JP2021153492A - Detection device - Google Patents

Abstract

To provide a detection device for detecting a substrate in a gas sample, by reaction between coenzyme and a substrate under presence of enzyme, capable of detecting the substrate without changing concentration of coenzyme according to concentration of the substrate in the gas sample.SOLUTION: A detection device comprises: a liquid channel in which a solution containing coenzyme can pass; a gas channel in which a gas sample can pass; an enzyme holding film whose one surface contacts a part of the liquid channel, the other surface contacts the gas channel, and is deformed according to a liquid pressure of the solution in the liquid channel, the enzyme holding film holding enzyme which catalyzes chemical reaction of a chemical substance in the gas sample by coupling to the coenzyme; a light radiating part for radiating excitation light which excites the coenzyme so as to traverse the liquid channel, toward the enzyme holding film; a detecting part for detecting fluorescence generated by excitation of the coenzyme; and a liquid pressure adjusting part for varying the liquid pressure of the solution in the liquid channel.SELECTED DRAWING: Figure 1

Description

The present invention relates to a detection device that detects a substance contained in a gas sample.

A detection device is known that detects a specific chemical substance contained in a sample by detecting a decrease in coenzyme associated with an enzymatic reaction.

For example, Patent Document 1 discloses a biosensor system that detects a chemical substance in a sample by utilizing the fact that when a coenzyme such as NADH is irradiated with a specific excitation light, it emits fluorescence. The biosensor system detects a decrease in the amount of fluorescence emitted by a coenzyme when the coenzyme decreases due to a reaction between a chemical substance such as ketones in a sample and the coenzyme in the presence of the enzyme. Chemical substances in the sample are detected and quantified.

Japanese Unexamined Patent Publication No. 2016-220573

For example, in the biosensor system as described above, if the concentration of the coenzyme in the solution used for detection is too low with respect to the concentration of the substrate in the gas sample, most of the coenzyme will be consumed and the substrate will be consumed. One of the problems is that it was necessary to change the concentration of coenzyme according to the concentration.

The present invention has been made in view of the above points, and is adjusted to the concentration of the substrate in the gas sample in a detection device that detects the substrate in the gas sample by the reaction between the coenzyme and the substrate in the presence of the enzyme. One of the purposes is to provide a detection device capable of detecting a substrate without changing the concentration of coenzyme.

The invention according to claim 1 is a detection device that detects a chemical substance in a gas sample, and is provided with a liquid flow path provided so that a solution containing a coenzyme can pass through and the gas sample can pass through. The gas flow path is provided so that one surface is in contact with a part of the liquid flow path and the other surface is in contact with the gas flow path, and is deformed according to the hydraulic pressure of the solution in the liquid flow path. A possible membrane that crosses the liquid flow path towards the enzyme-retaining membrane and an enzyme-retaining membrane that retains an enzyme that binds to the coenzyme to catalyze the chemical reaction of a chemical substance in the gas sample. A light irradiation unit that irradiates excitation light that excites the coenzyme, a detection unit that detects fluorescence generated when the coenzyme is excited, and a change in the hydraulic pressure of the solution in the liquid flow path. It is characterized by having a hydraulic pressure adjusting unit for causing the liquid pressure to be adjusted.

It is a block diagram which shows the structure of the detection apparatus which concerns on Example. It is sectional drawing which shows the structure of the biosensor which concerns on Example. It is an enlarged view of a part of FIG. It is an enlarged view of a part of FIG. It is an enlarged view of a part of FIG. It is a block diagram which shows the structure of the control device of the detection device which concerns on Example. It is a flowchart which shows an example of the routine executed in an Example.

Examples of the present invention will be described in detail below. In the following description and the accompanying drawings, the same reference numerals are given to substantially the same or equivalent parts.

FIG. 1 is a diagram schematically showing a configuration of a detection device 10 according to an embodiment of the present invention. The detection device 10 is a device that includes the biosensor 11 and operates the biosensor 11 to measure the concentration of the substrate in the gas sample.

The biosensor 11 is a device that indirectly detects the substrate by reacting the substrate contained in the gas sample with the coenzyme in the presence of the enzyme and detecting the fluorescence emitted by the coenzyme. In the presence of coenzymes such as NADH and NADPH, substrates such as aldehydes and ketones undergo a reverse reaction by dehydrogenase and become primary alcohols and secondary alcohols. By this reaction, NADH and NADPH are oxidized and changed to NAD + and NADP +.

For example, when secondary alcohol dehydrogenase (S-ADH) is used as the enzyme and NADH (reduced nicotinamide adenine dinucleotide) is used as the coenzyme, the substrate acetone is reduced to 2-propanol. The reaction formula of the above reaction is shown in Chemical formula 1.

In the above reaction, the enzyme S-ADH acts as a catalyst for the reaction in which the substrate acetone is reduced to 2-propanol. NADH, which is a coenzyme, is oxidized to NAD + (oxidized nicotinamide adenine dinucleotide) by the reverse reaction of dehydrogenase.

Further, for example, the reaction formula when alcohol dehydrogenase (ADH), acetaldehyde as a substrate, and NADH as a coenzyme is used is shown in Chemical formula 2. In this reaction, the substrate acetaldehyde is reduced to ethanol.

NADH and NADPH are excited by ultraviolet rays in the vicinity of 340 nm and emit fluorescence. On the other hand, NAD + and NADP + changed by the reaction are not excited by ultraviolet rays and do not fluoresce. Since the amount of fluorescence emitted by NADH and NADPH correlates with the amount of aldehydes and ketones, the amount of aldehydes and ketones can be estimated from the change in the amount of fluorescence before and after the reaction.

The biosensor 11 is provided with a porous membrane (enzyme holding membrane) in which an enzyme is immobilized, and is configured such that a gas sample and a coenzyme solution containing a coenzyme come into contact with each other through this membrane. ing. Therefore, when the chemical reaction proceeds in the vicinity of the enzyme-retaining membrane and the excitation light for exciting the coenzyme is irradiated in the vicinity of the enzyme-retaining membrane, fluorescence is generated. The biosensor 11 is configured to be able to detect the fluorescence.

The solution supply mechanism 13 is connected to the biosensor 11. The solution supply mechanism 13 is configured to mix a coenzyme solution containing a coenzyme with a buffer solution and supply it to the biosensor 11.

