Method and unit for detecting substance in gas phase

Abstract

To provide a method and a detection unit capable of preferably detecting a substance in a gas phase.SOLUTION: A method for detecting a substance in a gas phase comprises: a separation step for separating cells from a cell suspension being a solution where cells are suspended; an exposure step for exposing the cells separated from the cell suspension, to a substance in a gas phase; and an evaluation step for evaluating the cells exposed to the substance in the gas phase, in this order. A detection unit used for detecting the substance in the gas phase comprises: a channel member for defining a channel for causing the cell suspension to flow, which is the solution where the cells are suspended; and a trap member which is arranged in the channel for blocking the flow in the channel, the trap member having one or more openings which are configured to trap the cells while allowing passage of the solution.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to a method for detecting a substance in the gas phase and to a detection unit for detecting a substance in the gas phase. [Background technology]

[0002] Patent Document 1 describes a technique for dissolving an odorant, which is a poorly water-soluble organic compound, in an aqueous solution by spraying droplets containing bubbles with a diameter of 1 μm or less toward a gas containing the odorant. Patent Document 1 proposes a technique for dissolving an odorant, which is a poorly water-soluble organic compound, in an aqueous solution, based on the finding that cells for detecting odorants must be placed in a liquid and cannot be placed in air. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2019 / 156180 Summary of the Invention [Problem to be solved by the invention]

[0004] When detecting a substance in the gas phase using the above-mentioned method, the substance needs to be dissolved in an aqueous solution. However, since the solubility of different substances in aqueous solutions varies, there is room for improvement in terms of detection accuracy and the time required for detection.

[0005] The present invention aims to provide a method and a detection unit which allow good detection of substances in the gas phase. [Means for solving the problem]

[0006] The method for detecting a substance in a gas phase of the present invention is [1] "a method for detecting a substance in a gas phase, comprising, in this order, a separation step of separating cells from a cell suspension, which is a solution in which cells are suspended, an exposure step of exposing the cells separated from the cell suspension to a substance in the gas phase, and an evaluation step of evaluating the cells exposed to the substance in the gas phase."

[0007] In the method for detecting a substance in a gas phase of the present invention, cells separated from a cell suspension are exposed to a substance in a gas phase, so that the substance in the gas phase can be detected without depending on the solubility of the substance in a solution, as compared with the case where cells are exposed to a substance in a liquid phase. Furthermore, the present inventors' investigations have revealed that when cells are exposed to a substance in a gas phase, the cells respond to the substance with high sensitivity, as compared with the case where cells are exposed to the substance in a liquid phase. Thus, the method for detecting a substance in a gas phase of the present invention can detect a substance in a gas phase well. The present invention is based on the findings of the present inventors that the cells can survive to some extent even when they are separated from a cell suspension and placed in a gas phase.

[0008] The method for detecting a substance in a gas phase of the present invention may be [2] "the method according to [1], wherein the separation step includes separating the cells from the cell suspension by contacting the cell suspension with a cell trapping portion." In this case, the cells can be easily separated from the cell suspension.

[0009] The method for detecting a substance in a gas phase of the present invention may be [3] "the method according to [2], in which the cell trapping portion has one or more openings configured to trap the cells while allowing the solution to pass therethrough, and the separation step includes flowing the cell suspension through a flow path provided with the cell trapping portion." In this case, cells can be separated from the cell suspension by flowing the cell suspension through the flow path, and the cells can be easily separated from the cell suspension. In addition, the cells trapped in one or more openings can be exposed to a substance in the gas phase from both sides of the openings, and the cells can be efficiently exposed to the substance.

[0010] The method for detecting a substance in a gas phase of the present invention may be [4] "the method according to [3], wherein the cell trapping portion includes a trapping member at least a part of which is made of a porous body, and the one or more openings are holes in the porous body." In this case, cells can be easily separated from the cell suspension.

[0011] The method for detecting a substance in a gas phase of the present invention may be [5] "the method according to [3] or [4], wherein the maximum width of each of the one or more openings is 30 μm or less." In this case, cells can be suitably trapped in the openings.

[0012] The method for detecting a substance in a gas phase of the present invention may be [6] "the method according to any one of [2] to [5], wherein the cell trapping portion is configured to trap cells having a maximum width of 15 μm or more." In this case, cells having a maximum width of 15 μm or more can be trapped in the cell trapping portion.

[0013] The method for detecting a substance in a gas phase of the present invention may be [6-1] "the method according to any one of [2] to [5], wherein the cell trapping portion is configured to trap cells having a maximum width of 10 μm or more." In this case, cells having a maximum width of 10 μm or more can be trapped in the cell trapping portion.

[0014] The method for detecting a substance in a gas phase of the present invention may be [6-2] "the method according to any one of [2] to [5], wherein the cell trapping portion is configured to trap cells having a maximum width of 5 μm or more." In this case, cells having a maximum width of 5 μm or more can be trapped in the cell trapping portion.

[0015] The method for detecting a substance in a gas phase of the present invention may be [6-3] "the method according to any one of [2] to [5], wherein the cell trapping portion is configured to trap cells having a maximum width of 1 μm or more." In this case, cells having a maximum width of 1 μm or more can be trapped in the cell trapping portion.

[0016] The method for detecting a substance in a gas phase of the present invention may be [6-4] "the method according to any one of [2] to [5], wherein the cell trapping portion is configured to trap cells having a maximum width of 0.5 μm or more." In this case, cells having a maximum width of 0.5 μm or more can be trapped in the cell trapping portion.

[0017] The method for detecting a substance in a gas phase of the present invention may be [7] "the method according to [2], wherein the cell trapping section includes a trapping area formed on the surface of a flow path for flowing the cell suspension, the trapping area is configured such that when the cell suspension comes into contact with the trapping area, the solution is not trapped but the cells are trapped on the trapping area, and the separation step includes flowing the cell suspension through the flow path so as to come into contact with the trapping area." In this case, the cells can be separated from the cell suspension by flowing the cell suspension through the flow path, and the cells can be easily separated from the cell suspension.

[0018] The method for detecting a substance in a gas phase of the present invention may be [8] "a method according to any one of [2] to [7], which includes a step of removing at least one of the cells and the substance from the cell trapping portion by flowing a washing liquid so as to contact the cell trapping portion after the evaluation step." In this case, at least one of the cells and the substance can be removed from the cell trapping portion after the evaluation step.

[0019] In the method for detecting a substance in a gas phase of the present invention, [9] "the separation step includes flowing the cell suspension through a flow path provided with the cell trap portion, and the volume of the flow path is 7000 mm 3 The present invention may be a method according to any one of [2] to [8], which is as follows. Studies by the present inventors have revealed that if the volume of the cell culture space is small, the cells can survive for a long period of time in a gas phase culture environment. Therefore, if the volume of the flow channel is small, the cells can be exposed to a substance in the gas phase for a long period of time.

[0020] The method for detecting a substance in a gas phase of the present invention may be

[10] "the method according to any one of [1] to [9], wherein the exposure time in the exposing step is 1 minute or less." Studies by the present inventors have revealed that when cells are exposed to a substance in a gas phase, the cells respond to the substance with high sensitivity compared to when the cells are exposed to the substance in a liquid phase. Thus, the method for detecting a substance in a gas phase of the present invention can detect a substance in the gas phase even if the exposure time is short.

[0021] The method for detecting a substance in a gas phase of the present invention may be

[11] "a method according to any one of [1] to [9], wherein the exposure time in the exposing step is 1 minute or longer." Studies by the present inventors have revealed that cells can survive in a gas phase culture environment for 1 minute or longer. Thus, in the method for detecting a substance in a gas phase of the present invention, even if the exposure time is 1 minute or longer, cells can be exposed to a substance in a gas phase without a significant decrease in cell viability.

[0022] The method for detecting a substance in a gas phase of the present invention may be

[12] "a method according to any one of [1] to [9], wherein the exposure time in the exposing step is from 1 hour to 8 hours." Studies by the present inventors have revealed that cells can survive for 1 hour or more even in a gas phase culture environment. Thus, in the method for detecting a substance in a gas phase of the present invention, cells can be exposed to a substance in a gas phase without a significant decrease in cell viability, even if the exposure time is from 1 hour to 8 hours.

[0023] The method for detecting a substance in a gas phase of the present invention may be

[13] "a method according to any one of [1] to

[12] , in which the exposing step is carried out in a sealed environment." Studies by the present inventors have revealed that cells can survive for a longer period of time under gas phase culture conditions in a sealed environment, compared to an open environment. Thus, when the exposing step is carried out in a sealed environment, cells can be exposed to a substance in a gas phase without a significant decrease in cell viability.

[0024] The method for detecting a substance in a gas phase of the present invention may be

[14] "the method according to any one of [1] to

[13] , wherein the evaluation step includes detecting fluorescence emitted by the cells." In this case, the cells exposed to the substance in the gas phase can be evaluated by detecting the fluorescence emitted by the cells.

[0025] The method for detecting a substance in a gas phase of the present invention may be

[15] "the method according to any one of [1] to

[14] , wherein the cell is a cell expressing a cell membrane receptor that binds to the substance." In this case, the substance in the gas phase can be detected using the cell expressing a cell membrane receptor that binds to the substance.

[0026] The method for detecting a substance in a gas phase of the present invention may be

[16] "the method according to any one of [1] to

[15] , wherein the substance is a substance consisting of molecules with a molecular weight of 20 or more and 400 or less." In this case, the substance consisting of molecules with a molecular weight of 20 or more and 400 or less can be detected.

[0027] The detection unit for a substance in a gas phase of the present invention is

[17] "a detection unit used for detecting a substance in a gas phase, comprising a flow path member defining a flow path for flowing a cell suspension, which is a solution in which cells are suspended, and a trap member disposed within the flow path so as to interrupt the flow within the flow path, the trap member defining one or more openings configured to trap the cells while allowing the solution to pass through."

[0028] In the detection unit for a substance in a gas phase of the present invention, a cell suspension is passed through a flow path, whereby cells can be trapped in the trapping member. Then, a gas containing a substance is passed through the flow path, whereby cells separated from the cell suspension can be exposed to the substance in the gas phase. In this case, the substance in the gas phase can be detected without depending on the solubility of the substance in the solution, as compared with the case where cells are exposed to the substance in the liquid phase. Furthermore, the inventors' studies have revealed that when cells are exposed to a substance in the gas phase, the cells respond to the substance with high sensitivity, as compared with the case where cells are exposed to the substance in the liquid phase. Furthermore, in this detection unit for a substance in a gas phase, the cells trapped in one or more openings can be exposed to the substance in the gas phase from both sides of the opening, and the cells can be efficiently exposed to the substance. Thus, the detection unit for a substance in a gas phase of the present invention can detect the substance in the gas phase well. The present invention is based on the findings of the inventors that even when cells are separated from a cell suspension and placed in the gas phase, the cells can survive to a certain extent.

[0029] The detection unit for a substance in a gas phase of the present invention may be

[18] "the detection unit according to

[17] , in which at least a part of the trapping member is made of a porous body, and the one or more openings are holes in the porous body." In this case, cells can be easily separated from a cell suspension.

[0030] The detection unit for a substance in a gas phase of the present invention may be

[19] "the detection unit according to

[17] or

[18] , wherein the maximum width of each of the one or more openings is 30 μm or less." In this case, cells can be suitably trapped in the openings.

[0031] The detection unit for a substance in a gas phase of the present invention may be

[20] "the detection unit according to any one of

[17] to

[19] , wherein the trapping member is configured to trap cells having a maximum width of 0.5 μm or more, 1 μm or more, 5 μm or more, 10 μm or more, or 15 μm or more." In this case, the trapping member can trap cells having a maximum width of 0.5 μm or more, 1 μm or more, 5 μm or more, 10 μm or more, or 15 μm or more.

[0032] The detection unit for a substance in a gas phase of the present invention is,

[21] "wherein the volume of the flow path is 7000 mm 3 The present invention may be a detection unit according to any one of

[17] to

[20] below. Studies by the present inventors have revealed that if the volume of the cell culture space is small, the cells can survive for a long period of time in a gas-phase culture environment. Therefore, if the volume of the flow channel is small, the cells can be exposed to a substance in the gas phase for a long period of time.

