WO2025069702A1 - Flow path substrate, cartridge, detection system, and method for manufacturing flow path substrate - Google Patents

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

This disclosure relates to flow path substrates, etc.

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

Japanese Patent Application Publication No. 2020-112383

The flow path substrate according to one embodiment of the present disclosure includes a liquid receiving section that receives a liquid and a flow path that is 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.

The method for manufacturing a flow path substrate according to one embodiment 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 in a position different from the first region of the flow path.

FIG. 1 is a schematic diagram illustrating an example of a detection system according to the present disclosure. 1A and 1B are schematic diagrams showing an example of a configuration of a cartridge of the present disclosure, and schematic diagrams showing a window portion of the cartridge of the present disclosure as viewed from above. 1A to 1C are schematic diagrams for explaining examples of use of the cartridge and detection device of the present disclosure. FIG. 1 is a block diagram illustrating an example of a detection system of the present disclosure. 1 is a plan view illustrating an example of a flow path substrate of a cartridge according to the present disclosure. 3 is a schematic diagram showing an example of a first region and a second region arranged in a flow channel provided in a flow channel substrate according to the first embodiment of the present disclosure. FIG. 5 is a flowchart showing an example of a method for manufacturing a flow channel substrate according to the first embodiment of the present disclosure. 5A to 5C are schematic diagrams showing an example of a method of applying a reagent to a flow channel provided in the flow channel substrate according to the first embodiment of the present disclosure. 1A and 1B are schematic plan views showing an example of a flow channel to which a reagent is applied, and schematic views showing another example of a method of applying the reagent to the flow channel. FIG. 11 is a plan view showing an example of a flow path substrate according to a second embodiment of the present disclosure. 13 is a schematic diagram showing an example of the arrangement of detection reagents in a flow channel provided in a flow channel substrate according to a second embodiment of the present disclosure. FIG. 13A to 13C are schematic diagrams for explaining examples of use of a plurality of flow channels provided in a flow channel substrate according to a second embodiment of the present disclosure. 10 is a flowchart showing an example of a manufacturing method of a flow path substrate 4B according to a second embodiment of the present disclosure. FIG. 13 is a perspective view of a flow path substrate according to a third embodiment. 15 is a cross-sectional view of the flow path substrate according to the third embodiment taken along a plane including line AA in FIG. 14. 16 is a partial enlarged view of a flow channel substrate according to a third embodiment, in a region surrounded by a dotted line in FIG. 15 . 17 is a partial enlarged view of a flow path substrate according to a fourth embodiment, in a region corresponding to FIG. 16 . 17 is a partial enlarged view of a flow path substrate according to a fifth embodiment, in a region corresponding to FIG. 16 . 17 is a partial enlarged view of a flow path substrate according to a sixth embodiment, in a region corresponding to FIG. 16 . 17 is a partial enlarged view of a flow path substrate according to a seventh embodiment, in a region corresponding to that in FIG. 16 . FIG. 4 is a diagram showing the surface roughness of a first deposit and the surface roughness of a second deposit.

One aspect of the present disclosure realizes a flow path substrate that can detect multiple substances by the simple operation of introducing liquid into multiple flow paths. According to one aspect of the present disclosure, it is possible to detect multiple substances by the simple operation of introducing liquid into multiple flow paths.

[Detection System]
Fig. 1 is a schematic diagram showing an example of a detection system 1 of 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 a cartridge 2 and a detection device 3.

The cartridge 2 is a test kit that detects a target substance contained in a specimen collected from a subject. The cartridge 2 may be a test kit that is capable of amplifying 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 portion 21 and a bottle portion 22.

The subject is not limited to humans, but may be any living organism capable of carrying a virus or bacteria, such as a mammal, bird, reptile, or amphibian.

The specimen may be saliva, urine, sweat, nasal mucus, blood, cells, etc. collected from the subject's body, or it may be soil and water collected from the object being tested, such as the ground, a river, or the sea. Alternatively, the specimen may be an attachment collected from the surface of the object being tested, such as a handrail, a door, clothing, shoes, or a toilet.

The detection target may be, for example, a virus or bacteria. The type of virus or bacteria 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 types of virus include influenza virus, coronavirus (e.g., SARS-CoV-2), respiratory syncytial virus (RS virus), human metapneumovirus, norovirus, HIV (Human Immunoficiency Virus), herpes virus, streptococcus, and Mycoplasma pominis.

The bottle portion 22 may contain a specimen collected from a subject. The main body portion 21 may be equipped with a flow path substrate 4 (see FIG. 5) that receives a liquid containing the specimen contained in the bottle portion 22 and contains a reagent that reacts with nucleic acid derived from the specimen when the specimen contains the target of detection. The nucleic acid derived from the target of detection may be extracted in the bottle portion 22, as will be described in detail later.

The detection device 3 is a device that detects nucleic acid derived from the detection target when the nucleic acid is amplified in the cartridge 2. The detection device 3 may have a housing capable of receiving the cartridge 2.

The detection device 3 may determine whether a certain concentration or more of nucleic acid derived from the detection target is present. The detection device 3 may determine that the sample is positive when a certain concentration or more of nucleic acid derived from the detection target is present. 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 from the detection device 3 corresponding to the amount of nucleic acid detected by the detection device 3.

The specific configuration of the cartridge 2 and the detection device 3 will be described later.

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

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 the NASBA substrate and enzymes to the template RNA results in amplified antisense single-stranded RNA. Since the entire series of steps in the NASBA reaction proceeds isothermally, the nucleic acid amplification reaction of RNA can be carried out without complex temperature control. In addition, since the amplified product of the NASBA reaction is single-stranded RNA, sequence-specific detection can be performed using a detection probe without going through a denaturation step.

[Specific configuration of the 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 a window portion 211 of the cartridge 2 as viewed from above. Fig. 3 is a schematic diagram for explaining an example of use of the cartridge 2 and the detection device 3.

As shown 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. Also, as shown 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.

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

As shown by the arrow at 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 the storage section 46. A reagent may be arranged in the flow path 45. Also, as shown at 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.

The flow path substrate 4 may have four flow paths 45, 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 paths 45A to 45D. The internal standard 47 may be located in a region different from the flow path 45 and used for comparison with a 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.

As shown by reference numeral 1102 in FIG. 2, the main body 21 is formed with a window 211 so that the storage sections 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 has multiple storage sections 46 and multiple internal standards 47, the window 211 may be formed so that all of the storage sections 46 and all of the internal standards 47 can be seen. In other words, the storage sections 46 and the internal standards 47 may be arranged on the main body 21 so as to be optically exposed from the window 211.

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. In addition, a lid may be provided at the connection section of the bottle section 22 with the main body 21. This allows the inside 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.

As shown by reference numeral 1111 in FIG. 3, when collecting saliva as a sample, for example, the subject inserts the sample collection portion 231 into the subject's mouth, thereby attaching saliva to the sample collection portion 231. As shown by reference numeral 1112 in FIG. 3, the subject attaches saliva to the sample collection portion 231, and then inserts the sampler 23 into the bottle portion 22.

The bottle portion 22 may contain a buffer solution 24. An example of the buffer solution 24 is a NASBA dissolving 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 non-ionic surfactant. When a non-ionic surfactant is contained in the buffer solution 24, the sample and the buffer solution 24 are mixed, and nucleic acid derived from the detection target can be extracted in the bottle portion 22.

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 can be contained in the bottle portion 22.

As shown by reference numeral 1113 in FIG. 3, the detection device 3 may include a receiving section 36 capable of receiving the cartridge 2. As shown 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 shown by reference numeral 1114 in FIG. 3. As a result, the buffer solution 24 mixed with the sample flows under its own weight through the sampler 23 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 received by the liquid receiving section 41.

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 by 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 by the bottle portion 22, and an example of a liquid that can be received by the liquid receiving portion 41.

[Specific configuration of the detection device]
Fig. 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 the 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.

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 acid 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 breaks down the pathogens and allows the nucleic acid components to be released from the pathogens. It also deactivates the DNA (deoxyribonucleic acid) decomposing enzyme. The heating unit 31 may then maintain the cartridge 2 at a temperature of 37°C to 41°C.

The heating unit 31 may heat each part of the cartridge 2 at a different temperature. For example, the heating unit 31 may have a first heating unit that heats the bottle portion 22 at a temperature of 80°C to 95°C, and a second heating unit that heats the main body portion 21 at a temperature of 37°C to 41°C. This allows the pathogens to be crushed in the bottle portion 22, and nucleic acid components to be liberated from the pathogens. Also, the DNA degrading enzyme can be inactivated in the bottle portion 22. The liquid in the bottle portion 22 then flows into the flow path substrate 4 provided in the main body portion 21, which is maintained at a temperature of 37°C to 41°C, and the nucleic acid may be amplified in the flow path substrate 4.

