Inactivation performance and mechanism of Escherichia coli in aqueous system exposed to iron oxide...

Accepted Manuscript

Title: Inactivation performance and mechanism of Escherichiacoli in aqueous system exposed to iron oxide loaded graphenenanocomposites

Author: Can-Hui Deng Ji-Lai Gong Guang-Ming ZengCheng-Gang Niu Qiu-Ya Niu Wei Zhang Hong-Yu Liu

PII: S0304-3894(14)00346-XDOI: http://dx.doi.org/doi:10.1016/j.jhazmat.2014.05.011Reference: HAZMAT 15918

To appear in: Journal of Hazardous Materials

Received date: 2-2-2014Revised date: 4-5-2014Accepted date: 5-5-2014

Please cite this article as: <doi>http://dx.doi.org/10.1016/j.jhazmat.2014.05.011</doi>

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http://dx.doi.org/doi:10.1016/j.jhazmat.2014.05.011

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Highlights

Magnetic-graphene oxide (M-GO) with excellent antibacterial activity

is prepared.

The antibacterial activity of M-GO relies on concentration and mass

ratio of M/GO.

Synergetic antibacterial effect of M-GO is observed with M/GO mass

ratio of 9.09.

TEM images illustrate that M-GO has penetrated into the cytoplasm.

Synergetic mechanism accounts for the antibacterial activity of

M-GO.

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Inactivation performance and mechanism of Escherichia coli in aqueous system

exposed to iron oxide loaded graphene nanocomposites

Can-Hui Deng, Ji-Lai Gong*, Guang-Ming Zeng, Cheng-Gang Niu, Qiu-Ya Niu, Wei

Zhang and Hong-Yu Liu

College of Environmental Science and Engineering, Hunan University, Changsha,

410082, PR China

Key Laboratory of Environmental Biology and Pollution Control, Ministry of

Education, Hunan University, Changsha 410082, PR China

*Corresponding author. Tel: +86 731 88822829; Fax: +86 731 88822829

E-mail: [email protected] (Ji-Lai Gong)

Abstract

The challenge to achieve efficient disinfection and microbial control without

harmful disinfection byproducts calls for developing new technologies.

Magnetic-graphene oxide (M-GO) with magnetic iron oxide nanoparticles well

dispersed on graphene oxide (GO) nanosheets exerted excellent antibacterial activity

against Escherichia coli. The antibacterial performance of M-GO was dependent on

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the concentration and the component mass ratio of M/GO. The synergetic

antibacterial effect of M-GO was observed with M/GO mass ratio of 9.09. TEM

images illustrated the interaction between Escherichia coli cells and M-GO

nanocomposites. M-GO nanomaterials were possible to deposit on or penetrate into

cells leading to leakage of intercellular contents and loss of cell integrity. The

inactivation mechanism of E. coli by M-GO was supposed to result from both the

membrane stress and oxidation stress during the incubation period. M-GO with

excellent antibacterial efficiency against E. coli and separation-convenient property

from water could be potent bactericidal nanomaterials for water disinfection.

Keywords: graphene oxide; magnetic iron oxide; antibacterial; oxidative stress;

water disinfection

1. Introduction

One of the most ubiquitous and crucial event for people throughout the world is

to provide adequate safe potable water affordably from disinfecting water without

causing more problems during the disinfecting process itself. There have been a

number of conventional chemical disinfectants widely used for potable water

disinfection, including free chlorine [1], chloramines [2] and ozone [3], which can

efficiently inhibit some microbial pathogens. Embarrassingly, most of them can form

harmful disinfection byproducts (DBPs) when interacting with various components of

natural water, many of which are carcinogens [4, 5].

In recent years, alternative disinfection technologies using nanomaterials have

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attracted significant attention. Several nanomaterials have been used as antibacterial

agents including inorganic nanomaterials (such as silver nanoparticles (nAg) [6, 7],

zeolite-supported silver [8], silicalite-supported silver and gold [9], photocatalytic

TiO2 [10] and ZnO [11]), natural organic antimicrobial peptides [12], chitosan [13]

and natural organic lysozyme-layered double hydroxides nanocomposites

(LYZ-LDHs) [14]. In addition, carbon-based nanomaterials, such as fullerol [15],

aqueous fullerence (nC60) [16], and carbon nanotubes (CNTs) [17] have displayed

fascinating antibacterial activities.

Graphene oxide (GO), as a one-atom-thick sheet of sp2-bonded carbon atoms that

are tightly packed into a two-dimensional crystal [18], has attracted many scientists

attention, since the experimental discovery of Geim et al. [19]. GO nanosheets are a

chemically modified graphene with epoxide and phenol hydroxyl groups on their

basal planes and carboxyl groups at their edges [20]. Recently, it has been reported

that GO exerted antibacterial properties toward Escherichia coli through damaging

the cell membrane, leading to the efflux of intracellular contents [18, 20]. Moreover,

GO nanosheets have been used as support to disperse gold [21] or silver [22]

nanoparticles for catalytic and antibacterial applications.

Magnetic iron oxide nanoparticles have been used in magnetic resonance [23],

target-drug delivery [24] and magnetic separation of biological components [25, 26],

because of their unique magnetic properties [27] and biocompatibility [28].

Noticeably, concerns have been made in terms of their potential antibacterial property

[29, 30]. Auffan et al. [31] reported the toxic effects of iron-based nanoparticles (i. e.

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Fe3O4, γFe2O3 and Fe°) toward the Gram-negative bacterium Escherichia coli. They

proposed that the cytotoxic effects of iron oxide appeared to be associated with

different redox states. Taylor et al. [32] also investigated the antibacterial activity of

magnetic nanoparticles and found the numbers of Staphylococcus epidermidis

decreased when treated with magnetic iron oxide nanoparticles at the dosages equals

to or greater than100 μg/mL. Tran et al. [29] found that polyvinyl alcohol (PVA)

mediated iron oxide (IO) nanoparticles (referred as IO/PVA nanoparticles) inhibited

Staphylococcus aureus growth, and the bactericidal activity of IO/PVA was mainly

contributed to the concentration of the nanoparticles.

In this work, the introduction of iron oxide magnetic (M) nanoparticles into

graphene oxide was proposed to constitute a novel antibacterial nanomaterial, which

will combine the antibacterial properties of graphene oxide and the separation

convenience of magnetic nanoparticles. Magnetic-graphene oxide (M-GO) was

synthesized by depositing magnetic iron oxide nanoparticles on the surface of GO

nanosheets. Escherichia coli, a typical Gram-negative bacterium, was employed as a

model due to its well-known pathogen commonly involved in water contamination

and widely used in reference tests to measure bactericidal properties [33, 34]. The

interaction between E. coli cells and M-GO and antibacterial mechanism of M-GO

were also investigated.

