WO2018216192A1

WO2018216192A1 - Smell sensor

Info

Publication number: WO2018216192A1
Application number: PCT/JP2017/019679
Authority: WO
Inventors: 小川　薫; 洋臣後藤
Original assignee: 株式会社島津製作所
Priority date: 2017-05-26
Filing date: 2017-05-26
Publication date: 2018-11-29

Abstract

Provided is a smell sensor that obtains sensitivity resulting in more stable quantification and can be produced with inexpensive materials. This smell sensor 1 is provided with at least one pair of electrodes 2, a conductor 4, a G protein-coupled receptor 5, and a detection unit (ammeter A). The conductor 4 bridges the at least one pair of electrodes 2 and is electrified. The G protein-coupled receptor 5 is adhered to the conductor 4. The detection unit uses a change in the electrification state of the conductor 4 to detect a change in the three-dimensional structure of the G protein-coupled receptor 5 when the G protein-coupled receptor 5 receives a component to be measured.

Description

Odor sensor

The present invention relates to an odor sensor using a G protein-coupled receptor.

Of the five human senses such as sight, hearing, touch, smell, and taste, physical sensors that replace vision, hearing, and touch have been developed, but chemical sensors that replace smell and taste are still developing. is there. For example, as conventional olfactory sensors, various sensors such as an oxide semiconductor method, a crystal resonator method, and a micro-cantilever method have been proposed. Low. Further, in the conventional type olfactory sensor, even if the main odor to be detected does not change, the change of other odors affects all sensor values, so the reproducibility is low. Sensors using olfactory receptors have been developed as next-generation olfactory sensors that can solve these problems (for example,

Patent Documents

1 and 2 and Non-Patent Document 1 below).

In Patent Document 1, cells expressing insect olfactory receptor proteins and fluorescent proteins are held in a container provided on a substrate, and a sample containing an odorant to be detected is brought into contact with the cells to emit light. Thus, an odor sensor that detects the odorous substance by receiving the light with a sensor is disclosed. In this type of odor sensor, an olfactory sensor cell can be obtained by, for example, expressing an olfactory receptor, an olfactory receptor auxiliary protein, and a calcium sensitive protein in an insect cell. When the olfactory receptor receives an odorant substance, an ion channel formed by the olfactory receptor and the olfactory receptor auxiliary protein opens, and calcium ions flow into the cell. As a result, the calcium ions and the fluorescent protein are combined, and fluorescence is generated from the fluorescent protein. An odor can be detected by detecting this fluorescence with a sensor.

Non-Patent Document 1 discloses a device configuration in which conductive polymer nanowires or single-walled carbon nanotubes are coated on an electrode and an olfactory receptor is bonded to the surface. This technology uses a field effect transistor (FET) as the measurement principle, and detects the odor by detecting the charge that changes on the sensor when the olfactory receptor receives the odor substance. Be able to.

Patent Document 2 discloses a biosensor including a signal conversion unit and a signal sensing unit on the surface of a solid substrate. The signal conversion unit includes a nanowire and electrodes at both ends of the nanowire. The signal sensing unit exists between one nanowire and another nanowire, and a receptor (receptor) that binds to a target substance is attached. In this way, the receptor is not directly attached to the nanowire, but the receptor is attached to the substrate surface around the nanowire, thereby suppressing the material dependence of the nanowire and detecting the target substance with high sensitivity.

JP 2013-027376 A Special table 2009-532697

Since the odor sensor exemplified in Patent Document 1 uses cells, in order to always sense, it is necessary to devise to keep the cells alive. Therefore, this type of sensor is a wet-type sensor in which a solution is always placed in a container. Therefore, there is a problem that it is always necessary to manage the state of the solution such as the concentration and pH of the culture solution, and that it is difficult for the receptors in the culture solution to stably accept odorants in the atmosphere. Further, there are many stages between the time when the receptor receives the odor substance and the time when fluorescence is detected, and there is a possibility that the response at each stage may be lost. Due to these influences, this type of sensor has a problem that the sensitivity becomes unstable or becomes low.

The odor sensor exemplified in Non-Patent Document 1 is not a wet sensor as in Patent Document 1, but a dry sensor, so that it is not necessary to manage the state of the solution. However, since the FET principle is used, it is necessary to use a semiconductor type material, and there is a problem that the material is relatively expensive. In addition, the charge that changes on the sensor when the olfactory receptor receives the odor substance may have different positive and negative and valence depending on the odor substance, so it is difficult to make the sensor response quantitative. It is. Furthermore, it is expected that the sensitivity varies depending on the ease with which the substance is charged. Such a problem may also occur in the sensor exemplified in Patent Document 2.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an odor sensor that can obtain sensitivity with more stable quantitativeness and can be manufactured even with an inexpensive material.

