JP2021153451A - Detector - Google Patents

Abstract

To provide a detector capable of increasing the utilization efficiency of fluorescent light to excitation light.SOLUTION: A detector comprises: an excitation light optical system that irradiates an enzyme holding film from the gas flow path side of a reaction part with excitation light; and a light guide member disposed on one axis so as to receive fluorescent light, and forming a light guide path along the one axis. The excitation light optical system is configured to condense the excitation light in either area on the gas flow path side from the enzyme holding film on the one axis, or inside the light guide member on the one axis.SELECTED DRAWING: Figure 2

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 substance contained in a gas sample by detecting a decrease in coenzymes associated with an enzymatic reaction.

As such a detection device, for example, it is possible to detect a substrate contained in a gas sample by utilizing the fact that NADH, which is a coenzyme, decreases with the reaction of a ketone such as acetone or a substrate such as nonenal. The biosensor system to be performed is disclosed in Patent Document 1. Specifically, the biosensor system of Patent Document 1 detects a decrease in coenzyme by utilizing the fact that when a coenzyme is irradiated with a specific excitation light, it emits fluorescence. In the biosensor system of Patent Document 1, the coenzyme is irradiated with excitation light via an optical fiber probe in the vicinity of the enzyme whose coenzyme concentration changes due to the substrate, and the fluorescence emitted by the coenzyme is detected. There is. Patent Document 2 describes a detection device capable of improving the detection accuracy of a specific substance and downsizing the device by improving the utilization efficiency of excitation light and fluorescence, which has been a problem in the biosensor system. Is presented.

Japanese Unexamined Patent Publication No. 2016-220573 WO2019-188304 Gazette

The excitation light becomes unnecessary light that deteriorates the S / N ratio of the detection signal. Therefore, in order to reduce the rate at which unnecessary excitation light is received by the detection unit, in the technique described in Patent Document 2, the light receiving side collimating lens is defocused most at the focal position of the light receiving side collimating lens. Was placed.

However, when the amount of excitation light and fluorescence received by the detection unit is actually calculated, in the optical system described in Patent Document 2, the amount of excitation light received is set to be the minimum. It was also found that the amount of light received by fluorescence was greatly reduced. That is, from the viewpoint of improving the S / N ratio, there is a problem that the optical system described in Patent Document 2 cannot be said to have an optimum optical system configuration.

The present invention has been made in view of the above points, and one of the problems is to provide a detection device that enhances the utilization efficiency of fluorescence with respect to excitation light.

The detection device according to claim 1 of the present application includes a gas flow path through which a gas sample flows, a solution flow path through which a solution containing a coenzyme that emits fluorescence when irradiated with excitation light flows, and the solution and the gas sample. A reaction section including an enzyme-retaining membrane that is formed so as to separate the gas flow path and the solution flow path while being in contact with the gas sample and holds an enzyme that catalyzes a chemical reaction of the gas sample by binding to the coenzyme. Along one axis, an excitation photooptical system that irradiates the enzyme holding film with the excitation light from the gas flow path side of the reaction unit, and an excitation photooptical system that receives the fluorescence are provided on the one axis. The excitation optical optical system includes a light guide member that forms a light guide path along the one axis, and the excitation photooptical system is closer to the gas flow path than the enzyme holding film on the one axis, or the one axis. It is characterized in that it is configured to collect the excitation light in any one region in the light guide member.

It is sectional drawing which shows the structure of the detection apparatus which concerns on Example 1. FIG. It is an enlarged cross-sectional view of the reaction part of the detection apparatus which concerns on Example 1. FIG. It is explanatory drawing explaining the optical path of the excitation light of the detection apparatus which concerns on Example 1. FIG. It is an enlarged partial cross-sectional view explaining the main part M1 around the condensing point of the excitation light LT1 shown in FIG. Changes in the received amount S of fluorescence and the received amount R of excitation light on the detection surface of the photodetector when both the focusing position of the excitation light and the light receiving range of the fluorescence optical system are in the solution flow path according to the comparative example. It is a graph which shows. Changes in the amount of fluorescence received S and the amount of excitation light received R on the detection surface of the photodetector when the light-receiving collimating lens of the detection device according to Example 1 is moved from the position of the comparative example in one axial direction. It is a graph which shows. It is sectional drawing which shows the structure of the detection apparatus which concerns on Example 2 of this invention. It is an enlarged partial cross-sectional view explaining the main part M2 around the focusing point of the excitation light shown in FIG. 7. When the light receiving side collimating lens is moved from the Pc position of the comparative example (FIG. 5) to the P2 position in the AX direction of one axis so that the focused position of the excitation light is in the light guide member according to the second embodiment. It is a graph which shows the change of S (light receiving amount of fluorescence LT2) / R (light receiving amount of excitation light LT1) of fluorescence and excitation light. It is an enlarged partial cross-sectional view explaining the main part M3 of the condensed state of the excitation light in the gas flow path, the solution flow path and the enzyme holding membrane along one axis in the detection apparatus which concerns on Example 3 of this invention. It is sectional drawing along one axis of the biosensor as the detection device which concerns on Example 4 of this invention.

