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Fabrication and characterisation of protein fibril–elastomer composites
Acta Biomaterialia 6 (2010) 1337–1341
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Fabrication and characterisation of protein fibril–elastomer composites Tomas Oppenheim, Tuomas P.J. Knowles, Stéphanie P. Lacour, Mark E. Welland * Nanoscience Centre, University of Cambridge, J J Thomson Avenue, Cambridge CB3 0FF, UK
a r t i c l e
i n f o
Article history: Received 18 June 2009 Received in revised form 18 September 2009 Accepted 6 October 2009 Available online 14 October 2009 Keywords: Protein fibril Carbon nanotube Elastomer Rigidity Anisotropy
a b s t r a c t Protein fibrils are emerging as a novel class of functional bionanomaterials. In this paper we make use of their rigidity by combining lysozyme fibrils with a silicone elastomer and demonstrating that at a filling ratio of 10%, the protein fibril composite is at minimum 2 times stiffer than a CNT elastomeric composite of the same filling ratio. We also show that when the elastomer is patterned such that the lysozyme fibrils can be spatially modulated within the elastomer, anisotropic moduli varying by a factor of 2 is produced. By using shear mixing as the fabrication process, the modulus of a 2 wt.% insulin fibril composite is equivalent to a CNT composite with the same filling ratio. In conclusion, we have presented the fabrication and mechanical characterisation of a class of elastomer/protein fibril composite material. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Protein nanofibrils are rigid structures which can be self-assembled from a range of different peptides and proteins under mild conditions in aqueous solution [1–7]. In this paper, we present a class of material where such organic fibrils are combined with silicone elastomers to provide spatially modulated changes in material rigidity. In particular we show that at a 10% filling ratio the change in rigidity is superior to corresponding carbon nanotube composites. Furthermore we demonstrate that by controlling the spatial distribution of the fibrils within the elastomer matrix, materials with highly anisotropic elastic moduli can be produced. By incorporating nanomaterials into polymer matrices, novel multi-functional materials can be produced. One functionality of nanocomposites that is heavily examined is to improve their mechanical properties by including 1D nanomaterials into the matrix. Due to their high aspect ratio, a more efficient stress transfer should occur between the matrix and the nanofilament and hence a stiffer composite should be produced. Carbon nanotubes (CNT) are a popular class of 1D nanomaterials to include into polymer matrices due to their excellent mechanical properties. With a Young’s modulus of 1 TPa, CNT’s are the stiffest 1D nanomaterials [8]. Although CNT’s have excellent mechanical properties, their energy intensive fabrication process and their potentially high toxicity make them less attractive [9,10]. Protenacious filaments are emerging as attractive candidates for components in functional materials for several reasons. They are * Corresponding author. Tel.: +44 1223 760305; fax: +44 1223 760309. E-mail address: [email protected] (M.E. Welland).
based on renewable resources, are biodegradable and their fabrication is possible in aqueous solutions under mild conditions [11], in contrast to traditional plastics which are predominantly derived from petroleum and require multi-step chemical processes in their synthesis. When fully assembled, protein fibrils are highly ordered one dimensional nanostructures [12] consisting of an elongated stack of b-strands connected by a hydrogen-bonding network [3,5]. It has recently become apparent that these types of fibrillar proteinaceous materials also occur naturally, and in some cases possess a functional role in biology, for instance as catalytic scaffolds in the production of melanin [13,14] or as functional bacterial coatings [15]. Interestingly these materials appear stiffer than most intercellular filaments [5,1], a fact which makes them good candidates for modulation of elastic properties of macroscopic materials. While the material properties of individual protein nanofibrils have been studied in detail [5,16,1], it has proved to be challenging to fabricate macroscopic materials exploiting the mechanical properties of the fibrils. This problem is also present with other nanostructures such as carbon nanotubes; composite materials based on such nanostructures typically exhibit mechanical properties which are orders of magnitude below the extrapolations made from the characteristics of the individual structures. This discrepancy has been attributed to the fact that stress transfer from the nanotubes to the filler matrix is challenging to optimise due to the difficulty of introducing chemical functionality onto the outside of defect-free carbon nanotubes and achieving good dispersion [17,9,18,1]. It is interesting to speculate therefore that protein nanofibrils, although possessing lower values of Young’s modulus, might exhibit better stress transfer characteristics due to the soft and in some cases hydrophobic parts outside the fibrils core which can interact with
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other fibrils or extraneous components. Many of the fabrication methods used in producing CNT filled polymers cannot simply be applied to protein nanofibre composites as the solvents used are incompatible with biomolecular superstructures. By using a layered sandwich type structure, with thin alternating layers of protein fibrils and Polydimethylsiloxane (PDMS), we found that composite materials could be reliably produced. 2. Materials and methods 2.1. Protein nanofibres Lysozyme protein nanofibres were produced by dissolving lyophilized hen egg white lysozyme (Sigma Aldrich) with diluted 10 mM HCl incubating the solution at 65 °C for up to several weeks. Polymerisation can be monitored through the increased viscosity of the solution finally resulting in a hydrogel. Growth of the fibres were accelerated by adding 5% pre-formed fibres (seeding) to the solution. Insulin protein nanofibrils were produced by dissoliving lyophilized Bovine Insulin (Sigma Aldrich) with diluted 2 mM HCl incubating the solution at 65 °C for up to a week. Fibril films were formed by evaporating the solvent. 2.2. Fabrication of 2 wt.% protein film composite Two wt.% Insulin fibril films were mixed with uncured PDMS for 4 h at 800 RPM using a Velp Scientifica Stirrer Type BS. Cross-linker was then added and the solution was stirred again for 2 h. Just before the PDMS fully cross-linked, the mixture was poured into a dogbone shaped mould to make tensile samples and then heated at 60 °C to accelerate the cross-linking process. 2.3. Sandwich structure A schematic of the fabrication process is shown in Fig. 1A. A silicon wafer was cleaned and plasma oxidised in order to increase the wetability of the surface for 10 min using a Electronic Diener Femto plasma etcher. A water soluble release layer was used (Poly(4-styrenesulfonic acid) (PSS) 18 wt.% solution water, Sigma Aldrich) to allow the sandwich structure to be easily removed from the Si wafer at the end of the fabrication procedure. PSS was spin coated at 1500 RPM for 30 s and dried by heating at 60 °C.
