Flow path substrate, cartridge, detection system, and method for manufacturing flow path substrate

Abstract

This flow path substrate has a first flow path, a plurality of second flow paths, and a first branch wall. The plurality of second flow paths branch from the first flow path in a direction not overlapping a first imaginary straight line overlapping the first flow path. Each of the plurality of second flow paths is thinner than the first flow path. The first branch wall is located in a region where the plurality of second flow paths branch. The first branch wall is continuous with the second flow paths. The first branch wall overlaps the first imaginary straight line.

Description

Flow path substrate, cartridge, detection system, and method for manufacturing flow path substrate

[0001] The present disclosure relates to a flow path substrate and the like.

[0002] The microchannel chip of Patent Document 1 includes one liquid inlet, multiple reaction sections in which chemicals that react with liquid are placed, multiple distribution sections that communicate with the liquid inlet, and channels that communicate with each distribution section and each reaction section.

[0003] Japanese Patent Application Publication No. 2020-112383

[0004] A flow path substrate according to one aspect of the present disclosure includes a liquid receiving section that receives a liquid and a flow path connected to the liquid receiving section, and the flow path has a first region in which a first reagent containing a primer is disposed, and a second region in which a second reagent containing an enzyme that amplifies nucleic acid is disposed at a position different from the first region.

[0005] A method for manufacturing a flow path substrate according to one aspect of the present disclosure includes a forming step of forming a liquid receiving portion for receiving a liquid and a flow path connected to the liquid receiving portion on a substrate, a first disposing step of disposing a first reagent containing a primer in the flow path, and a second disposing step of disposing a second reagent containing an enzyme for amplifying nucleic acid at a position different from the first region of the flow path.

[0006] FIG. 1 is a schematic diagram showing an example of a detection system of the present disclosure. FIG. 1 is a schematic diagram showing an example of a configuration of a cartridge of the present disclosure, and a schematic diagram of a window portion of the cartridge of the present disclosure when viewed from above. FIG. 2 is a schematic diagram for explaining an example of use of the cartridge and detection device of the present disclosure. FIG. 3 is a block diagram showing an example of a detection system of the present disclosure. FIG. 4 is a plan view showing an example of a flow path substrate of a cartridge of the present disclosure. FIG. 5 is a schematic diagram showing an example of a first region and a second region arranged in a flow path provided in a flow path substrate according to a first embodiment of the present disclosure. FIG. 6 is a flowchart showing an example of a method for manufacturing a flow path substrate according to the first embodiment of the present disclosure. FIG. 7 is a schematic plan view showing an example of a flow path to which a reagent has been applied, and a schematic diagram showing another example of a method for applying a reagent to a flow path. FIG. 8 is a plan view showing an example of a flow path substrate according to a second embodiment of the present disclosure. FIG. 9 is a schematic diagram showing an example of an arrangement of a detection reagent in a flow path provided in a flow path substrate according to the second embodiment of the present disclosure. FIG. 10 is a schematic diagram for explaining an example of use of a plurality of flow paths provided in a flow path substrate according to the second embodiment of the present disclosure. FIG. 11 is a flowchart showing an example of a method for manufacturing a flow path substrate 4B according to the second embodiment of the present disclosure. 15 is a cross-sectional view of a flow path substrate according to a third embodiment, taken along a plane including line A-A in FIG. 14. FIG. 16 is a partially enlarged view of a flow path substrate according to a third embodiment, taken along a region surrounded by a dotted line in FIG. 15. FIG. 17 is a partially enlarged view of a flow path substrate according to a fourth embodiment, taken along a region corresponding to FIG. 16. FIG. 18 is a partially enlarged view of a flow path substrate according to a fifth embodiment, taken along a region corresponding to FIG. 16. FIG. 19 is a partially enlarged view of a flow path substrate according to a sixth embodiment, taken along a region corresponding to FIG. 16. FIG. 20 is a partially enlarged view of a flow path substrate according to a seventh embodiment, taken along a region corresponding to FIG. 16. FIG. 21 is a diagram showing the surface roughness of a first deposit and the surface roughness of a second deposit.

[0007] According to one aspect of the present disclosure, a flow path substrate is provided that can detect multiple substances by simply introducing a liquid into multiple flow paths. According to one aspect of the present disclosure, a flow path substrate is provided that can detect multiple substances by simply introducing a liquid into multiple flow paths.

[0008] [Detection System] Fig. 1 is a schematic diagram showing an example of a detection system 1 according to the present disclosure. Fig. 1 is a schematic diagram showing an example of the appearance of a cartridge 2 and an example of the appearance of a detection device 3. Fig. 2 is a block diagram showing an example of the detection system 1. As shown in Fig. 1, the detection system 1 may include the cartridge 2 and the detection device 3.

[0009] The cartridge 2 is a test kit for detecting a target substance contained in a specimen collected from a subject. The cartridge 2 may be a test kit that can amplify nucleic acid derived from the target substance using a reagent when the target substance is contained in a specimen inserted into the cartridge 2. The cartridge 2 may include a main body 21 and a bottle 22.

[0010] The subject is not limited to a human, but may be any living organism that can harbor a virus or bacterium, such as a mammal, bird, reptile, or amphibian.

[0011] The specimen may be saliva, urine, sweat, nasal mucus, blood, cells, etc. collected from the body of a subject, or may be soil and water collected from an object to be inspected, such as the ground, a river, the sea, etc. Alternatively, the specimen may be an attachment collected from the surface of an object to be inspected, such as a handrail, a door, clothing, shoes, or a toilet bowl.

[0012] The detection target may be, for example, a virus or a bacterium. The type of virus or bacterium contained in the sample is not limited to one type, and may be two or more types. When the detection target is a virus, examples of the type of virus include influenza virus, coronavirus (e.g., SARS-CoV-2), respiratory syncytial virus (RS virus), human metapneumovirus, norovirus, HIV (human immunodeficiency virus), herpes virus, streptococcus, and Mycoplasma pominis.

[0013] A specimen collected from a subject may be contained in the bottle portion 22. The main body portion 21 may include a flow path substrate 4 (see FIG. 5 ) that receives a liquid containing the specimen contained in the bottle portion 22 and, when the specimen contains a target substance, contains a reagent that reacts with nucleic acid derived from the target substance. The nucleic acid derived from the target substance may be extracted in the bottle portion 22, as will be described in detail below.

[0014] The detection device 3 is a device that detects nucleic acid derived from a detection target when the nucleic acid is amplified in the cartridge 2. The detection device 3 may have a housing that can receive the cartridge 2.

[0015] The detection device 3 may determine whether nucleic acid derived from the detection target is present at a certain concentration or above. The detection device 3 may determine that the sample is positive when nucleic acid derived from the detection target is present at a certain concentration or above. Alternatively, the detection device 3 may be a measurement device that measures the concentration of nucleic acid derived from the detection target. In this embodiment, the detection device 3 is described as determining whether the sample is positive. However, a determination device separate from the detection device 3 may determine whether the sample is positive by obtaining data corresponding to the amount of nucleic acid detected by the detection device 3 from the detection device 3.

[0016] The specific configurations of the cartridge 2 and the detection device 3 will be described later.

[0017] [NASBA Method] In the flow path substrate 4, nucleic acid derived from the detection target may be amplified by an isothermal nucleic acid amplification method. Examples of the isothermal nucleic acid amplification method include NASBA (Nucieic Acid Sequence-Based Amplification). Other examples include NEAR (Nicking Enzyme Amplification), LAMP (Loop-mediated Isothermal Amplification), and TMA (Transcription-mediated Amplification). In this embodiment, NASBA may be used as the isothermal nucleic acid amplification method.

[0018] The NASBA method is an isothermal nucleic acid amplification method that uses three types of enzymes (AMV reverse transcriptase, RNase H, and T7 RNA polymerase) and two types of primers. In the NASBA method, simply adding a NASBA substrate and enzyme to the template RNA results in amplified antisense single-stranded RNA. Because the entire series of steps in the NASBA reaction proceeds isothermally, RNA nucleic acid amplification can be performed without complex temperature control. Furthermore, because the amplification product of the NASBA reaction is single-stranded RNA, sequence-specific detection can be performed using a detection probe without a denaturation step.

[0019] [Specific Configuration of Cartridge] Reference numeral 1101 in Fig. 2 is a schematic diagram showing an example of the configuration of the cartridge 2. Reference numeral 1102 in Fig. 2 is a schematic diagram of the window portion 211 of the cartridge 2 as viewed from above. Fig. 3 is a schematic diagram for explaining an example of how the cartridge 2 and the detection device 3 are used.

[0020] As indicated by reference numeral 1101 in Fig. 2 , the main body 21 may include a flow path substrate 4. The flow path substrate 4 may include a liquid receiving portion 41 that receives liquid. Furthermore, as indicated by reference numeral 1102 in Fig. 2 , the flow path substrate 4 may include a storage portion 46 that stores liquid that has flowed through the flow path substrate 4. The housing of the main body 21 may be made of an opaque material. In this case, the main body 21 may include a window portion 211 so that the inside of the main body 21 can be seen.

[0021] The bottle portion 22 is a container capable of holding a liquid. The bottle portion 22 may be made of a transparent material, but may also be made of an opaque material.

[0022] As indicated by the arrow at reference numeral 1101 in Fig. 2 , the liquid receiving section 41 receives the liquid that has flowed in from the bottle section 22. The liquid received by the liquid receiving section 41 flows through a flow path 45 (see Fig. 5 ) arranged in the flow path substrate 4, and is then stored in a storage section 46. A reagent may be arranged in the flow path 45. Furthermore, as indicated by reference numeral 1102 in Fig. 2 , an internal standard (standard section) 47 may be provided near the storage section 46. The internal standard 47 may be used for optical comparison with the storage section 46.

[0023] The flow channel substrate 4 may include four flow channels 45A to 45D (see FIG. 5). A mixed fluid containing a sample and a labeling substance that reacts with the target substance contained in the sample may be flowed through at least one of the flow channels 45A to 45D. The internal standard 47 may be located in a region different from the flow channels 45 and used for comparison with the mixed fluid containing the sample and the labeling substance that reacts with the target substance contained in the sample. For example, the intensity of light (e.g., fluorescence) emitted by the internal standard 47 may be compared with the intensity of light (e.g., fluorescence) emitted by the sample and the labeling substance that reacts with the target substance contained in the sample.

[0024] As indicated by reference numeral 1102 in Fig. 2 , the main body 21 is formed with a window 211 so that the reservoirs 46 and internal standards 47 arranged on the flow path substrate 4 can be seen through the window 211 when viewed from above. When the flow path substrate 4 includes a plurality of reservoirs 46 and a plurality of internal standards 47, the window 211 may be formed so that all of the reservoirs 46 and all of the internal standards 47 can be seen. In other words, the reservoirs 46 and the internal standards 47 may be arranged on the main body 21 so as to be optically exposed from the window 211.

[0025] As shown by reference numeral 1112 in FIG. 3 , the sampler 23 may be attached to the main body 21. The sampler 23 may include a specimen collection section 231 and a fixing section 232. The specimen collection section 231 is a section for collecting a specimen. The specimen collection section 231 may be, for example, a pleated resin member. The fixing section 232 may fix the bottle section 22 to the main body 21 when the sampler 23 is inserted into the bottle section 22. The connection section between the main body 21 and the bottle section 22 may have, for example, a screw structure. Furthermore, a lid may be provided at the connection section of the bottle section 22 with the main body 21. This allows the interior of the bottle section 22 to be sealed. The lid may be made of aluminum. The subject may remove the lid and insert the sampler 23 into the bottle section 22.

[0026] As indicated by reference numeral 1111 in Fig. 3 , when collecting saliva as a specimen, for example, the subject inserts the specimen collection portion 231 into the subject's mouth to cause saliva to adhere to the specimen collection portion 231. As indicated by reference numeral 1112 in Fig. 3 , the subject applies saliva to the specimen collection portion 231 and then inserts the sampler 23 into the bottle portion 22.

[0027] The bottle portion 22 may contain a buffer solution 24. An example of the buffer solution 24 is a NASBA solution. The buffer solution 24 may contain, for example, a surfactant and an RNA (ribonucleic acid) degrading enzyme inhibitor. However, the RNA degrading enzyme inhibitor may be disposed on the flow path substrate 4. In this case, the buffer solution 24 may not contain an RNA degrading enzyme inhibitor. An example of a surfactant is Tween-20 (polysorbate 20), which is an example of a nonionic surfactant. When the buffer solution 24 contains a nonionic surfactant, nucleic acids derived from the detection target can be extracted in the bottle portion 22 by mixing the sample and the buffer solution 24.

[0028] When the subject inserts the sampler 23 into the bottle portion 22, the saliva adhering to the specimen collection portion 231 is mixed with the buffer solution 24. The buffer solution 24 mixed with the specimen may be an example of a liquid that the bottle portion 22 can contain.

[0029] As indicated by reference numeral 1113 in Fig. 3 , the detection device 3 may include a receiving section 36 that can receive the cartridge 2. As indicated by reference numeral 1113 in Fig. 3 , the subject attaches the cartridge 2 containing the sample to the receiving section 36, and then turns the detection device 3 upside down, as indicated by reference numeral 1114 in Fig. 3 . As a result, the buffer solution 24 mixed with the sample flows, due to its own weight, down the sampler 23 and into the flow path substrate 4 provided in the main body section 21. The buffer solution 24 mixed with the sample may be an example of a liquid that the liquid receiving section 41 receives.

[0030] As a negative control, only the buffer solution 24 may be poured into the flow path substrate 4. In this case, the buffer solution 24 itself is an example of a liquid that can be accommodated in the bottle portion 22, and an example of a liquid that can be received by the liquid receiving portion 41. Also, a substance that contains a pathogen other than the detection target may be mixed into the buffer solution 24. In this case, the buffer solution 24 mixed with the substance is an example of a liquid that can be accommodated in the bottle portion 22, and an example of a liquid that can be received by the liquid receiving portion 41.

[0031] 4 is a block diagram showing an example of the detection system 1. As shown in Fig. 4, the detection device 3 may be a device that detects nucleic acid amplified in a flow channel 45 provided in a flow channel substrate 4. The detection device 3 may include, for example, a heating unit 31, a pressurizing unit 32, a light irradiating unit 33, an imaging unit 34, and a control unit 35.

[0032] The heating unit 31 may be a member that heats the cartridge 2 inserted into the detection device 3. The heating unit 31 may heat the cartridge 2 to a temperature that promotes the reaction between the nucleic acid and the reagent in the flow path substrate 4. When amplifying nucleic acids by the NASBA method, the heating unit 31 may heat the cartridge 2 to a temperature of 37°C to 41°C. In this embodiment, the heating unit 31 first heats the cartridge 2 at a temperature of 80°C to 95°C for 3 to 10 minutes. This disrupts pathogens and liberates nucleic acid components from the pathogens. It also inactivates DNA (deoxyribonucleic acid) degrading enzymes. Thereafter, the heating unit 31 may maintain the cartridge 2 at a temperature of 37°C to 41°C.