Hereinafter, examples according to the present invention will be described with reference to the drawings. In the examples, components having substantially the same function and configuration are designated by the same reference numerals, so that duplicate description will be omitted.

FIG. 1 shows a cross section of a biosensor 10 as a detection device according to an embodiment of the present invention along one axis AX.

In FIG. 1, the biosensor 10 as a detection device is a device that detects the fluorescence emitted by the coenzyme that binds to the apoenzyme and detects the substrate to be detected.

Specifically, the coenzyme used for detecting the substrate of the biosensor 10 is excited by excitation light and fluoresces in one of the states before and after the reaction of the substrate. Therefore, the biosensor 10 detects the substrate by utilizing the fact that the amount of fluorescence emitted by the coenzyme changes depending on the reaction of the substrate.

For example, as shown in Chemical formula 1, when NADH (reduced nicotinamide adenine dinucleotide) is used as a coenzyme, the enzyme S-ADH catalyzes the reaction in which the substrate acetone is reduced to 2-propanol. do. At this time, the coenzyme NADH is oxidized to NAD + (oxidized nicotinamide adenine dinucleotide) by an enzymatic reaction.

Here, NADH receives excitation light of a predetermined wavelength and emits fluorescence, but does not emit fluorescence even if NAD + receives excitation light of the same wavelength. Therefore, the fluorescence intensity detected before and after this reaction fluctuates.

The biosensor 10 of this embodiment is an apparatus for measuring the concentration of a substrate by measuring the amount of fluorescence emitted by the NADH.

As shown in FIG. 1, the biosensor 10 has an excitation optical optical system 20, a flow cell 30, and a fluorescence optical system 50. In the biosensor 10, the cases C1 to C5 are connected by fastening screws (not shown) or the like to be integrated, and the light source LT, the excitation optical optical system 20, the flow cell 30, the light guide member 40, the fluorescent optical system 50, and the photodetector element 60 are integrated. It is formed in a tubular shape that covers each circumference. That is, the cases C1 to C5 accommodate the light source LT, the excitation optical optical system 20, the flow cell 30, the light guide member 40, the fluorescent optical system 50, and the light detection element 60 along one axis AX. Therefore, the biosensor 10 composed of the cases C1 to C5 is configured to prevent outside light from entering the inside thereof.

[Excitation optical optical system 20]
In FIG. 1, the light source LT housed in the case C1 is a light emitting device that emits excitation light. The light source LT is, for example, an ultraviolet light emitting diode that emits ultraviolet light having a peak wavelength of 340 nm as excitation light. The light emitting device 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.

The excitation optical optical system 20 housed in the case C2 whose one end is connected to the case C1 is an optical system that transmits the excitation light emitted from the light source LT on one axis AX. The excitation optical optical system 20 collects the excitation light toward one axis AX. One axis AX is the same axis as the optical axis of the excitation light in this embodiment.

The excitation optical optical system 20 includes a collimated lens 21 that makes the excitation light parallel, and a ball lens 22 that is a condensing lens that collects the excitation light made parallel by the collimated lens 21 on one axis AX. .. The ball lens 22 is configured to have at least a numerical aperture higher than the numerical aperture of the light guide member 40.

Since the ball lens 22 has a short focal length among the condensing lenses, the configuration of the biosensor 10 can be made smaller. Further, the ball lens 22 has a large numerical aperture (NA) among the condensing lenses, and can take in more excitation light.

