Micro Gas Analyzer Measurement of Nitric Oxide in Breath by Direct

Jul 15, 2009 - Breath samples were collected into a 5 L Mylar bag, and then these were supplied to both the micro gas analyzer and the CL instrument. ...
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Micro Gas Analyzer Measurement of Nitric Oxide in Breath by Direct Wet Scrubbing and Fluorescence Detection Kei Toda,*,† Takahiro Koga,† Junichi Kosuge,‡ Mieko Kashiwagi,‡ Hiroshi Oguchi,§ and Takemi Arimoto§ Department of Chemistry, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan, Tsukuba Research Institute, Sekisui Medical Co., Ltd., 3-3-1 Koyodai, Ryugasaki, Ibaraki 301-0852, Japan, and GASTEC Co., 8-8-6 Fukayanaka, Ayase, Kanagawa 252-1195, Japan A novel method is proposed to measure NO in breath. Breath NO is a useful diagnostic measure for asthma patients. Due to the low water solubility of NO, existing wet chemical NO measurements are conducted on NO2 after removal of pre-existing NO2 and conversion of NO to NO2. In contrast, this study utilizes direct measurement of NO by wet chemistry. Gaseous NO was collected into an aqueous phase by a honeycombpatterned microchannel scrubber and reacted with diaminofluorescein-2 (DAF-2). Fluorescence of the product was measured using a miniature detector, comprising a blue light-emitting diode (LED) and a photodiode. The response intensity was found to dramatically increase following addition of NO2 into the absorbing solution or air sample. By optimizing the conditions, the sensitivity obtained was sufficient to measure parts per billion by volume levels of NO continuously. The system was applied to real analysis of NO in breath, and the effect of coexisting compounds was investigated. The proposed system could successfully measure breath NO. Gaseous nitric oxide (NO) is an important precursor of atmospheric pollution. In addition, exhaled NO (eNO) in human breath reflects health conditions. Gaseous NO in breath was first identified in 1991 by Gustafsson et al.1 Following this finding, many sources of eNO have been reported, for example NO formed enzymatically by NO synthase (NOS) in alveoli,2 by NOS in paranasal sinuses,3 and in the oral cavity (mainly from NOS activity4). Increased eNO as a symptom in asthma patients has * To whom correspondence should be addressed. E-mail: todakei@ sci.kumamoto-u.ac.jp. Phone: +81-96-342-3389. † Kumamoto University. ‡ Sekisui Medical Co., Ltd. § GASTEC Co. (1) Gustafsson, L. E.; Leone, A. M.; Persson, M. G.; Wiklund, N. P.; Moncada, S. Biochem. Biophys. Res. Commun. 1991, 181, 852–857. (2) Kobzik, I.; Bredt, D. S.; Lowenstein, C. J.; Drazen, J.; Gaston, B.; Sugarbaker, D.; Stamler, J. S. Am. J. Respir. Cell Mol. Biol. 1993, 9, 371–377. (3) Lundberg, J. O.; Farkas-Szallasi, T.; Weitzberg, E.; Rinder, J.; Lidholm, J.; Anggaard, A.; Hokfelt, T.; Lundberg, J. M.; Alving, K. Nat. Med. 1995, 1, 370–373. (4) Rimeika, D.; Nyren, S.; Wiklund, N. P.; Koskela, L. R.; Torring, A.; Gustafsson, L. E.; Larsson, S. A.; Jacobsson, H.; Lindahl, S. G.; Wiklund, C. U. Am. J. Respir. Crit. Care Med. 2004, 170, 450–455. 10.1021/ac901131d CCC: $40.75  2009 American Chemical Society Published on Web 07/15/2009

