network – Non-slipping Fibril Bundle (N-NFB) model is put forward to examine one of the most challenging issues, the critical mechanical behavior of spider and silkworm silk fibers at the breakage point. At the nano-micro level, the silk fibers consist of a bundle of nano-fibrils with strong friction among them. At the nano-fibril level, -crystallites together and silk molecular chains constitute the molecular networks. According to the model, a better alignment of nano -crystallites, a larger number of -crystallites at one cross section of a nano-fibril and a smaller effective loading area of a peptide chain in -crystallites will eventually give rise to stronger silk fibers, in excellent agreement with our observations of both spider dragline and silkworm silks. Furthermore, the non-slipping fibrous contact of nano fibrils among the bundles serves as the crack-stopper, which restricts the propagation of cracks and significantly reinforces the silk fibers. The synergy between the molecular networks and the fibril bundle gives rise to the unusual mechanical performance of the silk fibers. The knowledge viii obtained will shed light on how to obtain ultra strong fibrous materials from the point of view of structures. In order to explain the formation of fibroin nano-fibrils, a model based on the electrostatic confinement is put forward to describe the one-dimensional growth of silk fibroin nano-fibrils formed in aqueous solutions. The electric fields generated by the silk fibroin nano-fibrils are much greater than thermal fluctuation. It follows that the local electric field is sufficient to cause the accumulation of fibroin molecules predominantly at the ends of the fibrils. Therefore, the electrostatic repulsion is able to confine the growth of fibrils in one dimension. The results acquired in this research may provide the guidelines for the control of the fibroin networks formation and even the silk fibers formation. Shear induced fluctuation enhancement (SIFE) effect is proposed to explain the shear induced fibril formation. The essence of the SIFE effect is the competition between the effect of shear and the thermal motion induced diffusion. The effect of shear is estimated by characterizing the rheological properties of fibroin solution with different concentrations. The diffusion is estimated by two different methods which are in agreement with each other. It is found that the effect of shear is slightly larger than diffusion, indicating that the shear flow may be strong enough to trigger SIFE effect. Based on these estimations, simulations are carried out to study the concentration fluctuation under shear. It found that a periodic structure of concentration fluctuation will ix solution under shear by small angle X-ray scattering (SAXS). I performed the SAXS experiments in SAXS station, Shanghai Synchrotron Radiation Facility. The wavelength of the X-ray is 0.124nm, with a beam size of 2mm×1mm. The shear device (Figure 4.6.a) is made by Kapton film which is transparent to X-ray. The inner part (diameter: 15mm) of the device can rotate, which is driven by a motor. The outer part (diameter: 21mm) is fixed on an iron base. The regenerated silk fibroin (RSF) solution (0.8%) is injected between the inner rotator and the outer cylinder (cf. Sub-section 4.3.2 for the preparation method of RSF solution). Figure 4.6 The shear device (a) and the scattering geometries (b). In this study, two different scattering geometries are applied (Figure 4.6.b). The left one is radial incident, the right one is tangential incident. 4.4.2 Results and discussion The scattering patterns of fibroin solution under different shear rates with 79 both radial incident and tangential incident are plotted in Figure 4.7. For radial incident, from the intensity profiles, it can be found that the intensity decreases while the shear rate increases (Figure 4.8.a). While the shear rate was larger than 35s-1, the scattering intensity would be too low to be detected. The five curves of tangential incident are almost overlapped with each other (Figure 4.8.b), so that the shear rate can reach the maximum of the device. The bursts in the horizontal direction were caused by some slit pairs in the X-ray path. They were not the signal from the fibroin solution. Figure 4.7 The scattering pattern of fibroin solution under different shear rates with both radial incident (a~c) and tangential incident (d~h). 