Polydimethylsiloxane (PDMS, Sylgard 184 from Dow Corning) was prepared by mixing 10 parts base and 1 part curing agent and then subjected to reduced pressure to remove any residual gas. The PDMS was spin coated at 6000 RPM for 30 s. The resulting 40 lm thick PDMS layer was cross-linked by heat treatment at 60 °C. Once dried, the PDMS layer was plasma oxidised for 10 s. Lysozyme hydrogel was then drop cast onto the PDMS surface and the solvent evaporated at room temperature leaving behind a protein fibril layer of approximately 4 lm in thickness. The process was then repeated typically ten times to yield a thin film which could be removed from the silicon wafer through dissolution of the PSS release layer after immersion in water. The fibril content was adjusted such that the final structure possessed 10 wt.% protein nanofibres. 2.4. Patterned sandwich structure A schematic of the fabrication process is shown in Fig. 2A. The patterned sandwich structure was fabricated in nearly the same way as the sandwich structure with the exception that the patterned sandwich structure was composed of only three layers: (1) Bottom PDMS layer, (2) Patterned PDMS layer with lysozyme fibrils inserted into the stamped channels, and (3) A covering PDMS layer. The patterned PDMS layer was prepared by first drop casting PDMS onto the bottom PDMS layer and then stamping the uncured PDMS with a patterned stamp. This configuration was held at 60 °C until the PDMS cross-linked. Hydrogel was then pippetted into the channels and the solvent evaporated at room temperature. 2.5. Fabrication of patterned stamp The master stamp for soft lithography was fabricated from photo lithographically patterned using a MJB4 mask aligner from SU8 2100 photoresist (MicroChem) spin coated at 1000 rpm for 30 s onto a silicon wafer. 2.6. Dynamic mechanical analysis A Q800 DMA was used to perform the tension testing in the axial and transverse directions as well as for dynamic mechanical testing. The sandwich structure and 2 wt.% insulin composite was tested in tensile mode at a constant temperature of 25 °C and a rate of strain increase of 10%/min. A Control PDMS sample was prepared either by repeating the sandwich structure preparation steps but omitting lysozyme hydrogel or by casting pure PDMS in a plexiglass mould. For the dynamic mechanical testing, the frequency of oscillation was 10 Hz, the amplitude of the dynamic strain was 0.1%, and the temperature was held constant at 25 °C. The prestress was varied from 0 to 1 MPa. Samples were prepared in the same manner as in the tensile test. 2.7. Statistical analysis Three samples were prepared for each mechanical test. CNT data was obtained from the literature. To determine the experimental variance of each test, the mechanical strength and modulus data were averaged and one times the standard deviation was used for the error bars. 3. Results 3.1. Mechanical properties of 2 wt.% insulin fibril composite
Fig. 1. Stress–strain curve for a 2 wt.% insulin fibril and CNT composite [17].
