JP6973772B2 - Smell sensor - Google Patents

Description

The present invention relates to a detection technique and to an odor sensor.

In recent years, analysis of the molecular mechanism and neural mechanism by which insects receive and discriminate odors has progressed, and the mechanism of odor discrimination of insects, which has high sensitivity and high discriminating power, is becoming clear. Therefore, it is becoming possible to artificially reproduce the olfactory function of insects. Insects identify odorants by a combination of response patterns of multiple sensory cells with different response selectivity to odorants. The odor response characteristics of olfactory cells are determined by the characteristics of the olfactory receptor proteins expressed in individual cells. Therefore, the information on the odorant is expressed as a combination of the response patterns of the olfactory receptors (see, for example, Patent Document 1).

On the other hand, research is underway to place cells on the electrodes of transistors and detect electrical changes in the cells with transistors. Here, aluminum is considered to be cytotoxic (see, for example, Non-Patent Documents 1 and 2). Therefore, a method of forming the electrode of the transistor with gold or coating it with a biocompatible substance has been proposed (see, for example, Non-Patent Document 3).

Japanese Unexamined Patent Publication No. 2013-27376

Plattet al., "Aluminium toxicity in the rat brain: Histochemical and immunocytochemical evidence", Brain Research Bulletin, 2001. Campbell et al., "Differential toxicity of aluminum salts in human cell lines of neural origin: implications for neurodegeneration", NeuroToxicology, 2001. Heer et al., "CMOS microelectrode array for the monitoring of electrogenic cells", Biosensors and Bioelectronics, 2004.

Forming the electrodes of the transistor on which the cells are placed with gold or coating with a biocompatible substance is costly. Therefore, one of the objects of the present invention is to provide a low-cost odor sensor.

According to an aspect of the present invention, a transistor having a gate electrode containing aluminum or aluminum oxide, an insect cell having an olfactory receptor placed on the gate electrode, and an electric current generated in the transistor when the insect cell reacts to an odor. An odor sensor is provided, comprising a detection device for detecting.

Also, according to aspects of the invention, a transistor with a gate electrode containing aluminum or aluminum oxide, a chamber on the transistor for containing olfactory receptor-bearing insect cells, and insect cells on the gate electrode. An olfactory sensor is provided that comprises a detector that detects the current generated by the transistor when the cell reacts to the odor.

In the above odor sensor, insect cells may express the olfactory receptor by the transgene.

In the above odor sensor, insect cells may express the olfactory receptor of an insect.

In the above odor sensor, the olfactory receptor may be an ion channel type receptor.

In the above odor sensor, the olfactory receptor may be selected from BmOR1, BmOR3, Or13a, Or56a, Or85b and PxOR1.

In the above odor sensor, the insect cell may be a moth-derived cell.

In the above odor sensor, insect cells may be selected from Sf21, Sf9, High Five and Tni-derived cells.

In the above odor sensor, the insect cell may be a cell derived from Drosophila.

In the above odor sensor, the insect cell may be a Drosophila S2 cell.

In the above odor sensor, insect cells may express a fluorescent protein whose fluorescence intensity changes according to the ion concentration.

In the above odor sensor, the transistor may be a field effect transistor.

In the above odor sensor, the gate electrode may be an extension gate electrode.

In the above odor sensor, aluminum or aluminum oxide may be exposed on the surface of the gate electrode.

The odor sensor described above may further include a Faraday cage covering the transistor.

According to the present invention, it is possible to provide an odor sensor at low cost.

