The capacity of airborne particulate matter to generate reactive oxygen species (ROS) has been correlated with the generation of oxidative stress both in vitro and in vivo. The cellular damage from oxidative stress, and by implication with ROS, is associated with several common diseases, such as asthma and chronic obstructive pulmonary disease (COPD), and some neurological diseases. Yet currently available chemical and in vitro assays to determine the oxidative capacity of ambient particles require large samples, analyses are typically done offline, and the results are not immediate.
Here we report the development of an online monitor of the oxidative capacity of aerosols (o-MOCA) to provide online, time-resolved assessment of the capacity of airborne particles to generate ROS. Our approach combines the Liquid Spot Sampler (LSS), which collects particles directly into small volumes of liquid, and a chemical module optimized for online measurement of the oxidative capacity of aerosol using the dithiothreitol (DTT) assay. The LSS uses a three-stage, laminar-flow water condensation approach to enable the collection of particles as small as 5 nm into liquid. The DTT assay has been improved to allow the online, time-resolved analysis of samples collected with the LSS but could be adapted to other collection methods or offline analysis of liquid extracts.
The o-MOCA was optimized and its performance evaluated using the
9,10-phenanthraquinone (PQ) as a standard redox-active compound. Laboratory
testing shows minimum interferences or carryover between consecutive
samples, low blanks, and a reproducible, linear response between the DTT
consumption rate (nmol min
Although there is ample evidence linking exposure to particulate air pollution to adverse health effects, the mechanisms that lead to those effects are not completely understood. A leading hypothesis is that inhaled ambient particulate matter generates reactive oxygen species (ROS), which in turn create cellular damage and induce oxidative stress (Fitzpatrick et al., 2014; Kelly, 2003; Birben et al., 2012; Xia et al., 2006). Oxidative stress is associated with many well-known, widely spread diseases such as Alzheimer's, atherosclerosis, diabetes, and myocardial infarction (Andersen et al., 2010; Block and Calderon-Garciduenas, 2009; Calderon-Garciduenas et al., 2008; Maritim et al., 2003; Singh and Jialal, 2006; Uttara et al., 2009). The oxidative potential of airborne particles is attributed not only to their chemical composition (organics, trace metals; Eiguren-Fernandez et al., 2010; Ercal et al., 2001; Jomova and Valko, 2011; Chien et al., 2009; Hawley et al., 2014) but also to their physical characteristics (particle size, shape, etc.; Bünger et al., 2000; Chien et al., 2009; de Haar et al., 2006; Wessels et al., 2010). Trace metals, such as copper and iron, can generate ROS via Fenton chemistry, while organics, including polycyclic aromatic hydrocarbons (PAHs) and quinones, generate ROS via metabolic transformations (Chung et al., 2006; Kumagai et al., 2012; Ercal et al., 2001; Valko et al., 2005).
To quantify the oxidative potential of airborne particulate matter, both in vivo and in vitro studies have been conducted. These studies have found correlations between the oxidative potential of airborne particles and the production of biological markers of ROS formation and oxidative stress (Bardet et al., 2014; Hawley et al., 2014; Li et al., 2004; Swanson et al., 2009; Acworth et al., 1999; Kim et al., 2001; Maier et al., 2008; Oberdorster, 2000). Yet the extent of these measurements is limited. Recently, efforts have been made to reduce and replace the use of animal models through cellular and chemical assays. Chemical assays are more rapid and less costly, and they offer a simpler means to screen airborne particulate matter toxicity. There are several assays used to assess airborne particle oxidative potential, each with different sensitivities to the ROS-generating compounds (Hedayat et al., 2014). The dihydroxybenzoate (DHBA) and ascorbic acid (AA) assays, which measures the ability of PM to deplete antioxidants, have been shown to be most sensitive to transition metals (Di Stefano et al., 2009; Janssen et al., 2014). The dithiothreitol (DTT) assay (Cho et al., 2005), which is based on the ability of redox-active compounds associated with particulate matter to catalyze the reduction by DTT of oxygen to superoxide, has been correlated with both organics and transition metals (Sauvain et al., 2015). The 2',7'-dichlorofluorescin diacetate (DCFH-DA) assay is based on the oxidation of DCFH based on the principle that non-fluorescent fluorescein derivatives will emit fluorescence after being oxidized by hydrogen peroxide (Hung and Wang, 2001; Huang et al., 2016).
