In Situ Observation of Hematite Nanoparticle Aggregates Using Liquid

Apr 29, 2016 - School of Environmental Sciences and Engineering, Peking University, Beijing, China, 100871. ‡ Beijing Institute of Nanoenergy and Na...
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In Situ Observation of Hematite Nanoparticle Aggregates Using Liquid Cell Transmission Electron Microscopy Juan Liu,*,† Zhiwei Wang,*,‡ Anxu Sheng,† Feng Liu,† Fuyu Qin,‡ and Zhong Lin Wang*,‡,§ †

School of Environmental Sciences and Engineering, Peking University, Beijing, China, 100871 Beijing Institute of Nanoenergy and Nanosystems, Chinese Academy of Sciences, Beijing, China, 100083 § School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0245, United States ‡

S Supporting Information *

ABSTRACT: Aggregation of nanoparticles impacts their reactivity, stability, transport, and fate in aqueous environments, but limited methods are available to characterize structural features and movement of aggregates in liquid. Here, liquid cell transmission electron microscopy (LCTEM) was utilized to directly observe the size, morphology, and motion of aggregates that were composed of 9 and 36 nm hematite nanoparticles, respectively, in water or NaCl solution. When mass concentrations were same, the aggregates of 9 nm nanoparticles were statistically more compact and slightly larger than those of 36 nm nanoparticles. Aggregates in both samples were typically nonspherical. Increasing ionic strength resulted in larger aggregates, and also enhanced the stability of aggregates under electron-beam irradiation. In water, small aggregates moved randomly and approached repeatedly to large aggregates before final attachment. In NaCl solution, small aggregates moved directly toward large aggregates and attached to the latter quickly. This observation provided a direct confirmation of the DLVO theory that the energy barrier to aggregation is higher in water than in salt solutions. This study not only presented the influences of particle size and ionic strength on aggregation state, but also demonstrated that LCTEM is a promising method to link aggregation state to dynamic processes of nanoparticles.

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INTRODUCTION Nanoparticles (NPs) are widespread in natural aqueous environments, resulting from diverse abiotic or biotic processes, as well as anthropogenic activities.1 In recent years, a lot of efforts have been made to investigate the size-dependent properties of NPs in biogeochemical processes.2−4 However, the tendency of NPs to aggregate in aqueous dispersion complicates these studies. Moreover, aggregation of NPs may evidently influence their reactivity, toxicity, transformation, transport, and fate in natural waters.5 Thus, understanding the evolution of aggregate size and morphology, as chemical properties of aqueous solutions change, is critical to predict the behavior of NPs in natural waters. Despite the importance of NP aggregation in many fields, advanced methods are still needed to better characterize the size and morphology evolution of NP aggregates in situ, or in vivo, or under environmentally relevant conditions. The prevailing experimental approaches for aggregation characterization are light scattering techniques, such as dynamic light scattering (DLS) and static light scattering (SLS). Up to date, within the field of environmental science, the work on NP aggregation in aqueous solution focused on aggregation kinetics. Very little was known about the morphology evolution of NP aggregates in solution. Moreover, the accuracy of light scattering techniques is limited by the presence of large clusters or particles, the concentration of primary particles, and aggregate shape.6 Transmission electron microscopy (TEM) based methods allow the investigation of the two-dimensional (2D) projected © 2016 American Chemical Society

image of each NP aggregate over a relatively large size range with down to subnanometer resolution.7 However, traditional TEM sample preparation methods usually lead to undesirable alteration of fragile aggregates. To avoid the alteration during sample drying, cryogenic TEM (cryo-TEM) has been introduced to preserve NP aggregates intact at cryogenic temperatures for direct imaging.8 Alternatively, a polymeric matrix or macromolecular agent can be used to “freeze” or stabilize NP aggregates at room temperature during their deposition on a TEM grid.9,10 However, these methods can only capture static snapshots of a dynamic process, which is difficult to represent the aggregation−disaggregation process. The size and morphology of NP aggregates are susceptible to subtle changes in solution conditions.8,11 The investigation of the dynamic aggregation− disaggregation process under various environmentally relevant conditions is crucial for evaluating mobility, bioavailability, and toxicity of NPs in aqueous environments. Recently, a burgeoning technique, in situ liquid-cell TEM (LCTEM), makes it possible to visualize the dynamic process of aggregation in fluid with sufficiently high spatial and temporal resolution. In a liquid cell, a layer of liquid containing NPs is sealed between two electron transparent windows in order to Received: Revised: Accepted: Published: 5606