It is a schematic diagram of the odor sensor which concerns on embodiment. It is a schematic cross-sectional view of the odor sensor which concerns on Example. It is a photograph of the odor sensor provided with the aluminum extension gate electrode according to the embodiment. It is a photograph of the odor sensor including the extension gate electrode which sputtered aluminum oxide according to the Example. The chemical formulas of Bombicole and Bombical. It is a chemical formula of 1-octen-3-ol. It is a graph which shows the detection result of the odor molecule using the cell which expresses the BmOR3 receptor which concerns on Example. It is a graph which shows the detection result of the odor molecule using the cell which expresses the Or13a receptor which concerns on Example. It is a graph which shows the detection result of the odor molecule using the cell which expresses the BmOR3 receptor which concerns on Example. It is a graph which shows the detection result of the odor molecule using the cell which expresses the Or13a receptor which concerns on Example. It is a micrograph which shows the time-dependent change of Sf21 cell on the aluminum substrate which concerns on Example. It is a graph which shows the time-dependent change of the survival rate of the Sf21 cell on the aluminum substrate which concerns on an Example. It is a micrograph which shows the time-dependent change of HEK293T cell on the aluminum substrate which concerns on Example. It is a graph which shows the increase curve of Sf21 cell and HEK293T cell on an aluminum substrate. It is a graph which shows the time change of the drain current of a MOSFET when the Faraday cage is installed and when it is not installed. It is a graph which shows the magnitude of the noise of the drain current of a MOSFET in the case where the Faraday cage is installed and the case where it is not installed. It is a graph which shows the detection result of the odor molecule at the time of using different flow rates using the cell expressing the BmOR3 receptor which concerns on Example. It is a graph which shows the result of having repeatedly detected an odor molecule using the cell expressing the BmOR3 receptor which concerns on Example.

An embodiment of the present invention will be described below. In the description of the following drawings, the same or similar parts are represented by the same or similar reference numerals. However, the drawings are schematic. Therefore, the specific dimensions and the like should be determined in light of the following explanations. In addition, it goes without saying that parts having different dimensional relationships and ratios are included between the drawings.

As shown in FIG. 1, the odor sensor according to the embodiment includes a transistor 10 having a gate electrode 14 containing aluminum or aluminum oxide, an insect cell 50 having an olfactory receptor arranged on the gate electrode 14, and an insect cell. A detection device 80 for detecting a current generated by the transistor 10 when the 50 reacts to an odor is provided.

The transistor 10 includes a semiconductor substrate 11. The transistor 10 is, for example, a field effect transistor (FET) and may be a MOSFET. A source electrode 12 and a drain electrode 13 are provided at intervals near the surface of the semiconductor substrate 11. A gate electrode 14 is arranged above the source electrode 12 and the drain electrode 13 of the semiconductor substrate 11 with an oxide insulating film interposed therebetween. The gate electrode 14 may be an extended gate electrode.

Aluminum or aluminum oxide is exposed on the surface of the gate electrode 14. For example, the gate electrode 14 is made of aluminum, and an _{oxide film made of aluminum oxide (Al 2} O ₃ ) is formed in the vicinity of the surface thereof. An oxide film made of aluminum oxide (Al ₂ O ₃ ) may not be formed in the vicinity of the surface. The gate electrode 14, which is an extension gate electrode, has a region in which the insect cell 50 is arranged. The size of the region in which the insect cell 50 is arranged is, for example, 100 μm × 100 μm, but is not limited thereto. The number of insect cells 50 arranged on the gate electrode 14 is, for example, 10 or less, but is not limited thereto.

The insect cell 50 is arranged on the gate electrode 14 of the transistor 10. By giving a solution containing the insect cell 50 on the gate electrode 14 and allowing it to stand for several tens of minutes to several hours, the insect cell 50 adheres to the gate electrode 14. Insect cell 50 expresses an olfactory receptor on the cell membrane. In the insect cell 50, the olfactory receptor may be naturally expressed or may be expressed by a transgene.