An excitation light bandpass filter EF that transmits the wavelength of the excitation light is provided between the collimating lens 21 and the ball lens 22. The band transmitted by the excitation light bandpass filter EF is a band including the wavelength of the excitation light excited by the coenzyme. Since NADH that absorbs ultraviolet rays of 340 nm and emits fluorescence is used as a coenzyme, the excitation light bandpass filter EF reduces visible light having a wavelength of 400 nm or more that interferes with the detection fluorescence band, for example.

[Flow cell 30]
On the other end side of the case C2, a flow cell 30 is formed by being connected to the case C3 on one axis AX. The flow cell 30 functions as a reaction unit on which the enzymatic reaction shown in Chemical formula 1 is carried out. The flow cell 30 has a gas flow path 31 through which a gas sample containing a substrate flows, a solution flow path 32 that holds a solution containing a coenzyme, and an enzyme holding film 33 that is a porous film that holds an enzyme.

The case C2 is formed with two through holes HA provided through the case C2 in a direction perpendicular to one axis AX. By inserting the gas flow path 31 through the two through holes HA, the gas flow path 31 can be mounted on one shaft AX. The gas flow path 31 has a translucent window W for transmitting excitation light on the light source LT side of the flow cell 30.

The case C3 is formed with two through holes HB provided so as to penetrate the case C3 in a direction perpendicular to one axis AX. By inserting the solution flow path 32 having a tubular structure into the two through holes HB, the solution flow path 32 can be mounted on one axis AX.

Here, the gas flow path 31 may have a structure such as a translucent tube that transmits an excitation light beam on at least one axis AX, or may be a translucent Petri dish-like container. Structure may be used. Further, the solution flow path 32 may have a structure such as a translucent tube through which a solution containing a coenzyme flows, for example, through which an excitation light beam on at least one axis AX is transmitted. It may have a structure like a light chalet-like container. In this case, the area other than the region where the excitation light beam of the solution flow path 32 is irradiated may be formed of a light-shielding member in order to reduce the stray light of the excitation light.

The substrate contained in the gas sample flowing through the gas flow path 31 contains either a ketone group or an aldehyde group.

FIG. 2 shows an enlarged cross section of a portion of the flow cell 30 which is a reaction portion along one axis AX.

As shown in FIG. 2, the solution flow path 32 includes an upstream connection portion provided on the upstream side in the solution flow direction, a downstream connection portion provided on the downstream side in the solution flow direction, and an upstream side. It has a curved portion (inner wall of the cylindrical portion CB) formed by being curved from the side connecting portion and the downstream connecting portion toward the light source LT side.

The inner wall of the cylindrical portion CB is a through hole H2 that is a part of the solution flow path 32 extending along one axis AX. The through hole H3 has a small diameter portion having a size equivalent to the outer diameter of the light guide member 40, and an enlarged diameter portion formed by expanding the diameter from the small diameter portion toward the fluorescence optical system 50.

Therefore, by fitting the light guide member 40 into the small diameter portion H3, the light guide member 40 is held by the flow cell 30 (case C3) as a part of the solution flow path 32.

The solution flowing through the solution flow path 32 contains a coenzyme and a buffer solution having a pH value in consideration of the enzyme and the optimum pH value of the coenzyme as components. As the coenzyme contained in the solution, 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 enzyme retention membrane 33 (that is, the porous membrane on which the enzyme is immobilized) that retains the enzyme is formed with the gas flow path 31 so as to close the through hole H1 of the gas flow path 31 and the through hole H2 of the solution flow path 32. It is provided in contact with both the solution flow path 32. That is, the enzyme retention film 33 is a diaphragm that allows the retained enzyme to come into contact with the solution in the solution flow path 32 and the gas sample in the gas flow path 31, and separates the solution flow path 32 and the gas flow path 31. ..

The enzyme-retaining membrane 33 is a membrane material in which an enzyme is immobilized on a porous membrane. Examples of the carrier of the enzyme retention film 33 include polytetrafluoroethylene, polydimethylsiloxane, polypropylene, polyethylene, polymethylmethacrylate, polystyrene, polyvinylidene fluoride, nitrocellulose, and cellulose.