[0033] The heating unit 31 may heat different parts of the cartridge 2 at different temperatures. For example, the heating unit 31 may have a first heating unit that heats the bottle unit 22 to a temperature of 80°C to 95°C, and a second heating unit that heats the main body unit 21 to a temperature of 37°C to 41°C. This allows pathogens to be crushed in the bottle unit 22, liberating nucleic acid components from the pathogens. Furthermore, the DNase can be inactivated in the bottle unit 22. The liquid in the bottle unit 22 then flows into the flow path substrate 4 provided in the main body unit 21, which is maintained at a temperature of 37°C to 41°C, and nucleic acid amplification may be performed in the flow path substrate 4.

[0034] The temperature to which the heating unit 31 heats may be the set temperature of the heating unit 31. Alternatively, it may be the liquid temperature of the liquid whose temperature is to be adjusted by the heating unit 31. When the temperature to which the heating unit 31 heats is the liquid temperature of the liquid whose temperature is to be adjusted by the heating unit 31, for example, the average temperature or central temperature of the liquid may be used. Here, the average temperature may be, for example, the average liquid temperature over a predetermined period of time. The central temperature may be, for example, the temperature at the center between the highest and lowest liquid temperatures. The central temperature may be calculated as the sum of the highest and lowest temperatures divided by 2. The detection device 3 may be equipped with a temperature sensor that detects the liquid temperature of the liquid whose temperature is to be adjusted by the heating unit 31. The heating unit 31 may heat the liquid whose temperature is to be detected by the temperature sensor so as to maintain the liquid temperature detected by the temperature sensor.

[0035] The pressurizing unit 32 may be a member that pressurizes the bottle portion 22 of the cartridge 2 inserted into the detection device 3. When the detection device 3 is configured to pressurize the bottle portion 22 by the pressurizing unit 32, the bottle portion 22 may be made of a material that is deformed by the pressure of the pressurizing unit 32. The bottle portion 22 is deformed by the pressure of the pressurizing unit 32, which makes it easier for the liquid in the bottle portion 22 to flow into the main body portion 21.

[0036] The pressurizing unit 32 may be a member that applies pressure to at least a portion of the side surface of the bottle part 22. The pressurizing unit 32 may be a member that clamps the side surface of the bottle part 22. The pressurizing unit 32 may be disposed in the detection device 3 at a position where it can apply pressure to the side surface on the bottom side of the bottle part 22, for example.

[0037] The light irradiating unit 33 may be a light source that irradiates the storage unit 46 and the internal standard 47 of the flow path substrate 4 with light through the window 211 of the main body 21. The light irradiating unit 33 irradiates, for example, excitation light onto the storage unit 46 and the internal standard 47.

[0038] A labeling substance that specifically binds to the nucleic acid amplified in the flow channel 45 may be disposed in the flow channel 45 of the flow channel substrate 4. The labeling substance may be optically observable, and may include, but is not limited to, a dye, a luminescent substance, or a fluorescent substance. If the labeling substance is a fluorescent substance, when the storage section 46 is irradiated with excitation light, the labeling substance bound to the nucleic acid emits fluorescence having a specific wavelength.

[0039] Examples of fluorescent substances contained in labeling substances include 6-caroxyfluorescein (FAM), Texas Red (TR) (registered trademark), and cyanine (CY)-based materials. When the fluorescent substance is 6-FAM, it emits fluorescence with a peak wavelength of 517 nm when irradiated with excitation light with a peak wavelength of 494 nm. When the fluorescent substance is Texas Red, it emits fluorescence with a peak wavelength of 615 nm when irradiated with excitation light with a peak wavelength of 596 nm. When the fluorescent substance is Cy3-carboxylic acid, it emits fluorescence with a peak wavelength of 570 nm when irradiated with excitation light with a peak wavelength of 555 nm.

[0040] The labeling substance may be, for example, a molecular beacon. A molecular beacon is a type of probe DNA. Each molecule of the probe DNA is modified with a fluorescent molecule and a quencher molecule. The quencher molecule is configured to absorb light in a wavelength band corresponding to the fluorescence wavelength of the fluorescent molecule, for example. The distance between the fluorescent molecule and the quencher molecule in the probe DNA that is not bound to the enzymatically amplified nucleic acid molecule is closer than the distance between the fluorescent molecule and the quencher molecule in the probe DNA that is bound to the enzymatically amplified nucleic acid molecule. The base sequence of the probe DNA may be appropriately designed based on the base sequence of the nucleic acid to be detected.

[0041] The internal standard 47 may be any substance that can be optically compared with the storage section 46, and may be, for example, but is not limited to, a dye, a luminescent substance, or a fluorescent substance. A fluorescent substance that emits fluorescence when irradiated with excitation light may be disposed as the internal standard 47. The fluorescent substance as the internal standard 47 and the fluorescent substance contained in the labeling substance may be the same type of fluorescent substance.

[0042] The imaging unit 34 is a component that images the storage unit 46 and the internal standard 47 through the window 211 of the main body 21. By imaging the storage unit 46 with the imaging unit 34, it may be possible to determine whether or not a certain concentration or more of nucleic acid derived from the detection target is present in the liquid stored in the storage unit 46 based on optical information measured from the labeling substance present in the storage unit 46. The optical information may be, for example, information obtained from a dye, a luminescent substance, or a fluorescent substance. The optical information may be, for example, information such as wavelength or luminance value. For example, the control unit 35 may determine whether a certain concentration or more of nucleic acid derived from the detection target is present in the liquid stored in the storage unit 46 based on the intensity of fluorescence emitted from the storage unit 46.

[0043] Furthermore, the image capturing unit 34 may capture an image of the internal standard 47, and the position of the internal standard 47 in the captured image may be identified based on optical information measured from the internal standard 47. The optical information may be, for example, information obtained from a dye, a luminescent substance, or a fluorescent substance. The optical information may be, for example, information such as wavelength or brightness value. For example, the control unit 35 may identify the position of the internal standard 47 in the captured image based on the fluorescence emitted from the internal standard 47. Then, the control unit 35 may identify the position of the flow path 45 in the captured image based on the identified position of the internal standard 47.

[0044] Furthermore, the image capturing unit 34 may analyze an image of the internal standard 47 to determine whether optical information equal to or greater than a predetermined value can be measured from the internal standard 47. The control unit 35 may determine that the image capturing unit 34 is operating normally if optical information equal to or greater than the predetermined value can be measured. For example, the control unit 35 may determine whether fluorescence of equal to or greater than a predetermined intensity can be received from the internal standard 47. The control unit 35 may determine that the image capturing unit 34 is operating normally if fluorescence of equal to or greater than the predetermined intensity can be received.

[0045] The determination of whether optical information equal to or greater than a predetermined value has been measured may be made once before measuring optical information obtained from the specimen after starting the detection device 3. For example, the determination of whether fluorescence equal to or greater than a predetermined intensity has been received, i.e., the measurement of the predetermined fluorescence intensity, may be made once after starting the detection device 3 and before measuring the intensity of fluorescence emitted by the specimen.

[0046] The control unit 35 may comprehensively control each component included in the detection device 3. The control unit 35 may include, for example, an intensity measurement unit 351 and a positive determination unit 352.

[0047] The intensity measurement unit 351 may measure optical information from the substance contained in the storage unit 46 and the internal standard 47. For example, the intensity measurement unit 351 may measure the intensity of fluorescence emitted by the substance contained in the storage unit 46 and the internal standard 47 upon receiving excitation light.

[0048] The control unit 35 may first control the light irradiation unit 33 to irradiate the storage unit 46 and the internal standard 47 with light. In this state, the control unit 35 may control the imaging unit 34 to capture an image including the storage unit 46 and the internal standard 47. The intensity measurement unit 351 may analyze the image captured by the imaging unit 34 and measure the brightness as optical information obtained from the substances contained in the storage unit 46 and the internal standard 47.

[0049] For example, the control unit 35 controls the light irradiating unit 33 to irradiate the storage unit 46 and the internal standard 47 with excitation light, and then acquires the luminance by analyzing the image captured by the imaging unit 34. In this way, the control unit 35 may measure the intensities of the fluorescence emitted by the substances contained in the storage unit 46 and the internal standard 47, respectively.

[0050] The intensity measurement unit 351 may measure optical information obtained from the reservoir 46 connected to the flow path 45 in which the reagent is disposed as optical information of the detection target. For example, the intensity measurement unit 351 may measure the intensity of fluorescence emitted from the reservoir 46 connected to the flow path 45 in which the reagent is disposed as the intensity of fluorescence of the detection target. Furthermore, the intensity measurement unit 351 may measure optical information emitted from the reservoir 46 connected to the flow path 45 for the negative control as optical information of the negative control. For example, the intensity measurement unit 351 may measure the intensity of fluorescence emitted from the reservoir 46 connected to the flow path 45 for the negative control as the intensity of fluorescence of the negative control. The flow path 45 for the negative control will be described later. The position of the flow path 45 in which the reagent is disposed and the position of the flow path 45 for the negative control may be determined in advance.

[0051] The positive determination unit 352 may determine whether or not a certain concentration or more of nucleic acid derived from the detection target is present in the specimen contained in the liquid stored in the storage unit 46, based on the optical information obtained from the storage unit 46 measured by the intensity measurement unit 351. For example, the positive determination unit 352 may determine whether a certain concentration or more of nucleic acid derived from the detection target is present in the specimen contained in the liquid stored in the storage unit 46, based on the intensity of fluorescence emitted from the storage unit 46 measured by the intensity measurement unit 351. The positive determination unit 352 may determine that the specimen is positive when it determines that a certain concentration or more of nucleic acid derived from the detection target is present.

[0052] For example, the positive determination unit 352 may determine whether a certain concentration or more of nucleic acid derived from the detection target is present by comparing the value indicated by the optical information obtained from the storage unit 46 with a threshold value. For example, the threshold value may be optical information associated with the minimum concentration of nucleic acid derived from the detection target that should be determined as positive. The threshold value may be set in advance through experiments, etc. For example, the positive determination unit 352 may determine whether a certain concentration or more of nucleic acid derived from the detection target is present by comparing the intensity of fluorescence emitted from the storage unit 46 with the threshold value.

[0053] The positive determination unit 352 may calculate a difference value by subtracting the value indicated by the optical information of the negative control from the value indicated by the optical information of the detection target. For example, the positive determination unit 352 may calculate a difference value by subtracting the fluorescence intensity of the negative control from the fluorescence intensity of the detection target. The positive determination unit 352 may compare the difference value with a threshold, and determine that the sample is positive if the difference value is equal to or greater than the threshold, and determine that the sample is negative if the difference value is less than the threshold.

[0054] Alternatively, the positive determination unit 352 may determine whether the sample is positive by comparing the difference value with the value indicated by the optical information obtained from the internal standard 47. For example, two internal standards 47 having different concentrations may be provided. The positive determination unit 352 may determine that the sample is positive if the difference value is within the range of values ​​indicated by the optical information obtained from the two internal standards 47. The optical information obtained from the two internal standards 47 may be optical information associated with the concentration of nucleic acid derived from the detection target to be determined as positive. The optical information obtained from the two internal standards 47 may be set in advance by experiment or the like.

[0055] For example, the positive determination unit 352 may determine whether the sample is positive by comparing the difference value with the intensity of the fluorescence emitted from the internal standard 47. The positive determination unit 352 may determine that the sample is positive if the difference value is within the range of the fluorescence intensities indicated by the two internal standards 47.

[0056] The control unit 35 may also create a calibration curve based on optical information obtained from the multiple internal standards 47. For example, the control unit 35 may create a calibration curve based on the intensities of fluorescence emitted by the multiple internal standards 47. In this case, the control unit 35 may use the calibration curve to calculate the concentration of nucleic acid corresponding to the difference value. The positive determination unit 352 may determine that the sample is positive if the calculated concentration of nucleic acid is equal to or greater than a reference value. The reference value may be a value associated with the concentration of nucleic acid derived from the detection target that should be determined to be positive. The reference value may be set in advance through experiments, etc.

[0057] In the following explanation, as an example, the labeling substance and internal standard 47 are fluorescent substances, and the control unit 35 measures the intensity of fluorescence as the optical information measured from the labeling substance and internal standard 47.

[0058] [First Form of Flow Channel Substrate] Fig. 5 is a plan view showing an example of a flow channel substrate 4A. The flow channel substrate 4A is an example of the flow channel substrate 4. Fig. 6 is a schematic diagram showing an example of a first region 451 and a second region 452 arranged in a flow channel 45 provided in the flow channel substrate 4A.

[0059] 5, the flow path substrate 4A may include a liquid receiving section 41 that receives a liquid, and a flow path 45 that connects to the liquid receiving section 41. The flow path 45 may have a first region 451 in which a first reagent 101 containing a primer is disposed, and a second region 452 in which a second reagent 102 containing an enzyme that amplifies nucleic acid is disposed at a position different from the first region 451. In the flow path 45 that communicates with the liquid receiving section 41, the space in which the first region 451 is located and the space in which the second region 452 is located may communicate with each other.

[0060] In this embodiment, as shown in FIG. 5 , the flow path substrate 4A may include a liquid receiving section 41, a branch flow path 42, a flow path 45, a storage section 46, and an internal standard 47. There may be one or more flow paths 45. There may also be one or more internal standards 47. In this embodiment, the flow paths 45 include four flow paths 45A to 45D, and the storage sections 46 include four storage sections 46A to 46D, but this is not limiting. In addition, the flow path substrate 4A includes five internal standards 47, but this is not limiting.

[0061] 4, the liquid receiving portion 41 is located above the flow path 45 and the storage portion 46. Therefore, the liquid flowing from the bottle portion 22 due to its own weight easily flows from the liquid receiving portion 41 to the storage portion 46 via the flow path 45.

[0062] The liquid receiving portion 41 may be an opening that receives the liquid flowing from the bottle portion 22. The branch flow path 42 may be a flow path that connects the liquid receiving portion 41 to each of the flow paths 45A to 45D. In other words, the flow paths 45A to 45D may branch from the branch flow path 42 that is connected to the liquid receiving portion 41. The branch flow path 42 may be in communication with the liquid receiving portion 41 and each of the flow paths 45A to 45D.

[0063] The flow path substrate 4A may include a filter unit 420. The filter unit 420 may be a portion having a narrower flow path width than other portions. The filter unit 420 may be located on the opposite side of the reservoir unit 46 from the position of the reagent disposed in the flow path 45. That is, the filter unit 420 may be located upstream of the position of the reagent disposed in the flow path 45. When solid impurities are contained in the liquid flowing from the liquid receiving unit 41, the possibility of the impurities flowing downstream of the filter unit 420 can be reduced. Furthermore, the filter unit 420 may be located upstream of the branching positions of the flow paths 45A to 45D. As shown in FIG. 5, the branch flow path 42 may include the filter unit 420. This reduces the possibility of the impurities flowing into the flow paths 45A to 45D when solid impurities are contained in the liquid flowing from the liquid receiving unit 41.

[0064] However, flow path substrate 4A may not be provided with branch flow paths 42. In this case, flow paths 45A to 45D may branch from liquid receiving section 41. When there is only one flow path 45, flow path 45 may be connected to liquid receiving section 41. Furthermore, the filter section may be located in each of flow paths 45A to 45D on the opposite side of reservoir section 46 from the position of the reagent placed in flow path 45. Each of flow paths 45A to 45D may have a filter section upstream of the position where first reagent 101 is placed or the position where first reagent 101 may be placed.