Insect cells may be cells derived from moths such as Spodoptera frugiperda and Trichoplusia ni. Examples of cells derived from Spodoptera frugiperda include Sf21 and Sf9. Sf21 cells are derived from ovarian cells. Sf21 cells can divide infinitely and establish a stable expression line that permanently expresses the introduced gene. In addition, Sf21 cells can survive in a wide temperature range of 18 ° C to 40 ° C and do not require carbon dioxide to adjust the pH of the culture medium. Although Sf21 cells do not originally have an olfactory receptor, it is possible to express the olfactory receptor by introducing a gene for the olfactory receptor. Sf9 cells are clones of Sf21. Examples of cells derived from cabbage looper include High Five and Tni. Tni-derived cells are derived from ovarian cells.

Alternatively, the insect cell may be a cell derived from Drosophila. Examples of Drosophila-derived cells include Drosophila S2 cells.

The olfactory receptor may be a G protein-coupled receptor or an ion channel receptor. The ion channel type receptor has a site that interacts with a ligand that is an odor molecule and a site through which ions flow. When the ion channel type receptor of the insect cell 50 binds to the ligand, cations such as sodium ion and calcium ion flow into the insect cell 50. In the insect cell 50, the influx of ions can occur within a few tens of milliseconds from the binding of the ligand. The amount of inflow to ion Many, for binding one ligand, the amount of ions flowing into the cell is said to be 10 ^7.

In general, certain types of olfactory receptors have specificity for certain odor molecules. In the insect cell 50, only one type of olfactory receptor corresponding to one type of odor molecule may be expressed, or a plurality of types of olfactory receptors corresponding to a plurality of types of odor molecules may be expressed. Further, the detection sensitivity of the odor sensor may be adjusted by adjusting the amount of the olfactory receptor to be expressed.

Examples of olfactory receptors are BmOR1, which is a receptor for Bombykol, which is a sex pheromone of Kaikoga, BmOR3, which is a receptor for Bombykal, which is a sex pheromone of Kaikoga, and a receptor for Drosophila. , Or13a, which is a receptor for 1-octen-3-ol, which has a musty odor, Or56a, which is a receptor for geosmin, which is a receptor for geosmin, which is a receptor for musty odor, and Or85b, which is a general odor receptor for oyster fly. And PxOR1, which is the sex pheromone receptor of Konaga, but is not limited thereto.

When the olfactory receptor is expressed in the insect cell 50 by genetic engineering, for example, a gene encoding the olfactory receptor is incorporated into a vector, and the constructed vector is transfected into a host cell. The gene encoding the olfactory receptor can be isolated, for example, by extracting mRNA from the olfactory organ of an insect and synthesizing cDNA. From the isolated cDNA, it is possible to amplify a part of the gene encoding the olfactory receptor by the PCR method using PCR primers.

A part of the gene encoding the olfactory receptor can also be obtained by incorporating the synthesized double-stranded cDNA into an appropriate vector and transforming Escherichia coli or the like with the vector to prepare a cDNA library. can. The cDNA can be incorporated into a vector by a conventional method using a restriction enzyme and a ligase, for example, by cutting the obtained cDNA with a restriction enzyme, inserting it into the restriction enzyme site of the vector DNA, and ligating it to the vector.

In the insect cell 50, the fluorescent protein may be expressed together with the olfactory receptor. For example, in the insect cell 50, when an odor molecule binds to an ion channel type olfactory receptor, ions such as calcium ions flow in the insect cell 50. Therefore, by introducing a gene that expresses a fluorescent protein whose fluorescence intensity changes according to ions into the insect cell 50, it is confirmed whether or not the insect cell 50 detects an odor molecule from the change in the fluorescence intensity. It becomes possible. Examples of fluorescent proteins include GCAMP3, GCAMP6s and aequorin.

At least a part of the gate electrode 14 of the odor sensor according to the embodiment is arranged in the chamber 60. The chamber 60 contains a solution 70 that may contain odor molecules to be detected. The solution 70 may appropriately contain a substance necessary for the survival of the insect cell 50. Further, a reference electrode 15 is arranged in the chamber 60 so as to be in contact with the solution 70.

