Concentration Dependent Effects of Bovine Serum Albumin on

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Environmental Processes

Concentration dependent effects of bovine serum albumin on graphene oxide colloidal stability in aquatic environment Binbin Sun, Yinqing Zhang, Wei Chen, Kunkun Wang, and Lingyan Zhu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06218 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 13, 2018

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Environmental Science & Technology

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Concentration dependent effects of bovine serum albumin on

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graphene oxide colloidal stability in aquatic environment

3

Binbin Sun, Yinqing Zhang, Wei Chen, Kunkun Wang, Lingyan Zhu*

4 5

Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of

6

Education), Tianjin Key Laboratory of Environmental Remediation and Pollution

7

Control, College of Environmental Science and Engineering, Nankai University,

8

Tianjin 300350, P. R. China

9 10 11 12 13 14 15

∗

16

+86-22-23500791. Fax: +86-22-23500791.

To whom correspondence should be addressed. E-mail: [email protected]. Phone:

ACS Paragon Plus 1 Environment


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ABSTRACT

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The impacts of a model globular protein (bovine serum albumin, BSA) on

19

aggregation kinetics of graphene oxide (GO) in aquatic environment were

20

investigated through time-resolved dynamic light scattering at pH 5.5. Aggregation

21

kinetics of GO without BSA as a function of electrolyte concentrations (NaCl, MgCl2

22

and CaCl2) followed the traditional Derjaguin-Landau-Verwey-Overbeek (DLVO)

23

theory, and the critical coagulation concentration (CCC) was 190, 5.41 and 1.61 mM,

24

respectively. As BSA was present, it affected the GO stability in a concentration

25

dependent manner. At fixed electrolyte concentrations below the CCC values, for

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example 120 mM NaCl, the attachment efficiency of GO increased from 0.08 to 1,

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then decreased gradually and finally reached up to zero as BSA concentration

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increased from 0 to 66.5 mg C/L. The low-concentration BSA depressed GO stability

29

mainly due to electrostatic binding between the positively charged lysine groups of

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BSA and negatively charged groups of GO, as well as double layer compression effect.

31

With the increase of BSA concentration, more and more BSA molecules were

32

adsorbed on GO, leading to strong steric repulsion which finally predominated and

33

stabilized the GO. These results provided significant information about the

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concentration dependent effects of natural organic matters on GO stability under

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environmentally relevant conditions.

36

KEYWORDS: graphene oxide (GO), aggregation kinetics, bovine serum albumin

37

(BSA), electrostatic interactions, (X)DLVO

2

ACS Paragon Plus Environment

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Introduction

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Graphene oxide nanoparticles (GO NPs) have been widely used as a chemically

40

tunable platform due to their unique electronic, thermal and mechanical properties.1-4

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GO features a thin two-dimensional structure and has many oxygen-containing

42

functional groups, such as carboxylic groups at its edges, epoxy and hydroxyl groups

43

on its basal planes.5 As a consequence, GO is usually better dispersed than other

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carbon nanomaterials, such as fullerene and carbon nanotubes, in aqueous solution.6

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The large-scale production and commercial application make it inevitably discharge

46

into natural aquatic environment, and has raised public concerns about its potential

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risks to the environments and human health. Recent studies indicated that GO may

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exert toxicities to zebrafish,7 bacteria,8 plant and human cells.9,10 The aggregation

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states of GO are correlated to its transport, bioavailability and toxicities.11,12 Thus,

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investigation on GO colloidal stability in naturally relevant aquatic environments is of

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great importance to understand its long-term adverse effects.

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Natural organic matters (NOMs) are a complex and heterogeneous mixture of

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different types of organic molecules, including humic substances (HSs, such as humic

54

acid and fulvic acid), polysaccharides and proteins.13 They are widely distributed in

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natural aquatic and soil environments and significantly affect the stability, dissolution

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and transport of nanomaterials.13-15 Many previous studies usually took HSs as

57

surrogates of NOMs to inspect their effects on GO environmental behaviors.11, 16-17

58

HSs are large molecules and contain many carboxylic and phenolic hydroxyl groups,

3



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which are usually negatively charged in natural aquatic environment. Generally, HSs

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could strongly enhance the transport and stability of GO mainly through increasing

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steric repulsive force between GO NPs.16,17

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Proteins are one of the major components of NOMs in biological wastewater and

