Aggregation Kinetics of Hematite Particles in the Presence of Outer

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Aggregation Kinetics of Hematite Particles in the Presence of Outer Membrane Cytochrome OmcA of Shewanella oneidenesis MR-1 Anxu Sheng, Feng Liu, Liang Shi, and JUAN LIU Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 20 Sep 2016 Downloaded from http://pubs.acs.org on September 20, 2016

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Environmental Science & Technology

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Aggregation Kinetics of Hematite Particles in the Presence of Outer

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Membrane Cytochrome OmcA of Shewanella oneidenesis MR-1

3 4

Anxu Shenga,†, Feng Liua,†, Liang Shib, c, Juan Liua,*

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a

9

b

College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China Department of Biological Sciences and Technology, School of Environmental Studies, China

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University of Geoscience in Wuhan, Hubei, 430074, China

11

c

Pacific Northwest National Laboratory, Richland, WA 99352, USA

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To be submitted to Environmental Science & Technology

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* Corresponding author.

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Address: College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China

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Phone: +86-10-62754292-808

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Email: [email protected]

22 23 24 25 26 27 28 29 30 31 32

†

Both authors contributed equally to this work

ACS Paragon Plus Environment


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Abstract

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The aggregation behavior of 9, 36, and 112 nm hematite particles were studied, respectively, in the

35

presence of OmcA, a bacterial extracellular protein, in aqueous dispersions at pH 5.7 through

36

time-resolved dynamic light scattering, electrophoretic mobility, and circular dichroism spectra. At low salt

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concentration, the attachment efficiencies of hematite particles in all sizes first increased, then decreased,

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and finally remained stable with the increase of OmcA concentration, indicating the dominant interparticle

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interaction changed along with the increase in the protein-to-particle ratio. Nevertheless, at high salt

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concentration, the attachment efficiencies of all hematite samples gradually decreased with increasing

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OmcA concentration, which can be attributed to the increasing steric force. Additionally, the aggregation

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behavior of OmcA-hematite conjugates was more correlated to total particle-surface area than primary

43

particle size. It was further established that OmcA could stabilize hematite nanoparticles more efficiently

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than bovine serum albumin (BSA), a model plasma protein, due to the higher affinity of OmcA to hematite

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surface. This study highlighted the effects of particle properties, solution conditions, and protein properties

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on the complicated aggregation behavior of protein-nanoparticle conjugates in aqueous environments.

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INTRODUCTION

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Nanoparticles (NPs) are ubiquitous in aqueous environments due to the widespread applications of

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emerging nanotechnologies or a variety of human activities and natural processes.1 The stability or

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aggregation state of NPs is a key factor that influences their mobility, fate, reactivity, nanotoxicity, and

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bioavailability in aquatic systems.2-4 As one of the major components of dissolved organic matter (DOM) in

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surface waters and the major biomacromolecules in extracellular polymeric substances (EPS),5 proteins

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have a high binding affinity to inorganic NPs and tend to immediately adsorb onto the surface of NPs,6

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which potentially leads to obvious changes in the interfacial behavior and aggregation state of NPs.7, 8

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Particularly, the proteins that are irreversibly bound to NPs comprise the “hard corona”, which is likely to

58

have a lasting effect on the stability of NPs owing to its long lifetime.9 Therefore, a better understanding of

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the aggregation behavior of NP-protein conjugates may improve our ability to predict the environmental

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reactivity and fate of NPs in natural waters, and also is indispensable for holistic evaluations of the role of

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proteins in bio-nano interactions.

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Many efforts have been devoted to elucidating the influence of proteins on NP aggregation, but still no

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conclusive generalization has been drawn, primarily because of the complexity of interactions involved.10, 11

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In addition to the van der Waals attraction and the electrostatic repulsion, non-DLVO

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(Derjaguin-Landau-Verwey-Overbeek) interactions, such as depletion attraction, steric repulsion, bridging

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flocculation, patch-charge attraction, etc., can also influence interfacial forces between protein-NP

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conjugates.12 The ability of proteins to stabilize or destabilize NPs suspensions has been shown to strongly

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depend on the concentration and physicochemical properties of proteins, the inherent properties of NPs,

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including size, shape, surface charge, and composition, etc., as well as solution conditions, such as pH, ionic

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strength, etc.8, 13 Due to the complexity of this topic, huge datasets including many different combinations of

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proteins, NPs, and solution conditions are required to gain sufficiently broad knowledge.14

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most previous studies were done with model plasma proteins, such as bovine serum albumin (BSA),

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immunoglobulin, fibrinogen, transferrin, and so on. Very few studies have addressed the impact of the

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proteins secreted from environmental bacteria on NP aggregation in aquatic environments. Moreau et al.7

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reported that extracellular proteins derived from sulfate-reducing bacteria induced the aggregation of

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biogenic ZnS NPs in the biofilms collected from lead–zinc mine waters. It implies that extracellular proteins

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may have a great impact on the stability and mobility of NPs in aqueous environments. More studies are


15

Moreover,


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needed on a larger scale to understand how extracellular proteins affect the aggregation state of NPs under

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environmentally relevant conditions.