When NADH or NADPH is used as the coenzyme, the enzyme retained in the enzyme retention membrane 33 is, for example, alanine dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, isocitrate dehydrogenase, uridine-5'-diphospho. -Glucose dehydrogenase, galactose dehydrogenase, formate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, glycerol dehydrogenase, glycerol-3-phosphate dehydrogenase, glucose dehydrogenase, glucose- 6-phosphate dehydrogenase, glutamate dehydrogenase, cholesterol dehydrogenase, sarcosine dehydrogenase, sorbitol dehydrogenase, carbonate dehydrogenase, lactic acid dehydrogenase, 3-hydroxybutyric acid dehydrogenase, pyruvate dehydrogenase Examples thereof include enzymes, fructose dehydrogenase, 6-phosphogluconic acid dehydrogenase, formaldehyde dehydrogenase, mannitol dehydrogenase, malic acid dehydrogenase, leucine dehydrogenase, etc., and in particular, NADH or NADPH. An enzyme that uses as an electron donor to reduce ketones (acetone, 2-butanone, 2-pentanone, etc.) or aldehydes (nonenal, etc.), more specifically, secondary alcohol dehydrogenase (S-ADH) (secondary). Alcohol dehydrogenase (secondary alcohol dehydrogenase) EC: 1.1.1.x), enone reductase (ER1) (enone reductase type 1, ER1) and the like can be used.

The case C2 is formed with an annular inner wall CC that projects so as to be in contact with the gas flow path 31 so as to face each other in a direction perpendicular to one axis AX. The annular inner wall CC is formed between the through hole HA and the through hole HB on one axis AX.

The case C3 is formed with a cylindrical portion CB that projects into the gas flow path 31 in a direction parallel to one axis AX so as to be connected to the gas flow path 31 via the enzyme retention membrane 33.

The enzyme-retaining membrane 33 is attached on one axis AX by covering the annular inner wall CC with the enzyme-retaining membrane 33 and fitting the cylindrical portion CB with the enzyme-retaining membrane 33 sandwiched therein. That is, the annular inner wall CC functions as an attachment portion that enables the enzyme holding membrane 33 to be attached to the cylindrical portion CB on one axis AX. As described above, the gas flow path 31, the liquid flow path 32, and the enzyme holding film 33 are detachable by the cases C2 and C3.

The flow cell 30 has a space SP between the side surface SS of the light guide member 40 extending along one axis AX and the wall portion of the enlarged diameter portion of the through hole H3. Assuming that the refractive index of the light guide member 40 is n ₁ and the refractive index of the space SP is n ₂ , the numerical aperture (NA) is given by the following equation.

Here, in order to maximize the numerical aperture (NA) under the condition that _{the refractive index n 1} of the light guide member 40 _{is fixed, it is necessary to set n 2} to 1, that is, the space SP to be air. Therefore, the numerical aperture (NA) of the light guide member 40 is maximized by forming an air layer around the side surface SS.

Here, the light guide path 41 of the light guide member 40 may be formed so as to reduce the amount of light guided by the excitation light. Specifically, the incident angle of the excitation light that has passed through the solution flow path 32 of the flow cell 30 with respect to at least a part of the light guide member 40 is from the maximum light receiving angle that can be totally reflected on the inner surface of the light guide path 41 of the light guide member 40. Should also be increased.

[Fluorescent optical system 50]
As shown in FIG. 1, the fluorescent optical system 50 housed in the case C4 having one end connected to the case C3 and the other end connected to the case C5 has a light guide member 40 that collects fluorescence and the like and a light guide member 40 that collects the fluorescence and the like in parallel light. It includes a light receiving side collimating lens 51 and a condensing lens 52 that collects the fluorescence made into parallel light by the light receiving side collimating lens 51 on one axis AX. The fluorescence optical system 50 is an optical system that mainly supplies the fluorescence emitted from the light guide member 40 to the photodetector element 60 (stored in the case C5) on one axis AX.

The light guide member 40 is provided on one axis AX in contact with the solution flow path 32 of the flow cell 30. The light guide member 40 is formed in a columnar shape in this embodiment. The light guide member 40 may have a shape other than a columnar shape, for example, a columnar shape having a non-planar end face, a polygonal columnar shape, or a truncated cone shape.