2. Materials and methods

2.1 Strain and chemicals

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The bacteria strain E. coli ATCC 25922 was purchased from the China Center for

Type Culture Collection (Beijing, China). Stock cultures were maintained on LB agar

slants at 4 ℃. Graphite powder was obtained from Shanghai Jin-Shan-Ting new

chemical factory (Shanghai, China). Multi-wall carbon nanotubes (MWNTs) with

outer diameter 40-60 nm and length 5-15 μm were obtained from Shenzhen Nanoport

Company (Shenzhen, China). Glutathione detection kit was obtained from Nanjing

Jiancheng Bioengineering Institute (Nanjing, China). Sodium hydroxide (NaOH),

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

hydrogen peroxide (H2O2), glutaraldehyde, glutathione (GSH), ferrous ammonium

sulfate [(NH4)2SO4·FeSO4·6H2O] and ammonium ferric sulfate [NH4Fe

(SO4)2·12H2O] were all purchased from Sinopharm chemical reagent Co., (Shanghai,

China). All the chemicals used in this study were of analytical reagent grade.

2.2 Preparation of GO

GO was prepared from graphite powder according to the method of Hummers

and Offeman [35] with some modification. Briefly, 1g graphite flakes, 23 mL H2SO4

and 0.5 g NaNO3 were added into a conical flask, and then mixed with 3 g KMnO4

under ice bath condition and magnetic stirring. Subsequently, the reaction was

controlled at a constant temperature of 35 ℃ for 1 h, followed by dilution with warm

de-ionized (DI) water. The reaction was continued at the temperature of 98 ℃ for 15

min and H2O2 was added to reduce the residual permanganate and manganese dioxide.

The resulting yellow suspension was filtered, centrifuged, and washed with DI water,

then freeze dried for 24 h to obtain graphite oxide powders. Finally, GO suspension

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was gained through ultrasonic exfoliation of the graphite oxide dispersed in DI water

for 45 min.

2.3 Synthesis of M-GO dispersions

The synthesis of M-GO was prepared on the basis of our previous report [36].

Firstly, 0.25 g graphite oxide was dispersed in 50 mL DI water with ultrasonication to

form suspension. Secondly, the iron oxide magnetic nanoparticles were prepared by

mixing ferric and ferrous solutions (molar ratio of 1.5 : 1 for Fe3+ and Fe2+,

respectively) with vigorous stirring under N2 atmosphere, with subsequent addition of

25 % aqueous ammonia to adjust pH at around 10. Then, a black precipitate was

allowed to age for 30 min at 85 ℃ to obtain M nanoparticles. Finally, GO

suspension was added dropwise to a certain amount of M dispersion at room

temperature with mild stirring for 45 min to obtain two kinds of M-GO with different

M/GO mass ratios of 5.56 for M1-GO and 9.09 for M2-GO (the mass ratio of M/GO

in M-GO nanocomposites was calculated by measuring weight percent of GO using

thermal gravimetric analysis (TGA)). Then, the M-GO nanocomposites were

separated using a magnet and thoroughly washed to neutral with DI water.

2.4 Cell preparation

Before each microbiological experiment, all samples and glassware were

sterilized at 121 ℃ for 15 min with autoclave. The bacterial strain (E. coli ATCC

25922) was grown in Luria-Bertani (LB) medium (tryptone 10 g, yeast extract 5 g,

and NaCl 5 g in 1L of DI water at pH of 7.0) at 37 ℃ for 24 h, on a rotary shaker at

approximately 120 rpm shaking speed. The cultures were harvested by centrifugation,

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washed three times to remove all traces of LB, and finally re-suspended in sterile DI

water. Bacterial cell suspensions were diluted to contain cells 106-107 CFU/mL.

2.5 Cell viability test

E. coli cells were incubated with various samples including GO, M, M1-GO and

M2-GO suspensions in DI water at 37 ℃ under 150 rpm shaking speed for 2 h at a

final cell concentration of 106-107 CFU/ml. For magnetic nanomaterials (i. e., M,

M1-GO and M2-GO), the mixture was first magnet separation. Then, the supernatants

were diluted to a series of 10-fold concentration gradient, and then 100 μL cell

dilutions were spread onto three LB plates per gradient solution, left to grow

overnight at 37 ℃. The ratio of the colony-forming units (CFU) between final

activated cells and the beginning cells of experiments was evaluated. The cells

suspension incubated without nanomaterials was used as control. All treatments were

prepared in triplicate.

2.6 TEM observation of E. coli cells

The cells treated and untreated with M-GO dispersion for 2 h were fixed with 3

% glutaraldehyde. The cells were washed three times by PBS, and then postfixed with

1 % osmium tetroxide for 2 h and washed again twice with PBS. The cells were then

dehydrated with 50, 70, 90 and 100 % ethanol for 10 min and embedded in Spurr’s

resin (polymerization at 60 ℃ overnight). The thin sections containing cells were

stained with 1 % uranyl acetate and Reynold’s lead citrate, air-dried, and then

examined under TEM.

2.7 Thiol oxidation and quantification

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The measurements of GSH oxidation by GO, M, M1-GO and M2-GO

nanomaterials were performed according to previous report with some modification

[37]. The concentration of thiols in GSH was quantified using assay kit.

Nanocomposites dispersion (250 μL) in 50 mM bicarbonate buffer (pH 8.6) was

added into 250 μL GSH (at the concentration of 0.4 mM in bicarbonate buffer) in

tubes to initiate the oxidation reaction. Then, the tubes described above were covered

with foil to prevent any illumination, placed on a shaker with a speed of 150 rpm at

room temperature (~25 ℃) for 2 h. After incubation, the nanomaterials were

separated by centrifugation with high speed for GO and by a magnet for M, M1-GO

and M2-GO. Then 100 μL aliquot of supernatant was withdrawn to place in a 96-well

plate, and then mixed with 100 μL buffer solution and 25 μL chromogenic agent

5,5’-dithio-bis-(2-nitroenzoic acid) (DTNB) to yield a yellow compound. Their

absorbance at 405 nm was measured on a microplate reader (Multiskan, USA). GSH

with bicarbonate buffer was used as a negative control and GSH with H2O2 (10 mM)

was used as a positive control. The loss of GSH was calculated using the method of

previous studies [20], where loss of GSH % = absorbance of (negative control –

sample) / absorbance of negative control × 100 %.

2.8 Characterization

The morphologies of the graphene-based nanomaterials and E. coli cells were

characterized using a field emission scanning election microscopy (FESEM)

(JSM-6700F LV microscope, Japan) and a transmission electron microscopy (TEM)

(JEM-3010). The structure phases of the synthesized antibacterial materials were

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analyzed by X-ray diffraction (XRD) (D/max 2550 X-ray diffractometer, Rigaku,

Japan). Infrared absorption spectra were measured on a Fourier transform infrared

(FTIR) spectroscope (IRAffinity-1, Shimadzu, Japan) at room temperature. And the

magnetization curve was recorded on vibrating sample magnetometer (Lake Shore

7410). ZRY-2P thermal analyzer was employed for TGA at temperature of 20-800 ℃

and heating rate of 20 ℃/min. The BET surface area was determined by Tristar 3020

volumetric analyzer (Micromeritics Instrument Corporation, USA). Raman spectra

were acquired on LabRAM-010 Laser Raman spectrometer (HORIBA Jobin Yvon,

France). Small-angle X-ray scattering experiments were performed with Anton Paar

SAXSess mc2. SAXS data were processed with SAXSquant program, where the

angular parameter (q) is defined as q = 4π sinθ/λ, whereθ and λ are the X-ray

scattering angle and wavelength, respectively. The obtained data were modified to

follow the Porod law, where the scattering intensity I (q) is proportional to q-2 for

moderate q values and to q-4 for large q values. The fractal dimension of the scattering

objects was calculated from the slop of the curve log I (q) vs. log (q).

