Mass Production of Biocompatible Graphene Using Silk Nanofibers

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Mass Production of Biocompatible Graphene Using Silk Nanofibers Xiaoyi Zhang,† Ling Wang,‡ Qiang Lu,*,†,§ and David L. Kaplan∥ †

National Engineering Laboratory for Modern Silk & Collaborative Innovation Center of Suzhou Nano Science and Technology, and §Key Laboratory of Stem Cells and Biomedical Materials of Jiangsu Province and Chinese Ministry of Science and Technology, Soochow University, Suzhou 215123, People’s Republic of China ‡ Biology Institute, Qilu University of Technology (Shandong Academy of Sciences), Shandong 250000, People’s Republic of China ∥ Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, United States Downloaded via DURHAM UNIV on July 2, 2018 at 14:04:00 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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ABSTRACT: Mass production of high-quality graphene dispersions under mild conditions impacts the utility of the material for biomedical applications. Various proteins have been used to prepare graphene dispersions, rare sources, and expensive prices for these proteins restrict their large-scale utility for the production of graphene. Here, inexpensive silk proteins as an abundant resource in nature were used for graphene exfoliation. The silk proteins were assembled into hydrophobic nanofibers with negative charge, and then optimized for the production of graphene. Significantly higher concentrations (>8 mg mL−1) and yields (>30%) of graphene dispersions under ambient aqueous conditions were achieved compared with previous protein-assisted exfoliation systems. The exfoliated graphene exhibited excellent stability in water and fetal bovine serum solution, cytocompatibility, and conductivity, suggesting a promising future in biomedical and bioengineering applications. KEYWORDS: graphene, silk nanofiber, exfoliation, mass production, biomedical application

1. INTRODUCTION Because of the outstanding mechanical, electrical, and thermal properties, as well as good cytocompatibility and high surface area,1−5 graphene has received extensive attention toward biological applications in tissue engineering, drug delivery, and bioelectrical and bioimaging devices.6−12 Facile processes for the large-scale production of pristine graphene under biologically benign conditions are prerequisites for these biological applications.8,9 Although exfoliation methods were developed to achieve the scalable production of graphene using organic solvents, 13,14 surfactants 15−17 and ionic liquids, 18,19 unaddressed challenges remain to prepare biocompatible and superior graphene products for economic and environmentally friendly processes. Ultrasonication-assisted exfoliation has been optimized to obtain defect-free graphene in aqueous solutions with surfactants.20−23 As alternatives to traditional surfactants, various proteins such as bovine serum albumin (BSA) have been used to facilitate the delamination of graphite platelets and inhibit the restacking of the graphene flakes, generating aqueous biographene dispersions.24−28 However, the limited scalability of these approaches remains a problem because increasing the production by increasing volume is not an option, resulting in low exfoliation efficiencies (500 mg were achieved by this simple mixing process.29−33 Further scaled production of the graphene was realized by tuning the volume of the blend solution, suggesting the feasibility of the method for future mass production in industry.32−34 The proteins used for exfoliating/stabilizing agents such as BSA were introduced to this shear-exfoliation process. Biocompatible graphene dispersions with higher concentration and exfoliation were prepared under gentle aqueous solutions at room temperature, implying a future in biological applications. Several studies confirmed that the charge density of the proteins was critical for effectively exfoliating and stabilizing the graphene dispersions.31−36 Increasing efforts were devoted to optimize the stability of these graphene dispersions in aqueous solution by choosing suitable proteins. For such scaling of production, proteins with higher charge density are required. Silk materials have been considered universal biomaterials because of their biocompatibility, mechanical properties, tunable biodegradability, and versatile processability36−40 and Received: March 23, 2018 Accepted: June 18, 2018 Published: June 18, 2018 A

DOI: 10.1021/acsami.8b04777 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

