Aggregation Kinetics and Self-Assembly Mechanisms of Graphene

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Aggregation Kinetics and Self-Assembly Mechanisms of Graphene Quantum Dots in Aqueous Solutions: Cooperative Effects of pH and Electrolytes Qingqing Li, Baoliang Chen, and Baoshan Xing Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04178 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 10, 2017

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Environmental Science & Technology

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Aggregation Kinetics and Self-Assembly Mechanisms of Graphene Quantum

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Dots in Aqueous Solutions: Cooperative Effects of pH and Electrolytes

3

Qingqing Li†, ‡, #, Baoliang Chen †, ‡, *, and Baoshan Xing#

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†Department of Environmental Science, Zhejiang University, Hangzhou 310058, China

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‡Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control, Hangzhou

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310058, China

9 10

#

Stockbridge School of Agriculture, University of Massachusetts, Amherst, Massachusetts 01003,

United States

11 12 13

* Corresponding Author E-mail: [email protected]

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Phone: 0086-571-88982587

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Fax: 0086-571-88982587

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1

ACS Paragon Plus Environment


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ABSTRACT

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The cooperative effects of pH and electrolytes on the aggregation of GQDs and the aggregate

20

morphologies are characterized. Because GQDs have an average size of 9 nm with abundant

21

O-functionalized edges, their suspension was very stable even in a high electrolyte

22

concentration and low pH solution. Divalent cations (Mg2+ and Ca2+) exceled at aggregating

23

the GQD nanoplates, while monovalent cations (Na+ and K+) did not disturb the stability. For

24

Na+ and K+, positive linear correlations were observed between the critical coagulation

25

concentration (CCC) and pH levels. For Mg2+ and Ca2+, negative, but nonlinear, correlations

26

between CCC and pH values could not be explained and predicted by the traditional DLVO

27

theory. Three-step mechanisms are proposed for the first time to elucidate the complex

28

aggregation of GQDs. The first step is the protonation/deprotonation of GQDs under different

29

pH values and the self-assembly of GQDs into GQD-water-GQD. The second step is the

30

self-assembly of small GQD pieces into large plates (graphene oxide-like) induced by the

31

co-existing Ca2+, and then conversion into 3D structures via π-π stacking. The third step is the

32

aggregation of the 3D-assembled GQDs into precipitates via the suppression of the electric

33

double layer. The self-assembly of GQDs prior to aggregation was supported by SEM and

34

HRTEM imaging. Understanding of the colloidal behavior of ultra-small nanoparticles like

35

GQDs is significantly important for the precise prediction of their environmental fate and risk.

2


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Table of Content (TOC)

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38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 3



60

INTRODUCTION

61

Graphene quantum dots (GQDs), one novel species of ultra-small graphene-based

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nanomaterials that possess exceptional physical and chemical properties, have inspired

63

intensive research efforts since being synthesized very recently.1-4 They are classified as a

64

zero-dimensional graphitic nanomaterial with lateral dimensions of less than 100 nm in a

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single to a few layers.5, 6 GQDs can be viewed from a dimensional perspective as a scaled

66

down version of graphene oxide (GO)3,

67

molecules, i.e., polycyclic aromatic hydrocarbons (PAHs).8-10 Correspondingly, these types of

68

nanoparticles (NPs) are generally derived through top-down nano-cutting methods3, 11 and

69

bottom-up organic synthesis routes.12 The surface morphology and edge oxygen-containing

70

groups can be further modified to gain controllable characteristics.13-16 Emerging as superior

71

and environmentally friendly functional materials, GQDs are attracting increasing attention in

72

various research areas of environmental, biological and energy-related applications.17-24

7

and also as an enlargement of benzene-based

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With such promising potential for many applications, GQDs would probably be released

74

into the environment and transported between environmental media, which may trigger

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uncertain ecological effects.25-29 For example, when serving as delivery vectors for medicines,

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GQDs have been shown to be readily taken up by cells and circulated in the blood system due

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to their smaller sizes. In the body fluids of higher animals, the exchange of cations such as

78

Na+, K+, Mg2+ and Ca2+ is highly dynamic, and all of these cations are vital to the regulation

79

of stable blood pressure and pH.30, 31 Excessive salt intake has been recognized as a major risk

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for triggering hypertension.32 The stability of delivery vectors, such as GQDs, could be

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influenced by blood salt concentrations, and their travel path and eventual fates could be

82

unintended. With such concerns, the response of GQDs to solutions with variable levels of

83

electrolytes and pH could be of vital importance, as the GQDs may encounter concentrated

84

cations, leading to variability in their transport and aggregation behavior.

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The stability of engineered nanoparticles is one of the vital factors that ultimately 4


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86

controls their environmental fate and ecological risks.33-36 Extensive research has been

87

conducted aiming to understand the stability and aggregation kinetics of nanoparticles in an

88

aqueous environment. In numerous publications, the Derjaguin-Landau-Verwey-Overbeek

89

(DLVO) theory has been commonly applied.37 The Schulze-Hardy rule was usually utilized to

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predict the minimum concentration of ions necessary to cause the rapid coagulation of

91

colloids.38 Attachment efficiency (α) was estimated on the basis of the inverse stability ratio

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(w). The successive suppression of the electric double layer (EDL) leads to the gradual

93

destabilization of nanoparticles, which is reflected by the lifting of attachment efficiency and

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the intensification of nanoparticle aggregation. The critical coagulation concentration (CCC)

95

value was accordingly determined and utilized as a quantitative indicator for the evaluation of

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the stability of nanoparticles in the aqueous phase. However, whether the aggregation of

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ultra-small nanoparticles, such as GQDs, obeys the traditional colloidal theory, which usually

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estimates nanoparticles with a spherical shape, remains unclear.

99

The colloidal behavior and aggregation mechanisms of GO have been studied previously.