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 to be temperature-adjusted by the heating unit 31. When the temperature to which the heating unit 31 heats is the liquid temperature of the liquid to be temperature-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 of the liquid temperature over a predetermined time. The central temperature may be, for example, the temperature at the center between the maximum and minimum liquid temperatures. The central temperature may be calculated as the sum of the maximum and minimum temperatures divided by 2. The detection device 3 may be equipped with a temperature sensor that detects the liquid temperature of the liquid to be temperature-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.

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 using the pressurizing unit 32, the bottle portion 22 may be made of a material that is deformed by the pressurization of the pressurizing unit 32. The bottle portion 22 is deformed by the pressurization 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.

The pressurizing unit 32 may be a member that pressurizes at least a portion of the side of the bottle portion 22. The pressurizing unit 32 may be a member that clamps the side of the bottle portion 22. The pressurizing unit 32 may be disposed in a position in the detection device 3 where it can pressurize the side of the bottom side of the bottle portion 22, for example.

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

The flow channel 45 of the flow channel substrate 4 may be provided with a labeling substance that specifically binds to the nucleic acid amplified within the flow channel 45. The labeling substance may be optically observable, and may be, for example, 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.

Examples of fluorescent substances contained in the labeling substance 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
In the case of an acid, it emits fluorescence with a peak wavelength of 570 nm when irradiated with excitation light with a peak wavelength of 555 nm.

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 quenching molecule. The quenching molecule is set 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 quenching molecule in the probe DNA that is not bound to the nucleic acid molecule amplified by the enzyme is closer than the distance between the fluorescent molecule and the quenching molecule in the probe DNA that is bound to the nucleic acid molecule amplified by the enzyme. The base sequence of the probe DNA may be appropriately designed based on the base sequence of the nucleic acid to be detected.

The internal standard 47 may be anything 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. The internal standard 47 may be a fluorescent substance that emits fluorescence when irradiated with excitation light. 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.

The imaging unit 34 is a member 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, the imaging unit 34 may 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 brightness 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 the fluorescence emitted from the storage unit 46.

Furthermore, the imaging unit 34 may image the internal standard 47, and identify the position of the internal standard 47 in the captured image 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. The control unit 35 may then identify the position of the flow path 45 in the captured image based on the identified position of the internal standard 47.

Furthermore, the image capturing unit 34 may analyze the image of the internal standard 47 to determine whether optical information of a predetermined value or more 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 of a predetermined value or more can be measured. For example, the control unit 35 may determine that fluorescence of a predetermined intensity or more 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 a predetermined intensity or more can be received.

The determination of whether optical information equal to or greater than a predetermined value has been measured may be performed once after starting the detection device 3 and before measuring the optical information obtained from the specimen. 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 performed once after starting the detection device 3 and before measuring the intensity of the fluorescence emitted by the specimen.

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

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

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.

For example, the control unit 35 controls the light irradiation unit 33 to irradiate the storage unit 46 and the internal standard 47 with excitation light, and then analyzes the image captured by the imaging unit 34 to obtain the luminance. As a result, the control unit 35 may measure the intensity of the fluorescence emitted by the substances contained in the storage unit 46 and the internal standard 47, respectively.

The intensity measuring unit 351 may measure the optical information obtained from the storage unit 46 connected to the flow path 45 in which the reagent is placed as the optical information of the detection target. For example, the intensity measuring unit 351 may measure the intensity of the fluorescence emitted from the storage unit 46 connected to the flow path 45 in which the reagent is placed as the intensity of the fluorescence of the detection target. The intensity measuring unit 351 may also measure the optical information emitted from the storage unit 46 connected to the flow path 45 for the negative control as the optical information of the negative control. For example, the intensity measuring unit 351 may measure the intensity of the fluorescence emitted from the storage unit 46 connected to the flow path 45 for the negative control as the intensity of the 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 placed and the position of the flow path 45 for the negative control may be determined in advance.

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 sample 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 sample contained in the liquid stored in the storage unit 46 based on the intensity of the fluorescence emitted from the storage unit 46 measured by the intensity measurement unit 351. If the positive determination unit 352 determines that a certain concentration or more of nucleic acid derived from the detection target is present, it may determine that the sample is positive.

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 the fluorescence emitted from the storage unit 46 with the threshold value.

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 value, and determine that the sample is positive if the difference value is equal to or greater than the threshold value, and determine that the sample is negative if the difference value is less than the threshold value.

In addition, the positive determination unit 352 may determine whether the sample is positive by comparing the difference value with a value indicated by optical information obtained from the internal standard 47. For example, two internal standards 47 having different concentrations may be arranged. 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 the nucleic acid derived from the detection target that should be determined to be positive. The optical information obtained from the two internal standards 47 may be set in advance by experiments, etc.

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. If the difference value is within the range of the fluorescence intensities indicated by the two internal standards 47, the positive determination unit 352 may determine that the sample is positive.

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 intensity 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 the nucleic acid corresponding to the above-mentioned difference value. The positive determination unit 352 may determine that the sample is positive if the calculated concentration of the nucleic acid is equal to or greater than a reference value. The reference value may be a value associated with the concentration of the nucleic acid derived from the detection target that should be determined to be positive. The reference value may be set in advance by an experiment or the like.

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.

[First Form of Flow Channel Substrate]
Fig. 5 is a plan view showing an example of a flow path substrate 4A. The flow path substrate 4A is an example of the flow path 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 path 45 included in the flow path substrate 4A.

As shown in FIG. 5, the flow path substrate 4A may include a liquid receiving section 41 that receives 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 be in communication with each other.

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. Also, there may be one or more internal standards 47. In this embodiment, the flow paths 45 are described as having four flow paths 45A to 45D, and the storage sections 46 are described as having four storage sections 46A to 46D, but this is not limited to this. Also, the flow path substrate 4A is described as having five internal standards 47, but this is not limited to this.

In the flow path substrate 4A, in the state shown by reference numeral 1114 in FIG. 4, the liquid receiving section 41 is located above the flow path 45 and the storage section 46. Therefore, the liquid flowing from the bottle section 22 due to its own weight easily flows from the liquid receiving section 41 through the flow path 45 to the storage section 46.

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

The flow path substrate 4A may have a filter section 420. The filter section 420 may be a section whose flow path width is narrower than other sections. The filter section 420 may be located on the opposite side of the reservoir section 46 from the position of the reagent placed in the flow path 45. In other words, the filter section 420 may be located upstream of the position of the reagent placed in the flow path 45. When solid impurities are contained in the liquid flowing from the liquid receiving section 41, the possibility of the impurities flowing downstream of the filter section 420 can be reduced. In addition, the filter section 420 may be located upstream of the branching position of the flow paths 45A to 45D. As shown in FIG. 5, the branch flow path 42 may have a filter section 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 section 41.

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

As shown in FIG. 5, all of the flow paths 45A-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 have multiple liquid receiving sections 41. In this case, each flow path 45 may be connected to each liquid receiving section 41, or one or multiple flow paths 45 may be connected to each liquid receiving section 41.

The flow paths 45A to 45D may be flow paths for flowing liquid. At least one of the flow paths 45A to 45D may be used to flow a mixed fluid containing a specimen and a labeling substance that reacts with the target substance contained in the specimen. Each of the flow paths 45A to 45D may be a flow path that receives liquid flowing from the liquid receiving section 41 and flows it to the storage sections 46A to 46D that are connected to each of the flow paths 45A to 45D. Each of the flow paths 45A to 45D may be connected from the liquid receiving section 41 to the storage sections 46A to 46D.

In at least one of the flow paths 45A to 45D, the reagent may be disposed on the opposite side of the liquid receiving section 41 with respect to the branching position of the flow paths 45A to 45D. In this way, if 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.

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

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

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

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 detection target. The primer may be added as necessary 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 primer may be, for example, a first primer and a second primer. For example, the first primer may be a forward primer that amplifies the nucleic acid molecule to be detected in the sense direction, and the second primer may be a reverse primer that amplifies the nucleic acid molecule in the antisense direction.

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

6, the first reagent 101 may be disposed in the first region 451 as a plurality of first attachments. In the first region 451, a dried product of a solution containing the above-mentioned reagent may be disposed as an attachment of the first reagent 101. This allows the first reagent 101 to dissolve in the liquid when the liquid that has flowed into the flow path 45 comes into contact with the first attachment. Furthermore, when the first reagent 101 is disposed in the flow path 45 independently as a plurality of attachments, the surface area of the first attachment can be increased. Therefore, the solubility of the first reagent 101 in the liquid can be increased. The first reagent 101 may be disposed in the first region 451 as a plurality of films. The number of first attachments may be more than one, and may be, for example, 5 or more, or 10 or more.

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 first reagent 101 is arranged in one row as the multiple first attachments, but they may be arranged in two or more rows. In addition, 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.

The first attachment may have a dot shape when viewed in a plane, as shown in FIG. 6. In addition, the first attachment may have a linear shape, an elliptical shape, or a polygonal shape such as a square, hexagon, or star shape when viewed in a plane.

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

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

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 average of the measurement results.

