Sniff-Cam (Bio-Fluorometric Gas-Imaging System) for Breath Acetaldehyde after Drinking

Abstract

Some volatile organic compounds (VOCs) in breath and skin gas are related to diseases and metabolisms, and non-invasively measuring a spatiotemporal distribution of human volatile concentration enables simple metabolism evaluation and identification of a volatile release site. Moreover it can be possible to obtain new knowledge in dermatology and metabolomics by in detail measuring the release site, release dynamics and concentration distribution of the volatile. Conventional analytical methods such as gas chromatography/mass spectrometry (GC-MS) have been applied for breath or skin gas measurement, it is challenging to accurately obtain the spatiotemporal information since it requires gas sampling and quick response for continuity. Recently we have developed a chemiluminescent imaging system of ethanol in the gas phase by exploiting an enzymatic reaction. Due to the use of an enzyme, the imaging system showed a high selectivity to a compound as well as a high sensitivity. In experiments we have demonstrated visualization of concentration distribution of gaseous ethanol in breath and skin gas after ingestion of alcohol. In this research, we aimed at quantitative imaging of a skin gas from human, and developed a bio-fluorometric gas-imaging system (sniff-cam) using an enzymatic reaction for gaseous acetaldehyde (AA), which is metabolite of ethanol. Here we report characteristics of the sniff-cam and result of quantitative imaging of gaseous AA. A reduced nicotinamide adenine dinucleotide (NADH) is consumed by an enzymatic reduction of AA with alcohol dehydrogenase (ADH), and its concentration is inversely proportional to that of gaseous AA (shown in additional figure (upper)). The concentration distribution of gaseous AA is quantitatively visualized with fluorescence emitted from NADH (λfl = 490 nm) by ultraviolet (UV) excitation (λex = 340 nm). The sniff-cam was developed as shown in additional figure (middle). In the system, an ADH-immobilized mesh was placed between an UV-light emitting diode (LED) array sheet (9×9 UV-LEDs, λ=340 nm, size 9×9 cm2, Dowa Electronics materials) that was designed to uniformly illuminate the ADH-immobilized mesh and a high-sensitive CCD camera in a dark box. Two band-pass filters for excitation light (BPFex, λ = 340 ± 42.5 nm, size 9×9 cm2, Edmund Optics) and fluorescence of NADH (BPFfl, λ = 490 ± 10 nm, φ25 mm, Asahi Spectra) were used to reduce the noise in the image. The ADH-immobilized mesh was prepared by crosslinking with glutaraldehyde (GA). A mixture solution of ADH (EC 1.1.1.1, 240 units, 369 units/mg solid, A7011, Sigma-Aldrich), bovine serum albumin (1.5 mg, 306-13383, Wako Pure Chemical Industries) and phosphate buffer (0.1 mol/L, 200 μL, pH 6.5) was dropped onto a cotton mesh substrate (100% cotton, thickness 1 mm, interval size 1 mm, size 2×2 cm2, Ohki Healthcare Holdings) and spread homogeneously, followed by storing the mesh substrate at 4 ºC for 1 hour. Then GA solution (2.5 vol%, 32 μL, 079-00533, Wako Pure Chemical Industries) was dropped onto the mesh substrate and crosslinking has taken place for 1 hour (additional figure (lower)). In characterization experiments, the ADH-immobilized mesh was soaked with NADH solution (500 μmol/L, 300 μL, in pH 6.5 Tris-HCl buffer) and then immediately placed in the dark box for measurement. The dynamic range of the sniff-cam was evaluated by using various concentrations (0.025–10 ppm) of gaseous AA blown on the ADH-immobilized mesh. As the results by blowing of gaseous AA, decrease of the fluorescence intensity at around the position of the gas outlet was observed. It validates that the sniff-cam allows to image gaseous AA concentration via fluorescence intensity. The dynamic range of the sniff-cam for gaseous AA was 0.1–10 ppm (r = 0.994) that encompasses typical AA concentration in human breath after drinking (1.2–6 ± 3.0 ppm). The selectivity of the sniff-cam was also evaluated by measuring typical chemical substances (acetaldehyde (5 ppm), ethanol (100 ppm), methanol (0.1 ppm), 2-propanol (0.1 ppm), acetone (0.6 ppm), methyl mercaptan (0.007 ppm)) in breath after intake of alcohol beverage. The result showed that no output signal was observed except for gaseous AA. In addition, the mixture of AA (5 ppm) and ethanol (100 ppm) was indicated same fluorescence intensity against single component of AA (5 ppm). These characteristics of the sniff-cam including a suitable dynamic range and high selectivity allow to accurately measure spatiotemporal distribution of gaseous AA contained in gas mixture such as the breath after intake of alcohol beverage. Figure 1