Article pubs.acs.org/est
Impact of Proteins on Aggregation Kinetics and Adsorption Ability of Hematite Nanoparticles in Aqueous Dispersions Anxu Sheng, Feng Liu, Nan Xie, and Juan Liu* School of Environmental Sciences and Engineering, Peking University, Beijing, China, 100871 S Supporting Information *
ABSTRACT: The initial aggregation kinetics of hematite nanoparticles (NPs) that were conjugated with two model globular proteinscytochrome c from bovine heart (Cyt) and bovine serum albumin (BSA)were investigated over a range of monovalent (NaCl) and divalent (CaCl2) electrolyte concentrations at pH 5.7 and 9. The aggregation behavior of Cyt-NP conjugates was similar to that of bare hematite NPs, but the additional electrosteric repulsion increased the critical coagulation concentration (CCC) values from 69 mM to 113 mM in NaCl at pH 5.7. An unsaturated layer of BSA, a protein larger than Cyt, on hematite NPs resulted in fast aggregation at low salt concentrations and pH 5.7, due to the strong attractive patch-charge interaction. However, the BSA-NP conjugates could be stabilized simply by elevating salt concentrations, owing to the screening of the attractive patch-charge force and the increasing contribution from steric force. This study showed that the aggregation state of protein-conjugated NPs is proved to be completely switchable via ionic strength, pH, protein size, and protein coverage. Macroscopic Cu(II) sorption experiments further established that reducing aggregation of hematite NPs via tailoring ionic strength and protein conjugation could promote the metal uptake by hematite NPs under harsh conditions.
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environment.9 On the other hand, some proteins were used as an efficient stabilizing agent to increase stability and biocompatibility of engineered NPs for applications in environmental remediation10 and biotechnology.11 With the growing use of NPs in biomedical and biotechnological applications, there is an increasing probability that NP−protein conjugates are released into the environment as consumer products.12 When NPs enter physiological environments, a protein coating, known as the “protein corona”, can form quickly on NP surfaces. In particular, the hard corona that is consisted of the proteins irreversibly bound to NP surfaces can evidently alter the aggregation state and surface properties of the NPs.13 Therefore, understanding the aggregation of NP− protein conjugates in aqueous dispersions is crucial to elucidate the bioavailability, reactivity, mobility, and fate of NPs in aqueous environments. There have been many attempts to understand the impact of proteins on NP stability, but still no conclusive generalization has been drawn. Both positive and negative influences of
INTRODUCTION Naturally occurring and anthropogenic nanoparticles (NPs) have been widely found in aqueous environments.1 The impacts of these NPs on natural and engineered waters, organisms and ecosystems, as well as human health, are greatly dependent on their stability and aggregation state.2−4 Many efforts have been made to relate aggregation state of NPs to their ability to uptake heavy metals,5 their reactivity and transport in natural waters,6 as well as their fate and toxicity in biological systems.2 Naturally occurring dissolved organic matter (DOM) in the aqueous environment can greatly change the aggregation state of NPs, leading to different behavior between field samples and their laboratory analogs. Proteins are one of the major components of DOM in surface waters.7 Owing to their hydrophobic moieties that favor their adsorption onto the inorganic colloidal surfaces, proteins have the potential to significantly alter the aggregation state of colloids/nanoparticles in aqueous environment.8 For example, in sulfate-reducing bacteria−dominated biofilms collected from a Pb and Zn Mine, aggregates of microbially derived extracellular proteins and biogenic zinc sulfide nanocrystals were found. This finding suggested that aggregation induced by extracellular proteins greatly limited the dispersal and mobility of NPs in natural © 2016 American Chemical Society
Received: Revised: Accepted: Published: 2228
