Fluorescent carbon dots derived from Maillard ... - ACS Publications

Subscriber access provided by READING UNIV

Article

Fluorescent carbon dots derived from Maillard reaction products: Their properties, biodistribution, cytotoxicity and antioxidant activity Dongmei Li, Xiaokang Na, Haitao Wang, Yisha Xie, Shuang Cong, Yukun Song, Bei-Wei Zhu, and Mingqian Tan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05643 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018

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.

Journal of Agricultural and Food Chemistry 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 34

Journal of Agricultural and Food Chemistry

1

Fluorescent carbon dots derived from Maillard reaction products: Their

2

properties, biodistribution, cytotoxicity and antioxidant activity

3

4

5

Dongmei Li1,2, Xiaokang Na1,2, Haitao Wang1,2, Yisha Xie1,2, Shuang Cong1,2, Yukun

6

Song1,2, Xianbing Xu1,2, Bei-Wei Zhu1,2 and Mingqian Tan1,2*

7

8

1

9

Seafood, Dalian Polytechnic University, Qinggongyuan 1, Ganjingzi District, Dalian

School of Food Science and Technology, National Engineering Research Center of

10

116034, Liaoning, People’s Republic of China

11

2

12

116034, Liaoning, People’s Republic of China

13

*Corresponding

14

[email protected], ORCID: 0000000275350035)

Engineering Research Center of Seafood of Ministry of Education of China, Dalian

author

(Tel

&

Fax:

+86-411-86318657,

ACS Paragon Plus Environment

E-mail:


15

Abstract

16

Food-borne nanoparticles have received great attention owing to their unique

17

physico-chemical properties and potential health risk. In this study, carbon dots (CDs)

18

formed during one of the most important chemical reactions in food processing field,

19

the Maillard reaction from the model system including glucose and lysine, was

20

investigated. The CDs purified from Maillard reaction products emitted a strong blue

21

fluorescence under ultraviolet light with a fluorescent quantum yield of 16.30%. In

22

addition, they were roughly spherical with sizes of around 4.3 nm and mainly

23

composed of carbon, oxygen, hydrogen, and nitrogen. Their surface groups such as

24

hydroxyl, amino and carboxyl groups were found to possibly enable CDs to scavenge

25

DPPH and hydroxyl radicals. Furthermore, the cytotoxicity assessment of CDs

26

showed that they could readily enter HepG2 cells while caused negligible cell death at

27

low concentration. However, high CDs concentrations were highly cytotoxic and led

28

to cell death via interference of glycolytic pathway.

29

30

Keywords: carbon dots, Maillard reaction, anti-oxidation, cell metabolism,

31

cytotoxicity


Page 2 of 34

Page 3 of 34


32

Introduction

33

The bio-effect of engineered nanomaterials in food items is an emerging research

34

topic in both academic and industrial communities1-3. Nanoscale materials (sizes: 1 -

35

100 nm) exhibit unique physico-chemical properties, such as high surface to volume

36

ratio, enhanced reactivity and distinct photocatalytic ability compared with their

37

respective bulk materials.4 Food supplemented with nanomaterials has a variety of

38

benefits, including extended shelf life and enhanced flavors.5 However, nanoscale

39

materials can penetrate cell and may cause adverse health effects due to their distinct

40

physico-chemical properties.6,7 Most current studies of nanomaterials in food mainly

41

focus on the assessments of properties and potential toxicity of engineered

42

nanomaterials that are added to food as food additives.8,9 These studies, however,

43

disregard nanomaterials that can be generated during food processing. It is important

44

to understand the origin of such foodborne nanomaterials, especially its potential

45

toxicity and biological impacts.

46

Carbon dots (CDs), which were first discovered in 2004,10 generally refer to the

47

spherical fluorescent nanoparticles with sizes of below 10 nm and are mainly

48

composed of carbon, oxygen, hydrogen, and/or nitrogen.11-14 The associations of CDs

49

with food have been reported. In 2012, Sk et. al.,15 reported the presence of CDs in

50

bread and suger; their formations were ascribed to the heating of carbohydrate. The

51

observation was further validated by Al-Hadi, who used human mesenchymal stem

52

cells as a model to prove the toxic effects of CDs found in bakery products.16 CDs

53

generated during food processing are the same as those engineered CDs produced by ACS Paragon Plus Environment


54

hydrothermal synthesis. However, the formation mechanisms, properties, and

55

potential health risks of CDs in food are not well understood.

56

Maillard reaction, also known as ‘non-enzymatic browning reaction’, is a

57

heat-induced browning reaction widely used in the food industry.17 Generally,

58

condensation reaction between reducing sugars and amino groups is the first step in

59

Maillard reaction. The reaction is then followed by the advanced stage, in which a

60

series of complex reactions, including dehydration, rearrangement, isomerization and

61

condensation reactions, take place to ultimately lead to the formation of brown

62

nitrogenous polymers, known as ‘melanoidins’.17-19 It is important to note that the

63

reactions that occur during the advanced stage of Maillard reaction, especially the

64

condensation reaction, are analogous to the hydrothermal synthesis process of CDs

65

prepared from molecular precursors, such as citric acid and ammonia solution.20,21

66

The products from Maillard reaction can emit fluorescence when irradiated with UV

67

light. Fluorescent products derived from Maillard reaction of glutathione and ascorbic

68

acid have been used as probes for detection of Hg2+ irons or small molecule.22 CDs,

69

which were thought to link with Maillard reaction, have recently been used in the

70

fabrication of tunable multicolor display.23 Nevertheless, because the exploration of

71

CDs formation mechanism is still needed, we are motivated to explore CDs derived

72

from the products of Maillard reaction in thermal food processing.

73

We have previously investigated the presence of CDs and their properties in

74

food items, such as instant coffee,24 beer25 and commercial beverages.26 In addition,

75

the formation mechanism of CDs in protein-based food items, such as pike eel27 and ACS Paragon Plus Environment

Page 4 of 34

Page 5 of 34


76

beef,28 has recently been demonstrated. These studies have shown that the CDs are

77

nitrogen-containing materials while their properties are closely linked to heating

78

temperature and raw materials. Moreover, the products generated from Maillard

79

reaction are found to highly correlate with types of food and heating temperature.29

80

Therefore, it is possible that Maillard reaction may be involved in the formation of

81

CDs in food.

