External ear biogas measuring device

Abstract

A biogas measurement device comprising: a biogas collector that is provided with an opening provided on a side thereof facing an external ear, a recessed part connected from the opening as a space for collecting a biogas released directly from the external ear, and a close contact part for separating the recessed part and the outside, the biogas collector thereby being mounted in close contact to the external ear; and a measurement instrument for measuring an object substance in a biogas that is derived from the external ear and is collected by the biogas collector, wherein outside air is actively not introduced into the recessed part.

Description

[Technical Field]

[0001] The present invention relates to a measuring device for measuring a target substance contained in a biological gas derived from the outer ear. [Background technology]

[0002] WO 2019 / 103130 A1 (paragraph

[0063] , Figure 18) discloses a biogas measuring device that can continuously collect biogas and measure target substances in the collected biogas both immediately and over time, and a biogas collector that can be used therewith. In particular, the document discloses a biogas measuring device in which a biogas collector is attached to a tight-fitting device shaped like an earphone that is attached to the ear canal.

[0003] This biological gas measuring device measures the amount of a target substance, such as ethanol, contained in skin gas released from the ear canal. The biological gas measuring device is provided with an inlet and an outlet. The inlet is connected to an inlet, and the outlet is connected to a carrier gas supply means. The outlet, through which the skin gas flows, is connected to a measuring instrument via a moisture sensor. Summary of the Invention [Problem to be solved by the invention]

[0004] When measuring target substances (e.g., ethanol) in skin gases by attaching a biological gas measuring device to the outer ear, there is a concern that the infusion of carrier gas into the outer ear may damage the eardrum, so attention must be paid to the flow rate and velocity of the carrier gas as well as the duration of use.

[0005] The objective of each aspect of the present application is to provide an outer ear skin gas measuring device that measures target substances from skin gas generated from the outer ear and that does not involve concerns about damage to the eardrum when supplying carrier gas. [Means for solving the problem]

[0006] (1) First aspect The first aspect of the present application is a biological gas measuring device for the outer ear, which comprises a biological gas collector that is attached in close contact with the outer ear and has an opening on the side facing the outer ear, a recessed portion connected to the opening as a space for collecting biological gases directly released from the outer ear, and a contact portion that separates the recessed portion from the outside, and a measuring instrument that measures target substances in biological gases derived from the outer ear and collected by the biological gas collector, and is characterized in that outside air is not actively introduced into the recessed portion.

[0007] The "biogas collector" may have any specific shape as long as it has an opening, a recess, and a contact portion. Specific shapes include, for example, an earphone type or a headphone type.

[0008] Here, "not actively introducing outside air into the recessed portion" means that outside air is not sent into the interior of the biogas collector using an air pump or the like. For example, the biogas collector may be configured so that the recessed portion is completely isolated from the outside, preventing any outside air from entering the recessed portion. However, a structure that allows outside air to passively enter, such as an opening communicating with the outside, may be provided. "Passive inflow of outside air" here means that outside air is not forced into the recessed portion through an opening communicating with the outside by any physical means (e.g., a pump), but outside air is allowed to flow into the recessed portion through this opening due to, for example, a pressure difference between the inside and outside or convection.

[0009] The term "measuring instrument" refers to any type of means capable of measuring the target substance in the biological gas, such as a biosensor that utilizes the enzyme reaction of the target substance or a semiconductor sensor that utilizes the antigen-antibody reaction of the target substance.

[0010] The "adhering means" can be of any type as long as it can seal the opening against the outer ear (specifically, the ear canal or the auricle, or both) and isolate the recess from the outside. For example, by attaching an elastic material to the edge of the opening and allowing it to be elastically deformed to adhere to the body, the opening can be directly attached to the outer ear.

[0011] With the above configuration, in this aspect, outside air is not actively introduced into the recessed portion, so concerns about damage to the eardrum that may accompany the active introduction of outside air are eliminated.

[0012] Since the amount of moisture contained in the biological gas released from the skin of the outer ear is extremely small compared to the amount of moisture contained in the biological gas released from other parts of the skin, in this embodiment, a moisture sensor and a correction means for correcting the measurement results based on the moisture detected by the moisture sensor are not required. However, it is acceptable to provide a moisture sensor and a correction means for more accurate measurements.

[0013] (2) Second aspect A second aspect of the present invention provides a biological gas measuring device for the outer ear that, in addition to the configuration of the first aspect, is sized to be inserted into the ear canal.

[0014] The external ear biogas measuring device of this embodiment has an earphone-type shape. In this embodiment, the biogas collector collects biogas emitted from the ear canal. The biogas collector is inserted into the ear canal like an earphone, with the outer periphery of the biogas collector tightly contacting the auricle by the contact portion and the recessed portion isolated from the outside.

[0015] (3) Third aspect The third aspect of the present invention is a biological gas measuring device for the outer ear, which is characterized in that, in addition to the configuration of the first aspect, the biological gas collector has a bowl-like shape large enough to cover the auricle, and the adhesive portion is provided on the edge of the bowl-like shape.

[0016] The external ear biogas measuring device of this embodiment has a headphone-like shape. In this embodiment, the biogas collector collects biogas emitted from the ear canal and the auricle. The biogas collector covers the auricle like headphones, with the outer periphery of the biogas collector tightly fitting around the auricle by the fitting portion, and the recessed portion is isolated from the outside.

[0017] (4) Fourth aspect A fourth aspect of the present invention is a biological gas measuring device for the external ear, which is characterized in that, in addition to the configuration of the second or third aspect, the biological gas collector has an outside air inlet which is an opening through which outside air is passively introduced, and a biological gas outlet which is connected to the measuring device via an outlet path, and an intake pump is connected to the biological gas outlet via the outlet path.

[0018] The "outside air inlet" is an opening that connects the internal space of the biogas collector to the outside. Outside air passively flows into the negatively pressurized recessed area, as described below. It is acceptable to provide a tube with an open end at this opening, or to provide a filter to block foreign matter from the outside.

[0019] The "biological gas outlet" is an opening through which the biological gas collected by the biological gas collector flows out from the recessed portion to the outside. This biological gas outlet is connected to a measuring instrument, for example, by a connecting pipe.