The chamber 60 may be provided with an introduction port for feeding a solution that may contain odor molecules into the chamber 60 and a discharge port for discharging the solution in the chamber 60. An introduction pump for feeding the solution into the chamber 60 is connected to the introduction port of the chamber 60. Further, a discharge pump for discharging the solution from the chamber 60 is connected to the discharge port of the chamber 60. As the introduction pump and the discharge pump, for example, a fixed-quantity liquid feed pump can be used.

When the solution 70 contains an odor molecule corresponding to the olfactory receptor of the insect cell 50, the insect cell 50 reacts with the odor molecule and the gate potential of the gate electrode 14 is displaced, so that the source electrode 12 and the drain electrode 13 have a odor molecule. Modulation occurs in the drain current flowing between them. Therefore, it is possible to detect the presence of odor molecules by detecting the modulation of the drain current of the transistor 10.

The detection device 80 is connected to, for example, the source electrode 12, the drain electrode 13, the back gate 16, and the reference electrode 15 of the transistor 10, and detects the modulation of the drain current of the transistor 10. As the detection device 80, a source measure unit (SMU) or the like can be used. A computer system 300 for analyzing the detected current and displaying it on a display may be connected to the detection device 80.

The reason why the gate potential of the gate electrode 14 is displaced by the reaction of the insect cell 50 with the odor molecule is considered to be that when the insect cell 50 reacts with the odor molecule, an inward ion flow is generated in the insect cell 50. It is not bound by this theory.

It is also possible to detect different odor molecules by arranging the odor sensors according to the embodiment in an array and expressing different olfactory receptors in the cells of each odor sensor.

The odor sensor according to the embodiment may include a Faraday cage covering the transistor 10. Since the Faraday cage shields the transistor 10 from the electric field, it is possible to reduce the noise of the drain current in the odor sensor.

Traditionally, aluminum and aluminum oxide have been considered harmful to cells. Therefore, conventionally, when manufacturing an odor sensor in which a transistor and a cell are combined, the electrode of the transistor is formed of gold or coated with a biocompatible substance, and the cell is arranged on the electrode. However, these methods are costly. On the other hand, after diligent research, the present inventors can survive for a long period of time without being damaged by aluminum or aluminum oxide even if the insect cells are placed on the electrode on which aluminum or aluminum oxide is exposed. I found that. Therefore, according to the embodiment, it is possible to provide an odor sensor with a low cost.

On the gate electrode 14 containing aluminum or aluminum oxide, the insect cell 50 can survive, for example, for 5 days or more.

The odor sensor according to the embodiment can be repeatedly reused in the portion of the transistor 10 by cleaning after use. As a cleaning method, for example, a commercially available detergent may be dropped on the surface of the transistor 10.

(Example)
(Manufacturing of transistors, etc.)
A 6-inch wafer having a plurality of MOSFETs formed with an aluminum extension gate electrode was cut by stealth dicing, and a chip containing the required number of MOSFETs was cut out. _{Further, an aluminum oxide (Al 2} O ₃ ) film as an ion-sensitive film was formed on the surface of the aluminum extension gate electrode using a sputtering device. The thickness of the aluminum oxide film was 40 nm to 80 nm. After that, the chip on which the ion-sensitive film was formed and the printed circuit board were connected by wire bonding. Further, epoxy resin was used to form a chamber surrounding the chip containing the extension gate electrode of the MOSFET, as shown in FIG.