[0033] The detection unit for a substance in a gas phase of the present invention may be

[22] "the detection unit according to any one of

[17] to

[21] , in which the trap member separates the space in the flow channel into a first portion and a second portion, and the flow channel member has a first opening connected to the first portion for supplying the cell suspension, a second opening connected to the first portion for supplying a gas containing the substance, and a third opening connected to the second portion for discharging the solution and the gas." In this case, after a cell suspension is supplied from the first opening to trap cells in the trap member, a gas containing the substance can be supplied from the second opening to expose the cells to the substance. In addition, the solution (cell suspension) and gas supplied from the first and second openings can be discharged from the third opening.

[0034] The detection unit for a substance in a gas phase of the present invention may be

[23] "the detection unit according to

[22] , in which the flow path member further has a fourth opening connected to the second part for supplying the gas." In this case, the gas containing the substance can be supplied not only from the second opening (first part side) but also from the fourth opening (second part side).

[0035] The detection unit for a substance in a gas phase of the present invention may be

[24] "the detection unit according to any one of

[17] to

[21] , wherein the trapping member includes a first trapping member and a second trapping member, the first trapping member separates a space in the flow path into a first portion and a second portion, the second trapping member separates a space in the flow path into the second portion and a third portion, and the flow path member has a first opening connected to the first portion for supplying the cell suspension, a second opening connected to the first portion for supplying a gas containing the substance, a third opening connected to the second portion for discharging the solution and the gas, a fourth opening connected to the third portion for supplying the cell suspension, and a fifth opening connected to the third portion for supplying the gas containing the substance." In this case, the substance can be detected at each position of the first trapping member and the second trapping member. Effect of the Invention

[0036] According to the present invention, it is possible to provide a method and a detection unit that can effectively detect a substance in a gas phase in the gas phase. [Brief description of the drawings]

[0037] [Figure 1] Fig. 1(a) is a perspective view of a detection unit according to an embodiment, and Fig. 1(b) is a cross-sectional view taken along line BB shown in Fig. 1(a). [Diagram 2] Fig. 2(a) is a plan view of the second substrate, and Fig. 2(b) is a cross-sectional view taken along line BB shown in Fig. 2(a). [Diagram 3] Fig. 3(a) is a plan view of the first substrate, and Fig. 3(b) is a cross-sectional view taken along line BB shown in Fig. 3(a). [Figure 4] 4(a) to (d) are plan views showing how cells are exposed to a gaseous substance after being separated from a cell suspension. [Diagram 5]5(a) and (b) are plan views showing how cells on a trapping member are washed with a solution. [Figure 6] 6(a) and (b) are plan views showing how cells on a trapping member are disrupted and removed with a cell disruption liquid. [Figure 7] 7(a) and (b) are plan views showing how cells on a trapping member are detached and removed from the trapping member using a cell detachment solution. [Figure 8] 8(a) to (d) are plan views showing an example of how cells on a trapping member are exposed to gas introduced through two openings. [Figure 9] 9(a) to (d) are plan views showing another example of how cells on a trapping member are exposed to gas introduced through two openings. [Figure 10] 10(a) to (e) are cross-sectional views showing modified examples of the trap member 30. FIG. [Figure 11] FIG. 11 is a perspective view of a detection unit according to a first modified example. [Figure 12] 12(a) and (b) are plan views showing how, in the first modified example, cells are separated from a cell suspension, then exposed to a substance in the gas, and fluorescence is measured at one detection position. [Figure 13] 13(a) and (b) are plan views showing how, in the first modified example, cells are separated from a cell suspension, then exposed to a substance in a gas, and fluorescence is measured at three detection positions. [Figure 14] 14(a) to (d) are plan views showing how cells are exposed to a substance in gas and fluorescence is measured at three detection positions while a washing step is performed repeatedly in the first modified example. [Figure 15] 15(a) to (d) are plan views showing how cells are exposed to a substance in gas and fluorescence is measured at three detection positions while a washing step is performed, in the first modified example. [Figure 16]Figures 16(a) to (c) are plan views showing how, in the first modified example, cells are exposed to a substance in a gas and fluorescence is measured at the detection position, each of which is a different detection position for each opening through which the gas is introduced. [Figure 17] Fig. 17(a) is a perspective view of a detection unit of a second modified example, and Fig. 17(b) is a partial cross-sectional view taken along line BB shown in Fig. 17(a). [Figure 18] Fig. 18(a) is a plan view of a second substrate of the second modified example, Fig. 18(b) is a plan view of a first member of the second modified example, and Fig. 18(c) is a plan view of a second member of the second modified example. [Figure 19] 19(a) to (f) are schematic diagrams showing the shape of a trap member according to a second modified example. [Figure 20] 20(a) and (b) are plan views showing how cells are exposed to a substance in gas after being separated from a cell suspension in the second modified example. [Figure 21] 21(a) and (b) are plan views showing how cells are trapped in the trapping member and separated from a cell suspension in the second modified example. [Figure 22] Fig. 22(a) is a plan view showing how gas is introduced into the flow channel and the cells trapped in the trapping member are exposed to a substance in the gas in the second modified example, and Fig. 22(b) is a plan view showing how the cells on the trapping member are washed in the second modified example. [Diagram 23] FIG. 23 is a plan view showing how a gas is introduced into the flow channel and the cells trapped in the trapping member are exposed to a substance in the gas in the second modified example. [Figure 24] FIG. 2 shows the dependence of cell survival rate on the volume of culture medium (culture supernatant) when a filter paper soaked in culture medium (control) or ethanol was placed in Example 1. [Diagram 25]Figures 25(a) to 25(g) show the dependence of cell survival rate on the volume of culture medium (culture supernatant) when filter paper soaked in 0% ethanol (100% culture medium), 20% ethanol (80% culture medium), 40% ethanol (60% culture medium), 50% ethanol (50% culture medium), 60% ethanol (40% culture medium), 80% ethanol (20% culture medium), and 100% ethanol (0% culture medium) was placed in Example 1. [Figure 26] Fig. 26(a) is a diagram showing the responsiveness of cells to substances in the gas phase when exposed to the gas phase after gas phase culture in Example 2. Fig. 26(b) is a diagram showing the responsiveness of cells to substances in the gas phase when exposed to the liquid phase after gas phase culture in Example 2. [Figure 27] FIG. 27 is a bar graph showing the results for exposure times of 0 and 1 minute in FIG. 26(a) and for exposure times of 1, 5 and 7 minutes in FIG. 26(b). [Figure 28] FIG. 28 shows the culture time-dependent changes in cell viability of HeLa cells cultured in a gas phase in Example 3. [Figure 29] FIG. 29 shows the culture time-dependent changes in cell viability of CHO-K1 cells cultured in a gas phase in Example 4. [Diagram 30] FIG. 30 shows the change in cell viability depending on the culture time for cells cultured in the gas phase in a closed or open environment in Example 5. [Diagram 31] Fig. 31(a) is a graph showing the culture time-dependent change in cell viability of cells cultured in a gas phase on a 12-well plate in Example 6. Fig. 31(b) is a graph showing the culture time-dependent change in cell viability of cells cultured in a gas phase on a 96-well plate in Example 6. [Diagram 32] Fig. 32(a) is a diagram showing fluorescent images of cells after gas-phase culture before histamine stimulation (0 seconds) and immediately after histamine stimulation (6 seconds) in Example 7. Fig. 32(b) is a diagram showing the results of displaying the time change in fluorescent intensity in the ROI shown in Fig. 32(a) normalized by the average brightness value in the ROI at the start of observation. [Diagram 33] FIG. 33 shows the ethanol concentration dependence of cell viability when cells on a non-permeable normal well plate or a permeable Transwell plate were exposed to ethanol in the gas phase in Example 8. [Diagram 34] Figure 34 is a plasmid map of the plasmid (pIB-Or13a-Orco plasmid) used to express Or13a protein and Orco protein in Example 9. [Diagram 35] Fig. 35(a) is a perspective view of the measurement container used in Example 9. Fig. 35(b) is an exploded perspective view of the measurement container used in Example 9. Fig. 35(c) is a top view of the lid of the measurement container used in Example 9. Fig. 35(d) is a bottom view of the dish body of the measurement container used in Example 9. [Diagram 36] Fig. 36(a) is a fluorescent image 0 seconds after the start of imaging in Example 9. Fig. 36(b) is a fluorescent image 60 seconds after the start of imaging in Example 9. Fig. 36(c) is a fluorescent image 180 seconds after the start of imaging in Example 9. [Figure 37] Figure 37 shows the time course of fluorescence intensity of Or13a-Orco-expressing Sf9 cells in Example 9. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. In the following description, the same or corresponding elements are designated by the same reference numerals, and duplicated description will be omitted.

[0039] The detection unit 1 (detection device) shown in Figures 1 to 3 is used to detect substances in the gas phase (Figures 4 to 9) using a cell 5 (Figure 4). A method for detecting substances in the gas phase using the detection unit 1 will be described later. First, the configuration of the detection unit 1 will be described below.

[0040] <Detection unit> As shown in Figures 1 to 3, the detection unit 1 includes a base body 2. The base body 2 includes a first substrate 10 and a second substrate 20. The first substrate 10 and the second substrate 20 are formed, for example, from glass in a rectangular plate shape and are optically transparent. The first substrate 10 has a flat surface 10a.

[0041] As shown in FIG. 3(a) and FIG. 3(b), a groove 11 is formed on the surface 10a of the first substrate 10. The groove 11 has a flow path portion 12, a first connection portion 13, a second connection portion 14, and a third connection portion 15. The flow path portion 12 is formed in a T-shape in a plan view (when viewed in a direction perpendicular to the surface 10a), and has a linear main body portion 12a and a branch portion 12b that branches from the middle portion of the main body portion 12a and extends linearly. The flow path portion 12 has a uniform cross-sectional shape along the extension direction. The cross-sectional shape is, for example, rectangular. In this example, in a cross section perpendicular to the extension direction, the width of the flow path portion 12 is 0.5 mm, and the depth of the flow path portion 12 is 0.2 mm.

[0042] The first connection portion 13 is connected to one end of the main body portion 12a. The third connection portion 15 is connected to the other end of the main body portion 12a. The second connection portion 14 is connected to one end of the branch portion 12b (the end opposite to the end connected to the main body portion 12a). The shapes of the first connection portion 13, the second connection portion 14, and the third connection portion 15 in a plan view are circular in this example, but are not limited to this and may be any shape.

[0043] The second substrate 20 is disposed on the surface 10a of the first substrate 10 and is in close contact with the surface 10a. Such close contact is achieved, for example, by using an adhesive. As a result, the opening of the flow path portion 12 of the groove 11 is blocked by the second substrate 20, and a flow path 40 is formed between the flow path portion 12 and the second substrate 20. In other words, the first substrate 10 and the second substrate 20 (base 2) are flow path members that define the flow path 40, and the second substrate 20 is a cover member that blocks the groove 11.

[0044] The flow path 40 has a shape corresponding to the above-mentioned flow path portion 12. The cross-sectional area of ​​the flow path 40 in a cross section perpendicular to the extending direction is not particularly limited, but is, for example, 0.0001 mm 2 Above 0.0003mm 2 Above 0.001mm 2 More than 0.003mm 2 Above 0.01mm 2 Above 0.03mm 2 Above 0.1mm 2 More than 0.3mm 2 Above 1.0mm 2 Above 3.0mm 2 Above 10mm 2 Above, 30mm 2 or more than 100mm 2 It may be more than 1000 mm 2 Below, 300mm 2 Below, 100mm 2 Below, 30mm 2 Below, 10mm 2 Below, 3.0mm 2 Less than or equal to 1.0mm 2 In one preferred embodiment, the cross-sectional area of ​​the flow passage 40 is 300 mm 2 The volume of the flow path 40 is not particularly limited, but is preferably 0.1 mm 3 More than 1mm 3 or more, 3mm 3 Above 10mm 3 Above, 30mm 3 Above 100mm 3 Above 300mm 3 Above 500mm 3 More than 1000mm 3 or more than 2000mm 3 It may be 100,000 mm or more. 3 Below, 30000mm 3 Below, 10000mm 3 Below, 7000mm 3 Below, 5000mm 3 Below, 3000mm 3 Below, 2000mm 3 Below, 1000mm 3 Below, 500mm 3Below, 400mm 3 Below, 100mm 3 Below, 50mm 3 Less than or equal to 10mm 3 It may be the following.