[0020] A suction pump is also connected to the biological gas outlet. This suction pump sucks the gas from the recess, and the gas reaches the measuring instrument. Furthermore, when the recess is under negative pressure, outside air passively flows into the recess through the outside air inlet.

[0021] With the above configuration, in this embodiment, outside air passively flows into the recessed portion, which has been made negative pressure by suction using the suction pump, from the outside air inlet, thereby avoiding damage to the eardrum that would otherwise be caused by actively introducing outside air.

[0022] When the biological gas released from the ear canal (in the second embodiment) or the ear canal and auricle (in the third embodiment) reaches the recess, it is sucked by the suction pump and flows from the biological gas outlet through the outlet path to the measuring instrument, where it is used to measure the target substance. The recess, which has become negative pressure due to the outflow of biological gas, is then filled with outside air through the outside air inlet. The flow rate of this outside air is just enough to eliminate the negative pressure in the recess, so damage caused by the outside air pressing against the eardrum can be avoided.

[0023] (5) Fifth aspect A fifth aspect of the present application's external ear biological gas measuring device has the same configuration as the fourth aspect, but is characterized in that the measuring device comprises a container that is attached so that at least a portion of it is adjacent to the outflow path and that contains a solution inside, an enzyme membrane that is attached to the container adjacent to the outflow path and is exposed to the biological gas in the outflow path, an irradiation unit that irradiates excitation light of a predetermined wavelength toward the solution in the container, and a light receiving unit that receives fluorescence, and an enzyme that catalyzes a chemical reaction of the target substance that involves a chemical change of a coenzyme contained in the solution is fixed to the enzyme membrane, and the coenzyme that has undergone the chemical change is excited by the excitation light and emits fluorescence.

[0024] The enzyme is selected depending on the target substance in the biogas. When primary alcohol dehydrogenase (ADH) is used as the enzyme, the target substance is ethanol (coenzyme: oxidized nicotinamide adenine dinucleotide (NAD) + )), acetaldehyde (coenzyme: reduced nicotinamide adenine dinucleotide (NADH)). When secondary alcohol dehydrogenase (S-ADH) is used as the enzyme, the target substances are acetone (coenzyme: NADH), 2-propanol (coenzyme: NAD + Furthermore, when aldehyde dehydrogenase (ALDH) is used as the enzyme, the target substance is acetaldehyde or 2-nonenal (coenzyme: NAD + ) and when formaldehyde dehydrogenase (FALDH) is used, the target substance is formaldehyde (coenzyme: NAD + )

[0025] According to this embodiment, the biological gas released from the ear canal (in the second embodiment) or the ear canal and auricle (in the third embodiment) flows through the recess into the outflow path and comes into contact with the enzyme membrane. The target substance contained in the biological gas undergoes a chemical reaction with the coenzyme by the enzyme immobilized on the enzyme membrane. The coenzyme that has undergone the chemical reaction is excited by the excitation light from the irradiation unit and emits fluorescence. By measuring this fluorescence with the light receiving unit, the target substance in the biological gas can be quantitatively measured.

[0026] (6) Sixth Aspect The sixth aspect of the biological gas measuring device for the external ear of the present invention is characterized in that, in addition to the features of the third aspect, the measuring device is built into the bowl-shaped interior, and air communication between the recess and the outside is blocked.

[0027] In other words, in this embodiment, the measuring device is built into the biological gas collector, and biological gas that reaches the recessed portion does not leak out to the outside. Therefore, there is no concern that the inflow of gas will damage the eardrum.

[0028] (7) Seventh aspect A seventh aspect of the external ear biological gas measuring device of the present invention has, in addition to the features of the sixth aspect, the measuring instrument comprises an enzyme membrane that faces the auricle in the recess and is exposed to biological gas, an irradiation unit that irradiates excitation light of a predetermined wavelength toward the enzyme membrane, and a light receiving unit that receives fluorescence, wherein a coenzyme and an enzyme that catalyzes a chemical reaction of the target substance that involves a chemical change of the coenzyme are fixed to the enzyme membrane, and the coenzyme that has undergone the chemical change is excited by the excitation light and emits fluorescence.

[0029] The "enzyme" and "coenzyme" in this embodiment are the same as those in the fifth embodiment described above.

[0030] According to this aspect, the biological gas released from the ear canal and the auricle comes into contact with the enzyme membrane at the recess. The target substance contained in the biological gas undergoes a chemical reaction with the enzyme immobilized on the enzyme membrane together with a coenzyme also immobilized on the enzyme membrane. The coenzyme undergoing the chemical reaction is excited by the excitation light from the irradiation unit and emits fluorescence. The fluorescence is measured by the light receiving unit, allowing the target substance in the biological gas to be quantitatively measured.

[0031] In this embodiment, the enzyme reaction occurs cumulatively in the enzyme membrane, which is isolated from the outside, and therefore the measured value of fluorescence measured by the light-receiving unit increases cumulatively within the quantitative constraints of the immobilized enzyme and coenzyme. By differentiating this cumulatively increasing measured value with respect to time, the change in fluorescence over time can be obtained.

[0032] (8) Eighth aspect An eighth aspect of the present invention is an external ear biological gas measuring device characterized in that, in addition to the features of any one of the first to seventh aspects, the biological gas collector is provided for each ear.

[0033] That is, the biogas measuring device of this embodiment includes a pair of biogas collectors, one for each ear. The pair of biogas collectors can be used, for example, to measure different target substances on the left and right. One biogas collector can be used for an enzyme reaction, and the other can be used for a control measurement in which no enzyme reaction is performed. Furthermore, one biogas collector can be used for an enzyme reaction, and the other can be connected to a moisture sensor for the purpose of correcting the moisture content of the biogas. [Effects of the Invention]

[0034] According to the present invention, it is possible to provide a skin gas measuring device for the outer ear that measures target substances from skin gas generated from the outer ear and that does not involve concerns about damage to the eardrum when supplying carrier gas. [Brief explanation of the drawings]

