Method and apparatus for measuring diacetyl

Abstract

To measure diacetyl by simple biosensing.SOLUTION: Provided is a method for measuring diacetyl, including: in a state of infiltrating an enzyme membrane having diol dehydrogenase immobilized on a support membrane with an acidic solution containing reduced nicotinamide dinucleotide (NADH) as a coenzyme, and bringing the membrane into contact with a specimen fluid assumed to contain diacetyl as a substrate, irradiating the membrane with excitation light that excites NADH; detecting fluorescence emitted from NADH that is reduced by the enzyme reaction; and analyzing the concentration of diacetyl in the specimen fluid on the basis of the degree of decrease in the intensity of the detected fluorescence.SELECTED DRAWING: Figure 11

Description

[Technical Field]

[0001] The present disclosure relates to a method and device for measuring diacetyl produced in a living body. [Background technology]

[0002] Living organisms emit various volatile organic compounds (VOCs) as biogases during their life activities. Biogases contain hundreds of different components, and it is necessary to measure the target component without being affected by other components. For example, it has been reported that from around the age of 40, Staphylococcus aureus and Staphylococcus epidermidis on the body surface use lactic acid in sweat as a precursor, pyruvic acid, and acetoin as an intermediate to produce "diacetyl" (the so-called middle-aged odor), an oily odor component that differs from the odor of aging. Diacetyl is the main odor component in fermented foods, and is also known as an off-flavor component that impairs the aroma of alcoholic beverages. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 5083768 Summary of the Invention [Problem to be solved by the invention]

[0004] Conventional methods for measuring these VOCs include using equipment such as gas chromatography mass spectrometry, but these instruments are expensive and large, making it difficult for individuals suffering from middle-aged odor to quantitatively determine the concentration of diacetyl emitted by themselves.

[0005] The inventors have previously developed a system for easily measuring VOCs using an NAD-dependent enzyme (Patent Document 1 above), but even with this, it was difficult to measure diacetyl using simple biosensing. [Means for solving the problem]

[0006] A diacetyl measurement method according to an embodiment of the present disclosure involves infiltrating an enzyme membrane having diol dehydrogenase (DDH) immobilized on a support membrane with an acidic solution containing reduced nicotinamide dinucleotide (NADH) as a coenzyme, allowing a sample fluid assumed to contain diacetyl as a substrate to flow through the membrane, irradiating the membrane with excitation light to excite NADH, detecting the fluorescence emitted from NADH that is reduced by the enzyme reaction, and analyzing the concentration of diacetyl in the sample fluid based on the degree of decrease in the intensity of the detected fluorescence.

[0007] In the above-mentioned diacetyl measurement method, it is desirable that the surface of the support membrane is first modified with polyethyleneimine, and then modified with glutaraldehyde, and then the DDH is immobilized in this state to form the enzyme membrane.

[0008] A diacetyl measuring device according to an embodiment of the present disclosure includes a coenzyme supplying device that infiltrates an enzyme membrane having DDH immobilized on a support membrane with an acidic solution containing reduced nicotinamide dinucleotide (NADH) as a coenzyme; a specimen supplying device that circulates a specimen fluid that is assumed to contain diacetyl as a substrate through the infiltrated enzyme membrane; an irradiation device that irradiates excitation light to excite NADH; a detection device that detects fluorescence emitted from NADH that is reduced by the enzyme reaction; and an analysis device that analyzes the concentration of diacetyl in the specimen fluid based on the degree of decrease in the intensity of the detected fluorescence.

[0009] In the diacetyl measuring device, it is desirable that the surface of the support membrane is surface-modified with polyethyleneimine, and then the enzyme membrane is formed by immobilizing the DDH in a state where the surface is surface-modified with glutaraldehyde. [Effects of the Invention]

[0010] According to the diacetyl measurement method and diacetyl measurement device of the aspects of the present disclosure, diacetyl can be measured by simple biosensing. [Brief explanation of the drawings]