The mixing tank 15 is a container capable of accommodating a mixed solution of a buffer solution and a coenzyme solution containing a coenzyme. The mixing tank 15 includes a buffer solution inflow port into which the buffer solution flows in, a coenzyme solution inflow port into which the coenzyme solution flows in, and a mixed solution outflow port from which the mixed solution flows out.

The buffer solution and the coenzyme solution that have flowed into the mixing tank 15 are mixed in the mixing tank 15 to form a mixed solution. For example, the mixing tank 15 may be provided with a mechanism for making the mixed liquid more uniform. For example, a stirring mechanism such as a stirring propeller or a magnetic stirrer may be provided inside the mixing tank 15. Further, for example, a static mixer may be adopted as the mixing tank 15, or a container having high resistance such as a filter container may be adopted.

The buffer solution supply system 17 is configured to supply the buffer solution to the mixing tank 15. The buffer solution supply system 17 includes a buffer solution server B1, a first pump P1, and a pipe L1.

The buffer solution server B1 is, for example, a bottle or a tank containing a buffer solution. The buffer solution server B1 is a source of the buffer solution in the detection device 10. The buffer server B1 contains, for example, a phosphate buffer solution.

The pipe L1 is a pipe connected to the buffer solution server B1 and the mixing tank 15 so that the buffer solution flows from the buffer solution server B1 to the mixing tank 15.

The first pump P1 is a liquid feeding pump connected to the pipe L1 so as to feed the buffer liquid from the buffer liquid server B1 toward the mixing tank 15. The first pump P1 is, for example, a piezo type (piezoelectric type) diaphragm pump. The first pump P1 is not limited to the piezo type diaphragm pump, and any liquid feeding pump such as a stepping motor type pump can be adopted.

The coenzyme solution supply system 19 is configured to supply a coenzyme solution containing a coenzyme to the mixing tank 15. The coenzyme solution supply system 19 includes a coenzyme solution server B2, a second pump P2, and a pipe L2.

The coenzyme solution server B2 is, for example, a bottle or tank containing a solution containing NADH as a coenzyme. The coenzyme solution server B2 is a source of the coenzyme solution in the detection device 10. For example, the coenzyme solution in the coenzyme solution server B2 is adjusted to have a pH of 10 to 11 in order to store the coenzyme in a stable state.

The pipe L2 is a pipe connected to the coenzyme solution server B2 and the mixing tank 15 so that the coenzyme solution flows from the coenzyme solution server B2 to the mixing tank 15.

The second pump P2 is a liquid feeding pump connected to the pipe L2 so as to feed the coenzyme solution from the coenzyme solution server B2 to the mixing tank 15. Like the first pump P1, the second pump P2 is a liquid feeding pump such as a piezo type diaphragm pump.

The mixed liquid supply system 21 supplies the mixed liquid in the mixing tank 15 to the biosensor 11. The mixing liquid supply system 21 includes a mixing tank 15, a third pump P3, and a pipe L3.

The pipe L3 is a pipe connected from the mixing tank 15 to the biosensor 11 so that the liquid in the mixing tank 15 can flow.

The third pump P3 is a liquid feeding pump connected to the pipe L3 so as to feed the mixed liquid from the mixing tank 15 toward the biosensor 11. In this embodiment, the case where the third pump P3 is a piezo type diaphragm pump (piezo pump) will be described. The third pump P3 is not limited to the piezo pump, and another liquid feeding pump capable of controlling the flow rate of the liquid can be adopted as the third pump P3.

The waste liquid container 22 is a container for accommodating the mixed liquid discharged from the biosensor 11. The pipe L4 is a pipe provided so that the mixed liquid discharged from the biosensor 11 can be distributed to the waste liquid container 22. The fourth pump P4 is a liquid feeding pump connected to the liquid outlet of the biosensor 11.

In this embodiment, the fourth pump P4 is a piezo type diaphragm pump like the third pump P3. As the fourth pump P4, another liquid feeding pump capable of controlling the flow rate of the liquid can be adopted.

The solution supply mechanism 13 may be provided with a valve (not shown) in addition to the above configuration. For example, an on-off valve may be provided at the buffer solution inlet, the coenzyme solution inlet, and the mixed solution outlet. Further, for example, a flow rate adjusting valve may be provided between the first pump P1 and the mixing tank 15 and between the second pump and the mixing tank 15.

The control device 23 is a computer that controls the operation of the solution supply mechanism 13. The control device 23 controls the buffer solution supply system 17 and the coenzyme solution supply system 19 of the solution supply mechanism 13 to adjust the concentration of the coenzyme in the mixed solution in the mixing tank 15.

The control device 23 is connected to the first pump P1, the second pump P2, the third pump P3, and the fourth pump P4 in a controllable manner. Further, the control device 23 controls the drive voltage of each of the first pump P1, the second pump P2, the third pump P3, and the fourth pump P4.

For example, the control device 23 controls the drive voltage of the first pump P1 and the second pump P2 to control the flow rate of the buffer solution and the coenzyme solution flowing into the mixing tank 15, and the mixed solution in the mixing tank 15. Adjust the coenzyme concentration, which is the concentration of the coenzyme in it. For example, the control device 23 changes the flow ratio of the buffer solution and the coenzyme solution without changing the total flow rate of the buffer solution and the coenzyme solution, thereby keeping the total amount of the liquid flowing into the mixing tank 15 constant. Adjust the coenzyme concentration while retaining.

For example, the control device 23 controls the third pump P3 to obtain the total flow rate of the mixed liquid flowing from the mixing tank 15 to the biosensor 11 and the total flow rate of the buffer liquid and the coenzyme solution flowing into the mixing tank 15. Adjust the flow rate of the mixture so that

Instead of mixing the buffer solution 17, the buffer solution supplied from the coenzyme solution supply system 19, and the coenzyme solution in the mixing tank 15, for example, the buffer solution and the coenzyme solution were mixed in advance. A mixed solution server containing a solution whose coenzyme concentration has been adjusted may be provided, and the mixed solution server and the third pump P3 may be connected via a pipe L3.

