Detection of NO and Snitrosocompounds using mid-IR
CRDS
Vitali Stsiapura1, Vincent K. Shuali1,
Angela Ziegler1, Kevin K. Lehmann1,
Benjamin M. Gaston2
1University
of Virginia; 2Case Western Reserve University
Biochemistry of NO-containing
compounds
• S-nitrosothiols (RS-NO) receiving attention in
biochemistry and medicine as donors of nitric oxide
(NO) and nitrosonium (NO+) - physiologically active
molecules involved in signal transduction through
transnitrosation of thiol protein groups[1][2]
• S-nitrosothiol signaling involved in various types of
cellular processes, diseases, e.g. cancer, asthma, cystic
fibrosis
[1]Lipton. A. J., Nature, 2001
[2]Arnell, D. R., Arch. Biochem.
Biophys., 1995
S-nitrosoglutathione, an S-nitrosothiol
• NO can be easily released
from S-nitrosothiols after
exposure to UV light (340
nm), quantum efficiency up
to 0.8 [3]
• S-nitrosothiols concentration
can be deduced by
measurement of released
NO amount:
• 𝑅𝑆𝑁𝑂
ℎν
[3]Veleeparampil,
𝑅𝑆 ∙ + .𝑁𝑂(𝑔)
M., Adv. Phys. Chem., 2009
[4]Balazy, M., J. Biol. Chem., 1998
Absorbance (arb. units)
NO and S-nitrosothiols
Wavelength (nm)
UV spectrum of synthetic GSNO[4]
Motivations
• Present methods of
detecting NO (g) (i.e.
chemiluminescence) not
sensitive enough to
measure concentrations
released from living cells,
at nanomolar levels
• Ability to differentiate
between isotope-labeled
NO will allow tracking of
NO compounds in cells
and biological tissues
[5]USGS
NO chemiluminescence apparatus[5]
Biogeochemistry of Carbon and Nitrogen in Aquatic Environments:
14NO and 15NO in 100 torr-18
air2
σ
of
ofcm
absorption cross-section,
´10
(10-18 cm2)
Mid-IR Spectroscopic detection of NO
3.5
14
R1/2(13/2)
for 14NO
3.0
14NO
15NO
R3/2(13/2)
for 14NO
2.5
NO
15
NO
H2O
100 torr He
296 K
2.0
1.5
R1/2(37/2)
for 15NO
R1/2(39/2)
for 15NO
1.0
0.5
0.0
1899
1900
1901
1902
-1
(cm )
1903
Frequency
-1
wavenumber, cm
Simulated from HITRAN
data [6] 1905
1904
Cavity Ring-down Spectroscopy
• Highly reflective mirrors (of 1- R < 10-4) allow light to bounce
many times in cavity, whose intensity decays in time at the rate
of:
(1  R)c
k0 
L
• Addition of sample with absorption coefficient α(υ)=Nσ(υ) yields:
k  k 0  c
• Thus ringdown time is used
to measure concentration N
To detector
IR from laser
Laser
RD cavity
detector
Description of External Cavity
Quantum Cascade Laser
• Model: Daylight
Solutions mid-IR
tunable ec-QCL
Tuning range:
70 cm-1
• Line width:
~ 6 MHz
Peak power to
cavity:
38 mW
Power (mW)
[7]
NO lines of interest
1940
Wavenumber (cm-1)
1880
Schematic of setup
InSb
detector
ec-QCL
(Laser)
AOM
Reference
cell
Internally-coupled
Etalon
InSb
detector
PCDAQ
Trigger
Mode-matching
optics
InSb
detector
Ring-down cavity
isolator
Cavity Ring-down scheme
Cavity
• AOM: R37040-3-5.4 (Gooch &
Housego)
• Laser deflected and freq shifted
by AOM to cavity, shut off of
AOM in ~ 150 ns
• 0th order to reference cell and
etalon for frequency calibration
Cavity Ring-down scheme
Cavity
• AOM: R37040-3-5.4 (Gooch &
Housego)
• Laser deflected and freq shifted
by AOM to cavity, shut off of
AOM in ~ 150 ns
• 0th order to reference cell and
etalon for frequency calibration
Optical isolation
Returning beam
blocked by polarizer
HV applied across crystal leads to
