Impact of Proteins on Aggregation Kinetics and Adsorption Ability of

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Impact of Proteins on Aggregation Kinetics and Adsorption Ability of Hematite Nanoparticles in Aqueous Dispersions Anxu Sheng, Feng Liu, Nan Xie, and JUAN LIU Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b05298 • Publication Date (Web): 29 Jan 2016 Downloaded from http://pubs.acs.org on February 2, 2016

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Environmental Science & Technology

Impact of Proteins on Aggregation Kinetics and Adsorption Ability of Hematite Nanoparticles in Aqueous Dispersions

Anxu Sheng, Feng Liu, Nan Xie, Juan Liu*

School of Environmental Sciences and Engineering, Peking University, Beijing, China, 100871

*corresponding author: Juan Lu Address: School of Environmental Sciences and Engineering, Peking University, Beijing, China, 100871 Phone: +86-10-62754292-808 Email: [email protected]

Submitted to ENVIRONMENTAL SCIENCE & TECHNOLOGY

Keywords: nanoparticles (NPs), iron oxide, aggregation kinetics, protein, metal uptake

ACS Paragon Plus Environment


1

ABSTRACT:

2

The initial aggregation kinetics of hematite nanoparticles (NPs) that were conjugated with

3

two model globular proteins - cytochrome c from bovine heart (Cyt) and bovine serum albumin

4

(BSA) - were investigated over a range of monovalent (NaCl) and divalent (CaCl2) electrolyte

5

concentrations at pH 5.7 and 9. The aggregation behavior of Cyt-NP conjugates was similar to

6

that of bare hematite NPs, but the additional electrosteric repulsion increased the critical

7

coagulation concentration (CCC) values from 69 mM to 113 mM in NaCl at pH 5.7. An

8

unsaturated layer of BSA, a protein larger than Cyt, on hematite NPs resulted in fast aggregation

9

at low salt concentrations and pH 5.7, due to the strong attractive patch-charge interaction.

10

However, the BSA-NP conjugates could be stabilized simply by elevating salt concentrations,

11

owing to the screening of the attractive patch-charge force and the increasing contribution from

12

steric force. This study showed that the aggregation state of protein-conjugated NPs is proved to

13

be completely switchable via ionic strength, pH, protein size, and protein coverage. Macroscopic

14

Cu(II) sorption experiments further established that reducing aggregation of hematite NPs via

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tailoring ionic strength and protein conjugation could promote the metal uptake by hematite NPs

16

under harsh conditions.

17 18 19 20 21 22 23 24 25 26 27 28


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INTRODUCTION

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Naturally occurring and anthropogenic nanoparticles (NPs) have been widely found in

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aqueous environments1. The impacts of these NPs on natural and engineered waters, organisms

32

and ecosystems, as well as human health, are greatly dependent on their stability and aggregation

33

state2-4. Many efforts have been made to relate aggregation state of NPs to their ability to uptake

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heavy metals5, their reactivity and transport in natural waters6, as well as their fate and toxicity in

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biological systems2.

36

Naturally occurring dissolved organic matter (DOM) in the aqueous environment can

37

greatly change the aggregation state of NPs, leading to different behavior between field samples

38

and their laboratory analogs. Proteins are one of the major components of DOM in surface

39

waters7. Owing to their hydrophobic moieties that favor their adsorption onto the inorganic

40

colloidal surfaces, proteins have the potential to significantly alter the aggregation state of

41

colloids/nanoparticles in aqueous environment8. For example, in sulfate-reducing bacteria–

42

dominated biofilms collected from a Pb and Zn Mine, aggregates of microbially derived

43

extracellular proteins and biogenic zinc sulfide nanocrystals were found. This finding suggested

44

that aggregation induced by extracellular proteins greatly limited the dispersal and mobility of

45

NPs in natural environment9. On the other hand, some proteins were used as an efficient

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stabilizing agent to increase stability and bio-compatibility of engineered NPs for applications in

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environmental remediation10 and biotechnology11. With the growing use of NPs in biomedical

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and biotechnological applications, there is an increasing probability that NP-protein conjugates

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are released into the environment as consumer products12. When NPs enter physiological

50

environments, a protein coating, known as the “protein corona”, can form quickly on NP surfaces.



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In particular, the hard corona that is consisted of the proteins irreversibly bound to NP surfaces

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can evidently alter the aggregation state and surface properties of the NPs13. Therefore,

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understanding the aggregation of NP-protein conjugates in aqueous dispersions is crucial to

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elucidate the bioavailability, reactivity, mobility, and fate of NPs in aqueous environments.