October 27, 2015 January 18, 2016 January 29, 2016 January 29, 2016 DOI: 10.1021/acs.est.5b05298 Environ. Sci. Technol. 2016, 50, 2228−2235
Article
Environmental Science & Technology
in the SI (section S1). Mass concentration of hematite in NP suspension was measured by acid digestion. 0.1 mL of NP suspension was dissolved in 9.9 mL of 5 M HCl and continuously shaken overnight. Then, 0.02 mL digested solution was diluted by using 4.98 mL 2% HNO3. Total Fe was determined with an inductively coupled plasma optical emission spectrometry (Prodigy High Dispersion ICP-OES, Teledyne Leeman Laboratories, Hudson, NH). The experiments were carried out in triplicates. Particle size and morphology of synthetic hematite NPs were measured on a field-emission transmission electron microscope (TEM, JEOL JEM-2100F) operated at 200 kV. TEM samples were prepared by placing a drop of diluted hematite suspension on a 400 mesh copper grid coated with ultrathin carbon layer and then drying it in air. Particle size distribution was obtained by analyzing more than 100 NPs on randomly selected areas on TEM images using ImageJ software.27 The TEM images of BSA-NP conjugates were taken by using the same instrument under the similar conditions. Hematite NP suspension was sonicated for about 5 min and added to 10 mM or 80 mM NaCl solution in the presence of 390 ng/cm2 BSA. After about an hour, TEM samples were prepared from the suspensions according to the method mentioned above. The crystalline phase of hematite particles was characterized by powder X-ray diffraction using a Rigaku D/MAX-2000 diffractometer with monochromatic CuKα radiation (λ = 0.15406 nm) at a scan rate of 0.02 2θ·s−1. Time-Resolved Dynamic Light Scattering (TR-DLS). The initial aggregation rates and resulting attachment efficiencies were determined by TR-DLS. The change of hydrodynamic diameters as a function of time was conducted on Zetasizer (Nano ZS90, Malvern, UK) operating with a He− Ne laser at a wavelength of 633 nm and a scattering angle of 90°. Before each measurement, hematite suspension was sonicated in a bath sonicator for 5 min. Then it was immediately diluted in a predetermined volume of pH 5.7 or 9 stock solution with desired concentrations of electrolyte and protein, respectively, in 10 mm diameter polystyrene cuvettes (Sarstedt, Germany). Additional information on the preparation of stock solutions and hematite suspensions is provided in SI Section 2. The concentration of hematite NPs in all experiments was fixed at 16 mg/L as the optimum condition for DLS measurements. Hydrodynamic diameter (Dh) was monitored every 5 s over a time period of 15−30 min. The z-average hydrodynamic radius of the aggregates was obtained from the autocorrelation function using the “general purpose mode”. No buffer was used in aggregation studies and adsorption experiments, because it may enhance the aggregation of NPs.28 No pH change was observed in all experiments at pH 5.7. In the case of pH 9, the pH values decreased to 7.6−8.0 at the end of DLS measurements due to the effect of CO2 in air. Nevertheless, the pH was still greater than or equal to pHiep. The small pH shift did not influence the comparison of the aggregation behavior under this condition to that at pH 5.7. Zeta Potential Measurements. The zeta potentials of bare hematite NPs and protein-NP conjugates as a function of NaCl concentrations at 25 °C were measured with a laser Doppler velocimetry setup (Nano ZS90, Malvern, UK). At least 5−10 measurements (15−30 cycles per measurement) were conducted for each sample. In the measurements of protein-NP conjugates, 16 mg/L hematite NPs was equilibrated with 8 mg/ L proteins in pH 5.7 or 9 stock solutions for at least 15 min before measurements. The zeta potential ζ was obtained from