recently been highlighted.5,6 As an endothelium-derived relaxing factor, NO causes vasodilation7,8 and it is also generated by NOS as a result of self-defense mechanisms. Therefore, eNO measurement is expected to be useful for noninvasive asthma diagnosis.9-11 Conventionally, a peak flow meter is used to monitor the timing of treatment for asthma patients. However, blowing strongly into a peak flow meter is hard, especially for the severe asthma sufferers. Breath analysis could potentially become a common protocol for noninvasive diagnosis of several diseases.12,13 The most popular method to determine gaseous NO is based on ozone induced chemiluminescence.14-16 Other methods used to identify eNO include gas chromatography/mass spectroscopy,17 selected ion flow tube mass spectroscopy (so-called SIFT-MS), and a quantum cascade laser-based spectroscopic analysis.18-20 However, development of smaller and more inexpensive instrumentation is required for both detailed atmospheric analysis and home (5) Alving, K.; Weitzberg, E.; Lundberg, J. M. Eur. Respir. J. 1993, 6, 1368– 1370. (6) Kharitonov, S. A.; Yates, D.; Robbins, R. A.; Logan-Sinclair, R.; Shinebourne, E. A.; Barnes, P. J. Lancet 1994, 343, 133–135. (7) Palmer, R. M.; Ferrige, A. G.; Moncada, S. Nature 1987, 327, 524–526. (8) Ignarro, L. J.; Buga, G. M.; Wood, K. S.; Byrns, R. E.; Chauduri, G. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 9265–9269. (9) Delgado-Coreoran, C.; Kisson, N.; Murphy, S. P.; Duckworth, L. J. Pediatr. Crit. Care Med. 2004, 5, 48–52. (10) Puckett, J. L.; George, S. C. Respir. Physiol. Neurobiol. 2008, 163, 166– 177. (11) Szefler, S.; Mitchell, H.; Sorkness, C. A.; Gergen, P. J.; O’Connor, G. T.; Morgan, W. J.; Katton, M.; Pongracic, J. A.; Teach, S. J.; Bloomberg, G. R.; Eggleston, P. A.; Gruchalla, R. S.; Kercsmar, C. M.; Liu, A. H.; Wildfire, J. J.; Curry, A. D.; Busse, W. Lancet 2008, 372, 1065–1072. (12) Amann, A.; Smith, D. Foreword. In Breath Analysis for Clinical Diagnosis and Therapeutic Monitoring; Amann, A., Smith, D., Eds. World Scientific Publishing: Singapore, 2005; pp xi-xviii. (13) van Rensen, E. L.; Straathof, K. C.; Veselic-Charavat, M. A.; Zwinderman, A. H.; Bel, E. H.; Sterk, P. J. Thorax 1999, 54, 403–408. (14) Fontijn, A.; Sabadell, A. J.; Ronco, R. J. Anal. Chem. 1970, 42, 575–579. (15) Toda, K.; Dasgupta, P. K. Chem. Eng. Commun. 2008, 195, 85–97. ¨ gren, E.; Szefler, S. J. J. (16) Silkoff, P. E.; Carlson, M.; Bourke, T.; Katial, R.; O Allergy Clin. Immunol. 2004, 114, 1241–1256. (17) Leone, A. M.; Gustafsson, L. E.; Francis, P. L.; Persson, M. G.; Wiklund, N. P.; Moncada, S. Biochem. Biophys. Res. Commun. 1994, 201, 883–887. (18) McCurdy, M. R.; Bakhirkin, Y. A.; Tittel, F. K. Appl. Phys. B: Laser Opt. 2006, 85, 445–452. (19) McCurdy, M. R.; Bakhirkin, Y.; Wysocki, G.; Tittel, F. K. J. Biomed. Opt. 2007, 12, 34034. (20) Heinrich, K.; Fritsch, T.; Hering, P.; Mu ¨ rtz, M. Appl. Phys. B: Laser Opt. 2009, 95, 281–286.

