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Light-enhanced Antibacterial Activity of Graphene Oxide Mainly via Accelerated Electron Transfer Yu Chong, Cuicui Ge, Ge Fang, Renfei Wu, He Zhang, Zhifang Chai, Chunying Chen, and Jun-Jie Yin Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00663 • Publication Date (Web): 03 Aug 2017 Downloaded from http://pubs.acs.org on August 7, 2017
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Light-enhanced Antibacterial Activity of Graphene Oxide
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Mainly via Accelerated Electron Transfer
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Yu Chong,†,⊥ Cuicui Ge,*,†,⊥ Ge Fang,† Renfei Wu,† He Zhang,† Zhifang Chai,†
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Chunying Chen*,‡ and Jun-Jie Yin*,⊥
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†
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Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University,
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Suzhou 215123, China
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‡
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Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Chinese
School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation
Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety & CAS Center for
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Academy of Sciences, Beijing 100190, China
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⊥
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Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug
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Administration, College Park, Maryland 20740, United States
Division of Bioanalytical Chemistry and Division of Analytical Chemistry, Office of
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ABSTRACT:
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Before graphene derivatives can be exploited as next generation antimicrobials, we
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must understand their behavior under environmental conditions. Here we demonstrate
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how exposure to simulated sunlight significantly enhances the antibacterial activity of
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graphene oxide (GO) and reveal the underlying mechanism. Our measurements of
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reactive oxygen species (ROS) showed only singlet oxygen (1O2) is generated by GO
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exposed to simulated sunlight, which contributes only slightly to the oxidation of
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antioxidant biomolecules. Unexpectedly, we find the main cause of oxidation is
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light-induced electron-hole pairs generated on the surface of GO. These light-induced
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electrons promote the reduction of GO, introducing additional carbon-centered free
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radicals which may also enhance the antibacterial activities of GO. We conclude that
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GO-mediated oxidative stress mainly is ROS-independent; simulated sunlight
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accelerates the transfer of electrons from antioxidant biomolecules to GO, thereby
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destroying bacterial antioxidant systems and causing the reduction of GO. Our
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insights will help support the development of graphene for antibacterial applications.
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Table of Contents
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INTRODUCTION The threat of bacterial infections that are untreatable due to antibiotic resistance
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is a deepening crisis worldwide. Therefore, it is highly desirable to develop novel
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antibacterial agents, particularly those which have mechanisms of action difficult for
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bacteria to overcome. The extraordinary electronic, mechanic, and optical
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characteristics of graphene structures and graphene derivatives suggest these could
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become the next generation of antimicrobial materials.1-3 The antibacterial action of
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graphene oxide (GO) arises from both physical and chemical activities.4-10 Physical
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damage is caused by the sharp edges of GO that shred bacterial membranes, damage
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RNA, and cause the destructive extraction phospholipid molecules.5-7 The primary
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type of chemical damage is believed to be oxidative stress, initiated by either
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reactive oxygen species (ROS) or by charge transfer.9,10
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To date, most reports have focused on the close relationship between
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antimicrobial activities and the physicochemical properties of GO.1,11,12 For instance,
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the antibacterial activity of GO is influenced by the size of the graphene sheets.4,13
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Data also indicate that the antimicrobial activity of GO can be enhanced by
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increasing the number of defects in GO nanosheets. Other characteristics which can
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alter GO antibacterial properties include layer number, morphology, dispersibility,
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surface charge, and oxygen content.14-17
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However, environmental conditions might also transform the physicochemical
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features of GO in ways that would alter their antibiotic effects.18,19 Several studies
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have shown that GO can be photochemically reduced by exposure to high-energy
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ultraviolet or laser light.20,21 Yang et al.22 reported such reduced GO could be used
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for in vivo photothermal therapy on tumors, due to its strong optical absorbance in
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the near-infrared (NIR) region. A study by Hou et al. in 2015 demonstrated that GO
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could behave as a semiconductor photocatalyst, generating electron−hole pairs able
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to reduce the oxygen content of GO structures.23 These findings leave open the
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question of whether exposure to sunlight could affect the antibacterial activity of GO.
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Further, it is not known whether data from previous explorations of GO exposed to
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specific durations of high intensity laser or other artificial light sources can provide
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adequate predictions about how photoreactions will occur under conditions of
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natural or simulated sunlight.
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Our current project examines these factors for the first time, systematically
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evaluating changes in the antibacterial activity of GO upon exposure to simulated
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sunlight. Then, we studied the oxidative stress pathway, including the generation of
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ROS, using electron spin resonance (ESR) techniques, to identify the mechanism
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responsible for that enhanced antibacterial activity. We investigated the possibility of
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ROS-independent oxidative stress and determined the production of light-induced
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electron-hole pairs. By using UV-vis spectrum and X-ray photoelectron
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spectroscopy (XPS) we were able to characterize the chemical transformation of GO
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in the presence of both simulated sunlight and reducing agents. Insights from these
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multiple approaches, enable us to provide a clear explanation for the observed
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enhancement of antibacterial activity of GO after exposure to simulated sunlight.
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MATERIALS AND METHODS
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Materials. Graphene oxide (GO) was purchased from Chengdu Organic
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Chemical Company, Chinese Academy of Science. The E. coli strain ATCC-25922
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was obtained from the American Type Culture Collection (ATCC, Rockville, MD,
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USA). 5,5'-Dithiobis (2-nitrobenzoic acid) (Ellman’s reagent, DTNB), 5,5-Dimethyl-
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1-pyrroline-N-oxide (DMPO), 5-tert-Butoxycarbonyl-5-methyl-1-pyrroline-N-oxide
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(BMPO) were obtained from Dojindo Laboratories (Kumamoto, Japan).