[0011] [Figure 1] This is a schematic diagram of a measuring device for attempting diacetyl measurement using DDH. [Figure 2] FIG. 2 is a schematic diagram showing the structure of the tip of a detection probe used in the measuring instrument of FIG. 1. [Figure 3A] FIG. 2 is a schematic cross-sectional view of a support membrane. [Figure 3B] FIG. 3B is a schematic cross-sectional view of the support membrane of FIG. 3A in a state where the surface of the support membrane has been treated with polyethyleneimine. [Figure 3C] FIG. 3C is a schematic cross-sectional view of the support membrane of FIG. 3B in a state where the surface of the support membrane has been treated with glutaraldehyde. [Figure 3D] FIG. 1 is a schematic cross-sectional view of an enzyme membrane. [Figure 4A] 1 is a graph showing diacetyl responsiveness when the molecular weight of polyethyleneimine used in the enzyme membrane is 2,000. [Figure 4B] 1 is a graph showing diacetyl responsiveness when the molecular weight of polyethyleneimine used in the enzyme membrane is 10,000. [Figure 4C] 1 is a graph showing diacetyl responsiveness when the molecular weight of polyethyleneimine used in the enzyme membrane is 25,000. [Figure 4D] 1 is a graph showing diacetyl responsiveness when the molecular weight of polyethyleneimine used in the enzyme membrane is 70,000. [Figure 4E] 1 is a graph showing diacetyl responsiveness when the molecular weight of polyethyleneimine used in the enzyme membrane is 750,000. [Figure 5] 1 is a graph showing the relationship between the molecular weight of polyethyleneimine used in the enzyme membrane and the stabilization time required when diacetyl is loaded. [Figure 6] 1 is a graph showing the relationship between the concentration of polyethyleneimine and glutaraldehyde used in the enzyme membrane and diacetyl responsiveness. [Figure 7A] 1 is a graph measuring diacetyl loading in an acidic environment. [Figure 7B] 1 is a graph showing the measurement of acetoin loading in an acidic environment. [Figure 7C] 1 is a graph showing the measurement of 1,2-butanediol loading in an alkaline environment. [Figure 7D] 1 is a graph showing the measurement of acetoin loading in an alkaline environment. [Figure 8] 1 is a graph measuring diacetyl using various buffers. [Figure 9] 1 is a graph showing the response to a diacetyl solution. [Figure 10] 1 is a graph showing quantitative characteristics for a diacetyl solution. [Figure 11] FIG. 1 is a schematic diagram showing the structure of a diacetyl measuring device according to the present disclosure. [Figure 12] FIG. 2 is a schematic diagram showing the structure of the tip of a detection probe used in the diacetyl measurement device of FIG. 1. [Figure 13A] 1 is a graph showing the responsiveness to diacetyl gas using a diacetyl measuring device. [Figure 13B] 13B is a graph showing an enlarged view of a low concentration portion of diacetyl gas in FIG. 13A. [Figure 14] 1 is a graph showing quantitative characteristics for diacetyl gas using a diacetyl measuring device. [Figure 15A] FIG. 1 is a schematic diagram showing how a head biogas sample is collected from a subject. [Figure 15B] FIG. 15B is a schematic diagram showing the flow path of the head biological gas sample collected in FIG. 15A. [Figure 16] FIG. 1 is a schematic diagram showing the flow path of a head biogas sample collected directly from a subject. [Figure 17] 1 is a graph showing an example of measuring diacetyl in head biogas. [Figure 18] FIG. 1 is a schematic diagram of a diacetyl gas imaging system. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. Note that the drawings referred to below show the configuration of each part in a schematic manner and do not reflect the actual size. Furthermore, symbols commonly used in each drawing refer to the same structure or member without any special explanation for each drawing.

[0013] (1) Principle of diacetyl fluorescence measurement using diol dehydrogenase Diacetyl, represented by the structural formula of Chemical Formula 1 below, undergoes an enzymatic reaction in an acidic environment by diol dehydrogenase (DDH) that moves from the left side to the right side of the reaction formula shown in Chemical Formula 2 below, producing acetoin, represented by the structural formula of Chemical Formula 3 below. At this time, the coenzyme, reduced nicotinamide dinucleotide (NADH), donates a proton to diacetyl, causing diacetyl to undergo reduction. Then, by releasing a proton, NADH is converted to oxidized nicotinamide dinucleotide (NADH). + ) is generated.

[0014] [ka]

[0015] [ka]

[0016] [ka]

[0017] When NADH is excited by ultraviolet light with a wavelength of 340 nm, it emits fluorescence with a wavelength of 490 nm. As NADH decreases through the reaction from the left side to the right side of the above chemical formula 2, the amount of fluorescence from NADH decreases. The amount of diacetyl used as a substrate can be quantified based on this decrease in fluorescence.

[0018] On the other hand, in an alkaline environment, acetoin represented by the structural formula of Chemical Formula 3 undergoes an enzymatic reaction by DDH, which moves from the right side to the left side of the reaction formula shown in Chemical Formula 2, producing diacetyl represented by the structural formula of Chemical Formula 1. At this time, acetoin reacts with the coenzyme NAD + By donating a proton to NAD, it is oxidized. + NADH is produced from the reaction from the right side to the left side of the above chemical formula 2. As NADH increases, the amount of fluorescence from NADH increases. This increase in fluorescence can be used to quantify the amount of diacetyl as a substrate.

