Accepted Manuscript Title: Inactivation performance and mechanism of Escherichia coli in aqueous system exposed to iron oxide loaded graphene nanocomposites Author: Can-Hui Deng Ji-Lai Gong Guang-Ming Zeng Cheng-Gang Niu Qiu-Ya Niu Wei Zhang Hong-Yu Liu PII: DOI: Reference:
S0304-3894(14)00346-X http://dx.doi.org/doi:10.1016/j.jhazmat.2014.05.011 HAZMAT 15918
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
2-2-2014 4-5-2014 5-5-2014
Please cite this article as: http://dx.doi.org/10.1016/j.jhazmat.2014.05.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights Magnetic-graphene
oxide (M-GO) with excellent antibacterial activity
is prepared. antibacterial activity of M-GO relies on concentration and mass
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The
Synergetic
cr
ratio of M/GO.
antibacterial effect of M-GO is observed with M/GO mass
images illustrate that M-GO has penetrated into the cytoplasm.
Synergetic
an
TEM
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ratio of 9.09.
mechanism accounts for the antibacterial activity of
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M
M-GO.
1
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Inactivation performance and mechanism of Escherichia coli in aqueous system exposed to iron oxide loaded graphene nanocomposites
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Can-Hui Deng, Ji-Lai Gong*, Guang-Ming Zeng, Cheng-Gang Niu, Qiu-Ya Niu, Wei
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Zhang and Hong-Yu Liu
College of Environmental Science and Engineering, Hunan University, Changsha,
an
410082, PR China
Key Laboratory of Environmental Biology and Pollution Control, Ministry of
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Education, Hunan University, Changsha 410082, PR China
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*Corresponding author. Tel: +86 731 88822829; Fax: +86 731 88822829
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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 2
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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
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nanocomposites. M-GO nanomaterials were possible to deposit on or penetrate into
cr
cells leading to leakage of intercellular contents and loss of cell integrity. The
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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
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excellent antibacterial efficiency against E. coli and separation-convenient property from water could be potent bactericidal nanomaterials for water disinfection.
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Keywords: graphene oxide; magnetic iron oxide; antibacterial; oxidative stress;
1. Introduction
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water disinfection
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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 3
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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
natural
organic
lysozyme-layered
double
hydroxides
nanocomposites
cr
and
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TiO2 [10] and ZnO [11]), natural organic antimicrobial peptides [12], chitosan [13]
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(LYZ-LDHs) [14]. In addition, carbon-based nanomaterials, such as fullerol [15], aqueous fullerence (nC60) [16], and carbon nanotubes (CNTs) [17] have displayed
an
fascinating antibacterial activities.
Graphene oxide (GO), as a one-atom-thick sheet of sp2-bonded carbon atoms that
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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
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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
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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. 4
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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
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magnetic nanoparticles and found the numbers of Staphylococcus epidermidis
cr
decreased when treated with magnetic iron oxide nanoparticles at the dosages equals
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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
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Staphylococcus aureus growth, and the bactericidal activity of IO/PVA was mainly contributed to the concentration of the nanoparticles.
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In this work, the introduction of iron oxide magnetic (M) nanoparticles into graphene oxide was proposed to constitute a novel antibacterial nanomaterial, which
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will combine the antibacterial properties of graphene oxide and the separation convenience of magnetic nanoparticles. Magnetic-graphene oxide (M-GO) was
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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 5
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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
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chemical factory (Shanghai, China). Multi-wall carbon nanotubes (MWNTs) with
cr
outer diameter 40-60 nm and length 5-15 μm were obtained from Shenzhen Nanoport
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Company (Shenzhen, China). Glutathione detection kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Sodium hydroxide (NaOH),
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potassium permanganate (KMnO4), sodium nitrate (NaNO3), sulfuric acid (H2SO4), hydrogen peroxide (H2O2), glutaraldehyde, glutathione (GSH), ferrous ammonium [(NH4)2SO4·FeSO4·6H2O]
and
ammonium
M
sulfate
ferric
sulfate
[NH 4 Fe
(SO 4 )2 ·12H2 O] were all purchased from Sinopharm chemical reagent Co., (Shanghai,
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China). All the chemicals used in this study were of analytical reagent grade. 2.2 Preparation of GO
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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 6
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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
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The synthesis of M-GO was prepared on the basis of our previous report [36].