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In this study, the outer membrane (OM) c-type cytochrome of Shewanella oneidenesis MR-1(MR-1),

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OmcA, was selected as a typical protein derived from environmental bacteria for several reasons. MR-1, as

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a model dissimilatory metal-reducing bacterium (DMRB), has attracted extensive interest, owning to its

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diverse metabolic capabilities and considerable potential for the bioremediation of radionuclide and metal

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contaminants.16-19 OmcA locates both on the OM and in the EPS of MR-1,20 serving as a terminal

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reductase of iron oxide minerals.21-23 The aggregation state of iron oxide particles conjugated to OmcA is

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key to diverse microbe-mineral interactions, such as extracellular electron transfer and particle mobility

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inside biofilms. In addition, OmcA can preferentially bind to hematite surface via a binding motif

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(Ser/Thr-Pro-Ser/Thr),24-26 so it has the potential to significantly influence the interfacial forces between

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hematite particles.

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The aggregation behavior of colloids has been extensively predicted by the classical DLVO theory, but

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it may not be applicable when the particle size is small enough.2 Electronic structure, surface charge behavior,

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and surface reactivity of NPs are likely to change with the decrease of particle size.27, 28 Correspondingly, the

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interparticle forces between NPs can be altered as a function of particle size and change the aggregation

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behavior.29 Besides, the adsorption behavior of proteins on NPs and the conformation of proteins adsorbed

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on NPs can also be affected by particle size.8 Therefore, the effect of primary particle size on aggregation

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state is probably even more prominent when proteins are present. To the best of our knowledge, this is the

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first study to systematically investigate particle size effect on the aggregation of NPs conjugated to proteins

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of environmental bacteria in aqueous dispersions.

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Our previous study presented the aggregation kinetics of 9 nm hematite NPs in the presence of the

100

model globular proteins, Cytochrome c from bovine heart (Cyt) or BSA, over a range of salt

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concentrations at pH 5.7 and 9, respectively.30 Based on the previous study, we further investigated the

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aggregation behavior of hematite particles in three different sizes conjugated with the proteins from a

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model environmental microbe. The object of this study is to quantify and compare the aggregation kinetics

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and colloidal stability of 9, 36, and 112 nm hematite particles as a function of ionic strength,

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protein-to-particle ratio, as well as protein concentration and binding affinity to hematite. Time-resolved

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dynamic light scattering (TR-DLS) was used to monitor aggregation behavior of hematite particles under


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different conditions. The obtained aggregation kinetics results, along with surface potentials measured by

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electrophoretic mobility, adsorption isotherms of proteins on particles, and the conformation of proteins

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adsorbed on particles obtained by the circular dichroism (CD) spectra, were used to elucidate the

110

aggregation mechanisms of protein-hematite conjugates. The effects of particle properties (primary particle

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size and surface area), solution conditions (ionic strength), and protein properties (concentration and

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affinity to particles) on the complicated aggregation behavior of protein-hematite conjugates were

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highlighted in this study.

114

MATERIALS AND METHODS

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Synthesis and Characterization of Hematite Particles. Hematite particles with three different sizes

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were synthesized by forced hydrolysis of Fe(III) salt solution according to the methods reported by

117

Schwertmann and Cornell.31 More details on the synthesis procedures are described in the Supporting

118

Information (section S1).

119

The particle concentration of hematite suspensions was determined by acid digestion. After 10 minute

120

sonication, 0.1 mL of particle suspension was added to 9.9 mL of 5 M HCl. The mixture was shaken

121

overnight. Then, 0.02 mL digested solution was diluted by 2% HNO3 for the determination of total Fe

122

concentration by inductively coupled plasma optical emission spectrometry (ICP-OES, Teledyne Leeman

123

Labs, Hudson, NH, USA). The mass concentration of hematite (Fe2O3) particles in suspensions was

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converted from the measured Fe concentration in the digested solution.32 The mean particle concentration

125

in each sample was determined from triplicate experiments. The shape and primary particle diameter of

126

synthetic hematite particles were examined with a field-emission transmission electron microscope (TEM,

127

JEOL JEM-2100F) operated at 200kV. TEM samples were prepared by depositing one drop of diluted

128

hematite suspension onto an ultrathin carbon coated copper grid (400 mesh) and then drying it in air. ImageJ

129

software (US National Institutes of Health) was used to determine the distribution of particle sizes by

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measuring more than 100 particles that were randomly selected in TEM images.33 The crystalline phase of

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synthetic particles was characterized by powder X-ray diffraction (XRD) using a Rigaku D/MAX-2000

132

diffractometer with monochromatic CuKα radiation (λ= 0.15406 nm) at a scan rate of 0.02 2θ·s-1. For XRD

133

measurements, concentrated suspensions were dried in air at ~ 40ºC and then loaded onto glass slides.