[0019] Acetoin, represented by the structural formula of Chemical Formula 3 above, undergoes an enzymatic reaction in an acidic environment by DDH, which shifts from the left side to the right side of the reaction formula shown in Chemical Formula 4 below, producing 1,2-butanediol, represented by the structural formula of Chemical Formula 5 below. At this time, the coenzyme NADH donates a proton to acetoin, causing the acetoin to undergo reduction. Then, by releasing a proton, NADH is converted to NAD + The reaction from the left side to the right side of the following chemical formula 4 reduces NADH, resulting in a decrease in the amount of fluorescence from NADH. This decrease in fluorescence can be used to quantify the amount of acetoin used as a substrate.

[0020] [ka]

[0021] [ka]

[0022] On the other hand, 1,2-butanediol represented by the structural formula of Chemical Formula 5 undergoes an enzymatic reaction in an alkaline environment by DDH, which shifts from the right side to the left side of the reaction formula shown in Chemical Formula 4, producing acetoin represented by the structural formula of Chemical Formula 3. At this time, 1,2-butanediol is converted into acetoin by the coenzyme NAD +By donating a proton to NAD, it is oxidized. + NADH is produced from the reaction from the right side to the left side of the above chemical formula 4. As NADH increases, the amount of fluorescence from NADH increases. This increase in fluorescence can be used to quantify the amount of 1,2-butanediol used as a substrate.

[0023] (2) Trial of diacetyl measurement using DDH Fig. 1 is a schematic diagram of a measuring instrument 5 for attempting to measure diacetyl using DDH. Fig. 2 is a schematic diagram showing the structure of the tip of a detection probe 50 used in the measuring instrument of Fig. 1.

[0024] The measuring instrument 5 includes an irradiation device 30 including an irradiation fiber 31 for irradiating excitation light, a detection device 40 including a detection fiber 41 for detecting fluorescence, and a detection probe 50 including a measurement fiber 51 that combines the irradiation fiber 31 and the detection fiber 41. A commercially available detection probe may be used as the detection probe 50, and for example, a combination of a 2-in-1 optical fiber assembly (BIF600-UV / VIS) commercially available from Ocean Optics Inc. (USA) and F100-9009 (manufactured by Ocean Optics Inc.) may be used.

[0025] In this disclosure, diacetyl in biogas is the target substance. As shown in the above-mentioned chemical formula 2, the measurement of diacetyl as the target substance is carried out by the reaction in which the substrate diacetyl is converted to acetoin by DDH, and the coenzyme NADH is consumed to convert the coenzyme NAD + In other words, the coenzyme NADH absorbs ultraviolet light with a wavelength of 340 nm as excitation light, becomes excited by this, and emits fluorescence with a wavelength of 491 nm. The diacetyl concentration before the reaction is measured by the degree to which the fluorescence decreases after the enzyme reaction.

[0026] The irradiation device 30 includes the above-described irradiation fiber 31, an ultraviolet light-emitting diode 32 connected to the irradiation fiber 31, and a low-bandpass filter 33 connected midway along the irradiation fiber 31. As described above, this embodiment utilizes the property of NADH that it absorbs ultraviolet light at 340 nm, and therefore, the ultraviolet light-emitting diode 32 used is one that excites ultraviolet light with a wavelength of 300 to 370 nm, preferably around 340 nm. Therefore, the low-bandpass filter 33 is a bandpass filter that passes ultraviolet light with a wavelength of 330 to 350 nm as irradiation light from the light source. Such a bandpass filter can be any commercially available one without particular limitation.

[0027] The detection device 40 includes the detection fiber 41, a photomultiplier tube 42 connected to the detection fiber 41, and a wideband bandpass filter 43 connected midway along the detection fiber 41. The detection fiber 41 receives fluorescence generated by excitation with excitation light irradiated from the ultraviolet light-emitting diode 32. The fluorescence generated by excitation has a specific wavelength different from the predetermined wavelength depending on the excitation phenomenon. In the case of NADH, this wavelength is 450 to 510 nm, more specifically, around 491 nm. Therefore, as shown in FIG. 1 , a bandpass filter that transmits only fluorescence with wavelengths of 450 to 510 nm, i.e., fluorescence of 400 nm or longer, is disposed as the wideband bandpass filter 43. As such a bandpass filter, any commercially available filter can be used without any particular limitation.

[0028] In the measuring instrument 5 of Figure 1, a computer system serving as an analysis device 60 is connected to the detection device 40 to analyze the fluorescence data detected by the detection device 40 and to analyze the concentration of diacetyl, which is the target substance of this embodiment.