80 Figure 4.8 The intensity profiles of fibroin solution under different shear rates with both radial incident (a) and tangential incident (b). When analyzing the results, to avoid the bursts, only the data from the azimuth 30~60º was selected. By Guinier plot [97], the radius of gyration can be calculated from the slope of the linear fitting showing in Figure 4.9.a~b, since ln I ( q )  ln I (0)  2 q R g . The calculated Rg is plotted in Figure 4.9.c. The results are quite close to those in Rössle et al’s experiments (5~7nm), although the range of shear rate in this experiment is much smaller [56]. In this case, the radius of gyration does not show any changes with the increase of the shear rate. Normally, the high q tail (q×Rg>1) can be expressed by power law: I (q)  q p . The exponent p is determined by the interface between the protein and the solvent. Larger p means the interface is sharper [97]. In this study, the exponent p slightly decreases while increasing the shear rate, means that the interface becomes more blurred when shear rate increasing (Figure 4.9.d). It may indicate that the protein is slightly unfolded under shear. Based on these studies, the effect of shear on individual fibroin molecule is 81 not obvious. It indicates that SIFE may play the major role in triggering the fibril formation under shear. Figure 4.9 The Guinier plot of both (a) radial incident and (b) tangential incident, as well as (c) the calculated Rg. And (d) the high q tail (q×Rg>>1) fitted by power law of both radial incident and tangential incident. 4.5 Summary The shear induced fluctuation enhancement (SIFE) effect was proposed to explain the shear induced fibril formation. The essence of the SIFE effect is the competition between shear energy S diffusion  /  and the free energy induced    2F S /  was estimated by characterizing the rheological .   82 properties of fibroin solution with different concentrations. estimated by two different methods. It was found that S  2F  was  /  is slightly   2F larger than , indicating that the shear energy may be large enough to  trigger SIFE effect. Thus simulations were carried out to study the SIFE effect in fibroin solution. It showed that a periodic structure of the enhanced fluctuation appeared in the solution with a spatial period at the order of 101m, thus light scattering with an infra-red laser as the light source can be a proper experimental schedule to examine the SIFE effect. In situ SAXS experiments were carried out to study the behavior of individual fibroin molecule in solution under shear. The radius of gyration and exponent of the high q tail of intensity profiles were analyzed as functions of shear rate and scattering geometry. These two quantities show few changes under shear. It implies that effect of shear flow on individual molecule is not obvious, so that the SIFE effect may play the major role in triggering the fibril formation under shear. 83 Chapter Conclusions and Outlook 5.1 Conclusions The aim of this thesis was to reveal some biophysical problems related to the formation, properties and applications of silkworm and spider silks. In Chapter 2, the N-NFB model based on the nano-molecular networking and twisted nano-fibril bundles was presented to describe the panorama of the hierarchical breakage of silk fibers. The breakage of the fiber occurs from molecular scale to micro scale: at molecular scale, the breaking is initiated by the splitting of β-crystallites; at the nano scale, a silk fibril breaks once the nano-molecular network fractures, and the breaking of a fibril forms a nano crack in the fibril bundle; at fibril bundle scale, silk fiber breaks only if enough cracks accumulate in one cross section of the fibril bundle. Based on the quantitative analysis by N-NFB model, the breaking stresses of spider silk 84 dragline fibers at different reeling rates were predicted, which are in excellent agreement with our measured ones. To the best of our knowledge, it was the first precise prediction of the breaking stress of silk fibers from their measurable structural parameters. This prediction verified the correctness of this model. In addition, the influence of the structural features on the breakage was discussed in detail. Furthermore, the twisted fibril bundle structure was found to have a crack-stopping property in blocking the propagation of cracks in both the transverse and the longitudinal directions. It suggests that the non-slipping fibril bundle has a structural advantage which can substantially enhance the strength of silk fibers. This study not only revealed the breaking mechanism and the