difference in refractive index between
x- and y- polarizations
Scan of cavity over
1.2 FSR
10
8
6
4
2
Cavity modes
with isolator
0
-2
CdTe EO crystal, used as ¼
wave plate
Cavity modes
w/o isolator
-4
0.00
0.02
0.04
0.06
time
0.08
0.10
0.12
0.14
Cavity Apparatus
“Super
mirrors”
Cavity surfaces
coated with inert
coating
(SilcoTekTM)
PZTs to scan
up to 1.4
cavity FSRs
Invar plate to fix
cavity length
• R = 0.99975 (F ~ 12000)
Mirror configuration: ZnSe mirrors with coating
(LohnStar)
• L = 0.35 m
• V= 350 mL
• FSR = 430 MHz
• τ0 = 4.6 μs
Gas delivery to cavity
7 μm
particle
filter
He flow
UV
Lamp
Flask
with
GSNO
sample
2.9ppm
NO in He
tank
manifold
Cold trap
(LN2 and
ethanol
slurry)
2 μm
particle
filter
Legend:
Vacuum
pump
NO flow
Ring-down cavity
Valve
Observed results
τ (μs)
NO line R3/2(13/2) at 1900.75 cm-1
0
300
600
900
Frequency detuning (MHz)
1200
Estimate of limit of detection
Allan deviation of k
Min σα: 7.8 × 10-10 cm-1
Isotopic measurement
0
0.3
0.6
0.9
1.2
1.5
1.8
0
0.3
0.6
0.9
1.2
1.5
1.8
0
0.3
0.6
0.9
1.2
1.5
1.8
Frequency detuning (GHz)
Conclusions
• Constructed compact RD system able to
measure sample concentration in seconds
• Obtained limit of detection of 30 pptv,
exceeding Kosterev’s limit of 0.7ppbv[10], goal
to exceed Mürtz’s[11] 7 pptv level
• Confirmed ability to measure 14NO and 15NO
levels in same scan
[10]Kosterev,
A., Appl. Optics, 2001
[11]Heinrich, K., Appl. Phys. B., 2009
Future plans
[9]
[9]Giusfredi,
G., Phys. Rev. Letters, 2010
Acknowledgments
• NIH and NSF: financial support
• Dr. Joseph Hodges (NIST): advice and
assistance on cavity length and frequency
stabilization
References
1.
Lipton, Andrew J., et al. "S-nitrosothiols signal the ventilatory response to hypoxia." Nature 413.6852
(2001): 171-174.
2. Arnelle, Derrick R., and Jonathan S. Stamler. "NO+, NO., and NO− donation by S-nitrosothiols:
implications for regulation of physiological functions by S-nitrosylation and acceleration of disulfide
formation." Archives of biochemistry and biophysics 318.2 (1995): 279-285.
3. Veleeparampil, Manoj M., Usha K. Aravind, and C. T. Aravindakumar. "Decomposition of S-Nitrosothiols
Induced by UV and Sunlight." Advances in Physical Chemistry 2009 (2010).
4. Balazy, Michael, et al. "S-Nitroglutathione, a product of the reaction between peroxynitrite and
glutathione that generates nitric oxide." Journal of Biological Chemistry 273.48 (1998): 32009-32015.
5. USGS Biogeochemistry of Carbon and Nitrogen in Aquatic Environments:
http://wwwbrr.cr.usgs.gov/projects/EC_biogeochemistry/facilities.htm
6. Rothman, Laurence S., et al. "The HITRAN 2004 molecular spectroscopic database." Journal of
Quantitative Spectroscopy and Radiative Transfer 96.2 (2005): 139-204.
7. Daylight Solutions, Inc. http://www.daylightsolutions.com
8. M. Reich, et al., Appl. Optics 25, 1986
9. Giusfredi, G., et al. "Saturated-absorption cavity ring-down spectroscopy." Physical review letters
104.11 (2010): 110801.
10. Kosterev, Anatoliy A., et al. "Cavity ringdown spectroscopic detection of nitric oxide with a continuouswave quantum-cascade laser." Applied optics 40.30 (2001): 5522-5529.
11. Heinrich, K., et al. "Infrared laser-spectroscopic analysis of 14NO and 15NO in human breath." Applied
Physics B 95.2 (2009): 281-286.