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However, limited information concerning GQDs is available. GO dispersion in aqueous

101

environments occurs in the form of single layers interacting via two predominate fundamental

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patterns: edge-to-edge and face-to-face.34, 39 Adding divalent cations could cause aggregations

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of GO nanosheets in edge-to-edge patterns, ascribed to their strong bridging/crosslinking and

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intercalating effects.34 Aggregation by lowering of the pH primarily occurs in the face-to-face

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pattern because of decreased electrostatic repulsion, increased van der Waals interactions, and

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possible π-π bond interactions. In turn, an increase in pH would stabilize GO dispersion due

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to the enhanced repulsion between the negatively charged GO nanosheets. The excessive

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addition of OH- could also cause aggregation, which is referred to as the “salting out” effect.40

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By adjusting the solution chemistry, conformational changes to 2D graphene materials could

110

occur,40-45 which would make their colloidal properties more complicated. Similar to their

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larger relative graphene oxide (GO), the amphiphilicity of GQDs is recognized to be pH 5



112

dependent due to the protonation/deprotonation of the oxygen-containing groups at the

113

edges.46-51 With large edge-to-area ratios and surface charge densities, GQDs are much more

114

hydrophilic than GO.39, 49, 52, 53 Recently, the self-recognition of GQDs has sparked attention

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in the field of smart and fine structure research, and the self-assembly of GQDs is thought to

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be pH dependent and affected by the coexisting cations and temperature.54 So far, the

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assembly behaviors of nanoparticles, such as GQDs, from molecule-like materials into large

118

particles before aggregation have been poorly studied. This knowledge gap must be filled to

119

fully recognize the colloidal behavior and nano-size effects of nanoparticles or nanosheets in

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the environment.

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The quantum confinement and nano-size effects have vested GQDs with exceptional

122

properties, and varied applications have been developed. However, there are almost no reports

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on the colloidal behavior and fate of GQDs in the environment. The overall objective of this

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work is to investigate the stability and aggregation kinetics of GQDs in aqueous systems with

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a wide range of water chemistries (i.e., cation type/valence, pH, and the combined effects of

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cations and pH). In this report, the cooperative effects of pH and electrolytes on the

127

self-assembly and aggregation mechanisms of GQDs are unveiled and characterized.

128 129

EXPERIMENTAL SECTION

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Synthesis of Graphene Quantum Dots.

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and cutting according to the methods described in an earlier report.11 Briefly, 6 g of

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commercial pitch-based carbon fibers (CF, micrometer-sized) were added to a mixture of

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concentrated H2SO4 (1200 mL) and HNO3 (400 mL). After two hours of sonication, the

134

mixture was put in an 80°C water bath and stirred for 24 h under reflux condensation. The

135

resulting solution was then cooled, and the pH was adjusted to 5 with Na2CO3. The final

136

product was dialyzed in a dialysis bag (with a retained molecular weight of 2000 Da) to

137

remove the extra acid and salts. The diluted and clear solution was then concentrated by

The GQDs were synthesized through oxidation

6


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evaporating water in a 75°C water bath to make the stock GQD solution. Concentrated HNO3

139

and NaOH were used to acquire GQD solutions at selected pH.

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GQD Characterization. The synthesized GQDs were observed by high resolution

141

transmission electron microscopy (HRTEM), atomic force microscopy (AFM) and field

142

emission scanning electric microscopy (SEM). The surface functional groups were further

143

characterized through Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy,

144

and X-ray photoelectron spectroscopy (XPS). The hydrodynamic diameter (Dh) and zeta

145

potential (ζ-potential) were measured with a ZetaSizer Nano ZS90 (Malvern Instruments,

146

Worcestershire, U.K.). The GQDs at selected pH values of 2, 4, and 12 were observed using

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HRTEM. The Ca(NO3)2-induced aggregation of GQDs was observed using SEM to monitor

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the surface morphology. Detailed procedures are presented in the Supporting Information (SI)

149

section.

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GQDs Aggregation Kinetics. The early stage aggregation kinetics of GQDs can be

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obtained from the initial rate of Dh change with time t. The time-resolved dynamic light

152

scattering (TR-DLS) method was applied. In the early aggregation stage, the initial

153

aggregation rate constant (kα) is proportional to the initial rate of increase in Dh and inversely

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proportional to the initial primary GQD concentrations in the suspension (N0). The early

155

aggregation stage is defined as the time period from the initiation of the experiment (t0) to the

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time when the measured Dh value exceeds 1.5-2 Dh,initial, which is also referred to as the

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reaction-limited regime. After that, the increase of Dh slows down until the aggregation rate

158

constant reaches 0, and this stage is referred to as the diffusion-limited regime.34

159

ଵ

݇ఈ ∝ ே ቀ బ

ௗ஽೓ ሺ௧ሻ ௗ௧

ቁ

௧→଴

(1)

160

Particle attachment efficiency (α) is used to quantify particle aggregation kinetics, and it is

161

defined as the initial aggregation rate constant (kα) normalized by the aggregation rate

162

constant measured under diffusion-limited (fast) conditions.34 7



163

ଵ

α=௪=௞

௞ഀ ഀ,೑ೌೞ೟

=

భ ೏ವ೓ ሺ೟ሻ ቀ ቁ ಿబ ೏೟ ೟→బ ೏ವ೓ ሺ೟ሻ భ ቀ ቁ ಿబ,೑ೌೞ೟ ೏೟ ೟→బ,೑ೌೞ೟

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(2)

164

The GQD concentrations across all samples were identical, allowing for a simplification of eq.

165

2 (i.e., N0 drops out). Therefore, α can be determined directly by normalizing the initial slope

166

of the aggregation profile for a specific background solution chemistry by the initial slope

167

under diffusion-limited (fast) conditions. The critical coagulation concentration (CCC) values

168

of the GQDs were determined from the intersection of the extrapolated lines through the

169

diffusion- and reaction-limited regimes.

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In the current study, a GQD concentration of 80 mg/L provided strong DLS signals and

171

was therefore used in all of the aggregation studies. Equal volumes (500 µL) of the GQD

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solutions with the selected pH and electrolyte solution (NaCl, KCl, CaCl2 or MgCl2) were

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pipetted into a DLS glass cuvette (Malvern Instruments, Worcestershire, U.K.) to achieve a

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specific electrolyte and GQD concentration at the selected pH (the pH variation of the mixing

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procedure was less than 0.2%). After being vortexed for 1 s, the cuvette was immediately

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placed in the DLS instrument. The intensity of scattered light was measured at 173°, and the

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autocorrelation function was allowed to accumulate for 15 s during the aggregation study. The

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Dh measurements were conducted over a period of 400 s. This time span was selected to

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ensure that the early-stage aggregation was complete.