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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
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mixing ferric and ferrous solutions (molar ratio of 1.5 : 1 for Fe3+ and Fe2+,
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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
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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
te
d
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
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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, 7
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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
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E. coli cells were incubated with various samples including GO, M, M1-GO and
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M2-GO suspensions in DI water at 37 ℃ under 150 rpm shaking speed for 2 h at a
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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
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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
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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
prepared in triplicate.
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suspension incubated without nanomaterials was used as control. All treatments were
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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 8
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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.
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Nanocomposites dispersion (250 μL) in 50 mM bicarbonate buffer (pH 8.6) was
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added into 250 μL GSH (at the concentration of 0.4 mM in bicarbonate buffer) in
us
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
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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
M
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
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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
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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 9
Page 9 of 47
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
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magnetization curve was recorded on vibrating sample magnetometer (Lake Shore
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7410). ZRY-2P thermal analyzer was employed for TGA at temperature of 20-800 ℃
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and heating rate of 20 ℃/min. The BET surface area was determined by Tristar 3020 volumetric analyzer (Micromeritics Instrument Corporation, USA). Raman spectra
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were acquired on LabRAM-010 Laser Raman spectrometer (HORIBA Jobin Yvon, France). Small-angle X-ray scattering experiments were performed with Anton Paar
M
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
d
scattering angle and wavelength, respectively. The obtained data were modified to
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follow the Porod law, where the scattering intensity I (q) is proportional to q-2 for
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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 10
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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
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GO matrix in M-GO nanocomposites. The histogram of the particle size distribution
cr
was presented in Fig. 1d. The reason that small amount of nanoparticles tended to
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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
an
previous report [38-40].
The X-ray diffraction (XRD) patterns of GO, M, M1-GO and M2-GO were
M
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
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d
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
Ac ce p
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 11
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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)
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was obviously reduced, and the GO peaks at 2θ = 42.3°(100) totally disappeared in
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the XRD patterns of M-GO, which could be caused by the reasons as follows: The
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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
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strong peaks of the M nanoparticles overwhelming the weak carbon peaks [48]. The results in our work were consistent with the previous studies [43].
M
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
d
cm-1 was ascribed to the stretching of O-H [49]. The peaks at 1725 cm-1, 1624 cm-1
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and 1399 cm-1 corresponded to carbonyl C=O stretching vibrations [43], aromatic
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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 12
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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
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M2-GO, respectively. In addition, the Raman G bands shifted from 1597 to 1591 cm-1
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and to 1592 cm-1 for M1-GO and M2-GO, respectively (see Fig. 4 b, c). For M-GO,
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the Raman G and D bands shifted to lower frequency in comparison with that of GO, indicating that GO was reduced [53, 54].
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Different surface roughness of materials could significantly influence the attachment of bacteria on the material surface at the period of interaction between
M
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
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calculated from N2 adsorption-desorption isotherms and BET equation, and the fractal dimension values of antibacterial nanomaterials as determined by SAXS data
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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 13
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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.
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The magnetic properties of the synthesized M-GO nanocomposites were
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recorded at room temperature (300 K) by VSM, as shown in Fig. 5. For the M1-GO,
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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
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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
M
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.
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3.2 Antibacterial activity of GO, M and M-GO dispersions Antibacterial activity of four types of nanomaterials obtained in this work was
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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)
14
Page 14 of 47
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,
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M, M1-GO, and M2-GO. GO dispersion exhibited an apparent antibacterial activity
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with the cell inactivation percentage at 77.49 ± 12.79 %. M dispersion displayed a
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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
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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
percentage up to 91.49 ± 2.82 %.
