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Development and collection efficiency of an electrostatic precipitator for in-vitro toxicity studies of nano- and submicron-sized aerosols
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Development and collection efficiency of an electrostatic precipitator for in-vitro toxicity studies of nano- and submicron-sized aerosols Ta-Chih Hsiao a,∗, Hsiao-Chi Chuang b, Chun-Wan Chen c, Tsun-Jen Cheng d, Ya-Chien Chang Chien a a
Graduate Institute of Environmental Engineering, National Central University, No. 300 Jhongda Rd., Jhongli City, Taoyuan County 32001, Taiwan School of Respiratory Therapy, Taipei Medical University, No. 250 Wu-Hsing Street, Taipei City 11031, Taiwan Institute of Labor, Occupational Safety and Health, Ministry of Labor, No. 99, Lane 407, Hengke Rd., Sijhih District, New Taipei City 22143, Taiwan d Institute of Occupational Medicine and Industrial Hygiene and Department of Public Health, National Taiwan University, No. 17, Shiujou Rd., Taipei 10055, Taiwan b c
a r t i c l e
i n f o
Article history: Received 24 April 2016 Revised 8 December 2016 Accepted 9 January 2017 Available online xxx Keywords: Nanoparticle exposure Air-liquid interface Dosimetry Electrostatic precipitator (ESP) Electrospray
a b s t r a c t The direct air-liquid interface (ALI) in vitro exposure method are used for high-throughput screening of nanoparticle toxicity, due to the relatively low capital required and the low cost of labor compared to animal and in vivo experiments. In this study, a new ALI exposure chamber using an electrostatic precipitator (ESP–ALI) was designed to improve the nano- and submicron-sized particle collection efficiency on the air-liquid exposure interface. Particle penetration tests were performed to characterize the performance under different operating conditions. The effects of different geometric dimensions and operating conditions were explored, and the similarity-scaling process was applied to reveal the hidden effects underlying the experimental data. The penetration results show that the developed electrostatic precipitator is able to efficiently collect particles with a size of up to 300 nm under a DC electric field of 5.0 kV/cm and at a flow rate of up to 1.5 lpm. The electrospray charging technique was also tested with this ESP– ALI system and proved to enhance the ALI collection efficiency without ozone generation. In addition, the particles collected on the exposure interface are uniformly distributed under various operating conditions, as supported by consistent dimensionless precipitation densities. © 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Nanotechnology has tremendous promise in various applications, such as medical imaging and drug delivery technologies. Numerous engineered nanoparticles, such as silver nanoparticles, have been manufactured and widely used in consumer and industrial products, as well as other novel technological applications, due to their physiochemical properties [1–5]. However, there are concerns about the potential adverse effects on the human body, when exposed to nanoparticles [1,2,6,7]. Therefore, research on nanotoxicity has gained considerable attention in recent years. To determine the toxicity of nanoparticles, the three methods of the exposure experiment has generally been used to evaluate the exposure-dose-response relationships: (1.) animal/in vivo testing, (2.) ex vivo studies of cells of bronchial lavage or biopsies, and (3.) in vitro systems of exposure of lung cells to pollutants under controlled conditions [8,9]. Although the in vitro system lacks the
∗
Corresponding author. E-mail addresses: [email protected], [email protected] (T.-C. Hsiao).
ability to clarify complex interactions between the different types of cells in their natural environment, this method enables investigators to examine the effects of inhaled toxins on specific cell types. The in vitro test thus offers valuable information that can be used to determine the potential cellular mechanisms mediating these responses [10]. In addition, in vitro tests are relatively inexpensive, compared to ex vivo studies or animal/in vivo experiments, and avoid the ethical issues surrounding animal testing. Therefore, in vitro tests are often used to study the health effects of particulate matter at the cellular and molecular level [11]. In a recent review of in vitro cell exposure studies by Paur et al., they further concluded that cell-based in vitro exposure studies might offer a new avenue for the toxicity screening of novel nanoparticles [12]. For inhalable particles exposure experiments meant to assess toxicity in the lungs, the most widely used in vitro method involves directly pipetting the particle suspension into the cell culture, which is then immersed in a fluid culture medium within a culture flask or within a culture dish on a supporting microporous membrane (transwell). The particle suspension is either a commercial product or made by suspending ambient-collected or lab-made aerosol particles in liquid. The exposure dosage, which is critical
http://dx.doi.org/10.1016/j.jtice.2017.01.003 1876-1070/© 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: T.-C. Hsiao et al., Development and collection efficiency of an electrostatic precipitator for invitro toxicity studies of nano- and submicron-sized aerosols, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.01.003
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for establishing the exposure-dose-response relationships of active substances, relates to the concentration of particles in the suspension [13–16]. However, it is unknown which fraction of the suspended particles will interact with the submersed cells cultured as a monolayer (Cohen et al. 2013). For primary contact organs such as the lungs, skin, or eyes, the aforementioned traditional in vitro testing method represents an unrealistic way of exposure, as the in vivo exposure occurs at the air-liquid interface instead of in fully immersed (submerged) conditions [17]. In addition, the process of pipetting particle suspensions sometimes allows cells to be exposed to all the particles at once, resulting in a very high dosage per unit time [18]. Moreover, the physiochemical properties of the particles, such as the size, surface area, morphology, and chemical composition, would be significantly altered during the suspension preparation and/or filter collection process. The physiochemical characteristics of aerosol particles are crucial for determining their toxic effects when in contact with cells [7,19,20]. Particle-cell interactions then shall be changed [21]. To avoid the potential issues arising from traditional in vitro exposure methods, the air-liquid interface (ALI) system, which directly guides the aerosol particles to deposit on the exposure surface/air-liquid interface without changing their intrinsic physiochemical properties, was suggested as a system that more realistically represents the actual exposure scenario. The reported ALI systems can be classified into two categories based on their particle-collection mechanisms: the aerodynamic ALI system and the electrostatic precipitating ALI (ESP–ALI) system. The aerodynamic ALI system primarily utilizes air flow to deliver particles that are deposited on the air-liquid interface (exposure interface). Gravitational settling, inertial impaction, and Brownian diffusion are the major particle-collection mechanisms. Among the different designs proposed previously [20–26], Tippe et al. is one of few research groups that has proposed a dosimetric method to quantitatively determine the convective transport and deposition due to Brownian diffusion and sedimentation [21]. In Tippe’s aerodynamic ALI, it was found that the collection efficiency is fairly constant but low (∼2%) for the particles with sizes ranging from 50 to 500 nm [27]. For the commercialized ALI system, CULTEX® module, it is only about 0.7% for 200 nm aerosol particles [18,28]. Although the collection efficiency of nano- and submicron-sized particles could be enhanced by increasing the working flow rate, it could place additional cellular stress on the culture cells and may even result in apoptosis. Therefore, aerodynamic ALI systems may not be suitable candidates for performing cell exposure tests for nano- or submicron-sized particles. By introducing an additional electrostatic force, ESP–ALI systems could dramatically improve the collection efficiency of nanoand submicron range particles. ESP–ALI systems also offer a slower deposition velocity towards the collection interface and alleviate the potential extracellular stress in exposure experiments [29]. The ESP–ALI system is only functional for charged particles and requires an added charging device. The external electric field in ESP and the charge release during particle deposition are generally not expected to affect in vitro cell tests. As suggested by Savi, Kalberer, Lang, Ryser, Fierz, Gaschen, Rička and Geiser [18], under the typical exposure concentration of challenging aerosols (about 104 particles/cm3 ), the resulting charge current due to particle deposition is in the sub-femtoampere range, which is much smaller than the typical threshold to damage the cells based on electroporation experiments. For the electric field, several ALI studies [11,18,30,31] have applied 1 to 5 kV/cm on cell cultures and do not observed any adverse effect. In addition, the controlled cell exposure units are operated in parallel to determine unknown influences. Several designs of ESP–ALI that can be used to collect nanoand submicron-sized particles efficiently on the exposure interface have been proposed recently [11,18,29,30,32]. Savi et al. designed