82

In this study, CDs formed during Maillard reaction of a model system: glucose

83

and lysine were investigated. The physic-chemical properties of CDs, such as particle

84

size, optical properties, surface group and elemental composition were characterized.

85

The reduction activity of CDs was assessed though DPPH- and hydroxyl-radicals

86

scavenging activities. Fe2+-chelating ability of CDs (through inhibition of Fenton

87

reaction) was studied to assess their antioxidant mechanism. The biodistribution of

88

CDs was investigated in onion epidermal and HepG2 cells. Toxicological risks of

89

CDs were studied in HepG2 cells. Finally, real-time analysis of cellular respiration

90

was conducted to evaluate the effects of CDs on mitochondrial and glycolytic

91

functions in HepG2 cells.

92

MATERIALS AND METHODS

93

Materials

94

Glucose, lysine, hydrogen peroxide, quinine sulfate (98%) and (ethylenedinitrilo)

95

tetraacetic acid (EDTA) disodium salt were purchased from Aladdin Technology

96

(Shanghai, China). Onion was purchased from a local grocery store in Dalian China.



97

2,2-Pipheugl-1-picrylhydrazyl

(DPPH)

and

3-(4,5-dimethyl-2-thiazoyl)-2,5-

98

diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis,

99

USA). Fetal bovine serum and Dulbecco’s minimum essential medium were

100

purchased from Hyclone (Logan, UT, USA). All chemicals were of analytical grade

101

and were used without further purification.

102

Separation of CDs from Maillard reaction products

103

Glucose (0.5 g) and lysine (0.5 g) were first dissolved in 5 mL of deionized water. The

104

solution was transferred to a 25 mL poly(tetrafluoroethylene) and then autoclaved at

105

180 oC for 10 h. The obtained crude product was purified using a D101 macroporous

106

adsorption resin column using water as an eluent. The fluorescent fractions were

107

collected, concentrated by a vacuum rotary evaporator to remove solvent, and

108

lyophilized to yield brown powder (0.25 g, 25%).

109

Instrumentation and characterization

110

The morphology of the CDs was examined by a JEM-2100 transmission electron

111

microscope (TEM) (JEOL, Tokyo, Japan) operation at 200 KV. Fluorescent spectra

112

were obtained by a F-2700 spectrophotometer (Hitachi, Tokyo, Japan) at ambient

113

conditions. Ultraviolet visible spectra were obtained by a Lambda 35 UV-vis

114

spectrophotometer (Perkin Elmer, Norwalk, USA). Lifetime was measured by Fluoro

115

Max-spectrofluorometer (Horiba Scientific Co, French) with a 450 nm laser as the

116

excitation source. The following equation was used to calculate the fluorescence

117

lifetime (τ) of CDs:


Page 6 of 34

Page 7 of 34


τ = ( A1τ1 + A2 τ 2 + A3 τ3 ) /( A1 + A2 + A3 )

118

119

Where Ai is the fractional contribution of time-resolved decay lifetime of τi.

120

The chemical groups on the surface of CDs were analyzed using a Fourier transform

121

infrared (FT-IR) spectrometer (Perkin Elmer, USA) with the KBr pellet technique

122

ranging from 500 to 4000 cm-1. X-ray photoelectron spectroscopy (XPS) spectra were

123

recorded by an Escalab 250Xi X-ray photoelectron spectrometer (ESCALAB250,

124

Thermo VG, Waltham, USA). X-ray diffraction (XRD) was measured on a

125

diffractometer (XRD-6100, Shimadzu, Kyoto, Japan).

126

Measurement of fluorescence quantum yields

127

The quantum yield (QY) of CDs was determined with quinine sulfate (dissolved in

128

0.1 M H2SO4, QY=55%) as a reference and calculated using the following equation:

129

A´ I n2 Ф=Ф´· I´ ·A · 2 n´

130

Where Φ is the QY of the CDs, I is the integrated emission intensity of CDs, n is the

131

refraction index, and A is the optical density. Ф´, A´, n´ and I´ are QY, optical density,

132

refraction index and integrated emission intensity of quinine sulfate, respectively. A

133

series of solutions of CDs and quinine sulfate were prepared with optical absorbance

134

values less than 0.1. The fluorescent spectra were recorded and their intensities were

135

integrated. QY was determined by comparison of the integrated fluorescent intensity

136

vs optical density. All the solutions were measured under the same instrumental

137

conditions.



138

Scavenging of DPPH free radicals

139

Scavenging capability of CDs for DPPH free radicals was estimated in this study.

140

Two milliliter of fresh prepared DPPH ethanol solution (50 µg mL-1) and 1 mL CDs

141

solution with different concentrations was mixed evenly, the absorbance at 519 nm

142

was recorded after incubation in dark for 60 min. Ethanol was used as the blank

143

control. DPPH radical scavenging capacity was calculated as follows:

144

DPPH radical scavenging activity %=(1-Asample/Acontrol)×100%

145

Scavenging of hydroxyl free radicals

146

Electron spin resonance (ESR) spectroscopy technology was used to determine the

147

hydroxyl radical scavenging activity according to the previous method.30 Briefly, 10

148

µL of CDs with different concentrations (1.56, 3.12, 6.25, 12.50, 25.00 and 50.00

149

mg/mL) were dispersed in deionized water (52 µL), 6 mM EDTA-Na2 (10 µL), 6%

150

H2O2 (8 µL), and 1 M 5,5-dimethyl-1-pyrroline N-oxide (DMPO, 10 µL). Ten

151

microliters of 6 mM FeSO4 was added to initiate the reaction. After incubated at

152

40 °C for 30 min, 40 µL of the mixture was transferred into a glass capillary tube

153

(Blaubrand intraMARK, Brand, Germany) for spectral acquisition with an ESR

154

spectrometer A200 (Bruker, Karisruhe, Germany). The amplitude of the second peak

155

of the spectrum represents the amount of DMPO-OH adducts, which is related to the

156

amount of hydroxyl radicals scavenged. The instrumental parameters were used as

157

receiver gain: 1.0 × 105, modulation amplitude: 1.0 G, microwave power: 74.8 mw,

158

microwave frequency: 9.44 GHz, time constant: 163.84 ms, conversion time: 480 ms


Page 8 of 34

Page 9 of 34


159

160

and modulation frequency: 100.00 kHz. Deionized water was used as a blank control.