The light guide member 40 is made of the same material such as glass having a uniform refractive index. The light guide member 40 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 40 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. A light guide path 41 formed along one axis AX is provided around the side surface of the light guide member 40.

An optical filter that transmits a band including a wavelength of fluorescence is provided between the light guide member 40 and the detection surface of the photodetector 60. For example, a fluorescence bandpass filter FF that transmits a band including a wavelength of fluorescence is provided between the light receiving side collimating lens 51 and the condensing lens 52. 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 range of wavelengths transmitted by the fluorescent bandpass filter FF is 440 to 510 nm.

A photodetector 60 for detecting fluorescence that has passed through the condenser lens 52 is provided on one axis AX. The photodetector 60 includes a photomultiplier tube, a photodiode detector, and a spectrophotometer including these, and detects the concentration of the substrate in the gas sample based on the detected fluorescence light intensity or spectroscopic characteristics.

[Ray tracing of excitation light of excitation optical optical system 20]
FIG. 3 shows an aspect of the excitation light emitted from the light source LT. In FIG. 3, the alternate long and short dash arrow indicates the excitation light LT1 (ultraviolet light). The excitation light LT1 emitted from the light source LT is made into parallel light by the collimating lens 21. FIG. 4 is an enlarged partial cross-sectional view illustrating the main part M1 around the focusing point of the excitation light LT1 shown in FIG.

The excitation light LT1 collimated with the one axis AX by the collimating lens 21 reduces light having a wavelength other than a predetermined band in the excitation light bandpass filter EF. The excitation light LT1 that has passed through the excitation light bandpass filter EF is focused by the ball lens 22 toward the solution flow path 32 of the flow cell 30.

In the first embodiment, the excitation light LT1 is focused toward the enzyme retention film 33 by the ball lens 22 so as to become divergent light in the solution of the solution flow path 32. This is on the light source LT side of the enzyme retention membrane 33 on the axis AX where the focusing position of the excitation light LT1 is one (either inside the gas flow path 31 or on the side close to the light source LT of the excitation light LT1 outside the gas flow path 31). This can be achieved. To adjust the focusing position of the excitation light LT1, change the position of the ball lens 22 on one axis AX, change the refractive index of the ball lens 22 to change the focusing position, or replace it with a lens having a short focal length f. This makes it possible.

As shown in FIG. 4, according to the excitation photooptical system 20 of Example 1, the excitation light LT1 is directed to the light source LT side of the enzyme holding film 33 (even in the gas flow path 31 and outside the excitation light LT1 light source LT1. Since the NADH group is excited in the state of the diffusion process in the solution of the solution flow path 32, the focusing rate on the detection surface of the light detection element 60 is as much as possible for the excitation light LT1. It can be adjusted to be low, and the fluorescent LT2 can be adjusted to be as high as possible.

In this way, when the excitation light LT1 is focused toward the enzyme holding film 33 by the ball lens 22, the focusing position is not between the enzyme holding film 33 and the solution in the solution flow path 32, but on the enzyme holding film 33. If it comes to the light source LT side (either inside the gas flow path 31 or near the light source LT of the excitation light LT1 outside the gas flow path 31), the excitation light LT1 becomes a divergent light state in the solution of the solution flow path 32. The excitation optical optical system 20 can be set so as to be. As a result, the collimation of the excitation light LT1 (light guide to the photodetector element 60) can be suppressed by arranging the collimating lens in the subsequent stage.

Further, since the ball lens 22 has a numerical aperture larger than the numerical aperture of the light guide member 40, the excitation light LT1 incident on the light guide path 41 at a sharp angle with respect to one axis AX increases. That is, the amount of excitation light LT1 incident on the light guide path 41 increases at an incident angle larger than the maximum light receiving angle of the light guide path 41. As a result, the amount of excitation light LT1 transmitted through the external light guide path 41 without guiding the light guide member 40 increases.

The excitation light LT1 made parallel by the light receiving side collimating lens 51 is reflected or absorbed by the fluorescent bandpass filter FF.

[Fluorescent ray tracing of fluorescent optical system 50]
In FIG. 3, the alternate long and short dash arrow indicates fluorescent LT2. When the coenzyme receives the excitation light LT1, it emits a fluorescent LT2. The fluorescent LT2 passes through the solution in the solution flow path 32 and enters the light guide member 40. The fluorescent LT2 that has guided the light guide member 40 is emitted from the emission end of the light guide member 40 toward the light receiving side collimating lens 51.