December 24, 2015 March 14, 2016 April 29, 2016 April 29, 2016 DOI: 10.1021/acs.est.5b06305 Environ. Sci. Technol. 2016, 50, 5606−5613

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Environmental Science & Technology

individual hematite NP aggregate in Milli-Q water and in NaCl solution were observed through time-lapse TEM images. Image analysis and trajectories tracking as a function of time were used to quantitatively describe the movement of NP aggregates. Our results provide new insights into the influence of primary particle size and ionic strength on the size, morphology, and movement of NP aggregates in aqueous dispersion.

isolate it from the high vacuum in TEM. The windows are typically two ultrathin silicon nitride (SiN) membranes supported on silicon microchips.12 The thickness of windows and the liquid layer needs to be sufficiently thin (tens of nanometers to a few micrometers) to transmit electrons, which is the main limiting factor of the spatial resolution. With the development of thin-film technology and microfabrication, a variety of liquid cells became commercially available.13 LCTEM is now becoming a routine technique for studying liquid-phase processes.14 A few observations have been done using LCTEM to study NP aggregation and coalescence.15−18 Grogan et al.15 reported the cluster growth of 5 nm Au NP aggregates in water using a custom-made liquid cell. The observed fractal dimension of the aggregates is consistent with three-dimensional diffusion limited aggregation (DLA). Chen et al.16 suggested that interaction potential between Au nanorods could be measured from their sampled trajectories observed by LCTEM. These studies reveal that in situ LCTEM can provide unprecedented capabilities to investigate particle−particle interactions, including the aggregation process. However, the application of LCTEM in aggregation study is still in its infancy. More LCTEM studies need to be done under environmentally relevant conditions on NPs that are ubiquitous in nature. Aggregation of NPs is controlled by particle diffusion in solution and interparticle interactions. Not only solution conditions, like pH and ionic strength, but also inherent properties of NPs, such as particle size and shape, can influence aggregation state. Classic Derjaguin−Landau−Verwey−Overbeak (DLVO) theory predicts that, at electrolyte concentrations below the critical coagulation concentration (CCC), elevated concentrations of ionic species can compress the electric double layer (EDL) and screen the electrostatic repulsion, leading to rapid aggregation. Besides, ionic strength can also influence the aggregate morphology. Legg et al. suggested that the presence of sodium nitrate induced denser clusters of ferrihydrite NPs, based on small-angle X-ray scattering (SAXS) and cryo-TEM results.8 Nonetheless, the process, how aggregate structure changed with increasing ionic strength, had not been observed. On the other hand, the influence of primary particle size on aggregation is still a subject of controversy. According to classic DLVO theory, interaction energy barrier decreases as particle size decreases, and accordingly smaller particles aggregate more readily than larger particles. This theory considers interacting surfaces as infinitely smooth and flat, which are definitely not consistent with actual NP surfaces. When primary particle size decreases to the nanoscale, the heterogeneity and curvature of NP surfaces may challenge the DLVO prediction.19 He et al. reported different aggregation behavior for 12, 32, and 65 nm hematite NPs at the same pH or ionic strength conditions.20 However, different mass and particle number concentrations of hematite NPs with three different sizes were used in the DLS measurements. The influence of particle concentration was coupled into the effect of particle size, which complicated the interpretation of DLS results. In this study, we report in situ observation of hematite NP aggregates in aqueous dispersions by using in situ LCTEM. Hematite NPs is one of the most widespread and stable iron oxide minerals in nature. It can be a reference nanomaterial for mirroring the properties of many other metal oxides.21 Moreover, aggregation of hematite NPs is related to many biotic and abiotic geochemical processes. In this study, the aggregate size and morphology of 9 and 36 nm hematite nanoparticles were statistically compared from static LCTEM images of 80−100 individual aggregates. The different stability and movement of

