Light-enhanced Antibacterial Activity of Graphene Oxide Mainly via

Aug 3, 2017 - We conclude that GO-mediated oxidative stress mainly is ROS-independent; simulated sunlight accelerates the transfer of electrons from a...
0 downloads 10 Views 866KB Size
Subscriber access provided by University of Florida | Smathers Libraries

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

Environmental Science & Technology

1

Light-enhanced Antibacterial Activity of Graphene Oxide

2

Mainly via Accelerated Electron Transfer

3

Yu Chong,†,⊥ Cuicui Ge,*,†,⊥ Ge Fang,† Renfei Wu,† He Zhang,† Zhifang Chai,†

4

Chunying Chen*,‡ and Jun-Jie Yin*,⊥

5



6

Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University,

7

Suzhou 215123, China

8



9

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

10

Academy of Sciences, Beijing 100190, China

11



12

Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug

13

Administration, College Park, Maryland 20740, United States

Division of Bioanalytical Chemistry and Division of Analytical Chemistry, Office of

ACS Paragon Plus Environment

Environmental Science & Technology

14

ABSTRACT:

15

Before graphene derivatives can be exploited as next generation antimicrobials, we

16

must understand their behavior under environmental conditions. Here we demonstrate

17

how exposure to simulated sunlight significantly enhances the antibacterial activity of

18

graphene oxide (GO) and reveal the underlying mechanism. Our measurements of

19

reactive oxygen species (ROS) showed only singlet oxygen (1O2) is generated by GO

20

exposed to simulated sunlight, which contributes only slightly to the oxidation of

21

antioxidant biomolecules. Unexpectedly, we find the main cause of oxidation is

22

light-induced electron-hole pairs generated on the surface of GO. These light-induced

23

electrons promote the reduction of GO, introducing additional carbon-centered free

24

radicals which may also enhance the antibacterial activities of GO. We conclude that

25

GO-mediated oxidative stress mainly is ROS-independent; simulated sunlight

26

accelerates the transfer of electrons from antioxidant biomolecules to GO, thereby

27

destroying bacterial antioxidant systems and causing the reduction of GO. Our

28

insights will help support the development of graphene for antibacterial applications.

29

ACS Paragon Plus Environment

Page 2 of 31

Page 3 of 31

30

Environmental Science & Technology

Table of Contents

31

ACS Paragon Plus Environment

Environmental Science & Technology

32 33

INTRODUCTION The threat of bacterial infections that are untreatable due to antibiotic resistance

34

is a deepening crisis worldwide. Therefore, it is highly desirable to develop novel

35

antibacterial agents, particularly those which have mechanisms of action difficult for

36

bacteria to overcome. The extraordinary electronic, mechanic, and optical

37

characteristics of graphene structures and graphene derivatives suggest these could

38

become the next generation of antimicrobial materials.1-3 The antibacterial action of

39

graphene oxide (GO) arises from both physical and chemical activities.4-10 Physical

40

damage is caused by the sharp edges of GO that shred bacterial membranes, damage

41

RNA, and cause the destructive extraction phospholipid molecules.5-7 The primary

42

type of chemical damage is believed to be oxidative stress, initiated by either

43

reactive oxygen species (ROS) or by charge transfer.9,10

44

To date, most reports have focused on the close relationship between

45

antimicrobial activities and the physicochemical properties of GO.1,11,12 For instance,

46

the antibacterial activity of GO is influenced by the size of the graphene sheets.4,13

47

Data also indicate that the antimicrobial activity of GO can be enhanced by

48

increasing the number of defects in GO nanosheets. Other characteristics which can

49

alter GO antibacterial properties include layer number, morphology, dispersibility,

50

surface charge, and oxygen content.14-17

51

However, environmental conditions might also transform the physicochemical

52

features of GO in ways that would alter their antibiotic effects.18,19 Several studies

53

have shown that GO can be photochemically reduced by exposure to high-energy

ACS Paragon Plus Environment

Page 4 of 31

Page 5 of 31

Environmental Science & Technology

54

ultraviolet or laser light.20,21 Yang et al.22 reported such reduced GO could be used

55

for in vivo photothermal therapy on tumors, due to its strong optical absorbance in

56

the near-infrared (NIR) region. A study by Hou et al. in 2015 demonstrated that GO

57

could behave as a semiconductor photocatalyst, generating electron−hole pairs able

58

to reduce the oxygen content of GO structures.23 These findings leave open the

59

question of whether exposure to sunlight could affect the antibacterial activity of GO.

