Method and device for detecting odor substances

Abstract

To provide a detection method and a detection device that can detect a target odor substance with high sensitivity, even when the sample contains interfering components that interfere with the response of the odor sensor. [Solution] A method for detecting a target odor substance contained in an aqueous sample includes an extraction step of mixing the aqueous sample with a volatile organic solvent to extract the target odor substance into the volatile organic solvent, and a detection step of volatilizing the volatile organic solvent containing the target odor substance obtained in the extraction step, and bringing the target odor substance contained in the volatilized material into contact with an odor sensor capable of detecting the target odor substance.

Description

Technical Field

[0001] The present invention relates to a method and a detection device for detecting a target odor substance contained in an aqueous specimen.

Background Art

[0002] The olfactory receptor of organisms is a protein established through a long evolutionary process, and selectively responds to specific odor substances by stereoscopically recognizing the molecular structure. The olfactory receptor has both high selectivity and sensitivity, and great expectations are placed on it as a next-generation odor sensor element.

[0003] As one of the detection methods of a biohybrid odor sensor using an olfactory receptor, there is a method using cultured cells expressing an olfactory receptor and a calcium sensor fluorescent protein. Such cultured cells are called sensor cells. The olfactory receptor of insects functions as a ligand-gated ion channel, and the sensor cells show the following responses to the target odor substance. First, when the target odor substance binds to the olfactory receptor localized on the cell membrane, the ion permeation pore of the receptor opens, and calcium ions (Ca 2+ ) flow into the cell from the outside. Next, as the Ca 2+ concentration in the cytoplasm increases, the fluorescence intensity of the calcium sensor fluorescent protein increases. Based on such a mechanism, it is possible to detect the target odor substance in the specimen by measuring the change in the fluorescence intensity of the sensor cells.

[0004] Conventional techniques for measuring the response of sensor cells to target odorants in solution samples have primarily involved observing cells on a glass-bottom petri dish with a fluorescence microscope or measuring cells seeded on a microplate with a fluorescence plate reader. In both cases, the sensor cells were present in an aqueous solution, and the response was measured by directly adding the liquid sample. However, if the sample contained components that interfered with the sensor cell response, detecting the target odorant could become difficult. Therefore, there was a need to develop new methods to either eliminate the influence of interfering substances or to detect the target odorant using sensor cells after separating it from the sample. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Sato K et al., Chemical vapor detection using a reconstituted insect olfactory receptor complex, Angew. Chem. Int. Ed. (2014) 53, 11798-11802 [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide a detection method and a detection device that can detect a target odor substance with high sensitivity, even when the sample contains interfering components that interfere with the response of the odor sensor.

[0007] As a result of diligent research, the inventors of the present invention have discovered that by first mixing an aqueous sample containing a target odor substance with a volatile organic solvent to extract the target odor substance into the volatile organic solvent, then volatilizing the volatile organic solvent containing the target odor substance, and then contacting this volatile substance with an odor sensor to detect the target odor substance, it is possible to detect the target odor substance with high sensitivity even when interfering components are present in the sample, thus completing the present invention.