180 181

RESULTS AND DISCUSSION

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Characterization of GQDs. The synthesized GQDs were examined by HRTEM, AFM and

183

SEM (Figure 1A-C). Small polygonal pieces were present under AFM and SEM. The physical

184

dimensions of the GQDs were between 3.9 and 26.8 nm (average square root of the area of

185

GQD nanoplates, Table 1), with an average value of 9.0 nm according to the AFM particle

186

analysis (Figure 1A). The inset diagram in Figure 1A reveals that GQDs were primarily

187

composed of one or two carbon domain layers with a thickness of approximately 1-2 nm. 8


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188

Clear lattice fringes (1120) with a lattice parameter of 0.242 nm were observed through

189

HRTEM imaging (Figure 1B), indicating the high crystallinity of the GQDs. The

190

corresponding fast Fourier transform (FFT) pattern is shown in the inset of Figure 1B. The

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edge orientation of the GQDs was predominantly consistent with the zigzag orientation,

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which was confirmed to possess excellent electrical and optical characteristics.11 SEM

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showed a slightly larger size distribution of GQDs (Figure 1C), which can be ascribed to the

194

platinum coating that improves the surface conductivity.

195 196

Figure 1. Characterization of GQDs. (A) AFM image of the GQDs, and the inset shows the

197

height analysis; (B) HRTEM image of the GQD nanoplate, and the inset represents the 2D

198

FFT pattern; (C) SEM image; (D) FTIR spectra; (E) Raman spectra; and high-resolution XPS

199

C1s spectra of carbon fibers (CF) (F) and GQDs (G).

200 201

The surface functional groups were further characterized through FTIR spectra, Raman

202

spectra, and XPS. As shown in the FTIR spectra (Figure 1D), the carbonyl, carboxyl,

203

hydroxyl, and epoxy groups were clearly present in the GQDs. Using the oxidation cutting

204

method, abundant oxygen-containing functional groups were introduced to the edges and onto 9



205

the basal planes of the GQDs in comparison with the CF. The Raman spectra of the CF and

206

GQDs were recorded (Figure 1E). The relative intensity of the “disorder” D band to that of

207

the crystalline G band (ID/IG) for the GQDs was 0.82, which was lower than the reported

208

value of 0.91 for the GQDs synthesized at 120 °C11 and much lower than the reported values

209

for GO and reduced GQ (rGO) synthesized through Hummer’s method.43, 55 The relatively

210

small ID/IG ratio reflected the high crystallinity of the GQDs, suggesting that the conjugated-π

211

network of the GQD basal planes was retained to a large extent. XPS measurements were also

212

taken, and the high-resolution spectra of C1s for the CF and GQDs are presented in Figure 1F

213

and G, respectively. The peak at 284.4 eV was attributed to the presence of C-C/C=C, which

214

is a dominant graphitic C1s peak. C-O (285.4 eV) and C=O (288.1 eV) were also identified at

215

the corresponding peaks. After the O-groups were introduced, O-C=O (288.7 eV) appeared in

216

the XPS spectra of the GQDs compared to that of the CF. The atomic ratios of C to O in the

217

CF and GQDs were 11.80 and 0.61, respectively. The very distinct C/O atomic ratios indicate

218

a high degree of oxidation of the GQDs. The relative contents of each bond were calculated

219

using the XPS-peak-differentiation-imitating analysis. In the spectra of CF, there was 78.50%

220

C-C/C=C, 15.21% C-O, and 6.31% C=O. Conversely for GQDs, there was 66.44% C-C/C=C,

221

18.62% C-O, 10.43% C=O, and 4.52% O-C=O. The presence of these O-groups make the

222

GQDs highly stable in water.11

223

Effects of Cation Types on GQD Aggregation. Attachment efficiencies (α) in the

224

presence of four selected cations (Na+, K+, Mg2+, and Ca2+) at an unadjusted pH (approximate

225

4) are presented in Figure 2A. There were significant aggregation capability gaps between

226

these cations, as the attachment efficiencies (α) were very distinct from each other without

227

overlapping. The aggregation of the GQDs in the four selected cations could be divided into

228

reaction-limited and diffusion-limited regimes according to the DLVO theory. In the

229

reaction-limited regime, GQDs were gradually destabilized, which could be a consequence of

230

the gradual elevation of the degree of charge screening and the reduction of the repulsive 10


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electrostatic interactions. In the diffusion-limited regime, the charge of the GQD nanoplates

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was completely screened, so aggregates formed.

233

234 235

Figure 2. Attachment efficiencies (α) of the GQDs as a function of electrolyte concentration

236

(NaCl, KCl, MgCl2, or CaCl2) at an unadjusted pH (approximately pH 4) (A); comparison

237

between the determined CCC values in the current study and the CCC values predicted by the

238

Schulze-Hardy rule (B); aggregation profiles of the GQDs with selected electrolytes at the

239

CCC (C).

240 11



241

The CCC values of the GQDs were determined to be 2900 mM (Na+), 1000 mM (K+), 70

242

mM (Mg2+), and 1.9 mM (Ca2+). The divalent cations (Mg2+ and Ca2+) were far more effective

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at aggregating GQDs. According to the Schulz-Hardy rule, CCC is positively proportional to

244

1/z6 (z represents the cation valence),38,

245

experiential model.34, 36 However, the CCC values determined with the same cation valence in

246

this study differed greatly, especially for the CCC determined with Ca2+, which deviated

247

largely from the predicted value (Figure 2B). This inconsistency suggests that there are other

248

interactions in addition to charge screening. The CCCs of the GQDs in monovalent

249

electrolytes (NaCl and KCl) were much higher than their environmentally relevant

250

concentrations (usually less than 10 mM)57 and even approached the upper limits of their

251

solubility in the case of NaCl.58 For graphene oxide, the reported CCC values were 44-200

252

mM for Na+ and 1.3-3.9 mM for Mg2+ at an unadjusted pH (approximate pH 5),34-36 which

253

was much lower than those of the GQDs at the unadjusted pH (~pH 4) measured in the

254

current study. This result indicates that it is much more difficult to aggregate GQDs, as they

255

are much smaller in size than GO. The CCC of GO was reported to be 0.7-2.6 mM for

256

Ca2+,34-36 similar to that of the GQDs (1.9 mM) in the current study. This result indirectly

257

verified the universal efficacy of Ca2+-aggregating nanoparticles, such as GO and GQDs.