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There have been many attempts to understand the impact of proteins on NP stability, but

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still no conclusive generalization has been drawn. Both positive and negative influences of

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proteins on NP stability have been reported. For example, the stabilization of iron oxide NPs by

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serum proteins was reported in biologically relevant conditions14, 15. On the other hand, Flynn et

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al. reported that BSA adsorption may either enhance or inhibit colloid mobility of iron-oxide

60

coated sand in saturated porous media, depending on protein coverage16. The discrepancy could

61

be attributed to the complex interactions between proteins and NPs. In addition to the van der

62

Waals attraction and the electrostatic repulsion, the interactions between protein-NP conjugates

63

may also include non-DLVO (Derjaguin–Landau–Verwey–Overbeek) interactions, such as

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depletion attraction, steric repulsion, or bridging flocculation17, etc. How proteins attach to

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NPsurfaces and the formation dynamics of the adsorbed protein layer can influence the

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non-DLVO forces18. Further complexity may arise from changeable surface properties of NPs

67

and potential protein deformation with solution chemistry19. So far most stability studies of

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protein-NP conjugates were conducted in biological media. There are still few studies on the

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stability of oxide NPs with proteins in aqueous dispersions under environmentally relevant

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conditions.

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In this study, Cytochrome c from bovine heart (Cyt) and Bovine serum albumin (BSA)

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were chosen as model proteins, primarily because they are globular proteins with high structural


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stability20. BSA is one of the most abundant proteins in serum and biological culture media. It is

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widely utilized in biotechnology due to its low cost, wide availability, and high

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structural/functional similarity to human serum albumin (HSA)21. Cyt was chosen to compare

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with BSA, in order to reveal how the inherent properties of proteins influence the aggregation of

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NP-protein conjugates. Compared to BSA, Cyt has the smaller molecular weight and the higher

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isoelectric point (Table S1). Moreover, cytochromes are redox metalloproteins, which can be

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used as redox catalysts or mediators for reductive dehalogenation reactions in environmental

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applications22. Besides, recent findings that outer membrane cytochromes assist the extracellular

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electron transfer between iron oxide and iron bacteria have encouraged the study on redox

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reactions between iron (oxyhydr)oxide NPs and purified cytochrome c of iron bacteria23-25. It is

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indispensable for studies in this area to understand the aggregation behavior of iron

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(oxyhydr)oxide NPs in the presence of cytochrome c.

85

Here, hematite NPs, one of the most abundant natural iron oxide minerals, were selected to

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study the impact of proteins on NP stability as a function of solution pH, ionic strength, cation

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valence, protein size, and molar ratios of proteins to NPs. The aggregation kinetics of hematite

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NPs with Cyt or BSA over a range of monovalent (NaCl) and divalent (CaCl2) electrolyte

89

concentrations were measured by time-resolved dynamic light scattering (DLS). Electrophoretic

90

mobility measurements were employed to investigate the change in surface potentials of hematite

91

NPs after the addition of proteins at varied pH and salt concentrations. In addition, Cu2+ ions

92

were used, as a surface probe species, to study how the adsorption ability of hematite NPs for

93

heavy metals changed, as the aggregation state of NPs was modulated by proteins.

94



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MATERIALS AND METHODS

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Hematite Synthesis and Characterization. Hematite NPs were synthesized by forced

97

hydrolysis of ferric nitrate solution according to the method reported by Schwertmann and

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Cornell26. More details on hematite synthesis are described in the supporting information

99

(section S1). Mass concentration of hematite in NP suspension was measured by acid digestion.

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0.1 mL of NP suspension was dissolved in 9.9 mL of 5 M HCl and continuously shaken

101

overnight. Then, 0.02 mL digested solution was diluted by using 4.98 mL 2% HNO3. Total Fe

102

was determined with an inductively coupled plasma optical emission spectrometry (Prodigy

103

High Dispersion ICP-OES, Teledyne Leeman Labs, Hudson, NH, USA). The experiments were

104

carried out in triplicates. Particle size and morphology of synthetic hematite NPs were measured

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on a field-emission transmission electron microscope (TEM, JEOL JEM-2100F) operated at

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200kV. TEM samples were prepared by placing a drop of diluted hematite suspension on a 400

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mesh copper grid coated with ultrathin carbon layer and then drying it in air. Particle size

108

distribution was obtained by analyzing more than 100 NPs on randomly selected areas on TEM

109

images using ImageJ software27. The TEM images of BSA-NP conjugates were taken by using

110

the same instrument under the similar conditions. Hematite NP suspension was sonicated for

111

about five minutes and added to 10 mM or 80 mM NaCl solution in the presence of 390 ng/cm2

112

BSA. After about an hour, TEM samples were prepared from the suspensions according to the

113

method mentioned above. The crystalline phase of hematite particles was characterized by

114

powder x-ray diffraction using a Rigaku D/MAX-2000 diffractometer with monochromatic

115

CuKα radiation (λ= 0.15406 nm) at a scan rate of 0.02 2θ∙s−1.