M
bacterial inactivation among the four kinds of nanomaterials, with the inactivation
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The disinfection activity of carbon-based nanocomposites including M1-GO, M2-GO and magnetic multi-walled carbon nanotubes (M-MWNTs) were also
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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 15
Page 15 of 47
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
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M2-GO at the mass ratio of GO/M 0.11, and decreased for M1-GO at 0.18 (displayed
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in Fig. 7a). It was concluded that the antibacterial activity of M-GO nanocomposites
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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
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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
M
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
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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
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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 16
Page 16 of 47
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
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between GO and M toward E. coli in our work was dependent on the mass ratio of
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M/GO in M-GO nanomaterials. The optimal mass ratio of M to GO and the
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mechanism of synergistic effect between GO and M will be investigated in our further studies.
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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
M
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
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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 ±
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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 17
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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
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were likely assigned to M-GO or M nanomaterials passed into cells via direct
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interaction with E. coli during incubation. Similar phenomena were also observed by
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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
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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
role in their antibacterial activity.
M
structures. Such irreversible damage of cells induced by M-GO may play a significant
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3.4 Oxidation ability of antibacterial materials The mechanism of oxidation damage was the most accepted explanation for the
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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 18
Page 18 of 47
[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
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M2-GO (at the same concentration of 100 μg/mL) for 2 h. The loss of GSH was
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quantified using a thiol quantization kit and the details of experiments were
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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
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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
M
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
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inactivation induced by the four nanomaterails. Therefore, it was concluded that the oxidative ability of the nanomaterials had a significant influence on their antibacterial
Ac ce p
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 19
Page 19 of 47
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
ip t
nanomaterials.
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3.5 Antibacterial mechanism of M-GO
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The correlation among antibacterial activities, surface roughness and GSH oxidation was summarized in Table 1. On the one hand, comparing GO and M2-GO,
an
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
M
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
d
possessed higher surface roughness with fractal dimension of 2.13 and surface area of
te
351.96 m2/g. Their distinct antibacterial activities indicated that surface property of
Ac ce p
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 20
Page 20 of 47
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
ip t
incubation period, membrane stress induced by direct interaction between sharp
cr
nanosheets and bacteria and the following superoxide anion-independent oxidation
us
toward intercellular components of cells. They suggested that the antimicrobial mechanism of graphene-based materials were contributed to the synergy of membrane
an
and oxidation stress.
Here, the synergetic mechanism was supposed to account for observations in our
M
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
te
d
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.
Ac ce p
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 21
Page 21 of 47
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
ip t
inducing ROS-independent oxidative stress toward E. coli cells. Tran et al. [29]
cr
confirmed metal oxide Fe3O4 inhibited the growth of S. aureus via oxidative stress
us
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.
an
The strong oxidation activity of M-GO toward GSH in our work supported that
M
M-GO was efficient to oxidize thiols or other intercellular contents.
4. Conclusions
te
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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
Ac ce p
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 22
Page 22 of 47
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
ip t
M-GO may be relying on both physical membrane puncture and chemical cellular
cr
matters oxidation, which were similar to the three-step antibacterial mechanisms of
us
CNTs. In views of these interesting properties of both strong magnetic property and outstanding antibacterial ability, M-GO nanomaterials have the potential applications
an
for environmental drinking water treatments. Acknowledgments
M
The authors are grateful for the financial supports from National Natural Science Foundation of China (51039001, 50978088, 50808070, 21275044 and 51108166),
te
d
Interdisciplinary Research Funds for Hunan University, the Natural Science Foundation of Hunan Province, China (Grant no. 12JJB003) and the Scientific
Ac ce p
Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
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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).
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Figure 2 XRD patterns of GO (a), M (b), M1-GO (c), and M2-GO (d).
cr
Figure 3 FTIR spectra of GO (a), M2-GO (b), and M1-GO (c).
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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
an
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
M
concentration of 100 μg/mL for 2 h at 37 ℃.
Figure 7 Comparison of antibacterial activity of M-GO nanomatrials with various
te
d
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
Ac ce p
(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. 33
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Scheme 1 Schematic diagram of M-GO synthesis.
Ac ce p
te
d
M
an
us
cr
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Scheme 2 Antibacterial mechanism of M-GO toward E. coli.
