Fluorometric Sniff-Cam (Gas-Imaging System) Utilizing Alcohol Dehydrogenase for Imaging Concentration Distribution of Acetaldehyde in Breath and Transdermal Vapor after Drinking

Understanding concentration distributions, release sites, and release dynamics of volatile organic compounds (VOCs) from the human is expected to lead to methods for noninvasive disease screening and assessment of metabolisms. In this study, we developed a visualization system (sniff-cam) that enabled one to identify a spatiotemporal change of gaseous acetaldehyde (AcH) in real-time. AcH sniff-cam was composed of a camera, a UV-LED array sheet, and an alcohol dehydrogenase (ADH)-immobilized mesh. A reverse reaction of ADH was employed for detection of gaseous AcH where a relationship between fluorescence intensity from nicotinamide adenine dinucleotide and the concentration of AcH was inversely proportional; thus, the concentration distribution of AcH was measured by detecting the fluorescence decrease. Moreover, the image differentiation method that calculated a fluorescence change rate was employed to visualize a real-time change in the concentration distribution of AcH. The dynamic range of the sniff-cam was 0.1–10 ppm which encompassed breath AcH concentrations after drinking. Finally, the sniff-cam achieved the visualization of the concentration distribution of AcH in breath and skin gas. A clear difference of breath AcH concentration was observed between aldehyde dehydrogenase type 2 active and inactive subjects, which was attributed to metabolic capacities of AcH. AcH in skin gas showed a similar time course of AcH concentration to the breath and a variety of release concentration distribution. Using different NADH-dependent dehydrogenases in the sniff-cam could lead to a versatile method for noninvasive disease screening by acquiring spatiotemporal information on various VOCs in breath or skin gas

Improved Sensitivity of Acetaldehyde Biosensor by Detecting ADH Reverse Reaction-Mediated NADH Fluoro-Quenching for Wine Evaluation

Acetaldehyde (AcH) is found in ambient air, foods, and the living body. This toxic substance is also contained in wine and known as an important ingredient affecting the quality of wine. Herein, we constructed and evaluated two different fiber-optic biosensors for measurement of AcH in the liquid phase (AcH biosensor) using aldehyde dehydrogenase (ALDH) or alcohol dehydrogenase (ADH). The AcH biosensor measured a concentration of AcH using fluorescence intensity of a reduced form of nicotinamide adenine dinucleotide (NADH) that was produced or consumed via catalytic reaction of the respective enzyme. In the AcH measurement system, an ultraviolet light emitting diode (UV-LED) and photomultiplier tube (PMT) were connected to a bifurcated optical fiber and were used to excite and detect NADH. A sensing region was developed using an optical fiber probe and an enzyme-immobilized membrane, buffer pH, and concentrations of a coenzyme in buffer solution for ALDH forward reaction and ADH reverse reaction were optimized, and the dynamic ranges were compared. ADH-mediated AcH biosensor showed higher sensitivity, wider dynamic range (1–500 μM), and capability of rapid measurement (less than 3 min) than ALDH-mediated AcH biosensor (5–200 μM). ADH biosensor also presented a high selectivity and allowed measurement of AcH in 9 different wine samples (5 red and 4 white wines). The determined concentrations were comparable to those measured by NADH absorbance method, which validated the accuracy of the ADH biosensor in AcH measurement

Fiber-Optic Bio-sniffer (Biochemical Gas Sensor) Using Reverse Reaction of Alcohol Dehydrogenase for Exhaled Acetaldehyde

Volatile organic compounds (VOCs) exhaled in breath have huge potential as indicators of diseases and metabolisms. Application of breath analysis for disease screening and metabolism assessment is expected since breath samples can be noninvasively collected and measured. In this research, a highly sensitive and selective biochemical gas sensor (bio-sniffer) for gaseous acetaldehyde (AcH) was developed. In the AcH bio-sniffer, a reverse reaction of alcohol dehydrogenase (ADH) was employed for reducing AcH to ethanol and simultaneously consuming a coenzyme, reduced form of nicotinamide adenine dinucleotide (NADH). The concentration of AcH can be quantified by fluorescence detection of NADH that was consumed by reverse reaction of ADH. The AcH bio-sniffer was composed of an ultraviolet light-emitting diode (UV-LED) as an excitation light source, a photomultiplier tube (PMT) as a fluorescence detector, and an optical fiber probe, and these three components were connected with a bifurcated optical fiber. A gas-sensing region of the fiber probe was developed with a flow-cell and an ADH-immobilized membrane. In the experiment, after optimization of the enzyme reaction conditions, the selectivity and dynamic range of the AcH bio-sniffer were investigated. The AcH bio-sniffer showed a short measurement time (within 2 min) and a broad dynamic range for determination of gaseous AcH, 0.02–10 ppm, which encompassed a typical AcH concentration in exhaled breath (1.2–6.0 ppm). Also, the AcH bio-sniffer exhibited a high selectivity to gaseous AcH based on the specificity of ADH. The sensor outputs were observed only from AcH-contained standard gaseous samples. Finally, the AcH bio-sniffer was applied to measure the concentration of AcH in exhaled breath from healthy subjects after ingestion of alcohol. As a result, a significant difference of AcH concentration between subjects with different aldehyde dehydrogenase type 2 (ALDH2) phenotypes was observed. The AcH bio-sniffer can be used for breath measurement, and further, an application of breath analysis-based disease screening or metabolism assessment can be expected due to the versatility of its detection principle, which allows it to measure other VOCs by using NADH-dependent dehydrogenases

Biochemical Gas Sensors (Biosniffers) Using Forward and Reverse Reactions of Secondary Alcohol Dehydrogenase for Breath Isopropanol and Acetone as Potential Volatile Biomarkers of Diabetes Mellitus

This study describes two biosniffers to determine breath acetone and isopropanol (IPA) levels and applies them for breath measurement in healthy subjects and diabetic patients. Secondary alcohol dehydrogenase (S-ADH) can reduce acetone and oxidize nicotinamide adenine dinucleotide (NADH to NAD⁺) in a weak acid environment. NADH can be excited by 340 nm excitation lights and subsequently emit 490 nm fluorescence. Therefore, acetone can be measured by the decrease in NADH fluorescence intensity. S-ADH can also oxidize IPA and reduce NAD⁺ to NADH when it is in an alkaline environment. Thus, IPA can be detected by the increase of fluorescence. The developed biosniffers show rapid response, high sensitivity and high selectivity. The breath acetone and IPA analysis in healthy subjects shows that the mean values were 750.0 ± 434.4 ppb and 15.4 ± 11.3 ppb. Both acetone and IPA did not show a statistical difference among different genders and ages. The breath acetone analysis for diabetic patients shows a mean value of 1207.7 ± 689.5 ppb, which was higher than that of healthy subjects (<i>p</i> < 1 × 10^–6). In particularly, type-1 diabetic (T1D) patients exhaled a much higher concentration of acetone than type-2 diabetic (T2D) patients (<i>p</i> < 0.01). The breath IPA also had a higher concentration in diabetic patients (23.1 ± 20.1 ppb, <i>p</i> < 0.01), but only T2D patients presented a statistical difference (23.9 ± 21.3 ppb, <i>p</i> < 0.01). These findings are worthwhile in the study of breath biomarkers for diabetes mellitus diagnosis. Additionally, the developed biosniffers provide a new technique for volatolomics research