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Protein Solutions. OmcA was expressed and purified from MR-1 strain LS331 with pLS147 as

135

described previously.23 The purity of isolated OmcA in the stock solution was confirmed by sodium



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dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and UV-Visible absorption spectroscopy

137

(UV-1800, Shimadzu, Japan). The aggregation behavior of OmcA was compared to that of BSA under the

138

same conditions. BSA lyophilized powder (≥ 96%) was purchased from Sigma-Aldrich and used as

139

received. The BSA stock solution was prepared by dissolving a certain amount of BSA lyophilized powder

140

in Milli-Q water and stored in 4ºC. The concentration of proteins in the stock solutions was determined by

141

UV–Vis spectroscopy, according to the absorbance at λ = 410nm (extinction coefficient EOmcA= 934 mM-1

142

cm-1) for OmcA and at λ = 280 nm (EBSA = 43.8 mM-1 cm-1) for BSA , respectively.34

143

Aggregation Kinetics. The aggregation kinetics of hematite particles with/without proteins was

144

quantified by using time-resolved dynamic light scattering (TR-DLS). The change of hydrodynamic

145

diameter (Dh) as a function of time was tracked on Zetasizer (Nano ZS90, Malvern, UK) operating with a

146

He−Ne laser at a wavelength of 633 nm and a scattering angle of 90º. Before each measurement, the

147

hematite suspension was sonicated in a bath sonicator for 5 minutes. Then, a certain volume of the

148

suspension was immediately added to the predetermined solution with desired concentrations of electrolyte

149

and protein. The final concentration of hematite particles was fixed at 16 mg/L that was the optimum

150

concentration for DLS measurements. The mixture was shaken shortly with a vortex mixer and then

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immediately transferred into a polystyrene cuvette (10 mm path length, Sarstedt, Germany) for

152

measurement. Dh was monitored at 5s intervals over a time period of 20-30 minutes. The initial

153

aggregation rate constant (k) of hematite particles was determined from a linear least squares regression

154

analysis of the increase in Dh with time (t):35 ݇∝

155

ଵ

ௗୈ౞ (௧)

ேబ

(

ௗ௧

)௧→଴

(1)

156

where N0 is the initial particle concentration. The details of the k determination have been described in our

157

previous study.30 No measurable protein aggregates were observed by DLS in NaCl solution at pH 5.7.

158

In this study, the attachment efficiency (α), known as the inverse stability ratio, was used to

159

quantitatively describe the aggregation behavior of hematite particles. It was calculated by normalizing the

160

measured k by the diffusion-limited aggregation rate constant (kfast) according to eq 2. The attachment

161

efficiency ranges from 0 to 1, representing the probability of an irreversible attachment resulting from the

162

collision of two particles.36

163

α=௞

௞

೑ೌೞ೟


(2)

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To calculate α in the presence of proteins, kfast was obtained from the diffusion-limited aggregation rate

165

constant determined under favorable aggregation conditions in the absence of proteins.37, 38

166

Zeta Potential Measurements. The zeta potentials ζ of bare hematite particles or protein-NP

167

conjugates in solutions with varying NaCl concentrations or protein concentrations at 25ºC were obtained

168

from the electrophoretic mobility µe using the generalized Smoluchowski equation. The electrophoresis

169

mobility of samples was measured on Nanosizer (Nano ZS90, Malvern, UK) with a laser Doppler

170

velocimetry setup. In all measurements, the particle concentration of hematite suspensions was fixed at 16

171

mg/L. To study the effect of protein concentration on the zeta potential, µe of protein-particle conjugates in

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10mM NaCl at pH 5.7 was measured as a function of protein concentration (0-6.5 mg/L). For each sample,

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at least 3 independent measurements were conducted, and no less than 15 cycles were collected for each run.

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Protein Adsorption on Hematite Particles. To compare the affinity of OmcA and BSA to hematite

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surface, adsorption isotherms of proteins on the smallest hematite NPs (HM1) were determined in 60mM

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NaCl solution at pH 5.7. After 10 minute sonication of the HM1 suspension, 35 µL of the suspension was

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added to the solution with varying protein concentrations. The samples were incubated in a shaker

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operating at 150 rpm for 1 h, and then centrifuged at 4000 × g for 10 minutes. The supernatants were

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transferred into new centrifuge tubes and centrifuged again at 4000 × g for 10 minutes to remove residual

180

hematite NPs.39 The concentration of OmcA in the final supernatants was measured by UV–Vis

181

spectroscopy as described above. BSA concentration was determined using Bradford protein assay kit.30

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The control experiment without hematite NPs indicated that no measurable proteins were settled or

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adsorbed on bottles during the absorption experiments or centrifugation. To obtain detectable amount of

184

proteins adsorbed by hematite particles, the particle concentration in the adsorption experiments was

185

increased to 200 mg/L. The equivalent adsorption amount for 16 mg/L HM1 NPs was presented.