[0029] As shown in Fig. 2, detection probe 50 has attached to its tip a cylindrical container 52 equipped with an enzyme membrane 70 having DDH immobilized thereon as an enzyme. Enzyme membrane 70, which will be described later, is fixed to the tip of container 52 by a short cylindrical membrane holder 54. Container 52 is immersed in buffer 15 contained in a beaker serving as coenzyme supplying device 10. Buffer 15, serving as a specimen fluid, contains a predetermined concentration of NADH as a coenzyme, and while being stirred by a stirrer 18 via a stirrer bar 18a placed in the beaker, specimen solution 17 containing diacetyl as a substrate is added by a pipette 16 to a predetermined concentration.

[0030] The enzyme membrane 70 refers to a membrane in which an enzyme is immobilized on a support membrane 71 (see FIG. 3A ), which is a membrane material. In the present disclosure, the enzyme is DDH. The support membrane 71 used can be made of any material conventionally used for immobilizing enzymes, without any particular limitations. Examples of such materials include resins such as polytetrafluoroethylene (PTFE), polydimethylsiloxane, polypropylene, polyethylene, polymethyl methacrylate, and polystyrene, and fibers such as cotton. The thickness of such a support is not particularly limited, but is preferably 100 nm to 200 μm, and more preferably 10 μm to 100 μm. The enzyme membrane 70 of the present disclosure was prepared as follows. Phosphate buffer solution (Kanto Chemical, 100 mM, pH 6.0, hereinafter referred to as “PB”) was used as a solvent for the following various reagents and as a cleaning solution.

[0031] 3A is a schematic cross-sectional view showing the support membrane 71. As the support membrane 71, a hydrophilic PTFE membrane (Omnipore, Merck Millipore) having a size of 2×2 cm and a thickness of 0.2 μm was used, which was washed with pure water.

[0032] Next, 1 mL of five types of 1 wt% polyethyleneimine PB solutions was dropped onto the washed support membrane 71, and after leaving it to stand for 5 minutes, it was washed three times with PB. As a result, as shown in Figure 3B, the surface of the support membrane 71 was modified with polyethyleneimine (hereinafter referred to as "PEI"), and amino groups were exposed on the surface. The five types of PEI used were as shown in Table 1 below.

[0033] [Table 1]

[0034] 300 μL of a 0.5 or 1% by volume glutaraldehyde (hereinafter referred to as "GA") solution was dropped onto the modified support membrane 71, and the membrane was left to stand for 5 minutes. After that, the membrane was washed three times with PB and then dried. As a result, the surface of the support membrane 71 was further modified with GA, and formyl groups were exposed on the surface, as shown in FIG. 3C.

[0035] Then, 32 μL of a PB solution of 0.1 mg / μL DDH (Chiralscreen OH E031, Daicel) was applied to the dried support membrane 71 and allowed to stand overnight at 4° C. As a result, DDH was immobilized on the surface of the support membrane 71 via the formyl group, forming an enzyme membrane 70, as shown in FIG.

[0036] This enzyme membrane 70 was cut into 0.5 cm squares and fixed to the tip of an optical fiber to prepare a biosensor for measuring diacetyl. Meanwhile, 300 μL of 1 mM NADH solution was placed in a cuvette, and while stirring with a stirrer, a diacetyl dilution solution was added every 200 seconds so that the diacetyl concentration in the cuvette changed from 10 μM to 1 mM. The initial fluorescence intensity was approximately 1.5 × 10 6 To obtain a fluorescence intensity of 100 cps, a DC voltage power supply (GS200, Yokogawa Electronics) was used to apply 30 V and 30–50 mA to the UV-LED, and the autofluorescence emitted by excited NADH was detected by a detector (H11890-210, Hamamatsu Photonics) via a biosensor inserted into the cuvette.

[0037] The above detection results are shown in the graphs in Figures 4A to 4E. Figure 4A shows the results using enzyme membrane A modified with PEI having a molecular weight of 2,000. Figure 4B shows the results using enzyme membrane B modified with PEI having a molecular weight of 10,000. Figure 4C shows the results using enzyme membrane C modified with PEI having a molecular weight of 25,000. Figure 4D shows the results using enzyme membrane D modified with PEI having a molecular weight of 70,000. Figure 4E shows the results using enzyme membrane E modified with PEI having a molecular weight of 750,000. As shown in Figures 4A to 4E, as the molecular weight of PEI increased, the noise decreased, and a concentration-dependent decay of fluorescence intensity—in other words, the decay of fluorescence intensity associated with the consumption of NADH in the enzymatic reaction of diacetyl—tended to become clearly apparent. Furthermore, when the diacetyl concentration in the cuvette was set to 100 μM using the enzyme membranes A to E, the time required for the measured value to stabilize was measured. As shown in the graph in Figure 5, the time required for the measured value to stabilize decreased as the molecular weight of PEI increased. From the above, it was found that the molecular weight of PEI used to prepare the enzyme membrane 70 was optimal at 750,000 among the above-mentioned PEIs used.