a co-direction ESP–ALI system with an AC electric field. The experimental results showed that the overall particle collection efficiency increased to 15–30%, with lower efficiencies for the smallest (50 nm) and largest (600 nm) particles [18]. To further increase the fraction of charged nanoparticles and improve the collection efficiency, the ESP–ALI systems reported by Sillanpää et al., Bruijne et al., and Volcken et al. incorporated an unipolar corona charger instead of a bipolar Kr85 charger [11,29,30]. Sillanpää et al. demonstrated that the collection efficiency can be greater than 90% for particles larger than 20 nm under a DC electric field of 5.3 kV/cm. Bruijne et al. reported an approximately 90% collection efficiency for “all particles” between 19 and 882 nm. In addition, compared to the low flow rates used in the ESP–ALIs with a Kr85 neutralizer (167 cm3 /min for EPDExS and 50 cm3 /min for Savi’s ESP–ALI), the corona charging ESP–ALIs system was operated at a higher flow rate (1.0∼4.0 lpm) while maintaining a good collection efficiency. It is noteworthy that these corona-charging based ESP–ALIs allow the direction of the electric field to be perpendicular to the direction of aerosol flow, which may induce non-uniform deposition on the exposure interface. However, few studies [17,27] have addressed the issue of the spatial uniformity of particle deposition over the exposed interface and its potential consequences. Moreover, Volckens et al. have reported the corona charging process releases ozone into the particle flow at a concentration of approximately 80 ppb in their system. The ozone released by the corona charger could introduce another significant confounding interference in exposure experiments. In this study, a new ESP–ALI exposure chamber was designed to operate at a moderate to high flow rate range (0.6∼1.5 lpm). The effects of different geometric dimensions and the operating conditions of the ESP–ALI chamber, such as the applied voltage, electrode spacing, and flow rate, were explored. The uniformity of the particle deposition pattern on the exposure interface was investigated by means of inspecting the deposition pattern of fluorescein salt. At last, instead of corona charging, the novel electrospray charging technique were applied to prevent the ozone generation in this ESP–ALI system. 2. Materials and methods Two experimental methods, the penetration test and the fluorescein tracing, were conducted to characterize the performance of the new ESP–ALI exposure chamber and to investigate the effects of different electrode spacing and operating conditions. The penetration test was implemented to evaluate the overall collection efficiency, which is a key performance indicator for ALI systems. However, as noted earlier, the regional wall losses inside the ALI chamber had not been taken into account, which may have led to overestimation of the exposure dose. In this study, the fluorescein tracing method was performed to inspect the fraction of particle deposition in different regions inside the ESP–ALI chamber and to quantify the uniformity of deposited particles. 2.1. Design of the new ESP–ALI exposure chamber The new ESP–ALI chamber is main component of the overall ESP–ALI system. It is a co-direction design with a DC electric field, and its schematic diagram is shown in Fig. 1a. The initial flow direction and the DC electric field are parallel, although the flow direction would be bended and perpendicular to the electric field near the surface of the collection media. This co-direction design was expected to preserve the uniformity of particle deposition on the exposure interface comparing to the cross-direction ESP–ALI design. The prototype basically consists of 4 coaxial components: the upper cylinder with an expansion inlet, the body cylinder, the
Please cite this article as: T.-C. Hsiao et al., Development and collection efficiency of an electrostatic precipitator for invitro toxicity studies of nano- and submicron-sized aerosols, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.01.003
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Fig. 1. Schematic diagram of the prototype ESP–ALI exposure chamber.
lower contraction base with an outlet, and the inside supporting cone. The gradual expansion configuration inside the upper cylinder was designed to smoothly guide the aerosol stream to the exposure interface and reduce the particle transportation loss. At the bottom of the upper cylinder, a perforated plate made by 1.0 mm copper was installed. There were over 240 small holes with 1.0 mm radius and creating about 70% opening area over the perforated metal plate. The perforated metal plate serves as the upper electrode, and introduces the aerosol particles under laminar flow to the following electric field, where the electrostatic force is applied. The petri dish was placed directly under the perforated plate, on a circular copper plate, which serves as the lower electrode. It was used for performance evaluation experiments to simulate the transwell for culturing cell. When a negative voltage is applied on the lower electrode, an effective electric field is formed to sandwich
the petri dish. Therefore, the charged particles are directed towards and deposited onto the air-liquid exposure interface. The distance between the perforated metal plate and the circular copper plate is the critical parameter for determining the effective electric field. Consequently, in order to investigate the effect of the electrode spacing (S), the current design allows the upper cylinder to be adjusted in the vertical direction (Fig. 1b). The operational flow rate of the prototype was in the range of 0.6∼1.5 lpm, and the electric field was varied from 0 up to 6.7 kV/cm. 2.2. Quantification of deposition and pattern 2.2.1. Penetration test A schematic diagram of the experimental setup for the penetration tests is shown in Fig. 2. Monodisperse sucrose particles with a single charge were produced to perform penetration tests for
Please cite this article as: T.-C. Hsiao et al., Development and collection efficiency of an electrostatic precipitator for invitro toxicity studies of nano- and submicron-sized aerosols, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.01.003
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Fig. 2. Schematic diagram of the experimental setup for the penetration test.
Table 1 Experimental parameters. Particle diametera , dp Electrode distance, S Flow rate, Q Applying voltageb , V a b
30 nm, 50 nm, 70 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm 15 mm, 20 mm 1.5 lpm, 0.6 lpm 0 kV, 2 kV, 4 kV, 6 kV, 8 kV, 10 kV
Only 50, 100 and 200 nm were tested for the fluorescein tracing experiments. The fluorescein tracing experiments only performed at 0, 0.25 and 0.5 kV.
the prototype ESP–ALI chamber. A constant output collision-type atomizer (TSI 3076) with a diffusion dryer was applied to generate polydisperse sucrose particles. Downstream of the diffusion dryer, a tandem electrostatic classifier system (EC, TSI 3071A) was used to classify the polydisperse particles, and result in the particle distribution within a narrow size range (the geometric standard deviations of the classified particles is about 1.10). Since the aerosol particles are passing through a radiative Kr85 neutralizer (TSI 3077) to reach Boltzmann charge distribution before delivering to the EC, the monodispersed particles classified by the tandem electrostatic classifier system are singly charged. 10 different electromobility sizes were selected for the tests. A scanning mobility particle sizer (SMPS, TSI 3934) was used to measure the particle size distributions upstream and downstream of the ESP–ALI chamber. The sheath flow rates of the EC and the DMA in the SMPS were both maintained at 6 lpm. The penetration at a selected particle size (λ(dp )) can be calculated by taking the ratio of the upstream and downstream distributions, as follows:
λ (d p ) =
Cout Cin
ferent surfaces and were then immersed in NaOH buffer (0.001 M) solutions. By comparing the fluorescence signals of the buffer solutions using a fluorometer (Turner Designs, Model 10-AU), the relative quantity of the particles deposited in different locations was retrieved. In this study, similar fluorescein tracing methods were employed to determine the particle loss fractions of different wall surfaces inside the ESP–ALI chamber, and to analyze the deposition uniformity on the petri dish. To investigate the deposition pattern and analyze the uniformity of the particles collected on the exposure interface, region 2 (petri dish) was divided into four concentric sub-regions (Fig. 1a). The concentric annular sub-regions 2-I to 2-III are where the cells will be cultured (i.e. the exposure region), and the sub-region 2-IV is the circular edge wall of the petri dish. The dimensionless precipitation density (β y ), is deployed to quantify the uniformity of the deposited particles. The β y is expressed as
βy =
MY /Ay
(M2−I + M2−II + M2−III + M2−IV )/Atotal
(2)
where My is the fluorometer reading of the buffer sample extracted from sub-region y (y = 2-I, 2-II, 2-III, 2-IV), and Ay and Atotal are area of the sub-region y and total area of region 2, respectively. The fluorescein deposition for each case was repeated twice, and the concentration of each extracted buffer sample was measured repeatedly using a fluorometer. For the fluorescein tracing experiments, only a subgroup of the experimental matrix for the penetration tests was performed. 3. Results and discussion
(1)
where Cdown and Cup are the downstream and upstream particle concentration, respectively, and the overall collection efficiency (η) is 1− λ(dp ). The detail experimental parameters conducted for evaluating the ESP–ALI performance were listed in Table 1. 2.2.2. Fluorescein tracing The fluorescein tracing method has been utilized in the literature for characterizing aerosol particle loss or deposition patterns [33–35]. In general, fluorescein salt has been used as the tracer particle. Monodisperse fluorescein particles, generated by a similar process as in the penetration experimental setup, were introduced to deposit in the testing components. After collecting enough fluorescein sodium, cotton swabs were used to carefully wipe the dif-
3.1. Effects of the particle size and applied voltage The experimental results of particle penetration tests at a flow rate of 1.5 lpm and electrode distance of 20 mm are shown in Fig. 3. It is apparent from Fig. 3 that the applied voltage and particle size both have strong influences on the penetration of the ESP–ALI chamber. The penetration curves for different particle sizes share a similar behavior, and almost all decrease quadratically with increasing applied voltage. In the case of 200 nm particles, the penetration dropped from 74% to 5% when the applied voltage increased from 1 to 6 kV. It was also found that the penetration decreases significantly with decreasing particle size. At the fixed applied voltage of 4 kV, which corresponds to an electric field of 2 kV/cm, the penetration drops from 67% to 0.1% when the particle
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Fig. 3. The penetration of monodispersed particles at various applied voltages (Q = 1.5 lpm and S = 20 mm).
