Highly Stretchable and Notch-Insensitive Hydrogel...

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Highly Stretchable and Notch-Insensitive Hydrogel Based on Polyacrylamide and Milk Protein Jinwoo Ma, ,Jaehun Lee, ,Sang Sub Han, Kyu Hwan Oh, Ki Tae Nam,* ,and Jeong-Yun Sun* ,,§ Department of Material Science and Engineering, Seoul National University, Seoul 151-742, South Korea § Research Institute of Advanced Materials (RIAM), Seoul National University, Seoul 151-744, South Korea * S Supporting Information ABSTRACT: Protein-based hydrogels have received attention for biomedical applications and tissue engineering because they are biocompatible and abundant. However, the poor mechanical properties of these hydrogels remain a hurdle for practical use. We have developed a highly stretchable and notch-insensitive hydrogel by integrating casein micelles into polyacrylamide (PAAm) networks. In the casein- PAAm hybrid gels, casein micelles and polyacrylamide chains synergistically enhance the mechanical properties. Casein-PAAm hybrid gels are highly stretchable, stretching to more than 35 times their initial length under uniaxial tension. The hybrid gels are notch-insensitive and tough with a fracture energy of approximately 3000 J/m 2 . A new mechanism of energy dissipation that includes friction between casein micelles and plastic deformation of casein micelles was suggested. KEYWORDS: tough hydrogels, polyacrylamide, casein, protein hydrogels, notch-insensitive hydrogels, energy dissipations P rotein-based hydrogels are attractive candidates for biomedical applications 1,2 and tissue engineering 35 because of their unique properties such as biocompatibility, responsiveness to various kinds of stimuli, and abundance. However, hydrogels based on protein generally show low stretchability and brittle properties with low toughness. 69 A few strategies have been attempted to enhance the mechanical properties of the protein-based hydrogels, such as force- unfolded protein hydrogels, 10 human tropoelastin-graphene oxide composite hydrogels, 11 and silk microber-reinforced silk hydrogel. 12 However, the mechanical properties of these gels are still insucient for applications requiring large deformations and high toughness. To toughen a hydrogel, researchers have suggested various mechanisms over the last few decades, such as double network hydrogels, 13 nanocomposite hydrogels, 14 macromolecular microsphere composite hydrogels, 15 and polyampholyte hydro- gels. 16 The double-network hydrogels are composed of primary networks (highly cross-linked) and secondary networks (loosely cross-linked). The secondary networks contribute shape retention, and the primary networks dissipate the energy applied to the gels. 17,18 The nanocomposite hydrogels exhibited high stretchability due to the high functionality of the exfoliated nanoclay. Multiple polymer chains are connected to two adjacent high-functionality cross-linkers, and these chains usually have nonuniform lengths. Relatively long chains maintain the elasticity of the nanocomposite hydrogels and the short chains participate in energy dissipation. Alternately, a macromolecular microsphere can be used as both an initiator and a cross-linker, similar to the nanocomposite. Polyampho- lyte hydrogels have both strong ionic bonds and weak ionic bonds between long ampholyte polymer chains. In this structure, the strong ionic bonds maintain the shape of the gels, and the weak ionic bonds dissipate energy by debonding. Therefore, the key factors to toughen a hydrogel are shape retention and energy dissipation. 1922 Casein is a major protein component of bovine milk, consisting of α s1 -, α s2 -, β-, and κ-caseins in approximate relative amounts of 4:1:3.5:1.5, respectively. Casein possesses several benecial characteristics suitable for biomaterials, such as good biodegradability and availability of reactive sites for chemical modication. 23,24 Casein forms a micellar structure in a hydrated state. Most of the κ-casein, which is a hydrophobic compound, is found on the surfaces of the casein micelles. 25 With acidication, which decreases the negative chargecharge repulsion on the casein micelle, casein forms a 3-dimensional (3D) coagulated structure by a hydrophobic association. A casein micelle consists of a number of submicelles which can be rearranged by applying a shear force. By this rearrangement of submicelles, a casein micelle can possibly deform plastically and as a result might have the capability to dissipate energy. Casein micelles would be good candidates for sacricial architecture in tough hydrogels. Previously, hydrogels composed of PAAm and casein protein have been reported for the purpose of drug release. 26 However, these hydrogels only possessed a PAAm Received: August 30, 2016 Accepted: October 17, 2016 Published: October 17, 2016 Letter www.acsami.org © 2016 American Chemical Society 29220 DOI: 10.1021/acsami.6b10912 ACS Appl. Mater. Interfaces 2016, 8, 2922029226

Transcript of Highly Stretchable and Notch-Insensitive Hydrogel...

