Queensland University of Technology
Discipline of Physics and Chemistry

THE APPLICATION OF A PROFLUORESCENT NITROXIDE PROBE
TO DETECT REACTIVE OXYGEN SPECIES DERIVED FROM
COMBUSTION-GENERATED PARTICULATE MATTER

Branka Miljevic

A THESIS SUBMITTED TO THE QUEENSLAND UNIVERSITY OF TECHNOLOGY IN
FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

September 2010



ABSTRACT
Particulate pollution has been widely recognised as an important risk factor
to human health. In addition to increases in respiratory and cardiovascular
morbidity associated with exposure to particulate matter (PM), WHO estimates that
urban PM causes 0.8 million premature deaths globally and that 1.5 million people
die prematurely from exposure to indoor smoke generated from the combustion of
solid fuels. Despite the availability of a huge body of research, the underlying
toxicological mechanisms by which particles induce adverse health effects are not
yet entirely understood. Oxidative stress caused by generation of free radicals and
related reactive oxygen species (ROS) at the sites of deposition has been proposed
as a mechanism for many of the adverse health outcomes associated with exposure
to PM. In addition to particle-induced generation of ROS in lung tissue cells, several
recent studies have shown that particles may also contain ROS. As such, they
present a direct cause of oxidative stress and related adverse health effects.
Cellular responses to oxidative stress have been widely investigated using

various cell exposure assays. However, for a rapid screening of the oxidative
potential of PM, less time-consuming and less expensive, cell-free assays are
needed. The main aim of this research project was to investigate the application of
a novel profluorescent nitroxide probe, synthesised at QUT, as a rapid screening
assay in assessing the oxidative potential of PM. Considering that this was the first
time that a profluorescent nitroxide probe was applied in investigating the oxidative
stress potential of PM, the proof of concept regarding the detection of PM–derived
ROS by using such probes needed to be demonstrated and a sampling methodology
needed to be developed. Sampling through an impinger containing profluorescent
nitroxide solution was chosen as a means of particle collection as it allowed
particles to react with the profluorescent nitroxide probe during sampling, avoiding
in that way any possible chemical changes resulting from delays between the
sampling and the analysis of the PM. Among several profluorescent nitroxide
probes available at QUT, bis(phenylethynyl)anthracene-nitroxide (BPEAnit) was
found to be the most suitable probe, mainly due to relatively long excitation and
emission wavelengths (λex= 430 nm; λem= 485 and 513 nm). These wavelengths are
i


long enough to avoid overlap with the background fluorescence coming from light
absorbing compounds which may be present in PM (e.g. polycyclic aromatic
hydrocarbons and their derivatives). Given that combustion, in general, is one of the
major sources of ambient PM, this project aimed at getting an insight into the
oxidative stress potential of combustion-generated PM, namely cigarette smoke,
diesel exhaust and wood smoke PM.
During the course of this research project, it was demonstrated that the
BPEAnit probe based assay is sufficiently sensitive and robust enough to be applied
as a rapid screening test for PM-derived ROS detection. Considering that for all
three aerosol sources (i.e. cigarette smoke, diesel exhaust and wood smoke) the
same assay was applied, the results presented in this thesis allow direct comparison

of the oxidative potential measured for all three sources of PM. In summary, it was
found that there was a substantial difference between the amounts of ROS per unit
of PM mass (ROS concentration) for particles emitted by different combustion
sources. For example, particles from cigarette smoke were found to have up to 80
times less ROS per unit of mass than particles produced during logwood
combustion. For both diesel and wood combustion it has been demonstrated that
the type of fuel significantly affects the oxidative potential of the particles emitted.
Similarly, the operating conditions of the combustion source were also found to
affect the oxidative potential of particulate emissions. Moreover, this project has
demonstrated a strong link between semivolatile (i.e. organic) species and ROS and
therefore, clearly highlights the importance of semivolatile species in particleinduced toxicity.

ii


KEYWORDS
Combustion aerosols, combustion-generated particulate matter, cigarette
smoke, wood smoke, diesel exhaust, health aspects of aerosol, health effects of
particulate matter, radicals, reactive oxygen species, ROS, oxidative stress, oxidative
potential, inflammatory potential, in vitro, profuorescent nitroxides; BPEAnit;
fluorescence, impinger, bubbling, collection efficiency

iii


STATEMENT OF ORIGINAL AUTHORSHIP
The work contained in this thesis has not been previously submitted to meet
requirements for an award at this or any other higher education institution. To the
best of my knowledge and belief, the thesis contains no material previously
published or written by another person except where due reference is made.


