WO2012090919A1

WO2012090919A1 - Chip for light sensor, light sensor, measurement system, and measurement method using same

Info

Publication number: WO2012090919A1
Application number: PCT/JP2011/080032
Authority: WO
Inventors: 健次浜地; 浩二三林; 寛之工藤
Original assignee: ローム株式会社; 国立大学法人東京医科歯科大学
Priority date: 2010-12-27
Filing date: 2011-12-26
Publication date: 2012-07-05

Abstract

The present invention is a chip (2) for a light sensor coupled to and used with an optical fiber (1), the chip for a light sensor characterized by comprising a first gas-permeable membrane (22) for covering a distal end of the optical fiber (1), and comprising a liquid chamber (21) for causing a liquid to flow to the surface of the first gas-permeable membrane (22) on the side opposite from the side that is coupled to the optical fiber (1).

Description

Optical sensor chip, optical sensor, measurement system, and measurement method using the same

The present invention relates to an optical sensor chip, an optical sensor, a measurement system, and a measurement method using the same.

So far, as biosensors, enzyme sensors for measuring glucose, lactic acid, cholesterol and the like have been developed and used in fields such as the medical and food industries. A biosensor refers to a sensor that utilizes the molecular recognition ability of biological materials such as microorganisms, enzymes, and antibodies, and applies the biological materials as molecular identification elements. In other words, the biosensor converts the reaction that occurs when the immobilized biological material recognizes the target substrate, the consumption of the enzyme due to the respiration of microorganisms, the enzymatic reaction, luminescence, etc. into an electrical signal or the like by the physicochemical device. To measure.

In the conventional general enzyme sensor, the electron acceptor produced by the reaction between the substrate and the enzyme contained in the sample liquid which is the specimen is reduced, and the reduction amount of the electron acceptor is reduced using a Clark-type oxygen electrode or the like. Quantitative analysis of specimens has been performed by electrochemical measurement.

In recent years, analysis methods using optical fiber sensors (optical sensors) have been developed in place of such electrochemical analysis. As an analysis device using an optical fiber sensor, an oxygen-sensitive optical fiber that measures the oxygen concentration by fixing the ruthenium complex to the tip of the optical fiber is used, which utilizes the phenomenon that the fluorescence reaction of the ruthenium complex is quenched by the surrounding oxygen concentration. Has been developed.

Patent Document 1 (Japanese Patent Laid-Open No. 2003-250516) discloses an optical sensor (odor sensor) in which an enzyme-immobilized film is brought into close contact with the tip of an oxygen-sensitive optical fiber. In the optical sensor disclosed in Patent Document 1, a buffer solution for washing the enzyme reaction product remaining on the enzyme-immobilized membrane is circulated around the optical fiber so that continuous measurement is possible. Yes.

However, in the optical sensor disclosed in Patent Document 1 shown in FIG. 13, eddy and stagnation are likely to occur in the flow of the buffer solution around the enzyme membrane, and the output may become unstable due to the disturbance of the flow. In addition, since the buffer solution is not in direct contact with the central part of the enzyme-immobilized membrane located at the tip of the optical fiber, the most affected part of the detection is not washed quickly, and the amount of substances in the environment outside the enzyme membrane changes. There was a problem that the responsiveness to becomes worse.

In addition, since the tip of the optical fiber is only covered with a dialysis membrane or the like on which an enzyme is immobilized, and environmental light may enter the optical fiber, there is a problem that it is susceptible to environmental light.

JP 2003-250516 A

An object of the present invention has been made in view of the above problems, and provides a chip for an optical sensor having excellent sensor characteristics that enables sensor output to be stabilized and continuous measurement with good responsiveness. That is. Another object of the present invention is to provide an optical sensor chip that enables measurement without being affected by ambient light.

The present invention is an optical sensor chip used in connection with an optical fiber,
A first gas permeable membrane covering the tip of the optical fiber;
An optical sensor chip comprising a liquid chamber for supplying a liquid to a surface of the first gas permeable membrane opposite to the side connected to the optical fiber.

The first gas permeable membrane is preferably an enzyme-immobilized membrane on which an enzyme that catalyzes a chemical reaction of the target substance is immobilized.

It is preferable to have at least one flow path that communicates the liquid chamber and the outside of the optical sensor chip.

It is preferable that the liquid chamber is a closed space not communicating with the outside of the optical sensor chip, or a space having a liquid inlet and an air hole.

The liquid chamber includes a second gas permeable membrane on a side opposite to the first gas permeable membrane,
It is preferable to have a stopper mechanism for making the distance between the tip of the optical fiber and the second gas permeable membrane constant when the optical fiber is connected.

The enzyme is preferably an oxidoreductase, a dehydrogenase or a luminescent enzyme.
The oxidoreductase is preferably an enzyme that consumes or generates oxygen by reacting with a substrate.

The optical fiber is preferably an oxygen sensitive optical fiber, a pH sensitive optical fiber or a light emission sensitive optical fiber.

It is preferable that the optical fiber is an oxygen-sensitive optical fiber, and a ruthenium organic complex is fixed to the tip of the optical fiber.

The present invention also relates to an optical sensor formed by connecting the optical sensor chip and an optical fiber.

The present invention also relates to a measurement system including at least one optical sensor.
The measurement system preferably includes a mechanism for replacing the liquid in the liquid chamber of the optical sensor chip.