116

Time-Resolved Dynamic Light Scattering (TR-DLS). The initial aggregation rates and


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resulting attachment efficiencies were determined by TR-DLS. The change of hydrodynamic

118

diameters as a function of time was conducted on Zetasizer (Nano ZS90, Malvern, UK)

119

operating with a He−Ne laser at a wavelength of 633 nm and a scattering angle of 90º. Before

120

each measurement, hematite suspension was sonicated in a bath sonicator for five minutes. Then

121

it was immediately diluted in a predetermined volume of pH 5.7 or 9 stock solution with desired

122

concentrations of electrolyte and protein, respectively, in 10 mm diameter polystyrene cuvettes

123

(Sarstedt, Germany). Additional information on the preparation of stock solutions and hematite

124

suspensions is provided in Supporting Information Section 2. The concentration of hematite NPs

125

in all experiments was fixed at 16 mg/L as the optimum condition for DLS measurements.

126

Hydrodynamic diameter (Dh) was monitored every 5 seconds over a time period of 15−30

127

minutes. The z-average hydrodynamic radius of the aggregates was obtained from the

128

autocorrelation function using the “general purpose mode”. No buffer was used in aggregation

129

studies and adsorption experiments, because it may enhance the aggregation of NPs28. No pH

130

change was observed in all experiments at pH 5.7. In the case of pH 9, the pH values decreased

131

to 7.6 - 8.0 at the end of DLS measurements due to the effect of CO2 in air. Nevertheless, the pH

132

was still greater than or equal to pHiep. The small pH shift did not influence the comparison of

133

the aggregation behavior under this condition to that at pH 5.7.

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Zeta Potential Measurements. The zeta potentials of bare hematite NPs and protein-NP

135

conjugates as a function of NaCl concentrations at 25ºC were measured with a laser Doppler

136

velocimetry setup (Nano ZS90, Malvern, UK). At least 5-10 measurements (15–30 cycles per

137

measurement) were conducted for each sample. In the measurements of protein-NP conjugates,

138

16 mg/L hematite NPs was equilibrated with 8 mg/L proteins in pH 5.7 or 9 stock solutions for at



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least 15 minutes before measurements. The zeta potential ζ was obtained from the electrophoretic

140

mobility µe using the generalized Smoluchowski equation.

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BSA Adsorption on hematite NPs. Adsorption isotherms of BSA on hematite NPs were

142

measured in solution at pH 5.7. After sonication for 10 minutes, 200 mg/L of hematite NP

143

suspension was added to the pH 5.7 stock solution with increasing amounts of BSA (0 - 200

144

mg/L) in 60 mM NaCl solution or with the fixed amount of BSA (100 mg/L) in the solution with

145

a range of NaCl concentration (10 -100 mM). The samples were incubated in a shaker operating

146

at 150 rpm and 25 ºC for 1 h, and then centrifuged for 10 minutes at 4000 g. The supernatants

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were transferred into new centrifuge tubes and again centrifuged for 10 minutes at 4000 g to

148

remove residual hematite NPs29. It was checked that no measurable BSA was settled or absorbed

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on bottles in the absence of hematite NPs under these conditions. BSA concentrations in

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supernatants were determined using Bradford protein assay kit (Beyotime Institute of

151

Biotechnology). The absorbance of samples at λ = 595 nm was measured by BioTek SynergyHT

152

multi-detection microplate reader, which was used to calculate the equilibrium concentration of

153

BSA left in supernatant.

154 155 156

Aggregation Kinetics. The initial aggregation rate constant (k) of hematite colloids was determined by monitoring the increase in Dh(t) with time (t)30: ଵ

k∝ே ቀ బ

ௗ஽೓ (௧) ௗ௧

ቁ

௧→଴

(1)

157

where N0 is the initial particle concentration. The value of (dDh(t)/dt)t→0 was obtained through a

158

linear least-squares regression analysis of the initial increase in Dh up to the point where Dh(t)

159

reaches 1.50Dh,031. At low electrolyte concentrations or in the presence of NPs stabilization

160

induced by proteins, however, the hydrodynamic diameter failed to reach 1.5Dh,0. Under such


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conditions, the linear regression was performed even if it ends before 1.5Dh,0, but the y-intercept

162

of the fitted line did not exceed 3 nm in excess of Dh,0 for all cases32.