(Preparation of cells)
From the start codon to the stop codon of the codon BmOrco gene derived from the antennal cDNA of Kaikoga was amplified with a primer containing the following gene-specific sequence to obtain the BmOrco gene (full length of ORF). The obtained BmOrco gene was inserted into a multi-cloning site of a pIB vector (manufactured by Invitrogen) to construct a pIB-BmOrco vector.
BmOrco:
Forward: 5'-ATGATGACCAAGGTCAAGACGC-3'
Reverse: 5'-CTACTTCAGTTGGATCAACACC-3'

Gene amplification by PCR uses forward and reverse primers at concentrations of 100 pmol / L, PrimeSTAR HS DNA polymerase (Takara Bio Inc. R010A), the reaction buffer attached to the polymerase, and dNTP, and attaches to the polymerase. I followed the protocol of. The PCR temperature conditions are a step of 94 ° C. for 1 minute, then a step of repeating a temperature cycle of 98 ° C. for 10 seconds, 55 ° C. for 15 seconds, 72 ° C. for 1.5 minutes for 30 cycles, and then at 72 ° C. The step was 5 minutes.

Similarly, the start codon to the stop codon of the receptor BmOR3 gene derived from the antennal cDNA of Kaikoga is amplified with a primer containing the following gene-specific sequence to obtain the BmOR3 gene (full length of ORF). rice field. The obtained BmOR3 gene was inserted into the multi-cloning site of the pIB vector to construct the pIB-BmOR3 vector.
BmOR3:
Forward: 5'-ATGATATTCGTCGACGATGCTG-3'
Reverse: 5'-TCATTCGGACACGGTACG-3'

Next, the protein expression cassette of the pIB-BmOR3 vector (the portion linked to the OpIE2 promoter (P (OpIE2)), BmOR3, and OpIE2 polyA addition signal (OpIE2pA)) was amplified and amplified to the BspH1 site of the pIB-BmOrco vector. The protein expression cassette was inserted to construct a dual expression vector pIB-BmOR3-BmOrco.

The constructed dual expression vector was introduced into Sf21 cells by a lipofection method (Cellfectin II; manufactured by Invitrogen) according to the attached manual of Cellfectin II. As a result, Sf21 cells expressing the BmOR3 receptor and the co-receptor BmOrco were obtained. In addition, Sf21 cells expressing Or13a receptor and co-receptor DmOrco were obtained by the same method.

(Manufacturing of odor sensor)
As described above, Sf21 cells expressing the BmOR3 receptor and the co-receptor BmOrco and Sf21 cells expressing the Or13a receptor and the co-receptor DmOrco were prepared. Each Sf21 cell was passaged for 1 to 3 days. The Sf21 cells were then dispersed in the assay buffer and the cell concentration in solution ^{was adjusted from 1.0 × 10 6} cells / mL to 1.5 × 10 ⁶ cells / mL using a cell counter. The composition of the assay buffer is 140 mmol / L NaCl, 5.6 mmol / L KCl, 4.5 mmol / L CaCl ₂ , 11.26 mmol / L MgCl ₂ , 11.32 _{mmol / L ו 4} , 9.4 mmol / L D-glucose. , And 5 mmol / L HEPES, and the pH of the assay buffer was 7.2.

Next, Sf21 cells were seeded on the extension gate electrode by dropping a solution containing 200 μL of Sf21 cells into a chamber surrounding the extension gate electrode of the MOSFET. Then, the cells were allowed to stand for 30 minutes, and Sf21 cells were adhered onto the extension gate electrode as shown in FIGS. 3 and 4. The odor sensor shown in FIG. 3 includes an aluminum (Al) extension gate electrode. Further, the odor sensor shown in FIG. 4 includes an aluminum oxide (Al ₂ O ₃ ) film formed by sputtering on an aluminum extension gate electrode as an ion sensitive film.

(Preparation of odorant-containing liquid)
Bombykal, an odor molecule that binds to the BmOR3 receptor, Bombykol, an odor molecule that binds to the BmOR1 receptor, and 1-octen-3-ol, an odor molecule that binds to the Or13a receptor. (Sigma-Aldrich) and prepared. Bombical and Bombicole were provided by Dr. Shigeru Matsuyama of the University of Tsukuba. The prepared odor molecules were dissolved in an assay buffer containing dimethyl sulfoxide (049-07213, Wako Pure Chemical Industries, Ltd.) at a concentration of 1 v / v% or less, respectively. The chemical formulas of Bombicole and Bombical are as shown in FIG. 5, and have very similar chemical structures. The chemical formula of 1-octen-3-ol is as shown in FIG.