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1-Hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) and 2,2,6,6-
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tetramethylpiperidine-1-oxyl (TEMPO) were purchased from Alexis Biochemicals
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(Enzo Life Sciences, Farmingdale, NY). All solutions were prepared using Milli-Q
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water (18 MΩ cm). Other chemicals were obtained from Sigma Aldrich (St. Louis,
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MO) and used as received. A 450 W Xenon lamp, filtered by an air mass (AM) 1.5 G
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solar simulation bandpass filter was used to provide simulated sunlight irradiation.
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The light path length was about 40 cm and the spectral irradiance of this source,
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monitored with a Newport Optical Meter, was determined to be 380 mW/cm2.
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Characterization of GO. The as-received sample was characterized by various
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techniques. Atomic force microscope (AFM) images were acquired from an AFM
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(Dimension Icon, Bruker) operating in the ScanAsyst mode. The UV-vis spectra
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were recorded on a Shimadzu UV-3600 UV-vis spectrophotometer. Raman spectra
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were obtained on a JY HR800 spectrometer with 532 nm wavelength incident laser
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light. We used a Horiba Fluorolog 3-221 spectrofluorometer with a 1 cm light path
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quartz cuvette to characterize photoluminescence of GO with or without antioxidants.
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The scans of excitation-emission matrix and its light scattering were corrected for
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the background subtraction and instrument configurations. The measurements were
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conducted at room temperature (about 22℃) in air (240 μM O2 concentration and
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pH=7.4) Assay for Antibacterial Activity of GO. The E. coli strain ATCC-25922 was
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cultured overnight in LB agar (Luria-Bertani) medium at 37ºC to promote an
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exponential growth phase. Bacterial suspensions were centrifuged (5000 rpm, 5 min)
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and washed twice with sterile saline to remove residual media components. Finally,
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each suspension was diluted with saline solution to 106-107 CFU/mL for the
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following experiments.
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First, GO was added to the bacterial suspension to reach final concentrations of
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25, 50, 75 and 100 𝜇g/mL, respectively. These suspensions were incubated at 37℃
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at 250 rpm shaking speed for 2 h. To assess the effects of simulated sunlight on the
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behavior of GO, 25 μg/mL GO exposed to simulated sunlight for periods ranging
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between 0-30 min under the following conditions: with either NaN3 or histone
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(which can scavenge singlet oxygen), or with no further addition (Control). Then,
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each bacterial suspension was plated on LB agar media and incubated overnight at
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37℃ for CFU enumeration.
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The antibacterial activity in each GO concentration/duration of light exposure
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was measured using LIVE/DEAD fluorescent staining as follows. After 2 h exposure
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to GO either with or without exposure to simulated sunlight, the cells were stained
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by adding SYTO9 and propidium iodide (PI) to each suspension. These samples
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were incubated for 30 min in the dark before 5 μL was pipetted to a microscope slide
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and then covered with a coverslip. Confocal laser microscopy (FV1200, OLYMPUS,
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Japan) was then used to take luminescence images.
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Morphological changes to the bacteria after treatment were visualized using a
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scanning electron microscope (SEM). After GO treatments, the cells were collected
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and fixed in a glutaraldehyde solution, dehydrated by a sequential series of ethanol
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solutions (25, 50, 75, 85, 95 and 100%) and dried in a desiccator to remove ethanol.
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Each sample was mounted onto an aluminum stub and imaged by SEM (S-4700,
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Hitachi, Japan).
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Direct ESR Detection of ROS. All ESR measurements were carried out at
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ambient temperature using an ESR spectrometer (EMX, Bruker, USA). The spin
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adducts were detected at the following settings, unless otherwise stated: 20 mW
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microwave power, 1 G field modulation and 100 G scan range. Fifty microliter
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aliquots of the sample solutions were put in separate quartz capillary tubes with
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internal diameters of 0.9 mm. The spin trap 4-oxo-TEMP was used to verify the
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presence of any singlet oxygen generated by GO upon exposure to simulated
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sunlight. The superoxide and hydroxyl radicals were determined using BMPO. The
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presence of holes and electrons were detected using the spin labels CPH and
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TEMPO.
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Assay for GSH and AA Oxidation. As described in our previous publications,
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UV-vis spectroscopy was used to determine the oxidation of glutathione (GSH) and
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ascorbic acid (AA) by GO exposed to simulated sunlight, as follows.28 50 µM GSH
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were added to the suspensions of GO (25 µg/mL) and exposed to simulated sunlight
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for 30 min. Adding 100 µM DNTB to the mixtures yielded a yellow product,
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quantified spectrophotometrically by measuring absorbance at 412 nm. To examine
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the effects of irradiated GO on AA, 25 µg/mL GO were mixed with 100 µM AA.
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During these 30 minutes of exposure to simulated sunlight, samples were collected
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every five minutes and the amount of remaining AA was determined by measuring
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the absorbance of AA at the 265 nm. We used NaN3 and histone separately to
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scavenge the singlet oxygen during photoexcitation of GO to further distinguish the
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effects of singlet oxygen on the oxidation of either AA or GSH.
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Characterization of GO Exposed to Simulated Sunlight in Presence of
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Antioxidants. Fluorescence spectra of 0.1 mg/mL GO in the presence or absence of
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0.2 mg/mL AA were acquired using an Edinburgh Instruments FLS 980
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spectrophotometer with a 1 cm light path quartz cuvette. Absorption spectra were
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recorded on a UV-vis spectrophotometer (UV-3600, Shimadzu, Japan). Milli-Q
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water (18 MΩ cm) was used as reference. The XPS measurements were performed
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on an X-ray photoelectron spectroscopy (ESCALAB 250Xi, Thermo Scientific, USA)
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using a monochromatic Al Ka (1486.6 eV) source.
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RESULTS AND DISCUSSION
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Characterization of GO. GO used in the present study was purchased from
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Chengdu Organic Chemical Company, Chinese Academy of Science. The
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characterization of this nanomaterial was shown in Figure S1, including Atomic
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force microscope (AFM) images, UV-vis spectra and Raman spectra. From the AFM
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height images, we observed GO have lateral dimension of several micrometers and
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thickness around 1 nm. The UV−vis absorption spectra showed that there are two
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peaks centered at around 231 and 280 nm in the UV region. Raman analysis
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displayed large D and G bands at 1345 and 1592 cm-1, respectively. All these
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characteristics agree well with other reports [13-15] and clearly demonstrate that the
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as-received sample is a fully exfoliated GO nanosheet.