The surface roughness Sa of the flow path 45 can be obtained from the measurement results of the entire flow path 45. In this case, the surface roughness Sa of the flow path 45 may be obtained from the measurement results of the entire bottom of the flow path 45. It may also be obtained from the measurement results of any part of the flow path 45. When multiple measurement results of any part of the flow path 45 are obtained, the surface roughness of the flow path 45 may be obtained by the arithmetic average of the measurement results.

Furthermore, the first attachment 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 attachment.

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

As shown in FIG. 6, the second reagent 102 may be disposed in the second region 452 as a plurality of second attachments. In the second region 452, a dried product of a solution containing the above-mentioned reagent may be disposed as an attachment of the second reagent 102. This allows the second reagent 102 to dissolve in the liquid when the liquid that has flowed into the flow path 45 comes into contact with the second attachment. When the second reagent 102 is disposed in the flow path 45 independently as a plurality of attachments, the surface area of the second attachment can be increased. Therefore, the solubility of the second reagent 102 in the liquid can be increased. The second reagent 102 may be disposed in the second region 452 as a plurality of films. The number of second attachments may be more than one, and may be, for example, 5 or more, or 10 or more.

As shown in FIG. 6, the multiple 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 multiple 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 multiple second attachments, but they may be arranged in two or more rows. In addition, the multiple 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.

The second attachment may have a dot shape when viewed in a plane, as shown in FIG. 6. In addition, the second attachment may have a linear shape, an elliptical shape, or a polygonal shape such as a square, hexagon, or star shape when viewed in a plane.

In FIG. 21, the figure indicated by the reference numeral 2103 is an enlarged photograph of the second adhesion, and the graph indicated by the reference numeral 2104 shows the surface height of the flow path 45 at the locations where the second adhesion is adhered and at the locations where the second adhesion is not adhered. The locations where the surface height is 0.000 μm are the surfaces of the flow path 45, and the locations where the surface height is greater than 0.000 μm are the surfaces where the second adhesion is adhered.

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

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 part of any second attachment. When multiple measurement results of any part of any second attachment are obtained, the surface roughness of the second attachment may be determined by the arithmetic average of the measurement results.

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

The surface roughness of the second attachment may be greater than the surface roughness of the flow path 45. In this case, the second attachment 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 attachment.

As shown in FIG. 5, the second region 452 may be disposed on the opposite side of the liquid receiving section 41 with respect to 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 then the second reagent 102.

As shown in FIG. 5, the second region 452 may be disposed closer to the storage section 46 than the first region 451. In other words, the first region 451, the second region 452, and the storage section 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 section 46.

If the sample contains the target of detection, the liquid containing the nucleic acid derived from the target of detection can first be brought into contact with the first reagent 101. This allows the nucleic acid derived from the target of detection to be annealed with the first primer and the second primer. After that, 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 of detection.

The first region 451 may contain a first primer and a second primer as the first reagent 101. Therefore, for example, dNTP and NTP may be placed in the second region 452 as the second reagent 102. Even with this arrangement, it is possible to amplify the nucleic acid derived from the detection target contained in the liquid flowing through the flow path 45.

Also, as shown in FIG. 6, the second attachment may be larger than the first attachment. For example, the first attachment and the second attachment may be disposed in 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.

The total area of the multiple first attachments can be determined by the amount of solution containing the first reagent 101. The total area of the multiple second attachments can be determined by the amount of solution containing the second reagent 102. For example, the total area of the multiple first attachments can be approximately twice the total area of the multiple second attachments.

The width of the flow path 45 may be, for example, 220 μm. In this case, if the first attachment is dot-shaped, its diameter may be smaller than the width of the flow path 45. The diameter of the first attachment may be, for example, 50 to 100 μm. The interval between the first attachments may be 100 to 200 μm. If the second attachment is dot-shaped, its diameter may be smaller than the width of the flow path 45. The diameter of the second attachment may be, for example, 80 to 200 μm. The dot interval between the second attachments may be 200 to 250 μm. Also, as shown in the graphs 2102 and 2104 in FIG. 21, the diameter of the second attachment may be larger than the first attachment. The heights shown in the graphs 2102 and 2104 can be considered to represent the heights of the first attachment and the second attachment, respectively. The diameter of the first deposit illustrated in the graph with reference numeral 2102 is 14.193 μm, whereas the diameter of the second deposit illustrated in the graph with reference numeral 2104 is 19.816 μm. In addition, the height of the second deposit may be greater than that of the first deposit.

For example, if the amount of liquid containing a specimen contained in the bottle portion 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 amount per attachment is approximately 0.7 nL, the number of first attachments will be 69 and the number of second attachments will be 34. The amounts of these liquids and solutions are less than when a PCR (Polymerase Chain Reaction) tube is used. In other words, by using the flow path substrate 4A, the amount of liquid containing the specimen and the application (printing amount) of the first reagent 101 and the second reagent 102 applied to the flow path 45 can be reduced.

The amounts of the first reagent 101 and the second reagent 102 may vary depending on the detection target. Therefore, the amount of solution of the first reagent 101, the number of first attachments, the amount of solution of the second reagent 102, and the number of second attachments may be changed depending on the detection target. In addition, 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 path 45 in the extension direction and the width of the flow path 45.

As described above, the nucleic acid contained in the liquid flowing in from the liquid receiving section 41 may first react with the first reagent 101 arranged in the first region 451, and then react with the second reagent 102 arranged in the second region 452. This is because the first reagent 101 anneals to the nucleic acid contained in the liquid in order to amplify the nucleic acid contained in the liquid with the second reagent 102. Also, as described above, when the cartridge 2 is once heated to a high temperature and then cooled to a temperature that promotes the amplification of the nucleic acid, the nucleic acid that reacted with the first reagent 101 may be cooled before reacting 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. Also, consider a case where the heating section 31 heats the bottle section 22 to a temperature of 80°C to 95°C and heats 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 flows from the bottle portion 22 through the liquid receiving portion 41 comes into contact with the second region 452 after the temperature of the liquid has sufficiently decreased.

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 reagent in one linear portion of the flow path 45, such as the first region 451 shown in FIG. 6, the reagent may be disposed in two or more linear portions.

The flow path 45 may further contain a labeling substance that specifically binds to the amplified nucleic acid. 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 the fluorescence emitted by the nucleic acid derived from the detection target by irradiating the storage section 46 with excitation light.

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 the nucleic acid amplified by the enzyme contained in the second reagent 102. The labeling substance may be a molecular beacon.

The liquid flowing through one of the flow paths 45A to 45D may function as a negative control. Figure 5 shows an example in which the flow path 45A functions as a negative control flow path.

The flow path 45A may not have the first region 451, but may have at least a labeling substance. In this embodiment, as shown in FIG. 5, the flow path 45A may have at least a second region 452 in which a labeling substance is disposed. This makes it possible to prevent the nucleic acid from reacting with a primer for amplifying the nucleic acid even if the liquid flowing through the flow path 45A contains nucleic acid derived from the detection target. Therefore, the detection device 3 can detect the intensity of fluorescence emitted by a substance other than the nucleic acid derived from the detection target 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 detection target that has been amplified by reacting with the first reagent 101 and the second reagent 102 in the flow path 45B, using the intensity of the fluorescence as a reference.

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 to detect nucleic acids derived from the detection target may be connected to the branch flow path 42. In addition to flow path 45A, flow paths 45C and 45D may also be flow paths that are not intended to detect nucleic acids derived from the detection target.

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 placed or the position where the reagent can be placed. This allows the liquid in which the reagent is dissolved to flow into 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 be reacted with the reagent efficiently.

It is sufficient that a bending region 453 is formed in the flow path 45 in which the first reagent 101 and the second reagent 102 are placed. In this embodiment, it is sufficient that a bending region 453B is formed at least in the flow path 45B.

The storage section 46 may be connected to the flow path 45. As shown in FIG. 5, the storage sections 46A to 46D may be connected to the flow paths 45A to 45D, respectively. The storage section 46 may store the liquid that flows from the flow path 45. The storage section 46 may be part of the flow path.

When the flow path 45 has a first region 451 and a second region 452, the storage section 46 may be located downstream of the second region 452 and store a liquid containing the amplified nucleic acid. In this embodiment, the flow path 45B has a first region 451 and a second region 452. Therefore, the storage section 46B may be located downstream of the second region 452B and store a liquid containing the amplified nucleic acid.

The storage section 46 can store the liquid that flows from the flow path 45. In the storage section 46 connected to the flow path 45 having the first region 451 and the second region 452, if the liquid contains nucleic acid derived from the detection target, the liquid containing the amplified nucleic acid can be stored. Therefore, the detection device 3 can image the amplified nucleic acid in a stable state.

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

As shown in FIG. 5, the first distance D1 between two adjacent storage sections 46 among the storage sections 46A-46D may be greater than the second distance D2 between the two flow paths 45 connected to each of the two storage sections 46. This allows the spacing between the two adjacent storage sections 46 to be relatively wide. Therefore, the detection device 3 can easily obtain the intensity of the fluorescence emitted from each storage section 46 through image analysis.