Signature:
Date:

iv


Table of Contents

ABSTRACT ..................................................................................................................i
KEYWORDS .............................................................................................................. iii
STATEMENT OF ORIGINAL AUTHORSHIP ............................................................... iv
LIST OF PUBLICATIONS ............................................................................................ ix
LIST OF TABLES ......................................................................................................... x
LIST OF FIGURES ....................................................................................................... x
ABBREVIATIONS ..................................................................................................... xv
ACKNOWLEDGEMENTS ........................................................................................ xvii
Chapter 1 ..................................................................................................................... 1
INTRODUCTION ....................................................................................................... 1
1.1.

Description of scientific problem investigated ............................................. 1

1.2.

Overall aims of the study .............................................................................. 2

1.3.

Specific objectives of the study .................................................................... 3


1.4.

Account of scientific progress linking the scientific papers ......................... 5

Chapter 2 ..................................................................................................................... 9
LITERATURE REVIEW ............................................................................................... 9
2.1.

Introduction – particles and health effects .................................................. 9

2.1.1.

Summary of epidemiological findings ................................................... 9

2.1.2.

Summary of toxicological findings ...................................................... 10

2.2.

Aerosol fundamentals and basic terminology ............................................ 12

2.3.

Combustion-generated PM ........................................................................ 15

2.3.1.

Cigarette smoke................................................................................... 16


2.3.2.

Diesel exhaust PM ............................................................................... 17

2.3.3.

Wood smoke particles ......................................................................... 20

2.4.

Particle characteristics relevant for health effects..................................... 22
v


2.4.1.

Size and surface area ........................................................................... 22

2.4.2.

Composition......................................................................................... 24

2.5.

Measurement of oxidative stress capacity of the PM ................................ 28

2.5.1.

In vitro studies ..................................................................................... 28


2.5.2.

Cell-free assays .................................................................................... 30

2.6.

Nitroxides .................................................................................................... 36

2.6.1.

Profluorescent nitroxides .................................................................... 37

2.7.

An overview of particle sampling approaches for toxicological studies .... 42

2.8.

Summary of research needs ....................................................................... 44

2.9.

References .................................................................................................. 46

Chapter 3 ................................................................................................................... 63
ON THE EFFICIENCY OF IMPINGERS WITH FRITTED NOZZLE TIP FOR COLLECTION
OF ULTRAFINE PARTICLES ..................................................................................... 63
Abstract .................................................................................................................. 65
3.1. Introduction .................................................................................................... 66

3.2. Experimental ................................................................................................... 67
3.3. Results and discussion .................................................................................... 70
3.4. Conclusion ....................................................................................................... 76
3.5. References ...................................................................................................... 77
Chapter 4 ................................................................................................................... 79
THE APPLICATION OF PROFLUORESCENT NITROXIDES TO DETECT REACTIVE
OXYGEN SPECIES DERIVED FROM COMBUSTION-GENERATED PARTICULATE
MATTER: CIGARETTE SMOKE – A CASE STUDY..................................................... 79
Abstract .................................................................................................................. 81
4.1. Introduction .................................................................................................... 82
4.2. Experimental ................................................................................................... 86
4.2.1. Materials................................................................................................... 86

vi


4.2.2. Cigarette smoke sampling ........................................................................ 86
4.2.3. Fluorescence measurements ................................................................... 89
4.2.4. Calibration curve ...................................................................................... 90
4.3. Results ............................................................................................................. 90
4.3.1. Mainstream cigarette smoke - linearity................................................... 90
4.3. 2. Sidestream cigarette smoke - sensitivity ................................................ 92
4.4. Discussion ....................................................................................................... 96
4.5. Conclusion....................................................................................................... 98
4.6. References ...................................................................................................... 99
4.7. Supplementary Information ......................................................................... 103
4.7.1. Reaction of BPEAnit with peroxyl radicals ............................................. 103
Chapter 5 ................................................................................................................. 105
PARTICLE EMISSIONS, VOLATILITY AND TOXICITY FROM AN ETHANOL
FUMIGATED COMPRESSION IGNITION ENGINE ................................................. 105