In the fluorescent LT2 collimated by the light receiving side collimating lens 51, light having a wavelength other than a predetermined band is reduced by the fluorescent bandpass filter FF. The fluorescent LT2 that has passed through the fluorescent bandpass filter FF is focused by the condenser lens toward the detection surface of the photodetector 60.

By providing the fluorescent bandpass filter FF in the fluorescent optical system 50, the amount of excitation light reaching the condenser lens 52 is reduced, but the attenuation rate of the fluorescent bandpass filter FF with respect to light other than the transmission band is 0. Therefore, the excitation light cannot be completely removed. Therefore, if the amount of excitation light reaching the detection surface of the photodetector 60 can be further reduced, the fluorescence measurement accuracy can be further improved. At this point, by moving the focusing point (in the gas flow path 31) of the excitation light LT1 away from the light source of the fluorescent LT2 (excited NADH group in the solution flow path 32) as described above, only the excitation light LT1 Can be defocused on the detection surface of the light detection element 60.

In Example 1 in which the focusing position of the excitation light LT1 is in front of the enzyme retention film 33, the effect is shown below using a comparative example.

As a comparative example, an optical system in which the positions of the excitation light condensing point and the fluorescent light source (excited NADH group) are very close to each other as in the prior art is used as a comparative example, and the excitation light condensing point is as in the comparative example and the present embodiment. For each of the optical systems away from the fluorescent light source, the amount of excitation light and fluorescence received by the light detection element when the light receiving side collimating lens 51 was moved along one axis AX direction was simulated.

FIG. 5 shows a comparative example on the detection surface of the photodetector element 60 when the light collecting position of the excitation light is in the solution flow path 32 and the light receiving side collimating lens 51 is moved in the direction of one axis AX. The changes in the light receiving amount S of the fluorescent LT2 and the light receiving amount R of the excitation light LT1 are shown in. Further, in FIG. 6, in the state of FIG. 4 according to the first embodiment (the focusing position of the excitation light LT1 exists on the light source LT side of the enzyme holding film 33), the light receiving side collimating lens 51 is moved in the direction of one axis AX. The changes in the light receiving amount S of the fluorescent LT2 and the light receiving amount R of the excitation light LT1 are shown on the detection surface of the photodetector element 60 at that time. In the case of the comparative example, the light receiving amount R of the excitation light LT1 is close to the bottom at the Pc position, but the light receiving amount S of the fluorescent LT2 is also at the bottom position, and the S / N ratio as an optical system is not optimal.

On the other hand, the ball lens 22 is moved from the Pc position of the comparative example in the direction of one axis AX to the side closer to the light source LT of the excitation light LT1 so that the focusing position of the excitation light LT1 comes in front of the enzyme retention film 33. In this state, changes in the amount of light received by the fluorescent LT2 and the excitation light LT1 when the light receiving side collimating lens 51 is moved in the direction of one axis AX are as shown in FIG. When the light receiving side collimating lens 51 is set to the position P1 in FIG. 6, the light receiving amount of the fluorescence LT2 is close to the maximum, and at the same time, the light receiving amount of the excitation light LT1 can be minimized. Since it is preferable that the fluorescence optical system 50 is set so that S (the amount of light received by the fluorescent LT2) / R (the amount of light received by the excitation light LT1) is maximized, the fluorescent optical system 50 does not undergo a chemical reaction. In the state, the ratio (S / R) of the light amount of the fluorescent LT2 to the light amount of the excitation light LT1 detected by the light detection element 60 is configured to be equal to or higher than a predetermined ratio.

As a result, according to the present embodiment, the fluorescence optical system 50 is configured such that the excitation light LT1 that has passed through the light guide member 40 is defocused on the detection surface of the photodetector element 60. The fluorescence optical system 50 is arranged along one axis AX, and at least a part of the fluorescence LT2 that has passed through the light guide member 40 is focused on the detection surface of the photodetector element 60. Therefore, it is possible to reduce the amount of light received by the excitation light LT1 detected on the detection surface of the photodetector element 60, and it is possible to improve the detection accuracy.