The measurement system is preferably for measuring odor.
Furthermore, the present invention also relates to a method for measuring a target substance using the measurement system.

During the measurement, it is preferable that the liquid in the liquid chamber does not move and the liquid in the liquid chamber moves between each measurement.

It is preferable to calculate the concentration of the target substance based on the differential value, integral value, integrated value or second-order differential value of the measured light output over time.

It is preferred that the liquid in the liquid chamber moves during the measurement.
The first gas permeable membrane is an enzyme-immobilized membrane on which an enzyme that catalyzes a chemical reaction of a target substance is immobilized.
As the enzyme, an oxidoreductase that consumes or generates oxygen by reacting with a substrate is used.
The substrate is methyl mercaptan, dimethyl sulfide, hydrogen sulfide, ammonia, or trimethylamine;
It is preferable to add ascorbic acid to the liquid.

The concentration of the ascorbic acid is preferably 10 mM or less.

According to the present invention, it is possible to provide a chip for an optical sensor having excellent sensor characteristics that enables a sensor output to be stabilized and continuous measurement with good responsiveness. In addition, according to the present invention, it is possible to provide an optical sensor chip that enables measurement without being affected by ambient light.

It is an enlarged view of the front-end | tip part of Embodiment 1 of the optical sensor of this invention. (A) is a longitudinal sectional view, (b) is a sectional view taken along the plane A-A ′, and (c) is a sectional view taken along the line B-B ′. It is an enlarged view of the front-end | tip part of Embodiment 2 of the optical sensor of this invention. (A) is a longitudinal sectional view, and (b) is a sectional view along the A-A ′ plane. It is an enlarged view of the front-end | tip part of the optical fiber in which the silicone overcoat was formed. It is the schematic of the measurement system of Embodiment 3. It is the schematic of the measurement system of Embodiment 4. 3 is a graph showing measurement results of Example 1. It is the elements on larger scale of the graph of FIG. FIG. 8 is a further partial enlarged view of FIG. 7. It is a graph which shows the calibration curve produced from the inclination of the graph of FIG. 8, and the density | concentration of each ethanol gas. It is a comparison figure of the measurement result of the optical sensor of this invention (Example 1), and the conventional semiconductor sensor. 6 is a graph showing measurement results of Reference Example 1. It is the graph which plotted the relationship between the addition density | concentration of sodium ascorbate in FIG. 11, and the sensor output in predetermined time. It is a schematic cross section of the conventional optical sensor.

<Optical sensor chip>
The optical sensor chip of the present invention is a member used in connection with an optical fiber,
A first gas permeable membrane on which an enzyme that catalyzes a chemical reaction of a target substance is immobilized;
A liquid chamber is provided for supplying a liquid to the surface of the first gas permeable membrane opposite to the side connected to the optical fiber.

(Optical fiber)
Examples of the optical fiber include an oxygen sensitive optical fiber, a pH sensitive optical fiber, and a light emission sensitive optical fiber. Oxygen-sensitive optical fiber is an optical fiber that can detect oxygen concentration by utilizing the phenomenon that fluorescence reaction is quenched by ambient oxygen concentration. PH-sensitive optical fiber and luminescence-sensitive optical fiber measure pH and luminescence, respectively. An optical fiber that can be used.

As the oxygen-sensitive optical fiber, an optical fiber in which the ruthenium organic complex is immobilized on the optical fiber can be suitably used by utilizing the phenomenon that the fluorescence reaction of the ruthenium organic complex is quenched by the surrounding oxygen concentration. As another oxygen-sensitive optical fiber, for example, an optical fiber in which an organic complex such as platinum, osmium, iridium, rhodium, rhenium and chromium is immobilized can be used.

Examples of organic complexes include ruthenium and the like, 2,2′-bipyridine, 1,10-phenanthroline, 4,7-diphenyl-1,10-phenanthroline, 4,7-dimethyl-1,10-phenanthroline, 4,7- Disulfonated diphenyl-1,10-phenanthroline, 2,2′-bi-2-thiazoline, 2,2′-bithiazole, 5-bromo-1,10-phenanthroline, 5-chloro-1,10-phenanthroline and the like And the like.

Among these, tris (2,2′-bipyridyl) ruthenium dichloride represented by the following chemical formula (1) is preferably used.

The method for fixing the ruthenium complex or the like to the tip of the optical fiber is not particularly limited, but can be fixed by, for example, a sol-gel method. Fluorescence reaction of ruthenium complex (excitation light: 470 nm, fluorescence: 600 nm) shows a quenching phenomenon depending on the oxygen and dissolved oxygen concentration in the gas phase and liquid phase in the presence of oxygen, so the measurement of oxygen concentration Is possible.

As the oxygen-sensitive optical fiber, a commercially available optical fiber can be used. For example, an optical fiber manufactured by Ocean Optisk Corporation can be used. The diameter of the optical fiber to be used can be selected according to the application, and a diameter of about 1.5 mm is usually used. However, the diameter of the optical fiber is not limited to this and is about 0.01 mm to 5.0 mm. Ranges can be used.