Abstract ................................................................................................................ 108
5.1. Introduction .................................................................................................. 109
5.2. Methodology ................................................................................................ 111
5.2.1. Engine, fuel and testing specifications................................................... 111
5.2.2. Particle measurement methodology ..................................................... 112
5.2.3. Particle volatility methodology .............................................................. 114
5.2.4. ROS concentration measurement – BPEAnit assay ............................... 115
5.3. Results ........................................................................................................... 116
5.3.1. Particle size distributions ....................................................................... 116
5.3.2. Particle volatility..................................................................................... 119
5.3.3. ROS concentration results...................................................................... 122
5.4. Discussion ..................................................................................................... 124
5.5. References .................................................................................................... 127
5.6. Supporting Information ................................................................................ 130

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Chapter 6 ................................................................................................................. 135
OXIDATIVE POTENTIAL OF LOGWOOD AND PELLET BURNING PARTICLES
ASSESSED BY A NOVEL PROFLUORESCENT NITROXIDE PROBE ......................... 135
Abstract ................................................................................................................ 138
6.1. Introduction .................................................................................................. 139
6.2. Experimental ................................................................................................. 141
6.2.1. Wood burners ........................................................................................ 141
6.2.2. Sampling setup and instrumentation ..................................................... 142
6.2.3. BPEAnit assay. ........................................................................................ 144
6.3. Results ........................................................................................................... 145
6.3.1. Particle emissions ................................................................................... 145
6.3.2. ROS from logwood burning particles. .................................................... 147

6.3.3. Correlation between ROS and organics. ................................................ 148
6.3.4. ROS from pellet burning particles .......................................................... 152
6.4. Discussion...................................................................................................... 153
6.5. References .................................................................................................... 157
6.6. Supporting information ................................................................................ 160
Chapter 7 ................................................................................................................. 163
CONCLUSIONS ..................................................................................................... 163
7.1. Principal significance of findings ................................................................... 164
7.2. Directions for future research ...................................................................... 171
7.3. References .................................................................................................... 173

viii


LIST OF PUBLICATIONS

1. Miljevic, B., Modini, R.L., Bottle, S.E., Ristovski, Z.D., 2009. On the efficiency
of impingers with fritted nozzle tip for collection of ultrafine particles.
Atmospheric Environment 43, 1372-1376.
2. Miljevic, B., Fairfull-Smith, K.E., Bottle, S.E., Ristovski, Z.D., 2009. The
application of profluorescent nitroxides to detect reactive oxygen species
derived from combustion-generated particulate matter: Cigarette smoke – a
case study. Atmospheric Environment 44, 2224-2230.
3. Surawski, N.C., Miljevic, B., Roberts, B.A., Modini, R.L., Situ, R., Brown, R.J.,
Bottle, S.E., Ristovski, Z.D., 2009. Particle emissions, volatility and toxicity
from an ethanol fumigated compression ignition engine. Environmental
Science & Technology 44, 229-235.
4. Miljevic, B., Heringa, M.F., Keller, A., Meyer, N.K., Good, J., Lauber, A.,
DeCarlo, P.F., Fairfull-Smith, K.E., Nussbaumer, T., Burtscher, H., Prevot,
A.S.H., Baltensperger, U., Bottle, S.E., Ristovski, Z.D., 2010. Oxidative

potential of logwood and pellet burning particles assessed by a novel
profluorescent nitroxide probe. Environmental Science & Technology 44,
6601-6607.

ix


LIST OF TABLES
Table 4-1. The amount of ROS per a) cigarette; b) puff (nsamples = 4). ....................... 92
Table 5-1. Speed, load and fuel settings used for both experimental campaigns. . 112

Table S 5-1. Test engine specifications. ................................................................... 130

LIST OF FIGURES
Figure 2-1.