size decreases from 400 to 150 nm. Taken together, these findings strongly suggest that electrostatic precipitation is the major particle collection mechanism in this ESP–ALI chamber. Therefore, the particle electrical mobility (Zp ) and drift velocity (Vc ) are two key parameters for determining the penetration of the ESP–ALI chamber. Known as the “electrical mobility”, the Zp describes the moving ability of a charged particle under the balance of electrostatic attraction and drag force. Zp is expressed as:
Zp =
neCc 3 π μd p
(3)
where n is the number of charges on the particle, e is the elementary charge (1.602 × 10 − 19 Coulomb), Cc is the Cunningham slip correction factor, μ is the gas viscosity, and dp is the electrical mobility equivalent particle diameter.
Vc = Z p × E
(4)
3.2. Effects of the flow rate and electrode distance In Fig. 4a (using Vc as the abscissa), the effect of the flow rate (Q) can also be clearly observed. The penetration curves for two different flow rates (Q = 1.5 lpm and 0.6 lpm) at an identical electrode distance (S = 20 mm) deviate from each other. The curve for the lower flow rate was located below the curve for the higher flow rate. The Q-effect is believed to be due to different particle residence times in the effective zone of the electric field. With an increased residence time, the influence of the electric field on the trajectories of the charged particles can be strengthened. Therefore, to achieve the same penetration, the flow rate of 0.6 lpm, which promotes a longer residence time, requires a much lower Vc than does the flow rate of 1.5 lpm. To further validate the postulation, the ratio of the electric drift velocity to an average flow velocity in the radial direction (Vc /Vavg,r ) was introduced as a new similarityscaling parameter in Fig. 4b. The average flow velocity in the radial direction (Vavg,r ) is defined as
Vavg,r = The Vc can be calculated by the product of Zp and the electric field (E = V/S) experienced. Thus, Vc is proportional to the applied voltage through the electric field (E) and is inversely proportional to the particle size through Zp . The Vc is the terminal velocity in the electric force field. In a typical electrostatic precipitator, for charged particles with identical Vc , their penetration through the ESP–ALI chamber ought to be the same. In other words, Vc can be a similarity-scaling parameter for correlating the ESP–ALI’s penetration curves. Therefore, the particle penetrations were re-plotted in Fig. 4a using Vc as the abscissa. As expected, the scattering penetration data points in Fig. 3 were then correlated by a quadratic fitting curve (the solid line) in Fig. 4a with a high r2 value (0.9934). This indicated that the behaviors/moving trajectories of particles in the ESP–ALI chamber would be alike when they share similarities (i.e. Vc ). In other words, the characteristic curve can be applied to predict the penetration of any charged particle in the ESP–ALI chamber, as long as the Vc of the particle is known. Moreover, the charged particles collected inside the ESP–ALI chamber were predominantly attributed to electrostatic precipitation. The effects of the particle size and applied voltage on the particle penetration of the ESP–ALI chamber are essentially embedded in the concept of the electric drift velocity (Vc ).
Q 2π L1 · h
(5)
where h is the gap between the acceleration (charged electrode) ring and the upper tip of the transwell. The reprocessed experimental results for the two different flow rates (the black dots in Fig. 4b) once again converged to form a characteristic fitting curve by using this dimensionless drift velocity, Vc /Vavg,r , as the new abscissa. In other words, the Q-effect is really a matter of particle residence time. However, the effect of the electrode distance is still discernable. In principle, varying the electrode distance could lead to two possible competing effects on the particle penetration. The penetration may be boosted by shortening the electrode distance, as the particle residence time may decrease. Conversely, shortening the electrode distance could also enhance the electric field under an identical applied voltage, and thus reduce penetration. However, the influence on the electric field is included in the calculation of Vc . The effect of the electrode distance is thus more related to the residence time than to the electric field in this study. Another dimensionless factor, h/L1 , was then added as a similarity-scaling parameter to normalize the effect of the electrode distance (Fig. 4c). L1 is the inner radius of the upper cylinder of the ESP–ALI chamber, and it was used as the characteristic length here. It can be
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Fig. 4. (a) The penetration vs. particle drift velocity (Vc ) for Q = 0.6 lpm and 1.5 lpm, (S = 20 mm). (b) The penetration vs. a dimensionless particle drift velocity (Vc /Vavg,r ) for S = 20 mm and 15 mm. (c) The final characteristic penetration curve for the ESP–ALI chamber.
Fig. 5. (a) . The effect of applying voltage on the dimensionless precipitation density, β (dp = 200 nm, Q = 0.6 lpm, S = 15 mm). (b) The effect of the flow rate on the dimensionless precipitation density, β (dp = 200 nm, S = 20 mm).
Please cite this article as: T.-C. Hsiao et al., Development and collection efficiency of an electrostatic precipitator for invitro toxicity studies of nano- and submicron-sized aerosols, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.01.003
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Fig. 6. SEM images of silver nanoparticles deposited on the transwell located inside the ESP–ALI chamber (50 0 0 0x).
clearly observed that upon applying (h/L1 )(Vc /Vavg,r ) as the final abscissa, all the experimental penetration results under different operational conditions can be well described by one final characteristic fitting curve. In other words, (h/L1 )(Vc /Vavg,r ) is the most important dimensionless parameter for determining particle penetration through the ESP–ALI chamber. The fitting expression for the final quadratic characteristic curve is
2 h h Vc Vc λ(d p , V, S, Q ) ≡ λ · = 6.535 × · − 4.936 Vavg,r L1 Vavg,r L1 ×
Vc
Vavg,r
·
h L1
+ 0.906, and r 2 = 0.9822.
(6)
3.3. Characterization of the spatial deposition uniformity The spatial deposition uniformity on the exposure interface can influence the results of the ALI exposure experiments; however,
7
few studies have explored this issue previously. For the ideal case of uniform deposition in this study, the β values and the coefficient of variance (C.V.) of the 3 concentric sub-regions (2-I, 2-II, and 2-III) should be equal to 0.33 and 0, respectively. However, when there is no electric field (0 kV), the fluorescein tracing results show a very non-uniform deposition pattern for 200 nm monodispersed particles, as supported by the C.V. of 0.899 (Fig. 5a). Most of the particles were deposited on the outer annular sub-region (2-III) and the circular edge wall of the petri dish (sub-region 2IV) due to their convective transport. This non-uniformity implies the inadequacy of conducting nano- or submicron particle exposure tests in aerodynamic ALI systems. On the other hand, once the voltage was applied, the particle deposition pattern became much more uniform, and the C.V. of β s dropped down to 0.109. This indicates that the ESP–ALI can effectively guide the particles to deposit uniformly onto the exposure area. It was also found that the deposition pattern becomes more uniform as the particle size increases from 50 to 200 nm, and less uniform deposition for the small particles could be due to their high Zp values. In addition, the fluorescein tracing results also showed a minor effect for the flow rate (Fig. 5b), and the deposition patterns for the two different flow rates were both quite uniform. These findings may both be caused by the predominant electrostatic attraction in the ESP–ALI chamber. The uniform effective electric field would guide the charged particles to uniformly deposit onto the exposure area, comparing to the flow field guidance (convective transport). The scanning electron microscope (SEM) investigation also validated the uniform distribution of silver nanoparticles deposited on the transwell located inside the ESP–ALI chamber (Fig. 6). Summing up the above, the ESP–ALI chamber developed in this study demonstrated fairly good spatial deposition uniformity. 3.4. Tests of the ESP–ALI system adapted with electrospray charging Currently, corona charging is widely used in ESP–ALI because of the high particle charging efficiency and the following high collection efficiency of nanoparticles. However, one well-known concern is the ozone generation in the corona discharging process. Although a 2-stage ESP configuration was used in the ESP–ALI system could alleviate ozone generation, the potential ozone released by the corona charger would still introduce confounding interferences in the exposure experiment. In this study, instead of corona
Fig. 7. The penetration curve of the ESP–ALI system tested with and without electrospray charging system (Q = 1.5 lpm, S = 20 mm).