3. Results and Discussion

3.1 Characterization of antibacterial materials

The preparation of M-GO nanocomposites was schematically illustrated in

Scheme 1. The typical morphology of GO were displayed in Fig. 1a and the images of

M-GO were observed using FESEM and TEM (shown in Fig. 1b, c, respectively). As

can be seen, the free-standing two dimensional GO sheets displayed flake-like shapes

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with high transparency and some wrinkles. Spherical magnetic particles with almost

uniform size were depicted in the SEM image (see Fig. 1b). As shown in Fig. 1c, M

nanoparticles with the average particle size of 11.64 nm were well-dispersed on the

GO matrix in M-GO nanocomposites. The histogram of the particle size distribution

was presented in Fig. 1d. The reason that small amount of nanoparticles tended to

anchor on the surface of GO with a high density can be explained by the magnetic

dipolar interaction among the M nanoparticles. Similar results were also observed in

previous report [38-40].

The X-ray diffraction (XRD) patterns of GO, M, M1-GO and M2-GO were

displayed in Fig. 2. It was observed that the two main diffraction peaks at 2θ = 10.0°

(001) and 42.3°(100) were attributed to the structure of GO nanosheets [41-43] in

the XRD pattern shown in Fig. 2a. The disappearance of the characteristic peak at 2θ

= 26.4° (002) in pristine graphite [44] was due to the introduction of

oxygen-containing groups on the surface of GO during oxidation process [45]. The

behavior of M nanoparticles was similar to M-GO in XRD pattern (Fig.2b, c, d). The

four main diffraction peaks at 2θ = 30.2°(220), 35.5°(311), 43.3°(400) and 57.2°

(511) that can be ascribed to maghemite or magnetite [46]. The two magnetic

composites have the similar crystal structure and it is difficult to distinguish according

to XRD patterns. The other two peaks at 2θ = 53.6° and 62.9°were assigned to the

(422) and (440) planes of hematite [47]. The relatively weak peak at 2θ = 18.3°(111)

was also observed due to the presence of goethite [36]. Therefore, iron oxides

nanoparticles in our work included magnetic magnetite (Fe3O4) and maghemite

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(γ-Fe2O3), and non-magnetic hematite (α-Fe2O3) and goethite (FeOOH). Compared to

M1-GO, M2-GO possessing a higher amount of M component produced more intense

M XRD peaks. It was noted that the characteristic peak of GO at 2θ = 10.0°(001)

was obviously reduced, and the GO peaks at 2θ = 42.3°(100) totally disappeared in

the XRD patterns of M-GO, which could be caused by the reasons as follows: The

weak peaks of carbon in M-GO resulted from the aggregation reduction of graphene

sheets and the increase of monolayer graphene in the presence of magnetite; the

strong peaks of the M nanoparticles overwhelming the weak carbon peaks [48]. The

results in our work were consistent with the previous studies [43].

The functional groups of GO, M1-GO and M2-GO nanocomposites were

investigated by FTIR spectra shown in Fig. 3. For GO, the absorption peak at 3417

cm-1 was ascribed to the stretching of O-H [49]. The peaks at 1725 cm-1, 1624 cm-1

and 1399 cm-1 corresponded to carbonyl C=O stretching vibrations [43], aromatic

C=C stretching, and carboxyl O=C-O stretching mode of sp2 carbon skeletal network,

respectively, while the bands at 1218 cm-1 and 1051 cm-1 were associated with

stretching of C-O of epoxy and alkoxy groups, respectively [50]. For M1-GO, the

peaks at 1625 and 1124 cm-1 were assigned to the aromatic C=C stretch and C-O

stretch, respectively. The stretching vibration of C=C in M2-GO appeared at 1624

cm-1. Moreover, the transmittance band around at 565 cm-1 in M-GO was mainly

assigned to the stretching vibration of Fe-O [51, 52].

Raman spectroscopy is one of the most sensitive and nondestructive techniques

to probe the ordered and disordered crystal structures of carbon materials. As shown

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in Fig. 4, Raman spectrum of GO displayed two prominent peaks at 1338 and 1597

cm-1, corresponding to the well-documented D band and G band, respectively. The

Raman D bands shifted from 1338 to 1324 cm-1 and to 1323 cm-1 for M1-GO and

M2-GO, respectively. In addition, the Raman G bands shifted from 1597 to 1591 cm-1

and to 1592 cm-1 for M1-GO and M2-GO, respectively (see Fig. 4 b, c). For M-GO,

the Raman G and D bands shifted to lower frequency in comparison with that of GO,

indicating that GO was reduced [53, 54].

Different surface roughness of materials could significantly influence the

attachment of bacteria on the material surface at the period of interaction between

materials and bacteria [55, 56]. The surface properties of four types of nanomaterials

including GO, M, M1-GO and M2-GO were investigated. The specific surface areas

calculated from N2 adsorption-desorption isotherms and BET equation, and the

fractal dimension values of antibacterial nanomaterials as determined by SAXS data

were presented in Table 1. The specific surface areas of antibacterial materials were

8.55, 73.34, 214.84 and 351.96 m2/g for GO, M, M1-GO and M2-GO, respectively.

The specific surface of M1-GO and M2-GO were higher than that of GO and M,

which was consistent with the fact that M nanoparticles were well dispersed on the

surface of GO nanosheets. Moreover, the fractal dimension of GO, M, M1-GO and

M2-GO was assigned to 2.09, 2.11, 2.11 and 2.13, respectively. It was well known

that the value of fractal dimension was determined by the degree of surface

roughness of materials [57]. The fractal dimension value of GO close to 2.09 was

inclined to be smoothed, which was in agreement with the two-dimensional structure

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of GO. However, the fractal dimension of M-GO (2.11 for M1-GO, and 2.13 for

M2-GO) was higher than that of GO indicating that M nanoparticles were deposited

on the surface of GO nanosheets resulting more irregularity or roughness.

The magnetic properties of the synthesized M-GO nanocomposites were

recorded at room temperature (300 K) by VSM, as shown in Fig. 5. For the M1-GO,

the reduction in the value of saturation magnetization (60.80 emu/g) as compared with

that of M2-GO (68.71 emu/g) could be attributed to the relatively lower amount of M

nanoparticles loaded on GO sheets. Both M1-GO and M2-GO dispersions could be

separated from aqueous solution by a magnet. It was reported that saturation

magnetization of 16.30 emu/g was sufficient for magnetic separation [58]. The

performance of magnetic separation for M-GO was shown in the insert of Fig. 5.