exceptional mechanical properties of silks, but the physical idea of the crack stopping mechanism and the synergy of the hierarchical structures might also provide a guide in developing novel techniques for the enhancement and design of the fibers in general. In Chapter 3, the significance of the electrostatic repulsion between fibril and the native fibroin molecules was explored. While pH=7, the electric potential energy of a single fibroin molecule was estimated to be twice larger than kBT. This estimation indicated that the electrostatic energy is strong enough to affect the Boltzmann distribution of the fibroin. Hence, the electrostatic repulsion is able to influence the shape of the aggregation. From the calculations carried out in this study, it was discovered that the electrostatic repulsion effect is a sufficient condition to confine the growth of 85 the fibril in one dimension, by keeping the growth rate at the end of fibril much larger than that at the middle. In this study, the ionization equilibrium of fibroin in aqueous solution and the screening effect of ions and water molecules were considered in detail. In general, this study not only revealed the mechanism of the one dimensional growth of fibroin fibril, but is also important in understanding the aggregation mechanism of other charged particles. In Chapter 4, the SIFE effect was proposed to explain the shear induced fibril formation. The essence of the SIFE effect is the competition between shear energy S S  /   2F and the thermal motion induced diffusion .   /  was estimated by characterizing the rheological properties of fibroin   2F solution with different concentrations. was estimated by two different  methods which are in agreement with each other. It was found that S is slightly larger than  /   2F , indicating that the shear energy may be large  enough to trigger SIFE effect. Based on these estimations, simulations were carried out to study the concentration fluctuation under shear. It found that a periodic structure of concentration fluctuation would appear, and this can be monitored by light scattering. In addition, SAXS experiments showed that the influence of shear on individual fibroin molecule is not obvious. 86 5.2 Outlook Based on my research, there are still several open areas worthwhile for further studies. 1. In Chapter 2, the friction is assumed to be infinite and any local slipping between fibrils can be prevented in N-NFB model. To further study the role of friction between fibrils, a partial slipping fibril bundle model is needed, in which the friction can be tuned from zero to infinity. 2. In Chapter 2, the splitting force of a β-crystallites Fβ(θ) is an immeasurable parameter. In this study, it was fitted by the breaking stress of silkworm silk collected at different reeling speeds. In future, this fitted result may need verification from other approaches. 3. In Chapter 3, it should be noted that the Stern layer which was caused by the Van der Waals adsorption of the charges was not considered independently in this study. This effect was considered as a part of the ionization of the protein. If referring to another material with strong Van de Waals adsorption, the Stern layer should be considered independently. 4. In Chapter 3, the electric field around a fibril can redistribute the charged fibroin molecules; and the redistribution can conversely affect the electric field. This effect was not included in this research, since what we studied was dilute fibroin solution, in which the charge density was low. For concentrated fibroin solution in silk spinning process, this effect is not neglectable. 5. In Chapter 4, there is only one order parameter included in SIFE theory, 87 the concentration of the solute. Actually, this theory can be modified by adding other parameters such as the orientation of the dissolved molecules, shape factors, etc. to describe the behavior of more complicated fluid under shear. 88 Reference: 1. Vollrath, F. and D.P. Knight, Liquid crystalline spinning of spider silk. Nature, 2001. 410(6828): p. 541-548. 2. Shao, Z. and F. Vollrath, Surprising strength of silkworm silk. Nature, 2002. 418(6899): p. 741-741. 3. Omenetto, F.G. and D.L. KapLan, A new route for silk. 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Uzunov, D.I., Introduction to the theory of critical phenomena : mean field, fluctuations and renormalization1993: World Scientific. 94 [...]