(1) The odor sensor according to the present invention includes a G protein-coupled receptor, a detection unit, and a measurement unit. The G protein coupled receptor is attached to a support. The detection unit detects a change in the three-dimensional structure of the G protein-coupled receptor when the measurement target component is received. The measurement unit measures the amount or concentration of the measurement target component based on the three-dimensional structure change of the G protein-coupled receptor detected by the detection unit.

According to such a configuration, when the G protein-coupled receptor attached to the support receives the measurement target component, the three-dimensional structure of the G protein-coupled receptor changes, and the measurement target component The amount or concentration of can be measured. Such a configuration detects a change in the three-dimensional structure of the receptor, not a charge that changes in the sensor when the receptor receives the substance to be measured, so that more stable and quantitative sensitivity can be obtained. Further, since the conductor is not limited to a semiconductor material, it can be realized by a relatively inexpensive dry sensor.

(2) The odor sensor may further include at least one pair of electrodes. In this case, the support may be a conductor that is bridged between the at least one pair of electrodes and energized. The detection unit may detect a change in the three-dimensional structure of the G protein-coupled receptor based on a change in the energization state of the conductor.

According to such a configuration, the three-dimensional structure of the G protein-coupled receptor changes when the G protein-coupled receptor attached to the conductor cross-linked between at least one pair of electrodes receives the component to be measured. By utilizing this, it is possible to detect the measurement target component by detecting the mechanical change based on the change in the energization state of the conductor.

(3) The odor sensor may further include an insulator or a semiconductor that coats the outside of the conductor. In this case, the G protein-coupled receptor may be disposed on the outside of the insulator or semiconductor, or may be disposed therebetween.

According to such a configuration, since the outside of the conductor is coated with the G protein-coupled receptor and the insulator or the semiconductor, it is possible to prevent the charge from entering the conductor from the substance to be detected. . Thereby, generation | occurrence | production of an electrical noise can be prevented, and since a measuring object component can be detected only based on the three-dimensional structure change of a mechanical receptor, the more stable sensitivity is obtained.

(4) The G protein-coupled receptor may be an olfactory receptor.

According to such a configuration, the measurement target component can be detected by using the fact that the olfactory receptor accepts the odorous substance as the measurement target component and changes the three-dimensional structure of the olfactory receptor at that time. . Thereby, as a chemical sensor capable of detecting an odor substance, it is possible to provide an odor sensor that can obtain a cheaper and more stable sensitivity.

(5) The conductor may include a nanotube.

According to such a configuration, a very small amount of a measurement target component can be detected with high sensitivity by using an extremely thin nanotube whose three-dimensional structure is easily changed as a conductor.

(6) The nanotube may be a conductive single-walled carbon nanotube to which the G protein-coupled receptor is attached.

According to such a configuration, it is possible to detect a very small amount of a measurement target component with high sensitivity by using, as a conductor, a single-walled carbon nanotube whose steric structure is easily changed compared to a multi-walled carbon nanotube.

(7) The nanotube may be a multi-walled nanotube having a conductive inner tube and an insulating or semiconductive outer tube disposed outside the inner tube. In this case, the G protein-coupled receptor may be attached to the outside of the outer tube.

According to such a configuration, the G protein conjugate attached to the outside of the outer tube by using the multi-walled nanotube in which the insulating or semiconductive outer tube is disposed outside the conductive inner tube as the conductor. It is possible to prevent charge from entering the inner tube from the substance received by the receptor. Thereby, generation | occurrence | production of an electrical noise can be prevented, and since a measuring object component can be detected only based on the three-dimensional structure change of a mechanical receptor, the more stable sensitivity is obtained.

According to the present invention, it can be realized by a dry-type sensor, and it detects not a charge that changes in the sensor when the receptor receives the substance to be measured, but a change in the mechanical three-dimensional structure, and thus more stable sensitivity. Is obtained.

It is the schematic perspective view which showed the structural example of the odor sensor which concerns on one Embodiment of this invention. It is the schematic diagram which showed the electrical structure of the odor sensor of FIG. It is a figure which shows the experimental result of the gas responsiveness using the odor sensor which has not made the receptor adhere to the conductor. It is a figure which shows the experimental result of the gas responsiveness using the odor sensor which attached mOR-M71 to the conductor. It is a figure which shows the experimental result of the gas responsiveness change of the odor sensor according to the density | concentration of mOR-M71.