[0035] [Figure 1]1 is a schematic diagram showing a first embodiment of a biological gas measuring device for an external ear according to the present invention. [Figure 2] 2 is an enlarged view of a main part of the external ear biological gas measuring device shown in FIG. 1. [Figure 3] 1 is a schematic diagram showing the appearance of a first example of the first embodiment. [Figure 4] 1 is a schematic cross-sectional view of a first example of the first embodiment. [Figure 5] 10 is a schematic diagram showing the appearance of a second example of the first embodiment. [Figure 6] 10 is a schematic cross-sectional view of a second example of the first embodiment. [Figure 7] 10 is a graph showing the change over time in the ethanol concentration in biological gas measured by the second example of the external ear biological gas measuring device. [Figure 8] A modification of the first example is shown. [Figure 9] A modified example of the second example is shown. [Figure 10] 1 is a schematic view showing the appearance of a second embodiment of a biological gas measuring device for the external ear according to the present invention. [Figure 11] 10 is a schematic cross-sectional view of a biological gas measuring device for an external ear according to a second embodiment. [Figure 12] 10 is a graph showing an example of the change over time in the total concentration of a target substance in a biological gas measured by the external ear biological gas measuring device of the second embodiment. [Figure 13] 13 is a graph obtained by differentiating the graph of FIG. 12 with respect to time. [Figure 14] 10 is a graph showing an example of the change over time in cumulative fluorescence intensity at the enzyme film in the external ear biological gas measuring device of the second embodiment. [Figure 15] 15 is a graph obtained by integrating the graph of FIG. 14 with respect to time. [Figure 16] 1 is a schematic diagram showing the experimental apparatus used in Example 1. [Figure 17] 1 is a graph showing the change over time in cumulative fluorescence intensity at the enzyme membrane for each ethanol concentration in the measurement gas. [Figure 18] The graph in FIG. 17 is shown in an enlarged form. [Figure 19]18 is a graph obtained by integrating each graph in FIG. 17 with respect to time. [Figure 20] The graph in FIG. 19 is shown in an enlarged form. [Figure 21] 1 is a graph showing the relationship between ethanol concentration and integrated cumulative fluorescence intensity for each measurement time. [Figure 22] 22 is a graph showing the coefficient of variation of cumulative fluorescence intensity for 11 ppb of ethanol at each time point in the graph of FIG. 21. [Figure 23] 10 is a graph showing an example of the results of a 4-minute measurement of the ethanol concentration in ear canal gas. [Figure 24] 24 is a graph showing an example of determining the ethanol concentration from the measurement results of FIG. 23. [Figure 25] 1 is a graph showing the results of measuring ethanol concentrations from ear canal gas (dashed line, circles) and exhaled breath (solid line, triangles) by actual subjects. [Figure 26] 1 is a graph showing the change over time in cumulative fluorescence intensity at the enzyme membrane for each ethanol concentration in the measurement gas. [Figure 27] 27 is a graph obtained by differentiating the graph of FIG. 26 with respect to time. [Figure 28] 28 is a graph showing the relationship between the peak values ​​(broken lines, squares) in each graph of FIG. 27 and the steady-state values ​​(solid lines, circles) of the fluorescence intensity in the graph of FIG. 26 and the ethanol concentration. DETAILED DESCRIPTION OF THE INVENTION

[0036] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. Hereinafter, "inside" refers to the side closest to the body when the biological gas collector 10 is attached to the outer ear, and "outside" refers to the opposite side. Note that common symbols in the drawings referred to in the following description indicate common components, and explanations of the components may be omitted in the descriptions of the drawings.

[0037] (1) First embodiment (1-1) Schematic configuration FIG. 1 is a diagram showing a schematic configuration of a first embodiment of a biological gas measuring device 1 for an external ear according to the present invention.

[0038] The external ear biological gas measuring device 1 of this embodiment comprises a biological gas collector 10 that is attached to the external ear and collects skin gas as biological gas derived from the external ear, a measuring instrument 100 that measures target substances in the skin gas collected by the biological gas collector 10, and an outflow path 40 through which the collected skin gas flows from the biological gas collector 10 to the measuring instrument 100.

[0039] In this embodiment, ethanol is the target substance in skin gas. The measurement of ethanol as the target substance is carried out by converting the substrate ethanol into acetaldehyde by alcohol dehydrogenase, which is accompanied by the coenzyme NAD + This utilizes the reaction in which the coenzyme NADH is produced from NADH. Specifically, the coenzyme NADH produced in the above reaction 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. Details of this will be described later.

[0040] (1-2) Measuring Instruments 100 The measuring instrument 100 has an optical fiber probe 113 which is a combination of an irradiation unit 111 made up of an optical fiber for inputting light and a light receiving unit 112 made up of an optical fiber for receiving light. Such an optical fiber probe 113 may be a commercially available one, 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.) or the like.

[0041] An ultraviolet light emitting diode 114 is connected to the irradiation unit 111 for emitting ultraviolet light, which is excitation light of a predetermined wavelength according to the excitation phenomenon to be used. A bandpass filter 116 is connected between the ultraviolet light emitting diode 114 and the optical fiber probe 113. In this embodiment, the ultraviolet light emitting diode 114 can be used as the light source, and the device can be simplified and manufactured more inexpensively than when a mercury lamp is used as the light source. In addition, it can be used as a portable device.

[0042] As described above, this embodiment utilizes the property of NADH to absorb ultraviolet light of 340 nm, and therefore, the ultraviolet light-emitting diode 114 used is one that excites ultraviolet light with a wavelength of 300 to 370 nm, preferably around 340 nm. Therefore, as shown in FIG. 1, it is preferable to connect a bandpass filter 116 between the ultraviolet light-emitting diode 114 and the optical fiber probe 113. The bandpass filter 116 refers to a filter that transmits only light of a specific wavelength from the light source. In this embodiment, for example, a bandpass filter that transmits ultraviolet light of 330 to 350 nm as incident light is used. Any commercially available bandpass filter 116 can be used without any particular restrictions as this type of bandpass filter 116.

[0043] In the external ear biogas measuring device 1 shown in FIG. 1, the light receiving unit 112 receives fluorescence generated by excitation with excitation light irradiated from an ultraviolet light-emitting diode 114. The light receiving unit 112 is connected to a detection unit 120 that detects the received fluorescence. Specifically, the detection unit 120 is a photodetector, such as a photomultiplier tube or a photodiode detector. 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, approximately 491 nm. Therefore, as shown in FIG. 1, it is preferable to provide a long-wavelength transmission filter 118 that transmits only fluorescence with wavelengths longer than the specific wavelength. In FIG. 1, a long-wavelength transmission filter 118 that transmits fluorescence with wavelengths of 400 nm or longer is used. Such a long-wavelength transmission filter 118 can be any commercially available filter without particular limitations. Although the biogas measuring device shown in FIG. 1 uses the long-wavelength transmission filter 118, a band-pass filter may be used instead. In this case, for example, a bandpass filter that transmits only fluorescence with a wavelength of 450 to 510 nm is used.

