Antibacterial [2-(Methacryloyloxy) ethyl] Trimethylammonium Chloride

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Antibacterial [2-(methacryloyloxy) ethyl] trimethylammonium chloride functionalized reduced graphene oxide/poly (ethyleneco-vinyl alcohol) multilayer barrier film towards food packaging Hualin Wang, minmin chen, Chongyang Jin, Baicheng Niu, Suwei Jiang, xingjiang li, and Shaotong jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04784 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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Journal of Agricultural and Food Chemistry

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Antibacterial

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functionalized reduced graphene oxide/poly (ethylene-co-vinyl alcohol) multilayer

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barrier film towards food packaging

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Hualin Wang†, § , Minmin Chen†, Chongyang Jin†, Baicheng Niu†, Suwei Jiang†, Xingjiang Li‡, §, Shaotong Jiang‡,

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†School of Chemistry and Chemical Engineering and ‡ School of Food Science and Engineering, Hefei University

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of Technology, 230009, Hefei, Anhui, P. R. China

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§

[2-(methacryloyloxy)

ethyl]

trimethylammonium

chloride

∗

§

Anhui Institute of Agro-Products Intensive Processing Technology, 230009, Hefei, Anhui, P. R. China

9 10

∗

Corresponding author. E-mail: [email protected] 1

ACS Paragon Plus Environment


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Abstract: The objective of present work was to construct antibacterial [2-(methacryloyloxy) ethyl]

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trimethylammonium chloride functionalized reduced graphene oxide/poly (ethylene-co-vinyl

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alcohol) (MTAC-rGO/EVOH) multilayer barrier films by using layer-by-layer assembly under a

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parallel electric field. Besides the barrier and mechanical properties, the film antibacterial

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activities and cytotoxicity of MTAC-rGO nanosheets were extensively investigated. The

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functionalization of rGO was achieved by grafting MTAC onto graphene framework through C

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(sp3)-C bonds. The assembly of MTAC-rGO on EVOH matrix not only significantly improved

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film mechanical strength, but also endowed the targeting film with outstanding moisture barrier

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even under a relative humidity of 99 % (e.g., 0.019 g m-2 s-1 atm-1 for (MTAC-rGO/EVOH)20 )

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besides good oxygen barrier (e.g., 0.07 cm3 m-2 d-1 atm-1 for (MTAC-rGO/EVOH)20). Among the

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testing films, MTAC-rGO/EVOH film had the best antibacterial activity, and the activity against S.

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aureus was better than E. coli. Meanwhile, the cytotoxicity of MTAC-rGO nanosheets was very

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low. Results suggest that MTAC-rGO/EVOH film may have great potential in food active

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packaging.

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KEYWORDS: layer-by-layer assembly, barrier, antibacterial activity, functionalized reduced

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graphene oxide, active packaging

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INTRODUCTION

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Barrier film with good antibacterial activity has attracted considerable attention in food

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packaging due to its synergistic effects in sustaining the safety and quality of packaged food

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products.1-4 Ethylene-co-vinyl alcohol (EVOH) has been widely used in food packaging due to its

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good oxygen barrier5 and mechanical strength.6,

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activity and its moisture barrier is poor due to the moisture sensitivity of –OH groups in the matrix,

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which have limited the application of EVOH in food packaging to a certain extent.

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However, EVOH film has no antibacterial

EVOH films can be endowed with antibacterial activity by introducing various antimicrobials

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such as silver,8 lysozyme

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emerged as a new effective approach to prevent microbial proliferation, indicating that the load of

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some metallic ions or metal oxides (e.g., Ag+, ZnO, and MoS2) on graphene could enhance the

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antibacterial activities of graphene;11-17 in addition, a few groups improved antibacterial activities

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of graphene by grafting some non-metallic antibacterial moieties (e.g., chlorophenyl18,

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guanidine,19, 20 quaternary phosphonium salts21 and N-halamines22) onto graphene frame. Known

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as the active moieties against microorganisms by interaction with the cell membrane, quaternary

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ammonium salts have displayed great potential application in food preservatives and drug delivery

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system.23-26 Representatively, Ye et al. fabricated reduced graphene oxide/quaternary ammonium

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salt (dodecyl dimethyl benzyl ammonium chloride and bromohexadecyl pyridine) nanocomposites

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with long-term antibacterial activity prepared by using non-covalent modification.25

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[2-(methacryloyloxy) ethyl] trimethylammonium chloride (MTAC) is a quaternary ammonium salt

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monomer with a reactive methacryloyl group, which is easily subjected to radical polymerization.