34
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Table 1 The correlation among antibacterial activities, surface roughness, and oxidative ability a
area (m2/g) c
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
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
(%)
ip t
b
2.11
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(%)
an
a
Loss of GSH Specific surface Fractal
cr
Loss of cells
Data extracted from Figure 6. b Data extracted from Figure 10a. c Calculated from N2
M
adsorption-desorption isotherms and BET equation. d As determined by SAXS data
Ac ce p
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d
under the Porod Law.
35
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Scheme 1
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Scheme 1
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Scheme 2
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Scheme 2
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S0304-3894(14)00346-X http://dx.doi.org/doi:10.1016/j.jhazmat.2014.05.011 HAZMAT 15918
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
2-2-2014 4-5-2014 5-5-2014
Please cite this article as: http://dx.doi.org/10.1016/j.jhazmat.2014.05.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights Magnetic-graphene
oxide (M-GO) with excellent antibacterial activity
is prepared. antibacterial activity of M-GO relies on concentration and mass
ip t
The
Synergetic
cr
ratio of M/GO.
antibacterial effect of M-GO is observed with M/GO mass
images illustrate that M-GO has penetrated into the cytoplasm.
Synergetic
an
TEM
us
ratio of 9.09.
mechanism accounts for the antibacterial activity of
Ac ce p
te
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M
M-GO.
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Inactivation performance and mechanism of Escherichia coli in aqueous system exposed to iron oxide loaded graphene nanocomposites
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Can-Hui Deng, Ji-Lai Gong*, Guang-Ming Zeng, Cheng-Gang Niu, Qiu-Ya Niu, Wei
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Zhang and Hong-Yu Liu
College of Environmental Science and Engineering, Hunan University, Changsha,
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410082, PR China
Key Laboratory of Environmental Biology and Pollution Control, Ministry of
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Education, Hunan University, Changsha 410082, PR China
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*Corresponding author. Tel: +86 731 88822829; Fax: +86 731 88822829
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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 2
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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
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nanocomposites. M-GO nanomaterials were possible to deposit on or penetrate into
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cells leading to leakage of intercellular contents and loss of cell integrity. The
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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
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excellent antibacterial efficiency against E. coli and separation-convenient property from water could be potent bactericidal nanomaterials for water disinfection.
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Keywords: graphene oxide; magnetic iron oxide; antibacterial; oxidative stress;
1. Introduction
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water disinfection
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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 3
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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
natural
organic
lysozyme-layered
double
hydroxides
nanocomposites
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and
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TiO2 [10] and ZnO [11]), natural organic antimicrobial peptides [12], chitosan [13]
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(LYZ-LDHs) [14]. In addition, carbon-based nanomaterials, such as fullerol [15], aqueous fullerence (nC60) [16], and carbon nanotubes (CNTs) [17] have displayed
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fascinating antibacterial activities.
Graphene oxide (GO), as a one-atom-thick sheet of sp2-bonded carbon atoms that
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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
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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
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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. 4
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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
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magnetic nanoparticles and found the numbers of Staphylococcus epidermidis
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decreased when treated with magnetic iron oxide nanoparticles at the dosages equals
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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
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Staphylococcus aureus growth, and the bactericidal activity of IO/PVA was mainly contributed to the concentration of the nanoparticles.
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In this work, the introduction of iron oxide magnetic (M) nanoparticles into graphene oxide was proposed to constitute a novel antibacterial nanomaterial, which
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will combine the antibacterial properties of graphene oxide and the separation convenience of magnetic nanoparticles. Magnetic-graphene oxide (M-GO) was
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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 5
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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
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chemical factory (Shanghai, China). Multi-wall carbon nanotubes (MWNTs) with
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outer diameter 40-60 nm and length 5-15 μm were obtained from Shenzhen Nanoport
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Company (Shenzhen, China). Glutathione detection kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Sodium hydroxide (NaOH),
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potassium permanganate (KMnO4), sodium nitrate (NaNO3), sulfuric acid (H2SO4), hydrogen peroxide (H2O2), glutaraldehyde, glutathione (GSH), ferrous ammonium [(NH4)2SO4·FeSO4·6H2O]
and
ammonium
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sulfate
ferric
sulfate
[NH 4 Fe
(SO 4 )2 ·12H2 O] were all purchased from Sinopharm chemical reagent Co., (Shanghai,
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China). All the chemicals used in this study were of analytical reagent grade. 2.2 Preparation of GO
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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 6
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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
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The synthesis of M-GO was prepared on the basis of our previous report [36].