FIG. 7 is a cross-sectional view showing the configuration of the detection device according to the second embodiment of the present invention. FIG. 8 is an enlarged partial cross-sectional view illustrating the main part M2 around the focusing point of the excitation light LT1 shown in FIG.

In this embodiment, as shown in FIGS. 7 and 8, the focusing position of the excitation light LT1 is placed on the light source LT side of the enzyme holding film 33, and the excitation light LT1 is in a divergent light state in the solution of the solution flow path 32. Instead of doing so, the optical system is configured so that the excitation light LT1 is in the state of the focusing process in the solution of the solution flow path 32, except that the focusing position of the excitation light LT1 is in the light guide member 40. It is the same as the first embodiment.

The excitation light LT1 of this embodiment adjusts the focusing point by changing the position of the ball lens 22, changing the refractive index of the ball lens 22 to change the focusing position, or replacing the lens with a lens having a long focal length f. , Can be achieved. By condensing the excitation light LT1 in the light guide member 40 and exciting the solution in the solution flow path 32 in the state of the condensing process, the condensing point of the excitation light LT1 is set to the light source of the fluorescent LT2 (excited NADH group). ), Only the excitation light LT1 can be defocused on the detection surface of the light detection element 60.

In FIG. 9, the ball lens 22 is moved so that the focused position of the excitation light LT1 is in the light guide member 40 according to the second embodiment, and then the light receiving side collimating lens 51 is moved to the Pc position of the comparative example (FIG. 5). The change of S (light reception amount of fluorescence LT2) / R (light reception amount of excitation light LT1) of fluorescence LT2 and excitation light LT1 when moved in the direction of one axis AX is shown. When the light receiving side collimating lens 51 is set to position P2 (near the maximum that receives the second largest amount of fluorescent light and the position that minimizes the amount of received excitation light), the amount of light received by the fluorescent LT2 is relative to the case of the comparative example (Pc position). S increases, and the light receiving amount R of the excitation light LT1 becomes close to the minimum. At this time, the received amount S of the fluorescent LT2 is lower than that of the first embodiment, but it is the same as that of the first embodiment in the sense that the received amount of the excitation light LT1 which is a deterioration factor of the S / N ratio is minimized. The effect of is obtained.

FIG. 10 describes a main part M3 of the focused state of the excitation light LT1 in the gas flow path 31, the solution flow path 32, and the enzyme retention film 33 along one axis AX in the detection device according to the third embodiment of the present invention. It is an enlarged partial cross-sectional view.

As shown in FIG. 10, this embodiment is the same as that of the second embodiment except that the excitation optical optical system 20 is arranged on one axis AX and includes a light-shielding film 70 capable of blocking the excitation light LT1. Is.

This example is a modification of Example 2 of the optical system in which the excitation light LT1 is in the state of condensing in the solution of the solution flow path 32, and the light guide member 40 is a solution on one axis AX. A light-shielding film 70 that blocks at least a part of the excitation light LT1 is provided on the flow path 32 side. That is, a light-shielding film 70 for shielding the excitation light LT1 may be provided on the light receiving surface of the light guide member 40 irradiated with the excitation light LT1.

According to this embodiment, the excitation light LT1 incident on the light guide member 40 can be reduced, so that the detection sensitivity of the fluorescent LT2 can be increased (the S / N ratio can be further increased).

The light-shielding film 70 may be an absorbent film or a reflective film. In the case of the reflective film, the coenzyme is excited again by the reflected excitation light LT1, so that the excitation efficiency of the excitation light LT1 can be increased.

The light-shielding film 70 of this embodiment also generates fluorescent LT2 that cannot be incident on the light guide member 40. However, as shown in the figure, fluorescent LT2 is synchrotron radiation emitted from each coenzyme in all directions. Some synchrotron radiation enters the light guide member 40 without being shielded by the light shielding film 70. On the other hand, since the excitation light LT1 is focused, the light in the portion toward the light-shielding film 70 cannot enter the light guide member 40.

Further, the light-shielding film 70 may cover the entire spot of the excitation light LT1 on the light receiving surface of the light guide member 40, or cover only a part including the center of the excitation light LT1 beam having a particularly high energy density. It may be hidden.