60

Further, it is not known whether data from previous explorations of GO exposed to

61

specific durations of high intensity laser or other artificial light sources can provide

62

adequate predictions about how photoreactions will occur under conditions of

63

natural or simulated sunlight.

64

Our current project examines these factors for the first time, systematically

65

evaluating changes in the antibacterial activity of GO upon exposure to simulated

66

sunlight. Then, we studied the oxidative stress pathway, including the generation of

67

ROS, using electron spin resonance (ESR) techniques, to identify the mechanism

68

responsible for that enhanced antibacterial activity. We investigated the possibility of

69

ROS-independent oxidative stress and determined the production of light-induced

70

electron-hole pairs. By using UV-vis spectrum and X-ray photoelectron

71

spectroscopy (XPS) we were able to characterize the chemical transformation of GO

72

in the presence of both simulated sunlight and reducing agents. Insights from these

73

multiple approaches, enable us to provide a clear explanation for the observed

74

enhancement of antibacterial activity of GO after exposure to simulated sunlight.

75

MATERIALS AND METHODS

ACS Paragon Plus Environment

Environmental Science & Technology

76

Materials. Graphene oxide (GO) was purchased from Chengdu Organic

77

Chemical Company, Chinese Academy of Science. The E. coli strain ATCC-25922

78

was obtained from the American Type Culture Collection (ATCC, Rockville, MD,

79

USA). 5,5'-Dithiobis (2-nitrobenzoic acid) (Ellman’s reagent, DTNB), 5,5-Dimethyl-

80

1-pyrroline-N-oxide (DMPO), 5-tert-Butoxycarbonyl-5-methyl-1-pyrroline-N-oxide

81

(BMPO) were obtained from Dojindo Laboratories (Kumamoto, Japan).

82

1-Hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) and 2,2,6,6-

83

tetramethylpiperidine-1-oxyl (TEMPO) were purchased from Alexis Biochemicals

84

(Enzo Life Sciences, Farmingdale, NY). All solutions were prepared using Milli-Q

85

water (18 MΩ cm). Other chemicals were obtained from Sigma Aldrich (St. Louis,

86

MO) and used as received. A 450 W Xenon lamp, filtered by an air mass (AM) 1.5 G

87

solar simulation bandpass filter was used to provide simulated sunlight irradiation.

88

The light path length was about 40 cm and the spectral irradiance of this source,

89

monitored with a Newport Optical Meter, was determined to be 380 mW/cm2.

90

Characterization of GO. The as-received sample was characterized by various

91

techniques. Atomic force microscope (AFM) images were acquired from an AFM

92

(Dimension Icon, Bruker) operating in the ScanAsyst mode. The UV-vis spectra

93

were recorded on a Shimadzu UV-3600 UV-vis spectrophotometer. Raman spectra

94

were obtained on a JY HR800 spectrometer with 532 nm wavelength incident laser

95

light. We used a Horiba Fluorolog 3-221 spectrofluorometer with a 1 cm light path

96

quartz cuvette to characterize photoluminescence of GO with or without antioxidants.

97

The scans of excitation-emission matrix and its light scattering were corrected for

ACS Paragon Plus Environment

Page 6 of 31

Page 7 of 31

Environmental Science & Technology

98

the background subtraction and instrument configurations. The measurements were

99

conducted at room temperature (about 22℃) in air (240 μM O2 concentration and

100 101

pH=7.4) Assay for Antibacterial Activity of GO. The E. coli strain ATCC-25922 was

102

cultured overnight in LB agar (Luria-Bertani) medium at 37ºC to promote an

103

exponential growth phase. Bacterial suspensions were centrifuged (5000 rpm, 5 min)

104

and washed twice with sterile saline to remove residual media components. Finally,

105

each suspension was diluted with saline solution to 106-107 CFU/mL for the

106

following experiments.