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charging, the novel electrospray charging technique were applied to avoid the ozone generation in this ESP–ALI system. Electrospray technique is generally used for fine aerosol generation [36] or ionization in mass spectrometry for chemical analysis [37], and recently it was also applied for air purification or pollution control [38,39]. When sufficiently high electric field applied to a conductive liquid stream delivered by a metallic capillary, the strong electrical force will pull out the front of liquid and create a liquid cone, a.k.a. Taylor cone. It will emit a liquid jet through the apex and produce highly charged fine droplets, which are employed to electrically charge challenging particles for the ESP–ALI system. In this experiment, a stainless capillary with an I.D. of 120 μm was selected. DI water was used as working fluid, and the operating flow rate is in the range of 1.0∼2.0 μl/min. The distance between the capillary and the ground is around 1.0 cm, and the applied voltage is between 4–5 kV. With its low voltage and current operation compared with those in traditional ESPs (when the electrical field strength is less than the electrical breakdown of carry air) no ozone will be produced in electrospray. Furthermore, the experimental results show that after electrospray charging the ALI collection efficiency of polydisperse aerosol had significantly improved for particles with electrical mobility size larger than 40 nm (Fig. 7).
4. Conclusions In this study, a new ESP–ALI in vitro cell exposure system was developed for the efficient deposition and controlled dosage of nano- and submicron-sized particles, and its performance was carefully characterized. The penetration results show that the ESP– ALI chamber is able to achieve over 95% collection efficiency for particles with sizes of up to 300 nm under a DC electric field of 5.0 kV/cm and at a flow rate of up to 1.5 lpm. It was found that Zp and Vc are the two key parameters for determining the penetration of the ESP–ALI chamber, which indicates that the electrostatic precipitation was the major particle collection mechanism. Furthermore, by virtue of similarity-scaling processes, the hidden effects of Vc and the residence time on the experimental penetration data were revealed, and all the experimental penetration results under different operating conditions were well correlated by one characteristic quadratic curve (using (h/L1 )(Vc /Vavg,r ) as the similarity-scaling parameter). Therefore, regardless of the particle size, the operational flow rate, the electrode distance, or the applied voltage, the particle penetration and overall collection efficiency of the ESP–ALI chamber can be predicted as long as the value of the similarity-scaling parameter of the particle is known. The reported expression for this characteristic curve enables estimation, evaluation, and controlled dosage when using the new ESP–ALI system. The new ESP–ALI chamber utilizes a design involving parallel flow and electric field directions to allow the particles to deposit more uniformly on the exposure interface. The uniformity of particle deposition in the exposure region was validated through the fluorescein tracing experiments. The examination results show consistent dimensionless precipitation densities and low C.V. values for the 3 concentric exposure sub-regions (2-I, 2-II, and 2-III) under various operating conditions. In addition, by comparing the cases with and without an applied voltage, it was found that the particle deposition uniformity can be effectively improved through electrostatic guidance. The SEM images further visually displayed uniform distribution of the deposited particles on the transwell located in the ESP–ALI chamber. In addition, the corona charger was replaced with a novel electrospray charger in this ESP–ALI system to prevent the ozone generation while keeps the high collection efficiency of nanoparticles. Therefore, the ESP–ALI system established and characterized in this study could be an appropriate
candidate for performing rapid toxicity screening experiments on novel nanoparticles.
Acknowledgments The authors gratefully appreciate the financial support for the ESP type air-liquid interface cell exposure chamber development provided by the Taiwan Institute of Occupational Safety and Health (IOSH-1013082, IOSH-1023037) and the Taiwan National Science Council (NSC101–2621–M–0 08–0 02). References [1] Maynard AD, Kuempel ED. Airborne nanostructured particles and occupational health. J Nanopart Res 2005;7:587–614. [2] Kreyling WG, Semmler-Behnke M, Möller W. Health implications of nanoparticles. J Nanopart Res 2006;8:543–62. [3] Gwinn MR, Vallyathan V. Nanoparticles: health effects—pros and cons, environ. Health Perspect 2006;114:1818. [4] Fadeel B, Garcia-Bennett AE. Better safe than sorry: understanding the toxicological properties of inorganic nanoparticles manufactured for biomedical applications. Adv Drug Deliv Rev 2010;62:362–74. [5] Marambio-Jones C, Hoek EMV. A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. J Nanopart Res 2010;12:1531–51. [6] Oberdörster G, Oberdörster E, Oberdörster J. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 2005;113:823. [7] Albanese A, Tang PS, Chan WCW. The effect of nanoparticle size, shape, and surface chemistry on biological systems. Annu Rev Biomed Eng 2012;14:1–16. [8] Rasmussen RE. In vitro systems for exposure of lung cells to NO2 and O3 . J Toxicol Environ Health A 1984;13:397–411. [9] Wallaert B, Fahy O, Tsicopoulos A, Gosset P, Tonnel AB. Experimental systems for mechanistic studies of toxicant induced lung inflammation. Toxicol Lett 20 0 0;112:157–63. [10] Devlin RB, Frampton ML, Ghio AJ. In vitro studies: what is their role in toxicology? Exp Toxicol Pathol 2005;57:183–8. [11] Volckens J, Dailey L, Walters G, Devlin RB. Direct particle-to-cell deposition of coarse ambient particulate matter increases the production of inflammatory mediators from cultured human airway epithelial cells. Environ Sci Technol 2009;43:4595–9. [12] Paur H-R, Cassee FR, Teeguarden J, Fissan H, Diabate S, Aufderheide M, et al. In-vitro cell exposure studies for the assessment of nanoparticle toxicity in the lung—a dialog between aerosol science and biology. J Aerosol Sci 2011;42:668–92. [13] Garrett NE, Campbell JA, Stack HF, Waters MD, Lewtas J. The utilization of the rabbit alveolar macrophage and chinese hamster ovary cell for evaluation of the toxicity of particulate materials: i. model compounds and metal-coated fly ash. Environ Res 1981;24:345–65. [14] Becker S, Soukup JM, Gilmour MI, Devlin RB. Stimulation of human and rat alveolar macrophages by urban air particulates: effects on oxidant radical generation and cytokine production. Toxicol Appl Pharm 1996;141:637–48. [15] Bayram H, Devalia JL, Sapsford RJ, Ohtoshi T, Miyabara Y, Sagai M, et al. The effect of diesel exhaust particles on cell function and release of inflammatory mediators from human bronchial epithelial cells in vitro. Am J Respir Cell Mol Biol 1998;18:441–8. [16] Boland S, Baeza-Squiban A, Fournier T, Houcine O, Gendron M-C, Chévrier M, et al. Diesel exhaust particles are taken up by human airway epithelial cells in vitro and alter cytokine production. Am J Physiol Lung Cell Mol Physiol 1999;276:L604–13. [17] Lenz AG, Karg E, Lentner B, Dittrich V, Brandenberger C, Rothen-Rutishauser B, et al. A dose-controlled system for air-liquid interface cell exposure and application to zinc oxide nanoparticles. Part Fibre Toxicol 2009;6:32. [18] Savi M, Kalberer M, Lang D, Ryser M, Fierz M, Gaschen A, et al. A novel exposure system for the efficient and controlled deposition of aerosol particles onto cell cultures. Environ Sci Technol 2008;42:5667–74. [19] Demokritou P, Büchel R, Molina RM, Deloid GM, Brain JD, Pratsinis SE. Development and characterization of a versatile engineered nanomaterial generation system (venges) suitable for toxicological studies. Inhal Toxicol 2010;22:107–16. [20] Cooney DJ, Hickey AJ. Cellular response to the deposition of diesel exhaust particle aerosols onto human lung cells grown at the air–liquid interface by inertial impaction. Toxicol in Vitro 2011;25:1953–65. [21] Tippe A, Heinzmann U, Roth C. Deposition of fine and ultrafine aerosol particles during exposure at the air/cell interface. J Aerosol Sci 2002;33:207–18. [22] Schreier H, Gagné L, Conary JT, Laurian G. Simulated lung transfection by nebulization of liposome Cdna complexes using a cascade impactor seeded with 2-Cfsmeo-Ce. J. Aerosol Med 1998;11:1–13. [23] Aufderheide M, Mohr U. CULTEX—an alternative technique for cultivation and exposure of cells of the respiratory tract to airborne pollutants at the air/liquid interface. Exp Toxicol Pathol 20 0 0;52:265–70.
