Concentration Dependent Effects of Bovine Serum Albumin on

Jun 12, 2018 - Here, we took NaCl at 120 mM as an example. The aggregation of GO in 120 mM NaCl electrolyte solution fell into three regimes. Speciall...
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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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Concentration dependent effects of bovine serum albumin on

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graphene oxide colloidal stability in aquatic environment

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

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Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of

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Education), Tianjin Key Laboratory of Environmental Remediation and Pollution

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

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Tianjin 300350, P. R. China

9 10 11 12 13 14 15



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

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

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ABSTRACT

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

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

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

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

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

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

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

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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

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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.

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

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

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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.

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

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(BSA), electrostatic interactions, (X)DLVO

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Introduction

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

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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

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

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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

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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

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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

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

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

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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,

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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

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

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that

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mercaptoundecyltrimethylammonium bromide coated Au NPs while stabilized the

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

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

BSA

at

2

nM

could

induce

the

aggregation

of

cationic

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

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concentrations on GO colloidal stability through time-resolved dynamic light

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

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characterization techniques were performed to disclose the interaction mechanisms

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

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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,

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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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

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

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

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

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

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functional groups on GO surface were obtained by Fourier transform infrared

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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

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

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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

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

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

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

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

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

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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

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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

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

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

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

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

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interference.30,31

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

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

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

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k∝

1 N0



dDh t dt



t→0



(1)

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

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

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

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

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

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

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constant kfast.22,33

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α=

1

= W k

k fast

dD t

=

 dth  t→0



(2)

dDh t  dt t→0, fast

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The critical coagulation concentration (CCC) was obtained by intersecting

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extrapolated lines through the RLCA and DLCA regimes.

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BSA adsorption on GO

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

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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

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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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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interactions between GO and BSA.

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RESULTS AND DISCUSSION

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Characterization of GO

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

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

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

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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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charged.

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Adsorption of BSA on GO and interactions between them

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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stacking interactions could contribute to the fluorescence quenching.

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Individual Aggregation of GO or BSA in the Presence of Electrolytes

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

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

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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

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