107

First, GO was added to the bacterial suspension to reach final concentrations of

108

25, 50, 75 and 100 𝜇g/mL, respectively. These suspensions were incubated at 37℃

109

at 250 rpm shaking speed for 2 h. To assess the effects of simulated sunlight on the

110

behavior of GO, 25 μg/mL GO exposed to simulated sunlight for periods ranging

111

between 0-30 min under the following conditions: with either NaN3 or histone

112

(which can scavenge singlet oxygen), or with no further addition (Control). Then,

113

each bacterial suspension was plated on LB agar media and incubated overnight at

114

37℃ for CFU enumeration.

115

The antibacterial activity in each GO concentration/duration of light exposure

116

was measured using LIVE/DEAD fluorescent staining as follows. After 2 h exposure

117

to GO either with or without exposure to simulated sunlight, the cells were stained

118

by adding SYTO9 and propidium iodide (PI) to each suspension. These samples

119

were incubated for 30 min in the dark before 5 μL was pipetted to a microscope slide

ACS Paragon Plus Environment

Environmental Science & Technology

120

and then covered with a coverslip. Confocal laser microscopy (FV1200, OLYMPUS,

121

Japan) was then used to take luminescence images.

122

Morphological changes to the bacteria after treatment were visualized using a

123

scanning electron microscope (SEM). After GO treatments, the cells were collected

124

and fixed in a glutaraldehyde solution, dehydrated by a sequential series of ethanol

125

solutions (25, 50, 75, 85, 95 and 100%) and dried in a desiccator to remove ethanol.

126

Each sample was mounted onto an aluminum stub and imaged by SEM (S-4700,

127

Hitachi, Japan).

128

Direct ESR Detection of ROS. All ESR measurements were carried out at

129

ambient temperature using an ESR spectrometer (EMX, Bruker, USA). The spin

130

adducts were detected at the following settings, unless otherwise stated: 20 mW

131

microwave power, 1 G field modulation and 100 G scan range. Fifty microliter

132

aliquots of the sample solutions were put in separate quartz capillary tubes with

133

internal diameters of 0.9 mm. The spin trap 4-oxo-TEMP was used to verify the

134

presence of any singlet oxygen generated by GO upon exposure to simulated

135

sunlight. The superoxide and hydroxyl radicals were determined using BMPO. The

136

presence of holes and electrons were detected using the spin labels CPH and

137

TEMPO.

138

Assay for GSH and AA Oxidation. As described in our previous publications,

139

UV-vis spectroscopy was used to determine the oxidation of glutathione (GSH) and

140

ascorbic acid (AA) by GO exposed to simulated sunlight, as follows.28 50 µM GSH

141

were added to the suspensions of GO (25 µg/mL) and exposed to simulated sunlight

ACS Paragon Plus Environment

Page 8 of 31

Page 9 of 31

Environmental Science & Technology

142

for 30 min. Adding 100 µM DNTB to the mixtures yielded a yellow product,

143

quantified spectrophotometrically by measuring absorbance at 412 nm. To examine

144

the effects of irradiated GO on AA, 25 µg/mL GO were mixed with 100 µM AA.

145

During these 30 minutes of exposure to simulated sunlight, samples were collected

146

every five minutes and the amount of remaining AA was determined by measuring

147

the absorbance of AA at the 265 nm. We used NaN3 and histone separately to

148

scavenge the singlet oxygen during photoexcitation of GO to further distinguish the

149

effects of singlet oxygen on the oxidation of either AA or GSH.

150

Characterization of GO Exposed to Simulated Sunlight in Presence of

151

Antioxidants. Fluorescence spectra of 0.1 mg/mL GO in the presence or absence of

152

0.2 mg/mL AA were acquired using an Edinburgh Instruments FLS 980

153

spectrophotometer with a 1 cm light path quartz cuvette. Absorption spectra were

154

recorded on a UV-vis spectrophotometer (UV-3600, Shimadzu, Japan). Milli-Q

155

water (18 MΩ cm) was used as reference. The XPS measurements were performed

156

on an X-ray photoelectron spectroscopy (ESCALAB 250Xi, Thermo Scientific, USA)

157

using a monochromatic Al Ka (1486.6 eV) source.