[0008] In other words, the present invention provides the following: (1) A method for detecting a target odor substance contained in an aqueous sample, An extraction step in which the aqueous sample is mixed with a volatile organic solvent to extract the target odor substance into the volatile organic solvent, A detection step involves volatilizing the volatile organic solvent containing the target odor substance obtained by the extraction step, and bringing the target odor substance contained in the volatilized material into contact with an odor sensor capable of detecting the target odor substance. A detection method that includes this. (2) The detection method according to (1), wherein the aqueous sample is a body fluid. (3) The detection method according to (2), wherein the bodily fluid is urine. (4) The detection method according to (1), wherein the volatile organic solvent is at least one selected from the group consisting of hexane, diethyl ether, methyl tert-butyl ether, benzene, and chloroform. (5) The detection method according to (4), wherein the volatile organic solvent is hexane. (6) The detection method described in (1), wherein multiple odor sensors are used to detect different odor substances. (7) The detection method according to (1), wherein the odor sensor is a sensor cell. (8) The detection method described in (7), wherein multiple sensor cells are used to detect different odor substances. (9) The detection method according to (7) or (8), wherein the sensor cells are used embedded in a hydrogel. (10) A detection device for detecting a target odor substance in an aqueous sample, comprising a sample container for containing a volatile organic solvent containing the target odor substance, and an odor sensor capable of detecting the target odor substance, wherein the sample container and the odor sensor are arranged in the same sealed space without contact with each other. (11) The detection device according to (10), wherein the sample containment section is hydrophilized. (12) The detection device according to (10), wherein multiple odor sensors for detecting different odor substances are arranged. (13) The detection device according to (10), wherein the odor sensor is a sensor cell. (14) The detection device according to (13), wherein multiple sensor cells for detecting different odor substances are arranged. (15) The detection device according to (13) or (14), wherein the sensor cells are used embedded in a hydrogel. (16) The detection device according to (15), wherein the sample containment portion is a recess provided in the lower substrate, and the hydrogel for embedding cells is contained in a microwell provided on the upper substrate, with the opening side of the microwell facing downward. [Effects of the Invention]

[0009] According to the detection method of the present invention, target odor substances can be detected with high sensitivity even when interfering components are present in the aqueous sample. [Brief explanation of the drawing]

[0010] [Figure 1] This figure shows a specific example of the detection device of the present invention. [Figure 2] This figure shows another specific example of the detection device of the present invention. [Figure 3] Figure 2 shows the upper substrate of the detection device, along with its usage instructions. [Figure 4] The following figures show the results of measuring acetophenone in the examples below. (a) Fluorescence recording images of the sensor element (sensor cell). Before and after reactions to hexane alone (control), hexane extract of HBSS / HEPES with added odorant (buffer), and hexane extract of artificial urine with added odorant (artificial urine). (b) Time evolution of the relative fluorescence response of the odor sensor device in (a). (c) Comparison of the maximum values ​​in (b). au: arbitrary units. [Figure 5] The following figure shows the relationship between extraction time and relative fluorescence response when acetophenone was measured in the example below. The graph shows the mean and standard deviation of 9 microwells. [Figure 6] A diagram showing the relationship between the mixing ratio (volume ratio) of hexane and artificial urine and the relative fluorescence response, where acetophenone was measured in the following examples. The average and standard deviation of 9 microwells are shown in the graph. [Figure 7] A diagram showing the relationship between the added amount of acetophenone and the relative fluorescence response, which was measured in the following examples. The average and standard deviation of 9 microwells are shown in the graph. [Figure 8] A diagram showing the relationship between the concentration of volatilized acetophenone and the relative fluorescence response, which was measured in the following examples. The average and standard deviation of 9 microwells are shown in the graph. [Figure 9] A diagram showing the relationship between the presence or absence of hydrophilization treatment of the sample chamber and the relative fluorescence response, which was measured in the following examples. The average and standard deviation of 9 microwells are shown in the graph. [Figure 10] A diagram showing the relative fluorescence responses in buffer and artificial urine measured in the following comparative examples. Buffer or artificial urine to which acetophenone was added was directly added to the buffer in which sensor cells arrayed using a microwell plate were immersed (the final concentration after addition of acetophenone was 100 μM), and the maximum values of the relative fluorescence responses of the sensor cells were compared. The average and standard deviation of 9 microwells are shown in the graph.

Embodiments for Carrying out the Invention

[0011] The aqueous sample to be used in the detection method of the present invention is a liquid sample at normal temperature (25 °C) and normal pressure, and is a sample in which liquid water is the main component (more than 50% by weight of the whole sample). Usually, it is an aqueous solution or an aqueous suspension using water as a medium. The aqueous sample is not limited at all as long as it may contain the target odor substance to be detected. Preferred examples include body fluids such as urine, saliva, blood (whole blood, serum, plasma), sweat, fecal suspension, tissue fluid, wiping fluid, etc., and tap water, sewage, groundwater, spring water, agricultural water, environmental water (river water, seawater, lake water, marsh water), etc. Among these, body fluids, particularly urine, are preferred.