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The aggregation attachment efficiency (α), i.e. the probability that two particles attach, is

164

used to quantify the initial aggregation kinetics of hematite colloids. It is calculated by

165

normalizing the k obtained in the solution of interest to the diffusion-limited aggregation rate

166

constant kfast determined under favorable aggregation30-33: α=

167

௞

௞೑ೌೞ೟

=

భ ೏ವ೓ (೟) ቀ ቁ ಿబ ೏೟ ೟→బ ೏ವ೓ (೟) భ ቀ ቁ (ಿబ )೑ೌೞ೟ ೏೟ ೟→బ,೑ೌೞ೟

(2)

168

To calculate α in the presence of proteins, (dDh(t)/dt)t→0,fast is obtained for the same type of

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hematite colloids in the absence of proteins, but in the same electrolyte under favorable

170

aggregation conditions31,

171

aggregation processes change from reaction-limited cluster aggregation (RLCA) to

172

diffusion-limited cluster aggregation (DLCA), were derived from the intersection of extrapolated

173

lines through both regimes.

33

. The critical coagulation concentrations (CCC), above which

174

Copper (II) adsorption. Cupper (II) adsorption on bare hematite NPs and BSA-NP

175

conjugates in solutions with different ionic strength was studied. The protein solution was

176

adjusted to the desired ionic strength (10mM, 40mM, or 100mM NaCl) at pH 5.7. The hematite

177

NP suspension was sonicated for ten minutes and then added to the protein solutions. The final

178

NP concentration was fixed at 80 mg/L and BSA concentration was 40 mg/L. Before spiking the

179

CuCl2 stock solution, the NP suspension with or without proteins was shaken for 30 minutes at

180

120 rpm. The initial concentration of Cu(II) in all experiments was fixed at 0.045mM. After

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shaking at 120 rpm for 4 hours at 25ºC, the copper-sorbed NPs were separated from the

182

suspension by 0.22 µm syringe filters (Millipore PES Membrane). The filtrates were then



183

acidified with 2% HNO3 for ICP-OES (Prodigy High Dispersion ICP-OES, Teledyne Leeman

184

Labs, Hudson, NH, USA) analysis. Results of control experiments showed that no measurable

185

amount of Cu(II) was adsorbed on the syringe filters or in the BSA solution without hematite

186

NPs. Additional control experiments were performed using Amicon Ultra-15 centrifugal filter

187

units (MWCO 3 kDa, Millipore) and 0.22 µm syringe filters, respectively, to filter NPs after

188

adsorption. The similar results indicated that 0.22 µm syringe filters were sufficient to remove

189

hematite NPs in these experiments.

190 191

RESULTS AND DISCUSSION:

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Characteristics of Hematite NPs. The powder X-ray diffraction (XRD) pattern of the

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synthetic nanoparticles (Figure S1) revealed that only hematite phase was present. The average

194

diameter of the particles measured by TEM was 9 ± 2 nm (Figure S2). The hydrodynamic

195

diameter of hematite NPs in aquesou solution (pH = 5.7, no NaCl addition) measured by DLS

196

was 125 ± 18 nm (Figure S2), indicating NPs tended to aggregate at this pH. The properties of

197

the synthetic hematite NPs and proteins used in this study are summarized in Table S1.

198

Effect of Ionic Strength on Stability of NP-Protein Conjugates. Apparent attachment

199

efficiencies of bare hematite NPs as a function of NaCl concentrations at pH 5.7 are shown in

200

Fig 1A. The corresponding aggregation profiles are presented in Figure S3A. The aggregation

201

behavior of bare hematite NPs in aqueous dispersions with NaCl (Fig 1A) and CaCl2 (Fig S4A)

202

at pH 5.7 were consistent with the prediction of the classic DLVO theory. The attachment

203

efficiency (α) of hematite nanoparticles increased with the increasing electrolyte concentrations,

204

and approached to 1 when the electrolyte concentration reached CCC. The faster aggregation


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rates at elevated electrolyte concentrations are attributed to the compression of the electric

206

double layer (EDL) and the decrease of surface potential. It was confirmed by the measured zeta

207

potentials (ζ potentials) of hematite NPs, which decreased with increasing NaCl concentrations

208

(Figure 2A). Since the CCC represents the minimum amount of electrolyte needed to eliminate

209

the repulsive energy barrier for rapid aggregation, it was used to assess the colloidal stability of

210

hematite NPs under various experimental conditions34. The CCCs of hematite NPs in the

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presence of NaCl and CaCl2 at pH 5.7 were 69 mM and 10 mM, respectively. It is consistent

212

with the prediction of the Schulze-Hardy Rule that the CCC is inversely proportional to the

213

valence of cations4.