A 2 wt.% insulin fibril composite was also fabricated by shear mixing insulin fibril films with PDMS to compare how the mechan-
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A
B
C
Fig. 2. The fabrication strategy for the lysozyme nanofibril/elastomer composite is illustrated in panel A. In panel B are shown the storage modulus of PDMS, of 10% CNT/ PDMS composite [19] and 10% lysozyme nanofibril/PDMS composite. Panel C shows typical samples used for the mechanical measurements: on the left hand side a film of pure PDMS which flexes under its own weight, and on the right a PDMS film of similar thickness but containing 10% lysozyme nanofibrils resulting in increased rigidity.
ical properties vary with fabrication process. Fig. 1 shows the resulting stress–strain curve after a tensile test for a 2 wt.% insulin fibril and CNT composite. Throughout the test, the 2 wt.% protein nanofibre stress data virtually lied on top of the 2 wt.% CNT curve. In addition, protein nanofibre stress data consistently exhibited average stress values approximately 1.2 times larger than the average stress data for the PDMS control. At larger strains, the experimental variance increased to approximately 100 MPa. This is in contrast to the experimental variance for the PDMS control data which showed a maximum standard deviation of about 50 MPa. 3.2. Mechanical properties of sandwich structure We measured the storage modulus under oscillating load for varying levels of pre-strain. The results show that the storage modulus of a lysozyme fibril-elastomer composite material is substantially increased compared with a film of PDMS alone, or in fact even compared with an isotropic carbon nanotube PDMS composite [19] with an identical filling ratio (10% in this case) as shown in Fig. 2. At a pre-stress of about 0.45 MPa, the sandwich structure exhibited an average modulus approximately 3 times larger than the CNT composite. The standard deviation for the protein nanofibre storage modulus data increased from approximately 0 MPa at a pre-stress of 0 MPa to a maximum of roughly 2 MPa at a pre-stress of 0.3 MPa. This is in contrast to the PDMS data in which the standard deviation increased from 0 MPa to a maximum of roughly 1 MPa at a pre-stress of 0.7 MPa. 3.3. Mechanical anisotropy in patterned sandwich structure This significant change that we have demonstrated in the Young’s modulus through doping PDMS elastomers with stiff protein fibrils prompted us to explore the possibilities of controlling
the spatial localisation of the fibril content. We illustrate this idea with the fabrication of a patterned sandwich structure using soft lithography (Fig. 3A). Prior to curing the elastomer, a patterned stamp was brought into contact with the PDMS layer; in this way channels of 150 lm in depth and 500 lm in width were formed. These channels then define the spatial distribution of the reinforcing lysozyme fibrils as the fibril containing hydrogel which is subsequently drop cast within these features. In order to quantify how much anisotropy in the elastic moduli were imparted through the patterned reinforcing fibril content, we performed tensile testing both in the perpendicular and parallel directions relative to the axis of the reinforcing patterns. Results of the tensile tests are shown in the stress–strain and modulus–strain curves in Fig. 3 B. Throughout the test, the average moduli data in the axial direction was approximately 2.5 times larger than in the transverse direction. Standard deviation values for stress and moduli in both the axial and transverse directions were relatively constant at strains larger than 2%. However, the standard deviation values for stress in the axial direction were consistently larger than the transverse direction. Stress standard deviation values in the axial direction were almost negligible while in the transverse direction were kept constant at approximately 50 MPa.
4. Discussion 4.1. Effect of fibril content on sandwich structure stiffness Even though no experimental variance was obtained for the CNT storage moduli data, the storage moduli for the protein nanofibre data were still substantially larger. The results indicate that the stress transfer in the protein nanofibre composite are more efficient than in the CNT composite.
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A
B
Fig. 3. Panel A shows the lithography process used for modulating the spatial localisation of the lysozyme nanofibril filler within the elastomer matrix. Panel B shows the measured stress as a function of strain in both the transverse and axial directions.
To determine the approximate theoretical maximum and minimum values for Young’s modulus for the protein nanofibre composite, the rule of mixtures equation is used:
Ef Em 6 E 6 ð1 f ÞEm þ fEf ð1 f ÞEf þ fEm
ð1Þ
where f is the volume fraction of the filler material and Em and Ef are the Young’s moduli of the matrix and filler material, respectively. When values for fibre volume fraction of f ¼ 0:1, PDMS Young’s modulus of Em 2 MPa, and protein nanofibre Young’s modulus of Ef 3 GPa [16,5] are used, the approximate theoretical moduli range for a protein nanofibre composite are between 2.2–300 MPa. Therefore our peak values of up to 7.5 MPa, although superior to the stiffness increase observed for a 10% doping of PDMS with CNTs, remain in the lower part of this interval, indicating that introducing alignment in the fibril layer and improving the stress transfer to the matrix can still potentially further increase the rigidity by up to an order of magnitude before the theoretical maximum is reached. 4.2. Effect of using shear mixing as a composite fabrication process The rule of mixtures, as discussed previously, indicates that the insulin fibril composite should have a lower modulus since the fibril modulus is substantially lower than the CNT’s. However, the data show that even within the range of the experimental variance, the moduli values for both composite materials are compatible. Unlike the sandwich structure, shear mixing does not easily distribute the film content throughout the elastomer. This in turn reduces the stress transfer efficiency when a load is applied and therefore reduces the potential of the composite of having a higher modulus. Nevertheless, the stress transfer from the PDMS to the protein nanofibre appears to be as efficient as for the CNT composite.