[0012] In the method of the present invention, an aqueous sample is mixed with a volatile organic solvent to extract the target odor substance into the volatile organic solvent. Here, the volatile organic solvent is an organic compound that is liquid at room temperature and atmospheric pressure, and can vaporize at room temperature and atmospheric pressure to exist as a gas in the atmosphere, and usually has a boiling point of 50°C to 260°C. Preferred examples of volatile organic solvents used in the present invention include, but are not limited to, hexane, diethyl ether, methyl tert-butyl ether, benzene, and chloroform. Of these, hexane is particularly preferred. Furthermore, the volatile organic solvent can be used alone or in combination with other volatile organic solvents.

[0013] The target odorants are substances that can be extracted into volatile organic solvents and are typically hydrophobic. Many odorants are hydrophobic. Examples of odorants include, but are not limited to, acetophenone, phenol, methylheptenone, and benzaldehyde. While a single target odorant may be used, it is also possible to detect multiple target odorants simultaneously (see below).

[0014] When mixing an aqueous suspension with a volatile organic solvent, stirring is preferable. The mixing ratio (v / v) of the aqueous suspension and the volatile organic solvent during mixing is usually about 0.5 to 100 parts aqueous sample to 1 part volatile organic solvent, preferably about 1 to 10 parts. The mixing time (extraction time) is usually about 1 minute to 2 hours, preferably about 2 minutes to 1 hour.

[0015] Next, the volatile organic solvent containing the target odor substance obtained in the extraction step is volatilized, and the target odor substance contained in the volatilized material comes into contact with an odor sensor capable of detecting the target odor substance. When the volatile organic solvent containing the target odor substance is volatilized, the target odor substance contained in the volatile organic solvent also volatilizes along with the volatile organic solvent and comes into contact with the odor sensor. Hereinafter, the solution containing the target odor substance in the volatile organic solvent may be referred to as the "sample." The sample and the odor sensor should be placed at a distance from each other such that the volatilized material from the sample comes into contact with the odor sensor, but to increase detection sensitivity, it is preferable to place the sample and the odor sensor in a small sealed space. Here, the volume of the sealed space is set appropriately according to the amount of sample, but is usually about 0.5 mL to 50 mL, preferably about 1 mL to 3 mL.

[0016] Various types of odor sensors are commercially available, and any of them can be used. After bringing the odor sensor into contact with the target odor substance, the target odor substance is detected using a method appropriate to each odor sensor.

[0017] While commercially available odor sensors can be preferably used as odor sensors, sensor cells, which have seen significant research and development in recent years, in which olfactory receptor proteins from organisms (especially insects such as mosquitoes) are supported on the cell membrane of the cell, can be particularly preferably used. In sensor cells, in the presence of the corresponding odor substance, the calcium ion channels of the olfactory receptors supported on the cell membrane open, allowing calcium ions to pass through and causing extracellular calcium ions to flow into the cell. Calcium sensor fluorescent proteins are present in the cytoplasm of the sensor cells, and as the calcium ion concentration in the cytoplasm increases, the calcium sensor fluorescent proteins bind to calcium ions and emit fluorescence. By measuring this fluorescence, the odor substance can be detected. The fluorescence intensity at this time correlates with the calcium ion concentration in the cytoplasm, and consequently, with the concentration of the target odor substance in the sample, so quantitative or semi-quantitative analysis of the target odor substance is also possible using sensor cells. Since quantitative and semi-quantitative analysis inevitably involves detection, "detection" in this invention also includes quantitative and semi-quantitative analysis. Various sensor cells corresponding to various odor substances can be produced as transient or stable expression cells, so these sensor cells can be preferably used. When detection is performed using sensor cells, the measurement time (time from sample addition to measurement) is set as appropriate, but is usually about 1 to 20 minutes, preferably about 2 to 12 minutes.

[0018] Furthermore, since various odor sensors (sensor cells) corresponding to various odor substances can be fabricated, it is possible to simultaneously measure multiple target odor substances in an aqueous sample by using multiple types of odor sensors that correspond to different odor substances.