The infrared (IR) spectra were measured using a Fourier transform-IR (FT-IR) spectrometer (Nicolet 6700 FT-IR, Thermo Scientific, FL, USA) where 64 scans were run in the wavenumber range 400−4000 cm−1 with a resolution of 4 cm−1. Raman spectra were recorded using a Raman spectrometer (Renishaw, 532 nm diode laser, New Mills, UK). A 532 nm laser was used with exposure time 10 s and laser power 50%. The microscope was calibrated using a typical Raman shift of Si-wafer sample at 520.7 cm−1. Graphene samples were dried on Si-wafers with ≈1 cm spot size. Thermogravimetric analysis (TGA) was carried out on a TGA/ DSC2 Star system (Mettler, Toledo, USA) with a heating rate of 10 °C min−1 in N2. The ζ-potentials of suspension were characterized by Zetasizer (Nano ZS, Malvern, Worcestershire, UK) at 25 °C. The electrical performance of the graphene materials was obtained using a CMT series JANDEL four-point probe at room temperature. After a high-speed centrifugation and resuspension in water, the aqueous graphene suspension was coated on a cellulose paper and dried in air to prepare conductive graphene papers. The conductivity was calculated by the following equation: σ = 1/tRs, where Rs is the sheet resistance measured by the four-point probe device and t is the film thickness. Also, a test with a light-emitting diode, battery, and the graphene sheet was performed to confirm the conductivity of the graphene materials. 2.4. Stability of Graphene Suspensions. The stability of aqueous graphene dispersions was assessed at various temperatures, pH values, and in the presence of FBS. After incubation for 30 days, the suspensions were measured with a BioTeK Synergy 4 spectrofluorometer (BioTeK, Winooski, VT). The change in concentration of the suspensions was calculated by comparing the values of absorbance at 660 nm. 2.5. Cytotoxicity Evaluation of Graphene Dispersions. Bone marrow mesenchymal stem cells (BMSCs) were seeded to 96-well plates (6 × 103 cells per well) and incubated for 24 h in 100 μL Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA) containing 10% (v/v) FBS (Invitrogen, Carlsbad, CA). The culture medium was replaced with fresh medium containing various amounts of graphene (0, 10, 20, 40, 60 μg mL−1). Cell Counting Kit-8 (CCK8, Beyotime) assays were conducted to evaluate the effect of graphene on cell viability according to the manufacturers protocol. Three samples were measured for each group (control, graphene with SNF, graphene without SNF). Confocal microscopy (Olympus FV10 inverted microscope, Nagano, Japan) was used to visualize cell morphology. After culturing with graphene for 24 h, the cells were stained with fluorescein isothiocyanate-phalloidin (Sigma-Aldrich, St. Louis, MO) and 4′,6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO) according to procedures reported previously.51 Representative fluorescence images of the stained samples were then obtained under excitation/emission at 358/462 nm and 494/518 nm, respectively. 2.6. Fabrication of Silk−Graphene Composite Films. Fresh silk solution and graphene dispersions were blended at different ratios and cast onto polystyrene Petri dishes (diameter 55 mm) to prepare transparent composite films. The solutions were dried at room temperature in a fume hood. Optical transmittance was measured with a UV−vis spectrometer (Cary 5000, Agilent, Santa Clara, USA) in the range of 400−1200 nm and extracted at a wavelength of 550 nm. The dried films were treated with methanol for 30 min to introduce water insolubility. The mechanical properties of the films were evaluated in hydrated conditions according to previous studies.52 The samples (10 mm in diameter and 5 mm in height) were first hydrated in distilled water for 4 h and then measured with a cross head speed of 2 mm min−1 at 25 °C using an Instron 3366 testing frame (Instron, Norwood, MA) with a 100 N loading cell. Five measurements were applied for every sample. 2.7. Statistical Analysis. SPSS v.18.0 software was used to perform statistical analyses. One-way ANOVA followed by the Student−Newman−Keuls test was used to compare the mean values of the data. Unless otherwise indicated, measurements are presented as means ± standard deviation.

have been used to prepare functional composite biomaterials because of strong interactions between silk proteins and various hydrophobic nanomaterials such as ferriferrous oxide, gold, and graphene.41−45 Compared with the proteins and peptides used in graphene exfoliation, including Vmh2,27 BSA,30 and I3C (IleIleIleCys),31 silk proteins are significantly less expensive and are already generated at an industrial scale (above 100 000 ton year−1 in China). Therefore, it seems reasonable to produce graphene with silk proteins as exfoliating and stabilizing agents. However, silk proteins are usually transformed into gels during the exfoliation process because of their metastability,46,47 resulting in the failure of utility of this protein in graphene production. Recently, hydrophobic silk nanofibers (SNFs) were prepared in aqueous solutions through tuning the assembly process, an approach that surmounts some of the inherent problems with stabilization of silk solutions.48,49 Here, SNFs were used to exfoliate graphene in aqueous solutions. These aqueous graphene dispersions were achieved with the nanofibers as exfoliating and stabilizing agents, showing higher graphene concentrations (>8 mg mL−1), yields (>30%), and production rates (>6 g h−1), as well as better stability in fetal bovine serum (FBS) solution than previously reported.29−33 SNFs could be removed through a centrifugation process to obtain graphene flakes with outstanding electrical properties. This simple mass production, low cost, and excellent performance through the use SNFs provides a useful approach for the scaled up production of graphene.