At the tip of the optical fiber, for example, a silicone overcoat 12 may be formed on a ruthenium organic complex 11 as shown in FIG. This has the effect of blocking ambient light that becomes measurement noise. Even when a silicone overcoat or the like is not formed at the tip of the optical fiber, for example, the same light-shielding effect can be obtained by forming the main body of the optical sensor chip coupled to the tip of the optical fiber from a material that is not light-transmissive. be able to. Although the presence of the silicone overcoat can block ambient light, the diffusion of oxygen is suppressed and the responsiveness is delayed. Therefore, the responsiveness is much faster when there is no overcoat.

(First gas permeable membrane)
The first gas permeable film is a film made of a material that transmits at least gas, and is provided so as to cover the tip of the optical fiber. Moreover, it is preferable that it is a material which does not permeate | transmit a liquid.

The first gas permeable membrane is preferably an enzyme-immobilized membrane on which an enzyme that catalyzes a chemical reaction of the target substance is immobilized. However, the first gas permeable membrane does not necessarily have an enzyme immobilized thereon, and the target substance can be measured by using an enzyme solution to which the enzyme is added as a buffer solution supplied into the liquid chamber. Can do.

As the base material of the first gas permeable membrane, dialysis membrane, hydrophilic polytetrafluoroethylene (H-PTFE) membrane, polycarbonate membrane, cellulose mixed ester membrane, cellulose acetate membrane, hydrophilic polyvinylidene fluoride (H-PVDF) ) Etc. are used. As the dialysis membrane and the H-PTFE membrane, commercially available ones are used without any limitation, and those having a film thickness of about 15 μm are usually used, but are not limited to those having a film thickness of about 15 μm. Specifically, for example, UC 36-32-100 (Viscose Corporation Inc., molecular weight cut off 14000, pore size 5 nm) is used as a dialysis membrane, and JGWP 14225 (Milipore Corporation, pore size 0.1 μm) is used as an H-PTFE membrane. Can be mentioned.

Examples of the enzyme that is immobilized when the first gas permeable membrane is an enzyme-immobilized membrane include oxidoreductase, dehydrogenase, and luminescent enzyme. The oxidoreductase used in the biosensor of the present invention is an enzyme that consumes or generates oxygen by reacting with a substrate. Such an oxidoreductase can be selected according to the target substance (substrate) to be measured. For example, alcohol oxidase is used to measure the concentration of ethanol, and flavin-containing monooxygenase is used to measure the concentrations of trimethylamine, methyl mercaptan and ammonia. Examples of other oxidoreductases include catalase, monoamine oxidase, glucose oxidase, uricase, invertase, mutarotase, phospholipase, choline oxidase, amino acid oxidase, ascorbate oxidase, creatine kinase, luciferase, lactate oxidase, cholesterol oxidase, methyl mercaptan oxidase Xanthine oxidase, tyrosinase, galactose oxidase and the like.

Examples of the method for preparing the enzyme-immobilized membrane include a comprehensive method using a photocrosslinkable resin, a crosslinking method, an adsorption method, and the like. Among them, a comprehensive method using a photocrosslinkable resin is generally used. Hereinafter, the inclusion method using a photocrosslinked resin will be described.

Examples of the photocrosslinkable resin include polyethylene glycol, polyvinyl alcohol, and the like. PVA-SbQ, Biosurfine-SPH, etc. manufactured by Toyo Gosei Kogyo Co., Ltd., in which polyvinyl alcohol is combined with a photosensitive group of SbQ, are used. Can do.

Using such a photocrosslinkable resin, a paste of a mixture containing an enzyme and a photocrosslinkable resin is applied to and impregnated on a substrate such as a dialysis membrane, and then the photocrosslinkable resin is crosslinked so that the enzyme becomes a substrate. An enzyme-immobilized membrane immobilized on can be prepared. Examples of the solvent used when producing the paste include a buffer solution, distilled water, and ion-exchanged water.

(Liquid chamber)
The optical sensor chip of the present invention includes a liquid chamber for supplying a liquid to the surface of the first gas permeable membrane opposite to the side connected to the optical fiber.

Here, the liquid is not particularly limited as long as it is a liquid that can clean the first gas permeable membrane at the tip of the optical fiber, and examples thereof include a buffer solution. When the first gas permeable membrane is an enzyme-immobilized membrane, it is preferable to use a buffer solution having a pH near the optimum pH of the enzyme immobilized on the enzyme-immobilized membrane, but the enzyme is immobilized. This restricts the movement of the protein, so that the optimum pH is likely to shift as compared with the enzyme in the solution. Due to the cleaning effect of the liquid, the target substance and the product by the enzyme reaction do not remain in the first gas permeable membrane at the tip of the optical fiber, and continuous or intermittent repeated measurement is possible.

Further, when the first gas permeable membrane is an enzyme-immobilized membrane, the buffer solution can contain additives necessary for the enzyme reaction of the enzyme immobilized on the enzyme-immobilized membrane. The additive can be appropriately selected according to the type of enzyme used and the type of target substance (substrate), and examples thereof include a reducing agent. For example, when measuring methyl mercaptan or the like using an oxidase such as monoamine oxidase, the amount of the enzyme reaction product is reduced by reducing the oxidized methyl mercaptan produced by the enzyme reaction with a reducing agent and subjecting it again to the enzyme reaction. The sensitivity can be increased by increasing the sensitivity. Examples of the reducing agent include ascorbic acid, dithiothreitol, NADH, and NADPH.