Typical urban PM number (A), surface (B) and volume (C) size

distributions. Taken from (Seinfeld and Pandis, 2006). ..................................... 13
Figure 2-2.

Typical engine exhaust size distribution; both mass and number

weightings are shown (Kittelson, 1998). ............................................................ 18
Figure 2-3. The fractional deposition and mechanism of deposition of inhaled
particles of different sizes in each region of the human respiratory tract (TB –
tracheobronchial, A – alveolar, NPL – Nasal Pharyngeal Laryngeal; TOTAL – sum
of all three curves) based on the ICRP Model (1994) assuming nose breathing.
The image is adopted from Oberdörster et al., (2007). ..................................... 23
Figure 2-4. Simplified mechanism of quinoid redox cycling (QH2 – catechol)

(Squadrito et al., 2001)....................................................................................... 27
Figure 2-5. Scheme of particulate matter (PM) catalysed DTT oxidation. ................ 32
Figure 2-6. Hydrolysis of DCFH-DA and ROS-induced oxidation of DCFH ................. 34
Figure 2-7. SOD–mimetic activity of 2,2,6,6-tetramethyl-piperidinoxyl (TPO). ........ 37
Figure 2-8. Reaction of trapping carbon-centred radiclas with nitroxide (3AP) and
derivatisation with NDA (Flicker and Green, 2001). ......................................... 40
Figure 2-9. Structures of some of the profluorescent nitroxides synthesised at QUT.
In these examples five membered nitroxide ring is covalently fused to: A) 9,10bis(phenylethynyl)anthracene (BPEA); B)9,10-diphenylanthracene and C)
phenanthrene..................................................................................................... 41

x


Figure 3-1. Schematic representation of the experimental set-up........................... 69
Figure 3-2. Droplet size distribution generated by bubbling particle-free air at 1 L
min-1 through 40 ml of solvent (water, 50% DMSO or cell media) in impinger
with frit porosity 1. ............................................................................................ 71
Figure 3-3. Particle losses on the fritted nozzle tip for the impingers used in this
study. .................................................................................................................. 72
Figure 3-4. Removal efficiency (A) and solvent capture efficiency (B) of the impinger
with the porosity grade 1 fritted nozzle tip operating at 1 L min-1 for two
different volumes (40 and 20 mL) of heptane and water.................................. 74
Figure 3-5. Removal efficiency (A) and solvent capture efficiency (B) of the
impingers with the porosity grade 1 and 2 operating at 1 L min-1 for two
different volumes (40 and 20 mL) of heptane. .................................................. 75
Figure 3-6. Removal efficiency (A) and solvent capture efficiency (B) of the SKC
midget impinger with the coarse pore size fritted nozzle tip operating at 1 L
min-1 and containing 10 mL of either heptane or water. .................................. 76
Figure 4-1. 9-(1,1,3,3-tetramethylisoindolin-2-yloxyl-5-ethynyl)-10-(phenylethynyl)
anthracene (BPEAnit) ......................................................................................... 85

Figure 4-2. Experimental set-up for the cigarette mainstream smoke sampling. .... 87
Figure 4-3. Experimental set up for sidestream cigarette smoke sampling. ............ 88
Figure 4-4. A) Fluorescence emission of BPEAnit when exposed to puffs of
mainstream cigarette smoke coming from one 3R4F cigarette (λex= 430 nm); B)
fluorescence intensity at 485 nm plotted against the number of puffs. ........... 91
Figure 4-5. A) fluorescence emission of BPEAnit when exposed to the gas phase of
sidestream smoke; B) fluorescence emission of BPEAnit when exposed to total
sidestream smoke; C) fluorescence intensity at 485 nm plotted against
sampling time (λex= 430 nm). ............................................................................. 93
Figure 4-6. Left y-axis: An example of size distribution of particles entering the
impinger and particles trapped in the impinger; Right y-axis: Collection