Hydroxyl free radicals scavenging activity %=(1- Asample/Acontrol)×100%

161

Fe2+-chelating ability

162

The Fe2+-chelating capacity was investigated by a modified method as described by

163

previous work.31 500 µL of CDs solutions with varying concentrations (1, 2, 4, 6, 8,

164

and 10 mg/mL) was mixed with 25 µL FeCl2 (2 mM) and 500 µL distilled water. After

165

5 min incubation, 50 µL of ferrozine (5 mM) was added and incubated at room

166

temperature for 5 min. After centrifuged at 2000 rpm for 10 min, the absorbance of

167

supernatant at 562 nm was measured. The capability of the sample to chelate Fe2+ was

168

calculated by the following equation:

169

Fe2+ Chelating ability(%) = [1 - Asample/Acontrol] × 100%.

170

Bio-distribution of CDs in onion epidermal cells

171

A small piece of the epidermal membrane of a fresh onion was treated with 1 mg/mL

172

CDs aqueous solution for 10 min. The membrane sample was laid flat on the surface

173

of a slide, and then covered with a thin glass coverslip ensuring that there was no air

174

bubble. The onion epidermal cells were then imaged by a fluorescence microscope

175

(Nikon, Tokyo, Japan) at the excitation wavelength of 405 nm.

176

Bio-distribution of CDs in HepG2 cells

177

HepG2 cells purchased from the Cell Bank of Type Culture Collection of the Chinese

178

Academy of Sciences, Shanghai, China were treated with 1.5 mg/mL CDs aqueous



179

solution at 37 °C for 24h.

Then the cells were thoroughly washed with phosphate

180

buffer saline (PBS) (500 µL each time) for three times and kept in PBS for optical

181

imaging. An inverted laser scanning confocal microscopy (SP8, Leica, Wetzlar,

182

Germany) with excitation wavelength of 408, 458 and 488 nm were used for blue,

183

green and red region images collection, respectively.

184

Cytotoxicity

185

The HepG2 cells were seeded in a 96-well plate with the density of 7000 cells/well

186

and incubated for 24 h. After incubation with different concentrations of CDs for 12 h,

187

20 µL of MTT (5 mg/mL) solution was added and incubated for another 4 h.32 The

188

HepG2 cells was further washed with PBS thrice and the resulted formazan crystal

189

was dissolved in DMSO. The absorbance of DMSO solution was recorded at 570 nm

190

using an Infinite 200 multi-mode microplate reader (Tecan, Hombrechtikon,

191

Switzerland). The cell viability was calculated as the following formula:

192

Cell viability (%) = Asample/ Ablank×100%

193

where Asample and Ablank are the optical density of the CDs and blank control,

194

respectively .

195

Real-time analysis of cellular respiration

196

The effects of CDs on mitochondrial and glycolytic function were studied using an

197

XFp Extracellular Flux Analyzer (Seahorse Bioscience, MA, USA), in which oxygen

198

consumption rate (OCR) and extracellular acidification rate (ECAR) of HepG2 cells


Page 10 of 34

Page 11 of 34


199

were measured in real-time. Briefly, sensor probes were incubated with seahorse XF

200

calibration solution at 37 °C for 12 h. HepG2 cells were seeded (in 50 µL medium) in

201

an 8-well cell culture plate with an initial cell density of 1x104 cells/well for 4 h. CDs

202

derived from Maillard reaction were diluted to 1, 2, 5, 10, and 20 mg/mL with the

203

medium supplemented with glucose (10 mM), sodium pyruvate (1 mM) and

204

glutamine (2 mM), pH 7.3 - 7.4. The cells were first incubated for 17 min and then

205

CD solutions were added. OCR and ECAR were then measured in real-time every

206

seven minutes, for a total of 48 min. Blank medium was used as a control.

207

Results and discussion

208

The hydrothermal reaction of glucose and lysine in a typical carbonyl ammonia

209

condensation reaction that takes place between glucose and lysine as a result of

210

Maillard reaction.33 Glucose possesses reactive carbonyl group, which reacts with the

211

nucleophilic amino group of the amino acid lysine, and forms a complex mixture of

212

Maillard reaction products. When irradiated with a UV light, strong blue fluorescence

213

was observed from Maillard reaction products prepared from glucose and lysine

214

through hydrothermal method, whereas that was not observed from glucose and lysine

215

solution prior to Maillard reaction (Fig. 1A, inset). The UV-vis absorption spectrum of

216

the fluorescent products (Fig. 1A) showed that two peaks associated with the π-π*

217

electronic transition in C=C and the n-π∗ transition in C=O were observed at 275 and

218

325 nm, respectively.34 The fluorescence spectra (Fig. 1B) exhibited the classical

219

bathochromic emission phenomenon with a maximum emission wavelength of 440

220

nm under an excitation wavelength of 360 nm. The result was similar to that of CDs ACS Paragon Plus Environment


221

that were previously reported to have excitation-dependent fluorescence behavior

222

(one of its characteristics),35 attributing to their size differences and multiple surface

223

emissive trap statuses.36,37 The CDs derived from Maillard reaction of glucose and

224

lysine had a fluorescence quantum yield of 16.30% in relative to the reference,

225

quinine sulfate. The TEM image displayed in Fig. 1C showed that the products were

226

composed of spherical nanoparticles with sizes ranging from 2.3 to 6.8 nm (Fig. S1).

227

In addition, it had an inherent characteristic with a fluorescence lifetime (time an

228

electron spends to return to its ground state) of 9.5 ns (Fig. 1D). These results strongly

229

suggest that Maillard reaction of glucose and lysine leads to the formation of

230

fluorescent nanoparticle products.

