1. Detection of NO and S- nitrosocompounds using mid-IR CRDS Vitali Stsiapura 1, Vincent K. Shuali 1, Angela Ziegler 1, Kevin K. Lehmann 1, Benjamin M. Gaston 2 1 University of Virginia; 2 Case Western Reserve University

2. 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 S-nitrosoglutathione, an S-nitrosothiol [1]Lipton. A. J., Nature, 2001 [2]Arnell, D. R., Arch. Biochem. Biophys., 1995

3. NO and S-nitrosothiols UV spectrum of synthetic GSNO [4] [3] Veleeparampil, M., Adv. Phys. Chem., 2009 [4] Balazy, M., J. Biol. Chem., 1998 Wavelength (nm) Absorbance (arb. units)

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 NO chemiluminescence apparatus [5] [5] USGS Biogeochemistry of Carbon and Nitrogen in Aquatic Environments:

5. Mid-IR Spectroscopic detection of NO 14 NO 15 NO R 1/2 (13/2) for 14 NO R 3/2 (13/2) for 14 NO R 1/2 (37/2) for 15 NO R 1/2 (39/2) for 15 NO Simulated from HITRAN data [6] Frequency (cm -1 ) σ of 14 NO and 15 NO in 100 torr of air (10 -18 cm 2 )

6. 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: Addition of sample with absorption coefficient α(υ)=Nσ(υ) yields: Thus ringdown time is used to measure concentration N IR from laser To detector RD cavity Laser detector

7. 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 19401880 Power (mW) Wavenumber (cm -1 ) [7] NO lines of interest

8. Schematic of setup ec-QCL (Laser) AOM Reference cell InSb detector Internally-coupled Etalon Ring-down cavity isolator InSb detector Mode-matching optics Trigger InSb detector PC- DAQ

9. Cavity Ring-down scheme AOM: R37040-3-5.4 (Gooch & Housego) Laser deflected and freq shifted by AOM to cavity, shut off of AOM in ~ 150 ns 0 th order to reference cell and etalon for frequency calibration Cavity

10. Cavity Ring-down scheme AOM: R37040-3-5.4 (Gooch & Housego) Laser deflected and freq shifted by AOM to cavity, shut off of AOM in ~ 150 ns 0 th order to reference cell and etalon for frequency calibration Cavity

11. Optical isolation CdTe EO crystal, used as ¼ wave plate 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 Cavity modes with isolator Cavity modes w/o isolator time

12. Cavity Apparatus R = 0.99975 (F ~ 12000) ZnSe mirrors with coating (LohnStar) L = 0.35 m V= 350 mL FSR = 430 MHz τ 0 = 4.6 μ s Mirror configuration: Invar plate to fix cavity length Cavity surfaces coated with inert coating (SilcoTek TM ) “Super mirrors” PZTs to scan up to 1.4 cavity FSRs

13. Frequency stabilization Internally coupled Fabry- Perot etalon Dithering of laser line around cavity mode Laser lineCavity mode Frequency [8] [8] Reich, 1986

14. Gas delivery to cavity UV Lamp Flask with GSNO sample He flow 7 μm particle filter Cold trap (LN2 and ethanol slurry) 2 μm particle filter manifold Ring-down cavity Vacuum pump 2.9ppm NO in He tank Legend: NO flow Valve

15. Observed results NO line R 3/2 (13/2) at 1900.75 cm -1 Frequency detuning (MHz) τ (μs) 0 300 600 1200 900

16. Estimate of limit of detection Allan deviation of k Min σ α : 7.8 × 10 -10 cm -1

17. Isotopic measurement 0 0.3 0.6 0.9 1.2 1.5 1.8 Frequency detuning (GHz)

18. 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 14 NO and 15 NO levels in same scan [10] Kosterev, A., Appl. Optics, 2001 [11] Heinrich, K., Appl. Phys. B., 2009

19. Future plans [9] [9] Giusfredi, G., Phys. Rev. Letters, 2010

20. Acknowledgments NIH and NSF: financial support Dr. Joseph Hodges (NIST): advice and assistance on cavity length and frequency stabilization

21. 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 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.comhttp://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 continuous- wave 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.

22. Statistics Transverse spacing: 135.2 MHz Pressure broadening: ~ 2.6 MHz/torr (self), ~ 2.2 MHz/torr (He) Transit time broadening: 180 kHz Etalon FSR: 750 MHz SNR of detector, sensitivity, etc. Thermal drift stuff

23. Gas delivery to cavity UV Photolysis Lamp Cold Trap (LN2 + ethanol) 3 Å Molecular Sieve He flow NO in He 2 μm particle filter To RD cell

24. Saturation Example Lamb dip [10] Lamb-dip Doppler- broadened line [9] [9] Giusfredi, 2010 [10] Taubman, 2004

25. Stabilization of cavity length HeNe AOM Frequency Comb IR Laser Look at beat freq Ring-down detector freq PDH Ideal beat freq Actual beat deviation freq PZT Ring-down cavity