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As a result, the antibacterial moiety MTAC can be grafted onto graphene by covalent

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and chitosan10. Antimicrobial graphene-based nanomaterials has

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modification.

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Graphene has displayed a great potential in the improvement of film barrier due to the large

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aspect ratio of nanosheet.27, 28 As it is well known, there are numerous hydrophilic groups (e.g.

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hydroxyl, epoxide, and carboxyl groups) on the graphene oxide (GO). Those hydrophilic groups

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are not beneficial in increasing the moisture barrier of films29. To overcome this drawback, we can

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use reduced graphene oxide (rGO) instead of GO30 or graft hydrophobic groups (e.g.,

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triethoxysilylpropyl,31 methoxybenzene,32 iodophenyl33 and aminophenyl groups34) on the

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graphene framework in the fabrication of graphene-based barrier film. Furthermore, multilayer

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structure is also an effective strategy to enhance the barrier of film. The layer-by-layer (LbL)

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assembly technique is a simple, inexpensive, and versatile process to construct highly ordered

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multilayer structures,35 which has been used for the assembly of graphene-based nano-platelets on

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polyethyleneimine substrate36, 37 and poly (ethylene terephthalate) substrate38 to obtain multilayer

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barrier films. As the driving forces for LbL assembly process are mainly from non-covalent

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interactions such as electrostatic attraction, hydrogen-bonding, van der Waals forces,

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charge-transfer complexes, and hydrophobic -hydrophobic attractions.39 Therefore, most of the

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substrates are restricted to some ionized or strong polar polymers. If the substrates are under

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parallel electric field at a high voltage, the charged nano-platelets in suspension can be assembled

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and oriented on them. Then the substrates may be extended to weak polar or non-polar polymers

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such as EVOH32.

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In order to achieve antibacterial and barrier synergistic effects for food packaging, the MTAC

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functionalized reduced graphene oxide/poly (ethylene-co-vinyl alcohol) (MTAC-rGO/EVOH)

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multilayer films were constructed in the present work. The LbL assembly of the positively charged 4


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MTAC-rGO on EVOH substrate was achieved under a parallel electric field. Besides barrier and

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mechanical properties, the antibacterial activities of MTAC-rGO/EVOH films and the cytotoxicity

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of the MTAC-rGO were extensively investigated.

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MATERIALS AND METHODS

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Materials. Poly (vinyl alcohol-co-ethylene) (EVOH, Soarnol®, containing 32 mol % of

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ethylene) was provided by The Nippon Synthetic Chemical Industry Co., Ltd. (Nippon Gohsei)

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(Osaka, Japan). [2-(methacryloyloxy) ethyl] trimethylammonium chloride (MTAC, purity 75 wt%

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in H2O) and hydrazine hydrate (purity ≥ 98%) were purchased from Aladdin Chemical Reagent

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Co., Ltd. (Shanghai, China). Graphite powder (average particle size ≤30 µm, purity ≥ 99.85%),

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sulfuric acid (H2SO4, 98%), potassium permanganate (KMnO4), sodium nitrate (NaNO3),

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hydrogen peroxide (H2O2, 30%), glacial acetic acid (99.5%) and N, N-dimethylformamide (DMF,

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99.8%) were available from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All the

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chemicals and reagents were of analytical grade and all solutions were prepared with Milli-Q

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water from a purification system (Millipore, Bedford, MA, USA).

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Peptone and beef extract were obtained from Aoboxing Product (Shanghai, China). Hank's

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balance salt solution (HBSS) was purchased from Beijing Solarbio Science & Technology Co. Ltd.

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(Beijing, China). Dulbecco's modified Eagle's medium (DMEM)/ high glucose, 0.25% trypsin 1X

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and fetal bovine serum (FBS) were provided by HyClone Laboratories Inc. (Utah, USA).