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MATERIALS AND METHODS Nanoparticle Synthesis. Hematite NPs in two sizes were synthesized by forced hydrolysis of ferric nitrate solution according to the methods reported by Schwertmann and Cornell.22 More details on synthesis are described in the Supporting Information (section S1). Nanoparticle Characterization. The crystalline phases of synthetic nanoparticles were characterized by powder X-ray diffraction using a Rigaku D/MAX-2000 diffractometer with monochromatic Cu Kα radiation (λ= 0.15406 nm) at a scan rate of 0.02 2θ·s−1. The samples for XRD measurements were prepared by drying the concentrated hematite suspensions in air at ∼40 °C and then loading onto a glass holder. Particle concentrations in nanoparticle suspensions were measured by acid digestion. The hematite NP suspension was sonicated for 10 min just before acid digestion. 0.1 mL of NP suspension was digested in 9.9 mL of 5 M HCl overnight on a shaker. Then, 0.02 mL digested solution was diluted using 4.98 mL of 2% HNO3 solution. Total Fe molar concentration in the solution was determined with an inductively coupled plasma optical emission spectrometry (Prodigy High Dispersion ICPOES, Teledyne Leeman Laboratories, Hudson, NH, USA). The experiments were carried out in triplicates to determine the mean and standard error of Fe2O3 NP concentration in suspensions. The particle size and morphology of HM1 and HM2 were determined by bright-field TEM imaging 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 from randomly selected areas on TEM images.2 Liquid Cell TEM (LCTEM). Liquid TEM experiments were carried out in a Poseidon 210 liquid fluid holder (Protochips inc.) with liquid cell E-chips containing silicon nitride amorphous windows of 30−50 nm thick. Prior to loading a liquid sample, the E-chips were rinsed in acetone and ethanol solvents (2 min for each) to remove the protective coatings, followed by an immediate canned air-dry. Subsequently, plasma cleaning (9:1 argon/oxygen mixture) was applied to the E-chips for about 5 min in IoN 40 plasma processing system (PVA TePla America Inc.), in order to change the silicon nitride membranes from hydrophobic to hydrophilic and also remove possible organic contaminants on the surfaces. LCTEM investigation was performed on a FEI F20 TEM operated at 200 kV. All the images present in this paper were recorded in parallel-beam TEM mode. Time-lapse TEM imaging was carried out generally with a dwell time of 0.25 s per frame and tens of frames at least for each sequence. For each experiment, hematite stock suspension was sonicated (150 W, 40 kHz) for 10 min and then diluted with Milli-Q water. The final concentration of hematite nanoparticles in all suspensions was 1.2 mM. To compare the movements of aggregates in water and in NaCl solution, fluid cell E-chips were used. Hematite NPs in Milli-Q water were first observed, and then the 93 mM NaCl solution was pumped through the fluid cell 5607

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was larger than that of HM2, which was consistent with the primary particle sizes observed by TEM (Figure 1). Structural Features of Hematite NP Aggregates. To obtain the general morphological properties of NP aggregates in suspension and diminish the electron beam (EB) effect on aggregates, the snapshot of each NP aggregate was taken quickly, and then immediately moved to next randomly selected area. Static images of 80−100 NP aggregates were taken in each sample. A direct measurement of the 2D projected image of each aggregate was conducted with the aid of image analysis software (see SI section 2). The statistical results of the measurements are summarized in Table 1. The morphology of aggregates in the NaCl solution was not measured, because most of them extended out of the field of view under the imaging conditions used in this study. The concentration of the NaCl solution, ∼ 93 mM, was larger than the CCC for 9 or 36 nm hematite NPs at near neutral pH,20 so NPs tended to form large aggregates quickly. The projected area equivalent diameter, or mobility-equivalent diameter (Dm), of an aggregate can be derived from its projected area (Aa) that is obtained from the binarized TEM image:26