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natural aquatic environment.13,18 The protein concentration in the secondary effluent

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of a wastewater treatment plant was 3.9-11.8 mg/L.19 When released into natural

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aquatic environment, proteins may significantly affect the aggregation states,

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toxicities and other behaviors of NPs.13,20 Different from HSs, proteins have abundant

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amino-acid residual groups which may be positively and negatively charged, and

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display complex secondary and tertiary structures. It is hypothesized that the impacts

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of proteins on environmental behaviors of NPs are different from HSs, but until now

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much less studies have been conducted. Limited studies reported contrary effects of

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proteins on the colloidal stabilities of NPs.21,22 For example, serum albumin induced

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aggregation of hematite NPs at low salt concentration through strong patch-charge

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attractive force.21 However, proteins were observed to enhance the stability of NPs,

74

such as graphene and MnO2 through steric repulsive force.22,23 These suggested that

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the interactions between proteins and NPs are more complicated and are related to the

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physicochemical properties of both NPs and proteins, as well as the media conditions.

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Bovine serum albumin (BSA) as a globular protein composed of 583 amino acids,

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is commonly used as a model protein due to its high percentages of sequence

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identities compared to human serum albumin (HSA, 76%), low cost and high

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structural stability.21,24 The isoelectric point of BSA is 4.6 and is negatively charged in 4


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natural aquatic environment (pH 5-9).24 However, BSA bears around 60 surface lysine

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(Lys) groups, which have an isoelectric point of 11 and are positively charged, thus

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could interact with negatively charged surface of NPs through electrostatic

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binding.24-26 There herein, BSA is an ideal model protein to examine the impacts of

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proteins on the stability of NPs.

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Previous studies observed that not only the physicochemical properties of NPs

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and NOMs, but also the concentrations of NOMs may affect the stability of NPs.21,27

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However, in most of the studies, NOM surrogates were always investigated at a few

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fixed concentrations. For example, Jiang et al reported that humic and fulvic acids at

90

1.7 and 2.7 mg/L could enhance the GO colloidal stability.17 Another study reported

91

that

92

mercaptoundecyltrimethylammonium bromide coated Au NPs while stabilized the

93

NPs at 10 µM.27 Very little studies have been conducted to fully understand the effects

94

of NOM varying in a wide range of concentrations on stability of NPs.

BSA

at

2

nM

could

induce

the

aggregation

of

cationic

95

The aims of this study were to investigate the impacts of BSA at a wide range of

96

concentrations on GO colloidal stability through time-resolved dynamic light

97

scattering at pH 5.5. The adsorption of BSA on GO was studied and a variety of

98

characterization techniques were performed to disclose the interaction mechanisms

99

between them. The aggregation kinetics of GO as a function of electrolyte

100

concentrations (NaCl, MgCl2 and CaCl2) with or without BSA were recorded. The

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impact mechanisms of BSA concentration on GO colloidal stability were

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systematically investigated. 5



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MATERIALS AND METHODS

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Materials and Chemicals

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GR-grade NaCl, MgCl2·6H2O, and anhydrous CaCl2 were purchased from

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Sinopharm Chemical Reagent Co. (Tianjin, China). Bovine serum albumin (BSA,

107

lyophilized powder, A2153) was purchased from Sigma-Aldrich Company (St. Louis,

108

MO, USA). All the other chemicals were of analytical grade or better and used as

109

obtained. The total organic carbon (TOC) content of BSA was measured to be 43.4%

110

through high-temperature oxidation (multi N/C 3100, Analytik Jena, Germany). The

111

BSA solution was freshly prepared before each set of experiments. All aqueous

112

samples were prepared with ultrapure water (>18.2 MΩ/ cm), then filtered through

113

cellulose acetate membrane filters (0.22 µm) to remove particulate impurities prior to

114

use and stored at 4 °C in the dark.