4.3. Effect of spatial distribution on mechanical anisotropy for patterned sandwich structure It is informative to compare the axial and transverse moduli with a computation of the axial Ek and transverse E? moduli for different filling factors as illustrated in the schema in Fig. 4. By using the rule of mixtures formula again, the customary isostrain approximation for the axial direction yields [20]: Ek ¼ n1 Em þ ð1 n1 Þ½n2 Em þ ð1 n2 Þ Ef and isostress in the transverse direction implies: E1 ? ¼ n1 =Em þð1 n2 Þ=ðn1 Em þ ð1 n1 ÞEf Þ. Therefore the anisotropy is given by:
Ek ½n1 þ ð1 n1 Þn2 Em þ ð1 n1 Þð1 n2 ÞEf ¼ h i1 E? n2 1n2 þ n1 Em þð1n Em 1 ÞE
ð2Þ
f
where n1 ¼ h1 =ðh1 þ h2 Þ and n2 ¼ w1 =ðw1 þ w2 Þ are the vertical and horizontal filling fractions. Fig. 3 shows the dependency of the anisotropy in the Young’s modulus for n1 ¼ 1=2 and n2 varying from 0 to 1. Interestingly, a filler with a modulus ratio of only Ef =Em ¼ 20 leads to anisotropies of over a factor of 3, whereas a void in the structure, corresponding to the smallest possible E1 =Em ¼ 0 ratio only leads to a smaller anisotropy. This demonstrates that the effective modulation of elastic properties to create significant anisotropies is not practical using only geometric features in a single material. Indeed, as demonstrated by the calculation discussed above, in order to obtain a significant effect, a very large fraction of such a material would need to be hollow, a fact which is not compatible with structural robustness. On the other hand, by using two materials and in particular through the inclusion of more rigid areas into elastomers, such as those which can be achieved with reinforcing protein nanofibrils, it is possible to achieve significant elastic anisotropy in a given direction without compromising structural integrity. The moduli data for the axial direction is at least 2 times larger than the transverse direction for the entire strain range. Thus,
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Fig. 4. Schema illustrating the anisotropy in the elastic modulus which can be achieved by geometric patterning of a single material and by combining two materials with different elastic moduli. Panel A shows the general geometry of a patterned sandwich structure as described in the text, and panel B contains the ratios of transverse to perpendicular moduli for filler to matrix ratios of 0 and 20.
given the result of the theoretical calculation for a filler fraction of zero, an experimentally determined axial to transverse moduli ratio of 2 indicates that significant mechanical anisotropy can indeed be produced. If the stress transfer from the PDMS to the fibres is improved, than the ratio of moduli could be significantly improved. 5. Conclusion In conclusion, we have presented the fabrication and mechanical characterisation of a class of elastomer/protein fibril composite material. The stiffening of the elastomer through the presence of 10% protein fibrils by using a sandwich structure was found to be significantly greater than for an isotropic 10% carbon nanotube PDMS composites reported in the literature. A 2 wt.% insulin fibril PDMS composite which was shear mixed produced a composite with an equivalent stiffness than a CNT PDMS composite with the same filling ratio. Furthermore, we have shown that the spatial distribution of protein fibrils within the material can be controlled, and that this strategy can be used to produce mechanically highly anisotropic films. This research was supported by the IRC in Nanotechnology, Nokia Research, St. John’s College Cambridge and the Royal Society.
References [1] Kol Nitzan, Adler-Abramovich Lihi, Barlam David, Shneck Roni Z, Gazit Ehud, Rousso Itay. Self-assembled peptide nanotubes are uniquely rigid bioinspired supramolecular structures. Nano Lett 2005;5:1343–6. [2] Cherny Izhack, Gazit Ehud. Amyloids: not only pathological agents but also ordered nanomaterials. Angew Chem Int Ed 2008;47:4062–9.