[0019] Next, a detection device suitable for performing the detection method of the present invention described above will be explained. The detection device of the present invention comprises a sample storage unit that contains a volatile organic solvent containing a target odor substance, and an odor sensor capable of detecting the target odor substance, wherein the sample storage unit and the odor sensor are arranged in the same sealed space without contact with each other.

[0020] Since odorants are typically hydrophobic, it is preferable to hydrophilize the sample containment section by oxygen plasma treatment or the like. Hydrophilizing the sample containment section makes the hydrophobic odorants more volatile, thereby increasing the detection sensitivity of the target odorant (see examples below).

[0021] A simple example will be explained with reference to Figure 1. The detection device shown in Figure 1 comprises a chamber 10. The chamber 10 is a sealed space 12, and within this sealed space 12, at the bottom of the chamber 10, is a sample storage section 14, which is an open container for storing a sample. An odor sensor 16 is positioned so as not to come into contact with the sample storage section 14.

[0022] When using the device, place the sample in the sample container 14 and leave it undisturbed. As shown by the dashed arrow in Figure 1, some of the volatile substances from the sample will come into contact with the odor sensor 16. By measuring the signal from the odor sensor 16, the target odor substance in the sample can be detected.

[0023] Next, a second specific example suitable for using sensor cells as odor sensors will be described based on Figures 2 and 3. The detection device shown in Figure 2 comprises a lower substrate 18 and an upper substrate 22 stacked on the lower substrate 18. The lower substrate 18 is provided with a sample storage section 20, which is a recess. The upper substrate 22 is provided with a recess 24 that works in cooperation with the sample storage section 20 to define a sealed space. When in use, the opening of the recess 24 faces downward, as shown in Figure 2. The bottom of the recess 24 (which becomes the top when in use) is provided with a plurality of microwells 26 for holding sensor cells.

[0024] Figure 3 shows only the upper substrate 22. The upper substrate 22 is provided with a recess 24, and at the bottom of the recess 24 are microwells 26 (9 in the illustrated example). The microwells 26 consist of cylindrical protrusions and are provided with four slits 28.

[0025] Before use, as a preparation step, the sensor cells 30 are first suspended in the hydrogel solution 32, and then a crosslinking agent is added and suspended again. After that, the sensor cell solution is pipetteed into each microwell 26 (see enlarged view at the bottom of Figure 3). At this time, because there are four slits 28, the hydrogel suspension of sensor cells can be easily placed into each microwell 26 without being obstructed by air bubbles. The hydrogel solution gels in the microwells 26, and the sensor cells 30 are held within the gelled hydrogel 30. After gelling, the hydrogel remains in the microwells 26 even if the substrate 22 is inverted. Next, the assay solution containing calcium ions is placed in the recesses 24. Store in this state under refrigeration until use.

[0026] During measurement, the sample is placed in the recess 20 of the lower substrate 18, while the upper substrate 22 is returned to room temperature, the assay solution is discarded, and the upper substrate 22 is inverted and stacked on top of the lower substrate 18. This defines a sealed space between the recess 20 in the lower substrate 18 and the recess 24 in the upper substrate 22. The dimensions of the lower substrate 18, recess 20, upper substrate 22, and recess 24 are designed so that a sealed space is formed between the recess 20 and recess 24 when the upper substrate 22 is inverted and stacked on top of the lower substrate 18. Although the recess 24 is not strictly necessary, forming the recess 24 and then forming the microwells 26 on top of it makes fabrication easier as it can be done only by cutting the plate. When left in this state, the sample volatilizes and the target odorant comes into contact with the sensor cells 30 in the hydrogel 32 held in the microwells 26 (hydrogel is permeable to odorants). Subsequently, the fluorescence from the sensor cells in each microwell 26 is measured using a fluorescence measuring device. The higher the fluorescence intensity, the higher the concentration of the target odor substance in the aqueous sample.

[0027] In the second specific example described above, if sensor cells that can detect different odor substances are placed in each microwell 26, it becomes possible to detect multiple target odor substances simultaneously.