using a syringe pump (Harvard Apparatus, Pump 11 Elite) at an infuse rate of 300 μL/h. All the TEM imaging was performed in static mode (no liquid flow) to avoid the effect of advection on the aggregation study. The parameters of aggregate size and morphology were measured from TEM images using the ImageJ software23 with FracLac plugin.24 Motion analyses of aggregates were performed using ImageJ with MTrackJ plugin.25 Additional information on TEM image processing and analysis is provided in the Supporting Information (section S2). Dissolution of hematite NPs induced by electron beam irradiation in Milli-Q water was observed in some cases. No recrystallization of dissolved ions was observed under the conditions used in this study. Whether or not dissolution can occur depends on many factors, such as dose rate, irradiation time, etc. It is beyond the scope of this paper, and will be separately addressed in a subsequent paper. All static images and movies in this paper were acquired under conditions when no obvious dissolution was observed. Dynamic Light Scattering (DLS). The hydrodynamic diameters (Dh) of hematite nanoparticles in Milli-Q water was measured on a Zetasizer (Nano ZS90, Malvern, UK) operating with a He−Ne laser at a wavelength of 633 nm and a scattering angle of 90°. The concentration of nanoparticles in all samples was fixed at 1.2 mM. The nanoparticle suspension was sonicated in a bath sonicator (150 W, 40 kHz) for 10 min before dilution to 1.2 mM, and then transferred to a 10 mm diameter polystyrene cuvette (Sarstedt, Germany) for DLS measurements. The preequilibrium time was 10 min. Each sample was measured at least three runs with 200 measurements for each run.

Dm = 2(Aa /π )1/2

(1)

When the NP concentration of HM1 and HM2 was 1.2 mM, the Dm of HM1 (153 ± 8 nm) was 1.3 times larger than that of HM2 (117 ± 14 nm) (Table 1, Figure S2). The two sample t-test indicated that the means of Dm for HM1 and HM2 were significantly different at the 0.05 level. The mean Dm measured from 2D projected TEM images was close to the Dh given by DLS (Table 1). Besides, Dh of HM1 was larger than that of HM2, which agreed with the trend of Dm. The larger NP aggregates of HM1 could be attributed to its higher particle concentration. The mean Dp of HM2 is 4.2 times bigger than that of HM1. That means the mass (M) of each HM2 particle is 74 times larger than that of HM1, according to the equation: M = 1/6πDp3·ρ, where ρ is the density of hematite. In this study, the same mass/molar concentration was used for HM1 and HM2, so the particle number concentration of HM1 NPs (PNHM1) was 74 times more than that of HM2 (PNHM2). The relatively larger PNHM1 caused the higher probability of particle collisions and consequently the larger Dh and Dm of aggregates.27 On the other hand, HM1 NPs had the higher surface energy and the smaller interaction energy barrier for aggregation, which could also lead to the larger aggregates.19 However, it is difficult to separate the contribution of different particle number concentration from the observed size effect. If the same particle number concentration was used for HM1 and HM2, the solid-to-solution ratio in HM2 would be 74 times larger than that in HM1. It might lead to a higher collision probability in HM2 and accordingly influence the aggregate size and morphology. The number of primary particles (N) in a NP aggregate can be calculated from the projected area (Aa) of the aggregate according to the following equation:7,28

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RESULTS AND DISCUSSION Characteristics of Synthetic Hematite NPs. The hematite NPs, HM1 and HM2, were synthesized similarly via forced hydrolysis of Fe(III) salt solution. The primary particle size (Dp) of HM1 and HM2 were 9 and 36 nm, respectively (Figure 1 and Table 1). TEM images show that HM1 NPs exhibited a rounded shape with rough surface, and HM2 NPs had a rhombohedral shape, in agreement with previous studies.20 XRD patterns (Figure S1) indicate that both HM1 and HM2 only exhibited the hematite phase. The full-width at half-maximum (fwhm) of HM1

⎛ A ⎞α N = ka⎜⎜ a ⎟⎟ ⎝ Ap ⎠

(2)

where Ap is the mean projected area of primary particles; ka is an empirical constant, and α is an empirical projected area exponent. The equation indicates that N increases exponentially with the increase of Aa or Dm (Figure S3A). In this study, we used the value ka = 1.10 and α = 1.08 to estimate N.28 Although these values were derived from the empirical studies of soot aggregates, they could be used to approximately estimate N of NP aggregates

Figure 1. TEM images and size distribution of HM1 (A and B) and HM2 (C and D) NPs. 5608