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

When either the first reagent 101 or the second reagent 102 is placed in the storage section 46, the first reagent 101 or the second reagent 102 different from the reagent placed in the storage section 46 may be placed in the flow path 45 located upstream compared to the storage section 46. For example, when the second reagent 102 is placed in the storage section 46, the first reagent 101 may be placed in the flow path 45 located upstream compared to the storage section 46. That is, the first region 451 may be located in the flow path 45 located upstream compared to the storage section 46, and the second region 452 may be located in the storage section 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 in that order.

(Other configurations)
In addition, the flow path substrate 4A may have a second storage portion 48 downstream of the storage portions 46A to 46D, which is connected to each 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 liquid flows into the storage portion 46 in an amount equal to or greater than the capacity of the storage portion 46, the liquid leaking out of the storage portion 46 can be stored in the second storage portion 48. This reduces the possibility of liquid flowing back from the storage portion 46 into the flow path 45.

Also, the second storage section 48 may be provided with a substance that functions as a positive control. For example, a substance that functions as a positive control may be provided only in the second storage section 48 of the flow path substrate 4A used as a positive control. The substance may be optically compared with the storage section 46, and may be, for example, a dye, a luminescent substance, or a fluorescent substance, but is not limited to this. In addition, in order to verify whether the detection system 1 functions normally, a sample that intentionally contains the detection target may be flowed through the flow path 45 via the liquid receiving section 41 before measuring the optical information of the sample to be determined, and the optical information corresponding to the nucleic acid derived from the detection target may be measured in the second storage section 48. As shown by the reference numeral 1102 in FIG. 2, the second storage section 48 can be viewed through the window section 211 of the main body section 21.

Furthermore, in at least one of the branched flow paths 45, the flow path substrate 4A may have a sensing substance located at a position farther from the liquid receiving section 41 than the storage section 46, which detects the passage of the liquid flowing through the flow path 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 is 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 arranged, the sensing substance dissolves. Therefore, the luminance of the sensing substance decreases. By checking the decrease in the luminance of the sensing substance, the progress of the liquid can be confirmed. In other words, based on whether the luminance of the sensing substance has decreased, it is possible to determine whether the liquid flowing through the flow path 45 is flowing normally. The detection substance is not particularly limited as long as it can confirm the passage of liquid flowing through the flow channel 45.

The second storage section 48 only needs to be connected to at least one of the storage sections 46A to 46D. However, the flow path substrate 4A does not need to have the second storage section 48.

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

(Reagent placement position)
In this embodiment, when the first reagent 101 and the second reagent 102 are placed in the flow path 45, at least a primer and an enzyme may be 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. Also, for example, the standard substance may be placed downstream of the flow path 45. For example, the standard substance may be placed in the storage section 46.

(Number of flow paths, etc.)
In this embodiment, the flow path substrate 4A includes four flow paths 45, but is not limited thereto. In this embodiment, the flow path substrate 4A may include at least one flow path 45 having a first region 451 and a second region 452. In this embodiment, when the flow path substrate 4A includes a plurality of flow paths 45 having a first region 451 and a second region 452, these flow paths 45 may function as flow paths used to detect the same detection target. That is, the first reagent 101 arranged in the plurality of flow paths 45 may be the same reagent, and the second reagent 102 arranged in the plurality of flow paths 45 may be the same reagent. In addition, when the flow path substrate 4A includes a plurality of flow paths 45 having a first region 451 and a second region 452, these flow paths 45 may function as flow paths used to detect different detection targets. That is, the first reagent 101 arranged in the plurality of flow paths 45 may be different reagents, and the second reagent 102 arranged in the plurality of flow paths 45 may be different reagents.

The flow path substrate 4A may have at least one flow path 45 for a positive control. That is, it may have a flow path 45 for flowing a liquid that is 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 that is intentionally containing a detection target that reacts with the first reagent 101 and the second reagent may be flowed in the flow path 45 for the positive control. However, the flow path 45 having the first region 451 and the second region 452 used for flowing a liquid containing a sample may be substituted as the flow path 45 for the positive control.

The number of storage sections 46 may be determined by the number of flow paths 45 to which they are connected.

(Position of internal standard)
In this embodiment, the internal standard 47 is disposed on the flow path substrate 4A, but is not limited to this. The internal standard 47 may be disposed on the surface of the housing of the cartridge 2, instead of on the flow path substrate 4A. In this case, the internal standard 47 may be disposed near the window portion 211 of the main body portion 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.

[Method of manufacturing the flow path substrate according to the first embodiment]
7 is a flow chart showing an example of a method for manufacturing the flow path substrate 4 A. This manufacturing method may be performed by a manufacturing apparatus for manufacturing the flow path substrate 4 A.

As shown in FIG. 7, first, the liquid receiving portion 41 and the flow path 45 may be formed on the substrate (S1; formation process). In this embodiment, in addition to the liquid receiving portion 41 and the flow path 45, a branch flow path 42, a storage portion 46, a second storage portion 48, and a discharge port 49 may be formed on the substrate.

The liquid receiving portion 41 and the flow path 45 may be formed, for example, as follows. For example, a mold in which the shapes of the liquid receiving portion 41 and the flow path 45 are patterned is placed on a substrate, and then resin is poured in. After the resin has hardened, the mold is removed. After the mold is removed, a lid is placed on the hardened resin, thereby forming the liquid receiving portion 41 and the flow path 45 on the substrate.

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 placement step). The first reagent 101 may be placed in the flow path 45B by applying the first reagent 101 to the flow path 45B. Also, in this embodiment, a second reagent 102 may be placed in the flow paths 45A and 45B (S3; second placement 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.

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 placed in the second storage section 48 by applying the fluorescent substance to the second storage section 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.

FIG. 8 is a schematic diagram showing an example of a method of applying a reagent 100 to a flow path 45 provided in a flow path substrate 4A. Reference numeral 1131 in FIG. 9 is a schematic plan view showing an example of a 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 of applying the reagent 100 to the flow path 45. The reagent 100 in FIG. 8 and FIG. 9 may be the first reagent 101 or the second reagent 102.

The application of the reagent 100 may be performed, for example, using an inkjet printer. Using an inkjet printer allows for 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.

The inkjet printer may apply the reagent 100 while moving the head 201 along the extension direction of the flow channel 45, thereby adhering multiple reagents 100 to the bottom 454 of the flow channel 45, as shown 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 the multiple deposits are placed 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 path 45.

As shown 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 multiple attachments are arranged independently side by side in a direction different from the extension direction of the flow channel 45. The multiple attachments may be arranged side by side, for example, in the width direction of the flow channel 45. Also, as shown by reference numeral 1132 in FIG. 9, new reagent 100 may be applied from above the dried reagent 100 arranged in the flow channel 45.

[Second embodiment of flow path substrate]
Fig. 10 is a plan view showing an example of a flow path substrate 4B. Fig. 11 is a schematic diagram showing an example of the arrangement of detection reagents in a flow path 45 included in the flow path substrate 4B. The flow path substrate 4B is an example of the flow path substrate 4.

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 storage sections 46A-46D connected to each of the plurality of flow paths 45. 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 makes it possible to detect a plurality of detection targets that are different from each other at once by the simple operation of introducing liquid into the flow path substrate 4B.

In this embodiment, as shown in FIG. 10, the flow path substrate 4B may include a liquid receiving section 41, a branch flow path 42, a plurality of flow paths 45A-45D, a plurality of storage sections 46A-46D, and an internal standard 47. The flow path substrate 4B may also include a second storage section 48 and an outlet 49.

The flow paths 45A-45D may branch off from a branch flow path 42 connected to the liquid receiving section 41. However, the flow path substrate 4B does not have to have a branch flow path 42. In this case, the flow paths 45A-45D may branch off from the liquid receiving section 41. By having the flow paths 45A-45D branch off 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 multiple flow paths 45A-45D. Therefore, multiple different detection targets can be detected at once without the need to introduce liquid into each of the multiple flow paths 45A-45D.

Furthermore, all of the flow paths 45A-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-45D. Therefore, multiple different detection targets can be detected at once by introducing the liquid once.

The detection reagent may be disposed on the opposite side of the liquid receiving section 41 from the branching position of the flow paths 45A to 45D. This reduces the possibility of different detection reagents mixing. For example, it reduces the possibility of the first detection reagent and the second detection reagent mixing.

Flow path substrate 4B differs from flow path substrate 4A in that the multiple flow paths 45 have a first flow path and a second flow path. In the following, only the differences between flow path substrate 4B and flow path substrate 4A will be described. In other words, the contents of flow path substrate 4B that were described for flow path substrate 4A will not be described in this embodiment.

In this 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.

As shown in FIG. 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.

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 there. 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 storage section 46. Here, "at least a portion of the detection reagent" may refer to some 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 storage section 46. Furthermore, substantially the same components of the detection reagent may be located in both the flow path 45 and 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 storage section 46.

