Fluorometric Bio-Sniffer (Gas Phase Biosensor) for Breath Acetone as a Volatile Product of Lipid Metabolism

Abstract

Breath analysis for healthcare and disease monitoring has become popular recently in global. There are hundreds of volatile organic compounds (VOCs) exist in human breath, and some of them can offer medical and biological information as the bloodstream. Particularly,acetone is one of the typical VOCs in exhaled breath, which is related to lipid metabolism in fasting, exercise and diabetes mellitus. The breath acetone concentrations have been previously shown to correlate strongly with blood acetone concentrations as well as with other ketones such as beta-hydroxybutyrate. Besides, there are many studies found the correlation between the breath acetone concentrations and blood glucose concentrations. Previous research indicated that the breath acetone concentration in healthy people shows the range from 200 to 900 ppb and higher concentration was found in breath of diabetic patients. Increase of the acetone concentration is usually a sign that cells cannot effectively use glucose as energy source, which often occurs in starvation, aerobic exercise, lack of insulin or resistance of insulin. Thus, acetone in exhaled breath has a potential to be a biomarker for non-invasive monitoring the progressive of diabetes. Typical quantitation of gaseous acetone usually uses gas chromatography with mass spectrometry (GC/MS) or proton transfer reaction mass spectrometry (PTR-MS). These methods are highly sensitivity but still have disadvantages in cost, measurement time and hard for continuity. Therefore, a rapid, convenient and highly selective method for acetone determination is strongly required. In this study, a highly sensitive acetone bio-sniffer (gas-phase biosensor) was developed and used to assess the acetone from exhale breath for evaluating the lipid metabolism. Nicotinamide adenine dinucleotide (NADH)-dependent secondary alcohol dehydrogenase (S-ADH) is a kind of enzyme that can reduce acetone to be isopropanol and meanwhile oxidase NADH to be NAD+ in acidic condition. It is known that NADH can be excited by UV (ultraviolet) light at central wavelength of 340 nm and release a fluorescent emission at peak of 490 nm simultaneously, while NAD+ do not have similar optical property. Thus, acetone concentration can be measured using the decreasing of NADH fluorescence intensity that is consumed by the enzymatic reaction of S-ADH. The fluorometric acetone bio-sniffer was composed of a NADH fluorescence measurement system, a flow-cell attaching on the fiber probe and an enzyme-immobilized membrane. The NADH measurement system consisted of a UV-LED as the excitation light source, band-pass filters ( λ = 340 ± 10 nm and λ = 490 ± 10 nm) for reducing noise, a photomultiplier tube (PMT) as fluorescence detector and bifurcated optical fibers for assemble. S-ADH was immobilized on the hydrophilic-PTFE membrane and equipped on the top of flow-cell by silicone O-ring. Phosphate buffer, which contained NADH, was circulated in the flow-cell to supply fresh co-enzyme and eliminate isopropanol and NAD+that produced by the enzyme membrane. When bio-sniffer contacted the gaseous acetone, enzymatic reaction that mentioned above occurred immediately and the change of fluorescence would detect by the PMT. This acetone bio-sniffer showed rapid reaction, highly sensitivity and selectivity. When the acetone vapors were supplied to the sensing region, fluorescent intensity decreased immediately and reached to a steady state. Then, the intensity would return to the initial value in short time when the acetone vapor supply was stopped. The relation between the concentration of acetone and the change of fluorescent intensity was investigated and the dynamic range was confirmed from 20 to 5300 ppb of acetone in the gas phase. This range encompasses the concentration of acetone vapors found in breath of healthy people and of those suffering from disorders of carbohydrate metabolism. The selectivity of the S-ADH was also characterized; several different VOCs were employed to compare the responses. The interference of 2-butanol and 2-pentanone were ignorable because they contained in healthy people in very low concentration. Finally, we applied the bio-sniffer to measure breath acetone for evaluation of lipid metabolism during long time fasting and an aerobic exercise test. The results showed that the mean breath acetone concentration of long time fasting healthy volunteers were higher than those who did not have fasting and display significant difference. Furthermore, the concentration of acetone in breath was observed significantly increase during aerobic exercise and it would recover to the initial value after long time rest. This highly sensitivity acetone bio-sniffer provides a new kind of analytical tool for non-invasive evaluation of human lipid metabolism and it is also expected to use for the clinical applications such as monitoring the progression of diabetes mellitus and diagnosis of other metabolism diseases.