Further, for example, the control device 23 controls the third pump P3 and the fourth pump P4 to adjust the discharge capacity of the third pump P3 and the fourth pump P4, and adjusts the discharge capacity of the third pump P3 and the fourth pump P4, and the liquid flow path in the biosensor 11 (not shown). ) Adjust the hydraulic pressure of the liquid. The third pump P3 and the fourth pump P4 are hydraulic pressure adjusting units for adjusting the hydraulic pressure in the liquid flow path.

For example, the control device 23 may adjust the hydraulic pressure by controlling the difference in discharge capacity between the third pump P3 and the fourth pump P4 to be a predetermined difference. Further, a pressure sensor (not shown) may be provided between the third pump P3 and the fourth pump P4, and the control device 23 may use the output of the pressure sensor to control the hydraulic pressure. good.

Further, the control device 23 is connected to the biosensor 11. The control device 23 acquires the amount of fluorescence detected by the biosensor 11. For example, the control device 23 controls the third pump P3 and the fourth pump P4 according to the acquired fluorescence amount to adjust the hydraulic pressure in the liquid flow path.

FIG. 2 is a cross-sectional view showing the configuration of the biosensor 11. For example, the biosensor 11 is configured such that each part is arranged in a cylindrical housing (not shown).

The light source 25 as a light irradiation unit is a light emitting device that emits excitation light that excites a coenzyme. The light source 25 is, for example, an ultraviolet light emitting diode that emits ultraviolet light having a peak wavelength of 340 nm. The light source 25 is not limited to the ultraviolet light emitting diode, and for example, an ultraviolet laser diode, a mercury lamp, or the like can be used. Let AX be the optical axis of the light source 25.

The excitation optical optical system 27 is an optical system provided on the optical axis AX. The excitation optical optical system 27 includes, for example, a collimating lens 28 provided around the optical axis AX, an excitation light bandpass filter 29, and a ball lens 30.

The collimating lens 28 is a collimating lens that makes the excitation light emitted from the light source 25 parallel light.

The excitation light bandpass filter 29 is an optical filter that transmits light in a specific wavelength band and blocks other light. The excitation light bandpass filter 29 transmits light in a band including a wavelength at which the coenzyme is excited. NADH used as a coenzyme in this example absorbs ultraviolet rays of 340 nm and emits fluorescence. Therefore, the range of wavelengths passed by the excitation light bandpass filter 29 is, for example, 330 to 350 nm.

The ball lens 30 is a condensing lens that collects the light transmitted through the excitation light bandpass filter 29 on the optical axis AX.

The flow cell 33 is a measuring container having an inlet and an outlet for the gas sample and having a structure capable of detecting the substrate in the gas sample at the central portion. The flow cell 33 is arranged with the optical axis AX as the center so that the excitation light from the ball lens 30 is incident on the central portion.

The flow cell 33 includes a gas flow path forming member 34, a liquid flow path forming member 35, and an enzyme holding membrane 36.

The gas flow path forming member 34 is a thick annular member, and has two through holes on the side surfaces facing each other in the radial direction of the annular member. In FIG. 2, a cross section of the gas flow path forming member 34 passing through the two through holes is shown. In addition, the direction in which the gas sample flows is indicated by an arrow.

The opening of the gas flow path forming member 34 on the light source 25 side is covered with a translucent sheet 34B. The translucent sheet 34B separates the gas sample from the space in which the excitation optical optical system 27 and the like are provided.

One of the two through holes of the gas flow path forming member 34 is the inlet of the gas sample, and the other is the outlet of the gas sample. The gas sample flowing in from one through hole flows out from the other through hole via the inside of the ring of the gas flow path forming member 34. Therefore, the space inside the two through holes of the gas flow path forming member 34 and the space inside the ring form the gas flow path 34A which is the flow path of the gas sample.

The gas flow path 34A, which is the flow path of the gas sample, may be formed without using the transparent sheet 34B. For example, by fitting the ball lens 30 into the gas flow path forming member without a gap, the gas flow path 34A, which is the flow path of the gas sample, can be formed.

The liquid flow path forming member 35 is a member having a shape in which a disk having a thickness smaller than the disk is stacked on a thick disk and having a through hole in the thickness direction.

The liquid flow path forming member 35 is formed with a communication hole that reaches from the side surface of the larger disk to the upper surface of the smaller disk. Two communication holes are provided at positions facing each other in the diameter direction of the disk shape.

In FIG. 2, the liquid flow path forming member 35 is arranged so that the centers of the two disk shapes overlap the optical axis AX. Further, FIG. 2 shows a cross section passing through the above two communication holes.

One of the two communication holes is an inlet of a solution containing a coenzyme, that is, a mixed solution of the coenzyme solution and the buffer solution, and is connected to the pipe L3 of the mixed solution supply system 21 and the third pump P3. The other communication hole is an outlet for a liquid containing a coenzyme, and is connected to a pipe L4 connected to a waste liquid container 22 and a fourth pump P4.

The mixed liquid flowing in from one communication hole flows out from the other through hole. In this way, the liquid flow path forming member 35 forms the liquid flow path 35A, which is a flow path through which the solution containing the coenzyme flows. For example, the liquid flow path forming member 35 is fitted with an annular support member 35B that is fitted to the smaller disk portion and supports the enzyme holding membrane 36 in a drumhead shape.

The enzyme retention film 36 is obtained by immobilizing an enzyme used for the reaction in the biosensor 11, that is, an apoenzyme that catalyzes a chemical reaction of a chemical substance in a gas sample, on a carrier such as a porous film. The enzyme retention membrane 36 is provided so as to be in contact with the liquid flow path 35A. The enzyme retention membrane 36 has elasticity and can be deformed in such a manner that the flow path width of the liquid flow path 35A changes.

For example, the enzyme retention film 36 is attached to the liquid flow path forming member 35 so as to partition the liquid flow path 35A and the gas flow path 34A and to penetrate the optical axis AX. The enzyme-retaining membrane 36 is hydrophilic and is well compatible with the mixed solution flowing through the liquid flow path 35A, and becomes transparent when the mixed solution permeates. The surface of the support member 35B of the liquid flow path forming member 35 on the light source side 25 and the enzyme retention film 36 exposed from the support member 35B are on the same plane.