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 necessary 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 the 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.

However, the first reagent 1012 may be a primer having a different sequence from 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 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 makes it possible to react the second detection target, which is different from the first detection target that reacts with the first reagent 1011, with the first reagent 1012. The first detection target and the second detection target are examples of detection targets.

Therefore, if the liquid flowing from the liquid receiving section 41 into the flow path 45B contains a first detection target, the first detection target can be amplified in the flow path 45B. If the liquid flowing from the liquid receiving section 41 into the flow path 45C contains a second detection target, the second detection target can be amplified in the flow path 45C. In other words, the liquid received in the liquid receiving section 41 can be introduced into multiple flow paths 45, and multiple different targets can be used as detection targets to amplify nucleic acids at once.

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

Also, as shown in FIG. 11, a second reagent 1021 may be placed in the second region 452B as the first detection reagent. A second reagent 1022 different from the second reagent 1021 may be placed in the second region 452C as the second detection reagent. 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.

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 the second reagent 1021.

This allows the second detection object, which is different from the first detection object that reacts with the second reagent 1021, to react with the second reagent 1022. Therefore, if the first detection object is contained in the liquid that has flowed from the liquid receiving section 41 into the flow path 45B, the first detection object can be amplified in the flow path 45B. If the second detection object is contained in the liquid that has flowed from the liquid receiving section 41 into the flow path 45C, the second detection object can be amplified in the flow path 45C.

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

The flow path 45 may further include a labeling substance disposed therein as a detection reagent. When the substances to be detected in each flow path 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 path 45B and the labeling substance disposed in flow path 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.

Furthermore, the flow path substrate 4B may have a flow path 45 that does not function as the first flow path or the second flow path. In other words, 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 may be connected to the branch flow path 42.

The flow path substrate 4B may include a negative control flow path 45 in which at least a labeling substance is arranged and at least one of the elements used in nucleic acid amplification, such as an enzyme that amplifies nucleic acids, is not arranged. In FIG. 10, the flow path 45A may be a negative control flow path in which at least a labeling substance is arranged. The flow path 45A may have a second region 452A in which a second reagent 102 containing a labeling substance is arranged. The flow path substrate 4B may also include a flow path 45 in which no detection reagent is arranged. In FIG. 10, the flow path 45D may be a flow path in which no detection reagent is arranged.

When the types of substances to be detected in each storage section 46 are different, 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 storage sections 46 need to be 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.

The same reagent may be located on the opposite side of the liquid receiving section 41 in the multiple flow paths 45 in which the different detection reagents are arranged. In other words, the same reagent may be located downstream of the position in the flow path 45 where the different detection reagents are arranged. For example, as described above, in the flow paths 45B and 45C, the enzyme as the same reagent may be located in the second region 452B and 452C located downstream of the first region 451B and 451C in which the primer is arranged. In addition, in the flow paths 45B and 45C, the dNTP and NTP as the same reagent may also be located in the second region 452B and 452C, for example. However, the dNTP, NTP, RNase inhibitor, trehalose, and surfactant as the same reagent may be located in the liquid receiving section 41 and/or the branch flow path 42.

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 delivery direction to the end on the storage section 46 side may be approximately the same. In other words, in each flow path 45, the length of the area in which the detection reagent is arranged 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 area in which the detection reagent is arranged 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.

As shown in FIG. 11, in this embodiment, lengths LB and LC in flow paths 45B and 45C may be approximately the same as each other. Length LB is the length from the end of first region 451B opposite storage section 46B to the storage section 46B side in second region 452B. Length LC is the length from the end of first region 451C opposite storage section 46C to the storage section 46C side in second region 452C. Length L1B of first region 451B in which first reagent 1011 is arranged and length L1C of first region 451C in which first reagent 1012 is arranged may be approximately the same as each other or may be different from each other. In addition, 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 approximately the same as each other or may be different from each other.

FIG. 12 is a schematic diagram for explaining an example of the use of multiple flow paths 45. As shown in FIG. 12, for example, flow path 45B may have a first region 451B in which first reagent 1011 is arranged, and a second region 452B in which second reagent 1021 is arranged. Flow path 45C may have a first region 451C in which first reagent 1012 is arranged, and a second region 452C in which second reagent 1022 is arranged. Flow path 45D may have a first region 451D in which first reagent 1013 is arranged, and a second region 452D in which second reagent 1023 is arranged. The first reagents 1011, 1012, and 1013 may be detection reagents different from each other. The second reagents 1021, 1022, and 1023 may be detection reagents different from each other. In addition, flow channel 45A may be a negative control flow channel in which at least a labeling substance is disposed, and at least one of the elements used in nucleic acid amplification, such as an enzyme that amplifies nucleic acids, is not disposed.

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 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 flow path 45C may be placed in flow path 45D instead of the first reagent 1013 and the second reagent 1023.

In other words, the flow path substrate 4B only needs to have 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 multiple flow paths 45.

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

The detection device 3 can therefore detect a first detection target by measuring the intensity of the fluorescence emitted from the storage section 46 connected to the first flow path. The detection device 3 can also detect a second detection target by measuring the intensity of the fluorescence emitted from the storage section 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.

The flow path substrate 4B may have 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 have a flow path 45X having a first region 451X in which a first reagent 1014 is arranged, and a second region 452X in which a second reagent 1024 is arranged. 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 equal to the number of flow paths 45 in which different detection reagents are arranged.

All of the first reagents 1011-1014 do not have to be different from each other. Any two of the first reagents 1011-1014 may be different detection reagents. In other words, some of the first reagents 1011-1014 may be the first detection reagent, and another part may be the second detection reagent. Also, all of the second reagents 1021-1024 do not have to be different from each other. Any two of the second reagents 1021-1024 may be different detection reagents. In other words, some of the second reagents 1021-1024 may be the first detection reagent, and another part may be the second detection reagent.

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

[Method of manufacturing a flow path substrate according to the second embodiment]
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.

As shown in FIG. 13, first, a flow path 45 and a storage section 46 may be formed in a substrate (S11). In this embodiment, in addition to the flow path 45 and the storage section 46, a liquid receiving section 41, a branch flow path 42, a second storage section 48, and an outlet 49 may be formed in the substrate.

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, which is one of the flow paths 45A to 45D and 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 storage section 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.

〔summary〕
The flow path substrate according to aspect 1 of the present disclosure comprises a liquid receiving section for receiving 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 for amplifying nucleic acid is disposed at a position different from the first region.

The flow path substrate according to aspect 2 of the present disclosure is the same as that of aspect 1, except that the second region is disposed on the opposite side of the liquid receiving section from the position where the first region is disposed.

In the flow path substrate according to aspect 3 of the present disclosure, in aspect 1 or 2, the flow path further includes a reservoir downstream of the second region for storing a liquid containing the amplified nucleic acid.

A flow path substrate according to a fourth aspect of the present disclosure is any one of the first to third aspects, wherein the first reagent is disposed in the first region as a plurality of first attachment objects.
A flow path substrate according to a fifth aspect of the present disclosure is similar to the fourth aspect, in that the surface roughness of the first attachment is greater than the surface roughness of the flow path.

The flow path substrate according to aspect 6 of the present disclosure is the same as in aspect 4 or 5, in which the first attachments are arranged as a coating.

The flow path substrate according to aspect 7 of the present disclosure is any one of aspects 4 to 6, in which the multiple first attachments are aligned in one direction.

The flow path substrate according to aspect 8 of the present disclosure is any one of aspects 4 to 7, in which the first attachments are disposed at the bottom of the flow path so as not to contact the inner wall of the flow path.

The flow path substrate according to aspect 9 of the present disclosure is any one of aspects 1 to 8, in which the second reagent is disposed in the second region as a plurality of second attachments.

The flow path substrate according to aspect 10 of the present disclosure is the same as aspect 9, in that the surface roughness of the second attachment is greater than the surface roughness of the flow path.

The flow path substrate according to aspect 11 of the present disclosure is the same as that of aspect 9 or 10, in which the second attachment is arranged as a coating.

The flow path substrate according to aspect 12 of the present disclosure is any one of aspects 9 to 11, in which the multiple second attachments are aligned in one direction.

The flow path substrate according to aspect 13 of the present disclosure is any one of aspects 9 to 12, in which the second attachments are disposed at the bottom of the flow path so as not to contact the inner wall of the flow path.

The 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 disposed in the second region as a plurality of second attachments, and the second attachments are larger than the first attachments.

The flow path substrate according to aspect 15 of the present disclosure is the flow path substrate according to claim 9, in which the first reagent is disposed 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.

The flow channel substrate according to aspect 16 of the present disclosure is any one of aspects 1 to 13, in which a labeling substance that specifically binds to the amplified nucleic acid is further disposed in the flow channel.

The flow path substrate according to aspect 17 of the present disclosure is the same as in aspect 16, in which the labeled substance is disposed downstream of the first region.

The flow path substrate according to aspect 18 of the present disclosure is the same as that of aspect 16 or 17, in which the second reagent includes the labeling substance.