258

Although the sizes of GO (~1 µm) and GQD (~9 nm) nanoparticles are quite different, their

259

colloidal behaviors are almost the same in the presence of Ca2+. This interesting observation

260

may be attributed to the self-assembly of GQDs into GO-like nanosheets with the assistance

261

of Ca2+, which will be explained later. The CCC values of several carbon-based nanomaterials

262

are listed in Table 1. Obviously, the CCC of the GQDs is greatest with the selected

263

electrolytes, demonstrating the high stability of this small nanomaterial.

56

and there have been studies that verified this

264 265 266 12


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Table 1 The Critical Coagulation Concentrations (CCC) of Several Carbon-based Nanoparticles in the Literature. CCC, mM KCl MgCl2

NPs

particle size

Concentration, mg/L

pH

NaCl

GO36

179.2 ± 111.5 nm a

40

5.5

44

35

not mentioned

10

5.2

200

0.7

35

not mentioned

10

5.2

30-35

0.7

GO34

582 ± 111.2 nm a

10

5

188

3.9

2.6

CNT33

17.6 ± 7.9 nm (diameter); 1.5 ± 1.5 µm (length)

2.54 TOC

6.0

25

1.5

2.6

5.92 TOC

7.5-8.5

160

8.0

6.1

80

4

2906

70

1.9

GO

rGO

C60

59

GQDs (current study)

55.7 ± 1.8 nm

b

average 9.0 nm (3.9-26.8 nm) a

1.3

1007

CaCl2 0.9

Note: a The average square root of the area; b hydrodynamic radius of the nanoparticles suspended in deionized water.

267 268

It is facile to correlate the aggregation behavior to the charging degree of GQDs and

269

attribute the effective destabilization of divalent cations to the reduced negativity of the

270

surface of the nanoparticles in the divalent electrolytes compared to the monovalent

271

electrolytes. The degree of difficulty or effectivity in aggregating the GQDs was reflected by

272

the CCC values. However, little attention has been paid to the aggregates forming during the

273

destabilization procedure. For this reason, the aggregation profiles of the GQDs in the

274

presence of the selected cations at their corresponding CCC are presented in Figure 2C.

275

Interestingly, the aggregation profiles were sorted into two groups: aggregation profiles in the

276

presence of KCl and CaCl2 and aggregation profiles in the presence of NaCl and MgCl2.

277

There was a larger increase in Dh in the presence of either KCl or CaCl2 than in the presence

278

of either NaCl or MgCl2. However, there were no significant differences between the results

279

with KCl and CaCl2 during the measured period, as well as between those with NaCl and

280

MgCl2. Correspondingly, the estimated kfast values for the selected cations were 0.92 (Na+),

281

2.15 (K+), 1.07 (Mg2+), and 2.42 (Ca2+). It appears as though a greater aggregation rate could

282

be achieved during the early aggregation stage in the presence of heavier cations (K+ and Ca2+,

283

with atomic weights of 39 and 40 g/mol, respectively) than in the presence of those with

284

relatively lighter weights (Na+ and Mg2+, with atomic weights of 23 and 24 g/mol, 13



Page 14 of 37

285

respectively). However, no differences in the kfast and hydrodynamic radius in the presence of

286

NaCl, MgCl2, and CaCl2 were observed in the aggregation kinetics of CNTs, GO and C60.33,34,

287

59

288

The inconsistency of the results in the current study may result from variations in the

289

dimensions of these examined nanoparticles. The small size of GQDs allowed them to be

290

easily disturbed and their movement to be more dynamic and random in an aqueous system.

291

Figure S-1C, F, G, and M represent the aggregation profiles of four selected cations at an

292

unadjusted pH. The differences in aggregation rates and aggregate sizes among the selected

293

cations are reflected in all of these tested samples. During the same period, the heavier cations

294

allowed the GQDs to aggregate faster and form larger aggregates. The aggregation rate and

295

the size of the aggregates in the early aggregation stage might be less dependent on the cation

296

valence but may have a strong correlation with the cation atomic weight. These results may

297

offer insight into the removal of various small nanoparticles in water, as the aggregates with

298

larger sizes are favorable for precipitation.

299

Note that the GQD particle size is 3.9-26.8 nm derived from AFM image (Figure 1A),

300

and the initial particle size derived from aggregation profiles at the corresponding CCC of

301

each cation (Figure 2C) is around 200 nm. This inconsistent suggested that the GQDs maybe

302

already form aggregates before the DLS measurement began because GQDs aggregated in a

303

relative fast mode at CCC of cations. In comparison, the hydrodynamic sizes

304

aggregates below CCC (i.e. ~20 nm at pH 7 in 0.50 mM Ca2+, Figure S-1) when the DLS

305

measurement started are comparable to the GQD particle size derived from AFM image. In

306

addition, before the DLS measurements begin, there was a 15-second autocorrection as

307

mentioned in the experimental section. Concerning this period time, the DLS measurement

308

was unable to record the initial part of entire aggregation process. Therefore, some

309

aggregation kinetic presented in the manuscript is just the partial of entire aggregation process.

310

Frankly, the very initial aggregation behaviors of nanomaterials are still existed as a big 14


of GQD

Page 15 of 37


311

puzzle or blind point. In this study, the morphologies of GQD aggregates were monitored to

312

interpret the possible aggregation process, which will be illustrated in the next.

313 314

Figure 3. Photo illustrating the influence of pH on the GQD solution (A); ζ-potentials of the

315

GQDs as a function of pH (B); morphology of the GQDs at pH 2 (C-E), pH 4 (F-H), and pH

316

12 (I-K). The scale bar corresponds to 20 nm in Figures C, F, and I and to 5 nm in Figures D,

317

E, G, H, J, and K.