Among these methods the dithiothreitol (DTT) assay (Cho et al., 2005)
developed to measure the oxidative capacity of airborne particles has
become widely adopted (Steenhof et al., 2011; Janssen et al.,
2014; Charrier et al., 2015; Godri et al., 2011). The acceptance of this
assay is based on several studies that show a high correlation between the
DTT assay and more specific biological markers of oxidative stress such as
heme oxygenase 1 (HO-1) and inflammatory markers such as interleukin-6 and
interleukin-8 (Li et al., 2003; Steenhof et al., 2011; Jiang et al., 2016),
and granulocyte macrophage colony-stimulating factor (GM-CSF; Hussain et
al., 2009; Uzu et al., 2011). Recent epidemiological studies have also found
an association between the DTT-measured oxidative potential and various
health end points such as asthma and congestive heart failure (Bates et
al., 2015; Fang et al., 2016; Yang et al., 2016). The DTT assay is based on
the ability of redox-active compounds associated with particulate matter to
catalyze the reduction by DTT of oxygen to superoxide. Currently the DTT
assay is done offline; particles collected on filters are incubated with
DTT over several periods between 10 and 45 min, and after each time
point the reaction between the DTT and the redox-active components is
quenched by the addition of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB),
forming a chromophore with an absorbance at 412 nm than can be measured to
determine the rate of DTT consumption over time. The total oxidative
capacity of the particulate matter is expressed as the rate of DTT consumed
per unit particle mass (nmol DTT min
Several online systems developed to monitor the oxidant capacity of airborne particles have been reported previously. King and Weber (2013) reported a method using a mist chamber to capture soluble gases and particles, coupled to automated analysis based on the DCFH assay. Venkatachari and Hopke (2008) and Wang et al. (2011) reported an automated DCFH assay method for measurement of ROS activity for the water-soluble component of ambient particles captured with a Particle into Liquid Sampler. Yet the DCFH assay is not as specific as the DTT assay described above, reporting also activity related to reactive nitrogen compounds. In addition, a study conducted by Chen et al. (2010) showed that photooxidation by the laser light utilized for fluorescence excitation in the DCFH assay can create false-positive results and background values increase with time. Moreover, the horseradish peroxidase (HRP) enzyme is used commonly in this assay to catalyze the generation of OH radicals and to improve the detection of target molecules (ROS). The presence of HRP in the reaction mixture induced a three-fold increase in DCFH oxidation, which can further lead to the overestimation of the measured oxidative potential (Pal et al., 2012.
A more recent online method using the DTT assay is reported by Sammeenoi et al., who coupled their analysis to a Particle into Liquid Sampler (Sameenoi et al., 2012). This sampler uses steam injection to condensationally enlarge the particles and impacts the droplets onto a surface continually washed by the water condensate, thus capturing the water-soluble components associated with the collected particles. As both water-soluble (Fang et al., 2016) and insoluble components (Shinyashiki et al., 2009; Verma et al., 2012; Wang et al., 2013; Li et al., 2015) of airborne particles have been associated with the oxidative potential, it is important to have the ability to measure the contribution of each fraction to the total oxidative potential. This is important as each fraction may have different physiological effects (Delfino et al., 2010).
In this paper, we present a new approach to provide online measurements of the oxidative capacity of airborne particles using the DTT assay and including both insoluble and soluble airborne particle constituents. Our approach uses the Liquid Spot Sampler (LSS) as our particle collector. This system enlarges particles through water condensation at moderate temperatures in a laminar flow and then directs the flow through a set of impaction jets to the surface of a liquid where the droplets are deposited inertially. Through choice of the jet configuration and flow rate, the insoluble particles remain suspended in the capturing liquid, and thus the LSS captures both insoluble and soluble components. Reported here is an online chemical module based on the DTT assay that coupled to the LSS enables in situ, time-resolved characterization of the ability of aerosols to generate ROS.