[0065] 5, all of the flow paths 45A to 45D may branch off from one liquid receiving section 41 or from a branch flow path 42 connected to one liquid receiving section 41. However, the flow path substrate 4A may be provided with a plurality of liquid receiving sections 41. In this case, each flow path 45 may be connected to each liquid receiving section 41, or one or more flow paths 45 may be connected to each liquid receiving section 41.

[0066] The flow paths 45A to 45D may be flow paths for flowing a liquid. A mixed fluid containing a specimen and a labeling substance that reacts with the target substance contained in the specimen may be flowed through at least one of the flow paths 45A to 45D. Each of the flow paths 45A to 45D may be a flow path that receives a liquid flowing from the liquid receiving section 41 and flows the liquid to the reservoirs 46A to 46D connected to the flow paths 45A to 45D, respectively. Each of the flow paths 45A to 45D may be in communication from the liquid receiving section 41 to the reservoirs 46A to 46D.

[0067] In at least one of the flow paths 45A to 45D, the reagent may be disposed on the opposite side of the branching position of the flow paths 45A to 45D from the liquid-receiving section 41. In this way, when the liquid received in the liquid-receiving section 41 contains nucleic acid derived from the detection target, the nucleic acid derived from the detection target can be amplified in the flow path 45.

[0068] In this embodiment, one of the flow paths 45A to 45D may have a first region 451 in which a first reagent 101 is disposed, and a second region 452 in which a second reagent 102 is disposed at a position different from the first region 451.

[0069] The liquid received in liquid receiving section 41 flows through each of flow paths 45A to 45D and is stored in reservoirs 46A to 45D. In this embodiment, as shown in FIG. 5 , flow path 45B has a first region 451 and a second region 452. Therefore, the liquid flowing through flow path 45B comes into contact with first reagent 101 and second reagent 102. If the sample contains a detection target, nucleic acid derived from the detection target can be amplified by first reagent 101 and second reagent 102.

[0070] Therefore, nucleic acid derived from the detection target can be amplified by a simple operation of flowing a liquid through the flow channel 45 without using a special tool such as a pipette.

[0071] The first reagent 101 may contain, for example, a primer having a sequence complementary to the sequence of a nucleic acid molecule derived from the target nucleic acid molecule. The primer may be added as needed as long as it is capable of amplifying the target nucleic acid molecule, and may be, for example, one type or three or more types. The primers may be, for example, a first primer and a second primer. For example, the first primer may be a forward primer that amplifies the target nucleic acid molecule in the sense direction, and the second primer may be a reverse primer that amplifies the nucleic acid molecule in the antisense direction.

[0072] In addition, the first reagent 101 may contain, for example, deoxynucleoside triphosphates (dNTPs), nucleoside triphosphates (NTPs), trehalose, and an RNase inhibitor. However, if the RNase inhibitor is contained in the bottle portion 22, the first reagent 101 does not need to contain the RNase inhibitor.

[0073] As shown in FIG. 6 , the first reagent 101 may be arranged in the first region 451 as a plurality of first attachments. A dried solution containing the above-described reagent may be arranged in the first region 451 as an attachment of the first reagent 101. This allows the first reagent 101 to dissolve in the liquid when the liquid flowing into the flow path 45 comes into contact with the first attachment. Furthermore, when the first reagent 101 is arranged in the flow path 45 independently as a plurality of attachments, the surface area of ​​the first attachment can be increased. This increases the solubility of the first reagent 101 in the liquid. The first reagent 101 may be arranged in the first region 451 as a plurality of films. The number of first attachments may be a plurality, for example, five or more, or ten or more.

[0074] As shown in FIG. 6 , the multiple first attachments may be arranged in one direction. The one direction may be along the flow path 45. This allows the first reagent 101 to be dissolved in the liquid sequentially along the flow of the liquid. The multiple first attachments may be arranged in one direction along the flow path 45. In this embodiment, the multiple first attachments 101 are arranged in one row, but they may also be arranged in two or more rows. Furthermore, the multiple first attachments may be arranged on the bottom 454 of the flow path 45 so as not to come into contact with the inner wall 455 of the flow path 45. This makes it easier to dissolve the first reagent 101 in the liquid.

[0075] The first attachment may have a dot shape when viewed in a plan view, as shown in Fig. 6. Alternatively, the first attachment may have a linear shape, an elliptical shape, or a polygonal shape such as a square, a hexagon, or a star when viewed in a plan view.

[0076] 21 is a diagram showing the surface roughness of the first deposit and the surface roughness of the second deposit (second reagent 102). The diagram indicated by reference numeral 2101 in FIG. 21 is an enlarged photograph of the first deposit (first reagent 101), and the graph indicated by reference numeral 2102 shows the surface height of the flow path 45 at locations where the first deposit is attached and at locations where the first deposit is not attached. Locations where the surface height is 0.000 μm are the surface of the flow path 45, and locations where the surface height is greater than 0.000 μm are the surfaces where the first deposit is attached.

[0077] As shown in FIG. 21 , the surface roughness of the first deposit may be greater than the surface roughness of the flow path 45. This can disrupt the flow of liquid flowing near the first deposit and promote dissolution of the first reagent 101. An example of a surface roughness index is the arithmetic mean roughness Sa (ISO 25178). The surface roughness (Sa) of the first deposit may be set to, for example, 0.6 to 3 μm, or 1 to 3 μm. The surface roughness of the first deposit illustrated in the graph indicated by reference numeral 2102 was 1.55 μm. The surface roughness (Sa) of the flow path may be set to, for example, less than 0.5 μm or less than 0.3 μm. The surface roughness of the flow path 45 illustrated in the graph indicated by reference numeral 2102 was 0.19 μm.

[0078] The surface roughness Sa of the first attachment can be determined from the measurement results of the entire first attachment. That is, it can be determined from the measurement results of the entire surface roughness of multiple first attachments. It may also be determined from the measurement results of any part of any first attachment. When multiple measurement results of any part of any first attachment are obtained, the surface roughness of the first attachment may be determined by the arithmetic mean of the measurement results.

[0079] Furthermore, the surface roughness Sa of the flow path 45 can be determined from the measurement results of the entire flow path 45. In this case, the surface roughness Sa of the flow path 45 may be determined from the measurement results of the entire bottom of the flow path 45. Alternatively, the surface roughness Sa of the flow path 45 may be determined from the measurement results of an arbitrary portion of the flow path 45. When multiple measurement results of an arbitrary portion of the flow path 45 are obtained, the surface roughness of the flow path 45 may be determined by the arithmetic mean of the measurement results.

[0080] In addition, the first adhesion may have a portion where the surface roughness is locally smaller than the surface roughness of the flow path 45, and the flow path 45 may have a portion where the surface roughness is locally larger than the surface roughness of the first adhesion.

[0081] The second reagent 102 may be, for example, an enzyme such as AMV-RT (Avian Myeloblastosis Virus), RNase H (Ribonuclease H), or T7 RNA polymerase. In addition, the second reagent 102 may contain, for example, trehalose and a surfactant.

[0082] As shown in FIG. 6 , the second reagent 102 may be disposed in the second region 452 as a plurality of second attachments. A dried product of a solution containing the above-described reagent may be disposed in the second region 452 as an attachment of the second reagent 102. This allows the second reagent 102 to dissolve in the liquid when the liquid flowing into the flow path 45 comes into contact with the second attachment. When the second reagent 102 is disposed in the flow path 45 as a plurality of independent attachments, the surface area of ​​the second attachment can be increased. This increases the solubility of the second reagent 102 in the liquid. The second reagent 102 may be disposed in the second region 452 as a plurality of films. The number of second attachments may be any number, for example, five or more, or ten or more.

[0083] As shown in FIG. 6 , the plurality of second attachments may be arranged in one direction. This allows the second reagent 102 to be dissolved in the liquid sequentially along the flow of the liquid. The plurality of second attachments may be arranged in one direction along the flow path 45. In this embodiment, the second reagent 102 is arranged in one row as the plurality of second attachments, but may be arranged in two or more rows. Furthermore, the plurality of second attachments may be arranged on the bottom 454 of the flow path 45 so as not to come into contact with the inner wall 455 of the flow path 45. This makes it even easier to dissolve the second reagent 102 in the liquid.

[0084] The second attachment may have a dot shape when viewed in a plan view, as shown in Fig. 6. Alternatively, the second attachment may have a linear shape, an elliptical shape, or a polygonal shape such as a square, a hexagon, or a star shape when viewed in a plan view.

[0085] 21, the diagram designated by the reference numeral 2103 is an enlarged photograph of the second deposit, and the graph designated by the reference numeral 2104 shows the surface height of the flow path 45 at locations where the second deposit is attached and at locations where the second deposit is not attached. Locations where the surface height is 0.000 μm are the surfaces of the flow path 45, and locations where the surface height is greater than 0.000 μm are the surfaces where the second deposit is attached.

[0086] The surface roughness of the first deposit and the surface roughness of the second deposit may be different. This allows the dissolution rates of the first deposit and the second deposit to differ. For example, as shown in the graphs denoted by reference numerals 2102 and 2104, the surface roughness of the first deposit may be greater than the surface roughness of the second deposit. This can disrupt the flow of liquid flowing near the first deposit and promote dissolution of the first reagent 101. An example of an index of surface roughness is the arithmetic mean roughness Sa (ISO 25178). The surface roughness (Sa) of the second deposit may be set, for example, to 0.1 to 0.5 μm or 0.2 to 0.4 μm. The surface roughness of the second deposit shown in the graph denoted by reference numeral 2104 was 0.27 μm.

[0087] The surface roughness Sa of the second attachment can be determined from the measurement results of the entire second attachment. That is, it can be determined from the measurement results of the entire surface roughness of multiple second attachments. It may also be determined from the measurement results of any portion of any second attachment. When multiple measurement results of any portion of any second attachment are obtained, the surface roughness of the second attachment may be determined by the arithmetic mean of the measurement results.

[0088] Furthermore, the second deposit may have a portion where the surface roughness is locally greater than the surface roughness of the first deposit, and the first deposit may have a portion where the surface roughness is locally less than the surface roughness of the second deposit.

[0089] The surface roughness of the second deposit may be greater than the surface roughness of the flow path 45. In this case, the second deposit may have a portion where the surface roughness is locally smaller than the surface roughness of the flow path 45, and the flow path 45 may have a portion where the surface roughness is locally greater than the surface roughness of the second deposit.

[0090] 5, the second region 452 may be disposed on the opposite side of the liquid receiving section 41 from the position where the first region 451 is disposed. In other words, the second region 452 may be disposed downstream of the first region 451. Therefore, the liquid flowing through the flow path 45B can be brought into contact with the first reagent 101 and the second reagent 102 in that order.

[0091] 5 , the second region 452 may be disposed closer to the storage portion 46 than the first region 451. In other words, the first region 451, the second region 452, and the storage portion 46 may be disposed in this order along the flow path 45. Therefore, the liquid flowing through the flow path 45B can be brought into contact with the first reagent 101 and the second reagent 102 in this order, and then stored in the storage portion 46.

[0092] When the sample contains a target substance, the liquid containing nucleic acid derived from the target substance can be first brought into contact with the first reagent 101. This allows the nucleic acid derived from the target substance to anneal with the first primer and the second primer. Thereafter, the liquid flowing through the flow path 45B comes into contact with the enzyme contained in the second reagent 102, thereby amplifying the nucleic acid derived from the target substance.

[0093] It is sufficient that a first primer and a second primer are disposed in the first region 451 as the first reagent 101. Therefore, for example, dNTP and NTP may be disposed in the second region 452 as the second reagent 102. Even with such an arrangement, nucleic acid derived from the detection target contained in the liquid flowing through the flow channel 45 can be amplified.

[0094] 6 , the second attachment may be larger than the first attachment. For example, the first attachment and the second attachment may be disposed on the flow path 45 by applying a solution of the first reagent 101 and a solution of the second reagent 102 to the flow path 45. The second reagent 102 may contain a surfactant. Therefore, the solution of the second reagent 102 spreads more easily on the bottom surface of the flow path 45 than the solution of the first reagent 101.

[0095] The total area of ​​the plurality of first deposits can be determined by the amount of solution containing the first reagent 101. The total area of ​​the plurality of second deposits can be determined by the amount of solution containing the second reagent 102. For example, the total area of ​​the plurality of first deposits may be approximately twice the total area of ​​the plurality of second deposits.

[0096] The width of the flow path 45 may be, for example, 220 μm. In this case, if the first deposits are dot-shaped, their diameter may be smaller than the width of the flow path 45. The diameter of the first deposits may be, for example, 50 to 100 μm. The spacing between the first deposits may be 100 to 200 μm. If the second deposits are dot-shaped, their diameter may be smaller than the width of the flow path 45. The diameter of the second deposits may be, for example, 80 to 200 μm. The spacing between the second deposits may be 200 to 250 μm. Furthermore, as shown in the graphs 2102 and 2104 in FIG. 21 , the diameter of the second deposits may be larger than that of the first deposits. The heights shown in the graphs 2102 and 2104 can be considered to represent the heights of the first deposits and the second deposits, respectively. The diameter of the first deposit illustrated in the graph indicated by reference numeral 2102 was 14.193 μm, whereas the diameter of the second deposit illustrated in the graph indicated by reference numeral 2104 was 19.816 μm. In addition, the height of the second deposit may be greater than that of the first deposit.

[0097] For example, if the volume of the liquid containing the specimen stored in the bottle 22 is approximately 50 nL, approximately 48 nL of the first reagent 101 solution and approximately 24 nL of the second reagent 102 solution are required to amplify the nucleic acid contained in the liquid. In this case, if the application volume per attachment is approximately 0.7 nL, the number of first attachments will be 69 and the number of second attachments will be 34. These liquid and solution volumes are smaller than when using a PCR (Polymerase Chain Reaction) tube. In other words, by using the flow path substrate 4A, the volume of the liquid containing the specimen and the application volume (printing volume) of the first reagent 101 and the second reagent 102 applied to the flow path 45 can be reduced.

[0098] The amounts of the first reagent 101 and the second reagent 102 may vary depending on the detection target. Therefore, the amount of the solution of the first reagent 101, the number of first attachments, the amount of the solution of the second reagent 102, and the number of second attachments may be changed depending on the detection target. Furthermore, the size of the first attachments, the size of the second attachments, the spacing between the first attachments, and the spacing between the second attachments may be adjusted depending on the length of the flow channel 45 in the extension direction and the width of the flow channel 45.

[0099] As described above, nucleic acids contained in the liquid flowing from the liquid receiving section 41 may first react with the first reagent 101 disposed in the first region 451 and then react with the second reagent 102 disposed in the second region 452. This is because the first reagent 101 anneals to the nucleic acids contained in the liquid in order to amplify the nucleic acids contained in the liquid with the second reagent 102. Furthermore, as described above, if the cartridge 2 is once heated to a high temperature and then cooled to a temperature that promotes nucleic acid amplification, the temperature may be lowered before the nucleic acids that have reacted with the first reagent 101 react with the second reagent 102. The distance between the first region 451 and the second region 452 may be determined taking these points into consideration. Consider a case in which the heating section 31 heats the bottle section 22 to a temperature of 80°C to 95°C and the main body section 21 to a temperature of 37°C to 41°C. In this case, the second region 452 may be positioned so that the liquid that has flowed in from the bottle portion 22 via the liquid receiving portion 41 comes into contact with the second region 452 after the temperature of the liquid has sufficiently dropped.