Highly Stretchable and Notch-Insensitive Hydrogel Based onPolyacrylamide and Milk ProteinJinwoo Ma,†,‡ Jaehun Lee,†,‡ Sang Sub Han,‡ Kyu Hwan Oh,‡ Ki Tae Nam,*,‡ and Jeong-Yun Sun*,‡,§

‡Department of Material Science and Engineering, Seoul National University, Seoul 151-742, South Korea§Research Institute of Advanced Materials (RIAM), Seoul National University, Seoul 151-744, South Korea

*S Supporting Information

ABSTRACT: Protein-based hydrogels have received attention for biomedicalapplications and tissue engineering because they are biocompatible and abundant.However, the poor mechanical properties of these hydrogels remain a hurdle forpractical use. We have developed a highly stretchable and notch-insensitive hydrogelby integrating casein micelles into polyacrylamide (PAAm) networks. In the casein-PAAm hybrid gels, casein micelles and polyacrylamide chains synergistically enhancethe mechanical properties. Casein-PAAm hybrid gels are highly stretchable, stretchingto more than 35 times their initial length under uniaxial tension. The hybrid gels arenotch-insensitive and tough with a fracture energy of approximately 3000 J/m2. A newmechanism of energy dissipation that includes friction between casein micelles andplastic deformation of casein micelles was suggested.

KEYWORDS: tough hydrogels, polyacrylamide, casein, protein hydrogels, notch-insensitive hydrogels, energy dissipations

Protein-based hydrogels are attractive candidates forbiomedical applications1,2 and tissue engineering3−5

because of their unique properties such as biocompatibility,responsiveness to various kinds of stimuli, and abundance.However, hydrogels based on protein generally show lowstretchability and brittle properties with low toughness.6−9 Afew strategies have been attempted to enhance the mechanicalproperties of the protein-based hydrogels, such as force-unfolded protein hydrogels,10 human tropoelastin-grapheneoxide composite hydrogels,11 and silk microfiber-reinforced silkhydrogel.12 However, the mechanical properties of these gelsare still insufficient for applications requiring large deformationsand high toughness.To toughen a hydrogel, researchers have suggested various

mechanisms over the last few decades, such as double networkhydrogels,13 nanocomposite hydrogels,14 macromolecularmicrosphere composite hydrogels,15 and polyampholyte hydro-gels.16 The double-network hydrogels are composed of primarynetworks (highly cross-linked) and secondary networks(loosely cross-linked). The secondary networks contributeshape retention, and the primary networks dissipate the energyapplied to the gels.17,18 The nanocomposite hydrogels exhibitedhigh stretchability due to the high functionality of the exfoliatednanoclay. Multiple polymer chains are connected to twoadjacent high-functionality cross-linkers, and these chainsusually have nonuniform lengths. Relatively long chainsmaintain the elasticity of the nanocomposite hydrogels andthe short chains participate in energy dissipation. Alternately, amacromolecular microsphere can be used as both an initiatorand a cross-linker, similar to the nanocomposite. Polyampho-

lyte hydrogels have both strong ionic bonds and weak ionicbonds between long ampholyte polymer chains. In thisstructure, the strong ionic bonds maintain the shape of thegels, and the weak ionic bonds dissipate energy by debonding.Therefore, the key factors to toughen a hydrogel are shaperetention and energy dissipation.19−22

Casein is a major protein component of bovine milk,consisting of αs1-, αs2-, β-, and κ-caseins in approximate relativeamounts of 4:1:3.5:1.5, respectively. Casein possesses severalbeneficial characteristics suitable for biomaterials, such as goodbiodegradability and availability of reactive sites for chemicalmodification.23,24 Casein forms a micellar structure in ahydrated state. Most of the κ-casein, which is a hydrophobiccompound, is found on the surfaces of the casein micelles.25