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Circular dichroism spectroscopy. The circular dichroism (CD) spectra were performed on the

187

suspensions of protein-particle conjugates in order to assess whether the secondary structure of proteins

188

changed upon adsorption onto hematite NPs or with the increase of NaCl concentration. After 15-minute

189

incubation of proteins conjugated with hematite NPs in the solution with varying NaCl or protein

190

concentrations at pH 5.7, CD spectra were recorded with a JASCO J-1500-150 spectropolarimeter (Japan)

191

under a constant stream of nitrogen gas. In order to reduce the interference of high salt concentration on

192

measurements, in the experiments with 100mM NaCl solution, the protein-NPs conjugates were filtered by



193

30 kDa Amicon Ultra centrifugal filter Devices (Millipore) and then resuspended in Milli-Q water.40 This

194

washing procedure was repeated for three times in order to remove most NaCl in suspensions just before

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CD measurements. The secondary structures of original proteins were determined by the CD spectra of

196

proteins diluted in Milli-Q water.

197 198 199 200

RESULTS AND DISCUSSION

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images showed that the primary particle sizes of the three synthetic particles were 9 ± 2 nm (HM1), 36 ± 6

202

nm (HM2) and 112 ± 12nm (HM3), respectively (Figure S1). All synthetic particles exhibited a nearly

203

isotropic shape. The geometric specific surface area (SSA) and zeta potential of the samples were listed in

204

the Table S1. The SSA ratio of HM1: HM2: HM3 was 12: 3: 1. At pH 5.7, all particles were positively

205

charged, which is consistent with the point of zero charge (7.2 – 9.5) reported in previous studies.28 The

206

power X-ray diffraction (XRD) patterns revealed that only hematite phase exhibited in the three synthetic

207

particles (Figure S2). The decreasing full-width at half-maximum (FWHM) from HM1 to HM3 was

208

consistent with the increasing primary particle size observed in TEM images (Figure S1).

Characteristics of Synthetic Hematite Particles. The particle-size distributions measured from TEM

209

Size-dependent Aggregation of Bare Hematite Particles. Attachment efficiencies of bare hematite

210

particles in three different sizes as a function of NaCl concentration at pH 5.7 are compared in Figure 1a.

211

The corresponding aggregation profiles are presented in SI Figure S3a. In the absence of proteins, all

212

hematite particles clearly exhibited DLVO type of aggregation profiles. The attachment efficiency

213

increased with the increase of salt concentration, until the NaCl concentration reached the critical

214

coagulation concentration (CCC). At the high ionic strength conditions ([NaCl] > CCC), the repulsive

215

energy barrier was completely screened, resulting in the diffusion-limited aggregation (DLA). It was

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confirmed by the lower zeta potentials of hematite particles at elevated NaCl concentrations (Figure 1b). In

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this scenario, a maximum aggregation rate was reached, and the further increase in electrolyte

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concentration had no effect on the attachment efficiency. According to the DLVO theory, the CCC value

219

represents the minimum concentration of electrolyte that is needed to completely destabilize the colloidal

220

suspension, so it has been widely used to assess the colloidal stability of NPs under different experimental

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conditions.28 When the same particle concentration was used, the CCC values of NaCl for HM1, HM2 and

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HM3 were 55mM, 45mM and 14mM, respectively. The observed higher CCC for the smaller particles was


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consistent with the computational results of the particle-size effect on CCC.41 A potential explanation is

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that the smaller NPs have the larger Debye length and accordingly the higher electrolyte concentration was

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needed for the complete compression of electrical double layer.41 Although He et al.28 reported a

226

decreasing trend in CCC values with the increase of hematite particle size, different particle concentrations

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were used for 12, 32, and 65 nm hematite NPs in that study. Thus, the reported difference in CCC values

228

for hematite NPs in different sizes could reflect a combined effect of particle concentration and primary

229

particle size. The effect of initial particle concentration on the aggregation kinetics and CCC values will be

230

discussed further below. In addition, it is worthy to mention that the zeta potential of HM3 in 5 mM NaCl

231

at pH 5.7 was obviously higher than the values of HM1 and HM2 (Fig 1b). This is consistent with the

232

results reported by He et al.28 At relatively low background salt concentrations, the smaller particles can be

233

less charged than the larger particles, as a result of the changes in the surface concentration of counterions,

234

electronic structure, surface reactivity, etc., with decrease in particle size.2

235

Effect of OmcA on the Stability of Hematite Particles. The effect of OmcA on the aggregation

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kinetics of hematite particles was studied at low salt concentration ([NaCl] = 10 mM < CCC for all

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particles) and high salt concentration ([NaCl] = 60mM > CCC for all particles), respectively. In 10mM

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NaCl solution without OmcA at pH 5.7, all hematite particles in different sizes were positively charged

239

(Figure 1b) and relatively stable owing to the electrostatic repulsion. The changing trend of attachment

240

efficiencies with increasing OmcA concentration ([OmcA]) for all particles in different sizes showed a

241

similar pattern (Figure 2a). The corresponding aggregation profiles are presented in SI Figure S3b.