[0100] The first region 451 and the second region 452 may be disposed in a linear portion of the flow path 45. In this case, the first reagent 101 and the second reagent 102 can be easily disposed in the flow path 45. When it is not possible to dispose the reagents in one linear portion of the flow path 45, like the first region 451 shown in FIG. 6 , the reagents may be disposed in two or more linear portions.

[0101] A labeling substance that specifically binds to the amplified nucleic acid may further be disposed in the flow path 45. If the labeling substance is a fluorescent substance, the labeling substance emits fluorescence when irradiated with excitation light. Therefore, the detection device 3 can measure the intensity of fluorescence emitted by the nucleic acid derived from the detection target by irradiating the storage section 46 with excitation light.

[0102] The labeling substance may be disposed downstream of the first region 451. For example, the labeling substance may be disposed in the second region 452 as the second reagent 102. This allows the labeling substance to efficiently bind to nucleic acid amplified by the enzyme contained in the second reagent 102. The labeling substance may be a molecular beacon.

[0103] The liquid flowing through one of the channels 45A to 45D may function as a negative control. Figure 5 shows an example in which the channel 45A functions as a channel for negative control.

[0104] The flow path 45A may not include the first region 451, but may include at least a labeling substance. In this embodiment, as shown in FIG. 5 , the flow path 45A may include at least a second region 452 including a labeling substance. This prevents the nucleic acid from reacting with a primer for amplifying the nucleic acid, even if the liquid flowing through the flow path 45A contains the nucleic acid. Therefore, the detection device 3 can detect the intensity of fluorescence emitted by substances other than the nucleic acid derived from the target nucleic acid by irradiating the liquid flowing through the flow path 45A and stored in the storage section 46A with excitation light. Therefore, the detection device 3 can accurately detect the intensity of fluorescence emitted by the nucleic acid derived from the target nucleic acid amplified by reaction with the first reagent 101 and the second reagent 102 in the flow path 45B, using the intensity of the fluorescence as a reference.

[0105] In this way, in the flow path substrate 4A, both the first reagent 101 and the second reagent 102 may not be arranged, and a flow path 45 that is not intended for detecting nucleic acids derived from the detection target may be connected to the branch flow path 42. In addition to the flow path 45A, the flow paths 45C and 45D may also be flow paths that are not intended for detecting nucleic acids derived from the detection target.

[0106] Each of the flow channels 45A to 45D may have a bending region 453A to 453D downstream of the flow channel 45. Each of the flow channels 45A to 45D may have a bending region 453A to 453D downstream of the position where the reagent is disposed or the position where the reagent can be disposed. This allows a liquid in which the reagent is dissolved to flow through the bending region 453. As this liquid flows through the bending region 453, the reagent and the liquid can be mixed. Therefore, the nucleic acid derived from the detection target can react with the reagent efficiently.

[0107] It is sufficient that the bent region 453 is formed in the flow channel 45 in which the first reagent 101 and the second reagent 102 are disposed. In this embodiment, it is sufficient that the bent region 453B is formed in at least the flow channel 45B.

[0108] The reservoir 46 may be connected to the flow path 45. As shown in Fig. 5, the reservoirs 46A to 46D may be connected to the flow paths 45A to 45D, respectively. The reservoir 46 may store the liquid that has flowed from the flow path 45. The reservoir 46 may be part of the flow path.

[0109] When the flow channel 45 has the first region 451 and the second region 452, the reservoir 46 may be located downstream of the second region 452 and may store a liquid containing amplified nucleic acid. In the present embodiment, the flow channel 45B has the first region 451 and the second region 452. Therefore, the reservoir 46B may be located downstream of the second region 452B and may store a liquid containing amplified nucleic acid.

[0110] The reservoir 46 can store the liquid flowing from the flow path 45. When the liquid contains nucleic acid derived from the detection target, the reservoir 46 can store the liquid containing the amplified nucleic acid, which is connected to the flow path 45 having the first region 451 and the second region 452. Therefore, the detection device 3 can stably image the amplified nucleic acid.

[0111] The width of the storage portion 46 may be larger than the width of the flow path 45. This can improve the measurement accuracy of the intensity of fluorescence emitted from the storage portion 46. The volume of the storage portion 46 may be a volume equivalent to the amount of liquid received by the liquid receiving portion 41. The volume of the storage portion 46 may be, for example, approximately 50 nL. In this way, the storage portion 46 may be formed on the flow path substrate 4A so that the volume of the storage portion 46 is relatively small.

[0112] 5, the first distance D1 between two adjacent reservoirs 46 among the reservoirs 46A to 46D may be greater than the second distance D2 between the two flow paths 45 connected to each of the reservoirs 46. This allows the spacing between the two adjacent reservoirs 46 to be relatively wide. Therefore, the detection device 3 can easily obtain the intensity of the fluorescence emitted from each reservoir 46 through image analysis.

[0113] A reagent may be placed in the reservoir 46. A reagent used for amplifying nucleic acid derived from the detection target may be placed in the reservoir 46. For example, either the first reagent 101 or the second reagent 102 may be placed in the reservoir 46. Also, for example, a portion of the first reagent 101 and / or the second reagent 102 may be placed in the reservoir 46.

[0114] When either the first reagent 101 or the second reagent 102 is placed in the reservoir 46, a first reagent 101 or a second reagent 102 different from the reagent placed in the reservoir 46 may be placed in a flow path 45 located upstream from the reservoir 46. For example, when the second reagent 102 is placed in the reservoir 46, the first reagent 101 may be placed in a flow path 45 located upstream from the reservoir 46. That is, the first region 451 may be located in the flow path 45 located upstream from the reservoir 46, and the second region 452 may be located in the reservoir 46. This allows the liquid flowing through the flow path 45B to come into contact with the first reagent 101 and then the second reagent 102.

[0115] (Other Configurations) Additionally, the flow path substrate 4A may have second storage portions 48 downstream of the storage portions 46A to 46D, each connected to one of the storage portions 46A to 46D. In other words, the second storage portion 48 may be disposed at a position farther away from the liquid receiving portion 41 than the storage portion 46. In this case, when a volume of liquid equal to or greater than the capacity of the storage portion 46 flows into the storage portion 46, the liquid leaking from the storage portion 46 can be stored in the second storage portion 48. This reduces the possibility of the liquid flowing back from the storage portion 46 into the flow path 45.

[0116] The second reservoir 48 may also contain a substance that functions as a positive control. For example, a substance that functions as a positive control may be placed only in the second reservoir 48 of the flow path substrate 4A used as a positive control. The substance may be optically compared with the substance in the reservoir 46, and may be, but is not limited to, a dye, a luminescent substance, or a fluorescent substance. To verify whether the detection system 1 is functioning properly, a sample intentionally containing the target substance may be passed through the liquid receiving section 41 into the flow path 45 before measuring the optical information of the target sample, and the optical information corresponding to the nucleic acid derived from the target sample may be measured in the second reservoir 48. As shown by reference numeral 1102 in FIG. 2 , the second reservoir 48 can be viewed through the window 211 of the main body 21.

[0117] Furthermore, in the flow path substrate 4A, a sensing substance that detects the passage of liquid flowing through the flow path 45 may be located at a position farther from the liquid receiving section 41 than the storage section 46 in at least one of the branched flow paths 45. The flow path 45 may be a space that branches at the branch flow path 42 and then communicates with the second storage section 48 via the storage section 46. For example, the position farther from the liquid receiving section 41 than the storage section 46 refers to the position where the liquid received by the liquid receiving section 41 reaches after flowing through the flow path 45 and passing through the storage section 46. The sensing substance may also be a fluorescent substance that is soluble in the liquid flowing through the flow path 45. In this configuration, when the liquid flowing through the flow path 45 passes the position where the sensing substance is located, the sensing substance dissolves. Therefore, the brightness of the sensing substance decreases. The progress of the liquid can be confirmed by confirming the decrease in brightness of the sensing substance. In other words, whether the liquid flowing through the flow path 45 is normal can be determined based on whether the brightness of the sensing substance decreases. The detection substance is not particularly limited as long as it can confirm the passage of the liquid flowing through the flow channel 45 .

[0118] The second reservoir 48 may be connected to at least one of the reservoirs 46 A to 46 D. However, the flow path substrate 4 A may not necessarily have the second reservoir 48 .

[0119] Furthermore, the flow path substrate 4A may have an outlet 49 connected to the flow path 45 downstream of the flow path 45. The outlet 49 may be connected to the storage portion 46 downstream of the storage portion 46. As shown in FIG. 5 , when the flow path substrate 4A has a second storage portion 48, the outlet 49 may be connected to the second storage portion 48 downstream of the second storage portion 48. The outlet 49 is a vent that discharges air or liquid in a space located between the liquid receiving portion 41 and the outlet 49 to the outside of the flow path substrate 4A. The space located between the liquid receiving portion 41 and the outlet 49, i.e., the space located within the flow path substrate 4A including the flow path 45, may be referred to as a flow path.

[0120] (Position of Reagents) In the present embodiment, when the first reagent 101 and the second reagent 102 are placed in the flow path 45, it is sufficient that at least a primer and an enzyme are placed in the flow path 45. Other reagents may be placed at positions other than the flow path 45. For example, dNTP, NTP, RNase inhibitor, trehalose, and surfactant may be placed upstream of the flow path 45. For example, these reagents may be placed at branching positions of the flow paths 45A to 45D. For example, these reagents may be placed in the liquid receiving section 41 and / or the branch flow path 42. Furthermore, for example, a standard substance may be placed downstream of the flow path 45. For example, the standard substance may be placed in the reservoir 46.

[0121] (Number of Flow Channels, etc.) In this embodiment, the flow channel substrate 4A has four flow channels 45, but this is not limited thereto. In this embodiment, the flow channel substrate 4A only needs to have at least one flow channel 45 having a first region 451 and a second region 452. In this embodiment, when the flow channel substrate 4A has a plurality of flow channels 45 each having a first region 451 and a second region 452, these flow channels 45 may function as flow channels used to detect the same target substance. That is, the first reagent 101 disposed in the plurality of flow channels 45 may be the same reagent, and the second reagent 102 disposed in the plurality of flow channels 45 may be the same reagent. Furthermore, when the flow channel substrate 4A has a plurality of flow channels 45 each having a first region 451 and a second region 452, these flow channels 45 may function as flow channels used to detect different target substances. That is, the first reagent 101 disposed in the plurality of flow channels 45 may be different reagents, and the second reagent 102 disposed in the plurality of flow channels 45 may be different reagents.

[0122] The flow path substrate 4A may include at least one flow path 45 for a positive control. That is, the flow path 45 may include a flow path 45 for flowing a liquid intentionally containing a detection target. The flow path 45 for the positive control may have a first region 451 and a second region 452 arranged from the upstream side. A liquid intentionally containing a detection target that reacts with the first reagent 101 and the second reagent may be flowed through the flow path 45 for the positive control. However, the flow path 45 having the first region 451 and the second region 452 and used for flowing a liquid containing a specimen may be substituted for the flow path 45 for the positive control.

[0123] The number of reservoirs 46 may be determined depending on the number of flow paths 45 to be connected.

[0124] (Position of Internal Standard) In the present embodiment, the internal standard 47 is arranged on the flow path substrate 4A, but this is not limiting. The internal standard 47 may be arranged on the surface of the housing of the cartridge 2, rather than on the flow path substrate 4A. In this case, the internal standard 47 may be arranged near the window 211 of the main body 21. The internal standard 47 may be located in a region different from the branch flow path 42, the flow path 45, and the storage portion 46.

[0125] [Method of Manufacturing Flow Channel Substrate According to First Embodiment] Fig. 7 is a flowchart showing an example of a method of manufacturing the flow channel substrate 4A. This manufacturing method may be performed by a manufacturing apparatus that manufactures the flow channel substrate 4A.

[0126] 7 , first, a liquid receiving section 41 and a flow path 45 may be formed on a substrate (S1; forming step). In this embodiment, in addition to the liquid receiving section 41 and the flow path 45, a branch flow path 42, a storage section 46, a second storage section 48, and an outlet 49 may be formed on the substrate.

[0127] The liquid receiving portion 41, the flow path 45, etc. may be formed, for example, as follows. For example, a template having the shapes of the liquid receiving portion 41, the flow path 45, etc. patterned thereon is placed on a substrate, and then a resin is poured into the substrate. After the resin has hardened, the template is removed. After the template is removed, a lid is placed on the hardened resin, thereby forming the liquid receiving portion 41, the flow path 45, etc. on the substrate.

[0128] Next, a reagent may be placed in the flow path 45. In this embodiment, a first reagent 101 may be placed in the flow path 45B (S2; first placing step). The first reagent 101 may be placed in the flow path 45B by applying the first reagent 101 to the flow path 45B. Furthermore, in this embodiment, a second reagent 102 may be placed in the flow paths 45A and 45B (S3; second placing step). The second reagent 102 may be placed in the flow paths 45A and 45B by applying the second reagent 102 to the flow paths 45A and 45B.

[0129] Next, an internal standard 47 may be placed on the substrate (S4). The internal standard 47 may be placed on the substrate by applying a fluorescent substance as the internal standard 47 to the substrate. Next, the fluorescent substance may be applied to the second reservoir 48, thereby placing the fluorescent substance in the second reservoir 48 (S5). The order of the processes from S2 to S5 does not matter. The processes from S2 to S5 may be performed in parallel.

[0130] Fig. 8 is a schematic diagram showing an example of a method for applying the reagent 100 to the flow path 45 provided in the flow path substrate 4A. Reference numeral 1131 in Fig. 9 is a schematic plan view showing an example of the flow path 45 to which the reagent 100 has been applied, and reference numeral 1132 in Fig. 9 is a schematic diagram showing another example of a method for applying the reagent 100 to the flow path 45. The reagent 100 in Figs. 8 and 9 may be the first reagent 101 or the second reagent 102.

[0131] The application of the reagent 100 may be performed using, for example, an inkjet printer. Use of an inkjet printer enables fine application of the reagent 100. The inkjet printer may include a head 201 that ejects the reagent 100. As shown by reference numeral 1121 in FIG. 8 , the inkjet printer may eject the reagent 100 from the head 201, thereby adhering the reagent 100 to the flow path 45 as shown by reference numeral 1122 in FIG. 8 . The reagent 100 that has adhered to the flow path 45 may be allowed to dry naturally.

[0132] The inkjet printer may apply the reagent 100 while moving the head 201 along the extension direction of the flow path 45, thereby adhering multiple reagents 100 to the bottom 454 of the flow path 45, as shown by reference numeral 1131 in Figure 9.

[0133] The inkjet printer may control the movement of the head 201 and the ejection of the reagent 100 so that the multiple deposits are disposed independently (separately) on the bottom 454. The inkjet printer may also control the movement of the head 201 and the ejection of the reagent 100 so that the deposits do not come into contact with the inner wall 455 of the flow channel 45.