[0038] Furthermore, the diacetyl responsiveness of the above-mentioned enzyme membrane E, when prepared with various combinations of PEI concentrations (mass%) and GA concentrations (volume%), was measured as described above. The results are shown in Figure 6. The combination of 1 mass% PEI and 0.5 volume% GA showed the highest responsiveness. The responsiveness decreased next in order: 5 mass% PEI and 0.25 volume% GA, 10 mass% PEI and 0.05 volume% GA, and 0.5 mass% PEI and 2.5 volume% GA. The responsiveness was lowest for the combination of 10 mass% PEI and 5 volume% GA. From the above, it was found that the PEI concentration range suitable for diacetyl responsiveness is 0.1 to 12.0 mass%, preferably 0.3 to 8.0 mass%, and more preferably 0.5 to 3.0 mass%. It was also found that the range of GA concentration suitable for diacetyl responsiveness is 0.03 to 8.0% by volume, preferably 0.05 to 3.0% by volume, and more preferably 0.2 to 0.8% by volume.

[0039] Based on the above results, the enzyme membrane E was attached to the tip of the detection probe 50 of the measuring instrument 5 shown in Figure 1, and diacetyl measurement was performed. The results are shown in the graph in Figure 7A. Here, buffer reservoir 11 contained 100 μM NADH buffer 15 (PB, pH 6.0), and diacetyl was added to buffer 15 so that the concentration became 10 μM after 2 minutes of measurement. The vertical axis of the graph shows the inverted decrease in fluorescence intensity (similar to Figure 7B described below). As this graph shows, in an acidic environment of pH 6.0, diacetyl can be detected by the reaction from the left side to the right side of the above equation 1.

[0040] The graph in Figure 7B shows the results of acetoin measurement using the same measuring instrument 5 as above. Here, buffer reservoir 11 contained 100 μM NADH buffer 15 (PB, pH 6.0), and acetoin was added to buffer 15 to make it 10 μM at the 2-minute measurement time. As this graph shows, in an acidic environment of pH 6.0, acetoin can be detected by the reaction from the left side to the right side of Equation 2 above.

[0041] Furthermore, the same measuring instrument 5 was used to measure 1,2-butanediol, and the results are shown in the graph in Figure 7C. Here, buffer reservoir 11 contained 100 μM NADH in buffer 15 (PB, pH 9.0), and 1,2-butanediol was added to buffer 15 to make it 10 μM at the 2-minute measurement time. The vertical axis of the graph shows the inverse increase in fluorescence intensity (similar to Figure 7D, described below). As this graph shows, in an alkaline environment of pH 9.0, 1,2-butanediol can be detected by the reaction from the right side to the left side of Equation 2 above.

[0042] The graph in Figure 7D shows the results of acetoin measurement using the same measuring instrument 5 as above. Here, buffer reservoir 11 contained 100 μM NADH buffer 15 (PB, pH 9.0), and acetoin was added to buffer 15 to make it 10 μM at the 2-minute measurement time. As shown in this graph, in an alkaline environment of pH 9.0, acetoin can be detected by the reaction from the right side to the left side of Equation 1 above.

[0043] Figure 8 is a graph showing the measurement of diacetyl using various buffers 15. This graph shows the results of measurements performed under the same conditions as those shown in Figure 7A, except for the different buffers 15 used. The buffers used, from top to bottom at the right end of the graph, were PB at pH 6.0, PB at pH 5.5, PB at pH 6.5, PB at pH 7.0, acetate buffer (AB) at pH 4.0, PB at pH 8.0, and bicarbonate buffer (BCB) at pH 10.0. As this graph shows, PB at pH 6.0 was found to be optimal for measuring diacetyl, given its high reaction responsiveness.

[0044] Next, using the above-mentioned enzyme membrane E as the enzyme membrane 70 and PB at pH 6.0 as the buffer 15, measurements were taken while gradually increasing the concentration of diacetyl added to the buffer 15. The results are shown in Figure 9. As shown in this graph, it was found that under these measurement conditions, a good concentration-dependent response was observed. The quantitative characteristics at this time are represented by the regression curve shown in the graph in Figure 10. Each plot in the graph is the average value of three measurements. This regression curve is approximated by Equation 1 below, with a correlation coefficient of 0.99. From this correlation curve, it was inferred that the quantitative characteristics were good within the shaded range in the figure, i.e., the range of 0.1 to 100 μM.

