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Presentation on theme: "MID-IR CAVITY RING-DOWN SPECTROMETER FOR BIOLOGICAL TRACE NITRIC OXIDE DETECTION Vincent Kan 2, Vitali Stsiapura 1,3, Ahmed Ragab 1, Kevin K. Lehmann 1,2,"— Presentation transcript:

1 MID-IR CAVITY RING-DOWN SPECTROMETER FOR BIOLOGICAL TRACE NITRIC OXIDE DETECTION Vincent Kan 2, Vitali Stsiapura 1,3, Ahmed Ragab 1, Kevin K. Lehmann 1,2, Ben Gaston 3 1 Dept. of Chemistry, 2 Dept. of Physics, 3 School 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 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

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

5 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. NO and S-nitrosothiols [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 Figure: Schematic of NO extraction

6 Detection of NO (state-of-the-art) For review of NO detection methods, see Elia, A. et al., 2011 MethodAdvantagesDisadvantagesLimit 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 ppbvV. Spagnolo, et al. (2010) Appl Phys B 100, 125–130 Faraday Modulation Spectroscopy Smaller optical path required Limited to small J values 0.38 ppbvR. Lewicki, et al., PNAS August 4, 2009 vol. 106 Cavity Ringdown Spectroscopy Compact and insensitive to laser power fluctuations Difficulty in alignment of cavity < 0.7 ppbvA. A. Kosterev, et al., Appl. Opt. 40, 5522 (2001)

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 14 NO/ 15 NO 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 IR from laser To detector 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 15 NO and 14 NO 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 19401880

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 Data simulated from HITRAN2004 and He broadening data from R. Pope, J. Wolff, J. Molec. Spectr. 208, 2001) R(9.5), Ω = 3/2 R(9.5), Ω = 1/2 R(1.5)

13 Absorption band (continued) 14 NO and 15 NO lines distinguishable with laser Region is absent of strong H 2 O and CO 2 lines R(19.5) for 15 NO R(20.5) for 15 NO R(7.5) for 14 NO

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 AOM: Isomet 1207B-6 1 st order to cavity 0 th order reference

16 Ring-down cavities 2-mirror cavity would have smaller volume but more susceptible to laser feedback 2-mirror: 4-mirror Cavity length: 0.45 m Volume: 228 mL Cavity length: 0.33 m Volume: 317 mL Mirror angle ~ 1°

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

18 Improving on Kosterev’s work Our laser’s linewidth is an order of magnitude less (~ 10 5 Hz vs. 10 6 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

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

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

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