The gas flow path forming member 34 is arranged so that the gas flow path forming member 34 is in contact with the upper surface of the support member 35B. Therefore, the opening on the lower surface side of the gas flow path forming member 34 is closed by the upper surface of the support member 35B and the enzyme retention membrane 36. In other words, in the annular inner portion of the gas flow path forming member 34, the translucent sheet 34B covering the opening of the surface of the gas flow path forming member 34 on the light source 25 side, the support member 35B, and the enzyme holding. A gas flow path 34A is formed by the membrane 36.

Therefore, the enzyme retention membrane 36 is in contact with the gas flow path 34A. Further, as described above, the liquid flow path 35A and the enzyme retention membrane 36 are in contact with each other. That is, the gas flow path 34A and the liquid flow path 35A are in contact with each other via the enzyme retention membrane 36. The enzyme retention membrane 36 is provided so that one surface is in contact with a part of the liquid flow path 35A and the other surface is in contact with the gas flow path 34A.

Therefore, in the presence of the enzyme immobilized on the enzyme retention membrane 36, the enzyme-catalyzed reaction between the substrate in the gas sample taken into the enzyme retention membrane 36 and the coenzyme flowing through the liquid flow path 35A A forward reaction or a reverse reaction can proceed.

As the carrier of the enzyme retention film 36, for example, polytetrafluoroethylene (PTFE), polydimethylsiloxane, polypropylene, polyethylene, polymethylmethacrylate, polystyrene, polyvinylidene fluoride, nitrocellulose, cellulose and the like can be used.

Further, in this example, the substrate contains either a ketone group or an aldehyde group. As the coenzyme, one that is excited by excitation light and emits fluorescence in one of the states before and after the reaction of the substrate is used. Examples of such coenzymes include NADH, NADPH (reduced nicotinamide adenine dinucleotide phosphate) and the like. The chemical structure of the coenzyme changes reversibly between the two states before and after the reaction of the substrate.

The enzymes immobilized on the enzyme retention membrane 36 are listed below with respect to the reaction using the substrate and coenzyme as described above. When NADH or NADPH is used as a coenzyme, the enzymes include, for example, alanine dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, isocitrate dehydrogenase, uridine-5'-diphospho-glucose dehydrogenase, and galactose dehydrogenase. Elementary enzyme, formate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, glycerol dehydrogenase, glycerol-3-phosphate dehydrogenase, glucose dehydrogenase, glucose-6-phosphate dehydrogenase, Glutamic acid dehydrogenase, cholesterol dehydrogenase, sarcosine dehydrogenase, sorbitol dehydrogenase, carbonate dehydrogenase, lactic acid dehydrogenase, 3-hydroxybutyric acid dehydrogenase, pyruvate dehydrogenase, fructose dehydrogenase, 6 -Phosphogluconic acid dehydrogenase, formaldehyde dehydrogenase, mannitol dehydrogenase, malic acid dehydrogenase, leucine dehydrogenase and the like can be mentioned.

In particular, as an enzyme immobilized on the enzyme retention membrane 36, a ketone (acetone, 2-butanone, 2-pentanone, ethyl vinyl ketone (enone), etc.) or aldehyde (acetaldehyde, nonenal, etc.) or aldehyde (acetone aldehyde, nonenal, etc.) using NADH or NADPH as an electron donor. An enzyme that reduces the amount of

Specifically, as enzymes immobilized on the enzyme retention membrane 36, alcohol dehydrogenase (ADH) (EC 1.1.1.2, etc.) and secondary alcohol dehydrogenase (S-ADH) (EC: 1.1.1. x), enone reductase type 1, ER1) and the like can be used.

The light guide member 37 is an optical member provided so as to be in contact with the liquid flow path 35A of the flow cell 33. For example, the light guide member 37 is arranged so as to be fitted into a through hole in the thickness direction along the optical axis AX of the liquid flow path forming member 35.

The light guide member 37 forms a light guide path that guides the light incident on the flow cell 33 via the excitation optical optical system 27 and the fluorescence emitted in the flow cell 33 along the optical axis AX. The light guide member 37 has, for example, a cylindrical shape. The shape of the light guide member 37 is not limited to a cylindrical shape, and may be, for example, a columnar shape having a non-planar end face, a polygonal columnar shape, or a truncated cone shape.

The light guide member 37 is made of the same material such as glass having a uniform refractive index. The light guide member 37 is not limited to the same material such as glass having a uniform refractive index, and may be composed of two materials, a core and a clad, which have different refractive indexes from each other, for example. Further, the light guide member 37 may be made of a material having little absorption with respect to the fluorescence wavelength to be measured, for example, having an absorption coefficient of 0.1 or less.

The light guide member 37 and the liquid flow path 35A may be separated by a translucent sheet (not shown).

The fluorescence optical system 39 is an optical system provided so that light from the light guide member 37 is incident on the optical axis AX. The fluorescence optical system 39 includes a collimating lens 40, a fluorescence bandpass filter 41, and a condenser lens 42.

The collimating lens 40 is a collimating lens that parallelizes the light from the light guide member 37.

The fluorescent bandpass filter 41 is an optical filter that transmits light in a specific wavelength band and blocks other light. The fluorescent bandpass filter 41 transmits light in a band including a wavelength of fluorescence emitted by excitation of a coenzyme.

The wavelength of fluorescence emitted by the excitation of NADH, which is a coenzyme, is 450 to 510 nm, and more specifically, around 491 nm. Therefore, in this embodiment, the wavelength range transmitted by the fluorescent bandpass filter 41 is, for example, 450 to 510 nm.

In addition, a long-pass filter, a short-pass filter, or the like (not shown) containing a wavelength of fluorescence emitted by excitation of NADH, which is a coenzyme, may be arranged in combination with the fluorescent bandpass filter 41.

The condensing lens 42 is a condensing lens that condenses the light transmitted through the fluorescent bandpass filter 41 on the optical axis AX.

The detector 43 as a detection unit is a detector that detects fluorescence that has passed through the fluorescence optical system 39. The detector 43 includes a photomultiplier tube, a photodiode detector, or a spectrophotometer including these, and measures the amount of detected fluorescence light.

Therefore, the detector 43 detects the fluorescence emitted by the coenzyme in the liquid flow path 35A of the flow cell 33. Further, the concentration of the substrate in the gas sample is calculated based on the amount of fluorescence detected by the detector 43.