1. Configuration of Odor Sensor FIG. 1 is a schematic perspective view showing a configuration example of an odor sensor 1 according to an embodiment of the present invention. FIG. 2 is a schematic diagram showing an electrical configuration of the odor sensor 1 of FIG. The odor sensor 1 includes a pair of electrodes 2. Each electrode 2 is formed of a highly conductive material such as gold, and is held by an insulating holding member 3 such as glass in a state of being separated so as not to contact each other.

Each electrode 2 is formed in a comb shape, for example. That is, each electrode 2 has a configuration in which a base portion 21 extending in a straight line and a plurality of comb portions 22 protruding from the base portion 21 in the same direction are integrally formed. Each electrode 2 is arranged such that the base portions 21 extend in parallel with each other, and the plurality of comb portions 22 of the other electrode 2 enter the spaces between the plurality of comb portions 22 of the one electrode 2. Thereby, the plurality of comb portions 22 provided in each electrode 2 are in close proximity so as not to contact each other. The distance between adjacent electrodes 2 (between the comb portions 22) is about several μm.

The conductor 4 is bridged between the pair of electrodes 2. In the present embodiment, for example, carbon nanotubes are used as the conductor 4. As an example, a carbon nanotube dispersion liquid using N, N-dimethylformamide as a solvent is applied so as to straddle between a plurality of comb portions 22 provided in each electrode 2. As a result, the conductor 4 made of an aggregate of a plurality of carbon nanotubes is bridged between the comb portions 22 facing each other, and electricity can be passed between the pair of electrodes 2 via the conductor 4. The resistance value of the conductor 4 is adjusted to be, for example, 60 to 200Ω.

The G protein-coupled receptor 5 (hereinafter simply referred to as “receptor 5”) is attached to the conductor 4. More specifically, a dried buffer solution containing the receptor 5 is attached to the conductor 4. However, the receptor 5 is not limited to a configuration in which the receptor 5 is dried and attached to the conductor 4, but the receptor 5 can be attached to the conductor 4 using various chemical or biological fixing methods. .

In this embodiment, an olfactory receptor is used as the receptor 5. As an example, a membrane fraction dispersion of Escherichia coli in which an olfactory receptor is expressed is dropped on the surface of the conductor 4 and dried under reduced pressure. The type of olfactory receptor is not particularly limited, but mOR-M71 (acetophenone receptor) is used in this embodiment.

In this embodiment, the insulator 6 is present around the conductor 4 and the receptor 5. That is, the outside of the conductor 4 is coated with the receptor 5 and the insulator 6 so that the substance to be detected does not directly contact the conductor 4. If the insulating material is mixed in the solution of the receptor 5, the entire outside of the conductor 4 can be coated with the receptor 5 and the insulator 6 when the receptor 5 is applied to the conductor 4. The insulating material constituting the conductor 6 is not particularly limited, such as a polymer or a biomaterial, but is preferably a material and a thickness that do not constrain the three-dimensional structure change (strain or the like) of the receptor 5. Examples of the insulating material include general surfactants such as sodium N-lauroyl sarcosine, polymer materials such as polyethylene glycol, liposomes and nanodisks.

Thus, the receptor 5 may be disposed between the insulators 6. In this case, the receptor 5 can be directly bonded to the conductor 4 and exposed to the surface of the odor sensor 1. A portion of the conductor 4 where the receptor 5 is not bonded is covered with an insulator 6. However, the receptor 5 is not limited to the configuration exposed on the surface of the odor sensor 1, and may be buried in a layer containing an insulating material. Moreover, the structure by which the receiver 5 is arrange | positioned on the outer side of the insulator 6 may be sufficient.

A DC voltage is applied from the DC power source 7 between the pair of electrodes 2. As a result, a current flows between the pair of electrodes 2 via the conductor 4, and the current is detected by the ammeter A. A change in the resistance value of the conductor 4 can be measured by detecting a current with the ammeter A while applying a constant voltage by the DC power source 7.

The olfactory receptor, which is the receptor 5, has a characteristic that the three-dimensional structure changes when a ligand (odor molecule) is received. The change is expected to be about 1 nm, and it is difficult to measure the three-dimensional structure change itself. In this embodiment, with the change in the three-dimensional structure of the receptor 5, the three-dimensional structure of the conductor 4 to which the receptor 5 is attached also changes, and the resistance value of the conductor 4 changes. Based on the current detected by the ammeter A, the change in the resistance value of the conductor 4 is detected by the detection unit 8 so that the change in the three-dimensional structure of the receptor 5 can be detected based on the change in the resistance value. it can. That is, the detection unit 8 detects the change in the three-dimensional structure of the receptor 5 when receiving the measurement target component (odor substance) based on the change in the energized state of the conductor 4. The measurement unit 9 measures the amount or concentration of the measurement target component based on the three-dimensional structure change of the receptor 5 detected by the detection unit 8.