proteins on NP stability have been reported. For example, the stabilization of iron oxide NPs by serum proteins was reported in biologically relevant conditions.14,15 On the other hand, Flynn et al. reported that BSA adsorption may either enhance or inhibit colloid mobility of iron-oxide coated sand in saturated porous media, depending on protein coverage.16 The discrepancy could be attributed to the complex interactions between proteins and NPs. In addition to the van der Waals attraction and the electrostatic repulsion, the interactions between protein-NP conjugates may also include non-DLVO (Derjaguin−Landau−Verwey−Overbeek) interactions, such as depletion attraction, steric repulsion, or bridging flocculation,17 etc. How proteins attach to NPsurfaces and the formation dynamics of the adsorbed protein layer can influence the nonDLVO forces.18 Further complexity may arise from changeable surface properties of NPs and potential protein deformation with solution chemistry.19 So far most stability studies of protein-NP conjugates were conducted in biological media. There are still few studies on the stability of oxide NPs with proteins in aqueous dispersions under environmentally relevant conditions. In this study, Cytochrome c from bovine heart (Cyt) and Bovine serum albumin (BSA) were chosen as model proteins, primarily because they are globular proteins with high structural stability.20 BSA is one of the most abundant proteins in serum and biological culture media. It is widely utilized in biotechnology due to its low cost, wide availability, and high structural/functional similarity to human serum albumin (HSA).21 Cyt was chosen to compare with BSA, in order to reveal how the inherent properties of proteins influence the aggregation of NP−protein conjugates. Compared to BSA, Cyt has the smaller molecular weight and the higher isoelectric point (Supporting Information (SI) Table S1). Moreover, cytochromes are redox metalloproteins, which can be used as redox catalysts or mediaors for reductive dehalogenation reactions in environmental applications.22 Besides, recent findings that outer membrane cytochromes assist the extracellular electron transfer between iron oxide and iron bacteria have encouraged the study on redox reactions between iron (oxyhydr)oxide NPs and purified cytochrome c of iron bacteria.23−25 It is indispensable for studies in this area to understand the aggregation behavior of iron (oxyhydr)oxide NPs in the presence of cytochrome c. Here, hematite NPs, one of the most abundant natural iron oxide minerals, were selected to study the impact of proteins on NP stability as a function of solution pH, ionic strength, cation valence, protein size, and molar ratios of proteins to NPs. The aggregation kinetics of hematite NPs with Cyt or BSA over a range of monovalent (NaCl) and divalent (CaCl2) electrolyte concentrations were measured by time-resolved dynamic light scattering (DLS). Electrophoretic mobility measurements were employed to investigate the change in surface potentials of hematite NPs after the addition of proteins at varied pH and salt concentrations. In addition, Cu2+ ions were used, as a surface probe species, to study how the adsorption ability of hematite NPs for heavy metals changed, as the aggregation state of NPs was modulated by proteins.
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MATERIALS AND METHODS Hematite Synthesis and Characterization. Hematite NPs were synthesized by forced hydrolysis of ferric nitrate solution according to the method reported by Schwertmann and Cornell.26 More details on hematite synthesis are described 2229
DOI: 10.1021/acs.est.5b05298 Environ. Sci. Technol. 2016, 50, 2228−2235
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The final NP concentration was fixed at 80 mg/L and BSA concentration was 40 mg/L. Before spiking the CuCl2 stock solution, the NP suspension with or without proteins was shaken for 30 min at 120 rpm. The initial concentration of Cu(II) in all experiments was fixed at 0.045 mM. After shaking at 120 rpm for 4 h at 25 °C, the copper-sorbed NPs were separated from the suspension by 0.22 μm syringe filters (Millipore PES Membrane). The filtrates were then acidified with 2% HNO3 for ICP-OES (Prodigy High Dispersion ICPOES, Teledyne Leeman Laboratories, Hudson, NH) analysis. Results of control experiments showed that no measurable amount of Cu(II) was adsorbed on the syringe filters or in the BSA solution without hematite NPs. Additional control experiments were performed using Amicon Ultra-15 centrifugal filter units (MWCO 3 kDa, Millipore) and 0.22 μm syringe filters, respectively, to filter NPs after adsorption. The similar results indicated that 0.22 μm syringe filters were sufficient to remove hematite NPs in these experiments.