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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
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mixing ferric and ferrous solutions (molar ratio of 1.5 : 1 for Fe3+ and Fe2+,
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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
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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
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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
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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, 7
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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
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E. coli cells were incubated with various samples including GO, M, M1-GO and
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M2-GO suspensions in DI water at 37 ℃ under 150 rpm shaking speed for 2 h at a
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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
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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
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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
prepared in triplicate.
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suspension incubated without nanomaterials was used as control. All treatments were
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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 8
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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.
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Nanocomposites dispersion (250 μL) in 50 mM bicarbonate buffer (pH 8.6) was
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added into 250 μL GSH (at the concentration of 0.4 mM in bicarbonate buffer) in
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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
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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
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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
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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
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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 9
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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
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magnetization curve was recorded on vibrating sample magnetometer (Lake Shore
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7410). ZRY-2P thermal analyzer was employed for TGA at temperature of 20-800 ℃
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and heating rate of 20 ℃/min. The BET surface area was determined by Tristar 3020 volumetric analyzer (Micromeritics Instrument Corporation, USA). Raman spectra
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were acquired on LabRAM-010 Laser Raman spectrometer (HORIBA Jobin Yvon, France). Small-angle X-ray scattering experiments were performed with Anton Paar
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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
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scattering angle and wavelength, respectively. The obtained data were modified to
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follow the Porod law, where the scattering intensity I (q) is proportional to q-2 for
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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 10
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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
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GO matrix in M-GO nanocomposites. The histogram of the particle size distribution
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was presented in Fig. 1d. The reason that small amount of nanoparticles tended to
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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
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previous report [38-40].
The X-ray diffraction (XRD) patterns of GO, M, M1-GO and M2-GO were
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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
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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
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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 11
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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)
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was obviously reduced, and the GO peaks at 2θ = 42.3°(100) totally disappeared in
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the XRD patterns of M-GO, which could be caused by the reasons as follows: The
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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
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strong peaks of the M nanoparticles overwhelming the weak carbon peaks [48]. The results in our work were consistent with the previous studies [43].
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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
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cm-1 was ascribed to the stretching of O-H [49]. The peaks at 1725 cm-1, 1624 cm-1
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and 1399 cm-1 corresponded to carbonyl C=O stretching vibrations [43], aromatic
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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 12
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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
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M2-GO, respectively. In addition, the Raman G bands shifted from 1597 to 1591 cm-1
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and to 1592 cm-1 for M1-GO and M2-GO, respectively (see Fig. 4 b, c). For M-GO,
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the Raman G and D bands shifted to lower frequency in comparison with that of GO, indicating that GO was reduced [53, 54].
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Different surface roughness of materials could significantly influence the attachment of bacteria on the material surface at the period of interaction between
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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
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calculated from N2 adsorption-desorption isotherms and BET equation, and the fractal dimension values of antibacterial nanomaterials as determined by SAXS data
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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 13
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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.
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The magnetic properties of the synthesized M-GO nanocomposites were
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recorded at room temperature (300 K) by VSM, as shown in Fig. 5. For the M1-GO,
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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
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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
M
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.
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3.2 Antibacterial activity of GO, M and M-GO dispersions Antibacterial activity of four types of nanomaterials obtained in this work was
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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,
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M, M1-GO, and M2-GO. GO dispersion exhibited an apparent antibacterial activity
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with the cell inactivation percentage at 77.49 ± 12.79 %. M dispersion displayed a
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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
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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
percentage up to 91.49 ± 2.82 %.
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bacterial inactivation among the four kinds of nanomaterials, with the inactivation
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The disinfection activity of carbon-based nanocomposites including M1-GO, M2-GO and magnetic multi-walled carbon nanotubes (M-MWNTs) were also
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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 15
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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
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M2-GO at the mass ratio of GO/M 0.11, and decreased for M1-GO at 0.18 (displayed
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in Fig. 7a). It was concluded that the antibacterial activity of M-GO nanocomposites
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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
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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
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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
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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
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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 16
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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
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between GO and M toward E. coli in our work was dependent on the mass ratio of
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M/GO in M-GO nanomaterials. The optimal mass ratio of M to GO and the
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mechanism of synergistic effect between GO and M will be investigated in our further studies.