FIG. 3 is an enlarged view of a portion A surrounded by a broken line in FIG. The reaction of the substrate, the enzyme and the coenzyme flowing through the gas flow path 34A will be described with reference to FIG. FIG. 3 schematically shows a state in which a solution containing the coenzyme NADH flows in the liquid flow path 35A and a gas sample containing a substrate S such as acetaldehyde flows in the gas flow path 34A. As described above, one surface of the enzyme retention membrane 36 is in contact with the liquid flow path 35A, and the other surface is in contact with the gas flow path 34A.

The enzyme-retaining membrane 36 is composed of, for example, a porous membrane 36A such as hydrophilic PTFE in contact with the gas flow path 34A and an enzyme-immobilizing material 36B in contact with the liquid flow path 35A. The enzyme-immobilized material 36B is obtained by immobilizing an enzyme EZ such as ADH on an immobilizing material such as PMEH (poly (MPC-co-EHMA)).

The substrate S in the gas sample permeates the porous membrane 36A to reach the enzyme fixative 36B and reaches the vicinity of the enzyme EZ. Further, the coenzyme NADH in the mixed solution flowing through the liquid flow path 35A reaches the vicinity of the enzyme EZ as the mixed solution permeates the enzyme-immobilizing material 36B. In the enzyme-immobilized material 36B, the enzyme and the coenzyme bind to each other and the enzyme-catalyzed reaction proceeds, and the substrate S is reduced to a product P such as ethanol. The coenzyme NADH is oxidized to NAD +.

The excitation light E is irradiated so as to cross the liquid flow path 35A along the optical axis AX. The coenzyme NADH that remains without being consumed by the enzyme-catalyzed reaction is excited by the excitation light E and fluoresces. NAD + produced by the enzyme-catalyzed reaction does not fluoresce even when irradiated with excitation light E.

For example, in the above enzyme-catalyzed reaction, when the amount of coenzyme in the mixed solution is larger than the amount (number of moles) of coenzyme NADH required for the total amount of substrate S in the gas sample to react, the reaction occurs. The unused coenzyme NADH remains. In this case, the fluorescence emitted by the residual coenzyme NADH can be detected, and the substrate S can be quantified.

However, if, for example, the amount of coenzyme NADH in the mixture is less than the amount of coenzyme NADH required for the total amount of substrate S in the gas sample to react, then most of the coenzyme NADH in the mixture will be Even if it is consumed for the reaction, the amount of the substrate S that has not been reacted due to the lack of the coenzyme NADH is unknown, so that the substrate S cannot be accurately quantified. Further, for example, when the amount of coenzyme NADH in the mixed solution is larger than the amount of coenzyme NADH required for the total amount of substrate S in the gas sample to react, most of the substrate S in the gas sample reacts. Even if this is the case, since the consumption rate of the coenzyme NADH in the mixed solution is low, the amount of decrease in fluorescence cannot be detected accurately, so that the substrate S cannot be accurately quantified.

It is considered that convection of the mixed liquid occurs in the portion of the liquid flow path 35A facing the enzyme retention membrane 36 (see FIG. 2). It is considered that the convection of the mixture frequently replaces NAD + produced by the enzyme-catalyzed reaction in the enzyme-immobilized material 36B with the coenzyme NADH that has not yet been used in the reaction.

Considering this, the amount of coenzyme NADH that can contribute to the enzyme-catalyzed reaction is the total amount of coenzyme NADH present in the entire volume of the flow path length of the portion of the liquid flow path 35A in contact with the enzyme-immobilizing material 36B. It can be considered that there is. Therefore, it is considered that the amount of coenzyme NADH that can contribute to the enzyme-catalyzed reaction can be increased by increasing the volume of the liquid flow path 35A at the flow path length.

FIG. 4 is an enlarged view of a portion B surrounded by a broken line in FIG. In FIG. 4, the portion of the liquid flow path 35A in contact with the enzyme retention membrane 36 is shown as a portion C surrounded by a broken line. FIG. 4 shows a state in which the pressure (hydraulic pressure) of the liquid in the liquid flow path 35A is made higher than a predetermined reference value, and the enzyme retention membrane 36 is deformed in a manner of expanding outward.

In FIG. 4, the width of the liquid flow path 35A through which the optical axis AX passes, that is, the portion where the excitation light is irradiated is shown as W1. The position of the inner surface of the enzyme retention membrane 36 when the pressure of the liquid is set as a predetermined reference value is shown by a broken line, and the flow path width in that case is shown as Wst. For example, the flow path width Wst is the flow path width when the enzyme retention membrane 36 is not deformed.

As shown in FIG. 4, in a state where the liquid pressure in the liquid flow path 35A is made higher than the reference value and the enzyme holding film 36 is deformed outward (flow path width W1), it is in contact with the enzyme holding film 36. The volume of the liquid flow path 35A in the portion C is larger than that when the hydraulic pressure in the liquid flow path 35A is used as a reference value (flow path width Wst). Therefore, the absolute amount of the coenzyme NADH present in the mixed solution in the flow path portion C of the liquid flow path 35A is larger when the liquid pressure is increased to obtain the flow path width W1.

In this way, by increasing the hydraulic pressure in the liquid flow path 35A and widening the flow path width of the liquid flow path 35A, it is possible to detect the concentration of the substrate S even in a gas sample having a high concentration of the substrate S. ..

For example, when the volume of the liquid flow path 35A in the partial C does not change, it is necessary to increase the concentration of the coenzyme NADH in the mixed solution in order to increase the absolute amount of the coenzyme NADH in the partial C.

In this embodiment, as shown in FIG. 4, by deforming the enzyme holding film 36 to widen the flow path width of the liquid flow path 35A, the flow without changing the concentration of the coenzyme in the mixed solution. The absolute amount of coenzyme NADH in part C of the tract can be increased. Therefore, it is possible to analyze a gas sample having a higher concentration of substrate S.

FIG. 5 is an enlarged view of a portion B surrounded by a broken line in FIG. In FIG. 5, as in the case of FIG. 4, the portion of the liquid flow path 35A in contact with the enzyme retention membrane 36 is shown as a portion C surrounded by a broken line. FIG. 5 shows a state in which the pressure (hydraulic pressure) of the liquid in the liquid flow path 35A is lowered below a predetermined reference value, and the enzyme retention membrane 36 is deformed in an inwardly recessed manner.