214

The attachment efficiencies of 16 mg/L hematite NPs in the presence of 8 mg/L Cyt with

215

increasing NaCl concentrations at pH 5.7 are presented in Figure 1A. It shows that α of NP-Cyt

216

conjugates increased as NaCl (Fig 1A) or CaCl2 (Fig S4A) concentrations increased. The trend is

217

similar to that of bare NPs, suggesting the principal interactions in this case were governed by

218

DLVO forces. However, the presence of Cyt increased the CCCs to 113 mM in NaCl and 48 mM

219

in CaCl2, respectively. It is probably due to the additional contribution from repulsive

220

electrosteric force originating from Cyt adsorption. The isoelectric point of Cyt is 10.37 (Table

221

S1). At pH 5.7, both hematite and Cyt possessed positively charged surfaces, but a certain level

222

of Cyt adsorption on hematite surface might occur due to the inhomogenous charge distribution

223

of Cyt or van der Waals attractive forces35. It is confirmed by the higher zeta potentials of

224

Cyt-NP conjugates (Fig 2A). Moreover, ζ potentials of Cyt-hematite conjugates decreased with

225

the increase of NaCl concentrations, suggesting the weakening of the electrosteric forces at

226

elevated salt concentrations.



227

On the contrary, increasing NaCl (Fig 1B) or CaCl2 concentrations (Fig S4B) decreased

228

attachment efficiencies of 16 mg/L hematite NPs in the presence of 8 mg/L BSA at pH 5.7. At

229

low salt concentration ([NaCl] < ~ 40mM, or [CaCl2] < ~8 mM), BSA induced fast aggregation

230

of hematite NPs and resulted in the higher attachment efficiencies than bare NPs. Nevertheless,

231

hematite NPs were totally stabilized (α → 0) by BSA at [NaCl] > 48 mM or [CaCl2] > 19 mM.

232

The deviation from DLVO theory is presumably attributed to non-DLVO forces originated from

233

BSA that adsorbed on NP surfaces. The pHiep of BSA is 4.76 in 1mM NaCl and 4.51 in 0.1M

234

NaCl36, so at pH 5.7 the adsorption of negatively charged BSA onto the positively charged

235

hematite surface is favorable. The negative zeta potentials of hematite-BSA conjugates at pH 5.7

236

(Figure 2A) also suggested the adsorption of BSA on hematite NPs. The charge reversal is a

237

most characteristic phenomenon, when charged polymers adsorb to oppositely charged

238

substrates37.

239

The BSA induced aggregation of hematite NPs at low salt concentration can be interpreted

240

with the attractive patch-charge interaction37. The adsorption of BSA on metal oxide surface

241

starts from the formation of a side-on monolayer (the minor axes of BSA perpendicular to the

242

oxide surface), and then proceeds to the generation of dimers as a result of protein-protein

243

interaction with the increase of BSA concentration29. The calculated amount of absorbed BSA

244

for a side-on monolayer on NPs is in the range of 223 ng/cm2 to 365 ng/cm2.29 In this study, the

245

initial concentrations of BSA and hematite NPs were 8 mg/L and 16 mg/L, respectively. Based

246

on the geometric specific surface area of 9 nm spherical hematite NPs (127 m2/g), the amount of

247

added BSA normalized by the NP surface area was 390 ng/cm2. Adsorption isotherm of BSA on

248

hematite surfaces at pH 5.7 as a function of BSA added was shown in Figure 3. When the


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amount of added BSA was 390 ng/cm2, the corresponding amount of BSA absorbed was

250

approximately 105 ng/cm2. Thus, under the conditions of the aggregation study, the amount of

251

adsorbed BSA on hematite NPs could be not enough to form a side-on monolayer (223 ng/cm2).

252

Moreover, disproportion and polarization of BSA could also lead to BSA-poor and BSA-rich

253

regions on NP surfaces 38. Besides, the adsorption of Cu2+ on BSA-NP aggregates (Figure 4) also

254

implied that BSA did not form a monolayer on NPs and a part of NP surface was exposed for

255

Cu2+ adsorption. Therefore, the negatively charged patches of BSA were likely to form on the

256

positively charged hematite surfaces under the conditions of the present study.

257

The morphology of BSA can be approximated by an equilateral triangular prism with sides

258

about 9 nm and a height of about 5.5 nm, so the thickness of a side-on monolayer of BSA is

259

about 2.5-5.5 nm39. When [NaCl] = 10mM, the Debye-Hüchel length (κ-1) is estimated to be 3

260

nm according to the following equation:

261

ఌఌ ௞ ்

ಳ ߢ ିଵ = ටଶேబ ூ௘ మ ಲ

(3)

262

where ε0 is the permittivity of vacuum, ε is the dielectric constant of water, T is temperature, NA

263

is Avogadro’s number, kB is the Boltzmann constant, e is the elementary charge, and I is the ionic