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diagnostics for asthma patients.21 Electrochemical sensors22-24 or miniature flow systems are one potential way to achieve this. However, wet chemical measurements require conversion of NO to NO2 after removal of any pre-existing NO2.25 Thus, the wet analysis system for NO is complicated, and results could be affected by both NO2 removal and conversion conditions. In order to overcome these problems, we attempted to develop a simple method to determine gaseous NO directly. Previously, we have developed a flow-based micro gas analysis system (µGAS) for on-site monitoring of atmospheric gases.26,27 This µGAS device has subsequently been applied to NO analysis. NO2 was measured using a Griess-Saltzman reaction, after removal of pre-existing NO2 and conversion of NO to NO2.28 Much attention has recently focused on carbon nanotube sensors due to their high sensitivity. Kuzmych et al. developed such a sensor for eNO, based on the same sequence (NO2 removal and conversion to NO2) and poly(ethylene imine) modified carbon nanotubes reactive to NO2.29 This system had a limit of detection (LOD) of 5 ppbv as NO, and the performance was optimized under high CO2 concentration. Unfortunately, the system has not been tested with real breath. Robinson et al. developed an alternative method and applied it to real breath analysis;30 chemiluminescence generated on the liquid-air interface was monitored on the hollow fibers. The reported performance was sufficient for breath analysis, with a LOD of 0.3 ppbv and response time of 2 s. However, this method also utilized NO2 removal and conversion of NO to NO2. In this work, we conducted a feasibility study on the measurement of eNO by combining a high performance gas collector with a superior NO probe reagent. To the best of our knowledge, this is the first report of direct measurement of gaseous NO with wet chemistry. An aqueous solution of diaminofluorescein-2 (DAF2), a reagent developed as a biochemical NO probe,31 was introduced through a honeycomb-shaped microchannel scrubber of µGAS. In the µGAS system, NO was extracted from airflow to react with DAF-2 in the aqueous phase. Whereas DAF-2 is not fluorescent due to photo induced electron transfer, it forms a triazolfluorescein product (DAF-2T) on reaction with NO. Fluorescence of the eluent from the microchannel scrubber was measured using a miniature detector comprising a blue lightemitting diode (LED) and a miniature photomultiplier tube module. (21) Ohira, S.; Toda, K. Anal. Chim. Acta 2008, 619, 143–156. (22) Gouma, P. I.; Kalyanasundaram, K. Appl. Phys. Lett. 2008, 93, Art. 244102. (23) Hemmingsson, T.; Linnarsson, D.; Gambert, R. J. Clin. Monit. Comput. 2004, 18, 379–387. (24) Maniscalco, M.; de Laurentiis, G.; Weitzberg, E.; Lundberg, J. O.; Sofia, M. Eur. J. Clin. Invest. 2008, 38, 197–200. (25) Japanese Standard Association. Continuous analyzers for oxides of nitrogen in ambient air, JIS B7953 (1981). JIS handbook Environmental Analysis; Japanese Standards Association: Tokyo, 1994; pp 683-698. (26) Toda, K.; Ohira, S.; Ikeda, M. Anal. Chim. Acta 2004, 511, 3–10. (27) Ohira, S.; Toda, K. Lab Chip 2005, 5, 1374–1379. (28) Toda, K.; Hato, Y.; Ohira, S.; Namihira, T. Anal. Chim. Acta 2007, 603, 60–65. (29) Kuzmych, O.; Allen, B. L.; Star, A. Nanotechnology 2007, 18, 375502– 375508. (30) Robinson, J. K.; Bollinger, M. J.; Birks, J. W. Anal. Chem. 1999, 71, 5131– 5136. (31) Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi, H.; Hirata, Y.; Nagano, T. Anal. Chem. 1998, 70, 2446–2453.