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Antimicrobial Activity of GO Exposed to Simulated Sunlight. First, we
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determined the toxicity of GO to bacteria without exposure to simulated sunlight. As
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shown in Figure S2, E. coli cells (106 to 107 CFU/mL) were incubated with various
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concentrations of GO for 2 h; colony count results indicated a considerable
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cytotoxicity, which increased in direct proportion to concentrations of GO. Scanning
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electron microscopy (SEM) images (Figure S3) clearly showed that GO treatment
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resulted in loss of bacterial membrane integrity. At this point we did not detect any
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trace of an ROS signal (Figure S4) using ESR, which is considered the most reliable
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and direct method to detect short-lived free radicals. However, found upon addition
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of GO, noticeable time-dependent GSH oxidation was observed (Figure S5), perhaps
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as a consequence of a direct charge transfer from GSH to GO. These findings
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indicate the antibacterial activity of GO may be caused by both direct membrane
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damage and ROS-independent oxidative stress; these observations are consistent
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with several earlier reports.1,4,14
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Next, we studied the effect of exposure to simulated sunlight on the antibacterial
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activities of GO. As shown in Figure 1a, 30 min simulated sunlight exposure alone
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had a negligible effect on E. coli cells compared to the set of control cells. Cell
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cultures treated with 25 μg/mL GO had a survival percentage of 68.8±9.2%;
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however when cultures containing that same amount of GO were exposed to
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simulated sunlight, the survival percentage was reduced to 24.9±5.9%,
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demonstrating that simulated sunlight significantly enhanced the antibacterial
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activity of GO (p < 0.01). We also investigated how duration of exposure to
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simulated sunlight could affect the antibiotic activity of GO. As shown in Figure 1b,
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as the length of exposure to simulated sunlight increased, the viability of E. coli in
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the presence of GO decreased in a stepwise fashion. To confirm this phenomenon we
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performed a fluorescence-based Live/Dead assay. As expected, no obvious cell
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apoptosis could be found in cultures exposed to just the simulated sunlight alone or
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to just the GO treatment alone. However, simultaneous treatment with GO and
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simulated sunlight markedly reduced viability, as indicated by a remarkable increase
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in the number of PI-permeable, red fluorescent cells (Figure 1c).
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ROS Generated by Light-irradiated GO. Previous research has established
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that, graphene-based nanomaterials (e.g., GO, rGO) exhibit a photothermal effect
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when irradiated by an NIR laser. This effect can kill both bacteria and tumor
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cells.18,22,25 However, exposure to simulated sunlight for 0 – 30 minutes does not
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significantly raise the temperature of GO-treated bacterial suspensions (data not
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shown); therefore photothermal effects can be excluded. Instead, we inferred that the
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light-enhanced antibacterial activities of GO might be a consequence of oxidative
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stress, resulting from light-induced generation of reactive oxygen species. To test
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this hypothesis, we used ESR spectroscopy to measure whether GO was able to
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produce ROS after exposure to simulated sunlight. We employed DMPO and BMPO
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as spin trap for the hydroxyl radical and superoxide anion, respectively, and
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4-oxo-TEMP to detect singlet oxygen. Based on previous research, we used an •OH
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generating Fenton reaction, enzymatic O2•− generating system, and the 1O2 generating
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compound Rose Bengal as references.26-28 Neither hydroxyl radical nor superoxide
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anion signals were observed (Figure S4), although a triplet spectrum, characteristic
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for the reaction between the 4-oxo-TEMP and singlet oxygen, was noted during the
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exposure of GO to simulated sunlight (Figure 2a). As a spin trap, 4-oxo-TEMP itself
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is ESR silent, but it can specifically capture 1O2 to yield a nitroxide radical,
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TEMPONE, which has an observable and stable ESR spectrum. In addition, only a
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negligible amount of hydrogen peroxide was generated during photoexcitation of GO
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by using the hydrogen peroxide assay kit (data not shown).
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These results demonstrate singlet oxygen could be generated by GO exposed to
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sunlight. To confirm this conclusion, we added sodium azide (NaN3), a scavenger
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capable of consuming generated 1O2, during the test. After adding 1 mM NaN3, only
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a negligible TEMPO signal could be detected (Figure 2a), indicating the triplet
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spectrum can be ascribed to the presence of singlet oxygen generated by GO. As
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superoxide anions cannot be generated under anaerobic conditions, we infer that this
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singlet oxygen must be generated by energy transfer from GO to the ground-state
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oxygen.
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GO-mediated Oxidative Stress and its Relationship with ROS. The overproduction of singlet oxygen will predictably impair the antioxidant defense
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systems of bacteria.29 To document this, we examined in vitro glutathione (GSH)
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oxidation by GO with or without exposure to simulated sunlight. GSH is an
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endogenous bacterial antioxidant, present in concentrations ranging between 0.1 and
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10 mM.14,30,31 The thiol groups in GSH, which can be quantified by the Ellman’s
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assay, can be oxidized to a disulfide bond, yielding the oxidized form, glutathione
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disulfide (GSSG). As shown in Figure 2b, 30 min exposure to either simulated
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sunlight or to GO alone caused extremely limited oxidation of glutathione, which
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agrees well with earlier findings.13,14,23 However, the extent of GSH oxidation was
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remarkably enhanced upon exposure to GO in combination with simulated sunlight;
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these effects increased as a function of exposure time, indicating that sunlight could
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accelerate GSH oxidation by GO. Next, we used ascorbic acid (AA) as another
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indicator of oxidative stress. AA is a water-soluble antioxidant which has an
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important biologic role regulating the intracellular redox state, through its interaction
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with GSH.32-34 The characteristic absorption maximum for AA is located at 265 nm.