264

strength. Therefore, at low salt concentration ([NaCl] < ~ 40mM, or [CaCl2] < ~8 mM), the

265

thickness of the adsorbed BSA layer was comparable to the Debye length. The BSA-rich regime

266

and hematite surface/the BSA-poor region possess opposite charges at pH 5.7, leading to a lateral

267

heterogeneity in surface charge on the hematite-BSA conjugates. When two BSA-NP conjugates

268

approach each other, attractive electrostatic interaction between the BSA-poor region of one

269

conjugate and the BSA-rich domain of the other could develop38, resulting in the patch-charge

270

attractive interaction. This attractive non-DLVO force is substantially stronger than the van der



271

Waals force37, especially for proteins with high molecular mass and at low salt levels, so

272

hematite-BSA conjugates aggregated more readily than bare hematite NPs at low ionic strength.

273

At high salt levels, the patch-charge attractive force is screened as a regular EDL force37, so the

274

steric repulsive force caused by BSA on NP surfaces stabilized NPs in aqueous dispersions.

275

Increasing NaCl concentration did not promote the adsorption of BSA on hematite NPs (Figure

276

S5). Therefore, the stabilization of BSA-NP conjugates at high ionic strength could not be

277

attributed to the stronger steric force resulting from a larger amount of BSA adsorbed on NP

278

surface. Depletion and bridging interactions may also lead to NP destabilization. However, the

279

bridging processes are rare at low salt levels37, and depletion interaction become important only

280

at high polyelectrolyte concentrations. Considering the low salt and protein concentrations used

281

in this study, the observed fast aggregation of BSA-NP conjugates could not be attributed to

282

these two kinds of non-DLVO interactions.

283

The size of surface heterogeneities is the key factor for the patch-charge attractive force37.

284

With decreasing molecular mass, the size of charged patches decreases and the additional

285

non-DLVO force tends to disappear. That is the reason why the patch-charge attraction was not

286

observed in the aggregation studies of hematite NPs in the presence of alginate or humic acid at

287

near neutral pHs32,

288

conditions though.

40

. HA/alginate and hematite NPs were oppositely charged under the

289

Effect of pH on hematite-protein aggregation. The stability of hematite NPs with BSA or

290

Cyt at pH 9 was shown in Figure 1 C. Bare hematite NPs were in the DLCA regime over the

291

entire range of NaCl concentrations (Fig 1C). It is related to the low ζ potentials of hematite

292

NPs at pH 9 (Figure 2B). The Nernst equation indicates that the surface potential (E) of a solid is


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293

directly proportional to the difference between the solution pH and its pHiep at room temperature4.

294

As pH increased from 5.7 to 9, it became closer to the pHiep of hematite, resulting the decrease of

295

ζ potentials.

296

The interaction energy of electrostatic repulsion between two equal-sized NPs is proportional

297

to E2. Consequently, the repulsive energy barrier dramatically decreased at pH 9 due to the

298

decrease of E, resulting in the fast aggregation.

299

At pH 9, the addition of Cyt hindered the aggregation of hematite NPs at low salt

300

concentrations. The trend of α for Cyt-NP conjugates as a function of salt concentrations at pH 9

301

was similar to that at pH 5.7, but the CCCs at pH 9 was obviously smaller than the value at pH

302

5.7(Figure 1). It could be related to the lower zeta potentials of Cyt-NPs at pH 9 (Figure 2B).

303

Considering pHiep of Cyt is 10.37 (Table S1), the surface potential of NP-Cyt conjugates at pH 9

304

is much less than that at pH 5.7. Thus, the contribution from electrosteric repulsion by adsorbed

305

Cyt at pH 9 was smaller than at pH 5.7. It implies that the stabilization of NPs by proteins is

306

more efficient at pH far from pHiep of proteins. In addition, it is worth to mention that the

307

maximum attachment efficiencies were less than 1 in the case with Cyt at both pH 5.7 and pH 9,

308

which suggested the contribution of steric stabilization by adsorbed Cyt41, 42.

309

Even though the aggregation of bare hematite NPs is more favorable at pH 9, BSA-hematite

310

conjugates presented a higher stability at pH 9 than at pH 5.7. As shown in Figure 1C, the

311

attachment efficiencies of hematite NPs with 390 ng/cm2 of BSA were around zero at pH 9 over

312

the entire range of salt concentrations. The addition of BSA decreased the zeta potentials of

313

hematite NPs at pH 9 down to -15 mV (Fig 2B), indicating the adsorption of BSA on hematite

314

NPs. The higher negative charge at pH 9 promoted electrosteric stabilization by adsorbed BSA.



