Investigation of Rheological Properties and Conformation of Silk

Mar 29, 2012 - ... at high shear rates showed behavior similar to that in native spinning, ... Binjia Zhang , Fengwei Xie , Julia L. Shamshina , Robin...
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Investigation of Rheological Properties and Conformation of Silk Fibroin in the Solution of AmimCl Qin Wang,† Yuhong Yang,*,‡ Xin Chen,† and Zhengzhong Shao*,† †

State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Laboratory of Advanced Materials, Fudan University, Shanghai 200433, People's Republic of China ‡ Research Centre for Analysis and Measurement, Fudan University, Shanghai 200433, People's Republic of China S Supporting Information *

ABSTRACT: The conformation and eventual morphology of silk fibroin (SF) chains are crucial for the mechanical properties of SF materials, and are strongly related to the solvation step as a key stage in their processing conditions. In this work, a novel SF/AmimCl (1-allyl-3-methylimidazolium chloride) solution with unique properties is reported and compared with conventional regenerated SF aqueous solutions, based on an investigation of its rheological properties. The steady shearing behavior suggested that AmimCl is a good solvent for SF molecules, and shear thinning of semidiluted SF/AmimCl solution at high shear rates showed behavior similar to that in native spinning, which is due to the rearrangement and orientation of SF molecular chains. Fitting of experimental dynamic viscoelastic data to the Rouse model provided an effective method to estimate the molecular weight of SF. We believe that this work not only provides a better understanding of the relationship between properties of silk protein and aggregation states of their molecular chains, but also provides tools to fabricate high-performance SF-based materials.

1. INTRODUCTION Silk fibroin (SF) from the Bombyx mori silkworm has potential applications in areas such as biomineralization, 1 tissue scaffolds,2 drug delivery,3 and so on, due to its unique combination of mechanical and biological properties, such as nontoxicity, biocompatibility, and biodegradability, as well as easy reconstitution.4 Besides natural silk fibers, SF materials are generally prepared from regenerated SF aqueous solutions to form films, gels, foams, and so on. Conventionally, the method to produce SF aqueous solutions is to dissolve degummed silk fibers in concentrated solutions of inorganic salts, such as saturated LiBr aqueous solution and CaCl2/ethanol/water solution5 and subsequently dialyzing in deionized water and then concentrating to the required level. However, the processes of both dialysis and concentration are time-consuming, and the solutions are unstable, with a shelf life of only around a few weeks before the protein becomes unstable and aggregates. In addition, without postdrawing, the as-spun fibers6,7 and films8 produced from these regenerated SF aqueous solutions are usually brittle. The poor mechanical properties of such SFbased materials are believed to result from not only the aggregation of SF molecules in aqueous solution,9 but also the degradation of fibroin molecules caused by the inorganic salt systems.10 These were also considered to be the reasons why rheological properties of regenerated SF solution and native SF dope are different.10 © 2012 American Chemical Society

Recently, the unique characteristics of ionic liquids (ILs), such as low melting point, extremely high vapor pressure, thermal stability, wide liquid range, high ionic conductivity, and easy renewability,11,12 have encouraged researchers to explore their application. To date, ILs have been widely applied not only in the fields of synthesis,13 catalysis,14 and electrochemistry,15,16 but also as a nonvolatile green solvent for biomacromolecules.17 After Swatloski et al. pioneered the use of IL for the preparation of cellulose solutions (up to concentration of 25% (w/w)),18 Phillips et al. found that the 1-butyl-3-methylimidazolium chloride (BmimCl) and 1-ethyl-3methylimidazolium chloride (EmimCl) could dissolve SF to high concentrations,19 which has been used to produce artificial silk fiber by wet-spinning20 and patterned silk film as scaffold.21 However, the melting points of ILs employed in these cases were relative high (65 °C for BmimCl and 89 °C for EmimCl, respectively), which might induce a greater risk of the degradation of SF molecular chains during the solvation procedure. As a result, the regenerated silk fiber spun from those solutions was quite poor in terms of the mechanical properties.20 It is widely accepted that the mechanical properties of SFbased materials are mainly determined by their molecular weight22 and morphology,8 which are closely related to the Received: March 13, 2012 Published: March 29, 2012 1875