The liquid in the liquid chamber is preferably in a state where it can directly contact the first gas permeable membrane. As a result, the target substance attached to the first gas permeable membrane can be quickly cleaned. In this case, since the remaining of the target substance on the first gas permeable film is suppressed, the responsiveness to the change in the concentration of the measurement object during continuous measurement is improved as compared with the conventional sensor as shown in FIG.

The optical sensor chip of the present invention preferably has at least one flow path that communicates the liquid chamber and the outside of the optical sensor chip. In this case, the flow path is preferably connected to a mechanism for replacing the liquid in the liquid chamber.

Alternatively, the liquid chamber may be a closed space that does not communicate with the outside of the optical sensor chip, or a space having a liquid inlet and an air hole. In addition, the airtightness of the closed space does not necessarily need to be airtight, and may be liquid tight. In this case, the liquid chamber can be filled in advance with a liquid and used as a disposable optical sensor chip. By using such a chip, a system for supplying a liquid at the time of measurement becomes unnecessary, and it becomes possible to carry out measurement easily.

In particular, in a reaction system that can use oxidoreductases such as methyl mercaptan, the oxide of the measurement target substance can be reduced using an optimal reducing agent, which can be recycled for measurement, and the sensor output can be amplified. Is possible. Therefore, it is preferable to amplify the output by storing a liquid containing ascorbic acid or the like in the liquid chamber for a certain time, and when the liquid chamber is a closed space or a space having a liquid inlet and an air hole, It can be used suitably.

<Optical sensor>
The present invention also relates to an optical sensor formed by connecting the optical sensor chip and the optical fiber. Specific combinations of enzymes and optical fibers used in the optical sensor of the present invention are exemplified below.

When measuring methyl mercaptan, a combination of monoamine oxidase (MAOA) and an oxygen-sensitive optical fiber can be used. The following MAOA representative reactions:
R—CH ₂ —NH ₂ + H ₂ O + O ₂ → R—CHO + NH ₃ + H ₂ O ₂
The amount of oxygen consumed is measured by an oxygen-sensitive optical fiber. In the case of measurement of methyl mercaptan, the following reaction is caused by the reaction diversity of MAOA:
2CH ₃ SH + O ₂ → CH ₃ SSCH ₃ + H ₂ O ₂
Is considered to be performed.

When measuring glucose in a solution, a combination of glucose oxidase (GOD) and an oxygen-sensitive optical fiber can be used. The amount of oxygen consumed by the oxidation reaction of glucose is measured with an oxygen-sensitive optical fiber.

When measuring alcohol in the solution, a combination of alcohol dehydrogenase and pH-sensitive optical fiber can be used. Since hydrogen is desorbed from alcohol by alcohol dehydrogenase and the pH in the solution changes, the pH change is detected by a pH-sensitive optical fiber.

As another combination for measuring the alcohol in the solution, a combination of alcohol oxidase, luminescent luciferase, and luminescence detection type optical fiber can be used. Luminescent luciferase measures hydrogen peroxide produced by the consumption of oxygen by alcohol oxidase based on the intensity of luminescence of the luminescent luciferase that consumes hydrogen peroxide to emit light.

When measuring an aldehyde such as acetaldehyde in a solution, a combination of an aldehyde dehydrogenase and a pH-sensitive optical fiber can be used. Since hydrogen is desorbed from the aldehyde by aldehyde dehydrogenase and the pH in the solution changes, the pH change is detected with a pH-sensitive optical fiber. In addition, formaldehyde can be measured by using formaldehyde dehydrogenase.

<Measurement system>
The measurement system of the present invention includes at least one optical sensor. One optical sensor may be provided, and when a plurality of optical sensors having different measurement targets are provided, a plurality of target substances can be detected simultaneously.

The measurement system preferably includes a mechanism for replacing the liquid in the liquid chamber of the optical sensor chip. As a mechanism for replacing the liquid, for example, a pump capable of feeding the liquid into the liquid chamber may be used.

The measurement target of the measurement system is not particularly limited, but can be suitably used for measuring odor.

<Measurement method>
The present invention also relates to a measurement method using the measurement system.

As an embodiment of the measurement method of the present invention, there is a method in which the liquid in the liquid chamber does not move during the measurement, and the liquid in the liquid chamber moves between each measurement. In this method, the liquid in the liquid chamber is stored during the measurement, and the enzyme reaction necessary for the measurement can be sufficiently advanced, so that the measurement sensitivity can be improved. In addition, when the measurement target substance is contained in the gas, the substance permeates and diffuses through the second gas permeable membrane, and the concentration in the stored liquid continues to increase. It is advantageous to make.

In this case, it is preferable to calculate the concentration of the target substance based on the differential value, integral value, integrated value, or second-order differential value of the measured light output with time. As a result, the measurement time can be shortened. It is desirable to construct a system that automatically performs such calculations using an analysis program. Here, “light output” is a concept including “intensity of light of a specific wavelength”, “shift amount of a specific wavelength”, and the like.

As another embodiment of the measurement method of the present invention, there is a method in which the liquid in the liquid chamber moves during the measurement. Used when you want to monitor the target substance continuously.