3.2 Antibacterial activity of GO, M and M-GO dispersions

Antibacterial activity of four types of nanomaterials obtained in this work was

evaluated using a model bacterium E. coli. The aqueous suspensions of GO, M,

M1-GO, and M2-GO with the same concentration (100 μg/mL) were incubated with

E. coli cell suspensions (106 to 107 CFU/mL) for 2 h at 37 ℃ and 150 rpm shaking

speed. A series of 100 μL 10-fold cell dilutions were spread onto LB agar plates and

grown in biochemical incubator at 37 ℃ for 24 h. The loss percent of viability was

calculated to quantify the antibacterial ability of nanomaterials.

the loss of viability, % = (1 – ) × 100%

where N is the colony number of activated cells (at the range of 106-107 CFU/mL)

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before each experiment. is the colony-forming units of the activated cells after

incubation with antibacterial nanomaterials for 2 h (CFU/mL).

Fig. 6 shows the antibacterial properties of various nanomaterials including GO,

M, M1-GO, and M2-GO. GO dispersion exhibited an apparent antibacterial activity

with the cell inactivation percentage at 77.49 ± 12.79 %. M dispersion displayed a

little bit weaker antibacterial activity, with the inactivation percentage at 53.00 ±

15.70 % compared to GO. But for M1-GO, the loss of E. coli viability reached to

62.26 ± 3.03 %, which was a little higher than that of M nanoparticles dispersion, but

a little lower than that of GO dispersion. However, M2-GO possessed the strongest

bacterial inactivation among the four kinds of nanomaterials, with the inactivation

percentage up to 91.49 ± 2.82 %.

The disinfection activity of carbon-based nanocomposites including M1-GO,

M2-GO and magnetic multi-walled carbon nanotubes (M-MWNTs) were also

investigated. The synthesis of M-MWNTs was prepared according to our previous

literature [46]. Results showed that M-MWNTs with the same concentration of

100μg/mL exerted much weaker inactivation ability (34.19 ± 5.06 %) against E. coli

than that of M1-GO (62.26 ± 3.03 %) and M2-GO (91.49 ± 2.82 %).

The effect of mass ratio of GO to M on antibacterial properties of nanomaterials

including GO, M, M1-GO, and M2-GO has been analyzed (shown in Fig. 7). The

total mass of four antibacterial nanomaterials was fixed. The ratios of GO/M were 0,

0.18 and 0.11 for M, M1-GO, and M2-GO, respectively. Noticeably, the bactericidal

ability of magnetic nanomaterials was enhanced in the presence of GO, which might

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be attributed to the moderate antibacterial properties of GO itself. However, the

antibacterial ability of GO-based nanomaterials was not proportional to GO mass

percentage in M-GO nanomaterials. The percent of cell viability loss increased for

M2-GO at the mass ratio of GO/M 0.11, and decreased for M1-GO at 0.18 (displayed

in Fig. 7a). It was concluded that the antibacterial activity of M-GO nanocomposites

was not only caused by GO component but also by M component. Additionally, the

bactericidal ability of magnetic nanomaterials was dependent on the ratio of M/GO

when addition of magnetite component into GO nanomaterials. The ratios of M/GO

were 0, 5.56 and 9.09 for M, M1-GO, and M2-GO, respectively. Cell viability loss

percent decreased for M1-GO at the mass ratio of M/GO 5.56 and increased for

M2-GO at 9.09. It was observed M1-GO with the M/GO mass ratio of 5.56 was not

beneficial to the cell viability loss compared with GO itself. On the contrary, M2-GO

exerted a higher antibacterial property with M/GO mass ratio of 9.09 when compared

to GO or M itself, illustrating that a synergistic antibacterial effect occurred between

GO and M (shown in Fig. 7b). Ma et al. [22] and Zhang et al. [59] reported that

silver-modified graphene materials displayed an excellent antibacterial activity

towards E. coli due to the synergistic effect of Ag nanoparticles and graphene oxide

(GO) or graphene nanosheets (GNS). Sreeprasad et al. [60] prepared a serial of

multifunctional graphene oxide/reduced graphene oxide (GO/RGO) based composites

by anchoring of native lactoferrin (NLf), chitosan (Ch) and Au clusters into GO/RGO,

such as RGO/GO-NLf-Ch and RGO/GO-Au@NLf-Ch. The composites exhibited

several folds higher antibacterial activity than GO/RGO itself, which was accounted

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for the synergetic effect of the combination of materials. Nangmenyi G et al. [61] also

reported a synergistic disinfection action between Fe2O3 and Ag on fiberglass when

compared to either Fe2O3 or Ag alone. Noticeably, synergistic antibacterial effect

between GO and M toward E. coli in our work was dependent on the mass ratio of

M/GO in M-GO nanomaterials. The optimal mass ratio of M to GO and the

mechanism of synergistic effect between GO and M will be investigated in our further

studies.

The concentration effect on the E. coli inactivation activity by M-GO was

presented in Fig. 8. The antibacterial property of M-GO (including M1-GO and

M2-GO) dispersions with diverse concentrations was investigated. The percent of cell

viability loss gradually went up with the increased concentration of M-GO. For

M1-GO, the loss of E. coli viability increased from 19.47 ± 1.20 % at the M1-GO

concentration of 30 μg/mL to 31.02 ± 15.70, 34.82 ± 15.00, 64.14 ± 5.00, 93.45 ±

1.76, and 99.84 ± 0.16 % after incubating with 40, 50, 100, 200, and 300 μg/mL

M1-GO suspensions, respectively. When the concentration of M1-GO achieved to 200

μg/mL, there were almost no living cells left. Compared with M1-GO, M2-GO

exhibited stronger antibacterial ability at the same concentration gradient. The

inactivation percent of E. coli by M2-GO increased from 24.65 ± 4.21 % at the

concentration of 30 μg/mL to 43.986 ± 7.83, 61.69 ± 7.89 % at the concentration of

40, and 50 μg/mL. There were 91.49 ± 2.82 % of E. coli cells were killed, when the

concentration of M2-GO reached to 100 μg/mL.

3.3 Interaction between E. coli cells and M-GO dispersion

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The interaction between E. coli cells and M-GO nanocomposites was illustrated

by TEM. Results revealed the cells treated with M-GO dispersion contained dark

granules around the outside cell wall and penetrated into the cytoplasm. These specks

were likely assigned to M-GO or M nanomaterials passed into cells via direct

interaction with E. coli during incubation. Similar phenomena were also observed by

Lee et al. [62] and Hu et al. [63]. Besides, most of E. coli cells lost their cellular

integrity, with significant destruction of the cell membrane and subsequent leakage of

cellular contents (Fig. 9b) after exposure to M-GO. This was similar to GO or rGO

[20], which induced membrane stress on E. coli cells, resulting in destruction of cell

structures. Such irreversible damage of cells induced by M-GO may play a significant

role in their antibacterial activity.