[0045] As shown in FIG. 2(a) and FIG. 2(b), the second substrate 20 has a first opening 21, a second opening 22, and a third opening 23 formed therein. The first opening 21, the second opening 22, and the third opening 23 penetrate the second substrate 20 in the thickness direction. The first opening 21, the second opening 22, and the third opening 23 are connected to the first connection portion 13, the second connection portion 14, and the third connection portion 15 of the first substrate 10, respectively. As a result, the flow path 40 is connected to the outside of the base body 2 at the first opening 21, the second opening 22, and the third opening 23. The shapes of the first opening 21, the second opening 22, and the third opening 23 in a plan view are circular in this example, but are not limited thereto and may be any shape.

[0046] A trapping member 30 (support, cell trapping portion) is disposed within the flow path 40. The trapping member 30 is formed, for example, in the shape of a rectangular plate from a porous body. The trapping member 30 is disposed within the flow path 40 so as to block the flow within the flow path 40. In this example, the trapping member 30 is disposed perpendicular to the flow direction within the flow path 40 (extension direction of the flow path 40). The trapping member 30 is disposed so that the outer edge of the trapping member 30 contacts the surface of the flow path 40 (without any gaps). The trapping member 30 is disposed at a portion located on the third connection portion 15 side with respect to the connection portion of the branch portion 12b in the main body portion 12a of the flow path portion 12.

[0047] The trap member 30 divides the space in the flow path 40 into a first portion 41 and a second portion 42. The first portion 41 corresponds to a portion of the main body 12a of the flow path portion 12 located on the first connection portion 13 side with respect to the trap member 30, and the branch portion 12b. The second portion 42 corresponds to a portion of the main body 12a of the flow path portion 12 located on the third connection portion 15 side with respect to the trap member 30. The first opening 21 and the second opening 22 are connected to the first portion 41, and the third opening 23 is connected to the second portion 42.

[0048] As shown in FIG. 1(b), the trapping member 30 has a plurality (a large number) of through-holes 31 (openings). In this example, the through-holes 31 are holes (fine pores) in the porous body constituting the trapping member 30. The through-holes 31 penetrate the trapping member 30 along the thickness direction (flow direction in the flow channel 40). The through-holes 31 are, for example, circular. When a cell suspension 3 (FIG. 4), which is a solution 6 in which cells 5 are suspended, is flowed, the through-holes 31 are configured to allow the solution 6 to pass through while trapping (capturing) the cells 5.

[0049] The maximum width of each through-hole 31 may be, for example, 30 μm or less, 25 μm or less, 20 μm or less, 15 μm or less, 10 μm or less, 7 μm or less, 4 μm or less, or 2 μm or less. When the maximum width of the through-hole 31 is equal to or less than the upper limit, the through-hole 31 does not allow the cell 5 to pass through, but allows the solution 6 to pass through. In the present disclosure, the "maximum width" means the maximum length of a line segment passing through any two points on the edge of the through-hole 31. For example, when the through-hole 31 is a perfect circle, the "maximum width" refers to the diameter of the perfect circle. Also, for example, when the through-hole 31 is an ellipse, the "maximum width" refers to the major axis of the ellipse. Also, when the through-hole 31 is a polygon, the "maximum width" means the length of the longest diagonal line of the polygon.

[0050] The trapping member 30 may be configured to trap cells having a maximum width of, for example, 0.5 μm or more, 1 μm or more, 5 μm or more, 10 μm or more, 15 μm or more, 20 μm or more, 25 μm or more, or 30 μm or more, and in a preferred embodiment is configured to trap cells having a maximum width of 15 μm or more.

[0051] The cells 5 (FIG. 4) trapped in the through-holes 31 of the detection unit 1 described above are not particularly limited as long as they are cells that can detect a substance in the gas phase by evaluating them after exposure to the substance in the gas phase, and may be either adherent cells or suspension cells. The cells 5 may be eukaryotic cells or prokaryotic cells (also called prokaryotes), and the prokaryotic cells may be microorganisms that belong to either bacteria or archaea.

[0052] In a preferred embodiment, the cell 5 is a cell expressing a cell membrane receptor that binds to a substance to be detected. Examples of such cells include a cell membrane receptor that has a function of increasing the calcium ion concentration in the cell when bound to a target substance, and CaTM-3, a calcium ion detection fluorescent probe (Kazuhisa Hirabayashi et al., "Development of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell-membranepermeability", Cell Calcium 60(4), 256-265 (2016)). When the cell 5 is exposed to a substance in the gas 4 (FIG. 4), the fluorescence intensity of CaTM-3 increases via an increase in the calcium ion concentration in the cell 5, thereby enabling the substance in the gas 4 to be detected. Cells expressing a cell membrane receptor that binds to a substance to be detected can be prepared by a method commonly performed by a person skilled in the art (for example, a lipofection method or an electroporation method).

[0053] In addition, in a preferred embodiment, the substance in the gas phase to be detected by the detection unit 1 using cells 5 may be, for example, a substance consisting of molecules with a molecular weight of 20 or more and 400 or less, or may be a substance consisting of molecules with a molecular weight of 30 or more and 300 or less, 50 or more and 200 or less, or 80 or more and 150 or less.

[0054] <Method for detecting substances in the gas phase> 4(a) to (d), an example of a method will be described in which a detection unit 1 is used to separate cells 5 from a cell suspension 3, which is a solution 6 in which cells 5 are suspended (separation process), expose the cells 5 to a gas 4 containing a substance to be detected (exposure process), and evaluate the cells 5 by detecting the fluorescence emitted by the cells 5 exposed to the substance (evaluation process).

[0055] In the present disclosure, "separating cells from a cell suspension" means separating the cells from the solution contained in the cell suspension, and means separating the cells from the solution to an extent that it is difficult to further remove the solution by conventional methods, even if some solution remains on the surface of the cells to be measured, for example.

[0056] The term "cell suspension" in the present disclosure means a solution in which cells are suspended (a liquid in which cells are dispersed). The cell suspension 3 used in the following examples can be prepared by a method commonly used by those skilled in the art. For example, when the cells 5 are adherent cells, the cell suspension 3 can be prepared by diluting the cells 5 detached from a dish in which the cells 5 are cultured using trypsin-EDTA or a cell scraper, etc., with a cell culture medium, etc.

[0057] In the following example, the cell suspension 3 is supplied to the flow path 40 from the first opening 21, and the gas 4 is supplied from the second opening 22. The cell suspension 3 and the gas 4 are discharged to the outside of the substrate from the third opening 23. As shown in FIG. 4, the supply of the cell suspension 3 from the first opening 21 and the supply of the gas 4 from the second opening 22 are performed using, for example, a syringe, but may also be performed using a tube or the like. The first opening 21 and the second opening 22 may be openable and closable using a valve. In the following description, closing the first opening 21 means that the liquid or gas is not supplied or discharged from the first opening 21, and opening the first opening 21 means that the liquid or gas is supplied or discharged from the first opening 21. This also applies to the second opening 22 and the third opening 23. The flow of the liquid or gas in the flow path 40 may be promoted by purging the first opening 21 or the second opening 22 using a pump, or by suction using a pump from the third opening 23, as necessary.

[0058] In the following example, cell 5 is an adherent cell that has a cell membrane receptor that has the function of increasing the calcium ion concentration within the cell when it binds to a substance in gas 4, and that has been stained with CaAM-3, and when cell 5 is exposed to the substance in gas 4, the intensity of the fluorescence emitted by cell 5 increases.

[0059] First, with the first opening 21 open and the second opening 22 closed, as shown in Fig. 4(b), the cell suspension 3 is introduced from the first opening 21. The cell suspension 3 introduced from the first opening 21 is introduced into the first portion 41 of the flow channel 40.

[0060] The cell suspension 3 introduced into the first portion 41 flows through the flow path 40 and reaches the trapping member 30. When the cell suspension 3 comes into contact with the trapping member 30, the cells 5 contained in the cell suspension 3 are trapped and remain on the trapping member 30, while the solution 6 passes through the through-holes 31 provided in the trapping member 30, reaches the second portion 42, and is finally discharged from the third opening 23. This separates the cells 5 from the cell suspension 3 (separation step). Thereafter, as necessary, the solution 6 may be introduced from the first opening 21 as shown in FIG. 4(c), and the cells 5 in the cell suspension 3 remaining in the first portion 41 are also swept away and trapped by the trapping member 30.

[0061] Next, the first opening 21 is closed, the second opening 22 is opened, and then, as shown in Fig. 4(d), the gas 4 containing the substance to be detected is introduced from the second opening 22. The gas 4 introduced from the second opening 22 is introduced into the first portion 41 of the flow path 40.

[0062] The gas 4 introduced into the first portion 41 flows through the flow path 40 and reaches the trapping member 30, and the cells 5 trapped on the trapping member 30 are exposed to the substance contained in the gas 4 (exposure step). The gas 4 that has reached the trapping member 30 then passes through the second portion 42 of the flow path 40 and is finally discharged from the third opening 23.

[0063] Finally, excitation light is irradiated onto the cells 5 on the trapping member 30 that have been exposed to the substance in the gas 4, and the presence or absence and / or concentration of the substance in the gas 4 is evaluated using the intensity of the fluorescence emitted by the cells 5 as an index (evaluation step). Since the first substrate 10 and the second substrate 20 are configured to be permeable to the excitation light and the fluorescence emitted by the cells 5, irradiation of the cells 5 with the excitation light and detection of the fluorescence can be performed from outside the first substrate 10 and the second substrate 20. Furthermore, the measurement of the fluorescence may be performed simultaneously with the exposure of the cells 5 to the substance contained in the gas 4, or may be performed after the exposure is completed.

[0064] The time (exposure time) for exposing the cell 5 to the gas 4 containing a substance to be detected is not particularly limited, and may be, for example, 0.1 seconds or more, 0.3 seconds or more, 1 second or more, 3 seconds or more, 10 seconds or more, 30 seconds or more, 1 minute or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, or 4 hours or more, or 24 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 1 hour or less, 30 minutes or less, 15 minutes or less, 10 minutes or less, 6 minutes or less, 4 minutes or less, 2 minutes or less, 1 minute or less, 45 seconds or less, 35 seconds or less, 30 seconds or less, 15 seconds or less, 10 seconds or less, 5 seconds or less, 3 seconds or less, 1 second or less, or 0.5 seconds or less. In a preferred embodiment, the exposure time is 1 minute or less. In another preferred embodiment, the exposure time is 1 minute or more. In another preferred embodiment, the exposure time is 1 hour or more and 8 hours or less. The exposure step may also be a step of repeatedly exposing the cells for the exposure time as described above, in which case the exposure step may include contacting the cells 5 with a liquid such as the solution 6 between each exposure.

[0065] Also, instead of being made openable and closable by using a valve, the first opening 21, the second opening 22, and the third opening 23 may be provided with a supply mechanism capable of supplying the cell suspension 3 and / or the gas 4 while keeping the inside of the flow path 40 sealed, and / or a discharge mechanism capable of discharging the cell suspension 3 and / or the gas 4 while keeping the inside of the flow path 40 sealed. In this case as well, the inside of the flow path 40 is sealed. Such a supply mechanism while sealing can be realized, for example, by piercing the injection needle of a syringe loaded with the cell suspension 3 or the gas 4 into the first opening 21, the second opening 22, or the third opening 23 sealed by a rubber plug, or by piercing the injection needle of a syringe loaded with the cell suspension 3 or the gas 4 into the first opening 21, the second opening 22, or the third opening 23 covered with an adhesive before solidification, and then solidifying the adhesive. In addition, the discharge mechanism while sealing can be realized, for example, by inserting an injection needle attached to one end of a hose into the first opening 21, the second opening 22, or the third opening 23, which is sealed by a rubber stopper, and connecting the other end of the hose to a pump or the like for suction.

[0066] In the above-described example, the evaluation step includes detecting the fluorescence emitted by the cells, but the cells can be evaluated by other methods. Examples of evaluation methods other than fluorescence detection include luminescence detection and a method of evaluating the cell fragments 8 (FIG. 6) or cells 5 recovered in the washing step described below.