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We consistently found that extremely significant AA oxidation occurred when GO
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and simulated sunlight were introduced; however, samples which did not contain GO
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or were not exposed to simulated sunlight only exhibited a minor depletion of AA
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(Figure 2c). These observations verify that GO exposed to sunlight could cause
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significant oxidation of biological antioxidants.
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The aforementioned results have suggested that only 1O2 could be determined,
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therefore we inferred the light-enhanced oxidation capacity of GO might arise
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primarily from the generated 1O2. In order to verify our hypothesis, we add NaN3 and
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histone (to scavenge the generated 1O2) into the GO dispersion before exposure to
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simulated sunlight. We had expected that the extent of GSH and AA oxidation by GO
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would be greatly inhibited, because the inducer, 1O2, would have been consumed.
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Surprisingly, the majority of GSH and AA still could be oxidized by GO that had been
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exposed to simulated sunlight even after the addition of scavengers. These findings
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suggest that generated 1O2 is not the dominant factor in the light-enhanced oxidation
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capacity of GO (Figure 2d). To confirm this hypothesis, we assessed the antibacterial
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activity of GO (after exposure to simulated sunlight) in the presence of 1O2
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scavengers. As expected, those conditions produced data comparable to those
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acquired without adding 1O2 scavengers (Figure S6). Given these results, we must
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conclude that oxidative stress caused by ROS is not the primary mechanism
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responsible for light-enhanced oxidation capacity of GO.
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Exploring the ROS-Independent oxidation capacity of GO to simulated
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sunlight. In order to identify a more likely mechanism, we investigated the
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photochemical fate of GO under biologically-relevant conditions. Previous research
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has shown GO has a large energy gap between π-state from its sp2 carbon sites and
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σ-state of its sp3-bonded carbons.35 Therefore, it is possible that electrons and holes
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are generated on the surface of GO when it is exposed to simulated sunlight. To
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explore this possibility, we investigated the light-induced formation of electron-hole
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pairs in situ, using ESR spectroscopy.28,36 Specifically, we used CPH to examine the
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oxidizing activity of photogenerated holes and used TEMPO to verify photogenerated
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electron. As shown in Figure 3a, CPH is ESR silent, therefore no reaction between
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CPH and GO was anticipated. Surprisingly, when the GO was exposed to simulated
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sunlight, an ESR spectrum of three lines with intensity ratios of 1:1:1 was observed.
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As the duration of exposure was extended, more CPH was oxidized to CP-nitroxide.
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In contrast to the standard behavior of CPH, TEMPO normally exhibits a stable
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triplet ESR spectrum, with relative intensities of 1:1:1, although it can be reduced to
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TEMPOH, which is ESR silent. We observed that the TEMPO signal intensity was
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unchanged in the presence of either GO alone or light alone. When both GO and
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simulated sunlight were present, we observed an obvious reduction in the ESR signal
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intensity, and that signal intensity continued to decrease as light exposure time was
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increased. These results confirm that GO exposed to simulated sunlight induces the
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generation of electron-hole pairs. However, due to the confinement of redox potential,
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those light-induced electron-hole pairs cannot react with surrounding H2O and O2 to
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respectively form •OH and O2•−.
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Our results demonstrate that 1O2, the only type of ROS determined in these
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experiments, could not be the primary cause of GSH oxidation. The oxidation activity
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of the light-induced holes led us to speculate that these holes might be the main factor
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driving the oxidation of antioxidant biomolecules. To verify this, we used
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fluorescence excitation-emission spectra characterization. As shown in Figure 3b, GO
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exhibited an inhomogeneous broadened fluorescence in the range of 410-750 nm,
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with a maximum at about 550 nm and excitation at 470 nm. Previous research has
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demonstrated that photoluminescence of GO is a consequence of radiative
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electron-hole pair recombination.35,37,38 Nonetheless, when we introduced AA, a
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remarkable decrease in photoluminescence intensity, even though the
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photoluminescence wavelength characteristics remained unchanged. This
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phenomenon demonstrates how antioxidant biomolecules could consume the
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light-induced holes, inhibit electron-hole pair recombination, and thereby suppress the
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photoluminescence of GO.
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Chemical Transformation of GO upon Exposure to Simulated Sunlight. The
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generation of light-induced holes could cause the oxidation of vital cellular
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components which mediate ROS-independent oxidative stress. This leads us to
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another question: if holes are responsible for oxidization of antioxidant biomolecules,
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what is the role of light-induced electrons? To investigate the fate of light-induced
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electrons, we added antioxidant biomolecules (GSH, AA) to GO dispersions, thereby
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consuming holes and leaving residual excited electrons. We characterized the
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changes of GO under a variety of conditions (including the presence or absence of
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antioxidants and with or without exposure to simulated sunlight) using UV-vis
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spectrum and X-ray photoelectron spectroscopy. Figure 4a illustrates a series of GO
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dispersions: no obvious color change was observed under conditions when only
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simulated sunlight or only antioxidant molecules were present, which confirms the
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slow reduction process of GO observed in previous reports.23,39,40 However, when
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both antioxidant molecules and simulated sunlight were present, the GO dispersions
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underwent very obvious color changes (from yellow to black), which indicate the
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tremendous reduction of GO under these conditions. These results demonstrate how
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the presence of antioxidants could induce sunlight-exposed GO to create significant
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amounts of excited residual electrons, which, in turn, would greatly accelerate the
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reduction of GO.