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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
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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
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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 ±
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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 17
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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
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were likely assigned to M-GO or M nanomaterials passed into cells via direct
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interaction with E. coli during incubation. Similar phenomena were also observed by
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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
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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
role in their antibacterial activity.
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structures. Such irreversible damage of cells induced by M-GO may play a significant
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3.4 Oxidation ability of antibacterial materials The mechanism of oxidation damage was the most accepted explanation for the
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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 18
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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
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M2-GO (at the same concentration of 100 μg/mL) for 2 h. The loss of GSH was
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quantified using a thiol quantization kit and the details of experiments were
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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
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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
M
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
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inactivation induced by the four nanomaterails. Therefore, it was concluded that the oxidative ability of the nanomaterials had a significant influence on their antibacterial
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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 19
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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
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nanomaterials.
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3.5 Antibacterial mechanism of M-GO
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The correlation among antibacterial activities, surface roughness and GSH oxidation was summarized in Table 1. On the one hand, comparing GO and M2-GO,
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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
M
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
d
possessed higher surface roughness with fractal dimension of 2.13 and surface area of
te
351.96 m2/g. Their distinct antibacterial activities indicated that surface property of
Ac ce p
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 20
Page 20 of 47
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
ip t
incubation period, membrane stress induced by direct interaction between sharp
cr
nanosheets and bacteria and the following superoxide anion-independent oxidation
us
toward intercellular components of cells. They suggested that the antimicrobial mechanism of graphene-based materials were contributed to the synergy of membrane
an
and oxidation stress.
Here, the synergetic mechanism was supposed to account for observations in our
M
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
te
d
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.
Ac ce p
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 21
Page 21 of 47
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
ip t
inducing ROS-independent oxidative stress toward E. coli cells. Tran et al. [29]
cr
confirmed metal oxide Fe3O4 inhibited the growth of S. aureus via oxidative stress
us
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.
an
The strong oxidation activity of M-GO toward GSH in our work supported that
M
M-GO was efficient to oxidize thiols or other intercellular contents.
4. Conclusions
te
d
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
Ac ce p
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 22
Page 22 of 47
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
ip t
M-GO may be relying on both physical membrane puncture and chemical cellular
cr
matters oxidation, which were similar to the three-step antibacterial mechanisms of
us
CNTs. In views of these interesting properties of both strong magnetic property and outstanding antibacterial ability, M-GO nanomaterials have the potential applications
an
for environmental drinking water treatments. Acknowledgments
M
The authors are grateful for the financial supports from National Natural Science Foundation of China (51039001, 50978088, 50808070, 21275044 and 51108166),
te
d
Interdisciplinary Research Funds for Hunan University, the Natural Science Foundation of Hunan Province, China (Grant no. 12JJB003) and the Scientific
Ac ce p
Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
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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).
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Figure 2 XRD patterns of GO (a), M (b), M1-GO (c), and M2-GO (d).
cr
Figure 3 FTIR spectra of GO (a), M2-GO (b), and M1-GO (c).
us
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
an
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
M
concentration of 100 μg/mL for 2 h at 37 ℃.
Figure 7 Comparison of antibacterial activity of M-GO nanomatrials with various
te
d
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
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(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. 33
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Scheme 1 Schematic diagram of M-GO synthesis.
Ac ce p
te
d
M
an
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cr
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Scheme 2 Antibacterial mechanism of M-GO toward E. coli.
34
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Table 1 The correlation among antibacterial activities, surface roughness, and oxidative ability a
area (m2/g) c
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
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
(%)
ip t
b
2.11
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(%)
an
a
Loss of GSH Specific surface Fractal
cr
Loss of cells
Data extracted from Figure 6. b Data extracted from Figure 10a. c Calculated from N2
M
adsorption-desorption isotherms and BET equation. d As determined by SAXS data
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d
under the Porod Law.
35
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Scheme 1
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