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EXPERIMENTAL SECTION Reagents and Gases. Fluorescent reagent DAF-2 (5 mM in DMSO, 0.55 mL in a bottle) was supplied as a gift from Sekisui Medical. A working solution of DAF-2 was prepared by dilution to 10 µM and alkalinity adjustment to 0.01 M NaOH. Standard gases NO and NO2 of a 100 ppmv concentration balanced with nitrogen were obtained from Japan Fine Products (Kawasaki). Investigation of interferences was conducted using source gases from standard cylinders of 100 ppmv of NH3, dimethyl sulfide (DMS), H2S, CH3SH, and CO, and 100% CO2. Organic vapors of acetone, acetaldehyde, isoprene, ethanol, and acetic acid were prepared by diffusion tubes made of glass kept in a thermally controlled chamber (Permeator, PD-1B-2, and diffusion tubes D-01) obtained from GASTEC (Ayase, Kanagawa). The temperature of the permeation chamber was maintained at 35 °C. Measurement System. The micro gas analyzer was made up of a microchannel scrubber and a fluorescence detector. The microchannel scrubber was fabricated on a polydimethylsiloxane (PDMS) plate on which a gas permeable membrane was pasted.27 A porous polytetrafluoroethylene (pPTFE) membrane was pasted on the PDMS microchannel. The channels were 200 µm wide and 100 µm deep and arranged in a honeycomb shape (Figure 1a). The DAF-2 solution was held in the microchannel while sample air was introduced to the opposite side of the membrane. NO gas molecules diffused through the membrane to react with DAF-2 in the solution. The DAF-2 solution, held in a 10 mL plastic syringe, was made to flow at 6-30 µL/min by a syringe pump (model 200, KD Scientific, Holliston, MA). After passing through the microchannel scrubber, the solution was introduced to the fluorescence detector. The detector included a transparent FEP tube (18 LW, Zeus, Orangeburg, SC) functioning as a fluorescence cell, a blue LED (NSPB500S (λmax 460 nm), Nichia, Anan, Tokushima, Japan), and a miniature photomultiplier tube (mPMT) module (H5784, Hamamatsu Photonics). A 520 nm cutoff filter film (SC-52, Fujifilm, Tokyo) was pasted on the optical window of the mPMT module. All parts in the liquid line were connected with small-bore Teflon tubing (AWG30, 0.3 mm i.d.). Sample air was aspirated by a miniature air pump (CM-15-12, Enomoto Micropump, Tokyo) and controlled at a flow rate of 200 mL/min by a rotameter equipped with a needle valve (RK200-V-B-6H-AIR-0.5 L/min, Kojima Instruments, Kyoto). Test gas was prepared by diluting standard NO gas twice with a house-made gas dilution system. The gas dilution system incorporated six mass flow controllers (SEC-E40MK3, Horiba STEC, Kyoto) and could prepare mixtures of three kinds of gases in parts per billion by volume levels. The prepared test gas was sampled by the described micro gas analyzer and concurrently by a chemiluminescence (CL) NOx analyzer (model 42i, Nippon Thermo, Kyoto) for comparison. Breath samples were collected into a 5 L Mylar bag, and then these were supplied to both the micro gas analyzer and the CL instrument. As the need arose, a soda lime column was used in collection of breath to the sample bag, a NO2 permeation tube was placed between the sample bag and analyzer during measurement, and an activated carbon column was used to clean the exhaust air by removing NO2 in the exhaust.

Figure 1. Photograph of main parts of the system (a) and diagram of the micro gas analyzer (b). Inset shows an enlarged picture of the honeycomb-patterned microchannel. MCS, microchannel scrubber; FD, fluorescence detector with a blue LED and a miniature PMT module; mAP, miniature airpump; FM, flow meter with a needle valve. A 500 JPY coin was placed on the MCS to indicate the dimensions.

RESULTS AND DISCUSSION Fluorescence Detector for DAF-2T. Diamino reagents, such as 2,3-diaminonaphthalene (DAN)32,33 and 3-amino-1,5-naphthalenedisulfonic acid (C-acid),34,35 are often utilized for fluorometric determination of nitrogen compounds. The measurement is generally based on the reaction of the diamino group with nitrite in a strongly acidic medium and the fluorescence measurement in an alkaline medium. Thus, an automated system using these reagents is complicated due to the necessary change in pH, particularly in the determination of gaseous NO2 where the pH must be adjusted three times for each stage of gas absorption, reaction. and fluorescence measurement. In contrast, DAF-2T fluorescence can be obtained over a wide pH range (7 < pH < 13).31 Therefore, complicated pH adjustment was not required for our NO measurement with DAF-2. We developed a miniature fluorescence detector for DAF-2T, in which DAF-2T (32) Misko, T. P.; Schilling, R. J.; Salvemini, D.; Moore, W. M.; Currie, M. G. Anal. Biochem. 1993, 214, 11–16. (33) Nussler, A. K.; Glanemann, M.; Schirmeier, A.; Liu, L.; Nu ¨ ssler, N. C. Nat. Protoc. 2006, 1, 2223–2226. (34) Motomizu, S.; Mikasa, H.; Toei, K. Talanta 1986, 33, 729–732. (35) Toda, K.; Hato, Y.; Mori, K.; Ohira, S.; Namihira, T. Talanta 2007, 71, 1652–1660.