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Our next assays monitored the reduction of GO using UV-vis spectroscopy. As
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with previous assays, the spectroscopic profile of GO did not change significantly
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when AA was added by itself, or if the GO was only exposed to simulated sunlight
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(Figure 4b). However, when both these factors were present, the maximum
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absorption peak of the GO dispersion gradually red-shifted from 231 nm to 263 nm
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and the optical absorption in the NIR region also markedly increased. We used XPS
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to characterize the reduction of GO under various conditions. As shown in Figure 4c,
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we detected three different peaks, centered at 284.5, 286.4 and 289.2 eV,
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corresponding to C=C/C–C in the aromatic rings, C–O of the epoxy, or the alkoxy
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groups and the COOH groups, respectively. Upon addition of AA, the intensities of
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all C 1s peaks of the carbons binding to the oxygen of the light-exposed GO
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decreased dramatically, indicating that most of the oxygen-containing functional
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groups had been removed. Because those light-induced electrons could be trapped by
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the oxygen functional groups, it is possible for oxygen-centered radicals or
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carbon-centered radicals to be introduced during the process of removing oxygen on
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the GO surface. As indicated in Figure 5, GO nanosheets exhibit a unique,
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symmetrical ESR signal at g = 2.002, close to the free electron g-value of 2.0023,
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representing carbon-centered radicals.41 We found that neither simulated sunlight
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alone nor antioxidant alone had any noticeable effects on the ESR signal intensity
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from the paramagnetic defects of GO. However, adding antioxidants in the presence
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of simulated sunlight resulted in a marked enhancement of the ESR signal. Notably,
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the ESR spectrum for the AA radical indicated that the oxidation of AA and the
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reduction of GO were simultaneous.
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Based on all the data, we conclude that exposure to simulated sunlight accelerate
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the electron transfer from antioxidant biomolecules to GO, and as a result, the
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antioxidant system is destroyed and GO itself is reduced accompanied by the
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introduction of carbon-centered free radicals. The oxidative stress, reduced GO and
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introduced carbon-centered free radicals co-contribute to the overall antibacterial
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efficacy.
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Implications. The antibacterial properties of graphene-based nanomaterials,
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such as GO, may offer specific advantages that surpass those of inorganic and
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polymeric nanomaterials.42-45 Yet, these cannot be exploited until we understand their
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behavior under environmental and other biologically-relevant conditions. Here we
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have demonstrated how GO exhibit impressive antibacterial upon exposure to
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simulated sunlight, resulting in oxidative stress that is mainly independent of ROS.
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Exposure to simulated sunlight accelerates the electron transfer from the innate
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antioxidant systems of E. coli to GO, thereby destroying the biomolecules that
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ordinarily provide protection from oxidative stressors. Meanwhile, the light-induced
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electrons promoted the reduction of GO, inducing additional carbon-centered free
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radicals, which may augment the antibacterial activities of GO. Our work provides
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the first full description of the antibacterial mechanisms of GO and offers guidance
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for developing highly-efficient graphene-based antibacterial materials.
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ASSOCIATED CONTENT
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Supporting Information Available: Additional experimental details and figures
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(Figures S1−S6), as described in the text. This material is available free of charge at
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http://pubs.acs.org.
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AUTHOR INFORMATION
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Corresponding Authors
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*E-mail:
[email protected] (C. Y. Chen);
[email protected] (J. J. Yin);
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[email protected] (C. C. Ge); Tel: +86-10-82545560.
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Notes
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The authors declare no competing financial interests.
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ACKNOWLEDGMENTS
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This work is partially supported by the National Basic Research Program of China
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(973 Program Grant No. 2014CB931900 and 2016YFA0201600), National Natural
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Science Foundation of China (11575123, 11621505 and 21320102003), Jiangsu
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Provincial Key Laboratory of Radiation Medicine and Protection, a project funded
377
by the Priority Academic Program Development of Jiangsu Higher Education
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Institutions (PAPD), and a regulatory science grant under the FDA Nanotechnology
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CORES Program. C. Chen appreciates the support from the NSFC Distinguished
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Young Scholars (11425520). C. Ge appreciates the support from the Open Project
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Program of Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety,
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Chinese Academy of Sciences (NSKF201611). Y. Chong appreciates the support
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from the China Scholarship Council (no. 1410100007). The authors thank Dr. Lili
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Fox Vélez for scientific writing and editing support. This article is not an official U.S.
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FDA guidance or policy statement. No official support or endorsement by the U.S.
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FDA is intended or should be inferred.
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b 120
120 **
100 *
80 60 40
**
20 0 Control
c
Control
Light
GO GO+Light
Light
E.coli Survival (%)
E.coli Survival (%)
a
GO+light
100
** *
80 60 40 20 0
Control
GO
0 10 20 30 Irradiation time (min)
GO+Light
387 388
Figure 1. Evidence of GO nanosheets killing E. coli with or without exposure to
389
simulated sunlight. (a) Antibacterial activity by GO under various conditions as
390
assessed by numbers of colony-forming units. Cultured E.coli cells were treated by
391
isotonic saline as control, simulated sunlight, or GO with or without exposure to
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simulated sunlight. (b) Antibacterial activities of GO are influenced by duration of
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exposure to simulated sunlight. The data shown are mean values and standard
394
deviations from a representative of three independent experiments. P values were
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calculated by the student's test: *p < 0.05, **p < 0.01. (c) Representative fluorescence
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images of live (green) and dead (red) cells after different treatments. Scale bars = 20
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μm.
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13000000
a
12000000
b
Control
11000000 10000000 9000000
GO
8000000 7000000 6000000 5000000 4000000
GO+NaN3
3000000
50
Loss of GSH (%)
14000000
2000000 1000000
c Loss of AA (%)
80
3320
Light3340 3360 GO GO with light
3380
3400
3420
40 20 0 5
10
15
20
Time (min) 399
30 20 10
5
10
15
25
30
20
25
30
Time (min)
d
AA GSH *
3440
100
X [G]
60
0
40
0
Loss of Antioxidant (%)
10 G 1003300
Light GO GO with light
0
GO+N2
0 -1000000
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80 60 40
*
20
0 GO+Light NaN3 Histone
+ -
+ + -
+ +
400
Figure 2. The generation of ROS and enhanced oxidizing activity by GO nanosheets
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exposed to simulated sunlight. (a) ESR spectra were obtained from samples
402
containing spin label (4-oxo-TEMP) and GO under different conditions, including
403
GO alone, addition of NaN3, and anaerobic conditions. ESR spectra were recorded
404
after 5 min of exposure to simulated sunlight. (b) In vitro GSH oxidation under three
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conditions: simulated sunlight alone, GO alone, and GO in the presence of simulated
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sunlight. (c) In vitro AA oxidation under the three conditions as above. (d) Loss of
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antioxidant agents (GSH or AA) by GO during exposure to simulated sunlight in
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either the absence or presence of NaN3 or histone (singlet oxygen scavengers). These
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data present the means and standard deviation from three experiments. P values
410
comparing differences between GO in the presence of simulated sunlight and other
411
conditions were calculated by the Student's T test: *p < 0.05.