158

RESULTS AND DISCUSSION

159

Characterization of GO. GO used in the present study was purchased from

160

Chengdu Organic Chemical Company, Chinese Academy of Science. The

161

characterization of this nanomaterial was shown in Figure S1, including Atomic

162

force microscope (AFM) images, UV-vis spectra and Raman spectra. From the AFM

163

height images, we observed GO have lateral dimension of several micrometers and

ACS Paragon Plus Environment

Environmental Science & Technology

164

thickness around 1 nm. The UV−vis absorption spectra showed that there are two

165

peaks centered at around 231 and 280 nm in the UV region. Raman analysis

166

displayed large D and G bands at 1345 and 1592 cm-1, respectively. All these

167

characteristics agree well with other reports [13-15] and clearly demonstrate that the

168

as-received sample is a fully exfoliated GO nanosheet.

169

Antimicrobial Activity of GO Exposed to Simulated Sunlight. First, we

170

determined the toxicity of GO to bacteria without exposure to simulated sunlight. As

171

shown in Figure S2, E. coli cells (106 to 107 CFU/mL) were incubated with various

172

concentrations of GO for 2 h; colony count results indicated a considerable

173

cytotoxicity, which increased in direct proportion to concentrations of GO. Scanning

174

electron microscopy (SEM) images (Figure S3) clearly showed that GO treatment

175

resulted in loss of bacterial membrane integrity. At this point we did not detect any

176

trace of an ROS signal (Figure S4) using ESR, which is considered the most reliable

177

and direct method to detect short-lived free radicals. However, found upon addition

178

of GO, noticeable time-dependent GSH oxidation was observed (Figure S5), perhaps

179

as a consequence of a direct charge transfer from GSH to GO. These findings

180

indicate the antibacterial activity of GO may be caused by both direct membrane

181

damage and ROS-independent oxidative stress; these observations are consistent

182

with several earlier reports.1,4,14

183

Next, we studied the effect of exposure to simulated sunlight on the antibacterial

184

activities of GO. As shown in Figure 1a, 30 min simulated sunlight exposure alone

185

had a negligible effect on E. coli cells compared to the set of control cells. Cell

ACS Paragon Plus Environment

Page 10 of 31

Page 11 of 31

Environmental Science & Technology

186

cultures treated with 25 μg/mL GO had a survival percentage of 68.8±9.2%;

187

however when cultures containing that same amount of GO were exposed to

188

simulated sunlight, the survival percentage was reduced to 24.9±5.9%,

189

demonstrating that simulated sunlight significantly enhanced the antibacterial

190

activity of GO (p < 0.01). We also investigated how duration of exposure to

191

simulated sunlight could affect the antibiotic activity of GO. As shown in Figure 1b,

192

as the length of exposure to simulated sunlight increased, the viability of E. coli in

193

the presence of GO decreased in a stepwise fashion. To confirm this phenomenon we

194

performed a fluorescence-based Live/Dead assay. As expected, no obvious cell

195

apoptosis could be found in cultures exposed to just the simulated sunlight alone or

196

to just the GO treatment alone. However, simultaneous treatment with GO and

197

simulated sunlight markedly reduced viability, as indicated by a remarkable increase

198

in the number of PI-permeable, red fluorescent cells (Figure 1c).