242

According to the variation tendency, the aggregation behavior can be divided into three regimes (Figure

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3a): (1) in Regime I, attachment efficiencies of hematite particles increased continuously with increasing

244

[OmcA] until the maximum value (α = 1) was reached at [OmcA] = 1.1 mg/L (Figure 2a). Correspondingly,

245

the zeta potential of OmcA-hematite conjugates decreased from 21.6 to 3 mV, as [OmcA] increased from 0

246

to 1.1 mg/L (Figure 4). At the point B in Figure 3a ([OmcA] = 1.1 mg/L), the α value reached the

247

maximum, and accordingly the zeta potential decreased to a value very close to zero. It implies that the

248

observed destabilization in this regime can be mainly attributed to the adsorption of negatively charged

249

OmcA (Figure S4) on the positive charged hematite surface (Figure 1b), resulting in surface charge

250

neutralization and decreasing electrostatic repulsion between OmcA-hematite conjugates. In Regime I, the

251

effect of ionic strength on α was studied at a randomly selected point A ([OmcA] = 0.5 mg/L, Figure 3a).



252

The attachment efficiency and the zeta potential at point A in 10 mM NaCl were 0.77 and 10 mV,

253

respectively. When [NaCl] was greater than 50mM, the α value increased to ~ 0.9 and inappreciably

254

fluctuated around this value (Figure 3b). It indicated that the screening effect of the increasing salt

255

concentration led to even faster aggregation. The value of α at the high ionic strength was a little less than

256

1, which might imply the minor contribution from steric repulsion.

257

(2) In Regime II, the increasing [OmcA] resulted in the decreasing α values of OmcA-hematite

258

conjugates. In addition, it was found that α values at the randomly selected point C ([OmcA] = 2.7 mg/L,

259

Figure 3a) in Regime II decreased with the increasing [NaCl] (Figure 3b). On the other hand, zeta potential

260

decreased as [NaCl] increased under this condition (Figure S5), indicating the weakening of electrostatic

261

repulsion. That means the higher ionic strength facilitated the stabilization of OmcA-hematite conjugates

262

in this case, so electrostatic repulsive force could not dominate the interparticle interaction in this regime.

263

The similar stabilization of protein-NP conjugates induced by increasing ionic strength has been reported

264

in our previous study,30 which can be related to the patch-charge attraction. This attraction was originated

265

from the electrostatic interaction between the negatively charged patches of OmcA adsorbed on the surface

266

of hematite particle clusters (the hydrodynamic diameter > 100 nm, Figure S3) and the exposed hematite

267

surfaces with the positive surface charge. The patch-charge attraction is substantial only when the

268

protein-rich domain and the protein-poor domain/particle surface possess opposite charges and at low salt

269

concentration. At high salt concentration, the patch-charge attractive force was screened, and the steric

270

repulsive force between OmcA adsorbed on NPs stabilized the OmcA-hematite conjugates. As the [OmcA]

271

increased in Regime II, the re-stabilization of hematite particles could be attributed to the increasing steric

272

repulsive forces. The adsorption isotherms of OmcA on HM1 NPs (Figure 5) showed that the amount of

273

OmcA adsorbed on hematite surface increased as more OmcA was added under the conditions of the

274

present study. The decreasing zeta potential of protein-NP conjugates with the increase of [OmcA] (Figure

275

4) also suggested the increasing adsorption of OmcA on hematite. The stronger steric repulsive force

276

provided by the more and more OmcA absorbed on hematite surface led to the decease of α values.

277

(3) In Regime III, the attachment efficiencies were around zero and independent of [OmcA] (Figure

278

3a). The zeta potentials of OmcA-hematite conjugates gradually decreased with the increase of [OmcA] in

279

Regime I and II (0 < [OmcA] < ~ 4.4 mg/L), but kept constant around -17 mV in regime III ([OmcA] > 4.4

280

mg/L) (Figure 4). At the point D ([OmcA] = 4.4 mg/L) in Regime III, the attachment efficiency remained


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around zero and was not influenced by ionic strength (Figure 3b). Adsorption isotherm of OmcA on HM1

282

NPs indicated that the concentration of adsorbed OmcA was 3.25 mg/L, when 4.4 mg/L OmcA was added

283

to the suspension with 16 mg/L hematite particles (Figure S6). Based on the geometric specific surface

284

area of 9 nm spherical hematite NPs (127 m2/g), the amount of adsorbed OmcA normalized by the NP

285

surface area was 160.6 ng/cm2 that is close to the reported amount of absorbed OmcA for forming a

286

monolayer on hematite surface (164 ng/cm2).42 Therefore, in Regime III, hematite particles were coated by

287

OmcA and completely stabilized by the repulsive steric force between adsorbed OmcA that is independent

288

of ionic strength. The patch-charge attraction due to the surface heterogeneity in this case became

289

inapplicable, because no discernible bare surface was exposed. Considering the irrelevance of α values and

290

ionic strength, the contribution of electrostatic repulsive force to the observed stabilization of particles was

291

negligible in this regime.