[0044] In the external ear biological gas measuring device 1 shown in Figure 1, a computer system 122 may be further connected as a visualization unit for analyzing the data detected by the detection unit 120 and visualizing the spatial distribution information of the concentration of ethanol, which is the target substance of this embodiment.Connecting such a computer system 122 makes it easier to analyze the data.

[0045] Next, the optical fiber probe 113 used in the external ear biogas measuring device 1 shown in FIG. 1 will be described. As shown in FIG. 2, the optical fiber probe 113 has a housing 140 attached to its tip as a gas-liquid diaphragm flow cell equipped with an enzyme membrane 144 on which an enzyme is immobilized. The enzyme membrane 144, which will be described later, is attached to the tip, which is one end of the housing 140. As shown in FIG. 1, the housing 140 may include a silicon tube 141 and a PMMA (polymethyl methacrylate) pipe 142 as components. Furthermore, as shown in FIG. 1, the enzyme membrane 144 may be fixed to the PMMA pipe 142 by an O-ring 143. A buffer solution is contained in a reaction section 145, which is the internal space of the housing 140. This buffer solution contains NAD as the coenzyme. + 1, is supplied to the reaction unit 145 via a buffer solution flow path 155 from a buffer solution reservoir 150, and circulates back to the buffer solution reservoir 150 via the buffer solution flow path 155. Meanwhile, an optical fiber probe 113, which combines an irradiation unit 111 and a light receiving unit 112, is inserted from the rear end, which is the other end of the container 140, and its tip reaches the reaction unit 145 and contacts the buffer solution, irradiating the excitation light in the buffer solution and receiving the fluorescence. As shown in FIG. 1, the tip, which is one end of the container 140, is attached adjacent to an outlet channel 40 through which a carrier gas (described below) containing skin gas captured by the biological gas collector 10 flows out. An enzyme membrane 144 attached to the tip of the container 140, which is adjacent to the tip, is exposed to the carrier gas. In other words, by attaching the tip, which is a part of the container 140, so that it is inserted into the outlet channel 40, the carrier gas and the buffer solution are separated by the enzyme membrane 144.

[0046] Next, the enzyme membrane 144 will be described. The enzyme membrane 144 refers to a membrane in which an enzyme is immobilized on a carrier, which is a membrane material. This enzyme catalyzes a chemical reaction of the target substance, which involves a chemical change of a coenzyme contained in the solution. Materials conventionally used for immobilizing enzymes can be used as the carrier without particular limitation. Examples of such materials include resins such as polytetrafluoroethylene, polydimethylsiloxane, polypropylene, polyethylene, polymethyl methacrylate, and polystyrene, and fibers such as cotton. The thickness of such a carrier is not particularly limited, but is preferably 100 nm to 200 μm, and more preferably 10 μm to 100 μm. A method for producing a membrane on which an enzyme is immobilized that can be used in the biogas measuring device of the present invention is described, for example, in JP 2009-168671 A, and materials that can be used as carriers are described in JP 2016-220573 A, the contents of which are incorporated herein by reference.

[0047] The external ear biogas measuring device 1 of this embodiment quantifies the concentration of NADH by the fluorescence generated when the coenzyme (specifically, NADH) undergoes the chemical change as excited by the excitation light incident from the ultraviolet light-emitting diode 114, and detects ethanol, which is a substrate of alcohol dehydrogenase that uses NADH as a coenzyme, using the NADH concentration as an indicator. Therefore, the substrate in the skin gas and the enzyme in the enzyme membrane 144 must react in the presence of the coenzyme. In the biogas measuring device shown in FIG. 1, the carrier gas containing skin gas flowing through the outflow channel 40 and the NADH flowing through the reaction section 145 react with each other. + By interposing the enzyme membrane 144 between the buffer solution containing NAD +The enzyme and the substrate can react in the presence of the carrier. Therefore, it is preferable that the carrier constituting the enzyme membrane 144 is porous. There are no particular restrictions on the size of the pores in the carrier as long as the enzyme reaction is possible, but the diameter is usually about 0.1 to 1 μm, and from the viewpoint of the efficiency of the enzyme reaction, it is preferably about 0.2 μm. Furthermore, it is preferable that the porosity of the carrier is 60 to 90%. Note that a mesh-like material can also be used as the carrier.

[0048] (1-3) Intake pump 160 In this embodiment, as shown in Fig. 1, an intake pump 160 that sucks skin gas from the biological gas collector 10 is provided downstream of the outlet channel 40. When the intake pump 160 sucks gas containing skin gas from the biological gas collector 10, air flows into the biological gas collector 10 from the outside air inlet 13, which serves as an opening. This inflowing air acts as a carrier gas and is mixed with the skin gas in the biological gas collector 10, and comes into contact with the enzyme membrane 144 at the tip of the container 140 in the outlet channel 40. At this time, when ethanol, which serves as an enzyme substrate contained in the skin gas, is oxidized by alcohol dehydrogenase, NAD contained in the buffer solution is oxidized. + is reduced, which emits fluorescence as described above, allowing the amount of ethanol in the skin gas to be measured. Such an intake pump 160 allows the skin gas to be continuously contacted with the enzyme membrane 144.

[0049] (1-4) Biogas collector 10 (1-4-1) Example 1 The biological gas collector 10 of the first example has an earphone-type appearance that fits inside the auricle P and is sized to be inserted into the ear canal C (see FIG. 4), as shown in FIG. 3. On the outside, an outside air inlet 13 and a biological gas outlet 14 are formed as openings. The biological gas outlet 14 is connected to the outflow path 40 described above.