The LSS is the front end of the system – it deposits
both soluble and insoluble components of airborne particulate matter into a
small volume of liquid. The LSS uses the three-stage water condensational
growth technology (Lewis and Hering, 2013; Hering et al., 2014; Eiguren
Fernandez et al., 2014) to enlarge submicrometer particles to form
micrometer-sized droplets that are subsequently deposited into a
water-filled vial. The supersaturation required for condensational growth is
created in a laminar flow through a wet-walled tube, the first portion of
which is cooled to
Flow diagram for the online monitor of the oxidative capacity of aerosols (o-MOCA).
The collection efficiency of the LSS was evaluated in our laboratory
(Berkeley, CA) using both hydrophilic (sulfate and nitrate) and highly
hydrophobic (sebacic acid) particles. Particle collection efficiency was
inferred through measurement of particle penetration through the sampler
after delivery into the liquid. For submicrometer particle sizes, the
aerosol was generated through atomization, mixing
The DTT assay is conducted in the chemical module after sample collection. The main components of the module are two precision syringe pumps (Cavro XLP6000, Tecan); a six-port analytical injection valve (EV-750, Alltech); a homemade heating/shaking system consisting of a aluminum block, a thermoelectric device, and a shaker (VWR), which contains the reaction vial where the sample and the DTT reagent (Sigma Aldrich) mix and react, a “mixing tee” (Upchurch) where the DTT-sample-containing solution and DTNB (Sigma-Aldrich) join and are mixed, and a diode array detector (DAD, Agilent 1100). PEEK and Teflon were selected as the materials for all liquid handling tubing to reduce unwanted chemical reactions.
The flow diagram of the o-MOCA is shown in Fig. 1. Although our chemical module has similarities to the semi-automated system developed by Fang et al. (2015), the o-MOCA has been improved to couple with a liquid particle collector, to eliminate unnecessary steps in the DTT assay, and to reduce sample cross contamination during online analysis.
As soon as the predetermined sample collection time ends, the whole sample
volume is transferred to the reaction vial using syringe pump 2. Once the
sample is transferred, the collection vial and lines are rinsed with
Chelex-treated water, and 500
The possibility of having particle loses during collection and transport of the suspension to the reaction vial has been considered. While not directly measured, losses to collection in the vial and during transport to the reaction vial are expected to be minimal. With our system both insoluble and soluble components or the particles are collected directly into water. Insoluble particles remain suspended in the water collection medium and have little opportunity to interact with vial and tube walls. In addition, suspended particles are close to being neutrally buoyant, rendering inertial forces negligible. A study conducted by Matas et al. (2004) has shown that neutrally buoyant particles are driven away from walls in a laminar tube flow when particle dimensions approach those of the tube diameter. Jochem et al. (2016) found that transport of particles in microfluidic devices and losses in comparable PEEK tubing were negligible. Based on these considerations and studies, we assume there is little opportunity for water-encapsulated particles to be lost to surfaces during both collection into water and transport.
System optimization was done using the 9,10-phenanthraquinone (PQ), a very
active redox compound commonly used for evaluating the DTT assay. Its
redox-active behavior has been well characterized, and several studies have shown
a linear relation (first-order reaction) between the rate of DTT loss and
the PQ concentration in solution (Charrier and Anastasio, 2012; Fang et
al., 2015). The carryover and cross contamination between injections were
examined by running consecutive samples containing different amounts of the
PQ standard. To evaluate if higher activity samples had an effect over
subsequent samples of lower activity, we measured the DTT loss rate of blanks
followed by high PQ concentrations (0.25
An important parameter to consider while developing the online system to
measure time-resolved oxidative capacity of airborne particles is the
sensitivity of the assay. A previous study conducted by Sameenoi et
al. (2012) showed that increasing the initial
DTT added to solution resulted in a reduction of the sensitivity of the
assay. Although their method of analysis was different to ours, we
considered this parameter when optimizing our online assay. The effect of
the initial amount of DTT in solution on the observed consumption rate was
evaluated through systematic increases in the mass of DTT added to the
reaction vial (50, 100, and 140 nmol) at a fixed PQ concentration
(0.25
Our long-term goal is to develop a field-deployable o-MOCA that can run
unattended for extended periods of time. However, as the DTT is a chemical
assay, and both the DTT and the DTNB are known to undergo degradation when
exposed to light and high temperatures, optimal conditions for maintaining the
integrity of these solutions for long sampling periods were determined.