292

At high salt concentration ([NaCl] = 60mM), the effect of [OmcA] on the stability of hematite

293

particles was different from that at low salt concentration. Figure 2b shows that, for all particles of different

294

sizes, the α values decreased with the increasing [OmcA] until α reached a plateau. The corresponding

295

aggregation profiles are presented in SI Figure S3c. The high salt concentration resulted in the screening of

296

electrostatic interactions, so patching-charge attraction and electrostatic repulsion that greatly impacted the

297

stability of hematite particles at low salt concentration were no longer applicable in this case. As [OmcA]

298

increased, the steric repulsive force gradually increased and overweighed the van der Walls attractive force,

299

resulting in the complete stabilization of hematite particles. Therefore, at high salt concentration, the

300

interparticle interaction between OmcA-hematite conjugates was dominated by the steric repulsive force

301

originated from OmcA adsorbed on hematite surface.

302

Effect of Primary Particle Size on the Aggregation of OmcA-hematite Conjugates. In addition to

303

the concentrations of OmcA and NaCl, the primary particle size of hematite also influenced the

304

aggregation state of OmcA-hematite conjugates. Although the attachment efficiencies of all particles in

305

different sizes presented the similar variation tendency with the increase of [OmcA], the extent to which

306

the same [OmcA] affected the aggregation state was different for the three samples and in the order HM3 >

307

HM2 > HM1 (Figure 2a). In this study, a fixed mass concentration of particles (16 mg/L) was used for the

308

three samples, so the total surface area of hematite particles was in the order HM1 > HM2 > HM3. As

309

discussed above, at low salt concentration, the concentration of OmcA at point B (the critical point



310

between Regime I and II) was close to the value for surface charge compensation, i.e. zero zeta potential.

311

The smaller particles had the larger surface area and correspondingly needed more sorbed OmcA for

312

surface charge compensation. It was confirmed by the zeta potential results (Figure 4) that 1.18, 0.48, and

313

0.08 mg/L OmcA, respectively, were needed to obtain the zero zeta potential for HM1, HM2, and HM3.

314

The ratio of [OmcA] needed for the surface charge neutralization was 14: 6: 1 for HM1: HM2: HM3. It is

315

consistent with the ratio of geometric specific surface areas, 12: 3: 1, for the three samples (Table S1).

316

Similarly, the transition between Regime II and III occurred around the critical point at which hematite

317

surface was fully covered by OmcA. The results of aggregation kinetics indicated that 4.4, 2.7, and 0.8

318

mg/L OmcA were, respectively, needed to completely stabilize 16 mg/L HM1, HM2, and HM3 (Figure 2).

319

These results agreed fairly well with the concentrations of OmcA, 4.4 mg/L for HM1, 2.7 mg/L for HM2,

320

and 0.5 mg/L for HM3, when the zeta potential attained a plateau value in 10 mM NaCl (Figure 4).

321

Similarly, in 60 mM NaCl, 2.7, 1.6, and 0.5 mg/L OmcA were needed to provide sufficient steric hindrance

322

to entirely stabilize HM1, HM2 and HM3, respectively (Figure 2b). Although these concentrations of

323

OmcA were smaller than the values for the complete stabilization at low salt concentration due to the lack

324

of patch-charge attraction, they were also in the same order of HM1 > HM2 > HM3. Therefore, the

325

size-dependent stability of hematite particles in the presence of OmcA mainly depends on the extent to

326

which hematite surface was changed by OmcA adsorption, when particle concentration, ionic strength, and

327

pH value were fixed.

328

In this study, the aggregation behavior of hematite particles in different sizes was compared by using

329

the same mass/molar concentration of particles. It is not uncommon, in the studies of size-dependent

330

reactivity, to use the same initial particle mass/molar concentration in order to keep the same ratio between

331

reactants in all experiments and compare the surface-area-normalized reaction rates of particles in different

332

sizes. To correlate the aggregation behavior observed in this study with the size-dependent reactivity

333

reported in other studies, the same mass/molar concentration of hematite particles was used. When the

334

particle mass/molar concentrations are same, the total surface area of the smaller particles should be much

335

larger than that of the bigger particles. For example, in this study, the total surface area of HM1 was 12

336

times more than the values of HM3, when the particle concentration was fixed at 16 mg/L. In order to

337

investigate the effect of surface area on the aggregation kinetics, we increased the particle concentration of

338

HM3 from 16 mg/L to 192 mg/L so that HM3 and HM1 have the same total surface area. In the absence of


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339

OmcA, increasing surface area of HM3 resulted in the increase of CCC from 14 mM to 30 mM that was

340

still smaller than the CCC of HM1 (Figure 6a). Thus, for the bare hematite particles, the trend that the

341

smaller particles have the larger CCC value was same no matter the same particle concentration or the

342

same total surface area was used. In both cases, the effect of different particle number concentrations

343

cannot be ruled out, but it only needs to be taken into account at extremely high particle concentration

344

when the presence of the particles surrounding two interacting particles reduces the total interaction energy

345

between them.43 Thus, the different particle number concentrations of HM1 and HM3 in this study would

346

not have a significant effect on CCC. On the other hand, in the presence of OmcA, increasing the particle

347

concentration of HM3 from 16 mg/L to 192 mg/L obviously altered the pattern of α versus [OmcA] for

348

HM3 from an abrupt change to the one that was quite similar to the pattern of 16 mg/L HM1 (Figure 6b). It

349

indicated that the aggregation state of OmcA-hematite conjugates was more correlated to surface area than

350

primary particle size. The extent to which the particle surface was changed by adsorbed proteins

351

determined the interparticle interaction and aggregation kinetics.