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solution can be stored for more than 1.5 years without any apparent instability effects, such as precipitation and gelation, similar to freshly prepared solution (Figure S1a). Furthermore, the flow curves of both freshly prepared and aged solutions showed similar trends and almost overlapped with each other over the whole range of shear rates (Figure S1b). In contrast, regenerated SF aqueous solution could be stored for only a few weeks due to the tendency for instability.27,28 As found in a previous report,29 the viscosity of SF aqueous solutions would change significantly, and gelled after storing for months. This suggests that there might be no obvious change of SF structure in AmimCl during the storage of SF/AmimCl solution. Owing to its easy storage, SF/AmimCl solution was superior to regenerated SF aqueous solutions, particularly from the industrial point of view. This important property could be helpful in the scaling up of the SF solution preparation. To evaluate the excellent solubility of SF in ionic liquid as well as the long-term stability of SF/AmimCl solution, the rheological properties of SF/AmimCl solution were investigated in the details, with particular interest in the macromolecular structure in solution. 3.2. Steady Shearing Measurement. Generally, the viscosity of SF/AmimCl solution increased with the amount of SF (Figure 1). However, the dependence of viscosity on

solvation process and the conformation of SF in the solution, as well as the post-treatments. Therefore, further understanding of the rheological properties of SF/ILs solution is crucial for the processing of high performance SF-based materials. In this study, we used 1-allyl-3-methylimidazolium chloride (AmimCl) with a lower melting point of 17 °C23 as the solvent and found it was capable of dissolving SF more rapidly at a relatively low temperature. Furthermore we investigated the rheological behavior of SF/AmimCl by steady and oscillatory shear measurement, which showed differences from that of conventional regenerated SF aqueous solutions. Further information on the structure of the macromolecular chains was acquired through further analysis on rheological properties.

2. EXPERIMENTAL SECTION 2.1. Materials. The ionic liquid (IL), 1-allyl-3-methylimidazolium chloride (AmimCl), was purchased from Lanzhou Institute of Chemical Physics, China. The IL was freeze-dried to remove water before use in all experiments. Bombyx mori cocoon silk was degummed twice with boiling in 0.5% Na2CO3 solution for 20 min and then rinsed with abundant distilled water to remove the sericin.24 The degummed silk fiber was dried at 30 °C in vacuum prior to use. 2.2. Sample Preparation. To prepare SF/AmimCl solution, the degummed silk fiber was mixed with AmimCl in a 25 mL conical flask and was stirred at 90 °C for 1.5 h to ensure the complete dissolution, during which gas bubbles were removed from the solutions by vacuum. The transparent SF/AmimCl solutions with various concentrations from 1 to 15 wt % (adjusted by the proportions of silk fiber and IL employed) were hermetically stored at room temperature and protected against moisture absorption. 2.3. Rheological Measurement. Rheological analysis was carried on a stress-controlled rheometer, Physical MCR-301 (Anton Paar Co.Inc.), protected with N2 through a H-PTD200 hood with peltier heating/cooling. A thin layer of low-viscosity silicon (η30 °C = 10 mPa·s) was placed around the borders of the measuring cell to prevent an uptake of moisture. For all the measurements, a parallel plate measuring geometry with 25 mm diameter was used. Shear viscosity η(γ̇) was measured with as a function of shear rate γ̇ in the range of 10−2 to 103 s−1 for the SF/AmimCl solutions with concentration lower than 6 wt % (C < 6 wt %) and 10−3 to 103 s−1 for the solutions with higher concentration (C > 6 wt %). In the oscillatory shear measurements, the storage and loss moduli, G′(ω) and G″(ω), were measured under a strain-control model as a function of frequency ω in the range of 1 × 102 to 6.81 × 10−2 rad/s for all SF/AmimCl solutions at 30 °C. The data for time−temperature superposition (TTS) of SF/ AmimCl solution (C = 15 wt %) was achieved at four temperatures (0, 10, 20, and 30 °C, respectively). Though N2 protection and a thin layer of low-viscosity silicon were used to prevent moisture absorption, we still need control every measurement to be less than 1.5 h at room temperature (around 20 °C) and 40% relative humidity.

