Measurement of Nitrogen Oxide in Expired Breath Using Porous Glass Analytical Chips

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Introduction Fractional exhaled nitric oxide (FeNO) is present in high concentrations in the breath of asthma patients with type 2 airway inflammation. In addition, FeNO concentration varies with asthma symptoms and treatments. The cutoff point for FeNO in adults with asthma is 20 - 25 ppb, and > 50 ppb is considered severe inflammation [1]. FeNO measurement is used worldwide for clinical examination of asthma [1-2]. However, FeNO measurement is limited to specific medical institutions. Therefore, patients need to return to the institution for exhaled breath analysis of FeNO concentration.We have developed an analytical chip—based on porous glass—for a simplified NO analysis [3-4]. This chip can detect ~ 19 ppb of FeNO in adults; ppb-level sensitivity is achieved by collecting exhaled breath in a sampling bag. However, the detection took several hours. In this study, low NO concentration was detected in tens of minutes by combining a porous glass analytical chip with a small sampling pump. We believe this method will enable at-home FeNO measurement. Method Preparation of porous glass analytical chip All chemical reagents were purchased from Tokyo-Kasei Co. (Japan) and FUJIFILM Wako Pure Chemical Corporation (Japan). The substrate of the analytical chip was a porous glass (Vycor #7930, Corning Co.) with a size of 8 mm × 8 mm × 1 mm, a specific surface area of 200 m2g-1, and an average pore size of 4 nm. The NO/NO2 conversion and NO2 analytical chips were prepared using our previously reported method [4]. Three different NO/NO2 conversion chips were prepared by immersing the porous glass in a 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO) ethanol solution (3.3 × 10–3, 33 × 10–3, and 160 × 10–3 mol L- 1), followed by drying. The NO2 analytical chip was prepared by immersing the porous glass in an alcohol solution of 2.5 × 10–3 mol L- 1 N, N-dimethyl-1-naphthyl-amine, and 2.0 × 10–2 mol L- 1 sulfanilamide and then drying it. Exposure to the NO atmosphere The exposure experiment was carried out by placing a NO/NO2 conversion chip and a NO2 analytical chip adjacent to each other in the chip holder. Using a small sampling pump (MP-∑30NII, SIBATA SCIENTIFIC TECHNOLOGY LTD., Japan), the NO atmosphere flowed through the two chips for 20 min at 0.05 L min- 1 (Fig. 1). The NO atmosphere (30, 75, 145, and 200 ppb) was prepared by mixing 100 ppm NO/NO2 gas with nitrogen gas in a Tedlar® bag. The relative humidity of the NO atmosphere was adjusted to 40% by vaporizing deionized water (235 - 280 mL) injected into the Tedlar® bag. The absorbance of the chips was measured using an ultraviolet-visible spectrophotometer (U-4100, HITACHI, Japan). The NO and NO2 concentrations in the NO atmosphere before and after passing through the chips were measured via a NO-NO2-NOx analyzer (42C, Nippon Thermo Co. Ltd., Japan). Results and Conclusions The NO/NO2 conversion efficiencies of the three prepared conversion chips (3.3 × 10–3, 33 × 10–3, and 160 × 10–3 mol L- 1) were 11%, 31%, and 61%, respectively. Increasing the PTIO content in the conversion chip improved the NO/NO2 conversion efficiency. Therefore, a low NO concentration measurement in an active system was evaluated using a conversion chip with an efficiency of 61%. Fig. 2 shows the relationship between the change in absorbance at 525 nm of the NO2 analytical chip and the initial NO concentration. The absorbance of the NO2 analytical chip at 525 nm increased due to the azo dye produced by the chemical reaction between NO2 and the detection reagents [4], and there was a positive linear relationship between the change in absorbance of the NO2 chip and NO concentration at 525 nm. Therefore, the formula for NO concentration measured at a flow rate of 0.05 L min- 1 for 20 min is as follows:[NO]0 = 4760 × ΔAbs525 where is the NO concentration (ppb) in the atmosphere, and is the change in absorbance at 525 nm of the NO2 analytical chip before and after exposure. This result suggests that FeNO can be measured in 20 min by using the analytical chip with an active method. References A. Hanania, M. Massanari, N. Jain, Measurement of fractional exhaled nitric oxide in real-word clinical practice alters asthma treatment decisions, Ann. Allergy Asthma Immunol.120 (2018) 414-418. doi: 10.1016/j.anai.2018.01.031.W. Pijnenburg, The Role of FeNO in Predicting Asthma, Front. Pediatr. 7 (2019) 1-5. doi: 10.3389/fped.2019.00041.Asanuma, S. Hino, Y. Y. Maruo, Development of an analytical chip for nitrogen monoxide detection using porous glass impregnated with 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl, Microchem. J. 151 (2019) 104251. doi: 10.1016/j.microc.2019.104251.Asanuma, K. Numata, Y. Y. Maruo, A colorimetric method for the measurement of ppb-level NO in exhaled air using porous glass analytical chip, Sensors and Actuators Reports 2 (2020) 100019. doi: 10.1016/j.snr.2020.100019. Figure 1