In the measurement method of the present invention, when the target substance (substrate) is methyl mercaptan, dimethyl sulfide, hydrogen sulfide, ammonia, or trimethylamine, an oxidoreductase that consumes or generates oxygen by reacting with the substrate is used as the enzyme. It is preferable to add ascorbic acid to the liquid. At this time, the concentration of ascorbic acid added is preferably 10 mM or less. Sensors and Actuators B: Chemical Volume 108, Issues 1-2, Pages 639-645 (July 22, 2005) have been released as related technologies for the effects of ascorbic acid concentration.

(Embodiment 1)
FIG. 1 shows an enlarged view of the tip portion of the photosensor according to Embodiment 1 of the present invention. In FIG. 1, the optical sensor chip 2 includes a chip body 20 including a liquid chamber 21, an enzyme-immobilized film (first gas permeable film) 22, and a second gas permeable film 23. As shown in FIG. 1, an enzyme-immobilized film 22 is in close contact with the tip of the optical fiber 1, and is fixed by a chip body 20 of a chip for optical sensors. The second gas permeable membrane 23 may be fixed to the tip of the chip body 20 with an O-ring or the like, or may be bonded and integrated.

As the enzyme-immobilized membrane 22, the same enzyme-immobilized membrane as described above can be used.
The second gas permeable membrane 23 is a membrane made of at least a gas permeable material, and is preferably a material that does not allow liquid to pass through when measuring a target substance in the gas. Specific examples include dialysis membranes, H-PTFE membranes, H-PVDF membranes, and the like. Further, the same enzyme as the enzyme immobilized on the enzyme immobilization membrane 22 may be immobilized on the second gas permeable membrane 23. Thereby, the production | generation of more enzyme reaction products and the substance consumption concerning an enzyme reaction are accelerated | stimulated, and it becomes possible to improve detection sensitivity.

In this embodiment, the introduction tube 211 and the discharge tube 212 are connected by a single liquid chamber 21 (flow path) that passes through the surface of the enzyme-immobilized membrane 22 on the side opposite to the optical fiber 1. As a result, the liquid can circulate stably and smoothly on the surface of the enzyme-immobilized film 22. The shape of the liquid chamber 21 is preferably a very simple one-way flow path or the like that suppresses the generation of swell and stagnation during liquid flow.

By making the surface of the enzyme immobilization film 22 in contact with the liquid in the liquid chamber 21, it is possible to efficiently remove (replace) reaction products and the like due to the enzyme reaction. Thus, a structure in which a flow path (liquid chamber) is provided between the enzyme-immobilized membrane 22 and the second gas permeable membrane 23 has not been known so far.

By producing the chip body 20 using a material that is not light transmissive (for example, black acrylic resin), it is possible to eliminate the influence of ambient light that enters the optical fiber 1 and becomes measurement noise. Further, even when a non-light-transmitting material is not used, the same light shielding effect can be obtained by making the structure of the chip body 20 less susceptible to ambient light around the enzyme-immobilized film 22 at the tip of the optical fiber 1. Can do.

The chip body 20 provided with the liquid chamber 21, the enzyme-immobilized film 22, and the second gas permeable film 23 are integrated into a single optical sensor chip 2, so that the entire chip is replaced for each measurement. Such a usage method (disposable) becomes possible, and preparation for measurement can be easily performed.

The optical sensor chip 2 preferably has a stopper mechanism for making the distance between the tip of the optical fiber 1 and the second gas permeable film 23 constant when the optical fiber 1 is connected. In FIG. 1A, the distance between the tip of the optical fiber 1 and the second gas permeable membrane 23 is kept constant. This is because the width of the liquid chamber 21 provided on the front end side of the chip body 20 is slightly narrower than the diameter of the hole provided for inserting the optical fiber 1 on the rear end side. This is because the optical fiber 1 stops at the position of FIG. 1A when inserted (FIG. 1B, which is a cross-sectional view taken along the line AA ′ of FIG. 1A, and FIG. FIG. 1C is a cross-sectional view taken along the line BB ′ of FIG.

The distance between the tip of the optical fiber 1 and the second gas permeable membrane 23 is preferably 0.05 to 0.6 mm. If the distance is too short, the liquid flow tends to be disturbed. On the other hand, if the distance is longer than this, the distance until the target substance reaches the enzyme-immobilized membrane 22 becomes longer, and it takes time to reach the target substance, resulting in poor responsiveness. In addition, since the target substance is diluted before reaching the enzyme-immobilized membrane 22, the measurement sensitivity decreases when compared in the same measurement time.

(Embodiment 2)
An optical sensor according to a second embodiment of the present invention will be described with reference to FIG. FIG. 2 is an enlarged view of the tip portion of the photosensor according to Embodiment 2 of the present invention. (A) is a longitudinal sectional view, and (b) is an AA ′ sectional view of FIG. 1 (a). The optical sensor of the present embodiment shown in FIG. 2 is different from the first embodiment in that the liquid chamber 21 (liquid reservoir) is a closed space that is not in communication with the outside. Since the other points are the same as those in the first embodiment, description thereof is omitted.

As shown in FIG. 2, the liquid chamber 21 is provided so as to surround the periphery of the enzyme-immobilized membrane (first gas permeable membrane) 22. The thickness of the liquid chamber 21 is not particularly limited, but is preferably about 2 mm or more. The volume of the liquid chamber 21 is not particularly limited, but is preferably about 20 μL or more. This is because if the amount of the solution accumulated in the liquid chamber is too small, the solution is dried immediately.