Thus, in this embodiment, the three-dimensional structure of the receptor 5 changes when the receptor 5 attached to the conductor 4 cross-linked between the pair of electrodes 2 receives the component to be measured. By detecting the three-dimensional structure change based on the change in the energized state of the conductor 4, it is possible to detect the measurement target component. Such a configuration detects not a charge that changes in the conductor 4 when the receptor 5 receives the component to be measured, but detects a change in the three-dimensional structure of the receptor 5, so that a more stable sensitivity can be obtained. Further, since the conductor 4 is not limited to a semiconductor material, it can be realized by a relatively inexpensive dry sensor.

In particular, in this embodiment, since the outside of the conductor 4 is coated with the receptor 5 and the insulator 6, it is possible to prevent the charge from entering the conductor 4 from the substance to be detected. Thereby, since generation | occurrence | production of an electrical noise is prevented and a measuring object component can be detected only based on the three-dimensional structure change of the mechanical receptor 5, the more stable sensitivity is obtained. However, instead of the insulator 6, the outside of the conductor 4 may be coated with a semiconductor.

In addition, when an olfactory receptor is used as a G protein-coupled receptor as in this embodiment, an odorant as a measurement target component is received by the olfactory receptor, and the three-dimensional structure of the olfactory receptor changes at that time. Can be used to detect the component to be measured. As a result, the odor sensor 1 can be provided as a chemical sensor capable of detecting an odor substance, which can be obtained at lower cost and with a stable sensitivity.

2. In order to confirm that the odor sensor 1 according to the present embodiment responds to a gas containing a measurement target component (acetophenone) and does not respond to a gas containing a component other than the measurement target component. The results of the experiment conducted in (1) will be described.

In this experiment, an odor sensor in which mOR-M71, which is an acetophenone receptor, was attached to a conductor was used. As the gas to be measured, in addition to gases containing odorous substances such as acetophenone, eugenol, and ethyl acetate, air supplied from an air cylinder (air) was also used. Further, an odor sensor in which the receptor is not attached to the conductor was prepared by dropping a buffer solution not containing the receptor onto the conductor and drying under reduced pressure, and an experiment was performed using this odor sensor.

FIG. 3 is a diagram showing the experimental results of gas responsiveness using an odor sensor in which a receptor is not attached to a conductor. On the other hand, FIG. 4 is a diagram showing an experimental result of gas responsiveness using an odor sensor in which mOR-M71 is attached to a conductor. As an experimental device, a dilution mixing device “FDL-1” (manufactured by Shimadzu Corporation) for an odor discriminating device was used, and the experiment was repeated several times while gradually reducing the gas concentration. The gas response evaluation results (waveforms) obtained as a result are shown in FIGS.

As shown in FIG. 3, when an odor sensor in which the receptor was not attached to the conductor was used, no response was confirmed for any of the gases containing acetophenone, eugenol, and ethyl acetate. In addition, no response was confirmed for air containing no odorous substances.

As shown in FIG. 4, when an odor sensor in which mOR-M71 was attached to a conductor was used, a response to a gas containing acetophenone was confirmed. On the other hand, for the gas containing eugenol and the gas containing ethyl acetate, the response to air was almost the same. Thus, it can be seen that by attaching mOR-M71 to a conductor, a highly selective odor sensor that specifically responds to acetophenone can be provided.

Next, the results of experiments conducted to confirm the relationship between the concentration of mOR-M71 and responsiveness to odorous substances will be described. In this experiment, mOR-M71 was diluted with a protein solution that did not express olfactory receptors to produce 100%, 75%, and 50% mOR-M71 solutions, and each solution was used as a conductor. An odor sensor was fabricated by attaching it. And each odor sensor confirmed the responsiveness with respect to the gas containing acetophenone.

FIG. 5 is a diagram showing experimental results of changes in gas responsiveness of the odor sensor according to the concentration of mOR-M71. According to the experimental results of FIG. 5, the responsiveness to acetophenone decreases as the concentration of mOR-M71 decreases. From this, it is considered that the response of the olfactory receptor to the odorous substance (acetophenone) can be detected.

3. Configuration Example of Conductor As the carbon nanotube that is an example of the conductor 4, for example, a multi-layer carbon nanotube including two layers of an inner tube and an outer tube is used.

The inner tube and the outer tube are cylindrical tubes each composed of a six-membered ring. The outer tube has a larger diameter than the inner tube, and is formed in a nested shape by being coaxially disposed outside the inner tube. The inner tube and the outer tube may be formed of different materials, or may be formed of the same material.