[0028] The present invention will be described in detail below based on examples and comparative examples. However, the present invention is not limited to the following examples.

[0029] Example 1 1. Method for extracting target odor substances from aqueous sample using a volatile organic solvent. To detect target odorants in the sample using an odor sensor device, the odorants were extracted using hexane, a volatile organic solvent. The sample used was either HBSS / HEPES solution (Hanks equilibrium salt solution (containing Ca and Mg, but without phenol red) buffered with 20 mM HEPES pH 7.2) or commercially available artificial urine. The composition of the artificial urine used was as follows:

[0030] [Table 1]

[0031] 1) Acetophenone was used as the target odorant and added to the sample to a final concentration of 0-100 μM. The target odorant was pre-dissolved in DMSO (Dimethyl sulfoxide) to prepare the sample so that the final concentration of DMSO in the sample was 0.1% (w / v). 2) Transfer 1 ml of the sample to which acetophenone has been added to a screw-cap tube, add 100 μL of hexane, and vortex vigorously for 10 to 60 minutes. 3) The mixture was centrifuged using a microcentrifuge at 15,000 rpm (21,600 × g) for 5 to 10 minutes to separate it into an upper layer (hexane fraction) and a lower layer (aqueous solution fraction). 4) Transfer the upper layer to another screw-cap tube, tightly close the cap, and store at -80°C until use.

[0032] 2. Fabrication of a sealed odor sensor device 2-1. Preparation of microwell plates and sample chambers The detection devices shown in Figures 2 and 3 were fabricated using the following method. A sealed odor sensor device consisting of an upper substrate (sometimes referred to as the "microwell plate") and a lower substrate (sometimes referred to as the "sample chamber") was fabricated (Figures 2 and 3). The microwell plate was used to array sensor cells encapsulated in hydrogel, and the sample chamber was used as the site for adding volatile organic solvents containing odor substances extracted from the sample. The external dimensions of the fabricated device were 24 mm in length and 36 mm in width. The microwell plate had nine microwells, each a cylindrical shape with a slit (outer diameter 1.6 mm, inner diameter 1.2 mm, height 1.0 mm), and were positioned at 0.4 mm intervals, 2.0 mm away from the wall of the large well (corresponding to recess 24 in Figures 2 and 3). The dimensions of the large well (recess 24) were 9.6 mm in length, 9.6 mm in width, and 3 mm in depth. The sample chamber has a large central chamber measuring 21.6 mm vertically, 21.6 mm horizontally, and 3 mm deep (capacity approximately 1.4 ml). The device was fabricated by cutting an acrylic plate with an NC precision machine. A transparent acrylic plate (4 mm thick) was used for the microwell plate, and a black acrylic plate (5 mm thick) was used for the sample chamber. To reduce the adsorption of odor substances (generally hydrophobic), the sample chamber was made hydrophilic by oxygen plasma treatment. The conditions for the oxygen plasma treatment were as follows: Plasma etching apparatus: FA-1 (Samco), oxygen plasma treatment (output 25W, oxygen gas flow rate 20 ml / min, 30 seconds).

[0033] 2-2. Method for introducing sensor cells into microwell plates The cells that responded to the target odorant (sensor cells) were insect cells ExpiSf9 (Thermo Fisher Scientific) that were modified to stably express the insect olfactory receptor, which consists of an olfactory receptor (OR) and a co-receptor (ORco), and calcium sensor fluorescent protein (GCaMP). The sensor cells were introduced into the microwell plates of the odor sensor device using the hydrogel TrueGel3D (True7, Sigma-Aldrich) as follows.

[0034] 1) The water, TrueGel3D buffer, and SLO-DEXTRAN included with the TrueGel3D product were mixed in the specified amounts in a microtube by pipetting, according to the manufacturer's protocol. 2) Sensor cells cultured in an Erlenmeyer flask with ExpiSf CD medium (Thermo Fisher Scientific) for 2-4 days at 27°C with shaking (120 rpm) were collected by centrifugation (300 × g, 5 minutes) and the final cell concentration was 0.9-1 × 10⁶. 8 Add the hydrogel solution prepared in 1) above to a cell / ml concentration, and gently suspend the cells by pipetting. 3) The specified amount of crosslinking agent for TrueGel3D was added to the sensor cell suspension described in 2) above, and the suspension was gently resuspended by pipetting. 4) Since the hydrogel begins to gel immediately and becomes difficult to pipette after about 10 minutes, 1.0 to 1.1 μL was dispensed into each microwell of the microwell plate using an electric pipette.