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Preparation of GO Suspension

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The solid GO (>98%) was supplied by Tianjin Plannano Energy Technologies

117

Co., Ltd (Tianjin, China). GO was dispersed in ultrapure water with stirring for one

118

night to activate the surface of GO,28 followed by ultra-sonication (600 W, 40 kHz) in

119

an ice bath for 4 h to get 600 mg/L stock suspension. The GO stock suspension could

120

be stable for as long as three months. UV-Vis spectroscopy of GO suspension from

121

200 to 700 nm were obtained on a Shimadzu UV-2600 spectroscopy (Japan). The

122

morphology of GO was examined by transmission electron microscopy (TEM,

123

JEM-2100F, JEOL, Japan) equipped with a Schottky field-emitter and an ultra

124

high-resolution. The TEM samples were prepared by depositing one drop of GO 6


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colloidal suspension on the copper grid, which was then air-dried under dust-free

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condition. Raman spectra of GO powder were recorded with a Renishaw inVia Raman

127

spectroscopy (RM2000) with an excitation wavelength of 532 nm in the range

128

500-2500 cm-1. The crystallinity and purity of GO powder were characterized by

129

X-ray diffraction (XRD, Bruker, D8 Advance, Germany) with monochromatized Cu

130

Kα radiation under 40 kV and 100 mA and the scanning range was from 5 to 80°. The

131

functional groups on GO surface were obtained by Fourier transform infrared

132

spectroscopy (FTIR, Bruker, TENSOR 37, Germany) in the range 400-4000 cm-1.

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KBr pellets were prepared by mixing the GO powder and dried KBr. The lateral size

134

was determined under atomic force microscope (AFM, Santa Barbara, CA). 5 µL GO

135

suspension was loaded on a cleaved mica plate with a diameter of 1 cm followed by

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air drying under dust-free condition.

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Aggregation Kinetics

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Aggregation experiments of GO were conducted by time-resolved dynamic light

139

scattering (TR-DLS). The intensity-weighted averaged hydrodynamic diameter (Dh)

140

of GO was measured at intervals of 15 s for 20 min to 2 h on a particle size and zeta

141

potential analyzer (Zetasizer Nano ZS90, Malvern Instruments, UK) with a He-Ne

142

laser (633 nm) at 90 °C scattering angle. The situation with polydispersity index

143

(PDI) > 0.7 occurred occasionally, and then was not taken into account when the

144

mean GO diameter was measured.29 0.5 mL of electrolyte solution was added in a 2

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mL sterile micro-centrifuge tube (Crystalgen, USA), which contained 0.5 mL of GO

146

suspension and 0.5 mL of water or BSA solution, to reach a total volume of 1.5 mL. 7



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The micro-centrifuge tube was immediately vortexed by hand for 1 s, and pipetted

148

into the DLS polystyrene cuvette (10 mm path length, Malvern, UK). The

149

concentration of GO was set at 10 mg/L for all the DLS measurements, unless stated

150

otherwise. The pH of GO suspensions was maintained at about 5.5 ± 0.2 by adjusting

151

with NaOH or HCl at 10 mmol/L, and no buffer was used to avoid any

152

interference.30,31

153

The initial aggregation period was defined as the time period from experimental

154

initiation (t0) to the time when measured Dh exceeded 1.5 times of its initial value

155

(Dh,initial).32 The initial aggregation rate constant (k) is proportional to (dDh(t)/dt)33:

156

k∝

1 N0

dDh t dt

t→0

(1)

157

where N0 is the initial GO concentration. Dh(t) is the intensity-weighted average

158

hydrodynamic diameter at time t. The particle attachment efficiency (α), also known

159

as the inverse stability ratio (1/W), is used to quantify the initial aggregation kinetics

160

of GO. For all the aggregation experiments, GO concentration (N0) is a constant. The

161

α is calculated by normalizing the measured k in the reaction-limited cluster

162

aggregation (RLCA) region to the diffusion-limited cluster (DLCA) aggregation rate

163

constant kfast.22,33

164

α=

1

= W k

k fast

dD t

=

dth t→0

(2)

dDh t dt t→0, fast

165

The critical coagulation concentration (CCC) was obtained by intersecting

166

extrapolated lines through the RLCA and DLCA regimes.

167

BSA adsorption on GO

168

Adsorption of BSA (0 to 260 mg C /L) on GO was conducted using batch 8


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169

experiments in 40 ml screw cap vials at pH 5.5 ± 0.2 and 25 °C. Sorption experiments

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were conducted with elevated GO concentration (200 mg/L) to accurately measure the

171

mass of BSA adsorbed on GO. All the sorption tests were conducted in duplicates.