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Journal of the Taiwan Institute of Chemical Engineers 0 0 0 (2017) 1–9
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Development and collection efficiency of an electrostatic precipitator for in-vitro toxicity studies of nano- and submicron-sized aerosols Ta-Chih Hsiao a,∗, Hsiao-Chi Chuang b, Chun-Wan Chen c, Tsun-Jen Cheng d, Ya-Chien Chang Chien a a
Graduate Institute of Environmental Engineering, National Central University, No. 300 Jhongda Rd., Jhongli City, Taoyuan County 32001, Taiwan School of Respiratory Therapy, Taipei Medical University, No. 250 Wu-Hsing Street, Taipei City 11031, Taiwan Institute of Labor, Occupational Safety and Health, Ministry of Labor, No. 99, Lane 407, Hengke Rd., Sijhih District, New Taipei City 22143, Taiwan d Institute of Occupational Medicine and Industrial Hygiene and Department of Public Health, National Taiwan University, No. 17, Shiujou Rd., Taipei 10055, Taiwan b c
a r t i c l e
i n f o
Article history: Received 24 April 2016 Revised 8 December 2016 Accepted 9 January 2017 Available online xxx Keywords: Nanoparticle exposure Air-liquid interface Dosimetry Electrostatic precipitator (ESP) Electrospray
a b s t r a c t The direct air-liquid interface (ALI) in vitro exposure method are used for high-throughput screening of nanoparticle toxicity, due to the relatively low capital required and the low cost of labor compared to animal and in vivo experiments. In this study, a new ALI exposure chamber using an electrostatic precipitator (ESP–ALI) was designed to improve the nano- and submicron-sized particle collection efficiency on the air-liquid exposure interface. Particle penetration tests were performed to characterize the performance under different operating conditions. The effects of different geometric dimensions and operating conditions were explored, and the similarity-scaling process was applied to reveal the hidden effects underlying the experimental data. The penetration results show that the developed electrostatic precipitator is able to efficiently collect particles with a size of up to 300 nm under a DC electric field of 5.0 kV/cm and at a flow rate of up to 1.5 lpm. The electrospray charging technique was also tested with this ESP– ALI system and proved to enhance the ALI collection efficiency without ozone generation. In addition, the particles collected on the exposure interface are uniformly distributed under various operating conditions, as supported by consistent dimensionless precipitation densities. © 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Nanotechnology has tremendous promise in various applications, such as medical imaging and drug delivery technologies. Numerous engineered nanoparticles, such as silver nanoparticles, have been manufactured and widely used in consumer and industrial products, as well as other novel technological applications, due to their physiochemical properties [1–5]. However, there are concerns about the potential adverse effects on the human body, when exposed to nanoparticles [1,2,6,7]. Therefore, research on nanotoxicity has gained considerable attention in recent years. To determine the toxicity of nanoparticles, the three methods of the exposure experiment has generally been used to evaluate the exposure-dose-response relationships: (1.) animal/in vivo testing, (2.) ex vivo studies of cells of bronchial lavage or biopsies, and (3.) in vitro systems of exposure of lung cells to pollutants under controlled conditions [8,9]. Although the in vitro system lacks the
∗
Corresponding author. E-mail addresses: [email protected], [email protected] (T.-C. Hsiao).
ability to clarify complex interactions between the different types of cells in their natural environment, this method enables investigators to examine the effects of inhaled toxins on specific cell types. The in vitro test thus offers valuable information that can be used to determine the potential cellular mechanisms mediating these responses [10]. In addition, in vitro tests are relatively inexpensive, compared to ex vivo studies or animal/in vivo experiments, and avoid the ethical issues surrounding animal testing. Therefore, in vitro tests are often used to study the health effects of particulate matter at the cellular and molecular level [11]. In a recent review of in vitro cell exposure studies by Paur et al., they further concluded that cell-based in vitro exposure studies might offer a new avenue for the toxicity screening of novel nanoparticles [12]. For inhalable particles exposure experiments meant to assess toxicity in the lungs, the most widely used in vitro method involves directly pipetting the particle suspension into the cell culture, which is then immersed in a fluid culture medium within a culture flask or within a culture dish on a supporting microporous membrane (transwell). The particle suspension is either a commercial product or made by suspending ambient-collected or lab-made aerosol particles in liquid. The exposure dosage, which is critical
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for establishing the exposure-dose-response relationships of active substances, relates to the concentration of particles in the suspension [13–16]. However, it is unknown which fraction of the suspended particles will interact with the submersed cells cultured as a monolayer (Cohen et al. 2013). For primary contact organs such as the lungs, skin, or eyes, the aforementioned traditional in vitro testing method represents an unrealistic way of exposure, as the in vivo exposure occurs at the air-liquid interface instead of in fully immersed (submerged) conditions [17]. In addition, the process of pipetting particle suspensions sometimes allows cells to be exposed to all the particles at once, resulting in a very high dosage per unit time [18]. Moreover, the physiochemical properties of the particles, such as the size, surface area, morphology, and chemical composition, would be significantly altered during the suspension preparation and/or filter collection process. The physiochemical characteristics of aerosol particles are crucial for determining their toxic effects when in contact with cells [7,19,20]. Particle-cell interactions then shall be changed [21]. To avoid the potential issues arising from traditional in vitro exposure methods, the air-liquid interface (ALI) system, which directly guides the aerosol particles to deposit on the exposure surface/air-liquid interface without changing their intrinsic physiochemical properties, was suggested as a system that more realistically represents the actual exposure scenario. The reported ALI systems can be classified into two categories based on their particle-collection mechanisms: the aerodynamic ALI system and the electrostatic precipitating ALI (ESP–ALI) system. The aerodynamic ALI system primarily utilizes air flow to deliver particles that are deposited on the air-liquid interface (exposure interface). Gravitational settling, inertial impaction, and Brownian diffusion are the major particle-collection mechanisms. Among the different designs proposed previously [20–26], Tippe et al. is one of few research groups that has proposed a dosimetric method to quantitatively determine the convective transport and deposition due to Brownian diffusion and sedimentation [21]. In Tippe’s aerodynamic ALI, it was found that the collection efficiency is fairly constant but low (∼2%) for the particles with sizes ranging from 50 to 500 nm [27]. For the commercialized ALI system, CULTEX® module, it is only about 0.7% for 200 nm aerosol particles [18,28]. Although the collection efficiency of nano- and submicron-sized particles could be enhanced by increasing the working flow rate, it could place additional cellular stress on the culture cells and may even result in apoptosis. Therefore, aerodynamic ALI systems may not be suitable candidates for performing cell exposure tests for nano- or submicron-sized particles. By introducing an additional electrostatic force, ESP–ALI systems could dramatically improve the collection efficiency of nanoand submicron range particles. ESP–ALI systems also offer a slower deposition velocity towards the collection interface and alleviate the potential extracellular stress in exposure experiments [29]. The ESP–ALI system is only functional for charged particles and requires an added charging device. The external electric field in ESP and the charge release during particle deposition are generally not expected to affect in vitro cell tests. As suggested by Savi, Kalberer, Lang, Ryser, Fierz, Gaschen, Rička and Geiser [18], under the typical exposure concentration of challenging aerosols (about 104 particles/cm3 ), the resulting charge current due to particle deposition is in the sub-femtoampere range, which is much smaller than the typical threshold to damage the cells based on electroporation experiments. For the electric field, several ALI studies [11,18,30,31] have applied 1 to 5 kV/cm on cell cultures and do not observed any adverse effect. In addition, the controlled cell exposure units are operated in parallel to determine unknown influences. Several designs of ESP–ALI that can be used to collect nanoand submicron-sized particles efficiently on the exposure interface have been proposed recently [11,18,29,30,32]. Savi et al. designed