In FIG. 5, the width of the liquid flow path 35A through which the optical axis AX passes, that is, the portion where the excitation light is irradiated is shown as W2. The position of the inner surface of the enzyme-retaining membrane when the pressure of the liquid is set as a predetermined reference value is shown by a broken line.

As shown in FIG. 5, in a state where the liquid pressure in the liquid flow path 35A is lower than the reference value and the enzyme holding film 36 is deformed inward (flow path width W2), it is in contact with the enzyme holding film 36. The volume of the liquid flow path 35A in the portion C is smaller than that when the liquid pressure in the liquid flow path 35A is used as a reference value (flow path width Wst).

Therefore, in the portion C of the liquid flow path 35A, the absolute amount of the coenzyme NADH present in the mixed liquid is smaller when the liquid pressure is lowered to obtain the flow path width W2.

By lowering the hydraulic pressure in the liquid flow path 35A and narrowing the flow path width of the liquid flow path 35A in this way, it is possible to accurately detect the concentration of the substrate S even in a gas sample having a low concentration of the substrate S. It becomes.

For example, when the volume of the liquid flow path 35A in the partial C does not change, it is necessary to lower the concentration of the coenzyme NADH in the mixed solution in order to reduce the absolute amount of the coenzyme NADH in the partial C.

In this embodiment, as shown in FIG. 5, by deforming the enzyme retention film 36 to narrow the flow path width of the liquid flow path 35A, the enzyme does not change the concentration of the coenzyme in the mixed solution. The absolute amount of coenzyme NADH in the portion C in contact with the retention film 36 can be reduced. Therefore, highly accurate analysis is possible for a gas sample having a low concentration of substrate S.

FIG. 6 is a block diagram showing the configuration of the control device 23 of the detection device 10. The control device 23 is, for example, a device in which each unit cooperates via the system bus 52.

The storage unit 53 is composed of, for example, a hard disk device, an SSD (solid state drive), a flash memory, or the like, and stores various programs related to the control of the liquid feed pump in the control device 23. In addition, the storage unit 53 stores a database such as the correlation between the amount of fluorescence and the concentration of coenzyme.

The various programs or databases may be acquired from other server devices or the like via a network (not shown), or may be recorded on a recording medium and read via various drive devices. You may.

Various programs stored in the storage unit 53 can be transmitted via a network, and can be recorded on a computer-readable recording medium and transferred.

The output unit 55 is an interface that outputs data to an external device of the control device 23. The output unit 55 is connected to each of the first pump P1, the second pump P2, the third pump P3, and the fourth pump P4 so as to be able to send a signal for adjusting the discharge capacity.

The input unit 57 is an interface for acquiring data from an external device of the control device 23. The input unit 57 is connected to the detector 43 of the biosensor 11. For example, the input unit 57 captures the fluorescence amount data detected by the detector 43.

The control unit 59 is composed of a CPU (Central Processing Unit) 59A, a ROM (Read Only Memory) 59B, a RAM (Random Access Memory) 59C, and the like, and functions as a computer. Then, the CPU 59A realizes various functions by reading and executing various programs stored in the ROM 59B and the storage unit 53.

For example, the control unit 59 controls the buffer solution supply system 17 and the coenzyme solution supply system 19. Specifically, the control unit 59 controls, for example, the discharge capacities of the first pump P1, the second pump P2, and the third pump P3 via the output unit 55. As a result, the control unit 59 controls the coenzyme concentration in the mixed solution by adjusting the supply flow rate of the buffer solution and the coenzyme solution to the mixing tank 15.

Further, for example, the control unit 59 acquires the detection result by the detection unit via the input unit 57, and controls the third pump P3 and the fourth pump P4 according to the detection result.

For example, when the concentration of the substrate in the gas sample is high and the amount of the coenzyme NADH in the mixed solution is insufficient, the control unit 59 changes, for example, the flow rates of the third pump P3 and the fourth pump P4. The hydraulic pressure of the mixed liquid in the liquid flow path 35A is controlled to be higher than a predetermined reference value. As a result, as shown in FIG. 4, the enzyme retention membrane 36 is deformed toward the outside of the liquid flow path 35A, and the width of the liquid flow path 35A is widened.

For example, when the concentration of the substrate in the gas sample is low and the amount of the coenzyme NADH in the mixed solution is excessive, the control unit 59 changes the flow rates of the third pump P3 and the fourth pump P4 to liquid. The hydraulic pressure of the mixed liquid in the flow path 35A is controlled to be lower than a predetermined reference value. As a result, as shown in FIG. 5, the enzyme retention membrane 36 is deformed toward the inside of the liquid flow path 35A, and the width of the liquid flow path 35A is narrowed.

The third pump P3 and the fourth pump P4 function as a hydraulic pressure adjusting unit that changes the hydraulic pressure of the solution in the liquid flow path 35A. Further, the control unit 59 functions as a control unit that controls the hydraulic pressure adjusting unit.

FIG. 7 is a flowchart showing the measurement routine RT1 which is an example of the routine executed by the control unit 59 of the control device 23 in the detection device 10. The measurement routine RT1 is executed, for example, in a state where a mixed solution of the buffer solution and the coenzyme solution is prepared at a predetermined concentration in the mixing tank 15.

When the measurement routine RT1 is started, the control unit 59 starts supplying the mixed solution, which is a solution containing the coenzyme in the mixing tank 15, to the biosensor 11 (step S101). In step S101, for example, the control unit 59 is a hydraulic pressure adjusting unit so that the hydraulic pressure of the mixed liquid in the liquid flow path 35A of the biosensor 11 becomes a predetermined standard hydraulic pressure (first hydraulic pressure). The third pump P3 and the fourth pump P4 are controlled as.

Further, for example, the control unit 59 makes the flow path width of the liquid flow path 35A a standard flow path width (first width) (for example, Wst in FIGS. 4 and 5 or W2 in FIG. 5). , The third pump P3 and the fourth pump P4 may be controlled. For example, a table in which the pump settings and the flow path width are associated in advance may be stored in the storage unit 53. The control unit 59 may control the third pump P3 and the fourth pump P4 based on the table.