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Table 1. Structural Features of Hematite NP Aggregates that Were Derived from Static LCTEM Images of Individual Aggregatesa sample

no. of observations

HM1 82 HM2 106 t-testd

statistics

Dp nm

Dm nm

Dh nm

p

Df

Rg nm

mean SDb SEMc mean SD SEM

9 2 0.1 36 6 0.7 Y

153 123 8 117 82 14 Y

120 5.2 0.2 110 5.1 0.2 Y

2.38 2.55 0.28 2.07 0.43 0.04 N

1.40 0.17 0.02 1.27 0.19 0.02 Y

25 19 2.1 59 43 4.2 Y

a

The plots of raw data are shown in the Supporting Information Figure S2 and S3). Dp: primary particle size. Dm: mobility-equivalent diameter. Dh: hydrodynamic diameters by DLS. p: aspect ratio. Df: fractal dimension. Rg: radius of gyration. bStandard deviation. cStandard error of the mean. d Two-sample t-test was used to test if the difference between the means of the two samples is significant at significance level of 0.05.

p values of HM1 and HM2 were insignificantly different from each other at the 0.05 level, one sample t test showed that both p values were significantly greater than 1 (Figure S3B). The influence of p on Dt can be expressed as31

in suspensions. When Dm = 153 and 117 were used for HM1 and HM2 (Table 1), respectively, the NHM1 was 1 order of magnitude larger than NHM2. Therefore, there were much more primary NPs in HM1 than in HM2 under the conditions of the present study. The fractal dimension (Df) is a key parameter to quantitatively describe aggregate morphology, indicating the degree of compactness, or openness, of an aggregate.29 The higher Df indicates the more compact structure of aggregates. In this study, the 2D fractal dimension of NP aggregates was derived from projected TEM images using a box counting method, which estimated Df of each aggregate from its own self-similarity properties.7,30 The mean Df of HM2 was smaller than that of HM1 (Table 1), suggesting that HM2 aggregates had slightly more open structure, when the same molar/mass concentration was used. In addition to Df, the radius of gyration (Rg) is also thought to be an important parameter to describe aggregate morphology by characterizing the spatial distribution of mass in an aggregate. It is defined as the mean square distance of particles from the center of the mass of the aggregate, which can be calculated by7 Rg =

LW β

Dt =

kT 3πηDt

(5)

where Ct is the end correction coefficient (Ct = 0.312 + 0.565/p − 0.100/p2. For a nonspherical aggregate that has a p value greater than 1, say p = 2, the term of (ln p + Ct) is 1.26. However, in DLS measurements, this term is assumed to be 1, as shown in eq 4. Thus, the calculated Dh from DLS could be evidently smaller than the actual value. The nonspherical shape of HM1 and HM2 aggregates observed by in situ LCTEM (Table 1) emphasized the inherent problem of DLS in determining the size of NP aggregates. Movement of NP Aggregates in Water. In addition to allow a direct measurement of morphological features of each collected NP aggregate, LCTEM also makes it possible to directly observe nanoscale movement of NP aggregates in liquid. The typical motion of HM1 and HM2 nanoparticle aggregates in water is shown in Supporting Information movies S1 and S2, respectively. Because the points of zero charge of bare SiN and hematite NPs are about pH 4.1 and pH 7.8, respectively,20,32 hematite NP aggregates initially adhered to the negatively charged SiN membrane in Milli-Q water (pH ≈ 6) by electrostatic attraction and van der Waals force. After several seconds of EB irradiation, some of small aggregates or isolated NPs detached and started to move in a quasi-two-dimensional plane close to the SiN membrane, where the focal plane was.16 The reason for the detachment could be the formation of positive electrical charges on the SiN membrane, as a result of secondary emission by EB irradiation,33 which repelled the positively charged hematite NPs. The van der Waals force between a membrane and a cluster can be expressed as

(3)

where L and W are the maximum and minimum projected lengths of aggregates, respectively; the parameter β is calculated from β = (kf/kLW)1/Df. kLW is a 2D structural factor, and kf is the fractal prefactor (or structural coefficient). In this study, β ≈ 2.34 was used to estimate Rg of each aggregate.7 It has been reported that Rg decreases linearly with the increase of compactness.26 Therefore, the relatively smaller mean of Rg for HM1 (Table 1) might also indicate the more compact structure of HM1 aggregates. It is known that Dh of an aggregate given by DLS is calculated from the translational diffusion coefficient by using the Stokes− Einstein equation:

Dh =

1 kT (ln p + C t) 3 πηL

F=− (4)

kAR 6z 2

(6)

where ka is the Hamaker constant of a value amounting to 1.5 × 10−19 J for water, R is the radius of the cluster, and z is the distance between the cluster and the membrane.33 It indicates that the larger aggregates are attracted more strongly by the membrane. As shown in movies S1 and S2, some large aggregates were standing still during the observation time. In these cases, the electrostatic repulsion force between SiN membrane and NPs was not strong enough to overcome van der Waals attraction force. Nevertheless, in both HM1 and HM2 samples, many small aggregates were found to move continuously in a random manner. Translational and rotational, as well as rotational−

where Dt is translational diffusion coefficient, k is Boltzmann’s constant, T is absolute temperature, η is the viscosity of the medium. The definition of Dh is the diameter of a sphere that has the same Dt as the measured aggregate. Therefore, the equation used for DLS is based on the assumption that the aggregates are nearly spherical. However, the measured aspect ratio (p) of hematite aggregates indicated that, in both HM1 and HM2, the typical aggregates were nonspherical. The aspect ratio of each aggregate was derived from the maximum (L) and the minimum (W) projected lengths of the aggregate (p = L/W). Although the 5609

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occurred. The slope of MSD plot can be used to estimate the 2D diffusion coefficients,35 according to Dt,2D = ⟨x2⟩/4t. Based on the MSD data in the first 20 s (Figure 2C), Dt,2D of the HM1 and HM2 aggregates were 4189 and 4496 nm2/s, respectively. The similar diffusion coefficients indicate that aggregate size affects the diffusion of aggregates more prominently than aggregate morphology or primary particle size. The calculated Dt of 35 nm nanoparticle in water (η = 0.89 mPa·s) at 25 °C is 2.1 × 107 nm2/ s, according to eq 4, which is 4 orders of magnitude larger than the measured diffusion coefficients from the MSD data. The same restricted movement of NPs has been observed in previous LCTEM studies.33,35 The reason could be hydrodynamic hindrance near the interface between water and SiN membrane. The diffusion coefficient of NPs moving parallel to a wall (D∥) can be expressed as33

translational coupling, movements of small aggregates were observed in both samples. The detachment of aggregates from the membrane could not be due to the beam-induced heating, because heating is typically insignificant in LCTEM.12,34 Figure 2A and B presents the TEM images of two NP aggregates in HM1 and HM2 samples respectively, which have

D = λ −1Dt

where λ∥−1 is the correction factor. It is directly proportional to the size of nanoparticles (Dm) and inversely proportional to the distance between the NP center and the wall (z). Figure 2 shows that the HM1 and the HM2 aggregates had the similar Dm. The image contrast indicates that they were close to the focal plane, so the values of z and the corresponding λ∥−1, as well as D∥, for these two aggregates could be very close. Thus, the observed Dt values and MSD plots for them were similar, as a result of the similar degree of hydrodynamic hindrance. During the last 5 s, the HM1 aggregate presented the relatively slower movement than the HM2 aggregate, because it attached to a neighboring aggregate (Figure 1A and movie S1). Influence of Electrolyte on Aggregate Movement. The effect of electrolyte on aggregation state was studied by pumping a sodium chloride solution through a liquid flow holder with HM1 sample. The typical TEM images of the same HM1 NP suspension before and after the addition of NaCl are shown in Figure S4. In NaCl solution, fewer small aggregates or isolated NPs were observed, and most NP aggregates became more compact. This suggests that the aggregate structure in this case was not determined by the DLA process that the more “open” aggregate structure forms at high ionic strength. The similar impact of ionic strength on aggregate structure was observed in the previous aggregation study of ferrihydrite NPs.8 Cryo-TEM and SAXS results showed that millimolar concentrations of NaNO3 are sufficient to trigger the growth and collapse of aggregates into denser clusters. Legg et al. attributed the aggregate structural changes to the decrease in suspension stability and the facilitation of nanoparticle deposition in salt solutions.8 It is worth mentioning that the stability of nanoparticle aggregates was obviously enhanced by adding the NaCl solution. Under the similar dose of EB irradiation, the NP aggregates in Milli-Q water moved fast out of the irradiated area, but those in the NaCl solution barely moved during the observation time (Figure 3). The fast movement of aggregates under the EB irradiation in water is presumably attributed to the charging of the SiN membrane and NPs induced by EB irradiation. The electrostatic repulsion between the charged NP aggregates and the membrane could be the driving force for the fast movement of aggregates in water. On the other hand, the presence of the concentrated NaCl solution could increase the conductivity of the solution and diminish the charging effect. Moreover, the relatively larger aggregates were formed in the NaCl solution, so the van der Waals attractive force between NP aggregates and the