318 319

Effect of pH on GQD Aggregation. Six vials of stable GQD suspensions at equal

320

concentrations (80 mg/L) with different pH levels were photographed two months after they

321

were first prepared (Figure 3A). No apparent aggregates were observed, but a darkening of

322

the color of the solution in accordance with an increase in pH was clearly demonstrated (light 15



323

brown → dark brown → brownish dark). Conversely, it has been reported that GO will form

324

aggregates at a pH of 2 due to the deceased negative charge of the GO sheets.34 The

325

corresponding ζ-potentials of these GQD suspensions were measured (Figure 3B). The

326

well-dispersed GQDs were all negatively charged, while both the addition of an acid and the

327

addition of a base unexpectedly increased the negative charge of GQDs. In the acidic

328

solutions, the ζ-potential increased in accordance with pH elevation, while in the alkaline

329

solutions, the ζ-potential decreased sharply in accordance with pH elevation. However, quite

330

differently, the variation trend of the ζ-potentials for most other carbonaceous nanomaterials

331

roughly declined with an increase in pH. It has been reported that GO at a pH of less than 5

332

was always destabilized and formed aggregates without the addition of extra salt.46, 51 The

333

hydrodynamic size distributions of the GQDs were measured (Figure S-2), and the GQD Dh

334

was relatively unchanged across the entire pH range, which indicates a high dispersibility of

335

GQDs at different pH levels as well.

336

The reason that ζ-potential increase in accordance with pH elevation in acid solution is

337

remain unknow, for further investigation, the morphology of the GQDs at selected pH levels

338

was observed using HRTEM. As shown in Figure 3, GQD nano-plates at different pH levels

339

exhibited different surface morphologies. To the best of our knowledge, this study is the first

340

to report the pH-dependent morphologies of carbon-based nanoparticles such as GQDs. At an

341

unadjusted pH (Figure 3F-H) and a high pH (Figure 3I-K), the aggregated GQDs presented

342

parallel lattice fringes; meanwhile, the GQDs in acidic solutions (pH 2) displayed whorl-like

343

surface fringes (Figure 3C-E). The whorl-like surface fringes of the GQDs were similar to

344

those of soot,60 which is derived from particulate emissions resulting from the incomplete

345

combustion of biomass and fossil fuels. Soot is believed to be composed of a crystallized

346

outer shell and an amorphous carbon-stacked inner core, which is considered to be highly

347

chemically reactive.60-63 The micro-structural change of GQD nano-plates could possibly

348

enhance the surface chemical reactivity and consequently alter the surface charging of these 16


Page 16 of 37

Page 17 of 37


349

GQD nano-plates. Hassanzadeh et al. found that GQDs with concentrations above their CAC

350

(critical association concentration) could self-assemble into porous, irregular, semi-spherical

351

particles through a nucleation-elongation process.54 Additionally, this self-assembly can be

352

tuned by adjusting the pH, ionic strength and concentration of the GQDs.54 The driving force

353

behind this cooperative self-assembly is believed to be the interplay of secondary interactions,

354

such as electrostatic, π-π, hydrogen bonding and/or hydrophobic interactions. Therefore, both

355

acidic and alkaline media favor the cooperative self-assembly and formation of multi-sheet

356

particles of GQDs. According to their results, secondary interactions enhanced with the

357

addition of acid in the GQD suspension and this could be the reason that ζ-potential decreased

358

with the increase of acidity of the solution. Basically, aggregation induced by either pH or

359

cations is a process of nucleation that involves radial growth of the assembly. In this study,

360

with the exception of the highly isolated scatter of the GQDs at a pH of 12 (Figure 3I-K),

361

GQDs at lower pH levels were all connected to each other to some degree (Figure 3 C-H), but

362

the connection didn’t precipitate GQDs out of the acid solution, instead it stabilized GQDs as

363

the measured hydrodynamic sizes were similar across the entire pH range and ζ-potentials

364

decreased in accordance with the pH decrease. It should be noted that due to the drying

365

process for TEM observation, the outcome photos showed instantaneous states of GQDs at

366

extreme high or low pH conditions rather than the accurate morphologies in the solution at the

367

selected pH levels.

368

Combined Effects of pH and Cation type on the Stability of GQDs. Charge variations

369

induced by pH could play an important role in nanomaterial aggregation.34, 36, 40. However, it

370

appears as though the stability of the GQDs in the current study was not pH dependent as

371

there was no obvious aggregates observed after quite a long-time period (Figure 3A), and the

372

resultant charge variations did not have a severe impact on GQD dispersibility (Figure S-2).

373

The hydrodynamic size (Dh) of GQD suspensions was relatively unchanged across the entire

374

pH range (Figure S-2). To further understand the pH-dependent stability of GQDs, the 17



375

aggregation kinetics were examined with all of the above-mentioned mono- and divalent

376

electrolytes (NaCl, KCl, MgCl2, and CaCl2). The results are summed up in Figure 4 with the

377

corresponding CCC values at different pH levels, which are presented as insets. Both

378

reaction- and diffusion-limited aggregation regimes were observed in the measurements. The

379

effects of pH on the CCC values of the GQDs were quite different for the monovalent and

380

divalent cations.

381 382

Figure 4. Attachment efficiencies (α) of GQDs as a function of electrolyte concentrations at

383

selected pH levels. The inset represents the relationship of the CCC values with the solution’s

384

pH levels for each given electrolyte. (A) NaCl, (B) KCl, (C) MgCl2, and (D) CaCl2.

385 386

For monovalent cations (Na+ and K+), positive linear correlations were observed between

387

the CCC and pH values (insets in Figure 4A and B), which indicate that the aggregation

388

behavior of GQDs is dominantly controlled by the protonation and deprotonation of the

389

oxygen-containing groups. This is consistent with the findings pertaining to GO in the 18


Page 18 of 37

Page 19 of 37


390

literature.34 It should be noted that we were unable to record the aggregation kinetics at higher

391

pH levels (>4) due to an insufficient count rate. The low count rate indicates that the GQDs

392

may act as highly stable regular salts in alkaline solutions. Moreover, no aggregates of GQDs

393

were observed at pH 7, pH 9, and pH 12 with monovalent cations, even when the added

394

electrolytes had reached their maximum concentrations. In addition, the determined CCC

395

values in the presence of NaCl at pH 2 (2729 mM), pH 3 (2785 mM) and pH 4 (2906 mM)

396

were already very close to the solubility of NaCl.58 Similarly, the CCC values in the presence

397

of KCl were determined to be 590 mM (pH 2), 813 mM (pH 3) and 1007 mM (pH 4). There

398

was an overlap of the pH-dependent aggregation kinetics in the presence of NaCl, but for now,

399

the exact mechanisms are unknown.

