Tandem Imaging of Breath Ethanol and Acetaldehyde Based on Multiwavelength Enzymatic Biofluorometry

Highly sensitive and selective imaging of human-borne volatile organic compounds (VOCs) enables an intuitive understanding of their concentrations and release sites. While multi-VOC imaging methods have the potential to facilitate step-by-step metabolic tracking and improve disease screening accuracy, no such system currently exists. In this study, we achieved simultaneous imaging of ethanol (EtOH) and acetaldehyde (AcH), the starting molecule and an intermediate metabolite of alcohol metabolism, using a multiwavelength VOC imaging system. The system employed alcohol dehydrogenase-catalyzed substrate oxidation (ADH_OX) and reduction (ADH_RD) reactions. The oxidation of EtOH by ADH_OX in the presence of NAD⁺ produced NADH, which was subsequently oxidized by diaphorase (DP) with resazurin, leading to the resorufin formation, characterized by red fluorescence (excitation at 560 nm and fluorescence at 590 nm). Reduction of AcH by ADH_RD consumed NADH, leading to a decrease in blue fluorescence (ex. 340 nm, fl. 490 nm). Meshes incorporating ADH_OX-DP or ADH_RD were arranged in tandem in front of a camera. Fluorescence images were captured, while a mixture of gaseous EtOH and AcH was applied by switching between two bandpass filters at 1 Hz. Each mesh exhibited selective responses to the target VOCs, with no significant impact on the dynamic range observed in either the single or tandem configurations (EtOH 1–300 ppm, AcH 0.2–5 ppm). The 90% response time was close after time–domain image differential analysis (EtOH = 26 s and AcH = 15 s). Furthermore, the system enabled simultaneous and quantitative imaging of EtOH and AcH concentrations in the breath after alcohol consumption. It also distinguished differences in alcohol metabolism based on the alcohol dehydrogenase 2 (ALDH2) activity, as indicated by the EtOH/AcH ratio (ALDH2 active vs nonactive: 120.9/0.71 ppm vs 129.2/1.99 ppm).