199

ROS Generated by Light-irradiated GO. Previous research has established

200

that, graphene-based nanomaterials (e.g., GO, rGO) exhibit a photothermal effect

201

when irradiated by an NIR laser. This effect can kill both bacteria and tumor

202

cells.18,22,25 However, exposure to simulated sunlight for 0 – 30 minutes does not

203

significantly raise the temperature of GO-treated bacterial suspensions (data not

204

shown); therefore photothermal effects can be excluded. Instead, we inferred that the

205

light-enhanced antibacterial activities of GO might be a consequence of oxidative

206

stress, resulting from light-induced generation of reactive oxygen species. To test

207

this hypothesis, we used ESR spectroscopy to measure whether GO was able to

ACS Paragon Plus Environment

Environmental Science & Technology

208

produce ROS after exposure to simulated sunlight. We employed DMPO and BMPO

209

as spin trap for the hydroxyl radical and superoxide anion, respectively, and

210

4-oxo-TEMP to detect singlet oxygen. Based on previous research, we used an •OH

211

generating Fenton reaction, enzymatic O2•− generating system, and the 1O2 generating

212

compound Rose Bengal as references.26-28 Neither hydroxyl radical nor superoxide

213

anion signals were observed (Figure S4), although a triplet spectrum, characteristic

214

for the reaction between the 4-oxo-TEMP and singlet oxygen, was noted during the

215

exposure of GO to simulated sunlight (Figure 2a). As a spin trap, 4-oxo-TEMP itself

216

is ESR silent, but it can specifically capture 1O2 to yield a nitroxide radical,

217

TEMPONE, which has an observable and stable ESR spectrum. In addition, only a

218

negligible amount of hydrogen peroxide was generated during photoexcitation of GO

219

by using the hydrogen peroxide assay kit (data not shown).

220

These results demonstrate singlet oxygen could be generated by GO exposed to

221

sunlight. To confirm this conclusion, we added sodium azide (NaN3), a scavenger

222

capable of consuming generated 1O2, during the test. After adding 1 mM NaN3, only

223

a negligible TEMPO signal could be detected (Figure 2a), indicating the triplet

224

spectrum can be ascribed to the presence of singlet oxygen generated by GO. As

225

superoxide anions cannot be generated under anaerobic conditions, we infer that this

226

singlet oxygen must be generated by energy transfer from GO to the ground-state

227

oxygen.

228 229

GO-mediated Oxidative Stress and its Relationship with ROS. The overproduction of singlet oxygen will predictably impair the antioxidant defense

ACS Paragon Plus Environment

Page 12 of 31

Page 13 of 31

Environmental Science & Technology

230

systems of bacteria.29 To document this, we examined in vitro glutathione (GSH)

231

oxidation by GO with or without exposure to simulated sunlight. GSH is an

232

endogenous bacterial antioxidant, present in concentrations ranging between 0.1 and

233

10 mM.14,30,31 The thiol groups in GSH, which can be quantified by the Ellman’s

234

assay, can be oxidized to a disulfide bond, yielding the oxidized form, glutathione

235

disulfide (GSSG). As shown in Figure 2b, 30 min exposure to either simulated

236

sunlight or to GO alone caused extremely limited oxidation of glutathione, which

237

agrees well with earlier findings.13,14,23 However, the extent of GSH oxidation was

238

remarkably enhanced upon exposure to GO in combination with simulated sunlight;

239

these effects increased as a function of exposure time, indicating that sunlight could

240

accelerate GSH oxidation by GO. Next, we used ascorbic acid (AA) as another

241

indicator of oxidative stress. AA is a water-soluble antioxidant which has an

242

important biologic role regulating the intracellular redox state, through its interaction

243

with GSH.32-34 The characteristic absorption maximum for AA is located at 265 nm.

244

We consistently found that extremely significant AA oxidation occurred when GO

245

and simulated sunlight were introduced; however, samples which did not contain GO

246

or were not exposed to simulated sunlight only exhibited a minor depletion of AA

247

(Figure 2c). These observations verify that GO exposed to sunlight could cause

248

significant oxidation of biological antioxidants.

249

The aforementioned results have suggested that only 1O2 could be determined,

250

therefore we inferred the light-enhanced oxidation capacity of GO might arise

251

primarily from the generated 1O2. In order to verify our hypothesis, we add NaN3 and

ACS Paragon Plus Environment

Environmental Science & Technology

252

histone (to scavenge the generated 1O2) into the GO dispersion before exposure to

253

simulated sunlight. We had expected that the extent of GSH and AA oxidation by GO

254

would be greatly inhibited, because the inducer, 1O2, would have been consumed.