With acidification, which decreases the negative charge−chargerepulsion on the casein micelle, casein forms a 3-dimensional(3D) coagulated structure by a hydrophobic association. Acasein micelle consists of a number of submicelles which can berearranged by applying a shear force. By this rearrangement ofsubmicelles, a casein micelle can possibly deform plastically andas a result might have the capability to dissipate energy. Caseinmicelles would be good candidates for sacrificial architecture intough hydrogels. Previously, hydrogels composed of PAAm andcasein protein have been reported for the purpose of drugrelease.26 However, these hydrogels only possessed a PAAm

Received: August 30, 2016Accepted: October 17, 2016Published: October 17, 2016

Letter

www.acsami.org

© 2016 American Chemical Society 29220 DOI: 10.1021/acsami.6b10912ACS Appl. Mater. Interfaces 2016, 8, 29220−29226

network without the 3D structure of casein. Hence, mechanicalenergy dissipation was not possible.In this study, we have presented a simple method for the

synthesis of tough casein-polyacrylamide hydrogels. The casein-PAAm hydrogel was prepared by an acidification of caseinmicelles and a radical polymerization of acrylamide (AAm). Weintroduced a new strategy for toughening a gel: deformationenergy was dissipated by friction between the micelles and byplastic deformation of the micelles. The process of synthesiswas described, and the mechanical properties of the synthesizedhydrogels were explored.

■ EXPERIMENTAL SECTIONCasein-PAAm hydrogels were prepared by a free radical polymer-ization of AAm and a coagulation of casein micelles, as shown inFigure S1. An aqueous solution of AAm with N,N′-methylenebis-(acrylamide) (MBAA) was prepared. A casein solution was preparedby dissolving casein powder in deionized (DI) water. The pH of thecasein solution was adjusted to 6.6 by the addition of NaOH solution.After the two stock solutions were mixed, glucono delta-lactone(GDL) powder was added as an acidifier to gradually decrease the pH.Ammonium persulfate (APS) as the radical initiator and N,N,N′,N′-tetramethylenediamine (TEMED) as the cross-linking accelerator forAAm were added to the solutions. Subsequently, the solutions werepoured into a glass mold. The solutions were then cured with 8 W, 254nm wavelength UV light for 2 h and left for 24 h for casein gelationbefore mechanical testing.Other details regarding the materials, the detail synthetic process,

the mechanical tests, the scanning electron microscope (SEM)

observation, the swelling experiments in DI water and phosphate-buffered saline (PBS), and the transmission electron microscope(TEM) observation are supplied in the Supporting Information.

■ RESULT AND DISCUSSIONWe have synthesized stretchable and notch-insensitive hydro-gels by integrating two types of polymers. Coagulation of thecasein micelle is followed by a radical polymerization to formpolyacrylamide, as shown in Figure S1. Casein shows a uniquemicellar structure in a hydrated state. κ-casein, which has anegatively charged hairy layer under neutral pH, is located onthe surface of the casein micelles, such that a repulsive forcebetween casein micelles contributes to the steric stabilization.With acidification, the net negative charge decreases, decreasingboth the electrostatic repulsion and the steric stabilization.Therefore, the charged hairs shrink and casein micellescoagulate through a local hydrophobic association. Due tothe hydrophobicity of the casein micelle, degree of the swellingof the hybrid gels decreased as the amount of the caseinincreased in the casein-PAAm hydrogels (Figure S2). It shouldbe noted that the PAAm chains were formed by radicalpolymerization before coagulation of the casein micelles toallow synthesis of the casein-PAAm hydrogel without globalphase separation.As shown in Figure 1, the casein-PAAm hydrogels were

highly stretchable and sufficiently tough to be used for complexdeformations. The deformation behaviors under various stressconditions were very stable even though the initial thickness of

Figure 1. Highly stretchable casein-polyacrylamide (PAAm) hydrogel. Casein-PAAm hydrogel was (a) knotted, and (b) stretched up to 13 times ofits initial length. (c, d) Square casein-PAAm hydrogel shows 2500% of areal expansion under biaxial tension. (e) Cylinder was covered with the gelmembrane. The cylinder was lubricated with silicone oil. (f) Stress−stretch curves of tensile tests for casein-PAAm hydrogels with various weightratio α. (g) Elastic modulus and rupture stretch of casein-PAAm gels (error bars, S.D.; n = 3). Casein-PAAm hydrogel samples with a polymer ratioof α = 40% (weight percentage of casein to casein plus acrylamide) were used for optical images.