[0035] To completely gel the hydrogel containing the sensor cells, the microwell plate described in 4) above was left to stand at 27°C for 25 minutes. Then, the larger wells (recesses 24) containing the microwells were filled with 200 μL of HBSS / PIPES / BSA solution (Hanks equilibrium salt solution (containing Ca and Mg, but without phenol red) buffered with 20 mM PIPES pH 6.2, to which 0.1% (w / v) bovine serum albumin (BSA) was added), and stored at 4°C until use.

[0036] 3. Detection of target odor substances using a sealed odor sensor device To measure target odorants extracted from a sample using hexane, or target odorants diluted by direct dissolution in hexane, detection using a sealed odor sensor device was performed using a fluorescence imager as follows.

[0037] 1) To return the microwell plates stored at 4°C as described in 2. above to room temperature, they were left to stand in a 27°C incubator for approximately 10 minutes. 2) To construct a sealed odor sensor device, 180 μL of HBSS / PIPES / BSA was removed by pipetting from the 200 μL of HBSS / PIPES / BSA filling the large well of a microwell plate and placed on the sample chamber. At this time, the microwell plate was placed upside down, with the side containing the microwells facing the sample chamber (see Figure 2). 3) The assembled odor sensor device was placed in a fluorescence imager (DP-T130z, Biotools), and the microwell plate and sample chamber were slightly offset to create a gap so that the sample could be dispensed by pipetting. 4) The green fluorescence (530 nm) of the sensor cells was detected under blue light (485 nm) excitation, and fluorescence image recording was started. To prevent fluorescence decolorization due to excessive irradiation of the excitation light, an intermittent timer (FT-022, Tokyo Glass Instruments) was used to perform 1.5-second exposures at 30-second intervals. 105 seconds after the start of recording, the door of the fluorescence imager was opened, 5 μL of hexane extract or hexane dilution containing the target odorant was dropped into the sample chamber, and the microwell plate and sample chamber were realigned and sealed. Fluorescence image recording was performed for a total of 540 seconds.

[0038] The recorded green fluorescence images were analyzed using the image analysis software ImageJ (NIH) to quantify and quantify the fluorescence intensity of the microwells. The changes in the quantified fluorescence intensity were normalized to a relative fluorescence response (ΔF / F0), where F0 represents the fluorescence intensity immediately after droplet placement and F represents the fluorescence intensity at other points in time. A graph was then created. Furthermore, the maximum relative fluorescence response (Max ΔF / F0) within the measurement time was extracted, and the measurement results from each experiment were compared.

[0039] 4. Detection of target odor substances using a sealed odor sensor device To detect target odor substances in samples, we investigated whether odor substances extracted from samples using volatile organic solvents could be measured using a fabricated sealed odor sensor device. HBSS / HEPES (buffer) or artificial urine was used as the sample, and acetophenone, the target odor substance, was added to a concentration of 100 μM. 100 μL of hexane was added to 1000 μL of the sample, and vortex extraction was performed for 10 minutes. The response of the odor sensor device to acetophenone in the hexane extract was measured (Figure 4). Measurements were performed for both buffer and artificial urine samples, as well as for hexane alone (control) which did not contain acetophenone.

[0040] Figure 4a shows fluorescence recording images of the sensor element (sensor cells arrayed in microwells). No significant changes were observed in the fluorescence images before and after the reaction in the control group. On the other hand, with the buffer or hexane extract of artificial urine, an increase in fluorescence intensity was observed in the images after the reaction, confirming the response of the odor sensor device to acetophenone.