255

Surprisingly, the majority of GSH and AA still could be oxidized by GO that had been

256

exposed to simulated sunlight even after the addition of scavengers. These findings

257

suggest that generated 1O2 is not the dominant factor in the light-enhanced oxidation

258

capacity of GO (Figure 2d). To confirm this hypothesis, we assessed the antibacterial

259

activity of GO (after exposure to simulated sunlight) in the presence of 1O2

260

scavengers. As expected, those conditions produced data comparable to those

261

acquired without adding 1O2 scavengers (Figure S6). Given these results, we must

262

conclude that oxidative stress caused by ROS is not the primary mechanism

263

responsible for light-enhanced oxidation capacity of GO.

264

Exploring the ROS-Independent oxidation capacity of GO to simulated

265

sunlight. In order to identify a more likely mechanism, we investigated the

266

photochemical fate of GO under biologically-relevant conditions. Previous research

267

has shown GO has a large energy gap between π-state from its sp2 carbon sites and

268

σ-state of its sp3-bonded carbons.35 Therefore, it is possible that electrons and holes

269

are generated on the surface of GO when it is exposed to simulated sunlight. To

270

explore this possibility, we investigated the light-induced formation of electron-hole

271

pairs in situ, using ESR spectroscopy.28,36 Specifically, we used CPH to examine the

272

oxidizing activity of photogenerated holes and used TEMPO to verify photogenerated

273

electron. As shown in Figure 3a, CPH is ESR silent, therefore no reaction between

ACS Paragon Plus Environment

Page 14 of 31

Page 15 of 31

Environmental Science & Technology

274

CPH and GO was anticipated. Surprisingly, when the GO was exposed to simulated

275

sunlight, an ESR spectrum of three lines with intensity ratios of 1:1:1 was observed.

276

As the duration of exposure was extended, more CPH was oxidized to CP-nitroxide.

277

In contrast to the standard behavior of CPH, TEMPO normally exhibits a stable

278

triplet ESR spectrum, with relative intensities of 1:1:1, although it can be reduced to

279

TEMPOH, which is ESR silent. We observed that the TEMPO signal intensity was

280

unchanged in the presence of either GO alone or light alone. When both GO and

281

simulated sunlight were present, we observed an obvious reduction in the ESR signal

282

intensity, and that signal intensity continued to decrease as light exposure time was

283

increased. These results confirm that GO exposed to simulated sunlight induces the

284

generation of electron-hole pairs. However, due to the confinement of redox potential,

285

those light-induced electron-hole pairs cannot react with surrounding H2O and O2 to

286

respectively form •OH and O2•−.

287

Our results demonstrate that 1O2, the only type of ROS determined in these

288

experiments, could not be the primary cause of GSH oxidation. The oxidation activity

289

of the light-induced holes led us to speculate that these holes might be the main factor

290

driving the oxidation of antioxidant biomolecules. To verify this, we used

291

fluorescence excitation-emission spectra characterization. As shown in Figure 3b, GO

292

exhibited an inhomogeneous broadened fluorescence in the range of 410-750 nm,

293

with a maximum at about 550 nm and excitation at 470 nm. Previous research has

294

demonstrated that photoluminescence of GO is a consequence of radiative

295

electron-hole pair recombination.35,37,38 Nonetheless, when we introduced AA, a

ACS Paragon Plus Environment

Environmental Science & Technology

296

remarkable decrease in photoluminescence intensity, even though the

297

photoluminescence wavelength characteristics remained unchanged. This

298

phenomenon demonstrates how antioxidant biomolecules could consume the

299

light-induced holes, inhibit electron-hole pair recombination, and thereby suppress the

300

photoluminescence of GO.