The optical sensor having such a liquid reservoir structure (liquid chamber 21) can improve the measurement sensitivity by collecting the reaction product of the dissolved target substance (such as odor substance) in the liquid reservoir. It becomes.

In this case, the measurement may be performed after waiting until the concentration of the reaction product reaches a steady state in the reservoir, but the rate of change in light output when the reaction product due to the enzyme reaction accumulates in the reservoir. By monitoring (differential value), measurement is possible even when the reaction product has a low concentration, and the measurement result can be obtained in a short time.

Similarly to the first embodiment, the chip body 20 including the liquid chamber 21 (liquid reservoir structure), the enzyme-immobilized film 22 and the second gas permeable film 23 are integrated into one chip. Thus, a method of use (disposable) in which the entire chip is replaced for each measurement becomes possible, and preparation for measurement can be easily performed.

Further, instead of using the liquid chamber 21 as a flow path as in the first embodiment, a liquid storage structure is provided, so that the liquid feeding system can be omitted and the measurement system can be greatly simplified. In the conventional method, when there is no liquid feeding system and there is no liquid reservoir structure, the enzyme-immobilized membrane starts to dry immediately, so that a sufficient sensor output cannot be obtained and the output is not stable. In addition, when there is a liquid delivery system, a buffer solution is supplied to the enzyme-immobilized membrane by the liquid delivery, and drying can be prevented, but a liquid delivery pump and piping are required, and the operation is not simple and downsized.・ It was disadvantageous for price reduction.

(Embodiment 3)
The present embodiment relates to a measurement system and a measurement method for measuring a target substance (such as an odor substance) in a sample gas using the optical sensor of the first embodiment. An outline of the measurement system of the present embodiment is shown in FIG.

The optical sensor chip 2 is attached to the tip of the optical fiber 1, and the excitation light emitted from the light source 31 reaches the rubidium organic complex or the like at the tip of the optical fiber 1, and the intensity according to the amount of oxygen bound to the complex Is excited. The excited fluorescence is measured by the spectrometer 32 through the optical fiber 1 and the intensity thereof is analyzed by the computer 33. For example, when oxygen is consumed by the enzymatic reaction of the target substance (substrate), the concentration of the target substance can be measured based on the amount of increase in fluorescence intensity after measurement relative to the fluorescence intensity before measurement.

The sample gas containing the target substance is sent into the reservoir 5 and loaded on the tip of the optical sensor chip 2. The sample gas may be continuously fed into the reservoir 5 at a constant speed, or may be stored in the reservoir 5 for a certain period of time after a certain amount of sample gas has been fed into the reservoir 5.

As the optical sensor chip 2, an optical sensor chip having a flow channel-like liquid chamber as described in the first embodiment with reference to FIG. 1 can be used. A liquid such as a phosphate buffer is fed into the flow channel-shaped liquid chamber of the optical sensor chip 2 by the pump 41. At this time, the liquid feeding amount is adjusted by the flow rate adjusting valve 42. The liquid feeding may be continuous or intermittent, and the liquid feeding amount can be appropriately set according to the measurement purpose.

(Embodiment 4)
The present embodiment relates to a measurement system and a measurement method for dissolving a target substance in a sample gas in a liquid and measuring the target substance in the liquid. An outline of the measurement system of the present embodiment is shown in FIG.

As in the third embodiment, the optical sensor chip 2 is attached to the tip of the optical fiber 1, and the excitation light emitted from the light source 31 reaches the rubidium organic complex or the like at the tip of the optical fiber 1 to reach the complex. Intensity fluorescence corresponding to the amount of oxygen binding is excited. The excited fluorescence is measured by the spectrometer 32 through the optical fiber 1 and the intensity thereof is analyzed by the computer 33.

As the optical sensor chip 2, an optical sensor chip having a flow channel-like liquid chamber as described in the first embodiment with reference to FIG. 1 can be used. The sample gas containing the target substance is sent into the liquid 6 such as a phosphate buffer solution through the air supply pipe 51, and the target substance is dissolved in the liquid 6. At this time, by continuously stirring the liquid 6 with the stirrer 61, the change in the concentration of the target substance in the sample gas to be blown in is quickly reflected in the dissolved amount of the target substance in the liquid 6. The liquid 6 in which the target substance is dissolved is sent by the pump 41 to the flow channel liquid chamber of the optical sensor chip 2. At this time, the liquid feeding amount is adjusted by the flow rate adjusting valve 42. The liquid feeding may be continuous or intermittent, and the liquid feeding amount can be appropriately set according to the measurement purpose. By measuring the concentration of the target substance in the liquid, the concentration of the target substance in the sample gas can be obtained.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

<Example 1>
(Production of enzyme-immobilized membrane and optical sensor)
Alcohol oxidase (AOD: alcohol oxidase, EC 1.1.3.13 A2404, 10-40 units / mg, Sigma-Aldrich) 15 μL and PVA-SbQ (SPP-H-1 (Bio) as photocrosslinking resin, Toyo Gosei Kogyo Co., Ltd.) 50 μL of the mixed solution was used as a paste. This paste was uniformly applied to an H-PTFE membrane (JGWP14225, Millipore Corporation, pore size 200 nm) cut to 30 × 30 mm ² . Next, after drying in a cool dark place at 4 ° C. for 6 hours, the mixture was allowed to stand for 2 hours under a fluorescent lamp, whereby alcohol oxidase was comprehensively immobilized by a photocrosslinking method to produce an enzyme-immobilized membrane. When preparing the enzyme-immobilized film, the paste was applied so as to be imprinted, and any excess enzyme after drying was scraped off.