[0045]

number

[0046] Furthermore, it was inferred that acetoin and 1,2-butanediol could also be measured in the same way as diacetyl using the above experimental system.

[0047] (3) Measurement of diacetyl gas Fig. 11 is a schematic diagram showing the structure of a diacetyl measuring device 1 that measures diacetyl gas. Fig. 12 is a schematic diagram showing the structure of the tip of a detection probe 50 used in the diacetyl measuring device 1 of Fig. 11. The diacetyl measuring device 1 comprises a measuring instrument 5, a coenzyme supplying device 10, and a specimen supplying device 20. The measuring instrument 5 comprises an irradiation device 30 including an irradiation fiber 31 for irradiating excitation light, a detection device 40 including a detection fiber 41 for detecting fluorescence, and a detection probe 50 including a measurement fiber 51 that combines the irradiation fiber 31 and the detection fiber 41. The irradiation device 30 and the detection device 40 are the same as those described above in (2), so further explanation will be omitted.

[0048] As shown in Fig. 12, the detection probe 50 has a cylindrical container 52 attached to its tip as a gas-liquid diaphragm flow cell equipped with an enzyme membrane 70 on which DDH is immobilized as an enzyme. The enzyme membrane 70 described above is attached to the tip of the container 52. As shown in Fig. 12, this container 52 encloses an inner tube 53 through which a measurement fiber 51 is inserted. As shown in Fig. 12, the enzyme membrane 70 is fixed to the container by a membrane holder 54 made of an O-ring. A buffer 15 flows through a reaction section 55, which is the internal space of the container 52. This buffer 15 contains NADH as a coenzyme, and is supplied to the reaction section 55 from a buffer reservoir 11 shown in Fig. 11 via a buffer flow path 12, and then released to the outside.

[0049] The measurement fiber 51 is inserted from the rear end side of the container 52, and its tip reaches the reaction section 55 and comes into contact with the buffer 15. In the buffer 15, excitation light is irradiated from the illumination fiber 31, and at the same time, fluorescence is received by the detection fiber 41.

[0050] As shown in FIG. 11 , the tip of container 52 is adjacent to sample gas flow path 24. Standard gas 25A is delivered to sample gas flow path 24 from standard gas flow path 24A via flow path selector 29 as sample gas 25. That is, diacetyl gas supplied at an arbitrary concentration from permeator 21 flows into flow path selector 29 as standard gas 25A at a predetermined flow rate by flow controller 22A. Also connected to flow path selector 29 are sample gas flow path 24B and clean air flow path 24C, which will be described later. Enzyme membrane 70, attached to the tip of container 52 adjacent to sample gas flow path 24, is exposed to sample gas 25. In other words, by attaching container 52 so that the tip is inserted into sample gas flow path 24, sample gas 25 and buffer 15 are separated by enzyme membrane 70. Specifically, diacetyl, a substrate contained in sample gas 25, is converted to acetoin through an enzymatic reaction with DDH immobilized on enzyme membrane 70, using NADH contained in buffer 15 as a coenzyme. At this time, NADH receives excitation light from illumination fiber 31 and emits fluorescence, which is detected by detection fiber 41. The higher the diacetyl concentration in sample gas 25, the greater the consumption of NADH as a coenzyme, resulting in a corresponding decrease in the fluorescence intensity from NADH. The diacetyl concentration in sample gas 25 is analyzed by analyzer 60 by applying the degree of decrease in fluorescence intensity to a predetermined calibration curve using standard gas 25A as sample gas 25.

[0051] Figures 13A and 13B are graphs showing the responsiveness to diacetyl gas using the diacetyl gas measuring device 1 of Figure 11. Note that Figure 13B shows an enlarged view of the low diacetyl gas concentration portion of Figure 13A. The NADH concentration in buffer 15 was 20 μM. For two minutes after the start of measurement, only carrier gas was delivered to sample gas flow path 24. Then, for five minutes from two minutes after the start of measurement, diacetyl gas was delivered to sample gas 25 to achieve the concentrations shown in the figure. As a result, as shown in the figure, a concentration-dependent decrease in fluorescence intensity was clearly observed. The quantitative characteristics at this time are represented by the regression curve shown in the graph of Figure 14. Each plot in the graph is the average value of three measurements. This regression curve is approximated by Equation 2 below, with a correlation coefficient (R) of 0.99. From this regression curve, it was inferred that the quantitative characteristics were good within the shaded range in the figure, i.e., the range of 2.1 to 108 ppb-v. This range includes the average diacetyl concentration emitted from the head, approximately 10 ppb-v.