[0134] As indicated by reference numeral 1131 in Fig. 9 , the inkjet printer may control the movement of the head 201 and the ejection of the reagent 100 so that a plurality of deposits are arranged independently side by side in a direction different from the extension direction of the flow channel 45. The plurality of deposits may be arranged side by side, for example, in the width direction of the flow channel 45. Furthermore, as indicated by reference numeral 1132 in Fig. 9 , new reagent 100 may be applied on top of the dried reagent 100 arranged in the flow channel 45.

[0135] [Second Form of Flow Channel Substrate] Fig. 10 is a plan view showing an example of a flow channel substrate 4B. Fig. 11 is a schematic diagram showing an example of the arrangement of detection reagents in the flow channels 45 provided in the flow channel substrate 4B. The flow channel substrate 4B is an example of the flow channel substrate 4.

[0136] As shown in FIG. 10 , the flow path substrate 4B may include a plurality of flow paths 45A-45D through which liquid flows, and a plurality of reservoirs 46A-46D connected to the plurality of flow paths 45, respectively. The plurality of flow paths 45 may include a first flow path in which at least a portion of a first detection reagent, which is a detection reagent for detecting a detection target, is located, and a second flow path in which at least a portion of a second detection reagent, which is a detection reagent for detecting a detection target different from the first detection reagent, is located. The detection reagent may be an example of a reagent that reacts with the detection target. The detection target may be, for example, a nucleic acid derived from the detection target. This allows multiple different detection targets to be detected at once by the simple operation of introducing liquid into the flow path substrate 4B.

[0137] 10 , the flow path substrate 4B may include a liquid receiving section 41, a branch flow path 42, a plurality of flow paths 45A to 45D, a plurality of storage sections 46A to 46D, and an internal standard 47. The flow path substrate 4B may include a second storage section 48 and an outlet 49.

[0138] The flow paths 45A to 45D may branch from a branch flow path 42 connected to the liquid receiving section 41. However, the flow path substrate 4B does not have to be provided with a branch flow path 42. In this case, the flow paths 45A to 45D may branch from the liquid receiving section 41. By having the flow paths 45A to 45D branch from the liquid receiving section 41 or the branch flow path 42, the liquid received in the liquid receiving section 41 can be introduced into the plurality of flow paths 45A to 45D. Therefore, it is possible to detect a plurality of different detection targets at once without needing to introduce a liquid into each of the plurality of flow paths 45A to 45D.

[0139] Furthermore, all of the flow paths 45A to 45D may branch off from a single liquid receiving section 41 or from a branch flow path 42 connected to a single liquid receiving section 41. This allows the liquid received in a single liquid receiving section 41 to be introduced into multiple flow paths 45A to 45D. Therefore, multiple different detection targets can be detected at the same time by introducing the liquid once.

[0140] Furthermore, the detection reagents may be disposed on the opposite side of the liquid receiving section 41 from the branching positions of the flow paths 45A to 45D, thereby reducing the possibility of different detection reagents mixing together, for example, reducing the possibility of the first detection reagent and the second detection reagent mixing together.

[0141] The flow path substrate 4B differs from the flow path substrate 4A in that the multiple flow paths 45 have a first flow path and a second flow path. Hereinafter, only the differences between the flow path substrate 4B and the flow path substrate 4A will be described. In other words, the same content as that described for the flow path substrate 4A will not be described in this embodiment.

[0142] In the present embodiment, the flow path substrate 4B may include a flow path 45B as the first flow path and a flow path 45C as the second flow path. As shown in Fig. 10 , a first region 451B and a second region 452B may be arranged in the flow path 45B, and a first region 451C and a second region 452C may be arranged in the flow path 45C.

[0143] 11 , a first reagent 1011 may be placed in the first region 451B as a first detection reagent. A first reagent 1012 different from the first reagent 1011 may be placed in the first region 451C as a second detection reagent. The first reagents 1011 and 1012 are examples of the first reagent 101. The first reagent 1011 may be the second detection reagent, and the first reagent 1012 may be the first detection reagent.

[0144] At least a portion of the detection reagent may be located in the flow path 45, or all of the detection reagent may be located therein. For example, a portion of the detection reagent may be located in the flow path 45, and the remainder of the detection reagent may be located in the reservoir 46. Here, "at least a portion of the detection reagent" may refer to some of the types of reagents when the detection reagent contains multiple types of reagents. For example, the first reagents 1011 and 1012 may be located in the flow path 45, and the second reagents 1021 and 1022 may be located in the reservoir 46. Furthermore, substantially the same components of the detection reagent may be located both in the flow path 45 and at a position other than the flow path 45. For example, substantially the same components of the detection reagent may be located in the flow path 45 and the reservoir 46.

[0145] The first reagents 1011 and 1012 may be primers having a sequence complementary to the sequence of a nucleic acid molecule derived from the detection target. The primers may be added as needed as long as they are capable of amplifying the target nucleic acid molecule, and may be, for example, one type or three or more types. In this embodiment, the first reagents 1011 and 1012 may be, for example, a first primer and a second primer. By arranging primers as detection reagents on the flow path substrate 4B, when the liquid received in the liquid receiving section 41 contains nucleic acid derived from the detection target, the nucleic acid derived from the detection target can be amplified in the flow path 45.

[0146] However, the first reagent 1012 may be a primer having a different sequence from that of the first reagent 1011. The first reagent 1012 may be a primer having a sequence complementary to the sequence of a nucleic acid molecule derived from a detection target different from that of the first reagent 1011. For example, the first reagent 1011 may be a primer having a sequence complementary to the sequence of a nucleic acid molecule derived from a first detection target, and the first reagent 1012 may be a primer having a sequence complementary to the sequence of a nucleic acid molecule derived from a second detection target different from the first detection target. This allows a second detection target different from the first detection target that reacts with the first reagent 1011 to react with the first reagent 1012. The first detection target and the second detection target are examples of detection targets.

[0147] Therefore, when the liquid flowing from liquid-receiving unit 41 into flow path 45B contains a first target substance, the first target substance can be amplified in flow path 45B. When the liquid flowing from liquid-receiving unit 41 into flow path 45C contains a second target substance, the second target substance can be amplified in flow path 45C. In other words, the liquid received in liquid-receiving unit 41 can be introduced into multiple flow paths 45, and multiple different targets can be used as target substances for nucleic acid amplification at the same time.

[0148] The first reagents 1011 and 1012 may contain, for example, dNTPs, NTPs, trehalose, and an RNase inhibitor. However, if the RNase inhibitor is contained in the bottle portion 22, the first reagents 1011 and 1012 may not contain the RNase inhibitor.

[0149] 11 , a second reagent 1021 may be disposed as the first detection reagent in the second region 452B. A second reagent 1022 different from the second reagent 1021 may be disposed as the second detection reagent in the second region 452C. The second reagents 1021 and 1022 are examples of the second reagent 102. The second reagent 1021 may be the second detection reagent, and the second reagent 1022 may be the first detection reagent.

[0150] The second reagents 1021 and 1022 may be, for example, enzymes such as AMV-RT (Avian Myeloblastosis Virus), RNase H (Ribonuclease H), and T7 RNA polymerase. However, the second reagent 1022 may be an enzyme with a different sequence from that of the second reagent 1021.

[0151] This allows a second detection target, which is different from the first detection target that reacts with second reagent 1021, to react with second reagent 1022. Therefore, when the liquid flowing from liquid receiving unit 41 into flow path 45B contains the first detection target, the first detection target can be amplified in flow path 45B. When the liquid flowing from liquid receiving unit 41 into flow path 45C contains the second detection target, the second detection target can be amplified in flow path 45C.

[0152] The second reagents 1021 and 1022 may contain, for example, trehalose and a surfactant.

[0153] A labeling substance may further be disposed in the flow channel 45 as a detection reagent. When the substances to be detected in each flow channel 45 are different from each other, the labeling substance may have a sequence corresponding to the different detection targets. In this embodiment, the labeling substance disposed in flow channel 45B and the labeling substance disposed in flow channel 45C may be different from each other. This allows the labeling substance to bind to the detection target in accordance with the type of detection target. In this embodiment, the labeling substance may be disposed in the second regions 452B and 452C.

[0154] Furthermore, the flow path substrate 4B may include a flow path 45 that does not function as the first flow path or the second flow path. That is, in the flow path substrate 4B, a detection reagent may not be disposed, and a flow path 45 that is not intended for detecting a detection target substance may be connected to the branch flow path 42.

[0155] The flow path substrate 4B may include a negative control flow path 45 in which at least a labeling substance is disposed, but at least one element used in nucleic acid amplification, such as an enzyme for amplifying nucleic acids, is not disposed. In FIG. 10 , the flow path 45A may be a negative control flow path in which at least a labeling substance is disposed. The flow path 45A may have a second region 452A in which a second reagent 102 containing a labeling substance is disposed. The flow path substrate 4B may also include a flow path 45 in which no detection reagent is disposed. In FIG. 10 , the flow path 45D may be a flow path in which no detection reagent is disposed.

[0156] When the types of substances to be detected in each reservoir 46 are different, it is sufficient that at least the sequences of the primers and labeling substances in the detection reagents placed in each of the flow paths 45 connected to these reservoirs 46 are different from each other. In other words, some of the detection reagents placed in each of the flow paths 45 may be the same reagent. By using the same reagent for different detection targets, the flow path substrate 4B can be manufactured more inexpensively. For example, the enzymes placed in each of the flow paths 45 may be the same reagent. Furthermore, the dNTPs and NTPs placed in each of the flow paths 45 may also be the same reagent. Furthermore, the RNase inhibitor, trehalose, and surfactant may also be the same reagent.

[0157] The same reagent may be located on the opposite side of the liquid-receiving section 41 in the multiple flow paths 45 in which different detection reagents are located. That is, the same reagent may be located downstream of the position in the flow path 45 where the different detection reagents are located. For example, as described above, in flow paths 45B and 45C, an enzyme as the same reagent may be located in second regions 452B and 452C located downstream of first regions 451B and 451C in which primers are located. Furthermore, in flow paths 45B and 45C, dNTP and NTP as the same reagent may also be located in second regions 452B and 452C, for example. However, dNTP, NTP, RNase inhibitor, trehalose, and surfactant as the same reagent may be located in the liquid-receiving section 41 and / or branch flow path 42.

[0158] Furthermore, in each of the multiple flow paths 45, the length of the multiple flow paths 45 from the end opposite the storage section 46 of the detection reagent along the liquid feed direction to the end on the storage section 46 side may be approximately the same. That is, in each flow path 45, the length of the region in which the detection reagent is disposed along the extension direction of the flow path 45 may be approximately the same. This makes it possible to make the time it takes for the liquid to pass through the region in which the detection reagent is disposed approximately the same in each flow path 45. Therefore, it is possible to make the reaction time between the detection target and the detection reagent approximately the same in each flow path 45.

[0159] As shown in FIG. 11 , in this embodiment, the lengths LB and LC of the flow paths 45B and 45C may be substantially the same. The length LB is the length from the end of the first region 451B opposite the reservoir 46B to the reservoir 46B side of the second region 452B. The length LC is the length from the end of the first region 451C opposite the reservoir 46C to the reservoir 46C side of the second region 452C. The length L1B of the first region 451B in which the first reagent 1011 is disposed and the length L1C of the first region 451C in which the first reagent 1012 is disposed may be substantially the same or different. Furthermore, in the flow paths 45B and 45C, the length L2B of the second region 452B in which the second reagent 1021 is disposed and the length L2C of the second region 452C in which the second reagent 1022 is disposed may be substantially the same or different.

[0160] FIG. 12 is a schematic diagram illustrating an example of use of multiple flow channels 45. As shown in FIG. 12, for example, flow channel 45B may have a first region 451B in which a first reagent 1011 is disposed and a second region 452B in which a second reagent 1021 is disposed. Flow channel 45C may have a first region 451C in which a first reagent 1012 is disposed and a second region 452C in which a second reagent 1022 is disposed. Flow channel 45D may have a first region 451D in which a first reagent 1013 is disposed and a second region 452D in which a second reagent 1023 is disposed. The first reagents 1011, 1012, and 1013 may be different detection reagents. The second reagents 1021, 1022, and 1023 may be different detection reagents. Furthermore, the channel 45A may be a channel for negative control in which at least a labeling substance is disposed, but at least one of the elements used for nucleic acid amplification, such as an enzyme for amplifying nucleic acid, is not disposed.

[0161] In this way, different detection reagents may be placed in each of the flow paths 45A to 45D. However, the same detection reagent, including the primer and the labeling substance, may be placed in each of some of the multiple flow paths 45. For example, the first reagent 1012 and the second reagent 1022 placed in the flow path 45C may be placed in the flow path 45D instead of the first reagent 1013 and the second reagent 1023.

[0162] That is, the flow path substrate 4B only needs to include at least one first flow path in which a first detection reagent is disposed and at least one second flow path in which a second detection reagent is disposed, as the flow paths 45. This makes it possible to detect multiple substances by the simple operation of introducing liquid into the multiple flow paths 45.

[0163] In this embodiment, liquid introduced from liquid receiving section 41 is branched by branch flow path 42, flows through each of flow paths 45A to 45D, and is stored in each of storage sections 46A to 46D. If the liquid contains a first detection target, the first detection target reacts with the first detection reagent as the liquid flows through the first flow path in which the first detection reagent is disposed. If the liquid contains a second detection target, the second detection target reacts with the second detection reagent as the liquid flows through the second flow path in which the second detection reagent is disposed.

[0164] Therefore, the detection device 3 can detect a first detection target by measuring the intensity of fluorescence emitted from the reservoir 46 connected to the first flow path. The detection device 3 can detect a second detection target by measuring the intensity of fluorescence emitted from the reservoir 46 connected to the second flow path. In other words, by arranging detection reagents capable of detecting different detection targets in different flow paths 45, the detection device 3 can detect multiple different detection targets at once with the simple operation of introducing liquid into the flow path substrate 4B.

[0165] The flow path substrate 4B may include at least two or more flow paths 45 that function as a first flow path and a second flow path. As shown in FIG. 12 , the flow path substrate 4B may include a flow path 45X having a first region 451X in which a first reagent 1014 is disposed and a second region 452X in which a second reagent 1024 is disposed. The first reagent 1014 may be different from the first reagents 1011, 1012, and 1013. The second reagent 1024 may be different from the second reagents 1021, 1022, and 1023. Therefore, the detection device 3 can simultaneously detect a plurality of different detection targets, the number of which corresponds to the number of flow paths 45 in which different detection reagents are disposed.

[0166] The first reagents 1011 to 1014 do not all have to be different from one another. Any two of the first reagents 1011 to 1014 may be detection reagents that are different from one another. That is, some of the first reagents 1011 to 1014 may be first detection reagents, and another part may be second detection reagents. Furthermore, the second reagents 1021 to 1024 do not all have to be different from one another. Any two of the second reagents 1021 to 1024 may be detection reagents that are different from one another. That is, some of the second reagents 1021 to 1024 may be first detection reagents, and another part may be second detection reagents.