231

The elemental analysis based on the XPS spectrum of CDs showed that three

232

predominant peaks at 285, 400 and 532 eV (Fig. 2A) were associated with elements C,

233

N and O, respectively; and with relative contents (calculated based on the integral

234

area) of ca. 69.5%, 8.6% and 21.6%, respectively. This result further signifies that the

235

nanoparticles from Maillard reaction products are carbon-rich fluorescent nanodots

236

that are highly analogous to the artificially synthesized CDs.38 The N/C atomic ratio

237

of CDs was calculated to be 12.3%, indicating high concentration of nitrogen.

238

High-resolution C1s XPS spectrum exhibited the characteristic peaks of C=C/C-C,

239

C-N, C-O and C=O at 284.6, 285.5, 286.4 and 287.4 eV, respectively (Fig. 2B). In

240

addition, high-resolution N1s peaks observed at around 399.2 and 400.8 eV can be

241

assigned to the pyridinic-like N and N-H groups, respectively (Fig. 2C). O1s XPS

242

spectrum was decomposed into peaks at 530.7, 531.4, 532.3 and 533.2 eV


Page 12 of 34

Page 13 of 34


243

corresponding to O-H, O=C-O, C-O and O=C-O, respectively (Fig. 2D). A broad peak

244

at around 21.82° (Fig. 2E) was due to highly disordered carbon atoms.

245

The surface groups of CDs were determined using FT-IR spectroscopy. As

246

shown in Fig. 2F, a broad peak centered at 3408 cm-1 was assigned to the stretching

247

vibrations of –OH and N-H bounds. A peak centered at 2936 cm-1 was due to the

248

vibration of -CH. A strong peak with high intensity at 1060 cm-1 indicated the

249

presence of aromatic alkoxy bonds. In addition, peaks at around 1600 cm-1 may be

250

due to the symmetric stretching vibration of C=O. These results indicate that there are

251

high contents of hydroxyl, amino and carbonyl groups on the surface of CDs.

252

As demonstrated in Fig. 3A, the effects of pH on the fluorescence intensity of

253

CDs can be neglected. While the fluorescence intensity was not influenced by ionic

254

strength of up to 10 mg/L NaCl (Fig. 3B), it decreased by about 10% under extremely

255

high ionic strength conditions (i.e. at higher than 8 mg/L NaCl). The effect of

256

different metal ions on the fluorescence intensity of CDs was also investigated.

257

Common metal ions, except for Cu2+ and Fe3+, had some influence on the

258

fluorescence intensity of CDs (Fig. 3C), incoherent with the previously reported

259

data.39 In addition, CDs exhibited excellent light stability when irradiated with UV

260

light (Fig 3D), as indicated by the fluorescence intensity that remained constant for up

261

to 1800 s.

262

Maillard reaction products have reportedly possess the characteristic reduction

263

activity against reactive oxygen species (ROS).40 In this study, two typical free

264

radicals, DPPH and hydroxyl radicals were used in experiments investigating the ACS Paragon Plus Environment


265

scavenging activity of ROS for the Maillard reaction-derived CDs. DPPH radicals are

266

stable free radicals because a lone pair electron on N atom is surrounded by three

267

benzene rings; however, a reaction with an antioxidant can result in the formation of a

268

stable DPPH-H complex together with the color change, from purple to light yellow.

269

As shown in Fig. 4A, the scavenging ability of CDs against DPPH increased with

270

increasing concentration of CDs, and a linear relation (with an equation: Y = 7.018 +

271

0.755X) between inhibition rate and CDs concentrations was observed. While CDs

272

had the half-maximal effective concentration (EC50) of 570 µg/mL, compared with

273

9.86 µg/mL for vitamin C (Vc), and the DPPH scavenging activity is however about

274

1/57th of Vc. The result demonstrated that although CDs had scavenging activity

275

against DPPH, the removal rate is relatively low compared with Vc.

276

Furthermore, the scavenging activity of CDs against hydroxyl radical was

277

investigated. Hydroxyl radicals are one of the most active chemical substances that

278

are harmful to human health due to their ability to attack primary biological

279

macromolecules, such as proteins, sugars, nucleic acids, lipids and other essential

280

molecules.41 Therefore, rate of hydroxyl radicals removal is a major indicator

281

indicating the capacity of an antioxidant. As shown in Fig. 4B, the signal intensity for

282

hydroxyl radicals decreased with increasing CDs concentration. The CDs from

283

Maillard reaction products had an EC50 towards hydroxyl radicals of 12.23 mg/mL.

284

This result further confirms the hydroxyl radicals’ scavenging ability of CDs derived

285

from Maillard reaction products. Nonetheless, the scavenging capability of CDs

286

against either DPPH or hydroxyl radicals is lower than those of N- and S-doped CDs


Page 14 of 34

Page 15 of 34


287

prepared from garlic.42

288

Antioxidants scavenge hydroxyl radicals in two ways: they can either directly

289

react with hydroxyl radicals (as discussed above) or indirectly inhibit the formation of

290

hydroxyl radicals by chelating metal ions involving in such formation. The data in Fig.

291

4C showed that the chelating ability increased with the increase of CDs concentration.

292

The EC50 of CDs towards hydroxyl radicals scavenging was determined to be 4.595

293

mg/mL, which is much higher than that of EDTA-Na2 (0.017 mg/mL). This suggests

294

that the metal ions involved in the formation of hydroxyl radicals are chelated by the

295

CDs, resulting in improved hydroxyl radicals scavenging capacity. These results

296

indicate that CDs derived from Maillard reaction products exhibit certain hydroxyl

297

radicals scavenging capacity through both the direct and the indirect mechanisms.

298

Bio-distribution of CDs in representative cells, onion epidermal cells and liver

299

cancer cells (HepG2), were examined. The inner skin of an onion was immersed in

300

CDs solution and then placed on glass slides. The distribution of CDs in onion cells

301

was visualized using a fluorescence microscope under an excitation wavelength of

302

405 nm. The bright-ﬁeld images in Fig. 5 showed that the cells before (Fig. 5A) and

303

after (Fig. 5D) treatment with CDs were not different. However, a blue fluorescence

304

was observed under the excitation wavelength of 405 nm in the epidermal cells

305

treated with CDs (Fig. 5E) in contrast with that in the control cells (Fig. 5B). The

306

fluorescence signal also indicated that CDs were distributed in the cell wall, but not in

307

the cytoplasm. The result is different from that previously reported for CDs obtained

308

from kvass.26 ACS Paragon Plus Environment


309

The bright-field and fluorescent images in Fig. 6 show that HepG2 cells

310

treated with CDs exhibit blue (Fig. 6B), green (Fig. 6C), and red (Fig. 6D)

311

fluorescence with excitation wavelengths of 408, 458 and 488 nm, respectively. The

312

fluorescence was found distributed mainly in the cytoplasm (not in nucleus), similarly

313

to the previous observation.26 This observation has also been seen for carbon quantum

314

dot prepared from cabbage, used as imaging probe in biomedical applications.43 The

315

results show that CDs from Maillard reaction products can easily enter both plant and

316

animal cells, and are distributed either inside the cell wall or the cytoplasm.