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10000000
8000000
6000000
TEMPO
CPH
a Control
Control
1 min
1 min
5 min
5 min
10 min
10 min
4000000
2000000
0
800
800
700.0
700
550.0 400.0 600
250.0 100.0 -50.00
500
400 350
412
400
450
Excitation wavelength(nm)
500
Emission wavelength (nm)
Emission wavelength(nm)
b
700.0
700
550.0 400.0
600
250.0 100.0 -50.00
500
400 350
400
450
500
Excitation wavelength (nm)
413
Figure 3. The generation of electron-hole pairs by GO exposed to simulated sunlight.
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(a) ESR spectra were obtained from samples containing different spin probes (CPH
415
and TEMPO) and GO exposed to simulated sunlight for different periods. The
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control represents the sample contained the spin probe alone, or exposed to
417
simulated sunlight, or the sample containing spin probe and GO before exposure to
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simulated sunlight. (b) Excitation−emission matrices (EEM) characterize the
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photoluminescence properties of GO by itself (left column) and GO upon addition of
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AA (right column).
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422 423
Figure 4. The chemical reduction of GO. (a) Photographs of GO dispersions under
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various conditions: sunlight alone, antioxidant (AA or GSH) alone, or antioxidant
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combined with sunlight. (b) UV-vis spectra of GO dispersions under different
426
conditions as above. The inset is an irradiation time-dependent UV-vis spectra of GO.
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Items 4A through 4G correspond to a range of exposure times (0-60 min). (c) XPS
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spectra of C 1s of GO in the presence of AA before and after exposure to simulated
429
sunlight.
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b
a 6
1x10 0
GO
6
g value=2.002
6
6
6
6
GO+GSH 30 min g value=2.002
6
1x10 0
GO+GSH+Light 30 min g value=2.002
6
-1x10
GO+AA 30 min g value=2.002
6
GO+AA+Light 30 min
1x10 0 6
g value=2.002
-1x10
-1x10
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g value=2.002
-1x10
-1x10
1x10 0
GO+Light
6
6
-1x10
1x10 0
1x10 0
3350 3360 3370 3380 3390 3400 Magnetic field (G)
3350 3360 3370 3380 3390 3400 Magnetic field (G)
432
Figure 5. The characterization of the carbon-centered free radicals formed by GO
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nanosheets under various conditions. (a) ESR spectra were obtained from samples
434
containing GO in the absence or presence of an antioxidant (GSH or AA). (b) ESR
435
spectra were obtained from samples containing GO in the absence or presence of an
436
antioxidant (GSH or AA) then exposed to simulated sunlight for 30 min.
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REFERENCES
438
(1) Hu, W. B.; Peng, C.; Luo, W. J.; Lv, M.; Li, X. M.; Li, D.; Huang, Q.; Fan, C. H.
439
Graphene-based antibacterial paper. ACS Nano 2010, 4, 4317-4323.
440
(2) Hegab, H. M.; ElMekawy, A.; Zou, L.; Mulcahy, D.; Saint, C. P.; Ginic-Markovic, M.
441
The controversial antibacterial activity of graphene-based materials. Carbon 2016, 105,
442
362-376.
443
(3) Ji, H. W.; Sun, H. J.; Qu, X. G. Antibacterial applications of graphene-based
444
nanomaterials: Recent achievements and challenges. Adv. Drug Deliv. Rev. 2016, 105,
445
176-189.
446
(4) Liu, S. B.; Hu, M.; Zeng, T. H.; Wu, R.; Jiang, R. R.; Wei, J.; Wang, L.; Kong, J.; Chen,
447
Y. Lateral dimension-dependent antibacterial activity of graphene oxide sheets. Langmuir
448
2012, 28, 12364-12372.
449
(5) Tu, Y. S.; Lv, M.; Xiu, P.; Huynh, T.; Zhang, M.; Castelli, M.; Liu, Z. R.; Huang, Q.;
450
Fan, C. H.; Fang, H. P.; Zhou, R. H. Destructive extraction of phospholipids from
451
Escherichia coli membranes by graphene nanosheets. Nat. Nanotechnol. 2013, 8, 594-601.
452
(6) Chen, J. N; Peng, H.; Wang, X. P.; Shao, F.; Yuan, Z. D.; Han, H. Y. Graphene oxide
453
exhibits broad-spectrum antimicrobial activity against bacterial phytopathogens and fungal
454
conidia by intertwining and membrane perturbation. Nanoscale 2014, 6, 1879-1889.
455
(7) Mao, J.; Guo, R. H.; Yan, L. T. Simulation and analysis of cellular internalization
456
pathways and membrane perturbation for graphene nanosheets. Biomaterials 2014, 35,
457
6069-6077.
458
(8) Zou, X. F.; Zhang, L.; Wang, Z. J.; Luo, Y. Mechanisms of the antimicrobial activities
ACS Paragon Plus Environment
Page 26 of 31
Page 27 of 31
Environmental Science & Technology
459
of graphene materials. J. Am. Chem. Soc. 2016, 138, 2064-2077.
460
(9) Gurunathan, S.; Han, J. W.; Dayem, A. A.; Eppakayala, V.; Kim, J. H. Oxidative
461
stress-mediated antibacterial activity of graphene oxide and reduced graphene oxide in
462
Pseudomonas aeruginosa. Int. J. Nanomed. 2012, 7, 5901-5914.