the electrophoretic mobility μ e using the generalized Smoluchowski equation. BSA Adsorption on hematite NPs. Adsorption isotherms of BSA on hematite NPs were measured in solution at pH 5.7. After sonication for 10 min, 200 mg/L of hematite NP suspension was added to the pH 5.7 stock solution with increasing amounts of BSA (0−200 mg/L) in 60 mM NaCl solution or with the fixed amount of BSA (100 mg/L) in the solution with a range of NaCl concentration (10−100 mM). The samples were incubated in a shaker operating at 150 rpm and 25 °C for 1 h, and then centrifuged for 10 min at 4000g. The supernatants were transferred into new centrifuge tubes and again centrifuged for 10 min at 4000g to remove residual hematite NPs.29 It was checked that no measurable BSA was settled or absorbed on bottles in the absence of hematite NPs under these conditions. BSA concentrations in supernatants were determined using Bradford protein assay kit (Beyotime Institute of Biotechnology). The absorbance of samples at λ = 595 nm was measured by BioTek SynergyHT multidetection microplate reader, which was used to calculate the equilibrium concentration of BSA left in supernatant. Aggregation Kinetics. The initial aggregation rate constant (k) of hematite colloids was determined by monitoring the increase in Dh(t) with time (t):30 k∝
1 ⎛ d D h (t ) ⎞ ⎜ ⎟ N0 ⎝ dt ⎠t → 0
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RESULTS AND DISCUSSION Characteristics of Hematite NPs. The powder X-ray diffraction (XRD) pattern of the synthetic nanoparticles (SI Figure S1) revealed that only hematite phase was present. The average diameter of the particles measured by TEM was 9 ± 2 nm (SI Figure S2). The hydrodynamic diameter of hematite NPs in aquesou solution (pH 5.7, no NaCl addition) measured by DLS was 125 ± 18 nm (SI Figure S2), indicating NPs tended to aggregate at this pH. The properties of the synthetic hematite NPs and proteins used in this study are summarized in SI Table S1. Effect of Ionic Strength on Stability of NP−Protein Conjugates. Apparent attachment efficiencies of bare hematite NPs as a function of NaCl concentrations at pH 5.7 are shown in Figure 1A. The corresponding aggregation profiles are presented in SI Figure S3A. The aggregation behavior of bare hematite NPs in aqueous dispersions with NaCl (Figure 1A) and CaCl2 (SI Figure S4A) at pH 5.7 were consistent with the prediction of the classic DLVO theory. The attachment efficiency (α) of hematite nanoparticles increased with the increasing electrolyte concentrations, and approached to 1 when the electrolyte concentration reached CCC. The faster aggregation rates at elevated electrolyte concentrations are attributed to the compression of the electric double layer (EDL) and the decrease of surface potential. It was confirmed by the measured zeta potentials (ζ potentials) of hematite NPs, which decreased with increasing NaCl concentrations (Figure 2A). Since the CCC represents the minimum amount of electrolyte needed to eliminate the repulsive energy barrier for rapid aggregation, it was used to assess the colloidal stability of hematite NPs under various experimental conditions.34 The CCCs of hematite NPs in the presence of NaCl and CaCl2 at pH 5.7 were 69 mM and 10 mM, respectively. It is consistent with the prediction of the Schulze-Hardy Rule that the CCC is inversely proportional to the valence of cations.4 The attachment efficiencies of 16 mg/L hematite NPs in the presence of 8 mg/L Cyt with increasing NaCl concentrations at pH 5.7 are presented in Figure 1A. It shows that α of NP-Cyt conjugates increased as NaCl (Figure 1A) or CaCl2 (SI Figure S4A) concentrations increased. The trend is similar to that of bare NPs, suggesting the principal interactions in this case were governed by DLVO forces. However, the presence of Cyt increased the CCCs to 113 mM in NaCl and 48 mM in CaCl2, respectively. It is probably due to the additional contribution
(1)
where N0 is the initial particle concentration. The value of (dDh(t)/dt)t→0 was obtained through a linear least-squares regression analysis of the initial increase in Dh up to the point where Dh(t) reaches 1.50Dh,0.31 At low electrolyte concentrations or in the presence of NPs stabilization induced by proteins, however, the hydrodynamic diameter failed to reach 1.5Dh,0. Under such conditions, the linear regression was performed even if it ends before 1.5Dh,0, but the y-intercept of the fitted line did not exceed 3 nm in excess of Dh,0 for all cases.32 The aggregation attachment efficiency (α), that is, the probability that two particles attach, is used to quantify the initial aggregation kinetics of hematite colloids. It is calculated by normalizing the k obtained in the solution of interest to the diffusion-limited aggregation rate constant kfast determined under favorable aggregation:30−33
α=
k k fast
=
1 N0 1 (N0)fast
( (
dDh(t ) dt dDh(t ) dt
) )
t→0
t → 0,fast
(2)
To calculate α in the presence of proteins, (dDh(t)/dt)t→0,fast is obtained for the same type of hematite colloids in the absence of proteins, but in the same electrolyte under favorable aggregation conditions.31,33 The critical coagulation concentrations (CCC), above which aggregation processes change from reaction-limited cluster aggregation (RLCA) to diffusionlimited cluster aggregation (DLCA), were derived from the intersection of extrapolated lines through both regimes. Copper(II) Adsorption. Cupper (II) adsorption on bare hematite NPs and BSA-NP conjugates in solutions with different ionic strength was studied. The protein solution was adjusted to the desired ionic strength (10 mM, 40 mM, or 100 mM NaCl) at pH 5.7. The hematite NP suspension was sonicated for 10 min and then added to the protein solutions. 2230
DOI: 10.1021/acs.est.5b05298 Environ. Sci. Technol. 2016, 50, 2228−2235
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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 five measurements of samples at each electrolyte concentration, and the error bars represent standard deviations.