317

In vitro cytotoxicity of CDs was evaluated in HepG2 cells, which were used as

318

model cells. The result in Fig. 7A shows that cell survival rate was 90% after 24 h

319

exposure to CDs of concentrations below 1 mg/mL. Further increasing CDs

320

concentration significantly decreased cell viability. Approximately 80% of HepG2

321

cells were dead when the concentration of CDs reached 10 mg/mL. These observation

322

demonstrates that the cytotoxicity of CDs is dose-dependent, which is consistent with

323

that previously reported.38 To further understand the cytotoxicity mechanism of CDs,

324

the effect of CDs on energy metabolism of HepG2 cells was examined.44 Mammalian

325

cells produce adenosine triphosphate via two major mechanisms: glycolysis and

326

oxidative phosphorylation (OXPHOS).45 Nevertheless, most cancer cells with intact

327

mitochondria convert OXPHOS to the less energetically favorable glycolysis.45

328

Specifically, the ECAR and OCR, indications of the glycolytic activity and

329

mitochondrial function, respectively, can be determined simultaneously within the

330

same small population of HepG2 cells. The result in Fig. 7B shows that OCR value


Page 16 of 34

Page 17 of 34


331

(including those at high concentration 20 mg/mL) remained unchanged compared

332

with control, and the mitochondrial respiration is almost unaffected by CDs derived

333

from Maillard reaction products. Unlike OCR, the value of ECAR dramatically

334

decreased when the concentration of CDs was higher than 1 mg/mL, in consistent

335

with the result from MTT assay. Moreover, such decrease suggests that CDs may

336

inhibit the glycolysis in HepG2 cells (Fig. 7C). The real-time data of OCR and ECAR

337

(Fig. 7D) further showed that while the OCR value was not substantially changed

338

compared with control prior to the addition of CDs, the ECAR value remarkably

339

decreased to lower than the base value. The ECAR value decreased to almost zero

340

when the CDs concentration was increased to 20 mg/mL. We can conjecture that CDs

341

may bind to certain key enzymes involving in glycolysis and inhibit their

342

complexes,46 nonetheless, further investigations are needed in order to better

343

understand such mechanism of actions.

344

In summary, we demonstrated that fluorescent CDs were the nanoparticles

345

derived from the products of Maillard reaction of glucose and lysine (a model system).

346

The fluorescent nanostructures were also found in real food like grilled fish. The

347

overall production yield of the nanostructures was about 1.67% as calculated based on

348

the weight of turbot flesh.47 Herein, the CDs derived from Maillard reaction are

349

mono-dispersed and exhibit the bathochromic emission phenomenon with a

350

fluorescent quantum yield of 16.30%. They are composed of four elements, including

351

C, H, O and N, with high contents of hydroxyl, amino and carbonyl/ carboxylate

352

groups on their surface. The CDs had unique antioxidant capacity as demonstrated by



353

free-radical scavenging activity. Importantly, these CDs were found to be mainly

354

distributed inside the cell wall of onion epidermal cells and the cytoplasm (not

355

nucleus) of HepG2 cells. Moreover, the CDs may inhibit the glycolysis of HepG2

356

cells, thus causing cytotoxicity. Further investigations are however needed to

357

elucidate such inhibition mechanism.

358

ASSOCIATED CONTENT

359

Supporting Information

360

The Supporting Information is available free of charge on the ACS Publications

361

website at DOI: XXXXXXX.

362

Size distribution histogram of the CDs formed in the Maillard reaction

363

(Fig.S1).

364

Acknowledgement:

365

This work was supported by the National Key Research and Development Program of

366

China (2017YFD0400103, 2016YFD0400404). We also thank Tao Liu from Liaoning

367

Bai Hao Biological Technology Co. Ltd. (Benxi, Liaoning, China) for his technical

368

assistant.


Page 18 of 34

Page 19 of 34


369

REFERENCES

370

(1)

Duncan, T.V. Applications of nanotechnology in food packaging and food

371

safety: barrier materials, antimicrobials and sensors. J Colloid Interf Sci. 2011,

372

363, 1-24.

373

(2)

Bradley, E.L.; Castle, L.; Chaudhry, Q. Applications of nanomaterials in food

374

packaging with a consideration of opportunities for developing countries.

375

Trends Food Sci. Tech. 2011, 22, 604-610.

376

(3)

Lim, J.H.; Sisco, P.N.; Mudalige, T.K.; Sanchez-Pomales, G.; Howard, P.C.;

377

Linder, S.W. Detection and characterization of SiO2 and TiO2 nanostructures

378

in dietary supplements. J. Agric. Food Chem. 2015, 63, 3144-3152.

379

(4)

a review of current toxicological data. Part Fibre Toxicol. 2013, 10,15.

380 381

Shi, H.; Magaye, R.; Castranova, V.; Zhao, J. Titanium dioxide nanoparticles:

(5)

Chen, H.; Seiber, J.N.; Hotze, M. ACS select on nanotechnology in food and

382

agriculture: a perspective on implications and applications. J. Agric. Food

383

Chem. 2014, 62, 1209-1212.

384

(6)

nanotubes. Accounts Chem. Res. 2013, 46,702-713.

385 386

Liu, Y.; Zhao, Y.; Sun, B.; Chen, C. Understanding the toxicity of carbon

(7)

Yan, L.; Gu, Z.; Zhao, Y. Chemical mechanisms of the toxicological properties

387

of nanomaterials: generation of intracellular reactive oxygen species. Chem.

