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Time-resolved and Online Determination of
Reactive Oxygen Species (ROS) in Ambient Air
Markus Kalberer, Stephen Fuller
Department of Chemistry
University of Cambridge, UK
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Overview
• Oxidative stress - a possible mechanism of particle-induced
health effects
• A new instrument measuring reactive oxygen species (ROS)
concentration on-line: design and instrument characterization
• First applications of the instrument
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Ambient particle cause negative biological/health effects
as observed in epidemiological and laboratory studies
Health effects of aerosols
London-Smog, 1952 Deaths SO2 aerosol particles (dust)
Six cities study, Dockery et al. 1993
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
What particle properties are relevant for
biological effects?
particles e.g.,
from combustion
sources
1m
potential damaging properties
- organic components
- metals
- mass, size, surface properties
effects in the cell
- ROS?
- oxidative stress?
- inflammation
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Oxidative stress in the lung
H2O2
O2-
OH
Respiratory
tract lining
fluid
Lung epicelial
cells
Circulatory
system
glutathione
vitamin C vitamin E
Antioxidants ROS
• Oxidative stress = disturbance of prooxidant- antioxidant balance leading to potential
biomolecular damage
• Oxidative stress can be induced by ROS in the lung lining layer
• Antioxidant depletion may cause damage to cell membranes, proteins
& DNA
Antioxidants
ROS
anti-ox
proteins org. peroxides & radicals
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
PM induced ROS in the lung
Formation routes of ROS from aerosol particles:
1. Particle bound ROS – e.g. H2O2, ROOH, radicals
= reactive and short-lived
2. (Catalytic) ROS production in the lung lining fluid by
redox active components; e.g., quinones and metals
3. Metabolism of organics (e.g. PAHs) may lead to ROS
formation
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Measuring ROS concentrations in solution
- the reaction system
• DCFH is oxidised by ROS in the presence of Horseradish
Peroxidase to the fluorescent product DCF; excited at 470 nm
and emitting at 520 nm.
• DCFH reacts with almost all ROS
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
HRP
HRP-I
HRP-II
ROS (ox) (H2O2)
DCF-H
DCF
DCF-H
DCF
ROS (red) (H2O)
• The fluorescence is directly proportional to the concentration of DCF
• While the DCFH assay gives a linear relationship with H2O2, organic
peroxides react much slower with HRP.
• This assay gives an indication of ROS activity related to an
equivalent H2O2 concentration. NOT an absolute ROS
concentration.
I [DCF] [H2O2]
(HRP = Horesradish Peroxidase)
Measuring ROS concentrations in solution
- the reaction system
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Measuring ROS concentrations online
• Many current aerosol studies measuring composition use
off-line filter collection techniques
• The time delay between collection and analysis possibly
allows for degradation and loss of reactive components
• Online analysis allows for fast analysis that minimizes
loss of reactive components and allows for higher time
resolution
• Online analysis allows for high time resolution
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Fast Online Quantification
of Oxidizing Particle Components
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Water Bath
37°C
hydrophobic
filter
Continuous
collection and
solvent
extraction
Connected to LabView program
0.25 ml / min
5 slpm
sampling
Aqueous
Aqueous
To vacuum
pump
HRP solution
To mix with
DCFH solution
Aerosol inlet
15 c
m
Fast Online Quantification
of Oxidizing Particle Components
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Particle into liquid sampling
0
0,2
0,4
0,6
0,8
1
0 100 200 300 400 500
Co
llecti
on
Eff
ecie
ncy
Aerodynamic Diameter / nm
Collection Effeciency
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
ROS quantification
Water Bath
37°C
Hydrophilic
filter
-Continuous
filter collection
and solvent
extraction
Connected to LabView program
0.25 ml / min
5 slpm
sampling
Aqueous
Aqueous
Fan cooled 5 W 470 nm LED
Spectrometer
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Experimental set up
Water Bath
37°C
Hydrophilic
filter
Continuous
filter collection
and solvent
extraction
Connected to LabView program
0.25 ml / min
5 slpm
sampling
Aqueous
Aqueous
Fan cooled 5 W 470 nm LED
Spectrometer
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Sensitivity calibration with H2O2
Fluorescence intensity after reaction has gone to completion after
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Normalised to equivalent
fluorescein fluorescence to
account for non linear
spectrometer behaviour at
higher concentrations
Sensitivity calibration with H2O2
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Sensitivity calibration with H2O2
R2 = 0.99
0
500
1000
1500
2000
0 100 200 300 400 500 600 700
[Flu
ore
sc
ien
] / n
M
[H2O2] / nM
Dynamic range:
ca. 2 orders of magnitude
Detection limit:
in liquid H2O2: ~ 10 nM
ambient air (5lpm):
4 nmol / m3
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Smog Chamber Experiments
• Experiments at Paul Scherrer Institut (PSI), Switzerland
• Primary moped emissions photochemically aged in smog
chamber
• Mopeds conforming to both Euro 1 and Euro 2 emissions
regulations
27 m3
Chamber
UV + NOx + O3
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Smog Chamber Experiments
Secondary Organic Aerosol Formation
Primary organic aerosol
(POA)
Soot
Gaseous volatile organic
compounds (VOCs)
Oxidation by ozone and
OH in the presence of
UV light and NOx
Oxidised VOCs
condense to form
secondary organic
aerosol (SOA)
Primary
Processing
Secondary
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Smog Chamber Experiments
Charcoal Denuder
Online ROS Instrument
Remove gaseous
oxidants
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Smog Chamber Experiments
With photochemical
SOA formation:
increase of ROS
ROS =
ca. 1 nmol μg-1 SOA
50
40
30
20
10
0
SO
A m
ass / µ
g m
-3
2.52.01.51.00.50.0
Time after lights on / hours
50
40
30
20
10
RO
S c
on
ce
ntr
atio
n /
nM
ole
s m
-3
Euro 1 SOA mass
Euro 2 SOA mass
Euro 1 ROS concentration
Euro 2 ROS concentration
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Smog Chamber Experiments: POA vs. SOA
ROS concentration in POA (before photochemical aging): negligible
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Smog Chamber Experiments: Euro1 vs. Euro2
ROS / g POA large differences between Euro 1 and Euro 2
8
6
4
2
RO
S c
on
ce
ntr
atio
n n
orm
alis
ed
to
PO
A m
ass
/ n
Mo
les m
-3 µ
g-1
2.52.01.51.00.50.0
Time after lights on / hours
Euro 1
Euro 2
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Ambient measurements
ROS in ambient urban air (Cambridge)
0
20
40
60
80
100
120
140
160
180
200
11:52:48 12:21:36 12:50:24 13:19:12 13:48:00 14:16:48 14:45:36 15:14:24 15:43:12
nM
ole
s m
-3
Time
clean air ambient air clean
air
F
luo
res
cen
ce s
ign
al
11:50 12:50 13:50 14:50 15:50
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Conclusions
• Design of online instrument to quantify ROS
• ROS “scavenged” within seconds: reactive ROS are not lost
• Time resolution: ca. 10min
• Detection limit: 10nmol (H2O2), 4 nmol / m3
• ROS only observed in SOA from moped emissions
ROS concentrations of ca. 1 nmol μg-1 SOA
• No ROS in primary moped emissions
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Acknowledgements
PSI, Villigen – Smog Chamber Experiments
Stephen Platt
Josef Dommen
Andre Prevot
Urs Baltensperger
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M. Kalberer AirMonTech, Barcelona, 25 April 2012
Thanks for your attention