the weakening of the electrosteric forces at elevated salt concentrations. On the contrary, increasing NaCl (Figure 1B) or CaCl2 concentrations (SI Figure S4B) decreased attachment efficiencies of 16 mg/L hematite NPs in the presence of 8 mg/L BSA at pH 5.7. At low salt concentration ([NaCl] < ∼ 40 mM, or [CaCl2] < ∼ 8 mM), BSA induced fast aggregation of hematite NPs and resulted in the higher attachment efficiencies than bare NPs. Nevertheless, hematite NPs were totally stabilized (α → 0) by BSA at [NaCl] > 48 mM or [CaCl2] > 19 mM. The deviation from DLVO theory is presumably attributed to nonDLVO forces originated from BSA that adsorbed on NP surfaces. The pHiep of BSA is 4.76 in 1 mM NaCl and 4.51 in 0.1 M NaCl,36 so at pH 5.7 the adsorption of negatively charged BSA onto the positively charged hematite surface is favorable. The negative zeta potentials of hematite-BSA conjugates at pH 5.7 (Figure 2A) also suggested the adsorption of BSA on hematite NPs. The charge reversal is a most characteristic phenomenon, when charged polymers adsorb to oppositely charged substrates.37 The BSA induced aggregation of hematite NPs at low salt concentration can be interpreted with the attractive patchcharge interaction.37 The adsorption of BSA on metal oxide surface starts from the formation of a side-on monolayer (the minor axes of BSA perpendicular to the oxide surface), and then proceeds to the generation of dimers as a result of protein−protein interaction with the increase of BSA
Figure 1. Comparison of attachment efficiencies of 16 mg/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.
from repulsive electrosteric force originating from Cyt adsorption. The isoelectric point of Cyt is 10.37 (SI Table S1). At pH 5.7, both hematite and Cyt possessed positively charged surfaces, but a certain level of Cyt adsorption on hematite surface might occur due to the inhomogeneous charge distribution of Cyt or van der Waals attractive forces.35 It is confirmed by the higher zeta potentials of Cyt-NP conjugates (Figure 2A). Moreover, ζ potentials of Cyt-hematite conjugates decreased with the increase of NaCl concentrations, suggesting 2231
DOI: 10.1021/acs.est.5b05298 Environ. Sci. Technol. 2016, 50, 2228−2235
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Environmental Science & Technology concentration.29 The calculated amount of absorbed BSA for a side-on monolayer on NPs is in the range of 223 ng/cm2 to 365 ng/cm2.29 In this study, the initial concentrations of BSA and hematite NPs were 8 mg/L and 16 mg/L, respectively. Based on the geometric specific surface area of 9 nm spherical hematite NPs (127 m2/g), the amount of added BSA normalized by the NP surface area was 390 ng/cm2. Adsorption isotherm of BSA on hematite surfaces at pH 5.7 as a function of BSA added was shown in Figure 3. When the amount of added
not form a monolayer on NPs and a part of NP surface was exposed for Cu2+ adsorption. Therefore, the negatively charged patches of BSA were likely to form on the positively charged hematite surfaces under the conditions of the present study. The morphology of BSA can be approximated by an equilateral triangular prism with sides about 9 nm and a height of about 5.5 nm, so the thickness of a side-on monolayer of BSA is about 2.5−5.5 nm.39 When [NaCl] = 10 mM, the Debye−Hüchel length (κ−1) is estimated to be 3 nm according to the following equation:
κ −1 =
εε0kBT 2NAIe 2
(3)
where ε0 is the permittivity of vacuum, ε is the dielectric constant of water, T is temperature, NA is Avogadro’s number, kB is the Boltzmann constant, e is the elementary charge, and I is the ionic strength. Therefore, at low salt concentration ([NaCl] < ∼ 40 mM, or [CaCl2] < ∼ 8 mM), the thickness of the adsorbed BSA layer was comparable to the Debye length. The BSA-rich regime and hematite surface/the BSA-poor region possess opposite charges at pH 5.7, leading to a lateral heterogeneity in surface charge on the hematite−BSA conjugates. When two BSA-NP conjugates approach each other, attractive electrostatic interaction between the BSA-poor region of one conjugate and the BSA-rich domain of the other could develop,38 resulting in the patch-charge attractive interaction. This attractive non-DLVO force is substantially stronger than the van der Waals force,37 especially for proteins with high molecular mass and at low salt levels, so hematiteBSA