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the samples was only 3 mm. It can be seen that knotted casein-PAAm hydrogels elongated to more than 13 times of theiroriginal length without fracture (Figure 1a, b). Furthermore,the areal strain of the casein-PAAm hydrogel exceeded 2500%(Figure 1c, d). The casein-PAAm hydrogel was also suitable forcomplex deformations. A gel with a diameter of only 90 mmwas used to cover a cylinder with a diameter of 70 mm and aheight of 210 mm (Figure 1e). Those experiments demon-strated that the casein-PAAm hydrogel was stable under thevarious deformation conditions without any distinct failure. Thecasein-PAAm hydrogels are extremely stretchable and can beapplied to various stress states.We performed a tensile test to observe the mechanical

properties of the hydrogels (Figure 1f inset). Casein-PAAmhydrogel could be stretched to over 30 times of its originallength without any visible rupture (Figure 1f). We studied thecasein-PAAm hydrogels with different casein to casein plusacrylamide weight ratios (Figure 1f, g). α is the weightpercentage of casein to casein plus acrylamide. The stretch atrupture increased dramatically in the case of 32 wt % of α. Arigid hydrogel cannot be formed for α > 40 wt % due to the lowAAm contents. There was little effect of variation ofcomposition on the elastic modulus of the casein-PAAmhydrogels at small strain. Because of the very low concentrationof cross-linker, the casein-PAAm hydrogels show low elasticmodulus (<12 kPa). To investigate the effects of chemicalcross-linking of PAAm, we measured mechanical properties forthe casein-PAAm hydrogel with various MBAA concentrations

(Figure S3a, b). As the concentration of MBAA increased, thestiffness of the casein-PAAm hydrogel increased. However, thestretchability of the casein-PAAm hydrogel decreased dramat-ically with increasing concentration of MBAA, showing thehighest stretchability with the minimum MBAA concentration.The hydrogels demonstrate a trade-off between the elasticmodulus and stretchability. In addition, notch-insensitivity ofcasein-PAAm hydrogels tended to decline as the concentrationof MBAA increased. Mechanical properties were highly affectedby the pH of the casein-PAAm hydrogels (Figure S4). Rupturestretch and strength of the casein-PAAm hydrogels decreased asthe pH of the casein stock increased. This occurs becausecasein micelle cannot build a strong network at high pHcondition. The low pH required for favorable synthesis of thehybrid gel would be a hurdle for applications underphysiological conditions. In Figure S5, we compared thecasein-PAAm hydrogel with other recently reported toughhydrogels which dissipate energy by physical association. Thecasein-PAAm hydrogels show higher stretchability but lowerstiffness compared to other tough hydrogels.27−31

A 90% strain compressive test was conducted to qualitativelyshow the toughness of casein-PAAm hydrogels, compared to itspure parent gels. Both pure acrylamide hydrogel and purecasein hydrogel were totally broken and crushed after the 90%strain compressive test (Figure 2a−f). However, there was novisible damage to the casein-PAAm hydrogel at 90%compression (Figure 2g−i). When compressive loading isapplied to a gel, cracks propagate by the highly concentrated

Figure 2. Compression tests for (a−c) PAAm, (d−f) casein, and (g−i) casein-PAAm hybrid gels up to 90% of compressive strain. The PAAm geland the casein gel showed brittle failures and plastic deformations after the compression, respectively. Casein-PAAm hybrid gels recovered theiroriginal lengths after 90% compression without failures. α of the hybrid gel was 40%.