3.4 Oxidation ability of antibacterial materials

The mechanism of oxidation damage was the most accepted explanation for the

antibacterial activity of graphene-based nanomaterials. Meanwhile, previous

researches reported that the M nanoparticles generated reactive oxygen species (ROS)

via Fenton reactions when interacting with bacteria, leading to protein oxidation and

DNA damage, and finally resulting in cells death [29, 51]. In view of this, the

oxidation ability of four antibacterial nanomaterials in this work was evaluated by

measuring the loss percentage of GSH to confirm the oxidative damage toward

bacteria. GSH is a small thiol containing tripeptide antioxidant in most Gram-negative

bacteria cells at levels of 0.1-10 mM [64]. The thiol groups (-SH) in GSH were

oxidized to disulfide bond (-S-S) sensitively when exposed to ROS or other oxidants

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[16]. Therefore, in this work, the oxidation degree of GSH was measured in vitro to

reflect indirectly the cellular oxidative destruction induced by four types of

nanomaterials. GSH (0.4 mM) was explored to incubate with GO, M, M1-GO and

M2-GO (at the same concentration of 100 μg/mL) for 2 h. The loss of GSH was

quantified using a thiol quantization kit and the details of experiments were

represented in the section of Materials and Methods.

Fig. 10a shows that 58.62 ± 0.54 % and 46.50 ± 4.77 % of GSH were oxidized

by GO and M after 2 h incubation, respectively. Compared to M (GSH oxidation

percentage 46.50 ± 4.77 %), the loss percent of GSH rose up to 51.11 ± 7.23 % for

M1-GO, and 58.55 ± 8.05 % for M2-GO, respectively. The oxidation tendency of

GSH by GO, M, M1-GO and M2-GO was similar to the tendency of E. coli

inactivation induced by the four nanomaterails. Therefore, it was concluded that the

oxidative ability of the nanomaterials had a significant influence on their antibacterial

properties.

Considering the concentration-dependent antibacterial activities of M-GO (see

Fig. 8), we speculated the oxidative ability of M-GO toward GSH would be

concentration-dependent as well. M-GO with different concentrations (30-300 μg/mL)

were incubated with 0.4 mM GSH for 2 h. Fig. 10b shows the fraction of GSH

oxidized by M1-GO or M2-GO was concentration dependent. GSH oxidation by

M-GO was37.60 ± 3.69 % and 64.64 ± 1.53 % for M1-GO and 40.86 ± 5.91 % and

79.34 ± 2.82 % for M2-GO at the M-GO concentration of 30 and 300 μg/mL,

respectively. It was obvious that M2-GO has relatively higher oxidation reactivity

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than M1-GO at the same concentration. Furthermore, GSH oxidation increased with

increasing concentration of M1-GO or M2-GO, which was consistent with the trend

that antibacterial activity increased with increasing concentration of M-GO

nanomaterials.

3.5 Antibacterial mechanism of M-GO

The correlation among antibacterial activities, surface roughness and GSH

oxidation was summarized in Table 1. On the one hand, comparing GO and M2-GO,

they possessed similar capacities in oxidizing GSH (GO at 58.62 ± 0.54 % vs M2-GO

at 58.55 ± 8.05 %); however, M2-GO dispersion exerted much higher bactericidal

activity (91.49 ± 2.82 %) than GO dispersion (77.49 ± 12.79 %). Their difference was

that GO was two-dimension nanosheets with surface area of 8.55 m2/g, while M2-GO

possessed higher surface roughness with fractal dimension of 2.13 and surface area of

351.96 m2/g. Their distinct antibacterial activities indicated that surface property of

antibacterial materials played an important role in the antibacterial mechanism. On the

other hand, comparing GO and M, GO (fractal dimension of 2.09 and surface area of

8.55 m2/g) was obviously smoother than M (fractal dimension of 2.11 and surface

area of 73.34 m2/g). However, the antibacterial activity of GO (77.49 ± 12.79 %) was

much higher than that of M (53.00 ± 15.70 %). This was obviously correlated with

their different GSH oxidation capacities. In addition, among the four types of

antibacterial materials, M2-GO with the highest surface roughness and oxidation

ability also exerted the highest antibacterial activity. Therefore, results shown in Table

1 suggested that the antibacterial activity of materials were ascribed to their surface

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property and oxidization ability.

Liu et al. [20] proposed a three-step antibacterial mechanism for graphene-based

materials including initial bacteria cells deposited on graphene-based materials during

incubation period, membrane stress induced by direct interaction between sharp

nanosheets and bacteria and the following superoxide anion-independent oxidation

toward intercellular components of cells. They suggested that the antimicrobial

mechanism of graphene-based materials were contributed to the synergy of membrane

and oxidation stress.

Here, the synergetic mechanism was supposed to account for observations in our

paper. The possible inactivation mechanism of M-GO toward bacteria in this work

was expressed clearly on Scheme 2. E. coli cells may first anchor on the surfaces of

M-GO during incubation in aqueous system. The shaking speed of 150 rpm used in

antibacterial assays had facilitated the suspension of M-GO in the aqueous solution.

Under the shaking condition, M-GO dispersion had more chances to interact with E.

coli for cell deposition.

After cells adhering to M-GO surfaces, the sharp edge of GO nanosheets may

destroy the integrity of cell membrane, then resulting in the leakage of intracellular

materials and finally cell death, as previously reported [18, 63]. It was likely that the

small size of M nanoparticles with average size of 11.64 nm maybe have

opportunities to penetrate into E. coli membranes. Lee et al [62] found that the reason

of zero-valent iron nanoparticles with sizes ranging from 10-80 nm had strong

bactericidal activity could be contributed to the small size nanoparticles penetration

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into cells. GSH oxidation assays (Fig. 9) demonstrated that the oxidation ability of

M-GO may play a vital role in bacteria inactivation when M-GO direct contacting

with cells. Liu et al. [20] illustrated that graphene-based materials were capable of

inducing ROS-independent oxidative stress toward E. coli cells. Tran et al. [29]

confirmed metal oxide Fe3O4 inhibited the growth of S. aureus via oxidative stress

generated by ROS. Therefore, it was possible that M-GO nanocomposites could also

oxidize bacterial components through mediating the oxidation ability of GO and M.

The strong oxidation activity of M-GO toward GSH in our work supported that

M-GO was efficient to oxidize thiols or other intercellular contents.

4. Conclusions

Four types of suspension (GO, M, M1-GO and M2-GO) were prepared to reveal

different antibacterial properties, specially the antibacterial activity of M-GO

nanocomposites. M-GO was synthesized by depositing magnetic iron oxide

nanoparticles on the surface of GO nanosheets, M nanoparticles could be supported

and stabilized on the GO surface resulting in excellent dispersion, and the

nanoparticles have an average size of 11.64 nm. The saturation magnetization was

60.80 emu/g for M1-GO and 68.71 emu/g for M2-GO, respectively. Therefore, M-GO

nanocomposites could be rapidly separated from aqueous solution using an external

magnetic field. Results showed that both bare and GO-coated magnetic iron oxide

nanocomposites were efficient in inhibiting the growth of E. coli. The various mass

ratios of M/GO in M-GO influenced the inactivation properties significantly. GO and

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M emerged synergistic effect on loss of cell viability, when M/GO mass ratio in

M-GO nanocomposites was adjusted to 9.09. And the antibacterial ability of M-GO

was concentration-dependent. The mechanisms of bacterial cytotoxicity caused by

M-GO may be relying on both physical membrane puncture and chemical cellular

matters oxidation, which were similar to the three-step antibacterial mechanisms of

CNTs. In views of these interesting properties of both strong magnetic property and

outstanding antibacterial ability, M-GO nanomaterials have the potential applications

for environmental drinking water treatments.