[0067] In addition to the separating step, exposing step, and evaluating step described above, the method for detecting a substance in a gas phase of the present invention may include, after the evaluating step, a step of removing at least one of the substance derived from the gas 4 and the cells 5 from the trapping member 30 by flowing a washing liquid so as to contact the trapping member 30. The washing liquid is a solution capable of removing at least the substance derived from the gas 4 from the trapping member 30, and may be, for example, the solution 6, the cell disruption liquid 7 (FIG. 6), or the cell detachment liquid 9 (FIG. 7).

[0068] In one embodiment, the washing step may be a step of removing the substance derived from the gas 4 without removing the cells 5 from the trapping member 30. In this case, since the substance derived from the gas 4 can be removed from the cells 5 on the trapping member 30 (resetting the cell state), the cells 5 on the trapping member 30 can be reused for the exposure step and the evaluation step. Below, with reference to Figures 5(a) and (b), the washing step performed after the evaluation step including detecting the fluorescence emitted by the cells 5 will be described using an example in which the substance derived from the gas 4 is a substance insoluble in the solution 6 and the washing liquid is the solution 6.

[0069] First, as shown in FIG. 5(a), after the evaluation step, the first opening 21 is opened, the second opening 22 is closed, and then the solution 6 is introduced from the first opening 21. The introduced solution 6 is introduced into the first portion 41 of the flow path 40 and reaches the trapping member 30. Substances originating from the gas 4 present on the surface of the trapping member 30 and on the surface of the cell 5 trapped in the trapping member 30 are swept away by the solution 6 and removed from the trapping member 30. Thereafter, as shown in FIG. 5(b), the solution 6 that has swept away the gas 4 passes through the through-hole 31 of the trapping member 30 and reaches the second portion 42, and is finally discharged from the third opening 23.

[0070] In the above example, the case where the substance derived from gas 4 is a substance insoluble in solution 6 and the washing liquid is solution 6 has been described as an example, but the combination of the substance derived from gas 4 and the washing liquid is not limited to this. For example, as in the above example, when the washing step is a step in which cells 5 are not removed from trapping member 30 and the substance derived from gas 4 is removed from trapping member 30, the washing liquid is not particularly limited as long as it is a solution that does not crush, solubilize, or peel cells 5 and dissolves the substance derived from gas 4.

[0071] The washing step may also be a step of removing both the substances derived from the gas 4 and the cells 5 from the trapping member 30. In this case, since the cells 5 and the substances derived from the gas 4 can be removed from the trapping member 30, the detection unit 1 can be repeatedly used in the detection method of the present invention. In this case, the washing liquid may be, for example, the cell disruption liquid 7 (FIG. 6) or the cell detachment liquid 9 (FIG. 7).

[0072] 6(a) and (b), an example of removing substances derived from cells 5 and gas 4 from the trapping member 30 will be described. In this specification, the cell disruption solution means a solution that can break the lipid bilayer of cells and liberate intracellular components by contacting the cells with the cell disruption solution 7. As the cell disruption solution 7 used for disrupting the cells 5, a solution containing a surfactant such as CHAPS or Triton-X100 can be used.

[0073] First, after the evaluation step, as shown in FIG. 6(a), the first opening 21 is opened and the second opening 22 is closed, and then the cell disruption solution 7 (washing solution) is introduced from the first opening 21. The introduced cell disruption solution 7 is introduced into the first part 41 of the flow path 40 and reaches the trapping member 30. The cells 5 trapped in the trapping member 30 are disrupted by the action of the cell disruption solution 7, and become cell fragments 8. Since the cell fragments 8 are smaller in size than the cells 5, the cell fragments 8 (cell fragments 8 that are small enough to pass through the through holes 31) pass through the through holes 31 provided in the trapping member 30, reach the second part 42, and are finally discharged from the third opening 23, as shown in FIG. 6(b).

[0074] In the above example, the cell disruption liquid 7 is introduced through the first opening 21 and the cell fragments 8 are discharged through the third opening 23, but the cell disruption liquid 7 may be introduced through the third opening 23 and the cell fragments 8 may be discharged through the first opening 21. In this case, since the cell fragments 8 do not need to be able to pass through the through-holes 31 provided in the trapping member 30, a trapping member 30 provided with smaller through-holes 31 may also be used.

[0075] The cell lysate 8 discharged in the washing step may be collected and evaluated to detect the substance in the gas 4. In other words, when the washing liquid is the cell lysate 7, the washing step may be performed between the exposure step and the evaluation step, and the evaluation step may be a step of evaluating the cell lysate 8 of the cells 5 removed in the washing step. The cell lysate 8 may be evaluated by a method that is commonly used by those skilled in the art when analyzing cell lysates (e.g., ELISA, immunoprecipitation, Western blotting, quantitative PCR, or high performance liquid chromatography), and the substance in the gas phase may be detected using as an indicator the presence or absence of a change in the composition of the cell lysate 8 caused by the exposure of the cells 5 to the substance in the gas 4, or the degree of such a change.

[0076] An example of removing substances derived from cells 5 and gas 4 from the trapping member 30 when the washing liquid used in the washing step is a cell detachment liquid 9 will be described with reference to Figs. 7(a) and (b). In this specification, the cell detachment liquid means a solution that can detach cells from a scaffold by contacting the cell detachment liquid with the cell detachment liquid in a state of adhering to the scaffold. As the cell detachment liquid 9 used to recover the trapped cells 5, for example, a trypsin-EDTA solution or the like can be used. The evaluation step in this case will be described below.

[0077] First, after the evaluation step, as shown in FIG. 7(a), the first opening 21 is opened and the second opening 22 is closed, and then the cell detachment solution 9 (cleaning solution) is introduced from the third opening 23. The introduced cell detachment solution 9 is introduced into the second portion 42 of the flow path 40 and reaches the trapping member 30. The cells 5 trapped in the trapping member 30 are detached from the trapping member 30 by the action of the cell detachment solution 9. The detached cells 5 flow through the first portion 41 along with the flow of the cell detachment solution 9, and are finally discharged from the first opening 21.

[0078] The cells 5 discharged in the washing step may be collected and evaluated to detect the substance in the gas 4. In other words, when the washing liquid is a cell detachment liquid 9, the washing step may be performed between the exposure step and the evaluation step, and the evaluation step may be a step of evaluating the cells 5 removed in the washing step. The collected cells 5 may be evaluated by a method that is commonly used by those skilled in the art when analyzing cells 5 (e.g., measuring cell viability by MTT assay or CCK-8 assay), and the substance in the gas phase may be detected using the degree of decrease in cell viability caused by exposure of the cells 5 to the substance in the gas 4 as an index.

[0079] Furthermore, although this example has been described assuming that cell 5 is an adherent cell, if cell 5 is a floating cell, cell 5 will not adhere to trapping member 30, and therefore cell 5 can be removed from trapping member 30 even if the solution introduced through third opening 23 is a solution other than cell detachment solution 9 (for example, a general cell culture medium such as solution 6).

[0080] Although an example in which the gas 4 is introduced from one opening (the second opening 22) has been described above, the gas may be introduced from two or more openings. In the following, with reference to Figs. 8(a) to (d), an example in which the first gas 4A is introduced from the second opening 22 and the second gas 4B is introduced from the fourth opening 24 will be described. In the example of Figs. 8(a) to (d), the detection unit 1 further has a fourth opening 24 for supplying the second gas 4B, which is connected to the second part 42 of the flow path 40, in addition to the first opening 21, the second opening 22, and the third opening 23. In this case, not only the first opening 21 and the second opening 22, but also the third opening 23 and the fourth opening 24 may be opened and closed using valves, and in the following example, it is assumed that valves are provided. The first gas 4A and the second gas 4B may be gases having the same composition, or may be gases having different compositions.

[0081] First, as shown in FIG. 8(a), the first opening 21 is opened, the second opening 22 and the fourth opening 24 are closed, and then the cell suspension 3 is introduced from the first opening 21 to trap the cells 5 in the trapping member 30 (separation step). Next, as shown in FIG. 8(b), the first opening 21 is closed and the second opening 22 is opened, and then the first gas 4A is introduced from the second opening 22 to expose the cells 5 on the trapping member 30 to the substances in the first gas 4A (first exposure step), while detecting the fluorescence emitted by the cells (first evaluation step). Next, as shown in FIG. 8(c), the first opening 21 is opened and the second opening 22 is closed, and then the solution 6 is introduced from the third opening 23 to remove the substances derived from the first gas 4A from the trapping member 30 without removing the cells 5 (washing step, resetting the cell state). Finally, as shown in FIG. 8(d), the third opening 23 is closed and the fourth opening 24 is opened, and then the second gas 4B is introduced through the fourth opening 24 to expose the cells 5 on the trapping member 30 to the substances in the second gas 4B (second exposure step), while detecting the fluorescence emitted by the cells (second evaluation step).

[0082] In the above example, the solution 6 is introduced from the third opening 23 in the cleaning step, but the solution 6 may be introduced from the first opening 21 in the cleaning step.

[0083] Furthermore, a cleaning process in which a cleaning solution is flowed from the third opening 23 toward the first opening 21 without removing the cells 5, as shown in FIG. 8(c), can be suitably applied in cases where the cells 5 are adherent cells, for example, and the cells 5 are not washed away from the trap member 30 to the first portion 41 by the cleaning solution.

[0084] In the above example, a cleaning step was included after the first exposure step and evaluation step and before the second exposure step and evaluation step. However, as shown in Figures 9(a) to (d), after the first exposure step and evaluation step (Figure 9(b)), instead of the cleaning step, a step of introducing a small amount of second gas 4B to wash away the solution 6 remaining in flow path 40 and removing first gas 4A from flow path 40 (Figure 9(c)) may be performed, and then a second exposure step and evaluation step (Figure 9(d)) may be performed.

[0085] <Action and Effects> As described above, in the detection unit 1 and the method for detecting a substance in a gas phase using the same, the cells 5 separated from the cell suspension 3 are exposed to a substance in the gas 4. Therefore, compared to the case where the cells are exposed to a substance in the liquid phase, the substance in the gas phase can be detected without depending on the solubility of the substance in the gas 4 in the solution 6. Furthermore, the present inventors' study revealed that when the cells are exposed to a substance in the gas phase, the cells respond to the substance with high sensitivity compared to the case where the cells are exposed to the substance in the liquid phase. Therefore, according to the detection unit 1 and the method for detecting a substance in a gas phase using the same, the substance in the gas 4 can be detected well. Note that, in order to maintain the survival state of the cells, it is essential to pay attention to drying, and it was previously thought that the exposure of the cells to the gas phase is preferably several seconds to several tens of seconds. However, the present invention is based on the finding by the present inventors that the cells can survive to some extent even when they are separated from the cell suspension and placed in the gas phase.

[0086] In the detection unit 1 and the method for detecting a substance in a gas phase using the same, the detection unit 1 includes the trapping member 30, so that the cells 5 can be separated from the cell suspension 3 by contacting the cell suspension 3 with the trapping member 30. This makes it possible to easily separate the cells 5 from the cell suspension 3. Such trapping of the cells 5 by the trapping member 30 can be more suitably performed when the trapping member 30 is made of a porous body, the through-holes 31 are holes in the porous body, the maximum width of each of the through-holes 31 is equal to or less than a predetermined upper limit, and / or the trapping member 30 is configured to trap cells whose maximum width is equal to or greater than a predetermined lower limit.

[0087] In the detection unit 1 and the method for detecting a substance in a gas phase using the same, the volume of the flow path 40 is set to a predetermined upper limit or less (for example, 7000 mm 3 The inventors' studies have revealed that if the volume of the cell culture space is small, the cells can survive for a long time in a gas-phase culture environment. Therefore, if the volume of the flow channel 40 is small, the cells 5 can be exposed to the substance in the gas 4 for a long time, and therefore the device can be suitably used for continuous measurement and detection of low-concentration substances in the gas phase.

[0088] Furthermore, the inventors' investigations have revealed that when cells are exposed to a substance in the gas phase, the cells respond to the substance with higher sensitivity than when the cells are exposed to the substance in the liquid phase. Therefore, in the method for detecting a substance in the gas phase using the detection unit 1, the substance in the gas phase can be detected even if the exposure time in the exposure step is short (for example, 1 minute or less).

