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.

Detection of NO and S-nitrosocompounds 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

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

NO and S-nitrosothiols• 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:

UV spectrum of synthetic GSNO[4]

[3]Veleeparampil, M., Adv. Phys. Chem., 2009[4]Balazy, M., J. Biol. Chem., 1998

Wavelength (nm)

Abso

rban

ce (a

rb. u

nits

)

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:

Mid-IR Spectroscopic detection of NO

14NO15NO

R1/2(13/2) for 14NO

R3/2(13/2) for 14NO

R1/2(37/2) for 15NO

R1/2(39/2) for 15NO

Simulated from HITRAN data [6]

Frequency (cm-1)

σ of

14N

O a

nd 15

NO

in 1

00 to

rr o

f air

(10-1

8 cm

2 )

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 usedto measure concentration N

L

cRk

)1(0

ckk 0

IR from laser

To detector

RD cavityLaser detector

Description of External Cavity Quantum Cascade Laser

• Model: Daylight Solutions mid-IR tunable ec-QCL Tuning range:70 cm-1

• Line width:~ 6 MHzPeak power tocavity:38 mW

1940 1880

Pow

er (m

W)

Wavenumber (cm-1)

[7]

NO lines of interest

Schematic of setup

ec-QCL (Laser)

AOM

Reference cell

InSb detector

Internally-coupledEtalon

Ring-down cavity

isolatorInSb detector

Mode-matchingoptics

Trigger

InSb detector

PC-DAQ

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

• 0th order to reference cell and etalon for frequency calibration

Laser

Cavity

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

• 0th order to reference cell and etalon for frequency calibration

Laser

Cavity

Optical isolation

CdTe EO crystal, used as ¼ wave plate

Returning beamblocked by polarizer

HV applied across crystal leads todifference in refractive index between x- and y- polarizations

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14

-4

-2

0

2

4

6

8

10 Scan of cavity over1.2 FSR

Cavity modes with isolator

Cavity modes w/o isolator

time

Cavity Apparatus

• R = 0.99975 (F ~ 12000)ZnSe mirrors with coating (LohnStar)

• L = 0.35 m• V= 350 mL• FSR = 430 MHz• τ0 = 4.6 μs

Mirror configuration:

Invar plate to fixcavity length

Cavity surfacescoated with inertcoating (SilcoTekTM)

“Supermirrors” PZTs to scan

up to 1.4 cavity FSRs

Gas delivery to cavity

UV Lamp

Flask with

GSNO sample

He flow7 μm

particle filter

Cold trap(LN2 and ethanol slurry)

2 μm particle

filtermanifold

Ring-down cavity

Vacuum pump

2.9ppm NO in He

tank

Legend:NO flow

Valve

Observed resultsNO line R3/2(13/2) at 1900.75 cm-1

Frequency detuning (MHz)

τ (μ

s)

0 300 600 1200900

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

References1. 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.com8. M. Reich, et al., Appl. Optics 25, 19869. 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.