The flow path substrate according to aspect 19 of the present disclosure is any one of aspects 16 to 18, in which the labeling substance is a molecular beacon.

The cartridge according to aspect 20 of the present disclosure comprises a flow path substrate according to any one of aspects 1 to 19 and a container capable of holding a liquid.

The detection system according to aspect 21 of the present disclosure includes the cartridge according to aspect 20 and a detection device that detects the nucleic acid amplified in the flow path.

The method of manufacturing a flow path substrate according to aspect 22 of the present disclosure includes a forming step of forming a liquid receiving section for receiving a liquid and a flow path connected to the liquid receiving section 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 in a position on the flow path different from the position where the first reagent is disposed.

The method of manufacturing a flow path substrate according to aspect 23 of the present disclosure is, in aspect 22, the first placement step is to place the first reagent in the flow path by applying a solution containing the first reagent to the flow path, and the second placement step is to place the second reagent in the flow path by applying a solution containing the second reagent to the flow path.

A flow path substrate according to Aspect 24 of the present disclosure is any one of Aspects 1 to 19, wherein the flow path is a plurality of flow paths, and the flow path substrate further includes a plurality of reservoirs connected to each of the plurality of flow paths;
The multiple flow paths include 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.

The flow channel substrate according to aspect 25 of the present disclosure is any one of aspects 1 to 19, in which the flow channel is a flow channel through which a mixed fluid containing a specimen and a labeling substance that reacts with the detection target contained in the specimen flows, and further includes a standard portion that is located in a region different from the flow channel and is compared with the mixed fluid.

The flow path substrate according to aspect 26 of the present disclosure is, in aspect 1, a plurality of flow paths through which a liquid flows, and includes a plurality of reservoirs connected to 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.

The flow path substrate according to aspect 27 of the present disclosure is the same as in aspect 26, in which the multiple flow paths branch off from the liquid receiving section or a flow path connected to the liquid receiving section.

The flow path substrate according to aspect 28 of the present disclosure is the same as in aspect 27, in which all of the multiple flow paths branch off from one of the liquid receiving sections or from a flow path connected to the one liquid receiving section.

The flow path substrate according to aspect 29 of the present disclosure is the same as that of aspect 27 or 28, in which the detection reagent is disposed on the opposite side of the liquid receiving section with respect to the branching position of the multiple flow paths.

In the flow path substrate according to aspect 30 of the present disclosure, in any one of aspects 26 to 29, the detection reagent includes a primer having a sequence corresponding to the detection target.

The flow path substrate according to aspect 31 of the present disclosure is the same as in aspect 30, in which the second detection reagent includes the primer having a different sequence from the first detection reagent.

The flow path substrate according to aspect 32 of the present disclosure is any one of aspects 26 to 31, in which the same reagent is arranged in the multiple flow paths.

The flow path substrate according to aspect 33 of the present disclosure is the same as that of aspect 32, in which the same reagent includes an enzyme that amplifies nucleic acid.

The flow path substrate according to aspect 34 of the present disclosure is in aspect 32 or 33, and further includes a liquid receiving section for receiving liquid, and the same reagent is located on the opposite side of the flow path in which the detection reagent is disposed from the liquid receiving section.

In the flow path substrate according to aspect 35 of the present disclosure, in any of aspects 26 to 34, the lengths of the flow paths from the end of the detection reagent opposite the storage portion to the end of the storage portion along the liquid transport direction are approximately the same for each of the multiple flow paths.

The flow path substrate according to aspect 36 of the present disclosure is any one of aspects 26 to 35, further comprising 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.

The flow path substrate according to aspect 37 of the present disclosure is the same as in aspect 36, except that the filter section is disposed upstream of the branching position of the multiple flow paths.

In the flow path substrate according to aspect 38 of the present disclosure, in any of aspects 26 to 36, the first distance between two adjacent storage sections among the plurality of storage sections is greater than the second distance between the two flow paths connected to each of the two storage sections.

The flow path substrate according to aspect 39 of the present disclosure is any one of aspects 26 to 38, in which each of the multiple flow paths has multiple bending regions downstream of the detection reagent.

<Other embodiments>
Conventional flow path substrates have room for improvement in terms of fluid control. According to the present disclosure, it is possible to improve the control of fluid in a flow path substrate.

Other embodiments of the present disclosure will be described below with reference to the drawings. In this specification, unless otherwise specified, "A to B" indicating 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 ease 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 as the downward direction, the positive direction of the X axis as the rightward direction, and the negative direction of the X axis as the leftward direction.

Third Embodiment
Hereinafter, the flow path substrate 1A according to the third embodiment will be described 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 specimen. A flow path 10 is formed inside the flow path substrate 1A. When a specimen is introduced inside the flow path substrate 1A, the specimen and the reagent are mixed inside the flow path substrate 1A. The specimen mixed with the reagent is temporarily stored in the detection unit 13. For example, "storage" refers to the specimen remaining in a certain area relative to the flow inside the flow path 10, but is not limited to this.

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, a dog, a cat, or a 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.

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 a probe DNA or a probe RNA designed to detect the target substance or a nucleic acid contained in the target substance. For example, the reagent may include a primer DNA or an enzyme for amplifying the target substance or a nucleic acid contained in the target substance.

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, spherical, concave, or convex surface in part. For example, the flow path substrate 1A may be located in whole or in part 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.

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 branch section 15A. The inlet hole 11, the reagent dissolving section 12, the detection section 13, the outlet hole 14, and the branch section 15A are part of the flow path 10. The inlet hole 11 can introduce a specimen into the flow path substrate 1A. The reagent dissolving section 12 is where the reagent is located and can dissolve the reagent in the specimen. The detection section 13 can store the specimen mixed with the reagent. The flow path substrate 1A has a plurality of detection sections 13. The outlet hole 14 can discharge the specimen 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 a plurality of outlet holes 14. The branch section 15A branches the flow path 10 into a plurality of paths.

The following is an overview of detecting a target substance contained in a sample using the flow path substrate 1A. First, the sample is introduced into the flow path from the introduction hole 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 the 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.

The flow path substrate 1A further has a filter section 16 located between the introduction hole 11 and the branch section 15A. By having the filter section 16, the flow path substrate 1A can separate solids or air bubbles contained in the specimen. As a result, the risk that solids or air bubbles contained in the specimen will interfere with the detection of the target substance in the detection section 13 can be reduced.

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

For example, the material of the flow path substrate 1A may be resin, glass, ceramic, or metal. For example, the material of the flow path substrate 1A may be cyclic olefin polymer, cyclic olefin copolymer, acrylic resin, or unstretched polypropylene. The flow path substrate 1A may be composed 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.

(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 into one of the faces of the flow path substrate 1A. The introduction hole 11 opens into a first face 17 of the flow path substrate 1A.

The shape of the introduction hole 11 may be selected from any shape. For example, the space inside the introduction hole 11 has a truncated cone shape. For example, the space 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, which is opposite the first surface 17. For example, the opening of the introduction hole 11 is approximately circular. For example, the opening of the introduction hole 11 may be elliptical, rectangular, hexagonal, or octagonal.

(Reagent dissolving section 12)
The reagent dissolving section 12 can dissolve the reagent in the specimen. The reagent dissolving section 12 can dissolve the reagent in the specimen flowing through the flow path. In the reagent dissolving section 12, the reagent is located on the bottom surface of the flow path. In the reagent dissolving section 12, the reagent may be located on the side surface of the flow path. The reagent dissolving section 12 communicates with the flow path D155 of the branching section 15A on the side opposite to the flow path C154.

The shape of the reagent dissolving section 12 may be selected arbitrarily. For example, the reagent dissolving section 12 has a straight or curved shape. The curved shape of the reagent dissolving section 12 may be an arc or L-shape. For example, the reagent dissolving section 12 of this embodiment has a shape in which arc shapes and straight shapes are repeated alternately. 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 change. For example, the width of the flow path of the reagent dissolving section 12 may be wider only in a portion.

(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 on the first surface 17 side and the surface on the second surface 18 side of the detection unit 13 may be translucent. The detection unit 13 communicates with the flow path D155 of the branching unit 15A on the side opposite to the flow path C154.

(Outlet hole 14)
The outlet hole 14 can lead the analyte from inside the flow path substrate 1A to the outside of the flow path substrate 1A. The outlet hole 14 can lead the analyte 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 faces of the flow path substrate 1A. The outlet hole 14 opens to a first face 17 of the flow path substrate 1A.

The shape of the outlet hole 14 may be selected arbitrarily. For example, the space inside the outlet hole 14 has a truncated cone shape. For example, the space inside the outlet hole 14 may have a triangular pyramid shape, a square pyramid shape, a cylindrical shape, a square prism shape, or the like. The outlet hole 14 has a shape in which the cross-sectional area of the outlet hole 14 decreases from the first surface 17 toward the second surface 18, which is opposite the first surface 17. The opening of the outlet hole 14 is approximately circular in shape. For example, the opening of the outlet hole 14 may be a square shape, a hexagon shape, an octagon shape, or the like.