conjugates aggregated more readily than bare hematite NPs at low ionic strength. At high salt levels, the patch-charge attractive force is screened as a regular EDL force,37 so the steric repulsive force caused by BSA on NP surfaces stabilized NPs in aqueous dispersions. Increasing NaCl concentration did not promote the adsorption of BSA on hematite NPs (SI Figure S5). Therefore, the stabilization of BSA−NP conjugates at high ionic strength could not be attributed to the stronger steric force resulting from a larger amount of BSA adsorbed on NP surface. Depletion and bridging interactions may also lead to NP destabilization. However, the bridging processes are rare at low salt levels,37 and depletion interaction become important only at high polyelectrolyte concentrations. Considering the low salt and protein concentrations used in this study, the observed fast aggregation of BSA-NP conjugates could not be attributed to these two kinds of non-DLVO interactions. The size of surface heterogeneities is the key factor for the patch-charge attractive force.37 With decreasing molecular mass, the size of charged patches decreases and the additional non-DLVO force tends to disappear. That is the reason why the patch-charge attraction was not observed in the aggregation studies of hematite NPs in the presence of alginate or humic acid at near neutral pHs.32,40 HA/alginate and hematite NPs were oppositely charged under the conditions though. Effect of pH on Hematite-Protein Aggregation. The stability of hematite NPs with BSA or Cyt at pH 9 was shown in Figure 1C. Bare hematite NPs were in the DLCA regime over the entire range of NaCl concentrations (Figure 1C). It is related to the low ζ potentials of hematite NPs at pH 9 (Figure 2B). The Nernst equation indicates that the surface potential (E) of a solid is directly proportional to the difference between the solution pH and its pHiep at room temperature.4 As pH
Figure 3. BSA adsorption isotherms on hematite NPs plotted as a function of BSA added after 1 h 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.
BSA was 390 ng/cm2, the corresponding amount of BSA absorbed was approximately 105 ng/cm2. Thus, under the conditions of the aggregation study, the amount of adsorbed BSA on hematite NPs could be not enough to form a side-on monolayer (223 ng/cm2). Moreover, disproportion and polarization of BSA could also lead to BSA-poor and BSArich regions on NP surfaces.38 Besides, the adsorption of Cu2+ on BSA-NP aggregates (Figure 4) also implied that BSA did
Figure 4. 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 4 h adsorption experiments. 2232
DOI: 10.1021/acs.est.5b05298 Environ. Sci. Technol. 2016, 50, 2228−2235
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Environmental Science & Technology increased from 5.7 to 9, it became closer to the pHiep of hematite, resulting the decrease of ζ potentials. The interaction energy of electrostatic repulsion between two equal-sized NPs is proportional to E2. Consequently, the repulsive energy barrier dramatically decreased at pH 9 due to the decrease of E, resulting in the fast aggregation. At pH 9, the addition of Cyt hindered the aggregation of hematite NPs at low salt concentrations. The trend of α for Cyt-NP conjugates as a function of salt concentrations at pH 9 was similar to that at pH 5.7, but the CCCs at pH 9 was obviously smaller than the value at pH 5.7(Figure 1). It could be related to the lower zeta potentials of Cyt-NPs at pH 9 (Figure 2B). Considering pHiep of Cyt is 10.37 (SI Table S1), the surface potential of NP-Cyt conjugates at pH 9 is much less than that at pH 5.7. Thus, the contribution from electrosteric repulsion by adsorbed Cyt at pH 9 was smaller than at pH 5.7. It implies that the stabilization of NPs by proteins is more efficient at pH far from pHiep of proteins. In addition, it is worth to mention that the maximum attachment efficiencies were less than 1 in the case with Cyt at both pH 5.7 and pH 9, which suggested the contribution of steric stabilization by adsorbed Cyt.41,42 Even though the aggregation of bare hematite NPs is more favorable at pH 9, BSA-hematite conjugates presented a higher stability at pH 9 than at pH 5.7. As shown in Figure 1C, the attachment efficiencies of hematite NPs with 390 ng/cm2 of BSA were around zero at pH 9 over the entire range of salt concentrations. The