a co-direction ESP–ALI system with an AC electric field. The experimental results showed that the overall particle collection efficiency increased to 15–30%, with lower efficiencies for the smallest (50 nm) and largest (600 nm) particles [18]. To further increase the fraction of charged nanoparticles and improve the collection efficiency, the ESP–ALI systems reported by Sillanpää et al., Bruijne et al., and Volcken et al. incorporated an unipolar corona charger instead of a bipolar Kr85 charger [11,29,30]. Sillanpää et al. demonstrated that the collection efficiency can be greater than 90% for particles larger than 20 nm under a DC electric field of 5.3 kV/cm. Bruijne et al. reported an approximately 90% collection efficiency for “all particles” between 19 and 882 nm. In addition, compared to the low flow rates used in the ESP–ALIs with a Kr85 neutralizer (167 cm3 /min for EPDExS and 50 cm3 /min for Savi’s ESP–ALI), the corona charging ESP–ALIs system was operated at a higher flow rate (1.0∼4.0 lpm) while maintaining a good collection efficiency. It is noteworthy that these corona-charging based ESP–ALIs allow the direction of the electric field to be perpendicular to the direction of aerosol flow, which may induce non-uniform deposition on the exposure interface. However, few studies [17,27] have addressed the issue of the spatial uniformity of particle deposition over the exposed interface and its potential consequences. Moreover, Volckens et al. have reported the corona charging process releases ozone into the particle flow at a concentration of approximately 80 ppb in their system. The ozone released by the corona charger could introduce another significant confounding interference in exposure experiments. In this study, a new ESP–ALI exposure chamber was designed to operate at a moderate to high flow rate range (0.6∼1.5 lpm). The effects of different geometric dimensions and the operating conditions of the ESP–ALI chamber, such as the applied voltage, electrode spacing, and flow rate, were explored. The uniformity of the particle deposition pattern on the exposure interface was investigated by means of inspecting the deposition pattern of fluorescein salt. At last, instead of corona charging, the novel electrospray charging technique were applied to prevent the ozone generation in this ESP–ALI system. 2. Materials and methods Two experimental methods, the penetration test and the fluorescein tracing, were conducted to characterize the performance of the new ESP–ALI exposure chamber and to investigate the effects of different electrode spacing and operating conditions. The penetration test was implemented to evaluate the overall collection efficiency, which is a key performance indicator for ALI systems. However, as noted earlier, the regional wall losses inside the ALI chamber had not been taken into account, which may have led to overestimation of the exposure dose. In this study, the fluorescein tracing method was performed to inspect the fraction of particle deposition in different regions inside the ESP–ALI chamber and to quantify the uniformity of deposited particles. 2.1. Design of the new ESP–ALI exposure chamber The new ESP–ALI chamber is main component of the overall ESP–ALI system. It is a co-direction design with a DC electric field, and its schematic diagram is shown in Fig. 1a. The initial flow direction and the DC electric field are parallel, although the flow direction would be bended and perpendicular to the electric field near the surface of the collection media. This co-direction design was expected to preserve the uniformity of particle deposition on the exposure interface comparing to the cross-direction ESP–ALI design. The prototype basically consists of 4 coaxial components: the upper cylinder with an expansion inlet, the body cylinder, the
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Fig. 1. Schematic diagram of the prototype ESP–ALI exposure chamber.
lower contraction base with an outlet, and the inside supporting cone. The gradual expansion configuration inside the upper cylinder was designed to smoothly guide the aerosol stream to the exposure interface and reduce the particle transportation loss. At the bottom of the upper cylinder, a perforated plate made by 1.0 mm copper was installed. There were over 240 small holes with 1.0 mm radius and creating about 70% opening area over the perforated metal plate. The perforated metal plate serves as the upper electrode, and introduces the aerosol particles under laminar flow to the following electric field, where the electrostatic force is applied. The petri dish was placed directly under the perforated plate, on a circular copper plate, which serves as the lower electrode. It was used for performance evaluation experiments to simulate the transwell for culturing cell. When a negative voltage is applied on the lower electrode, an effective electric field is formed to sandwich
the petri dish. Therefore, the charged particles are directed towards and deposited onto the air-liquid exposure interface. The distance between the perforated metal plate and the circular copper plate is the critical parameter for determining the effective electric field. Consequently, in order to investigate the effect of the electrode spacing (S), the current design allows the upper cylinder to be adjusted in the vertical direction (Fig. 1b). The operational flow rate of the prototype was in the range of 0.6∼1.5 lpm, and the electric field was varied from 0 up to 6.7 kV/cm. 2.2. Quantification of deposition and pattern 2.2.1. Penetration test A schematic diagram of the experimental setup for the penetration tests is shown in Fig. 2. Monodisperse sucrose particles with a single charge were produced to perform penetration tests for
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Fig. 2. Schematic diagram of the experimental setup for the penetration test.
Table 1 Experimental parameters. Particle diametera , dp Electrode distance, S Flow rate, Q Applying voltageb , V a b
30 nm, 50 nm, 70 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm 15 mm, 20 mm 1.5 lpm, 0.6 lpm 0 kV, 2 kV, 4 kV, 6 kV, 8 kV, 10 kV
Only 50, 100 and 200 nm were tested for the fluorescein tracing experiments. The fluorescein tracing experiments only performed at 0, 0.25 and 0.5 kV.
the prototype ESP–ALI chamber. A constant output collision-type atomizer (TSI 3076) with a diffusion dryer was applied to generate polydisperse sucrose particles. Downstream of the diffusion dryer, a tandem electrostatic classifier system (EC, TSI 3071A) was used to classify the polydisperse particles, and result in the particle distribution within a narrow size range (the geometric standard deviations of the classified particles is about 1.10). Since the aerosol particles are passing through a radiative Kr85 neutralizer (TSI 3077) to reach Boltzmann charge distribution before delivering to the EC, the monodispersed particles classified by the tandem electrostatic classifier system are singly charged. 10 different electromobility sizes were selected for the tests. A scanning mobility particle sizer (SMPS, TSI 3934) was used to measure the particle size distributions upstream and downstream of the ESP–ALI chamber. The sheath flow rates of the EC and the DMA in the SMPS were both maintained at 6 lpm. The penetration at a selected particle size (λ(dp )) can be calculated by taking the ratio of the upstream and downstream distributions, as follows:
λ (d p ) =
Cout Cin
ferent surfaces and were then immersed in NaOH buffer (0.001 M) solutions. By comparing the fluorescence signals of the buffer solutions using a fluorometer (Turner Designs, Model 10-AU), the relative quantity of the particles deposited in different locations was retrieved. In this study, similar fluorescein tracing methods were employed to determine the particle loss fractions of different wall surfaces inside the ESP–ALI chamber, and to analyze the deposition uniformity on the petri dish. To investigate the deposition pattern and analyze the uniformity of the particles collected on the exposure interface, region 2 (petri dish) was divided into four concentric sub-regions (Fig. 1a). The concentric annular sub-regions 2-I to 2-III are where the cells will be cultured (i.e. the exposure region), and the sub-region 2-IV is the circular edge wall of the petri dish. The dimensionless precipitation density (β y ), is deployed to quantify the uniformity of the deposited particles. The β y is expressed as
βy =
MY /Ay
(M2−I + M2−II + M2−III + M2−IV )/Atotal
(2)
where My is the fluorometer reading of the buffer sample extracted from sub-region y (y = 2-I, 2-II, 2-III, 2-IV), and Ay and Atotal are area of the sub-region y and total area of region 2, respectively. The fluorescein deposition for each case was repeated twice, and the concentration of each extracted buffer sample was measured repeatedly using a fluorometer. For the fluorescein tracing experiments, only a subgroup of the experimental matrix for the penetration tests was performed. 3. Results and discussion
(1)
where Cdown and Cup are the downstream and upstream particle concentration, respectively, and the overall collection efficiency (η) is 1− λ(dp ). The detail experimental parameters conducted for evaluating the ESP–ALI performance were listed in Table 1. 2.2.2. Fluorescein tracing The fluorescein tracing method has been utilized in the literature for characterizing aerosol particle loss or deposition patterns [33–35]. In general, fluorescein salt has been used as the tracer particle. Monodisperse fluorescein particles, generated by a similar process as in the penetration experimental setup, were introduced to deposit in the testing components. After collecting enough fluorescein sodium, cotton swabs were used to carefully wipe the dif-
3.1. Effects of the particle size and applied voltage The experimental results of particle penetration tests at a flow rate of 1.5 lpm and electrode distance of 20 mm are shown in Fig. 3. It is apparent from Fig. 3 that the applied voltage and particle size both have strong influences on the penetration of the ESP–ALI chamber. The penetration curves for different particle sizes share a similar behavior, and almost all decrease quadratically with increasing applied voltage. In the case of 200 nm particles, the penetration dropped from 74% to 5% when the applied voltage increased from 1 to 6 kV. It was also found that the penetration decreases significantly with decreasing particle size. At the fixed applied voltage of 4 kV, which corresponds to an electric field of 2 kV/cm, the penetration drops from 67% to 0.1% when the particle
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Fig. 3. The penetration of monodispersed particles at various applied voltages (Q = 1.5 lpm and S = 20 mm).