[0089] Furthermore, the inventors' investigations have revealed that cells can survive for a long period of time in a gas-phase culture environment. Therefore, in the method for detecting a substance in a gas phase using the detection unit 1, cells can be exposed to a substance in a gas phase without a significant decrease in cell viability, even if the exposure time in the exposure step is long (for example, 1 minute or more, or 1 hour or more and 8 hours or less).

[0090] Furthermore, the inventors' study revealed that cells can survive for a longer period of time under gas-phase culture conditions in a sealed environment compared to an open environment. Therefore, in the method for detecting a substance in a gas phase using the detection unit 1, when the exposure step is performed in a sealed environment, cells can be exposed to a substance in the gas phase without a significant decrease in cell viability, and therefore the method can be suitably used for continuous measurement and detection of a substance in a gas phase at a low concentration.

[0091] In the detection unit 1 and the method for detecting a substance in the gas phase using the same, the first substrate 10 and the second substrate 20 of the detection unit 1 are optically transparent, and therefore it is possible to detect the fluorescence emitted by the cells 5. Therefore, it is possible to detect the gas 4 based on the intensity of the fluorescence emitted by the cells 5.

[0092] The trapping member 30 separates the space in the flow path 40 into a first portion 41 and a second portion 42. The first substrate 10 and the second substrate 20 (flow path member) have a first opening 21 connected to the first portion 41 for supplying the cell suspension 3, a second opening 22 connected to the first portion 41 for supplying the gas 4 containing the substance, and a third opening 23 connected to the second portion 42 for discharging the solution 6 and the gas 4. This allows the cell suspension 3 to be supplied from the first opening 21 to trap the cells in the trapping member 30, and then the gas 4 containing the substance to be supplied from the second opening 22 to expose the cells 5 to the substance. In addition, the solution 6 (cell suspension 3) and the gas 4 supplied from the first opening 21 and the second opening 22 can be discharged from the third opening 23.

[0093] <Modification> In the above-described example, both the first substrate 10 and the second substrate 20 are described as being made of light-transmitting glass, but the material of the first substrate 10 and the second substrate 20 is not limited thereto. For example, when the evaluation step includes detecting the fluorescence emitted by the cells, if at least one of the first substrate 10 and the second substrate 20 is light-transmitting, irradiation of the cells with excitation light and detection of the fluorescence are possible. Therefore, one of the first substrate 10 and the second substrate 20 may be formed to be transparent to the light emitted by the cells, and the other may be formed to be non-transmitting (or may be formed of a material with low light transmittance such as a silicon wafer). In addition, when the evaluation step includes evaluating the cell fragments 8 or the cells 5 recovered in the washing step, both the first substrate 10 and the second substrate 20 may be formed to be non-transmitting to the light emitted by the cells (or may be formed of a material with low light transmittance such as a silicon wafer).

[0094] The trapping member 30 may have a shape as shown in Figs. 10(a) to 10(e). In the example shown in Fig. 10(a), one circular through-hole 31 (opening) is provided in the trapping member 30. In the example shown in Fig. 10(b), a large number of through-holes 31 (openings) are provided in the trapping member 30 in a striped pattern. In the example shown in Fig. 10(a), an outlet port other than the through-hole 31 may be further formed in the trapping member 30. When one through-hole 31 is provided as in the example shown in Fig. 10(a), if a cell 5 is trapped in the through-hole 31, the cell suspension 3, the gas 4, and the solution 6 may not be able to pass through the trapping member 30. However, by providing an outlet port, the cell suspension 3, the gas 4, and the solution 6 can pass through the trapping member 30 even after the cell 5 is trapped in the through-hole 31.

[0095] In the examples shown in Figs. 10(c) to (e), the trap member 30 is disposed so as to block a part of the flow in the flow path 40, and a gap 32 (opening) is formed between the surface of the flow path 40 and the trap member 30. More specifically, in the example of Fig. 10(c), the trap member 30 is in contact with the surface of the flow path 40 at one side, and one gap 32 (opening) is formed outside the contact portion. In the example of Fig. 10(d), the trap member 30 is in contact with the surface of the flow path 40 at two opposing sides, and two gaps 32 (opening) are formed outside the contact portion. In the example of Fig. 10(e), the trap member 30 is in contact with the surface of the flow path 40 at three sides, and one gap 32 (opening) is formed outside the contact portion (the remaining side). As another example, the trap member 30 may be in contact with the surface of the flow path 40 at four sides, and a triangular gap 32 (opening) may be formed at a corner of the trap member 30.

[0096] 1(b), the multiple (large number) through holes 31 are regularly arranged, but the multiple through holes 31 may be irregularly (randomly) arranged. The sizes of the multiple through holes 31 do not have to be uniform.

[0097] In the trapping member 30 shown in FIG. 1(b), the shape of the through-hole 31 is uniform in the thickness direction, and the cells 5 are trapped on the surface of the trapping member 30, but the shape of the through-hole 31 does not have to be uniform in the thickness direction, and the cells 5 may be trapped inside the trapping member 30. The trapping member 30 may be, for example, a depth filter or a membrane filter, as a trapping member in which the shape of the through-hole 31 is not uniform in the thickness direction. The pores provided in the depth filter have a larger diameter closer to the filter surface and a smaller diameter the further inside the filter. Therefore, when the trapping member 30 is a depth filter, the trapping member 30 can trap the cells 5 not only on its surface but also inside. The trapping member 30 may be fibrous or composed of multiple layers. In the example of FIG. 1(b), the porous body constituting the trap member 30 has a structure in which the through holes 31 (pores) extend in a regular direction (thickness direction), but it may have a structure in which the through holes 31 extend in irregular directions and are irregularly distributed in three dimensions (irregular porous structure).

[0098] The detection unit 1A of the first modified example shown in FIG. 11 does not have the trap member 30. In the detection unit 1A, the surface of the flow channel part 12 and / or the contact surface of the second substrate 20 with the flow channel 40 are configured so that when the cell suspension 3 comes into contact with the surface, the solution 6 is not trapped, but the cells 5 are trapped (i.e., the surface 40a of the flow channel 40 corresponds to the trap region 33). Such a trap region 33 can be achieved by selecting an appropriate material (e.g., glass), or by treating (e.g., hydrophilizing) or modifying (e.g., collagen coating, polylysine coating, or fibronectin coating) the surface 40a of the flow channel 40, and the treatment or modification in this case may be for increasing the adhesiveness of the adherent cells, or for adhering the floating cells to the substrate. The trap region 33 constitutes a cell trap part for trapping the cells 5.

[0099] Furthermore, in the case where the contact surface of the second substrate 20 with the flow channel 40 is configured such that when the cell suspension 3 comes into contact with the contact surface, the solution 6 is not trapped but the cells 5 are trapped, the detection unit 1A may be used such that the second substrate 20 is vertically below the first substrate 10. In this case, the cells 5 are likely to adhere to the contact surface of the second substrate 20 with the flow channel 40 due to gravity, and therefore, if a cover glass is used as the second substrate 20, for example, the cells 5 exposed to the substance in the gas phase in the exposure step can be easily observed for fluorescence (evaluation step) by an inverted microscope or the like.

[0100] In the separation step when the detection unit 1A is used, as shown in FIG. 12(a), the first opening 21 is opened and the second opening 22 is closed, and the cell suspension 3 is introduced from the first opening 21, and the cell suspension 3 is left stationary or slowly flowed in the flow path 40 for a certain period of time (for example, 5 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 4 hours or more, 8 hours or more, or 16 hours or more). As a result, the cells 5 adhere to the surface 40a of the flow path 40. Next, as shown in FIG. 12(b), the second opening 22 is opened and the first opening 21 is closed, and then the gas 4 is introduced from the second opening 22, whereby the solution 6 is removed from the flow path 40, while the cells 5 adhering to the surface 40a remain in the flow path 40. As a result, the cells 5 are separated from the solution 6 (separation step), and instead the cells 5 are exposed to the substances in the gas 4 (exposure step), so that the substances in the gas 4 can be detected. In FIG. 12, detection (separation, exposure, and evaluation) of the cells 5 is performed at only one detection position 50, but as shown in FIG. 13, detection of the cells 5 may be performed at a plurality of detection positions 50.

[0101] A method for detecting a substance in the gas phase using the detection unit 1A may include, as shown in Figures 14(a) to (d), a step of introducing a first gas 4A from the second opening 22 and measuring the fluorescence intensity at multiple detection positions 50 (Figure 14(b)), a cleaning step (Figure 14(c)), and a step of introducing a second gas 4B from the second opening 22 and measuring the fluorescence intensity at multiple detection positions 50 (Figure 14(d)).

[0102] 14, the second opening 22 is an opening for introducing the first gas 4A and the second gas 4B, but the second opening 22 may be an opening for introducing the first gas 4A, the second gas 4B, and the solution 6. In this case, in the cleaning step, the solution 6 is introduced from the second opening 22 instead of the first opening 21, thereby removing the first gas 4A remaining in the second opening 22. Therefore, even if a valve is not provided, it is possible to prevent the first gas 4A and the second gas 4B from being mixed (contamination) when the second gas 4B is introduced from the second opening 22 after the cleaning step.

[0103] Furthermore, the detection unit 1A may further have a fourth opening 24 for supplying a gas, which is connected to the second portion 42. In this case, a method for detecting a substance in a gas phase using the detection unit 1A may include, as shown in Figures 15(a) to 15(d), a step of introducing a first gas 4A from the second opening 22 and measuring the fluorescence intensity at a plurality of detection positions 50 (Figure 15(b)), a cleaning step (Figure 15(c)), and a step of introducing a second gas 4B from the fourth opening 24 and measuring the fluorescence intensity at a plurality of detection positions 50 (Figure 15(d)).

[0104] The detection unit 1A may also have a fourth opening 24 for supplying gas, connected to the second portion 42, a first detection position 51 located between a portion connected to the second opening 22 on the flow path 40 and a portion connected to the fourth opening 24, and a second detection position 52 located between a portion connected to the fourth opening 24 on the flow path 40 and the third opening 23. In this case, as shown in Figs. 16(a) to (c), in the method for detecting a substance in a gas phase using the detection unit 1A, after the separation step (Fig. 16(a)), in the exposure step and evaluation step, the cell 5 located at the first detection position 51 is exposed to a substance in the first gas 4A introduced from the second opening 22 (Fig. 16(b)). Then, the cell 5 located at the second detection position 52 is exposed to a substance in the second gas 4B introduced from the fourth opening 24 (Fig. 16(c)). This allows the cells 5 to be exposed to different substances in a plurality of gases.

[0105] In the detection unit 1A described above, the flow path 40 may be configured so that the entire surface 40a is configured so that when the cell suspension 3 comes into contact with it, the solution 6 is trapped while the cells 5 are trapped (i.e., the entire surface 40a is the trapping region 33), or only a portion of the surface 40a (for example, only the portion corresponding to the detection position 50) may be configured in a similar manner (i.e., only a portion of the surface 40a is the trapping region 33).

[0106] In the detection unit 1A, the surface 40a of the flow path 40 is configured so that when the cell suspension 3 comes into contact with it, the solution 6 is trapped while the cells 5 are trapped, so that the cells 5 can be separated from the cell suspension 3 by flowing the cell suspension 3 through the flow path 40, and the cells 5 can be easily separated from the cell suspension 3.

[0107] The base 2 of the detection unit 1B of the second modified example shown in FIG. 17 has a first member 10A, a second member 10B, and a second substrate 20. As shown in FIG. 18(a), the second substrate 20 is configured similarly to that described for the detection unit 1A shown in FIG. 1. The first member 10A and the second member 10B are formed, for example, from glass in a rectangular shape and are optically transparent. The first member 10A has a flat surface 10a.

[0108] As shown in FIG. 18(b), the first member 10A has a flow path opening 11A and a third connection opening 15A. The flow path opening 11A and the third connection opening 15A penetrate in the thickness direction. The flow path opening 11A has a first flow path main body 12A, a flow path branching portion 12B, a first connection opening 13A, a second connection opening 14A, and a first trap holding portion 16A. The first flow path main body 12A is linear, and the flow path branching portion 12B branches from the middle portion of the first flow path main body 12A and extends linearly. The first flow path main body 12A has a uniform cross-sectional shape along the extension direction. The cross-sectional shape is, for example, rectangular. In this example, in a cross section perpendicular to the extension direction, the width of the first flow path main body 12A is 0.5 mm, and the height of the flow path portion 12 is 0.2 mm. The shape of the third connection opening 15A in a plan view is circular in this example, but is not limited to this and may be any shape.