301

Chemical Transformation of GO upon Exposure to Simulated Sunlight. The

302

generation of light-induced holes could cause the oxidation of vital cellular

303

components which mediate ROS-independent oxidative stress. This leads us to

304

another question: if holes are responsible for oxidization of antioxidant biomolecules,

305

what is the role of light-induced electrons? To investigate the fate of light-induced

306

electrons, we added antioxidant biomolecules (GSH, AA) to GO dispersions, thereby

307

consuming holes and leaving residual excited electrons. We characterized the

308

changes of GO under a variety of conditions (including the presence or absence of

309

antioxidants and with or without exposure to simulated sunlight) using UV-vis

310

spectrum and X-ray photoelectron spectroscopy. Figure 4a illustrates a series of GO

311

dispersions: no obvious color change was observed under conditions when only

312

simulated sunlight or only antioxidant molecules were present, which confirms the

313

slow reduction process of GO observed in previous reports.23,39,40 However, when

314

both antioxidant molecules and simulated sunlight were present, the GO dispersions

315

underwent very obvious color changes (from yellow to black), which indicate the

316

tremendous reduction of GO under these conditions. These results demonstrate how

317

the presence of antioxidants could induce sunlight-exposed GO to create significant

ACS Paragon Plus Environment

Page 16 of 31

Page 17 of 31

Environmental Science & Technology

318

amounts of excited residual electrons, which, in turn, would greatly accelerate the

319

reduction of GO.

320

Our next assays monitored the reduction of GO using UV-vis spectroscopy. As

321

with previous assays, the spectroscopic profile of GO did not change significantly

322

when AA was added by itself, or if the GO was only exposed to simulated sunlight

323

(Figure 4b). However, when both these factors were present, the maximum

324

absorption peak of the GO dispersion gradually red-shifted from 231 nm to 263 nm

325

and the optical absorption in the NIR region also markedly increased. We used XPS

326

to characterize the reduction of GO under various conditions. As shown in Figure 4c,

327

we detected three different peaks, centered at 284.5, 286.4 and 289.2 eV,

328

corresponding to C=C/C–C in the aromatic rings, C–O of the epoxy, or the alkoxy

329

groups and the COOH groups, respectively. Upon addition of AA, the intensities of

330

all C 1s peaks of the carbons binding to the oxygen of the light-exposed GO

331

decreased dramatically, indicating that most of the oxygen-containing functional

332

groups had been removed. Because those light-induced electrons could be trapped by

333

the oxygen functional groups, it is possible for oxygen-centered radicals or

334

carbon-centered radicals to be introduced during the process of removing oxygen on

335

the GO surface. As indicated in Figure 5, GO nanosheets exhibit a unique,

336

symmetrical ESR signal at g = 2.002, close to the free electron g-value of 2.0023,

337

representing carbon-centered radicals.41 We found that neither simulated sunlight

338

alone nor antioxidant alone had any noticeable effects on the ESR signal intensity

339

from the paramagnetic defects of GO. However, adding antioxidants in the presence

ACS Paragon Plus Environment

Environmental Science & Technology

340

of simulated sunlight resulted in a marked enhancement of the ESR signal. Notably,

341

the ESR spectrum for the AA radical indicated that the oxidation of AA and the

342

reduction of GO were simultaneous.

343

Based on all the data, we conclude that exposure to simulated sunlight accelerate

344

the electron transfer from antioxidant biomolecules to GO, and as a result, the

345

antioxidant system is destroyed and GO itself is reduced accompanied by the

346

introduction of carbon-centered free radicals. The oxidative stress, reduced GO and

347

introduced carbon-centered free radicals co-contribute to the overall antibacterial

348

efficacy.

349

Implications. The antibacterial properties of graphene-based nanomaterials,

350

such as GO, may offer specific advantages that surpass those of inorganic and

351

polymeric nanomaterials.42-45 Yet, these cannot be exploited until we understand their

352

behavior under environmental and other biologically-relevant conditions. Here we

353

have demonstrated how GO exhibit impressive antibacterial upon exposure to

354

simulated sunlight, resulting in oxidative stress that is mainly independent of ROS.

355

Exposure to simulated sunlight accelerates the electron transfer from the innate

356

antioxidant systems of E. coli to GO, thereby destroying the biomolecules that

357

ordinarily provide protection from oxidative stressors. Meanwhile, the light-induced

358

electrons promoted the reduction of GO, inducing additional carbon-centered free

359

radicals, which may augment the antibacterial activities of GO. Our work provides

360

the first full description of the antibacterial mechanisms of GO and offers guidance

361

for developing highly-efficient graphene-based antibacterial materials.