[0041] Figure 4b shows the time evolution of the normalized relative fluorescence response in the odor sensor device shown in Figure 4a. The sample was added to the odor sensor device 105 seconds after the start of recording. The curves in the graph show the mean and standard deviation of the nine sensor elements. In the control, no significant increase in fluorescence response was observed after the addition of the sample. In contrast, with the buffer or hexane extract of artificial urine, the increase in fluorescence response began approximately 60 seconds after sample addition and reached its maximum value at approximately 400 seconds.

[0042] Figure 4c shows a comparison of the maximum values ​​of the relative fluorescence response over time in Figure 4b. The fluorescence response to hexane extracts of buffer or artificial urine was confirmed to be more than five times higher than that of the control.

[0043] Example 2: Investigation of extraction time in extraction using hexane To investigate the experimental conditions for the hexane extraction method, the effect of extraction time was evaluated. Artificial urine was used as the sample, and acetophenone was added to a concentration of 100 μM. 100 μL of hexane was added to 1000 μL of the sample, and vortexing was performed for 10, 20, 30, and 60 minutes, respectively. The response of an odor sensor device to acetophenone contained in the hexane extract was measured (Figure 5). Comparing the maximum values ​​of the relative fluorescence response, no significant difference was observed in the extraction efficiency of acetophenone from artificial urine using hexane at the 10-60 minute extraction times investigated.

[0044] Example 3: Investigation of the mixing volume ratio of hexane to sample in extraction using hexane. To investigate the experimental conditions for the hexane extraction method, the effect of the hexane-to-sample mixing volume ratio was evaluated. Artificial urine was used as the sample, and acetophenone was added to a concentration of 10 μM. 1000 μL, 500 μL, 200 μL, and 100 μL of hexane were mixed with 1000 μL of sample (hexane:artificial urine volume ratios of 1:1, 1:2, 1:5, and 1:10), and the response of an odor sensor device to acetophenone contained in the hexane extract was measured (Figure 6). The vortex extraction operation was performed for 20 minutes. By comparing the maximum values ​​of the relative fluorescence response, it was confirmed that the extraction efficiency of acetophenone from artificial urine using hexane improved as the proportion of artificial urine increased for the hexane:artificial urine mixing volume ratios investigated (1:1 to 1:10).

[0045] Example 4: Concentration-dependent detection of odor substances contained in a sample. To evaluate the quantitative accuracy of the hexane extraction method and the fabricated odor sensor device, the concentration-dependent detection of odor substances in the sample was investigated. Artificial urine was used as the sample, and acetophenone was added at concentrations of 0.1, 1, 10, and 100 μM. Hexane and the sample were mixed in a volume ratio of 1:10, and vortex extraction was performed for 20 minutes. The response of the odor sensor device to acetophenone in the hexane extract was measured (Figure 7). By comparing the maximum values ​​of the relative fluorescence response, it was confirmed that the hexane extraction method and the fabricated odor sensor device, when used in combination, showed linearity in the range of 1 to 100 μM for acetophenone added to artificial urine.

[0046] Example 5: Concentration-dependent detection of volatile odor substances To evaluate the quantitative accuracy of the fabricated odor sensor device, the concentration-dependent detection of odorants in their volatile state was investigated. Acetophenone was used as the odorant and diluted with hexane to achieve volatile concentrations of 0.01, 0.1, 1, 10, 100, and 1000 ppm in the sample chamber. Equal amounts of hexane-diluted acetophenone were added to the sample chamber, and the response of the odor sensor device to the volatile acetophenone was measured (Figure 8). By comparing the maximum relative fluorescence responses, it was confirmed that the fabricated odor sensor device showed linearity with respect to volatile acetophenone in the range of 1 to 100 ppm, and that linearity was lost above 1000 ppm.