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stress at small internal or external cracks. Single networkhydrogels, such as the pure PAAm and the pure caseinhydrogels in this study, are susceptible to crack propagationbecause there is no mechanism to relieve the concentratedstress in those materials. That is, the small cracks in thosehydrogels can easily turn into running cracks. In contrast, acrack in the casein-PAAm hydrogel cannot turn into runningcrack but it is blunted immediately. When stress is applied tothe gels, hydrophobic associations between the casein micellesstart to break, and the casein micelles might deform plasticallyby shear force. This energy dissipation makes the crack edgelessand the hydrogels tough.We stretched the casein-PAAm hydrogel with 5 notches (3

internal notches, 2 external notches, 5 mm length each) todemonstrate the notch-insensitivity of casein-PAAm hydrogel(Figure 3a). The notched casein-PAAm hydrogel was stretchedto over 28 times of its initial length. When casein-PAAmhydrogel was stretched, the notch tips were blunted, and theyremained in a stable state. Additionally, we prepared the casein-PAAm hydrogels with notch sizes ranging from 15 to 60 mm(Figure 3b) and measured the extension properties of thosehydrogels. The tensile load−extension curves were adoptedinstead of the conventional stress−stretch curve. Because thedeformations of notched samples were not homogeneous, thestress field of the sample was nonlinear through the entire area.It was surprising that the rupture stretch of casein-PAAmhydrogel is almost constant, insensitive to the notch length.As notch-insensitivity results from the high fracture energy

and large plastic zone near the crack tips of the casein-PAAmhydrogels, we tried to measure the fracture energies of casein-

PAAm hydrogels. The work, U, done by the applied force tostretch the gel to a length L was calculated by integrating thearea under the tensile load−extension curve in Figure S6. Thework U was then plotted against the notch length c for thecasein-PAAm hydrogels with different casein to casein plusacrylamide weight ratios. L was defined as the length stretchedwhen the notch turned into a running crack. In the presentwork, the notch became a running crack at the end of stretchingand crack growth occurs just before the fracture of the notchedgels. Thus, L was directly determined as the increase in thelength approaching the end of the stretching process of the gel.The fracture energy Γ was calculated from eq 1 according tothe method of Rivlin and Thomas (Figure 3c, d).

Γ = ∂∂

⎜ ⎟⎛⎝

⎞⎠b

Uc

1

L0 (1)

where b0 is the initial thickness of the casein-PAAm hydrogel

sample (3 mm) and ∂∂( )U

c Lis the slope of the U vs c curve at

specified L. Calculated fracture energies increased drastically at32 wt % casein because of its high stretchability and notch-insensitivity. The specific values of L for various α are shown inFigure S6.We suggest that the internal structure and intermolecular

behaviors of casein-PAAm hydrogels (Figure 4a) are respon-sible for the high toughness and notch-insensitivity of casein-PAAm hydrogels, To investigate possible cross-links betweenthe two types of polymers, we analyzed the casein gel,polyacrylamide gel, and casein-PAAm hydrogels with variouscasein to (casein plus acrylamide) weight ratio, α (Figure S7)

Figure 3. Notch-insensitive fracture in a casein-PAAm hydrogel. (a) Strip of the casein-PAAm hydrogel was pulled uniaxially with 5 initial notches.Each notch was 5 mm in length. Casein-PAAm hydrogel samples with a polymer ratio of α = 40% were used. (b) Load−displacement curves fromfracture tests with various initial crack sizes. An edge crack was introduced to a casein-PAAm hydrogel samples with a polymer ratio of α = 40%. Theinitial length between the two clamps was L0 = 5 mm. (c) Total energy, U, stored in the test piece at an extension ΔL was calculated by integratingthe area under the load-extension curves. Various polymer ratios, α = 0, 16, 32, and 40% were tested. The load-extension curves for α = 0, 16, and32% are shown in Figure S6. (d) Fracture energy of casein-PAAm hydrogels as a function of polymer ratio α (error bars, S.D.; n = 3).

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using Fourier transform infrared (FT-IR) spectroscopy. Theabsence of new peaks in the FT-IR spectra of casein-PAAmhydrogels suggested that there was no strong intermolecularcovalent bonding between the PAAm chains and caseinmicelles. However, hydrogen bonding might exist betweentwo networks. In the casein-PAAm hydrogels, the caseinmicelles are integrated into the PAAm network. The acrylamidechains form covalent cross-links with MBAA, and the caseinmicelles agglomerated into the 3D structure by hydrophobicassociations. When a deformation was applied to the casein-PAAm hydrogels, friction between casein micelles and a plastic

deformation of the casein micelle dissipated the energy (Figure4b).To demonstrate the dissipation mechanism of the casein

micelles, we changed the amount of casein in the casein-PAAmhydrogels at a constant PAAm concentration (Figure 4c). Wefixed the amount of PAAm at 7.5 wt %, and the amount ofcasein was increased from 0 wt % to 6 wt %. As shown in Figure4c, the stretchability of the casein-PAAm hydrogel dramaticallyincreased between 2 and 4 wt % casein in the gel. Thisindicated that the amount of casein highly affected the cross-linking density and the amount of energy dissipated. We believethere was a threshold amount of casein to obtain enough