Photodecomposition was eliminated by keeping the reagent containing
reservoirs in the dark (wrapped in aluminum foil). The effect of ambient
temperature on the stability of the reagent was evaluated in two different
ways: (i) by measuring the consumption rate of a blank solution for
consecutive runs over a 7 h period and (ii) by measuring and comparing
daily the absorbance of the DTT–DTNB mixture at
Collection efficiency for laboratory-generated salt and sebacic
acid aerosols with the Liquid Spot Sampler:
Once the chemical module was optimized, the system was run continuously for several hours to measure the DTT consumption rate and linearity for different concentrations of PQ. Further validation of the online method was conducted using a diesel exhaust particle extract (DEP; courtesy of Arthur Cho and Debra Schmitz, University of California Los Angeles (UCLA)). This DEP was used to develop the original DTT assay and later as a control for the improved method (Cho et al., 2005). It has been used as a positive control when measuring the activity of ambient particulate matter in all the studies conducted by Arthur Cho's group. It is also used by Ning Li (Michigan State University) as a positive control for in vitro assays when measuring biomarkers of oxidative stress.
The fully automated o-MOCA was tested for continuous online measurement of
ambient aerosols in our laboratory (ADI, Berkeley, CA). Our prototype o-MOCA
ran unattended, without operator interaction, over a 3-day period. Ambient
particulate matter samples collected at 3 L min
The collection efficiency for mixed salt particle size and sampling flows,
and highly hydrophobic sebacic acid are shown in Fig. 2. Hydrophobic
particles showed collection efficiencies of 80 % for 15 nm particles,
increasing to > 90 % for larger particle sizes (Fig. 2a). For
hydrophilic salts collection efficiencies > 90 % were obtained
for all particle sizes and flows (Fig. 2a and b). Higher collection
efficiencies (> 97 %) were observed for sampling flow rates
varying between 1.5 and 2.5 L min
During these experiments, the volume of the collecting liquid was adjusted
according to the sampling flow. For lower flow rates, smaller volumes could
be used (down to 200
In our online system, we use a continuous-flow DAD instead of a batch
fixed-wavelength (412 nm) UV–Vis detector. Our first step was to evaluate the
equivalence between the readings of the benchtop UV–Vis detector
(Spectronic 20D, Milton-Roy) and the online method with the DAD detector
for the same sample. We ran the standard offline DTT assay (Cho et al.,
2005) using PQ as our standard compound, and the absorbance of the same
solution after 0, 5, 10, and 20 min reaction was measured with both
detectors. Good linear relation was observed between the detectors
(
DTT consumption rate (nmol min
As in many liquid-based online systems, the potential for carryover from
injection to injection and sample to sample is of concern. Although the
internal volume of the lines has been minimized, the dead volume of the
syringe pumps could lead to cross contamination. To eliminate interferences
and cross contamination between consecutive injections, we added a 150
After this improvement, we tested the new configuration for possible
carryover using different standard amounts. The DTT consumption rate (nmol min
Peak absorbance for water blanks and PQ (0.25
Figure 4 shows the average consumption rates (
When reagents were kept at ambient conditions, a slow decrease in the
absorbance peak of the DTT–DTNB mixture, estimated for
DTT consumption rate (nmol min
Figure 5 illustrates how the oxidative capacity of the samples is obtained
with the o-MOCA system. Shown are data obtained for duplicate blanks and PQ
samples. Figure 5a shows the raw absorbance data for each of four different
quantities of PQ after addition of 500
Laboratory evaluation and performance of the automated system for
different PQ solutions and blanks:
Figure 5c presents the regression equation for DTT consumption for this range of PQ concentrations. As expected, a linear correlation is obtained with near-zero intercept, indicating a good system response to this standard compound. The small standard deviation and consistency of these data which span an order-of-magnitude variation in the PQ concentrations indicate that the online DTT method works properly.