addition of BSA decreased the zeta potentials of hematite NPs at pH 9 down to −15 mV (Figure 2B), indicating the adsorption of BSA on hematite NPs. The higher negative charge at pH 9 promoted electrosteric stabilization by adsorbed BSA. The attractive patch-charge interactions were not observed at pH 9, probably because the surface potential of hematite NPs was close to zero. It is substantial only when the patches and the substrate are oppositely charged. The lack of this attractive interaction at pH 9 also facilitated the stabilization of NPs by BSA. Aggregation of NPs through cation bridging, especially with Ca2+, has been widely observed for NPs with fulvic acids, humic acids, polysaccharides, and any other DOMs.7 Moreover, Cabridging is more sufficient at alkaline pH, owning to the lack of the competitive adsorption of protons7. However, the aggregation behavior of hematite NPs with proteins in CaCl2 solution (SI Figure S4) at pH 5.7 or 9 was similar to that in NaCl solution (Figure 1). It indicated that CaCl2, over the concentration range studied (up to 150 mM), did not lead to interparticle bridging of bare hematite NPs or protein-hematite conjugates. Effect of Protein Size and Concentration on Stability of Hematite NPs. The attachment efficiencies of protein-NP conjugates as a function of protein concentration are presented in Figure 5. When the NP concentration was fixed, increasing protein concentration led to the decreasing α of NPs over the range of 0−1200 ng/cm2. Protein surface coverage and the thickness of protein coatings are positively related to the initial protein concentration.43 When the NPs surface is totally coated by thick protein layers, the attractive patch-charge interactions could be eliminated, and the steric repulsion between adsorbed proteins could totally stabilize NPs. However, in some cases, high protein surface coverage or excess proteins in dispersions might also drive aggregation of NPs by a bridging mechanism43 or depletion attraction.18 In this study, relatively low concentrations of proteins and NPs were used in order to
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 25 °C. The concentration of added NPs was 16 mg/L for all experiments.
study the aggregation behavior of protein-NP conjugates under environmentally relevant conditions. As shown in Figure 5, about 32 mg/L Cyt was needed to stabilize 16 mg/L hematite NPs in 60 mM (>CCC) NaCl at pH 5.7. Nevertheless, only about 9 mg/L BSA was sufficient to stabilize the same amount of NPs. At pH 5.7, the surface potential of Cyt-NP conjugates was higher than that of BSA-NP conjugates (Figure 2A), but BSA presented the higher ability to stabilize NPs. It implied that steric repulsive force is more efficient than electrostatic repulsive force in stabilizing NPs. The size of BSA is 5.5 × 5.5 × 9 nm,29 while that of Cyt is 3 × 3.4 × 3.4 nm.44 Thus, a higher molar ratio of proteins to NPs is needed for Cyt to form a monolayer on NP surface. The thickness of adsorbed protein layer (δ) is proportional to the size of proteins, so the BSA monolayer is thicker than the Cyt monolayer. Under the conditions studied, NPs were stabilized at high salt concentrations mainly by steric repulsive force. Compared to Cyt, BSA is a better stabilizer for hematite NPs, owing to the fewer proteins needed for completely covering NP surfaces and the formation of sufficiently thick coating to maintain a high steric free energy of interaction at high salt concentrations. Effect of Aggregation State on Metal Uptake. In order to investigate the effects of protein adsorption and NP aggregation on heavy metal ion sequestration, copper uptake experiments were performed on bare hematite NPs and BSAhematite conjugates over a range of NaCl concentrations at pH 5.7 (Figure 4). As NaCl concentration increased from 10 to 100 mM, the percentage of Cu2+ adsorbed on bare hematite surfaces decreased from 72.5 ± 4% to 59.1 ± 0.5%. It indicates that the aggregation of hematite NPs induced at elevated salt concentrations could evidently reduce copper uptake, probably due to the decreasing amount of reactive surface sites available for Cu2+ adsorption. Previous studies showed that characteristics of iron oxyhydroxide NP aggregates, such as morphological, structural, surface charge, and surface area differences have a noticeable effect on copper retention.5,45 In the presence of 390 ng/cm2 BSA, the percentage of Cu adsorbed on NPs increased from 58.0 ± 2.4% to 71.2 ± 3.3%, as NaCl concentration increased from 10 to 100 mM. Control experiments showed that no measurable Cu adsorption was observed in the BSA solution. The higher capacity of NPs for Cu adsorption at elevated salt concentrations might be 2233