[0167] The flow path substrate 4B may include at least one positive control flow path 45. A detection reagent that reacts with a liquid that has been intentionally made to contain a detection target may be placed in the positive control flow path 45. However, the flow path 45 that functions as the first flow path and / or the second flow path used to flow a liquid containing a specimen may be substituted for the positive control flow path 45. In this case, a liquid that has been intentionally made to contain a detection target that reacts with the detection reagent placed in the flow path 45 may be flowed in the flow path 45.

[0168] 13 is a flowchart showing an example of a method for manufacturing the flow path substrate 4B. This manufacturing method may be performed by a manufacturing apparatus for manufacturing the flow path substrate 4B.

[0169] 13 , first, a flow path 45 and a storage portion 46 may be formed on a substrate (S11). In this embodiment, in addition to the flow path 45 and the storage portion 46, a liquid receiving portion 41, a branch flow path 42, a second storage portion 48, and an outlet 49 may be formed on the substrate.

[0170] Next, a detection reagent may be placed in the flow path 45. In this embodiment, a first detection reagent may be placed as a detection reagent in a first flow path, which is any one of the flow paths 45A to 45D (S12). A second detection reagent different from the first detection reagent may be placed in a second flow path, among the flow paths 45A to 45D, different from the first flow path in which the first detection reagent is placed (S13). Next, a fluorescent substance may be placed on the substrate as an internal standard 47 (S14). Next, a fluorescent substance may be placed in the second reservoir 48 (S15). The order of the processes of S12 to S15 does not matter. The processes of S12 to S15 may be performed in parallel.

[0171] [Summary] The flow path substrate according to aspect 1 of the present disclosure comprises a liquid receiving section that receives a liquid and a flow path connected to the liquid receiving section, and the flow path has a first region in which a first reagent containing a primer is disposed, and a second region in which a second reagent containing an enzyme that amplifies nucleic acid is disposed at a position different from the first region.

[0172] A flow path substrate according to a second aspect of the present disclosure is the same as that of the first aspect, wherein the second region is disposed on the opposite side of the liquid receiving section relative to the position where the first region is disposed.

[0173] A flow path substrate according to a third aspect of the present disclosure is the flow path substrate of the first or second aspect, wherein the flow path further includes a reservoir downstream of the second region for storing a liquid containing amplified nucleic acid.

[0174] A flow path substrate according to Aspect 4 of the present disclosure is any one of Aspects 1 to 3, wherein the first reagent is disposed in the first region as a plurality of first attachments. A flow path substrate according to Aspect 5 of the present disclosure is the flow path substrate according to Aspect 4, wherein the surface roughness of the first attachments is greater than the surface roughness of the flow path.

[0175] A flow path substrate according to a sixth aspect of the present disclosure is the same as that of the fourth or fifth aspect, wherein the plurality of first attachments are arranged as a film.

[0176] A flow path substrate according to Aspect 7 of the present disclosure is any one of Aspects 4 to 6, wherein the plurality of first attachments are aligned in one direction.

[0177] A flow path substrate according to aspect 8 of the present disclosure is any one of aspects 4 to 7, wherein the plurality of first attachments are arranged at the bottom of the flow path so as not to contact the inner wall of the flow path.

[0178] A flow path substrate according to Aspect 9 of the present disclosure is any one of Aspects 1 to 8, wherein the second reagent is disposed in the second region as a plurality of second attachments.

[0179] A flow path substrate according to aspect 10 of the present disclosure is the same as aspect 9, wherein the surface roughness of the second deposit is greater than the surface roughness of the flow path.

[0180] A flow path substrate according to an eleventh aspect of the present disclosure is the same as that of the ninth or tenth aspect, wherein the second attachment is arranged as a film.

[0181] A flow path substrate according to Aspect 12 of the present disclosure is any one of Aspects 9 to 11, wherein the plurality of second attachments are aligned in one direction.

[0182] A flow path substrate according to aspect 13 of the present disclosure is any one of aspects 9 to 12, wherein the plurality of second attachments are arranged at the bottom of the flow path so as not to contact the inner wall of the flow path.

[0183] A flow path substrate according to aspect 14 of the present disclosure is any one of aspects 4 to 8, in which the second reagent is arranged in the second region as a plurality of second attachments, and the second attachments are larger than the first attachments.

[0184] A flow path substrate according to aspect 15 of the present disclosure is a flow path substrate as described in aspect 9, wherein in aspect 9, the first reagent is arranged in the first region as a plurality of first attachments, and the surface roughness of the second attachments is smaller than the surface roughness of the first attachments.

[0185] A flow channel substrate according to Aspect 16 of the present disclosure is any one of Aspects 1 to 13, wherein a labeling substance that specifically binds to the amplified nucleic acid is further disposed in the flow channel.

[0186] A flow path substrate according to Aspect 17 of the present disclosure is the same as Aspect 16, wherein the labeling substance is disposed downstream of the first region.

[0187] A flow path substrate according to Aspect 18 of the present disclosure is the same as Aspect 16 or 17, wherein the second reagent includes the labeling substance.

[0188] A nineteenth aspect of the present disclosure provides the flow path substrate of any one of the sixteenth to eighteenth aspects, wherein the labeling substance is a molecular beacon.

[0189] A cartridge according to Aspect 20 of the present disclosure includes the flow path substrate according to any one of Aspects 1 to 19 and a container capable of containing a liquid.

[0190] A detection system according to aspect 21 of the present disclosure includes the cartridge according to aspect 20 and a detection device that detects nucleic acid amplified in the flow channel.

[0191] A method for manufacturing a flow path substrate according to aspect 22 of the present disclosure includes a forming step of forming a liquid receiving portion for receiving a liquid and a flow path connected to the liquid receiving portion on a substrate, a first disposing step of disposing a first reagent containing a primer in the flow path, and a second disposing step of disposing a second reagent containing an enzyme for amplifying nucleic acid at a position on the flow path different from the position where the first reagent is disposed.

[0192] In a method for manufacturing a flow path substrate according to aspect 23 of the present disclosure, in aspect 22, in the first placement step, the first reagent is placed in the flow path by applying a solution containing the first reagent to the flow path, and in the second placement step, the second reagent is placed in the flow path by applying a solution containing the second reagent to the flow path.

[0193] A flow path substrate according to aspect 24 of the present disclosure is any one of aspects 1 to 19, wherein the flow paths are a plurality of flow paths, and further includes a plurality of reservoirs connected to each of the plurality of flow paths, and the plurality of flow paths include a first flow path in which at least a portion of a first detection reagent, which is a detection reagent for detecting a detection target, is located, and a second flow path in which at least a portion of a second detection reagent, which is a detection reagent for detecting a detection target different from the first detection reagent, is located.

[0194] A flow path substrate according to aspect 25 of the present disclosure is any of aspects 1 to 19, wherein the flow path is a flow path through which a mixed fluid containing a sample and a labeled substance that reacts with a target substance contained in the sample flows, and further includes a standard section located in a region different from the flow path and for comparison with the mixed fluid.

[0195] A flow path substrate according to aspect 26 of the present disclosure is, in aspect 1, a flow path that is a plurality of flow paths through which a liquid flows, and that includes a plurality of reservoirs connected to the plurality of flow paths, and the plurality of flow paths includes a first flow path in which at least a portion of a first detection reagent, which is a detection reagent that detects a detection target, is located, and a second flow path in which at least a portion of a second detection reagent, which is a detection reagent that detects a detection target different from the first detection reagent, is located.

[0196] A flow path substrate according to Aspect 27 of the present disclosure is the flow path substrate of Aspect 26, wherein the plurality of flow paths branch off from the liquid receiving section or a flow path connected to the liquid receiving section.

[0197] A flow path substrate according to Aspect 28 of the present disclosure is the same as Aspect 27, wherein all of the plurality of flow paths branch off from one of the liquid receiving sections or from a flow path connected to the one liquid receiving section.

[0198] A flow path substrate according to Aspect 29 of the present disclosure is the flow path substrate of Aspect 27 or 28, wherein the detection reagent is disposed on the opposite side of the liquid receiving section with respect to a branching position of the plurality of flow paths.

[0199] A flow path substrate according to Aspect 30 of the present disclosure is any one of Aspects 26 to 29, wherein the detection reagent includes a primer having a sequence corresponding to the detection target.

[0200] A flow path substrate according to Aspect 31 of the present disclosure is based on Aspect 30, wherein the second detection reagent includes the primer having a different sequence from that of the first detection reagent.

[0201] A flow path substrate according to Aspect 32 of the present disclosure is any one of Aspects 26 to 31, wherein the same reagent is arranged in the plurality of flow paths.

[0202] A flow path substrate according to Aspect 33 of the present disclosure is the flow path substrate of Aspect 32, wherein the same reagent includes an enzyme that amplifies nucleic acid.

[0203] The flow path substrate of aspect 34 of the present disclosure, in aspect 32 or 33, further comprises a liquid receiving portion for receiving a liquid, and the same reagent is located on the opposite side of the liquid receiving portion in the flow path in which the detection reagent is arranged.

[0204] In the flow path substrate of aspect 35 of the present disclosure, in any of aspects 26 to 34, the lengths of the multiple flow paths from the end of the detection reagent opposite the storage section along the liquid flow direction to the end on the storage section side are approximately the same for each of the multiple flow paths.

[0205] The flow path substrate according to aspect 36 of the present disclosure, in any of aspects 26 to 35, further comprises a filter section in which the flow path width is narrower than other sections, and the filter section is located on the opposite side of the storage section from the position of the detection reagent.

[0206] A flow path substrate according to Aspect 37 of the present disclosure is the same as Aspect 36, wherein the filter section is disposed upstream of a branching position of the plurality of flow paths.

[0207] A flow path substrate according to aspect 38 of the present disclosure is any of aspects 26 to 36, wherein a first distance between two adjacent reservoirs among the plurality of reservoirs is greater than a second distance between two flow paths connected to each of the two reservoirs.

[0208] A flow path substrate according to Aspect 39 of the present disclosure is any one of Aspects 26 to 38, wherein each of the plurality of flow paths has a plurality of bending regions downstream of the detection reagent.

[0209] <Regarding Other Embodiments> Conventional flow path substrates have room for improvement in fluid control. According to the present disclosure, it is possible to improve the fluid control in a flow path substrate.

[0210] Other embodiments according to the present disclosure will be described below with reference to the drawings. In this specification, unless otherwise specified, "A to B" representing a numerical range means "greater than or equal to A and less than or equal to B." In this specification, unless otherwise specified, "A / B" means "the value obtained by dividing A by B." In this specification, "fluid" includes not only liquids but also liquids containing solids and gases. For convenience of explanation, in Figures 15, 16, 17, 18, 19, and 20, the positive direction of the Y axis will be described as the upward direction, the negative direction of the Y axis will be described as the downward direction, the positive direction of the X axis will be described as the rightward direction, and the negative direction of the X axis will be described as the leftward direction.

[0211] Third Embodiment A flow path substrate 1A according to a third embodiment will be described below with appropriate reference to the drawings. The flow path substrate 1A is a substrate used for detecting or quantifying a target substance contained in a sample. A flow path 10 is formed inside the flow path substrate 1A. When a sample is introduced into the flow path substrate 1A, the sample and a reagent are mixed inside the flow path substrate 1A. The sample mixed with the reagent is temporarily stored in a detection unit 13. For example, "storage" refers to the sample remaining in a certain region relative to the flow in the flow path 10, but is not limited to this.

[0212] For example, the specimen may include a substance derived from a living organism. For example, the specimen may include a substance derived from a mammal, such as a human, dog, cat, or cow. For example, the specimen may include a substance excreted from a living organism or a substance extracted from a living organism. For example, the specimen may include urine, blood, sweat, saliva, or nasal secretions. For example, the target substance may be a virus, a bacterium, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a protein.

[0213] For example, the reagent may include a substrate that reacts with the target substance or a nucleic acid or enzyme contained in the target substance. For example, the reagent may include a substrate that reacts with the target substance or a nucleic acid or enzyme contained in the target substance and emits fluorescence. For example, the reagent may include probe DNA, probe RNA, or the like designed to detect the target substance or a nucleic acid contained in the target substance. For example, the reagent may include primer DNA, an enzyme, or the like for amplifying the target substance or a nucleic acid contained in the target substance.

[0214] The configuration of the flow path substrate 1A will be described with reference to Figures 14, 15, and 16. As shown in Figure 14, the flow path substrate 1A has a rectangular parallelepiped shape. For example, the flow path substrate 1A may have a curved surface, a spherical surface, a concave surface, or a convex surface in part. For example, the flow path substrate 1A may be located in part or in whole inside a housing made of resin, metal, or the like. For example, the flow path substrate 1A may be made of a single member. For example, the flow path substrate 1A may be made by combining two or more members.

[0215] As shown in FIG. 15 , the flow path substrate 1A has a flow path 10 therein. The flow path substrate 1A has an inlet hole 11, a reagent dissolving section 12, a detection section 13, an outlet hole 14, and a branching section 15A. The inlet hole 11, the reagent dissolving section 12, the detection section 13, the outlet hole 14, and the branching section 15A are part of the flow path 10. The inlet hole 11 can introduce a sample into the flow path substrate 1A. The reagent dissolving section 12 is where a reagent is located and can dissolve the reagent in the sample. The detection section 13 can store the sample mixed with the reagent. The flow path substrate 1A has multiple detection sections 13. The outlet hole 14 can discharge the sample introduced from the inlet hole 11, which is introduced after the detection section 13 is filled, to the outside of the flow path substrate 1A. The flow path substrate 1A has multiple outlet holes 14. The branching section 15A branches the flow path 10 into multiple paths.

[0216] An overview of detecting a target substance contained in a sample using the flow path substrate 1A will be described. First, the sample is introduced into the flow path through the inlet 11. The sample moving through the flow path 10 is distributed to multiple branched flow paths at the branching section 15A. The sample moving through each flow path is mixed with a reagent in the reagent dissolving section 12. The sample mixed with the reagent in this manner is stored in the detection section 13. The target substance is detected by observing the sample stored in the detection section 13. For example, the reagent may contain a substrate that reacts with the target substance and emits fluorescence. In this case, the sample may be observed by irradiating the detection section 13 with excitation light. When observing the sample by irradiating the detection section 13 with excitation light, the target substance may be detected based on the fluorescence emitted from the substrate in the sample.

[0217] The flow path substrate 1A further includes a filter section 16 located between the introduction hole 11 and the branch section 15A. The flow path substrate 1A includes the filter section 16, which allows it to separate solids or bubbles contained in the sample. As a result, it is possible to reduce the risk that solids or bubbles contained in the sample will interfere with the detection of the target substance in the detection section 13.

[0218] The flow path substrate 1A has a first surface 17 which is the front surface of the flow path substrate 1A, and a second surface 18 which faces the first surface 17. For example, the second surface 18 is the back surface of the flow path substrate 1A relative to the first surface 17. The top surface of the flow path 10 is the surface of the flow path 10 which is located on the first surface 17 side. The bottom surface of the flow path 10 is the surface of the flow path 10 which is located on the second surface 18 side. The side surface of the flow path 10 is the surface of the flow path 10 which is inclined with respect to the top surface and the bottom surface.