After executing step S101, the control unit 59 determines whether or not a gas sample has been introduced into the gas flow path 34A of the flow cell 33 (step S102). For example, in step 102, the control unit 59 determines whether or not the gas sample has been introduced based on the opening and closing of a valve (not shown) for introducing the gas sample.

In step S102, the control unit 59 determines that the gas sample has not been introduced (step S102: NO), repeats step S102, and determines again whether or not the gas sample has been introduced.

When the control unit 59 determines in step S102 that the gas sample has been introduced (step S102: YES), the control unit 59 measures the amount of fluorescence by the detector 43 (step S103). In step S103, the control unit 59 acquires the fluorescence amount (first fluorescence amount) emitted by the coenzyme in the flow cell 33 from the detector 43.

After executing step S103, the control unit 59 controls the third pump P3 and the fourth pump P4 so that the hydraulic pressure of the mixed liquid in the liquid flow path 35A rises (step S104). In step S104, for example, in the control unit 59, the hydraulic pressure of the mixed liquid in the liquid flow path 35A becomes higher than the predetermined standard hydraulic pressure (first hydraulic pressure), and the flow of the liquid flow path 35A. The third pump P3 and the fourth pump P4 as the hydraulic pressure adjusting unit are controlled so that the path width becomes a flow path width (second width) larger than the standard flow path width (first width).

After executing step S104, the control unit 59 measures the amount of fluorescence again (step S105). In step S105, the control unit 59 acquires the fluorescence amount (second fluorescence amount) emitted by the coenzyme in the flow cell 33 from the detector 43.

After executing step S105, the control unit 59 determines whether or not the change in the amount of fluorescence is greater than a predetermined value (step S106). In step S106, the control unit 59 compares the amount of fluorescence acquired in step S105 with the amount of fluorescence acquired in step S103, and acquires a change in the amount of fluorescence.

When the control unit 59 determines in step S106 that the change in the amount of fluorescence is not greater than the predetermined value, that is, within the predetermined value (step S106: NO), the control unit 59 returns to step S104 and mixes in the liquid flow path 35A. The third pump P3 and the fourth pump P4 as the hydraulic pressure adjusting unit are controlled so that the hydraulic pressure of the liquid is further increased, that is, the flow path width is larger than the second width.

In step S106, the fact that the change in the amount of fluorescence is within a predetermined value is, for example, a coenzyme in the mixed solution rather than an appropriate amount of the coenzyme NADH for accurately detecting the concentration of the substrate S in the gas sample. It can be considered to indicate that the amount of NADH is small. In this case, the amount of coenzyme NADH is insufficient to accurately quantify the substrate. Therefore, it is considered that the amount of coenzyme NADH in the liquid flow path 35A in contact with the enzyme retention membrane 36 can be increased by widening the flow path width in step S104.

When the control unit 59 determines in step S106 that the change in the amount of fluorescence is greater than a predetermined value (step S106: YES), the control unit 59 calculates the substrate concentration in the gas sample (step S107).

In step S107, the control unit 59 calculates the substrate concentration in the gas sample based on, for example, the amount of fluorescence acquired in the latest step S105 and the hydraulic pressure of the mixture (in other words, the flow path width). For example, the storage unit 53 may store a table in which the relationship between the hydraulic pressure (or flow path width) of the mixed solution, the combination of the amount of fluorescence, and the substrate concentration is associated in advance. The control unit 59 may calculate the substrate concentration based on the table. The calculated substrate concentration may be displayed on an operation screen (not shown) connected to the control device 23.

After executing step S107, the control unit 59 controls the third pump P3 and the fourth pump P4 so that the hydraulic pressure of the mixed liquid in the liquid flow path 35A becomes the original standard hydraulic pressure (step S108). After that, the sample measurement subroutine is terminated.

In step S101, step S104 and step S108, the third pump P3 and the fourth pump P4 function as a hydraulic pressure adjusting unit for changing the hydraulic pressure of the solution in the liquid flow path 35A, and the control unit 59 adjusts the hydraulic pressure. It functions as a control unit that controls the unit.

In the above measurement routine RT1, before introducing the gas sample, the amount of fluorescence is detected in a state where the solution containing the coenzyme is circulated in the liquid flow path 35A, and the flow path width is confirmed by the amount of fluorescence. May be good.

As described in detail above, in the detection device of this embodiment, one surface is in contact with a liquid flow path through which a solution containing a coenzyme flows, and the other surface is in contact with a gas flow path through which a gas sample containing a substrate flows. It has an enzyme-retaining film that can be deformed in such a manner that the width of the liquid flow path changes according to the hydraulic pressure. Further, it has a hydraulic pressure adjusting unit for adjusting the hydraulic pressure and a control unit for controlling the hydraulic pressure adjusting unit.

Further, in this example, a substrate containing a ketone group or an aldehyde group in a gas sample is reacted with a coenzyme NADH or NADPH in the presence of an enzyme, and the fluorescence emitted by the coenzyme is detected to detect these. An example of a detection device for detecting the concentration of the substrate has been described. The combination of coenzyme and substrate is not limited to these. The idea of this embodiment can also be applied to, for example, a detection device that uses NAD + or NADP + as a coenzyme and detects the concentration of a substrate containing a hydroxy group such as ethanol.

According to the detection device of this example, in the reaction between the coenzyme and the substrate in the presence of the enzyme, the liquid flow path facing the enzyme-retaining membrane in which the enzyme-catalyzed reaction occurs according to the concentration of the substrate in the gas sample. The width can be changed, and the total amount of coenzyme present in the liquid flow path facing the enzyme holding membrane can be changed. Therefore, according to the detection device of this embodiment, it is possible to measure a gas sample having an unknown concentration or a gas sample having a wide concentration range.

Further, according to the detection device of this example, it is not necessary to change the concentration in the solution containing the coenzyme according to the concentration of the substrate in the gas sample. For example, it is not necessary to re-prepare or replace the mixed solution of the solution containing the coenzyme and the buffer solution.

Therefore, in the detection device that detects the substrate in the gas sample by the reaction between the coenzyme and the substrate in the presence of the enzyme, the substrate is detected without changing the concentration of the coenzyme according to the concentration of the substrate in the gas sample. It is possible to provide a detection device which can be used.

