Method and apparatus for measuring biological gas

Abstract

To provide a biological gas measurement method capable of simultaneously and accurately determining the concentrations of two different target substances in a biological gas.SOLUTION: A biological gas measurement method includes: passing a sample gas, presumed to contain a first substrate and a second substrate, through a first mesh, in which at least a first enzyme is immobilized on a fibrous mesh and which is infiltrated with a solution containing at least a first coenzyme that is the coenzyme of the first enzyme, and a second mesh, in which at least a second enzyme is immobilized on a fibrous mesh and which is infiltrated with a solution containing at least a second coenzyme that is the coenzyme of the second enzyme; irradiating the first mesh and second mesh with first excitation light that excites a first reactant varying based on the enzyme reaction of the first substrate, and second excitation light that excites a second reactant varying based on the enzyme reaction of the second substrate; video-recording first fluorescence due to the first excitation light and the second fluorescence due to the second excitation light: and analyzing the concentrations of the first and second substrates in the sample gas based on the video-recorded images.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method and an apparatus for measuring a target substance contained in a biogas. [Background technology]

[0002] Biogas contains volatile compounds derived from metabolism and diseases, and it is believed that non-invasive measurement will enable metabolic function evaluation and disease screening. The technology described in the following Patent Document 1 is disclosed as a technology for collecting and measuring volatile components in biogas generated from the skin. In addition, the technology described in the following Patent Document 2 is disclosed as a technology for collecting and measuring target substances in biogas released from the outer ear. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO 2019 / 103130 A1 [Patent Document 2] WO 2022 / 092219 A1 Summary of the Invention [Problem to be solved by the invention]

[0004] In the above-mentioned prior art, the substance generated by the enzyme reaction of the target substance in the biogas is irradiated with excitation light of a specific wavelength, and the intensity of the fluorescence of the specific wavelength generated is measured to calculate the concentration of the target substance. On the other hand, when trying to measure multiple target substances in the biogas using the above-mentioned prior art, it was necessary to divide the biogas sample into multiple parts and subject each of them to an enzyme reaction according to the target substance.

[0005] The present disclosure provides a biological gas measuring method and device that can simultaneously and accurately grasp the concentrations of two types of target substances in a biological gas. [Means for solving the problem]

[0006] The biological gas measurement method disclosed herein comprises: passing a sample gas assumed to contain a first substrate and a second substrate through a first mesh having at least a first enzyme immobilized thereon and being infiltrated with a solution containing at least a first coenzyme that is a coenzyme of the first enzyme; and passing a second mesh having at least a second enzyme immobilized thereon and being infiltrated with a solution containing at least a second coenzyme that is a coenzyme of the second enzyme through the first mesh and the second mesh, irradiating the first mesh and the second mesh with a first excitation light that excites a first reactant that increases or decreases based on an enzyme reaction of the first substrate, and a second excitation light that excites a second reactant that increases or decreases based on an enzyme reaction of the second substrate, capturing a video of the first fluorescence due to the first excitation light and the second fluorescence due to the second excitation light, and analyzing the concentrations of the first substrate and the second substrate in the sample gas based on the captured video.

[0007] The biological gas measuring device disclosed herein comprises a first mesh having at least a first enzyme immobilized on a fibrous mesh and then soaked with a solution containing at least a first coenzyme that is a coenzyme of the first enzyme; a second mesh having at least a second enzyme immobilized on a fibrous mesh and then soaked with a solution containing at least a second coenzyme that is a coenzyme of the second enzyme; a gas distribution device that distributes a sample gas assumed to contain a first substrate and a second substrate through the first mesh and the second mesh; an irradiation device that irradiates the first mesh and the second mesh with a first excitation light that excites a first reactant whose amount increases or decreases based on an enzyme reaction of the first substrate and a second excitation light that excites a second reactant whose amount increases or decreases based on an enzyme reaction of the second substrate; an imaging device that captures a video of a first fluorescence due to the first excitation light and a second fluorescence due to the second excitation light; and an analysis device that analyzes the concentrations of the first substrate and the second substrate in the sample gas based on the captured video.

[0008] In the biogas measuring method of the present disclosure, the concentrations of two types of substrates, a first substrate and a second substrate, in a biogas are analyzed simultaneously. The first substrate undergoes an enzymatic reaction involving a first enzyme and a first coenzyme, thereby increasing or decreasing the first reactant. The second substrate undergoes an enzymatic reaction involving a second enzyme and a second coenzyme, thereby increasing or decreasing the second reactant. Here, the first reactant and the second reactant are substances that increase or decrease due to the enzymatic reaction of the first substrate and the second substrate, respectively, and may be substances that are directly purified by the enzymatic reaction of the first substrate or the second substrate, or may be substances that increase or decrease indirectly due to the enzymatic reaction of the first substrate or the second substrate. In addition, the first reactant and the second reactant may be a first coenzyme and a second coenzyme, respectively, or substances that have a different redox state from these. Here, the increase or decrease of the first reactant and the second reactant means that they may be produced by the enzymatic reaction and increase, or may be consumed by the enzymatic reaction and decrease. The first enzyme and the second enzyme may be different enzymes or the same enzyme.

[0009] The first and second meshes are immobilized with the first and second enzymes, respectively, and may also contain other substances depending on the substrate and enzyme reaction. Furthermore, the first and second meshes on which the first and second enzymes are immobilized are soaked with a solution containing the first and second coenzymes, respectively, before (preferably immediately before) passing the sample gas through them. This solution may also contain other substances depending on the substrate and enzyme reaction.

[0010] The specimen gas is a gas specimen that is assumed to contain a first substrate and a second substrate. Examples of the specimen gas include the breath or skin gas of a subject. The specimen gas is passed through the first mesh and the second mesh. The passing of the specimen gas is performed, for example, by a gas passing device that includes a gas flow path and a pump.