Figure 2. Spectra of DAF-2T fluorescence, the blue LED emission, and the cutoff filter transmittance. Tested solution for the fluorescence was 100 nM DAF-2T in 0.01 M NaOH.

can be excited by the blue LED (Figure 2). Emitted fluorescence was measured selectively from the reflection of excitation light with the help of the cutoff filter. A good signal-to-noise ratio was obtained with a 520 nm cutoff filter. Maximum wavelengths of excitation and emission spectra were in close proximity to each Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

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Figure 3. Response chart for NO. Actual NO concentrations are shown by the CL signal in the bottom panel. The absorbing/reaction solution used for µGAS was 10 µM DAF-2 in 0.01 M NaOH with a flow rate of 10 µL/min.

other. However, a low background signal from the scattering of excitation light was obtained using shorter range excitation and longer range emission lights. Collection of NO. Effective collection of an appropriate amount of NO into the solution is key to this work. The honeycomb-patterned microchannel scrubber was developed 4 years ago.27 This pPTFE/PDMS device has a preconcentration factor 20 000 times higher than conventional impingers. This has previously enabled highly sensitive measurement of H2S and SO2. These acidic gases can be collected effectively using alkaline solution. However, as NO is neither acidic nor basic, its absorption cannot be accelerated by an acid-base reaction. Furthermore, Henry’s law constant (KH) of NO is only 0.0019 M/atm.36 This is much smaller than those of other gases measured with wet chemistry, such as HCHO (KH 3000), NH3 (KH 56), HONO (KH 50), SO2 (KH 1.2), H2S (KH 0.098), and NO2 (KH 0.012). Thus, measurement of NO after collection of an aqueous phase is challenging. However, response to NO could be obtained using the microchannel scrubber and DAF-2 (Figure 3) without removal of pre-existing NO2 or conversion of NO to NO2. The NO concentrations were altered from 250 to 50 ppbv, and responses from the NO free baseline were recorded. To the best of our knowledge, this is the first demonstration of direct NO measurement with wet chemistry. However, the response intensity and response speed were not sufficient for breath measurements at that time. Because the absorbing time decreases with increases in liquid flow rate, the response intensity was inversely proportional to liquid flow rate. Slower flow rates increase the sensitivity but are detrimental to the response time. The flow rate should be reduced to 10 µL/ min in order to obtain reasonable response intensities at the sacrifice of the response time (Figure 3). Therefore, many attempts were made to improve the sensitivity. First, conditions for NO absorption were investigated. The response intensity altered when different mediums were used for pH adjustment, these included 0.1 M phosphate buffer (pH 7.4), 0.01 M NaHCO3 (pH 7.8), 0.01 M borax (pH 9.1), 0.01 M Na2CO3 (pH 10.5), 0.001, 0.01, and 0.1 M NaOH (pH 10.8, 11.8, 12.7) and 3% triethanolamine (TEA) (pH 10.8). Of the above (36) Lide, D. R., Frederikse, H. P. R., Eds. CRC Handbook of Chemistry and Physics, 76th ed.; CRC Press, Inc.: Boca Raton, FL, 1995.

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solutions, 0.01 M NaOH optimized the response intensity. Furthermore, the concentration of NaOH also affected the response intensity, with 0.001 and 0.1 M NaOH producing only 1/10 and 1/2 the intensity of 0.01 M NaOH, respectively. TEA solution, which is usually used for NO2 absorption, did not work for NO collection. Addition of organic solvents to the DAF-2 solution were also tested: 50% acetonitrile, 20% isopropyl alcohol (IPA), and 5% N,N-dimethylformamide. The alkalinity of all the solutions was maintained as 0.01 M NaOH. Only IPA enhanced the response intensity, producing a response 1.3 times that without the solvent. Since the solvent effect was not high, it was decided not to use an organic solvent to simplify the measurement. Addition of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) was also tested because NO is known to react with DAF-2 in the presence of dissolved oxygen. Therefore, PTIO might help in the reaction as an oxidizer. Actually, PTIO is often used for atmospheric NO analysis especially in passive samplers,37 as well as in biological NO scavenging.38 However, opposite to the expected enhancement, the signal for NO decreased to 39% on addition of PTIO. Probably, NO was depleted by PTIO with formation of nitrite ions.39 In consideration of the results of these investigations, the DAF-2 solution used included 0.01 M NaOH. Enhancement of Response to NO. The optimization of conditions discussed above was not sufficient for breath analysis. Therefore, we reconsidered the mechanism of the reaction between NO and DAF-2. NO reacts with DAF-2 in the presence of dissolved oxygen, details of this reaction in the aqueous phase are as follows (eqs 1-4).40,41 In aqueous solution, two molecules of NO react with dissolved O2 to form two molecules of NO2, the formed NO2 reacts with further NO to form N2O3. The N2O3 is a strong nitroso agent and reacts with DAF-2 to form the triazolfluorescein DAF-2T. NOg a NOaq

