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Hybrid silk fibers dry-spun from regenerated silk fibroin/graphene oxide aqueous solutions Chao Zhang, Yaopeng Zhang, Huili Shao, and Xuechao Hu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11245 • Publication Date (Web): 19 Jan 2016 Downloaded from http://pubs.acs.org on January 22, 2016
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Hybrid silk fibers dry-spun from regenerated silk fibroin/graphene
†
oxide
aqueous
solutions
†
Chao Zhang (Co-first author), Yaopeng Zhang*, (Co-first author), Huili Shao, Xuechao Hu
State Key Laboratory for Modication of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai201620, P.R. China. E-mail:
[email protected] KEYWORDS: Regenerated silk fibroin, graphene oxide, dry-spinning, nano-confined, interphase, reinforced.
†
C. Zhang and Y. Zhang contributed equally to this work.
ABSTRACT: Regenerated silk fibroin (RSF)/graphene oxide (GO) hybrid silk fibers were dryspun from a mixed dope of GO suspension and RSF aqueous solution. It was observed that the presence of GO greatly affect the viscosity of RSF solution. The RSF/GO hybrid fibers showed lower β-sheet content compared to pure RSF fibers from FTIR result. The result of Synchrotron Radiation Wide Angle X-ray Diffraction showed that the addition of GO confined the crystallization of silk fibroin, leading to the decrease of crystallinity, the smaller crystallite size and new formation of interphase zones in the artificial silks. Synchrotron Radiation Small Angle X-ray Scattering also proved that GO sheets in the hybrid silks and blended solutions were coated with a certain thickness of interphase zones due to the complex interaction between the
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two components. A low addition of GO, together with the mesophase zones formed between GO and RSF, enhanced the mechanical properties of hybrid fibers. The highest breaking stress of the hybrid fibers can be reached 435.5±71.6 MPa, 23% improvement in comparison to degummed silk and 72% larger than pure RSF silk fiber. The hybrid RSF/GO materials with good biocompatibility and enhanced mechanical properties may have potential applications in tissue engineering, bioelectronic devices or energy storage.
Introduction Silk fibroin (SF), produced by silkworms (e.g. Bombyx mori), is considered to be a better biomaterial owing to its excellent mechanical properties, biocompatibility, low immunogenicity and biodegradability.1-3 Based on different requirments, silk fibroin can be fabricated to fibers4, gels5, films6 and sponge7. Over the last two decades, SF biomaterial has been extensively used in biomedical field as tissue engineering scaffold for bone8, skin9 and vascular repair10. Recently, many researches focused on the potential use of SF for bone tissue repair since the basic requirement of tissue engineering scaffolds should be biocompatible and easily implantable with less infection risk.11-12 However, neat silk fibroin materials normally are still not strong enough for bone repair. For this, many inorganic particles, including hydroxyapatite (HAP)13-15, montmorillonite(MMT)16 and diopside17, were introduced to improve the mechanical properties of SF-based materials. Furthermore, there are also some reports about adding organic materials to improve flexibility18, compressive modulus8 of SF materials and enhance cell proliferation and attachment19 on it. Generally, the method of producing regenerated silk fibroin (RSF) fibers is wet-spinning, electrospinning and dry-spinning. The artificial silk fibers or mats produced by wet-spinning and eletrospinning have better mechanical properties, but the two methods usually involve with
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organic solvent or coagulation bath, which might lead to serious environmental problems. As for dry-spinning method, it can avoid all the problems described above with employing water as solvent. In order to further improve the mechanical properties of artificial silks, many reinforcement methods, such as post-treatment, adding metal ions, have been carried out.20-21 The mechanical properties of silk-based materials can be improved by using various kind of fillers, including micro-sized silk fiber, human hair-derived keratin8, carbon tube22, hydroxyapatite (HAp)23, cellulose nanocrystals (CNCs)24. However, a key problem is that which kind of fillers can effectively transfer the intrinsic mechanical characteristics to polymer matrix. In fact, the reinforcing effect of nanofiller depends on its shape and dimension. For example, nanodiamond and carbon nanotube (CNT) interact with polymer matrix at 0-D point and 1-D linear contact, while 2-D graphene nanosheet exhibits higher specific surface area and larger aspect ratio, which make it more effective to reinforce composite. Among carbon-based fillers, graphene oxide (GO), a two-dimensional carbon material, has good optical, thermal, mechanical properties, which recently draws more and more attention.25-26 Conventional fillers, such as clay sheet, metal oxide, which have high strength and modulus, were often used as fillers. However, most of these inorganic particles are brittle and have high weight densities. GO is a material with a light weight and great flexibility, which makes it a perfect nano-filler. The mechanical properties of composites could be obviously improved at a much lower addition of GO than other fillers.27-28 In addition, GO slightly affects the spinnability of blended solution.29 The greatly reduced cost of GO also boosts it further applications. The abundant oxygenic functional groups make GO sheets disperse well in water and polymer matrix. Therefore, GO is widely used as a filler to improve the mechanical properties of
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composites due to its nanoscale morphology and high lateral dimensions. For example, Hu et al. 30
prepared GO-SF nanomembranes through spin-assisted layer-by-layer(SA-LbL) technique.