Next, an optical sensor having a structure as shown in FIG. 1 was produced using the produced enzyme-immobilized film. An H-PTFE membrane was used as the second gas permeable membrane. The H-PTFE membrane was fixed to the tip of the chip body 20 using a silicon O-ring. The enzyme-immobilized membrane was attached so as to be in contact with the sensitive portion at the tip of an oxygen-sensitive optical fiber (FOXY-R, od: 1.5 mm manufactured by Ocean Optics).

(experimental method)
Using the optical sensor produced as described above, a standard gas generator (0.1 mol / L phosphate buffer, pH 8.0) was fed into a liquid chamber using a pump. Permeater, TYPE PD-1B-2 (manufactured by Gastec Co., Ltd.) was used to generate ethanol gas of a predetermined concentration, and the generated ethanol gas was loaded at the tip of the sensor at a flow rate of 200 mL / min.

Spectrophotometer (MODEL S4000-FL, manufactured by Ocean Optics) and A / D conversion are used as the sensor output intensity, which is the amount of increase in fluorescence intensity due to the decrease in oxygen concentration due to the oxidation reaction of oxygen (oxygen consumption reaction) by ethanol. Measured with a computer via a device (DAQ Card-700, PCMCIA-type A / D card, manufactured by National Instruments).

The concentration of ethanol gas is 0 (air), 0.7, 1.1, 2.2, 4.4, 12.4, 22.5, 31.0, 49.6, 82.6, 124. The above measurement was performed for each case of 0.0 ppm.

Further, the flow rate of ethanol gas and the flow rate of buffer solution were controlled as follows. First, during the period from the start of the experiment to 2 minutes after the start, loading with air (air) with a flow rate of 0.2 L / min was performed, and the buffer solution was sent at 1.5 mL / min. Next, during the period of 2 to 14 minutes after the start of the experiment, ethanol gas of each concentration was loaded at 0.2 L / min, and the buffer solution feeding was stopped. Furthermore, for 14 to 20 minutes after the start of the experiment, loading with 0.2 L / min of air (air) was performed, and the buffer solution was sent at 1.5 mL / min.

The experimental results are shown in FIG. FIG. 6 is a graph showing the relationship between the sensor output intensity and the ethanol gas concentration. In FIG. 6, the horizontal axis indicates the elapsed time from the start of measurement, and the vertical axis indicates the sensor output intensity. The sensor output intensity starts to increase according to the concentration of each ethanol gas 2 minutes after the start of measurement when ethanol gas starts to be loaded on the tip of the sensor. It can be seen that when the ethanol gas load is stopped approximately 14 minutes after the start of the measurement and the buffer solution is resumed, the sensor output intensity rapidly decreases and quickly returns to the initial state.

Fig. 7 shows an enlarged view of 1 to 5 minutes after the start of measurement in the graph of Fig. 6. Further, FIG. 8 shows an enlarged view of 4 to 5 minutes after the start of measurement in which linearity is recognized in the graph in FIG. From the graph shown in FIG. 8, a relational expression between the time (x) from the start of measurement and the sensor output intensity (y) was determined for each concentration of ethanol gas. The results are shown in Table 1.

FIG. 9 shows a calibration curve prepared from this slope (coefficient of x in the above relational expression) and the concentration of each ethanol gas. As shown in FIG. 9, the slope and the concentration of ethanol gas have a good correlation, and it was shown that ethanol gas can be measured from this slope. Further, as shown in FIG. 10, in the case of a conventional semiconductor sensor, it reacts in addition to ethanol. However, according to the optical sensor of the present invention, ethanol can be selectively measured.

(Reference Example 1)
<Production of enzyme-immobilized membrane and electrochemical sensor>
A mixed solution of 25 μL of MAO-A (Monoamine Oxidase type A, manufactured by Japan BD) and 80 μL of PVA-SbQ (SPP-H-1 (Bio), manufactured by Toyo Gosei Co., Ltd.) was used as a paste. This paste was uniformly applied to a dialysis membrane (UC 36-32-100, Viscose Corporation Inc., molecular weight cut off 14000, pore diameter 5 nm) cut into 40 × 50 mm ² . Next, after drying for 60 minutes in a cool dark place at 4 ° C., the mixture was allowed to stand for 30 minutes under a fluorescent lamp, whereby monoamine oxidase was comprehensively immobilized by a photocrosslinking method to prepare an enzyme-immobilized membrane. When preparing the enzyme-immobilized film, the paste was applied so as to be imprinted, and any excess enzyme after drying was scraped off.

Next, using the prepared enzyme-immobilized membrane, nylon net and silicon O— are used so that the surface on which the enzyme is not directly applied is in contact with the sensitive part of the Clark oxygen electrode (BO-P, Able Co., Ltd.). An electrochemical sensor was fabricated by fixing with a ring.