[0050] As shown in the cross-sectional view of Figure 4, the biological gas collector 10 is composed of a cylindrical collector 15 with a bottom and a contact part 20 that covers the bottom side of the collector 15. An opening 11 facing the ear canal C is provided inside the collector 15, and a recessed part 12 is formed as an internal space connected to the opening 11. The recessed part 12 is a space that collects skin gas that is directly released from the skin of the ear canal C. The contact part 20 is made of a highly deformable material such as elastomer or silicone rubber, and is in close contact with the auricle P to separate the recessed part 12 from the outside. An outside air inlet 13 and a biological gas outlet 14 open through the contact part 20 on the outside of the collector 15, and the biological gas outlet 14 is connected to the outflow path as described above.

[0051] The outside air inlet 13 connects the recessed portion 12 to the outside. In the recessed portion 12, air flowing in from the outside through the outside air inlet 13 mixes with skin gas generated from the skin in the ear canal C. As described above, the skin gas mixed with the air is guided from the outlet 40 to the measuring instrument 100 shown in FIG. 1 by suction using the suction pump 160. At this time, negative pressure is created in the recessed portion 12, and external air flows into the recessed portion 12 through the outside air inlet 13. In other words, outside air is not actively introduced into the recessed portion 12 by a pump or the like, but is passively introduced by the negative pressure generated by the suction pump 160.

[0052] (1-4-2) Second example The biological gas collector 10 of the second example is a headphone-type device having a bowl-like shape large enough to cover the auricle P, as shown in Fig. 5. An outside air inlet 13 and a biological gas outlet 14 are formed on the surface as openings. The biological gas outlet 14 is connected to the outflow path 40 described above.

[0053] As shown in the cross-sectional view of Figure 6, the biological gas collector 10 is composed of a bowl-shaped collector 15 and a contact part 20 that covers the periphery of the collector 15. An opening 11 facing the ear canal C is provided inside the collector 15, and a recessed part 12 is formed as an internal space connected to the opening 11. The recessed part 12 is a space that collects skin gases that are released directly from the auricle P and the skin of the ear canal C. The contact part 20 is made of a highly deformable material such as elastomer or silicone rubber, and is in close contact with the periphery of the auricle P to separate the recessed part 12 from the outside. An outside air inlet 13 and a biological gas outlet 14 open through the contact part 20 on the outside of the collector 15, and the biological gas outlet 14 is connected to the outflow path as described above.

[0054] The outside air inlet 13 connects the recessed portion 12 to the outside. In the recessed portion 12, air flowing in from the outside through the outside air inlet 13 mixes with skin gas generated from the skin of the auricle P and the ear canal C. As described above, the skin gas mixed with the air is guided from the outlet 40 to the measuring instrument 100 shown in FIG. 1 by suction using the suction pump 160. At this time, negative pressure is created in the recessed portion 12, and outside air flows into the recessed portion 12 through the outside air inlet 13. In other words, outside air is not actively introduced into the recessed portion 12 by a pump or the like, but is passively introduced by the negative pressure generated by the suction pump 160.

[0055] (1-5) Measurement using measuring instrument 100 As described above, the mixed gas of the skin gas and the carrier gas contacts the tip of the container 140 in the outlet channel 40. The tip of the container 140 is fitted with an enzyme membrane 144 on which alcohol dehydrogenase is immobilized, and in a reaction section 145 sandwiching the enzyme membrane 144, NAD, a coenzyme of alcohol dehydrogenase, is reacted with the enzyme membrane 144. + The enzyme membrane 144 is wetted with the buffer solution, and if the skin gas in contact with this enzyme membrane 144 contains ethanol as a substrate, the ethanol is converted to acetaldehyde by alcohol dehydrogenase. +On the other hand, in the buffer solution of the reaction section 145, excitation light with a wavelength of 340 nm is constantly irradiated from the irradiation section 111, but NAD + The NADH converted from absorbs the excitation light, becomes excited, and emits fluorescence with a wavelength of 491 nm. This fluorescence is received by the light receiving unit 112 and is quantified by the detection unit 120 as, for example, fluorescence intensity.

[0056] In this measurement, it is desirable to measure gases with known ethanol concentrations in advance and obtain a calibration curve showing the correlation with the fluorescence intensity.

[0057] Figure 7 shows the ethanol concentration over time in the skin gas (left vertical axis) and exhaled breath (right vertical axis) of a seated subject wearing the second example biogas collector 10 on the auricle of the subject, who was then inhaled 0.4 g of ethanol per kg of body weight within 5 minutes. The curved line in Figure 7 indicates the ethanol concentration (ppb) in the skin gas. The bar graph indicates the ethanol concentration (ppm) measured from the exhaled breath collected by a mask-shaped biogas collector worn over the subject's nose and mouth using a measuring device 100 similar to that shown in Figure 1. The triangle symbol indicates the ethanol concentration (ppm) in the exhaled breath measured using an alcohol detection tube. The air suction rate by the intake pump 160 was 200 ml / min.

[0058] As a result, the ethanol concentration in breath peaked 40 minutes after ingestion using both measurement methods, while the ethanol concentration in skin gas peaked 60 minutes after ingestion. Therefore, although the ethanol concentration in outer ear skin gas was about 20 minutes behind that in breath, the patterns of rise and fall were highly correlated, indicating that ethanol detection from outer ear skin gas is as effective as that from breath.

[0059] In this way, in the first embodiment of the external ear biological gas measuring device 1 of the present invention, skin gas comes into contact with the enzyme membrane 144 moistened with a buffer solution, and ethanol is measured. Here, since there are almost no sweat glands in the auricle P and the ear canal C, the spikes in output caused by the release of skin gas components from the sweat glands due to sweating are not a problem. + It is also possible to detect other substrates using other dehydrogenases that utilize NAD. + It is also possible to detect other substrates by enzyme reactions using other coenzymes other than ethanol. In other words, enzymes and coenzymes can be used depending on the substrate to be detected, which is not limited to ethanol, and is a target substance in biogas.