[3] Dobson Christopher M. Protein folding and misfolding. Nature 2003;426:884–90. [4] Chiti Fabrizio, Dobson Christopher M. Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem 2006;75:333–66. [5] Knowles Tuomas PJ, Fitzpatrick Anthony W, Meehan Sarah, Mott Helen R, Vendrusculo Michele, Dobson Christopher M, et al. Role of intermolecular forces in defining material properties of protein nanofibrils. Science 2007;318:1900–3. [6] Dobson Christopher M. Protein misfolding, evolution, and disease. Trends Biochem Sci 1999;24:329–32. [7] Zhang Shuguang. Fabrication of novel biomaterials through molecular selfassembly. Nat Biotechnol 2003;21:1171–7. [8] Treacy MM, Ebbesen TW, Gibson TM. Exceptionally high young’s modulus observed for individual carbon nanotubes. Nature 1996;381:678–80. [9] Coleman Jonathan N, Khan Umar, Gunko Yurii K. Mechanical reinforcement of polymers using carbon nanotubes. Adv Mater 2006;18:689–706. [10] Porter Alexandra E et al. Direct imaging of single-walled carbon nanotubes in cells. Nat Nanotechnol 2007;2:713–7. [11] Zhang Shuguang, Marini Davide M, Hwang Wonmuk, Santoso Steve. Design of nanostructured biological materials through self-assembly of peptides and proteins. Curr Opin Chem Biol 2002;6:865–71. [12] Knowles TPJ, Smith JF, Craig A, Dobson CM, Welland ME. Spatial persistence of angular correlations in amyloid fibrils. Phys Re Lett 2006;96:223801–5. [13] Fowler Douglas M, Koulov Atanas V, Alory-Jost Christelle, Marks Michael S, Balch William E, Kelly Jeffery W. Functional amyloid formation within mammalian tissue. PLoS Biol 2006;4:100–7. [14] Fowler Douglas M, Koulov Atanas V, Balch William E, Kelly Jeffery W. Functional amyloid from bacteria to humans. Trends Biochem Sci 2007;32:217–24. [15] Chapman Matthew R. Role of Escherichia coli curli operons in directing amyloid fiber formation. Science 2002;295:851–4. [16] Smith Jeffrey F, Knowles Tuomas PJ, Dobson Christopher M, MacPhee Cait E, Welland Mark E. Characterization of the nanoscale properties of individual amyloid fibrils. PNAS 2006;103:15806–11. [17] Huang YY, Terentjev EM. Dispersion and rheology of carbon nanotubes in polymers. Int J Mater Form 2006;1:63–74. [18] Baughman Ray H, Zakhidov Anvar A, de Heer Walt A. Carbon nanotubes the route toward applications. Science 2002;297:787–92. [19] Paul J et al. Mechanics of prestressed polydimethylsiloxane–carbon nanotubecomposite. Appl Phys Lett 2006;89:184101–3. [20] Hull D, Clyne TW. An introduction to composite materials. second ed. Cambridge University Press; 1996.
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Fabrication and characterisation of protein fibril–elastomer composites Tomas Oppenheim, Tuomas P.J. Knowles, Stéphanie P. Lacour, Mark E. Welland * Nanoscience Centre, University of Cambridge, J J Thomson Avenue, Cambridge CB3 0FF, UK
a r t i c l e
i n f o
Article history: Received 18 June 2009 Received in revised form 18 September 2009 Accepted 6 October 2009 Available online 14 October 2009 Keywords: Protein fibril Carbon nanotube Elastomer Rigidity Anisotropy
a b s t r a c t Protein fibrils are emerging as a novel class of functional bionanomaterials. In this paper we make use of their rigidity by combining lysozyme fibrils with a silicone elastomer and demonstrating that at a filling ratio of 10%, the protein fibril composite is at minimum 2 times stiffer than a CNT elastomeric composite of the same filling ratio. We also show that when the elastomer is patterned such that the lysozyme fibrils can be spatially modulated within the elastomer, anisotropic moduli varying by a factor of 2 is produced. By using shear mixing as the fabrication process, the modulus of a 2 wt.% insulin fibril composite is equivalent to a CNT composite with the same filling ratio. In conclusion, we have presented the fabrication and mechanical characterisation of a class of elastomer/protein fibril composite material. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Protein nanofibrils are rigid structures which can be self-assembled from a range of different peptides and proteins under mild conditions in aqueous solution [1–7]. In this paper, we present a class of material where such organic fibrils are combined with silicone elastomers to provide spatially modulated changes in material rigidity. In particular we show that at a 10% filling ratio the change in rigidity is superior to corresponding carbon nanotube composites. Furthermore we demonstrate that by controlling the spatial distribution of the fibrils within the elastomer matrix, materials with highly anisotropic elastic moduli can be produced. By incorporating nanomaterials into polymer matrices, novel multi-functional materials can be produced. One functionality of nanocomposites that is heavily examined is to improve their mechanical properties by including 1D nanomaterials into the matrix. Due to their high aspect ratio, a more efficient stress transfer should occur between the matrix and the nanofilament and hence a stiffer composite should be produced. Carbon nanotubes (CNT) are a popular class of 1D nanomaterials to include into polymer matrices due to their excellent mechanical properties. With a Young’s modulus of 1 TPa, CNT’s are the stiffest 1D nanomaterials [8]. Although CNT’s have excellent mechanical properties, their energy intensive fabrication process and their potentially high toxicity make them less attractive [9,10]. Protenacious filaments are emerging as attractive candidates for components in functional materials for several reasons. They are * Corresponding author. Tel.: +44 1223 760305; fax: +44 1223 760309. E-mail address: [email protected] (M.E. Welland).