[0011] The first excitation light is a light beam having a wavelength that excites the first reactant. When the first excitation light is irradiated onto the first reactant, a first fluorescence is emitted from the excited first reactant. The wavelength of the first fluorescence is desirably different from the wavelength of the first excitation light. The second excitation light is a light beam having a wavelength that excites the second reactant. When the second excitation light is irradiated onto the second reactant, a second fluorescence is emitted from the excited second reactant. The wavelength of the second fluorescence is desirably different from the wavelength of the second excitation light. Furthermore, it is desirably that the wavelengths of the first excitation light and the second excitation light are different, and it is desirably that the wavelengths of the first fluorescence and the second fluorescence are different. The first excitation light and the second excitation light are generated, for example, by an irradiation device capable of generating light beams of these wavelengths. In this case, the first excitation light and the second excitation light may be generated from separate irradiation devices, or may be generated from the same irradiation device.

[0012] If the sample gas contains a first substrate, it undergoes an enzymatic reaction with the immobilized first enzyme and the first coenzyme contained in the infiltrating solution as it flows through the first mesh, and the first reactant increases or decreases based on this. When the sample gas is irradiated with the first excitation light, the intensity of the first fluorescence increases or decreases in accordance with the increase or decrease in the first reactant. On the other hand, if the sample gas contains a second substrate, it undergoes an enzymatic reaction with the immobilized second enzyme and the second coenzyme contained in the infiltrating solution as it flows through the second mesh, and the second reactant increases or decreases based on this. When the sample gas is irradiated with the second excitation light, the intensity of the second fluorescence increases or decreases in accordance with the increase or decrease in the second reactant.

[0013] The first and second fluorescence change with changes in the respective concentrations of the first and second substrates in the flowing sample gas. These changes are captured as video by a photographing device such as a video camera, and the concentrations of the first and second substrates in the sample gas are analyzed based on the captured video. This analysis is performed, for example, by an analyzing device formed of a computer that stores calibration curve data obtained from video images captured with known concentrations of the first and second substrates.

[0014] For example, when the first reactant (or the second reactant) is a substance that is generated as the enzymatic reaction of the first substrate (or the second substrate) proceeds, the intensity of the first fluorescence (or the second fluorescence) increases, and the greater the degree of this increase, the greater the concentration of the first substrate (or the second substrate) in the sample gas.

[0015] On the other hand, if the second reactant (or the first reactant) is a substance that is consumed as the enzymatic reaction of the second substrate (or the first substrate) proceeds, the intensity of the second fluorescence (or the first fluorescence) decreases. Therefore, the greater the degree of this decrease, the greater the concentration of the second substrate (or the first substrate) in the sample gas.

[0016] In the above-mentioned method for measuring a biological gas, for example, the first coenzyme is oxidized nicotinamide adenine dinucleotide (NAD + ), and the first enzyme is NAD + as a coenzyme to produce reduced nicotinamide adenine dinucleotide (NADH), the first substrate is a substance that undergoes an enzymatic reaction with the first enzyme, the second coenzyme is NADH, and the second enzyme uses NADH as a coenzyme to produce NAD + The second substrate is a substance that undergoes an enzymatic reaction by the second enzyme, and the first mesh contains, together with the first enzyme, NAD using NADH as a coenzyme. + and the first mesh has an additional enzyme immobilized thereon that produces NAD + the first excitation light has a wavelength that excites the first reactant produced from the additional substrate in an enzymatic reaction by the additional enzyme, the second coenzyme is NADH, and the second enzyme uses NADH as a coenzyme to produce NAD + the second substrate is a substance that undergoes an enzymatic reaction by the second enzyme, and the second excitation light has a wavelength that excites NADH.

[0017] For example, the first substrate is ethanol, the first enzyme is alcohol dehydrogenase (ADH), the second substrate is acetaldehyde, the second enzyme is ADH, the additional enzyme is diaphorase, the additional substrate is resazurin, and the first reactant is resorufin. In this case, the biogas measurement method includes the steps of: (a) measuring a biological gas containing ethanol; (b) measuring a biological gas containing oxidized nicotinamide adenine dinucleotide (NAD); + ) in which diaphorase is immobilized on the first mesh together with ADH as the first enzyme, and NAD as the first coenzyme is immobilized on the first mesh. + The present invention can be applied to a case in which the cell is infiltrated with a solution containing resazurin, which is a substrate of diaphorase, together with the first excitation light, and the first substrate is acetaldehyde, the second enzyme is ADH, the second coenzyme is reduced nicotinamide adenine dinucleotide (NADH), and the second excitation light has a wavelength that excites NADH.

[0018] When the first substrate is ethanol, in an alkaline environment, the first substrate, ethanol, is converted to acetaldehyde by the enzymatic action of the first enzyme, ADH, immobilized on the first mesh, as shown in the following chemical formula 1. Then, the first coenzyme, NAD, contained in the solution that permeates the first mesh, + is reduced to NADH. Furthermore, the enzyme diaphorase, which is immobilized on the first mesh together with ADH, + Using the reduced NADH as a coenzyme, it converts resazurin, a substrate contained in the solution that infiltrates the first mesh, into resorufin.

[0019] [ka]

[0020] That is, resorufin is the first reactant because it is a substance that increases based on the enzymatic reaction of the first substrate. When resorufin is exposed to the first excitation light with a wavelength of 560 nm, it emits a first fluorescence with a wavelength of 590 nm. In other words, as the reaction of the first substrate progresses, the first reactant increases, and the intensity of this first fluorescence increases. In other words, the greater the degree of increase in this first fluorescence, the higher the concentration of ethanol, which is the first substrate.

