1. 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

2. 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
R: cysteine, protein
Lipton, A.J. et al. //Nature 2001; Arnelle D. R. and Stamler J. S. // Arch. Biochem. Biophys. 1995; Gaston, B. et al. //PNAS 1993

3. Motivations
S-nitrosothiol signaling is involved in many different types of disease. For example:
Cancer: Lim et al. Nature 452:646, 2008
Asthma: Que et al., Science 308:1618, 2005
Cystic Fibrosis: Marozkina et al., PNAS USA 107:11393, 2010
Apnea: Lipton et al., Nature 413:171,2003
Ischemia: Singel et al., Nature 430:297, 2004
Shock: Liu et al., Cell 116:617, 2004
Parkinson’s: Lipton et al., Science 308:1870, 2005
Alzheimer’s: Cho et al., Science 324:102, 2009 3

4. Motivations (continued)
Present methods of detecting S-nitrosothiols (i.e. chemiluminescence method) not sensitive enough to accurately measure concentrations in living cells, which are at nanomolar levels
Ability to differentiate between isotope-labeled S-nitrosothiols will allow tracking of S-nitrosothiols in cells and biological tissues
Picture of gas extraction from sample, convert picomole to partial pressure of NO

5. 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 L-Cysteine+CuCl mixture[1]
S-nitrosothiols concentration can be deduced by measurement of released NO amount.
Napthlene dryer between S-nitrosothiol and CRD cell
Put in schematic of photodissociation of NO and action of carrier gas to cavity, solenoid valves that fill cell and pump out cell
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 S-Nitrosothiols Induced by UV and Sunlight,” Advances in Physical Chemistry, vol. 2009

6. 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, vol. 106
Cavity Ringdown Spectroscopy
Compact and insensitive to laser power fluctuations
Difficulty in alignment of cavity
< 0.7 ppbv
A. A. Kosterev, et al., Appl. Opt. 40, 5522 (2001)
The advantages of our setup over Kosterev: narrower laser linewidth (they had 3 MHz), shorter shutdown time with AOM vs. Shutting off laser current. Also say how with a L cavity this is equivalent to 1.2 pmol assuming 0.7 ppbv
For review of NO detection methods, see Elia, A. et al., 2011

7. 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 real-time with high sensitivity and determine 14NO/15NO ratio

8. Cavity Ring-down Spectroscopy
Highly reflective mirrors (of 1- R < 10-4) allow light to bounce many times in cavity, decaying in
Addition of sample with absorption coefficient α(υ)=Nσ(υ) yields
Thus ringdown time is used to measure concentration N
Make cavity ringdown mirrors look curved, and make arrow to detector squigly to show it as « leakage »
To detector
IR from laser
High number of passes due to
high reflectivity of mirrors
Time

9. 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

10. Schematic of cw-CRDS instrument

11. 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
Our line width will be better than Kosterev’s by an order of magnitude
1940
1880

12. 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
R(1.5)
R(9.5), Ω = 1/2
R(9.5), Ω = 3/2
Also talk about being away from Q branch which is hard to resolve, pronounce NO15 as “N15O”
Data simulated from HITRAN2004 and He broadening data from R. Pope, J. Wolff, J. Molec. Spectr. 208, 2001)

13. Absorption band (continued)
14NO and 15NO lines distinguishable with laser
Region is absent of strong H2O and CO2 lines
R(7.5) for 14NO
Blow up H20 and CO2 lines so we can see which NO lines are best to detect
R(19.5) for 15NO
R(20.5) for 15NO

14. Principal scheme of cw-CRDS-instrument

15. Ge Acousto-optic modulator
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:
IR from laser
Measured value around 250 ns, determined by speed of sound. This is several times less than the shutoff time in Kosterev’s set up which used laser current modulation.
1st order
to cavity
AOM: Isomet 1207B-6
0th order
reference

16. Ring-down cavities 2-mirror: 4-mirror
Cavity length: 0.45 m
Volume: 228 mL
4-mirror
Talk about smaller volume leading to smaller minimum moles detectable, so want long but narrow cavity for max optical path length
Calculate effective path length based on mirror’s reflectivity (99.975%). Put in picture of mirror and how reflective they are. So 4000 passes = ~ 1.7 km
Cavity length: 0.33 m
Volume: 317 mL
Mirror angle ~ 1°
2-mirror cavity would have smaller volume but more susceptible to laser feedback

17. Optical isolator Isolation may not be necessary with
4-mirror cavity design
EO crystal is CdTe, used as ¼ wave plate

18. 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 s)
Members of group have obtained relative errors in t measurements of Given the absorption cross section of NO lines in region, this corresponds to ~ 6 pptv

19. 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
Current progress Under 1 minute of data collection (multiple ringdowns)
Put in expected limit of detection

20. Acknowledgments
NSF Instrument Development for Biological Research Program
The NIH’s National Heart, Lung, and Blood Institute (1P01 HL101871, 3R01 HL59337)
National Institutes of Health’s National Heart, Lung, and Blood Institute