[0052]

number

[0053] FIG. 15A is a schematic diagram showing how a head biogas sample is collected from a subject. Specifically, gas collection bag 26a connected to sample bag 26 is attached directly to the subject's head H, and collected gas 25B flowing in from gas collection bag 26a is collected into sample bag 26. This sample bag 26 is stored until a subsequent measurement is performed. FIG. 15B is a schematic diagram showing the flow path of the head biogas sample collected in FIG. 15A. Collected gas 25B is sent as sample gas 25 from collected gas flow path 24B to specimen gas flow path 24 via flow path switch 29. Specifically, sample bag 26, into which collected gas 25B has been collected in advance, is connected to flow rate controller 22B, and collected gas 25B flows into flow path switch 29 via collected gas flow path 24B at a predetermined flow rate controlled by flow rate controller 22B. Also connected to flow path selector 29 is clean air flow path 24C, into which clean air 25C sent by flow rate controller 22C from clean air source 27 flows. As described above, standard gas flow path 24A is also connected to flow path selector 29. This allows flow path selector 29 to appropriately switch between standard gas 25A, collected gas 25B, and clean air 25C and send them to sample gas flow path 24 as sample gas 25, and also allows the diacetyl concentration to be measured from collected gas 25B in sample bag 26.

[0054] 16 is a schematic diagram showing the flow path of a head biogas sample directly collected from a subject. Specifically, a gas collector 28 connected to a clean air source 27 at its upstream side is directly attached to the subject's head H, and its downstream side is connected to a flow rate controller 22B. Head biogas is collected by the gas collector 28 using clean air 25C as a carrier gas, and the collected gas flows into a flow path switch 29 as collected gas 25B via a collected gas flow path 24B at a predetermined flow rate by the flow rate controller 22B. Similarly to FIG. 15B, the flow path switch 29 is also connected to a clean air flow path 24C into which clean air 25C sent from the clean air source 27 by the flow rate controller 22C flows, and further to a standard gas flow path 24A. This allows flow path switch 29 to appropriately switch between standard gas 25A, sampled gas 25B, and clean air 25C and send them to sample gas flow path 24 as sample gas 25, and also makes it possible to measure in real time diacetyl released from the head as sampled gas 25B collected by gas collector 28. Furthermore, by changing the position at which gas collector 28 is attached, it is possible to measure the amount of diacetyl released from each part of the head.

[0055] 17 is a graph showing an example of diacetyl measurement from analyte gas 25 obtained from the head. Specifically, nitrogen gas was flowed as a carrier gas through analyte gas flow path 24 for the first five minutes, analyte gas 25 was flowed through analyte gas flow path 24 for the next five minutes, and then carrier gas was again flowed through analyte gas flow path 24. In the graph, open square symbols represent measurement results obtained by flowing carrier gas instead of analyte gas 25, open triangle symbols represent measurement results obtained by flowing analyte gas 25 containing 8 ppb-v of diacetyl, open circle symbols represent measurement results obtained by flowing analyte gas 25 collected from the head using sample bag 26, filled square symbols represent measurement results obtained by flowing analyte gas 25 containing 14.5 ppb-v of diacetyl, filled triangle symbols represent measurement results obtained by flowing analyte gas 25 collected directly from gas collector 28, and filled circle symbols represent measurement results obtained by flowing analyte gas 25 containing 26.3 ppb-v of diacetyl. The subject from whom sample gas 25 was collected from the head was a male (51), who was instructed to use unscented shampoo and treatment for one week prior to the measurement and was prohibited from washing his hair for more than 24 hours prior to the measurement.

[0056] As a result, a concentration-dependent decrease in fluorescence intensity of diacetyl was observed. For the sample gas 25 collected from the head, the diacetyl concentration of sample gas 25 collected directly from gas collector 28 was found to be between 14.5 and 26.3 ppb-v, which is consistent with the range of diacetyl concentrations in head gases reported previously. The diacetyl concentration of sample gas 25 collected in sample bag 26 was approximately half that of sample gas collected directly from gas collector 28. This is presumably due to adsorption of diacetyl to the inner surface of sample bag 26 or the influence of moisture contained in sample gas 25.

[0057] As described above, the diacetyl measuring device 1 of this embodiment comprises a coenzyme supplying device 10 that infiltrates an enzyme membrane 70 having DDH immobilized on a support membrane 71 with an acidic solution containing reduced nicotinamide dinucleotide (NADH) as a coenzyme; a specimen supplying device 20 that circulates a specimen fluid that is assumed to contain diacetyl as a substrate through the enzyme membrane 70 after the infiltration; an irradiation device 30 that irradiates with excitation light that excites NADH; a detection device 40 that detects fluorescence emitted from NADH that is reduced by the enzyme reaction; and an analysis device 60 that analyzes the concentration of diacetyl in the specimen fluid based on the degree of decrease in the intensity of the detected fluorescence.