315

The attractive patch-charge interactions were not observed at pH 9, probably because the surface

316

potential of hematite NPs was close to zero. It is substantial only when the patches and the

317

substrate are oppositely charged. The lack of this attractive interaction at pH 9 also facilitated the

318

stabilization of NPs by BSA.

319

Aggregation of NPs through cation bridging, especially with Ca2+, has been widely

320

observed for NPs with fulvic acids, humic acids, polysaccharides, and any other DOMs7.

321

Moreover, Ca-bridging is more sufficient at alkaline pH, owning to the lack of the competitive

322

adsorption of protons7. However, the aggregation behavior of hematite NPs with proteins in

323

CaCl2 solution (Fig S4) at pH 5.7 or 9 was similar to that in NaCl solution (Figure 1). It indicated

324

that CaCl2, over the concentration range studied (up to 150 mM), did not lead to inter-particle

325

bridging of bare hematite NPs or protein-hematite conjugates.

326

Effect of Protein Size and Concentration on Stability of Hematite NPs. The attachment

327

efficiencies of protein-NP conjugates as a function of protein concentration are presented in

328

Figure 5. When the NP concentration was fixed, increasing protein concentration led to the

329

decreasing α of NPs over the range of 0 - 1200 ng/cm2. Protein surface coverage and the

330

thickness of protein coatings are positively related to the initial protein concentration43. When the

331

NPs surface is totally coated by thick protein layers, the attractive patch-charge interactions

332

could be eliminated, and the steric repulsion between adsorbed proteins could totally stabilize

333

NPs. However, in some cases, high protein surface coverage or excess proteins in dispersions

334

might also drive aggregation of NPs by a bridging mechanism43 or depletion attraction18. In this

335

study, relatively low concentrations of proteins and NPs were used in order to study the

336

aggregation behavior of protein-NP conjugates under environmentally relevant conditions.


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337

As shown in Figure 5, about 32 mg/L Cyt was needed to stabilize 16 mg/L hematite NPs in

338

60 mM (> CCC) NaCl at pH 5.7. Nevertheless, only about 9 mg/L BSA was sufficient to

339

stabilize the same amount of NPs. At pH 5.7, the surface potential of Cyt-NP conjugates was

340

higher than that of BSA-NP conjugates (Figure 2A), but BSA presented the higher ability to

341

stabilize NPs. It implied that steric repulsive force is more efficient than electrostatic repulsive

342

force in stabilizing NPs. The size of BSA is 5.5 × 5.5 × 9 nm29, while that of Cyt is 3 × 3.4 × 3.4

343

nm44. Thus, a higher molar ratio of proteins to NPs is needed for Cyt to form a monolayer on NP

344

surface. The thickness of adsorbed protein layer (δ) is proportional to the size of proteins, so the

345

BSA monolayer is thicker than the Cyt monolayer. Under the conditions studied, NPs were

346

stabilized at high salt concentrations mainly by steric repulsive force. Compared to Cyt, BSA is

347

a better stabilizer for hematite NPs, owing to the fewer proteins needed for completely covering

348

NP surfaces and the formation of sufficiently thick coating to maintain a high steric free energy

349

of interaction at high salt concentrations.

350

Effect of Aggregation State on Metal Uptake. In order to investigate the effects of protein

351

adsorption and NP aggregation on heavy metal ion sequestration, copper uptake experiments

352

were performed on bare hematite NPs and BSA-hematite conjugates over a range of NaCl

353

concentrations at pH 5.7 (Figure 4). As NaCl concentration increased from 10 mM to 100 mM,

354

the percentage of Cu2+ adsorbed on bare hematite surfaces decreased from 72.5 ± 4 % to 59.1

355

± 0.5%. It indicates that the aggregation of hematite NPs induced at elevated salt concentrations

356

could evidently reduce copper uptake, probably due to the decreasing amount of reactive surface

357

sites available for Cu2+ adsorption. Previous studies showed that characteristics of iron

358

oxyhydroxide NP aggregates, such as morphological, structural, surface charge, and surface area

359

differences have a noticeable effect on copper retention5, 45. In the presence of 390 ng/cm2 BSA,

360

the percentage of Cu adsorbed on NPs increased from 58.0 ± 2.4 % to 71.2 ± 3.3%, as NaCl

361

concentration increased from 10 mM to 100 mM. Control experiments showed that no



362

measurable Cu adsorption was observed in the BSA solution. The higher capacity of NPs for Cu

363

adsorption at elevated salt concentrations might be attributed to inhibition of NP aggregation by

364

BSA. In the experiments of both bare hematite and BSA-NP conjugates, the trends of capacity

365

for Cu adsorption as a function of salt concentrations were consistent with the changes of

366

aggregation state. It implies the importance of aggregation state in copper uptake on hematite

367

NPs. The NP aggregation was inhibited by the addition of BSA at high salt concentration, which

368

could increase the surface area for Cu adsorption. The decrease of BSA adsorption on NPs with

369

the increase of salt concentration (Figure S5) could also contribute to the higher capacity for Cu

370

adsorption at higher NaCl concentration. However, the TEM images of BSA-NP conjugates

371

(Figure S6) showed that the aggregates of BSA-NP conjugates in 10 mM NaCl solution

372

obviously had a more compact structure than those in 80 mM NaCl solution. Some internal

373

surface area became accessible for Cu adsorption, when the aggregate structure turned to an open

374

and loose structure. It could be the main reason for the higher adsorption capacity of BSA-NP

375

conjugates at the higher ionic strength.