[0109] The first connection opening 13A is connected to one end of the first flow path main body 12A. The first trap holding portion 16A is connected to the other end of the first flow path main body 12A. The second connection opening 14A is connected to one end of the flow path branch portion 12B (the end opposite to the connection end with the first flow path main body 12A). The shapes of the first connection opening 13A, the second connection opening 14A, and the first trap holding portion 16A in a plan view are circular in this example, but are not limited to this and may be any shape.

[0110] As shown in FIG. 18(c), a groove 11B is formed on the surface 10a of the second member 10B. The groove 11B has a second flow path main body 12C, a second trap holder 16B, and a third connection portion 15. The second flow path main body 12C is linear and exists parallel to the first flow path main body 12A. The second flow path main body 12C has a uniform cross-sectional shape along the extension direction. The cross-sectional shape is, for example, rectangular. In this example, in a cross section perpendicular to the extension direction, the second flow path main body 12C is 0.5 mm, and the depth of the second flow path main body 12C is 0.2 mm.

[0111] The second trap holding portion 16B is connected to one end of the second flow path main body 12C. The third connection portion 15 is connected to the other end of the second flow path main body 12C. In this example, the shape of the second trap holding portion 16B and the third connection portion 15 in a plan view is circular, but is not limited to this and may be any shape.

[0112] The first member 10A is disposed on the surface 10a of the second member 10B and is in close contact with the surface 10a. The second substrate 20 is in close contact with the surface of the first member 10A opposite to the second member 10B. This is achieved by, for example, using an adhesive. As a result, the opening of the first flow path main body 12A is blocked by the second member 10B and the second substrate 20, and the opening of the second flow path main body 12C is blocked by the first member 10A, and a flow path 40 is formed between the first flow path main body 12A and the second member 10B and the second substrate 20, and between the second flow path main body 12C and the first member 10A. That is, the first member 10A, the second member 10B, and the second substrate 20 are flow path members that define the flow path 40.

[0113] The positional relationship between the openings and grooves provided in the first member 10A, the second member 10B, and the second substrate 20 will be described. The first connection opening 13A of the first member 10A is connected to the first opening 21 of the second substrate 20. The second connection opening 14A of the first member 10A is connected to the second opening 22 of the second substrate 20. The first trap holding portion 16A of the first member 10A is connected to the second trap holding portion 16B of the second member 10B. The third connection opening 15A of the first member 10A is connected to the third opening 23 of the second substrate 20 and the third connection portion 15 of the second member 10B.

[0114] A trapping member 30 (cell trapping portion) is disposed between the first trap retaining portion 16A and the second trap retaining portion 16B so as to separate them. In this example, the trapping member 30 is circular. The shape of the trapping member 30 may be the same as that of the trapping member 30 in the detection unit 1 already described, as shown in Figs. 19(a) to (f). That is, as shown in Figs. 19(a) to (c), the trapping member 30 may have a through-hole 31 (opening) penetrating the trapping member 30 along the thickness direction (flow direction in the flow channel 40), or as shown in Figs. 19(d) to (f), a gap 32 (opening) may be formed between the trapping member 30 and the surface of the flow channel 40.

[0115] In the detection unit 1B, the trap member 30 also divides the space in the flow path 40 into a first portion 41 and a second portion 42. The first portion 41 is a portion located between the first flow path main body 12A and the second member 10B and the second substrate 20. The second portion 42 is a portion located between the second flow path main body 12C and the first member 10A.

[0116] As with the detection unit 1 already described, such a detection unit 1B can be used in a method for detecting a substance in the gas 4, for example, as shown in Figures 20(a) and (b). Note that a step of introducing a solution 6 from the first opening 21 may be included between the step shown in Figure 20(a) and the step shown in Figure 20(b). In this case, the cells 5 in the cell suspension 3 remaining in the first portion 41 are also swept away and trapped by the trapping member 30, so that in the exposure step, it is possible to prevent cells exposed to the substance in the gas 4 and cells not exposed to the substance from being mixed in the flow channel 40 due to the cells 5 remaining in the first portion 41.

[0117] As a modified example, as shown in FIGS. 21 to 23, the detection unit 1B may be configured so as to perform measurements at cell trapping portions provided at a plurality of locations. In the example of Figures 21 to 23, the detection unit 1B is provided with four cell trapping portions (first trapping member 30A, second trapping member 30B, third trapping member 30C, and fourth trapping member 30D), four openings (first opening 21A, fourth opening 21B, sixth opening 21C, and eighth opening 21D) for introducing the cell suspension 3 so as to contact any of the cell trapping portions, four openings (second opening 22A, fifth opening 22B, seventh opening 22C, and ninth opening 22D) for introducing the first gas 4A so as to contact any of the cell trapping portions, four openings (tenth opening 24A, eleventh opening 24B, twelfth opening 24C, and thirteenth opening 24D) for introducing the second gas 4B so as to contact any of the cell trapping portions, and a third opening 23 for discharging the solution 6 derived from the cell suspension 3, the first gas 4A, and the second gas 4B. The flow path 40 is divided into five parts (first part 41, second part 42, third part 43, fourth part 44 and fifth part 45) by four trapping members (first trapping member 30A, second trapping member 30B, third trapping member 30C and fourth trapping member 30D). Of these, the second part 42 faces all of the four trapping members (first trapping member 30A, second trapping member 30B, third trapping member 30C and fourth trapping member 30D) and is connected to the third opening 23 for discharging the solution 6 derived from the cell suspension 3, the first gas 4A and the second gas 4B. The first part 41 faces only the first trapping member 30A out of the four trapping members and is connected to the first opening 21A for introducing the cell suspension 3, the second opening 22A for introducing the first gas 4A and the tenth opening 24A for introducing the second gas 4B. The third portion 43 faces only the second trapping member 30B out of the four trapping members, and is connected to a fourth opening 21B for introducing the cell suspension 3, a fifth opening 22B for introducing the first gas 4A, and an eleventh opening 24B for introducing the second gas 4B.The fourth portion 44 faces only the third trapping member 30C among the four trapping members, and is connected to the sixth opening 21C for introducing the cell suspension 3, the seventh opening 22C for introducing the first gas 4A, and the twelfth opening 24C for introducing the second gas 4B. The fifth portion 45 faces only the fourth trapping member 30D among the four trapping members, and is connected to the eighth opening 21D for introducing the cell suspension 3, the ninth opening 22D for introducing the first gas 4A, and the thirteenth opening 24D for introducing the second gas 4B. An example of a method for detecting a substance in a gas 4 using such a detection unit 1B will be described below with reference to FIGS. 21 to 23.

[0118] First, as shown in Fig. 21(a), the first opening 21A, the fourth opening 21B, the sixth opening 21C, the eighth opening 21D and the third opening 23 are opened, and the other openings provided in the flow path 40 are closed, and then the cell suspension 3 is introduced from the first opening 21A, the fourth opening 21B, the sixth opening 21C and the eighth opening 21D. The introduced cell suspension 3 reaches the first trapping member 30A, the second trapping member 30B, the third trapping member 30C or the fourth trapping member 30D, and as shown in Fig. 21(b), the cells 5 are trapped by these trapping members, while the solution 6 passes through them and is discharged from the third opening 23 (separation process). Next, the first opening 21A, the fourth opening 21B, the sixth opening 21C, and the eighth opening 21D are closed, and the second opening 22A, the fifth opening 22B, the seventh opening 22C, and the ninth opening 22D are opened, and then the first gas 4A is introduced from the second opening 22A, the fifth opening 22B, the seventh opening 22C, and the ninth opening 22D. As shown in FIG. 22(a), when the introduced first gas 4A passes through the first part 41, the third part 43, the fourth part 44, or the fifth part 45 of the flow path 40 and reaches the first trapping member 30A, the second trapping member 30B, the third trapping member 30C, or the fourth trapping member 30D, the cells 5 trapped by these trapping members are exposed to the substance in the first gas 4A (first exposure step). As a result, the fluorescence emitted by the cells is measured (first evaluation step), and the substance in the first gas 4A can be detected. The second gas 4B that passes through these trapping members and reaches the second portion 42 of the flow path 40 is finally discharged from the third opening 23. Next, the second opening 22A, the fifth opening 22B, the seventh opening 22C, and the ninth opening 22D are closed, and the first opening 21A, the fourth opening 21B, the sixth opening 21C, and the eighth opening 21D are opened, and then, as shown in Fig. 22(b), a solution 6 is introduced from the first opening 21A, the fourth opening 21B, the sixth opening 21C, and the eighth opening 21D to remove substances in the gas 4 from the first trapping member 30A, the second trapping member 30B, the third trapping member 30C, or the fourth trapping member 30D and the cells 5 trapped therein (cleaning step).Finally, the first opening 21A, the fourth opening 21B, the sixth opening 21C and the eighth opening 21D are closed, and the tenth opening 24A, the eleventh opening 24B, the twelfth opening 24C and the thirteenth opening 24D are opened. Then, as shown in Fig. 23, the second gas 4B is introduced from the tenth opening 24A, the eleventh opening 24B, the twelfth opening 24C and the thirteenth opening 24D, so that the cells 5 trapped in the first trapping member 30A, the second trapping member 30B, the third trapping member 30C or the fourth trapping member 30D are exposed to the substance in the second gas 4B (second exposure step). This allows the substance in the second gas 4B to be detected by measuring the fluorescence emitted by the cells (second evaluation step).

[0119] In the example described above, the cells 5 trapped in the four trapping members were exposed to one type of first gas 4A in the first exposure step and to one type of second gas 4B in the second exposure step, but the gas to which each of the cells 5 trapped in the four trapping members is exposed may be different. In this case, substances in the gases can be detected for four types of gases in each exposure step, allowing detection with higher throughput.

[0120] In the above example, cells are placed in all four trapping members, but cells 5 may be trapped in only some of the trapping members and used for the exposure step and evaluation step. In this case, for example, by opening the first opening 21 and the third opening 23 and closing the other openings, and introducing the cell suspension 3 through the first opening 21, cells can be trapped in only the first trapping member 30A among the four trapping members.

[0121] 21(a), a step of introducing the solution 6 from the first opening 21A, the fourth opening 21B, the sixth opening 21C, and the eighth opening 21D may be included. In this case, the cells 5 in the cell suspension 3 remaining in the first portion 41, the third portion 43, the fourth portion 44, and the fifth portion 45 are also swept away and trapped in the first trapping member 30A, the second trapping member 30B, the third trapping member 30C, and the fourth trapping member 30D. This makes it possible to prevent cells exposed to the substance in the gas phase and cells not exposed to the substance from being mixed in the flow channel 40 due to the cells 5 remaining in the first portion 41, the third portion 43, the fourth portion 44, and the fifth portion 45 in the exposure step.

[0122] In the above example, the second opening 22A, the fifth opening 22B, the seventh opening 22C, and the ninth opening 22D are openings for introducing the first gas 4A, but these openings may be openings for introducing the first gas 4A and the solution 6. In this case, in the cleaning step, by introducing the solution 6 from the second opening 22A, the fifth opening 22B, the seventh opening 22C, and the ninth opening 22D instead of the first opening 21A, the fourth opening 21B, the sixth opening 21C, and the eighth opening 21D, the first gas 4A remaining in the second opening 22A, the fifth opening 22B, the seventh opening 22C, and the ninth opening 22D can also be removed, so that even if no valve is provided, the first gas 4A and the second gas 4B can be prevented from being mixed (contamination) in the second exposure step. In addition, from the viewpoint of preventing contamination, first, a first exposure step and a first evaluation step may be performed by introducing a second gas 4B through the second opening 22A, the fifth opening 22B, the seventh opening 22C, and the ninth opening 22D located upstream of the flow path 40, a cleaning step may be performed by introducing a solution 6 through the first opening 21A, the fourth opening 21B, the sixth opening 21C, and the eighth opening 21D, and then a second exposure step and a second evaluation step may be performed by introducing a first gas 4A through the first opening 21A, the fourth opening 21B, the sixth opening 21C, and the eighth opening 21D located downstream of the flow path 40.