[0058] Furthermore, the diacetyl measuring device 1 can implement a diacetyl measuring method in which an enzyme membrane 70 having DDH immobilized on a support membrane 71 is infiltrated with an acidic solution containing reduced nicotinamide dinucleotide (NADH) as a coenzyme, and in contact with a sample fluid assumed to contain diacetyl as a substrate, excitation light is irradiated to excite NADH, fluorescence emitted from NADH that is reduced by the enzyme reaction is detected, and the concentration of diacetyl in the sample fluid is analyzed based on the degree of decrease in the intensity of the detected fluorescence.

[0059] Furthermore, it is preferable that the surface of the support membrane 71 is first modified with polyethyleneimine, and then modified with glutaraldehyde, and then the DDH is immobilized thereon to form the enzyme membrane 70. The molecular weight of polyethyleneimine is preferably 8,000 to 10,000,000, and more preferably 100,000 to 1,000,000. Furthermore, the support membrane 71 is preferably a polytetrafluoroethylene membrane.

[0060] (4) Imaging 18 is a schematic diagram of a diacetyl gas imaging system. This imaging system includes the enzyme membrane 70 soaked in an acidic solution containing NADH, a sample gas flow path 24 through which a sample gas assumed to contain acetaldehyde and ethanol flows through the enzyme membrane 70, an ultraviolet light-emitting diode 32 that irradiates the enzyme membrane 70 with excitation light having a wavelength of 340 nm, the low-pass filter 33, a high-pass filter 43 that transmits fluorescence having a wavelength of 491 nm emitted from the excited NADH, and a camera 44 that captures video of the transmitted fluorescence. Based on the captured video of the fluorescence, the dynamics of diacetyl in the sample gas 25 can be analyzed in real time. [Industrial Applicability]

[0061] The present disclosure can be used for a method and device for measuring diacetyl produced in a living body. [Explanation of symbols]

[0062] 1 Diacetyl measuring device 5 Measuring instruments 10 coenzyme supply device 11 buffer reservoir 12 buffer flow path 15 Buffer 16 Pipette 17 Sample solution 18 Stirrer 18a Stir bar 20 Sample supply device 21 Permeator 22A, 22B, 22C flow controller 24 Sample gas flow path 24A Standard gas flow path 24B Collected gas flow path 24C Clean air flow path 25 Sample gas 25A Standard gas 25B Collected gas 25C Clean air 26 Sample bag 26a Gas collection bag 27 Clean air source 28 Gas collector 29 Flow path switch 30 Irradiation device 31 Irradiation fiber 32 Ultraviolet light emitting diode 33 Low-band bandpass filter 40 Detector 41 Detection fiber 42 Photomultiplier tube 43 High-bandwidth bandpass filter 44 Camera 50 detection probe 51 measurement fiber 52 container 53 inner tube 54 membrane holder 55 reaction section 60 Analyzer 70 Enzyme membrane 71 Support membrane 72 Polyethyleneimine 73 Glutaraldehyde 74 Diol dehydrogenase H head

Claims

1. A diacetyl measurement method in which an enzyme membrane having diol dehydrogenase immobilized on a support membrane is infiltrated with an acidic solution containing reduced nicotinamide dinucleotide (NADH) as a coenzyme, and in contact with a sample fluid assumed to contain diacetyl as a substrate, excitation light that excites NADH is irradiated, fluorescence emitted from NADH that decreases due to the enzyme reaction is detected, and the concentration of diacetyl in the sample fluid is analyzed based on the degree of decrease in the intensity of the detected fluorescence.

2. 2. The method for measuring diacetyl according to claim 1, wherein the surface of the support membrane is surface-modified with polyethyleneimine, and then surface-modified with glutaraldehyde, and the diol dehydrogenase is immobilized in this state to form the enzyme membrane.

3. a coenzyme supplying device for infiltrating an enzyme membrane having a support membrane on which diol dehydrogenase is immobilized with an acidic solution containing reduced nicotinamide dinucleotide (NADH) as a coenzyme; a sample supplying device for supplying a sample fluid that is assumed to contain diacetyl as a substrate to the infiltrated enzyme membrane; an irradiation device that irradiates excitation light that excites NADH; a detection device that detects fluorescence emitted from NADH that is reduced by the enzyme reaction; and an analyzer that analyzes the concentration of diacetyl in the sample fluid based on the degree of decrease in the intensity of the detected fluorescence.

4. The diacetyl measuring device according to claim 3, wherein the surface of the support membrane is surface-modified with polyethyleneimine, and then surface-modified with glutaraldehyde, and the diol dehydrogenase is immobilized in this state to form the enzyme membrane.

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