376

Environmental Implications. This study showed the different impact of two model

377

globular proteins on NP aggregation in aqueous dispersions under environmentally relevant

378

conditions. Results from this study suggested that the aggregation state of NPs in the presence of

379

proteins depended on both solution properties, like pH and ionic strength, and protein properties,

380

such as molecular weight, pHiep, protein concentration, etc. Compared to Cyt, BSA with the

381

larger molecular weight were more efficient to stabilize NPs, considering the thicker protein

382

layers could be formed by the less amount of BSA. On the other hand, the attractive patch-charge

383

interaction could induce fast NP aggregation, when BSA formed a laterally heterogeneous layer

384

on hematite NPs. This non-DLVO force is substantially stronger than the van der Waals force37,

385

and especially important for proteins with high molecular mass at low salt levels. Under certain

386

conditions, other non-DLVO forces, including depletion and bridging forces, could become

387

important to the aggregation of NP-protein conjugates. Further work is needed to elucidate the


Page 18 of 29

Page 19 of 29


388

precise conditions where these non-DLVO forces play a key role in determining NP aggregation.

389

Proteins are important biomacromolecules in all organisms and also widespread in surface

390

waters. The adsorption of proteins on NP can induce or inhibit NP aggregation, depending on

391

both solution conditions and protein properties. The correlation between NP aggregation and

392

capacity for metal uptake observed in this study suggested that protein-NP conjugates could be

393

efficient materials for metal removal from wastewater under harsh conditions. Understanding the

394

influence of proteins on NP aggregation, as shown in this study, is essential to evaluate the

395

reactivity and toxicity of NPs in biogeochemical processes, and to assess the influence of NPs on

396

the fate and transport of pollutants in aqueous environments, as well as to assist the

397

functionalization of NPs for a large variety of environmental and biotechnological applications.

398 399

ASSOCIATED CONTENT

400

Supporting Information. Additional figures and details for Materials and Methods and Results

401

and Discussion are presented. This material is available free of charge via the Internet at

402

http://pubs.acs.org.

403 404

AUTHOR INFORMATION

405

Corresponding Author

406

*Phone:

407

Environmental Sciences and Engineering, Peking University, Beijing, China, 100871

(+86)010-62754292-808;

email:

[email protected];

408 409

ACKNOWLEDGEMENTS


address:

School

of


410

This work was financially supported by National Basic Research Program of China (973

411

Program, 2014CB846001) and National Natural Science Foundation of China (41472306).

412


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413 414 415 416 417

Figure 1. Comparison of attachment efficiencies of 16mg/L bare hematite NPs and hematite NPs in the presence of (A) 8 mg/L Cyt at pH 5.7, (B) 8 mg/L BSA at pH 5.7, and (C) 8 mg/L BSA or Cyt at pH 9 as a function of NaCl concentration.



418 419 420 421 422 423

Figure 2. Zeta potential of bare hematite NPs and protein-conjugated NPs as a function of NaCl concentrations at pH 5.7(A) and pH 9 (B). Each data point represents the mean of at least 5 measurements of samples at each electrolyte concentration, and the error bars represent standard deviations.


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424 425 426 427 428 429

Figure 3 BSA adsorption isotherms on hematite NPs plotted as a function of BSA added after 1 hour of adsorption time in 60 mM NaCl solution at pH 5.7. The dotted horizontal line indicates the calculated amount of BSA needed for one side-on monolayer to cover the surface area of NPs.



430 431 432 433 434 435

Figure 4. The uptake of aqueous divalent copper ions by bare hematite NPs (cross-hatching) and hematite-BSA conjugates (diagonal) in 10, 40, and 100 mM NaCl solutions at pH 5.7 after four-hour adsorption experiments.


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436 437 438 439 440 441 442

Figure 5. Attachment efficiencies of NP-protein conjugates in 60 mM NaCl solutions at pH 5.7 as a function of the concentration of Cyt and BSA, respectively, at 25ºC. The concentration of added NPs was 16 mg/L for all experiments.



443

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