The membranes exhibited outstanding ultimate stress, tensile modulus, and breaking energy, which was 300 MPa, 145 GPa and 2.2 MJ m-3, respectively. Huang et al.31 also prepared a strong RSF film by casting silk fibroin-GO hydrogels. The tensile stress and modulus were improved to 221±16 MPa and 17.2±1.9 GPa, respectively. It is known that different nano-particles have different interaction behavior with RSF leading to different crystallization of RSF. MWNT is favorable for the formation of β-sheet structure of RSF, while TiO2 nanoparticles cause the nanoconfined crystallization of RSF.32-33 However, few researches focus on RSF/GO fibers and comprehensively explain the influence of GO on the crystalline structure and secondary structure of silk fibroin in the composite. As RSF may have severe interaction with the two-dimensional GO flake, it is necessary to investigate the enhancement mechanism of RSF/GO composite. Encouraged by GO or graphene being used as fillers to reinforce polymer fibers, such as chitosan fibers, cellulose fibers and PA6, we fabricated RSF/GO hybrid fibers to investigate the effect of GO on silk fibroin. The structure and the complex interaction between silk fibroin and GO were characterized by synchrotron radiation wide angle X-ray diffraction (SR-WAXD) and synchrotron radiation small angle X-ray scattering (SR-SAXS) .
Experimental section Preparation of regenerated silk fibroin/graphene oxide fibers RSF solutions were prepared according to the method described in our previous work.34 B. mori cocoons (produced in 2014 and in Tongxiang city, Zhejiang Province, China) were
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degummed twice by immersing it in 0.5wt% aqueous Na2CO3 solutions at 100 ℃ for 30 min and washed with deionized water. After being dried overnight, the extracted silks were dissolved in 9.0 M LiBr aqueous solution at 40°C for 2 h. Then the solution was filtered and dialyzed against deionized water for 3 days to obtain aqueous RSF solution. When the solution was concentrated to 20wt% under forced airflow at 10°C, GO aqueous dispersion (0.5 mg/ml, the Sixth Elements Hi-Tech Development Co., Ltd., China) was blended with the RSF solution. The mass ratio of GO/RSF was 0/1000, 0.5/1000, 1/1000, 1.5/1000 and 2/1000, respectively. The samples were designated as RSF and RSF/GO-X, for example, RSF/GO-1.0 means GO/RSF=1/1000, degummed silk was designated as De-silk. The spinning dope was transferred to a syringe when the total weight concentration of RSF and GO was about 45wt%. The syringe was kept at 7°C for 4h to discharge the bubbles in the spinning dope. Then the dry-spinning was conducted by using a custom-built equipment at 25°C and 45% relative humidity in air (See Figure S1). A flow rate was set at 0.5 µL/min using a syringe pump (Model 210P, KD Scientific Inc. USA). The distance between spinneret and take-up roll was 10 cm and the spinning speed was 3 cm/s. The as-spun fibers were then stored in a sealed dryer for 48 h. A single as-spun fiber was firstly immersed in 80vol% ethanol aqueous solution for 30-40 s. Then the post-treatment was carried out by drawing the fibers to four times length in the 80vol% ethanol aqueous solution and keeping them in the solution for 2 h with external tension. Rheological tests The rheological measurements were conducted on an RS150 rheometer (Thermo-Haake, Germany) with a 20 mm parallel plate (Ti, gap 0.3 mm) at 22±1°C. Mechanical properties test
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The average diameter of each sample was obtained from more than 5 points distributed along the fiber axis in optical microphotographs (BX-51, Olympus, Japan). The diameter was used to calculate the cross section area of the fiber with circular cross section. As the cross section of degummed cocoon silk is irregular, we embedded the fibers in Spurr’s resin for utralmicrotomy and observed the cross-section using Hitachi S-3000N scanning electron microscope (SEM). The breaking stress of fiber can be determined by dividing the actual load by the cross-section area of each specimen. Instron 5565 material testing machine was used to measure the mechanical properties of the single fiber fixed to a paper frame at (25 ± 2) °C and (45 ± 5) % of relative humidity (RH). The experiment was performed at an extension rate of 2 mm/min with a gauge length of 10 mm. Twenty single fibers for each sample were tested.35-36 The mechanical properties were statistically analyzed using student’s t-test.37 The results of RSF fibers were used as benchmark for comparison with RSF/GO hybrid fibers. For each test, the significance level was set p