Figure 1. Flow curves for pure AmimCl and SF/AmimCl solution with various concentration of SF at 30 °C.

shear rate, that is, lower shear thinning (at lower shear rate), Newtonian flow (at intermediate shear rate), and greater shear thinning (at higher shear rate) exhibited different trends in the range of concentrations investigated. Flow curves of SF/ AmimCl solution with low concentration showed lower shear thinning, followed by Newtonian flow in the subsequent shear rate range, which was similar to that of pure AmimCl solution. As the concentration of SF increased, the region of lower shear thinning became less evident or even disappeared, meanwhile a higher rate shear thinning region emerged. It was apparent that pseudoplastic flow occurred for SF/AmimCl solutions with high concentrations, that was the Newtonian region, followed by the higher rate shear thinning region, which was similar to that of typical polymer solutions.30 3.2.1. Concentration Dependence of Viscosity, Intrinsic Viscosity, and Huggins Constant of SF/AmimCl Solution. The effect of concentration on the viscosity in the Newtonian region was further investigated and shown in Figure 2. It was noticeable that zero shear viscosity, η0, showed a power-law dependence on concentration with the equation as follows:

3. RESULTS AND DISCUSSION 3.1. Preparation and Storage of SF/AmimCl Solution. Although BmimCl has been widely used as a solvent for biomacromolecules,18,19,25,26 the long dissolving time at high temperature limited its further application due to the disadvantages mentioned in the Introduction. In this study, AmimCl was selected to dissolve silk fiber, and the solubility of SF in AmimCl and BmimCl were compared. A 15 wt % SF solution was achieved after dissolving in AmimCl at 90 °C for 1.5 h, whereas only a 12 wt % SF solution was obtained in BmimCl at 100 °C for 4 h (the details are shown in Table S1). Evidently, AmimCl exhibited a stronger solvation capability for SF molecules, perhaps due to its lower melting point. Interestingly, if sealed at room temperature, SF/AmimCl 1876

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Biomacromolecules η0 ∝ C m

Article

(1)

Figure 3. Concentration dependence of ηsp/C for SF/AmimCl solutions at 30 °C.

The Huggins approach34,35 based on a general formula correlating specific and intrinsic viscosities provided the information on both intrinsic viscosity and the Huggins constant. It allowed the use of viscosity over a large range of polymer concentrations, both in the dilute and semidilute states. Here a truncated version of the general Huggins equation, defined in eq 4,29 was applied.

Figure 2. Dependence of Newtonian viscosity on concentration for SF/AmimCl solutions at 30 °C.

In the double logarithmic coordinates, the dependence of η0 on concentration showed two different trends with slopes (m) of 1.1 and 3.8, belonging to dilute and semidilute regions, respectively, according to Doi−Edwards theory.31 Meanwhile, the critical concentration C* was identified as about 5 wt %. Due to a common relationship of the concentration dependence on different states of macromolecules in solution,32 the possible conformations of SF molecular chain were summarized schematically in the inset of Figure 2. In the dilute solution region (C < C*), the protein exists mainly as individual chains (inset a of Figure 2) and the excluded volume between chains keeps them apart from each other and makes them adopt a somewhat expanded selfavoiding walk conformation.33 As the concentration of SF is raised, the conformations of individual chains start to contact each other at the critical overlap concentration C* (inset b of Figure 2). Further increasing the SF concentration to a semidilute solution (C > C*), association occurred among the crowed molecules chains, making the chains behave as though it were in a polymer melt (inset c of Figure 2).32 The intrinsic viscosity was correlated with the hydrodynamic volume of molecular solute, thus reflecting the salvation quality of solvent from a thermodynamic point of view. Intrinsic viscosity, [η], was determined by the conventional method, as given in eqs 2 and 3.

ηsp = (η − ηs)/ηs

(2)

[η] = lim(ηsp /C)C → 0

(3)

ηsp = C[η] + KH(C[η])2 + A(C[η])h

(4)

where KH is the Huggins constant. Figure 4 showed the correlation between ηsp and C[η], where [η] = 880 mL/g. In the double logarithmic coordinate, the plot

Figure 4. Specific viscosity, as a function of C[η] for SF/AmimCl solution at 30 °C.

of ηsp against C[η] resulted in a linear relationship in the range of larger C[η]. From this linear regression, the parameter, h, was obtained as about 4.1. Then, through a polynomial fitting of ηsp versus C[η], the Huggins constant KH was estimated as about 0.1. Although quantitative analysis and discussion of the Huggins constant are not important here since the thermodynamic meaning of its absolute value remains rather ambiguous,36 it was empirically used as a criterion for judging the solvation ability of a solvent. Generally, KH is sensitive to chain aggregation and the association among polymer chains always results in high KH.37 It is widely accepted that a solvent with KH less than 0.5 (to a solute) could dissolved such specific substance well, therefore, AmimCl was judged here to be a good solvent for silk fibroin. In other words, the silk fibroin molecular chains were expected to exist freely in AmimCl solution rather than aggregate as they do in pure water.9,38 The aggregations of SF in water was demonstrated by AFM observations shown in Figure S2. Many clusters with the diameter around several tens of nanometers were observed,

