Biofluorometric Real-Time Image Sensing of Transcutaneously Emitted Ethanol Vapor By Enzyme-Immobilized Mesh Sheet

Abstract

Introduction Human derived volatile organic compounds (H-VOCs) had been attracted as relating to disease and metabolism since the era of ancient Greece. Because some H-VOCs, which reflect internal body conditions, are dissolved in the blood and unconsciously released in breath and/or transcutaneously, measurement of H-VOCs realize non-invasive disease screening and assessment of metabolism [1]. In this work, targeting transcutaneously emitted ethanol (TrEtOH) vapor as a typical H-VOC, we developed a biofluorometric real-time image sensing system with high sensitivity (skin-gas cam). Alcohol dehydrogenase (ADH), which enzymatically reduces nicotinamide adenine dinucleotide (NAD) using EtOH as another substrate, was utilized for real-time quantitative detection of TrEtOH. Furthermore, we examined the spatiotemporal and quantitative imaging of TrEtOH at various body sites when the subject took alcohol orally. Detectability and sensitivity in relation to each skin tissue were precisely analyzed. Materials and Methods ADH was immobilized on a cotton mesh, which was shaped to fit with a body shape for equalizing a distance between the body surface and the mesh. The ADH was covalently cross-linked by glutaraldehyde on a cotton mesh. The ADH-enzymatic reaction of oxidized NAD+ and EtOH was employed on the skin-gas cam (Fig.1a). The gas-imaging was achieved utilizing fluorescence of the reduced product—NADH (λex = 340 nm, λem = 490 nm). The optical system was constructed with a highly-sensitive CMOS camera, a UV-LED ring-light (λcenter = 340 ± 5 nm), and two bandpass filters (Fig. 1b).A dynamic range of the skin-gas cam was evaluated over 0.005–300 ppm EtOH vapor prepared by a standard gas generator at a constant flow rate of 100 mL/min for 20 sec. EtOH-specific selectivity was also tested with various kinds of VOCs contained in transcutaneous gas at 1 ppm each.The human subject experiment was approved by Tokyo Medical and Dental University (code: 2018-M160). Subjects took distilled liquor at the amount of 0.4 g of EtOH per 1 kg of body weight within 15 min. And then, TrEtOH was measured by the skin-gas cam for 45 min. Results and Discussions The spatiotemporal distribution of EtOH concentration was quantitatively detected by the fluorescent light intensity on the ADH-immobilized mesh. A time-domain image analysis computed a rate of ADH-mediated reaction based on the NADH fluorescence video [2]. The enzymatic reaction rate reached a maximal value at 30 sec after standard EtOH vapor application. A correlation between the maximal values of reaction rates and EtOH vapor concentrations was obtained over 0.02–300 ppm, which covered the concentration range of TrEtOH after alcohol intake in the human subject experiment. Moreover, the ADH-immobilized mesh sensor showed significant specificity to EtOH.The high sensitivity and specificity of the skin-gas cam to EtOH vapor enabled the detection of partially different concentrations of TrEtOH between the palm and wrist (Fig. 1c). Spatiotemporally higher concentrations of TrEtOH were frequently observed at the palms than the wrists, which seemed to be related to sweating,. It is known that the density of sweat gland, which reacts not only to thermal stimuli but also to mental stimuli, is higher in a palm than a wrist [3]. We are now also working on simultaneous measurements of sweating and TrEtOH to better understand this initial finding. Conclusions Biofluorometric real-time imaging of transcutaneously emitted EtOH vapor was achieved by the developed skin-gas cam system based on the ADH-immobilized cotton mesh sensor. The spatiotemporal change of EtOH vapor concentration was measured quantitatively. Under the time-domain image differential analysis, the response time was 30 sec. Furthermore, the skin-gas cam showed sufficient sensitivity (0.02–300 ppm) and high selectivity to TrEtOH for practical use. Finally, the time and site-specific emission profile of TrEtOH was obtained from subjects who took alcohol. Measurement other VOCs such as acetaldehyde, acetone, and 2-propanol is the next target to realize non-invasive disease screening and assessment of metabolism.