400

The characteristics of GQD stability in the presence of divalent electrolytes were very

401

different and completely unexpected. According to the ζ-potential data (Figure 3B), the GQDs

402

were more stable in the solutions with higher pH levels, which could be attributed to the

403

enhanced hydrophilicity of GQD nanoplates derived from their functional groups with

404

deprotonated edges.64 However, as shown in Figures 4C and 4D, the CCC variation trend as a

405

function of pH was completely reversed for divalent cations. Negative, but nonlinear,

406

correlations were observed between the CCC and pH values, which could not be predicted by

407

the traditional DLVO theory. The measured CCC values in the presence of CaCl2 were

408

determined to be 81 mM (pH 2), 5.5 mM (pH 3), 1.9 mM (pH 4), 1.3 mM (pH 7), 0.92 mM

409

(pH 9), and 0.65 mM (pH 12). A sharp increase in CCC values occurred when the pH

410

decreased from 4 to 2, and this result was consistent with the ζ-potential variation trend of the

411

GQDs (Figure 3B). The decrease in CCC from pH2 to pH 4 for Ca2+ was 42.8-fold, which

412

was much larger than that from pH 4 to pH 12 (2.9-fold). The commonly used simulated body

413

fluid (SBF) contains Na+/K+/Mg2+/Ca2+ in concentrations (mM) of 142.0/5.0/1.5/2.5, with the

414

pH adjusted to 7.4.65 According to the results of our current study, the CCC of Ca2+ at pH 7

415

was determined to be 1.3 mM. Therefore, Ca2+ in SBF may be concentrated enough to 19



416

precipitate the GQDs if used as a drug vector. In this case, GQDs may not be acceptable

417

candidates for the delivery of therapeutics. This finding calls for the evaluation of the

418

colloidal properties and aggregation behaviors of certain GQDs under various pH conditions

419

before they can safely be used as carriers for therapeutics because the pH values of the human

420

body can be variable.

421

It should be noted that when the measurements were taken in the presence of MgCl2 in

422

the acidic solutions, the count rates were so low that the suspensions were not suitable for

423

DLS measurement, which also suggests that GQD nanoplates are well dispersed in acidic

424

solutions in the presence of MgCl2. The measured CCC values in the presence of MgCl2 were

425

determined to be 69 mM (pH 4), 25 mM (pH 7), 6.5 mM (pH 9), and 1.0 mM (pH 12). No

426

GQD aggregates were observed at pH 2 and pH 3 with the addition of MgCl2 at maximum

427

concentrations. The variation trend of pH dependency for the divalent cations was reversed

428

compared to the results for the monovalent cations, and the trend was also contrary to what

429

was predicted based on the ζ-potential results (Figure 3B). These unusual observations could

430

be partially attributed to the limitations of the ζ-potential measurements in describing particle

431

stability, as it only reflects the surface charge density on the ζ-plane. It is possible that

432

additional processes or novel mechanisms could be involved in the aggregation of GQDs.

433

To take a clear look at the effect of pH on the aggregates, the aggregation profiles of the

434

GQDs at different pH values with the selected cations (at the CCC) were selected and are

435

compared in Figure 5. All of the aggregation profiles are also available in Figure S-1 of the

436

Supporting Information. In the presence of monovalent cations (Figure S-1A-F), the sizes of

437

the aggregates were not significantly affected by pH. Only a slight decline in accordance with

438

the pH increase was observed for NaCl (Figure 5A). This decline is clearly shown in Figure

439

5B for KCl. On the contrary, a large increase in aggregate size with pH elevation was

440

observed in the case of the divalent cations (Figure S-1G-P). As is clearly shown in Figure 5C,

441

the aggregate size at pH 12 was distinctly large. This is because Mg2+ primarily exists as a 20


Page 20 of 37

Page 21 of 37


442

Mg(OH)2 colloid at pH 12, which enhanced the heteroaggregation of the GQD nanoplates and

443

Mg(OH)2 colloids. Similarly, Chowdhury et al. recently studied the complex roles of divalent

444

cations, pH, and NOM in the aggregation and stability of rGO.35 An increase in the rGO

445

aggregate size with pH elevation was observed in the presence of NaCl with CaCl2/MgCl2.

446

The authors attributed this phenomenon to the stronger binding of Ca2+ with GO functional

447

groups and the increased surface charge of GO.

448 449

Figure 5. Aggregation profiles of the GQDs at selected pH levels with the various electrolytes;

450

NaCl (A), KCl (B), MgCl2 (C), and CaCl2 (D).

451 452

Furthermore, aggregations were observed using SEM with and without the addition of

453

CaCl2 at selected pH levels (Figure 6). Prior to the addition of the divalent cation Ca2+, the

454

GQDs at lower pH levels were scattered across the sample plates (Figure 6 A, and B), while

455

those at pH 12 were clustered due to the “salting out” effect induced by the extremely

456

concentrated alkaline solution (Figure 6C). After the addition of Ca2+, all three of these

457

samples at different pH values formed aggregates with separated metal salts (Figure 6D-F). 21



458

Interestingly, the aggregates at pH 12 displayed two patterns: large aggregations with metal

459

salts of large sizes (Figure 6F) and small clusters without apparent metal salts separated out

460

(Figure 6I). Performing the aggregation observations using TEM was more difficult due to the

461

weak photo-permeability of the dense aggregates (Figure S-3). As the pH increased, the

462

aggregates became densely interconnected. The aggregates at a pH of 2 formed two patterns:

463

aggregates with specifically polygonal structures and aggregates with interconnected

464

structures. Both of these two patterns existed universally. The different surface morphologies

465

were used to reflect the potential aggregation mechanism, which will be described later.

466 467 468

Figure 6. Morphologies of GQDs determined using SEM imaging without (A-C) and with

469

(D-I) the addition of CaCl2 at different pH values of 2 (A, D, G), 4 (B, E, H) and 12 (C, F, I).