(Smell sensor setup)
A reference electrode was placed to contact the solution in the chamber. Further, in the examples shown in FIGS. 7 to 10, a peristaltic tube pump (MP-2010: Tokyo Rika Kikai Co., Ltd.) was connected to each of the introduction port and the discharge port of the chamber. Then, in FIGS. 7 to 9, the flow rate at the inlet and the outlet was set to 140 μL / min, and an assay buffer containing dimethyl sulfoxide was flowed into the chamber at a concentration of 1 v / v% or less. In FIG. 10, the flow velocity at the inlet and the outlet is set to 250 μL / min. In the embodiments shown in FIGS. 15 to 18, a perista biomini pump (AC-2120: ATTO) was connected to each of the chamber inlet and outlet. Then, the flow rates at the inlet and the outlet were set to 670 μL / min and 1500 μL / min, and an assay buffer containing dimethyl sulfoxide was flowed into the chamber at a concentration of 1 v / v% or less.

The source electrode, drain electrode, backgate and reference electrode of the MOSFET are connected to the source measure unit (SMU, B2902A, KeySight Technology), and using SMU, 2.5V to the drain electrode and -2 to the source electrode. A voltage of 5 V and 0 V was applied to the reference electrode. Then, it was allowed to stand for 500 to 1000 seconds until the drain current detected by the SMU became stable.

(Odor detection using cells expressing BmOR3 receptor on aluminum oxide)
An assay buffer containing dimethylsulfoxide at a concentration of 1 v / v% or less described above is flowed into the chamber of an odor sensor comprising Sf21 cells expressing the BmOR3 receptor, and then Bombicole is contained at a concentration of 30 μmol / L. The assay buffer was run for 100 seconds, followed by a 100 second run of assay buffer containing Bombical at a concentration of 30 μmol / L. As mentioned above, Bombicole specifically binds to other receptors and Bombicall binds to the BmOR3 receptor.

As a result, as shown in FIG. 7, no modulation exceeding the background noise was observed in the drain current during the flow of the assay buffer containing no odor molecule and the flow of the bombicole. On the other hand, when Bombical was applied, the drain current increased by 0.5 μA. As described above, Bombicole and Bombical have similar chemical structures and have similar molecular weight differences, but it has been shown that the odor sensor according to the embodiment can specifically detect Bombical. rice field.

(Odor detection using cells expressing Or13a receptor on aluminum oxide)
An assay buffer containing dimethylsulfoxide at a concentration of 1 v / v% or less described above is passed through the chamber of an odor sensor containing Sf21 cells expressing the Or13a receptor, and then an assay containing bombicals at a concentration of 30 μmol / L. The buffer was run for 120 seconds and then the assay buffer containing 1-octen-3-ol at a concentration of 30 μmol / L was run for 120 seconds. As mentioned above, Bombicard specifically binds to other receptors and 1-octen-3-ol binds to the Or13a receptor.

As a result, as shown in FIG. 8, no modulation exceeding the background noise was observed in the drain current during the flow of the assay buffer containing no odor molecule and the flow of the bombicard. On the other hand, when 1-octen-3-ol was passed, the drain current increased by 0.1 μA.

(Cleaning of odor sensor)
White 7-AL (alkaline, non-foaming, Ui Kasei Co., Ltd.) was diluted to 2% with ultrapure water (milliQ) to prepare a cleaning solution. 1 mL of the cleaning liquid was put into the chamber of the odor sensor, pipetting was performed, and the cleaning liquid after pipetting was discarded. The washing was repeated 5 times. Next, 1 mL of ultrapure water was put into the chamber, pipetting was performed, and the water after pipetting was discarded. The washing was repeated 3 times. Then, the extension gate electrode was dried using an air blow.