KH ) 0.0019 M atm-1

2 NOaq + O2 aq f 2 NO2 aq

(1)

k ) 2.8 ∼ 11.6 × 106 M-2s-1 (2)

NOaq + NO2 aq f N2O3 aq

k ) 1.1 × 109 M-1s-1 (3)

N2O3 aq + DAF-2 f DAF-2T(fluorescent)

(4)

The first reaction in the solution (eq 2) is the rate-determining step; it requires two molecules of NO and has a small rate constant. If the original solution contains NO2, dissolved NO could react directly with NO2 aq (eq 3) to allow the whole cascade of reactions to occur smoothly. According to this hypothesis, we (37) Hauser, C. D.; Battle, P.; Mace, N. Atmos. Environ. 2009, 43, 1823–1826. (38) Hooper, D. C.; Bagasra, O.; Marini, J. C.; Zborek, A.; Ohnishi, S. T.; Kean, R.; Champion, J. M.; Sarker, A. B.; Bobroski, L.; Farber, J. L.; Akaike, T.; Maeda, H.; Koprowski, H. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2528– 2533. (39) Goldstein, S.; Russo, A.; Samunil, A. J. Biol. Chem. 2003, 51, 50949–50955. (40) Ignarro, L. J.; Fukuto, J. M.; Griscavage, J. M.; Rogers, N. E.; Byrns, R. E. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 8103–8107. (41) Takahama, U.; Hirota, S.; Oniki, T. Chem. Res. Toxicol. 2006, 19, 1066– 1073.

performed a trial by adding NO2 to the absorbing solution to accelerate the reaction. In accordance with our previous attempts for in situ gas generation,42 chemical generation of NO2 was attempted by mixing nitrite or nitrate with ascorbic acid and phosphoric acid. In a batch test in a test tube, this generated a brown gas (NO2). We then tested flowing and mixing these reagents together, followed by introduction to the honeycomb-patterned microchannel gas desorber.28,42 The gases generated were monitored with the CL NOx analyzer, which revealed that NO was generated along with NO2. Alternative NO2 generation methods have been reported, including electrochemical generation43 and photodecomposition of nitrate with an ∼300 nm UV light.44,45 The photodecomposition system was simplified to be suitable for our purposes, but again both NO and NO2 were observed in the generated gas. Problems arose with side reactions46 in both photoinduced and chemical generation; consequently, we concluded that neither method was suitable for NO2 generation in the aqueous phase. Next, we attempted to introduce NO2 into the solution prior to the NO collection. This was achieved by passing the solution through small Teflon tubing (AWG 30) in the permeation tube chamber. NO2 gas permeated into the DAF-2 solution and successfully enhanced the NO response. Hence, “aqueous NO2” accelerates the reaction of NO with DAF-2. This experiment helped confirm the reaction mechanism. The next step was to add gaseous NO2 into the air sampling line, as this was easier than introducing it directly into the DAF-2 solution. Figure 4a shows the responses with and without NO2. The liquid flow rate was increased 3 times over that used previously, resulting in a very small response intensity without NO2 but an improved response speed. In contrast, the response intensity increased 10 times with the addition of ∼200 ppbv of NO2. However, the absorbing/reaction solution must be alkaline to capture NO2. The amplification rate increased with increasing NO2 concentrations but this effect diminished at higher concentrations (Figure 4b). Consequently, we decided to use a NO2 concentration of 200 ppbv. In this test, the various concentrations of NO2 were prepared from the standard gas cylinder. However, practically, it was easier to put the permeation tube device in the sampling air line. Ultimately, the permeation tube device was placed between the sample airbag and the microchannel gas collector. Response output under different NO concentrations was investigated, and a calibration curve from the data was compared with that obtained without NO2 addition (Figure 5). Without NO2 addition, it can be seen that under low concentrations no response to NO was obtained. In contrast, with NO2 addition, the response was proportional to the NO concentration and the calibration curve was linear and passed through the origin. NO2 addition improved the response dramatically, especially in the low concentration range. The LODs without and with the addition of NO2 were 25 ppbv and 0.82 ppbv, respectively, (42) Ohira, S.; Someya, K.; Toda, K. Anal. Chim. Acta 2007, 588, 147–152. (43) Yoshimori, T.; Kawahara, H.; Hara, T.; Ikeda, A. Anal. Chim. Acta 1978, 98, 171–175. (44) Dubowski, Y.; Colussi, A. J.; Hoffmann, M. R. J. Phys. Chem. A 2001, 105, 4928–4932. (45) Boxe, C. S.; Colussi, A. J.; Hoffmann, M. R.; Murphy, J, G,; Wooldridge, P. J.; Bertram, T. H.; Cohen, R. C. J. Phys. Chem. A 2005, 109, 8520–8525. (46) Chu, L.; Anastasio, C. J. Phys. Chem. A 2003, 107, 9594–9602.