352

It is worth to mention that previous studies have reported the dependence of protein adsorption on the

353

primary particle size of NPs.9 The smaller NPs with the higher curvature are expected to increase the

354

deflection angle between adsorbed proteins, so the proteins adsorbed on smaller NPs may undergo fewer

355

changes in conformation. To check the particle size effect on the conformation of OmcA adsorbed on

356

hematite particles, the CD spectroscopy was performed on OmcA conjugated with HM1, HM2, and HM3 in

357

10 mM and 100 mM NaCl, respectively, at pH 5.7 (Figure S7). At the low salt concentration, the CD

358

spectra of native OmcA and the OmcA conjugated to the three kinds of hematite particles were very similar

359

(Figure S7a). That means no distinct changes in the conformation of OmcA after adsorption on hematite

360

particles in different sizes were observed through the CD measurements. At the high salt concentration,

361

compared to the native OmcA, the β-sheets content of the adsorbed OmcA increased slightly (Figure S7b),

362

but this subtle change happened on OmcA conjugated to all hematite particles in different sizes to the same

363

extent. Thus, the size-dependent aggregation kinetics described above could not be related to the different

364

conformation of the protein layer on hematite particles in different sizes. In addition, the CD spectra of

365

OmcA right after exposed to NPs and after 20-minute incubation were compared (data not shown). No

366

obvious changes were observed in the CD spectra of samples with different incubation time, so there were

367

no detectable structural changes in OmcA during the time period of aggregation measurements. Shi et al



368

(2006) also reported that no change was observed in the UV-visible adsorption spectra of OmcA after

369

adsorption onto hematite in 140 mM KCl solution, which confirmed no major conformation change of

370

OmcA under the similar conditions.23

371

Effect of Protein Properties on the Stability of Protein-particle Conjugates. In order to investigate

372

the effect of binding affinity of proteins on the stability of NPs, the attachment efficiencies of 16 mg/L HM1

373

NPs in the presence of OmcA and BSA were compared in 60mM NaCl (> CCC) at pH 5.7 (Figure 5a). Both

374

BSA and OmcA were negatively charged in NaCl solution at pH 5.7, so the increasing concentration of the

375

proteins could gradually decrease the measured zeta potential down to a negative plateau value (Figure 4).30

376

BSA has the molecular weight of 66 kDa with the size of 5.5 × 5.5 × 9 nm3 that is similar to the size of

377

OmcA (85 kDa, 5 × 6 × 9.5 nm3).42, 44, 45 No apparent conformation changes were observed by CD

378

measurements in BSA or OmcA after the adsorption on hematite particles (Figure S7). Despite of the

379

similarity between these two kinds of proteins, Figure 5 showed that only 2.7 mg/L OmcA was sufficient to

380

completely stabilize 16 mg/L HM1 NPs in 60mM NaCl, but 9 mg/L BSA was needed under the same

381

conditions. As described above, at high ionic strength, hematite NPs were stabilized by the steric repulsive

382

force originated from the proteins adsorbed on surface. The results indicated that, before the complete

383

stabilization of NPs, OmcA could stabilize hematite NPs more effectively than the same amount of BSA.

384

The different abilities of BSA and OmcA to stabilize hematite NPs could be attributed, at least partly, to

385

their different affinity to hematite surface. The adsorption isotherms of OmcA and BSA on HM1 NPs in 60

386

mM NaCl at pH 5.7 and room temperature (Figure 5b) showed that a larger percentage of OmcA was

387

adsorbed on hematite than BSA, when the same concentration of proteins was added. It implies that OmcA

388

has the higher affinity to hematite NPs than BSA. Lower et al. reported that the hematite-binding motif

389

(Ser/Thr-Pro-Ser/Thr) near the terminal heme-binding domain (heme 9 & 10) of OmcA was expected to

390

facilitate the OmcA adsorption onto hydroxylated hematite surface via the hydrogen bonding.25, 44 At high

391

salt concentration, the aggregation kinetics of HM1 NPs was mainly related to the van der Waals attraction

392

and the steric repulsion originated from proteins that rapidly adsorbed onto NPs, because the patch-charge

393

attraction and the electrostatic interaction were screened. The adsorption of proteins onto hematite NPs

394

needs to be quick enough to form a steric stabilizing layer against aggregation by particle contact. Protein

395

concentration and protein adsorption kinetics are the key parameters to determine the formation of the steric

396

stabilizing layer.9 In addition to increase the protein concentration, the higher protein affinity to particle


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397

surface can also facilitate the fast protein adsorption onto NPs. Therefore, OmcA can strongly and rapidly

398

adsorb onto the surface of hematite NPs by means of the hematite binding motif, and accordingly exhibit a

399

relatively high ability to stabilize hematite NPs.