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Escherichia coli (E. coli, 8099) and Staphylococcus aureus (S. aureus, ATCC 6538) were provided

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by the China Center of Industrial Culture Collection (Beijing, China). HeLa cells were obtained as

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a gift from the Research Institute of Toxicology at School of Food Science and Engineering in

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Hefei University of Technology (Hefei, Anhui). 5



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Preparation of functionalized graphene nanosheets. Reduced graphene oxide (rGO) was

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prepared according our previous work.32 Briefly, the graphene oxide (GO) was firstly prepared

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from graphite powders by the modified Hummers method, and then dispersed in a sodium

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dodecylsulfate (SDS, surfactant) aqueous solution (1 wt%) to form a concentration of 1 mg/ml

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dispersion. After being reduced by hydrazine hydrate at 80 °C for 24 h, the mixture was

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centrifuged and the filter cake was washed with Milli-Q water and acetone three times before

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dried in a vacuum oven (60 °C, 24 h). Subsequently, the cake was ground by using an agate mortar

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to obtain reduced graphene oxide (rGO).

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The as-prepared rGO (30 mg) was dispersed in 30 ml Milli-Q water (Millipore, Bedford, MA,

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USA) by gently stirring for 1 h and then sonicated for 10 min. The functionalization agent

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[2-(methacryloyloxy) ethyl] trimethylammonium chloride (MTAC) (13.3 g) and Milli-Q water (40

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ml) were added into the suspension at room temperature. After stirring for 30 min, the system was

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purged under a gentle stream of nitrogen for 30 min to remove oxygen. Then, 100 mg of

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(NH4)2S2O8 (80 wt%, dissolved in Milli-Q water) was slowly drop-wise added into the flask

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through a dropping funnel. After reacted at 60 °C for 48 h, the MTAC functionalized rGO

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(MTAC-rGO) were obtained. In order to remove the residue functionalization agent, the mixture

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was centrifuged and the filter cake was washed with Milli-Q water and acetone three times. After

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being dried in a vacuum oven (60 °C, 24 h), the cake was ground by using an agate mortar and

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pestle to a powder of MTAC-GO (33-35 mg).

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LbL assembly of MTAC-rGO/EVOH flims. The LbL assembly of MTAC-rGO/EVOH flim

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was performed under a parallel electric field generated by a high voltage power supply

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(LBZ-120kV/2mA, Shanghai Liubao Electric Appliance Co., Ltd., Shanghai, China) according to 6


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our previous work.32 Briefly, a stainless steel disk substrate (140 mm in diameter and 3 mm in

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thickness) was first dipped in EVOH solution (3 wt %, glacial acetic acid: H2O = 9:1, v/v) for 1

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min, and then dried in oven (40 °C, 4 h). Owing to the positively charged MTAC-rGO, the

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substrate was subjected to the LbL assembly process in the suspension (16 mg MTAC-rGO

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powder in 160 ml DMF, pH 7) at cathode plate under the electric field (45 kV, 10 min) (Figure

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1A), and then the substrate was dried in an oven (40 °C, 1h) before the next cycle (Figure 1B). For

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the comparison of antibacterial activity, we prepared rGO/EVOH film under the same process

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parameters by exchanging the electrodes due to the negatively charged rGO.

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Zeta potential. The surface charge of rGO and MTAC-rGO were investigated by zeta

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potential performed on a Zetasizer Nano-ZS90 apparatus (Malvern Instruments, Worcestershire,

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UK). The testing suspensions (1%, w/v) were prepared by dispersing rGO and MTAC-rGO

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powders in DMF respectively at 35 oC for 1 h, and then cooled to room temperature. The

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suspensions (1ml) were injected into a plug-type electrode using needle tubing, and then the

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electrode was placed in the test cell. Triplicate measurements were taken for each sample at room

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temperature.