Figure 4. Response to NO with and without the addition of NO2 (a) and effect of NO2 addition on the response to NO (b). In (b), the NO concentration was maintained at 100 ppbv and NO2 concentration was changed from 0 to 300 ppbv.

Figure 5. Calibration curves with and without NO2 addition. The DAF-2 solution flow rates were different, 30 µL/min and 10 µL/min in the cases with and without NO2 addition, respectively.

with the same high liquid flow rate of 30 µL/min. NO concentrations in the breath of healthy subjects and asthma patients are typically 5-20 and 30-80 ppbv, respectively, when sampled with a suitable blowing resistance (threshold level of 27.2 ppbv).47 Hence, the proposed system has sufficient sensitivity to determine the NO concentration in human breath. Interferences from Coexisting Species in Breath. There are many gaseous species in breath. Twenty-nine typical compounds with different functional groups were selected and tested in the solution phase; these included alcohols, aromatic hydro(47) Tsuburai, T.; Tsurikisawa, N.; Morita, S.; Hasunuma, H.; Kanegae, H.; Ishimaru, Y.; Fukutomi, Y.; Tanimoto, H.; Ono, E.; Oshikata, C.; Sekiya, K.; Otomo, M.; Maeda, Y.; Taniguchi, M.; Ikehara, K.; Akiyama, K. Allergol. Int. 2008, 57, 223–229.

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Table 1. Results of Interference Tests for Gaseous Species

gas nitric oxide acetone acetaldehyde ammonia isoprene carbon monoxide ethanol acetic acid dimethyl sulfidec hydrogen sulfide carbon dioxide

CO2 with soda lime column

NO (CH3)2CO CH3CHO NH3 C5H8 CO C2H5OH CH3COOH (CH3)2S H2S CO2

concentration (ppmv)

relative response (%)a

0.2 630 7.1 1.0 2300 200 2.1 1.0 1.0 1.0 0.0 2.0 3.0 4.0 5.0 5.0

100 NDb ND ND ND ND ND ND ND ND 100 96 61 34 20 98

Figure 6. Responses to 50 ppbv NO standard and three kinds of breath samples.

a Relative response compared with that for 0.2 ppmv NO. b ND: not detectable. c Relative response for 0.05 ppmv NO compared with data without CO2.

carbons, olefins, halogenated hydrocarbons, ketones, thiols, esters, aldehydes, ethers, amines, heterocyclic hydrocarbons, nitriles, and nitro compounds. For preliminary investigation, an excess of the target species (25 mM) was added into 1.7 µM DAF-2 and fluorescence increases were compared with that from the NO generator NOC 7 composed of 1-hydroxy-2-oxo-3-(Nmethyl-3-aminopropyl)-3-methyl-1-triazene.48,49 Among the tested compounds, thioacetate, acetaldehyde, n-hexanal, acetic acid, m-toluic acid, benzyl amine, di-n-butylamine, and furan were found to slightly increase fluorescence. However, after 30 min of reaction with DAF-2, the fluorescence intensities of these compounds were less than 2% of NO, in which the concentration estimated from the amount of added NOC 7 was 2.5 mM (1/10 of the test compounds). In the aqueous experiments, increases in fluorescence intensity from these compounds were negligible. Gas-based experiments were also conducted to investigate possible interference (Table 1). Most of the compounds did not show serious interference. Acetone,50 acetaldehyde, ammonia,51 isoprene,52 carbon monoxide, acetic acid, dimethyl sulfide,53 and hydrogen sulfide, which are typically found in breath, gave no interference even in high concentrations. After water vapor, CO2 is the second major gas contained in breath. CO2 itself did not show any interference. However, it did affect the NO signal when measured with NO2 addition. Response intensities for NO decreased with increases in CO2 concentration; the response was only 1/5 at 5% CO2, a level typically present in breath. This interference was caused by CO2 neutralizing the absorbing/ reaction solution to prevent dissolution of NO2. It could be (48) Adachi, Y.; Hashimoto, K.; Ono, N.; Yoshida, M.; Suzuki-Kusaba, M.; Hisa, H.; Satoh, S. Eur. J. Pharmacol. 1997, 324, 223–226. (49) Product information of NOC, http://www.dojindo.com/home/index.php?page) shop.product_details&flypage)flypage.tpl&product_id)340&category_id) 84&keyword)triazene&option)com_virtuemart&Itemid)58. (50) Teshima, N.; Li, J.; Toda, K.; Dasgupta, P. K. Anal. Chim. Acta 2005, 535, 189–199. (51) Toda, K.; Li, J.; Dasgupta, P. K. Anal. Chem. 2006, 78, 7284–7291. (52) Ohira, S.; Li, J.; Lonneman, W. A.; Dasgupta, P. K.; Toda, K. Anal. Chem. 2007, 79, 2641–2649. (53) Azad, M. A. K.; Ohira, S.; Toda, K. Anal. Chem. 2006, 78, 6252–6259.