Figure 2. Comparison of the motion of HM1 and HM2 aggregates. TEM images of the HM1 (A) and the HM2 (B) aggregates at the start time with their movement trajectories in 25 s shown in colored curves. The color corresponds to the time. The red dotted circle indicates the position of the aggregate in the image. (C) Mean-square displacement, MSD, vs time of the HM1 (square) and HM2 (circle) aggregates.

similar Dm (30−40 nm). The trajectories of the aggregates were extracted from movies S1 and S2, which are indicated as colored lines in Figure 2A and B. It can be seen from the trajectories that the aggregates moved without a directional preference within the observation time span. The random walk or Brownian motion of NP aggregates in horizontal direction was analyzed quantitatively using the mean square displacement (MSD), which is defined as MSD =

1 n

n

xi (τ ) − ⇀ xi (0))2 ∑ (⇀ i=1

(8)

(7)

xi (0) and ⇀ xi (τ ) are the positions of the trajectory i at time where ⇀ 0 and time τ, respectively. n is the number of trajectories.33 Figure 2C shows the plots of the MSD(⟨x2⟩) vs time (t) for the two aggregates in Figure 2A and B. The MSD approximately linearly increased with time, except within the first several seconds when the initial detachment of aggregates from SiN membrane 5610

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Figure 3. (upper row) Fast movement of the HM1 aggregate with time under the EB irradiation with an estimated dose rate of 73 electrons/(s Å2) in water. The aggregate was repelled from the irradiated area by the electrostatic repulsion due to charging of the membrane and particles. (lower row) HM1 aggregate staying stable with time under EB irradiation with an estimated dose rate of 83 electrons/(s Å2) in NaCl solution. The arrows in the upper left corner of images indicate the subtle change of the nanoparticles on the edge of the aggregate with time.

Figure 4. Cluster−cluster aggregation of HM1 sample in water (upper) and NaCl (lower) solution. (A and D) TEM images of HM1 aggregates in water and in NaCl solution, respectively, at the start time with the movement trajectories (colored curves) in 25 s. The tracked small aggregates are indicated by the red dotted circles. The red × in A and D indicates the position of the reference points. (B and E) TEM images of the aggregates at t = 25 s. (C and F) Distance between the tracked aggregate and the reference point vs time in water and in NaCl solution, respectively.

4A−C showed the attachment of a small cluster to the bigger aggregate in water (also see movie S1). The trajectory in Figure 4A indicates that the small cluster diffused in a nondirectional manner. We arbitrarily chose a point on the large aggregate that was nearly static in the observation as a reference point. The distance between the center of the small cluster and the reference point was measured as a function a time (Figure 4C). The distance to the reference point unregularly changed with time, which suggests the random walk of the small cluster in water before the attachment. On the contrary, in the NaCl solution, three small clusters moved directly toward the large aggregate prior to the attachment (Figure 4D and E, movie S3). The

SiN membrane became even stronger (eq 6). It inhibited the detachment and movement of aggregates. This finding implies that the immobilization of NP aggregates in LCTEM studies could be achieved by elevating electrolyte concentration. A common artifact in LCTEM experiments is repulsion of free diffusion NPs instantly from the field of view.36 In order to slow down the movement of NPs, a water−glycerol mixture was widely used in LCTEM studies.33,35 Thus, adding electrolyte could be an alternative method to mitigate this artifact, when glycerol might be an interference factor. Another interesting effect of electrolyte on NP aggregation is that it changed the process of cluster−cluster aggregation. Figure 5611