[0219] For example, the material of the flow path substrate 1A may be resin, glass, ceramic, metal, or the like. For example, the material of the flow path substrate 1A may be cyclic olefin polymer, cyclic olefin copolymer, acrylic resin, unstretched polypropylene, or the like. The flow path substrate 1A may be made of a plurality of materials. For example, the flow path substrate 1A may be formed by injection molding, resin cutting, photolithography, or the like. The cross section perpendicular to the first surface 17 of the flow path 10 has a rectangular shape. For example, the cross section perpendicular to the first surface 17 of the flow path 10 may have a shape such as an approximately circular shape, an elliptical shape, a trapezoidal shape, or a triangular shape.

[0220] (Introduction hole 11) The introduction hole 11 can introduce a specimen into the inside of the flow path substrate 1A. The introduction hole 11 can introduce a specimen into the flow path 10. The introduction hole 11 connects the outside of the flow path substrate 1A to the flow path 10. The introduction hole 11 is one end of the flow path 10. The introduction hole 11 opens to one of the surfaces of the flow path substrate 1A. The introduction hole 11 opens to a first surface 17 of the flow path substrate 1A.

[0221] The shape of the introduction hole 11 may be selected arbitrarily. For example, the space located inside the introduction hole 11 has a truncated cone shape. For example, the space located inside the introduction hole 11 may have a triangular pyramid shape, a square pyramid shape, a cylindrical shape, a square prism shape, or the like. The introduction hole 11 has a shape in which the cross-sectional area of ​​the introduction hole 11 decreases from the first surface 17 toward the second surface 18 opposite the first surface 17. For example, the opening of the introduction hole 11 is substantially circular. For example, the opening of the introduction hole 11 may be elliptical, rectangular, hexagonal, octagonal, or the like.

[0222] (Reagent dissolving unit 12) The reagent dissolving unit 12 can dissolve a reagent in a specimen. The reagent dissolving unit 12 can dissolve a reagent in a specimen flowing through a flow path. In the reagent dissolving unit 12, the reagent is located on the bottom surface of the flow path. In the reagent dissolving unit 12, the reagent may be located on the side surface of the flow path. The reagent dissolving unit 12 is connected to flow path D155 of branching unit 15A on the side opposite to flow path C154.

[0223] The shape of the reagent dissolving section 12 may be selected arbitrarily. For example, the reagent dissolving section 12 has a linear shape or a curved shape. The curved shape of the reagent dissolving section 12 may be an arc shape or an L-shape. For example, the reagent dissolving section 12 of this embodiment has a shape in which arc shapes and linear shapes are alternately repeated. In order to dissolve the reagent in the sample in the reagent dissolving section 12, the reagent dissolving section 12 does not have to have a curved shape. For example, the width of the flow path of the reagent dissolving section 12 is constant. For example, the width of the flow path of the reagent dissolving section 12 may vary. For example, the width of the flow path of the reagent dissolving section 12 may be wider only in a portion.

[0224] (Detection Unit 13) The detection unit 13 can store a specimen. The detection unit 13 can store a specimen mixed with a reagent. For example, when a target substance is detected by light using the flow path substrate 1A, the surface of the detection unit 13 on the first surface 17 side and the surface of the detection unit 13 on the second surface 18 side may be light-transmitting. The detection unit 13 is connected to the flow path D155 of the branching unit 15A on the side opposite to the flow path C154.

[0225] (Outlet hole 14) The outlet hole 14 can lead the specimen from the inside of the flow path substrate 1A to the outside of the flow path substrate 1A. The outlet hole 14 can lead the specimen from the flow path 10 to the outside of the flow path substrate 1A. The outlet hole 14 is one end of the flow path of the flow path substrate 1A. The outlet hole 14 opens to one of the surfaces of the flow path substrate 1A. The outlet hole 14 opens to a first surface 17 of the flow path substrate 1A.

[0226] The shape of the lead-out hole 14 may be selected arbitrarily. For example, the space located inside the lead-out hole 14 has a truncated cone shape. For example, the space located inside the lead-out hole 14 may have a triangular pyramid shape, a square pyramid shape, a cylindrical shape, a square prism shape, or the like. The lead-out hole 14 has a shape in which the cross-sectional area of ​​the lead-out hole 14 decreases from the first surface 17 toward the second surface 18 opposite the first surface 17. The opening of the lead-out hole 14 has a substantially circular shape. For example, the opening of the lead-out hole 14 may have a square shape, a hexagonal shape, an octagonal shape, or the like.

[0227] (Branch Section 15A) The branch section 15A branches the flow path of the flow path substrate 1A into multiple paths. For example, the branch section 15A branches the flow path of the flow path substrate 1A into four paths. The number of branches is not limited to this and can be set to any number. The branch section 15A branches the flow path of the flow path substrate 1A to the left and right. "Branching the flow path to the left and right" refers to, but is not limited to, the flow path extending from the branch section 15A having a first flow path extending vertically, a second flow path extending to the right of the first flow path, and a third flow path extending to the left of the first flow path. For example, "branching the flow path to the left and right" also includes the case where the flow path extending from the branch section 15A has a first flow path extending horizontally, a second flow path extending above the first flow path, and a third flow path extending below the first flow path.

[0228] The branching section 15A may be located between the introduction hole 11 and the detection section 13. In this embodiment, the branching section 15A is located between the introduction hole 11 and the reagent dissolving section 12. For example, the branching section 15A may be located between the reagent dissolving section 12 and the detection section 13. By having the branching section 15A, the flow path substrate 1A can distribute the same sample to multiple parts within the flow path 10. Therefore, detection of a target substance under multiple conditions can be easily performed.

[0229] 16 , the branching portion 15A has a flow path A151, a flow path B152, and a first branching wall 153. The branching portion 15A has a plurality of flow paths B152. In this embodiment, the branching portion 15A has two flow paths B152. The number of flow paths B152 included in the branching portion 15A is not limited to this and can be set to any number.

[0230] In the branching portion 15A, the width of each of the multiple flow paths B152 is narrower than the width of the flow path A151. For example, the ratio of the width of the flow path A151 to the width of the flow path B152 (width of flow path B152 / width of flow path A151) may be 0.800 or less. As long as the width of the flow path B152 is narrower than the width of the flow path A151, the width and depth of the flow path A151 and the width and depth of the flow path B152 may be set to any values. For example, the width of the flow path A151 may be 200 μm to 1000 μm. For example, the depth of the flow path A151 may be 25 μm to 200 μm. For example, the width of the flow path B152 may be 20.0 μm to 500 μm. For example, the depth of the flow path B152 may be 25.0 μm to 200 μm.

[0231] Flow path A151 is a flow path connected to the liquid receiving section 41. Flow path A151 can pass a specimen introduced from the introduction hole 11. Flow path A151 is connected to the filter section 16. Flow path A151 can pass a specimen that has flowed out from the filter section 16. The width of flow path A151 is constant. The width of flow path A151 may vary. Here, in this specification, "thickness" refers to the cross-sectional area perpendicular to the extension direction of the flow path. "Extension direction of the flow path" refers to the direction in which the spaces formed by the flow path are continuously located. For example, "extension direction of the flow path" refers to the direction in which the fluid flows when the fluid is made to flow through the flow path, but is not limited to this.

[0232] The multiple flow paths B152 are flow paths of the multiple flow paths. The flow path B152 is a flow path branched from the flow path A151. The flow path B152 can pass the sample flowing out from the flow path A151. The flow path B152 can pass a portion of the sample flowing out from the flow path A151. The width of the flow path B152 is constant. The width of the flow path B152 may vary.

[0233] The multiple flow paths B152 are flow paths branched off from the flow path A151 in a direction that does not overlap with a first virtual straight line that overlaps with the flow path A151. Here, the "first virtual straight line that overlaps with the flow path A151" refers to a straight line that extends from a point located within the flow path A151 in the extension direction of the flow path A151. The multiple flow paths B152 are flow paths branched off to the left and right from the flow path A151.

[0234] The thicknesses of the multiple flow paths B152 may be the same or different. The sum of the thicknesses of the multiple flow paths B152 is smaller than the thickness of the flow path A151. For example, the ratio of the thickness of the flow path A151 to the sum of the thicknesses of the flow paths B152 (sum of the thicknesses of the flow paths B152 / thickness of the flow path A151) may be 0.900 or less. The sum of the thicknesses of the multiple flow paths B152 is larger than the thickness of the flow path C154. For example, the ratio of the thickness of the flow path C154 to the sum of the thicknesses of the flow paths B152 (sum of the thicknesses of the flow paths B152 / thickness of the flow path A151) may be 1.50 or more.

[0235] The first branch wall 153 is located in a region where the multiple flow paths B152 branch off. The first branch wall 153 is located between two flow paths B152. The first branch wall 153 is connected to the flow path B152. In this embodiment, the first branch wall 153 is continuous with the side surface of the flow path B152. In this embodiment, the first branch wall 153 is a surface of the side surface of the flow path 10 that is continuous with the side surface of the flow path B152 and is located upstream of the inlet of the flow path B152. The first branch wall 153 is not continuous with the side surface of the flow path A151.

[0236] The first branch wall 153 overlaps with a first virtual line that overlaps with the flow path A151. In this embodiment, the length of the first branch wall 153 is greater than the width of the flow path A151. For example, the length of the first branch wall 153 may be smaller than the width of the flow path A151. The first branch wall 153 has a curved surface or a flat surface. The first branch wall 153 of this embodiment has a flat surface and a curved surface. The first branch wall 153 of this embodiment has a curved surface located between two flat surfaces. For example, the first branch wall 153 may have only two flat surfaces. For example, the first branch wall 153 may have only one curved surface.

[0237] The branching portion 15A further includes a flow path C154. The branching portion 15A includes a plurality of flow paths C154. In the present embodiment, the branching portion 15A includes two flow paths C154. The branching portion 15A includes the same number of flow paths C154 as the plurality of flow paths B152. The number of flow paths C154 included in the branching portion 15A may be different from the number of flow paths B152 included in the branching portion 15A.

[0238] The flow path C154 is connected to the flow path B152. Each of the multiple flow paths C154 is connected to each of the multiple flow paths B152. Each of the multiple flow paths C154 may be connected to only a portion of the multiple flow paths B152. The flow path C154 is a flow path extending from the flow path B152. The flow path C154 can pass the sample that has flowed out from the flow path B152. Each of the multiple flow paths C154 can pass the sample that has flowed out from each of the multiple flow paths B152.

[0239] The width of the flow path C154 may be thicker or thinner than the width of the flow path A151. In this embodiment, the width of the flow path C154 is thinner than the width of the flow path A151. In this embodiment, the width of each of the multiple flow paths C154 is thinner than the width of the flow path A151. For example, the ratio of the width of the flow path A151 to the width of the flow path C154 (width of flow path C154 / width of flow path A151) may be 0.600 or less. In this embodiment, the sum of the widths of the multiple flow paths C154 is smaller than the width of the flow path A151. For example, the ratio of the width of the flow path A151 to the sum of the widths of the flow paths C154 (sum of the widths of flow paths C154 / width of flow path A151) may be 0.950 or less.

[0240] The width of the flow path C154 may be thicker or thinner than the width of the flow path B152. In this embodiment, the width of the flow path C154 is thicker than the width of the flow path B152. In this embodiment, the width of each of the multiple flow paths C154 is thicker than the width of each of the multiple flow paths B152. For example, the ratio of the width of the flow path B152 to the width of the flow path C154 (width of the flow path C154 / width of the flow path B152) may be 1.05 or more.

[0241] For example, the width of the flow path C154 may be 20.0 μm to 500 μm. For example, the depth of the flow path C154 may be 25.0 μm to 200 μm. In this embodiment, the width of the flow path C154 is constant. The thickness of the flow path C154 may vary. The thickness of the multiple flow paths C154 may be the same or different. In this embodiment, the thickness of the multiple flow paths C154 is the same.

[0242] Branching section 15A further includes flow path D155. Flow path D155 is a flow path branched from flow path C154. Flow path D155 can pass the specimen flowing out from flow path C154. Flow path D155 can pass a portion of the specimen flowing out from flow path C154.

[0243] The branching section 15A has a plurality of flow paths D155. In the present embodiment, the branching section 15A has four flow paths D155. The number of flow paths D155 included in the branching section 15A is not limited to four and can be set to any number. The plurality of flow paths D155 are flow paths branched from the flow path C154. The plurality of flow paths D155 are flow paths branched from the flow path C154 in a direction that does not overlap with a second imaginary line that overlaps with the flow path C154. Here, the "second imaginary line that overlaps with the flow path C154" refers to a line that extends from a point located within the flow path C154 in the extension direction of the flow path C154. The plurality of flow paths D155 are flow paths branched to the left and right from the flow path C154.

[0244] The width of the flow path D155 may be thicker or thinner than the width of the flow path A151. In this embodiment, the width of the flow path D155 is thinner than the width of the flow path A151. In this embodiment, the width of each of the multiple flow paths D155 is thinner than the width of the flow path A151. For example, the ratio of the width of the flow path A151 to the width of the flow path D155 (width of the flow path D155 / width of the flow path A151) may be 0.800 or less.

[0245] The width of flow path D155 may be thicker or thinner than the width of flow path B152. In this embodiment, the width of flow path D155 is thinner than the width of flow path B152. In this embodiment, the width of each of the multiple flow paths D155 is thinner than the width of each of the multiple flow paths B152. For example, the ratio of the width of flow path A151 to the width of flow path D155 (width of flow path D155 / width of flow path A151) may be 0.980 or less. For example, the width of flow path D155 may be the same as that of flow path B152.

[0246] The width of the flow path D155 may be thicker or thinner than the width of the flow path C154. In this embodiment, the width of the flow path D155 is thinner than the width of the flow path C154. In this embodiment, the width of each of the multiple flow paths D155 is thinner than the width of the flow path C154. For example, the ratio of the width of the flow path C154 to the width of the flow path D155 (width of the flow path D155 / width of the flow path C154) may be 0.950 or less.

[0247] For example, the width of the flow path D155 may be 20.0 μm to 500 μm. For example, the depth of the flow path D155 may be 25.0 μm to 200 μm. In this embodiment, the width of the flow path D155 is constant. The width of the flow path D155 may vary. The thickness of the multiple flow paths D155 may be the same or different. In this embodiment, the thickness of the multiple flow paths D155 is the same.

[0248] In the branching portion 15A, the sum of the lengths of the plurality of flow paths B152 and the plurality of flow paths C154 is smaller than the sum of the lengths of the flow paths A151. In the branching portion 15A, the sum of the lengths of the plurality of flow paths B152 and the plurality of flow paths C154 is smaller than the sum of the lengths of the plurality of flow paths D155 and the length of the reagent dissolving portion 12.

[0249] In the branching section 15A of this embodiment, the angle formed by two flow paths B152 is defined as a first angle. The first angle may be, for example, 90.0 degrees or greater. In the branching section 15A of this embodiment, the angles formed by adjacent flow paths D155 among the four flow paths D155 are defined as a second angle and a third angle. The second angle and the third angle may be, for example, 90.0 degrees or less. For example, the first angle is greater than the second angle and the third angle. For example, the sum of the second angle and the third angle is greater than the first angle. The second angle and the third angle may be the same angle or different angles.