[0021] On the other hand, when the second substrate is acetaldehyde, in an acidic environment, the second substrate, acetaldehyde, is converted to ethanol by the enzymatic action of the second enzyme, ADH, immobilized on the second mesh, as shown in the following chemical formula 2. Then, the second coenzyme, NADH, contained in the solution that permeates the second mesh is oxidized to NAD + As a result, NADH is consumed and decreased.

[0022] [ka]

[0023] That is, the second coenzyme NADH is a substance that decreases due to the enzymatic reaction of the second substrate, and is therefore also a second reactant. When NADH receives second excitation light with a wavelength of 340 nm, it emits second fluorescence with a wavelength of 490 nm. In other words, as the reaction of the second substrate progresses, the second reactant decreases, and the intensity of this second fluorescence decreases. In other words, the greater the degree of decrease in this second fluorescence, the higher the concentration of the second substrate, acetaldehyde.

[0024] In the above example, the biogas measuring method of the present disclosure includes immobilizing ADH and diaphorase on a fibrous mesh and then measuring NAD +A sample gas assumed to contain ethanol and acetaldehyde is passed through a first mesh impregnated with a resazurin-containing solution and a second mesh having ADH immobilized on a fibrous mesh and then impregnated with an NADH-containing solution, and the first mesh and the second mesh are irradiated with a first excitation light having a wavelength that excites resorufin produced from resazurin by an enzymatic reaction with diaphorase, and a second excitation light having a wavelength that excites NADH. A video of the first fluorescence due to the first excitation light and the second fluorescence due to the second excitation light are captured, and the concentrations of ethanol and acetaldehyde in the sample gas are analyzed based on the captured video.

[0025] Furthermore, in the biogas measuring device of the present disclosure, the first coenzyme is oxidized nicotinamide adenine dinucleotide (NAD + ), and the first enzyme is NAD + as a coenzyme to produce reduced nicotinamide adenine dinucleotide (NADH), the first substrate is a substance that undergoes an enzymatic reaction with the first enzyme, the second coenzyme is NADH, and the second enzyme uses NADH as a coenzyme to produce NAD + The second substrate is a substance that undergoes an enzymatic reaction by the second enzyme, and the first mesh contains, together with the first enzyme, NAD using NADH as a coenzyme. + and the first mesh has an additional enzyme immobilized thereon that produces NAD + the first excitation light has a wavelength that excites the first reactant produced from the additional substrate in an enzymatic reaction by the additional enzyme, the second coenzyme is NADH, and the second enzyme uses NADH as a coenzyme to produce NAD + the second substrate is a substance that undergoes an enzymatic reaction by the second enzyme, and the second excitation light can be formed to have a wavelength that excites NADH.

[0026] For example, the first substrate is ethanol, the first enzyme is alcohol dehydrogenase (ADH), the second substrate is acetaldehyde, the second enzyme is ADH, the additional enzyme is diaphorase, the additional substrate is resazurin, and the first reactant is resorufin. In this case, the biogas measuring device of the present disclosure immobilizes ADH and diaphorase on a fibrous mesh and then detects NAD + and a first mesh impregnated with a resazurin-containing solution; a second mesh having ADH immobilized on a fibrous mesh and then impregnated with an NADH-containing solution; a gas distribution device for distributing a sample gas assumed to contain acetaldehyde and ethanol through the first mesh and the second mesh; an imaging device for irradiating the first mesh and the second mesh with first excitation light having a wavelength that excites resorufin produced from resazurin by an enzyme reaction with diaphorase and second excitation light having a wavelength that excites NADH, and taking a video of the first fluorescence due to the first excitation light and the second fluorescence due to the second excitation light; and an analysis device for analyzing the concentrations of acetaldehyde and ethanol in the sample gas based on the taken video.

[0027] In order to simultaneously capture the first fluorescence and the second fluorescence of the same biological gas as a moving image, it is desirable that the first mesh and the second mesh are superimposed along the flow direction of the sample gas.

[0028] In addition, when using resazurin as an additional substrate, detecting the first fluorescence at 590 nm from resorufin, which is the first reactant produced in the enzymatic reaction with diaphorase as the additional enzyme, and detecting the second fluorescence at 490 nm from NADH as the second coenzyme, the following substrates can also be measured.

[0029] For example, the first substrate may be (E)-2-nonenal and the second substrate may be acetaldehyde, in which case the first enzyme is aldehyde dehydrogenase (ALDH) and the second enzyme is alcohol dehydrogenase (ADH).

[0030] That is, as shown in the following chemical formula 3, the first substrate (E)-2-nonenal becomes (E)-2-nonanoic acid methyl ester by the enzymatic action of the first enzyme, ALDH, immobilized on the first mesh. + is reduced to NADH. Furthermore, the enzyme diaphorase, which is immobilized on the first mesh together with ADH, + Using the reduced NADH as a coenzyme, resazurin, a substrate contained in the solution infiltrating the first mesh, is converted to resorufin, a first reactant. When resorufin is exposed to a first excitation light with a wavelength of 560 nm, it emits a first fluorescence with a wavelength of 590 nm. In other words, as the reaction of the first substrate progresses, the first reactant increases, and the intensity of this first fluorescence increases. In other words, the greater the degree of increase in this first fluorescence, the higher the concentration of the first substrate, (E)-2-nonenal.