388

Asian J. 2013, 8, 2342-2353.

389 390

(8)

Huang, C.;Sun, M.Y.; Yang, Y.; Wang, F.; Ma, X.Q.; Li, J.Q.; Wang, Y.L.; Ding, Q.R.; Ying, H.; Song, H.Y.; Wu, Y.N.; Jiang, Y.G.; Jia, X.D.; Ba, Q.;



391

Wang, H. Titanium dioxide nanoparticles prime a specific activation state of

392

macrophages. Nanotoxicology. 2017, 11, 737-750.

393

(9)

Peters, R.; Kramer, E.; Oomen, A.G.; Rivera, Z.E.; Oegema, G.; Tromp, P.C.;

394

Fokkink, R.; Rietveld, A.; Marvin, H.J.; Weigel, S.; Peijnenburg, A.A.;

395

Bouwmeester, H. Presence of nano-sized silica during in vitro digestion of

396

foods containing silica as a food additive. Acs Nano. 2012, 6, 2441-2451.

397

(10)

Xu, X.Y.; Ray, R.; Gu, Y.L.; Ploehn, H.J.; Gearheart, L.; Raker, K.; Scrivens,

398

W.A. Electrophoretic analysis and purification of fluorescent single-walled

399

carbon nanotube fragments. J. Am. Chem. Soc. 2004, 126,12736-12737.

400

(11)

RSC Adv. 2014, 4, 27184-27200.

401 402

Song, Y.B; Zhu, S.J.; Bai, Y. Bioimaging based on fluorescent carbon dots.

(12)

Zhu, S.J.; Song, Y.B.; Zhao, X.H.; Shao, J.R.; Zhang, J.H.; Bai, Y. The

403

photoluminescence mechanism in carbon dots (graphene quantum dots, carbon

404

nanodots, and polymer dots): Current state and future perspective. Nano Res.

405

2015, 8, 355-381.

406

(13)

Cayuela, A.; Soriano, M. L.; Carrillo‐Carrion, C.; Valcarcel, M. ChemInform

407

abstract: semiconductor and carbon‐based fluorescent nanodots: the need for

408

consistency. Chem. Commun. 2016, 47,1311.

409

(14)

Chem. Soc. Rev. 2015, 44, 362-381.

410 411 412

Lim, S.Y.; Shen, W.; Gao, Z. Carbon quantum dots and their applications.

(14)

Sk, M.P.; Jaiswal, A.;Paul, A.;Ghosh, S.S.; Chattopadhyay, A. Presence of amorphous carbon nanoparticles in food caramels. Sci. Rep. 2012, 2, 383.


Page 20 of 34

Page 21 of 34


413

(16)

Al-Hadi, A.M.; Periasamy, V. S.; Athinarayanan, J.; Alshatwi, A.A. The

414

presence of carbon nanostructures in bakery products induces metabolic stress

415

in human mesenchymal stem cells through CYP1A and p53 gene expression.

416

Environ. Toxicol. Phar. 2016, 41,103-112.

417

(17)

Martins, S.I.F.S.; Jongen, W.M.F.; Boekel, M.A.J.S.V. A review of Maillard

418

reaction in food and implications to kinetic modelling. Trends Food Sci.

419

Technor. 2000, 11, 364-373.

420

(18)

consumed foods. J. Agr. Food Chem. 2012, 60, 7071-7079.

421 422

(19)

Smuda, M.; Glomb, M.A. Fragmentation pathways during Maillard-induced carbohydrate degradation. J. Agr. Food Chem. 2013, 61, 10198-10208.

423 424

Degen, J.; Hellwig, M.; Henle, T. 1,2-dicarbonyl compounds in commonly

(20)

Zhang, Y.; Wang, Y.L.; Feng, X.T.; Zhang, F.; Yang, Y.Z.; Liu, X.G. Effect of

425

reaction

426

nitrogen-doped carbon dots. Appl. Surf. Sci. 2016, 387,1236-1246.

427

(21)

temperature

on

structure

and

fluorescence

properties

of

Hu, Y.P.; Yang, J.; Tian, J.W.; Yu, J.S. How do nitrogen-doped carbon dots

428

generate from molecular precursors? An investigation of the formation

429

mechanism and a solution-based large-scale synthesis. J. Mater. Chem. B.

430

2015, 3, 5608-5614.

431

(22)

Dong, J.X.; Song, X.F.; Shi, Y.; Gao, Z.F.; Li, B.L.; Li, N.B.; Luo, H.Q. A

432

potential fluorescent probe: Maillard reaction product from glutathione and

433

ascorbic acid for rapid and label-free dual detection of Hg(2+) and biothiols.

434

Biosens. Bioelectron. 2016, 81, 473-479.



435

(23)

Wei,

W.;

Xu,

C.;

Wu,

L.;

Wang,

J.;

Page 22 of 34

Ren,

J.;

Qu,

X.

436

Non-enzymatic-browning-reaction: a versatile route for production of

437

nitrogen-doped carbon dots with tunable multicolor luminescent display. Sci.

438

Rep. 2014, 4, 3564.

439

(24)

Jiang, C.; Wu, H.; Song, X.; Ma, X.; Wang, J.; Tan, M. Presence of

440

photoluminescent carbon dots in Nescafe(R) original instant coffee:

441

applications to bioimaging. Talanta. 2014, 127, 68-74.

442

(25)

Wang, Z.Y.; Liao, H.; Wang, B.; Zhao, H.; Tan, M. Fluorescent carbon dots

443

from beer for breast cancer cell imaging and drug delivery. Int. J. Numer.

444

Anal. Met. 2015, 7, 8911-8917.

445

(26)

Liao, H.; Jiang, C.; Liu, W.; Vera, J.M.; Seni, O.D.; Demera, K.; Yu, C.; Tan,

446

M. Fluorescent nanoparticles from several commercial beverages: their

447

properties and potential application for bioimaging. J. Agr. Food Chem. 2015,

448

63, 8527-8533.

449

(27)

Bi, J.; Li, Y.; Wang, H.; Song, Y.; Cong, S.; Yu, C.; Zhu, B.W.; Tan, M.