xi


efficiency of the impinger used in this study (pore 1) when bubbling particles
from 50 to 380 nm in diameter through 20 mL of DMSO at 1 L min-1............... 94
Figure 4-7. ROS concentration related to sidestream smoke particles. Error bars
present one standard deviation. ........................................................................ 95
Figure S 4-1. Time course for the reaction of BPEAnit with peroxyl radicals..... .....104
Figure 5-1. Schematic representation of the experimental configuration used in this
study. ................................................................................................................ 113
Figure 5-2. SMPS derived particle number distributions at intermediate speed (1700
rpm), full load, for neat diesel (E0) and 40% ethanol (E40) engine operation.
Error bars denote ± one standard error ........................................................... 117
Figure 5-3. Correlation of particle size (CMD) with the ethanol substitution
percentage for tests conducted at 2000 rpm, full load ( =-0.939). Error bars
denote ± one standard error. ........................................................................... 118
Figure 5-4. Brake-specific PM2.5 emissions at intermediate speed (1700 rpm) with
various load settings and ethanol substitutions.


Error bars denote ± one

standard error. ................................................................................................. 119
Figure 5-5. Volume fraction remaining (VFR) versus thermodenuder temperature at
intermediate speed (1700 rpm). (a) 100% load E0 and E40. (b) 25% load E0 and
E20. Note well the linear scale on the ordinate for (a) and the logarithmic scale
on the ordinate for (b). Error bars are calculated using the uncertainties in the
diameter measurement. .................................................................................. 120
Figure 5-6. Percentage of volatile particles at intermediate speed (1700 rpm) and
50%, 25% load and idle mode for various ethanol substitutions. Error bars have
been calculated using the statistical uncertainty in the counts

. ..... 121

Figure 5-7. Fluorescence spectra of BPEAnit control (HEPA filtered) and test samples
for neat diesel (E0) and 40% ethanol (E40) at intermediate speed (1700 rpm)
and full load. ..................................................................................................... 122
Figure 5-8. ROS concentrations at intermediate speed (1700 rpm) with various load
settings and ethanol substitutions. Error bars denote ± one standard error. 123
xii


Figure S 5-1. Particle size distribution for different thermodenuder temperatures
(

. (a) 100% load E0. (b) 100% load E40. (c) 50% load E0. (d) 50% load

E40....................................................................................................................131
Figure S 5-2. The volume percentage of volatile material that coats non-volatile

particles at intermediate speed (1700 rpm) and for various load settings and
ethanol substitutions. Error bars are calculated using the uncertainties in the
diameter measurement....................................................................................132
Figure S 5-3. A calculation of air available for combustion at intermediate speed
(1700 rpm), half load, for various ethanol substitutions.

Error bars are

calculated based using the uncertainties in the fuel consumption and gaseous
emissions measurements.................................................................................132
Figure S 5-4. OH radical emissions from an AVL Boost simulation conducted at 2000
rpm, full load....................................................................................................133
Figure 6-1. A schematic representation of the experimental setup. ..................... 144
Figure 6-2. Examples of mass concentration (A), number concentration (B) and
geometric mean diameter (D(GMD); C) for particles from logwood burning in the
traditional logwood stove (black) and the automatic pellet boiler (grey). The
graphs show the values after dilution. Dotted lines denote the time periods at
which samples for each phase of logwood burning were normally taken. ..... 147
Figure 6-3. Average ROS concentrations of logwood burning particle emissions for
cold and warm start. Warm start 1 and 2 present sampling after refilling the
stove for the first and second time. Error bars present one standard error (n=4).
.......................................................................................................................... 148
Figure 6-4. Fluorescence emission of BPEAnit when sampling with and without a
thermodenuder. ............................................................................................... 149
Figure 6-5. Correlation between the amount of ROS and the amount of organics for
start-up phase of cold-start (A), stable phase of cold-start (B) and warm-start
(C) logwood burning......................................................................................... 151