<Experiment method>
In FIG. 5, an experiment for measuring the methyl mercaptan concentration in the solution was performed using a measurement system in which the air supply pipe 51 was not provided. First, phosphate buffer (0.1 M, pH 8.5) to which 0, 1, 3, 5, 10, 15 or 20 mM ascorbic acid (L (+)-sodium ascorbate, Wako Pure Chemical Industries, Ltd.) was added The sensitive part of the sensor was immersed in 50 mL. Methyl mercaptan solution is added dropwise so that the final concentration in the phosphate buffer is 1 mM, and the amount of dissolved oxygen reduced by the MAO-A enzyme reaction (oxygen consumption reaction) accompanying the addition of the methyl mercaptan solution is reduced to oxygen. Detected with electrodes.

<Experimental result>
FIG. 11 is a graph of sensor output (output value in which the amount of decrease in the electrical signal accompanying the decrease in dissolved oxygen is a positive value) measured using the electrochemical sensor when ascorbic acid of each concentration is added. Show. The horizontal axis of the graph indicates the elapsed time from the start of measurement, and methyl mercaptan was added 2 minutes after the start of measurement. An increase in sensor output was observed when sodium ascorbate was added to the phosphate buffer.

FIG. 12 is a graph plotting the relationship between the sodium ascorbate concentration in the phosphate buffer and the sensor output 30 minutes after the start of measurement. As shown in FIG. 12, it can be seen that the highest sensor output is exhibited when the sodium ascorbate concentration is 10 mM. When the ascorbic acid concentration is 15 mM and 20 mM, the sensor output is lower than that of 10 mM.

Although this experiment is an experiment conducted using an electrochemical sensor, from this result, even in the measurement method of the present invention, when measuring methyl mercaptan or the like, the concentration of ascorbic acid added to the buffer solution is 10 mM or less. It is considered to be preferable.

It should be considered that the embodiments and examples disclosed herein are illustrative and non-restrictive in every respect. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

1 optical fiber, 11 ruthenium organic complex, 12 silicone overcoat, 2 optical sensor chip, 20 chip body, 21 liquid chamber, 211 introduction tube, 212 discharge tube, 22 enzyme-immobilized membrane (first gas permeable membrane), 23 Second gas permeable membrane, 31 light source, 32 spectrometer, 33 computer, 41 pump, 42 flow control valve, 5 reservoir, 51 air supply pipe, 6 liquid, 61 stirrer.

Claims

An optical sensor chip (2) used in connection with an optical fiber (1),
A first gas permeable membrane (22) covering the tip of the optical fiber (1);
An optical sensor chip comprising a liquid chamber (21) for supplying a liquid to the surface of the first gas permeable membrane (22) opposite to the side connected to the optical fiber (1). .
The optical sensor chip according to claim 1, wherein the first gas-permeable membrane (22) is an enzyme-immobilized membrane (22) on which an enzyme that catalyzes a chemical reaction of a target substance is immobilized.
2. The optical sensor chip according to claim 1, further comprising at least one flow path communicating between the liquid chamber (21) and the outside of the optical sensor chip (2).
The optical sensor chip according to claim 1, wherein the liquid chamber (21) is a closed space not communicating with the outside of the optical sensor chip (2), or a space having a liquid inlet and an air hole. .
The liquid chamber (21) includes a second gas permeable membrane (23) on the opposite side of the first gas permeable membrane (22),
The said optical fiber (1) has a stopper mechanism for making the distance of the front-end | tip of the said optical fiber (1), and the said 2nd gas permeable film (23) constant, when connected. Chip for optical sensor.
The optical sensor chip according to claim 2, wherein the enzyme is an oxidoreductase, a dehydrogenase or a luminescent enzyme.
The optical sensor chip according to claim 6, wherein the oxidoreductase is an enzyme that consumes or generates oxygen by reacting with a substrate.
The optical sensor chip according to claim 1, wherein the optical fiber (1) is an oxygen-sensitive optical fiber (1), a pH-sensitive optical fiber (1), or a light-emitting sensitive optical fiber (1).
The optical sensor chip according to claim 1, wherein the optical fiber (1) is an oxygen-sensitive optical fiber (1), and a ruthenium organic complex is fixed to a tip portion thereof.
An optical sensor formed by connecting the optical sensor chip (2) according to claim 1 and an optical fiber (1).
A measurement system comprising at least one optical sensor according to claim 10.
The measurement system according to claim 11, comprising a mechanism for replacing the liquid in the liquid chamber (21) of the optical sensor chip (2).
The measurement system according to claim 11, which is for measuring odor.
A method for measuring a target substance using the measurement system according to claim 11.
The measurement method according to claim 14, wherein the liquid in the liquid chamber (21) does not move during measurement, and the liquid in the liquid chamber (21) moves between measurements.
15. The measurement method according to claim 14, wherein the concentration of the target substance is calculated based on a differential value, an integral value, an integrated value, or a second-order differential value of the measured light output with time.
The measurement method according to claim 14, wherein the liquid in the liquid chamber (21) moves during the measurement.
The first gas permeable membrane (22) is an enzyme-immobilized membrane (22) on which an enzyme that catalyzes a chemical reaction of a target substance is immobilized.
As the enzyme, an oxidoreductase that consumes or generates oxygen by reacting with a substrate is used,
The measurement method according to claim 14, wherein the substrate is methyl mercaptan, dimethyl sulfide, hydrogen sulfide, ammonia, or trimethylamine, and ascorbic acid is added to the liquid.
The measurement method according to claim 18, wherein the concentration of ascorbic acid added is 10 mM or less.