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The BSA solution was added into the GO suspension with 80 mM NaCl. All the vials

173

were incubated in a rotary shaking incubator for 1 h, and then centrifuged at 4500 g

174

for 15 min. The supernatant was transferred into a new centrifuge tube and again

175

centrifuged to separate the free BSA from the solution. Preliminary experiments

176

indicated that no measurable BSA was settled or adsorbed on the bottles in the control

177

experiments (without GO colloids). The BSA concentration was determined using

178

Bradford protein assay kit (Beijing Solarbio Science & Technology Co.) by

179

BioTekSynergy H4 at λ =595 nm. The precipitates obtained due to centrifugation were

180

freeze-dried, and subjected to FTIR analysis in the range 400-4000 cm-1.

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Characterization of the interactions between BSA and GO

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The zeta potentials of GO in the absence and presence of BSA at 2 mg C/L over

183

a wide range of electrolyte concentrations were measured on a Malvern Zetasizer with

184

at least five repeats. The zeta potential of GO changed as a function of BSA

185

concentration (0 to 30 mg C/L) was also measured in the presence and absence of

186

electrolytes at fixed concentration. The zeta potentials of GO were calculated by the

187

electrophoretic motilities (EPMs) using Smoluchowski expression for plate-like

188

particles.34 Fluorescence emissions spectra (LS-55, Perkin-Elmer, USA) equipped

189

with Xe lamp were also recorded with excitation at 280 nm, and emission wavelength

190

in the range of 290-500 nm. The secondary structure change within BSA during its 9



191

interaction with GO was determined using a J-715 circular dichroism spectrometer

192

(CD, JASCO, Japan) over a range of 190-260 nm with three replicates. FTIR was

193

used to characterize the spectra and secondary structures of BSA before and after

194

incubation with GO. The morphologies of GO with and without BSA in the presence

195

of NaCl were observed by TEM. The particle−particle interaction energy profiles

196

were calculated under different solution conditions using (X)DLVO theory (detailed

197

equations and parameters are provided in the Supporting Information) to reveal the

198

interactions between GO and BSA.

199

RESULTS AND DISCUSSION

200

Characterization of GO

201

A series of characterization techniques were applied to reveal the size,

202

morphology and surface chemistry properties of GO. The UV-Vis spectra of GO

203

(Figure 1A) indicates that the maximum absorption peak of GO was at 228 nm, which

204

was corresponding to the π-π* transition. The ratio of ID (intensity of D band, at 1335

205

cm−1) / IG (intensity of G band, at 1596 cm−1) in the Raman spectra was 0.869 (Figure

206

1B). The ratio was lower than that reported in a previous study (1.55−1.78),35

207

indicating that the GO used in present study had high degree of graphitization.36 A

208

strong peak in the XRD pattern was observed at 2θ = 10.5 ° (Figure 1C), which was

209

the characteristic diffraction peak of GO. The result was consistent with that in

210

previous study.37 The FTIR spectra (Figure 1D) revealed that there were abundant

211

chemically reactive functional groups on the outermost surface and defect sites of GO,

212

such as C=O (1720 cm−1), 1630 cm-1 (C=C), C−O (1042 cm−1), C−O−C (1252 cm−1) 10


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and C−OH (1405 cm−1).35,38 The relatively broad peak at 3427 cm−1 could be ascribed

214

to the water molecules adsorbed on GO.38 The GO thickness measured by atomic

215

force microscopy (AFM) was 0.7−1.8 nm, the lateral dimension was in the range of

216

200 nm to 1.2 µm (Figure S1), and the average surface area was 208.6 m2/g.39

217

Although the GO was not spherical, DLS was usually applied to characterize the Dh of

218

GO, which was treated as spherical particles that had the same average translational

219

diffusion coefficient.16,40,41 Dilute GO suspension at 10 mg/L with narrow lateral size

220

distribution was used for DLS measurement. The average Dh of GO stock suspension

221

was 200 ± 7.5 nm with very narrow size distribution and the PDI was 0.178,

222

indicating that GO was well dispersed in aqueous solution. The zeta potential of GO

223

at 10 mg/L (1 mmol/L NaCl, pH 5.5) was −46.4 ± 0.46 mV and its electrophoretic

224

mobility (EPM) was −3.48 ± 0.04, indicating the surface of GO was highly negatively

225

charged.