xiii



Figure 6-6. Fluorescence intensity of BPEAnit after sampling emissions from the
automatic pellet boiler. .................................................................................... 152
Figure 6-7. Examples of the temperature in the logwood stove (black) and pellet
boiler (grey) during combustion. The dips in the logwood stove temperature
are due to refilling of the stove........................................................................ 154
Figure 6-8. Correlation between the combustion chamber temperature and ROS
concentration for logwood burning. Error bars present one standard deviation.
.......................................................................................................................... 155
Figure S 6-1. Size dependent collection efficiency of the impinger used in this study
when bubbling aerosol through 20 ml of DMSO and at flow rate of 1 L min-1
(adopted from Miljevic et al., 2010 )................................................................161

xiv


ABBREVIATIONS
AAPH – 2,2’-azo-bis-(2-amidinopropane) dihydrochloride
3AP – 3-amino-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy
ATP – adenosine triphosphate
BPEAnit – BPEA-nitroxide or 9,10-bis(phenylethynyl)anthracene-nitroxide
DCFH – 2’,7’– dichlorodihydrofluorescein
DCFH-DA – 2’,7’– dichlorodihydrofluorescein diacetate
DEP – diesel exhaust particles
DHR-6G – dihydrorhodamine–6G
DMPO – 5,5-dimethyl-1-pyrroline-N-oxide
DMSO – dimethyl sulphoxide
DNA – deoxyribonucleic acid
DTNB – 5,5’-dithiobis-2-nitrobenzoic acid
DTT – dithiothreitol

EPR – electron paramagnetic resonance
ETS – environmental tobacco smoke
GC-MS – gas chromatography-mass spectrometry
GM-CSF – granulocyte-macrophage colony-stimulating factor
GSH – glutathione
GSSG – glutathione disulfide
GST – glutathione-S-transferase
HC – hydrocarbons
HPLC – high performance liquid chromatography
IL – interleukin
xv


LC-MS – liquid cromatography-mass spectrometry
MAPK – mitogen-activated protein kinase
MS – mainstream smoke
NAD(P)H – nicotinamide adenine dinucleotide (phosphate)
NDA – naphthalenedicarboxaldehyde
NFκB – nuclear factor κB
NOx – oxides of nitrogen
NQO1 – NADPH quinine oxidoreductase
OH-1 – heme oxygenase-1
8-oxodG – 8-oxo-7,8-dihydro-2′-deoxyguanosine
Q – quinone
QH. – semiquinone
QH2 – hydroquinone
QUT – Queensland University of Technology
PM – particulate matter
POHPAA – p-hydroxyphenylacetic acid
ROS – reactive oxygen species

SOA – secondary organic aerosol
SOD – superoxide dismutase
SS – sidestream smoke
TNF-α – tumor necrosis factor α
TPO – 2,2,6,6- tetramethyl-piperidinoxyl
VOC – volatile organic compounds
WHO – World Health Organisation
xvi


ACKNOWLEDGEMENTS
My deepest gratitude goes to my supervisors, A/Prof Zoran Ristovski and
Prof Steven Bottle for providing me with an opportunity to work under their
supervision and learn from them. Zoran and Steve, thank you for giving me the
freedom to explore my own ideas, make decisions and work independently, while
still giving me all the supervision and support I needed. Thank you for your patience,
friendship and continuous encouragement during my PhD journey.
I would like to thank Queensland University of Technology for awarding me
with the Australian government funded International Postgraduate Research
Scholarship. It was an honour to be a recipient of this scholarship.
I would also like to thank the European Comission project EUROCHAMP for
the financial support during the wood burning campaign in Horw, Switzerland. I am
grateful to Prof Urs Baltensperger for providing me with an opportunity to
participate in this campaign.
I am thankful to Dr Kathryn Fairfull-Smith for always having the nitroxide
ready for me.
I would also like to acknowledge the support, friendship and invaluable
advices of my colleagues from ILAQH and Bottle group.
I thank my parents, sister and grandparents for their love, encouragement
and support; I thank my friends back home in Croatia for constantly reminding me

that there's no such place as far away. A special thanks goes to all the members of
Sundac and Osterman family for all the help, support and friendship they have given
me during my time in Australia.
At last, but far from least, I would like to express my innermost gratefulness
to my husband Senad. Dear Seni, if it wasn’t for your love, support and persistent
faith in me, my passion for research, for this project, would be drowned by
loneliness and nostalgia. I dedicate this thesis to you.

xvii



Chapter 1

INTRODUCTION

1.1.