[0031] [ka]

[0032] On the other hand, when the second substrate is acetaldehyde, as shown in the above chemical formula 2, in an acidic environment, the second substrate acetaldehyde is converted to ethanol by the enzymatic action of the second enzyme ADH immobilized on the second mesh. Then, the second coenzyme NADH contained in the solution that permeates the second mesh is oxidized to NAD + Therefore, NADH as the second reactant is consumed and decreased. When NADH receives the second excitation light with a wavelength of 340 nm, it emits a second fluorescence with a wavelength of 490 nm. In other words, as the reaction of the second substrate progresses, the second reactant decreases, and the intensity of this second fluorescence decreases. In other words, the greater the degree of decrease in this second fluorescence, the higher the concentration of the second substrate, acetaldehyde.

[0033] In another example, the first substrate is 2-propanol and the second substrate is acetone, in which case the first enzyme is a secondary alcohol dehydrogenase (S-ADH) and the second enzyme is also an S-ADH.

[0034] That is, as shown in the following chemical formula 4, the first substrate, 2-propanol, is converted to acetone by the enzymatic action of the first enzyme, S-ADH, immobilized on the first mesh. + is reduced to NADH. Furthermore, the enzyme diaphorase, which is immobilized on the first mesh together with ADH, + Using the reduced NADH as a coenzyme, resazurin, a substrate contained in the solution that permeates the first mesh, is converted into resorufin, a first reactant. When resorufin is exposed to a first excitation light with a wavelength of 560 nm, it emits a first fluorescence with a wavelength of 590 nm. In other words, as the reaction of the first substrate progresses, the first reactant increases, and the intensity of this first fluorescence increases. In other words, the greater the degree of increase in this first fluorescence, the higher the concentration of the first substrate, 2-propanol.

[0035] [ka]

[0036] On the other hand, the second substrate, acetone, is converted to 2-propanol by the enzymatic action of the second enzyme, S-ADH, immobilized on the second mesh in an acidic environment, as shown in Chemical Formula 5 below. Then, the second coenzyme, NADH, contained in the solution that permeates the second mesh is oxidized to NAD + Therefore, NADH as the second reactant is consumed and decreased. When NADH receives the second excitation light with a wavelength of 340 nm, it emits a second fluorescence with a wavelength of 490 nm. In other words, as the reaction of the second substrate progresses, the second reactant decreases, and the intensity of this second fluorescence decreases. In other words, the greater the degree of decrease in this second fluorescence, the higher the concentration of acetone, the second substrate.

[0037] [ka]

[0038] Furthermore, the first substrate may be ethanol and the second substrate may be acetone, in which case the first enzyme is alcohol dehydrogenase (ADH) and the second enzyme is secondary alcohol dehydrogenase (S-ADH).

[0039] That is, as shown in the above formula 1, the first substrate, ethanol, is converted to acetaldehyde by the enzymatic action of the first enzyme, ADH, immobilized on the first mesh. + is reduced to NADH. Furthermore, the enzyme diaphorase, which is immobilized on the first mesh together with ADH, + Using the reduced NADH as a coenzyme, resazurin, a substrate contained in the solution infiltrating the first mesh, is converted into resorufin, a first reactant. When resorufin is exposed to a first excitation light with a wavelength of 560 nm, it emits a first fluorescence with a wavelength of 590 nm. In other words, as the reaction of the first substrate progresses, the first reactant increases, and the intensity of this first fluorescence increases. In other words, the greater the degree of increase in this first fluorescence, the higher the concentration of ethanol, the first substrate.

[0040] On the other hand, as shown in Chemical Formula 5, the second substrate, acetone, becomes 2-propanol in an acidic environment by the enzymatic action of the second enzyme, S-ADH, immobilized on the second mesh. Then, the second coenzyme, NADH, contained in the solution that permeates the second mesh is oxidized to NAD+, and NADH as the second reactant is consumed and reduced. When NADH is exposed to the second excitation light with a wavelength of 340 nm, it emits a second fluorescence with a wavelength of 490 nm. In other words, as the reaction of the second substrate progresses, the second reactant is reduced, and the intensity of this second fluorescence decreases. In other words, the greater the degree of reduction in this second fluorescence, the higher the concentration of acetone, the second substrate.

[0041] As the additional enzyme and additional substrate, in addition to the above-mentioned diaphorase and resazurin, an enzyme and substrate that uses NADH as a coenzyme, as represented by the following reaction formula, can be used.

[0042] Substrate (reduced state) + NADH + H + ←→Substrate (oxidation state) + NAD +

[0043] For example, an example of an additional substrate in which an enzymatic reaction is mediated using diaphorase as an additional enzyme is a substance represented by the following chemical formula 6. From this additional substrate, rhodamine 110 represented by chemical formula 7 is produced as a first reactant through an enzymatic reaction mediated by diaphorase using NADH as a coenzyme. This first reactant absorbs the first excitation light of 498 nm and emits the first fluorescence of 521 nm.

[0044] [ka]

[0045] [ka]

[0046] Another additional substrate in which an enzymatic reaction is mediated using diaphorase as an additional enzyme is WST-8, represented by the following chemical formula 8. From this additional substrate, WST-8 formazan, represented by the chemical formula 9, is produced as a first reactant through an enzymatic reaction mediated by diaphorase using NADH as a coenzyme. When irradiated with white light, this first reactant absorbs orange light at 460 nm.

[0047] [ka]

[0048] [ka]

[0049] Furthermore, DCI-MQ, represented by the following chemical formula 10, is used as an additional substrate, and DCI-MQH, represented by the chemical formula 11, is generated as a first reactant by an oxidation reaction with NADH. This first reactant absorbs the first excitation light of 668 nm and emits the first fluorescence of 660 nm.