[0123] In the detection unit 1B described above, the first trapping member 30A separates the space within the flow path 40 into a first portion 41 and a second portion 42, the second trapping member 30B separates the space within the flow path 40 into a second portion 42 and a third portion 43, the third trapping member 30C separates the space within the flow path 40 into a second portion 42 and a fourth portion 44, and the fourth trapping member 30D separates the space within the flow path 40 into the second portion 42 and a fifth portion 45. The base 2 (flow path member) has a first opening 21A connected to the first portion 41 for introducing the cell suspension 3, a second opening 22A connected to the first portion 41 for introducing the first gas 4A, a third opening 23 connected to the second portion 42 for discharging the solution 6 and the first gas 4A, a fourth opening 21B connected to the third portion 43 for introducing the cell suspension 3, a fifth opening 22B connected to the third portion 43 for introducing the first gas 4A, a fourth opening 21B connected to the third portion 43 for introducing the cell suspension 3, a fifth opening 22B connected to the third portion 43 for introducing the first gas 4A, an eighth opening 21D connected to the fifth portion 45 for introducing the cell suspension 3, and a ninth opening 22D connected to the fifth portion 45 for introducing the first gas 4A. Thus, a substance can be detected at each position of the first trapping member 30A to the fourth trapping member 30D. It should be noted that the fourth portion 44 and the fifth portion 45 may be omitted. EXAMPLES

[0124] The present invention will be described in more detail below using examples, but the present invention is not limited to the following examples.

[0125] <Cell culture> HeLa cells (human cervical cancer cells) were cultured in Dulbecco's modified Eagle's medium (Nacalai tesque #08456-65, hereafter referred to as DMEM) supplemented with 10 vol% inactivated fetal bovine serum (MP Biomedicals #2916754, hereafter referred to as FBS). CHO-K1 cells (Chinese hamster ovary cells) were cultured in Ham's F-12 Nutrient Mix, GlutaMAX (Gibco #31765-035) supplemented with 10% inactivated FBS (Gibco #10099-141). 0.25% Trypsin-EDTA (Gibco #25200-056) was used to detach the cells. The incubator used was a CO2 incubator (Panasonic #MCO-170AICUVD-PJ) or a culture system (Tokai Hit #STXG-IX3WX) on a microscope (Olympus #IX83) set to 37°C, 5% CO2, and natural evaporation using a humidified tray.

[0126] <Culture container, filter paper> The 96-well plates had flat polystyrene bottoms (TPP #92096), the 12-well plates had flat polystyrene bottoms (IWAKI #3815-012), the triple-well dishes had flat glass bottoms (IWAKI #3970-103), the 96-well plates with integrated Transwell inserts had 1.0 μm pore size inserts made of polyester membrane (CORNING #3380), and the filter paper (ADVANTEC #00021185) was cut to a size that covered the wells and UV-sterilized.

[0127] <Cell viability measurement> The cells were cultured for 2 hours in a culture medium containing 10% Cell Count Reagent SF (Nacalai tesque #07553-44), a reagent for measuring the number of live cells, and then the absorbance at 450 nm was measured using a microplate reader (Thermo Scientific #Varioskan Lux).

[0128] <Separation of cells and solution> To remove the culture medium from the cells, an aspirator (Nitto Kohki Co., Ltd. #LV-435A) equipped with a micropipette tip (Nikkyo Technos Co., Ltd. #NT-200G) was used.

[0129] Example 1: Cellular response to substances in the gas phase upon exposure in the gas and liquid phases 0.5 x 10 per well 4 HeLa cells were seeded on a 96-well plate and left to stand until the next day to allow the cells to adhere. The volume of culture medium (culture supernatant) was adjusted to 0-35 μL, and filter paper soaked in culture medium (control), ethanol (Fujifilm Wako Pure Chemical Corporation #057-00456), or a mixture of these was placed on the top of the plate. The plate was covered with a lid and left to stand in a CO2 incubator for 1 minute to expose it to volatile components. The filter paper was removed, the volume of culture medium was adjusted to 100 μL, and the plate was left to stand until the next day, after which the cell viability was measured.

[0130] The results when filter paper soaked in culture solution (control) or ethanol was placed are shown in Figure 24. Under conditions of 0 μL of culture solution, the survival rate of the ethanol group was lower than that of the control group, whereas under conditions of 5 μL or more of culture solution, no decrease in survival rate was observed in the ethanol group. This demonstrated that the cells exhibited high responsiveness to substances in the gas phase when exposed to 0 μL of culture solution, i.e., when exposed to the gas phase.

[0131] The results when filter paper soaked in culture solution, ethanol, or a mixture of these in various ratios was placed are shown in Figures 25(a) to (g). Figure 25(a) shows the results for 0% ethanol (100% culture solution). Figure 25(b) shows the results for 20% ethanol (80% culture solution). Figure 25(c) shows the results for 40% ethanol (60% culture solution). Figure 25(d) shows the results for 50% ethanol (50% culture solution). Figure 25(e) shows the results for 60% ethanol (40% culture solution). Figure 25(f) shows the results for 80% ethanol (20% culture solution). Figure 25(g) shows the results for 100% ethanol (0% culture solution). Of these, as shown in Figure 25(g), a decrease in cell viability was observed when filter paper soaked in 100% ethanol was placed under the condition of 0 μL of culture solution (culture supernatant).

[0132] Example 2: Responsiveness of cells to substances in the gas phase when exposed to gas and liquid phases after gas phase culture 0.5 x 10 per well 4 HeLa cells were seeded onto a 96-well plate and left to stand until the next day to allow the cells to adhere. The culture medium was removed, the plate was covered with a lid and left to stand in a CO2 incubator for 3 hours. The culture medium volume was adjusted to 0 μL for gas phase exposure and 100 μL for liquid phase exposure, and filter paper containing culture medium (control) or ethanol was placed on the top of the plate. The plate was covered with a lid and left to stand in a CO2 incubator for 1 to 10 minutes to expose to volatile components. The filter paper was removed, the culture medium volume was adjusted to 100 μL, and the plate was left to stand until the next day, after which cell viability was measured.

[0133] The results for gas phase exposure are shown in Figure 26(a). The results for liquid phase exposure are shown in Figure 26(b). Figure 27 shows the results for exposure times of 0 and 1 minute in Figure 26(a) and for exposure times of 1, 5, and 7 minutes in Figure 26(b). First, in both Figures 26(a) and (b), the cells responded to ethanol exposure, demonstrating that the cells survived even after 3 hours of gas phase culture and maintained responsiveness to substances in the gas phase.

[0134] 26(a), (b) and 27, a significant decrease in viability was observed with shorter exposure times in the gas phase compared to exposure in the liquid phase, and even with an exposure time of 1 minute, the cell viability decreased to about 20%. This demonstrated that exposure of cells to gas phase substances exhibits higher sensitivity than exposure in liquid phase, and that substances in the gas phase can be detected based on the cell response even with exposure times of 1 minute or less.

[0135] <Example 3: Change in cell viability depending on gas phase culture time> 0.5 x 10 per well 4 HeLa cells were seeded in a 96-well plate and left to stand until the next day to allow the cells to adhere. The culture medium was removed and the plate was left to stand in a CO2 incubator for 0 to 8 hours. The volume of the culture medium was adjusted to 100 μL and the plate was left to stand until the next day, after which the cell viability was measured.

[0136] The results are shown in Figure 28. No significant decrease in cell viability was observed when the gas phase culture time was 8 hours or less. This suggests that exposure of cells to substances in the gas phase can be carried out for at least 8 hours, provided that the substances do not induce cell death.

[0137] Example 4: Cell type dependence of changes in cell viability in gas phase culture 0.5 x 10 per well 4 CHO-K1 cells were seeded in a 96-well plate and left to stand until the next day to allow the cells to adhere. The culture medium was removed and the plate was left to stand in a CO2 incubator for 0 to 8 hours. The volume of the culture medium was adjusted to 100 μL and the plate was left to stand until the next day, after which the cell viability was measured.

[0138] The results are shown in Figure 29. In the case of CHO-K1 cells, nearly 60% of the cells survived after 8 hours of gas phase culture, suggesting that long-term gas phase exposure experiments can be performed with cell types other than the HeLa cells used in Example 3.

[0139] <Example 5: Comparison of changes in cell viability during gas phase culture in a closed environment / open environment> 0.5 x 10 per well 4 CHO-K1 cells were seeded in a 96-well plate and left to stand until the next day to allow the cells to adhere. The culture medium was removed, and the plate was left to stand in a CO2 incubator for 0 to 8 hours with or without a lid. The volume of the culture medium was adjusted to 100 μL, and the plate was left to stand until the next day, after which the cell viability was measured.

[0140] The results are shown in Figure 30. When cells were cultured for a long time in an open environment, the cell viability was lower than when they were cultured in a closed environment. This suggests that long-term gas-phase exposure experiments can be more appropriately performed in a closed environment.

[0141] Example 6: Dependence of cell viability on culture volume in gas phase culture 0.5 x 10 per well 4 HeLa cells were cultured in a 96-well plate (volume per well: approximately 400 mm 3 ) or 0.2 × 10 per well 5 HeLa cells were cultured in a 12-well plate (volume per well: approximately 7000 mm 3 ) and left to stand until the next day to allow the cells to adhere. The culture medium was removed and the plate was left to stand in a CO2 incubator for ~7 hours. The culture medium volume was adjusted to 100 μL for the 96-well plate and 500 μL for the 12-well plate, and the plate was left to stand until the next day, after which the cell viability was measured.

[0142] The results when a 12-well plate was used are shown in Figure 31(a). The results when a 96-well plate was used are shown in Figure 31(b). Compared to the use of a 12-well plate with a large volume per well, the cell viability after long-term culture was maintained at a high level when a 96-well plate with a small volume per well was used. This demonstrated that gas-phase exposure experiments to substances in the gas phase can be more suitably carried out in an environment with a small volume of cell culture space.

[0143] Example 7: Fluorescence-based assessment of the activity of cell membrane receptors expressed in gas-cultured cells 0.5 x 10 per well 4 HeLa cells were seeded in a triple-well dish and left to attach until the next day. The culture medium was removed, and the dish was left to stand in a CO2 incubator for 3 hours, after which the receptor activity of the cell membrane was evaluated.

[0144] The receptor activity of the cell membrane was evaluated by fluorescent observation of the change in intracellular calcium concentration due to histamine stimulation. The cells were left in the gas phase with HBSS(-) (Fujifilm Wako Pure Chemicals #085-09355) (hereafter referred to as buffer solution) supplemented with 2% 1M HEPES (Gibco #15630-080), washed three times, then buffer solution containing 3μM CaTM-3AM (Goryo Chemicals #GC507) was added and left in a CO2 incubator for 30 minutes. After washing three times with buffer solution, the cells were placed in a culture system on a microscope. Live imaging was started at 2-second intervals (initial observation image: 0 seconds), and 5 seconds after the start of observation, buffer solution containing histamine (Sigma-Aldrich #H7125) was added to a final concentration of 1μM (observation image immediately after addition: 6 seconds), and observation was continued for 10 minutes.

[0145] Fluorescence images taken before histamine stimulation (0 seconds) and immediately after histamine stimulation (6 seconds) are shown in Figure 32(a). Figure 32(b) shows the time course of the fluorescence intensity in the ROI shown in Figure 32(a), normalized by the average brightness value in the ROI at the start of observation. Even in cells placed in the gas phase, calcium oscillations in response to histamine binding to the histamine receptor could be detected by fluorescence, demonstrating that the cell membrane receptors expressed in cells cultured in the gas phase maintain their activity and that this activity can be detected by fluorescence.

[0146] Example 8: Cellular response when exposed to gas phase on a porous cell trap 1.0 x 10 per well 4CHO-K1 cells were seeded onto a 96-well plate (non-permeable) or a 96-well plate with a Transwell insert (permeable) and left to stand for 1 hour to allow the cells to adhere. The culture medium was removed, and 10 μL of a mixture of culture medium and ethanol was placed into the container, which was then left to stand for 1 minute in a CO2 incubator to expose the cells to volatile components. The cells were harvested using trypsin-EDTA and seeded onto a new 96-well plate, left to stand until the next day, and the cell viability was measured.

[0147] The results are shown in Figure 33. When the permeable Transwell plate was used, the cell viability decreased even when exposed to a lower concentration of ethanol compared to when the non-permeable normal well plate was used. This demonstrated that cells on the porous cell trap showed a relatively high response to the substance when exposed to the substance in the gas phase.

