midirnitricoxidecrds-2.pptx
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MID-IR CAVITY RING-DOWN
SPECTROMETER FOR
BIOLOGICAL TRACE NITRIC
OXIDE DETECTION
Vincent Kan2, Vitali Stsiapura1,3,
Ahmed Ragab1, Kevin K.
Lehmann1,2, Ben Gaston3
1Dept.
of Chemistry, 2Dept. of Physics, 3School of Medicine
Motivations
• S-nitrosothiols (RS-NO) have received
much attention in biochemistry and
medicine as donors of nitric oxide (NO)
and nitrosonium (NO+) - physiologically
active molecules involved in signal
transduction through transnitrosation.
RS-NO + R’S-H RS-H + R’S-NO
Lipton, A.J. et al. //Nature 2001; Arnelle D. R. and Stamler J. S. //
Arch. Biochem. Biophys. 1995; Gaston, B. et al. //PNAS 1993
Motivations
S-nitrosothiol signaling is involved in many different types
of disease. For example:
•
•
•
•
•
•
•
•
Cancer:
Asthma:
Cystic Fibrosis:
Apnea:
Ischemia:
Shock:
Parkinson’s:
Alzheimer’s:
Lim et al. Nature 452:646, 2008
Que et al., Science 308:1618, 2005
Marozkina et al., PNAS USA 107:11393, 2010
Lipton et al., Nature 413:171,2003
Singel et al., Nature 430:297, 2004
Liu et al., Cell 116:617, 2004
Lipton et al., Science 308:1870, 2005
Cho et al., Science 324:102, 2009
Motivations (continued)
• Present methods of detecting Snitrosothiols (i.e. chemiluminescence
method) not sensitive enough to
accurately measure concentrations in
living cells, which are at nanomolar levels
• Ability to differentiate between isotopelabeled S-nitrosothiols will allow tracking of
S-nitrosothiols in cells and biological
tissues
NO and S-nitrosothiols
• NO can be easily released
from S-nitrosothiols
1) after exposure to UV light
(340 nm), ϕ up to 0.8
2) or reaction with LCysteine+CuCl mixture[1]
• S-nitrosothiols
concentration can be
deduced by measurement
of released NO amount.
Figure: Schematic of NO extraction
[1]
L.A. Palmer, B. Gaston, Methods Enzymol. 2008
[2] M. M. Veleeparampil, U.K. Aravind, and C. T. Aravindakumar, “Decomposition of SNitrosothiols Induced by UV and Sunlight,” Advances in Physical Chemistry, vol. 2009
Detection of NO (state-of-the-art)
Method
Advantages
Disadvantages Limit of
sensitivity
References
Laser
absorption
spectroscopy
Absolute
measurement
of
concentration
High number of
passes needed
to detect
change in laser
power over
laser noise
< 1 ppbv
J.B. McManus et
al (2006) Appl Phys
B 85, 235–
241.
Photo-acoustic
spectroscopy
Ease and
tolerance of
alignment
High power
laser required,
not absolute
method
15 ppbv
V. Spagnolo, et al.
(2010) Appl Phys B
100,
125–130
Faraday
Modulation
Spectroscopy
Smaller optical
path required
Limited to small
J values
0.38 ppbv
R. Lewicki, et al.,
PNAS August 4,
2009 vol. 106
Cavity
Compact and
Difficulty in
< 0.7 ppbv
Ringdown
insensitive
to
alignment
•
For review of NO
detection methods,
see Elia, A. etofal., 2011
Spectroscopy
laser power
cavity
fluctuations
A. A. Kosterev, et
al., Appl. Opt. 40,
5522 (2001)
We are building a cw-CRDS
instrument to:
• Accurately detect and measure
concentration of nitric oxide, released from
S-nitrosothiols, down to pptv levels using a
cavity ringdown technique
• Develop a portable CRDS system that can
measure NO in a gaseous sample in realtime with high sensitivity and determine
14NO/15NO ratio
Cavity Ring-down Spectroscopy
• Highly reflective mirrors (of 1- R < 10-4) allow light
L
to bounce many times in cavity, decaying in
(1 R)c
• Addition of sample with absorption coefficient
L
α(υ)=Nσ(υ) yields
c(1 R L)
• Thus ringdown time is used to measure
concentration N
To detector
IR from laser
High number of passes due to
high reflectivity of mirrors
Time
Main advantages of CRDS
• High sensitivity (projected to be up to 2
orders of magnitude better than
chemiluminescence)
• Being a direct absorption method, does
not require concentration calibration
• High path length with small sample volume
compared to multipass LAS techniques
• Ability to distinguish concentrations of
15NO and 14NO separately
Schematic of cw-CRDS instrument
Description of External Cavity
Quantum Cascade Laser
• Model: Daylight
Solutions mid-IR
tunable ec-QCL
Center λ:
1916 cm-1
• Tuning range:
70 cm-1
• Line width:
~ 100 kHz
Peak power:
60 mW
1940
1880
Absorption band
• rotationally resolved lines in the vibrational
fundamental transition near 5.2 µm
• R-branch lines of both 3/2 and 1/2 magnetic
electronic substates distinguishable
14
NO in He (100 torr)
R(1.5)
R(9.5), Ω = 1/2
2
cm )
3
R(9.5), Ω = 3/2
(in 10
-18
2
1
0
1880
1890
1900
1910
1920
1930
-1
Frequency (cm )
Data simulated from HITRAN2004 and He broadening data from R. Pope, J. Wolff, J.
Molec. Spectr. 208, 2001)
1940
Absorption band (continued)
• 14NO and 15NO lines distinguishable with laser
• Region is absent of strong H2O and CO2 lines
NO
NO
H2O, x100 times
15
2
Cross section cm
14
3
CO2, x100 times
Absorption cross section
14
15
of NO and NO in 100 torr
of air
R(7.5) for 14NO
2
R(20.5) for 15NO
R(19.5) for 15NO
1
0
1900
1901
1902
1903
1904
1905
1906
-1
Frequency (cm )
1907
1908
1909
1910
Principal scheme of cw-CRDS-instrument
Ge Acousto-optic modulator
IR from laser
AOM: Isomet 1207B-6
• PZT on AOM driven
by RF amp whose RF
source is shut off on
demand (extinction of
70 dB of laser intensity
into cavity)
• Shutdown time:
250 ns (measured)
Theoretical:
order
1st
to cavity
0th order
reference
Ring-down cavities
2-mirror:
Cavity length: 0.45 m
Volume: 228 mL
4-mirror
Cavity length: 0.33 m
Volume: 317 mL
Mirror
angle ~
1°
2-mirror cavity would have smaller volume but more
susceptible to laser feedback
Optical isolator
Isolation may not be necessary with
4-mirror cavity design
EO crystal is CdTe, used as ¼ wave plate
Improving on Kosterev’s work
• Our laser’s linewidth is an order of
magnitude less (~ 105 Hz vs. 106 Hz)
• Our laser shutoff time is controlled by the
AOM, also shorter (~ 10-7 s vs. 10-6 s)
• Members of group have obtained relative
errors in measurements of 10-5. Given
the absorption cross section of NO lines
in region, this corresponds to ~ 6 pptv
Future (long term)
• Optimize instrument’s optical components
to reach close to ppt levels in under 1 min
• Build an inlet system that can take in NO
from S-nitrosothiol sampling system and
feed into cavity
• Development of portable CRDS device
• Generalize system to work with breath NO
intake
Acknowledgments
• NSF Instrument Development for
Biological Research Program
• The NIH’s National Heart, Lung, and Blood
Institute (1P01 HL101871, 3R01 HL59337)