based on renewable resources, are biodegradable and their fabrication is possible in aqueous solutions under mild conditions [11], in contrast to traditional plastics which are predominantly derived from petroleum and require multi-step chemical processes in their synthesis. When fully assembled, protein fibrils are highly ordered one dimensional nanostructures [12] consisting of an elongated stack of b-strands connected by a hydrogen-bonding network [3,5]. It has recently become apparent that these types of fibrillar proteinaceous materials also occur naturally, and in some cases possess a functional role in biology, for instance as catalytic scaffolds in the production of melanin [13,14] or as functional bacterial coatings [15]. Interestingly these materials appear stiffer than most intercellular filaments [5,1], a fact which makes them good candidates for modulation of elastic properties of macroscopic materials. While the material properties of individual protein nanofibrils have been studied in detail [5,16,1], it has proved to be challenging to fabricate macroscopic materials exploiting the mechanical properties of the fibrils. This problem is also present with other nanostructures such as carbon nanotubes; composite materials based on such nanostructures typically exhibit mechanical properties which are orders of magnitude below the extrapolations made from the characteristics of the individual structures. This discrepancy has been attributed to the fact that stress transfer from the nanotubes to the filler matrix is challenging to optimise due to the difficulty of introducing chemical functionality onto the outside of defect-free carbon nanotubes and achieving good dispersion [17,9,18,1]. It is interesting to speculate therefore that protein nanofibrils, although possessing lower values of Young’s modulus, might exhibit better stress transfer characteristics due to the soft and in some cases hydrophobic parts outside the fibrils core which can interact with
1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.10.013
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other fibrils or extraneous components. Many of the fabrication methods used in producing CNT filled polymers cannot simply be applied to protein nanofibre composites as the solvents used are incompatible with biomolecular superstructures. By using a layered sandwich type structure, with thin alternating layers of protein fibrils and Polydimethylsiloxane (PDMS), we found that composite materials could be reliably produced. 2. Materials and methods 2.1. Protein nanofibres Lysozyme protein nanofibres were produced by dissolving lyophilized hen egg white lysozyme (Sigma Aldrich) with diluted 10 mM HCl incubating the solution at 65 °C for up to several weeks. Polymerisation can be monitored through the increased viscosity of the solution finally resulting in a hydrogel. Growth of the fibres were accelerated by adding 5% pre-formed fibres (seeding) to the solution. Insulin protein nanofibrils were produced by dissoliving lyophilized Bovine Insulin (Sigma Aldrich) with diluted 2 mM HCl incubating the solution at 65 °C for up to a week. Fibril films were formed by evaporating the solvent. 2.2. Fabrication of 2 wt.% protein film composite Two wt.% Insulin fibril films were mixed with uncured PDMS for 4 h at 800 RPM using a Velp Scientifica Stirrer Type BS. Cross-linker was then added and the solution was stirred again for 2 h. Just before the PDMS fully cross-linked, the mixture was poured into a dogbone shaped mould to make tensile samples and then heated at 60 °C to accelerate the cross-linking process. 2.3. Sandwich structure A schematic of the fabrication process is shown in Fig. 1A. A silicon wafer was cleaned and plasma oxidised in order to increase the wetability of the surface for 10 min using a Electronic Diener Femto plasma etcher. A water soluble release layer was used (Poly(4-styrenesulfonic acid) (PSS) 18 wt.% solution water, Sigma Aldrich) to allow the sandwich structure to be easily removed from the Si wafer at the end of the fabrication procedure. PSS was spin coated at 1500 RPM for 30 s and dried by heating at 60 °C.
Polydimethylsiloxane (PDMS, Sylgard 184 from Dow Corning) was prepared by mixing 10 parts base and 1 part curing agent and then subjected to reduced pressure to remove any residual gas. The PDMS was spin coated at 6000 RPM for 30 s. The resulting 40 lm thick PDMS layer was cross-linked by heat treatment at 60 °C. Once dried, the PDMS layer was plasma oxidised for 10 s. Lysozyme hydrogel was then drop cast onto the PDMS surface and the solvent evaporated at room temperature leaving behind a protein fibril layer of approximately 4 lm in thickness. The process was then repeated typically ten times to yield a thin film which could be removed from the silicon wafer through dissolution of the PSS release layer after immersion in water. The fibril content was adjusted such that the final structure possessed 10 wt.% protein nanofibres. 2.4. Patterned sandwich structure A schematic of the fabrication process is shown in Fig. 2A. The patterned sandwich structure was fabricated in nearly the same way as the sandwich structure with the exception that the patterned sandwich structure was composed of only three layers: (1) Bottom PDMS layer, (2) Patterned PDMS layer with lysozyme fibrils inserted into the stamped channels, and (3) A covering PDMS layer. The patterned PDMS layer was prepared by first drop casting PDMS onto the bottom PDMS layer and then stamping the uncured PDMS with a patterned stamp. This configuration was held at 60 °C until the PDMS cross-linked. Hydrogel was then pippetted into the channels and the solvent evaporated at room temperature. 2.5. Fabrication of patterned stamp The master stamp for soft lithography was fabricated from photo lithographically patterned using a MJB4 mask aligner from SU8 2100 photoresist (MicroChem) spin coated at 1000 rpm for 30 s onto a silicon wafer. 2.6. Dynamic mechanical analysis A Q800 DMA was used to perform the tension testing in the axial and transverse directions as well as for dynamic mechanical testing. The sandwich structure and 2 wt.% insulin composite was tested in tensile mode at a constant temperature of 25 °C and a rate of strain increase of 10%/min. A Control PDMS sample was prepared either by repeating the sandwich structure preparation steps but omitting lysozyme hydrogel or by casting pure PDMS in a plexiglass mould. For the dynamic mechanical testing, the frequency of oscillation was 10 Hz, the amplitude of the dynamic strain was 0.1%, and the temperature was held constant at 25 °C. The prestress was varied from 0 to 1 MPa. Samples were prepared in the same manner as in the tensile test. 2.7. Statistical analysis Three samples were prepared for each mechanical test. CNT data was obtained from the literature. To determine the experimental variance of each test, the mechanical strength and modulus data were averaged and one times the standard deviation was used for the error bars. 3. Results 3.1. Mechanical properties of 2 wt.% insulin fibril composite
Fig. 1. Stress–strain curve for a 2 wt.% insulin fibril and CNT composite [17].