ACS Paragon Plus Environment

Page 18 of 31

Page 19 of 31

Environmental Science & Technology

362

ASSOCIATED CONTENT

363

Supporting Information Available: Additional experimental details and figures

364

(Figures S1−S6), as described in the text. This material is available free of charge at

365

http://pubs.acs.org.

366

AUTHOR INFORMATION

367

Corresponding Authors

368

*E-mail: [email protected] (C. Y. Chen); [email protected] (J. J. Yin);

369

[email protected] (C. C. Ge); Tel: +86-10-82545560.

370

Notes

371

The authors declare no competing financial interests.

372

ACKNOWLEDGMENTS

373

This work is partially supported by the National Basic Research Program of China

374

(973 Program Grant No. 2014CB931900 and 2016YFA0201600), National Natural

375

Science Foundation of China (11575123, 11621505 and 21320102003), Jiangsu

376

Provincial Key Laboratory of Radiation Medicine and Protection, a project funded

377

by the Priority Academic Program Development of Jiangsu Higher Education

378

Institutions (PAPD), and a regulatory science grant under the FDA Nanotechnology

379

CORES Program. C. Chen appreciates the support from the NSFC Distinguished

380

Young Scholars (11425520). C. Ge appreciates the support from the Open Project

381

Program of Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety,

382

Chinese Academy of Sciences (NSKF201611). Y. Chong appreciates the support

383

from the China Scholarship Council (no. 1410100007). The authors thank Dr. Lili

ACS Paragon Plus Environment

Environmental Science & Technology

384

Fox Vélez for scientific writing and editing support. This article is not an official U.S.

385

FDA guidance or policy statement. No official support or endorsement by the U.S.

386

FDA is intended or should be inferred.

ACS Paragon Plus Environment

Page 20 of 31

Page 21 of 31

Environmental Science & Technology

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

392

simulated sunlight. (b) Antibacterial activities of GO are influenced by duration of

393

exposure to simulated sunlight. The data shown are mean values and standard

394

deviations from a representative of three independent experiments. P values were

395

calculated by the student's test: *p < 0.05, **p < 0.01. (c) Representative fluorescence

396

images of live (green) and dead (red) cells after different treatments. Scale bars = 20

397

μm.

398

ACS Paragon Plus Environment

Environmental Science & Technology

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

Page 22 of 31

80 60 40

*

20

0 GO+Light NaN3 Histone

+ -

+ + -

+ +

400

Figure 2. The generation of ROS and enhanced oxidizing activity by GO nanosheets

401

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

405

conditions: simulated sunlight alone, GO alone, and GO in the presence of simulated

406

sunlight. (c) In vitro AA oxidation under the three conditions as above. (d) Loss of

407

antioxidant agents (GSH or AA) by GO during exposure to simulated sunlight in

408

either the absence or presence of NaN3 or histone (singlet oxygen scavengers). These

409

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.

ACS Paragon Plus Environment

Page 23 of 31

Environmental Science & Technology

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.

414

(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

416

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

418

simulated sunlight. (b) Excitation−emission matrices (EEM) characterize the

419

photoluminescence properties of GO by itself (left column) and GO upon addition of

420

AA (right column).

421

ACS Paragon Plus Environment

Environmental Science & Technology

422 423

Figure 4. The chemical reduction of GO. (a) Photographs of GO dispersions under

424

various conditions: sunlight alone, antioxidant (AA or GSH) alone, or antioxidant

425

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.

427

Items 4A through 4G correspond to a range of exposure times (0-60 min). (c) XPS

428

spectra of C 1s of GO in the presence of AA before and after exposure to simulated

429

sunlight.

430

ACS Paragon Plus Environment

Page 24 of 31

Page 25 of 31

Environmental Science & Technology

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

431

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

433

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.

ACS Paragon Plus Environment

Environmental Science & Technology

437

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.

ACS Paragon Plus Environment