The limit of detection (LOD) of the system, defined as 5 times the standard
deviation of the slope obtained for our blanks (
Comparison of consumption rates (nmol min
To validate our improved method, we ran the online DDT assay using DEP extract (in DMSO) obtained from UCLA (courtesy of
Arthur Cho and Debra Schmitz). We measured the DTT consumption rate for
three different initial amounts of DEP (
These results indicate that our improved assay is comparable to the original DTT method and that the reported consumption rates will be directly comparable to laboratory-based assays with equal accuracy.
DTT consumption rate (nmol min
The interface of the chemical module with the Liquid Spot Sampler was done connecting a port from one of the syringe pumps to the sample collection vial. The line is connected to the bottom of the collection vial to remove the entire particle-containing suspension. The liquid suspension, containing both water-soluble and water-insoluble material, is then delivered to the reaction vial where the reaction with DTT takes place. After sample delivery, the sample collection vial is rinsed twice with double-distilled water (DDW) (Chelex) and filled again for the next sample collection. All these steps are conducted automatically. After some preliminary testing, the original collection vial was modified slightly to provide more complete sample transfer into the chemical module. We also found that suspension transfer was aided by turning the sampling flow off during the transfer and rinsing steps. A solenoid valve under software control was added to enable automatic switching off/on of the sample flow. The improved and optimized o-MOCA interface was initially evaluated using different standard solutions of PQ (as shown in Fig. 5). Finally, to accommodate the analysis of an air sample containing insoluble as well as soluble particulate matter, we added a stainless-steel frit (Upchurch) in the line through which the sample extracted from the reaction vial is delivered to the injection valve. This way, particles are physically present in the suspension while conducting the reaction but do not reach the injection valve, the mixing tee, or the detector, avoiding interferences and clogging of the lines. No differences were observed in DTT consumption rates for PQ solution with and without the frit.
The fully automated o-MOCA was tested for continuous online measurement of
ambient aerosols in our laboratory. Our prototype o-MOCA ran unattended over
a 3-day period. Ambient PM
Figure 6 shows the oxidative capacity of the consecutive 3 h samples
measured as the consumption rate of DTT nmol min
The measured activity for airborne particles collected in Berkeley, reported
as the DTT consumption rate (nmol min
We present a method for the online, time-resolved measurement of the
oxidative potential of ambient particulate matter. The o-MOCA is a prototype
system which allows automated collection and analysis of ambient particles
by combining a Liquid Spot Sampler with a newly developed online chemical
module. The LSS collects both soluble and insoluble components of airborne
particles as concentrated samples of water suspensions. The chemical module
has been developed and optimized to streamline the DTT analysis of the
oxidative potential of the sample. Laboratory-based tests using the
9,10-phenanthraquinone and DEP extracts as the redox-active compounds show
that the newly developed chemical module for streamlined DTT assay reports
similar responses to the standard benchtop DTT assay. The o-MOCA was run
continuously and unattended for 3 days in our laboratory, collecting 3 h
samples of ambient PM
All the data presented in this study are available from theauthors upon request.
The authors declare that they have no conflict of interest.
Aerosol Dynamics Inc. holds several patents covering the particle collection technology utilized in this study and has licensed this technology for particle collection to Aerosol Devices Inc., Fort Collins, CO.
The authors gratefully acknowledge financial support for this work, which was funded by the National Institutes of Health through a Phase I SBIR grant (R43ES025468). Edited by: P. Laj Reviewed by: two anonymous referees