A 2 wt.% insulin fibril composite was also fabricated by shear mixing insulin fibril films with PDMS to compare how the mechan-
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A
B
C
Fig. 2. The fabrication strategy for the lysozyme nanofibril/elastomer composite is illustrated in panel A. In panel B are shown the storage modulus of PDMS, of 10% CNT/ PDMS composite [19] and 10% lysozyme nanofibril/PDMS composite. Panel C shows typical samples used for the mechanical measurements: on the left hand side a film of pure PDMS which flexes under its own weight, and on the right a PDMS film of similar thickness but containing 10% lysozyme nanofibrils resulting in increased rigidity.
ical properties vary with fabrication process. Fig. 1 shows the resulting stress–strain curve after a tensile test for a 2 wt.% insulin fibril and CNT composite. Throughout the test, the 2 wt.% protein nanofibre stress data virtually lied on top of the 2 wt.% CNT curve. In addition, protein nanofibre stress data consistently exhibited average stress values approximately 1.2 times larger than the average stress data for the PDMS control. At larger strains, the experimental variance increased to approximately 100 MPa. This is in contrast to the experimental variance for the PDMS control data which showed a maximum standard deviation of about 50 MPa. 3.2. Mechanical properties of sandwich structure We measured the storage modulus under oscillating load for varying levels of pre-strain. The results show that the storage modulus of a lysozyme fibril-elastomer composite material is substantially increased compared with a film of PDMS alone, or in fact even compared with an isotropic carbon nanotube PDMS composite [19] with an identical filling ratio (10% in this case) as shown in Fig. 2. At a pre-stress of about 0.45 MPa, the sandwich structure exhibited an average modulus approximately 3 times larger than the CNT composite. The standard deviation for the protein nanofibre storage modulus data increased from approximately 0 MPa at a pre-stress of 0 MPa to a maximum of roughly 2 MPa at a pre-stress of 0.3 MPa. This is in contrast to the PDMS data in which the standard deviation increased from 0 MPa to a maximum of roughly 1 MPa at a pre-stress of 0.7 MPa. 3.3. Mechanical anisotropy in patterned sandwich structure This significant change that we have demonstrated in the Young’s modulus through doping PDMS elastomers with stiff protein fibrils prompted us to explore the possibilities of controlling
the spatial localisation of the fibril content. We illustrate this idea with the fabrication of a patterned sandwich structure using soft lithography (Fig. 3A). Prior to curing the elastomer, a patterned stamp was brought into contact with the PDMS layer; in this way channels of 150 lm in depth and 500 lm in width were formed. These channels then define the spatial distribution of the reinforcing lysozyme fibrils as the fibril containing hydrogel which is subsequently drop cast within these features. In order to quantify how much anisotropy in the elastic moduli were imparted through the patterned reinforcing fibril content, we performed tensile testing both in the perpendicular and parallel directions relative to the axis of the reinforcing patterns. Results of the tensile tests are shown in the stress–strain and modulus–strain curves in Fig. 3 B. Throughout the test, the average moduli data in the axial direction was approximately 2.5 times larger than in the transverse direction. Standard deviation values for stress and moduli in both the axial and transverse directions were relatively constant at strains larger than 2%. However, the standard deviation values for stress in the axial direction were consistently larger than the transverse direction. Stress standard deviation values in the axial direction were almost negligible while in the transverse direction were kept constant at approximately 50 MPa.
4. Discussion 4.1. Effect of fibril content on sandwich structure stiffness Even though no experimental variance was obtained for the CNT storage moduli data, the storage moduli for the protein nanofibre data were still substantially larger. The results indicate that the stress transfer in the protein nanofibre composite are more efficient than in the CNT composite.
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A
B
Fig. 3. Panel A shows the lithography process used for modulating the spatial localisation of the lysozyme nanofibril filler within the elastomer matrix. Panel B shows the measured stress as a function of strain in both the transverse and axial directions.