Description of scientific problem investigated
A great number of epidemiological and laboratory studies have shown

strong associations between levels of ambient particulate matter (PM) and
increased respiratory and cardiovascular disease morbidity and mortality,
particularly among individuals with pre-existing cardiopulmonary diseases (Englert,
2004). To develop methods that could help to mitigate the adverse health
outcomes induced by PM, it is important to know the PM properties and the
mechanism(s) that are responsible for PM toxicity. Identification of the PM
properties that are the most relevant for promoting adverse health effects is crucial
not only for our mechanistic understanding but also for the implementation of
strategies for improving air quality. Despite the availability of a huge body of

research, the underlying toxicological mechanisms by which particles induce
adverse health effects are not yet entirely understood. Recently, it has become
evident that those particles have the ability to generate free radicals and related
reactive oxygen species (ROS). These species are responsible for driving oxidative
stress at sites of deposition and thereby triggering a cascade of events associated
with inflammation and, at higher concentrations, cell death.
One of the important aspects of environmental sciences in the last decade
was to indentify the physical and chemical characteristics of ambient PM
responsible for its health effects and within that scope, particle size, surface area
and chemical components, such as metals and certain classes of organics (e.g.
quinones) have been implicated in PM-induced health effects and more specifically,
in the generation of ROS.
ROS can be formed endogenously, by the lung tissue cells, during the
phagocytic processes initiated by the presence of PM in the lungs, or by particle1


related chemical species that have the potential to generate ROS. In addition to the
particle-induced generation of ROS, several recent studies have shown that
particles may also contain ROS (so called, exogenous ROS). As such, they present a
direct cause of oxidative stress and related adverse health effects and the
hypothesis that particles contain or produce ROS is the driving force for this
research project.
It is a reasonable assumption that exogenous ROS can cause the same
responses in the cell as endogenously formed ROS. Therefore, a rapid screening
assay able to evaluate PM oxidative potential in terms of their inherent ROS would
be beneficial for gaining better understanding about the nature of the particles
most relevant for their negative health impact. Such a screen would also provide a
helpful tool in efforts to further improve air quality and protect public health.
Cellular responses to oxidative stress have been widely investigated using
various cell exposure assays. However, in order to provide a rapid screening test for

the oxidative potential of PM, less time-consuming and cheaper, cell-free (or
acellular) assays are necessary. Several cell-free approaches have been used to
explore oxidative potential of PM in a quantitative manner. They all have certain
limitations, do not provide directly comparable results and, to date, none of these
assays has been acknowledged as the best acellular assay and none have yet been
widely adopted for investigation of potential PM toxicity.

1.2.

Overall aims of the study
Taking into account the research problem introduced in the previous

section, the main aim of this research project was to develop a methodology that
would enable us to apply a novel profluorescent nitroxide probe, synthesised at
Queensland University of Technology (QUT), for the detection and quantification of
ROS present on and derived from the surface of combustion-generated PM.
QUT’s profluorescent nitroxide probes have been used in monitoring polymer
degradation and investigating cellular redox status, but have never been applied in
2


investigating airborne particulate matter. Profluorescent nitroxides are a type of
compound consisting of a fluorophore linked to a nitroxide-containing ring. They
have a very low fluorescence emission due to inherent quenching of fluoreophore’s
fluorescence by the nitroxide group, but upon radical trapping or redox activity, a
strong fluorescence is observed. Previously published literature regarding nitroxide
chemistry led to the idea of applying profluorescent nitroxides in assessing the
oxidative stress potential of PM. Using this methodology ROS are not measured
directly; their amount in the sample is expressed as the amount of the nitroxide
probe that has been transformed to a fluorescent product (calculated using