226

Adsorption of BSA on GO and interactions between them

227

To reveal the mechanisms of interactions between GO and BSA, sorption

228

experiments, and several characterization methods such as zeta potentials, FTIR, CD

229

spectra, were performed. BSA adsorption on GO was evaluated at different BSA

230

concentrations (0 to 260 mg C/L) in the presence of 80 mM NaCl at pH 5.5, and the

231

adsorption isotherm is shown in Figure S2. The amount of BSA adsorbed on GO

232

increased with the increase of initial concentration (C0) of BSA until maximum

233

adsorption was reached at a C0 of 174 mg C /L. The adsorption capacity of GO to

234

BSA was about868 mg C/L (2000 mg/g), which was lower than that reported by Gan 11



235

et al (4080 mg/g) but one order of magnitude higher than that reported in another

236

study (105 mg/g).31,42 In the absence or presence of electrolyte at fixed concentrations,

237

the absolute value of GO zeta potential decreased initially with the increasing

238

concentration of BSA, and then reached steady values (Figure 2A). At BSA

239

concentration of 30 mg C/L, the zeta potential of GO in the absence of electrolyte was

240

−34.7 mV. Although the amphiphilic BSA is negatively charged at pH 7.0, there are

241

large amount of positively charged Lys residues on its surface. Previous simulations

242

and experiments demonstrated that the positively charged domain of BSA could bind

243

with negatively charged surfaces, such as silica and citrate-coated gold NPs.24-26,43

244

Under the experimental pH (5.5 ± 0.2), the positively charged Lys groups of BSA

245

could interact with the negatively charged groups of GO (i.e., carboxyl groups) via

246

strong electrostatic attraction, thus reducing the absolute value of GO zeta potential.

247

Previous studies indicated that hydrophobic interactions and π−π stacking contributed

248

to the adsorption of BSA on GO.31,42,44 The hydrophilic surface of BSA could also

249

interact with the functional groups of GO via hydrogen bonding, thus exposing the

250

hydrophobic core to the surface and leading to lower stability of GO.31,45

251

The FTIR results (Figure 2B) shed light on the nature of sorptive interactions

252

between GO and BSA. The FTIR of GO (200 mg/L) after sorption of BSA at low (50

253

mg C/L) and high (200 mg C/L) concentrations were conducted. The absorption bands

254

for proteins in the FTIR were commonly referred to as three specific amide regions.46

255

The FTIR spectra at 1655 and 1542 cm−1 are due to amide I band (C=O stretching

256

vibration) and amide II (mainly N−H bending vibration, coupled to C=O and C=C 12


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stretching), respectively. Amide III region (1200-1350 cm-1) has multiple

258

smallerbands, but is primarily composed of CH2 scissoring motion. The absorption at

259

1400 cm-1 revealed the side-chain COO- groups of BSA. After sorption of BSA, the

260

spectra of GO became similar to that of free BSA as the BSA concentration increased

261

from 50 to 200 mg C/L, indicating strong sorption and coverage of BSA on GO. The

262

absorbance intensity ratio of amide I and amide II was often used to qualitatively

263

assess orientation change of BSA.45,47,48 The ratio of free BSA was 1.17, and

264

increased to 1.51-1.75 after incubation of BSA (200 and 50 mg C/L) with GO (200

265

mg/L), suggesting that N−H group was involved in the adsorption of BSA on GO

266

surface.45,48 The CD spectra (Figure 2C) provided additional information for the

267

secondary change of protein. After incubation with GO, the α-helical (208 and 222 nm)

268

of BSA noticeably decreased while its shape and position remained constant. This

269

implied that GO interacted with the amino acid residues of BSA but did not

270

completely unfold the α-helical of BSA.31,49 The fluorescence intensity of BSA

271

(Figure 2D) decreased as the GO concentration increased, which also supported the

272

high affinity of GO to the aromatic amino acid residues of BSA, such as tryptophan.

273

Besides the oxygen-containing groups, GO also contained aromatic moieties, thus π−π

274

stacking interactions could contribute to the fluorescence quenching.

275

Individual Aggregation of GO or BSA in the Presence of Electrolytes

276

The GO stock colloid solution was stable for as long as three months without

277

significant change in Dh. The aggregation profiles of GO as functions of electrolyte

278

concentrations are presented in Figure S3. GO displayed similar aggregation 13



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behaviors in the three electrolytes. The aggregation rate increased with the increase of

280

the electrolyte concentration at relatively low concentration regime (i.e.,

Concentration Dependent Effects of Bovine Serum Albumin on

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