(Branch portion 15A)
The branching section 15A branches the flow path of the flow path substrate 1A into a plurality of paths. For example, the branching section 15A branches the flow path of the flow path substrate 1A into four paths. The number of paths to be branched is not limited to this, and can be set to any number. The branching 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 the flow path extending from the branching section 15A having a first flow path extending up and down, 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, but is not limited thereto. For example, "branching the flow path to the left and right" also includes the case where the flow path extending from the branching section 15A has a first flow path extending to the left and right, a second flow path extending above the first flow path, and a third flow path extending below the first flow path.

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 locations within the flow path 10. This makes it possible to easily detect a target substance under multiple conditions.

As shown in FIG. 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 that the branching portion 15A has is not limited to this, and can be set to any number.

In the branching portion 15A, the width of each of the multiple flow paths B152 is narrower than the width of flow path A151. For example, the ratio of the width of flow path A151 to the width of 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 flow path B152 is narrower than the width of flow path A151, the width and depth of flow path A151 and the width and depth of flow path B152 may be set to any value. For example, the width of flow path A151 may be 200 μm to 1000 μm. For example, the depth of flow path A151 may be 25 μm to 200 μm. For example, the width of flow path B152 may be 20.0 μm to 500 μm. For example, the depth of flow path B152 may be 25.0 μm to 200 μm.

The flow path A151 is a flow path connected to the liquid receiving section 41. The flow path A151 can pass a specimen introduced from the introduction hole 11. The flow path A151 is connected to the filter section 16. The flow path A151 can pass a specimen that has flowed out from the filter section 16. The width of the flow path A151 is constant. The width of the 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. The "extension direction of the flow path" refers to the direction in which the spaces formed by the flow paths are continuously located. For example, the "extension direction of the flow path" refers to the direction in which the fluid flows when the fluid is made to flow in the flow path, but is not limited to this.

The multiple flow paths B152 are flow paths of the multiple flow paths. The flow path B152 is a flow path branched off from the flow path A151. The flow path B152 can pass the specimen flowing out from the flow path A151. The flow path B152 can pass a part of the specimen 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.

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

The thickness 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 flow path A151. For example, the ratio of the thickness of flow path A151 to the sum of the thicknesses of flow paths B152 (sum of the thicknesses of flow paths B152/thickness of 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 flow path C154. For example, the ratio of the thickness of flow path C154 to the sum of the thicknesses of flow paths B152 (sum of the thicknesses of flow paths B152/thickness of flow path A151) may be 1.50 or more.

The first branch wall 153 is located in the 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 flow path B152. In this embodiment, the first branch wall 153 is continuous with the side surface of flow path B152. In this embodiment, the first branch wall 153 is a surface of the side surface of flow path 10 that is continuous with the side surface of flow path B152 and is located upstream of the inlet of flow path B152. The first branch wall 153 is not continuous with the side surface of flow path A151.

The first branch wall 153 overlaps with a first virtual straight 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 less 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 in this embodiment has a flat surface and a curved surface. The first branch wall 153 in 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.

The branching portion 15A further has a flow path C154. The branching portion 15A has a plurality of flow paths C154. In this embodiment, the branching portion 15A has two flow paths C154. The branching portion 15A has the same number of flow paths C154 as the plurality of flow paths B152. The number of flow paths C154 that the branching portion 15A has may be different from the number of flow paths B152 that the branching portion 15A has.

Flow path C154 is connected to 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. Flow path C154 is a flow path extending from flow path B152. Flow path C154 can pass a sample that has flowed out from flow path B152. Each of the multiple flow paths C154 can pass a sample that has flowed out from each of the multiple flow paths B152.

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

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

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.

The branching portion 15A further includes a flow path D155. The flow path D155 is a flow path branched off from the flow path C154. The flow path D155 can pass the specimen flowing out from the flow path C154. The flow path D155 can pass a portion of the specimen flowing out from the flow path C154.

The branching section 15A has a plurality of flow paths D155. In this embodiment, the branching section 15A has four flow paths D155. The number of flow paths D155 in the branching section 15A is not limited to this and can be set to any number. The plurality of flow paths D155 are flow paths branched off from the flow path C154. The plurality of flow paths D155 are flow paths branched off 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 off to the left and right from the flow path C154.

The width of flow path D155 may be thicker or thinner than the width of flow path A151. In this embodiment, the width of flow path D155 is thinner than the width of flow path A151. In this embodiment, the width of each of the multiple flow paths D155 is thinner than the width of flow path A151. 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 0.800 or less.

The width of flow path D155 may be thicker or thinner than that of flow path B152. In this embodiment, the width of flow path D155 is thinner than that of flow path B152. In this embodiment, the width of each of the multiple flow paths D155 is thinner than that 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.

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

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.

In the branching section 15A, the sum of the lengths of the multiple flow paths B152 and the multiple flow paths C154 is smaller than the sum of the lengths of the flow paths A151. In the branching section 15A, the sum of the lengths of the multiple flow paths B152 and the multiple flow paths C154 is smaller than the sum of the lengths of the multiple flow paths D155 and the reagent dissolving section 12.

In the branching section 15A of this embodiment, the angle formed by the two flow paths B152 is defined as the first angle. The first angle may be, for example, 90.0 degrees or more. 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 the second angle and the 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 may be different angles.

(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 in the flow channel 10.

(Flow of sample at branching portion 15A)
The flow of the specimen in the branching section 15A will be described below. In the flow path substrate 1A, the multiple flow paths B152 branch off from the flow path A151 in a direction that does not overlap with the first virtual straight line. Therefore, the risk that most of the specimen flowing out of the flow path A151 will directly flow into the flow path B152 is low. In addition, in the flow path substrate 1A, the first branching wall 153 overlaps with the first virtual straight line. Therefore, most of the specimen flowing out of the flow path A151 will collide with the 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 straight line and when there is no first branching wall, the risk that the specimen will flow only into one of the multiple flow paths B152 can be reduced.

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

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 straight line. Also, in the flow path substrate 1A of this embodiment, the first branch wall 153 overlaps with the first virtual straight line. Also, 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 any of the multiple flow paths B152, and will flow only in the other flow paths B152.

Fourth Embodiment
The flow path substrate 2A according to the fourth embodiment will be described below. The flow path substrate 2A is different from the flow path substrate 1A in the configuration of the branching section 25A. As shown in FIG. 17, the thicknesses of the multiple flow paths B252 are different. Of the two flow paths B252, one is thinner than the other. The thicknesses of the multiple flow paths D255 are different. Of the four flow paths D255, the thicknesses of the two flow paths D255 connected to one flow path B252 are thinner than the thicknesses of the two flow paths D255 connected to the other flow path B252. Of the four flow paths D255, the thicknesses of the two flow paths D255 connected to a flow path B252 thinner than one flow path B252 are thinner than the thicknesses of the two flow paths D255 connected to the other flow path B252. In the flow path substrate 2A of this embodiment, it is also possible to facilitate the flow of the specimen into all the flow paths B252.

Fifth Embodiment
The flow path substrate 3A according to the fifth embodiment will be described below. 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 a configuration 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. In the flow path substrate 3A of this embodiment, it is also possible to facilitate the flow of the specimen into all of the flow paths B352A.

Sixth Embodiment
The flow path substrate 4A according to the sixth embodiment will be described below. 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 corresponding to the flow path C154 in the flow path substrate 1A. The branching section 45E has a plurality of flow paths D455E branched 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. In the flow path substrate 4A of this embodiment, it is also possible to make it easier for the specimen to flow into all of the flow paths B452E. In addition, in the flow path substrate 4A of this embodiment, it is also possible to make it easier for the specimen to flow into all of the flow paths D455E.

Seventh Embodiment
The flow path substrate 5A according to the seventh embodiment will be described below. 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, the multiple flow paths D555 are branched from only one flow path C554. The two flow paths D555 are branched from only one flow path C554. The multiple flow paths D555 are connected only to one flow path C554. The length of one flow path C554 is longer than the length of the other flow path C554. In the flow path substrate 5A of this embodiment, it is also possible to facilitate the flow of the specimen into all the flow paths B552.

The invention according to this disclosure has been described above based on the drawings and examples. However, the invention according to this disclosure is not limited to the above-mentioned embodiments. In other words, the invention according to this disclosure can be modified in various ways within the scope of this 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 this disclosure. In other words, it should be noted that a person skilled in the art can easily make various modifications or corrections based on this disclosure. It should also be noted that these modifications or corrections are included in the scope of this disclosure.