[0050] [ka]

[0051] [ka]

[0052] The biogas measuring method and biogas measuring device disclosed herein may include those capable of measuring three or more types of substrates as long as they can measure two types of substrates, the first substrate and the second substrate. For example, if a third fluorescence generated from a substance that increases or decreases in an enzyme reaction of a third substrate, which is different from the first substrate and the second substrate, can be detected separately from the first fluorescence and the second fluorescence, three types of substrates can be simultaneously measured. The same applies to the fourth substrate and subsequent substrates. Effect of the Invention

[0053] According to the present disclosure, there is provided a biological gas measuring method and device capable of simultaneously and accurately determining the concentrations of two types of target substances in a biological gas. [Brief description of the drawings]

[0054] [Figure 1] 1 shows a schematic overview of a biological gas measuring device according to an embodiment. [Diagram 2] 1 shows a front view of a rotary filter. [Diagram 3] The switching of the bandpass filter is shown in a time chart. [Figure 4]1 is a graph showing the change in fluorescence intensity over time for ethanol (upper part) and acetaldehyde (lower part) when a sample gas containing ethanol and acetaldehyde is measured using a biological gas measuring device. [Diagram 5] 5 is a graph showing the time course of the rate of change in fluorescence based on the graph in FIG. 4 for ethanol (upper part) and acetaldehyde (lower part). [Figure 6] 1 is a graph showing quantitative measurement of ethanol in the biogas measuring device of the embodiment. [Figure 7] 1 is a graph showing quantitative measurement of acetaldehyde in the biological gas measuring device of the embodiment. [Figure 8] 1 is a graph showing the change in the fluorescent spectrophotometer of ethanol over time when ethanol and acetaldehyde in breath are measured simultaneously based on moving images. [Figure 9] 1 is a graph showing the change in the fluorescent spectrophotometer of acetaldehyde over time when ethanol and acetaldehyde in breath are simultaneously measured based on moving images. [Figure 10] This graph compares the results of measuring ethanol (top row) and acetaldehyde (bottom row) in the breath of a subject with ALDH2 activity (left side) and a subject without ALDH2 activity (right side) using a biogas measuring device of an embodiment (top graph of each row) and a commercially available gas detection tube (bottom graph of each row). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] Hereinafter, a biological gas measurement method and a biological gas measurement device according to the present embodiment will be described with reference to the drawings. Note that the drawings referred to below show schematic configurations of the respective components and do not reflect the actual sizes.

[0056] FIG. 1 is a schematic diagram showing an overview of the biological gas measuring device of this embodiment. The biological gas measuring device 10 of FIG. 1 includes a first mesh 20 having at least a first enzyme immobilized on a fibrous mesh and then infiltrated with a solution containing at least a first coenzyme that is a coenzyme of the first enzyme; a second mesh 30 having at least a second enzyme immobilized on a fibrous mesh and then infiltrated with a solution containing at least a second coenzyme that is a coenzyme of the second enzyme; a gas distribution device 40 that distributes a sample gas that is assumed to contain a first substrate and a second substrate through the first mesh 20 and the second mesh 30; an irradiation device 50 that irradiates the first mesh and the second mesh with a first excitation light E1 that excites a first reactant that increases or decreases based on an enzyme reaction of the first substrate and a second excitation light E2 that excites a second reactant that increases or decreases based on an enzyme reaction of the second substrate; an imaging device 60 that captures a video of a first fluorescence F1 due to the first excitation light E1 and a second fluorescence F2 due to the second excitation light E2; and an analysis device 70 that analyzes the concentrations of the first substrate and the second substrate in the sample gas based on the captured video.

[0057] Specifically, the biogas measuring device 10 in FIG. 1 is a device in which alcohol dehydrogenase (ADH) and diaphorase are immobilized on a fibrous mesh, and oxidized nicotinamide adenine dinucleotide (NAD + the first mesh 20 and the second mesh 30 are each formed by immobilizing ADH on a fibrous mesh and then infiltrating the mesh with a solution containing reduced nicotinamide adenine dinucleotide (NADH); a gas distribution device 40 that distributes a sample gas assumed to contain acetaldehyde and ethanol through the first mesh 20 and the second mesh 30; an imaging device 60 that irradiates the first mesh 20 and the second mesh 30 with first excitation light E1 having a wavelength that excites resorufin produced from resazurin by an enzyme reaction caused by diaphorase and second excitation light E2 having a wavelength that excites NADH, and captures a video of a first fluorescence F1 due to the first excitation light E1 and a second fluorescence F2 due to the second excitation light E2; and an analysis device 70 that analyzes the concentrations of acetaldehyde and ethanol in the sample gas based on the captured video.

[0058] In addition to the first enzyme ADH, an additional enzyme diaphorase is immobilized on the first mesh 20. Also, the second enzyme ADH is immobilized on the second mesh 30. For example, the enzymes can be immobilized on the meshes as follows. First, each enzyme is sprayed on a cotton mesh with an opening width of 2 mm, and the mesh is left to stand at 4°C for 60 minutes and dried. Next, the mesh is immersed in glutaraldehyde and left to stand at 4°C for 90 minutes again to crosslink the enzymes with glutaraldehyde. Finally, the mesh is washed with water to wash away the unimmobilized enzymes and glutaraldehyde, and then dried to obtain the first mesh 20 and the second mesh 30. The first mesh 20 and the second mesh 30 are overlapped along the flow direction of the sample gas.

[0059] The first mesh 20 is used to measure NAD, a coenzyme for ADH, immediately before measurement. + The second mesh 30 is infiltrated with an alkaline solution containing resazurin, which is a substrate (i.e., additional substrate) for diaphorase, and the second mesh 30 is infiltrated with an acidic solution containing NADH, which is a coenzyme for ADH, immediately before measurement.