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The present study demonstrates that LCTEM could be used to directly determine the size and structure of fragile NP aggregates in aqueous dispersion, and also facilitate real-time observations of the interaction between individual aggregates. This capability of LCTEM allows a mechanistic investigation of dynamic processes involving NP aggregates. Moreover, the flow through liquid cell allows in situ studies on how solution properties affect NP aggregation with the high resolution of the electron microscope. LCTEM is a promising technique to in situ observe a range of different specimens encompassing geochemical and environmental science research, but artifacts, such as contamination, charging, unwanted crystal growth or dissolution, etc., must be taken into consideration for the interpretation of observations.36

distance between the small clusters and the reference point on the large aggregate linearly decreased with time (After ∼13 s, the small clusters attached to the large one, so their distance to the reference point did not change any more). One reason for the direct attachment in the NaCl solution was probably related to the larger interaction force between the clusters and the membrane. As mentioned above, EB irradiation could result in the electrostatic repulsive force between hematite NPs and the membrane in water. The addition of NaCl could weaken this electrostatic repulsive force and decrease the distance (z) between them. According to eq 6, the interaction force between the membrane and the clusters is inversely proportional to z2, so in the NaCl solution the stronger resistance from the membrane probably limited the random motion of aggregates. Another reason could be that the elevated ionic strength lowered the energy barrier for the cluster−cluster aggregation. In water, each aggregate is enveloped by a repulsive and anisotropic “cloud” of a radius. Only when the repulsive clouds of two aggregates experience the least overlap, they can come close to each other.16 It can be seen from movie S1 and Figure 4A that the small cluster moved in close proximity to the large aggregate and approached to it from several different orientations before the final attachment. The small cluster might be repulsed by the repulsive “cloud” of the large aggregate and try different ways to overcome the interaction energy barrier. In NaCl solution, the electrostatic repulsion was screened, and the short-range interaction between aggregates is dominated by the van der Waals attraction. It facilitated the final attachment process. When the distance between two aggregates was larger than the range of the van der Waals force, the long-range attraction, which is commonly observed between identically charged particles in confined geometries37 (i.e., the fluid cell in this study), could be the driving force for two aggregates to move together. The underlying mechanism of the like-charge attraction is still controversial. A potential explanation is that confining walls redistribute ions and couterions in solution around particles, which mediate a longrange attraction.37,38 Therefore, the long-range like-charge attraction and the short-range van der Waals attraction worked in concert to drive the direct attachment of small aggregates to the large aggregates in the NaCl solution.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b06305. Additional figures and details for Materials and Methods and Results and Discussion (PDF) Example of HM1 aggregates moving in Milli-Q water, recorded at a rate of 4 frames/s with an estimated dose rate of 0.7 electrons/(s Å2) (AVI) Example of HM2 aggregates moving in Milli-Q water, recorded at a rate of 4 frames/s with an estimated dose rate of 49 electrons/(s Å2) (AVI) Example of HM1 aggregates moving in NaCl solution, recorded at a rate of 4 frames/s and with an estimated dose rate of 22.8 electrons/(s Å2) (AVI)

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AUTHOR INFORMATION

Corresponding Authors

*Phone: (+86)010-62754292-808. E-mail: [email protected] (J.L.). *E-mail: [email protected] (Z.L.W.). *E-mail: [email protected] (Z.W.). Notes

The authors declare no competing financial interest.

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ENVIRONMENTAL IMPLICATIONS The observations presented here indicate that the smaller NPs tended to form more compact and larger aggregates in water, when the mass/molar concentrations of NPs were same. This finding suggests that the effect of aggregation state on particle reactivity and stability could be more prominent for small NPs than for their bulk counterparts, because more surface area became inaccessible in the larger and more compact aggregates. Increasing ionic strength not only induced the larger and more compact aggregates, but also promoted the cluster−cluster aggregation by changing the process how they came together. This finding is of particular importance to understand the behavior of NPs in estuaries and groundwater. Both size and structure of NP aggregates significantly influence a variety of reactions on NPs, such as metal uptake,11 redox reactions,39 dissolution,3,40 etc. and also determine transport and transformation of NPs in aqueous environments.5 Therefore, novel techniques for characterizing the size and morphology evolution of NP aggregates in situ are critical for understanding the relationship between aggregation state and environmental impact of NPs.

ACKNOWLEDGMENTS This work was financially supported by National Basic Research Program of China (973 Program, 2014CB846001), National Natural Science Foundation of China (41472306), and the “thousands talents” program for pioneer researcher and his innovation team, China.

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REFERENCES

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