REFERENCE SIGNS LIST 1 Detection system 2 Cartridge 3 Detection device 4, 4A, 4B Flow path substrate 22 Bottle portion (container)
41 Liquid receiving portion 42 Branch flow path (flow path)
45 flow path (first flow path, second flow path)
46 Reservoir 47 Internal standard (standard part)
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 section 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 Branch section 151 Flow path A
152, 252, 352A, 452E, 552 Flow path B
153 First branch wall 154, 354 Flow path C
155, 255, 455E, 555 Channel D
157 Second branch wall 16 Filter section

Claims (54)

A liquid receiving portion for receiving liquid;
A flow path connected to the liquid receiving portion,
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. The flow path substrate according to claim 1, wherein the second region is disposed on the opposite side of the liquid receiving portion with respect to the position where the first region is disposed. The flow channel substrate according to claim 1 or 2, wherein the flow channel further comprises a reservoir downstream of the second region for storing a liquid containing the amplified nucleic acid. The flow path substrate according to any one of claims 1 to 3, wherein the first reagent is disposed in the first region as a plurality of first attachments. The flow path substrate according to claim 4, wherein the surface roughness of the first attachment is greater than the surface roughness of the flow path. The flow path substrate according to claim 4 or 5, wherein the plurality of first attachments are arranged as a film. The flow path substrate according to any one of claims 4 to 6, wherein the first attachments are arranged in one direction. The flow path substrate according to any one of claims 4 to 7, wherein the first attachments are disposed at the bottom of the flow path so as not to contact the inner wall of the flow path. The flow path substrate according to any one of claims 1 to 8, wherein the second reagent is disposed in the second region as a plurality of second attachments. The flow path substrate according to claim 9, wherein the surface roughness of the second attachment is greater than the surface roughness of the flow path. The flow path substrate according to claim 9 or 10, wherein the second attachment is disposed as a coating. The flow path substrate according to any one of claims 9 to 11, wherein the second attachments are arranged in one direction. The flow path substrate according to any one of claims 9 to 12, wherein the second attachments are disposed at the bottom of the flow path so as not to contact the inner wall of the flow path. the second reagent is disposed in the second region as a plurality of second deposits;
The flow path substrate according to claim 4 , wherein the second attachment is larger than the first attachment. the first reagent is disposed on the first region as a plurality of first deposits;
The flow path substrate according to claim 9 , wherein a surface roughness of the second deposit is smaller than a surface roughness of the first deposit. The flow channel substrate according to any one of claims 1 to 13, further comprising a labeling substance disposed in the flow channel that specifically binds to the amplified nucleic acid. The flow path substrate according to claim 16, wherein the labeling substance is disposed downstream of the first region. The flow channel substrate according to claim 16 or 17, wherein the second reagent includes the labeling substance. The flow channel substrate according to any one of claims 16 to 18, wherein the labeling substance is a molecular beacon. A flow path substrate according to any one of claims 1 to 19;
A cartridge comprising: a container capable of containing a liquid. A cartridge according to claim 20;
a detection device for detecting amplified nucleic acid in the flow path. A forming step of forming a liquid receiving portion for receiving a liquid and a flow path connected to the liquid receiving portion on the substrate;
a first disposing step of disposing a first reagent including a primer in the flow channel;
and a second placing step of placing a second reagent containing an enzyme for amplifying nucleic acid at a position in the flow channel different from the position at which the first reagent is placed. In the first disposing step, a solution containing the first reagent is applied to the flow path to dispose the first reagent in the flow path;
The method for manufacturing a flow path substrate according to claim 22 , wherein in the second disposing step, the second reagent is disposed in the flow path by applying a solution containing the second reagent to the flow path. The flow path is a plurality of flow paths,
Further comprising a plurality of reservoirs connected to the plurality of flow paths,
The plurality of flow paths include
a first flow path in which at least a portion of a first detection reagent that is a detection reagent for detecting a detection target is located;
The flow path substrate according to claim 1 , further comprising: a second flow path in which at least a portion of a second detection reagent that is a detection reagent for detecting a detection target different from the first detection reagent is located. the flow path is a flow path through which a mixed fluid containing a specimen and a labeling substance that reacts with a detection target contained in the specimen flows;
The flow path substrate according to claim 1 , further comprising a standard portion located in an area different from the flow path and for comparing with the mixed fluid. The flow path is a plurality of flow paths through which a liquid flows,
A plurality of reservoirs connected to the plurality of flow paths,
The plurality of flow paths include
a first flow path in which at least a portion of a first detection reagent that is a detection reagent for detecting a detection target is located;
The flow path substrate according to claim 1 , further comprising: a second flow path in which at least a portion of a second detection reagent that is a detection reagent for detecting a detection target different from the first detection reagent is located. 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. The flow path substrate according to claim 27, wherein all of the multiple flow paths branch off from one of the liquid receiving sections or from a flow path connected to the one liquid receiving section. The flow path substrate according to claim 27, wherein the detection reagent is disposed on the opposite side of the liquid receiving portion with respect to the branching position of the plurality of flow paths. The flow channel substrate according to claim 26, wherein the detection reagent includes a primer having a sequence corresponding to the substance to be detected. The flow channel substrate according to claim 30, wherein the second detection reagent includes a primer having a different sequence from the first detection reagent. The flow path substrate according to claim 26, wherein the same reagent is disposed in the multiple flow paths. The flow channel substrate according to claim 32, wherein the same reagent includes an enzyme that amplifies nucleic acid.
The flow path substrate according to claim 32 , wherein the identical reagent is located on an opposite side to the liquid receiving section in the flow path in which the detection reagent is located. The flow path substrate according to claim 26, wherein the lengths of the detection reagent from the end opposite the reservoir to the end on the reservoir side along the liquid delivery direction of the detection reagent are substantially the same for each of the multiple flow paths. The filter portion is a portion having a narrower flow path width than other portions,
The flow path substrate according to claim 26 , wherein the filter section is located on an opposite side of the storage section with respect to a position of the detection reagent. The flow path substrate according to claim 36, wherein the filter section is disposed upstream of a branching position of the plurality of flow paths. The flow path substrate according to claim 26, wherein a first distance between two adjacent storage portions among the plurality of storage portions is greater than a second distance between two flow paths connected to each of the two storage portions. The flow path substrate according to claim 26, wherein each of the multiple flow paths has multiple bend regions downstream of the detection reagent. A flow path A;
A plurality of flow paths B branching off from the flow path A in a direction not overlapping a first virtual line overlapping the flow path A;
a first branch wall located in a region where the plurality of flow paths B branch off and continuous with the flow path B;
The flow paths B each have a smaller width than the flow path A, and the first branch wall overlaps with the first virtual line,
The flow path A is a flow path connected to the liquid receiving portion,
The flow path substrate according to claim 27 , wherein the plurality of flow paths B are the plurality of flow paths. The flow path substrate according to claim 40, wherein the sum of the widths of the multiple flow paths B is smaller than the width of the flow path A. The liquid ejection device further includes a plurality of flow paths C located on the opposite side of the flow paths A with respect to the plurality of flow paths B and extending from each of the plurality of flow paths B,
The flow path substrate according to claim 40 , wherein each of the flow paths C is larger in width than each of the flow paths B. The flow path substrate according to claim 42, wherein the sum of the widths of the multiple flow paths B is greater than the width of each of the flow paths C. The flow path substrate according to claim 42, wherein the sum of the widths of the multiple flow paths C is smaller than the width of the flow path A. Further, a plurality of flow paths D branch off from the flow path C in a direction not overlapping with a second virtual line overlapping with the flow path C,
The flow path substrate according to claim 42 , wherein each of the flow paths D has a width smaller than a width of the flow path C, and a second branching wall located in the region where the multiple flow paths D branch off overlaps with the second virtual straight line. The flow path substrate according to claim 45, wherein the width of each of the flow paths D is smaller than the width of each of the flow paths B. The flow path substrate according to claim 45, further comprising a reagent dissolving section located on the opposite side of the flow paths C with respect to the plurality of flow paths D, extending from each of the plurality of flow paths D, and in which a reagent to be dissolved in the fluid is located. The liquid supply device further includes a plurality of detection units capable of storing a fluid,
The flow path substrate according to claim 45 , wherein each of the plurality of detection units is in communication with the flow path D on an opposite side to the flow path C. The flow path substrate according to claim 47, wherein the sum of the length of flow path B and the length of flow path C is smaller than the sum of the length of flow path A and the sum of the length of flow path D and the length of the reagent dissolving section. The plurality of flow paths B are two flow paths B branched 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 branching off to the left and right from the plurality of flow paths C. The angle between the two flow paths B is a first angle,
In the four flow paths D, when the angle of one of the angles formed by the adjacent flow paths D is defined as a second angle and the angle of the other angle is defined as a third angle,
The flow path substrate of claim 50 , wherein the first angle is greater than the second angle and the third angle. The flow path substrate of claim 51, wherein the sum of the second angle and the third angle is greater than the first angle. the first angle is greater than 90 degrees;
52. The flow path substrate of claim 51, wherein the second angle and the third angle are less than 90 degrees. Further comprising a plurality of flow paths D branching off from each of the plurality of flow paths B,
The flow path substrate according to claim 40 , wherein each of the flow paths D is thinner than each of the flow paths B.

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