... silk fiber formation In the process of animal silk fiber formation, a phase transition of the spinning dope happens in a very fast manner at room temperature and atmospheric pressure [1, 56] In contrast with the artificial fiber in petroleum industry, the formation of natural silks is less energy consumable and more environmental friendly Hence, the spinning process of silkworm silks and spider silks attracts... diamonds indicate the f, nβ, A values from silkworm and spider silks, respectively The influence of three structural parameters (d)f, (e)nβ, (f)A on the breaking stress of silk fibers The solid and hollow circles indicate the f, nβ , A values from silkworm and spider silks, respectively .39 Figure 2.9 Fracture of (a) the Bulk Network (BN) model and (b) the Slippery Fibril Bundle (SFB) model... =0.938nm, b =0.949nm, c =0.698nm for silkworm B mori silks [15], and ˆ ˆ ˆ a =1.03nm, b =0.944nm, c =0.695nm for spider Nephila dragline silks [16], The β-structure sequence is GAGAGSGAAS(GAGAGS)n, n=1~11 for B mori silkworm silks and GAGA(A)n, n=4~6 for N pilipes spider silks (G: Glycine, A: Alanine, S:Serine) (Figure 1.1.e) X-ray diffraction (XRD) [17, 18] and Fourier transform infrared spectroscopy (FTIR)... molecules in the β-conformations (including both intra  -sheets and nano  -crystallites) [14] remain to be 46% for spider silks and 48% for silkworm silks, even at different reeling/spinning speeds Due to the fact that there is much difference in  -crystallinity (cf the results above), it can be expected more intra-molecular β-sheets in spider silks (21%) than in silkworm silks (7%) [14] 1.3 The... model 31 Figure 2.5 Stress-strain curves of (a) the silkworm silks and (c) the spider silks reeled at the indicated speeds in a controlled and steady manner (b) Comparison between the measured breaking stresses with the fitted ones of the silkworm silks, and (d) comparison between the measured breaking stresses with the predicted ones of spider silks based on the N-NFB model 33 Figure 2.6 p(σs) for different... (AFM) results revealed that both spider dragline silk and silkworm silk are 4 composed of numerous nano-fibrils of a diameter around 30nm for Bombyx mori silkworm silks and around 35nm for Nephila pilipes spider dragline silks [14] Thus it can be estimated that the number of nano-fibrils across a spider silk fiber and a silkworm fiber are~1.1×104, and ~5.6×104, respectively Both are in the same order... performance of silks is caused by the internal structure evolution [14, 23] The typical breaking stress is 1200MPa and 650MPa for spider dragline silk and silkworm silk reeled at their natural reeling speed, respectively Figure 1.2 Representative stress-strain curves of Nephila pilipes spider dragline silks (green curve: by forced silking at natural reeling speed 10 mms-1) and Bombyx mori silkworm silks (blue... resist deformation and failure (Reproduced from [30]) (c) The rough surface of fibril was demonstrated to be critical to prevent the slipping between the fibrils (Reproduced from [31]) Studies by Analytical Theory: Considering the composition rather than the exact structures, Porter et al analyzed the breaking mechanism by analyzing the energy dissipation and predicted the stress-strain curve and the fracture. .. will branch and entangle with each other to form nano-fibrous networks [54, 55] In some cases, the fibril formation should be avoided, such as in transparent fibroin film; in other cases, the fibril formation should be utilized, such as in hydrogel In this regard, to understand the mechanism controlling the growth 12 of fibroin nano-fibrils is a relevant step in controlling fibrous materials formation. .. spectroscopy (FTIR) [19] were applied to determine the secondary structures of silks Typically, the content of  -crystallites (crystallinity c%) is about 25% for spider draglines and 41% for silkworm silks, respectively The crystallite sizes along a, b, c directions La×Lb×Lc are around 2×3×6 nm for spider silks and 2×3×11nm for silkworm 5 silks The ordering of the  -crystallites plays a key role in the mechanical . ANIMAL SILKS: FRACTURE MECHANISM AND FORMATION MECHANISM Gong Li (B. Sc, Sichuan University) A THESIS SUBMITTED FOR THE. silkworm silks and (c) the spider silks reeled at the indicated speeds in a controlled and steady manner. (b) Comparison between the measured breaking stresses with the fitted ones of the silkworm silks, . fibers. The solid and hollow circles indicate the f, n β , A values from silkworm and spider silks, respectively. 39 Figure 2.9 Fracture of (a) the Bulk Network (BN) model and (b) the Slippery Fibril