2. MATERIALS AND METHODS 2.1. Preparation of SNFs. SNFs were prepared via procedures reported previously.50 Fresh silk solution (6 wt %) obtained via traditional processing was slowly concentrated to above 20 wt % over 24 h to form metastable nanoparticles. The nanoparticle solutions were diluted to 0.5 wt % with distilled water and incubated for >24 h in sealed beakers at 60 °C until nanofiber formation. 2.2. Preparation of SNF-Stabilized Aqueous Graphene Dispersions. Natural graphite (purity >99%, 300 mesh) was purchased from Qingdao Haida Inc., China. Calculated amounts of graphite and SNF were added in a kitchen blender at specific blade speeds (5, 15, 30, 45 krpm) and volumes (100, 200, 400, 800 mL) to perform the exfoliation. The solution temperature was controlled to be below 40 °C during the exfoliation process. Samples were centrifuged at 1500 rpm for 45 min to remove unexfoliated graphite particles. The supernatant graphene dispersion was collected for further investigation, and the graphene concentration was determined by measuring absorbance at 660 nm (A660) of the suspension (Figure S1).14−34 Most of free SNFs were separated from the graphene dispersion by centrifuging twice at 14 000 rpm for 20 min. The SNFfree graphene was re-dispersed in water after sonication treatment. 2.3. Characterization of the Graphene Dispersions. The morphology of the samples was examined by atomic force microscopy (AFM, NanoScope V, Veeco, NY, USA), scanning electron microscopy (SEM, S-4800, Hitachi, Tokyo, Japan), and transmission electron microscopy (TEM, FEI, Hillsboro, USA). For AFM experiments, graphene suspensions were diluted to below 0.001 wt % to show the original morphology and 2 μL of the diluted solution was spin-coated onto freshly cleaved mica surfaces. The morphology of graphene was observed by AFM in air. A 225 μm long silicon cantilever with a spring constant of 3 N m−1 was used in tapping mode at 0.5 Hz−1 scan rate. The morphology of samples was also observed with SEM at 3 kV. Before investigation, 2 μL samples were added directly onto conductive tape, dried in air, and coated with platinum for 60 s. Samples for TEM were prepared by placing samples onto copper TEM grids and dried for 2 h in air. The samples were imaged at 200 kV. B


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3. RESULTS AND DISCUSSION 3.1. Graphite Exfoliation with SNF Solutions. Graphene exfoliation with a kitchen blender under benign conditions provides a promising approach for achieving large-scale production of graphene materials for biological applications. Studies have confirmed that the exfoliated graphene sheets are stabilized by electrostatic repulsive interactions with surfactants.30−35,53 Therefore, different biocompatible proteins that provide sufficient interactions with graphene and enough surface charge are suitable as exfoliating and stabilizing agents for producing graphene dispersions. Hydrophobic SNFs are mainly composed of beta-sheet structure, endowing superior stability and interactions with hydrophobic graphene.49 SNFs could also be dispersed in aqueous solution because of strong repulsive interactions from high surface charge density.49 Considering the multiple advantages of SNFs (hydrophobic features, water dispersibility, stability, high charge density, and biocompatibility) (Figure S2), SNFs were considered useful exfoliating and stabilizing agents for graphene production.24−28,49 Figure 1 shows a schematic of the process for the exfoliation of graphene using SNFs.