Acknowledgments

The authors are grateful for the financial supports from National Natural Science

Foundation of China (51039001, 50978088, 50808070, 21275044 and 51108166),

Interdisciplinary Research Funds for Hunan University, the Natural Science

Foundation of Hunan Province, China (Grant no. 12JJB003) and the Scientific

Research Foundation for the Returned Overseas Chinese Scholars, State Education

Ministry.

References:

[1] G.P. Winward, L.M. Avery, T. Stephenson, B. Jefferson, Chlorine disinfection of

grey water for reuse: Effect of organics and particles, Water Res. 42 (2008) 483-491.

[2] W. Lee, P. Westerhoff, Formation of organic chloramines during water disinfection

- chlorination versus chloramination, Water Res. 43 (2009) 2233-2239.

[3] P. Xu, M.L. Janex, P. Savoye, A. Cockx, V. Lazarova, Wastewater disinfection by

of 47

Accep

ted

Man

uscr

ipt

24

ozone: main parameters for process design, Water Res. 36 (2002) 1043-1055.

[4] M.A. Shannon, P.W. Bohn, M. Elimelech, J.G. Georgiadis, B.J. Marinas, A.M.

Mayes, Science and technology for water purification in the coming decades, Nature

452 (2008) 301-310.

[5] S.D. Richardson, M.J. Plewa, E.D. Wagner, R. Schoeny, D.M. DeMarini,

Occurrence, genotoxicity, and carcinogenicity of regulated and emerging disinfection

by-products in drinking water: A review and roadmap for research, Mutat. Res-Rev

Mutat. 636 (2007) 178-242.

[6] V.K. Sharma, R.A. Yngard, Y. Lin, Silver nanoparticles: Green synthesis and their

antimicrobial activities, Adv. Colloid Interface Sci. 145 (2009) 83-96.

[7] Q. Bao, D. Zhang, P. Qi, Synthesis and characterization of silver nanoparticle and

graphene oxide nanosheet composites as a bactericidal agent for water disinfection, J.

Colloid Interface Sci. 360 (2011) 463-470.

[8] R. Guerra, E. Lima, M. Viniegra, A. Guzman, V. Lara, Growth of Escherichia coli

and Salmonella typhi inhibited by fractal silver nanoparticles supported on zeolites,

Microporous Mesoporous Mater. 147 (2012) 267-273.

[9] R. Guerra, E. Lima, A. Guzman, Antimicrobial supported nanoparticles: Gold

versus silver for the cases of Escherichia coli and Salmonella typhi, Microporous

Mesoporous Mater. 170 (2013) 62-66.

[10] O. Akhavan, E. Ghaderi, Photocatalytic Reduction of Graphene Oxide

Nanosheets on TiO2 Thin Film for Photoinactivation of Bacteria in Solar Light

Irradiation, J. Phys. Chem. C 113 (2009) 20214-20220.

of 47

Accep

ted

Man

uscr

ipt

25

[11] M.A. Gondal, M.A. Dastageer, A. Khalil, K. Hayat, Z.H. Yamani, Nanostructured

ZnO synthesis and its application for effective disinfection of Escherichia coli micro

organism in water, J. Nanopart Res. 13 (2011) 3423-3430.

[12] E. Gazit, Self-assembled peptide nanostructures: the design of molecular building

blocks and their technological utilization, Chem. Soc. Rev. 36 (2007) 1263-1269.

[13] M.M. Lou, B. Zhu, I. Muhammad, B. Li, G.L. Xie, Y.L. Wang, H.Y. Li, G.C. Sun,

Antibacterial activity and mechanism of action of chitosan solutions against apricot

fruit rot pathogen Burkholderia seminalis, Carbohyd. Res. 346 (2011) 1294-1301.

[14] Q.Z. Yang, Y.Y. Chang, H.Z. Zhao, Preparation and antibacterial activity of

lysozyme and layered double hydroxide nanocomposites, Water Res. 47 (2013)

6712-6718.

[15] A.R. Badireddy, E.M. Hotze, S. Chellam, P. Alvarez, M.R. Wiesner, Inactivation

of Bacteriophages via photosensitization of fullerol nanoparticles, Environ. Sci.

Technol. 41 (2007) 6627-6632.

[16] D.Y. Lyon, P.J.J. Alvarez, Fullerene Water Suspension (nC60) Exerts Antibacterial

Effects via ROS-Independent Protein Oxidation, Environ. Sci. Technol. 42 (2008)

8127-8132.

[17] S. Kang, M. Herzberg, D.F. Rodrigues, M. Elimelech, Antibacterial effects of

carbon nanotubes: Size does matter, Langmuir 24 (2008) 6409-6413.

[18] O. Akhavan, E. Ghaderi, Toxicity of Graphene and Graphene Oxide Nanowalls

Against Bacteria, ACS Nano 4 (2010) 5731-5736.

[19] K.S. Novoselov, A.K. Geim, S.V. Morozov, D. Jiang, Y. Zhang, S.V. Dubonos,

of 47

Accep

ted

Man

uscr

ipt

26

I.V. Grigorieva, A.A. Firsov, Electric field effect in atomically thin carbon films,

Science 306 (2004) 666-669.

[20] S.B. Liu, T.H. Zeng, M. Hofmann, E. Burcombe, J. Wei, R.R. Jiang, J. Kong, Y.

Chen, Antibacterial Activity of Graphite, Graphite Oxide, Graphene Oxide, and

Reduced Graphene Oxide: Membrane and Oxidative Stress, ACS Nano 5 (2011)

6971-6980.

[21] P.V. Kamat, Graphene-Based Nanoarchitectures. Anchoring Semiconductor and

Metal Nanoparticles on a Two-Dimensional Carbon Support, J. Phys. Chem. Lett. 1

(2009) 520-527.

[22] J.Z. Ma, J.T. Zhang, Z.G. Xiong, Y. Yong, X.S. Zhao, Preparation,

characterization and antibacterial properties of silver-modified graphene oxide, J.

Mater. Chem. 21 (2011) 3350-3352.

[23] C. Sun, J.S.H. Lee, M.Q. Zhang, Magnetic nanoparticles in MR imaging and

drug delivery, Adv. Drug Deliver. Rev. 60 (2008) 1252-1265.

[24] O. Veiseh, J.W. Gunn, M.Q. Zhang, Design and fabrication of magnetic

nanoparticles for targeted drug delivery and imaging, Adv. Drug Deliver. Rev. 62

(2010) 284-304.