[0060] Gas distribution device 40 distributes specimen gas, which is assumed to contain ethanol and acetaldehyde, through gas flow path 46 to first mesh 20 and second mesh 30. Specifically, specimen gas is drawn by diaphragm pump 41 from sample bag 45 containing specimen gas collected from a subject. The drawn specimen gas is sent to gas flow path 46 at a predetermined flow rate by first flow path adjustment valve 42. Meanwhile, in the gas flow path, air as a carrier gas is sent to gas flow path 46 from compressor 43 at a predetermined flow rate by second flow path adjustment valve 44. That is, gas flow path 46 continuously distributes specimen gas mixed with air through first mesh 20 and second mesh 30.

[0061] The irradiation device 50 of this embodiment is composed of a first irradiation device 51 that irradiates the first mesh 20 from the upstream side of the gas flow path 46 with first excitation light E1 having a wavelength of 560 nm that excites resorufin, and a second irradiation device 52 that irradiates the second mesh 30 from the downstream side of the gas flow path 46 with second excitation light E2 having a wavelength of 340 nm that excites NADH.

[0062] If resorufin is present on the first mesh 20, it is excited by the first excitation light E1 and emits a first fluorescence F1 at 590 nm. If NADH is present on the second mesh 30, it is excited by the second excitation light E2 and emits a second fluorescence F2 at 490 nm. The first fluorescence F1 and the second fluorescence F2 are captured as a moving image by the imaging device 60 through the rotating filter 80.

[0063] 2 is a front view showing a schematic diagram of the rotary filter 80. The rotary filter 80 is a device that rotates a wheel 83, to which two filters are attached, around a rotation shaft 84 of a motor. One of the two filters is a first bandpass filter 81 that transmits light with a wavelength of 590 nm, which is the first fluorescence F1, and the other is a second bandpass filter 82 that transmits light with a wavelength of 490 nm, which is the second fluorescence F2.

[0064] In order to capture the first fluorescence F1 and the second fluorescence F2 with high resolution, it is desirable for the imaging device 60 to have an ultra-high sensitivity. An example of such an imaging device 60 is an ultra-high sensitivity CMOS camera.

[0065] The rotation of the rotary filter 80 is controlled by a motor controller 85 to which a synchronous signal is input from the image capture device 60. Specifically, every time a synchronous signal corresponding to a predetermined reference time (for example, 1 second) is input, the motor controller 85 rotates the wheel 83 half a turn, and the filter covering the lens of the image capture device 60 is switched from the first bandpass filter 81 to the second bandpass filter 82, and from the second bandpass filter 82 to the first bandpass filter 81. This state is shown in the time chart of FIG. 3. The first excitation light E1 (second from the top) having a wavelength of 560 nm from the first irradiation device 51 and the second excitation light E2 (top) having a wavelength of 340 nm from the second irradiation device 52 are constantly emitted during imaging. On the other hand, the first bandpass filter 81 (second from the bottom) that passes the first fluorescence F1 having a wavelength of 590 nm and the second bandpass filter 82 (bottom) that passes the second fluorescence F2 having a wavelength of 490 nm are switched every predetermined time (for example, 1 second). As a result, the moving images actually captured by the imaging device 60 are recorded as an image in which the first fluorescence F1 and the second fluorescence F2 are switched every predetermined time (for example, 1 second), as shown in the middle part of FIG.

[0066] Based on the moving images thus captured by the imaging device 60, the analysis device 70 analyzes the concentrations of the first substrate (i.e., ethanol) and the second substrate (i.e., acetaldehyde). Specifically, the analysis device 70 can be configured as a computer having a CPU (Central Processing Unit), a ROM (Read Only Memory), a RAM (Random Access Memory), and a storage device. The analysis device 70 divides the moving images captured by the imaging device 60 into predetermined time periods (e.g., 1 second) and measures the fluorescence intensity of each portion.

[0067] In order to eliminate the influence of external light when photographing the fluorescence from the first mesh 20 and the second mesh 30, the first mesh 20 and the second mesh 30, the first illumination device 51 and the second illumination device 52, the rotating filter 80 and the photographing device 60 are placed in a dark box 11 which does not allow entry of external light.

[0068] Next, gas containing predetermined concentrations of ethanol and acetaldehyde was artificially prepared as a sample gas, and the results of this gas being measured using the biological gas measuring device of Fig. 1 are shown. The measurement results for ethanol as the first substrate are shown in the upper graph of Fig. 4, and the measurement results for acetaldehyde as the second substrate are shown in the lower graph of Fig. 4. The shaded areas in each graph indicate that sample gas containing 100 ppm ethanol and 5 ppm acetaldehyde was passed through the first mesh 20 and the second mesh 30 during the period from 24 seconds to 44 seconds after the start of the measurement.

[0069] The graph for ethanol in the upper part of Fig. 4 shows that the fluorescence intensity gradually increases after ethanol as the first substrate is captured by the first mesh 20, but the rate of increase slows down around 100 seconds from the start of the measurement and then reaches a plateau. On the other hand, the graph for acetaldehyde in the lower part of Fig. 4 shows that the fluorescence intensity gradually decreases after acetaldehyde as the second substrate is captured by the second mesh 30, but the rate of decrease slows down around 100 seconds from the start of the measurement and then reaches a plateau.

[0070] The graphs in the upper and lower parts of Figure 5 are plots of the differential values ​​at each time point from the graphs in the upper and lower parts of Figure 4, respectively. In the graph of the differential value of ethanol in the upper part of Figure 5, a peak value is observed around 65 seconds from the start of measurement. In other words, the rate of increase in fluorescence intensity was maximum at this point. On the other hand, in the graph of the differential value of acetaldehyde in the lower part of Figure 5, a peak value is observed around 56 seconds from the start of measurement. In other words, the rate of decrease in fluorescence intensity was maximum at this point.