above 100 mL, stable graphene concentrations were achieved without a fall-off with 800 mL. According to the tendency, stable high graphene concentrations should be remained at higher volumes, which will be clarified in our following study. The result in terms of yield was of interest as it indicated the possibility for scaled up production of graphene in the future, which was superior to other exfoliating strategies including sonication and high shear mixers.32−34 At an optimal SNF concentration of 5 mg mL−1 and graphite concentration of 20 mg mL−1, a graphene dispersion with a concentration above 8 mg mL−1 was achieved at the rotation 45 krpm for 1 h and centrifugation at 1500 rpm for 45 min, significantly higher than that reported previously.32−34 The yields of the graphene dispersions under optimal conditions were calculated at different liquid volumes and were above 30%, which was also significantly higher than the maximum yield obtained by other protein-assisted exfoliation processes.24,30,35 Different studies achieved slight inferior concentrations and yields for the aqueous graphene dispersions, respectively,18,30−34 yet to the best of our knowledge, the present study obtained the highest concentrations and yields simultaneously, a production rate of 6 g of graphene h−1. Owing to the improved concentration and yield of aqueous graphene dispersions, as well as the inexpensive cost for the silk, SNFs were the better choice for graphite exfoliation compared with traditional proteins reported previously.24,30,35 3.2. Characterization of Graphene Suspensions. The changes in concentration of aqueous graphene dispersions under various temperatures and pH values were investigated to evaluate stability (Figure 3). Here, SNFs were used as the stabilizing agent. Because previous studies revealed superior stability of the nanofibers in serum, certain pHs, and temperatures,38,49 the stability of graphene dispersions was assessed in various conditions. The graphene dispersion (8 mg mL−1) in SNF solution showed outstanding stability, without significant decrease of concentration even after storage at 4 °C for above 1 month (Figure 3A). For some biological uses of graphene dispersions, the storage stability of graphene in 50% FBS solution at 4 °C was also checked at month and there was no detectable precipitation or aggregation (Figure 3B). There was also no reduction in the concentration of graphene at increasing temperatures from 4 to 60 °C (Figure 3C), indicating tolerance to temperature. Zeta potential was used to reveal the influence of pH on graphene stability; zeta potential changed from +10 to −40 mV when the pH was increased from 3 to 13 (Figure 3D). An isoelectric point for the dispersions was noted at about 4, which is similar to the isoelectric point of silk.55 The zeta potential results suggested that the observed negative charge of the dispersions was derived from the SNFs adhered on graphene surface (Table S1). When the graphene dispersions were incubated at room temperature for 1 week, the concentrations of graphene remained unchanged over a range of pHs (5−13) but decreased substantially because of aggregate formation at pH 4. The phenomenon confirmed the crucial role of electrostatic repulsion among SNFs in stabilizing the graphene dispersions. Although SNFs endowed the graphene dispersion with stability, it is necessary to remove the nanofibers when the graphene is used in various electrical applications.54 Highspeed centrifugation (14 000 rpm) was effective to destroy the physical interactions between the silk and graphene flakes. After centrifugation, TEM and SEM images showed the absence of SNFs (Figure 4A). FT-IR spectra (Figure 4B)

Figure 1. (A) Schematic of the process for exfoliation of graphene using SNFs and (B) type of blades used to generate the shear forces.

To test the feasibility of SNFs as exfoliating agents, shear exfoliation of pristine graphite was carried out with a kitchen blender in an aqueous solution of the SNFs. On the basis of previous studies,30,54 centrifugation at 1500 rpm for 45 min was used to remove the unexfoliated graphite, keeping the exfoliated graphene in the supernatant. The graphene concentration in the stable dispersions was calculated based on previous protocols (Figure S1).14−34 Different conditions, such as SNF concentration, graphite concentration, solution volume, shearing time, and shearing rates were optimized to achieve stable aqueous dispersions of graphene with higher concentrations (Figure 2). With increased SNF and graphite concentrations, the graphene concentration initially increased, peaked at a SNF concentration of 5 mg mL−1 and graphite concentration of 20 mg mL−1, and then decreased, similar to what occurred in other surfactant-assisted exfoliation processes.33,34 The graphene concentration also increased steadily with mixing time and blade rotation rate for all data sets studied. Considering application economy and limited speed options in a kitchen blender, 1 h and a rotation rate of 45 krpm were determined to produce suitable graphene dispersions. The effect of liquid volume was investigated between the range of 100−800 mL (graphite 20 mg mL−1, SNF 5 mg mL−1, rotation time 1 h, rotation rate 45 krpm). After the volume was C


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Figure 2. Concentration of graphene dispersions as a function of: (A) SNF concentration, when the graphite concentration is 20 mg mL−1, blade speed is 45 krpm, exfoliation time is 60 min, and the volume is 200 mL; (B) graphite concentration, when the SNF concentration is 5 mg mL−1, blade speed is 45 krpm, exfoliation time is 60 min, and the volume is 200 mL; (C) blade speed, when the SNF concentration is 5 mg mL−1, graphite concentration is 20 mg mL−1, exfoliation time is 60 min, and the volume is 200 mL; (D) exfoliation time, when the SNF concentration is 5 mg mL−1, graphite concentration is 20 mg mL−1, blade speed is 45 krpm, and the volume is 200 mL; (E) volume, when the SNF concentration is 5 mg mL−1, graphite concentration is 20 mg mL−1, blade speed is 45 krpm, and exfoliation time is 60 min. (n = 3, measurements are presented as means ± standard deviation).