The cells were seeded on the MOSFET expressing the BmOR3 receptor, the odor was detected, and the MOSFET was washed four times. As shown in FIG. 9, even if the washing was repeated multiple times. It was confirmed that the odor sensor specifically responds to Bombical.

(Odor detection using cells expressing Or13a receptor on aluminum)
An odor sensor with Sf21 cells expressing the Or13a receptor was produced, which is similar to the above except that the surface of the extension gate electrode is aluminum _{instead of aluminum oxide (Al 2} O _3). The assay buffer containing dimethylsulfoxide at a concentration of 1 v / v% or less described above is flowed into the chamber of the sensor, and then an assay buffer containing decanol at a concentration of 100 μmol / L is flowed for 120 seconds, and then 100 μmol / L. The assay buffer containing 1-octen-3-ol at a concentration was run for 120 seconds.

As a result, as shown in FIG. 10, no modulation exceeding the background noise was observed in the drain current during the flow of the assay buffer containing no odor molecule and the flow of decanol. On the other hand, when 1-octen-3-ol was passed, the drain current increased by 1.2 μA.

(Percentage of cell survival on aluminum substrate)
An aluminum film having a film thickness of 500 nm was formed on the surface of the silicon substrate by a sputtering device to obtain an aluminum substrate. In addition, wild-type Sf21 cells into which no olfactory receptor was introduced were prepared.

As shown in FIG. 11, the life or death of the Sf21 cells on the aluminum substrate was determined every day for 5 days after the Sf21 cells were seeded on the aluminum substrate. A dye exclusion test method using trypan blue (Wako Pure Chemical Industries, Ltd.) was used to determine whether the patient was alive or dead. According to the dye exclusion test method, dead cells are stained blue by the dye, so that the proportion of viable cells can be determined by microscopic observation.

As a result, as shown in FIG. 12, it was confirmed that 97% or more of Sf21 cells survived on the aluminum substrate over 5 days. Furthermore, from the results of the microscopic observation shown in FIG. 11, it was confirmed that the Sf21 cells also grew on the aluminum substrate and the cell density increased.

Next, HEK293T cells derived from human fetal kidney were prepared. As shown in FIG. 13, the life or death of HEK293T cells on the aluminum substrate was determined every day for 5 days after the HEK293T cells were seeded on the aluminum substrate. A dye exclusion test method using trypan blue (Wako Pure Chemical Industries, Ltd.) was used to determine whether the patient was alive or dead. As a result, it was observed that the HEK293T cells grew slowly on the aluminum substrate over time, and the cells died due to the corrosion of the aluminum substrate.

The increase curves of Sf21 cells and HEK293T cells on an aluminum substrate are shown in FIG. It was confirmed that Sf21 cells grew on an aluminum substrate. On the other hand, the number of HEK293T cells increased / decreased on the aluminum substrate, and the number of cells was not stable.

These results indicate that insect cells are compatible with the aluminum substrate, while mammalian cells are not compatible with the aluminum substrate.

(Smell detection using Faraday cage)
The noise level of the drain current when the Faraday cage is installed around the MOSFET on which the aluminum oxide (Al ₂ O ₃ ) film is formed, and the noise level of the drain current when the Faraday cage is not installed around the MOSFET. Was measured. As a result, as shown in FIGS. 15 and 16, it was confirmed that the noise level of the drain current was significantly reduced to about 1/3 when the Faraday cage was installed.

(Evaluation of the effect of flow rate on odor detection)
An assay buffer containing dimethyl sulfoxide at a concentration of 1 v / v% or less described above is passed through the chamber of an odor sensor containing Sf21 cells expressing the BmOR3 receptor, and then an assay containing Bombicall at a concentration of 10 μmol / L. The buffer was run at a flow rate of 670 μL / min for about 60 seconds or 1500 μL / min for about 30 seconds. As mentioned above, Bombicard binds to the BmOR3 receptor. The odor sensor was placed on a tabletop vibration isolation table. The odor sensor was also equipped with a Faraday cage.