463
(10) Li, J. H.; Wang, G.; Zhu, H. Q.; Zhang, M.; Zheng, X. H.; Di, Z. F.; Liu, X. Y.; Wang, X.
464
Antibacterial activity of large-area monolayer graphene film manipulated by charge transfer.
465
Sci. Rep. 2014, 4, 4359.
466
(11) Pham, V. T. H.; Truong, V. K.; Quinn, M. D. J.; Notley, S. M.; Guo, Y. C.; Baulin, V. A.;
467
Al Kobaisi, M.; Crawford, R. J.; Ivanova, E. P. Graphene induces formation of pores that kill
468
spherical and rod-shaped bacteria. ACS Nano 2015, 9, 8458-8467.
469
(12) Zhang, N.; Li, X. Y.; Ye, H. C.; Chen, S. M.; Ju, H. X.; Liu, D. B.; Lin, Y.; Ye, W.;
470
Wang, C. M.; Xu, Q.; Zhu, J. F.; Song, L.; Jiang, J.; Xiong, Y. J. Oxide defect engineering
471
enables to couple solar energy into oxygen activation. J. Am. Chem. Soc. 2016, 138,
472
8928-8935.
473
(13) Perreault, F.; de Faria, A. F.; Nejati, S.; Elimelech, M. Antimicrobial properties of
474
graphene oxide nanosheets: why size matters. ACS Nano 2015, 9, 7226-7236.
475
(14) Liu, S. B.; Zeng, T. H.; Hofmann, M.; Burcombe, E.; Wei, J.; Jiang, R. R.; Kong, J.;
476
Chen, Y. Antibacterial activity of graphite, graphite oxide, graphene oxide, and reduced
477
graphene oxide: membrane and oxidative stress. ACS Nano 2011, 5, 6971-6980.
478
(15) Wang, J. L.; Wei, Y. J.; Shi, X. H.; Gao, H. J. Cellular entry of graphene nanosheets: the
479
role of thickness, oxidation and surface adsorption. RSC Adv. 2013, 3, 15776-15782.
480
(16) Akhavan, O.; Ghaderi, E. Toxicity of graphene and graphene oxide nanowalls against
ACS Paragon Plus Environment
Environmental Science & Technology
481
bacteria. ACS Nano 2010, 4, 5731-5736.
482
(17) Hui, L. W.; Piao, J. G.; Auletta, J.; Hu, K.; Zhu, Y. W.; Meyer, T.; Liu, H. T.; Yang, L.
483
H. Availability of the basal planes of graphene oxide determines whether it is
484
antibacterial. ACS Appl. Mater. Interfaces 2014, 6, 13183-13190.
485
(18) Wu, M. C.; Deokar, A. R.; Liao, J. H.; Shih, P. Y.; Ling, Y. C. Graphene-based
486
photothermal agent for rapid and effective killing of bacteria. ACS Nano 2013, 7,
487
1281-1290.
488
(19) Hu, X. G.; Zhou, M.; Zhou, Q. X. Ambient water and visible-light irradiation drive
489
changes in graphene morphology, structure, surface chemistry, aggregation, and
490
toxicity. Environ. Sci. Technol. 2015, 49, 3410-3418.
491
(20) Matsumoto, Y.; Koinuma, M.; Ida, S.; Hayami, S.; Taniguchi, T.; Hatakeyama, K.;
492
Tateishi, H.; Watanabe, Y.; Amano, S. Photoreaction of graphene oxide nanosheets in water.
493
J. Phys. Chem. C 2011, 115, 19280-19286.
494
(21) Gengler, R. Y. N.; Badali, D. S.; Zhang, D. F.; Dimos, K.; Spyrou, K.; Gournis, D.;
495
Miller, R. J. D. Revealing the ultrafast process behind the photoreduction of graphene
496
oxide. Nat. Commun. 2013, 4, 2560.
497
(22) Yang, K.; Zhang, S.; Zhang, G. X.; Sun, X. M.; Lee, S. T.; Liu, Z. Graphene in mice:
498
ultrahigh in vivo tumor uptake and efficient photothermal therapy. Nano Lett. 2010, 10,
499
3318-3323.
500
(23) Hou, W. C.; Chowdhury, I.; Goodwin, D. G.; Henderson, W. M.; Fairbrother, D. H.;
501
Bouchard, D.; Zepp, R. G. Photochemical transformation of graphene oxide in
502
sunlight. Environ. Sci. Technol. 2015, 49, 3435-3443.
ACS Paragon Plus Environment
Page 28 of 31
Page 29 of 31
Environmental Science & Technology
503
(24) Chong, Y.; Ge, C. C.; Yang, Z. X.; Garate, J. A.; Gu, Z. L.; Weber, J. K.; Liu, J. J.; Zhou,
504
R. H. Reduced cytotoxicity of graphene nanosheets mediated by blood-protein coating. ACS
505
Nano 2015, 9, 5713-5724.
506
(25) Wang, Y. W.; Fu, Y. Y.; Peng, Q. L.; Guo, S. S.; Liu, G.; Li, J.; Yang, H. H.; Chen, G. N.
507
Dye-enhanced graphene oxide for photothermal therapy and photoacoustic imaging. J. Mater.
508
Chem. B 2013, 1, 5762-5767.
509
(26) Yin, J. J.; Lao, F.; Fu, P. P.; Wamer, W. G.; Zhao, Y. L.; Wang, P. C.; Qiu, Y.; Sun, B. Y.;
510
Xing, G. M.; Dong, J. Q. Liang, X. J. Chen, C. Y. The scavenging of reactive oxygen species
511
and the potential for cell protection by functionalized fullerene materials. Biomaterials 2009,
512
30, 611-621.
513
(27) Liu, Y.; Wu, H. H.; Li, M.; Yin, J. J.; Nie, Z. H. pH dependent catalytic activities of
514
platinum nanoparticles with respect to the decomposition of hydrogen peroxide and
515
scavenging of superoxide and singlet oxygen. Nanoscale 2014, 6, 11904-11910.