[25] Q.A. Pankhurst, J. Connolly, S.K. Jones, J. Dobson, Applications of magnetic

nanoparticles in biomedicine, J. Phys. D: Appl. Phys. 36 (2003) R167-R181.

[26] D. Horak, M. Babic, H. Mackova, M.J. Benes, Preparation and properties of

magnetic nano- and microsized particles for biological and environmental separations,

J. Sep. Sci. 30 (2007) 1751-1772.

of 47

Accep

ted

Man

uscr

ipt

27

[27] T.A. Liu, P. Spincemaille, L. de Rochefort, R. Wong, M. Prince, Y. Wang,

Unambiguous identification of superparamagnetic iron oxide particles through

quantitative susceptibility mapping of the nonlinear response to magnetic fields,

Magn. Reson. Imaging 28 (2010) 1383-1389.

[28] T.K. Jain, M.K. Reddy, M.A. Morales, D.L. Leslie-Pelecky, V. Labhasetwar,

Biodistribution, clearance, and biocompatibility of iron oxide magnetic nanoparticles

in rats, Mol. Pharm. 5 (2008) 316-327.

[29] N. Tran, A. Mir, D. Mallik, A. Sinha, S. Nayar, T.J. Webster, Bactericidal effect

of iron oxide nanoparticles on Staphylococcus aureus, Int. J. Nanomed. 5 (2010)

277-283.

[30] S. Chatterjee, A. Bandyopadhyay, K. Sarkar, Effect of iron oxide and gold

nanoparticles on bacterial growth leading towards biological application, J.

Nanobiotechnol. 9 (2011).

[31] M. Auffan, W. Achouak, J. Rose, M.A. Roncato, C. Chaneac, D.T. Waite, A.

Masion, J.C. Woicik, M.R. Wiesner, J.Y. Bottero, Relation between the redox state of

iron-based nanoparticles and their cytotoxicity toward Escherichia coli, Environ. Sci.

Technol. 42 (2008) 6730-6735.

[32] E.N. Taylor, T.J. Webster, The use of superparamagnetic nanoparticles for

prosthetic biofilm prevention, Int. J. Nanomed. 4 (2009) 145-152.

[33] O. Akhavan, E. Ghaderi, Escherichia coil bacteria reduce graphene oxide to

bactericidal graphene in a self-limiting manner, Carbon 50 (2012) 1853-1860.

[34] P.S.M. Dunlop, J.A. Byrne, N. Manga, B.R. Eggins, The photocatalytic removal

of 47

Accep

ted

Man

uscr

ipt

28

of bacterial pollutants from drinking water, J. Photoch. Photobio. A: Chem 148 (2002)

355-363.

[35] W.S Hummers Jr., R.E. Offeman, Preparation of Graphitic Oxide, J. Am. Chem.

Soc. 80 (1958) 1339.

[36] J.H. Deng, X.R. Zhang, G.M. Zeng, J.L. Gong, Q.Y. Niu, J. Liang, Simultaneous

removal of Cd(II) and ionic dyes from aqueous solution using magnetic graphene

oxide nanocomposite as an adsorbent, Chem. Eng. J. 226 (2013) 189-200.

[37] C.D. Vecitis, K.R. Zodrow, S. Kang, M. Elimelech,

Electronic-Structure-Dependent Bacterial Cytotoxicity of Single-Walled Carbon

Nanotubes, ACS Nano 4 (2010) 5471-5479.

[38] M.J. Hu, Y. Lu, S. Zhang, S.R. Guo, B. Lin, M. Zhang, S.H. Yu, High yield

synthesis of bracelet-like hydrophilic Ni-Co magnetic alloy flux-closure nanorings, J.

Am. Chem. Soc. 130 (2008) 11606-11607.

[39] S.L. Pan, Z.G. An, J.J. Zhang, G.Z. Song, Synthesis and hierarchical assembly of

CoNi flowery particles, Mater. Chem. Phys. 124 (2010) 342-346.

[40] S. Bai, X.P. Shen, G.X. Zhu, M.Z. Li, H.T. Xi, K.M. Chen, In situ Growth of

NixCo100-x Nanoparticles on Reduced Graphene Oxide Nanosheets and Their

Magnetic and Catalytic Properties, ACS Appl. Mater. Interfaces 4 (2012) 2378-2386.

[41] J.J. Shi, J.J. Zhu, Sonoelectrochemical fabrication of Pd-graphene nanocomposite

and its application in the determination of chlorophenols, Electrochim. Acta 56 (2011)

6008-6013.

[42] Q.Y. Wang, X.Q. Cui, W.M. Guan, W.T. Zheng, J.L. Chen, X.L. Zheng, X.M.

of 47

Accep

ted

Man

uscr

ipt

29

Zhang, C. Liu, T.Y. Xue, H.T. Wang, Z. Jin, H. Teng, Synthesis of flower-shape

palladium nanostructures on graphene oxide for electrocatalytic applications, J. Phys.

Chem. Solids 74 (2013) 1470-1474.

[43] Y.J. Yao, S.D. Miao, S.Z. Liu, L.P. Ma, H.Q. Sun, S.B. Wang, Synthesis,

characterization, and adsorption properties of magnetic Fe3O4@graphene

nanocomposite, Chem. Eng. J. 184 (2012) 326-332.

[44] G.X. Zhao, J.X. Li, X.M. Ren, C.L. Chen, X.K. Wang, Few-Layered Graphene

Oxide Nanosheets As Superior Sorbents for Heavy Metal Ion Pollution Management,

Environ. Sci. Technol. 45 (2011) 10454-10462.

[45] M.J. McAllister, J.L. Li, D.H. Adamson, H.C. Schniepp, A.A. Abdala, J. Liu, M.

Herrera-Alonso, D.L. Milius, R. Car, R.K. Prud'homme, I.A. Aksay, Single sheet

functionalized graphene by oxidation and thermal expansion of graphite, Chem. Mater.

19 (2007) 4396-4404.

[46] J.L. Gong, B. Wang, G.M. Zeng, C.P. Yang, C.G. Niu, Q.Y. Niu, W.J. Zhou, Y.

Liang, Removal of cationic dyes from aqueous solution using magnetic multi-wall

carbon nanotube nanocomposite as adsorbent, J. Hazard. Mater. 164 (2009)

1517-1522.

[47] M.A. Legodi, D. de Waal, The preparation of magnetite, goethite, hematite and

maghemite of pigment quality from mill scale iron waste, Dyes Pigments. 74 (2007)

161-168.

[48] X. Yang, C.L. Chen, J.X. Li, G.X. Zhao, X.M. Ren, X.K. Wang, Graphene

oxide-iron oxide and reduced graphene oxide-iron oxide hybrid materials for the

of 47

Accep

ted

Man

uscr

ipt

30

removal of organic and inorganic pollutants, RSC Adv. 2 (2012) 8821-8826.

[49] J.F. Shen, Y.Z. Hu, M. Shi, N. Li, H.W. Ma, M.X. Ye, One Step Synthesis of

Graphene Oxide-Magnetic Nanoparticle Composite, J. Phys. Chem. C 114 (2010)

1498-1503.