where ηs and η are the viscosities of the pure solvent and the solution, respectively, C is the concentration, and ηsp is the specific viscosity of the solution, which allows exclusion of the solvent contribution to the viscosity of solutions. The relationship between ηsp/C and C for SF/AmimCl solutions was presented in Figure 3. The inset of Figure 3 showed data for dilute solutions, from which the intrinsic viscosity was determined to be around 880 mL/g as the extrapolated value of ηsp/C as the silk concentration approaches zero. It should be noted that further discussion of the intrinsic viscosity would be meaningful here only if a direct comparison of intrinsic viscosities was made for the same SF solute dissolving in different solvents. 1877

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probably due to interaction among the hydrophobic blocks of silk molecules.39 In AmimCl, an increasing number of freely expanding silk chains could overlap and associate with each other with increases in the concentration of SF solution. This could explain that the relationship between η0 and C in our work was similar to the classic power-law rule as discussed above (Figure 2). In recent years, great advances have been made in the study of structure−property relationships of ILs.40,41 IL has a unique structure, especially in its cationic part. A common cationic structure is composed of two parts, which are a large imidazolium ring with positive charge as head and a nonpolar long alkane chain as tail. Such structure endows IL with similar properties to a surfactant. Thus, some applications have been explored in fields such as controlled release42 and emulsion polymerization,43 where the nonpolar tail of an IL was found to interact with either apolar molecules or apolar parts of a molecule through solvatophobic interaction. This interaction can prevent apolar molecules from aggregation. Similarly, the unique structure of IL molecules may be responsible for its good solvency for SF. The nonpolar alkane chain of imidazolium cation would interact with the hydrophobic blocks of SF molecules (highly preserved GAGAGS or GAGAGAGS sequences), while the polar imidazolium ring was exposed on the outside to decrease the possibility of interaction among hydrophobic blocks, aroused by either electrostatic repulsion or steric hindrance of adjacent imidazolium rings. Therefore, the tendency to form β-sheet structure was highly inhibited, and the SF/AmimCl solution could be stably stocked for much longer than SF aqueous solutions. 3.2.2. Shear Rate Dependence of η in SF/AmimCl Solution. Low and high rate shear-thinning regions of SF/AmimCl solutions were shown in Figure 1, corresponding to the dilute and semidilute solutions, respectively. For the shear thinning behavior, the shear viscosity changed according to a power law variation, η = K(γ̇)n, where the exponent n is the slope of the shear thinning in double logarithmic coordinates, reflecting the degree of the structure’s sensitivity to shearing. The scaling values of n were summarized in Figure 5 for different concentrations of SF/AmimCl solution. It could be seen that n decreased gradually with the SF concentration and even disappeared subsequently in the low shear rate region; contrarily, n increased with the SF concentration in high shear rate region.

Shear-thinning behavior at low shear rate, so-called yielding, resulted from the gradual breaking of weak physical networks by small shear stress,44 and the existence of the hydrogenbonded network in dialkylimidazolium halide has been reported previously.45 It could be speculated that such yielding behavior only existing in dilute SF/AmimCl solutions was mainly induced by those “free” IL molecules without interactions with the silk fibroin chains. The hydrogen-bonded network formed by those ILs gradually disappeared with the increase of SF because of the reduction of “free” IL. Therefore, the shear thinning at low shear rates resulted from the contribution of solvent. In the high shear rate region, the shear-thinning which could be found in many classic polymer melt or solution was considered to be induced by the rearrangement and orientation of macromolecular chains along the shear direction.30 The exponent n is strongly influenced by intermolecular interactions, either among chains or solvent-to-chain interactions.46 In our case, the variation of n for semidilute solutions was probably due to the interaction between SF chains. As mentioned above, freely expanded SF chains tended to overlap and associated with each other with the increase of SF in good solvents, leading to the increase in sensitivity to shearing. In addition, another parameter γ̇C, the critical shear rate from the Newtonian to the power law regions, was also shown in the inset of Figure 5 and shows that γ̇C moved to the lower shear rates region as the concentration of SF solution increased, similar to the performance of classic polymer solutions.35 This implied that the shear-sensitivity of structure was enhanced by the increase of SF, coincident with the results shown for the changes in n. In summary, the rearrangement and orientation of SF chains became easier with increases in SF concentration in AmimCl solution. 3.2.3. Schematic for SF Structure in Solutions. Based on the observations presented above in the context of the classic rheological behavior of polymer melts and solutions10,30,35,47 and in comparison with the behavior of regenerated SF aqueous solutions, a schematic illustration was shown in Figure 6 for a proposal about the molecular chain structure of SF in AmimCl solution at different deformation rates. Numerous rheological studies of regenerated SF aqueous solutions have been done previously.10,24,29,48 Those solutions almost always behaved like Newtonian fluids in all shear regions, except for a slight shear thinning at low shear rates,