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Structure and morphology. Fourier transform infrared spectroscopy (FTIR) spectra were

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conducted on a Nicolet 6700 spectrometer (Thermo Nicolet, Madison, WI, USA), and the powders

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were subjected to FTIR spectroscopy in the range of 4000-400 cm-1 using KBr pellets. Raman

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spectra were recorded using a confocal micro-Raman spectrometer (LabRAM HR-800, Horiba

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Jobin Yvon, France) with a He-Ne laser excitation at 633 nm. X-ray diffraction (XRD) patterns

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were obtained using an X-ray diffractometer (X’Pert Pro MPD, Philips, Netherlands) with Cu Kα

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radiation (λ=0.15406 nm) and operated at 40 kV and 40 mA. X-ray photoelectron spectroscopy 7



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(XPS) of samples was performed by Mg Kα radiation with an ESCALAB 250 (Thermo-VG

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Scientific, USA) X-ray photoelectron spectrometer. Atomic force microscopy (AFM) images of

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the MTAC-GO after one cycle in parallel electric field were observed by Bruker Dimension Icon

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scanning probe microscope (Bruker Co., Germany). The microstructure and morphology of

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MTAC-GO nanosheets were characterized by a transmission electron microscopy (TEM,

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JEM-2100, JEOL, Japan). MTAC-GO powders (1.0 mg) were suspended in 10 ml DMF by

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sonication for 10 min at room temperature. The suspensions were dropped on a lacey carbon

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support film for TEM observation.

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The fracture surface MTAC-GO/EVOH film was observed using scanning electron

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microscopy (SEM, SU8020, Hitachi, Japan). Prior to examination, the tearing fracture surfaces of

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films were coated with sputter-gold.

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Barrier properties for water vapor and oxygen. The films (MTAC-rGO surface exposure to the

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wet environment ), sealed on beakers containing silica gel (0% RH), were placed in an artificial

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climate incubator (BIC 250, Shanghai Boxun industry & Commerce Co., Ltd, Shanghai, China).

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The temperature and relative humidity of the incubator were adjusted to 25 oC and the high

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detection limit (99%, RH), respectively. The moisture absorbed was estimated by periodical

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weighing of beakers at 6 h intervals during 5 days. WVTR (g m-2 d-1atm-1) was determined for

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three replicate specimens each type, as follows:

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(1)

WVTR= w / A× t × ∆P

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where w is the weight gain of beaker (g), A is the area of exposed film (m2), t is the time of weight

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gain (s), and ∆P is the water vapor partial pressure difference (atm) across the two sides of film

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calculated on the basis of relative humidity. 8


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Oxygen transmission rate (OTR, according to ASTMD1434) of films was determined at 23

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o

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Guangzhou, China). The open testing area of each sample in three parallel measurements was

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approximately 50 cm2, respectively. Film thickness was measured with a hand-held micrometer

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(BC Ames Co., Waltham, MA, USA).

C and 0% RH on a N500 gas permeameter (Guangzhou Biaoji packaging equipment Co., Ltd

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Mechanical properties. Tensile measurements were carried out according to the procedure

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outlined in ASTM method D882-91 with an average of five measurements taken for each ﬁlm (at

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least two ﬁlms per formulation). Each specimen was cut into rectangular strip about 1 cm × 10 cm,

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and then mounted between the grips of TA-XTPlus Texture Analyser (Stable Micro Systems, Co.,

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UK). The initial grip separation was adjusted to 50 mm and the crosshead speed was set at 0.5

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mm/s. The tensile strength and elongation at break of each specimen were calculated directly on

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the basis of the stress–strain curves by using OriginPro 8 software (Origin Lab Corporation, USA),

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correspondingly, the Young's modulus was determined from the slope of the initial linear portion.

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Antibacterial activity assay. In the present work, the typical (MTAC-rGO/EVOH)20 and

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(rGO/EVOH)20 films were subjected to the test of antibacterial activities, using bare EVOH film as

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control. The antibacterial activities of films were evaluated by the plate-counting method. E. coli

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and S. aureus strains were selected as Gram-negative and Gram-positive bacteria models,

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respectively.

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E.coli and S. aureus strains were cultured overnight in Luria-Bertani (LB) culture medium on

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a shaker platform (37 °C, 200 rpm). Then, the bacterial suspension was diluted in fresh culture

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medium until reached log phase (107 CFU ml-1). In order to assure the film contacting with

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bacterial suspension the completely, an excessive dosage of bacterial suspension (100 µl) was 9



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spread on the active surface of film (1×1 cm2) and then incubated at 37 °C for 1 h, using bare

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EVOH film as control. The excess bacterial suspension was then discarded and the film was

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washed by 5 ml of PBS solution (0.2 mol l-1) to remove the non-contact bacteria on the surface.