[0071] The above-mentioned analysis was performed on sample gases with various substrate concentrations (specifically, 1-300 ppm for ethanol and 0.5-5 ppm for acetaldehyde), and the peak values ​​of the respective differential values ​​(i.e., peak values ​​of the rate of change of fluorescence intensity) are plotted on the vertical axis and the substrate concentration on the horizontal axis in Figure 6 (ethanol) and Figure 7 (acetaldehyde). However, the horizontal axis in Figure 6 plots the substrate concentration in logarithm. These graphs show that the logarithm of the ethanol concentration in the sample gas has a high linear regression with the peak value of the rate of change of fluorescence intensity (Figure 6), and that the acetaldehyde concentration in the sample gas has a high linear regression with the peak value of the rate of change of fluorescence intensity (Figure 7).

[0072] From the above, it has been shown that if the ethanol concentration in the sample gas is in the range of 1 to 300 ppm and the acetaldehyde concentration is in the range of 0.2 to 5 ppm, the substrate concentration in the sample gas can be analyzed from the peak value of the rate of change of the fluorescence intensity using the graphs in Figures 6 and 7 as calibration curves. The analysis device 70 stores these graphs in the storage device as, for example, regression line data, calculates the peak value of the rate of change of the fluorescence intensity calculated from the moving image, and applies this data to analyze the substrate concentration in the sample gas.

[0073] Next, the results of measurement of a sample gas derived from a living body using the biogas measuring device of the present disclosure are shown. In advance, one subject with aldehyde dehydrogenase 2 (ALDH2) (ALDH2[+]) and one subject without ALDH2 (ALDH2[-]) were identified through patch testing. Each subject was a healthy adult who agreed to have their breath gas collected. Each subject had not consumed alcohol or medicine within 72 hours, was fasting for 4 hours or more, and ingested 0.4 g of alcohol per kg of body weight within 15 minutes. Then, 30 minutes after the completion of the intake, the subject blew breath into the sample bag 45 to collect the sample gas.

[0074] The rate of change in fluorescence intensity over time for ethanol and acetaldehyde in sample gas from an ALDH2[-] subject is shown in Figure 8 and Figure 9, respectively. When the peak values ​​observed in Figures 8 and 9 were fitted to the calibration curves in Figures 6 and 7, respectively, the ethanol concentration in the breath was found to be 129.2 ppm, and the acetaldehyde concentration was found to be 1.99 ppm. These figures demonstrate that ethanol and acetaldehyde can be measured simultaneously without any problems in sample gas collected from actual subjects.

[0075] FIG. 10 shows the ethanol concentration (upper row) and acetaldehyde concentration (lower row) in the sample gas collected under the above-mentioned conditions for an ALDH2[+] subject (left) and an ALDH2[-] subject (right). In each graph, the upper bar graph shows the measurement value using the biogas measuring device of the present disclosure, and the lower bar graph shows the measurement value using a commercially available gas detector tube (No. 112L (ethanol), No. 92L (acetaldehyde), Gastec). The detectable range of this gas detector tube is 50-2,000 ppm for ethanol and 1-20 ppm for acetaldehyde in 100 mL of sample gas.

[0076] From FIG. 10, it was found that the biogas measuring device of the present disclosure has the same detection sensitivity as a commercially available gas detector tube with respect to the ethanol concentration in the breath. In addition, the value of the acetaldehyde concentration in the breath of the ALDH2[+] subject was smaller than that of the ALDH2[-] subject, so it was found that the presence or absence of ALDH was clearly reflected in the measured value. And, the biogas measuring device of the present disclosure was able to detect the acetaldehyde concentration of the ALDH2[+] subject, which was below the detection limit of the commercially available gas detector tube. From this, it was inferred that the biogas measuring device of the present disclosure can detect ethanol and acetaldehyde in the breath with higher accuracy than the commercially available gas detector tube.

[0077] It has been found that the biogas measuring device of the present disclosure described above can be used to implement a biogas measuring method in which a sample gas assumed to contain a first substrate and a second substrate is passed through a first mesh 20 having at least a first enzyme immobilized on a fibrous mesh and then infiltrated with a solution containing at least a first coenzyme that is a coenzyme of the first enzyme, and a second mesh 30 having at least a second enzyme immobilized on a fibrous mesh and then infiltrated with a solution containing at least a second coenzyme that is a coenzyme of the second enzyme, the first mesh 20 and the second mesh 30 are irradiated with a first excitation light that excites a first reactant that increases or decreases based on an enzyme reaction of the first substrate, and a second excitation light that excites a second reactant that increases or decreases based on an enzyme reaction of the second substrate, the first fluorescence due to the first excitation light and the second fluorescence due to the second excitation light are recorded as videos, and the concentrations of the first substrate and the second substrate in the sample gas are analyzed based on the recorded video.

[0078] Specifically, ADH and diaphorase were immobilized on a fibrous mesh and then NAD was measured using the biogas measuring device of the present disclosure. + It has been found that a method for measuring biological gases can be implemented in which a sample gas assumed to contain ethanol and acetaldehyde is passed through a first mesh 20 impregnated with a resazurin-containing solution and a second mesh 30 having ADH immobilized on a fibrous mesh and then impregnated with an NADH-containing solution, the first mesh 20 and the second mesh 30 are irradiated with a first excitation light having a wavelength that excites resorufin produced from resazurin by an enzymatic reaction with diaphorase, and a second excitation light having a wavelength that excites NADH, the first fluorescence due to the first excitation light and the second fluorescence due to the second excitation light are recorded as videos, and the concentrations of ethanol and acetaldehyde in the sample gas are analyzed based on the recorded video. [Industrial Applicability]