450

Presence

451

nanostructures from roasted Pike Eel (Muraenesox cinereus). J. Agr. Food

452

Chem. 2017, 10.1021/acs.jafc.7b02303.

453

(28)

and

formation

mechanism

of

foodborne

carbonaceous

Li, Y.; Bi, J.; Liu, S.; Wang H; Yu, C.; Li, D.; Zhu, B.W.; Tan, M. Presence

454

and formation of fluorescence carbon dots in a grilled hamburger. Food

455

Funct. 2017, 8, 2558-2565.

456

(29)

Wang, H.; Qian, H.; Yao, W. Melanoidins produced by the Maillard reaction:


Page 23 of 34


structure and biological activity. Food Chem. 2011, 128, 573-584.

457 458

(30)

Qi, H.; Ji, X.L.; Liu, S.; Feng, D.D.; Dong, X.F.; He, B.Y.; Srinivas, J.; Yu,

459

C.X. Antioxidant and anti-dyslipidemic effects of polysaccharidic extract

460

from sea cucumber processing liquor. Electron J. Biotechn. 2017, 28, 1-6.

461

(31)

Huang, D.J.; Chen, H. J.; Hou, W.C.; Lin, C.D.; Lin, Y.H. Active recombinant

462

thioredoxin h protein with antioxidant activities from sweet potato (Ipomoea

463

batatas [L.] Lam Tainong 57) storage roots. J. Agr. Food Chem. 2004, 52,

464

4720-4724.

465

(32)

Zhao, S.J.; Lan, M.Y.; Zhu, X.Y.; Xue, H.T.; Ng, T.W.; Meng, X.M.; Lee,

466

C.S.; Wang, P.F.; Zhang, W.J. Green synthesis of bifunctional fluorescent

467

carbon dots from garlic for cellular imaging and free radical scavenging. Acs

468

Appl. Mater. Inter. 2015, 7, 17054-17060.

469

(33)

Assoumani, MB; Maxime, D; Nguyen, NP. Evaluation of a lysine-glucose

470

Maillard model system using three rapid analytical methods. In Maillard

471

reactions in chemistry, food and health, 1 edition.; Editors: Labuza, TP.,

472

Reineccius, VM., Baynes, J., Eds.; Royal Society of Chemistry: Cambridge,

473

UK, 1994; 151, 43-50.

474

(34)

Du, F.Y.; Li, J.N.; Hua, Y.; Zhang, M.M.; Zhou, Z.; Yuan, J.; Wang, J.; Peng,

475

W.X.; Zhang, L.; Xia, S.; Wang, D.Q.; Yang, S.M.; Xu, W.R.; Gong, A.H.;

476

Shao, Q.X. Multicolor nitrogen-doped carbon dots for live cell imaging. J.

477

Biomed. Nano. technol. 2015, 11, 780-788.

478

(35)

Chen, X.X.; Jin, Q.Q.; Wu, L.Z.; Tung, C.H.; Tang, X.J. Synthesis and unique



479

photoluminescence properties of nitrogen-rich quantum dots and their

480

applications. Angew. Chem. Int. Edit.2014, 53, 12542-12547.

481

(36)

Dong, Y.; Pang, H.; Yang, H.; Guo, C.; Shao, J.; Chi, Y.; Li, C.; Yu, T.

482

Carbon-based dots co-doped with nitrogen and sulfur for high quantum yield

483

and excitation-independent emission. Angew. Chem. Int. Edit. 2013, 52,

484

7800-7804.

485

(37)

Hou, J.; Wang, L.; Zhang, P.; Xu, Y.; Ding, L. Facile synthesis of carbon dots

486

in an immiscible system with excitation-independent emission and thermally

487

activated delayed fluorescence. Chem. Commun. 2015, 51, 17768-17771.

488

(38)

Sachdev, A.; Gopinath,P. Green synthesis of multifunctional carbon dots from

489

coriander leaves and their potential application as antioxidants, sensors and

490

bioimaging agents. Analyst. 2015, 140, 4260-4269.

491

(39)

Gao, Z.; Wang, L.B.; Su, R.X.; Huang, R.L.; Qi, W.; He, Z.M. A carbon

492

dot-based "off-on" fluorescent probe for highly selective and sensitive

493

detection of phytic acid. Biosens Bioelectron. 2015, 70, 232-238.

494

(40)

Kitts, D.D.; Chen, X.M.; Jing, H. Demonstration of antioxidant and

495

anti-inflammatory bioactivities from sugar-amino acid maillard reaction

496

products. J. Agr. Food Chem. 2012, 60, 6718-6727

497

(41)

Abramov, A.Y.; Scorziello, A.; Duchen, M.R.Three distinct mechanisms

498

generate oxygen free radicals in neurons and contribute to cell death during

499

anoxia and reoxygenation. J. Neurosci. 2007. 27,1129-1138.

500

(42)

Shen, J.; Shang, X.M.; Chen, X.Y.; Wang, D.; Cai, Y. Highly fluorescent N,


Page 24 of 34

Page 25 of 34


501

S-co-doped carbon dots and their potential applications as antioxidants and

502

sensitive probes for Cr (VI) detection. Sensors Actuat B-Chem. 2017, 248,

503

92-100.

504

(43)

Alam, A.M.; Park, B.Y.; Ghouri, Z.K.; Yong, K.H. Synthesis of carbon

505

quantum

506

photoluminescence properties: excellent imaging agent for biomedical

507

application. Green Chem. 2015, 17,3791-3797.

508

(44)

dot

from

cabbage

with

down-

and

up-conversion

Sheng, Y.; Fotso, S.; Serrill, J.D.; Shahab, S.; Santosa, D. A.; Ishmael, J. E.;

509

Proteau, P.J.; Zabriskie, T.M.; Mahmud, T . Succinylated apoptolidins from

510

amycolatopsis sp. ICBB 8242. Org. Lett. 2015, 17, 2526-2529.

511

(45)

Zhang, J.; Nuebel, E.; Wisidagama, D.R.; Setoguchi, K.; Hong, J.S.; Van

512

Horn , C.M.; Imam, S.S.; Vergnes, L.; Malone, C.S.; Koehler, C.M.; Teitell,

513

M.A. Measuring energy metabolism in cultured cells, including human

514

pluripotent stem cells and differentiated cells. Nat. Protoc. 2012, 7,

515

1068-1085.