For example, the detection device of this embodiment can be used to detect aldehydes in human exhaled breath. For example, the state of drinking can be confirmed from the aldehyde concentration in the exhaled breath.

Further, for example, using the detection device of this embodiment, ketones such as acetone in the exhaled breath of a person can be detected, and a health state such as a metabolic state can be confirmed. For example, the detection device of this embodiment can be applied for medical purposes.

Further, for example, the detection device of the present embodiment can be mounted on the moving body to check the drinking state and the health state of the user who uses the moving body.

In the above embodiment, an example of adjusting the hydraulic pressure in the liquid flow path by using the third pump P3 and the fourth pump P4 as the hydraulic pressure adjusting unit has been described, but the present invention is not limited to this. For example, the hydraulic pressure is controlled by controlling only the pump on the upstream side (only the third pump P3) or only the downstream pump (only the fourth pump P4) from the portion where the liquid flow path is in contact with the enzyme retention membrane. You may adjust.

In the above embodiment, when the liquid feed pump is used as the hydraulic pressure adjusting unit, it may be used in a mode of suppressing the pulsation of the pump. For example, the pulsation may be suppressed by controlling the drive timing of two pumps such as the third pump P3 and the fourth pump P4. In addition, even if the fluorescence amount is detected only when the hydraulic pressure is maximized by the maximum pressure of the pump, the average value of multiple times is used as the fluorescence amount measurement result, and the influence of pulsation on the fluorescence amount measurement value is eliminated. good.

Further, the means for adjusting the hydraulic pressure in the hydraulic pressure adjusting unit is not limited to the pump. For example, the hydraulic pressure may be adjusted by using a flow rate adjusting valve instead of the fourth pump P4 in this embodiment.

In the above embodiment, an example of measuring the amount of fluorescence of the coenzyme fluorescence that changes due to the enzyme-catalyzed reaction while flowing the solution containing the coenzyme through the liquid flow path 35A of the flow cell 33 has been described, but the present invention is limited to this. No. For example, a solution containing a coenzyme may be retained in the liquid flow path 35A of the flow cell 33, and the amount of fluorescence of the coenzyme may be measured. Specifically, the liquid pressure in the liquid flow path is adjusted by using the third pump P3 and the fourth pump P4, the flow path width of the liquid flow path 35A is adjusted to a desired width, and then the third pump P3 and The drive of the fourth pump P4 is temporarily stopped. In this state, a gas sample is introduced into the gas flow path 34A of the flow cell 33, the enzyme catalytic reaction is allowed to proceed for a predetermined time, and the amount of fluorescence of the coenzyme is measured. When the measurement is completed, the third pump P3 and the fourth pump P4 may be driven again to replace the solution in the liquid flow path 35A of the flow cell 33 to perform the next measurement. As a result, the reaction time of the enzyme-catalyzed reaction can be sufficiently secured, so that the consumption of the solution used for the measurement can be reduced.

The configuration in the above-described embodiment is merely an example, and can be appropriately selected and changed according to the intended use and the like.

10 Detection device 11 Biosensor 13 Solution supply mechanism 15 Mixing tank 17 Buffer solution supply system 19 Coenzyme solution supply system 21 Mixing solution supply system 22 Waste liquid container 23 Control device 25 Light source 27 Excitation photooptical system 33 Flow cell 34 Gas flow path forming member 34A Gas flow path 34B Translucent sheet 35 Liquid flow path forming member 35A Liquid flow path 35B Support member 36 Enzyme holding film 37 Light guide member 39 Fluorescent optical system 43 Detector 52 System bus 53 Storage unit 55 Output unit 57 Input unit 59 Control unit

Claims (7)

A detector that detects chemical substances in gas samples.
A liquid flow path provided to allow a solution containing a coenzyme to pass through,
A gas flow path provided so that the gas sample can pass through, and
One surface is provided so as to be in contact with a part of the liquid flow path and the other surface is in contact with the gas flow path, and the width of the liquid flow path is increased according to the hydraulic pressure of the solution in the liquid flow path. An enzyme-retaining membrane that is deformable in a changing manner and retains an enzyme that catalyzes the chemical reaction of a chemical substance in the gas sample by binding to the coenzyme.
A light irradiation unit that irradiates an excitation light that excites the coenzyme so as to cross the liquid flow path toward the enzyme retention membrane.
A detection unit that detects fluorescence generated when the coenzyme is excited, and
A detection device including a hydraulic pressure adjusting unit that changes the hydraulic pressure of the solution in the liquid flow path. The detection device according to claim 1, wherein the hydraulic pressure adjusting unit includes a pump connected to at least one of upstream and downstream of the enzyme holding membrane of the liquid flow path. A control unit that controls the hydraulic pressure adjusting unit is included.
According to any one of claims 1 or 2, the control unit changes the hydraulic pressure of the solution in the liquid flow path according to the amount of fluorescence of the fluorescence detected by the detection unit. The detector described. After acquiring the first fluorescence amount, which is the amount of the fluorescence when the hydraulic pressure adjusting unit is controlled so that the width of the liquid flow path becomes the first width, the control unit obtains the first fluorescence amount from the detection unit. The second fluorescence amount, which is the amount of fluorescence when the hydraulic pressure adjusting unit is controlled so that the width of the liquid flow path becomes a second width larger than the first width, is acquired from the detection unit. ,
When the difference between the first fluorescence amount and the second fluorescence amount is within a predetermined value, the hydraulic pressure adjusting unit is controlled so that the width of the liquid flow path becomes larger than the second width. The detection device according to claim 3, wherein the detection apparatus is used. When the hydraulic pressure of the solution in the liquid flow path becomes high, the enzyme retention film deforms so as to swell outward to widen the width of the liquid flow path, and the hydraulic pressure of the solution in the liquid flow path becomes low. The detection device according to any one of claims 1 to 4, wherein the liquid flow path is deformed so as to be recessed inward to narrow the width of the liquid flow path. The detection device according to any one of claims 1 to 5, wherein the chemical substance is an aldehyde or a ketone. The detection device according to any one of claims 1 to 6, wherein the chemical substance is acetaldehyde.

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