Figure 5. Scaling exponent, n, varied with the concentration of SF at 30 °C. The inset displayed the critical shear rate decreasing with increasing concentration.

Figure 6. Possible mechanism of shear thinning (a) regenerated SF aqueous solutions; (b) diluted SF/AmimCl solutions; (c) semidiluted SF/AmimCl solutions. 1878

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which was commonly interpreted as the alignment of SF molecules.29 According to the classic rheology of polymer solution, the alignment of molecules should be reflected by the shear thinning at higher shear rate.30 However, this rheological behavior has not yet been reported in regenerated SF aqueous solutions even if the concentration of SF reached to 18.5%.10 As mentioned before, the shear thinning at low shear rate is generally ascribed to the destruction of a weak physical network.44 Thus, a possible structure of regenerated SF in aqueous solution under zero shear is proposed in Figure 6a. The solute molecules tend to aggregate into nanoscale clusters by hydrophobic interaction, due to the amphiphilic structure of SF macromolecules, and the nanoscale clusters linked together through hydrogen bonds between clusters, forming a weak physical network in the static state. When the shear stress is imposed and further increased, the physical network among clusters is gradually perturbed and destrupted, and the clusters separated into free units. However, it is unlikely that significant dissociation of the clusters is compatible the rearrangement and orientation of silk molecular chains. Obviously, this sheds the light on why the wet-spinning of as-spun fiber from regenerated SF aqueous solutions is always brittle, and postdrawing is necessary to induce crystallite alignment for the enhancement of toughness.6 In contrast to aqueous solutions, AmimCl has been shown here to be a good solvent for SF, in which silk molecules act as random walk chains. For dilute SF/AmimCl solutions (Figure 6b), the remaining “free” AmimCl molecules form physical network by hydrogen bonding between themselves, as discussed above. When the shear stress is gradually increased to the yield stress, this network is heavily disrupted and a yield behavior is observed, namely, shear thinning followed by Newtonian flow. When the concentration of SF is further increased into the semidilute region (Figure 6c), much more AmimCl molecules interact with silk fibroin, resulting in the disappearance of the physical network among AmimCl molecules. Moreover, silk fibroin molecular chains tend to overlap and associate with each other in semidilute solution. Consequently, the whole solution moves together along the shear direction as a Newtonian fluid in the low shear rate region, while the macromolecules undergo a realignment process at high shear rates to reduce the intermolecular friction to move easily along the shear direction, reflected by the appearance of a power-law shear thinning at high shear rates. Interestingly, the orientation of SF molecules in AmimCl solution is quite similar to that in native Bombyx mori dope,10,49−51 which encourages the application of SF/AmimCl solutions, especially for spinning. 3.3. Viscoelastic Properties of SF/AmimCl Solution. The angular frequency dependences of storage (G′) and loss moduli (G″) for SF/AmimCl solutions varied with the concentrations, as shown in Figure 7a. For lower concentrations, G′ was almost independent of ω initially and a plateau was observed, subsequently followed by a typical plastic flow behavior, which was characterized by the increase of G′ and G″ in line with ω on logarithmic scales with slopes of 2 and 1, respectively. For higher concentrations (over 7 wt %), the plateau of G′ at low frequencies gradually disappeared with increases in concentration, while G′ and G″ did not simply increase with ω, but converged at high frequencies. Furthermore, it was found that G′ and G″ for the 15 wt % solution tended to cross at high frequencies, similar to the dynamic viscoelastic behavior of native silk dope.10,49

Figure 7. Storage, G′, and loss modulus, G″, as a function of frequency, increasing with concentration for SF/AmimCl solutions (a) and pure AmimCl (b) at 30 °C.