[0047] Example 6: Effects of hydrophilization of the sample chamber Odor substances are generally hydrophobic and tend to adsorb easily onto hydrophobic surfaces such as acrylic. To improve the detection sensitivity of the developed odor sensor device, the effect of hydrophilization of the sample chamber was investigated. Hydrophilization was performed by oxygen plasma treatment. Acetophenone was used as the odor substance and diluted with hexane to a volatilization concentration of 1 ppm in the sample chamber. Acetophenone diluted with hexane was dropped into both a hydrophilized and an untreated sample chamber, and the response of the odor sensor device to the volatilized acetophenone was measured (Figure 9). Comparing the maximum values ​​of the relative fluorescence response, the response to 1 ppm acetophenone using the untreated sample chamber was approximately 1.7 times that of 0 ppm acetophenone. In contrast, the response to 1 ppm acetophenone using the hydrophilized sample chamber was approximately 5.9 times that of 0 ppm acetophenone. These results confirm that hydrophilization of the sample chamber improves the detection sensitivity of the developed odor sensor device.

[0048] Comparative Example 1: Example of target odor substance detection when an aqueous solution sample is directly added to an odor sensor device. Conventionally, in the detection of odor substances using sensor cells as sensor elements, the main method has been to directly add a sample containing the odor substance (such as artificial urine) to the buffer (such as HBSS / HEPES / BSA) in which the sensor cells are immersed. Similar to conventional methods, experiments were conducted in which a buffer containing the target odor substance or artificial urine was added directly to the large wells of the microwell plate of this device (without removing the HBSS / HEPES / BSA filling the large wells). As a result, it was observed that the response of sensor cells to odor substances in artificial urine was nearly three times lower compared to the response to odor substances in the buffer (HBSS / HEPES) (Figure 10). It is thought that some component of the artificial urine inhibits the sensor cell response, but the identification of this inhibitory component has not yet been performed. Similar problems are expected to occur in the measurement of odor substances in bodily fluids such as real urine. [Explanation of Symbols]

[0049] 10 chambers 12 Closed space 14 Sample storage section 16 Odor Sensor 18 Lower circuit board 20 Sample storage section 22 Upper circuit board 24 recesses 26 microwells 28 slits 30 Sensor Cells 32 Hydrogels

Claims

1. A method for detecting a target odor substance contained in an aqueous sample, An extraction step in which the aqueous sample is mixed with a volatile organic solvent to extract the target odor substance into the volatile organic solvent, A detection step involves volatilizing the volatile organic solvent containing the target odor substance obtained by the extraction step, and bringing the target odor substance contained in the volatilized material into contact with an odor sensor capable of detecting the target odor substance. A detection method that includes this.

2. The detection method according to claim 1, wherein the aqueous sample is a body fluid.

3. The detection method according to claim 2, wherein the bodily fluid is urine.

4. The detection method according to claim 1, wherein the volatile organic solvent is at least one selected from the group consisting of hexane, diethyl ether, methyl tert-butyl ether, benzene, and chloroform.

5. The detection method according to claim 4, wherein the volatile organic solvent is hexane.

6. The detection method according to claim 1, wherein multiple odor sensors are used to detect different odor substances.

7. The detection method according to claim 1, wherein the odor sensor is a sensor cell.

8. The detection method according to claim 7, wherein multiple sensor cells are used to detect different odor substances.

9. The detection method according to claim 7 or 8, wherein the sensor cells are used in a state embedded in a hydrogel.

10. A detection device for detecting a target odor substance in an aqueous sample, comprising a sample containment section for containing a volatile organic solvent containing the target odor substance, and an odor sensor capable of detecting the target odor substance, wherein the sample containment section and the odor sensor are arranged in the same sealed space without contact with each other.

11. The detection device according to claim 10, wherein the sample containment section is subjected to hydrophilic treatment.

12. The detection device according to claim 10, wherein multiple odor sensors for detecting different odor substances are arranged.

13. The detection device according to claim 10, wherein the odor sensor is a sensor cell.

14. The detection device according to claim 13, wherein multiple sensor cells, each detecting a different odor substance, are arranged.

15. The detection device according to claim 13 or 14, wherein the sensor cells are used in a state embedded in a hydrogel.

16. The detection device according to claim 15, wherein the sample containment portion is a recess provided in the lower substrate, and the hydrogel for embedding cells is contained in a microwell provided on the upper substrate, with the opening side of the microwell facing downward.

Images