470 471

In a previous report, the mechanical properties of GO sheets were significantly enhanced

472

by modification with a small amount of Mg2+ and Ca2+, two divalent alkaline earth-metal

473

ions.61 The results were rationalized in terms of the chemical cross-linking between the

474

functional groups of the GO sheets and the divalent metal ions. Additionally, the edge 22


Page 22 of 37

Page 23 of 37


475

bonding contributed to the mechanical enhancement.66 In another closely related study,

476

Medhekar et al. found that the large-scale properties of GO sheets were controlled by

477

networks of hydrogen bonds between neighboring GO interlayers.67 The extent and collective

478

strength of these interlayers of hydrogen bond networks were controlled by the water content.

479

Thus, the mechanical properties can be intensified by increasing the density of the functional

480

groups, which can lead to an increase in hydrogen bonding.67 Herein, chemical cross-linking

481

and hydrogen bonding could control the edge-to-edge and face-to-face interactions of the GO

482

sheets and further control their aggregation and assembly. A GQD is a micro-version of GO,

483

and the aggregation behavior and assembly process would be governed by the

484

above-mentioned two forces (chemical cross-linking and hydrogen bonding). The combined

485

effects of pH and divalent cations could be the main factors that lead to the anomalous

486

aggregation behavior of GQDs.

487

Based on the current evidence, it is obvious that both pH and cation type are significantly

488

involved in controlling the aggregation behavior of GQDs in an aqueous environment. To

489

gain insight into the complex impact on GQD stability, a comparative analysis of the

490

aggregation kinetics in the presence of cations with equal valence at different pH levels was

491

carried out (Figure 7) to exclude the effects of ionic strength. Due to the impact of the CCC

492

on amplification and deamplification, the CCC gaps were successively narrowed while the pH

493

levels were increased in both the monovalent and divalent cation solutions (Figure 7). With

494

increasing pH, the difference in aggregation capacity between the cations with equal valence

495

was reduced, especially for the divalent cations (Mg2+ and Ca2+). This could be attributed to

496

the increased ionic strength derived from the extra salts used to adjust the pH in the alkaline

497

solution. However, the bridging of Ca2+ to the deprotonated carboxyl groups was enhanced at

498

higher pH levels. The simultaneous narrowing of the CCC gaps among the cations with equal

499

valence led to the expansion of the CCC gaps between the mono- and divalent cations in

500

accordance with increases in the pH (Figure S-4). 23



501 502

Figure 7. Attachment efficiencies (α) of GQDs as a function of cation concentration at

503

selected pH levels with the addition of monovalent cations at pH 2 (A), pH 3 (B), and pH 4 (C)

504

and with the addition of divalent cations at pH 4 (D), pH 7 (E), pH 9 (F), and pH 12 (G). 24


Page 24 of 37

Page 25 of 37


505

Aggregation Mechanisms of GQDs. In the current study, complicated aggregation of

506

GQDs was observed by adjusting the cation type and pH of the solutions. GQDs have been

507

shown to be self-recognizable at the molecular level and can potentially self-assemble into

508

different 3D structures.54 The secondary interactions controlled by the solution chemistry

509

could interplay and either favor or disfavor the formation of 3D structures. From this point of

510

view, the complex aggregations of GQDs under variable solution chemistries can be divided

511

into three steps, which are proposed in this study. A diagram of the schematic mechanism of

512

the colloidal behavior of GQDs is presented in Figure 8. The first step is the

513

protonation/deprotonation of GQDs at different pH values, and the self-assembly of GQD

514

ultra-small nanoparticles is controlled by the primary charge interaction. The second step is

515

the self-assembly of small pieces of GQD into large plates, which is induced by the

516

co-existing Ca2+, and the plates are then converted into 3D structures by secondary

517

interactions, such as π-π stacking. The third step is the aggregation of the 3D-assembled

518

GQDs into precipitates, which is controlled by primary and secondary interactions.

519

The adjustment of the pH of the solution first changed the surface charge of the GQDs

520

and made them act differently in acidic and alkaline solutions. With abundant O-functional

521

groups on the edges, the protonation/deprotonation of the GQD nanosheets was evident in

522

successive procedures. In pace with the protonation, the polycyclic aromatic network of

523

GQDs became more hydrophobic, which facilitated the extensive hydrogen bonding induced

524

by the self-recognition of the neighboring GQD nanosheets, and the GQD-water-GQD

525

sandwich-like structure was therefore assembled (Figure 8A).53,

526

observed interconnection of protonated GQD nanoplates at pH 2 (Figure 3C-E), GQD

527

aggregates with composite structures stably existed in water instead of precipitating in a

528

face-to-face pattern, which is an energetically favorable interaction mode.53 The protonation

529

of the carboxyl groups facilitated GQD-water-GQD self-assembly, which could be a

530

reasonable explanation for the high stability of the GQDs at low pH levels (Figure 3A). The 25


54, 67

Consistent with the


531

differences between the stabilization in acid/alkaline solutions was due mainly to the

532

differences in the dominant secondary interactions. In acidic solutions, hydrogen bonding

533

created distance between the neighboring GQD nanoplates (Figure 8A), while in alkaline

534

solutions, the connections between the GQDs were isolated by electrostatic repulsion (Figure

535

8D). These results were verified by observing the isolated GQD nanoplates at a pH of 12

536

(Figure 3I-K).

537 538

Figure 8. Proposed three-step mechanisms for the aggregation behavior of GQDs with and

539

without the addition of salt under acidic (protonation) and alkaline (deprotonation) conditions.

540

The red ‘+’ symbol surrounding the particles of A, B, and C indicates a positive charge; the

541

blue ‘-’ symbol surrounding the particles of D, E, F, and G indicates a negative charge; and

542

the yellow point denotes the Ca2+ cation.