400

Environmental Implications.

401

The study presented here showed that both positive and negative influences of proteins on NP

402

stability in aqueous dispersions can be realized, which depends on the inherent properties of NPs and

403

proteins, the protein-to-NP ratios, as well as solution conditions. Most previous studies about aggregation

404

behavior and interfacial interaction of protein-NP complex focus on the blood proteins from the

405

perspectives of nanomedicine and nanotoxicity. In this study, the outer membrane cytochrome, OmcA, of a

406

model iron microbe from aqueous environment, Shewanella oneidenesis MR-1, was investigated and

407

compared to BSA, a widely studied serum albumin protein. Their different abilities to stabilize hematite

408

NPs imply that the findings reported in the previous studies of blood proteins are not necessarily applicable

409

to extracellular proteins by environmental bacteria. More studies need to be conducted in order to gain

410

sufficiently broad knowledge of how extracellular proteins modulate the aggregation state of NPs in

411

natural waters, which is indispensable to thoroughly understand a wide variety of biogeochemical

412

processes involving NPs, such as extracellular respiration, cell uptake, biomineralization, etc.

413

On the other hand, the relationship between the inherent properties of NPs and proteins, protein

414

adsorption onto NP surfaces, and the structure of protein corona remains unclear. Typically, a rapid

415

adsorption of protein on NP surface can alter the interfacial properties and aggregation state of NPs, and

416

also give them new biological identity that can be distinct from the original properties of NPs. Different

417

from bare NPs, the stability of NPs in the presence of proteins is not only related to salt concentration, but

418

also to many other factors, such as protein concentration, NP surface area, the affinity of proteins to NP

419

surfaces, etc. The results in this study showed that the aggregation state was more correlated to the ratio of

420

protein concentration to NP surface area than the primary particle size. Therefore, the complex aggregation

421

behavior of NPs in the presence of biomacromolecules needs to be systematically studied and carefully

422

considered in the size-dependent studies of NPs in biotic reactions.

423 424

ASSOCIATED CONTENT

425

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



426

Discussion are presented. This material is available free of charge via the Internet at http://pubs.acs.org.

427 428

AUTHOR INFORMATION

429

Corresponding Author

430

*Phone: (+86)010-62754292-808; email: [email protected]; address: College of Environmental Sciences

431

and Engineering, Peking University, Beijing 100871, China

432 433

ACKNOWLEDGEMENTS

434

This work was financially supported by National Natural Science Foundation of China (41472306) and

435

National Basic Research Program of China (973 Program, 2014CB846001). We also thank Xinxin Wang for

436

the help in the measurements of CD spectra.

437


Page 16 of 26

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438 439

Figure 1. (a) attachment efficiencies and (b) zeta potentials of 9 nm (HM1), 36 nm (HM2), and 112nm

440

(HM3) bare hematite particles as a function of NaCl concentrations at pH 5.7.

441



442

443 444

Figure 2. The effect of OmcA concentrations on attachment efficiencies of hematite particles in different

445

sizes in 10 mM (a) and 60 mM (b) NaCl.

446


Page 18 of 26

Page 19 of 26


447 448 449

Figure 3. (a) Attachment efficiencies of 16 mg/L HM1 NPs in 10mM NaCl as a function of OmcA

450

concentration. According to the changing trend of attachment efficiencies, the effect of OmcA

451

concentration was divided into three regimes. (b) Comparison of attachment efficiencies of HM1 NPs at

452

the A-D points in the three regimes as a function of NaCl concentrations. The different trends at the points

453

indicate that different interparticle forces dominated the interactions between the OmcA-hematite

454

conjugates in the three regimes.



455 456 457

Figure 4. Zeta potential of OmcA-hematite conjugates as a function of OmcA concentration in 10mM

458

NaCl.

459 460 461


Page 20 of 26

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462 463

Figure 5. (a) The change of attachment efficiencies of 16 mg/L HM1 NPs in the presence of OmcA or

464

BSA in 60 mM NaCl as a function of protein concentration. (b) Adsorption isotherms of OmcA and BSA,

465

respectively, on 16 mg/L HM1 NPs plotted as a function of protein concentration added in 60 mM NaCl

466

solution at pH 5.7. The solid dots represent the concentration of proteins adsorbed on HM1 NPs, and the

467

open dots represent the adsorption percentage of added proteins.

468



469 470

Figure 6. (a) Comparison of attachment efficiencies of bare HM1 and HM3 as a function of NaCl

471

concentration, when HM3 had the same mass concentration or total surface area as HM1.(b) Comparison

472

of attachment efficiencies of HM1 and HM3 as a function of OmcA concentration, when HM3 had the

473

same mass concentration or total surface area as HM1.

474


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475

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Aggregation Kinetics of Hematite Particles in the Presence of Outer

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