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Subsequently, the film was dipped into a centrifuge tube containing 5 ml PBS and sonicated for 1

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h. The bacteria suspension was spread on LB agar plate and incubated at 37 °C for 24 h for

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counting units.

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Scanning electron microscopy (SEM, SU8020, Hitachi, Japan) were used to observe the

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morphologies of bacteria after exposure to EVOH and (MTAC-rGO/EVOH)20 films, Prior to

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examination, the surfaces of specimens were coated with sputter-gold. After contacting with

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bacterial suspension, the specimens were washed with PBS solution and then fixed with

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Karnovsky’s fixative (2% paraformaldehyde, 2.5% glutaraldehyde in 0.2 mol l-1 PBS at pH 7.4)

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for 4 h before dehydrated by a sequential immersion in ethanol aqueous solutions (30, 50, 70, 80,

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90, and 100 %) for 10 min, respectively. Subsequently, the specimens were freeze-dried overnight.

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Cytotoxicity assay of MTAC-rGO. HeLa cells were cultured in Dulbecco's modified Eagle's

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medium (DMEM) supplemented with FBS (10 %) and penicillin and streptomycin solution (1%)

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and then incubated in a CO2 incubator (37 °C, 5% CO2). In order to evaluate the cytotoxicity of

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MTAC-rGO nanosheets, HeLa cells were selected to culture with them. Hela cells after three

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passages were washed three times with HBSS and then digested with 0.25 % trypsin 1X before

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being centrifuged for 10 min (100 g, 4 °C) for the further experiment.

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Hela cells were plated in the 96-well plates (100 µl per well, ~5 × 103 cells ml−1) and incubated

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for 24 h. The culture medium was then discarded, and the testing specimens were separately

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injected to the plates with concentrations at 0.0, 10.0, 20.0, 30.0, 40.0, and 50.0 µg ml−1 in 100 µl 10


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fresh culture mediums, respectively. After an incubation of 24 h, the culture medium was removed

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from plates, followed by washing three times with HBSS. Subsequently, HeLa cells were reacted

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with 100 µl of CCK-8 in each well and incubated in the CO2 incubator for 2 h. After then, the

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culture mediums were measured at 450 nm optical density (OD450) with a microplate reader

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(Bio-Rad, USA) to evaluate the cell viability (CV). The values of CV were calculated as follows:

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CV =

Test Blank OD450 − OD450 Ctrl Blank OD450 − OD450

(2)

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Test Ctrl Blank where OD450 , OD450 and OD450 are the OD450 of graphene, control, and blank (without Hela

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cells and MTAC-rGO nanosheets) culture mediums, respectively.

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Fluorescent images were taken to observe the live and attached Hela cells exposure to

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specimens by using fluorescent microscopy (Olympus BX43, Japan) with excitation at 488 nm

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provided by a Multi Ar laser on BWA detection. Hela cells were plated in 24-well plates with

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slides (500 µl per well, ~5 × 103 cells ml−1) and incubated for 24 h. After removing the culture

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medium from each well, MTAC-rGO nanosheets with fresh culture medium (500 µl) at

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concentrations of 0.0, 10.0, 20.0, 30.0, 40.0, and 50.0 µg ml−1 was separately injected to the wells

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and incubated for 24 h. After then, the culture medium was removed from the plate and the cells

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were washed three times with HBSS. To dye the cells, 200 µl rhodamine 123 (0.001 mol l-1,

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dissolved in DMSO）was injected to the wells and incubated for 45 min. After being washed three

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times with HBSS, the cells were fixed on the slides by using 10 % formalin solution before

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subjected to the observation of fluorescent microscope.

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Statistical analysis. Each experiment was repeated three times. Statistical analysis was

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performed using the unpaired Student’s t-test, and the results were expressed as the means ±

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standard deviation (SD). A value of p

Antibacterial [2-(Methacryloyloxy) ethyl] Trimethylammonium Chloride

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