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Figure 7. Correlation of NO values obtained by a µGAS and a conventional CL instrument.

eliminated by placing a soda lime column in the collection line before the bag or in the measurement sampling line. Therefore, breath air was sampled into a bag through the soda lime column in real breath analyses. NO2 coexisting in breath could interfere due to acceleration of NO collection. In the original conditions, namely without NO2 addition, the interference from NO2 was successfully eliminated by a soda lime column impregnated with triethanolamine. In addition, excess of NO2 (200 ppbv) was added in the final conditions and the effect of originally existing NO2 would be negligible. Actually, in our measurements, the NO2 levels in breath were less than 5 ppbv, even NO2 in the room was higher. Measurement of Breath NO. After optimizing the conditions, breath NO measurements were performed. Examples are shown in Figure 6. Standards (50 ppbv NO) or breath samples were collected in a Mylar bag through a soda lime column, and the bag was then attached to the sampling line. The sample air went to both the µGAS and the CL NOx analyzer concurrently, and both instrument signals were monitored. The performance of the µGAS was dramatically improved, and the response speed and sensitivity were comparable to the CL instrument. The µGAS was much smaller and simpler. Responses to breath samples were successfully obtained and NO concentrations were measured from the response intensity. We measured breath samples 30 times from 11 healthy people, and the data from the two different methods agreed well (Figure 7). The NO level ranged from 6.3 to 27.1 ppbv. The correlation coefficient

(R2) of 0.970 was obtained under the final conditions, while R2 was 0.434 for 10 samples without NO2 addition. The relationship between the data by µGAS (PPB µGAS) and by chemiluminescence (PPB CL) is indicated below with 95% confidence intervals for the slope and intercept. PPB µGAS ) 0.976((0.032)PPB CL - 0.2628((0.475), R2 ) 0.970

(5)

Thus, the proposed system has the potential to measure NO levels contained in breath. CONCLUSIONS In spite of the weak water solubility of NO gas, ppbv levels of NO could be measured successfully using a micro gas analysis system (µGAS) coupled with the NO fluorescence probe reagent DAF-2. To the best of our knowledge, this was the first demonstration of direct NO analysis with wet chemistry, without conversion to NO2. The performance was dramatically improved by addition of NO2 either into absorbing solution or sample air. Finally, the NO contained in breath could be measured,

and data produced by the proposed system agreed with those from a stationary conventional CL instrument. The proposed system is simple and small. The cost for the instrument is much lower (∼1/10) than the conventional NO analyzers. Consequently, it could be useful for home diagnosis of asthma and in small hospitals, if the liquid supply and data processing were simplified further. The proposed system has the strong potential for point of care measurements. Of course, the system can also be applied to atmospheric NO monitoring. ACKNOWLEDGMENT The authors wish to thank Japan Science and Technology Agency for support of Collaborative Development of Innovative Seeds (Potentiality Verification Stage). As well, they thank Mr. Nobuyoshi Ebata (Sekisui Medical Co.) for his help and arrangement in starting this project.

Received for review May 24, 2009. Accepted June 30, 2009. AC901131D

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