As shown in Figure 7b, the plateau of G′ at lower frequencies could also be found in pure AmimCl solution (not observed by Kuang, probably due to the limitation of their rheometer sensitivity52). Existence of a weak network in the solution53 could be inferred from this plateau, which was in good agreement with the discussion above from the data from steady shear measurements. It could also be concluded that such a weak network was formed by hydrogen bonds of the “free” AmimCl molecules in dilute solutions. The time−temperature superposition (TTS) principle was introduced into our study to overcome the limitations of the rheometer of measuring dynamic information at high frequencies (ω larger than 100 rad/s). The master curves of storage and loss modulus at various temperatures (with reference temperature Tf = 30 °C) for 15% SF/AmimCl solution are shown in Figure 8. To estimate the feasibility of TTS, the curve of horizontal shift factor (aT) versus the inverse of temperature is calculated, as displayed in the inset of Figure 8. The linear form of the plot indicated that the temperaturedependence of aT could be described by an Arrhenius-type equation. Therefore, the linear relationship of lnaT against 1/T and the overlapping of the master curve proved that TTS hold well for the viscoelastic data of the SF/AmimCl solution, and the homogeneity of the solution under our experimental conditions was also indicated. aT =

η(T )T0 η(T0)T

(5)

As the master curve in Figure 8 shows, on logarithmic scales, G′ and G″ increased with frequency with the curve slopes of 1.8 and 1, respectively, and then merged into one curve with the 1879

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years), more simple sample preparation process, and the steady shearing behavior and dynamic viscoelastic behavior of the semidilute SF/AmimCl solution were more like those of native dope. The Huggins constant, KH, of SF in AmimCl was estimated to be far less than 0.5, indicating that AmimCl is a good solvent for silk fibroin. Therefore, the semidilute solution state of the SF/AmimCl enable us to use the Rouse model to describe its dynamic viscoelastic behavior, which also provide an effective method to estimate the molecular weight of SF. Furthermore, the alignment and orientation of SF molecular chains in AmimCl solvent at the high shear rates were also similar to that in native silk solutions. We not only fabricated a SF/IL system closer to native dope, compared with aqueous solution, but further demonstrated the differences between these two. We believe that this work will make it possible to prepare high-performance SF base materials in the future.

Figure 8. Time-temperature superposition for 15 wt % SF/AmimCl solution (Tf = 30 °C). The inset shows the shift factor (aT) with different temperatures.

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ASSOCIATED CONTENT

* Supporting Information S

Regenerated SF aqueous solution, flow curves of both fresh and aged SF/AmimCl solutions, and tapping AFM image for regenerated SF aqueous solution. This material is available free of charge via the Internet at http://pubs.acs.org.

slope of 0.5 at high frequencies, behaving like the Rouse model. Because the Rouse model describes the dynamic rheological behavior of a macromolecular solute in good or θ solvents,54,55 it could also be inferred that AmimCl solution was a good solvent for SF. Moreover, this angular frequency dependence of modulus proved that 15 wt % SF/AmimCl solution was still in a semidilute unentangled concentration regime. It should be noticed that this viscoelastic behavior was different from that of native Bombyx mori dope,46,51 which showed an obvious crossover between G′ and G″ and a pseudoplateau modulus at high angular frequency, like the typical entangled polymer solutions or melts.32 The breakage of molecular chains during the degumming in Na2CO3 solution and solvation in inorganic salt solution, as well as the inadequate concentration may contribute to this unentangled state of SF in AmimCl. According to eqs 6−8 for the Rouse model, listed below, there is a specific relationship between molecular weight (Mw) and modulus. Thus, it provided a means to calculate molecular weight of a given solution by fitting our experimental viscoelastic data to Rouse model.

p=1

1 + ω 2τp2

N

G″ = (ρRT /M ) ∑ p=1

*E-mail: [email protected]; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundations of China (NSFC 21034003 and NSFC 20874018) and 973 Project of Chinese Ministry of Science and Technology (No. 2011CB605700), as well as State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, DongHua University. The authors additionally thank Dr. Zuguang Gong and Mr. Tao Xu at Fudan University for their valuable suggestions and discussions.