[0060] (1-6) Variations In a modified example of the first example shown in FIG. 8, a pair of earphone-type biogas collectors 10 are provided for each ear. Similarly, in a modified example of the second example shown in FIG. 9, a pair of headphone-type biogas collectors 10 are provided for each ear, and these are connected by a headband 16. With an external ear biogas measuring device 1 equipped with a pair of biogas collectors 10 for each ear, it is possible to simultaneously measure different target substances in the left and right ears, such as ethanol concentration in the right ear and acetaldehyde concentration in the left ear. Furthermore, by configuring one biogas collector 10 to introduce skin gas into a measuring instrument 100 with enzymes and the other biogas collector 10 to introduce skin gas into a measuring instrument 100 without enzymes, it is possible to measure environmental noise with the other biogas collector 10 and use that noise to correct the measurement results of the target substance in the skin gas of one biogas collector 10. Furthermore, it is possible to measure the target substance using skin gas from one biological gas collector 10 while measuring moisture using skin gas from the other biological gas collector 10, and correct the measurement results of the target substance using the measured moisture.

[0061] In a headphone-type biological gas collector 10 as shown in FIG. 9, a power source and a miniaturized measuring device 100 may be attached to the headband 16.

[0062] (2) Second embodiment 10 is a diagram showing a schematic view of the external appearance of a second embodiment of the external ear biogas measuring device 1 of the present invention. In this embodiment, similar to the external ear biogas measuring device 1 of the first embodiment, the biogas collector 10 is formed in the form of headphones that cover the auricle. Furthermore, a pair of biogas collectors 10 are provided, one for each ear, and these are connected by a headband 16. A power supply 17 and a circuit 18 for operating and controlling the measuring device 100, which will be described later, are attached to this headband 16.

[0063] As shown in the cross-sectional view of Figure 11, the biological gas collector 10 of this embodiment is composed of a bowl-shaped collector 15 and a contact part 20 that covers the periphery of the collector 15. An opening 11 facing the ear canal C is provided inside the collector 15, and a recessed part 12 is formed as an internal space connected to the opening 11. The recessed part 12 is a space that collects skin gas that is released directly from the auricle P and the skin of the ear canal C. The contact part 20 is made of a highly deformable material such as elastomer or silicone rubber, and contacts the periphery of the auricle P to separate the recessed part 12 from the outside.

[0064] In this embodiment, an enzyme membrane 144 is attached to the recess 12. An irradiation unit 111 that irradiates the enzyme membrane 144 with excitation light and a light receiving unit 112 that receives fluorescence emitted from the enzyme membrane 144 are attached to the inner surface of the collector 15. The irradiation unit 111, the light receiving unit 112, and the enzyme membrane 144 constitute the measuring device 100. That is, in this embodiment, the measuring device 100 is built into the inside of the collector 15, which has a bowl-shaped structure, and air communication between the recess 12 and the outside air is blocked. Note that a small gap is generated between the contact portion 20 and the periphery of the auricle P, and it is acceptable for a small amount of outside air to flow into the recess 12 through this gap.

[0065] The material of the enzyme membrane 144 is the same as that of the first embodiment. As in the first embodiment, alcohol dehydrogenase as an enzyme is immobilized on the enzyme membrane 144. In this embodiment, NAD as a coenzyme of alcohol dehydrogenase is further immobilized on the enzyme membrane 144. + is also fixed.

[0066] The enzyme membrane 144 is attached to the recess 12 in a state where it is wetted with a buffer solution. If the skin gas that comes into contact with this enzyme membrane 144 contains ethanol as a substrate, the ethanol is converted into acetaldehyde by alcohol dehydrogenase. At this time, the coenzyme NAD + On the other hand, in the buffer solution of the reaction section 145, excitation light with a wavelength of 340 nm is constantly irradiated from the irradiation section 111, but NAD + The NADH converted from absorbs the excitation light, becomes excited, and emits fluorescence with a wavelength of 491 nm. The fluorescence is received by the light-receiving unit 112, and the obtained data is transmitted to the external detection unit 120 via a wireless transmitter (not shown). The data is then processed by the computer system 122, and is quantified, for example, as fluorescence intensity. The light-receiving unit 112 and detection unit 120 may be connected by wire.

[0067] In this embodiment, there is no flow of outside air into or out of the recess 12. Therefore, skin gases released from the auricle P and the ear canal C accumulate in the recess 12. Therefore, NADH produced as a result of the enzyme reaction of the enzyme membrane 144 with ethanol in the skin gas accumulates over time, and the fluorescence emitted by excited NADH continues to increase over time, as shown in the graph in Figure 12. By differentiating this graph with respect to time, a graph of the increase or decrease in ethanol concentration over time is obtained, as shown in Figure 13.

[0068] Since the reaction in the enzyme membrane accumulates over time, the cumulative fluorescence intensity shown on the vertical axis is expressed as a graph that monotonically increases over time, as in Figure 14. Then, by integrating this graph over time to create a graph like Figure 15 (i.e., the area of ​​the graph in Figure 14 up to a specific time point), for each gas with a known substrate concentration, it is possible to measure the concentration of the substrate (e.g., ethanol) in the skin gas by using a calibration curve in which the fluorescence intensity values ​​are plotted at a specific time point (e.g., 4 minutes).

[0069] In addition to ethanol, the external ear biological gas measuring device 1 of the present disclosure can also measure acetone and other target substances. In the case of acetone, it is excreted from the body as skin gas via the blood, and measuring its concentration makes it possible to evaluate the fat burning status, the progression of diabetes, and the like. Furthermore, it is possible to measure nonenal, a substance that causes body odor associated with aging, and evaluate changes in metabolic function associated with aging. In addition, by selecting an enzyme and a coenzyme and accordingly selecting the wavelength of the excitation light from the irradiating unit 111 and the wavelength of the fluorescence incident on the light receiving unit 112, it is also possible to measure other target substances that may be contained in skin gas. [Example]

[0070] (1) Example 1 (1-1) Experimental equipment In Example 1, an ethanol-containing gas simulating skin gas from the ear canal (hereinafter referred to as "ear canal gas") was measured using an experimental device shown in Fig. 16. The experimental device shown in Fig. 16 uses the biological gas collector 10 of Fig. 11, and a funnel 170 is attached to the opening 11. The cross-sectional area of ​​the maximum diameter part of the funnel 170 is 1385 mm 2 The cross-sectional area of ​​the smallest diameter is 13 mm 2The funnel 170 has a smallest diameter part to which an air vent 180 of the same diameter is connected. The irradiation unit 111 incorporates an ultraviolet LED drive circuit, and the enzyme membrane 144 is irradiated with excitation light of 340 nm wavelength. The light receiving unit 112 is composed of a wireless CMOS camera capable of receiving 491 nm fluorescence emitted by the enzyme membrane 144. Data on the fluorescence received by the light receiving unit 112 is wirelessly transmitted to an external tablet terminal equipped with the light receiving unit 112 and the detection unit 120, where the fluorescence intensity is measured.