size decreases from 400 to 150 nm. Taken together, these findings strongly suggest that electrostatic precipitation is the major particle collection mechanism in this ESP–ALI chamber. Therefore, the particle electrical mobility (Zp ) and drift velocity (Vc ) are two key parameters for determining the penetration of the ESP–ALI chamber. Known as the “electrical mobility”, the Zp describes the moving ability of a charged particle under the balance of electrostatic attraction and drag force. Zp is expressed as:
Zp =
neCc 3 π μd p
(3)
where n is the number of charges on the particle, e is the elementary charge (1.602 × 10 − 19 Coulomb), Cc is the Cunningham slip correction factor, μ is the gas viscosity, and dp is the electrical mobility equivalent particle diameter.
Vc = Z p × E
(4)
3.2. Effects of the flow rate and electrode distance In Fig. 4a (using Vc as the abscissa), the effect of the flow rate (Q) can also be clearly observed. The penetration curves for two different flow rates (Q = 1.5 lpm and 0.6 lpm) at an identical electrode distance (S = 20 mm) deviate from each other. The curve for the lower flow rate was located below the curve for the higher flow rate. The Q-effect is believed to be due to different particle residence times in the effective zone of the electric field. With an increased residence time, the influence of the electric field on the trajectories of the charged particles can be strengthened. Therefore, to achieve the same penetration, the flow rate of 0.6 lpm, which promotes a longer residence time, requires a much lower Vc than does the flow rate of 1.5 lpm. To further validate the postulation, the ratio of the electric drift velocity to an average flow velocity in the radial direction (Vc /Vavg,r ) was introduced as a new similarityscaling parameter in Fig. 4b. The average flow velocity in the radial direction (Vavg,r ) is defined as
Vavg,r = The Vc can be calculated by the product of Zp and the electric field (E = V/S) experienced. Thus, Vc is proportional to the applied voltage through the electric field (E) and is inversely proportional to the particle size through Zp . The Vc is the terminal velocity in the electric force field. In a typical electrostatic precipitator, for charged particles with identical Vc , their penetration through the ESP–ALI chamber ought to be the same. In other words, Vc can be a similarity-scaling parameter for correlating the ESP–ALI’s penetration curves. Therefore, the particle penetrations were re-plotted in Fig. 4a using Vc as the abscissa. As expected, the scattering penetration data points in Fig. 3 were then correlated by a quadratic fitting curve (the solid line) in Fig. 4a with a high r2 value (0.9934). This indicated that the behaviors/moving trajectories of particles in the ESP–ALI chamber would be alike when they share similarities (i.e. Vc ). In other words, the characteristic curve can be applied to predict the penetration of any charged particle in the ESP–ALI chamber, as long as the Vc of the particle is known. Moreover, the charged particles collected inside the ESP–ALI chamber were predominantly attributed to electrostatic precipitation. The effects of the particle size and applied voltage on the particle penetration of the ESP–ALI chamber are essentially embedded in the concept of the electric drift velocity (Vc ).
Q 2π L1 · h
(5)
where h is the gap between the acceleration (charged electrode) ring and the upper tip of the transwell. The reprocessed experimental results for the two different flow rates (the black dots in Fig. 4b) once again converged to form a characteristic fitting curve by using this dimensionless drift velocity, Vc /Vavg,r , as the new abscissa. In other words, the Q-effect is really a matter of particle residence time. However, the effect of the electrode distance is still discernable. In principle, varying the electrode distance could lead to two possible competing effects on the particle penetration. The penetration may be boosted by shortening the electrode distance, as the particle residence time may decrease. Conversely, shortening the electrode distance could also enhance the electric field under an identical applied voltage, and thus reduce penetration. However, the influence on the electric field is included in the calculation of Vc . The effect of the electrode distance is thus more related to the residence time than to the electric field in this study. Another dimensionless factor, h/L1 , was then added as a similarity-scaling parameter to normalize the effect of the electrode distance (Fig. 4c). L1 is the inner radius of the upper cylinder of the ESP–ALI chamber, and it was used as the characteristic length here. It can be
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Fig. 4. (a) The penetration vs. particle drift velocity (Vc ) for Q = 0.6 lpm and 1.5 lpm, (S = 20 mm). (b) The penetration vs. a dimensionless particle drift velocity (Vc /Vavg,r ) for S = 20 mm and 15 mm. (c) The final characteristic penetration curve for the ESP–ALI chamber.
Fig. 5. (a) . The effect of applying voltage on the dimensionless precipitation density, β (dp = 200 nm, Q = 0.6 lpm, S = 15 mm). (b) The effect of the flow rate on the dimensionless precipitation density, β (dp = 200 nm, S = 20 mm).
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Fig. 6. SEM images of silver nanoparticles deposited on the transwell located inside the ESP–ALI chamber (50 0 0 0x).
clearly observed that upon applying (h/L1 )(Vc /Vavg,r ) as the final abscissa, all the experimental penetration results under different operational conditions can be well described by one final characteristic fitting curve. In other words, (h/L1 )(Vc /Vavg,r ) is the most important dimensionless parameter for determining particle penetration through the ESP–ALI chamber. The fitting expression for the final quadratic characteristic curve is
2 h h Vc Vc λ(d p , V, S, Q ) ≡ λ · = 6.535 × · − 4.936 Vavg,r L1 Vavg,r L1 ×
Vc
Vavg,r
·
h L1
+ 0.906, and r 2 = 0.9822.
(6)
3.3. Characterization of the spatial deposition uniformity The spatial deposition uniformity on the exposure interface can influence the results of the ALI exposure experiments; however,
7
few studies have explored this issue previously. For the ideal case of uniform deposition in this study, the β values and the coefficient of variance (C.V.) of the 3 concentric sub-regions (2-I, 2-II, and 2-III) should be equal to 0.33 and 0, respectively. However, when there is no electric field (0 kV), the fluorescein tracing results show a very non-uniform deposition pattern for 200 nm monodispersed particles, as supported by the C.V. of 0.899 (Fig. 5a). Most of the particles were deposited on the outer annular sub-region (2-III) and the circular edge wall of the petri dish (sub-region 2IV) due to their convective transport. This non-uniformity implies the inadequacy of conducting nano- or submicron particle exposure tests in aerodynamic ALI systems. On the other hand, once the voltage was applied, the particle deposition pattern became much more uniform, and the C.V. of β s dropped down to 0.109. This indicates that the ESP–ALI can effectively guide the particles to deposit uniformly onto the exposure area. It was also found that the deposition pattern becomes more uniform as the particle size increases from 50 to 200 nm, and less uniform deposition for the small particles could be due to their high Zp values. In addition, the fluorescein tracing results also showed a minor effect for the flow rate (Fig. 5b), and the deposition patterns for the two different flow rates were both quite uniform. These findings may both be caused by the predominant electrostatic attraction in the ESP–ALI chamber. The uniform effective electric field would guide the charged particles to uniformly deposit onto the exposure area, comparing to the flow field guidance (convective transport). The scanning electron microscope (SEM) investigation also validated the uniform distribution of silver nanoparticles deposited on the transwell located inside the ESP–ALI chamber (Fig. 6). Summing up the above, the ESP–ALI chamber developed in this study demonstrated fairly good spatial deposition uniformity. 3.4. Tests of the ESP–ALI system adapted with electrospray charging Currently, corona charging is widely used in ESP–ALI because of the high particle charging efficiency and the following high collection efficiency of nanoparticles. However, one well-known concern is the ozone generation in the corona discharging process. Although a 2-stage ESP configuration was used in the ESP–ALI system could alleviate ozone generation, the potential ozone released by the corona charger would still introduce confounding interferences in the exposure experiment. In this study, instead of corona
Fig. 7. The penetration curve of the ESP–ALI system tested with and without electrospray charging system (Q = 1.5 lpm, S = 20 mm).