DOI: 10.1021/acs.est.5b05298 Environ. Sci. Technol. 2016, 50, 2228−2235
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attributed to inhibition of NP aggregation by BSA. In the experiments of both bare hematite and BSA-NP conjugates, the trends of capacity for Cu adsorption as a function of salt concentrations were consistent with the changes of aggregation state. It implies the importance of aggregation state in copper uptake on hematite NPs. The NP aggregation was inhibited by the addition of BSA at high salt concentration, which could increase the surface area for Cu adsorption. The decrease of BSA adsorption on NPs with the increase of salt concentration (SI Figure S5) could also contribute to the higher capacity for Cu adsorption at higher NaCl concentration. However, the TEM images of BSA-NP conjugates (SI Figure S6) showed that the aggregates of BSA-NP conjugates in 10 mM NaCl solution obviously had a more compact structure than those in 80 mM NaCl solution. Some internal surface area became accessible for Cu adsorption, when the aggregate structure turned to an open and loose structure. It could be the main reason for the higher adsorption capacity of BSA-NP conjugates at the higher ionic strength. Environmental Implications. This study showed the different impact of two model globular proteins on NP aggregation in aqueous dispersions under environmentally relevant conditions. Results from this study suggested that the aggregation state of NPs in the presence of proteins depended on both solution properties, like pH and ionic strength, and protein properties, such as molecular weight, pHiep, protein concentration, etc. Compared to Cyt, BSA with the larger molecular weight were more efficient to stabilize NPs, considering the thicker protein layers could be formed by the less amount of BSA. On the other hand, the attractive patchcharge interaction could induce fast NP aggregation, when BSA formed a laterally heterogeneous layer on hematite NPs. This non-DLVO force is substantially stronger than the van der Waals force,37 and especially important for proteins with high molecular mass at low salt levels. Under certain conditions, other non-DLVO forces, including depletion and bridging forces, could become important to the aggregation of NP− protein conjugates. Further work is needed to elucidate the precise conditions where these non-DLVO forces play a key role in determining NP aggregation. Proteins are important biomacromolecules in all organisms and also widespread in surface waters. The adsorption of proteins on NP can induce or inhibit NP aggregation, depending on both solution conditions and protein properties. The correlation between NP aggregation and capacity for metal uptake observed in this study suggested that protein−NP conjugates could be efficient materials for metal removal from wastewater under harsh conditions. Understanding the influence of proteins on NP aggregation, as shown in this study, is essential to evaluate the reactivity and toxicity of NPs in biogeochemical processes, and to assess the influence of NPs on the fate and transport of pollutants in aqueous environments, as well as to assist the functionalization of NPs for a large variety of environmental and biotechnological applications.
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Article
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-10-62754292-808; e-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by National Basic Research Program of China (973 Program, 2014CB846001) and National Natural Science Foundation of China (41472306).
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REFERENCES
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b05298. Additional figures and details for Materials and Methods and Results and Discussion are presented (PDF) 2234
DOI: 10.1021/acs.est.5b05298 Environ. Sci. Technol. 2016, 50, 2228−2235
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DOI: 10.1021/acs.est.5b05298 Environ. Sci. Technol. 2016, 50, 2228−2235