To determine the approximate theoretical maximum and minimum values for Young’s modulus for the protein nanofibre composite, the rule of mixtures equation is used:
Ef Em 6 E 6 ð1 f ÞEm þ fEf ð1 f ÞEf þ fEm
ð1Þ
where f is the volume fraction of the filler material and Em and Ef are the Young’s moduli of the matrix and filler material, respectively. When values for fibre volume fraction of f ¼ 0:1, PDMS Young’s modulus of Em 2 MPa, and protein nanofibre Young’s modulus of Ef 3 GPa [16,5] are used, the approximate theoretical moduli range for a protein nanofibre composite are between 2.2–300 MPa. Therefore our peak values of up to 7.5 MPa, although superior to the stiffness increase observed for a 10% doping of PDMS with CNTs, remain in the lower part of this interval, indicating that introducing alignment in the fibril layer and improving the stress transfer to the matrix can still potentially further increase the rigidity by up to an order of magnitude before the theoretical maximum is reached. 4.2. Effect of using shear mixing as a composite fabrication process The rule of mixtures, as discussed previously, indicates that the insulin fibril composite should have a lower modulus since the fibril modulus is substantially lower than the CNT’s. However, the data show that even within the range of the experimental variance, the moduli values for both composite materials are compatible. Unlike the sandwich structure, shear mixing does not easily distribute the film content throughout the elastomer. This in turn reduces the stress transfer efficiency when a load is applied and therefore reduces the potential of the composite of having a higher modulus. Nevertheless, the stress transfer from the PDMS to the protein nanofibre appears to be as efficient as for the CNT composite.
4.3. Effect of spatial distribution on mechanical anisotropy for patterned sandwich structure It is informative to compare the axial and transverse moduli with a computation of the axial Ek and transverse E? moduli for different filling factors as illustrated in the schema in Fig. 4. By using the rule of mixtures formula again, the customary isostrain approximation for the axial direction yields [20]: Ek ¼ n1 Em þ ð1 n1 Þ½n2 Em þ ð1 n2 Þ Ef and isostress in the transverse direction implies: E1 ? ¼ n1 =Em þð1 n2 Þ=ðn1 Em þ ð1 n1 ÞEf Þ. Therefore the anisotropy is given by:
Ek ½n1 þ ð1 n1 Þn2 Em þ ð1 n1 Þð1 n2 ÞEf ¼ h i1 E? n2 1n2 þ n1 Em þð1n Em 1 ÞE
ð2Þ
f
where n1 ¼ h1 =ðh1 þ h2 Þ and n2 ¼ w1 =ðw1 þ w2 Þ are the vertical and horizontal filling fractions. Fig. 3 shows the dependency of the anisotropy in the Young’s modulus for n1 ¼ 1=2 and n2 varying from 0 to 1. Interestingly, a filler with a modulus ratio of only Ef =Em ¼ 20 leads to anisotropies of over a factor of 3, whereas a void in the structure, corresponding to the smallest possible E1 =Em ¼ 0 ratio only leads to a smaller anisotropy. This demonstrates that the effective modulation of elastic properties to create significant anisotropies is not practical using only geometric features in a single material. Indeed, as demonstrated by the calculation discussed above, in order to obtain a significant effect, a very large fraction of such a material would need to be hollow, a fact which is not compatible with structural robustness. On the other hand, by using two materials and in particular through the inclusion of more rigid areas into elastomers, such as those which can be achieved with reinforcing protein nanofibrils, it is possible to achieve significant elastic anisotropy in a given direction without compromising structural integrity. The moduli data for the axial direction is at least 2 times larger than the transverse direction for the entire strain range. Thus,
T. Oppenheim et al. / Acta Biomaterialia 6 (2010) 1337–1341
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Fig. 4. Schema illustrating the anisotropy in the elastic modulus which can be achieved by geometric patterning of a single material and by combining two materials with different elastic moduli. Panel A shows the general geometry of a patterned sandwich structure as described in the text, and panel B contains the ratios of transverse to perpendicular moduli for filler to matrix ratios of 0 and 20.
given the result of the theoretical calculation for a filler fraction of zero, an experimentally determined axial to transverse moduli ratio of 2 indicates that significant mechanical anisotropy can indeed be produced. If the stress transfer from the PDMS to the fibres is improved, than the ratio of moduli could be significantly improved. 5. Conclusion In conclusion, we have presented the fabrication and mechanical characterisation of a class of elastomer/protein fibril composite material. The stiffening of the elastomer through the presence of 10% protein fibrils by using a sandwich structure was found to be significantly greater than for an isotropic 10% carbon nanotube PDMS composites reported in the literature. A 2 wt.% insulin fibril PDMS composite which was shear mixed produced a composite with an equivalent stiffness than a CNT PDMS composite with the same filling ratio. Furthermore, we have shown that the spatial distribution of protein fibrils within the material can be controlled, and that this strategy can be used to produce mechanically highly anisotropic films. This research was supported by the IRC in Nanotechnology, Nokia Research, St. John’s College Cambridge and the Royal Society.
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