Figure 4. (A) SEM and TEM images of graphene in the presence or removal of SNFs; (B) FT-IR spectra for different samples: graphite, pristine graphite; graphene, the exfoliated graphene after the removal of SNFs; SNFs, silk nanofibers; SNF-stabilized graphene, the exfoliated graphene before the removal of SNFs.

Figure 3. Changes with graphene dispersions when stored in: (A) deionized water, (B) 50% FBS, (C) different temperatures (0, 20, 40, 60 °C). (D) Zeta potential changes of graphene dispersions at different pHs. (n = 3, measurements are presented as means ± standard deviation).

confirmed the dislodging of the SNFs where the typical silk peaks at 1527 and 1630 cm−1 were almost gone from the spectra of graphene materials after the centrifugation treatment. The results indicated that the present strategy with SNFs as exfoliating and stabilizing agents, and their removal, was effective. Raman spectroscopy was used to characterize the graphene materials.4,12,22,53 Compared with the parent graphite particles, the exfoliated samples exhibited significantly different intensity and shapes at G (1579 cm−1), D (1346 cm−1), and 2D (2705 cm−1) bands in the spectra (Figure 5), suggesting the formation of typical graphene flakes. The shift of the 2D band is used to estimate the layers of graphene flakes.15,22 In contrast to the 2D band of the graphite with unsymmetrical

Figure 5. Raman spectra of exfoliated graphene and graphite: (A) overall Raman spectra of exfoliated graphene and graphite, (B) comparison of 2D peak between graphene and graphite.

shape, a broader and symmetric peak with the shift from 2714 to 2705 cm−1 appeared for the exfoliated graphene, which implied thin graphene flakes with fewer layers.54,56 Further increases in density of I2D/IG from 0.4 to 0.6 confirmed the presence of the few-layered graphene. The ratios of D band to G band (ID/IG) were used to evaluate the defect content in the graphene.15,22,56,57 Similar to several polymer-assisted-exfoliated graphene,30−34 an ID/IG intensity ratio of about 0.5 was D


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ACS Applied Materials & Interfaces achieved, indicating high quality for the graphene formed in the process, with few defects. The type of defects was deduced via the ratio of ID/ID′. The value of ID/ID′ in the exfoliated graphene was about 2.6, in the range of edge-type defects rather than sp3 defects.30,34 These results suggested that there was no oxidation during the exfoliation process, which would endow the graphene with high electrical properties. Therefore, the Raman results supported the effectiveness of the exfoliation process of graphite into few-layered graphene with high quality and few defects. Microscopy studies, including AFM and TEM, revealed the size and layers of graphene materials (Figure 6), showing thin

Figure 7. Performance of graphene dispersions: (A) fluorescent images of cells cultured with graphene for 24 h, scale bar = 150 μm; (B) CCK-8 results of the cells cultured with graphene for 24 h (n = 5, measurements are presented as means ± standard deviation, no significant difference); (C) interface and surface SEM images of graphene-coated paper; (D) conductivity of paper illustrated with an light-emitting diode circuit. Figure 6. Microscopic analysis of nanosheets found in graphene dispersions: (A) TEM image of graphene nanosheets, (B) lateral size distribution of graphene sheets, (C) AFM images of graphene nanosheets, and (D) thickness distribution calculated from AFM.

of the exfoliated graphene flakes were measured using standard four-point probe resistivity after the graphene dispersions were coated onto a cellulose paper to form homogeneous layers with a thickness of about 10 μm (Figure 7C). The conductivity of the graphene layer was calculated to be about 1100 S m−1 with a resistance (Rs) of 90 Ω sq−1. A test with a light-emitting diode, battery, and the graphene sheet confirmed the high conductivity of the graphene materials (Figure 7D). Although the conductivity of the graphene flakes was inferior to the graphene prepared via BSA-assisted exfoliation process, partly because of the existence of few defects and tiny remaining SNFs,30 the samples had superior features to most reduced graphene oxide materials reported in the literature and should be high enough for various biomedical applications.31,58 The graphene dispersions were blended with silk solutions prepared via traditional procedures36 to prepare composite silk films, confirming potential utility of the dispersions in biomedical fields. The films were transparent and retained 86% UV transmittance at 550 nm when containing 0.31 wt % graphene, which suggested homogeneous dispersion of the graphene inside silk matrix without aggregation (Figure 8A,B). Compared with graphene-free silk films, the composite films containing only 0.31 wt % graphene flakes exhibited significantly higher mechanical properties, with tensile strength and elongation at break increased from 14.5 MPa and 165.8% to 22.4 MPa and 260.9%, respectively (Figure 8C and Table S2). The mechanical properties of the composite films then decreased when more graphene sheets were added to the films (Table S2), which is consistent with that happened in other graphene−polymer composite materials.34,56 It is a common