[0079] The present disclosure can be used in a biogas measurement method and a biogas measurement device. [Explanation of symbols]

[0080] 10 Biogas measuring device 11 Dark box 20 First mesh 30 Second Mesh 40 gas flow device 41 diaphragm pump 42 first flow path adjustment valve 43 Compressor 44 Second flow control valve 45 Sample bag 46 Gas flow path 50 Irradiation device 51 First irradiation device 52 Second irradiation device 60 Imaging Equipment 70 Analyzer 80 Rotating Filter 81 First bandpass filter 82 Second bandpass filter 83 Wheel 84 Rotating shaft 85 Motor controller E1 First excitation light E2 Second excitation fluorescence F1 First fluorescence F2 Second fluorescence

Claims

1. a first mesh having at least a first enzyme immobilized on a fibrous mesh and then infiltrated with a solution containing at least a first coenzyme that is a coenzyme of the first enzyme; a second mesh having at least a second enzyme immobilized on a fibrous mesh and then infiltrated with a solution containing at least a second coenzyme that is a coenzyme of the second enzyme; A sample gas that is assumed to contain a first substrate and a second substrate is passed through the sample gas; Irradiating the first mesh and the second mesh with a first excitation light that excites a first reactant that increases or decreases based on an enzyme reaction of the first substrate and a second excitation light that excites a second reactant that increases or decreases based on an enzyme reaction of the second substrate; taking a video of the first fluorescence due to the first excitation light and the second fluorescence due to the second excitation light; The concentration of the first substrate and the second substrate in the sample gas is analyzed based on the captured video image.

2. The first coenzyme is an oxidized nicotinamide adenine dinucleotide (NAD ＋ ) and The first enzyme is NAD ＋ is an enzyme that produces reduced nicotinamide adenine dinucleotide (NADH) using as a coenzyme, the first substrate is a substance that undergoes an enzymatic reaction by the first enzyme; the second coenzyme is NADH, The second enzyme uses NADH as a coenzyme to convert NAD ＋ is an enzyme that produces the second substrate is a substance that undergoes an enzymatic reaction by the second enzyme; The first mesh contains the first enzyme and NADH, which acts as a coenzyme to produce NAD. ＋ An additional enzyme that produces The first mesh is a NAD ＋ and infiltrating the substrate with a solution containing an additional substrate which is a substrate for the additional enzyme; the first excitation light has a wavelength that excites the first reactant generated from the additional substrate in an enzyme reaction by the additional enzyme, the second coenzyme is NADH, The second enzyme uses NADH as a coenzyme to convert NAD ＋ is an enzyme that produces the second substrate is a substance that undergoes an enzymatic reaction by the second enzyme; The second excitation light has a wavelength that excites NADH. The method for measuring a biological gas according to claim 1.

3. the first substrate is ethanol; the first enzyme is alcohol dehydrogenase (ADH); the second substrate is acetaldehyde; the second enzyme is ADH, the additional enzyme is diaphorase; said further substrate being resazurin; The first reactant is resorufin. The method for measuring a biological gas according to claim 2.

4. 4. The biological gas measuring method according to claim 1, wherein the first mesh and the second mesh are overlapped along a flow direction of the sample gas.

5. a first mesh having at least a first enzyme immobilized on a fibrous mesh and then infiltrated with a solution containing at least a first coenzyme that is a coenzyme of the first enzyme; a second mesh having at least a second enzyme immobilized on a fibrous mesh and then infiltrated with a solution containing at least a second coenzyme that is a coenzyme of the second enzyme; a gas flow device that flows a sample gas, the sample gas being assumed to contain a first substrate and a second substrate, through the first mesh and the second mesh; an irradiation device that irradiates the first mesh and the second mesh with a first excitation light that excites a first reactant that increases or decreases based on an enzyme reaction of the first substrate, and a second excitation light that excites a second reactant that increases or decreases based on an enzyme reaction of the second substrate; an imaging device that captures a video of a first fluorescence due to the first excitation light and a second fluorescence due to the second excitation light; and an analyzer that analyzes the concentrations of the first substrate and the second substrate in the sample gas based on the captured video image.

6. The first coenzyme is an oxidized nicotinamide adenine dinucleotide (NAD ＋ ) and The first enzyme is NAD ＋ is an enzyme that produces reduced nicotinamide adenine dinucleotide (NADH) using as a coenzyme, the first substrate is a substance that undergoes an enzymatic reaction by the first enzyme; the second coenzyme is NADH, The second enzyme uses NADH as a coenzyme to convert NAD ＋ is an enzyme that produces the second substrate is a substance that undergoes an enzymatic reaction by the second enzyme; The first mesh contains the first enzyme and NADH, which acts as a coenzyme to produce NAD. ＋ An additional enzyme that produces The first mesh is a NAD ＋ and infiltrating the substrate with a solution containing an additional substrate which is a substrate for the additional enzyme; the first excitation light has a wavelength that excites the first reactant generated from the additional substrate in an enzyme reaction by the additional enzyme, the second coenzyme is NADH, The second enzyme uses NADH as a coenzyme to produce NAD ＋ is an enzyme that produces the second substrate is a substance that undergoes an enzymatic reaction by the second enzyme; The second excitation light has a wavelength that excites NADH. The biological gas measuring device according to claim 5.

7. the first substrate is ethanol; the first enzyme is alcohol dehydrogenase (ADH); the second substrate is acetaldehyde; the second enzyme is ADH, the additional enzyme is diaphorase; said further substrate being resazurin; The first reactant is resorufin. The biological gas measuring device according to claim 6.

8. 8. The biological gas measuring device according to claim 5, wherein the first mesh and the second mesh are overlapped along a flow direction of the sample gas.

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