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(6)

τp = 6η0M /(π 2p2 RT )

REFERENCES

(1) Cheng, C.; Shao, Z. Z.; Vollrath, F. Adv. Funct. Mater. 2008, 18, 2172−2179. (2) Kim, H. J.; Kim, U. J.; Vunjak-Novakovic, G.; Min, B. H.; Kaplan, D. L. Biomaterials 2005, 26, 4442−4452. (3) Hofmann, S.; Foo, C.; Rossetti, F.; Textor, M.; VunjakNovakovic, G.; Kaplan, D. L.; Merkle, H. P.; Meinel, L. J. Controlled Release 2006, 111, 219−227. (4) Shao, Z. Z.; Vollrath, F. Nature 2002, 418, 741−741. (5) Chen, X.; Knight, D. P.; Shao, Z. Z.; Vollrath, F. Polymer 2001, 42, 9969−9974. (6) Zhou, G. Q.; Shao, Z. Z.; Knight, D. P.; Yan, J. P.; Chen, X. Adv. Mater. 2009, 21, 366−370. (7) Yan, J. P.; Zhou, G. Q.; Knight, D. P.; Shao, Z. Z.; Chen, X. Biomacromolecules 2010, 11, 1−5. (8) Yin, J. W.; Chen, E. Q.; Porter, D.; Shao, Z. Z. Biomacromolecules 2010, 11, 2890−2895. (9) Greving, I.; Dicko, C.; Terry, A.; Callow, P.; Vollrath, F. Soft Matter 2010, 6, 4389−4395. (10) Holland, C.; Terry, A. E.; Porter, D.; Vollrath, F. Polymer 2007, 48, 3388−3392. (11) Wilkes, J. S.; Zaworotko, M. J. J. Chem. Soc., Chem. Commun. 1992, 965−967. (12) Rogers, R. D.; Seddon, K. R. Science 2003, 302, 792−793.

ωτp 1 + ω 2τp2

AUTHOR INFORMATION

Corresponding Author

ω 2τp2

N

G′ = (ρRT /M ) ∑

■

(7) (8)

Mw of SF obtained in from our measurements was calculated to be around 144 kDa with a polydispersity index (Mw/Mn) of 1.95. The detailed investigations into the effect of degumming and solvation processes on molecular weight of silk fibroin are under study and will be reported later.

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CONCLUSIONS The instability of regenerated SF aqueous solution as well as poor mechanical properties of SF materials directly prepared from aqueous solution restricts the wider applications of SF based materials. In this work, we developed a novel SF solution, that is, SF/AmimCl solution, which showed a number of unique properties compared with regenerated SF aqueous solution, such as longer sample storage time (over one and half 1880