nanosheets with submicron lateral sizes. Figure 6B represents the histogram of the sheet sizes measured from 100 sheets from different TEM images. The broader distribution from 100 to 800 nm appeared for the graphene, which was similar to few-layered graphene prepared by other turbulence-assisted exfoliation processes.30,32−34 Figure 6C is a typical AFM image of graphene flakes, indicating lateral sizes of 100−800 nm. About 100 randomly selected sheets were measured to calculate thickness, and the majority of the graphene sheets were 1−4 nm, which suggested the few-layered structures. Superior mass production, biological friendly fabrication, and defect-free few-layered structures of graphene dispersions suggest promising utility in biomedical applications.6−12 Previous studies have confirmed that SNFs had good cytocompatibility and have been used in various tissue regeneration and drug delivery studies.37−40,48,49 BMSCs were cultured with the graphene dispersions here, to evaluate cytocompatibility in vitro. Figure 7A shows excellent cell viability after culturing with the graphene samples in the presence and absence of SNFs for 24 h. The CCK-8 assays indicated that the BMSCs remained >80% viable at concentrations of 60 μg mL−1 (Figure 7B), which is higher than graphene oxide (GO),31 confirming cytocompatibility. Compared with graphene oxide materials, the pristine graphene usually had better conductivity.53 The conductivity E


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TGA curve of SNF-stabilized graphene; calibration plot used to calculate the absorbance (660 nm) of the biofunctionalized graphene; SEM, AFM images, and FTIR spectra of SNFs; mechanical properties of composite films containing various ratios of graphene; and zeta potential of SNF, SNF-stabilized graphene, and graphene dispersions (PDF)

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Corresponding Author

*E-mail: [email protected]. Phone: (+86)-51267061649. ORCID

Qiang Lu: 0000-0003-4889-5299 David L. Kaplan: 0000-0002-9245-7774

Figure 8. Performance of silk−graphene composite films: (A) macrograph images of composite films containing different graphene contents of 0, 0.15, and 0.31 wt %, respectively; (B) transmittance of composite films at 550 nm, and (C) stress−strain curves of pure silk films and composite films containing 0.31 wt % graphene in the wet state.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the National Key Research and Development Program of China (2016YFE0204400), NIH (R01NS094218, R01AR070975) and the AFOSR.

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phenomenon for the composite materials where redundant fillers would reduce the mechanical properties. Therefore, further study is necessary to clarify the optimal content of the graphene sheets in silk films. Mass production of high-quality graphene under mild conditions using inexpensive raw materials and apparatuses without toxic chemicals or solvents is desired for biomedical applications. The graphite was successfully exfoliated into graphene dispersions with SNFs as exfoliating and stabilizing agents, achieving optimization of both concentration and yield. The exfoliated graphene showed desirable few-layered structures, cytocompatibility, and feasibility for developing composite materials, implying potential utility in biological optical and electronic devices. The combination of these attributes, in addition to the abundance and low cost of silk proteins, makes the SNF-assisted exfoliation system promising for further development.

4. CONCLUSIONS Inexpensive silk proteins were assembled into hydrophobic nanofibers with a high negative charge density for optimizing mass production of aqueous graphene dispersions. The highquality graphene dispersions with concentrations >8 mg mL−1 and yields of 30% were achieved under mild aqueous environments with a kitchen blender, suggesting the future potential for scale up. These dispersions also showed long-term stability under various conditions, consisted of a few-layered structures with fewer defects, and satisfied electrical and cytocompatible properties, most of which were superior to widely used GO/reduced GO. Considering the cost and abundance silk, as well as the inexpensive apparatus and absence of organic or toxic solvents and chemicals, the exfoliation system with SNFs provides a promising avenue for producing graphene on a larger scale.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b04777. F


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Mass Production of Biocompatible Graphene Using Silk Nanofibers

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