calibration curve; for description see page 88). It should be noted that, in addition
to ROS and other free radicals, profluorescent nitroxides can react with strong
oxidants and reductants and if such species are present in PM sample, they might
contribute to the overall rise of fluorescence.
This was the first time that profluorescent nitroxide probes were applied as
a tool for the assessment of PM’s potential to cause oxidative stress and the key
task was primarily to demonstrate the proof of concept regarding the detection of
PM–derived ROS by using a profluorescent nitroxide probe.
Given that combustion, in general, is one of the major sources of ambient
PM affecting in this manner both indoor (e.g. tobacco smoking, wood burning
stoves and fireplaces) and outdoor (e.g. vehicle emissions, biomass burning) air
quality, this project aimed at getting an insight into oxidative stress potential of PM
coming from different combustion sources.

1.3.

Specific objectives of the study

The specific objectives of the study can be summarised as follows:
•

To develop a sampling approach suitable for toxicological assessment. The
main requirement for this type of analysis was to conserve the chemical and
surface properties of PM (for example, to avoid aggregation of particles
during sampling). Considering the reactivity of ROS, there was also a need to
3


avoid any possible chemical changes resulting from delays between the
sampling and the analysis of PM and to efficiently capture as much of the

particle-related ROS as possible during the sampling. It was also important
to have a sampling system that is appropriate for field use.
•

To select a model combustion-generated aerosol (i.e. PM source) needed to
demonstrate proof of concept regarding the detection of PM–derived ROS
by using a profluorescent nitroxide probe. The main requirement for this
objective was to have an aerosol source convenient for laboratory use.

•

Among several profluorescent probes available at QUT, to select the one
that is the most appropriate in the assessment of combustion aerosols. The
key aspect for this objective was to select a probe that has the excitation
and emission wavelength long enough to avoid overlapping with the
background fluorescence coming from optically active compounds which
may be present in PM.

•

To perform validation of the probe, especially in terms of linearity of
fluorescence response. To obtain meaningful quantitative measurements of
aerosol activity, the linearity of the fluorescence response of the probe is of
crucial importance.

•

To apply profluorescent nitroxide probe in assessing the oxidative potential
of particles produced by other combustion sources and:
-


to investigate oxidative stress potential in relation to different
combustion conditions and different fuel characteristics

-

to investigate oxidative stress potential in relation to the organic content
of PM

-

to provide a comparison of oxidative stress potential of particles coming
from different combustion sources

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1.4.

Account of scientific progress linking the scientific papers
This thesis contains a collection of papers in which the specific aims of the

project were addressed. These papers have been published or submitted for
publication in refereed journals.
As stated in the previous section, to perform PM sampling for toxicological
assays, such as oxidative stress assays, it is important to choose the appropriate
sampling approach. For this study, a liquid impingement was thought to be a good
sampling approach as it would allow particles to react with the profluorescent
nitroxide probe in a sampling liquid during sampling. This avoids any possible
chemical changes resulting from delays between the sampling and the analysis of

PM. Therefore, it was decided to sample PM into an impinger containing a
profluorescent nitroxide solution. Since liquid impingement is not 100% efficient in
capturing all of the PM, it was anticipated to increase collection efficiency somehow
and for that purpose an impinger with a fritted nozzle tip was chosen. A fritted
nozzle tip increases the contact surface between the aerosol and the liquid and
should, therefore, increase the collection efficiency of the impinger. In order to
conduct quantitative chemical analysis on the particles collected by the impingers,
it is important to know the portion of the particles being collected in the liquid (i.e.
collection efficiency). A paper focused on the determination of the efficiency of
impingers with fritted nozzle tip for collection of ultrafine and near-ultrafine
(diameter < 220 nm) particles and factors influencing the collection efficiency is
presented in Chapter 3. It is entitled “On the efficiency of impingers with fritted
nozzle tip for collection of ultrafine particles” and has been published in the journal
“Atmospheric Environment” as a technical paper. Although the collection
efficiencies presented in this paper are only for particles smaller than 220 nm, the
same experimental procedure was used to determine collection efficiency for
particles larger than 220 nm (up to 600 nm). The collection efficiency of particles
larger than 220 nm in the impinger was needed for other studies presented in this
thesis.

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