Abstract
Methanol (MeOH) in exhaled breath has potential for non-invasive assessment of intestinal flora. In this study, we have developed a biochemical gas sensor (bio-sniffer) for MeOH in the gas phase using fluorometry and a cascade reaction with two enzymes, alcohol oxidase (AOD) and formaldehyde dehydrogenase (FALDH). In the cascade reaction, oxidation of MeOH was initially catalyzed by AOD to produce formaldehyde, and then this formaldehyde was successively oxidized via FALDH catalysis together with reduction of oxidized form of β-nicotinamide adenine dinucleotide (NAD+). As a result of the cascade reaction, reduced form of NAD (NADH) was produced, and MeOH vapor was measured by detecting autofluorescence of NADH. In the development of the MeOH bio-sniffer, three conditions were optimized: selecting a suitable FALDH for better discrimination of MeOH from ethanol in the cascade reaction; buffer pH that maximizes the cascade reaction; and materials and methods to prevent leaking of NAD+ solution from an AOD-FALDH membrane. The dynamic range of the constructed MeOH bio-sniffer was 0.32–20 ppm, which encompassed the MeOH concentration in exhaled breath of healthy people. The measurement of exhaled breath of a healthy subject showed a similar sensorgram to the standard MeOH vapor. These results suggest that the MeOH bio-sniffer exploiting the cascade reaction will become a powerful tool for the non-invasive intestinal flora testing.
Highlights
Intestinal flora has been gaining its attention year by year after several reports were made regarding influences of the intestinal flora to general health conditions [1,2]
It is expected that the alcohol oxidase (AOD)-formaldehyde dehydrogenase (FALDH) fluorescence is applicable to the non-invasive measurement of MeOH in such body fluids as urine or saliva, but it would be quicker and easier if the same assessment could be made with exhaled breath
Waterproofing was made on the gas phase side of the membrane, which was opposite to the side of AOD and FALDH being immobilized, because the AOD-FALDH membrane was hydrophilic, and the NAD+ solution in the flow-cell had leaked into the gas phase (Figure 2)
Summary
Intestinal flora has been gaining its attention year by year after several reports were made regarding influences of the intestinal flora to general health conditions [1,2]. According to a study by Lindinger et al, eating pectin-contained fruits, such as apples, lead to the increase in breath MeOH concentration [20] These reports suggest the potential for non-invasive assessment of intestinal bacteria activity by measuring MeOH in exhaled breath. Broek et al introduced a MeOH sensor that was composed of a separation column and Pd-doped SnO2 chemoresistive sensor This combination made the size compact and made it possible to measure MeOH vapor even in the presence of much higher concentration of ethanol within 2 min [33]. Due to the cascade reaction, the AOD-FALDH fluorosensor was able to measure MeOH with high selectivity, especially from other aliphatic alcohols, including ethanol Based on these results, it is expected that the AOD-FALDH fluorescence is applicable to the non-invasive measurement of MeOH in such body fluids as urine or saliva, but it would be quicker and easier if the same assessment could be made with exhaled breath. Our biochemical gas sensor (bio-sniffer) has shown those required characteristics in previous studies for ethanol, acetaldehyde, isopropanol, and acetone [38,39,40,41], and in this study, we combined the advantages of the bio-sniffer and AOD-FALDH cascade reaction to measure MeOH in exhaled breath
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