[0148] <Example 9: Detection of a target substance in the gas phase using cells expressing a cell membrane receptor that binds to the target substance> We investigated whether a target substance in the gas phase can be detected by cells expressing a heterocomplex of Or13a protein (an olfactory receptor in Drosophila melanogaster) and Orco protein (a co-receptor in Bombyx mori) on the cell membrane, which functions as a cell membrane receptor that binds to the target substance. Sf9 cells, which are ovarian cells of the fall armyworm, were used as the cells. For the evaluation, since the Or13a-Orco olfactory receptor is a ligand-gated ion channel that promotes calcium influx into cells upon ligand binding, we exposed the volatile odor chemical 1-Octen-3-ol (Sigma-Aldrich #O5284) to Sf9 cells expressing the Or13a-Orco olfactory receptor in a gas phase culture environment, and examined the change in intracellular calcium concentration when the odor chemical bound to the olfactory receptor on the cell membrane by fluorescent observation.

[0149] [Generation of Or13a-Orco expressing Sf9 cells] Sf9 cells expressing a heterocomplex of Or13a and Orco proteins on the cell membrane (Or13a-Orco expressing Sf9 cells) were prepared according to the method described in the literature (ManeeratTermtanasombat et al., "Cell-Based Odorant Sensor Array for OdorDiscrimination Based on Insect Odorant Receptors", Journal of ChemicalEcology volume 42, 716-724 (2016).).

[0150] 1. Synthesis of artificial genes The following genes were artificially synthesized by Eurofins Genomics, in which a Not I restriction enzyme sequence (GCGGCCGC) and a Kozak sequence (GCCGCC) were added upstream of the start codon (ATG) of the Or13a gene (Odorant receptor 13a of the fruit fly (Drosophila melanogaster), NCBI Gene ID: 32562) or Orco gene (Odorant receptor coreceptor of the silkworm (Bombyx mori), GenBank accession number: AB100454.1), and an Xba I restriction enzyme sequence (TCTAGA) was added downstream of the stop codon (TAA or TAG). The sequence of the artificial gene Not I-Or13a-Xba I created by adding this to the Or13a gene is shown in SEQ ID NO: 1. The sequence of the artificial gene Not I-Orco-Xba I created by adding this to the Orco gene is shown in SEQ ID NO: 2.

[0151] 2. Construction of expression vector A plasmid (pIB-Or13a) was prepared by inserting Not I-Or13a-Xba I (SEQ ID NO: 1) into the Not I-Xba I site of the pIB / V5-His vector (Invitrogen #V802001), which is an expression vector for recombinant proteins in insect cells, and a plasmid (pIB-Orco) was prepared by inserting Not I-Orco-Xba I (SEQ ID NO: 2) into the same site. Next, a BspH I-Or13a-Pci I fragment of pIB-Or13a was inserted into the BspH I site of pIB-Or13a to prepare a plasmid (pIB-Or13a-Orco plasmid) for expressing Or13a and Orco proteins. The plasmid map of the pIB-Or13a-Orco plasmid is shown in FIG. 34.

[0152] 3. Generation of Or13a-Orco expressing Sf9 cells Sf9 cells were transformed with pIB-Or13a-Orco plasmid by electroporation using a gene transfer device (BTX #ECM630). Sf9 cells expressing a heterocomplex of Or13a and Orco proteins on the cell membrane (Or13a-Orco-expressing Sf9 cells) were obtained using blasticidin S resistance as an indicator. The Or13a-Orco-expressing Sf9 cells thus produced were cultured in a culture medium containing Grace's Insect medium (Gibco #11605094) supplemented with inactivated 10% fetal bovine serum (HyClone #SH30070.03), 10μg / ml gentamicin (Gibco #15710064) and 10μg / ml blasticidin S (Gibco #A1113903). The incubator used was a cool incubator (As One Corporation #ICI-100) set to 27℃.

[0153] [Preparation of measurement vessel for gas-phase exposure] A perspective view of the measurement container used in this example is shown in Fig. 35(a), and an exploded perspective view of the measurement container is shown in Fig. 35(b). As shown in Fig. 35(a) and (b), the measurement container used in this example consists of a lid and a dish body, which were fabricated from a collagen I-coated glass base dish (hereinafter referred to as glass dish, IWAKI #4970-011, container diameter 35 mm, glass bottom diameter 27 mm).

[0154] As shown in the top views of Figures 35(a), (b) and (c), a circular hole with a diameter of 22 mm was made in the glass dish lid, and the hole was covered with a polypropylene film. The film was recessed from the top surface of the lid so that the lower surface of the film (the side facing the bottom surface of the dish body) was 2 mm high from the bottom surface of the dish body and parallel to the bottom surface of the dish body. In this case, a circular hole with a diameter of 6 mm and a rectangular hole with a size of 3 mm x 7 mm were made in the film, as shown in Figure 35(c). Furthermore, a 15 mm square filter paper (ADVANTEC #00021185) was attached from below to the surface of the film parallel to the bottom surface of the dish body, and a circular hole with a diameter of 6 mm was made in the filter paper so as to overlap with the circular hole in the film. As a result, the space inside the measurement container communicated with the outside through a circular hole (solution introduction hole) with a diameter of 6 mm, which was used to add ionomycin solution. The space inside the measurement container was also in contact with the outside at a rectangular hole of 3 mm x 7 mm provided in the film, separated by a sheet of filter paper, and the part separated by the filter paper (exposed part of the filter paper) was used to introduce the substance to be detected into the gas phase inside the measurement container by impregnating the filter paper with a solution containing the substance to be detected.

[0155] An example of the underside view of the dish body with cells attached is shown in Figure 35(d). The collagen I-coated glass bottom surface was further coated with Biocompatible Anchor for cell Membrane (hereinafter referred to as BAM, Yuka Sangyo Co., Ltd. #SUNBRIGHT OE-020CS).

[0156] [Detection of gas phase substances] Or13a-Orco expressing Sf9 cells were detached with a cell scraper (IWAKI #9020-250) and collected by centrifugation. After washing twice with imaging buffer (5mM HEPES pH7.2, 9.4mM D-Glucose, 140mM NaCl, 5.6mM KCl, 4.5mM CaCl2, 11.26mM MgCl2, 11.32mM MgSO4), imaging buffer containing 3μM CaTM-3 AM (Goryo Chemical Co., Ltd. #GC507) was added and the cells were stained by leaving it in a cool incubator for 30 minutes. After washing three times with buffer, the cells were seeded on the BAM-coated glass of the dish body and left in a cool incubator for 10 minutes to allow the stained cells to adhere to the bottom. The cells were filled with imaging buffer and left on a microscope to stabilize for 1 hour.

[0157] The imaging buffer was removed, and the lid of the measurement container for gas-phase exposure was placed on the dish body. The measurement container was reinstalled on the microscope, and live imaging was started at 2-second intervals. The fluorescence image at the start of imaging is shown in Figure 36(a). 20 seconds after the start of imaging, 50 μl of imaging buffer containing 25 μM 1-Octen-3-ol was soaked into the filter paper at the exposed part of the filter paper, and Or13a-Orco expressing Sf9 cells were exposed to volatilized 1-Octen-3-ol (an odorant). The fluorescence image 60 seconds after the start of imaging (40 seconds after exposure to 1-Octen-3-ol) is shown in Figure 36(b). 140 seconds after the start of imaging, 200 μl of imaging buffer containing 5 μM ionomycin (calcium ionophore, Nacalai tesque #19444-91) was added to the glass dish through the solution introduction hole, allowing calcium to flow into the cells. A fluorescent image taken 180 seconds after the start of imaging (40 seconds after the addition of ionomycin) is shown in Fig. 36(c). In addition, Fig. 37 shows the results of normalizing the average fluorescence intensity over time in the ROIs shown in Fig. 36(a) to (c) by the average intensity in the ROI at the start of observation.

[0158] 36 and 37, when cells were exposed to the target substance (1-Octen-3-ol) in the gas phase, an increase in fluorescence intensity due to calcium influx was observed, similar to the case of adding ionomycin to cells. From the above, it became clear that it is possible to detect substances in the gas phase by exposing cells separated from a solution to a substance in the gas phase and then evaluating it. [Explanation of symbols]

[0159] 1, 1A, 1B...detection unit, 2...substrate (flow path member), 3...cell suspension, 4...gas (gas containing substance), 5...cell, 6...solution, 7...cell disruption solution (cleaning solution), 9...cell detachment solution (cleaning solution), 10...first substrate (flow path member), 10A...first member (flow path member), 10B...second member (flow path member), 20...second substrate (flow path member), 21, 21A...first opening, 21B...fourth opening, 22, 22A...second opening, 22B...fifth opening, 23...third opening, 30...trapping member (cell trapping section), 31...through hole (opening), 32...gap (opening), 33...trapping area (cell trapping section), 40...flow path, 40a...surface, 41...first part, 42...second part.

Claims

1. 1. A method for detecting a substance in a gas phase, comprising: A separation step of separating the cells from a cell suspension, which is a solution in which the cells are suspended; an exposing step of exposing the cells separated from the cell suspension to a substance in the gas phase; and evaluating the cells exposed to the substance in the gas phase; A method comprising, in order:

2. The method of claim 1 , wherein the separating step comprises separating the cells from the cell suspension by contacting the cell suspension with a cell trapping portion.

3. the cell trapping portion having one or more openings configured to trap the cells while allowing the solution to pass therethrough; The method according to claim 2 , wherein the separating step comprises flowing the cell suspension through a flow path provided with the cell trapping portion.

4. The method according to claim 3 , wherein the cell trapping portion includes a trapping member at least partially made of a porous body, and the one or more openings are holes in the porous body.

5. The method of claim 3 , wherein the maximum width of each of the one or more openings is less than or equal to 30 μm.

6. The method of claim 2 , wherein the cell trapping portion is configured to trap cells having a maximum width of 5 μm or more.

7. the cell trapping section includes a trapping area formed on a surface of a flow path for flowing the cell suspension, and the trapping area is configured such that when the cell suspension comes into contact with the trapping area, the solution is not trapped, but the cells are trapped on the trapping area; The method of claim 2 , wherein the separating step comprises flowing the cell suspension through the flow channel so as to contact the trapping area.

8. The method according to claim 2 , further comprising, after the evaluation step, a step of removing at least one of the cells and the substance from the cell trapping portion by flowing a washing solution so as to contact the cell trapping portion.

9. the separating step includes flowing the cell suspension through a flow path provided with the cell trapping portion, The volume of the flow path is 7000 mm ３ 3. The method of claim 2, wherein:

10. The method according to any one of claims 1 to 9, wherein the exposure time in the exposure step is 1 minute or less.

11. The method according to any one of claims 1 to 9, wherein the exposure time in the exposure step is 1 minute or longer.

12. The method according to any one of claims 1 to 9, wherein the exposure time in the exposure step is from 1 hour to 8 hours.

13. A detection unit for use in detecting a substance in a gas phase, comprising: A flow path member that defines a flow path for flowing a cell suspension, which is a solution in which cells are suspended; and A trap member disposed in the flow path so as to interrupt the flow in the flow path. Equipped with The trapping member defines one or more openings configured to trap the cells while allowing the solution to pass through. A detection unit.

14. The trap member separates a space within the flow path into a first portion and a second portion, The detection unit according to claim 13, wherein the flow path member has: a first opening connected to the first portion for supplying the cell suspension; a second opening connected to the first portion for supplying a gas containing the substance; and a third opening connected to the second portion for discharging the solution and the gas.

15. The detection unit according to claim 14 , wherein the flow path member further comprises a fourth opening connected to the second portion and for supplying the gas.

16. The trap member includes a first trap member and a second trap member, the first trap member separates a space within the flow path into a first portion and a second portion, and the second trap member separates a space within the flow path into the second portion and a third portion, The detection unit according to claim 13, wherein the flow path member has: a first opening connected to the first portion for supplying the cell suspension; a second opening connected to the first portion for supplying a gas containing the substance; a third opening connected to the second portion for discharging the solution and the gas; a fourth opening connected to the third portion for supplying the cell suspension; and a fifth opening connected to the third portion for supplying a gas containing the substance.

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