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(13) Welton, T. Chem. Rev. 1999, 99, 2071−2083. (14) Dupont, J.; de Souza, R. F.; Suarez, P. A. Z. Chem. Rev. 2002, 102, 3667−3691. (15) Ohno, H. Electochemical Aspects of Ionic Liquids; WileyInterscience: Hoboken, 2005. (16) Armand, M.; Endres, F.; MacFarlane, D. R.; Ohno, H.; Scrosati, B. Nat. Mater. 2009, 8, 621−629. (17) Fu, C. J.; Shao, Z. Z.; Fritz, V. Chem. Commun. 2009, 6515− 6529. (18) Swatloski, R. P.; Spear, S. K.; Holbrey, J. D.; Rogers, R. D. J. Am. Chem. Soc. 2002, 124, 4974−4975. (19) Phillips, D. M.; Drummy, L. F.; Conrady, D. G.; Fox, D. M.; Naik, R. R.; Stone, M. O.; Trulove, P. C.; De Long, H. C.; Mantz, R. A. J. Am. Chem. Soc. 2004, 126, 14350−14351. (20) Phillips, D. M.; Drummy, L. F.; Naik, R. R.; De Long, H. C.; Fox, D. M.; Trulove, P. C.; Mantz, R. A. J. Mater. Chem. 2005, 15, 4206−4208. (21) Gupta, M. K.; Khokhar, S. K.; Phillips, D. M.; Sowards, L. A.; Drummy, L. F.; Kadakia, M. P.; Naik, R. R. Langmuir 2007, 23, 1315− 1319. (22) Cao, H.; Chen, X.; Huang, L.; Shao, Z. Z. Mater. Sci. Eng., C 2009, 29, 2270−2274. (23) Zhang, H.; Wu, J.; Zhang, J.; He, J. S. Macromolecules 2005, 38, 8272−8277. (24) Chen, X.; Shao, Z. Z.; Marinkovic, N. S.; Miller, L. M.; Zhou, P.; Chance, M. R. Biophys. Chem. 2001, 89, 25−34. (25) Xie, H. B.; Li, S. H.; Zhang, S. B. Green Chem. 2005, 7, 606− 608. (26) Xie, H. B.; Zhang, S. B.; Li, S. H. Green Chem. 2006, 8, 630− 633. (27) Ayub, Z. H.; Arai, M.; Hirabayashi, K. Biosci., Biotechnol., Biochem. 1993, 57, 1910−1912. (28) Kim, U. J.; Park, J. Y.; Li, C. M.; Jin, H. J.; Valluzzi, R.; Kaplan, D. L. Biomacromolecules 2004, 5, 786−792. (29) Zainuddin; Le, T. T.; Park, Y.; Chirila, T. V.; Halley, P. J.; Whittaker, A. K. Biomaterials 2008, 29, 4268−4274. (30) Tam, K. C.; Tiu, C. J. Rheol. 1989, 33, 257−280. (31) Doi, M.; Edward, S. F. J. Chem. Soc., Faraday Trans II 1978, 74, 1818−1832. (32) Colby, R. H. Rheol. Acta 2010, 49, 425−442. (33) Rubinstein, M .; Colby, R. H. Polymer Physics; Oxford University Press: New York, 2003. (34) Huggins, M. L. J. Am. Chem. Soc. 1942, 64, 2716−2718. (35) Kulicke, W. M.; Kniewske, R. Rheol. Acta 1984, 23, 75−83. (36) Tamai, N.; Tatsumi, D.; Matsumoto, T. Biomacromolecules 2004, 5, 422−432. (37) Schoff, C. K. Polymer Handbook; 4th ed.; John Wiley & Sons: New York, 1999. (38) Hossain, K. S.; Ochi, A.; Ooyama, E.; Magoshi, J.; Nemoto, N. Biomacromolecules 2003, 4, 350−359. (39) Roseman, M. A. J. Mol. Biol. 1988, 200, 513−522. (40) Kuang, Q. L.; Zhang, J.; Wang, Z. G. J. Phys. Chem. B 2007, 111, 9858−9863. (41) Ueno, K.; Tokuda, H.; Watanabe, M. Phys. Chem. Chem. Phys. 2010, 12, 1649−1658. (42) Shen, Y. F.; Zhang, Y. J.; Kuehner, D.; Yang, G. F.; Yuan, F. Y.; Niu, L. Chemphyschem 2008, 9, 2198−2202. (43) Yan, F.; Texter, J. Chem. Commun. 2006, 2696−2698. (44) Pham, K. N.; Petekidis, G.; Vlassopoulos, D.; Egelhaaf, S. U.; Poon, W. C. K.; Pusey, P. N. J. Rheol. 2008, 52, 649−676. (45) Dong, K.; Zhang, S. J.; Wang, D. X.; Yao, X. Q. J. Phys. Chem. A 2006, 110, 9775−9782. (46) Terry, A. E.; Knight, D. P.; Porter, D.; Vollrath, F. Biomacromolecules 2004, 5, 768−772. (47) Milas, M.; Rinaudo, M.; Knipper, M.; Schuppiser, J. L. Macromolecules 1990, 23, 2506−2511. (48) Zhu, J.; Zhang, Y.; Shao, H.; Hu, X. Polymer 2008, 49, 2880− 2885.

(49) Holland, C.; Terry, A. E.; Porter, D.; Vollrath, F. Nat. Mater. 2006, 5, 870−874. (50) Kojic, N.; Bico, J.; Clasen, C.; Mckinley, G. H. J. Exp. Biol. 2006, 209 (21), 4355−4362. (51) Moriya, M.; Roschzttardtz, F.; Nakahara, Y.; Saito, H.; Masubuchi, Y.; Asakura, T. Biomacromolecules 2009, 10, 929−935. (52) Kuang, Q. L.; Zhao, J. C.; Niu, Y. H.; Zhang, J.; Wang, Z. G. J. Phys. Chem. B 2008, 112, 10234−10240. (53) Mahaut, F.; Chateau, X.; Coussot, P.; Ovarlez, G. J. Rheol. 2008, 52, 287−313. (54) Ferry, J. D. Viscoelastic Properties of Polymers, 3rd ed.; John Wiley and Sons, Inc.: New York, 1980. (55) Hair, D. W.; Amis, E. J. Macromolecules 1989, 22, 4528−4536.

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dx.doi.org/10.1021/bm300387z | Biomacromolecules 2012, 13, 1875−1881