543 544

After the addition of the divalent cations, the second step of complex aggregation

545

proceeded. In the acidic solution, the added cations may intercalate into the basal planes of the

546

GQDs and compress the GQD-water-GQD self-assembly structures into denser ones. These

547

compressed GQD supramolecules could further interact with each other through crosslinking

548

of the GQD edges (Figure 8B). However, the conditions in the alkaline solution were vastly 26


Page 26 of 37

Page 27 of 37


549

different and can be further divided into two substeps. The formerly isolated GQD nanoplates

550

were interconnected by the crosslinking/bridging of divalent cations at the edges of the GQDs

551

(Figure 8E), and the repulsive forces between the negatively charged GQDs decreased

552

simultaneously. The edge-to-edge bridging could enlarge the size of the GQD composite, like

553

knitting the small pieces of GQDs into large graphene-like nanosheets with cation-made pins

554

(Figure 8F). After forming this crosslinking-enhanced self-assembly, the GQD composites

555

with large lateral sizes would bend, fold and stack through π-π interactions (Figure 8F),

556

maybe perhaps with more cations intercalating between the basal planes. These two substeps

557

proceeded in totally different patterns. During the first substep, the GQDs interconnected in

558

an edge-to-edge pattern, while during the second substep, the GQD self-assembly stacked in a

559

face-to-face pattern. The ordered surface morphologies of the self-assembled GQDs under

560

alkaline conditions (Figure 3H and 3K) supports the graphene-like π-π stacking mechanisms

561

of assembled GQDs, while the disordered morphologies of the assembled GQDs under acidic

562

conditions (Figure 3D) reflect the GQD-water-GQD interactions. The differences between the

563

second step in acidic and alkaline solutions can be attributed to the strength of the

564

crosslinking, which was reflected by the differences in aggregate size at the selected pH levels

565

(Figure 5D).

566

After the self-recognition involved in the first two steps, the third and last step of

567

aggregation began with the growth of the GQD self-assemblies into irregular sphere-like

568

structures (Figure 8C and G). According to the DLVO theory, the sphere-like GQD

569

composites would eventually aggregate and precipitate out of the solutions, similar to other

570

spherical nanoparticles. The growth of the GQD self-assemblies is presented in Figure S-3.

571

After the addition of CaCl2, polygonal and interconnected assemblies were clearly present at

572

pH 2 (Figure S-3A-C) and pH 4 (Figure S-3D), and densely assembled aggregates were

573

present at pH 12 (Figure S-3E). In contrast, GQDs could have difficulty assembling into

574

supermolecular composites with the addition of monovalent cations due to the lack of the 27



575

crosslinking/bridging effect. They aggregate in another path after gradual EDL suppression

576

due to increased ion strength.

577

The cooperative effects of pH and electrolytes on GQD aggregation are highly dependent

578

on the cation type. For example, the cooperative effects adhere to the DLVO theory in the

579

presence of monovalent cations, which is reflected by GQD colloids that become more stable

580

in alkaline solutions (Figure 4A, and B). In contrast, when co-existing with divalent cations,

581

GQD nanoparticles are heavily aggregated in solutions with a high pH (Figure 4C, and D),

582

which reveals a discrepancy with predictions based on the DLVO theory. Based on the

583

proposed three-step aggregation mechanism, this colloidal discrepancy is attributed to the

584

heavy self-assembly of GQDs (second step) induced by divalent cations, especially Ca2+,

585

under alkaline conditions.

586

Furthermore, the aggregation of nanoparticles, especially those with 2D structures, was

587

always accompanied by conformational changes.43, 45 During aggregation, the nanoparticles

588

wrinkled, bended, folded, and stacked.40-43, 64, 68 A GQD is classified as a zero-dimension

589

graphite nanomaterial, but small GQDs can be assembled into lateral GQD-composites with

590

increased sizes, which could further bend, fold, and stack into large and dense aggregates. If

591

the O-functional groups on the opposite sides of the GQD nanosheet were unevenly

592

distributed, this could induce the basal planes to bend away from the negative field in the

593

inner space of the bending sheet.42 The bending would result in GQD composites that are

594

similar to micro-envelopes filled with a negative charge, which can ensnare positively

595

charged ions within them. Assisted by the crosslinking/bridging effect of divalent cations, the

596

aggregation of bended GQD nanosheets could be enhanced in alkaline systems. The

597

surface-cation interactions have inspired potential applications for GQDs in drug delivery and

598

waste material capture.40

599

In conclusion, GQDs is considered as a type of anisotropic nano-size composite, but its

600

aggregation behavior in the aqueous solution is missing. It is a great challenge to elucidate the 28


Page 28 of 37

Page 29 of 37


601

fundamental mechanisms of GQD aggregation behavior based on the traditional colloidal

602

science. The first time investigation on GQD aggregation behavior in the current study

603

suggested whether the colloidal approach and knowledge can be translate to this unique

604

nanomaterial regime or not is highly depended on the suspension system. The complex

605

influences of pH and cation valence on GQD aggregation were investigated, and a three-step

606

mechanism of self-assembly that involved aggregation was proposed for the first time. In

607

addition to the primary charge interaction, the interplay of several secondary interactions led

608

to different interaction modes under varied solution chemistries. The adjustment of pH levels

609

and coexisting cations could lead to anomalous aggregation behavior due to the

610

self-recognition triggered and enhanced by the above-mentioned adjustments. Our

611

understanding of the assembly of GQD aggregates should be updated, as there could be

612

aggregates that are stable in solutions, as well as aggregates that would precipitate out of

613

solutions. These are significantly important findings related to the safety of GQDs as delivery

614

vectors for therapeutics and the health risks of GQDs released into the environment.

615 616

ASSOCIATED CONTENT

617

Supporting Information

618

Sample preparation for microscopy is presented. Aggregation profiles (Figure S-1),

619

hydrodynamic size distribution (Figure S-2), morphology (Figure S-3), and attachment

620

efficiencies of GQDs (Figure S-4) are presented. This information is available free of charge

621

via the Internet at http://pubs.acs.org.

622 623

AUTHOR INFORMATION

624

Corresponding Author

625

*Phone: 0086-571-88982587; fax: 0086-571-88982587; e-mail: [email protected]. 29



626

Notes

627

The authors declare no competing ﬁnancial interest.

628 629

ACKNOWLEDGEMENTS

630

This project was supported by the National Natural Science Foundation of China (Grant

631

21425730, 21537005, and 21621005), the National Basic Research Program of China (Grant

632

2014CB441106), the Doctoral Fund of Ministry of Education China (Grant J20130039) and

633

USDA-NIFA Hatch program (MAS 00475).

634 635

REFERENCES

636

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637

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