[0250] (Filter section 16) The filter section 16 can separate solids or air bubbles contained in the sample. The filter section 16 is located between the introduction hole 11 and the branching section 15A. For example, the filter section 16 may be located between the branching section 15A and the reagent dissolving section 12. For example, the filter section 16 has the thickest region of the flow path 10.

[0251] (Flow of Sample in Branching Section 15A) The flow of sample in branching section 15A will now be described. In flow path substrate 1A, the multiple flow paths B152 branch off from flow path A151 in a direction that does not overlap with the first virtual line. Therefore, there is little risk that the majority of the sample flowing out of flow path A151 will flow directly into flow path B152. Furthermore, in flow path substrate 1A, first branching wall 153 overlaps with the first virtual line. Therefore, most of the sample flowing out of flow path A151 will collide with first branching wall 153 and then flow into each of the multiple flow paths B152. As a result, compared to when the first branching wall does not overlap with the first virtual line or when there is no first branching wall, there is less risk that the sample will flow into only one of the multiple flow paths B152.

[0252] Furthermore, in the flow path substrate 1A, the width of each of the multiple flow paths B152 is thinner than the width of the flow path A151. Therefore, resistance is generated when the sample flowing out of the flow path A151 flows into one of the flow paths B152. As a result, compared to when the width of each of the multiple flow paths B is thicker than the width of the flow path A, the risk of the sample flowing into only one of the multiple flow paths B152 can be reduced.

[0253] In the flow path substrate 1A of this embodiment, the multiple flow paths B152 branch off from the flow path A151 in a direction that does not overlap with the first virtual line. Furthermore, in the flow path substrate 1A of this embodiment, the first branch wall 153 overlaps with the first virtual line. Furthermore, in the flow path substrate 1A of this embodiment, the width of each of the multiple flow paths B152 is thinner than the width of the flow path A151. As a result, it is possible to reduce the risk that the sample will not flow in one of the multiple flow paths B152 and will flow only in the other flow paths B152.

[0254] Fourth Embodiment A flow path substrate 2A according to a fourth embodiment will now be described. The flow path substrate 2A differs from the flow path substrate 1A in the configuration of the branching section 25A. As shown in FIG. 17 , the widths of the multiple flow paths B252 are different. Of the two flow paths B252, one is thinner than the other. The widths of the multiple flow paths D255 are different. Of the four flow paths D255, the widths of the two flow paths D255 connected to one flow path B252 are thinner than the two flow paths D255 connected to the other flow path B252. Of the four flow paths D255, the widths of the two flow paths D255 connected to a flow path B252 that is thinner than the one flow path B252 are thinner than the two flow paths D255 connected to the other flow path B252. The flow path substrate 2A of this embodiment also facilitates the flow of specimens into all of the flow paths B252.

[0255] Fifth Embodiment A flow path substrate 3A according to the fifth embodiment will now be described. The flow path substrate 3A is different from the flow path substrate 1A in the configuration of the branching section 35A. As shown in FIG. 18 , the branching section 35A does not have configurations corresponding to the flow path D155 and the second branching wall 157 in the flow path substrate 1A. The branching section 35A has four flow paths C354. Of the four flow paths C354, two flow paths C354 are connected to one flow path B352A. The flow path substrate 3A of this embodiment can also facilitate the flow of specimens into all of the flow paths B352A.

[0256] Sixth Embodiment A flow path substrate 4A according to the sixth embodiment will now be described. The flow path substrate 4A is different from the flow path substrate 1A in the configuration of the branching section 45E. As shown in FIG. 19 , the branching section 45E does not have a configuration equivalent to the flow path C154 in the flow path substrate 1A. The branching section 45E has a plurality of flow paths D455E branching from a plurality of flow paths B452E. In the branching section 45E, the flow path D455E branches from the flow path B452E. The width of each of the plurality of flow paths D455E is thinner than the width of each of the plurality of flow paths B452E. The flow path substrate 4A of this embodiment can also facilitate the flow of analytes into all of the flow paths B452E. Furthermore, the flow path substrate 4A of this embodiment can also facilitate the flow of analytes into all of the flow paths D455E.

[0257] Seventh Embodiment A flow path substrate 5A according to the seventh embodiment will now be described. The flow path substrate 5A is different from the flow path substrate 1A in the configuration of the branching section 55A. As shown in FIG. 20 , in the branching section 55A, multiple flow paths D555 branch off from only one flow path C554. Two flow paths D555 branch off from only one flow path C554. Multiple flow paths D555 are connected to only one flow path C554. The length of one flow path C554 is longer than the length of the other flow path C554. The flow path substrate 5A of this embodiment can also make it easier for the sample to flow into all of the flow paths B552.

[0258] The invention according to the present disclosure has been described above based on the drawings and examples. However, the invention according to the present disclosure is not limited to the above-described embodiments. In other words, the invention according to the present disclosure can be modified in various ways within the scope of the present disclosure, and embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the invention according to the present disclosure. In other words, it should be noted that a person skilled in the art could easily make various modifications or corrections based on the present disclosure. It should also be noted that these modifications or corrections are included in the scope of the present disclosure.

[0259] DESCRIPTION OF SYMBOLS 1 Detection system 2 Cartridge 3 Detection device 4, 4A, 4B Flow path substrate 22 Bottle section (container) 41 Receiving section 42 Branch flow path (flow path) 45 Flow path (first flow path, second flow path) 46 Storage section 47 Internal standard (standard section) 101, 1011 to 1014 First reagent (detection reagent, first detection reagent, second detection reagent) 102, 1021 to 1024 Second reagent (detection reagent, first detection reagent, second detection reagent) 420 Filter section 451 First region 452 Second region 453 Bending region 454 Bottom 455 Inner wall D1 First distance D2 Second distance 1A, 2A, 3A, 4A, 5A Flow path substrate 10 Flow path 11 Inlet hole 12 Reagent dissolving section 13 Detection section 14 Outlet hole 15A Branching portion 151 Flow path A 152, 252, 352A, 452E, 552 Flow path B 153 First branching wall 154, 354 Flow path C 155, 255, 455E, 555 Flow path D 157 Second branching wall 16 Filter portion

Claims

1. A liquid receiving section that accepts liquid, The system includes a flow path connected to the liquid receiving section, The channel substrate has a first region where a first reagent containing a primer is arranged, and a second region where a second reagent containing an enzyme for nucleic acid amplification is arranged at a position different from the first region.

2. The flow path substrate according to claim 1, wherein the second region is located on the opposite side from the liquid receiving portion with respect to the position where the first region is located.

3. The channel substrate according to claim 1 or 2, wherein the channel further comprises a storage section downstream of the second region for storing a liquid containing amplified nucleic acids.

4. The flow channel substrate according to claim 1 or 2, wherein the first reagent is arranged in the first region as a plurality of first deposits.

5. The channel substrate according to claim 4, wherein the surface roughness of the first deposit is greater than that of the channel surface roughness.

6. The flow channel substrate according to claim 4, wherein the plurality of first deposits are arranged as a coating.

7. The flow channel substrate according to claim 4, wherein the plurality of first deposits are arranged in one direction.

8. The flow channel substrate according to claim 4, wherein the plurality of first deposits are arranged at the bottom of the flow channel so as not to come into contact with the inner wall of the flow channel.

9. The flow channel substrate according to claim 1, wherein the second reagent is arranged in the second region as a plurality of second deposits.

10. The channel substrate according to claim 9, wherein the surface roughness of the second deposit is greater than that of the channel surface roughness.

11. The flow channel substrate according to claim 9, wherein the second deposit is arranged as a film.

12. The flow channel substrate according to claim 9, wherein the plurality of second deposits are arranged in one direction.

13. The flow channel substrate according to claim 9, wherein the plurality of second deposits are arranged at the bottom of the flow channel so as not to come into contact with the inner wall of the flow channel.

14. The second reagent is arranged in the second region as a plurality of second deposits. The flow channel substrate according to claim 4, wherein the second deposit is larger than the first deposit.

15. The first reagent is arranged in the first region as a plurality of first deposits. The channel substrate according to claim 9, wherein the surface roughness of the second deposit is smaller than that of the first deposit.

16. The channel substrate according to claim 1, wherein a labeling substance that specifically binds to amplified nucleic acids is further disposed in the channel.

17. The flow channel substrate according to claim 16, wherein the labeled material is located downstream of the first region.

18. The flow channel substrate according to claim 16, wherein the second reagent includes the labeling substance.

19. The channel substrate according to claim 16, wherein the labeling substance is a molecular beacon.

20. The flow channel substrate according to claim 1, A cartridge comprising a container capable of holding liquid.

21. The cartridge according to claim 20, A detection system comprising a detection device for detecting nucleic acids amplified in the aforementioned channel.

22. A forming step of forming a liquid receiving section and a flow channel connected to the liquid receiving section on a substrate, A first arrangement step involves placing a first reagent containing a primer in the aforementioned flow path, A method for manufacturing a channel substrate, comprising: a second arrangement step of arranging a second reagent containing an enzyme for nucleic acid amplification at a position in the channel different from the position where the first reagent is arranged.

23. In the first placement step, the first reagent is placed in the flow channel by applying a solution containing the first reagent to the flow channel. The method for manufacturing a channel substrate according to claim 22, wherein in the second placement step, the second reagent is placed in the channel by applying a solution containing the second reagent to the channel.

24. The aforementioned flow path consists of multiple flow paths, The system further comprises a plurality of storage units connected to each of the plurality of flow paths, The aforementioned plurality of channels are A first channel in which at least a portion of the first detection reagent, which is a detection reagent for detecting the target object, is located, The channel substrate according to claim 1, comprising: a second channel in which at least a portion of a second detection reagent, which is a detection reagent for detecting a target object different from the first detection reagent, is located.

25. The aforementioned channel is a channel through which a mixed fluid containing a sample and a labeling substance that reacts with the target to be detected contained in the sample flows. The flow channel substrate according to claim 1, further comprising a standard section located in a region different from the flow channel and for comparison with the mixed fluid.

26. The aforementioned flow path is a plurality of flow paths through which liquid flows, It comprises a plurality of storage units connected to the plurality of flow paths, The aforementioned plurality of channels are A first channel in which at least a portion of the first detection reagent, which is a detection reagent for detecting the target object, is located, The channel substrate according to claim 1, comprising: a second channel in which at least a portion of a second detection reagent, which is a detection reagent for detecting a target object different from the first detection reagent, is located.

27. The flow path substrate according to claim 26, wherein the plurality of flow paths branch off from the liquid receiving section or a flow path connected to the liquid receiving section.

28. The flow path substrate according to claim 27, wherein all of the plurality of flow paths branch off from one of the liquid receiving units, or a flow path connected to one of the liquid receiving units.

29. The flow channel substrate according to claim 27, wherein the detection reagent is located on the opposite side of the liquid receiving portion with respect to the branching position of the plurality of flow channels.

30. The flow channel substrate according to claim 26, wherein the detection reagent includes a primer having an arrangement corresponding to the object to be detected.

31. The channel substrate according to claim 30, wherein the second detection reagent includes the primer having a different sequence from the first detection reagent.

32. The channel substrate according to claim 26, wherein the same reagent is arranged in the plurality of channels.

33. The channel substrate according to claim 32, wherein the same reagent includes an enzyme that amplifies nucleic acids.

34. The channel substrate according to claim 32, wherein the same reagent is located in the channel on the opposite side from the liquid receiving section in the channel where the detection reagent is arranged.

35. The flow path substrate according to claim 26, wherein in each of the plurality of flow paths, the lengths of the plurality of flow paths from the end opposite to the storage section to the end on the storage section side of the detection reagent along the direction of liquid delivery are substantially the same.

36. It further includes a filter section, which has a narrower flow path width compared to other parts. The flow channel substrate according to claim 26, wherein the filter portion is located on the opposite side of the location of the detection reagent from the storage portion.

37. The flow path substrate according to claim 36, wherein the filter section is located upstream of the branching points of the plurality of flow paths.

38. The flow channel substrate according to claim 26, wherein the first distance between two adjacent storage sections among the plurality of storage sections is greater than the second distance between two flow channels connected to each of the two storage sections.

39. The channel substrate according to claim 26, wherein each of the plurality of channels has a plurality of bent regions downstream of the detection reagent.

40. Flow path A and Multiple flow paths B branch off from flow path A in a direction that does not overlap with the first virtual straight line that overlaps with flow path A, Located in a region where the plurality of flow paths B branch off, and having a first branch wall continuous with the flow paths B, Each channel B has a diameter that is narrower than the diameter of channel A, and the first branch wall coincides with the first virtual straight line. The aforementioned flow path A is a flow path connected to the liquid receiving section, The flow path substrate according to claim 27, wherein the plurality of flow paths B are the plurality of flow paths.

41. The channel substrate according to claim 40, wherein the sum of the diameters of the plurality of channels B is smaller than the diameter of the channel A.

42. The system further has a plurality of flow paths C located on the opposite side of the flow path A from the plurality of flow paths B, and extending from each of the plurality of flow paths B. The channel substrate according to claim 40, wherein each channel C has a diameter greater than the diameter of channel B.

43. The channel substrate according to claim 42, wherein the sum of the diameters of the plurality of channels B is greater than the diameter of each of the channels C.

44. The channel substrate according to claim 42, wherein the sum of the diameters of the plurality of channels C is smaller than the diameter of the channel A.

45. The system further has a plurality of channels D that branch off from the channel C in a direction that does not overlap with the second virtual straight line that overlaps with the channel C, The channel substrate according to claim 42, wherein each channel D has a diameter narrower than the diameter of the channel C, and a second branch wall located in the region where the plurality of channels D branch coincides with the second virtual straight line.

46. The channel substrate according to claim 45, wherein the diameter of each channel D is narrower than the diameter of each channel B.

47. The channel substrate according to claim 45, further comprising a reagent dissolving section located on the opposite side of the channel C from the plurality of channels D, extending from each of the plurality of channels D, and containing a reagent to be dissolved in a fluid.

48. It further has multiple detection units capable of storing fluid, The flow path substrate according to claim 45, wherein each of the plurality of detection units communicates with respect to the flow path D on the side opposite to the flow path C.

49. The channel substrate according to claim 47, wherein the sum of the lengths of channel B and channel C is smaller than the sum of the lengths of channel A and the sum of the lengths of channel D and the reagent dissolution section.

50. The aforementioned multiple flow paths B are two flow paths B that branch off to the left and right from the flow path A. The flow path substrate according to claim 45, wherein the plurality of flow paths D are four flow paths D that branch off to the left and right from the plurality of flow paths C.

51. Let the angle formed by the two flow paths B be the first angle. In the four flow channels D described above, when the angle formed by adjacent flow channels D is defined as the second angle (the second angle) and the angle formed by the other angle (the third angle), The flow channel substrate according to claim 50, wherein the first angle is greater than the second angle and the third angle.

52. The flow channel substrate according to claim 51, wherein the sum of the second angle and the third angle is greater than the first angle.

53. The first angle is greater than 90 degrees. The flow channel substrate according to claim 51, wherein the second angle and the third angle are less than 90 degrees.

54. The system further comprises multiple flow paths D branching off from each of the aforementioned multiple flow paths B, The channel substrate according to claim 40, wherein each channel D has a diameter narrower than the diameter of channel B.