[50] V. Chandra, J. Park, Y. Chun, J.W. Lee, I.C. Hwang, K.S. Kim, Water-Dispersible

Magnetite-Reduced Graphene Oxide Composites for Arsenic Removal, ACS Nano 4

(2010) 3979-3986.

[51] B.S. Inbaraj, T.Y. Tsai, B.H. Chen, Synthesis, characterization and antibacterial

activity of superparamagnetic nanoparticles modified with glycol chitosan, Sci.

Technol. Adv. Mat. 13 (2012).

[52] B.S. Inbaraj, T.H. Kao, T.Y. Tsai, C.P. Chiu, R. Kumar, B.H. Chen, The synthesis

and characterization of poly(gamma-glutamic acid)-coated magnetite nanoparticles

and their effects on antibacterial activity and cytotoxicity, Nanotechnology 22 (2011).

[53] Y.S. Fu, P. Xiong, H.Q. Chen, X.Q. Sun, X. Wang, High Photocatalytic Activity

of Magnetically Separable Manganese Ferrite-Graphene Heteroarchitectures, Ind. Eng.

Chem. Res. 51 (2012) 725-731.

[54] Y.S. Fu, H.Q. Chen, X.Q. Sun, X. Wang, Combination of cobalt ferrite and

graphene: High-performance and recyclable visible-light photocatalysis, Appl. Catal.

B: Environ. 111 (2012) 280-287.

[55] K. Shellenberger, B.E. Logan, Effect of molecular scale roughness of glass beads

on colloidal and bacterial deposition, Environ. Sci. Technol. 36 (2002) 184-189.

[56] C. Vasilescu, P. Drob, E. Vasilescu, I. Demetrescu, D. Ionita, M. Prodana, S.I.

of 47

Accep

ted

Man

uscr

ipt

31

Drob, Characterisation and corrosion resistance of the electrodeposited

hydroxyapatite and bovine serum albumin/hydroxyapatite films on Ti-6Al-4V-1Zr

alloy surface, Corros. Sci. 53 (2011) 992-999.

[57] E. Lima, A. Guzmán-Vargas, J. Méndez-Vivar, H. Pfeiffer, J. Fraissard,

Fe-ZSM-5 Catalysts: Preparation in Organic Media, Fe-particle Morphology and NOx

Reduction Activity, Catal. Lett. 120 (2008) 244-251.

[58] J.L. Gong, X.Y. Wang, G.M. Zeng, L. Chen, J.H. Deng, X.R. Zhang, Q.Y. Niu,

Copper (II) removal by pectin-iron oxide magnetic nanocomposite adsorbent, Chem.

Eng. J. 185 (2012) 100-107.

[59] Z. Zhang, J. Zhang, B.L. Zhang, J.L. Tang, Mussel-inspired functionalization of

graphene for synthesizing Ag-polydopamine-graphene nanosheets as antibacterial

materials, Nanoscale 5 (2013) 118-123.

[60] T.S. Sreeprasad, M.S. Maliyekkal, K. Deepti, K. Chaudhari, P.L. Xavier, T.

Pradeep, Transparent, Luminescent, Antibacterial and Patternable Film Forming

Composites of Graphene Oxide/Reduced Graphene Oxide, ACS Appl. Mater.

Interfaces 3 (2011) 2643-2654.

[61] G. Nangmenyi, X.A. Li, S. Mehrabi, E. Mintz, J. Economy, Silver-modified iron

oxide nanoparticle impregnated fiberglass for disinfection of bacteria and viruses in

water, Mater. Lett. 65 (2011) 1191-1193.

[62] C. Lee, J.Y. Kim, W.I. Lee, K.L. Nelson, J. Yoon, D.L. Sedlak, Bactericidal effect

of zero-valent iron nanoparticles on Escherichia coli, Environ. Sci. Technol. 42 (2008)

4927-4933.

of 47

Accep

ted

Man

uscr

ipt

32

[63] W.B. Hu, C. Peng, W.J. Luo, M. Lv, X.M. Li, D. Li, Q. Huang, C.H. Fan,

Graphene-Based Antibacterial Paper, ACS Nano 4 (2010) 4317-4323.

[64] R.C. Fahey, W.C. Brown, W.B. Adams, M.B. Worsham, Occurrence of

glutathione in bacteria, J. Bacteriol. 133 (1978) 1126-1129.

of 47

Accep

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Man

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Figure Captions

Figure 1 TEM image of GO (a), SEM image of M-GO (b), TEM image of M-GO (c),

and the size distribution of iron oxide nanoparticles (d).

Figure 2 XRD patterns of GO (a), M (b), M1-GO (c), and M2-GO (d).

Figure 3 FTIR spectra of GO (a), M2-GO (b), and M1-GO (c).

Figure 4 Raman spectra of GO (a), M1-GO (b) and M2-GO (c).

Figure 5 Magnetization curves of M2-GO (a) and M1-GO (b) at 300 K. The insert

shows the M-GO nanocomposites dispersion and magnetic separation.

Figure 6 Inactivation of E. coli by GO, M, M1-GO and M2-GO, at the same

concentration of 100 μg/mL for 2 h at 37 ℃.

Figure 7 Comparison of antibacterial activity of M-GO nanomatrials with various

GO/M mass ratios (a) and M/GO mass ratios (b).

Figure 8 Antibacterial activity of M-GO with various concentrations. 5 mL of M-GO

(at 30, 40, 50, 100, 200, 300 μg/mL) was incubated with 5 mL E. coli (106-107

CFU/mL) for 2 h at 37 ℃.

Figure 9 TEM images of E. coli cells treated with DI water (a) and M-GO (b) at 37

℃ for 2 h.

Figure 10 (a) Oxidation of GSH (0.4 mM) by GO, M, M1-GO and M2-GO, at the

same concentration of 100 μg/mL. (b) GSH (0.4 mM) was incubated with various

concentrations of M-GO for 2 h. GSH oxidized by H2O2 (10 mM) was used as a

positive control and GSH with bicarbonate buffer (50 mM) was used as a negative

control.

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Scheme 1 Schematic diagram of M-GO synthesis.

Scheme 2 Antibacterial mechanism of M-GO toward E. coli.

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Table 1

The correlation among antibacterial activities, surface roughness, and oxidative ability

Loss of cells a

(%)

Loss of GSH

b (%)

Specific surface

area (m2/g) c

Fractal

dimension d

GO 77.49 ± 12.79 58.62 ± 0.54 8.55 2.09

M 53.00 ± 15.70 46.50 ± 4.77 73.34 2.11

M1-GO 62.26 ± 3.03 51.11 ± 7.23 214.84 2.11

M2-GO 91.49 ± 2.82 58.55 ± 8.05 351.96 2.13

a Data extracted from Figure 6. b Data extracted from Figure 10a. c Calculated from N2

adsorption-desorption isotherms and BET equation. d As determined by SAXS data

under the Porod Law.

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Figure 10

Figure 10

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Scheme 1

Scheme 1

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Scheme 2

Scheme 2

Inactivation performance and mechanism of Escherichia coli in aqueous system exposed to iron oxide...

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