[0071] The enzyme membrane 144 was prepared as follows: First, 1 cm of the enzyme membrane was placed in an ethanol solution containing 15% PMEH (a copolymer of MPC (2-methacryloxyethylphosphorylcholine) and EHMA (2-ethylhexyl methacrylate)). 2 A polymer solution was prepared by adding ADH so that the concentration was 60.1 U per unit volume. This polymer solution was coated on the surface of a hydrophilic polytetrafluoroethylene (OMNIPORE MEMBRANE FILTER, MILLIPORE) fiber cut into 2 cm squares, and the fiber was left to dry for 180 minutes at 4°C. Immediately before measurement, 500 μM NAD + 50 μL of the solution was added dropwise and allowed to penetrate.

[0072] (1-2) Measurement conditions After setting this enzyme membrane 144 in the biological gas collector 10, air containing a predetermined concentration of ethanol (hereinafter referred to as "measurement gas") was supplied to the biological gas collector 10 from the ventilation pipe 180 through the funnel 170. The flow rate of the measurement gas in the ventilation pipe 180 was 100 mL / min / cm. 2 The flow rate of the measurement gas when it is supplied from the funnel 170 to the opening 11 is 1 mL / min / cm 2 The ethanol concentrations in the measurement gas were 11 ppb, 50 ppb, 224 ppb, 1 ppm, 5 ppm, 25.7 ppm, 129 ppm, 222 ppm, and 444 ppm.

[0073] The results of measurements taken under the above conditions for 5 minutes are shown in the graph of Figure 17. Furthermore, the graph of Figure 18 shows an enlarged view of the area below the dashed line in the graph of Figure 17. For the measurement gases of all ethanol concentrations, the NAD + Because the responses are cumulative, the graph generally shows a monotonically increasing trend.

[0074] The graph in Figure 19 shows the results of integrating each graph in Figure 17 with respect to time. Furthermore, the graph in Figure 20 shows an enlarged view of the area below the dashed line in Figure 19. This integration process resulted in each graph in Figure 17 drawing a smoother curve. It was also inferred that the degree of increase in each graph depends on the ethanol concentration.

[0075] Therefore, Figure 21 plots the relationship between ethanol concentration and integrated cumulative fluorescence intensity at measurement times of 1, 2, 3, 4, and 5 minutes from the graph in Figure 19. Note that each point in Figure 21 is the average of three measurements, and the upper and lower points indicate the standard deviation. It was found that the graphs at each time point all drew curves with similar shapes.

[0076] The graph in Figure 22 shows the coefficient of variation of the cumulative fluorescence intensity for 11 ppb of ethanol at each time point in the graph in Figure 21. As shown in this graph, the coefficient of variation was smallest at a measurement time of 4 minutes, which was thought to be the most stable measurement condition with little variance in the measurements. Therefore, the measurement time was set at 4 minutes.

[0077] Based on the above, the ethanol concentration in ear canal gas is measured using the following procedure. For example, if the measurement results for 4 minutes are as shown in the graph in Figure 23, the area of ​​the shaded portion in the figure can be calculated as the integral value by integrating over 4 minutes. If this integral value is 0.2 x 10 10 If the graph shown in FIG. 24, which is the graph for 4 minutes in FIG. 21, is used as the calibration curve, the ethanol concentration corresponding to this integral value is 10 4 It is calculated in ppb, or 10 ppm.

[0078] (1-3) Actual measurement The results of measurements taken under the above conditions by actual subjects are shown below. The subjects were two people, as shown in Table 1 below. The "ALDH2" column in the table indicates whether each subject has type 2 aldehyde dehydrogenase (+) or not (-).

[0079] [Table 1]

[0080] After confirming that each subject had not taken any medication or consumed alcohol within the 72 hours prior to the test, they ingested 0.4g of alcohol per kg of body weight within 5 minutes. Immediately after ingestion, they were fitted with the external ear biogas measuring device shown in Figure 11, and external ear canal gas was measured for 4 minutes. This measurement was repeated every 20 minutes up to 120 minutes, starting immediately after ingestion as 0 minutes.

[0081] In parallel with the measurement of ear canal gas, the ethanol concentration in the exhaled breath was also measured. Specifically, the subject was asked to take a deep breath and hold it for 10 seconds. Then, they were asked to exhale slowly for 3 seconds to remove dead space, and the end-tidal air was collected in a sample bag. The ethanol concentration was measured from this collected exhaled air according to the standard method.

[0082] The above results are shown in the graph in Figure 25. In Figure 25, the upper graph shows the results for subject A, and the lower graph shows the results for subject B. In both the upper and lower graphs, the dashed line shows the measurement results for ear canal gas, and the solid line shows the measurement results for exhaled breath. Furthermore, the vertical axis on the left shows the ethanol concentration in ear canal gas, and the vertical axis on the right shows the ethanol concentration in exhaled breath.

[0083] The graph in Figure 25 confirms that for all subjects, the peak of ethanol concentration in ear canal gas appeared later than the peak of ethanol concentration in exhaled breath. This is presumably because the barrier between the blood vessels and alveolar lumen in the alveoli is approximately 2.2 μm thick, while the eardrum separating the outer ear from the middle ear is 50 to 100 μm thick, which means that it takes longer for biogas to diffuse in the outer ear.

[0084] Furthermore, it was confirmed that the peak ethanol concentrations in ear canal gas and exhaled breath of subject A came earlier than those of subject B, and that the respective peak values ​​were lower in subject A than in subject B. This result indicates that subject A, who has ALDH2, metabolizes alcohol in his body more quickly than subject B. These results confirm that measuring ethanol concentration using ear canal gas accurately reflects the condition of the subject, and that it is a useful and practical method for measuring actual ethanol concentration.