516

(46)

Wang, Z.Y.; Wang, N.; Chen, J.P.; Shen, J.G. Emerging glycolysis targeting

517

and drug discovery from chinese medicine in cancer therapy. Evid-based

518

Compl. Alt. 2012,2012,1-13.

519

(47)

Bi, J.; Wang, H.; Kamal, T.; Zhu, B-W.; Tan, M. A fluorescence turn-off-on

520

chemosensor based on carbon nanocages for detection of ascorbic acid, RSC

521

Adv. 2017, 7, 30481–30487.

522



523

FIGURE CAPTIONS.

524

Figure 1. (A) UV-visible absorption spectrum of CDs formed in the Maillard reaction

525

using glucose and lysine as a model system. Insets show the photographs of glucose

526

and lysine solution (1, 1’), and CDs (2, 2’) formed in the Maillard reaction under the

527

illumination of visible (1, 2) and UV light (1’, 2’), (B) fluorescence spectra, (C) TEM

528

image (Inset shows the high resolution TEM image) and (D) fluorescence decay curve

529

of the CDs formed in the Maillard reaction.

530

Figure 2. (A) XPS survey, high resolution spectra of (B) C1s, (C) N1s, (D) O1s for the

531

CDs formed in the Maillard reaction. (E)XRD pattern and (F) FTIR spectra the CDs

532

formed in the Maillard reaction.

533

Figure 3. Effect of (A) pH, (B) ionic strength, (C) metal ions, and (D) continuous UV

534

radiation on fluorescence intensity of the CDs formed in the Maillard reaction. F.L.

535

represents fluorescence.

536

Figure 4. (A) Scavenging ability of the CDs formed in the Maillard reaction and

537

vitamin C (inset) against DPPH. (B) ESR spectra of DMPO/•OH adducts at different

538

concentrations of the CDs formed in the Maillard reaction. (C) Chelating ability of the

539

CDs formed in the Maillard reaction and EDTA-Na2 (inset) to Fe2+.

540

Figure 5. Bright field and fluorescence microscope images of onion epidermal cells at

541

the illustration of (A) visible light and (B) excitation wavelength of 405 nm, (C)

542

overlay of (A) and (B). Bright field and fluorescence microscope images of onion

543

epidermal cells incubated with CDs at the illustration of (D) visible light and (E)


Page 26 of 34

Page 27 of 34


544

excitation wavelength of 405 nm. (F) overlay of (D) and (E). Scale bar = 100 µm.

545

Figure 6. (A) Bright-field and fluorescent images of HepG2 cells incubated with CDs

546

at the excitation wavelengths of (B) 408 nm, (C) 458 nm, and (D) 488 nm,

547

respectively. Scale bar=30 µm.

548

Figure 7. (A) Cell viability of HepG2 cells by MTT assay after incubation with CDs

549

formed in the Maillard reaction. (B) Relative OCR (oxygen consumption rate) and (C)

550

ECAR (extracellular acidification rate) of HepG2 cells in the addition of CDs. (D)

551

Real-time analysis of OCR and ECAR in HepG2 cells upon adding CDs (20 mg/mL)

552

at 17 min for a total of 48 min. (Mean ± SD, n = 3).

553

554

555



556

557

Fig.1

558

559

560


Page 28 of 34

Page 29 of 34


Intensity (a.u.)

N1s

200 400 600 800 Binding Energy (eV)

D

N-H 400.8

pyridinic N 399.3

398 400 402 404 Binding Energy (eV)

E

F

100 50 0 10

20

30

40

50

*O=C-O 531.4

60

2θ(Degree)

O-H 530.7

O=C-O* 533.2

Glu

CDs

Lys

200 C=N N-H 150 100

C=C N-H

50 C-O-C 0

O-H

C-H

C=O

3500 3000 2500 2000 1500 1000 500

-1 Wavenumber (cm )

561

562

532.3 C-O

526 528 530 532 534 536 538 Binding Energy (eV)

21.82o

150

C-N 285.4 C-O 286.3 C=O 287.6

280 282 284 286 288 290 292 Binding Energy (eV)

Transmittance (%)

Intensity (a.u.)

C

396

C-C/C=C 284.5

Intensity (a.u.)

0

Intensity (a.u.)

B

O1s

C1s

Intensity (a.u.)

A

Fig. 2.

563

564

565



0.5

0.0

F.L. Intensity (a.u.)

3

4

5

6

7

8

9

** ** ** ** ** ** **

0.5

0.0 0

1

2

3

4

5

6

7

8

9 10

Concentration (mg/mL)

D

1.0

1200

** ** **

0.5

**

0.0

*

10 11

pH

C

567

** **

2

566

B 1.0

**


** ** **

**



A 1.0

Page 30 of 34

900 600 300 0

+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ 2+ trol 2 Con Mg Mn Zn Ca Co Fe Ni Cu Fe

0

300

600

900

1200

Time (s)

Ion species Fig. 3.

568

569

570

571

572

573


1500

1800

Page 31 of 34


574 575

Fig. 4.

576

577

578 579

Fig. 5.



580

581 582

Fig. 6.

583

584

585


Page 32 of 34

Page 33 of 34


120

0.8

*

**

0.6 0.4 ** 0.2 0.0 0

0.25 0.5

1

2

5

90 60 30 0 0

10

1

C

20

120 **

100

100

OCR (pmol/min)

Normalized ECAR (%)

10

OCR

80

60 40

** **

20

2

5

10

**

100 **

80

60

20

20 **

0

20

6

**

**

**

**

12 18 24 30 36 42 48

Time (min)

CDs (mg/mL)

60 40

40

0

1

**

Injection:CDs (20 mg/mL)

** 0

**

ECAR

80

0

587

5

D 120

120

586

2

CDs (mg/mL)

CDs (mg/mL)

Fig. 7.

588

589

590

591

592

593

594

595


0

ECAR (mpH/min)

Cell viability

Normalized OCR (%)

B 150

A 1.0


596

TOC

597 598

599

600

601

602

603


Page 34 of 34

Fluorescent carbon dots derived from Maillard ... - ACS Publications

Recommend Documents