In this embodiment, the inner tube is made of conductive carbon, and the outer tube is made of an insulating or semiconductive material. The receptor 5 is attached to the outside of the outer tube, and the penetration of charge from the substance received by the receptor 5 into the inner tube is prevented. Thereby, since generation | occurrence | production of an electrical noise is prevented and a measuring object component can be detected only based on the three-dimensional structure change of the mechanical receptor 5, the more stable sensitivity is obtained.

4). Modified Example of Conductor The conductor 4 is not limited to a carbon nanotube using carbon as a conductive material, but may be a nanotube using another conductive material (for example, metal). By using extremely thin and flexible nanotubes as the conductor 4, a very small amount of a measurement target component can be detected with high sensitivity.

Further, the conductor 4 is not limited to two layers, and may be a multi-layer nanotube of three or more layers, or may be a single-wall nanotube composed of only one layer. In this case, if the receptor 5 is attached to the outside of the conductive single-walled nanotube, the flexible single-walled nanotube is used as the conductor 4 as compared with the multi-walled nanotube, and a minute amount of the measurement target component is detected with high sensitivity. be able to.

However, the conductor 4 is not limited to the nanotube, and can be formed using various materials having conductivity other than the nanotube.

5). Other Modifications In the embodiment described above, the configuration in which the current flowing between the pair of electrodes 2 is detected by the ammeter A to detect a change in the energization state of the conductor 4 has been described. However, the present invention is not limited to such a configuration, and for example, a configuration in which a change in the energization state of the conductor 4 is detected by detecting a voltage with a voltmeter while passing a constant current through the conductor 4 may be used. . In addition, the odor sensor 1 is not limited to a configuration in which a pair of electrodes 2 is provided, but may have a configuration in which a plurality of pairs of electrodes 2 are provided.

The acceptor 5 is not limited to the configuration in which the acceptor 5 is attached to the conductor 4 via the insulator 6, and may be a constitution in which the acceptor 5 is directly attached to the conductor 4 without providing the insulator 6, for example. The receptor 5 is not limited to the olfactory receptor, and the measurement target component is not limited to the odor substance.

The receptor 5 is not limited to the structure attached to the conductor 4, but may be a structure attached to another support. In this case, the odor sensor 1 is not limited to the configuration in which the conductor 4 is cross-linked between at least one pair of electrodes 2 and the receptor 5 is attached to the conductor 4, but for example, an optical detection method or the like. A configuration that detects a change in the three-dimensional structure of the receptor 5 by other than the electrical detection method may be used.

As the support, for example, it is conceivable to use a composite nanostructure using gold nanoparticles. In this case, by attaching the receptor 5 to the composite nanostructure holding a plurality of gold nanoparticles, it is possible to detect the three-dimensional structure change of the composite nanostructure when the receptor 5 receives the odorant. There is sex. It is also conceivable to optically detect a change in the three-dimensional structure of the receptor 5 using Raman spectroscopy.

DESCRIPTION OF SYMBOLS 1 Odor sensor 2 Electrode 3 Holding member 4 Conductor 5 G protein conjugate receptor 6 Insulator 7 DC power supply 8 Detection part 21 Base part 22 Comb part A Ammeter

Claims

A G protein coupled receptor attached to a support;
A detection unit that detects a change in the three-dimensional structure of the G protein-coupled receptor when receiving a component to be measured;
An odor sensor comprising: a measurement unit that measures the amount or concentration of the measurement target component based on a three-dimensional structure change of the G protein-coupled receptor detected by the detection unit.
Further comprising at least one pair of electrodes;
The support is a conductor that is bridged between the at least one pair of electrodes and energized;
2. The odor sensor according to claim 1, wherein the detection unit detects a three-dimensional structure change of the G protein-coupled receptor based on a change in an energized state of the conductor.
Further comprising an insulator or a semiconductor coating the outside of the conductor;
The odor sensor according to claim 1, wherein the G protein-coupled receptor is disposed between or outside the insulator or the semiconductor.
The odor sensor according to claim 1, wherein the G protein-coupled receptor is an olfactory receptor.
The odor sensor according to claim 1, wherein the conductor includes a nanotube.
The odor sensor according to claim 5, wherein the nanotube is a conductive single-walled carbon nanotube to which the G protein-coupled receptor is attached.
The nanotube is a multi-walled nanotube having a conductive inner tube and an insulating or semi-conductive outer tube disposed outside the inner tube;
The odor sensor according to claim 5, wherein the G protein-coupled receptor is attached to the outside of the outer tube.