[0085] (2) Example 2 (2-1) Experimental equipment In Example 2, an ethanol-containing gas simulating external ear canal gas was measured using the same experimental equipment as in Example 1. However, the enzyme membrane 144 was prepared as follows. A cotton mesh with an opening of approximately 1 mm was cut into a 2 cm square. 2 An ADH solution was added dropwise to the membrane so that the concentration was 60.1 U per unit volume, and the membrane was left to dry for 60 minutes at 4°C. Next, a 2.5 v / v% glutaraldehyde solution was added dropwise, and the membrane was left to dry for 90 minutes at 4°C. After that, the membrane was washed with Tris-HCl solution to remove excess enzyme. Immediately before measurement, 10 mM NAD + 80 μL of the solution was dropped and allowed to penetrate. The enzyme membrane 144 used in this example had higher breathability than the enzyme membrane 144 used in Example 1.

[0086] (2-2) Measurement conditions After this enzyme membrane 144 was set in the biological gas collector 10, the same measurement gas as in Example 1 was supplied to the biological gas collector 10 from the ventilation pipe 180 through the funnel 170. The flow rate of the measurement gas in the ventilation pipe 180 was 100 mL / min / cm. 2 The flow rate of the measurement gas when it is supplied from the funnel 170 to the opening 11 is 1 mL / min / cm 2 The ethanol concentrations in the measurement gas were 10 ppb, 50 ppb, 100 ppb, 500 ppb, 1 ppm, 3 ppm, and 10 ppm.

[0087] The results of measurements under the above conditions are shown in the graph in Figure 26. For all ethanol concentrations, the fluorescence intensity showed a monotonically increasing trend from approximately 60 seconds to approximately 180 seconds after the start of measurement, after which it plateaued. Figure 27 shows the fluorescence intensity curves in Figure 26 differentiated with respect to time. The vertical axis in Figure 27 represents the slope of the fluorescence intensity curve in Figure 26 at each time point. From Figure 27, it can be inferred that the maximum value of the slope of the fluorescence intensity curve (i.e., the peak value of each graph) depends on the ethanol concentration in the measurement gas.

[0088] Figure 28 plots the logarithm of the peak values ​​of each graph in Figure 27 and the logarithm of the fluorescence intensity at the peak in Figure 26 against the logarithm of the ethanol concentration. The right-hand vertical axis represents the logarithm of the peak value (dashed line), and the left-hand vertical axis represents the logarithm of the fluorescence intensity (solid line). Figure 28 shows that the peak value, i.e., the logarithm of the maximum slope of the fluorescence intensity curve, has a linear relationship with the logarithm of the ethanol concentration. Furthermore, the logarithm of the fluorescence intensity at the maximum slope also has a linear relationship with the logarithm of the ethanol concentration. Therefore, by using the graph in Figure 28 as a calibration curve and applying the peak values ​​obtained by differentiating the graph measuring the ethanol concentration in ear canal gas with respect to time to this calibration curve, it was inferred that ethanol concentrations in the range of 10 ppb to 10 ppm can be measured. [Industrial Applicability]

[0089] The present invention can be used as a biological gas measuring device for the outer ear.

Claims

1. A biological gas collector that is attached to the outer ear in close contact with the outer ear, the biological gas collector having an opening provided on the side facing the outer ear, a recessed portion connected to the opening as a space for collecting biological gases directly emitted from the outer ear, and a contact portion separating the recessed portion from the outside, and a measuring instrument for measuring a target substance in the biological gas derived from the outer ear collected by the biological gas collector; A biological gas measuring device comprising: The biological gas collector is sized to be inserted into the ear canal, The biological gas collector has an outside air inlet, which is an opening through which outside air is passively introduced, and a biological gas outlet connected to the measuring device via an outlet path, and an intake pump is connected to the biological gas outlet via the outlet path.

2. A biological gas collector that is attached in close contact with the outer ear, comprising an opening provided on the side facing the outer ear, a recessed portion connected to the opening as a space for collecting biological gases that are directly emitted from the outer ear, and a contact portion that separates the recessed portion from the outside, and a measuring instrument for measuring a target substance in the biological gas derived from the outer ear collected by the biological gas collector; A biological gas measuring device comprising: The biological gas collector has a bowl-like shape large enough to cover the auricle, The contact portion is provided on a periphery of the bowl-shaped shape, The biological gas collector has an outside air inlet, which is an opening through which outside air is passively introduced, and a biological gas outlet connected to the measuring device via an outlet path, and an intake pump is connected to the biological gas outlet via the outlet path.

3. The measuring instrument is a container attached to the outlet channel so that at least a portion of the container is adjacent to the outlet channel and contains a solution therein; an enzyme membrane attached to a portion of the container adjacent to the outflow passage and exposed to biological gas in the outflow passage; an irradiation unit that irradiates the solution in the container with excitation light of a predetermined wavelength; a light receiving unit that receives the fluorescent light, an enzyme that catalyzes a chemical reaction of the target substance accompanied by a chemical change of a coenzyme contained in the solution is immobilized on the enzyme membrane; 3. The biological gas measuring device for external ear according to claim 1, wherein the coenzyme that has undergone the chemical change is excited by the excitation light and emits fluorescence.

4. A biological gas collector that is fitted closely to the outer ear and includes an opening on the side facing the outer ear, a recessed portion connected to the opening as a space for collecting biological gases that are directly emitted from the outer ear, and a contact portion that separates the recessed portion from the outside, and a measuring instrument for measuring a target substance in the biological gas derived from the outer ear collected by the biological gas collector; A biological gas measuring device comprising: The biological gas collector has a bowl-like shape large enough to cover the auricle, The contact portion is provided on a periphery of the bowl-shaped shape, The measuring device is built into the bowl-shaped portion, A biological gas measuring device for the outer ear, characterized in that air communication between the recessed portion and the outside is blocked.

5. The measuring instrument is An enzyme membrane that faces the auricle in the recess and is exposed to biological gas; an irradiation unit that irradiates the enzyme membrane with excitation light of a predetermined wavelength; a light receiving unit that receives the fluorescent light, a coenzyme and an enzyme that catalyzes a chemical reaction of the target substance that involves a chemical change of the coenzyme are immobilized on the enzyme membrane; 5. The biological gas measuring device for external ear according to claim 4, wherein the coenzyme that has undergone the chemical change is excited by the excitation light and emits fluorescence.

6. 6. The biological gas measuring device for the external ear according to claim 1, wherein the biological gas collector is provided for each of the two ears.

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