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charging, the novel electrospray charging technique were applied to avoid the ozone generation in this ESP–ALI system. Electrospray technique is generally used for fine aerosol generation [36] or ionization in mass spectrometry for chemical analysis [37], and recently it was also applied for air purification or pollution control [38,39]. When sufficiently high electric field applied to a conductive liquid stream delivered by a metallic capillary, the strong electrical force will pull out the front of liquid and create a liquid cone, a.k.a. Taylor cone. It will emit a liquid jet through the apex and produce highly charged fine droplets, which are employed to electrically charge challenging particles for the ESP–ALI system. In this experiment, a stainless capillary with an I.D. of 120 μm was selected. DI water was used as working fluid, and the operating flow rate is in the range of 1.0∼2.0 μl/min. The distance between the capillary and the ground is around 1.0 cm, and the applied voltage is between 4–5 kV. With its low voltage and current operation compared with those in traditional ESPs (when the electrical field strength is less than the electrical breakdown of carry air) no ozone will be produced in electrospray. Furthermore, the experimental results show that after electrospray charging the ALI collection efficiency of polydisperse aerosol had significantly improved for particles with electrical mobility size larger than 40 nm (Fig. 7).
4. Conclusions In this study, a new ESP–ALI in vitro cell exposure system was developed for the efficient deposition and controlled dosage of nano- and submicron-sized particles, and its performance was carefully characterized. The penetration results show that the ESP– ALI chamber is able to achieve over 95% collection efficiency for particles with sizes of up to 300 nm under a DC electric field of 5.0 kV/cm and at a flow rate of up to 1.5 lpm. It was found that Zp and Vc are the two key parameters for determining the penetration of the ESP–ALI chamber, which indicates that the electrostatic precipitation was the major particle collection mechanism. Furthermore, by virtue of similarity-scaling processes, the hidden effects of Vc and the residence time on the experimental penetration data were revealed, and all the experimental penetration results under different operating conditions were well correlated by one characteristic quadratic curve (using (h/L1 )(Vc /Vavg,r ) as the similarity-scaling parameter). Therefore, regardless of the particle size, the operational flow rate, the electrode distance, or the applied voltage, the particle penetration and overall collection efficiency of the ESP–ALI chamber can be predicted as long as the value of the similarity-scaling parameter of the particle is known. The reported expression for this characteristic curve enables estimation, evaluation, and controlled dosage when using the new ESP–ALI system. The new ESP–ALI chamber utilizes a design involving parallel flow and electric field directions to allow the particles to deposit more uniformly on the exposure interface. The uniformity of particle deposition in the exposure region was validated through the fluorescein tracing experiments. The examination results show consistent dimensionless precipitation densities and low C.V. values for the 3 concentric exposure sub-regions (2-I, 2-II, and 2-III) under various operating conditions. In addition, by comparing the cases with and without an applied voltage, it was found that the particle deposition uniformity can be effectively improved through electrostatic guidance. The SEM images further visually displayed uniform distribution of the deposited particles on the transwell located in the ESP–ALI chamber. In addition, the corona charger was replaced with a novel electrospray charger in this ESP–ALI system to prevent the ozone generation while keeps the high collection efficiency of nanoparticles. Therefore, the ESP–ALI system established and characterized in this study could be an appropriate
candidate for performing rapid toxicity screening experiments on novel nanoparticles.
Acknowledgments The authors gratefully appreciate the financial support for the ESP type air-liquid interface cell exposure chamber development provided by the Taiwan Institute of Occupational Safety and Health (IOSH-1013082, IOSH-1023037) and the Taiwan National Science Council (NSC101–2621–M–0 08–0 02). References [1] Maynard AD, Kuempel ED. Airborne nanostructured particles and occupational health. J Nanopart Res 2005;7:587–614. [2] Kreyling WG, Semmler-Behnke M, Möller W. Health implications of nanoparticles. J Nanopart Res 2006;8:543–62. [3] Gwinn MR, Vallyathan V. Nanoparticles: health effects—pros and cons, environ. Health Perspect 2006;114:1818. [4] Fadeel B, Garcia-Bennett AE. Better safe than sorry: understanding the toxicological properties of inorganic nanoparticles manufactured for biomedical applications. Adv Drug Deliv Rev 2010;62:362–74. [5] Marambio-Jones C, Hoek EMV. A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. J Nanopart Res 2010;12:1531–51. [6] Oberdörster G, Oberdörster E, Oberdörster J. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 2005;113:823. [7] Albanese A, Tang PS, Chan WCW. The effect of nanoparticle size, shape, and surface chemistry on biological systems. Annu Rev Biomed Eng 2012;14:1–16. [8] Rasmussen RE. In vitro systems for exposure of lung cells to NO2 and O3 . J Toxicol Environ Health A 1984;13:397–411. [9] Wallaert B, Fahy O, Tsicopoulos A, Gosset P, Tonnel AB. Experimental systems for mechanistic studies of toxicant induced lung inflammation. Toxicol Lett 20 0 0;112:157–63. [10] Devlin RB, Frampton ML, Ghio AJ. In vitro studies: what is their role in toxicology? Exp Toxicol Pathol 2005;57:183–8. [11] Volckens J, Dailey L, Walters G, Devlin RB. Direct particle-to-cell deposition of coarse ambient particulate matter increases the production of inflammatory mediators from cultured human airway epithelial cells. Environ Sci Technol 2009;43:4595–9. [12] Paur H-R, Cassee FR, Teeguarden J, Fissan H, Diabate S, Aufderheide M, et al. In-vitro cell exposure studies for the assessment of nanoparticle toxicity in the lung—a dialog between aerosol science and biology. J Aerosol Sci 2011;42:668–92. [13] Garrett NE, Campbell JA, Stack HF, Waters MD, Lewtas J. The utilization of the rabbit alveolar macrophage and chinese hamster ovary cell for evaluation of the toxicity of particulate materials: i. model compounds and metal-coated fly ash. Environ Res 1981;24:345–65. [14] Becker S, Soukup JM, Gilmour MI, Devlin RB. Stimulation of human and rat alveolar macrophages by urban air particulates: effects on oxidant radical generation and cytokine production. Toxicol Appl Pharm 1996;141:637–48. [15] Bayram H, Devalia JL, Sapsford RJ, Ohtoshi T, Miyabara Y, Sagai M, et al. The effect of diesel exhaust particles on cell function and release of inflammatory mediators from human bronchial epithelial cells in vitro. Am J Respir Cell Mol Biol 1998;18:441–8. [16] Boland S, Baeza-Squiban A, Fournier T, Houcine O, Gendron M-C, Chévrier M, et al. Diesel exhaust particles are taken up by human airway epithelial cells in vitro and alter cytokine production. Am J Physiol Lung Cell Mol Physiol 1999;276:L604–13. [17] Lenz AG, Karg E, Lentner B, Dittrich V, Brandenberger C, Rothen-Rutishauser B, et al. A dose-controlled system for air-liquid interface cell exposure and application to zinc oxide nanoparticles. Part Fibre Toxicol 2009;6:32. [18] Savi M, Kalberer M, Lang D, Ryser M, Fierz M, Gaschen A, et al. A novel exposure system for the efficient and controlled deposition of aerosol particles onto cell cultures. Environ Sci Technol 2008;42:5667–74. [19] Demokritou P, Büchel R, Molina RM, Deloid GM, Brain JD, Pratsinis SE. Development and characterization of a versatile engineered nanomaterial generation system (venges) suitable for toxicological studies. Inhal Toxicol 2010;22:107–16. [20] Cooney DJ, Hickey AJ. Cellular response to the deposition of diesel exhaust particle aerosols onto human lung cells grown at the air–liquid interface by inertial impaction. Toxicol in Vitro 2011;25:1953–65. [21] Tippe A, Heinzmann U, Roth C. Deposition of fine and ultrafine aerosol particles during exposure at the air/cell interface. J Aerosol Sci 2002;33:207–18. [22] Schreier H, Gagné L, Conary JT, Laurian G. Simulated lung transfection by nebulization of liposome Cdna complexes using a cascade impactor seeded with 2-Cfsmeo-Ce. J. Aerosol Med 1998;11:1–13. [23] Aufderheide M, Mohr U. CULTEX—an alternative technique for cultivation and exposure of cells of the respiratory tract to airborne pollutants at the air/liquid interface. Exp Toxicol Pathol 20 0 0;52:265–70.
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