516
(28) Chong, Y.; Ge, C.; Fang, G.; Tian, X.; Ma, X.; Wen, T.; Wamer, W. G.; Chen, C.; Chai,
517
Z.; Yin, J. J. Crossover between anti-and pro-oxidant activities of graphene quantum dots in
518
the absence or presence of light. ACS Nano 2016, 10, 8690-8699.
519
(29) Kasperczyk, S.; Dobrakowski, M.; Kasperczyk, J.; Ostałowska, A.; Zalejska-Fiolka, J.;
520
Birkner, E. Beta-carotene reduces oxidative stress, improves glutathione metabolism and
521
modifies antioxidant defense systems in lead-exposed workers. Toxicol. Appl. Pharmacol.
522
2014, 280, 36-41.
523
(30) Fahey, R. C.; Brown, W. C.; Adams, W. B.; Worsham, M. B. Occurrence of glutathione
524
in bacteria. J. Bacteriol. 1978, 133, 1126-1129.
ACS Paragon Plus Environment
Environmental Science & Technology
525
(31) Huang, Y. Z.; Chen, X. L.; Shi, S. G.; Chen, M.; Tang, S. H.; Mo, S. G.; Wei, J. P.;
526
Zheng, N. F. Effect of glutathione on in vivo biodistribution and clearance of
527
surface-modified small Pd nanosheets. Sci. China Chem. 2015, 58, 1753-1758.
528
(32) Winkler, B. S.; Orselli, S. M.; Rex, T. S. The redox couple between glutathione and
529
ascorbic acid: a chemical and physiological perspective. Free Radic. Biol. Med. 1994, 17,
530
333-349.
531
(33) Du, J.; Cullen, J. J.; Buettner, G. R. Ascorbic acid: chemistry, biology and the treatment
532
of cancer. Biochim. Biophys. Acta. 2012, 1826, 443-457.
533
(34) He, M.; Lu, L. Y.; Zhang, J. C.; L, D. Z. Facile preparation of l-ascorbic acid-stabilized
534
copper-chitosan nanocomposites with high stability and antimicrobial properties. Sci. Bull.
535
2015, 60, 227-234.
536
(35) Loh, K. P.; Bao, Q. L.; Eda, G.; Chhowalla, M. Graphene oxide as a chemically tunable
537
platform for optical applications. Nat. Chem. 2010, 2, 1015-1024.
538
(36) He, W. W.; Kim, H. K.; Wamer, W. G.; Melka, D.; Callahan, J. H.; Yin, J. J.
539
Photogenerated charge carriers and reactive oxygen species in ZnO/Au hybrid
540
nanostructures with enhanced photocatalytic and antibacterial activity. J. Am. Chem. Soc.
541
2014, 136, 750-757.
542
(37) Chien, C. T.; Li, S. S.; Lai, W. J.; Yeh, Y. C.; Chen, H. A.; Chen, I. S.; Chen, L. C.;
543
Chen, K. H.; Nemoto, T.; Isoda, S.; Chen, M. W.; Fujita, T.; Eda, G.; Yamaguchi, H.;
544
Chhowalla, M.; Chen, C. W. Tunable photoluminescence from graphene oxide. Angew.
545
Chem., Int. Ed. 2012, 51, 6662-6666.
546
(38) Eda, G.; Lin, Y. Y.; Mattevi, C.; Yamaguchi, H.; Chen, H. A.; Chen, I. S.; Chen, C. W.;
ACS Paragon Plus Environment
Page 30 of 31
Page 31 of 31
Environmental Science & Technology
547
Chhowalla, M. Blue photoluminescence from chemically derived graphene oxide. Adv.
548
Mater. 2010, 22, 505-509.
549
(39) Zhang, J. L.; Yang, H. J.; Shen, G. X.; Cheng, P.; Zhang, J. Y.; Guo, S. W. Reduction of
550
graphene oxide via L-ascorbic acid. Chem. Commun. 2010, 46, 1112-1114.
551
(40) Pham, T. A.; Kim, J. S.; Kim, J. S.; Jeong, Y. T. One-step reduction of graphene oxide
552
with L-glutathione. Colloids Surf. A Physicochem. Eng. Asp. 2011, 384, 543-548.
553
(41) Marciano, O.; Gonen, S.; Levy, N.; Teblum, E.; Yemini, R.; Nessim, G. D.; Ruthstein,
554
S.; Elbaz, L. Modulation of oxygen content in graphene surfaces using
555
temperature-programmed reductive annealing: electron paramagnetic resonance and
556
electrochemical study. Langmuir 2016, 32, 11672-11680.
557
(42) Zhao, R.; Wang, H.; Ji, T.; Anderson, G.; Nie, G.; Zhao, Y. Biodegradable cationic
558
ε-poly-L-lysine-conjugated polymeric nanoparticles as a new effective antibacterial
559
agent. Sci. Bull. 2015, 60, 216-226.
560
(43) Setyawati, M. I.; Kutty, R. V.; Tay, C. Y.; Yuan, X.; Xie, J.; Leong, D. T. Novel
561
theranostic DNA nanoscaffolds for the simultaneous detection and killing of Escherichia
562
coli and Staphylococcus aureus. ACS Appl. Mater. Interfaces. 2014, 6, 21822-21831.
563
(44) Lv, X.; Wang, P.; Bai, R.; Cong, Y.; Suo, S.; Ren, X.; Chen, C. Inhibitory effect of silver
564
nanomaterials on transmissible virus-induced host cell infections. Biomaterials 2014, 35,
565
4195-4203.
566
(45) Qiao, Y.; Zhai, Z.; Chen, L.; Liu, H. Cytocompatible 3D chitosan/hydroxyapatite
567
composites endowed with antibacterial properties: toward a self-sterilized bone tissue
568
engineering scaffold. Sci. Bull. 2015, 60, 1193-1202.
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