Figure 4. Schematic illustration of internal structure and toughening mechanism of casein-PAAm hydrogels. (a) Schematic illustration of internalstructure of casein-PAAm hydrogels. (b) Casein micelles form a coagulated structure in a PAAm network due to hydrophobic association. Energydissipation may occur between the coagulated casein micelles during a deformation. (c) Concentration of the casein micelle influences themechanical properties of casein-PAAm hydrogels. The concentration of PAAm was fixed as 7.5 wt %. (d) Bright-field transmission electronmicroscopy (TEM) images of casein micelles. (e) Cyclic loading of casein-PAAm hydrogel showing energy dissipation during a deformation. (f)After a cyclic loading with a maximum stretch of 7, the gel was immediately reloaded to rupture.

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energy dissipation between casein micelles. As the size of acasein micelle is approximately 150 nm (Figure 4d), theintermicelle distance highly affects the bonding density and thetotal bonding energy. We carried out a rheological character-ization to investigate the viscoelastic properties of the gels(Figure S8). The loss modulus of the casein hydrogel wasrelatively higher than that of the PAAm hydrogel. This impliesthat the casein micelle behaved plastically and dissipated energyin the hybrid gels. Additionally, the fracture energy of thecasein-PAAm hydrogels decreased at high stretching rates(Figure S9). This result suggested that the area of the energydissipating zone around the crack tip shrank under rapiddeformation. The casein-PAAm hydrogels dissipated energyeffectively, as shown by the hysteresis (Figure 4e). The casein-PAAm hydrogel displayed stress softening at the secondloading. Stress softening on reloading indicates that energy wasdissipated and that casein micelles were plastically deformedduring the first tensile cycle. However, rupture stretch did notchange, although the gels were unloaded from a stretch of 7 andreloaded immediately (Figure 4f). This meant that the flawsensitivity of the gel was not changed by cycling.In summary, we present a simple method to synthesize

highly stretchable, notch-insensitive and tough casein-PAAmhydrogels. Casein-PAAm hydrogel was formed by the acid-ification of casein micelles and UV cross-linking of acrylamide.Although pure casein and pure PAAm hydrogels fractured in abrittle manner, the casein-PAAm hydrogels can be deformedand stretched over 35 times their original length and exhibited ahigh fracture energy (∼3000 J/m2). We suggest that the shaperetention of the acrylamide network and the energy dissipationof casein micelles contributed to these enhanced mechanicalproperties of the gels. The energy dissipation could occur bytwo mechanisms: friction between casein micelles and plasticdeformation of casein submicelles. This novel energydissipation mechanism provides a better understanding ofhow to design a tough hydrogel. Thus, it can broaden currenthydrogel studies and applications.

■ ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsami.6b10912.

Materials, detail synthetic proceess, mechanical tests,scanning electron microscope (SEM) observation,swelling experiments in DI water and phosphate-bufferedsaline (PBS), transmission electron microscope (TEM)observation, FT-IR spectroscopy, and rheological char-acterization of the gels (PDF)

■ AUTHOR INFORMATION

Corresponding Authors*E-mail: [email protected].*E-mail: [email protected].

Author Contributions†J.M. and J.L. contributed equally. The manuscript was writtenthrough the contributions of all authors. All authors have givenapproval to the final version of the manuscript.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This work was supported by the National Research Foundationof Korea (NRF) Grant funded by the Korean Government(MSIP) (2015R1A5A1037668 and No.2016R1C1B2007569).J.-Y.S. and J.M. acknowledge the support from the sourcetechnology and materials funded by Ministry of Trade, Industryand Energy of Korea (MOTIE) (10052783).

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ACS Applied Materials & Interfaces Letter

DOI: 10.1021/acsami.6b10912ACS Appl. Mater. Interfaces 2016, 8, 29220−29226

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