As described above, according to the biosensor 10 according to the present embodiment, the excitation light LT1 traveling straight on one axis AX can be blocked. Further, the incident angle of the excitation light LT1 incident on the light guide member 40 can be increased, and a large amount of the excitation light LT1 can be prevented from being guided in the light guide path 41. As a result, the amount of light received by the excitation light LT1 detected on the detection surface of the photodetector 60 can be reduced, and the detection accuracy can be improved.

FIG. 11 shows a cross section of the biosensor 10 as the detection device according to the fourth embodiment of the present invention along one axis AX.

In this embodiment, as shown in FIG. 11, the wall thickness of the plano-convex lens 22a or the like having a plane for emitting the excitation light LT1 instead of the thick ball lens 22 of the condenser lens in the excitation optical optical system 20 is thin. It is the same as the above embodiment except that it is replaced with a condenser lens.

Due to the shape of the ball lens 22, the lens becomes thicker and the back focus distance becomes shorter, so that the moving range in the AX direction of one axis becomes narrower. According to this embodiment, by replacing the ball lens with the plano-convex lens 22a, it is possible to improve the problem that the moving range of the condenser lens is narrowed.

Further, it is considered that the plano-convex lens 22a is more cost effective than the ball lens 22. At this time, by making the focal length f longer than that of the ball lens 22 by the condensing lens 22a of the plano-convex lens, the change in the condensing state of the excitation light LT1 when moved on one axis AX becomes gentle. Easy to adjust.

The equipment, the configuration of the device, etc. in the above-described embodiment are merely examples, and can be appropriately selected or changed depending on the intended use and the like. For example, this device is a biosensor that selectively detects a small amount of organic volatile components contained in a gas using an enzyme, and is used for environmental measurement, medical biodiagnosis, exercise physiology measurement, production line measurement, etc. Will be done.

10 Biosensor 20 Excitation optical optical system 30 Flow cell 31 Gas flow path 32 Solution flow path 33 Enzyme holding film 40 Light guide member 41 Light guide path 50 Fluorescent optical system 51 Light receiving side collimating lens 52 Condensing lens 60 Light detection element 70 Light shielding film AX One axis

Claims (9)

A gas flow path through which a gas sample flows, a solution flow path through which a solution containing a coenzyme that emits fluorescence when irradiated with excitation light flows, and the gas flow path and the solution flow path while being in contact with the solution and the gas sample. A reaction section including an enzyme-holding membrane that is formed so as to separate the two and holds an enzyme that catalyzes the chemical reaction of the gas sample by binding to the coenzyme.
An excitation optical optical system that irradiates the enzyme holding membrane with the excitation light from the gas flow path side of the reaction unit along one axis.
A light guide member provided on the one axis so as to receive the fluorescence and forming a light guide path along the one axis.
With
The excitation photooptical system collects the excitation light on the gas flow path side of the enzyme holding film on the one axis or in any one region in the light guide member on the one axis. A detection device characterized in that it is configured to. A fluorescence optical system arranged along the one axis and condensing at least a part of the fluorescence that has passed through the light guide path on the detection surface of the photodetector is provided.
The detection device according to claim 1, wherein the fluorescence optical system is configured such that the excitation light passing through the light guide path is defocused on the detection surface. The fluorescence optical system is configured such that the ratio of the amount of fluorescence light to the amount of excitation light detected by the photodetector is equal to or greater than a predetermined ratio in a state where the chemical reaction does not occur. The detection device according to claim 2, wherein the detection device is characterized. The detection device according to any one of claims 1 to 3, wherein the excitation optical optical system is composed of a numerical aperture larger than the numerical aperture of the light guide member. The detection device according to any one of claims 1 to 4, wherein a space is provided along a side surface of the light guide member extending in a direction along the one axis. The detection device according to any one of claims 1 to 5, wherein the enzyme-retaining membrane is a porous membrane. The detection device according to any one of claims 1 to 6, wherein the excitation optical optical system has a plano-convex lens having a plane for irradiating the enzyme holding film with the excitation light. The detection device according to any one of claims 1 to 7, wherein the light guide member has a light-shielding film that blocks at least a part of the excitation light on the solution flow path side on the one axis. .. The detection device according to any one of claims 1 to 8, wherein an optical filter that transmits a band including a wavelength of fluorescence is provided between the light guide member and the detection surface.

Images