As a result, as shown in FIG. 17, when the flow rate was about 670 μL / min, the drain current did not return to the base value after the response detection of Bombical was detected. On the other hand, when the flow rate was about 1500 μL / min, the drain current returned to the base value after the response detection of Bombical was detected. The appropriate flow rate may vary depending on the size, shape, sensitivity, and the like of the odor sensor. Therefore, an appropriate flow rate may be appropriately set according to the size, shape, sensitivity, and the like of the odor sensor.

(Repeated odor detection using cells expressing BmOR3 receptor on aluminum oxide)
An assay buffer containing dimethylsulfoxide at a concentration of 1 v / v% or less is flowed through the chamber of an odor sensor containing Sf21 cells expressing the BmOR3 receptor, and the same assay buffer as the solution being flowed as a control is flowed for about 30 seconds. bottom. The assay buffer containing Bombical was then run at a concentration of 10 nmol / L for about 30 seconds. Then, an assay buffer containing no odor molecule was flown to remove the odor molecule from the odor sensor, and the drain current was returned to the base value. After that, an assay buffer containing Bombical was run at a concentration of 100 nmol / L for about 30 seconds. The flow rate of the solution at this time was set to be about 1500 μL / min. The odor sensor was placed on a tabletop vibration isolation table. The odor sensor was also equipped with a Faraday cage. As a result, as shown in FIG. 18, it was shown that the odor molecule can be repeatedly detected.

10 ... Transistor, 11 ... Semiconductor substrate, 12 ... Source electrode, 13 ... Drain electrode, 14 ... Gate electrode, 15 ... Reference electrode, 16 ... Back gate, 50. .. Insect cells, 60 ... chamber, 70 ... solution, 80 ... detector, 300 ... computer system

Claims (15)

A semiconductor substrate, a transistor comprising a semiconductor substrate being arranged on oxide insulating film, a gate electrode made of the disposed on the oxide insulating film of aluminum, a,
Insect cells having olfactory receptors placed on the gate electrode,
A detection device that detects the current generated by the transistor when the insect cell reacts to the odor.
Equipped with an odor sensor. The odor sensor according to claim 1 , further comprising a chamber for containing the insect cells, which is arranged on the transistor. The odor sensor according to claim 1 or 2, wherein the insect cell expresses an olfactory receptor by an introduction gene. The odor sensor according to claim 1 or 2, wherein the insect cell expresses an olfactory receptor of an insect. The odor sensor according to any one of claims 1 to 4, wherein the olfactory receptor is an ion channel type receptor. The odor sensor according to any one of claims 1 to 5, wherein the olfactory receptor is selected from BmOR1, BmOR3, Or13a, Or56a, Or85b and PxOR1. The odor sensor according to any one of claims 1 to 6, wherein the insect cell is a cell derived from moth. The odor sensor according to claim 7, wherein the insect cell is selected from Sf21, Sf9, High Five, and Tni-derived cells. The odor sensor according to any one of claims 1 to 6, wherein the insect cell is a cell derived from Drosophila. The odor sensor according to claim 9, wherein the insect cell is a Drosophila S2 cell. The odor sensor according to any one of claims 1 to 10, wherein the insect cell expresses a fluorescent protein whose fluorescence intensity changes according to an ion concentration. The odor sensor according to any one of claims 1 to 11, wherein the transistor is a field effect transistor. The odor sensor according to any one of claims 1 to 12, wherein the gate electrode is an extension gate electrode. The odor sensor according to any one of claims 1 to 13, wherein aluminum is exposed on the surface of the gate electrode. The odor sensor according to any one of claims 1 to 14, further comprising a Faraday cage covering the transistor.

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